A traditional Chinese medicine composition, a preparation method therefor and application thereof

Abstract

The application discloses a traditional Chinese medicine composition and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine. The traditional Chinese medicine composition is mainly composed of the following medicinal materials in parts by weight: 3-35 parts of Semen Euryae, 3-35 parts of kidney-tonifying medicinal materials, 5-35 parts of Salvia miltiorrhiza, 3-35 parts of blood-activating and stasis-removing medicinal materials, 5-60 parts of Astragalus membranaceus, 1-20 parts of Pericarpium Citri Reticulatae and 6-35 parts of Dioscorea opposita. The kidney-tonifying medicinal materials are Dipsacus asper or Morinda officinalis, and the blood-activating and stasis-removing medicinal materials are Spatholobus suberectus or Panax notoginseng. The traditional Chinese medicine composition has the functions of tonifying kidney and activating blood, is aimed at recurrent pregnancy loss (RPL) of the type of kidney deficiency and blood stasis, has the bidirectional regulating function of anticoagulation and hemostasis, and can be used for preventing RPL combined with prethrombotic state (PTS) and metrorrhagia during pregnancy before pregnancy, and treating RPL combined with PTS and metrorrhagia during pregnancy after pregnancy, and has a great application prospect.

Description

TECHNICAL FIELD

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

[0002] Recurrent pregnancy loss (RPL) refers to the loss of embryo before 28 weeks of pregnancy for 2 times or more, including biochemical pregnancy. RPL is a common and difficult disease in women of childbearing age, and the incidence rate is as high as 1-5%. With the increase of the number of abortions and age, the probability of abortion of women will also increase.

[0003] Prethrombotic state (PTS) is one of the important causes of PRL, and accounts for about 20% in maternal causes. PTS refers to a high blood coagulation state or a high thromboembolic tendency caused by coagulation, anticoagulation dysfunction or disorder, etc. This state can cause local microthrombosis of the placenta, and further cause microvascular obstruction of the placenta, insufficient blood and oxygen supply, abnormal angiogenesis, impaired blood perfusion, and ultimately cause poor embryo development and stillbirth and other adverse pregnancy outcomes. Based on the pathological mechanism of PTS, antithrombotic therapy is considered to be an effective treatment for RPL combined with PTS. Among them, the anticoagulation and platelet inhibition therapy of low molecular weight heparin or aspirin is the main treatment scheme, but there are many adverse reactions such as bleeding and liver damage.

[0004] Common adverse reactions are: (1) bleeding: bleeding is the most common adverse reaction of anti-platelet and anticoagulant therapy, which can cause disability and even endanger the life of the patient. Common bleeding includes subcutaneous ecchymosis, nosebleed, oral mucosa bleeding, gum bleeding, ocular conjunctival bleeding, gastrointestinal bleeding, bladder bleeding, vaginal bleeding, etc. Therefore, the medical history should be inquired in detail before antithrombotic therapy, and the risk of bleeding should be evaluated. (2) Liver damage: liver damage is a common adverse reaction of low molecular weight heparin, with an incidence of 4% to 13%. Therefore, liver function should be monitored regularly during the use of low molecular weight heparin. Once liver damage is found, viral hepatitis should be ruled out. (3) Allergic reaction: since low molecular weight heparin belongs to animal-derived multi-component mixture, allergy is a common adverse reaction of low molecular weight heparin, and common symptoms include skin itching, skin rash, urticaria, local redness, etc. A study on the incidence of itching after using low molecular weight heparin during pregnancy showed that the incidence of heparin was 6.6%, enoxaparin was 9.7%, and nartograstim was 30.9%; the incidence of skin rash showed that the incidence of heparin was 4.4%, enoxaparin was 0%, and nartograstim was 4.6%; the incidence of local redness showed that the incidence of heparin was 2.2%, enoxaparin was 1.8%, and nartograstim was 13.8%; the incidence of induration showed that the incidence of heparin was 13.3%, enoxaparin was 20.3%, and nartograstim was 42.1%. Rare adverse reactions include acute laryngeal edema, anaphylactic shock, etc. Moreover, there have been reports of patients' allergy and even death caused by heparin.

[0005] Meanwhile, over-treatment and non-standard use of low molecular weight heparin or aspirin are common in clinical practice, and the adverse reactions and other problems caused by the treatment come one after another, especially when PTS is combined with pregnancy and intrauterine hemorrhage, the treatment is the most difficult, and there is a contradiction between anticoagulation and hemostasis.

[0006] Traditional Chinese medicine treatment for preventing miscarriage has a long history, and its safety and effectiveness have been tested and proved by long-term clinical practice. At present, there are some Chinese medicine preparations for treating RPL on the market, but their therapeutic effect is not stable, and they do not have good effect on a part of patients. SUMMARY

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

[0008] In one aspect, the present application discloses a traditional Chinese medicine composition, wherein the active ingredients are mainly composed of the following weight parts of medicinal materials:

[0009] Cuscuta 3-35 parts, kidney tonic 3-35 parts, Salvia miltiorrhiza 5-35 parts, blood-activating and stasis-removing medicine 3-35 parts, Astragalus 5-60 parts, dried tangerine or orange peel 1-20 parts, Chinese yam 6-35 parts;

[0010] The kidney tonic is Dipsacus or Morinda officinalis, and the blood-activating and stasis-removing medicine is Spatholobus suberectus or Panax notoginseng.

[0011] The inventors found in the investigation that intrauterine blood accumulation is a common ultrasonic manifestation in early and middle pregnancy, and part of it can last to late pregnancy, with an incidence of 0.46%-39.5%. Intrauterine blood accumulation can be related to adverse pregnancy outcomes such as spontaneous abortion and placental abruption. The proportion of PTS in patients with intrauterine blood accumulation is significantly increased, suggesting that PTS can be one of the causes of intrauterine blood accumulation. At present, the use of anticoagulant drugs to prevent PTS will increase the incidence of subplacental hematoma (a type of intrauterine blood accumulation) during pregnancy. In addition, there are also pregnant women with heart valve thrombosis treated with thrombolytic drugs who have developed massive subplacental hematoma during pregnancy. However, anticoagulant and antiplatelet therapy can increase the incidence of intrauterine blood accumulation during pregnancy. Therefore, there is a contradiction in the treatment of RPL combined with PTS by Western medicine.

[0012] However, in traditional Chinese medicine, RPL belongs to the category of "sliding abortion" and "miscarriage", and its main mechanism is damage to the Chong and Ren channels, unstable fetal essence, and repeated pregnancy and miscarriage. The common syndromes of RPL in clinical practice 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" in traditional Chinese medicine. The root pathogenesis is "kidney deficiency", and the important pathogenesis is "blood stasis". Blood does not flow due to blood stasis, blood stasis blocks the blood, blood does not follow the meridians, and then the blood spills outside the meridians, and the blood outside the meridians is blood stasis, which further aggravates bleeding and forms an adverse cycle. Treatment should be combined with disease and syndrome, and the damage should be prevented, bleeding should be stopped first, blood stasis should be removed, and blood stasis and fetal protection should be promoted simultaneously. Therefore, the inventors consider that the method of tonifying the kidney and activating the blood can be one of the methods to break through the bottleneck of anticoagulation and hemostasis in Western medicine treatment.

[0013] The inventors found in the investigation that most of the traditional Chinese medicine compositions currently on the market for treating RPL are aimed at patients with "spleen and kidney deficiency", "kidney qi deficiency", and "kidney deficiency and blood heat" syndromes, and there is a lack of drugs for treating RPL with "kidney deficiency and blood stasis" syndrome, and there is a lack of drugs with bidirectional regulation of thrombosis and bleeding. This can be one of the reasons for the poor therapeutic effect of traditional Chinese medicine preparations for treating RPL.

[0014] On the basis of the investigation, the inventors believe that the disease is fundamentally caused by kidney deficiency and blood stasis. Therefore, the treatment should be combined with disease and syndrome, and the damage should be prevented. First, blood stasis should be stopped, and then blood stasis should be removed. In addition, both blood stasis and fetal security should be considered. Based on this, the inventors designed the prescription with the idea of tonifying kidney and activating blood. First, they considered using Cuscuta, Dipsacus (or Radix Morindae Officinalis) to tonify kidney and secure the uterus, and using Salvia miltiorrhiza, Spatholobus suberectus (or Radix Notoginseng) to activate blood and remove blood stasis. However, it was found that the nourishment of postnatal QI and blood can better play the role of promoting pregnancy and securing the fetus, so the inventors added Astragalus and Dioscorea to tonify Qi and invigorate the spleen. In addition, the inventors found that for blood-activating and blood-stasis-removing drugs, there are certain safety problems with Chuanxiong and safflower, so they chose Salvia miltiorrhiza and Spatholobus suberectus (or Radix Notoginseng). Compared with similar drugs, the latter is safer while having the effect of nourishing blood and activating blood. Similarly, for the selection of drugs, the inventors also considered licorice, but practice has proved that Pericarpium Citri Reticulatae can regulate Qi and stomach, and pregnant women have better effects.

[0015] Finally, after repeated groping and trying, the inventors proposed a kidney-tonifying and blood-activating prescription, which comprises Cuscuta, Dipsacus or Radix Morindae Officinalis, Salvia miltiorrhiza, Spatholobus suberectus or Radix Notoginseng, Astragalus, Pericarpium Citri Reticulatae, and Dioscorea. Among them, Cuscuta and Astragalus are the monarch drugs, Cuscuta tonifies kidney yin and kidney yang, Astragalus tonifies Qi and invigorates the spleen, and nourishes blood and secures the uterus. The two drugs together tonify Qi and blood, and balance water and earth. Dipsacus (or Radix Morindae Officinalis), Spatholobus suberectus (or Radix Notoginseng), and Salvia miltiorrhiza are the ministerial drugs, Dipsacus or Radix Morindae Officinalis tonifies liver and kidney and strengthens tendons and bones; Spatholobus suberectus or Radix Notoginseng nourishes blood and removes blood stasis; and Salvia miltiorrhiza activates blood and removes blood stasis, cools blood and regulates the uterus, and tonifies kidney and removes blood stasis. Dioscorea is the auxiliary drug, which tonifies the spleen and kidney, benefits the stomach and generates saliva, and cooperates with the monarch drug to enhance the effect of tonifying kidney and invigorating the spleen; and Pericarpium Citri Reticulatae is the ministerial drug, which regulates Qi and stomach and harmonizes various drugs.

[0016] Specifically, Cuscuta, Dipsacus (or Radix Morindae Officinalis), etc. can tonify kidney yang and benefit kidney essence, so that kidney Qi is strong, the uterus is consolidated, and the fetus is tied; Salvia miltiorrhiza, Spatholobus suberectus (or Radix Notoginseng) can disperse blood stasis, so that blood stasis is removed, new blood is generated, Qi and blood are abundant, blood vessels are unobstructed, and the fetus is nourished; Astragalus and Dioscorea are added to tonify Qi and invigorate the spleen; and Pericarpium Citri Reticulatae moderates the drug properties and harmonizes various drugs.

[0017] Further, the inventors also conducted animal research and clinical research on the above-mentioned traditional Chinese medicine composition.

[0018] In animal studies, the present application first selected recurrent miscarriage mice (CBA / J x DBA / 2) as the experimental system to test the efficacy of different groups and different preparation methods of the traditional Chinese medicine composition (Shenjing Huoxue recipe) of the present application. The results showed that among the different Shenjing Huoxue recipes, different Shenjing Huoxue recipes 1-7 all had the effect of reducing the embryo loss rate and improving the morphology of the uterine embryo, but Shenjing Huoxue recipe 1 (corresponding to the formula: 12 parts of Semen Eurycoma, 10 parts of Radix Sophorae, 15 parts of Radix Salviae Miltiorrhizae, 15 parts of Caulis Spatholobi, 20 parts of Radix Astragali, 5 parts of Pericarpium Citri Reticulatae, and 15 parts of Rhizoma Dioscoreae) had the most advantages in reducing the embryo loss rate and improving the morphology of the uterine embryo and was relatively low in cost. Therefore, Shenjing Huoxue recipe 1 was selected for further investigation.

[0019] Then, KM mice were selected as the experimental system for coagulation time detection. The coagulation time of each dose of Shenjing Huoxue recipe 1 was significantly prolonged, suggesting that the traditional Chinese medicine composition (Shenjing Huoxue recipe 1) of the present application has an anticoagulant effect.

[0020] Next, the recurrent miscarriage mice (CBA / J x DBA / 2) were used as the experimental system to compare the efficacy of Shenjing Huoxue recipe 1 of the present application with different marketed antenatal Chinese medicines, to explore the dose-effect of Shenjing Huoxue recipe and the pregnancy-promoting effect of pre-pregnancy administration. The results showed that compared with each antenatal Chinese medicine and dydrogesterone, the medium dose of Shenjing Huoxue recipe 1 (i.e. referring to the clinical human dose) had comprehensive advantages in reducing the embryo loss rate of recurrent miscarriage mice, increasing the uterine wet weight and average fetal weight, and reducing placental maternal surface bleeding. At the same time, Shenjing Huoxue recipe 1 of the present application significantly increased the levels of CG and P in the serum of pregnant mice, and significantly decreased the level of D-dimer in the serum of pregnant mice, suggesting that it can regulate sex hormone levels and improve the maternal hypercoagulable state; at the same time, the Shenjing Huoxue recipe 1 group can inhibit the occurrence of placental fibrosis and Fib deposition, suggesting that it can promote the normal development of the placenta. In addition, pre-pregnancy administration or continuous administration of Shenjing Huoxue recipe 1 of the present application before and after pregnancy significantly reduced the embryo loss rate of recurrent miscarriage mice and improved the morphology of the miscarriage mouse uterus containing fetuses.

[0021] In clinical research, the present application collected information of 111 cases of early and middle pregnancy with intrauterine hemorrhage patients from the gynecology and obstetrics of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (outpatient and inpatient departments), and used self-control study before and after treatment to statistically analyze the changes in the area of intrauterine hemorrhage after 2 weeks and 4 weeks of treatment with Shenjing Huoxue recipe 1 combined with conventional western medicine treatment. The results showed that the combination of Shenjing Huoxue recipe 1 reduced the area of intrauterine hemorrhage after 2 weeks and 4 weeks, and the longer the combined treatment time, the greater the reduction in the area of intrauterine hemorrhage. This result suggests that Shenjing Huoxue recipe 1 can effectively improve early and middle pregnancy with intrauterine hemorrhage.

[0022] In summary, the traditional Chinese medicine composition (the kidney-tonifying and blood-activating formula) of the present application reduces the embryo loss rate of RPL mice, improves the hypercoagulable state of pregnant mice, and reduces the hematometra area of patients with early and middle pregnancy combined with hematometra, indicating that the formula has the bidirectional regulation effect of anticoagulation and hemostasis. Therefore, the present application not only makes up for the lack of traditional Chinese medicine compositions for RPL patients of the "kidney deficiency and blood stasis type" on the market, but also breaks through the bottleneck of western medicine treatment of hematometra and PTS, and can be well applied to the treatment of RPL combined with PTS, pregnancy hematometra and other diseases, and can be used for pre-pregnancy assisted reproduction and post-pregnancy fetal protection.

[0023] In some of the schemes, the active ingredients of the traditional Chinese medicine composition mainly consist of the following weight parts of medicinal materials:

[0024] Cuscuta 6-12 parts, Dipsacus 9-15 parts, Salvia 10-15 parts, Spatholobus 9-15 parts, Astragalus 9-30 parts, Tangerine Peel 3-10 parts, Dioscorea 15-30 parts;

[0025] Alternatively, Cuscuta 6-12 parts, Radix Morindae Officinalis 3-10 parts, Salvia 10-15 parts, Spatholobus 9-15 parts, Astragalus 9-30 parts, Tangerine Peel 3-10 parts, Dioscorea 15-30 parts;

[0026] Alternatively, Cuscuta 6-12 parts, Dipsacus 9-15 parts, Salvia 10-15 parts, Sanchi 3-9 parts, Astragalus 9-30 parts, Tangerine Peel 3-10 parts, Dioscorea 15-30 parts;

[0027] Alternatively, Cuscuta 6-12 parts, Radix Morindae Officinalis 3-10 parts, Salvia 10-15 parts, Sanchi 3-9 parts, Astragalus 9-30 parts, Tangerine Peel 3-10 parts, Dioscorea 15-30 parts.

[0028] In some of the schemes, the active ingredients of the traditional Chinese medicine composition mainly consist of the following weight parts of medicinal materials:

[0029] Cuscuta 12 parts, Dipsacus 10 parts, Salvia 15 parts, Spatholobus 15 parts, Astragalus 20 parts, Tangerine Peel 5 parts, Dioscorea 15 parts;

[0030] Alternatively, Cuscuta 12 parts, Radix Morindae Officinalis 9 parts, Salvia 15 parts, Spatholobus 15 parts, Astragalus 20 parts, Tangerine Peel 5 parts, Dioscorea 15 parts;

[0031] Alternatively, Cuscuta 12 parts, Dipsacus 10 parts, Salvia 15 parts, Sanchi 9 parts, Astragalus 20 parts, Tangerine Peel 5 parts, Dioscorea 15 parts;

[0032] Alternatively, Cuscuta 12 parts, Radix Morindae Officinalis 9 parts, Salvia 15 parts, Sanchi 9 parts, Astragalus 20 parts, Tangerine Peel 5 parts, Dioscorea 15 parts.

[0033] In some of the schemes, the Semen cuscutae is salt Semen cuscutae, and the Radix leonuri is salt Radix leonuri. It can be understood that the above salt Semen cuscutae or salt Radix leonuri, etc. can be prepared according to the corresponding traditional Chinese medicine processing method.

[0034] In another aspect, the application also discloses a preparation method of the traditional Chinese medicine composition, comprising the following steps: extracting the medicinal materials by a solvent extraction method to obtain the traditional 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 of the schemes, the solvent extraction method comprises decoction, reflux extraction, immersion or percolation.

[0037] In some of the schemes, the solvent is selected from water and an ethanol aqueous solution.

[0038] It can be understood that in the above extraction method, the water extraction of the traditional water extraction and alcohol precipitation method also belongs to the above solvent extraction method, which can be prepared according to the conventional Chinese medicine extraction process. In the extraction solvent, the volume percentage concentration of ethanol in the ethanol aqueous solution or the water extraction and alcohol precipitation 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] The medicinal materials of the prescription amount are weighed, the solvent is added according to the predetermined solid-liquid ratio, extraction is carried out by heating, filtration is carried out, the solvent in the filtrate is recovered, and the traditional Chinese medicine composition is obtained.

[0041] In some of the schemes, the preparation method comprises the following steps: the medicinal materials of the prescription amount are weighed, the solvent is added according to the predetermined solid-liquid ratio, extraction is carried out by heating, filtration is carried out, the solvent in the filtrate is recovered, and the traditional Chinese medicine composition is obtained.

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

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

[0044] In some of the schemes, the extraction time is 0.5-3 hours, for example, 1-2 hours.

[0045] In some of the schemes, the extraction temperature is 80-100°C, for example, 90-100°C.

[0046] In some of the schemes, the filtrate is concentrated to a relative density of 1.10-1.30, 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 60-75%, for example, 65%.

[0048] In some of the schemes, the standing time after the addition of ethanol to the concentrated filtrate is 8-24 hours, for example, 12 hours.

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

[0050] The step of preparing the traditional Chinese medicine composition into a freeze-dried powder is as follows: taking the filtrate, concentrating to obtain an extract, and drying the extract by freeze-drying to obtain a freeze-dried powder.

[0051] The step of preparing the traditional Chinese medicine composition into granules is as follows: taking the filtrate, concentrating to an extract with a relative density of 1.05-1.10, drying, adding a filler, and wet granulating to obtain granules, which are obtained; further, the filler is maltodextrin, and the relative density is measured at 50-70℃, for example, at 60℃.

[0052] The application further discloses a medicine, which comprises the traditional Chinese medicine composition and pharmaceutically acceptable excipients, and further, the traditional Chinese medicine composition is administered orally, and further, the dosage form of the traditional Chinese medicine composition is a capsule, granules, tablets, oral liquid, mixture or syrup.

[0053] It can be understood that the pharmaceutically acceptable excipients include carriers, excipients and / or fillers, etc., which can be selected and adjusted according to the requirements of the dosage form, and include any such material that, when combined with an active ingredient, allows the ingredient to remain biologically active and does not react with the immune system of the subject. The above different dosage forms can be prepared according to the conventional methods in the art.

[0054] In another aspect, the application further discloses the traditional Chinese medicine composition and the medicine in the preparation of a medicine for tonifying the kidney and activating blood.

[0055] In some of the schemes, the medicine for tonifying the kidney and activating blood is used for preventing and / or treating recurrent pregnancy loss (RPL).

[0056] In some of the schemes, the medicine for tonifying the kidney and activating blood is used for pre-pregnancy assisted pregnancy and / or post-pregnancy anti-abortion, for example, the pre-pregnancy assisted pregnancy medicine is used for preventing RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases; the post-pregnancy medicine is used for treating RPL combined with PTS, intrauterine hemorrhage during pregnancy and other diseases.

[0057] In some of the schemes, the kidney-tonifying and blood-activating drug is used for recurrent pregnancy loss (RPL) combined with pre-thrombotic state (PTS) and hematorrhagia.

[0058] The above "treatment" means alleviating symptoms associated with a disease, disorder or condition, or halting further development or worsening of these symptoms. "Treatment" as used herein can include one or more of curative, palliative, and prophylactic treatment, depending on the disease and condition of the patient. Treatment can also include administration of the pharmaceutical preparation of the present application in conjunction with other treatments.

[0059] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any way, i.e. to obtain the preferred embodiments of the present application.

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

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

[0062] The positive progress effect of the present application is that:

[0063] The traditional Chinese medicine composition of the present application, aiming at RPL of "kidney deficiency and blood stasis" syndrome type, plays a bidirectional regulating role of anticoagulation and hemostasis, can treat PRL combined with PTS, hematorrhagia during pregnancy, etc., has good curative effect, such as can significantly prolong the coagulation time of normal KM mice; at the same time, can significantly reduce the spontaneous abortion rate of abortion model mice, can improve the PTS of CBA / JxDBA / 2 abortion model, and plays a role in protecting pregnant mice. Clinically, the area of hematorrhagia in patients with hematorrhagia can be significantly reduced after administration of the prescription for 2 weeks and 4 weeks, etc.

[0064] Moreover, the traditional Chinese medicine composition can be used before pregnancy to prevent RPL combined with PTS, hematorrhagia during pregnancy, etc., and can be used after pregnancy to treat RPL combined with PTS, hematorrhagia during pregnancy, etc. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 Morphology of mouse uterine (containing embryo) tissue of different kidney-tonifying and blood-activating prescriptions in Example 3.

[0066] Figure 2The images show the morphological characteristics of the uterus and placenta in mice treated with the following formulas in Example 5: Kidney-tonifying and Blood-activating Formula, Pregnancy-preserving Capsule, Pregnancy-enhancing Oral Liquid, Donkey-hide Gelatin Blood-tonifying Granules, and Dydrogesterone.

[0067] Figure 3 Images of the uterus of mice in each group in Example 6.

[0068] Figure 4 Images of mouse placentas and HE-stained tissue sections from each group in Example 6 are shown.

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

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

[0071] Figure 7 This is the drug administration flowchart in Example 8.

[0072] Figure 8 The images show the morphological features of the uterine (including embryo) tissues of each group of mice in Example 8. Detailed Implementation

[0073] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0074] Example 1

[0075] This embodiment prepares various traditional Chinese medicine compositions (i.e., kidney-tonifying and blood-activating formulas 1, 2, 3, 4, 5, 6, and 7), which have the effects of tonifying the kidney and activating blood. Their formulas are as follows:

[0076] (1) Kidney-tonifying and blood-activating formula 1: 12g of salted dodder seed, 10g of teasel root, 15g of salvia miltiorrhiza, 15g of chicken blood vine, 20g of astragalus root, 5g of dried tangerine peel, and 15g of yam.

[0077] Weigh out 10 times the prescribed amount of medicinal materials, add 10 times the amount of water at a ratio of 10 ml water per gram of medicinal materials, and decoct for 1 hour. Filter the solution. Add 8 times the amount of water to the residue and decoct for 1 hour. Filter the solution again. Combine the filtrates, concentrate the filtrate into a thick paste, and then freeze-dry it to make a freeze-dried powder.

[0078] 1g of the resulting lyophilized powder is equivalent to 3.43g of crude drug, which is stored at 4℃ for later use.

[0079] (2) Kidney-tonifying and blood-activating formula 2: Cuscuta chinensis 12g, Dipsacus asper 10g, Salvia miltiorrhiza 15g, Spatholobus suberectus 15g, Astragalus membranaceus 20g, Citrus reticulata 5g, Dioscorea opposita 15g.

[0080] Take 10 times the amount of medicinal materials in the prescription, add 10 times the amount of water, and decoct for 1 hour. Filter the mixture. Add 8 times the amount of water to the residue, and decoct for 1 hour. Filter the mixture. Combine the filtrates, and concentrate the mixture into a thick paste. Dry the thick paste by freeze-drying to obtain a freeze-dried powder.

[0081] The obtained 1 g of the freeze-dried powder is equivalent to 3.24 g of crude drug, and is stored in a refrigerator at 4°C for later use.

[0082] (3) Bushen Huoxue Decoction 3: Semen Eurycoma 3 g, Radix Dipsaci 3 g, Radix Salviae Miltiorrhizae 35 g, Caulis Spatholobi 35 g, Radix Astragali 5 g, Pericarpium Citri Reticulatae 20 g, and Rhizoma Dioscoreae 35 g.

[0083] Take 10 times the amount of medicinal materials in the prescription, add 10 times the amount of water, and decoct for 1 hour. Filter the mixture. Add 8 times the amount of water to the residue, and decoct for 1 hour. Filter the mixture. Combine the filtrates, and concentrate the mixture into a thick paste. Dry the thick paste by freeze-drying to obtain a freeze-dried powder.

[0084] The obtained 1 g of the freeze-dried powder is equivalent to 3.24 g of crude drug, and is stored in a refrigerator at 4°C for later use.

[0085] (4) Bushen Huoxue Decoction 4: Semen Eurycoma 35 g, Radix Dipsaci 35 g, Radix Salviae Miltiorrhizae 5 g, Caulis Spatholobi 5 g, Radix Astragali 60 g, Pericarpium Citri Reticulatae 1 g, Rhizoma Dioscoreae 6 g.

[0086] Take 10 times the amount of medicinal materials in the prescription, add 10 times the amount of water, and decoct for 1 hour. Filter the mixture. Add 8 times the amount of water to the residue, and decoct for 1 hour. Filter the mixture. Combine the filtrates, and concentrate the mixture into a thick paste. Dry the thick paste by freeze-drying to obtain a freeze-dried powder.

[0087] The obtained 1 g of the freeze-dried powder is equivalent to 3.24 g of crude drug, and is stored in a refrigerator at 4°C for later use.

[0088] (5) Bushen Huoxue Decoction 5: Semen Eurycoma 12 g, Radix Dipsaci 10 g, Radix Salviae Miltiorrhizae 15 g, Radix Notoginseng 9 g, Radix Astragali 20 g, Pericarpium Citri Reticulatae 5 g, and Rhizoma Dioscoreae 15 g.

[0089] Take 10 times the amount of medicinal materials in the prescription, add 10 times the amount of water, and decoct for 1 hour. Filter the mixture. Add 8 times the amount of water to the residue, and decoct for 1 hour. Filter the mixture. Combine the filtrates, and concentrate the mixture into a thick paste. Dry the thick paste by freeze-drying to obtain a freeze-dried powder.

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

[0091] (6) Bushen Huoxue Recipe 6: Semen cuscutae 12g, Radix morindae officinalis 9g, Radix salviae miltiorrhizae 15g, Caulis sargentodoxae 15g, Radix astragali 20g, Pericarpium citri reticulatae 5g, Rhizoma dioscoreae 15g.

[0092] Take 10 times the amount of the prescription of medicinal materials, add 10 times the amount of water (1g of medicinal materials: 10ml of water) to decoct for 1h, filter; add 8 times the amount of water to decoct for 1h, filter. Combine the filtrate, concentrate to a clear extract with a relative density of 1.05-1.10 (60°C), dry, add maltodextrin in an appropriate amount, mix well, wet granulation, dry, and you get granules.

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

[0094] (7) Bushen Huoxue Recipe 7: Semen cuscutae 12g, Radix morindae officinalis 9g, Radix salviae miltiorrhizae 15g, Radix notoginseng 9g, Radix astragali 20g, Pericarpium citri reticulatae 5g, Rhizoma dioscoreae 15g.

[0095] Take 10 times the amount of the prescription of medicinal materials, add 10 times the amount of water (1g of medicinal materials: 10ml of water) to decoct for 1h, filter; add 8 times the amount of water to decoct for 1h, filter. Combine the filtrate, concentrate to a clear extract with a relative density of 1.05-1.10 (60°C), dry, add maltodextrin in an appropriate amount, mix well, wet granulation, dry, and you get granules.

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

[0097] Example 2

[0098] This example prepares a granule that can be used to tonify the kidney and promote blood circulation. The formula is as follows, in terms of weight parts of raw medicinal materials:

[0099] Semen cuscutae 3-35 parts, kidney-tonifying medicinal 3-35 parts, Radix salviae miltiorrhizae 5-35 parts, blood-activating and stasis-removing medicinal 3-35 parts, Radix astragali 5-60 parts, Pericarpium citri reticulatae 1-20 parts, Rhizoma dioscoreae 6-35 parts;

[0100] The kidney-tonifying medicinal is Radix dipsaci or Radix morindae officinalis, and the blood-activating and stasis-removing medicinal is Caulis sargentodoxae or Radix notoginseng, which can be replaced and adjusted.

[0101] The granule is prepared as follows:

[0102] Take the prescription amount of medicinal materials, add 10 times the amount of water (1g of medicinal materials: 10ml of water) in proportion to decoct for 1h, filter; add 8 times the amount of water to decoct for 1h, filter. Combine the filtrate, concentrate to a clear extract with a relative density of 1.05-1.10 (60°C), dry, add maltodextrin in an appropriate amount, mix well, wet granulation, dry, and you get granules.

[0103] Example 3

[0104] The effects of various traditional Chinese medicine compositions prepared in Example 1 (i.e., Bushen Huoxue Formula 1, 2, 3, 4, 5, 6, and 7) on CBA / J x DBA / 2 abortion mice were compared.

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

[0106] 1. Materials and methods

[0107] 1.1 Materials

[0108] 1.1.1 Experimental system

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

[0110] 1.1.2 Test drugs

[0111] Traditional Chinese medicine compositions Bushen Huoxue Formula 1, 2, 3, 4, 5, 6, and 7 prepared in Example 1.

[0112] 1.1.3 Experimental reagents and instruments

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

[0114] 1.2 Methods

[0115] 1.2.1 Animal modeling and grouping

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

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

[0118] The next day at 9:00, the vaginal plug was observed, and the female mice that had mated were considered to be pregnant and were recorded as G 0.5 days. If the female mice were not pregnant, they were kept for the next mating. The male mice could mate with the female mice in the same day multiple times, and they needed to rest for one day before the next mating.

[0119] The RSA model mice were randomly divided into the following 9 groups on G 0.5 days: model control group, Bushen Huoxue Decoction 1, 2, 3, 4, 5, 6, 7, and dydrogesterone group, with 10 mice in each group.

[0120] 1.2.2 Dosing regimen

[0121] Based on the recommended human dose (body weight of 60 kg), the conversion factor between adult and mouse was 9.01, and the equivalent dose conversion formula was: mouse dose = (recommended human dose / 60 kg) x 9.01. From G 0.5 days to G 13.5 days, the normal control group was given pure water at 0.1 mL / 10 g / d, and the Bushen Huoxue Decoction groups were given the corresponding solution at 0.1 mL / 10 g / d. The mice were given the solution once a day, and the dosages of the groups are shown in Table 1.

[0122] Table 1 Dosage of each group

[0123]

[0124] 1.2.3 Solution preparation

[0125] (1) Bushen Huoxue Decoction 1: 1 g of Bushen Huoxue Decoction 1 lyophilized powder is equivalent to 3.43 g of crude drug. The dosage of crude drug for mice is 13.82 g of crude drug / kg / d, so the dosage of Bushen Huoxue Decoction 1 lyophilized powder for mice is 4.03 g of lyophilized powder / kg / d. The solution needs to be prepared at a concentration of 0.403 g / mL for gavage at 0.1 mL / 10 g / d. According to the average weight of mice of 30 g, 10 animals need 3.0 mL of solution. Therefore, 4 mL of Bushen Huoxue Decoction 1 solution is prepared. Specifically, 1.612 g of Bushen Huoxue Decoction 1 lyophilized powder is accurately weighed and placed in an EP tube, and an appropriate amount of pure water is added for dissolution. The volume is then adjusted to 4 mL. The tube is inverted 5 times, and the lyophilized powder is completely dissolved by ultrasonic heating at 60°C (about 10 min). The solution is prepared and used immediately.

[0126] (2) Bushen Huoxue Recipe 2: 1 g of Bushen Huoxue Recipe 2 water extract alcohol precipitation extract is equivalent to 4.01 g of crude drug, the crude drug dosage of mice is 13.82 g of crude drug / kg / d, so the dosage of Bushen Huoxue Recipe 2 mouse extract is 3.45 g of extract / kg / d, and the concentration of 0.345 g / mL needs to be prepared for gavage at 0.1 mL / 10 g / d. According to the average weight of mice of 30 g, 10 animals need 3.0 mL of solution. Now prepare 4 mL of Bushen Huoxue Recipe 2 solution. The specific operation is to accurately weigh 1.380 g of Bushen Huoxue Recipe 2 water extract alcohol precipitation extract into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 4 mL. Shake up and down 5 times, and heat to completely dissolve the extract (about 10 min) at 60°C ultrasonic. Prepare and use immediately.

[0127] (3) Bushen Huoxue Recipe 3: 1 g of Bushen Huoxue Recipe 3 freeze-dried powder is equivalent to 3.12 g of crude drug, and the crude drug dosage of mice is 20.42 g of crude drug / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 3 mouse freeze-dried powder is 6.55 g of freeze-dried powder / kg / d, and the water extract solution concentration of 0.655 g / mL needs to be prepared for gavage at 0.1 mL / 10 g / d. According to the average weight of mice of 30 g, 10 animals need 3.0 mL of solution. Now prepare 4 mL of Bushen Huoxue Recipe 3 solution. The specific operation is to accurately weigh 2.62 g of Bushen Huoxue Recipe 3 freeze-dried powder into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 4 mL. Shake up and down 5 times, and heat to completely dissolve the freeze-dried powder (about 10 min) at 60°C ultrasonic. Prepare and use immediately.

[0128] (4) Bushen Huoxue Recipe 4: 1 g of Bushen Huoxue Recipe 4 freeze-dried powder is equivalent to 3.05 g of crude drug, and the crude drug dosage of mice is 22.07 g of crude drug / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 4 mouse freeze-dried powder is 7.24 g of freeze-dried powder / kg / d, and the water extract solution concentration of 0.724 g / mL needs to be prepared for gavage at 0.1 mL / 10 g / d. According to the average weight of mice of 30 g, 10 animals need 3.0 mL of solution. Now prepare 4 mL of Bushen Huoxue Recipe 4 solution. The specific operation is to accurately weigh 2.896 g of Bushen Huoxue Recipe 4 freeze-dried powder into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 4 mL. Shake up and down 5 times, and heat to completely dissolve the freeze-dried powder (about 10 min) at 60°C ultrasonic. Prepare and use immediately.

[0129] (5) Bushen Huoxue Recipe 5: 1 g of Bushen Huoxue Recipe 5 lyophilized powder is equivalent to 3.24 g of crude drugs. The crude drug dosage for mice is 12.91 g of crude drugs / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 5 lyophilized powder for mice is 3.98 g of lyophilized powder / kg / d. The solution concentration needs to be 0.398 g / mL for gavage administration at 0.1 mL / 10 g / d. According to the average weight of mice of 30 g, 10 animals need 3.0 mL of solution. Now prepare 4 mL of Bushen Huoxue Recipe 5 solution. The specific operation is to accurately weigh 1.592 g of Bushen Huoxue Recipe 5 lyophilized powder into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 4 mL. Invert 5 times, heat to completely dissolve the lyophilized powder at 60°C (about 10 min), and use it immediately.

[0130] (6) Bushen Huoxue Recipe 6: 1 g of Bushen Huoxue Recipe 6 lyophilized powder is equivalent to 3.36 g of crude drugs. The crude drug dosage for mice is 13.67 g of crude drugs / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 6 lyophilized powder for mice is 4.06 g of lyophilized powder / kg / d. The solution concentration needs to be 0.406 g / mL for gavage administration at 0.1 mL / 10 g / d. According to the average weight of mice of 30 g, 10 animals need 3.0 mL of solution. Now prepare 4 mL of Bushen Huoxue Recipe 6 solution. The specific operation is to accurately weigh 1.624 g of Bushen Huoxue Recipe 6 lyophilized powder into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 4 mL. Invert 5 times, heat to completely dissolve the lyophilized powder at 60°C (about 10 min), and use it immediately.

[0131] (7) Bushen Huoxue Recipe 7: 1 g of Bushen Huoxue Recipe 7 lyophilized powder is equivalent to 3.32 g of crude drugs. The crude drug dosage for mice is 12.76 g of crude drugs / kg / d. Therefore, the dosage of Bushen Huoxue Recipe 7 lyophilized powder for mice is 3.84 g of lyophilized powder / kg / d. The solution concentration needs to be 0.384 g / mL for gavage administration at 0.1 mL / 10 g / d. According to the average weight of mice of 30 g, 10 animals need 3.0 mL of solution. Now prepare 4 mL of Bushen Huoxue Recipe 7 solution. The specific operation is to accurately weigh 1.536 g of Bushen Huoxue Recipe 7 lyophilized powder into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 4 mL. Invert 5 times, heat to completely dissolve the lyophilized powder at 60°C (about 10 min), and use it immediately.

[0132] (8) Dydrogesterone solution: The dosage of dydrogesterone in mice was 3.00 mg / kg / day, administered by gavage at a rate of 0.1 mL / 10 g / day. The required drug solution concentration was 0.300 mg / mL. Based on an average mouse weight of 30 g, 3.0 mL of solution was needed for 10 animals. To prepare 4.0 mL of dydrogesterone solution, 1.2 mg of dydrogesterone was required. Each tablet contained 10 mg of dydrogesterone, and the average tablet weight was 0.1426 g. The specific procedure was to accurately weigh 17.11 mg of dydrogesterone powder (i.e., 1.2 mg of dydrogesterone), place it in an EP tube, add an appropriate amount of pure water to dissolve, and bring the volume to 4 mL. The tube was inverted 5 times and sonicated for 5 minutes to ensure complete dissolution. The solution was prepared and used immediately.

[0133] 1.2.4 Indicator Detection

[0134] Mice were anesthetized with 5% isoflurane on day G13.5. Blood was collected from the abdominal aorta after anesthesia and stored in centrifuge tubes without sodium citrate to obtain serum. After blood collection, the mice were quickly dissected. The uterus was removed and photographed to record its appearance. The uterus was then dissected 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 Data Statistics

[0136] All data were entered into SPSS 26.0 for statistical analysis. Count data for each group, such as embryo resorption rate, were analyzed using the χ² method for four-fold tables. 2 Statistical analysis was performed using the exact Fisher test and / or precise probability method. The mean ± standard deviation (x ± s) of all measurement data was calculated. An analysis of variance (F-test) was performed between experimental groups before comparisons were made. When the variances were homogeneous, the Student-T test (unpaired T-test) was used for statistical analysis. When the variances were unequal, a corrected Student-T test was used. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly significant.

[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 formulas reduced the embryo loss rate in mice with recurrent miscarriage.

[0140] Table 2 Embryo loss rate in each group of mice ( 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] 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 modeling was successful; compared with the model control group, the embryo loss rate of the kidney and blood circulation promoting prescription groups 1, 2, 5, 6 and 7 was extremely significantly decreased (P<0.01), and the embryo loss rate of the kidney and blood circulation promoting prescription groups 3 and 4 was significantly decreased (P<0.05).

[0144] 2.2 Uterus (including embryo) morphology

[0145] The uterus (including embryo) tissue morphology of each group of traditional Chinese medicine composition (i.e. kidney and blood circulation promoting prescription groups 1, 2, 3, 4, 5, 6 and 7) mice is shown in Table 2.2. Figure 1

[0146] It can be seen that the embryos of the normal group were light red, thick and uniform, like beads; the model group appeared black-brown absorbed embryos, visible blood stasis with uneven size, and some or even all embryos disappeared; the embryos of each kidney and blood circulation promoting prescription group were full and light red, and occasionally black-brown absorbed embryos were seen.

[0147] Combining the results of 2.1 and 2.2, it can be found that kidney and blood circulation promoting prescription groups 1, 2, 5, 6 and 7 are better in down-regulating the embryo loss rate, and can improve the uterus embryo morphology. At the same time, the cost of preparing kidney and blood circulation promoting prescription group 1 is lower, so kidney and blood circulation promoting prescription group 1 is selected for investigation in the following examples.

[0148] Example 4

[0149] This example investigates the effect of the pharmaceutical composition prepared in Example 1 (kidney and blood circulation promoting prescription group 1) on the coagulation time of normal Kunming (KM) mice.

[0150] 1. Materials and methods

[0151] 1.1 Materials

[0152] 1.1.1 Experimental system

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

[0154] 1.1.2 Test drugs

[0155] ​The Chinese medicine composition prepared in Example 1 is Shengen Huoxue Decoction 1 lyophilized powder.

[0156] Enoxaparin: State Food and Drug Administration No. J20180036, Sanofi Winthrop Industrie, specification 0.6 mL: 6000 AXal IU, 2 bottles per box.

[0157] 1.1.3 Experimental reagents and instruments

[0158] Electronic balance: JJ3000, Changshu Shuangjie Testing Instrument Factory; analytical balance: EL204, Mettler-Toledo Instrument (Shanghai) Co., Ltd.; vortex: XW-80A, Haimen Qilinkailai Instrument Manufacturing Co., Ltd.

[0159] 1.2 Method

[0160] 1.2.1 Animal grouping

[0161] 50 SPF KM mice were randomly divided into 5 groups according to body weight: normal control group, Shengen Huoxue Decoction low-dose group, Shengen Huoxue Decoction medium-dose group, Shengen Huoxue Decoction high-dose group, and low molecular heparin group, 10 mice in each group.

[0162] 1.2.2 Dosing regimen

[0163] Taking the recommended dose of human body (body weight 60 kg) as the standard, the conversion coefficient of adult and mouse is 9.01, and the equivalent dose conversion formula is: mouse dose = (recommended dose of human body / 60 kg) x 9.01, and the intragastric administration or subcutaneous injection is performed.

[0164] The normal control group was given pure water by intragastric administration at 10 mL / kg; the Shengen Huoxue Decoction low-dose group was given Shengen Huoxue Decoction aqueous solution by intragastric administration, the dose was half of the human dose (46 g crude drug per person per day), and the converted mouse crude drug dose was 6.91 g crude drug / kg / d; the Shengen Huoxue Decoction medium-dose group was given Shengen Huoxue Decoction aqueous solution by intragastric administration, the dose was the human dose (92 g crude drug per person per day), and the converted mouse crude drug dose was 13.82 g crude drug / kg / d; the Shengen Huoxue Decoction high-dose group was given Shengen Huoxue Decoction aqueous solution by intragastric administration, the dose was twice the human dose (184 g crude drug per person per day), and the converted mouse crude drug dose was 27.63 g crude drug / kg / d. The low molecular heparin group was given enoxaparin injection by subcutaneous injection, the dose was the recommended human dose (4000 AXalU / time / day), and the converted mouse subcutaneous dose was 6.00 AXalU / 10 g.

[0165] 1.2.3 Solution preparation

[0166] Example 1 The self-made 1 g of lyophilized powder is equivalent to 3.43 g of Shenshu Huoxue Fang crude drug. The mouse gavage administration volume is 0.1 mL / 10 g / d. Therefore, the high-dose Shenshu Huoxue Fang mouse lyophilized powder administration dose is 8.06 g of lyophilized powder / kg / d, and the solution concentration to be prepared is 0.806 g / mL; the medium-dose Shenshu Huoxue Fang mouse lyophilized powder administration dose is 4.03 g of lyophilized powder / kg / d, and the solution concentration to be prepared is 0.403 g / mL; the low-dose Shenshu Huoxue Fang mouse lyophilized powder administration dose is 2.01 g of lyophilized powder / kg / d, and the water extract concentration to be prepared is 0.201 g / mL. The recommended clinical human dose of enoxaparin is 4000 AXal U / each / time / day for subcutaneous administration, and the mouse dosage is 600 AXal U / kg / d.

[0167] (1) High-dose Shenshu Huoxue Fang: according to the average mouse body weight of 30 g, 10 animals need 3.0 mL of solution. Since the low-dose and medium-dose Shenshu Huoxue Fang are prepared by gradient dilution of the high-dose Shenshu Huoxue Fang, 7 mL of high-dose Shenshu Huoxue Fang solution is prepared. The specific operation is to accurately weigh 5.642 g of Shenshu Huoxue Fang lyophilized powder into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 7 mL. Invert 5 times, heat to completely dissolve the lyophilized powder at 60°C by ultrasonic (about 10 min), and prepare for immediate use.

[0168] (2) Medium-dose Shenshu Huoxue Fang: according to the average mouse body weight of 30 g, 10 animals need 3.0 mL of solution. 4 mL of medium-dose Shenshu Huoxue Fang solution is prepared. The specific operation is to accurately transfer 2.0 mL of high-dose Shenshu Huoxue Fang solution into an EP tube, add 2.0 mL of pure water, vortex for 30 s, and mix well, and prepare for immediate use.

[0169] (3) Low-dose Shenshu Huoxue Fang: according to the average mouse body weight of 30 g, 10 animals need 3.0 mL of solution. 4 mL of low-dose Shenshu Huoxue Fang solution is prepared. The specific operation is to accurately transfer 2.0 mL of medium-dose Shenshu Huoxue Fang into an EP tube, add 2.0 mL of pure water, vortex for 30 s, and mix well, and prepare for immediate use.

[0170] (4) Low-dose enoxaparin sodium solution: according to the average mouse body weight of 30 g, 10 animals need 3.0 mL of solution. 4.0 mL of enoxaparin sodium solution is prepared. The specific operation is to take 24 μL (240 AXal U, 10000 AXal U / mL) of pre-filled enoxaparin sodium injection into a 5 mL EP tube, add 3976 μL of 5% glucose injection, mix well, and obtain 4000 μL (240 AXal U, 60 AXal U / mL) of solution, and prepare for immediate use.

[0171] 1.2.4 Index detection

[0172] One hour after a single drug administration, blood was collected from the orbit using a capillary tube. Timing began from the moment blood flowed into the tube. Once the tube was full, it was placed flat on a glass dish. After 30 seconds, one end of the capillary tube was broken (starting from the blood-collecting end), and the tube was slowly pulled to the left and right. This process was repeated every 10 seconds after 50 seconds. When blood streaks appeared at the break point, the other end was broken for verification. The time it took for blood streaks to appear at both ends was recorded as the clotting time (s).

[0173] 1.2.5 Data Processing

[0174] Same as Example 3.

[0175] 2. Results

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

[0177] Table 3 shows that the kidney-tonifying and blood-activating formula significantly prolonged the clotting time in 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 clotting time of the test drug (Kidney-tonifying and Blood-activating Formula) group was significantly prolonged (P<0.01), suggesting that the Kidney-tonifying and Blood-activating Formula has an antithrombotic effect.

[0181] Example 5

[0182] This embodiment examines the effects of the drug composition (Kidney-tonifying and Blood-activating Formula 1) prepared in Example 1 on CBA / J×DBA / 2 aborted mice compared with different traditional Chinese medicines for preventing miscarriage.

[0183] Based on the CBA / J×DBA / 2 recurrent miscarriage mouse model, this study investigated the effects of a kidney-tonifying and blood-activating formula and different traditional Chinese medicine (TCM) preparations for preventing miscarriage (Baotailing Capsules, Yunkang Oral Liquid, and Ejiao Buxue Granules) on embryo loss rate, uterine and placental tissue morphology. Baotailing Capsules are a TCM preparation used to treat "kidney deficiency" and "spleen and kidney deficiency" syndromes in RPL; Yunkang Oral Liquid is a TCM preparation used to treat "kidney deficiency and blood heat" syndromes in RPL; and Ejiao Buxue Granules are a TCM preparation used to treat "qi and blood deficiency" syndromes in 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 No.: SCXK(Beijing)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(Guangdong)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 Capsule: 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 Blood Tonifying 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 Capsule group, the Yunkang Oral Liquid group, the Ejiao Blood Tonifying 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 (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. Gavage administration was carried out.

[0201] The normal control group was given pure water by gavage at a dose of 10 mL / kg; the Bushenhuoxuefang group was given Bushenhuoxuefang solution by gavage at a dose of 13.82 g crude drug / kg / d, which was converted from the human dose (92 g crude drug / person / d); the Baotailing capsule group was given Baotailing capsule solution by gavage at a dose of 0.6758 g / kg / d, which was converted from the human dose (4.5 g / person / d); the Yikang oral liquid group was given Yikang oral liquid by gavage at a dose of 9.01 mL / kg / d, which was converted from the human dose (60 mL / person / d); the Ejiao buxue granules group was given Ejiao buxue granules solution by gavage at a dose of 1.201 g / kg / d, which was converted from the human dose (8.0 g / person / d); the dydrogesterone group was given dydrogesterone solution by gavage at a dose of 3.00 mg / kg / d, which was converted from the human dose (20 mg / person / d).

[0202] From G0.5 days to G13.5 days, each group was given the corresponding solution at a concentration of 0.1 mL / 10 g / d once a day.

[0203] 1.2.3 Solution preparation

[0204] The gavage volume of the mice in the experiment was 0.1 mL / 10 g / d. The 1 g of Bushenhuoxuefang freeze-dried powder prepared in Example 1 was equivalent to 3.43 g of Bushenhuoxuefang crude drug, so the gavage dose of the Bushenhuoxuefang mouse freeze-dried powder was 4.03 g freeze-dried powder / kg / d, and the water extract solution concentration needed to be 0.403 g / mL; the gavage dose of the Baotailing capsule group was 0.6758 g / kg / d, i.e. 0.068 g / mL; the gavage dose of the Yikang oral liquid group was 9.01 mL / kg / d, and the drug oral liquid was directly given; the gavage dose of the Ejiao buxue granules group was 1.201 g / kg / d, i.e. 0.120 g / mL; the gavage dose of the dydrogesterone group was 3.00 mg / kg / d, i.e. 0.300 mg / mL.

[0205] (1) Bushenhuoxuefang: according to the average body weight of 30 g of mice, 3.0 mL of solution was needed for 10 animals. 4 mL of Bushenhuoxuefang solution was prepared. The specific operation was to accurately weigh 1.612 g of Bushenhuoxuefang freeze-dried powder into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 4 mL. Invert 5 times, heat to completely dissolve the freeze-dried powder (about 10 min) at 60°C by ultrasonic, and prepare for immediate use.

[0206] (2) Baotailing Capsule solution: according to the average weight of mice 30 g, 10 animals need solution 3.0 mL. Now prepare 4.0 mL Baotailing Capsule solution. The specific operation is to accurately weigh 0.272 g Baotailing Capsule powder into an EP tube, add appropriate amount of pure water to dissolve, and then make up to 4 mL. Invert 5 times, ultrasonic for 5 min, fully dissolved, and prepare for immediate use.

[0207] (3) Ejiao Buxue Granules solution: according to the average weight of mice 30 g, 10 animals need solution 3.0 mL. Now prepare 4.0 mL Ejiao Buxue Granules solution. The specific operation is to accurately weigh 0.480 g Ejiao Buxue Granules into an EP tube, add appropriate amount of pure water to dissolve, and then make up to 4 mL. Invert 5 times, ultrasonic for 5 min, fully dissolved, and prepare for immediate use.

[0208] (4) Dydrogesterone solution: the dose of dydrogesterone group mice is 3.00 mg / kg / d, and the concentration of drug solution needed for gavage is 0.300 mg / mL. Now prepare 4.0 mL dydrogesterone solution, which needs 1.2 mg dydrogesterone. Each dydrogesterone tablet contains 10 mg dydrogesterone, and the average tablet weight of dydrogesterone tablet is 0.1426 g. Accurately weigh 17.11 mg dydrogesterone tablet powder (i.e. 1.2 mg dydrogesterone) into an EP tube, add appropriate amount of pure water to dissolve, and make up to 4 mL. Invert 5 times, ultrasonic for 5 min, fully dissolved, and prepare for immediate use.

[0209] 1.2.4 Index detection

[0210] On G13.5 days, the mice were anesthetized with 5% isoflurane, and blood was taken from the abdominal aorta after anesthesia. The blood was stored in a centrifuge tube without sodium citrate to obtain serum. After blood collection, the uterus was quickly dissected, the appearance was recorded by photographing, and the weight of the uterus was weighed. Then the uterus was cut along the longitudinal axis at the uterine ligament, the total embryo number, the number of aborted embryos were counted, and the embryo loss rate was calculated: embryo loss rate = aborted embryo number / total embryo number x 100%.

[0211] 1.3 Data statistics

[0212] The same as Example 3.

[0213] 2. Results

[0214] 2.1 Mouse embryo loss rate

[0215] The results are shown in the following table, which shows that Bushen Huoxue Decoction, Baotailing Capsule, Pregnancy Oral Liquid and dydrogesterone can all reduce the embryo loss rate of mice with recurrent spontaneous abortion.

[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, compared with the normal control group, the embryo loss rate of the model control group was significantly increased (P<0.01), indicating that the modeling was successful; compared with the model control group, the embryo loss rate of the Bushen Huoxue Decoction and dydrogesterone group was extremely significantly decreased (P<0.01), and the embryo loss rate of the BaoTuiLing Capsule and the GongKang 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 following table. Compared with different traditional Chinese medicine for preventing miscarriage, Bushen Huoxue Decoction and dydrogesterone both up-regulated the uterine wet weight and average embryo weight of recurrent miscarriage mice.

[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 of the model control group were significantly decreased (P<0.01), and the uterine wet weight of the dydrogesterone group was significantly increased (P<0.01); compared with the model control group, the uterine wet weight and average embryo weight of the Bushen Huoxue Decoction group and the dydrogesterone group were significantly increased (P<0.01).

[0227] 2.3 Uterine and placental tissue morphology

[0228] The uterine and placental tissue morphology of mice in the Bushen Huoxue Decoction, BaoTuiLing Capsule, GongKang Oral Liquid, Ejiao Blood-Enriching Granules and dydrogesterone groups is shown in the following table. Figure 2

[0229] ​It can be seen that the normal group embryos are light red, thick and uniform, like beads; the model group appears black-brown absorption embryos, visible blood stasis, uneven size, and some or even complete disappearance of the embryo; most of the embryos of the BaoTuiLing capsule and the GongKang oral liquid are full and light red, occasionally appearing black-brown absorption embryos, while the embryos of the ShuShenHuoxue recipe and the dydrogesterone group are full and light red, and no black-brown absorption embryos appear.

[0230] Under the observation of the body microscope, it is found that compared with the placental maternal surface edge of the normal group, the model group appears bleeding; the placental maternal surface edge bleeding of the ShuShenHuoxue recipe and the BaoTuiLing capsule group is significantly reduced or disappeared; the placental maternal surface edge bleeding of the GongKang oral liquid, the Ejiao blood granules and the dydrogesterone group is not reduced. Figure 2

[0231] The results show that the ShuShenHuoxue recipe, the BaoTuiLing capsule, the GongKang oral liquid and the dydrogesterone can improve the uterine and placental tissue morphology of each group to a certain extent, and the ShuShenHuoxue recipe has the best effect in all aspects.

[0232] Example 6

[0233] This example studies the dose-effect relationship of the ShuShenHuoxue recipe 1 prepared in Example 1 in the treatment of CBA / J×DBA / 2 abortion mice.

[0234] Based on the CBA / J×DBA / 2 recurrent abortion mouse model, the effects of different doses of the ShuShenHuoxue recipe on the embryo loss rate, uterine and placental tissue morphology of the model, the changes of serum chorionic gonadotropin (CG), progesterone (P), estradiol (E2), fibrinogen (FIB) and D-dimer (D-D) indexes of mice were detected by ELISA method, the collagen deposition in the decidua area of mouse placenta was observed by Masson staining, and the expression of fibrinogen (Fib) protein in the decidua area of mouse placenta was observed by immunohistochemical method.

[0235] 1. Materials and methods

[0236] 1.1 Materials

[0237] 1.1.1 Experimental system

[0238] ​Balb / c male mice 5, DBA / 2 male mice 25, CBA / J female mice 60, 8 weeks old, SPF level, purchased from Beijing Vantoll Life Science and Technology Co., Ltd. (License No. SCXK (Jing) 2021-0011). After the animals were purchased, they were placed in the SPF animal laboratory of the Experimental Center of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (License No. SYXK (Yue) 2023-0092) and were housed in separate cages. All experimental operations comply with the relevant provisions of animal welfare.

[0239] 1.1.2 Test substance

[0240] The traditional Chinese medicine composition prepared in Example 1, Bushen Huoxue Fang 1 lyophilized powder, hereinafter referred to as Bushen Huoxue Fang.

[0241] Drospirenone tablets: GMP code HJ20170221, Abbott Biologicals B.V., each containing 10 mg of drospirenone.

[0242] 1.1.3 Experimental reagents and instruments

[0243] The same as Example 3.

[0244] 1.2 Method

[0245] 1.2.1 Animal modeling and grouping

[0246] The modeling method was the same as 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, Bushen Huoxue Fang low-dose group, Bushen Huoxue Fang medium-dose group, Bushen Huoxue Fang high-dose group, and positive drug group (drospirenone), 10 mice in each group.

[0247] 1.2.2 Dosing regimen

[0248] Taking the recommended dose for the human body (body weight 60 kg) as the standard, the conversion factor for adults and mice was 9.01, and the equivalent dose conversion formula was: mouse dose = (recommended dose for human body / 60 kg) x 9.01, and gavage was performed.

[0249] The normal control group was given pure water by gavage at 10 mL / kg; the low-dose group of Bushen Huoxue Decoction was given Bushen Huoxue Decoction solution by gavage, with the dosage being half of the human dosage (46 g crude drug per person per day), and the dosage of the mouse crude drug was converted to be 6.91 g crude drug / kg / d; the medium-dose group of Bushen Huoxue Decoction was given Bushen Huoxue Decoction solution by gavage, with the dosage being the human dosage (92 g crude drug per person per day), and the dosage of the mouse crude drug was converted to be 13.82 g crude drug / kg / d; the high-dose group of Bushen Huoxue Decoction was given Bushen Huoxue Decoction solution by gavage, with the dosage being twice of the human dosage (184 g crude drug per person per day), and the dosage of the mouse crude drug was converted to be 27.63 g crude drug / kg / d; the dydrogesterone group was given dydrogesterone aqueous solution by gavage, with the dosage being the human dosage for habitual abortion (20 mg per person per day), and the dosage of the mouse dydrogesterone was converted to be 3.00 mg / kg / d.

[0250] From G0.5 days to G13.5 days, each group was given the corresponding concentration of solution by gavage at 0.1 mL / 10 g / d, once a day.

[0251] 1.2.3 Solution preparation

[0252] Since 1 g of self-made Bushen Huoxue freeze-dried powder is equivalent to 3.43 g of Bushen Huoxue Decoction crude drug, the gavage volume of the mouse is 0.1 mL / 10 g / d. Therefore, the gavage dosage of the high-dose Bushen Huoxue Decoction mouse freeze-dried powder is 8.06 g freeze-dried powder / kg / d, and the water extract solution concentration needs to be 0.806 g / mL; the gavage dosage of the medium-dose Bushen Huoxue Decoction mouse freeze-dried powder is 4.03 g freeze-dried powder / kg / d, and the water extract solution concentration needs to be 0.403 g / mL; the gavage dosage of the low-dose Bushen Huoxue Decoction mouse freeze-dried powder is 2.01 g freeze-dried powder / kg / d, and the water extract solution concentration needs to be 0.201 g / mL. The gavage dosage of the mouse dydrogesterone group is 3.00 mg / kg / d, i.e. 0.300 mg / mL.

[0253] High-dose Bushen Huoxue Decoction: according to the average body weight of the mouse of 30 g, 10 animals need 3.0 mL of solution. Since the low-dose and medium-dose Bushen Huoxue Decoction are prepared by gradient dilution of the high-dose Bushen Huoxue Decoction, 7 mL of high-dose Bushen Huoxue Decoction solution is prepared. The specific operation is to accurately weigh 5.642 g of Bushen Huoxue Decoction freeze-dried powder into an EP tube, add an appropriate amount of pure water to dissolve, and then make up to 7 mL. Invert 5 times, and heat to completely dissolve the freeze-dried powder at 60°C by ultrasonic (about 10 min), and prepare it immediately for use.

[0254] Medium-dose Bushen Huoxue Decoction: according to the average body weight of the mouse of 30 g, 10 animals need 3.0 mL of solution. 6 mL of medium-dose Bushen Huoxue Decoction solution is prepared. The specific operation is to accurately transfer 3.0 mL of high-dose Bushen Huoxue Decoction solution into an EP tube, add 3.0 mL of pure water, vortex for 30 s, and mix thoroughly, and prepare it immediately for use.

[0255] Low-dose Bushen Huoxue Decoction: According to the average body weight of 30 g of mice, 10 animals require 3.0 mL of solution. Prepare 4 mL of low-dose Bushen Huoxue Decoction solution. The specific operation is to accurately transfer 2.0 mL of medium-dose Bushen Huoxue Decoction into an EP tube, add 2.0 mL of pure water, vortex for 30 s, and mix thoroughly. Prepare and use immediately.

[0256] Drospirenone solution: The dosage of drospirenone for mice in the drospirenone group is 3.00 mg / kg / d, and the drug solution concentration required for gavage administration is 0.300 mg / mL at 0.1 mL / 10 g / d. Prepare 4.0 mL of drospirenone solution, which requires 1.2 mg of drospirenone. Each tablet contains 10 mg of drospirenone, and the average tablet weight of drospirenone tablets is 0.1426 g. The specific operation is to accurately weigh 17.11 mg of drospirenone tablet powder (i.e., 1.2 mg of drospirenone) into an EP tube, dissolve with an appropriate amount of pure water, and dilute to 4 mL. Invert 5 times, ultrasonic for 5 min, and dissolve thoroughly. Prepare and use immediately.

[0257] 1.2.4 Index detection

[0258] On G13.5 days, mice were anesthetized with 5% isoflurane, and blood was taken from the abdominal aorta after anesthesia. The serum was obtained by storing the blood in a centrifuge tube without sodium citrate. After blood collection, the uterus was quickly dissected, the appearance was recorded by photographing, and the weight of the uterus was weighed. Then, the uterus was cut along the longitudinal axis at the uterine ligament, the total embryo number, the number of aborted embryos, and the embryo loss rate were counted: embryo loss rate = aborted embryo number / total embryo number x 100%. The contents of CG, E2, P, and D-D in the serum were detected using a kit, and the pathological changes (placenta) were observed by HE staining.

[0259] 1.3 Data statistics

[0260] The same as Example 3.

[0261] 2. Results

[0262] 2.1 Embryo loss rate

[0263] The results are shown in the following table. Different doses of Bushen Huoxue Decoction significantly reduced the embryo 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] Results showed that compared with the normal control group, the embryo loss rate of the model control group was significantly higher (P<0.01); compared with the model control group, the embryo loss rate of different doses of Bushen Huoxue Decoction and dydrogesterone group was significantly decreased (P<0.01).

[0268] 2.2 Mouse uterine and placental tissue morphology

[0269] The results are shown in the following table, and the Bushen Huoxue Decoction can regulate the serum hormone levels and coagulation-related factor content of mice. Figures 3-4 Figure 3 are the uterine pictures of mice in each group, Figure 4 are the placenta pictures and placenta HE staining tissue sections of mice in each group. The results showed that Bushen Huoxue Decoction can significantly improve the uterine and placental tissue morphology of mice in each group.

[0270] As can be seen from Figure 3 , the normal group embryos are light red, thick and uniform, like beads; the model group appears black-brown absorbed embryos, visible blood stasis, uneven in size, and some or even complete disappearance of embryos; the Bushen Huoxue Decoction group and the dydrogesterone group are full of embryos, with light red color.

[0271] Figure 4 The upper picture is the photo under the body type microscope (red arrow is the significant bleeding site), and the observation found that compared with the placental maternal surface edge of the normal group, the model group showed bleeding; the placental maternal surface edge of the low, medium and high dose Bushen Huoxue Decoction group was reduced or disappeared; the placental maternal surface edge of the dydrogesterone group did not significantly reduce the bleeding phenomenon.

[0272] Figure 4 The lower picture is the HE staining section, and the observation of pathological tissue structure is consistent with the above results. The placental spongiotrophoblast (SP) and labyrinthine layer (Lab) structure of the normal group of mice developed well, the structure between the layers was clear and distinct, and the vascular structure was full. The SP layer and Lab layer structure of the model group developed poorly, the structure between the layers was not clear, the vascular structure atrophied or even disappeared, and there was a large area of bleeding in the SP layer. Compared with the model group, the placental SP layer and Lab layer structure of the dydrogesterone group of mice developed well, the boundary became clearer, and the vascular structure tended to be full, but there was still bleeding; the placental SP layer and Lab layer structure of the low, medium and high dose Bushen Huoxue Decoction group of mice developed well, the boundary became clearer, and the vascular structure tended to be full, and the placental SP layer bleeding was reduced or disappeared.

[0273] 2.3 Serum hormone levels and coagulation factor content of mice

[0274] The results are shown in the following table, and the Bushen Huoxue Decoction can regulate the serum hormone levels and coagulation-related factor content 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 CG, P and FIB levels of the model control group were reduced (P < 0.05), and the D-D level was increased (P < 0.01); compared with the model group, the serum CG level of the low-, medium- and high-dose Shenshu Huoxue Formula groups was increased (P < 0.05), the P level was increased (P < 0.01), and the FIB level was increased (P < 0.01); the serum D-D level of the medium-dose Shenshu Huoxue Formula group was reduced (P < 0.05), the serum D-D level of the high-dose Shenshu Huoxue Formula group was reduced (P < 0.01), and the serum CG level of the positive drug dydrogesterone group was increased (P < 0.05), the P level was increased (P < 0.01), the FIB level was increased (P < 0.01), and the D-D level was reduced (P < 0.01).

[0279] 2.4 Collagen deposition and Fib protein expression in the decidua basalis of mice

[0280] Figure 5 (40x magnification) is the result of collagen deposition in the decidua basalis of mice. Masson staining will stain collagen into blue, which can be used to judge 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 process of tissues. Excessive deposition of collagen can lead to the occurrence of fibrosis, and further lead to abnormal development of the placenta.

[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 was significantly increased (P < 0.01), indicating the occurrence of placental fibrosis. Compared with the model group, although there was no significant change in collagen deposition in the placenta of mice in the positive drug dydrogesterone group, the collagen deposition in the placenta of mice in different dose Shenshu Huoxue Formula groups was significantly reduced, and the relative area of collagen was significantly reduced (P < 0.01). This result suggests that different doses of Shenshu Huoxue Formula can improve the occurrence of placental fibrosis and improve the development of embryos.

[0282] Figure 6The results of immunohistochemical method and the results of collagen protein were mutually confirmed. The Fib protein positive expression was dyed brown by immunohistochemical method. Fib deposition is one of the pathological features of abortion mice, which can cause thrombosis, leading to placental vascular obstruction, affecting oxygen and nutrient supply. The results showed that compared with the normal group, the Fib protein expression in the model group was significantly increased (P<0.01); compared with the model group, the Fib protein expression in the different dose of Bushen Huoxue Decoction group was significantly reduced (P<0.01), which indicated that different doses of Bushen Huoxue Decoction group could inhibit the deposition of Fib in the placenta and promote the normal development of the placenta.

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

[0284]

[0285] Note: All data were normalized to the normal control group. Compared with 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 Bushen Huoxue Decoction in the treatment of early and middle pregnancy combined with intrauterine hemorrhage.

[0288] Early and middle pregnancy combined with intrauterine hemorrhage patients were used as research objects, and self-control study was used. The changes of intrauterine hemorrhage area within 2 weeks and 4 weeks after the treatment of conventional western medicine combined with Bushen Huoxue Decoction were statistically analyzed to evaluate the effectiveness of Bushen Huoxue Decoction in the treatment of early and middle pregnancy combined with intrauterine hemorrhage. The ethical approval number of this experiment was K-2024-119.

[0289] 1. Research objects and related standards

[0290] 1.1 Research objects

[0291] From January 2020 to February 2023, pregnant women with early and middle pregnancy combined with intrauterine hemorrhage who visited the gynecology and obstetrics center of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (outpatient and inpatient department) were included in the study.

[0292] 1.2 Diagnostic criteria

[0293] 1.2.1 Early intrauterine pregnancy

[0294] Refer to the 9th edition of Obstetrics and Gynecology (People's Medical Publishing House, Xie Jinxing, Kong Beihua) for the description. Early intrauterine pregnancy refers to early ultrasound findings of intrauterine gestational sac or germinal bud, blood hCG level consistent with gestational age, and ultrasound examination shows primitive heart tube pulsation, indicating fetal survival.

[0295] 1.2.2 Threatened abortion

[0296] Refer to the relevant standards of the 9th edition of Obstetrics and Gynecology. Threatened abortion refers to the appearance of a small amount of vaginal bleeding before 28 weeks of intrauterine pregnancy, often dark red or bloody discharge, no pregnancy discharge, followed by paroxysmal lower abdominal pain or back pain. Gynecological examination shows that the cervical orifice is not open, the fetal membrane is not broken, and the size of the uterus is consistent with the number of missed periods.

[0297] 1.2.3 Hematometra

[0298] Refer to the proposed ultrasound atlas in Obstetrics and Gynecology. Hematometra is an ultrasound that suggests the presence of a liquid dark area in the uterine cavity, between the gestational sac and the uterine wall, and between the placenta and the uterine wall, showing a triangular, crescent or ring shape.

[0299] 1.3 Inclusion criteria

[0300] (1) Intrauterine pregnancy, single live fetus in utero, gestational age <28 weeks at the time of presentation;

[0301] (2) At the same time, meet the diagnostic criteria for threatened abortion and hematometra;

[0302] (3) Found hematometra after taking Bushen Huoxue Decoction or its modified version;

[0303] (4) Complete research data.

[0304] 1.4 Exclusion criteria

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

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

[0307] (3) Cervical polyps, cervicitis and other causes of vaginal bleeding.

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

[0309] 2. Research program

[0310] 2.1 Research category and data collection method

[0311] This study is a self-control study. By checking the patient's outpatient and inpatient medical records, the use of traditional Chinese medicine and western medicine during diagnosis and treatment, and ultrasound information, the changes of intrauterine hematometra within 2 weeks and 4 weeks were observed. Only patients taking Bushen Huoxue Decoction or its modified prescription (the medicinal ingredients remain unchanged, and the medicinal amount is adjusted within a small range) were included in this study, and the western medicine treatment plan was not limited. The prescription of Bushen Huoxue Decoction is as follows: Radix Cuscutae 12 g, Radix Dipsaci 10 g, Radix Salviae Miltiorrhizae 15 g, Caulis Spatholobi 15 g, Radix Astragali 20 g, Pericarpium Citri Reticulatae 5 g, and Rhizoma Dioscoreae 15 g.

[0312] 2.2 Hematometra area measurement: The longest diameter and the widest diameter perpendicular to it of intrauterine hematometra under ultrasound were taken as length and width respectively, and the hematometra area = length × width.

[0313] 2.3 Ultrasound information recording time: The interval between the previous ultrasound before taking Bushen Huoxue Decoction and the current ultrasound was 2 weeks (1-14 days) or 4 weeks (15-24 days).

[0314] 2.4 Efficacy index: The changes of hematometra area within 2 weeks and 4 weeks were taken as the main efficacy index.

[0315] 3. Statistical method

[0316] The hematometra area before the first visit, within 2 weeks of medication, and within 4 weeks of medication was entered into Excel software for arrangement, and SPSS 25.0 statistical software package was used for statistical analysis. According to whether the hematometra area before and after the intervention met the normal distribution, paired sample t test or paired sample Wilcoxon signed rank test was used. P<0.05 was considered statistically significant.

[0317] 4. Research results

[0318] A total of 111 patients with early and middle pregnancy combined with intrauterine hematometra were collected in this study, among which 31 patients did not have a follow-up ultrasound after the first visit, 82 patients had ultrasound results within 2 weeks, and 66 patients had ultrasound results within 2 weeks and 4 weeks.

[0319] 4.12-week intrauterine hematometra area

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

[0321] Table 9 Changes of intrauterine hematometra area within 2 weeks of medication M (P25-P75)

[0322]

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

[0324] The results show that the combined use of the kidney and blood circulation prescription for 2 weeks can reduce the area of blood accumulation in the uterine cavity.

[0325] 4.2 Area of blood accumulation in the uterine cavity within 4 weeks

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

[0327] Table 10 Changes in the area of blood accumulation in the uterine cavity after 4 weeks of administration M (P25-P75)

[0328]

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

[0330] The results show that the combined use of the kidney and blood circulation prescription for 4 weeks can reduce the area of blood accumulation in the uterine cavity.

[0331] 4.3 Comparison of the difference in the area of blood accumulation in the uterine cavity

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

[0333] Table 11 Comparison of the difference in the area of blood accumulation in the uterine cavity after 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 P<0.001, which did not meet the normality distribution. The two paired sample Wilcoxon signed rank test was used, P<0.001, and the difference was statistically significant.

[0336] It can be seen that the difference in the change of blood accumulation area between 2 weeks and 4 weeks after treatment with the combined kidney and blood circulation prescription has statistical significance (Z=-4.381, P<0.001), indicating that the longer the combined treatment time, the greater the reduction in the area of blood accumulation in the uterine cavity.

[0337] Example 8

[0338] This example studies the pregnancy-promoting effect of the drug composition prepared in Example 1 (kidney and blood circulation prescription 1) on CBA / J x DBA / 2 abortion mice treated with pre-pregnancy administration.

[0339] Based on the CBA / J x DBA / 2 recurrent abortion mouse model, the effects of different pre-pregnancy administration of the kidney and blood circulation prescription (pre-pregnancy administration, pre-pregnancy administration to G7.5 days, pre-pregnancy administration to G13.5 days) on the pregnancy rate, embryo loss rate, and uterine tissue morphology of the model were investigated.

[0340] 1. Materials and methods

[0341] 1.1 Material

[0342] 1.1.1 Experimental system

[0343] DBA / 2 male mice 20, CBA / J female mice 40, 8 weeks old, SPF level, purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd. (License No.: SCXK (Jing) 2021-0011). After purchasing the animals, 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) for caging and feeding. All experimental operations comply with the relevant provisions of animal welfare.

[0344] 1.1.2 Test substance

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

[0346] 1.1.3 Experimental reagents and instruments

[0347] The same as Example 3.

[0348] 1.2 Method

[0349] 1.2.1 Animal modeling and grouping

[0350] The modeling method is the same as Example 3. 40 mature unmated female CBA / J mice in proestrus were selected by vaginal smear and randomly divided into the following 4 groups: model control group, pre-pregnancy drug administration group, pre-pregnancy drug administration to G7.5 days group, and pre-pregnancy drug administration to G13.5 days group, 10 in each group.

[0351] 1.2.2 Dosing regimen

[0352] Taking the recommended dose for the human body (body weight 60 kg) as the standard, the conversion factor of adult and mouse is 9.01, and the equivalent dose conversion formula is: mouse dose = (recommended dose for human body / 60 kg) x 9.01, for gavage administration.

[0353] Pure water was administered at 10 mL / kg; the dose of Bushen Huoxue Decoction solution was the human dose (92 g crude drug per person per day), and after conversion, the mouse crude drug administration dose was 13.82 g crude drug / kg / d, administered at 10 mL / kg.

[0354] Model control group: gavage with pure water for 1 week, once a day, and after successful cohabitation (G0.5 days), continue to give pure water to G13.5 days.

[0355] Pre-pregnancy drug administration group: gavage with Bushen Huoxue Decoction at least 7 days before cohabitation, once a day, and after successful cohabitation (G0.5 days), start continuous administration of pure water to G13.5 days, once a day.

[0356] Pre-pregnancy administration to G7.5 days group: intragastrically administered ShuJenHuoXue recipe at least 7 days before mating, once a day, after successful mating (G0.5 days), continuously administered ShuJenHuoXue recipe to G7.5 days, and continuously administered pure water to G13.5 days from G8.5 days.

[0357] Pre-pregnancy administration to G13.5 days group: intragastrically administered ShuJenHuoXue recipe at least 7 days before mating, once a day, continuously administered ShuJenHuoXue recipe to G13.5 days after successful mating (G0.5 days).

[0358] The specific administration procedure of each group is shown in the following figure Figure 7 After each group was administered with the corresponding liquid for 7 days, the female mice in estrus were screened by vaginal smear, and mating was performed at a ratio of 2:1 (female:male). The vaginal plugs were observed at 8:00 the next day to detect the pregnant mice, which were recorded as G0.5 days. The female mice with successful mating were separated from those without successful mating, and the former were not mated again, while the latter were continuously mated and administered with the corresponding test drug solution. If mating was still unsuccessful after 2 estrus cycles, it was considered as mating failure. All mice in this experiment were successfully mated to form the experimental groups.

[0359] 1.2.3 Preparation of test drug solution

[0360] The same as Example 3.

[0361] 1.2.4 Index detection

[0362] At G13.5 days, the mice were anesthetized with 5% isoflurane, and blood was taken from the abdominal aorta after anesthesia. The serum was obtained by storing the blood in a centrifuge tube without sodium citrate. After blood collection, the uterus was quickly dissected, and the appearance and morphology were recorded by photography. The uterus was then cut along the longitudinal axis at the uterine ligament, and the total embryo number, abortion embryo number, 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 x 100%;

[0364] Embryo loss rate = abortion embryo number / total embryo number x 100%.

[0365] 1.3 Data statistics

[0366] The same as Example 3.

[0367] 2. Results

[0368] 2.1 Pregnancy rate

[0369] The results are shown in the following table. Pre-pregnancy administration of ShuJenHuoXue recipe in different ways 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 following table. Pre-pregnancy administration of the kidney-tonifying and blood-activating recipe in different ways significantly reduced the embryo 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 Uterus (including embryos) morphology

[0378] The uterus (including embryo) tissue morphology of mice administered with the kidney-tonifying and blood-activating recipe before pregnancy in different ways is shown in the following table. Figure 8

[0379] The model group showed black-brown absorbed embryos, visible blood stasis, uneven size, and partial or even complete disappearance of embryos; the uterine congestion of each pre-pregnancy administration group was significantly reduced, the total number of embryos increased, most of the embryos were full and uniform, and showed light red color, with occasional black-brown absorbed embryos.

[0380] The above results show that pre-pregnancy administration of the kidney-tonifying and blood-activating recipe can promote pregnancy and improve pregnancy rate, and post-pregnancy administration of the recipe can reduce embryo loss rate. The kidney-tonifying and blood-activating recipe can both assist pregnancy before pregnancy and promote fetal comfort after pregnancy.​

Claims

1. A traditional Chinese medicine composition, characterized in that, The active ingredients thereof are composed of the following weight parts of medicinal materials: Cuscuta 6~12 parts, Dipsacus 9~15 parts, Salvia 10~15 parts, Spatholobus 9~15 parts, Astragalus 9~30 parts, Tangerine Peel 3~10 parts, Yam 15~30 parts; Or, Cuscuta 6~12 parts, Radix Morindae Officinalis 3-10 parts, Salvia 10~15 parts, Spatholobus 9~15 parts, Astragalus 9~30 parts, Tangerine Peel 3~10 parts, Yam 15~30 parts; Or, Cuscuta 6~12 parts, Dipsacus 9~15 parts, Salvia 10~15 parts, Sanchi 3-9 parts, Astragalus 9~30 parts, Tangerine Peel 3~10 parts, Yam 15~30 parts; Or, Cuscuta 6~12 parts, Radix Morindae Officinalis 3-10 parts, Salvia 10~15 parts, Sanchi 3-9 parts, Astragalus 9~30 parts, Tangerine Peel 3~10 parts, Yam 15~30 parts.

2. The traditional Chinese medicine composition of claim 1, wherein, The active ingredients thereof are composed of the following weight parts of medicinal materials: Cuscuta 12 parts, Dipsacus 10 parts, Salvia 15 parts, Spatholobus 15 parts, Astragalus 20 parts, Tangerine Peel 5 parts, Yam 15 parts; Or, Cuscuta 12 parts, Radix Morindae Officinalis 9 parts, Salvia 15 parts, Spatholobus 15 parts, Astragalus 20 parts, Tangerine Peel 5 parts, Yam 15 parts; Or, Cuscuta 12 parts, Dipsacus 10 parts, Salvia 15 parts, Sanchi 9 parts, Astragalus 20 parts, Tangerine Peel 5 parts, Yam 15 parts; Or, Cuscuta 12 parts, Radix Morindae Officinalis 9 parts, Salvia 15 parts, Sanchi 9 parts, Astragalus 20 parts, Tangerine Peel 5 parts, Yam 15 parts.

3. The traditional Chinese medicine composition of any one of claims 1-2, wherein, The Cuscuta is salt Cuscuta, and the Radix Morindae Officinalis is salt Radix Morindae Officinalis.

4. The preparation method of the traditional Chinese medicine composition according to any one of claims 1-2, characterized in that, The method comprises the following steps: extracting the medicinal materials by a solvent extraction method to obtain the traditional Chinese medicine composition; and the solvent is selected from water and an ethanol aqueous solution.

5. The preparation method of the traditional Chinese medicine composition according to claim 4, characterized in that, The solvent extraction method comprises decoction, reflux extraction, immersion, and percolation.

6. The preparation method of the traditional Chinese medicine composition according to claim 4, characterized in that, The method comprises the following steps: The medicinal materials are weighed according to a prescription amount, a solvent is added according to a predetermined solid-liquid ratio, and the solvent in the filtrate is recovered after filtration and concentration to obtain the traditional Chinese medicine composition; Or, the medicinal materials are weighed according to a prescription amount, a solvent is added according to a predetermined solid-liquid ratio, and the solvent in the filtrate is recovered after filtration, concentration, and addition of ethanol, and then the precipitate is removed to obtain the traditional Chinese medicine composition.

7. The preparation method of the traditional Chinese medicine composition according to claim 6, characterized in that, The solid-liquid ratio is 1 g of medicinal materials: 10±5 ml of solvent; And / or, the extraction is performed 1-3 times; And / or, the extraction is performed for 0.5-3 hours; And / or, the extraction is performed at a temperature of 80-100 ℃; And / or, the filtrate is concentrated to a relative density of 1.10-1.30, and then ethanol is added; And / or, the filtrate is concentrated, and then ethanol is added to a volume percentage of 60-75%; And / or, the standing time after the addition of ethanol is 8-24 hours.

8. The preparation method of the traditional Chinese medicine composition according to claim 7, characterized in that, The extraction is performed 2 times, the solid-liquid ratio for the first extraction is 1 g: 10±3 ml, and the solid-liquid ratio for the second extraction is 1 g: 8±3 ml; And / or, the extraction is performed for 1-2 hours; And / or, the extraction is performed at a temperature of 90-100 ℃; And / or, the filtrate is concentrated to a relative density of 1.15, and then ethanol is added; And / or, the filtrate is concentrated, and then ethanol is added to a volume percentage of 65%; And / or, the standing time after the addition of ethanol is 12 hours.

9. The preparation method of the traditional Chinese medicine composition according to claim 6, characterized in that, The method further comprises the step of preparing the traditional Chinese medicine composition into a freeze-dried powder or granules, The step of preparing the traditional Chinese medicine composition into a freeze-dried powder is as follows: taking the filtrate, concentrating to obtain an extract, and drying the extract by freeze-drying to obtain a freeze-dried powder. The step of preparing the traditional Chinese medicine composition into granules is as follows: taking the filtrate, concentrating to obtain a clear extract with a relative density of 1.05-1.10, drying, adding a filler, and wet granulating to obtain granules.

10. The preparation method of the traditional Chinese medicine composition according to claim 9, characterized in that, The filler is malt dextrin, and the relative density is measured at 60°C.

11. A medicament, characterized by comprising: The application further comprises the traditional Chinese medicine composition of any one of claims 1-3 and pharmaceutically acceptable excipients.

12. The medicament according to claim 11, wherein The administration route of the medicine is oral.

13. The medicament according to claim 12, wherein The dosage form of the medicine is capsules, granules, tablets, oral liquids, mixtures or syrup.

14. Use of the traditional Chinese medicine composition of any one of claims 1-3 or the medicine of any one of claims 11-13 in the preparation of a medicine for tonifying kidney and activating blood.

15. The use according to claim 14, wherein the compound is ###0005### The medicine for tonifying kidney and activating blood is used for preventing and / or treating recurrent pregnancy loss.

16. The use of claim 15, wherein, The medicine for tonifying kidney and activating blood is used for pre-pregnancy assisted pregnancy and / or post-pregnancy anti-miscarriage.

17. The use of claim 15, wherein the compound is ###00010### 15 The medicine for tonifying kidney and activating blood is used for recurrent pregnancy loss combined with pre-thrombotic state and intrauterine hemorrhage. The medicine for tonifying kidney and activating blood is used for recurrent pregnancy loss combined with pre-thrombotic state and intrauterine hemorrhage.

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