FIELD OF THE INVENTION
The present invention relates to the preparation of industrially viable phytopharmaceutical composition consisting of 4-Hydroxyisoleucine (12-13%) and Trigonelline (7-8%) and also other bioactive compounds such as Pinitol and Raffinose and isolating 95% pure 4-HIL from the seeds of Trigonella foenum graecum (fenugreek) without using any column chromatography techniques or ion exchange resins and prevention or treatment of Polycystic Ovary Syndrome (PCOS) and its related symptoms by 4-HIL, Trigonelline and their composition.

BACKGROUND OF THE INVENTION
Polycystic ovarian syndrome (PCOS) is a gynecological disorder and results by metabolic and endocrine disturbance. PCOS contributes to infertility and deteriorates various other physiological functions. The prevalence of this disorder among infertile women can be nearly 10-12% in India and represent the unmet medical need and invite attention (Dasgupta and Reddy, J. Postgrad. Med. 54, 115-125, 2008). PCOS can occur in the adolescent and adult women, which is debilitating issue for the child desiring women as well as other aged women. This not only affects the reproductive functions, but also other woman's long-term health in various ways such as insulin resistance, diabetes, heart-attack or stroke and risk of developing endometrial cancer (Daniilidis and Dinas, Hippokratia. 13, 90-92, 2009). This is the consequence of endocrinopathy where the androgens are excessively upregulated, which disturbs the milieu of intra-ovarian as well other physiological functions posing a complicated situation in addition to infertility.
The treatment for the polycystic ovary syndrome in not available; however, insulin sensitizers are being prescribed and laparoscopic ovarian drilling also recommended (Chitra et al., ur. J. Obstet. Gynecol. Reprod. Biol. 221, 135-138, 2018). For child desiring women, the priming with insulin sensitizers are administered before the ovulation induction by clomiphene citrate (El et al., Reprod. Biomed. Online. 29, 119-124, 2014), follicular stimulating hormone (FSH), human chorionic gonadotropin (hCG), as an adjunct therapy (Vitek et al., Minerva Ginecol. 68, 450-457, 2016). However, case is reported as nonresponders and emphasizes the further understanding of new regiments /specific drugs or natural products for the PCOS treatment.
In line of new PCOS therapies, our group has been working on the metabolic syndromes and PCOS is one of them. Our earlier finding 4-hydroxyisolucine (4HIL) isolated from Trigonella feonum graecum has shown ameliorating effect by improving the insulin sensitivity associated with metabolic syndrome. As the metabolic syndrome is growing due to change in the life style, we need the management strategies at the same time and it may be advantageous if we can utilize the already documented human consumed plants or derivatives for the medicinal/ therapeutic value. Polycystic ovarian syndrome (PCOS) is a gynecological disorder and results by metabolic and endocrine disturbance. Trigonella feonum-graecum commonly known as fenugreek belongs to the Leguminosae family, which is one of the widely used herbs in food and medicine (http://cdn.naturaldispensary.com/downloads/A_Research_Review_of_Fenugreek.pdf). Recent research work from our group demonstrated its use in hypoglycemic, hypocholesterolemic and galactagogue properties.
The leaves and seeds of the fenugreek plant are used as powders and extracts for medicine use. Fenugreek seeds contain 45-60% carbohydrates, most of which is a mucilaginous fiber which is 30% soluble and 20% insoluble fiber. It also contains about 20-30% proteins that are high in lysine and tryptophan, a small amount of oils (5-10%), a small amount of pyridine alkaloids (mostly trigonelline), and a few flavonoids, free amino acids, sapogenins, vitamins and volatile oils.
Constituents in fenugreek that are thought to be responsible for its hypoglycemic effects include the testa and endosperm of the defatted seeds called the A-subfraction, the 4-hydroxyisoleucine and the fiber. It is also thought that the saponins in the seeds are transformed in the gastrointestinal tract into sapogenins and this is responsible for the lipid lowering effects.
Reference may be made to an article, Christophe et al., discovered that the major isomer, that is, 2S,3R,4S of 4-hydroxyisoleucine induces insulin secretion through a direct effect on pancreatic ß-cells in rats and humans. 4-Hydroxyisoleucine has shown to possess antidiabetic and antidyslipidemic effects in animal models. Furthermore, 4-HIL was proven to possess glucose dependent insulinotropic and insulin sensitizing effects in in-vivo rodent models. It ameliorates fatty acid-induced insulin resistance and inflammatory response in skeletal muscle cells and also improves insulin resistance by promoting mitochondrial biogenesis and act through AMPK and AKT dependent pathway.
Reference may be made to an article, Fowden isolated 4-HIL for the first time using ion exchange resin column chromatography from the hydro-alcoholic extract of fenugreek seeds (Leslie Fowden Helen M.Pratt Alfred Smith, Phytochemistry 12, 1707-1711, 1973).
Reference may be made to an article, Hajimehdipoor and co-workers reported 0.4% 4-HIL in Iranian fenugreek seeds in their fractions obtained through defatted seeds extraction with hydroalcohol followed by ion exchange resin chromatography (2010 J. Med. Plants, 9(6)) and Y.Sauvaire and co-workers obtained 0.6 grams of 4-HIL from 100 grams of seeds by defatting with hexane followed by aqueous alcohol extraction and subsequent ion exchange resin column chromatography (Yves Sauvaire and Gerard Ribes, US 5,470,879, 1995). A chemical method has been also reported, which involves the treatment of extract with various aromatic aldehydes to convert 4-HIL into its aminal form and hydrolyzing the resultant aminal to 4-HIL, which is not economical and tedious (Tokumasu, EP 2123629 A1, 2009).Total synthesis of 4-HIL also has been reported using eight steps, which is also not useful for large scale synthesis with respect to time and expenditure (Qian Wang, European Journal of Organic Chemistry 834-839, 2002). Another approach is used for the total synthesis of 4-HIL using five steps which involve 1,3-Dipolar cycloaddition of a chiral nitrone derived from (-) -menthone to (E)-1,4-dichlorobut-2-ene was the key step in synthesis of (2S,3S,4R)-4-hydroxyisoleucine, obtained in 21% overall yield with high enantiopurity (Kaïss Aouadi, Tetrahedron Letters 53, 2817-2821, 2012), which is also economically and industrially not viable.
A microbial method has also been explored for the production of 4-HIL, which involves the reaction of L-isoleucine in aqueous solvent in the presence of L-isoleucine dioxygenase and isolate (2S, 3R, 4S) 4-hydroxyl-isoleucine (Tomohiro Kodera, (US8273562 B2, 2012). However, the scaling of production is not yet established. Thus, the existing methods for purification and synthesis of 4-HIL and microbial production have several limitations; therefore, an efficient process is required to purify large quantities in simple and cost effective manner.
Due to above mentioned problems, several groups prepared the 4-HIL containing fractions by multistep process such as defatting the seeds powder with non-polar solvents like hexane or petroleum ether and extracting with various ratios of hydroalcohols and loading on expensive ion-exchange resin containing column-chromatography and eluting with toxic ammonia containing solutions. These methods also have several disadvantages such as expensive, time consuming, laborious, environmental and scale-up problems. For example Ma Yaqiong and Yongqiang Zhao obtained 105 grams of fraction from 5 kg of Trigonella seeds, which contains 20.4% of 4-HIL after repeated column chromatography using ion exchange resins (CN110117232 A, CN110117231A). Further Zhang Keqin and co-workers obtained 4.32 grams of 14.47% of 4-HIL containing fraction from 200 grams of seeds after fractionation with various solvents such as hexane, ethylacetate, butanol and CC using ion exchange resins (CN 1397545 A). Indus Biotech Pvt. Ltd ( 252/MUM/2007) obtained 9 to 10 grams of fraction, in which trigonelline contains 25-26% and remaining 4-HIL and other amino acids from defatted seeds powder (1kg) extraction with alcohols followed by ion exchange resin column chromatography. Innovassynth.Tech. Ltd (421/MUM/2005) and Lee Steve and co-workers could prepare 10-70% 4-HIL containing fractions by repeated column chromatography and ion exchange resins usage (Steve S Lee, PCTUS0314408[WO03/094948 A1], 2003). The 4-HIL concentration increase is proportional to the number of steps, more time and cost. All these processes have less scope for industrial viability. An Indian patent 901/Del/2009 filed Amity University, developed a fraction which contains 40% of saponin and 10% 4-HIL with reference to the seeds.
Recently Srinivasa Reddy developed a new process for the preparation of fenugreek powder with high trigonelline, saponin, 4-hydroxyisoleucine, galactomannans contents, wherein the process comprising steps of raw seed optimization, seed treatment, soaking of seed in water, incubation in growth chamber, termination of seed growth and powder preparation (WO2019/17134/A1); however, the quantity of 4-HIL or Trigonelline or Galactomanan increased in raw sprout material with reference to the raw seeds not determined. Moreover the quantity of active constituents such as 4-HIL, Trigonelline and Galactomannan in raw sprout material will be extremely very low, when compared with extracts prepared by water or organic solvents such as alcohol or hydroalcohols from seeds powder. The patients have to take large quantities of raw sprout material to get the desired biological response due to very low concentration of active constituents.
Trigonelline, a major alkaloid component of fenugreek, has hypoglycemic, hypolipidemic, neuroprotective, antimigraine, sedative, memory-improving, antibacterial, antiviral, and anti-tumor activities, and it has been shown to reduce diabetic auditory neuropathy and platelet aggregation. It acts by affecting ß-cell regeneration, insulin secretion, activities of enzymes related to glucose metabolism, reactive oxygen species, axonal extension, and neuron excitability (Zhou et al., Curr. Med. Chem. 19, 3523-3531, 2012).
Pinitol, a compound isolated from fenugreek plants, has been reported to possess insulin-like properties. The hypoglycemic activity of pinitol was recognized in recent years, pinitol played a positive role in regulating insulin-mediated glucose uptake in the liver through translocation and activation of the PI3K/ Akt signaling pathway in T2DM rats (Gao et al., J. Agric. Food Chem. 63, 6019-6026, 2015). This is the reason that there is a growing interest in the use of pinitol as a food supplement because of its reported efficacy in lowering blood glucose levels with no side effects and nil toxicity. The synthetic approach for the preparation of pinitol is also used which involves starting from the optically pure 7-oxanorbornenic sulfone (+)-. Microbial oxidation approach was also used for the synthesis of (+) – pinitol from benzene involving six steps which gives overall yield of 35%.
Hassanzadeh Bashtian M and co-workers demonstrated about the adjuvant therapy of the fenugreek seeds extract (with metformin) in PCOS women improved the sonographic results and menstrual cyclicity (Hassanzadeh et al., Iran J. Pharm. Res. 12, 475-481, 2013); however, the active principles and quantity required to manage PCOS activity was not studied.
Reference may be made to Patent No: WO 2008107909 entitled: Pharmaceutical compositions comprising trigonelline and 4-hydroxyisoleucine and a process thereof” described about the preparation of a pharmaceutical composition having dopaminergic activity and predicted to be beneficial in other related pharmaceutical activities including PCOS. This invention also focuses on the 4-hydroxyisoleucine and trigonolline or their composition dopaminergic activity, but not to treat or manage PCOS.
Reference may be made to an article, Swaroop et al., Int. J. Med. Sci. 12, 825-831, 2015 reported the beneficial effect of Trigonella foenum-graecum seed extract (fenugreek seed extract, Furocyst, 2 capsules of 500 mg each/day), enriched in approximately 40% furostanolic saponins in an open label, one-arm, non-randomized, post-marketing surveillance study in 50 premenopausal women (18-45 years, BMI<42) diagnosed with PCOS over a period of 90 consecutive days. The furocyst is a saponin enriched fraction devoid of 4-hydroxyisoleucine and trigonelline.
Reference may be made to Patent No: US 20140170128 entitled: Insulinotropic compositions comprising proteins and peptides for the management of glycated hemoglobin and blood glucose describes about the composition with peptides (more than two amino acids) containing 4-hydroxyisoleucine along chromium, magnesium and antioxidant containing composition. Here also the assignee predicted the composition containing peptides could be beneficial for PCOS, but not with 4-hydroxyisoelcueine (Vijai K.Pasupuleti, US 20140170128, 2014).
In view of above, attempts have been made to study the beneficial effect of 4-HIL and Trigonelline for the management of PCOS and development of a phytopharmaceutical composisition consisting of 4-hydroxysisoleucine, trigonelline, pinitol and raffinose in simple method and also 95% pure 4-hydroxyisoleucine without using any column chromatography, HPLC or ion exchange resins

OBJECTIVE OF THE INVENTION
Main objective of the invention is to isolate the >95% pure 4-Hydroxyisoleucine (4-HIL), and prepare 12-13% of 4-Hydroxyisolucine containing fraction along with 7-8% of Trigonelline, Pinitol and Raffinose from Trigonella feonum-graecum seed as a phytopharmaceutical product with much simpler way for the effective and safe management of PCOS and ovulatory induction attributes who desire to bear children along with unmarried or young females without the intent of child desire. At the same time, improving the (normal ovarian folliculogenesis along with corpora lutea health, and ovulation potential) of PCOS in hyperandrogenized.
Another objective of the present invention is to provide an effective and safe inclusion complex for treating polycystic ovary syndrome (PCOS), which is a cause of female sterility, menstrual disorder, acne, excess hair growth, obesity, or the like.
Yet another main objective is to induce ovulation in the PCOS condition with either 4-Hydroxyisoleucine, or Trigonelline, or 4-HIL, Trigonelline, Pinitol and Raffinose containing fraction from the seeds of fenugreek.
Yet another objective is to induce antral follicles associated with hypernadrogenized PCOS using 4-HIL isolated from the fenugreek seeds
Yet another objective is to treat female infertility associated with hypernadrogenized PCOS using Trigonelline isolated from the fenugreek seeds.
Yet another objective is to improve the corpora lutea health in the PCOS by 4-HIL containg fraction from fenugreek seeds.
Yet another objective to ameliorating effect of 4-HIL on the corpora lutea health in the PCOS condition.
Yet another objective is to ameliorating effect of Trigonelline on the corpora lutea health in the PCOS condition
Yet another objective includes to ameliorating effect of 4-HIL on the ovulation rate for the PCOS related anovulation management.
Yet another objective is to provide ovulation potential enhancement by Trigonelline for the management of PCOS associated anovulation.
Yet another objective is to provide ovarian reserve, preantral follicles (primordial and primary follicles) enhancement by 4-HIL.
Yet another objective is providing ovulation potential enhancement by 4-HIL, Trigonelline, Pinitol and Raffinose containing fraction for the management of PCOS associated anovulation.
Yet another objective is to provide a process of preparing 4-Hydroxyisoleucine (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction from the extracts of fenugreek seeds with much simpler way.
Yet another objective is to provide >95% pure 4-Hydroxyisoleucine from the fenugreek seeds extract without using ion exchange resins or column chromatography or HPLC.
Yet another objective is to provide a process of purifying trigonelline from the fenugreek seeds extract.
Yet another objective is to prepare the novel formulation of the 4-Hydroxyisolucine (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction to achieve the better efficacy.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure-1. Purification of 4HIL from the fenugreek seeds without column chromatography or ion exchange resins or HPLC.
Figure-2. Preparation of phytopharmaceutical composition with 12-13% of 4-HIL and with other bioactive compounds such as Trigonelline (7-8%), Pinitol and Raffinose from the fenugreek seeds without column chromatography or ion exchange resins or HPLC (method-1).
Figure-3. Preparation of phytopharmaceutical composition with 12-13% of 4-HIL and 7-8% of Trigonelline and Pinitol and Raffinose from the fenugreek seeds without column chromatography or ion exchange resins or HPLC (method-2).
Figure-4. 1H-NMR spectra of ethylacetate extract
Figure-5. 13C-NMR spectra of ethylacetate extract
Figure-6. 1H-NMR spectra of ethanol extract
Figure-7. 13C-NMR spectra of ethanol extract
Figure-8. 1H-NMR spectra of EtOAc fraction
Figure-9. 13C-NMR spectra of EtOA fraction
Figure-10. 1H-NMR spectra of Phytopharmaceutical composition which contains 4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose
Figure-11. 13C-NMR spectra of Phytopharmaceutical composition which contains 4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose
Figure-12. 1H-NMR spectra of 4-HIL
Figure -13. 13C-NMR spectra of 4-HIL
Figure-14. Mass spectra of 4-HIL
Figure-15. 1H-NMR spectra of Trigonelline
Figure-16. 13C-NMR spectra of Trigonelline
Figure-17. Mass spectra of Trigonelline
Figure-18. 1H-NMR spectra of Pinitol
Figure-19. 13C-NMR spectra of Pinitol
Figure-20. Mass spectra of Pinitol
Figure-21. 1H-NMR spectra of Raffinose
Figure-22. 13C-NMR spectra of Raffinose
Figure-23. Mass spectra of Raffinose
Figure 24: Total ion chromatogram of compound 4-hydroxy isoleucine and trigonelline
Figure 25: Extracted ion chromatogram of 4-hydroxy isoleucine
Figure 26: Extracted ion chromatogram of trigonelline
Figure 27: MS/MS spectrum of trigonelline
Figure 28: MS/MS spectrum of 4-hydroxyisoleucine
Figure-29: Ovarian histology (A) and histological analysis (B) of cystic follicles in the ovary from Non-PCOS, PCOS, PCOS+ Phytopharmaceutical composition (4-HIL, Trigonelline, Pinitol and Raffinose containing fraction) and PCOS+ metformin.
Figure-30: Ovarian histology (A) and histological analysis (B) of atretic follicles in the ovary from Non-PCOS, PCOS, PCOS+ Phytopharmaceutical composition (4-HIL, Trigonelline, Pinitol and Raffinose containing fraction) and PCOS+ metformin.
Figure-31: Ovarian histology (A) and histological analysis (B) of Graafian follicles in the ovary from Non-PCOS, PCOS, PCOS+ Phytopharmaceutical composition (4-HIL, Trigonelline, Pinitol and Raffinose containing fraction) and PCOS+ metformin.
Figure-32: Ovarian histology (A) and histological analysis (B) of Corpus Luteum in the ovary from Non-PCOS, PCOS, PCOS+ Phytopharmaceutical composition (4-HIL, Trigonelline, Pinitol and Raffinose containing fraction) and PCOS+ metformin.
Figure-33: Ovarian histology (A) and histological analysis (B) of cystic follicles in the ovary from Non-PCOS, PCOS, PCOS+4-hydroxyisolucine, PCOS+trigonelline and PCOS+ metformin.
Figure-34: Ovarian histology (A) and histological analysis (B) of atretic follicles in the ovary from Non-PCOS, PCOS, PCOS+4-hydroxyisolucine, PCOS+trigonelline and PCOS+ metformin.
Figure-35: Ovarian histology (A) and histological analysis (B) of antral follicles in the ovary from Non-PCOS, PCOS, PCOS+4-hydroxyisolucine, PCOS+trigonelline and PCOS+ metformin.
Figure-36: Ovarian histology (A) and histological analysis (B) of ovulation indicator, corpus luteum in the ovary from Non-PCOS, PCOS, PCOS+4-hydroxyisolucine, PCOS+trigonelline and PCOS+ metformin.
Figure-37: Ovarian histology (A) and histological analysis (B) of ovarian follicular cysts and corpus luteum from Non-PCOS, PCOS, PCOS+4-hydroxyisolucine, PCOS+trigonelline, PCOS+ metformin and PCOS +Phytopharmaceutical composition (4-HIL, Trigonelline, Pinitol and Raffinose containing fraction) + formulations.
Figure-38: Ovulation rate determination by counting the ovulated oocyte in the DHEA induced-PCOS, DHEA/PCOS+ 4HIL, DHEA/PCOS+ trigonelline, DHEA/PCOS+ phytopharmaceutical composition and non-PCOS rat model.
Figure-39. Ovulated eggs analysis from Non-PCOS, PCOS, PCOS+4-HIL enriched fraction, PCOS+ 4-HIL, PCOS+Trigonelline, phytopharmaceutical composition and PCOS+ metformin.
Figure-40. The preantral follicles, primordial and primary follicle were increased by 4-HIL treatment in the prematurely failed ovarian SD rat model.

BIOLOGICAL RESOURCES USED IN THE INVENTION:
The Fenugreek seeds [scientific name :-- Trigonella foenumgraecum] were purchased from the local market Ms Ashok Kumar & Kanhaiah Kumar, Rakabganj, Lucknow, Uttar Pradesh, India 226004 and used to make the phytopharmaceutical composition.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a process of purification for 4-Hydroxyisoleucine and phytopharmaceutical composition with 12-13% of 4-Hydroxyisoleucine and other bioactive compounds such as Trigonelline (7-8%), Pinitaol and Raffinose from the Trigonella foenum-graecum seeds (Fenugreek seeds) in a simple method without using column chromatography techniques and its formulation thereof as a therapeutic agents for the treatment of PCOS and associated problems, hyperandrogen, ovarian follicular health, corpora lutea, ovulation for not only child desring females but also for unmarried or young females.
The present invention discloses the beneficial effect of 4-HIL and Trigonelline at 50 mg/kg body weight dose for the management of Polycystic Ovary Syndrome (PCOS) in animal models for the first time. Subsequently 12 to 13% of 4-HIL containing fraction was prepared which also contain other compounds such as Trigonelline (7.3% of the fraction), Pinitol and Raffinose. The phytopharmaceutical composition i.e. 4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction found active at 150 mg/kg dose in the same animal model to reduce the cystic follicle and improve the ovulation potential in the PCOS condition. The formulation prepared by us enhanced the efficacy of phytopharmaceutical composition 4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction.
Method of 4-HIL purification without any column chromatography or ion exchange resins or HPLC
A method of isolating pure 4-HIL from the alcoholic extract of fenugreek seeds, including the steps of (Figure-1):
(a) Grinding the seeds of fenugreek plant portions to provide a powder;
(b) Extracting the fenugreek seeds powder with ethylacetate followed by aliphatic alcohols;
(c) Drying the fenugreek ethylacetate and alcoholic extract
(d) Removing fatty acids content from alcoholic extract using ethylacetate by repeated trituration
(e) Removing the moisture content by adding alcohol to defatted material and evaporating the alcohol repeatedly
(f) Re-dissolving the fraction in aliphatic alcohols and filtering the alcohol insoluble compound
(g) Washing the compound repeatedly with aliphatic alcohols to give pure 4-HIL (1.2 to 1.4 g from 1 Kg of seeds powder)
Preparation of phytopharmaceutical composition consisting of 12-13% 4-HIL, Trigonelline (7-8%), Pinitol and Raffinose containing fraction:

A method of making a bioactive phytopharmaceutical composition, comprising a 4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose including the steps of (Figure 2 to 3):
Method 1:
(a) Grinding the seeds of fenugreek to provide a powder;
(b) Extracting the fenugreek seeds powder with ethylacetate;
(c) Evaporating the ethylacetate to provide a fenugreek ethylacetate extract
(d) Percolation of ethylacetate extracted powder with aliphatic alcohols
(e) Evaporating the aliphatic alcohols to give alcoholic extracts
(f) Removing fatty acids content from alcoholic extracts using ethylacetate by repeated
trituration
(g) Dissolving the extract in triple distilled water and filtering
(h) Concentrating the water soluble portion to give 12-13% 4-HIL containing fraction with
Trigonelline (7-8%), Pinitol and Raffinose
Method 2:
(a) Grinding the seeds of fenugreek to provide a powder;
(b) Extracting the fenugreek seeds powder with ethylacetate;
(c) Evaporating the ethylacetate to provide a fenugreek ethylacetate extract
(d) Percolation of ethylacetate extracted powder with aliphatic alcohols
(e) Evaporating the aliphatic alcohols to give alcoholic extracts
(g) Dissolving the extracts in triple distilled water and ethylacetate (70:30 ratio)
(h) Separating the water layer after ethylacetate evaporation
(h) Concentrating the water soluble portion to give 12-13% 4-HIL containing fraction with Trigonelline (7-8%), Pinitol and Raffinose
Quantification of Phytopharmaceutical composition (4-HIL and Trigonelline in fraction) by HPLC-QTOF-MS/MS
HPLC-QTOF-MS/MS was used to quantify the composition. The 4-Hydroxyisoleucine is present in an amount of about 12.9% and Trigonelline is present in an amount of about 7.3% by weight based on the total weight of the composition.
Chemicals and materials
Acetonitrile, methanol (LC-MS grade) and formic acid (analytical grade) were purchased from Fluka, Sigma-Aldrich (St. Louis, MO, USA). Milli-Q Ultra-pure water was obtained from Millipore water purification system (Millipore, Milford, MA, USA). Reference standards of 4-hydroxy isoleucine and trigonelline were obtained by isolation.
Sample preparation
Dried residues of plant sample (approximately 1 mg) were weighed accurately and dissolved in 1 mL of 100% methanol using ultrasonicator. The solutions were filtered through 0.22 µm syringe filter. The filtrates were further diluted with methanol to final working concentration. 100 µL aliquot was injected into the MS system via direct infusion for MS and MS/MS parameter development and optimization. 2 µL aliquot was injected into the UPLC–MS/MS system for analysis.
Preparation of calibration standards solution
Primary stock solutions of compounds 4-hydroxyisoleucine and trigonelline were individually prepared by dissolving the compounds in methanol to achieve desired concentration of 1 mg/mL. A mixed standard stock solution containing both compounds was also prepared in methanol. Then the working standard solutions were prepared by diluting the mixed standard stock solution with methanol to a series of concentrations within the ranges 10, 20, 30, 50, 100, 250, 500, 750 and 1000 ng/mL for plotting calibration curves. The calibration curves were constructed by plotting the value of peak areas versus the value of concentrations of each compound. All stock solutions were stored at -20 °C until use.
Instrumentation and analytical conditions
An acquity ultra-performance liquid chromatography (UPLC) system consisting of an auto sampler and a binary pump equipped with a 10µL loop was used. The compounds were separated on an Acquity UPLC phenyl column (1.7 µm, 2.1×100 mm, Waters, Milford, MA) analytical column at 30?C. The UPLC system was interfaced with hybrid linear ion trap triple-quadrupole mass spectrometer (API 4000 QTRAP™ MS/MS system from AB Sciex). ESI in positive ion mode were used to optimize the parameters, which were as follows: the ion spray voltage was 5500 V, the turbo spray temperature, 550?C; nebulizer gas, 50 psi; heater gas, 50 psi; collision gas, and curtain gas at 50 psi. Optimization of the mass spectrometric conditions were carried out by infusing 100 ng/mL solutions of the compounds dissolved in methanol at 10 µL/min flow rate using a Harvard syringe pump (Harvard Apparatus, South Natick, MA, USA). The full range scan from m/z 100 to 500 in +ESI-MS analysis was recorded. The precursor ion, product ion, corresponding declustering potential (DP), entrance potential (EP), collision energy (CE) and cell exit potential (CXP) were optimized and the most intense product ion from precursor ion of each compound was chosen for the MRM (Table 2). Analyst 1.6.2 software (AB Sciex) was used for data acquisition and data processing.

Table 1: UPLC condition (isocratic)
Time (min.) % A % B Flow rate (mL/min)
Initial 80.00 20.00 0.250
0.5 80.00 20.00 0.250
3.00 80.00 20.00 0.250
A; 0.1% (v/v) formic acid aqueous solution, B; methanol, injection volume; 2µl,

Table 2: Compound dependent parameters (MRM) of 4-Hydroxyisoleucine and Trigonelline
Compound 4-Hydroxy isoleucine Trigonelline
Retention time (min) 0.87 1.03
Precursor (Q1) mass (Da) 137.9 148.2
Product (Q3) mass (Da) 92.0 74.0
Dwell time 200 200
Declustering Potential (eV) 70 35
Entrance Potential (eV) 7.2 7.5
Collision Energy (eV) 31.5 19
Cell Exit Potential (eV) 13 9.5

The structural identification of each compound was carried out on the basis of their accurate mass, molecular formula, and retention time and MS spectra by HPLC–QTOF–MS/MS. 4-HIL and Trigonelline were unambiguously identified by comparing their retention time with authentic standards. The retention time, molecular formulas are summarized in (Table 2). These studies confirmed the presence of 12.9% of 4-HIL and 7.3% of Trigonelline in the Phytopharmaceutical composition (Table 3).
Table 3: Contents (in mg/g) of 4-Hydroxyisoleucine and Trigonelline in Phytopharmaceutical composition (n=3)
Sample 4-hydroxy isoleucine Trigonelline
4-HIL, Trigonelline, Pinitol and Raffinose containing fraction 129.8 mg/g; (12.9%) 73.2 mg/g; (7.3%)
Beneficial effect of Phytopharmaceutical composition and 4-HIL and Trigonelline in Polycystic Ovary Syndrome (PCOS) studies
Results
Ameliorating effect of Phytopharmaceutical composition (4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction), on the ovarian follicular health improvement in PCOS. In PCOS condition, there is an increase in cystic follicles; hence, we determined herein. We administered the 150mg/kg b.wt. 4-HIL containing phytopharmaceutical composition in DHEA-induced PCOS (Ubba et al., Reprod. Sci. 24, 738-752, 2017) in SD rats for 3 weeks and did the histological analysis (Fig. 29). Histologically, the cystic follicles were increased in PCOS condition, one of the characterstics of PCOS, but suppressed in the 150 mg/kg body weight phytopharmaceutical composition treated group for three weeks, suggesting the restoration of normal health of the ovary post-PCOS condition (Fig. 29 A and B).
Phytopharmaceutical composition (4-HIL (12-13%, Trigonelline (7-8%), Pinitol and Raffinose containing fraction) on the ovarian follicular health in PCOS. In the ovarian histological follicular analysis, the atretic follicles are non-responding follicles in the follicular dynamics and is increased in the PCOS, which interestingly were restored to SHAM level by the phytopharmaceutical composition treatment for three weeks (Fig.30 A and B).
Phytopharmaceutical composition (4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction) improves ovarian follicular health, preovulatory follicle; Graafian follicle, in PCOS. In the ovarian histological follicular analysis, in the DHEA induced PCOS group, the Graafian follicles were reduced (Fig.31), but restored in 150 mg/kg body weight phytopharmaceutical composition treatment for three weeks.
Phytopharmaceutical composition (4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction) improves corpus luteum numbers in PCOS. In the PCOS the ovulation is hampered and attains an oligo or anovulatory state; therefore, we assessed the ovulation potential of the 150 mg/kg body weight phytopharmaceutical composition treatment group for three weeks in the PCOS animal group (Fig.32A and B). We assessed the corpus luteum to know the ovulated follicles. Sham showed good number of the corpus luteum, which were reduced upon DHEA induced PCOS condition. The 150 mg/kg body weight dose of 4-HIL treatment increased the numbers of corpus luteum (Fig.32 A and B).
Ameliorating effect of isolated 4-hydroxyisolucine (>95%) and trigonelline (>95%) compounds on the ovarian follicular health, cystic follicles. We examined the isolated pure compound 4-HIL and Trigonelline treatment in a 50 mg/kg body weight dose for 2 weeks on the ovarian follicles in the DHEA-induced PCOS condition. The increased cystic follicle in the PCOS were reduced by said compounds individually (Fig.33 A and B).
Ameliorating effect of isolated 4-Hydroxyisolucine (>95%) compound on the ovarian follicular health, atretic follicles. In the PCOS, the number of atretic follicles were increased by the treatment of the 4-HIL for 2 weeks reduced the same, but not by the Trigonelline (Fig.34 A and B).
Ameliorating effect of isolated 4-Hydroxyisolucine (>95%) and Trigonelline (>95%) compounds on the ovarian follicular health, antral follicles. Antral follicles is seen reduced during PCOS and those are restored by both the compounds at the dose of 50 mg/kg body weight treatment for 2 weeks (Fig.35 A and B).
Ameliorating effect of isolated 4-Hydroxyisolucine (>95%) and Trigonelline (>95%) compounds on the ovulation potential indicator corpus luteum. Further, we checked the response of 4-Hydroxyisolucine and Trigonelline compounds in ovulation indicator, corpora lutea in the DHEA-induced PCOS condition. We found that sham group showed the highest number of corpus luteum, while DHEA group showed the lowest number of luteum. Administration of the compounds 4-hydroxyisolucine and trigonelline compounds increased the number of corpus luteum in comparison to DHEA group as well as metformin group (standard) (Fig. 36 A and B).
Ameliorating effect of Phytopharmaceutical composition (4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction) with formulation on the ovarian follicular health. We determined the ameliorating effect of 4-HIL containing phytopharmaceutical composition (500mg/kg body weight) with 2-Hydroxypropyl Cyclodextrin complex formulation (1:2 wt/wt ratio) on PCOS orally for two weeks post-PCOS development. Interestingly, we found the 4-HIL containing phytopharmaceutical composition in 500mg/kg body weight with formulation in 2-Hydroxypropyl Cyclodextrin complex was equally effective to reduce the cystic ovarian follicles. On the other hand, the ovulation indicator, corpus luteum was increased significantly by administration of 4-hydroxyisolucine containing phytopharmaceutical composition and 2-Hydroxypropyl Cyclodextrin complex in PCOS, which was more potent than then as standard drug, metformin (Fig. 37 A, B and C).
Formulation of the obtained 4-HIL enriched fraction with 2-hydroxypropyl cyclodextrin
A formulation of the obtained 4-HIL enriched fraction with 2-hydroxypropyl cyclodextrin in different wt/wt ratio was prepared by kneading method. The 4-HIL enriched fraction/2-Hydroxypropyl cyclodextrin complex obtained was subjected to measurement of zeta potential, size and PDI analysis Malvern Zetasizer Nano ZS. Percent entrapment efficiency (% EE) of 4 HIL was found to be 93.63 ± 1.42 % as determined by using UV-Vis spectrophotometer and the data is as presented in Table 4.
Table 4: Characterization of 4-HIL enriched fraction/2-Hydroxypropyl cyclodextrin complex
Weight ratio
(4-HIL enriched fraction: 2-hydroxypropyl cyclodextrin) Zeta potential
(mV) Size
(nm) PDI % EE
1:1 -25 ± 4.3 173 ± 5.02 0.46 ± 0.076 78.63 ± 1.92
1:2 -26 ± 3.1 111 ± 0.13 0.25 ± 0.014 93.63 ± 1.42
1:4 -24 ± 4.2 164 ± 4.01 0.41 ± 0.026 91.01 ± 1.15
1:5 -24 ± 3.5 258 ± 7.6 0.38 ± 0.019 85.2 ± 1.77

Ameliorating effect of 4-HIL and trigonelline on the ovulation potential during PCOS. In the DHEA induced-PCOS, the ovulation is dysregulatd and attains an oligo or anovulatory state. Hence, we assessed the ovulation potential of 4HIL and Trigonelline treatment groups for three weeks in the PCOS animal group (Fig.38 A). We assessed the ovulated eggs/oocyte in the fallopian tubes. Sham showed good number (~20) of oocyte in the fallopian tube, which were reduced (~ 6) upon DHEA-induced PCOS condition. The 50 mg/kg body weight dose of 4-HIL treatment increased the numbers of ovulated oocytes (~15). Similarly, trigonelline (50mg/kg b. wt.) exerted the effect (Fig. 38 A and B).

Ameliorating effect of 4-HIL enriched fraction on the ovulation potential during PCOS. In the DHEA induced-PCOS, the ovulation is dysregulated and attains an oligo or anovulatory state. Therefore, we assessed the ovulation potential of 4-HIL enriched fraction along with 4-HIL, and Trigonelline along with a standard (comparator) compound, Metformin treatment groups for three weeks in the PCOS animal group (Fig.39 A). We assessed the ovulated eggs/oocyte in the fallopian tubes. Sham showed good number (~12) of oocyte in the fallopian tube, which were reduced (<1) upon DHEA-induced PCOS condition. The 50 mg/kg body weight dose of 4-HIL treatment increased the numbers of ovulated oocytes (~5). Similarly, Trigonelline (50mg/kg b. wt.) exerted the effect (7). The 4-HIL enriched fraction increased the ovulated eggs to ~4. Metformin group demonstrated (~1.3) oocyte in the oviduct (Fig. 39 A and B).
Ovarian prenatal follicles (primordial and primary follicle) are promoted by 4-HIL in prematurely failed ovary. To assess 4-HIL preantral follicles promoting effect we chose 4-vinylcyclohexene diepoxide based prematurely failed ovarian (premenopausal conditions) SD rat model (Ref). The sham groups showed normal preantral follicles, which were reduced by VCD-induced premature ovarian failure (POF) case and increased by oral treatment of 4-HIL for 3weeks (Fig. 4 A and B), demonstrating the ovarian reserve promoting ability of 4-HIL.
EXAMPLES
Following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.
Example 1:
Preparation of formulations of 4-HIL enriched fraction
4-HIL enriched fraction/2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method, using 4-HIL enriched fraction and 2-Hydroxypropyl cyclodextrin in a weight ratio of 1:1. Briefly, approximately 30 mg of 2-2-Hydroxypropyl Cyclodextrin was dissolved in 10 mL of hydro-alcoholic solution (ethanol:distilled water, 20:80 v/v) at 65 °C for 30 min. 4-HIL enriched fraction in ethanol (10 mL) was added to the hydro-alcoholic solution of 2-Hydroxypropyl cyclodextrin (30 mg) with continuous agitation. Then the mixture was continuously kneaded in a mortar for 45 minutes to form inclusion complex. The resulting mass was kept in vacuum desiccators overnight for evaporation of solvents; dried residue was re-dissolved in water and filtered under vacuum. The sample was lyophilized, weighed, sealed and stored at room temperature for further use.
Example 2
Preparation of formulations of phytopharmaceutical composition (4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction)
4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction/ 2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method, using phytopharmaceutical composition and 2-Hydroxypropyl Cyclodextrin in a weight ratio of 1:1. Briefly, approximately 30 mg of 2-2-Hydroxypropyl Cyclodextrin was dissolved in 10 mL of ethanol and distilled water (20: 80 v/v) at 65 °C for 30 min. 4-HIL containing phytopharmaceutical composition in ethanol was added to the hydro-alcoholic solution of 2-Hydroxypropyl Cyclodextrin with continuous agitation. Then the mixture was continuously kneaded in a mortar for 45 minutes to form inclusion complex. The resulting mass was kept in vacuum desiccators overnight for evaporation of solvents; dried residue was re-dissolved in water and filtered under vacuum. The sample was lyophilized, weighed, sealed and stored at room temperature for further use.
Results:
2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method was subjected to zeta potential, size and PDI analysis. Zeta potential, size and PDI of the formulation were obtained as -25±4.3 mV, 173±5.02 nm and 0.46±0.076 respectively. Percent entrapment efficiency (% EE) was determined by using UV-Vis spectrophotometer and it was found to be 78.63±1.92%.
Example 3
Preparation of formulations of Phytopharmaceutical composition (4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction)/2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method, using 4-HIL containing phytopharmaceutical composition and 2-Hydroxypropyl Cyclodextrin in a weight ratio of 1:2. Briefly, approximately 60 mg of 2-Hydroxypropyl Cyclodextrin was dissolved in 10 mL of ethanol and distilled water (20: 80 v/v) at 65 °C for 30 min. 4-HIL containing phytopharmaceutical composition in ethanol was added to the hydro-alcoholic solution of 2-Hydroxypropyl Cyclodextrin with continuous agitation. Then the mixture was continuously kneaded in a mortar for 45 minutes to form inclusion complex. The resulting mass was kept in vacuum desiccators overnight for evaporation of solvents; dried residue was re-dissolved in water and filtered under vacuum. The sample was lyophilized, weighed, sealed and stored at room temperature.
Results:
2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method was subjected to zeta potential, size and PDI analysis. Zeta potential, size and PDI of the formulation were obtained as -26±3.1 mV, 111±0.13 nm and 0.25±0.014 respectively. Percent entrapment efficiency (% EE) was determined by using UV-Vis spectrophotometer and it was found to be 93.63±1.42%.
Example 4
Preparation of formulations of Phytopharmaceutical composition (4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction)/2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method, using 4-HIL containing phytopharmaceutical composition and 2-Hydroxypropyl Cyclodextrin in a weight ratio of 1:4. Briefly, approximately 120 mg of 2-2-Hydroxypropyl Cyclodextrin was dissolved in 10 mL of ethanol and distilled water (20: 80 v/v) at 65 °C for 30 min. 4-HIL containing phytopharmaceutical composition in ethanol was added to the hydro-alcoholic solution of 2-Hydroxypropyl Cyclodextrin with continuous agitation. Then the mixture was continuously kneaded in a mortar for 45 minutes to from inclusion complex. The resulting mass was kept in vacuum desiccators overnight for evaporation of solvents; dried residue was re-dissolved in water and filtered under vacuum. The sample was lyophilized, weighed, sealed and stored at room temperature.
Results:
2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method was subjected to zeta potential, size and PDI analysis. Zeta potential, size and PDI of the formulation were obtained as -24±4.2 mV, 164±4.01 nm and 0.41±0.026 respectively. Percent entrapment efficiency (% EE) was determined by using UV-Vis spectrophotometer and it was found to be 91.01±1.15%.
Example 5
Preparation of formulations of Phytopharmaceutical composition (4-HIL (12-13%), Trigonelline (7-8%), Pinitol and Raffinose containing fraction)/2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method, using 4-HIL containing phytopharmaceutical composition and 2-Hydroxypropyl Cyclodextrin in a weight ratio of 1:5. Briefly, approximately 150 mg of 2-2-Hydroxypropyl Cyclodextrin was dissolved in 10 mL of ethanol and distilled water (20: 80 v/v) at 65 °C for 30 min. 4-HIL containing fraction in ethanol was added to the hydro-alcoholic solution of 2-Hydroxypropyl Cyclodextrin with continuous agitation. Then the mixture was continuously kneaded in a mortar for 45 minutes to form inclusion complex. The resulting mass was kept in vacuum desiccators overnight for evaporation of solvents; dried residue was re-dissolved in water and filtered under vacuum. The sample was lyophilized, weighed, sealed and stored at room temperature.
Results:
2-Hydroxypropyl Cyclodextrin complex was obtained by kneading method was subjected to zeta potential, size and PDI analysis. Zeta potential, size and PDI of the formulation were obtained as -24±3.5 mV, 258±7.6 nm and 0.38±0.019 respectively. Percent entrapment efficiency (% EE) was determined by using UV-Vis spectrophotometer and it was found to be 85.2±1.77%.
Experimental conditions of biological studies:
Example 6
Experimental PCOS induction in animals by dehydroepiandrosterone and dosing regiments: Rattus norvegicus, Sprague Dawley strain (aged 21 days) was used for this study. The animals were housed in a temperature-controlled facility (25 ± 1°C) with required illumination (12 h light and 12 dark) at CDRI, Lucknow, India. Free access to food and water were provided to the animals. Pre-pubertal female rat (strain Sprague Dawley), 21 days old were divided into four groups having four animals in each group. The PCOS/DHEA group received DHEA (6mg/100g body weight, dissolved in 10% of 95% ethanol and diluted in corn oil) through subcutaneous injection (25 gauge) for 35days. Metformin, 4-HIL and trigonelline animal groups first received DHEA for 21 days and then 22 day onwards oral dose of metformin (30mg/100g body weight, dissolved in sterile water with 1% gum acacia), 4-HIL (50mg/kg body weight, dissolved in sterile water 1% gum acacia) , trigonelline (50mg/kg body weight, dissolved in sterile water 1% gum acacia ) and 4-HIL containing fraction (150mg/kg body weight, dissolved in sterile water 1% gum acacia) using cannula (16 gauge) for three weeks more along with daily injection of DHEA. The control/SHAM group rat received oral or subcutaneous injections of saline or corn oil only (Ubba et al., 2017b).
Example 7
Induction of prematurely failed ovary (POF) model in SD rat: POF was induced in immature female or prepubertal SD rats (21-days old) by 4-Vinylcyclohexene diepoxide (VCD) (Sigma-Aldrich, India) administered intra-peritoneally at a dose of 160mg/kg body weight for a period of 21-days (once daily) (Haas et al., Comp Med. 57, 443-449, 2007; Battiston et al., J Steroid Biochem. Mol. Biol. 165, 170-181, 2017). 4-HIL (50mg/kg body weight, dissolved in sterile water 1% gum acacia) was fed orally using cannula (16 gauge) for three weeks more along with daily injection of VCD.
Example 8
Ovarian tissue histology. We used the haematoxylin and eosin staining method to check the histopathology of the developed PCOS model and treated rat model groups. Ovarian tissues were fixed in 4% PFA (Paraformaldehyde) for 1 hour at 4°C after washing with PBS (Phosphate Buffered Saline). Then dehydrated ovarian tissues using 50% isopropanol for 20 minutes, followed by 70% isopropanol for 20 minutes, 90% isopropanol for 20 minutes and then in 100% isopropanol for 20 minutes, 15 minutes and 15 minutes. After the dehydration process, ovarian tissues were kept in xylene+isopropanol (3:1) solution for 10 minutes and cleaned three times with xylene for 20 minutes each. The tissue fixed in Wax+Xylene (1:1) in cassettes for 20 minutes at 58°C in the Ultrasonic cleaning bath with a heater (Fisher Scientific, Rockford, USA). We incubated the tissues in liquified paraffin wax to allow its permeabialization into the tissue for 2:30 hours. Paraffin blocks were prepared using module and ring (Yorco Sales Pvt. Ltd, New Delhi, India).
Ovarian sections of 5µm were cut serially using microtome (Leica Biosystem, Germany) and mounted on poly-L-lysine coated glass slides. Sections were deparaffinized with two changes of xylene (10 min each) and rehydrated with subsequent changes of absolute alcohol (two times, five min each), 95% (two minute) and 70% alcohol (two minute). Sections were briefly washed with distilled water and stained with haematoxylin solution for one min. After staining, sections were again washed and rinsed in 95% alcohol (10 repeats) and counterstained with 0.5% eosin for 30 second. This was followed by dehydration through 95% and absolute alcohol two times for five minutes each. Finally, the slides were cleared in xylene, two times for five min each and mounted with DPX mount. The tissue sections were imaged under the light microscope (CKX41 Trinocular with Cooled CCD Camera Model Q Imaging MP5.0-RTV-CLR-10-C from Olympus, Tokyo, Japan)(Ubba et al., 2017b).
Example 9
Ovarian follicles characterization. Follicles of every stage were classified according to the following criteria: primordial follicles were identified as follicles with oocytes surrounded by less differentiated squamous 4-8 granulosa cells (GCs) and primary follicles were identified as those with oocytes surrounded by a single layer of cuboidal GCs (Markholt et al., 2012). Secondary follicles were identified as those with oocytes surrounded by two to four complete GC layers and theca cells. Antral follicles were identified as those with oocytes surrounded by complete GC layers and theca cells with visible antrum. Graafian follicles were identified as those with oocytes surrounded by complete GC layer and theca cells with large antrum. Corpus luteum was identified as those with giant luteal cells. Atretic follicles were identified as those with compactly lacked arranged granulosa cells and presence or absence of oocyte. Empty follicles were identified as those without having oocyte (Rodgers and Irving-Rodgers, 2010). Cystic follicles were identified as those with almost lost of granulosa cells and oocytes surrounded by thick theca cells layer (Kafali et al., 2004).
Example 10
Ovulation rate determination. Sprague Dawley strain was used for this particular study. Pre-pubertal female rat (strain Sprague Dawley), 21 days old were divided into four groups having four animals in each group. The PCOS/DHEA group received DHEA (6mg/100g body weight, dissolved in 10% of 95% ethanol and diluted in corn oil) through subcutaneous injection (25 gauge) for 35days. 4-HIL enriched fraction (150mg/kg body weight), Metformin, trigonelline and 4-HIL groups first received DHEA for 21 days followed by continuation of DHEA and oral dose of metformin (30mg/100g body weight, dissolved in sterile water with 1% gum acacia), trigonelline (50mg/kg body weight, dissolved in sterile water 1% gum acacia) and 4-HIL (50mg/kg body weight, dissolved in sterile water 1% gum acacia) respectively using cannula (16 gauge) for 2 more week. The control/SHAM group rat received oral or subcutaneous injections of saline or corn oil only (Ubba et al., 2017b). The PCOS alone group received s.c. dose of DHEA for 35 days. All the animal irrespective of the treatment groups, at 36 day of the animal age, 20 IU of PMSG was administered intra-peritoneally, which was followed by hCG (25 IU) treatment (i.p) after 48 hrs of PMSG. Post-hCG (12 hrs), the ovulation was determined by punchering the ampularry region on the petridish under the inverted phase contrast microscope to count the ovulated oocytes. The procedure was as described earlier (Hirabayashi et al., Exp. Anim. 50, 365-369, 2001) (Popova et al., Mol. Reprod. Dev. 63, 177-182, 2002) (Kon et al., Exp. Anim. 54, 185-187, 2005).

ADVANTAGES OF THE INVENTION
1. Present invention provides a simple and economic process to isolate 4-Hydroxyisoleucine enriched fraction from Trigonella feonum-graecum seeds as a phytopharmaceutical product for the effective and safe management of PCOS and ovulatory induction attributes in females.
2. Present invention provides an effective and safe therapeutic agent for treating polycystic ovary syndrome (PCOS), which is a cause of female sterility, menstrual disorder, acne, excess hair growth, obesity, or the like.
3. Present invention provides a method to induce ovulation in the PCOS condition with either 4-Hydroxyisoleucine, or Trigonelline, or 4-HIL enriched fraction from the seeds of fenugreek.
4. Present invention also provides a novel formulation of the 4-hydroxyisoleucine enriched fraction to achieve better efficacy.
CLAIMS:We Claim:
1. A process for isolation of 4-hydroxyisoleucine (4-HIL) enriched fraction comprising 12-13% by weight of 4-hydroxyisoleucine based on the total weight of the fraction, 7-8% by weight of trigonelline based on the total weight of the fraction, pinitol and raffinose from Trigonella Foenum-graecum (fenugreek) seeds, wherein the process comprises the steps of:
a) collecting and grinding the fenugreek seeds to obtain a powder;
b) percolating the powdered seeds as obtained in step (a) in a first solvent, at least four times under mechanical stirring for a time period in the range of 20 to 24, followed by filtration to obtain solvent insoluble portion as a first extract;
c) percolating the first extract as obtained in step (b) in a second solvent at least four times under mechanical stirring for a time period in the range of 20 to 24h, followed by solvent evaporation to obtain a syrupy second extract;
d) macerating the syrupy extract as obtained in step (c) in a solvent system at least twice to obtain a syrupy mixture;
e) macerating the syrupy mixture as obtained in step (d) in organic solvent or water at least twice, followed by filtration and concentration to obtain water soluble 4-hydroxyisoleucine (4-HIL) or its enriched fraction respectively.

2. The process as claimed in claim 1, wherein the first solvent is ethylacetate and the second solvent is selected from methanol, ethanol or propanol.
3. The process as claimed in claim 1, wherein the solvent system used in step (d) is straight chain alcohols or ethylacetate
4. The process as claimed in claim 1, wherein the aqueous solvent used in step (e) is distilled water.
5. The process as claimed in claim 1, wherein the 4-hydroxyisoleucine (4-HIL) enriched fraction is used in the range of 150-500 mg/kg body weight for the treatment of polycystic ovarian syndrome (PCOS).
6. The process as claimed in claim 1, wherein the 12-13% by weight of 4-hydroxyisoleucine is used in the range of 30-100 mg/kg body weight for the treatment of for the treatment of polycystic ovarian syndrome (PCOS). The process as claimed in claim 1, wherein the 7-8% by weight of trigonelline is used in the range of 30-100 mg/kg body weight for the treatment of for the treatment of polycystic ovarian syndrome (PCOS).
7. An inclusion complex comprising 4-hydroxyisoleucine (4-HIL) enriched fraction as obtained in claim 1 with 2-hydroxypropyl cyclodextrin for the treatment of polycystic ovarian syndrome (PCOS).
8. A process to obtain the inclusion complex as claimed in claim 7, comprising the steps of:
a) dissolving 2-hydroxypropyl cyclodextrin in ethanol:distilled water (20:80 v/v) at 65 °C for 30 min to obtain solution 1.
b) dissolving 4-HIL enriched fraction as obtained in claim 1 in ethanol to obtain solution 2;
c) mixing solution 1 as obtained in step (a) and solution 2 as obtained in step (b) at room temperature with continuous agitation to obtain a reaction mixture; and
d) kneading the reaction mixture as obtained in step (c) continuously at room temperature using mortar for 45 minutes, followed by vacuum desiccation, re-dissolution in water, filtration and lyophilization to obtain the inclusion complex.
9. The inclusion complex as claimed in claims 7 and 8, wherein the ratio of 4-HIL enriched fraction: 2-hydroxypropyl cyclodextrin is in the range of 1:1 to 1:5 (w/w).
10. The inclusion complex as claimed in claims 7-8, wherein the inclusion complex is used in the range of 50 mg/kg to 500mg/kg body weight.
11. The inclusion complex as claimed in claims 7-8, wherein the size of the inclusion complex is in the range of 110-265 nm, zeta potential is in the range of -20 to -30 mV and % entrapment efficacy is in the range of 75-95% .
12. The inclusion complex as claimed in claims 7-8, wherein inclusion complex is administered by oral route for the treatment of polycystic ovarian syndrome (PCOS) where the cystic follicles and atretic follicles were reduced, and at the same time, Graafian follicles and corpus luteum, and ovulation potential is increased. Further, the preantral follicles are promoted by 4-HIL even in the premature ovarian failed condition.