Use of icariin in the preparation of drugs for the prevention and treatment of cerebral infarction hemorrhage

Abstract

This invention belongs to the pharmaceutical field, specifically relating to the use of icariin in the preparation of drugs for the prevention and treatment of hemorrhagic transformation in cerebral infarction. Icariin not only significantly reduces the risk of primary hemorrhagic transformation, asymptomatic hemorrhagic transformation, and secondary hemorrhagic transformation following the use of thrombolytic, anticoagulant, or antiplatelet drugs in patients with cerebral infarction, but also significantly shortens bleeding time and reduces bleeding volume. While exerting the above effects, it does not alter coagulation parameters or complete blood count values, nor does it affect the thrombolytic efficacy of thrombolytic drugs. Furthermore, it has a significant preventive effect on the occurrence of hemorrhagic infarction that can be determined based on the initial head CT / MRI scan, and is of great significance for improving the short-term and long-term efficacy of thrombolytic therapy for infarct-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the use of icariin in the preparation of drugs for the prevention and treatment of cerebral infarction hemorrhage. Background Technology

[0002] Currently, cardiovascular and cerebrovascular diseases have become major threats to the health and lives of Chinese people. Among them, stroke has been the second leading cause of death in my country for five consecutive years, and also the second leading cause of death worldwide. By 2010, the Chinese Guidelines for Secondary Prevention of Ischemic Stroke and Transient Ischemic Attack (2010 Edition) clearly stated that cerebrovascular disease has become the leading cause of disability and death in both urban and rural areas of my country, and with the aging population, the incidence of stroke is expected to increase year by year. Therefore, early prevention and treatment of various types of ischemic infarction, and reducing sequelae, have become key issues in modern medicine.

[0003] Thrombolytic therapy is currently the only evidence-based treatment for acute ischemic infarction and is included in the prevention and treatment guidelines for cerebrovascular diseases in various countries. For example, the American Stroke Association specifically recommends intravenous recombinant tissue plasminogen activator (rt-PA) thrombolysis within 3 hours of onset, and for occlusion of large cranial arteries, including the vertebrobasilar artery or middle cerebral artery, intra-arterial rt-PA thrombolysis is recommended within 6 hours of onset. The results of my country's national "Ninth Five-Year Plan" key project, "A Multicenter, Randomized, Double-Blind, Placebo-Controlled Clinical Study of Early Thrombolytic Therapy for Acute Cerebral Infarction," also indicate that intravenous thrombolysis with urokinase (UK) within 6 hours of onset of acute cerebral infarction is effective and safe. The principle of thrombolytic therapy lies in the fact that thrombolytic drugs can rapidly dissolve thrombi, open occluded blood vessels, restore blood supply to the infarcted area, rescue the ischemic penumbra, and relieve neurological deficit symptoms and signs, thereby saving lives and reducing sequelae.

[0004] Although thrombolytic therapy is currently the only evidence-based and universally recognized effective treatment, it is a double-edged sword. While dissolving blood clots, it can also lead to various serious complications such as hemorrhagic transformation, and it has a strict time window, severely limiting its clinical application. Hemorrhagic transformation (HT) after thrombolysis refers to the leakage of blood from ischemic arteries after thrombolytic therapy in acute ischemic stroke, due to the restoration of blood flow to the ischemic area, and even rupture of the artery under pressure. Currently, Western medicine treats various complications after thrombolysis by addressing individual links or factors, without achieving satisfactory results. Therefore, it is of great significance to fully utilize the advantages of traditional Chinese medicine, which emphasizes holistic regulation and multi-link comprehensive treatment, and to seek effective treatments and prescriptions that can reduce or eliminate the risk of various hemorrhagic transformations while dissolving blood clots, thereby improving the short-term and long-term efficacy of thrombolytic therapy for infarct-related diseases.

[0005] Epimedium is the dried stem and leaves of several plants in the Berberidaceae family, including Epimedium brevicornum Maxim., Epimedium sagittatum Maxim., Epimedium pubescens Maxim., and Epimedium koreanum Nakai. Clinically, it is mainly used for kidney yang deficiency, impotence, frequent urination, infertility; rheumatic pain, numbness and contracture of limbs, weakness of muscles and bones, difficulty walking; and kidney yang deficiency, wheezing, cough, and shortness of breath.

[0006] Icaritin (IT) is a polyhydroxy flavonoid monomer from *Epimedium brevicornu*, a plant belonging to the genus *Epimedium* in the family Berberidaceae. Pharmacological studies have shown that IT has a stronger anti-osteoporosis effect than other flavonoid glycosides in *Epimedium brevicornu*, and in vitro, it promotes osteoblast activity and inhibits osteoclast activity. In recent years, icariin and icariin, as important active components of *Epimedium brevicornu*, have received increasing attention from pharmaceutical researchers. For example, patent application CN101637467A discloses the application of icariin in the preparation of drugs for treating osteoporosis. Patent US6399579 discloses the use of icariin in the treatment of sexual dysfunction.

[0007] There are currently no literature reports on the prevention and treatment of hemorrhage transformation by icariin. Summary of the Invention

[0008] To prevent or treat hemorrhagic transformation of cerebral infarction, this invention provides the use of icariin in the preparation of drugs for the prevention and treatment of hemorrhagic transformation.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] Use of icariin in the preparation of drugs for the prevention and treatment of cerebral infarction hemorrhage.

[0011] The aforementioned transformation of cerebral infarction hemorrhage into ischemic cerebral infarction secondary hemorrhage, primary hemorrhage, or asymptomatic hemorrhage is one or more of the following:

[0012] The aforementioned cerebral infarction hemorrhage transforms into hemorrhagic cerebral infarction. The aforementioned secondary hemorrhage transforms into hemorrhagic transformation caused by one or more treatment methods, including thrombolytic drugs, antithrombotic drugs, or endovascular therapy.

[0013] Furthermore, the thrombolytic drug is alteplase, and the antithrombotic drug is one or more of an anticoagulant or an antiplatelet drug, wherein the anticoagulant is heparin sodium and the antiplatelet drug is aspirin.

[0014] The aforementioned primary hemorrhage transformed into spontaneous hemorrhage after acute cerebral infarction.

[0015] Pharmacological Examples 1-2: Effects of Icariin on Bleeding Time in Mice Induced by Heparin Sodium and Aspirin. The results showed that Icariin could significantly shorten the bleeding time in mice induced by heparin sodium and aspirin, and did not change the blood routine and coagulation parameters while exerting a procoagulant effect.

[0016] The effects of icariin on the thrombolytic effect of thrombolytic drugs in Example 3 and on the effects of icariin on hemorrhagic transformation induced by thrombolytic drugs in Example 4 showed that icariin can significantly improve hemorrhagic transformation induced by thrombolytic drugs, and while preventing hemorrhagic transformation and reducing bleeding, it does not affect the thrombolytic effect of thrombolytic drugs on thrombus dissolution, nor does it affect blood routine values.

[0017] Icariin also has a significant preventive and therapeutic effect on bleeding 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 bleeding transformation and asymptomatic bleeding transformation.

[0018] In addition to its significant preventive and therapeutic effects on hemorrhagic transformation following cerebral infarction, icariin also has a significant preventive and therapeutic effect on hemorrhagic infarction that can be identified based on the first cranial CT / MRI scan.

[0019] Icariin can be administered via gastrointestinal and non-gastrointestinal routes. Non-gastrointestinal routes include, but are not limited to, subcutaneous, intradermal, arterial, venous, intramuscular, joint, intrathecal, intracranial, pleural, and intraperitoneal injections or infusions, as well as nasal, buccal, sublingual, tracheal, urethral, ​​rectal, or local administration to the lesion. It can be taken concurrently with other medications or as a preventative measure.

[0020] In addition to providing the use of icariin in the preparation of drugs for the prevention and treatment of hemorrhage transformation, this invention also provides a pharmaceutical preparation for the treatment of the above-mentioned diseases, which contains icariin and pharmaceutically acceptable excipients.

[0021] Pharmaceutical preparations include, but are not limited to, injections, powder injections, capsules, tablets, microemulsions, pellets, and enteric-coated soft capsules.

[0022] The pharmaceutical excipients used in the formulations of this invention are commonly used excipients known to those skilled in the art.

[0023] Suitable pharmaceutical excipients are described in detail in *Complete Guide to Pharmaceutical Excipients* (page 123, Sichuan Science and Technology Press, 1993, edited by Luo Mingsheng and Gao Tianhui). For example, commonly used pharmaceutical excipients for preparing microemulsion formulations include, but are not limited to, soybean oil, polyoxyethylene-23-lauryl ether, 1,2-propanediol, hydrogenated coconut oil glyceride, lauroyl polyethylene glycol-32-glyceride, polyethylene glycol 3350, safflower oil, cottonseed oil, and decaglyceryl monostearate; commonly used pharmaceutical excipients for preparing pellet formulations include, but are not limited to, polyethylene glycol 6000 and polyethylene glycol 1000; commonly used pharmaceutical excipients for preparing capsule formulations include, but are not limited to, lactose and corn starch. Commonly used pharmaceutically acceptable carriers for preparing soft capsule formulations include, but are not limited to, medium-chain fatty acid glycerides, polyoxyethylene castor oil, and 1,2-propanediol.

[0024] Those skilled in the art can select suitable pharmaceutical excipients according to actual needs and formulate the formulations of the present invention using methods known in the art. The formulations include, but are not limited to, solids, liquids, oils, emulsions, gels, aerosols, inhalers, sprays, capsules, pills, patches, and suppositories.

[0025] Preferably, the dosage of the drug for the prevention and treatment of hemorrhage transformation is 0.02 mg / kg to 200 mg / kg; more preferably, the dosage of the drug for the prevention and treatment of hemorrhage transformation is 0.2 mg / kg to 20 mg / kg.

[0026] Compared with existing technologies, icariin has the following advantages in the prevention and treatment of hemorrhage transformation:

[0027] (1) Significant therapeutic effect

[0028] It can significantly shorten the duration of bleeding in mice induced by heparin sodium and aspirin; and effectively prevent hemorrhagic transformation caused by thrombolytic drugs.

[0029] (2) It does not change the values ​​of coagulation parameters and blood routine, and does not affect the thrombolytic effect of thrombolytic drugs.

[0030] (3) Safe and non-toxic, with comprehensive effects

[0031] Epimedium glycoside is an extract from traditional Chinese medicine. Compared with Western medicine, it not only has the advantages of being safe and non-toxic, but also plays a role in overall regulation and comprehensive treatment of multiple aspects. Specific Implementation

[0032] The present invention is further described below through specific embodiments, but the present invention is not limited to the following embodiments.

[0033] Formulation Example 1: Icariin Microemulsion Formulation

[0034]

[0035] Preparation process: Weigh the prescribed amounts of soybean oil, polyoxyethylene-23-lauryl ether, and 1,2-propanediol, mix them thoroughly, and then add icariin to dissolve. Ultrasonic treatment can also be used to accelerate dissolution, yielding a clear solution, which is the icariin microemulsion formulation. The particle size was measured using a laser particle size analyzer, with an average particle size of 15 nm.

[0036] Formulation Example 2: Icariin Microemulsion Formulation

[0037]

[0038] Preparation process: Weigh the prescribed amounts of hydrogenated coconut oil glyceride, lauroyl polyethylene glycol-32-glyceride, 1,2-propanediol, and polyethylene glycol 3350, mix them thoroughly, and then add icariin to dissolve. Ultrasonic treatment can also be used to accelerate dissolution, yielding a clear solution, which is the icariin microemulsion formulation. The particle size was measured using a laser particle size analyzer, with an average particle size of 40 nm.

[0039] Formulation Example 3: Icariin Injection

[0040]

[0041] 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% activated carbon for injection, stir, decarbonize, and the product is obtained.

[0042] Formulation Example 4: Icariin Injection

[0043]

[0044] Preparation process: Mix the prescribed amount of ethanol and Tween-80 evenly, 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 final product.

[0045] Formulation Example 5: Icariin Injection

[0046] Epimedium aglycone 1g

[0047] 3.3L of ethanol

[0048] Add water for injection to 10L

[0049] 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.

[0050] Formulation Example 6: Epimedium Acid Glycoside Droplets Formulation

[0051]

[0052] Polyethylene glycol-6000 14.5g

[0053] Polyethylene glycol-1000 5.0g

[0054] Make 1000 pills

[0055] Preparation process: Weigh the prescribed amount of icariin 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 thoroughly to make it uniform, pour it into a dropper bottle, and drop it at 95±2℃; drop it into a glass condenser column containing 4-6 mL of methyl silicone oil, remove it after it has solidified, and use absorbent paper to remove the adhering methyl silicone oil to obtain the final product.

[0056] Formulation Example 7: Icariin Enteric-coated Soft Capsule Formulation

[0057] Contents prescription:

[0058] Epimedium aglycone 10g

[0059] 10g of anhydrous ethanol

[0060] 1,2-Propanediol 10g

[0061] 50g of polyoxyethylene castor oil

[0062] 20g of medium-chain triglycerides

[0063] Rubber prescription:

[0064] 10g of gelatin

[0065] 5g of glycerin

[0066] 10g of purified water

[0067] Enteric coating solution prescription:

[0068]

[0069]

[0070] Preparation process: Weigh the prescribed amounts of medium-chain fatty acid glycerides, polyoxyethylene castor oil, 1,2-propanediol, and anhydrous ethanol, mix and stir evenly, then add icariin to dissolve. Ultrasonic treatment can also be used to accelerate dissolution, yielding a clear concentrate, which is the icariin microemulsion concentrate. Dilute the obtained microemulsion concentrate with water at a weight ratio of 1:10-20 to a clear solution, obtaining the soft capsule microemulsion contents. Weigh the prescribed amounts of gelatin, glycerin, and purified water, mix evenly, and press into a rubber sheet. Then weigh the prescribed amounts of Eudragit L30D-55, triethyl citrate, talc, and purified water, mix evenly to prepare an enteric coating solution. Encapsulate the soft capsule microemulsion contents containing icariin with the rubber sheet to form soft capsules, and then coat the soft capsules with an enteric coating to obtain enteric soft capsules.

[0071] Formulation Example 8: Epimedium Acid Capsule Formulation

[0072]

[0073] 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, then fill into 1000 capsule shells.

[0074] Formulation Example 9: Icariin Tablets

[0075]

[0076] Preparation process: Icariin and excipients microcrystalline cellulose and sodium carboxymethyl starch are mixed evenly, and an appropriate amount of starch slurry is added to form a soft mass, which is then granulated through a 16-mesh sieve. The wet granules are dried at 60℃, and the dried granules are sieved through a 20-mesh sieve to remove fine powder. This powder is then mixed with magnesium stearate, and then mixed with the dry granules again. The mixture is then compressed into tablets, each containing approximately 200mg.

[0077] Formulation Example 10: Epimedium Aglycone Powder for Injection

[0078]

[0079] Preparation process: Weigh the prescribed amount of icariin for injection and dissolve it in an appropriate amount of water for injection. Then, add the specified amount of pre-sterilized and pyrogen-free treated material and mix well, then add water for injection to the specified volume of 1000 ml. Add 5 g of activated charcoal for injection to the above solution, heat at 60-80℃ for 30 minutes, filter through a filter membrane, and collect the filtrate. Filter the filtrate under aseptic conditions using a sterile filter under positive pressure, and then filter through a 0.22 μM microporous membrane. Perform pyrogen and semi-finished product content checks on the filtrate, and then dispense into vials. Pre-freeze in a dedicated freeze-drying chamber at -40℃ for 1.5-3.5 hours, sublimate under vacuum until 90% of the free water is removed, and then heat to dry (maximum temperature not exceeding 35℃). After freeze-drying, icariin powder for injection is obtained.

[0080] Pharmacological Example 1: Effect of icariin on prolonged bleeding time in mice induced by heparin sodium

[0081] 1. Drugs and their sources

[0082] Heparin sodium injection

[0083] Source: Jiangsu Wanbang Biochemical Pharmaceutical Group Co., Ltd.

[0084] Batch number: 51701106

[0085] Ethylphenidyl sulfonamide injection

[0086] Source: Shandong Fangming Pharmaceutical Group Co., Ltd.

[0087] Batch number: 18060672

[0088] 2. Experimental animals and grouping

[0089] Seventy-two 5-6 week old ICR mice, weighing 19-21g, were used, with half being male and half female. The mice were quarantined for 7 days after entering the laboratory, and healthy mice were selected as test animals. They were weighed on the first and last days of quarantine.

[0090] Based on their weight at the end of quarantine, male and female animals were divided into six groups using a simplified randomization method: normal group (C), model group (V), low, medium, and high dose groups of icariin (2, 6, and 18 mg / kg, L, M, and H), and positive control group (200 mg / kg ethamsylate, P).

[0091] 3. Pre-drug administration and model establishment

[0092] After grouping, the normal group and model group were administered 10 ml / kg of icariin solution by gavage. The solution was prepared by adding 12 g of hydroxypropyl methylcellulose E5 and 0.1 g of sodium dodecyl sulfate to 1 L of water. The icariin dosage groups were administered 2, 6, and 18 mg / kg of icariin by gavage, once daily for five consecutive days. The positive control group received an intraperitoneal injection of 200 mg / kg ethamsylate injection once daily for five consecutive days. One hour after the last pre-administration, the model group and all treatment groups were injected with 150 U / kg heparin sodium injection via tail vein to establish the model, while the normal group received an injection of 10 ml / kg sodium chloride injection via tail vein.

[0093] 4. Detection indicators

[0094] 4.1 Bleeding time

[0095] Fifteen minutes after modeling, the tails of each mouse were clipped to measure the bleeding time.

[0096] 4.2 Complete blood count and coagulation panel

[0097] After determining the bleeding time, mice were anesthetized with sodium pentobarbital, and blood was collected from the abdominal main vein to test complete blood count and coagulation parameters (PT, APTT, TT, FIB).

[0098] 5. Test Results

[0099] 5.1 Bleeding time

[0100] The bleeding time of mice in each group is shown in Table 1.

[0101] Table 1. Effect of icariin on heparin sodium-induced bleeding time in mice.

[0102] 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 ﹩﹩& ]]>

[0103] Note: Compared with the normal group ## P<0.01;

[0104] Compared with the model group, ﹩ P<0.05, ﹩﹩ P<0.01;

[0105] Compared with the positive control group, & P<0.05.

[0106] As shown in the table, 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 treatment group was significantly shortened, indicating that icariin can shorten the bleeding time of bleeding mice. The shortening of bleeding time is more significant with increasing dose, indicating that the procoagulant effect of icariin is dose-dependent. Furthermore, the high-dose group showed a significant difference compared with the positive control group, indicating that the procoagulant effect of icariin is better than that of the positive control.

[0107] 5.2 Complete blood count and coagulation panel

[0108] The results of routine blood tests and coagulation parameters for each group of mice are shown in Tables 2 and 3, respectively.

[0109] Table 2. Results of routine blood tests in mice of each group

[0110]

[0111]

[0112] Table 3 Results of coagulation tests in mice of each group

[0113]

[0114] As can be seen from the table above, there were no significant differences in the blood routine and coagulation parameters among the groups of mice, indicating that icariin had no significant effect on blood routine and coagulation parameters when it shortened the bleeding time prolongation caused by heparin sodium.

[0115] Pharmacological Example 2: Effect of Icariin on Aspirin-Induced Prolongation of Bleeding Time in Mice

[0116] 1. Drugs and their sources

[0117] aspirin

[0118] Source: Shantou Jinshi Pharmaceutical Factory Co., Ltd.

[0119] Batch number: 1903012

[0120] Ethylphenidyl sulfonamide injection

[0121] Source: Shandong Fangming Pharmaceutical Group Co., Ltd.

[0122] Batch number: 1905032

[0123] 2. Experimental animals and grouping

[0124] Ninety-six 5-6 week old ICR mice, weighing 19-21g, were used, with half being male and half female. The mice were quarantined for 7 days after entering the laboratory, and healthy mice were selected as test animals. They were weighed on the first and last days of quarantine.

[0125] Based on their weight at the end of quarantine, male and female animals were divided into six groups using a simplified randomization method: normal group (C), model group (V), low, medium, and high dose groups of icariin (2, 6, and 18 mg / kg, L, M, and H), and positive control group (200 mg / kg ethamsylate, P).

[0126] 3. Pre-drug administration and model establishment

[0127] After grouping, the normal group and model group were administered 10 ml / kg of icariin solvent by gavage. The icariin dosage groups were administered 2, 6, and 18 mg / kg of icariin by gavage, respectively, once daily for five consecutive days. The positive control group was injected intraperitoneally with 200 mg / kg ethamsylate injection once daily for five consecutive days. One hour after the last pre-administration, the model group and all drug-administered groups were administered 20 mg / kg of aspirin by gavage to induce the model, while the normal group was administered an equal volume of physiological saline by gavage.

[0128] 4. Bleeding time detection

[0129] Fifteen minutes after modeling, the tails of each mouse were clipped to measure the bleeding time.

[0130] 5. Test Results

[0131] The bleeding time of mice in each group is shown in Table 4. Compared with the normal group, the bleeding time of mice in the model group was significantly prolonged, indicating successful modeling. Compared with the model group, the bleeding time of mice in each treatment group was significantly shortened, indicating that icariin can shorten the bleeding time of bleeding mice. The shortening of bleeding time is more significant with increasing dose, indicating that the procoagulant effect of icariin is dose-dependent. Furthermore, the high-dose group showed a significant difference compared with the positive control group, indicating that the procoagulant effect of icariin is better than that of the positive control.

[0132] Table 4. Effect of icariin on aspirin-induced bleeding time in mice.

[0133]

[0134]

[0135] Note: Compared with the normal group ## P<0.01;

[0136] Compared with the model group, ﹩ P<0.05, ﹩﹩ P<0.01;

[0137] Compared with the positive control group, & P<0.05.

[0138] Pharmacological Example 3: Effect of Icariin on the Thrombolytic Effect of Thrombolytic Drugs

[0139] 1. Construction of a rat abdominal aortic thrombosis model

[0140] A rat abdominal aortic thrombosis model was established as follows: Rats were anesthetized by intraperitoneal injection of 40 mg / kg of 1.5% sodium pentobarbital. The rats were shaved and skinned, and fixed in a supine position on a rat board. The abdominal skin was incised to isolate the blood vessels and surrounding tissues, exposing the abdominal aorta. The abdominal aorta was carefully dissected using a glass needle. A 0.7 cm × 1.5 cm strip of tin foil was placed under the abdominal aorta, and then a 0.5 cm × 1.0 cm strip of filter paper soaked in 35% FeCl3 solution was wrapped around the abdominal aorta. After 30 minutes, the filter paper was removed. At this time, the blood vessel wall was significantly darkened, and emboli were visible under a microscope. Pathological sections showed that the blood vessel was blocked by thrombus.

[0141] 2. Grouping and Dosing

[0142] SD rats were randomly divided into three groups (n=10 per group) according to body weight: a thrombosis model group, a lumbrokinase capsule group, and a lumbrokinase capsule + icariin group. Administration of the drug was performed 5 minutes after model establishment, with the lumbrokinase capsule administered at a dosage of 30 × 10⁻⁶ g / L. 4 Icariin was administered via direct injection into the duodenum at a dose of U / kg, while icariin was administered via gavage at a dose of 18 mg / kg. The thrombosis model group was treated with an equal volume of distilled water instead.

[0143] 3. Get materials

[0144] Clamp both ends of the abdominal aorta with hemostatic forceps, completely remove the vascular segment from the modeling site, accurately cut off the 0.5cm vascular segment wrapped in filter paper, weigh it, and preserve the remaining vascular segment in 10% formaldehyde.

[0145] 4. Determination of rat thrombus weight

[0146] Weigh the removed blood vessel segment using an electronic balance. After removing the thrombus, weigh the blood vessel again. The wet weight of the thrombus = total weight of the thrombus and blood vessel - weight of the blood vessel.

[0147] The removed thrombus is dried in an oven at 80°C, left overnight, and weighed to obtain the dry weight of the thrombus.

[0148] 5. Test Results

[0149] The thrombus weights of rats in each group are shown in the table below. As can be seen from the table, the thrombus weight in the lumbrokinase group was significantly lower than that in the model group, indicating that lumbrokinase has a significant thrombolytic effect. Compared with the lumbrokinase group, there was no significant difference in the thrombus weight in the lumbrokinase + icariin group, indicating that icariin does not affect the thrombolytic effect of lumbrokinase on rat thrombi.

[0150] Table 5. Effects of epimedium glycoside on thrombus weight in rats of different groups.

[0151] Model group 9 2.98±0.54 0.31±0.12 Lumbrokinase 10 <![CDATA[1.01±0.22 ## ]]> <![CDATA[0.04±0.01 ## ]]> Lumbrokinase + Icariin 10 1.24±0.19 0.04±0.02

[0152] Note: Compared to the model group, ## P<0.01.

[0153] Pharmacological Example 4: Effect of Icariin on Hemorrhagic Transformation Induced by Thrombolytic Drugs

[0154] 1. Preparation of animal models

[0155] A rat model of autologous thromboembolism in the middle cerebral artery was prepared as follows: Rats were anesthetized by intraperitoneal injection of 10% chloric acid hydrate at 350 mg / kg body weight, and fixed supine on the operating table. Following aseptic procedures, routine skin disinfection and draping were performed. A 3.0 cm incision was made in the midline of the neck. The muscles and subcutaneous tissue were bluntly dissected, and the right CCA, ECA, and ICA were separated, taking care not to damage the vagus nerve. The internal carotid artery was dissected to the skull base, and the ECA was ligated at the CCA bifurcation. Using a 1 ml syringe, 0.1 ml of arterial blood was collected from the rat's femoral artery. 4 U of thrombin was aspirated, mixed, and allowed to stand for 8-10 minutes until the blood coagulated. This coagulated blood was then injected into a 24G intravenous catheter to create an embolus approximately 1 cm long, which was then connected to a 1-2 ml saline syringe. Then, the indwelling needle is inserted into the CCA anterior to the bifurcation of the ECA and ICA, and further into the ICA by about 8-10 mm anteromedially. The needle core is then removed, and the syringe plunger and saline solution are quickly flushed into the ICA. The ICA is then ligated and sutured. In the sham surgery group (control group), all surgical procedures were the same as those in the thrombolysis model groups, except for the injection of saline solution instead of the plunger.

[0156] 2. Animal inclusion criteria

[0157] Two hours after modeling, observe the symptoms and signs of the rats. The presence of the following symptoms and signs indicates that the model has been successfully established: Horner's sign on the ipsilateral side of ischemia, walking in circles on the opposite side of ischemia; tilting to the opposite side; when the rat is lifted by its tail, the forelimb on the opposite side of cerebral ischemia is flexed, raised, adducted, and extended at the elbow.

[0158] 2. Experimental grouping and drug administration

[0159] One hundred and two dozen rats were randomly divided into three groups: a sham-operated group, an rt-PA thrombolysis group (thrombolysis group), and low, medium, and high rt-PA thrombolysis + icariin groups. The sham-operated group consisted of 18 rats, and the other groups each contained 21 rats. Each group was further subdivided according to different sacrifice time points (3, 6, and 24 hours after thrombolysis). At each time point, the sham-operated group had 6 rats, while the other groups had 9 rats at 3 hours and 6 rats at other time points. An additional 20 rats were reserved for replacement if the model failed (due to death or a score of 0).

[0160] Except for the sham-operated group, all other groups were given alteplase (rt-PA) (5 mg / kg, diluted to 1 ml with sterile water for rt-PA) via the femoral vein 3 hours after modeling. The sham-operated group was given an equal volume of normal saline.

[0161] The treatment group received two doses, administered by gavage immediately after model establishment and immediately after thrombolytic therapy. The doses were 2, 6, and 18 mg / kg, respectively. The sham-operated group received an equal volume of normal saline.

[0162] 4. Methods for Observing and Measuring Indicators

[0163] 4.1 Overall condition comparison and routine blood tests

[0164] 4.1.1 Compare the overall condition of rats in the sham-operated group, thrombolysis group, and thrombolysis group + icariin group.

[0165] 4.1.2 Blood routine tests were performed on rats in the sham-operated group, thrombolysis group, and thrombolysis group + icariin group.

[0166] 4.2 Measurement of cerebral infarction volume (rate)

[0167] Except for the sham-operated group, three rats from each group were sacrificed 3 hours after thrombolysis. Brain tissue was flash-frozen at -20°C for 20 minutes, and 2mm thick coronal slices were prepared. These slices were then immersed in 2% TTC solution, incubated in a light-protected water bath at 37°C for 30 minutes, and fixed in 4% paraformaldehyde solution for 24 hours. After photographing, infarct volume was measured using image analysis software (Image-Pro Plus 6.0). Infarct volume = volume of the non-ischemic hemisphere - volume of the non-infarcted area of ​​the ischemic hemisphere. The infarct volume percentage was calculated as a percentage (%) of the infarct volume to the volume of the non-ischemic hemisphere.

[0168] 4.3 Measurement of intracranial hemorrhage volume:

[0169] (1) Draw the standard curve

[0170] Four healthy rats were euthanized under overdose anesthesia and their blood was removed via cardiac perfusion. Eight cerebral hemispheres were collected, and 0, 0.5, 1, 2, 4, 8, 16, 32, and 50 μL of male SD rat arterial blood were added sequentially to each hemisphere. PBS (pH 7.4) was added to a total volume of 3 ml, homogenized for 30 seconds, and then sonicated to lyse the lysate. The mixture was centrifuged at 13000 rpm for 30 min at 4°C. 0.8 ml of Drabkin's reagent was mixed with 0.2 ml of the supernatant from each sample. The mixture was incubated at room temperature in the dark for 10 min, and the absorbance was measured at 540 nm (absorbance of samples without added blood was set to 0). A standard curve was plotted with absorbance on the ordinate and the amount of blood in each sample on the abscissa.

[0171] (2) Measurement of hemorrhage volume in the infarcted hemisphere

[0172] Tissue from the infarcted hemisphere of rats with cerebral infarction was added to PBS (pH 7.4) to a total volume of 3 ml, homogenized for 30 seconds, and then sonicated to lyse. The homogenate was centrifuged at 13000 rpm for 30 min. 0.8 ml of Drabkin's reagent was mixed with 0.2 ml of the supernatant from each sample, and the mixture was incubated at room temperature in the dark for 10 min. The absorbance was then measured at 540 nm (the absorbance of bloodless brain tissue samples was set to 0, and measurements were performed on the same batch). The blood volume corresponding to the absorbance on the standard curve is the amount of hemorrhage.

[0173] 5. Statistical processing

[0174] All experimental data are expressed as mean ± standard deviation and were processed using PASW Statistics 17.0 software. One-way ANOVA was used for comparisons between and within groups. If variances were homogeneous, the LSD test was used; otherwise, the Dunnett T3 nonparametric test was used. Correlation analysis was performed using the two-tailed Pearson product-moment correlation coefficient test for linear correlation, with a two-tailed P-value < 0.05 considered statistically significant.

[0175] 6. Experimental Results

[0176] 6.1 Overall condition comparison and routine blood test

[0177] 6.1.1 Overall situation comparison,

[0178] The rats in the sham-operated group were in good mental condition, and their fur was glossy and normal. The rats in the thrombolysis group and the thrombolysis group plus icariin at all doses showed a significant decline in mental condition after surgery compared to before surgery. The manifestations were: dull fur luster, reduced spontaneous activity, and relatively sluggish response to external stimuli. The thrombolysis group was the worst, while the thrombolysis group plus the high dose of icariin showed the best mental condition.

[0179] 6.1.2 Complete Blood Count (CBC)

[0180] Blood routine test results showed that there was no significant difference in blood routine values ​​between 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-operated group.

[0181] 6.2 Measurement of cerebral infarction volume (rate)

[0182] TTC staining can be used to observe the extent of cerebral ischemia. Staining results: In the sham-operated group and other groups, the non-infarcted brain tissue showed a uniform red color, while the infarcted brain tissue was significantly edematous, pale white, and dull. After TTC staining, the infarct area appeared pale white. The results showed that the cerebral infarction volume rate was (38.84±3.67)% 3 hours after rat modeling. TTC staining of the brain tissue in the sham-operated group showed no change. Compared with the model group, TTC staining of the thrombolytic group and the thrombolytic drug + icariin groups showed a significant reduction in the pale white area of ​​the infarct area. Cerebral infarction volume measurement revealed that the reduction rate of cerebral infarction volume in the thrombolytic drug + icariin groups was comparable to that in the thrombolytic group. This result indicates that icariin does not affect the thrombolytic effect of the thrombolytic drug.

[0183] 6.3 Comparison of cerebral hemorrhage volume in different groups of rats at different time points

[0184] The amount of cerebral hemorrhage in rats at different time points is shown in the table below. The table shows that the hemorrhage amount in the thrombolysis group was significantly greater than that in the sham-operated group, indicating that the thrombolytic drug induced hemorrhagic transformation in rats with cerebral infarction, and the hemorrhage amount increased significantly with time. Compared with the thrombolysis group, the hemorrhage amount was significantly reduced in all dose groups of the thrombolytic drug + icariin combination, and the hemorrhage amount did not increase with time. This indicates that icariin can effectively prevent hemorrhagic transformation induced by thrombolytic drugs.

[0185] Table 6 Comparison of cerebral hemorrhage volume in rats at different time points.

[0186]

[0187] Note: Compared with the sham surgery group. ## P<0.01;

[0188] Compared with the thrombolysis group, ﹩ P<0.05, ﹩﹩ P<0.01.

Claims

1. The use of icariin in the preparation of drugs for the prevention and treatment of cerebral infarction hemorrhage, characterized in that, The cerebral infarction hemorrhage is transformed into a hemorrhage caused by the use of thrombolytic or antithrombotic drugs; the antithrombotic drug is one or more of anticoagulants or antiplatelet drugs.

2. The use as described in claim 1, characterized in that, The thrombolytic drug mentioned is alteplase.

3. The use as described in claim 1, characterized in that, The anticoagulant is heparin sodium; the antiplatelet drug is aspirin.

4. The use as described in claim 1, characterized in that, The drug for preventing and treating cerebral infarction hemorrhage is a pharmaceutical preparation containing icariin.

5. The use as described in claim 4, characterized in that, The pharmaceutical preparation is a solid or liquid preparation.

6. The use as described in claim 5, characterized in that, The pharmaceutical preparation is an oil, emulsion, gel, aerosol, inhaler, spray, capsule, pill, patch, or suppository.

7. The use as described in claim 1, characterized in that, The dosage of the drug for preventing and treating cerebral infarction hemorrhage is 0.02 mg / kg to 200 mg / kg.

8. The use as described in claim 7, characterized in that, The dosage of the drug for preventing and treating cerebral infarction hemorrhage is 0.2 mg / kg to 20 mg / kg.

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