阅读说明：本技术 荞麦碱在制备抗卵巢衰老药物中的应用 (Application of buckwheat alkali in preparing anti-ovarian-aging medicine ) 是由 韦敏 宋萍萍 钱怡云 颜露 吕晔 陈海萍 于 2020-01-06 设计创作，主要内容包括：本发明公开了荞麦碱在制备抗卵巢衰老药物中的应用。荞麦碱通过上调c-fos mRNA及其表达产物蛋白水平而治疗多种卵巢衰退疾病,如多囊卵巢综合征、功能失调性子宫出血、卵巢早衰或绝经期综合征等具有显著效果。本发明是将荞麦碱制成用于治疗卵巢衰退疾病的口服给药、经皮给药或注射给药。(The invention discloses application of buckwheat alkaloid in preparing an anti-ovarian-senescence medicament. The fagopyrum esculentum alkali has obvious effect of treating various ovarian failure diseases such as polycystic ovarian syndrome, dysfunctional uterine bleeding, premature ovarian failure or menopausal syndrome and the like by up-regulating the protein level of c-fos mRNA and an expression product thereof. The invention relates to an oral administration, a transdermal administration or an injection administration for treating ovarian failure diseases, which is prepared by using the buckwheat alkali.)

1. Application of buckwheat alkali in preparing medicine for resisting ovarian aging is provided.

2. Use according to claim 1, characterized in that the symptoms of ovarian senescence are polycystic ovarian syndrome, dysfunctional uterine bleeding, premature ovarian failure or menopausal syndrome.

3. Use according to claim 1, characterized in that the use is effected by up-regulating the protein levels of c-fos mRNA and its expression products.

4. The use of claim 1, wherein the medicament comprises 1-1000mg of buckwheat alkaloid and a pharmaceutically acceptable carrier.

5. The use of claim 1, wherein the medicament is in any pharmaceutically acceptable form.

6. The use as claimed in claim 5, wherein the dosage form of the medicament comprises granules, tablets, granules, soft capsules, drop pills, ointments or injections.

Technical Field

The invention relates to a medical application of buckwheat alkali, namely the application of buckwheat alkali in a medicament for effectively treating various ovarian failure diseases.

Background

Ovarian aging is a complex biological process of multifactorial interactions, progressive accumulation. As follicles are continuously consumed in the course of the year, the decrease in the number of follicles is the main cause of ovarian senescence; metabolic products in the organism accumulate microenvironment changes in the ovary, such as damage of free radicals and glycosylation end products to follicles, continuous accumulation of factors such as mitochondrial DNA mutation and telomere shortening, and the quality of the remaining follicles is reduced at the same time; the expression and function of a series of genes in the Ovary are changed, and the secretion of H-P-O Axis (Hypothalamus-Pituitary-Ovary Axis) hormone regulates the function of the Ovary precisely. Ovarian Failure, ovarian senescence, is manifested by menopause, known as Premature Ovarian Failure (POF) before age 40, pathological ovarian senescence. Ovarian dysfunction can cause ovarian dysfunction diseases (such as polycystic ovarian syndrome, dysfunctional uterine bleeding and the like), and then perimenopausal syndrome due to failure, wherein the diseases successively cause aging diseases of each organ system: the incidence of coronary heart disease and cerebrovascular disease in the circulatory system is obviously improved, presenile dementia and depression are easy to occur in the central nervous system, osteoporosis is high due to the rapid increase of calcium loss, and the like. In recent years, with the pace of life increasing and the learning pressure becoming excessive, there has been a tendency toward a marked increase in ovarian failure disease-related diseases occurring in women. Since each family has young, middle-aged or elderly women, the above diseases caused by ovarian aging have become social problems of common concern, and the research on the diseases has become a hotspot and difficulty in the reproductive development world at home and abroad.

At present, at home and abroad researches mostly consider that ovarian aging is related to factors such as heredity, immunity, metabolism, virus infection, iatrogenic factors and the like. The following aspects are summarized: 1 chromosome karyotype abnormality, some syndrome genes are mainly concentrated on X chromosome and autosome through research methods of patients with chromosome abnormality ovarian aging, the abnormality of chromosome karyotype is related to family inheritance, and in addition, gene mutation caused by environmental factors is also a key factor for causing the disease. 2 immune factors, possibly together with cytokines, induce the expression of antigens in granulocytes and luteal cells, mainly in the histocompatibility complex (MHC), which induce an autoimmune reaction in the body, causing granulocytes and ovaThe bubbles are destroyed. In addition, ovarian failure is thought to be associated with the production of ovarian autoantibodies and autoimmune diseases associated with other endocrine glands or systems, such as autoimmune thyroiditis, systemic lupus erythematosus, myasthenia gravis, parathyroid gland failure, rheumatoid arthritis, idiopathic thrombocytopenic purpura, diabetes, and the like. Abnormalities in 3 gonadotropins and their receptors, and deficiencies in transmission of the gonadotropins FSH, LH and their receptors (FSHR, LHR) may cause a decline in ovarian function. Enzyme 4 deficiency can cause galactosemia associated with the development of ovarian failure. Galactose can directly damage oocytes, its metabolites can in turn cause damage to ovarian parenchyma, and the infiltration of galactose molecules can also alter gonadotropin activity, thereby causing ovarian follicular depletion. 5 ovarian failure factors, with the increasing frequency of female social activities, the ovaries can be damaged by the fact that the ovaries are unintentionally exposed to the stimulation of reflecting rays for a long time or the ovaries are subjected to radiation with large dose or long time due to work, diseases and accidents. 6 other related high risk factors, smoking, depression, prolonged exposure to exogenous E in large quantities₂And the puerpera has close relationship with ovarian failure. The research on the influence of heavy metal environmental hormones on ovarian function decline is blank, and with the rapid development of economy in China, the environmental factors can have long-term adverse effects on reproductive health of people in China.

Hormone Replacement Therapy (HRT) is an important method for treating symptoms and diseases related to ovarian aging in modern medicine at present, but the existing research obviously shows that the HRT has multiple side effects and long-term carcinogenicity, so that the clinical application of the HRT is obviously limited. In response to the state, China follows the theory of traditional Chinese medicine, advocates 'tonifying kidney and nourishing yin, soothing liver and nourishing blood' to delay ovarian aging, adopts a traditional Chinese medicine formula for treatment, but under the condition that the etiology and the pathology of the traditional Chinese medicine are not clearly understood at present, the misdistricts of indiscriminate medication and indiscriminate treatment exist, and the compound medicine treatment has the defects of unclear effective substance basis and unknown action mechanism, so that the traditional Chinese medicine cannot be modernized and internationalized.

Buckwheat (Fagomine), which is a polyhydroxypyridine alkaloid, is present in buckwheat seeds, mulberry, wolfberry, chestnut, black bean leaves, broussonetia papyrifera leaves and chicken mulberry leaves, and is a mild glycosidase inhibitor. Buckwheat alkali, molecular formula: C6H13NO3, molecular weight: 147.18, the structure of which is as formula I, there is no report on the application of buckwheat alkali in preparing anti-ovarian-aging medicine.

（Ⅰ）。

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of the buckwheat alkaloid in preparing the anti-ovarian-aging medicine. The buckwheat alkali (Fagomine) has the following structure:

the invention relates to application of Fagomine in resisting ovarian aging diseases, in particular to application of Fagomine in resisting ovarian aging diseases, which has obvious curative effects on diseases such as polycystic ovarian syndrome, dysfunctional uterine bleeding, premature ovarian failure or menopausal syndrome and the like, and has the characteristics of safe use, long-term taking and the like when being used for the application. Therefore, the fagopyrum tataricum can be applied to preparing the anti-ovarian-aging medicine, and the medicine has the application of treating various ovarian failure diseases.

The chromatin co-immunoprecipitation technology is used for exploring the changes of genes and protein levels of the upstream and downstream elements of c-fos before and after the action of the buckwheat alkali under the aging condition, so that the buckwheat alkali can be used for up-regulating the mRNA expression of the c-fos and the protein level of an expression product thereof, which is the mechanism of the medicine for treating various ovarian failure diseases.

The buckwheat alkaloid can be obtained from commercial sources or prepared by the prior art.

The buckwheat alkaloid applied by the invention can be prepared into a medicament with any pharmaceutically allowed auxiliary material or pharmaceutical excipient, and the preparation can be any pharmaceutically allowed dosage form, including but not limited to liquid preparation, granules, tablets, medicinal granules, soft capsules, dripping pills, ointment or injection.

The administration form of the medicament provided by the invention mainly comprises oral administration, transdermal administration or injection administration.

The amount of the buckwheat alkaloid to be administered in the present invention varies depending on the condition, body weight, administration form and the like of the patient. The recommended dose is 0.5-200 mg/kg-d, characterized in that the medicament contains 1-1000mg of buckwheat alkaloid and a pharmaceutically acceptable carrier.

Compared with the prior art, the invention has the beneficial effects that: the fagopyrum esculentum alkali has obvious proliferation effect on isolated ovarian granular cells, can obviously improve the secretion level of progestogen of the isolated ovarian granular cells, can obviously increase the secretion of the progestogen of a naturally aging rat, has obvious anti-apoptosis effect on the ovarian granular cells, can reduce the levels of FSH, LH and T of a polycystic ovary syndrome model rat, and has sex hormone regulation effect on a cyclophosphamide-induced premature ovarian failure rat. The effect of the buckwheat alkali is realized by up-regulating the protein level of c-fosmRNA and an expression product of the c-fosmRNA. Therefore, the fagopyrum tataricum can be applied to the preparation of the anti-ovarian-aging medicine, in particular to the preparation of the medicine for resisting the premature ovarian failure and the ovarian resistance syndrome of the ovarian aging symptom. The invention provides a new clinical application of the buckwheat alkaloid and enlarges the application range of the buckwheat alkaloid.

Detailed Description

The present invention will be further described with reference to specific examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental animal is purchased from Shanghai Sphere-BiKai experimental animals Co., Ltd, and estradiol valerate with better clinical curative effect is selected as a positive control. The buckwheat alkaloid reference substance is purchased from Sigma company, has a purity of more than or equal to 98 percent and is dissolved in water to prepare the administration concentration.

[ example one ] buckwheat growth on isolated ovarian granulosa cells (MTT method)

Collecting female 22-27 days old rats, killing by conventional feeding for 48 h and cervical dislocation, aseptically picking bilateral ovaries of rats, crushing ovaries to release granular cells, diluting with 2mL of DMEM-F12 culture solution containing penicillin and streptomycin, and gently blowing to disperse into single cells. The granular cells were collected by centrifugal filtration. Will be provided withThe cell suspension with a certain concentration is inoculated in a 24-well plate, each well is 80 mul, each well is added with 120 mul of culture solution to adjust the cell density to 200 mul/well, and each group is provided with 6 parallel wells. It was placed in 5% CO₂Culturing in an incubator for 24 h. Taking CO₂Culturing granular cell suspension for 24 hr in incubator, sequentially adding molding concentration cadmium chloride and different concentration Fagopyrine samples (2.5 mg. multidot.mL) into each well^-1、0.5mg·mL^-1、0.1mg·mL^-1、0.02mg·mL^-1、0.004mg·mL^-1) In parallel, a normal control group (physiological saline) and a model group (cadmium chloride and physiological saline) were set. After 20 h of conventional culture, MTT solution (5 mg. multidot.mL) was added to each well^-1) And continuously culturing for 4h by 20 muL, adding 80 muL of triple liquid into each hole, culturing overnight, selecting a wavelength of 570 nm, and measuring the absorbance of each hole on an enzyme-linked immunosorbent assay (ELISA) detector, wherein the result is shown in table 1. The results show that the model group and the normal control group have significant difference (p)<0.01), the proliferation effect of the isolated ovarian granular cells after the action of the positive drug and different doses of the buckwheat alkali is obviously different from that of the model group (average p)<0.01）。

EXAMPLE two estroprogestative effects of Fagopyrine on isolated ovarian granulosa cells (radioimmunoassay)

Collecting female 22-27 days old rats, killing by conventional feeding for 48 h and cervical dislocation, aseptically picking bilateral ovaries of rats, crushing ovaries to release granular cells, diluting with 2ml DMEM-F12 culture solution containing penicillin and streptomycin, and gently blowing to disperse into single cells. The granular cells were collected by centrifugal filtration. The cell suspension with a certain concentration is inoculated in a 24-well plate, each well is 80 mul, each well is added with 120 mul of culture solution to adjust the cell density to 200 mul/well, and each group is provided with 6 parallel wells. It was placed in 5% CO₂Culturing in an incubator for 24 h. Taking CO₂Culturing granular cell suspension for 24 hr in culture box, sequentially adding molded concentration cadmium chloride and different concentration Fagopyrine samples into each well, and setting normal control group (normal saline) and model group (cadmium chloride and normal saline) in parallel). It was placed in 5% CO₂Culturing in an incubator for 24 h. Centrifuging to obtain cell supernatant, and determining E with radioimmunoassay kit₂P content, results are shown in table 2. The results show that the model group and the normal control group have significant difference (p)<0.01), the secretion level of the progestogen of the isolated ovarian granular cells after the action of the positive drug and different doses of the buckwheat alkali is obviously different from that of the model group (p)<0.01) and the estrogen secretion level of the isolated ovarian granulosa cells after the action of different doses of the buckwheat alkali has no significant difference from that of the model group.

EXAMPLE III Effect of buckwheat alkaloid on the secretion of female progestogen in spontaneously aging rats (radioimmunoassay)

Adopts a model of natural aging. Selecting 14-month-old female SD rats with the weight (270 +/-30) g, adaptively feeding the animals for one week, making vaginal cytological smears, continuously observing 4 estrus cycles, and successfully taking the animals as female old models when the vaginal cytological appearance shows that the estrus cycles are prolonged, then the estrus is continued and the pseudopregnant cells are repeated. The natural aging rats are randomly divided into a model group, a buckwheat alkali (10mg/kg, 30mg/kg, 50mg/kg) group and a positive medicine group, 10 animals in each group are respectively taken, 11 female rats with the age of 4 months are taken as a normal control group, the stomach is perfused for 1 time every day, the normal control group and the model group are given with physiological saline with equal volume, blood is taken from orbital venous plexus of the rats before and after the last administration after 30 days of continuous administration, centrifugation and serum taking are carried out, and the contents of estrogen and progesterone in the serum are respectively determined by a radioimmunoassay, and the results are shown in a table 3. The results show that the model group and the normal control group have significant difference (p is less than 0.01), the estrogen and the progestogen have significant difference (p is less than 0.01) after the positive drug is infused for 30 days, the secretion of the progestogen is improved after the buckwheat (high, medium and low doses) is significantly different from the model group (p is less than 0.01), and the estrogen secretion has no significant difference from the model group.

EXAMPLE four Effect of buckwheat in apoptosis of ovarian granulosa cells in spontaneously aging rats (flow cytometry)

An initial model of natural aging is used. Selecting 14-month-old female SD rats with the weight (270 +/-30) g, adaptively feeding the animals for one week, making vaginal cytological smears, continuously observing 4 estrus cycles, and successfully taking the animals as female old models when the vaginal cytological appearance shows that the estrus cycles are prolonged, then the estrus is continued and the pseudopregnant cells are repeated. The natural aging rats are randomly divided into a model group, a buckwheat alkali (10mg/kg, 30mg/kg and 50mg/kg) group and a positive medicine group, each group comprises 10 animals, 11 female rats with the age of 4 months are additionally taken as a normal control group, the stomach is perfused for 1 time every day, the normal control group and the model group are given with physiological saline with equal volume, after 30 days of continuous administration, ovarian granulosa cells are collected after the last administration, flow cytometry detection is carried out, and the apoptosis relation between the administration and the ovarian granulosa cells is analyzed, and the result is shown in table 4. The result shows that the anti-apoptosis effect of the positive drug and the buckwheat alkali (high, medium and low dose) on the ovarian granular cells is obvious after 30 days.

EXAMPLE five Effect of buckwheat in polycystic ovary syndrome model rats

24d old immature SD female rats, 30-40g weight, 0.5mg 18-methylnorethindrone 1 times daily subcutaneous injection, and at 27d old HCG 1.5IU 2 times daily subcutaneous injection for 21d total injection. After the model building is successful, the model group, the sample group (high, medium and low dose) and the positive drug group (0.018 g/d of metformin) are randomly divided into 10 groups, each group is provided with another 10 SD female rats of 45d age as normal control groups, the stomach is irrigated for 1 time every day, the normal control groups and the model groups are given with physiological saline with equal volume, after 15 days of continuous administration and the last administration, blood is taken to detect the concentration of FSH, LH and T in blood serum, and the result is shown in table 5. The results show that the model group and the normal control group have significant difference (p is less than 0.01), and the FSH, LH and T water levels after the positive drug and the buckwheat (high, medium and low dose) are administrated for 15 days have different significant difference from the model group.

EXAMPLE sixthly Effect of buckwheat in the sex hormone levels in premature ovarian failure rats

9d SD female rats are injected with 8 mg/kg d cyclophosphamide for 14 days to the abdominal cavity for molding. After the model building is successful, the model group, the sample group (high, medium and low dose) and the positive drug group (0.075 mg/kg of beaumetone) are randomly divided into 10 groups, each group is provided with another 10 SD female rats with age of 23d as normal control groups, the stomach is irrigated for 1 time every day, the normal control groups and the model groups are given with equal-volume normal saline, after 35 days of continuous administration and the last administration, blood is taken for blood test to detect FSH and E in serum₂The results are shown in Table 6. The results show that the model group and the normal control group have significant difference (p)<0.01), FSH and E35 days after filling positive drug and buckwheat alkali (high and medium dose)₂The water mean was different from the model group in significance.

[ example seven ] study of mechanism of action of fagomine on ovary

Measuring a target gene regulated by c-fos in an ovarian granulosa cell by using a Chromatin Immunoprecipitation (ChIP); the changes of the gene and protein level of the upstream and downstream elements of the c-fos under the aging condition before and after the action of the buckwheat alkali are explored, so that the preliminary mechanism of the function decline action of the buckwheat alkali on the ovarian function with the c-fos as a target is discussed. The results show that fagomine up-regulates c-fos mRNA expression and its expression product protein levels.

[ example eight ] Fagopyrine capsules

100g of buckwheat alkali, 80g of starch, 24g of sodium carboxymethyl starch, 17g of dextrin and a proper amount of 70% ethanol, and 1000 granules (each granule contains 100mg of buckwheat alkali).

[ example nine ] Fagopyrine capsules

15g of buckwheat alkali, 10g of starch, 4g of sodium carboxymethyl starch, 12g of dextrin and a proper amount of 70% ethanol, and 1000 granules (each granule contains 15mg of buckwheat alkali).

[ example ten ] buckwheat alkali tablet

1000g of buckwheat alkali, 55g of lactose, 15g of compressible starch, 10g of hydroxypropyl cellulose, 5g of magnesium stearate and a proper amount of 70% ethanol, and 1000 tablets (each tablet contains 1000mg of buckwheat alkali).

[ example eleven ] granular buckwheat base

6g of buckwheat alkali, 800g of lactose powder, 194g of magnesium stearate and a proper amount of 70% ethanol, and 1000g of granules are prepared (each g of granules contains 6mg of buckwheat alkali).

[ example twelve ] Sopholidine Soft capsules

20g of buckwheat alkali, 10g of soybean oil, 5g of gelatin, 5g of glycerol and 7g of distilled water, and 1000 granules (each granule contains 20mg of buckwheat alkali).

[ example thirteen ] buckwheat alkali cream

1g of buckwheat alkali, 100g of stearic acid, 20g of cetyl alcohol, 10g of glycerin monostearate, 10g of liquid paraffin, 0.8g of methylparaben, 0.2g of butylparaben, 140g of glycerol, 5g of potassium hydroxide, 10g of ethanol and the balance of distilled water to 1000 g.

[ example fourteen ] buckwheat alkali cream

0.5g of buckwheat alkali, 100g of stearic acid, 20g of cetyl alcohol, 10g of glycerin monostearate, 10g of liquid paraffin, 0.8g of methylparaben, 0.2g of butyl hydroxybenzoate, 140g of glycerol, 5g of potassium hydroxide, 10g of ethanol and the balance of distilled water to 1000 g.

[ example fifteen ] buckwheat alkali gel

0.5g of buckwheat alkali, PEG400450g, 80g of hexanediol, 8020g of Tween, 30g of carbomer, 6ml of ethanolamine, 10g of benzyl alcohol and distilled water added to 1000 g.

[ example sixteen ] buckwheat alkali injection

Dissolving buckwheat alkali 25.0g in 1000ml of water for injection, fully stirring for dissolving, adding water for injection to 2000ml, adding activated carbon 2.5g for injection, heating to 60 ℃, stirring for 30 minutes, filtering by a carbon rod, filtering the filtrate by a 0.25 mu m microporous filter membrane for sterilization, subpackaging in 1000 penicillin bottles with the content of 2.0ml per bottle, sealing and sterilizing to obtain the buckwheat alkaloid injection.