Use of total alkaloids of mulberry twig in the manufacture of medicine for treating polycystic ovary syndrome

Abstract

Use of mulberry extract in the manufacture of products for treating and / or improving polycystic ovary syndrome. Experiments have demonstrated that it can reduce the ovarian weight of rats with polycystic ovary syndrome, improve the ovarian pathology, regulate the estrous cycle of rats with polycystic ovary syndrome in the intervention process, and significantly regulate the secretion level of sex hormones in rats with polycystic ovary syndrome.

Description

[Technical field]

[0001] The present invention relates to the field of medicine, specifically to the use of total alkaloids of mulberry twig in the manufacture of a drug for treating polycystic ovary syndrome (PCOS). [Background technology]

[0002] Polycystic ovarian syndrome (PCOS) is a common endocrine disorder in women of reproductive age. Clinically, it manifests as hyperandrogenism, oligo- or anovulation, and changes in the ovaries to polycystic morphology. PCOS causes abnormal secretion of sex hormones in the body due to various factors, resulting in the growth of follicles being stopped midway, the follicular walls becoming hyperplastic, and the follicular follicles remaining in the ovaries becoming cysts. This not only increases the incidence of gynecological diseases such as infertility and cervical cancer, but also causes various mental disorders such as depression. In addition, research has shown that pregnant women with PCOS not only increase the risk of related pregnancy complications, but also increase the risk of their offspring suffering from various "mental disorders" and metabolic diseases such as PCOS due to epigenetics. Statistics show that the risk of daughters of PCOS pregnant women suffering from PCOS is five times higher. PCOS is a reproductive endocrine disorder caused by various factors, and is highly heterogeneous, with large differences in pathophysiological characteristics among patients. Currently, medical treatment for polycystic ovary syndrome is still limited to treating its clinical symptoms, such as taking oral contraceptives to regulate the menstrual cycle, using ethinyl estradiol cyproterone tablets to reduce androgen levels, and ovulation induction treatment with Clomid (CC) or Letrozole for patients who wish to become pregnant. However, clinical treatment has limited effectiveness, while taking contraceptives is likely to cause fluid retention, liver dysfunction, and mental disorders such as depression, and ovulation induction treatment with Clomiphene or Clomid (CC) is likely to cause ovarian hyperstimulation syndrome (OHSS), and is accompanied by many side effects. Currently, laparoscopic ovarian drilling and ovarian wedge resection are commonly used surgical treatments, but laparoscopic ovarian drilling may not be effective, may cause intrapelvic adhesions, and may reduce ovarian function, and ovarian wedge resection has disadvantages such as a large surgical wound and adhesion of tissues around the ovaries after surgery, so it is not often performed in clinical practice. These treatments for PCOS are not ideal and only provide partial relief while having serious side effects.Therefore, how to reduce the risk to life and improve fertility by researching and developing safe and effective PCOS treatments has become a major scientific challenge that needs to be solved urgently worldwide. Summary of the Invention [Problem to be solved by the invention]

[0003] The object of the present invention is to provide a new medicinal use of mulberry extract or its main active ingredients. [Means for solving the problem]

[0004] The new use of the mulberry extract or its main active ingredients provided by the present invention is any one of the following (a1) to (a4). (a1) Use of a mulberry extract in the manufacture of a product for treating polycystic ovary syndrome; (a2) Use of a mulberry extract in the manufacture of a product for improving polycystic ovary syndrome; (a3) Use of mulberry extract in the treatment of polycystic ovary syndrome; (a4) Use of mulberry extract in improving polycystic ovary syndrome.

[0005] The treatment and / or amelioration of polycystic ovary syndrome is manifested by at least one of the following: 1) Regulating the secretion levels of sex hormones in patients with polycystic ovary syndrome, 2) Regulating the menstrual cycle in patients with polycystic ovary syndrome; 3) To improve the polycystic ovarian condition in patients with polycystic ovary syndrome.

[0006] Among these, the sex hormones are selected from at least one of T (testosterone), E2 (estradiol), LH (luteinizing hormone), PRL (prolactin), P (progesterone), SHBG (sex hormone binding globulin) and AMH (anti-Mullerian hormone).

[0007] The present invention also relates to the use of a mulberry extract or a main active ingredient thereof in at least one of the following uses (b1) to (b6): (b1) manufacturing a product that regulates the secretion levels of sex hormones in patients with polycystic ovary syndrome; (b2) manufacturing products to regulate the menstrual cycle in women with polycystic ovary syndrome; (b3) manufacturing a product to improve the polycystic ovarian condition of patients with polycystic ovary syndrome; (b4) regulating the secretion levels of sex hormones in patients with polycystic ovary syndrome; (b5) Regulating the menstrual cycle in patients with polycystic ovary syndrome; (b6) Improving the polycystic state of the ovaries in patients with polycystic ovary syndrome.

[0008] The product is a drug or a drug formulation.

[0009] The mulberry extract is an extract of mulberry branches, an extract of mulberry bark and / or an extract of mulberry leaves. Alternatively, the mulberry extract can be provided as commercially available mulberry twig total alkaloids tablets (National Pharmaceutical Standard Z20200002).

[0010] In one embodiment, the mulberry extract or its main active ingredients of the present invention can improve apoptosis of ovarian granulosa cells in PCOS patients.

[0011] In one embodiment, the mulberry extract of the present invention or its main active ingredients can reduce body weight and ovarian weight in PCOS patients.

[0012] The mulberry extract can be prepared by referring to the method described in CN 113143997 A. The specific preparation method is as follows: 1) producing a crude extract of a mulberry plant; 2) separating the crude extract with a cationic resin and / or, if necessary, an anionic resin to obtain the mulberry extract.

[0013] The method further comprises: 3) subjecting the effluent separated by the resin in step 2) to alcohol precipitation and collecting the supernatant; 4) subjecting the supernatant to a concentration and drying treatment.

[0014] The method may further include a step of subjecting the effluent separated by the resin in step 2) to a concentration and drying treatment.

[0015] The mulberry extract acts on humans or mammals.

[0016] The mulberry family plant may be selected from Mulberry Tree, Log Mulberry, Morus Alba, Peony Mulberry, Japanese Mulberry or hybrid mulberry, and the hybrid mulberry is preferably Yue Sang No. 11, Gui Sang You No. 62 or Mulberry Special You No. 2. Various parts of the plant may be used, such as leaves, roots, branches, bark, buds, stems, fruits, etc.

[0017] The mulberry extract may be an extract of mulberry alkaloids.

[0018] In one embodiment of the invention, the mulberry extract comprises alkaloids, polysaccharides, flavonoids and amino acids.

[0019] Preferably, the alkaloid is 1-deoxynojirimycin (DNJ), N-methly-1-deoxynojirimycin, fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-O-(α- The compound contains at least one of 2-O-(α-D-Galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.

[0020] In the present invention, the weight percentage of DNJ is 30% or more of the total alkaloids. Preferably, the weight percentage of DNJ is 40% or more of the total alkaloids. More preferably, the weight percentage of DNJ is 50% or more of the total alkaloids.

[0021] Preferably, for the mulberry extract, the weight content of each component is as follows: Alkaloids 3~99% Polysaccharide 0.2~70% Flavonoids 0-10% Amino acids 0-50% Other ingredients 0~25%

[0022] Preferably, for the mulberry extract, the weight content of each component is as follows: Alkaloids 30~99% Polysaccharide 0.2~35% Flavonoids 0~2% Amino acids 0-30% Other ingredients 0~20%

[0023] More preferably, for said mulberry extract, the weight content of each component is as follows: Alkaloids 50~99% Polysaccharides 0.2~35% Flavonoids 0~2% Amino acids 0-30% Other ingredients 0~20%

[0024] The alkaloid content may be 60 to 70% or 70 to 80%.

[0025] More preferably, the weight content of each component in said mulberry extract is as follows: Alkaloids 50~99% Polysaccharide 0.2~25% Flavonoids 0~1% Amino acids 0-20% Other ingredients 0~20%

[0026] In one embodiment, the preparation of the mulberry extract comprises the steps of preparing a crude extract, optionally separating with a cationic resin and / or an anionic resin, optionally carrying out an alcohol precipitation process on the effluent separated by the resin, and optionally concentrating and drying. Preferably, the preparation of the mulberry extract comprises step 1) of preparing a crude extract, step 2) of separating with a cationic resin and / or optionally an anionic resin, optionally carrying out an alcohol precipitation process on the effluent separated by the resin of step 2), step 3) of optionally carrying out an alcohol precipitation process on the effluent separated by the resin of step 2), and optionally concentrating and drying step 4).

[0027] In one embodiment, the mulberry extract is prepared according to the following procedure: mulberry branches, mulberry leaves or mulberry bark are crushed, extracted by heating under reflux with water and / or alcohol solution or acidic water, the amount of solvent is 3-20 times the amount of the drug substance, the extraction is repeated 1-3 times, the extract is combined, concentrated, and poured into a cation exchange resin, the unadsorbed impurities are washed off with distilled water, eluted with 0.2-3N ammonia water, the eluate is concentrated, and poured into anion exchange resin, the unadsorbed part is collected, ethanol is added, and the impurities are removed by precipitation, centrifuged, and the supernatant is concentrated under reduced pressure, or spray-dried, or freeze-dried to obtain the extract.

[0028] In one embodiment, the mulberry extract is prepared according to the following procedure: mulberry branches, mulberry leaves or mulberry bark are crushed, and extracted by heating under reflux with water and / or alcohol solution or acidic water, the amount of the solvent is 3-20 times the amount of the drug substance, the extraction is repeated 1-3 times, the extract is combined, concentrated, and poured into a cation exchange resin, the unadsorbed impurities are washed off with distilled water, eluted with 0.2-3N ammonia water, the eluate is concentrated, and poured into an anion exchange resin, the unadsorbed portion is collected, and concentrated under reduced pressure, or spray-dried, or freeze-dried to obtain an extract.

[0029] In one embodiment, the mulberry extract is prepared according to the following procedure: mulberry branches, mulberry leaves or mulberry bark are crushed, and extracted by heating under reflux with water and / or alcohol solution or acidic water, the amount of the solvent is 3 to 20 times the amount of the raw drug, the extraction is repeated 1 to 3 times, the extract is combined and concentrated, and then poured into a cation exchange resin, the unadsorbed impurities are washed off with distilled water, and the extract is eluted with 0.2 to 3N ammonia water, and the eluate is concentrated under reduced pressure, or spray-dried, or freeze-dried to obtain the extract.

[0030] In one embodiment, the mulberry extract is prepared according to the following procedure: mulberry branches, mulberry leaves or mulberry bark are crushed, extracted by heating under reflux with water, the amount of solvent is 3-20 times (preferably 4-15 times) the amount of the drug substance, the extraction is repeated 1-3 times (the extraction time is preferably 0.5-3 h / time), the extract is combined, concentrated, and poured into a cation exchange resin, the unadsorbed impurities are washed off with distilled water, eluted with 0.2-3N ammonia water, the eluate is concentrated, and poured into anion exchange resin, the unadsorbed part is collected, ethanol is added, and the impurities are removed by precipitation, centrifuged, and the supernatant is concentrated under reduced pressure, or spray-dried, or freeze-dried to obtain the extract.

[0031] Preferably, after the cationic resin is packed in a column, it is activated by passing an acidic solution, a basic solution, and an acidic solution in this order. Preferably, the basic solution is passed until the pH of the eluate reaches 8.0 to 9.5, preferably 8.5 to 9.5. Preferably, the basic solution is selected from an aqueous ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution, or a sodium carbonate solution. Preferably, the concentration of the basic solution is 0.5 to 4 mol / L. Preferably, the acidic solution is passed until the pH of the eluate reaches 3.0 to 7.0, preferably 4.5 to 6.5. Preferably, the acidic solution is selected from a hydrochloric acid solution, a phosphoric acid solution, and a disodium hydrogen phosphate-citrate buffer solution. If necessary, the cationic resin may be washed with deionized water in an amount 3 to 5 times the column volume after the final passage of the acidic solution. Preferably, the cation resin is 732 type strongly acidic styrenic cation exchange resin, 734 type strongly acidic styrenic cation exchange resin and D001 type macroporous strongly acidic styrenic cation exchange resin.

[0032] Preferably, the weight ratio of the amount of the cationic resin used to the amount of the plant raw material input is 1:2-20. After the crude plant extract is injected into the cationic resin, elution is performed on the injected cationic resin with an eluent. Preferably, the concentration of the eluent is 0.5-2.5 mol / L. Preferably, the flow rate of the eluent is 5-10 BV / h.

[0033] Preferably, the anion resin is 717 type strongly basic styrene-based anion exchange resin, D201 type macroporous strongly basic styrene-based anion exchange resin, and D218 type macroporous strongly basic acrylic-based anion exchange resin. Preferably, the weight ratio of the amount of the anion resin used to the amount of the plant raw material input is 1:1-32. When the liquid flows out of the anion resin, collection is started. Preferably, collection is stopped when the volume of the collected liquid is 0.1-5 times the weight of the plant raw material input.

[0034] Preferably, the weight ratio of ethanol used in the alcohol precipitation treatment to the amount of plant raw material added is 1:20 to 300. In the alcohol precipitation treatment, the stirring speed is 40 to 500 rpm.

[0035] Preferably, the drug further comprises a pharma- ceutically acceptable carrier. The carrier is an inert ingredient that is suitable for the route or method of administration and has no toxic effects on the human body. The carrier may be a solid excipient or a liquid excipient. Examples of solid excipients include microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, crosslinked povidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch and talc. Examples of liquid excipients include water, ethanol, syrup and glycerin.

[0036] Preferably, the drug is in an oral dosage form, more preferably, the drug is in the form of tablets, capsules, oral liquids, oral emulsions, pills, granules, syrups and powders.

[0037] The present invention further provides a method for preventing and / or treating polycystic ovary syndrome, comprising the step of administering said mulberry extract or its main active ingredient to an animal or human subject, thereby preventing and / or treating polycystic ovary syndrome.

[0038] In the present invention, the animal may be a mammal. Effect of the Invention

[0039] PCOS is a metabolic disease caused by multiple factors, and traditional Chinese medicines have the advantages of containing multiple ingredients and exerting medicinal effects on multiple targets, as well as having fewer side effects and a high level of safety.

[0040] The present invention has been demonstrated by pharmacological tests as follows. 1. SZ-A can reduce ovarian weight and improve ovarian pathology in PCOS rats. 2. The SZ-A administration group regulated the estrous cycle of PCOS rats through the intervention process. 3. SZ-A drug can significantly regulate the secretion levels of sex hormones in PCOS rats.

[0041] In order to more clearly describe the technical aspects of the specific embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the specific embodiments or the prior art. However, the drawings in the following description are only some embodiments of the present invention, and it is obvious that a person skilled in the art can derive other drawings based on these drawings without any creative ingenuity. [Brief description of the drawings]

[0042] [Figure 1] (A) is a photograph of the ovarian morphology of animals in the blank group, model group, and treatment group 21 days after administration, (C) is the body weight of animals in the blank group, model group, and treatment group 21 days after administration, and (B) is the result of HE-stained sections of ovarian tissue 21 days after administration. [Diagram 2]1 shows serum testosterone (T) levels in animals of the blank group, model group, and administration group 21 days after administration in Experimental Example 9. [Diagram 3] 4 shows serum estradiol (E2) levels in animals in the blank group, model group, and administration group 21 days after administration in Experimental Example 9. [Figure 4] 1 shows serum luteinizing hormone (LH) levels in animals of the blank group, model group and administration group 21 days after administration in Experimental Example 9. [Diagram 5] 1 shows serum prolactin (PRL) levels in animals of the blank group, model group, and administration group 21 days after administration in Experimental Example 9. [Figure 6] 1 shows serum progesterone (P) levels in animals of the blank group, model group, and administration group 21 days after administration in Experimental Example 9. [Figure 7] 13 shows serum AMH levels in animals in the blank group, model group, and administration group 21 days after administration in Experimental Example 9. [Figure 8] 1 shows serum SHBG levels in animals of the blank group, model group, and administration group 21 days after administration in Experimental Example 9. [Figure 9] These are serum T levels of animals in the blank group, model group, and administration group 21 days after administration in Experimental Example 10. # indicates P<0.05 for the model group compared to the blank group, ## indicates P<0.01 for the model group compared to the blank group, * indicates P<0.05 for the administration group compared to the model group, and ** indicates P<0.01 for the administration group compared to the model group. [Figure 10] These are serum T levels, SHBG levels, E2 levels, and P levels in animals from the blank group, model group, and treatment group 21 days after administration in Experimental Example 11. # indicates P<0.05 for the model group compared to the blank group, ## indicates P<0.01 for the model group compared to the blank group, * indicates P<0.05 for the treatment group compared to the model group, and ** indicates P<0.01 for the treatment group compared to the model group. [Figure 11] 1 shows pathological sections of the ovaries of animals in the blank group, model group, and administration group in Experimental Example 11, 21 days after administration. [Figure 12] 4 shows the number of follicles and the number of corpora lutea in animals of the blank group, model group, and administration group 21 days after administration in Experimental Example 11. [Figure 13] 11 is an analysis graph of the estrous cycle of rats during the treatment period in Experimental Example 11, (A) is a representative estrous cycle of each experimental group, (B) is a quantitative analysis of the estrous cycle of each experimental animal, where M / D represents metestrus / dyestrus, E represents estrus, and P represents proestrus. # represents P<0.05 for the model group compared to the blank group, ## represents P<0.01 for the model group compared to the blank group, * represents P<0.05 for the treatment group compared to the model group, and ** represents P<0.01 for the treatment group compared to the model group. [Figure 14] 11 shows graphs of the analysis of the estrous cycle of rats during the treatment period after the expansion of the experimental scale (18 rats / group) in Experimental Example 11, where (A) is a representative estrous cycle of each experimental group, and (B) is a quantitative analysis of the estrous cycle of each experimental animal, where M / D represents metestrus / dyestrus, E represents estrus, and P represents proestrus. # represents P<0.05 for the model group compared to the blank group, ## represents P<0.01 for the model group compared to the blank group, * represents P<0.05 for the treatment group compared to the model group, and ** represents P<0.01 for the treatment group compared to the model group. ★ represents P<0.05 for the SZ-A group compared to the Diane-35 group, and ★★ represents P<0.01 for the SZ-A group compared to the Diane-35 group. [Figure 15] These are serum T levels and E2 levels in animals of the blank group, model group, and administration group 21 days after administration in Experimental Example 12. # indicates P<0.05 for the model group compared to the blank group, ## indicates P<0.01 for the model group compared to the blank group, * indicates P<0.05 for the administration group compared to the model group, and ** indicates P<0.01 for the administration group compared to the model group. [Figure 16] 1 shows pathological sections of the ovaries of animals in the blank group, model group, and administration group in Experimental Example 12, 21 days after administration. [Figure 17]The figures show the number of follicles and corpora lutea in the blank, model, and treatment groups 21 days after administration in Experimental Example 12. # indicates P<0.05 for the model group compared to the blank group, ## indicates P<0.01 for the model group compared to the blank group, * indicates P<0.05 for the treatment group compared to the model group, and ** indicates P<0.01 for the treatment group compared to the model group. [Figure 18] These are serum T levels, AMH levels, GnRH levels, and SHBG levels in animals of the blank group, model group, and treatment group 21 days after administration in Experimental Example 13. # represents P<0.05 for the model group compared to the blank group, ## represents P<0.01, * represents P<0.05 for the treatment group compared to the model group, and ** represents P<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The present invention will be described in more detail below with reference to the following examples. These illustrative examples will make the features and advantages of the present invention more clear. However, the present invention is not limited to the following examples. The above methods are all conventional unless otherwise specified. The above raw materials are available from public commercial channels unless otherwise specified.

[0044] The word "exemplary" herein means "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" should not be construed as superior to other embodiments.

[0045] Furthermore, the configurations in the respective embodiments of the present invention described below can be combined unless they are mutually inconsistent.

[0046] The component contents of the present invention are measured by published methods (see the methods described in patents with publication numbers CN111077247A and CN110393738A).

[0047] The abbreviations used in the section verifying the effectiveness of mulberry extract are as follows: T (testosterone), E2 (estradiol), LH (luteinizing hormone), P (progesterone), PRL (prolactin), LEP (leptin), SHBG (sex hormone-binding globulin), AMH (anti-Mullerian hormone), GnRH (gonadotropin-releasing hormone).

[0048] The methods for measuring each hormone are as follows: E2: Estradiol radioimmunoassay kit instruction manual - Atom High-Tech Co., Ltd. (National Pharmaceutical Standard S20033010); LH: Iodine [ 125 I] Human luteinizing hormone radioimmunoassay kit (National Pharmaceutical Association S10950161); PRL: Iodine [ 125 I] Prolactin radioimmunoassay kit instruction manual (National Pharmaceutical Association S10950161); T: Iodine [ 125 I] Testosterone radioimmunoassay kit instruction manual (S10940093); Serum ADP, GnRH, SHBG and AMH of each animal are measured using Elisa kits. P: Progesterone radioimmunoassay kit instruction manual - Tianjin Kyowa Pharmaceutical Technology Co., Ltd. (National Pharmaceutical Association S10950202); EXAMPLES

[0049] 1. Example of mulberry extract production

[0050] Example 1 Preparation of Mulberry Extract 1 1000 kg of fresh mulberry branches (Mulberry no. 11) were crushed, 4000 L of water was added, and extraction was performed by heating under reflux for 2 hours. The extracts were combined and filtered to remove insoluble matter to obtain a crude extract. The crude extract was then thermally concentrated until the solid mass percentage content was 4%, and the mixture was kept at 50°C to be injected into the cationic resin column.

[0051] 150 kg of D113 type macroporous weakly acidic allylbenzene cationic resin was packed in a column, and 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and the column was further activated by washing with deionized water in an amount 5 times the column volume. The concentrated extract was injected and eluted with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. When the pH of the effluent from the cationic column was detected to be > 7, the eluate was collected, and when the collected liquid reached 900 L, collection was stopped, and the collected liquid was directly purified in the anionic column.

[0052] A column was filled with 62.5 kg of D218 type macroporous strongly basic acrylic anion resin, and activated by passing 1.5 mol / L sodium hydroxide solution through the column until the pH of the eluate reached 9.0, 1.5 mol / L hydrochloric acid solution through the column until the pH of the eluate reached 3.5, and 1.5 mol / L sodium hydroxide solution through the column until the pH of the eluate reached 9.0. The collected eluate from the cationic resin was poured into the anionic resin, and the effluent was collected until the effluent reached 870 L.

[0053] The collected liquid was centrifuged to remove impurities, then concentrated using a reverse ion osmosis membrane, and the liquid density after concentration was 1.25. This was transferred to an alcohol precipitation tank, and 25 L of absolute ethanol was added under the condition of a stirring blade at 500 rpm. After the addition of ethanol was completed, the stirring was stopped and the alcohol precipitation was carried out for 24 hours, and the supernatant was concentrated under reduced pressure to obtain the extract.

[0054] The effluent was concentrated under reduced pressure to obtain a mulberry branch extract, in which the mass percentage content of alkaloids was 52% (in the alkaloids, the mass percentage content of DNJ was 69.5%, the mass percentage content of DAB was 11.5%, and the mass percentage content of FAG was 15%), the mass percentage content of polysaccharides was 22%, the mass percentage content of flavonoids was 0.8%, and the mass percentage content of amino acids was 20%.

[0055] Example 2 Preparation of Mulberry Extract 2 After crushing 10 kg of fresh mulberry twigs (Sanguo No. 2), 150 L of water was added in two batches, and each time, extraction was performed for 3 hours by decoction method. The extract was combined and filtered to remove insoluble matter. The extract was thermally concentrated until the mass percent content of solid matter was 8%, and then transferred to an alcohol precipitation tank, and 2367.9 g (3 L) of anhydrous ethanol was added under the condition of a stirring blade of 300 rpm. After the addition of ethanol was completed, the stirring was stopped, and alcohol precipitation was performed for 24 hours, and the supernatant was used as the injection liquid for the cationic resin column. 5 kg of 002SC type strong acid styrene-based cationic resin was filled into the column, and the cationic resin was activated according to the method of Example 1. The concentrated and alcohol-precipitated extract was poured into the column, and then eluted with 100 L of 5 mol / L potassium chloride at a rate of 5 BV / h. The effluent was measured with 20% silicotungstic acid. When a white precipitate formed, collection was started. When the collected liquid reached 25 L, collection was stopped and the collected liquid was directly purified using an anion column.

[0056] 10 kg of 711 type strong basic styrene-based anion resin was packed in a column, and the anion resin was activated according to the method of Example 1. The collected eluate of the cationic resin was poured into the anion resin, and the effluent was collected. When the effluent reached 15 L, the collection was stopped. The collected effluent was poured into the cationic resin again, and separation was performed twice again in the order of the cationic resin and the anion resin according to the above method.

[0057] The collected liquid obtained after three column separations was centrifuged to remove impurities, then concentrated using a reverse ion osmosis membrane. The liquid density after concentration was 1.25, which was then transferred to an alcohol precipitation tank and 125 g of absolute ethanol was added at a stirring blade of 1000 rpm. After the addition of ethanol was completed, the stirring was stopped, and the mixture was allowed to settle in alcohol for 24 hours, and the supernatant was concentrated under reduced pressure to obtain an extract. In addition, fresh mulberry bark and mulberry leaves (Mulberry Special No. 2) were extracted using the same extraction method and conditions as the above method.

[0058] The obtained mulberry twig extract had an alkaloid mass percentage content of 98%, a polysaccharide mass percentage content of 0.2%, a flavonoid mass percentage content of 0.05%, and an amino acid mass percentage content of 0.

[0059] The obtained mulberry bark extract had an alkaloid content of 95% by mass, a polysaccharide content of 2% by mass, a flavonoid content of 0.1% by mass, and an amino acid content of 1% by mass.

[0060] The resulting mulberry leaf extract had an alkaloid content of 90%, a polysaccharide content of 4%, a flavonoid content of 0.1%, and an amino acid content of 3% by weight.

[0061] Example 3 Preparation of Mulberry Extract 3 1000 kg of fresh mulberry branches (Kantongwa) were crushed, 11500 L of water was added, and the mixture was heated under reflux for 2 hours to extract. The extracts were combined and filtered to remove insoluble matter to obtain a crude extract. The crude extract was centrifuged to remove impurities, and then concentrated by reverse ion osmosis until the solid mass percentage content was 1%, and used as the injection liquid for the cation exchange resin column.

[0062] 300 kg of D001 type macroporous strong acid styrene-based cationic resin was packed in a column, and the cationic resin was activated according to the method of Production Example 1. The concentrated crude extract was poured into the column, and eluted with 5000 L of 0.04 mol / L ammonium nitrate at an elution rate of 5 BV / h. The effluent was measured with 20% silicotungstic acid. When a white precipitate was formed, collection was started, and when the collected liquid reached 1000 L, collection was stopped.

[0063] The collected liquid obtained after separation using a cation column was concentrated using a nanofiltration membrane and then concentrated under reduced pressure to obtain an extract.

[0064] The resulting mulberry twig extract had an alkaloid content of 15% by mass, a polysaccharide content of 20% by mass, a flavonoid content of 7% by mass, and an amino acid content of 45% by mass.

[0065] Example 4 Preparation of Mulberry Extract 4 333 kg of dried mulberry branches (Guangdong mulberry No. 11) were crushed, and then 4000 L of water was added. Extraction was carried out twice by heating and refluxing for 1 hour each time. The extracts were combined and filtered, and the extract was concentrated to 1 kg herbal medicine amount / L.

[0066] 150 kg of D113 type macroporous weakly acidic allylbenzene cationic resin was packed in a column, and 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and the column was further activated by washing with deionized water in an amount 5 times the column volume. The concentrated extract was injected and eluted with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. When the pH of the effluent from the cationic column was detected to be > 7, the eluate was collected, and when the collected liquid reached 900 L, collection was stopped, and the collected liquid was directly purified in the anionic column.

[0067] 125 kg of D218 type macroporous strongly basic acrylic anion resin was packed in a column, and 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, and 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, for activation. The collected eluate from the cationic resin was poured into the anionic resin, and the effluent with a pH higher than 8 was collected until the effluent reached 870 L.

[0068] The collected liquid obtained through separation by anion column was filtered through a microfiltration membrane to remove impurities, and then concentrated through a reverse ion osmosis membrane. The liquid density after concentration was 1.1, which was transferred to an alcohol precipitation tank and 15 kg of anhydrous ethanol was added under the condition of a stirring blade of 400 rpm. After the addition of ethanol was completed, the stirring was stopped, and the alcohol precipitation was carried out for 24 hours, and the supernatant was concentrated under reduced pressure to obtain an extract of mulberry branches. The content of the sample was 80% by mass percent of alkaloids, 5% by mass percent of polysaccharides, 0.1% by mass percent of flavonoids, and 4% by mass percent of amino acids.

[0069] Example 5 Preparation of Mulberry Extract 5 After crushing 400 kg of dried mulberry branches (Guangdong mulberry No. 11), 4000 L of water was added and extracted twice by heating and refluxing, each time for 1 hour. The extracts were combined and filtered, and the extract was concentrated to 1 kg herbal medicine / L.

[0070] A column was filled with 62.5 kg of D218 type macroporous strongly basic acrylic anion resin, and activated by passing 1.5 mol / L sodium hydroxide solution through it until the pH of the eluate reached 9.0, 1.5 mol / L hydrochloric acid solution through it until the pH of the eluate reached 3.5, and 1.5 mol / L sodium hydroxide solution through it until the pH of the eluate reached 9.0. The collected extract concentrate was poured into the anion resin, and the effluent was collected.

[0071] The collected liquid obtained through anion column separation was filtered through a microfiltration membrane to remove impurities, then concentrated through a reverse ion osmosis membrane, and further concentrated under reduced pressure and dried to obtain a mulberry branch extract.The sample contents were alkaloid mass percentage 3%, polysaccharide mass percentage 70%, flavonoid mass percentage 10%, and amino acid mass percentage 10%.

[0072] Example 6 Preparation of Mulberry Extract 6 1500 kg of fresh mulberry branches (Mulberry no. 11) were crushed, and then 6000 L of water was added and extracted by heating under reflux for 2 hours. The extracts were combined and filtered to remove insoluble matter to obtain a crude extract. The crude extract was then thermally concentrated until the solid mass percentage content was 4%, and then kept at 50°C to be injected into the cationic resin column.

[0073] 100 kg of D113 type macroporous weakly acidic allylbenzene cationic resin was packed in a column, and 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and the column was further activated by washing with deionized water in an amount 5 times the column volume. The concentrated extract was injected and eluted with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. When the pH of the effluent from the cationic column was detected to be > 7, the eluate was collected, and when the collected liquid reached 900 L, collection was stopped, and the collected liquid was directly purified in the anionic column.

[0074] 62.5 kg of D218 type macroporous strongly basic acrylic anion resin was packed in a column, and 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, and 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, and the resin was activated. The collected eluate from the cationic resin was poured into the anionic resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain an extract of mulberry branches, of which the mass percent content of alkaloids was 30%, the mass percent content of polysaccharides was 35%, the mass percent content of flavonoids was 2%, and the mass percent content of amino acids was 25%.

[0075] Example 7 Preparation of Mulberry Extract 7 1000 kg of fresh mulberry branches (Mulberry no. 11) were crushed, 4000 L of water was added, and extraction was performed by heating under reflux for 2 hours. The extracts were combined and filtered to remove insoluble matter to obtain a crude extract. The crude extract was then thermally concentrated until the solid mass percentage content was 4%, and the mixture was kept at 50°C to be injected into the cationic resin column.

[0076] 100 kg of D113 type macroporous weakly acidic allylbenzene cationic resin was packed in a column, and 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and the column was further activated by washing with deionized water in an amount 5 times the column volume. The concentrated extract was injected and eluted with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. When the pH of the effluent from the cationic column was detected to be > 7, the eluate was collected, and when the collected liquid reached 900 L, collection was stopped, and the collected liquid was directly purified in the anionic column.

[0077] 62.5 kg of D218 type macroporous strongly basic acrylic anion resin was packed in a column, and 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, and 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, and the resin was activated. The collected eluate from the cationic resin was poured into the anionic resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain an extract of mulberry branches, of which the mass percent content of alkaloids was 40%, the mass percent content of polysaccharides was 25%, the mass percent content of flavonoids was 0.5%, and the mass percent content of amino acids was 25%.

[0078] Example 8 Preparation of Mulberry Extract 8 333 kg of dried mulberry branches (Guangdong mulberry No. 11) were crushed, and then 4000 L of water was added. Extraction was carried out twice by heating and refluxing for 1 hour each time. The extracts were combined and filtered, and the extract was concentrated to 1 kg herbal medicine amount / L.

[0079] 150 kg of D113 type macroporous weakly acidic allylbenzene cationic resin was packed in a column, and 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and the column was further activated by washing with deionized water in an amount 5 times the column volume. The concentrated extract was injected and eluted with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. When the pH of the effluent from the cationic column was detected to be > 7, the eluate was collected, and when the collected liquid reached 900 L, collection was stopped, and the collected liquid was directly purified in the anionic column.

[0080] 62.5 kg of D218 type macroporous strongly basic acrylic anion resin was packed in a column, and 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, and 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, for activation. The collected eluate from the cationic resin was poured into the anionic resin, and the effluent with a pH higher than 8 was collected until the effluent reached 870 L.

[0081] The collected liquid obtained through separation by anion column was filtered through a microfiltration membrane to remove impurities, and then concentrated through a reverse ion osmosis membrane. The liquid density after concentration was 1.1, which was transferred to an alcohol precipitation tank and 15 kg of anhydrous ethanol was added under the condition of a stirring blade of 400 rpm. After the addition of ethanol was completed, the stirring was stopped, and the alcohol precipitation was carried out for 24 hours, and the supernatant was concentrated under reduced pressure to obtain an extract of mulberry branches. The content of the sample was 63% by mass percent of alkaloids, 23% by mass percent of polysaccharides, 1% by mass percent of flavonoids, and 5% by mass percent of amino acids.

[0082] 2. Verification of the effectiveness of mulberry extract

[0083] Experimental Example 9: Pharmacodynamic study of mulberry extract for the prevention and / or treatment of polycystic ovary syndrome (PCOS) 1. Experimental scheme 1.1 Experimental animals Nine female SD rats, newly weaned at 23 days of age, in good health and with shiny fur, were raised at the SPF level.

[0084] 1.2 Experimental drugs DHEA (dehydroepiandrosterone): 30 mg / mL, 30 mg of DHEA was dissolved in 100 μL of absolute ethanol, followed by the addition of 900 μL of soybean oil. 1% CMC: 1 g of CMC was weighed out and added to 100 mL of pure water, then placed on a magnetic stirrer and stirred at 60°C and 500 r / min until the CMC was completely dissolved and the solution became transparent. The mulberry extract SZ-A prepared in Example 1 was prepared to different concentrations and dissolved in 1% CMC solution.

[0085] 1.3 Modeling and Dosing The blank group (3 rats) was subcutaneously injected with the same amount of oil in the back of the neck, and the model group (6 rats) was subcutaneously injected with DHEA solution (0.2mL / 100g (60mg / kg) in the back of the neck. The injections were given once a day for a total of 21 days.

[0086] From the 11th day of modeling, rat vaginal secretions were collected at 8:00 a.m. every morning, and the rats were subjected to vaginal smear examinations for two cycles (about 10 days) to monitor the estrous cycle. On the 21st day of modeling, the testosterone (T) levels were measured and the models were evaluated. After 21 days of modeling, the model group showed disrupted estrous cycles and high androgens, indicating that PCOS modeling was successful.

[0087] After the modeling was successful, the animals were randomly divided into a blank group, a model group, and a group administered mulberry extract SZ-A (based on the total alkaloids in mulberry branches, 100mg / kg / d), and during the administration period, the blank group and the model group were given equal amounts of saline. On the 21st day of administration, each biochemical index was measured.

[0088] 1.4 Index observation and measurement methods (1) During the administration period, the body weight and mental state of the animals were measured. (2) Vaginal smear (measurement of one cycle; rats usually have one estrus cycle of 5 days, and measurements were taken from the 15th to 20th day after administration). (3) Expression of related hormones in serum: T, E2, LH, FSH, P, PRL, GnRH, SHBG and AMH were measured 21 days after administration. (4) After 21 days of treatment, the rats were killed and ovarian tissues were collected for HE staining. (5) Body weight and ovarian weight were observed. Ovarian weight: Peripheral blood from the ovarian tissue was aspirated using filter paper, and then the weight was measured. (6) Statistical analysis: Analysis was performed using SPSS software.

[0089] 2.Measurement results (1) Effect of drugs on rat body weight The experimental results after 21 days of administration are shown in Figure 1(C). Compared with the blank group, the body weight of the model group increased significantly, and the body weight of the model rats significantly decreased after the administration intervention. This indicates that the drug can significantly regulate the body weight of PCOS rats.

[0090] (2) Effect of drugs on the estrous cycle in PCOS rats Vaginal smear results showed that the rats in the normal control group showed a regular estrous cycle in the order of proestrus, estrus, metestrus, and diestrus, while the rats in the PCOS model group showed irregular estrous cycles. In the treatment group, the estrous cycle changes were improved during the intervention process.

[0091] (3) Effects of drugs on the ovaries of PCOS rats The ovarian morphology was observed, and the results are shown in Figure 1(A). When observing the external appearance of the ovarian tissue of rats in each experimental group, the ovarian surface of the rats in the blank group was bright red, with no obvious enlarged follicles, while the ovarian surface of the rats in the model group was pale and had multiple enlarged follicles of different sizes. In the treated group, the ovarian surface was healthy and shiny, and the symptom of pale ovaries was significantly improved compared to the model group.

[0092] The results of stained sections of ovarian tissue are shown in Figure 1(B). The ovarian tissue of the normal control rats had a normal structure as a whole under an optical microscope, and follicles at different stages of development were observed under the microscope. The follicles nearing maturity contained oocytes and corona radiata cells, the number of theca cell layers was small, there were no cystic follicles, and the ovarian interstitial cells were densely and orderly arranged. The ovarian tissue of the model rats had obvious abnormalities as a whole under an optical microscope, with obvious changes to multicystic morphology, no oocytes were observed in the follicles nearing maturity, the granulosa cells were loosely arranged, the number of layers was reduced and some were peeled off, the corona radiata cells were not observed, and theca cell layers were thickened. In rats in the drug intervention group (SZ-A), ovarian pathology was significantly improved, with corpora lutea and multiple follicles in all ovaries, oocytes and corona radiata cells in follicles approaching maturity, granulosa cells arranged in a dense and orderly fashion, an increased number of layers, and a thinner cell layer of the theca.

[0093] (4) Effects of drugs on serum sex hormone levels in PCOS rats The results are shown in Figures 2-8. Compared with the blank group, the expression of serum T, LH, E2.PRL and AMH all increased, while the expression of SHBG and progesterone P decreased in the model group. Compared with the model group, after 21 days of administration, the levels of hormones such as T (testosterone), E2 (estradiol), LH (luteinizing hormone), PRL (prolactin) and AMH decreased to different degrees, and SHBG increased slightly in the administration group. This indicates that the drug can significantly regulate the secretion levels of sex hormones in PCOS rats.

[0094] Experimental Example 10: Pharmacodynamic study of mulberry extract on LZ-induced PCOS model 1. Drug Preparation Letrozole (simply called LZ) Preparation concentration: 0.25 mg / mL, Dosage: 0.4 mL / 100 g. 1% CMC: 1 g of CMC was weighed out and added to 100 mL of pure water, then placed on a magnetic stirrer and stirred at 60°C and 500 r / min until the CMC was completely dissolved and the solution became transparent. The mulberry extract SZ-A prepared in Example 1 was prepared to different concentrations and dissolved in 1% CMC solution.

[0095] 2. Experimental Grouping

[0096] [Table 1]

[0097] 3. Experimental Method Eighteen 6-week-old female SD rats in good health with shiny fur were selected, and were adaptively reared for 3 days, and then divided into a blank group, a model group, and an administration group. The rats in the blank group were fed with basal feed, while the rats in the model group and administration group were fed with basal feed and were forcibly administered LZ (1 mg / kg / d) for 6 weeks to create a rat PCOS model. After successful model evaluation, the administration group was administered according to the dosage and administration method shown in Table 1, and the blank group and model group were administered an equal amount of physiological saline. Samples were taken 21 days after administration, and efficacy evaluation was performed using the same measurement and analysis methods as in Experimental Example 9.

[0098] 4.Results In the model group (vs. blank group), testosterone (T) levels were significantly increased, and T levels were significantly decreased after treatment with SZ-A (100 mg / kg) (see Figure 9).

[0099] Experimental Example 11: Pharmacodynamics study of mulberry extract on LZ+HFD-induced PCOS model 1. Drug Preparation The same procedure was followed as in Experimental Example 10.

[0100] 2. Experimental Grouping

[0101] [Table 2]

[0102] 3. Experimental Method Eighteen 3-week-old female SD rats in good health with shiny fur were selected, and were adaptively raised for 3 days, and randomly divided into blank group, model group, and treatment group. After high-fat feed was administered to the model group and treatment group for 3 weeks, LZ (1 mg / kg / d) was forcibly administered orally for 6 weeks, and HFD (high-fat feed (research diets D12492)) was administered to create a rat PCOS model. After the model evaluation was successful, the treatment group was administered according to the dosage and administration method shown in the table, and during the administration period, the blank group and model group were given an equal amount of physiological saline. From the 10th to the 19th day after treatment, two estrus cycles were measured, and on the 21st day after treatment, samples were taken and efficacy evaluation was performed. The specific measurement and analysis methods were the same as those in Experimental Example 9.

[0103] 4.Results 1.SHBG (sex hormone binding globulin), E2 (estradiol), P (progesterone) in the model group (vs blank group) were significantly decreased, and were significantly improved after SZ-A (100mg / kg) was administered, and SZ-A could also decrease the T (testosterone) expression level (see Figure 10).

[0104] 2. Ovarian conditions The results are shown in Figures 11 and 12. 1) The ovarian tissue of the blank group had a normal structure overall, with follicles at each stage being visible, the structure within the follicles being clear, there being abundant and orderly arranged granular layers, there being no dilated cysts, and the corpus luteum structure being clear. 2) The number of expandable cysts in the model group increased significantly, and the number of corpora lutea decreased. 3) After treatment with SZ-A, the number of dilated cysts was significantly decreased, the number of corpora lutea was significantly increased, and the ovarian pathology was significantly improved.

[0105] 3. Effect of drugs on the estrous cycle in PCOS rats The results are shown in Figure 13. As a result, the estrous cycle of the rats in the blank group was regular, about 4-5 days per cycle, while the rats in the model group had a long metestrus or diestrus period. After treatment with SZ-A, the estrous cycle of the PCOS rats was significantly regulated, the duration of metestrus and diestrus was significantly shortened and tended to normalize, and the estrus state was significantly improved.

[0106] In addition to the above experiments, the experimental scale (18 mice / group) was expanded for the LZ+HFD-induced PCOS model without changing the modeling and administration methods, and positive drug Daine-35 and metformin (Met.) groups, as well as low-, medium-, and high-dose groups of SZ-A were designed. The specific group divisions and administration are shown in Table 3.

[0107] [Table 3]

[0108] On the 10th day of treatment, the estrus state of the rats was monitored for 10 days (Figure 14). The results showed that the estrus cycle of the rats in the blank group was regular, about 4-5 days / cycle (Figure 14 A-blank), of which proestrus (P) was about 17%, estrus (E) was about 24%, and metestrus / dysestrus (M / D) was about 59% (Figure B-blank). In contrast, the estrus cycle of the rats in the model group was irregular, and they were almost in metestrus / dysestrus for a long period (97%) (Figure A / B-blank). After drug treatment, the duration of metestrus / dysestrus was shortened in each treatment group compared to the model group, and regular estrus cycles were observed in the late treatment period. The efficacy of SZ-A gradually increased with increasing dose, and SZ-AL showed an improvement effect equivalent to that of the positive drug Diane-35 (rate of diestrus / metaestrus: 79% vs. 80%). The efficacy of SZ-AH was significantly superior to that of Diane-35 (rate of diestrus / metaestrus: 72% vs. 80%), but slightly superior to that of Met. (rate of diestrus / metaestrus: 72% vs. 74%).

[0109] Experimental Example 12: Pharmacodynamic study of mulberry extract on DHEA-induced PCOS model 1. Drug Preparation DHEA (dehydroepiandrosterone): 30 mg / mL. 30 mg of DHEA was dissolved in 1 mL of soybean oil, placed on a magnetic stirrer, and stirred at 50°C and 500 r / min until the DHEA was completely dissolved. 1% CMC: 1 g of CMC was weighed out and added to 100 mL of pure water, then placed on a magnetic stirrer and stirred at 60°C and 500 r / min until the CMC was completely dissolved and the solution became transparent. The mulberry extract SZ-A prepared in Example 1 was prepared to different concentrations and dissolved in 1% CMC solution.

[0110] 2. Experimental Grouping

[0111] [Table 4]

[0112] 3. Experimental Method Eighteen 3-week-old female SD rats in good health with shiny fur were selected, and were adaptively reared for 3 days. They were then randomly divided into blank group, model group, and treatment group, with 6 rats in each group. The rats in the blank group were fed with basal chow and subcutaneously injected with the solvent (60 mg / kg / d), while the rats in the model group and treatment group were fed with normal chow and subcutaneously injected with DHEA oil solution (60 mg / kg / d) for 42 days to create a rat PCOS model. After successful modeling, the rats in the blank group continued to be fed with basal chow and were force-fed with saline (100 mg / kg / d), while the rats in the model group and treatment group continued to be fed with basal chow and were force-fed with saline (100 mg / kg / d), and the treatment group was force-fed with SZ-A (100 mg / kg / d). After 21 days of treatment, samples were taken and efficacy evaluation was performed.

[0113] 4.Results 1. Sex hormone index In rats of the model group (vs. blank group), both T and E2 levels were significantly increased, and administration of SZ-A (100 mg / kg) was able to significantly improve T and E2 expression (the results are shown in FIG. 15).

[0114] 2. Ovarian conditions The results are shown in Figures 16 to 17. As can be seen from Figures 16 to 17, in the blank group, follicles at each stage were observed, the structure within the follicles was clear, the granular layer was abundant and arranged in an orderly manner, and the corpus luteum structure was clear. In the model group, the number of expandable cysts significantly increased and the number of corpora lutea decreased. After administration of SZ-A, the number of expandable cysts significantly decreased and the ovarian condition was significantly improved.

[0115] Experimental Example 13: Pharmacodynamics study of mulberry extract on DHEA+HFD-induced PCOS model 1. Drug Preparation DHEA (dehydroepiandrosterone): 30 mg / mL. 30 mg of DHEA was dissolved in 1 mL of soybean oil, placed on a magnetic stirrer, and stirred at 50°C and 500 r / min until the DHEA was completely dissolved. 1% CMC: 1 g of CMC was weighed out and added to 100 mL of pure water, then placed on a magnetic stirrer and stirred at 60°C and 500 r / min until the CMC was completely dissolved and the solution became transparent. The mulberry extract SZ-A prepared in Example 1 was prepared to different concentrations and dissolved in 1% CMC solution.

[0116] 2. Experimental Grouping

[0117] [Table 5]

[0118] 21-day-old weaned female SD rats in good health with shiny fur were selected and were adaptively reared for 3 days, then randomly divided into blank, model, SZ-AL and SZ-AM groups, with 6 rats per group. The rats in the blank group were fed basal chow and subcutaneously injected with the solvent (60 mg / kg / d), while the rats in the model and treatment groups were fed HFD (high fat chow (research diets D12492)) and subcutaneously injected with DHEA oil solution (60 mg / kg / d), and modeling was continuously induced for 7 weeks to create a rat PCOS model.

[0119] After the model evaluation was successful, the rats in the blank group were fed with basal feed and were forcibly administered saline (100mg / kg / d), the rats in the model and treatment groups were fed with high-fat feed and were forcibly administered saline (100mg / kg / d), the rats in the model group were forcibly administered SZ-A (50mg / kg / d), and the rats in the SZ-AM group were forcibly administered SZ-A (100mg / kg / d). After 21 days of treatment, samples were taken and the efficacy was evaluated using the same measurement and analysis methods as in Experimental Example 9.

[0120] 4.Results 1) In the model group (vs. blank group), T, AMH and GnRH levels increased significantly, and T and AMH expression was significantly decreased by administration of SZ-A (50, 100mg / kg). GnRH expression was also significantly decreased by administration of SZ-A (100mg / kg), and a tendency for GnRH expression to decrease was observed by administration of SZ-A (50mg / kg). Detailed results are shown in Figure 18. 2) In the model group (vs. blank group), SHBG levels were significantly decreased, and SHBG expression was significantly increased by administration of SZ-A (50, 100 mg / kg). Specific results are shown in Figure 18.

[0121] The above-mentioned efficacy evaluation results demonstrated that SZ-A exhibits a certain therapeutic effect against PCOS. [Industrial Applicability]

[0122] In the present invention, the pharmacological test proves that SZ-A can reduce the ovarian weight and improve the ovarian pathology of PCOS rats, the SZ-A administration group can regulate the estrous cycle of PCOS rats in the intervention process, and the SZ-A drug can significantly regulate the secretion level of sex hormones in PCOS rats. From the above results, it can be found that SZ-A can be developed as a product for treating and / or improving polycystic ovary syndrome.

Claims

1. 1. Use of a mulberry extract or a main active ingredient thereof, which is any one of the following uses (a1) to (a4): (a1) use of a mulberry extract in the manufacture of a product for treating polycystic ovary syndrome; (a2) use of a mulberry extract in the manufacture of a product for preventing polycystic ovary syndrome; (a3) the use of a mulberry extract in the treatment of polycystic ovary syndrome; (a4) Use of mulberry extract in the prevention of polycystic ovary syndrome.

2. The treatment and / or amelioration of polycystic ovary syndrome includes: 1) Regulating the secretion levels of sex hormones in patients with polycystic ovary syndrome; 2) Regulating the menstrual cycle in patients with polycystic ovary syndrome; 3) Improve the polycystic state of the ovaries in patients with polycystic ovary syndrome 2. The use according to claim 1, characterized in that it is represented by at least one of the following:

3. Use of a mulberry extract or its main active ingredients in at least one of the following (b1) to (b6): (b1) producing a product for regulating the secretion levels of sex hormones in patients with polycystic ovary syndrome; (b2) manufacturing a product for regulating the menstrual cycle in patients with polycystic ovary syndrome; (b3) producing a product that improves the polycystic ovarian condition of a patient with polycystic ovary syndrome; (b4) regulating the secretion levels of sex hormones in patients with polycystic ovary syndrome; (b5) regulating the menstrual cycle in patients with polycystic ovary syndrome; (b6) Improving the polycystic ovarian condition in patients with polycystic ovary syndrome.

4. 4. The use according to claim 2 or 3, characterized in that the sex hormone is selected from at least one of T (testosterone), E2 (estradiol), LH (luteinizing hormone), PRL (prolactin), P (progesterone), SHBG (sex hormone binding globulin) and AMH (anti-Mullerian hormone).

5. the product is a drug or drug formulation; The use according to any one of claims 1 to 4, characterized in that the mulberry extract is an extract of mulberry branches, an extract of mulberry bark and / or an extract of mulberry leaves.

6. The method for producing the mulberry extract comprises: 1) producing a crude extract of a mulberry plant; 2) separating the crude extract on a cationic resin and / or, if necessary, an anionic resin to obtain the mulberry extract.

7. The method further comprises: 3) subjecting the effluent separated by the resin in step 2) to alcohol precipitation and collecting the supernatant; 4) subjecting the supernatant to a concentration and drying treatment; Alternatively, the use according to claim 6, further comprising a step of concentrating and drying the effluent separated by the resin in step 2).

8. The use according to any one of claims 1 to 7, characterized in that the main active ingredients of the mulberry extract are at least one of 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin, fagomine, 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol, calystegine B2, calystegine C1, 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.

9. Use according to any one of claims 1 to 8, characterized in that the mulberry extract acts on humans or mammals.

10. A method for preventing and / or treating polycystic ovary syndrome, comprising administering to an animal or human subject a mulberry extract or its main active ingredients according to any one of claims 1 to 8, thereby preventing and / or treating polycystic ovary syndrome.

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