ANNEXUREI

Identification of Cleome gynandra L. by B.S.I., Kolkafa

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Dr.- o u r a n i a n Roy Head of t h e I e p t . of C h e m i s t r y C a c h a r C o l l e a e S i l c h a r 786 U Assam

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. . i t h r e t e r e i ^ c e t o yo.ir l e t t e r No.cc/ l" 1-3 d a t e a L-.9.2000 a l o n g . i t h a ' s a m p l e packet . cujn.ai ju .ny a d r y , p r e ; , ed p l a n t m a t e r i a l h a n d e d o v e r t o t h i s o f f i c e on 21s t c t . 2 0 0 0 for f a v o u r of i d e n t i f i c a t i o n , I am t o inform, you t h a t t h e specimen, h a s b e e n i d e n t i f i e d a £ : Cleome gynandra L i n n . C«= G y n a n d r o p s i s p e n t a p h y l l a (L inn , DC.) of t he fami ly C l e o m a c e a e .

The r r e i - t of r u p e e s f i f t y i s e n c l o s e d h e r e w i t h specimen fo r your'pfijcui: .:il«.,„_..... ....,„

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ANNEXURE II

Abstract of Poster presented at IUPAC International Conference on Biodiversity and Natural Products, 2004

ICOB-4 &ISCNP-24 IUPAC International Conference

on Biodiversity and Natural Products:

Chemistry and Medical Applications 26-31 January 2004, New Delhi (India)

www.bnp2004.com

ABSTRACTS CONFERENCE PROGRAMME

Organized by DEPARTMENT OF CHEMISTRY, UNIVERSITY OF DELHI

&

COUNCIL OF SCIENTIFIC ANIj) INDUSTRIAL RESEARCH

P-294

CHEMICAL AND MEDICINAL PERSPECTIVES OF THE SEED OIL OF CLEOME GYNANDRA L.

S.B. Paul and S. Roy

Department, of Chemistry,Assam University, Silchar - 788011, India Fax : +91-3842-270802; Email: sbpaull@rediffmail.com

Cleome gyanandrL (Cleomaceae) is a wild and semi-cultivated annual herb growing throughout the Warmer parts of '.lie globe and having ethnic use by tribals of Arunachal Pradesh and Bodo tribals of Assam as well as used in traditional system of medicine. The bruised leaves are used in headache, neuralgia, rheumatism and other local pains. Seeds are anthelmintic and seed oil is used in curing skin diseases. Decoction of root is used in expelling round worms. Essential oil has anti-tick property and leaf extract possess antifeedant and insecticidal property. It is a popular leafy vegetable in many parts of Africa.

Keeping this in view, this communication highlights the chemical and medicinal properties of the seed oil hitherto not used for edible or industrial purposes. Physico-chemical characterisation such as colour, odour, density, refractive index, viscosity, saponification value, iodine value, acid value, peroxide value and NSM are evaluated. A simplified method of analysis of sterols in the seed oil is also described. Toxicological evaluation of safety of the seed oil by feeding swiss albino mice ad libitum followed by examination of serum cholesterol level and histological examination of vital organs are reported. Antifungal property of the seed oil evaluated against a few fungal species on CDA medium is also incorporated.

The results revealed acceptable edible oil characteristics of the seed oil of cleome gynandra L. to encourage fanners in its exploitation. Moreover, this may provide breeders a better tool in his selection.

ANNEXURE III

Paper Presented in National Seminar on Biodiversity Conservation, April 2006.

N A 1 1 0 IN A I,, S I<; IV! I I N A R on

B1 GDI V E R S I T Y C O N S E R V A LION ] 1 II' P O S T - R I O S C E N A R I O IN I N D I A

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April 19-21,2006

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Orj;nm/.cd by Department of Ecology and Environmental Science

Assam University, Silchar - 788 Oil Assam

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Sponsored by Ministry of Environment and Forests, New Delhi

and Department of Science and Technology, New Delhi

13. A study on Tall Grassland Habitat in Laokhowa Wildlife Sanctuary, Assam, India

S. K. Nath' & S. K. Sarma2

'Deptt. of Botany, Dhing College, Dhing, Nagaon 2Deptt. of Botany, Guwahati University, Guwahati

Grasslands along with their associated fauna was widely distributed in Indian sub-continent but at present these are restricted only to some protested areas of India. In Assam, Laokhowa Wildlife Sanctuary is one of such sanctuary where grassland covers 35% of the total area. In the present paper, an attempt has been made to identify the dominant grass species and their associated fauna. The grasslands are studied according to the relative abundance of different dominant tall grass species. The grassland areas arc dominated by grasses viz. Imperata cylindrica, Saccharumiponluneum, Er.ianthus ravannae, Slerostachyafusca etc. Cutting and extensive grazing affect the distribution of grasslands in Laokhowa Wildlife Sanctuary. Immediate conservation strategies are required to save the grassland habitat of the sanctuary, which is the major habitat for one horned Rhinoceros, wild Buffalo, wild Pig etc.

Key words : Dominant arasses, Lrokhowa Wildlife Sanctuary.

0A. Biodiversity and Natural Products : The Talc of Cleome Gynandra Linn.

S. B. Paul* & S. Roy

Department of Chemistry, Assam University, Silchar - 788011 *E-mail : sbpaul@rediffmail.com

Nature's ingenuity in creating variety of enchanting skeletons with generous sprinkling of functionalities and stereochemical delicacies is thrilling. Natural products will continue to play a crucial role in meeting the demand of novel molecular diversity for pharmaceuticals, nutraceuticals and agrochemicals.

Apart from a large number of cultivated plants with their wide diversity, the Indian region is very rich floristically, which may not have an obvious economic value at present but in the face of ever changing patterns of our needs regarding food, fuel, fibre, fodder, shelter, medicare, etc. one cannot foretell as to what species would be needed when.

Growing throughout th^ warmer parts of the globe, Cleome gynandra L. (Cleomaceae) is a wild and seiviicultivaled annual herb used in traditional system of medicine. In view of this, a few products from the various part of the plant, growing in Barak Valley have been isolated, chemically characterised & investigated their toxicological antifungal and anthelmintic properties.

11

Biodiversity and Natural Products : The Tale of Cleome Gynandra Linn.

S.B.Paul* & S. Roy Department of Chemistry, Assam University, Silchar - 788011

*e-mail : sbpaul@rediffmail.com

Abstract

Nature's ingenuity in creating variety of enchanting skeletons with generous sprinkling of functionalities and stereochemical delicacies is thrilling. Natural products will continue to play a crucial role in meeting the demand of novel molecular diversity for pharmaceuticals, nutraceuticals and agrochemicals.

Apart from a large number of cultivated plants with their wide diversity, the Indian region is very rich llonstically and many may not have an obvious economic value at present. But in the face of ever changing patterns of our needs regarding food, fuel, fibre, fodder, shelter, medicare, etc. one cannot foretell as to what species would be needed when.

Growing throughout the warmer parts of the globe, Cleome gynandra L. (Cleomaccae) is a wild and semi-cultivated annual herb used in traditional system of medicine, (in view of this, a few products from the various parts of the plant, growing in Barak Valley have been isolated, chemically characterized and their toxicological. antifungal and anthelmintic properties investigated.

Since the plant under study grows in abundance even in wastelands, it may be economically viable source of some useful pharmaceuticals, neutraceuticals and agrochemicals. But study on the whole gamut of parameters is needed to achieve the goal of conservation of the plant.

Key-words : molecular diversity, cleomaceae, natural products.

Introduction :

Biodiversity is intricately linked with diverse natural products in the form of pharmaceuticals, neutraceuticals, agrochemicals, botanicals etc. which have so far been isolated and will continue to be harnessed for the benefit of mankind. Nature is infinitely more inventive than chemists and natural molecules have withstood many tough tests of survival for millions of years1. Nature's ingenuity in creating variety of structures with generous sprinkling of functionalities and stereochemical delicacies is thrilling. This, was highlighted by unraveling of many new and enchanting natural products from plant, marine and fungal sources having exotic properties2.' Cellulose (1) the most abundant organic molecule and most abundant single polymer of glucose in the biosphere having (3-1, 4-linkage is the structural material while the a-1, 4-linked polymer starch (2) is a storage material. Both the ultrabitter (ca. 4.000.000) centapicrin (3) from century plant and supersweet (ca. 100) glycyrrhizinic acid (4) from licorice root have carbohydrate moiety. Cytotoxic dimeric alkaloid dramacidin (5) from carribian sponge, insecticidal isodomic acid A (6) from Japanese red algae and extreme toxin palyt.oxin from coelenterate.s having 64 chiral centers are a few of them. In contrast to terrestrial monoterpenes virtually all marine monoterpenes are halogenated .

Out oi 2000 medicines described in Indian system of Medicine about 200 are of animal origin, another about 200 are of mineral origin and remaining 1500 are of plant origin. About 15000 plants of medicinal value are anticipated to be available in India. This richness is attributed to climatic conditions and geographical locations4. Insecticides of diverse structures such as pyrethnns (7), wtenons (8) etc. from over 2000 species belonging to some sixty families5, antioxidants effecting holistically in complicated disorders like diabetes' and

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biopesticides like azadiractin (9) with adjuvants (10) having enhanced pesticidal property and biodegradability are examples of molecular diversity. Bioactive natural products being considered as template in time-tested strategy for development of more active but safer products justify the need of exploring this diversity.

Cleome gynandra L. (Fam. Cleomaceae) syn Gynandropsis gynandra Briq., Gynandropsis pentaphylla D.C. commonly known as Cat's whiskers, spider herb is an annual herb etc. that grows upto lm tall and common in tropical and sub-tropical climatic region8. The leaves are used in Africa as vegetable having high protein content, seeds and oils reported to be anti-nematode and the plant has been used in traditional medicine for treatment of rheumatism, headache, stomachache, severe infection of threadworms and as fish poison9. Anti-tick property of essential oil of the plant have been reported10. It finds use in Indian system of medicine and ethnomedicine 2. A few chemicals have been isolated from the plant "" ". Biological activities of extract of plant parts reported17'8. [3-sitosterol (11) was found as the major phytosterol in seed oil16. Nutritive value and safety aspects of the seed oil was evaluated and it was found to have hypocholesteraemic effects in experimental mice1 . GC-MS analysis of the hydrodistillate of aerial parts of the plant growing in Barak Valley showed significant difference from that of Nairobi sample20.

In this paper isolation of a flavonoid and anthelmintic property of some extracts from the plant collected locally are reported.

Materials and Methods :-

Isolation of flavonoid - Fresh flowers (50g) of Cleome gynandra L growing to a height of about lm were subjected to cold maceration with methanol (100ml x 2), extract collected by filtration was subjected to concentration in vacuo. The concentrate (50ml) was shaken successively with petroleum ether (40 - 60°C) and diethyl ether, 25ml each and the aqueous fraction collected using separating funnel. To this 50ml of excess diethyl ether was added and kept in freezer for 15 days. After this period deposits were collected and repeatedly recrystallised from 50% aqueous methanol. The crystals analysed by chromatography, co-chromatography and spectroscopy.

Anthelmintic study - Whole plant was air dried and separated into aerial part and root part. Mature seeds were collected and air dried. These parts were crushed and subjected to soxhlet extraction. Extracts were evaporated in vacuo.

Live parasites (nematode : Helerakis gallinarum, Cestode : Raillietina eclrinoboflvida from domestic fowl and trematode : Fasciolopsis buski from freshly slaughtered pig at an abattoir in Shillong) were collected in 0.9% phosphate buffered saline (PBS) and incubated at 37 ± 1°C in media containing no extract (control) or crude extract at 10, 20 and 50 mg/ml PBS with 1% dimethyl sulphoxide (DMSO). Five replicates were used for each concentration and time required for loss of movement and death were recorded. Mean time and SD values were calculated using standard methods.

3

Results and discussion :

Flavonoid - Recrystallised pale yellow needles, yield 0.2% fresh flower basis, m. pt. 186-187°C, P.C. Rf 0.52 in BAW (4:1 : 5) shows characteristic UV spectra with A.max

(MeOH) 259, 266, 299 and 359 nm, effect of shift reagents, colour reaction and Molisch test. The identity of the compound as rutin (Vitamin P) (12) confirmed by Co-chromatography and NMR spectroscopy21.

The significance of isolation of the flavonol rutin are that it is scavanger of oxygen free radicals, suppressor of tumor growth, inhibitor of ascorbic acid oxidase and it prevents lipid oxidation by chelating transition metal ions, atherosclerosis and intramascular thrombosis. Rutin with vitamin C gives cold tolerance22. Flavonoid identification is useful in chanotaxonomic studies of plants23.

Anthelmintic study -

The results of the observation on time for loss of movement (P) and death (D), Table 1 shows that there is an orderly decline in time with concentration. Whereas F. buski in control survived 21.55 ± 1.15 h its paralysis and death occurred at 1.36 ± 0.67 and 1.45 ± 0.70 h respectively using aerial defatted methaniolic extract (402) at 50 mg/ml concentration. Almost similar result was obtained using root ethanolic extract (501).

For H. gallinarum surviving 27.25 ± 2.35 h in control, root ethanolic extract (501) at 50 mg/ml concentration gives maximum response, i.e. paralysis and death at 1.45 ± 0.85 and 1.98 +0.57 h respectively.

For R. echinobolhrida paralysis and death occurred at a minimum time of 1.53 + 0.68 and 2.05 ± 0.95 h using 50 mg/ml concentration of root ethanolic extract (501) although defatted methanolic extract of aerial part (402) shows quickest response at lower concentration of 10 mg/ml.

Anthelmintic efficacy of the extracts in this study compares very well with previously reported results..24'25

Conclusion :

In view^of the occurance of bioactjve constituents in Cleome gynandra L. and its world wide distribution further work in respect of chemical, biological and ecological aspects are warranted. Attempts in respect of its promotion and conservation may also be undertaken.

Acknowledgement :-

The authors gratefully acknowledge help and guidance by Prof. V. Tandon, Department of Zoology, NEHU Shillong in conducting the anthelmintic study, Director, RSIC, Shillong for providing spectra and UGC (NER) for granting MRP to S. Roy.

4

Table 1 : Efficacy of crude extracts (Seed, root, stem bark) of Cleome gynandra on R. echinobothrida, H. gallinarium and F. buski.

Treatment (nig/ml)

Time (h) taken for complete loss of movement (P) and death (D) Fasciolopsis buski Heterakis gallinarum Raillietina echinobothrida

402, Arial part methanolic, defatted Controls 10

20

50

21.55 ± 1.15 P 3.95 ±0.68 D 6.15 ±0.74 P 2.64 ±0.85 D 4.65 ±0.27 P 1.36 ±0.67 D 1.45 ±0.70

27.25 ±2.39 P 4.65 ±1.05 D 5.55 ±0.56 P 2.35 ±0.56 D 3.25 ±0.75 P 0.75 ±0.26 D 2.45 ± 0.65

70.65 ± 0.95 P 7.35 ±0.85 D 8.65 ±0.98 P 5.55 ±0.85 D 7.25 ±0.45 P 1.15 ±0.54 D 3.35 ±0.65

501, Root ethanoiic Controls 10

20

50

21.55 ± 1.15 P 5.30 ±1.15 D 6.85 ±1.36 P 2.85 ±0.69 D 3.64 ±0.58 P 1.31 ±0.53 D 1.42 ±0.68

27.25 ±2.35 P 4.95 ±0.96 D 6.65 ±0.87 P 2.58 ±0.63 D 3.85 ±0.35 P 1.45 ±0.85 D 1.98 ±0.57

70.65 ±0.95 P 8.05 ±0.68 D 9.45 ±0.38 P 5.85 ±0.60 D 6.38 ±0.93 P 1.53 ±0.68 D 2.05 ±0.95

215, Seed methalonic, defatted Controls 10

20

50

21.55 ± 1.15 P 7.69 ±1.24

D 13.09 ±0.95 P 5.65 ±0.86 D 8.09 ±0.89 P 3.12 ±0.54 D 4.65 ±0.95

27.25 ±2.35 P 6.58 ±0.68 D 7.95 ±1.35 P 3.46 ±0.59 D 4.67 ± 0.89 P 1.34 ±0.52 D 2.68 ±0.45

70.65 ±0.95 P 14.98 ± 1.65 D 17.05 ±2.08 P 9.75 ± 0.67

D 12.14 ±0.92 P 1.75 ±0.95 D 2.68 ±0.53

232, Seed ethalonic, defatted Controls 10

20

50

21.55 ±1.15 P 4.54 ±0.69 D 6.38 ±0.95 P 3.45 ±0.62 D4.71 ±0.92 P 1.54 ±0.68 D 2.06 ±0.90

27.25 ±2.35 P 5.65 ±0.96 D 8.52 ±0.68 P2.61 ±0.36 D 4.09 ±0.58 P 0.69 ±0.35 D 2.29 ±0.61

70.65 ±0.95 P 8.91 ±0.74 D 9.05 ±0.35 P6.16±0.39 D 8.59 ±0.94 P 1.36 ±0.86 D 3.29 ± 0.60

401, Stem bark Controls 10

20

50

21.55 ± 1.15 P 4.68 ±0.42 D 6.26 ±0.59 P 2.45 ±0.61 D4.10±0.85 P 2.57 ±0.62 D 2.36 ±0.69

27.25 ±2.35 P 5.63 ±0.97 D 8.56 ±0.35 P 2.61 ±0.94 D 4.35 ±0.68 P 0.76 ±0.89 D 2.64 ±0.39

70.65 ±0.95 P 8.93 ±1.56 D 9.39 ±0.92 P 6.69 ±0.85 D 8.95 ± 0.26 P 1.39 ±0.43 D 3.69 ±0.52

5

References :

1. Valiathan, M.S., Curr. Sci., 75(11), p. 1122-6 (1998).

2. Srikrishna, A., Ph.D. Thesis, University of Hyderabad, p. 3 (1981). 3. Thomson, R.H. Ed. The Chemistry of Natural Products, Blackie Academic &

Professional, London, p. -243, 400 (1993). 4. Jain, S.K., Medicinal Plants, NBT, New Delhi, p. 1-3 (1995). 5. Dev, S. and Koul, O, Insecticides of Natural Origin, Harwood Academic Publishers,

Amsterdam, p. 4-7 (1997). 6. Tiwari, A.K. & Rao, J.M., Curr. Sci. 83(1) p. 30-38 (2002). 7. Gupta, G.P. & Raghuraman, M; Neem for Sustainable Pest Management of

Boldworms in Cotton, IUPAC, Conference on Biodiversity and Natural Products : Chemistry and Medical Applications, p. 29 (2004).

8. Elfers, J. et. al., Flora of Tropical East Africa, Whitefriars Press, London, p. 18 (1964).

9. Chweya, J.A. & Mnzava, N.A., Promoting the Conservation and Uses of Underutilized and Neglected Crops. 11, IPGRI, Rome (1997).

10. Lwande. W. et. al. Gynandropsis gynandra Essential Oil and Its Constituents as Tick Repellants. Plytochemistry, 50, p. 401-405 (1999).

•>11. Chopra, R.N. et. al., Glossary of Indian Medicinal Plants, PID, CSIR, New Delhi, p. 129(1956).

12. Bhuyan, D.K., Ph.D. Thesis, Gauhati University, p. 220-221 (1989). 13. Gupta, R.K. et. al. Indian. J. Pharm. 30, p. 127 (1968). 14. Jain, A.C. & Gupta, S.M. J. Nat. Prod. 48, p. 332-333 (1985). 15. Chatterjee, A. et. al. J. Nat. Prod., 62, p. 618-618 (1999). 16. Paul, S.B. & Roy, S. Isolation of sterols from seed oil of North-East Indian Cleome

gynandra L. by Argentation TLC, Res. J. Chem. Environ, (communicated).

17. Ajaiyeoba, E.O. et. al. Pharm. Biol., 39(3), p. 217 (2001). 18. Rao, R. et. al. Antibacterial activity of root of Gynandopsis gynandra L., Indian

Drugs, 35(11), p. 721-722 (1998). 19. Paul, S.B. & Roy, S., Chemical and Medicinal Perspectives of seed oil of Cleome

gynandra L. IUPAC Conference on Biodiversity and Natural Products : Chemistry and Medical Applications, p. 294 (2004).

20. Paul, S.B. & Roy, S., Constituents of Hydro-distillate of Cleome gynandra L., Int. J. Chem. Sci. (Communicated).

21.Mabry, T. J. et. al. The Systematic Identification of Flavonoids, Springer, N.Y. (1970).

22. Robak. J. & Gryglewski, R.J., Bioactivity of Flavonoids, Pol. J. Pharmacol., 48, p. 555-564(1996).

23. Harborne, J.B., The Flavonoids, Chapman & Hall, London, p. 1056-1095 (1975). 24. Tandon, V. et. al. In vitro Anthelmintic Activity of Tuber Root Extract of F. vestila,

Parasilol. Res. (83), p. 492-498 (1997). 25. Roy, B. & Tandon, V., Flukicidal Activity of A. nigra (Zingiberaceae) against

nematode, F. buski, Biomedical letters, 60, p. 23-29 (1999).

6

ANNEXURE IV

Short Communication : Published in Research Journal of Chemistry and Environment, September 2006

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Research Journal Of Chemistry And Environment. .Vol. 10 (3) Sept. (2006) Res. J. Chem. Environ.

Short Communication:

Isolation of Sterols from Seed Oil of North East Indian Cleome gynandra L. by Argentation-TLC

S.B. Paul* and S. Roy

Department of Chemistry, Assam University, Silchar 788011 (Assam), INDIA *au_examina tion@yahoo.com

Abstract

^-Sitosterol is isolated as the major phytosterol from seed oil of Cleome gynandra L. by Argentation - TLC.

Keywords: Cleome gynandra, argentation-TLC, P-Sitosterol.

Introduction Cleome gynndra L (Cleomaceae) is an annual weed in

tropical countries1 cultivated as popular leafy vegetable in Africa2. It finds use in the Indian medicine3 and as indigenous medicine4. In our investigation on the nutritive and safety aspects of the seed oil, we observed lowering of serum cholesterol in experimental mice.5 Phytosterols are reported to impart hypocholesteraemic effects in humans6 and phytosterol fed chicken produced significant lowering of cholesterol in the yolks of consumable eggs7. Phytosterols possess anti-inflamatory, antibacterial, antifungal, antiulcerative & antitumor activity8"11. The profile of plytosterols is characteristic of taxonomic classification & detection of adulteration12. This communication deals with isolation of phytosterols from the seed oil of Cleome gynandra.

Material and Methods Mature seeds of the herb collected from Barak Valley

of North East India in Feb.-March, 2006 were air-dried at ambient temperature and ground. Oil was extracted from the ground seeds (100 g) by stirring for 3 hr. at ambient temperature in 400 ml hexane, suction filtration through Whatman No. 1 paper and solvent removed by rotary vacuum evaporation. TLC of seed oil on aluminium-backed, pre-coated silica gel 60F thin layer chromatography plate, 0.2 mm thickness using hexane-dicthyl ether - acetic acid (40:10:1) as developing solvent revealed eight fractions after spraying with 10% ethanolic phosphomolybdic acid and charring13. Preparative TLC of the seeds oil was done on standard silica get G plate using hexane - diethyl ether - acetic acid (40:10:1) as developing solvent. Iodine was used for visualisation of separated lipid zones. Elution of the band at Rf0.30 with hexane - ether (1:1) gives free sterol mixture (FSM). Unsaponifiable fraction (USF) of the oil was prepared by cold saponification14, extraction with ether, washing and vacuum evaporation.

Results and Discussion Argentation-TLC of FSM and USF was performed on

standard Silica gel plate previously impregnated with saturated

95% methanolic AgN03and activated for 30 min at 110°C.'5

Chloroform - diethyl ether-acetic acid (97:2.3:0.5) was used as developing solvent and 0.2% ethanolic 2', 7' - dichlorofluorescein (DCF) as visualisation agent under UV. After localisation, the sterol fractions are recovered with hexane-diethyl ether (1:1), silver ions and DCF removed16. Each sterol fraction was recrystallised from aq. MeOH, characterised by IR spectra and estimated as complex with acid-FeCl3.'7 P-sitosterol was identified as the major phytosterol in FSM (55.6%) as well as in USF (41.7%), it's amount being 800-824 mg/lOOg of the oil. In spite of common occurrence of sitosterol14'18 its isolation is significant in view of its reported biological properties.

References 1. Elfers J. et al, Flora of Tropical East Africa, 18 (1964)

2. Imbamba S.K., East Afr. Agr. Forest J. 38, 246, (1973)

3. Chopra R.N., Nayar S.L. and Chopra I.C., Glossary of Indian Mdicinal Plants, PID, CSIR, New Delhi, 129 (1956)

4. Usher G., Dictionary of Plants used by Man (1973)

5. Paul S.B. and Roy S., Chemical and Medicinal Perspectives of the seed oil of Cleome gynandra L, IUPAC Conference on Biodiversity and Natural Products: Chemistry and Medical Applications, 294, (2004)

6. Jones PJ.H. et al, Can. J. Physiol. Pharmacol. 75, 217-27, (1997).

7. Ramsamy P. et al, Investigation of the phytosterols from Dioscorea alar a L for possible triglyceride and cholesterol lowering effects, IUPAC Conference on Biodiversity and Natural Products, 298, (2004)

8. Akihisa T. et al, J. Agric. Food Chem., 48, 2313-19 (2000)

9. Ling W.H. and Jones P.J.H., Life ScL, 57, 195-206, (1995)

10. Pegel K.H., South Afr. J. ScL, 93, 263-8, (1997)

11. Awad et al, The Journals of Nutrition, 130, 2127-30 (2000)

12. Cercaci L et al, J. Chromatogr A., 985, 211-20 (2003)

13. StahlE., Thin-Layer Chromatography, Springer-Verlag, N Y (1969)

14. Beveridge T.H.J, et. al., /. agric. Food Chem., 50, 745, (2002)

15. Marinetti G.V. Ed., Lipid Chromatographic Analysis, 471 (1976)

16. Henderson R.J. and Tocher D.R., Thin-Layer Chromatography, In: Hamilton R.J. et al, Lipid Analysis - A Practical Approach, 85, (1991)

17. Das S.K. and Banerjee A.B., Ind. J. Expt. Biol, 18, 969 (1980)

18. Kalo P. and Kuuranne T, J. Chromatogr A., 935, 237-48 (2001)

(Received 5th June 2006, accepted 2nd July 2006)

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