DESC:FIELD OF THE INVENTION

The present invention relates to an extraction process from seeds of Trigonella foenum -graecum. The present invention also provides formulation of the active compounds extracted and its therapeutic use. The invention relates to 12-Z-octadecenoic acid and octadec-14-enoic acid. The active compounds are isolated from T. foenum seeds, and its pharmaceutically acceptable salts and pro-drugs thereof, to processes for their preparation, to pharmaceutical compositions comprising the same and to their use for preventing and treating disorders of carbohydrate or lipid metabolism, including diabetes mellitus (type 1 and type 2 diabetes), pre-diabetes, and Metabolic Syndrome.

BACKGROUND OF THE INVENTION

Diabetes mellitus (DM) is a metabolic disorder resulting from a defect in insulin secretion, insulin action, or both leading to chronic hyperglycemia. It is often accompanied with disturbances of carbohydrate, fat and protein metabolism and severe diabetic complications such as retinopathy, neuropathy, nephropathy, cardiovascular complications, and ulceration. WHO projects diabetes to be the 7th 54 leading cause of death afflicting up to 366 million globally with 79.4 million individuals being affected by 2030. An effective therapeutic approach for management of diabetes and obesity is to decrease hyperglycemia by retarding and reducing the digestion of ingested carbohydrates. Inhibition of carbohydrate degrading enzymes significantly reduces postprandial increase in blood glucose after a meal by delaying starch hydrolysis. This suppression of postprandial hyperglycemia delays the progression of vascular complications associated with DM. One such enzyme, human pancreatic a-amylase (HPA, a-1,4-glucan-4-glucanohydrolase, E.C. 3.2.1.1) plays a pivotal role in DM. It catalyses the initial step in hydrolysis of starch to maltose which is eventually degraded to glucose by a-glucosidases. Hence, retardation of starch digestion by HPA inhibition plays a key role in the control of postprandial hyperglycemia in type-II DM. By inhibiting HPA in the small intestines, the rate of hydrolysis of starch is, decreased delaying the digestion process. This spreading of digestion process reduces the amount of glucose generated and released in the blood and is one of the effective strategies in lowering postprandial hyperglycemia. The currently available treatments have side effects such as hypoglycemia, weight gain and other complications, which necessitate the need for the development of new anti-diabetic targets and therapies for glycemic control. The inability of current therapies to control hyperglycemia without any side effects along with its high cost and poor availability impels the search towards traditional herbal remedies, which may provide valuable leads and therapeutic strategies. Also, HPA inhibitors have been, reported to be devoid of side effects. The use of natural plant products as a complementary approach for management of DM is growing with >1200 plants being reported to have anti-diabetic effects. The key obstacles, which have restricted the utilization of alternative medicines, are their lack of proper documentation, stringent quality control, identification of key bioactive components and their mechanism of action. Moreover, only a few comprehensive studies on scientific validation of traditional antidiabetic medicinal plants are known and thus offer an attractive source of HPA inhibitors.

Trigonella foenum-graecum is also known as Fenugreek. Fenugreek (Trigonella foenum-graecum) commonly known as Bird's foot, Greek hayseed, trigonella, bockshornsame, Methi, and huluba, is herb known in the art of integrative medicine.

The fenugreek plant belongs to the leguminous family and is an annual plant. The plant is native to Western Asia and has since spread widely over Asia, Europe, North Africa and the Middle East. Fenugreek has been cultivated since ancient times and has many uses in traditional medicine.

Fenugreek is used both as a herb (the leaves) and as the seeds. Fenugreek and products thereof are traditionally used as a demulcent, laxative, lactation stimulant. Fenugreek is a common constituent in the Ayurvedic medicine. Fenugreek and products thereof have been proposed for treatment conditions as diverse as alopecia, arthritis, cancer, diabetes, gastrointestinal disorders, high cholesterol, induce childbirth, infections, inflammation, stimulation of lactation, lymphadenitis, muscle pain, promote urination, skin ulcers, wound healing. The mechanism of action is not well characterized.

According to references cited at Memorial Sloan-Kettering Cancer Center's site regarding use of herbs in integrative medicine, the following substances are identified in fenugreek: Alkaloids: Trigonelline (yields nicotinic acid with roasting), gentianine, carpaine, choline; Proteins and amino acids: 4-Hydroxyisoleucine, histidine, lysine, arginine; Flavonoids: Apigenin, luteolin, orientin, vitexin, quercetin; Saponins: Graecunins, fenugrin B, fenugreekine, trigofoenosides A-G; Steroidal sapogenins: yamogenin, diosgenin, smilagenin, sarsasapogenin, tigogenin, neotigogenin, gitogenin, neogitogenin, yuccagenin; Fiber: Gum, neutral detergent fiber; Other: coumarin, lipids, vitamins, minerals.

One of the constituents of fenugreek, saponin is a mild detergent and used in applications such gently clean ancient manuscripts and textiles. In research, the membrane permeabilizing properties are used in intracellular histo-chemistry staining applications to allow antibody access to intracellular proteins due to the membrane.

In WO 2009/057125, extracts from fenugreek seeds are used to provide a dietary supplement. In this case, the hydrocolloids are extracted together with proteins from crushed seeds using various solvents and/or aqueous solutions.

Allen Robert et al. had performed the extraction isomerization during hydrogenation. The isomerization that takes place during the catalytic hydrogenation of linoleic acid and Me linoleate produces cis and trans 9-, 10-, 11-, and 12-monoenes. The double bond at the 12-position appears to hydrogenate slightly faster than that in the 9-position. More octadecanoic acids with double bonds at 10- or 11-positions are produced during "selective" (high temp., low pressure) hydrogenation than in a nonselective process. Although the degree of selectivity of the hydrogenation is determined by the amount of isomerization of the original pentadiene system to a conjugated diene, only part of the methylene-interrupted diene goes through this type of isomerization even during a highly selective hydrogenation. The half hydrogenation-dehydrogenation reaction mechanism is applied to explain the simultaneous positional and geometrical isomerizations

WO2014207038 describes process for the catalytic hydrogenation of vegetable oils wherein the oil is placed in contact with molecular hydrogen in the presence of a catalyst comprising supported metal Palladium, characterized in that said process is performed in the presence of an amount of water comprised between 5: 1 and 100: 1 with respect to the weight of metal Palladium.

EP2417446 had disclosed a method for characterizing phytochemicals present in an extract, said method-comprising steps of: a) sample preparation for extraction of phytochemicals; and metabolites b) Liquid chromatography and Mass spectrometry.

In 1973, Fowden et al., in Phytochemistry 12:1707-1711, 1973, reported the presence of (2S,3R,4R)-4-hydroxy-3-methylpentanoic acid (4-hydroxyisoleucine) in the seeds of fenugreek (Trigonella foenum-graecum), an annual herbaceous plant that is widespread in regions of Asia, Africa, and Europe. Its absolute configuration was subsequently restudied and corrected as being (2S,3R,4S) by Alcock et al. in Phytochemistry 28:1835-1841, 1989. It has been demonstrated that (2S,3R,4S)-4-hydroxyisoleucine possesses insulinotropic and insulin sensitizing activities (see Broca et al., Am. J. Physiol. 277:E617-E623, 1999; Broca et al., Eur. J. Pharmacol. 390:339-345, 2000; Broca et al., Am. J Physiol. Endocrinol. Metab.287:E463-E471, 2004) and that compound has since been developed for the treatment of diabetes (U.S. Pat. No. 5,470,879; PCT publication Nos.WO 97/32577, WO 01/15689, and WO-2005/039626). Although methods for the preparation of (2S,3R,4S)-4-hydroxyisoleucine have been described, see for example U.S. Patent Application Publication No. US 2003/0219880; Rolland-Fulcrand et al., Eur. J. Org. Chem. 873-877, 2004; and Wang et al., Eur. J. Org. Chem. 834-839, 2002, no one has ever disclosed synthetic analogs of 4-Hydroxyisoleucine, let alone analogs useful for the prevention and/or treatment of metabolic diseases such as diabetes.

However, there is a need in the society for a simple and feasible extraction process for the extraction of active compounds from seeds of Trigonella foenum-graecum and formulation of the same.

The present invention relates to the specific extraction process from seeds from Trigonella foenum-graecum and formulation of the same as described herein the specification.

In view of the above, there is an important need for alternative and improved compounds for preventing and treating disorders of carbohydrate or lipid metabolism, particularly diabetes.

There is also a need for pharmaceutical compositions and therapeutic methods of stimulating glucose uptake and/or of stimulating insulin secretion.

The present invention provides such compounds along with methods for their use. Accordingly, the present invention fulfills the above-mentioned needs and also other needs as it will be apparent to those skilled in the art upon reading the following specification.

SUMMARY OF THE INVENTION

A primary object of the present invention is to provide a novel process for extraction of the active compound(s) from seeds of Trigonella foenum-graecum, which is simple, and yet provide good yield and purity.

The object of the present invention is to provide a novel process for extraction of pure active compound(s) 12-Z-octadecenoic acid and octadec-14-enoic acid from seeds of Trigonella foenum-graecum.

Another object of the present invention is to provide a formulation prepared from the extracted compound(s) from seeds of Trigonella foenum-graecum.

Another object of the present invention is to provide a formulation of isolated 12-Z-octadecenoic acid, the active compound from T. foenum seeds, as a-amylase inhibitor have therapeutic applications.

Another object of the present invention is to provide a formulation of isolated 12-Z-octadecenoic acid, the active compound from T. foenum seeds, as ?-amylase inhibitor which can be administered by any suitable method, such as oral, parenteral (for example, intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), inhalation spray, nasal, vaginal, rectal, sublingual, urethral (for example, urethral suppository) or topical routes of administration (for example, gel, ointment, cream, aerosol, etc.).

Another object of the present invention is to provide a formulation of isolated octadec-14-enoic acid, the active compound from T. foenum seeds, as a-amylase inhibitor have therapeutic applications as hypoglycemic agents.

Another object of the present invention is to provide a formulation of isolated 12-Z-octadecenoic acid and octadec-14-enoic acid, the active compound from T. foenum seeds, as a-amylase inhibitor have therapeutic applications as hypoglycemic agents and also facilitate in weight loss by inhibiting carbohydrate absorption, enhancing lipolysis, and normalizing the metabolism of glucose in a human.

Another object of the present invention relates to 12-Z-octadecenoic acid and octadec-14-enoic acid, pharmaceutically acceptable salts, and prodrugs thereof isolated from seeds of Trigonella foenum-graecum, to processes for their preparation, and to pharmaceutical compositions comprising the same.

Another object of the present invention wherein 12-Z-octadecenoic acid and octadec-14-enoic acid isolated from seeds of Trigonella foenum-graecum stimulate both glucose uptake and insulin secretion are useful for the prevention and treatment of disorders of carbohydrate or lipid metabolism, including diabetes mellitus (type 1 and type 2 diabetes), pre-diabetes, Metabolic Syndrome and obesity

DETAILED DESCRIPTION OF TH DRAWINGS

Fig.1: Analytical HPLC chromatogram of bioactive fraction obtained from column Chromatography.
Fig 2: Sigmoidal plot of HPA relative enzyme inhibition (%) versus varying concentration Z-Octadec-12-enoic acid.
Fig 3: LineWeaver-Burk plot for HPA inhibition at varying Z-octa-12-enoic acid concentration with starch as substrate.
Fig. 4 Secondary plot for HPA inhibition at varying Z-octa-12-enoic acid concentration.
Fig. 5 LineWeaver-Burk plot for HPA inhibition at varying Z-octa-12-enoic acid concentration with maltopentaose as substrate.
Fig. 6 Bowden plot for HPA inhibition at varying concentration of Z-octa-12-enoic acid with maltopentaose as substrate.
Fig. 7. Cytotoxicity and pancreatic inhibition in AR42J cell line.
Fig.8 Changes in serum biochemical parameters on oral administration of BDMC, C.longa isopropanol extract, azadiradione and gedunin to diabetic rats.
Fig 9. Changes in serum biochemical parameters on oral administration of BDMC, C.longa isopropanol extract, azadiradione and gedunin to diabetic rats.
Fig 10. Changes in serum biochemical parameters on oral administration of BDMC, C.longa isopropanol extract, azadiradione and gedunin to diabetic rat.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of the present invention is all about an extraction method from seeds of Trigonella foenum-graecum, which yields, to a pure active compound or mixture of compounds.

Main embodiment of the present invention is to provide extraction process from seeds of Trigonella foenum-graecum.

Final extraction product may contain crude extract, mixture of specific compounds or a single pure compound. Preferably Z-octa-12-enoic acid and Octadec-14-enoic acid are present in pure form or in the form of mixture. The extraction can be done from any part of Trigonella foenum-graecum plant, however preferably from seeds.

The term “disorder of carbohydrate metabolism” is meant a metabolic disorder in which the subject having the disorder cannot properly metabolize sugars. Examples of such disorders include, for example, diabetes mellitus (type 1 and type 2), pre-diabetes, hyperglycemia, impaired glucose tolerance, Metabolic Syndrome, glycosuria, diabetic neuropathy and nephropathy, obesity, and eating disorders.

The term “disorder of lipid metabolism” is meant a metabolic disorder in which the subject having the disorder cannot properly metabolize, distribute and/or store fat. Examples of such disorders include, but are not limited to type 2 diabetes, pre-diabetes, and Metabolic Syndrome.

The term “effective amount” is meant the amount of a compound required to treat or prevent a disorder of carbohydrate or lipid metabolism, such as, for example, diabetes and Metabolic Syndrome. The effective amount of active compound(s) used to practice the present invention for therapeutic or prophylactic treatment of conditions caused by or contributed to by a disorder of carbohydrate or lipid metabolism varies depending upon the manner of administration, and the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. An effective amount can also be that which provides some amelioration of one or more symptoms of the disorder or decreases the likelihood of incidence of the disorder.

The term “elution step” used herein is the process of extracting one material from another by washing with a solvent.

One aspect of the invention relates to the preparation of an extract of plant material of a least one plant of the genus Trigonella. In the preferred embodiment said plant material is obtained from Trigonellafoenum-graecum.

The method of preparing said extract according to the invention comprises:
1. preparing a mixture of plant material and liquid,
2. incubating said mixture,
3. heating of said mixture,
4. recovering a liquid extract from mixture eg. by separating remaining plant material from the mixture.

The plant material may be whole plant, leaves, seeds or roots of said plant or combinations of said plant materials. The plant material may be fresh, frozen, dried or combinations thereof. The preferred embodiment the plant material is seeds of Trigonella foenum-graecum, most preferably dried seeds of said plant.

The extraction process as used herein is performed on the seed part of Trigonella foenum-graecum plant. Dried form of seeds of Trigonella foenum-graecum to be used for the extraction process and needs to be cleaned properly before using. Dried seed of Trigonella foenum-graecum is to be milled or ground before using for extraction process by a known method of milling or grinding.

In order to facilitate the extraction of the active ingredients of the plant material, said plant material is soaked in a liquid preferably water. The mixture of liquid and plant material is incubated for at least 3 hours, more preferably at least 6 hours, preferably at least 12 hours, such as at least 24 hours. The incubation is usually performed at temperatures between 0 and 45°C, suitably at temperatures between 10 and 40 °C.

Subsequently, the mixture comprising the plant material soaked in a liquid is heated, preferably to a temperature above 65°C. In a certain aspect, the mixture is boiled.

The mixture according to the invention comprises plant material and a liquid. The ratio by weight of said plant material and said liquid in said mixture 1 to 1, or preferably less plant material by weight such as 1 to 2, or less plant material by weight such as 1 to 3, or less plant material by weight such as 1 to 4, or less plant material by weight such as 1 to 5, or less plant material by weight such as 1 to 6, or less plant material by weight such as 1 to 7, or less plant material by weight such as 1 to 8, or less plant material by weight such as 1 to 9, or less plant material by weight such as 1 to 10. In a preferred embodiment, the ratio by weight of said plant material and said liquid is 1 to 6.

During the heating of the mixture additional liquid may be added at least once in order to compensate for evaporated liquid and liquid taken up by the plant material. The liquid is heated for at least 5 minutes, such as 10 to 45 minutes, more preferably 20 to 30 minutes, such as 20 minutes. The heating may be terminated when the embryo is released from the seeds, which is associated with increased viscosity of the mixture.

The extraction process involves extraction of seeds by polar and non-polar solvents. The solvent which can be used for the extraction process of the present invention can be selected from the group consisting of aliphatic alcohols such as methanol, ethanol and butanol and water.

The extract of the invention may be purified to isolate the active ingredient(s) by any appropriate method. Thus, the extract may be fractioned using column chromatography filtration, extraction, precipitation, etc. In a presently useful method the extract is fractioned using HPLC. In a specific method the active ingredient(s) is included in an extract fraction obtainable by column chromatography (silica gel,60-120 mesh, column size 30cm and eluted successively with cyclohexane, dichloromethane, ethyl acetate and methanol (30ml each) in a binary gradient starting from cyclohexane and DCM (1:0 to 0:1), DCM and Ethyl acetate(1:0 to 0:1) followed by Ethyl acetate and methanol (1:0 to 0:1) and collecting the fraction at the time interval between 5 and 10 min.

The extract obtained by the method according to the invention is particularly useful for the indications described herein, which are believed to reflect the profile of the active ingredient(s) released from the plant material to the extract in term of quality and quantity.

The plant material (seed) to be extracted in polar to non-polar solvent on an increasing degree of non-polarity. The different extracts obtained sequentially were cold water, hot water, and methanol or any other combination of polar to non-polar solvent.

The polar solvent may be 2-methyl-i-propanol, methyl isoamyl ketone, n-butyl acetate, methyl isobutyl ketone, tetrahydrofuran, 2,6-lutidine, ethyl acetate, isopropanol, chloroform, cyclohexanone, methyl ethyl ketone, methyl n-propyl ketone, 2-picoline, dioxane, ethanol, nitroethane, pyridine, acetone, methoxy ethanol, acetic acid, acetonitrile, methanol, nitromethane, m-cresol; and/or water.

The nonpolar solvent may be squalane, isooctane, n-decane, 1,1,2-trichlorotrifluoroethane, cyclohexane, n-hexane, pentane, cyclopentane, heptane, petroleum ether, carbon disulfide, n-butyl chloride, carbon tetrachloride, dibutyl ether, triethylamine, diisopropyl ether, toluene, o-xylene, p-xylene, methyl t-butyl ether, bromobenzene, chiorobenzene, iodobenzene, o-dichlorobenzene, diethyl ether, benzene, dichloromethane, ethyl bromide, fluorobenzene, ethylene dichloride, isopentanol, ethylene chloride, 2-propanol, n-butanol, n-propanol, and/or tert-butanol.

Active compounds as used herein in the specification means the compound and mixture of compound resulted after the extraction process. The active compound can be either single compound or mixture of compounds which are having some therapeutic activity. The active compounds are preferably Z-octa-12-enoic acid and Octadec-14-enoic acid

In one embodiment, the present invention also provides a formulation of the active compounds extracted and its therapeutic use.

In another embodiment, formulations comprising active compounds described herein can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The formulations can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active compounds into formulation ready to use.

Active compounds can be formulated in the compositions per se, or in the form of a prodrug, hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as described herein. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.

In another embodiment, a formulation comprises active compounds and at least one excipient selected from diluent, binder, disintegrant, lubricant, preservative, emulsifier, anti-oxidant, thickener, stabilizer or mixtures thereof.

Formulation of the present invention may be administered by any suitable method, such as oral, parenteral (for example, intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), inhalation spray, nasal, vaginal, rectal, sublingual, urethral (for example, urethral suppository) or topical routes of administration (for example, gel, ointment, cream, aerosol, etc.).

Active compounds may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic acceptable carriers, adjuvants, excipients and vehicles appropriate for each route of administration. In addition, the formulation of the present invention can be used for treating humans.

The final product of extraction process is further characterized and analyzed by different parameters and models.

In one embodiment, the present invention also provides the method of treating diabetes in human and animals by administration of extracts alone or formulation of extracts in an appropriate form.

The following examples illustrate the invention in specific ways. These examples are not intended to be a limitation on the scope of the invention in any respect and should not be so construed. In addition, the following examples are encompassed by the claims and their equivalents.

EXAMPLES
Example 1 : Isolation and Extraction the bioactive component from T. foenum

The plant material (100g) was successively extracted in polar to non-polar solvent on an increasing degree of non-polarity. The different extracts obtained sequentially were cold water, hot water, and methanol. Methanol extract was further solvent fractionated with hexane. Distilled water was added to the plant material in a ratio of 1:4 (w/v) and kept at 300C (24 h) and 550C (2 h) at 130 rpm for cold and hot water extracts, respectively. For each solvent, the extract was filtered and centrifuged at 10,000 rpm at 40C and the residue collected for subsequent solvent extraction. The organic solvent methanol was added in a ratio of 1:3 (w/v) and refluxed with the residue for 3 h at their respective boiling temperatures. Hexane was added to 60 % of methanol extract in a ratio of (1:3 (v/v) and extracted. Each filtered extract was concentrated in vacuo in a rotary evaporator at 500C and stored at -200C. Stock solutions for inhibition assay were prepared by dissolving upto50 mg of each extract in 1 mL of DMSO and appropriately diluted before use a-amylase inhibition assay.
Hexane fraction (1 gram) was subjected to column chromatography (silica gel,60-120 mesh, column size 30cm and eluted successively with cyclohexane, dichloromethane, ethyl acetate and methanol (30ml each) in a binary gradient starting from cyclohexane and DCM (1:0 to 0:1),DCM and Ethyl acetate(1:0 to 0:1) followed by Ethyl acetate and methanol(1:0 to 0:1) . These elute were loaded on TLC. Toluene: Ethylacetate:Formic acid (5:1:3) was used as mobile phase and the spots were visualized by phosphomolybdic acid stain. Fractions showing similar profiles on TLC were pooled, dried, and then reconstituted in DMSO for bioactivity. For initial analysis, bioactive fractions (0.34 mg) was loaded into analytical HPLC C-18 column (25cm X 4.6mm X 5µ) with Acetonitrile: water (90:10) as the mobile phase. The flow rate was maintained at 0.8ml/min and PDA detector was set at 203nm.The column was maintained at 26 °C. Obtained peaks were collected and checked for bioactivity.

Example 2 : Characterization of the isolated bioactive compound
HR-ESI-MS was recorded on an LC system (Dionex Ultimate 3000, Thermofisher scientific, Germany) consisting of an Acquity High-Performance LC and electrospray ionization tandem mass spectrometer (ESI-MS/MS; Bruker Daltonik GmbH, Germany) was used. ESI-MS/MS was performed in the positive mode: capillary. NMR spectra were acquired using a Varian INOVA 500 spectrometer operating at 500 MHz for 1H and 125 MHz for 13C nuclei. Chemical shifts were given in ppm using tetramethylsilane (TMS) as an internal reference. All NMR data processing was done using Mnova software (Mestrelab Research S.L.). Samples for NMR analysis were prepared by dissolving the pure compound in 500 µL of CD3OD (Sigma) and placing the solutions in 5-mm NMR tubes.

Example 3 : Amylase inhibition assay
a-amylase catalyses the endo-hydrolysis of 1,4-a-D glucosidic linkages in the polysaccharides containing three or more 1,4-a-linked D-glucose units into an oligosaccharide mixture including maltose, maltotriose and a number of other a-(1-6) and a-(1-4) oligo dextrans. Maltose one of the products is a reducing sugar can be estimated by DNSA. 3, 5 dinitro salicylic acids are reduced in presence of free carbonyl (C=O) present in reducing sugar to 3-amino-5-nitrosalicylic under alkaline conditions which give absorbance maxima at 540 nm. Thus maltose, the product and in turn the enzyme activity in presence and absence of inhibitor was quantified using the maltose standard graph. The inhibition assay was performed using the chromogenic DNSA method. The total assay mixture composed of 500 µl of 0.02 M sodium phosphate buffer (pH 6.9 containing 6 mM sodium chloride), 0.04 units of PPA / 0.2 units of HPA solution and extracts/lead inhibitor were incubated at 370C for 10 min. After pre-incubation, 500 µl of 1% (v/v) starch solution in the above buffer was added to each tube and incubated at 370C for 15 min. The reaction was terminated with 1.0 mL DNSA reagent, placed in boiling water bath for 5 min, cooled to room temperature, diluted and the absorbance measured at 540nm. The control reaction representing 100 % enzyme activity did not contain any plant extract. To eliminate the absorbance produced by plant extract/lead inhibitor, appropriate controls were also included. One unit of enzyme activity is defined as the amount of enzyme required to release one µM of maltose from starch per min under the assay conditions.

For the determination of the inhibitor concentration at which 50 % inhibition of enzyme activity occurs (IC50), the assay was performed as above except that the inhibitor/plant extract concentrations were varied. The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and calculated by logarithmic regression analysis from the mean inhibitory values. Acarbose was used as a positive control at a concentration range of 6.5 µg-32.8 µg. The IC50 values were defined as the concentration of the extract, containing the a-amylase inhibitor that inhibited 50 % of the PPA/HPA activity.
The other quantifiers were calculated as follows:
% Relative enzyme activity = (enzyme activity of test/enzyme activity of control)*100.
% inhibition in the a-amylase activity = (100 - % relative enzyme activity).

Table 1: Percent yield and PPA inhibition of sequential solvent extracts.
Trigonellafoenumgraceum % Yield PPA Inhibitionat (34 mg/ml)
Cold Water Extract 39.6% NI
Hot Water Extract 9.57% NI
Methanol Extract 4.75% 22%
Residual Weight 4.87% NI
Hexane fraction 1% 82%

Of the sequential solvent extracts, methanol extract exhibited 22 % inhibition. Further solvent fractionation with hexane extract exhibited 82 % inhibition.

Example 4; Silica column chromatography
1 gram of hexane fraction was subjected to column chromatography (silica gel,60-120 mesh,column size 30cm and eluted successively with cyclohexane, Dichloromethane, Ethyl acetate and methanol(30ml each) in a binary gradient starting from Cyclohexane and DCM(1:0 to 0:1),DCM and Ethyl acetate(1:0 to 0:1) followed by Ethyl acetate and methanol(1:0 to 0:1) . These elute were loaded on TLC (image). Toluene: Ethylacetate: Formic acid (5:1:3) was used as mobile phase and the spots were visualized by phosphomolybdic acid stain. Fractions showing similar profiles on TLC were pooled, dried, and then reconstituted in DMSO and bioactivity was tested. The fraction eluted in 80% DCM and 20% EA gradient showed similar profile and 73% inhibition whereas other fractions collected did not show any inhibition.

Example 5: Analytical HPLC
For initial analysis, bioactive fraction(0.34 mg)was loaded into analytical HPLC C-18 column (25cm X 4.6mm X 5µ) with Acetonitrile:water (90:10) as mobile phase. The flow rate was maintained at 0.8ml/min and PDA detector was set at 203nm.The column was maintained at 26 °C. Obtained peaks were collected and checked for bioactivity. The collected peak was further re-injected into analytical column to check its profile. Graph shown in Fig.1

Table 2.Peak profile and PPA inhibition of Analytical HPLC chromatogram.
Peaks Retention time PPA inhibition (34 mg/ml)
1 4.889 NI
2 7.149 NI
3 8.776 70%
4 11.907 82%
5 14.781 NI
Example 6: Kinetics of enzyme inhibition
The mode of inhibition of HPA by the lead inhibitors was determined using Michaelis-Menton and Lineweaver-Burk equations. Starch (1-5 mg/ml) and maltopentaose (0.05-0.4 mM) were incubated with inhibitors-HPA for 10 and 2.5 min, respectively and the residual enzyme activity determined by DNSA method for starch and Nelson-Somogyi’s method for maltopentaose. The inhibitor constants (Ki) were determined on the basis of mode of inhibition.

Example 7: In vivo animal studies
All the pre-clinical animal studies were conducted at National Toxicology Centre, Pune, India. Five week old male Sprague-Dawley rats weighing 120–170 g were used for experiments. The animal room environment was controlled with a temperature of 22 ± 2°C and day/night cycles of 12 hours light, 12 hours dark (19:00–7:00). Filtered tap water and regular animal non-purified diet from Maharashtra Chakan Food Mill, Pune were supplied. Animals were allowed to acclimate to the laboratory environment for 10 days, weighed 3 times per week, and randomly assigned to the different study groups (6 animals per group ) The protocols were approved by the following ethics committee: Institutional Animal Ethics Committee (IAEC), National Toxicology Centre, APT Research Foundation, Pune, Maharashtra, India

Example 8: Antidiabetic activity of the crude extract and lead inhibitors
Forty-two animals randomly divided into seven groups were given the extract/lead inhibitor molecule as mentioned in table 2.1 for 28 days. Body weight and fasting blood glucose was measured once in every week using the Accu-Chek® Advantage-II Glucose meter (Roche Diagnostics, Manheim, Germany). A sample (1 mL) of the blood from each animal was collected on 0, 14 and 28th day in EDTA-containing tubes (1.5 mg mL-1) to be used for hematology measurement. The remaining blood samples were used to prepare sera that were stored at -80°C until used. The biochemical serum parameters like total protein (TP), triglycerides, cholesterol, high density lipoprotein (HDL), urea, creatinine, serum glutamic oxaloacetic transaminase (SGOT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), total bilirubin and a-amylase from days 0, 14 and 28th days were measured in NTC using Pathozyme and Coral clinical system diagnostic kits on a semi-automated Biochemistry Analyser Smart-7 from Pathozyme. After 28 days the animals were sacrificed by decapitation under light ether anesthesia.

Example 9: Statistical analysis
Each in vitro experiment was repeated as 3 independent sets with each set in triplicates. Results are expressed as means S.E.M. with a number of observations (n). The best-fit values were achieved by applying either linear fit or non-linear least square regression using the software, Microcal Origin 6.0 (Microcal Software Inc., Northampton, USA). Data was analyzed by student’s t-test using SPSS statistical package SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at p < 0.05. For in vivo experiments, n=6 was taken. The data was analyzed by Bonferroni’s Multiple comparison tests with 95 % confidence limit. Differences were considered statistically significant at p < 0.05.

As given in Fig 8, Fig 9 & Fig 10, Changes in serum biochemical parameters on oral administration of partially purified hexane extract (HE) and 12-Z-octadecenoic acid were taken. Changes in A. Blood glucose level, B. Total proteins, C. Triglycerides, D. cholesterol, E. LDL, F. Urea, G. Creatinine, H. SGOT, I. Alkaline phosphatase, J. Total Bilirubin, K. Lactate dehydrogenase, L. a-amylase on the administration of the partially purified hexane and purified compound for a period of 28 days. n=6; (p < 0.05) were considered to be significant.

The graph indicates as follows:
Normal Control Disease control Acarbose treated Partially purified hexane extract Changes in 14-A. Blood glucose level, 14-B. Total proteins, 14-C. Triglycerides, 14-D. cholesterol, 14-E. HDL, 14-F. Urea, 14-G. Creatinine, 14-H. SGOT, 14-I. Alkaline phosphatase, 14-J. Total Bilirubin, 14-K. Lactate Dehydrogenase, 14-L. a-amylase.
,CLAIMS:We claim,

[Claim 1]. A process for extraction of 12-Z-octadecenoic acid and octadec-14-enoic acid from Trigonella foenum-graecum, said process comprising;
(a) preparing a mixture of plant material and liquid by providing plurality of plant material milled / ground Trigonella foenum-graecum and soaked in liquid;
(b) the said mixture is incubated for 3 hours to 24 hours at temperatures between 0 and 45°C;
(c) heating the said mixture at temperatures between 40 and 100°C for 5 minutes to 45 minutes;
(d) the said mixture heating is terminated on the release of the embryo from the seeds;
(e) the liquid is added to maintain the ratio of plant material and a liquid;
(f) extraction of the Trigonella foenum-graecum plant using a polar or non-polar solvent and;
(g) Purification by column chromatography.

[Claim 2]. A process for extraction of 12-Z-octadecenoic acid and octadec-14-enoic acid from Trigonella foenum-graecum as in claim 1, wherein the primary extraction is carried out
(a) at a temperature range between 10 degree Celsius and 45 degree Celsius;
(b) for a duration between 3 hours to 24 hours, wherein a;
(c) the ratio of seeds to the solvent for each extraction ranges is between 1:1 to 1:10 (w/v); and
(d) a concentration of the extract is carried out by heating at a temperature between 50 degree Celsius to 65 degree Celsius.

[Claim 3]. The process as in claim 2, wherein the polar solvent may be 2-methyl-i-propanol, methyl isoamyl ketone, n-butyl acetate, methyl isobutyl ketone, tetrahydrofuran, 2,6-lutidine, ethyl acetate, isopropanol, chloroform, cyclohexanone, methyl ethyl ketone, methyl n-propyl ketone, 2-picoline, dioxane, ethanol, nitroethane, pyridine, acetone, methoxy ethanol, acetic acid, acetonitrile, methanol, nitromethane, m-cresol; and/or water.

[Claim 4]. The process as in claim 2, wherein the nonpolar solvent may be squalane, isooctane, n-decane, 1,1,2-trichlorotrifluoroethane, cyclohexane, n-hexane, pentane, cyclopentane, heptane, petroleum ether, carbon disulfide, n-butyl chloride, carbon tetrachloride, dibutyl ether, triethylamine, diisopropyl ether, toluene, o-xylene, p-xylene, methyl t-butyl ether, bromobenzene, chlorobenzene, iodobenzene, o-dichlorobenzene, diethyl ether, benzene, dichloromethane, ethyl bromide, fluorobenzene, ethylene dichloride, isopentanol, ethylene chloride, 2-propanol, n-butanol, n-propanol, and/or tert-butanol.

[Claim 5]. The process as in claim 2, wherein the extraction can be done from whole plant, leaves, seeds or roots of said plant or combinations of said plant materials. The plant material may be fresh, frozen, dried or combinations thereof, most preferably dried seeds of said plant.

[Claim 6]. The process for the purification of 12-Z-octadecenoic acid and octadec-14-enoic acid by column chromatography, comprising:
(a) Extracting the components with solvent;
(b) fractionating the extract obtained in the preceding step on the basis of polarity wherein said elute fractions are obtained by chromatography using a silica gel column (column size, 30 cm; 60-120 mesh size);
(c) cyclohexane, dichloromethane, ethyl acetate and methanol gradient system, wherein the gradient system is based on an initial elution step of loading said column with cyclohexane and DCM, and on intermediate elution steps with DCM and Ethyl acetate gradually Ethyl acetate and methanol on a terminal elution step loading said column with Toluene : Ethylacetate : Formic acid; and the spots were visualized by phosphomolybdic acid stain.

[Claim 7]. A formulation prepared from the active compound of extracted compound(s) from seeds of Trigonella foenum-graecum wherein the extract consists isolated 12-Z-octadecenoic acid and octadec-14-enoic acid, the active compound from T. foenumseeds, as a-amylase inhibitors have therapeutic applications as oral hypoglycemic agents.

[Claim 8]. The formulation claimed in claim 9, wherein 12-Z-octadecenoic acid and octadec-14-enoic acid isolated from seeds of Trigonella foenum-graecum stimulate both glucose uptake and insulin secretion.

[Claim 9]. The formulation claimed in claim 9, wherein the formulation is used for the prevention and treatment of disorders of carbohydrate or lipid metabolism, including diabetes mellitus (type 1 and type 2 diabetes), pre-diabetes, and Metabolic Syndrome.

[Claim 10]. The formulation as claimed in claim 9, wherein the said formulation is administered by oral, parenteral inhalation spray, nasal, vaginal, rectal, sublingual, urethral or topical routes.