Pharmaceutical composition comprising herbal medicine and metformin as active ingredients for prevention or treatment of polycystic ovary syndrome

Abstract

The present invention relates to a pharmaceutical composition comprising herbal medicine (Siryung-Tang) and metformin as active ingredients for the prevention or treatment of polycystic ovary syndrome (PCOS)-related diseases. Effects such as: inhibition of nitric oxide production; regulation of secretion (expression) of inflammation-related factors; reduction of cystic follicles in ovaries; increase in mature follicles; decrease in blood levels of testosterone, estradiol, and luteinizing hormone; increase in blood levels of follicle-stimulating hormone, etc. were more significantly observed when Siryung-Tang and metformin are administered in combination than alone, whereby the ingredients can be advantageously utilized as a composition for preventing, treating, or alleviating polycystic ovary syndrome or diseases related thereto.

Description

Pharmaceutical composition for preventing or treating polycystic ovary syndrome containing herbal medicine and metformin as active ingredients

[0001] The present invention relates to a pharmaceutical composition for preventing or treating polycystic ovary syndrome, comprising a herbal medicine (Siryung-Tang) and metformin as active ingredients.

[0002] Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder that affects 4-12% of women of reproductive age. The diagnostic criteria for PCOS, established by the National Institutes of Health in 1990, require both chronic anovulation and clinical or biochemical hyperandrogenism. In contrast, the 2003 revised European / American Joint Committee on Reproductive Endocrinology (JEC) criteria define PCOS as two or more of the following three criteria: chronic anovulation, clinical or biochemical hyperandrogenism, and the presence of a bead-like pattern of about ten small follicles along the margin of an enlarged ovary.

[0003] Symptoms of PCOS include oligomenorrhea, amenorrhea, hyperandrogenism, hyperandrogenemia, and polycystic ovary morphology. In addition, based on a survey of age and weight, approximately 50% of women with PCOS have common biochemical features such as serum luteinizing hormone hypersecretion, insulin resistance, and compensatory hyperinsulinemia compared to controls.

[0004] Premature ovarian failure, an example of a condition related to polycystic ovary syndrome, occurs when ovarian function does not function properly. It is classified into irregular ovulatory cycles, luteal dysfunction, and anovulatory cycles. Anovulatory cycles can occur physiologically during puberty and menopause, but can also cause infertility in women of childbearing age. Luteal dysfunction is characterized by rapid regression of luteal function, leading to infertility and miscarriage. Because irregular ovulatory cycles are characterized by abnormalities only in the length of the cycle, they are believed to be due to an abnormality in the length of the follicular phase. While ovarian failure can manifest as menopausal symptoms in older women approaching menopause, it can also occur abnormally in women under 40, making it difficult to conceive. If symptoms worsen in this condition, it can lead to premature menopause.

[0005] The cause of PCOS remains unclear. Like other complex diseases, both genetic and environmental factors are believed to play a role in its development. As research techniques have advanced, the focus of primary pathophysiology has shifted from the ovaries to the hypothalamic-pituitary axis, and later to defects in insulin action. While research findings linking various genetic factors to PCOS have been reported, clinically applicable genetic tests remain lacking. Consequently, efforts are urgently needed to develop therapeutics and diagnostics for PCOS.

[0006] The purpose of the present invention is to provide a composition for preventing, treating or improving polycystic ovary syndrome.

[0007] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating polycystic ovary syndrome or a disease related thereto, comprising Siryeong-Tang and metformin as active ingredients.

[0008] In addition, the present invention provides a health functional food composition for preventing or improving polycystic ovary syndrome or a disease related thereto, comprising Siryeong-Tang and metformin as active ingredients.

[0009] In addition, the present invention provides a pharmaceutical composition for preventing or treating ovotoxity, comprising Siryeong-Tang and metformin as active ingredients.

[0010] In addition, the present invention provides a health functional food composition for preventing or improving ovotoxity, which contains Siryeong-Tang and metformin as active ingredients.

[0011] According to the present invention, when Siryeong-Tang and metformin are administered in combination, the effects of suppressing nitric oxide production; regulating the secretion (expression) of inflammatory factors; reducing the number of cystic follicles in the ovary; increasing the number of mature follicles; reducing the concentrations of testosterone, estradiol, and luteinizing hormone in the blood; and increasing the concentration of follicle-stimulating hormone in the blood are more significant than when Siryeong-Tang and metformin are administered alone, and thus the present invention can be usefully utilized as a composition for preventing, treating, or improving polycystic ovary syndrome or diseases related thereto.

[0012] Figure 1 shows the results of analyzing the effects of Siryeong-Tang (hereinafter referred to as SRT) and metformin (hereinafter referred to as Met) (hereinafter referred to as samples) on dystoxemia. ###p<0.001 (vs normal group) and *p<0.05, ***p<0.001 (vs VCD only treatment group).

[0013] Figure 2 shows the results of analyzing the effect of the sample on nitric oxide (NO) production. ###p<0.001 (vs. normal group) and *p<0.05, ***p<0.001 (vs. LPS only treatment group).

[0014] Figure 3 shows the results of measuring the secretion of IL-1β and IL-6 by ELISA analysis to confirm the effect of the sample on the secretion of proinflammatory cytokines. ###p<0.001 (vs normal group) and *p<0.05, ***p<0.001 (vs LPS only treatment group).

[0015] Figure 4 shows the results of analyzing the effect of the sample on the expression of inflammation-related factors.

[0016] Figure 5 shows the results of measuring the amount of estradiol secreted by ELISA analysis to confirm the effect of the sample on the production of steroid hormones in granulosa cells. ###p<0.001 (vs normal group) and *p<0.05, ***p<0.001 (vs FSH and AD treatment groups).

[0017] Figure 6 shows the results of analyzing the effects of the sample on the production of cystic follicles and graafian follicles in the ovary. #p<0.05, ###p<0.001 (vs. normal group) and *p<0.05, ***p<0.001 (vs. control group).

[0018] Figure 7 shows the results of analyzing the effects of samples on blood testosterone, estradiol, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations. ##p<0.01, ###p<0.001 (vs. normal group) and **p<0.01, ***p<0.001 (vs. control group).

[0019] Figure 8 shows the results of analyzing the effect of the sample on blood AST (aspartate aminotransferase) and ALT (alanine aminotransferase) concentrations.

[0020] Hereinafter, the present invention will be described in more detail.

[0021]

[0022] The present invention provides a pharmaceutical composition for preventing or treating polycystic ovary syndrome or a disease related thereto, or ovotoxity, comprising Siryeong-Tang and metformin as active ingredients.

[0023] The above Siryeong-tang may include, but is not limited to, Bupleurum falcatum Linne, Atractylodes orientale Juzepzuk, Atractylodes macrocephala Koidzumi, Polyporus umbellatus Fries, Poria cocos Wolf, Pinellia ternata Breitenbach, Scutellaria baicalensis Georgi, Panax ginseng C. A. Meyer, Glycyrrhiza uralensis Fischer, Cinnamomum cassia J. Presl, and Ginger (Zingiber officinale Roscoe).

[0024] Siryeong-tang is a prescription for treating diarrhea with fever and dry mouth, and for treating atopic dermatitis with excessive heat and little cold, resulting in dry mouth, chest tightness, and anxiety. Siryeong-tang's pharmacological effects include anti-inflammatory, steroid-like, and immunosuppressive effects. It has also been reported to improve lymphedema after radiation therapy, breast cancer surgery, or hip replacement.

[0025] Metformin is a compound represented by the following chemical formula 1, and the IUPAC name of the compound is 3-(diaminomethylidene)-1,1-dimethylguanidine, C4H 11 N5). Metformin, an insulin sensitizer and widely used as a treatment for type 2 diabetes, is used to reduce insulin resistance in women with polycystic ovary syndrome, thereby reducing blood insulin and androgen levels and treating anovulation.

[0026] [Chemical Formula 1]

[0027]

[0028] The diseases associated with the above polycystic ovary syndrome may be one or more selected from the group consisting of hyperandrogenism, amenorrhea, oligomenorrhea, dysfunctional uterine bleeding, premature menopause, infertility, and endometriosis, but are not limited thereto.

[0029] The above toxicity may be induced by VCD (4-vinylcyclohexene diepoxide).

[0030] The above pharmaceutical composition can inhibit nitric oxide production.

[0031] In addition, the pharmaceutical composition can suppress the expression of one or more selected from the group consisting of IL-1β (Interleukin-1β), IL-6 (Interleukin-6), iNOS (Inducible nitric oxide synthase), COX-2 (cyclooxygenase-2), and estradiol, but is not limited thereto.

[0032] Additionally, the pharmaceutical composition may decrease the concentration of testosterone, estradiol or luteinizing hormone in the blood, or increase the concentration of follicle-stimulating hormone in the blood.

[0033] Additionally, the pharmaceutical composition may reduce the number of cystic follicles or increase the number of mature follicles (graafian follicles).

[0034] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0035] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0036] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0037] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.

[0038] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0039] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.

[0040] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0041] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, administration route, etc. of the pharmaceutical composition. A person skilled in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0042]

[0043] In addition, the present invention provides a health functional food composition for preventing or improving polycystic ovary syndrome or a disease related thereto, or ovotoxity, comprising Siryeong-Tang and metformin as active ingredients.

[0044] The present invention can be generally used as a commonly used food.

[0045] The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with functional properties useful to the human body as defined in the Health Functional Food Act. "Functionality" refers to ingestion for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects.

[0046] The above health functional food composition may contain conventional food additives, and its suitability as the above “food additive” is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0047] Items listed in the above “Food Additives Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0048] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a composition according to the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by mixing a composition according to the present invention with additives such as excipients and filling a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

[0049] The definitions of terms for the above excipients, binders, disintegrants, lubricants, flavoring agents, etc. are described in documents known in the art and include those with the same or similar functions. There is no particular limitation on the type of food, and all health functional foods in the conventional sense are included.

[0050] In the present invention, the term “prevention” refers to any act of suppressing or delaying the disease or symptom by administering the composition according to the present invention.

[0051] In the present invention, the term “treatment” refers to any act of improving or beneficially changing the disease or symptom by administering a composition according to the present invention.

[0052] In the present invention, the term “improvement” refers to any act of improving the bad condition of the disease or symptom by administering the composition according to the present invention.

[0053] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0054]

[0055] [Experimental Example 1] Experimental Preparation

[0056] 1-1. Sample

[0057] SRT (Siryung-Tang) used as a sample of the present invention was prepared as follows. The constituent herbs of Siryung-Tang [Bupleurum falcatum Linne, Atractylodes orientale Juzepzuk, Atractylodes macrocephala Koidzumi, Polyporus umbellatus Fries, Poria cocos Wolf, Pinellia ternata Breitenbach, Scutellaria baicalensis Georgi, Ginseng (Panax ginseng C. A. Meyer), Glycyrrhiza uralensis Fischer, Cinnamomum cassia J. Presl, and Zingiber officinale Roscoe] were purchased from Human Herb Co., Ltd., and were selected and refined before use.

[0058] Approximately 66 g (12 g of Siho, 9.8 g of Taesa, 5.6 g of Baekchul, 5.6 g of Jeolyeong, 5.6 g of Bokryeong, 5.2 g of Pinellia pinnata, 4.6 g of Hwanggeum, 4.6 g of Insam, 4.6 g of Gamcho, 2.2 g of Yukgae, and 6 g of Geongang), equivalent to 2 doses of Siryeongtang, was placed in a round flask with 800 ml of distilled water and heated under reflux for 4 hours. The extract was cooled to room temperature, filtered twice with filter paper, and the filtered solution was concentrated under reduced pressure and freeze-dried to obtain 12.7 g of water extract (yield 19.3%).

[0059] Additionally, Met (metformin) used as a sample of the present invention was purchased from Sigma-Aldrich (USA).

[0060]

[0061] 1-2. Cell model

[0062] COV434 cells, a human granulosa cell line used as a cell model of the present invention, were purchased from Sigma-Aldrich (St. Louis, MO, USA), and RAW 264.7 cells, a mouse macrophage cell line, were purchased from the Korea Cell Line Bank (KCLB, Seoul, Korea). The cells were each seeded in DMEM (Dulbecco's Modified Eagle's Medium, WELGENE) medium containing 10% FBS (fetal bovine serum, WELGENE, Daegu, Korea) and 1% penicillin / streptomycin (penicillin / streptomycin, Thermo Fisher, GrandIsland, NY, USA), and cultured in an incubator (Thermo Fisher Scientific, Langenselbold, Germany) at 37°C and 5% CO2. COV434 cells were cultured under the above culture conditions with the addition of L-glutamine (2 mM) to the culture medium.

[0063]

[0064] 1-3. Animal models

[0065] Six-week-old female SD (Sprague-Dawley) rats used as animal models for the present invention were purchased from Young Bio Co., Ltd. (Seongnam, Korea). The animals were acclimated to the laboratory environment for one week with sufficient food and water before being used in the experiments. The laboratory environment was maintained at a temperature of 22±2°C with a 12-hour day-night cycle until the end of the experiment.

[0066]

[0067] [Experimental Example 2] Statistical Analysis

[0068] The results of cell experiments were expressed as mean ± standard deviation (SD), and the results of animal experiments were expressed as mean ± standard error of the mean (SEM). To test for significance, one-way analysis of variance (ANOVA) was performed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA), and then analyzed with Tukey's multiple comparison test at the p<0.05 level.

[0069]

[0070] [Example 1] Cell protection against toxicity

[0071] To determine the effect of the samples on toxicity, an MTT assay was performed. COV434 cells (1 × 10⁴ cells / well) were seeded in 96-well plates and serum-starved for 4 h. Cells were then pretreated with SRT (10, 50, or 100 μg / ml) or / and Met (1 mM) for 1 h, and sequentially treated with VCD (4-vinylcyclohexene diepoxide, 0.5 mM, Sigma-Aldrich). The cells were then cultured at 37°C and 5% CO2 for 24 h. After that, 10 μL of MTT solution (2 mg / mL) was added per well and reacted for 2 hours in an incubator at 37°C and 5% CO2. After completely removing the MTT solution and culture medium, the formazan crystals formed within the cells were dissolved with DMSO (dimethyl sulfoxide, 100 μL) and the absorbance was measured at 540 nm using an ELISA plate reader (Tecan Austria GmbH, Grodig, Austria). The cell viability relative to the normal group (sample and VCD untreated group) was expressed as a percentage.

[0072] As a result, as shown in Fig. 1, the cell viability significantly decreased in the control group (VCD only treatment group) compared to the normal group (sample and VCD untreated group), and the cell viability in the SRT only treatment group significantly increased compared to the control group starting from the treatment concentration of 50 μg / ml. In addition, among the sample treatment groups, the SRT and Met combination treatment group showed a higher cell viability than the SRT only treatment group (based on the same SRT treatment concentration). From the above results, it was confirmed that the SRT and Met combination treatment exhibited a cytoprotective effect against nematode toxicity.

[0073]

[0074] [Example 2] NO production analysis

[0075] To determine the effect of the samples on nitric oxide (NO) production, RAW 264.7 cells cultured in serum-free medium for more than 6 h were pretreated with SRT (10, 100, 300, or 500 μg / ml) or / and Met (1 mM) for 2 h, treated with LPS (Lipopolysaccharide, 100 ng / mL), and cultured for 18 h. Afterwards, the culture medium was centrifuged, and an equal amount of Griess reagent was added to 100 μL of the supernatant, and the cells were incubated at room temperature for 10 min. The absorbance was measured at a wavelength of 540 nm using a microplate reader. The amount of NO production was expressed as a fold increase compared to the vehicle-treated control group. Dexamethasone (Dex, 5 μM) was used as a positive control.

[0076] As a result, as shown in Fig. 2, the amount of NO production significantly increased in the control group (LPS only treatment group) compared to the normal group (sample and LPS untreated group), and the amount of NO production significantly decreased in the sample treatment group compared to the control group. In particular, among the sample treatment groups, the amount of NO production was lower in the SRT and Met combination treatment group compared to the SRT only treatment group (based on the same SRT treatment concentration). From the above results, it was confirmed that the SRT and Met combination treatment suppressed NO production.

[0077]

[0078] [Example 3] Analysis of proinflammatory cytokines

[0079] To determine the effect of the sample on the secretion of proinflammatory cytokines, ELISA analysis was performed to measure the secretion of IL-1β and IL-6. 100 μL of the supernatant used in Example 2 was measured using Cymax TM Experiments were performed according to the protocols provided for the Mouse IL-1β ELISA Kit (AbFrontier, Seoul, Korea) and the Mouse IL-6 ELISA Kit (Elabscience), and absorbance was measured at a wavelength of 450 nm using a microplate reader. Dexamethasone (Dex, 5 μM) was used as a positive control.

[0080] As a result, as shown in Fig. 3, the secretion of IL-1β and IL-6 significantly increased in the control group (LPS only treatment group) compared to the normal group (sample and LPS untreated group), and the secretion of IL-1β and IL-6 significantly decreased in the sample treatment group compared to the control group. In particular, among the sample treatment groups, the secretion of IL-1β and IL-6 was lower in the SRT and Met combination treatment group compared to the SRT only treatment group (based on the same SRT treatment concentration). From the above results, it was confirmed that the SRT and Met combination treatment suppressed the secretion of IL-1β and IL-6.

[0081]

[0082] [Example 4] Analysis of expression of inflammation-related factors

[0083] To determine the effect of the samples on the expression of inflammatory factors, Western blot was performed to measure the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). RAW 264.7 cells cultured in serum-free medium for more than 6 hours were pretreated with SRT (100 μg / ml) or / and Met (1 mM) for 2 hours, treated with 100 ng / mL LPS, and then cultured for 18 hours. For protein extraction, the cells were washed twice with PBS, lysed with RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA), centrifuged at 12,000 × g for 30 minutes, and the supernatant was collected. Protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific), and protein samples were prepared using sample buffer (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to a PVDF membrane. To block nonspecific binding of antibodies, the membrane was treated with blocking buffer (5% non-fat milk) for 1 hour and washed with PBST (Phosphate-Buffered Saline with Tween 20) solution containing 0.1% Tween 20.Afterwards, protein antibodies (COX-2, iNOS, and β-actin) were used for treatment overnight at 4°C, and HRP-linked anti-rabbit or anti-mouse secondary antibodies were used. After reacting with enhanced chemiluminosescence solution (Amersham, Piscataway, NJ, USA), the expression level of each protein was observed and images were acquired using a Fusion Solo-2 image analyzer (Vilber Lourmat, Paris, France).

[0084] As a result, as shown in Fig. 4, iNOS and COX-2 expression significantly increased in the control group (LPS only treatment group) compared to the normal group (sample and LPS untreated group), and iNOS and COX-2 expression significantly decreased in the sample treatment group compared to the control group, and in particular, among the sample treatment groups, iNOS and COX-2 expression was lower in the SRT and Met combination treatment group compared to the SRT only treatment group (based on the same SRT treatment concentration). From the above results, it was confirmed that SRT and Met combination treatment reduced iNOS and COX-2 expression.

[0085]

[0086] [Example 5] Estradiol analysis

[0087] To determine the effect of the samples on the steroid hormone production of granulosa cells, estradiol secretion was measured by ELISA analysis. COV434 cells cultured in serum-free medium for more than 24 h were pretreated with SRT (10, 100, or 300 μg / ml) or / and Met (1 mM) for 2 h, and then treated with FSH (follicle-stimulating hormone, 30 ng / mL) and AD (androstenedione, 10 μM) to induce estradiol secretion, followed by incubation for 24 h. Afterwards, 100 μL of the supernatant obtained by centrifugation of the culture medium was applied to the E2 (Estradiol) ELISA Kit (Elabscience) and measured according to the provided protocol.

[0088] As a result, as shown in Fig. 5, the amount of estradiol secretion was significantly increased in the control group (FSH and AD treated group) compared to the normal group (sample, FSH and AD untreated group), and the amount of estradiol secretion was significantly decreased in the sample treated group compared to the control group. In particular, among the sample treated groups, the amount of estradiol secretion was lower in the SRT and Met combined treatment group compared to the SRT alone treatment group (based on the same SRT treatment concentration). From the above results, it was confirmed that the SRT and Met combined treatment suppressed estradiol secretion.

[0089]

[0090] [Example 6] Analysis of the effect of improving polycystic ovary syndrome

[0091] To determine the effects of the sample on polycystic ovary syndrome (PCOS), animal experiments were conducted. The experimental groups were set up as follows. All animal experiments were conducted in accordance with guidelines approved by the Dongguk University Animal Experiment Ethics Committee (Approval Number: 2023-03248).

[0092] 1) Normal group: Normal rats + tap water

[0093] 2) Control group: PCOS rats + tap water

[0094] 3) Sample treatment group 1 (SRT only treatment group): PCOS rats + SRT (1000 mg / kg)

[0095] 4) Sample treatment group 2 (Met only treatment group): PCOS rats + Met (100 mg / kg)

[0096] 5) Sample treatment group 3 (SRT and Met combined treatment group): PCOS rats + SRT (1000 mg / kg) + Met (100 mg / kg)

[0097] The control and sample treatment groups were injected intramuscularly once with 4 mg of estradiol valerate (Sigma-Aldrich, USA) dissolved in 0.2 ml of sesame oil (Sigma-Aldrich, USA) to induce PCOS. The normal group was injected intramuscularly once with 0.2 ml of regular sesame oil. Six weeks after the injection of estradiol valerate, the sample treatment group was orally administered SRT (total dose: 1000 mg / kg) or / and Met (total dose: 100 mg / kg) five times a week for five weeks (SRT and Met were dissolved in tap water and then administered to the animal model). The normal and control groups were orally administered tap water instead of the sample five times a week for five weeks. Thereafter, changes in follicles according to the sample treatment were analyzed.

[0098] For histopathological analysis, the animal model was sacrificed, one ovary and uterus were removed, fixed in 10% formalin, embedded in paraffin, and 5 μm thin sections were prepared and stained with hematoxylin-eoxin (H&E). The slides were scanned with a whole slide scanner (3DHISTECH Ltd, Budapest, Hungary) and analyzed using CaseViewer. TMImages were acquired and analyzed using software (3DHISTECH Ltd).

[0099] For serum analysis, blood was obtained from the animal model on the last day of the experiment by cardiac puncture, centrifuged at 3000 rpm for 15 minutes using a centrifuge (Beckman Coulter, Fullerton, CA), and the supernatant was collected. To measure the concentrations of testosterone, estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in the obtained serum, ELISA kits were purchased from Elabscience (USA). To measure the concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are indicators of liver damage, Asanset GOT (glutamic oxaloacetic transaminase) and GPT (glutamic pyruvic transaminase) reagents (Asan Pharmaceutical Co., Ltd., Seoul, Korea) were purchased and measured according to the manufacturer's manual.

[0100] As a result, as shown in Fig. 6, the number of cystic follicles (Cyst No.) in the ovaries significantly increased and the number of mature follicles (graafian follicles) significantly decreased in the control group (PCOS) compared to the normal group (Normal), whereas the number of cystic follicles in the ovaries significantly decreased and the number of mature follicles significantly increased in the sample-treated group compared to the control group. In particular, the decrease in the number of cystic follicles in the ovaries and the increase in the number of mature follicles were more significant in the sample-treated group (M+S) compared to the sample-only treated group (M and S).

[0101] In addition, as shown in Fig. 7, the blood testosterone, estradiol, and LH concentrations significantly increased and the blood FSH concentration significantly decreased in the control group compared to the normal group (Con), whereas the blood testosterone, estradiol, and LH concentrations significantly decreased and the blood FSH concentration increased in the sample treatment group compared to the control group. In particular, the decrease in the blood testosterone, estradiol, and LH concentrations and the increase in the blood FSH concentration were more significant in the sample combination treatment group (M+S) compared to the sample-only treatment group (M and S).

[0102] Additionally, as shown in Figure 8, there was no significant difference in blood AST and ALT concentrations among all experimental groups.

[0103] From the above results, it was confirmed that combined treatment with SRT and Met improved PCOS without showing hepatotoxicity.

[0104]

[0105] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating polycystic ovary syndrome or diseases related thereto, containing Siryeong-Tang and metformin as active ingredients.

2. A pharmaceutical composition according to claim 1, characterized in that the siryeong-tang contains Bupleurum falcatum Linne, Alisma orientale Juzepzuk, Atractylodes macrocephala Koidzumi, Polyporus umbellatus Fries, Poria cocos Wolf, Pinellia ternata Breitenbach, Scutellaria baicalensis Georgi, Ginseng (Panax ginseng C. A. Meyer), Glycyrrhiza uralensis Fischer, Cinnamomum cassia J. Presl, and Ginger (Zingiberofficinale Roscoe).

3. A pharmaceutical composition according to claim 1, characterized in that the disease associated with polycystic ovary syndrome is at least one selected from the group consisting of hyperandrogenism, amenorrhea, oligomenorrhea, dysfunctional uterine bleeding, premature menopause, infertility, and endometriosis.

4. A pharmaceutical composition according to claim 1, characterized in that it inhibits the production of nitric oxide.

5. In the first paragraph, the pharmaceutical composition is characterized in that it inhibits the expression of at least one selected from the group consisting of IL-1β (Interleukin-1β), IL-6 (Interleukin-6), iNOS (Inducible nitric oxide synthase), COX-2 (cyclooxygenase-2), and Estradiol.

6. In the first paragraph, the pharmaceutical composition is characterized in that it reduces the concentration of testosterone, estradiol or luteinizing hormone in the blood, or increases the concentration of follicle-stimulating hormone in the blood.

7. A health functional food composition for preventing or improving polycystic ovary syndrome or diseases related thereto, containing Siryung-Tang and metformin as active ingredients.

8. A pharmaceutical composition for preventing or treating ovotoxity, comprising Siryeong-Tang and metformin as active ingredients.

9. A pharmaceutical composition according to claim 8, characterized in that the poor toxicity is induced by VCD (4-vinylcyclohexene diepoxide).

10. A health functional food composition for preventing or improving ovotoxity, containing Siryeong-Tang and metformin as active ingredients.

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