Chapter I - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/24322/1/sawti thesis chapters 2009.pdftaken over by Romans and Arabs from whom after its enrichment with Chinese and

Chapter I

Introduction

2

The man since antiquity had to depend upon nature and plants for

sustenance and survival. Nature like mother has always nourished

humanity in her lap. Man as savage must have known by experience how

to relieve his sufferings by the use of plants growing around him. Co-

existence of death, diseases and decay of human being has lead to the

study of diseases and their treatment which has been contemporary with

the dawn of human intellect.

The history of medicinal plants dates back to Vedic period about

4500-1600B.C. after that, the Egyptians (1), Babylonians (2), Greeks,

Romans, Chinese and Indians developed their characteristics materia

medica respectively. According to Wahid and Siddiqui (1961), modern

medicine is supposed to be derived from Greek medicine which was

taken over by Romans and Arabs from whom after its enrichment with

Chinese and Indian medicine was taken over by Europeans.

INDIA THE CRADLE OF MATERIA MEDICA

The history of medicine in India can be traced back to the oldest

repository of human knowledge. Charka (1800 B.C) and Sushrutra (1800

B.C), the eminent physician in Indian medicine have described about 700

plant species as therapeutic agents about 500 are beings mentioned in

Indian flora and few of them come from Rig-Veda (4500 B.C.), and

Atharvanaveda (2000 B.C.-1500 B.C.) (3, 4), Chopra et al., (1969) have

included about 1000 plant species in his book titled “The glossary of

Indian medicinal plants”.

India has a rich herbal heritage and awareness on the medicinal

value of the plant in global market showing upward trend at alarming

rates. Medicinal plants as a group comprise approximately 8000 species

Chapter - I

3

and account for around 50% of the higher flowering plant species of

India. There are about 400 families in the world of flowering plants, at

least 315 are represented in India.

Of India’s total turn over of Rs 3,100 crores the trade in medicinal

plants in India is estimated to the tune of Rs. 675 crores per year.

whereas Ayurvedic and herbal products, major OTC (over the counter)

product contribute around Rs.1,700 crores, Ayurvedic ethical

formulations constitutes around Rs.850 crores and Ayurvedic classical

formulations constitute remaining Rs.550 crores. India exports have

steadily grown at the rate of 65%, since 1991-92 and grew upto Rs.215

crores in 2000-01 from Rs.130 crores in 1991-92. The clinical use of

artemisinin, etoposide and taxol has once more focussed attention on

plants as source of novel drug entities (5). According to WHO about 80%

of world inhabitants rely mainly on traditional medicine and also 20,000

plant species out of total 2, 50,000 species are in use all over the world.

In India, we have 18,000 flowering plants and out of which around 8,000

are medicinal plants.

There are few Indian medicinal plants having immense medicinal values

and can cure disease like Cancer. Few anti-cancer plants are as follows:

I. The active principle compound Jatropholone A, B, C and

Crotofolin A of Jatropha gossypifolia Linn possesses anti-cancer

property (6, 7).

II. Among the best known anti-cancer constituents are the so

called vinca alkaloids i.e. vinblastine and vincristine isolated

from Catharanthus roseus linn (8).

III. Plants like Ostodes paniculata (9) B.I., Feddiea fisheri,

Soulamea soulameoides (10), Dirca occidentalis (11), Taxus (12)

and Passerine vulgaris (13) showed anti-cancer activity. Taxol

4

from Taxus brevifolia Nutt. or Taxus baccata Linn. is the latest

addition of anti-cancer drugs especially in ovarian cancer (14).

India with its wide climatic conditions and geographical features,

possess a vast natural resources including herbs and other plants with

marginal activities. The hilly areas, valleys, dry and wet lands forest and

climatic adaptation differing from tropical to temperate zones provided

this advantage. With these factors, the Indian medicinal flora is the

richest and biggest one with high therapeutic potentialities.

A list of some important medicinal plants with their active principles is given in Table 1.1

Table 1.1 Some Important Pharmacological active plants isolates

S. No. Name of the plant Name of the

compound

Reference

No.

1. Jatropha gosspifolia l. Jatropholone A,B

and C

6, 7

2. Catharanthus roseus

(l.) g.don

Vinblastine and

Vincristine

8

3. Taxus brevifolia nutt Taxol analogue of

different side chains

15, 16

4. Artemisia annua l. Artemisinin 17

5. Ephedra sinaica stapf. Ephedrine 18

6. Papaver somnifera

Linn.

Morphine, Codeine 19, 20

7. Piper longum Linn. And

piper nigum Linn.

Piperine 21

8. Phytoalexins of sweet

potato

Caffeic acid,

Cholorogenic acid

and Isocholorogenic

acid

22

5

CO 2

HOH

N H

O(Me) 3CO

Artemisinin

Taxol

………..{

Analogue of taxol having different Side chain

O

O

O

OO

H

CH3

CH3H

HCH3

Ph NH O

O Ph

OH

O

AcO O

OH

CH3

AcO O

CH3

CH3

CH3

BzO

OH

6

Ephedrine

Morphine R = H

Codeine R = Me

Vinblastine R = Me

Vincristine R = CH

NH

CH3

CH3

OH

OR

O

OH

H

N Me

NH

NOH

CH3

MeOOC

N

N

RH

OHCOOMe

H

MeOOC

Me

MeO

7

Piperine

R =

Isochlorogenic acid

N

H H

H H O

O

O

OR

OH

OH COOH

OR

CH3

OH

OH

O

8

Jatropholone-A

Jatropholone-B

Jatropholone-C

CH3

CH3

CH3

OR

CH2

O

CH3

CH3

CH3

CH3

OH

CH2

O

CH3

CH3

CH3

CH3

CH2

O

CH3

MeOOC

9

COOHOH

OH

Caffeic acid

Chlorogenic acid

O

OH

OHOH

O

OH

OH

10

SUBJECT

A study on Cyperus species from Uttarakhand

Uttarakhand is particularly rich in medicinal herb and other plants

of great economic importance, which has been inadequately explored. A

number of species are still being exploited commercially for their use in

chemical industries and medicinal preparation. About 279 aromatic

plants belonging to 141 genera and 54 natural orders have been reported

to be distributed in the region (23).

For the present work Cyperus species belonging to family

Cyperaceae have been taken for detailed studies which have its own

medicinal and economical importance.

BOTANICAL ASPECT AND ECONOMICAL IMPORTANCE OF

FAMILY CYPERACEAE

A large cosmopolitan family of mostly herbaceous plants,

Cyperaceae, occurs primarily in moist temperate to wet tropical regions

of the world; several species are of economic importance. The family

comprises about 104 genera and more than 5000 species world wide,

although estimates of numbers vary greatly due to differing taxonomic

concepts of individual researchers. The largest genus is Carex with about

2000 species world wide, followed by Cyperus with about 550 species.

Sedges have featured in literature since antiquity. The family is well

circumscribed and uncontroversial. It was formally described by De

Jussieu in 1789, the name is derived from the genus Cyperus, originally

from the Greek Kupeiros, meaning sedge. Spikelet and inflorescence

structure, together with other evidence, forms the basis for classification

within the family (24).

11

Although it’s a very large family, not many species of the

Cyperaceae have an economic importance. To mention some

concrete examples, the tubers of Cyperus esculentus are edible and

known as chufas or tiger nuts. Cyperus involucrates is used as

fodder which is grazed by domestic stocks in Kenya.

In Gabon the rhizomes of Cyperus articulates L. are used in

the treatment of migraine (25). In north-eastern Thailand Cyperus

corymbosus is used in the production of mats of various types and

sizes. Some Cyperus species are sometimes been referred to as the

world’s worst weed (26). The family is discussed in Chapter - II

more elaborately.

12

Chapter II

Review Of Literature

13

Cyperaceae as previously discussed is a large cosmopolitan family

of mostly herbaceous plants. It occurs primarily in moist to wet tropical

regions of the world; several species are of economic importance. Many

plants of the genus Cyperus have been the subject of chemical studies

for a long time. Volatile constituents as well as higher boiling compounds

have been investigated to such an extent that some generalization can be

done.

The genus mainly consists of prenylated quinones such as

breviquinone (27), cyperaquinone (28), scabequinone (29) and

conicaquinone (30). However methylaurones have also been isolated from

Cyperus capitatus (31). The main hydrocarbon in most of the species is

cyperene (32). In a multidisciplinary research programme, in a search

for anti-malarial natural product we came across the alleged anti-

malarial activity of Cyperus rotundus Linn. tubers reported in the

guidelines of Thai medicinal plants used in primary health care.

Some anti-feedents were isolated from the basal stem of Cyperus

nipponicus and C. distans, and identified by their spectral analysis as

coumarin, remirol, furoquinones, cyperaquinones and scabequinone

(33). Four metabolites named carexanes I-L (34) and one previously

unknown tetrastilbene (cis-miyabenol A) and two known oligostilbenes

(kobophenol B and cis-miyabenol C (35) has been isolated from Carex

species.

A report on essential oil constituents from tubers of C. scariosus

have shown to contain cyperene (13.91%), caryophyllane (12.45%), iso-

patchoul-4(5)-en-3-one (12.25%), trans-pinocerveol (7.24%), rotundene

(5.76%), eudesma-4 (14)-11diene (4.55%), rotundone (4.32%) and

Chapter - II

14

guaiazulene (3.21%) as its main constituents (36), while resveratol

oligomers, nepalesinol A, B and C (37) and nepalesinol D-G (38) were

reported from the stem of Kobresia nepalensis (Cyperaceae). Some recent

reports on C. rotundus have shown the presence of three new

sesquiterpene hydrocarbons (-)-isorotundene, (-)-cypera-2, 4(15)-diene, (-

)-norrutendene and a ketone (+)-cyperadione (39), while another report

shows the isolation of patchoulene, caryophylene-α-oxide, 10, 12-

peroxycalamenene and 4, 7-dimethyl-1-tetralone were isolated from C.

rotundus (40).

Three new compounds, the hydrocarbon, (-)-eudesma-2,4(15)-11-

triene, the sesquiterpene alcohol (-)-eudesma-3,11-dien-5-ol and the

diterpene hydrocarbon (-)-dolabella-3,7,18-triene (41) along with a

benzoquinone, named as alopecuquinone (42) was isolated from

C.alopecuroides while 3-methylaurones were isolated from C. capitatus

(43)

Two new prenylflavans 7,3-dihydroxy-5,5-dimethoxy-8-

prenylflavan and 5,7,3-trihydroxy-5-methoxy-8-prenylflavan were

isolated from Cyperus conglomeratus (44). Insect juvenile hormones (JHs)

are structurally related sesquiterpenoids. In 1998, insect juvenile

hormone (JHiii), methyl 10R-11—epoxy-3, 7, 11- trimethyl 2E, 6E

dodecadienoate and its metabolic precursor in insects, methyl farnesoate

were first reported in the sedges of Cyperus iria L. and Cyperus aromatics

(45).

Cyperus rotundus (Motha) was investigated for antibacterial activity

against Staphylococcus aureus, Escheria coli, Bacillus subtitis etc. (46)

and was effective in all kinds of arthritis (47). It is also a constituent of

New Diarex and Renalka syrup for the treatment of diarrohoea (48) and

urinary tract infection respectively (49). The natural quinone, hydroxyl-

dietrichequinone(3-heptadec-8-enyl-2-hydroxy-5-methoxy-{1,4}

15

benzoquinone), is a secondary metabolite Cyperus javanicus was found

to inhibit mitochondrial respiration and photosynthesis in their electron

transport system (50). Kobusone and isokobusone have been isolated

from Cyperus rotundus (51), while Nyasse et al have isolated α-corymbol,

β-corymbol, mandassidone and mustakone from Cyperus articulates (52,

53).

Among Cyperus species Cyperus rotundus is the most investigated

specie. The essential as well as polar extract resulting in isolation and

characterization of many compounds including scariodione (54),

cyperolone (55), 4α-5α-epoxy-11-eudesmen-3-α-ol (56), α-rotunol and β-

rotunol (57), pachoulenone, cyperenal and patchoulenol (58). A report

on essential oil from the tubers of Cyperus esculentus shows the

presence of α-pinene and α-thujene as the major constituents (59).

Studies have suggested that Chinese prescription, Kagen-karyu could

play a protective role against hypercholestromia through the regulation

of cholesterol levels and inhibition of lipid peroxidation. It comprises of

six crude drugs, out which one is Cyperus rotundus (rhizome) (60).

However the methanolic extract of rhizomes of Cyperus articulatus, a

plant possessing anticonvulsant activity protected mice against maximal

electroshock (MES) and pentyleneterazol (PTZ) - induced seizures. It also

delayed the onset of seizures induced by isonicotinic acid hydrazide and

strongly antagonized N-methyl-D-aspartate induced turning behavior

(61) and also essential oil from the rhizome of Cyperus scarious was

analysed by GC-MS. Among the 31 compounds identified, the major

constituents were alpha-pinene, beta-pinene, caryophyllene oxide,

copaene, longiverbenone, myrtenal, spathulenol and trans-pinocarveol

(62).

16

Some important sesquiterpenes isolated so far from

Cyperus species are:

Cyperene

Mandassidone

Kobusone

CH3

O

CH3 CH2

O

CH3

CH3

H

H

O

CH3

CH3

O

CH3

CH3

CH3CH3

17

Isokobusone

α - Corymbolol

β- Corymbolol

CH2

CH3

CH3

CH3

OH

OH

CH2

CH3CH3

H

O

H

OH

CH2

CH3

CH3

CH3

OH

OH

18

Scariodione

Cyperolone

Patchoulenone

CH3

OCH3

CH3

CH2

OH

O

CH3

O

CH3

CH3

CH3

CH3

CH3

O

CH3

CH3

19

CH3

CH3

CH3

CH3

OH

Patchoulenol

HOH 2C

CH3

CH3

CH3

Cyperenol

α-Rotunol, β- Rotunol

CH2

CH3

CH3

OOH

OH

20

4α-, 5α-Epoxy-11-eudesmen-3-α-ol

Mustakone

CH2

CH3

CH3

CH3O

OH

OH

CH3

OCH3 CH3

CH3

21

Chapter III

Chemical Analysis

Of

Cyperus paniceus

22

3.1 INTRODUCTION

In a bid to investigate the chemical constituents of Cyperus species

we present here the results of our investigation on Cyperus paniceus

collected from Kumaun regions of Uttarakand Himalaya. To the best of

our knowledge no work has been reported so far on the chemical

constituents of mentioned Cyperus species collected from the region.

Taxonomy and distribution:

Cyperus paniceus (Rottb.) Boech., a perennial rhizomatous herb;

rhizome slender, stoloniferous. Stems tufted, up to 60 cm high,

trigonous, usually thickened into nodule at base. Leaves shorter or

longer than the stem, up to 4 mm wide. Umbel simple; rays 3-7, 0-4 cm

long, terminating in dense cylindrical spikes 5-18mm long, bracts 4-6,

up to 20 cm long , leaf-like, spikelets 1-flowered, acute, often recurved.

Glumes 4; the 2 lowest empty, persistent, the third fertile, ovate, striate,

the fourth empty, lancoelate, with a long subulate tip. Stamens 3, achne

2mm long, oblong-ellipsoid, often slightly curved, trigonous, and pale-

brown.

Plant collection and identification:

In the Himalaya, altitudinal limits demarcate the various

vegetation types and floristic boundaries (63) .The rhizomes of Cyperus

paniceus (Rottb.) Boech were collected from Kumaon region of

Uttarakhand, India in August 2000. The Identity of the plant specimen

was confirmed from B.S.I. Northern Circle, Dehradun (Ref No. 9/2003-

04/Tech/575(2). The voucher specimen was deposited at Chemistry

Department, Kumaon University, Almora

Chapter - III

23

3.2 EXPERIMENTAL

3.2.1 General Remarks :

The solvents were used after proper distillation and purification.

Column chromatography was applied with silica gel BDH (60-120 mesh)

and E.Merc (230-400 mesh, ASTM). The TLC was conducted on layers of

silica gel containing 13% gypsum as binder. The visualization of spots

was achieved by UV lamp or by spraying different reagents viz.

(a). Combination of ethyl alcohol, anisaldehyde and conc. H2SO4.

(b). Combination of Vanilin and conc. H2SO4.

Melting Point : These were recorded on Tempo melting point apparatus

IR Spectra : Perkin Elmer - 298

UV Spectra : Hitachi - 220

HPLC : Water HPLC with variable UV-Vis and RI

detectors, column; µ- porasil and nucleosil (300

x 7.8 nm)

GC : Varian Vista 6000 controlled by Varian DS-604

data processor using fused silica Capillary

column (DB-5, 60mx0.25m Id., 0.4 µm coating),

N2 as carrier gas

GC-MS : Thermoquest Trace GC-2000 interfaced with Polaris - Q (Finnigan Mat) Ion Trap mass spectrometer

13CNMR and1HNMR : Bruker DRX-300 (300MHz FT NMR with low

and high temperature facility -90˚ C to 80˚ C

24

3.2.2 Extraction of the oil:

The essential oil has been extracted through steam distillation

method in a copper still fitted with a glass condenser. The rhizomes (4.0

kg) so collected were washed properly, dried in shade and crushed before

subjecting to steam distillation. The condensate was treated with n-

hexane, after shaking the layer with dissolved oil it was dried over

anhydrous Na2SO4. The solvent was removed with a rotary thin-film

evaporator at 35˚ C. The yield was (0.04%) which was then subjected to

different separation techniques.

3.2.3 GC and GC-MS analysis:

The oil sample was analyzed by gas chromatography using Flame

Ionization Detector (GC Fig 3.1). The temperature programming used

was as given below:

i. Injection Temperature : 240˚ C

ii. Detector Temperature : 280˚ C

iii. Initial Oven Temperature : 60˚ C

iv. Programming Rate : 3˚ C/min

v. Final Oven Temperature : 210˚

vi. Total Run Time : 70 min

GC-MS analysis was performed under identical conditions on a

Thermoquest Trace GC-2000 interfaced with Polaris Q (Finnigan Mat)

Ion Trap mass spectrometer, using helium as a carrier gas (flow rate 1.0

ml/min).

25

26

3.2.4 Fractionation of the oil and isolation of major constituent:

The oil was chromatographed over silica gel (60-120 mesh, BDH) in

glass column. The solvent used were n-hexane and mixture of n-hexane-

diethyl ether (5 to 20% ether in n-hexane, 1000ml) and finally washed

with diethyl ether (50 ml). The fractions were examined on silica gel TLC

plates. The fractions which are almost identical were mixed and

subjected to column chromatography with silica gel (230-400 ASTM, E.

Merc) to separate more identical fractions. The fractions were collected

and examined by their gas chromatography under isothermal and

column temperature programmed conditions.

The fractions with similar constituents were mixed further

reducing the number of fractions which were worked up for isolation of

Compound Cb-1 and Cb-2. The flow chart [Scheme 1] for the isolation of

Cb-1 and Cb -2 has been drawn.

27

10% ether(83-96)

Plant Material (Rhizome) 4 Kg

1. Washed, dried & crushed2. Condensate treated with n-hexane3. Layer separated & dried over anhydrous

Na2SO4

Main Oil (5 ml)

1. Subjected to column chromatography(60-120 mesh)

2. Solvent: Hexane & ether

Column I (1-37) fractions collected)


1. Fractions (6-17) were found useful2. Again subjected to column

chromatography

1. TLC was done for column I a& column II2. Similar fractions i.e. 22-23 of column &

19-26 of column II mixed3. Column chromatography (230-400 mesh)

Column III

100% Hexane(1-14)

3% ether(15-40)

5% ether(41-59)

8% ether(60-82)

Conducting TLC (43 to 48) fractions found identical

Conducting TLC (73 to 81) fractions found identical

Cb-1 Cb-2

Scheme I

28

3.2.5 Spectral data of the compounds

Compound Cb-1 : Liquid with odour

IR γmaxfilm (cm -1) : 3048, 2932, 2850, 1713,1600, 1570 and 1490

MS m/z (%) : 284 (M+,23), 269 (75), 227 (10),185 (B.P.),

143(31), 129(12) and 91(5)

1HNMR (CDCl3) δ ppm : Table: 3.1, Fig: 3.2

13CNMR (CDCl3) δ ppm : Table: 3.2, Fig: 3.3


IR γmaxfilm (cm -1) : 3345, 2825, 2927 and 1740

MS m/z (%) : 286(M+, 17), 271 (57) , 253 (B.P.), 211(42), 183

(27), 159 (29) 129 (22) and 117 (12)



29

Table: 3.1. 1HNMR spectral data of compound Cb-1 in δ ppm:

Chemical shift Proton count Probable assignments

0.84 3 H-13

1.10 3 H-11

1.14 6 H-10

1.47 1 H-2 <′>

1.54 1 H-5 <′>

1.57 1 H-2 <″>

1.59 1 H-1 <′>

1.60 1 H-3 <′>

1.64 1 H-5 <″>

1.69 1 H-1 <″>

1.70 1 H-3 <″>

1.83 1 H-12

2.83 1 H-9

2.91 1 H-4 <″>

2.97 1 H-4 <′>

6.78 1 H-6

7.03 1 H-8

7.14 1 H-7

9.66 1 H-14 (-CHO)

30

Table: 3.2. 13CNMR spectra data of compound Cb-1 in δppm:

Chemical shift CHn Probable assignment

18.6 CH3 C-19

19.9 CH2 C-5

22.0 CH3 C-18

23.1 CH2 C-10

23.9 CH3 C-16

30.5 CH2 C-8

32.4 CH2 C-6

33.4 CH C-15

39.8 CH2 C-3

41.2 C C-1

48.6 C C-4

50.4 C C-2

124.5 CH C-14

124.8 CH C-13

127.8 CH C-12

135.6 C C-7

145.5 C C-11

145.6 C C-9

203.6 CH C-20 (-CHO)

31

Table: 3.3. 1HNMR spectral data of compound Cb-2 in δppm:


0.90 3 H-13

1.08 3 H-10

1.14 6 H-15

1.42 1 H-3 <′>

1.49 1 H-4 <′>

1.49 1 H-6 <′>

1.51 1 H-2 <″>

1.52 1 H-3 <″>

1.53 1 H-11

1.59 1 H-4 <″>

1.59 1 H-6 <″>

1.61 1 H-2 <″>

2.68 1 H-1 (-OH)

2.83 1 H-14

2.84 1 H-5 <″>

2.90 1 H-5 <′>

3.50 1 H-12 <′>

3.70 1 H-12 <″>

6.68 1 H-7

6.91 1 H-9

7.03 1 H-8

32

Table: 3.4. 13CNMR spectra data of compound Cb-2 in δppm:


22.4 CH3 C-15

23.7 CH2 C-5

23.9 CH3 C-19

24.2 CH3 C-20

24.2 CH2 C-10

29.7 CH3 C-17

30.8 CH C-18

36.2 CH2 C-6

33.4 C H2 C-8

38.6 C C-4

39.6 C H2 C-3

39.8 C C-1

51.5 CH2 C-2

70.2 CH2 C-16

123.7 CH C-13

124.2 CH C-14 (C-OH)

126.7 CH C-12

135.3 C C-7

144.2 C C-9

145.5 C C-11

33

34

35

36

37

3.3: Results and Discussion:

The oil so collected was subjected to constituent analysis having

yield 0.04 %. The GC of the oil shows more than 40 peaks, 33 among

these were identified The IR indicates the presence of hydroxyl, aldehyde

group in the separated fractions. The hydrocarbons mainly appear in

fraction 1 which was identified on the basis of their mass spectra in GC-

MS analysis (Table 3.5). Almost all the compounds were in the

oxygenated monoterpene/ sequiterpene region of the chromatogram.

Among hydrocarbons cyperene (3.8%), Allo-aromadendrene (2.7%) and

δ-cadinene (1.6%) were found as the major constituents, while, camphor

(4.2%), thymol (3.6%), occidentalol (3.2%), spathulenol (4.4%),

dehydroabietal (24.5%) and dehydroabietol (4.0%) were reported as the

main oxygenated constituents of the oil.

38

Table: 3.5. Essential oil constituents identified on the basis of GC retention data

S.No. Compound R I % in oil 1. p-Cymene 1026 T

2. Limonene 1031 T 3. 1,8-Cineole 1033 2.1 4. Undecane 1099 0.3 5. Camphor 1143 4.2 6. Terpin-4-ol 1177 0.8 7. α - Terpineol 1189 0.1 8. Thymol 1290 3.6 9. α - Copaene 1376 0.6 10. Cyperene 1378 3.8 11. β- Caryophyllene 1418 0.4 12. α – Neo-Clovene 1454 0.1 13. Allo-aromadendrene 1461 2.7 14. Germacerene-D 1480 0.4 15. Epi-cubebol 1493 0.3 16. α- Muurolene 1499 T 17. Cuparene 1502 0.4 18. Cubebol 1514 T 1.2 19. δ - Cadinene 1524 1.6 20. α- Colacorene 1542 0.3 21. Occidentalol 1548 3.2 22. Spathulenol 1576 4.4 23. β- Copaen-4α -ol 1584 0.7 24. 1-epi-cubenol 1627 0.5 25. Epi-α- cadinol 1627 1.4 26. α-muurolol 1645 0.4 27. α-eudesmol 1652 0.3 28. Epi-laurenene 1891 0.1 29. Cembrene 1942 0.2 30. Bifloratriene 1974 1.7 31. Seselin 1992 0.5 32. Dehydro-abietal 2263 24.5 33. Dehydroabietol 2359 4.0

39

(i) Characterization of Cb- 1

It was obtained from polar fraction of the oil. The molecular formula of

compound was established as C20H280 by its mass spectrum, showing

M+ at m/z 284 (23). The other fragment ion peaks were recorded at m/z

269 (75), 227 (10), 185(B.P.), 145 (31), 129 (12), and 91 (5).

The IR spectrum of the compound showed the presence of a –CHO

group as it have shown a strong band at 1713 cm-1 due to >C=O

stretching in combination with a doublet at 2850-2715 cm-1, due to

aldehydic C-H stretching. The peak at 3080 cm-1 (aromatic C-H

stretching) and strong peaks below 900 cm-1 indicate that it has an

aromatic ring in the structure. However, the band at 2932 and 2850 cm-1

also point the presence of aliphatic C-H (CH3, CH2 and CH) stretch.

These absorption coupled with the presence of C stretching

absorption at 1610, 1495 and 1430 cm-1 confirm the presence of

aromatic ring in its structure.

The structural feature of compound was established primarily from

its 1HNMR spectral data (Table 3.1). The presence of an aldehydic group

was further confirmed by the presence of a highly deshielded proton at δ

9.66 ppm, while aromatic protons were recorded at δ 6.78, 7.03 and 7.14

ppm. Presence of an isopropyl moiety was shown by its absorption at δ

1.14 (6H, d) and 2.83 (1H, m) ppm, while the angular methyl was

recorded at δ 1.10 and the forth methyl at cyclohexane ring at 0.84 ppm.

The structure was further supported by its 13CNMR spectral data

(Table 3.2), as it showed a downfield shift at δ 203.6 ppm, confirm the

presence of a carbonyl (- CHO) carbon in its structure. The aromatic

carbons were reported at δ 124.5, 124.8, 127.8, 135.6, 145.5 and 145.6

ppm. The presence of an isopropyl group was confirmed by its absorption

at δ 23.9 ppm due to two gem dimethyls, while an angular methyl was

shown by its absorption at δ 22.0 ppm.

40

All these observations obtained from 13CNMR, 1HNMR, MS and IR

analysis proposed to establish the Structure (1) for Dehydroabietal.

Dehydroabietal

CH3CHO

CH3

CH3

CH3

H

41

(ii) Characterization of compound Cb- 2

The molecular formula of the compound was established as

C20H300 by mass spectroscopy showing M+ at m/z (%) 286 (17). The other

important fragment ion peaks were recorded at 271 (57), 253 (B.P.), 211

(42), 183 (27), 159 (29), 129 (22), and 117 (12).

The IR spectrum revealed the presence of an alcoholic (-OH) group

as it shows absorption at 3345 cm-1, the aromatic C-H stretching was

recorded at 3080, 1405, 1602 and 1610 cm-1, while, the aliphatic C-H

stretching (CH3 ,CH2 and CH) was reported at 2825 and 2927 cm-1.

The main structural feature of the compound is evident from the 1HNMR spectral data (Table 3.3). The deshielded protons at δ 7.03 (1H,

s), 6.91 (1H, s) and 6.68 (1H, s) ppm indicates the presence of an

alcoholic group was confirmed by its absorption at δ 2.68 due to –OH

protons. Since it is a primary alcohol the shift between δ 3.50 -3.70 ppm

indicate the presence of two protons on the carbons attach to –OH group

(- CH2-OH). The presence of an isopropyl moiety was shown by its

absorption at δ 1.14 (6H d) due to the presence of two gem

dimethyl on a tertiary carbon ( ) observed at δ 2.83 (1H, m)

ppm. The presence of one angular methyl among two

other methyl groups were observed by its absorption at δ 1.08 ppm,

while, the forth methyl was reported at δ 0.90 ppm.


(Table 3.4). The presence of (-OH) hydroxyl group at C-20 carbon of

cyclohexane moiety was shown by its absorption a shift at δ 70.2 ppm.

Geminal methyls of isopropyl moiety were observed at δ 23.9 ppm. The

presence of angular –CH3 group at C-15 was observed at δ 22.4 ppm,

while the presence of aromatic ring was confirmed by downfield shifts of

aromatic carbons between δ 123.7 – 145.5 ppm.

42

All these observations proposed to establish the structure Cb-2 for

abietol.

Dehydroabietol

CH3

CH3 OH

CH3

CH3

H

43

3.4.1 Solvent Extraction and Isolation:

The powdered, dried roots of the plants were successively extracted

with hexane, CHCl3 and MeOH in soxhlet extractor. The Chloroform

fraction was found to be useful which was then collected for further

investigation.

The chloroform extract was subjected to column chromatography

(silica gel-60-120 mesh, BDH). The column was eluted using a stepwise

gradient of EtOAc 0-100% in hexane. A total of eighty, 50 ml fraction

were collected. Fractions of similar composition as determined by TLC

were pooled together out of which two fractions were almost pure and

separated as Cb-3 and Cb-4.

The two compounds so collected were subjected to constituent

analysis. The whole process can be explained through Scheme 2.

44

Conducting TLC (41-45) fractionsfound identical

Plant Material (2Kg)

1. Washed, dried & Crushed2. Extracted with hexane, MeOH, CHCL3

n – hexane soluble

MeOHsoluble

CHCL3 soluble

Scheme II

1. Found Useful2. Column chromatography 3. Eluted using stepwise gradient of

EtOAc– 0-100% in hexane4. 80 fractions collected and TLC conducted

2% EtOAc

Following fraction found identical and pooled together

Cb-3 Cb-4

TLC was conducted (14-18) fractions found identical

5% EtOAc

45

3.4.2 Spectral data of compounds

Compound Cb-3 : Yellow crystals

IR γmaxfilm (cm -1) : 3400, 3080, 2927,2854,1733,1635, 1230.

MS m/z (%) : 248 (M+, 72.4), 233 [100), 215(22.0), 205(9.0),

191(13), 177(9), 105(3).


13CNMR(CDCl3) δ ppm : Table: 3.7, Fig: 3.7


IR γmaxfilm (cm -1) : 3112, 2848, 1706, 1294, 1029

MS m/z (%) : 242[M+, 100), 213 (16), 197(34), 185(24),

167(21), 157(19), 149(46), 57(74).



46



1.64 3 H-7

2.48 3 H-5

3.11 1 H-3 <′>

3.21 1 H-3 <″>

3.87 3 H-6

4.89 1 H-4

5.04 1 H-8

5.21 1 H-9

6.00 2 H-2

13.38 1 H-1 (-OH)

47

Table: 3.7. 13CNMR spectral data of compound Cb-3 in δppm:


17.0 CH3 C-14

31.0 CH2 C-7

32.3 CH3 C-10

55.5 CH3 C-11

85.8 CH C-4

88.0 CH C-8

103.0 C C-1

106.5 C C-5

112.5 CH2 C-13

144.7 C C-12

159.5 C C-6

160.3 C C-3 (-C-OH)

167.7 C C-2

202.3 C C-9 (C=O)

48



1.88 3 H-4

2.35 3 H-3

4.89 1 H-5

5.12 1 H-6

6.71 1 H-1

7.55 1 H-2

49

Table: 3.9. 13CNMR spectral data of compound Cb-4 in δppm:


9.0 CH3 C-11

22.2 CH3 C-14

102.8 CH C-7

112.5 CH2 C-13

123.0 C C-1

125.7 C C-9

127.7 C C-5

134.7 CH C-10

142.4 C C-12

154.0 C C-6

154.1 C C-8

154.7 C C-2

162.6 C C-4

174.9 C C-3

50

51

52

53

54

3.5 Results and Discussion

The chloroform extract from the tubers of Cyperus paniceus afforded

large amount of the carmine pigments, Cyperaquinone (Cb- 4) and its

precursor Remirol (Cb- 3).

(i) Characterization of Cb- 3

Compound Cb-3 was obtained as yellow crystalline compound. The

molecular formula of the compound was established by mass

spectroscopy as C14H16O4 showing M+ at m/z (%) 248 (72). The fragment

ion peaks were separated at m/z 233 (100), 215 (22), 205 (9), 191 (13),

177 (9) and 105 (3).

The IR spectrum revealed the presence of a hydroxyl (-OH) function

as it showed absorption at 3400 cm-1. The aromatic C-H stretching was

shown by its absorption at 3080 cm-1 and C stretching at 1615, 1498

and 1435 cm-1. However, the band at 2854 and 2927 cm-1 also point out

the presence of an aliphatic C-H stretching. A strong carbonyl (>C=O)

band was recorded at 1733 cm-1 along with vinylic (>C=C<) stretching at

1635 cm-1. Absorption at 1230 cm-1 showed the presence of an ether

linkage in its structure.

The structural feature of Compound Cb-3 was characterized by its 1HNMR spectral data (Table 3.6), which showed the presence of a highly

deshielded phenolic proton at δ13.38 ppm (1H, ѕ), with a single aromatic

proton at δ6.0 ppm (1H, ѕ). The presence of a methoxy function (–O-CH3)

was confirmed by the absorption of CH3 protons at δ 3.87 ppm, while the

presence of acetyl group (-CO-CH3) was shown by absorption of –CH3

protons at δ 2.48 ppm. Two methylene protons of isopropenyl moiety

were reported at δ 5.04 (1H, s) and 5. 21(1H, dd) ppm.

The structure Cb-3 was further supported by 13CNMR spectral

observation (Table 3.7). The downfield shift at 202.3 ppm shows the

55

presence of a >C= O group, while at 160.3 ppm showed a hydroxyl

substitution on C-3. Presence of a furan moiety in its structure was

confirmed by its chemical shift at δ 167.7 (C-2) and 103.0 (C-1) ppm. The

presence of vinylic carbons have shown by the chemical shift at δ 144.7

(C-12) and 112. 5 (C-13) ppm, while methyl carbon of methoxy (–O-CH3)

group was observed at δ 55.5 ppm.

The above analysis of the compound from 1HNMR, 13CNMR, MS

and IR spectral data confirm to establish the structure of Cb- 3 as

Remirol (C14H1604).

Remirol

O

C H 3

OC H 3

OOH

C H 2

C H 3

56

(ii) Characterisation of Cb 4

It is the major component of the chloroform extract of plant

rhizomes. The molecular formula C14H10O4 was established from its mass

spectrum M+; m/z (%) 242 (100), the other fragment ion peaks were at

213 (16), 197 (34), 185 (24), 167 (21), 151 (19), 149 (46), and 57 (74).

The IR spectrum of the compound showed the presence of an

aromatic C-H stretching at 3112 cm-1 and C stretch at 1435, 1500

and 1615 cm-1. Presence of carbonyl linkage was confirmed by its

absorption at 1706 cm-1. The vinylic (>C=C< ) stretching was observed at

1630 cm-1, while aliphatic C-H stretching was observed at 2928 and

2825 cm-1.

The structure was characterized by its 1HNMR spectral data (Table:

3.8), which shows two deshielded aromatic protons of furan ring at δ

7.55 (1H,d) and 6.71 ppm (1H,s) on C-10 and C-7 respectively,

suggesting the presence of two heterocyclic rings in the compound. The

downfield shifts of two methylene protons at δ 4.89 and 5.12 ppm and a

upfield shift of –CH3 protons at δ 1.88 ppm indicates the presence of a

side chain isopropenyl function in its structure. The downfield shift of

another methyl group at δ 2.35 ppm (3H, d) suggested a side chain on

aromatic furan ring.

The structure was further supported by 13CNMR spectral data

(Table 3.9). The downfield shifts at δ 174.9 and 162.6 ppm were due to

the presence of two (>C=O) carbonyl linkage at C-3 and C-4 respectively,

indicates the presence of a benzoquinone nucleus in the compound. The

methylene carbons of isopropyl moiety were recorded at δ 142.4 and

112.5 ppm, while, the aromatic carbons were recorded at δ102.82,

123.06, 125.73, 127.78, 134.72, 154.09, 154.12 and 154.70 ppm, also

confirmed the presence of a benzoquinone and furanoid ring in its

structure

57

Cyperaquinone

O O

O

OC H 3

C H 2

C H 3

58

Chapter IV

Chemical Analysis

Of

Cyperus niveus

59

4.1 INTRODUCTION

In continuation of our work with Cyperus species, we present here

our investigation on chemical constituents of a new Cyperus species from

Himalayan region of Uttarakhand, the Cyperus niveus. Literature repots

have shown that the concern species so far was not explored for its

chemical constituents.


Perennial, culm bases swollen and fused into a horizontal rhizome,

roots slender, culms crowded and often growing in a straight line;

inflorescence a solitary usually globose head of 5-50 spikelets, white

when young turning pale reddish brown; in dry grassland.

The species is not very much chemically analyzed, but its

distribution has been a matter of studies mainly in Garhwal Himalayan.

Competition has been an important evolutionary force that has led to

niche separation, specialization and diversification (64).

To know the distribution of plant, the study site was divided into

three sites A, B and C and competition between the different sites were

studied. At site C, Capillipedium perviflorum vs Cyperus niveus exhibited

maximum niche overlap but at this site competition as well as stable

equilibrium was found between dominating and co-dominating specie.

Niche overlap may also vary with species, densities and inter or

interspecific competition (65).

Chapter - IV

60

Plant collection and identification:

The rhizomes of Cyperus niveus Retz. were collected from Kumaon

region of Uttaranchal, India in the month of July. The identity of the

plant specimen was confirmed from B.S.I. Northern Circle, Dehradun

(Ref No. 9/2003-04/Tech./374). The voucher specimen was deposited at

Chemistry Department, Kumaon University, Almora.

4.2 EXPERIMENTAL

4.2.1 General Remarks: The solvents were used after proper purification and distillation.

TLC was conducted on silica gel G and visualization of spots was

achieved by spraying different reagents viz.

(i) Combination of ethyl alcohol, anisaldehyde and conc.H2SO4

(ii) Combination of Vanilin & conc. H2SO4

The IR Spectra, UV spectra, HPLC, GC, GC-MS, 1HNMR and 13CNMR were recorded as discussed in the previous chapter.

4.2.2 Extraction of the oil:

The extraction of oil was carried out as discussed in chapter III. The

rhizomes so collected were subjected to steam distillation and the

condensate is treated with n-hexane, the n-hexane layer with dissolved

oil was separated through separating funnel and the separated layer was

dried over anhydrous Na2SO4. The solvent was removed with a rotatory

thin-film evaporator at 35˚C. The yield was (0.02%).

4.2.3 Gas chromatographic analysis:

The oil sample was analyzed by gas chromatography using Flame

Ionization Detector and DB Wax columns and OV-101 (GC Fig. 4.1). The

temperature programming used was as given below:

61

i. Injection Temperature : 240˚C

ii. Detector Temperature : 280˚C

iii. Initial Oven Temperature : 60˚C

iv. Programming Rate : 3˚C /min

v. Final Oven Temperature : 210˚

vi. Total Run Time : 70 min

vii. Column (fused silica) : DB wax (60 m x 0.25 mm)

OV-101(30 m x 0.25 mm)

The GC-MS analysis was performed under same condition as

mentioned in Chapter III.

62

63

4.2.4 Fractionation of the oil and isolation of major constituents:

The oil was chromatographed over silica gel (60-120 mesh, BDH) in

a glass column. The solvent used were n-hexane and mixture of n-

hexane – ether (5 to 20% ether in n-hexane 100 ml) and finally washed

with ethyl acetate, 50 ml.

The fractions were examined on silica gel TLC plates. The fractions

which are almost identical were pooled in and subjected to column

chromatography with silica gel (230-400 mesh, ASTM) to separate more

identical fractions. The fractions so collected were subjected to gas

chromatographic analysis under isothermal and column temperature

programme conditions.

The fractions with similar constituents were mixed and send for

spectral analysis. The process can be explained through Scheme III.

64

Scheme IIIPlant Material (Rhizome) 4

Kg

1. Washed, dried & Crushed2. Condensate treated with n-hexane3. Layer separated & dried over anhydrous Na2SO4


5% ether 6-13 found useful

3% ether(2-13)

Conducting TLC found identical

Cb-5

Main Oil

Column II (1-43) fractions collected)

1. TLC was conducted for column I & II

2. Fractions (45-55) & (56 to 61) of column I were mixed

3. Column chromatography (230-400 mesh)

8% ether(27-51)

5% ether(14-26)

Cb-6

5% ether 26-31 found useful

65

4.2.5. Spectral data of the compounds

Compound Cb-5 : Colourless liquid

IR γmaxfilm (cm -1): : 3445, 2920, 2860, 1630 and 1380

MS m/z (%) : 220 [M+], 205(36), 202(29), 187(31), 177(13),

159(24), 145(33), 138(77), 131(52), 124(45),

105(60), 91(61), 67(47), 55(57) and 41(B.P.)

1HNMR (CDCl3) δ ppm : Table: 4.1

13CNMR (CDCl3) δ ppm : Table: 4.2


IR γmaxfilm (cm -1) : 2927, 2825, 2715, 1724 and 1635

MS m/z (%) : 218[M+, B.P.], 203(76), 185(25), 175(40),

161(17), 147(32), 133(40), 119(35), 105(48),

91(56), 79(39), 67(34), 55(26) and 41(22).

1HNMR (CDCl3) δ ppm : Table: 4.3

13CNMR (CDCl3) δ ppm : Table: 4.4

66

Table: 4.1. 1HNMR Spectral data of Compound Cb-5 in δ ppm:


1.13 3 H-10

1.41 1 H-5<″>

1.67 1 H-3<′>

1.69 3 H-11

1.77 1 H-3<″>

1.80 1 H-5<′>

1.87 3 H-9

1.89 1 H-7<″>

1.89 1 H-2<″>

1.91 1 H-6

1.92 1 H-7<′>

1.92 1 H-2<′>

2.24 1 H-8<′>

2.34 1 H-8<″>

2.81 1 H-1

4.00 1 H-4

4.68 1 H-13

4.83 1 H-12

67

Table: 4.2. 13CNMR Spectral data of Compound Cb- 5 in δ ppm:


14.9 CH3 C-11

20.8 CH3 C-15

23.2 CH3 C-12

23.4 CH2 C-10

27.7 CH2 C-5

29.2 CH2 C-7

35.1 C C-1

36.2 CH2 C-3

40.5 CH2 C-9

43.2 CH C-8

72.0 CH C-6

108.6 CH2 C-14

134.4 C C-4

135.8 C C-2

149.4 C C-13

68

Table: 4.3. 1HNMR Spectral data of Compound Cb-6 in δ ppm:


0.77 3 H-1

0.8 6 H-9

1.04 1 H-3<'>

1.14 1 H-3<">

1.52 1 H-5

1.63 1 H-4<">

1.63 1 H-4<'>

2.02 1 H-7<'>

2.07 1 H-2

2.12 1 H-7<">

2.39 1 H-6<'>

2.49 1 H-6<">

2.75 1 H-8<'>

2.85 1 H-8<">

10.07 1 H-10

69

Table: 4.4. 13CNMR Spectral data of Compound Cb- 6 in δ ppm:


18.1 CH3 C-1

22.7 CH3 C-13

22.7 CH3 C-14

26.0 CH2 C-9

28.6 CH2 C-5

29.1 CH2 C-10

29.9 CH2 C-4

34.4 CH2 C-7

38.4 CH C-3

43.8 C C-12

44.1 CH C-6

60.7 C C-2

135.8 C C-11

172.6 C C-8

187.6 CH C-15

70


The oil yield from the fresh plant material (rhizomes) was

determined as 0.02 % the GC of the oil showed more than 40 peaks.

Most of the major constituents were reported in oxygenated region of

the chromatogram (Fig. 4.1).

Column chromatography and GC determination revealed only 7-8

% hydrocarbon and more than 90.0 % were oxygenated compounds.

Identified components accounts for about 87.0 % of the oil (Table: 4.5).

The hydrocarbons mainly separated by hexane in fractions 1-4. Two

oxygenated compounds of the oil were separated as Cb-5 an alcohol

and Cb-6 an aldehyde from the oxygenated fractions.

71

Table: 4.5. Essential oil constituents of Cyperus niveus identified on the basis of GC retention data

S. No. Compound RI % in oil 1. Limonene 1031 0.1 2. 1,8 Cineole 1036 0.1 3. Menthofuron 1164 0.2 4. Terpinene- 4-ol 1177 0.4 5. α-Terpineol 1189 0.6 6. α-Copaene 1376 0.12 7. β-Patchoulene 1380 0.12 8. β-Cubebene 1390 0.4 9. Cyperene 1398 2.6 10. β-Caryophyllene 1418 0.3 11. Aromadendrene 1439 0.4 12. γ-Patchoulene 1441 0.1 13. α-Himachalene 1447 0.2 14. α-Patchoulene 1456 1.1 15. β-Chamigrene 1475 1.0 16. Germacrene-D 1483 1.2 17. Epi-Cubibol 1493 0.8 18. t-β-Guainene 1500 0.7 19. Cubebol 1514 0.6 20. δ-Cadinene 1524 1.0 21. Acor-4-ene(6,11-oxido) 1531 1.6 22. α-Colacorene 1542 0.4 23. β-Copaen-4α-ol 1584 0.2 24. Viridiflorol 1590 3.2 25. Cubenol 1614 2.1 26. Cedranone 1618 1.8 27. Cyperol 1626 5.8 28. Cyperenal 1660 33.6 29. Occidentalol acetate 1678 16.2 30. Ambroxide 1756 1.2 31. 14-oxy- α-muurolene 1764 2.3 32. Occidol 1832 2.4 33. Cis-n nuciferol acetate 1835 2.0 34. Occidol acetate 1970 2.4

72

(i) Characterization of Cb- 5:

The spectral data of compound has same pattern as of isocyperol

which has been isolated from Cyperus rotundus (66). The molecular

formula of the compound is assigned as C15H240, showing M+ at m/z

(%) 220 (55). The other fragment ion-peaks were separated at m/z (%)

205 (36), 202 (29), 187 (31), 177 (13), 159 (24), 145 (33), 138 (77),

131(52), 124 (45), 105 (60), 91 (61), 67 (47), 55 (57) and 41 (100).

The IR spectrum revealed the presence of a hydroxyl (OH) group as

showing absorption at 3445 cm-1. Aliphatic C-H stretching (CH3, CH2

and CH) was observed at 2920 and 2860 cm-1. An olefinic >C=C<

stretching was observed at 1630 cm-1.

The main structural feature of the compound was evident from its 1HNMR spectral data (Table 4.1), concluded that it is a secondary

alocohol, since the proton vicinal to the hydroxy group was reported at δ

4.00 ppm. The presence of an isopropenyl group is indicated by the

signal of two vinylic protons at δ4.67 ppm, showing coupling

correlations with an olefinic Me-group (δ 1.69 ppm). Further the protons

of angular methyl were observed as singlet at δ 1.13 ppm, while olefinic

methyl group at cyclohexene ring was observed at δ 1.77 ppm.


(Table 4.2). The methylene carbons of isopropyl moiety were recorded at

δ 108.6 and 149.4 ppm with a -CH3 carbon at δ 20.8 ppm indicate the

presence of a carbon (C-6) bearing a –OH group. However, the presence

of an endocyclic double bond (=) in its structure was confirmed by its

absorption at δ 134.4 and 135.8 ppm.

73

The above characterization supports the proposed structure (Cb-5) for

Cyperol.

Cyperol

O H

C H 3

C H 3

C H 2

C H 3

74

(ii)Characterization of Cb- 6:

The investigation of the essential oil has enabled the identification

of the sesquiterpenoid which has been reported earlier (67). The

compound was assigned the molecular formula C15H22O showing M+ at

m/z (%) 218(100). The other fragment peaks were observed at m/z (%)

203 (76), 185 (25), 175 (40), 161 (17), 147 (32), 133 (40), 119 (35), 105

(48), 91 (56), 79 (39), 67 (34), 55 (26) and 41 (22).

An observation of its IR spectrum revealed the presence of an

aldehyde function (-CHO), as it showed a normal carbonyl (>C=O) stretch

at 1724 cm-1 with a Fermi doublet between 2825 and 2715 cm-1 due to

aldehyde C-H stretch in its structure. The aliphatic C-H stretch was

shown by its absorption at 2927 and 2854 cm-1, while a vinylic >C=C<

stretch was recorded at 1635 cm-1.

The main structural feature of the compound was evident from its 1HNMR spectral data (Table 4.3). The compound has three methyl groups

occurring at δ 0.80 and 0.77 ppm. The absence of fourth methyl and

presence of an aldehydic proton showing a downfield shift at δ 10.0 ppm

confirmed the identity of Cyperenal. The structure was further supported

by its 13CNMR spectral data (Table 4.4), in which aldehydic carbon was

reported at δ 187.6 ppm. The endocyclic olefinic carbons were reported

at δ 135.8 and 172.6 ppm, while two gem-dimethyl on C-12 were at δ

22.7 ppm and a third methyl on C-1 was recorded at δ 18.1 ppm.

75

The above spectral values support the structure (Cb- 6) and molecular

formula of the mentioned compound.

Cyperenal

CH3CH3

CH3

H

O

76

4.4.1: Solvent Extraction and Isolation

The powdered, dried roots of the plants were extracted with hexane,

CHCL3 and MeOH in soxhlet extractor.

The chloroform fraction was found to be useful, which was then

collected for further investigation.

The extract was subjected to Column Chromatography (60-120

mesh, BDH). The Column was eluted using a step-wise gradient

Ethylacetate (0-100%) in hexane. TLC was conducted; similar fractions

were pooled together out of which only one pure fraction was

differentiated as Cb-7. The compound so separated was sent for

constituent analysis. The whole process can be explained through

Scheme IV.

77


1. Washed, dried & Crushed2. Extracted with hexane, , CHCL3, MeOH


MeOHsoluble

CHCL3 soluble

Scheme IV

10% EtOAc

Cb-7

TLC was conducted(73-77) fractions Mixed together

1. Found useful2. Eluted used stepwise gradient of ELOAc

0-100% in n-heaxane3. 80 fractions were collected and TLC

conducted4. Following fractions found identical &

Pooled together

78

4.4.2 Spectral data of the compound


IR γmaxfilm (cm -1) : 3078, 2920, 2860, 1685, 1615, 1570, 1500

MS m/z (%) : 174(M+, 80), 159 (B.P), 146(26), 132(52),

118(37), 91(20) and 77(80).

1HNMR(CDCl3)δ ppm : Table: 4.6, Figure: 4.2

13CNMR(CDCl3) δ ppm : Table: 4.7, Figure: 4.3

79

Table: 4.6.1 HNMR Spectral data of Compound Cb-7 in δ ppm:


1.35 3 H-7

1.84 1 H-4 <">

2.18 1 H-4 <">

2.34 3 H-8

2.54 1 H-5<">

2.73 1 H-5<">

3.03 1 H-6

7.08 1 H-1

7.10 1 H-3

7.59 1 H-2

80

Table: 4.7.13 CNMR spectral data of compound Cb-7 in δ ppm:


20.3 CH3 C-12

20.6 CH3 C-11

30.6 CH2 C-6

32.8 CH C-4

36.3 C H2 C-8

124.9 CH C-9

125.8 CH C-5

127.1 CH C-3

135.1 C C-7

136.0 C C-2

144.8 C C-10

199.4 C C-1 (>C=O)

81

82

83

4.5 Results and Discussion

(i) Characterization of Cb- 7:

It is an anti-malarial which is previously reported in Cyperus

rotundus and Lavender oil (40). The molecular formula C12H14O was

supported by its mass spectral data showing M+ at m/z (%)174(80) and

BP at 159 (100).The other important fragment ion peaks were reported at

m/z (%)146 (26), 132 (52), 118 (37), 91(20) and 77(50).

The IR spectra revealed the presence of a carbonyl (>C=O) linkage

at 1685 cm-1. The aliphatic C-H stretching (CH3, CH2 and CH) was

observed at 2920 cm-1 and 2860 cm-1, while, the presence of an

aromatic nucleus was indicated by the absorption at 3078, 1615, 1570

and 1500 cm-1 showed aromatic C-H and C stretch.

The characterization of Compound Cb-7 was explained by its 1HNMR spectral data (Table 4.6), which indicates the presence of

aromatic protons as it is absorbed at δ7.08 (1H , s), 7.10 (1H, s) and

7.59 (1H, s) ppm which is also supported by its 13CNMR absorption

(Table 4.7) at δ 124.9, 125.8, 127.1, 135.1, 136.0 and 144.8 ppm. A

doublet was recorded at δ 1.35 ppm was due to a –CH3 on a

cyclohexanone ring at C-4, while a deshielded -CH3 at δ 2.34 ppm

present as a side chain moiety on aromatic nucleus at C-7.

The presence of >C=O linkage was further confirmed by its 13CNMR

spectral data as it showed a downfield shift at δ 199.4 ppm, while -CH2

carbons of C-8 and C-6 were reported at δ 36.3 and 30.6 ppm

respectively. Two methyl carbons at C-4 and C-7 were recorded at δ 20.6

and 20.3 ppm respectively.

All these spectral data confirm the structure 5 for compound 4, 7-

Dimethyl- 1- tetralone having molecular formulae C12H14 O.

84

4, 7-Dimethyl -1tetralone

CH3

CH3

O

85

Chapter V

Chemical Analysis

Of

Cyperus brevifolius

86

5.1 INTRODUCTION

In a bid to reinvestigate the essential oil constituents of Himalayan

Cyperus species we present here the results of our investigation on

Cyperus brevifolius (Rottb) H. To the best of our knowledge no work has

been reported on the essential oil of Cyperus brevifolius (Rottb.)H.


Cyperus brevifolius (Rottb.) H. (syn. Kyllinga brevifolius) is a

slender perennial herb from a short horizontal rhizome. Culms crowded,

5-30 cm long and 0.3-0.4 mm thick (but wider across the leaf-sheaths),

triangular, glabrous. Leaves from the lower 8cm only, 3-4 per culm, only

2-3 perfecting leaf-blades; lower sheaths pale reddish brown, upper

greenish, all glabrous. Inflorescence a single terminal globose whitish

congested anthela about 3mm in diameter. Invocular bracts usually 3,

foliaceaous, erect or spreading; the largest 2-6 cm long and 1.0-1.4mm

wide. Largest glume 1.5-1.7mm long, transparent (as young) with a green

slightly excurrent smooth midrib; the whole glume turning reddish

brown when fruiting.

Although the genus Kyllinga Rottb. was incorporated in Cyperus

more than 100 years ago it was until 100 years later that it was proven

beyond doubt that this is actually correct. It is therefore a great shame

that we now in the twentieth century are stuck with a generic reference

book where Kyllinga and many other genera actually belonging to

Cyperus are recognized as separate genera.

Chapter - V

87

Not much work has been recorded on the species. A small account

of Indigenous practice of treating human liver disorders in Assam of

Kyllinga brevifolia is found to be unique in the present study (68).

Plant collection and Identification:

The rhizomes of Cyperus brevifolius were collected from Kumaon

region of Uttarakhand (2000m) India in August. The identity of plant

specimen was confirmed from B.S.I., Northern Circle Dehradun (Ref.

BSI/Tech/545). The voucher specimen was deposited at Chemistry

Department, Kumaon University, Almora.

5.2 EXPERIMENTAL 5.2.1 GC and GC/MS analysis:

The GC analysis was performed on a Varian Vista-6000 GC

controlled by a Varian DS-604 data processor using fused silica Capillary

column (DB-5, 60m x 0.25 Id., 0.4µm coating), at a temperature

programming 60˚ C→220˚ C at the rate of 3˚ C /min., with injector and

detector temperature at 210˚ C and 230˚ C respectively and nitrogen as

carrier gas (flow rate 1.0 ml/min., at a pressure 4.0 kg/cm2). GC-MS

analysis was performed under identical conditions on a Thermoquest

Trace GC-2000 interfaced with Polaris-Q (Finnigan Mat) Ion Trap mass

spectrometer, using helium as a carrier gas (flow rate 1.0 ml/min). The

components were identified by comparison of mass spectral data with

those of literature and by retention indices (Table 5.1).

The GC and GC-MS (Fig.5.1) determination revealed about 18%

hydrocarbons and more than 80% oxygenated compounds. Among

hydrocarbons β-pinene (1.1%), β-cubebene (1.1%), cyperene (3.9%), β-

caryophyllene (11.6%) and germacrene-D (3.3%) while, among

oxygenated 1, 8-cineole (2.8%), linalool (5.3%), terpin-4-ol (4.9%), α-terpineol (4.7%) and α-eudesmol (8.8%) were reported as the major

constituents of the oil.

88

Table: 5.1. Essential oil constituents of Cyperus brevifolius identified on the basis of GC retention data

S.No. Compound RI % in oil 1. α-Fenchene 951 0.3 2. β-Pinene 981 1.1 3. Myrcene 995 0.5 4. 1,8-Cineole 1036 2.8 5. β-Ocimene 1050 0.4 6. Linalool 1098 5.3 7. cis-Tnujone 1102 0.2 8. Terpi-4-ol 1177 4.9 9. α-Terpineol 1189 4.7 10. Myrtenol 1194 2.9 11. Thymol methyl ether 1235 1.6 12. Carvacrol methyl ether 1244 2.4 13. Thymoquinone 1249 1.2 14. Isomenthyl acetate 1306 1.8 15. β-Cubenene 1390 1.1 16. Cyperene 1398 3.9 17. β-Caryophyllene 1418 11.6 18. 2,5-Dimethoxy-para-cymene 1423 0.5 19. Aromadendrene 1439 0.4 20. Z-β-Farnesene 1443 5.1 21. α -Humulene 1454 0.3 22. β-Chamigrene 1475 1.6 23. Germacrene D 1483 3.3 24. Valencene 1491 1.7 25. trans-β-Guaiene 1500 0.2 26. Germacrene A 1503 0.1 27. Epi-α-salinene 1517 1.4 28. trans-Calamene 1521 0.2 29. Spathulenol 1576 0.8 30. Caryophyllene Oxide 1581 2.9 31. Epi-α-Cadinol 1640 3.9 32. Cubenol 1642 0.2 33. α -Eudesmol 1652 8.8 34. Cyperenal 1662 0.3

89

90

5.2.2 Solvent extraction and isolation:

The powdered, dried roots of the plants were successively extracted

with hexane, CHCl3 and MeOH in soxhlet extractor. The Chloroform

fraction was found to be useful which was then collected for further

investigation.

The chloroform extract was subjected to column chromatography

(silica gel-60-120 mesh, BDH). The column was eluted using a stepwise

gradient of EtOAc 0-100% in hexane. A total of eighty, 50 ml fraction

were collected. Fractions of similar composition as determined by TLC

were pooled together out of which two fractions were almost pure and

separated as Cb-8 and Cb-9.

The two compounds so collected were subjected to constituent

analysis. The whole process can be explained through Scheme 5.

91

Conducting TLC (48-54) found fractionsidentical


1. Washed, dried & Crushed2. Extracted with hexane, MeOH, CHCL3


MeOHsoluble

CHCL3 soluble

Scheme V

1. Found Useful2. Column chromatography 3. Eluted using stepwise gradient of

EtOAc – 0-100% in hexane4. 90 fractions collected and TLC conducted

2% EtOAc

Following fractions found identical and pooled together

Cb-8 Cb-9

TLC was conducted (20-28) fractionsfound identical

5% EtOAc

92

5.2.3 Spectral data of the compounds:


IR γmaxfilm (cm -1): : 3072, 2980, 2870, 1765, 1620, 1460 and 1495

MS m/z (%) : 328(M+, 3), 285 (B.P.), 242(62), 227(12), 215(8),

169(5) and 77(4)




IR γmaxfilm (cm -1): : 3350, 3080, 2975, 2868, 1605, 1210 cm-1

MS m/z (%) : 286(M+, 35), 271 (B.P.), 253(12), 187(78), 145(30),

117(14) and 91(9)



93

Table: 5.2.1HNMR spectral data of compound Cb-8 in δ ppm:

Chemical shift Proton count Probable assignment

cis trans cis trans cis trans

1.06 1.06 3 3 H-11 H-11

1.14 1.14 6 6 H-13 H-13

1.21 1.21 6 6 H-10 H-10

1.41 1.41 1 1 H- 3 <″> H-3 <'>

1.48 1.48 1 1 H-1 <″> H-1 <'>

1.50 1.50 1 1 H-2 <'> H-2 <'>

1.51 1.51 1 1 H-3<″> H-3 <″>

1.56 1.56 1 1 H-5 <'> H-5 <'>

1.58 1.58 1 1 H-1 <″> H-1<″>

1.60 1.60 1 1 H-2 <'> H-2 <'>

1.61 1.61 1 1 H-12 H-12

1.66 1.66 1 1 H-5 <″> H-5 <″>

2.39 2.39 3 3 H-8 H-8

2.81 2.81 1 1 H-4 <″> H-4 <″>

2.87 2.87 1 1 H-4 <'> H-4 <'>

3.01 3.01 1 1 H-9 H-9

6.62 6.62 1 1 H-6 H-6

6.79 6.79 1 1 H-7 H-7

94

Table: 5.3.13CNMR spectral data of compound Cb-8 in δ ppm:



19.2 20.6 CH2 CH2 C-5 C-5

21.0 21.0 CH3 CH3 C-17 C-17

23.0 21.3 CH3 CH3 C-19 C-15

23.0 23.0 CH3 CH3 C-20 C-19

24.8 23.0 CH3 CH3 C-15 C-20

26.6 24.0 CH2 CH2 C-10 C-10

27.2 26.4 CH CH3 C-18 C-21

27.5 26.4 CH3 CH3 C-21 C-22

27.5 27.2 CH3 CH C-22 C-18

30.0 30.8 CH2 CH2 C-8 C-8

33.3 34.5 C C C-4 C-4

37.6 37.2 C CH2 C-1 C-3

38.8 38.7 CH2 CH2 C-3 C-6

41.7 40.8 CH2 C C-6 C-1

50.1 54.3 CH C C-2 C-2

118.0 118.0 CH CH C-14 C-14

126.9 127.6 CH CH C-12 C-12

133.1 132.6 C C C-7 C-7

136.6 136.6 C C C-11 C-11

146.2 146.2 C C C-13 C-13

148.8 147.1 C C C-9 C-9

170.0 170.0 C C C-17 C-16

95

Table: 5.4. 1 HNMR spectral data of in compound Cb-9 δ ppm:

Chemical shift Proton count Probable assignment


1.06 1.06 3 3 H-11 H-11

1.12 1.21 6 6 H-10 H-10

1.14 1.14 6 6 H-13 H-13

1.41 1.41 1 1 H- 4<’> H- 4<’>

1.48 1.48 1 1 H-2 <’> H-2 <’>

1.50 1.50 1 1 H-3<’> H-3<’>

1.51 1.51 1 1 H-4<″> H-4<″>

1.56 1.56 1 1 H-6<’> H-6<’>

1.58 1.58 1 1 H-2 <″> H-2 <″>

1.60 1.60 1 1 H-3 <″> H-3 <″>

1.61 1.61 1 1 H-12 H-12

1.66 1.66 1 1 H-6 <’> H-6 <’>

2.72 2.72 1 1 H-9 H-9

2.81 2.81 1 1 H-5 <″> H-5 <″>

2.87 2.87 1 1 H-5 <’> H-5 <’>

4.42 4.42 1 1 H-1 H-1

6.63 6.63 1 1 H-8 H-8

6.79 6.79 1 1 H-7 H-7

96

Table: 5.5.13CNMR spectral data of compound Cb-9 in δ ppm:


trans cis trans cis trans cis

20.6 19.2 CH2 CH2 C-5 C-5

21.3 22.5 CH3 CH3 C-20 C-16

22.5 22.5 CH3 CH3 C-16 C-17

22.5 24.8 CH3 CH3 C-17 C-20

24 26.4 CH2 CH C-10 C-15

26.4 26.6 CH CH2 C-15 C-10

26.4 27.5 CH3 CH3 C-18 C-18

26.4 27.5 CH3 CH3 C-19 C-19

30.8 30 CH2 CH2 C-8 C-8

34.4 33.3 C C C-4 C-4

37.2 37.6 CH2 C C-3 C-1

38.7 38.8 CH2 CH2 C-6 C-3

40.7 41.7 C CH2 C-1 C-6

54.2 50.1 C CH C-2 C-2

110.7 110.7 CH CH C-14 C-14

125.7 125.7 CH CH C-12 C-12

127.3 126 C C C-7 C-7

132 132 C C C-11 C-11

146.4 145.3 C C C-9 C-9

151 151 C C C-13 C-13

97

98

99

100

101


The oil yield from the plant material (rhizome) was estimated

0.07%. The gas chromatogram of the oil from Cyperus brevifolius showed

the presence of more than 40 constituents among these, 34 constituents

were identified on the basis of their MS and GC retention data (Table

5.1). While the repeated column chromatography of chloroform extract

from the rhizomes leads to the isolation of Cb-8 and Cb-9.

(i) Characterisation of Cb- 8

It was obtained as liquid from the chloroform extract of the roots.

The molecular formula of the compound C22H32O2 was established by its

mass spectrum data as it showed molecular ion peak at m/z- 328 ( %),

with a base peak at 285. The other fragment ion peaks were recorded at

242(62), 227(12), 215(8), 169(5) and 77(4).

In the IR spectrum of the compound, absorption at 3072 cm-1

shows an aromatic C-H stretching. The carbonyl (>C=O) of ester linkage

have been observed at 1765 cm-1, while, aromatic >C C< stretching

have been observed at 1460, 1495 and1620 cm-1. However, the band at

2980 and 2870 cm-1 also point out the presence of aliphatic C-H

stretching.

The structural feature of compound Cb-8 was also studied on the

basis of its 1HMNR spectral data (Table 5.2) which shows absorption at δ

2.39 ppm due to O-CO-CH3 An angular methyl was reported at δ1.06

ppm. The two geminal methyls of isopropyl moiety at benzene ring have

been observed at δ 1.21 ppm. The aromatic protons were reported at δ

6.62 and 6.79 ppm.

The structure was further supported by its 13 CNMR spectral

observations (Table 5.3). It revealed the presence of a phenolic ester

102

(-O-CO-CH3) as it showed absorption at δ 170.0 ppm due to carbonyl

carbon (-C0-). The aromatic carbons were recorded at δ 148.8, 146.2,

136.6, 133.1 127.9 and 118.0 ppm. The gem-dimethyl carbons of

isopropyl moiety were observed at δ 23.0 ppm.

The cis-configuration of the compound was indicated by the

comparison of 13 CNMR data with its trans-isomer. The ring carbons C-1

and C-2 of the trans-isomer are less shielded i.e. δ 40.77(C-1) and 54.26

(C-1) ppm than the corresponding rings carbons of the cis-isomers which

were recorded at δ 37.6 (C-1) and 50.1 (C-2) ppm, which was also shown

by two carbons being shared by both the cyclohexane and the aromatic

ring ( C-7 and C-9) possess different values in the both the isomers. The

C-7 and C-9 carbons of the cis-isomer were recorded at δ148.8 and

133.1 ppm respectively whereas in trans-isomer the C-7 and C-9 carbons

are observed at δ 147.1 and 132.6 ppm.

It is expected as observed, that the chemical shift for the carbons

as mentioned will be upfield than that of trans-isomers.

CH3

CH3 CH3

CH3

CH3

O-CO-CH3

H

CH3

CH3 CH3

CH3

CH3

O-CO-CH3

H

cis- Ferreginol acetate trans- Ferreginol acetate

103

(ii) Charaterisation of Cb- 9

This is another constituents separated from the chloroform extract

of rhizomes. The molecular formula of the compound is C20H30O was

established by its mass spectrum data as its showed molecular ion-peak

at m/z (%) 286 (35) where the base peak is at 271. The fragment ion

peaks are at 253(12), 187(78), 145(30), 117(14) and 91(9).

The IR spectrum of the compound revealed the presence of a

phenolic –OH as it shows absorption at 3350 cm-1. Aromatic C-H

stretching was observed at 3080 cm-1, and a broad band at 1210 cm-1

due to C-O stretching. The aliphatic C-H stretching was found at 2975

and 2868 cm-1.

The 1HNMR spectral data was also studied to establish the

structure of the compound Cb-9 (Table 5.4). The presence of phenolic –

OH which was observed at δ 4.62 ppm. The aromatic protons were

reported at δ 6.79 and 6.63 ppm. An angular methyl of trans-

configuration were recorded at δ 1.06 ppm. The geminal methyls of iso-

propyl moiety were observed at δ 1.12 ppm.

The structure was also supported by its 13CNMR spectra (Table

5.5). A shift at δ 151.0 ppm revealed the presence of a phenolic –OH at

C-13. The aromatic carbons were recorded at δ 132.0, 127.3, 125.7, and

110.7 ppm. The geminal dimethyl carbons of isopropyl moiety were at δ

22.5 ppm.

The trans- configuration of the compound was further confirmed

by the comparison of 13 CNMR data with its cis-isomers, where in the ring

carbon C-3 and C-2 of cis-isomer were reported at δ 37.6 and 50.1 ppm

respectively, which are more shielded than the corresponding ring

carbons of trans-isomer, being reported at δ 40.7 and 54.2 ppm of

carbons C-1 and C-2 respectively.

104

Similarly two carbons being shared by the cyclohexane and

aromatic moiety of both the isomers possess different values. The

carbons of cis-isomers at C-7 and C-9 are recorded at δ 126.0 and 145.3

ppm, whereas in trans-isomer they were observed at δ 127.0 and 146.4

ppm respectively.

CH3

CH3

CH3 CH3

OH

CH3

H

CH3

CH3 CH3

CH3

CH3

OH

H

trans-Ferruginol cis- Ferruginol

105

Chapter VI

Conclusion

106

Conclusion

Among the members of Cyperaceae family, Cyperus rotundus , have been

explored extensively for its medicinal values so far, whereas no such work have

been reported in their other relative species. Therefore the present attempt has

been undertaken especially for the species collected from the Himalayan region

of Uttrakhand, to determine and establish its medicinal and economic value in

various streams of pharmacopeias.

The constituents so separated are collected from the oil obtained by the

steam distillation of the rhizomes of the concern species i. e. Cyperus paniceus,

C. niveus and C. brevifolius. The first such attempt on the concerned species to

explore its medicinal and economic value confirms the presence of nine

constituents as Dehydroabietal, Abietol, Remirol, Cyperaquinone, Cyperol,

Cyperenal, 4, 7-Dimethyl-1-tetralone, ferreginol acetate and ferruginol. The

present study revealed that out of the nine constituents so isolated, Cyperol,

Cyperenal and 4, 7-Dimethyl-1-tetralone, the three have already been known

(from other members of the family) for their anti-malarial as well as insecticidal

properties. Thus it has been confirmed that the nominate species of Cyperus

also possess the same properties.

However, the six constituents that have been isolated for the first time

from the rhizomes of the Cyperaceae family needs to be further investigated for

the use in various pharmacological practices, so that, it could be used

frequently for the various ailments prevailing in the present world.

107

Chapter VII

References

108

1. Martinetz, D. and Hartwig, R. (1998). Taschenbuch der Riechstoffe,

Verlag Harri Deutsch, pp. 5-15.

2. Bhattacharjee, K. S. (1998). Handbook of Medicinal plants, Pointers

Publishers, Jaipur, India.

3. Dev, S. (1999). Environment Health Perspect, 107, 783.

4. Fallarino, M. (1994). Herbal Gram., 31, 38.

5. Phillipson David, J. (2001). Phytochemistry, 56, 237.

6. Torrance, S. J. (1976). Journal of Organic Chemistry, 41, 1855.

7. Purshothaman, K. K. (1979). Tetrahedron Letters, 11, 979.

8. Bhakuni, D. S. (1990). Drug From Plants Science Reporter, 12.

9. Handa, S. S., Kingdom, A. D., Cordell G. A. and Farnsworth N. R.

(1983). J. Nat. Prod., 46, 123.

10. Handa. S.S., Kingdom A.D., Cordell G.A. and Farnsworth N.R.,

(1983). J. Nat. Prod., 46, 359.

11. Badwi, M. M., Handa S. S., Kingdom A. D., Cordell G. A. and

Farnsworth N. R. (1983). J. Pharma Sci., 72, 1285.

12. Kingdom, A. D. (1982). Plants Pharmacy Internat., 3,326.

13. Xian G., Handa S. S., Pezzuto J. M., Kingdom, A. D.and

Faransworth N. R. (1984). Planta Medica, 51, 358.

14. Christoper, J. (1993). Biosciences, 43, 3.

15. Wani, M. C., Taylor, H. I., Wall, M.E., Coggen, P. and Mc Phail,

A.T. (1971). J. Am. Chem. Soc., 93, 2325.

109

16. Denis, J. N., Grecne, A. E., Gvenard, D. and Potier, P. (1988). J.

Am. Chem. Soc., 110, 5917.

17. Clark, A.M. (1996). Pharm. Res., 13, 1133.

18. Chen, K. K. and Schmidt, C. F. (1930). Medicine, 9, 1.

19. Terry, C. E. and Pellens, M. (1928). Bureau of Social Hygiene, New

York.

20. Musto, D. F. (1973). The American Diseases, Yale University

Press, New Haven.

21. Atal, C. K., Zutshi, U. and Rao, P. G. (1981). J. of Ethano

Pharmacology, 4, 229.

22. Muller, K. O. and Borger, H. (1940). Arh. Biol., 23, 189.

23. Sinha, G. K. (1977). Advances in Essential Oil Industry A

Symposium, Eds., L. D. Kapoor and R. Krishan, Today and

Tomorrow, Delhi, pp.47.

24. Archer C. (1998). Plant Life, 18, 14.

25. Inglis, C. (1994). Cyperaceae Newsletter, 4, 13.

26. Simpson, D. A. (1992). Cyperaceae Newsletter, 2, 10.

27. Allan, R. D, Wells, R. J. and Macleod, J. K. (1973). Tetrahedron

Letters, 36, 7.

28. Allan, R. D., Corell, R. L. and Wells, R. J. (1969). Tetrahedron

Letters, 32, 4669.

29. Allan, R. D., Dunlop, R. W., Kendall, M. J. and Wells, R. J.

(1972). Tetrahedron Letters, 36, 241.

110

30. Macleod, J. K., Worth, B. R. and Wells, R. J. (1972). Tetrahedron

Letters, 35, 241.

31. Seabra, R. M., Andrade, P. B., Ferreres, F. and Morerira, M.

(1997). Phytochemistry ,45, 839.

32. Trivedi, B., Motl, O., Smolikova, J. and Sorm, F. (1964).

Tetrahedron Letters, pp.1197.

33. Morimoto, M., Fuji, Y. and Komai K. (1999). Phytochemistry, 51,

605.

34. Fiorentino, A., D’ Abrosca, B., Pacifico, S., Natale, A. and Monaco

P. (2006). Phytochemistry, 67, 971.

35. Meng Y., Bourne, P. C., Whiting, P., Sik, V. and Dinan L. (2001).

Phytochemistry, 57, 393.

36. Pandey, A. K. and Chowdhary, A. R. (2002). Indian Perfumer, 46,

325.

37. Yamda, M., Hayashi, K., Hiroshi, H. Ikeda, S., Hoshino, T.,

Tsutsui, K., Tsutsui, K., Iinuma, M. and Nozaki, H. (2006).

Phytochemistry, 67, 307.

38. Yamda, M., Hayashi, K., Hiroshi, H., Tsuji, R., Kakumoto, K.,

Ikeda, S., Hoshino, T., Tsutsui, K., Tsutsui, K., Ito, T., Iinuma, M.

and Nozaki, H. (2006). Chem. Pharm. Bull., 54, 354.

39. Sonawa, M. M., Wilfried, K. A. (2001). Phytochemistry, 58, 799.

40. Thebtaranonth, C., Thebtaranonth, Y., Wanauppathamkul, S.

and Yuthavong, Y. (1995). Phytochemistry, 40, 125.

111

41. Sonwa, M. M. and Wilfried, K. A. (2001). Photochemistry, 56, 321.

42. Nassar, M. I., Abdel-Razik, A. F., EI-Khrisy, Ezz EI- Din A. M.,

Dawidar, A. M., Bystrom A. and Mabry, T. J. (2002).

Photochemistry, 60, 385.

43. Seabra, R. M., Silva, A. M. S., Andrade, P. B. and Moreira, M. M.

(1998). Phytochemistry, 48, 1429.

44. Abdel-Razik, A. F., Nassar, M. I., EI-Khirs, E. D. A., Dawidar,

A.A.M.and Mabry, T. J. (2005). Fitoterapia, 76, 762.

45. Toong, Y.C., Schooley, D.A. and Baker, F.C. (1998). Nature,

333,170.

46. Puratchikody, A., Jaswanth, A., Nagalakshmi, A., Anagumeenal,

P.K. and Ruckmani, K. (2001). Indian Journal of Pharmaceutical

Sciences, 63, 326.

47. Singh, V., Abbas, S. S. and Singh, N. (2003). 2nd World Congress

on “Biotechnological Developments of Herbal Medicine” NBRI ,

Lucknow pp.33.

48. Kulkarni, K. and Joshi, V. K. (2002). Indian Journal Of Clinical

Practice, 12, 37.

49. Pandey, K. K., Vandana and Dwivedi, M. (2001). Antiseptic, 98,

295.

50. Morimoto, M., Shimomura, Y., Mizuno, R. and Komai, K. (2001).

Bioscience, Biotechnology and Biochemistry, 65, 1849.

112

51. Joseph-Nathan, P., Martinez, E., Santillan, R. I., Wesener, J. R.

and Gunther, H. (1984). Organic Magnetic Resonance, 2, 308.

52. Nyasse B., Tih, R.G., Sodengam, B.L., Martin, M.T.and Bodo, B.

(1988). Phytochemistry, 27, 179.

53. Nyasse, B., Tih, R. G., Sodengam, B. L., Martin, M.T. and Bodo,

B. (1988). Phytochemistry, 27, 3319.

54. Nerali, S. B., Kalai, P. S., Chakravarti, K.K. and Batacharya, S.C.

(1965). Tetrahedron Letters, 4053.

55. Kapadia, V. H., Naik, V. G., Wadia, M. S. and Dev, S. (1967).

Tetrahedron Letters, 4661.

56. Hikino, H. and Aota, K. (1976). Phytochemistry, 15, 1265.

57. Hikino, H., Aota, K., Kuwano, D. and Takemoto, T. (1971).

Tetrahedron Letters, 27, 4831.

58. Nerali, S. B. and Chakravati, K. K. (1967). Tetrahedron Letters,

2447.

59. Kubmarawa, D., Ogunwande, I. A., Korie, D. A., Olawore, N.O.

and Kasali, A. A. (2005). Flavour and Fragrance Journal, 20, 640.

60. Yokozawa, T., Cho, E. J., Sasaki, S., Satoh, A., Okamoto, T. and

Sei, Y. (2006). Biological & Pharmeceutical Bulletein, 29, 760.

61. Singh, J., Mishra, N. P., Joshi, G., Singh, S.C., Sharma, A. and

Khanuja, S. P. S. (2005). Journal of Medicinal and Aromatic Plant

Sciences, 27, 344.

113

62. Chowdhary, J. U., Yusuf, M. and Hossain, M. M. (2005). Indian

Perfumer, 49, 103.

63. Mehar-Homji, V. M. (1978). Environmental physiology and ecology

of plants, Bishan Singh and Mahendra Pal Singh. (eds.),

Dehradun.

64. Young, K. A. (2004). Ecology, 85 (2), 342.Morris, D. W. (1999).

Evolutionary Ecology research, 1, 3.

65. Hikino, H., Aota, K. and Takemoto, T. (1967). Chemical and

pharmaceutical Bulletin, 15, 1929.

66. Takano, S. and Kawaminami, S. (1988). Phytochemistry, 27,

1197.

67. Lye, K. A. and Cheek, M. (2006). Nord. J. Bot., 24: 273.

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