Medical Uses of Epimedin

Abstract

The invention discloses a use of icariin in preparing a drug for preventing and treating hemorrhagic diseases, which belongs to the field of medicine. Icariin can improve platelet dysfunction, shorten thromboplastin time, promote blood coagulation, and can be used for the prevention and treatment of hemorrhagic diseases, especially the treatment of hemorrhagic transformation of cerebral infarction, gastrointestinal bleeding caused by thrombolytic drugs or antithrombotic drugs for cerebral infarction, or bleeding complications caused by thrombolytic drugs or antithrombotic drugs for myocardial infarction.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and relates to the medical use of icariin, and specifically to the use of icariin in preparing medicines for preventing or treating hemorrhagic diseases. Background Art

[0002] When a human blood vessel is damaged, blood may flow out or seep out of the blood vessel. At this time, the body will stop the bleeding through a series of physiological reactions, which is called hemostasis. The hemostasis process involves many factors and includes a series of complex physiological and biochemical reactions. Diseases characterized by spontaneous or persistent bleeding after blood vessel damage caused by defects in hemostasis are called hemorrhagic diseases.

[0003] Bleeding disorders can be divided into the following main types according to their causes and pathogenesis:

[0004] (I) Caused by abnormal platelet function. Including hemorrhagic transformation of cerebral infarction, gastrointestinal bleeding caused by thrombolytic drugs or antithrombotic drugs for cerebral infarction, or hemorrhagic complications of thrombolytic drugs or antithrombotic drugs for myocardial infarction. Among them, hemorrhagic transformation of cerebral infarction is secondary hemorrhagic transformation of ischemic cerebral infarction, primary hemorrhagic transformation or asymptomatic hemorrhagic transformation. Thrombolytic drugs or anticoagulant drugs are currently effective methods for the treatment of acute ischemic infarction, but while dissolving thrombi, they can also lead to a variety of serious complications such as hemorrhagic transformation, which seriously limits their clinical application. At present, Western medicine only prevents and treats various complications after treatment with thrombolytic drugs or anticoagulants from a single link or a single factor, and has not yet achieved satisfactory results. Therefore, it is of great significance to give full play to the advantages of traditional Chinese medicine in focusing on overall regulation and multi-link comprehensive treatment, and to seek effective treatment methods and prescriptions that can dissolve thrombi while reducing or eliminating the risks of various types of hemorrhagic transformation, and improve the short-term and long-term efficacy of thrombolytic treatment of infarction diseases. (II) Caused by abnormal blood vessel walls. Including congenital or hereditary and acquired. Congenital diseases include hereditary hemorrhagic telangiectasia, familial simplex purpura, and congenital connective tissue diseases. (III) Caused by abnormal coagulation. Including congenital, hereditary and acquired. Congenital coagulation abnormalities refer to hemophilia A, B and hereditary FXI deficiency; hereditary prothrombin, FV, FVII, FX deficiency, hereditary fibrinogen deficiency and reduction, hereditary FXII deficiency and reduction. Hereditary coagulation abnormalities refer to hepatic coagulation disorders; vitamin K deficiency; anti-factor VIII, IX antibody formation; uremic coagulation abnormalities, etc. (IV) Caused by abnormal anticoagulation and fibrinolysis. Mainly acquired diseases, including but not limited to hemorrhagic diseases caused by overdose of heparin or its pharmaceutically acceptable salts, overdose of coumarin drugs, increase in immune-related anticoagulants or overdose of thrombolytic drugs.

[0005] Epimedium is the dried stems and leaves of Epimedium brevicornum Maxim., Epimedium sagittatum Maxim., Epimedium pubescens Maxim. or Epimedium koreanum Nakai, which are plants of the Berberidaceae family. It is mainly used clinically for kidney yang deficiency, impotence, frequent urination, infertility; rheumatic pain, numbness and cramps of limbs, weakness of tendons and bones, and difficulty in walking; kidney yang deficiency, wheezing, coughing and shortness of breath.

[0006] Icariin (IT) is a polyhydroxyflavonoid monomer component in the plant Epimedium of the Berberidaceae family. Pharmacological studies have shown that IT has a stronger anti-osteoporosis effect than other flavonoid glycosides in Epimedium, and has the effect of promoting osteoblast activity and inhibiting osteoclast activity in vitro. In recent years, icariin and icariin, as important active ingredients in Epimedium, have attracted more and more attention from medical workers. For example, patent application CN101637467A discloses the use of icariin in the preparation of drugs for the treatment of osteoporosis. Patent US6399579 discloses the use of icariin in the treatment of sexual dysfunction. Summary of the invention

[0007] During the research process, the applicant found that icariin can improve platelet dysfunction, shorten thromboplastin time, and promote coagulation. It can be used for the prevention and treatment of platelet dysfunction and hemorrhagic diseases caused by it, hemorrhagic diseases caused by coagulation abnormalities, hemorrhagic diseases caused by vascular wall abnormalities, or hemorrhagic diseases caused by anticoagulation and fibrinolysis.

[0008] In one aspect, the present application provides the use of icariin in a drug for preventing or treating a disease, wherein the disease is platelet dysfunction and bleeding diseases caused by it, bleeding diseases caused by coagulation abnormalities, bleeding diseases caused by vascular wall abnormalities, or bleeding diseases caused by anticoagulation and fibrinolysis.

[0009] In one embodiment, the hemorrhagic disease caused by abnormal platelet function is selected from hemorrhagic transformation of cerebral infarction, gastrointestinal bleeding caused by thrombolytic drugs or antithrombotic drugs for cerebral infarction, or bleeding complications of thrombolytic drugs or antithrombotic drugs for myocardial infarction.

[0010] In one embodiment, the hemorrhagic transformation of cerebral infarction is secondary hemorrhagic transformation of ischemic cerebral infarction, primary hemorrhagic transformation or asymptomatic hemorrhagic transformation.

[0011] In one embodiment, the secondary hemorrhagic transformation is hemorrhagic transformation caused by one or more treatment methods selected from thrombolytic drugs, antithrombotic drugs, and endovascular therapy.

[0012] In one embodiment, the thrombolytic drug is selected from urokinase, streptokinase, reteplase, alteplase, tenecteplase or lanoteplas. In one embodiment, the antithrombotic drug is one or more of an anticoagulant and an antiplatelet drug. In one embodiment, the anticoagulant is selected from heparin or a pharmaceutically acceptable salt thereof, a low molecular weight heparin or a pharmaceutically acceptable salt thereof, and recombinant hirudin. In one embodiment, the antiplatelet drug is selected from aspirin, clopidogrel or ticlopidine.

[0013] In one embodiment, the primary hemorrhage transforms into spontaneous hemorrhagic transformation after acute cerebral infarction.

[0014] In one embodiment, the primary hemorrhage transforms into spontaneous hemorrhagic transformation after acute cerebral infarction.

[0015] In one embodiment, the thrombolytic drug for myocardial infarction or the antithrombotic drug for bleeding complications is selected from urokinase, streptokinase, reteplase, alteplase, tenecteplase or lanoteplase; the antithrombotic drug is an antithrombotic treatment for the onset of myocardial infarction and the primary and secondary prevention periods; the antithrombotic drug is one or more of an anticoagulant or antiplatelet drug;

[0016] In one embodiment, the antiplatelet drug is selected from aspirin, clopidogrel or ticlopidine, and the anticoagulant drug is selected from heparin or its pharmaceutically acceptable salt, low molecular weight heparin or its pharmaceutically acceptable salt, and recombinant hirudin;

[0017] In one embodiment, the bleeding complication is one or more of subcutaneous hemorrhage, intracranial hemorrhage, upper gastrointestinal bleeding or gingival bleeding.

[0018] In one embodiment, the thrombolytic drug in the gastrointestinal bleeding caused by the thrombolytic drug or antithrombotic drug for cerebral infarction is one or more of urokinase, streptokinase, reteplase, alteplase, tenecteplase or lanoteplase; the antithrombotic drug is one or more of anticoagulant drugs or antiplatelet drugs.

[0019] In one embodiment, the platelet dysfunction is abnormal platelet activation function or aggregation function; the platelet dysfunction is congenital platelet defect or acquired platelet defect;

[0020] In one embodiment, the congenital platelet defect is hereditary giant platelet syndrome, hereditary thrombasthenia or congenital connective tissue disease; the acquired platelet defect is caused by drugs or diseases;

[0021] In one embodiment, the drug is one or more of an antibacterial drug, an antitumor drug or heparin, and the disease is one or more of uremia, diabetes, nephrotic syndrome, coronary heart disease or leukemia.

[0022] In one embodiment, the bleeding disorder caused by abnormal coagulation is hemophilia.

[0023] In one embodiment, the hemophilia is hemophilia caused by genetic factors or secondary hemophilia; the secondary hemophilia is caused by drugs or autologous diseases.

[0024] In one embodiment, the autologous disease includes but is not limited to liver disease, hemolysis, acquired immunodeficiency disease, Evans syndrome, chronic lymphocytic leukemia, various acute leukemias, lymphoma, systemic lupus erythematosus, rheumatoid arthritis or hyperthyroidism; the liver disease is caused by any one of chronic persistent hepatitis and cirrhosis; the drugs include but are not limited to alkylating agents, antimetabolites, cytotoxic preparations chlorothiazide drugs and their synergists.

[0025] In one embodiment, the hemophilia is any one of hemophilia A, hemophilia B or hemophilia C.

[0026] In one embodiment, the hemorrhagic disease caused by abnormal anticoagulation and fibrinolysis is an acquired disease, including but not limited to overdose of heparin or its pharmaceutically acceptable salts, overdose of coumarin drugs, increase in immune-related anticoagulants or overdose of thrombolytic drugs.

[0027] Efficacy Example 1 Effect of icariin on platelet activation function The results showed that 3 days after icariin was administered, the peripheral anticoagulated blood of the animals was activated under the same conditions of adenosine diphosphate, and the expression of CD62p in all platelets marked by CD61 increased significantly, that is, the activation rate of platelets activated by adenosine diphosphate increased, which was significantly different from the basal value before administration. It is suggested that under the experimental conditions, intragastric administration of icariin can significantly promote platelet activation induced by adenosine diphosphate (ADP) in the peripheral anticoagulated blood of rats.

[0028] The results of the study on the effect of icariin on platelet aggregation function in vitro showed that all concentrations of icariin have the effect of promoting adenosine diphosphate-induced platelet aggregation activity, and the promoting effect increases with increasing concentration, indicating that the promoting effect of icariin on adenosine diphosphate-induced platelet aggregation activity is dose-dependent.

[0029] Efficacy Example 2 The test on the effect of icariin on hemorrhagic transformation after cerebral ischemia-reperfusion caused by aspirin or clopidogrel showed that icariin can significantly inhibit the hemorrhagic transformation after cerebral ischemia-reperfusion caused by aspirin or clopidogrel.

[0030] Efficacy Example 3 Effect of icariin on the bleeding time of mice induced by sodium heparin The results showed that icariin can significantly shorten the bleeding time of mice induced by sodium heparin, and does not change the blood routine and four coagulation items while exerting a procoagulant effect.

[0031] Efficacy Example 4 Effect of icariin on the bleeding time of mice induced by aspirin The results showed that icariin can significantly shorten the bleeding time of mice induced by aspirin.

[0032] The results of Example 5 of the effect of icariin on the thrombolytic effect of thrombolytic drugs and Example 6 of the effect of icariin on hemorrhagic transformation caused by thrombolytic drugs show that icariin can significantly improve the hemorrhagic transformation caused by thrombolytic drugs, and while preventing hemorrhagic transformation and reducing the amount of bleeding, it does not affect the dissolving effect of thrombolytic drugs on thrombi, and does not affect the blood routine values. Icariin 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 reducing effect on the risk of spontaneous hemorrhagic transformation and asymptomatic hemorrhagic transformation. In addition to having a significant preventive and therapeutic effect on the hemorrhagic transformation after the above-mentioned cerebral infarction, icariin also has a significant preventive and therapeutic effect on the occurrence of hemorrhagic infarction that can be determined based on the first cranial CT / MRI.

[0033] Pharmacological Example 7: The preventive effect of icariin on gastrointestinal bleeding caused by aspirin. Pharmacological Example 8: The preventive effect of icariin on gastric bleeding caused by thrombolytic drugs. The test results show that icariin can not only reduce the incidence of gastrointestinal bleeding caused by thrombolytic drugs, but also a high dose of icariin has a significant preventive effect on gastrointestinal bleeding caused by thrombotic diseases themselves, and the above effects are all dose-dependent.

[0034] Efficacy Example 9 Effect of icariin on hemorrhagic complications caused by thrombolytic drugs The experimental results show that icariin can significantly reduce the incidence of hemorrhagic complications of myocardial infarction caused by thrombolytic drugs, and its simultaneous use with thrombolytic drugs does not affect the thrombolytic effect of thrombolytic drugs.

[0035] Efficacy Example 10 Effect of icariin on the bleeding time of hemophiliac mice with coagulation factor IX gene knockout The results showed that icariin can effectively shorten the bleeding time of hemophiliac mice with IX gene knockout, and there was no significant difference in the blood routine test of each group of mice.

[0036] Efficacy Example 11 Effect of icariin on activated partial thromboplastin time in acquired hemophilia A The results showed that icariin can significantly shorten the activated partial thromboplastin time of hemophilia patients and promote blood coagulation in hemophilia patients.

[0037] Icariin has a significant therapeutic effect not only on hemophilia caused by genetic factors, but also on secondary hemophilia.

[0038] When icariin is used for the prevention and treatment of hemorrhagic diseases, the administration routes include gastrointestinal and parenteral routes, and the parenteral routes include but are not limited to subcutaneous, intradermal, arterial, venous, muscle, joint, intrathecal, intracranial, thoracic, intraperitoneal injection or instillation, nasal, buccal, sublingual, tracheal, urethral, ​​rectal or local administration of lesions, etc. It can be taken at the same time as other drugs, or taken in advance for preventive medication.

[0039] In addition to providing the use of icariin in preparing drugs for preventing and treating hemorrhagic diseases, the present invention also provides a pharmaceutical preparation used in treating the above diseases, which comprises icariin and pharmaceutically acceptable excipients.

[0040] The pharmaceutical excipients used in the preparation of the present invention are common excipients known to those skilled in the art.

[0041] Suitable pharmaceutical excipients are described in detail in the "Compendium of Pharmaceutical Excipients" (page 123, Sichuan Science and Technology Press, published in 1993, edited by Luo Mingsheng and Gao Tianhui). For example, pharmaceutical excipients commonly used in the preparation of microemulsion preparations include but are not limited to soybean oil, polyoxyethylene-23-lauryl ether, 1,2-propylene glycol, hydrogenated coconut oil glyceride, lauroyl polyethylene glycol-32-glyceride, polyethylene glycol 3350, safflower oil, cottonseed oil, and decaglycerol monostearate; pharmaceutical excipients commonly used in the preparation of drop pill preparations include but are not limited to polyethylene glycol 6000 and polyethylene glycol 1000; pharmaceutical excipients commonly used in the preparation of capsule preparations include but are not limited to lactose and corn starch. Pharmaceutically acceptable carriers commonly used in the preparation of soft capsule preparations include but are not limited to medium-chain fatty acid glycerides, polyoxyethylene castor oil, 1,2-propylene glycol, etc.

[0042] Those skilled in the art can select suitable pharmaceutical excipients according to actual needs and prepare the preparation of the present invention by methods known in the art. The preparation includes but is not limited to solid, liquid, oil, emulsion, gel, aerosol, inhalant, spray, capsule, pill, patch and suppository, etc.

[0043] The icariin of the present invention can improve platelet dysfunction, shorten thromboplastin time, promote blood coagulation, and can be used for the prevention and treatment of platelet dysfunction and hemorrhagic diseases caused by it, hemorrhagic diseases caused by abnormal coagulation, hemorrhagic diseases caused by abnormal blood vessel walls, or hemorrhagic diseases caused by anticoagulation and fibrinolysis. DETAILED DESCRIPTION

[0044] The present invention is further described below through specific implementation modes, and the present invention is not limited to the following embodiments.

[0045] Preparation Example 1 Epimedin Microemulsion Preparation

[0046]

[0047] Preparation process: Weigh the prescribed amount of soybean oil, polyoxyethylene-23-lauryl ether, and 1,2-propylene glycol, mix and stir evenly, then add icariin to dissolve, or ultrasonic treatment can be used to accelerate dissolution, to obtain a clear solution, which is the icariin microemulsion preparation. The particle size is measured by a laser particle size analyzer, and the average particle size is 15nm.

[0048] Preparation Example 2 Epimedin Microemulsion Preparation

[0049]

[0050]

[0051] Preparation process: Weigh the prescribed amount of hydrogenated coconut glyceride, lauroyl polyethylene glycol-32-glyceride, 1,2-propylene glycol, and polyethylene glycol 3350, mix and stir evenly, then add icariin to dissolve, or ultrasonic treatment can be used to accelerate dissolution, to obtain a clear solution, which is the icariin microemulsion preparation. The particle size is measured by a laser particle size analyzer, and the average particle size is 40nm.

[0052] Preparation Example 3 Epimedin Injection

[0053]

[0054] Preparation process: add icariin to the prescribed amount of PEG-400, stir to dissolve, add 0.9% sodium chloride solution to 10L, stir evenly, add 0.5% needle-grade activated carbon, stir, and decarbonize to obtain.

[0055] Preparation Example 4 Epimedin Injection

[0056]

[0057] Preparation process: Evenly mix the prescribed amount of ethanol and Tween-80, add icariin, stir to dissolve, add water for injection to 10L, stir evenly, add 0.5% activated carbon for injection, stir, and decarbonize to obtain the product.

[0058] Preparation Example 5 Epimedin Injection

[0059] Epimedin 1g Ethanol 3.3L

[0060] Add water for injection to 10L

[0061] Preparation process: Add the prescribed amount of ethanol to icariin, stir to dissolve, add water for injection to 10L, stir evenly, add 0.5% activated carbon for injection, stir, and decarbonize to obtain the product.

[0062] Preparation Example 6 Epimedin Drop Pill Preparation

[0063] Epimedin 5.0g Macrogol-6000 14.5g

[0064] Macrogol-1000 5.0g 1000 tablets

[0065] Preparation process: Weigh the prescribed amount of epimedin that has passed through a 100-mesh sieve, add it to a mixture of polyethylene glycol 6000 and polyethylene glycol 1000 that has been heated and melted in a water bath, stir it thoroughly to make it uniform, put it into a dropping bottle, and drip it at 95±2℃; drip it into a glass condensation column containing 4-6mL of methyl silicone oil, take it out after it is formed, and use absorbent paper to absorb the adhering methyl silicone oil to obtain the product.

[0066] Preparation Example 7 Epimedin Enteric-coated Soft Capsule Preparation

[0067] Contents prescription:

[0068]

[0069]

[0070] Rubber prescription:

[0071] Gelatin 10g Glycerin 5g

[0072] Purified water 10g

[0073] Enteric coating solution prescription:

[0074]

[0075] Preparation process: Weigh 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 icariin to dissolve, and ultrasonic treatment can also be used to accelerate dissolution, so as to obtain the soft capsule microemulsion content. Weigh the prescribed amount of gelatin, glycerin, and purified water, mix evenly, and press into a rubber skin, then weigh the prescribed amount of Eudragit L30D-55, triethyl citrate, talcum powder, and purified water and mix evenly to obtain an enteric coating solution. The soft capsule microemulsion content containing icariin is wrapped with a rubber skin to prepare a soft capsule, and an enteric coating is applied on the soft capsule to obtain an enteric soft capsule.

[0076] Preparation Example 8 Epimedin Capsule Preparation

[0077]

[0078] Preparation process: Mix 100g of icariin, 120g of lactose and 130g of corn starch in a mixer for 10-15 minutes, add 5g of magnesium stearate and mix for 1-3 minutes, and fill into 1000 capsule shells.

[0079] Preparation Example 9 Epimedin Tablets

[0080]

[0081] Preparation process: evenly mix epimedin and auxiliary materials microcrystalline cellulose and sodium carboxymethyl starch, add appropriate amount of starch slurry to make soft material, and then sieve through 16 mesh sieve for granulation. Dry the wet granules at 60℃, sieve the dry granules through 20 mesh sieve, sieve out the fine powder in the dry granules, mix with magnesium stearate, and then mix with the dry granules, press into tablets, each tablet is about 260mg, and the product is obtained.

[0082] Preparation Example 10 Epimedin Powder Injection

[0083]

[0084] Preparation process: Weigh the prescribed amount of icariin for injection raw material, add an appropriate amount of water for injection to dissolve. Then, add the prescribed amount of pre-sterilized and pyrogen-free solution and mix well, then add water for injection to the prescribed 10L; add 50g of activated carbon for injection to the above liquid, heat at 60-80℃ for 30 minutes, filter with a filter membrane, and collect the filtrate. The above filtrate is sterilized by positive pressure sterilization filtration with a sterilizing filter according to the aseptic operation method, filtered with a 0.22μM microporous filter membrane, and the filtrate is tested for pyrogens and semi-finished product content before being packaged in vials. In a special freeze-drying box, pre-freeze at below -40℃ for 1.5-3.5 hours, sublimate under vacuum, and heat and dry after 90% of the free water is removed (the maximum temperature shall not exceed 35℃), and the icariin powder injection can be prepared after freeze-drying.

[0085] Efficacy Example 1 Effect of Icariin on Platelet Activation Function and Platelet Aggregation Function in Vitro

[0086] 1. Effect of icariin on platelet activation function

[0087] 1.1 Experimental animals

[0088] SD rats, SPF grade, weighing 250-300g, half male and half female, female rats are not pregnant.

[0089] 1.2 Flow cytometry detection of platelet activation

[0090] 0.5 mL of blood was collected from the rat orbit and anticoagulated with 3.8% sodium citrate (the ratio of blood to anticoagulant was 9:1) to prepare anticoagulated blood. 50 ul of anticoagulated blood was taken from each animal for determination of spontaneous platelet activation rate; another 50 ul of anticoagulated blood was added with 50 ul of adenosine diphosphate with a final concentration of 10 μmol for activation. 1.0-1.5 μL of CD62p PE / Cy7 and CD61PE were added to the above two anticoagulated blood, respectively, to label all platelets with CD61 and activated platelets with CD62p. 50 μL of anticoagulated blood was added with 50 μL of adenosine diphosphate with a final concentration of 10 μmol for activation. G2a-PE / Cy7 and G-PE double labeling were used as isotype controls. The above tubes were incubated at 4°C in the dark for 20 min; then 0.5 mL of 4% paraformaldehyde was added for fixation for 10 min. Take 50 μL of the fixed blood sample, add 1 mL of diluent to dilute and analyze on the machine. Delineate the platelet cell population in the CD61 PE / side angle light scatter (SSC) dual parameter scatter plot, count 5000 platelets, further count the number of cells positively expressing CD61 and CD62p in the CD61 / CD62p scatter plot, and reflect the platelet activation rate (%) as the percentage of CD62p positive expression rate to CD61 expression rate.

[0091] 1.3 Effect of icariin on adenosine diphosphate-induced platelet activation in rats

[0092] 24 rats were divided into 3 groups, 8 rats in each group, and 3, 6, and 18 mg / kg of icariin were administered by gavage, once a day, for 3 consecutive days. 1.0 mL of blood was collected from the eye sockets before administration and 20 minutes after the last administration, of which 0.5 mL was used to determine the blood routine test and 0.5 mL was used to prepare anticoagulated blood. After processing according to the above-mentioned flow cytometry detection method for platelet activation, the platelet activation rate activated by adenosine diphosphate before administration (basic value) and the platelet activation rate activated by adenosine diphosphate after administration were detected, and the two were compared. At the same time, 50ul of rat anticoagulated blood was taken before administration without adding adenosine diphosphate, and the spontaneous activation rate of animal anticoagulated blood was determined by the same method. The animals were fasted for 16-24 hours before each blood collection.

[0093] 1.4 Test items and test results

[0094] (1) Effects on adenosine diphosphate-induced platelet activation in rats

[0095] After the peripheral anticoagulated blood of animals was activated by adenosine diphosphate before administration, the proportion of activated platelets in all platelets marked with CD61 increased significantly compared with the spontaneous activation rate, that is, the expression of CD62p increased significantly, indicating that adenosine diphosphate can induce an increase in platelet activation rate. Three days after the administration of icariin, the peripheral anticoagulated blood of animals was activated by adenosine diphosphate under the same conditions, and the expression of CD62p increased significantly in all platelets marked with CD61, that is, the activation rate of platelets activated by adenosine diphosphate increased, which was significantly different from the basal value before administration. It is suggested that under the experimental conditions, intragastric administration of icariin can significantly promote platelet activation induced by adenosine diphosphate in the peripheral anticoagulated blood of rats. See Table 1 for details.

[0096] Table 1 Comparison of platelet activation rate after adenosine diphosphate activation before and after administration in vivo

[0097]

[0098] (2) Blood routine

[0099] There was no significant difference in the blood routine values ​​of each icariin dose group compared with those before administration, especially in the number of platelets. This result shows that icariin does not affect the number of platelets while increasing platelet activity. The results are shown in Table 2.

[0100] Table 2 Effect of icariin on blood routine test of rats induced by adenosine diphosphate

[0101] <![CDATA[Platelet count (10 9 / L)]]> <![CDATA[White blood cell count (10 9 / L)]]> <![CDATA[Red blood cell count (10 12 / L)]]> Hemoglobin concentration (g / L) Before administration 1203±127 1.759±0.213 6.457±0.675 154.5±10.9 3mg / kg 1124±209 1.832±0.154 7.213±0.554 180.5±11.2 6mg / kg 1397±143 1.653±0.421 6.783±0.871 179.0±9.2 18mg / kg 1249±166 1.761±0.335 7.235±0.656 181.6±9.5

[0102] 2. Effect of icariin on platelet aggregation in vitro

[0103] 2.1 In vitro platelet aggregation activity assay

[0104] After local anesthesia with procaine, the rabbits were surgically separated from the common carotid artery to collect blood, and the blood was anticoagulated with 3.8% sodium citrate and centrifuged at 800r / min for 10min to prepare platelet-rich plasma (PRP). The remaining part was centrifuged at 300r / min for 10min to prepare platelet-poor plasma (PPP) for platelet aggregation experiment. PRP 265ul and icariin 30ul (to make the final concentrations 0.1mmol / L, 0.5mmol / L and 5mmol / L, respectively) were added to the test tube, incubated for 5min, and 5ul adenosine diphosphate (final concentration 25umol / L) was used as an inducer to observe and record the maximum aggregation rate within 5min. Dimethyl sulfoxide with a mass fraction of 1% was used as a control to observe the promoting effect of icariin at various concentrations on adenosine diphosphate-induced platelet aggregation.

[0105] 2.2 Experimental Results

[0106] The promoting effect of icariin at different concentrations on adenosine diphosphate-induced platelet aggregation in vitro is shown in Table 3. It can be seen from the table that all concentrations of icariin have the effect of promoting adenosine diphosphate-induced platelet aggregation activity, and the promoting effect increases with increasing concentration, indicating that the promoting effect of icariin on adenosine diphosphate-induced platelet aggregation activity is dose-dependent.

[0107] Table 3 Epimedin promotes ATP-induced platelet aggregation activity in vitro

[0108] Group concentration Aggregation promotion rate (%) Icariin low dose group 0.1mmol / L 23.3±4.5 Icariin medium dose group 0.5mmol / L 38.9±8.7 High dose of icariin group 5mmol / L 52.2±9.0

[0109] Efficacy Example 2 Effect of Icariin on Hemorrhagic Transformation after Cerebral Ischemia-Reperfusion Induced by Aspirin or Clopidogrel

[0110] 1. Experimental Procedure

[0111] 1.1 Model making and experimental methods

[0112] Male ICR mice, 150, Jinan Pengyue Experimental Animal Breeding Co., Ltd., SPF grade, weight 27-30g. Animal Certificate Number: No.1107262011001201, No.1107262011001347.

[0113] Mice were quarantined for 3-7 days after entering the room, and healthy (female nulliparous) mice were selected as test animals. The main inspection contents during the quarantine period include but are not limited to: whether the number and quality indicators of animals required at the time of application are consistent; general condition; body weight; animals that fail the above quarantine will not be included in this experiment. See Table 4 for details.

[0114] Table 4 Grouping and drug administration of mice

[0115]

[0116] Note: The day when drug administration started was defined as Day 1.

[0117] 5-week-old ICR male mice were raised to 6 weeks of age and divided into control group, model group, icariin-3 mg / kg group, icariin-6 mg / kg group, icariin-12 mg / kg group, and icariin-12 mg / kg group according to body weight. On days 1-3 after grouping, the icariin groups were gavaged with 3, 6, and 12 mg / kg icariin, the icariin group was gavaged with 12 mg / kg icariin, once a day, and the other groups were gavaged with the corresponding solvents; except for the control group, the other groups were gavaged with aspirin (100 mg / kg) or clopidogrel (15 mg / kg) once a day; the gavage volume was 10 mL / kg.

[0118] Ten minutes after administration on the third day, the mice were anesthetized by intraperitoneal injection of 70 mg / kg sodium pentobarbital and underwent cerebral ischemia-reperfusion (MCAO) surgery, with ischemia for 1 hour and reperfusion for 23 hours.

[0119] 24 hours after surgery was the end point of the experiment.

[0120] 1.2 Detection indicators

[0121] Bleeding rate: Mice were anesthetized by intraperitoneal injection of 70 mg / kg sodium pentobarbital, brain tissue was taken, cerebral hemorrhage was recorded, and the cerebral hemorrhage rate of mice in each group was calculated. Cerebral hemorrhage rate (%) = number of animals with cerebral hemorrhage / number of surviving animals in each group × 100.

[0122] 2. Data processing

[0123] All data were entered into Excel documents and statistically analyzed using SPSS17.0, and expressed as mean ± standard deviation. One-way ANOVA and t test were used to compare the data of each group.

[0124] 3. Test results

[0125] Effect on bleeding rate

[0126] The experimental results showed that in the hemorrhagic transformation model induced by aspirin combined with cerebral ischemia-reperfusion, the bleeding rate of the model group was 85.7%. The bleeding rate decreased significantly after the icariin was given in advance for prevention. The bleeding rates of the icariin-3 mg / kg group, the icariin-6 mg / kg group, and the icariin-12 mg / kg group were 37.5%, 25.0%, and 22.2%, respectively. The bleeding rate was not significantly improved after the icariin was given in advance for prevention. The results are shown in Table 5.

[0127] In the hemorrhagic transformation model induced by clopidogrel combined with cerebral ischemia-reperfusion, the bleeding rate of the model group was 80%. After the prevention of icariin in advance, the bleeding rate decreased significantly. The mortality rates of the icariin-3 mg / kg group, icariin-6 mg / kg group, and icariin-12 mg / kg group were 42.9%, 25.0%, and 25.0%, respectively. The bleeding rate was not significantly improved after the prevention of icariin in advance. The results are shown in Table 6.

[0128] Table 5 Effect of icariin on the bleeding rate of hemorrhagic transformation after cerebral ischemia induced by aspirin

[0129]

[0130]

[0131] Table 6 Effect of icariin on the bleeding rate of hemorrhagic transformation after cerebral ischemia induced by clopidogrel

[0132]

[0133] In this study, we selected antiplatelet aggregation drugs (aspirin or clopidogrel) and combined them with cerebral ischemia-reperfusion to successfully establish a hemorrhagic transformation model according to the literature, and investigated the preventive effect of icariin on the hemorrhagic transformation animal model through the bleeding situation.

[0134] This study successfully replicated the hemorrhagic transformation mouse animal model, and the bleeding rate was significantly increased. After the prevention of icariin, the above indicators were significantly reduced, indicating that icariin can significantly inhibit the hemorrhagic transformation after cerebral ischemia-reperfusion caused by aspirin or clopidogrel.

[0135] Efficacy Example 3 Effect of Icariin on Prolonged Bleeding Time of Mice Induced by Heparin Sodium

[0136] 1. Drugs and sources

[0137] Heparin Sodium Injection (Source: Jiangsu Wanbang Biochemical Pharmaceutical Group Co., Ltd.; Batch No.: 51701106)

[0138] Ethamsulfonamide injection (Source: Shandong Fangming Pharmaceutical Group Co., Ltd.; Batch No.: 18060672)

[0139] 2. Experimental animals and groups

[0140] 72 ICR mice aged 5-6 weeks, weighing 19-21g, half male and half female. Mice were quarantined for 7 days after entering the room, and healthy mice were selected as test animals. They were weighed on the first and last day of quarantine. According to the weight on the last day of quarantine, the male and female animals were divided into six groups by simplified random method, namely normal group (C), model group (V), low, medium and high dose groups of icariin (2, 6, 18 mg / kg, L, M, H) and positive drug group (200 mg / kg ethylsulfonamide, P).

[0141] 3. Pre-medication and modeling

[0142] After grouping, the normal group and the model group were gavaged with 10ml / kg of icariin 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 icariin was gavaged with 2, 6, and 18mg / kg of icariin once a day for five consecutive days; the positive drug group was intraperitoneally injected with 200mg / kg of phensulfonamide injection once a day for five consecutive days. One hour after the last pre-administration, the model group and each drug group were injected with 150U / kg of sodium heparin injection through the tail vein to establish the model, and the normal group was injected with 10ml / kg of sodium chloride injection through the tail vein.

[0143] 4. Detection indicators

[0144] 4.1 Bleeding time

[0145] 15 minutes after modeling, the tail was cut to measure the bleeding time of each mouse.

[0146] 4.2 Routine blood test and four coagulation tests

[0147] After the bleeding time was tested, the mice were anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta to test routine blood test and four coagulation items (prothrombin time, thrombin time, thrombin time, and fibrinogen).

[0148] 5. Test results

[0149] 5.1 Bleeding time

[0150] The bleeding time of mice in each group is shown in Table 7.

[0151] Table 7 Effect of icariin on bleeding time of mice induced by heparin sodium

[0152] Group Number of animals Bleeding time (min) C 12 16.2±3.3 V 12 <![CDATA[35.5±9.8 ## ]]> P 12 <![CDATA[21.4±6.7 ＄ ]]> L 12 <![CDATA[26.4±8.8 ＄ ]]> M 12 <![CDATA[22.7±9.1 ＄ ]]> H 12 <![CDATA[15.9±6.6 ＄＄& ]]>

[0153] Note: Compared with the normal group, ## P<0.01; compared with the model group, ＄ P<0.05, ＄＄ P<0.01; compared with the positive control group, & P<0.05.

[0154] 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 model was successfully established; compared with the model group, the bleeding time of the mice in each drug-treated group was significantly shortened, indicating that icariin can shorten the bleeding time of bleeding mice, and the shortening of bleeding time is more significant with the increase of the dose, indicating that the procoagulant effect of icariin 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 icariin is better than that of the positive control drug.

[0155] 5.2 Routine blood test and four coagulation tests

[0156] The results of routine blood tests and four coagulation tests of mice in each group are shown in Tables 8 and 9, respectively.

[0157] Table 8 Blood routine examination results of mice in each group

[0158] Group <![CDATA[Platelet count (10 9 / L)]]> <![CDATA[White blood cell count (10 9 / L)]]> <![CDATA[Red blood cell count (10 12 / L)]]> Hemoglobin concentration (g / L) C 1117±181 1.989±0.213 7.034±0.675 132.4±9.9 V 1056±161 1.896±0.197 7.135±0.554 140.5±8.7 P 1097±114 1.903±0.221 6.998±0.871 139.0±7.2 L 1046±136 1.895±0.207 7.109±0.656 141.6±8.5 M 1110±128 1.934±0.199 7.111±0.634 153.7±9.3 H 1059±170 1.888±0.223 7.090±0.761 133.5±7.6

[0159] Table 9 Results of four blood coagulation tests in each group of mice

[0160] Group Prothrombin time (sec) Thromboplastin time (sec) Thrombin time (sec) Fibrinogen (g / L) C 10.02±1.34 23.23±2.09 26.66±1.34 1.99±0.34 V 9.99±1.02 24.12±1.89 25.51±1.78 1.92±0.29 P 10.03±1.24 22.78±2.34 24.34±1.37 1.86±0.40 L 10.12±1.34 25.19±1.90 22.23±2.01 1.83±0.22 M 11.23±1.45 25.09±2.22 24.09±1.99 1.82±0.56 H 9.98±1.23 24.55±2.66 25.54±1.78 2.01±0.52

[0161] As can be seen from the above table, there were no significant differences in the blood routine and four coagulation parameters among the mice in each group, indicating that icariin had no significant effect on the blood routine and four coagulation parameters when shortening the prolonged bleeding time caused by sodium heparin.

[0162] Efficacy Example 4 Effect of Icariin on Prolonged Bleeding Time of Mice Induced by Aspirin

[0163] 1. Drugs and sources

[0164] Aspirin (Source: Shantou Yushi Pharmaceutical Factory Co., Ltd.; Batch No.: 1903012)

[0165] Ethamsulfonamide injection (Source: Shandong Fangming Pharmaceutical Group Co., Ltd.; Batch No.: 1905032)

[0166] 2. Experimental animals and groups

[0167] 96 ICR mice aged 5-6 weeks, weighing 19-21g, half male and half female. The mice were quarantined for 7 days after entering the room, and healthy mice were selected as test animals. They were weighed on the first and last days of quarantine.

[0168] The male and female animals were divided into six groups according to their body weight at the last day of quarantine by a simplified random method, namely the normal group (C), model group (V), low-, medium- and high-dose groups of icariin (2, 6, 18 mg / kg, L, M, H) and a positive drug group (200 mg / kg phensulfonamide, P).

[0169] 3. Pre-medication and modeling

[0170] After grouping, the normal group and model group were given 10ml / kg icariin solvent by gavage, and the icariin groups were given 2, 6, and 18mg / kg icariin by gavage, once a day for five consecutive days; the positive drug group was intraperitoneally injected with 200mg / kg phensulfonamide injection once a day for five consecutive days. One hour after the last pre-administration, the model group and each drug group were given 20mg / kg aspirin by gavage to establish the model, and the normal group was given an equal volume of normal saline by gavage.

[0171] 4. Bleeding time test

[0172] 15 minutes after modeling, the tail was cut to measure the bleeding time of each mouse.

[0173] 5. Test results

[0174] The bleeding time of mice in each group is shown in Table 10. Compared with the normal group, the bleeding time of mice in the model group was significantly prolonged, indicating that the model was successfully established; compared with the model group, the bleeding time of mice in each drug-treated group was significantly shortened, indicating that icariin can shorten the bleeding time of bleeding mice, and the shortening of bleeding time is more significant with the increase of the dose, indicating that the procoagulant effect of icariin is dose-dependent, and there is a significant difference between the high-dose group and the positive control group, indicating that the procoagulant effect of icariin is better than that of the positive control drug.

[0175] Table 10 Effect of icariin on aspirin-induced bleeding time in mice

[0176] Group Number of animals Bleeding time (min) C 12 19.2±4.5 V 12 <![CDATA[37.6±7.8 ## <!-- 12 -->]]> P 12 <![CDATA[26.4±6.4 ＄ ]]> L 12 <![CDATA[28.4±9.8 ＄ ]]> M 12 <![CDATA[24.7±7.3 ＄ ]]> H 12 <![CDATA[16.7±7.2 ＄＄& ]]>

[0177] Note: Compared with the normal group, ## P<0.01; compared with the model group, ＄ P<0.05, ＄＄ P<0.01; compared with the positive control group, & P<0.05.

[0178] Efficacy Example 5 Effect of Icariogenin on the Thrombolytic Effect of Thrombolytic Drugs

[0179] 1. Construction of the rat abdominal aorta thrombosis model

[0180] The rat abdominal aorta thrombosis model was prepared as follows. Rats were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital 40 mg / kg. The rats were shaved and skinned on the abdomen and fixed on a mouse board in a supine position. The rat abdominal skin was cut open, the blood vessels and surrounding tissues were isolated, the abdominal aorta was exposed, and the abdominal aorta was carefully separated with a glass needle. A 0.7 cm × 1.5 cm tin foil strip was placed under the abdominal aorta, and then the abdominal aorta was wrapped with a filter paper strip (0.5 cm × 1.0 cm) soaked in 35% FeCl3 solution. The filter paper strip was removed after 30 minutes. At this time, the color of the blood vessel wall became significantly darker, and emboli were formed in the blood vessels under the microscope. Pathological sections showed that the blood vessels were blocked by thrombi.

[0181] 2. Grouping and Dosing

[0182] SD rats were randomly divided into three groups according to their body weight, with 10 rats in each group, namely, thrombosis model group, lumbrokinase capsule group, and lumbrokinase capsule + icariin group. 4 U / kg was directly injected into the duodenum, icariin was intragastrically administered at 18 mg / kg, and the thrombosis model group was replaced with an equal volume of distilled water.

[0183] 3. Get materials

[0184] The two ends of the abdominal aorta were clamped with hemostatic forceps, the vascular segment at the modeling site was completely removed, and a 0.5 cm vascular segment wrapped with filter paper was accurately cut and weighed. The remaining vascular segment was preserved in 10% formaldehyde.

[0185] 4. Determination of Thrombus Weight in Rats

[0186] The weight of the removed blood vessel segment was weighed with an electronic balance, and the blood vessel weight was weighed again after the thrombus was removed. The wet weight of the thrombus = the total weight of the thrombus and the blood vessel - the weight of the blood vessel.

[0187] The removed thrombus was dried in an oven at 80°C, left overnight, and weighed to obtain the dry weight of the thrombus.

[0188] 5. Test results

[0189] The thrombus weight of rats in each group is shown in the table below. It can be seen from the table that compared with the model group, the thrombus weight of the lumbrokinase group was significantly reduced, indicating that lumbrokinase has a significant thrombolytic effect; compared with the lumbrokinase group, there was no significant difference in the thrombus in the lumbrokinase + icariin group, indicating that epimedium does not affect the dissolving effect of lumbrokinase on rat thrombus.

[0190] Table 11 Effect of icariin on thrombus weight in rats of each group

[0191] Group Number of cases Thrombus wet weight (mg) Thrombus dry weight (mg) Model Group 9 2.98±0.54 0.31±0.12 Lumbrokinase group 10 <![CDATA[1.01±0.22 ## ]]> <![CDATA[0.04±0.01 ## ]]> Lumbrokinase + Icariin combination 10 1.24±0.19 0.04±0.02

[0192] Note: Compared with the model group, ## P<0.01.

[0193] Efficacy Example 6 Effect of Icariin on Hemorrhagic Transformation Caused by Thrombolytic Drugs

[0194] 1. Preparation of Animal Model

[0195] The rat middle cerebral artery autologous thromboembolism model was prepared according to the following method: the rat was anesthetized by intraperitoneal injection of 10% hydrated chloric acid at 350 mg / kg body weight, fixed on the operating table in supine position, and routine skin disinfection and draping were performed according to the aseptic operation procedures. A 3.0 cm incision was made in the midline skin of the neck, and the muscles and subcutaneous tissues were bluntly separated. The right bilateral common carotid arteries, external carotid arteries and internal carotid arteries were separated to avoid damaging the vagus nerve. The internal carotid arteries were separated to the base of the skull, and the external carotid arteries were ligated at the bifurcation of the bilateral common carotid arteries. 0.1 ml of arterial blood was collected from the femoral artery of the rat using a 1 ml syringe, 4 U of thrombin was inhaled, mixed and allowed to stand for 8-10 minutes, and after the blood coagulated, it was injected into a 24G intravenous indwelling needle to form an embolus about 1 cm long, followed by a 1-2 ml syringe of normal saline. Then the indwelling needle was inserted into the bilateral common carotid arteries in front of the bifurcation of the external carotid artery and the internal carotid artery, and then advanced forward and medially into the internal carotid artery for about 8-10 mm. The needle core was pulled out, and the embolus and saline in the syringe were quickly flushed into the internal carotid artery, and then ligated and sutured. In the sham operation group and control group, except for the injection of saline instead of embolus, the rest of the surgical steps were the same as those of the above thrombolytic model groups.

[0196] 2. Animal inclusion criteria

[0197] The symptoms and signs of the rats were observed 2 hours after the modeling operation. The following symptoms and signs indicated that the model was successfully made: Homer sign on the ipsilateral side of ischemia, circling toward the contralateral side of ischemia when walking; leaning toward the contralateral side; when the rats were suspended by their tails, the forelimbs on the contralateral side of cerebral ischemia were flexed, raised, shoulders adducted, and elbows extended.

[0198] 3. Experimental Grouping and Drug Administration

[0199] 102 rats were randomly divided into sham operation group, rt-PA thrombolysis group (thrombolysis group), rt-PA thrombolysis + icariin low, medium and high groups, including 18 rats in the sham operation group and 21 rats in each of the other groups. Each group was evenly divided according to different killing time points (3, 6, and 24 hours after thrombolysis), with 6 rats in the sham operation group at each time point, 9 rats in each of the other groups at 3 hours, and 6 rats in each of the other time points. Another 20 rats were reserved for random addition in case of model failure (death or score 0).

[0200] All groups except the sham operation group were given alteplase (rt-PA) (5 mg / kg, add sterile water prepared for rt-PA to 1 ml) via femoral vein 3 hours after modeling. The sham operation group was given an equal volume of normal saline.

[0201] The treatment group was given drugs twice, one by gavage immediately after modeling and the other after thrombolytic therapy. The doses were 2, 6, and 18 mg / kg, respectively. The sham operation group was given an equal volume of normal saline.

[0202] 4. Index observation and measurement methods

[0203] 4.1 Overall condition comparison and blood routine examination

[0204] 4.1.1 Compare the overall conditions of rats in the sham operation group, thrombolysis group, and thrombolysis + icariin group.

[0205] 4.1.2 The blood routine tests of rats in the sham operation group, thrombolysis group, and thrombolysis + icariin group were performed.

[0206] 4.2 Determination of cerebral infarction volume (rate)

[0207] Except for the sham operation group, 3 rats were killed in each group at 3 hours after thrombolysis, and the brain tissue was placed in a -20℃ refrigerator for quick freezing for 20 minutes, and then 2mm thick coronal brain slices were prepared, immersed in 2% thrombin time C solution, and placed in a 37℃ light-proof water bath for 30 minutes, and fixed in 4% paraformaldehyde solution for 24 hours. After taking pictures, the infarct volume was determined using image analysis software (Image-Pro Plus6.0). Infarct volume = non-ischemic hemisphere volume minus the non-infarcted area volume of the ischemic hemisphere. The calculation of the infarct volume rate was expressed as the percentage (%) of the infarct volume and the non-ischemic hemisphere volume.

[0208] 4.3 Determination of cerebral hemorrhage volume:

[0209] (1) Draw a standard curve

[0210] Four healthy rats were taken, and after being killed by overdose of anesthesia, blood was removed by cardiac perfusion. Eight cerebral hemisphere tissues were taken, and 0, 0.5, 1, 2, 4, 8, 16, 32, and 50ul of male SD rat arterial blood were added to each hemisphere tissue in turn, and then PBS with pH = 7.4 was added to a total volume of 3mi. After homogenization for 30s, ultrasonic lysis was performed and centrifuged at 13000r / min for 30min at 4°C. 0.8ml of drabkin reagent was mixed with 0.2ml of the supernatant of each sample, and the sample was placed at room temperature and away from light for 10min, and the absorbance was measured at a wavelength of 540nm (the absorbance of the sample without blood was set to 0). The standard curve was drawn with the absorbance as the ordinate and the amount of blood in each sample as the abscissa.

[0211] (2) Determination of hemisphere hemorrhage volume on the infarcted side

[0212] Add PBS with pH=7.4 to the infarcted hemisphere tissue of rats with cerebral infarction to a total volume of 3 ml, homogenize for 30 seconds, then ultrasonically lyse and centrifuge at 13000 r / min for 30 minutes. Take 0.8 ml of drabkin reagent and mix with 0.2 ml of supernatant of each sample, place at room temperature and avoid light for 10 minutes, and then measure the absorbance at a wavelength of 540 nm (the absorbance of brain tissue samples without blood is set as 0, and the same batch is measured). The blood volume corresponding to the absorbance on the standard curve is the amount of bleeding.

[0213] 5. Statistical analysis

[0214] All experimental data were expressed as mean ± standard deviation, and the data were processed using PASW Statistics 17.0 statistical software package. One-way analysis of variance was used for comparison between and within groups. If the variance was homogeneous, the LSD test was used. If the variance was unequal, the Dunne thrombin time T3 nonparametric test was used. The linear correlation two-tailed Pearson product moment correlation coefficient test was used for correlation analysis, and a bilateral P value of < 0.05 was considered statistically significant.

[0215] 6. Experimental Results

[0216] 6.1 Overall condition comparison and blood routine test

[0217] 6.1.1 Overall Comparison

[0218] The sham operation group rats had a good mental state and normal fur color. The mental state of the rats in the thrombolysis group and the thrombolysis group + icariin group after surgery was significantly worse than before surgery, as shown by dull fur color, reduced spontaneous activity, and relatively slow response to the outside world. The thrombolysis group was the worst, and the thrombolysis group + icariin high-dose group had the best mental state.

[0219] 6.1.2 Routine blood test

[0220] The results of routine blood tests showed that there was no significant difference in the blood routine values ​​of rats in the thrombolysis group and the thrombolysis group + icariin group, and there was no significant difference between the thrombolysis group, the thrombolysis group + icariin group and the sham operation group.

[0221] 6.2 Determination of cerebral infarction volume (rate)

[0222] Thrombin time C staining can be used to observe the scope of cerebral ischemia. Staining results: The non-infarcted brain tissue of the sham operation group and the other groups was uniformly red, while the infarcted brain tissue was obviously edematous, pale, and dull. After thrombin time C staining, the infarcted area was pale. The results showed that the cerebral infarction volume rate was (38.84±3.67)% 3h after the model was taken from the spare rats. There was no change in the thrombin time C staining of the brain tissue of the sham operation group rats. Compared with the model group, the thrombin time C staining of the rats in the thrombolytic group and the thrombolytic drug + icariin groups showed a significant reduction in the pale area of ​​the infarcted area. The cerebral infarction volume was measured and found that the reduction rate of the cerebral infarction volume in the thrombolytic drug + icariin groups was equivalent to that in the thrombolytic group. This result shows that icariin does not affect the thrombolytic effect of thrombolytic drugs.

[0223] 6.3 Comparison of cerebral hemorrhage volume in rats of different groups at different time points

[0224] The amount of cerebral hemorrhage in rats of each group at different time points is shown in the following table. It can be seen from the table that the amount of bleeding in the thrombolytic group was significantly greater than that in the sham operation group, indicating that the thrombolytic drug caused the hemorrhagic transformation of rats with cerebral infarction, and the amount of bleeding increased significantly with the extension of time; compared with the thrombolytic group, the amount of bleeding in the thrombolytic drug + icariin group at each dose was significantly reduced, and the amount of bleeding did not increase with the extension of time. This shows that icariin can effectively prevent the hemorrhagic transformation caused by thrombolytic drugs.

[0225] Table 12 Comparison of cerebral hemorrhage in rats in each group at different time points

[0226] Grouping Number of cases 3 hours 6 hours 24 hours Sham operation group 6 0.04±0.02 0.04±0.02 0.04±0.02 Thrombolysis group 6 <![CDATA[6.43±0.89 ## ]]> <![CDATA[9.78±1.01 ## ]]> <![CDATA[15.32±2.01 ## ]]> Thrombolytic drugs + low-dose icariin group 6 <![CDATA[3.09±0.78 ＄ ]]> <![CDATA[3.34±0.99 ＄＄ ]]> <![CDATA[4.22±1.66 ＄＄ ]]> Thrombolytic drugs + icariin medium dose group 6 <![CDATA[2.22±0.71 ＄＄ ]]> <![CDATA[3.09±1.15 ＄＄ ]]> <![CDATA[2.98±2.34 ＄＄ ]]> Thrombolytic drugs + high-dose icariin group 6 <![CDATA[1.01±0.54 ＄＄ ]]> <![CDATA[1.02±0.11 ＄＄ ]]> <![CDATA[0.97±0.56 ＄＄ ]]>

[0227] Note: Compared with the sham operation group, ## P<0.01; compared with the thrombolytic group, ＄ P<0.05, ＄＄ P<0.01.

[0228] Efficacy Example 7 Preventive Effect of Icariin on Gastrointestinal Bleeding Induced by Aspirin

[0229] 1. Modeling and grouping

[0230] A gastrointestinal bleeding model was established in 24 SD rats by intragastric administration of aspirin at 500 mg / kg, and another 12 SD rats were given an equal volume of normal saline by intragastric administration as normal control. The bleeding model rats were randomly divided into a model group and an icariin group. The icariin group was given icariin at 18 mg / kg by intragastric administration while the model was established, and the model group was given an equal volume of normal saline by intragastric administration.

[0231] 2. Dissect and observe the bleeding of gastric mucosal ulcer

[0232] 24 hours after intragastric administration, the rats were anesthetized and killed, and the surface bleeding, erosion and ulcer of the gastric mucosa were observed after dissection.

[0233] 3. Experimental results and discussion

[0234] The occurrence of gastric mucosal bleeding in rats in each group is shown in Table 13. It can be seen from the table that: no bleeding occurred in the normal control group, the bleeding incidence in the model group was 66.7%, and compared with the model group, the gastric mucosal bleeding in the icariin group was significantly milder, and the bleeding incidence was significantly lower than that in the model group, which was statistically significant (P < 0.01), indicating that icariin can significantly reduce the incidence of gastrointestinal bleeding caused by aspirin.

[0235] Table 13 Comparison of gastric mucosal bleeding in rats in each group

[0236] Group Number of animals (n) Number of bleeding (n) Bleeding rate (%) Normal control group 12 0 - Model Group 12 8 66.7 Icariin group 12 2 <![CDATA[16.7 ## ]]>

[0237] Compared with the model group, ## P<0.01.

[0238] Efficacy Example 8 Preventive Effect of Icariin on Gastric Bleeding Induced by Thrombolytic Drugs

[0239] 1. Modeling and grouping

[0240] 120 SD rats were randomly divided into sham operation group, model group, urokinase group, high dose of icariin + urokinase group, medium dose of icariin + urokinase group, and low dose of icariin + urokinase group by random number method, with 20 rats in each group; icariin of each dose group was intragastrically administered 4 days before surgery, with the doses of 2 mg / kg, 6 mg / kg, and 18 mg / kg, respectively, and the sham operation group, model group, and urokinase group were intragastrically administered with equal volumes of normal saline; except for the sham operation group, the other groups were administered with 5000u / kg urokinase at a concentration of 200u / μL and a volume of 20μL through the regional artery via catheter, and the model group and icariin group were administered with equal volumes of normal saline through the regional artery. The animal model of thromboembolic cerebral ischemia was prepared by occluding the middle cerebral artery with autologous thrombus combined with suture, and the catheter was indwelling.

[0241] 2. Observation indicators and measurement methods

[0242] 2.1 Calculation of bleeding rate

[0243] 24 hours after arterial administration, the rats in each group were anesthetized and dissected, and the number of bleeding cases was counted under naked eye and light microscope, and the ratio of the number of bleeding cases to the total number of rats was calculated.

[0244] 2.2 Pathological observation of gastric tissue:

[0245] The left lung and gastric antrum were removed and fixed with 100g / L neutral formaldehyde solution, dehydrated, transparent, waxed, embedded, sliced, stained, and observed under a light microscope. The degree of damage was graded from mild to severe according to the degree of tissue congestion, edema and cell degeneration: grade I 0%-25%, grade II 26%-50%, grade III 51%-75%, grade IV>75% (bleeding was seen under the microscope).

[0246] 3. Experimental results and discussion

[0247] 3.1 Comparison of bleeding rates

[0248] Comparison of gastric bleeding rates in rats of each group is shown in Table 14. It can be seen from the table that gastric bleeding occurred in the model group compared with the sham operation group, indicating that cerebral ischemia can cause gastric bleeding complications to a certain extent; the bleeding rate in the urokinase group was significantly increased compared with the model group, with a significant difference (P < 0.01), indicating that thrombolytic drugs can cause gastrointestinal bleeding complications when performing thrombolytic treatment on thrombotic diseases; the bleeding rates in each dose group of urokinase + icariin were significantly reduced compared with the urokinase group, with a significant difference (P < 0.01), indicating that icariin has the effect of reducing the gastrointestinal bleeding rate caused by thrombolytic drugs, and this effect is dose-dependent.

[0249] There was a significant difference between the high-dose icariin group and the model group (P < 0.01), indicating that icariin also has a certain preventive effect on gastrointestinal bleeding caused by thrombotic diseases themselves.

[0250] Table 14 Comparison of gastric bleeding rates in rats of each group

[0251]

[0252]

[0253] Compared with the sham operation group, ＄＄ P<0.01; compared with urokinase group, ## P<0.01; compared with the model group, & P<0.05.

[0254] 3.2 Pathological observation of gastric tissue

[0255] The results of gastric tissue pathological observation of rats in each group are shown in Table 15. It can be seen from the table that the gastric injury rate in the urokinase group was significantly increased compared with the model group, with a significant difference (P < 0.01), indicating that thrombolytic drugs can cause complications such as gastric tissue congestion, edema, cell degeneration and even bleeding when performing thrombolytic treatment on thrombotic diseases; the incidence of the above complications in each dose group of urokinase + icariin was significantly reduced compared with the urokinase group, with a significant difference (P < 0.01 or P < 0.05), indicating that icariin has the effect of reducing complications such as gastrointestinal tissue congestion, edema, cell degeneration and even bleeding caused by thrombolytic drugs, and compared with the model group, the incidence of the above complications in the high-dose group of urokinase + icariin was significantly reduced. The above results show that icariin can not only reduce the incidence of gastrointestinal bleeding caused by thrombolytic drugs, but also alleviate the symptoms of gastrointestinal tissue congestion, edema and cell degeneration, and play a good recovery role in gastrointestinal damage.

[0256] In addition, the comparison results with the model group showed that icariin also has a certain preventive and therapeutic effect on digestive tract damage caused by ischemic diseases themselves.

[0257] Table 15 Comparison of gastric tissue pathological grading results of rats in each group

[0258]

[0259] Compared with the sham operation group, ＄＄ P<0.01; compared with urokinase group, ## P<0.01; compared with the model group, & P<0.05.

[0260] Efficacy Example 9 Effect of Icariin on Bleeding Complications Induced by Thrombolytic Drugs

[0261] 1. Model Preparation

[0262] Sixty Wistar male rats (SPF grade), weighing 220-260g, were randomly divided into a myocardial infarction model group (MI) and a control group according to a random number table. The control group had 12 rats and the model group had 48 rats. The rats were housed in cages in a conventional manner, with 4 rats in each cage. After recording normal electrocardiograms, rats in the myocardial infarction model group were subcutaneously injected with 150mg·kg of isoproterenol (ISO). - 1 .d -1 , at a volume of 0.2mL / 100g, at intervals of 24 hours, for 2 consecutive days. The control group was subcutaneously injected with an equal amount of normal saline (0.2mL / 100g) for 2 consecutive days. The electrocardiogram was monitored 24 hours after the second subcutaneous injection of isoproterenol. After the rats were anesthetized with ether, the six-lead (limb lead) electrocardiogram of the rats was recorded before and after the injection, the heart rate was measured, and the degree of Q wave and ST segment deviation was observed to evaluate whether the model was successful. The ST segment elevation of lead II greater than 0.2mV or Q waveform became the standard for myocardial infarction.

[0263] 2. Grouping and Dosing

[0264] Thirty-six modeling rats were selected and randomly divided into model group, thrombolytic group and thrombolytic drug + icariin group according to the random number table. The thrombolytic group was given rt-PA at 5 mg / kg via the femoral vein, the thrombolytic drug + icariin group was given icariin (18 mg / kg) by gavage in addition to the same dose of thrombolytic drug, and the model group was given a full volume of normal saline via the femoral vein.

[0265] 3. Detection indicators

[0266] 3.1 ECG monitoring of rats in each group

[0267] Rats were anesthetized by ether inhalation. When the rats entered the anesthetized state, breathing became stable, righting reflex disappeared, and pain disappeared, the rats were fixed on the experimental table in supine position, needle electrodes were inserted into the subcutaneous part of the rats' limbs (be careful to avoid inserting into the muscles), and the electrocardiogram of the rats was monitored using limb leads. The right upper limb was red, the left upper limb was yellow, the left lower limb was green, and the right lower limb was black. The electrocardiograph was turned on, the paper feed speed was 50 mm / s, and the voltage was 1 mv. After the electrocardiogram was completed, the animals were returned to the cage.

[0268] 3.2 Observation of cardiac tissue morphology in rats in each group

[0269] After the last administration, the animals were killed and fixed in a supine position on a mouse board. 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 large blood vessels were cut and the heart was taken out, the blood remaining in the heart cavity was washed out with 4°C 0.9% sodium chloride solution, the left and right atrial appendages and residual large blood vessels were cut off and filtered dry with filter paper, the lung mass value and heart mass value of the rat were weighed and calculated using an electronic balance, the actual weight of the heart (wet weight) was weighed, and its ratio to body weight (HWI = heart weight / body weight) was calculated.

[0270] 3.3 Observation of bleeding complications in rats in each group

[0271] The bleeding conditions of rats in each group were observed, including subcutaneous bleeding points. The bleeding conditions of internal organs and intracranial hemorrhage were observed after dissection.

[0272] 4. Experimental results and discussion

[0273] 4.1 ECG results of each group

[0274] Compared with the control group, the rats in the model group developed pathological Q waves, indicating that the model was successful; compared with the model group, the pathological Q waves disappeared in the thrombolytic group and the thrombolytic + icariin group, indicating that thrombolytic drugs can significantly improve the myocardial infarction state of rats with myocardial infarction, and icariin does not affect the thrombolytic effect of thrombolytic drugs.

[0275] 4.2 Comparison of the gross morphology of the heart in each group of rats

[0276] The gross morphology of the hearts of rats in each group was observed by naked eyes. The heart volume of the model group was significantly larger than that of the control group, the left ventricular cavity was significantly enlarged, the myocardial transmural necrosis in the infarct area was pale and thin. The degree of heart volume increase and myocardial transmural necrosis in the thrombolytic group and the thrombolytic drug + icariin group were significantly milder than those in the model group. The heart weight of rats in each group and the ratio of heart weight to body weight are shown in the following table. It can be seen from the table that the ratio of heart weight to body weight in the model group was significantly different from that in the control group (P < 0.01), indicating that the model was successfully established; the thrombolytic group and the thrombolytic drug + icariin group were significantly different from the model group (P < 0.01), and there was no significant difference between the thrombolytic group and the thrombolytic drug + icariin group, indicating that thrombolytic drugs can significantly improve the myocardial infarction state of rats with myocardial infarction, and icariin does not affect the effect of thrombolytic drugs.

[0277] Table 16 Comparison of the gross morphology of the heart of rats in each group

[0278]

[0279] Compared with the control group, ## P<0.01; compared with the model group, ＄＄ P<0.01.

[0280] 4.3 Comparison of bleeding complications in rats in the drug-treated groups

[0281] Table 17 Comparison of bleeding complications in rats in the drug-treated groups

[0282]

[0283] Compared with the thrombolytic group, ## P<0.01.

[0284] As can be seen from the above table, the bleeding percentage in the thrombolytic group was significantly higher than that in the thrombolytic drug + icariin group, which was statistically significant (P < 0.01). This result shows that icariin can significantly reduce the incidence of bleeding complications caused by thrombolytic drugs.

[0285] Efficacy Example 10 Effect of Icariin on Bleeding Time of Coagulation Factor IX Gene Knockout Hemophiliac Mice

[0286] 1. Experimental Animals

[0287] IX gene knockout hemophilia mice were provided by Beijing Biocytogen Biotech Co., Ltd.

[0288] 2. Animal Grouping and Dosing

[0289] 40 IX gene-deficient hemophilia mice were randomly divided into four groups, 10 mice in each group. The low, medium and high dose groups (B, C and D) of icariin were given by gavage at 3 mg / kg, 6 mg / kg and 18 mg / kg, respectively, once a day for 11 consecutive days. The control group (A) was given an equal volume of normal saline by gavage.

[0290] 3. Detection indicators

[0291] 3.1 Bleeding time determination

[0292] One hour after the last administration, the tail of each mouse was cut to measure the bleeding time.

[0293] 3.2 Routine blood test

[0294] After the last bleeding time test, the mice were anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta to test routine blood tests.

[0295] 4. Test results

[0296] 4.1 Bleeding time determination

[0297] The bleeding time of mice in each group is shown in the following table. It can be seen from the table that compared with the control group, the bleeding time of mice in each dose group of icariin was significantly shortened, which was statistically significant ( # P<0.05 or ## P<0.01), indicating that icariin can effectively shorten the bleeding time of IX gene knockout hemophilia mice.

[0298] Table 18 Effect of icariin on bleeding time of IX gene knockout hemophilia mice

[0299] Group Number of animals Bleeding time (min) Control group (A) 16 37.8±12.4 B 16 <![CDATA[25.5±8.9 # ]]> C 16 <![CDATA[20.4±6.9 # ]]> D 16 <![CDATA[16.5±9.7 ## ]]>

[0300] Compared with the control group, # P<0.05 or ## P<0.01.

[0301] 4.2 Routine blood test

[0302] There was no significant difference in the blood routine values ​​of mice in each icariin dose group and the control group, indicating that icariin shortened the bleeding time of hemophilia mice without affecting the blood routine values.

[0303] Efficacy Example 11 Effect of Icariin on Activated Partial Thromboplastin Time in Acquired Hemophilia A

[0304] 1. Experimental Materials and Animals

[0305] Activated partial thromboplastin time assay: The kit was purchased from Shanghai Sun Biotechnology Co., Ltd.; plasma from patients with acquired hemophilia A was provided by Linyi People's Hospital; healthy New Zealand rabbits used in the experiment were provided by China Bingfeng Animal Husbandry Cattle and Sheep Breeding Research Base.

[0306] 2. Preparation and administration of acquired hemophilia A animal model

[0307] Twenty-four healthy New Zealand rabbits were randomly divided into a control group, a model group, and a treatment group of 8 rabbits each, each weighing about 1.5 kg. The rabbits were kept in stainless steel cages at room temperature of 18-25℃. All groups were given a normal diet without any special treatment. One week later, the rabbits were anesthetized by 2% pentobarbital (1.5 ml / kg) through the ear vein, and then the left femoral vein of the rabbits was isolated and exposed for injection. Plasma from patients with acquired hemophilia A (diagnosed by the Department of Hematology of Linyi People's Hospital) was intravenously injected into the model group and the treatment group at 2 ml / kg rabbit body weight. After the injection, the treatment group was immediately given 18 mg / kg of icariin by gavage, and the model group was given an equal amount of normal saline by gavage; the control group rabbits were intravenously injected with an equal amount of normal human plasma, and blood was drawn from the ear vein 30 minutes before the injection of plasma and 30 minutes, 60 minutes, 90 minutes, and 120 minutes after the injection of plasma, respectively. The blood was anticoagulated with 3.8% 1:9 sodium citrate, and the anticoagulated blood was centrifuged at 3000 r / m to separate the plasma.

[0308] 3. Determination of thromboplastin time and statistical methods

[0309] Referring to the instructions of the kit, take 0.1 ml of rabbit plasma, then add 0.1 ml of thromboplastin time reagent, mix and incubate at 37°C for 2 min, then add 0.1 ml of 0.025 M calcium chloride preheated at 37°C, mix well and immediately start the stopwatch to measure the thromboplastin time value at each time point. Repeat the thromboplastin time twice for each time point and take the average of the results.

[0310] SPSS11.0 software was used for statistical analysis of the data. The data were expressed as mean ± standard deviation, and normality test and variance homogeneity test were performed. The t test was used for comparison between groups.

[0311] 4. Test results

[0312] The comparison of thromboplastin time at each time point in each group is shown in Table 19. It can be seen from the table that the thromboplastin time value of the control group rabbits did not change significantly before and after the injection of plasma. Compared with the thromboplastin time value before injection, the thromboplastin time of the model group rabbit plasma at 30min, 60min, 90min, and 120min after injection was significantly prolonged, and the prolongation of the thromboplastin time showed a time-dependent trend, while the treatment group could significantly shorten the thromboplastin time at each time point. This shows that icariin can significantly shorten the activated partial thromboplastin time of hemophilia patients and promote blood coagulation in hemophilia patients.

[0313] Table 19 Comparison of thromboplastin time in each group at each time point

[0314] Group Number of cases (n) -30 0 60 90 120 Control group 8 39.3±3.76 38.7±1.09 36.6±2.98 38.3±4.87 39.6±5.98 Model Group 8 37.6±1.23 <![CDATA[48.3±2.09 # ]]> <![CDATA[52.9±3.44 ## ]]> <![CDATA[65.4±3.78 ## ]]> <![CDATA[69.9±5.23 ## ]]> Treatment Group 8 36.8±4.12 <![CDATA[40.4±3.23 ＄ ]]> <![CDATA[39.9±4.09 ＄＄ ]]> <![CDATA[40.4±5.67 ＄＄ ]]> <![CDATA[41.3±6.21 ＄＄ ]]>

[0315] Compared with the control group, # P<0.05, ## P<0.01; compared with the model group, ＄ P<0.05, ＄＄ P<0.01.

Claims

1. Use of icariin in the preparation of a medicament for preventing or treating hemorrhagic diseases, characterized in that The hemorrhagic disease is platelet dysfunction and the hemorrhagic disease caused by it; The platelet dysfunction is abnormal platelet activation function or aggregation function; The hemorrhagic disease caused by abnormal platelet function is hemorrhagic transformation of cerebral infarction, gastrointestinal bleeding caused by thrombolytic drugs or antithrombotic drugs for cerebral infarction, or bleeding complications of thrombolytic drugs or antithrombotic drugs for myocardial infarction.

2. The use according to claim 1, characterized in that The hemorrhagic transformation of cerebral infarction is secondary hemorrhagic transformation of ischemic cerebral infarction, primary hemorrhagic transformation or asymptomatic hemorrhagic transformation.

3. The use according to claim 2, characterized in that The secondary hemorrhagic transformation is caused by one or more treatment methods selected from thrombolytic drugs, antithrombotic drugs and endovascular treatment; The thrombolytic drug is selected from urokinase, streptokinase, reteplase, alteplase, tenecteplase or lanoteplase; The antithrombotic drug is one or more of an anticoagulant drug and an antiplatelet drug; The anticoagulant drug is selected from heparin or its pharmaceutically acceptable salt, low molecular weight heparin or its pharmaceutically acceptable salt, and recombinant hirudin; The antiplatelet drug is selected from aspirin, clopidogrel or ticlopidine.

4. The use according to claim 2, characterized in that The primary hemorrhage transforms into spontaneous hemorrhage after acute cerebral infarction.

5. The use according to claim 1, characterized in that The thrombolytic drug for myocardial infarction or the thrombolytic drug for bleeding complications of the antithrombotic drug is selected from urokinase, streptokinase, reteplase, alteplase, tenecteplase or lanoteplase; the antithrombotic drug is an antithrombotic treatment for the onset of myocardial infarction and the primary and secondary prevention stages; or the antithrombotic drug is one or more of an anticoagulant or antiplatelet drug.

6. The use according to claim 5, characterized in that The antiplatelet drug is selected from aspirin, clopidogrel or ticlopidine, and the anticoagulant drug is selected from heparin or its pharmaceutically acceptable salt, low molecular weight heparin or its pharmaceutically acceptable salt, and recombinant hirudin.

7. The use according to claim 5, characterized in that The bleeding complications are one or more of subcutaneous hemorrhage, intracranial hemorrhage, and upper gastrointestinal bleeding.

8. The use according to claim 1, characterized in that The thrombolytic drugs in the gastrointestinal bleeding caused by the thrombolytic drugs or antithrombotic drugs for cerebral infarction are one or more of urokinase, streptokinase, reteplase, alteplase, tenecteplase or lanoteplase; the antithrombotic drugs are one or more of anticoagulant drugs or antiplatelet drugs.

9. The use according to claim 8, characterized in that The antiplatelet drug is selected from aspirin, clopidogrel or ticlopidine, and the anticoagulant drug is selected from heparin or its pharmaceutically acceptable salt, low molecular weight heparin or its pharmaceutically acceptable salt, and recombinant hirudin.

10. The use according to claim 1, characterized in that The platelet dysfunction is an acquired platelet defect.

11. The use according to claim 10, characterized in that The acquired platelet deficiency is caused by drugs or diseases.

12. The use according to claim 11, characterized in that The drug is one or more of an antibacterial drug, an antitumor drug or heparin, and the disease is one or more of uremia, diabetes, nephrotic syndrome, coronary heart disease or leukemia.

13. The use according to any one of claims 1 to 12, characterized in that The pharmaceutical preparation containing icariin is injection, powder injection, capsule, tablet, microemulsion or pill.

14. The use according to claim 13, characterized in that The capsule is an enteric-coated soft capsule.

15. The use according to any one of claims 1 to 12, characterized in that The dosage of the drug is 0.01 mg / kg-100 mg / kg.

16. The use according to claim 15, characterized in that: The dosage of the drug is 0.1 mg / kg-10 mg / kg.

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