FIELD OF INVENTION:
This invention relates to the therapeutic ingredients of seeds of Fenugreek (Trigonella foenum greacum), and more particularly to an extraction process for obtaining a composition with a defined purity of total saponins.
BACKGROUND AND PRIOR ART:
Fenugreek (Trigonella foenum graecum), an annual herb of family Leguminosea, has a long history as both a culinary and medicinal herb. The seeds of Fenugreek are commonly used as a spice in food preparations due to the strong flavour and aroma. The seeds are reported to have restorative and nutritive properties. Fenugreek seeds are used in remedies for diabetes and hypercholesterolaemia in Indian, Arabic and Chinese medicine. Its utility has been proved experimentally in diabetic humans (Sharma and Raghuram, 1990). Seeds are aromatic, bitter, carminative, galactogouge, antibacterial and may be eaten raw or cooked. Bulk of the seed is dietary fiber (50%) and protein (30%) both of which have no taste or flavor. Bitterness is mainly due to the oil, steroidal saponins and alkaloids. For 100 g of mature seeds, there is 30 g protein, 30 g (gel-forming) soluble fiber (15% galactomannan in the endosperm), 20 g insoluble fiber, 7.5 g lipids (6.3 g neutral- mainly triglycerides, 450 mg phospholipids- mainly phosphotidyl choline & phosphotidyl ethanolamine, 135 mg soluble lecithin), 2 g sapogenins (diosgenin, gitogenin, furastanol, yamogenin etc.), 160 mg Ca, 1.5 mg ionizable Fe (total Fe 14 mg), 370 mg phosphorous (phytin 157 mg), 19 mg Na, 530 mg K, 33 mg Cu, 100 meg Cr, 1550 mg Mn, 160 mg Mg, 7 mg Zn, 16 mg S, 165 mg CI, 50 mg choline, 380 mg trigonelline, traces of gentianine & carpaine, 43 mg ascorbic acid, 96 meg carotene, 340 meg thiamine, 290 meg riboflavin, 1100 meg nicotinic acid, 84 meg folic acid of which 14.5 meg is free folic acid and 120 U of heat-labile trypsin inhibitor.
Literature available so far reveals that Fenugreek seeds possess a plethora of benefits under various experimental conditions. The seeds possess significant antidiabetic (Sharma et al., 1996), antiatherosclerotic (Sharma et al., 1996), antiinflammatory (Thakur et al., 1994), antinociceptive (Puri, 1998),
antiulcerogenic (Suja Pandian et al., 2002) and antineoplastic effects (Sur et al, 2001).
Damage to the liver can be successfully prevented or controlled by supplementation with antioxidant substances of plant origin. Fenugreek seeds have antioxidant activity and have been shown to produce beneficial effects such as neutralization of free radicals and enhancement of antioxidant apparatus (Anuradhaand Ravikumar, 1998, 2001). Furthermore, the polyphenols fraction of the seeds was found to inhibit peroxide-induced oxidative damage and prevent haemolysis of erythrocytes in vitro (Kaviarasan et al., 2003). It has been observed that the administration of Fenugreek seeds protects rat liver from alcohol-induced oxidative stress (Thirunavukkarasu et al., 2003). Detailed studies to explore the protective role of the seeds, the effect of the polyphenols extract of the seeds on ethanol-induced cytotoxicity in Chang liver cells and compared it with a standard hepatoprotective drug silymarin. and evaluated this by measuring cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolism, lactate dehydrogenase (LDH) leakage, the ratio of reduced glutathione and oxidized glutathione (GSH/GSSG), thiobarbituric acid reactive substances (TBARS) formation, intracellular reactive oxygen species (ROS) production and apoptosis in Chang liver cells exposed to ethanol in the presence and absence of FPEt and or silymarin. This study provides evidence that fenugreek seeds offer a strong protective effect against ethanol-induced cytotoxicity in Chang liver cells.
Fenugreek contains approximately 4 to 8 % saponins, the main therapeutic ingredient, along others, with 4-hydroxy-isolucein. Saponin-rich extract induces release of testosterone in males, increases secretory functions, induces uterine contractions in females. Biological enlargement of breast tissue has been claimed with the use of whole seeds. Fenugreek saponins are reported to reduce cholesterol, albeit small, via hormone synthesis without effect on triglycerides and bind to dietary lipids. Trigonellin, an alkaloid, is thought to reduce glycosuria in diabetes.
In view of above, attempts have been made to prepare Fenugreek extract rich in
saponins. Reference may be made to the
http://www.kbcincusa.com/Naturallv/Naturally98-01-01.htm. where a
combination of organic solvents and physical conditions may be used to separate oleoresin from seed powder. A novel method (US Patent issued to Vitamed, 1997) uses non-toxic, completely removable solvent to separate the stabilized-defatted-fiber-protein (SDF) powder and the oleoresin in commercial scale.
As per United States Patent 5997877 Fenugreek seeds were converted to flakes and flakes were extracted by solvent under agitator. The solvent used is 95% ethanol. The extraction was conducted with the ratio of fenugreek to solvent 1:3 w/v (12 liters of solvent were used, for 4kg of fenugreek flakes), at 60° C. for 2 hr.
The resultant slurry was centrifuged at 9000 g for 15 main. The supernatant was filtered through a Whatman No. 4 filter paper. The oleoresins were recovered by removing the solvent from filtrate in a rotoevaporator at a temperature of 65 to 70 degrees Celsius. The spent material after extraction, being extracted fenugreek flakes, was dried at room temperature, yielding 3.4 kg of dry flakes and 470.4 grams of oleoresin were yielded, with a diosgenin content of 1.4%.
100 grams of the first extracted dry flakes was combined with an additional 300 ml ot 95% ethanol for a second extraction in an agitator for 120 minutes at 60 degrees Celsius, The slurry was again centrifuged to separate the solids from the solvent portion. After drying the solids at room temperature, 92.9 grams of second extracted dry flakes were yielded.
The second filtrate was rotoevaporated again at 65 to 70 degrees Celsius, yielding an additional 4.0 g of oleoresin.
The second example of the aforesaid patent refers that Fenugreek seeds were tempered, over-fraction, were extracted in an agitator for 4 hours at 60 degrees Celsius, using water as the solvent at a ratio of 1:30 w/v. As a sulfiting agent, sodium bisulfite was used (containing 58.5% SO2), and was added to the hot water at a volume of 0.13%.
After this extraction, the slurry was centrifuged, to yield heavy and light components. The light phase from the centrifuge was precipitated by mixing
with an equal amount of anhydrous ethanol, the precipitated gum produced thereby then recovered by further centrifugation.
The recovered crude gum (heavy phase from the centrifugation of the precipitated solvent) was then mixed with ten times the amount of starting fenugreek over-fraction of a washing solution, being anhydrous ethanol at a low speed using a warring blender for one minute. The washed gum solids were then recovered by centrifugation or filtration. The washing step was carried out twice. The recovered clean gum solids were then dried at room temperature with continuous agitation. Various drying methods including oven drying, freeze drying and vacuum drying were attempted but it was found that agitation drying at room temperature yielded the whitest product at the lowest cost. 39.5 grams of dry product was yielded.
However the third example of the aforesaid patent refers recovery of soluble fiber from previously extracted Fenugreek Seed.
US Patent No. 5,997,877 relates to a process for the recovery of substantially pure extracts (oleoresins and saponins) from fenugreek seed using solvent. The solvent used is a polar alcohol, for example an alkyl alcohol such as methanol, ethanol, propanol, isopropanol and their solvent water mixtures. The ratio of fenugreek flakes to solvent is in the range of 1:2 to 1:20 (w/v).
But the present invention is directed to obtain an composition containing saponins and 4- hydroxyl isoleucine
United States Patent 7338675 , titled "Fenugreek seed bio-active compositions and methods for extracting same", describes a method for extracting a composition of bio-active compounds from fenugreek seeds, wherein the method comprises the steps of: (1) providing a plurality of fenugreek seeds; (2) preparing the fenugreek seeds; and (3) extracting a composition of bio-active compounds from the fenugreek seeds, wherein the bio-active compounds comprise 4-hydroxyisoleucine and one or more amino acids selected from the group consisting of arginine, aspartate, threonine, serine, glutamate, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tryptophan, phenylalanine, ornithine, lysine, histidine and gamma-amino butyrate. The composition of bio-active compounds and methods for extraction of same
preferably include between about 10% and 70% of 4-hydroxyisoleucine and between about 20% and 40% of the amino acids.
Francis et al in his article entitled "The biological action of saponins in animal systems: a review" published in British Journal of Nutrition (2002), 88, 587-605 talks about different strategies available for the isolation of Saponins which usually begins with the extraction of the plant material with aqueous methanol or ethanol. Further processing of the extract is carried out after evaporation under reduced pressure, dissolution in a small amount of water and phase separation into n-butanol. However he also mentions it is currently recognized that this step is sometimes undesirable, since only those saponins with short oligosaccharide side chains will eventually be extracted into the butanolic phase.
Similarly Makkar et al in his paper entitled "Plant secondary metabolites" talks about secondary metabolites of plant origin particularly saponin extraction using 50% aqueous methanol, chloroform and the extract is then treated with aqueous solution with equal volume of n-butanol.
US Publication No. 20050238738 and WO2005084323 relates to compositions of bio-active compounds derived, isolated, and/or extracted from Fenugreek containing between about 10% and about 90% of the 4-hydroxyisoleucine, other amino acids and one or more members selected from the group consisting of alkaloids, glycosides, volatile oils, saponins, sapogenins, mannans, flavonoids, fatty acids, vitamins and provitamins, minerals, and carbohydrates.
Thus as can be seen from the above prior art, there is a need for an extraction process that results into a composition containing 40% saponins and 10 % 4-hydroxyl isoleucine which has many therapeutic applications.
OBJECTS OF THE INVENTION:
The principal object of the present invention is to provide an extraction process for the production of free flowing powder of Fenugreek saponins with 4- hydroxyl isoleucine.
Another object of the present invention is to provide produce an extract of the defined content of saponins using the aforesaid process.
Yet another object of the present invention is to provide a process that
reduces the long operation span.
Still another object of the present invention is to provide a cost effective
process.
SUMMARY:
Accordingly the present invention provides an extraction process for
producing Fenugreek extract containing not less than 40% saponins along with
4-hydroxy isoleucine from Fenugreek seeds. The extract is rich in constituents
responsible for protection of cell structure and function from the toxic effects of
ethanol and the utility of Fenugreek seeds in the treatment of alcoholic liver
disease. The process provides composition with enhanced acceptability effects
for the user and as pharmaceutical and nutritional products thereof.
In a preferred embodiment of the present invention, an extraction process for a composition containing not less than 40% of Saponins and not less 10 % 4-hydroxyisoleucine wherein the said process comprises the following steps:
(a) grind sun dried fenugreek seeds, sieve the powdered mass to obtain fine
material;
(b) extract the fine material of step (a) with 80% low molecular weight alcohol repeat this step thrice at a temperature;
(c) filter the extract obtained in step (b) and mix ;
(d) distill the extract of step (c) under vacuum;
(e) extract the aqueous phase obtained in step(d) with n-butanol;
(f) separate out the mixture obtained in step (e) into aqueous and butanol phase by leaving the mixture at 10° C for 3 hours time duration and distill the aqueous phase;
(g) dry the soft mass obtained in step (f).
In another embodiment of the invention an extraction process wherein the amount of solvent used for extraction is four times of the starting material.
In yet another embodiment of the invention an extraction process wherein the solvent used for extraction is selected from the group comprising low molecular alcohol such as methanol, ethanol, propanol.
In still another embodiment of the invention, an extraction process wherein
the temperature for the extraction step is ranging from 20 to 35°C.
In another embodiment of the present invention, an extraction process
wherein the temperature for the distillation of alcoholic extract is ranging from
40 to 55°C.
In yet another embodiment of the present invention, an extraction process
wherein the amount of n-butanol used for treating the aqueous phase is ranging
from 250 ml to 500 ml.
In still another embodiment of the present invention, an extraction process
wherein the temperature for the distillation of aqueous phase is ranging from 60
to75°C.
In another embodiment of the present invention, an extraction process
wherein the said process yields a composition containing 40-44% saponins and
10-11% 4-hydroxy isoleucine.
BRIEF DESCRIPTION OF TABLES:
It is to be noted, however, that the appended drawings and tables illustrate only
typical embodiments of this invention and are therefore not to be considered for
limiting of its scope, for the invention may admit to other equally effective
embodiments.
Table 1 showing comparative summary of the examples.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention discloses a method for the production of extract having 40% Fenugreek saponins with 4-hydroxyisoleucine of 10% concentration from Fenugreek seeds.
Fenugreek {Trigonella foenum graecum) Hindi Name: Methi Dana is an annual herb that belongs to the family Leguminosea. The seeds obtained for the present invention from the INA Market, New Delhi.
4-Hydroxyisoleucine is an amino acid extracted from the Fenugreek seeds which is devoid of characteristic smell and bitter taste that of Fenugreek seeds
and leaves. As Fenugreeks extract it is a mixture of sterio-isomers, lactones with other amino acids present in Fenugreek seeds. These natural active chemicals extracted from Fenugreek seed are highly unstable when exposed to air and moisture, starts to degrade. These are stabilized for long shelf life and packed under Nitrogen atmosphere. Anti-diabetic properties of fenugreek seed are linked to the presence of this free amino acid (4-hydroxyisoleucine). It has been proved as significant insulin-promoting material of benefit to anyone who desires to regulate blood glucose. In non insulin-dependent diabetics, standardized 4-hydroxyisoleucine extracts can help to maintain a stable glucose level. Additionally, 4-hydroxyisoleucine is of use to those who wish to reduce weight gain, and for athletes whose insulin demands typically exceed those of non-athletes.
In the present invention pass the seeds of Fenugreek through Aptrition Mill. Separate the flour as fine powder and fiber, by sieving through 10 mesh sieve. Extract the powdered mass with a combination of water and low molecular weight primary alcohol at a temperature in the range of 20-35° C, with 4 times solvent of the starting material (w/v). Repeat this step thrice. It was established that under aforesaid operating parameters after third extraction the herb completely exhaust for saponins content.
Filter the extract and mix. Then distill the filtrate at a temperature in the range of 40-55° C under vacuum, till foaming appears. Extract the aqueous phase with n-butanol at once under agitation, at 10°C. Allow the solution to stand for 3 hours at the same temperature and n-butanol phase separates out. Distill the aqueous phase at temperature to 60-75° C, till the dissolved solids are higher than 70%. Dry the resulted soft-mass under vacuum at 60-75°C, for 8-12 hours. The bright yellow free flowing powder has saponins more than 40% and 4-hydroxyisoleucin not less than 10%.
Analysis of powder was done: The extract was analysed by gravimetric method. Gravimetric analysis was done by the following method: weigh accurately the dry extract powder, add methanol-LR grade, and reflux on water bath for 1 hour. Cool and filter. Repeat the process two times more in the same manner. Combine all the three filtrates, reduce to 15 to 20 ml, by distillation and add the same drop-wise with continuous stirring to acetone in a pre-weighed dish. Allow the precipitate to settle for an hour. Decant the upper layer and dry
the precipitate portion on a Water bath, with avoiding bumping. Finally dry in an oven at 80°C to 90°C. Cool it in desiccators and weigh, percentage of saponin content (w/w) is calculated, against the weight taken in the beginning.
TLC Examination of isomers of 4-Hydroxy-isoleucine; The standard of 4-hydroxyisoleucine was obtained from Chromadex (Product ID ASB 000008504-005). Both sample and standard were dissolved in water, accurately weighted. Samples and standard were spotted on plates of silica gel G type 60 (Merck) 250u. thick, different concentration, using Hamilton micro syringe. Plate was developed either solvent A [n-propanol: ammonia solution 34% (7:3)] or B [phenol: water (3:1)]. Mobile phase was allowed to run for the distance of 15cm, TLC plate was dried at 105°C and spots were developed with ninhydrin (0.3 gm in 100ml n-butanol and 3ml glacial acetic acid). The evaluation was made on the basis of spot intensity and area against the standard preparation.
The invention is described in detail with reference to the example given below. The example is provided just to illustrate the invention and therefore, should not be construed to limit the scope of the invention.
EXAMPLE 1:
2.0 Kg Fenugreek seeds, previously dried on Sun light at day temperature 35-37°C, were grounded to a powder, using Aptrition Mill. The powdered mass was sieved through 10 mesh to separate fiber and fine material. The fine material was extracted with water having 80% low molecular weight alcohol, especially ethanol, at 28°C, with 8 Liter solvent at each, at thrice. The extract was filtered and mixed. The filtrate was distilled out at 50° C temperature, under vacuum, till foaming appears. The aqueous phase was extracted with 250 ml of n-butanol, under agitation at 10°C and solution was allowed to stand for 3 hours at the same temperature and n-butanol phase was separated out. The aqueous phase was distilled at temperature to 60° C, till dissolved solids were 72.04%. Dry the resulted soft-mass under vacuum at 60° C, for 12 hours. The bright yellow free flowing powder, 125.8 gm, has saponins 43.6% and 4-hydroxyisoluecine 10.76%
For analysis of saponins; weigh accurately 3 gm of the dry extract powder, add methanol-LR grade, 50ml and reflux on water bath for 1 hour. Cool and filter. Repeat the process two times more in the same manner. Combine all the three
filtrates, reduce to 15 to 20 ml, by distillation and add the same drop-wise with continuous stirring to 50ml acetone in a pre-weighed dish. Allow the precipitate to settle for an hour. Decant the upper layer and dry the precipitate portion on a Water bath, with avoiding bumping. Finally dry in an oven at 80°C to 90°C. Cool it in desiccator and weigh, percentage of saponin content (w/w) is calculated, against the weight taken in the beginning.
TLC Examination of isomers of 4-Hydroxyisoleucine: Both sample and standard were dissolved in water, separately, previously accurately weighted. Different cone, of Sample preparation and standard preparation were spotted on plates of silica gel G type 60 (Merck) 250u-thick, using Hamilton micro syringe. Plate was developed either solvent A [n-propanol: ammonia solution 34% (7:3)] or B [phenol: water (3:1)]. Mobile phase was allowed to run for the distance of 15cm, TLC plate was dried at 105°C and spots were developed with ninhydrin (0.3 gm in 100ml n-butanol and 3ml glacial acetic acid). The evaluation was made on the basis of spot intensity and area against the nearest matching spot of standard preparation}.
EXAMPLE 2:
4.0 Kg Fenugreek seeds, previously dried on sun light temp 35-37°C are grounded to powder, using Aptrition Mill. The powdered mass was sieved through 10 mesh to separate fiber and fine material. The fine material was extracted with water having 80% low molecular weight alcohol, especially ethanol, at 35°C, with 16 Liter solvent at each, at thrice. The extract was filtered and mixed. The filtrate was distilled out at 55°C temperature, under vacuum, till foaming appears. The aqueous phase was extracted with 500 ml of n-butanol, under agitation at 10°C and solution was allowed to stand for 3 hours at the same temperature and n-butanol phase was separated out. The aqueous phase was distilled at temperature to 75°C, till dissolved solids were 76%. Dry the resulted soft-mass under vacuum at 70°C, for 9 hours. The bright yellow free flowing powder, 244 gm, has saponins 43.41% and 4-hydroxyisoleucine 10.57%
For analysis of saponins weigh accurately 3 gm of the dry extract powder, add methanol-LR grade, 50ml and reflux on water bath for 1 hour. Cool and filter. Repeat the process two times more in the same manner. Combine all the three filtrates, reduce to 15 to 20 ml, by distillation and add the same drop-wise with
continuous stirring to 50ml acetone in a pre-weighed dish. Allow the precipitate to settle for an hour. Decant the upper layer and dry the precipitate portion on a Water bath, with avoiding bumping. Finally dry in an oven at 80°C to 90°C. Cool it in desiccator and weigh, percentage of saponin content (w/w) is calculated, against the weight taken in the beginning.
TLC Examination of isomers of 4-Hydroxyisoleucine: Both sample and standard were dissolved in water, separately, previously accurately weighted. Different cone, of Sample preparation and standard preparation were spotted on plates of silica gel G type 60 (Merck) 250u thick, using Hamilton micro syringe. Plate was developed either solvent A [n-propanol: ammonia solution 34% (7:3)] or B [phenol: water (3:1)]. Mobile phase was allowed to run for the distance of 15cm, TLC plate was dried at 105°C and spots were developed with ninhydrin (0.3 gm in 100ml n-butanol and 3ml glacial acetic acid). The evaluation was made on the basis of spot intensity and area against the nearest matching spot of standard preparation}.
EXAMPLE 3:
3.0 Kg Fenugreek seeds, previously dried on sun light temp35- 37°C were grounded to a powder, using Aptrition Mill. The powdered mass was sieved through lOmesh to separate fiber and fine material. The fine material was extracted with water having 80% low molecular weight alcohol, especially ethanol, at 20°C, with 12 Liter solvent at each, at thrice. The extract was filtered and mixed. The filtrate was distilled out at 40° C temperature, under vacuum, till foaming appears. The aqueous phase was extracted with 375 ml of n-butanol, under agitation at 10°C and solution was allowed to stand for 3 hours at the same temperature and n-butanol phase was separated out. The aqueous phase was distilled at temperature to 70° C, till dissolved solids were 74.3 %. Dry the resulted soft-mass under vacuum at 68° C, for 10 hours. The bright yellow free flowing powder, 186.8 gm, has saponins 42.87% and 4-hydroxyisoleucine 10.55%
For analysis of saponins weigh accurately 3 gm of the dry extract powder, add methanol-LR grade, 50ml and reflux on water bath for 1 hour. Cool and filter. Repeat the process two times more in the same manner. Combine all the three filtrates, reduce to 15 to 20 ml, by distillation and add the same drop-wise with continuous stirring to 50mI acetone in a pre-weighed dish. Allow the precipitate
to settle for an hour. Decant the upper layer and dry the precipitate portion on a Water bath, with avoiding bumping. Finally dry in an oven at 80°C to 90°C. Cool it in desiccator and weigh, percentage of saponin content (w/w) is calculated, against the weight taken in the beginning.
TLC Examination of isomers of 4-Hydroxyisoleucine: Both sample and standard were dissolved in water, separately, previously accurately weighted. Different cone, of Sample preparation and standard preparation were spotted on plates of silica gel G type 60 (Merck) 250u. thick, using Hamilton micro syringe. Plate was developed either solvent A [n-propanol: ammonia solution 34% (7:3)] or B [phenol: water (3:1)]. Mobile phase was allowed to run for the distance of 15cm, TLC plate was dried at 105°C and spots were developed with ninhydrin (0.3 gm in 100ml n-butanol and 3ml glacial acetic acid). The evaluation was made on the basis of spot intensity and area against the nearest matching spot of standard preparation}.
EXAMPLE 4:
2.0 Kg Fenugreek seeds, previously dried on sun light temp 35- 37°C were grounded to a powder, using Aptrition Mill. The powdered mass was sieved through 10 mesh to separate fiber and fine material. The fine material was extracted with water having 80% low molecular weight alcohol, especially ethanol, at 30°C, with 8 Liter solvent at each, at thrice. The extract was filtered and mixed. The filtrate was distilled out at 40° C temperature, under vacuum, till foaming appears. The aqueous phase was extracted with 250 ml of n-butanol, under agitation at 10°C and solution was allowed to stand for 3 hours at the same temperature and n-butanol phase was separated out. The aqueous phase was distilled at temperature to 68° C, till dissolved solids were 72.07 %. Dry the resulted soft-mass under vacuum at 75° C, for 8 hours. The bright yellow free flowing powder. 126.02 gm, has saponins 42.48% and 4-hydroxy isoleucine 10.6%
For analysis of saponins weigh accurately 3 gm of the dry extract powder, add methanol-LR grade, 50ml and reflux on water bath for 1 hour. Cool and filter. Repeat the process two times more in the same manner. Combine all the three filtrates, reduce to 15 to 20 ml, by distillation and add the same drop-wise with continuous stirring to 50ml acetone in a pre-weighed dish. Allow the precipitate to settle for an hour. Decant the upper layer and dry the precipitate portion on a
Water bath, with avoiding bumping. Finally dry in an oven at 80°C to 90°C. Cool it in desiccator and weigh, percentage of saponin content (w/w) is calculated, against the weight taken in the beginning.
TLC Examination of isomers of 4-Hydroxyisoleucine: Both sample and standard were dissolved in water, separately, previously accurately weighted. Different cone, of Sample preparation and standard preparation were spotted on plates of silica gel G type 60 (Merck) 250u thick, using Hamilton micro syringe. Plate was developed either solvent A [n-propanol: ammonia solution 34% (7:3)] or B [phenol: water (3:1)]. Mobile phase was allowed to run for the distance of 15cm, TLC plate was dried at 105°C and spots were developed with ninhydrin (0.3 gm in 100ml n-butanol and 3ml glacial acetic acid). The evaluation was made on the basis of spot intensity and area against the nearest matching spot of standard preparation}.
Table-1: Comparative summary of the aforesaid examples

(Table Removed)
Numerous modifications and adaptations of the system of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the true spirit and scope of this invention. REFERENCES:
1. Anuradha, C. V. and Ravikumar, P. (1998) Anti lipid peroxidative activity of seeds of fenugreek (Trigonella foenum graecum). Medical Science Research 26, 317-321.
2. Anuradha, C. V. and Ravikumar, P. (2001) Restoration of tissue antioxidants by fenugreek (Trigonella foenum graecum) seeds in alloxan-diabetic rats. Indian Journal of Physiology and Pharmacology 45, 408-420.
3. Kaviarasan, S., Vijayalakshmi, K. and Anuradha, C. V. (2004) Polyphenol-rich extract of fenugreek seeds protect erythrocytes from oxidative damage. Plant Foods for Human Nutrition 59, 143-147 .
4. Puri, D. (1998) Therapeutic potential of fenugreek. Indian Journal of
Physiology and Pharmacology 42, 423-424.
5. Sharma, R. D. and Raghuram, T. C. (1990) Hypoglycaemic effect of fenugreek seeds in non-insulin dependent diabetic subjects. Nutrition Research 10, 731-739.
6. Sharma, R. D., Sarkar, A., Hazra, D. K. et al. (1996) Hypolipidaemic effect of fenugreek seeds: a chronic study in non-insulin dependent diabetic patients. Phytotherapy Research 10, 332-334.
7. Suja Pandian, R., Anuradha, C. V. and Viswanathan, P. (2002)
Gastroprotective effect of fenugreek seeds (Trigonella foenum graecum) on
experimental gastric ulcer in rats. Journal of Ethnopharmacology 81, 393-397.
8. Thakur, A. M., Sarvaiya. J. G., Bhavsar, S. K. et al. (1994) Studies on anti
inflammatory activities of trigonella in rats. In Update Ayurveda 75, Bombay,
India.

WE CLAIM:
1. An extraction process for a composition containing not less than 40% of
Saponins and not less 10 % 4-hydroxy isoleucine wherein the said process
comprises
(a) grind sun dried fenugreek seeds, sieve the powdered mass to obtain fine material;
(b) extract the fine material of step (a) with 80% low molecular weight alcohol repeat this step thrice at a temperature;
(c) filter the extract obtained in step (b) and mix ;
(d) distill the extract of step (c) under vacuum;
(e) extract the aqueous phase obtained in step(d) with n-butanol;
(f) separate out the mixture obtained in step (e) into aqueous and butanol phase by leaving the mixture at 10°C for 3 hours time duration and distill the aqueous phase;
(g) dry the soft mass obtained in step (f).

2. The extraction process as claimed in claim 1 wherein the amount of solvent used for extraction is four times of the starting material.
3. The extraction process as claimed in claim 1 wherein the solvent used for extraction is selected from the group comprising low molecular alcohol such as methanol, ethanol, propanol.
4. The extraction process as claimed in claim 1 wherein the temperature for the extraction step is ranging from 20 to 35 ° C.
5. The extraction process as claimed in claim 1 wherein the temperature for the distillation of alcoholic extract is ranging from 40 to 55° C.
6. The extraction process as claimed in claim 1 process wherein the amount of n-butanol used for treating the aqueous phase is ranging from 250 ml to 500 ml.
7. The extraction process as claimed in claim 1 wherein the temperature for the distillation of aqueous phase is ranging from 60 to 75 ° C.
8. The extraction process as claimed in claim 1 wherein the said process yields an composition containing 40-44% saponins and 10-11% 4-hydroxy isoleucine.
9. An extraction process for a composition containing not less than 40% of Saponins and not less 10 % 4-hydroxyisoleucine and composition substantially as herein described with reference to the tables and examples accompanying this specification.