WO2023005130A1 - 一种治疗不孕症的中药组合物及其制备方法、应用 - Google Patents

一种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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WO2023005130A1
WO2023005130A1 PCT/CN2021/141259 CN2021141259W WO2023005130A1 WO 2023005130 A1 WO2023005130 A1 WO 2023005130A1 CN 2021141259 W CN2021141259 W CN 2021141259W WO 2023005130 A1 WO2023005130 A1 WO 2023005130A1
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chinese medicine
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traditional chinese
medicine composition
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徐有志
路文杰
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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    • 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
    • A61K36/232—Angelica
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/076—Poria
    • 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)
    • 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)
    • A61K36/28—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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/288—Taraxacum (dandelion)
    • 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)
    • A61K36/36—Caryophyllaceae (Pink family), e.g. babysbreath or soapwort
    • 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)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • A61K36/428—Trichosanthes
    • 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)
    • A61K36/58—Meliaceae (Chinaberry or Mahogany family), e.g. Azadirachta (neem)
    • 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)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • 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)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/89—Cyperaceae (Sedge family)
    • A61K36/8905—Cyperus (flatsedge)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

Definitions

  • the invention belongs to the technical field of research on traditional Chinese medicine for infertility, and in particular relates to a traditional Chinese medicine composition for treating infertility, a preparation method and application thereof.
  • Hyperprolactinemia refers to a syndrome characterized by elevated prolactin, amenorrhea, galactorrhea, anovulation, and infertility caused by internal and external environmental factors.
  • the incidence rate in women of childbearing age is about 6%-10%. Based on their chaotic endocrine state and continuous anovulation, a considerable number of patients have reproductive disorders, and the incidence of infertility in these patients is very high. According to According to statistics, the resulting infertility accounts for about 75% of women's anovulatory infertility.
  • hyperprolactinemia to the categories of "amenorrhea”, “spontaneous milk production”, and “hypomenorrhea”.
  • the main cause of hyperprolactinemia is kidney deficiency and liver stagnation. If the dredging is neglected, and the qi and blood are in disorder, the milk will come out automatically.
  • the treatment methods of hyperprolactinemia recognized at home and abroad mainly include the following: bromocriptine, levodopa (Levo-Dopa), octahydrobenzoquinoline ( CV205-502 ), vitamin B6, etc. Ovulation therapy, etc., are all expensive and have not yet been clearly supported by evidence-based medical evidence.
  • Traditional Chinese medicine takes a long time and generally needs to be used continuously for three menstrual cycles (that is, three consecutive months), but it has obvious advantages in terms of effectiveness compared with Western medicine.
  • Kaiyu Zhongyu decoction has shown a good effect in the treatment of liver depression type infertility, but its treatment for hyperprolactinemia type infertility, insufficient follicular tone and stunting and retardation has not been reported.
  • the present invention provides a traditional Chinese medicine composition for treating infertility, which can reduce the level of prolactin hormone, increase the thickness of endometrium and restore the normal development of follicles, and finally effectively assist pregnancy.
  • the invention provides a traditional Chinese medicine composition for treating infertility, which is composed of the following raw materials in parts by weight: 10-15 parts of angelica, 15-25 parts of fried Atractylodes macrocephala, 20-25 parts of fried white peony root, and 8 parts of fried aconite -12 parts, Moutan bark 10-12 parts, Poria cocos 12-15 parts, Trichosanthes 7-9 parts, Neem 4-6 parts, Passepartout 10-12 parts, Wangbuliuxing 6-12 parts, dandelion 8- 12 servings.
  • it consists of the following raw materials in parts by weight: 10 parts of angelica, 15 parts of fried Atractylodes macrocephala, 20 parts of fried white peony root, 12 parts of fried aconite, 12 parts of Moutan cortex, 15 parts of poria cocos, 7 parts of trichosanthes, neem fruit 6 servings, 10 servings of Passepartout, 12 servings of Wang Buliuxing, and 8 servings of Dandelion.
  • the stir-fried Atractylodes macrocephala is stir-fried with soil
  • the stir-fried white peony is stir-fried with rice wine
  • the stir-fried aconite is stir-fried with vinegar
  • the neem and wangbuliuxing are stir-fried.
  • the present invention also provides a preparation method of the above-mentioned traditional Chinese medicine composition for treating infertility, which includes the following steps: weighing each Chinese medicine raw material according to parts by weight, adding water for decoction after mixing, collecting the decoction liquid, and concentrating to obtain the traditional Chinese medicine combination.
  • the specific steps of adding water and decocting are: mixing the raw materials of traditional Chinese medicines, adding water equivalent to 6-8 times the total weight of the medicinal materials, soaking for 30 ⁇ 5min, decocting for 30 ⁇ 5min, filtering, and collecting the first
  • the filtrate and the filter residue are added with water equivalent to 4-6 times the total weight of the medicinal materials, continue to decoct for 30 ⁇ 5min, filter, and collect the second filtrate; combine the two filtrates to obtain the decoction.
  • the concentration is to concentrate the decoction until the crude drug content is 2 g/ml.
  • the present invention also provides the application of the above-mentioned traditional Chinese medicine composition for treating infertility in the preparation of medicine for treating hyperprolactinemia type infertility.
  • Another object of the present invention is to provide a traditional Chinese medicine preparation for treating hyperprolactinemia-type infertility, which uses the above-mentioned traditional Chinese medicine composition as an active ingredient, and is supplemented with pharmaceutically acceptable auxiliary materials and carriers.
  • the traditional Chinese medicine composition for treating infertility provided by the invention, white peony root is reused in the prescription, nourishes blood and regulates the liver, relieves stagnation; Moutan cortex, bitter and slightly cold, enters the heart and liver blood, is dedicated to cooling blood and dissipating blood stasis, and has Cooling blood without leaving blood stasis, promoting blood without leaving blood stasis, these two medicines are the king; angelica and Cyperus cyperi, nourishing blood and soothing the liver, relieving stagnation and softening the liver, are ministers; supplemented with Atractylodes macrocephala to cultivate the spleen and strengthen the spleen, restricting the liver and restraining the spleen soil, In addition, Poria cocos invigorates the spleen and calms the heart; it is best to use trichosanthin to nourish yin and promote body fluid, and then to support liver yin; newly added vaccarina, paspartout, and
  • the traditional Chinese medicine composition provided by the present invention has the functions of soothing the liver and relieving stagnation, nourishing yin and nourishing blood, clearing heat and eliminating troubles, and is used for premenstrual tension syndrome and infertility accompanied by increased serum prolactin, which belongs to liver stagnation.
  • Heat, yin and blood deficiency, symptoms include irregular menstruation or amenorrhea, or long-term infertility after marriage, chest pain or breast pain, dizziness, restlessness, insomnia, dreaminess, dry mouth and throat, soreness of the waist and knees, Red tongue with thin yellow or thin white fur and dry, stringy and thready pulse; clinical test results show that it is safe, effective, and has no toxic or side effects on the human body.
  • the traditional "Kaiyu Zhongyu Decoction” is mainly used to soothe the liver and relieve depression, regulate menstruation seeds, and treat infertility caused by stagnation of liver qi in women; the prescription of the present invention is mainly aimed at lactation
  • Liver depression patients with high levels of hormone hormones are unable to conceive successfully. They are mainly adjusted by nourishing blood and soothing the liver, softening the liver and invigorating the spleen, and periodically and rationally taking medication according to the key time points of the patient's menstrual cycle to achieve high prolactin levels in the patient.
  • the symptoms of blood signs disappear, the endometrium thickens, and the follicles develop and mature successfully for the purpose of conception until successful delivery.
  • Figure 1 is the result of inducing the proliferation of primary mouse endometrial epithelial MEEC cells in vitro;
  • Figure 2 is the result of inducing the proliferation of human umbilical vein endothelial cells in vitro
  • Fig. 3 is the impact on mouse body weight, ovary weight, uterus weight; A, body weight; B, uterus weight; C, ovary weight;
  • Fig. 4 is the H&E staining result of PRL model mouse ovary tissue; A, normal control group; B, model group; C, comparative example 1 group; D, comparative example 2 groups; E, prescription of the present invention;
  • Fig. 5 is the clinical B-ultrasound detection result of a female patient of childbearing age with hyperprolactinemia infertility; A, before treatment; B, after treatment by the prescription of the present invention.
  • the invention provides a traditional Chinese medicine composition for treating infertility, which is composed of the following raw materials in parts by weight: 10-15 parts of angelica, 15-25 parts of fried Atractylodes macrocephala, 20-25 parts of fried white peony root, and 8 parts of fried aconite -12 parts, Moutan bark 10-12 parts, Poria cocos 12-15 parts, Trichosanthes 7-9 parts, Neem 4-6 parts, Passepartout 10-12 parts, Wangbuliuxing 6-12 parts, dandelion 8- 12 servings.
  • the above-mentioned traditional Chinese medicine composition can treat hyperprolactinemia type infertility, and is used 10 days before menstruation.
  • a traditional Chinese medicine composition for treating infertility which is composed of the following raw materials: 12g angelica, 15g fried Atractylodes macrocephala, 20g fried white peony, 8g fried aconite, 10g cortex moutan, 12g poria cocos, 9g trichosanthes, 4g azalea, and Lulu Tong 10g, Wang Buliuxing 6g, Dandelion 8g.
  • a traditional Chinese medicine composition for treating infertility which is composed of the following raw materials: 15g angelica, 25g fried Atractylodes macrocephala, 25g fried white peony, 12g fried aconite, 10g cortex moutan, 15g poria cocos, 9g trichosanthes, 6g neem, and Lulu Tong 12g, Wangbuliuxing 12g, Dandelion 12g.
  • a traditional Chinese medicine composition for treating infertility which is composed of the following raw materials: 10g angelica, 15g fried Atractylodes macrocephala, 20g fried white peony, 12g fried aconite, 12g cortex moutan, 15g poria cocos, 7g trichosanthes, 6g neem, Lulu Tong 10g, Wangbuliuxing 12g, dandelion 8g.
  • a traditional Chinese medicine composition for treating infertility which is composed of the following raw materials: 12g angelica, 20g fried Atractylodes macrocephala, 22g fried white peony, 10g fried aconite, 12g cortex moutan, 15g poria cocos, 8g trichosanthes, 5g neem, Lulu Tong 12g, Wang Buliuxing 8g, Dandelion 8g.
  • a traditional Chinese medicine composition for treating infertility which is composed of the following raw materials: 15g angelica, 25g fried Atractylodes macrocephala, 20g fried white peony, 8g fried aconite, 12g cortex moutan, 15g poria cocos, 9g trichosanthes, 6g azalea, and Lulu Tong 10g, Wangbuliuxing 12g, dandelion 8g.
  • a traditional Chinese medicine composition for treating infertility which is composed of the following raw materials: 12g angelica, 18g fried Atractylodes macrocephala, 25g fried white peony, 12g fried aconite, 10g cortex moutan, 12g poria cocos, 9g trichosanthes, 4g neem, and Lulu Tong 10g, Wang Buliuxing 6g, Dandelion 10g.
  • a traditional Chinese medicine composition for treating infertility which is composed of the following raw materials: 30g of Radix Paeoniae Alba, 9g of Cyperus Cyperi, 15g of Angelica, 15g of Stir-fried Atractylodes Rhizoma Atractylodes Rhizome, 9g of Cortex Moutan, 9g of Poria cocos, and 6g of Trichosanthum Fen.
  • a traditional Chinese medicine composition for treating infertility which is composed of the following raw materials: 10g of angelica, 15g of fried Atractylodes macrocephala, 20g of fried white peony root, 12g of fried aconite, 12g of Moutan cortex, 15g of Poria cocos, 7g of trichosanthes, 12g of buliuxin, Dandelion 8g.
  • the preparation methods of the Chinese medicine compositions of the above-mentioned Examples 1-6 and Comparative Examples 1-2 are all carried out according to the following steps: after weighing each Chinese medicine raw material by weight and mixing them in a non-iron boiler, adding an amount equivalent to the total amount of medicinal materials Soak in 6-8 times the weight of water for 30 ⁇ 5 minutes, decoct for 30 ⁇ 5 minutes, filter, and collect the filtrate for the first time; add water equivalent to 4-6 times the total weight of the medicinal materials to the filter residue, and continue to decoct for 30 ⁇ 5 minutes , filter, and collect the second filtrate; combine the two filtrates and concentrate until the crude drug content is 2g/ml.
  • MEEC mouse endometrial epithelial cells
  • the mouse uterine tissues washed with PBS were collected under aseptic conditions, cut into small pieces of about 1 mm 3 , and digested with 1 mg/ml collagenase and 2 ng/ml DNase in the Digest at 37°C for 30 minutes, then stop the digestion with DMEM medium containing 10% FBS; continue to use a 40 ⁇ m sterile cell filter to collect the cell suspension in a centrifuge tube, and wash the cell filter with serum-free medium before collecting the cells Put the adenoid mass in the filter into a centrifuge tube, centrifuge at 500 ⁇ g for 5 minutes, discard the supernatant, collect the precipitate, add 1ml 0.25% trypsin (containing EDTA) and incubate on a shaker at 37°C for 10 minutes, add 9ml medium to terminate Digest, centrifuge at 500 ⁇ g for 5 minutes, discard the supernatant, collect MEEC cells into a cell culture dish, and culture them overnight in
  • HUVEC Human umbilical vein endothelial cells HUVEC (ATCC, USA) and primary mouse endometrial epithelial cells (MEEC) were selected and cultured in DMEM medium containing 10% FBS, penicillin 100U/ml, and streptomycin 100U/ml.
  • CCK-8 method was used to detect the proliferation activity of induced human umbilical vein endothelial cells and primary mouse endometrial epithelial cells in vitro
  • Example 3 was better than that of Comparative Example 1 and Comparative Example 2, and induced MEEC cells in a concentration-dependent manner. proliferative activity.
  • the traditional Chinese medicine composition provided by embodiment 3 can well induce the proliferative activity of human umbilical vein endothelial cell HUVEC, for example, after 5.0mg/ml treats HUVEC cell for 48h, its effect on cell proliferation The induction rate reaches 64.65%, which proves that the prescription of the present invention has the effect of promoting cell proliferation and angiogenesis.
  • the invention improves the mouse hyperprolactinemia syndrome caused by metoclopramide in vivo
  • mice The 10-week-old female Kunming mice (Anhui Experimental Animal Center, Anhui) were randomly divided into normal control group and PRL model group; the mice in the model group were subcutaneously injected with 0.1ml Metoclopramide (metoclopramide hydrochloride injection, Sinopharm Group), 24mg/kg, normal control group was given 0.1ml normal saline, twice a day, continuous injection for 6 days. On the next day after the last administration, the mice were weighed, and 10% chloral hydrate was anesthetized by intraperitoneal injection (i.p.) at 3ml/kg, and the mouse serum prolactin (PRL) was measured by ELISA after abdominal aortic puncture and blood collection. )level. The mouse model with elevated serum prolactin was successfully established as a PRL mouse model.
  • Metoclopramide metaloclopramide hydrochloride injection, Sinopharm Group
  • the successful PRL model mice were randomly divided into model group, comparative example 1 group, comparative example 2 group, 10 each of the embodiment of the present invention 3 groups, and 10 normal control groups were taken; each group of mice had Routine feeding under the same conditions.
  • the treatment group was administered with the traditional Chinese medicine composition prepared in Example 3 of the present invention; Group 1 of Comparative Example, Group 2 of Comparative Example and Group 3 of Example 3 of the present invention were treated with 20.0 g crude drug/kg by oral gavage, and the model group was given the same amount of physiological Saline was administered orally; each group was administered continuously for 20 days, and the clinical manifestations of the animals were observed.
  • mice dissect the mice in each group to collect arterial blood for serum prolactin (PRL) level determination, collect and weigh the uterus, ovary and other organ tissues of the mice and fix them in 10% formalin solution for preservation , to prepare specimens for histological examination.
  • PRL serum prolactin
  • pathological sections includes steps such as dehydration, wax immersion, embedding, sectioning, staining, sealing, etc., and finally placed under a microscope for observation. Specific steps are as follows:
  • Wax immersion and embedding immerse the fixed tissue in melted paraffin (55-60°C) for 40 minutes, repeat 2-3 times. Then for embedding, place the wax block mold on the cold plate of the embedding machine, first drop a small amount of melted paraffin, and before the paraffin is completely solidified, place the tissue soaked in wax in the mold until the paraffin at the bottom of the mold is completely After solidification, continue to add new liquid paraffin to submerge the entire tissue block, then reversely fix the embedding frame on the mold, continue to add paraffin into the embedding frame, and remove the mold after the entire wax block is completely solidified, that is, Embedded wax blocks;
  • H&E staining First, stain the tissue section with 0.5% hematoxylin staining solution for 3 minutes, rinse the floating color with tap water, turn blue with 1% ammonia water, then rinse with tap water, differentiate with 1% hydrochloric acid ethanol, and remove the parts that should not be stained The color was removed, and the cell nuclei were stained by washing with tap water for more than 30 minutes. Second, counterstain with 0.5% eosin solution for 10 minutes to stain the cytoplasm red, then pass through 95% ethanol twice, 1 minute each time, and 100% ethanol twice, 5 minutes each time, for thorough dehydration . Again, immerse the slices in xylene for 2 times, each time for 10 minutes for transparent treatment;
  • the weight of the uterus of the mice in the 1st group of the comparative example, the 2nd group of the comparative example, and the 3rd group of the embodiment of the present invention all returned to normal levels, and the recovery effect of the 3rd group of the embodiment of the present invention was compared with the 1st group of the comparative example, and the 2nd group of the comparative example Better (**P ⁇ 0.01, B in Fig. 3);
  • the mouse ovary weights of the comparative example 1 group, the comparative example 2 group, and the embodiment of the present invention group 3 were all higher than the model group, wherein the present invention Example 3 group had the best effect (*P ⁇ 0.05, C in Fig. 3 ), and there was no statistically significant difference from the normal control group; indicating that the administration of Example 3 group of the present invention had a better effect.
  • Prolactin Prolactin (PRL).
  • Example 3 of the present invention As can be seen from Table 1, compared with the normal control group, the PRL model group mice serum prolactin (PRL) level significantly increased, and there was significant statistical significance (P ⁇ 0.05); compared with the model group, the results of Example 3 of the present invention The level of serum PRL in the mice with the prescription was significantly lowered, and the improvement effect of the prescription of Example 3 of the present invention was significantly better than that of the group 1 and group 2, both of which were statistically significant (P ⁇ 0.05).
  • PRL serum prolactin
  • prolactin prolactin
  • Table 2 the clinical detection of female patients with hyperprolactinemia infertility found that prolactin (PRL) was significantly increased.
  • prolactin PRL was significantly down-regulated to the normal level range, and there was significant statistical significance (P ⁇ 0.05), suggesting that the prescription of the present invention is beneficial to restore the normal development of follicles and improve the success rate of conception.

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Abstract

本发明属于不孕症中药研究技术领域，具体涉及一种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：当归10-15份、炒白术15-25份、炒白芍20-25份、炒香附子8-12份、牡丹皮10-12份、茯苓12-15份、天花粉7-9份、苦楝子4-6份、路路通10-12份、王不留行6-12份、蒲公英8-12份。本发明提供的治疗不孕症的中药组合物，能够降低泌素激素水平、增长子宫内膜厚度并恢复卵巢卵泡正常发育，最终有效助孕。

Description

一种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07月26日提交中国专利局、申请号为2021108455151、发明名称为“一种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
本发明属于不孕症中药研究技术领域，具体涉及一种治疗不孕症的中药组合物及其制备方法、应用。
背景技术
高泌乳素血症是指由内外环境因素引起的，以催乳素升高、闭经、溢乳、无排卵和不孕为特征的综合征。在育龄期间，备受关注的除了月经的异常外，应该就是高泌乳素血症所致的排卵功能障碍性不孕症了。在生育年龄妇女中的发病率约为6％-10％，基于其混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。
中医将高泌乳素血症归属于“闭经”、“乳汁自出”、“月经过少”等范畴。引起高泌乳素血症的原因主要是肾虚肝郁，肾为先天之本，主生殖、藏精，肾精亏虚则冲任失调，血海空虚，无血可下而致闭经，若肝郁疏泄失职，气血逆乱，则乳汁自出。
西医研究中，高泌乳素血症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。但迄今都尚未能够形成较为完整而成熟的理论。
目前国内外公认的高泌乳素血症的治疗方法主要有以下几种：溴隐亭、左旋多巴(Levo-Dopa)、八氢苯并喹啉(CV205-502)、维生素B 6等及促排卵治疗等，但都价格昂贵，且目前都尚未获得循证医学证据的明确支持。中药用时长，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。开郁种玉汤在治疗肝郁型不孕症显示出较好效果，但是其针对高泌乳素血症型不孕症、卵泡张力不足及 发育不良迟缓症的治疗未见报道。
发明内容
针对以上技术问题，本发明提供一种治疗不孕症的中药组合物，能够降低泌乳素激素水平、增长子宫内膜厚度并恢复卵泡正常发育，最终有效助孕。
本发明提供一种治疗不孕症的中药组合物，其按重量份数计由以下原料组成：当归10-15份、炒白术15-25份、炒白芍20-25份、炒香附子8-12份、牡丹皮10-12份，茯苓12-15份、天花粉7-9份、苦楝子4-6份、路路通10-12份、王不留行6-12份、蒲公英8-12份。
优选地，其按重量份数计由以下原料组成：当归10份、炒白术15份、炒白芍20份、炒香附子12份、牡丹皮12份、茯苓15份、天花粉7份、苦楝子6份、路路通10份、王不留行12份、蒲公英8份。
优选地，所述炒白术为土炒制，所述炒白芍为黄酒炒制，所述炒香附子为醋炒制，所述苦楝子及所述王不留行均为清炒。
本发明还提供了上述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，混合后加水煎煮，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步骤是：将各中药原料混合，加入相当于药材总重量6-8倍量的水浸泡30±5min后，煎煮30±5min，过滤，收集第一次滤液；滤渣再次加入相当于药材总重量4-6倍量的水，继续煎煮30±5min，过滤，收集第二次滤液；合并两次滤液，即得所述煎煮液。
优选地，所述浓缩是将所述煎煮液浓缩至含生药量为2g/ml。
本发明还提供了上述治疗不孕症的中药组合物在制备治疗高泌乳素血症型不孕症的药物中的应用。
本发明的另一个目的是提供一种用于治疗高泌乳素血症型不孕症的中药制剂，其以上述中药组合物为活性成分，并辅以药学上可接受的辅料及载体。
本发明提供的治疗不孕症的中药组合物，方中重用白芍，养血调肝、解其郁；牡丹皮，苦辛微寒，入心、肝血分，专功凉血散瘀，且具有凉血而不留瘀，活血而不妄行的特点，此二药为君；当归、香附子，养血疏肝、解郁柔肝，为臣；佐以白术培土健脾，制约肝克脾土，又以茯苓健脾宁心； 使以天花粉滋阴生津，再助养肝阴为最妙；新增王不留行、路路通、蒲公英，三药同入肝胃经，通利血脉、通经络、散结消肿，加入少量苦楝子，此四药为使，四药联用，更增强化解肝气郁结，辅助臣药养血疏肝、解郁柔肝的功效，降低泌乳素功效倍增。全方具有疏肝解郁、滋阴养血、清热除烦的功效，用于经前期紧张综合症、不孕症伴有血清泌乳素增高，症属肝郁化热、阴血亏虚者。
对比现有技术，本发明的有益效果为：
1、本发明提供的中药组合物，全方具有疏肝解郁、滋阴养血、清热除烦、用于经前期紧张综合症、不孕症伴有血清泌乳素增高，症属肝郁化热、阴血亏虚者，症见月经不调或闭经，或婚后久不受孕，胸胁或乳房胀痛，头晕目眩，心烦易怒，失眠，多梦，口燥咽干，腰膝酸软，舌红苔薄黄或薄白而干，脉弦细而数；临床验用结果表明安全、有效、对人体无毒副作用。
2、与传统“开郁种玉汤”比较，临床应用和基础实验结果显示，本方剂可以通过降低高泌素激素水平、增长子宫内膜厚度及恢复卵泡正常发育，最终有效助孕。
3、传统“开郁种玉汤”主要用于疏肝解郁、调经种子，治妇人肝气郁结所致的不孕症；本发明组方治疗高泌乳素血症型不孕症主要针对泌乳素激素水平较高的肝郁型患者无法成功受孕，主要通过养血疏肝、柔肝健脾的方式进行调理，且根据患者月经周期关键时间节点进行周期性合理用药，达到让患者高泌乳素血征的症状消失、子宫内膜增厚、卵泡发育成熟后成功受孕直至成功生产的目的。
说明书附图
图1是在体外诱导原代小鼠子宫内膜上皮MEEC细胞的增殖结果；
图2是在体外诱导人脐静脉血管内皮细胞的增殖结果；
图3是对小鼠体重、卵巢重量、子宫重量的影响；A、体重；B、子宫重量；C、卵巢重量；
图4是PRL模型小鼠卵巢组织的H&E染色结果；A、正常对照组；B、模型组；C、对比例1组；D、对比例2组；E、本发明组方；
图5是高泌乳素血症不孕症的育龄女性患者的临床B超检测结果；A、 治疗前；B、本发明方治疗后。
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。
本发明提供一种治疗不孕症的中药组合物，其按重量份数计由以下原料组成：当归10-15份、炒白术15-25份、炒白芍20-25份、炒香附子8-12份、牡丹皮10-12份、茯苓12-15份、天花粉7-9份、苦楝子4-6份、路路通10-12份、王不留行6-12份、蒲公英8-12份。
上述中药组合物能够治疗高泌乳素血症型不孕症，于月经前期10天使用。
下面结合具体实施例对本发明提供的中药组合物进行说明：
实施例1
一种治疗不孕症的中药组合物，其由以下原料组成：当归12g、炒白术15g、炒白芍20g、炒香附子8g、牡丹皮10g、茯苓12g、天花粉9g、苦楝子4g、路路通10g、王不留行6g、蒲公英8g。
实施例2
一种治疗不孕症的中药组合物，其由以下原料组成：当归15g、炒白术25g、炒白芍25g、炒香附子12g、牡丹皮10g、茯苓15g、天花粉9g、苦楝子6g、路路通12g、王不留行12g、蒲公英12g。
实施例3
一种治疗不孕症的中药组合物，其由以下原料组成：当归10g、炒白术15g、炒白芍20g、炒香附子12g、牡丹皮12g、茯苓15g、天花粉7g、苦楝子6g、路路通10g、王不留行12g、蒲公英8g。
实施例4
一种治疗不孕症的中药组合物，其由以下原料组成：当归12g、炒白术20g、炒白芍22g、炒香附子10g、牡丹皮12g、茯苓15g、天花粉8g、苦楝子5g、路路通12g、王不留行8g、蒲公英8g。
实施例5
一种治疗不孕症的中药组合物，其由以下原料组成：当归15g、炒白术25g、炒白芍20g、炒香附子8g、牡丹皮12g、茯苓15g、天花粉9g、苦楝子6g、路路通10g、王不留行12g、蒲公英8g。
实施例6
一种治疗不孕症的中药组合物，其由以下原料组成：当归12g、炒白术18g、炒白芍25g、炒香附子12g、牡丹皮10g、茯苓12g、天花粉9g、苦楝子4g、路路通10g、王不留行6g、蒲公英10g。
对比例1
一种治疗不孕症的中药组合物，其由以下原料组成：白芍30g、香附子9g、当归15g、炒白术15g、牡丹皮9g、茯苓9g、天花粉6g。
对比例2
一种治疗不孕症的中药组合物，其由以下原料组成：当归10g、炒白术15g、炒白芍20g、炒香附子12g、牡丹皮12g、茯苓15g、天花粉7g、王不留行12g、蒲公英8g。
上述实施例1-6及对比例1-2的中药组合物的制备方法均是按照以下步骤进行：按重量份数称取各中药原料后混合于非铁质煮器后，加入相当于药材总重量6-8倍量的水浸泡30±5min后，煎煮30±5min，过滤，收集第一次滤液；滤渣再次加入相当于药材总重量4-6倍量的水，继续煎煮30±5min，过滤，收集第二次滤液；合并两次滤液，浓缩至含生药量为2g/ml。
下面对本发明提供的中药组合物的治疗效果进行说明：
一、对人源细胞和鼠源原代细胞增殖的影响
1、分离和培养小鼠原代子宫内膜上皮细胞(MEEC)
动物饲养、检疫、处死解剖后，于无菌条件下收集用PBS清洗后的小鼠子宫组织，剪成约1mm 3小碎块，用含1mg/ml胶原酶、2ng/ml DNase的消化液于37℃消化30分钟，再用含10％FBS的DMEM培养基终止消化；继续用40μm无菌细胞过滤器收集细胞悬液于离心管中，并用无血清培养基反洗细胞过滤器后，收集细胞过滤器中的腺团块于离心管中，500×g离心5分钟，弃上清，收集沉淀，加入1ml 0.25％胰酶(含EDTA)于37℃摇床孵育10分钟，加9ml培养基终止消化，500×g离心5分钟， 弃上清，收集MEEC细胞至细胞培养皿中，于37℃、5％CO 2细胞培养箱中培养过夜，开展MEEC纯度鉴定用于后续实验。
2、CCK-8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)、原代小鼠子宫内膜上皮细胞(MEEC)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，用对比例1组(基础组方)、对比例2组(基础方加味王不留行和蒲公英组方)、本发明实施例3(基础方加味王不留行、蒲公英、路路通、苦楝子组方)制备得到的中药组合物分别对细胞(设1.25、2.5、5.0、10.0、15.0、20.0mg/ml等5个浓度梯度，每个浓度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CKK-8溶液50μl/孔后，置于37℃培养箱内避光孵育2h-4h，用全波长酶标仪(Thermo Lab systems，USA)测定各孔在450nm波长的光吸收值(OD450)，计算细胞增殖率值。
3、结果
3.1、CCK-8法检测在体外诱导人脐静脉血管内皮细胞、原代小鼠子宫内膜上皮细胞的增殖活性
结果如图1-2所示。
CCK-8实验结果显示(参照图1)，应用对比例1组(即传统“开郁种玉汤”组)、对比例2组(基础方加味王不留行和蒲公英组方)、本发明实施例3提供的中药组合物处理原代小鼠子宫内膜上皮MEEC细胞48h后，本发明实施例3组效果优于对比例1组、对比例2组，呈浓度依赖式地诱导了MEEC细胞的增殖活力。
此外，结果显示(参照图2)，实施例3提供的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，5.0mg/ml处理HUVEC细胞48h后，其对细胞增殖的诱导率达到64.65％，证明本发明方具有促进细胞增殖及血管新生的作用。
二、小鼠体内药效学分析实验
1.本发明方在体内改善甲氧氯普胺所致小鼠高泌乳素血症综合征
1.1高泌乳素血症(PRL)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10周龄雌性昆明小鼠(安徽实验动物中心，安徽)随机分为正常对照组、PRL模型组；模型组小鼠经背部皮下注射0.1ml灭吐灵(即盐酸甲氧氯普胺注射液，国药集团)，24mg/kg，正常对照组给予0.1ml生理盐水，每日二次，连续注射6天。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法测定小鼠血清泌乳素(PRL)水平。小鼠血清泌乳素升高者为PRL小鼠模型造模成功。
随后，将造模成功的PRL模型小鼠随机分为模型组、对比例1组、对比例2组、本发明实施例3组各10只，并取正常对照组10只；各组小鼠均常规同条件喂养。治疗组使用本发明实施例3制备得到的中药组合物给药；对比例1组、对比例2组及本发明实施例3组经口服灌胃治疗20.0g生药/kg，模型组给予等量生理盐水口服灌胃；各组均连续给药20天，并观察动物临床表现。按照上述实验方法称重，解剖各组小鼠采集动脉血进行血清学泌乳素(PRL)水平测定，收集称重小鼠子宫、卵巢等脏器组织后于10％福尔马林溶液中固定保存，制备标本用于组织学检测。
1.2组织标本的制备
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。具体步骤如下：
(1)脱水：取出固定好的小鼠脏器组织，自来水流水下充分冲洗，然后采用梯度浓度的乙醇对固定好的组织进行脱水，即乙醇浓度由低到高，逐层将组织中水分以乙醇置换出来，将组织按照梯度乙醇的浓度为50％处理2h、70％处理2h、80％处理2h、95％(2次)处理各1h、100％(2次)处理个1h的顺序处理；
(2)透明：将脱水处理后的组织浸入二甲苯Ⅰ处理60min和二甲苯Ⅱ处理60min透明；
(3)浸蜡与包埋：将固定好的组织浸入融化的石蜡(55-60℃)中处理40分钟，重复2-3次。然后进行包埋，将蜡块模具放置到包埋机制冷板上，先少量滴加融化的石蜡，在石蜡未完全凝固前，将浸蜡充分的组织立于模具内，待模具底部的石蜡完全凝固后，继续加入新的液态石蜡，淹没整个组织块，然后将包埋框反向固定在模具上，包埋框中继续加入石蜡， 待整个蜡块完全凝固后，将模具取下，即为包埋好的蜡块；
(4)切片：将蜡块表面修理平整，直到切下的石蜡薄膜上面可见少量组织时，再将切片机的切片厚度调整为5μm，继续休整蜡块，待肉眼观察切下的石蜡薄膜所涵组织面完整，即可进行切片，切片厚度为4-5μm。将切片薄膜平摊在摊片机中(温度55-60℃)展平组织切片，应用载玻片缓缓向上捞出薄膜切片，使薄膜附着在载玻片中央位置后，将载玻片置烘片机上烘烤2h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，用0.5％苏木素染色液染色组织切片3分钟，自来水冲洗浮色后，1％氨水反蓝，再用自来水冲洗后，以1％盐酸乙醇分化，将不应着色部分的颜色脱掉，自来水冲洗30分钟以上，对细胞核进行染色。其次，用0.5％伊红溶液进行复染10分钟，使胞质染成红色后，经95％的乙醇2次，每次1分钟，100％的乙醇2次，每次5分钟，进行彻底脱水。再次，将切片浸入二甲苯中2次，每次10分钟进行透明处理；
(7)封片：用中性树胶对染色好的切片进行封片。在连接照相系统的生物显微镜下观察组织的病理变化并拍照。
1.4结果
1.4.1对小鼠体重、卵巢重量、子宫重量的影响
结果如图3所示。
由图3可知，与正常对照组比较，模型组小鼠体重无较大变化(P＞0.05，图3中A)、子宫重量降低(##P<0.01，图3中B)、卵巢重量降低(#P<0.05，图3中C)。与模型组比较，本发明实施例3组小鼠体重并无变化(P＞0.05)，且与正常对照组比较无统计学差异，说明本发明组方用药具有较好的初步安全性。
与模型组比较，对比例1组、对比例2组、本发明实施例3组小鼠子宫重量均恢复正常水平，且本发明实施例3组较对比例1组、对比例2组的恢复效果更好(**P＜0.01，图3中B)；与模型组比较，对比例1组、对比例2组、本发明实施例3组小鼠卵巢重量均较模型组升高，其中本发明实施例3组效果最佳(*P＜0.05，图3中C)，与正常对照组无统计学 显著性差别；说明本发明实施例3组用药具有较好的效果。
1.4.2对PRL模型小鼠卵巢组织形态学变化的影响
结果如图4所示。
H&E染色结果显示，与正常对照组比较，模型组小鼠的卵巢呈弥漫性囊样重度改变，可见较多大小不一的裹腔，卵泡膜细胞层较厚并可见部分脱落，粒层细胞多数呈扁平或立方形的不完整形态(图4中A-B；50×)；与模型组、对比例1组、对比例2组比较，本发明实施例3组小鼠卵巢中有黄体和多个卵泡，近成熟期卵泡有卵细胞、放射冠，颗粒细胞排列紧密，较为明显地改善了卵巢的病变损伤(图4中C-E；50×)。
1.4.3血清性激素水平影响的测定结果
结果如表1所示。
表1对小鼠血清泌乳素(PRL)性激素水平的影响结果
组别 数量(n) PRL(μg/L) SD
正常对照组 10 14.65 0.98
模型组 10 32.61 1.24
对比例1组 10 20.71 0.99
对比例2组 10 18.73 0.61
本发明实施例3组 10 13.89 0.78
注：泌乳素：Prolactin(PRL)。
由表1可知，与正常对照组比较，PRL模型组小鼠血清泌乳素(PRL)水平显著升高，且有显著的统计学意义(P＜0.05)；与模型组比较，本发明实施例3组方小鼠血清PRL水平显著降低，而且本发明实施例3组方的改善作用显著优于对比例1组、对比例2组，均有统计学意义(P＜0.05)。
三、本发明方对高泌乳素血症临床患者治疗效果分析
1、高泌乳素血症临床患者治疗有效率统计分析
对88个患有高泌乳素血症女性(24岁-40岁)患者的临床检测结果发现，于患者每个月的月经前10天开始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1-3个疗程后，有效率达到约96％以上。
2、临床患者激素水平统计
结果如表2所示。
表2对高泌乳素血症临床患者血清泌乳素(PRL)水平的影响结果
组别 数量(n) 泌乳素PRL(ng/mL) SEM
治疗前 88 34.83 2.86
治疗后 88 16.65 2.63
由表2可知，对高泌乳素血症不孕症的女性患者的临床检测发现，泌乳素(PRL)明显升高，使用本申请提供的中药组合物1-3个疗程后，临床检测结果表明，泌乳素PRL明显被下调到正常水平范围，且有显著的统计学意义(P＜0.05)，提示本发明方有利于恢复卵泡正常发育提高受孕成功率。
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
结果如图5所示。
对高泌乳素血症不孕症的育龄女性患者的临床检测结果发现，患者由于泌乳素增高导致了双侧卵巢多囊样声像改变、子宫内膜薄型发展(有的子宫内膜厚度仅约为5mm)，使用本申请提供的中药组合物1-3个疗程后，子宫形态大小恢复正常、子宫内膜增厚，卵巢功能能恢复到正常水平，较大程度增强卵泡发育并形成发育成熟的卵泡、宫内妊娠，成功受孕并顺利生产。
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 (10)

  1. 一种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：当归10-15份、炒白术15-25份、炒白芍20-25份、炒香附子8-12份、牡丹皮10-12份、茯苓12-15份、天花粉7-9份、苦楝子4-6份、路路通10-12份、王不留行6-12份和蒲公英8-12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：当归10份、炒白术15份、炒白芍20份、炒香附子12份、牡丹皮12份、茯苓15份、天花粉7份、苦楝子6份、路路通10份、王不留行12份和蒲公英8份。
  3. 根据权利要求1或2所述的中药组合物，其特征在于，所述炒白术为土炒制，所述炒白芍为黄酒炒制，所述炒香附子为醋炒制，所述苦楝子和所述王不留行均为清炒。
  4. 一种权利要求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，混合后加水煎煮，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步骤是：将各中药原料混合，加入相当于药材总重量6-8倍量的水浸泡30±5min后，煎煮30±5min，过滤，收集第一次滤液；滤渣再次加入相当于药材总重量4-6倍量的水，继续煎煮30±5min，过滤，收集第二次滤液；合并两次滤液，即得所述煎煮液。
  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. 一种权利要求1～3任一项所述的中药组合物在制备治疗高泌乳素血症型不孕症的药物中的应用。
  8. 一种用于治疗高泌乳素血症型不孕症的中药制剂，其特征在于，所述中药制剂以权利要求1～3任一项所述的中药组合物为活性成分，并辅以药学上可接受的辅料及载体。
  9. 一种用于治疗高泌乳素血症型不孕症的方法，其特征在于，包括以下步骤：于月经前期10天使用权利要求1～3任一项所述的中药组合物 或权利要求4～6任意一项所述制备方法得到的重要组合物或权利要求8所述中药制剂。
  10. 根据权利要求9所述的方法，其特征在于，所述使用包括以10天为一疗程，连续使用1-3疗程；
    每天服用一剂。
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