Collection of the Authenticatd Sample from Leading Manufacturers, Natural Remedies, Bangalore

6. / Brief resume of the intended work:
6.1: Need to the study:
Trigonella foenum grecum Linn belonging to family leguminoseae is widely used in traditional system of medicine and is reported to possess anti-dyslipidemic7, anti-hyperglycemic6-7, anti-inflammatory8, anti-pyretic8, gastro protective activities9. The seed is considered to be potential economic source of diosgenin and yamogenin for the steroid industry. As it is known that sapogenins vary not only in the percentage content but also in their nature with the place of origin of seed.
Numbers of studies are reported on the bioactivities for the pharmacological actions from the extracts of Trigonella foenum seeds, but limited studies have been reported on the isolation and estimation of major constituent. So the present study was aimed at the investigation of major constituents towards standardization. Hence it is an important plant for the scientific studies and is useful to consolidate the new findings.
6.2: Review of literature.
The ethanolic extract of Trigonella foenum yielded a mixture of flavonoid components and steroidal saponins. The flavonoid part on column chromatography using butanol: acetic acid: water:: (4:1 : 5) gives two flavonoid aglycones i.e. quarcetin and luteolin in pure form. The saponin part on hydrolysis yields two steroidal sapogenins i.e. diosgenin and gitogenin which we are identify by paper chromatography, mixed melting point and IR spectra1.
Diosgenin and its epimer 25β-spirosta epimer, yamogenin were isolated from west Pakistan and Moroccan seeds of Trigonella foenum graceum, by hydrolyzing with HCL and extracted with petrol in soxhlet for 2 hours which gave 3 extracts where first and second extracts yielded Diosgenin and its epimer and 3rd extract gives mixture of diosgenin and gitogenin, which were identified by TLC and separated by column chromatography eluting with Benzene : chloroform :: (7 : 3) and confirmed by melting point and UV spectra. Similarly sterols i.e. β-sitosterol and cholesterol were isolated by saponification, extraction with petrol, chromatograped and identified by IR and GLC data2.
The defatted ethanolic extract of Trigonella foenum graceum on successive extraction with different solvents like light petroleum, chloroform, acetone, carbon tetrachloride yielded a mixture of steroidal saponin which on column chromatography on neutral alumina using light petroleum acetone and methanol as a eluents gave 5 saponins named as graecunins B, C, D, E and G. The IR spectra of all saponin showed the absorption of spirostanol side chain i.e. ir ν max (KBr) 980, 920, 900, 860cm-1. These results indicate that all the saponins are spirostanol glycoside. Each saponin on acid hydrolysis yielded diosgenin and sugar which were identified by PC, MP, IR and GLC data3.
Two new furostanol glycosides, trigofoenosides F and G have been isolated has their methyl ethers from the methanolic extract of Trigonella foenum graceum seeds by droplet current chromatography using chloroform : methanol : water
(7 : 13 : 8), butanol : pyridine : water :: (6 : 4 : 3), hexane : ethyl acetate as solvents, and identified by TLC, IR, H-NMR studies4.
6 new furastanol types steroid saponins called trigoneosides were isolated from the methanolic extract of seeds of Egyptian Trigonella foenum graceum. The methanolic extract on Diaion HP-20 column chromatography using water, methanol and acetone gave three fractions which on reverse phase silica gel column chromatography and HPLC yielded trigoneosides Xa, Xb, XIb, XIIa, XIIB, and XIIIa the compound were identified by IR, H-NMR, C13-NMR and Mass spectroscopic analysis5.
The hypoglycemic effect of seeds of Trigonella foenum graceum was investigated in both normal and alloxan induced diabetic rats. The alcoholic extract showed significant reduction in blood sugar levels from 74.33 to 60,56mg% in normal rats and 201.25 to 121.25 mg% in diabetic rats. In addition the extract also showed a favorable effect on glucose disposition in glucose fed hyper glycemic rats6.
From the seed of Trigonella foenum an unusual amino acid, 4-hydroxyisoleucine has been isolated by extract with alcohol, chloroform and butanol and chromatographed using ethyl acetate and methanol in 90 : 10 ratio showed significant decrease in plasma triglyceride level by 33% and total cholesterol by 22% and free acid by 14%, accompanied by an increase in HDL-C/TC ratio by 39% in the dyslipidemic hamster models7.
Anti-inflammatory and anti-pyretic effects of the Trigonella foenum leaves extract showed significant decrease in formalin induced edema in a singe dose of 1000mg/kg and also significantly reduced hyperthermia induced by brewers yeast in 1 and 2 hr after their administration. The result indicated that Trigonellafoenum leaves extract possess anti-inflammatory and anti-pyretic properties in both i.p and p.o administration. Phytochemical studies indicated that alkaloids, cardiac glycosides and phenols as major component in the extract and suggest NSAID-like mechanism for it8.
The gastro protective effect of Trigonella foenum seed compared to omeprazole was studied on ethanol induced gastric ulcer. The aqueous extract and a gel fraction isolated from the seed showed significant ulcer protective effect. The cytoprotective effect is found not only due to the anti-secretary action but also to the effects on mucosal glycoprotein. The fenugreek seeds also prevented the rise in lipid peroxidation induced by ethanol presumably by enhancing anti-oxidant potential of the gastric mucosa there by lowering mucosal injury. These observations show that Trigonella foenum seeds possess anti-ulcer activity9.
6.3: Main objectives of the study:
Extraction of chemical constituents using different solvents.
TLC/HPTLC/HPLC analysis of the extracts and identifying the bands.
Running column chromatography to isolate the major constituents.
Elucidation of structures of the isolated constituents by various spectroscopic methods.
7.
8. / Materials and methods:
7.1: Source of the data:
The required data will be obtained from:
Electronic data (Internet).
Published research papers.
Review articles from journals.
Library of IISC, Govt. College of Pharmacy
Library of Natural Remedies.
7.2 Methods of collection of the data (including sampling procedure if any)

Collection of the authenticatd sample from leading manufacturers, natural remedies, Bangalore.
TLC/HPLC analysis to get data about Column Chromatography elutes.
After these strategic approaches the components, which are separated, will be identified by spectral analysis.

7.3: Does the study require any investigations or intervention to be conducted on patients other human or animals? If so, please describe briefly:
- No –
7.4: Has ethical clearance been obtained from your institute in case of 7.3
-- Not applicable--
List of references:

Varshney IP, Sharma SC. Saponins and Sapogenins : Part XXXII. Studies on Trigonella foenum-grecum Linn Seeds. Jour Indian Chem. Soc. 1966;43(8):564-67.
Fazli FRY, Roland Hardman. Isolation and characterization of steroids and other constituents from Trigonella foenum-graecum. Phytochemistry. 1971;10:2497-2503.
Varhiney IP, Jain DC. Saponins from Trigonella foenum-Graecum leaves. J Natural products. 1984;47(1):44-46.
Rajesh K Gupta, Dharam C Jain, Raghunath S Thakur. Furostanol Glycosides from Trigonela foenum-graecum seeds. Phytochemistry. 1984;23(11):2605-07.
Toshiyuki Murakami, Akinobu Kishi, Hisashi Matsuda, Masayuki Yoshikawa. Medicinal Foodstuffs.XVII. Fenugreek seed.(3): structures of new Furostanol-type steroid saponins, Trigoneosides Xa, Xb, XIb, XIIa, XIIb and XIIIa, from the seeds of Egyptian Trigonella foenum-graecum L. Chem. Pharm. Bull. 2000;48(7):994-1000.
Vats V, Grover JK, Rathi SS. Evaluation of anti-hyperglycemic and hypoglycemic effect of Trigonella foenum-graecum Linn, Ocimum sanctum Linn and Pterocrpus marsupium Linn in normal and alloxanized diabetic rats. J Ethnopharmacol. 2002;79:95-100.

Tadigoppula Narender, Anju Puri, Shweta, Tanvir Khaliq Rashmi Saxena, Geetika Bhatia,et-al. 4-Hydroxyisoeucine an unusual amino acid as antidyslipidemic and anti-hyperglycemic agent. Bioorganic and medicinal chemistry letters. 2006;16:293-296.
Ahmadiani A, Javan M, Semnanian S, Barat E, Kamalinejad M. Anti-inflammatory and anti-pyretic effects of Trigonella foenum-graecum leaves extract in the rat. J Ethnopharmacol. 2001;75:283-286.
Suja Pandian R, Anuradha CV, Viswanathan P. Gastroprotective effect of fenugreek seeds (Trigonella foenum graecum) on experimental gastric ulcer in rats. J Ethnopharmacol. 2002;81:393-97.