Use of natural plant active ingredients in prevention and / or treatment of ovarian dysfunction diseases

Abstract

The invention provides an application of a natural plant active component in preparation of a medicine for preventing and / or treating ovarian dysfunction diseases in a subject. The natural plant active component comprises an active component of safflower and an optional active component of salvia miltiorrhiza and / or an active component of epimedium.

Description

Technical Field

[0001] The present invention relates to a method and a medicine for preventing and / or treating ovarian dysfunction diseases. Background Art

[0002] The ovaries are important reproductive and endocrine organs in female animals, and their functions include ovulation and secretion of hormones (such as steroid hormones, estrogen and progesterone). When ovarian function is disordered, such as abnormal follicular maturation or reduced steroid hormone secretion, it will cause a series of ovarian dysfunction diseases, such as follicular development, maturation or ovulation disorders. Patients may show infertility, spontaneous abortion and premature aging, which will also affect the endocrine function of the ovaries.

[0003] At present, in clinical practice, the treatment of ovarian dysfunction is mainly based on hormone replacement therapy and assisted reproductive therapy. However, estrogen has the following side effects. First, estrogen can stimulate endometrial lesions. Women's estrogen, androgen, progesterone, etc. interact and restrict each other. If estrogen is secreted excessively under the action of drugs and the body's pituitary gland, the endometrium will cause abnormal proliferation under the action of estrogen for a long time, thereby causing endometrial lesions. Secondly, the uterus will cause uterine fibroids under the action of estrogen for a long time, and the breast will also develop lesions under the action of estrogen, leading to breast hyperplasia and even breast cancer. Then estrogen will also affect the central nervous system of the human body. Taking too much estrogen can cause dizziness, nausea, and even high blood pressure, diabetes, etc. At the same time, in clinical practice, patients have a certain psychological burden on hormone therapy or refuse to use hormone therapy. For assisted reproductive therapy, it is expensive, and most patients with anovulation or rare ovulation are still powerless. Importantly, estrogen can only improve endocrine, but cannot increase the number of follicles. At present, there is no clear and effective drug or strategy to prevent and delay ovarian dysfunction.

[0004] Therefore, there is an urgent need to find a new drug for preventing or treating ovarian dysfunction diseases, and the drug has the advantages of obvious effect and low side effects. Summary of the invention

[0005] In order to solve the above technical problems existing in the prior art, the present disclosure provides a drug and a treatment method for preventing and / or treating ovarian dysfunction. The drug treatment of the present disclosure can effectively restore ovarian function, promote follicle development and ovulation.

[0006] According to one aspect of the present disclosure, there is provided a use of natural plant active ingredients in the preparation of a medicament for preventing and / or treating ovarian dysfunction diseases in a subject, wherein the natural plant active ingredients include active ingredients of safflower.

[0007] In some embodiments, the active ingredient of safflower includes hydroxysafflor yellow A or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

[0008] In some embodiments, the natural plant active ingredients further include active ingredients of Salvia miltiorrhiza and / or Epimedium;

[0009] In some embodiments, the active ingredients of Salvia miltiorrhiza include salvianolic acid and / or tanshinone, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof; preferably, the active ingredients of Salvia miltiorrhiza include salvianolic acid B and / or tanshinone IIA, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof.

[0010] In some embodiments, the active ingredients of Epimedium include icariin, icariin and / or epimedin C, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof.

[0011] In some embodiments, the ovarian dysfunction disease may include, but is not limited to, one or more of premature ovarian insufficiency (POI), premature ovarian failure (POF), diminished ovarian reserve (DOR), poor ovarian response (POR), early menopause, ovarian function damage, ovarian insufficiency and ovarian dysfunction, decreased reproductive function, infertility, perimenopausal syndrome or menopausal syndrome.

[0012] In some embodiments, the ovarian dysfunction disease may present symptoms such as hormonal imbalance, menstrual (estrus) cycle disorder, infrequent ovulation or anovulation, and may cause other diseases such as decreased reproductive function, infertility, perimenopausal syndrome, and menopausal syndrome.

[0013] In some embodiments, the ovarian dysfunction disease is caused by genetic factors, iatrogenic factors, immune factors, environmental factors, advanced physiological age or other factors.

[0014] In some embodiments, the natural plant active ingredient has at least one of the following uses: (1) increasing ovarian reserve; (2) restoring sex hormone secretion; (3) restoring and / or promoting ovulation; (4) restoring ovarian function; (5) promoting follicular development; (6) increasing the level of anti-Mullerian hormone (AMH) in serum; (7) alleviating oxidative stress damage; (8) increasing the level of superoxide dismutase (SOD) in serum; (9) increasing the level of reduced glutathione (GSH) and / or total antioxidant capacity (T-AOC) in serum, (10) reducing the level of malondialdehyde (MDA) in serum; (11) increasing the number of ovarian follicles at all levels (including primordial, primary, secondary, and mature follicles) and corpora lutea, and / or reducing the number of atretic follicles; (12) increasing the number of offspring; (13) restoring the estrous cycle.

[0015] In some embodiments, the natural plant active ingredients can increase ovarian reserve and elevate the level of anti-Mullerian hormone (AMH), thereby treating or preventing ovarian dysfunction.

[0016] According to some embodiments, the natural plant active ingredients can increase the level of superoxide dismutase (SOD) in serum, increase the level of reduced glutathione (GSH) and total antioxidant capacity (T-AOC), and reduce the level of malondialdehyde (MDA) in serum. This can improve the body's antioxidant capacity and reduce the degree of oxidative stress, thereby preventing or treating ovarian dysfunction.

[0017] According to some embodiments, the natural plant active ingredients can restore ovulation in subjects suffering from ovarian disorders.

[0018] In some embodiments, the active ingredients of Carthamus tinctorius, the active ingredients of Salvia miltiorrhiza and / or the active ingredients of Epimedium are administered simultaneously or sequentially.

[0019] In some embodiments, the mass ratio of the active ingredients of safflower to the active ingredients of salvia miltiorrhiza is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:1.

[0020] In some embodiments, the mass ratio of the active ingredient of safflower to the active ingredient of epimedium is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:1.

[0021] According to another aspect of the present disclosure, a composition for preventing or treating ovarian disorders is provided, wherein the composition comprises active ingredients of safflower.

[0022] In some embodiments, the active ingredient of safflower includes hydroxysafflor yellow A or a solvate, hydrate or pharmaceutically acceptable salt thereof.

[0023] In some embodiments, the composition further comprises active ingredients of Salvia miltiorrhiza and / or Epimedium.

[0024] In some embodiments, the active ingredients of Salvia miltiorrhiza include salvianolic acid and / or tanshinone, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof; preferably, the active ingredients of Salvia miltiorrhiza include salvianolic acid B and / or tanshinone IIA, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof.

[0025] In some embodiments, the active ingredients of Epimedium include icariin, icariin and / or epimedin C, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof.

[0026] In some embodiments, the mass ratio of the active ingredients of safflower to the active ingredients of salvia miltiorrhiza is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:1.

[0027] In some embodiments, the mass ratio of the active ingredient of safflower to the active ingredient of epimedium is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:1.

[0028] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0029] In some embodiments, the pharmaceutically acceptable excipients may include, but are not limited to, one or more of pharmaceutically acceptable carriers, diluents, adjuvants and excipients.

[0030] In some embodiments, the dosage form of the composition includes, but is not limited to, tablets, capsules, solutions, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, patches, sustained-release preparations, controlled-release preparations, or targeted preparations.

[0031] In some embodiments, the composition can be administered, for example, by injection, oral administration, rectal administration, etc.

[0032] In some embodiments, the active ingredients of Carthamus tinctorius, the active ingredients of Salvia miltiorrhiza, and / or the active ingredients of Epimedium are formulated to be administered in the same dosage form or in separate dosage forms.

[0033] According to a fourth aspect of the present disclosure, a method for treating and / or preventing ovarian dysfunction in a subject is provided, the method comprising administering a preventively or therapeutically effective amount of the natural plant active ingredient to a subject in need thereof.

[0034] The inventors of the present disclosure used a mouse model of premature ovarian failure to verify that oral administration or injection of the active ingredients of safflower, the active ingredients of salvia miltiorrhiza and / or the active ingredients of epimedium can increase the anti-Mullerian hormone level of mice, increase the number of ovarian follicles at all levels (primitive, primary, secondary, mature follicles) and corpora lutea, reduce the number of atretic follicles, and increase the number of litters. Moreover, the number of mice whose estrous cycles were restored was significantly increased compared with the control group, and the results of mouse serum test showed that the degree of oxidative stress was reduced and the antioxidant index was enhanced. These results show that the active ingredients of safflower, the active ingredients of salvia miltiorrhiza and / or the active ingredients of epimedium have significant effects in treating ovarian disorders and protecting fertility. In addition, in the present disclosure, the active ingredients of safflower, the active ingredients of salvia miltiorrhiza and / or the active ingredients of epimedium are components of the natural herbal medicine safflower, which will not produce adverse reactions when used, are safe to use, and have small side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The weight changes of mice in each administration group of Example 1 before modeling, after modeling and after treatment are shown.

[0036] Figure 2 The weight changes of mice in each administration group of Example 2 before modeling, after modeling and after treatment are shown.

[0037] Figure 3 The weight changes of mice in each administration group of Example 3 before modeling, after modeling and after treatment are shown.

[0038] Figure 4 The weight changes of mice in each administration group of Example 4 before modeling, after modeling and after treatment are shown.

[0039] Figure 5 The cell morphology of different estrous cycles of mice is shown, scale bar: 50 μm.

[0040] Figure 6 The results of observation of the estrous cycle of mice in each administration group in Example 2 are shown.

[0041] Figure 7 The results of observation of the estrous cycle of mice in each administration group in Example 3 are shown.

[0042] Figure 8 Shows the Figure 7 The statistical proportion of each phase of the estrous cycle.

[0043] Fig. 9 Shows the Figure 7 The number of estrus periods counted.

[0044] Fig.10The results of observation of the estrous cycle of mice in each administration group in Example 5 are shown.

[0045] Fig.11 The test results of ovarian reserve hormone and antioxidant capacity in Example 6 are shown. A is the level of anti-Mullerian hormone (AMH); B is the level of total antioxidant capacity (T-AOC); C is the level of reduced glutathione (GSH); D is the level of serum malondialdehyde (MDA) in mice after administration in Example 1; E is the level of serum superoxide dismutase (SOD). * indicates a significant difference (P<0.05); ** indicates a significant difference (P<0.01); *** indicates a significant difference (P<0.001); **** indicates a significant difference (P<0.0001).

[0046] Fig.12 The ovarian tissue sections and follicle classification and counting results in Example 7 are shown. A is a representative ovarian tissue section; B is the classification and counting results of follicles at various levels: primordial follicles, primary follicles, secondary follicles and atretic follicles. * indicates a significant difference (P<0.05)

[0047] Fig.13 The effect of the drug treatment on ovulation of mice in each drug-dosing group of Example 1 is shown.

[0048] Fig.14 The effect of the drug treatment on ovulation of mice in each drug administration group of Example 2 is shown.

[0049] Fig.15 The effect of the drug treatment on ovulation of mice in each drug administration group of Example 4 is shown.

[0050] Fig.16 The results of mitochondrial JC-10 staining of postovulated oocytes in Example 9 are shown.

[0051] Fig.17 Shows the Fig.16 The red / green ratio statistical results are shown in Figure 2. The red / green ratio represents the high and low membrane potential. ** indicates a significant difference (P<0.01)

[0052] Fig.18 A representative picture of mouse litter size of Example 4 is shown.

[0053] Fig.19 The litter size of mice two months after treatment in Example 4 is shown.

[0054] Fig. 20 The protective effect of drugs on the chemotherapy-damaged ovarian cell model is shown. A is the inhibitory effect curve of 4-HC on cells, and B and C are the protective dose gradients of HSYA and SAB on cell viability, respectively.

[0055] Fig.21 The results of expression detection of genes Lhcgr, Sf1, and Cdkn2a in KK1 cells after 4-HC treatment and different drug administrations are shown.

[0056] Fig. 22 The results of gene Ncoa4 and Gpx4 expression detection in KK1 cells after 4-HC treatment and different drug administration are shown.

[0057] Fig.23 The results of gene Fshr and Cdkn2a expression in KK1 cells after 4-HC treatment and different drug administrations are shown.

[0058] Figure 21-23 In the table, * indicates significant difference (P<0.05); ** indicates significant difference (P<0.01); *** indicates significant difference (P<0.001); **** indicates significant difference (P<0.0001). DETAILED DESCRIPTION

[0059] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0060] definition

[0061] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0062] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0063] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0064] The term "premature ovarian insufficiency (POI)" used in this article is a serious ovarian dysfunction disease, which refers to the loss of ovarian function in women before the age of 40 due to follicle depletion. The clinical manifestations are amenorrhea for 4 months, infrequent or frequent menstruation, and follicle-stimulating hormone (FSH) twice tested more than 25U / L with an interval of more than 4 weeks, with or without fluctuating decreases in estrogen levels. In the long term, there will be risks such as congenital heart disease, intellectual disability, emotional abnormalities, osteoporosis, adrenal and thyroid dysfunction, diabetes, and recurrent miscarriage. POI patients suffer from severe ovulatory disorders due to ovarian dysfunction, and their fertility is significantly reduced or lost. The incidence of POI in patients with primary amenorrhea is as high as 10-28%, and the overall incidence is about 3.7%, and it is showing an increasing trend year by year. Clinically, POI patients may present with primary amenorrhea or even absent puberty. Most of them suffer from secondary amenorrhea, which is caused by natural aging, chromosomal or gene defects, autoimmune diseases, environmental factors and iatrogenic factors.

[0065] The term "premature ovarian failure (POF)" used in this article refers to the occurrence of amenorrhea for more than 4 to 6 months before the age of 40 in women, with two tests of follicle-stimulating hormone (FSH) exceeding 40U / L at an interval of more than 4 weeks, accompanied by decreased estrogen and menopausal symptoms. Premature ovarian failure is one of the main causes of female infertility. The American Society for Reproductive Medicine divides the disease process into normal, latent, biochemical abnormality and clinical abnormality stages based on FSH levels, fertility and menstrual status.

[0066] The term "ovarian reserve" used in this article refers to the primordial follicles contained in the female ovarian cortex. After birth, the number of primordial follicles no longer increases, and the number of primordial germ cells in the ovarian cortex will no longer increase. Diminished / decreased ovarian reserve (DOR), which can also be called low ovarian reserve function, reduced ovarian reserve function, decreased ovarian reserve, decreased ovarian reserve, etc., refers to the number of follicles contained in the ovaries being less than the expected number of follicles, and the ovarian responsiveness and fertility of women of childbearing age are reduced. This will make conception more difficult and reduce the chances of receiving in vitro fertilization (IVF) and other fertility treatments. Compared with the miscarriage rate of women without DOR, DOR patients can also lead to a higher chance of miscarriage. The number and / or quality of oocytes in the ovaries of DOR patients decreases, accompanied by increased FSH levels.

[0067] As used herein, the term "premature menopause" refers to menopause occurring earlier than the normal age of menopause, before the age of 45 years.

[0068] The term "poor ovarian response (POR)" used in this article, also known as poor ovarian response, refers to the low response of the ovaries to stimulating drugs and the low number of eggs obtained when women undergo controlled superovulation for assisted reproductive technology. Ovarian poor responsiveness is considered an early sign of diminished ovarian reserve.

[0069] The term "gonadotropin (Gn)" as used herein is a glycoprotein hormone that regulates the development of vertebrate gonads and promotes the production and secretion of sex hormones. For example, the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secreted by the anterior pituitary gland can stimulate the development of germ cells in the ovaries or testicles and the production and secretion of sex hormones; the human chorionic gonadotropin (HCG) secreted by the human placenta can promote the secretion of progesterone by the corpus luteum of pregnancy.

[0070] The term "estrogen" as used herein is mainly secreted by the ovaries and mainly includes estradiol (E 2 ), estrone, estriol, etc. Estrogen has the function of promoting the maturation of the accessory reproductive organs, vagina, uterus, fallopian tubes, etc. of adolescent girls. Estradiol itself is a steroidal estrogen, secreted by the granulosa cells of the ovarian follicles. There are two types of estradiol, α and β, of which α type E2 has a stronger physiological effect. In patients with premature ovarian failure, E 2 The level dropped.

[0071] The term "follicle stimulating hormone (FSH)" as used herein refers to a heterodimeric glycoprotein hormone composed of different subunits α and β secreted by the anterior pituitary basophils. It regulates the growth, development, puberty sexual maturity of the human body, and a series of physiological processes related to reproduction through the female hypothalamus-pituitary-ovarian axis. FSH is secreted in the human body in a pulsatile manner, and women change with the menstrual cycle. The main function of FSH is to cause granulosa cell proliferation, endometrial cell differentiation, follicular fluid formation, and follicular cavity expansion, thereby causing follicle growth and development, improving the effectiveness of mature follicles, and then improving ovulation rate and pregnancy rate. In patients with premature ovarian failure, the FSH level in serum increases.

[0072] Female reproductive function is regulated by the positive and negative feedback mechanism of hormone signals between the hypothalamus-pituitary-ovary. This mechanism is called the "gonad axis", which determines the cyclic response of the ovaries and further determines the cyclic response of the uterus. Ovarian dysfunction or decreased reserve function is often accompanied by an increase in early ovarian follicular FSH. This is due to a decrease in the secretory function of the ovaries. This change causes the pituitary gland to secrete more stimulating hormones to improve ovarian secretion. The adaptive response of poor ovarian response leads to a compensatory increase in gonadotropin-releasing hormone (GnRH), which directly stimulates the pituitary gland to secrete FSH, leading to an increase in FSH. It is generally believed that as FSH increases, it indicates a decrease in ovarian reserve, poor ovarian responsiveness, a small number of developing follicles and eggs obtained, and decreased ovarian function.

[0073] The term "iatrogenic factors" used in this article mainly includes ovarian dysfunction caused by surgery, radiotherapy and chemotherapy drugs, such as ovarian function damage.

[0074] The term "pharmaceutically acceptable" as used herein refers to an agent that can be applied to humans and / or other animals as subjects and does not produce excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). The term "excipient" refers to an auxiliary material that is present in a pharmaceutical preparation at the same time as the active ingredient and does not produce excessive adverse reactions or side effects, including carriers, osmotic pressure regulators, pH regulators, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. The term "pharmaceutically acceptable excipient" refers to a high-safety excipient that is suitable for a specific pharmaceutical preparation and is routinely used in pharmacy. The term "carrier" includes, but is not limited to, liposomes, ethosomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

[0075] The term "pharmaceutically acceptable salt" used herein may include alkali metal salts (eg, sodium salts or potassium salts), alkaline earth metal salts (eg, calcium salts or magnesium salts), and salts formed with suitable organic ligands (eg, quaternary ammonium salts).

[0076] The term "estrus cycle", also known as the estrus cycle, used in this article refers to a regular physiological change in female placental mammals, which is induced by sex hormones. The estrus cycle of mice or rats is repeated rhythmically every 4 to 5 days. According to the follicular development rules and reproductive endocrine changes in the estrus cycle, the estrus cycle can be divided into proestrus, estrus, metestrus and diestrus. Mice or rats are used as an animal model to study their estrus cycle, and are widely used in experiments to explore the pathogenesis of diseases related to the female menstrual cycle, evaluate the effects of drugs, and develop corresponding drugs.

[0077] As used herein, the term "prevention" refers to preventive treatment of a subclinical disease state, intended to reduce the probability of the clinical disease state occurring. "Prevention" can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment of a subject who has not yet presented with a clinical disease state, while secondary prevention is defined as preventing a secondary occurrence of the same or similar clinical disease state.

[0078] As used herein, the term "treat" refers to the treatment of a disease, symptom or condition, including: 1) inhibiting the development of the disease, symptom or condition; and / or, 2) delaying or alleviating the disease, symptom or condition.

[0079] The safflower (Carthamus tinctorius L.) mentioned in this article belongs to the annual herbaceous plant of the genus Carthamus in the family Asteraceae, also known as thorny safflower, red and blue flower, and grass safflower. Hydroxysafflor yellow A is the most effective water-soluble component of safflower pharmacological effects and has the following structure:

[0080]

[0081] The salvia miltiorrhiza Bunge mentioned in this article is a perennial upright herb belonging to the genus Salvia of the family Lamiaceae. It is also known as blood ginseng, purple salvia, five-phoenix flower, and blood-activating root. The main active ingredients of salvia include salvianolic acid (salvianolic acid A, salvianolic acid B, tanshinone, caffeic acid, and rosmarinic acid) and tanshinone (including tanshinone IIA, tanshinone ⅡA, tanshinone ⅡB, cryptotanshinone, and isocrypttanshinone). Among them, salvianolic acid B has the following structure:

[0082]

[0083] Tanshinone IIA has the following structure:

[0084]

[0085] The epimedium (Epimediumbrevicornu Maxim.) mentioned in this article is a perennial herbaceous plant of the genus Epimedium of the Berberidaceae family, also known as short-horned epimedium. The main active ingredients of epimedium include icariin, icariin and / or epimedin C, wherein icariin has the following structure:

[0086]

[0087] Icaritin has the structure shown below:

[0088]

[0089] Epimedin C has the structure shown below:

[0090]

[0091] Biomarkers mentioned herein, such as anti-Müllerian hormone (AMH), superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH) and total antioxidant capacity (T-AOC), etc., can be detected using methods generally known in the art. Detection methods generally encompass methods for quantifying biomarker levels in samples (quantitative methods). The following methods are generally known to those skilled in the art for qualitative and / or quantitative detection of biomarkers. Samples can be conveniently determined and are commercially available.

[0092] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0093] Example

[0094] Example 1

[0095] 1. Preparation of Animal Model

[0096] C57 mice (6 mice per group, 7 weeks old) were intraperitoneally injected with a mixture of 120 mg / kg / d cyclophosphamide and 30 mg / kg / d busulfan for 4 consecutive days, and 75 mg / kg / d cyclophosphamide alone was intraperitoneally injected on the 5th, 6th, and 7th days to establish the ovarian insufficiency (POI) model.

[0097] 2. Method of administration

[0098] After modeling, mice in each group were treated with medication starting on the 8th day and continued for 14 days.

[0099] Dosage group:

[0100] (1) HSYA+EpiC: 20 mg / kg / d hydroxysaffron yellow yellow A (HSYA) + 20 mg / kg / d epimedin C (EpiC);

[0101] (2) Tiia+SAB: 30 mg / kg / d of tanshinone IIA (Tiia) + 30 mg / kg / d of salvianolic acid B (SAB);

[0102] (3) HSYA+EpiC+Tiia+SAB: 20 ​​mg / kg / d hydroxysaffron yellow yellow A+20 mg / kg / d epimedin C and 30 mg / kg / d Tiia+30 mg / kg / d SAB;

[0103] (4) Vehicle: Model group control, an equal amount of normal saline (containing 2.5% DMSO)

[0104] (5) Ctrl: normal control group, an equal amount of physiological saline (containing 2.5% DMSO).

[0105] 3. Daily vaginal smear cytology observation

[0106] Expose the mouse vagina, drip about 30 μL of physiological saline, gently aspirate it onto a glass slide, and observe it under a microscope. If necessary, perform HE staining: fix with 95% ethanol for 10 minutes, stain with hematoxylin aqueous solution for 5 to 8 minutes, rinse with running water for separation, then stain with eosin for 3 minutes, dry the slide, and seal with neutral gum.

[0107] 4. Weight measurement

[0108] Body weight was measured before modeling (start of treatment), after modeling, and after treatment.

[0109] 5. Hormone and oxidative stress index detection

[0110] After the treatment, the mice were killed, and their blood was collected, allowed to stand, and separated by centrifugation at 4500 rpm to obtain serum, which was stored in a -80°C refrigerator for later use in measuring hormone and oxidative stress indicators in mice.

[0111] 6. Oocyte Detection

[0112] One side of the collected ovaries was fixed with 4% paraformaldehyde, and then sliced ​​and stained with HE; the other side of the ovary was stored in a -80℃ refrigerator for future use. A sterile 1ml syringe was used to cut the ampulla of the fallopian tube to collect the mouse oocytes, and the collected oocytes were observed and counted under a microscope, and mitochondrial and nuclear staining was performed.

[0113] 7. Detection of fertility and littering

[0114] After the treatment, these mice were caged with male mice in a 2:1 ratio, and the birth rate was recorded.

[0115] Weight measurement results Figure 1 And as shown in Table 1.

[0116] Table 1

[0117] Before modeling Before treatment (after modeling) After treatment Ctrl 18.77±0.26 19.40±0.16 20.27±0.18 Vehicle Group 18.66±0.30 16.02±0.49 18.67±1.39 Tiia+SAB group 18.95±0.21 16.55±0.42 18.92±0.39 HSYA+Epi C group 19.02±0.14 16.55±0.30 19.32±0.34 HSYA+EpiC+Tiia+SAB group 18.93±0.16 16.28±0.21 19.20±0.35

[0118] Depend on Figure 1 As shown in the results of Table 1, the three drug combinations are effective in maintaining body weight, among which HSYA+EpiC has the best effect.

[0119] Example 2

[0120] 1. Preparation of Animal Model

[0121] The ovarian insufficiency (POI) model was established in the same manner as in Example 1.

[0122] 2. Method of administration

[0123] After modeling, mice in each group were treated with medication starting on the 8th day and continued for 14 days.

[0124] Dosage group:

[0125] (1) Vehicle: Model group control, normal saline

[0126] (2) SAB: 30 mg / kg / d salvianolic acid B (SAB);

[0127] (3) EpiC: 20 mg / kg / d EpiC;

[0128] (4) HSYA: 20 mg / kg / d hydroxysafflor yellow A (HSYA);

[0129] (5) SAB+HSYA: 30 mg / kg / d salvianolic acid B (SAB) + 20 mg / kg / d hydroxysaffron yellow A (HSYA);

[0130] (6) SAB+EpiC+HSYA: 30 mg / kg / d salvianolic acid B (SAB) + 20 mg / kg / d epimedin C (EpiC) + 20 mg / kg / d hydroxysaffron yellow A (HSYA). 3. Body weight measurement

[0131] Body weight was measured before modeling (start of treatment), after modeling, and after treatment.

[0132] Weight measurement results Figure 2 As shown, different administration groups all had effective effects on body weight maintenance, among which the HSYA administration group had the best effect.

[0133] Example 3

[0134] 1. Preparation of Animal Model

[0135] The POI model was established by intraperitoneal injection of 120 mg / kg cyclophosphamide into C57 mice (6 mice in each group, 7 weeks old) on days 1, 13, 20 and 28, respectively.

[0136] 2. Dosage Grouping

[0137] Treatment was carried out for 14 days after modeling, and the administration components were:

[0138] (1) Salvianolic acid B (SAB) treatment group: 60 mg / kg / d of salvianolic acid B;

[0139] (2) Hydroxysafflor yellow A (HSYA) treatment group: 40 mg / kg / d hydroxysafflor yellow A;

[0140] (3) Salvianolic acid B and hydroxysafflor yellow A (SAB+HSYA) combined treatment group: 60 mg / kg / d of salvianolic acid B and 40 mg / kg / d of hydroxysafflor yellow A mixed solution were used respectively;

[0141] (4) Control group: an equal amount of normal saline;

[0142] (5) Vehicle group: an equal amount of normal saline.

[0143] 3. Weight measurement

[0144] Body weight was measured before modeling (start of treatment), after modeling, and after treatment.

[0145] Weighing results see Figure 3 , body weight began to decrease significantly 2 days after cyclophosphamide administration, and the effect of cyclophosphamide in the body lasts about 7 days. Figure 3 It shows that hydroxysafflor yellow A can effectively alleviate the weight loss of mice caused by cyclophosphamide.

[0146] Example 4

[0147] 1. Preparation of Animal Model

[0148] The POI model is established in the same manner as in Example 1.

[0149] 2. Dosage Grouping

[0150] After modeling, the groups were treated for 7 days (intraperitoneal injection), and the administration components were:

[0151] (1) Hydroxysafflor yellow A treatment group: 40 mg / kg / d Hydroxysafflor yellow A;

[0152] (2) Icariin (ICA) treatment group: 40 mg / kg / d icariin;

[0153] (3) Hydroxysafflor yellow A and icariin combined treatment group: 40 mg / kg / d of hydroxysafflor yellow A and 40 mg / kg / d of icariin mixture;

[0154] (4) Vehicle group: an equal amount of normal saline.

[0155] 3. Weight measurement

[0156] Body weight was measured before modeling (start of treatment), after modeling, and after treatment.

[0157] Weighing results see Figure 4 The HSYA group and the HSYA and ICA combined group both had good effects on body weight, and the HSYA and ICA combined group had a significantly better effect than the other groups.

[0158] Example 5. Observation results of the estrous cycle of modeling and treatment mice

[0159] During the estrous cycle, a series of histological and physiological changes occur in the reproductive and endocrine systems of mice. Based on these changes, the estrous cycle can be divided into proestrus (P), estrus (E), metestrus (M) and diestrus (D).

[0160] Vaginal smear cytology observation was performed in the four periods of the estrous cycle (P: proestrus; E: estrus; M: metestrus; D: diestrus). The mouse vagina was exposed, and about 30 μL of physiological saline was dripped into it, and it was gently aspirated onto a glass slide and observed under a microscope. If necessary, staining was performed, and after the slide was dried, it was stained with a hematoxylin aqueous solution for several minutes, washed with running water, and then stained with alcohol eosin for several seconds, and sealed with neutral gum. Figure 5 The cell morphology of different estrous cycles in mice is schematically shown.

[0161] Vaginal smear and cytology observations were performed continuously to plot the estrous cycle of mice (each curve represents the estrous cycle of a mouse). The estrous cycle of mice is about 4 to 5 days, which is divided into four periods. If the estrous cycle graph shows "N", it indicates that the estrous cycle is regular or has returned to normal. We regard an estrous cycle that lasts for more than 4 consecutive days after treatment as a cycle stagnation. During the drug administration period, the proportion of each period of the estrous cycle of each group of mice was counted.

[0162] The exemplary observation results of the estrous cycle of mice in each administration group of Example 2 are as follows: Figure 6 As shown, it can be seen that there are more mice with estrous cycle arrest in the Vehicle group, while there are fewer mice with estrous cycle arrest in the other groups.

[0163] The observation results of the estrous cycle of mice in each administration group of Example 3 are as follows: Figure 7 As shown in the figure, the statistical comparison of each phase of the estrous cycle is as follows Figure 8 As shown in Figure 2, the proportion of estrus (P+E+M) in the HSYA group and the SAB group increased, while the proportion of diestrus (D) decreased. Fig. 9 As shown, it can be seen that the number of estrus periods in the HSYA group and the SAB group increased.

[0164] The observation results of the estrous cycles of the mice in each administration group of Example 4 are as follows: Fig.10As shown (the red dotted line represents the endpoint of modeling), all 5 mice in the Vehicle group were arrested, 2 out of 5 mice in the HSYA group were arrested, 2 out of 5 mice in the ICA group were arrested, and only 3 out of 6 mice in the HSYA+ICA group were arrested. It can be seen that there were fewer mice with estrous cycle arrest in each drug-treated group.

[0165] The above results all indicate that the estrus period of mice in the HSYA and / or SAB, HSYA and / or ICA administration groups was prolonged, and the diestrus period was shortened, indicating that it has a protective effect on maintaining the estrus cycle of mice injured by chemotherapy.

[0166] Example 6. Detection of serum hormones and antioxidant indicators

[0167] Anti-Müllerian hormone (AMH) is one of the important reproductive physiological regulatory factors in women. It is mainly secreted by the preantral follicles and small antral follicle granulosa cells of the ovaries and is not regulated by gonadotropins. In terms of evaluating ovarian reserve function, AMH reflects the trend of ovarian reserve decline with age earlier than FSH, estradiol (E2), inhibin B (inhB) and antral follicle count (AFC). It can quickly and effectively determine the level of ovarian reserve capacity and is the most accurate biomarker of ovarian aging.

[0168] The oxidative stress hypothesis holds that the reduction of antioxidant components in the body leads to a weakened ability to scavenge free radicals, which in turn leads to oxidative damage to biomacromolecules, an imbalance between cellular oxidation and antioxidant functions, and oxidative stress, which in turn leads to disease and aging. Oxygen free radicals act on unsaturated fatty acids in lipids to generate peroxidized lipids, including malondialdehyde (MDA); the antioxidant defense system that scavenge free radicals includes superoxide dismutase (SOD). Recently, it has been reported that oxidative stress is associated with ovarian aging and POI in primates.

[0169] After the treatment, the mice were killed, and the blood was collected, centrifuged at 4500 rpm, and separated into serum, which was stored in a -80°C refrigerator for later use to measure the hormone and oxidative stress indicators of the mice.

[0170] This example shows the levels of hormones and redox indices in the blood of each modeled and treated mouse in Examples 1-4.

[0171] The expression level of AMH was detected by ELISA (human anti-Mullerian hormone (AMH) detection kit, purchased from Quanzhou Ruixin Biotechnology Co., Ltd.), and it was found that the content was the highest in the SAB+HSYA group and the HSYA group ( Fig.11 A).

[0172] The total antioxidant capacity (T-AOC) detection kit (purchased from Solebao) was used to detect the level of T-AOC in serum. The results of the two batches of experiments showed that the highest level was in the SAB+HSYA group and the highest level was in the HSYA group ( Fig.11 B). After treatment, serum malondialdehyde levels in mice in the HSYA group were significantly reduced ( Fig.11 C), the level of reduced glutathione increased significantly ( Fig.11 D) The level of superoxide dismutase in the HSYA group was also higher than that in the other three groups ( Fig.11 E).

[0173] Example 7. Detection of follicles at various levels, corpus luteum and atretic follicles

[0174] The mice in each group after treatment in Example 3 were killed, and the ovarian tissue was taken. One ovary was fixed with 4% paraformaldehyde, embedded in paraffin, and then serially sectioned (each section was 5 μm, and one section was collected every 5 sections). The 5 sections containing the largest surface of each ovary were stained with HE, and the primordial follicles, primary follicles, secondary follicles, corpora lutea and atretic follicles were counted respectively.

[0175] Exemplary results are Fig.12 As shown in A and 12B, compared with the control group (Vehicle group), the number of primordial follicles, primary follicles and corpus luteum increased in the HSYA group, while the number of primary follicles and secondary follicles increased in the SAB+HSYA group, indicating that HSYA alone or in combination with SAB has the effect of increasing follicles.

[0176] Example 8. Effects of natural herbal medicine components or their combination on ovulation in POI mice

[0177] For the POI mice modeled and treated in Example 1, Example 2 and Example 4, after superovulation (injection of chorionic gonadotropin 48 hours after injection of pregnant mare serum), the ampulla of the mouse oviduct was obtained 14 to 16 hours later, and the number of eggs obtained was recorded.

[0178] Example 1 The number of ovulations in each medication group is as follows Fig.13 As shown, the results showed that there was no significant difference in the number of induced ovulations in each treatment group compared with the Vehicle group, among which the HSYA+EpiC group was the best.

[0179] Example 2: The exemplary ovulation number of each dosing group is as follows Fig.14 As shown, the results showed that for HSYA and SAB, the SAB+HSYA group had the highest number of ovulations.

[0180] Example 4 The number of ovulations in each medication group is as follows Fig.15 As shown, the results showed that for HSYA and ICA, the number of ovulations in the HSYA group was the highest.

[0181] Example 9. Effects of natural plant drug components or their combination on mouse oocyte quality

[0182] The mitochondrial membrane potential of the oocytes discharged in Example 3 was detected using a JC-10 kit (purchased from Biyuntian Biological Company). In normal cells, JC-10 selectively aggregates in the mitochondrial matrix to form a reversible red fluorescent polymer (Ex=540nm, Em=590nm); due to the decline or loss of membrane potential in unhealthy mitochondria, JC-10 changes from a polymer to a monomer form and exists in the cytoplasm, producing green fluorescence (Ex=490nm, Em=525nm). The change in color can directly reflect the change in mitochondrial membrane potential, and the degree of mitochondrial depolarization can be measured by the ratio of red / green fluorescence intensity.

[0183] The results are as follows Fig.16 and Fig.17 The results showed that compared with the model group, all drug administrations could alleviate the decrease in oocyte membrane potential, especially HSYA, which had the most significant difference (P<0.01) ( Fig.16 ).

[0184] Example 10. Protective effect of natural herbal medicine components or their combination on fertility and litter birth in POI mice

[0185] The POI female mice after modeling and drug treatment in Example 4 were co-housed with wild-type C57 male mice at a ratio of 2:1 and observed for 2 months. The birth and littering conditions were recorded. Representative figures are shown in Fig.18 The results are as follows: three mice in the Vehicle group gave birth to 0, 1, and 3 pups; two mice in the ICA group gave birth to none; three mice in the HSYA group gave birth to 5, 8+2 (two litters), and 11 pups; three mice in the HSYA+ICA group gave birth to 1, 5+3 (two litters), and 6 pups. Fig.19 The results showed that both the HYSA administration group and the HSYA+ICA administration group could increase the number of litters born by mice.

[0186] Example 11. Protective effect of natural plant drug components on ovarian granulosa cells of mice damaged by chemotherapy

[0187] 4-HC (4-hydroxyperoxide cyclophosphamide), an active metabolite of cyclophosphamide, can cross-link DNA and induce T cell apoptosis independently of caspase receptor activation, activating the mitochondrial death pathway by generating reactive oxygen species (ROS).

[0188] The mouse KK1 granulosa cell line was treated with 8μM 4-HC for 24h to construct a cell model of chemotherapy-induced premature ovarian insufficiency. The experimental results showed that the IC50 of 4-HC-induced mouse KK1 granulosa cells was 8μM at 24h and 4μM at 48h. Fig. 20 A).

[0189] Dosage group:

[0190] After modeling, the drug was added and treated for 24 hours, and then the cell viability was detected using Cell Counting Kit-8 (CCK-8), Cell Proliferation-Toxicity Detection Kit (Dojindo, Japan). The drug administration components were:

[0191] (1) HSYA group: hydroxysafflor yellow A (HSYA);

[0192] (2) SAB group: salvianolic acid (SAB);

[0193] (3) Model group: 4-HC.

[0194] The effects of hydroxysafflor yellow A (HSYA) and salvianolic acid (SAB) on cell survival in a cell model of chemotherapy-induced premature ovarian insufficiency are shown in Fig. 20 B and Fig. 20 C. The experimental results showed that HSYA had a protective effect on the cell survival of chemotherapy-induced POI cell model at a concentration of 100 μM. SAB had a significant protective effect on cells at a concentration of 25 μM and above, and the effect increased in a dose-dependent manner.

[0195] Example 12. Effects of natural plant drug components on gene expression in ovarian granulosa cells of chemotherapy-damaged mice

[0196] Lhcgr is the receptor gene for luteinizing hormone, which can activate the cAMP signaling pathway; Sf1 is a nuclear transcription factor, also known as Nr5a1, which is involved in steroidogenesis, etc. Fshr is the receptor gene for follicle-stimulating hormone. Cdkn2a (P16) is an aging marker gene.

[0197] Ferroptosis is an iron-dependent programmed cell death mediated by lipid peroxidation. Increasing evidence shows that ferroptosis in oocytes and granulosa cells is strongly associated with the occurrence of POI and other reproductive diseases. The chemotherapy drug cyclophosphamide (CTX) is converted into its active metabolite 4-hydroxycyclophosphamide (4-HC) in vivo and produces more reactive oxygen species (ROS), which are involved in the initiation of ferroptosis in ovarian granulosa cells (GC) after exposure to CTX.

[0198] Glutathione peroxidase 4 (GPX4) is considered to be one of the main signs of ferroptosis. Under normal circumstances, GPX4 catalyzes the conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG); when GPX4 activity is inhibited, it promotes the accumulation of lipid peroxidation product malondialdehyde (MDA), leading to ferroptosis. Studies have shown that CTX induces ferroptosis in GCs by inhibiting the expression of GPX4, depletion of GSH and increase of MDA; and by regulating the generation of intracellular ROS and oxidative stress response, it blocks the occurrence of ferroptosis through the GPX4 pathway, protects GCs, and thus improves the number and quality of oocytes. Autophagy is a self-renewal process of eukaryotic cells to remove dysfunctional proteins and organelles. Excessive autophagy of GCs induced by CTX can lead to follicular atresia and ovarian dysfunction. NCOA4 plays a key role in the selective autophagy of ferritin. Studies have shown that knocking out NCOA4 can block the Fenton reaction and inhibit the occurrence of ferroptosis. Therefore, CTX can not only promote the occurrence of ovarian ferroptosis, but also induce excessive autophagy in ovarian GCs. Studies have reported that NCOA4-mediated iron autophagy can cause granulosa cell senescence.

[0199] Based on the above, this example used QPCR (the kit was purchased from Dakoway Biotechnology Co., Ltd.) to detect the expression levels of Lhcgr, Sf1, Fshr, Cdkn2a, and Ncoa4 and Gpx4 of ferroptosis and autophagy-related pathways in each medication group.

[0200] 1. For HSYA and icariin (ICT), KK1 cells were treated with 4 μM 4-HC and drugs for 24 h, and the drug administration groups were as follows:

[0201] (1) HYSA: 1 μM

[0202] (2) ICT: 1 μM

[0203] (3) HYSA+ICT: 1 μM each

[0204] (4) Control: equal volume of DMSO

[0205] (5) Vehicle: equal volume of DMSO

[0206] The experimental results are as follows Fig.21As shown in the figure, compared with the Control group, the mRNA levels of Lhcgr and Sf1 in the Vehicle group were significantly reduced, indicating that the model can reduce the mRNA expression levels of Lhcgr and Sf1 in mouse granulosa cells, and the model is established. Compared with the Vehicle group, the HSYA group can significantly increase the expression of the Sf1 gene and reduce the mRNA expression of the aging marker gene Cdkn2a (P16); compared with the Vehicle group, the ICT group and the HSYA+ICT group can also increase the expression of Sf1 and reduce the expression of Cdkn2a.

[0207] Fig. 22 The results showed that compared with the Control group, the mRNA expression level of Ncoa4 in the Vehicle group was upregulated (P<0.05), and the mRNA expression level of Gpx4 was downregulated. Compared with the Vehicle group, the mRNA expression level of Ncoa4 in the HSYA group was downregulated, and the mRNA expression level of Gpx4 was upregulated. Compared with the Vehicle group, the mRNA expression level of Ncoa4 in the ICT group was upregulated, and the mRNA expression level of Gpx4 was upregulated (P<0.05). Compared with the Vehicle group, the mRNA expression level of Ncoa4 in the HSYA+ICT group was downregulated (P<0.01; P<0.05), and the mRNA expression level of Gpx4 was upregulated. The above results indicate that HSYA and / or ICT can increase cell viability.

[0208] 2. For HSYA and SAB, 4 μM 4-HC was used to treat KK1 cells simultaneously with the drugs for 24 h. The drug administration groups were as follows:

[0209] (1) HYSA: 1 μM

[0210] (2)SAB: 1 μM

[0211] (3) HYSA+SAB: 1 μM each

[0212] (4) Control: equal volume of DMSO

[0213] (5) Vehicle: equal volume of DMSO

[0214] The experimental results are as follows Fig.23 As shown, the results showed that the HSYA group could significantly increase the expression of Fshr gene and reduce the expression of Cdkn2a gene, and SAB also had the above effects.

[0215] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of natural plant active ingredients in the preparation of a drug for preventing and / or treating ovarian dysfunction in a subject, wherein the natural plant active ingredients include active ingredients of safflower.

2. The use according to claim 1, characterized in that: The active ingredient of safflower includes hydroxysafflor yellow A or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

3. The use according to claim 1 or 2, characterized in that: The natural plant active ingredients also include active ingredients of Salvia miltiorrhiza and / or Epimedium; Preferably, the active ingredients of Danshen include salvianolic acid and / or tanshinone, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof; more preferably, the active ingredients of Danshen include salvianolic acid B and / or tanshinone IIA, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof; Preferably, the active ingredients of Epimedium include icariin, icariin and / or epimedin C, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof.

4. The use according to any one of claims 1 to 3, characterized in that: The ovarian dysfunction disease includes one or more of premature ovarian insufficiency, premature ovarian failure, diminished ovarian reserve function, ovarian hyporesponsiveness, early menopause, ovarian function damage, ovarian insufficiency, ovarian hypofunction, reproductive function decline, infertility, perimenopausal syndrome or menopausal syndrome; Preferably, the ovarian dysfunction disease is caused by genetic factors, iatrogenic factors, immune factors, environmental factors, advanced physiological age or other factors.

5. The use according to any one of claims 1 to 4, characterized in that: The natural plant active ingredients have at least one of the following uses: (1) increasing ovarian reserve; (2) restoring sex hormone secretion; (3) restoring and / or promoting ovulation; (4) restoring ovarian function; (5) promoting follicular development; (6) increasing the level of anti-Mullerian hormone (AMH) in serum; (7) alleviating oxidative stress damage; (8) increasing the level of superoxide dismutase (SOD) in serum; (9) increasing the level of reduced glutathione (GSH) and / or total antioxidant capacity (T-AOC) in serum, (10) reducing the level of malondialdehyde (MDA) in serum; (11) increasing the number of ovarian follicles and corpora lutea at all levels, and / or reducing the number of atretic follicles; (12) increasing the number of offspring; and (13) restoring the estrous cycle.

6. The use according to any one of claims 2 to 5, characterized in that: The active ingredients of Carthamus tinctorius, the active ingredients of Salvia miltiorrhiza and / or the active ingredients of Epimedium are applied simultaneously or sequentially.

7. The use according to any one of claims 2 to 6, characterized in that: The mass ratio of the active ingredient of safflower to the active ingredient of salvia miltiorrhiza is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:1; and / or The mass ratio of the active ingredient of safflower to the active ingredient of epimedium is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:

1.

8. A composition for preventing or treating ovarian disorders, characterized in that: The composition comprises an active ingredient of safflower, Preferably, the active ingredient of safflower includes hydroxysafflor yellow A or a solvate, hydrate or pharmaceutically acceptable salt thereof.

9. The composition according to claim 8, characterized in that The composition further comprises active ingredients of Danshen and / or Epimedium, Preferably, the active ingredients of Danshen include salvianolic acid and / or tanshinone, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof; more preferably, the active ingredients of Danshen include salvianolic acid B and / or tanshinone IIA, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof; Preferably, the active ingredients of the epimedium include icariin, icariin and / or epimedin C, or pharmaceutically acceptable salts, esters, hydrates or solvates thereof; Preferably, the mass ratio of the active ingredient of safflower to the active ingredient of salvia miltiorrhiza is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:1; and / or The mass ratio of the active ingredient of safflower to the active ingredient of epimedium is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:

1.

10. The composition according to any one of claims 8 or 9, characterized in that The composition further comprises a pharmaceutically acceptable excipient, Preferably, the pharmaceutically acceptable excipients include one or more of a pharmaceutical carrier, a diluent, an adjuvant and an excipient; Preferably, the composition is a tablet, capsule, solution, granule, pill, powder, ointment, pill, suspension, powder, injection, suppository, cream, spray, patch, sustained-release preparation, controlled-release preparation or targeted preparation.

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