Pharmaceutical composition for preventing or treating reproductive dysfunction of elderly women and application of pharmaceutical composition

Abstract

The invention relates to the technical field of medicine application, and discloses a medicine composition for preventing or treating reproductive dysfunction of elderly women and application of the medicine composition. The invention provides a new therapeutic drug and means for prevention and treatment of reproductive dysfunction of elderly women. By constructing an animal model of reproductive dysfunction of the elderly women and taking the ovulation number of animals, hormone level related to reproduction, pregnancy rate and progeny development condition as detection indexes, the pharmacodynamic effect and safety of the pharmaceutical composition and the influence of the pharmaceutical composition on progeny development quality and genetic variability are evaluated for the first time; the effectiveness and safety of the pharmaceutical composition for treating reproductive dysfunction of elderly women are fully proved.

Description

Technical Field

[0001] The present application relates to the technical field of drug use, and specifically to a drug composition for preventing or treating reproductive dysfunction in elderly women and its use. Background Art

[0002] In recent years, the phenomenon of late marriage and late childbearing has become increasingly common, and the group of elderly infertility patients has gradually increased. With the increase of age, the fertility of elderly women decreases, the hormone levels in the body become disordered, the ovarian function declines, the number and quality of follicles decrease, and the abnormal separation and structural and quantitative abnormalities of the eggs increase. Data show that the abnormal separation and structural and quantitative abnormalities of the eggs of elderly women are also the main causes of spontaneous abortion and the high incidence of fetal malformations. At present, the treatment methods for reproductive dysfunction in elderly women are still limited. How to improve the reproductive function of elderly pregnant women and the healthy development of their offspring has become a problem that needs to be solved urgently. Summary of the invention

[0003] In view of this, in order to solve the problem that the treatment methods for reproductive dysfunction in elderly women are relatively limited in the prior art, the present application provides a pharmaceutical composition for preventing or treating reproductive dysfunction in elderly women and its use. Experiments have proved that the pharmaceutical composition has a good pharmacodynamic effect of preventing and treating reproductive dysfunction in elderly women and / or offspring development.

[0004] In a first aspect, the present application provides a pharmaceutical composition for preventing or treating reproductive dysfunction in elderly women.

[0005] In some specific embodiments, the raw materials of the pharmaceutical composition include the following Chinese herbal medicine pieces by weight:

[0006] 5-15 parts of velvet antler, 10-30 parts of epimedium, 10-40 parts of curculigo, 10-30 parts of dipsaccharum, 10-30 parts of mistletoe, 15-50 parts of dodder seeds, 10-30 parts of wolfberry, 10-30 parts of raspberry, 30-60 parts of yam, 10-30 parts of lotus seeds, 15-50 parts of poria, 10-30 parts of astragalus, 10-50 parts of white peony root, 10-30 parts of spiny jujube seeds, 10-30 parts of Uncaria rhynchophylla, 15-50 parts of salvia miltiorrhiza, 15-50 parts of red peony root, and 30-60 parts of Millettia reticulata.

[0007] In some specific embodiments, the raw material composition of the pharmaceutical composition is:

[0008] 5 parts of velvet antler, 10 parts of roasted epimedium, 10 parts of curculigo, 10 parts of dipsaci, 10 parts of parasitic plant, 15 parts of dodder seeds, 10 parts of wolfberry fruits, 10 parts of raspberries, 30 parts of yam, 10 parts of lotus seeds, 15 parts of poria, 10 parts of astragalus, 15 parts of white peony root, 10 parts of stir-fried spiny jujube seeds, 10 parts of Uncaria rhynchophylla, 15 parts of salvia miltiorrhiza, 15 parts of red peony root, and 30 parts of Millettia reticulata.

[0009] In a second aspect, the present application provides a pharmaceutical preparation.

[0010] The pharmaceutical preparation comprises the pharmaceutical composition and / or its active extract.

[0011] In some embodiments, the pharmaceutical preparation further comprises a pharmaceutically acceptable excipient;

[0012] Optionally, the auxiliary materials include one or more of solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, stabilizers, flavoring agents, preservatives, suspending agents, coating materials, fragrances and thickeners.

[0013] Exemplarily, the raw materials are respectively passed through a 90-mesh sieve and then mixed into powder, 54 g of refined honey is added to every 100 g of powder to make honeyed pills, which are then dried below 50° C. and coated with peach gum or corn protein.

[0014] In some embodiments, the dosage form of the pre-drug preparation includes any pharmaceutically acceptable preparation;

[0015] Optionally, the dosage form of the pharmaceutical preparation is tablets, pills, decoctions, capsules, powders, drops, lyophilized products, granules, ointments, patches, suspensions, syrups, oral solutions, injections, suppositories or any combination thereof.

[0016] In a third aspect, the present application provides a method for preparing the pharmaceutical preparation.

[0017] In some specific embodiments, the method for preparing the drug includes the steps of weighing a certain amount of raw material components for compounding or extracting active ingredients and then compounding.

[0018] In some specific embodiments, the pharmaceutical preparation is a pill for regulating menstruation and promoting pregnancy. The prescription composition of the pill for regulating menstruation and promoting pregnancy is: 5g of velvet antler (dehaired), 10g of epimedium (roasted), 10g of curculigo, 10g of diaspermum, 10g of mistletoe, 15g of dodder seed, 10g of wolfberry, 10g of raspberry, 30g of yam, 10g of lotus seed (de-cored), 15g of tuckahoe, 10g of astragalus, 15g of white peony root, 10g of spiny jujube seed (fried), 10g of Uncaria rhynchophylla, 15g of salvia miltiorrhiza, 15g of red peony root, and 30g of Millettia reticulata. The above 18 raw materials are crushed into fine powder, sieved, and mixed. For every 100g of powder, add 40-50g of refined honey and an appropriate amount of water to make pills, dry them, make water-honey pills, and coat them with gelatin.

[0019] In a fourth aspect, the present application provides the use of the pharmaceutical composition and / or its active extract or the pharmaceutical preparation for preparing a drug for increasing the expression of genes associated with ovarian aging.

[0020] In some specific embodiments, the gene includes at least one of the Hfm-1 gene, the Gdf-9 gene, the Mtor gene, the Bax gene, and the Bcl-2 gene.

[0021] The pharmaceutical composition and / or its active extract or pharmaceutical preparation can increase the expression of genes related to ovarian aging and slow down ovarian aging.

[0022] In some embodiments, the drug for increasing the expression of genes associated with ovarian aging also has at least one of the following effects (1)-(3):

[0023] (1) Increase the level of estradiol E2 in serum;

[0024] (2) Reduce the level of follicle-stimulating hormone (FSH) in serum;

[0025] (3) Regulate the levels of progesterone P and / or luteinizing hormone LH in serum.

[0026] In some specific embodiments, the pharmaceutical composition and / or its active extract or pharmaceutical preparation is used to prepare a drug for preventing or treating reproductive dysfunction in elderly women and / or improving offspring development.

[0027] In some specific embodiments, the improving offspring development comprises increasing at least one of pregnancy rate, litter size, and live birth rate.

[0028] In some specific embodiments, the symptoms of reproductive dysfunction in advanced-age women include at least one of disordered hormone levels in the body, ovarian aging, and decreased number and / or quality of follicles.

[0029] Ovarian aging refers to the rapid decline in reproductive function in women over 35 years old, with clinical symptoms such as a decrease in the number and quality of eggs produced and a decrease in embryonic development potential.

[0030] In some embodiments, the subjects of application include humans and animals other than humans.

[0031] In some specific embodiments, the drug preparation (menstrual regulation and pregnancy promotion pills) is used orally; the adult dosage is 5 g once, twice a day, for 20 consecutive days starting from the fifth day of the menstrual cycle; those without cycles can take it for 20 consecutive days each month for three months or as directed by a doctor.

[0032] Compared with the prior art, the beneficial effects of this application are:

[0033] The present application provides a pharmaceutical composition for preventing or treating reproductive dysfunction in elderly women and its use. Provide new therapeutic drugs and means for the prevention and treatment of reproductive dysfunction in elderly women, thereby providing technical support for the rational application of drugs. By constructing an animal model of reproductive dysfunction in elderly women, using the number of animal ovulations, fertility-related hormone levels, pregnancy rate and offspring development as detection indicators, a detailed, intuitive and in-depth study was conducted to evaluate the pharmacodynamic effects and safety of the above-mentioned pharmaceutical composition for the first time, and the effects on the quality of offspring development and genetic variability. The results showed that the pharmaceutical composition has the effect of improving the reproductive function of elderly infertility model animals, increasing the pregnancy rate, litter size and live birth rate, and the potential mechanism of action is to regulate hormone levels and gene expression levels in reproductive organs that are closely related to ovarian function. For the first time, the effectiveness and safety of the pharmaceutical composition for reproductive dysfunction in elderly women have been fully demonstrated. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1a Comparison of Hfm-1 gene in ovaries and uteri of aged female mice and mice of appropriate age;

[0036] Figure 1b Comparison of Gdf-9 gene in ovaries and uteri of aged female mice and mice of appropriate age;

[0037] Figure 1c Comparison of Bax gene in ovaries and uteri of aged female mice and mice of appropriate age;

[0038] Figure 1d Comparison of Bcl-2 gene in ovaries and uteri of aged female mice and mice of appropriate age;

[0039] Figure 1e Comparison of Mtor gene in ovaries and uteri of aged female mice and mice of appropriate age;

[0040] Figure 1a-1e In the table, ovary refers to the ovary, and uterus refers to the uterus; 13M refers to 13-month-old female mice, and 3M refers to 3-month-old female mice.

[0041] Figure 2 The effect of the pharmaceutical composition of the present application on the serum levels of relevant hormones (estradiol E2, progesterone P, follicle-stimulating hormone FSH, and luteinizing hormone LH) in aged mice after one week of intervention;

[0042] Figure 3 The effect of the pharmaceutical composition of the present application on the serum levels of relevant hormones (estradiol E2, progesterone P, follicle-stimulating hormone FSH, and luteinizing hormone LH) in aged mice after 2 weeks of intervention;

[0043] Figure 4 Effect of the pharmaceutical composition of the present application on the serum levels of relevant hormones (estradiol E2, progesterone P, follicle-stimulating hormone FSH, and luteinizing hormone LH) in aged mice after 3 weeks of intervention;

[0044] Figure 5a Comparison of Hfm-1 gene in mouse ovaries and uteri after 1 week of drug administration;

[0045] Figure 5b Comparison of Gdf-9 gene in mouse ovary and uterus after 1 week of drug administration;

[0046] Figure 5c Comparison of Bax gene in mouse ovary and uterus after 1 week of drug administration;

[0047] Figure 5d Comparison of Bcl-2 gene in mouse ovary and uterus after 1 week of drug administration;

[0048] Figure 5e Comparison of Mtor gene in mouse ovary and uterus after 1 week of drug administration;

[0049] Figure 5a-5e Ovary refers to ovary, uterus refers to uterus; H-13M refers to female mice in the high-dose group, L-13M refers to female mice in the low-dose group, and C-13M refers to female mice in the control group;

[0050] Figure 6a Comparison of Hfm-1 gene in mouse ovaries and uteri after 2 weeks of drug administration;

[0051] Figure 6b Comparison of Gdf-9 gene in mouse ovary and uterus after 2 weeks of drug administration;

[0052] Figure 6c Comparison of Bax gene in mouse ovary and uterus after 2 weeks of drug administration;

[0053] Figure 6d Comparison of Bcl-2 gene in mouse ovary and uterus after 2 weeks of drug administration;

[0054] Figure 6e Comparison of Mtor gene in mouse ovary and uterus after 2 weeks of drug administration;

[0055] Figure 6a-6eIn the table, ovary refers to the ovary, uterus refers to the uterus; H-13M refers to the female mice in the high-dose group, L-13M refers to the female mice in the low-dose group, and C-13M refers to the female mice in the control group;

[0056] Figure 7a Comparison of Hfm-1 gene in mouse ovaries and uteri after 3 weeks of drug administration;

[0057] Figure 7b Comparison of Gdf-9 gene in mouse ovary and uterus after 3 weeks of drug administration;

[0058] Figure 7c Comparison of Bax gene in mouse ovary and uterus after 3 weeks of drug administration;

[0059] Figure 7d Comparison of Bcl-2 gene in mouse ovary and uterus after 3 weeks of drug administration;

[0060] Figure 7e Comparison of Mtor gene in mouse ovary and uterus after 3 weeks of drug administration;

[0061] Figure 7a-7e L-13M refers to female mice in the low-dose group, and C-13M refers to female mice in the control group;

[0062] Figure 8 The results of the thrombus detection experiment after mating of mice;

[0063] Fig. 9 It is the comparison result of DNA sequence sequencing. DETAILED DESCRIPTION

[0064] The following examples are provided for a better understanding of the present application, but are not limited to the best implementation mode described, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the protection scope of the present application.

[0065] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0066] In the present application, the selection scope involving "and / or", "or / and", and "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

[0067] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0069] In some embodiments, the menstruation regulating and pregnancy promoting pills can also be used to prepare drugs for preventing or treating reproductive dysfunction in elderly women.

[0070] When the Tiaojing Cuyan Pills are the only active ingredient in the drug for preventing or treating reproductive dysfunction in elderly women, the usage of the Tiaojing Cuyan Pills is to take them orally, 5g at a time, twice a day, for 20 consecutive days starting from the fifth day of the menstrual cycle. For those without a cycle, take them for 20 consecutive days every month for three months or follow the doctor's advice.

[0071] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0072] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0073] Example 1

[0074] The drug for preventing or treating reproductive dysfunction in elderly women described in this embodiment comprises the following ingredients:

[0075] Deer antler (dehaired) 5g, roasted epimedium 10g, curculigo 10g, dipsacus 10g, mistletoe 10g, dodder seed 15g, wolfberry 10g, raspberry 10g, yam 30g, lotus seed (decored) 10g, poria 15g, astragalus 10g, white peony 15g, stir-fried spiny jujube kernel 10g, Uncaria rhynchophylla 10g, salvia miltiorrhiza 15g, red peony root 15g, Millettia reticulata 30g.

[0076] The preparation method for preventing or treating reproductive dysfunction in elderly women described in this embodiment comprises:

[0077] The above raw materials were crushed into fine powder, passed through a 90-mesh sieve, and mixed evenly. For every 100g of powder, 40g of refined honey and appropriate amount of water were added to make pills, dried, and made into water-honey pills.

[0078] Example 2

[0079] The drug for preventing or treating reproductive dysfunction in elderly women described in this embodiment includes the following ingredients: 5g of pilose antler, 30g of epimedium, 10g of curculigo, 30g of dipsacus, 10g of mistletoe, 50g of dodder seed, 10g of wolfberry, 30g of raspberry, 30g of yam, 30g of lotus seed, 15g of poria, 30g of astragalus, 10g of white peony root, 30g of spiny jujube seed, 10g of Uncaria rhynchophylla, 50g of salvia miltiorrhiza, 15g of red peony root, and 60g of Millettia reticulata.

[0080] The preparation method for preventing or treating reproductive dysfunction in elderly women described in this embodiment comprises: crushing the above raw materials into fine powder, passing through a 90-mesh sieve, mixing, and pressing into tablets to form tablets.

[0081] Example 3

[0082] The drug for preventing or treating reproductive dysfunction in elderly women described in this embodiment comprises the following ingredients:

[0083] Deer antler 15g, epimedium 10g, curculigo 40g, dipsaccharum 10g, mistletoe 30g, dodder seed 15g, wolfberry 30g, raspberry 10g, yam 60g, lotus seed 10g, poria 50g, astragalus 10g, white peony 50g, spiny jujube seed 10g, Uncaria rhynchophylla 30g, salvia miltiorrhiza 15g, red peony root 50g, Millettia reticulata 30g.

[0084] The preparation method for preventing or treating reproductive dysfunction in elderly women described in this embodiment comprises: crushing the above raw materials into fine powder, passing through a 200-mesh sieve, mixing, making a soft material with an appropriate amount of 80% ethanol, granulating, drying, and making capsules.

[0085] Experimental Example 1

[0086] A mouse model of reproductive dysfunction in elderly women was established by selecting 12-13 month old mice, and the drug composition prepared in the present application was administered to the mice, and the pharmacodynamics, safety and mechanism of action of the drug composition prepared in the present application were evaluated.

[0087] 1. Model establishment

[0088] 1.1 Establishment and grouping of wild-type advanced-age infertile mouse model

[0089] In this experiment, mice aged 12 to 13 months were selected to simulate clinical elderly infertile female patients.

[0090] Experimental animals: BALB / c mice (albino laboratory mice) were obtained from the Institute of Laboratory Animal Resources, China Food and Drug Inspection Institutes. The experimental animal production license number is SCXK (Beijing) 2022-0002, and the experimental animal use license number is SYXK (Beijing) 2022-0014.

[0091] Age: 12-13 months, Number: 90, Female;

[0092] 3 months old, 3 females.

[0093] The animal grouping and experimental environment adaptation period is 5 to 7 days. During this period, the mice were fed SPF grade pellet feed and free to drink sterilized tap water. The room temperature was 20 to 26°C, the relative humidity was 40% to 70%, and the lighting was 12 hours light / 12 hours dark.

[0094] Three wild-type female mice aged 12-13 months and three wild-type female mice aged 3 months were randomly selected to verify the mRNA levels of five genes related to ovarian aging: Hfm-1, Mtor, Gdf-9, Bcl-2, and Bax. The results are as follows: Figure 1a-1e (* indicates P < 0.05, ** indicates P < 0.01) Figure 1a-1e Comparison of ovarian and uterine-related regulatory genes between mice with ovarian dysfunction and mice of appropriate age; ovary refers to ovary, uterus refers to uterus; 13M refers to 13-month-old female mice, and 3M refers to 3-month-old female mice of appropriate age; Figure 1a Comparison of Hfm-1 gene in ovaries and uteri of aged female mice and mice of appropriate age; Figure 1b Comparison of Gdf-9 gene in ovaries and uteri of aged female mice and mice of appropriate age; Figure 1c Comparison of Bax gene in ovaries and uteri of aged female mice and mice of appropriate age; Figure 1d Comparison of Bcl-2 gene in ovaries and uteri of aged female mice and mice of appropriate age; Figure 1e This is a comparison of the Mtor gene in the ovaries and uterus of elderly female mice and mice of appropriate age. The results showed that the expression levels of the five genes selected, Hfm-1, Mtor, Gdf-9, Bcl-2, and Bax, in the ovaries and uterus of wild-type elderly mice were significantly lower than those in the ovaries and uterus of mice of appropriate age. The data showed that the elderly mice selected for the experiment did show symptoms of ovarian dysfunction, and the screening results of related genes were also consistent with the expected downregulation of gene expression levels. The results showed that 12-13 month old wild-type female mice can be used for modeling, and the above genes can be used for the next step of testing.

[0095] 2. Grouping and Dosing

[0096] 2.1 Dosing information for establishing a wild-type elderly infertile mouse model

[0097] In this experiment, female mice aged 12 to 13 months were divided into 3 groups, a high-dose group, a low-dose group and a control group. The pharmaceutical composition prepared in Example 1 was mixed with pure water to form a suspension, and the high and low doses were 10.275 g / kg and 2.055 g / kg, respectively; the control group was given pure water, as shown in Table 1. The animals were given medication 3 weeks before mating, and the medication was given by gavage daily, 5 days a week, and the medication cycle was 3 weeks.

[0098] Table 1 Grouping and dosing information of the wild-type advanced-age infertile mouse model experiment

[0099] Group Number of animals Dosage (g / kg) High dose group (H) 50 10.275 Low dose group (L) 50 2.055 Control group (C) 50 /

[0100] 3. Sample collection and indicator testing

[0101] 3.1 Test methods

[0102] Peripheral blood was collected from the eye sockets of wild-type elderly infertility model mice in each group 1 week, 2 weeks, and 3 weeks after oral administration. The mouse blood was centrifuged at 3000 rpm / min for 15 min to separate the serum. The levels of estradiol (E2), progesterone (PROG), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum were determined by enzyme-linked immunosorbent assay using an enzyme-linked immunosorbent assay kit according to the instructions. The uterus and ovaries were also collected, and the uterine and ovarian functional genes were detected using q-PCR technology.

[0103] After 3 weeks of administration, 10 female mice were randomly selected from each group and mated with male mice in a 1:1 ratio. The mating was carried out every other day and the vaginal plug was recorded. The female mice were put together at 17:00 every day (marked as 0d). The vaginal plug of the female mice was checked at 9:00 the next day (marked as 0+1d). The female mice with successful plug detection were kept in a single cage for delivery, and the plug detection rate was recorded. After 14 days of plug detection, the female mice waiting to deliver were selected to confirm the pregnancy rate. After the expected delivery date, the late pregnancy rate, the number of litters born by female mice, the number of weaned and live fetuses, and the genetic variation of the newborn mice were further recorded; the physiological and biochemical indicators and organ index indicators of the mice before and after administration were measured.

[0104] 4. Experimental Results

[0105] 4.1 Effects of drug composition on sex hormone levels in model animals

[0106] After one week of administration, the serum levels of estradiol E2, progesterone P, follicle-stimulating hormone FSH, and luteinizing hormone LH were measured in 3 mice in each of the high-dose, low-dose, and control groups. The specific results are as follows: Figure 2 As shown (* indicates P < 0.05, ** indicates P < 0.01). Figure 2 It can be seen that compared with the control group, the serum estradiol E2 content and progesterone P index in the high-dose group and the low-dose group increased, and the follicle-stimulating hormone FSH and luteinizing hormone LH content decreased; in the luteinizing hormone LH index, there were extremely significant differences between the high-dose and low-dose groups compared with the control group (P<0.01), and there were significant differences between the low-dose group and the high-dose group (P<0.05), and there was a dose effect.

[0107] After 2 weeks of administration, the serum levels of estradiol E2, progesterone P, follicle-stimulating hormone FSH, and luteinizing hormone LH were measured in 3 mice in each of the high-dose, low-dose, and control groups. The specific results are as follows: Figure 3 As shown (* indicates P < 0.05, ** indicates P < 0.01). Figure 3 It can be seen that compared with the control group, the serum estradiol E2 content and progesterone P index in the high-dose and low-dose groups increased, the follicle-stimulating hormone FSH decreased, and the luteinizing hormone content remained basically the same; there was a fluctuation trend but the difference was not significant.

[0108] After 3 weeks of administration, the serum levels of estradiol E2, progesterone P, follicle-stimulating hormone FSH, and luteinizing hormone LH in the 6 low-dose mice and the 6 control mice were measured. The specific results are as follows: Figure 4 As shown (* indicates P < 0.05, ** indicates P < 0.01). Figure 4 It can be seen that compared with the control group, the serum estradiol E2 content in the low-dose group increased and there was a very significant difference (P<0.01); the progesterone P content remained basically the same, and the follicle-stimulating hormone FSH and luteinizing hormone LH contents decreased.

[0109] 4.2 Effects of the drug composition on ovarian and uterine gene expression levels in model animals

[0110] After one week of administration, the expression levels of each gene in the ovarian and uterine tissues of the three groups of mice were detected. Figure 5a-5e (* indicates P < 0.05, ** indicates P < 0.01), where ovary refers to ovary, uterus refers to uterus; H-13M refers to female mice in the high-dose group, L-13M refers to female mice in the low-dose group, and C-13M refers to female mice in the control group; Figure 5a Comparison of Hfm-1 gene in mouse ovaries and uteri after 1 week of drug administration; Figure 5b Comparison of Gdf-9 gene in mouse ovary and uterus after 1 week of drug administration; Figure 5c Comparison of Mtor gene in mouse ovary and uterus after 1 week of drug administration; Figure 5d Comparison of Bax gene in mouse ovary and uterus after 1 week of drug administration; Figure 5eThis is a comparison of Bcl-2 gene in the ovary and uterus of mice after 1 week of administration. Compared with the control group, the expression of Hfm-1 gene increased in the ovary and uterus of the high-dose group, and the expression level of ovary in the low-dose group was basically the same, and the expression level of uterus increased; for Gdf-9 gene and Mtor gene, compared with the control group, the expression level of ovary in the high-dose group and the low-dose group was basically the same, and the gene expression level of uterus was upregulated; compared with the control group, the relative expression of Bax gene was slightly lower in the ovary of the high-dose group and the low-dose group than the control group, still indicating the characteristics of functional impairment; the expression level of uterus in the high-dose group and the low-dose group was basically the same; the relative expression of Bcl-2 gene, compared with the control group, the expression level of ovary and uterus in the high-dose group and the low-dose group was high and low, and the reason needs further testing. In summary, the test substance can increase the expression level of genes related to reproductive organ dysfunction, indicating that the test substance may have a positive regulatory effect on inhibiting functional impairment.

[0111] After 2 weeks of administration, the expressions of related genes in the ovaries and uteri of the three groups of mice were detected. Figure 6a -e (* indicates P < 0.05, ** indicates P < 0.01), ovary refers to ovary, uterus refers to uterus; H-13M refers to female mice in the high-dose group, L-13M refers to female mice in the low-dose group, and C-13M refers to female mice in the control group. Figure 6a Comparison of Hfm-1 gene in mouse ovaries and uteri after 2 weeks of drug administration; Figure 6b Comparison of Gdf-9 gene in mouse ovary and uterus after 2 weeks of drug administration; Figure 6c Comparison of Mtor gene in mouse ovary and uterus after 2 weeks of drug administration; Figure 6d Comparison of Bax gene in mouse ovary and uterus after 2 weeks of drug administration; Figure 6e Comparison of Bcl-2 gene in the ovary and uterus of mice 2 weeks after administration; the expression of Hfm-1 gene in the ovary and uterus of the high-dose group and the low-dose group was lower than that in the control group, and the expression level in the uterus showed a dose effect, showing that the expression level in the high-dose group was higher than that in the low-dose group; the expression levels of Gdf-9 and Mtor genes in the ovary and uterus of the high-dose group and the low-dose group were basically the same and upregulated compared with the control group; the expression level of Bax gene in the ovary of the high-dose group and the low-dose group was slightly lower than that in the control group, and the expression characteristics of the functional impairment model still appeared; the expression level in the uterus of the high-dose group and the low-dose group was higher than that in the control group; the relative expression of Bcl-2 gene, the expression level in the ovary of the high-dose group increased and was statistically different from that in the low-dose group, and the expression level in the uterus of the low-dose group was significantly higher than that in the control group (P<0.05); comprehensive analysis showed that the effect of the test substance on the expression of related genes still existed 2 weeks after administration.

[0112] To further confirm the trend of gene expression level changes, the sample size was increased. After 3 weeks of administration, the ovaries and uteri of 6 mice in the low-dose group and 3 mice in the control group were randomly selected for gene mRNA expression level detection. The results are as follows: Figure 7a-7e (* indicates P < 0.05, ** indicates P < 0.01), H-13M refers to the female mice in the high-dose group, L-13M refers to the female mice in the low-dose group, and C-13M refers to the female mice in the control group.

[0113] Figure 7a Comparison of Hfm-1 gene in mouse ovaries and uteri after 3 weeks of drug administration; Figure 7b Comparison of Gdf-9 gene in mouse ovary and uterus after 3 weeks of drug administration; Figure 7c Comparison of Mtor gene in mouse ovary and uterus after 3 weeks of drug administration; Figure 7d Comparison of Bax gene in mouse ovary and uterus after 3 weeks of drug administration; Figure 7e Comparison of Bcl-2 gene in mouse ovary and uterus after 3 weeks of administration. The expression of Hfm-1 gene in the ovary and uterus of the low-dose group was lower than that in the control group; the relative expression of Gdf-9 gene, Mtor gene, Bax and Bcl-2 gene, the expression level of low-dose ovary and uterus was up-regulated compared with the control group; the expression characteristics of ovarian and uterine function recovery of the functional decline model were shown, indicating that the pharmaceutical composition of the present application has a positive regulatory effect in correcting functional decline.

[0114] 4.3 Effects of the pharmaceutical composition of the present application on the reproductive function of model animals and the development of offspring

[0115] Three weeks after administration, 10 mice were randomly selected from the high-dose group, 15 mice were randomly selected from the low-dose group and 15 mice were randomly selected from the control group according to the number of animals. They were mated with male mice at a 1:1 ratio, and the thrombus detection rate, pregnancy rate, weaning rate and litter size were recorded.

[0116] See the pictures of thrombus detection experiment Figure 8 , the left picture shows that the thrombus detection was unsuccessful, and the right picture shows that the thrombus detection was successful. In the high-dose group, 10 out of 10 female mice successfully detected thrombi, in the low-dose group, 11 out of 15 female mice successfully detected thrombi, and in the control group, 10 out of 15 female mice successfully detected thrombi, and the thrombus detection success rates (%) were 100%, 73.3%, and 66.7%, respectively. After 20 days of pregnancy, the detection statistics showed that 4 female mice in the high-dose group were successfully pregnant, 3 female mice in the low-dose group were successfully pregnant, and 3 female mice in the control group were successfully pregnant, and the pregnancy rates were 40%, 27.3%, and 30.0%, respectively. After 21 days of pregnancy, one female mouse in the high-dose group gave birth smoothly, and 3 pups were born and survived and weaned smoothly. The female mice in the low-dose group and the control group all died of dystocia. The pregnancy parameters are shown in Table 2. The experimental results show that the pharmaceutical composition of the present application can improve the thrombus detection rate, pregnancy rate, and promote normal production in elderly mice.

[0117] Table 2 Pregnancy parameters of female mice in each group after mating after drug administration

[0118]

[0119] 4.4 Effect of the drug composition of the present application on the safety of use and genetic variation of offspring

[0120] Considering that the organ coefficients and blood physiological and biochemical parameters of experimental animals are often used in preclinical toxicology studies of drugs, 6 female mice were randomly selected from the high-dose group, low-dose group and control group for blood physiological and biochemical indexes and organ coefficient index determination after 3 weeks of administration. The results of body weight and organ coefficient determination are shown in Table 3, the overall mean results of biochemical indicators are shown in Table 4, and the overall mean results of blood physiological indicators are shown in Table 5.

[0121] Table 3 Absolute index of organs of female mice in each group after administration (g)

[0122] organ High dose group Low dose group Control group weight 23.21±0.92 24.99±2.45 23.52±1.89 heart 0.12±0.01 0.11±0.01 0.11±0.02 liver 0.98±0.04 0.95±0.09 1.00±0.13 brain 0.44±0.01 0.41±0.03 0.42±0.02 spleen 0.10±0.02 0.09±0.02 0.10±0.02 kidney 0.28±0.02 0.27±0.03 0.28±0.04 Thymus 0.03±0.01 0.04±0.01 0.03±0.01 Lungs (including bronchi) 0.18±0.01 0.18±0.04 0.20±0.04 Adrenal glands 0.01±0.00 0.01±0.00 0.01±0.00 Ovaries 0.01±0.00 0.01±0.00 0.02±0.00 Uterus 0.12±0.04 0.15±0.14 0.17±0.08

[0123] Table 4 Blood biochemical indexes test results

[0124]

[0125]

[0126] Table 5 Blood physiological index measurement results

[0127]

[0128]

[0129] Table 3 Body mass and organ coefficient measurement results The absolute value results of organs showed that after 3 weeks of administration, there was no significant difference in the mass of various organs of female mice in the high-dose group, low-dose group and control group.

[0130] As can be seen from the results in Table 4 , there were no statistical differences in blood biochemical indices among mice in each group after 3 weeks of administration;

[0131] From the results in Table 5, it can be seen that there is no statistical difference in the blood physiological data of mice in each group after 3 weeks of administration.

[0132] The above experiments show that the pharmaceutical composition of the present application does not cause damage to the liver, kidneys, and other organs of the recipient mice and has good safety.

[0133] One litter of offspring (including unviable offspring) and their parents from the high-dose group, low-dose group and control group was randomly selected, and genomic DNA was extracted to detect the variation of 10 SNP sites. PCR amplification and direct sequencing were used to perform forward and reverse sequencing of the 10 SNP sites. The comparison results of some DNA sequence sequencing are shown in Fig. 9 No mutations were found after DNA sequence comparison between the offspring and parents in the three groups, indicating that the test substance did not cause gene mutations or chromosomal aberrations in the offspring.

[0134] The drug composition used in the above test can be replaced by the menstruation regulating and pregnancy promoting pills of equal quality (purchased from Tong Ren Tang Pharmaceutical Factory of Beijing Tong Ren Tang Co., Ltd., 22035305), with the same effect, which will not be described here.

[0135] In summary, this experiment adopted an advanced-age infertile mouse model to conduct a pharmacodynamic evaluation of the pharmaceutical composition of the present application. First, after the pharmaceutical composition of the present application intervened in wild-type advanced-age mice, the pregnancy rate and weaning rate were improved and the elderly female mice successfully gave birth to healthy pups; secondly, the study showed that its mechanism of action was related to the regulation of animal hormone levels and ovarian and uterine function gene expression at different stages; finally, the safety test based on the wild-type advanced-age infertile model mouse model showed that the pharmaceutical composition of the present application did not cause abnormalities in the weight, organ index, physiological and biochemical indicators of the mother mice after administration, and no adverse results were found in the genetic detection indicators, indicating that the drug safety is good.

[0136] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection created by this application.

Claims

1. A pharmaceutical composition for preventing or treating reproductive dysfunction in elderly women, characterized in that: The raw materials of the pharmaceutical composition include the following Chinese herbal medicine pieces by weight: 5-15 parts of velvet antler, 10-30 parts of epimedium, 10-40 parts of curculigo, 10-30 parts of dipsaccharum, 10-30 parts of mistletoe, 15-50 parts of dodder seeds, 10-30 parts of wolfberry, 10-30 parts of raspberry, 30-60 parts of yam, 10-30 parts of lotus seeds, 15-50 parts of poria, 10-30 parts of astragalus, 10-50 parts of white peony root, 10-30 parts of spiny jujube seeds, 10-30 parts of Uncaria rhynchophylla, 15-50 parts of salvia miltiorrhiza, 15-50 parts of red peony root, and 30-60 parts of Millettia reticulata.

2. The pharmaceutical composition according to claim 1, characterized in that The raw material composition of the pharmaceutical composition is: 5 parts of velvet antler, 10 parts of roasted epimedium, 10 parts of curculigo, 10 parts of dipsaci, 10 parts of parasitic plant, 15 parts of dodder seeds, 10 parts of wolfberry fruits, 10 parts of raspberries, 30 parts of yam, 10 parts of lotus seeds, 15 parts of poria, 10 parts of astragalus, 15 parts of white peony root, 10 parts of stir-fried spiny jujube seeds, 10 parts of Uncaria rhynchophylla, 15 parts of salvia miltiorrhiza, 15 parts of red peony root, and 30 parts of Millettia reticulata.

3. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 1 or 2 and / or its active extract.

4. The pharmaceutical preparation according to claim 3, characterized in that The pharmaceutical preparation also includes pharmaceutically acceptable excipients; Optionally, the auxiliary materials include one or more of solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, stabilizers, flavoring agents, preservatives, suspending agents, coating materials, fragrances and thickeners.

5. The pharmaceutical preparation according to claim 3 or 4, characterized in that The dosage form of the pharmaceutical preparation includes pharmaceutically acceptable preparations; Optionally, the dosage form of the pharmaceutical preparation includes tablets, pills, decoctions, capsules, powders, drops, lyophilized products, granules, ointments, patches, suspensions, syrups, oral solutions, injections, suppositories or any combination thereof.

6. A method for preparing the pharmaceutical preparation according to any one of claims 3 to 5, characterized in that: The preparation method of the pharmaceutical preparation comprises the steps of weighing a certain amount of raw material components for compounding or extracting active ingredients for compounding.

7. Use of the pharmaceutical composition according to claim 1 or 2 and / or its active extract or the pharmaceutical preparation according to any one of claims 3 to 5 for preparing a drug for increasing the expression of genes associated with ovarian aging, wherein the genes include at least one of the Hfm-1 gene, the Gdf-9 gene, the Mtor gene, the Bax gene, and the Bcl-2 gene.

8. The use according to claim 7, characterized in that The drug for increasing the expression of genes associated with ovarian aging also has at least one of the following effects (1)-(3): (1) Increase the level of estradiol E2 in serum; (2) Reduce the level of follicle-stimulating hormone (FSH) in serum; (3) Regulate the levels of progesterone P and / or luteinizing hormone LH in serum.

9. The use according to claim 8, characterized in that Use of the pharmaceutical composition and / or active extract thereof according to claim 1 or 2 or the pharmaceutical preparation according to any one of claims 3 to 5 for preparing a drug for preventing or treating reproductive dysfunction in elderly women and / or improving offspring development.

10. The use according to claim 9, characterized in that The symptoms of reproductive dysfunction in advanced-age women include at least one of disordered hormone levels, ovarian aging, and decreased number and / or quality of follicles; And / or, the improving offspring development includes increasing at least one of pregnancy rate, litter size, and live birth rate.

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