Application of a traditional Chinese medicine composition in the preparation of antithrombotic drugs

Abstract

The present invention belongs to the field of medical technology, and specifically relates to the use of a traditional Chinese medicine composition in the preparation of an antithrombotic drug. The present invention relates to the use of a traditional Chinese medicine composition in the preparation of an antithrombotic drug, wherein the components of the traditional Chinese medicine composition include Cuscuta australis root, Isatis indigotica root, gypsum, honeysuckle, bupleurum root, scutellaria baicalensis, and liquorice. Animal experimental results show that the traditional Chinese medicine composition of the present invention has superior antithrombotic ability to aspirin and can effectively prevent and treat thrombosis caused by carrageenan. The traditional Chinese medicine composition of the present invention can significantly prolong coagulation time, has a good anticoagulant effect, and has strong fibrinolytic activity and excellent thrombolytic effect, showing significant application prospects in the treatment of thrombotic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the application of a traditional Chinese medicine composition in the preparation of an antithrombotic drug. Background Art

[0002] Thrombosis occurs when formed elements in circulating blood form abnormal clots within blood vessels, or deposits form on the heart or blood vessel walls. Thrombosis-induced vascular obstruction is the primary mechanism leading to cardiovascular and cerebrovascular emergencies, and antithrombotic therapy has become a key strategy in the treatment of cardiovascular and cerebrovascular diseases.

[0003] Antithrombotic drugs can be divided into anticoagulants, antiplatelet aggregation drugs, and thrombolytic drugs. Clinically, antiplatelet drugs such as aspirin and clopidogrel are primarily used for the prevention and treatment of thromboembolic diseases, with prevention being the primary approach. Although these drugs have some effects, their therapeutic effects are limited and unsatisfactory.

[0004] Chinese invention patent CN104474021A discloses a Chinese medicine composition for treating superficial fever and its preparation method. The composition uses natural edible botanicals as raw materials and utilizes a carefully selected combination of herbs to effectively treat superficial fever by strengthening the body's constitution, clearing away heat and detoxifying. Studies have shown that the composition has a good analgesic effect on writhing mice, a good inhibitory effect on croton oil-induced ear swelling in mice, and can inhibit Escherichia coli endotoxin-induced fever in rabbits. The composition also has a certain antibacterial effect and can degrade endotoxins in vitro. It has been clinically used to treat colds, fever, upper respiratory tract infections, sore throats, tonsillitis, mumps, acute pharyngitis, acute bronchitis, and simple pneumonia, achieving good results. However, to date, there have been no reports on the use of the composition in antithrombotic settings. Summary of the Invention

[0005] The present invention aims to solve the problems in the prior art and provides an application of a traditional Chinese medicine composition in the preparation of an antithrombotic drug.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The invention discloses an application of a traditional Chinese medicine composition in the preparation of an antithrombotic drug. The components of the traditional Chinese medicine composition include Cudrania tricuspidata root, Isatis indigotica root, Gypsum Fibrosum, Honeysuckle, Bupleurum Root, Scutellaria Baicalensis and Licorice.

[0008] Preferably, the traditional Chinese medicine composition comprises the following components by weight: 400-600 parts of Cudrania root, 200-300 parts of Isatis indigotica root, 250-400 parts of Gypsum, 160-240 parts of Honeysuckle, 130-170 parts of Bupleurum, 130-170 parts of Scutellaria, and 40-60 parts of Licorice.

[0009] More preferably, the Chinese medicine composition comprises the following components by weight: 450-550 parts of Cudrania root, 200-250 parts of Isatis indigotica root, 300-350 parts of gypsum, 180-220 parts of honeysuckle, 150-170 parts of bupleurum, 160-170 parts of scutellaria, and 45-55 parts of liquorice.

[0010] Further preferably, the Chinese medicine composition comprises the following components in parts by weight: 500 parts of Cudrania root, 250 parts of Isatis indigotica root, 333 parts of gypsum, 200 parts of honeysuckle, 167 parts of Bupleurum, 167 parts of Scutellaria, and 50 parts of Licorice.

[0011] Preferably, the method for preparing the active ingredient of the traditional Chinese medicine composition comprises the following steps:

[0012] (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, clean and select, and grind the roots of Cuscuta australis, Radix Isatidis, Gypsum, Flos Lonicerae, Radix Bupleuri, Radix Scutellariae, and Radix Glycyrrhizae into coarse powder respectively;

[0013] (2) extracting the southern isatis root coarse powder with ethanol, combining the extracts, filtering, and using the residue and filtrate for later use;

[0014] (3) combining the medicinal residue from step (2) with the remaining coarse powder, adding water to boil, combining the decoctions, filtering, concentrating the filtrate, adding ethanol, and combining the filtrate with the filtrate from step (2), allowing to stand, filtering, and obtaining a clear paste;

[0015] (4) Finally, the clear paste of step (3) is concentrated and dried to obtain dry paste powder, that is, the active ingredient of the traditional Chinese medicine composition.

[0016] Preferably, the volume fraction of the ethanol in step (2) is 50-70%, the volume of the ethanol is 1.5-3 times the weight of the southern isatis root powder, the number of extractions is 1-3 times, and the extraction time is 1-3 hours.

[0017] Preferably, the volume of water added in step (3) is 1.5-3 times the total weight of the medicinal residue and the remaining coarse powder, the number of decoctions is 2-4 times, the decoction time is 1.5-2.5 hours, the filtrate is concentrated to a relative density of 1.38-1.40 measured at 70-90°C, the filtrate is concentrated and then cooled to room temperature, ethanol is added to make the alcohol content reach 50-70%, and the standing time is 20-26 hours.

[0018] Preferably, the concentration in step (4) is performed to a relative density of 1.38-1.40 when measured at 70-80°C.

[0019] Preferably, the dosage form of the drug is granules, capsules, tablets, powders, oral liquids, pills, tinctures, syrups, suppositories, gels, sprays or injections.

[0020] Preferably, the preparation method of the granules comprises the following steps:

[0021] (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, radix bupleuri, radix scutellariae, and radix glycyrrhizae into coarse powder;

[0022] (2) Add 1.5-3 times the amount of 50-70% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for 1-3 times, each time for 1-3 hours. Combine the extracts, filter, and use the residue and filtrate for later use;

[0023] (3) combining the medicinal residue from step (2) with the remaining coarse powder, adding 1.5-3 times the amount of water and decocting for 2-4 times, each time for 1.5-2.5 hours, combining the decoctions, filtering, concentrating the filtrate to a relative density of 1.38-1.40 measured at 70-90° C., cooling to room temperature, adding ethanol to make the alcohol content reach 50-70%, combining with the filtrate from step (2), standing for 24 hours, filtering, and obtaining a clear paste;

[0024] (4) Concentrating the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 70-80° C., drying to obtain a dry paste powder for later use;

[0025] (5) Add appropriate pharmaceutically acceptable excipients to the dry paste powder of step (4), granulate, and dry to obtain the product.

[0026] Preferably, the preparation method of the capsule comprises the following steps:

[0027] (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, radix bupleuri, radix scutellariae, and radix glycyrrhizae into coarse powder;

[0028] (2) Add 1.5-3 times the amount of 50-70% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for extraction twice, each time for 2 hours. Combine the extracts, filter, and use the residue and filtrate for later use;

[0029] (3) combining the medicinal residue of step (2) with coarse powder of Cudrania tricuspidata root, gypsum, honeysuckle, bupleurum root, scutellaria baicalensis, and liquorice, adding 1.5-3 times the amount of water and boiling for 3 times, each time for 2 hours, combining the decoctions, filtering, and concentrating the filtrate to a clear paste having a relative density of 1.38-1.40 measured at 80° C., cooling to room temperature, adding ethanol to make the alcohol content reach 50-70%, combining with the filtrate of step (2), standing for 24 hours, filtering, and obtaining a clear paste for use;

[0030] (4) Concentrating the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 80° C., drying to obtain a dry paste powder for later use;

[0031] (5) Add appropriate pharmaceutically acceptable excipients to the dry paste powder obtained in step (4) to granulate, dry, mix, and put into capsules to obtain the product.

[0032] Preferably, the method for preparing the pills comprises the following steps:

[0033] (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, radix bupleuri, radix scutellariae, and radix glycyrrhizae into coarse powder;

[0034] (2) Add 1.5-3 times the amount of 50-70% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for extraction twice, each time for 2 hours. Combine the extracts, filter, and use the residue and filtrate for later use;

[0035] (3) combining the medicinal residue of step (2) with coarse powder of Cudrania tricuspidata root, gypsum, honeysuckle, bupleurum root, scutellaria baicalensis, and liquorice, adding 1.5-3 times the amount of water and boiling for 3 times, each time for 2 hours, combining the decoctions, filtering, and concentrating the filtrate to a clear paste having a relative density of 1.38-1.40 measured at 80° C., cooling to room temperature, adding ethanol to make the alcohol content reach 50-70%, combining with the filtrate of step (2), standing for 24 hours, filtering, and obtaining a clear paste for use;

[0036] (4) Concentrating the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 80° C., drying to obtain a dry paste powder for later use;

[0037] (5) The dry paste powder obtained in step (4) is made into pills according to a conventional method.

[0038] Preferably, the excipient is selected from one or more of sucrose, mannitol, microcrystalline cellulose, starch, lactose, dextrin, polyvinyl pyrrolidone, Tween 80, calcium phosphate, polyethylene glycol, silicon dioxide, magnesium stearate, soluble starch stevioside, aspartame, protein sugar, acetamidopropyl betaine, xylitol and fructose.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The present invention provides an application of a traditional Chinese medicine composition in the preparation of an antithrombotic drug. Animal experimental results show that the traditional Chinese medicine composition of the present invention has an antithrombotic ability superior to that of aspirin, can effectively prevent and treat thrombosis caused by carrageenan, can significantly prolong coagulation time, has a good anticoagulant effect, and has an excellent thrombolytic effect.

[0041] (2) The fibrinolytic activity experimental results show that the Chinese herbal composition of the present invention has strong fibrinolytic activity within the prescription range. Only the specific Chinese herbal composition prescription of the present invention can have excellent fibrinolytic activity. The Chinese herbal composition of the present invention has significant application prospects in the treatment of thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The black tail status of mice in the model group at 72 hours.

[0043] Figure 2 The black tail status of mice taking the traditional Chinese medicine composition of the present invention at 72 hours.

[0044] Figure 3 The black tail status of mice in the aspirin group at 72 hours. DETAILED DESCRIPTION

[0045] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0046] Example 1

[0047] Prescription: 500 parts of Cudrania root, 250 parts of Isatis indigotica root, 333 parts of Gypsum, 200 parts of Honeysuckle, 167 parts of Bupleurum, 167 parts of Scutellaria, and 50 parts of Licorice.

[0048] Preparation: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice into coarse powder.

[0049] (2) Add twice the amount of 60% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for extraction twice, each time for 2 hours. Combine the extracts, filter, and set aside the residue and filtrate.

[0050] (3) The medicinal residue of step (2) is combined with coarse powder of Cudrania tricuspidata root, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice, and decocted three times with twice the amount of water, each time for 2 hours. The decoctions are combined and filtered. The filtrate is concentrated to a clear paste having a relative density of 1.38-1.40 measured at 80°C, cooled to room temperature, and ethanol is added to make the alcohol content reach 60%. The mixture is combined with the filtrate of step (2), allowed to stand for 24 hours, filtered, and a clear paste is obtained for use.

[0051] (4) Concentrate the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 80° C., dry it to obtain a dry paste powder for later use.

[0052] (5) Add 666.8 g of sucrose and 166.7 g of dextrin to the dry powder obtained in step (4), granulate, dry, mix, and bag. The above prescription can be used to make 1000 g of granules.

[0053] Example 2

[0054] Prescription: 400 parts of Cudrania root, 200 parts of Isatis indigotica root, 250 parts of Gypsum, 160 parts of Honeysuckle, 130 parts of Bupleurum, 130 parts of Scutellaria, and 40 parts of Licorice.

[0055] Preparation: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice into coarse powder.

[0056] (2) Add 1.5 times the amount of 50% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for extraction three times, each time for 1 hour. Combine the extracts, filter, and set aside the residue and filtrate.

[0057] (3) The medicinal residue of step (2) is combined with coarse powder of Cudrania tricuspidata root, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice, and decocted 4 times with 1.5 times the amount of water, each time for 1.5 hours. The decoctions are combined, filtered, and the filtrate is concentrated to a clear paste having a relative density of 1.38-1.40 measured at 70°C. The mixture is cooled to room temperature, ethanol is added to make the alcohol content reach 50%, and the mixture is combined with the filtrate of step (2), allowed to stand for 24 hours, filtered, and a clear paste is obtained for use.

[0058] (4) Concentrate the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 70° C., dry it to obtain a dry paste powder for later use.

[0059] (5) Add 100 g of starch, 200 g of microcrystalline cellulose, 16 g of magnesium stearate, and 550 g of sucrose to the dry powder obtained in step (4), granulate the mixture, dry it, mix it, and bag it. The above prescription can be used to make 1000 g of granules.

[0060] Example 3

[0061] Prescription: 600 parts of mulberry tree roots, 300 parts of southern isatis root, 400 parts of gypsum, 240 parts of honeysuckle, 170 parts of bupleurum, 170 parts of scutellaria, and 60 parts of licorice.

[0062] Preparation: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice into coarse powder.

[0063] (2) Add 3 times the amount of 70% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for extraction twice, each time for 1 hour. Combine the extracts, filter, and set aside the residue and filtrate.

[0064] (3) The medicinal residue of step (2) is combined with coarse powder of Cudrania tricuspidata root, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice, and decocted twice with 3 times the amount of water, each time for 2.5 hours. The decoctions are combined and filtered. The filtrate is concentrated to a clear paste having a relative density of 1.38-1.40 measured at 90°C, cooled to room temperature, and ethanol is added to make the alcohol content reach 70%. The mixture is combined with the filtrate of step (2), allowed to stand for 24 hours, filtered, and a clear paste is obtained for use.

[0065] (4) Concentrate the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 90° C., dry it to obtain a dry paste powder for later use.

[0066] (5) Add 600 g of soluble starch stevia, 180 g of sucrose, 10 g of calcium phosphate, and 10 g of xylitol to the dry paste powder obtained in step (4), granulate, dry, mix, and bag. The above prescription can be made into 1000 g of granules.

[0067] Comparative Example 1

[0068] Prescription: 200 parts of Cudrania root, 500 parts of Isatis indigotica root, 100 parts of Gypsum, 100 parts of Honeysuckle, 50 parts of Bupleurum, 50 parts of Scutellaria, and 20 parts of Licorice.

[0069] Preparation: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice into coarse powder.

[0070] (2) Add twice the amount of 60% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for extraction twice, each time for 2 hours. Combine the extracts, filter, and set aside the residue and filtrate.

[0071] (3) The medicinal residue of step (2) is combined with coarse powder of Cudrania tricuspidata root, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice, and decocted three times with twice the amount of water, each time for 2 hours. The decoctions are combined and filtered. The filtrate is concentrated to a clear paste having a relative density of 1.38-1.40 measured at 80°C, cooled to room temperature, and ethanol is added to make the alcohol content reach 60%. The mixture is combined with the filtrate of step (2), allowed to stand for 24 hours, filtered, and a clear paste is obtained for use.

[0072] (4) Concentrate the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 80° C., dry it to obtain a dry paste powder for later use.

[0073] (5) Add 657 g of sucrose and 190 g of dextrin to the dry powder obtained in step (4), granulate, dry, mix, and bag. The above prescription can be made into 1000 g of granules.

[0074] Comparative Example 2

[0075] Prescription: 800 parts of mulberry tree roots, 100 parts of southern isatis root, 200 parts of gypsum, 200 parts of honeysuckle, 100 parts of bupleurum, 100 parts of scutellaria, and 80 parts of licorice.

[0076] Preparation: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice into coarse powder.

[0077] (2) Add twice the amount of 60% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for extraction twice, each time for 2 hours. Combine the extracts, filter, and set aside the residue and filtrate.

[0078] (3) The medicinal residue of step (2) is combined with coarse powder of Cudrania tricuspidata root, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice, and decocted three times with twice the amount of water, each time for 2 hours. The decoctions are combined and filtered. The filtrate is concentrated to a clear paste having a relative density of 1.38-1.40 measured at 80°C, cooled to room temperature, and ethanol is added to make the alcohol content reach 60%. The mixture is combined with the filtrate of step (2), allowed to stand for 24 hours, filtered, and a clear paste is obtained for use.

[0079] (4) Concentrate the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 80° C., dry it to obtain a dry paste powder for later use.

[0080] (5) Add 615 g of sucrose and 180 g of dextrin to the dry powder obtained in step (4), granulate, dry, mix, and bag. The above prescription can be used to make 1000 g of granules.

[0081] Comparative Example 3

[0082] Prescription: 573 parts of honeysuckle, 477 parts of bupleurum, 477 parts of scutellaria, and 140 parts of licorice.

[0083] Preparation: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; grind honeysuckle, bupleurum, scutellaria, and liquorice into coarse powder.

[0084] (2) Combine the coarse powders of honeysuckle, bupleurum, scutellaria, and liquorice, add twice the amount of water and boil for three times, each time for 2 hours. Combine the decoctions, filter, and concentrate the filtrate to a clear paste with a relative density of 1.38-1.40 at 80°C. Cool to room temperature, add ethanol to make the alcohol content reach 60%, let it stand for 24 hours, filter, and obtain a clear paste for use.

[0085] (3) Concentrate the clear paste obtained in step (2) to a thick paste having a relative density of 1.38-1.40 measured at 80° C., dry it to obtain a dry paste powder for later use.

[0086] (4) Add 668 g of sucrose and 170 g of dextrin to the dry powder obtained in step (3), granulate, dry, mix, and bag. The above prescription can be used to make 1000 g of granules.

[0087] Experiment 1 Effect of the Chinese herbal composition of the present invention on carrageenan-induced thrombosis in mice

[0088] 1. Experimental Materials

[0089] 1.1 Mice: 35 male, SPF Kunming mice, provided by the Experimental Animal Center of Sichuan Academy of Traditional Chinese Medicine (Production License No. SCXK(Chuan)2018-19), weighing 17-20 g. They were housed in individual cages at the Experimental Animal Center of Sichuan Academy of Traditional Chinese Medicine with free access to food and water. Bedding was changed regularly at room temperature of 22-25°C, relative humidity of 50 ± 5%, and a 12-h light and dark cycle.

[0090] 1.2 Chinese herbal composition of the present invention: A dry paste powder was prepared according to the raw material formula and preparation process of Example 1. The yellow powder was obtained. 1 g of dry paste powder was equivalent to 10.01 g of the original drug. The dosage in this experiment was calculated based on g / kg of the original drug. Before use, the Chinese herbal composition prepared in Example 1 was dissolved in a 0.5% sodium carboxymethyl cellulose aqueous solution to prepare a suspension of the desired concentration for later use.

[0091] 1.3 0.9% sodium chloride injection: batch number L122070408, purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0092] 1.4 Sodium carboxymethyl cellulose: Batch number 2014102701, purchased from Chengdu Kelong Chemical Reagent Factory. Immediately before use, prepare a 0.5% sodium carboxymethyl cellulose aqueous solution with deionized water.

[0093] 1.5 Carrageenan: Batch No. SLBL2288V, purchased from Sigma. Dissolve in 0.9% sodium chloride injection immediately before use.

[0094] 1.6 Aspirin: Batch No. BJ70805, 100 mg / tablet, purchased from Bayer HealthCare Co., Ltd. Before use, grind and prepare a suspension of the desired concentration with 0.5% sodium carboxymethylcellulose aqueous solution.

[0095] 1.7 Waterproof digital caliper: model IP54, measuring range 0-150mm, accuracy ±0.03mm, purchased from Chengdu Xinchengliang Tools Co., Ltd.

[0096] 2. Modeling method

[0097] Thirty-five male Kunming mice weighing 17-20 g were fed for one day and then randomly divided according to body weight into a model group, a group receiving the Chinese herbal composition of the present invention (6.23 g / kg), and an aspirin group (20 mg / kg). Each group consisted of 11-12 mice. Each group was gavaged with the vehicle or the corresponding drug twice daily for five consecutive doses at a rate of 0.1 mL / 10 g. After the third dose, the mice were subcutaneously injected with carrageenan in a saline solution (40 mg / kg, 0.1 mL / 10 g) in the lumbar region. The room temperature was maintained below 18°C. After 72 hours, the tails of the mice were observed for thrombosis formation, and the thrombus formation rate (%) and relative thrombus length (%) were calculated. Data were expressed as x ± s, and one-way analysis of variance was performed using SPSS 18.0 software.

[0098] The calculation formulas for thrombosis rate and relative thrombus length are as follows:

[0099] Thrombosis rate (%) = number of mice with thrombosis ÷ number of mice in each group × 100%

[0100] Relative thrombus length (%) = thrombus length ÷ tail length × 100%

[0101] 3. Experimental Results

[0102] The experimental results are shown in Table 1 and Figure 1-3 . Figure 1 The black tail status of mice in the model group at 72 h; Figure 2 The black tail status of mice in the Chinese medicine composition group at 72 hours; Figure 3 The black tail status of mice in the aspirin group at 72 hours.

[0103] From Table 1 and Figure 1-3 It can be seen that after the carrageenan was injected, visible thrombi gradually formed on the tails of the mice, and the relative length of the thrombi reached 47%; one mouse in the Chinese medicine composition group did not form thrombi, and the relative lengths of the thrombi of the remaining 11 mice were significantly reduced, which was statistically significant compared with the model group. In addition, the anti-thrombotic ability of the Chinese medicine composition group was better than that of the aspirin group, and the group could effectively prevent and treat thrombosis caused by carrageenan.

[0104] Table 1 Effect of the Chinese medicine composition of the present invention on carrageenan-induced thrombosis in mice

[0105]

[0106] Note: Compared with the model group, * Indicates P<0.05.

[0107] Experiment 2 Effect of the Chinese herbal composition of the present invention on anticoagulant activity

[0108] 1. Experimental Materials

[0109] 1.1 Mice: 46 male, SPF Kunming mice, provided by the Experimental Animal Center of Sichuan Academy of Traditional Chinese Medicine, production license number SCXK(Chuan)2018-19, weighing 17-20 g. They were housed in individual cages at the Experimental Animal Center of Sichuan Academy of Traditional Chinese Medicine with free access to food and water, and regular bedding changes. The room temperature was 22-25°C, the relative humidity was 50 ± 5%, and the light and dark cycles were 12 h each.

[0110] 1.2 Chinese herbal composition of the present invention: A dry paste powder was prepared according to the raw material formula and preparation process of Example 1. The yellow powder was obtained. 1 g of dry paste powder was equivalent to 10.01 g of the original drug. The dosage in this experiment was calculated based on g / kg of the original drug. Before use, the Chinese herbal composition prepared in Example 1 was dissolved in a 0.5% sodium carboxymethyl cellulose aqueous solution to prepare a suspension of the desired concentration for later use.

[0111] 1.3 0.9% sodium chloride injection: batch number L122070408, purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0112] 1.4 Sodium carboxymethyl cellulose: Batch number 2014102701, purchased from Chengdu Kelong Chemical Reagent Factory. Immediately before use, prepare a 0.5% sodium carboxymethyl cellulose aqueous solution with deionized water.

[0113] 2. Modeling method

[0114] 46 male Kunming mice weighing 17-20g were fed adaptively for 1 day and then randomly divided into a control group, a low-dose group of the Chinese medicine composition of the present invention (6.23g / kg), a medium-dose group of the Chinese medicine composition of the present invention (12.46g / kg) and a high-dose group of the Chinese medicine composition of the present invention (24.92g / kg). Each group had 11-12 mice. Each group was gavaged with a solvent or corresponding drug, 2 times / d, for 5 consecutive times, 0.1mL / 10g. The control group was gavaged with an equal amount of normal saline for 4 consecutive weeks. One day after the gavage, the mice were anesthetized with ether, 0.2mL of blood was collected from the tail tip, and the clotting time was calculated. The data were used One-way ANOVA was performed using SPSS 18.0 software.

[0115] 3. Experimental Results

[0116] The experimental results are shown in Table 2. As can be seen from Table 2, the three dose groups of mice gavaged with the drug can significantly prolong the coagulation time, all reaching a significant difference level. Among them, the high-dose group of the Chinese herbal composition of the present invention has the best anticoagulant effect, which is 3.06 times that of the control group. The anticoagulant effect of the medium-dose group of the Chinese herbal composition of the present invention is 2.05 times that of the control group. The anticoagulant effect of the low-dose group of the Chinese herbal composition of the present invention is 1.21 times that of the control group.

[0117] Table 2 Effect of the anticoagulant activity of the Chinese medicine composition of the present invention

[0118]

[0119] Note: Compared with the control group, * indicates P < 0.05, ** Indicates P<0.01.

[0120] Experiment 3 Effect of the Chinese herbal composition of the present invention on fibrinolytic activity

[0121] 1. Experimental Materials

[0122] 1. Experimental Materials

[0123] 1.1 Sample: According to the raw material formula and preparation process of Examples 1-3 and Comparative Examples 1-3, dry paste powder was prepared. The yellow powder was obtained. 1g of dry paste powder was equivalent to 10.01g of native drug, 9.78g of native drug, 9.75g of native drug, 6.67g of native drug, 7.71g of native drug, and 9.73g of native drug, respectively. Before use, the dry paste powder prepared in Example 1 and Comparative Examples 1-3 was added to a triangular flask according to high, medium, and low doses. 20mL of pure water was added, and the mixture was ultrasonically soaked for 5min. The supernatant was centrifuged and used for fibrinolytic activity detection.

[0124] 1.2 Solution A: Weigh 121.1 g of tris (hydroxymethyl)aminomethane (Tris) and dissolve it in deionized water. Adjust the pH to 8.0 with concentrated hydrochloric acid. Finally, dilute to volume with 1 L of deionized water to obtain the mother solution. Take 10 mL of the mother solution and dilute to 500 mL with deionized water to obtain Solution A.

[0125] 1.3B solution: 0.9% sodium chloride injection, batch number L122070408, purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0126] 1.4 Fibrinogen solution: Weigh 1 g of fibrinogen powder (batch number 140626-202213, purchased from China Food and Drug Inspection Institutes) and dissolve it in 20 mL of Solution A until fully dissolved to obtain a 50 mg / mL fibrinogen solution.

[0127] 1.5 Thrombin solution: Dissolve 1000 U of thrombin (batch number 140625-202114, purchased from China Food and Drug Inspection Institute) in 10 mL of solution A and allow it to fully dissolve to obtain 100 U / mL thrombin solution.

[0128] 1.6 Standard urokinase solution: Dissolve urokinase standard (1560 IU / bottle, batch number 140604-202125, purchased from the China Food and Drug Administration) in 2 mL of sterile saline (0.9% w / v) to prepare a 780 IU / mL urokinase standard solution. Transfer 1 mL of this solution to another vial containing 1 mL of 0.9% sodium chloride injection to prepare a 390 IU / mL solution. This was then diluted with 0.9% sodium chloride injection to prepare 195 IU / mL, 97.5 IU / mL, 48.75 IU / mL, and 24.375 IU / mL standard urokinase solutions, respectively.

[0129] 2. Experimental Methods

[0130] 2.1 Preparation of Fibrinogen Plate: Weigh 0.15 g agar (0.75%), 0.1755 g NaCl (0.15 M), and 20 mL of Solution A into a 50 mL Erlenmeyer flask and dissolve the agar. Stopper the flask and heat in a microwave oven to dissolve. When the flask cools to 50°C, add 1 mL of fibrinogen solution and 100 μL of thrombin solution. Mix thoroughly and quickly pour into a sterile plate. Let stand at room temperature for 30 min to form a fibrin clot.

[0131] 2.2 Draw a urokinase standard curve: Use a hole puncher to punch 7 holes on a fibrin plate and mark them; take 10 μL of standard urokinase solution of each concentration into the corresponding hole, let it stand for 10 minutes, and then transfer it to a 37°C constant temperature incubator for reaction. After 18 hours, take it out and measure the two perpendicular diameters of each dissolution circle; set three parallel groups for the above operation, and take the average value after measurement; then use the common logarithm of the product of the perpendicular diameters (S) as the horizontal axis and the common logarithm of the standard urokinase concentration as the vertical axis to make a urokinase enzyme activity standard curve.

[0132] 2.3 Determination of Thrombolytic Activity of Test Samples: Take 10 μL of sample and, following the procedure described in 2.2 above, load onto the same plate. Incubate at 37°C. After 18 hours, measure the two perpendicular diameters of the lysis zone. Calculate the enzyme activity using the standard curve and the dilution concentration. Data are expressed as x ± s, and one-way analysis of variance was performed using SPSS 18.0 software.

[0133] 3. Experimental Results

[0134] The results are shown in Table 3. It can be seen from the results in Table 3 that the fibrinolytic activity of the high-dose group of the traditional Chinese medicine composition in Example 1 of the present invention is 1338 IU / g, the fibrinolytic activity of the medium-dose group is 771 IU / g, and the fibrinolytic activity of the low-dose group is 318 IU / g. The fibrinolytic activities of the low-dose, medium-dose, and high-dose groups of the traditional Chinese medicine compositions in Examples 1-3 were significantly different compared with the control group and Comparative Examples 1-3. Among them, compared with Example 1, the only difference in Comparative Examples 1-2 is the number of parts of the traditional Chinese medicine flavors in the prescription, but the fibrinolytic activity of Example 1 is higher than that of Comparative Examples 1-2, indicating that only the specific number of parts of the traditional Chinese medicine flavors of the present invention has excellent fibrinolytic activity; the difference between Comparative Example 3 and Example 1 is only the different flavors, and similarly, the fibrinolytic activity of Example 1 is also higher than that of Comparative Example 3, indicating that the flavors of the specific traditional Chinese medicine composition of the present invention have stronger fibrinolytic activity and can effectively prevent and treat thrombosis formation.

[0135] Table 3 Influence of the fibrinolytic activity of the traditional Chinese medicine composition of the present invention ( n = 3)

[0136]

[0137] Note: Compared with the control group, ** indicates P < 0.01; compared with Comparative Example 1, aa indicates P < 0.01; compared with Comparative Example 2, bb indicates P < 0.01; compared with Comparative Example 3, cc indicates P < 0.01.

[0138] Experiment 4 Influence of the traditional Chinese medicine composition of the present invention on thrombolytic activity

[0139] 1. Experimental materials

[0140] 1.1 Rats: 60 SD rats, half male and half female, SPF grade, provided by the Experimental Animal Center of Sichuan Academy of Traditional Chinese Medicine, production license number SCXK (Sichuan) 2018-19, body weight 17-20 g. They were housed in separate cages at the Experimental Animal Center of Sichuan Academy of Traditional Chinese Medicine, with free access to food and water, and the bedding was changed regularly. The room temperature was 22-25 °C, the environmental relative humidity was 50 ± 5%, and the light and dark periods were each 12 h.

[0141] 1.2 The traditional Chinese medicine composition of the present invention: The dry extract powder was prepared according to the raw material formula and preparation process of Example 2, yellow powder, 1 g of dry extract powder is equivalent to 8.01 g of crude drug, and the dosage in this experiment was calculated according to g / kg of crude drug. Before use, the traditional Chinese medicine composition prepared in Example 2 was dissolved in a 0.5% sodium carboxymethylcellulose aqueous solution to prepare a suspension with the required concentration for standby.

[0142] 1.3 0.9% sodium chloride injection: batch number L122070408, purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0143] 1.4 Sodium carboxymethyl cellulose: Batch number 2014102701, purchased from Chengdu Kelong Chemical Reagent Factory. Immediately before use, prepare a 0.5% sodium carboxymethyl cellulose aqueous solution with deionized water.

[0144] 2. Modeling method

[0145] After 1 day of adaptive feeding, SD rats were randomly divided into sham operation group, model group, low-dose group of the Chinese medicine composition of the present invention (6.23 g / kg), medium-dose group of the Chinese medicine composition of the present invention (12.46 g / kg) and high-dose group of the Chinese medicine composition of the present invention (24.92 g / kg) according to body weight, with 11-12 mice in each group. After fasting for 12 hours, 350 mg / kg of 10% chloral hydrate was injected intraperitoneally for anesthesia. The hair on the neck was removed and the rats were fully disinfected with iodine. An incision of about 3 cm was made on the skin. The carotid artery was isolated and fully exposed. A 0.5×0.5 cm 2 The filter paper was soaked in 30% ferric chloride solution for 30 seconds and then wrapped around the carotid artery. Tin foil was placed under the filter paper to separate the filter paper from the surrounding tissue to prevent the ferric chloride from damaging the surrounding tissue. The sham operation group was treated with filter paper soaked in normal saline. After 3 minutes, the filter paper was removed and the wrapped artery was rinsed twice with normal saline. The wound was sutured and disinfected with iodine. One day after the operation, each group was gavaged with solvent or corresponding drugs. The control group was gavaged with an equal amount of normal saline once a day for 4 consecutive weeks. One day after the end of gavage, 0.5 cm of the carotid artery at the surgical site was cut, and the filter paper absorbed excess water and weighed to evaluate the thrombolytic effect. The data were used One-way ANOVA was performed using SPSS 18.0 software.

[0146] 3. Experimental Results

[0147] The results are shown in Table 4. The experimental results showed that the blood vessel weight in the sham-operated group was 5.21 g, and in the model group was 14.8 g, demonstrating successful thrombosis modeling. The carotid artery vascular volume of rats in the low-dose, medium-dose, and high-dose groups of the Chinese medicinal composition of the present invention was significantly reduced compared to the model group, demonstrating a thrombolytic effect. The high-dose group had the most pronounced thrombolytic effect, with the blood vessel weight being only 6.83 g.

[0148] Table 4 Effect of the thrombolytic effect of the Chinese medicine composition of the present invention

[0149]

[0150]

[0151] Note: Compared with the control group, * indicates P < 0.05, ** Indicates P<0.01.

[0152] Experiment 5 Effect of the Chinese herbal composition of the present invention on carrageenan-induced paw swelling model in rats

[0153] 1. Experimental Materials

[0154] 1.1 Rats: Male SD rats, SPF grade, provided by the Experimental Animal Center of Sichuan Academy of Traditional Chinese Medicine, production license number SCXK(Chuan)2018-19; or provided by Chengdu Enswell Biotechnology Co., Ltd., production license number SCXK(Xiang)2019-0004; or provided by Beijing Huafukang Biotechnology Co., Ltd., production license number SCXK(Beijing)2019-0008.

[0155] 1.2 Chinese herbal composition of the present invention: A dry paste powder was prepared according to the raw material formula and preparation process of Example 3. The yellow powder was obtained. 1 g of dry paste powder was equivalent to 12.01 g of the original drug. The dosage in this experiment was calculated based on g / kg of the original drug. Before use, the Chinese herbal composition prepared in Example 3 was dissolved in a 0.5% sodium carboxymethyl cellulose aqueous solution to prepare a suspension of the desired concentration for later use.

[0156] 1.3 0.9% sodium chloride injection: batch number L122070408, purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0157] 1.4 Sodium carboxymethyl cellulose: Batch number 2014102701, purchased from Chengdu Kelong Chemical Reagent Factory. Immediately before use, prepare a 0.5% sodium carboxymethyl cellulose aqueous solution with deionized water.

[0158] 1.5 Carrageenan: Batch No. SLBL2288V, purchased from Sigma. Dissolve in 0.9% sodium chloride injection immediately before use.

[0159] 1.6 Prednisone acetate tablets: 5 mg / tablet, batch number LA21250, Zhejiang Xianjun Pharmaceutical Co., Ltd. Grind the tablets immediately before use and prepare a suspension of the desired concentration with 0.5% sodium carboxymethylcellulose aqueous solution.

[0160] 1.7 Toe volume measurement instrument: Model PV-200, purchased from Chengdu Taimeng Technology Co., Ltd.

[0161] 2. Modeling method

[0162] Fifty male SD rats weighing 160-180 g were taken. After adaptive feeding for 2 days, they were randomly divided into a model group, a low-dose group of the Chinese medicine composition of the present invention (4.98 g / kg), a medium-dose group of the Chinese medicine composition of the present invention (9.97 g / kg), a high-dose group of the Chinese medicine composition of the present invention (19.94 g / kg) and a prednisone acetate group (8 mg / kg) according to body weight. Each group had 10 mice. Each group was gavaged with a solvent or corresponding drug, 2 times / d, for 5 consecutive times, 10 mL / 100 g. The volume of the right hind paw of the rat was measured 30 minutes after the last administration. Subsequently, 0.1 mL / rat of 1% carrageenan saline solution was injected under the plantar fascia of the right hind paw of each group except the control group to induce inflammation. The rats in the control group were injected with an equal volume of saline at the same site. The paw volume was measured 0.5 h, 1 h, 2 h, 3 h, 4 h and 6 h after inflammation, and the degree of paw swelling (%) was calculated. The data were used One-way ANOVA was performed using SPSS 18.0 software.

[0163] The formula for calculating foot swelling is as follows:

[0164] Foot swelling (%) = (post-inflammatory foot volume - pre-inflammatory foot volume) / pre-inflammatory foot volume × 100%.

[0165] 3. Experimental Results

[0166] The experimental results are shown in Table 5. As shown in Table 5, the initial paw volume of the model rats was 2.04±0.07 mL. After carrageenan was injected under the plantar fascia, the paws gradually swelled. Compared with the model group, 6 hours after inflammation, the medium-dose and high-dose groups of the Chinese medicinal composition of the present invention showed a significant reduction in paw swelling in the rats. It is speculated that the Chinese medicinal composition of the present invention may improve blood rheology, regulate coagulation and fibrinolysis, and inhibit thrombus formation, thereby unblocking the meridians and ensuring the flow of Qi and blood, significantly reducing paw swelling in rats.

[0167] Table 5 Effects of the Chinese medicine composition of the present invention on carrageenan-induced rat foot swelling model

[0168]

[0169] Note: Compared with the model group, * indicates P < 0.05, ** Indicates P<0.01.

[0170] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a Chinese medicine composition in the preparation of an antithrombotic drug, characterized in that: The Chinese medicine composition is composed of the following components by weight: 400-600 parts of Cudrania root, 200-300 parts of Isatis indigotica root, 250-400 parts of Gypsum, 160-240 parts of Honeysuckle, 130-170 parts of Bupleurum, 130-170 parts of Scutellaria, 40-60 parts of Licorice, The preparation method of the active ingredient of the traditional Chinese medicine composition comprises the following steps: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, clean and select, and grind the roots of Cuscuta australis, southern isatis root, gypsum, honeysuckle, bupleurum, scutellaria baicalensis and liquorice into coarse powder respectively; (2) Extract the southern isatis root powder with ethanol, combine the extracts, filter, and set aside the residue and filtrate; (3) combining the medicinal residue from step (2) with the remaining coarse powder, adding water to boil, combining the decoction, filtering, concentrating the filtrate, adding ethanol, and combining it with the filtrate from step (2), allowing it to stand, filtering, and obtaining a clear paste; (4) Finally, the clear paste of step (3) is concentrated and dried to obtain dry paste powder, that is, the active ingredient of the traditional Chinese medicine composition.

2. The use according to claim 1, characterized in that The traditional Chinese medicine composition is composed of the following components by weight: 450-550 parts of Cudrania root, 200-250 parts of Isatis indigotica root, 300-350 parts of gypsum, 180-220 parts of honeysuckle, 150-170 parts of bupleurum, 160-170 parts of scutellaria, and 45-55 parts of liquorice.

3. The use according to claim 1, characterized in that The volume fraction of ethanol in step (2) is 50-70%, the volume of ethanol is 1.5-3 times the weight of southern isatis root powder, the number of extractions is 1-3 times, and the extraction time is 1-3 hours.

4. The use according to claim 1, characterized in that The volume of water in step (3) is 1.5-3 times the total weight of the medicinal residue and the remaining coarse powder, the number of decoctions is 2-4 times, the decoction time is 1.5-2.5 hours, the filtrate is concentrated to a relative density of 1.38-1.40 at 70-90°C, the filtrate is concentrated and then cooled to room temperature, ethanol is added to make the alcohol content reach 50-70%, and the standing time is 20-26 hours.

5. The use according to claim 1, characterized in that The concentration in step (4) is performed until the relative density is measured at 70-80° C. to be 1.38-1.

40.

6. The use according to any one of claims 1 to 5, characterized in that The dosage form of the medicine is granules, capsules, tablets, powders, oral liquids, pills, tinctures, syrups, suppositories, gels, sprays or injections.

7. The use according to claim 6, characterized in that The preparation method of the granules comprises the following steps: (1) Weigh the Chinese medicinal materials according to the weight ratio of the raw materials and select them; crush the roots of Cuscuta australis, Radix Isatidis, gypsum, honeysuckle, bupleurum, scutellaria baicalensis, and liquorice into coarse powder; (2) Add 1.5-3 times the amount of 50-70% ethanol to the crude powder of Radix Isatidis indigotica and heat under reflux for 1-3 times, each time for 1-3 hours. Combine the extracts, filter, and set aside the residue and filtrate. (3) Combine the dregs from step (2) with the remaining coarse powder, add 1.5-3 times the amount of water and boil for 2-4 times, each time for 1.5-2.5 hours, combine the decoctions, filter, and concentrate the filtrate to a relative density of 1.38-1.40 measured at 70-90°C, cool to room temperature, add ethanol to make the alcohol content reach 50-70%, combine with the filtrate from step (2), let it stand for 24 hours, filter, and obtain a clear paste; (4) Concentrating the clear paste obtained in step (3) to a thick paste having a relative density of 1.38-1.40 measured at 70-80°C, drying to obtain a dry paste powder for later use; (5) Add appropriate pharmaceutically acceptable excipients to the dry paste powder of step (4), granulate, and dry to obtain the product.

8. The use according to claim 7, characterized in that The auxiliary material is selected from one or more of sucrose, mannitol, microcrystalline cellulose, starch, lactose, dextrin, polyvinyl pyrrolidone, Tween 80, calcium phosphate, polyethylene glycol, silicon dioxide, magnesium stearate, soluble starch stevioside, aspartame, protein sugar, acetamidopropyl betaine, xylitol and fructose.

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