Medicinal Uses of Icaritin

Abstract

Use of icaritin in the manufacture of a drug for preventing and treating bleeding disorders, which belongs to the pharmaceutical field. Icaritin can improve platelet dysfunction, shorten thromboplastin time, and promote blood coagulation, and is useful for the prevention and treatment of bleeding disorders, particularly for the treatment of hemorrhagic transformation of cerebral infarction, gastrointestinal bleeding caused by thrombolytic or antithrombotic drugs for cerebral infarction, or bleeding complications caused by thrombolytic or antithrombotic drugs for myocardial infarction.

Description

[Technical field]

[0001] The present invention belongs to the field of medicine and relates to the pharmaceutical use of Icaritin, in particular in the manufacture of a medicament for preventing or treating bleeding disorders. [Background technology]

[0002] When blood vessels in the human body are damaged, blood can leak or seep out of the vessels. At this time, the body stops the bleeding through a series of physiological reactions, which is called hemostasis. The hemostasis process involves various factors and a series of complex physiological and biochemical reactions. Diseases characterized by spontaneous bleeding due to defective hemostatic function or bleeding after vascular injury are called bleeding disorders.

[0003] Bleeding disorders can be divided into several main types depending on their etiology and mechanism of development:

[0004] (1) Abnormal platelet function, including hemorrhagic transformation of cerebral infarction, gastrointestinal bleeding caused by thrombolytic or antithrombotic drugs for cerebral infarction, or bleeding complications caused by thrombolytic or antithrombotic drugs for myocardial infarction. Among them, hemorrhagic transformation of cerebral infarction includes secondary hemorrhagic transformation of ischemic cerebral infarction, primary hemorrhagic transformation, or asymptomatic hemorrhagic transformation. At present, treatment with thrombolytic or anticoagulant drugs is an effective method for treating acute ischemic infarction, but it causes various serious complications such as hemorrhagic transformation while dissolving thrombus, which significantly limits its clinical application. At present, Western medicine prevents and treats various complications after treatment with thrombolytic drugs or anticoagulants by focusing on each individual part or element, but the therapeutic effects are still not satisfactory. Therefore, if the advantages of overall adjustment and multi-part comprehensive treatment of traditional Chinese medicine can be fully exerted, it is of great significance to seek effective treatment methods and prescription drugs that can dissolve thrombus and at the same time reduce or eliminate the risks of various hemorrhagic transformations, and improve the near- and long-term therapeutic effects of thrombolytic treatment of infarction-type diseases.

[0005] (2) Abnormalities of the blood vessel walls, including congenital, genetic and acquired diseases. Congenital diseases include hereditary hemorrhagic telangiectasia, familial purpura simplex and congenital connective tissue disease.

[0006] (3) Blood coagulation abnormalities, including congenital, hereditary and acquired. Congenital blood coagulation abnormalities include hemophilia A, B, hereditary FV deficiency, hereditary prothrombin, FV, FVII, FX deficiency, hereditary fibrinogen deficiency and hypofibrinogen, and hereditary FXII deficiency and hypofibrinogen. Hereditary blood coagulation abnormalities include hepatic disease-related blood coagulation disorders, vitamin K deficiency, anti-factor VIII, anti-factor IX antibody formation, uremic blood coagulation abnormalities, etc.

[0007] (4) Anticoagulation and fibrinolysis disorders: These are mainly acquired disorders, including but not limited to bleeding disorders caused by excessive doses of heparin or its pharmacologic salts, excess coumarin drugs, increased immune-mediated anticoagulants, or excess thrombolytic drugs.

[0008] Epimedium brevicornum Maxim, Epimedium sagiatum Maxim, Epimedium pubescens Maxim, or Korean Epimedium koreanum Nakai are the dried stems and leaves of the Epimedium brevicornum Maxim, Epimedium sagiatum Maxim, Epimedium pubescens Maxim, or Korean Epimedium koreanum Nakai plants of the Epimedium family. Clinically, it is mainly used for kidney yang deficiency, impotence, frequent urination, infertility, rheumatoid paralysis and pain, limb paralysis and convulsions, muscular and bone atrophy, difficulty walking, and wheezing and irritability.

[0009] Icaritin (IT) is a polyhydroxyflavonoid monomer component in E. oleracea, a plant of the E. oleracea family, Berberaceae. Pharmacological studies have shown that the anti-osteoporosis effect of IT is higher than that of other flavone glycoside compounds in E. oleracea, and IT has the effect of promoting osteoblast activity and inhibiting osteoclast activity in vitro. In recent years, icariin and icaritin, which are important active ingredients in E. oleracea, have been increasingly attracting the attention of medical practitioners. For example, patent application CN101637467A discloses the application of icaritin in the manufacture of drugs for treating osteoporosis. Patent US6399579 discloses the use of icaritin in treating sexual dysfunction. Summary of the Invention [Means for solving the problem]

[0010] During the course of research, the applicant discovered that Icaritin can improve platelet dysfunction, shorten the thromboplastin time, and promote blood coagulation, and is useful for the prevention and treatment of platelet dysfunction and bleeding disorders resulting therefrom, bleeding disorders resulting from blood coagulation disorders, bleeding disorders resulting from vascular wall abnormalities, or bleeding disorders resulting from anticoagulation and fibrinolysis disorders.

[0011] In one aspect, the present application provides the use of icaritin in the manufacture of a medicament for preventing or treating a disease, the disease being a platelet function disorder and a bleeding disorder resulting therefrom, a bleeding disorder resulting from a blood coagulation disorder, a bleeding disorder resulting from a vascular wall disorder, or a bleeding disorder resulting from an anticoagulation and fibrinolysis disorder.

[0012] In one embodiment, the bleeding disorder due to platelet dysfunction is selected from hemorrhagic transformation of cerebral infarction, gastrointestinal bleeding due to a thrombolytic or antithrombotic agent for cerebral infarction, or bleeding complications due to a thrombolytic or antithrombotic agent for myocardial infarction.

[0013] In one embodiment, the cerebral infarction hemorrhagic transformation is secondary ischemic cerebral infarction hemorrhagic transformation, primary hemorrhagic transformation or asymptomatic hemorrhagic transformation.

[0014] In one embodiment, the secondary hemorrhagic transformation is hemorrhagic transformation due to the use of one or more of the following therapeutic methods: thrombolytic drugs, antithrombotic drugs, and endovascular therapy.

[0015] In one embodiment, the thrombolytic agent is selected from urokinase, streptokinase, reteplase, alteplase, tenecteplase, and lanoteplase. In one embodiment, the antithrombotic agent is one or more of an anticoagulant and an antiplatelet agent. In one embodiment, the anticoagulant is selected from heparin or a pharma- ceutically acceptable salt thereof, low molecular weight heparin or a pharma- ceutically acceptable salt thereof, and recombinant hirudin. In one embodiment, the antiplatelet agent is selected from aspirin, clopidogrel, or ticlopidine.

[0016] In one embodiment, the primary hemorrhagic transformation is spontaneous hemorrhagic transformation after acute cerebral infarction.

[0017] In one embodiment, in the case of bleeding complications due to the thrombolytic or antithrombotic drugs for myocardial infarction, the thrombolytic drug is selected from urokinase, streptokinase, reteplase, alteplase, tenecteplase, and lanoteplase. The antithrombotic drug is an antithrombotic treatment in the onset stage, primary prevention stage, and secondary prevention stage of myocardial infarction. The antithrombotic drug is one or more of anticoagulants or antiplatelet drugs.

[0018] In one embodiment, the antiplatelet agent is selected from aspirin, clopidogrel or ticlopidine, and the anticoagulant is selected from heparin or a pharma- ceutically acceptable salt thereof, low molecular weight heparin or a pharma- ceutically acceptable salt thereof, or recombinant hirudin.

[0019] In one embodiment, the bleeding complication is one or more of subcutaneous bleeding, intracranial bleeding, upper gastrointestinal bleeding, or gingival bleeding.

[0020] In one embodiment, in the case of gastrointestinal bleeding caused by a thrombolytic or antithrombotic drug for cerebral infarction, the thrombolytic drug is one or more of urokinase, streptokinase, reteplase, alteplase, tenecteplase, and lanoteplase, and the antithrombotic drug is one or more of an anticoagulant or an antiplatelet drug.

[0021] In one embodiment, the platelet dysfunction is a platelet activation or aggregation dysfunction, and the platelet dysfunction is a congenital platelet deficiency or an acquired platelet deficiency.

[0022] In one embodiment, the congenital platelet deficiency is hereditary giant platelet syndrome, hereditary thrombasthenia or a congenital connective tissue disease and the acquired platelet deficiency is due to a drug or disease.

[0023] In one embodiment, the drug is one or more of an antibacterial agent, an antitumor agent, or heparin, and the disease is one or more of uremia, diabetes, nephrotic syndrome, coronary heart disease, or leukemia.

[0024] In one embodiment, the bleeding disorder caused by a blood clotting disorder is hemophilia.

[0025] In one embodiment, the hemophilia is genetic hemophilia or secondary hemophilia, wherein the secondary hemophilia is due to a drug or an autoimmune disease.

[0026] In one embodiment, the autoimmune disease includes, but is not limited to, liver lesions, hemolysis, acquired immune deficiency, Evans syndrome, chronic lymphocytic leukemia, various acute leukemias, lymphomas, systemic lupus erythematosus, rheumatoid arthritis, or hyperthyroidism. The liver lesions are any one of liver lesions caused by chronic persistent hepatitis and liver cirrhosis, and the drugs include, but are not limited to, alkylating agents, metabolic inhibitors, cytotoxic agents, chlorothiazides and their synergists.

[0027] In one embodiment, the hemophilia is any one of hemophilia A, hemophilia B or hemophilia C.

[0028] In one embodiment, the bleeding disorder due to an anticoagulation and fibrinolysis disorder is an acquired disorder, including but not limited to, due to an excess dose of heparin or a pharmacologic salt thereof, an excess of coumarin drugs, an increase in immune-mediated anticoagulants, or an excess of thrombolytic drugs.

[0029] In the first pharmaceutical example, the research results of the effect of Icaritin on platelet activation function show that after Icaritin is administered for 3 days, the peripheral anticoagulant blood of animals is activated under the same conditions of adenosine diphosphate, and then, in all platelets labeled with CD61, the expression of CD62p is obviously increased, that is, the platelet activation rate activated by adenosine diphosphate is increased, and there is a significant difference compared with the basal value before administration.In this experimental condition, it is presented that intragastric administration of Icaritin can obviously promote the activation of platelets induced by adenosine diphosphate (ADP) in peripheral anticoagulant blood of rats.

[0030] According to the results of a study on the effect of icaritin on platelet aggregation function in vitro, all concentrations of icaritin have the effect of promoting platelet aggregation activity induced by adenosine diphosphate, and the promoting effect increases with increasing concentration, indicating that the promoting effect of icaritin on platelet aggregation activity induced by adenosine diphosphate exhibits a dose-dependent relationship.

[0031] In Efficacy Example 2, a test of the effect of Icaritin on hemorrhagic transformation after cerebral ischemia-reperfusion caused by aspirin or clopidogrel shows that Icaritin can significantly suppress hemorrhagic transformation after cerebral ischemia-reperfusion caused by aspirin or clopidogrel.

[0032] In Pharmacological Example 3, the results of the effect of Icaritin on the bleeding time of mice induced by heparin sodium show that Icaritin can significantly shorten the bleeding time of mice induced by heparin sodium, and exerts a coagulation-promoting effect while not changing the four parameters of blood tests and blood coagulation.

[0033] In Drug Efficacy Example 4, the results of the effect of Icaritin on the bleeding time of mice induced by aspirin show that Icaritin can significantly shorten the bleeding time of mice induced by aspirin.

[0034] The results of the effect of icaritin on the thrombolytic effect of thrombolytic drugs in Example 5 and the effect of icaritin on hemorrhagic transformation caused by thrombolytic drugs in Example 6 show that icaritin can significantly improve hemorrhagic transformation caused by thrombolytic drugs, and can prevent hemorrhagic transformation and reduce the amount of bleeding, while not affecting the dissolving effect of thrombolytic drugs on thrombus and not affecting the values ​​of blood tests. Icaritin also has a significant preventive and therapeutic effect on hemorrhagic transformation caused by thrombolytic drugs, anticoagulants and antiplatelet drugs other than those listed in the above examples, and also has a significant risk reduction effect on spontaneous hemorrhagic transformation and asymptomatic hemorrhagic transformation. In addition to having a significant preventive and therapeutic effect on hemorrhagic transformation after the occurrence of the above-mentioned cerebral infarction, icaritin also has a significant preventive and therapeutic effect on the occurrence of hemorrhagic infarction that can be determined based on the initial cranial CT / MRI.

[0035] In Pharmacological Example 7, the test results on the preventive effect of Icaritin against gastrointestinal bleeding caused by aspirin, and in Pharmacological Example 8, the test results on the preventive effect of Icaritin against gastric bleeding caused by thrombolytic drugs, showed that Icaritin can not only reduce the incidence of gastrointestinal bleeding caused by thrombolytic drugs, but also that high doses of Icaritin have a significant preventive effect against gastrointestinal bleeding caused by thrombotic diseases themselves, and both of the above effects show a dose-dependent relationship.

[0036] In Pharmaceutical Example 9, the experimental results of the effect of icaritin on bleeding complications caused by thrombolytic drugs show that icaritin can significantly reduce the incidence of myocardial infarction bleeding complications caused by thrombolytic drugs, and does not affect the thrombolytic effect of thrombolytic drugs even when used simultaneously with thrombolytic drugs.

[0037] In Efficacy Example 10, the results of the effect of Icaritin on the bleeding time of hemophilic mice with deleted blood coagulation factor IX gene show that Icaritin can effectively shorten the bleeding time of hemophilic mice with deleted blood coagulation factor IX gene, and there is no significant difference in the blood tests of mice in each group.

[0038] In Pharmacological Example 11, the results of the effect of Icaritin on the activated partial thromboplastin time of acquired hemophilia A show that Icaritin can significantly shorten the activated partial thromboplastin time of hemophilia patients and promote blood coagulation in hemophilia patients.

[0039] Icaritin not only has a significant therapeutic effect on hemophilia caused by genetic factors, but also has a significant therapeutic effect on secondary hemophilia.

[0040] When Icaritin is used for the prevention and treatment of bleeding disorders, the administration route includes the digestive tract route and the parenteral route, and the parenteral route includes, but is not limited to, subcutaneous, intradermal, arterial, venous, muscular, joint, intrathecal, intracranial, thoracic, intraperitoneal injection or drip infusion, nasal, buccal, sublingual, tracheal, urethral, ​​rectal or local administration to the lesion, etc. Icaritin may be administered simultaneously with other drugs, or may be administered in advance for preventive medication.

[0041] The present invention not only provides the use of icaritin in the manufacture of a medicament for the prevention and treatment of bleeding disorders, but also provides a pharmaceutical formulation comprising icaritin and pharma- ceutical auxiliary materials for use in treating the above-mentioned disorders.

[0042] The pharmaceutical auxiliary materials used in the formulations of the present invention are common auxiliary materials known to those skilled in the art.

[0043] Suitable pharmaceutical auxiliary materials have already been described in detail in "Complete Guide to Pharmaceutical Auxiliary Materials" (page 123, published by Sichuan Science and Technology Publishing House, 1993, edited by Luo Mingsheng and Gao Tianhui). For example, common pharmaceutical auxiliary materials for producing microemulsion formulations include but are not limited to soybean oil, polyoxyethylene-23-lauryl ether, 1,2-propylene glycol, hydrogenated coconut oil glycerin, lauroyl polyethylene glycol-32-monoglyceride, polyethylene glycol 3350, safflower oil, cottonseed oil, decaglycerin monooleate ester. Common pharmaceutical auxiliary materials for producing drop formulations include but are not limited to polyethylene glycol 6000, polyethylene glycol 1000. Common pharmaceutical auxiliary materials for producing capsule formulations include but are not limited to lactose and corn starch. Common pharma-ceutically acceptable carriers for producing soft capsule formulations include but are not limited to medium chain fatty acid glycerides, polyoxyethylene castor oil, 1,2-propylene glycol, etc.

[0044] Those skilled in the art can select suitable pharmaceutical auxiliary materials according to actual needs and prepare the formulations of the present invention by methods known in the art, including but not limited to solids, liquids, oils, emulsions, gels, aerosols, inhalants, sprays, capsules, pills, patches and suppositories.

[0045] The Icaritin of the present invention can improve platelet dysfunction, shorten the thromboplastin time, and promote blood coagulation, and is useful for the prevention and treatment of platelet dysfunction and bleeding disorders resulting therefrom, bleeding disorders resulting from blood coagulation disorders, bleeding disorders resulting from vascular wall abnormalities, or bleeding disorders resulting from anticoagulation and fibrinolysis disorders. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present invention will be further described below with reference to specific embodiments, and the present invention is not limited to the following examples.

[0047] Formulation Example 1 Microemulsion formulation of Icaritin Ikaritin 10g Soybean oil 35g Polyoxyethylene-23-lauryl ether 60g 1,2-propylene glycol 30g Preparation process: take the prescribed amount of soybean oil, polyoxyethylene-23-lauryl ether, 1,2-propylene glycol, mix and stir evenly, then add icaritin to dissolve, and can be ultrasonicated to accelerate dissolution, obtain a clear solution, that is, icaritin microemulsion preparation. Its particle size is measured by laser particle sizer, and the average particle size is 15nm.

[0048] Formulation Example 2 Microemulsion formulation of Icaritin Icaritin 0.1g Hydrogenated coconut oil monoglyceride 5g Lauroyl polyethylene glycol-32-monoglyceride 20g 1,2-propylene glycol 5g Polyethylene glycol 3350 20g Preparation process: take the prescribed amount of hydrogenated coconut oil monoglyceride, lauroyl polyethylene glycol-32-monoglyceride, 1,2-propylene glycol, polyethylene glycol 3350, mix and stir evenly, then add icaritin to dissolve, optionally ultrasonicate to accelerate dissolution, obtain a clear solution, that is, icaritin microemulsion preparation. Its particle size is measured by laser particle sizer, the average particle size is 40nm.

[0049] Formulation Example 3 Icaritin Injection Ikaritin 500g PEG-400 2L Add ethanol 0.5L 0.9% sodium chloride solution up to 10L. The manufacturing process is as follows: add icaritin to the prescribed amount of PEG-400, stir to dissolve, add 0.9% sodium chloride solution up to 10L, stir evenly, add 0.5% activated carbon for injection, stir, and decarbonize to obtain the product.

[0050] Formulation Example 4 Icaritin Injection Icaritin 10g Ethanol 2L Tween-80 1500g Add water for injection up to 10L The manufacturing process is to mix the prescribed amount of ethanol and Tween-80 uniformly, add Icaritin and stir to dissolve, add water for injection up to 10L, stir uniformly, add 0.5% activated carbon for injection, stir and decarbonize to obtain.

[0051] Formulation Example 5 Icaritin Injection Icaritin 1g Ethanol 3.3L Add water for injection up to 10L The manufacturing process is to add the prescribed amount of ethanol to Icaritin, stir and dissolve it, add water for injection up to 10L, stir evenly, add 0.5% activated carbon for injection, stir and decarbonize to obtain.

[0052] Formulation Example 6 Icaritin Drop Formulation Icaritin 5.0g Polyethylene glycol-6000 14.5g Polyethylene glycol-1000 5.0g 1000 tablets Manufacturing process: A prescribed amount of Icaritin is weighed and added to a mixture containing the prescribed amount of polyethylene glycol 6000 and polyethylene glycol 1000, which is heated and melted in a water bath, and then thoroughly stirred to homogenize it, and then placed in a dropping bottle and dropped at 95±2°C. The mixture is dropped into a glass condensation column containing 4-6mL of methyl silicone oil, and after molding, it is removed and the attached methyl silicone oil is absorbed with absorbent paper to obtain the product.

[0053] Formulation Example 7 Icaritin enteric soft capsule formulation Prescription of contents, Icaritin 10g Absolute ethanol 10g 1,2-Propylene glycol 10g Polyoxyethylene castor oil 50g Medium chain fatty acid glycerides 20g Capsule coating formulation, Gelatin 10g Glycerin 5g Purified water 10g Enteric coating formulation, EudragitL30D-55 100g Triethyl citrate 3g Talc 7.5g Purified water 200g Preparation process: take the prescribed amount of medium chain fatty acid glyceride, polyoxyethylene castor oil, 1,2-propylene glycol, and anhydrous ethanol, mix and stir evenly, then add icaritin and dissolve, optionally ultrasonicate to accelerate dissolution, to obtain soft capsule microemulsion content. Take the prescribed amount of gelatin, glycerin, and purified water, mix evenly, and press into capsule shell, take the prescribed amount of Eudragit L30D-55, triethyl citrate, talc, and purified water, mix evenly, to obtain enteric coating solution. Coat the soft capsule microemulsion content containing icaritin with capsule shell to produce soft capsule, and coat the soft capsule with enteric coating to produce enteric soft capsule.

[0054] Formulation Example 8 Icaritin Capsule Formulation Icaritin 50g Lactose 120g Cornstarch 130g Magnesium stearate 5g Manufacturing process: 100g of Icaritin, 120g of lactose and 130g of cornstarch are mixed in a mixer for 10-15 minutes, 5g of magnesium stearate is added and mixed for 1-3 minutes, and then the mixture is put into 1000 capsule shells.

[0055] Formulation Example 9 Icaritin Tablets Icaritin 50g Microcrystalline Cellulose 200g Sodium carboxymethyl starch 8g Magnesium stearate 1.5g Appropriate amount of 8% starch slurry Manufacturing process: Icaritin is mixed with the auxiliary materials microcrystalline cellulose and sodium carboxymethyl starch evenly, and an appropriate amount of starch slurry is added to produce a soft material, which is then sieved through a 16 mesh sieve to granulate. The wet particles are dried at 60°C, the dry particles are sieved through a 20 mesh sieve to granulate, the fine powder in the dry particles is sieved, and the powder is mixed evenly with magnesium stearate, then mixed evenly with the dry particles, and compressed into tablets, and each tablet is about 260mg in weight.

[0056] Formulation Example 10 Icaritin Powder Injection Icaritin 10g Glucose 500g Add water for injection up to 10L A total of 2000 freeze-dried products will be produced. Manufacturing process: A prescribed amount of Icaritin raw material for injection is weighed and dissolved by adding an appropriate amount of water for injection. Next, a predetermined amount of glucose that has been previously sterilized and heat-removed is added and mixed uniformly, and then water for injection is added up to the specified amount of 10L. 50g of activated carbon for injection is added to the above drug solution, heated at 60-80°C for 30 minutes, filtered through a filter membrane, and the filtrate is collected. The above filtrate is subjected to positive pressure sterilization filtration with a sterilization filter according to the aseptic operation method, filtered through a 0.22μM microporous filter membrane, and the filtrate is subjected to heat source inspection and semi-finished product content inspection, and then dispensed into penicillin bottles. In a dedicated freeze-drying box, the powder is frozen in advance at -40°C or less for 1.5-3.5 hours, and 90% of the free water is sublimated and removed at a vacuum, and then heated and dried (the maximum temperature does not exceed 35°C), and when the freeze-drying is completed, the Icaritin powder injection can be produced.

[0057] Drug Efficacy Example 1: Effect of Icaritin on Platelet Activation Function and In Vitro Platelet Aggregation Function 1. Effect of Icaritin on platelet activation function 1.1 Experimental animals SD rats, SPF level, weighing 250-300 g, half male and half female, and the female rats were non-pregnant.

[0058] 1.2 Flow cytometric detection of platelet activation 0.5 mL of blood is taken from the orbit of a rat, and 3.8% sodium citrate is added for anticoagulation (the ratio of blood to anticoagulant is 9:1) to prepare anticoagulated blood. 50 μL of anticoagulated blood is taken from each animal and used to measure the platelet spontaneous activation rate. Another 50 μL of anticoagulated blood is taken and 50 μL of adenosine diphosphate with a final concentration of 10 μmol is added to activate it. 1.0-1.5 μL of CD62pPE / Cy7 and CD61PE are added to the above two types of anticoagulated blood respectively to perform double labeling, labeling all platelets with CD61 and labeling activated platelets with CD62p. 50 μL of anticoagulated blood is taken and 50 μL of adenosine diphosphate with a final concentration of 10 μmol is added to activate it. G2a-PE / Cy7 is administered intragastrically for double labeling, and G-PE is administered intragastrically as a homogenous control. Each tube is incubated at 4°C in the dark for 20 min. Then, 0.5 mL of 4% paraformaldehyde is added to fix for 10 min. 50 μL of the fixed blood sample is taken, diluted with 1 mL of diluent, and analyzed by flow cytometer. Platelet cell population is defined in the dual parameter scatter plot of CD61PE / side angle light scattering (SSC), 5000 platelets are counted, and the number of cells positively expressing CD61 and CD62p is counted in the CD61 / CD62p scatter plot, and the platelet activation rate (%) is reflected by the percentage of CD61 expression that is occupied by CD62p positive expression.

[0059] Effect of icaritin on 1.3 adenosine diphosphate-induced platelet activation in rats in vivo 24 rats were taken and divided into 3 groups, 8 rats in each group, and 3, 6, and 18 mg / kg of Icaritin were administered intragastrically, once a day, for 3 consecutive days. 1.0 mL of blood was taken from the orbit before administration and 20 min after the final administration, of which 0.5 mL was used to measure blood tests, and 0.5 mL was used to prepare anticoagulated blood, which was processed by the above-mentioned flow cytometry detection method for platelet activation and then analyzed by a flow cytometer to detect the platelet activation rate (basal value) activated by adenosine diphosphate before administration and the platelet activation rate activated by adenosine diphosphate after administration, and the two were compared. At the same time, 50 μl of rat anticoagulated blood was taken before administration, and the same method was used to detect without adding adenosine diphosphate, and the spontaneous activation rate of the animal anticoagulated blood was measured. The animals were fasted for 16 to 24 h before each blood collection.

[0060] 1.4 Detection items and test results (1) Effect of adenosine diphosphate on the induction of in vivo platelet activation in rats Before administration, the peripheral anticoagulant blood of the animals was activated by adenosine diphosphate, and compared with the spontaneous activation rate, the ratio of activated platelets was obviously increased in all platelets labeled with CD61, that is, the expression of CD62p was obviously increased, indicating that adenosine diphosphate can induce an increase in the platelet activation rate. After administration of Icaritin for 3 days, the peripheral anticoagulant blood of the animals was activated under the same conditions with adenosine diphosphate, and the expression of CD62p was obviously increased in all platelets labeled with CD61, that is, the platelet activation rate activated by adenosine diphosphate was increased, and there was a significant difference compared with the basal value before administration. Under the present experimental conditions, Icaritin was administered intragastrically and was shown to be able to obviously promote the platelet activation induced by adenosine diphosphate in rat peripheral anticoagulant blood. Specifically, it is shown in Table 1.

[0061] [Table 1]

[0062] (2) Blood tests The blood test values ​​for each Icaritin dose group showed no significant differences compared to before administration, especially in platelet counts. This result explains that Icaritin increases platelet activity while not affecting platelet counts. The results are shown in Table 2.

[0063] [Table 2]

[0064] 2. Effect of Icaritin on platelet aggregation function in vitro 2.1 Experimental method for promoting in vitro platelet aggregation activity After the rabbits were locally anesthetized with procaine, the common carotid artery was isolated by surgery to collect blood, which was then anticoagulated with sodium citrate with a mass fraction of 3.8%, centrifuged at 800 r / min for 10 min to produce platelet-rich plasma (PRP), and the remaining portion was centrifuged at 300 r / min for 10 min to produce platelet-poor plasma (PPP) for a platelet aggregation experiment. 265 μl of PRP and 30 μl of icaritin (final concentrations of 0.1 mmol / L, 0.5 mmol / L, and 5 mmol / L, respectively) were added to a measurement tube, incubated for 5 min, and 5 μl of adenosine diphosphate (final concentration 25 μmol / L) was used as an inducer, and the maximum aggregation rate within 5 min was observed and recorded. Dimethyl sulfoxide with a mass fraction of 1% was used as a control, and the promoting effect of each concentration of icaritin on platelet aggregation induced by adenosine diphosphate was observed.

[0065] 2.2 Experimental results The promoting effect of different concentrations of Icaritin on platelet aggregation activity induced by adenosine diphosphate in vitro is as shown in Table 3. As can be seen from the table, each concentration of Icaritin has the effect of promoting platelet aggregation activity induced by adenosine diphosphate, and the promoting effect increases with increasing concentration, illustrating that the promoting effect of Icaritin on platelet aggregation activity induced by adenosine diphosphate exhibits a dose-dependent relationship.

[0066] [Table 3]

[0067] Drug Efficacy Example 2 Effect of Icaritin on hemorrhagic transformation after cerebral ischemia-reperfusion by aspirin or clopidogrel 1. Experimental Procedure 1.1 Model creation and experimental method Male ICR mice, 150 mice, Jinan Pengyue Laboratory Animal Breeding Co., Ltd., SPF level, weight 27-30g. Animal certificate number, No. 1107262011001201, No. 1107262011001347.

[0068] Mice are quarantined for 3-7 days after entering the facility, and healthy (non-pregnant female) mice are selected as test animals. The main inspection contents during the quarantine period include, but are not limited to, whether the number of animals and quality indicators meet those requested at the time of application, their general condition, and weight. Animals that fail the above quarantine will not be included in this study. The details are shown in Table 4.

[0069] [Table 4]

[0070] Five-week-old ICR male mice were raised until they reached six weeks of age, and divided into a control group, a model group, an icaritin-3 mg / kg group, an icaritin-6 mg / kg group, an icaritin-12 mg / kg group, and an icariin-12 mg / kg group according to their body weight. After being divided into groups, in the first to third days, 3, 6, and 12 mg / kg of icaritin were intragastrically administered to each icaritin group, and 12 mg / kg of icariin was intragastrically administered to the icariin group, once a day, and the corresponding solvent was intragastrically administered to each of the remaining groups. In addition to the control group, aspirin (100 mg / kg) or clopidogrel (15 mg / kg) was intragastrically administered to each of the remaining groups, once a day. The volume of intragastric administration was 10 mL / kg in each case.

[0071] On the third day, 10 min after administration, the mice were anesthetized by intraperitoneal injection of 70 mg / kg sodium pentobarbital, and intraluminal middle cerebral artery occlusion (MCAO) surgery was performed, followed by 1 h of ischemia and 23 h of reperfusion.

[0072] 24 h after surgery is the experimental end point.

[0073] 1.2 Detection indicators Hemorrhage rate: Mice were anesthetized by intraperitoneal injection of 70mg / kg sodium pentobarbital, brain tissue was taken, and the cerebral hemorrhage status was recorded, and the cerebral hemorrhage rate of mice in each group was calculated as follows: cerebral hemorrhage rate% = number of animals with cerebral hemorrhage / number of surviving animals in each group × 100.

[0074] 2. Data processing All data were entered into an Excel document, and statistical analysis was performed using SPSS 17.0, and the results were expressed as mean ± SD. One-way ANOVA and ttest were used to statistically analyze and compare the data of each group.

[0075] 3. Test results Impact on bleeding rate According to the experimental results, in a hemorrhagic transformation model induced by cerebral ischemia-reperfusion combined with aspirin, the bleeding rate in the model group was 85.7%, and the bleeding rate was significantly reduced after icaritin was administered in advance for prevention. The bleeding rates in the icaritin-3 mg / kg group, icaritin-6 mg / kg group, and icaritin-12 mg / kg group were 37.5%, 25.0%, and 22.2%, respectively, and although icaritin was administered in advance for prevention, the bleeding rate did not obviously improve. The results are shown in Table 5.

[0076] In a hemorrhagic transformation model induced by cerebral ischemia-reperfusion in combination with clopidogrel, the bleeding rate in the model group was 80%, and the bleeding rate was significantly reduced after pre-administration of iniocanin for prevention. The bleeding rates in the icaritin-3 mg / kg group, icaritin-6 mg / kg group, and icaritin-12 mg / kg group were 42.9%, 25.0%, and 25.0%, respectively, and although icariin was pre-administered for prevention, the bleeding rate did not improve obviously. The results are shown in Table 6.

[0077] [Table 5]

[0078] [Table 6]

[0079] In this study, we refer to the literature to select an antiplatelet aggregation drug (aspirin or clopidogrel) in combination with cerebral ischemia-reperfusion to successfully establish a hemorrhagic transformation model, and consider the preventive effect of icaritin on hemorrhagic transformation animal models depending on the bleeding situation.

[0080] This study successfully replicated the animal model of hemorrhagic transformed mice, and demonstrated that when the bleeding rate significantly increased, after administration of icaritin for prevention, all of the above indicators significantly decreased, indicating that icaritin can significantly inhibit hemorrhagic transformation after cerebral ischemia-reperfusion caused by aspirin or clopidogrel.

[0081] Drug Efficacy Example 3: Effect of Icaritin on the Prolongation of Mouse Bleeding Time Caused by Heparin Sodium 1. Drugs and Sources Heparin sodium injection (source: Jiangsu Wabang Biochemical Pharmaceutical Group Co., Ltd., lot number: 51701106) Etamsilate injection (source: Shandong Fangming Pharmaceutical Group Co., Ltd.; batch number: 18060672)

[0082] 2. Test animals and grouping Seventy-two 5-6 week old ICR mice, weighing 19-21g, half male and half female. Mice were quarantined for 7 days after entering the facility, and healthy mice were selected as test animals. They were weighed on the first and last days of quarantine. A simplified randomization method was used based on the weight on the last day of quarantine, and male and female animals were divided into six groups: normal group (C), model group (V), Icaritin low, medium, and high dose groups (2, 6, 18mg / kg, L, M, H), and positive drug group (200mg / kg ethamsylate, P).

[0083] 3. Pre-dosing and modeling After grouping, the normal group and the model group were intragastrically administered 10ml / kg of Icaritin solvent, which was prepared by adding 12g of hydroxypropyl methylcellulose E5 and 0.1g of sodium dodecyl sulfate to 1L of water. Each dose group of Icaritin was intragastrically administered 2, 6, and 18mg / kg of Icaritin, once a day for five consecutive days. The positive drug group was intraperitoneally injected with 200mg / kg of ethamsilate injection, once a day for five consecutive days. One hour after the last pre-administration, the model group and each administration group were modeled by injecting 150U / kg of heparin sodium injection into the tail vein, and the normal group was injected with 10ml / kg of sodium chloride injection into the tail vein.

[0084] 4. Detection Indicators 4.1 Bleeding time After 15 min of modeling, the tail is cut to measure the bleeding time of each mouse.

[0085] 4.2 Blood tests and blood coagulation After completing the detection of bleeding time, the mice are anesthetized with sodium pentobarbital, blood is taken from the abdominal main vein, and blood tests and four parameters of blood coagulation (prothrombin time, thromboplastin time, thrombin time, fibrinogen) are detected.

[0086] 5. Test Results 5.1 Bleeding time The bleeding times for each group of mice are shown in Table 7.

[0087] [Table 7]

[0088] As can be seen from the table, compared with the normal group, the bleeding time of the mice in the model group was significantly prolonged, indicating that the modeling was successful. Compared with the model group, the bleeding time of the mice in each administration group was significantly shortened, indicating that icaritin can shorten the bleeding time of bleeding mice, and the shortening of bleeding time is more significant with increasing dosage, indicating that the procoagulant effect of icaritin is dose-dependent, and the high-dose group has a significant difference compared with the positive control drug group, indicating that the procoagulant effect of icaritin is higher than that of the positive control drug.

[0089] 5.2 Blood tests and blood coagulation The results of the four blood tests and blood coagulation tests for the mice in each group are shown in Tables 8 and 9, respectively.

[0090] [Table 8]

[0091] [Table 9]

[0092] As can be seen from the table above, there were no significant differences in the blood tests and four blood coagulation parameters of the mice in each group, which indicates that when Icaritin shortens the prolongation of bleeding time caused by heparin sodium, it has no significant effect on the blood tests and four blood coagulation parameters.

[0093] Drug Efficacy Example 4: Effect of Icaritin on Aspirin-Induced Prolongation of Bleeding Time in Mice 1. Drugs and Sources Aspirin (source, Shantou Jinshi Pharmaceutical Bench Co., Ltd., lot number, 1903012) Ethamsilate injection (source: Shandong Fangming Pharmaceutical Group Co., Ltd.; batch number: 1905032)

[0094] 2. Test animals and grouping Ninety-six 5-6 week old ICR mice, weighing 19-21g, half male and half female. Mice are quarantined for 7 days after entering the facility, and healthy mice are selected as test animals. They are weighed on the first and last day of quarantine.

[0095] Randomization was simplified according to the body weight on the last day of quarantine, and male and female animals were divided into six groups: normal group (C), model group (V), low, medium and high dose icaritin groups (2, 6, 18 mg / kg, L, M, H), and positive drug group (200 mg / kg ethamsilate, P).

[0096] 3. Pre-dosing and modeling After grouping, the normal group and model group were intragastrically administered 10ml / kg of Icaritin solvent, and each dose group of Icaritin was intragastrically administered 2, 6, and 18mg / kg of Icaritin, respectively, once daily for five consecutive days. The positive drug group was intraperitoneally injected with 200mg / kg of etamsilate injection, once daily for five consecutive days. One hour after the final pre-administration, the model group and each administration group were intragastrically administered 20mg / kg of aspirin to model, and the normal group was intragastrically administered an equal volume of saline.

[0097] 4. Bleeding time detection After 15 min of modeling, the tail is cut to measure the bleeding time of each mouse.

[0098] 5. Test Results The bleeding time of the mice in each group is shown in Table 10. Compared with the normal group, the bleeding time of the mice in the model group was significantly prolonged, indicating that the modeling was successful. Compared with the model group, the bleeding time of the mice in each administration group was significantly shortened, indicating that icaritin can shorten the bleeding time of bleeding mice, and the shortening of bleeding time is more significant with increasing dosage, indicating that the procoagulant effect of icaritin is dose-dependent, and there is a significant difference between the high-dose group and the positive control drug group, indicating that the procoagulant effect of icaritin is higher than that of the positive control drug.

[0099] [Table 10]

[0100] Drug Efficacy Example 5: Effect of Icaritin on the thrombolytic effect of thrombolytic drugs 1. Establishment of a rat abdominal aortic thrombosis model A rat abdominal aortic thrombosis model was created in the following manner. Anesthetize the rat by intraperitoneally injecting 40 mg / kg of 1.5% pentobarbital sodium. Shave the hair on the abdomen of the rat to prepare the skin, and fix it on a mouse plate in a supine position. Incise the abdominal skin of the rat, isolate the blood vessels and surrounding tissue, expose the abdominal aorta, and carefully separate the abdominal aorta with a glass needle. Place a 0.7 cm x 1.5 cm tin foil strip under the abdominal aorta, then wrap the abdominal aorta with a filter paper strip (0.5 cm x 1.0 cm) soaked in 35% FeCl3 solution, and remove the filter paper strip after 30 min. At this time, the color of the vascular wall becomes obviously darker, and it can be seen under a microscope that an embolism is formed in the blood vessel. The pathological section shows that the blood vessel is occluded by a thrombus.

[0101] 2. Grouping and Dosing SD rats were randomly divided into three groups according to body weight, with 10 rats in each group: thrombosis model group, lumbrokinase capsule group, and lumbrokinase capsule + icaritin group. 5 min after the model, lumbrokinase capsule was administered at 30 × 104 The animals were administered by direct injection into the duodenum at a dose of 18 U / kg, and Icaritin was administered intragastrically at a dose of 18 mg / kg. For the thrombus model group, the animals were substituted with an equal volume of distilled water.

[0102] 3. Interview Close both ends of the abdominal aorta with a hemostat, take the entire vessel segment at the position to be modeled, precisely cut out a 0.5 cm vessel segment covered with filter paper, weigh it, and store the remaining vessel segment in 10% formaldehyde.

[0103] 4. Measurement of Rat Clot Weight The weight of the vessel segment taken with an electronic balance was measured, and the vessel was then weighed after removing the thrombus. The wet weight of the thrombus=the total weight of the thrombus and vessel--the weight of the vessel.

[0104] The removed thrombus is dried in an oven at 80° C., left overnight, and weighed to obtain the dry weight of the thrombus.

[0105] 5. Test Results The weight of the rat thrombus in each group is shown in the table below. As can be seen from the table, compared with the model group, the weight of the thrombus in the lumbrokinase group was significantly reduced, which explains that lumbrokinase has a significant thrombolytic effect. Compared with the lumbrokinase group, there was no significant difference in the thrombus in the lumbrokinase + icaritin group, which explains that icaritin does not affect the lysis effect of lumbrokinase on rat thrombus.

[0106] [Table 11]

[0107] Drug Efficacy Example 6: Effect of Icaritin on hemorrhagic transformation caused by thrombolytic drugs 1. Preparation of Animal Model A rat cerebral middle artery autothrombosis embolism model is prepared by the following method. The rat is anesthetized by intraperitoneal injection of 10% chloral hydrate at 350 mg / kg body weight, fixed supine on the operating table, and conventional skin disinfection is performed according to aseptic operation procedures, followed by laying a surgical drape. A notch with a cut length of about 3.0 cm is made in the midline of the neck skin, the muscle and subcutaneous tissue are inactively separated, the right bilateral common carotid arteries, the external carotid artery and the internal carotid artery are separated, the internal carotid artery is separated to the base of the skull so as not to damage the vagus nerve, and the external carotid arteries are ligated at the bilateral branching points of the common carotid arteries. The rat's femoral artery is selected with a 1 ml syringe, 0.1 ml of arterial blood is taken, 4 U thrombin is aspirated, uniformly mixed and left to stand for 8 to 10 min, and after the blood has coagulated, it is injected into a 24 g venous indwelling needle tube to produce an embolism with a length of about 1 cm, which is then connected to a 1 to 2 ml saline syringe. Next, the needle is inserted into both common carotid arteries in front of where the external carotid artery and the internal carotid artery branch, and inserted about 8-10 mm into the internal carotid artery toward the anterior medial side, the needle core is removed, and the embolus and saline in the syringe are quickly injected into the internal carotid artery, which is then ligated and sutured. In the sham operation control group, the other operation steps are the same as those of the above thrombolysis model groups, except that saline is injected instead of the embolus.

[0108] 2. Animal Intake Criteria The rats were observed for symptoms and signs 2 hours after the modeling operation, and the following symptoms and signs indicated that the model was successfully created: Horner's syndrome on the same side as ischemia, in which the rat rotates to the opposite side of the ischemia when running, leans to the opposite side, and when the rat is suspended in the air with its tail raised, the forelimb on the opposite side of the cerebral ischemia is flexed and lifted, with shoulder adduction and elbow extension.

[0109] 3. Experimental Grouping and Dosing 102 rats were randomly divided into sham operation group, rt-PA thrombolysis group (thrombolysis group), rt-PA thrombolysis + Icaritin low, medium, and high dose groups, of which sham operation group had 18 rats, and each of the remaining groups had 21 rats. Here, each group was further grouped according to different lethal time points (3, 6, and 24 hours after thrombolysis) in average, with 6 rats per time point in sham operation group, 9 rats per group at 3 hours for each of the remaining groups, and 6 rats per group at other time points. In addition, 20 rats were taken for use, and randomly supplemented if modeling failed (dead or scored 0), for example.

[0110] All other groups except the sham-operated group were administered alteplase (rt-PA) (5 mg / kg, rt-PA mixed with sterile water was added to 1 ml) via the femoral vein after 3 h of modeling. The sham-operated group was administered an equal volume of saline.

[0111] The treatment group was administered with two separate doses, one immediately after modeling and the other immediately after thrombolytic treatment, and the doses were 2, 6, and 18 mg / kg, respectively. The sham-operated group was administered with an equal volume of saline.

[0112] 4. Indicator observation and measurement methods 4.1 Overall condition comparison and blood test 4.1.1 Compare the overall status of rats in each group: sham operation group, thrombolysis group, and thrombolysis + Icaritin group.

[0113] 4.1.2 Detect blood tests of rats in each group: sham operation group, thrombolysis group, and thrombolysis + Icaritin group.

[0114] 4.2 Measurement of cerebral infarction volume (rate) In addition to the sham operation group, three rats were sacrificed for each group during 3 hours of thrombolysis, and the brain tissue was placed in a -20℃ refrigerator for 20 minutes to be flash frozen. Then, 2mm-thick coronal brain slices were prepared, immersed in 2% thrombin solution, placed in a 37℃ light-shielded water bath for 30 minutes, and fixed in 4% paraformaldehyde solution for 24 hours. After photographing, the infarct volume was measured using image analysis software (Image-Pro Plus 6.0). Infarct volume = non-ischemic hemisphere volume - ischemic hemisphere non-infarct area volume. The infarct volume ratio was calculated as the percentage (%) of infarct volume and non-ischemic hemisphere volume.

[0115] 4.3 Measurement of cerebral hemorrhage volume (1) Preparation of standard curve Four more healthy rats were taken, and blood was taken by cardiac perfusion after being killed by overanesthesia. Eight brain hemisphere tissues were taken, and 0, 0.5, 1, 2, 4, 8, 16, 32, and 50 μl of male SD rat arterial blood was added to each hemisphere tissue in order, and PBS with a pH of 7.4 was added to the total volume of 3 ml, and the tissues were homogenized for 30 s, then sonicated, and centrifuged at 13,000 r / min at 4°C for 30 min. 0.8 ml of Drabkin's reagent was taken and mixed with 0.2 ml of the supernatant of each sample, and the samples were left at room temperature in the dark for 10 min, after which the absorbance was measured at a wavelength of 540 nm (the absorbance of the sample without blood was set to 0). The absorbance was taken as the ordinate, and the blood volume in each sample was taken as the abscissa, and a standard curve was drawn.

[0116] (2) Measurement of the amount of hemorrhage in the infarcted hemisphere Add PBS (pH=7.4) to the infarcted hemisphere tissue of the cerebral infarcted rats up to a total volume of 3ml, homogenize for 30s, then sonicate and centrifuge at 13000r / min for 30min. Take 0.8ml of Drabkin's reagent and mix with 0.2ml of the supernatant of each sample, leave it at room temperature in the dark for 10min, and measure the absorbance at a wavelength of 540nm (the absorbance of the brain tissue sample without blood is set to 0 and measured with the same lot). The corresponding blood volume on the obtained absorbance standard curve is the hemorrhage volume.

[0117] 5.Statistical Processing All experimental data are expressed as mean ± standard deviation, and data processing is performed with PASWStatistics17.0 statistical software package. Comparison between groups and within groups is performed by single-factor analysis of variance, and LSD test is used if variance is uniform, and Dunne thrombin time T3 nonparametric test is used if variance is different. Correlation analysis is performed by linear correlation two-tailed Pearson product difference correlation coefficient test, and a two-sided P value < 0.05 is considered to have statistical significance.

[0118] 6. Experimental Results 6.1 Overall situation comparison and blood test detection 6.1.1 Overall situation comparison The mental state of the rats in the sham operation group was good, and the color and luster of the fur was normal. The mental state of the rats in the thrombolysis group and the thrombolysis group + Icaritin group was significantly worse than that before surgery, as described below: the fur color was dark, spontaneous activity was reduced, and the reaction to the outside world was relatively slow. The m-thrombolysis group had the worst mental state, and the thrombolysis group + high-dose Icaritin group had the best mental state.

[0119] 6.1.2 Blood test detection The results of the blood tests showed that the thrombolysis group had no significant differences compared to the thrombolysis group + Icaritin group, and there were no significant differences between the thrombolysis group and the thrombolysis group + Icaritin group compared to the sham surgery group.

[0120] 6.2 Cerebral infarction volume (rate) measurement The extent of cerebral ischemia was observed by thrombin time C staining. According to the staining results, the non-infarcted lateral brain tissue of the sham operation group and each group was uniform and consistent red, while the infarcted lateral brain tissue was obviously edematous, pale and dull, and the infarcted area was pale after staining with thrombin time C. According to the research results, the cerebral infarct volume rate was measured to be (38.84±3.67)% 3h after modeling the preliminary rats, and there was no change in the rat brain tissue thrombin time C staining of the sham operation group. Compared with the model group, the rat thrombin time C staining of the thrombin time C ...

[0121] 6.3 Comparison of the amount of cerebral hemorrhage in rats of each group at different time points The amount of cerebral hemorrhage of rats in each group at different time points is shown in the table below. As can be seen from the table, the amount of hemorrhage in the thrombolysis group is significantly greater than that in the sham operation group, indicating that thrombolysis causes hemorrhagic transformation in cerebral infarction rats, and the amount of hemorrhage increases significantly with time. Compared with the thrombolysis group, the amount of hemorrhage in each dose group of thrombolysis + icaritin is significantly reduced, and the amount of hemorrhage does not increase with time. It is explained that icaritin can effectively prevent hemorrhagic transformation caused by thrombolysis.

[0122] [Table 12]

[0123] Medicinal Efficacy Example 7 Preventive effect of Icaritin on gastrointestinal bleeding caused by aspirin 1. Modeling and Grouping A gastrointestinal bleeding model was created by intragastric administration of 500 mg / kg of aspirin to 24 SD rats, and an equal volume of saline was administered intragastrically to 12 SD rats to serve as normal controls.The bleeding model rats were randomly divided into a model group and an Icaritin group, and the Icaritin group was administered intragastric administration of 18 mg / kg at the same time as the model was created, and the model group was administered intragastric administration of an equal volume of saline.

[0124] 2. Dissection and observation of gastric mucosal ulcer bleeding After 24 hours of intragastric administration, the rats are anesthetized and sacrificed, and then dissected to observe the state of bleeding, erosion, and ulcers on the surface of the gastric mucosa.

[0125] 3. Test results and discussion The incidence of gastric mucosal bleeding in rats in each group is shown in Table 13. As can be seen from the table, no bleeding occurred in the normal control group, and the incidence of bleeding in the model group was 66.7%. Compared with the model group, the gastric mucosal bleeding of rats in the Icaritin group was significantly milder, and the incidence of bleeding was significantly reduced compared with the model group, P<0.01, indicating that Icaritin can significantly reduce the incidence of gastrointestinal bleeding caused by aspirin.

[0126] [Table 13]

[0127] Drug Efficacy Example 8: Preventive effect of Icaritin on gastric bleeding caused by thrombolytic drugs 1. Modeling and Grouping 120 SD rats were divided into sham operation group, model group, urokinase group, icaritin high dose + urokinase group, icaritin medium dose + urokinase group, icaritin low dose + urokinase group by random number method, each group has 20 rats. Among them, each dose group of icaritin was intragastrically administered 4 days before operation, the dose was 2mg / kg, 6mg / kg, 18mg / kg, respectively, and sham operation group, model group and urokinase group were intragastrically administered with equal volume of physiological saline. In addition to sham operation group and model group, the remaining groups were administered from regional artery via catheter, the dose of urokinase was 5000u / kg, the concentration was 200u / μL, and the administration volume was 20μL, and sham operation group and model group were administered with equal volume of physiological saline from regional artery. An animal model of thromboembolic cerebral ischemia is created by binding a thrombus with an autologous thrombus to occlude a middle cerebral artery, and a catheter is placed.

[0128] 2. Observation indicators and measurement methods 2.1 Bleeding rate calculation 24 hours after the intraarterial administration, the rats in each group were anesthetized and dissected, and the number of bleeding cases was observed with the naked eye and an optical microscope, and the ratio of the number of bleeding cases to the total number was calculated.

[0129] 2.2 Pathological observation of gastric tissue, The left lung and gastric antrum were excised and fixed in 100g / L neutral formaldehyde solution, respectively, and then dehydrated, cleared, wax-immersed, embedded, sectioned, and stained by conventional methods, and pathological morphological changes were observed under an optical microscope. The degree of damage was graded from mild to severe according to the degree of tissue congestion, edema, and cell degeneration, with grade I being 0%-25%, grade II being 26%-50%, grade III being 51%-75%, and grade IV >75% (bleeding was observed under a microscope).

[0130] 3. Experimental results and discussion 3.1. Bleeding rate comparison The comparison of the gastric bleeding rate of rats in each group is shown in Table 14. As can be seen from the table, the model group showed gastric bleeding compared with the sham operation group, indicating that cerebral ischemia can cause gastric bleeding complications to a certain extent. The urokinase group showed a significant increase in bleeding rate compared with the model group, with a significant difference (P<0.01), indicating that thrombolytic drugs can cause gastrointestinal bleeding complications when used for thrombolytic treatment of thrombotic diseases. The urokinase + icaritin dose groups showed a significant decrease in bleeding rate compared with the urokinase group, with a significant difference (P<0.01), indicating that icaritin has the effect of reducing the gastrointestinal bleeding rate caused by thrombolytic drugs, and this effect shows a dose-dependent relationship.

[0131] The high dose group of Icaritin had a significant difference (P<0.01) compared with the model group, indicating that Icaritin has a certain preventive effect against gastrointestinal bleeding due to thrombotic diseases themselves.

[0132] [Table 14]

[0133] 3.2 Pathological observation of gastric tissue The pathological observation results of the stomach tissue of rats in each group are shown in Table 15. As can be seen from the table, the rate of gastric damage in the urokinase group is significantly improved compared with the model group, with a significant difference (P<0.01), which shows that thrombolytic drugs can cause complications such as gastric tissue congestion, edema and cell degeneration, and even bleeding when used for thrombolytic treatment of thrombotic diseases. The incidence rates of the above complications in each dose group of urokinase + icaritin are significantly reduced compared with the urokinase group, with a significant difference (P<0.01 or P<0.05), which shows that icaritin has the effect of reducing complications such as gastrointestinal tissue congestion, edema and cell degeneration, and even bleeding caused by thrombolytic drugs, and the incidence rate of the above complications in the high dose group of urokinase + icaritin is significantly reduced compared with the model group. According to the above results, icaritin can not only reduce the incidence rate of gastrointestinal bleeding caused by thrombolytic drugs, but also reduce symptoms such as gastrointestinal tissue congestion, edema and cell degeneration, and play a good recovery effect on gastrointestinal damage.

[0134] Furthermore, the results obtained in comparison with the model group show that icaritin has a certain preventive and therapeutic effect on gastrointestinal damage caused by ischemic disease itself.

[0135] [Table 15]

[0136] Drug Efficacy Example 9: Effect of Icaritin on bleeding complications caused by thrombolytic drugs 1. Creating the Model Sixty male Wistar rats (SPF grade) weighing 220-260 g were randomly divided into a myocardial infarction modeling group and a control group according to a randomization table, with 12 rats in the control group and 48 rats in the modeling group. They were generally housed in separate cages, with 4 rats in each cage. After normal electrocardiograms were recorded, the rats in the myocardial infarction modeling group were administered 150 mg kg -1 .d -1The rats were subcutaneously injected with 1000 mg of isoprenaline (ISO) at a volume of 0.2 mL / 100 g, with a 24-hour interval for two consecutive days. The control group was subcutaneously injected with an equal amount of saline (0.2 mL / 100 g) in the same manner for two consecutive days. The electrocardiogram was monitored for 24 hours after the second subcutaneous injection of isoprenaline, and the rats were anesthetized with ether and the synchronous six-lead (limb-body lead) electrocardiogram was recorded before and after administration, the heart rate was measured, and the degree of Q wave and ST segment offset was observed to evaluate whether the modeling was successful or not. The elevation of the ST segment in lead II greater than 0.2 mV or the formation of a Q wave was regarded as the standard for myocardial infarction.

[0137] 2. Grouping and Dosing Thirty-six rats were selected for modeling and randomly divided into a model group, a thrombolysis group, and a thrombolysis drug + Icaritin group according to a randomization number table. The thrombolysis group was administered 5 mg / kg of rt-PA via the femoral vein, the thrombolysis drug + Icaritin group was administered the same dose of thrombolysis drug and Icaritin (18 mg / kg) was administered intragastrically at the same time, and the model group was administered an equal volume of normal saline via the femoral vein.

[0138] 3. Detection indicators 3.1 Electrocardiogram monitoring of rats in each group The rat is anesthetized by inhaling ether. After the rat enters an anesthetized state, breathing is stable, righting reflex disappears, and pain sensation disappears, the rat is placed in a supine position and fixed on the test table. Needle electrodes are inserted subcutaneously into the rat's four limbs (avoiding insertion into muscles), and limb leads are adopted to monitor the rat's electrocardiogram. The electrocardiogram machine is started with the paper feed speed of 50mm / s and the voltage of 1mV. After the electrocardiogram is completed, the animal is returned to its cage.

[0139] 3.2 Cardiac histomorphometric observation of rats in each group After the last administration, the animals were sacrificed, fixed supine on a mouse plate, the skin was disinfected with 75% alcohol, the chest was quickly opened, the skin and subcutaneous tissue were cut along the midline of the sternum with tissue scissors, the ribs on the left side of the sternum were cut, the great vessels were cut to remove the heart, the blood remaining in the heart cavity was washed with 4°C 0.9% sodium chloride solution, the left and right atrial appendages and the remaining great vessels were cut off, filtered through filter paper and dried, and the rats were weighed on an electronic balance to calculate the body mass and heart mass of the rats, and the actual weight (wet weight) of the heart was weighed, and the ratio of this to the body weight (HWI=heart weight / body mass) was calculated.

[0140] 3.3 Observation of bleeding complications in rats of each group The rats in each group were observed for bleeding conditions, including subcutaneous bleeding points, and the bleeding conditions of each internal organ and intracranial bleeding points were observed after dissection.

[0141] 4. Test results and discussion 4.1 Electrocardiogram status of each group Compared with the control group, pathological Q waves appeared in the rats of the model group, indicating that the modeling was successful. Compared with the model group, pathological Q waves disappeared in both the thrombolysis group and the thrombolysis + Icaritin group, indicating that the thrombolysis drug can significantly improve the myocardial infarction state of the rats with myocardial infarction, and Icaritin does not affect the thrombolysis effect of the thrombolysis drug.

[0142] 4.2 Comparison of the overall cardiac morphology of rats in each group Observe the general shape of the heart of each group of rats with the naked eye, and the model group has a significant increase in heart volume, a significant expansion of the left ventricular cavity, and necrosis of the myocardial wall permeability in the infarcted area, and the color is pale and pale.Compared with the model group, the degree of increase in heart volume and the degree of myocardial wall permeability necrosis in the thrombolysis group and the thrombolysis drug + icaritin group are significantly less than the model group.The heart weight and the ratio of heart weight to body mass of each group of rats are shown in the table below.As can be seen from the table, the model group has a significant difference (P<0.01) in the ratio of heart weight to body mass compared with the control group, which indicates that the modeling was successful. The thrombolysis group and the thrombolysis drug + Icaritin group had significant differences (P<0.01) compared with the model group, and the thrombolysis group had no significant differences compared with the thrombolysis drug + Icaritin group, indicating that the thrombolysis drug can significantly improve the myocardial infarction state of rats with cardiac infarction, and the Icaritin group does not affect the effect of the thrombolysis drug.

[0143] [Table 16]

[0144] 4.3 Comparison of bleeding complications in rats treated with this drug

[0145] [Table 17]

[0146] As can be seen from the above table, the bleeding percentage in the thrombolysis group was significantly higher than that in the thrombolysis + icaritin group, with statistical significance (P<0.01). This result shows that icaritin can obviously reduce the incidence of bleeding complications caused by thrombolysis.

[0147] Drug Efficacy Example 10: Effect of Icaritin on bleeding time in hemophilic mice with deleted blood coagulation factor IX gene 1. Test Animals The IX gene-deleted hemophilic mice were provided by Beijing Baiosaitu Gene Biology Co., Ltd.

[0148] 2. Animal Grouping and Dosing Forty IX gene-deficient hemophilic mice were randomly divided into four groups, with 10 mice in each group. The low, medium and high dose groups (B, C and D) were administered 3mg / kg, 6mg / kg and 18mg / kg of Icaritin intragastrically once daily for 11 consecutive days. The control group (A) was administered an equal volume of saline intragastrically.

[0149] 3. Detection indicators 3.1 Bleeding time measurement One hour after the last administration, the bleeding time of each mouse is measured by cutting the tail.

[0150] 3.2 Blood test measurements After completing the final bleeding time detection, the mice are anesthetized with sodium pentobarbital, and blood is collected from the abdominal vein to detect blood tests.

[0151] 4. Test Results 4.1 Bleeding time measurement The bleeding time of the mice in each group is shown in the table below. As can be seen from the table, compared with the control group, the bleeding time of the mice in each Icaritin dose group was significantly shortened, with statistical significance (#P<0.05 or ## P<0.01), demonstrating that icaritin can effectively shorten the bleeding time of hemophilia mice with IX gene deletion.

[0152] [Table 18]

[0153] 4.2 Blood tests Blood tests of mice in each Icaritin dose group showed no significant difference from the control group, explaining that Icaritin shortens the bleeding time of hemophilic mice without affecting the blood test values.

[0154] Drug Efficacy Example 11: Effect of Icaritin on Activated Partial Thromboplastin Time in Acquired Hemophilia A 1. Test materials and animals Measurement of activated partial thromboplastin time, the kit was purchased from Shanghai Sun Biotechnology Co., Ltd., plasma from patients with acquired hemophilia A was provided by Linyi People's Hospital, and healthy experimental New Zealand rabbits were provided by China Bingfeng Pastoral Cattle and Sheep Breeding Research Base.

[0155] 2. Creation and administration of an animal model of acquired hemophilia A 24 healthy New Zealand rabbits were selected and randomly divided into a control group, a model group and a treatment group, each weighing about 1.5 kg. They were kept in separate cages in stainless steel cages at room temperature of 18-25°C, and each group was fed a normal diet without any special treatment. After one week, the rabbits were anesthetized with 2% pentobarbital (1.5 ml / kg) through the ear margin vein, and then the left femoral vein of the rabbits was isolated and exposed for injection. The plasma of a patient with acquired hemophilia A (diagnosis confirmed by the Hematology Department of Linyi People's Hospital) was intravenously injected into the rabbits in the model group and treatment group according to 2 ml / kg of rabbit body weight, and after the injection was completed, the treatment group was immediately administered 18 mg / kg of icaritin by intragastric administration, and the model group was intragastricly administered with an equal amount of saline. The rabbits in the control group were intravenously injected with an equal amount of normal human plasma, and blood samples were taken from the marginal ear vein 30 min before and 30, 60, 90, and 120 min after plasma injection, respectively, and anticoagulated with 3.8% 1:9 sodium citrate. The anticoagulated blood was centrifuged at 3000 r / m, and then the plasma was separated.

[0156] 3. Thromboplastin time measurement and statistical methods Refer to the kit instructions, take 0.1 ml of rabbit plasma, then add 0.1 ml of thromboplastin time reagent, mix and place in a warm bath at 37°C for 2 minutes, add 0.1 ml of 0.025 M calcium chloride at 37°C preheated and mix evenly, then immediately start a stopwatch to measure the thromboplastin time value at each time point, repeat the thromboplastin time at each time point twice, and take the average result.

[0157] The data were statistically analyzed using SPSS11.0 software. Data were expressed as mean ± standard deviation, and normality and variance tests were performed. Intergroup comparisons were performed using t-tests.

[0158] 4. Test Results The comparison of thromboplastin time at each time point of each group is shown in Table 19. As can be seen from the table, the rabbit thromboplastin time value of the control group has no obvious change before and after plasma injection; the rabbit plasma thromboplastin time of the model group is obviously prolonged at 30min, 60min, 90min and 120min after injection compared with the thromboplastin time value before injection, and the prolongation of thromboplastin time shows a time-dependent trend; the treatment group can obviously shorten the thromboplastin time at each time point. It explains that Icaritin can obviously shorten the thromboplastin time of the activated part of hemophilia patients and promote the blood coagulation of hemophilia patients.

[0159] [Table 19]

Claims

1. A pharmaceutical composition comprising icaritin for preventing or treating a bleeding disorder, The bleeding disorder is a bleeding disorder caused by a platelet dysfunction disorder, a bleeding disorder caused by a blood coagulation disorder, or a bleeding disorder caused by an anticoagulation and fibrinolysis disorder, The bleeding disease caused by abnormal platelet function is hemorrhagic transformation of cerebral infarction, gastrointestinal bleeding caused by a thrombolytic or antithrombotic drug for cerebral infarction, or bleeding complications caused by a thrombolytic or antithrombotic drug for myocardial infarction, The bleeding disorder caused by blood coagulation abnormality is hemophilia, which is any one of hemophilia A, hemophilia B, and hemophilia C; The pharmaceutical composition, characterized in that the bleeding disorder due to anticoagulation and fibrin lysis abnormality is an acquired disorder, including, but not limited to, those caused by an excess of heparin, an excess of coumarin drugs, an increase in immune-related anticoagulants, or an excess of thrombolytic drugs.

2. The cerebral infarction hemorrhagic transformation is a secondary ischemic cerebral infarction hemorrhagic transformation, a primary hemorrhagic transformation, or an asymptomatic hemorrhagic transformation; Preferably, the secondary hemorrhagic transformation is hemorrhagic transformation by using one or more of the following therapeutic methods: thrombolytic drugs, antithrombotic drugs, and endovascular therapy; More preferably, the thrombolytic agent is selected from urokinase, streptokinase, reteplase, alteplase, tenecteplase, and lanoteplase; More preferably, the antithrombotic agent is one or more of an anticoagulant and an antiplatelet agent; More preferably, the anticoagulant is selected from heparin or a pharma- ceutically acceptable salt thereof, low molecular weight heparin or a pharma- ceutically acceptable salt thereof, and recombinant hirudin; More preferably, the antiplatelet agent is selected from aspirin, clopidogrel, and ticlopidine; The pharmaceutical composition according to claim 1, characterized in that the primary hemorrhagic transformation is spontaneous hemorrhagic transformation after acute cerebral infarction.

3. In the case of bleeding complications due to the thrombolytic or antithrombotic drug for myocardial infarction, the thrombolytic drug is selected from urokinase, streptokinase, reteplase, alteplase, tenecteplase, and lanoteplase, and the antithrombotic drug is used for antithrombotic treatment in the onset stage, primary prevention stage, and secondary prevention stage of myocardial infarction, or the antithrombotic drug is one or more of anticoagulants or antiplatelet drugs; Preferably, the antiplatelet agent is selected from aspirin, clopidogrel and ticlopidine, and the anticoagulant is selected from heparin or a pharma- ceutically acceptable salt thereof, low molecular weight heparin or a pharma- ceutically acceptable salt thereof and recombinant hirudin; The pharmaceutical composition according to claim 1, characterized in that the bleeding complication is preferably one or more of subcutaneous bleeding, intracranial bleeding, upper gastrointestinal bleeding or gingival bleeding.

4. In the case of gastrointestinal bleeding caused by the thrombolytic drug for cerebral infarction or the antithrombotic drug, the thrombolytic drug is one or more of urokinase, streptokinase, reteplase, alteplase, tenecteplase, and lanoteplase, and the antithrombotic drug is one or more of anticoagulants or antiplatelet drugs; The pharmaceutical composition according to claim 1, characterized in that the antiplatelet drug is selected from aspirin, clopidogrel, and ticlopidine, and the anticoagulant is selected from heparin or a pharma- ceutically acceptable salt thereof, low molecular weight heparin or a pharma- ceutically acceptable salt thereof, and recombinant hirudin.

5. The platelet dysfunction is a platelet activation dysfunction or aggregation dysfunction, Preferably, the platelet dysfunction is a congenital platelet deficiency or an acquired platelet deficiency, Preferably, the congenital platelet deficiency is hereditary giant platelet syndrome, hereditary thrombasthenia or congenital connective tissue disease, and the acquired platelet deficiency is due to a drug or disease; More preferably, the drug is one or more of an antibacterial agent, an antitumor agent or heparin, and the disease is one or more of uremia, diabetes, nephrotic syndrome, coronary heart disease or leukemia.

6. The hemophilia is genetic hemophilia or secondary hemophilia, Preferably, the secondary hemophilia is due to a drug or an autoimmune disease, More preferably, said autoimmune disease includes, but is not limited to, liver lesions, hemolysis, acquired immune deficiency, Evans syndrome, chronic lymphocytic leukemia, various acute leukemias, lymphomas, systemic lupus erythematosus, rheumatoid arthritis or hyperthyroidism; More preferably, the liver lesion is caused by chronic persistent hepatitis or liver cirrhosis; More preferably, the pharmaceutical composition according to claim 1, characterized in that the drug includes, but is not limited to, alkylating agents, metabolic inhibitors, cytotoxic agents, chlorothiazide class drugs and their synergists.

7. The pharmaceutical composition according to any one of claims 1 to 6, which is in the form of an injection, a powder injection, a capsule, a tablet, a microemulsion, a drop or an enteric-coated soft capsule.

8. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that the dosage of Icaritin is 0.01 mg / kg to 100 mg / kg.

9. The pharmaceutical composition according to claim 8, characterized in that the dosage of Icaritin is 0.1 mg / kg to 10 mg / kg.

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