Rhizoma menispermi extract effective part, preparation method thereof and application of rhizoma menispermi extract effective part in preparation of medicine for preventing or treating polycystic ovarian syndrome

Abstract

The invention belongs to the technical field of natural medicinal chemistry, and particularly relates to an effective part of a rhizoma menispermi extract, a preparation method of the effective part and application of the effective part to preparation of a medicine for preventing or treating polycystic ovarian syndrome (PCOS). The effective part of the rhizoma menispermi extract is mainly a mixture of dauricine (DAU) and dauricine methyl ester (DAU-Me), and the weight percentage of the effective part is 75%-95%. The effective part of the rhizoma menispermi extract, provided by the invention, can be used for remarkably improving the estrus cycle disorder, insulin resistance, blood sex hormone disorder and ovarian polycystic degeneration of PCOS model mice, does not cause obvious reproductive toxicity, and can be used for preparing a medicine for preventing or treating the polycystic ovarian syndrome.

Description

Technical Field

[0001] The present invention relates to the field of natural drug chemistry, and in particular to the medicinal plant Pteris dahurica ( Menispermum dauricum DC.) dry root extract effective part and preparation method thereof and application in preparing medicine for preventing or treating polycystic ovary syndrome. Background Art

[0002] Decreased fertility has become a serious social problem. The global infertility rate has reached 10%~15%. Reproductive endocrine disorders are currently the leading cause of female infertility. Polycystic ovary syndrome (PCOS) is one of the most common causes of anovulatory infertility. PCOS is a syndrome of endocrine and metabolic disorders characterized by hyperandrogenemia, abnormal ovulation, and polycystic ovarian changes, often accompanied by insulin resistance. However, the pathogenesis of PCOS is still unclear, and it is usually treated symptomatically based on clinical manifestations. Due to the high heterogeneity of its clinical manifestations, the treatment of PCOS has become a hot and difficult issue of common concern in the fields of gynecology and endocrinology.

[0003] Clinically, hyperandrogenemia and insulin resistance are the two major characteristics of polycystic ovary syndrome. According to statistics, 50%-70% of patients also have insulin resistance. At present, there is a lack of radical treatment for PCOS. Western medicine mainly uses hormone regulating drugs for treatment. For example, ethinyl estradiol cyproterone acetate tablets (Diane-35) are suitable for patients with clinical manifestations of hyperandrogenism or hyperandrogenemia, and cyclical progesterone withdrawal or other short-acting oral contraceptives are usually used to adjust the menstrual cycle. However, due to the large side effects and many contraindications in clinical practice, the clinical application of hormone therapy is limited to a certain extent. In addition, some drugs that reduce insulin (such as metformin, etc.) are also used for auxiliary treatment.

[0004] The intestinal microecology mainly includes intestinal bacteria, fungi, viruses and metabolites, among which the research on intestinal bacteria is relatively in-depth. Recent studies have confirmed that the human intestinal microecology is closely related to the occurrence and development of PCOS. Intestinal bacteria can affect the host's natural immune system and systemic metabolic homeostasis by converting bile acid or producing metabolites such as agmatine, thereby regulating insulin resistance and affecting the occurrence and development of PCOS. Therefore, taking the human microecology as the research entry point and developing corresponding microecological preparations and drugs can provide new paths and more effective measures for the treatment and prevention of PCOS.

[0005] Bat Kudzu Menispermum dauricumDC.) The dried rhizome is called the root of the bean. The root of the bean is bitter and cold in nature; it is slightly toxic. It enters the lung, stomach, and large intestine meridians. It has the effects of clearing away heat and detoxifying, promoting dampness and reducing swelling. It is mainly used to treat acute pharyngitis, tonsillitis, swollen and painful gums, lung heat cough, damp heat, jaundice, constipation, etc. At present, no research has shown that the extract of the bean and its structural analogs can relieve or treat polycystic ovary syndrome. Summary of the invention

[0007] In order to solve the problem of insufficient or poor efficacy of drugs for treating PCOS in clinic, the present invention provides an effective part of the extract of the root of Sophora flavescens and its preparation method and its application in preparing drugs for preventing or treating polycystic ovary syndrome (PCOS). The present invention provides a new idea for preventing or treating polycystic ovary syndrome in clinic and for the modernization of traditional Chinese medicine.

[0008] The technical solution for achieving the purpose of the present invention is as follows: In a first aspect, the present invention provides a method for preparing an effective fraction of a Radix Sophorae Flavescentis extract having the effect of preventing or treating polycystic ovary syndrome, comprising the following steps: (1) Take the clean rhizome of the root of the northern bean, dry it thoroughly and then extract it; (2) preparing an ethanol:methanol:water extract in a volume ratio of (0.5-3):(0.5-2):(1-2), adjusting the temperature to 10-100° C., mixing the extract with the northern bean root in step (1) and shaking for 1-12 hours; (3) After the extraction in step (2) is completed, solid-liquid separation is performed to obtain a crude extract, which is then rotary evaporated at 30 to 60° C. for 1 to 6 hours to allow the organic solvent to fully evaporate, thereby obtaining a concentrated solution. At this time, the extract still contains a large amount of water; (4) subjecting the concentrated solution obtained in step (3) to vacuum freeze drying at -1.0 MPa to -0.10 MPa for 1 to 18 hours to obtain a dry light yellow to black powder; (5) dissolving the powder obtained in step (4) in ethanol, filtering to obtain a clear solution, concentrating the solution, and evaporating to dryness to obtain a crude extract of the effective part of the target Radix Sophorae Flavescentis extract; (6) Using high performance liquid chromatography to track the activity of the crude extract of the effective part in step (5), the effective component obtained by eluting for 6 to 7 minutes has the best effect, and the effective part of the northern bean root extract is obtained after concentration; (7) Liquid chromatography-tandem mass spectrometry was performed to analyze that the effective part of the northern bean root extract was mainly a mixture of dauroyl amine (DAU) and dauroyl amine methyl ester (DAU-Me), accounting for 75%-95% by mass percentage.

[0009] As an optional manner, in the above method for preparing the effective fraction of the Radix Sophorae Flavescentis extract, the Radix Sophorae Flavescentis in step (1) should be chopped into small pieces of 2 mm.

[0010] As an optional manner, in the above-mentioned method for preparing the effective part of the northern bean root extract, in step (2), the volume ratio of ethanol:methanol:water is 3:1:2, the solid-liquid ratio is 1 g:6-10 mL, the extraction temperature is 50-60°C, and the oscillation extraction time is 4-5 hours.

[0011] As an optional manner, in the above method for preparing the effective fraction of the northern bean root extract, the step (3) is performed by rotary evaporation at 50° C. for 4 to 6 hours.

[0012] As an optional manner, in the above method for preparing the effective fraction of the northern bean root extract, in step (4), freeze-drying is carried out at -0.70 MPa for 12 to 18 hours.

[0013] As an optional manner, in the above method for preparing the effective fraction of the northern bean root extract, a 0.22 μm filter membrane is used for filtration in step (5).

[0014] As an optional manner, in the above-mentioned method for preparing the effective part of the northern bean root extract, the chromatographic column used in step (6) is: a 5 μm particle size C18 chromatographic column, 4.6×250 mm; the mobile phase is: by volume percentage, A: 80% water + 20% acetonitrile, B: 80% acetonitrile + 20% isopropanol, and the liquid phase conditions are: 0-4 min, 30% B; 4-10 min, 30% B-90% B; 10-12 min, 90% B; 12-13 min, 90% B-30% B, 13-15 min, 30% B.

[0015] As an optional method, in the above-mentioned method for preparing the effective part of the extract of the root of northern bean, the root of northern bean is selected from the plant of Menispermaceae, Pueraria lobata ( Menispermum dauricum DC. ), the collection time is limited to spring and autumn, and the origin is one or more regions in Northeast China, North China, East China, Shaanxi, Ningxia, Gansu, and Shandong.

[0016] In the second aspect, the present invention provides an effective part of a root extract of Sophora flavescens having the effect of preventing or treating polycystic ovary syndrome, prepared by the preparation method described in the first aspect. The effective part of the root extract of Sophora flavescens is mainly a mixture of Dauricine (DAU) and Dauricine methyl ester (DAU-Me), accounting for 75%-95% by mass.

[0017] In a third aspect, the present invention provides use of the effective fraction of the extract of the root of Sophora flavescens described in the second aspect in the preparation of a dipeptidyl peptidase-4 (DPP4) inhibitor.

[0018] In a fourth aspect, the present invention provides use of the effective fraction of the Rhizoma Coptidis extract described in the second aspect in the preparation of a drug for preventing or treating polycystic ovary syndrome.

[0019] As an optional mode, in the above application, the drug also comprises other DPP4 inhibitors commonly used in clinical practice.

[0020] Preferably, the other DPP4 inhibitor is selected from one or more of the following: sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, gemigliptin or teneligliptin.

[0021] More preferably, the other DPP4 inhibitor is selected from sitagliptin.

[0022] As an optional mode, in the above application, the dosage form of the drug is selected from granules, capsules, tablets, powders, oral solutions, suspensions or emulsions.

[0023] As an optional mode, in the above application, the dosage form of the drug is enteric-coated tablets.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The effective fraction of the root of northern soybean extract provided by the present invention significantly improves estrous cycle disorder, insulin resistance, blood hormone imbalance and polycystic ovary in PCOS model mice without causing obvious reproductive toxicity, can be used for preparing drugs for preventing or treating polycystic ovary syndrome, and has good clinical application prospects and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 :Effects of various components of the extract of Sophora flavescens root on the estrous cycle of DHEA mice. The figures are shown as mean ± SD. M / D: metestrus / diestrus; E: estrus; P: proestrus. One-way ANOVA was used to evaluate the significance of each group. Compared with the model group, P < 0.05. Compared with the model group, P < 0.01. Compared with the model group, P < 0.001. Compared with the model group, P < 0.0001. Component 7 showed the most significant improvement, followed by DAU.

[0026] Figure 2:Effects of various components of the root extract of Sophora flavescens on the results of glucose tolerance test (GTT) in DHEA mice. The figures are shown as mean ± SD. Since it is difficult to distinguish the differences between the groups in the line graph, only the significance of component 7 and the model group is shown. In order to show the differences between the groups more clearly, the area under the curve was calculated and the significance of each group was evaluated using one-way ANOVA. Compared with the model group, P < 0.05. Compared with the model group, P < 0.01. Compared with the model group, P < 0.001. Compared with the model group, P < 0.0001. Component 7 showed the most significant improvement, followed by DAU.

[0027] Figure 3 :Effects of various components of the extract of Sophora flavescens root on the results of insulin tolerance test (ITT) in DHEA mice. The figures are shown as mean ± SD. Since it is difficult to distinguish the differences between the groups in the line graph, only the significance of component 7 and the model group is shown. Compared with the model group, P < 0.001. Component 7 showed the most significant improvement.

[0028] Figure 4 :Effects of various components of the extract of the root of Sophora flavescens on blood sex hormones in DHEA mice. The figures are shown as mean ± SD. One-way ANOVA was used to evaluate the significance of each group. Compared with the model group, P < 0.05. Compared with the model group, P < 0.01. Compared with the model group, P < 0.001. Compared with the model group, P < 0.0001. Component 7 showed the most significant improvement, followed by DAU.

[0029] Figure 5 :Effects of Sophora flavescens root extract on polycystic ovarian changes in DHEA mice. Figures are shown as mean ± SD. One-way ANOVA was used to evaluate the significance of each group. Compared with the model group, P < 0.05. Compared with the model group, P < 0.01. Compared with the model group, P < 0.001. Compared with the model group, P < 0.0001. Component 7 showed the most significant improvement, followed by DAU.

[0030] Figure 6 : Effects of the effective fraction of the extract of Sophora flavescens root (component 7) on the estrous cycle of DHEA mice. The figures are shown as mean ± SD. M / D: metestrus / diestrus; E: estrus; P: proestrus. One-way ANOVA was used to evaluate the significance of each group. Compared with the model group, P < 0.05. Compared with the model group, P < 0.01. Compared with the model group, P < 0.001. Compared with the model group, P < 0.0001. Among them, sitagliptin + component 7 showed the most obvious improvement, followed by component 7.

[0031] Figure 7 :Effects of the effective fraction of the extract of the root of Sophora flavescens (component 7) on GTT in DHEA mice. The figures are shown as mean ± SD. Since the line graph is not easy to distinguish the differences between the groups, the area under the curve was calculated and the significance of each group was evaluated using one-way ANOVA. Compared with the model group, P < 0.05. Compared with the model group, P < 0.01. Compared with the model group, P < 0.001. Compared with the model group, P < 0.0001. Among them, sitagliptin + component 7 showed the most obvious improvement, followed by component 7.

[0032] Figure 8 :Effects of the effective fraction of the extract of Sophora flavescens root (Component 7) on ITT in DHEA mice. The figures are shown as mean ± SD. Since it is difficult to distinguish the differences between the groups in the line graph, only the significance of sitagliptin + component 7 and the model group is shown. Compared with the model group, P < 0.001. Among them, sitagliptin + component 7 showed the most obvious improvement, followed by component 7.

[0033] Fig. 9 :Effects of the effective fraction of the extract of the root of Sophora flavescens (component 7) on blood sex hormones in DHEA mice. The figures are shown as mean ± SD. One-way ANOVA was used to evaluate the significance of each group. Compared with the model group, P < 0.05. Compared with the model group, P < 0.01. Compared with the model group, P < 0.001. Compared with the model group, P < 0.0001. Among them, sitagliptin + component 7 showed the most obvious improvement, followed by component 7.

[0034] Fig.10 :Effects of the effective fraction of Radix Sophorae Fructus (Component 7) on polycystic ovarian changes in DHEA mice. The figures are shown as mean ± SD. One-way ANOVA was used to evaluate the significance of each group. Compared with the model group, P < 0.05. Compared with the model group, P < 0.01. Compared with the model group, P < 0.001. Compared with the model group, P < 0.0001. Among them, sitagliptin + component 7 showed the most obvious improvement, followed by component 7. DETAILED DESCRIPTION

[0036] After extensive and in-depth research and experiments, the inventors found that a specific effective part of the root extract of Sophora flavescens can inhibit the activity of bacterial DPP4 and has the effect of preventing and treating polycystic ovary syndrome. The effective part of the root extract of Sophora flavescens was fed to experimental subjects and found that it can improve insulin resistance, estrous cycle disorders, hormone secretion disorders and ovarian polycystic changes in polycystic ovary syndrome model mice. The present invention was completed on this basis.

[0037] As used herein, the term "comprising" indicates that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting mainly of" and "consisting of" are included in the term "comprising".

[0038] The present invention provides the use of an effective part of a root extract of northern soybean for preventing or treating polycystic ovary syndrome. Mice are given high androgen stimulation to create a polycystic ovary syndrome model, and the effective part of the root extract of northern soybean has the ability to improve insulin resistance, estrous cycle disorders and polycystic ovary-like changes in the model animals. According to a preferred embodiment of the present invention, DHEA-induced PCOS model mice are treated with an effective part of a root extract of northern soybean, and the estrous cycle, sex hormones and ovarian morphology can be kept normal compared with an untreated control group. Therefore, the effective part of the root extract of northern soybean can be used to prevent and treat polycystic ovary syndrome.

[0039] The drug of the present invention can be administered in the form of any conventional pharmaceutical preparation in the art, wherein the pharmaceutical preparation includes excipients, pharmaceutically acceptable media and carriers, which can be selected according to the administration route. The drug of the present invention can further include auxiliary components.

[0040] The pharmaceutically acceptable excipients of the present invention include conventional solvents in the pharmaceutical field (such as water, ethanol, propylene glycol, injection oil, etc.), diluents (such as starch, powdered sugar, dextrin, lactose, pregelatinized starch, microcrystalline cellulose, inorganic calcium salts (such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, etc.), mannitol, etc., vegetable oil, polyethylene glycol, etc.), binders (such as water, ethanol, starch slurry, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, hydroxypropyl methyl cellulose, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, etc.), lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, magnesium lauryl sulfate, etc.), absorption accelerators (such as surfactants, Azone (laurorazepam), EDTA, salicylic acid, amino acid ethylamine derivatives, acetoacetate, β-dicarboxylate, aromatic acid compounds, aliphatic acids, etc.), preservatives (such as benzoic acid, butyl hydroxypropyl ester, methyl hydroxypropyl ester, phenol, m-cresol, etc.), flavoring agents (such as sucrose, stevioside, etc.), etc.

[0041] In addition, as a preferred embodiment, the drug of the present invention can be produced in an enteric coating preparation by various well-known methods so that the active ingredients of the drug can pass through the stomach smoothly without being destroyed by gastric acid.

[0042] The pharmaceutical composition of the present invention can be made into enteric-coated tablets for oral use. The term "enteric coating" in the present application includes all coatings allowed for use in conventional drugs, which are not degraded by gastric acid but can be fully decomposed in the small intestine and quickly release the active ingredients of the present invention. The enteric coating of the present invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as HCl solution with pH=1, and preferably decomposed within 1.0 hour in synthetic intestinal fluid such as buffer with pH=7.0.

[0043] The enteric coating of the present invention is coated with about 16 to 30 mg per tablet, preferably 16 to 25 mg, and more preferably 16 to 20 mg. The thickness of the enteric coating of the present invention is 5 to 100 μm, and the ideal thickness is 20 to 80 μm. The enteric coating component is a conventional polymer in the field of pharmaceutical preparations.

[0044] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified. The purchase of some raw materials and reagents is as follows: The root of northern bean used in the present invention is selected from the plant of Menispermaceae, kudzu vine ( Menispermum dauricum DC.), the collection time is limited to spring and autumn, and the origin is one or more regions in Northeast China, North China, East China, Shaanxi, Ningxia, Gansu, and Shandong.

[0046] Example 1: Preparation of Radix Sophorae Flavescentis Extract Take the clean rhizomes of the root of the northern bean root produced in Heilongjiang Province, dry them thoroughly, and then cut them into pieces for extraction. Prepare an ethanol: methanol: water extract in a volume ratio of 3: 1: 2, adjust the temperature to 60 ° C, mix the extract with the northern bean root and shake for 5 hours. Solid-liquid separation to obtain a crude extract, rotary evaporation at 50 ° C for 4 hours to fully evaporate the organic solvent to obtain a concentrated solution, at which time there is still a lot of water in the extract. The concentrate is vacuum freeze-dried at -0.7 MPa for 18 hours to obtain a dry light yellow to black powder. The powder is dissolved in ethanol, and a clear solution is obtained after filtration. The solution is concentrated and evaporated to dryness to obtain a crude extract of the effective part of the target northern bean root extract.

[0047] High performance liquid chromatography was used to separate the above crude extracts and explore the key effective parts. The chromatographic column used was: 5 μm particle size C18 chromatographic column, 4.6×250 mm; the mobile phase was: by volume percentage, A: 80% water + 20% acetonitrile, B: 80% acetonitrile + 20% isopropanol, and the liquid phase conditions were: 0-4 min, 30% B; 4-10 min, 30% B-90% B; 10-12 min, 90% B; 12-13 min, 90% B-30% B, 13-15 min, 30% B. The liquid phase method took a total of 15 minutes, and the effluent was collected every minute, and a total of 15 effluents were obtained. Each effluent was rotary evaporated to dryness at 50°C, weighed, and re-dissolved in methanol to obtain components 1-15. An equal amount of dried crude extract of northern bean root was dissolved in methanol to obtain component 16.

[0048] Example 2: Inhibitory activity of the extract of Sophora flavescens root and its components on bacterial DPP4 PCOS can be effectively improved by regulating abnormal glucose tolerance. Recent studies have found that bacterial DPP4 activity can regulate blood sugar, and thus has the potential to become a new bacterial target for PCOS. However, it is not yet clear what the main components in the northern bean root extract that inhibit bacterial DPP4 activity are. Therefore, by separating the components of the northern bean root extract by liquid chromatography, 16 components were obtained (see Example 1), and then the activity tracking strategy was used to evaluate the inhibitory effect of each component on bacterial DPP4. The bacterial DPP4 was purified from an in vitro expression system independently constructed by the inventor's team. The inhibitory effects of the 16 components on bacterial DPP4 are shown in Table 1.

[0049] Table 1: Inhibitory activity of the extract from the root of Sophora flavescens and its components against bacterial DPP4

[0050] Among them, component 16 is a crude extract, and components 1-15 are components separated by liquid chromatography according to retention time. Component 7 shows the most obvious DPP4 inhibitory activity.

[0051] Example 3: Analysis of the main components of the effective part of the extract of the root of Sophora flavescens (Component 7) LC-MS was used to analyze the main components and contents in component 7, and the inhibitory effect of each component on DPP4 was evaluated. The results are shown in Table 2.

[0052] Table 2: Analysis of the main components of component 7 and the inhibitory effect of each component on DPP4

[0053] Through LC-MS analysis, it was determined that the main component of component 7 was daurine DAU, accounting for about 60.3%, the minor component was daurine methyl ester DAU-Me, accounting for about 23.2%, and the other components were daurine analogs, all of which had DPP4 inhibitory activity. In addition, the activity of component 7 was higher than that of any single component, indicating that there was a synergistic effect between the components.

[0054] Example 4: Comparison of the improvement effects of single components, component 7 and crude extracts in the effective components of Radix Sophorae Fructus on the PCOS mouse model induced by dehydroepiandrosterone (DHEA) Materials: The test substance is the extracts of various components of the roots of Sophora flavescens collected from North China, which are extracted, purified and dried to obtain powder. The dosage of the drug for gavage is 10 mg / kg body weight, and the frequency is once a day.

[0055] C57 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with a temperature of 20-24°C, a constant humidity of 50-60%, a light period of 12 hours (8:00-20:00), sound insulation, free access to food and water, and experiments were conducted after one week of adaptation. Blood glucose test strips (Roche), blood glucose meter (Roche), dehydroepiandrosterone (Sigma), and sex hormone detection kits.

[0056] Methods: Forty-two female C57 mice aged 3-4 weeks and weighing 10-12 g were randomly divided into (1) normal control group, (2) model control group, (3) DAU group, (4) DAU-Me group, (5) other DAU analogs of component 7 group, (6) component 7 group, and (7) crude extract group, with 6 mice in each group. Groups (2) to (7) were given subcutaneous injection of DHEA. Mice in each group were gavaged with 10 mg / kg of the corresponding drug for 4 consecutive weeks. The control group and the model group were given an equal amount of normal saline. The estrous cycle was tested daily 10 days after DHEA injection. The estrous cycle of mice in the model group was significantly disturbed, indicating that the model was successfully established. Oral glucose tolerance and insulin tolerance were tested before the last administration. After the last administration, blood was collected and centrifuged at 3000 rpm at 4℃ to determine serum sex hormone levels. Ovarian sections were sliced ​​to observe pathological changes.

[0057] Results: Compared with the model group, the treatment with component 7 of the root of northern bean and other extracts could significantly improve the related indicators of insulin resistance, the ovarian status and sex hormone levels of mice, with component 7 being the most significant, followed by DAU. Figure 1-5 .

[0058] Example 5: Improvement effect of the effective ingredient of Radix Sophorae Fructus (Component 7) on the DHEA-induced PCOS mouse model Materials: The test substance is the component 7 of the extract of the root of Sophora flavescens collected from North China. After extraction, purification and drying, a light yellow to dark yellow powder is obtained. The oral administration amount is 10 mg / kg body weight, and the frequency is once a day. Sitagliptin is selected as the positive control drug, and the oral administration amount is 10 mg / kg body weight, and the frequency is once a day.

[0059] C57 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with a temperature of 20-24°C, a constant humidity of 50-60%, a light period of 12 hours (8:00-20:00), sound insulation, free access to food and water, and experiments were conducted after one week of adaptation. Blood glucose test strips (Roche), blood glucose meter (Roche), dehydroepiandrosterone (Sigma), and sex hormone detection kits.

[0060] Methods: Thirty female C57 mice aged 3-4 weeks and weighing 10-12 g were randomly divided into (1) normal control group, (2) model control group, (3) component 7 group, (4) sitagliptin group, and (5) component 7 + sitagliptin group, with 6 mice in each group. Groups (2) to (5) were given subcutaneous injection of DHEA. Mice in each group were gavaged with 10 mg / kg of the corresponding drug, and the combination group was gavaged with component 7 (10 mg / kg) and sitagliptin (10 mg / kg) for 4 consecutive weeks. The control group and the model group were given an equal amount of normal saline. The estrous cycle was tested daily 10 days after DHEA injection. The estrous cycle of mice in the model group was significantly disturbed, indicating that the model was successfully established. Oral glucose tolerance and insulin tolerance were tested before the last administration. After the last administration, blood was collected and centrifuged at 3000 rpm at 4℃ to determine serum sex hormone levels. Ovarian sections were sliced ​​to observe pathological changes.

[0061] Results: Compared with the model group, component 7, sitagliptin, and component 7+sitagliptin can significantly improve insulin resistance-related indicators, restore normal estrous cycle, and improve the ovarian status and sex hormone levels of mice. Component 7+sitagliptin has the most significant effect, followed by component 7. Figure 6-10 .

[0062] Example 6: Safety of the effective part of the extract of the root of Sophora flavescens (Component 7) for pregnant women during the whole cycle Materials: The test substance was component 7 of the extract of the root of Sophora flavescens collected from North China. After extraction, purification and drying, a light yellow to dark yellow powder was obtained. The oral administration amount of the drug was 10 mg / kg body weight, and the frequency was once a day. Sitagliptin was selected as the positive control drug, and the oral administration amount of the drug was 10 mg / kg body weight, and the frequency was once a day. The combined drug group was gavaged with component 7 (10 mg / kg) and sitagliptin (10 mg / kg), respectively, once a day.

[0063] C57 male and female mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The temperature was 20-24°C, the humidity was 50-60%, the light was 12 hours (8:00-20:00), and the mice were soundproofed. They were allowed to eat and drink freely. The experiments were conducted after one week of acclimatization.

[0064] Methods: Twenty-four female C57 mice, 8-10 weeks old, weighing 22-26 g, were randomly divided into (1) normal control group, (2) group 7, (3) sitagliptin group, and (4) sitagliptin + group 7 groups, with 6 mice in each group. Group (1) was given PBS every day, and groups (2)-(4) were given corresponding drug treatment every day. One week later, they were mated with healthy male mice (10-12 weeks old, 1:1), and the vaginal plug was checked the next morning to determine the mating date. The drug-treated group continued to be given drugs every day until 1 day before delivery, and the control group was given an equal amount of PBS. The weight changes of pregnant mice were recorded. Pregnant mice were killed one day before delivery, and blood was collected and centrifuged at 4℃ and 3000 rpm to determine serum sex hormone levels. Ovarian sections were sliced ​​to observe pathological changes. Pregnancy rate, fetal weight, number of live births, number of stillbirths, and number of resorbed fetuses were statistically analyzed.

[0065] Results: Compared with the control group, the effective part of the northern bean root extract (component 7), sitagliptin and their combined use did not cause obvious reproductive toxicity, and did not change the weight and number of offspring. The results are shown in Table 3-4.

[0066] Table 3: Effects of the effective fraction of the extract from the root of Sophora flavescens (component 7) on the live birth rate and sex ratio of mouse offspring

[0067] Table 4: Effects of the effective fraction of the extract from the root of Sophora flavescens (component 7) on the weight and body length of mouse offspring

[0068] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an effective fraction of a root extract of Sophora flavescens for preventing or treating polycystic ovary syndrome, characterized in that: The following steps are involved: (1) Take the clean rhizome of the root of the northern bean, dry it thoroughly and then extract it; (2) preparing an ethanol:methanol:water extract in a volume ratio of (0.5-3):(0.5-2):(1-2), adjusting the temperature to 10-100° C., mixing the extract with the northern bean root in step (1) and shaking for 1-12 hours; (3) After the extraction in step (2) is completed, solid-liquid separation is performed to obtain a crude extract, which is then rotary evaporated at 30 to 60° C. for 1 to 6 hours to allow the organic solvent to fully evaporate, thereby obtaining a concentrated solution. At this time, the extract still contains a large amount of water; (4) subjecting the concentrated solution obtained in step (3) to vacuum freeze drying at -1.0 MPa to -0.10 MPa for 1 to 18 hours to obtain a dry light yellow to black powder; (5) dissolving the powder obtained in step (4) in ethanol, filtering to obtain a clear solution, concentrating the solution, and evaporating to dryness to obtain a crude extract of the effective part of the target Radix Sophorae Flavescentis extract; (6) Using high performance liquid chromatography to track the activity of the crude extract of the effective part in step (5), the effective component obtained by eluting for 6 to 7 minutes has the best effect, and the effective part of the northern bean root extract is obtained after concentration; (7) Liquid chromatography-tandem mass spectrometry was performed to analyze that the effective part of the northern bean root extract was mainly a mixture of dauroyl amine (DAU) and dauroyl amine methyl ester (DAU-Me), accounting for 75%-95% by mass percentage.

2. The method for preparing the effective fraction of the Radix Sophorae Fructus extract according to claim 1, characterized in that: In step (1), the bean roots should be chopped into 2 mm pieces.

3. The method for preparing the effective fraction of the Radix Sophorae Flavescentis extract according to claim 1, characterized in that: In step (2), the volume ratio of ethanol:methanol:water is 3:1:2, the solid-liquid ratio is 1 g:6-10 mL, the extraction temperature is 50-60°C, and the oscillation extraction time is 4-5 hours.

4. The method for preparing the effective fraction of the Radix Sophorae Fructus extract according to claim 1, characterized in that: In step (3), the mixture is subjected to rotary evaporation at 50° C. for 4 to 6 hours; in step (4), the mixture is freeze-dried at -0.70 MPa for 12 to 18 hours; and in step (5), the mixture is filtered using a 0.22 μm filter membrane.

5. The method for preparing the effective fraction of the Radix Sophorae Fructus extract according to claim 1, characterized in that: The chromatographic column used in step (6) is: 5 μm particle size C18 chromatographic column, 4.6×250 mm; the mobile phase is: by volume percentage, A: 80% water + 20% acetonitrile, B: 80% acetonitrile + 20% isopropanol, and the liquid phase conditions are: 0-4 min, 30% B; 4-10 min, 30% B-90% B; 10-12 min, 90% B; 12-13 min, 90% B-30% B, 13-15 min, 30% B.

6. The method for preparing the effective fraction of the Radix Sophorae Fructus extract according to claim 1, characterized in that: The northern bean root is selected from the Menispermaceae plant kudzu vine ( Menispermum dauricum DC. ), the collection time is limited to spring and autumn, and the origin is one or more regions in Northeast China, North China, East China, Shaanxi, Ningxia, Gansu, and Shandong.

7. The effective fraction of the extract of Radix Sophorae Fructus having the effect of preventing or treating polycystic ovary syndrome prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The effective part of the northern bean root extract is mainly a mixture of daurine (DAU) and daurine methyl ester (DAU-Me), accounting for 75%-95% by mass percentage.

8. Use of the effective fraction of the root extract of Sophora flavescens according to claim 7 in the preparation of a dipeptidyl peptidase-4 (DPP4) inhibitor.

9. Use of the effective fraction of the extract of the Radix Sophorae Flavescentis according to claim 7 in the preparation of a medicament for preventing or treating polycystic ovary syndrome.

10. The use according to claim 8 or claim 9, characterized in that: The dosage form of the drug is selected from granules, capsules, tablets, powders, oral solutions, suspensions or emulsions.

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