WRRC Bulletin 46

Aquatic Plants from Minnesota Part I - Chemical Survey

by

K Lee Su

and

E John Staba

Department ofPharmaeognosy

University of Minnesota

The work upon which this publication is based was

supported in part by funds provided by the

United States Department of the Interior as

authorized under the Water Resources Research

Act of 1964 Public Law 88-379

FEBRUARY 1972

Minneapolis Minnesota

WATER RESOURCES HESEARCH CENTER

UNIVERSITY OF MINNESOTA

PageCONTENTS

D Saponins 30

E Steroids 30

F Lipids 35

Page

II INTRODUCTION 1

A Objective of the Study 1 IV REFERENCES 112 B Phytochemical Screenings 1

1 Alkaloid 2 2 Flavonoid 2 3 Tannin 3 4 Saponin 4 5 Steroid 4 6 Lipid 5

C Review of the Literature 6

1 General considerations 6 2 Alkaloids 6 3 Flavonoids bull 9 4 Tannins saponins steroids and lipids 9

II MATERIALS AND METHODS 9

A Plant Collection and Identification 9

B Extraction 11

C Detection for Alkaloids 12

1 Purification of extracts 12 2 TIlin-layer chromatography 12

D Detection for Flavonoids 13

E Detection for Tannins 13

F Detection for Saponins 14

1 Hemolysis test 14 2 Froth test 15

G Detection for Steroids 15

H Lipid Analysis 15

1 Gravimetric determination of total lipids 15 2 Systemic analysis of lipid distribut ion 16 3 Fatty acid constituents of triglycerides isolated

from selected plant extracts 16 a Isolation of trig1ycerides 16 b Methanolysis bull bull bull bull 17 c Hydrogenation bull bull 17 d Gas-liquid chromatography (GLC) 17

III RESULTS AND DISCUSSIONS 19

A Alkaloids 19

B Flavonoids 23

C Tannins 23

iiiIi

ILLUSTRATIONS

Figure Page

1 Purification of Skellysolve F Chloroform and 80 ethanol

extracts for the alkaloid detection

2 Procedure for the detection of tannins

13

14

3 Standard graph of log retention time vs carbon number of

reference fattyacids bullbullbullbullbullbullbull 18

Table

1

2

3

4

5

6

7

8

9

10

11

TABLES

Page

Composition of Fatty Acids from Some Aquatic Plants (GLC

Area )(75) bullbullbull 7

List of Plant Collected 8

Thin-layer Fluorescent and Alkaloid Patterns 20

Thin-layer Fluorescent and Flavonoid Patterns 24

Presence of Tffi1nins in Minnesotan Aquatic Plants 28

Detection of Saponins by Homolysis and Froth Tests 31

Thin-layer Fluorescent and Steroid Patterns 32

Gravimetric Determination of Total Lipids 34

Systemic Analysis of Lipid Distribution bull 38

Constituent Fatty Acids of Triglycerides Derived From Selected

Aquatic Plants 40

Comparison of Major Fatty Acids in Aquatic and Terrestrial

Plants (Linseed and Soybean) bull 41

iv v

FOREWORD

This Bulletin is published in furtherance of the purposes of the Water Resources Research Act of 1964 The purpose of the Act is to stimulate sponsor provide for and supplement present programs for the conduct of research investigations experiments and the training of scientists in the field of water and resources which affect water The Act is promoting a more adequate national program of water resources research by furnishing financial assistance to non-Federal research

The Act provides for establishment of Water Resources Research Centers at Universities throughout the Nation On September 1 1964 a Water Reshysources Research Center was established in the Graduate School as an intershydisciplinary component of the University of Minnesota The Center has the responsibility for unifying and stimulating University water resources reshysearch through the administration of funds covered in the Act and made avail shyable by other sources coordinating University research with water resources programs of local State and Federal agencies and private organizations throughout the State and assisting in training additional scientists for work in the field of water resources through research

This Bulletin is number 46 in a series of publications designed to present information bearing on water resources research in Minnesota and the results of some of the research sponsored by the Center The Bulletin is concerned with the results of a survey of selected aquatic plants in Minnesota conducted in anticipation of finding compowlds which might be useful in medicine The survey is part of a research project aimed at findshying a nutritional medicinal or industrial use for the unwanted aquatic plants in lake shoreline areas It is possible that some aquatic plants may contain industrially useful g~q-mucilages or new useful antimicrobial anticoagulant or antineoplastic therapeutic principles If a good indusshytrial medicinal or nutritional use for aquatic plants can be discovered the results of the research could provide an economic incentive for aquatic plant collection and control The successful completion of the project may significantly assist the State and Nation in partially solving their lake pollution problems

This Bulletin is related to the following research project

OWRR Project No A-025-Minn

Project Title Alleviation of Lake Pollution by Utilization of Aquatic Plants for Nutritional Medicinal or Industrial Purposes

Principal Investigator E John Staba Dept of Pharmacognosy College of Pharmacy University of Minnesota

Project Began July 1 1970 Scheduled Completion June 30 1973

FCST Research Category V-E

in Minnesota have not been surveyed medicinally for useful chemical compounds A study was conducted with a reasonable antici shypation of finding compounds such as alkaloids flavonoids tannins saponins steroids and lipids which might be useful in medicine Examination of chemshyical constituents was accomplished on the following plants collected from

vi

Taxonomic identification of aquatic plants was made and exhaustive exshytraction using solvents ranging from the non-polar to polar type was followshyed to determine the nature of the various constituents present in the aquatic plants Detection for compounds involved purification of extracts thinshylayer chromatography hemolysis test froth test gravimetric determination methanolysis and hydrogenation

Thin-layer chromatographic detection studies indicated original extracts did not appear to contain alkaloids Several plant species demonstrated Dragendorff positive spots Flavonols were most wideIv distributed in the plant extracts studies Tannins especially the condensed type were widely distributed in the plants screened Five species of plants are saponin posishytive Beta-sitosterol was tentatively identified as being present 8 species The lipid contents of 3 species may be considered for their nutritional value

Publication Descriptors Aquatic Plants Chemical Compounds Medicine Minnesota Alkaloids Flavonoids Tannins Saponins Steroids Lipids Phytochemical Screening Chromatography

Publication Identifiers Hemolysis Test Froth Test Methanolysis Hydrogenation Dragendorff Positive Spots Beta-sitosterol

vii

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I INTRODUCTION

A Objective of the Study

Higher and lower forms of plants have and arc contributing important drugs for the physicians use (1) Higher plants are being studied for their anti-neoplastic (2-5) antimicrobial (6-22) and other pharmacologishycal activities (2324) The lower forms such as the plankton algae are known to produce antibiotic substances (2526) the blue-green algae are believed to produce a cyclic polypeptide endotoxin (27) Aquatic plants can be considered in some ways to be botanically and ecologically related to both the higher plants and the algae They may also be considered nuisances which often bring about biological excesses that arc inimical to recreational and other water uses (28)

The aquatic plants in general and the Minnesotan aqlkltic plants in particular have not been surveyed medicinally for useful chemical comshypounds Therefore this series study was done with a reasonable anticishypation of finding compounds (such as alkaloids flavonoids tannins saponins steroids and lipids) which might be useful in medicine

eolExamination of chemieal constituents were

leeted from various lakes done on the following plants

B Phytoehemieal Screenings

Phytoehemieal screening programs have most often been direeted toward finding alkaloids flavonoids tannins saponins steroids and lipids The extraction sehemes for screening are normally one of the following two

i) Selective sequential extraction dependant upon the polarity of the solvent For example the dried powdered plant materials are extrActed first with petroleum ether (non-polar solvent) then with chloroform ethashynol and finally with water (polar solvent)

i1) Non-seleetive extraetion with hydroalcoholic solvents sueh as 50 or 80ethanol and subsequent fractionation of the ethanolie extract with organie solvents under different conditions

Artifacts caused by thermo-oxidation chemical oxidations and enzymati c hydrolysis should be avoided during the extraction

In order to explain the complexity of screening programs an introshyduction to the chemistry of constituents most often found in plants together with the problems whieh might be encountered in the screening are presented

1

1 Alkaloid

Alkaloids are widely distributed in the plant kingdom (29-35) and even in some bacteria (36) Wall (37-40) Webb (35) and Hultin (4142) are among those who have extensively screened vascular plants for alkashyloids

Alkaloids are natural products that are physiologically active posshysess basic or sometimes neutral properties and often contain heterocyclic nitrogen The definition is broad and great variations in chemical strucshytures are possible ranging from simple primary amines to very complex inshydole compounds The chemical groups of alkaloids include phenylalkylashymine (i e ephedrine) purine (caffeine) pyridine (evonine) pyrrol idine (tropinone) pyridine-pyrrolidine (nicotine) condensed piperidine-pyrrolishydine (atropine) quino ine (cinchonine) isoquinol ine (morphine) and indole (ergoclavine)

Therefore in general the plant materials are extracted either with acidic water to extract the alkaloids as salts or by addition of base to the plant material in order to extract the free base by an organic solvent Plants containing weakly basic or neutral alkaloids such as rutaecarpine colchicine and ricinine would not be extracted into the organic solvent and special approaches must be developed for those alkaloids Further purification of the plant extract may be necessary to avoid the extraction of false-positive reacting alkaloids such as those reported LJr some proteins and non-nitrogenous compounds (1314)

Alkaloid color reagents are used to spray on chromatograms in order to visualize alkaloid spots The most commonly used ones are Dragendorffs iodoplatinate antimony trichloride and cerium sulfate (in sulfuric acid or in phosphoric acid) (45)

2 Flavonoid

The flavonoid compounds in the plant kingdom are confined almost enshytirely to the flowering plants and ferns (6) Chemically flavonoids may be described as plant pigments containing two C6 groups (substituted benzene rings) connected by a three carbon aliphatic chain (C6-C Examples of the chemical classes of flavonoids are the catechLns leucoanthocyanidines flavanones flavanonols flavones flavonols and anthocyanidins A slight variation of C6-C 3-C6 patterns is seen in chalshycones dihydrochalcones aurones and isoflavones where the central pyran ring is open modified into a bem~alcoumaranone ring or the ring substishytution is shifted [rom C2 to Biologically flavonoids possess extremeshyly diverse pharmacological for instance quercitrin has an antishyviral effect (47) rutin is a capillary antihemorrhagic eupatomin has antishycancer activity (48) and flavone has bactericidal properties (9)

Flavonoids are often extracted by methanol aqueous ethanol of actone (50) Many color reactions can be used to characterize the different classes of [lavonoids and many of those color reactions have been reviewed by Seikel (51)

2

3 Tannin

According to Swain (52) the term tannin II can be applied to naturally occurring compound of high molecular weight (between about to 3000) containing a sufficiently large number of phenolic hydroxyl or other suitable groups (1-2 per 100 MW) to enable it to form effective cross-links between proteins and other macromolecules

Tannins may be divided st ructurally into the following two dist inc t classes

i) Hydrolyzable tannins consist of a polyhydric alcohol esterified with gallic acid or derivatives of gallic acid such as ellagic acid Tannins having this structure can readily be hydrolyzed by acids bases or enzymes (tannin acylhydrolases) Gallotannins and ellagitannins are included in this group

ii) Condensed tannins cont ain phenolic nuclei which when treated with the above hydrolytic reagents do not hvdrolyze but instead polymerize to yield insoluable amorphous and often red colored phlobaphenes An exshyample for tannins of this type is catechin a polymer of flavin-3-ols

Both the hydrolyzable and condensed tannins are widely distributed in nature An extensive survey of the occurrence of tannins in the plant kingdom was done hy Bath-Smith (53) One interesting observation found was that ellagic acid is absent from non-vascular plants ferns gy~losperms and monocotyledons

Tannins may be precipitated from their solutions hy different salts Salts of heavy metals (Ag 7n Cu Sn) precipitate tannins indiscriminashytively neutral lead salts precipitate tannins possessing adjacent phenolic hydroxyl groups while lead sub acetate precipitates tannins wi th non-adj ashycent hydroxyl groups (51) Wall (37) found that preCipitation with lead acetate occurred in every plant extract examined whether or not tannin was found by other tests Therefore tannins are separated from other plant constituents by the solvent cxtraction method Tannins are often exshytracted from plant materials with hot water and then salted out with sodshyium choloride Wall (37) detected tannins in the hot aqucous extracts preshypared from the dried 95 alcoholic extract Pcrsinos et al (55) directly used the 80 alcohol extract for tannin detection in their pharmacognostishycal study of Nigerian plants

Tannins can be detected by using a 1 solution of gelatin containing 10 sodium chloride (56) This test is based on the protein-binding capashycity of the tannin Since the condition of pH and ionic strength are crishytical Farnsworth (57) modified the test by using buffered gelatin-sal t reagent Another detecting agent is 1 ferric chloridc solution which either precipitates tannins or forms different colors ranging from blue blue-black green to bluc-green It should be recognizcd that many other phenolic compounds form colors with fcrric choloride solution Because of tannins polymcr and high molecular weight nature it usually runs as a streak on paper or thin-layer chromatograms (58) As an alternativc the nature of tannin is determined by acid or alkaline hydrolysis of the tannin extract and subsequent chromatographic detection of the building uni ts (gallic ellagic acid etc) in the hydrolysate

3

4 Saponin

Saponins are widely distributed among higher plants (59) Bioshygenetically saponins can be divided into two groups

i) Glycosides of triterpenoid alcohols (normally at the 3-position) such as oleanane

ii) Glycosides of a particular steroid structure having a spiroketal side chain at the E and F ring juncture A few of the steroidal sapongenins are distinguished by having a ring juncture

Due to the sugar residue at 3-beta-hydroxyl group both types of saposhynins are soluble in water and ethanol but insoluble in ether Their aglyshycones (sapogenins) without the sugar moiety possess the solubilitv charshyacteristics of other sterols Thus saponins are most conveniently~ extracted from plants with 70-95 hot ethanol or isopropanol

Saponins are most often detected by

i) Foam test Saponins are presumed to be present when the charactershyistic honeycomb froth persists for at least 30 minutes after shaking an aqueous boiled (3-5 minutes) plant material

ii) Hemolytic test Saponin containing plant extracts mixed with defibrinated blood in a buffered physiological saline solution cause the hemolysis of red blood cells Digitonin a saponin available in crystalshyline form is generally used as the standard Tannins will interfere with the hemolytic action of saponins as they form a protective coating around the red blood cells The removal of tannins with magnesium salt previous to performing the hemolytic test was suggested by Farnsworth (57)

Both triterpenoid and steroid saponins behave the same in those two tests Tf necessary the Liebermann-Burchard test may be used to differshyentiate them red pink or purple colors are developed with triterpenoid saponins whereas blue or blue-green colors are formed with steroidal saponins (60)

5 Steroid

Steroids are derivatives of the tetracyclic hydrocarbon cyclopentanshyoperhydrophenanthrene The steroids thus far discovered in plants include sterols certain sapogenins alkaloids cardenolides and hormones (61)

i) Sterols are hydroxysteroids of C27 C28 and C29 series (62) An example of C27 sterol is cholesterol which is not only present in animals but has also been isolated from algae by Tsuda et al (63) from potato and Dioscorea plant by Johnson et al (64) A common sterol is ergoshyC28 sterol and widely distributed C29 sterols are beta-sitosterol and stigmasshyterol

ii) Alkaloids The occurrence of steroidal alkaloids is limited mostly to Solanum Veratrum and Holarhena (65) species They contain a 5-mernbered E ring and a 6-mernbered nitrogen containing F ring

4

iii) Cardiac glycosides They are glycosides of either C23 (cardenoshyJides) or C24 (bufadienolides) steroids with potent cardiac activity They are structurally characterized by an unsaturated lactone ring at Cl7

uncture of ring C and D a l4-beta-hydroxy group and by the pecushysugars (such as D-digitoxose D-digitalose D-cymarose and D-sarmenshy

tose) attached at

iv) Hormones Pregnane (C2l) androstane (C19 ) (66) and estrane (C18 ) (67) derivatives have been isolated from plants

Steroids can be extracted with benzene or ether and the chromatographic technique is of great value to detect plant materials for steroids A very good article reviewing the application of TLC for steroid detection has been written by Neher (68) Among the reagents most used and most generally apshyplicable are suI furic aci d-alcohol chlorosul phonic acid-acetic acid and molybdophosphoric acid Anisaldehyde-sulfuric acid reagent is used for deshytection of many steroids it is non-specific but with high sensitivity Dinitrobenzoic acid (Kedde reagent) and m-dinitrobenzene are specific for the detection of cardenolides and 17-ketosteroids respectively cerium sulfate is specific for the detection of nitrogen-containing steroids Keller (ferric chloride-acetic acid) and Kiliani (ferric sulfate-sulfuric acid) are specific for the deoxy sugar moiety of cardiac glycosides Frerejacque and DeGraeve (69) had tabulated all the cardiac glycoside deshytecting agents and their methods of preparation The use of iodine as a non-destructive location reagent for steroids in TIC has been introduced by Stevens (70) This technique is very valuable for preparative thin- and thick-layer chromatography

6 Lipid

Since triglycerides have been studied in this thesis only the saponishyfiable lipids will be discussed The saponifiable lipids are classified according to their structures into the fo11owing categories fatty acids fatty acid esters (triglycerides waxes etc) phospholipids and glycoshylipids

Non-polar lipids are extracted with petroleum ether or benzene and polar lipids are extracted with isopropanol isopropanol-chloroform methshyanol-chloroform ethanol-chloroform or ethanol-ether In addition to the avoidance of thermo- chemical and enzymatic oxidation and hydrolysis lipid extraction should be carried out under nitrogen to prevent the oxishydation of unsaturated compounds The extracts can be fractionated by TLC into different lipid classes According to Mangold (71) lipids can be visualized on the chromatograms with iodine vapour 2 7-dichlorofluorshyescein Rhodamine B of 6 G chromic acid-concentrated sulfuric acid and 50 sulfuric acid followed by charring The last reagent reveals differshyent color changes for different lipids during the heating process for example cholesterol and its esters turn red first then violet brown and finally black

Gas-liquid chromatography has been successfully employed in the separshyation of fatty acid mono- di- and triglycerides (72-74) The technique is also commonly used to analyze the fatty acid content of the triglycerides after methanolysis of the triglycerides The application of GTC in lipid

5

screening has been reported by Schlenk et a1 (75) in their search for arachidonic 5111417-eicosatetraenoic and related acids in plants

C Review of the Literature

1 General Considerations

Most studies on aquatic plants are urientated toward either ecology or limnology (76-80) Minerals (81-86) sugars (87-91) organic acids (9293) chlorophyll and xanthophyll pigments (94) have often been studied for their relationship to plant physiology

Chara sp are reported to contain amino acids (95) and uronic acid containing polysaccharides (96) contained many of the important amino acids but none with sulfur presence of vitamin B12 in Chara may be due to bacteria living epiphytically or in proximity to the algae Vitamins B2 and C were found in some aquatic plants of Moldavia (97)

The branch chain sugar D-apiose was first found in Leman by Duff in 1963 (98) and is believed to be a constituent of the cell wall (99) The biosynthesis of D-apiose was studied by feeding l4C02 or myo-inositol-2_l4C to the plant and observing its localization in the cell wall (100shy102)

2 Alkaloids

From the rhizome of Nuphar luteum five thioalkaloids ie thiobishynupharidine allothiobinupharidine pseudothiobinupharidine thiobisdeoxyshynupharidine and neothiobinupharidine were isolated (103) It also conshytains nupharine (104) Mass spectroscopy of their structures were recordshyed and structures assigned according to an NMR study (105106)

From the rhizome of Nuphar japonicum the following lupine alkaloids were isolated deoxynupharidine (107) nupharamine (108-110) nupharane (Ill) nupharidine (112) nuphamine (113) and an unstable base dehydroshydeoxynupharidine (114) The abolute configuration of the identified strucshytures have been reported (115-117) Arata (118119) succeeded in syntheshysizing dl-deoxynupharidine

A piperidine alkaloid nuphenine has been isolated from Nuphar variegatum (120)

Paper chromatographic analysis of Nymphaea alkaloids to be present in the leaves and flowers TIleir structures remain undetermined but all are the ~uphar alkaloids

extracts showed two one in the root (121)

believed different from

Carex contains the indole alkaloids brevicolline harman brevicarine four other alkaloids (122-124) The strucshyture of brevicolline was studied and determined by Terenteva (125126)

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3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

PageCONTENTS

D Saponins 30

E Steroids 30

F Lipids 35

Page

II INTRODUCTION 1

A Objective of the Study 1 IV REFERENCES 112 B Phytochemical Screenings 1

1 Alkaloid 2 2 Flavonoid 2 3 Tannin 3 4 Saponin 4 5 Steroid 4 6 Lipid 5

C Review of the Literature 6

1 General considerations 6 2 Alkaloids 6 3 Flavonoids bull 9 4 Tannins saponins steroids and lipids 9

II MATERIALS AND METHODS 9

A Plant Collection and Identification 9

B Extraction 11

C Detection for Alkaloids 12

1 Purification of extracts 12 2 TIlin-layer chromatography 12

D Detection for Flavonoids 13

E Detection for Tannins 13

F Detection for Saponins 14

1 Hemolysis test 14 2 Froth test 15

G Detection for Steroids 15

H Lipid Analysis 15

1 Gravimetric determination of total lipids 15 2 Systemic analysis of lipid distribut ion 16 3 Fatty acid constituents of triglycerides isolated

from selected plant extracts 16 a Isolation of trig1ycerides 16 b Methanolysis bull bull bull bull 17 c Hydrogenation bull bull 17 d Gas-liquid chromatography (GLC) 17

III RESULTS AND DISCUSSIONS 19

A Alkaloids 19

B Flavonoids 23

C Tannins 23

iiiIi

ILLUSTRATIONS

Figure Page

1 Purification of Skellysolve F Chloroform and 80 ethanol

extracts for the alkaloid detection

2 Procedure for the detection of tannins

13

14

3 Standard graph of log retention time vs carbon number of

reference fattyacids bullbullbullbullbullbullbull 18

Table

1

2

3

4

5

6

7

8

9

10

11

TABLES

Page

Composition of Fatty Acids from Some Aquatic Plants (GLC

Area )(75) bullbullbull 7

List of Plant Collected 8

Thin-layer Fluorescent and Alkaloid Patterns 20

Thin-layer Fluorescent and Flavonoid Patterns 24

Presence of Tffi1nins in Minnesotan Aquatic Plants 28

Detection of Saponins by Homolysis and Froth Tests 31

Thin-layer Fluorescent and Steroid Patterns 32

Gravimetric Determination of Total Lipids 34

Systemic Analysis of Lipid Distribution bull 38

Constituent Fatty Acids of Triglycerides Derived From Selected

Aquatic Plants 40

Comparison of Major Fatty Acids in Aquatic and Terrestrial

Plants (Linseed and Soybean) bull 41

iv v

FOREWORD

This Bulletin is published in furtherance of the purposes of the Water Resources Research Act of 1964 The purpose of the Act is to stimulate sponsor provide for and supplement present programs for the conduct of research investigations experiments and the training of scientists in the field of water and resources which affect water The Act is promoting a more adequate national program of water resources research by furnishing financial assistance to non-Federal research

The Act provides for establishment of Water Resources Research Centers at Universities throughout the Nation On September 1 1964 a Water Reshysources Research Center was established in the Graduate School as an intershydisciplinary component of the University of Minnesota The Center has the responsibility for unifying and stimulating University water resources reshysearch through the administration of funds covered in the Act and made avail shyable by other sources coordinating University research with water resources programs of local State and Federal agencies and private organizations throughout the State and assisting in training additional scientists for work in the field of water resources through research

This Bulletin is number 46 in a series of publications designed to present information bearing on water resources research in Minnesota and the results of some of the research sponsored by the Center The Bulletin is concerned with the results of a survey of selected aquatic plants in Minnesota conducted in anticipation of finding compowlds which might be useful in medicine The survey is part of a research project aimed at findshying a nutritional medicinal or industrial use for the unwanted aquatic plants in lake shoreline areas It is possible that some aquatic plants may contain industrially useful g~q-mucilages or new useful antimicrobial anticoagulant or antineoplastic therapeutic principles If a good indusshytrial medicinal or nutritional use for aquatic plants can be discovered the results of the research could provide an economic incentive for aquatic plant collection and control The successful completion of the project may significantly assist the State and Nation in partially solving their lake pollution problems

This Bulletin is related to the following research project

OWRR Project No A-025-Minn

Project Title Alleviation of Lake Pollution by Utilization of Aquatic Plants for Nutritional Medicinal or Industrial Purposes

Principal Investigator E John Staba Dept of Pharmacognosy College of Pharmacy University of Minnesota

Project Began July 1 1970 Scheduled Completion June 30 1973

FCST Research Category V-E

in Minnesota have not been surveyed medicinally for useful chemical compounds A study was conducted with a reasonable antici shypation of finding compounds such as alkaloids flavonoids tannins saponins steroids and lipids which might be useful in medicine Examination of chemshyical constituents was accomplished on the following plants collected from

vi

Taxonomic identification of aquatic plants was made and exhaustive exshytraction using solvents ranging from the non-polar to polar type was followshyed to determine the nature of the various constituents present in the aquatic plants Detection for compounds involved purification of extracts thinshylayer chromatography hemolysis test froth test gravimetric determination methanolysis and hydrogenation

Thin-layer chromatographic detection studies indicated original extracts did not appear to contain alkaloids Several plant species demonstrated Dragendorff positive spots Flavonols were most wideIv distributed in the plant extracts studies Tannins especially the condensed type were widely distributed in the plants screened Five species of plants are saponin posishytive Beta-sitosterol was tentatively identified as being present 8 species The lipid contents of 3 species may be considered for their nutritional value

Publication Descriptors Aquatic Plants Chemical Compounds Medicine Minnesota Alkaloids Flavonoids Tannins Saponins Steroids Lipids Phytochemical Screening Chromatography

Publication Identifiers Hemolysis Test Froth Test Methanolysis Hydrogenation Dragendorff Positive Spots Beta-sitosterol

vii

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I INTRODUCTION

A Objective of the Study

Higher and lower forms of plants have and arc contributing important drugs for the physicians use (1) Higher plants are being studied for their anti-neoplastic (2-5) antimicrobial (6-22) and other pharmacologishycal activities (2324) The lower forms such as the plankton algae are known to produce antibiotic substances (2526) the blue-green algae are believed to produce a cyclic polypeptide endotoxin (27) Aquatic plants can be considered in some ways to be botanically and ecologically related to both the higher plants and the algae They may also be considered nuisances which often bring about biological excesses that arc inimical to recreational and other water uses (28)

The aquatic plants in general and the Minnesotan aqlkltic plants in particular have not been surveyed medicinally for useful chemical comshypounds Therefore this series study was done with a reasonable anticishypation of finding compounds (such as alkaloids flavonoids tannins saponins steroids and lipids) which might be useful in medicine

eolExamination of chemieal constituents were

leeted from various lakes done on the following plants

B Phytoehemieal Screenings

Phytoehemieal screening programs have most often been direeted toward finding alkaloids flavonoids tannins saponins steroids and lipids The extraction sehemes for screening are normally one of the following two

i) Selective sequential extraction dependant upon the polarity of the solvent For example the dried powdered plant materials are extrActed first with petroleum ether (non-polar solvent) then with chloroform ethashynol and finally with water (polar solvent)

i1) Non-seleetive extraetion with hydroalcoholic solvents sueh as 50 or 80ethanol and subsequent fractionation of the ethanolie extract with organie solvents under different conditions

Artifacts caused by thermo-oxidation chemical oxidations and enzymati c hydrolysis should be avoided during the extraction

In order to explain the complexity of screening programs an introshyduction to the chemistry of constituents most often found in plants together with the problems whieh might be encountered in the screening are presented

1

1 Alkaloid

Alkaloids are widely distributed in the plant kingdom (29-35) and even in some bacteria (36) Wall (37-40) Webb (35) and Hultin (4142) are among those who have extensively screened vascular plants for alkashyloids

Alkaloids are natural products that are physiologically active posshysess basic or sometimes neutral properties and often contain heterocyclic nitrogen The definition is broad and great variations in chemical strucshytures are possible ranging from simple primary amines to very complex inshydole compounds The chemical groups of alkaloids include phenylalkylashymine (i e ephedrine) purine (caffeine) pyridine (evonine) pyrrol idine (tropinone) pyridine-pyrrolidine (nicotine) condensed piperidine-pyrrolishydine (atropine) quino ine (cinchonine) isoquinol ine (morphine) and indole (ergoclavine)

Therefore in general the plant materials are extracted either with acidic water to extract the alkaloids as salts or by addition of base to the plant material in order to extract the free base by an organic solvent Plants containing weakly basic or neutral alkaloids such as rutaecarpine colchicine and ricinine would not be extracted into the organic solvent and special approaches must be developed for those alkaloids Further purification of the plant extract may be necessary to avoid the extraction of false-positive reacting alkaloids such as those reported LJr some proteins and non-nitrogenous compounds (1314)

Alkaloid color reagents are used to spray on chromatograms in order to visualize alkaloid spots The most commonly used ones are Dragendorffs iodoplatinate antimony trichloride and cerium sulfate (in sulfuric acid or in phosphoric acid) (45)

2 Flavonoid

The flavonoid compounds in the plant kingdom are confined almost enshytirely to the flowering plants and ferns (6) Chemically flavonoids may be described as plant pigments containing two C6 groups (substituted benzene rings) connected by a three carbon aliphatic chain (C6-C Examples of the chemical classes of flavonoids are the catechLns leucoanthocyanidines flavanones flavanonols flavones flavonols and anthocyanidins A slight variation of C6-C 3-C6 patterns is seen in chalshycones dihydrochalcones aurones and isoflavones where the central pyran ring is open modified into a bem~alcoumaranone ring or the ring substishytution is shifted [rom C2 to Biologically flavonoids possess extremeshyly diverse pharmacological for instance quercitrin has an antishyviral effect (47) rutin is a capillary antihemorrhagic eupatomin has antishycancer activity (48) and flavone has bactericidal properties (9)

Flavonoids are often extracted by methanol aqueous ethanol of actone (50) Many color reactions can be used to characterize the different classes of [lavonoids and many of those color reactions have been reviewed by Seikel (51)

2

3 Tannin

According to Swain (52) the term tannin II can be applied to naturally occurring compound of high molecular weight (between about to 3000) containing a sufficiently large number of phenolic hydroxyl or other suitable groups (1-2 per 100 MW) to enable it to form effective cross-links between proteins and other macromolecules

Tannins may be divided st ructurally into the following two dist inc t classes

i) Hydrolyzable tannins consist of a polyhydric alcohol esterified with gallic acid or derivatives of gallic acid such as ellagic acid Tannins having this structure can readily be hydrolyzed by acids bases or enzymes (tannin acylhydrolases) Gallotannins and ellagitannins are included in this group

ii) Condensed tannins cont ain phenolic nuclei which when treated with the above hydrolytic reagents do not hvdrolyze but instead polymerize to yield insoluable amorphous and often red colored phlobaphenes An exshyample for tannins of this type is catechin a polymer of flavin-3-ols

Both the hydrolyzable and condensed tannins are widely distributed in nature An extensive survey of the occurrence of tannins in the plant kingdom was done hy Bath-Smith (53) One interesting observation found was that ellagic acid is absent from non-vascular plants ferns gy~losperms and monocotyledons

Tannins may be precipitated from their solutions hy different salts Salts of heavy metals (Ag 7n Cu Sn) precipitate tannins indiscriminashytively neutral lead salts precipitate tannins possessing adjacent phenolic hydroxyl groups while lead sub acetate precipitates tannins wi th non-adj ashycent hydroxyl groups (51) Wall (37) found that preCipitation with lead acetate occurred in every plant extract examined whether or not tannin was found by other tests Therefore tannins are separated from other plant constituents by the solvent cxtraction method Tannins are often exshytracted from plant materials with hot water and then salted out with sodshyium choloride Wall (37) detected tannins in the hot aqucous extracts preshypared from the dried 95 alcoholic extract Pcrsinos et al (55) directly used the 80 alcohol extract for tannin detection in their pharmacognostishycal study of Nigerian plants

Tannins can be detected by using a 1 solution of gelatin containing 10 sodium chloride (56) This test is based on the protein-binding capashycity of the tannin Since the condition of pH and ionic strength are crishytical Farnsworth (57) modified the test by using buffered gelatin-sal t reagent Another detecting agent is 1 ferric chloridc solution which either precipitates tannins or forms different colors ranging from blue blue-black green to bluc-green It should be recognizcd that many other phenolic compounds form colors with fcrric choloride solution Because of tannins polymcr and high molecular weight nature it usually runs as a streak on paper or thin-layer chromatograms (58) As an alternativc the nature of tannin is determined by acid or alkaline hydrolysis of the tannin extract and subsequent chromatographic detection of the building uni ts (gallic ellagic acid etc) in the hydrolysate

3

4 Saponin

Saponins are widely distributed among higher plants (59) Bioshygenetically saponins can be divided into two groups

i) Glycosides of triterpenoid alcohols (normally at the 3-position) such as oleanane

ii) Glycosides of a particular steroid structure having a spiroketal side chain at the E and F ring juncture A few of the steroidal sapongenins are distinguished by having a ring juncture

Due to the sugar residue at 3-beta-hydroxyl group both types of saposhynins are soluble in water and ethanol but insoluble in ether Their aglyshycones (sapogenins) without the sugar moiety possess the solubilitv charshyacteristics of other sterols Thus saponins are most conveniently~ extracted from plants with 70-95 hot ethanol or isopropanol

Saponins are most often detected by

i) Foam test Saponins are presumed to be present when the charactershyistic honeycomb froth persists for at least 30 minutes after shaking an aqueous boiled (3-5 minutes) plant material

ii) Hemolytic test Saponin containing plant extracts mixed with defibrinated blood in a buffered physiological saline solution cause the hemolysis of red blood cells Digitonin a saponin available in crystalshyline form is generally used as the standard Tannins will interfere with the hemolytic action of saponins as they form a protective coating around the red blood cells The removal of tannins with magnesium salt previous to performing the hemolytic test was suggested by Farnsworth (57)

Both triterpenoid and steroid saponins behave the same in those two tests Tf necessary the Liebermann-Burchard test may be used to differshyentiate them red pink or purple colors are developed with triterpenoid saponins whereas blue or blue-green colors are formed with steroidal saponins (60)

5 Steroid

Steroids are derivatives of the tetracyclic hydrocarbon cyclopentanshyoperhydrophenanthrene The steroids thus far discovered in plants include sterols certain sapogenins alkaloids cardenolides and hormones (61)

i) Sterols are hydroxysteroids of C27 C28 and C29 series (62) An example of C27 sterol is cholesterol which is not only present in animals but has also been isolated from algae by Tsuda et al (63) from potato and Dioscorea plant by Johnson et al (64) A common sterol is ergoshyC28 sterol and widely distributed C29 sterols are beta-sitosterol and stigmasshyterol

ii) Alkaloids The occurrence of steroidal alkaloids is limited mostly to Solanum Veratrum and Holarhena (65) species They contain a 5-mernbered E ring and a 6-mernbered nitrogen containing F ring

4

iii) Cardiac glycosides They are glycosides of either C23 (cardenoshyJides) or C24 (bufadienolides) steroids with potent cardiac activity They are structurally characterized by an unsaturated lactone ring at Cl7

uncture of ring C and D a l4-beta-hydroxy group and by the pecushysugars (such as D-digitoxose D-digitalose D-cymarose and D-sarmenshy

tose) attached at

iv) Hormones Pregnane (C2l) androstane (C19 ) (66) and estrane (C18 ) (67) derivatives have been isolated from plants

Steroids can be extracted with benzene or ether and the chromatographic technique is of great value to detect plant materials for steroids A very good article reviewing the application of TLC for steroid detection has been written by Neher (68) Among the reagents most used and most generally apshyplicable are suI furic aci d-alcohol chlorosul phonic acid-acetic acid and molybdophosphoric acid Anisaldehyde-sulfuric acid reagent is used for deshytection of many steroids it is non-specific but with high sensitivity Dinitrobenzoic acid (Kedde reagent) and m-dinitrobenzene are specific for the detection of cardenolides and 17-ketosteroids respectively cerium sulfate is specific for the detection of nitrogen-containing steroids Keller (ferric chloride-acetic acid) and Kiliani (ferric sulfate-sulfuric acid) are specific for the deoxy sugar moiety of cardiac glycosides Frerejacque and DeGraeve (69) had tabulated all the cardiac glycoside deshytecting agents and their methods of preparation The use of iodine as a non-destructive location reagent for steroids in TIC has been introduced by Stevens (70) This technique is very valuable for preparative thin- and thick-layer chromatography

6 Lipid

Since triglycerides have been studied in this thesis only the saponishyfiable lipids will be discussed The saponifiable lipids are classified according to their structures into the fo11owing categories fatty acids fatty acid esters (triglycerides waxes etc) phospholipids and glycoshylipids

Non-polar lipids are extracted with petroleum ether or benzene and polar lipids are extracted with isopropanol isopropanol-chloroform methshyanol-chloroform ethanol-chloroform or ethanol-ether In addition to the avoidance of thermo- chemical and enzymatic oxidation and hydrolysis lipid extraction should be carried out under nitrogen to prevent the oxishydation of unsaturated compounds The extracts can be fractionated by TLC into different lipid classes According to Mangold (71) lipids can be visualized on the chromatograms with iodine vapour 2 7-dichlorofluorshyescein Rhodamine B of 6 G chromic acid-concentrated sulfuric acid and 50 sulfuric acid followed by charring The last reagent reveals differshyent color changes for different lipids during the heating process for example cholesterol and its esters turn red first then violet brown and finally black

Gas-liquid chromatography has been successfully employed in the separshyation of fatty acid mono- di- and triglycerides (72-74) The technique is also commonly used to analyze the fatty acid content of the triglycerides after methanolysis of the triglycerides The application of GTC in lipid

5

screening has been reported by Schlenk et a1 (75) in their search for arachidonic 5111417-eicosatetraenoic and related acids in plants

C Review of the Literature

1 General Considerations

Most studies on aquatic plants are urientated toward either ecology or limnology (76-80) Minerals (81-86) sugars (87-91) organic acids (9293) chlorophyll and xanthophyll pigments (94) have often been studied for their relationship to plant physiology

Chara sp are reported to contain amino acids (95) and uronic acid containing polysaccharides (96) contained many of the important amino acids but none with sulfur presence of vitamin B12 in Chara may be due to bacteria living epiphytically or in proximity to the algae Vitamins B2 and C were found in some aquatic plants of Moldavia (97)

The branch chain sugar D-apiose was first found in Leman by Duff in 1963 (98) and is believed to be a constituent of the cell wall (99) The biosynthesis of D-apiose was studied by feeding l4C02 or myo-inositol-2_l4C to the plant and observing its localization in the cell wall (100shy102)

2 Alkaloids

From the rhizome of Nuphar luteum five thioalkaloids ie thiobishynupharidine allothiobinupharidine pseudothiobinupharidine thiobisdeoxyshynupharidine and neothiobinupharidine were isolated (103) It also conshytains nupharine (104) Mass spectroscopy of their structures were recordshyed and structures assigned according to an NMR study (105106)

From the rhizome of Nuphar japonicum the following lupine alkaloids were isolated deoxynupharidine (107) nupharamine (108-110) nupharane (Ill) nupharidine (112) nuphamine (113) and an unstable base dehydroshydeoxynupharidine (114) The abolute configuration of the identified strucshytures have been reported (115-117) Arata (118119) succeeded in syntheshysizing dl-deoxynupharidine

A piperidine alkaloid nuphenine has been isolated from Nuphar variegatum (120)

Paper chromatographic analysis of Nymphaea alkaloids to be present in the leaves and flowers TIleir structures remain undetermined but all are the ~uphar alkaloids

extracts showed two one in the root (121)

believed different from

Carex contains the indole alkaloids brevicolline harman brevicarine four other alkaloids (122-124) The strucshyture of brevicolline was studied and determined by Terenteva (125126)

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3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

ILLUSTRATIONS

Figure Page

1 Purification of Skellysolve F Chloroform and 80 ethanol

extracts for the alkaloid detection

2 Procedure for the detection of tannins

13

14

3 Standard graph of log retention time vs carbon number of

reference fattyacids bullbullbullbullbullbullbull 18

Table

1

2

3

4

5

6

7

8

9

10

11

TABLES

Page

Composition of Fatty Acids from Some Aquatic Plants (GLC

Area )(75) bullbullbull 7

List of Plant Collected 8

Thin-layer Fluorescent and Alkaloid Patterns 20

Thin-layer Fluorescent and Flavonoid Patterns 24

Presence of Tffi1nins in Minnesotan Aquatic Plants 28

Detection of Saponins by Homolysis and Froth Tests 31

Thin-layer Fluorescent and Steroid Patterns 32

Gravimetric Determination of Total Lipids 34

Systemic Analysis of Lipid Distribution bull 38

Constituent Fatty Acids of Triglycerides Derived From Selected

Aquatic Plants 40

Comparison of Major Fatty Acids in Aquatic and Terrestrial

Plants (Linseed and Soybean) bull 41

iv v

FOREWORD

This Bulletin is published in furtherance of the purposes of the Water Resources Research Act of 1964 The purpose of the Act is to stimulate sponsor provide for and supplement present programs for the conduct of research investigations experiments and the training of scientists in the field of water and resources which affect water The Act is promoting a more adequate national program of water resources research by furnishing financial assistance to non-Federal research

The Act provides for establishment of Water Resources Research Centers at Universities throughout the Nation On September 1 1964 a Water Reshysources Research Center was established in the Graduate School as an intershydisciplinary component of the University of Minnesota The Center has the responsibility for unifying and stimulating University water resources reshysearch through the administration of funds covered in the Act and made avail shyable by other sources coordinating University research with water resources programs of local State and Federal agencies and private organizations throughout the State and assisting in training additional scientists for work in the field of water resources through research

This Bulletin is number 46 in a series of publications designed to present information bearing on water resources research in Minnesota and the results of some of the research sponsored by the Center The Bulletin is concerned with the results of a survey of selected aquatic plants in Minnesota conducted in anticipation of finding compowlds which might be useful in medicine The survey is part of a research project aimed at findshying a nutritional medicinal or industrial use for the unwanted aquatic plants in lake shoreline areas It is possible that some aquatic plants may contain industrially useful g~q-mucilages or new useful antimicrobial anticoagulant or antineoplastic therapeutic principles If a good indusshytrial medicinal or nutritional use for aquatic plants can be discovered the results of the research could provide an economic incentive for aquatic plant collection and control The successful completion of the project may significantly assist the State and Nation in partially solving their lake pollution problems

This Bulletin is related to the following research project

OWRR Project No A-025-Minn

Project Title Alleviation of Lake Pollution by Utilization of Aquatic Plants for Nutritional Medicinal or Industrial Purposes

Principal Investigator E John Staba Dept of Pharmacognosy College of Pharmacy University of Minnesota

Project Began July 1 1970 Scheduled Completion June 30 1973

FCST Research Category V-E

in Minnesota have not been surveyed medicinally for useful chemical compounds A study was conducted with a reasonable antici shypation of finding compounds such as alkaloids flavonoids tannins saponins steroids and lipids which might be useful in medicine Examination of chemshyical constituents was accomplished on the following plants collected from

vi

Taxonomic identification of aquatic plants was made and exhaustive exshytraction using solvents ranging from the non-polar to polar type was followshyed to determine the nature of the various constituents present in the aquatic plants Detection for compounds involved purification of extracts thinshylayer chromatography hemolysis test froth test gravimetric determination methanolysis and hydrogenation

Thin-layer chromatographic detection studies indicated original extracts did not appear to contain alkaloids Several plant species demonstrated Dragendorff positive spots Flavonols were most wideIv distributed in the plant extracts studies Tannins especially the condensed type were widely distributed in the plants screened Five species of plants are saponin posishytive Beta-sitosterol was tentatively identified as being present 8 species The lipid contents of 3 species may be considered for their nutritional value

Publication Descriptors Aquatic Plants Chemical Compounds Medicine Minnesota Alkaloids Flavonoids Tannins Saponins Steroids Lipids Phytochemical Screening Chromatography

Publication Identifiers Hemolysis Test Froth Test Methanolysis Hydrogenation Dragendorff Positive Spots Beta-sitosterol

vii

middotctZt$rMtk

I INTRODUCTION

A Objective of the Study

Higher and lower forms of plants have and arc contributing important drugs for the physicians use (1) Higher plants are being studied for their anti-neoplastic (2-5) antimicrobial (6-22) and other pharmacologishycal activities (2324) The lower forms such as the plankton algae are known to produce antibiotic substances (2526) the blue-green algae are believed to produce a cyclic polypeptide endotoxin (27) Aquatic plants can be considered in some ways to be botanically and ecologically related to both the higher plants and the algae They may also be considered nuisances which often bring about biological excesses that arc inimical to recreational and other water uses (28)

The aquatic plants in general and the Minnesotan aqlkltic plants in particular have not been surveyed medicinally for useful chemical comshypounds Therefore this series study was done with a reasonable anticishypation of finding compounds (such as alkaloids flavonoids tannins saponins steroids and lipids) which might be useful in medicine

eolExamination of chemieal constituents were

leeted from various lakes done on the following plants

B Phytoehemieal Screenings

Phytoehemieal screening programs have most often been direeted toward finding alkaloids flavonoids tannins saponins steroids and lipids The extraction sehemes for screening are normally one of the following two

i) Selective sequential extraction dependant upon the polarity of the solvent For example the dried powdered plant materials are extrActed first with petroleum ether (non-polar solvent) then with chloroform ethashynol and finally with water (polar solvent)

i1) Non-seleetive extraetion with hydroalcoholic solvents sueh as 50 or 80ethanol and subsequent fractionation of the ethanolie extract with organie solvents under different conditions

Artifacts caused by thermo-oxidation chemical oxidations and enzymati c hydrolysis should be avoided during the extraction

In order to explain the complexity of screening programs an introshyduction to the chemistry of constituents most often found in plants together with the problems whieh might be encountered in the screening are presented

1

1 Alkaloid

Alkaloids are widely distributed in the plant kingdom (29-35) and even in some bacteria (36) Wall (37-40) Webb (35) and Hultin (4142) are among those who have extensively screened vascular plants for alkashyloids

Alkaloids are natural products that are physiologically active posshysess basic or sometimes neutral properties and often contain heterocyclic nitrogen The definition is broad and great variations in chemical strucshytures are possible ranging from simple primary amines to very complex inshydole compounds The chemical groups of alkaloids include phenylalkylashymine (i e ephedrine) purine (caffeine) pyridine (evonine) pyrrol idine (tropinone) pyridine-pyrrolidine (nicotine) condensed piperidine-pyrrolishydine (atropine) quino ine (cinchonine) isoquinol ine (morphine) and indole (ergoclavine)

Therefore in general the plant materials are extracted either with acidic water to extract the alkaloids as salts or by addition of base to the plant material in order to extract the free base by an organic solvent Plants containing weakly basic or neutral alkaloids such as rutaecarpine colchicine and ricinine would not be extracted into the organic solvent and special approaches must be developed for those alkaloids Further purification of the plant extract may be necessary to avoid the extraction of false-positive reacting alkaloids such as those reported LJr some proteins and non-nitrogenous compounds (1314)

Alkaloid color reagents are used to spray on chromatograms in order to visualize alkaloid spots The most commonly used ones are Dragendorffs iodoplatinate antimony trichloride and cerium sulfate (in sulfuric acid or in phosphoric acid) (45)

2 Flavonoid

The flavonoid compounds in the plant kingdom are confined almost enshytirely to the flowering plants and ferns (6) Chemically flavonoids may be described as plant pigments containing two C6 groups (substituted benzene rings) connected by a three carbon aliphatic chain (C6-C Examples of the chemical classes of flavonoids are the catechLns leucoanthocyanidines flavanones flavanonols flavones flavonols and anthocyanidins A slight variation of C6-C 3-C6 patterns is seen in chalshycones dihydrochalcones aurones and isoflavones where the central pyran ring is open modified into a bem~alcoumaranone ring or the ring substishytution is shifted [rom C2 to Biologically flavonoids possess extremeshyly diverse pharmacological for instance quercitrin has an antishyviral effect (47) rutin is a capillary antihemorrhagic eupatomin has antishycancer activity (48) and flavone has bactericidal properties (9)

Flavonoids are often extracted by methanol aqueous ethanol of actone (50) Many color reactions can be used to characterize the different classes of [lavonoids and many of those color reactions have been reviewed by Seikel (51)

2

3 Tannin

According to Swain (52) the term tannin II can be applied to naturally occurring compound of high molecular weight (between about to 3000) containing a sufficiently large number of phenolic hydroxyl or other suitable groups (1-2 per 100 MW) to enable it to form effective cross-links between proteins and other macromolecules

Tannins may be divided st ructurally into the following two dist inc t classes

i) Hydrolyzable tannins consist of a polyhydric alcohol esterified with gallic acid or derivatives of gallic acid such as ellagic acid Tannins having this structure can readily be hydrolyzed by acids bases or enzymes (tannin acylhydrolases) Gallotannins and ellagitannins are included in this group

ii) Condensed tannins cont ain phenolic nuclei which when treated with the above hydrolytic reagents do not hvdrolyze but instead polymerize to yield insoluable amorphous and often red colored phlobaphenes An exshyample for tannins of this type is catechin a polymer of flavin-3-ols

Both the hydrolyzable and condensed tannins are widely distributed in nature An extensive survey of the occurrence of tannins in the plant kingdom was done hy Bath-Smith (53) One interesting observation found was that ellagic acid is absent from non-vascular plants ferns gy~losperms and monocotyledons

Tannins may be precipitated from their solutions hy different salts Salts of heavy metals (Ag 7n Cu Sn) precipitate tannins indiscriminashytively neutral lead salts precipitate tannins possessing adjacent phenolic hydroxyl groups while lead sub acetate precipitates tannins wi th non-adj ashycent hydroxyl groups (51) Wall (37) found that preCipitation with lead acetate occurred in every plant extract examined whether or not tannin was found by other tests Therefore tannins are separated from other plant constituents by the solvent cxtraction method Tannins are often exshytracted from plant materials with hot water and then salted out with sodshyium choloride Wall (37) detected tannins in the hot aqucous extracts preshypared from the dried 95 alcoholic extract Pcrsinos et al (55) directly used the 80 alcohol extract for tannin detection in their pharmacognostishycal study of Nigerian plants

Tannins can be detected by using a 1 solution of gelatin containing 10 sodium chloride (56) This test is based on the protein-binding capashycity of the tannin Since the condition of pH and ionic strength are crishytical Farnsworth (57) modified the test by using buffered gelatin-sal t reagent Another detecting agent is 1 ferric chloridc solution which either precipitates tannins or forms different colors ranging from blue blue-black green to bluc-green It should be recognizcd that many other phenolic compounds form colors with fcrric choloride solution Because of tannins polymcr and high molecular weight nature it usually runs as a streak on paper or thin-layer chromatograms (58) As an alternativc the nature of tannin is determined by acid or alkaline hydrolysis of the tannin extract and subsequent chromatographic detection of the building uni ts (gallic ellagic acid etc) in the hydrolysate

3

4 Saponin

Saponins are widely distributed among higher plants (59) Bioshygenetically saponins can be divided into two groups

i) Glycosides of triterpenoid alcohols (normally at the 3-position) such as oleanane

ii) Glycosides of a particular steroid structure having a spiroketal side chain at the E and F ring juncture A few of the steroidal sapongenins are distinguished by having a ring juncture

Due to the sugar residue at 3-beta-hydroxyl group both types of saposhynins are soluble in water and ethanol but insoluble in ether Their aglyshycones (sapogenins) without the sugar moiety possess the solubilitv charshyacteristics of other sterols Thus saponins are most conveniently~ extracted from plants with 70-95 hot ethanol or isopropanol

Saponins are most often detected by

i) Foam test Saponins are presumed to be present when the charactershyistic honeycomb froth persists for at least 30 minutes after shaking an aqueous boiled (3-5 minutes) plant material

ii) Hemolytic test Saponin containing plant extracts mixed with defibrinated blood in a buffered physiological saline solution cause the hemolysis of red blood cells Digitonin a saponin available in crystalshyline form is generally used as the standard Tannins will interfere with the hemolytic action of saponins as they form a protective coating around the red blood cells The removal of tannins with magnesium salt previous to performing the hemolytic test was suggested by Farnsworth (57)

Both triterpenoid and steroid saponins behave the same in those two tests Tf necessary the Liebermann-Burchard test may be used to differshyentiate them red pink or purple colors are developed with triterpenoid saponins whereas blue or blue-green colors are formed with steroidal saponins (60)

5 Steroid

Steroids are derivatives of the tetracyclic hydrocarbon cyclopentanshyoperhydrophenanthrene The steroids thus far discovered in plants include sterols certain sapogenins alkaloids cardenolides and hormones (61)

i) Sterols are hydroxysteroids of C27 C28 and C29 series (62) An example of C27 sterol is cholesterol which is not only present in animals but has also been isolated from algae by Tsuda et al (63) from potato and Dioscorea plant by Johnson et al (64) A common sterol is ergoshyC28 sterol and widely distributed C29 sterols are beta-sitosterol and stigmasshyterol

ii) Alkaloids The occurrence of steroidal alkaloids is limited mostly to Solanum Veratrum and Holarhena (65) species They contain a 5-mernbered E ring and a 6-mernbered nitrogen containing F ring

4

iii) Cardiac glycosides They are glycosides of either C23 (cardenoshyJides) or C24 (bufadienolides) steroids with potent cardiac activity They are structurally characterized by an unsaturated lactone ring at Cl7

uncture of ring C and D a l4-beta-hydroxy group and by the pecushysugars (such as D-digitoxose D-digitalose D-cymarose and D-sarmenshy

tose) attached at

iv) Hormones Pregnane (C2l) androstane (C19 ) (66) and estrane (C18 ) (67) derivatives have been isolated from plants

Steroids can be extracted with benzene or ether and the chromatographic technique is of great value to detect plant materials for steroids A very good article reviewing the application of TLC for steroid detection has been written by Neher (68) Among the reagents most used and most generally apshyplicable are suI furic aci d-alcohol chlorosul phonic acid-acetic acid and molybdophosphoric acid Anisaldehyde-sulfuric acid reagent is used for deshytection of many steroids it is non-specific but with high sensitivity Dinitrobenzoic acid (Kedde reagent) and m-dinitrobenzene are specific for the detection of cardenolides and 17-ketosteroids respectively cerium sulfate is specific for the detection of nitrogen-containing steroids Keller (ferric chloride-acetic acid) and Kiliani (ferric sulfate-sulfuric acid) are specific for the deoxy sugar moiety of cardiac glycosides Frerejacque and DeGraeve (69) had tabulated all the cardiac glycoside deshytecting agents and their methods of preparation The use of iodine as a non-destructive location reagent for steroids in TIC has been introduced by Stevens (70) This technique is very valuable for preparative thin- and thick-layer chromatography

6 Lipid

Since triglycerides have been studied in this thesis only the saponishyfiable lipids will be discussed The saponifiable lipids are classified according to their structures into the fo11owing categories fatty acids fatty acid esters (triglycerides waxes etc) phospholipids and glycoshylipids

Non-polar lipids are extracted with petroleum ether or benzene and polar lipids are extracted with isopropanol isopropanol-chloroform methshyanol-chloroform ethanol-chloroform or ethanol-ether In addition to the avoidance of thermo- chemical and enzymatic oxidation and hydrolysis lipid extraction should be carried out under nitrogen to prevent the oxishydation of unsaturated compounds The extracts can be fractionated by TLC into different lipid classes According to Mangold (71) lipids can be visualized on the chromatograms with iodine vapour 2 7-dichlorofluorshyescein Rhodamine B of 6 G chromic acid-concentrated sulfuric acid and 50 sulfuric acid followed by charring The last reagent reveals differshyent color changes for different lipids during the heating process for example cholesterol and its esters turn red first then violet brown and finally black

Gas-liquid chromatography has been successfully employed in the separshyation of fatty acid mono- di- and triglycerides (72-74) The technique is also commonly used to analyze the fatty acid content of the triglycerides after methanolysis of the triglycerides The application of GTC in lipid

5

screening has been reported by Schlenk et a1 (75) in their search for arachidonic 5111417-eicosatetraenoic and related acids in plants

C Review of the Literature

1 General Considerations

Most studies on aquatic plants are urientated toward either ecology or limnology (76-80) Minerals (81-86) sugars (87-91) organic acids (9293) chlorophyll and xanthophyll pigments (94) have often been studied for their relationship to plant physiology

Chara sp are reported to contain amino acids (95) and uronic acid containing polysaccharides (96) contained many of the important amino acids but none with sulfur presence of vitamin B12 in Chara may be due to bacteria living epiphytically or in proximity to the algae Vitamins B2 and C were found in some aquatic plants of Moldavia (97)

The branch chain sugar D-apiose was first found in Leman by Duff in 1963 (98) and is believed to be a constituent of the cell wall (99) The biosynthesis of D-apiose was studied by feeding l4C02 or myo-inositol-2_l4C to the plant and observing its localization in the cell wall (100shy102)

2 Alkaloids

From the rhizome of Nuphar luteum five thioalkaloids ie thiobishynupharidine allothiobinupharidine pseudothiobinupharidine thiobisdeoxyshynupharidine and neothiobinupharidine were isolated (103) It also conshytains nupharine (104) Mass spectroscopy of their structures were recordshyed and structures assigned according to an NMR study (105106)

From the rhizome of Nuphar japonicum the following lupine alkaloids were isolated deoxynupharidine (107) nupharamine (108-110) nupharane (Ill) nupharidine (112) nuphamine (113) and an unstable base dehydroshydeoxynupharidine (114) The abolute configuration of the identified strucshytures have been reported (115-117) Arata (118119) succeeded in syntheshysizing dl-deoxynupharidine

A piperidine alkaloid nuphenine has been isolated from Nuphar variegatum (120)

Paper chromatographic analysis of Nymphaea alkaloids to be present in the leaves and flowers TIleir structures remain undetermined but all are the ~uphar alkaloids

extracts showed two one in the root (121)

believed different from

Carex contains the indole alkaloids brevicolline harman brevicarine four other alkaloids (122-124) The strucshyture of brevicolline was studied and determined by Terenteva (125126)

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3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

FOREWORD

This Bulletin is published in furtherance of the purposes of the Water Resources Research Act of 1964 The purpose of the Act is to stimulate sponsor provide for and supplement present programs for the conduct of research investigations experiments and the training of scientists in the field of water and resources which affect water The Act is promoting a more adequate national program of water resources research by furnishing financial assistance to non-Federal research

The Act provides for establishment of Water Resources Research Centers at Universities throughout the Nation On September 1 1964 a Water Reshysources Research Center was established in the Graduate School as an intershydisciplinary component of the University of Minnesota The Center has the responsibility for unifying and stimulating University water resources reshysearch through the administration of funds covered in the Act and made avail shyable by other sources coordinating University research with water resources programs of local State and Federal agencies and private organizations throughout the State and assisting in training additional scientists for work in the field of water resources through research

This Bulletin is number 46 in a series of publications designed to present information bearing on water resources research in Minnesota and the results of some of the research sponsored by the Center The Bulletin is concerned with the results of a survey of selected aquatic plants in Minnesota conducted in anticipation of finding compowlds which might be useful in medicine The survey is part of a research project aimed at findshying a nutritional medicinal or industrial use for the unwanted aquatic plants in lake shoreline areas It is possible that some aquatic plants may contain industrially useful g~q-mucilages or new useful antimicrobial anticoagulant or antineoplastic therapeutic principles If a good indusshytrial medicinal or nutritional use for aquatic plants can be discovered the results of the research could provide an economic incentive for aquatic plant collection and control The successful completion of the project may significantly assist the State and Nation in partially solving their lake pollution problems

This Bulletin is related to the following research project

OWRR Project No A-025-Minn

Project Title Alleviation of Lake Pollution by Utilization of Aquatic Plants for Nutritional Medicinal or Industrial Purposes

Principal Investigator E John Staba Dept of Pharmacognosy College of Pharmacy University of Minnesota

Project Began July 1 1970 Scheduled Completion June 30 1973

FCST Research Category V-E

in Minnesota have not been surveyed medicinally for useful chemical compounds A study was conducted with a reasonable antici shypation of finding compounds such as alkaloids flavonoids tannins saponins steroids and lipids which might be useful in medicine Examination of chemshyical constituents was accomplished on the following plants collected from

vi

Taxonomic identification of aquatic plants was made and exhaustive exshytraction using solvents ranging from the non-polar to polar type was followshyed to determine the nature of the various constituents present in the aquatic plants Detection for compounds involved purification of extracts thinshylayer chromatography hemolysis test froth test gravimetric determination methanolysis and hydrogenation

Thin-layer chromatographic detection studies indicated original extracts did not appear to contain alkaloids Several plant species demonstrated Dragendorff positive spots Flavonols were most wideIv distributed in the plant extracts studies Tannins especially the condensed type were widely distributed in the plants screened Five species of plants are saponin posishytive Beta-sitosterol was tentatively identified as being present 8 species The lipid contents of 3 species may be considered for their nutritional value

Publication Descriptors Aquatic Plants Chemical Compounds Medicine Minnesota Alkaloids Flavonoids Tannins Saponins Steroids Lipids Phytochemical Screening Chromatography

Publication Identifiers Hemolysis Test Froth Test Methanolysis Hydrogenation Dragendorff Positive Spots Beta-sitosterol

vii

middotctZt$rMtk

I INTRODUCTION

A Objective of the Study

Higher and lower forms of plants have and arc contributing important drugs for the physicians use (1) Higher plants are being studied for their anti-neoplastic (2-5) antimicrobial (6-22) and other pharmacologishycal activities (2324) The lower forms such as the plankton algae are known to produce antibiotic substances (2526) the blue-green algae are believed to produce a cyclic polypeptide endotoxin (27) Aquatic plants can be considered in some ways to be botanically and ecologically related to both the higher plants and the algae They may also be considered nuisances which often bring about biological excesses that arc inimical to recreational and other water uses (28)

The aquatic plants in general and the Minnesotan aqlkltic plants in particular have not been surveyed medicinally for useful chemical comshypounds Therefore this series study was done with a reasonable anticishypation of finding compounds (such as alkaloids flavonoids tannins saponins steroids and lipids) which might be useful in medicine

eolExamination of chemieal constituents were

leeted from various lakes done on the following plants

B Phytoehemieal Screenings

Phytoehemieal screening programs have most often been direeted toward finding alkaloids flavonoids tannins saponins steroids and lipids The extraction sehemes for screening are normally one of the following two

i) Selective sequential extraction dependant upon the polarity of the solvent For example the dried powdered plant materials are extrActed first with petroleum ether (non-polar solvent) then with chloroform ethashynol and finally with water (polar solvent)

i1) Non-seleetive extraetion with hydroalcoholic solvents sueh as 50 or 80ethanol and subsequent fractionation of the ethanolie extract with organie solvents under different conditions

Artifacts caused by thermo-oxidation chemical oxidations and enzymati c hydrolysis should be avoided during the extraction

In order to explain the complexity of screening programs an introshyduction to the chemistry of constituents most often found in plants together with the problems whieh might be encountered in the screening are presented

1

1 Alkaloid

Alkaloids are widely distributed in the plant kingdom (29-35) and even in some bacteria (36) Wall (37-40) Webb (35) and Hultin (4142) are among those who have extensively screened vascular plants for alkashyloids

Alkaloids are natural products that are physiologically active posshysess basic or sometimes neutral properties and often contain heterocyclic nitrogen The definition is broad and great variations in chemical strucshytures are possible ranging from simple primary amines to very complex inshydole compounds The chemical groups of alkaloids include phenylalkylashymine (i e ephedrine) purine (caffeine) pyridine (evonine) pyrrol idine (tropinone) pyridine-pyrrolidine (nicotine) condensed piperidine-pyrrolishydine (atropine) quino ine (cinchonine) isoquinol ine (morphine) and indole (ergoclavine)

Therefore in general the plant materials are extracted either with acidic water to extract the alkaloids as salts or by addition of base to the plant material in order to extract the free base by an organic solvent Plants containing weakly basic or neutral alkaloids such as rutaecarpine colchicine and ricinine would not be extracted into the organic solvent and special approaches must be developed for those alkaloids Further purification of the plant extract may be necessary to avoid the extraction of false-positive reacting alkaloids such as those reported LJr some proteins and non-nitrogenous compounds (1314)

Alkaloid color reagents are used to spray on chromatograms in order to visualize alkaloid spots The most commonly used ones are Dragendorffs iodoplatinate antimony trichloride and cerium sulfate (in sulfuric acid or in phosphoric acid) (45)

2 Flavonoid

The flavonoid compounds in the plant kingdom are confined almost enshytirely to the flowering plants and ferns (6) Chemically flavonoids may be described as plant pigments containing two C6 groups (substituted benzene rings) connected by a three carbon aliphatic chain (C6-C Examples of the chemical classes of flavonoids are the catechLns leucoanthocyanidines flavanones flavanonols flavones flavonols and anthocyanidins A slight variation of C6-C 3-C6 patterns is seen in chalshycones dihydrochalcones aurones and isoflavones where the central pyran ring is open modified into a bem~alcoumaranone ring or the ring substishytution is shifted [rom C2 to Biologically flavonoids possess extremeshyly diverse pharmacological for instance quercitrin has an antishyviral effect (47) rutin is a capillary antihemorrhagic eupatomin has antishycancer activity (48) and flavone has bactericidal properties (9)

Flavonoids are often extracted by methanol aqueous ethanol of actone (50) Many color reactions can be used to characterize the different classes of [lavonoids and many of those color reactions have been reviewed by Seikel (51)

2

3 Tannin

According to Swain (52) the term tannin II can be applied to naturally occurring compound of high molecular weight (between about to 3000) containing a sufficiently large number of phenolic hydroxyl or other suitable groups (1-2 per 100 MW) to enable it to form effective cross-links between proteins and other macromolecules

Tannins may be divided st ructurally into the following two dist inc t classes

i) Hydrolyzable tannins consist of a polyhydric alcohol esterified with gallic acid or derivatives of gallic acid such as ellagic acid Tannins having this structure can readily be hydrolyzed by acids bases or enzymes (tannin acylhydrolases) Gallotannins and ellagitannins are included in this group

ii) Condensed tannins cont ain phenolic nuclei which when treated with the above hydrolytic reagents do not hvdrolyze but instead polymerize to yield insoluable amorphous and often red colored phlobaphenes An exshyample for tannins of this type is catechin a polymer of flavin-3-ols

Both the hydrolyzable and condensed tannins are widely distributed in nature An extensive survey of the occurrence of tannins in the plant kingdom was done hy Bath-Smith (53) One interesting observation found was that ellagic acid is absent from non-vascular plants ferns gy~losperms and monocotyledons

Tannins may be precipitated from their solutions hy different salts Salts of heavy metals (Ag 7n Cu Sn) precipitate tannins indiscriminashytively neutral lead salts precipitate tannins possessing adjacent phenolic hydroxyl groups while lead sub acetate precipitates tannins wi th non-adj ashycent hydroxyl groups (51) Wall (37) found that preCipitation with lead acetate occurred in every plant extract examined whether or not tannin was found by other tests Therefore tannins are separated from other plant constituents by the solvent cxtraction method Tannins are often exshytracted from plant materials with hot water and then salted out with sodshyium choloride Wall (37) detected tannins in the hot aqucous extracts preshypared from the dried 95 alcoholic extract Pcrsinos et al (55) directly used the 80 alcohol extract for tannin detection in their pharmacognostishycal study of Nigerian plants

Tannins can be detected by using a 1 solution of gelatin containing 10 sodium chloride (56) This test is based on the protein-binding capashycity of the tannin Since the condition of pH and ionic strength are crishytical Farnsworth (57) modified the test by using buffered gelatin-sal t reagent Another detecting agent is 1 ferric chloridc solution which either precipitates tannins or forms different colors ranging from blue blue-black green to bluc-green It should be recognizcd that many other phenolic compounds form colors with fcrric choloride solution Because of tannins polymcr and high molecular weight nature it usually runs as a streak on paper or thin-layer chromatograms (58) As an alternativc the nature of tannin is determined by acid or alkaline hydrolysis of the tannin extract and subsequent chromatographic detection of the building uni ts (gallic ellagic acid etc) in the hydrolysate

3

4 Saponin

Saponins are widely distributed among higher plants (59) Bioshygenetically saponins can be divided into two groups

i) Glycosides of triterpenoid alcohols (normally at the 3-position) such as oleanane

ii) Glycosides of a particular steroid structure having a spiroketal side chain at the E and F ring juncture A few of the steroidal sapongenins are distinguished by having a ring juncture

Due to the sugar residue at 3-beta-hydroxyl group both types of saposhynins are soluble in water and ethanol but insoluble in ether Their aglyshycones (sapogenins) without the sugar moiety possess the solubilitv charshyacteristics of other sterols Thus saponins are most conveniently~ extracted from plants with 70-95 hot ethanol or isopropanol

Saponins are most often detected by

i) Foam test Saponins are presumed to be present when the charactershyistic honeycomb froth persists for at least 30 minutes after shaking an aqueous boiled (3-5 minutes) plant material

ii) Hemolytic test Saponin containing plant extracts mixed with defibrinated blood in a buffered physiological saline solution cause the hemolysis of red blood cells Digitonin a saponin available in crystalshyline form is generally used as the standard Tannins will interfere with the hemolytic action of saponins as they form a protective coating around the red blood cells The removal of tannins with magnesium salt previous to performing the hemolytic test was suggested by Farnsworth (57)

Both triterpenoid and steroid saponins behave the same in those two tests Tf necessary the Liebermann-Burchard test may be used to differshyentiate them red pink or purple colors are developed with triterpenoid saponins whereas blue or blue-green colors are formed with steroidal saponins (60)

5 Steroid

Steroids are derivatives of the tetracyclic hydrocarbon cyclopentanshyoperhydrophenanthrene The steroids thus far discovered in plants include sterols certain sapogenins alkaloids cardenolides and hormones (61)

i) Sterols are hydroxysteroids of C27 C28 and C29 series (62) An example of C27 sterol is cholesterol which is not only present in animals but has also been isolated from algae by Tsuda et al (63) from potato and Dioscorea plant by Johnson et al (64) A common sterol is ergoshyC28 sterol and widely distributed C29 sterols are beta-sitosterol and stigmasshyterol

ii) Alkaloids The occurrence of steroidal alkaloids is limited mostly to Solanum Veratrum and Holarhena (65) species They contain a 5-mernbered E ring and a 6-mernbered nitrogen containing F ring

4

iii) Cardiac glycosides They are glycosides of either C23 (cardenoshyJides) or C24 (bufadienolides) steroids with potent cardiac activity They are structurally characterized by an unsaturated lactone ring at Cl7

uncture of ring C and D a l4-beta-hydroxy group and by the pecushysugars (such as D-digitoxose D-digitalose D-cymarose and D-sarmenshy

tose) attached at

iv) Hormones Pregnane (C2l) androstane (C19 ) (66) and estrane (C18 ) (67) derivatives have been isolated from plants

Steroids can be extracted with benzene or ether and the chromatographic technique is of great value to detect plant materials for steroids A very good article reviewing the application of TLC for steroid detection has been written by Neher (68) Among the reagents most used and most generally apshyplicable are suI furic aci d-alcohol chlorosul phonic acid-acetic acid and molybdophosphoric acid Anisaldehyde-sulfuric acid reagent is used for deshytection of many steroids it is non-specific but with high sensitivity Dinitrobenzoic acid (Kedde reagent) and m-dinitrobenzene are specific for the detection of cardenolides and 17-ketosteroids respectively cerium sulfate is specific for the detection of nitrogen-containing steroids Keller (ferric chloride-acetic acid) and Kiliani (ferric sulfate-sulfuric acid) are specific for the deoxy sugar moiety of cardiac glycosides Frerejacque and DeGraeve (69) had tabulated all the cardiac glycoside deshytecting agents and their methods of preparation The use of iodine as a non-destructive location reagent for steroids in TIC has been introduced by Stevens (70) This technique is very valuable for preparative thin- and thick-layer chromatography

6 Lipid

Since triglycerides have been studied in this thesis only the saponishyfiable lipids will be discussed The saponifiable lipids are classified according to their structures into the fo11owing categories fatty acids fatty acid esters (triglycerides waxes etc) phospholipids and glycoshylipids

Non-polar lipids are extracted with petroleum ether or benzene and polar lipids are extracted with isopropanol isopropanol-chloroform methshyanol-chloroform ethanol-chloroform or ethanol-ether In addition to the avoidance of thermo- chemical and enzymatic oxidation and hydrolysis lipid extraction should be carried out under nitrogen to prevent the oxishydation of unsaturated compounds The extracts can be fractionated by TLC into different lipid classes According to Mangold (71) lipids can be visualized on the chromatograms with iodine vapour 2 7-dichlorofluorshyescein Rhodamine B of 6 G chromic acid-concentrated sulfuric acid and 50 sulfuric acid followed by charring The last reagent reveals differshyent color changes for different lipids during the heating process for example cholesterol and its esters turn red first then violet brown and finally black

Gas-liquid chromatography has been successfully employed in the separshyation of fatty acid mono- di- and triglycerides (72-74) The technique is also commonly used to analyze the fatty acid content of the triglycerides after methanolysis of the triglycerides The application of GTC in lipid

5

screening has been reported by Schlenk et a1 (75) in their search for arachidonic 5111417-eicosatetraenoic and related acids in plants

C Review of the Literature

1 General Considerations

Most studies on aquatic plants are urientated toward either ecology or limnology (76-80) Minerals (81-86) sugars (87-91) organic acids (9293) chlorophyll and xanthophyll pigments (94) have often been studied for their relationship to plant physiology

Chara sp are reported to contain amino acids (95) and uronic acid containing polysaccharides (96) contained many of the important amino acids but none with sulfur presence of vitamin B12 in Chara may be due to bacteria living epiphytically or in proximity to the algae Vitamins B2 and C were found in some aquatic plants of Moldavia (97)

The branch chain sugar D-apiose was first found in Leman by Duff in 1963 (98) and is believed to be a constituent of the cell wall (99) The biosynthesis of D-apiose was studied by feeding l4C02 or myo-inositol-2_l4C to the plant and observing its localization in the cell wall (100shy102)

2 Alkaloids

From the rhizome of Nuphar luteum five thioalkaloids ie thiobishynupharidine allothiobinupharidine pseudothiobinupharidine thiobisdeoxyshynupharidine and neothiobinupharidine were isolated (103) It also conshytains nupharine (104) Mass spectroscopy of their structures were recordshyed and structures assigned according to an NMR study (105106)

From the rhizome of Nuphar japonicum the following lupine alkaloids were isolated deoxynupharidine (107) nupharamine (108-110) nupharane (Ill) nupharidine (112) nuphamine (113) and an unstable base dehydroshydeoxynupharidine (114) The abolute configuration of the identified strucshytures have been reported (115-117) Arata (118119) succeeded in syntheshysizing dl-deoxynupharidine

A piperidine alkaloid nuphenine has been isolated from Nuphar variegatum (120)

Paper chromatographic analysis of Nymphaea alkaloids to be present in the leaves and flowers TIleir structures remain undetermined but all are the ~uphar alkaloids

extracts showed two one in the root (121)

believed different from

Carex contains the indole alkaloids brevicolline harman brevicarine four other alkaloids (122-124) The strucshyture of brevicolline was studied and determined by Terenteva (125126)

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3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

middotctZt$rMtk

I INTRODUCTION

A Objective of the Study

Higher and lower forms of plants have and arc contributing important drugs for the physicians use (1) Higher plants are being studied for their anti-neoplastic (2-5) antimicrobial (6-22) and other pharmacologishycal activities (2324) The lower forms such as the plankton algae are known to produce antibiotic substances (2526) the blue-green algae are believed to produce a cyclic polypeptide endotoxin (27) Aquatic plants can be considered in some ways to be botanically and ecologically related to both the higher plants and the algae They may also be considered nuisances which often bring about biological excesses that arc inimical to recreational and other water uses (28)

The aquatic plants in general and the Minnesotan aqlkltic plants in particular have not been surveyed medicinally for useful chemical comshypounds Therefore this series study was done with a reasonable anticishypation of finding compounds (such as alkaloids flavonoids tannins saponins steroids and lipids) which might be useful in medicine

eolExamination of chemieal constituents were

leeted from various lakes done on the following plants

B Phytoehemieal Screenings

Phytoehemieal screening programs have most often been direeted toward finding alkaloids flavonoids tannins saponins steroids and lipids The extraction sehemes for screening are normally one of the following two

i) Selective sequential extraction dependant upon the polarity of the solvent For example the dried powdered plant materials are extrActed first with petroleum ether (non-polar solvent) then with chloroform ethashynol and finally with water (polar solvent)

i1) Non-seleetive extraetion with hydroalcoholic solvents sueh as 50 or 80ethanol and subsequent fractionation of the ethanolie extract with organie solvents under different conditions

Artifacts caused by thermo-oxidation chemical oxidations and enzymati c hydrolysis should be avoided during the extraction

In order to explain the complexity of screening programs an introshyduction to the chemistry of constituents most often found in plants together with the problems whieh might be encountered in the screening are presented

1

1 Alkaloid

Alkaloids are widely distributed in the plant kingdom (29-35) and even in some bacteria (36) Wall (37-40) Webb (35) and Hultin (4142) are among those who have extensively screened vascular plants for alkashyloids

Alkaloids are natural products that are physiologically active posshysess basic or sometimes neutral properties and often contain heterocyclic nitrogen The definition is broad and great variations in chemical strucshytures are possible ranging from simple primary amines to very complex inshydole compounds The chemical groups of alkaloids include phenylalkylashymine (i e ephedrine) purine (caffeine) pyridine (evonine) pyrrol idine (tropinone) pyridine-pyrrolidine (nicotine) condensed piperidine-pyrrolishydine (atropine) quino ine (cinchonine) isoquinol ine (morphine) and indole (ergoclavine)

Therefore in general the plant materials are extracted either with acidic water to extract the alkaloids as salts or by addition of base to the plant material in order to extract the free base by an organic solvent Plants containing weakly basic or neutral alkaloids such as rutaecarpine colchicine and ricinine would not be extracted into the organic solvent and special approaches must be developed for those alkaloids Further purification of the plant extract may be necessary to avoid the extraction of false-positive reacting alkaloids such as those reported LJr some proteins and non-nitrogenous compounds (1314)

Alkaloid color reagents are used to spray on chromatograms in order to visualize alkaloid spots The most commonly used ones are Dragendorffs iodoplatinate antimony trichloride and cerium sulfate (in sulfuric acid or in phosphoric acid) (45)

2 Flavonoid

The flavonoid compounds in the plant kingdom are confined almost enshytirely to the flowering plants and ferns (6) Chemically flavonoids may be described as plant pigments containing two C6 groups (substituted benzene rings) connected by a three carbon aliphatic chain (C6-C Examples of the chemical classes of flavonoids are the catechLns leucoanthocyanidines flavanones flavanonols flavones flavonols and anthocyanidins A slight variation of C6-C 3-C6 patterns is seen in chalshycones dihydrochalcones aurones and isoflavones where the central pyran ring is open modified into a bem~alcoumaranone ring or the ring substishytution is shifted [rom C2 to Biologically flavonoids possess extremeshyly diverse pharmacological for instance quercitrin has an antishyviral effect (47) rutin is a capillary antihemorrhagic eupatomin has antishycancer activity (48) and flavone has bactericidal properties (9)

Flavonoids are often extracted by methanol aqueous ethanol of actone (50) Many color reactions can be used to characterize the different classes of [lavonoids and many of those color reactions have been reviewed by Seikel (51)

2

3 Tannin

According to Swain (52) the term tannin II can be applied to naturally occurring compound of high molecular weight (between about to 3000) containing a sufficiently large number of phenolic hydroxyl or other suitable groups (1-2 per 100 MW) to enable it to form effective cross-links between proteins and other macromolecules

Tannins may be divided st ructurally into the following two dist inc t classes

i) Hydrolyzable tannins consist of a polyhydric alcohol esterified with gallic acid or derivatives of gallic acid such as ellagic acid Tannins having this structure can readily be hydrolyzed by acids bases or enzymes (tannin acylhydrolases) Gallotannins and ellagitannins are included in this group

ii) Condensed tannins cont ain phenolic nuclei which when treated with the above hydrolytic reagents do not hvdrolyze but instead polymerize to yield insoluable amorphous and often red colored phlobaphenes An exshyample for tannins of this type is catechin a polymer of flavin-3-ols

Both the hydrolyzable and condensed tannins are widely distributed in nature An extensive survey of the occurrence of tannins in the plant kingdom was done hy Bath-Smith (53) One interesting observation found was that ellagic acid is absent from non-vascular plants ferns gy~losperms and monocotyledons

Tannins may be precipitated from their solutions hy different salts Salts of heavy metals (Ag 7n Cu Sn) precipitate tannins indiscriminashytively neutral lead salts precipitate tannins possessing adjacent phenolic hydroxyl groups while lead sub acetate precipitates tannins wi th non-adj ashycent hydroxyl groups (51) Wall (37) found that preCipitation with lead acetate occurred in every plant extract examined whether or not tannin was found by other tests Therefore tannins are separated from other plant constituents by the solvent cxtraction method Tannins are often exshytracted from plant materials with hot water and then salted out with sodshyium choloride Wall (37) detected tannins in the hot aqucous extracts preshypared from the dried 95 alcoholic extract Pcrsinos et al (55) directly used the 80 alcohol extract for tannin detection in their pharmacognostishycal study of Nigerian plants

Tannins can be detected by using a 1 solution of gelatin containing 10 sodium chloride (56) This test is based on the protein-binding capashycity of the tannin Since the condition of pH and ionic strength are crishytical Farnsworth (57) modified the test by using buffered gelatin-sal t reagent Another detecting agent is 1 ferric chloridc solution which either precipitates tannins or forms different colors ranging from blue blue-black green to bluc-green It should be recognizcd that many other phenolic compounds form colors with fcrric choloride solution Because of tannins polymcr and high molecular weight nature it usually runs as a streak on paper or thin-layer chromatograms (58) As an alternativc the nature of tannin is determined by acid or alkaline hydrolysis of the tannin extract and subsequent chromatographic detection of the building uni ts (gallic ellagic acid etc) in the hydrolysate

3

4 Saponin

Saponins are widely distributed among higher plants (59) Bioshygenetically saponins can be divided into two groups

i) Glycosides of triterpenoid alcohols (normally at the 3-position) such as oleanane

ii) Glycosides of a particular steroid structure having a spiroketal side chain at the E and F ring juncture A few of the steroidal sapongenins are distinguished by having a ring juncture

Due to the sugar residue at 3-beta-hydroxyl group both types of saposhynins are soluble in water and ethanol but insoluble in ether Their aglyshycones (sapogenins) without the sugar moiety possess the solubilitv charshyacteristics of other sterols Thus saponins are most conveniently~ extracted from plants with 70-95 hot ethanol or isopropanol

Saponins are most often detected by

i) Foam test Saponins are presumed to be present when the charactershyistic honeycomb froth persists for at least 30 minutes after shaking an aqueous boiled (3-5 minutes) plant material

ii) Hemolytic test Saponin containing plant extracts mixed with defibrinated blood in a buffered physiological saline solution cause the hemolysis of red blood cells Digitonin a saponin available in crystalshyline form is generally used as the standard Tannins will interfere with the hemolytic action of saponins as they form a protective coating around the red blood cells The removal of tannins with magnesium salt previous to performing the hemolytic test was suggested by Farnsworth (57)

Both triterpenoid and steroid saponins behave the same in those two tests Tf necessary the Liebermann-Burchard test may be used to differshyentiate them red pink or purple colors are developed with triterpenoid saponins whereas blue or blue-green colors are formed with steroidal saponins (60)

5 Steroid

Steroids are derivatives of the tetracyclic hydrocarbon cyclopentanshyoperhydrophenanthrene The steroids thus far discovered in plants include sterols certain sapogenins alkaloids cardenolides and hormones (61)

i) Sterols are hydroxysteroids of C27 C28 and C29 series (62) An example of C27 sterol is cholesterol which is not only present in animals but has also been isolated from algae by Tsuda et al (63) from potato and Dioscorea plant by Johnson et al (64) A common sterol is ergoshyC28 sterol and widely distributed C29 sterols are beta-sitosterol and stigmasshyterol

ii) Alkaloids The occurrence of steroidal alkaloids is limited mostly to Solanum Veratrum and Holarhena (65) species They contain a 5-mernbered E ring and a 6-mernbered nitrogen containing F ring

4

iii) Cardiac glycosides They are glycosides of either C23 (cardenoshyJides) or C24 (bufadienolides) steroids with potent cardiac activity They are structurally characterized by an unsaturated lactone ring at Cl7

uncture of ring C and D a l4-beta-hydroxy group and by the pecushysugars (such as D-digitoxose D-digitalose D-cymarose and D-sarmenshy

tose) attached at

iv) Hormones Pregnane (C2l) androstane (C19 ) (66) and estrane (C18 ) (67) derivatives have been isolated from plants

Steroids can be extracted with benzene or ether and the chromatographic technique is of great value to detect plant materials for steroids A very good article reviewing the application of TLC for steroid detection has been written by Neher (68) Among the reagents most used and most generally apshyplicable are suI furic aci d-alcohol chlorosul phonic acid-acetic acid and molybdophosphoric acid Anisaldehyde-sulfuric acid reagent is used for deshytection of many steroids it is non-specific but with high sensitivity Dinitrobenzoic acid (Kedde reagent) and m-dinitrobenzene are specific for the detection of cardenolides and 17-ketosteroids respectively cerium sulfate is specific for the detection of nitrogen-containing steroids Keller (ferric chloride-acetic acid) and Kiliani (ferric sulfate-sulfuric acid) are specific for the deoxy sugar moiety of cardiac glycosides Frerejacque and DeGraeve (69) had tabulated all the cardiac glycoside deshytecting agents and their methods of preparation The use of iodine as a non-destructive location reagent for steroids in TIC has been introduced by Stevens (70) This technique is very valuable for preparative thin- and thick-layer chromatography

6 Lipid

Since triglycerides have been studied in this thesis only the saponishyfiable lipids will be discussed The saponifiable lipids are classified according to their structures into the fo11owing categories fatty acids fatty acid esters (triglycerides waxes etc) phospholipids and glycoshylipids

Non-polar lipids are extracted with petroleum ether or benzene and polar lipids are extracted with isopropanol isopropanol-chloroform methshyanol-chloroform ethanol-chloroform or ethanol-ether In addition to the avoidance of thermo- chemical and enzymatic oxidation and hydrolysis lipid extraction should be carried out under nitrogen to prevent the oxishydation of unsaturated compounds The extracts can be fractionated by TLC into different lipid classes According to Mangold (71) lipids can be visualized on the chromatograms with iodine vapour 2 7-dichlorofluorshyescein Rhodamine B of 6 G chromic acid-concentrated sulfuric acid and 50 sulfuric acid followed by charring The last reagent reveals differshyent color changes for different lipids during the heating process for example cholesterol and its esters turn red first then violet brown and finally black

Gas-liquid chromatography has been successfully employed in the separshyation of fatty acid mono- di- and triglycerides (72-74) The technique is also commonly used to analyze the fatty acid content of the triglycerides after methanolysis of the triglycerides The application of GTC in lipid

5

screening has been reported by Schlenk et a1 (75) in their search for arachidonic 5111417-eicosatetraenoic and related acids in plants

C Review of the Literature

1 General Considerations

Most studies on aquatic plants are urientated toward either ecology or limnology (76-80) Minerals (81-86) sugars (87-91) organic acids (9293) chlorophyll and xanthophyll pigments (94) have often been studied for their relationship to plant physiology

Chara sp are reported to contain amino acids (95) and uronic acid containing polysaccharides (96) contained many of the important amino acids but none with sulfur presence of vitamin B12 in Chara may be due to bacteria living epiphytically or in proximity to the algae Vitamins B2 and C were found in some aquatic plants of Moldavia (97)

The branch chain sugar D-apiose was first found in Leman by Duff in 1963 (98) and is believed to be a constituent of the cell wall (99) The biosynthesis of D-apiose was studied by feeding l4C02 or myo-inositol-2_l4C to the plant and observing its localization in the cell wall (100shy102)

2 Alkaloids

From the rhizome of Nuphar luteum five thioalkaloids ie thiobishynupharidine allothiobinupharidine pseudothiobinupharidine thiobisdeoxyshynupharidine and neothiobinupharidine were isolated (103) It also conshytains nupharine (104) Mass spectroscopy of their structures were recordshyed and structures assigned according to an NMR study (105106)

From the rhizome of Nuphar japonicum the following lupine alkaloids were isolated deoxynupharidine (107) nupharamine (108-110) nupharane (Ill) nupharidine (112) nuphamine (113) and an unstable base dehydroshydeoxynupharidine (114) The abolute configuration of the identified strucshytures have been reported (115-117) Arata (118119) succeeded in syntheshysizing dl-deoxynupharidine

A piperidine alkaloid nuphenine has been isolated from Nuphar variegatum (120)

Paper chromatographic analysis of Nymphaea alkaloids to be present in the leaves and flowers TIleir structures remain undetermined but all are the ~uphar alkaloids

extracts showed two one in the root (121)

believed different from

Carex contains the indole alkaloids brevicolline harman brevicarine four other alkaloids (122-124) The strucshyture of brevicolline was studied and determined by Terenteva (125126)

6

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sect M Ul o Ii o u

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o N N

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lt ltlt (1j (1j C C (1j C

0 o 0 ltIl lttl (1j w (1j m jJ (j) jI

rlt U middotrl 0 -llt i( cJ (l) cJ (jQ)middotrlOJ-llt-llt~ f]J Vl Q) CIl (fl C ill (f) U) tfl C J c Q) OJ G) ltIlltIlCClttlCCltIlC(1jCCCCC GJ w(l)w~~w~~w~w~(l)~~~~

~ H~H~~H~~H~H~~~~~~

c cJ 0

rlt 0

~ gtlt w

~ rlt C H 0 - 0 0 0 0 c X (l) C C

c cJc C C (l) M W rurl r-I ~rlU U Q)~ooc J c rlt ~rltJ PlGJCO

oct ~~~~HH~~H~HH~~W~~

H M (1j fJ

(J) 8 ~ s

J rtj m f) rlt ltIl gt 0 0 gt Ul 0

Ul -11 ~M rl ltIl Ul C M jl J ~ p C 4-l Ul rlt mM~4middotMucrj 0 ltIl 0 rlt H (1j rlt (j) H wOjJjJMmiddotrlQ)U 4-1 l t )l H m J w

rlt (1j lttl~(JlWO~H~C~lttlltllUW U M OJ r- J J M ro (Ij H 0 rl rj w c w ~ U l (j) M cwrlumJcQ)C~jIUUooHJM P gt gtltIl(1j(1jS~ltIlr~S(1j(j)~OgtMC

Cfl U M Po lttl cJ ltIl (1j C p H N (j) ~ (1j

(1j C C C C C Cl (1j rlt o 0 000 S S

r M rlt U1 H wwww+-JJ1 H H H rlt (j) W(l)Q)c)CJr-rl (1j (1j (1j (1j H C MMbOMbGC C wu cmiddotrimr) o 0 000 ltIl (1j

lttl ww(1j-ucc~(1jrrSSMbOlttl gt H ~~~~2~~~~22~~~~~~ C lttl GJ c Cl (1j (1j Cl M ~ C (1j (j) 0 0 0 0 0 Po Po ~ u U Cf) () U U W N lt ~ ~ P-1 PI p -j p UJ en f-

(j) (j) ltIl ltIl III (j) U U ltlJ III

III (j) (1j ltIl (1j ltIl W lttl Cl w W ltll (j) C (j) ltll (j) ltlJ rl -r- ltJ) Q) C) (U OJ U cJ lttl OJ U U (1j ltIl (j) H H ltlJ lttl ltIl ltIl (1j (1j (1j (1j (j)

M ltIl (1j ltIl (l) (j) (1j (1j (1j (1j OJ ltll (l) (j) (j) ~ ~ (1j ~ ltlJ +J jI ill U U Q)C ~ ill U U U U U = c Q)

~ U (1j (1j (1j (1j (1j C U U U (1j (1j (1j (1j (1j lttl (1j U ~ M Cl SaOJHH~OO(1jOOOooMMCl 0 rlt H ~~~~~~~~~~~~~~~~~ a (1j W oj c MMH~~H~~OJoj(1j(1j(1j(1jPoP~ Ul ~ U ~octoctuuu~~~ZZZZZVlCfl~

rlt I ~

N u o Ul

(l) Ul (j) U c M Ul lttl o 0 0 (1j M CO (1j H M o ltlJ ~ u oct ~M

8

(1j (1j (1j (1j ltltltlt C C C C 000 0 +Jww+- (l) GJ (l) (l) C C C C C C C C

rl middotri rl r ~~~~

P (j) -

C GJ H (j) C Q) bO-r-

bull ~ C ltIl H - I-J r1

(J)

C r (l) gt 9 u W ltIl (J) ltIl Ul

Ul ltll M 0 HoGJH Q) rl -r- Q) S ltIl H 0 ltll - (1j gt

0 (j) w

S gt S

M gt M ~

c gt ltIl Poc (l) o Po H (1j W 0 ltIlc (1j rl c Po H H Po S

~pound~t2

III ltIl (j) U ltIl (l) (j) ltll

M ltIl lttl (1j III III III gt U U U

c (1j (1j (1j PMOJ (l) o ltIl (1j Cl WHcc (1j 0 p p H J S (j) ltIl gt gt u~zz

I ltlJ gt u o U C 0 CIO

~ i 00

]1 w

J M J ~

0 C C rlt ltIl

GJ (1j 0

~ 0 H (l) C 0 0 gt rlt

w cJ (l) 0

U

lt H 0 -l

H (j) (j) Ul w ltIl (J)

cJ lttl I

C C 0 z w Po (j) 00 U X 0 (j) (j) M

w 0 U H (j) ltlJ (j)

~~-sect gt 0 (l) w U w

Po ltlJ (l)

OHVl C ltlJ ltIl C

rlt 0 (Jl (j) III III W gt0 U ltIl Cl III (l) 8 C o 0 0 U C -1

lttl w OJ U

~ ~ ~ 3 t1l rl

W 0 Ul U w C I (1j MClt P 0 Ul

ltll H (1j H (l) w ltlJ W o c Ul ~ ~ - - M N

3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

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III IV V I II

a b c a b c a b c --------

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lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

1 Alkaloid

Alkaloids are widely distributed in the plant kingdom (29-35) and even in some bacteria (36) Wall (37-40) Webb (35) and Hultin (4142) are among those who have extensively screened vascular plants for alkashyloids

Alkaloids are natural products that are physiologically active posshysess basic or sometimes neutral properties and often contain heterocyclic nitrogen The definition is broad and great variations in chemical strucshytures are possible ranging from simple primary amines to very complex inshydole compounds The chemical groups of alkaloids include phenylalkylashymine (i e ephedrine) purine (caffeine) pyridine (evonine) pyrrol idine (tropinone) pyridine-pyrrolidine (nicotine) condensed piperidine-pyrrolishydine (atropine) quino ine (cinchonine) isoquinol ine (morphine) and indole (ergoclavine)

Therefore in general the plant materials are extracted either with acidic water to extract the alkaloids as salts or by addition of base to the plant material in order to extract the free base by an organic solvent Plants containing weakly basic or neutral alkaloids such as rutaecarpine colchicine and ricinine would not be extracted into the organic solvent and special approaches must be developed for those alkaloids Further purification of the plant extract may be necessary to avoid the extraction of false-positive reacting alkaloids such as those reported LJr some proteins and non-nitrogenous compounds (1314)

Alkaloid color reagents are used to spray on chromatograms in order to visualize alkaloid spots The most commonly used ones are Dragendorffs iodoplatinate antimony trichloride and cerium sulfate (in sulfuric acid or in phosphoric acid) (45)

2 Flavonoid

The flavonoid compounds in the plant kingdom are confined almost enshytirely to the flowering plants and ferns (6) Chemically flavonoids may be described as plant pigments containing two C6 groups (substituted benzene rings) connected by a three carbon aliphatic chain (C6-C Examples of the chemical classes of flavonoids are the catechLns leucoanthocyanidines flavanones flavanonols flavones flavonols and anthocyanidins A slight variation of C6-C 3-C6 patterns is seen in chalshycones dihydrochalcones aurones and isoflavones where the central pyran ring is open modified into a bem~alcoumaranone ring or the ring substishytution is shifted [rom C2 to Biologically flavonoids possess extremeshyly diverse pharmacological for instance quercitrin has an antishyviral effect (47) rutin is a capillary antihemorrhagic eupatomin has antishycancer activity (48) and flavone has bactericidal properties (9)

Flavonoids are often extracted by methanol aqueous ethanol of actone (50) Many color reactions can be used to characterize the different classes of [lavonoids and many of those color reactions have been reviewed by Seikel (51)

2

3 Tannin

According to Swain (52) the term tannin II can be applied to naturally occurring compound of high molecular weight (between about to 3000) containing a sufficiently large number of phenolic hydroxyl or other suitable groups (1-2 per 100 MW) to enable it to form effective cross-links between proteins and other macromolecules

Tannins may be divided st ructurally into the following two dist inc t classes

i) Hydrolyzable tannins consist of a polyhydric alcohol esterified with gallic acid or derivatives of gallic acid such as ellagic acid Tannins having this structure can readily be hydrolyzed by acids bases or enzymes (tannin acylhydrolases) Gallotannins and ellagitannins are included in this group

ii) Condensed tannins cont ain phenolic nuclei which when treated with the above hydrolytic reagents do not hvdrolyze but instead polymerize to yield insoluable amorphous and often red colored phlobaphenes An exshyample for tannins of this type is catechin a polymer of flavin-3-ols

Both the hydrolyzable and condensed tannins are widely distributed in nature An extensive survey of the occurrence of tannins in the plant kingdom was done hy Bath-Smith (53) One interesting observation found was that ellagic acid is absent from non-vascular plants ferns gy~losperms and monocotyledons

Tannins may be precipitated from their solutions hy different salts Salts of heavy metals (Ag 7n Cu Sn) precipitate tannins indiscriminashytively neutral lead salts precipitate tannins possessing adjacent phenolic hydroxyl groups while lead sub acetate precipitates tannins wi th non-adj ashycent hydroxyl groups (51) Wall (37) found that preCipitation with lead acetate occurred in every plant extract examined whether or not tannin was found by other tests Therefore tannins are separated from other plant constituents by the solvent cxtraction method Tannins are often exshytracted from plant materials with hot water and then salted out with sodshyium choloride Wall (37) detected tannins in the hot aqucous extracts preshypared from the dried 95 alcoholic extract Pcrsinos et al (55) directly used the 80 alcohol extract for tannin detection in their pharmacognostishycal study of Nigerian plants

Tannins can be detected by using a 1 solution of gelatin containing 10 sodium chloride (56) This test is based on the protein-binding capashycity of the tannin Since the condition of pH and ionic strength are crishytical Farnsworth (57) modified the test by using buffered gelatin-sal t reagent Another detecting agent is 1 ferric chloridc solution which either precipitates tannins or forms different colors ranging from blue blue-black green to bluc-green It should be recognizcd that many other phenolic compounds form colors with fcrric choloride solution Because of tannins polymcr and high molecular weight nature it usually runs as a streak on paper or thin-layer chromatograms (58) As an alternativc the nature of tannin is determined by acid or alkaline hydrolysis of the tannin extract and subsequent chromatographic detection of the building uni ts (gallic ellagic acid etc) in the hydrolysate

3

4 Saponin

Saponins are widely distributed among higher plants (59) Bioshygenetically saponins can be divided into two groups

i) Glycosides of triterpenoid alcohols (normally at the 3-position) such as oleanane

ii) Glycosides of a particular steroid structure having a spiroketal side chain at the E and F ring juncture A few of the steroidal sapongenins are distinguished by having a ring juncture

Due to the sugar residue at 3-beta-hydroxyl group both types of saposhynins are soluble in water and ethanol but insoluble in ether Their aglyshycones (sapogenins) without the sugar moiety possess the solubilitv charshyacteristics of other sterols Thus saponins are most conveniently~ extracted from plants with 70-95 hot ethanol or isopropanol

Saponins are most often detected by

i) Foam test Saponins are presumed to be present when the charactershyistic honeycomb froth persists for at least 30 minutes after shaking an aqueous boiled (3-5 minutes) plant material

ii) Hemolytic test Saponin containing plant extracts mixed with defibrinated blood in a buffered physiological saline solution cause the hemolysis of red blood cells Digitonin a saponin available in crystalshyline form is generally used as the standard Tannins will interfere with the hemolytic action of saponins as they form a protective coating around the red blood cells The removal of tannins with magnesium salt previous to performing the hemolytic test was suggested by Farnsworth (57)

Both triterpenoid and steroid saponins behave the same in those two tests Tf necessary the Liebermann-Burchard test may be used to differshyentiate them red pink or purple colors are developed with triterpenoid saponins whereas blue or blue-green colors are formed with steroidal saponins (60)

5 Steroid

Steroids are derivatives of the tetracyclic hydrocarbon cyclopentanshyoperhydrophenanthrene The steroids thus far discovered in plants include sterols certain sapogenins alkaloids cardenolides and hormones (61)

i) Sterols are hydroxysteroids of C27 C28 and C29 series (62) An example of C27 sterol is cholesterol which is not only present in animals but has also been isolated from algae by Tsuda et al (63) from potato and Dioscorea plant by Johnson et al (64) A common sterol is ergoshyC28 sterol and widely distributed C29 sterols are beta-sitosterol and stigmasshyterol

ii) Alkaloids The occurrence of steroidal alkaloids is limited mostly to Solanum Veratrum and Holarhena (65) species They contain a 5-mernbered E ring and a 6-mernbered nitrogen containing F ring

4

iii) Cardiac glycosides They are glycosides of either C23 (cardenoshyJides) or C24 (bufadienolides) steroids with potent cardiac activity They are structurally characterized by an unsaturated lactone ring at Cl7

uncture of ring C and D a l4-beta-hydroxy group and by the pecushysugars (such as D-digitoxose D-digitalose D-cymarose and D-sarmenshy

tose) attached at

iv) Hormones Pregnane (C2l) androstane (C19 ) (66) and estrane (C18 ) (67) derivatives have been isolated from plants

Steroids can be extracted with benzene or ether and the chromatographic technique is of great value to detect plant materials for steroids A very good article reviewing the application of TLC for steroid detection has been written by Neher (68) Among the reagents most used and most generally apshyplicable are suI furic aci d-alcohol chlorosul phonic acid-acetic acid and molybdophosphoric acid Anisaldehyde-sulfuric acid reagent is used for deshytection of many steroids it is non-specific but with high sensitivity Dinitrobenzoic acid (Kedde reagent) and m-dinitrobenzene are specific for the detection of cardenolides and 17-ketosteroids respectively cerium sulfate is specific for the detection of nitrogen-containing steroids Keller (ferric chloride-acetic acid) and Kiliani (ferric sulfate-sulfuric acid) are specific for the deoxy sugar moiety of cardiac glycosides Frerejacque and DeGraeve (69) had tabulated all the cardiac glycoside deshytecting agents and their methods of preparation The use of iodine as a non-destructive location reagent for steroids in TIC has been introduced by Stevens (70) This technique is very valuable for preparative thin- and thick-layer chromatography

6 Lipid

Since triglycerides have been studied in this thesis only the saponishyfiable lipids will be discussed The saponifiable lipids are classified according to their structures into the fo11owing categories fatty acids fatty acid esters (triglycerides waxes etc) phospholipids and glycoshylipids

Non-polar lipids are extracted with petroleum ether or benzene and polar lipids are extracted with isopropanol isopropanol-chloroform methshyanol-chloroform ethanol-chloroform or ethanol-ether In addition to the avoidance of thermo- chemical and enzymatic oxidation and hydrolysis lipid extraction should be carried out under nitrogen to prevent the oxishydation of unsaturated compounds The extracts can be fractionated by TLC into different lipid classes According to Mangold (71) lipids can be visualized on the chromatograms with iodine vapour 2 7-dichlorofluorshyescein Rhodamine B of 6 G chromic acid-concentrated sulfuric acid and 50 sulfuric acid followed by charring The last reagent reveals differshyent color changes for different lipids during the heating process for example cholesterol and its esters turn red first then violet brown and finally black

Gas-liquid chromatography has been successfully employed in the separshyation of fatty acid mono- di- and triglycerides (72-74) The technique is also commonly used to analyze the fatty acid content of the triglycerides after methanolysis of the triglycerides The application of GTC in lipid

5

screening has been reported by Schlenk et a1 (75) in their search for arachidonic 5111417-eicosatetraenoic and related acids in plants

C Review of the Literature

1 General Considerations

Most studies on aquatic plants are urientated toward either ecology or limnology (76-80) Minerals (81-86) sugars (87-91) organic acids (9293) chlorophyll and xanthophyll pigments (94) have often been studied for their relationship to plant physiology

Chara sp are reported to contain amino acids (95) and uronic acid containing polysaccharides (96) contained many of the important amino acids but none with sulfur presence of vitamin B12 in Chara may be due to bacteria living epiphytically or in proximity to the algae Vitamins B2 and C were found in some aquatic plants of Moldavia (97)

The branch chain sugar D-apiose was first found in Leman by Duff in 1963 (98) and is believed to be a constituent of the cell wall (99) The biosynthesis of D-apiose was studied by feeding l4C02 or myo-inositol-2_l4C to the plant and observing its localization in the cell wall (100shy102)

2 Alkaloids

From the rhizome of Nuphar luteum five thioalkaloids ie thiobishynupharidine allothiobinupharidine pseudothiobinupharidine thiobisdeoxyshynupharidine and neothiobinupharidine were isolated (103) It also conshytains nupharine (104) Mass spectroscopy of their structures were recordshyed and structures assigned according to an NMR study (105106)

From the rhizome of Nuphar japonicum the following lupine alkaloids were isolated deoxynupharidine (107) nupharamine (108-110) nupharane (Ill) nupharidine (112) nuphamine (113) and an unstable base dehydroshydeoxynupharidine (114) The abolute configuration of the identified strucshytures have been reported (115-117) Arata (118119) succeeded in syntheshysizing dl-deoxynupharidine

A piperidine alkaloid nuphenine has been isolated from Nuphar variegatum (120)

Paper chromatographic analysis of Nymphaea alkaloids to be present in the leaves and flowers TIleir structures remain undetermined but all are the ~uphar alkaloids

extracts showed two one in the root (121)

believed different from

Carex contains the indole alkaloids brevicolline harman brevicarine four other alkaloids (122-124) The strucshyture of brevicolline was studied and determined by Terenteva (125126)

6

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3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

4 Saponin

Saponins are widely distributed among higher plants (59) Bioshygenetically saponins can be divided into two groups

i) Glycosides of triterpenoid alcohols (normally at the 3-position) such as oleanane

ii) Glycosides of a particular steroid structure having a spiroketal side chain at the E and F ring juncture A few of the steroidal sapongenins are distinguished by having a ring juncture

Due to the sugar residue at 3-beta-hydroxyl group both types of saposhynins are soluble in water and ethanol but insoluble in ether Their aglyshycones (sapogenins) without the sugar moiety possess the solubilitv charshyacteristics of other sterols Thus saponins are most conveniently~ extracted from plants with 70-95 hot ethanol or isopropanol

Saponins are most often detected by

i) Foam test Saponins are presumed to be present when the charactershyistic honeycomb froth persists for at least 30 minutes after shaking an aqueous boiled (3-5 minutes) plant material

ii) Hemolytic test Saponin containing plant extracts mixed with defibrinated blood in a buffered physiological saline solution cause the hemolysis of red blood cells Digitonin a saponin available in crystalshyline form is generally used as the standard Tannins will interfere with the hemolytic action of saponins as they form a protective coating around the red blood cells The removal of tannins with magnesium salt previous to performing the hemolytic test was suggested by Farnsworth (57)

Both triterpenoid and steroid saponins behave the same in those two tests Tf necessary the Liebermann-Burchard test may be used to differshyentiate them red pink or purple colors are developed with triterpenoid saponins whereas blue or blue-green colors are formed with steroidal saponins (60)

5 Steroid

Steroids are derivatives of the tetracyclic hydrocarbon cyclopentanshyoperhydrophenanthrene The steroids thus far discovered in plants include sterols certain sapogenins alkaloids cardenolides and hormones (61)

i) Sterols are hydroxysteroids of C27 C28 and C29 series (62) An example of C27 sterol is cholesterol which is not only present in animals but has also been isolated from algae by Tsuda et al (63) from potato and Dioscorea plant by Johnson et al (64) A common sterol is ergoshyC28 sterol and widely distributed C29 sterols are beta-sitosterol and stigmasshyterol

ii) Alkaloids The occurrence of steroidal alkaloids is limited mostly to Solanum Veratrum and Holarhena (65) species They contain a 5-mernbered E ring and a 6-mernbered nitrogen containing F ring

4

iii) Cardiac glycosides They are glycosides of either C23 (cardenoshyJides) or C24 (bufadienolides) steroids with potent cardiac activity They are structurally characterized by an unsaturated lactone ring at Cl7

uncture of ring C and D a l4-beta-hydroxy group and by the pecushysugars (such as D-digitoxose D-digitalose D-cymarose and D-sarmenshy

tose) attached at

iv) Hormones Pregnane (C2l) androstane (C19 ) (66) and estrane (C18 ) (67) derivatives have been isolated from plants

Steroids can be extracted with benzene or ether and the chromatographic technique is of great value to detect plant materials for steroids A very good article reviewing the application of TLC for steroid detection has been written by Neher (68) Among the reagents most used and most generally apshyplicable are suI furic aci d-alcohol chlorosul phonic acid-acetic acid and molybdophosphoric acid Anisaldehyde-sulfuric acid reagent is used for deshytection of many steroids it is non-specific but with high sensitivity Dinitrobenzoic acid (Kedde reagent) and m-dinitrobenzene are specific for the detection of cardenolides and 17-ketosteroids respectively cerium sulfate is specific for the detection of nitrogen-containing steroids Keller (ferric chloride-acetic acid) and Kiliani (ferric sulfate-sulfuric acid) are specific for the deoxy sugar moiety of cardiac glycosides Frerejacque and DeGraeve (69) had tabulated all the cardiac glycoside deshytecting agents and their methods of preparation The use of iodine as a non-destructive location reagent for steroids in TIC has been introduced by Stevens (70) This technique is very valuable for preparative thin- and thick-layer chromatography

6 Lipid

Since triglycerides have been studied in this thesis only the saponishyfiable lipids will be discussed The saponifiable lipids are classified according to their structures into the fo11owing categories fatty acids fatty acid esters (triglycerides waxes etc) phospholipids and glycoshylipids

Non-polar lipids are extracted with petroleum ether or benzene and polar lipids are extracted with isopropanol isopropanol-chloroform methshyanol-chloroform ethanol-chloroform or ethanol-ether In addition to the avoidance of thermo- chemical and enzymatic oxidation and hydrolysis lipid extraction should be carried out under nitrogen to prevent the oxishydation of unsaturated compounds The extracts can be fractionated by TLC into different lipid classes According to Mangold (71) lipids can be visualized on the chromatograms with iodine vapour 2 7-dichlorofluorshyescein Rhodamine B of 6 G chromic acid-concentrated sulfuric acid and 50 sulfuric acid followed by charring The last reagent reveals differshyent color changes for different lipids during the heating process for example cholesterol and its esters turn red first then violet brown and finally black

Gas-liquid chromatography has been successfully employed in the separshyation of fatty acid mono- di- and triglycerides (72-74) The technique is also commonly used to analyze the fatty acid content of the triglycerides after methanolysis of the triglycerides The application of GTC in lipid

5

screening has been reported by Schlenk et a1 (75) in their search for arachidonic 5111417-eicosatetraenoic and related acids in plants

C Review of the Literature

1 General Considerations

Most studies on aquatic plants are urientated toward either ecology or limnology (76-80) Minerals (81-86) sugars (87-91) organic acids (9293) chlorophyll and xanthophyll pigments (94) have often been studied for their relationship to plant physiology

Chara sp are reported to contain amino acids (95) and uronic acid containing polysaccharides (96) contained many of the important amino acids but none with sulfur presence of vitamin B12 in Chara may be due to bacteria living epiphytically or in proximity to the algae Vitamins B2 and C were found in some aquatic plants of Moldavia (97)

The branch chain sugar D-apiose was first found in Leman by Duff in 1963 (98) and is believed to be a constituent of the cell wall (99) The biosynthesis of D-apiose was studied by feeding l4C02 or myo-inositol-2_l4C to the plant and observing its localization in the cell wall (100shy102)

2 Alkaloids

From the rhizome of Nuphar luteum five thioalkaloids ie thiobishynupharidine allothiobinupharidine pseudothiobinupharidine thiobisdeoxyshynupharidine and neothiobinupharidine were isolated (103) It also conshytains nupharine (104) Mass spectroscopy of their structures were recordshyed and structures assigned according to an NMR study (105106)

From the rhizome of Nuphar japonicum the following lupine alkaloids were isolated deoxynupharidine (107) nupharamine (108-110) nupharane (Ill) nupharidine (112) nuphamine (113) and an unstable base dehydroshydeoxynupharidine (114) The abolute configuration of the identified strucshytures have been reported (115-117) Arata (118119) succeeded in syntheshysizing dl-deoxynupharidine

A piperidine alkaloid nuphenine has been isolated from Nuphar variegatum (120)

Paper chromatographic analysis of Nymphaea alkaloids to be present in the leaves and flowers TIleir structures remain undetermined but all are the ~uphar alkaloids

extracts showed two one in the root (121)

believed different from

Carex contains the indole alkaloids brevicolline harman brevicarine four other alkaloids (122-124) The strucshyture of brevicolline was studied and determined by Terenteva (125126)

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3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

screening has been reported by Schlenk et a1 (75) in their search for arachidonic 5111417-eicosatetraenoic and related acids in plants

C Review of the Literature

1 General Considerations

Most studies on aquatic plants are urientated toward either ecology or limnology (76-80) Minerals (81-86) sugars (87-91) organic acids (9293) chlorophyll and xanthophyll pigments (94) have often been studied for their relationship to plant physiology

Chara sp are reported to contain amino acids (95) and uronic acid containing polysaccharides (96) contained many of the important amino acids but none with sulfur presence of vitamin B12 in Chara may be due to bacteria living epiphytically or in proximity to the algae Vitamins B2 and C were found in some aquatic plants of Moldavia (97)

The branch chain sugar D-apiose was first found in Leman by Duff in 1963 (98) and is believed to be a constituent of the cell wall (99) The biosynthesis of D-apiose was studied by feeding l4C02 or myo-inositol-2_l4C to the plant and observing its localization in the cell wall (100shy102)

2 Alkaloids

From the rhizome of Nuphar luteum five thioalkaloids ie thiobishynupharidine allothiobinupharidine pseudothiobinupharidine thiobisdeoxyshynupharidine and neothiobinupharidine were isolated (103) It also conshytains nupharine (104) Mass spectroscopy of their structures were recordshyed and structures assigned according to an NMR study (105106)

From the rhizome of Nuphar japonicum the following lupine alkaloids were isolated deoxynupharidine (107) nupharamine (108-110) nupharane (Ill) nupharidine (112) nuphamine (113) and an unstable base dehydroshydeoxynupharidine (114) The abolute configuration of the identified strucshytures have been reported (115-117) Arata (118119) succeeded in syntheshysizing dl-deoxynupharidine

A piperidine alkaloid nuphenine has been isolated from Nuphar variegatum (120)

Paper chromatographic analysis of Nymphaea alkaloids to be present in the leaves and flowers TIleir structures remain undetermined but all are the ~uphar alkaloids

extracts showed two one in the root (121)

believed different from

Carex contains the indole alkaloids brevicolline harman brevicarine four other alkaloids (122-124) The strucshyture of brevicolline was studied and determined by Terenteva (125126)

6

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Ul ~

p

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~ o CJ)

i o -lt

4-1

Ul 0 M u ltl

gtshyIJ amp 4-1 o

sect M Ul o Ii o u

Q)

~ 1-lt

o N N

ro

N

ro

ro

o ro

o

-0

N

-0

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o -0

o

o t

~ ~ ~ oj p

ro t

t

o

-0

o o N

o

o

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j

00

N

t

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M

M

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t

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-0

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t

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o

t

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t

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N

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00

o

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o

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co t t

co

ro

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oj IJ o

p ~

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ro

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lt ltlt (1j (1j C C (1j C

0 o 0 ltIl lttl (1j w (1j m jJ (j) jI

rlt U middotrl 0 -llt i( cJ (l) cJ (jQ)middotrlOJ-llt-llt~ f]J Vl Q) CIl (fl C ill (f) U) tfl C J c Q) OJ G) ltIlltIlCClttlCCltIlC(1jCCCCC GJ w(l)w~~w~~w~w~(l)~~~~

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c cJ 0

rlt 0

~ gtlt w

~ rlt C H 0 - 0 0 0 0 c X (l) C C

c cJc C C (l) M W rurl r-I ~rlU U Q)~ooc J c rlt ~rltJ PlGJCO

oct ~~~~HH~~H~HH~~W~~

H M (1j fJ

(J) 8 ~ s

J rtj m f) rlt ltIl gt 0 0 gt Ul 0

Ul -11 ~M rl ltIl Ul C M jl J ~ p C 4-l Ul rlt mM~4middotMucrj 0 ltIl 0 rlt H (1j rlt (j) H wOjJjJMmiddotrlQ)U 4-1 l t )l H m J w

rlt (1j lttl~(JlWO~H~C~lttlltllUW U M OJ r- J J M ro (Ij H 0 rl rj w c w ~ U l (j) M cwrlumJcQ)C~jIUUooHJM P gt gtltIl(1j(1jS~ltIlr~S(1j(j)~OgtMC

Cfl U M Po lttl cJ ltIl (1j C p H N (j) ~ (1j

(1j C C C C C Cl (1j rlt o 0 000 S S

r M rlt U1 H wwww+-JJ1 H H H rlt (j) W(l)Q)c)CJr-rl (1j (1j (1j (1j H C MMbOMbGC C wu cmiddotrimr) o 0 000 ltIl (1j

lttl ww(1j-ucc~(1jrrSSMbOlttl gt H ~~~~2~~~~22~~~~~~ C lttl GJ c Cl (1j (1j Cl M ~ C (1j (j) 0 0 0 0 0 Po Po ~ u U Cf) () U U W N lt ~ ~ P-1 PI p -j p UJ en f-

(j) (j) ltIl ltIl III (j) U U ltlJ III

III (j) (1j ltIl (1j ltIl W lttl Cl w W ltll (j) C (j) ltll (j) ltlJ rl -r- ltJ) Q) C) (U OJ U cJ lttl OJ U U (1j ltIl (j) H H ltlJ lttl ltIl ltIl (1j (1j (1j (1j (j)

M ltIl (1j ltIl (l) (j) (1j (1j (1j (1j OJ ltll (l) (j) (j) ~ ~ (1j ~ ltlJ +J jI ill U U Q)C ~ ill U U U U U = c Q)

~ U (1j (1j (1j (1j (1j C U U U (1j (1j (1j (1j (1j lttl (1j U ~ M Cl SaOJHH~OO(1jOOOooMMCl 0 rlt H ~~~~~~~~~~~~~~~~~ a (1j W oj c MMH~~H~~OJoj(1j(1j(1j(1jPoP~ Ul ~ U ~octoctuuu~~~ZZZZZVlCfl~

rlt I ~

N u o Ul

(l) Ul (j) U c M Ul lttl o 0 0 (1j M CO (1j H M o ltlJ ~ u oct ~M

8

(1j (1j (1j (1j ltltltlt C C C C 000 0 +Jww+- (l) GJ (l) (l) C C C C C C C C

rl middotri rl r ~~~~

P (j) -

C GJ H (j) C Q) bO-r-

bull ~ C ltIl H - I-J r1

(J)

C r (l) gt 9 u W ltIl (J) ltIl Ul

Ul ltll M 0 HoGJH Q) rl -r- Q) S ltIl H 0 ltll - (1j gt

0 (j) w

S gt S

M gt M ~

c gt ltIl Poc (l) o Po H (1j W 0 ltIlc (1j rl c Po H H Po S

~pound~t2

III ltIl (j) U ltIl (l) (j) ltll

M ltIl lttl (1j III III III gt U U U

c (1j (1j (1j PMOJ (l) o ltIl (1j Cl WHcc (1j 0 p p H J S (j) ltIl gt gt u~zz

I ltlJ gt u o U C 0 CIO

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GJ (1j 0

~ 0 H (l) C 0 0 gt rlt

w cJ (l) 0

U

lt H 0 -l

H (j) (j) Ul w ltIl (J)

cJ lttl I

C C 0 z w Po (j) 00 U X 0 (j) (j) M

w 0 U H (j) ltlJ (j)

~~-sect gt 0 (l) w U w

Po ltlJ (l)

OHVl C ltlJ ltIl C

rlt 0 (Jl (j) III III W gt0 U ltIl Cl III (l) 8 C o 0 0 U C -1

lttl w OJ U

~ ~ ~ 3 t1l rl

W 0 Ul U w C I (1j MClt P 0 Ul

ltll H (1j H (l) w ltlJ W o c Ul ~ ~ - - M N

3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

(1j (1j (1j

lt ltlt (1j (1j C C (1j C

0 o 0 ltIl lttl (1j w (1j m jJ (j) jI

rlt U middotrl 0 -llt i( cJ (l) cJ (jQ)middotrlOJ-llt-llt~ f]J Vl Q) CIl (fl C ill (f) U) tfl C J c Q) OJ G) ltIlltIlCClttlCCltIlC(1jCCCCC GJ w(l)w~~w~~w~w~(l)~~~~

~ H~H~~H~~H~H~~~~~~

c cJ 0

rlt 0

~ gtlt w

~ rlt C H 0 - 0 0 0 0 c X (l) C C

c cJc C C (l) M W rurl r-I ~rlU U Q)~ooc J c rlt ~rltJ PlGJCO

oct ~~~~HH~~H~HH~~W~~

H M (1j fJ

(J) 8 ~ s

J rtj m f) rlt ltIl gt 0 0 gt Ul 0

Ul -11 ~M rl ltIl Ul C M jl J ~ p C 4-l Ul rlt mM~4middotMucrj 0 ltIl 0 rlt H (1j rlt (j) H wOjJjJMmiddotrlQ)U 4-1 l t )l H m J w

rlt (1j lttl~(JlWO~H~C~lttlltllUW U M OJ r- J J M ro (Ij H 0 rl rj w c w ~ U l (j) M cwrlumJcQ)C~jIUUooHJM P gt gtltIl(1j(1jS~ltIlr~S(1j(j)~OgtMC

Cfl U M Po lttl cJ ltIl (1j C p H N (j) ~ (1j

(1j C C C C C Cl (1j rlt o 0 000 S S

r M rlt U1 H wwww+-JJ1 H H H rlt (j) W(l)Q)c)CJr-rl (1j (1j (1j (1j H C MMbOMbGC C wu cmiddotrimr) o 0 000 ltIl (1j

lttl ww(1j-ucc~(1jrrSSMbOlttl gt H ~~~~2~~~~22~~~~~~ C lttl GJ c Cl (1j (1j Cl M ~ C (1j (j) 0 0 0 0 0 Po Po ~ u U Cf) () U U W N lt ~ ~ P-1 PI p -j p UJ en f-

(j) (j) ltIl ltIl III (j) U U ltlJ III

III (j) (1j ltIl (1j ltIl W lttl Cl w W ltll (j) C (j) ltll (j) ltlJ rl -r- ltJ) Q) C) (U OJ U cJ lttl OJ U U (1j ltIl (j) H H ltlJ lttl ltIl ltIl (1j (1j (1j (1j (j)

M ltIl (1j ltIl (l) (j) (1j (1j (1j (1j OJ ltll (l) (j) (j) ~ ~ (1j ~ ltlJ +J jI ill U U Q)C ~ ill U U U U U = c Q)

~ U (1j (1j (1j (1j (1j C U U U (1j (1j (1j (1j (1j lttl (1j U ~ M Cl SaOJHH~OO(1jOOOooMMCl 0 rlt H ~~~~~~~~~~~~~~~~~ a (1j W oj c MMH~~H~~OJoj(1j(1j(1j(1jPoP~ Ul ~ U ~octoctuuu~~~ZZZZZVlCfl~

rlt I ~

N u o Ul

(l) Ul (j) U c M Ul lttl o 0 0 (1j M CO (1j H M o ltlJ ~ u oct ~M

8

(1j (1j (1j (1j ltltltlt C C C C 000 0 +Jww+- (l) GJ (l) (l) C C C C C C C C

rl middotri rl r ~~~~

P (j) -

C GJ H (j) C Q) bO-r-

bull ~ C ltIl H - I-J r1

(J)

C r (l) gt 9 u W ltIl (J) ltIl Ul

Ul ltll M 0 HoGJH Q) rl -r- Q) S ltIl H 0 ltll - (1j gt

0 (j) w

S gt S

M gt M ~

c gt ltIl Poc (l) o Po H (1j W 0 ltIlc (1j rl c Po H H Po S

~pound~t2

III ltIl (j) U ltIl (l) (j) ltll

M ltIl lttl (1j III III III gt U U U

c (1j (1j (1j PMOJ (l) o ltIl (1j Cl WHcc (1j 0 p p H J S (j) ltIl gt gt u~zz

I ltlJ gt u o U C 0 CIO

~ i 00

]1 w

J M J ~

0 C C rlt ltIl

GJ (1j 0

~ 0 H (l) C 0 0 gt rlt

w cJ (l) 0

U

lt H 0 -l

H (j) (j) Ul w ltIl (J)

cJ lttl I

C C 0 z w Po (j) 00 U X 0 (j) (j) M

w 0 U H (j) ltlJ (j)

~~-sect gt 0 (l) w U w

Po ltlJ (l)

OHVl C ltlJ ltIl C

rlt 0 (Jl (j) III III W gt0 U ltIl Cl III (l) 8 C o 0 0 U C -1

lttl w OJ U

~ ~ ~ 3 t1l rl

W 0 Ul U w C I (1j MClt P 0 Ul

ltll H (1j H (l) w ltlJ W o c Ul ~ ~ - - M N

3 Flavonoids

Carexidin (a 3-deoxyan thocyanidine) was discovered in (127) Flavonoid A (5734

and flavonoid B (luteolin) were isolated by means of a polyamide column from Lerona minor (128129)

4 Tannins saponins steroids and lipids

Very few studies have been done on aquatic plants for tannins saposhynins steroids or lipids Tannin was reported to be present in

Nuphar and (ell agic acid) (130) but absent (131) Trace amount steroidal sapogenins were found in sp (132) Stigmasterol and beta-sitosterol were found in the the flower (13-3) of Nupha~~ luteulTl A 1 ipoprotein complex was in ElodClt3 (131)

The acid composition of some aquatic plants have been analyzed by Schlenk (75 by means of the gas-liquid chromatographic te~hnique The results are summarized in Table 1

II Materials and Methods

A Plant Collection and Identification

Plant materials used in this study were collected from various lakes in Minnesota during August and September 1968 Specimens representing the collections were pressed between blotters and carefully dried at 50middotC Taxonomic identification was made by Dr Robert C Bright Assistant Proshyfessor in Limnology University of Minnesota in the field and later conshyfirmed by Dr Gerald B Ownbey Professor and Curator of Herbarium Unishyversity of Minnesota One voucher herbarium for each plant has been deshyposited at the Botanical Museum Harvard University Cambridge Massachushysetts Representative plants were also preserved in jars containing water 95 ethanol formaldehyde (631) and 5 glycerine Copper ion at 002 ppm was added to help retain the original color of plants

A list of those plants collected representing one algae seventeen monocots and four dicots is shown in Table 2 The gross appearance and general ecology of the plants are discussed alphabetically Abbreviations in the parentheses will be utilized in tables throughout this dissertation instead of the full names of plants

AnachilEis (Michx) Rich (Ac) branched that form large masses usually three in each whorl

Stems are so are whorled

(Water arum) (Cp) Emergent Perennial herbs with and solitary spathes leave~s are either ovate or subrotund (5-10 cm)

9

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

Carex lacustris Willd (Cl) Emergent plants grown in swamps and marsh~eaves are stout usually with conspicuous cross-septate and with numerous elevated nerves

Ceratophyllum demersum L (Coontail) (Cd) Submerged Stems with whorls of stiff forked leaves and leaflets with toothed or sershyrated margins on one side only They are freely branched forming large masses and are found in quiet water

Chara vulgaris (Cv) Submerged green algae (Muskgrass) The plant possesses a musky odor and is made up of stems bearing whorled smooth brittle branches easily snapped with a slight pressure

Eleocharis smallii L (Spike rush) (Es) Erect and emergent Rhizomes are often conspicuous and the leaf sheaths are obliquely trunshycate and firm

Lemna minor L (Duckweed) (Lm) They represent the smallest of the aquatic plants (2-4 x 15-3 mm) They have no true leaves nor stems but the floating green plant body usually possessing a tiny root that peneshytrates the water They may grow sufficiently dense to prohibit sunlight from penetrating the water thus killing algae and other aquatic plants

Myriophyllum exalbescens (Fern) Jeps (Water-mil foil) (Me) Subshymerged herbs in quiet water Leaves are feather-like with one c~ntral axis and branches in whorls around the stem

Nuphar variegatum Engelm (Yellow water lily) (Nv) Floating leaves are heart-shaped with veins radiating from the mid-rib nearly to the margin without forking The floating flowers are attractive and yellow

Nymphaea tuberosa Paine (Water lily) (Nt) Floating circular leaves possess much-forked veins radiating to the margin Flowers with green sepals and white petals and are long-petioled (usually striped)

Potamogeton amplifolius Tuckerm (Pa) Those belonging to Potamogeton sp (Pondweed) are plants with usually both floating and submerged leaves scattered along the stem and with midribs evident when held against bright light For Potamogeton amplifolius the submerged leaves (8-20 cm) are falcately folded and floating leaves (5-10 cm) are elliptic

Potamogeton natans L (Pn) Stems are simple or sparingly branched Submerged leaves are phyllodial narrowly linear (1-4 cm x 102 mm) floatshying leaves are elliptic (5-10 x 2-45 cm) Petiole usually exceeding the blade and flexibly attached to it

Potamogeton pectinatus L (Pp) Leaves are all submerged narrowly linear (3-10 cm x 05-15 mm) Lower part of stem is simple or sparingly branched with elongated internotes while the upper part is freely dichoshytamously branched Therefore the appearence of a bounch of rounded treadshylike leaves as they float in the water is very characteristic

Potamogeton richardsonii (Benn) Rybd (Pr) Stems are freely branched and densely leafy Leaves are lanceolate to nearly linear (3-12 cm x 5-20 mm)

10

Potamogeton zosteriformis Fern (pz) Stems are freely branched flattened and winged (1-3 mm wide) Leaves are linear (1-2 cm x 2-5 mm) They are most usually found in slow streams

Sagittaria cuneata Sheldon (Water plantain) (Sc) Emergent plants rooted to the substratum and extending upward out of the water Leaves are long-petioled and sagittate with variable sizes (6-18 x 1-10 cm)

Sagittaria latifolia Willd (Arrow-head) (Sl) Emergent plants usushyally found in swamps or ponds leaves are sagittate (5-40 x 2-25 cm)

Sparganium eurycarpum Engelm (Se) Stout and emergent (5-12 dm) Leaves are shallowly and broadly triangular in cross section (8 dm x 6-12 mm) They are grown in mud or shallow water

Sparganium fluctuans (Morong) Robins (Sf) Floating plants with slender elongate stems (15 dm) leaves are flat thin alternate transshylucent with cross reticulate and with sheathing bases

~ angustifolia L (Cat-tail) (Ta) Erect colonial herbs with long linear leaves sheathing at the base Flowers are densely crowded in long cylindric terminal spikes They are grown in marshes

Vallisneria americana Michx (Va) Perennial herbs with very thin long ribbon-like basal submersed leaves (2 m x 3-10 mm) They are found in quiet water area

Zizania equatica L (Wild rice) (Za) Robust annual grasses usually 2-3 m high They are found in marshes and shallow water with tall culms and wide flat blades

B Extraction

Plants were rinsed thoroughly and adherring foreign materials such as sand leeches and other plant species were removed The plants were then spread on metal screens and dried in a well-ventilated oven at 50degC When dry each plant species was milled (Fitz Mill Model D Chicago Ill) to pass a No 14 seive weighed and stored in a tightly closed polyethyshylene bag until being extracted

In order to study the nature of the various constituents present in those aquatic plants exhaustive extraction using solvents ranging from the non-polar to polar type was followed The solvent sequence used was skellysolve F (bp 30-60degC) chloroform 80 ethanol acidic water and lastly basic water For each extraction 150 gm of dried powdered plant material was used All the concentrated extracts prepared were stored unshyder nitrogen in amber colored bottles sealed with paraffin and freezed

Dried powdered plant material was extracted continuously with skellyshysolve F and then with chloroform in a Soxhlet extractor until the fresh extract no longer had a green color Each extract was concentrated to a volume of 20 ml in a flash evaporator

11

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

The skellysolve F and chloroform extracted plant material was air dried and placed in a Waring blender containing 1500 ml of 80 ethanol After homogenizing twice for 30 seconds the material was allowed to macerate overnight The mixture was then vacuum filtered through a layer of cheesecloth and then through a Whatman No 1 filter paper TIle residshyual plant material was transferred to the Waring blender and the extracshytion procedure repeated The filtrates were then combined and concenshytrated to a volume of 40 ml in a flash evaporator

The solvent exhausted plant material was then macerated for 24 hours with warm (60 0 e) water that had been adjusted with 10 Hel to pH 3 The mi xture was vacuum filtered and flash evaporated to a volume of 20 ml The same procedure was followed for basic water extraction except that 10 KOH was used to adjust the mixture to pH 10

C Detection for Alkaloids

1 Purification of extracts

Skellysolve F chloroform and 90 ethanol extracts were purified according to the flow chart in Fig 1 before spotting on the silica gel H plates

2 Thin-layer chromatography

Preparation of plates

Cleaned and dried glass plates (10 x 20 em) were coated (025 mm) with silica gel H (prepared according to Stahl for thin-layer chromatograshyphy E Merck Ag Darmstadt Germany) on a DeSaga apparatus (Brinkmann Instruments Inc Great Neck Long Island) The slurry was made by mixing 25 gm of si1ica gel H with 75 ml of the mixture of methanol and water (31) for 20 seconds The plates were air dried for 20 minutes activated at 110degC for 30 minutes and stored over calcium sulfate in a desiccator

Each purified extract (10 ~l) was applied to the silica gel H plate and developed in chloroformacetonediethylamine (541) for the skellysolve F extracts or in n-butanol acetic acidwater (411) for the chloroform and 80 ethanol extracts The following alkaloids which represent different chemical classes were used as standards at a concenshytration of 10 mgml in 50 ethanolcaffeine (purine) L-hyoscyarnine (troshy

and ergonovine maleate (indole)

Detection

The following methods were used for the visualization of colorshyless substances on chromatograms i) Observation under ultraviolet light (254 m~) ii) Spraying with Dragendorffs reagent (134) iii) Spraying with 1 p-dimethylaminobenzaldehyde (PDAB Ehrlichs reagent) followed by freshly prepared 01 NaNO in 50 ethanol (135)

12

Figure 1 Purification of skellysolve F chloroform and 80 ethanol extracts for the alkaloid detection

Skelly F CHC1 3 or 80 EtOH ext (1 ml)

i) For skelly F or CHC1 3 ext add 1 rnl of or ii) For 80 EtOH ext reshymove any alcohol present in a water bath filter add 1 ml of eHel] to the filtrate And then add 1 HCl (1 ml)

l Acidic Aq

10 NH40H to pH 90 CHe13 (1 ml)

tlayer Alkaline aq

(discard)

D Detection for Flavonoids

Skellysolve F and chloroform extracts were applied (10 Ill) to silica gel H thin-layer plates prepared as previously described (p 50) developed in chloroformmethanol (955) whereas 80 ethanol extracts were applied (10 ~I) on 20 x 20 em cellulose sheets (No 606 l Eastman Kodak Co N Y) and developed in n-butanolacetic acidwater (415) The chromatograms were examined by i) Direct observat ion of yellow to pink compounds ii) Flushyorescent pattern (254 mil) iii) Exposure to ammonia vapour and iv) Spraying with p-nitrobenzene diazonium fluoroborate (05 aqueous) The standards (4 mgml in methanol) used were rutin umbe 11 i ferone visnagin and provisshymine

E Detection for Tannins

An aliquot (02 rnI) of 80 ethanol acidi c water or basic water exshytract was mixed with distilled water (18 rnl) filtered and the filtrate tested for the presence of tannins Aqueous tannic acid USP (1) was used as the standard The test procedure used is shown in Fig 2 The reagents used were i) Buffered gelatin salt solution- dissolve (1 gm) and NaCl (5 grn) in acid phthalate buffer (pH 30 100 ii) 10 M Urea- dissolve urea (30 gm) in water (50 ml) and iii) ous ferric chloride solution

13

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

Figure 2 Procedure for the detection of tannins

Sample (025 ml)

I Buffered gelatin-salt solution (5 gtts)

Ppt (positive tannin test)

t Centrifuge for 10 min

Residue

t 10 M urea (15 ml)

Soluble produce

(positive tannin test) 9 (3 gtts)

Blue-black or green color

(positive tannin tes t)

F Detection for Saponins

1 HemolysiS test

As aliquot (05 ml) of 80 ethanol acidic water or basic water extracts was freed of tannin by adding 80 ethanol (45 ml) or 09 normal saline for water extracts filtered and heated at 70degC for 15 minutes Magnesium oxide (1 gm) was added to the filtrate and heated at 70 0 e for 15 minutes to form a tannin-MgO complex Ethanol (95 5 ml) was then added and the tannin-free filtrate used for the hemolysis test A digishytonin solution (01 gmml in 80 ethanol) was used as the standard

The hemolysis test consists of mixing 1 ml of detanned filtrate with 1 ml of standarized red blood cells The hemolytic activity was recorded as the time in minutes required to completely hemolyze the red blood cells On each experimental day an aliquot of stock red blood cells (5 ml) was withdrawn and standarized with 05 ml of digitonin solution (01 mgml) Indication of hemolysis of the red blood cells was observed microshyscopically and by centrifugation after a ten-minute reaction period A colorless upper layer after centrifugation indicated a negative saponin test whereas a red upper layer indicated a positive saponin test The stock red blood cells were diluted with isotonic phosphate buffer (pH 74) in a dilution of 15 to obtain a positive digitonin hemolysis test

The standarized red blood cells were prepared by defibrinating fresh human whole blood (6 ml) with sealed fine capillary tubes The fibrinshyfree blood was suspended in normal saline (35 ml) and centrifuges (speed

14

six serial No 37618 P-2 International Equipment Co Needham Hts Mass) The supernatant was discarded and the packed red blood cells washed three times with normal saline The washed packed red blood cells were resuspended in normal saline (100 ml) stored at 10degC overshynight and used within 2-3 days The isotonic phosphate buffer used was prepared by dissolving 044 gm of NaCl in the mixture of 20 ml of sodium biphosphate solution (08 in water) and 80 ml of sodium phosphate solushytion (094 in water)

2 Froth test

A freshly prepared hot water extract was made by heating a mixshyture of 1 gm of dried plant material and 15 ml of distilled water on a water bath After cooling the mixture was filtered through four layers of cheese-cloth and the filtrate was shaken vigorously for exactly one minute The honeycomb froth geight formed after exactly 5 and 30 minutes were recorded Digitonin (1 ml 01 mgml) was used as the standard

G Detection for Steroids

Skellysolve F chloroform and 80 ethanol extracts were applied (10 Ill) to silica gel H thin-layer plates as previously described (p 50) developed in cyclohexaneethylacetate (11) The plates were examined by i) Fluorescent pattern (254 mil) it) Heating at 1000e for 15 minutes after spraying with freshly prepared anisaldehyde reagent (1 vv of anisaldehyde in 2 vv concentrated sulfuric acid in glacial acetic acid) (137) and iii) Spraying with Kedde reagent (1 35-dinitrobenzoic acid in 05 N of 50 vv aqueous methanolic KOH) (138) followed by Zimmermann reagent (mix 1 vol of 2 m-dinitrobenzene in ethanol with 1 vol of 125 N of ethanolic KOH) (139) Androstandiol digitoxigenin digitOXin progesterone and betashysitosterol (10 mgml in 11 misture of methanol and chloroform) were used as standards

H Lipid Analysis

1 Gravimetric determination of total lipids

Skellysolve F and chloroform extracts representing 11025 gm and 10275 gm of dry plant material respectively were brought up to a volume of 25 ml with their respective solvents An aliquot of 1 ml was transferred to a preweighed aluminum dish which was then freed of solvent in a high vacuum desiccator until a constant weight was achieved Weight percent (ww) content of lipids extractable by skellysolve F or chloroform with respect to the original dried powdered plant material was obtained by the following formula

concentration (gml) x (ml)

11025 (gm for skellysolve F ext) or 10275 (gm for CHC1 3 ext) x 100

15

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

2 Systemic analysis of lipid distribution

Thin-layer chromatography

5kellysolve F and chloroform extracts (5 IJI each) were applied to thin-layer Chromagram Sheets (20 x 20 cm 6061 silica gel without flushyorescent indicator Eastman Kodak Co NY) The solvent system solve Fether (955) was used for the skellysolve F extracts and that skellysolve Betherglacial acetic acid (70301) for the chloroform exshytracts TIle lipids were detected by exposing the chromatograms to iodine vapour

The reference standard used was lipid mixture 58 (Lipids Preparation Laboratory The Hormel Institute Austin Minn) which consists of oleic acid methyl oleate alkyl diglyceride (primarily dioleate) triolein oleyl palmitate octadecene-9 neutral plasmalogen cholesterol and cholesshyterol oleate

3 Fatty acid constituents of triglycerides isolated from selected plant extracts

Triglycerides were first separated from the skellysolve F and chloroform extracts of Nymphaea tuberosa canadensis and Carex lacustris Methyl esters acids obtained by methanolysis of pure triglycerides were analyzed by GLe Conshyfirmation of chain lengths of fatty acids and their degree of unsaturation was achieved by hydrogenation with subsequent GLC analysis of hydrogenated methyl esters of fatty acids

a Isolation of triglycerides

Altquots of skellysolve F and chloroform extracts of each plant were applied as a narrow band (5 111l11) on sil ica gel G plates (1 mm) under a stream of nitrogen A C-16 triglyceride standard was spotted on both sides of the band The plates were developed in the solvent system of skellysolve Fdiethyl ether (8020) Triglyceride appeared as a darker band on the chromatograms which was easily seen by observing the plates against a strong light source in a dark room The standard triglyceride co-chromatographed on both sides served as an additional guide-line

The triglyceride fractions from the skellysolve F and chloroform exshytracts were combined and transferred to a screw-capped vial and extracted three times with peroxide-free diethyl ether previously saturated with oxygen-free water The ethereal extracts were brought down to almost dryness (trace of water left) under a stream of nitrogen A small amount of skellysolve F was then added and the extract dried over anhydrous sodium sulfate and under nitrogen to remove the water If necessary a second solvent system of skellysolve Fdiethyl ether (9010) was used for the thin-layer chromatographic purification of triglyceride

16

b Methanolysis

The pure triglyceride (S mg) and 25 m1 of reaction mixture (absolute methanol benzene and concentrated sulfuric acid 86104) were reacted under nitrogen at 80-90middotC for 2 hours to form the fatty acid methyl derivatives Water (40 ml) was added at the end of the reshyaction period and the mixture extracted three times with hexane (5 ml) The combined hexane extract was dried over anhydrous sodium sulfate and stored under nitrogen until ready for gas-liquid chromatographic analysis

TLC of methyl esters of fatty acids which after spraying with 02 of 27-dichlorofluorescene in 95 ethanol form a characteristic yellow fluorescent spots under ultraviolet light

c Hydrogenation

Hydrogenated methyl esters of fatty acids were prepared by mixing 4 ml of methyl esters in skellysolve F (1 with a few crysshytals of platinum dioxide and then subjected to hydrogenation for 3 hours at 40 pounds in a hydrogenator (Parr Instrument Company Inc Moline Ill )

d Gas-liquid chromatography (GLC)

The instrument employed was a BeckmanC~-2 Gas Chromatograph (Beckman Scientific Instrument Division Fullerton Calif) equipped with a flame ionization detector The aluminum column used (6 feet in length and 18 inch LD) was packed with 20 ww diethylene glycol succinate (DEGS) on Anakrom A (100110 mesh) and purchased [rom Analab Inc Hamshyden Conn The column temperature used was 175degC the injection-port temperature was 200degC and helium at 25 psi was used as the carrier gas

The standard used was GLC Reference Mixture No 1 (Iipids Preparation Laboratory The Hormel Institute Austin Minn) which is a mixture of methyl esters of palmitic (160) stearic (180) oleic (181) linoleic (182) and linoleic acid (183)

Sample peaks were identified with the aid of the standard graph of log retention time vs carbon number (Fig 3) Assignment of the carbon chair length of each unknown peak was made possible through its retention time The area of each peak was calculated by multiplying its peak height with one-half of its base width and the relative percentage of each fatty acid ester or that of its hydrogenated compound was obtained by dividing its peak area with total peak areas on the chromatograph

17

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

III RESULTS AND DISCUSSIONS

A Alkaloids

The resid ts for the thin-layer chromatographic detection of al kaloids are shown in Table 3

Most of the original extracts did not appear to contain alkaloids after examining the TLC of 10 III aliquots of the extracts (equivalent to 750 mg of dry plant powder for skellysolve F and chloroform extracts and 375 mg for 80 ethanol extracts) Therefore the extracts were further purified to remove water soluble organic materials which might have inshy

I lON terfered with the alkaloidal detection

0 demonstrated Dragendorff positive spots

demersum Carex lacustris Lerona mInor -j

f reactions Dragendorff positive spots in concentrations

~ japonicum and ~ been reported to contain lupine (107-119) and (103105106) reshy

spectively The nature of two alkaloids (l2l) known However the alkaloid in Nymphaea in a positive Ehrlich reaction and is suspected to indole-type alkaloid Although indole alkaloids are reported present in 022-126) they are not present in Carex lacu~tris

Dragendorffs reagent can detect very small concentrations of alka]oids (140) by forming an orange to pink metal complex Farnsworth (44) found that conjugated carbonyl (ketone or aldehyde) or lactone functions was the

L~~~~--------i n IS M ~I minimum structural requirement for a Dragendorff reaction to occur By this criterium natural products such as coumarins anthraquinones etc will also give an alkaloid-like reaction Ehrlichs reagent has been widely used for the detect ion of indole compounds Any electron-withdrawing group (eg -eN -C=O) decreases the electron density around the 2-carbon atom of the indole nucleus and will repell the attacking electrophilic aldehyde Ehrlichs reagent will react with simple indoles (tryptophan 5-0H tryptoshyphan etc) aromatic amines (anthranilic acid) ureides (thiourea) and phloshyroglucinol (141) The color of the condensation products formed between

Ftgure 3 Standard graph of log retention time vs carbon number of indole and aldehydes may be purple pink blue green to brown orange or reference fatty acids yellow Most indole alkaloids will form a purple b]ue to gray co]or with

p-dimethylaminobenzaldehyde and these colors may be stabilized by freshly prepared 01 NaN02 1n ethanol solution

The interpretation symbols (+t+ ++ + or t) for the Dragendorff reshyactions are only approximate as the surface area represented by a single alkaloid spot increases with an increase in Rf value

J

1918

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

V

c

Table 3 Thin-layer Fluorescent and Alkaloid Patterns I

Ac-l

Ac-2

Cd

Cl

Cp

Cv

Es

Lm

Me

Nt Iv

Nt st

Nv-l Iv

Nv-l st

Nv-2 Iv

1II4 IV v II3

a b c a b c a

S 030 blye + 030 C A 040 bl ++ 008 pi + 049 S 009 or ++ C A 008 ye ++ 001 or t S 073 bi +

082 re + C 002 ye ++ 002

030 ye ++ A 037 pu + 016 pi + S 080 C 002 pi + A 016 or br + S C 043 bl +

062 bl + A 002 bl ++ 052 pi t

042 bl ++ s 076 re + C 079 pi t 079 A 070 or + 070 S 003 gr + C A S C 048 bl + 060 or t A 041 bl + 004 pi + 5 004 wh ++

015 br + C A S 036 re + C A 043 bl + 060 pu ++ 060 S C A 039 pu ++ 005 or + 066

049 bl ++ 066 pu gy + 5 C A 044 or + 030 5 007 or + C 043 or ++ A 020 gr ye + 021 or +++ S C 024 gr ye + 023 or ++ A 040 or pi ++

20

(Table 3 continued)

I

Nv-2 st

Pa

Pn

Pp

Pr

pz

Sc

Se

5f

51

Ta-l

Ta-2

III II

IV

b

gr ye

br

pu bl

gy

ye gr ye

gy

gy

yg

c

+

+

t

t

+ +

+

+

+

S

C A S C A 5 C A

5 C A S

C

A 5 C A 5 C A S C

A 5

C A

s C A S C A S

C A

a

005 085 070 020 073

043

048 044 053 015

043 080

043 052

041

043 006 025 044 055 061 003 007 015 075

046 058 068 005

036 066

044 008 064

043

b c

ye + ye +

gr ye + bl ye ++

re t

bi +

bl + bl + bI + br +

bl t

re +

bI t

re t

bl +

bl ++ gr ye + bl + bl +

gr ye + gr bl +

pu + ye + ye + bl +

bl + ye +

gr bI + pi +

bl t

ye wh +++

bI + gr ++ bl +

bI ++

a

028 020 073

053

067

011

048 067 072 048

005

077

009 036

21

b

or or or

br

br

pi

or or pi pi

or pi

pi

pi br

c

+ +++ +

+

t

+

+ + t

+

+

t

+ t

a

070

059

072

072

077

046

060 062

b

ye

br

ye

br

ye

br

bl pu pu

+

t

+

+

+

+

t t

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

3 continued)

III IV V I II

a b c a b c a b c --------

Va S 006 ye ++ 023 or + 046 ye t 006 gr or +

C 002 by + A 030 bl +

046 bl + Za S

C A 043 bl + 059 br t

Std 3 5) Er a) 005 pu ++ 002 or ++ 002 gy +

b) 029 pu ++ 029 gy or ++ 029 pu ++ Hy a) 019 ye or ++

b) 024 or ++ Ca a) 052 or ++

b) 028 or +

lRoman numerials 1- Plant names 11- Extracts 111- Fluorescent pattern (254 m~) IV- Dragendorffs positive spots V- p-Dimethylaminobenzaldeshyhyde positive spots

2Plant names Ac- Anacharis canadensis (1- collected at Lake Minnetonka 2- collected at Lake Itasca) Cd- Ceratophyllum demersum Cl- Carex lacustris Cp- Calla palustris Cv- Chara vulgaris Es- Eleocharis smal1ii Lm- Lerona minor Me- Myriophyllum exalbescens Nt- Nymphaea tuberosa (lv- leaf st- stem) Nv- Nuphar variegatum (1- collected at Lake Minnetonka 2- collected at the Pine Lake) Pa- Potamogeton folius Pn- ~ Pp-~ pectinatus Pr-~ richardsonii pzshyzosteriformis Sagittaria cuneata Se- Sparganium eurycarpum Sparganium fluctuans Sl- Sagittaria latifolia Ta- Typha angustifolia (1- collected at the Silver Lake 2- collected at the Pine Lake) VashyVallisneria americana Za- Zizania aquatica

3Extracts and solvent systems A- 80 Ethanol C- Chloroform S- Skellyshysolve F a)- Solvent system - chloroformacetonediethylamine (541) for Skellysolve F extracts b) Solvent system- n-butanolacetic acid water (411) for Chloroform and 80 ethanol extracts

4a- Rf values b- Color bl-blue br-brown gr-green gy-gray or-orange pi-pink pu-purple re-red wh-white ye-yellow c- Intensity +++=high ~ medium += low t= trace

5Standards Er- Ergonovine maleate Hy-Hyoscyamine Ca-Caffeine

22

B Flanonoids

The results for the thin-layer chromatographic detection for flavoshyno ids are shown in Table 4

Flavonols were most widely distributed in the plant extracts studied These compounds were present in Calla palustris Lemna Myriophyllum exalbescens Nuphar variegatum natans ~ ~ richardsonni ~ zosteriformis cuneata ~ Typha angustifolia and Zizania aquatica appear to present in Potamogeton amplifolius ~ natans ~ pectinatus ~ zosteriformis Sagitshytaria cuneata ~ latifolia Sparganium fluctuans and Vallisneria americana Flavones were present in minor Myriophyllum exalbescens and Nymphaea 1 tuberosa Anthocyanidine aurones are not present in 80 ethanol exshytracts as indicated by the absence of visible orange to pink spots on the chromatograms Catechin was present in lacustris and Potamogeton richardsonii The flavonol spots fluorescent blue-purpoe before ammonia vapour treatment turned yellow instantly in the ammonia vapour chamber and their fluorescence intensified According to the patterns of ring subshystitutions the Rf values for those flavonols varies from 044 to 076 in the solvent system of n-butanolacetic acidwater (415) The pale blue fluorescent spots (Rf values 088-090 in BAW 415) of flavanones showed no conspicuous color change by ammonia vapour or only being slightly enshyhanced The orange-yellow fluorescent spots of flavones (Rf value 035 in BAW 415) were intensified to dull brown or bright yellow after fuming with ammonia The blue fluorescent spots of catechins (Rf values 090-094 in BAW 415) appear only after ammonia vapour treatment The flavonOid 3-deoxyanthocyanidine previously found in Carex riparia and f (127) was not in C whereas the presence of the flavone minor reported (128 was evident

With the exception of catechins and leukoanthocyanines the flavonoid compounds fluorescent under ultraviolet light and their fluorescence may be intensified or changed by exposing the chromatogram to ammonia fumes The action of ammonia on flavonoids is reversible p-Nitrobenbenzene diashyzonium f1uoroborate is a very effective coupling reagent forming with pheshynols yellow orange or brown colors The spray is non-specific and will not only detect flavonoids but also aryl amines tannins etc (142)

C Tannins

The results for the color detection of tannins are shown in Table 5

Tannins especially the condensed type were widely distributed in the aquatic plants screened The following plant species were found to contain condensed tannins Eleocharis smallii Lemna minor Myriophyllum exalbescens amplifolius ~ natans ~ richardsonii and Sparganium fluctuans extracts formed precipitates with gelatin-salt solution and the resulting urea solubilized precipishytates showed a blue-green or green-yellow color with the ferric chloride reagent

Nuphar variegatum and Numphea tuberosa contain hydrolyzable tannins which after solubilization with urea formed a blue-black or purple-blue color with the ferric chloride reagent Acidic and basic water extracts

23

VI

r Table 4 Thin-layer Fluorescent and Flavonoid Patterns l bull

I

Ac-l

Ac-2

Cd

C1

s C

A

S C

A

S C

A

s C

I II

Cp

Cv

Es

A

S C

A

S C

A S

C

Lm

A

s C

A

V31 III Daylight uv Daylight UV

043 ye + 067 re t 060 bl t gr + 092 ye t ye +

008 040 or + 060 bl t bl + 092 or + or + 078 017 043 ye + 066 re t 041 ye + br + 060 bl + + 076 015

ye + re t

gr ye

023 ye + 040 or ++ 072 054

re bl

t t

ye + bi t

072 b1 + bl + 090 bI + 051 ye + 017 gy gr + 045 036

ye ++ br +

059 066 bl t

ye + hI +

085 ye + 006 re or + 044 ye +

052 bi + 077 ye + 026 b] + 044 ye + 050 bl + 073 re t 041 052 068 ye ++

ye bi

t +

ye pu

t

+

007 O ]6 gr + 035 062 086

pu pu bl

+ ++ +

ye ye

++ ++

br br pu

+ +

24

VI

or +

or +

ye t

or +

or + br t

br +

or +++

br + br +

br + br +

ye + ye +

or +

br + hr +

(Table 4 continued)

I II III

Me S 040 C 013 A 035

057 062

Nt S 037 Iv C 026

041 050 080

A 035 071

Nt S 037 st C 021

A 035 071

Nv-l S 043 Iv C 040

A 035 057 066

Nv-l S 020 st C 021

A 035 057 066

Nv-2 S 043 Iv 068

C 003 045

A 035 057 066

Nv-2 S 085 st C 024

051 080

A 035 057

Pa S 078 C 052 A 090

IV

Daylight

ye +

ye ++

ye +

ye ++

ye ++ ye ++

ye + ye +

ye + ye ++

ye ++

ye +

re ++ re gr +

UV

ye pu bl

re

re or pu

or pu

ye pu bl

ye pu bl

ye

ye pu bi re

re ye

bl

25

t

+ +

++

+ + t

t

+

t

++ +

t

++ t

t

t

+ t

+

t

t

t

V

Daylight UV

ye

br

br

ye

or

ye

br

br

br br

or

ye

br

br

t

+

+

+

+

++

+

+

+ ++

+

+

t

t

ye

ye ye

ye

ye

+

++ t

++

+

ye

pu

or pu

ye pu

or pu h]

br pu

pu

b]

+

t

+ t

+ +

+ ++ +

+ ++

++

t

(Table 4 continued)

I II III

Pn S 066 C 007

045 A 057

066 090

Pp S C 008 A 066

090 Pr S 076

084 c 045

067 A 057

094 pz S 038

C 021 A 057

090 Sc S 082

C 042 A 066

090 Se S 046

076 C A 067

Sf S C 044

048 058

A 090 S 049

084 C 003 A 066

089 Ta-l S 082

C 035 A 044

057 074

IV

Daylight uv

V

(Table 4 continued)

re or

or

re ye ye re

br

or or

ye gy

ye

ye ye

Daylight uv

++

++

+ + + +

+

++ ++

+ +

+

++ ++

pu t pu + pu ++

pu t

bl +

pu t

re t

pu + bl t

pu + bl t

ye + wh +

gr bl ++

ye + pu + bl t

ye t ye gr +

pu + bl t

pu ++

26

ye ye

ye ye

ye

ye

ye

ye

ye

ye ye ye

+ ++ +

t

t

t

++

++

+

+

t

t

++

pu bl bl

bl

pu bl pu

pi

pi bl

gr bl

br bl

br br br

+ ++ ++

+

+ +

+

+ t

++

+ +

t t

++

VI

or +

br t br + br ++

or ++

br +

br ++

br +

or + br + br +

br + br +

ye +

hr +

br +

br + br + br + or +

br + ye br ++

I

Ta-2

Va

Za

Std Ru Ru Urn Vi Pr

II

S C A S C A

S C A

a) b) a) a) a)

Daylight uv Daylight

021 ye + (Identical with those for Ta-l A) 011 br t

056 re t

088 ye + 081 or + 050 or + 045 ye wh ++ 057 bl t ye t

066 bl + ye +

002 gr ye ++ ye ++ 066 ye t pu ++ ye ++ 031 hI ++ 053 gr ++ 074 pu ++

IV V III VI

uv

or +

or +++

ye br +

bl t ye + hI t

pu +++ br ++ pu +++ br +

or ++ hr +++ ye +

11- Plant names (refer to Table 3 footnote 2) 11- Extracts S- skelshylysolve F C- chloroform A- 80 ethanol 111- Rf values IV- visihle and fluorescent patterns without any treatment V- visible and fluorshyescent patterns after exposure to ammonia vapour VI- Nitrobenzene diashyzonium fluorohorate positive spots

2Solvent systems for samples and standards a) Chloroformmethanol (955) for skellysolve F and chloroform extracts b) n-Butanolacetic acidwater (415) for 80 ethanol extracts Standards Ru- Rutin UmshyUmbelliferone Vi- Visnagin Pr- Provismine

3Color and intensity of spots under daylight and ultraviolet light hlshyblue br- hrown gr- green gy- gray or- orange pi- pink pu- purple re- red wh- white ye- yellow +H= high ++= medium += low t= trace

27

Table 5 Presence of Tannins in Minnesotan Aquatic Plants l

III IV 2 III IV l

I 11 I II a b c a b c a b c a b c

Ac-l A t wh t Nt A + br + ~ bl +++ AW st AW gr + BW BW pu +++

Ac-2 A Nv-l A gr br + bu ~ ++ AW Iv AW + gr + BW + gr + BW + gr +

Cd A t wh t Nv-l A t gr t ye + AW + ye + st AW BW + ye + BW t bl + + pu gr +

Cl A + br +++ br gr + Nv-2 A + ye br ++ gy bl +++ AW gr br + Iv AW + gr ++ BW + br ++ BW + gr ++

Cp A Nv-2 A N 00 AW st AW

BW gr br t BW + ye gr + Cv A Pa A + br + gr +

AW AW t br + + br ~ +++ BW BW + gr br ++

Es A + ye gr + ~~ + Pn A + gr + ~~ +++ AW gr t AW + gr + gr +++ BW gr + BW + gr br ++

Lm A + br gr ++ ye gr ++ Pp A t wh + +~ AW + gr ++ AW + gr + bl ~ ++ BW + gr br ++ BW + gr +++

Me A + gr + + ~~ +++ Pr A + gr ye + ~~ + AW gr br + AW gr + BW + gr br ++ BW + gr br +

Nt A + br +++ +++ pz A Iv AW gr ++ AW + ye +

BW + br pu +++ BW

(Table 5 continued)

III IV III IV I II I 11

a b c a b c a b c a b c

Sc A Ta-l A + br gr + ~ + AW + gr ye + AW BW + gr ++ BW br t

Se A + br + ~ + Ta-2 A + wh br + br ~ ++ AW gr t AW ye t BW + gr br + BW + br ++

Sf A + br + ~~ + Va A + ye gr + ~ + AW gr t AW + gr + BW + gr br ++ BW + gr +

Sl A Za A + gr br + ye t AW + gr ++ AW BW gr ++ BW + br +

N -0 11- Plant names (refer to Table 3 footnote 2 11- Extracts A- 80 Ethanol AW- Acidic water BWshy

Basic water 111- Gelatin-salt solution a- Ppt (+) no ppt (blank) trace ppt (t) b- color bl shyblue br- brown gr- green gy- gray pu- purple wh- white ve- yellow c- Intensity +++= high ++= medium += low t trace IV- Ferric chloride test

2 Underlined colors represent those precipitants dissolved by urea (positive tannin test)

of variegatum and Nymphaea tuberosa gave a positive ferric chloride hydrolysis products of the action of the acid or the base on

tannins gallic or ellagic acid account for the blue or green ferric chloride test

D Saponins

The results from hemolysis and froth tests for the detection of saponins are shown in Table 6

Only Nuphar variegatum (stem collected at Lake Minnetonka) Potamoshygeton pectinatus R richardsonii and angustifolia failed to hemolyze standarized red blood cells in 10 minutes However the hemolytic activity of the other plant species may be due to the presence of non-saponin hemolyshytic plant constituents such as amines rancid fats or plant acids (57) which affect the permeability andor membrane integrity of the red blood cells The following species demonstrated a hemolytic activity in less than 5 minutes and a characteristic honeycomb froth height of more than 5 mm in 5 minutes and therefore are saponin positive Nuphar varieshygaturn (collected at the Pine Lake) Potamogeton Sparganium fluctuans and Sagittaria ~~~~~

E Steroids

The results for the thin-layer chromatographic detection of steroids are shown in Table 7

Beta-sitosterol is tentatively identified as being present in Cerashytophyllum demersum Carex lacustris Nuphar variegaturn Potamogeton amplishyfolius R richardsonii f zosteriformis Sparganium fluctuans and Typha angustifolia This interpretation is based upon its Rf values of 050-056 in 11 cyclohexaneethyl acetate and its reaction with anisaldehyde reagent Beta-sitosterol has also been reported (133) as being present in the rhizome and flower of Nuphar luteum Sagittaria latifolia may contain a hydroxy steroid (Rf value 038 in 11 cyclohexaneethyl acetate) because of its color reaction with anisal dehyde reagent Cardenol ides 3- or 17-oxostershyoids are totally absent in the plant species studied although brown KeddeshyZimmermann reactions were observed

Anisaldehyde reagent is a general detecting reagent with high sensishytivity for steroids terpenes carbonyl compounds etc (137) Beta-sitoshysterol gives purple color with anisaldehyde reagent 16-hydroxy-steroids and many hydroxy-derivatives of progesterone give yellow or yellow-brown color and Il-ketosteroids give red or carmin color (143) Kedde reagent forms a blue-violet color with alpha beta-unsaturated 5-ring lactones (cardenolides) (144) Zimmermann reagent is specific for ketonic steroids with an ortho unsat urated metbylene group (144) The m-dini trobenzene reshyacts with the methylene group which is activated by the oxe-function and 3-oxo-steroids appear immediately as blue spots whereas l7-oxo-steroids with an unsaturated 16 pOSition give violet color after 3 to 6 minutes

30

Table 6 Detection of Saponins by Homolysis and Froth Tests l

IV2 IV I II III v I II III v

a b a b

Ac-l A AW

5 40 4 2 Nv-2

st A AW

4 6 4 66

BW BW 60 Ac-2 A

AW 5

29 3 1 Pa A

AW 1

6 4 66 BW BW

Cd A Pn A AW 6 1 AW 4 1 1 BW BW 60

CI A Pp A AW 1 AW 3 1 BW 8 BW

Cp A AW 6

Pr A AW 43 3 2

BW BW Cv A

AW 8

8 3 37 pz A

AW 5 1

BW BW Es A

AW 5 Sc A

AW 4

17 4 2

BW 10 BW Lm A Se A

AW 4 8 4 50 AW 5 BW BW 60

Me A AW

4 16 3

Sf A AW

2 8 4 50

BW BW 60 Nt Iv

A AW 6

Sl A AW

5 29 8 4 50

BW BW Nt A 5 Ta-1 A 5 st AW 4 1 AW

BW BW Nv-l Iv

A AW

12 12 6 50

Ta-2 A AW 15 3 2

BW 58 BW 50 Nv-l st

A AW

2 1

Va A AW

3 12 4 2

BW 12 BW 7 Nv-2 Iv

A AW

2 10 6 60

Za A AW 9

6 4 66

BW BW Digitonin 3 3 12 66

11- Plant names (refer to Table 3 footnote 2) 11- Extracts A- 80 ethanol AW- acidic water BW- basic water 111- Hemolytic activity The time in min required to completely hemolyze the RBCs IV- Froth height (mm) V- Froth persistency- ratio of froth height of the fresh hot water extract in 30 minutes as compared to that in 5 minutes

2Froth height at a- 5 minutes b- 30 minutes

31

(Table 4 continued)

I II III

Pn S 066 C 007

045 A 057

066 090

Pp S C 008 A 066

090 Pr S 076

084 c 045

067 A 057

094 pz S 038

C 021 A 057

090 Sc S 082

C 042 A 066

090 Se S 046

076 C A 067

Sf S C 044

048 058

A 090 S 049

084 C 003 A 066

089 Ta-l S 082

C 035 A 044

057 074

IV

Daylight uv

V

(Table 4 continued)

re or

or

re ye ye re

br

or or

ye gy

ye

ye ye

Daylight uv

++

++

+ + + +

+

++ ++

+ +

+

++ ++

pu t pu + pu ++

pu t

bl +

pu t

re t

pu + bl t

pu + bl t

ye + wh +

gr bl ++

ye + pu + bl t

ye t ye gr +

pu + bl t

pu ++

26

ye ye

ye ye

ye

ye

ye

ye

ye

ye ye ye

+ ++ +

t

t

t

++

++

+

+

t

t

++

pu bl bl

bl

pu bl pu

pi

pi bl

gr bl

br bl

br br br

+ ++ ++

+

+ +

+

+ t

++

+ +

t t

++

VI

or +

br t br + br ++

or ++

br +

br ++

br +

or + br + br +

br + br +

ye +

hr +

br +

br + br + br + or +

br + ye br ++

I

Ta-2

Va

Za

Std Ru Ru Urn Vi Pr

II

S C A S C A

S C A

a) b) a) a) a)

Daylight uv Daylight

021 ye + (Identical with those for Ta-l A) 011 br t

056 re t

088 ye + 081 or + 050 or + 045 ye wh ++ 057 bl t ye t

066 bl + ye +

002 gr ye ++ ye ++ 066 ye t pu ++ ye ++ 031 hI ++ 053 gr ++ 074 pu ++

IV V III VI

uv

or +

or +++

ye br +

bl t ye + hI t

pu +++ br ++ pu +++ br +

or ++ hr +++ ye +

11- Plant names (refer to Table 3 footnote 2) 11- Extracts S- skelshylysolve F C- chloroform A- 80 ethanol 111- Rf values IV- visihle and fluorescent patterns without any treatment V- visible and fluorshyescent patterns after exposure to ammonia vapour VI- Nitrobenzene diashyzonium fluorohorate positive spots

2Solvent systems for samples and standards a) Chloroformmethanol (955) for skellysolve F and chloroform extracts b) n-Butanolacetic acidwater (415) for 80 ethanol extracts Standards Ru- Rutin UmshyUmbelliferone Vi- Visnagin Pr- Provismine

3Color and intensity of spots under daylight and ultraviolet light hlshyblue br- hrown gr- green gy- gray or- orange pi- pink pu- purple re- red wh- white ye- yellow +H= high ++= medium += low t= trace

27

Table 5 Presence of Tannins in Minnesotan Aquatic Plants l

III IV 2 III IV l

I 11 I II a b c a b c a b c a b c

Ac-l A t wh t Nt A + br + ~ bl +++ AW st AW gr + BW BW pu +++

Ac-2 A Nv-l A gr br + bu ~ ++ AW Iv AW + gr + BW + gr + BW + gr +

Cd A t wh t Nv-l A t gr t ye + AW + ye + st AW BW + ye + BW t bl + + pu gr +

Cl A + br +++ br gr + Nv-2 A + ye br ++ gy bl +++ AW gr br + Iv AW + gr ++ BW + br ++ BW + gr ++

Cp A Nv-2 A N 00 AW st AW

BW gr br t BW + ye gr + Cv A Pa A + br + gr +

AW AW t br + + br ~ +++ BW BW + gr br ++

Es A + ye gr + ~~ + Pn A + gr + ~~ +++ AW gr t AW + gr + gr +++ BW gr + BW + gr br ++

Lm A + br gr ++ ye gr ++ Pp A t wh + +~ AW + gr ++ AW + gr + bl ~ ++ BW + gr br ++ BW + gr +++

Me A + gr + + ~~ +++ Pr A + gr ye + ~~ + AW gr br + AW gr + BW + gr br ++ BW + gr br +

Nt A + br +++ +++ pz A Iv AW gr ++ AW + ye +

BW + br pu +++ BW

(Table 5 continued)

III IV III IV I II I 11

a b c a b c a b c a b c

Sc A Ta-l A + br gr + ~ + AW + gr ye + AW BW + gr ++ BW br t

Se A + br + ~ + Ta-2 A + wh br + br ~ ++ AW gr t AW ye t BW + gr br + BW + br ++

Sf A + br + ~~ + Va A + ye gr + ~ + AW gr t AW + gr + BW + gr br ++ BW + gr +

Sl A Za A + gr br + ye t AW + gr ++ AW BW gr ++ BW + br +

N -0 11- Plant names (refer to Table 3 footnote 2 11- Extracts A- 80 Ethanol AW- Acidic water BWshy

Basic water 111- Gelatin-salt solution a- Ppt (+) no ppt (blank) trace ppt (t) b- color bl shyblue br- brown gr- green gy- gray pu- purple wh- white ve- yellow c- Intensity +++= high ++= medium += low t trace IV- Ferric chloride test

2 Underlined colors represent those precipitants dissolved by urea (positive tannin test)

of variegatum and Nymphaea tuberosa gave a positive ferric chloride hydrolysis products of the action of the acid or the base on

tannins gallic or ellagic acid account for the blue or green ferric chloride test

D Saponins

The results from hemolysis and froth tests for the detection of saponins are shown in Table 6

Only Nuphar variegatum (stem collected at Lake Minnetonka) Potamoshygeton pectinatus R richardsonii and angustifolia failed to hemolyze standarized red blood cells in 10 minutes However the hemolytic activity of the other plant species may be due to the presence of non-saponin hemolyshytic plant constituents such as amines rancid fats or plant acids (57) which affect the permeability andor membrane integrity of the red blood cells The following species demonstrated a hemolytic activity in less than 5 minutes and a characteristic honeycomb froth height of more than 5 mm in 5 minutes and therefore are saponin positive Nuphar varieshygaturn (collected at the Pine Lake) Potamogeton Sparganium fluctuans and Sagittaria ~~~~~

E Steroids

The results for the thin-layer chromatographic detection of steroids are shown in Table 7

Beta-sitosterol is tentatively identified as being present in Cerashytophyllum demersum Carex lacustris Nuphar variegaturn Potamogeton amplishyfolius R richardsonii f zosteriformis Sparganium fluctuans and Typha angustifolia This interpretation is based upon its Rf values of 050-056 in 11 cyclohexaneethyl acetate and its reaction with anisaldehyde reagent Beta-sitosterol has also been reported (133) as being present in the rhizome and flower of Nuphar luteum Sagittaria latifolia may contain a hydroxy steroid (Rf value 038 in 11 cyclohexaneethyl acetate) because of its color reaction with anisal dehyde reagent Cardenol ides 3- or 17-oxostershyoids are totally absent in the plant species studied although brown KeddeshyZimmermann reactions were observed

Anisaldehyde reagent is a general detecting reagent with high sensishytivity for steroids terpenes carbonyl compounds etc (137) Beta-sitoshysterol gives purple color with anisaldehyde reagent 16-hydroxy-steroids and many hydroxy-derivatives of progesterone give yellow or yellow-brown color and Il-ketosteroids give red or carmin color (143) Kedde reagent forms a blue-violet color with alpha beta-unsaturated 5-ring lactones (cardenolides) (144) Zimmermann reagent is specific for ketonic steroids with an ortho unsat urated metbylene group (144) The m-dini trobenzene reshyacts with the methylene group which is activated by the oxe-function and 3-oxo-steroids appear immediately as blue spots whereas l7-oxo-steroids with an unsaturated 16 pOSition give violet color after 3 to 6 minutes

30

Table 6 Detection of Saponins by Homolysis and Froth Tests l

IV2 IV I II III v I II III v

a b a b

Ac-l A AW

5 40 4 2 Nv-2

st A AW

4 6 4 66

BW BW 60 Ac-2 A

AW 5

29 3 1 Pa A

AW 1

6 4 66 BW BW

Cd A Pn A AW 6 1 AW 4 1 1 BW BW 60

CI A Pp A AW 1 AW 3 1 BW 8 BW

Cp A AW 6

Pr A AW 43 3 2

BW BW Cv A

AW 8

8 3 37 pz A

AW 5 1

BW BW Es A

AW 5 Sc A

AW 4

17 4 2

BW 10 BW Lm A Se A

AW 4 8 4 50 AW 5 BW BW 60

Me A AW

4 16 3

Sf A AW

2 8 4 50

BW BW 60 Nt Iv

A AW 6

Sl A AW

5 29 8 4 50

BW BW Nt A 5 Ta-1 A 5 st AW 4 1 AW

BW BW Nv-l Iv

A AW

12 12 6 50

Ta-2 A AW 15 3 2

BW 58 BW 50 Nv-l st

A AW

2 1

Va A AW

3 12 4 2

BW 12 BW 7 Nv-2 Iv

A AW

2 10 6 60

Za A AW 9

6 4 66

BW BW Digitonin 3 3 12 66

11- Plant names (refer to Table 3 footnote 2) 11- Extracts A- 80 ethanol AW- acidic water BW- basic water 111- Hemolytic activity The time in min required to completely hemolyze the RBCs IV- Froth height (mm) V- Froth persistency- ratio of froth height of the fresh hot water extract in 30 minutes as compared to that in 5 minutes

2Froth height at a- 5 minutes b- 30 minutes

31

Table 5 Presence of Tannins in Minnesotan Aquatic Plants l

III IV 2 III IV l

I 11 I II a b c a b c a b c a b c

Ac-l A t wh t Nt A + br + ~ bl +++ AW st AW gr + BW BW pu +++

Ac-2 A Nv-l A gr br + bu ~ ++ AW Iv AW + gr + BW + gr + BW + gr +

Cd A t wh t Nv-l A t gr t ye + AW + ye + st AW BW + ye + BW t bl + + pu gr +

Cl A + br +++ br gr + Nv-2 A + ye br ++ gy bl +++ AW gr br + Iv AW + gr ++ BW + br ++ BW + gr ++

Cp A Nv-2 A N 00 AW st AW

BW gr br t BW + ye gr + Cv A Pa A + br + gr +

AW AW t br + + br ~ +++ BW BW + gr br ++

Es A + ye gr + ~~ + Pn A + gr + ~~ +++ AW gr t AW + gr + gr +++ BW gr + BW + gr br ++

Lm A + br gr ++ ye gr ++ Pp A t wh + +~ AW + gr ++ AW + gr + bl ~ ++ BW + gr br ++ BW + gr +++

Me A + gr + + ~~ +++ Pr A + gr ye + ~~ + AW gr br + AW gr + BW + gr br ++ BW + gr br +

Nt A + br +++ +++ pz A Iv AW gr ++ AW + ye +

BW + br pu +++ BW

(Table 5 continued)

III IV III IV I II I 11

a b c a b c a b c a b c

Sc A Ta-l A + br gr + ~ + AW + gr ye + AW BW + gr ++ BW br t

Se A + br + ~ + Ta-2 A + wh br + br ~ ++ AW gr t AW ye t BW + gr br + BW + br ++

Sf A + br + ~~ + Va A + ye gr + ~ + AW gr t AW + gr + BW + gr br ++ BW + gr +

Sl A Za A + gr br + ye t AW + gr ++ AW BW gr ++ BW + br +

N -0 11- Plant names (refer to Table 3 footnote 2 11- Extracts A- 80 Ethanol AW- Acidic water BWshy

Basic water 111- Gelatin-salt solution a- Ppt (+) no ppt (blank) trace ppt (t) b- color bl shyblue br- brown gr- green gy- gray pu- purple wh- white ve- yellow c- Intensity +++= high ++= medium += low t trace IV- Ferric chloride test

2 Underlined colors represent those precipitants dissolved by urea (positive tannin test)

of variegatum and Nymphaea tuberosa gave a positive ferric chloride hydrolysis products of the action of the acid or the base on

tannins gallic or ellagic acid account for the blue or green ferric chloride test

D Saponins

The results from hemolysis and froth tests for the detection of saponins are shown in Table 6

Only Nuphar variegatum (stem collected at Lake Minnetonka) Potamoshygeton pectinatus R richardsonii and angustifolia failed to hemolyze standarized red blood cells in 10 minutes However the hemolytic activity of the other plant species may be due to the presence of non-saponin hemolyshytic plant constituents such as amines rancid fats or plant acids (57) which affect the permeability andor membrane integrity of the red blood cells The following species demonstrated a hemolytic activity in less than 5 minutes and a characteristic honeycomb froth height of more than 5 mm in 5 minutes and therefore are saponin positive Nuphar varieshygaturn (collected at the Pine Lake) Potamogeton Sparganium fluctuans and Sagittaria ~~~~~

E Steroids

The results for the thin-layer chromatographic detection of steroids are shown in Table 7

Beta-sitosterol is tentatively identified as being present in Cerashytophyllum demersum Carex lacustris Nuphar variegaturn Potamogeton amplishyfolius R richardsonii f zosteriformis Sparganium fluctuans and Typha angustifolia This interpretation is based upon its Rf values of 050-056 in 11 cyclohexaneethyl acetate and its reaction with anisaldehyde reagent Beta-sitosterol has also been reported (133) as being present in the rhizome and flower of Nuphar luteum Sagittaria latifolia may contain a hydroxy steroid (Rf value 038 in 11 cyclohexaneethyl acetate) because of its color reaction with anisal dehyde reagent Cardenol ides 3- or 17-oxostershyoids are totally absent in the plant species studied although brown KeddeshyZimmermann reactions were observed

Anisaldehyde reagent is a general detecting reagent with high sensishytivity for steroids terpenes carbonyl compounds etc (137) Beta-sitoshysterol gives purple color with anisaldehyde reagent 16-hydroxy-steroids and many hydroxy-derivatives of progesterone give yellow or yellow-brown color and Il-ketosteroids give red or carmin color (143) Kedde reagent forms a blue-violet color with alpha beta-unsaturated 5-ring lactones (cardenolides) (144) Zimmermann reagent is specific for ketonic steroids with an ortho unsat urated metbylene group (144) The m-dini trobenzene reshyacts with the methylene group which is activated by the oxe-function and 3-oxo-steroids appear immediately as blue spots whereas l7-oxo-steroids with an unsaturated 16 pOSition give violet color after 3 to 6 minutes

30

Table 6 Detection of Saponins by Homolysis and Froth Tests l

IV2 IV I II III v I II III v

a b a b

Ac-l A AW

5 40 4 2 Nv-2

st A AW

4 6 4 66

BW BW 60 Ac-2 A

AW 5

29 3 1 Pa A

AW 1

6 4 66 BW BW

Cd A Pn A AW 6 1 AW 4 1 1 BW BW 60

CI A Pp A AW 1 AW 3 1 BW 8 BW

Cp A AW 6

Pr A AW 43 3 2

BW BW Cv A

AW 8

8 3 37 pz A

AW 5 1

BW BW Es A

AW 5 Sc A

AW 4

17 4 2

BW 10 BW Lm A Se A

AW 4 8 4 50 AW 5 BW BW 60

Me A AW

4 16 3

Sf A AW

2 8 4 50

BW BW 60 Nt Iv

A AW 6

Sl A AW

5 29 8 4 50

BW BW Nt A 5 Ta-1 A 5 st AW 4 1 AW

BW BW Nv-l Iv

A AW

12 12 6 50

Ta-2 A AW 15 3 2

BW 58 BW 50 Nv-l st

A AW

2 1

Va A AW

3 12 4 2

BW 12 BW 7 Nv-2 Iv

A AW

2 10 6 60

Za A AW 9

6 4 66

BW BW Digitonin 3 3 12 66

11- Plant names (refer to Table 3 footnote 2) 11- Extracts A- 80 ethanol AW- acidic water BW- basic water 111- Hemolytic activity The time in min required to completely hemolyze the RBCs IV- Froth height (mm) V- Froth persistency- ratio of froth height of the fresh hot water extract in 30 minutes as compared to that in 5 minutes

2Froth height at a- 5 minutes b- 30 minutes

31

of variegatum and Nymphaea tuberosa gave a positive ferric chloride hydrolysis products of the action of the acid or the base on

tannins gallic or ellagic acid account for the blue or green ferric chloride test

D Saponins

The results from hemolysis and froth tests for the detection of saponins are shown in Table 6

Only Nuphar variegatum (stem collected at Lake Minnetonka) Potamoshygeton pectinatus R richardsonii and angustifolia failed to hemolyze standarized red blood cells in 10 minutes However the hemolytic activity of the other plant species may be due to the presence of non-saponin hemolyshytic plant constituents such as amines rancid fats or plant acids (57) which affect the permeability andor membrane integrity of the red blood cells The following species demonstrated a hemolytic activity in less than 5 minutes and a characteristic honeycomb froth height of more than 5 mm in 5 minutes and therefore are saponin positive Nuphar varieshygaturn (collected at the Pine Lake) Potamogeton Sparganium fluctuans and Sagittaria ~~~~~

E Steroids

The results for the thin-layer chromatographic detection of steroids are shown in Table 7

Beta-sitosterol is tentatively identified as being present in Cerashytophyllum demersum Carex lacustris Nuphar variegaturn Potamogeton amplishyfolius R richardsonii f zosteriformis Sparganium fluctuans and Typha angustifolia This interpretation is based upon its Rf values of 050-056 in 11 cyclohexaneethyl acetate and its reaction with anisaldehyde reagent Beta-sitosterol has also been reported (133) as being present in the rhizome and flower of Nuphar luteum Sagittaria latifolia may contain a hydroxy steroid (Rf value 038 in 11 cyclohexaneethyl acetate) because of its color reaction with anisal dehyde reagent Cardenol ides 3- or 17-oxostershyoids are totally absent in the plant species studied although brown KeddeshyZimmermann reactions were observed

Anisaldehyde reagent is a general detecting reagent with high sensishytivity for steroids terpenes carbonyl compounds etc (137) Beta-sitoshysterol gives purple color with anisaldehyde reagent 16-hydroxy-steroids and many hydroxy-derivatives of progesterone give yellow or yellow-brown color and Il-ketosteroids give red or carmin color (143) Kedde reagent forms a blue-violet color with alpha beta-unsaturated 5-ring lactones (cardenolides) (144) Zimmermann reagent is specific for ketonic steroids with an ortho unsat urated metbylene group (144) The m-dini trobenzene reshyacts with the methylene group which is activated by the oxe-function and 3-oxo-steroids appear immediately as blue spots whereas l7-oxo-steroids with an unsaturated 16 pOSition give violet color after 3 to 6 minutes

30

Table 6 Detection of Saponins by Homolysis and Froth Tests l

IV2 IV I II III v I II III v

a b a b

Ac-l A AW

5 40 4 2 Nv-2

st A AW

4 6 4 66

BW BW 60 Ac-2 A

AW 5

29 3 1 Pa A

AW 1

6 4 66 BW BW

Cd A Pn A AW 6 1 AW 4 1 1 BW BW 60

CI A Pp A AW 1 AW 3 1 BW 8 BW

Cp A AW 6

Pr A AW 43 3 2

BW BW Cv A

AW 8

8 3 37 pz A

AW 5 1

BW BW Es A

AW 5 Sc A

AW 4

17 4 2

BW 10 BW Lm A Se A

AW 4 8 4 50 AW 5 BW BW 60

Me A AW

4 16 3

Sf A AW

2 8 4 50

BW BW 60 Nt Iv

A AW 6

Sl A AW

5 29 8 4 50

BW BW Nt A 5 Ta-1 A 5 st AW 4 1 AW

BW BW Nv-l Iv

A AW

12 12 6 50

Ta-2 A AW 15 3 2

BW 58 BW 50 Nv-l st

A AW

2 1

Va A AW

3 12 4 2

BW 12 BW 7 Nv-2 Iv

A AW

2 10 6 60

Za A AW 9

6 4 66

BW BW Digitonin 3 3 12 66

11- Plant names (refer to Table 3 footnote 2) 11- Extracts A- 80 ethanol AW- acidic water BW- basic water 111- Hemolytic activity The time in min required to completely hemolyze the RBCs IV- Froth height (mm) V- Froth persistency- ratio of froth height of the fresh hot water extract in 30 minutes as compared to that in 5 minutes

2Froth height at a- 5 minutes b- 30 minutes

31

I c

Table 7 Thin-layer Fluorescent and Steroid Patterns l

Ac-1

Ac-2

Cd

C1

Cp

Cv

Es

Lm

Me

Nt Iv

III2 IV23 II

a b c a b

S C 049 pu A 001 wh ++ 001 ye

007 pu gr S C 047 bl A 001 ye + 007 gr

021 pu s 061 re pu

079 br C 022 b1 gr

051 pu 078 re ye

A 002 ye S 050 pu C 010 ye

026 gy 052 pu 082 re pu

A 001 ye ++ 004 re br 008 bl +

s 071 re t 001 ye 028 pu ye 081 ye

C 077 re pu A 002 br S 062 pu

086 pu C 071 gr ye

084 br A 001 wh ++ 001 gr ye S 040 bl + 047 pu C 045 bl + 031 gy

053 re br 082 pu

A 001 br 005 or 010 gr or

S 077 re C A 001 ye

007 re S C A 012 ye + 012 gr ye S C 026 ye pu A 020 re +

32

c

+ + +

+ + t

+ + + + + + + + + + + +

+ + + +

+++ + t

+ t

++ + + + + ++ ++ ++ +

++ t

++ ++

(Table 7 continued)

III

a b c

032 re +

001 ye ++

001 ye ++ 046 re t

024 ye t

002 gr ye +

001 wh ++

I

Nt st

Nv-l Iv

Nv-1 st

Nv-2 Iv

Nv-2 st

Pa

Pn

Pp

Pr

pz

II

s C A S C A S C A S

C

A

S

C A

s

C

A S C A S C A S

C

A S

C A

33

IV

a

009

074

075

009 050 067 027 074 001 017 010 051 077 019 001 020 056 080 019 027 052 081 001 076

002

007 020 052 082 039 059 071 084 001 001 050 072

002

b

ye pu

re br

re

ye pu pu

pu ye gy

re pu gr ye

pu pu ye

pu ye br

pu ye pu pu br gy gy pu

re pu ye re

ye

re bl pu pu re pu

re pu gr ye pu ye ye pu

re pu

ye

+

+

t

t

+ + + + ++ t t

+ + + ++ t

+ + + + + + ++ +

t t

+ t + + + + ++ ++ + t

++

(Table 7 continued)

I II a

Sc 5 C A

045

001

Se

Sf

5 C A 5

C

012 011

005 053 015 052

A 5

015

C A 021

Ta-l 5

C A

044 069 073 007

Ta-2

Va

Za

An Dg Dx Pr 5i

5 C A 5 C A 5 C A

020 007

001

001

F Lipids

The results for the gravimetric determination of total lipids obtained for each botanical family studied are summarized in Table 8 those for the systemic analysis of lipid distribution are shown in Table 9 and those for the fatty acid constituents of triglycerides are shown in Table 10

Total lipids extractable by skellysolve F and chloroform range from 068 (Chara vulgaris) to 667 natans) of dry plant material There is a wide variation of contents among Najadaceae (150shy489) Hydrocharitaceae (143-4 and Alismataceae (478-658) No reliable difference in total contents was found in Cyperaceae (118shy1 73) Sparganiaceae (074-1 and Typhaceae (108-1 73) because of the small number of plants within the genus studied

and Nymphaea with respectively may conshy

sidered for nutritional value but the presence of alkaloid in N might be a drawback to its usefulness shy

Iodine vapour is a sensitive (less than 1 gamma) detector for all unshysaturated lipids and some saturated nitrogenous lipids (71) Identificashytion of lipid classes in the plant extracts is tentatively obtained by comparing with the S8 standard (145) Free fatty acid (Rf value 000 in 955 skellysolve Fdiethyl ether) is not present in the extracts studied free sterol (Rf values 003-006 in 955 skellysolve Fdiethyl ether) is

common and only absent from fatty acid esters values 043-044 in 955 ether) are found in

Chara vulgaris Sagittaria ~~~~~ Eleocharis smallii Zizania ~77~~~_~~~~0~~~~= osa hydrocarbons (Rf value sent only in Nuphar ~~~~~ are identical in the Hydrocharitaceae very similar in Cyperaceae and Numphaeaceae but quite different in Alismataceae and Sparganiaceae Only a few plant extracts showed the presence of triglycershyides (Rf values 010-012 in skellysolve Fdiethyl etheracetic acid 70301) by TLC This is due to the lower concentration of triglyceride as compared to other lipid classes in aquatic plants

As shown in Table 11 the major fatty acids of triglycerides are 160 (carbon nurnbernumber of unsaturation) 181 161 and 182 for Anacharis

203 241 182 and 160 for Ceratophyllum 183 and 160 for 160 240 18 1 for

(leaves 0 183 and 240 for ~~~~ High content of not very common 240 fatty

and 203 241 fatty acids in are compared to the fatty acid contents soyshy

bean oils (cf Table 11) the aquatic plants studied also indicated the lower concentrations of stearic acid With the exception of lacusshy

palmitic acid in aquatic plants studied was present in whereas unsaturated fatty acids (oleic linoleic and linolenic) in lower pershy

centage than those found in linseed or soybean Composition of fatty acids from other aquatic plants reported (Table 1) also showed a lower content of stearic acid but with higher percentage of palmitic acid as compared to those found in linseed and soybean oils Nevertheless the unsaturated C-18 fatty acid contents are comparable to those for linseed and soybean

35

III

b

re

ye

ye gr bl

bl bl

gr bl bl

gr

re

bl wh ye ye

bl ye gr

wh

ye

IV

c

t

++

+ ++

+ + ++ ++

+

+

+ +++

++ +

+ +

++

++

a

086 076 001 059

001 019 056 051 063 075 001 008 050

050 078

001 007 050 053 005 020

001

074 001 029 014 002 040 055

b

br ye re pu

ye pu

ye pu pu pu

gr ye re pu br

gr re gr re

br ye pu br

br re br pu pu

re br br

ye

re pu br br gr bl ye pu

c

+ + ++ t

+lshy

t t

++ + ++ ++ t

+

++ t + +

++ + + + + +

++

++ ++ + ++

+++ + ++

11- Plant names (refer to Table 3 footnote 2) and standards Anshyandrostan-diol Dg- digitoxigenin Dx- digitoxin Pr- progesterone 5i- beta-sitosterol 11- Extracts S- 5kellysolve C- chloroform Ashy80 ethanol 111- Fluorescence pattern (254 m~) IV- Anisaldehyde positive spots

2a- Rf values b- Color and c- Intensity (refer to Table 4 footnote 4) 3Underlined Rf values represent a positive Kedde-Zimmermann test

34

(Table 7 continued)

I II a

Sc 5 C A

045

001

Se

Sf

5 C A 5

C

012 011

005 053 015 052

A 5

015

C A 021

Ta-l 5

C A

044 069 073 007

Ta-2

Va

Za

An Dg Dx Pr 5i

5 C A 5 C A 5 C A

020 007

001

001

F Lipids

The results for the gravimetric determination of total lipids obtained for each botanical family studied are summarized in Table 8 those for the systemic analysis of lipid distribution are shown in Table 9 and those for the fatty acid constituents of triglycerides are shown in Table 10

Total lipids extractable by skellysolve F and chloroform range from 068 (Chara vulgaris) to 667 natans) of dry plant material There is a wide variation of contents among Najadaceae (150shy489) Hydrocharitaceae (143-4 and Alismataceae (478-658) No reliable difference in total contents was found in Cyperaceae (118shy1 73) Sparganiaceae (074-1 and Typhaceae (108-1 73) because of the small number of plants within the genus studied

and Nymphaea with respectively may conshy

sidered for nutritional value but the presence of alkaloid in N might be a drawback to its usefulness shy

Iodine vapour is a sensitive (less than 1 gamma) detector for all unshysaturated lipids and some saturated nitrogenous lipids (71) Identificashytion of lipid classes in the plant extracts is tentatively obtained by comparing with the S8 standard (145) Free fatty acid (Rf value 000 in 955 skellysolve Fdiethyl ether) is not present in the extracts studied free sterol (Rf values 003-006 in 955 skellysolve Fdiethyl ether) is

common and only absent from fatty acid esters values 043-044 in 955 ether) are found in

Chara vulgaris Sagittaria ~~~~~ Eleocharis smallii Zizania ~77~~~_~~~~0~~~~= osa hydrocarbons (Rf value sent only in Nuphar ~~~~~ are identical in the Hydrocharitaceae very similar in Cyperaceae and Numphaeaceae but quite different in Alismataceae and Sparganiaceae Only a few plant extracts showed the presence of triglycershyides (Rf values 010-012 in skellysolve Fdiethyl etheracetic acid 70301) by TLC This is due to the lower concentration of triglyceride as compared to other lipid classes in aquatic plants

As shown in Table 11 the major fatty acids of triglycerides are 160 (carbon nurnbernumber of unsaturation) 181 161 and 182 for Anacharis

203 241 182 and 160 for Ceratophyllum 183 and 160 for 160 240 18 1 for

(leaves 0 183 and 240 for ~~~~ High content of not very common 240 fatty

and 203 241 fatty acids in are compared to the fatty acid contents soyshy

bean oils (cf Table 11) the aquatic plants studied also indicated the lower concentrations of stearic acid With the exception of lacusshy

palmitic acid in aquatic plants studied was present in whereas unsaturated fatty acids (oleic linoleic and linolenic) in lower pershy

centage than those found in linseed or soybean Composition of fatty acids from other aquatic plants reported (Table 1) also showed a lower content of stearic acid but with higher percentage of palmitic acid as compared to those found in linseed and soybean oils Nevertheless the unsaturated C-18 fatty acid contents are comparable to those for linseed and soybean

35

III

b

re

ye

ye gr bl

bl bl

gr bl bl

gr

re

bl wh ye ye

bl ye gr

wh

ye

IV

c

t

++

+ ++

+ + ++ ++

+

+

+ +++

++ +

+ +

++

++

a

086 076 001 059

001 019 056 051 063 075 001 008 050

050 078

001 007 050 053 005 020

001

074 001 029 014 002 040 055

b

br ye re pu

ye pu

ye pu pu pu

gr ye re pu br

gr re gr re

br ye pu br

br re br pu pu

re br br

ye

re pu br br gr bl ye pu

c

+ + ++ t

+lshy

t t

++ + ++ ++ t

+

++ t + +

++ + + + + +

++

++ ++ + ++

+++ + ++

11- Plant names (refer to Table 3 footnote 2) and standards Anshyandrostan-diol Dg- digitoxigenin Dx- digitoxin Pr- progesterone 5i- beta-sitosterol 11- Extracts S- 5kellysolve C- chloroform Ashy80 ethanol 111- Fluorescence pattern (254 m~) IV- Anisaldehyde positive spots

2a- Rf values b- Color and c- Intensity (refer to Table 4 footnote 4) 3Underlined Rf values represent a positive Kedde-Zimmermann test

34

Table 8 Gravimetric Determination of Total Lipids

Family

Characeae

Alismataceae

Araceae

Cyperaceae

Gramineae

Hydrocharitaceae

Lemnaceae

Najadaceae

Sparganiaceae

Plantl 1

Cv

Sc

Sl

Cp

C1

Es

7a

Ac-l

Ac-2

Va

Lm

Pa

Pn

Jp

Pr

pz

Se

Sf

Ext 2

S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C

Wt (ww) 3

011 043 248 1 37 219 305 1 73 209 035 058 046 091 037 050 025 089 060 293 230 029 106 192 021 086 331 204 018 043 037 118 016 098 106 034 039 103

(Table 8 continued)

Wt TotalTotal Family Plant l Ext 21iDids4 (ww)3

054 Typhaceae Ta-l S 046 087C 041

385 Ta-2 S 028 107C 079 524 Ceratophy1laceae Cd S 021 100C 079 382 Haloragaceae Me S 032 099

C 067 093 Nymphaeaceae Nv-l S 1 89 355Iv C 166 1 37 Nv-1 S 094

1 83st C 089 087 Nv-2 S 117

1 96Iv C 079 114 Nv-2 S 064 119st C 055 353 Nt S 225 391Iv C 166 2 59 Nt S 083

277Bt C 1 94

298

107 lPlant name- (refer to Table 3 footnote 3) 2Extract S- skellysolve F C- chloroform

535 3Weight of lipid extractable by skellysolve F or chloroform 4Total weight of lipid extractable by skellysolve F and shloroform

061

1 55

114

1 40

1 42

3736

Table 9 Systemic Analysis of Lipid Distribution

Family

Characeae

Alismataceae

Araceae

Cyperaceae

Gramineae

Hydrocharitaceae

Najadaceae

Sparganiaceae

Extract2

Plant ll Skelly F Chloroform

Cv

Sc

Sl

Cp

Cl

Es

Za

Ac-l

Ac-2

Va Pa

Pn Pp

Rf

006 043 003 011 018 030 040 054 063 003 006 008 021 033 043 006 043

006 043

005 038 044 005

005

010 005 008

Col

br br br gr br br br br br br br br 1gtr br br br br

br br

br br br br

br

br br br

Int

+ t +lshy

++shy++shy++shy

+ + + + +Ishy

+ t

t +Ishy

+ t

+ +Ishy

+ + + +

+

+Ishy

+Ishy

+

Rf

003 011 006 033

006

003 011 030

003 011 032 053 003 01] 032 037 011

003 011 037 003 011 037 003 003 011 034

Col

br gr ye br

ye br

br gr br

br gr br br br gr br br gr

br gr br br gr br br br gr br

Int

+ +Ishy

+ t

+

+ +lshy

t

+ + + t + + + t

t

+ + + + + + + + + t

Pr (Identical with those of Pa result) pz

Se 005 br + Sf 005 br + 003 br +

011 gr + 014 br + 021 br t 033 br + 063 br t

38

(Table 9 continued)

Extract

Family Plant Skelly F Chloroform

Rf Col lnt Rf Col lnt

Typhaceae Ta-l 005 br + 011 gr + 021 br +

Ta-2 005 br + 011 gr + 021 br +

Ceratophyllaceae Cd 005 br + 011 gr ye + 020 br + 026 br t 034 br +

lIaloragaceae Me 005 br + 003 br + 011 br gr + 036 br +

Nymphaeaceae Nv-l 005 br +Ishy 003 br + Iv 011 br ye + 011 br +

023 br t 038 br + Nv-2 Iv 037 br +Ishy 056 br +

044 br + Nt Iv 060 br +Ishy

Nv-l st 005 br + 003 br + 011 br ye + 011 gr + 044 br t 038 br + 060 br t

Nv-2 st Nt st (Identical those for Nv-l st)

S8 standard 000 br + 021 br + 005 br + 038 br + 012 br + 056 br +Ishy019 br + 023 br + 038 br t 044 br + 060 br +

1P1ant names- (refer to Table 3 footnote 2) 2Rf- Rf values Col- Color br- brown gr- green ye- yellow Int

Intensity +++= high ++= medium += low t~ trace

39

Table 10 Constituent Fatty Acids of Triglycerides Derived From Selected Aquatic Plants

Plant Chain length amp numshyber of double bonds

As As hydrogenated methyl esters

140 150 160 161 180 181 182

487 700

2970 1291

375 1750 1084

314 290

2588

4219

Carex 1acustris

150 160 161 180 181 182 183 203 240 241 140 160 170 180 181 182 183 220 240

()

(7)

Trace 781 318 1 46 673

1236 463

2905 724

2346 113 794 522 690

1045 2357 2680 108 101

Trace 1874

3918

1218 2363

113 1677 479

5974

476 NymEhaea 140 509 321 tuberosa (Iv) 150 Trace Trace

160 2824 1444 161 402 17 0 Trace Trace 180 450 21 97 181 946 182 954 183 857

NymEhaea tuberosa (st) 150 Trace Trace

160 2232 5381 161 210 170 100 211 180 289 3617 181 421 182 4396 183 1648 240 112

40

Table 11

Linseed

Soybean

Ac

Cd

Cl

Nt Iv

Nt st

Comparison of Major Fatty Acids Plants (Linseed and Soybean)

Chain length amp number of unsat 2

160 180 181 182 183

72 34 185 170 552

135 70 259 480 66

297 32 175 108 75

78 15 67 124 46

79 69 105 236 268

292 45 95 95 86

223 29 42 440 165

in Aquatic and Terrestrial

Total of unsat 3 Ref

907

805

358

237

609

276

647

(146)

(147)

1P1ant names Ac- Anacharis Cl- Carex lacustris Nt Iv- ~~~~a ~~~~ (ste~

2Percent contents of fatty acids were listed under each fatty acid column

3Total of unsaturated fatty acid (181 182 and 183)

41

IV REFERENCES

1 Farnsworth NR et a1 1966 Biological and phytochemical evaluashytion of plants I Biological test procedures and results from two hundred accessions L10ydia 101-122

2 Goto M and H Sato 1969 Determination of antitumor activity in rat ascites hepatomas by agar diffusion technique Yakugaku Zasshi 89 821-827

3 Hartwell JL 1960 Plant remedies for cancer Cancer Chemother Rep 19-24

4 Bianchi B and JR Cole 1969 Antitumor agents from Agave schottH (AmarylUdaceae) J Pharm Sci 58 589-591

5 Cole JR E Bianchi and ER Trumbull 1969 Antitumor agents from Bursera microphyl1a (Burseraceae) II Isolation of a new lignanshyburseran J Pharm Sci~ 175-176

6 Pates AL and GC Madsen 1955 Occurrence of antimicrobial substances in chlorophyl10se plants growing in Florida II Bot Gaz

250-261

7 Hughes JE 1952 Survey of antibiotics in the wild green plants of southern California Antibiot Chemother 2 487-491

8 Azarowicz EN JE Hughes and CL Perkins 1952 Anti-biotics in plants of southern California active against Mycobacterium tubershy

607 and niger Antibiot Chemother 2 532-536

9 Bushnell OA M Fukuda and T Makinodan 1950 The antibacterial properties of some plants found in Hawaii Pac Sci 4 167-183

10 Hayes LE 1947 Survey of higher plants for presence of anti shybacterial substances Bot Gaz 108 408-414

11 Carlson HJ HD Bissell and MG Mueller 1946 Antimalarial and antibacterial substances separated from higher plants J Bacteriol 52 155-168

12 Carlson HJ HG Douglas and J Robertson 1948 Screening methshyods for determining antibiotic activity of higher plants J Bactershyiol 55 235-240

13 Carlson HJ HG Douglas and J Robertson 1948 Antibacterial substances separated from plants J Bacteriol 55 241-248

14 Sanders DW P Weatherwax and LS McClung 1945 Antibacteria] substances from plants collected in Indiana J Bacteriol 49 611shy615

15 Nickell LC 1959 Antimicrobial activity of vascular plants Econ Bot Q 281-318

16 Skinner FA 1955 Antibiotics In K Peach and MW Tracey (ed) Modern Methods of Plant Analysis voT 3 Springer-Verlag Berlin pp 626-744

17 Burlage HM ME Jones GF McKenna and A Taylor 1952 Studies on toxic plants for antibacterial effects Tex Rep BioI Med 10 803-815

42

18 Maleszadeh F 1968 Antimicrobial activity of Lawsonia ~~==~ L Appl Microbiol 16 663-664

19 McCleary JA and DL Walkington 1964 Antimicrobial activity of the Cactaceae Bull Torrey Bot Garden 91 177-181

20 Wolters B 1968 Saponins as plant fungistatic compounds On the antibiotic action of saponins III P1anta 79 77-83

21 Ma TS and R Roper 1968 Microchemical investigation of medicinal plants I The antituberculosis principle in Prunus mume and chinensis Mikrochim Acta 2 167-181--------- shy

22 Nagy JG and R Tengerdy 1968 Antibacterial actions of essenshytial oils of Artemisia as an ecological factor II Antibacterial actions of Artemisia tridenta bacteria from the rumen of mule deer Appl Microbiol 1amp 441-444

23 Farnsworth NR 1966 Biological and phytochemical screening of plants J Pharm Sci 225-276

24 Jiu J 1966 A survey of some medicinal plants of Mexico for selected biological activities Lloydia 29 250-259

25 Noemi G M Nadal and LV Rodriguez 1963 Sarganin and chonalgin new antibiotic substances from Puerto Rico Antimicrob Ag Chemother 3 68-72

26 Noemi G and M Nadal 1964 Isolation and characterization of sarganin complex a new broad spectrum antibiotic isolated from marine algae Antimicrob Ag Chemother 131-] 34

27 Gorham PR 1962 The toxin produced by waterblooms of the blueshygreen algae Amer J Pub Health 52 2100-2105

28 Holm LG LW Weldon and RD Blackburn 1969 Aquatic yeneeds Science 699-709

29 Manske RHF and HL Holmes 1951-1965 The alkaloids Eight vols Academic Press NY

30 Henry TA ]939 The plant alkaloids Blakiston Phila

31 Bentley KW 1957 In the Alkaloids Interscience Publishers NY

32 Willaman JJ and BG Schubert 1955 Alkaloid hunting Econ Bot 9 141-150

33 Willaman JJ and BG Schubert 1955 Alkaloid hunting suppleshymental table of Genera US Department of Agriculture ARS ARSshy73-1

34 Wil1aman JJ and BG Schubert 1961 Alkaloid-bearing plants and their contained alkaloids Technical bulletin No 1234 US Department of Agriculture Washington DC

35 Webb LJ 1952 An australian phytochemical survey II Alkaloids in Queensland flowering plants Aust Common Sci Ind Res Organ Bul1 Melbourne No 268 5-99

36 Massingill JL Jr and JE Hodgkins 1967 Alkaloids of bacteria Phytochemistry i 977-982

43

Table 9 Systemic Analysis of Lipid Distribution

Family

Characeae

Alismataceae

Araceae

Cyperaceae

Gramineae

Hydrocharitaceae

Najadaceae

Sparganiaceae

Extract2

Plant ll Skelly F Chloroform

Cv

Sc

Sl

Cp

Cl

Es

Za

Ac-l

Ac-2

Va Pa

Pn Pp

Rf

006 043 003 011 018 030 040 054 063 003 006 008 021 033 043 006 043

006 043

005 038 044 005

005

010 005 008

Col

br br br gr br br br br br br br br 1gtr br br br br

br br

br br br br

br

br br br

Int

+ t +lshy

++shy++shy++shy

+ + + + +Ishy

+ t

t +Ishy

+ t

+ +Ishy

+ + + +

+

+Ishy

+Ishy

+

Rf

003 011 006 033

006

003 011 030

003 011 032 053 003 01] 032 037 011

003 011 037 003 011 037 003 003 011 034

Col

br gr ye br

ye br

br gr br

br gr br br br gr br br gr

br gr br br gr br br br gr br

Int

+ +Ishy

+ t

+

+ +lshy

t

+ + + t + + + t

t

+ + + + + + + + + t

Pr (Identical with those of Pa result) pz

Se 005 br + Sf 005 br + 003 br +

011 gr + 014 br + 021 br t 033 br + 063 br t

38

(Table 9 continued)

Extract

Family Plant Skelly F Chloroform

Rf Col lnt Rf Col lnt

Typhaceae Ta-l 005 br + 011 gr + 021 br +

Ta-2 005 br + 011 gr + 021 br +

Ceratophyllaceae Cd 005 br + 011 gr ye + 020 br + 026 br t 034 br +

lIaloragaceae Me 005 br + 003 br + 011 br gr + 036 br +

Nymphaeaceae Nv-l 005 br +Ishy 003 br + Iv 011 br ye + 011 br +

023 br t 038 br + Nv-2 Iv 037 br +Ishy 056 br +

044 br + Nt Iv 060 br +Ishy

Nv-l st 005 br + 003 br + 011 br ye + 011 gr + 044 br t 038 br + 060 br t

Nv-2 st Nt st (Identical those for Nv-l st)

S8 standard 000 br + 021 br + 005 br + 038 br + 012 br + 056 br +Ishy019 br + 023 br + 038 br t 044 br + 060 br +

1P1ant names- (refer to Table 3 footnote 2) 2Rf- Rf values Col- Color br- brown gr- green ye- yellow Int

Intensity +++= high ++= medium += low t~ trace

39

Table 10 Constituent Fatty Acids of Triglycerides Derived From Selected Aquatic Plants

Plant Chain length amp numshyber of double bonds

As As hydrogenated methyl esters

140 150 160 161 180 181 182

487 700

2970 1291

375 1750 1084

314 290

2588

4219

Carex 1acustris

150 160 161 180 181 182 183 203 240 241 140 160 170 180 181 182 183 220 240

()

(7)

Trace 781 318 1 46 673

1236 463

2905 724

2346 113 794 522 690

1045 2357 2680 108 101

Trace 1874

3918

1218 2363

113 1677 479

5974

476 NymEhaea 140 509 321 tuberosa (Iv) 150 Trace Trace

160 2824 1444 161 402 17 0 Trace Trace 180 450 21 97 181 946 182 954 183 857

NymEhaea tuberosa (st) 150 Trace Trace

160 2232 5381 161 210 170 100 211 180 289 3617 181 421 182 4396 183 1648 240 112

40

Table 11

Linseed

Soybean

Ac

Cd

Cl

Nt Iv

Nt st

Comparison of Major Fatty Acids Plants (Linseed and Soybean)

Chain length amp number of unsat 2

160 180 181 182 183

72 34 185 170 552

135 70 259 480 66

297 32 175 108 75

78 15 67 124 46

79 69 105 236 268

292 45 95 95 86

223 29 42 440 165

in Aquatic and Terrestrial

Total of unsat 3 Ref

907

805

358

237

609

276

647

(146)

(147)

1P1ant names Ac- Anacharis Cl- Carex lacustris Nt Iv- ~~~~a ~~~~ (ste~

2Percent contents of fatty acids were listed under each fatty acid column

3Total of unsaturated fatty acid (181 182 and 183)

41

IV REFERENCES

1 Farnsworth NR et a1 1966 Biological and phytochemical evaluashytion of plants I Biological test procedures and results from two hundred accessions L10ydia 101-122

2 Goto M and H Sato 1969 Determination of antitumor activity in rat ascites hepatomas by agar diffusion technique Yakugaku Zasshi 89 821-827

3 Hartwell JL 1960 Plant remedies for cancer Cancer Chemother Rep 19-24

4 Bianchi B and JR Cole 1969 Antitumor agents from Agave schottH (AmarylUdaceae) J Pharm Sci 58 589-591

5 Cole JR E Bianchi and ER Trumbull 1969 Antitumor agents from Bursera microphyl1a (Burseraceae) II Isolation of a new lignanshyburseran J Pharm Sci~ 175-176

6 Pates AL and GC Madsen 1955 Occurrence of antimicrobial substances in chlorophyl10se plants growing in Florida II Bot Gaz

250-261

7 Hughes JE 1952 Survey of antibiotics in the wild green plants of southern California Antibiot Chemother 2 487-491

8 Azarowicz EN JE Hughes and CL Perkins 1952 Anti-biotics in plants of southern California active against Mycobacterium tubershy

607 and niger Antibiot Chemother 2 532-536

9 Bushnell OA M Fukuda and T Makinodan 1950 The antibacterial properties of some plants found in Hawaii Pac Sci 4 167-183

10 Hayes LE 1947 Survey of higher plants for presence of anti shybacterial substances Bot Gaz 108 408-414

11 Carlson HJ HD Bissell and MG Mueller 1946 Antimalarial and antibacterial substances separated from higher plants J Bacteriol 52 155-168

12 Carlson HJ HG Douglas and J Robertson 1948 Screening methshyods for determining antibiotic activity of higher plants J Bactershyiol 55 235-240

13 Carlson HJ HG Douglas and J Robertson 1948 Antibacterial substances separated from plants J Bacteriol 55 241-248

14 Sanders DW P Weatherwax and LS McClung 1945 Antibacteria] substances from plants collected in Indiana J Bacteriol 49 611shy615

15 Nickell LC 1959 Antimicrobial activity of vascular plants Econ Bot Q 281-318

16 Skinner FA 1955 Antibiotics In K Peach and MW Tracey (ed) Modern Methods of Plant Analysis voT 3 Springer-Verlag Berlin pp 626-744

17 Burlage HM ME Jones GF McKenna and A Taylor 1952 Studies on toxic plants for antibacterial effects Tex Rep BioI Med 10 803-815

42

18 Maleszadeh F 1968 Antimicrobial activity of Lawsonia ~~==~ L Appl Microbiol 16 663-664

19 McCleary JA and DL Walkington 1964 Antimicrobial activity of the Cactaceae Bull Torrey Bot Garden 91 177-181

20 Wolters B 1968 Saponins as plant fungistatic compounds On the antibiotic action of saponins III P1anta 79 77-83

21 Ma TS and R Roper 1968 Microchemical investigation of medicinal plants I The antituberculosis principle in Prunus mume and chinensis Mikrochim Acta 2 167-181--------- shy

22 Nagy JG and R Tengerdy 1968 Antibacterial actions of essenshytial oils of Artemisia as an ecological factor II Antibacterial actions of Artemisia tridenta bacteria from the rumen of mule deer Appl Microbiol 1amp 441-444

23 Farnsworth NR 1966 Biological and phytochemical screening of plants J Pharm Sci 225-276

24 Jiu J 1966 A survey of some medicinal plants of Mexico for selected biological activities Lloydia 29 250-259

25 Noemi G M Nadal and LV Rodriguez 1963 Sarganin and chonalgin new antibiotic substances from Puerto Rico Antimicrob Ag Chemother 3 68-72

26 Noemi G and M Nadal 1964 Isolation and characterization of sarganin complex a new broad spectrum antibiotic isolated from marine algae Antimicrob Ag Chemother 131-] 34

27 Gorham PR 1962 The toxin produced by waterblooms of the blueshygreen algae Amer J Pub Health 52 2100-2105

28 Holm LG LW Weldon and RD Blackburn 1969 Aquatic yeneeds Science 699-709

29 Manske RHF and HL Holmes 1951-1965 The alkaloids Eight vols Academic Press NY

30 Henry TA ]939 The plant alkaloids Blakiston Phila

31 Bentley KW 1957 In the Alkaloids Interscience Publishers NY

32 Willaman JJ and BG Schubert 1955 Alkaloid hunting Econ Bot 9 141-150

33 Willaman JJ and BG Schubert 1955 Alkaloid hunting suppleshymental table of Genera US Department of Agriculture ARS ARSshy73-1

34 Wil1aman JJ and BG Schubert 1961 Alkaloid-bearing plants and their contained alkaloids Technical bulletin No 1234 US Department of Agriculture Washington DC

35 Webb LJ 1952 An australian phytochemical survey II Alkaloids in Queensland flowering plants Aust Common Sci Ind Res Organ Bul1 Melbourne No 268 5-99

36 Massingill JL Jr and JE Hodgkins 1967 Alkaloids of bacteria Phytochemistry i 977-982

43

Table 10 Constituent Fatty Acids of Triglycerides Derived From Selected Aquatic Plants

Plant Chain length amp numshyber of double bonds

As As hydrogenated methyl esters

140 150 160 161 180 181 182

487 700

2970 1291

375 1750 1084

314 290

2588

4219

Carex 1acustris

150 160 161 180 181 182 183 203 240 241 140 160 170 180 181 182 183 220 240

()

(7)

Trace 781 318 1 46 673

1236 463

2905 724

2346 113 794 522 690

1045 2357 2680 108 101

Trace 1874

3918

1218 2363

113 1677 479

5974

476 NymEhaea 140 509 321 tuberosa (Iv) 150 Trace Trace

160 2824 1444 161 402 17 0 Trace Trace 180 450 21 97 181 946 182 954 183 857

NymEhaea tuberosa (st) 150 Trace Trace

160 2232 5381 161 210 170 100 211 180 289 3617 181 421 182 4396 183 1648 240 112

40

Table 11

Linseed

Soybean

Ac

Cd

Cl

Nt Iv

Nt st

Comparison of Major Fatty Acids Plants (Linseed and Soybean)

Chain length amp number of unsat 2

160 180 181 182 183

72 34 185 170 552

135 70 259 480 66

297 32 175 108 75

78 15 67 124 46

79 69 105 236 268

292 45 95 95 86

223 29 42 440 165

in Aquatic and Terrestrial

Total of unsat 3 Ref

907

805

358

237

609

276

647

(146)

(147)

1P1ant names Ac- Anacharis Cl- Carex lacustris Nt Iv- ~~~~a ~~~~ (ste~

2Percent contents of fatty acids were listed under each fatty acid column

3Total of unsaturated fatty acid (181 182 and 183)

41

IV REFERENCES

1 Farnsworth NR et a1 1966 Biological and phytochemical evaluashytion of plants I Biological test procedures and results from two hundred accessions L10ydia 101-122

2 Goto M and H Sato 1969 Determination of antitumor activity in rat ascites hepatomas by agar diffusion technique Yakugaku Zasshi 89 821-827

3 Hartwell JL 1960 Plant remedies for cancer Cancer Chemother Rep 19-24

4 Bianchi B and JR Cole 1969 Antitumor agents from Agave schottH (AmarylUdaceae) J Pharm Sci 58 589-591

5 Cole JR E Bianchi and ER Trumbull 1969 Antitumor agents from Bursera microphyl1a (Burseraceae) II Isolation of a new lignanshyburseran J Pharm Sci~ 175-176

6 Pates AL and GC Madsen 1955 Occurrence of antimicrobial substances in chlorophyl10se plants growing in Florida II Bot Gaz

250-261

7 Hughes JE 1952 Survey of antibiotics in the wild green plants of southern California Antibiot Chemother 2 487-491

8 Azarowicz EN JE Hughes and CL Perkins 1952 Anti-biotics in plants of southern California active against Mycobacterium tubershy

607 and niger Antibiot Chemother 2 532-536

9 Bushnell OA M Fukuda and T Makinodan 1950 The antibacterial properties of some plants found in Hawaii Pac Sci 4 167-183

10 Hayes LE 1947 Survey of higher plants for presence of anti shybacterial substances Bot Gaz 108 408-414

11 Carlson HJ HD Bissell and MG Mueller 1946 Antimalarial and antibacterial substances separated from higher plants J Bacteriol 52 155-168

12 Carlson HJ HG Douglas and J Robertson 1948 Screening methshyods for determining antibiotic activity of higher plants J Bactershyiol 55 235-240

13 Carlson HJ HG Douglas and J Robertson 1948 Antibacterial substances separated from plants J Bacteriol 55 241-248

14 Sanders DW P Weatherwax and LS McClung 1945 Antibacteria] substances from plants collected in Indiana J Bacteriol 49 611shy615

15 Nickell LC 1959 Antimicrobial activity of vascular plants Econ Bot Q 281-318

16 Skinner FA 1955 Antibiotics In K Peach and MW Tracey (ed) Modern Methods of Plant Analysis voT 3 Springer-Verlag Berlin pp 626-744

17 Burlage HM ME Jones GF McKenna and A Taylor 1952 Studies on toxic plants for antibacterial effects Tex Rep BioI Med 10 803-815

42

18 Maleszadeh F 1968 Antimicrobial activity of Lawsonia ~~==~ L Appl Microbiol 16 663-664

19 McCleary JA and DL Walkington 1964 Antimicrobial activity of the Cactaceae Bull Torrey Bot Garden 91 177-181

20 Wolters B 1968 Saponins as plant fungistatic compounds On the antibiotic action of saponins III P1anta 79 77-83

21 Ma TS and R Roper 1968 Microchemical investigation of medicinal plants I The antituberculosis principle in Prunus mume and chinensis Mikrochim Acta 2 167-181--------- shy

22 Nagy JG and R Tengerdy 1968 Antibacterial actions of essenshytial oils of Artemisia as an ecological factor II Antibacterial actions of Artemisia tridenta bacteria from the rumen of mule deer Appl Microbiol 1amp 441-444

23 Farnsworth NR 1966 Biological and phytochemical screening of plants J Pharm Sci 225-276

24 Jiu J 1966 A survey of some medicinal plants of Mexico for selected biological activities Lloydia 29 250-259

25 Noemi G M Nadal and LV Rodriguez 1963 Sarganin and chonalgin new antibiotic substances from Puerto Rico Antimicrob Ag Chemother 3 68-72

26 Noemi G and M Nadal 1964 Isolation and characterization of sarganin complex a new broad spectrum antibiotic isolated from marine algae Antimicrob Ag Chemother 131-] 34

27 Gorham PR 1962 The toxin produced by waterblooms of the blueshygreen algae Amer J Pub Health 52 2100-2105

28 Holm LG LW Weldon and RD Blackburn 1969 Aquatic yeneeds Science 699-709

29 Manske RHF and HL Holmes 1951-1965 The alkaloids Eight vols Academic Press NY

30 Henry TA ]939 The plant alkaloids Blakiston Phila

31 Bentley KW 1957 In the Alkaloids Interscience Publishers NY

32 Willaman JJ and BG Schubert 1955 Alkaloid hunting Econ Bot 9 141-150

33 Willaman JJ and BG Schubert 1955 Alkaloid hunting suppleshymental table of Genera US Department of Agriculture ARS ARSshy73-1

34 Wil1aman JJ and BG Schubert 1961 Alkaloid-bearing plants and their contained alkaloids Technical bulletin No 1234 US Department of Agriculture Washington DC

35 Webb LJ 1952 An australian phytochemical survey II Alkaloids in Queensland flowering plants Aust Common Sci Ind Res Organ Bul1 Melbourne No 268 5-99

36 Massingill JL Jr and JE Hodgkins 1967 Alkaloids of bacteria Phytochemistry i 977-982

43

IV REFERENCES

1 Farnsworth NR et a1 1966 Biological and phytochemical evaluashytion of plants I Biological test procedures and results from two hundred accessions L10ydia 101-122

2 Goto M and H Sato 1969 Determination of antitumor activity in rat ascites hepatomas by agar diffusion technique Yakugaku Zasshi 89 821-827

3 Hartwell JL 1960 Plant remedies for cancer Cancer Chemother Rep 19-24

4 Bianchi B and JR Cole 1969 Antitumor agents from Agave schottH (AmarylUdaceae) J Pharm Sci 58 589-591

5 Cole JR E Bianchi and ER Trumbull 1969 Antitumor agents from Bursera microphyl1a (Burseraceae) II Isolation of a new lignanshyburseran J Pharm Sci~ 175-176

6 Pates AL and GC Madsen 1955 Occurrence of antimicrobial substances in chlorophyl10se plants growing in Florida II Bot Gaz

250-261

7 Hughes JE 1952 Survey of antibiotics in the wild green plants of southern California Antibiot Chemother 2 487-491

8 Azarowicz EN JE Hughes and CL Perkins 1952 Anti-biotics in plants of southern California active against Mycobacterium tubershy

607 and niger Antibiot Chemother 2 532-536

9 Bushnell OA M Fukuda and T Makinodan 1950 The antibacterial properties of some plants found in Hawaii Pac Sci 4 167-183

10 Hayes LE 1947 Survey of higher plants for presence of anti shybacterial substances Bot Gaz 108 408-414

11 Carlson HJ HD Bissell and MG Mueller 1946 Antimalarial and antibacterial substances separated from higher plants J Bacteriol 52 155-168

12 Carlson HJ HG Douglas and J Robertson 1948 Screening methshyods for determining antibiotic activity of higher plants J Bactershyiol 55 235-240

13 Carlson HJ HG Douglas and J Robertson 1948 Antibacterial substances separated from plants J Bacteriol 55 241-248

14 Sanders DW P Weatherwax and LS McClung 1945 Antibacteria] substances from plants collected in Indiana J Bacteriol 49 611shy615

15 Nickell LC 1959 Antimicrobial activity of vascular plants Econ Bot Q 281-318

16 Skinner FA 1955 Antibiotics In K Peach and MW Tracey (ed) Modern Methods of Plant Analysis voT 3 Springer-Verlag Berlin pp 626-744

17 Burlage HM ME Jones GF McKenna and A Taylor 1952 Studies on toxic plants for antibacterial effects Tex Rep BioI Med 10 803-815

42

18 Maleszadeh F 1968 Antimicrobial activity of Lawsonia ~~==~ L Appl Microbiol 16 663-664

19 McCleary JA and DL Walkington 1964 Antimicrobial activity of the Cactaceae Bull Torrey Bot Garden 91 177-181

20 Wolters B 1968 Saponins as plant fungistatic compounds On the antibiotic action of saponins III P1anta 79 77-83

21 Ma TS and R Roper 1968 Microchemical investigation of medicinal plants I The antituberculosis principle in Prunus mume and chinensis Mikrochim Acta 2 167-181--------- shy

22 Nagy JG and R Tengerdy 1968 Antibacterial actions of essenshytial oils of Artemisia as an ecological factor II Antibacterial actions of Artemisia tridenta bacteria from the rumen of mule deer Appl Microbiol 1amp 441-444

23 Farnsworth NR 1966 Biological and phytochemical screening of plants J Pharm Sci 225-276

24 Jiu J 1966 A survey of some medicinal plants of Mexico for selected biological activities Lloydia 29 250-259

25 Noemi G M Nadal and LV Rodriguez 1963 Sarganin and chonalgin new antibiotic substances from Puerto Rico Antimicrob Ag Chemother 3 68-72

26 Noemi G and M Nadal 1964 Isolation and characterization of sarganin complex a new broad spectrum antibiotic isolated from marine algae Antimicrob Ag Chemother 131-] 34

27 Gorham PR 1962 The toxin produced by waterblooms of the blueshygreen algae Amer J Pub Health 52 2100-2105

28 Holm LG LW Weldon and RD Blackburn 1969 Aquatic yeneeds Science 699-709

29 Manske RHF and HL Holmes 1951-1965 The alkaloids Eight vols Academic Press NY

30 Henry TA ]939 The plant alkaloids Blakiston Phila

31 Bentley KW 1957 In the Alkaloids Interscience Publishers NY

32 Willaman JJ and BG Schubert 1955 Alkaloid hunting Econ Bot 9 141-150

33 Willaman JJ and BG Schubert 1955 Alkaloid hunting suppleshymental table of Genera US Department of Agriculture ARS ARSshy73-1

34 Wil1aman JJ and BG Schubert 1961 Alkaloid-bearing plants and their contained alkaloids Technical bulletin No 1234 US Department of Agriculture Washington DC

35 Webb LJ 1952 An australian phytochemical survey II Alkaloids in Queensland flowering plants Aust Common Sci Ind Res Organ Bul1 Melbourne No 268 5-99

36 Massingill JL Jr and JE Hodgkins 1967 Alkaloids of bacteria Phytochemistry i 977-982

43

37 Wall ME et al 1954 Steroidal sapogenins XII Survey of plants for steroidal and other constituents J Amer Pharm Ass Sci Ed 43

38 Wall ME 1955 Steroidal sapogenins XXV Survey of plants for steroidal sapongenins and other constituents T Amer Pharm Ass Sci Ed 44 438-440

39 Wall ME et a] 1957 Steroidal sapogenins XLITI Survey of plants for steroidal sapongenins and other constituents J Amer Pharm Ass Sci Ed 46 653-684

40 Wall ME et a1 1959 Steroidal sapongenins LV Survey of plants for steroidal sapongenins and other constituents J Amer Pharm Ass Sci Ed 48 695-722

41 Hultin E and K Torssel1 1965 Alkaloid-screening of Swedish plants Phytochemistry 425-433

42 Hu1tin E ]965 Alkaloid screening of plants from Boyce Thompson southwestern arboretum Acta Chern Scand 19 1297-1300

43 Farnsworth NR and KL Euler 1962 An alkaloid screening proshycedure utilizing thin-layer chromatography Lloydia 186-195

44 Farnsworth NR NA Pilewski and FJ Draus 1962 Studies on false-positive alkaloid reactions with Dragendorffs reagent Lloydia 25 312-319

45 Stahl E 1969 Thin-layer Chromatography 2nd Ed Springer-Verlag Berlin Heidelberg NY p 423

46 Geissman TA 1961 The Chemistry of Flavonoid Compounds MacMilshylan NY

47 Cutting WC et al 1951 Antiviral chemotherapy V Further reshyport on f1avonoids Stanford Med Bull 9 236-242

48 Kupchan SM JR Knox and MS Udayamurthy ]965 Tumor inhibishytors VIII Eupatorin new cytotoxic flavone from ====-==== ~ J Pharm Sci 929-930

49 Wi1laman J T 1955 Some biological effects of the flavonoids J Amer Pharm Ass Sci Ed 44 404-408

50 Wall ME et al 1954 Steroidal sapogenins VII Survey of plants for steroidal sapogenins and other constituents J Amer Pharm Ass Sci Ed 1-7

51 Seikel MK 1962 Geissman (ed) The Chemistry of Flavoshynoid Compounds The Co NY p 34

52 Swain T Bonner J and IE Varner (ed) Plant Bioshychemistry Press NY and London p 552

53 Bate-Smith EC 1962 J Linn Soc London Bot 58 95

54 Ramstad E 1959 Modern Pharmacognosy Blakiston Division McGrawshyHill Book Company Inc

55 Persinos GJ and JW Schermerhorn 1964 A preliminary study of ten Nigerian plants Econ Bot 18 329- 341

44

56 Wilson JA and HB Merrill 1931 Analysis of Leather and Materials Used in Making It 1st ed The McGraw-Hill Book Co Inc NY p 290 and p 293

57 Segelman AB and NR Farnsworth and MW Quimby 1969 Biologishycal and phytochemical evaluation of plants III False-negative saposhynin test results induced by the presence of tannins Lloydia 32 52-58

58 Brown BR PE Brown and WT Pike 1966 The leaf tannin of willow-berb (Chamaenerion (L) Scop) BiochembullT

733-738

59 Wall ME et al 1961 Steroidal sapongenins LX Survey of plants for steroidal sapongenins and other constituents T Pharm Sci 50 1001-1034

60 Simes JJH et al 1959 An Australian phytochemical survey III Saponins in Eastern Australian flowering plants Aust Common Sci Ind Res Organ Bull Melbourne No 5-31

61 Heftmann E 1965 Steroids J Bonner and IE Varner (ed) Plant Biochemistry Academic Press NY and London p 693

62 K1yne W 1957 The naturally occurring steroids I In W Klyne (ed) The Chemistry of Steroids John Wiley N Y p iDs

63 Tsuda K et a1 1958 Unterschungen Uber steroide IX Die sterine aus meeres-algen Chem Pharm Bull (Tokyo) 6 724-727

64 Johnson DF RD Bennett and E Heftmann 1963 Cholesterol in higher plants Science 140 198-199

65 leger O and V Prelog 1960 RHF Manski (ed) Alkaloids Academic Press NY pp

66 Zalkow LH NI Burke and G Keen 1964 The occurrence of 5shyalpha-androstane-3-beta l6-alpha 17-alpha-triol in Rayless Goldenshyrod (Aplopappus Blank) Tetrahedron Lett 4 217-221

67 Bradbury RB and DE White 1954 Estrogens and related subshystances in plants Vitam Horm 12 207-233

68 Neher R 1969 TLC of steroids and related compounds In E Stahl (ed) Thin-layer Chromatography A Laboratory Handbook 2nd ed Springer-Verlag Berlin Heidelberg NY p 311

69 Frerejacque M and P DeGraeve 1963 Reaction colorees et reacshytions de fluorescence des digitaliques Ann Pharm Fr 21 509-528

70 Stevens PF and AB Turner 1969 The use of iodine as a nonshydestructive location reagent for steroids in TLC J Chromatogr 43 282-286

71 Mangold HK 1961 Thin-layer chromatography of lipids J Amer Oil Chern Soc 38 708-727

72 Novitskaya GV 1969 The chromatographic separation of higher fatty acid monoglycerides according to chain length and unsaturation J ChromatogrgtQ 422-430

73 Jamieson GR And EH Reid 1969 The leaf lipids of some members of the Boraginaceae family Phytochemistry 8 1489-1494

45

74 Watts D 1969 Separation of mono- and diglycerides by gas-liquid chromatography J Lipid Res 33-40

75 Schlenk H and JL Gellerman 1965 Arachidonic 5111417shyeicosatetraenoic and related acids in plants Identification of unshysaturated fatty acids J Am Oil Chemists Soc 42 504-511

76 Fish GR and GM Will 1966 Fluctuations in the chemical composhysition of two lake weeds from New Zealand Weed Res 346-349

77 1967 ~~~~~_~~~~~

Morphological and embryological studies in NymphaeashyDOrb and stellata Willd Bot

78 Mauve AA 1967 Water-lilies in south Africa N lotus N capensis N Through BA-49 52818shy

79 Salageanu N and L Tipa 1967 The diurnal course of photosyntheshysis in higher aquatic plants Rev Roum BioI Ser Bot 12 295shy318 Through BA 49 52895

80 Forsberg C 1966 Sterile germination requirements of seeds of some water plants Physiol Plant 1105-1109

81 Haraszti E 1961 The importance of the mineral contents of sour grasses in feeding Acta Vet Acad Sci Hung 393-399 Through CA 57 17153h bull

82 Paribok TA 1966 Content of some chemical elements in the wild plants of the polar Urals as related to the problem of the serpentine vegetation Bot Zh 339-353 Through CA 64 20565a

83 Boichenko EA 1964 Compounds of Mn and iron in plants Dokl Akad Nauk SSSR 158 464-466 Through CA 2l l6438e

84 Saenko GM 1968 Distribution of some metals in plants Fizio1 Rast 15 139-144 Through CA 93492d

85 Oborn ET 1964 Intracellular and extracellular concentrations of manganese and other elements by aquatic organisms US Geol Surv Water Supply Papers 1667c 18 Through CA 1662g

86 Allenby KG 1967 The Mn and Ca contents of some aquatic plants and the water in which they grow Hydrobiologica 239-244 Through CA 8689k

87 Esipova IV 1962 Cromatographic examination of sugars of some plants gorwing in winter in the south Kyzyl-Kum region Uzbeksk BioI Zh 6 27-32 Through CA l4438f

88 Hodgson RH 1966 Growth and carbohydrate status of Sago pond-weed Weeds 263-268

89 Stich G 1957 Glycosides of some Alismaceae Rev Gen Bot 64 549-571 Through CA 7455i

90 Watanabe T 1960 Honey and pollen III Sugar composition of pollen of Nippon Nogei Kaguku Kaisha 34 704-708 Through shy

91 Khan NA 1965 Cereals and cereal products III Starch varieties in certain and food waste in East Pakistan Sci Res (Dacca 11-19 Through CA l2224e

46

92 Pigulevskaya LV 1957 Chemical composition of peat-forming materials and their effect on peat composition Trudy Inst Torfa Inst Torfa Akad Nauk Beloruss SSR 3-11 Through CA 54 2698h

93 Ermakova TA 1960 Composition and food value of some types of water vegetation in the Kara-Kum canal Izvest Adad Nauk Turkmen SSR Ser BioI Nauk 51-58 Through CA l1689c

94 Ballester A 1966 Critique of the spectrophotometric and chromashytographic methods for the study of plankton pigments Invest Pesquera 30 613-630 Through CA l8907h

95 Vaidya BS 1960 Amino acids in Chara brachypus J Univ Bombay BioI Sci 29 151-153 Through CA 57 l2902b

96 Anderson DMW and NJ King 1961 Polysaccharides of the Characeae TIT The carbohydrate content of australis Biochim Biophys Acta 449-454

97 Dyachenko NI 1962 Vitamin content characteristics of some aquatic plants of Moldavia Izvest Akad Nauk Moldavsk SSR 6 32-37 Through CA 8118a

98 Duff RB 1963 Occurrence of apiose in Lernna and other angioshysperms Biochem J 33-34p

99 Beck E 1965 Apiose as a constituent of the cell wall of higher plants Z Naturforsch 62-67 Through CA 62 l5072a

100 Mendicino J 1965 Biosynthesis of the branched chain sugar-Dshyapiose in Lernna and parsley J BioI Chern 240 2797-2805

101 Roberts RM 1967 Inositol metabolism in plants IV Biosynthesis of apiose in Lernna and Plant Physio1 ~ 659-666

102 Beck E 1966 Iosotopic studies on the biosynthesis of apiose in Lernna Z Pflanzenphysiol 71-84 Through CA 65 l4115e

103 Achmatowicz O and Z Be1len 1962 Alkaloids of Nuphar luteum (L) SM Tso1ation of alkaloids containing sulphur Tetrahedron Lett 1121-1124

104 Novikova SI 1960 Methodology of the production of the alkaloid nupharine Mikrobiol 7h Akad Nauk Ukr RSR 22 67 Through CA 2041g

105 Achmatowicz O and JT Wrobel 1964 Alkaloids from Nuphar III A new alkaloid neo-thiobinupharidine Spectroscopic on the structure of thiobinupharidine and neothiobinupharidine Tetrahedron Lett 129-136

106 Achmatowicz 0 H Banaszek G Spite11er and JT Wrobel 1964 Alkaloids from Nuphar luteum IV Mass spectroscopy of thiobinupharishydine neothiobinupharidine and their desu1furation products Tetrashyhedron Lett 16 927-934

107 Arata Y N Hazama and Y Kojima 1962 Constituents of rhizoma Absolute configuration of deoxynupharidine I Yakushy

326-328

108 Ohashi T 1959 Constituents of rhizoma Nuphari~ XIV Constitushytion of nupharamine I Yakugaku Zasshi 79 729- 34

47

109 Kotake M 1963 Alkaloids japonicum Isolation of minor alkaloids nupharamine and nupharamine ethyl ether Nippon Kagaku 2asshi 160-162 Through CA 60 6891a

1l0 Kawasaki I 1963 Absolute configuration of nupharamine Bull Chern Soc Japan 36 1474-1477

111 Arata Y 1947 Constituents of rhizoma Nupharis synthesis of nupharane Kanazawa Daigaku Yakugakubu Kenkyu Nempo 7 49-51 Through CA 53 195c

112 Kusumoto S 1956 Nupharidine an alkaloid from ___---- ~co-Nippon Kagaku Zasshi 12 1302-1304 Through CA

113 Arata Y 1965 Nuphamine a new alkaloid of Nuphar L===

Chern Pharm Bull (Tokyo) 392-393

114 Arata Y 1964 Dehydrodeoxynupharidine a new alkaloid of japonicum Chern Pharm Bull (Tokyo) 1l 1394-1395

115 Kotake M I Kawasaki and T Okamoto 1962 The absolute configshyuration of deoxynupharidine Bull Chern Soc Japan ll 1335-1341

116 Arata Y 1965 Constituents of Nuphar japonicum XXII Structure of nuphamine Chern Pharm Bull (Tokyo) 11 1247-1251

117 Arata Y 1967 Constituents of rhizoma Nupharis XXIV Structure of a new alkaloid anhydronupharamine Yakugaku Zasshi 1094shy1l02

118 Arata Y 1960 Synthesis of dl-deoxynupharidine Yakugaku 2asshi 80 855-856

119 Arata Y T Nakanishi and Y Asaoka 1962 Constituents of Nuphar japonicum XVIII Synthesis of alkaloids from_~~~~_~~~~~ I Synthesis of dl-deoxynupharidine Chern Pharm 10 675-679

120 Barchet R 1965 Alkaloids of Nuphar variegatum Tetrahedron Lett 47 4229-4232

121 Bukowiecki H 1964 Chromatographic analysis of alkaloids from Polish water lilies Acta Polon Pharm 21 121-126 Through CA 62 12152b

122 Terenteva IV 1957 Alkaloid-bearing sedge of Moldavia Referat Zhur Khim BioI Khim Abstra No 20181 Through CA 3932f

123 Terenteva IV 1957 Alkaloids from Carex brevicollis Zhur Obshchei Khim 27 3170-3173 TIlrough CA 2l 9l73e

124 Terenteva IV 1960 Alkaloids of Carex brevicollis Alkaloidoshynosyne Rast Moldavii Moidavsk Filial Akad Nauk SSSR Inst Khim pp 41-47 Through CA~ 2476g

125 Terenteva IV 1960 The structure of brevicolline- the alkaloid of Carex brevicollis Alkaloidonosyne Rast Moldavii Moldavsk Filial Akad Nauk SSR Inst Khim pp 21-33 Through CA 4607f

126 Terenteva IV and VA Bolyak 1962 Spectrophotometric detershymination of brevicolline Izv Akad Nauk Moldavsk SSSR pp 71shy74 Through CA l599le

48

127 Clifford HT and JB Harborne 1969 Flavonoid in the sedges (Cyperaceae) Phytochemistry~

128 Tikhonov 01 1965 F1avonoids of the lesser duckweed minor) I Preliminary studies Farmatsevt 2h 20 63-65 CA 64 8639h

129 Tikhonov 01 1965 Flavonoids minor II Farmatsevt 2h 20 53-55 Through CA

130 Naya Y 1965 A constituent of the rhizomes of sp Nippon Kagaku Zasshi~ 313-315 Through CA 63

131 Cordes WC 1960 Response of idioblasts to environmental changes temperature and light Plant 13 187-191 Through CA jL 3788d

132 Altman RFA 1956 Chemical studies of Amazonian plants II Plants containing steroidal sapogenins Bo1 tee inst agron norte 31 67-80 Through CA 506b

133 Arata Y 1961 Constituents of rhizoma Nupharis XVI Isolation of beta-sitosterol palmitic acid and oleic acid Kanazawa Daigaku Yakugakubu Kenkyu Nempo 35-39 Through CA 21 l554lab

134 Bobbitt JM 1968 Introduction to Chromatorgraphy Reinhold Book Corp NY Amsterdam amp London p 70

135 Staba EJ and P Laursen 1966 Morning glory tissue cultures Growth and examination for indole alkaloids J Pharm Sci 1099-1104

136 USP 16th ed p 929

137 Kirchner JG 1967 Technique of Organic Chemistry (A Weissshyberger ed) vol XII chromatography Interscience Publishers NY London and Sydney p 638

138 Lewbart ML W Wehrli and T Reichstein 1963 Helv Chim Acta 46 505

139 Kirchner JG Technique of Organic Chemistry (A Weissshyberger ed) Publishers NY London and Sydney vol XII p 159

140 Martello R and NR Farnsworth 1962 Observation on the sensishytivity of several common alkaloid precipitating reagents L10ydia 25 176-185

141 Durkee AB and JC Sirois 1964 The detection of some indoles and related chromatography on paper chromatograms J Chromatogr 13 173-180

142 Cheronis ND and JB Entrikin (ed) 1957 Semi-micro Qualitashytive Organic Analysis Interscience Publishers Inc NY and London p 237

143 Lisboa BP 1964 Characterization of thin-layer chromatograms by in situ togr~ 136-151

144 Neher R 1964 Steroid chromatography (2nd revised and enlarged ed) Elseview Publishing Co Amsterdam London and NY p125

49

145 Spener FK 1969 Personal communications

146 Vereshchagin AG and GV Novitskaya 1965 The triglyceride composition of linseed oil J Amer Oil Chem Soc 43 970-974

147 Sietz FG 1965 Die Kennzah1en des Sojaoles Fette Seifen Anstrichmitte1 67 411-412 Through CA 63 13587e

50

74 Watts D 1969 Separation of mono- and diglycerides by gas-liquid chromatography J Lipid Res 33-40

75 Schlenk H and JL Gellerman 1965 Arachidonic 5111417shyeicosatetraenoic and related acids in plants Identification of unshysaturated fatty acids J Am Oil Chemists Soc 42 504-511

76 Fish GR and GM Will 1966 Fluctuations in the chemical composhysition of two lake weeds from New Zealand Weed Res 346-349

77 1967 ~~~~~_~~~~~

Morphological and embryological studies in NymphaeashyDOrb and stellata Willd Bot

78 Mauve AA 1967 Water-lilies in south Africa N lotus N capensis N Through BA-49 52818shy

79 Salageanu N and L Tipa 1967 The diurnal course of photosyntheshysis in higher aquatic plants Rev Roum BioI Ser Bot 12 295shy318 Through BA 49 52895

80 Forsberg C 1966 Sterile germination requirements of seeds of some water plants Physiol Plant 1105-1109

81 Haraszti E 1961 The importance of the mineral contents of sour grasses in feeding Acta Vet Acad Sci Hung 393-399 Through CA 57 17153h bull

82 Paribok TA 1966 Content of some chemical elements in the wild plants of the polar Urals as related to the problem of the serpentine vegetation Bot Zh 339-353 Through CA 64 20565a

83 Boichenko EA 1964 Compounds of Mn and iron in plants Dokl Akad Nauk SSSR 158 464-466 Through CA 2l l6438e

84 Saenko GM 1968 Distribution of some metals in plants Fizio1 Rast 15 139-144 Through CA 93492d

85 Oborn ET 1964 Intracellular and extracellular concentrations of manganese and other elements by aquatic organisms US Geol Surv Water Supply Papers 1667c 18 Through CA 1662g

86 Allenby KG 1967 The Mn and Ca contents of some aquatic plants and the water in which they grow Hydrobiologica 239-244 Through CA 8689k

87 Esipova IV 1962 Cromatographic examination of sugars of some plants gorwing in winter in the south Kyzyl-Kum region Uzbeksk BioI Zh 6 27-32 Through CA l4438f

88 Hodgson RH 1966 Growth and carbohydrate status of Sago pond-weed Weeds 263-268

89 Stich G 1957 Glycosides of some Alismaceae Rev Gen Bot 64 549-571 Through CA 7455i

90 Watanabe T 1960 Honey and pollen III Sugar composition of pollen of Nippon Nogei Kaguku Kaisha 34 704-708 Through shy

91 Khan NA 1965 Cereals and cereal products III Starch varieties in certain and food waste in East Pakistan Sci Res (Dacca 11-19 Through CA l2224e

46

92 Pigulevskaya LV 1957 Chemical composition of peat-forming materials and their effect on peat composition Trudy Inst Torfa Inst Torfa Akad Nauk Beloruss SSR 3-11 Through CA 54 2698h

93 Ermakova TA 1960 Composition and food value of some types of water vegetation in the Kara-Kum canal Izvest Adad Nauk Turkmen SSR Ser BioI Nauk 51-58 Through CA l1689c

94 Ballester A 1966 Critique of the spectrophotometric and chromashytographic methods for the study of plankton pigments Invest Pesquera 30 613-630 Through CA l8907h

95 Vaidya BS 1960 Amino acids in Chara brachypus J Univ Bombay BioI Sci 29 151-153 Through CA 57 l2902b

96 Anderson DMW and NJ King 1961 Polysaccharides of the Characeae TIT The carbohydrate content of australis Biochim Biophys Acta 449-454

97 Dyachenko NI 1962 Vitamin content characteristics of some aquatic plants of Moldavia Izvest Akad Nauk Moldavsk SSR 6 32-37 Through CA 8118a

98 Duff RB 1963 Occurrence of apiose in Lernna and other angioshysperms Biochem J 33-34p

99 Beck E 1965 Apiose as a constituent of the cell wall of higher plants Z Naturforsch 62-67 Through CA 62 l5072a

100 Mendicino J 1965 Biosynthesis of the branched chain sugar-Dshyapiose in Lernna and parsley J BioI Chern 240 2797-2805

101 Roberts RM 1967 Inositol metabolism in plants IV Biosynthesis of apiose in Lernna and Plant Physio1 ~ 659-666

102 Beck E 1966 Iosotopic studies on the biosynthesis of apiose in Lernna Z Pflanzenphysiol 71-84 Through CA 65 l4115e

103 Achmatowicz O and Z Be1len 1962 Alkaloids of Nuphar luteum (L) SM Tso1ation of alkaloids containing sulphur Tetrahedron Lett 1121-1124

104 Novikova SI 1960 Methodology of the production of the alkaloid nupharine Mikrobiol 7h Akad Nauk Ukr RSR 22 67 Through CA 2041g

105 Achmatowicz O and JT Wrobel 1964 Alkaloids from Nuphar III A new alkaloid neo-thiobinupharidine Spectroscopic on the structure of thiobinupharidine and neothiobinupharidine Tetrahedron Lett 129-136

106 Achmatowicz 0 H Banaszek G Spite11er and JT Wrobel 1964 Alkaloids from Nuphar luteum IV Mass spectroscopy of thiobinupharishydine neothiobinupharidine and their desu1furation products Tetrashyhedron Lett 16 927-934

107 Arata Y N Hazama and Y Kojima 1962 Constituents of rhizoma Absolute configuration of deoxynupharidine I Yakushy

326-328

108 Ohashi T 1959 Constituents of rhizoma Nuphari~ XIV Constitushytion of nupharamine I Yakugaku Zasshi 79 729- 34

47

109 Kotake M 1963 Alkaloids japonicum Isolation of minor alkaloids nupharamine and nupharamine ethyl ether Nippon Kagaku 2asshi 160-162 Through CA 60 6891a

1l0 Kawasaki I 1963 Absolute configuration of nupharamine Bull Chern Soc Japan 36 1474-1477

111 Arata Y 1947 Constituents of rhizoma Nupharis synthesis of nupharane Kanazawa Daigaku Yakugakubu Kenkyu Nempo 7 49-51 Through CA 53 195c

112 Kusumoto S 1956 Nupharidine an alkaloid from ___---- ~co-Nippon Kagaku Zasshi 12 1302-1304 Through CA

113 Arata Y 1965 Nuphamine a new alkaloid of Nuphar L===

Chern Pharm Bull (Tokyo) 392-393

114 Arata Y 1964 Dehydrodeoxynupharidine a new alkaloid of japonicum Chern Pharm Bull (Tokyo) 1l 1394-1395

115 Kotake M I Kawasaki and T Okamoto 1962 The absolute configshyuration of deoxynupharidine Bull Chern Soc Japan ll 1335-1341

116 Arata Y 1965 Constituents of Nuphar japonicum XXII Structure of nuphamine Chern Pharm Bull (Tokyo) 11 1247-1251

117 Arata Y 1967 Constituents of rhizoma Nupharis XXIV Structure of a new alkaloid anhydronupharamine Yakugaku Zasshi 1094shy1l02

118 Arata Y 1960 Synthesis of dl-deoxynupharidine Yakugaku 2asshi 80 855-856

119 Arata Y T Nakanishi and Y Asaoka 1962 Constituents of Nuphar japonicum XVIII Synthesis of alkaloids from_~~~~_~~~~~ I Synthesis of dl-deoxynupharidine Chern Pharm 10 675-679

120 Barchet R 1965 Alkaloids of Nuphar variegatum Tetrahedron Lett 47 4229-4232

121 Bukowiecki H 1964 Chromatographic analysis of alkaloids from Polish water lilies Acta Polon Pharm 21 121-126 Through CA 62 12152b

122 Terenteva IV 1957 Alkaloid-bearing sedge of Moldavia Referat Zhur Khim BioI Khim Abstra No 20181 Through CA 3932f

123 Terenteva IV 1957 Alkaloids from Carex brevicollis Zhur Obshchei Khim 27 3170-3173 TIlrough CA 2l 9l73e

124 Terenteva IV 1960 Alkaloids of Carex brevicollis Alkaloidoshynosyne Rast Moldavii Moidavsk Filial Akad Nauk SSSR Inst Khim pp 41-47 Through CA~ 2476g

125 Terenteva IV 1960 The structure of brevicolline- the alkaloid of Carex brevicollis Alkaloidonosyne Rast Moldavii Moldavsk Filial Akad Nauk SSR Inst Khim pp 21-33 Through CA 4607f

126 Terenteva IV and VA Bolyak 1962 Spectrophotometric detershymination of brevicolline Izv Akad Nauk Moldavsk SSSR pp 71shy74 Through CA l599le

48

127 Clifford HT and JB Harborne 1969 Flavonoid in the sedges (Cyperaceae) Phytochemistry~

128 Tikhonov 01 1965 F1avonoids of the lesser duckweed minor) I Preliminary studies Farmatsevt 2h 20 63-65 CA 64 8639h

129 Tikhonov 01 1965 Flavonoids minor II Farmatsevt 2h 20 53-55 Through CA

130 Naya Y 1965 A constituent of the rhizomes of sp Nippon Kagaku Zasshi~ 313-315 Through CA 63

131 Cordes WC 1960 Response of idioblasts to environmental changes temperature and light Plant 13 187-191 Through CA jL 3788d

132 Altman RFA 1956 Chemical studies of Amazonian plants II Plants containing steroidal sapogenins Bo1 tee inst agron norte 31 67-80 Through CA 506b

133 Arata Y 1961 Constituents of rhizoma Nupharis XVI Isolation of beta-sitosterol palmitic acid and oleic acid Kanazawa Daigaku Yakugakubu Kenkyu Nempo 35-39 Through CA 21 l554lab

134 Bobbitt JM 1968 Introduction to Chromatorgraphy Reinhold Book Corp NY Amsterdam amp London p 70

135 Staba EJ and P Laursen 1966 Morning glory tissue cultures Growth and examination for indole alkaloids J Pharm Sci 1099-1104

136 USP 16th ed p 929

137 Kirchner JG 1967 Technique of Organic Chemistry (A Weissshyberger ed) vol XII chromatography Interscience Publishers NY London and Sydney p 638

138 Lewbart ML W Wehrli and T Reichstein 1963 Helv Chim Acta 46 505

139 Kirchner JG Technique of Organic Chemistry (A Weissshyberger ed) Publishers NY London and Sydney vol XII p 159

140 Martello R and NR Farnsworth 1962 Observation on the sensishytivity of several common alkaloid precipitating reagents L10ydia 25 176-185

141 Durkee AB and JC Sirois 1964 The detection of some indoles and related chromatography on paper chromatograms J Chromatogr 13 173-180

142 Cheronis ND and JB Entrikin (ed) 1957 Semi-micro Qualitashytive Organic Analysis Interscience Publishers Inc NY and London p 237

143 Lisboa BP 1964 Characterization of thin-layer chromatograms by in situ togr~ 136-151

144 Neher R 1964 Steroid chromatography (2nd revised and enlarged ed) Elseview Publishing Co Amsterdam London and NY p125

49

145 Spener FK 1969 Personal communications

146 Vereshchagin AG and GV Novitskaya 1965 The triglyceride composition of linseed oil J Amer Oil Chem Soc 43 970-974

147 Sietz FG 1965 Die Kennzah1en des Sojaoles Fette Seifen Anstrichmitte1 67 411-412 Through CA 63 13587e

50

109 Kotake M 1963 Alkaloids japonicum Isolation of minor alkaloids nupharamine and nupharamine ethyl ether Nippon Kagaku 2asshi 160-162 Through CA 60 6891a

1l0 Kawasaki I 1963 Absolute configuration of nupharamine Bull Chern Soc Japan 36 1474-1477

111 Arata Y 1947 Constituents of rhizoma Nupharis synthesis of nupharane Kanazawa Daigaku Yakugakubu Kenkyu Nempo 7 49-51 Through CA 53 195c

112 Kusumoto S 1956 Nupharidine an alkaloid from ___---- ~co-Nippon Kagaku Zasshi 12 1302-1304 Through CA

113 Arata Y 1965 Nuphamine a new alkaloid of Nuphar L===

Chern Pharm Bull (Tokyo) 392-393

114 Arata Y 1964 Dehydrodeoxynupharidine a new alkaloid of japonicum Chern Pharm Bull (Tokyo) 1l 1394-1395

115 Kotake M I Kawasaki and T Okamoto 1962 The absolute configshyuration of deoxynupharidine Bull Chern Soc Japan ll 1335-1341

116 Arata Y 1965 Constituents of Nuphar japonicum XXII Structure of nuphamine Chern Pharm Bull (Tokyo) 11 1247-1251

117 Arata Y 1967 Constituents of rhizoma Nupharis XXIV Structure of a new alkaloid anhydronupharamine Yakugaku Zasshi 1094shy1l02

118 Arata Y 1960 Synthesis of dl-deoxynupharidine Yakugaku 2asshi 80 855-856

119 Arata Y T Nakanishi and Y Asaoka 1962 Constituents of Nuphar japonicum XVIII Synthesis of alkaloids from_~~~~_~~~~~ I Synthesis of dl-deoxynupharidine Chern Pharm 10 675-679

120 Barchet R 1965 Alkaloids of Nuphar variegatum Tetrahedron Lett 47 4229-4232

121 Bukowiecki H 1964 Chromatographic analysis of alkaloids from Polish water lilies Acta Polon Pharm 21 121-126 Through CA 62 12152b

122 Terenteva IV 1957 Alkaloid-bearing sedge of Moldavia Referat Zhur Khim BioI Khim Abstra No 20181 Through CA 3932f

123 Terenteva IV 1957 Alkaloids from Carex brevicollis Zhur Obshchei Khim 27 3170-3173 TIlrough CA 2l 9l73e

124 Terenteva IV 1960 Alkaloids of Carex brevicollis Alkaloidoshynosyne Rast Moldavii Moidavsk Filial Akad Nauk SSSR Inst Khim pp 41-47 Through CA~ 2476g

125 Terenteva IV 1960 The structure of brevicolline- the alkaloid of Carex brevicollis Alkaloidonosyne Rast Moldavii Moldavsk Filial Akad Nauk SSR Inst Khim pp 21-33 Through CA 4607f

126 Terenteva IV and VA Bolyak 1962 Spectrophotometric detershymination of brevicolline Izv Akad Nauk Moldavsk SSSR pp 71shy74 Through CA l599le

48

127 Clifford HT and JB Harborne 1969 Flavonoid in the sedges (Cyperaceae) Phytochemistry~

128 Tikhonov 01 1965 F1avonoids of the lesser duckweed minor) I Preliminary studies Farmatsevt 2h 20 63-65 CA 64 8639h

129 Tikhonov 01 1965 Flavonoids minor II Farmatsevt 2h 20 53-55 Through CA

130 Naya Y 1965 A constituent of the rhizomes of sp Nippon Kagaku Zasshi~ 313-315 Through CA 63

131 Cordes WC 1960 Response of idioblasts to environmental changes temperature and light Plant 13 187-191 Through CA jL 3788d

132 Altman RFA 1956 Chemical studies of Amazonian plants II Plants containing steroidal sapogenins Bo1 tee inst agron norte 31 67-80 Through CA 506b

133 Arata Y 1961 Constituents of rhizoma Nupharis XVI Isolation of beta-sitosterol palmitic acid and oleic acid Kanazawa Daigaku Yakugakubu Kenkyu Nempo 35-39 Through CA 21 l554lab

134 Bobbitt JM 1968 Introduction to Chromatorgraphy Reinhold Book Corp NY Amsterdam amp London p 70

135 Staba EJ and P Laursen 1966 Morning glory tissue cultures Growth and examination for indole alkaloids J Pharm Sci 1099-1104

136 USP 16th ed p 929

137 Kirchner JG 1967 Technique of Organic Chemistry (A Weissshyberger ed) vol XII chromatography Interscience Publishers NY London and Sydney p 638

138 Lewbart ML W Wehrli and T Reichstein 1963 Helv Chim Acta 46 505

139 Kirchner JG Technique of Organic Chemistry (A Weissshyberger ed) Publishers NY London and Sydney vol XII p 159

140 Martello R and NR Farnsworth 1962 Observation on the sensishytivity of several common alkaloid precipitating reagents L10ydia 25 176-185

141 Durkee AB and JC Sirois 1964 The detection of some indoles and related chromatography on paper chromatograms J Chromatogr 13 173-180

142 Cheronis ND and JB Entrikin (ed) 1957 Semi-micro Qualitashytive Organic Analysis Interscience Publishers Inc NY and London p 237

143 Lisboa BP 1964 Characterization of thin-layer chromatograms by in situ togr~ 136-151

144 Neher R 1964 Steroid chromatography (2nd revised and enlarged ed) Elseview Publishing Co Amsterdam London and NY p125

49

145 Spener FK 1969 Personal communications

146 Vereshchagin AG and GV Novitskaya 1965 The triglyceride composition of linseed oil J Amer Oil Chem Soc 43 970-974

147 Sietz FG 1965 Die Kennzah1en des Sojaoles Fette Seifen Anstrichmitte1 67 411-412 Through CA 63 13587e

50

145 Spener FK 1969 Personal communications

146 Vereshchagin AG and GV Novitskaya 1965 The triglyceride composition of linseed oil J Amer Oil Chem Soc 43 970-974

147 Sietz FG 1965 Die Kennzah1en des Sojaoles Fette Seifen Anstrichmitte1 67 411-412 Through CA 63 13587e

50