CN114949099B

CN114949099B - Traditional Chinese medicine composition for resisting premature ovarian failure and aging and application thereof

Info

Publication number: CN114949099B
Application number: CN202210511029.0A
Authority: CN
Inventors: 杨慧龄; 李娇; 周本杰; 何滔
Original assignee: Guangdong Medical University
Current assignee: Guangdong Medical University
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-04-28
Anticipated expiration: 2042-05-11
Also published as: CN114949099A

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for resisting premature ovarian failure and aging and application thereof. The traditional Chinese medicine composition for resisting premature ovarian failure and aging comprises, by mass, 10-50 parts of prepared rehmannia root, 20-50 parts of semen cuscutae, 1-50 parts of Chinese angelica, 10-17 parts of medlar, 1-50 parts of epimedium, 1-50 parts of morinda officinalis, 10-50 parts of Chinese yam, 1-50 parts of poria cocos, 10-50 parts of red sage root, 1-50 parts of ligusticum wallichii, 1-50 parts of radix bupleuri, 1-50 parts of nutgrass galingale rhizome, 1-50 parts of moutan bark and 1-10 parts of liquorice. The traditional Chinese medicine composition for resisting premature ovarian failure and aging can effectively improve the estrus cycle disorder of an organism and the endocrine dysfunction of the ovary of the organism, thereby effectively improving the symptoms of premature ovarian failure and aging.

Description

Traditional Chinese medicine composition for resisting premature ovarian failure and aging and application thereof

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for resisting premature ovarian failure and aging and application thereof.

Background

Premature ovarian failure (premature ovarian failure, POF) refers to a group of gynaecological diseases characterized by elevated concentrations of serum follicle stimulating hormone (follicle stimulating hormone, FSH) and luteinizing hormone (luteinizing hormone, LH) and reduced concentrations of Estradiol (E2) in women before age 40, manifested by prolonged menstrual cycle and even amenorrhea, resulting in infertility due to reduced reproductive function. Most patients can see low-stimulin symptoms such as flushing of the face, decreased libido, hot flushes, hyperhidrosis, infertility and the like. The occurrence of the disease not only affects the normal life and health of the patient, but also greatly affects the psychological of the patient, and various serious complications such as heart disease, osteoporosis and the like can be caused along with the development of the disease. In recent years, the incidence of POF has been increasing and there has been a trend toward younger development, with the incidence of women under 40 being 1-3% and the incidence of women under 30 being 0.1%.

POF is currently thought to be the result of multi-factor, multi-pathway coaction, including oxidative stress, autoimmune disease, altered telomere length, altered telomerase activity, deregulation of the neuro-endocrine system, and the like; hormone replacement therapy is often adopted by Western medicine, and although the curative effect is satisfactory, serious complications such as uterine bleeding, endometrial cancer and breast cancer can be caused by long-term large-dose use of estrogen.

Therefore, there is a need for an effective agent against premature ovarian failure and aging with little complications.

Disclosure of Invention

Aiming at the problems, the invention aims to provide an anti-POF or anti-ovarian aging traditional Chinese medicine composition and various applications thereof, wherein the traditional Chinese medicine composition can effectively improve the disorder of the estrus cycle of an organism and the endocrine dysfunction of the ovary of the organism, thereby effectively improving the symptoms of premature ovarian failure and ovarian aging.

The invention provides a traditional Chinese medicine composition for resisting premature ovarian failure and aging, which comprises, by mass, 10-50 parts of prepared rehmannia root, 20-50 parts of semen cuscutae, 1-50 parts of Chinese angelica, 10-17 parts of medlar, 1-50 parts of epimedium herb, 1-50 parts of morinda officinalis, 10-50 parts of Chinese yam, 1-50 parts of poria cocos, 10-50 parts of red sage root, 1-50 parts of ligusticum wallichii, 1-50 parts of radix bupleuri, 1-50 parts of rhizoma cyperi, 1-50 parts of moutan bark and 1-10 parts of liquorice.

In some specific embodiments, the traditional Chinese medicine composition for resisting premature ovarian failure and aging can comprise, by mass, 18-22 parts of prepared rehmannia root, 13-17 parts of semen cuscutae, 8-12 parts of Chinese angelica, 13-17 parts of medlar, 8-12 parts of epimedium herb, 8-12 parts of morinda officinalis, 13-17 parts of Chinese yam, 8-12 parts of poria cocos, 13-17 parts of red sage root, 8-12 parts of ligusticum wallichii, 8-12 parts of radix bupleuri, 8-12 parts of rhizoma cyperi, 8-12 parts of moutan bark and 4-8 parts of liquorice.

In some specific embodiments, the above-mentioned traditional Chinese medicine composition for resisting premature ovarian failure and aging may include, by mass, 20 parts of radix rehmanniae Preparata, 15 parts of semen Cuscutae, 10 parts of radix Angelicae sinensis, 15 parts of fructus Lycii, 10 parts of herba Epimedii, 10 parts of radix Morindae officinalis, 15 parts of rhizoma Dioscoreae, 10 parts of Poria, 15 parts of radix Salviae Miltiorrhizae, 10 parts of rhizoma Chuanxiong, 10 parts of radix bupleuri, 10 parts of rhizoma Cyperi, 10 parts of cortex moutan, and 6 parts of radix Glycyrrhizae.

In some embodiments, the preparation method of the traditional Chinese medicine composition for resisting premature ovarian failure and aging comprises the following steps:

the invention also provides a medicament containing the traditional Chinese medicine composition for resisting premature ovarian failure and aging, and the medicament can also comprise pharmaceutically acceptable auxiliary materials.

In certain embodiments, the dosage form of the medicament may be any of a tablet, powder, capsule, granule, or nano-formulation.

The invention provides an application of the traditional Chinese medicine composition in preparation of a preparation for treating premature ovarian failure and delaying ovarian aging.

The invention also provides application of the traditional Chinese medicine composition for resisting premature ovarian failure and aging in preparation of a preparation for improving the estrus cycle disorder of an organism.

The invention also provides application of the traditional Chinese medicine composition for resisting premature ovarian failure and aging in preparation of a preparation for improving endocrine dysfunction of the ovary of the organism.

In certain embodiments, the formulation may be in the form of any of a tablet, powder, capsule, granule, or nano-formulation.

The invention has the beneficial effects that: (1) The traditional Chinese medicine composition for resisting premature ovarian failure and aging can enable the wet weight and the ovarian index of the ovarian injury model rat caused by the chemotherapeutic medicine to be recovered to be normal; (2) The traditional Chinese medicine composition for resisting premature ovarian failure and aging can effectively improve the estrus cycle disorder of an organism and recover the normal development of follicles; (3) The traditional Chinese medicine composition for resisting premature ovarian failure and aging can effectively improve the disorder of ovarian endocrine function caused by a chemotherapeutic medicine and promote the normal development of follicles; (4) The traditional Chinese medicine composition for resisting premature ovarian failure and aging has better treatment effect than western medicine estrogen through in-vitro and in-vivo ovarian injury cells and rat ovarian injury models, and has good safety.

Drawings

FIG. 1 shows the effect of different doses of DDP on COV434 cells.

FIG. 2 shows the effect of different concentrations of rat serum blanks on COV434 cells.

FIG. 3 shows the effect of different concentrations of drug-containing serum on COV434 cells.

FIG. 4 is a comparison of the effect of each drug-containing serum on DDP-injured COV434 granulosa cells.

FIG. 5 shows the detection of NC-group serum cells by flow cytometry.

FIG. 6 is a graph showing the effect of flow cytometry on a model of DDP-damaged COV434 cells from 15% blank serum.

FIG. 7 shows how flow cytometry detects the effect of high-dose drug-containing serum of 15% anti-POF traditional Chinese medicine formulation on DDP injury COV434 cell model.

FIG. 8 shows the effect of low-dose serum containing 15% anti-POF traditional Chinese medicine composition on DDP injury COV434 cell model by flow cytometry.

FIG. 9 flow cytometry was performed to examine the effect of 15% estradiol valerate drug-containing serum on a DDP injury COV434 cell model.

FIG. 10 flow cytometry detects apoptosis improvement effect of each drug-containing serum on DDP injury COV434 cell model.

FIG. 11 shows protein expression on the PI3K/Akt pathway of DDP-damaged COV434 cells with each drug-containing serum.

FIG. 12 is a comparison of the effect of each drug-containing serum on E2 hormone levels in the supernatant of DDP-injured COV434 cells.

FIG. 13 is a comparison of the effect of each drug-containing serum on FSH hormone levels in the supernatant of DDP-injured COV434 cells.

FIG. 14 is a comparison of the effect of each drug-containing serum on LH hormone levels in the supernatant of DDP-injured COV434 cells.

Figure 15 is a comparison of rat body weight monitoring.

FIG. 16 shows the results of HE staining (x 100) with a vaginal abscission cell smear from pre-estrus SD female rats.

FIG. 17 shows the results of HE staining (x 100) with vaginal abscission cell smears from estrus SD female rats.

FIG. 18 shows the results of HE staining (x 100) with a vaginal abscission cell smear from estrus SD female rats.

FIG. 19 shows the results of HE staining (x 100) with vaginal abscission cell smears from estrus-later SD female rats.

Figure 20 is the effect of NC group on the estrus cycle of rats.

Fig. 21 is a cycle of estrus in POF rats.

Figure 22 is the effect of estradiol valerate on the estrus cycle of POF rats.

FIG. 23 is the effect of a kidney nourishing and embryo raising pellet on the estrus cycle of POF rats.

Figure 24 is the effect of high doses of anti-POF traditional Chinese medicine formulation on the estrus cycle of POF rats.

Fig. 25 is the effect of dose in anti-POF traditional Chinese medicine formulation on the estrus cycle of POF rats.

Figure 26 is the effect of low doses of anti-POF traditional Chinese medicine formulation on the estrus cycle of POF rats.

FIG. 27 is a comparison of serum E2 from each group of rats.

FIG. 28 is a comparison of serum FSH from rats in each group.

FIG. 29 is a comparison of serum LH from rats in each group.

In fig. 1, P <0.0001; in fig. 3, NC:15% blank serogroup, HH:15% of anti-POF traditional Chinese medicine formula high-dose drug-containing serogroup, HL:15% anti-POF traditional Chinese medicine formula low-dose medicated serogroup, HW:15% estradiol valerate drug-containing serogroup, P <0.001; in fig. 4, NC group: 15% blank serum control, DDP group: cisplatin+15% blank serum model group, HH: cisplatin+15% anti-POF traditional Chinese medicine formula high-dose drug-containing serogroup, HL: cisplatin+15% anti-POF traditional Chinese medicine formulation low dose drug-containing serogroup, HW: cisplatin+15% estradiol valerate drug-containing serogroup. * p <0.05, < p <0.0001vs NC group; * P <0.01, p <0.0005; in fig. 11, NC group: 15% blank serum control, DDP group: ddp+15% blank serum model group, HH: DDP+15% anti-POF traditional Chinese medicine formula high-dose drug-containing serogroup, HL: ddp+15% anti-POF traditional Chinese medicine formulation low dose drug-containing serogroup, HW: ddp+15% estradiol valerate drug-containing serogroup; in fig. 12 to 14, NC group: 15% blank serum control, DDP group: ddp+15% blank serum model group, HH: DDP+15% anti-POF traditional Chinese medicine formula high-dose drug-containing serogroup, HL: ddp+15% anti-POF traditional Chinese medicine formulation low dose drug-containing serogroup, HW: ddp+15% estradiol valerate drug-containing serogroup. * P <0.0001, p <0.001, p <0.01, p <0.05; in fig. 27 to 29, NC group: normal rat group, POF group: CTX induced rat POF model group, pof+h: ctx+high dose anti-POF traditional Chinese medicine formula rat group, pof+m: ctx+ medium dose anti-POF traditional chinese medicine formula rat group, pof+l: ctx+low dose anti-POF traditional Chinese medicine formula rat group, pof+w: ctx+estradiol valerate drug rat group, pof+z: ctx+kidney and fetus nourishing pill rat group is p <0.0001, p is less than or equal to 0.0005, p=0.0012, p <0.006 or p <0.05.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.

In recent years, the traditional Chinese medicine has development potential by carrying out POF treatment through modes of meridian prescription, proved prescription, acupuncture, combination of traditional Chinese medicine and western medicine and the like, but the traditional Chinese medicine has the characteristic of dialectical treatment, and each doctor has flexible prescription compatibility, lacks a system and quantitative analysis of the prescription, and is difficult to carry out innovation development and utilization of the traditional Chinese medicine through the way, so the traditional Chinese medicine composition is qualitatively and quantitatively analyzed in batches, and the prescription is optimized to obtain the POF-resistant traditional Chinese medicine composition. In addition, the therapeutic effect of the POF-resistant traditional Chinese medicine composition is better than western medicine estrogen through in-vivo and in-vitro ovarian injury cells and a rat ovarian injury model, and the safety is good.

Preferably, the traditional Chinese medicine composition for resisting premature ovarian failure and aging can comprise, by mass, 18-22 parts of prepared rehmannia root, 13-17 parts of semen cuscutae, 8-12 parts of Chinese angelica, 13-17 parts of medlar, 8-12 parts of epimedium herb, 8-12 parts of morinda officinalis, 13-17 parts of Chinese yam, 8-12 parts of poria cocos, 13-17 parts of red sage root, 8-12 parts of ligusticum wallichii, 8-12 parts of radix bupleuri, 8-12 parts of rhizoma cyperi, 8-12 parts of moutan bark and 4-8 parts of liquorice.

Preferably, the traditional Chinese medicine composition for resisting premature ovarian failure and aging can comprise 20 parts by weight of prepared rehmannia root, 15 parts by weight of semen cuscutae, 10 parts by weight of Chinese angelica, 15 parts by weight of medlar, 10 parts by weight of epimedium, 10 parts by weight of morinda officinalis, 15 parts by weight of Chinese yam, 10 parts by weight of poria cocos, 15 parts by weight of salvia miltiorrhiza, 10 parts by weight of ligusticum wallichii, 10 parts by weight of radix bupleuri, 10 parts by weight of rhizoma cyperi, 10 parts by weight of tree peony bark and 6 parts by weight of liquorice.

Specifically, the above-mentioned Chinese medicinal composition for resisting premature ovarian failure and senility can be prepared by selecting different preparation methods according to dosage form requirements, in some specific implementations, can be directly selected from water decoction, and can also be prepared in the form of extract, pill or other nano preparation.

The invention also provides a medicament containing the traditional Chinese medicine composition for resisting premature ovarian failure and aging, and the medicament also comprises a pharmaceutically acceptable auxiliary material.

Specifically, in the above-mentioned Chinese medicinal composition for resisting premature ovarian failure and senility, some medicinal auxiliary materials can be added, or made into different dosage forms, or for other different effects, such as sweetener for improving administration resistance, for example, auxiliary materials for inhibiting bitter taste but not affecting medicinal effect.

Further, the dosage form of the medicament is any one of a tablet, powder, capsule or granule or a nano preparation.

Specifically, the above Chinese medicinal composition for resisting premature ovarian failure and aging can be prepared into tablets, powder, capsules or granules which are convenient to take and carry by adding the auxiliary materials besides decocting with water.

It is noted that the above-mentioned Chinese medicinal composition for resisting premature ovarian failure and senility has high-concentration toxic side effect, and the maximum quantity of active component unit of the above-mentioned Chinese medicinal composition for resisting premature ovarian failure and senility can not exceed its toxic side effect quantity, and its toxic side effect quantity is greater than 17.29g/kg. Therefore, the highest single dose cannot be greater than 17.29g/kg; of course, in actual use, the dosage of a single dose is adjusted according to the treatment course, and the dosage of a single dose is low, but the daily dosage of the treatment course is not suitable to be more than 17.29g/kg.

The invention also provides application of the traditional Chinese medicine composition in preparation of preparations for treating premature ovarian failure and aging.

The invention also provides application of the traditional Chinese medicine composition in preparation of preparations for resisting premature ovarian failure and aging.

In still another aspect, the invention provides an application of the traditional Chinese medicine composition in preparation of a preparation for improving the estrus cycle disorder of an organism.

In still another aspect, the invention provides an application of the traditional Chinese medicine composition in preparation of a preparation for improving endocrine dysfunction of an ovary of a human body.

Further, in the preparation for treating premature ovarian failure and aging, the preparation for improving the estrus cycle disorder of the organism or the preparation for improving the endocrine function disorder of the ovary of the organism, the preparation is any one of a tablet, powder, capsule, granule or nano preparation.

Specifically, the traditional Chinese medicine composition for resisting premature ovarian failure and aging improves the development condition of the ovary by improving the estrus cycle disorder and the endocrine function disorder of the ovary of the organism, so that the traditional Chinese medicine composition for resisting premature ovarian failure has a remarkable effect.

In the same way, in the preparation for treating premature ovarian failure and aging, the preparation for improving the estrus cycle disorder or the preparation for improving the endocrine function disorder of the ovary of the organism, the active ingredients of the traditional Chinese medicine composition for treating premature ovarian failure and aging cannot be too high, toxic and side effects can occur, and the amount of a single dose needs to be controlled according to the treatment program.

For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.

In the following examples (including cell assays and animal assays), the ELISA detection method is specifically described as follows:

(1) The required strips were removed from the aluminium foil bags after 60min equilibration at room temperature and the remaining strips were sealed with a self-sealing bag and returned to 4 ℃.

(2) Setting a standard substance hole and a sample hole, wherein 50 mu L of standard substances with different concentrations are added into the standard substance hole;

(3) The sample to be measured is added into the sample hole by 50 mu L, and the blank hole is not added.

(4) In addition to the blank wells, 100. Mu.L of horseradish peroxidase (HRP) -labeled detection antibody was added to each of the standard wells and sample wells, and the reaction wells were sealed with a sealing plate membrane and incubated for 60min in a 37℃water bath or incubator.

(5) Removing liquid, beating to dry on absorbent paper, filling washing liquid (350 mu L) in each hole, standing for 1min, throwing off the washing liquid, beating to dry on absorbent paper, and repeating the plate washing for 5 times.

(6) After adding 50. Mu.L of each substrate A, B to each well and incubating at 37℃for 15min in the absence of light, 50. Mu.L of a stop solution was added for 15min, and the OD of each well was measured at a wavelength of 450 nm.

(7) And (3) calculating an experimental result: and drawing a standard curve on a piece of coordinate paper or by using related software by taking the OD value of the measured standard substance as an abscissa and the concentration value of the standard substance as an ordinate, obtaining a linear regression equation, substituting the OD value of the sample into the equation, and calculating the concentration of the sample.

1. Cell assay

In the following examples, 24 female SD rats were bred in pure form, SPF grade, body weight 200-230g,7-8 weeks old, 45-52 days old, purchased from university of southern medical science laboratory animal management center, and bred in university of Guangdong medical science animal experiment center; COV434 human ovarian granule tumor cells were purchased from the Living technologies Co., ltd, and cultured in the department of medicine precision laboratory attached to the seventh hospital at the university of Zhongshan.

In the following examples, each of the formulation drug-containing serum was prepared according to the following methods:

(1) Firstly, after the 24 female rats of 45-52 days old are adaptively bred for 1 week, dividing the female rats into 4 groups according to random number arrangement, wherein 6 female rats in each group are respectively a blank control group, an anti-POF traditional Chinese medicine formula low-dose group, an anti-POF traditional Chinese medicine formula high-dose group and an oestradiol valerate group;

(2) Then, the following processes were performed, respectively: (1) blank control (NC) (6): feeding common feed and equivalent physiological saline to irrigate the stomach (1 ml/100 g); (2) anti-POF traditional Chinese medicine formulation low dose group (HL) (6): feeding common feed and anti-POF traditional Chinese medicine prescription, wherein the low dose of the traditional Chinese medicine decoction freeze-dried powder is 0.451g/100g for stomach irrigation; (3) anti-POF traditional Chinese medicine formulation high dose group (HH) (6): feeding a common feed and anti-POF traditional Chinese medicine prescription, decocting the traditional Chinese medicine decoction freeze-dried powder with high dosage 1.8057g/100g and pouring the stomach; (4) estradiol valerate positive drug control (HW) (6): normal feed plus 0.0104mg/100g gastric lavage is fed.

(3) The above groups were each intragastrically administered 1 time every 24h, 6d continuously, 2 times of 7d repeated administration, interval time 2h, and after the last intragastrically administered 1h, anesthetized rats were injected with afo Ding Fuqiang and abdominal aortic blood collection was performed using a blood collection tube;

(4) Standing the blood of each group at room temperature for 3h, transferring to 4deg.C, standing for 1h, centrifuging at 3500rpm at normal temperature for 25min, carefully absorbing supernatant, sterilizing with 0.22 μm filter on an ultra-clean bench, inactivating in 56 deg.C water bath for 30min, packaging, and storing in-80deg.C refrigerator.

In the following examples, the COV434 cell ovarian injury cell model used was constructed as follows:

(1) COV434 cells were cultured at 1X 10 ⁵ The density of each hole is paved on a 96-well plate, the culture is carried out by using a complete culture medium, and after the cells are attached and the cell density reaches 70%, the following treatments are respectively carried out in groups: (1) background group: DEME medium; (2) blank control group: COV434 cells + DEME medium; (3) cisplatin (DDP) action experimental group x 5: COV434 cells + different concentrations (6.25Cisplatin (DDP) +DEME medium at mg/L, 12.5mg/L, 25mg/L, 50mg/L, and 100 mg/L;

(2) After 24 hours of the above groups, 10% of CCK-8 reagent is added, OD value is measured after 2 hours, and proper cisplatin (DDP) action concentration is screened, and as shown in a result of a figure 1, COV434 cell viability is reduced to 34.14% when the DDP action concentration is 25mg/L, and cell growth is obviously inhibited; when the DDP action concentration is 6.25mg/L, the activity of COV434 cells is reduced to 75.46%, so that only slight damage can be caused to the cells, and the subsequent study is not facilitated; therefore, the concentration of the active agent was 12.5mg/L as the DDP molding concentration, at which time the cell viability was 57.92%.

Prior to performing the following examples, the CCK-8 method was used to compare the optimal working concentration of each group of drug-containing sera, as follows:

(1) COV434 cells were cultured at 1X 10 ⁵ The density of the cells/well was spread on a 96-well plate, cultured using a complete medium, and after the cells were attached and the cell density reached 70%, the cells were grouped as follows: (1) background group: DEME medium; (2) blank control group: COV434 cells + DEME medium; (3) rat blank serum effect experimental group x 3: COV434 cells + rat blank serum at different concentrations (10%, 15%, 20%) + DEME medium;

(2) After 24 hours of each group, 10% of CCK-8 reagent is added, OD value is measured after 2 hours, and proper rat blank serum action concentration is screened, so that when the rat blank serum addition proportion is 20%, the cell activity is reduced to 75.44%, and toxic action (P < 0.001) is obviously shown on COV434 cells;

(3) Then, 15% of the serum containing the drug of each of the above formulations was applied to COV434 cells for 24 hours, and the cell viability was measured by CCK-8 method, and the results are shown in FIG. 3, and the serum containing 15% of the drug was used as the addition ratio in the following examples, as compared with the normal cell group, without significant difference.

In the following examples, the specific procedure for cellular protein extraction, i.e., western blotting experiments, was as follows:

(1) Extraction of total cell proteins: after the cell fusion degree of the 6-hole plate reaches 80%, the 6-hole plate is treated by proper medicines, the PBS is pre-cooled and rinsed three times, 150 μl of 1 xRIPA cell lysate (containing 1.5 μl of protease inhibitor and 1.5 μl of phosphatase inhibitor) is added after the PBS buffer is sucked, the mixture is repeatedly blown until the foam completely covers cells, the mixture is fully lysed on ice for 20min, the lysed cell suspension is collected by scraping the cells in a 1.5ml sterilization EP tube, the mixture is centrifuged at 12000rpm and 4 ℃ for 10min, and the supernatant (protein sample) is taken into a new sterilization EP tube and placed at the temperature of-80 ℃ for standby.

(2) BCA method to determine the concentration of total protein: 200 μl BCA working solution (solution A: solution B=50:1) was prepared for each well of the 96-well plate, 5 μl sample+15 μl PBS dilution was placed in a 37℃incubator for 30min, the absorbance at 562nm was measured by a microplate reader, the concentration of the sample was calculated by using a standard curve measured by a standard substance, and the volume containing 30 μg protein loading was calculated by the protein concentration of the sample.

(3) Protein denaturation: taking out the protein sample into a 1.5ml EP tube, adding 5 Xprotein loading buffer solution to dilute to 1X, uniformly mixing, heating in 96 ℃ water bath for denaturation for 7-8 min, and placing at-20 ℃ for standby.

(4) Preparation of SDS-PAGE gel: aligning the lower ends of the long and short glass plates, clamping the long and short glass plates, placing the short glass plates outwards on a glue distributing clamp, and filling ddH ₂ O leak detection is carried out for 5min. Preparing SDS-PAGE separating gel with different concentrations according to the molecular weight of protein, mixing separating gel reagent, adding between two plates, and adding ddH ₂ And (3) flattening the separation gel. Solidifying for about 10-15 min, and discarding ddH ₂ O, adding the prepared concentrated gel reagent, inserting a tooth comb, and solidifying for 10-15 min.

(5) Electrophoresis: and adding electrophoresis buffer solution into an electrophoresis tank, wherein the constant pressure is 60-70V when electrophoresis concentration is carried out, and setting the constant pressure to be 100-120V when bromophenol blue indication strips run to the separation gel. According to the position of the required protein in the marker, the time can be estimated to stop electrophoresis and power is turned off.

(6) Transferring: PVDF film was cut to 1X 6cm and immersed in methanol for 2min. Placing the black surface of the transfer film clamp under the bottom, placing the transparent surface on the top, placing the sponge, the filter paper, the glue, the PVDF film, the filter paper and the sponge which are soaked in the transfer film liquid in sequence from bottom to top, clamping the transfer film clamp, placing the transfer film clamp into a groove, pouring the transfer film liquid, covering a cover, placing the transfer film clamp into a low-temperature system filled with ice cubes, and transferring the transfer film with constant pressure of 60-110V for 45-75 min.

(7) Closing: after the membrane transfer is finished, the PVDF membrane is taken out and placed in the protein-free rapid sealing liquid, and the sealing is carried out by a normal-temperature low-speed shaking table

20min。

(8) Incubating the antibody: (1) incubation resistance: PVDF film containing target protein is placed on the corresponding primary antibody, and the dilution ratio is 1:1000. incubate overnight at 4 ℃. (2) Secondary antibody incubation: the membrane was washed 3 times with TBST for 10min each. Adding a secondary antibody corresponding to the primary antibody species, and diluting the mixture in a proportion of 1: incubate at room temperature for 1h at 5000. TBST was washed 3 times for 10min each.

(9) Developing: chemiluminescent reaction (ECL): and placing a PVDF film on the bottom plate, adding a chemiluminescent liquid, and performing exposure imaging by an exposure instrument to obtain a target protein band image.

(10) And (5) scanning and storing the result.

Example 1 preparation of anti-POF traditional Chinese medicine composition

The following examples were prepared by decocting 20g of prepared rehmannia root, 15g of dodder seed, 10g of Chinese angelica, 15g of medlar, 10g of epimedium herb, 10g of morinda root, 15g of Chinese yam, 10g of poria cocos, 15g of red sage root, 10g of szechuan lovage rhizome, 10g of bupleurum root, 10g of nutgrass galingale rhizome, 10g of tree peony bark and 6g of liquorice with water to obtain a decoction.

Example 2CCK-8 method to observe the effects of each group of medicated serum

COV434 ovarian cell injury model cells were treated at 1X 10 ⁵ The density of each hole is paved on a 96-well plate, the culture is carried out by using a complete culture medium, and after the cells are attached to the wall and reach 70%, the cells are respectively divided into the following groups: (1) background group: DEME medium. (2) Normal control group (group a): COV434 cells + rat blank serum + DEME medium; (3) model control group (group B): COV434 cells + DDP + rat blank serum + DEME medium; (4) anti-POF traditional Chinese medicine formula drug-containing serum high dose group (group C): COV434 cells+DDP+high-dose group rat drug-containing serum+DEME medium; (5) anti-POF traditional Chinese medicine formula medicated serum low dose group (group D): COV434 cells+DDP+low-dose group rat medicated serum+DEME medium; (6) estradiol valerate drug group (group E): COV434 cells+DDP+estradiol valerate group rat medicated serum+DEME medium;

After each group is acted for 24 hours, 10% of CCK-8 reagent is added, OD value is measured after 2 hours, and cell viability of each group is detected, so that compared with a normal control group, the cell viability of a DDP model group is reduced to 56.48% (. Times.P < 0.0001); compared with the DDP model group, the high-dose drug-containing serogroup cell viability of the anti-POF traditional Chinese medicine formula (the formula in example 1) is improved to 94.68% (< 0.0005) and the estradiol valerate drug-containing serogroup cell viability is improved to 84.73% (< 0.001), and no obvious difference is seen from the normal group comparison; the anti-POF traditional Chinese medicine formulation (formulation in example 1) improved the cell viability of the low-dose drug-containing serogroup to 80.49% (< P < 0.001), but there was still a difference compared to the normal group (< P < 0.05).

Example 3 flow assay of apoptosis in ovarian injury cell models by drug-containing serum of groups

COV434 cells were grown at 2X 10 ⁶ The density was plated in six well plates, cultured using complete medium, and after COV434 cell confluence reached 70%, the following groupings were made and treated separately as follows: (1) blank cell group x 3: COV434 cells + DMEM medium; (2) model group: COV434 cells + DDP + DMEM medium; (3) anti-POF traditional Chinese medicine formula drug-containing serum high-dose experimental group: COV434 cells+DDP+high-dose group rat medicated serum+DMEM medium; (4) anti-POF traditional Chinese medicine formula medicated serum low dose experimental group: COV434 cells+DDP+low-dose group rat medicated serum+DMEM medium; (5) drug-containing serum estradiol valerate experimental group: COV434 cells+DDP+estradiol valerate group rat medicated serum+DMEM medium;

(2) After 24h of action, the flow pretreatment was carried out as follows: (1) gently sucking the stock culture solution into a clean centrifuge tube, centrifuging at 2000rpm for 5min, digesting COV434 adherent cells with pancreatin without EDTA, adding DMEM complete medium to stop digestion, centrifuging at 2000rpm for 5min, and collecting each group of cells; (2) washing the cells with PBS 2 times, centrifuging at 2000rpm for 5min, and collecting 1-5×10 ⁵ A cell; (3) adding 500 μl Binding Buffer to resuspend cells; (4) the blank cell group has 3 groups, which are respectively set as blank tube, annexin V single-dyeing tube and PI single-dyeing tube, in which the blank tube is voltage-regulated, and does not make dyeing treatment, and 5 mul Annexin V-FITC is added into the Annexin V single-dyeing tube to make dyeing, and PI single-dyeingTube addition 5 μ l Propidium lodide staining; after 5 mu l of Annexin V-FITC and 5 mu l Propidium lodide are added into the other groups for dyeing treatment, the reaction is carried out at room temperature for 10min in a dark place; within 1 hour, observation and detection were performed using a flow cytometer.

The flow cytometer detection conditions are shown in fig. 5, 6, 7, 8 and 9, and then the apoptosis is counted, as shown in fig. 10, and compared with the normal control a group, the DDP model B group COV434 has higher apoptosis rate (P < 0.01), which indicates that DDP has apoptosis promoting effect on COV434 cells. Compared with the DDP model of the B group, the apoptosis rate of the COV434 in the high-dose drug-containing serum group of the C group anti-POF traditional Chinese medicine composition is obviously reduced (P is less than 0.0001), the apoptosis rate of the COV434 in the low-dose drug-containing serum group of the D group anti-POF traditional Chinese medicine composition is obviously reduced (P is less than 0.0005), and the apoptosis rate of the COV434 in the estradiol valerate-containing serum group of the E group is obviously reduced (P is less than 0.0001) and all have statistical differences. In addition, the high-dose drug-containing serogroup of the anti-POF traditional Chinese medicine composition in group C has lower apoptosis rate (P < 0.05) than the low-dose drug-containing serogroup COV434 of the anti-POF traditional Chinese medicine composition in group D, and has statistical difference.

Example 4Western Blot detection of PI3K, foxO a, p-FoxO3a, akt, p-Akt, bax, bcl-2 protein expression

The protein was extracted from each cell line in example 3, and western blotting was performed as described above, and the results are shown in fig. 11:

wherein, bax, bcl-2: compared with a normal cell group, the expression of Bax protein of the DPP model group is obviously increased, and the expression of Bcl-2 protein is obviously reduced; after the effect of 78 medicated serums, the expression of Bax protein is reduced, and the expression of Bcl-2 protein is increased, wherein the high-dose medicated serums of the anti-POF traditional Chinese medicine formula have the most obvious effect, and the low-dose medicated serums of the anti-POF traditional Chinese medicine formula may have dose-dependent effect.

Akt, p-Akt: compared with the normal cell group, the expression quantity of the Akt proteins in each group has no obvious difference; the p-Akt protein of the DDP model group is not expressed, the expression level of the p-Akt protein is obviously increased after the action of each drug-containing serum, wherein the expression of the p-Akt protein of the high-dose drug-containing serum group of the anti-POF traditional Chinese medicine formula is most obvious, and the p-Akt protein of the high-dose drug-containing serum group of the anti-POF traditional Chinese medicine formula and the low-dose drug-containing serum group of the anti-POF traditional Chinese medicine formula possibly have a dose-dependent effect.

FoxO3a, p-FoxO3a: the FoxO3a protein expression levels of each group were not significantly different from those of the normal cell group. The expression level of p-FoxO3a protein in the DDP model group is reduced, and after the effect of each drug-containing serum, the expression level of p-FoxO3a protein in each group is obviously increased. Wherein, the expression of p-FoxO3a protein of the high-dose medicated serum group of the anti-POF traditional Chinese medicine composition is most obvious, and the low-dose medicated serum group of the anti-POF traditional Chinese medicine composition can have a dose-dependent effect.

PI3K: compared with the normal cell group, the DDP model group has the advantages that the PI3K protein expression level is reduced, and after the action of each drug-containing serum, the PI3K protein expression level of each drug-containing serum group is increased, but the expression level is not obvious, and the expression level is greatly different from that of the normal group.

Above, the anti-POF traditional Chinese medicine formula drug-containing serum can improve COV434 granulosa cell apoptosis caused by DDP, restore cell viability, further verify that PI3K, p-Akt, p-Foxo3a and Bcl-2 protein expression can be up-regulated through PI3K/Akt signal paths, bax protein expression can be down-regulated, COV434 granulosa cell apoptosis caused by DDP can be improved together, E2, FSH and LH hormone levels of ovarian granulosa cells can be restored, damage of ovarian granulosa cells after chemotherapy can be effectively prevented and treated, premature exhaustion of follicular reserve can be reduced, recovery of ovarian function can be promoted, and a certain degree of concentration dependency can exist between high-dose groups and low-dose groups.

EXAMPLE 5ELISA detection of the differences in E2, LH, FSH levels in supernatants of groups of drug-containing serum-affected ovarian injury cell models

The difference in E2, LH, FSH levels in supernatants of each group of drug-containing serum-acting ovarian injury cell models was detected using ELISA, and the results are shown in fig. 12, 13 and 14:

compared with the normal control group, the level of E2 in the DDP model group is obviously reduced (P < 0.0005), and the levels of FSH and LH are obviously increased (P < 0.0005);

Compared with the DDP model group, the high-dose drug-containing serogroup extracellular fluid E2 level of the anti-POF traditional Chinese medicine composition is obviously increased (P < 0.01), the FSH level is obviously reduced (P < 0.0001), and the LH level is obviously reduced (P < 0.0005); the low-dose drug-containing serogroup extracellular fluid E2 level of the anti-POF traditional Chinese medicine composition is increased (P < 0.05), the FSH level is reduced (P < 0.001), and the LH level is reduced (P < 0.05); estradiol valerate-containing serogroup extracellular fluid E2 levels were significantly elevated (P < 0.0005), FSH levels were significantly reduced (P < 0.0001), LH levels were significantly reduced (P < 0.0001).

The level of extracellular fluid E2 of each drug-containing serogroup is not obviously different from that of the normal group; the anti-POF traditional Chinese medicine formula low-dose medicine-containing serogroup extracellular fluid FSH level is higher than that of a high-dose medicine-containing serogroup (P < 0.05), estradiol valerate medicine-containing serogroup (P < 0.0005), the estradiol valerate medicine-containing serogroup extracellular fluid FSH level is lower than that of a normal group (P < 0.05), and the comparison between the high-dose medicine-containing serogroup and the low-dose medicine-containing serogroup of the anti-POF traditional Chinese medicine formula and the normal group is not obviously different; the level of the extracellular fluid LH of each drug-containing serogroup is not obviously different from that of a normal group, and compared with that of the drug-containing serogroup containing estradiol valerate, the level of the FSH of the low-dose drug-containing serogroup of the anti-POF traditional Chinese medicine composition is obviously higher (P is less than 0.01), and the high-dose drug-containing serogroup of the anti-POF traditional Chinese medicine composition is not obviously different.

2. Animal test section

In the following examples, 70 female adult SD rats (clean grade, 7-8 weeks old, healthy, weight 170-190 g) were purchased from the university laboratory animal management center of southern Guangzhou medical science (animal production pass number: 44002100031527).

In the following examples, the establishment of the POF model used was: the establishment of CTX induced rat POF model, namely dividing the 70 rats into 10 normal groups according to the weight-combined random number table method, and constructing 60 groups. Except for the normal group, CTX was injected intraperitoneally at a first dose of 50mg/kg, and continuous intraperitoneal injection was performed for 14d at 8mg/kg to establish a model of POF or ovarian injury.

In the following examples, rats were subjected to animal groups and different treatments, respectively: grouping the molded rats according to a random number table method, wherein 8 rats are arranged in each group: (1) blank control group: feeding common feed and equivalent physiological saline for gastric lavage; (2) model control group: feeding common feed and equivalent physiological saline for gastric lavage; (3) low dose group of anti-POF traditional Chinese medicine formulation: feeding common feed and anti-POF traditional Chinese medicine in a prescription, wherein the traditional Chinese medicine is decocted with water to obtain a low dosage; (4) dosage group in anti-POF traditional Chinese medicine formulation: feeding common feed and the traditional Chinese medicine decoction of the anti-POF traditional Chinese medicine formula; (5) high dose group of anti-POF traditional Chinese medicine formula: feeding common feed and self-made anti-POF traditional Chinese medicine prescription traditional Chinese medicine water decoction with high dosage; (6) traditional Chinese medicine control group: feeding common feed and kidney nourishing and embryo culturing pill water solution, and lavaging; (7) western medicine control group: feeding common feed and estradiol valerate water solution for stomach irrigation; each of the above groups was intragastrically 1 time every 24 hours for 30 consecutive days.

In the above animal grouping and the different treatments, the doses administered were as follows: (1) the amount of the traditional Chinese medicine decoction of the anti-POF traditional Chinese medicine formula is as follows: according to the equivalent dose conversion method of human and animal administration (daily administration dose (g/kg) of rats=daily administration dose (g)/60 kg×6.25 of human, the low, medium and high dose groups are respectively 1\2 times and 1 times and 2 times of the clinically applicable dose, namely, the stomach-resisting POF traditional Chinese medicine formula water decoction is respectively infused according to 8.645g/kg, 17.29g/kg and 34.58g/kg of body mass; (2) kidney nourishing and embryo culturing pill: the dosage of adult administration is 5g once and 3 times a day, the total dosage is 15g, and the dosage is 1.5625g/kg (15 g/60kg×6.25=1.5625 g/kg) in terms of rat dosage by referring to the equivalent dose rate table of conversion of body surface area between human and animal; (3) estradiol valerate tablet: the dose of 1mg daily for adults was 0.104mg/kg (1 mg/60kg×6.25=0.104 mg/kg) in terms of rats.

Example 6 weight and wet weight of ovaries of rats of each group

As a result of weight recording of the rats in each group, as shown in fig. 15, the rats in the POF model group showed no significant weight gain, and a part of the rats showed weight loss, and the rats gradually showed a duller reaction, listlessness, decreased activity, a dull body Mao Andan, a large and thin stool, a hair erection, a back curling, a aversion to cold, an ice-cold limbs, and a camping pile within 14d of the molding period, compared with the normal group. After the drug treatment is given, the weight of the rats is gradually increased, and after 2 weeks of the drug treatment, compared with the rats in the model control group, the rats in each treatment group have the advantages of ingestion, increased water intake, weight increase, bright and moist hair and more sensitive activity.

After the end of the 30 th day of administration, the rats were fasted and not water-inhibited for 12 hours, and the mass of the rats was weighed, and the results are shown in the following table 1.

TABLE 1 anti-cancerInfluence of POF traditional Chinese medicine formula on model rat weight

Group of	Animals (only)	Body weight (g)
			Normal control group	5	225.7±19.25
POF model control group	5	190.8±8.574****
			Traditional Chinese medicine control group	5	209.8±12.12**
Western medicine control group	5	208.9±12.71*
			High-dosage group of traditional Chinese medicine	5	205.3±10.84**
Chinese medicine dosage group	5	201.3±8.125***
			Low-dosage group of traditional Chinese medicine	5	218.0±16.96****

Note that: in table 1, compared to the normal control group: * P <0.0001, P <0.0001;

p <0.001, P <0.05 compared to POF model control; p <0.05 compared with the dosage in the traditional Chinese medicine.

As can be seen from table 1, rats in the POF model group had a slow body weight gain compared to the normal group (< 0.0001); compared with the POF model group, estradiol valerate (P < 0.05), kidney-nourishing and fetus-nourishing pill (P < 0.005) and low-dose anti-POF traditional Chinese medicine formula (P < 0.0001) can lead the weight of the POF rats to rise, the difference has statistical significance, and the high-dose and medium-dose anti-POF traditional Chinese medicine formulas have no obvious difference.

After weighing the weights of the rats in each group, the rats were anesthetized by injection with afos Ding Fuqiang; taking abdominal aortic blood, standing at room temperature for 1h, centrifuging at 3-000 rpm with a low-temperature refrigerated centrifuge for 25min, separating serum, sub-packaging, and freezing at-80deg.C for hormone analysis. Ovarian tissues were removed, and the ovarian wet weight was weighed by a precision electronic balance, and the ovarian index (ovarian index/% =ovarian wet weight/rat mass×100%) was calculated, and the results are shown in table 2 below.

TABLE 2 influence of anti-POF traditional Chinese medicine formulation on model ovarian wet weight and ovarian index

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Note that: in table 2, compared to the normal control group: * P <0.005, P <0.05; p <0.05, P <0.01 compared to POF model control group.

As can be seen from table 2, the wet weight of the ovaries of the rats in the model group significantly decreased (P < 0.005) and the ovaries index decreased (P < 0.05) compared to the normal group, and the difference was statistically significant. Compared with the POF model group, the traditional Chinese medicine formulas of estradiol valerate (P < 0.005), kidney-nourishing and fetus-nourishing pill (P < 0.005) and high (P < 0.005) and middle (P < 0.005) anti-POF can increase the ovarian wet weight of the model rat, the difference has statistical significance, and the traditional Chinese medicine formulas of high-dose anti-POF have no obvious difference. Compared with the POF model group, the estradiol valerate (P < 0.05), the kidney-nourishing and fetus-nourishing pill (P < 0.05) and the middle (P < 0.005) and low (P < 0.005) anti-POF traditional Chinese medicine formulas can increase the ovary index of the model rat, the difference has statistical significance, and the high-dose anti-POF traditional Chinese medicine formulas have no obvious difference.

Example 7 estrus cycle observations in rats

Starting vaginal abscission cell smear from the 1 st day of molding, ending the administration of the vaginal abscission cell smear before 14 days, and observing the estrus periodic change of the molding group rat in 2 continuous periods (14 days). The specific operation method comprises the following steps: vaginal smear is started at 10:00AM every day, a proper amount of physiological saline is sucked by a 200ul pipette, and the vaginal part of the rat is gently blown and sucked and then blown to the middle of a glass slide to prevent falling off. After the smear is air-dried, the smear is fixed in 95% ethanol solution for 0.5h, and then the smear is placed in hematoxylin dye and soaked for 3min. Then soaking in the differentiation liquid to differentiate, and controlling the time to be about 10 s. After rinsing gently under running water for 5s, transfer to eosin dye for 1min. Gently washing under running water for 5s, soaking in 95% ethanol solution, observing under microscope, judging specific estrus cycle of rats according to the form and type of the exfoliated cells in the smear, and recording change condition of estrus cycle of each rat.

The results are shown in fig. 16, 17, 18 and 19, pre-estrus (Proestrus): some of the nucleated elliptic epithelial cells were most abundant, see fig. 16; estrus (Estrus): the large, flat, flaky, coreless keratinized squamous epithelial cells are most numerous with uneven edges, see fig. 17; estrus interval (Metestrus): leukocyte predominance, see figure 18; estrus late (Diestrus): all three types of cells were present but without specific ratios, see figure 19.

Estrus cycle analysis was performed on each of the above groups of rats, and the results are shown in table 3 below.

TABLE 3 influence of anti-POF traditional Chinese medicine formulation on estrus cycle in POF rats

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Note that: comparison to the normal control group: * P <0.0001; p <0.001, < P <0.0001, compared to the POF model control group.

In table 3 above, compared with the normal group, the POF group rats showed periodic disorders such as cycle extension or cycle arrest, and the differences were statistically significant (< 0.0001); compared with the POF group after the drug treatment, the estrus cycle of the anti-POF traditional Chinese medicine formula of estradiol valerate group (P < 0.001), kidney nourishing and fetus raising pill group (P < 0.001), high (P < 0.001), middle (P < 0.001) and low (P < 0.0001) is shortened, normal contrast period level is restored, and the difference has statistical significance; there was no significant difference between each treatment group and the normal group.

Analysis of the development cycle of each group of rats showed that the vaginal smear of the POF group showed a dramatic decrease in the number of keratinocyte-shedding cells, leukocytosis, nucleated epithelial cytopenia, and some smears were accompanied by a large amount of mucus in the estrus and later phases, as compared to the normal group, as shown in fig. 20, 21, 22, 23, 24, 25 and 26; after treatment, the vaginal abscission cell reduction condition of each treatment group is improved, and the estrus cycle of rats is gradually restored to be normal.

EXAMPLE 8ELISA method for determination of serum E2, FSH, LH hormone levels

Serum E2, FSH, LH hormone levels were measured following the above-described ELISA assay procedure and the results are shown in figures 27, 28 and 29:

the differences were statistically significant in serum E2 concentrations lower than those in the normal group (< P < 0.0001), serum FSH (< P < 0.001), AMH, LH (< P < 0.0005) than in the normal group, as compared to the normal group. Compared with the POF model group, the serum E2 concentration of each drug treatment group is increased, in addition to the high-dose group of the anti-POF traditional Chinese medicine composition, estradiol valerate group (P < 0.0001), kidney nourishing and fetus raising pill group (P < 0.0005) and low-dose group of the anti-POF traditional Chinese medicine composition (P < 0.0001) have statistical significance, and compared with the normal control group, the E2 can be restored to normal level, wherein the low-dose group of the anti-POF traditional Chinese medicine composition is superior to the medium-dose group (P < 0.005), the difference has statistical significance, and the difference has no obvious statistical difference from other treatment groups.

Compared with the POF model group, the serum FSH concentration of each drug treatment group is reduced, and besides the high-dose group of the anti-POF traditional Chinese medicine composition, the estradiol valerate group (P < 0.0001), the kidney nourishing and fetus raising pill group (P < 0.0005) and the low-dose group of the anti-POF traditional Chinese medicine composition (P < 0.0001) have statistical differences, and the medium-dose group (P < 0.001) have no obvious differences compared with the normal group, so that the FSH can be restored to the normal level, and the groups have no obvious differences.

Compared with the POF model group, the serum LH concentration of each drug treatment group is reduced, and besides the high-dose group of the anti-POF traditional Chinese medicine composition, the estradiol valerate group (P < 0.0001), the kidney nourishing and fetus raising pill group (P < 0.05) and the low-dose group of the anti-POF traditional Chinese medicine composition (P < 0.0005) have statistical differences, and the medium-dose group (P < 0.001) have no obvious differences compared with the normal group, so that the LH can be restored to the normal level, and the groups have no obvious differences.

After the cyclophosphamide acts on the rat, the wet weight and the ovary index of the rat are obviously reduced compared with those of a normal control group, which proves that the cyclophosphamide can damage the ovarian tissue of the rat and the ovarian injury model is successfully established. The middle-and low-dose anti-POF traditional Chinese medicine formulas can enable the wet weight and the ovarian index of the POF model rats caused by cyclophosphamide to be recovered to be normal, and have no obvious difference with the wet weight and the ovarian index of the rats in normal groups, wherein the low-dose anti-POF traditional Chinese medicine formula has the strongest improvement effect and the effect superior to the estradiol valerate, and the high-dose anti-POF traditional Chinese medicine formula has no improvement effect, which indicates that the high-concentration toxic and side effects exist.

After the action of Cyclophosphamide (CTX), the estrus cycle abnormality of rats appears to different degrees, the estrus cycle is prolonged, and the rats are stopped at estrus intervals or at estrus later stages, which indicates that continuous intraperitoneal injection of cyclophosphamide for 2 weeks can cause estrus cycle disorder of ovaries of female SD rats and influence normal development of follicles, thereby indirectly representing changes of morphology of vaginal abscission cells. After the treatment of medicines such as estradiol valerate, high, medium and low-dose anti-POF traditional Chinese medicine formulas, kidney nourishing and fetus raising pills and the like, the vaginal abscission cell reduction condition of rats is improved, the estrus cycle is gradually restored to be normal, and the anti-POF traditional Chinese medicine formulas can be presumed to improve the estrus cycle disorder caused by cyclophosphamide and restore the normal development of follicles.

Above, after 2 weeks of continuous intraperitoneal injection of Cyclophosphamide (CTX), the serum E2 levels were significantly reduced (P < 0.001) and serum FSH, LH levels were significantly increased (P <0.005 or 0.05) in the model group compared to the normal group. It is known that cyclophosphamide can damage the endocrine function of the ovary, leading to a significant decrease in the level of E2, and a significant increase in the levels of FSH and LH. After the medicine treatment, other treatment groups except the high-dose anti-POF traditional Chinese medicine formula can effectively recover E2, FSH and LH to normal levels, wherein the low-dose anti-POF traditional Chinese medicine formula has the best improving effect and better effect than oestradiol valerate; the anti-POF traditional Chinese medicine composition can improve the disorder of the endocrine function of the ovary caused by cyclophosphamide and promote the normal development of the follicle, but has certain high-concentration toxic and side effects.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims

1. The traditional Chinese medicine composition for resisting premature ovarian failure is characterized by being prepared from, by mass, 18-22 parts of prepared rehmannia root, 13-17 parts of semen cuscutae, 8-12 parts of Chinese angelica, 13-17 parts of medlar, 8-12 parts of epimedium, 8-12 parts of morinda officinalis, 13-17 parts of Chinese yam, 8-12 parts of poria cocos, 13-17 parts of red sage root, 8-12 parts of ligusticum wallichii, 8-12 parts of radix bupleuri, 8-12 parts of rhizoma cyperi, 8-12 parts of moutan bark and 4-8 parts of liquorice.

2. The anti-premature ovarian failure traditional Chinese medicine composition according to claim 1, wherein the composition is prepared from 20 parts by mass of prepared rehmannia root, 15 parts by mass of semen cuscutae, 10 parts by mass of Chinese angelica, 15 parts by mass of medlar, 10 parts by mass of epimedium, 10 parts by mass of morinda officinalis, 15 parts by mass of Chinese yam, 10 parts by mass of poria cocos, 15 parts by mass of salvia miltiorrhiza, 10 parts by mass of ligusticum wallichii, 10 parts by mass of radix bupleuri, 10 parts by mass of rhizoma cyperi, 10 parts by mass of tree peony bark and 6 parts by mass of liquorice.

3. The anti-premature ovarian failure traditional Chinese medicine composition according to claim 1, further comprising pharmaceutically acceptable auxiliary materials.

4. The composition of claim 3, wherein the formulation is any one of a tablet, powder, capsule or granule.

5. Use of a traditional Chinese medicine composition according to any one of claims 1 to 2 for the preparation of a formulation for the treatment of premature ovarian failure.

6. Use of a traditional Chinese medicine composition according to any one of claims 1 to 2 for the preparation of a formulation for the treatment of premature ovarian failure.

7. The use according to any one of claims 5 to 6, wherein the formulation is in the form of any one of a tablet, powder, capsule or granule.