Formulations for the treatment and / or prevention of polycystic ovary syndrome and methods for producing the same

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 belongs to the pharmaceutical field, and specifically relates to the use of total alkaloids from mulberry twigs in the manufacture of therapeutic agents for polycystic ovary syndrome (PCOS).

Background Art

[0002] Polycystic ovarian syndrome (PCOS) is one of the endocrine disorders commonly seen in reproductive-age women. Clinically, hyperandrogenism, oligovulation or anovulation, and changes in the morphological form of the ovaries into polycystic occur. PCOS causes abnormal secretion of the body's sex hormones due to various factors. As a result, the growth of follicles stops halfway, the follicle walls are over-formed, and they remain in the ovaries and become cysts, which not only increases the incidence of gynecological diseases such as infertility and cervical cancer, but also causes various mental disorders such as depression. Also, according to research, if a pregnant woman has PCOS, not only does the risk of related pregnancy complications increase, but epigenetics also increases the risk of the offspring suffering from various "mental disorders" and metabolic diseases such as PCOS. According to statistics, the risk of a daughter of a PCOS pregnant woman suffering from PCOS has increased by as much as five times. PCOS is a reproductive endocrine disorder caused by various factors, with high heterogeneity and large differences in pathophysiological characteristics among patients. Currently, the medical treatment of polycystic ovarian syndrome still remains at the stage of treating its clinical symptoms, such as taking oral contraceptives to regulate the menstrual cycle, using ethinyl estradiol cyproterone tablets, etc. to reduce androgen levels, and inducing ovulation treatment with clomiphene citrate (CC) or letrozole for patients who desire pregnancy. However, while clinical treatment has limitations in effectiveness, taking contraceptives is likely to cause mental disorders such as fluid retention, abnormal liver function, and depression, and in the case of ovulation induction treatment with clomiphene citrate or clomiphene citrate (CC), ovarian hyperstimulation syndrome (OHSS) is likely to occur, accompanied by many side effects. As for surgical treatment, currently, laparoscopic ovarian drilling and ovarian wedge resection are common. However, laparoscopic ovarian drilling may not achieve a treatment effect, may cause pelvic adhesions, or may reduce ovarian function, and ovarian wedge resection has disadvantages such as a large surgical wound and adhesion of the tissues around the ovaries after the operation, so it is not commonly performed clinically. The treatment of PCOS by these treatment methods is not ideal, with severe side effects and only being able to relieve some symptoms.Therefore, how to research and develop safe and effective PCOS treatment drugs to reduce the risk to life safety and improve reproductive ability has become a major scientific issue that needs to be urgently solved globally.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The object of the present invention is to provide a new pharmaceutical use of mulberry extract or its main active ingredient.

Means for Solving the Problems

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

[0005] The treatment and / or improvement of the polycystic ovary syndrome is represented by at least one of the following. 1) Adjusting the secretion level of sex hormones in patients with polycystic ovary syndrome, 2) Regularizing the menstrual cycle of patients with polycystic ovary syndrome, 3) Improving the polycystic state of the ovaries in patients with polycystic ovaries.

[0006] Among them, 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-Müllerian hormone).

[0007] The present invention also relates to the use of a mulberry extract or its main active ingredient in at least one of the following (b1) to (b6). (b1) Manufacturing a product for regulating the secretion level of sex hormones in patients with polycystic ovary syndrome, (b2) Manufacturing a product for regularizing the menstrual cycle in patients with polycystic ovary syndrome, (b3) Manufacturing a product for improving the polycystic state of the ovaries in patients with polycystic ovaries, (b4) Regulating the secretion level of sex hormones in patients with polycystic ovary syndrome, (b5) Regularizing the menstrual cycle in patients with polycystic ovary syndrome, (b6) Improving the polycystic state of the ovaries in patients with polycystic ovaries.

[0008] The product is a drug or a pharmaceutical preparation.

[0009] The mulberry extract is an extract of mulberry twigs, an extract of mulberry root bark, and / or an extract of mulberry leaves. Alternatively, the mulberry extract can also be provided as a commercially available total alkaloid tablet of mulberry twigs (National Medicine Approval Code Z20200002).

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

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

[0012] The mulberry extract can be manufactured with reference to the method described in CN 113143997 A. The specific manufacturing method is 1) The step of manufacturing a crude extract of Moraceae plants, and 2) Separating the crude extract with a cation resin and / or, if necessary, an anion resin to obtain the mulberry extract.

[0013] The method further comprises 3) performing an alcohol precipitation treatment on the effluent separated by the resin in step 2) and collecting the supernatant; 4) performing a concentration and drying treatment on the supernatant. It may include these steps.

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

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

[0016] The Moraceae plant can be selected from Morus cathayana, Morus australis, Morus atropurpurea, Morus wittiorum, Morus mongolica or hybrid mulberry. The hybrid mulberry is preferably Morus atropurpurea cv. Guangsang 11, Morus atropurpurea cv. Guisangyou 62 or Morus atropurpurea cv. Sangteyou 2. Various parts of the plant such as leaves, roots, branches, barks, buds, stems, fruits can be used.

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

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

[0019] Preferably, the alkaloid includes at least one of 1-deoxynojirimycin (1-deoxynojirimycin or DNJ), N-methyl-1-deoxynojirimycin (N-methly-1-deoxynojirimycin), fagomine (fagomine or FAG), 3-epi-fagomine (3-epi-fagomine), 1,4-dideoxy-1,4-imino-D-arabinitol (1,4-dideoxy-1,4-imino-D-arabinitol or DAB), calystegin B2 (calysteginB2), calystegin C1 (calysteginC1), 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin (2-O-(α-D-Galactopyranosyl)-1-deoxynojirimycin), 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin (6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin), 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol (1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol).

[0020] Among them, the weight percentage of DNJ is 30% or more of the total alkaloid. Preferably, the weight percentage of DNJ is 40% or more of the total alkaloid. More preferably, the weight percentage of DNJ is 50% or more of the total alkaloid.

[0021] Preferably, for the mulberry extract, the weight content of each component is as follows. Alkaloid 3 - 99% Polysaccharide 0.2 - 70% Flavonoid 0 - 10% Amino acid 0 - 50% Other components 0 - 25%

[0022] Preferably, for the mulberry extract, the weight content of each component is as follows. Alkaloid 30 - 99% Polysaccharides 0.2 - 35% Flavonoid 0 - 2% Amino acid 0 - 30% Other components 0 - 20%

[0023] More preferably, for the extract of the mulberry, the weight content of each component is as follows. Alkaloid 50 - 99% Polysaccharides 0.2 - 35% Flavonoid 0 - 2% Amino acid 0 - 30% Other components 0 - 20%

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

[0025] Even more preferably, for the extract of the mulberry, the weight content of each component is as follows. Alkaloid 50 - 99% Polysaccharides 0.2 - 25% Flavonoid 0 - 1% Amino acid 0 - 20% Other components 0 - 20%

[0026] In one embodiment, the production of the extract of the mulberry includes the steps of producing a crude extract, separating it with a cation resin and / or an anion resin if necessary, performing an alcohol precipitation treatment on the effluent separated by the resin if necessary, and performing a concentration and drying treatment step if necessary. Preferably, the production of the extract of the mulberry includes step 1) of producing a crude extract, step 2) of separating it with a cation resin and / or an anion resin if necessary, step 3) of performing an alcohol precipitation treatment on the effluent separated by the resin in step 2) if necessary, and step 4) of performing a concentration and drying treatment step if necessary.

[0027] In one embodiment, the mulberry extract is produced according to the following procedure. Branches, leaves or white mulberry bark of mulberry are pulverized, and extracted by heating under reflux with water and / or an alcohol solution or acidic water, with the amount of solvent being 3 to 20 times that of the crude drug material. The extraction is repeated 1 to 3 times, the extraction liquids are combined, concentrated, poured into a cation exchange resin, the impurities not adsorbed are washed off with distilled water, eluted with 0.2 to 3N ammonia water, the eluate is concentrated, poured into an anion exchange resin, the non-adsorbed portion is collected, ethanol is added, precipitated to remove impurities, centrifuged, 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 produced according to the following procedure. Branches, leaves or white mulberry bark of mulberry are pulverized, and extracted by heating under reflux with water and / or an alcohol solution or acidic water, with the amount of solvent being 3 to 20 times that of the crude drug material. The extraction is repeated 1 to 3 times, the extraction liquids are combined, concentrated, poured into a cation exchange resin, the impurities not adsorbed are washed off with distilled water, eluted with 0.2 to 3N ammonia water, the eluate is concentrated, poured into an anion exchange resin, the non-adsorbed portion is collected, concentrated under reduced pressure, or spray-dried, or freeze-dried to obtain the extract.

[0029] In one embodiment, the mulberry extract is produced according to the following procedure. Branches, leaves or white mulberry bark of mulberry are pulverized, and extracted by heating under reflux with water and / or an alcohol solution or acidic water, with the amount of solvent being 3 to 20 times that of the crude drug material. The extraction is repeated 1 to 3 times, the extraction liquids are combined, concentrated, poured into a cation exchange resin, the impurities not adsorbed are washed off with distilled water, eluted with 0.2 to 3N ammonia water, 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 produced according to the following procedure. The branches, leaves or white mulberry bark of mulberry are crushed, heated under reflux with water for extraction, with the solvent amount being 3 to 20 times (preferably 4 to 15 times) that of the crude drug material, and the extraction is repeated 1 to 3 times (the extraction time is preferably 0.5 to 3 h / time). The extraction solutions are combined, concentrated, poured into a cation exchange resin, and the impurities not adsorbed are washed off with distilled water, eluted with 0.2 to 3 N aqueous ammonia, the eluate is concentrated, poured into an anion exchange resin, and the unadsorbed part is collected, ethanol is added, precipitation is carried out to remove impurities, centrifugation is performed, and the supernatant is concentrated under reduced pressure, or spray-dried, or freeze-dried to obtain the extract.

[0031] Preferably, after filling the cation resin into the column, it is activated in the order of passing an acidic solution, passing a basic solution, and passing an acidic solution. Preferably, the basic solution is passed until the pH of the eluate is 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 is 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, a sodium hydrogen phosphate-citric acid buffer solution. If necessary, after passing the acidic solution for the last time, the cation resin may be further washed with 3 to 5 times the column volume of deionized water. Preferably, the cation resin is a 732-type strongly acidic styrene-based cation exchange resin, a 734-type strongly acidic styrene-based cation exchange resin and a D001-type macroporous strongly acidic styrene-based cation exchange resin.

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

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

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

[0035] Preferably, the drug further contains a pharmaceutically acceptable carrier. The carrier is an inert component suitable for the administration route or administration method and having no toxic effect on the human body. The carrier may be a solid excipient or a liquid excipient. Examples of the solid excipient 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 the liquid excipient include water, ethanol, syrup, and glycerin.

[0036] Preferably, the drug is in an oral dosage form, and more preferably, the drug is 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, which includes a step of preventing and / or treating polycystic ovary syndrome by administering the mulberry extract or its main active ingredient to a target animal or human.

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

Advantages of the Invention

[0039] PCOS is a metabolic disease caused by multiple factors. Chinese herbal medicines have multiple components and can exert medicinal effects on multiple targets, and have advantages such as few side effects and high safety.

[0040] The present invention has proven the following through pharmacological tests. 1. SZ-A can reduce the ovarian weight of PCOS rats and improve the ovarian pathological conditions. 2. The SZ-A administration group can regulate the estrus cycle of PCOS rats during the intervention process. 3. The SZ-A drug can significantly regulate the secretion level of sex hormones in PCOS rats.

[0041] To more clearly explain the specific embodiments of the present invention or the technical aspects in the prior art, the following briefly introduces the drawings that need to be used in the description of the specific embodiments or the prior art. The drawings in the following description are some embodiments of the present invention, and it will be obvious to those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0043] Hereinafter, the present invention will be described in more detail with reference to examples. These exemplary explanations will make the features and advantages of the present invention clearer. However, the present invention is not limited to the following examples. Unless otherwise specified, all the above methods are conventional methods. Unless otherwise specified, the above raw materials are available from publicly disclosed commercial channels.

[0044] The term "exemplary" here means "used as an example, embodiment, or explanatory". Any embodiment described as "exemplary" here should not be construed as being superior to other embodiments.

[0045] In addition, the configurations in each embodiment of the present invention described below can be combined as long as they do not conflict with each other.

[0046] The component content according to the present invention is measured by a publicly disclosed method (see the methods described in the 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 macroporous weakly acidic allylbenzene cation resin was packed into a column, and a 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5. Then, a 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5. After that, a 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and further washed with 5 times the column volume of deionized water to activate it. The concentrated extract was injected, and 1000 L of 2.5 mol / L aqueous ammonia was used to elute at an elution rate of 6 BV / h. When the pH of the effluent from the cation column was detected to be >7, the eluate was collected. When the collected liquid reached 900 L, the collection was stopped, and the collected liquid was directly purified with an anion column.

[0052] 62.5 kg of D218 macroporous strongly basic acrylic anion resin was packed into a column, and a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0. Then, a 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5. After that, a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0 to activate it. The eluate of the collected cation resin was injected into the anion resin, and the effluent was collected until the effluent reached 870 L.

[0053] After centrifuging the collected liquid to remove impurities, concentration was carried out with a reverse ion osmosis membrane until the specific gravity of the concentrated liquid reached 1.25. This was transferred into 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, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was concentrated under reduced pressure to obtain the extract extract.

[0054] The effluent was concentrated under reduced pressure to obtain the extract extract of mulberry branches. Among them, the mass percentage content of alkaloids was 52% (in 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: Production of Mulberry Extract 2 After pulverizing 10 kg of fresh mulberry branches (Mulberry Super Elite No. 2), 150 L of water was added in two portions, and each portion was extracted by decoction for 3 h. The extracts were combined and filtered to remove insoluble substances. The extract was concentrated by heating until the mass percentage content of the solid matter reached 8%, and then transferred into an alcohol precipitation tank. Under the condition of a stirring blade at 300 rpm, 2367.9 g (3 L) of absolute ethanol was added. After the addition of ethanol was completed, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was used as the injection liquid into the cation resin column. 5 kg of 002SC type strongly acidic styrene-based cation resin was packed into the column, and the cation resin was activated according to the method of Example 1. The concentrated and alcohol-precipitated extract was injected, and then eluted at an elution rate of 5 BV / h using 100 L of 5 mol / L potassium chloride. The effluent was measured with 20% silicotungstic acid. When a white precipitate occurred, collection was started, and when the collected liquid reached 25 L, collection was stopped. The collected liquid was directly purified with an anion column as it was.

[0056] 10 kg of 711 type strongly basic styrene-based anion resin was packed into the column, and the anion resin was activated according to the method of Example 1. The eluate of the collected cation resin was injected into the anion resin, and the effluent was collected. When the effluent reached 15 L, collection was stopped. The collected liquid was injected into the cation resin again, and separated twice again in the order of cation resin and anion resin according to the above method.

[0057] The collected liquid obtained after column separation was centrifuged to remove impurities, and then concentrated by reverse ion osmosis membrane. After concentration, the specific gravity of the liquid became 1.25. This was transferred into an alcohol precipitation tank, and 125 g of absolute ethanol was added under the condition of a stirring blade at 1000 rpm. After the addition of ethanol was completed, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was concentrated under reduced pressure to obtain the extract. Also, extraction of fresh mulberry bark and mulberry leaves (Mulberry Super Elite No. 2) was carried out under the same extraction method and conditions as the above method.

[0058] The obtained extract of mulberry twigs 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] In the obtained extract of mulberry root bark, the alkaloid mass percentage content was 95%, the polysaccharide mass percentage content was 2%, the flavonoid mass percentage content was 0.1%, and the amino acid mass percentage content was 1%.

[0060] In the obtained extract of mulberry leaves, the alkaloid mass percentage content was 90%, the polysaccharide mass percentage content was 4%, the flavonoid mass percentage content was 0.1%, and the amino acid mass percentage content was 3%.

[0061] Example 3 Preparation of Mulberry Extract 3 After crushing 1000 kg of fresh mulberry twigs (Canton Kwai), 11500 L of water was added, and extraction was carried out by heating under reflux for 2 h. The extraction liquids were combined, filtered to remove insoluble substances, and a crude extract was obtained. After centrifuging the crude extract to remove impurities, it was concentrated by reverse ion osmosis membrane until the mass percentage content of the solid matter reached 1%, and used as the injection liquid for the cation resin column.

[0062] 300 kg of D001 type macroporous strongly acidic styrene-based cation resin was filled into the column, and the cation resin was activated according to the method of Production Example 1. The concentrated crude extract was injected, and elution was carried out at an elution rate of 5 BV / h using 5000 L of 0.04 mol / L ammonium nitrate. The effluent was measured with 20% silicotungstic acid, and collection was started when a white precipitate occurred, and collection was stopped when the collected liquid reached 1000 L.

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

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

[0065] Example 4 Preparation of Mulberry Extract 4 After pulverizing 333 kg of dried mulberry branches (Mulberry Variety Yue Sang 11), 4000 L of water was added, and extraction was carried out in two portions by the method of heating under reflux, with each extraction for 1 h. The extraction liquids were combined, filtered, and the extraction liquid was concentrated to 1 kg of crude drug amount / L.

[0066] 150 kg of D113 type macroporous weakly acidic allylbenzene cation resin was packed into a column, and a 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, then a 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, then a 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and further washed with 5 times the column volume of deionized water to activate it. The concentrated extraction liquid was injected, and elution was carried out at an elution rate of 6 BV / h using 1000 L of 2.5 mol / L aqueous ammonia. When it was detected that the pH of the effluent from the cation column > 7, the eluate was collected. When the collected liquid reached 900 L, the collection was stopped, and the collected liquid was directly purified with an anion column.

[0067] 125 kg of D218 type macroporous strongly basic acrylic anion resin was packed into a column, and a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, then a 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, then a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0 to activate it. The eluate of the collected cation resin was injected into the anion resin, and the effluent with a pH higher than 8 was collected until the effluent reached 870 L.

[0068] After filtering the collected solution obtained through separation by an anion column with a precision filtration membrane to remove impurities, it was concentrated by a reverse ion osmosis membrane until the specific gravity of the concentrated liquid reached 1.1. Then, this was transferred into an alcohol precipitation tank, and 15 kg of absolute ethanol was added under the condition of a stirring blade at 400 rpm. After the addition of ethanol was completed, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was concentrated under reduced pressure to obtain an extract of mulberry twigs. The content of the sample was that the mass percentage content of alkaloids was 80%, the mass percentage content of polysaccharides was 5%, the mass percentage content of flavonoids was 0.1%, and the mass percentage content of amino acids was 4%.

[0069] Example 5 Preparation of Mulberry Extract 5 After pulverizing 400 kg of dried mulberry twigs (Yuesang No. 11), 4000 L of water was added, and extraction was carried out in two portions by the heating reflux method, with each extraction for 1 h. The extraction solutions were combined, filtered, and the extraction solution was concentrated to 1 kg of crude drug amount / L.

[0070] 62.5 kg of D218 type macroporous strongly basic acrylic anion resin was filled into a column, and a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0. Then, a 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, and then a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0 for activation. The collected extracted and concentrated solution was injected into the anion resin, and the effluent was collected.

[0071] After filtering the collected solution obtained through separation by an anion column with a precision filtration membrane to remove impurities, it was concentrated by a reverse ion osmosis membrane, and further concentrated under reduced pressure and dried to obtain an extract of mulberry twigs. The content of the sample was that the mass percentage content of alkaloids was 3%, the mass percentage content of polysaccharides was 70%, the mass percentage content of flavonoids was 10%, and the mass percentage content of amino acids was 10%.

[0072] Example 6 Preparation of Mulberry Extract 6 After crushing 1500 kg of fresh mulberry branches (Morus atropurpurea Roxb. cv. Yuesang 11), 6000 L of water was added, and extraction was carried out by the heating reflux method for 2 h. The extraction liquids were combined, filtered to remove insoluble substances, and a crude extraction liquid was obtained. The crude extraction liquid was heat-concentrated until the mass percentage content of the solid substance reached 4%, and was kept at 50 °C as the injection liquid into the cation resin column.

[0073] A column was filled with 100 kg of D113 macroporous weakly acidic allylbenzene cation resin. A 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, a 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, a 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and then it was activated by washing with deionized water five times the column volume. The concentrated extraction liquid was injected, and elution was carried out at an elution rate of 6 BV / h using 1000 L of 2.5 mol / L aqueous ammonia. When it was detected that the pH of the effluent from the cation column > 7, the eluate was collected. When the collected liquid reached 900 L, the collection was stopped, and the collected liquid was directly purified by an anion column.

[0074] A column was filled with 62.5 kg of D218 macroporous strongly basic acrylic anion resin. A 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, a 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, and it was activated. The eluate of the collected cation resin was injected into the anion 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. Among them, the mass percentage content of alkaloids was 30%, the mass percentage content of polysaccharides was 35%, the mass percentage content of flavonoids was 2%, and the mass percentage content of amino acids was 25%.

[0075] Example 7 Preparation of Mulberry Extract 7 After crushing 1000 kg of fresh mulberry branches (Morus atropurpurea Roxb. cv. Yuesang 11), 4000 L of water was added, and extraction was carried out by heating under reflux for 2 h. The extraction liquids were combined, filtered to remove insoluble substances, and a crude extract was obtained. The crude extract was concentrated by heating until the mass percentage content of solids reached 4%, and was kept at 50 °C as the injection liquid for the cation resin column.

[0076] A column was filled with 100 kg of D113 macroporous weakly acidic allylbenzene cation resin. A 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, then a 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, then a 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and it was further washed with 5 times the column volume of deionized water to activate it. The concentrated extract was injected, and elution was carried out at an elution rate of 6 BV / h using 1000 L of 2.5 mol / L aqueous ammonia. When it was detected that the pH of the effluent from the cation column > 7, the eluate was collected. When the collected liquid reached 900 L, the collection was stopped, and the collected liquid was directly purified with an anion column.

[0077] A column was filled with 62.5 kg of D218 macroporous strongly basic acrylic anion resin. A 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, then a 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, then a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0 to activate it. The eluate of the collected cation resin was injected into the anion 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. Among them, the mass percentage content of alkaloids was 40%, the mass percentage content of polysaccharides was 25%, the mass percentage content of flavonoids was 0.5%, and the mass percentage content of amino acids was 25%.

[0078] Example 8 Preparation of Mulberry Extract 8 After drying and pulverizing 333 kg of mulberry branches (Yuesang 11), 4000 L of water was added, and extraction was carried out in two portions by the method of heating under reflux, with each extraction lasting 1 h. The extraction solutions were combined, filtered, and the extraction solution was concentrated to 1 kg of crude drug amount / L.

[0079] 150 kg of D113 macroporous weakly acidic allylbenzene cation resin was packed into a column, and a 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, then a 1 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 8.5, then a 2 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 4.5, and further washed with 5 times the column volume of deionized water to activate it. The concentrated extraction solution was injected, and then eluted at an elution rate of 6 BV / h using 1000 L of 2.5 mol / L aqueous ammonia. When it was detected that the pH of the effluent from the cation column was >7, the eluate was collected. When the collected solution reached 900 L, the collection was stopped, and the collected solution was directly purified with an anion column.

[0080] 62.5 kg of D218 macroporous strongly basic acrylic anion resin was packed into a column, and a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0, then a 1.5 mol / L hydrochloric acid solution was passed through until the pH of the eluate reached 3.5, then a 1.5 mol / L sodium hydroxide solution was passed through until the pH of the eluate reached 9.0 to activate it. The eluate of the collected cation resin was injected into the anion resin, and the effluent with a pH higher than 8 was collected until the effluent reached 870 L.

[0081] After filtering the collected solution obtained through separation by an anion column with a precision filtration membrane to remove impurities, it was concentrated with an anion permeation membrane until the specific gravity of the concentrated liquid reached 1.1. This was then transferred into an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added under the condition of a stirring blade speed of 400 rpm. After the addition of ethanol was completed, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was concentrated under reduced pressure to obtain an extract of mulberry twigs. The content of the sample was that the mass percentage content of alkaloids was 63%, the mass percentage content of polysaccharides was 23%, the mass percentage content of flavonoids was 1%, and the mass percentage content of amino acids was 5%.

[0082] II. Verification of the effects of the mulberry extract

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

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

[0085] 1.3 Modeling and administration An equal amount of oil was subcutaneously injected into the back of the neck of the blank group (3 rats), and 0.2 mL / 100 g (60 mg / kg) of the DHEA solution was subcutaneously injected into the back of the neck of the model group (6 rats). The number of administrations was once a day, and the injection was carried out for a total of 21 days.

[0086] From the 11th day of modeling, rat vaginal secretions were collected every morning at 8 o'clock, and vaginal smear examinations were performed on the rats for two cycles (about 10 days) to monitor the estrous cycle. On the 21st day of modeling, the testosterone (T) was measured to evaluate the model. After 21 days of modeling, disturbances in the estrous cycle and high androgen were observed in the model group, indicating the success of PCOS modeling.

[0087] After the success of modeling, the rats were randomly divided into a blank group, a model group, and a Morus extract SZ-A administration group (100 mg / kg / d based on the total alkaloids of Morus twigs). During the administration period, an equal amount of physiological saline was given to the blank group and the model group. 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 the mental state of the animals were measured. (2) Vaginal smear (measured for one cycle. Usually, rats have an estrous cycle of 5 days, and the measurement was started from the 15th to 20th day after administration). (3) Expression of related hormones in serum: On the 21st day after administration, T, E2, LH, FSH, P, PRL, GnRH, SHBG, and AMH were measured. (4) After 21 days of administration, the rats were sacrificed to collect ovarian tissues, and HE staining was performed. (5) The body weight and ovarian weight were observed. Ovarian weight: After aspirating the peripheral blood of the ovarian tissue with filter paper, it was weighed and statistically analyzed. (6) Statistical analysis: Analyzed using SPSS software.

[0089] 2. Measurement Results (1) Effect of the drug on the body weight of rats 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 after intervention by administration, the body weight of the model rats decreased significantly. It was shown that the drug could significantly adjust the body weight of PCOS rats.

[0090] (2) Effect of the drug on the estrous cycle of PCOS rats As a result of vaginal smears, rats in the normal control group showed regular estrous cycles in the order of proestrus, estrus, metestrus, and diestrus, while the estrous cycles of the PCOS model group showed irregular changes. In the administration group, the changes in estrous cycles were improved during the intervention process.

[0091] (3) Effect of drugs on the ovaries of PCOS rats The morphology of the ovaries was observed, and the results are shown in Figure 1(A). When observing the external morphology of the ovarian tissues of rats in each experimental group, the ovarian surface of rats in the blank group was bright red, and no obviously enlarged follicles were seen. The ovarian surface of the model group was bluish-white, and multiple enlarged follicles of different sizes were seen. In the administration group, the ovarian surface had good blood color and luster, and the bluish-white symptom of the ovaries was significantly improved compared with the model group.

[0092] The results of the stained sections of ovarian tissues are shown in Figure 1(B). The ovarian tissues of rats in the normal control group had a normal structure as a whole under an optical microscope. Follicles at different developmental stages were seen under the microscope. Oocytes and corona radiata cells were seen in follicles close to the mature stage. The number of cell layers in the follicular membrane was small, there were no cystic follicles, and the interstitial cells of the ovaries were densely and neatly arranged. The ovarian tissues of rats in the model group showed obvious abnormalities as a whole under an optical microscope, and the change to a polycystic morphology was obvious. No oocytes were seen in follicles close to the mature stage. The granulosa cells were loosely arranged, the number of layers decreased, some were peeled off, no corona radiata cells were seen, and the cell layer of the follicular membrane was thickened. In rats in the drug intervention group (SZ-A), the pathological conditions of the ovaries were significantly improved. Corpora lutea and multiple follicles were seen in all ovaries. Oocytes and corona radiata cells were seen in follicles close to the mature stage. The granulosa cells were densely and neatly arranged, the number of layers increased, and the cell layer of the follicular membrane was thinned.

[0093] (4) Effect of drugs on serum hormone levels in PCOS rats The results are shown in Figures 2 to 8. Compared with the blank group, in the model group, the expressions of T, LH, E2, PRL, and AMH in the serum all increased, while the expressions of SHBG and progesterone P decreased. Compared with the model group, 21 days after administration, in the administration group, the levels of hormones such as T (testosterone), E2 (estradiol), LH (luteinizing hormone), PRL (prolactin), and AMH decreased to varying degrees, and SHBG increased slightly. It was shown that the drug could 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. Preparation of drugs The preparation concentration of letrozole (simply referred to as LZ): 0.25 mg / mL, dosage: 0.4 mL / 100 g. 1% CMC: Weighed 1 g of CMC, added it to 100 mL of pure water, placed it on a magnetic stirrer, and stirred at a temperature of 60 °C and a rotation speed of 500 r / min until CMC was completely dissolved and became transparent. The mulberry extract SZ-A prepared in Example 1 was prepared at different concentrations and dissolved in a 1% CMC solution.

[0095] 2. Grouping of experiments

[0096]

Table 1

[0097] 3. Experimental method Eighteen 6-week-old female SD rats with good health and shiny hair were selected, adaptively bred for 3 days, and divided into a blank group, a model group, and an administration group. The rats in the blank group were given basal diet, while the rats in the model group and the administration group were given basal diet and LZ (1 mg / kg / d) by forced oral administration for 6 weeks to establish 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 the model group were administered an equal amount of physiological saline. Samples were collected 21 days after administration, and pharmacodynamic evaluation was carried out by the same measurement and analysis methods as in Experimental Example 9.

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

[0099] Experimental Example 11: Pharmacodynamic study of mulberry extract on LZ + HFD-induced PCOS model 1. Preparation of drugs The same method as in Experimental Example 10 was used.

[0100] 2. Grouping of experiments

[0101] [Table 2]

[0102] 3. Experimental method Eighteen 3-week-old female SD rats with good health and shiny hair were selected, adaptively bred for 3 days, and randomly divided into a blank group, a model group, and an administration group. After administering a high-fat diet to the model group and the administration group for 3 weeks, LZ (1 mg / kg / d) was forcibly administered orally for 6 weeks, and HFD (high-fat diet (research diets D12492)) was administered to establish a rat PCOS model. After successful model evaluation, the administration group was administered according to the dosage and administration method shown in the table. During the administration period, an equal amount of physiological saline was given to the blank group and the model group. From the 10th to the 19th day after treatment, the estrous cycle of two cycles was measured. On the 21st day after treatment, samples were collected and a drug efficacy evaluation was carried out. 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), and P (progesterone) in the model group (vs the blank group) significantly decreased. After administration of SZ-A (100 mg / kg), it was significantly improved. In addition, SZ-A could reduce the expression level of T (testosterone) (see Figure 10).

[0104] 2. Ovarian conditions The results are shown in Figures 11 - 12. 1) The ovarian tissue of the blank group had a normal structure as a whole, follicles at each stage were observed, the structure within the follicles was clear, the granulosa layer was numerous and arranged neatly, there were no dilated cysts, and the corpus luteum structure was clear. 2) The number of dilated cysts in the model group increased significantly, and the number of corpus luteum decreased. 3) After treatment with SZ - A, the number of dilated cysts decreased significantly, the number of corpus luteum increased significantly, and the ovarian condition improved significantly.

[0105] 3. Effects of drugs on the estrous cycle of 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 / cycle. For the rats in the model group, the late estrus or anestrus period was long. After treatment with SZ - A, the estrous cycle of PCOS rats could be significantly regulated, the periods of late estrus and anestrus were significantly shortened and tended to normalize, and the estrous state was significantly improved.

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

[0107]

Table 3

[0108] On the 10th day of treatment, the estrous state of the rats was monitored for 10 days (Figure 14). As a result, the estrous cycle of the rats in the blank group was regular, about 4 - 5 days / cycle (Figure 14 A - blank), among which, the proestrus (P) was about 17%, the estrus (E) was about 24%, and the late estrus / anestrus (M / D) was about 59% (Figure 14(B - blank). In contrast, the estrous cycles of the rats in the model group were irregular, and they were in the late estrus / estrus diestrus stage for a long period (97%) (Figure 14 (A / B - blank). After treatment with the drug, compared with the model group, the period of late estrus / estrus diestrus in each administration group was shortened, and a regular estrous cycle was observed in the late administration stage. With the increase in the dosage of SZ - A, the drug efficacy gradually became stronger. SZ - A - L showed an improvement effect equivalent to that of the positive drug Diane - 35 (ratio of estrus diestrus / late estrus: 79% vs 80%), and the drug efficacy of SZ - A - H was significantly better than that of Diane - 35 (ratio of estrus diestrus / late estrus: 72% vs 80%), but it was slightly better than that of Met. (ratio of estrus diestrus / late estrus: 72% vs 74%).

[0109] Experimental Example 12: Pharmacodynamic study of the mulberry extract on the DHEA - induced PCOS model 1. Preparation of drugs DHEA (Dehydroepiandrosterone): 30 mg / mL, prepared by dissolving 30 mg of DHEA in 1 mL of soybean oil, placing it on a magnetic stirrer, and stirring at 50 °C and a rotation speed of 500 r / min until DHEA was completely dissolved. 1% CMC: Weighed 1 g of CMC, added it to 100 mL of pure water, placed it on a magnetic stirrer, and stirred at a temperature of 60 °C and a rotation speed of 500 r / min until CMC was completely dissolved and became transparent. The mulberry extract SZ - A produced in Example 1 was prepared at different concentrations and dissolved in a 1% CMC solution.

[0110] 2. Grouping of experiments

[0111]

Table 4

[0112] 3. Experimental method Eighteen 3-week-old female SD rats with good health and shiny hair were selected, adaptively bred for 3 days, and randomly divided into a blank group, a model group, and an administration group, with 6 rats in each group. The rats in the blank group were given basal diet and subcutaneously injected with a solvent (60 mg / kg / d). The rats in the model group and the administration group were given normal diet and subcutaneously injected with a DHEA oil solution (60 mg / kg / d) for 42 days to establish a rat PCOS model. After the success of modeling, the rats in the blank group continued to be given basal diet and were force-fed with physiological saline (100 mg / kg / d). The rats in the model group and the administration group continued to be given basal diet. The rats in the model group were force-fed with physiological saline (100 mg / kg / d), and the rats in the administration group were force-fed with SZ-A (100 mg / kg / d). After 21 days of treatment, samples were collected and the drug efficacy was evaluated.

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

[0114] 2. Ovarian pathological conditions The results are shown in Figures 16 - 17. As can be seen from Figures 16 - 17, in the blank group, follicles at each stage were observed, the structure inside the follicles was clear, the granulosa layer was numerous and arranged neatly, and the corpus luteum structure was clear. In the model group, the number of enlarged cysts was significantly increased, and the number of corpora lutea decreased. After the administration of SZ-A, the number of enlarged cysts was significantly decreased, and the ovarian pathological conditions were significantly improved.

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

[0116] 2. Grouping of experiments

[0117]

Table 5

[0118] Female SD rats that had just been weaned at 21 days of age, were in good health, and had shiny hair were selected. After adaptively breeding for 3 days, they were randomly divided into a blank group, a model group, an SZ-A-L group, and an SZ-A-M group, with 6 rats in each group. The rats in the blank group were given a basal diet and subcutaneously injected with a solvent (60 mg / kg / d). The rats in the model group and the administration groups were given an HFD (high-fat diet (research diets D12492)) and subcutaneously injected with a DHEA oil solution (60 mg / kg / d), and continuously induced modeling for 7 weeks to create a rat PCOS model.

[0119] After successful model evaluation, the rats in the blank group continued to be given a basal diet and forced oral administration of physiological saline (100 mg / kg / d). The rats in the model and administration groups continued to be given a high-fat diet. The rats in the model group were forced oral administration of physiological saline (100 mg / kg / d), the SZ-A-L group was forced oral administration of SZ-A (50 mg / kg / d), and the SZ-A-M group was forced oral administration of SZ-A (100 mg / kg / d). After continuous treatment for 21 days, samples were collected, and the drug efficacy was evaluated by the same measurement and analysis methods as in Experimental Example 9 specifically.

[0120] 4. Results 1) In the model group (vs blank group), the levels of T, AMH, and GnRH were significantly increased. Administration of SZ-A (50, 100 mg / kg) could significantly decrease the expressions of T and AMH. In addition, administration of SZ-A (100 mg / kg) could also significantly decrease the GnRH expression, and a decreasing trend of GnRH expression was observed after administration of SZ-A (50 mg / kg). The detailed results are shown in Figure 18. 2) In the model group (vs blank group), the SHBG level was significantly decreased. Administration of SZ-A (50, 100 mg / kg) significantly increased the SHBG expression. The specific results are shown in Figure 18.

[0121] The measurement results of the above-mentioned efficacy evaluation proved that SZ-A showed a certain therapeutic effect on PCOS.

Industrial Applicability

[0122] In the present invention, through pharmacodynamic tests, it was proved that SZ-A could reduce the ovarian weight of PCOS rats and improve the ovarian pathological conditions. The SZ-A administration group could regulate the estrus cycle of PCOS rats during the intervention process, and the SZ-A drug could significantly regulate the secretion levels of sex hormones in PCOS rats. From the above results, it was found that SZ-A could be developed as a product for treating and / or improving polycystic ovary syndrome.

Claims

1. A preparation for treating and / or preventing polycystic ovary syndrome, comprising a mulberry extract as an active ingredient, wherein the weight content of each component in the mulberry extract is alkaloid 30 - 99%, polysaccharide 0.2 - 35%, flavonoid 0 - 2%, amino acid 0 - 30%, other components 0 - 20%, and the alkaloid contains 1 - deoxynojirimycin, and the weight percentage of the 1 - deoxynojirimycin is 30% or more of the total alkaloid.

2. The treatment and / or prevention of polycystic ovary syndrome 1) regulating the secretion level of sex hormones in patients with polycystic ovary syndrome, 2) regularizing the menstrual cycle of patients with polycystic ovary syndrome, 3) improving the polycystic state of the ovaries in patients with polycystic ovaries is characterized by being represented by at least one of the above, and the preparation according to Claim 1.

3. The sex hormone is selected from at least one of testosterone, estradiol, luteinizing hormone, prolactin, progesterone, sex hormone - binding globulin, and anti - Müllerian hormone, and the preparation according to Claim 2.

4. The mulberry extract acts on humans or mammals, and the preparation according to Claim 1.

5. The mulberry extract is an extract of mulberry branches, mulberry white bark, or mulberry leaves, and the preparation according to Claim 1.

6. A method for manufacturing the preparation according to any one of Claims 1 to 5, comprising 1) a step of manufacturing a crude extract of Moraceae plants, and 2) a step of separating the crude extract with a cation resin and / or, if necessary, an anion resin to obtain the mulberry extract.

7. The method further comprises 3) a step of performing alcohol precipitation treatment on the effluent separated by the resin in step 2) and collecting the supernatant, and 4) a step of performing concentration and drying treatment on the supernatant, and the method for manufacturing the preparation according to Claim 6.

8. The method further comprises a step of performing concentration and drying treatment on the effluent separated by the resin in step 2), and the method for manufacturing the preparation according to Claim 6.

9. The weight content of each component in the mulberry extract is alkaloid 40 - 99%, polysaccharide 0.2 - 35%, flavonoid 0 - 2%, amino acid 0 - 30%, other components 0 - 20%, The preparation according to claim 1, characterized in that it is so.

10. The weight content of each component in the extract of mulberry is alkaloid 50 to 99%, polysaccharide 0.2 to 35%, flavonoid 0 to 2%, amino acid 0 to 30%, other components 0 to 20%, and, or the weight content of each component in the extract of mulberry is alkaloid 50 to 99%, polysaccharide 0.2 to 25%, flavonoid 0 to 1%, amino acid 0 to 20%, other components 0 to 20%, The preparation according to claim 1.

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