Concentrated Asparagus Racemosus Root Composition and Method for its Preparation

Abstract

This invention discloses a synergistically concentrated asparagus racemosus root extract composition comprising shatavalin I, dehydroshatavalin I, shatavalin IV, and shatavalin IX; a method for its preparation; and methods for treating and using the composition for women's health benefits.

Description

[Technical Field]

[0001] The present invention relates to a synergistically concentrated asparagus racemosus root composition comprising (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV and (iv) shatavalin IX; a method for preparing the same; and a method for using the same for women's health benefits. [Background technology]

[0002] Hormones are chemical messengers produced by various endocrine glands in the body, which are necessary for maintaining various metabolic processes. Imbalance occurs when there is too much or too little of a hormone, and can disrupt many aspects of an individual's life. Women are more prone to such imbalances than men. Fluctuations in hormone secretion are more pronounced during puberty, perimenopause, and menopause. Several factors, including drug treatments, chemotherapy, thyroiditis, and environmental pollutants, can contribute to hormonal imbalances in women, which can lead to infertility, premenstrual syndrome (PMS), polycystic ovary syndrome (PCOS), and polycystic ovary disease (PCOD). Symptoms of hormonal imbalance in women include irregular periods, severe periods, acne, weight gain, hirsutism (excessive facial hair growth), eating disorders, stress, anxiety, diabetes, mood swings, fatigue, and irritability. Pharmacological treatment options for women with hormonal imbalances include hormone replacement therapy (to reduce facial flushing and night sweats), antiandrogens, letrozole (to stimulate ovulation in PCOS conditions), and eflornithine (to slow excessive facial hair growth). However, most of these long-term treatment options have several side effects on the body. Therefore, alternative approaches such as vitamins, nutrients, minerals, and herbal therapies are gaining popularity because they are effective with fewer side effects.

[0003] Hormone therapy, such as estrogen therapy, progesterone therapy, or estrogen-progesterone combination therapy, has proven to be the most effective. However, there are clear risks associated with hormone therapy, including an increased risk of breast cancer, stroke, and heart disease.

[0004] Patent Document 1 discloses an oral dosage form of shatavari containing a mixture of an alcohol extract and an aqueous extract of shatavari (asparagus racemosus) root uniformly impregnated in sugar globules, with a concentration of the active ingredient shatavariin IV of 0.14-0.18%.

[0005] Another patent document, Document 2, discloses a method for solubilizing water-insoluble bioactive compounds and their metabolites using a natural triterpene glycoside as a solubilizing agent. It discloses shatavali glycoside obtained from Asparagus racemosus, which is standardized to 4% w / w (HPLC) or higher for shatavalin IV.

[0006] Patent Document 3 discloses an oligospirostanoside of formula 1,3-0-[α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→4)-0-β-D-glucopyranosyl]-25(S)-5β-spirostan-3β-ol isolated from Asparagus racemosus, and a pharmaceutical composition comprising an effective amount of the oligospirostanoside 3-0-[α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1Δ4)-0-β-D-glucopyranosyl]-25(S)-5β-spirostan-3β-ol contained in a pharmaceutically acceptable carrier.

[0007] Non-patent document 1 discloses a method for the quantitative analysis of saponin glycoside bioactive components in Asparagus racemosus. High-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS / MS) was developed and validated for the simultaneous determination of five saponin glycosides—asparacoside, shatavalin IX, shatavalin IV, asparanin A, and shatavalin V—in A. racemosus extracted with 70% MeOH.

[0008] However, there are no reported extracts of Asparagus racemosus root containing concentrated active phytochemicals. Therefore, in the art, there is still a need to provide improved products of Asparagus racemosus root containing concentrated active phytochemicals, thereby enhancing its efficacy in the problem of women's health treatment with reduced side effects. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Indian Patent Publication No. 202121017695 [Patent Document 2] U.S. Patent Application Publication No. 2014303258 [Patent Document 3] U.S. Patent Publication No. 6649745 [Non-patent literature]

[0010] [Non-Patent Document 1] Churanya Onloma et al(Natural Product Communications,2017;Vol.12(1)) [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The main object of the present invention is to provide an asparagus racemosus root composition comprising (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV, and (iv) shatavalin IX for the health benefits of women; and a method for preparing the same.

[0012] Another object of the present invention is to provide a formulation of a concentrated asparagus racemosus root composition and a method for preparing the same.

[0013] A further object of the present invention is to provide a method and use of a concentrated asparagus racemosus root composition for the health benefits of women. [Means for solving the problem]

[0014] Accordingly, in one embodiment, the present invention provides a synergistically concentrated asparagus racemosus root extract comprising (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV and (iv) shatavalin IX for the health benefits of women.

[0015] In another aspect, the Disclosure provides, for the health benefits of women, a synergistically concentrated asparagus racemosus root extract comprising (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV, and (iv) shatavalin IX; in which shatavalin I is in the range of 0.5 to 15%, dehydroshatavalin I is in the range of 0.1 to 25%, shatavalin IV is in the range of 2 to 25%, and shatavalin IX is in the range of 0.001 to 1.0%.

[0016] In yet another aspect, the present invention provides a method for preparing a synergistically concentrated asparagus racemosus root extract comprising (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV, and (iv) shatavalin IX, the method comprising: (a) concentrating an aqueous extract of asparagus racemosus (shatavari) root by re-extraction with a polar organic solvent, or by loading the aqueous extract onto a macroporous resin and eluting it with aqueous alcohol to obtain a concentrated extract; (b) extracting used or remaining asparagus racemosus (shatavari) root with a polar organic solvent to obtain a second extract; (c) combining the extracts obtained from steps (a) and (b) to obtain a concentrated asparagus racemosus composition; and (d) optionally blending the concentrated asparagus racemosus composition with at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers, and diluents.

[0017] In another aspect, the present disclosure provides a composition / formulation of a synergistically concentrated Asparagus racemosus root extract comprising (i) Shatavarin I (ii) Dehydroshatavarin I (iii) Shatavarin IV (iv) Shatavarin IX, and at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents, or as a food.

[0018] In another aspect, the present invention provides a method for preparing a composition of a synergistically concentrated Asparagus racemosus root extract comprising (i) Shatavarin I (ii) Dehydroshatavarin I (iii) Shatavarin IV (iv) Shatavarin IX, the method comprising the step of blending with at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents, or as a food.

[0019] Also, another aspect of the present disclosure is a method of obtaining at least one female reproductive and / or sexual health benefit selected from promoting or regulating the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances, alleviating symptoms of menopausal disorders including perimenopause, premenstrual syndrome (PMS), menstrual cycle-related discomfort, dysmenorrhea, polycystic ovary syndrome (PCOS), polycystic ovary disease (PCOD), gynecological disorders, and symptoms of urogenital or genital infections; supporting ovarian function, maintaining libido, reducing dyspareunia, and improving lactation; comprising supplementing a human subject in need thereof with an effective dose of a synergistically concentrated Asparagus racemosus root extract composition comprising (i) Shatavarin I in the range of 0.5 - 15%; (ii) Dehydroshatavarin I in the range of 0.1 - 25%; (iii) Shatavarin IV in the range of 2 - 25% and (iv) Shatavarin IX in the range of 0.001 - 1.0%; (v) optionally, at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents, or as a food.

[0020] Another aspect of the present invention involves (i) shatavarin I in the range of 0.5 to 15%; (ii) dehydroshatavarin I in the range of 0.1 to 25%; (iii) shatavarin IV in the range of 2 to 25% and (iv) shatavarin IX in the range of 0.001 to 1.0%; optionally, promoting or regulating the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances, reducing symptoms of perimenopause, premenstrual syndrome (PMS), menstrual cycle-related discomfort, menstrual pain, polycystic ovary syndrome (PCOS), The present invention provides the use of a synergistically concentrated asparagus racemosus root extract composition comprising at least one component selected from pharmaceutically or nutritionally, or dietarily acceptable, excipients, carriers, and diluents for obtaining at least one female reproductive and sexual health benefit selected from: alleviating symptoms of polycystic ovary disease (PCOD), gynecological disorders, and genitourinary or genitourinary tract infections; supporting ovarian function, and maintaining libido, reducing dyspareunia, and improving lactation.

[0021] In a further aspect, the present invention relates to a novel shatavarin not previously reported, i.e., formula; JPEG2026519309000001.jpg43153 Dehydroshatavalin I, 1H NMR(d6-DMSO): δ 5.16(1H,d,J=4.4 Hz),5.13(1H,d,J=4.0 Hz),4.90(1H,d,J=4.8 Hz),4.81-4.85(4H,m),4.72(1H,s),4.62-4.68(4H,m),4.59(1H,d,J=5.2 Hz),4.47(1H,d,J=8.0 Hz),4.41(1H,d,J=5.6 Hz),4.37(1H,t,J=6.4 Hz),4.33(1H,d,J=7.6 Hz),4.27-4.32(1H,m),4.16(1H,t,J=5.6 Hz),4.08(1H,d,J=8.0 Hz),3.64-3.67(3H,m),3.61(1H,brs),3.57-3.58(1H,m),3.37-3.51(7H,m),3.03-3.19(9H,m),2.92-3.01(2H ,m),2.01-2.13(3H,m),1.63-1.84(5H,m),1.55(3H,s),1.36-1.52(9H,m),1.15-1.28(5H,m),1.10(3H,d,J=6.4 Hz),1.06-1.09(2H,m),0.95-1.05(2H,m),0.90(3H,s),0.89(3H,d,J=6.4 Hz), 0.62 (3H,s) (mass (Q-tof, anion mode): m / z 1047.5400 (MH) - ) It provides a shatavarin characterized by the following. [Brief explanation of the drawing]

[0022] [Figure 1] The chemical structure of shatabalin isolated from the root of Asparagus racemosus is shown. [Figure 2] HPLC chromatogram of a concentrated extract composition of Asparagus racemosus. Detailed description of the invention

[0023] The present invention will be described in detail in relation to certain preferred and optional embodiments so that its various aspects may be more fully understood and recognized.

[0024] Unless otherwise specified, all technical and scientific terms disclosed herein, including but not limited to chemical structures, process steps, or materials, are intended, as well as their equivalents as commonly understood by those skilled in the art to which the invention pertains. For the purpose of describing the invention, certain terms are specifically defined herein as follows:

[0025] Throughout this specification, the terms “concentrated asparagus racemosus root extract,” “concentrated asparagus racemosus root extract composition,” “concentrated asparagus racemosus composition,” or “synergistically concentrated asparagus racemosus root extract composition,” as used interchangeably herein, refer to and include asparagus racemosus root extracts containing (i) shatavarin I, (ii) dehydroshatavarin I, (iii) shatavarin IV, and (iv) shatavarin IX.

[0026] Chemical compounds, or simply compounds, may be identified by any of their chemical structures, chemical names, or common names as used herein. Compounds used herein may contain one or more chiral centers and / or double bonds, and therefore may exist as isomers. The terms “methods to obtain women’s health functions” or “improvements” mean inhibiting, preventing, or blocking the onset of a medical condition (disease, disorder, or state), and / or causing reduction, remission, or regression of the condition. Those skilled in the art will understand that various methodologies and assays can be used to assess the onset of a medical condition, and similarly, various methodologies and assays can be used to assess the reduction, remission, or regression of a medical condition.

[0027] geographical origin The sources of the medicinal herbs used in this invention are as follows: The asparagus racemosus roots were sourced from a vendor in Mumbai, Maharashtra, India.

[0028] Asparagus racemosus: Also known as Shatavari, Satavari, or Satabar, it belongs to the Asparagaceae family. It is known as the "Queen of Herbs" and is one of the famous medicinal herbs in Ayurveda. It is considered a hormone balancer and general tonic for enhancing the health and vitality of both men and women. Its medical value has been reported in traditional Indian medicine such as Ayurveda, Unani, and Siddha. "Shatavari is very effective in problems relating to the female reproductive system. The ancient text Charak Samhita speaks of the use of Asparagus racemosus to treat disorders related to women's health." In modern Ayurvedic practice, the root of the plant is considered effective as an antispasmodic, appetite stimulant, stomach tonic, aphrodisiac, galactogog, astringent, antidiarrheal, anti-erythrorhizal, laxative, anticancer, anti-inflammatory, blood purifier, anti-tuberculosis, and anti-epileptic drug, as well as for relieving night blindness, kidney problems, jaundice, and throat complaints. Furthermore, it is also known as "Medhya"—a plant that enhances intelligence and promotes learning and memory. As a potent immunostimulant, Shatavari helps increase the body's resistance during normal and immunosuppressed states, boosts immunity during disease states, and aids in the recovery of the immune system. Pharmacologically, the extract has been reported as an antioxidant, anti-stress, anti-ulcer agent, wound healer, cough suppressant, gastroprotective, and neuroprotective agent, among others.

[0029] Asparagus racemosus extracts are available on the market through various extraction processes, such as aqueous and alcohol extracts, along with standardization of total saponins by gravimetric analysis. Scientific literature has isolated shatavalin I-X and characterized their chemical structures by spectroscopic analysis (one paper standardized a methanol extract for shatavalin IV, as shown in Table 1). However, there are no reports on the standardization of extracts for compositions containing multiple phytochemical markers present in Asparagus racemosus, particularly dehydroshatavalin I or dehydroshatavalin I.

[0030] Table 1: Marker content in methanol extracts reported in the literature [Table 1]

[0031] Generally, Shatavari roots have been extracted with water or aqueous ethanol for industrial and other research purposes. However, these solvents do not extract the total shatavarin (the active ingredient) present in the roots. Therefore, a more general process is required for the complete extraction of the active ingredient.

[0032] Therefore, the inventors conducted several experiments and, surprisingly, discovered a novel two-step extraction process that yields optimal concentrations of all active phytochemicals. In one embodiment, the method began with macerating the raw material in water for 16 hours, then extracting the used raw material after water extraction again with 90% aqueous ethanol to obtain a used 90% ethanol extract (AR-4). The aqueous extract was subjected to concentration for the total shatavari compound using one of two processes: 1) obtaining a concentrated extract (AR-3) by re-extracting the aqueous extract with 90% aqueous ethanol, or 2) obtaining a concentrated extract (AR-11) by treating the aqueous extract on a macroporous resin or subjecting it to membrane filtration, or subjecting it to membrane filtration (AR-12). In the final step, the used 90% ethanol extract (AR-4) was combined with one of the concentrated aqueous extracts (AR-3, AR-11, or AR-12) to obtain a novel shatavari extract composition containing all active phytochemicals. Similarly, the inventors demonstrated the present invention using a variety of suitable polar solvents, such as ethanol, n-butanol, methanol, acetone, aqueous ethanol, aqueous methanol, aqueous n-butanol, aqueous acetone, and mixtures thereof, as well as aqueous alcohols such as aqueous methanol, aqueous ethanol, and aqueous n-butanol, to obtain a novel Shatavari extract composition containing all active phytochemicals, as demonstrated in the examples.

[0033] Concentration with Macroporous Resins: Macroporous resins refer to a type of polymer material that has the ability to adsorb or capture selected substances on its surface. They are commonly used in various industries for the purification, separation, and removal of specific molecules or ions from mixtures. They are widely used for the extraction, purification, and concentration of natural compounds from plant extracts, herbal medicines, and other natural sources. Adsorption of target compounds from crude extracts onto macroporous (adsorbent) resins, followed by their elution using organic solvents, has proven to be an effective and efficient strategy for the purification of several natural products.

[0034] Therefore, an aqueous extract of Asparagus racemosus was passed through a macroporous resin to bind total shatavalin, the resin was washed with water, and finally the column was eluted with an aqueous organic solvent such as aqueous ethanol, and the solvent was evaporated to obtain a concentrated extract. The used raw material after aqueous extraction was re-extracted with a polar organic solvent (Examples 8-11).

[0035] Next, the concentrated asparagus racemosus extract composition of the present invention was analyzed by analytical HPLC using the isolated compound to obtain a novel, standardized concentrated asparagus racemosus extract composition, and the results are shown in Table 2 below.

[0036] Table 2: Estimated four shatavarins in extracts obtained according to the methods of Examples 1 and 8 (from 100 g of raw material each), by HPLC analysis. [Table 2]

[0037] The method of the present invention yields a higher yield of four shatavalin in total (2.48 g and 2.86 g according to the methods of Example-1 and Example-8, respectively, compared to 1.24 g and 1.40 g in water and aqueous ethanol, respectively). Thus, the new two-step method (both Example 1 and Example 8) significantly increases the total recovery rate of shatavalin. Other solvents such as butanol, methanol, and acetone were also used instead of 90% aqueous ethanol to obtain concentrated asparagus racemosus root extract (Examples 2-7, 9-10). For comparison, water extracts and 90% aqueous ethanol extracts were also prepared separately (Comparative Examples 1-2).

[0038] In addition to increasing the yield of a total of four shatavarins, the novel two-step extraction process also significantly increases the content of dehydroshatavarin I and shatavarin IV in the extract. Dehydroshatavarin I was observed as an entirely new compound isolated from shatavarin root, which had not been isolated from any other herb or species. Shatavarin IV is one of the key active ingredients of shatavarin root. In Examples 1 and 8, the shatavarin IV content in the extracts was 1.1 g and 1.3 g, respectively, compared to 225 mg and 226 mg in the water and aqueous ethanol extracts, respectively. The two-step method of the present invention (both Examples 1 and 8) unexpectedly increased the shatavarin IV recovery rate.

[0039] In one embodiment, the present invention discloses a novel two-step extraction process for shatavari root, comprising sequential extraction using both water and hydroalcohol. This process not only yields a higher yield of the extract with respect to the active ingredient, but also yields a wider range of active shatavarin, making the extract more potent and effective than extracts produced using conventional extraction methods.

[0040] Advantages of this method compared to conventional methods: • Higher yield of total shatavalin. • The novel compound dehydroshatavalin-I was isolated and concentrated. • Higher shatavalin IV content.

[0041] In another embodiment, the present invention discloses a concentrated asparagus racemosus extract comprising (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV and (iv) shatavalin IX for the health benefits of women.

[0042] In another aspect, the Disclosure provides, for the health benefits of women, a concentrated asparagus racemosus extract comprising (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV, and (iv) shatavalin IX; in which shatavalin I is in the range of 0.5 to 15%, dehydroshatavalin I is in the range of 0.1 to 25%, shatavalin IV is in the range of 2 to 25%, and shatavalin IX is in the range of 0.001 to 1.0%.

[0043] In another embodiment, the disclosure provides a concentrated asparagus racemosus composition comprising (i) shatavarin I; (ii) dehydroshatavarin I; (iii) shatavarin IV; and (iv) shatavarin IX; and (v) at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents.

[0044] In another embodiment, the Disclosure provides a concentrated asparagus racemosus composition comprising (i) shatavarin I; (ii) dehydroshatavarin I; (iii) shatavarin IV; and (iv) shatavarin IX together with (v) at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents, wherein the asparagus racemosus composition varies in the range of 90% to 10% by weight, and the excipient in the composition varies in the range of 10% to 90% by weight.

[0045] In yet another embodiment, the present invention provides a concentrated asparagus racemosus composition for the health benefits of women, comprising (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV and (iv) shatavalin IX (shatavalin I in the range of 0.5 to 15%; dehydroshatavalin I in the range of 0.1 to 25%; shatavalin IV in the range of 225%; and shatavalin IX in the range of 0.001 to 1.0%) together with at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents.

[0046] In yet another embodiment, the present invention provides a concentrated asparagus racemosus composition, which is also standardized to total saponins in the range of 20-90% by gravimetric analysis.

[0047] In another embodiment, the present invention provides a concentrated asparagus racemosus composition comprising (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV and (iv) shatavalin IX; and optionally containing at least one additional component selected from shatavalin II, shatavalin III, shatavalin V, shatavalin VI, shatavalin VII, shatavalin VIII, shatavalin IX, asparacoside, asparanin and immunoside or mixtures thereof.

[0048] In another embodiment, the present invention provides a concentrated asparagus racemosus composition comprising (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV and (iv) shatavalin IX; and optionally, at least one extract or fraction selected from Nigella sativa, Cinnamomum zeylanicum, Foeniculum vulgare, Trigonella foenum-graecum, Glycyrrhiza glabra and Linum usitatissimum, or at least one phytochemical selected from inositols such as myo-inositol or D-chiro-inositol or a mixture thereof.

[0049] In yet another embodiment, the present invention provides the above-described concentrated asparagus racemosus composition, the asparagus racemosus extract obtained from at least one plant part selected from the group including roots, leaves, stems, soft stems, soft twigs, aerial parts, whole fruits, fruit peel, fruit rind, seeds, flower heads, bark, hardwood, rhizomes, or whole plants or mixtures thereof.

[0050] In another embodiment, the present invention provides a method for preparing a concentrated asparagus racemosus extract comprising (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV; and (iv) shatavalin IX. (a) A step of extracting asparagus racemosus root powder with water, (b) A step of evaporating the aqueous extract to a minimum volume to obtain a concentrate, (c) Step (b) A step of re-extracting the concentrate with a polar organic solvent, (d) A step of evaporating the polar organic solvent-soluble extract to obtain a concentrated extract, (e) A step in which the aqueous extract from step (a) is alternately passed through an adsorbent resin and eluted with aqueous alcohol, (f) A step of evaporating the aqueous alcohol eluent to obtain a concentrated extract, (g) A step of extracting the asparagus racemosus root residue from step (a) with a polar organic solvent, (h) A step of evaporating the polar organic solvent extract from step (g) to obtain a second extract, (i) a step of combining the extracts from step (d) or step (f) and step (h) to obtain a concentrated asparagus racemosus extract composition; The polar solvent used in the method for preparing a concentrated asparagus racemosus extract composition is selected from, but is not limited to, n-butanol, ethanol, methanol, aqueous ethanol, aqueous methanol, aqueous n-butanol, and mixtures thereof.

[0051] In another embodiment, the present invention provides an alternative method for preparing a concentrated asparagus racemosus composition containing (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV and (iv) shatavalin IX by loading an aqueous extract of asparagus racemosus onto a macroporous resin column, eluting the column through an aqueous organic solvent such as aqueous ethanol, and combining the fractions.

[0052] In another embodiment, the present invention provides a method for preparing a composition of concentrated asparagus racemosus root extract comprising (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV, and (iv) shatavalin IX, comprising the step of blending the extract with at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents.

[0053] In another embodiment, the present invention provides a method for obtaining at least one female reproductive and / or sexual health benefit selected from supporting ovarian function and maintaining libido, reducing dyspareunia and / or sexual health, and improving lactation; the method comprising supplementing a human subject in need with an effective dose of an asparagus racemosus composition comprising (i) shatavarin I; (ii) dehydroshatavarin I; (iii) shatavarin IV; (iv) shatavarin IX, and (v) optionally, at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers, and diluents.

[0054] In another embodiment, the Disclosure provides the use of an asparagus racemosus composition comprising (i) shatavarin I; (ii) dehydroshatavarin I; (iii) shatavarin IV; (iv) shatavarin IX together with (v) optionally, promoting or regulating the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances; reducing symptoms of perimenopause disorders, including perimenopause, premenstrual syndrome (PMS), menstrual cycle-related discomfort; alleviating symptoms of polycystic ovary syndrome (PCOS), polycystic ovary disease (PCOD), gynecological disorders, and genitourinary or genital infections; supporting ovarian function; and obtaining at least one female reproductive and sexual health benefit selected from: (i) shatavarin I; (ii) dehydroshatavarin I; (iii) shatavarin IV; (iv) shatavarin IX together with at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers, and diluents, to obtain at least one female reproductive and sexual health benefit selected from: (i) shatavarin I; (ii) dehydroshatavarin I; (iii) shatavarin IV; (iv) shatavarin IX together with (v) optionally, promoting or regulating the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances; reducing symptoms of perimenopause disorders, including perimenopause, premenstrual syndrome (PMS), menstrual cycle-related discomfort; alleviating symptoms of polycystic ovary syndrome (PCOS), polycystic ovary disease (PCOD), gynecological disorders, and genitourinary infections; supporting ovarian function; and maintaining libido, reducing dyspareunia, and improving lactation.

[0055] How to use: Estrogen receptor-α (ER-α): The cyclical secretion of estrogen and its interaction with related receptors, estrogen receptor-α and β (ER-α and ER-β), play a significant role in regulating ovarian function, endometrial proliferation, and differentiation. Recently, G protein-coupled estrogen receptors (GPERs) have been described as mediating non-genomic estrogen signaling. Alterations in the ER-α signaling pathway affect cellular activities such as ovulation, cell cycle phases, cell proliferation, migration, and invasion. Furthermore, chronic imbalances in female hormones affect ovarian function, leading to cyst formation in the ovaries and resulting in polycystic ovary syndrome (PCOS). Similarly, endocrine and metabolic abnormalities associated with PCOS can increase the risk of endometrial hyperplasia and cancer in women. Therefore, infertility in patients with PCOS can result not only from anovulation but also from endometrial dysfunction. Selective estrogen receptor modulators (SERMs), which are nonsteroidal anti-steroidal drugs, are widely used in clinical and therapeutic applications in the management of hormonal imbalances and PCOS in women. SERMs can act as both ER agonists and antagonists. However, sustained activation of ER-α by ER agonists has several side effects, including ER-α-positive cancer. Alternatively, safer options, including phytoestrogens such as isoflavones, prenylflavonoids, coumestans, and lignans, are increasing for managing hormonal imbalances that result in several women's health conditions, including PCOS.

[0056] The ability of four compounds—shatavalin I, dehydroshatavalin I, shatavalin IV, and shatavalin IX—and their combinations, to enhance pER-α (estrogen receptor alpha phosphorylated at serine 118) expression in the human breast tumor cell line MCF-7 was evaluated, and the results are shown in Table 3. pER-α represents the active form of the hormone receptor. Unexpectedly, the combination of the four markers showed better efficacy in increasing pER-α expression compared to the corresponding individual markers.

[0057] Table 3: Normalized pER-α expression for four markers and their combinations. [Table 3]

[0058] Therefore, pER-α expression with shatavalin I, dehydroshatavalin I, shatavalin IV, and shatavalin IX at concentrations of 0.2 μg / mL, respectively, showed increases of 21.43%, 60.71%, 42.86%, and 35.71% compared to the vehicle control; compared to the other markers, the novel marker dehydroshatavalin I showed better efficacy, which is a surprising result. The combination of all four markers of the present invention at the same concentration showed a 93.46% increase in pER-α expression compared to the individual markers, which is a surprising and unexpected result of ER-α activation.

[0059] Furthermore, the pER-α expression activity of asparagus racemosus root water extract (Comparative Example 1), 90% aqueous ethanol (Comparative Example 2), and the composition of the present invention (Example 1) was screened, and the results are shown in Table 4. Unexpectedly, the composition of the present invention showed better efficacy in pER-α expression compared to the corresponding water extract and 90% aqueous ethanol extract.

[0060] Table 4: Normalized pER-α expression of aqueous extract of Asparagus racemosus root, 90% aqueous ethanol extract, and the composition of the present invention. [Table 4]

[0061] Therefore, pER-α expression from the 0.2 μg / mL aqueous extract (AR-1) and the 90% aqueous ethanol extract of Asparagus racemosus (AR-2) showed increases of 23.08% and 30.77%, respectively, compared to the vehicle control (Table 4). On the other hand, the extract composition of the present invention, comprising a concentrated aqueous extract of Asparagus racemosus obtained using 90% aqueous ethanol and a used Asparagus racemosus raw material extract (Comp-1) obtained using the same concentration of 90% aqueous ethanol, showed a 57.69% increase in pER-α expression, which is a surprising and unexpected result regarding the increase in ER-α activation in cells.

[0062] Prostaglandin E2 (PGE2): Prostaglandins (PGs) are lipid compounds called eicosanoids, derived from arachidonic acid released from lipid membranes after activation of the phospholipase A2 enzyme. Cyclooxygenases-1 and 2 (COX-1 and COX-2) are enzymes responsible for prostaglandin E2 (PGE2) production. Higher PGE2 levels increase the sensitivity of peripheral nociceptive primary afferent neurons and central nociceptive neurons, which can lead to the development of uterine spasms resulting in menstrual pain (dysmenorrhea). Pharmacological therapy for dysmenorrhea involves the use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, which block PGE2 production by inhibiting the COX enzyme. However, long-term use of NSAIDs can lead to undesirable side effects. Therefore, alternative strategies such as sacral reflex therapy and natural supplements are being explored to manage dysmenorrhea.

[0063] Therefore, the aqueous extract of Asparagus racemosus root (AR-1) and the composition of the present invention (Comp-1) were screened for their PGE2 inhibitory activity, and the results are shown in Table 5. Unexpectedly, the composition of the present invention showed better efficacy in inhibiting PGE2 production compared to the corresponding aqueous extract.

[0064] Table 5: PGE2 inhibition by Asparagus racemosus aqueous extract and the composition of the present invention [Table 5]

[0065] Therefore, an aqueous extract of Asparagus racemosus (AR-1) at 10 μg / mL showed a 15.23% reduction in PGE2 production; on the other hand, the composition of the present invention, comprising a concentrated aqueous extract of Asparagus racemosus obtained using 90% aqueous ethanol, and used Asparagus racemosus raw material (comp-1) obtained using the same treatment concentration of 90% aqueous ethanol, showed an inhibition of 39.71 (Table 5), which is 24.48% higher activity than AR-1, and this is a surprising and unexpected result of PGE2 inhibition.

[0066] Estrone: Perimenopause or the transition to perimenopause is traditionally classified into early and late stages. Understanding the biological mechanisms of perimenopause is a complex process. Menopausal physiological changes are primarily due to endocrine changes during perimenopause. A decrease in the number of follicles leads to a decline in levels of inhibin B, anti-Müllerian hormone, and ovarian estrogens [estrone (E1), 17β-estradiol (E2), estriol (E3)]. Estrogen is known to provide natural protection during the premenopausal stage. Women in perimenopause have very low circulating levels of estrogen. Estrone is one of the estrogens that converts to 17β-estradiol (E2) and helps balance hormones. Several pharmacological strategies and herbal supplements (containing phytoestrogens) targeting perimenopause / menopause and associated metabolic disorders have focused on hormone balance, particularly estrogen and progesterone (demonstrated in Table 6 of Example 7).

[0067] Evaluation of supplementation with Asparagus racemosus concentrated extract composition to total follicle count in female Sprague Dawley rats: Ovarian follicles contain oocytes and are surrounded by granulosa cells. Four different types of follicles are observed in the ovary based on their developmental stage: primordial, primary, secondary, and tertiary (vestibulal). The number of primordial follicles, which represent true ovarian reserve, is determined in the fetus and decreases throughout life. However, primordial follicles cannot be visualized by ultrasound due to their small size (less than 0.05 mm in diameter). Vestibular or Graafian follicles are more easily identifiable by ultrasound when they reach a diameter of 2 mm. It is recommended that follicle counting be performed during the early follicular phase of the menstrual cycle. However, given the intracycle variability of ovarian reserve, follicle counting can be performed at any point in the menstrual cycle. Pre-follicles (AF) or Graafian follicles (GF) are considered the best clinical markers for evaluating indicators of functional ovarian reserve and fertilization ability. This helps predict the response to gonadotropin stimulation and the chances of pregnancy after in vitro fertilization (IVF). The number of GFs decreases with age and may help predict the timing of spontaneous menopause. The efficacy of the extract composition of the present invention (comp-1) was compared with a simple aqueous extract (AR-1) in an in vivo experiment in female Sprague Dawley rats, as described in Example 28. Unexpectedly, the data from the in vivo study showed better efficacy for the composition in increasing the total number of follicles compared to the aqueous extract of Asparagus racemosus, suggesting that the concentrated Asparagus racemosus extract tends to show better efficacy (as demonstrated in Table 16 of Example 28).

[0068] Therefore, the concentrated asparagus racemosus root extract composition of the present invention, containing shatavalin I, dehydroshatavalin I, shatavalin IV, and shatavalin IX, was obtained by combining a concentrated aqueous extract of asparagus racemosus obtained using 90% aqueous ethanol with a used asparagus racemosus raw material extract obtained using 90% aqueous ethanol, and showed better efficacy in pER-α expression and PGE2 inhibition compared to ordinary aqueous extracts and 90% aqueous ethanol extracts. Furthermore, surprisingly, the concentrated asparagus racemosus extract composition showed better efficacy in increasing the total number of follicles in experimental rats.

[0069] composition Several compositions were prepared, each containing (i) shatavalin I in the range of 0.5–15%, (ii) dehydroshatavalin I in the range of 0.1–25%, (iii) shatavalin IV in the range of 2–25%, and (iv) shatavalin IX in the range of 0.001–1.0%.

[0070] For example, Asparagus racemosus obtained with 90% aqueous ethanol Various compositions of concentrated water extracts of the root (AR-3) and used or remaining Asparagus racemosus (Shatavari) root extract (AR-4) obtained with 90% aqueous ethanol were prepared as shown in Example 13. Similarly, various compositions of concentrated water extracts of Asparagus racemosus root obtained with polar organic solvents and used or remaining Asparagus racemosus (Shatavari) root obtained with polar organic solvents were prepared as shown in Examples 14-19.

[0071] Furthermore, various compositions of concentrated aqueous extract of Asparagus racemosus root (AR-11) obtained through a macroporous resin process by elution with 90% aqueous ethanol and used or residual Asparagus racemosus root extract (AR-4) obtained with 90% aqueous ethanol were prepared as shown in Example 20. Similarly, various compositions of concentrated aqueous extract of Asparagus racemosus root obtained by a macroporous resin process by elution with polar organic solvents and used or residual Asparagus racemosus root extract obtained using other polar organic solvents were prepared as shown in Examples 21-22.

[0072] Furthermore, various compositions of (i) an aqueous extract of Asparagus racemosus root (AR-1), a concentrated aqueous extract of Asparagus racemosus root obtained by a macroporous resin process by elution with 90% aqueous ethanol (AR-11), and a 90% aqueous ethanol extract of used or remaining Asparagus racemosus root (AR-4) were prepared as shown in Example 23.

[0073] Furthermore, these compositions (1-25) were evaluated for women's health benefits selected from promoting or regulating the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances; reducing symptoms of perimenopause, including perimenopause, premenstrual syndrome (PMS), menstrual cycle-related discomfort, and menstrual pain; alleviating symptoms of polycystic ovary syndrome (PCOS), polycystic ovary disease (PCOD), gynecological disorders, and genitourinary or genital infections; supporting ovarian function; and maintaining libido, reducing dyspareunia, and improving lactation.

[0074] pER-α expression activity: These compositions (1-25) were evaluated for normalized pER-α expression activity compared to their corresponding individual components (Example 26). Data from in vitro pER-α expression revealed that these compositions showed unexpectedly better efficacy compared to their corresponding individual components, and revealed that compositions containing (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV and (iv) shatavalin IX; and compositions obtained by combining (a) a concentrated aqueous extract of Asparagus racemosus root obtained by either a solvent extraction process or a macroporous resin column chromatography process with (b) a polar organic solvent extract of used or residual Asparagus racemosus (shatavari) root tended to show synergistic effects.

[0075] For example, an aqueous extract of Asparagus racemosus root concentrated with 0.16 μg / mL of 90% aqueous ethanol (AR-3), and an extract of spent raw material in 90% aqueous ethanol at 0.04 μg / mL (AR-4) showed pER-α expression of 0.42 and 0.09, respectively. Composition-1, containing these two extracts in a 4:1 ratio, showed pER-α expression of 0.72 at a concentration of 0.2 μg / mL, which is significantly better than the additive effect from these two components (0.42 + 0.09 = 0.51), suggesting a synergistic effect between the aqueous extract of Asparagus racemosus root concentrated with 90% aqueous ethanol (AR-3) and the extract of spent raw material in 90% aqueous ethanol (AR-4). Compositions 2-5, obtained by combining these two components in ratios of 3:1, 2:1, 1:1, and 1:2, respectively, also showed synergistic effects on pER-α expression compared to the expression shown by their corresponding individual components, as summarized in Table 8. Similarly, the other compositions (6-25) also showed synergistic effects on pER-α expression compared to the expression shown by their corresponding individual components, as summarized in Tables 9-11.

[0076] Prostaglandin E2 (PGE2) inhibitory activity: These compositions (1-25) were evaluated for PGE2 inhibitory activity compared to their corresponding individual components (Example 27). Data from in vitro PGE2 inhibition revealed that these compositions showed unexpectedly better inhibition compared to their corresponding individual components, and that compositions containing (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV and (iv) shatavalin IX; and compositions obtained by combining (a) a concentrated aqueous extract of Asparagus racemosus root produced by either a solvent extraction process or a macroporous resin column chromatography process with (b) a polar organic solvent extract of used or residual Asparagus racemosus (Shatavari) root tended to show synergistic effects.

[0077] For example, a concentrated 90% aqueous ethanol (AR-3) extract obtained from an aqueous extract of Asparagus racemosus root at a concentration of 8.0 μg / mL and a 90% aqueous ethanol (AR-4) extract from spent raw materials at a concentration of 2.0 μg / mL showed inhibition of PGE2 by 18.66% and 5.94%, respectively. Composition-1 containing these two extracts in a 4:1 ratio showed inhibition of PGE2 by 39.71% at a concentration of 10 μg / mL, which was significantly better than the additive effect from these two components (18.66 + 5.94 = 24.60%), suggesting a synergistic effect between the 90% aqueous ethanol (AR-3) extract obtained from the aqueous extract of Asparagus racemosus root and the 90% aqueous ethanol (AR-4) extract from spent raw materials. Compositions 2-5, obtained by combining these two components in ratios of 3:1, 2:1, 1:1, and 1:2, respectively, also showed synergistic effects in PGE2 inhibition compared to the inhibition shown by their corresponding individual components, as summarized in Table 12. Similarly, the other compositions (6-25) also showed synergistic effects in PGE2 inhibition compared to the inhibition shown by their corresponding individual components, as summarized in Tables 13-15.

[0078] Formulation: The synergistically concentrated compositions described, comprising (i) 0.5–15% shatavarin I; (ii) 0.1–25% dehydroshatavarin I; (iii) 2–25% shatavarin IV and (iv) 0.001–1.0% shatavarin IX, may be formulated using excipients, carriers, and diluents that are pharmaceutically or nutritionally acceptable or dietarily acceptable, selected from monosaccharides, disaccharides, polysaccharides, dextrins, polyhydric alcohols or sugar alcohols, cellulosic derivatives, silicates, metal stearates, organic acids, fatty acid esters, calcium pantothenate, amino acids, proteins, organometallic salts, natural pigments, flavorings, Class I and Class II preservatives and mixtures thereof.

[0079] Monosaccharides are selected from, but are not limited to, glucose, dextrose, fructose, and galactose; disaccharides are selected from, but are not limited to, sucrose, maltose, lactose, lactulose, trehalose cellobiose, and chitobiose; polysaccharides are selected from, but are not limited to, starches and modified starches, such as sodium starch glycolate, pregelatinized starch, soluble starch, ultrasperse A, and other modified starches; dextrins include yellow dextrin, white dextrin, maltodextrin, glucidex 12D, rice maltodextrin, and Tapioca / Cassava. The following are selected from, but are not limited to, maltodextrin; the polyhydric alcohol or sugar alcohol is selected from, but is not limited to, sorbitol, mannitol, inositol, xylitol, and isomalt; the cellulosic derivative is selected from, but is not limited to, microcrystalline cellulose, hydroxypropyl methylcellulose, and hydroxyethylcellulose; the silicate is selected from, but is not limited to, neucillin, beegum, talc, colloidal silicon dioxide, and siloid; the metal stearate is selected from, but is not limited to, calcium stearate, magnesium stearate, and zinc stearate; the organic acid is selected from, but is not limited to, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, and L-ascorbic acid; Natural gums include acacia, carrageenan, guar gum, xanthan gum, gum garti, biopolymers / biopolysaccharides such as sodium alginate, calcium alginate, and alginic acid, but are not limited to these; proteins include whey protein, whey isolate, casein, gelatin, pectin, and agar, but are not limited to these; inorganic metal salts include sodium chloride, calcium chloride, dicalcium phosphate, zinc sulfate, and zinc chloride, but are not limited to these; natural sweeteners include licorice, stevia, stevioside, such as rebaudioside A, palm sugar, and natural sweet proteins such as blazein, monellin, curculin, mavinlin, miraculin, and pentazin, but are not limited to these.

[0080] For example, comp-26 was prepared by combining AR-3 and AR-4 with acacia rubber and colloidal silicon dioxide. Similarly, other compositions (comp 27-29) were prepared as shown in Example 25.

[0081] Method: A method for preparing a synergistically concentrated asparagus racemosus composition comprising: (i) 0.5–15% shatavalin I; (ii) 0.1–25% dehydroshatavalin I; (iii) 2–25% shatavalin IV and (iv) 0.001–1.0% shatavalin IX; (v) optionally together with at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers and diluents; the method comprising the following steps; (a) A step of extracting asparagus racemosus root powder with water, (b) A step of evaporating the aqueous extract to a minimum volume to obtain a concentrate, (c) Step (b) A step of re-extracting the concentrate with a polar organic solvent, (d) A step of evaporating the polar organic solvent-soluble extract to obtain a concentrated extract, (e) The aqueous extract from step (a) is passed alternately through an adsorbent resin, and then the resin is eluted with aqueous ethanol or subjected to membrane filtration. (f) A step of evaporating the aqueous alcohol eluent to obtain a concentrated extract, (g) A step of extracting the asparagus racemosus root residue from step (a) with a polar organic solvent, (h) A step of evaporating the polar organic solvent extract from step (g) to obtain a second extract, (i) A step of combining the extracts from step (d) or step (f) and step (h) to obtain a synergistically concentrated asparagus racemosus extract composition; (j) The step of optionally blending the combined extract from step (i) with at least one component selected from pharmaceutically or nutritionally, or dietarily acceptable excipients, carriers, and diluents.

[0082] The polar organic solvent used in the method for preparing a synergistically concentrated asparagus racemosus extract composition is selected from, but is not limited to, ethanol, n-butanol, methanol, acetone, aqueous ethanol, aqueous methanol, aqueous acetone, and mixtures thereof.

[0083] Evaluation of synergistically concentrated extracts of Asparagus racemosus (comp-26, comp-27, comp-28 and 90% aqueous ethanol extract (AR-2)) in letrozole-induced PCOS in female Sprague Dawley rats:

[0084] Oral glucose tolerance test (OGTT): The OGTT measures circulating glucose levels after glucose intake, and this test is widely used as an indicator of insulin resistance. PCOS is known to cause metabolic dysfunction, including insulin resistance. Letrozole-induced rats exhibit impaired glucose tolerance and elevated circulating blood glucose, which closely mimics the clinical features of impaired glucose tolerance or insulin resistance in PCOS.

[0085] The area under the curve (AUC) was calculated using serum glucose concentration as a function of time (minutes). AUC values ​​increased in letrozole-induced (G2) rats compared to vehicle-controlled (G1) rats, indicating impaired glucose tolerance or increased insulin resistance. AUC values ​​in AR-2 and Comp-26, Comp-27, and Comp-28 supplemented rats showed improvements of 9.70%, 13.51%, 16.62%, and 20.05%, respectively, compared to G2 rats. These improvements indicated increased glucose tolerance in Comp-26, Comp-27, and Comp-28 supplemented rats, and these effects were significantly better than in the AR-2 group of rats (Table 17).

[0086] Serum hormone levels: Letrozole is an aromatase inhibitor; it increases serum testosterone levels and decreases estrogen levels, leading to hyperandrogenism or a state of excessive androgens or male hormones. Hyperandrogenism is a common feature and is prevalent in over 80% of women with PCOS.

[0087] Estradiol: 17β-estradiol (E2) is a female hormone produced in the ovaries that affects women's physical and psychological well-being. E2 plays a crucial role in the development of the female reproductive system, exerting its effects on target organs and cells through multiple estrogen receptors (ER), ERα, ERβ, and G protein-coupled estrogen receptors (GPER, also known as GPR30) to maintain various stages of normal development in the human ovaries and uterus. E2 levels change dramatically during hormonal transitions, such as puberty, menopause, and the postpartum period. During these transitions, women are at high risk of psychological and physical distress. Women have higher levels of E2 during their reproductive years and gradually decrease after menopause. Balancing / improving E2 levels may be useful in managing perimenopause and conditions such as polycystic ovary syndrome (PCOS).

[0088] Testosterone: In women, androgens, including testosterone, are produced by the ovaries and adrenal glands, as well as by the conversion of less potent androgens to more potent ones in the periphery. Women with PCOS exhibit androgen hypertrichosis, with significantly elevated levels of testosterone, free testosterone, androstenedione, dehydroepiandrosterone (DHEA), and DHEA sulfate (DHEAS) compared to ovulating and non-hirsutistic women. Androgen hypertrichosis results in a wide range of symptom manifestations, including mild acne, increased terminal (coarse) hair growth on midline structures (face, neck, abdomen), and androgenic changes in body habitus, accompanied by a waist-to-hip ratio. The primary goal of treatment in all forms is to suppress insulin-promoting LH-driven androgen production, including testosterone. Therefore, balancing or improving the E2 and testosterone ratio will alleviate the clinical symptoms of ovarian dysfunction, including PCOS.

[0089] Serum samples from all experimental rats were analyzed for 17β-estradiol and testosterone. In letrozole-induced PCOS rats (G2), serum estradiol levels decreased (32.97±13.89 pg / ml in G1 vs. 41.35±23.93 pg / ml) and testosterone levels increased (3.18±2.16 pg / ml in G1 vs. 0.26±0.10). Comp 26, 27, and 28 supplemented rats showed improvements of 10.74%, 13.95%, and 19.20% in serum 17β-estradiol, and 43.08%, 60.38%, and 70.44% in serum testosterone levels compared to G2 rats. AR-2 increased 17β-estradiol by 7.24% and decreased testosterone levels by 30.50% compared to G2 rats. These data indicate that supplementation with comp 26, 27, or 28 helps improve hormone balance in experimental PCOS rats, and these improvements are significantly better than those in AR-2 supplemented rats. Serum hormone analysis data are shown in Table 18.

[0090] Total number of follicular cysts and follicles: PCOS is a heterogeneous disorder affecting at least 7% of adult women. Ovarian cyst formation is one of the symptoms of PCOS, along with elevated androgen levels and irregular or skipped periods. The term "polycystic" means "many cysts" and refers to the formation of many small, fluid-filled sacs that grow along the outer edge of the ovary. These sacs are follicles containing immature eggs that do not mature and induce ovulation. The lack of ovulation alters estrogen, progesterone, FSH, and LH levels and increases androgen levels. Balancing hormone levels in conditions such as PCOS, perimenopause, or perimenopause, along with improving insulin sensitivity, may improve ovulation and potentially reduce cyst formation.

[0091] In experimental animals, ovarian sections were examined for the total number of follicles, including follicular cysts and Graafian follicles. Letrozole significantly reduced the total number of follicles and Graafian follicles and increased the number of cysts in the ovaries of experimental rats. Supplementation with comp 26, 27, and 28 increased the total number of follicles by 30.67%, 46.92%, and 49.59%, respectively, the number of Graafian follicles by 28.29%, 38.05%, and 70.73%, respectively, and decreased the number of cysts by 38.06%, 46.82%, and 50.66%, respectively, compared to letrozole-induced PCOS rats. In contrast, AR-2 supplemented rats showed an increase of 19.56% and 16.59% in total follicle and Graafian follicle numbers, respectively, and a decrease of 27.38% in the number of follicular cysts compared to G2 rats. These improvements in comp 26, 27, and 28 supplemented rats were significantly better than in AR-2 supplemented rats. The observations regarding the total number of follicles, including follicular cysts and Graafian follicles, are summarized in Table 19.

[0092] Therefore, these findings suggest that comp 26, 27, 28, and AR-2 improve glucose tolerance in letrozole-induced PCOS rats. The incidence of hyperglycemia due to glucose tolerance or insulin resistance is highly correlated in humans and is a prominent feature of PCOS. These findings also suggest that AR-2 and comp 26, 27, and 28 significantly alleviated clinical symptoms of PCOS, such as the number of follicles and cysts, in experimental rats. Furthermore, the test products (compositions and conventional extracts) are effective in improving hormonal balance (estrogen vs. testosterone) and Graafian follicle count in experimental animals. It should be noted that a higher number of Graafian follicles or mature follicles is an established clinical marker of improved fertility. Importantly, comp 26, 27, and 28 are significantly superior to conventional extracts in improving ovarian function, alleviating PCOS symptoms (number of cysts and follicles), reducing androgen hypersensitivity, and improving hormonal balance and fertility.

[0093] Therefore, a synergistically concentrated asparagus racemosus (shatavari) root extract composition comprising: (i) 0.5-15% shatavarin I; (ii) 0.1-25% dehydroshatavarin I; (iii) 2-25% shatavarin IV and (iv) 0.001-1.0% shatavarin IX; and (v) optionally, at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers, and diluents; demonstrated health benefits for women.

[0094] Therefore, in key embodiments, the present invention provides a synergistically concentrated asparagus racemosus root extract comprising (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV, and (iv) shatavalin IX for the health benefits of women.

[0095] In one preferred embodiment, the present invention relates to a novel Shatavari compound not previously reported, namely of formula; JPEG2026519309000007.jpg48153 Provides the dehydroshatavalin compound I, 1 H NMR(d6-DMSO): δ 5.16(1H,d,J=4.4 Hz),5.13(1H,d,J=4.0 Hz),4.90(1H,d,J=4.8 Hz),4.81-4.85(4H,m),4.72(1H,s),4.62-4.68(4H,m),4.59(1H,d,J=5.2 Hz),4.47(1H,d,J=8.0 Hz),4.41(1H,d,J=5.6 Hz),4.37(1H,t,J=6.4 Hz),4.33(1H,d,J=7.6 Hz),4.27-4.32(1H,m),4.16(1H,t,J=5.6 Hz),4.08(1H,d,J=8.0 Hz),3.64-3.67(3H,m),3.61(1H,brs),3.57-3.58(1H,m),3.37-3.51(7H,m),3.03-3.19(9H,m),2.92-3.01(2H ,m),2.01-2.13(3H,m),1.63-1.84(5H,m),1.55(3H,s),1.36-1.52(9H,m),1.15-1.28(5H,m),1.10(3H,d,J=6.4 Hz),1.06-1.09(2H,m),0.95-1.05(2H,m),0.90(3H,s),0.89(3H,d,J=6.4 Hz), 0.62 (3H,s) (mass (Q-tof, anion mode): m / z 1047.5400 (MH) - )

[0096] In a preferred embodiment, the present invention provides a synergistically concentrated asparagus racemosus extract composition comprising (i) 0.5 to 15% shatavalin I; (ii) 0.1 to 25% dehydroshatavalin I; (iii) 2 to 25% shatavalin IV and (iv) 0.001 to 1.0% shatavalin IX for the benefit of women's health.

[0097] In a preferred embodiment, the present invention provides a synergistically concentrated asparagus racemosus extract composition comprising (i) 0.5 to 15% shatavalin I; (ii) 0.1 to 25% dehydroshatavalin I; (iii) 2 to 25% shatavalin IV and (iv) 0.001 to 1.0% shatavalin IX; the composition is produced by combining (a) a concentrated aqueous extract of asparagus racemosus (shatavari) root obtained by either polar organic solvent extraction or purification with a macroporous resin, and (b) a used or residual polar organic solvent extract of asparagus racemosus (shatavari) root.

[0098] In a preferred embodiment, the present invention provides a synergistically concentrated asparagus racemosus root extract composition comprising (i) 0.5 to 15% shatavarin I; (ii) 0.1 to 25% dehydroshatavarin I; (iii) 2 to 25% shatavarin IV; and (iv) 0.001 to 1.0% shatavarin IX; and (v) optionally at least one additional component selected from the group consisting of biological agents and nootropes; pharmaceutically acceptable active ingredients, vitamins, minerals; and pharmaceutically or nutritionally or dietarily acceptable excipients, carriers, or diluents.

[0099] In another embodiment, the present invention provides a synergistically concentrated asparagus racemosus extract composition comprising (i) 0.5 to 15% shatavalin I; (ii) 0.1 to 25% dehydroshatavalin I; (iii) 2 to 25% shatavalin IV and (iv) 0.001 to 1.0% shatavalin IX; and (v) at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents, wherein the asparagus racemosus root extract varies in the range of 90% to 10% by weight, and the excipients in the composition vary in the range of 10% to 90% by weight.

[0100] In another embodiment, the present invention provides a synergistically concentrated asparagus racemosus extract composition comprising (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV and (iv) shatavalin IX; and optionally containing at least one additional component selected from shatavalin II, shatavalin III, shatavalin V, shatavalin VI, shatavalin VII, shatavalin VIII, shatavalin IX, asparacoside, asparanin and immunoside or mixtures thereof.

[0101] In another embodiment, the present invention provides a synergistically concentrated asparagus racemosus extract composition comprising (i) shatavalin I; (ii) dehydroshatavalin I; (iii) shatavalin IV and (iv) shatavalin IX; and (v) optionally, at least one phytochemical selected from Nigella sativa, Cinnamom zeiranicum, Phoenicula vulgare, Trigonella phoenum grecum, Glycyrrhiza glabra and Linum ucithachissimum, or myo-inositol or D-chiro-inositol or a mixture thereof.

[0102] In another embodiment, the present invention relates to a method for preparing a synergistically concentrated asparagus racemosus extract composition comprising: (i) 0.5–15% shatavalin I; (ii) 0.1–25% dehydroshatavalin I; (iii) 2–25% shatavalin IV and (iv) 0.001–1.0% shatavalin IX; (v) optionally, at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers and diluents; and a method comprising the following steps: (a) A step of extracting asparagus racemosus root powder with water, (b) A step of evaporating the aqueous extract to a minimum volume to obtain a concentrate, (c) Step (b) A step of re-extracting the concentrate with a polar organic solvent, (d) A step of evaporating the polar organic solvent-soluble extract from step (c) to obtain a concentrated extract, (e) The water extract from step (a) is passed alternately through an adsorbent resin, and then the resin is eluted with aqueous ethanol, (f) A step of evaporating the aqueous alcohol eluent from step (e) to obtain a concentrated extract, (g) A step of extracting the asparagus racemosus root residue from step (a) with a polar organic solvent, (h) A step of evaporating the polar organic solvent extract from step (g) to obtain a second extract, (i) A step of combining the extracts from step (d) or step (f) and step (h) to obtain a synergistically concentrated asparagus racemosus extract composition; (j) The step of optionally blending the synergistically concentrated asparagus racemosus root extract composition from step (i) with at least one component selected from pharmaceutically or nutritionally, or dietarily acceptable excipients, carriers, and diluents.

[0103] The polar organic solvent used in the method for preparing a synergistically concentrated asparagus racemosus extract composition is selected from, but is not limited to, ethanol, n-butanol, methanol, acetone, aqueous ethanol, aqueous methanol, aqueous n-butanol, aqueous acetone, and mixtures thereof.

[0104] In yet another embodiment, the present invention provides, for the health benefits of women, a synergistically concentrated asparagus racemosus extract composition comprising (i) shatavalin I, (ii) dehydroshatavalin I, (iii) shatavalin IV and (iv) shatavalin IX, the composition being prepared by a method comprising: (a) concentrating an aqueous extract of asparagus racemosus (shatavari) root by re-extracting with a polar organic solvent or by loading the aqueous extract onto a macroporous resin and eluting with aqueous alcohol to obtain a concentrated extract; (b) extracting used or remaining asparagus racemosus (shatavari) root with a polar organic solvent to obtain a second extract; (c) combining the extracts obtained from steps (a) and (b); and (d) optionally blending with at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers and diluents.

[0105] In yet another embodiment, an aqueous extract of Asparagus racemosus (Shatavari) root is also concentrated by membrane filtration.

[0106] In another embodiment, the present invention provides, for the health benefits of women, a synergistically concentrated asparagus racemosus extract composition comprising (i) 0.5-15% shatavalin I; 0.1-25% dehydroshatavalin I; 2-25% shatavalin IV and 0.001-1.0% shatavalin IX, wherein the composition is obtained by (a) extracting asparagus racemosus roots with water and (b) re-extracting with a polar organic solvent, or by loading the water extract onto a macroporous resin and dissolving it with aqueous alcohol. The preparation is carried out by a method comprising: (c) concentrating an aqueous extract of Asparagus racemosus (Shatavari) root by extracting it with a polar organic solvent to obtain a second extract; (d) combining the extracts obtained from steps (b) and (c); and (e) optionally blending it with at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers and diluents.

[0107] In another important embodiment, the present invention promotes or modulates the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances; reduces symptoms of perimenopause, premenstrual syndrome (PMS), menstrual cycle-related discomfort; alleviates symptoms of polycystic ovary syndrome (PCOS), polycystic ovary disease (PCOD), gynecological disorders, and genitourinary or reproductive system infections; supports ovarian function, maintains libido, reduces dyspareunia, and improves lactation. The present invention provides a method for obtaining female reproductive and / or sexual health benefits, comprising supplementing a subject in need of it with an effective dose of a synergistically concentrated asparagus racemosus composition comprising (i) 0.5–15% shatavarin I; 0.1–25% dehydroshatavarin I; 2–25% shatavarin IV and 0.001–1.0% shatavarin IX; and (v) optionally, at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers, and diluents.

[0108] In another embodiment, the Disclosure provides (i) shatavarin I in the range of 0.5–15%; (ii) dehydroshatavarin I in the range of 0.1–25%; (iii) shatavarin IV in the range of 2–25% and (iv) shatavarin IX in the range of 0.001–1.0%; (v) optionally, promoting or regulating the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances, reducing symptoms of perimenopause, premenstrual syndrome (PMS), menstrual cycle-related discomfort, polycystic ovary syndrome (PCOS) The present invention provides the use of a synergistically concentrated asparagus racemosus composition containing at least one component selected from pharmaceutically or nutritionally, or dietarily acceptable, excipients, carriers, and diluents to obtain at least one female reproductive and sexual health benefit selected from: alleviating symptoms of PCOS, polycystic ovary disease (PCOD), gynecological disorders, and genitourinary or genital infections; supporting ovarian function; and maintaining libido, reducing dyspareunia, and improving lactation.

[0109] In another embodiment, the composition disclosed above; the phytochemical reference marker compound or pharmacologically active marker or group of markers may be estimated by several analytical techniques known in the art, such as HPLC, gravimetric analysis, UV, GC, LC-MS, and ICP-mass, but are not limited to those described above.

[0110] In another embodiment, the synergistically concentrated asparagus racemosus composition disclosed above is formulated into a dosage form selected from any suitable form, such as a dry powder, a liquid, a beverage, a food, a dietary supplement, or a tablet, capsule, a soft chewable tablet, a gummy, or a gummy bar.

[0111] In another embodiment, the synergistically concentrated asparagus racemosus composition disclosed above may be formulated into nutritional / dietary supplements that can be intended / made into dosage forms of health foods or foods for specific health uses, such as solid foods like chocolate or nutrition bars, semi-solid foods like creams, jams or gels, or beverages such as soft drinks, lactic acid drinks, drops, candies, chewing gum, gummy candies, yogurt, ice cream, pudding, sweet bean jelly, jelly, cookies, tea, soft drinks, juices, milk, coffee, cereals, snack bars, etc.

[0112] In another embodiment, the synergistically concentrated asparagus racemosus composition disclosed above is formulated into controlled-release tablets using a controlled-release polymer-based coating by techniques including nanotechnology, microencapsulation, colloidal carrier systems, and other drug delivery systems for obtaining the desired therapeutic benefit. [Examples]

[0113] Comparative Example 1 (AR-1): Aqueous extract of Asparagus racemosus root. Asparagus racemosus roots (100g) were crushed and macerated for 4 hours, then extracted with water (800mL) at ambient temperature. The aqueous extract was filtered, and this process was repeated for 4 hours using water (2 × 600mL) under similar extraction conditions. The combined aqueous extracts were evaporated under reduced pressure to obtain the product as a brown solid (AR-1, 45g).

[0114] Comparative Example 2 (AR-2): 90% aqueous ethanol extract of Asparagus racemosus root. Asparagus racemosus (100g) was crushed and extracted with 90% aqueous ethanol (800mL) at 80°C for 4 hours. The contents were cooled to room temperature and filtered. The extraction process was repeated under the same extraction conditions using 90% aqueous ethanol (800mL). The combined 90% aqueous ethanol extract was evaporated under reduced pressure to obtain the product as a brown solid (AR-2, 42.0g).

[0115] Example 1: Concentration of water extract with 90% aqueous ethanol and extraction with used 90% aqueous ethanol. AR-3: Asparagus racemosus root (100g) was crushed and extracted with water (800mL) under maceration at ambient temperature for 16 hours. The aqueous extract was filtered, and this process was repeated with water (2 × 600mL) under similar extraction conditions for 4 hours. The combined aqueous extracts were evaporated under reduced pressure to obtain an aqueous extract. 90% aqueous ethanol (250mL) was added to this aqueous extract and stirred at a higher temperature for 2 hours. The 90% aqueous ethanol layer was separated and evaporated under reduced pressure to obtain the product as a brown solid (23g, AR-3).

[0116] AR-4: The used raw material after water extraction was extracted with 90% aqueous ethanol (600 mL) at a higher temperature for 2 hours. The mixture was cooled to room temperature and filtered. The extraction process was repeated twice using 90% aqueous ethanol (2 × 600 mL) under the same extraction conditions. The combined 90% aqueous ethanol solution was evaporated under reduced pressure to obtain the product as a brown solid (5.17 g, AR-4).

[0117] The solids from the two extracts (AR-3 and AR-4) were combined and ground to obtain a concentrated asparagus racemosus root extract as a homogeneous brown solid. The concentrated asparagus racemosus root extract of the present invention was subjected to column chromatography using a solvent that increased polarity with ordinary silica gel. The fractions were further subjected to preparative HPLC using a reversed-phase C18 silica column to obtain the pure compound. The structure of the pure compound is as follows: 1 HNMR, 13 The compounds were established by analyzing 13C NMR and mass spectrometry data. By comparing the spectral data with those reported in the literature, three of the pure compounds were identified as shatavarin I, shatavarin IV, and shatavarin IX. The fourth compound was identified as dehydroshatavarin I, which, surprisingly, is a novel compound not reported in the literature.

[0118] Shatavalin I:1 H NMR(d6-DMSO): δ5.17(1H,d,J=4.4Hz),5.14(1H,d,J=2.4Hz),5.00(1H,s),4.82- 4.90(5H,brs),4.72(1H,s),4.59-4.63(3H,m),4.47(1H,d,J=7.6Hz),4.38-4.43 (3H,m),4.33(1H,d,J=7.6Hz),4.16(1H,t,J=5.6Hz),4.09(1H,d,J=7.6Hz),3.92 (2H,brs),3.88(1H,dd,J=6.0,9.6Hz),3.65-3.69(3H,m),3.57-3.61(2H,m),3.42 -3.51(8H,m),3.16-3.19(3H,m),3.11-3.14(2H,m),3.05-3.10(3H,m),2.93-3.0 0(2H,m),1.79-1.93(3H,m),1.74(2H,brs),1.58-1.69(5H,m),1.35-1.55(11H,m ), 1.16-1.24 (2H,m), 1.06-1.15 (2H,m), 1.01 (3H,d,J=6.0Hz), 0.95-0.98 (1H,m), 0.90-0.91 (3H,m), 0.90 (3H,s), 0.88 (3H,d,J=6.4Hz), 0.80 (1H,s), 0.72 (3H,s). Mass (Q-tof, Yin Immobile): m / z 1065.5480 (MH) - .

[0119] デヒドロシャタバリンI: 11H NMR (d6-DMSO): δ 5.16 (1H, d, J = 4.4 Hz), 5.13 (1H, d, J = 4.0 Hz), 4.90 (1H, d, J = 4.8 Hz), 4.81 - 4.85 (4H, m), 4.72 (1H, s), 4.62 - 4.68 (4H, m), 4.59 (1H, d, J = 5.2 Hz), 4.47 (1H, d, J = 8.0 Hz), 4.41 (1H, d, J = 5.6 Hz), 4.37 (1H, t, J = 6.4 Hz), 4.33 (1H, d, J = 7.6 Hz), 4.27 - 4.32 (1H, m), 4.16 (1H, t, J = 5.6 Hz), 4.08 (1H, d, J = 8.0 Hz), 3.64 - 3.67 (3H, m), 3.61 (1H, brs), 3.57 - 3.58 (1H, m), 3.37 - 3.51 (7H, m), 3.03 - 3.19 (9H, m), 2.92 - 3.01 (2H, m), 2.01 - 2.13 (3H, m), 1.63 - 1.84 (5H, m), 1.55 (3H, s), 1.36 - 1.52 (9H, m), 1.15 - 1.28 (5H, m), 1.10 (3H, d, J = 6.4 Hz), 1.06 - 1.09 (2H, m), 0.95 - 1.05 (2H, m), 0.90 (3H, s), 0.89 (3H, d, J = 6.4 Hz), 0.62 (3H, s). Mass (Q-tof, negative ion mode): m / z 1047.5400 (M - H) - .

[0120] Shatabarin IX: 1 1H NMR (d6-DMSO): δ 5.49 (1H, brs), 5.16 (2H, brs), 5.00 (1H, s), 4.94 - 4.96 (1H, m), 4.82 (4H, brs), 4.59 - 4.63 (1H, m), 4.38 - 4.51 (4H, m), 4.29 (1H, d, J = 7.2 Hz), 4.09 (1H, d, J = 8.0 Hz), 3.92 (2H, brs), 3.64 - 3.69 (5H, m), 3.42 - 3.51 (3H, m), 2.93 - 3.20 (8H, m), 1.80 - 1.93 (3H, m), 1.72 (2H, brs), 1.58 - 1.69 (5H, m), 1.35 - 1.55 (10H, m), 1.09 - 1.24 (7H, m), 0.90 (3H, s), 0.85 - 0.90 (6H, m), 0.72 (3H, s). Mass (Q-tof, positive ion mode): m / z 903.1861 (M + H)+ .

[0121] Shatavalin IV: 1 H NMR (d6-DMSO): δ5.11(1H,d,J=3.6Hz),5.07(1H,d,J=3.6Hz),4,82(1H,d,J=4.0Hz),4.48(1H,d,J=4.0Hz),4.74(1H,s),4.53-4.56( 3H,m),4.48(1H,d,J=7.6Hz),4.32(3H,brs),4.09(1H,brs),3.79-3.93(3H,m),3.59-3.69(3H,m),3.43-3.51(6H,m),3.36(2H,t,J=8 .4Hz),3.21(2H,brs),3.09-3.13(2H,m),3.00(1H,brs),1.91(2H,brs),1.75-1.84(5H,m),1.67-1.69(3H,m),1.28-1.56(12H,m),1. 16(3H,d,J=6.4Hz),1.12(3H,d,J=6.4Hz),1.01-1.10(2H,m),1.02(3H,d,J=6.8Hz),0.94(3H,d,J=6.8Hz),0.91(3H,s),0.72(3H,s). LC-MS (cation mode): m / z 909(M+Na) + .

[0122] Next, the extracts were standardized using pure compounds as reference markers. Pure compounds of Asparagus racemosus were evaluated using analytical HPLC, and their retention times (Rt) are summarized in Table 6 below. Representative HPLC chromatograms of Asparagus racemosus extract compositions are shown in Figure 2.

[0123] Table 6: Retention time of the four markers [Table 6]

[0124] Example 2: Concentration of the aqueous extract with 90% aqueous butanol and extraction with used 90% aqueous ethanol. AR-5: Asparagus racemosus root (100g) was crushed and extracted with water (800mL) under maceration at ambient temperature for 16 hours. The aqueous extract was filtered, and this process was repeated with water (2 × 600mL) under similar extraction conditions for 4 hours. The combined aqueous extract was evaporated under reduced pressure to a weight of 60g. 90% aqueous n-butanol (250mL) was added to this residue and stirred at 80°C for 2 hours. The 90% aqueous n-butanol layer was separated and evaporated under reduced pressure to obtain the product as a brown solid (9g, AR-5).

[0125] AR-4: The used raw material after water extraction was extracted as described in Example 1, and the product was obtained as a brown solid (5.17 g, AR-4).

[0126] Example 3: Concentration of the aqueous extract with n-butanol and extraction with used 90% aqueous ethanol. AR-6: Asparagus racemosus root (100g) was crushed and extracted with water (800mL) under maceration at ambient temperature for 16 hours. The aqueous extract was filtered, and this process was repeated with water (2 × 600mL) under similar extraction conditions for 4 hours each. The combined aqueous extract was evaporated under reduced pressure to a volume of 200mL. n-butanol (200mL) was added to this solution and stirred at ambient temperature for 30 minutes. The n-butanol layer was separated, and the process was repeated twice with n-butanol (2 × 200mL). The combined n-butanol layer was evaporated under reduced pressure to obtain the product as a brown solid (11g, AR-6).

[0127] AR-4: The used raw material after water extraction was extracted as described in Example 1, and the product was obtained as a brown solid (5.17 g, AR-4).

[0128] Example 4: Concentration of the aqueous extract with methanol and extraction with used 90% aqueous ethanol. AR-7: Asparagus racemosus roots (100g) were crushed and extracted with water (800mL) under maceration at ambient temperature for 16 hours. The aqueous extract was filtered, and this process was repeated with water (2 × 600mL) under similar extraction conditions for 4 hours. The combined aqueous extracts were evaporated under reduced pressure to a weight of 60g. Methanol (250mL) was added to this residue and stirred at a higher temperature for 2 hours. The methanol layer was separated and evaporated under reduced pressure to obtain the product as a brown solid (34g, AR-7).

[0129] AR-4: The used raw material after water extraction was extracted as described in Example 1, and the product was obtained as a brown solid (5.17 g, AR-4).

[0130] Example 5: Concentration of the aqueous extract with 80% aqueous acetone and extraction with used 90% aqueous ethanol. AR-8: Asparagus racemosus roots (100g) were crushed and extracted with water (800mL) under maceration at ambient temperature for 16 hours. The aqueous extract was filtered, and this process was repeated with water (2 × 600mL) under similar extraction conditions for 4 hours. The combined aqueous extracts were evaporated under reduced pressure to a weight of 60g. 80% aqueous acetone (250mL) was added to this residue and stirred at a higher temperature for 2 hours. The 80% aqueous acetone layer was separated and evaporated under reduced pressure to obtain the product as a brown solid (8g, AR-8).

[0131] AR-4: The used raw material after water extraction was extracted as described in Example 1, and the product was obtained as a brown solid (5.17 g, AR-4).

[0132] Example 6: Concentration of aqueous extract with methanol and extraction with used 90% aqueous methanol.

[0133] AR-7: Asparagus racemosus root (100g) was crushed, extracted with water, and concentrated with methanol using the procedure described in Example 4 to obtain the product as a brown solid (34g, AR-7).

[0134] AR-9: The used raw material after water extraction was extracted with 90% aqueous methanol (600 mL) at high temperature for 2 hours. It was cooled to room temperature and filtered. The extraction process was repeated twice under the same extraction conditions using 90% aqueous methanol (2 × 400 mL). The combined 90% aqueous methanol extract was evaporated under reduced pressure to obtain the product as a brown solid (6.5 g, AR-9).

[0135] Example 7: Concentration of aqueous extract with 80% aqueous acetone and extraction with used 80% aqueous acetone. AR-8: Asparagus racemosus root (100g) was crushed, extracted with water, and concentrated with 80% aqueous acetone using the procedure described in Example 5 to obtain the product as a brown solid (8g, AR-8).

[0136] AR-10: The used raw material after water extraction was extracted with 80% aqueous acetone (600 mL) at high temperature for 2 hours. The contents were cooled to room temperature and filtered. The extraction process was repeated twice using 80% aqueous acetone (2 × 400 mL) under the same extraction conditions. The combined 80% aqueous acetone extract was evaporated under reduced pressure to obtain the product as a brown solid (4.1 g, AR-10).

[0137] Example 8: Concentration of water extract using macroporous resin and extraction with used 90% aqueous ethanol. AR-11: Asparagus racemosus root (100g) was crushed and extracted with water (800mL) under maceration at ambient temperature for 16 hours. The aqueous extract was filtered, and this process was repeated with water (2 × 600mL) under the same extraction conditions for 4 hours each. The combined aqueous extract was loaded onto PA-800 macroporous resin (125mL, loading time 1 BV / h). After loading was complete, the column was eluted with water (250mL, 2 BV / h). Subsequently, the column was further eluted with 90% aqueous ethanol (750mL, 2 BV / h), and the 90% aqueous ethanol eluate was evaporated under reduced pressure to obtain the product as a brown solid (5.0g, AR-11).

[0138] AR-4: The used raw material after water extraction was extracted with 90% aqueous ethanol as described in Example 1, and the product was obtained as a brown solid (5.17 g, AR-4).

[0139] The solids from the two extracts mentioned above (AR-11 and AR-4) were combined and ground to obtain a concentrated asparagus racemosus root extract as a homogeneous brown solid.

[0140] Example 9: Concentration of aqueous extract using macroporous resin and extraction with used 90% aqueous methanol. AR-11: Asparagus racemosus root (100g) was crushed, extracted with water, and concentrated in a macroporous resin using the procedure described in Example 8 to obtain the product as a brown solid (5.0g, AR-11). AR-9: The used raw material after water extraction was extracted with 90% aqueous methanol as described in Example 6, and the product was obtained as a brown solid (6.5g, AR-9).

[0141] Example 10: Concentration of water extract using macroporous resin and extraction with used 80% aqueous acetone. AR-11: Asparagus racemosus root (100g) was crushed, extracted with water, and concentrated in a macroporous resin using the procedure described in Example 8 to obtain the product as a brown solid (5.0g, AR-11). AR-10: The used raw material after water extraction was extracted with 80% aqueous acetone as described in Example 7, and the product was obtained as a brown solid (4.1g, AR-10).

[0142] Example 11: Aqueous extraction of Asparagus racemosus root, concentration of the aqueous extract with a macroporous resin, and extraction with used 90% aqueous ethanol. AR-1: Asparagus racemosus roots (40 kg) were crushed and extracted with water (320 L) at ambient temperature under maceration for 16 hours. The aqueous extract was filtered, and this process was repeated with water (2 × 240 L) under similar extraction conditions for 4 hours. The combined aqueous extract was divided into two parts. Part 1 of the solution was evaporated under reduced pressure to obtain the product as a brown solid (8 kg, AR-1).

[0143] AR-11: Part 2 of the solution was loaded onto PA-800 macroporous resin (25 L, loading time 1 BV / h). After loading was complete, the column was eluted with water (50 L, 2 BV / h). Subsequently, the column was further eluted with 90% aqueous ethanol (150 L, 2 BV / h), and the 90% aqueous ethanol eluate was evaporated under reduced pressure to obtain the product as a brown solid (1.1 kg, AR-11).

[0144] AR-4: The used raw material after water extraction was extracted with 90% aqueous ethanol (240 L) at high temperature for 2 hours. It was cooled to room temperature and filtered. The extraction process was repeated twice using 90% aqueous ethanol (2 × 160 L) under the same extraction conditions. The combined 90% aqueous ethanol extract was evaporated under reduced pressure to obtain the product as a brown solid (2.2 kg, AR-4).

[0145] Example 12: Concentration of water extract using membrane filtration and extraction with used 90% aqueous ethanol. AR-12: Asparagus racemosus roots (100g) were crushed and extracted with water (800mL) under maceration at ambient temperature for 16 hours. The aqueous extract was filtered, and this process was repeated with water (2 × 600mL) under the same extraction conditions for 4 hours. The combined aqueous extracts were filtered through a 0.5 micron filter and passed through an ultrafiltration (10,000 Da) membrane. The ultrafiltration permeate was collected and evaporated under reduced pressure to obtain the product as a brown solid (20.16g, AR-12).

[0146] AR-4: Used R / M after water extraction was extracted with 90% aqueous ethanol (600 mL) under a continuous extraction process at 80°C for 12 hours. The aqueous ethanol extract after continuous extraction was evaporated under reduced pressure to obtain the product as a brown solid (4.13 g, AR-4).

[0147] Example 13: Preparation of various compositions of asparagus racemosus root-containing aqueous extract concentrated with 90% aqueous ethanol (AR-3) and consumed 90% aqueous ethanol extract (AR-4) in the following ratios. Comp-1(C-1): C-1 is prepared by combining AR-3 and AR-4 in a 4:1 ratio; Comp-2(C-2): C-2 is prepared by combining AR-3 and AR-4 in a 3:1 ratio; Comp-3(C-3): C-3 is prepared by combining AR-3 and AR-4 in a 2:1 ratio; Comp-4(C-4): C-4 is prepared by combining AR-3 and AR-4 in a 1:1 ratio; Comp-5(C-5):C-5 was prepared by combining AR-3 and AR-4 in a 1:2 ratio.

[0148] Example 14: Preparation of various compositions of aqueous extract containing asparagus racemosus root concentrated with 90% aqueous butanol (AR-5) and consumed 90% aqueous ethanol extract (AR-4) in the following ratios. Comp-6 (C-6): C-6 is prepared by combining AR-5 and AR-4 in a 2:1 ratio; Comp-7 (C-7): C-7 was prepared by combining AR-5 and AR-4 in a 1:1 ratio; Comp-8 (C-8): C-8 was prepared by combining AR-5 and AR-4 in a 1:2 ratio.

[0149] Example 15: Preparation of various compositions of aqueous extract containing asparagus racemosus root concentrated with n-butanol (AR-6) and consumed 90% aqueous ethanol extract (AR-4) in the following ratios. Comp-9 (C-9): C-9 was prepared by combining AR-6 and AR-4 in a 1:1 ratio.

[0150] Example 16: Preparation of various compositions of aqueous extract containing asparagus racemosus root concentrated with methanol (AR-7) and consumed 90% aqueous ethanol extract (AR-4) in the following ratios. Comp-10 (C-10): C-10 was prepared by combining AR-7 and AR-4 in a 1:1 ratio.

[0151] Example 17: Preparation of various compositions of aqueous extract containing asparagus racemosus root concentrated with 80% aqueous acetone (AR-8) and consumed 90% aqueous ethanol extract (AR-4) in the following ratios. Comp-11(C-11): C-11 was prepared by combining AR-8 and AR-4 in a 1:1 ratio.

[0152] Example 18: Preparation of various compositions of aqueous extract containing asparagus racemosus root concentrated with methanol (AR-7) in the following ratios and the consumed 90% aqueous methanol extract (AR-9). Comp-12(C-12): C-12 was prepared by combining AR-7 and AR-9 in a 1:1 ratio.

[0153] Example 19: Preparation of various compositions of aqueous extract containing asparagus racemosus root concentrated with 80% aqueous acetone (AR-8) and consumed 80% aqueous acetone extract (AR-10) in the following ratios. Comp-13(C-13): C-13 was prepared by combining AR-8 and AR-10 in a 1:1 ratio.

[0154] Example 20: Preparation of various compositions of asparagus racemosus root-containing aqueous extract concentrated with the following ratios of macroporous resin (AR-11) and consumed 90% aqueous ethanol extract (AR-4). Comp-14 (C-14): C-14 was prepared by combining AR-11 and AR-4 in a 2:1 ratio; Comp-15 (C-15): C-15 was prepared by combining AR-11 and AR-4 in a 1:1 ratio; Comp-16 (C-16): C-16 was prepared by combining AR-11 and AR-4 in a 1:2 ratio;

[0155] Example 21: Preparation of various compositions of aqueous extract containing asparagus racemosus root concentrated with macroporous resin (AR-11) and consumed 90% aqueous methanol extract (AR-9) in the following ratios. Comp-17 (C-17): C-17 was prepared by combining AR-11 and AR-9 in a 2:1 ratio; Comp-18 (C-18): C-18 was prepared by combining AR-11 and AR-9 in a 1:1 ratio; Comp-19 (C-19): C-19 was prepared by combining AR-11 and AR-9 in a 1:2 ratio.

[0156] Example 22: Preparation of various compositions of asparagus racemosus root-containing aqueous extract concentrated with the following ratios of macroporous resin (AR-11) and consumed 80% aqueous acetone extract (AR-10). Comp-20 (C-20): C-20 was prepared by combining AR-11 and AR-10 in a 2:1 ratio; Comp-21 (C-21): C-21 was prepared by combining AR-11 and AR-10 in a 1:1 ratio; Comp-22 (C-22): C-22 was prepared by combining AR-11 and AR-10 in a 1:2 ratio.

[0157] Example 23: Preparation of various compositions of aqueous extracts concentrated with asparagus racemosus root-containing aqueous extract (AR-1), macroporous resin (AR-11) in the following ratios, and consumed 90% aqueous ethanol extract (AR-10). Comp-23 (C-23): C-23 was prepared by combining AR-1, AR-11, and AR-4 in a ratio of 8:1:2; Comp-24 (C-24): C-24 is prepared by combining AR-1, AR-11, and AR-4 in a 1:1:1 ratio; Comp-25 (C-25): C-25 was prepared by combining AR-1, AR-11, and AR-4 in a ratio of 1:3:3.

[0158] Example 24: Standardization of Asparagus racemosus root extract Various compositions of asparagus racemosus root extract were standardized against shatavalin I, dehydroshatavalin I, shatavalin IV, and shatavalin IX by analytical HPLC, and the results are summarized in Table 7.

[0159] Table 7: Estimated four shatavarins in compositions (1-24) by HPLC analysis. [Table 7]

[0160] Example 25: Formulation of the composition Comp-26: A blend of acacia rubber (180g) and water was stirred at room temperature for 15-30 minutes, followed by the addition of concentrated water extract AR-3 (640g). Stirring was continued at room temperature for 5-10 minutes. Then, used 90% ethanol extract, AR-4 (160g), was gradually introduced at room temperature and stirred for approximately 30 minutes until a homogeneous mixture was obtained. The resulting contents were then dried under reduced pressure to obtain flakes. These flakes were uniformly blended with colloidal silicon dioxide (20g) in a polyethylene bag or a suitable blender. The mixture was further ground and sieved through a #40 mesh to obtain the composition as a fine powder (Comp-26).

[0161] Comp-27: A blend of acacia rubber (1.26 kg) and water was stirred at room temperature for 15-30 minutes, followed by the addition of AR-1 (3.59 kg). Stirring was continued at room temperature for 5-10 minutes. Then, AR-11 (0.64 kg) and AR-4 (1.37 kg) were gradually introduced at room temperature, and the mixture was stirred for approximately 30 minutes until a homogeneous mixture was obtained. The resulting contents were then dried under reduced pressure to obtain flakes. These flakes were uniformly blended with colloidal silicon dioxide (0.14 kg) in a polyethylene bag or a suitable blender. The mixture was further ground and sieved through a #40 mesh to obtain the composition as a fine powder (Comp-27).

[0162] Comp-28: A blend of acacia rubber (1.08 kg) and water was stirred at room temperature for 15-30 minutes, followed by the addition of AR-11 (1.44 kg). Stirring was continued at room temperature for 5-10 minutes. Then, AR-4 (3.36 kg) was slowly added at room temperature and stirred for approximately 30 minutes until a homogeneous mixture was obtained. The resulting contents were dried under reduced pressure to obtain flakes. These flakes were uniformly blended with colloidal silicon dioxide (0.12 kg) in a polyethylene bag or a suitable blender. The mixture was further ground and sieved through a #40 mesh to obtain the composition as a fine powder (Comp-28).

[0163] Comp-29: A blend of acacia rubber (0.12 kg) and water was stirred at room temperature for 15-30 minutes, followed by the addition of AR-11 (1.1775 kg). Stirring was continued at room temperature for 5-10 minutes. Then, AR-4 (0.1725 kg) was slowly added at room temperature, and the mixture was stirred for approximately 30 minutes until a homogeneous mixture was obtained. The resulting contents were dried under reduced pressure to obtain flakes. These flakes were uniformly blended with colloidal silicon dioxide (0.030 kg) in a polyethylene bag or a suitable blender. The mixture was further ground and sieved through a #40 mesh to obtain the composition as a fine powder (Comp-29).

[0164] Example 25: Standardization of formulated asparagus racemosus root extract composition Various formulated compositions of asparagus racemosus root extract were standardized against shatavalin I, dehydroshatavalin I, shatavalin IV, and shatavalin IX by analytical HPLC, and the results are summarized in Table 8.

[0165] Table 8: Estimated four shatavarins in compositions (26-29) by HPLC analysis [Table 8]

[0166] Example 26: Evaluation of estrogen receptor alpha (ER-α) phosphorylation by Western blot assay. Human MCF-7 cells (ATCC catalog number HTB-22; 5 × 10 in 3 mL) 5 Cells (100 / well) were seeded into a 6-well cell culture plate and maintained in DMEM medium containing 10% FBS at 37°C under a humidified atmosphere of 5% CO2. After 48 hours of seeding, the cells were washed twice with serum-free DMEM medium (without phenol red) and maintained in the same medium for a further 24 hours. Cells were treated with different test samples (0.2 μg / mL) in serum-free DMEM medium and incubated in a CO2 incubator at 37°C for 4 hours. Untreated cells were used as the vehicle control.

[0167] Western blotting: After incubation, cell culture plates were placed on an ice tray and washed twice with cold 1XPBS. 80 microliters of lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 μg / mL aprotinin, 10 μg / mL leupeptin, 1% Triton X-100, 1 mM NaF, 1 mM Na3VO4, 0.5% sodium deoxycholate, and 1 μM pepstatin) were added to each well and sonicated for 30 seconds. After homogenization, the samples were centrifuged at 16128 × g for 10 minutes at 4°C to precipitate undisturbed cells and nuclei. The supernatant was collected and proteins were quantified using the Pierce BCA protein assay kit (Thermo Scientific catalog number 23225). Protein samples were subjected to SDS-PAGE, and the separated proteins were transferred onto a nitrocellulose membrane using the wet blotting method. Briefly, 10 μg of protein was loaded onto an acrylamide gel (7.5% dissolved) and run at 100 V for approximately 1 hour and 40 minutes. At the end of the run, the protein was transferred to the nitrocellulose membrane by placing the transfer system in a 4°C chamber (100 V, 2 hours). After transfer, the membrane was washed with 0.1 M Tris-buffered saline containing 0.05% Tween 20 (TBST) and blocked with Superblock blocking buffer (Thermo Scientific catalog no. 37535) for 30 minutes with constant agitation. The membrane was incubated with a primary antibody that recognizes phospho-ER-α (phosphorylation site: Ser118) in TBST at 4°C for 16 hours with agitation. The membranes were washed three times and incubated with peroxidase-affinity goat anti-rabbit secondary antibody (Jackson ImmunoResearch catalog no. 111-035-045; 1:10000 dilution) at room temperature for 1 hour with agitation. They were then washed three times in TBST for 5 minutes each. Immunoreactive bands were detected using a chemiluminescent substrate (Thermo Scientific catalog no. 34080), and images were captured using a Bio-Rad Molecular imager (model: ChemiDOC MP).The phospho-ER-α antibody was removed from the membrane and reprobed with anti-β-actin antibody (Sigma Chemical Co., USA; 1:10000 dilution) by incubation at room temperature for 2 hours, and images were captured. The intensities of the phospho-ER-α and actin protein bands were calculated and normalized using Carestream MI software. Normalized pER-α expression (in arbitrary units) was measured using the following formula: JPEG2026519309000011.jpg13163

[0168] Tables 9-11 summarize the normalized pER-α expression results in cells treated with individual extracts and their compositions.

[0169] Table 9: Normalized pER-α expression in cells treated with compositions of asparagus racemosus root-containing aqueous extract concentrated with 90% aqueous ethanol (AR-3) and consumed 90% aqueous ethanol extract (AR-4). [Table 9]

[0170] Table 10: Compositions of asparagus racemosus root-containing aqueous extract concentrated with consumed 90% aqueous butanol (AR-5) and 90% aqueous ethanol extract (AR-4); and normalized pER-α expression of asparagus racemosus root-containing aqueous extract compositions concentrated with macroporous resin (AR-11) and consumed 90% aqueous ethanol extract (AR-4). [Table 10]

[0171] Table 11: Normalized pER-α expression of compositions of water-containing aqueous extract of Asparagus racemosus root (AR-1), aqueous extract concentrated with macroporous resin (AR-11), and consumed 90% aqueous ethanol extract (AR-10). [Table 11]

[0172] Example 27: In vitro cell-based assay for prostaglandin E2 (PGE2) inhibition Human blood from healthy volunteers was collected from peripheral veins using a syringe containing 2 mM EDTA. Plasma was separated by centrifugation at 1000 rpm for 10 minutes, and the residual blood was diluted in RPMI medium supplemented with 10% FBS and 2 mM EDTA in a 1:3 ratio. 30 ml of blood was carefully overlaid on 15 mL of Ficoll / Lymphoprep in a 50 mL Falcon tube in the dark, and the tube was centrifuged at 350 × g for 30 minutes at an acceleration of 9 without using the brake. The buffy coat (interface between the medium and Ficoll) containing peripheral blood mononuclear cells (PBMCs) was carefully collected in 25 mL of cold 1 × phosphate-buffered saline (PBS) and centrifuged at 1200 rpm for 10 minutes. Residual RBCs found in the PBMC pellet were removed by treatment with ACK lysis buffer (Gibco catalog no. A10492-01) and washed with fresh 1 × PBS. The PBMCs were then 0.1 × 10⁶ 6 Cells were seeded in 96-well plates with a cell density of 10 cells / well and treated with test samples of different concentrations. Cells containing 0.2% DMSO were used as a vehicle control. The plates were incubated in a CO2 incubator at 37°C for 2 hours. Finally, the vehicles were removed and the cells were induced with LPS (final concentration of 10 ng / mL) for 4 hours by maintaining the plates at 37°C in a CO2 incubator. Cells treated with LPS alone were used as an induction control. The plates were centrifuged at 1200 rpm for 5 minutes, and 120 μL of cell-free supernatant was collected. PGE2 quantification was performed using an ELISA kit (Cayman Chemicals catalog number 514010) according to the manufacturer's instructions. Absorbance was measured at 412 nm in dynamic mode for 30 minutes using a Spectramax 2e plate reader. PGE2 inhibition was calculated using the following formula. PGE2 inhibition % = [(PGE2 concentration during induction) - (PGE2 concentration in test sample)] / (PGE2 concentration during induction) × 100

[0173] The inhibition rates of PGE2 production by individual extracts and their compositions are summarized in Tables 12-15.

[0174] Table 12: PGE2 inhibition by Asparagus racemosus aqueous extract and the composition of the present invention [Table 12]

[0175] Table 13: Inhibition of PGE2 by compositions of asparagus racemosus root-containing aqueous extract concentrated with 90% aqueous ethanol (AR-3) and consumed 90% aqueous ethanol extract (AR-4). [Table 13]

[0176] Table 14: Compositions of asparagus racemosus root-containing aqueous extract concentrated with 90% aqueous butanol (AR-5) and consumed 90% aqueous ethanol extract (AR-4); and inhibition of PGE2 by compositions of asparagus racemosus root-containing aqueous extract concentrated with macroporous resin (AR-11) and consumed 90% aqueous ethanol extract (AR-4). [Table 14]

[0177] Table 15: Inhibition of PGE2 by compositions of asparagus racemosus root-containing aqueous extract (AR-1), aqueous extract concentrated with macroporous resin (AR-11), and consumed 90% aqueous ethanol extract (AR-10). [Table 15]

[0178] Example 28: In vivo efficacy of AR-1 and Comp-1 in alleviating cyclophosphamide-induced primary ovarian dysfunction in Sprague Dawley rats.

[0179] After a 7-day acclimatization period, female Sprague Dawley rats (age: 10-12 weeks; body weight: 230-290g) were randomly divided into four groups (n=8): vehicle control (G1), cyclophosphamide (CYP) mg / kg (G2), AR-1 (100mg / kg; G3), and comp-1 (100mg / kg; G4). On day 8 of the experiment, each rat in groups G2, G3, and G4 received an (ip) dose of 200mg / kg of CYP, followed by an additional 8mg / kg / day of CYP on days 9-21. G1 and G2 rats were administered 0.5% carboxymethylcellulose sodium (CMC-Na), while G3 and G4 rats received the test supplements (AR-1 and comp-1) daily via oral gastric tube feeding for 21 consecutive days. On day 22, all animals were sacrificed using CO2 inhalation. Ovaries were harvested, fixed in 10% formalin, and embedded in paraffin wax. The paraffin-embedded tissue specimens were cut into 5 μm thick sections, placed on clean glass slides, and stained with hematoxylin and eosin (HE). The stained tissue sections were examined under a 20x objective lens using an optical microscope (Axio Scope A1, Carl-Zeiss, Germany). Twenty randomly selected fields were analyzed for primordial follicles, primary follicles, antral follicles, Graafian follicles, and atresia follicles, and the average total number of follicles present in each tissue section was calculated. The data are shown in Table 16.

[0180] For example, 100 mg doses of Asparagus racemosus aqueous extract (AR-1) and Asparagus racemosus concentrated extract composition (comp-1) showed improvements of 32.68% and 51.15%, respectively, in total follicle count compared to the cyclophosphamide control (G2). Surprisingly, the concentrated Asparagus racemosus extract composition showed higher efficacy (Table 16). A reduction in follicle count is a major feature of primary ovarian dysfunction, also known as premature ovarian failure, which causes hormonal imbalances and disrupts regular menstrual cycles, infertility, and menopausal symptoms.

[0181] Table 16: Evaluation of Asparagus racemosus concentrate supplementation for total follicle count in female Sprague Dawley rats [Table 16]

[0182] Example 29: Efficacy of Comp-26, Comp-27, and Comp-28 supplementation compared to 90% aqueous ethanol extract of Shatavari (AR-2) in letrozole-induced PCOS in female Sprague Dawley rats.

[0183] After a 7-day acclimatization period, female Sprague Dawley rats (age: 10–12 weeks, body weight 250–300 g) were randomly assigned to six groups (n=7): vehicle control (G1), letrozole (G2; 1 mg / kg), AR-2 (G3; 20 mg / kg), Comp-26 (G4; 20 mg / kg), Comp-27 (G5; 20 mg / kg), and Comp-28 (G6; 20 mg / kg).

[0184] The study was conducted in two phases: an induction phase and a replacement phase. During the induction phase, all animals except G1 were administered letrozole enterally to induce PCOS for 21 days. During the replacement phase (days 22–48), all rats were supplemented with either a vehicle or one of the test items once daily. An oral glucose tolerance test (OGTT) was performed on day 49 in rats fasted overnight. Blood samples were collected on day 48. Serum was separated from the blood samples by centrifugation at 3200xg for 15 minutes at 4°C and stored in aliquots at -80°C for biomarker analysis. On day 50, all rats were euthanized using CO2 asphyxiation and their ovaries were collected. For histopathological examination, the ovaries were fixed with 10% normal-buffered formalin. The fixed tissues were paraffin-embedded, sectioned using a microtome (5–7 μm), and stained with hematoxylin-eosin according to a standard protocol. Stained tissue sections were examined under a light microscope (Carl Zeiss, Germany) at 10x magnification.

[0185] Tables 17 and 18 show the results of oral glucose tolerance tests (OGTT), serum hormone levels such as 17β-estradiol, and testosterone. Furthermore, Table 19 shows the total follicles, Graafian follicles, and follicular cysts.

[0186] Table 17: Evaluation of Comp-26, Comp-27, and Comp-28 supplementation compared to 90% aqueous ethanol extract of Shatavari (AR-2) in oral glucose tolerance tests (OGTT) in female Sprague Dawley rats. [Table 17]

[0187] Table 18: Evaluation of Comp-26, Comp-27, and Comp-28 supplementation compared to 90% aqueous ethanol extract of Shatavari (AR-2) on serum hormone levels, including 17β-estradiol and testosterone, in female Sprague Dawley rats. [Table 18]

[0188] Table 19: Evaluation of Comp-26, Comp-27, and Comp-28 supplementation compared to 90% aqueous ethanol extract of Shatavari (AR-2) on total follicles, Graaf follicles, and follicular cysts in female Sprague Dawley rats. [Table 19]

[0189] It will be understood that the above description is illustrative and not limiting. Embodiments will be apparent to those skilled in the art upon consideration of the above description. Therefore, the scope of the present invention should not be determined by reference to the above description, but rather by the appended claims, together with the entire scope of equivalents to which such claims are entitled.

Claims

1. A synergistically concentrated asparagus racemosus extract composition comprising, for the health benefits of women, (i) 0.5 to 15% shatavalin I; (ii) 0.1 to 25% dehydroshatavalin I; (iii) 2 to 25% shatavalin IV and (iv) 0.001 to 1.0% shatavalin IX.

2. The synergistically concentrated extract composition according to claim 1, optionally containing at least one additional component selected from the group consisting of excipients, carriers, and diluents that are pharmaceutically or nutritionally acceptable, selected from monosaccharides, disaccharides, polysaccharides, dextrins, polyhydric alcohols or sugar alcohols, cellulosic derivatives, silicates, metal stearates, organic acids, fatty acid esters, calcium pantothenate, amino acids, proteins, organometallic salts, natural pigments, flavorings, Class I and Class II preservatives and mixtures thereof.

3. The monosaccharide is selected from, but is not limited to, glucose, dextrose, fructose, and galactose; the disaccharide is selected from, but is not limited to, sucrose, maltose, lactose, lactulose, trehalose cellobiose, and chitobiose; the polysaccharide is selected from, but is not limited to, starch and modified starch, such as sodium starch glycolate, pregelatinized starch, soluble starch, ultrasperse A, and other modified starches; the dextrin is selected from, but is not limited to, yellow dextrin, white dextrin, maltodextrin, glucidex 12D, rice maltodextrin, Tapioca / Cassava Selected from, but not limited to, maltodextrin; polyhydric alcohol or sugar alcohol selected from, but not limited to, sorbitol, mannitol, inositol, xylitol, isomalt; cellulosic derivative selected from, but not limited to, microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose; silicate selected from, but not limited to, neucillin, beegum, talc, colloidal silicon dioxide, siloid; metal stearate selected from, but not limited to, calcium stearate, magnesium stearate, zinc stearate; organic acid, citric acid, The synergistically concentrated extract composition according to claim 2, wherein the natural gum is selected from, but is not limited to, tartaric acid, malic acid, succinic acid, lactic acid, and L-ascorbic acid; the natural gum is selected from, but is not limited to, acacia, carrageenan, guar gum, xanthan gum, gum ghati, and biopolymers / biopolysaccharides, such as sodium alginate, calcium alginate, and alginic acid; the protein is selected from, but is not limited to, whey protein, whey isolate, casein, gelatin, pectin, and agar; and the organometallic salt is selected from, but is not limited to, sodium chloride, calcium chloride, dicalcium phosphate, zinc sulfate, and zinc chloride. Natural sweeteners, such as licorice, stevia, stevioside, such as rebaudioside, palm sugar, and natural sweet proteins, such as but not limited to blazein, monellin, curculin, mavinlin, miraculin, and pentazin.

4. The synergistically concentrated extract composition according to claim 2, wherein the concentrated asparagus racemosus extract composition varies in the range of 90% to 10% by weight, and the excipient in the composition varies in the range of 10% to 90% by weight.

5. The synergistically concentrated extract composition according to claim 1, wherein the concentrated asparagus racemosus extract composition optionally contains at least one additional component selected from the group consisting of shatavalin II, shatavalin III, shatavalin V, shatavalin VI, shatavalin VII, shatavalin VIII, shatavalin IX, asparacoside, asparanin, and immunoside or mixtures thereof.

6. The synergistically concentrated extract composition according to claim 1, wherein the concentrated asparagus racemosus extract composition optionally contains at least one extract or fraction selected from the group consisting of at least one phytochemical selected from Nigella sativa, Cinnamom zeiranicum, Phoenicula vulgare, Trigonella phoenum grecum, Glycyrrhiza glabra and Linum ucithossimum, or inositol such as myo-inositol or D-chiro-inositol or a mixture thereof.

7. A synergistically concentrated asparagus racemosus extract composition comprising (i) 0.5–15% shatavalin I; (ii) 0.1–25% dehydroshatavalin I; (iii) 2–25% shatavalin IV and (iv) 0.001–1.0% shatavalin IX for the benefit of women's health; and (a) concentrating the aqueous extract of asparagus racemosus (shatavari) root by re-extraction with a polar organic solvent, or by loading the aqueous extract onto a macroporous resin and eluting it with aqueous alcohol to obtain a concentrated extract. An asparagus racemosus extract composition prepared by a method comprising: (a) extracting consumed or remaining asparagus racemosus (shatavari) roots with a polar organic solvent to obtain a second extract; (c) combining the extracts obtained from steps (a) and (b) to obtain a concentrated asparagus racemosus composition; and (d) optionally blending the concentrated asparagus racemosus composition with at least one component selected from pharmaceutically or nutritionally acceptable excipients, carriers, and diluents.

8. (a) A step of extracting asparagus racemosus root powder with water, (b) A step of evaporating the aqueous extract to a minimum volume to obtain a concentrate, (c) The step of re-extracting the concentrate from step (b) with a polar organic solvent, (d) A step of evaporating the polar organic solvent-soluble extract to obtain a concentrated extract, (e) A step in which the aqueous extract from step (a) is alternately passed through an adsorbent resin and eluted with aqueous alcohol, (f) A step of evaporating the aqueous alcohol eluent to obtain a concentrated extract, (g) A step of extracting the asparagus racemosus root residue from step (a) with a polar organic solvent, (h) A step of evaporating the polar organic solvent extract from step (g) to obtain a second extract, (i) a step of combining the extracts from step (d) or step (f) and step (h) to obtain a synergistically concentrated asparagus racemosus extract composition; (j) The step of optionally blending the synergistically concentrated extract composition of Asparagus racemosus from step (i) with at least one component selected from pharmaceutically or nutritionally, or dietarily acceptable excipients, carriers and diluents. A method for preparing the synergistically concentrated asparagus racemosus extract composition according to claim 7, comprising the above.

9. The method according to claim 8, wherein the polar organic solvent is selected from ethanol, n-butanol, methanol, acetone, aqueous ethanol, aqueous methanol, aqueous acetone, and mixtures thereof, and the aqueous alcohol is selected from methanol, ethanol, and n-butanol.

10. The synergistically concentrated asparagus racemosus extract composition according to claim 2, which is formulated into a dosage form selected from any suitable form such as a dry powder, liquid, beverage, food, dietary supplement, or tablet, capsule, soft chewable tablet, gummy, or gummy bar.

11. The synergistically concentrated asparagus racemosus extract composition according to claim 2, which can be formulated into a nutritional / dietary supplement in the form of a health food or food for a specific health use, such as a solid food like chocolate or a nutrition bar, a semi-solid food like cream, jam or gel, or a beverage like a soft drink, lactic acid drink, drop, candy, chewing gum, gummy candy, yogurt, ice cream, pudding, sweet bean jelly, jelly, cookies, tea, soft drink, juice, milk, coffee, cereal, snack bar, etc.

12. The synergistically concentrated asparagus racemosus extract composition according to claim 2, formulated into a controlled-release tablet using a controlled-release polymer-based coating by techniques including nanotechnology, microencapsulation, colloidal carrier systems, and other drug delivery systems for obtaining the desired therapeutic benefit.

13. To promote or regulate the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances; to reduce symptoms of perimenopause, including premenstrual syndrome (PMS), menstrual cycle-related discomfort; to alleviate symptoms of polycystic ovary syndrome (PCOS), polycystic ovary disease (PCOD), gynecological disorders, and genitourinary or reproductive system infections; to support ovarian function and maintain libido, reduce dyspareunia, and improve lactation, for at least one female reproductive and / or sexual health as selected from: A method for obtaining health benefits, comprising supplementing a subject in need of it with an effective dose of a synergistically concentrated extract composition of Asparagus racemosus, comprising: (i) 0.5 to 15% shatavalin I; (ii) 0.1 to 25% dehydroshatavalin I; (iii) 2 to 25% shatavalin IV and (iv) 0.001 to 1.0% shatavalin IX; and (v) optionally, at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers, and diluents.

14. The use of a synergistically concentrated extract composition of Asparagus racemosus, comprising: (i) 0.5–15% shatavalin I; (ii) 0.1–25% dehydroshatavalin I; (iii) 2–25% shatavalin IV and (iv) 0.001–1.0% shatavalin IX; (v) optionally promoting or regulating the production of reproductive hormones to alleviate hormone deficiencies and / or hormonal imbalances, reducing symptoms of perimenopause, including perimenopause, premenstrual syndrome (PMS), and menstrual cycle-related discomfort. Use of a synergistically concentrated extract composition of Asparagus racemosus, comprising at least one component selected from pharmaceutically or nutritionally or dietarily acceptable excipients, carriers, and diluents, for the purpose of alleviating symptoms of polycystic ovary syndrome (PCOS), polycystic ovary disease (PCOD), gynecological disorders, and genitourinary or genital infections; supporting ovarian function, and obtaining at least one female reproductive and sexual health benefit selected from maintaining libido, reducing dyspareunia, and improving lactation.

15. １ H NMR (d ６ -DMSO): δ 5.16 (1H, d, J = 4.4 Hz), 5.13 (1H, d, J = 4.0 Hz), 4.90 (1H, d, J = 4.8 Hz), 4.81 - 4.85 (4H, m), 4.72 (1H, s), 4.62 - 4.68 (4H, m), 4.59 (1H, d, J = 5.2 Hz), 4.47 (1H, d, J = 8.0 Hz), 4.41 (1H, d, J = 5.6 Hz), 4.37 (1H, t, J = 6.4 Hz), 4.33 (1H, d, J = 7.6 Hz), 4.27 - 4.32 (1H, m), 4.16 (1H, t, J = 5.6 Hz), 4.08 (1H, d, J = 8.0 Hz), 3.64 - 3.67 (3H, m), 3.61 (1H, brs), 3.57 - 3.58 (1H, m), 3.37 - 3.51 (7H, m), 3.03 - 3.19 (9H, m), 2.92 - 3.01 (2H, m), 2.01 - 2.13 (3H, m), 1.63 - 1.84 (5H, m), 1.55 (3H, s), 1.36 - 1.52 (9H, m), 1.15 - 1.28 (5H, m), 1.10 (3H, d, J = 6.4 Hz), 1.06 - 1.09 (2H, m), 0.95 - 1.05 (2H, m), 0.90 (3H, s), 0.89 (3H, d, J = 6.4 Hz), 0.62 (3H, s) (Mass (Q-tof, anion mode): m / z 1047.5400 (M - H) － ), characterized by the formula; Dehydroshatavalin I.

16. Dehydroshatavalin I according to claim 15, obtained from the root extract of Asparagus racemosus.

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