chapter i - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/24322/1/sawti thesis chapters...
TRANSCRIPT
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)
Column I (1-62) 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
Compound Cb-2 : Liquid with odour
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)
1HNMR (CDCl3) δ ppm : Table: 3.3, Fig: 3.4
13CNMR (CDCl3) δ ppm : Table: 3.4, Fig: 3.5
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:
Chemical shift Proton count Probable assignments
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:
Chemical shift CHn Probable assignment
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.
The structure was further supported by its 13CNMR spectral data
(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).
1HNMR (CDCl3) δ ppm : Table: 3.6, Fig: 3.6
13CNMR(CDCl3) δ ppm : Table: 3.7, Fig: 3.7
Compound Cb-4 : Liquid with odour
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).
1HNMR (CDCl3) δ ppm : Table: 3.8, Fig: 3.8
13CNMR (CDCl3) δ ppm : Table: 3.9, Fig: 3.9
46
Table: 3.6. 1HNMR spectral data of compound Cb-3 in δppm:
Chemical shift Proton count Probable assignments
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:
Chemical shift CHn Probable assignment
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
Table: 3.8. 1HNMR spectral data of compound Cb-4 in δppm:
Chemical shift Proton count Probable assignments
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:
Chemical shift CHn Probable assignment
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.
Taxonomy and distribution:
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
Column I (1-81) fractions collected)
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
Compound Cb-6 : Colourless liquid
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:
Chemical shift Proton count Probable assignments
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:
Chemical shift CHn Probable assignment
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:
Chemical shift Proton count Probable assignments
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:
Chemical shift CHn Probable assignment
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
4.3: Results and Discussion:
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.
The structure was further supported by its 13CNMR spectral data
(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
Plant Material (2Kg)
1. Washed, dried & Crushed2. Extracted with hexane, , CHCL3, MeOH
n – hexane soluble
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
Compound Cb-7 : Colourless liquid
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:
Chemical shift Proton count Probable assignments
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:
Chemical shift CHn Probable assignment
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.
Taxonomy and distribution:
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
Plant Material (2Kg)
1. Washed, dried & Crushed2. Extracted with hexane, MeOH, CHCL3
n – hexane soluble
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:
Compound Cb-8 : Colourless liquid
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)
1HNMR (CDCl3) δ ppm : Table: 5.2, Fig: 5.2
13CNMR (CDCl3) δ ppm : Table: 5.3, Fig: 5.3
Compound Cb-9 : Colourless liquid
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)
1HNMR (CDCl3) δ ppm : Table: 5.4, Fig: 5.4
13CNMR (CDCl3) δ ppm : Table: 5.5, Fig: 5.5
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:
Chemical shift CHn Probable assignment
cis trans cis trans cis trans
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
cis trans cis trans cis trans
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:
Chemical shift CHn Probable assignment
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
5.3: Results and Discussion:
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.