WO2022227814A1 - 一种用于治疗不孕症的中药组合物及其制备方法、应用 - Google Patents

一种用于治疗不孕症的中药组合物及其制备方法、应用 Download PDF

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WO2022227814A1
WO2022227814A1 PCT/CN2022/076993 CN2022076993W WO2022227814A1 WO 2022227814 A1 WO2022227814 A1 WO 2022227814A1 CN 2022076993 W CN2022076993 W CN 2022076993W WO 2022227814 A1 WO2022227814 A1 WO 2022227814A1
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chinese medicine
parts
traditional chinese
fried
medicine composition
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French (fr)
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路文杰
徐有志
徐明祥
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安徽医科大学
合肥兜率宫医药科技有限公司
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Priority to ZA2023/10045A priority Critical patent/ZA202310045B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A61K36/804—Rehmannia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A—HUMAN NECESSITIES
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    • A61K36/18—Magnoliophyta (angiosperms)
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/284—Atractylodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/34—Campanulaceae (Bellflower family)
    • A61K36/344—Codonopsis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K36/18—Magnoliophyta (angiosperms)
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    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

Definitions

  • the invention belongs to the technical field of traditional Chinese medicine, and specifically discloses a traditional Chinese medicine composition for treating infertility, a preparation method and an application thereof.
  • Infertility is a reproductive disorder. At present, the incidence of infertility in China is 7%-10%, and it is increasing year by year. Infertility can be caused by a variety of causes, such as the influence of environmental drugs, ovulation problems, and organic problems of the body, which can all lead to infertility.
  • PCOS Polycystic ovary syndrome
  • PCOS In addition to causing female infertility, oligomenorrhea, amenorrhea, and hirsutism, PCOS also has far-reaching effects on the endometrium, glucose and lipid metabolism, and cardiovascular system. Endometrial cancer, breast cancer, obesity, coronary heart disease, The risk of atherosclerosis, diabetes, etc. increases, and some patients also have psychological barriers due to oligomenorrhea, amenorrhea, infertility, acne, obesity, hirsutism and many other problems. Therefore, it is of great significance to pay attention to the pathogenesis and treatment progress of PCOS, to improve the pregnancy rate of polycystic ovary syndrome and ovulatory dysfunction infertility patients, to reduce their abortion rate, and to reduce their treatment costs.
  • the pathophysiology and pathogenesis of PCOS are very complex and still inconclusive, which may be related to many factors such as hyperfunction of the adrenal glands, abnormal insulin-like growth factor, neuroendocrine dysfunction and genetics.
  • the abnormality of the hypothalamus-pituitary-ovarian axis affects the normal secretion of gonadotropin and luteinizing hormone, and the ovarian secretion of excessive androgens;
  • the membrane island Steroid resistance is an important metabolic abnormality in PCOS patients;
  • the lack of ovarian and adrenal hormones increases the conversion of steroids to androgens.
  • hypotheses such as the adrenal sprouting hypothesis, the "double defect" hypothesis, etc., but so far they have not been able to form a relatively complete and mature theory.
  • the treatment methods of polycystic ovary recognized at home and abroad mainly include the following: high-dose estrogen therapy, intrauterine infusion of granulocyte colony-stimulating factor (g-CSF), oral aspirin, vitamin E, sildenafil and uterine Endoscopy, etc., but they have not yet been clearly supported by evidence-based medical evidence.
  • g-CSF granulocyte colony-stimulating factor
  • traditional Chinese medicine is used for a long time, it generally requires continuous medication for three menstrual cycles (that is, three consecutive months), but it has obvious advantages in effectiveness compared with Western medicine.
  • Perimenopause syndrome also known as menopausal syndrome (MPS) refers to a group of syndromes with neuropsychological symptoms mainly caused by the fluctuation or reduction of sex hormones in women before and after menopause.
  • natural menopause refers to the exhaustion of follicles in the ovary, or the loss of response of the remaining follicles to gonadotropins, the follicles no longer develop and secrete estrogen, and cannot stimulate endometrial growth, leading to menopause.
  • the etiology is complex and there are immune factors. , genetic factors, infectious factors, endocrine factors, anatomical factors, etc.
  • Perimenopause dysfunction is also a common clinical type of gynecological disease. According to research reports, the overall effective rate of Yijing Decoction in the treatment of patients with perimenopausal dysfunction is 90%, and the endometrial thickness of patients changes. The changes of related hormone indexes in serum were significantly lower than the previous ones, but there are few related reports on the treatment of polycystic ovary syndrome-type infertility, follicular hypotonia and dysplasia.
  • the present invention provides a traditional Chinese medicine composition for treating infertility, which has the curative effects of promoting endometrial thickness increase, enhancing follicle tension, restoring normal follicle development, and finally effectively assisting pregnancy.
  • the traditional Chinese medicine composition for treating infertility is composed of the following raw materials in parts by weight: 25-30 parts of Rehmannia glutinosa, 12-15 parts of Angelica sinensis, 25-35 parts of fried Atractylodes Rhizoma, 15-20 parts of fried yam servings, 10-12 servings of fried white peony root, 9-12 servings of raw sour jujube seed, 6-10 servings of Danpi, 9-12 servings of North Radix Ginseng, 8-10 servings of fried Eucommia ulmoides, 20-25 servings of Codonopsis Radix, Bupleurum 6- 10 servings, 8-12 servings of fried fragrant aconite and 6-9 servings of power.
  • it is composed of the following raw materials in parts by weight: 30 parts of Rehmannia glutinosa, 15 parts of Angelica sinensis, 30 parts of fried Atractylodes, 15 parts of fried yam, 10 parts of fried white peony, 10 parts of raw sour jujube seeds, 10 parts of Danpi, 10 servings of Astragalus, 10 servings of Fried Eucommia, 20 servings of Codonopsis Radix, 10 servings of Bupleurum, 10 servings of Fried Aconite and 6 servings of Su Stem.
  • the present invention also provides the preparation method of the above-mentioned traditional Chinese medicine composition for treating infertility, comprising the following steps: weighing each traditional Chinese medicine raw material by weight, mixing, adding water for decocting, collecting the decocting liquid, and concentrating to obtain the The traditional Chinese medicine composition.
  • the specific process of adding water for decocting is as follows: after mixing the various traditional Chinese medicine raw materials, adding water equivalent to 2-4 times the total weight of the medicinal materials for the first time, soaking for 20-30 minutes, decocting for 20-30 minutes, filtering, Collect the filtrate; add water equivalent to 3-4 times the weight of the medicinal material to the filter residue, continue to decoct for 20-30min, filter, and combine the two filtrates.
  • the concentration is to concentrate the decoction liquid to a crude drug content of 2 g/ml.
  • the above-mentioned traditional Chinese medicine composition for treating infertility can be used to prepare a medicine for treating polycystic ovary infertility, insufficient follicular tone and dysplasia of liver depression and spleen deficiency type.
  • the present invention also provides a traditional Chinese medicine preparation for treating infertility, which uses the above-mentioned traditional Chinese medicine composition as an active ingredient, and is supplemented with pharmaceutically acceptable auxiliary materials, such as diluents, excipients, fillers, adhesives commonly used in pharmacy , wetting agent, disintegrant, absorption enhancer, surfactant, adsorption carrier, lubricant, etc.
  • pharmaceutically acceptable auxiliary materials such as diluents, excipients, fillers, adhesives commonly used in pharmacy , wetting agent, disintegrant, absorption enhancer, surfactant, adsorption carrier, lubricant, etc.
  • the above-mentioned traditional Chinese medicine preparations are oral preparations, such as granules, pills, capsules, tablets, oral liquids, etc., or injections by intraperitoneal, intravenous, intramuscular injection, and the traditional Chinese medicine preparations can be used to treat liver depression and spleen deficiency. Cystic ovarian infertility, follicular hypotonia, and dysplasia.
  • kidney stores the essence of reproduction.
  • the formation, growth, development and reproduction of human beings are mainly realized by the metaplasia of kidney essence.
  • the rise and fall of kidney qi determines whether the kidney essence is full or not.
  • the reproductive capacity is dominated by the rise and fall of kidney qi. It is directly related to the excess and deficiency of kidney qi.
  • Menstruation and pregnancy are the most important physiological characteristics of women. They are all dominated by the uterus.
  • the kidney mainly stores the sperm and the uterus.
  • the rise and fall of kidney qi also determines whether the uterus functions normally. Qi deficiency cannot nourish the uterus normally, and the dysfunction of the uterus affects the growth of the endometrium, which leads to the failure of embryo implantation and the occurrence of infertility.
  • Rehmannia glutinosa is warm and rich in flavor, moisturizing and rich in liquid, nourishes blood, nourishes yin, invigorates essence and nourishes marrow, and is a monarch medicine; Angelica sinensis, white peony root, soothes liver and nourishes blood; Codonopsis, Codonopsis, Atractylodes and Chinese yam invigorating the spleen and nourishing the source are the ministers; Radix Astragali is the assistant, benefiting the lung qi, nourishing the yin of the lung and stomach, nourishing the upper source of water, and restricting the warm and dry properties of various medicines; Eucommia ulmoides nourishes the liver and kidney; Heart yin, calms the heart and calms the mind, peony bark cools blood and dissipates blood stasis; Bupleurum soothes the liver and regulates qi; Xi
  • the traditional "Yijing Decoction” is mainly used to treat women with qi stagnation in the heart, liver and spleen meridians, women with menopausal syndrome such as those who are not seven or seven years old, and menstrual water will break first before they are seven or seven years old. Due to the first failure of kidney qi, insufficient essence and blood, and qi stagnation and blood stagnation; and the traditional Chinese medicine composition provided by the invention for the treatment of polycystic ovary syndrome infertility is mainly aimed at patients with liver stagnation and spleen deficiency.
  • Soothing the liver and strengthening the spleen are used for conditioning, and periodic and rational drug use is carried out according to the key time nodes of the patient's menstrual cycle to achieve the disappearance of the symptoms of polycystic ovary syndrome, the thickening of the endometrium, and the mature follicles. Production;
  • Fig. 1 is a graph showing the pro-proliferation effect of the medicine provided in the embodiment of the present invention on HUVEC and MOGC cells detected by MTT method;
  • Figure 2 is the effect of the medicine provided in the embodiment of the present invention on improving the body weight, uterus and ovarian index of model mice induced by DHEA; compared with the normal control group, *P ⁇ 0.05; compared with the model group, #P ⁇ 0.05 ;
  • A mouse weight;
  • B mouse uterus weight;
  • C mouse ovary weight;
  • Fig. 3 is the effect that the medicine provided in the embodiment of the present invention improves the number of growing follicles, the number of mature follicles and the number of corpus luteum in model mice induced by dehydroepiandrosterone; compared with the normal control group, **P ⁇ 0.01, ***P ⁇ 0.001; compared with the model group, #P ⁇ 0.05; A, the number of growing follicles; B, the number of mature follicles; C, the number of corpus luteum;
  • Fig. 4 is a graph of improving the implantation disorder of model mice caused by mifepristone by drugs provided in the embodiment of the present invention; (a) normal control group; (b) mifepristone modeling group; (c) mifepristone The drug treatment group using the basic formula after modeling; (d) the drug treatment group using the prescription of the present invention after mifepristone modeling;
  • FIG. 5 is a diagram showing the situation that the traditional Chinese medicine composition provided in the embodiment of the present invention can repair the endometrial lesions of alcohol-induced model mice; (a) normal control group; (b) ethanol modeling group; (c) ethanol modeling Houbasi prescription drug treatment group; (d) the present invention drug treatment group; hematoxylin and eosin (H&E) staining (100 ⁇ );
  • FIG. 6 is a diagram of immunohistochemical staining of Vimentin protein, a marker of endometrial proliferation and angiogenesis in model mice, which can be repaired by the traditional Chinese medicine composition provided in the embodiment of the present invention; (a) normal control group; (b) ethanol modeling group; (c) the basic formula drug treatment group after ethanol modeling; (d) the present invention formula drug treatment group; immunohistochemical staining (IHC) Vimentin protein (100 ⁇ );
  • Figure 7 is the B-ultrasound test results of a typical clinical patient before treatment; a and b represent the B-ultrasound test results of different cases, and the same row of diagrams represents the B-ultrasound test results of the same case;
  • Figure 8 is the B-ultrasound detection results of typical clinical patients after 1-4 courses of treatment; a and b represent the B-ultrasound detection results of different cases, and the same row of diagrams represents the B-ultrasound detection results of the same case.
  • the invention provides a traditional Chinese medicine composition for treating infertility, which is composed of the following raw materials in parts by weight: 25-30 parts of Rehmannia glutinosa, 12-15 parts of Angelica sinensis, 25-35 parts of fried Atractylodes Rhizoma, and 15-35 parts of fried yam.
  • the present invention also provides the preparation method of the above-mentioned traditional Chinese medicine composition for treating infertility, comprising the following steps:
  • the above-mentioned traditional Chinese medicine composition can be added with commonly used auxiliary materials, such as diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. It can be prepared into granules, capsules, tablets, oral liquids or injections by the above conventional methods.
  • auxiliary materials such as diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. It can be prepared into granules, capsules, tablets, oral liquids or injections by the above conventional methods.
  • a traditional Chinese medicine composition for treating infertility comprising the following raw materials: 25g of Rehmannia glutinosa, 12g of Angelica sinensis, 25g of fried Atractylodes, 15g of fried yam, 10g of fried white peony root, 9g of raw sour jujube kernel, 6g of Danpi, Radix Radix et Rhizoma 9g, Fried Eucommia 8g, Codonopsis 20g, Bupleurum 6g, Fried Aconite 8g and Su Stem 6g.
  • a traditional Chinese medicine composition for treating infertility comprising the following raw materials: 30g of Rehmannia glutinosa, 15g of Angelica sinensis, 35g of fried Atractylodes, 20g of fried yam, 12g of fried white peony root, 12g of raw sour jujube seed, 10g of Danpi, Radix Radix et Rhizoma 12g, Fried Eucommia 10g, Codonopsis 25g, Bupleurum 10g, Fried Aconite 12g and Su Stem 9g.
  • a traditional Chinese medicine composition for treating infertility comprising the following raw materials: 30g of Rehmannia glutinosa, 15g of Angelica sinensis, 30g of fried Atractylodes, 15g of fried yam, 10g of fried white peony root, 10g of raw sour jujube seed, 10g of Danpi, Radix Radix et Rhizoma 10g, Fried Eucommia 10g, Codonopsis 20g, Bupleurum 10g, Fried Aconite 10g and Su Stem 6g.
  • Example 3 Since the therapeutic effects of the medicines prepared in Examples 1-3 are basically the same, the following only takes the medicine prepared in Example 3 as an example to illustrate the therapeutic effects of the medicines for treating infertility provided by the present invention.
  • mice 6-8-week-old female C57BL/6 mice (Anhui Provincial Laboratory Animal Center, Anhui) were raised in SPF animal laboratory and quarantined, and then subcutaneously injected with PMSG (horse chorionic gonadotropin) 40IU/mice, 48 hours later After the mice were killed by CO 2 inhalation anesthesia, the mouse ovaries were dissected and collected under aseptic conditions, washed in pre-cooled sterile PBS (containing 3% 100U/ml penicillin, 100 ⁇ g/ml streptomycin), and then stabbed under a dissecting microscope.
  • PMSG human chorionic gonadotropin
  • the granulosa cells were released in pre-cooled DMEM medium and made into a single cell suspension, then digested with 1 ml of 0.25% trypsin (containing 0.02% EDTA) for 40 minutes in a 37°C, 5% CO 2 incubator -1 hours (subject to the digestion as mucus and the cell clumps are separated), add medium containing 15% FBS to terminate the digestion, filter with a 200-mesh cell sieve, centrifuge at 500 ⁇ g for 5 minutes, discard the supernatant to collect cells and inoculate them in a 60 mm culture dish Incubate at 37 °C, 5% CO 2 overnight, and after changing the medium to discard non-adherent cells, the purity of MOGC was identified for subsequent experiments.
  • trypsin containing 0.02% EDTA
  • HUVEC Human umbilical vein endothelial cells HUVEC (ATCC, USA) and primary mouse ovarian granulosa cells (MOGC) were selected and cultured in DMEM medium containing 10% FBS, 100U/ml penicillin, and 100U/ml streptomycin according to 5 ⁇ 10 3 cells (3 ⁇ 10 3 HUVEC cells)/well were inoculated in a 96-well plate and cultured overnight, and the cells were treated with the traditional Chinese medicine composition prepared in Example 3 of the present invention (set 5 concentration gradients/5 duplicate wells, And set up solvent control group) after 48 hours of treatment, discard the medium of the well plate, add 50 ⁇ l/well of 5 mg/ml MTT solution, place it in a 37°C incubator and incubate in the dark for 2h-4h, then add 150 ⁇ l/well DMSO solution , placed on a shaker until the crystals melted, and a full-wavelength microplate reader (Thermo Lab systems, USA) was used to measure
  • MTT assay induces the proliferation of human vascular endothelial cells and primary mouse ovarian cancer granulosa cells in vitro
  • the results of the MTT experiment showed that the application of the traditional Chinese medicine composition provided in Example 3 of the present invention (represented by the formula group of the present invention) after treating the primary mouse ovarian granulosa cells MOGC for 48 hours, induced the proliferation activity of MOGC cells in a concentration-dependent manner .
  • the results show that the traditional Chinese medicine composition can better induce the proliferation activity of human umbilical vein endothelial cells HUVEC.
  • the traditional Chinese medicine composition of the present invention has the effect of promoting angiogenesis.
  • PCOS polycystic ovary syndrome
  • mice The 25-day-old C57BL/6J mice (Shanghai Slack Laboratory Animal Co., Ltd.) that were reared in the SPF barrier system animal room and quarantined were randomly divided into normal control group and PCOS model group; Subcutaneous injection of 0.2ml dehydroepiandrosterone (DHEA, Shanghai McLean Biochemical Technology Co., Ltd.) 70mg/kg oil (the solvent is glycerol, Sinopharm Chemical Reagent Co., Ltd.), the normal control group was given 0.2ml glycerol, once a day, Continuous injection for 20 days.
  • DHEA dehydroepiandrosterone
  • mice were anesthetized by intraperitoneal injection (i.p.) of 10% chloral hydrate at 3 ml/kg, and the serum luteinizing survivin was measured by ELISA after blood collection through abdominal aorta puncture. , testosterone level, anatomically collected and weighed mouse ovary and uterus and other organs and tissues, and then fixed in 10% formalin solution for preservation.
  • i.p. intraperitoneal injection
  • Vaginal smears of mice always show that they are in the interestrus phase, with a large number of white blood cells and a small amount of nucleated epithelial cells and vaginal mucus. There is no complete estrous cycle.
  • the increase in serum luteinizing survivin and testosterone is a sign of successful modeling of the PCOS model. .
  • the PCOS model mice that were successfully modeled were randomly divided into the model group, the basic formula group, the traditional Chinese medicine composition group of Example 3, 10 mice in each, and 10 mice in the normal control group; the mice in each group were routinely fed under the same conditions.
  • the dosage is obtained, the treatment group is administered with the traditional Chinese medicine composition prepared in Example 3 of the present invention; the basic formula group is administered with traditional Yijing Decoction (
  • the specific composition of the basic recipe is: Rehmannia glutinosa 30g, fried Atractylodes 30g, fried yam 15g, Angelica 15g, fried white peony root 10g, raw sour jujube seed 10g, Cortex Moutan 10g, Beisha ginseng 10g, Bupleurum 10g, fried Eucommia 10g and ginseng 20g ).
  • the basic formula group and the treatment group were given 25.0 g crude drug/kg by gavage, and the normal control group and the model group were given the same amount of normal saline by gavage.
  • the weight of the mice in the model group increased (P ⁇ 0.05, A in Figure 2), decreased uterine weight (P ⁇ 0.01, B in Figure 2), and increased ovarian weight (P ⁇ 0.05, Figure 2).
  • C) in Figure 2 consistent with obesity or overweight and enlarged ovaries in most PCOS clinical patients.
  • the weight of the mice in the prescription group of the present invention decreased (P ⁇ 0.05), and there was no statistical difference compared with the normal control group; compared with the model group, the weight of the uterus of the mice in the basic prescription group and the prescription group of the present invention returned to normal.
  • the prescription group of the present invention has the best effect (P ⁇ 0.05, C in Figure 2), and there is no statistically significant difference with the normal control group; it shows that the prescription group of the present invention has a better effect.
  • the number of mature follicles can also be increased to a level close to or better than normal, with significant statistical differences (B in Figure 3, P ⁇ 0.001); compared with the model group, the traditional Chinese medicine composition of the present invention can The number of corpus luteum increased to the normal level, and the effect was significantly better than that of the basic formula group (C in Figure 3, P ⁇ 0.001).
  • Testosterone Testosterone(T); Luteinizing Hormone: Luteinizing hormone(LH); Follicle-stimulating Hormone: Follicle-stimulating hormone(FSH); Estradiol: Estradiol(E2)
  • mice 6-8 week old C57BL/6 mice (sex ratio of male and female mice: 2:1, Shanghai Slack Animal Center, Shanghai) were selected and raised in SPF animal laboratory.
  • the female mice were randomly divided into the normal control group and the mifepristone model group, and then the female and male mice were co-caged at a ratio of 2:1 for breeding (the appearance of vaginal suppository was recorded as the first day of pregnancy),
  • the model group was given a single subcutaneous injection of 0.1ml mifepristone (0.08mg mifepristone dissolved in 0.1ml propylene glycol) on the 4th day of pregnancy, and the normal control group was given 0.1ml propylene glycol, and the experimental animals were sacrificed on the 12th day.
  • Uterine and ovarian tissues were collected to observe the number of embryo implantation in each group to confirm that the mifepristone-induced embryo implantation disorder mouse model was successfully constructed.
  • the female mice after modeling were randomly divided into a model control group, a basic formula treatment group, and the inventive formula treatment group, and a normal control group was set.
  • the corresponding experimental groups were treated with normal saline (normal control group and model control group), the basic formula and the formula of the present invention respectively for 12 days and the clinical symptoms of the experimental animals were observed once a day.
  • the experimental mice were sacrificed, and the uterus and ovary of each group of experimental animals were collected to observe the embryo implantation, and conduct morphological and histological analysis.
  • the purchase, quarantine and feeding of experimental animals are as above.
  • the female mice were randomly divided into a normal control group and a model group.
  • the model group was treated with 0.5ml of alcohol (95% ethanol) through the uterine horn of the mouse uterus for 5 minutes during the estrus period of the female mouse's physiological cycle.
  • the uterine cavity was washed with normal saline (N.S.) and the residual ethanol was sucked out;
  • the normal control group was treated with the same volume of PBS for uterine cavity perfusion and N.S. washing; 6 hours after modeling, the animals were sacrificed and dissected to observe the endometrial injury of each group.
  • mice with successful modeling were divided into a modeling control group, a basic formula group, and a formula group of the present invention, and a normal control group (10 experimental mice in each group) was set up. mice) for 12 consecutive days. During the period, the clinical symptoms of experimental animals were observed daily. After the experiment, the mice were sacrificed, and the uteruses of the mice in each group were collected for histomorphological observation.
  • histopathological sections includes steps such as dehydration, wax immersion, embedding, sectioning, staining, and mounting, and finally placed under a microscope for observation.
  • steps such as dehydration, wax immersion, embedding, sectioning, staining, and mounting, and finally placed under a microscope for observation.
  • the specific operation steps are as follows:
  • Wax immersion and embedding Immerse the transparently treated tissue in paraffin wax at 55-60°C for full wax immersion, then place the tissue in the mold and then embed the tissue in paraffin on the cooling plate of the embedding machine;
  • H&E staining First, the sections were stained with 0.5% hematoxylin for 3 minutes, rinsed with tap water, 1% ammonia inverse blue, rinsed with tap water, differentiated with 1% hydrochloric acid ethanol, rinsed with tap water for more than 30 minutes, and stained the cell nuclei. Next, after counterstaining with 0.5% eosin for 10 minutes to stain the cytoplasm red, the sections were dehydrated with 95% ethanol twice (1 minute/time) and 100% ethanol twice (5 minutes/time), and then dehydrated. Immerse in xylene for 2 times (10 minutes/time) for transparent treatment. Finally, the sections were mounted with neutral gum, and the pathological changes of the tissue were observed and photographed under a microscope connected to a camera system.
  • Color development use DAB chromogenic reagent to develop color, and observe the section under a microscope to determine the staining time, rinse with tap water to terminate the color development; Rinse with tap water to stop color development and return to blue;
  • H&E staining is a relatively common histopathological technique and one of the gold standards for clinical medical diagnosis. It can be used for the diagnosis of various diseases and the monitoring of organ lesions.
  • the main organs such as ovary, uterus, heart, liver, spleen, lung and kidney of each group of mice were collected at the end of the experiment, and stored in 4. % PFA solution for H&E staining. The results are shown in Figure 5.
  • ethanol modeling can significantly reduce the expression of Vimentin in the uterine tissue cells of the model mice, while the expression of Vimentin in the traditional Chinese medicine composition group provided in Example 3 of the present invention has recovered, indicating that the present invention is implemented.
  • the traditional Chinese medicine composition provided in Example 3 has the effect of promoting animal endometrial cell proliferation and angiogenesis in vivo.
  • Table 2 Chinese medicine composition provided by the present invention improves serum sex hormone levels in PCOS clinical patients
  • Luteinizing hormone Luteinizing hormone (LH); follicle-generating hormone: Follicle-stimulating hormone (FSH).
  • the clinical detection result to the above-mentioned female patient of childbearing age suffering from PCOS infertility finds that using 1-4 courses of treatment of the Chinese medicine composition provided by the application (take one dose every day before the monthly menstruation or after the menstruation is clean, decoction, After 10 consecutive days, counted as a course of treatment), ovarian function can basically return to normal level, follicles grow from 3-8 millimeters (mm) in diameter to 16-19 mm, follicular tension is significantly enhanced and mature follicles are gradually formed, which is relatively Greatly enhanced follicle development and improved pregnancy success rate, the results are shown in Figure 7 (before treatment) and Figure 8 (after treatment).

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Abstract

本发明属于中药技术领域，具体公开一种用于治疗不孕症的中药组合物及其制备方法、应用。该中药组合物按重量份数计由以下原料组成：熟地黄25-30份、当归12-15份、炒白术25-35份、炒山药15-20份、炒白芍10-12份、生酸枣仁9-12份、丹皮6-10份、北沙参9-12份、炒杜仲8-10份、党参20-25份、柴胡6-10份、炒香附子8-12份和苏梗6-9份。本发明提供的用于治疗不孕症的中药组合物，能够增加对子宫内膜厚度增长、增强卵泡张力、恢复卵泡正常发育的疗效，最终有效助孕。

Description

一种用于治疗不孕症的中药组合物及其制备方法、应用
本申请要求于2021年04月29日提交中国专利局、申请号为CN202110488991.2、发明名称为“一种用于治疗不孕症的中药组合物及其制备方法、应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于中药技术领域，具体公开一种用于治疗不孕症的中药组合物及其制备方法、应用。
背景技术
不孕症是一种生育障碍状态，目前中国不孕症的发病率为7％-10％，且呈逐年上升趋势。不孕症可由多种病因导致，如环境药物的影响、排卵问题及身体器质性问题等均可导致不孕的发生。
多囊卵巢综合征(polycystic ovary syndrome，PCOS)是指一种以高雄激素血症及持续性无排卵为主要特征的多起因临床表现多态性的复杂的生殖内分泌综合征。PCOS在生育年龄妇女中的发病率约为6％-10％，基于其紊乱的内分泌状态及持续的无排卵，导致了相当一部分患者发生了生殖障碍，此类患者不孕症发病率很高，据统计由此导致的不孕症约占到女性无排卵性不孕症的75％之多。而且PCOS患者的自然流产率约达20％-41％。PCOS除了可导致女性不孕、月经稀发、闭经、多毛外，还对子宫内膜、糖脂代谢及心血管系统等有着较为深远的影响，子宫内膜癌、乳腺癌、肥胖、冠心病、动脉粥样硬化、糖尿病等发病风险増加，有些患者还会因为月经稀发、闭经、不孕、痤疮、肥胖、多毛等诸多问题引发心理障碍。所以关注PCOS的发病及治疗进展，提高多囊卵巢综合征排卵功能障碍性不孕症患者的妊娠率，减少其流产率，降低其治疗费用等具有非常重要的意义。
PCOS的病理生理及发病机制非常的复杂，至今仍无定论，可能与肾上腺功能初现时功能过盛、膜岛素样生长因子异常、神经内分泌功能失调及遗传等诸多因素相关。目前比较公认的是以下三点假说：(1)下丘脑－垂体－卵巢轴的异常，影响了促性腺激素及促黄体生成素的正常分泌，及 卵巢分泌过量的雄激素；(2)膜岛素抵抗是PCOS患者的重要代谢异常；(3)卵巢及肾上腺激素的缺乏，增加了类固醇向雄激素的转化。另外也有一些假说，比如肾上腺萌动假说、“双重缺陷”假说等，但迄今都尚未能够形成较为完整而成熟的理论。
目前国内外公认的多囊卵巢的治疗方法主要有以下几种：大剂量雌激素治疗、粒细胞集落刺激因子(g-CSF)宫腔内灌注、口服阿司匹林、维生素E、西地那非及宫腔镜等，但目前都尚未获得循证医学证据的明确支持。中药用时虽长，一般需要持续用药三个月经周期(即连续三个月时间)，但其相对西药在有效性方面具有明显优势。
围绝经期综合征又称更年期综合征(MPS)，指妇女绝经前后出现性激素波动或减少所致的一系列以自主神经系统功能紊乱为主，伴有神经心理症状的一组症候群。其中，自然绝经指卵巢内卵泡用尽，或剩余的卵泡对促性腺激素丧失了反应，卵泡不再发育和分泌雌激素，不能刺激子宫内膜生长，导致绝经，其病因复杂，有免疫性因素、遗传性因素、感染性因素、内分泌性因素、解剖因素等。
围绝经期功能失调性也是临床常见的妇科疾病类型，有研究报道结果显示，应用益经汤对围绝经期功能失调性患者进行治疗后的总体有效率为90％，患者的子宫内膜厚度变化、血清中相关激素指标的变化等均显著较之前的降低，但是其针对多囊卵巢综合症型不孕症、卵泡张力不足及发育不良迟缓症的治疗鲜有相关报道。
发明内容
为了解决上述问题，本发明提供一种用于治疗不孕症的中药组合物，其具有促进子宫内膜厚度增长、增强卵泡张力、恢复卵泡正常发育的疗效，最终有效助孕。
本发明提供的用于治疗不孕症的中药组合物，按重量份数计由以下原料组成：熟地黄25-30份、当归12-15份、炒白术25-35份、炒山药15-20份、炒白芍10-12份、生酸枣仁9-12份、丹皮6-10份、北沙参9-12份、炒杜仲8-10份、党参20-25份、柴胡6-10份、炒香附子8-12份和苏梗6-9份。
优选地，按重量份数计由以下原料组成：熟地黄30份、当归15份、 炒白术30份、炒山药15份、炒白芍10份、生酸枣仁10份、丹皮10份、北沙参10份、炒杜仲10份、党参20份、柴胡10份、炒香附子10份和苏梗6份。
本发明还提供了上述用于治疗不孕症的中药组合物的制备方法，包括以下步骤：按重量份数称取各中药原料后混合，加水煎煮，收集煎煮液，浓缩，即得所述中药组合物。
优选地，所述加水煎煮的具体过程为：将各中药原料混合后，第一次加入相当于药材总重量2-4倍量的水浸泡20-30min后，煎煮20-30min，过滤，收集滤液；滤渣再加入相当于药材重量3-4倍量的水，继续煎煮20-30min，过滤，合并两次滤液即可。
优选地，所述浓缩是将所述煎煮液浓缩至含生药量为2g/ml。
上述用于治疗不孕症的中药组合物能够用于制备治疗肝郁脾虚型多囊卵巢不孕症、卵泡张力不足及发育不良迟缓症的药物。
本发明还提供一种治疗不孕症的中药制剂，以上述中药组合物为活性成分，辅以药学上可接受的辅料，如药学上常用的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
优选地，上述中药制剂为口服制剂，如颗粒剂、丸剂、胶囊剂、片剂、口服液等，或经腹腔、静脉、肌肉注射的注射剂，该中药制剂能够被用于治疗肝郁脾虚型多囊卵巢不孕症、卵泡张力不足及发育不良迟缓症。
中医认为肾贮藏着生殖之精，人的形成、生长发育、生殖主要靠肾精的化生来实现，肾气盛衰与否决定肾精是否充盛，生殖能力以肾气的强弱盛衰为主导，与肾气之有余不足有直接关系，女子的生理特点中月经、妊娠最重要，均由胞宫所主，肾主藏精系胞宫，肾气的盛衰同时决定着胞宫功能是否正常，肾气虚不能正常濡养胞宫，胞宫功能失常影响内膜的生长，从而导致胚胎种植失败而致不孕的发生。
本发明提供的用于治疗不孕症的中药组合物，熟地黄甘温味厚、质润多液，养血滋阴、补精益髓，为君药；当归、白芍，柔肝滋血；党参、白术、山药健脾而养化源为臣；沙参为佐，益肺气、养肺胃之阴，滋水之上源，并制约诸药温燥之性；杜仲补益肝肾；酸枣仁养心阴、宁心安神，牡丹皮凉血散瘀；柴胡疏肝理气；香附子疏肝解郁调经；性平气香，能和血 中之气，通行十二经、八脉气分；苏梗理气宽中、通畅气机，此五药为使，不仅分入心、肝、脾、肾四经，且兼顾他经，即解心肝脾之气郁，又散血分之郁。全方旨在补肾疏肝、健脾、解郁。诸药合用，具有温补肾阳之效，且补而不腻、温而不燥，补阳药与补阴药熟地黄并用，阳得阴助而生化无穷。
对比现有技术，本发明的有益效果为：
1、传统“益经汤”主要用于治疗妇女心、肝、脾经气郁，年未七七，经水先断者等妇女绝经期综合征的患者，年未至七七而经水先断，缘于肾气先衰，精血不足，加之气郁血滞；而本发明提供的中药组合物治疗多囊卵巢综合症型不孕症主要针对肝郁脾虚型患者无法成功受孕，是通过补肾、疏肝、健脾方式进行调理，且根据患者月经周期关键时间节点进行周期性合理用药，达到让患者多囊卵巢综合征的症状消失、子宫内膜增厚、卵泡发育成熟后成功受孕，直至成功生产；
2、与传统益经汤比较，临床应用和基础实验结果显示，本发明提供的中药组合物能够增加其对子宫内膜厚度增长、增强卵泡张力、恢复卵泡正常发育的疗效，并最终有效助孕。
附图说明
图1是MTT法检测本发明实施例提供的药物对HUVEC和MOGC细胞的促增殖作用图；
图2是本发明实施例提供的药物改善脱氢表雄酮诱导的模型小鼠体重、子宫和卵巢指数的影响；与正常对照组比较，*P＜0.05；与模型组比较，#P＜0.05；A、小鼠体重；B、小鼠子宫重量；C、小鼠卵巢重量；
图3是本发明实施例提供的药物改善脱氢表雄酮诱导的模型小鼠生长卵泡数、成熟卵泡数和黄体数的影响；与正常对照组比较，**P＜0.01，***P＜0.001；与模型组比较，#P＜0.05；A、生长卵泡数；B、成熟卵泡；C、黄体数；
图4是本发明实施例提供的药物改善米非司酮所致模型小鼠着床障碍图；(a)正常对照组；(b)米非司酮造模组；(c)米非司酮造模后使用基础方药物治疗组；(d)米非司酮造模后使用本发明方药物治疗组；
图5是本发明实施例提供的中药组合物可修复酒精诱导的模型小鼠损伤的子宫内膜病变情况图；(a)正常对照组；(b)乙醇造模组；(c)乙 醇造模后基础方药物治疗组；(d)本发明方药物治疗组；苏木素和伊红(H&E)染色(100×)；
图6是本发明方实施例提供的中药组合物可修复模型小鼠损伤的子宫内膜增值与血管新生标志物Vimentin蛋白免疫组化染色图；(a)正常对照组；(b)乙醇造模组；(c)乙醇造模后基础方药物治疗组；(d)本发明方药物治疗组；免疫组化染色(IHC)Vimentin蛋白(100×)；
图7是典型临床患者治疗前的B超检测结果；a、b代表不同病例的B超检测结果，同一行示图表示同一病例的B超检测结果；
图8是典型临床患者治疗1-4个疗程后的B超检测结果；a、b代表不同病例的B超检测结果，同一行示图表示同一病例的B超检测结果。
具体实施方式
下面通过实施例进一步描述本发明，但是本发明不受这些实施例的限制。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
本发明提供一种用于治疗不孕症的中药组合物，按重量份数计由以下原料组成：熟地黄25-30份、当归12-15份、炒白术25-35份、炒山药15-20份、炒白芍10-12份、生酸枣仁9-12份、丹皮6-10份、北沙参9-12份、炒杜仲8-10份、党参20-25份、柴胡6-10份、炒香附子8-12份和苏梗6-9份。
本发明还提供了上述用于治疗不孕症的中药组合物的制备方法，包括以下步骤：
按重量份数称取各中药原料后混合，第一次加入相当于药材总重量8-10倍量的水浸泡20-30min后，煎煮20-30min，过滤，收集滤液；滤渣再加入相当于药材重量6-8倍量的水，继续煎煮20-30min，过滤，合并两次滤液，所得药液浓缩至含生药量为2g/ml，即得所述中药组合物。
上述中药组合物能够加入常用的辅料，如稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等，并按照药学上常规的方法制备成颗粒剂、胶囊剂、片剂、口服液或注射剂。
下面列举几个实施例对本发明提供的治疗不孕症的药物进行说明。
实施例1
一种用于治疗不孕症的中药组合物，由以下原料组成：熟地黄25g、当归12g、炒白术25g、炒山药15g、炒白芍10g、生酸枣仁9g、丹皮6g、北沙参9g、炒杜仲8g、党参20g、柴胡6g、炒香附子8g和苏梗6g。
实施例2
一种用于治疗不孕症的中药组合物，由以下原料组成：熟地黄30g、当归15g、炒白术35g、炒山药20g、炒白芍12g、生酸枣仁12g、丹皮10g、北沙参12g、炒杜仲10g、党参25g、柴胡10g、炒香附子12g和苏梗9g。
实施例3
一种用于治疗不孕症的中药组合物，由以下原料组成：熟地黄30g、当归15g、炒白术30g、炒山药15g、炒白芍10g、生酸枣仁10g、丹皮10g、北沙参10g、炒杜仲10g、党参20g、柴胡10g、炒香附子10g和苏梗6g。
由于实施例1-3制备得到的药物的治疗效果基本相同，故以下仅以实施例3制备的药物为例对本发明提供的治疗不孕症的药物的治疗效果进行说明。
一、对人源细胞和鼠源原代细胞的增殖的影响
1.1小鼠原代卵巢颗粒细胞(MOGC)的分离与培养
6-8周龄雌性C57BL/6小鼠(安徽省实验动物中心，安徽)饲养于SPF级动物实验室经检疫后，皮下注射给予PMSG(马绒毛膜促性腺激素)40IU/只，48小时后CO 2吸入麻醉法处死小鼠后，无菌条件下解剖收集小鼠卵巢于预冷无菌PBS中(含3％100U/ml青霉素，100μg/ml链霉素)清洗后，在解剖显微镜下刺破卵泡后释放颗粒细胞于预冷DMEM培养基并制成单细胞悬液后，应用1ml的0.25％胰蛋白酶(含0.02％EDTA)于37℃、5％CO 2培养箱中消化40分钟-1小时(以消化为黏液状、细胞团块分离为准)，加含15％FBS的培养基终止消化，200目细胞筛过滤，500×g离心5分钟，弃上清收集细胞接种于60mm培养皿中于37℃、5％CO 2培养箱中过夜，换液弃去未贴壁细胞后，进行MOGC的纯度鉴定用于后续实验。
1.2 MTT法检测细胞增殖的活性
选取人脐静脉血管内皮细胞HUVEC(ATCC，美国)和原代小鼠卵巢颗粒细胞(MOGC)培养于含10％FBS，青霉素100U/ml，链霉素100U/ml的DMEM培养基中，按照5×10 3个细胞(3×10 3HUVEC细胞)/孔接种于96孔板培养过夜后，应用本发明实施例3制备得到的中药组合物处理细胞(设5个浓度梯度/5个复孔，并设溶剂对照组)处理48h后，弃去孔板培养基后，加入5mg/ml MTT溶液50μl/孔后，置于37℃培养箱内避光孵育2h-4h后，加入150μl/孔DMSO溶液，置于摇床摇至结晶物融解，应用全波长酶标仪(Thermo Lab systems，USA)测定各孔在570nm波长的光吸收值(OD 570)，计算抑制率和计算IC 50值。
1.3结果
1.3.1 MTT检测法在体外诱导人血管内皮细胞、原代小鼠卵巢癌颗粒细胞的增殖
以不同浓度该中药组合物处理原代小鼠卵巢颗粒细胞MOGC(A)和人源血管内皮细胞HUVEC(B)细胞48h后，应用MTT法检测细胞的增殖情况。结果如图1所示。
MTT实验结果显示，应用本发明实施例3提供的中药组合物(图中以本发明方组表示)处理原代小鼠卵巢颗粒细胞MOGC 48h后，呈浓度依赖式地诱导了MOGC细胞的增殖活力。此外，结果显示，该中药组合物可以较好诱导人脐静脉血管内皮细胞HUVEC的增殖活力，例如，与对照组比较，2.5mg/ml处理HUVEC细胞48h后，其对细胞增殖的诱导率达到49.93％，证明本发明所述中药组合物具有促进血管新生的作用。
二、小鼠体内药效学分析实验
1.在体内改善脱氢表雄酮所致小鼠多囊卵巢综合征
1.1 PCOS(多囊卵巢综合征)小鼠模型的造模及给药方法
将饲养于SPF级屏障系统动物室并经检疫后的25日龄C57BL/6J小鼠(上海斯莱克实验动物责任有限公司)随机分为正常对照组、PCOS模型组；模型组小鼠经颈背部皮下注射0.2ml脱氢表雄酮(DHEA，上海麦克林生化科技有限公司)70mg/kg油剂(溶剂为甘油，国药集团化学试剂有限公司)，正常对照组给予0.2ml甘油，每日一次，连续注射20天。于每日晨将浸有生理盐水的小棉签插入小鼠阴道口采集阴道脱落细胞于 载玻片上晾干后，应用95％乙醇处理15分钟、5％美蓝染色10分钟，冲洗并晾干后，于显微镜下进行细胞学检查确定小鼠的动情周期。此外，于末次给药的次日，称量小鼠体重，10％水合氯醛按照3ml/kg经腹腔注射(i.p.)麻醉小鼠，经腹主动脉穿刺采血后ELISA法测定血清促黄体生存素、睾酮水平，解剖收集并称重小鼠卵巢和子宫等脏器组织后于10％福尔马林溶液中固定保存。
小鼠阴道涂片始终显示处于动情间期，可见大量白细胞，另有少量有核上皮细胞及阴道粘液，无完整动情周期出现，血清促黄体生存素、睾酮升高者为PCOS模型造模成功标志。
随后，将造模成功的PCOS模型小鼠随机分为模型组、基础方组、实施例3中药组合物组各10只、正常对照组10只；各组小鼠均常规同条件喂养。根据“人和动物间体表面积折算的等效剂量比率表”得出给药剂量，治疗组使用本发明实施例3制备得到的中药组合物给药；基础方组使用传统益经汤给药(基础方具体组成为：熟地黄30g、炒白术30g、炒山药15g、当归15g、炒白芍10g、生酸枣仁10g、牡丹皮10g、北沙参10g、柴胡10g、炒杜仲10g和人参20g)。基础方组、治疗组灌胃25.0g生药/kg，正常对照组、模型组给予等量生理盐水灌胃；各组均连续灌服12天，观察动物临床表现。
按照上述实验方法称重，采集各组小鼠阴道脱落细胞进行细胞学检查、解剖采集动脉血进行血清学激素测定(睾酮、促黄体生成素、卵泡生成素、雌二醇)，收集称重小鼠卵巢、子宫等脏器组织，计算各级卵泡总成熟卵泡数、黄体数。
1.2结果
1.2.1本发明所述药物组合物对小鼠体重、卵巢指数、子宫指数变化
结果如图2所示。
由图2可知，与正常对照组比较，模型组小鼠体重增加(P<0.05，图2中A)、子宫重量降低(P<0.01，图2中B)、卵巢重量增加(P<0.05，图2中C)，与大多数PCOS临床患者肥胖或体重超标，卵巢增大相符。与模型组比较，本发明方组小鼠体重减轻(P＜0.05)，且与正常对照组比较无统计学差异；与模型组比较，基础方组、本发明方组小鼠子宫重量均 恢复正常水平，且本发明方组较基础方组的恢复效果更好(P＜0.05，图2中C)；与模型组比较，基础方组、本发明方组小鼠卵巢指数均较模型组降低，其中本发明方组效果最佳(P＜0.05，图2中C)，与正常对照组无统计学显著性差别；说明本发明方组用药有较好的效果。
1.2.2本发明所述中药组合物对小鼠卵巢卵泡数及黄体数变化
结果如图3所示。
由图3可知，与正常对照组比较，模型组小鼠的生长卵泡数显著地增加(图3中A，P<0.01)，成熟卵泡数及黄体数均显著减少(图3中B-C，P<0.001)，提示PCOS造模成功；与模型组、基础方组比较，本发明所述中药组合物能使生长卵泡数恢复至正常水平，具有较显著的统计学差异(图3中A，P<0.01)，也能使成熟卵泡数增高至接近及优于正常水平，均有较显著地统计学差异(图3中B，P<0.001)；与模型组比较，本发明所述中药组合物能使黄体数增高至正常水平，且效果明显优于基础方组(图3中C，P<0.001)。
1.3.3血清性激素水平影响的测定结果
结果如表1所示。
表1药物改善脱氢表雄酮诱导的模型小鼠血清性激素水平
Figure PCTCN2022076993-appb-000001
注：睾酮：Testosterone(T)；促黄体生成素：Luteinizing hormone(LH)；卵泡生成素：Follicle-stimulating hormone(FSH)；雌二醇：Estradiol(E2)
由表1可知，与正常对照组比较，PCOS模型组小鼠血清睾酮(T)、促黄体生成素(LH)和卵泡生成素(FSH)水平均显著升高，雌二醇(E2)水平显著降低，且有显著的统计学意义(P＜0.05)；与模型组比较，本发 明方组小鼠血清T、LH、FSH水平显著降低，E2水平显著升高，均有统计学意义(P＜0.01)；而且本发明方组的改善作用显著优于基础方组，均有统计学意义(P＜0.05)。
2在体内改善米非司酮所致小鼠着床障碍
2.1造模及给药方法
选用6-8周龄C57BL/6小鼠(雌雄小鼠的性别比为2:1，上海斯莱克动物中心，上海)饲养于SPF级动物实验室。经检疫后，将雌性小鼠随机分为正常对照组和米非司酮模型组，随后按2:1将雌性和雄性小鼠合笼进行繁育后(出现阴道栓记为妊娠第1天)，模型组在小鼠妊娠第4天单次给予皮下注射0.1ml米非司酮(0.08mg米非司酮溶于0.1ml丙二醇)，正常对照组给予0.1ml丙二醇，于第12天处死实验动物后收集子宫和卵巢组织用于观察各组胚胎着床数，确定米非司酮诱导的胚胎着床障碍小鼠模型构建成功。
随后，按上述方法造模(同时设正常对照)后，随机将造模后的雌性小鼠随机分成模型对照组、基础方治疗组、本发明方治疗组，并设正常对照组。分别应用生理盐水(正常对照组和模型对照组)、基础方、本发明方连续治疗处理对应的实验组12天并每天一次观察实验动物临床症状。实验结束后处死实验小鼠收集各组实验动物子宫和卵巢后观察胚胎着床情况，及进行形态学与组织学分析。
2.2实验结果
结果如图4所示。
由图4可知，米非司酮诱导的胚胎着床障碍模型小鼠经本发明提供的药物治疗或生理盐水(正常对照组)处理12天后，未发现本发明方组实验小鼠明显异常；实验结束后对收集各组实验动物的子宫大小、内膜厚度、卵巢形态变化观察结果显示，本发明方提供的药物治疗后可较显著地改善米非司酮诱导的小鼠胚胎着床障碍。
3.改善乙醇诱导的子宫内膜损伤
3.1动物造模及治疗方法
实验动物购买、检疫与饲养等如上述。将雌性小鼠随机分为正常对照组、模型组，模型组于雌性小鼠生理周期的动情期用0.5ml酒精(质量分 数95％乙醇)经小鼠子宫角进行宫腔灌注处理5分钟后，应用生理盐水(N.S.)冲洗宫腔后吸出残余乙醇；正常对照组应用同体积的PBS进行宫腔灌注及N.S.冲洗处理后；于造模6小时后处死并解剖观察各组实验动物子宫内膜损伤情况，确定乙醇诱导的子宫内膜损伤模型被成功构建后，将造模成功的小鼠分为造模对照组、基础方组、本发明方组，并设置正常对照组(每组10只实验小鼠)，连续治疗处理12天。期间每日观察实验动物的临床症状。实验结束后处死小鼠，收集各组小鼠的子宫用于用于组织形态学观察。
3.2 H&E染色
组织病理切片的制备包括脱水、浸蜡、包埋、切片、染色、封片等步骤，最后置于显微镜下观察。具体操作步骤如下：
(1)脱水：取出固定好的小鼠脏器组织，自来水流水冲洗，然后应用浓度由低到高的梯度浓度乙醇(50％处理2小时、70％处理2小时、80％处理2小时、95％(2次)各处理1小时、100％(2次)各处理1小时的顺序)对组织进行脱水处理，逐层将组织中水分以乙醇置换出来；
(2)透明：将脱水后的组织分两次浸入二甲苯中进行透明处理，每次1小时；
(3)浸蜡与包埋：将透明处理后的组织浸入55-60℃的石蜡中处理进行充分浸蜡后，将组织立于模具内后于包埋机制冷板上进行组织石蜡包埋；
(4)切片：将蜡块组织表面修理平整后，将切片机的切片厚度调整为4-5μm进行切片后，将组织切片平摊在55-60℃水浴摊片机中展平，应用载玻片缓缓向上捞出组织切片，将载玻片置烘片机60℃烘烤2小时以上；
(5)脱蜡：二甲苯Ⅰ和Ⅱ各处理20分钟后，梯度乙醇(100％、95％、80％、70％、50％)，每次5分钟，最后于蒸馏水中浸泡5分钟；
(6)H&E染色：首先，应用0.5％苏木素染色切片3分钟，自来水冲洗，1％氨水反蓝，自来水冲洗，1％盐酸乙醇分化，自来水冲洗30分钟以上，对细胞核进行染色。其次，用0.5％伊红复染10分钟使胞质染成红色后，将切片经95％乙醇2次(1分钟/次)，100％乙醇2次(5分钟/ 次)进行脱水处理，再浸入二甲苯中2次(10分钟/次)进行透明处理。最后，用中性树胶封片,在连接照相系统的显微镜下观察组织的病理变化并拍照。
3.3免疫组化(IHC)染色
(1)动物组织的固定、透明、石蜡包埋、切片、脱蜡等方法见上述3.2；
(2)封闭：质量分数3％H 2O 2溶液封闭内源性过氧化物酶(湿盒避光孵育10分钟)；
(3)抗原修复：室温下去离子水漂洗切片3次后，将切片置于含枸橼酸钠缓冲液的抗原修复盒中于高压锅中煮沸(上汽3分钟)进行抗原修复处理；取出抗原修复盒，待切片自然冷却后，将切片置于PBS中洗3次，再将切片置于0.1％TritonX-100中，摇床慢摇震荡15分钟，PBS中再冲洗3次，每次5分钟；
(4)抗体孵育：应用将质量分数5％脱脂牛奶溶液于37℃孵箱中封闭切片40分钟后，应用滤纸吸去残留的牛奶封闭液，稀释(1:500稀释)并滴加一抗anti-Vimentin(Elabscience，武汉)于切片后置于湿盒内避光4℃冰箱孵育过夜后，取出湿盒于室温静置待切片恢复至室温(约30-40分钟)，应用1×PBS漂洗3次(5分钟/次)；滴加含1％BSA稀释的生物素标记的二抗于37℃孵育10-30分钟后，应用PBS漂洗3次(5分钟/次)；滴加应用1×PBS稀释的辣根过氧化物酶标记的链霉卵白素后于37℃孵育10-30分钟，应用PBS漂洗3次(5分钟/次)；
(5)显色：应用DAB显色剂显色，并将切片置于显微镜下观察确定染色时间后，自来水冲洗终止显色；苏木素染色细胞核后，将切片置于显微镜下观察确定染色时间，应用自来水冲洗终止显色和返蓝；
(6)梯度乙醇洗脱(80％乙醇，5分钟；95％乙醇，5分钟；100％乙醇，5分钟)；二甲苯脱水透明，5分钟；应用中性树胶封片、晾干后，将切片置于显微镜下观察并拍照后进行分析统计。
3.4实验结果
(1)H&E染色
H&E染色是比较常见的一项组织病理学技术，是临床上医疗诊断的 金标准之一，可用于多种疾病的诊断、脏器病变监测等。为评估本发明提供的中药组合物对实验动物子宫内膜的修复作用，在实验终点收集各组小鼠的卵巢、子宫、心、肝、脾、肺和肾等主要脏器，并保存于4％PFA溶液中，进行H&E染色。结果如图5所示。
结果显示，应用乙醇对小鼠进行子宫灌注造模后，实验小鼠的子宫内膜出现了严重受损脱落，应用本发明实施例3提供的中药组合物对模型动物进行连续12天的治疗后，乙醇造模诱导的子宫内膜损伤得到了显著地改善，说明本发明提供的中药组合物具有修复模型小鼠损伤的子宫内膜的功效。
免疫组化染色
为了进一步验证本发明提供的中药组合物于体内促进子宫内膜细胞增殖、血管新生效果，在实验终点时，收集动物的子宫组织，将其固定与4％PFA溶液中固定并切片后应用细胞增殖与血管新生相关标志物Vimentin进行免疫组化染色。结果如图6所示。
结果显示，乙醇造模(AM)可显著降低模型小鼠子宫组织细胞中Vimentin的表达量，而在本发明实施例3提供的中药组合物组中的Vimentin表达量有所恢复，表明本发明实施例3提供的中药组合物在体内具有促进动物子宫内膜细胞增殖和血管新生的作用。
三、对PCOS临床患者治疗效果分析
1.1 PCOS临床患者治疗有效率统计分析
对58个患有肝郁脾虚型多囊卵巢综合症(PCOS)育龄女性(24岁-40岁)患者的临床检测结果与观察发现，于患者每个月的月经干净后连续使用本申请提供的中药组合物10天(每天一剂)，计算为一个疗程，连续治疗1-4个疗程后，有效率达到约95％以上。
1.2临床患者激素水平统计
对上述患有PCOS不孕症的育龄女性患者的临床检测结果发现，促黄体生成激素(LH)、卵泡雌激素(FSH)明显升高(如有患者的LH分别高于正常水平的下限15.41倍和上限2.94倍)，使用本申请提供的中药组合物1-4个疗程(于每月的月经前或者月经干净后每天一剂，煎服，连续服用10天，计为一个疗程)后，临床检测促黄体生成激素(LH)、卵泡 雌激素(FSH)明显被下调到正常水平范围，有利于恢复卵泡正常发育，结果如表2所示。
表2本发明提供的中药组合物改善PCOS临床患者血清性激素水平
Figure PCTCN2022076993-appb-000002
注：促黄体生成素：Luteinizing hormone(LH)；卵泡生成素：Follicle-stimulating hormone(FSH)。
1.3恢复卵巢功能、卵泡正常发育能力并增强卵泡张力的统计
对上述患有PCOS不孕症的育龄女性患者的临床检测结果发现，使用本申请提供的中药组合物1-4个疗程(于每月的月经前或者月经干净后每天服用一剂，煎服，连续10天，计为一个疗程)后，卵巢功能基本能恢复到正常水平，卵泡由原来直径3-8毫米(mm)增长至16mm-19mm，卵泡张力明显增强并逐渐形成发育成熟的卵泡，较大程度增强卵泡发育及提高受孕成功率，结果如图7(治疗前)和图8(治疗后)所示。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其它实施例，这些实施例都属于本发明保护范围。

Claims (14)

  1. 一种用于治疗不孕症的中药组合物，其特征在于，按重量份数计由以下原料组成：熟地黄25-30份、当归12-15份、炒白术25-35份、炒山药15-20份、炒白芍10-12份、生酸枣仁9-12份、丹皮6-10份、北沙参9-12份、炒杜仲8-10份、党参20-25份、柴胡6-10份、炒香附子8-12份和苏梗6-9份。
  2. 根据权利要求1所述的中药组合物，其特征在于，按重量份数计由以下原料组成：熟地黄30份、当归15份、炒白术30份、炒山药15份、炒白芍10份、生酸枣仁10份、丹皮10份、北沙参10份、炒杜仲10份、党参20份、柴胡10份、炒香附子10份和苏梗6份。
  3. 根据权利要求1或2所述的中药组合物，其特征在于，所述炒白术为蜜炙，所述炒山药、所述炒白芍及所述炒杜仲均为盐水炒制，所述炒香附子为醋炒制。
  4. 根据权利要求1～3任一项所述的中药组合物的制备方法，其特征在于，包括以下步骤：按重量份数称取各中药原料后混合，加水煎煮，收集煎煮液，浓缩，即得所述中药组合物。
  5. 根据权利要求4所述的制备方法，其特征在于，所述加水煎煮的具体过程为：将各中药原料混合后，第一次加入相当于药材总重量8-10倍量的水浸泡20-30min后，煎煮20-30min，过滤，收集滤液；滤渣再加入相当于药材重量6-8倍量的水，继续煎煮20-30min。
  6. 根据权利要求4或5所述的制备方法，其特征在于，所述浓缩是将所述煎煮液浓缩至含生药量为2g/ml。
  7. 根据权利要求1-3任一项所述的中药组合物在制备治疗肝郁脾虚型多囊卵巢不孕症、卵泡张力不足及发育不良迟缓症的药物中的应用。
  8. 一种治疗不孕症的中药制剂，其特征在于，其以权利要求1-3任一项所述的中药组合物或权利要求4～6所述制备方法得到的中药组合物为活性成分，辅以药学上可接受的辅料。
  9. 根据权利要求8所述的中药制剂，其特征在于，所述辅料选自稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体和润滑剂。
  10. 根据权利要求8所述的中药制剂，其特征在于，所述中药制剂为口服制剂或注射剂。
  11. 根据权利要求10所述的中药制剂，其特征在于，所述口服制剂包括颗粒剂、胶囊剂、片剂和口服液。
  12. 一种治疗不孕症的方法，其特征在于，患者摄入权利要求1-3任一项所述的中药组合物或权利要求8～11任一项所述的中药制剂。
  13. 权利要求1-3任一项所述的中药组合物或权利要求8～11任一项所述的中药制剂在改善米非司酮所致胚胎着床障碍中的应用。
  14. 权利要求1-3任一项所述的中药组合物或权利要求8～11任一项所述的中药制剂在改善乙醇诱导的子宫内膜损伤中的应用。
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