CN113082147A

CN113082147A - Traditional Chinese medicine composition for treating infertility and preparation method and application thereof

Info

Publication number: CN113082147A
Application number: CN202110488991.2A
Authority: CN
Inventors: 路文杰; 徐有志; 徐明祥
Original assignee: Hefei Douliugong Medical Technology Co ltd; Anhui Medical University
Current assignee: Hefei Douliugong Medical Technology Co ltd; Anhui Medical University
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-07-09
Also published as: ZA202310045B; AU2021102869A4; WO2022227814A1

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly discloses a traditional Chinese medicine composition for treating infertility and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 25-30 parts of prepared rehmannia root, 12-15 parts of angelica, 25-35 parts of fried bighead atractylodes rhizome, 15-20 parts of fried Chinese yam, 10-12 parts of fried white paeony root, 9-12 parts of raw wild jujube kernel, 6-10 parts of cortex moutan, 9-12 parts of radix glehniae, 8-10 parts of fried eucommia bark, 20-25 parts of codonopsis pilosula, 6-10 parts of radix bupleuri, 8-12 parts of fried cyperus rotundus and 6-9 parts of caulis perllae. The traditional Chinese medicine composition for treating infertility provided by the invention can increase the curative effects on endometrial thickness increase, follicular tension enhancement and restoration of normal follicular development, and finally effectively assist pregnancy.

Description

Traditional Chinese medicine composition for treating infertility and preparation method and application thereof

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly discloses a traditional Chinese medicine composition for treating infertility and a preparation method and application thereof.

Background

Infertility is a fertility impairment state, the current incidence rate of infertility in China is 7% -10%, and the infertility tends to rise year by year. Infertility can be caused by various causes, such as influence of environmental drugs, ovulation problem, physical and organic problems and the like.

Polycystic ovary syndrome (PCOS) is a complex reproductive endocrine syndrome characterized by hyperandrogenism and persistent anovulation with multiple clinically manifested polymorphisms. The incidence of PCOS in women of childbearing age is about 6% to 10%, and based on its disturbed endocrine status and continuous anovulation, a significant proportion of patients develop reproductive disorders, and the incidence of infertility in such patients is high, and it is statistically estimated that the resulting infertility accounts for as much as 75% of anovulatory infertility in women. Moreover, the natural abortion rate of PCOS patients reaches about 20% -41%. Besides causing female infertility, thin menstruation, amenorrhea and hirsutism, PCOS has profound influence on endometrium, glycolipid metabolism, cardiovascular system and the like, the risk of endometrial cancer, breast cancer, obesity, coronary heart disease, atherosclerosis, diabetes and the like is increased, and some patients cause psychological disorders due to a plurality of problems of thin menstruation, amenorrhea, infertility, acne, obesity, hirsutism and the like. Therefore, the method has very important significance for paying attention to the attack and treatment progress of PCOS, improving the pregnancy rate of patients with the ovulation dysfunction infertility caused by polycystic ovarian syndrome, reducing the abortion rate and lowering the treatment cost.

The pathophysiology and pathogenesis of PCOS are very complex, so far, the PCOS is not definite, and may be related to a plurality of factors such as excessive function at the early appearance of adrenal gland function, abnormal membrane island-like growth factor, neuroendocrine dysfunction, heredity and the like. Currently, the following three hypotheses are accepted: (1) abnormalities in the hypothalamic-pituitary-ovarian axis affect the normal secretion of gonadotropins and luteinizing hormone, and the ovarian secretion of excess androgen; (2) peninsulin resistance is an important metabolic abnormality in PCOS patients; (3) deficiency of ovarian and adrenal hormones increases the conversion of steroids to androgens. There are also some hypotheses, such as the adrenal gland sprouting hypothesis, the "double defect" hypothesis, etc., but so far, no complete and mature theory has been developed.

At present, the generally accepted treatment methods for the polycystic ovary at home and abroad mainly comprise the following methods: large dose estrogen therapy, granulocyte colony stimulating factor (g-CSF) intrauterine infusion, oral aspirin, vitamin E, sildenafil, hysteroscope and the like, but at present, clear support of evidence-based medical evidence is not obtained. Although the traditional Chinese medicine is long in use and generally needs to be continuously taken for three menstrual cycles (namely three continuous months), the traditional Chinese medicine has obvious advantages in effectiveness compared with western medicines.

Perimenopausal syndrome, also known as climacteric syndrome (MPS), refers to a group of syndromes caused by sex hormone fluctuation or decrease before and after menopause of women, mainly dysfunction of autonomic nervous system, accompanied with neuropsychological symptoms. Among them, natural menopause refers to menopause caused by complicated etiology including immune factors, hereditary factors, infectious factors, endocrine factors, anatomical factors, etc. due to exhaustion of follicles in ovaries or loss of response of remaining follicles to gonadotropin, no longer developing and secreting estrogen, and failing to stimulate growth of endometrium.

Perimenopausal dysfunction is also a common clinical gynecological disease type, and research report results show that the total effective rate of perimenopausal dysfunction patients treated by the Yijing decoction is 90%, the change of endometrial thickness of the patients, the change of related hormone indexes in serum and the like are obviously reduced compared with the prior art, but relevant reports are rarely made on the treatment of polycystic ovarian syndrome type infertility, insufficient follicular tension and dysplasia retardation.

Disclosure of Invention

The invention provides a traditional Chinese medicine composition for treating infertility, which can increase the curative effects on endometrial thickness increase, follicular tension enhancement and follicle normal development recovery, and finally effectively assist pregnancy.

The invention provides a traditional Chinese medicine composition for treating infertility, which comprises the following raw materials in parts by weight: 25-30 parts of prepared rehmannia root, 12-15 parts of angelica, 25-35 parts of fried bighead atractylodes rhizome, 15-20 parts of fried Chinese yam, 10-12 parts of fried white paeony root, 9-12 parts of raw wild jujube kernel, 6-10 parts of cortex moutan, 9-12 parts of radix glehniae, 8-10 parts of fried eucommia bark, 20-25 parts of codonopsis pilosula, 6-10 parts of radix bupleuri, 8-12 parts of fried cyperus rotundus and 6-9 parts of caulis perllae.

Preferably, the feed additive comprises the following raw materials in parts by weight: 30 parts of prepared rehmannia root, 15 parts of angelica, 30 parts of fried bighead atractylodes rhizome, 15 parts of fried Chinese yam, 10 parts of fried white peony root, 10 parts of raw wild jujube kernel, 10 parts of cortex moutan, 10 parts of radix glehniae, 10 parts of fried eucommia bark, 20 parts of codonopsis pilosula, 10 parts of radix bupleuri, 10 parts of fried cyperus rotundus and 6 parts of caulis perllae.

The invention also provides a preparation method of the traditional Chinese medicine composition for treating infertility, which comprises the following steps: weighing the traditional Chinese medicine raw materials according to the parts by weight, mixing, adding water for decoction, collecting decoction, and concentrating to obtain the traditional Chinese medicine composition.

Preferably, the specific process of decocting with water is as follows: mixing the Chinese medicinal materials, adding water 2-4 times of the total weight of the medicinal materials for the first time, soaking for 20-30min, decocting for 20-30min, filtering, and collecting filtrate; adding 3-4 times of water into the residue, decocting for 20-30min, filtering, and mixing the filtrates.

Preferably, the concentration is to concentrate the decoction until the crude drug content is 2 g/ml.

The traditional Chinese medicine composition for treating infertility can be used for preparing medicines for treating liver-stagnation and spleen-deficiency type polycystic ovarian infertility, insufficient follicular tension and hypoplasia.

The invention also provides a traditional Chinese medicine preparation for treating infertility, which takes the traditional Chinese medicine composition as an active ingredient and is supplemented with pharmaceutically acceptable auxiliary materials, such as pharmaceutically common diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like.

The Chinese medicinal preparation is oral preparation, such as granule, pill, capsule, tablet, oral liquid, etc., or injection via intraperitoneal, intravenous or intramuscular injection, and can be used for treating liver-stagnation and spleen-deficiency type polycystic ovarian infertility, follicular tension insufficiency and dysplasia retardation.

The traditional Chinese medicine considers that the kidney stores the essence of the genitals, the formation, growth and development and reproduction of a person are mainly realized by the metaplasia of the kidney essence, whether the kidney essence is abundant or not is determined by the abundance or insufficiency of the kidney qi, the reproductive capacity is mainly determined by the abundance or insufficiency of the kidney qi, and has a direct relation with the surplus and insufficiency of the kidney qi, the menstruation and pregnancy in the physiological characteristics of a woman are mainly dominated by the uterus, the kidney mainly stores the essence system uterus, the abundance or insufficiency of the kidney qi simultaneously determines whether the function of the uterus is normal, the kidney qi can not normally nourish the uterus, and the dysfunction of the uterus influences the growth of the intima, thereby causing the occurrence of infertility caused by the implantation failure of embryos.

The traditional Chinese medicine composition for treating infertility provided by the invention is prepared from middle-aged rehmannia root which is sweet, warm and thick in taste, moist and abundant in liquid, and is a monarch drug for nourishing blood, nourishing yin, replenishing essence and benefiting marrow; angelica, white peony root, radix angelicae sinensis, liver-nourishing and blood-nourishing; the codonopsis pilosula, the bighead atractylodes rhizome and the Chinese yam are used as ministers to strengthen the spleen and nourish the source; the root of straight ladybell is used as an adjuvant for tonifying lung qi, nourishing yin of lung and stomach and nourishing the upper source of water, and restricts the warm and dry property of the medicines; the eucommia bark can tonify the liver and kidney; semen Ziziphi Spinosae can nourish heart yin, tranquilize mind, and cortex moutan can cool blood and dissipate blood stasis; the bupleurum root soothes liver and regulates qi; cyperus rotundus for soothing liver, relieving depression and regulating menstruation; neutral in nature and fragrant in smell, it can harmonize qi in blood and circulate qi of twelve meridians and eight meridians; the five medicines are used for leading the qi to enter the heart, the liver, the spleen and the kidney and also for other meridians, namely relieving the qi depression of the heart, the liver and the spleen and dispersing the depression of the blood. The whole formula aims at tonifying the kidney, soothing the liver, strengthening the spleen and relieving depression. The combination of the above medicines has the effects of warming and invigorating kidney yang, and tonifying but not greasy, warming but not dry, and the combination of yang-tonifying medicine and yin-tonifying medicine radix rehmanniae Preparata can help yang yin and make biochemistry infinite.

Compared with the prior art, the invention has the beneficial effects that:

1. the traditional 'Yijing decoction' is mainly used for treating women menopausal syndrome patients with qi stagnation of heart, liver and spleen channels, which is not seven years old, and people with first break of menstrual water, and the like, the first break of menstrual water is caused by kidney qi deficiency, insufficiency of essence and blood, and qi stagnation and blood stagnation; the traditional Chinese medicine composition for treating polycystic ovary syndrome type infertility mainly aims at the problem that a patient with liver depression and spleen deficiency type cannot successfully conceive, is conditioned by tonifying kidney, soothing liver and strengthening spleen, and is periodically and reasonably taken according to key time nodes of a menstrual cycle of the patient, so that the symptoms of polycystic ovary syndrome of the patient disappear, endometrium is thickened, and follicle is developed and matured to be successfully conceive until the polycystic ovary syndrome type infertility is successfully produced;

2. compared with the traditional Yijing decoction, the clinical application and basic experimental results show that the traditional Chinese medicine composition provided by the invention can increase the curative effects of the traditional Chinese medicine composition on endometrial thickness increase, follicular tension enhancement and follicle normal development recovery, and finally effectively assist pregnancy.

Drawings

FIG. 1 is a graph showing that the MTT method detects the proliferation promoting effect of the drug provided by the embodiment of the invention on HUVEC and MOGC cells;

FIG. 2 is a graph showing the effect of drugs provided in the examples of the present invention on the improvement of the body weight, uterine and ovarian indices of dehydroepiandrosterone-induced model mice; p <0.05 compared to normal control; compared with the model group, # P < 0.05; A. mouse body weight; B. mouse uterine weight; C. mouse ovarian weight;

FIG. 3 is a graph showing the effect of drugs provided in the examples of the present invention on the improvement of dehydroepiandrosterone-induced growth follicle count, mature follicle count, and corpus luteum count in a model mouse; p <0.01, P <0.001, compared to normal controls; compared with the model group, # P < 0.05; A. number of follicles growing; B. mature follicles; C. the number of corpus luteum;

FIG. 4 is a graph of the drug provided by the present invention for improving mifepristone-induced implantation disorder in a model mouse; (a) a normal control group; (b) a mifepristone making module; (c) treating the group with a basic prescription drug after mifepristone molding; (d) the medicine treatment group of the formula is used after mifepristone molding;

FIG. 5 is a graph showing the effects of a Chinese medicinal composition of the present invention on repairing endometriotic lesions in an alcohol-induced mouse model; (a) a normal control group; (b) an ethanol molding module; (c) a basal prescription drug treatment group after ethanol molding; (d) the invention is a medicine treatment group; hematoxylin and eosin (H & E) staining (100 ×);

FIG. 6 is a graph of endometrium proliferation and immunohistochemical staining of angiogenesis marker Vimentin protein for repairing injury of a model mouse by the traditional Chinese medicine composition provided by the embodiment of the invention; (a) a normal control group; (b) an ethanol molding module; (c) a basal prescription drug treatment group after ethanol molding; (d) the invention is a medicine treatment group; (ii) a Immunohistochemical staining (IHC) Vimentin protein (100 ×);

FIG. 7 is the results of a B-mode ultrasound examination of a typical clinical patient prior to treatment; a. b and c represent B ultrasonic detection results of different cases, and the same row diagram represents the B ultrasonic detection result of the same case;

FIG. 8 is the results of a type-B ultrasonic test after 1-4 treatment sessions in a typical clinical patient; a. b and c represent the B ultrasonic detection results of different cases, and the same line diagram represents the B ultrasonic detection result of the same case.

Detailed Description

The present invention is further described below by way of examples, but the present invention is not limited by these examples. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The preparation method of the traditional Chinese medicine composition for treating infertility comprises the following steps:

weighing the traditional Chinese medicine raw materials according to the parts by weight, mixing, adding water which is 8-10 times of the total weight of the medicinal materials for the first time, soaking for 20-30min, decocting for 20-30min, filtering, and collecting filtrate; adding water 6-8 times the weight of the medicinal materials into the residue, decocting for 20-30min, filtering, mixing the filtrates, and concentrating the medicinal liquid to obtain the Chinese medicinal composition with crude drug content of 2 g/ml.

The above Chinese medicinal composition can be mixed with common adjuvants such as diluent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, surfactant, adsorption carrier, lubricant, etc., and made into granule, capsule, tablet, oral liquid or injection according to conventional pharmaceutical method.

The following examples are provided to illustrate the infertility treatment drug of the present invention.

Example 1

A traditional Chinese medicine composition for treating infertility is composed of the following raw materials: 25g of prepared rehmannia root, 12g of angelica, 25g of fried bighead atractylodes rhizome, 15g of fried Chinese yam, 10g of fried white peony root, 9g of raw wild jujube kernel, 6g of cortex moutan, 9g of radix glehniae, 8g of fried eucommia bark, 20g of codonopsis pilosula, 6g of radix bupleuri, 8g of fried cyperus rotundus and 6g of caulis perllae.

Example 2

A traditional Chinese medicine composition for treating infertility is composed of the following raw materials: 30g of prepared rehmannia root, 15g of Chinese angelica, 35g of fried bighead atractylodes rhizome, 20g of fried Chinese yam, 12g of fried white paeony root, 12g of raw wild jujube kernel, 10g of cortex moutan, 12g of radix glehniae, 10g of fried eucommia bark, 25g of codonopsis pilosula, 10g of radix bupleuri, 12g of fried nutgrass galingale rhizome and 9g of caulis perllae.

Example 3

A traditional Chinese medicine composition for treating infertility is composed of the following raw materials: 30g of prepared rehmannia root, 15g of Chinese angelica, 30g of fried bighead atractylodes rhizome, 15g of fried Chinese yam, 10g of fried white paeony root, 10g of raw wild jujube kernel, 10g of cortex moutan, 10g of radix glehniae, 10g of fried eucommia bark, 20g of codonopsis pilosula, 10g of radix bupleuri, 10g of fried nutgrass galingale rhizome and 6g of caulis perllae.

Since the therapeutic effects of the drugs prepared in examples 1 to 3 are substantially the same, the therapeutic effect of the drug for treating infertility, which is provided by the present invention, will be described below by taking the drug prepared in example 3 as an example.

First, Effect on proliferation of human-derived cells and murine-derived Primary cells

1.1 isolation and culture of mouse Primary ovarian granulosa cells (MOGC)

6-8 week-old female C57BL/6 mouse (Anhui laboratory center, Anhui province, Anhui) is housed in SPF animal laboratory, and after quarantine, PMSG (horse chorionic gonadotropin) 40 IU/mouse is subcutaneously injected, and CO is administered after 48 hours₂After sacrifice by inhalation anesthesia, the mouse ovaries were aseptically dissected and harvested and washed in pre-chilled sterile PBS (containing 3% penicillin 100U/ml, streptomycin 100. mu.g/ml)Then, after puncturing follicles under a dissecting microscope to release granulosa cells in a pre-cooled DMEM medium and preparing a single cell suspension, 1ml of 0.25% trypsin (containing 0.02% EDTA) was applied at 37 ℃ and 5% CO₂Digesting for 40 min-1 hr in incubator (based on digestion into mucus and cell mass separation), adding 15% FBS-containing culture medium to terminate digestion, filtering with 200 mesh cell sieve, centrifuging at 500 Xg for 5 min, discarding supernatant, collecting cells, inoculating into 60mm culture dish, culturing at 37 deg.C and 5% CO₂And (4) staying overnight in the incubator, replacing the culture solution, discarding the non-adherent cells, and performing MOGC purity identification for subsequent experiments.

1.2MTT assay for cell proliferation Activity

Human umbilical vein endothelial cells HUVEC (ATCC, USA) and primary Mouse Ovarian Granular Cells (MOGC) are selected and cultured in DMEM medium containing 10% FBS, penicillin 100U/ml and streptomycin 100U/ml according to the ratio of 5 x 10³One cell (3X 10)³HUVEC cells/wells are inoculated in a 96-well plate for overnight culture, the cells (5 concentration gradients/5 multiple wells and a solvent control group) treated by the traditional Chinese medicine composition prepared in the embodiment 3 of the invention are treated for 48h, the well plate culture medium is discarded, 50 mul/well of 5mg/ml MTT solution is added, the mixture is placed in an incubator at 37 ℃ and incubated for 2h to 4h in the absence of light, 150 mul/well of DMSO solution is added, the mixture is placed in a shaking table and shaken until crystals are dissolved, the light absorption value (OD570) of each well at the wavelength of 570nm is measured by using a full-wavelength microplate reader (Thermo Lab systems, USA), and the inhibition rate and the IC50 value are calculated.

1.3 results

1.3.1MTT assay method for inducing proliferation of human vascular endothelial cells, Primary mouse ovarian carcinoma granulosa cells in vitro

After primary mouse ovarian granulosa cells MOGC (A) and human vascular endothelial cells HUVEC (B) are treated by the traditional Chinese medicine composition at different concentrations for 48 hours, the proliferation condition of the cells is detected by applying an MTT method. The results are shown in FIG. 1.

MTT experimental results show that after primary mouse ovarian granulosa cells MOGC are treated by the traditional Chinese medicine composition provided by the embodiment 3 for 48 hours, the proliferation activity of the MOGC cells is induced in a concentration-dependent manner. In addition, the result shows that the traditional Chinese medicine composition can well induce the proliferation activity of human umbilical vein endothelial cells HUVEC, for example, compared with a control group, after HUVEC cells are treated by 2.5mg/ml for 48 hours, the induction rate of the HUVEC cells on cell proliferation reaches 49.93 percent, and the formula of the invention is proved to have the effect of promoting angiogenesis.

Second, pharmacodynamics analysis experiment in vivo of mouse

1. In vivo improvement of mouse polycystic ovary syndrome caused by dehydroepiandrosterone

1.1 modeling and dosing methods for PCOS (polycystic ovary syndrome) mouse model

Randomly dividing a 25-day-old C57 BL/6J mouse (Shanghai Si Laike laboratory animal responsibility Co., Ltd.) bred in an animal room of an SPF-grade barrier system and quarantined into a normal control group and a PCOS model group; the mice in model group were injected with 70mg/kg oil (solvent is glycerol, national drug group chemical reagent limited) of 0.2ml dehydroepiandrosterone (DHEA, Shanghai Michelin Biochemical technology limited) subcutaneously via neck and back, and the mice in normal control group were administered with 0.2ml glycerol once a day for 20 days. The mouse is characterized in that a small cotton swab soaked with physiological saline is inserted into a mouse vaginal orifice every morning to collect vaginal cast-off cells, the vaginal cast-off cells are aired on a glass slide, treated for 15 minutes by using 95% ethanol and dyed for 10 minutes by using 5% methylene blue, and after the vaginal cast-off cells are washed and aired, cytological examination is carried out under a microscope to determine the estrus cycle of the mouse. On the day following the last administration, the mice were weighed, anesthetized with 10% chloral hydrate at 3ml/kg by intraperitoneal injection (i.p.), blood was collected by abdominal aorta puncture, and then serum levels of luteinizing survivin and testosterone were measured by ELISA method, dissected, collected, weighed, and fixed in 10% formalin solution.

A mouse vaginal smear is always in an estrus interval, a large number of white blood cells can be seen, a small number of nucleated epithelial cells and vaginal mucus are additionally arranged, a complete estrus cycle does not appear, and a mark for successfully modeling a PCOS model is shown for a person with serum luteinizing survivin and testosterone rising.

Then, the PCOS model mice successfully modeled were randomly divided into 10 mice each of the model group, the basic formula group, the Chinese medicinal composition group of example 3, and 10 mice of the normal control group; each group of mice was fed under the same conditions as usual. The administration dose is obtained according to an equivalent dose ratio table converted from the surface areas of human and animal bodies, and the treatment group is administered with the traditional Chinese medicine composition prepared in the embodiment 3 of the invention; the basic prescription group is administrated by using a traditional Yijing decoction (the basic prescription specifically comprises 30g of prepared rehmannia root, 30g of fried bighead atractylodes rhizome, 15g of fried Chinese yam, 15g of Chinese angelica, 10g of fried white paeony root, 10g of raw wild jujube kernel, 10g of tree peony bark, 10g of coastal glehnia root, 10g of Chinese thorowax root, 10g of fried eucommia bark and 20g of ginseng). The basic formula group and the treatment group are intragastrically filled with 25.0g crude drug/kg, and the normal control group and the model group are intragastrically filled with the same amount of normal saline; the animals were drenched continuously for 12 days for observation of clinical manifestations.

Weighing according to the experimental method, collecting vaginal cast-off cells of each group of mice to perform cytological examination, dissecting and collecting arterial blood to perform serological hormone measurement (testosterone, luteinizing hormone, follitropin and estradiol), collecting and weighing organ tissues of ovaries, uteri and the like of the mice, and calculating the total mature follicle number and corpus luteum number of each stage of follicles.

1.2 results

1.2.1 the formula of the invention changes the body weight, ovarian index and uterine index of mice

The results are shown in FIG. 2.

As can be seen from FIG. 2, the weight gain (P <0.05, FIG. 2A), the weight reduction of uterus (P <0.01, FIG. 2B), and the weight increase of ovary (P <0.05, FIG. 2C) in the model group mice compared with the normal control group are consistent with obesity or overweight and increased ovary in most clinical PCOS patients. Compared with the model group, the weight of the mice in the square group is reduced (P is less than 0.05), and the mice have no statistical difference compared with the normal control group; compared with the model group, the uterus weights of the mice of the basic and the invention square groups are recovered to normal level, and the recovery effect of the invention square group is better than that of the basic square group (P is less than 0.05, figure 2C); compared with the model group, the ovary indexes of the mice of the basic group and the invention group are reduced compared with the ovary indexes of the model group, wherein the effect of the invention group is best (P is less than 0.05, figure 2C), and the invention group has no statistical significance difference with the normal control group; the prescription of the invention has better effect.

1.2.2 the changes of the prescription of the invention on the ovarian follicle number and the corpus luteum number of mice

The results are shown in FIG. 3.

As can be seen from fig. 3, the number of growing follicles in the model group mice was significantly increased (fig. 3A, P <0.01), and the number of mature follicles and the number of corpus luteum were significantly decreased (fig. 3B-C, P <0.001), compared to the normal control group, indicating that the PCOS modeling was successful; compared with a model group and a basic group, the method can restore the number of the growing follicles to a normal level, has more obvious statistical difference (figure 3A, P is less than 0.01), also can increase the number of the mature follicles to be close to or better than the normal level, and has more obvious statistical difference (figure 3B, P is less than 0.001); compared with the model group, the invention can increase the corpus luteum number to the normal level, and the effect is obviously better than that of the basic group (figure 3C, P is less than 0.001).

1.3.3 measurement of the Effect of serum sex hormone levels

The results are shown in Table 1.

TABLE 1 drug improvement of serum sex hormone levels in dehydroepiandrosterone-induced model mice

Effect of the invention on serum sex hormone levels in PCOS model mice (Table 1)

Note: testosterone: testosterone (T); luteinizing hormone: luteinizing Hormon (LH); folliculin hormone: folliculcle-stimulating hormone (FSH); estradiol: estradiol (E2).

As can be seen from Table 1, compared with the normal control group, the serum testosterone (T), Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) levels of the mice in the PCOS model group are all obviously increased, the estradiol (E2) level is obviously reduced, and the statistical significance is significant (P is less than 0.05); compared with a model group, the levels of T, LH and FSH in the serum of the mice in the square group are obviously reduced, and the level of E2 is obviously increased, which have statistical significance (P is less than 0.01); the improvement effect of the formula of the invention is obviously better than that of the basic formula, and the formula has statistical significance (P is less than 0.05).

2 improving implantation disorder of mice caused by mifepristone in vivo

2.1 Molding and administration methods

The 6-8 week old C57BL/6 mice (male and female mice with a sex ratio of 2:1, Shanghai Si Laike animal center, Shanghai) were selected and bred in SPF-class animal laboratories. After quarantine, female mice are randomly divided into a normal control group and a mifepristone model group, then the female and male mice are bred in a cage according to the ratio of 2:1 (vaginal suppository appears and is recorded as the 1 st day of pregnancy), the model group is injected with 0.1ml of mifepristone (0.08mg of mifepristone is dissolved in 0.1ml of propylene glycol) in a single time at the 4 th day of pregnancy of the mice, the normal control group is injected with 0.1ml of propylene glycol, uterus and ovary tissues are collected after the experimental animals are killed at the 12 th day for observing the implantation number of embryos of each group, and the success construction of the mifepristone-induced embryo implantation disorder mouse model is determined.

Then, after modeling (setting normal control at the same time) according to the method, the female mice after modeling are randomly divided into a model control group, a basic formula treatment group and a formula treatment group of the invention, and a normal control group is set. The physiological saline (normal control group and model control group), the basic formula and the formula of the invention are respectively applied to continuously treat the corresponding experimental group for 12 days and observe the clinical symptoms of the experimental animals once a day. After the experiment is finished, the experimental mice are sacrificed, the implantation condition of the embryo is observed after the uterus and the ovary of each group of experimental animals are collected, and the morphological and histological analysis is carried out.

2.2 results of the experiment

The results are shown in FIG. 4.

As can be seen from FIG. 4, after mifepristone-induced embryo implantation disorder model mice are treated by the drug therapy or normal saline (normal control group) provided by the invention for 12 days, no obvious abnormality of experimental mice of the invention is found; observation results of the change of the uterus size, the endometrial thickness and the ovary morphology of each group of experimental animals are collected after the experiment is finished show that the medicament provided by the invention can obviously improve the mouse embryo implantation disorder induced by mifepristone after treatment.

3. Ameliorating ethanol-induced endometrial injury

3.1 animal modeling and treatment methods

Purchasing, quarantine and breeding of experimental animals are as described above. Randomly dividing the female mice into a normal control group and a model group, carrying out uterine cavity perfusion treatment on the model group by 0.5ml of alcohol (ethanol with the mass fraction of 95%) through uterine horns of the mice for 5 minutes in the estrus of the physiological period of the female mice, and then flushing the uterine cavity by using physiological saline (N.S.) and sucking out residual ethanol; the normal control group is subjected to uterine cavity perfusion and N.S. flushing treatment by using PBS with the same volume; killing the mice after 6 hours of model building, dissecting and observing the endometrial injury condition of each experimental animal group, dividing the mice successfully modeled into a model building control group, a basic square group and a square group of the invention after determining that the ethanol-induced endometrial injury model is successfully built, setting a normal control group (10 experimental mice in each group), and continuously treating for 12 days. During which the clinical symptoms of the experimental animals were observed daily. Mice were sacrificed after the experiment was completed and the uteri of each group of mice were collected for histomorphological observation.

3.2H & E staining

The preparation of the histopathological section comprises the steps of dehydration, wax dipping, embedding, slicing, dyeing, mounting and the like, and finally, the histopathological section is observed under a microscope. The specific operation steps are as follows:

(1) and (3) dehydrating: taking out fixed mouse organ tissues, flushing with running water, then dehydrating the tissues by using gradient concentration ethanol (the sequence of 50% for 2 hours, 70% for 2 hours, 80% for 2 hours, 95% (2 times), 1 hour for each treatment, and 100% (2 times) for 1 hour) with the concentration from low to high, and replacing water in the tissues by ethanol layer by layer;

(2) and (3) transparency: immersing the dehydrated tissue into dimethylbenzene for two times for carrying out transparent treatment, wherein each time is 1 hour;

(3) wax dipping and embedding: immersing the tissue after transparent treatment in paraffin of 55-60 ℃ for treatment and full paraffin immersion, standing the tissue in a mould, and then embedding the tissue in paraffin on an embedding mechanism cold plate;

(4) slicing: repairing and flattening the surface of a wax block tissue, adjusting the slice thickness of a slicer to 4-5 mu m for slicing, flattening the tissue slice in a water bath slice spreading machine at 55-60 ℃, slowly and upwards fishing out the tissue slice by applying a glass slide, and baking the glass slide for more than 2 hours at 60 ℃;

(5) dewaxing: after 20 minutes of each treatment of xylene I and xylene II, gradient ethanol (100%, 95%, 80%, 70%, 50%) is added for 5 minutes each time, and finally, the mixture is soaked in distilled water for 5 minutes;

(6) h & E staining: first, the sections were stained with 0.5% hematoxylin for 3 minutes, washed with tap water, 1% ammonia water to turn blue, washed with tap water, differentiated with 1% hydrochloric acid ethanol, and washed with tap water for more than 30 minutes to stain the nuclei. Next, after staining cytoplasm with red color by counterstaining with 0.5% eosin for 10 minutes, the sections were dehydrated with 95% ethanol 2 times (1 minute/time), 100% ethanol 2 times (5 minutes/time), and then soaked in xylene for 2 times (10 minutes/time) to be cleared. Finally, the tissues were mounted with neutral gum, and the pathological changes were observed and photographed under a microscope connected to a photographic system.

3.3 Immunohistochemical (IHC) staining

(1) The methods of fixing, transparentizing, paraffin embedding, slicing, dewaxing and the like of animal tissues are shown in the 3.2;

(2) and (3) sealing: mass fraction 3% H₂O₂Solution blocking of endogenous peroxidase (wet box incubation for 10 min in the dark);

(3) antigen retrieval: rinsing the slices with deionized water at room temperature for 3 times, placing the slices in an antigen retrieval box containing sodium citrate buffer solution, and boiling (steaming for 3 minutes) in a pressure cooker to perform antigen retrieval treatment; taking out the antigen retrieval box, after the section is naturally cooled, placing the section in PBS for washing for 3 times, then placing the section in 0.1% TritonX-100, shaking the section by a shaking table slowly for 15 minutes, and washing in PBS for 3 times, wherein each time is 5 minutes;

(4) antibody incubation: sealing the slices in an incubator at 37 ℃ for 40 minutes by using a skim milk solution with the mass fraction of 5%, then sucking residual milk sealing liquid by using filter paper, diluting (1:500 dilution), dropwise adding anti-vismentin (Elapscience, Wuhan) into the slices, placing the slices in a wet box in a dark place at 4 ℃ for incubation overnight, taking out the wet box, standing the slices at room temperature to recover to the room temperature (about 30-40 minutes), and rinsing 3 times (5 minutes/time) by using 1 XPBS; after dropwise addition of biotin-labeled secondary antibody diluted with 1% BSA at 37 ℃ for 10-30 minutes, rinsing 3 times (5 min/time) with PBS; dripping 1 × PBS diluted streptavidin labeled with horseradish peroxidase, incubating at 37 deg.C for 10-30 min, and rinsing with PBS 3 times (5 min/time);

(5) color development: applying DAB color developing agent for color development, placing the section under a microscope for observation, and after determining the dyeing time, flushing with tap water to stop color development; after hematoxylin stains cell nucleuses, the section is placed under a microscope for observation to determine staining time, and tap water is used for washing to stop color development and blue returning;

(6) gradient ethanol elution (80% ethanol, 5 min; 95% ethanol, 5 min; 100% ethanol, 5 min); dehydrating and transparent xylene for 5 minutes; sealing the slices with neutral gum, air drying, observing the slices under a microscope, taking pictures, and analyzing and counting.

3.4 results of the experiment

(1) H & E staining

H & E staining is a common histopathological technique, is one of the gold standards for clinical medical diagnosis, and can be used for diagnosis of various diseases, organ lesion monitoring and the like. In order to evaluate the repairing effect of the traditional Chinese medicine composition on the endometrium of an experimental animal, the major organs of ovaries, uteruses, hearts, livers, spleens, lungs, kidneys and the like of each group of mice are collected at the end point of the experiment, stored in 4% PFA solution and subjected to H & E staining. The results are shown in FIG. 5.

The results show that after the ethanol is used for carrying out uterine perfusion molding on the mice, the endometrium of the experimental mice is seriously damaged and falls off, and after the traditional Chinese medicine composition provided by the embodiment 3 of the invention is used for continuously treating model animals for 12 days, the endometrium damage induced by ethanol molding is obviously improved, which indicates that the traditional Chinese medicine composition provided by the invention has the effect of repairing the damaged endometrium of the model mice.

(2) Immunohistochemical staining

In order to further verify the effects of the traditional Chinese medicine composition on promoting endometrial cell proliferation and angiogenesis in vivo, at the end point of an experiment, uterine tissues of an animal are collected, fixed in a 4% PFA solution and sliced, and then immunohistochemical staining is carried out by using a cell proliferation and angiogenesis related marker Vimentin. The results are shown in FIG. 6.

The results show that ethanol modeling (AM) can significantly reduce the expression level of Vimentin in uterine tissue cells of a model mouse, while the expression level of Vimentin in the traditional Chinese medicine composition group provided by the embodiment 3 of the invention is recovered, which indicates that the traditional Chinese medicine composition provided by the embodiment 3 of the invention has the effects of promoting the proliferation of endometrial cells and angiogenesis of animals in vivo.

Third, analysis of treatment effect of PCOS clinical patients

1.1 statistical analysis of treatment effectiveness of PCOS clinical patients

Clinical test results and observation of 58 women (24-40 years old) with liver depression and spleen deficiency type polycystic ovary syndrome (PCOS) of childbearing age show that after every month of menstruation of the patients is clean, the traditional Chinese medicine composition provided by the application is continuously used for 10 days (one dose per day), the calculation is one treatment course, and after 1-4 treatment courses, the effective rate reaches more than about 95%.

1.2 clinical patient hormone level statistics

Clinical test results of female patients with PCOS infertility of childbearing age show that Luteinizing Hormone (LH) and follicle-stimulating hormone (FSH) are obviously increased (if LH of the patients is respectively 15.41 times higher than the lower limit and 2.94 times higher than the upper limit of the normal level), after 1-4 courses of treatment (one dose is taken before every month before menstruation or after every day after the menstruation is clear, decocted and continuously taken for 10 days, counted as a course of treatment), the clinically detected Luteinizing Hormone (LH) and follicle-stimulating hormone (FSH) are obviously reduced to the normal level range, which is beneficial to restoring normal development of follicles, and the results are shown in Table 2.

TABLE 2 the Chinese medicinal composition provided by the present invention improves serum sex hormone levels of PCOS clinical patients

Effect of the inventive formulation on serum sex hormone levels in clinical patients with PCOS (Table 2)

Note: luteinizing hormone: luteinizing Hormon (LH); folliculin hormone: folliculcle-stimulating hormone (FSH).

1.3 statistics for restoration of ovarian function, normal follicular development and enhancement of follicular tone

Clinical test results of female patients with PCOS infertility in childbearing age show that, after 1-4 treatment courses (one dose is taken before each month of menstruation or after each day of clean menstruation, decocted and taken continuously for 10 days, counted as a treatment course), the ovarian function can be basically recovered to a normal level, the ovarian follicles grow to 16-19 mm from the original diameter of 3-8 mm, the follicular tension is obviously enhanced and mature follicles are gradually formed, the follicular development is greatly enhanced and the success rate of conception is improved, and the results are shown in fig. 7 (before treatment) and fig. 8 (after treatment).

The above disclosure is only for the specific embodiment of the present invention, but the embodiment of the present invention is not limited thereto, and any variations that can be made by those skilled in the art should fall within the scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating infertility is characterized by comprising the following raw materials in parts by weight: 25-30 parts of prepared rehmannia root, 12-15 parts of angelica, 25-35 parts of fried bighead atractylodes rhizome, 15-20 parts of fried Chinese yam, 10-12 parts of fried white paeony root, 9-12 parts of raw wild jujube kernel, 6-10 parts of cortex moutan, 9-12 parts of radix glehniae, 8-10 parts of fried eucommia bark, 20-25 parts of codonopsis pilosula, 6-10 parts of radix bupleuri, 8-12 parts of fried cyperus rotundus and 6-9 parts of caulis perllae.

2. The traditional Chinese medicine composition for treating infertility according to claim 1, which is characterized by comprising the following raw materials in parts by weight: 30 parts of prepared rehmannia root, 15 parts of angelica, 30 parts of fried bighead atractylodes rhizome, 15 parts of fried Chinese yam, 10 parts of fried white peony root, 10 parts of raw wild jujube kernel, 10 parts of cortex moutan, 10 parts of radix glehniae, 10 parts of fried eucommia bark, 20 parts of codonopsis pilosula, 10 parts of radix bupleuri, 10 parts of fried cyperus rotundus and 6 parts of caulis perllae.

3. The traditional Chinese medicine composition for treating infertility according to claim 1 or 2, wherein the parched white atractylodes rhizome is roasted with honey, the parched yam rhizome, the parched white peony root and the parched eucommia bark are all roasted with saline water, and the parched cyperus rotundus is roasted with vinegar.

4. The method for preparing a Chinese medicinal composition for the treatment of infertility according to claim 1, comprising the steps of: weighing the traditional Chinese medicine raw materials according to the parts by weight, mixing, adding water for decoction, collecting decoction, and concentrating to obtain the traditional Chinese medicine composition.

5. The method for preparing a Chinese medicinal composition for treating infertility according to claim 4, wherein the water decoction comprises the following steps: mixing the Chinese medicinal materials, adding water with an amount of 8-10 times of the total weight of the medicinal materials for the first time, soaking for 20-30min, decocting for 20-30min, filtering, and collecting filtrate; adding water 6-8 times the weight of the medicinal materials into the residue, decocting for 20-30min, filtering, and mixing the filtrates.

6. The method for preparing a Chinese medicinal composition for the treatment of infertility according to claim 4 or 5, wherein the concentration is to concentrate the decoction to a crude drug content of 2 g/ml.

7. Use of the Chinese medicinal composition for treating infertility according to any of claims 1 to 3 in the preparation of a medicament for treating liver-stagnation and spleen-deficiency type polycystic ovarian infertility, follicular tension insufficiency and dysplasia retardation.

8. A Chinese medicinal preparation for treating infertility, which is characterized in that the Chinese medicinal composition of any one of claims 1 to 3 is used as an active ingredient, and pharmaceutically acceptable auxiliary materials are added.

9. The traditional Chinese medicine preparation according to claim 8, which is an oral preparation or an injection.