(a) Review of proteins, oils, carbohydtales, trace nufu,,:.:~·- c''IJ ic•.\ic: •.·dions.

(:.) Review of chromatographic techniques of analysi'l!_; ·· .:i;;o c:cic's, fc:tly acids,

c"'rbohydretes and cations.

(c) Literature Survey.

(r!) Problem taken and work done.

t~UAI•'t'EU - I

INTRODUCTION:

Cell is the basic unit of all living organisms. It is responsible for all the vital activities

oflife in plants as we)! as in animal kingdom. Cell is mainly made up of proteins, lipids

and carbohydrates.

As Proteins, Carbohydrates, Fats and Oils are the chief sources of nutrients of food

and feed, their chemical investigations were considered essential. These nutrients and their

functions are briefly described below :

PROTEINS:

Proteins are main constituents of cell, tissues, and muscles and other body fluids

like blood and gastro intestinal fluid etc. of living being of both ruminants and non

ruminants. They play aeveral important and diverse roles which govern various biochemical

processes responsible for existence of life. Nucleo proteins of the genes regulates cell division

and heredity in living organisms. As enzymes proteins catalyze and carry out various

biochemical reactions responsible for growth and maintenance of cell life. Proteins govern

activities like cell inte-raction, cell motility, gene repression, mitogenetic and intercellular

transport in plants. Plants and microbes are capable of converting inorganic nitrogenous

substances like ammonium salts, nitrites, nitrates of soil and even nitrogen of air into

amino acids and proteins required for their growth and maintenance.

1.

Living beings both herbivorous and carnivorous need ready made proteins, or their

intermediate, or their ultimate breakdown products "amino acids", (often called body

building stones or blocks), as elaborated from other form oflife (plant or microbial origin)

for synthesis and maintenance of their tissues. Thus human beings and animals solely

meet their protein requirements through direct or indirect consumption of plant or microbial

or animal proteins of good quality in adequate quantity.

The need for protein to build new tissues and to maintain and repair the old continue><

throughout life. Proteins as enzymes and hormones catalyze various biochemical reaction

involved in the digestion of food, major components like fats, carbohydrates and protein~

etc." Antibodies" defend.~ living beings against infections are also proteinaceous in natur.,.

Various biochemical processes oflife involve continuous ,;ynthesis and degradation of tis.~u••

proteins and thus tissues are not metabolically inert. Body tissues of adults slowly wash•

away due to "Wrar a11d 7Par". To compen><at" tlus lo>-.s, amino acids as proteins of good

2 quality are supplied daily to both human beings and animals, which is a constant feature

of the process oflife. Protein requirements of infants for the formation of new body tissues

and their developments, are greater in comparison to metabolically quiescent adults. Normal

adult daily needs lg of protein per kg of body weight for maintaining nitrogen equilibria of

the body.

Enzymes and hormones are proteinaceous in structure. They act as biological catalyst

and carry out various biochemical decomposition reactions involved with digestion of major

components of food like carbohydrates, fats and proteins. Fats and carbohydrates can not

be utilized as sources of heat in the absence of enzymes.

The proteins consumed are not assimilated as such. Proteolytic enzymes (viz. pepsin,

trypsin, chromotrypsin and erypsin etc.) of the digestive juices or fluids of the

gastrointestinal tract, in acidic medium decompose dietary proteins into their component

amino acids at body temperature. Besides food proteins other tissue constituents and even

hydrolytic enzymes too undergo hydrolysis to form amino acids. Free amino acids thus

liberated after digestion, enter the blood stream from intestine and the blood carries them

to liver and other vital parts of the body, where they are used for various tissue protein

synthesis. The unutilized amino acids are oxidized and converted into urea, uric acid etc.

and excreted out in urine.

Unless all indispensable amino acids are present in balanced proportion in the diet,

it can not be used for the synthesis of body tissue proteins. 18 - 20 amino acids are of

common occurrence in dietary proteins of vegetable or animal origin. Block suggested

nutritive classification of amino acids which is as under:

CLASSIFICATION OF AMINO ACIDS:

Indispensable or Essential Amino Acids :

Indispensable or essential amino acids include histidine, lysine, threonine,

phenylalanine, valine, methionine, leucine, isoleucine and tryptophan. Living beings are

incapable of synthesizing these amino acids at a rate sufficient for their optimum growth

from materials normally available in diet. These are called indispensable or essential amino

acids. Their presence in dietary protein~< in prop!!r or balanced proportion is a must nnd

their presence determines the quality of proteins. lnfact protein nutrition is essentially

indispensable, or essential amino acid nutrition and an adequate supply of these eRSential

amino acids in food and feeds of living beings is necessary.

3 Semi Indispensable or Semi Essential Amino Acids :

Amino acids like cystine, tyrosine, glycine, serine and arginine can partly replace

some indispensable amino acids. This class of amino acids are called semi-indispensable or

semi essential amino acids.

Dispensable or Non-Essential Amino Acids:

These are being synthesized by living beings at a rate sufficient for their growth

from materials ordinarily available in the diet. This group of amino acids include aspartic

acid, glutamic acid, glycine, alanine, proline, hydroxyproline and isomeric amino butyric

acids etc. and called dispensable or non essential amino acids.

In tabular form Block's1 nutritive classification of amino acids is as given below:

TABLE -NO.I

Nutritive Classification of Amino Acids

Indispensable/Essential Semi-Indispensable Dispensable/Non-Essential

Lysine Gl . ++ ycme Aspratic acid Tryptophan Serine Glutamic acid Methionine Cystine Alanine Threonine Proline Valine Tyrosine Hydroxy proline Leucine Isoleucine Phenylalanine Histidine

Arginine++

++ Arginine and glycine are essential for chicks and turkeys. Serine possess sparing,

effect for glycine. Recent investigations have shown that arginine and histidine are

non- essential for human beings.

Common Sources of Proteins :

Most of the food and feed materials of vegetable or animal or microbial origin contam

proteins in widely varying ranges of concentration. Among plant foods, pulses or legumes

in particular, protein percentage rangeR between IIi- :JII':'r-, Roy a hean iRan exception which

contains nearly 43'7r of proteins. Nuts have higher protein contents, approximate rang<'

lies between 20 - 3o% or so. Defatted oil seeds may contain up to nO% of proteins. Cereals

like rice, maize, barley and wheat are poor sources of proteins, its concentration vary

between 7.15 - 12.15'!"n. Rice contains 7.15% proteins and wheat 12.15%. Outer coat of cereals

contain higher percentage of protein in contrast to inner kernel. Animal foods like meat,

fish and eggs etc. are rich in proteins. Recently microbial sources like yeast and other

fungi, species ofPenicillum, Aspergillus, Fusariusm, Rhizopus Cheatomium, Agaricus and

many bacteria too are reported to be non-toxic and rich sources of edible grade proteins of

quality superior to that of cereals. Many microbes contain higher lysc;ine contents than

cereals or legumes. Closer the amino acid make up of food proteins to those of tissues, the

greater is its nutritive value and quality.

Detailed description of proteins and amino acids have been given in many standard

texts. (Neurath and Bai!y2; Greenberi; Rajlakshmi4

; Wilson etal5; Harper6

; Mateles and

Tannenbaun7

; Norton8 and Dixon and Webb\

Two third of the entire world population is suffering from protein deficiency diseases10

which includes everlasting damages like stunted growth, mental retardation, and idiocy in

children and adolescent. It causes reduction in weight, greater susceptibility to infection,

frequent loose stools, incapacity to sustained hard work, lethargy, delayed healing of wounds

and Oedema etc. in adults.

It was considered worthwhile to examine the quality of proteins of various seeds of

Madhya Pradesh origin by chromatographic techniques as no work is reported in literature.

Oils and Fats:

Oils and fats are triestors of glycerol and higher fatty acids. They occur both in

plants and animals either as glycerides (X) or as phospholipids like lecithin (Y) or as ester

of monohydric alcohols (Z) like waxes which are esters of cetyl alcohol (C H OHJ with 16 33

lauric, palmitic and myristic acids.

CH .OOC.R I 2

CHOOC.R'

I Cll .OOC.R"

2

(XJ

CH200CR

I CH.OOC.R'

I CII - 0.\'!0IIJ.OB

2 \

0

!Yl

R-COOR

!Z J

5 Where R, R' and R" are alkyl groups alike or different and B stands for organic base

like ch1ol.ine. In plants, oils and fats occur in spores, seeds, fruits, leaves, roots and other

vegetative organs in varying amounts. In spores, seeds and tubers oil functions as food

reserve to be used during germination and early growth period of the plant.

Oil extracted from seeds of plants of temperate climate are drying or semi-drying

type, since they contain more of unsaturation. Oils of non drying type are of common

occurrence in seeds of plants, of tropical regions.

Oils are better sources of energy than protein and carbohydrate. Energy supplied by

oil (9callgm) is greater than protein (4cal/gm) and carbohydrate (4 cal/gm). Detailed

description of oils and fats and their utilization have been described by Hilditch and . . 11 12 13 14 WJI!tams , Gunstone ; Coon and Rathmann etc.

Carbohydrates :

Carbohydrates are poly hydroxy aldehydes or ketones and their derivatives including

deoxy sugars, amino sugars, sugar alcohols and acids. Monosaccharides like glucose,

galactose, fructose, arabinose, rhamnose etc; oligosaccharides eg. sucrose, maltose, cellibiose,

raffinose, stachyose and verbacose etc; and polysaccharides like cellulose, starch, insulin,

chitin and glycogen etc. are of common and frequent occurrence in plants and animal tissues.

Their detailed description have been given by Feiser and Feiser15

, Pigman16

, Pigman and

(, 11 H 18 B k d. 19 d S. l . 20 ,oepp , arper , o a 1a an , tnc a1r etc.

Mineral Nutrients and Toxic Cations :

Animals and human beings also need mineral elements like calcium, phosphorous,

sodium, chlorine, potassium, magnesium and sulphur in appreciable amounts; others like

iron, manganese, copper, iodine, zinc, cobalt and molybdenum in traces for growth and

maintenance of good health. Functions, utility and daily dietary requirements of various

mineral elements for maintenance of good health have been described in various texts of

Nutrition eg. Rajlakshmi4

, Wilson5

eta!, Brock21, and Underwood22 etc.

Elements like mercury, cadmium, lead, chromium, zinc, copper, arsenic, nickel,

selenium, tin, antimony, beryllium, cobalt and manganese are reported to be toxic in natur<·.

Their toxicology and control have been descriued uy Dara23

, De24, Berman25

, and Mance2r.

etc.

The study of natural product.' or plant products involve their hydrolytic, reductive

or oxidative degradation into simple compounds and investigation of colour reactions of

these compounds, often lead to their structure elucidation. Various techniques of

chromatography in conjunction with spectrophotometric determination of colours or ultra

violet spectroscopy applied to the study of plant products are briefly reviewed below

CHROMATOGRAPHY:

Chromatography is a versatile technique of achieving separations of a mixture into

its component fractions. In all chromatographic processes, the forces of adsorption partition

or the ion exchange are in action, often all three together. But one or the other of these may

be predominant. In usual classification of chromatographic process into adsorption and

partition chromatography the system consists of pairs of solid, liquid and gas phases. Several . 27 28 . . 29 30

reviews by Lederer and Lederer , Block eta! , Smith and Steakins , Stahl ,

.Rauvinderath31, T.rutor32

, Bobbit33, Mariani and Bettolo

34, Calvin

35, .and Mcomie and

Polland36 have described chromatography both from theory and practical application point

of view.

Amino acids, ultimate breakdown products of proteins, sugars and their derivatives,

fatty acids obtained on hydrolysis of oils and fats, and methyl esters of fatty acids etc.,

were separated into components by paper chromatography on Whatman chromatographic

grade filter No. 1. Resolution of amino acids can also be achieved by paper electrophoresis

a combination of paper chromatography and paper electrophoresis, paper chromatography

on special ion exchange resin papers and thin layer chromatography plates of cellulose.

silica gel - G, silica gel H, ion exchange resins etc. Amino acids can also be separated bv

adsorption, partition or ion exchange resin column chromatographic techniques.

Chromatography is the most important analytical tool in the hands of chemists engaged in

phytochemical studies, for this technique facilitates the separations of minor components

not necessarily coloured, for there are many instrumental spectrographic methods of

identifying them after achieving their separations. Presently chromatography has become

a common and efficient method of separation, purification and characterization of

constituents of natural or synthetic origin.

Paper and Thin Layer Chromatography :

lR - 20 amino acids have been separated into its components on Whatman

chromatographic grade filter paper by Con:-den, Gorden and ~fartin 37 ; Levy and C'hunl:!1•.

D t 39 \".11. d Ki. b 40 K. b d,... . 41 R 42 43 ..• en ; •YI 1am an r y ; 1r y an '.am ; utter ; :\farchal and Mittwar ·;Gin". ,... · · 14.S R df. ~r, 49 . 41' . 49 . .~n ; "lTI eta ; e wld ; Hausman , M artm ; Matha1as ; Bo1ssonnas ; Deck(•r '.

Wieland~2 ; Block~ 3 ; Gross and Roland~4 ; Rockland and Dunn~~; Shrivastava·1:

7 .Joshi eta!58' 6\ using different solvents and ascending, descending, horizontal or two

dimensional development techniques. C:onsden eta!37

confirmed the presence of 22 amino

acids in wool protein hydrolysate by applying two dimensional paper chromatographic

ll'chnique. Bickel 1111d Souchonr,2 l'xnminl'd cht'omatogt'nphicall.Y on Whatma11

chromatographic filter paper the amino acid composition of urine and blood of healthy and

diseased children. Special ion exchange resin papers were used by Knight63

'64

; Peterson

and Butler65; Rmit and Rtockenr,6 for separating amino acids. Heathcoate etal

67; Atfield

and Morris68; VonARX and Neher69

; Smith eta!70; Wollen webber

7\ Frodyma and Free

72

and recently .Joshi 78-75 eta! worked out thin layer chromatographic techniques for resolving

amino acids on cellulose, silica gel or ion exchange resin TLC: plates using different solvents

for development.

Paper and thin layer chromatography of carbohydrates have been described by

Glegg76; Shellard77

; Partridge 78; Boggs 79

; Wilson80

; William and Kirby8

\ Radha-

Kr- h h 82 p 'dh 83 K 84 G. . 181\ p 'h R6 H t 187 s h . RR IS namurt y ; r1 am ; awerau ; :drl eta ; ar1 ar ; aye a ; ...__ c we1ger , 89 90 . 91 92 . 93 . 94

Wolfram eta!; Prey eta!; Tate and Bishop ; Gee ; Deferral eta! ; Menzies ; Berry

and Mitche!l95; Shrtvastava96

; Joshi and Nigam!l7; and.Joshi and Shrivastava98

•

Fatty acids were chromatographed by reversed phase chromatographic technique on

liquid paraffin impregnated filter paper by Kaufman 99

; Buchman 100

. Derivatives of fatty

acids have been separated on filter paper by Michael101

; Hiryama102

; Inouye 103

; Fink and

F . k1o4 J hi1o5-tos t Th- 1 h h. h . r 'd ·r . r .d m ; • os · e c. m ayer c romatograp IC tee mques or 1 ent1 ymg a tty ac1 s

h 107 107-112

and t eir derivatives were developed by Kaufman and coworkers ; Purdy and 113 .114

Trutor ; and Room1 eta!.

Paper and thin layer chromatography of cations have been described by Mcomie and

P ll d36 . Ill\ . 116 . 117 II~

o ar ; Bhatnagar and Pooma ; Pfeil ; Elbe1h and Gabra ; Kertes and Lederer ;

M k 119 h 120 121 .123 124 f!r us ; B atnagar and Bhatacharya ; Hunt eta] ; .Joshi ' eta! etc.

Quantitation by Extractive Spectrophotometric Technique :

Colours of individual components from chromatographically resolved paper or TLC'

chromatogram were extracted with some suitable solvent like water, alcohol or water

saturated butanol clc. and it.R optical denHity menHured at Huitnble wave length and compan·d

with known stand11rd using a photoelectric colorimeter or a spectrophotometer. Tin~

d h b . dL Bl kl2o B II' 12r. 127 '•" proce ure as een rev1ewe y oc ; o mg eta!; Souchon ; Rockland and Dunn .

L d 'h 3R h . 12R , 129 130 131 l'l'' cvy an (. ung ; Ne enng ; Crreok ; Bode ; Schwedtfeger ; Woiwood · ·, l'op•·

d "'t 133 R 134 I ,., 11 . 13:. c· _ .13fi I an ., evens ; aven eta ; ne mgton ; rln eta etc.

Automatic Amino Acid Analyzer Technique:

Moore 137 eta} described Beckman analyzer, consisting of Beckman's resin W-2 packed

in a glass column (32 X 9 em) was used for resolving amino acids. The loaded column was

successively elutec:l with the buffer of pH 3.25, 4.12, 6.40 at 70ml!hour flow rate for 60,30

and 90 minutes respectively, for complete elution of all amino acids in a certain definite

sequence. Ninhydrin flow rate was set at 3fi ml/hour and the column temperature was

maintained at fiO' C, Standard mixture of amino acids was also run under identical conditions

for comparison and quantitation of various amino acids. This teclmique quantitates protein

hydrolysate in 4- 5 pours. Recently a rapid method of quantitating amino acids of protein

hydrolysate have been developed by Bidling Meyer13R eta!. This process involved conversion

of ai1Jino acid to their phenylthio carbamyl derivatives on a PICO Tag column and these

derivatives chroma to graphed on model ALC 204liquid chromatograph (Water and ASSOC;

which consisted of two waters M 6000 A solvent delivery system and M 440 fixed wave

length UV detector (254 nm) controlled with an M 720 controller. The temperature was ' controlled within:!: 1 C with a column heater (Water and Assoc.). Samples were injected in

volumes ranging 1. 401 using auto injector. Eluents were kept in an atmosphere of helium.

The solvent system consisted of two eluents: (A) an aqueous buffer and (B) 60% aceto nitrite

in water. The typical buffer was 0.14 M sodium acetate containing 0.5 mlll TEA and titrated

to pH 6.35 with glacial acetic acid. A gradient which was run for separation consisted of

10% B transversing to fil% Bin 10 minutes using a convex curve. After this a column

washing step was programmed to 100% B so that any residual sample components would

Le cleaned from the column. This method quantitates amino acids of the protein hydrolysate

inju~t 12 minutes or so.

Gas Liquid Chromatography :

This technique was developed by .James and Martin 139, and involves partitioning of

volatile components of a mixture between a mobile gas phase (carrier) and a stationary

non volatile liquid or a solid phase. Non volatile components after conversion into vola til<•

derivatives may be chromatographed Loth qualitatively and quantitatively Ly c;u· technique. The technique has been reviewed by .James140

; lloffman 141; Keulemans 1 '1 ~;

K 143 144 14~ • 14rl . napman ; Brenner ; Bayer ; and (,oloy etc. Th1s technique baH been applied lor

investigations of amino acids as their trinuoro acetyl ester denvativcs.

9 Literature Survey :

A survey of the work done reviews that a large number of workers have

phytochemically investigated several plants for the presence of both free and protein bound

amino acids, adopting various chromatographic techniques. Some of the important citations

are contributed by Ekpenyoung 147,Shavart148, Azimov 149, Nahid and Zaidi150

, Sadek and 151 152 11\3 154 d dd J!\F, Wilson , Kasai eta! , Kapoor eta! , Dardemie eta! , N arayanmurthy an Re Y ,

!56 11\7 158 d N · 1-o~ Pant and Bishnoi , Gaveshili eta! , Watson and Fowden , Rao an Igam , 160 lfi1 . 162 d co· hJI;'<

Bhatnap;ar eta! , Awasthi and Thaker , Cautam and Purohtt , Pant an ,...,mg , · · tn-1 · · lnr. • I d t 'I 11;6 J k' d 'l'h· k If;; .Jo:-;lu and N1gnrn ,Bnnet:JCL' nnd N1gntn , (,an: 111 an , 1opra , . »n 1 an d er ,

. HiH . W9 170 , 171 . dro 171 Bax1 and Thaker , Das and Sakia , Kawatra eta! , 1 andon eta! , Smha an uupta , 173 , . 174 d K 175 R kr. h d Chopra , Ramkrishna and Subramaman , Pant an apoor , am IS na an

176 177 ' 178 . .179 ' 180 181 T d Sankara , Bhowan eta] , Sharma eta! , P1ch_1 , (xu pta eta! , Kapoor eta! , an on 182 183-186 . 187 . 188

eta! , Udayasekhara Rao , Prattbha and Reddy , Laxmmarayan etal , Khatta 189 .190 191 192

eta! , Yadav and Bhardwaj , Bhattacharya eta! , Longvah and Deosthale , Mohan 193-194 . 191\ . 196 I d and ,J anardhanan , Arulmozh1 and ,J anardhanan , Ruales and N mr , Temp e an

. 197 . 198-199 . 200 d Ojobe , RaJaram and .Janadhanan , Smgh and Eggusm , Dhan Prakash an

. h 201 Bh dChr' - 202 C'h 1203 W d '204 N k I 205 N d Mts ra , atty an , 1stison , , ang eta , an aw1 , wo o o , nanna an

Phillips206, Geervani and Eggum207, Baker etal208, Achinewhu209

, Bradbear and 210 - 211 212 213 Boulter , Badr and GeoglV , Khader and Venkatrao , Bhatnagar eta! , Chavan fWd

M 214 N' . d K t· 21° K . d N. . 21 r; AI. d Q d 217 D. d ager , IranJan an a 1yar , atlyar an IranJan , 1 an a ry , 111 a

218 - 219 220 . 221 222 -and Saha , Dmda eta! , Garg and Oswal , Ansan eta! , Saxena eta! , Behan and

Andh. 1223 B . 1224 D' d d" h 22!\ D. d 1226 1wa , artana eta , m a an uu a , m a eta .

Oils of different seeds were examined for their fatty acid composition by reser\'ed

phase paper and cellulose thin layer chromatography offatty acids and their methyl ester

and also by gas liquid chromatography of methyl esters by Khanna etai227, Patnaik and

R t 22R S'dd' . t 1229 T. . 12ao M h . 231 . 232 au , 1 Iqlll e a , 1wan eta , o an Murtl eta! , Tiwan eta! , Badam1

t ·1233 K t !234 B d . d p .1235 B d . d D .236 d- 237-~:l" e a , apoor e a , a amt an at1 , a am1 an esa1 , Be 1 and A tal .

D d (' h 239 . 240 . 241 . 242 utta an r ash , Ra1kar and Magar , Qaz1 eta! , Ansan eta] , Pant and 'l'ul .243 R d N' 244 ..,. h dB . .2-tfi . z-tr. ··1 ; S1an1 , ao an 1gam , .~mg an aJpal , Awasth1 and Thaker , Ahmad etar .

B d - J24R ro I . ]249 M . 2!\0 " , or, I ?fi2 •',·, a am1 eta , "e p1 eta , once , ,-,engupta and (,upta- , Arora· , Umarav eta!· .

I' l B 1 d d B'll 204 S'dd' · t2"" · l zr.r. 2"7 '"•" \..rlR 1ana a 1a ur 1111 1 a , . 1 1qu1 eta , Sa t!In eta I , C~arg · , :-;;engupta l•lal- .

B d. d 12fi9 2611 - 261 2fi2 2f•1 e 1 an Ata , Dorrell , Appealv1st , Rankov etal , (;umstone eta} , Chaw!.,

t 121>1 R. . d S 1 zr.s K' 1 d . 2fifi 2fi7 e a , lZVl an , u tana , 1ma an Laxmmarayana , Khabir eta] , Dinda ,11lfl

Sl 1268 K . d Bl . 2m S d d l 270 o;- 1 • 1a , at1yar an 1at1a , , Oil ~n Rat 1ore , Rathee and Kaushal- , Ti" an .llHl

. 2i2 2i:J • ,. ' 27·t 'l7~ M1shra , Mehta and Upadhaya , llwan eta! , Kamel eta]- ·, Laxminarava 11 ,11 1.1

2if1 . 27/ . T 'l';'R ·l-•) • eta! , Naras1mha !{eddy eta! , Hukmam and l day~Pkhara Hao· , Hukmani·'·. S,·khon

etal2

R0

, Worthington etal2

R1, Badami etal

2"

2·2"

4, Haheja £•tal 2

"", Siddiqui etal 2"r., .Jot<h1 12R7-2RR I h' d ' . 2R~ ' - 290 . 291 29?

eta , • os 1 an Shnvasta\'a , Sumah etal , AhuJa eta! , Sharma da] •.

}0 293-295 296 297 - 29R ,Jain etal , Nagaraj , Rao etal and Azeemuddm etal etc.

Carbohydrate composition of ethanolic extracts of defatted seeds were analysed by

paper and thin layer chromatographic teclmiques by .Jindal and MukheJ:jee299

, Chakraborty 300 301 302 303 304 J k" 305

and Rao , Saunders , Sequeira and Lew , Bourne eta! , Pant , a tmor , 306 307 - 308 J h' d Vijailaxroi and Chauhan , Rao and Nigam , Kapoor and MukherJee , os 1 an

_ 309 _ _ 310-311 312 -1 d M 3!3 t N1gam , .Josh1 and Shnvastava , Varshney eta! , and Patl an agar e c.

Problem Taken and Work done by Author :

In phytochemistry compositional data of a particular plant varies with variation in

climate, soil and fertilizer conditions, growth, variety, maturity stage etc. Various techniques

adopted for extraction of plant constituents and their subsequent qualitative and

quantitative determipations may give varying compositional data.

Because of litUe or no published information on seeds of various plants grown in

Raipur and its surrounding areas, determinations involving calorie contents, nutritional

value or characteristics of proteins, fats, carbohydrates and essential trace nutrients by

various chomatographic techniques were undertaken and are reported in this thesis.

A large number of seeds namely, Cleome viscosa, Anacardium occidentale,k

Buchanania latifolia, Glycine Linn, Guizotia abyssinica, Trigonella foenum graecum, Acacia

arabia, Archis hypogea, Dolichos biflorus, Phaseolus vulgaris, Cymopsis tetragonoloba Linn,

Coriandrum sativum, Leucaena leucocephala of Madhya Pradesh origin were collected from

different areas and were screened for their proteins, fats, carbohydrates and calorie contents.

Maximum protein percentage was observed in Glycine Linn and minimum in Cleo me viscosa

seeds. Phaseolus vulgaris seeds contains minimum of fats 0. 7'7c and calories 390 cals/lOOg,

and maximum fats fi7 .fi% and calories 6fi0 cals/1\l\lg in Buchanania latifolia seeds.

Acid protein hydrolysate of various defatted seeds and synthetic mixture of authentic

amino acids were chromatographed on Whatman filter paper No. 1 and E. Merck Cellulose

TLC plates by various unidimensional circular and two dimensional chromatographic

techniques in different solvents using various specific and multiple staining reagents for

their identification. Presence of cystine, lysine, histidine, arginine, aspartic acid, glycine,

serine, glutamic acid, threonine, alanine, proline, tyroRine, valine, methionint•,

phenylalanine, leucine and isoleucine in varying amount." waR confirmed in all the Rel'd~

examined.

Ninhydrin colours of re,oh·ed ammo acids from two dimensionally dP\'l'lopPd

II chromatograms were quantitated by extractive spectrophotometric method at 570 nm using

Carl Zeiss Spectrophotometer. Proline colours were measured at 440 nm. Tryptophan

estimations were rnade from alkaline protein hydrolysates of various seed meals.

Acid protein hydrolysates of C:leome viscosa, Guizotia abyssinica, Glycine Linn and

Phaseolus vulgaris seeds were also quantitated by rapid HPLC: technique after derivatisation

into their phenyl thio carbamyl derivatives using ALC:- 204 liquid chromatograph. These

techniques gave comparable results for various amino acids except leucine.

All ihe see<l!; examined coniuined Hi- I H umino ncida including nine esseniinl amino

acids in varying a1nounts. None of the seeds investigated was found to contain essential

amino acids in balanced proportion or in proportion comparable to whole egg proteins.

Cleome viscosa, Acacia arabica and Dolichos biforus seeds were found to contain lysine

contents 2.3, 4.5, p.nd 4.8% respectively and thus are deficient in lysine. Other seeds are

rich in lysine, its concentration ranges between 5.1'fe in Arachis hypogaea to 8.2% maximum

in Phaseolus vulg11ris seeds respectively. High methionine + cystine contents, percentage

3.90 and 3.8 were observed in Trigonella foenum graecum seeds and Buchanania latifolia

seeds. Methionine+ cystine contents of other seeds ranges between 0.5 to 2. 7%. All seeds

examined are found to be deficient in tryptophan except Glycine Linn, Trigonella foenum

graecum and cyarnopsis tetragonoloba Linn S{•eds. Other essential amino acids viz. -

histidine, threcnine, valine, phenylalanine + tyrosine, leucine +isoleucine are present in

good proportion in 1111 the seeds chromatographically examined. Presence of serine, glycine,

tyrosine, cystine, !lrginine, glutamic acid, aspratic acid, alanine and proline in varying

amounts have be*'n observed in all the seeds of Madhya Pradesh origin currently

investigated.

Seeds ofBuchanania, latifolia, Anacardium occidentale, Arachis hypogaea, Guizotia

abyssinica, Cleome viscosa, Glycine Linn, Acacia arabica, Leucaena leucocephala and

C:yamopsis tetragonoloba Linn contains oil 57.5%, 48.5, 40.0, 3fi.5, 28.0, 22.0, 8.0, 7.fi, 2.H

percent respectively. Other seeds ofDolichos species and Phasecolus vulgaris contain lei's

than 2% of oil. Reversed phase paper and cellulose thin layer chromatography of fatty

acids and their methyl esters in conjunction with GLC: of methyl esters confirmed the

presence of palmit ·ic, stearic, oleic and linoleic acids as main components of various seed

oils examined.

Quantitation of fatty acids as their methyl esters by (; LC - technique reveall'd the

presence oflinoleic acid (PUFAJ, percentage: :Jfi.ll, fil.5, fifi.7 and 7l.fi in Arachis hypog;wa.

(;\ycine Linn, C:leome Vil'cofm and (;uizotia ahyRRinica Reed oiiR rcRpecli\'ely and tlm.~ tht··'''

oils are of edible grade. Linoleic acid contents of Buchanania latifolia, Anacard1um

occidentale and C:yamopsis tetragonoloha seed oil is fi.4, 17 .fi and 25.Wi- respecti\'ely

12. Oleic acid concentration was found to be maximum in Ana cardium occidentale and

minimum in Guizoti~ abyssinica seed oils. Saturated fatty acids, both palmitic and stearic

ranges between 12.7% (minimum) in Glycine Linn to 37.4% in Buchanania latifolia seed

oil respectively. Presence oflinolenic acid 5.23%, 0.5% was observed in Glycine Linn and

Cyamopsis tetragonoloba oils.

( ~onccntrates of ethanolic extracts ofvariouR dcf atted Reeds were co-chromatographcd

with authentic sugars on Whatman filter paper No. 1 and E. Merck cellulose T.L.C. plates

in various solvents. Presence ofverbacose, stachycose, raffinose, sucrose, glucose and fructose

were found to be common in Guizotia abyssinica, Anacardium occidentale, Arachis hypogaea,

trigonella foenum graecum and Buuchanania latifolia seeds in varying amounts. Verbacose

was found to be absC)nt in Phaseolus vulgaris. It contains stachyose, raffinose, sucrose,

glucose, and fructosC). Cleo me viscosa gave positive spots for verbacose, raffinose, sucrose,

glucose and fructose. Spots of xylose and rhamnose were observed only in Arachis hypogea

seed extracts. Anilin!" phthalate colours of resolved sugar were quantitated by extractive

spectrophotometric method at 410 n.m. using Carl Zeiss's spectrophotometer.

Seperations ofCu, Ni, Co, Zn, Mn, Fe etc. from their synthetic mixture and solutions

of analysed alloys on Whatman filter paper No. 1, were achieved chromatographically.

Resolved Cu, Ni, Co and Zn ions as their pyridyl azo naphthol complexes were quantitated

by extractive spectrophotometric technique using Carl Zeiss spectrophotometer.

Rolutions of ash of various seeds in10'Yn HCJ acid were chromatographed qualitatively

on Whatman filter paper No.1. Presence ofNi, Cu, CC), Mn, Zn, and Fe was confirmed in

various seeds analysed. Quantitation of various cations was carried out by atomic absorption

spectra · meter Varian Techtron (Australia) Model ABQ 57 fi. Concentration of Cu, Co,

Ni, Mn, Zn and Fe ions in limits much below their toxic level was confirmed in seeds of

Phaseolus vulgaris, Cleome viscosa, Guizotia abyssinica, Anacardium occidentale, Dolichos

uniflorous, Glycine Linn, Arachis hypogaea, Trigonella foenum graecum, Buchanania

latifolia, Cyamopsis tetragonoloba and Do)ichos biflorus .

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