Effective fraction of Rhizoma Menispermi extract, preparation method thereof and application in preparing medicine for preventing or treating polycystic ovary syndrome

Abstract

The present invention belongs to the technical field of natural medicine chemistry, and specifically relates to an effective part of Menispermi Rhizoma extract, a preparation method thereof, and an application thereof in the preparation of a drug for preventing or treating polycystic ovary syndrome (PCOS). The effective part of the Menispermi Rhizoma extract is mainly a mixture of daurisoline (DAU) and methyl daurisolate (DAU-Me), and in terms of mass percentage, the proportion is 75%-95%. The effective part of the Menispermi Rhizoma extract provided by the present invention significantly improves the estrous cycle disorder, insulin resistance, blood sex hormone disorder and ovarian polycystic-like changes of PCOS model mice, and does not cause obvious reproductive toxicity, and can be used for the preparation of a drug for preventing or treating polycystic ovary syndrome.

Description

Technical Field

[0001] The present invention relates to the field of natural medicine chemistry. Specifically, the present invention relates to an effective part of the dried root extract of the medicinal plant Menispermum dahuricum DC., its preparation method, and its application in the preparation of a drug for preventing or treating polycystic ovary syndrome. Menispermum dauricum DC.), and its application in the preparation of a drug for preventing or treating polycystic ovary syndrome. Background Art

[0002] The decline in fertility has become a serious social problem, and the global infertility rate reaches 10% - 15%. Reproductive endocrine disorders are currently the leading cause of female infertility. Polycystic ovary syndrome (PCOS for short) is one of the most common causes of anovulatory infertility. PCOS is a syndrome of endocrine and metabolic disorders, characterized by hyperandrogenism, abnormal ovulation, and polycystic ovarian changes, often accompanied by insulin resistance. However, the pathogenesis of PCOS remains unclear, and it is usually treated symptomatically according to 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, hyperandrogenism and insulin resistance are two major characteristics of polycystic ovary syndrome. According to statistics, 50% - 70% of patients have insulin resistance at the same time. Currently, there is a lack of radical treatment for PCOS. Western medicine mainly uses hormone-regulating drugs for treatment. For example, ethinylestradiol and cyproterone acetate tablets (Diane-35) are suitable for patients with clinical manifestations of hyperandrogenism or hyperandrogenemia, and the menstrual cycle is usually adjusted by cyclic progesterone withdrawal or other short-acting oral contraceptives. However, due to the relatively large side effects of hormone therapy and many clinical contraindications, its clinical application is restricted to a certain extent. In addition, some drugs that reduce insulin (such as metformin, etc.) are also used for adjuvant treatment.

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

[0005] Menispermum dahuricum Menispermum dauricumThe dried rhizome of Menispermum dauricum DC. is called Beidougen. Beidougen is bitter in taste and cold in nature; it is slightly poisonous. It belongs to the lung, stomach, and large intestine meridians. It has the effects of clearing heat and detoxifying, and promoting diuresis and detumescence. It is mainly used to treat acute pharyngitis, tonsillitis, gingival swelling and pain, cough due to lung heat, damp heat, jaundice, constipation, etc. Currently, there is no research indicating that the extract of Menispermum dauricum DC. and its structural analogs can relieve or treat polycystic ovary syndrome. Summary of the Invention

[0006] In order to solve the problems of insufficient drugs or poor efficacy in the clinical treatment of PCOS, the present invention provides an effective part of the extract of Menispermum dauricum DC., its preparation method, and its application in the preparation of drugs for preventing or treating polycystic ovary syndrome (PCOS). The present invention provides a new idea for the clinical prevention or treatment of polycystic ovary syndrome and the modernization of traditional Chinese medicine.

[0007] The technical solutions for achieving the object of the present invention are as follows:

[0008] In the first aspect, the present invention provides a preparation method of an effective part of the extract of Menispermum dauricum DC. with the function of preventing or treating polycystic ovary syndrome, including the following steps:

[0009] (1) Take the clean rhizome of Menispermum dauricum DC., thoroughly dry it and then carry out extraction;

[0010] (2) Prepare an ethanol: methanol: water extract according to a volume ratio of (0.5 - 3):(0.5 - 2):(1 - 2), adjust the temperature to 10 - 100 °C, mix the extract with the Menispermum dauricum DC. in step (1) and shake for 1 - 12 hours;

[0011] (3) After the extraction in step (2) is completed, carry out solid - liquid separation to obtain a crude extract, rotary evaporate at 30 - 60 °C for 1 - 6 hours to fully volatilize the organic solvent and obtain a concentrated solution. At this time, there is still a large amount of water in the extract;

[0012] (4) Carry out vacuum freeze - drying on the concentrated solution obtained in step (3) at - 1.0 MPa to - 0.10 MPa for 1 - 18 hours to obtain a dry light yellow to black powder;

[0013] (5) Dissolve the powder obtained in step (4) in ethanol, filter to obtain a clear solution, concentrate the solution and evaporate to dryness to obtain the crude extract of the effective part of the target extract of Menispermum dauricum DC.;

[0014] (6) Use high - performance liquid chromatography to conduct activity tracking on the crude extract of the effective part in step (5). The effective components eluted in 6 - 7 minutes have the best effect. After concentration, the effective part of the extract of Menispermum dauricum DC. is obtained;

[0015] (7) Perform liquid chromatography-tandem mass spectrometry detection. It is analyzed that the effective part of the Menispermi Rhizoma extract is mainly a mixture of dauricine (DAU) and methyl dauricine (DAU-Me), and in terms of mass percentage, the proportion is 75% - 95%.

[0016] As an optional method, in the preparation method of the effective part of the Menispermi Rhizoma extract above, in step (1), the Menispermi Rhizoma should be chopped into small sections of 2 mm.

[0017] As an optional method, in the preparation method of the effective part of the Menispermi Rhizoma extract above, in step (2), the volume ratio of ethanol:methanol:water is 3:1:2, the material-liquid ratio is 1 g: 6 - 10 mL, the extraction temperature is 50 - 60 °C, and the oscillation extraction time is 4 - 5 hours.

[0018] As an optional method, in the preparation method of the effective part of the Menispermi Rhizoma extract above, in step (3), rotary evaporation is carried out at 50 °C for 4 - 6 hours.

[0019] As an optional method, in the preparation method of the effective part of the Menispermi Rhizoma extract above, in step (4), freeze-drying is carried out at -0.70 MPa for 12 - 18 hours.

[0020] As an optional method, in the preparation method of the effective part of the Menispermi Rhizoma extract above, in step (5), filtration is carried out using a 0.22 μm filter membrane.

[0021] As an optional method, in the preparation method of the effective part of the Menispermi Rhizoma extract above, the chromatographic column used in step (6) is: a C18 chromatographic column with a particle size of 5 μm, 4.6×250 mm; the mobile phase is: in terms of 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.

[0022] As an optional method, in the preparation method of the effective part of the Menispermi Rhizoma extract above, the Menispermi Rhizoma is selected from the dry rhizome of Menispermum dauricum ( Menispermum dauricum DC. ), and the collection time is limited to spring and autumn, and the production areas are one or more of Northeast China, North China, East China, and Shaanxi, Ningxia, Gansu, and Shandong.

[0023] In a second aspect, the present invention provides an effective fraction of Menispermi Rhizoma extract prepared by the preparation method described in the first aspect above, which has the effect of preventing or treating polycystic ovary syndrome. The effective fraction of Menispermi Rhizoma extract is mainly a mixture of dauricine (DAU) and methyl dauricine (DAU-Me), and the proportion is 75%-95% by mass percentage.

[0024] In a third aspect, the present invention provides the use of the effective fraction of Menispermi Rhizoma extract described in the second aspect above in the preparation of a dipeptidyl peptidase-4 (DPP4) inhibitor.

[0025] In a fourth aspect, the present invention provides the use of the effective fraction of Menispermi Rhizoma extract described in the second aspect above in the preparation of a drug for preventing or treating polycystic ovary syndrome.

[0026] As an optional manner, in the above application, the drug further comprises other clinically commonly used DPP4 inhibitors.

[0027] Preferably, the other DPP4 inhibitors are selected from one or more of the following: sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, gemigliptin or teneligliptin.

[0028] More preferably, the other DPP4 inhibitor is selected from sitagliptin.

[0029] As an optional manner, in the above application, the dosage form of the drug is selected from granules, capsules, tablets, powders, oral solutions, suspensions or emulsions.

[0030] As an optional manner, in the above application, the dosage form of the drug is enteric-coated tablets.

[0031] The present invention has the following beneficial effects compared with the prior art:

[0032] The effective fraction of Menispermi Rhizoma extract provided by the present invention significantly improves the estrous cycle disorder, insulin resistance, blood sex hormone imbalance and ovarian polycystic-like changes in PCOS model mice, and does not cause obvious reproductive toxicity. It can be used to prepare drugs for preventing or treating polycystic ovary syndrome, and has good clinical application prospects and commercial value. Description of the Drawings

[0033] Figure 1: Effects of each component of Rhizoma Menispermi extract on the estrous cycle of DHEA mice. The pictures show mean ± SD. M / D: late estrus / diestrus; E: estrus; P: proestrus. One-way ANOVA was used to evaluate the significance among groups, indicating P < 0.05 compared with the model group, indicating P < 0.01 compared with the model group, indicating P < 0.001 compared with the model group, indicating P < 0.0001 compared with the model group. Among them, component 7 showed the most obvious improvement, followed by DAU.

[0034] Figure 2 : Effects of each component of Rhizoma Menispermi extract on the results of glucose tolerance test (GTT) in DHEA mice. The pictures show mean ± SD. Since it is not easy to distinguish the differences among groups in the line graph, only the significance between component 7 and the model group was shown. To more clearly show the differences among groups, the area under the curve was calculated and one-way ANOVA was used to evaluate the significance among groups, indicating P < 0.05 compared with the model group, indicating P < 0.01 compared with the model group, indicating P < 0.001 compared with the model group, indicating P < 0.0001 compared with the model group. Among them, component 7 showed the most obvious improvement, followed by DAU.

[0035] Figure 3 : Effects of each component of Rhizoma Menispermi extract on the results of insulin tolerance test (ITT) in DHEA mice. The pictures show mean ± SD. Since it is not easy to distinguish the differences among groups in the line graph, only the significance between component 7 and the model group was shown. indicating P < 0.001 compared with the model group. Among them, component 7 showed the most obvious improvement.

[0036] Figure 4 : Effects of each component of Rhizoma Menispermi extract on the blood sex hormones in DHEA mice. The pictures show mean ± SD. One-way ANOVA was used to evaluate the significance among groups, indicating P < 0.05 compared with the model group, indicating P < 0.01 compared with the model group, indicating P < 0.001 compared with the model group, indicating P < 0.0001 compared with the model group. Among them, component 7 showed the most obvious improvement, followed by DAU.

[0037] Figure 5 : Effects of Rhizoma Menispermi extract on polycystic ovary-like changes in DHEA mice. The pictures show mean ± SD. One-way ANOVA was used to evaluate the significance among groups, Indicates P < 0.05 compared with the model group, Indicates P < 0.01 compared with the model group, Indicates P < 0.001 compared with the model group, Indicates P < 0.0001 compared with the model group. Among them, component 7 shows the most obvious improvement, followed by DAU.

[0038] Figure 6 : Effects of the effective part of Rhizoma Menispermi extract (component 7) on the estrous cycle of DHEA mice. The pictures show mean ± SD. M / D: late estrus / diestrus; E: estrus; P: proestrus. One-way ANOVA was used to evaluate the significance among groups, Indicates P < 0.05 compared with the model group, Indicates P < 0.01 compared with the model group, Indicates P < 0.001 compared with the model group, Indicates P < 0.0001 compared with the model group. Among them, sitagliptin + component 7 shows the most obvious improvement, followed by component 7.

[0039] Figure 7 : Effects of the effective part of Rhizoma Menispermi extract (component 7) on the GTT of DHEA mice. The pictures show mean ± SD. Since it is not easy to distinguish the differences among groups in the line graph, the area under the curve was calculated and one-way ANOVA was used to evaluate the significance among groups, Indicates P < 0.05 compared with the model group, Indicates P < 0.01 compared with the model group, Indicates P < 0.001 compared with the model group, Indicates P < 0.0001 compared with the model group. Among them, sitagliptin + component 7 shows the most obvious improvement, followed by component 7.

[0040] Figure 8 : Effects of the effective part of Rhizoma Menispermi extract (component 7) on the ITT of DHEA mice. The pictures show mean ± SD. Since it is not easy to distinguish the differences among groups in the line graph, only the significance between sitagliptin + component 7 and the model group is shown. Indicates P < 0.001 compared with the model group. Among them, sitagliptin + component 7 shows the most obvious improvement, followed by component 7.

[0041] Figure 9 : Effects of the effective part of Rhizoma Menispermi extract (component 7) on the blood sex hormones of DHEA mice. The pictures show mean ± SD. One-way ANOVA was used to evaluate the significance among groups, Indicates P < 0.05 compared with the model group, Indicates P < 0.01 compared with the model group, Indicates P < 0.001 compared with the model group, Indicates P < 0.0001 compared with the model group. Among them, sitagliptin + component 7 showed the most obvious improvement, followed by component 7.

[0042] Figure 10 : Effects of the effective part of Rhizoma Menispermi (component 7) on polycystic ovarian-like changes in DHEA mice ovaries. The pictures show mean ± SD. One-way ANOVA was used to evaluate the significance among groups. Indicates P < 0.05 compared with the model group. Indicates P < 0.01 compared with the model group. Indicates P < 0.001 compared with the model group. Indicates P < 0.0001 compared with the model group. Among them, sitagliptin + component 7 showed the most obvious improvement, followed by component 7. Detailed implementation mode

[0043] Through extensive and in-depth research and experiments, the inventor found that the effective part of a specific Rhizoma Menispermi extract can inhibit the activity of DPP4 of bacterial origin and has the effect of preventing and treating polycystic ovary syndrome. Feeding the experimental subjects with the effective part of the Rhizoma Menispermi extract, it was found that it can improve insulin resistance, estrous cycle disorder, hormone secretion disorder and ovarian polycystic-like changes in polycystic ovary syndrome model mice. On this basis, the present invention was completed.

[0044] As used herein, the term "comprising" means that various components can be applied together in the mixtures or compositions of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

[0045] The present invention provides the use of the effective part of Rhizoma Menispermi extract in preventing or treating polycystic ovary syndrome. Mice were given high androgen stimulation to create a polycystic ovary syndrome model, and the effective part of the Rhizoma Menispermi extract has the ability to improve insulin resistance, estrous cycle disorder and polycystic ovarian-like changes in the model animals. According to a preferred example of the present invention, treating DHEA-induced PCOS model mice with the effective part of Rhizoma Menispermi extract can maintain normal estrous cycle, sex hormones and ovarian morphology compared with the untreated control group. Therefore, the effective part of the Rhizoma Menispermi extract can be used to prevent and treat polycystic ovary syndrome.

[0046] The medicament of the present invention can be administered in any conventional pharmaceutical preparation form in the art, and the pharmaceutical preparation includes excipients, pharmaceutically acceptable media and carriers, and these substances can be selected according to the administration route. The medicament in the present invention may further contain auxiliary components.

[0047] 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 paste, 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 polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, etc.), lubricants (such as magnesium stearate, colloidal silicon dioxide, talc, hydrogenated vegetable oil, polyethylene glycols, magnesium lauryl sulfate, etc.), absorption promoters (such as surfactants, Azone (laurocapram), EDTA, salicylic acid, aminoethylamine derivatives, acetyl acetate esters, β-dicarboxylic esters, aromatic acidic compounds, aliphatic acids, etc.), preservatives (such as benzoic acid, propylparaben, methylparaben, phenol, m-cresol, etc.), flavoring agents (such as sucrose, steviol glycosides, etc.).

[0048] In addition, as a preferred embodiment, the drug of the present invention can be produced in enteric-coated preparations by a variety of well-known methods so that the active ingredient of the drug can pass through the stomach smoothly without being destroyed by gastric acid.

[0049] The pharmaceutical composition of the present invention can be made into enteric-coated tablets for oral use. The term "enteric coating" in this application includes all coatings allowed for conventional drugs, which are not degraded by gastric acid but can be fully decomposed and rapidly release the active ingredient of the present invention in the small intestine. The enteric coating of the present invention can maintain for more than 2 hours at 36-38 °C in synthetic gastric acid such as HCl solution with pH = 1, and preferably decompose within 1.0 hour in synthetic intestinal fluid such as buffer solution with pH = 7.0.

[0050] The enteric coating of the present invention is coated at about 16-30 mg per tablet, preferably 16-25 mg, more preferably 16-20 mg. The thickness of the enteric coating in the present invention is 5-100 μm, and the ideal thickness is 20-80 μm. The components of the enteric coating are conventional polymers in the field of pharmaceutical preparations.

[0051] The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0052] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified. Among them, the purchase situations of some raw materials and reagents are as follows:

[0053] Menispermum dahuricum used in the present invention is selected from the plant Menispermum dahuricum DC. of the family MenispermaceaeMenispermum dauricum DC. The dried rhizomes of Menispermum dahuricum (Turcz.) DC., with the collection time limited to spring and autumn, are produced in one or more regions of Northeast China, North China, East China, and Shaanxi, Ningxia, Gansu, and Shandong.

[0054] Example 1: Preparation of Menispermum dahuricum Extract

[0055] Take the clean rhizomes of Menispermum dahuricum from Heilongjiang Province, cut them into pieces after thorough drying for extraction. Prepare an ethanol:methanol:water extractant with a volume ratio of 3:1:2, adjust the temperature to 60 °C, mix the extractant with Menispermum dahuricum and shake for 5 hours. Separate the solid from the liquid to obtain the crude extract, perform rotary evaporation at 50 °C for 4 hours to fully volatilize the organic solvent, and obtain a concentrated solution. At this time, there is still a large amount of water in the extract. Perform vacuum freeze-drying on the concentrated solution at -0.7 MPa for 18 hours to obtain a dry light yellow to black powder. Dissolve this powder in ethanol, filter to obtain a clear solution, concentrate the solution, and evaporate to dryness to obtain the crude extract of the effective part of the target Menispermum dahuricum extract.

[0056] Use high-performance liquid chromatography to perform active separation on the above crude extract to explore the key effective parts. The chromatographic column used is: a C18 chromatographic column with a particle size of 5 μm, 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. The liquid phase method takes a total of 15 minutes, collect the effluent every minute, and a total of 15 portions of effluent are obtained. Evaporate each portion of the effluent to dryness at 50 °C, weigh it, and redissolve it with methanol to obtain components 1 - 15. Dissolve an equal amount of the dried crude extract of Menispermum dahuricum in methanol to obtain component 16.

[0057] Example 2: Inhibitory Activity of Menispermum dahuricum Extract and Each Component against Bacterial DPP4

[0058] By regulating abnormal glucose tolerance, PCOS can be effectively improved. Recent studies have found that the activity of bacterial DPP4 can regulate blood sugar, and thus has the potential to become a new bacterial target for PCOS. However, it is not yet clear which are the main components in the Menispermum dahuricum extract that inhibit the activity of bacterial DPP4. Therefore, each component of the Menispermum dahuricum extract is separated by liquid chromatography to obtain 16 components (see Example 1), and then the active tracking strategy is used to evaluate the inhibitory effect of each component on bacterial DPP4. The bacterial DPP4 is 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.

[0059] Table 1: Inhibitory Activity of Menispermum dahuricum Extract and Each Component against Bacterial DPP4

[0060]

[0061] Among them, component 16 is the crude extract, and components 1-15 are the components separated by liquid chromatography according to the retention time. Component 7 shows the most obvious DPP4 inhibitory activity.

[0062] Example 3: Analysis of the main components of the effective part (component 7) of Menispermi Rhizoma extract

[0063] The main components and contents in component 7 were analyzed by LC-MS, and the inhibitory effects of each component on DPP4 were evaluated. The results are shown in Table 2.

[0064] Table 2: Analysis of the main components of component 7 and the inhibitory effects of each component on DPP4

[0065]

[0066] Through LC-MS analysis, it was determined that the main component in component 7 is dauricine (DAU), accounting for about 60.3%, the secondary component is dauricine methyl ester (DAU-Me), accounting for about 23.2%, and the other components are dauricine analogs, all of which have DPP4 inhibitory activity. Moreover, the activity of component 7 is higher than that of any single component, indicating a synergistic effect among the components.

[0067] Example 4: Comparison of the improvement effects of single components, component 7 and crude extract in the effective components of Menispermi Rhizoma on the PCOS mouse model induced by dehydroepiandrosterone (DHEA)

[0068] Materials: The test substances were the extracts of each component of Menispermi Rhizoma collected from North China, and the powders obtained after extraction, purification and drying. The dosage of the intragastric administration drug was 10 mg / kg body weight, and the frequency was once a day.

[0069] C57 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The temperature was 20-24 °C, the humidity was kept at 50-60%, the light was on for 12 hours (8:00-20:00), sound insulation was provided, and the mice had free access to food and water. The experiment was carried out after one week of environmental adaptation. Blood glucose test strips (Roche), blood glucose meters (Roche), dehydroepiandrosterone (Sigma), and sex hormone detection kits.

[0070] Method: 42 female C57 mice, 3 - 4 weeks old, 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 analog group of fraction 7, (6) fraction 7 group, (7) crude extract group, with 6 mice in each group. Groups (2) - (7) were given subcutaneous injection of DHEA. Mice in each group were intragastrically administered the corresponding drug at a dose of 10 mg / kg body weight for 4 consecutive weeks. The control group and the model group were given an equal amount of normal saline. The estrous cycle was detected daily 10 days after DHEA injection. The estrous cycle of the mice in the model group was significantly disordered, indicating successful establishment of the model. Oral glucose tolerance and insulin tolerance were detected before the last administration. After the last administration, blood was collected, centrifuged at 3000 rpm at 4°C, and the serum sex hormone levels were measured. Ovarian sections were observed for pathological changes.

[0071] Results: Compared with the model group, treatment with fraction 7 of Rhizoma Menispermi and other extract groups could significantly improve the insulin resistance - related indicators, improve the ovarian status and sex hormone levels of mice. Fraction 7 was the most significant, followed by DAU. The results are shown in Figures 1-5 .

[0072] Example 5: Improvement effect of the active ingredient (fraction 7) of Rhizoma Menispermi on the PCOS mouse model induced by DHEA

[0073] Materials: The test substance was fraction 7 of the extract of Rhizoma Menispermi collected from North China, which was obtained as a light yellow to dark yellow powder after extraction, purification and drying. The intragastric administration dose 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 intragastric administration dose of the drug was 10 mg / kg body weight, and the frequency was once a day.

[0074] C57 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The temperature was 20 - 24°C, the humidity was kept at 50 - 60%, the light was on for 12 hours (8:00 - 20:00), the environment was sound - insulated, and the mice had free access to food and water. The experiment was carried out after one week of environmental adaptation. Blood glucose test strips (Roche), blood glucose meters (Roche), dehydroepiandrosterone (Sigma), and sex hormone detection kits.

[0075] Method: 30 female C57 mice, 3 - 4 weeks old, 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. The mice in groups (2) - (5) were given subcutaneous injection of DHEA. The mice in each group were intragastrically administered the corresponding drug at a dose of 10 mg / kg body weight. The combined - medication group was intragastrically administered component 7 (10 mg / kg) and sitagliptin (10 mg / kg). This was done continuously for 4 weeks. The control group and the model group were given an equal volume of normal saline. The estrous cycle was detected daily 10 days after DHEA injection. The estrous cycle of the mice in the model group was significantly disordered, indicating successful establishment of the model. Oral glucose tolerance and insulin tolerance were detected before the last administration. After the last administration, blood was collected, centrifuged at 3000 rpm at 4°C, and the serum sex hormone levels were measured. Ovarian sections were observed for pathological changes.

[0076] Results: Compared with the model group, the three treatment methods of component 7, sitagliptin, and component 7 + sitagliptin could significantly improve the insulin - resistance - related indicators, restore the normal estrous cycle, improve the ovarian status and sex hormone levels of the mice. The effect of component 7 + sitagliptin was the most significant, followed by component 7. The results are shown in Figures 6-10 .

[0077] Example 6: Safety of the effective part of Rhizoma Menispermi extract (component 7) in the whole - cycle treatment of pregnant and lying - in women

[0078] Materials: The test substance was the component 7 of Rhizoma Menispermi extract collected from North China, which was obtained as a light - yellow to dark - yellow powder after extraction, purification, and drying. The intragastric administration dose of the drug was 10 mg / kg body weight, with a frequency of once a day. Sitagliptin was selected as the positive - control drug, and the intragastric administration dose of the drug was 10 mg / kg body weight, with a frequency of once a day. The combined - medication group was intragastrically administered component 7 (10 mg / kg) and sitagliptin (10 mg / kg), once each per day.

[0079] C57 male and female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The temperature was 20 - 24°C, the humidity was kept at 50 - 60%, the light was on for 12 hours (8:00 - 20:00), the environment was sound - insulated, and the mice had free access to food and water. The experiment was carried out after the mice had adapted to the environment for one week.

[0080] Method: 24 female C57 mice, 8 - 10 weeks old, weighing 22 - 26 g, were randomly divided into (1) normal control group, (2) component 7 group, (3) sitagliptin group, and (4) sitagliptin + component 7 group, with 6 mice in each group. The mice in group (1) were given PBS daily, and the mice in groups (2) - (4) were given corresponding drug treatments daily. After one week, they were mated with healthy male mice (10 - 12 weeks old, 1:1), and the vaginal plugs were checked the next morning to determine the mating date. The drug - treated groups continued to be given drugs daily until the day before parturition, and the control group was given an equal amount of PBS. The weight changes of pregnant mice were recorded. On the day before parturition, the pregnant mice were sacrificed, blood was taken, centrifuged at 3000 rpm at 4°C, and the serum sex hormone levels were measured. Ovarian sections were observed for pathological changes. Indicators such as pregnancy rate, fetal weight, number of live births, number of dead fetuses, and number of resorbed fetuses were statistically analyzed.

[0081] Results: Compared with the control group, the effective part of Rhizoma Menispermi extract (component 7), sitagliptin, and their combination did not cause obvious reproductive toxicity and did not change the weight and number of offspring. The results are shown in Tables 3 - 4.

[0082] Table 3: Effects of the effective part of Rhizoma Menispermi extract (component 7) on the live - birth rate and sex ratio of mouse offspring

[0083]

[0084] Table 4: Effects of the effective part of Rhizoma Menispermi extract (component 7) on the weight and body length of mouse offspring

[0085]

[0086] 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 equivalent technologies, the present invention is also intended to include these changes and modifications.

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

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

Images