Traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition is mainly prepared from the following medicinal materials in parts by weight: 3 to 35 parts of semen cuscutae, 3 to 35 parts of kidney tonifying medicine, 5 to 35 parts of radix salviae miltiorrhizae, 3 to 35 parts of blood circulation promoting and blood stasis removing medicine, 5 to 60 parts of radix astragali seu hedysari, 1 to 20 parts of pericarpium citri reticulatae and 6 to 35 parts of rhizoma dioscoreae. The kidney tonifying medicine is teasel root or morinda officinalis, and the blood circulation promoting and blood stasis removing medicine is suberect spatholobus stem or pseudo-ginseng. The traditional Chinese medicine composition has the effects of tonifying the kidney and promoting blood circulation, has the two-way regulation effects of anticoagulation and hemostasis aiming at kidney deficiency and blood stasis type repeated pregnancy loss (RPL), can be used for assisting pregnancy before pregnancy to prevent diseases such as RPL combined with prethrombotic state (PTS) and gestation period uterine cavity hematocele, can be used for treating the diseases such as RPL combined with PTS and gestation period uterine cavity hematocele after pregnancy, and has a relatively great application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine composition and a preparation method and application thereof. Background Art

[0002] Recurrent pregnancy loss (RPL) refers to two or more consecutive embryo losses before 28 weeks of pregnancy, including biochemical pregnancies. RPL is a common and difficult disease in women of childbearing age, with an incidence rate of up to 1-5%. As the number of miscarriages and age increase, the probability of another miscarriage in women also increases.

[0003] Prethrombotic state (PTS) is one of the important causes of PRL, accounting for about 20% of maternal causes. PTS refers to a hypercoagulable state of blood or a high tendency of thromboembolism caused by coagulation or anticoagulation disorders or impairments. This state can lead to local microthrombosis in the placenta, which in turn causes microvascular obstruction of the placenta, insufficient blood supply and oxygen supply, abnormal angiogenesis, and impaired blood perfusion, ultimately leading to adverse pregnancy outcomes such as embryonic maldevelopment and stillbirth. Based on the pathological mechanism of PTS, antithrombotic therapy is considered to be an effective treatment for RPL combined with PTS. Among them, anticoagulation and platelet inhibition therapy with low molecular weight heparin or aspirin are the main treatment options, but there are many adverse reactions such as bleeding and liver damage.

[0004] Common adverse reactions include: (1) Bleeding: Bleeding is the most common adverse reaction to antiplatelet and anticoagulant therapy. In severe cases, it can cause disability or even endanger the patient's life. Common bleeding includes subcutaneous bruising, nosebleeds, oral mucosal bleeding, gingival bleeding, conjunctival bleeding, gastrointestinal bleeding, bladder bleeding, vaginal bleeding, etc. Therefore, a detailed medical history should be inquired before antithrombotic treatment to assess the risk of bleeding. (2) Liver damage: Liver damage is a common adverse reaction of low molecular weight heparin, with an incidence of 4% to 13%. Therefore, during the use of low molecular weight heparin, liver function should be monitored regularly. Once liver damage is found, viral hepatitis should be ruled out. (3) Allergic reaction: Since low molecular weight heparin is a multi-component mixture of animal origin, allergy is a common adverse reaction of low molecular weight heparin, and common ones include skin itching, rash, urticaria, local redness and swelling, etc. A study has statistically analyzed the incidence of pruritus after the use of low molecular weight heparin during pregnancy. The results showed that the incidence of pruritus after the use of low molecular weight heparin during pregnancy was 6.6% for heparin, 9.7% for enoxaparin, and 30.9% for nadroparin. The statistical results of the incidence of rash showed that the incidence of pruritus after the use of low molecular weight heparin during pregnancy was 4.4% for heparin, 0% for enoxaparin, and 4.6% for nadroparin. The statistical results of the incidence of local redness and swelling showed that the incidence of pruritus after the use of low molecular weight heparin during pregnancy was 2.2% for heparin, 1.8% for enoxaparin, and 13.8% for nadroparin. The statistical results of the incidence of induration showed that the incidence of pruritus after the use of low molecular weight heparin during pregnancy was 13.3% for heparin, 20.3% for enoxaparin, and 42.1% for nadroparin. Rare adverse reactions include acute laryngeal edema and anaphylactic shock. In addition, there have been reports of allergic reactions and even death in patients caused by the use of heparin internationally.

[0005] At the same time, excessive treatment and irregular use of low molecular weight heparin or aspirin are common in clinical practice, and adverse reactions and other problems caused by this treatment follow one after another. Especially when PTS is combined with intrauterine hematoma during pregnancy, the treatment is the most difficult, and there is a contradiction between anticoagulation and hemostasis treatment.

[0006] Traditional Chinese medicine has a long history of treating pregnancy, and its safety and effectiveness have been tested and proven in long-term clinical practice. Currently, there are some traditional Chinese medicine preparations on the market for the treatment of RPL, but their therapeutic effects are not stable and do not have a good effect on some patients. Summary of the invention

[0007] In view of the above problems, the present invention provides a traditional Chinese medicine composition, which has the effects of tonifying the kidney and activating blood circulation, and has the bidirectional regulating effects of anticoagulation and hemostasis for patients with "kidney deficiency and blood stasis type" RPL. The traditional Chinese medicine composition can be used before pregnancy to assist pregnancy and prevent RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases, and can also be used after pregnancy to treat RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases.

[0008] In one aspect, the present invention discloses a Chinese medicine composition, the active ingredients of which are mainly composed of the following medicinal materials in parts by weight:

[0009] 3-35 parts of Cuscuta seeds, 3-35 parts of kidney-tonifying drugs, 5-35 parts of Salvia miltiorrhiza, 3-35 parts of blood-activating and stasis-removing drugs, 5-60 parts of Astragalus, 1-20 parts of dried orange peel, and 6-35 parts of Chinese yam;

[0010] The kidney-tonifying medicine is Dipsacus asper or Morinda officinalis, and the blood-activating and stasis-removing medicine is Millettia reticulata or Panax notoginseng.

[0011] The inventors analyzed and found in the investigation that intrauterine hemorrhage is a common ultrasound manifestation in early and mid-pregnancy, and some of them can last until late pregnancy, with an incidence of 0.46% to 39.5%. Intrauterine hemorrhage may be associated with adverse pregnancy outcomes such as spontaneous abortion and placental abruption. The proportion of PTS in patients with intrauterine hemorrhage is significantly increased, suggesting that PTS may be one of the causes of intrauterine hemorrhage. The current use of anticoagulants to prevent PTS will increase the occurrence of subchorionic hematoma (a type of intrauterine hemorrhage) during pregnancy. In addition, pregnant women who use thrombolytic drugs to treat heart valve thrombosis also develop huge subchorionic hematomas during pregnancy. However, anticoagulant and antiplatelet therapy may increase the incidence of intrauterine hemorrhage during pregnancy. Therefore, there is a contradiction in the Western medicine treatment of RPL combined with PTS.

[0012] In traditional Chinese medicine, RPL belongs to the category of "slippery abortion" and "frequent miscarriage". Its main mechanism is damage to Chong and Ren meridians, unstable fetus, and repeated miscarriage. Clinically, the common syndromes of RPL are spleen and kidney deficiency (39.96%), kidney qi deficiency (25.86%), qi and blood deficiency (15.53%), kidney deficiency and blood stasis (9.82%), kidney deficiency and blood heat (2.62%), and other syndromes (6.21%). Among them, RPL combined with PTS belongs to the category of "kidney deficiency and blood stasis syndrome" in traditional Chinese medicine, which takes "kidney deficiency" as the fundamental pathogenesis and "blood stasis" as the important pathogenesis. If qi does not circulate blood, blood stasis is blocked inside, and blood does not circulate through the meridians, then blood will overflow outside the veins, and the blood that leaves the meridians is blood stasis, which then aggravates bleeding and forms a bad cycle. Treatment should be combined with disease and syndrome, pre-cultivation of damage, hemostasis first, blood stasis removal as the key, and blood stasis removal and fetal stagnancy should be carried out simultaneously. Therefore, the inventors consider that the method of invigorating the kidney and activating blood circulation may be one of the methods to break through the bottleneck of anticoagulation and hemostasis in western medicine treatment.

[0013] The inventors have found through research that the Chinese medicine compositions currently used to treat RPL on the market are mostly for patients with "spleen and kidney deficiency", "kidney qi deficiency" and "kidney deficiency and blood heat" syndromes, and there is a lack of effective drugs for the treatment of "kidney deficiency and blood stasis" syndrome RPL, and a lack of drugs with a bidirectional regulatory effect on thrombosis and bleeding. This may be one of the reasons why the current Chinese medicine preparations used to treat RPL have a poor therapeutic effect.

[0014] On the basis of this investigation, the inventor believes that the disease is based on kidney deficiency as the fundamental pathogenesis, and blood stasis is an important pathogenesis. During treatment, the disease and syndrome should be combined, the damage should be pre-cultivated, hemostasis should be the first, blood stasis should be removed, and blood stasis and miscarriage should be taken simultaneously. Based on this, the inventor takes tonifying the kidney and promoting blood circulation as the formula design idea, first considers using Cuscuta chinensis and Dipsacus asper (or Morinda officinalis) to play the role of tonifying the kidney and consolidating the Chong and the role of miscarriage consolidating the Chong, and using Salvia miltiorrhiza and Millettia spatholobi (or Panax notoginseng) to play the role of promoting blood circulation and removing blood stasis. However, it is found that the acquired qi and blood nourishment can better play the role of assisting pregnancy and miscarriage, so the qi-tonifying and spleen-strengthening drugs Astragalus and Chinese yam are added. And the inventor found that for drugs for promoting blood circulation and removing blood stasis, Chuanxiong, safflower, etc. have certain safety issues, so Salvia miltiorrhiza and Millettia spatholobi (or Panax notoginseng) are selected. Compared with similar medicinal flavors, the latter has the effect of nourishing blood and promoting blood circulation and is safer. Similarly, for the selection of the drug, licorice has also been considered, but practice has proved that dried tangerine peel can promote qi and stomach, and pregnant women use it more effectively.

[0015] Finally, after repeated exploration and attempts, the inventor proposed a kidney-tonifying and blood-activating prescription, including Cuscuta, Radix Euphorbiae or Morinda officinalis, Salvia miltiorrhiza, Millettia reticulata or Panax notoginseng, Astragalus, Tangerine peel, and Chinese yam. Among them, Cuscuta and Astragalus are monarch drugs, Cuscuta chinensis tonifies kidney yin and kidney yang, and Astragalus invigorates qi and spleen, nourishes blood and consolidates Chong, and the two drugs play a double role in qi and blood, and pay equal attention to water and soil. Radix Euphorbiae (or Morinda officinalis), Millettia reticulata (or Panax notoginseng) and Salvia miltiorrhiza are ministerial drugs, Radix Euphorbiae or Morinda officinalis tonifies liver and kidney, and strengthens tendons and bones; Millettia reticulata or Panax notoginseng invigorates blood and nourishes blood, removes blood stasis and relieves stagnation; Salvia miltiorrhiza activates blood circulation and removes blood stasis, cools blood and regulates Chong, and nourishes kidney and removes blood stasis. Chinese yam is an adjuvant, which invigorates spleen and consolidates kidney, benefits stomach and produces body fluid, and cooperates with monarch drugs to enhance the function of invigorating kidney and strengthening spleen; Tangerine peel is a messenger drug, which promotes qi and stomach, and harmonizes all drugs.

[0016] Specifically, Cuscuta australis and Dipsacus asper (or Morinda officinalis) can both replenish kidney yang, benefit kidney essence, make kidney qi strong, consolidate Chong and Ren meridians, and ensure the fetus is well supported; Salvia miltiorrhiza and Millettia reticulata (or Panax notoginseng) can dissipate blood stasis, allow blood stasis to proceed, new blood to be generated, qi and blood to be abundant, blood vessels to be unobstructed, and the fetus to be supported and nourished; Astragalus membranaceus and yam are added to the combination to replenish qi and strengthen the spleen; Tangerine peel moderates the medicinal properties and harmonizes the various medicines.

[0017] Furthermore, the inventors also conducted animal studies and clinical studies on the above-mentioned Chinese medicine composition.

[0018] In terms of animal research, the present invention first selected recurrent abortion mice (CBA / J×DBA / 2) as the experimental system, and tested the pharmacodynamic effects of the Chinese medicine composition (Bu Shen Huo Xue Fang) obtained by different prescriptions and different preparation methods of the present invention. The results showed that among the Chinese medicine compositions of Bu Shen Huo Xue Fang, different Bu Shen Huo Xue Fangs 1 to 7 all had the effect of lowering the embryo loss rate and improving the uterine embryo morphology, but Bu Shen Huo Xue Fang 1 (corresponding formula: 12 parts of Cuscuta, 10 parts of Dipsacus, 15 parts of Salvia miltiorrhiza, 15 parts of Millettia reticulata, 20 parts of Astragalus, 5 parts of Tangerine Peel, 15 parts of Chinese Yam) had the most advantages in lowering the embryo loss rate and improving the uterine embryo morphology and had a lower cost. Therefore, Bu Shen Huo Xue Fang 1 was selected for the remaining investigations.

[0019] Then, KM mice were selected as the experimental system to test the blood coagulation time. The blood coagulation time of each dose of Bushen Huoxue Recipe 1 was significantly prolonged, indicating that the Chinese medicine composition (Bushen Huoxue Recipe 1) of the present invention has an anticoagulant effect.

[0020] Then, recurrent abortion mice (CBA / J×DBA / 2) were used as the experimental system to compare the pharmacodynamic effects of the Bu Shen Huo Xue Fang 1 of the present invention with different Chinese patent medicines for tocolysis on the market, and to explore the dose-effect effect of the Bu Shen Huo Xue Fang and the pregnancy-aiding effect of pre-pregnancy medication. The results showed that compared with various Chinese patent medicines for tocolysis and dydrogesterone, the medium dose of Bu Shen Huo Xue Fang 1 (i.e., referring to the clinical human dose) had comprehensive advantages in reducing the embryo loss rate of recurrent abortion mice, increasing uterine wet weight and average fetal weight, and reducing placental maternal and fetal bleeding. At the same time, the Bu Shen Huo Xue Fang 1 of the present invention significantly increased the CG and P levels in the serum of pregnant mice, and significantly decreased the D-dimer level in the serum of pregnant mice, indicating that it can regulate sex hormone levels and improve maternal hypercoagulable state; at the same time, the Bu Shen Huo Xue Fang 1 group can inhibit the occurrence of placental fibrosis and Fib deposition, indicating that it can promote normal placental development. In addition, the invigorating kidney and activating blood circulation prescription 1 of the present invention can significantly reduce the embryo loss rate of recurrent abortion mice and improve the morphology of the fetus-containing uterus of abortion mice when administered before pregnancy or continuously before and after pregnancy.

[0021] In terms of clinical research, the present invention collected information on 111 patients with early and mid-term pregnancy complicated with intrauterine hemorrhage who visited the Department of Gynecology and Obstetrics (outpatient and inpatient departments) of the Women and Children's Center of the First Affiliated Hospital of Guangzhou University of Chinese Medicine, and adopted a self-controlled before-after study to statistically analyze the changes in the intrauterine hemorrhage area 2 weeks and 4 weeks after conventional Western medicine treatment combined with Bushen Huoxue Recipe 1. The results showed that the combined use of Bushen Huoxue Recipe 1 reduced the intrauterine hemorrhage area by 2 weeks and 4 weeks, and the longer the combined treatment time, the greater the reduction in the intrauterine hemorrhage area. The results suggest that Bushen Huoxue Recipe 1 can effectively improve early and mid-term pregnancy complicated with intrauterine hemorrhage.

[0022] In summary, the Chinese medicine composition (Bu Shen Huo Xue Fang) of the present invention lowered the embryonic loss rate of RPL mice, improved the hypercoagulable state of pregnant mice, and reduced the blood accumulation area of ​​clinical early and mid-term pregnancy patients with intrauterine hemorrhage, indicating that the prescription has a bidirectional regulatory effect of anticoagulation and hemostasis. Therefore, the present invention not only makes up for the lack of Chinese medicine compositions for "kidney deficiency and blood stasis type" RPL patients on the market, but also breaks through the bottleneck of intrauterine hemorrhage and PTS in Western medicine treatment, and can be well applied to the treatment of RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases, and can be used for both pre-pregnancy pregnancy assistance and post-pregnancy fetal maintenance.

[0023] In some of the schemes, the active ingredients of the Chinese medicine composition are mainly composed of the following medicinal materials in parts by weight:

[0024] 6-12 parts of Cuscuta, 9-15 parts of Dipsacus asper, 10-15 parts of Salvia miltiorrhiza, 9-15 parts of Millettia reticulata, 9-30 parts of Astragalus, 3-10 parts of Tangerine peel, 15-30 parts of Dioscorea opposita;

[0025] Or, 6-12 parts of Cuscuta seeds, 3-10 parts of Morinda officinalis, 10-15 parts of Salvia miltiorrhiza, 9-15 parts of Millettia reticulata, 9-30 parts of Astragalus, 3-10 parts of Citrus reticulata, and 15-30 parts of Dioscorea opposita;

[0026] Or, 6-12 parts of Cuscuta australis, 9-15 parts of Dipsacus asper, 10-15 parts of Salvia miltiorrhiza, 3-9 parts of Panax notoginseng, 9-30 parts of Astragalus, 3-10 parts of Citrus reticulata, and 15-30 parts of Dioscorea opposita;

[0027] Alternatively, 6-12 parts of Cuscuta australis, 3-10 parts of Morinda officinalis, 10-15 parts of Salvia miltiorrhiza, 3-9 parts of Panax notoginseng, 9-30 parts of Astragalus, 3-10 parts of dried tangerine peel, and 15-30 parts of yam.

[0028] In some of the schemes, the active ingredients of the Chinese medicine composition are mainly composed of the following medicinal materials in parts by weight:

[0029] 12 parts of Cuscuta seeds, 10 parts of Dipsacus asper, 15 parts of Salvia miltiorrhiza, 15 parts of Millettia reticulata, 20 parts of Astragalus, 5 parts of Citrus reticulata, 15 parts of Dioscorea opposita;

[0030] Or, 12 parts of Cuscuta seeds, 9 parts of Morinda officinalis, 15 parts of Salvia miltiorrhiza, 15 parts of Millettia reticulata, 20 parts of Astragalus, 5 parts of Citrus reticulata, and 15 parts of Dioscorea opposita;

[0031] Or, 12 parts of Cuscuta chinensis, 10 parts of Dipsacus asper, 15 parts of Salvia miltiorrhiza, 9 parts of Panax notoginseng, 20 parts of Astragalus, 5 parts of Citrus reticulata, and 15 parts of Dioscorea opposita;

[0032] Or, 12 parts of Cuscuta australis, 9 parts of Morinda officinalis, 15 parts of Salvia miltiorrhiza, 9 parts of Panax notoginseng, 20 parts of Astragalus, 5 parts of Citrus reticulatae, and 15 parts of Dioscorea oppositae.

[0033] In some of the solutions, the dodder seed is salt dodder seed, and the Morinda officinalis is salt Morinda officinalis. It can be understood that the salt dodder seed or salt Morinda officinalis can be prepared according to the corresponding traditional Chinese medicine preparation method.

[0034] On the other hand, the present invention also discloses a method for preparing the above-mentioned Chinese medicine composition, comprising the following steps: extracting the medicinal materials by solvent extraction method to obtain the Chinese medicine composition.

[0035] It can be understood that the above preparation method can be prepared according to the traditional Chinese medicine extraction method.

[0036] In some embodiments, the solvent extraction method includes: decoction, reflux extraction, maceration or percolation.

[0037] In some embodiments, the solvent is selected from: water, ethanol aqueous solution.

[0038] It is understandable that, among the above-mentioned extraction methods, the water extraction of the traditional water extraction and alcohol precipitation method also belongs to the above-mentioned solvent extraction method, and can be carried out according to the conventional Chinese medicine extraction process. Among them, the volume percentage concentration of the ethanol aqueous solution or ethanol during the water extraction and alcohol precipitation used as the extraction solvent also belongs to the category that can be adjusted according to the extraction efficiency, such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.

[0039] In some of the schemes, the preparation method comprises the following steps:

[0040] Weigh a prescribed amount of medicinal materials, add a solvent according to a predetermined material-liquid ratio, heat for extraction, filter, and recover the solvent in the filtrate to obtain the product.

[0041] In some of the schemes, the preparation method includes the following steps: weighing a prescribed amount of medicinal materials, adding a solvent according to a predetermined material-liquid ratio, heating for extraction, filtering, concentrating the filtrate and adding ethanol, allowing to stand, removing the precipitate and recovering the solvent in the filtrate to obtain the product.

[0042] In some of the schemes, the material-liquid ratio is 1 g of medicinal material: 10±5 ml of solvent.

[0043] In some schemes, the number of extractions is 1-3 times, for example, 2 times, the solid-liquid ratio used in the first extraction is 1:10±3, and the solid-liquid ratio used in the second extraction is 1:8±3.

[0044] In some embodiments, the extraction time is 0.5-3 hours, such as 1-2 hours.

[0045] In some embodiments, the extraction temperature is 80-100°C, such as 90-100°C.

[0046] In some of the schemes, the filtrate is concentrated to a relative density of 1.10-1.30, for example 1.15, and then ethanol is added.

[0047] In some of the schemes, the filtrate is concentrated and then ethanol is added to a volume percentage of ethanol of 60-75%, for example 65%.

[0048] In some embodiments, the filtrate is concentrated and then the ethanol is added and then the standing time is 8-24 hours, for example, 12 hours.

[0049] In some of the schemes, the preparation method further comprises the step of preparing the Chinese medicine composition into a freeze-dried powder or granules.

[0050] The steps of preparing the Chinese medicine composition into freeze-dried powder are: taking the filtrate, concentrating it to obtain an extract, and drying the extract by freeze-drying to obtain freeze-dried powder;

[0051] The steps of preparing the Chinese medicine composition into granules are: taking the filtrate, concentrating it to a clear paste with a relative density of 1.05-1.10, drying it, adding a filler, and wet granulating it to obtain granules; further, the filler is maltodextrin, and the relative density is measured at 50-70°C, for example, at 60°C.

[0052] The present invention also discloses a medicine, comprising the above-mentioned Chinese medicine composition and pharmaceutically acceptable excipients. Further, the Chinese medicine composition is administered orally. Furthermore, the dosage form of the Chinese medicine composition is capsules, granules, tablets, oral liquids, mixtures or syrups.

[0053] It is understood that the pharmaceutically acceptable excipients include carriers, excipients and / or fillers, etc., which can be selected and adjusted according to the dosage form requirements, and include any such materials that, when combined with the active ingredient, allow the ingredient to maintain biological activity and not react with the subject's immune system. The above different dosage forms can be prepared according to conventional methods in the art.

[0054] On the other hand, the present invention also discloses the use of the above-mentioned Chinese medicine composition and the above-mentioned medicine in preparing medicine for invigorating the kidney and activating blood circulation.

[0055] In some of the embodiments, the kidney-tonifying and blood-activating drug is used to prevent and / or treat recurrent pregnancy loss (RPL).

[0056] In some of these schemes, the kidney-tonifying and blood-activating drugs are used to assist pregnancy before pregnancy and / or to maintain pregnancy after pregnancy. For example, the drugs are used to assist pregnancy before pregnancy to prevent RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases; and the drugs are used after pregnancy to treat RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases.

[0057] In some of these schemes, the kidney-tonifying and blood-activating drugs are used for recurrent pregnancy loss (RPL) combined with prothrombotic state (PTS) and intrauterine hemorrhage.

[0058] The above-mentioned "treatment" means to slow down the symptoms associated with a disease, disorder or condition, or to stop the further development or deterioration of these symptoms. Depending on the disease and condition of the patient, "treatment" as used herein may include one or more of cure, alleviation and preventive treatment. Treatment may also include administering the pharmaceutical preparation of the present invention in combination with other treatments.

[0059] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0060] In the above formula, the content of each component is prepared by raw material medicine. When the raw material medicine is prepared, the effective ingredient can be extracted from the mixture of raw material medicines in the prescribed amount. The extraction may include: decoction, reflux extraction, immersion or percolation, etc. However, it can be understood that in the present invention, the pharmaceutical composition can also be a mixed granule obtained by mixing the concentrated granules made by processing and decoction of a single Chinese herbal medicine, but the content of each medicinal flavor in the mixed granule should be reduced to the weight of the corresponding raw material medicine for formulation. That is, the above components can be granules. If a decoction-free granule is used, it can be directly dissolved in water according to the proportion and then taken.

[0061] The reagents and raw materials used in the present invention are commercially available.

[0062] The positive and progressive effects of the present invention are:

[0063] A Chinese medicine composition of the present invention plays a bidirectional regulatory role of anticoagulation and hemostasis for RPL of "kidney deficiency and blood stasis" syndrome, can treat PRL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases, and has good curative effect, such as significantly prolonging the coagulation time of normal KM mice; at the same time, it can significantly reduce the spontaneous abortion rate of abortion model mice, improve PTS of CBA / J×DBA / 2 abortion model, and play a role in protecting pregnant mice. Clinically, the prescription can significantly reduce the intrauterine hemorrhage area of ​​patients with intrauterine hemorrhage after 2 weeks and 4 weeks of administration.

[0064] Furthermore, the Chinese medicine composition can be used before pregnancy to assist pregnancy and prevent RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases, and can also be used after pregnancy to treat RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a diagram of the histological morphology of the mouse uterus (including embryos) in different kidney-tonifying and blood-activating prescriptions in Example 3.

[0066] Figure 2These are the histological morphologies of the uterus and placenta of mice in the Bushen Huoxue Recipe, Baotailing Capsules, Yunkang Oral Liquid, Ejiao Buxue Granules, and Dydrogesterone groups in Example 5.

[0067] Figure 3 These are pictures of the uterus of mice in each group in Example 6.

[0068] Figure 4 These are pictures of the placenta of each group of mice in Example 6 and pictures of HE-stained tissue sections of the placenta.

[0069] Figure 5 This is a diagram of collagen deposition in the placenta decidua region of each group of mice in Example 6.

[0070] Figure 6 This is a graph showing the expression of Fib protein in the placenta decidua region of each group of mice in Example 6.

[0071] Figure 7 This is a flow chart of the administration in Example 8.

[0072] Figure 8 The diagram is a histological morphology diagram of the uterus (including embryos) of each group of mice in Example 8. DETAILED DESCRIPTION

[0073] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0074] Example 1

[0075] This embodiment prepares a variety of Chinese medicine compositions (i.e., kidney-tonifying and blood-activating prescriptions 1, 2, 3, 4, 5, 6, and 7), which have the effects of kidney-tonifying and blood-activating, and their formulas are respectively:

[0076] (1) Kidney-tonifying and blood-activating prescription 1: Cuscuta australis 12g, Dipsacus asper 10g, Salvia miltiorrhiza 15g, Millettia reticulata 15g, Astragalus 20g, Tangerine peel 5g, Chinese yam 15g.

[0077] Weigh 10 times the amount of the prescribed medicinal material, add 10 times the amount of water according to the material-liquid ratio of 10 ml of water per gram of medicinal material, decoct for 1 hour, and filter; add 8 times the amount of water to the residue and decoct for 1 hour, and filter. Combine the filtrate, concentrate the filtrate into a thick paste, and then freeze-dry it to make freeze-dried powder.

[0078] 1 g of the obtained freeze-dried powder is equivalent to 3.43 g of crude drug and is stored in a refrigerator at 4°C for future use.

[0079] (2) Kidney-tonifying and blood-activating prescription 2: Salt dodder seed 12g, Dipsacus asper 10g, Salvia miltiorrhiza 15g, Millettia reticulata 15g, Astragalus 20g, Tangerine peel 5g, Chinese yam 15g.

[0080] Weigh 10 times the amount of the prescription medicinal material, add 10 times the amount of water according to the material-liquid ratio of 10 ml of water per gram of medicinal material, decoct for 1 hour, and filter; add 8 times the amount of water to the filter residue and decoct for 1 hour, and filter. Combine the filtrates, decompress and concentrate them to a relative density of 1.15, cool, add 95% ethanol while stirring, so that the alcohol content reaches 65%, stir well, let stand overnight, filter, recover ethanol from the filtrate until there is no alcohol taste, and dry to obtain the water-extracted and alcohol-precipitated extract of the Bushen Huoxue Recipe.

[0081] 1 g of the water-extracted and alcohol-precipitated extract obtained is equivalent to 4.01 g of the crude drug and is stored in a refrigerator at 4°C for future use.

[0082] (3) Kidney-tonifying and blood-activating prescription 3: Cuscuta australis 3g, Dipsacus asper 3g, Salvia miltiorrhiza 35g, Millettia reticulata 35g, Astragalus 5g, Tangerine peel 20g, Chinese yam 35g.

[0083] Weigh 10 times the amount of the prescribed medicinal material, add 10 times the amount of water according to the material-liquid ratio of 10 ml of water per gram of medicinal material, decoct for 1 hour, and filter; add 8 times the amount of water to the residue and decoct for 1 hour, and filter. Combine the filtrate, concentrate the filtrate into a thick paste, and then freeze-dry it to make freeze-dried powder.

[0084] 1 g of the obtained freeze-dried powder is equivalent to 3.12 g of crude drug and is stored in a refrigerator at 4°C for future use.

[0085] (4) Recipe 4 for nourishing the kidney and activating blood circulation: 35g of Cuscuta australis, 35g of Dipsacus asper, 5g of Salvia miltiorrhiza, 5g of Millettia reticulata, 60g of Astragalus, 1g of Citrus reticulata, and 6g of Dioscorea opposita.

[0086] Weigh 10 times the amount of the prescribed medicinal material, add 10 times the amount of water according to the material-liquid ratio of 10 ml of water per gram of medicinal material, decoct for 1 hour, and filter; add 8 times the amount of water to the residue and decoct for 1 hour, and filter. Combine the filtrate, concentrate the filtrate into a thick paste, and then freeze-dry it to make freeze-dried powder.

[0087] 1 g of the obtained freeze-dried powder is equivalent to 3.05 g of crude drug and is stored in a refrigerator at 4°C for future use.

[0088] (5) Kidney-tonifying and blood-activating prescription 5: Cuscuta australis 12g, Dipsacus asper 10g, Salvia miltiorrhiza 15g, Panax notoginseng 9g, Astragalus 20g, Tangerine peel 5g, Chinese yam 15g.

[0089] Weigh 10 times the amount of the prescribed medicinal material, add 10 times the amount of water according to the material-liquid ratio of 10 ml of water per gram of medicinal material, decoct for 1 hour, and filter; add 8 times the amount of water to the residue and decoct for 1 hour, and filter. Combine the filtrate, concentrate the filtrate into a thick paste, and then freeze-dry it to make freeze-dried powder.

[0090] 1 g of the obtained freeze-dried powder is equivalent to 3.24 g of crude drug and is stored in a refrigerator at 4°C for future use.

[0091] (6) Recipe 6 for nourishing the kidney and activating blood circulation: 12g salt dodder seed, 9g salt Morinda officinalis, 15g salvia miltiorrhiza, 15g Millettia reticulata, 20g astragalus, 5g tangerine peel, 15g yam.

[0092] Weigh 10 times the amount of the prescribed medicinal material, add 10 times the amount of water according to the material-liquid ratio of 10 ml of water per gram of medicinal material, decoct for 1 hour, and filter; add 8 times the amount of water to the residue and decoct for 1 hour, and filter. Combine the filtrate, concentrate the filtrate into a thick paste, and then freeze-dry it to make freeze-dried powder.

[0093] 1 g of the obtained freeze-dried powder is equivalent to 3.36 g of crude drug and is stored in a refrigerator at 4°C for future use.

[0094] (7) Kidney-tonifying and blood-activating formula 7: Cuscuta australis 12g, Morinda officinalis 9g, Salvia miltiorrhiza 15g, Panax notoginseng 9g, Astragalus 20g, Tangerine peel 5g, Chinese yam 15g.

[0095] Weigh 10 times the amount of the prescribed medicinal material, add 10 times the amount of water according to the material-liquid ratio of 10 ml of water per gram of medicinal material, decoct for 1 hour, and filter; add 8 times the amount of water to the residue and decoct for 1 hour, and filter. Combine the filtrate, concentrate the filtrate into a thick paste, and then freeze-dry it to make freeze-dried powder.

[0096] 1 g of the obtained freeze-dried powder is equivalent to 3.32 g of crude drug and is stored in a refrigerator at 4°C for future use.

[0097] Example 2

[0098] This embodiment prepares a granule which can be used to nourish the kidney and promote blood circulation. The formula thereof is calculated in parts by weight of the raw materials:

[0099] 3-35 parts of Cuscuta seeds, 3-35 parts of kidney-tonifying drugs, 5-35 parts of Salvia miltiorrhiza, 3-35 parts of blood-activating and blood-stasis-removing drugs, 5-60 parts of Astragalus, 1-20 parts of dried orange peel, and 6-35 parts of Chinese yam;

[0100] The kidney-tonifying medicine is Dipsacus asper or Morinda officinalis, and the blood-activating and blood-stasis-removing medicine is Millettia reticulata or Panax notoginseng, which can be replaced and adjusted.

[0101] The granules are prepared according to the following method:

[0102] Weigh the medicinal materials in the formula, add 10 times the amount of water (1g medicinal materials: 10ml water) according to the proportion, decoct for 1 hour, filter; add 8 times the amount of water to the residue, decoct for 1 hour, filter. Combine the filtrate, concentrate to a clear paste with a relative density of 1.05-1.10 (60°C), dry, add an appropriate amount of maltodextrin, mix well, wet granulate, dry, and obtain granules.

[0103] Example 3

[0104] This example examines the comparative effects of various traditional Chinese medicine compositions (i.e., Bushen Huoxue Fang 1, 2, 3, 4, 5, 6, 7) prepared in Example 1 on CBA / J×DBA / 2 abortion mice.

[0105] Based on the CBA / J×DBA / 2 recurrent spontaneous abortion mouse model, the effects of Bushen Huoxue Fang with different formulas, ingredients, and extraction processes on the embryo loss rate and uterine (including embryo) tissue morphology of this model were investigated.

[0106] 1. Materials and Methods

[0107] 1.1 Materials

[0108] 1.1.1 Experimental System

[0109] 5 BALB / C male mice, 45 DBA / 2 male mice, and 100 CBA / J female mice, 8 weeks old, SPF grade, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK(Jing)2021-0011). After the animals were purchased, they were placed in the SPF animal laboratory of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (License No.: SYXK(Yue)2023-0092) and housed in separate cages. All experimental operations complied with the relevant regulations on animal welfare.

[0110] 1.1.2 Test Drugs

[0111] The traditional Chinese medicine compositions Bushen Huoxue Fang 1, 2, 3, 4, 5, 6, 7 prepared in Example 1.

[0112] 1.1.3 Experimental Reagents and Instruments

[0113] Normal saline: Batch No. 140911A, Foshan Shuanghe Pharmaceutical Co., Ltd.; Isoflurane: Batch No. 20221201, Jiangsu Hengfengqiang Biotechnology Co., Ltd.; Electronic balance: Model JJ3000, Changshu Shuangjie Testing Instrument Factory; Analytical balance: Model EL204, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; Vortex mixer: Model XW-80A, Qilinbeier Instrument Manufacturing Co., Ltd., Haimen; Microplate reader: Model EPOCH, BioTek Instruments, Inc., USA.

[0114] 1.2 Methods

[0115] 1.2.1 Animal Modeling and Grouping

[0116] Normal pregnant mice: Estrous CBA / J female mice were caged with BALB / C male mice at a ratio of 2:1 to establish 10 normal pregnant mouse models.

[0117] RSA model mice: Estrous CBA / J female mice were caged with DBA / 2 male mice at a ratio of 2:1 to establish an RSA model.

[0118] Observe the vaginal plug at 9:00 the next day after the cage is put together. If the vaginal plug is detected (mated), it is considered pregnant and recorded as G 0.5 days. If not pregnant, the female mouse will continue to be kept and wait for the next cage to conceive. Male mice can mate with the female mice in the cage multiple times on the same day. After mating, they need to rest for one day before arranging the next cage mating.

[0119] On the 0.5th day of pregnancy, the RSA model mice were randomly divided into the following 9 groups: model control group, Bushen Huoxue recipe 1, 2, 3, 4, 5, 6, 7, and dydrogesterone group, with 10 mice in each group.

[0120] 1.2.2 Dosage regimen

[0121] Taking the recommended dose for humans (weight 60 kg) as the standard, the conversion coefficient between adults and mice was 9.01, and the equivalent dose conversion formula was: mouse dose = (recommended dose for humans / 60 kg) × 9.01, and intragastric administration was performed. From G0.5 days to G13.5 days, the normal control group was intragastrically administered with 0.1 mL / 10 g / d of pure water, and each group of the Bushen Huoxue prescription was intragastrically administered with 0.1 mL / 10 g / d of the corresponding solution, once a day. The dosage of each group is shown in Table 1.

[0122] Table 1 Dosage of each group

[0123]

[0124] 1.2.3 Solution preparation

[0125] (1) Bu Shen Huo Xue Fang 1: 1g Bu Shen Huo Xue Fang 1 freeze-dried powder is equivalent to 3.43g crude drug. The dosage of crude drug for mice is 13.82g crude drug / kg / d. Therefore, the dosage of Bu Shen Huo Xue Fang 1 freeze-dried powder for mice is 4.03g freeze-dried powder / kg / d. The concentration required for gavage is 0.403g / mL at 0.1mL / 10g / d. Based on the average weight of mice of 30g, 10 animals need 3.0mL of solution. Prepare 4mL of Bu Shen Huo Xue Fang 1 solution. The specific operation is to accurately weigh 1.612g Bu Shen Huo Xue Fang 1 freeze-dried powder and place it in an EP tube. Add appropriate amount of pure water to dissolve it, and then adjust the volume to 4mL. Invert it upside down 5 times, ultrasonically heat it at 60℃ until the freeze-dried powder is completely dissolved (about 10min), and prepare it for use.

[0126] (2) Bushen Huoxue Recipe 2: 1g of Bushen Huoxue Recipe 2 water-extracted alcohol-precipitated extract is equivalent to 4.01g of crude drug. The dosage of crude drug for mice is 13.82g of crude drug / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 2 mouse extract is 3.45g of extract / kg / d. The concentration required for oral administration is 0.345g / mL at 0.1mL / 10g / d. Based on the average weight of mice of 30g, 3.0mL of solution is required for 10 animals. Now prepare 4mL of Bushen Huoxue Recipe 2 solution. The specific operation is to accurately weigh 1.380g of Bushen Huoxue Recipe 2 water-extracted alcohol-precipitated extract and place it in an EP tube, add an appropriate amount of pure water to dissolve it, and then adjust the volume to 4mL. Invert it upside down 5 times, and ultrasonically heat it at 60℃ until the extract is completely dissolved (about 10min). Prepare it and use it immediately.

[0127] (3) Bushen Huoxue Recipe 3: 1g of Bushen Huoxue Recipe 3 freeze-dried powder is equivalent to 3.12g of crude drug. The dosage of crude drug for mice is 20.42g of crude drug / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 3 freeze-dried powder for mice is 6.55g freeze-dried powder / kg / d. The concentration of the water extract required is 0.655g / mL. Based on the average weight of mice of 30g, 3.0mL of solution is required for 10 animals. Now prepare 4mL of Bushen Huoxue Recipe 3 solution. The specific operation is to accurately weigh 2.62g of Bushen Huoxue Recipe 3 freeze-dried powder and place it in an EP tube. Add appropriate amount of pure water to dissolve it, and then adjust the volume to 4mL. Invert it upside down 5 times, ultrasonically heat it at 60℃ until the freeze-dried powder is completely dissolved (about 10min), and use it immediately.

[0128] (4) Bu Shen Huo Xue Fang 4: 1g Bu Shen Huo Xue Fang 4 freeze-dried powder is equivalent to 3.05g crude drug. The dosage of crude drug for mice is 22.07g crude drug / kg / d. Therefore, the dosage of Bu Shen Huo Xue Fang 4 freeze-dried powder for mice is 7.24g freeze-dried powder / kg / d. It is administered by gavage at 0.1mL / 10g / d. The concentration of the water extract required is 0.724g / mL. Based on the average weight of mice of 30g, 10 animals need 3.0mL of solution. Now prepare 4mL of Bu Shen Huo Xue Fang 4 solution. The specific operation is to accurately weigh 2.896g of Bu Shen Huo Xue Fang 4 freeze-dried powder and place it in an EP tube. Add appropriate amount of pure water to dissolve it, and then adjust the volume to 4mL. Invert it upside down 5 times, ultrasonically heat it at 60℃ until the freeze-dried powder is completely dissolved (about 10min), and use it immediately.

[0129] (5) Bushen Huoxue Recipe 5: 1g of Bushen Huoxue Recipe 5 freeze-dried powder is equivalent to 3.24g of crude drug. The dosage of crude drug for mice is 12.91g of crude drug / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 5 freeze-dried powder for mice is 3.98g of freeze-dried powder / kg / d. It is administered by oral gavage at 0.1mL / 10g / d, and the concentration of the water extract required is 0.398g / mL. Based on the average weight of mice of 30g, 10 animals need 3.0mL of solution. Now prepare 4mL of Bushen Huoxue Recipe 5 solution. The specific operation is to accurately weigh 1.592g of Bushen Huoxue Recipe 5 freeze-dried powder and place it in an EP tube, add appropriate amount of pure water to dissolve it, and then adjust the volume to 4mL. Invert it upside down 5 times, ultrasonically heat it at 60℃ until the freeze-dried powder is completely dissolved (about 10min), and use it immediately.

[0130] (6) Bu Shen Huo Xue Fang 6: 1g Bu Shen Huo Xue Fang 6 freeze-dried powder is equivalent to 3.36g crude drug. The dosage of crude drug for mice is 13.67g crude drug / kg / d. Therefore, the dosage of Bu Shen Huo Xue Fang 6 freeze-dried powder for mice is 4.06g freeze-dried powder / kg / d. It is administered by gavage at 0.1mL / 10g / d. The concentration of the water extract required is 0.406g / mL. Based on the average weight of mice of 30g, 10 animals need 3.0mL of solution. Now prepare 4mL of Bu Shen Huo Xue Fang 6 solution. The specific operation is to accurately weigh 1.624g Bu Shen Huo Xue Fang 6 freeze-dried powder and place it in an EP tube. Add appropriate amount of pure water to dissolve it, and then adjust the volume to 4mL. Invert it upside down 5 times, ultrasonically heat it at 60℃ until the freeze-dried powder is completely dissolved (about 10min), and use it immediately.

[0131] (7) Bushen Huoxue Recipe 7: 1g of Bushen Huoxue Recipe 7 freeze-dried powder is equivalent to 3.32g of crude drug. The dosage of crude drug for mice is 12.76g of crude drug / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 7 freeze-dried powder for mice is 3.84g of freeze-dried powder / kg / d. The concentration of the water extract required is 0.384g / mL. Based on the average weight of mice of 30g, 3.0mL of solution is required for 10 animals. Prepare 4mL of Bushen Huoxue Recipe 7 solution. The specific operation is to accurately weigh 1.536g of Bushen Huoxue Recipe 7 freeze-dried powder and place it in an EP tube. Add appropriate amount of pure water to dissolve it, and then adjust the volume to 4mL. Invert it upside down 5 times, ultrasonically heat it at 60℃ until the freeze-dried powder is completely dissolved (about 10min), and use it immediately.

[0132] (8) Dydrogesterone solution: The dosage of mice in the dydrogesterone group was 3.00 mg / kg / d, and the drug solution concentration required was 0.300 mg / mL. Based on the average weight of 30 g of mice, 3.0 mL of solution is required for 10 animals. Now 4.0 mL of dydrogesterone solution is prepared, which requires 1.2 mg of dydrogesterone. Each tablet contains 10 mg of dydrogesterone, and the average weight of dydrogesterone tablets is 0.1426 g. The specific operation is to accurately weigh 17.11 mg of dydrogesterone tablet powder (i.e. 1.2 mg of dydrogesterone), place it in an EP tube, add an appropriate amount of pure water to dissolve, and dilute to 4 mL. Invert upside down 5 times, sonicate for 5 minutes, fully dissolve, and prepare for use.

[0133] 1.2.4 Index detection

[0134] Mice were anesthetized with 5% isoflurane on G13.5 days. After anesthesia, blood was collected from the abdominal aorta and stored in a centrifuge tube without sodium citrate to obtain serum. After blood collection, the uterus was quickly dissected and the uterus was removed. The appearance was recorded by photographing. The uterus was then opened along the longitudinal axis at the uterine frenulum, the total number of embryos and the number of aborted embryos were counted, and the embryo loss rate was calculated: embryo loss rate = number of aborted embryos / total number of embryos × 100%.

[0135] 1.3 Statistics

[0136] All data were input into SPSS26.0 for statistical analysis. The count data of each group, such as embryo absorption rate, were analyzed using the chi-square test of the four-cell table data. 2 The test and / or the exact Fisher's exact probability method were used for statistical analysis; the measurement data of each group were calculated as the mean ± standard deviation (x ± s), and the inter-group variance analysis (F-test) was performed before the inter-group comparison of each experimental group. When the inter-group variances were equal, the inter-group comparison was statistically analyzed using the Student-T test (unpaired T test), and when the inter-group variances were unequal, the corrected Student-T test was used for statistical analysis. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.

[0137] 2. Results

[0138] 2.1 Mouse embryo loss rate

[0139] The results are shown in the table below. The results show that all the kidney-tonifying and blood-activating prescriptions can reduce the embryonic loss rate of mice with recurrent miscarriage.

[0140] Table 2 Embryo loss rate of mice in each group ( n=10)

[0141]

[0142] Note: Compared with the normal control group, **P < 0.01; compared with the model control group, # P < 0.05, ## P < 0.01

[0143] As can be seen from the above results, compared with the normal control group, the embryo loss rate in the model control group was significantly increased (P < 0.01), indicating successful modeling; compared with the model control group, the embryo loss rates in the Bushen Huoxue Formula 1, 2, 5, 6, and 7 groups were extremely significantly decreased (P < 0.01), and the embryo loss rates in the Bushen Huoxue Formula 3 and Bushen Huoxue Formula 4 groups were significantly decreased (P < 0.05).

[0144] 2.2 Uterus (including embryo) morphology

[0145] The tissue morphology of the uterus (including embryo) of mice in each group of the traditional Chinese medicine composition (i.e., Bushen Huoxue Formula 1, 2, 3, 4, 5, 6, and 7) was as Figure 1 shown.

[0146] It can be seen that the embryos in the normal group were light red, thick and uniform, like a string of beads; in the model group, there were black-brown absorbed embryos, visible stasis could be seen with the naked eye, the sizes were uneven, and some embryos even completely disappeared; the embryos in each Bushen Huoxue Formula group were plump, light red, and occasionally there were black-brown absorbed embryos.

[0147] Combining the results of 2.1 and 2.2, it can be found that Bushen Huoxue Formula 1, 2, 5, 6, and 7 were better in down-regulating the embryo loss rate and could all improve the uterine embryo morphology. At the same time, the cost of preparing Bushen Huoxue Formula 1 was lower, so Bushen Huoxue Formula 1 was selected for investigation in the subsequent examples.

[0148] Example 4

[0149] This example investigated the effect of the pharmaceutical composition (Bushen Huoxue Formula 1) prepared in Example 1 on the clotting time of normal Kunming (KM) mice.

[0150] 1. Materials and methods

[0151] 1.1 Materials

[0152] 1.1.1 Experimental system

[0153] 50 male Kunming mice, SPF grade, were purchased from the Guangdong Provincial Medical Experimental Animal Center (license number: SCXK(Guangdong)2022 - 0002). After the animals were purchased, they were placed in the SPF animal laboratory of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (license number: SYXK(Guangdong)2018 - 0092) and housed separately in cages. All experimental operations complied with the relevant regulations on animal welfare.

[0154] 1.1.2 Test drugs

[0155] The Chinese medicine composition Bushen Huoxue Recipe 1 freeze-dried powder prepared in Example 1.

[0156] Enoxaparin: National Medicine Standard No. J20180036, Sanofi Winthrop Industrie, specification 0.6mL: 6000AXalIU, 2 vials per box.

[0157] 1.1.3 Experimental reagents and instruments

[0158] Electronic balance: Model JJ3000, Changshu Shuangjie Testing Instrument Factory; Analytical balance: Model EL204, Mettler-Toledo Instrument (Shanghai) Co., Ltd.; Vortex analyzer: Model XW-80A, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.

[0159] 1.2 Methods

[0160] 1.2.1 Animal grouping

[0161] Fifty SPF KM mice were randomly divided into five groups according to their body weight: normal control group, low-dose Bushen Huoxue prescription group, medium-dose Bushen Huoxue prescription group, high-dose Bushen Huoxue prescription group, and low-molecular-weight heparin group, with 10 mice in each group.

[0162] 1.2.2 Dosage regimen

[0163] Taking the recommended dose for humans (body weight 60kg) as the standard, the conversion factor between adults and mice is 9.01, and the equivalent dose conversion formula is: mouse dose = (recommended dose for humans / 60kg) × 9.01, and the drug is administered by gavage or subcutaneous injection.

[0164] The normal control group was gavaged with pure water at 10mL / kg; the low-dose group of the Bushen Huoxue Recipe was gavaged with the Bushen Huoxue Recipe aqueous solution, the dose was half of the human dose (46g crude drug / person / d), and the crude drug dose for mice was 6.91g crude drug / kg / d after conversion; the medium-dose group of the Bushen Huoxue Recipe was gavaged with the Bushen Huoxue Recipe aqueous solution, the dose was the human dose (92g crude drug / person / d), and the crude drug dose for mice was 13.82g crude drug / kg / d after conversion; the high-dose group of the Bushen Huoxue Recipe was gavaged with the Bushen Huoxue Recipe aqueous solution, the dose was twice the human dose (184g crude drug / person / d), and the crude drug dose for mice was 27.63g crude drug / kg / d after conversion. The low-molecular-weight heparin group was subcutaneously injected with enoxaparin injection at the clinically recommended human dose (4000AXalU / time / day), and the subcutaneous dose for mice was 6.00AXalU / 10g after conversion.

[0165] 1.2.3 Solution preparation

[0166] Example 1 1g of homemade freeze-dried powder is equivalent to 3.43g of the raw medicine of the Bushen Huoxue prescription, and the volume of oral administration to mice is 0.1mL / 10g / d. Therefore, the dosage of freeze-dried powder for high-dose Bushen Huoxue prescription mice is 8.06g freeze-dried powder / kg / d, and the concentration of the solution to be prepared is 0.806g / mL; the dosage of freeze-dried powder for medium-dose Bushen Huoxue prescription mice is 4.03g freeze-dried powder / kg / d, and the concentration of the solution to be prepared is 0.403g / mL; the dosage of freeze-dried powder for low-dose Bushen Huoxue prescription mice is 2.01g freeze-dried powder / kg / d, and the concentration of the water extract to be prepared is 0.201g / mL. The recommended clinical human dose of enoxaparin is 4000AXalU / time / day for subcutaneous administration, which is converted to 600AXalU / kg / d for mice.

[0167] (1) High-dose Bushen Huoxue Recipe: Based on the average weight of 30g of mice, 3.0mL of solution is required for 10 animals. Since low- and medium-dose Bushen Huoxue Recipes are prepared by gradient dilution of high-dose Bushen Huoxue Recipe, 7mL of high-dose Bushen Huoxue Recipe solution is now prepared. The specific operation is to accurately weigh 5.642g of Bushen Huoxue Recipe freeze-dried powder and place it in an EP tube, add an appropriate amount of pure water to dissolve, and then adjust the volume to 7mL. Invert it upside down 5 times, ultrasonically heat it at 60℃ until the freeze-dried powder is completely dissolved (about 10min), and use it immediately after preparation.

[0168] (2) Medium-dose Bushen Huoxue Recipe: Based on the average weight of 30 g of mice, 3.0 mL of solution is required for 10 animals. Prepare 4 mL of medium-dose Bushen Huoxue Recipe solution. The specific operation is to accurately transfer 2.0 mL of high-dose Bushen Huoxue Recipe solution into an EP tube, add 2.0 mL of pure water, vortex for 30 seconds, mix thoroughly, and prepare and use immediately.

[0169] (3) Low-dose Bushen Huoxue Recipe: Based on the average weight of 30 g of mice, 3.0 mL of solution is required for 10 animals. Prepare 4 mL of low-dose Bushen Huoxue Recipe solution. The specific operation is to accurately transfer 2.0 mL of medium-dose Bushen Huoxue Recipe into an EP tube, add 2.0 mL of pure water, vortex for 30 seconds, mix thoroughly, and use immediately after preparation.

[0170] (4) Enoxaparin sodium solution: Based on the average weight of 30 g of mice, 3.0 mL of solution is required for 10 animals. Prepare 4.0 mL of enoxaparin sodium solution. The specific operation is to draw 24 μL (240 AXalu, 10000 AXalu / mL) of pre-filled enoxaparin sodium injection into a 5 mL EP tube, add 3976 μL of 5% glucose injection, mix thoroughly, and obtain 4000 μL (240 AXalu, 60 AXalu / mL) solution, which is prepared and used immediately.

[0171] 1.2.4 Index detection

[0172] One hour after a single dose, blood was collected from the eye socket using a capillary tube. The timer started from the time blood flowed into the tube. After the tube was filled with blood, the capillary glass tube was placed flat on a glass dish. After 30 seconds, one end of the capillary glass tube was broken (the other end that received blood was broken), and it was slowly pulled to the left and right. After 50 seconds, it was broken every 10 seconds. When blood streaks appeared at the break, the other end was broken for verification. The time when blood streaks appeared at both ends was recorded as the coagulation time (s).

[0173] 1.2.5 Data processing

[0174] Same as Example 3.

[0175] 2. Results

[0176] The results are shown in the following table.

[0177] Table 3 Bushen Huoxue prescription significantly prolonged the coagulation time of mice ( n=10)

[0178]

[0179] Note: Compared with the normal control group, ** P<0.01

[0180] The results showed that compared with the normal control group, the coagulation time of the test drug (Bu Shen Huo Xue Fang) group was significantly prolonged (P<0.01), indicating that the Bu Shen Huo Xue Fang had an anti-thrombotic effect.

[0181] Example 5

[0182] This example investigates the effects of the drug composition (Bu Shen Huo Xue Fang 1) prepared in Example 1 and different Chinese patent medicines for prenatal care on CBA / J×DBA / 2 aborted mice.

[0183] Based on the CBA / J×DBA / 2 recurrent spontaneous abortion mouse model, the effects of the Bushen Huoxue recipe and different Chinese patent medicines for prenatal care (Baotailing capsule, Yunkang oral liquid, and Ejiao Buxue granules) on the embryonic loss rate, uterine and placental tissue morphology of the model were investigated. Among them, Baotailing capsule is a Chinese patent medicine used to treat the "kidney deficiency" and "spleen and kidney deficiency" syndromes of RPL, Yunkang oral liquid is a Chinese patent medicine used to treat the "kidney deficiency and blood heat" syndrome of RPL, and Ejiao Buxue granules are a Chinese patent medicine used to treat the "qi and blood deficiency" syndrome of RPL.

[0184] 1. Materials and Methods

[0185] 1.1 Materials

[0186] 1.1.1 Experimental system

[0187] Five male Balb / c mice, 30 male DBA / 2 mice, and 70 female CBA / J mice, 8 weeks old, SPF grade, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License number: SCXK(Jing)2021-0011). After purchase, the animals were placed in the SPF animal laboratory of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (License number: SYXK(Yue)2023-0092) and housed separately in cages. All experimental operations complied with the relevant regulations on animal welfare.

[0188] 1.1.2 Test drugs

[0189] The freeze-dried powder of the traditional Chinese medicine composition Bushen Huoxue Fang 1 prepared in Example 1, hereinafter referred to as Bushen Huoxue Fang.

[0190] Baotailing Capsules: National Drug Approval Number Z20050821, Shaanxi Dongtai Pharmaceutical Co., Ltd., specification 0.5 g / capsule, 36 capsules per box.

[0191] Yunkang Oral Liquid: National Drug Approval Number Z10970068, Huiyinbi Group Zhejiang Qiji Pharmaceutical Co., Ltd., specification 10 mL / branch, 6 branches per box.

[0192] Ejiao Buxue Granules: National Drug Approval Number Z37021196, Dong'e Ejiao Co., Ltd., specification 4 g / bag, 30 bags per box.

[0193] Dydrogesterone Tablets: National Drug Approval Number HJ20170221, Abbott Biologicals B.V., each tablet contains 10 mg of dydrogesterone.

[0194] 1.1.3 Experimental reagents and instruments

[0195] Same as Example 3.

[0196] 1.2 Methods

[0197] 1.2.1 Animal modeling and grouping

[0198] Modeling was carried out according to the method of Example 3. Subsequently, on the 0.5th day of pregnancy, the RSA model mice were randomly divided into the following 6 groups: namely, the model control group, the Bushen Huoxue Fang group, the Baotailing Capsules group, the Yunkang Oral Liquid group, the Ejiao Buxue Granules group, and the positive drug group (dydrogesterone), with 10 mice in each group.

[0199] 1.2.2 Administration plan

[0200] Based on the recommended dose for humans (body weight calculated as 60 kg), the conversion coefficient between adults and mice is 9.01. The equivalent dose conversion formula is: mouse dose = (human recommended dose / 60 kg) × 9.01, and gavage administration was carried out.

[0201] The normal control group was gavaged with pure water at 10 mL / kg; the Bushen Huoxue Fang group was gavaged with Bushen Huoxue Fang solution at the human dose (92 g crude drug / person / d), and the converted crude drug dose for mice was 13.82 g crude drug / kg / d; the Baotailing Capsule group was gavaged with Baotailing Capsule aqueous solution at the clinically recommended human dose (4.5 g / person / d), and the converted mouse dose was 0.6758 g / kg / d; the Yunkang Oral Liquid group was gavaged with Yunkang Oral Liquid at the clinically recommended human dose The mice were given an oral administration of 9.01 mL / kg / d of Ejiao Buxue Granules water extract at a dose of the clinically recommended human dose (8.0 g / person / d), and the mice were given an oral administration of 1.201 g / kg / d of Ejiao Buxue Granules water extract at a dose of the clinically recommended human dose (20 mg / person / d), and the mice were given an oral administration of 3.00 mg / kg / d of dydrogesterone.

[0202] From G0.5 to G13.5, each group was intragastrically administered with solutions of corresponding concentrations at 0.1 mL / 10 g / d, once a day.

[0203] 1.2.3 Solution preparation

[0204] The volume of oral administration of mice in this experiment was 0.1mL / 10g / d. Example 1: 1g of self-made Bu Shen Huo Xue lyophilized powder is equivalent to 3.43g of Bu Shen Huo Xue Fang crude drug, so the dosage of Bu Shen Huo Xue Fang mouse lyophilized powder is 4.03g lyophilized powder / kg / d, and the concentration of water extract is 0.403g / mL; the dosage of mice in Bao Tai Ling capsule group is 0.6758g / kg / d, that is, 0.068g / mL; the dosage of mice in Yun Kang oral liquid group is 9.01mL / kg / d, and the oral liquid is directly administered; the dosage of mice in E Jiao Bu Xue Granule group is 1.201g / kg / d, that is, 0.120g / mL; the dosage of mice in Dydrogesterone group is 3.00mg / kg / d, that is, 0.300mg / mL.

[0205] (1) Bushen Huoxue Recipe: Based on the average weight of 30g of mice, 3.0mL of solution is required for 10 animals. Prepare 4mL of Bushen Huoxue Recipe solution. The specific operation is to accurately weigh 1.612g of Bushen Huoxue Recipe freeze-dried powder and place it in an EP tube, add an appropriate amount of pure water to dissolve, and then adjust the volume to 4mL. Invert it upside down 5 times, and ultrasonically heat it at 60℃ until the freeze-dried powder is completely dissolved (about 10 minutes), and use it immediately after preparation.

[0206] (2) Baotailing Capsule Solution: Based on the average weight of 30g of mice, 3.0mL of solution is required for 10 animals. Prepare 4.0mL of Baotailing Capsule Solution. The specific operation is to accurately weigh 0.272g of Baotailing Capsule powder and place it in an EP tube, add an appropriate amount of pure water to dissolve, and then adjust the volume to 4mL. Invert it upside down 5 times, sonicate for 5 minutes, fully dissolve, and use it immediately after preparation.

[0207] (3) Donkey-hide gelatin blood-enriching granule solution: Based on the average weight of 30 g of mice, 3.0 mL of solution is required for 10 animals. Prepare 4.0 mL of donkey-hide gelatin blood-enriching granule solution. The specific operation is to accurately weigh 0.480 g of donkey-hide gelatin blood-enriching granules and place them in an EP tube, add an appropriate amount of pure water to dissolve, and then adjust the volume to 4 mL. Invert it upside down 5 times, sonicate for 5 minutes, and fully dissolve it. Prepare and use it immediately.

[0208] (4) Dydrogesterone solution: The dosage of mice in the dydrogesterone group was 3.00 mg / kg / d, and the drug solution concentration required was 0.300 mg / mL. Now prepare 4.0 mL of dydrogesterone solution, which requires 1.2 mg of dydrogesterone. Each tablet contains 10 mg of dydrogesterone, and the average weight of dydrogesterone tablets is 0.1426 g. Accurately weigh 17.11 mg of dydrogesterone tablet powder (i.e. 1.2 mg of dydrogesterone), place it in an EP tube, add appropriate amount of pure water to dissolve, and make up to 4 mL. Invert upside down 5 times, sonicate for 5 minutes, fully dissolve, and prepare for use.

[0209] 1.2.4 Index detection

[0210] Mice were anesthetized with 5% isoflurane on G13.5 days. After anesthesia, blood was collected from the abdominal aorta and stored in a centrifuge tube without sodium citrate to obtain serum. After blood collection, the uterus was quickly dissected, the uterus was removed, the appearance was recorded by photographing, and the weight of the uterus with the fetus was weighed. The uterus was then opened along the longitudinal axis at the uterine frenulum, the total number of embryos and the number of aborted embryos were counted, and the embryo loss rate was calculated: embryo loss rate = number of aborted embryos / total number of embryos × 100%.

[0211] 1.3 Statistics

[0212] Same as Example 3.

[0213] 2. Results

[0214] 2.1 Mouse embryo loss rate

[0215] The results are shown in the table below. The results show that Bushen Huoxue recipe, Baotailing capsule, Yunkang oral liquid and dydrogesterone all reduced the embryo loss rate of recurrent miscarriage mice.

[0216] Table 4 Embryo loss rate of mice in each group ( n=10)

[0217]

[0218] Note: Compared with the normal control group, ** P<0.01; compared with the model control group, # P<0.05, ## P<0.01

[0219] From the above results, it can be seen that compared with the normal control group, the embryo loss rate of the model control group was significantly increased (P<0.01), indicating that the model was successful; compared with the model control group, the embryo loss rate of the Bushen Huoxue Decoction and Dydrogesterone groups was extremely significantly decreased (P<0.01), and the embryo loss rate of the Baotailing Capsule and Yunkang Oral Liquid was significantly decreased (P<0.05).

[0220] 2.2 Uterine wet weight and average embryo weight

[0221] The results are shown in the table below. Compared with different Chinese patent medicines for tocolysis, both the Bushen Huoxue recipe and dydrogesterone upregulated the uterine wet weight and average embryo weight of mice with recurrent miscarriage.

[0222] Table 5 Uterine wet weight and average embryo weight of mice in each group ( n=10)

[0223]

[0224]

[0225] Note: Compared with the normal control group, ** P<0.01; compared with the model control group, ## P<0.01

[0226] Compared with the normal control group, the uterine wet weight and average embryo weight in the model control group were significantly decreased (P<0.01), and the uterine wet weight in the dydrogesterone group was significantly increased (P<0.01); compared with the model control group, the uterine wet weight and average embryo weight in the Bushen Huoxue prescription group and the dydrogesterone group were significantly increased (P<0.01).

[0227] 2.3 Uterine and placental tissue morphology

[0228] The morphology of uterus and placenta tissues of mice in the Bushen Huoxue prescription, Baotailing capsule, Yunkang oral liquid, Ejiao Buxue granules and dydrogesterone groups was as follows: Figure 2 shown.

[0229] It can be seen that the embryos in the normal group were light pink, thick and uniform, like beads; the model group had dark brown absorbed embryos, with visible blood stasis, uneven sizes, and some or even completely disappeared embryos; most of the embryos in the Baotailing Capsules and Yunkang Oral Liquid were plump and light pink, with occasional dark brown absorbed embryos, while in the Bushen Huoxue Recipe and Dydrogesterone groups, the embryos were plump and light pink, and no dark brown absorbed embryos appeared.

[0230] Observation under a stereomicroscope revealed that compared with the normal group, the model group had bleeding on the maternal side of the placenta; the bleeding on the maternal side of the placenta in the Bushen Huoxue Recipe and Baotailing Capsules groups was significantly reduced or disappeared; the bleeding on the maternal side of the placenta in the Yunkang Oral Liquid, Ejiao Buxue Granules, and Dydrogesterone groups did not reduce ( Figure 2 ).

[0231] The results showed that the Bushen Huoxue recipe, Baotailing capsules, Yunkang oral liquid and dydrogesterone could improve the uterine and placental tissue morphology of each group of mice to a certain extent, among which the Bushen Huoxue recipe had the best effect in all aspects.

[0232] Example 6

[0233] In this example, the dose-effect relationship of the drug composition (Bu Shen Huo Xue Fang 1) prepared in Example 1 in treating CBA / J×DBA / 2 aborted mice was studied.

[0234] Based on the CBA / J×DBA / 2 recurrent abortion mouse model, the effects of different doses of Bushen Huoxue recipe on the embryo loss rate, uterine and placental tissue morphology were investigated. The changes of serum chorionic gonadotropin (CG), progesterone (P), estradiol (E2), fibrinogen (FIB) and D-dimer (DD) in mice were detected by ELISA. Masson staining was used to observe the collagen deposition in the decidua region of the mouse placenta. Immunohistochemistry was used to observe the expression of fibrinogen (Fib) protein in the decidua region of the mouse placenta.

[0235] 1. Materials and Methods

[0236] 1.1 Materials

[0237] 1.1.1 Experimental system

[0238] Five male Balb / c mice, 25 male DBA / 2 mice, and 60 female CBA / J mice, 8 weeks old, SPF grade, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK(Jing)2021-0011). After purchase, the animals were placed in the SPF animal laboratory of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (License No.: SYXK(Yue)2023-0092) and housed separately in cages. All experimental operations complied with the relevant regulations on animal welfare.

[0239] 1.1.2 Test article

[0240] The freeze-dried powder of the traditional Chinese medicine composition Bushen Huoxue Fang 1 prepared in Example 1, hereinafter referred to as Bushen Huoxue Fang for short.

[0241] Dydrogesterone tablets: National Medicine Approval No. HJ20170221, Abbott Biologicals B.V., each tablet contains 10 mg of dydrogesterone.

[0242] 1.1.3 Experimental reagents and instruments

[0243] Same as Example 3.

[0244] 1.2 Methods

[0245] 1.2.1 Animal model establishment and grouping

[0246] The method for establishing the model was the same as that in Example 3. On the 0.5th day of pregnancy (G0.5), the RSA model mice were randomly divided into the following 5 groups: model control group, low-dose Bushen Huoxue Fang group, medium-dose Bushen Huoxue Fang group, high-dose Bushen Huoxue Fang group, and positive drug group (dydrogesterone), with 10 mice in each group.

[0247] 1.2.2 Administration regimen

[0248] Based on the recommended dose for humans (calculated based on a body weight of 60 kg), the conversion coefficient between adults and mice is 9.01, and the equivalent dose conversion formula is: mouse dose = (human recommended dose / 60 kg) × 9.01. Gavage administration was carried out.

[0249] The normal control group was gavaged with pure water at 10 mL / kg; the low-dose group of the Bushen Huoxue recipe was gavaged with the Bushen Huoxue recipe solution, the dose was half of the human dose (46 g crude drug / person / d), and the crude drug dose for mice after conversion was 6.91 g crude drug / kg / d; the medium-dose group of the Bushen Huoxue recipe was gavaged with the Bushen Huoxue recipe solution, the dose was the human dose (92 g crude drug / person / d), and the crude drug dose for mice after conversion was 13.82 g crude drug / kg / d; the high-dose group of the Bushen Huoxue recipe was gavaged with the Bushen Huoxue recipe solution, the dose was twice the human dose (184 g crude drug / person / d), and the crude drug dose for mice after conversion was 27.63 g crude drug / kg / d; the dydrogesterone group was gavaged with the dydrogesterone aqueous solution, the dose was the clinically recommended human dose for habitual abortion (20 mg human / d), and the dydrogesterone dose for mice after conversion was 3.00 mg / kg / d.

[0250] From G0.5 to G13.5, each group was intragastrically administered with solutions of corresponding concentrations at 0.1 mL / 10 g / d, once a day.

[0251] 1.2.3 Solution preparation

[0252] Since 1g of homemade Bu Shen Huo Xue lyophilized powder is equivalent to 3.43g of Bu Shen Huo Xue Fang crude drug, the volume of oral administration to mice is 0.1mL / 10g / d. Therefore, the dosage of high-dose Bu Shen Huo Xue Fang lyophilized powder to mice is 8.06g lyophilized powder / kg / d, and the concentration of water extract is 0.806g / mL; the dosage of medium-dose Bu Shen Huo Xue Fang lyophilized powder to mice is 4.03g lyophilized powder / kg / d, and the concentration of water extract is 0.403g / mL; the dosage of low-dose Bu Shen Huo Xue Fang lyophilized powder to mice is 2.01g lyophilized powder / kg / d, and the concentration of water extract is 0.201g / mL. The dosage of mice in the dydrogesterone group is 3.00mg / kg / d, that is, 0.300mg / mL.

[0253] High-dose Bushen Huoxue Recipe: Calculated based on the average weight of 30g of mice, 10 animals need 3.0mL of solution. Since low- and medium-dose Bushen Huoxue Recipes are prepared by gradient dilution of high-dose Bushen Huoxue Recipes, 7mL of high-dose Bushen Huoxue Recipe solution is now prepared. The specific operation is to accurately weigh 5.642g of Bushen Huoxue Recipe freeze-dried powder and place it in an EP tube, add an appropriate amount of pure water to dissolve, and then adjust the volume to 7mL. Invert upside down 5 times, ultrasonically heat at 60℃ until the freeze-dried powder is completely dissolved (about 10min), and prepare it for immediate use.

[0254] Medium-dose Bushen Huoxue Recipe: Based on the average weight of 30g mice, 3.0mL of solution is required for 10 animals. Prepare 6mL of medium-dose Bushen Huoxue Recipe solution. The specific operation is to precisely pipette 3.0mL of high-dose Bushen Huoxue Recipe solution into an EP tube, add 3.0mL of pure water, vortex for 30s, mix thoroughly, and prepare for immediate use.

[0255] Low-dose Bushen Huoxue Recipe: Based on the average weight of 30g mice, 3.0mL of solution is required for 10 animals. Prepare 4mL of low-dose Bushen Huoxue Recipe solution. The specific operation is to precisely pipette 2.0mL of medium-dose Bushen Huoxue Recipe into an EP tube, add 2.0mL of pure water, vortex for 30s, mix thoroughly, and prepare and use immediately.

[0256] Dydrogesterone solution: The dosage of mice in the dydrogesterone group was 3.00 mg / kg / d, and the drug solution concentration required was 0.300 mg / mL. Now prepare 4.0 mL of dydrogesterone solution, which requires 1.2 mg of dydrogesterone. Each tablet contains 10 mg of dydrogesterone, and the average weight of dydrogesterone tablets is 0.1426 g. The specific operation is to accurately weigh 17.11 mg of dydrogesterone tablet powder (i.e. 1.2 mg of dydrogesterone), place it in an EP tube, add an appropriate amount of pure water to dissolve, and dilute to 4 mL. Invert upside down 5 times, ultrasonicate for 5 minutes, fully dissolve, and prepare and use it now.

[0257] 1.2.4 Index detection

[0258] Mice were anesthetized with 5% isoflurane on G13.5 days. After anesthesia, blood was collected from the abdominal aorta, and the blood was stored in a centrifuge tube without sodium citrate to obtain serum. After the blood was collected, the uterus was quickly dissected, the uterus was separated, the appearance was recorded by photographing, and the weight of the uterus with the fetus was weighed. Then, the uterus was cut open along the longitudinal axis at the uterine frenulum, the total number of embryos and the number of aborted embryos were counted, and the embryo loss rate was calculated: embryo loss rate = number of aborted embryos / total number of embryos × 100%. The levels of CG, E2, P, and DD in serum were detected using a kit, and pathological changes (placenta) were observed by histopathological HE staining.

[0259] 1.3 Statistics

[0260] Same as Example 3.

[0261] 2. Results

[0262] 2.1 Embryo loss rate

[0263] The results are shown in the table below. Different doses of the Bushen Huoxue recipe significantly reduced the embryonic loss rate of mice.

[0264] Table 6 Embryo loss rate of mice in each group ( n=10)

[0265]

[0266] Note: Compared with the normal control group, ** P<0.01; compared with the model control group, ## P<0.01

[0267] The results showed that compared with the normal control group, the embryo loss rate in the model control group was significantly increased (P<0.01); compared with the model control group, the embryo loss rate in the different doses of Bushen Huoxue prescription and dydrogesterone groups was significantly decreased (P<0.01).

[0268] 2.2 Mouse uterus and placenta tissue morphology

[0269] The results are as follows Figure 3-4 As shown, Figure 3 The pictures of the uterus of mice in each group are shown below. Figure 4 The pictures of the placenta and HE-stained tissue sections of the placenta in each group of mice are shown in Figure 1. The results show that the Bushen Huoxue recipe can significantly improve the morphology of the uterus and placenta tissues in each group of mice.

[0270] from Figure 3 It can be seen that the embryos in the normal group were light pink, thick and uniform, like beads; the model group had dark brown absorbed embryos, with visible blood stasis, uneven sizes, and some or even completely disappeared embryos; the embryos in the Bushen Huoxue Recipe group and the Dydrogesterone group were plump and light pink.

[0271] Figure 4 The above picture is a photo taken under a stereomicroscope (the red arrow indicates the site of significant bleeding). It was found that compared with the maternal edge of the placenta in the normal group, bleeding occurred in the model group; the bleeding at the maternal edge of the placenta in the low, medium, and high doses of the Bushen Huoxue prescription groups was reduced or disappeared; the bleeding at the maternal edge of the placenta in the dydrogesterone group was not significantly reduced.

[0272] Figure 4 The following figure is a HE-stained section. The findings of the pathological tissue structure observation corroborate the above results. The spongiotro-phoblast (SP) and labyrinthine layer (Lab) structures of the placenta of mice in the normal group were well developed, with clear boundaries between the structures and full vascular structures. The SP and Lab layers of the model group were poorly developed, with unclear boundaries between the structures, atrophic or even disappeared vascular structures, and large-scale bleeding in the SP layer. Compared with the model group, the SP and Lab layers of the placenta of mice in the dydrogesterone group tended to develop well, the boundaries became clearer, and the vascular structure tended to be full, but bleeding still occurred; the SP and Lab layers of the placenta of mice in the low, medium, and high doses of the Bushen Huoxue prescription groups tended to develop well, the boundaries became clearer, the vascular structure tended to be full, and the bleeding of the SP layer of the placenta was reduced or disappeared.

[0273] 2.3 Serum hormone levels and coagulation factor contents in mice

[0274] The results are shown in the table below. The Bushen Huoxue recipe can regulate the serum hormone levels and coagulation-related factor contents of mice.

[0275] Table 7 Serum hormone levels and coagulation-related factor contents of mice in each group ( n=10)

[0276]

[0277] Note: Compared with the normal control group, * P<0.05, ** P<0.01; compared with the model control group, # P<0.05, ## P<0.01

[0278] Compared with the normal group, the levels of CG, P and FIB in the model control group were decreased (P<0.05), and the level of DD was increased (P<0.01). Compared with the model group, the serum CG level was increased in the low, medium and high doses of Bushen Huoxue Fang groups (P<0.05), the P level was increased (P<0.01), and the FIB level was increased (P<0.01). The serum DD level was decreased in the medium dose of Bushen Huoxue Fang group (P<0.05), and decreased in the high dose of Bushen Huoxue Fang group (P<0.01). The serum CG level was increased (P<0.05), the P level was increased (P<0.01), the FIB level was increased (P<0.01), and the DD level was decreased (P<0.01) in the positive drug dydrogesterone group.

[0279] 2.4 Collagen deposition and Fib protein expression in the decidual region of mouse placenta

[0280] Figure 5 (Magnification, 40×) shows the collagen deposition in the decidual region of the mouse placenta. Masson staining will dye collagen blue, which can be used to determine the degree of tissue fibrosis. Collagen can maintain the structural integrity of tissues and organs, and plays a key role in the development of the placenta and the repair of tissues. Excessive deposition can lead to fibrosis, which in turn leads to abnormal placental development.

[0281] The results showed that there was almost no collagen deposition in the normal group; compared with the normal pregnancy group, obvious collagen deposition was observed in the model group, and the relative area of ​​collagen increased significantly (P < 0.01), indicating the occurrence of placental fibrosis. Compared with the model group, although there was no significant change in the placental collagen deposition in the positive drug dydrogesterone group, the placental collagen deposition in the mice in the different doses of the Bushen Huoxue prescription group was significantly reduced, and the relative area of ​​collagen was significantly reduced (P < 0.01). The results suggest that different doses of the Bushen Huoxue prescription can improve the occurrence of placental fibrosis and improve embryonic development.

[0282] Figure 6(Magnification, 40×) is the expression result of Fib protein in the decidual region of the mouse placenta. The results of immunohistochemistry confirm the results of collagen. Immunohistochemistry will dye the positive expression of Fib protein brown. Fib deposition is one of the pathological characteristics of aborted mice, which may cause thrombosis, leading to placental vascular obstruction, affecting oxygen and nutrient supply. The results showed that compared with the normal group, the expression of Fib protein in the model group was significantly increased (P < 0.01); compared with the model group, the expression of Fib protein in different doses of Bushen Huoxue prescription groups was significantly reduced (P < 0.01), indicating that different doses of Bushen Huoxue prescription groups can inhibit placental Fib deposition and promote the normal development of the placenta.

[0283] Table 8 Relative area of ​​collagen expression and Fib protein expression in each group of mice ( n=10)

[0284]

[0285] Note: All data were normalized to the normal control group. ** P<0.01; compared with the model control group, ## P<0.01; compared with the dydrogesterone group, aa P<0.01

[0286] Example 7

[0287] This example evaluates the clinical efficacy of the Bushen Huoxue recipe in the treatment of early and mid-term pregnancy complicated with intrauterine hemorrhage.

[0288] Patients with early and mid-term pregnancy complicated with intrauterine hemorrhage were selected as the research subjects. A self-controlled before-after study was conducted to statistically analyze the changes in intrauterine hemorrhage area within 2 weeks and 4 weeks after conventional Western medicine treatment combined with Bushen Huoxue prescription treatment, and to evaluate the effectiveness of Bushen Huoxue prescription in treating early and mid-term pregnancy complicated with intrauterine hemorrhage. The ethics approval number of this experiment is K-2024-119.

[0289] 1. Research subjects and related standards

[0290] 1.1 Research subjects

[0291] Pregnant women with early or mid-term pregnancy complicated with intrauterine hemorrhage who visited the Department of Gynecology and Obstetrics (outpatient and inpatient) of the Women and Children's Center of the First Affiliated Hospital of Guangzhou University of Chinese Medicine from January 2020 to February 2023.

[0292] 1.2 Diagnostic criteria

[0293] 1.2.1 Early intrauterine pregnancy

[0294] The method is formulated with reference to the relevant description in the 9th edition of Obstetrics and Gynecology (People's Medical Publishing House, edited by Xie Xing and Kong Beihua). Early intrauterine pregnancy refers to the early discovery of an intrauterine gestational sac or embryo by ultrasound, the blood hCG level is basically consistent with the gestational age, and the ultrasound examination shows the pulsation of the primitive heart tube, indicating that the embryo is alive.

[0295] 1.2.2 Threatened abortion

[0296] The standard is formulated with reference to the 9th edition of Obstetrics and Gynecology. Threatened abortion refers to a small amount of vaginal bleeding before 28 weeks of intrauterine pregnancy, which is usually dark red or bloody leucorrhea, without discharge of pregnancy products, followed by paroxysmal lower abdominal pain or back pain. Gynecological examination shows that the cervix is ​​not open, the fetal membranes are not ruptured, and the size of the uterus is consistent with the number of weeks of amenorrhea.

[0297] 1.2.3 Hematometra

[0298] Refer to the "Ultrasound Atlas of Obstetrics and Gynecology". Intrauterine hemorrhage refers to the presence of dark liquid areas in the uterine cavity, between the gestational sac and the uterine wall, and between the placenta and the uterine wall under ultrasound, which are triangular, crescent or ring-shaped.

[0299] 1.3 Inclusion criteria

[0300] (1) Intrauterine pregnancy, with a single live fetus in the uterus and a gestational age of less than 28 weeks;

[0301] (2) Meet the diagnostic criteria for threatened abortion and intrauterine hemorrhage;

[0302] (3) Taking Bushen Huoxue prescription or its modified prescriptions after intrauterine hemorrhage was found;

[0303] (4) The research data is complete.

[0304] 1.4 Exclusion criteria

[0305] (1) Intrauterine pregnancy combined with extrauterine pregnancy;

[0306] (2) Pregnancy complicated with placental abruption;

[0307] (3) Those with vaginal bleeding caused by cervical polyps, cervical inflammation, etc.

[0308] Anyone who meets any of the above conditions can be excluded.

[0309] 2. Research plan

[0310] 2.1 Research Type and Data Collection Method

[0311] This study is a self-controlled before-after study. By reviewing the outpatient and inpatient medical records of the patients, recording the use of Chinese medicine compound and Western medicine during the diagnosis and treatment process and ultrasound information, the changes in intrauterine blood accumulation within 2 weeks and 4 weeks were observed. This study only included patients who took the Bushen Huoxue Recipe or its modified recipe (the taste of the medicine remained unchanged, and the dosage was adjusted in a small range), and the Western medicine treatment plan was not limited. The Bushen Huoxue Recipe was composed of 12g of salt dodder seed, 10g of Dipsacus asper, 15g of Salvia miltiorrhiza, 15g of Millettia reticulata, 20g of Astragalus, 5g of dried tangerine peel, and 15g of Chinese yam.

[0312] 2.2 Measurement of blood accumulation area: The longest diameter of the intrauterine blood accumulation under ultrasound and the widest diameter perpendicular to it are taken as the length and width respectively, and the blood accumulation area = length × width.

[0313] 2.3 Ultrasound information recording time: The time interval between the last ultrasound and the last ultrasound before taking the Bushen Huoxue prescription was 2 weeks (1 to 14 days) and 4 weeks (15 to 24 days).

[0314] 2.4 Efficacy indicators: The changes in the area of ​​hemorrhage at 2 weeks and 4 weeks were used as the main efficacy indicators.

[0315] 3. Statistical methods

[0316] The hemorrhage area before the visit, within 2 weeks of medication, and within 4 weeks of medication was entered into Excel software for sorting, and statistical analysis was performed using SPSS25.0 statistical software package. Paired sample t test or paired sample Wilcoxon sign test was used according to whether the hemorrhage area before and after the intervention conformed to the normal distribution. P < 0.05 was considered statistically significant.

[0317] 4. Research Results

[0318] This study collected information on 111 patients with early and mid-term pregnancy complicated by intrauterine hematoma, of whom 31 had no follow-up ultrasound examination after consultation, 82 had ultrasound results within 2 weeks, and 66 had ultrasound results within 2 weeks and 4 weeks.

[0319] 4.12 weeks intrauterine blood accumulation area

[0320] The results are shown in the following table.

[0321] Table 9 Changes in the area of ​​intrauterine blood accumulation after 2 weeks of medication (P25~P75)

[0322]

[0323] Note: The normality test results of the hemorrhage area before treatment and 2 weeks after administration were both P < 0.001, which did not meet the normal distribution. The two-paired sample Wilcoxon signed rank test was used, P < 0.001, and the difference was statistically significant.

[0324] The results showed that the combined use of the Bushen Huoxue recipe for 2 weeks could reduce the area of ​​blood accumulation in the uterine cavity.

[0325] 4.2 Intrauterine blood accumulation area within 4 weeks

[0326] The results are shown in the following table.

[0327] Table 10 Changes in the area of ​​intrauterine blood accumulation after 4 weeks of medication (P25~P75)

[0328]

[0329] Note: The normality test results of the hemorrhage area before treatment and after 4 weeks of treatment were both P < 0.001, which did not meet the normal distribution. The two-paired sample Wilcoxon signed rank test was used, P < 0.001, and the difference was statistically significant

[0330] The results showed that the combined use of the Bushen Huoxue recipe for 4 weeks could reduce the area of ​​blood accumulation in the uterine cavity.

[0331] 4.3 Comparison of the difference in intrauterine blood area

[0332] The results are shown in the following table.

[0333] Table 11 Comparison of the difference in intrauterine blood area between 2 weeks and 4 weeks M (P25~P75)

[0334]

[0335] Note: The normality test results of the 2-week difference and the 4-week difference were both P < 0.001, which did not meet the normal distribution. The two-paired sample Wilcoxon signed rank test was used, and P < 0.001, indicating a statistically significant difference

[0336] It can be seen that after the combined treatment with Bushen Huoxue prescription, the difference in the change of blood accumulation area between 2 weeks and 4 weeks was statistically significant (Z=-4.381, P<0.001), indicating that the longer the combined treatment time, the greater the reduction in the area of ​​uterine intrauterine blood accumulation.

[0337] Example 8

[0338] In this example, the efficacy of the drug composition (Bu Shen Huo Xue Fang 1) prepared in Example 1 in treating CBA / J×DBA / 2 aborted mice before pregnancy was studied.

[0339] Based on the CBA / J×DBA / 2 recurrent miscarriage mouse model, the effects of different pre-pregnancy administration of Bushen Huoxue prescription (pre-pregnancy administration, pre-pregnancy administration until G7.5 days, and pre-pregnancy administration until G13.5 days) on the pregnancy rate, embryonic loss rate, and uterine tissue morphology of the model were investigated.

[0340] 1. Materials and Methods

[0341] 1.1 Materials

[0342] 1.1.1 Experimental System

[0343] Twenty 8-week-old SPF-grade male DBA / 2 mice and forty 8-week-old SPF-grade female CBA / J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK(Jing)2021-0011). After purchase, the animals were placed in the SPF-grade animal laboratory of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (License No.: SYXK(Yue)2023-0092) and housed separately in cages. All experimental operations complied with the relevant regulations on animal welfare.

[0344] 1.1.2 Test Substances

[0345] The freeze-dried powder of the traditional Chinese medicine composition Bushen Huoxue Fang 1 prepared in Example 1, hereinafter simply referred to as Bushen Huoxue Fang.

[0346] 1.1.3 Experimental Reagents and Instruments

[0347] Same as Example 3.

[0348] 1.2 Methods

[0349] 1.2.1 Animal Modeling and Grouping

[0350] The modeling method was the same as that in Example 3. Forty mature unmated female CBA / J mice in the proestrus stage were screened by vaginal smear and randomly divided into the following 4 groups: model control group, pre-pregnancy administration group, pre-pregnancy administration until G7.5 group, and pre-pregnancy administration until G13.5 group, with 10 mice in each group.

[0351] 1.2.2 Administration Scheme

[0352] Based on the recommended dose for humans (calculated based on a body weight of 60 kg), the conversion coefficient between adults and mice is 9.01. The equivalent dose conversion formula is: mouse dose = (human recommended dose / 60 kg) × 9.01, and gavage administration was carried out.

[0353] Pure water was gavaged at a dose of 10 mL / kg; the dose of the Bushen Huoxue Fang solution was the human dose (92 g of crude drug / human / d). After conversion, the crude drug administration dose for mice was 13.82 g of crude drug / kg / d and was gavaged at a dose of 10 mL / kg.

[0354] Model control group: Gavage with pure water for 1 week, once a day. After successful co-habitation (G0.5 day), pure water was continuously administered until day 13.5.

[0355] Pre-pregnancy administration group: Gavage with Bushen Huoxue Fang for at least 7 days before co-habitation, once a day. After successful co-habitation (G0.5 day), pure water was continuously administered until G13.5, once a day.

[0356] The group that received medication before pregnancy until G7.5 days: Administered Bushen Huoxue recipe by gavage once a day for at least 7 days before cage integration. After successful cage integration (G0.5 days), began to administer Bushen Huoxue recipe continuously until G7.5 days. From G8.5 days, pure water was continuously administered until G13.5 days.

[0357] The group that received medication before pregnancy until G13.5 days: The Bu Shen Huo Xue recipe was given by gavage once a day for at least 7 days before cage integration. After successful cage integration (G0.5 days), the Bu Shen Huo Xue recipe was given continuously until G13.5 days.

[0358] The specific drug administration flow chart of each group is shown in Figure 7 . After each group was given the corresponding drug solution for 7 days, female mice in estrus were screened by vaginal smears and caged at a female:male ratio of 2:1. Vaginal plugs were observed at 8:00 the next day after caged. Those with vaginal plugs or sperm in vaginal smears were considered pregnant and recorded as G0.5 days. Female mice that were successfully caged and those that were not successfully caged were raised separately in each group. Female mice that were successfully caged were no longer caged; female mice that were not successfully caged continued to be caged and given the corresponding test drug solution. If caged at the end of two estrus cycles, it was considered a failure. All mice in this experiment were successfully caged to form the experimental group.

[0359] 1.2.3 Preparation of test drug solution

[0360] Same as Example 3.

[0361] 1.2.4 Index detection

[0362] Mice were anesthetized with 5% isoflurane on G13.5 days. After anesthesia, blood was collected from the abdominal aorta and stored in a centrifuge tube without sodium citrate to obtain serum. After blood collection, the uterus was quickly dissected, the uterus was removed, the appearance was recorded by photographing and the weight of the uterus with the fetus was weighed. Then, the uterus was opened along the longitudinal axis at the uterine frenulum, the total number of embryos and the number of aborted embryos were counted, and the pregnancy rate and embryo loss rate were calculated:

[0363] Pregnancy rate = number of pregnant mice in the group / total number of mice in the group × 100%;

[0364] Embryo loss rate = number of aborted embryos / total number of embryos × 100%.

[0365] 1.3 Statistics

[0366] Same as Example 3.

[0367] 2. Results

[0368] 2.1 Pregnancy rate

[0369] The results are shown in the table below. Different methods of administering the Bushen Huoxue recipe before pregnancy all increased the pregnancy rate of mice.

[0370] Table 12 Pregnancy rate of mice in each group

[0371]

[0372] 2.2 Embryo loss rate

[0373] The results are shown in the table below. Different methods of administering the Bushen Huoxue recipe before pregnancy significantly reduced the embryonic loss rate of mice (P<0.01).

[0374] Table 13 Pregnancy rate and embryo loss rate of mice in each group ( n=10)

[0375]

[0376] Note: Compared with the model control group, ## P<0.01

[0377] 2.3 Uterine (containing embryo) morphology

[0378] The histological morphology of the uterus (including embryos) of mice given Bushen Huoxue prescription in different ways before pregnancy Figure 8 shown.

[0379] In the model group, dark brown absorbed embryos appeared, with visible congestion, uneven sizes, and some or even complete disappearance of embryos. In the pre-pregnancy medication groups, uterine congestion was significantly reduced, the total number of embryos increased, most of the embryos were plump and uniform, light red, and dark brown absorbed embryos were occasionally seen.

[0380] The above results show that giving the Bushen Huoxue recipe before pregnancy can promote pregnancy and increase the pregnancy rate, and giving the Bushen Huoxue recipe after pregnancy can reduce the embryo loss rate. The Bushen Huoxue recipe can not only help pregnancy before pregnancy, but also promote pregnancy after pregnancy.

Claims

1. A Chinese medicine composition, characterized in that: The active ingredients are mainly composed of the following medicinal materials in parts by weight: 3-35 parts of Cuscuta seeds, 3-35 parts of kidney-tonifying drugs, 5-35 parts of Salvia miltiorrhiza, 3-35 parts of blood-activating and blood-stasis-removing drugs, 5-60 parts of Astragalus, 1-20 parts of dried orange peel, and 6-35 parts of Chinese yam; The kidney-tonifying medicine is Dipsacus asper or Morinda officinalis, and the blood-activating and stasis-removing medicine is Millettia reticulata or Panax notoginseng.

2. The Chinese medicine composition according to claim 1, characterized in that Its active ingredients are mainly composed of the following medicinal materials in parts by weight: 6-12 parts of Cuscuta australis, 9-15 parts of Dipsacus asper, 10-15 parts of Salvia miltiorrhiza, 9-15 parts of Millettia reticulata, 9-30 parts of Astragalus, 3-10 parts of Tangerine peel, 15-30 parts of Dioscorea opposita; Or, 6-12 parts of Cuscuta seeds, 3-10 parts of Morinda officinalis, 10-15 parts of Salvia miltiorrhiza, 9-15 parts of Millettia reticulata, 9-30 parts of Astragalus, 3-10 parts of Citrus reticulata, and 15-30 parts of Dioscorea opposita; Or, 6-12 parts of Cuscuta australis, 9-15 parts of Dipsacus asper, 10-15 parts of Salvia miltiorrhiza, 3-9 parts of Panax notoginseng, 9-30 parts of Astragalus, 3-10 parts of Citrus reticulata, and 15-30 parts of Dioscorea opposita; Alternatively, 6-12 parts of Cuscuta seeds, 3-10 parts of Morinda officinalis, 10-15 parts of Salvia miltiorrhiza, 3-9 parts of Panax notoginseng, 9-30 parts of Astragalus, 3-10 parts of dried tangerine peel, and 15-30 parts of yam.

3. The Chinese medicine composition according to claim 1, characterized in that: It is mainly composed of the following medicinal materials in parts by weight: 12 parts of Cuscuta seeds, 10 parts of Dipsacus asper, 15 parts of Salvia miltiorrhiza, 15 parts of Millettia reticulata, 20 parts of Astragalus, 5 parts of Citrus reticulata, 15 parts of Dioscorea opposita; Or, 12 parts of Cuscuta seeds, 9 parts of Morinda officinalis, 15 parts of Salvia miltiorrhiza, 15 parts of Millettia reticulata, 20 parts of Astragalus, 5 parts of Citrus reticulata, and 15 parts of Dioscorea opposita; Or, 12 parts of Cuscuta chinensis, 10 parts of Dipsacus asper, 15 parts of Salvia miltiorrhiza, 9 parts of Panax notoginseng, 20 parts of Astragalus, 5 parts of Citrus reticulatae, and 15 parts of Dioscorea oppositae; Or, 12 parts of Cuscuta australis, 9 parts of Morinda officinalis, 15 parts of Salvia miltiorrhiza, 9 parts of Panax notoginseng, 20 parts of Astragalus, 5 parts of Citrus reticulatae, and 15 parts of Dioscorea oppositae.

4. The Chinese medicine composition according to any one of claims 1 to 3, characterized in that: The dodder seed is salt dodder seed, and the Morinda officinalis is salt Morinda officinalis.

5. The method for preparing the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: extracting the medicinal material by a solvent extraction method to obtain the traditional Chinese medicine composition.

6. The method for preparing the Chinese medicine composition according to claim 5, characterized in that: The solvent extraction method includes: decoction, reflux extraction, maceration or percolation; and / or, the solvent is selected from: water, ethanol aqueous solution.

7. The method for preparing the Chinese medicine composition according to claim 5, characterized in that: The following steps are involved: Weigh the prescribed amount of medicinal materials, add solvent according to the predetermined material-liquid ratio, heat and extract, filter, and recover the solvent in the filtrate to obtain; Alternatively, the medicinal material of the prescription amount is weighed, a solvent is added according to a predetermined material-liquid ratio, the mixture is heated for extraction, filtered, ethanol is added after the filtrate is concentrated, the mixture is allowed to stand, the precipitate is removed, and the solvent in the filtrate is recovered to obtain the product; Furthermore, the material-liquid ratio is 1g medicinal material: 10±5ml solvent; And / or, the number of extractions is 1-3 times, for example, 2 times, the solid-liquid ratio used in the first extraction is 1:10±3, and the solid-liquid ratio used in the second extraction is 1:8±3; And / or, the extraction time is 0.5-3 hours, for example 1-2 hours; And / or, the extraction temperature is 80-100° C., for example, 90-100° C.; and / or, the filtrate is concentrated to a relative density of 1.10-1.30 and then ethanol is added, for example, 1.15; and / or, after concentrating the filtrate, adding ethanol until the volume percentage of ethanol is 60-75%, for example 65%; And / or, the filtrate is concentrated and then the ethanol is added and then allowed to stand for 8-24 hours, for example, 12 hours.

8. The method for preparing the Chinese medicine composition according to claim 7, characterized in that: The method further comprises the step of preparing the Chinese medicine composition into freeze-dried powder or granules. The steps of preparing the Chinese medicine composition into freeze-dried powder are: taking the filtrate, concentrating it to obtain an extract, and drying the extract by freeze-drying to obtain freeze-dried powder; The steps of preparing the Chinese medicine composition into granules are: taking the filtrate, concentrating it to a clear paste with a relative density of 1.05-1.10, drying it, adding a filler, and wet granulating it to obtain granules; further, the filler is maltodextrin, and the relative density is measured at 50-70°C, for example, at 60°C.

9. A drug, characterized in that The Chinese medicine composition comprises the Chinese medicine composition according to any one of claims 1 to 4, and pharmaceutically acceptable excipients. Further, the Chinese medicine composition is administered orally. Furthermore, the Chinese medicine composition is in the form of capsules, granules, tablets, oral liquids, mixtures or syrups.

10. Use of the Chinese medicine composition according to any one of claims 1 to 4 and the medicine according to claim 9 in the preparation of a medicine for tonifying the kidney and activating blood circulation, further, the medicine for tonifying the kidney and activating blood circulation is used to prevent and / or treat recurrent pregnancy loss syndrome, for example, the medicine for tonifying the kidney and activating blood circulation is used to assist pregnancy before pregnancy and / or to maintain pregnancy after pregnancy, and is particularly suitable for recurrent pregnancy loss combined with prethrombotic state and intrauterine hemorrhage.

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