Application of resina draconis and resina draconis phenolic extract in antithrombosis and application of monomeric compound of resina draconis phenolic extract in antiplatelet aggregation or cerebral infarction protection

Abstract

The invention provides application of resina draconis and resina draconis phenolic extracts in antithrombosis and application of monomeric compounds of the resina draconis phenolic extracts in resisting platelet aggregation or cerebral infarction protection, and belongs to the technical field of medicine. The invention provides application of resina draconis or resina draconis phenolic extracts in preparation of antithrombotic drugs, provides application of the resina draconis phenolic extracts or six monomeric compounds in the resina draconis phenolic extracts in preparation of antithrombotic or antiplatelet aggregation drugs, and further provides application of loureirin D in preparation of cerebral infarction protection drugs. The invention relates to application of resina draconis phenolic extract or resina draconis phenolic ethyl acetate extract in preparation of medicines for improving Evans'blue leakage, medicines for protecting blood-brain barrier, medicines for reducing brain water content or medicines for reducing cerebral hemorrhage. The resina draconis, the resina draconis phenolic extract and monomer compounds in the resina draconis phenolic extract are natural active ingredients and are good in safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of Dragon's blood and phenolic extract of Dragon's blood in anti-thrombosis and the application of monomer compounds of Dragon's blood in anti-platelet aggregation or cerebral infarction protection. BACKGROUND

[0002] Arterial thrombosis has become a major problem endangering human health worldwide. The formation of arterial thrombosis depends on the activation and aggregation of platelets, which is mediated by surface receptors (such as GPIIb / IIIa and P2Y12) to adhere and release active substances (such as ADP and TXA2), triggering the coagulation cascade, and finally forming a mixed thrombus mainly composed of fibrin and blood cells. Among them, the combination of ADP and platelet P2Y12 receptor is a key step in regulating platelet aggregation, which not only accelerates thrombus expansion, but also enhances thrombin generation through positive feedback, forming a vicious cycle. The key to current anti-thrombotic therapy is the use of anti-platelet drugs. Clopidogrel, as a first-line anti-platelet aggregation drug, achieves therapeutic purposes by inhibiting the combination of ADP and P2Y12 receptors on the surface of platelets. Long-term use has the risk of bleeding transformation. In clinical practice, 10-30% of patients will experience nosebleeds, gum bleeding, and even serious gastrointestinal bleeding, intracranial hemorrhage, and other adverse reactions, which greatly limit its clinical application.

[0003] In the face of such a difficult situation, exploring new treatment strategies has become a top priority. Natural products are a part of compounds with pharmacological activity found in plants, animals, or microorganisms. They are widely used in drug development due to their good safety and relatively small side effects. Traditional Chinese medicine is an important source of new drug discovery, such as the discovery of typical drugs such as paclitaxel and artemisinin. Therefore, discovering lead compounds with good anti-thrombotic activity from traditional Chinese medicine and ethnic medicine is one of the fast and effective ways to explore new treatment strategies. Dragon's blood has the functions of promoting blood circulation to remove blood stasis, relieving inflammation and pain, and promoting tissue regeneration and wound healing. Currently, there is no related technology report on isolating compounds with good anti-thrombotic or anti-platelet aggregation activity from Dragon's blood.

[0004] Ischemic stroke is a disease with high mortality and high disability, which seriously endangers human life and health. Ischemic stroke is mainly caused by blood vessel obstruction, which suddenly reduces blood supply or interrupts blood flow, thus depriving the ability to transport oxygen and nutrients to the brain. Current treatment methods for ischemic stroke include surgical mechanical thrombectomy and recombinant tissue plasminogen activator (rt-PA), which is the only drug approved by the US Food and Drug Administration for the treatment of acute ischemic stroke. However, rt-PA is only suitable for a small number of patients due to its limited therapeutic window and the risk of bleeding after thrombolysis. The risk of hemorrhagic transformation after thrombolysis is closely related to the damage to the blood-brain barrier after stroke. Therefore, the key to finding new treatment methods is to develop drugs that can treat ischemic stroke while reducing blood-brain barrier damage caused by thrombolysis. SUMMARY

[0005] The application aims to provide the application of dragon's blood and dragon's blood phenolic extract in antithrombosis and the application of monomer compounds in anti-platelet aggregation or cerebral infarction protection. The dragon's blood and dragon's blood phenolic extract have excellent antithrombotic activity, and the dragon's blood phenolic extract and the monomer compounds separated from the dragon's blood phenolic extract have excellent antithrombotic and anti-platelet aggregation effects or protection of the blood-brain barrier, wherein the monomer compound Loureirin D has excellent cerebral infarction reduction effect. The dragon's blood, dragon's blood phenolic extract and the monomer compounds thereof are natural active ingredients and have good safety.

[0006] In order to achieve the above application purposes, the application provides the following technical solutions. The application provides the application of dragon's blood or dragon's blood phenolic extract in the preparation of an antithrombotic drug, wherein the dragon's blood phenolic extract includes one or more of total dragon's blood phenolic extract, dragon's blood phenolic ethyl acetate extract and dragon's blood phenolic methanol extract.

[0007] The application provides the application of dragon's blood phenolic extract or monomer compounds in the dragon's blood phenolic extract in the preparation of an antithrombotic drug or an anti-platelet aggregation drug, wherein the dragon's blood phenolic extract includes one or more of total dragon's blood phenolic extract, dragon's blood phenolic ethyl acetate extract and dragon's blood phenolic methanol extract, and the monomer compounds are any one of compounds with structures shown in formulae I-VI. Formula I; Formula II; Formula III; Formula IV; Formula V; Formula VI.

[0008] Preferably, the thrombus comprises an arterial thrombus.

[0009] Preferably, the arterial thrombus comprises a common carotid artery thrombus or a middle cerebral artery thrombus.

[0010] Preferably, the common carotid artery thrombus comprises a FeCl3-induced common carotid artery thrombus.

[0011] Preferably, the middle cerebral artery thrombus comprises a photochemical-induced middle cerebral artery thrombus.

[0012] Preferably, the platelet aggregation comprises adenosine diphosphate-induced platelet aggregation.

[0013] The application provides application of a monomer compound in a phenolic extract of dragon's blood in preparation of a cerebral infarction protective drug, the monomer compound being dracorhodin D, having the structure shown in formula V. Formula V.

[0014] Preferably, the cerebral infarction comprises a cerebral infarction caused by middle cerebral artery embolism.

[0015] The application provides application of the phenolic extract of dragon's blood or the ethyl acetate phenolic extract of dragon's blood in preparation of an Evans' blue leakage improving drug, a blood-brain barrier protective drug, a cerebral water content reducing drug or a cerebral hemorrhage reducing drug.

[0016] Beneficial effects: the application provides application of dragon's blood or a phenolic extract of dragon's blood in preparation of an antithrombotic drug, the phenolic extract of dragon's blood comprising one or more of a total phenolic extract of dragon's blood, an ethyl acetate phenolic extract of dragon's blood and a methanol phenolic extract of dragon's blood. The dragon's blood or the phenolic extract of dragon's blood has excellent antithrombotic activity, and specifically, the dragon's blood and the total phenolic extract of dragon's blood can reduce the embolus weight of a FeCl3-damaged common carotid artery of a rat.

[0017] Moreover, the application also provides the use of the dragon's blood phenolic extract or the monomer compound in the dragon's blood phenolic extract in the preparation of an anti-thrombus drug or an anti-platelet aggregation drug. The dragon's blood phenolic extract has excellent anti-thrombus and anti-platelet aggregation effects, and the total dragon's blood phenolic extract can reduce the FeCl3 injury of the carotid artery of a rat, increase the blood flow of the part, and has anti-platelet aggregation. The ethyl acetate extract of the dragon's blood phenolic extract (denoted as EA extract) and the methanol extract of the dragon's blood phenolic extract (denoted as MeOH extract) can not only reduce the FeCl3 injury of the carotid artery of a rat, increase the blood flow of the part, but even the dragon's blood phenolic extract or the EA extract can significantly improve the Evans' blue leakage, protect the blood-brain barrier, reduce the brain water content, and reduce the risk of cerebral hemorrhage. The dragon's blood phenolic extract is a natural active ingredient and has good safety.

[0018] The monomer compound is specifically denoted as compound 1 (formula I), compound 2 (formula II), compound 7 (formula III), compound 12 (formula IV), compound 17 (formula V, loganin D), and compound 24 (formula VI). The monomer compound has excellent anti-platelet aggregation effect, and the compound 1, the compound 2, the compound 7, the compound 12, the compound 17 (loganin D), and the compound 24 can all improve the platelet aggregation effect stimulated by an adenosine diphosphate (ADP) solution. The results of the examples show that the compound 1, the compound 2, the compound 7, the compound 12, the compound 17, and the compound 24 can significantly reduce the platelet aggregation rate, and the effect is equivalent to that of the positive control drug ticagrelor, and the anti-platelet IC 50 The anti-platelet IC50values of the compound 1, the compound 2, the compound 7, the compound 12, the compound 17, and the compound 24 are 4.31 μM, 9.75 μM, 8.04 μM, 7.96 μM, 4.21 μM, and 8.93 μM, respectively. The monomer compound can be separated from the dragon's blood phenolic extract and has good safety.

[0019] Meanwhile, the application provides the use of the monomer compound in the dragon's blood phenolic extract in the preparation of a cerebral infarction protection drug, and the monomer compound is loganin D. The loganin D has excellent anti-thrombus and anti-platelet aggregation effects, and also has a cerebral protection effect on a cerebral infarction rat caused by a middle cerebral artery embolism, and the effect is specifically reflected in the improvement of the neurological score, the motor ability, the protection of cerebral edema, and the blood-brain barrier of the cerebral infarction rat. The results of the examples show that the loganin D can significantly reduce the blood-brain barrier leakage and the brain water content of the pMCAO rat, has a blood-brain barrier protection effect, and can significantly improve the motor dysfunction of the cerebral infarction rat. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a DB anti-thrombus, DBE anti-thrombus, and DBE anti-platelet aggregation activity screening result diagram. Figure 2 Figure for the anti-thrombosis activity screening results of different extracts in DBE; Figure 3 Figure for the efficacy comparison of EA extract and MeOH extract in the photothrombotic model; Figure 4 Figure for the anti-platelet aggregation activity screening results of 25 monomer compounds separated from the EA extract; Figure 5 Figure for the anti-platelet aggregation activity screening results of 25 monomer compounds separated from the EA extract; Figure 6 Figure for the blood-brain barrier efficacy comparison of the Loureirin D in the pMCAO cerebral infarction model. DETAILED DESCRIPTION

[0021] Dragon's blood has the effects of activating blood circulation, relieving inflammation and pain, and promoting tissue healing, and has the pharmacological effects of protecting the cardiovascular system, nervous system, and anti-tumor. The chemical components include phenols, terpenes, sterols, etc., and the active ingredients are mainly phenols. The present application separates and extracts the phenolic extract of dragon's blood from different polarities based on the anti-thrombosis activity. The purpose is to clarify the specific effective components of the anti-thrombosis activity and anti-platelet aggregation, to provide a theoretical basis and experimental basis for the research and development of safe and efficient new anti-thrombosis and anti-platelet aggregation drugs, and to bring new breakthroughs to the field of arterial thrombosis treatment. The following will be described in detail.

[0022] The present application provides the application of dragon's blood or phenolic extract of dragon's blood in the preparation of anti-thrombosis drugs, wherein the phenolic extract of dragon's blood includes one or more of the total phenolic extract of dragon's blood, the ethyl acetate extract of phenolic extract of dragon's blood, and the methanol extract of phenolic extract of dragon's blood.

[0023] Different kinds of extracts can be further prepared from the total phenolic extract of dragon's blood, including the petroleum ether extract of phenolic extract of dragon's blood, the dichloromethane extract of phenolic extract of dragon's blood, the ethyl acetate extract of phenolic extract of dragon's blood, and the methanol extract of phenolic extract of dragon's blood. The specific preparation method includes the following steps: The total phenolic extract of dragon's blood is separated by silica gel column chromatography, and the eluents used are petroleum ether, dichloromethane, ethyl acetate, and methanol in sequence. The eluent corresponding to the petroleum ether, dichloromethane, ethyl acetate, and methanol elution is collected respectively, and the solvents are removed to obtain the petroleum ether extract, dichloromethane extract, ethyl acetate extract, and methanol extract of the total phenolic extract of dragon's blood.

[0024] As an embodiment of the present application, the thrombus can be an arterial thrombus; the arterial thrombus can include a common carotid artery thrombus or a middle cerebral artery thrombus; the common carotid artery thrombus can include a FeCl3-induced common carotid artery thrombus, and the middle cerebral artery thrombus can include a photochemical-induced middle cerebral artery thrombus.

[0025] As an embodiment of the present application, the anti-thrombus drug includes an active ingredient and a pharmaceutically acceptable carrier, the active ingredient is dragon's blood and / or a phenolic extract of dragon's blood, the content of the active ingredient in the anti-thrombus drug can independently be 0.1-99.9wt%, further can be 1-99wt%, and more further can be 2-50wt%; the pharmaceutically acceptable carrier includes one or several of diluents, binders, disintegrants and solvents.

[0026] As an embodiment of the present application, the administration mode of the anti-thrombus drug can include oral administration, injection or local administration; the dosage form of the anti-thrombus drug can include tablets, capsules, injections, granules, pills, suspensions, dispersions, syrups, transdermal preparations, enteric-coated tablets, sprays or lozenges.

[0027] The present application provides an application of a phenolic extract of dragon's blood or a monomer compound in the phenolic extract of dragon's blood in preparing an anti-thrombus drug or an anti-platelet aggregation drug, the phenolic extract of dragon's blood includes one or several of a total phenolic extract of dragon's blood, an ethyl acetate extract of phenolic of dragon's blood and a methanol extract of phenolic of dragon's blood, and the monomer compound is any one of compounds with structures shown in formulae I-VI: Formula I; Formula II; Formula III; Formula IV; Formula V; Formula VI.

[0028] As an embodiment of the present application, the specific type of the thrombus is consistent with the above technical solution, and will not be repeated here.

[0029] As an embodiment of the present application, the platelet aggregation includes adenosine diphosphate-induced platelet aggregation.

[0030] As an embodiment of the present application, the anti-thrombotic drug and the anti-platelet aggregation drug independently comprise an active ingredient and a pharmaceutically acceptable carrier, the active ingredient being one or more of the total extract of phenolic compounds from dragon's blood, the ethyl acetate extract of phenolic compounds from dragon's blood, the methanol extract of phenolic compounds from dragon's blood, and the compound having the structure shown in Formula I-VI, and the content of the active ingredient in the anti-thrombotic drug and the anti-platelet aggregation drug can independently be 0.1-99.9wt%, further 1-99wt%, more further 2-50wt%, and again further 2.36-20wt%.

[0031] As an embodiment of the present application, the anti-thrombotic drug and the anti-platelet aggregation drug can have the same pharmaceutically acceptable carrier, dosage form, and administration mode as the above technical solution, and will not be described herein again.

[0032] As an embodiment of the present application, the anti-thrombotic drug and the anti-platelet aggregation drug can have the same administration mode and dosage form as the anti-thrombotic drug, and will not be described herein again.

[0033] The present application provides an application of a monomer compound in the total extract of phenolic compounds from dragon's blood in the preparation of a cerebral infarction protective drug, the monomer compound being dracorhodin D, having the structure shown in Formula V: Formula V.

[0034] As an embodiment of the present application, the cerebral infarction includes cerebral infarction caused by middle cerebral artery embolism.

[0035] As an embodiment of the present application, the cerebral infarction protective drug comprises an active ingredient and a pharmaceutically acceptable carrier, the active ingredient being the compound having the structure shown in Formula V (dracorhodin D), and the content of the active ingredient in the anti-platelet aggregation drug can be 0.1-99.9wt%, further 1-99wt%, more further 2-50wt%, and again further 2.36-20wt%.

[0036] As an embodiment of the present application, the anti-thrombotic drug and the anti-platelet aggregation drug can have the same pharmaceutically acceptable carrier, dosage form, and administration mode as the above technical solution, and will not be described herein again.

[0037] The present application provides an application of the total extract of phenolic compounds from dragon's blood or the ethyl acetate extract of phenolic compounds from dragon's blood in the preparation of an Evans' blue leakage improving drug, a blood-brain barrier protective drug, a cerebral water content reducing drug, or a cerebral hemorrhage reducing drug.

[0038] As an embodiment of the present application, the Evans' blue leakage medicine, the blood-brain barrier protecting medicine, the brain water content reducing medicine and the brain hemorrhage reducing medicine comprise an active ingredient and a pharmaceutically acceptable carrier, the content of the active ingredient in the Evans' blue leakage medicine, the blood-brain barrier protecting medicine, the brain water content reducing medicine and the brain hemorrhage reducing medicine can independently be 0.1-99.9wt%, further can be 1-99wt%, and more further can be 2-50wt%; the type, dosage form and administration mode of the pharmaceutically acceptable carrier in the Evans' blue leakage medicine, the blood-brain barrier protecting medicine, the brain water content reducing medicine and the brain hemorrhage reducing medicine can be consistent with the above technical solution, and will not be described here.

[0039] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present application.

[0040] Preparation Example 1 Four extracts with different polarities were separated from the phenolic extract of dragon's blood (DBE, 25g, Jiangsu Kangle Pharmaceutical Co., Ltd.), including the following steps: The phenolic extract of dragon's blood was separated by silica gel column chromatography, and the eluents used were petroleum ether, dichloromethane, ethyl acetate and methanol in turn, and four extracts with different polarities were obtained in turn, which were denoted as petroleum ether (PE) extract, dichloromethane (CH2Cl2) extract, ethyl acetate (EA) extract and methanol (MeOH) extract after removing the solvents.

[0041] Preparation Example 2 Twenty-five monomer compounds were separated from the EA extract obtained in Preparation Example 1, including the following steps: The EA extract described in Preparation Example 1 was separated by normal phase silica gel column chromatography (the eluents used were dichloromethane and ethyl acetate, gradient elution was used, and the volume ratio of dichloromethane to ethyl acetate was 95:5→0:1), and nine components were obtained in turn according to the TLC plate, which were denoted as components A-I after removing the solvents. The B component (1.0 g) was separated by Sephadex LH-20 column chromatography (eluent: MeOH and H2O, volume ratio 95:5) to obtain two components, which were denoted as B1 component and B2 component after removing the solvent, respectively; the B1 component was separated by semi-preparative liquid column chromatography (eluent: MeOH and H2O, volume ratio 69:31) to obtain compound 20 (10.0 mg, t R = 23 min), compound 21 (11.0 mg, t R = 27 min) and compound 24 (9.0 mg, t R = 21 min), respectively; the B2 component was separated by semi-preparative liquid column chromatography (eluent: MeOH and H2O, volume ratio 69:31) to obtain compound 12 (12.0 mg, t R = 32.1 min) and compound 19 (3.0 mg, t R = 32.7 min), respectively; The D component (4.0 g) was first separated by Sephadex LH-20 column chromatography (eluent: MeOH) to obtain two components (denoted as D1 component and D2 component after removing the solvent) and compound 25 (12.0 mg), respectively; the D1 component was separated by semi-preparative liquid column chromatography (eluent: ACN and H2O, volume ratio 25:75) to obtain compound 7 (3.0 mg, t R = 25 min) and compound 17 (10.0 mg, t R = 28 min), respectively; the D2 component was separated by 6 times of silica gel normal phase column chromatography (eluent: CH2Cl2 and EtOAc, gradient elution, volume ratio 96:4→80:20) to obtain 5 components according to the TLC plate, which were denoted as D2a~D2e components after removing the solvent, respectively; the D2a component was separated by semi-preparative liquid column chromatography (eluent: ACN and H2O, volume ratio 38:62) to obtain compound 14 (6.0 mg, t R = 45 min) and compound 15 (7.0 mg, t R = 47 min), respectively; the D2b component was separated by semi-preparative liquid column chromatography (eluent: ACN and H2O, volume ratio 38:62) to obtain compound 3 (3.0 mg, t R= 38 min); the D2c fraction was separated by semi-preparative liquid column chromatography (eluent ACN and H2O, volume ratio 38:62) to give compound 6 (6.0 mg, t R = 24 min); the D2d fraction was separated by semi-preparative liquid column chromatography (eluent ACN and H2O, volume ratio 38:62) to give a mixture of compound 8 (7.0 mg, t R = 24 min) and compound 9 (3.0 mg, t R = 24 min); the D2e fraction was separated by semi-preparative liquid column chromatography (eluent MeOH and H2O, volume ratio 54:46) to give compound 4 (4.0 mg, t R = 46 min), compound 10 (10.0 mg, t R = 30 min) and compound 11 (7.0 mg, t R = 26 min); The F fraction (1.0 g) was separated by semi-preparative liquid column chromatography (eluent ACN and H2O, volume ratio 28:72) to give compound 2 (32.0 mg, t R = 22 min), compound 16 (21.0 mg, t R = 27 min) and compound 18 (19.0 mg, t R = 36 min); The G fraction was separated by silica gel normal phase column chromatography (eluent CH2Cl2and MeOH, gradient elution, volume ratio 99:1→95:5) for three times, followed by Sephadex LH-20 column chromatography (eluent MeOH) to give three fractions, which were recorded as G1-G3 fractions after removal of the solvents; the G1 fraction was separated by semi-preparative liquid column chromatography (eluent ACN and H2O, volume ratio 25:75) to give compound 5 (6.0 mg, t R = 18 min); the G2 fraction was separated by semi-preparative liquid column chromatography (eluent ACN and H2O, volume ratio 30:70) to give compound 13 (7.0 mg, t R = 41 min); the G3 fraction was separated by silica gel column chromatography (eluent MeOH and H2O, volume ratio 95:5) to give compound 23 (40.0 mg); The I component was separated by Sephadex LH-20 column chromatography (the eluent was MeOH), to obtain two components, which were denoted as I1 component and I2 component after removing the solvent; the I1 component was recrystallized by dichloromethane to obtain compound 1 (80.0 mg), and the I2 component was recrystallized by ethyl acetate to obtain compound 22 (7.0 mg).

[0042] From the above, the EA extract was separated to obtain 25 monomer compounds, the specific structural formula and characterization results are as follows: .

[0043] The English names of the 25 monomer compounds are shown in Table 1: Table 1 English names of 25 monomer compounds

[0044] The specific structures of compound 1, compound 2, compound 7, compound 12, compound 17 and compound 24 are as follows: 1 The H NMR data is shown in Table 2, 13 The C NMR data is shown in Table 3.

[0045] Table 2 H NMR data of 6 monomer compounds 1 H NMR data

[0046] Note: The deuterated reagent of compound 1, compound 2, compound 7 and compound 17 is deuterated methanol, and the deuterated reagent of compound 12 and compound 24 is deuterated chloroform.

[0047] Table 3 C NMR data of 6 monomer compounds 13 C NMR data

[0048] Note: The deuterated reagent of compound 1, compound 2, compound 7 and compound 17 is deuterated methanol, and the deuterated reagent of compound 12 and compound 24 is deuterated chloroform.

[0049] Example 1 Anti-thrombus and anti-platelet aggregation efficacy evaluation of DB and DBE Carotid artery thrombosis model induced by FeCl3: Male SD rats weighing 260-280 g were used in the experiment. The left common carotid artery was wrapped with filter paper soaked in 40% FeCl3 solution for 5 min to induce carotid artery thrombosis. Aspirin was used as a positive drug. The experiment was divided into 5 groups: Sham, Model (CMC-Na), DB (800 mg / kg), DBE (300 mg / kg), and Aspirin (100 mg / kg). DB is dragon's blood, and DBE is the phenolic extract of dragon's blood used in Preparation Example 1. The samples were taken after 5 days of intragastrical administration. Experimental parameters: After anesthesia induction with 3% isoflurane, the right common carotid artery was exposed through a midline incision in the neck, and the operation was performed under a microscope. Sham group: The same surgical procedure was used, and the right common carotid artery was wrapped with filter paper soaked in normal saline for 5 min. The physiological state of the rats was monitored daily during the experiment. The blood flow in the ischemic core of the rat carotid artery was measured using a laser Doppler flowmeter. The blood flow difference was evaluated by comparing the blood flow before and 5 days after administration. Thrombus weight detection: The wet and dry weights of the thrombus were measured to evaluate the efficacy of different extracts.

[0050] Photochemical-induced middle cerebral artery thrombosis model: Male C57 mice weighing 20-25 g were anesthetized with anesthetic Xylazine. After positioning the animals, the skull was exposed through a midline skin incision. A cold light source (UltraNail, 100 mW, 540 nm) was placed above the right frontal cortex (relative to the anterior horn point, anterior-posterior: ±1.5 mm, medial-lateral: 0-2 mm). Five minutes before light exposure, rose Bengal (50 mg / kg, dissolved in 0.9% NaCl solution, Sigma) was injected through the tail vein. Subsequently, a 5-minute light exposure was performed through the intact skull area to establish a unilateral thrombotic model of the right hemisphere. The physiological state of the mice was monitored daily throughout the experiment. Clopidogrel (CLP) was used as a positive drug. The experiment was divided into 5 groups: Sham, Model (CMC-Na), DBE (300 mg / kg), DBE (600 mg / kg), and CLP (40 mg / kg). The dose was determined based on the content ratio of each component in DBE. Continuous intragastrical administration was performed for 3 days.

[0051] Anti-platelet aggregation experiment: The eyeballs of the mice in the photothrombotic model were removed, and the blood samples were placed in 2 mL EP tubes containing sodium citrate solution (concentration of 3.8%), with a volume ratio of 1:9 between the sodium citrate solution and the blood sample. The platelet-rich plasma (PRP) was obtained by first centrifugation (800 rpm, 10 min). The PRP was subjected to high-speed centrifugation (3000 rpm, 10 min) to separate and purify the platelets. The obtained platelets were counted, and the concentration of the platelets was calculated. The concentration of the platelets was adjusted to 4.8×10 8Platelet aggregation was evaluated using an AggRAM analyzer (Helena Laboratories, USA). After calibration of the instrument, 225 μL of platelet samples were added to 25 μL of agonists adenosine diphosphate (ADP, 3 μM), arachidonic acid (AA, 5 μM), thrombin (1 U) under static conditions (37 °C, 5 min) to stimulate the platelet aggregation, and the anti-platelet aggregation effect of DBE on different agonists was detected.

[0052] Figure 1 Figure for the screening results of DB anti-thrombus, DBE anti-thrombus and DBE anti-platelet aggregation activity; wherein A is the carotid blood flow; B is the carotid thrombus weight; C is the effect of different agonists on platelet aggregation rate (light plug model), from left to right corresponding to adenosine diphosphate, thrombin and arachidonic acid. The results show that DB and DBE can significantly increase the blood flow at the common carotid artery of FeCl3 injured rats and reduce the thrombus weight, and are comparable to the effect of the positive drug aspirin. In addition, in the light plug model, DBE can effectively inhibit ADP-induced platelet aggregation.

[0053] Example 2 Anti-thrombotic efficacy evaluation of different extracts in DBE The FeCl3 induced common carotid artery thrombosis model of Example 1 was established, and the experiment was set up with Sham, Model (CMC-Na), PE (0.96 mg / kg), CH2Cl2(21.6 mg / kg), EA (171 mg / kg), MeOH (102 mg / kg), and DBE (300 mg / kg) in total 7 groups, wherein DBE is the phenolic extract of dragon's blood used in Preparation Example 1, PE, CH2Cl2, EA and MeOH are different polarity extracts obtained after silica gel column chromatography separation of DBE in Preparation Example 1, i.e. PE extract, CH2Cl2 extract, EA extract and MeOH extract; the dosages are determined according to the content proportion of each extract in DBE. After 5 days of gavage, the blood flow and plug weight were detected.

[0054] Figure 2 Figure for the screening results of anti-thrombotic activity of different extracts in DBE, wherein A is the carotid blood flow, and B is the carotid thrombus weight. The results show that the EA extract and the MeOH extract can significantly increase the blood flow at the common carotid artery of FeCl3 injured rats and reduce the thrombus weight, and are comparable to the effect of DBE.

[0055] Example 3 Protective effect of EA extract and MeOH extract on light plug induced brain injury in mice The middle cerebral artery thrombosis model induced by photochemistry was established as in Example 1, and the experiment was set up with Sham, Model (CMC-Na), EA (342 mg / kg), MeOH (204 mg / kg), and DBE (600 mg / kg). The dosages were determined according to the content of each extract in DBE. The animals were administered intragastrically for 3 consecutive days.

[0056] Cerebral artery blood flow detection: The cerebral artery blood flow was monitored before and after the operation and 3 days after administration by a laser speckle blood flow instrument. An incision was made on the scalp to expose the skull, and a computer-controlled optical scanner was vertically placed 15 cm above the skull. After setting the parameters, the automatic focusing function was started to obtain high-resolution images, and the cerebral artery blood flow data at different time points were collected and analyzed using the Moor FLPI Review software.

[0057] mNSS neurological score: The neurological function damage of the mice with photothrombosis-induced brain injury was evaluated as follows: Motor function (total score 4 points): Normal movement (0 points): The animal can walk, run normally, and the limbs move coordinately without any abnormal gait or limb weakness; for example, the mouse or rat can move freely and quickly in an open field, and the limbs move smoothly without dragging limbs, shaking or falling, etc. Mild motor dysfunction (1 point): The animal shows slight gait abnormalities when walking, such as slight limb incoordination or slight instability when walking, but can still walk independently and maintain balance; there may be slight limb dragging, but it does not affect normal activity. Moderate motor dysfunction (2 points): The animal has difficulty walking, with obvious gait instability and poor limb coordination; there may be frequent limb dragging, and even occasional falls, but it can continue to walk with assistance (such as a light push). Severe motor dysfunction (3 points): The animal can hardly walk independently, with severely limited limb movement, and is mostly in a lying state, only weak limb movement can be induced by strong stimulation (such as pain stimulation). Limb paralysis (4 points): The animal completely loses limb movement ability and has no limb movement response to any stimulation.

[0058] Sensory function (total score 3 points): Normal sensation (0 points): the animal's response to stimuli such as touch, pain, and proprioception is normal; for example, when the animal's limbs or body is lightly touched with a cotton swab, the animal can quickly respond, such as avoiding, turning its head, etc.; a slight needle prick (using appropriate needle, controlling the depth and force of the needle prick) can produce normal pain response, such as limb withdrawal, squeaking, etc.; when placed on an inclined plane or raised limbs, the animal can maintain body balance through proprioception. Mild sensory impairment (1 point): the animal's response to touch, pain, or proprioception is slightly dull; for example, the response to light touch is delayed, the pain response to needle prick is weaker than normal, and the ability to maintain balance on an inclined plane is slightly worse, but the animal can still perceive the stimulus and respond to some extent. Moderate sensory impairment (2 points): the animal's response to sensory stimuli is significantly dull; stronger touch stimuli (such as pressing) are needed to elicit a response, pain response is significantly reduced, balance on an inclined plane is difficult to maintain, and proprioception is significantly impaired. Severe sensory impairment (3 points): the animal has almost no response to touch, pain, and proprioception stimuli, and may have only a weak response or no response to very strong stimuli.

[0059] Reflex function (total score 3 points): Normal reflex (0 points): including corneal reflex, ear reflex, righting reflex, etc. physiological reflex is normal; for example, when the cornea is lightly touched with a fine cotton thread, the animal will quickly blink; when the ear is slightly stimulated by sound or air flow, the animal will have ear twitching response; when the animal is inverted in the air, it can quickly turn over its body to restore normal posture. Mild reflex reduction (1 point): partial reflex reduction; for example, corneal reflex or ear reflex is slightly delayed, righting reflex is slightly slow, but the reflex action can still be completed. Moderate reflex reduction (2 points): reflex is significantly reduced; strong stimulation is needed to elicit reflex, and the reflex action is incomplete or significantly delayed; for example, the corneal reflex may only have slight eye movement, and the righting reflex may take a long time to complete.

[0060] Severe reflex reduction or disappearance (3 points): most reflexes are lost, and it is difficult to elicit reflex action even with strong stimulation.

[0061] Score range and significance: The total score of mNSS is 0-10 points, and the higher the score, the more severe the neurological dysfunction. 0-3 points: generally considered normal or only slightly impaired, the animal's daily life activities (such as eating, drinking, and self-activity) are basically not affected. 4-6 points: indicates moderate neurological dysfunction, the animal may have obvious motor, sensory or reflex abnormalities, and has some impact on normal life activities, such as reduced activity range, slightly difficult to eat, etc. 7-10 points: indicates severe neurological dysfunction, the animal's quality of life is severely decreased, and may need special care, such as unable to eat and drink independently, severely limited activity, etc.

[0062] Evan’s Blue leakage: The blood brain barrier (BBB) permeability of the mice with photothrombotic brain injury was determined by Evan’s Blue (EB) dye method. 90 min before the experiment, 2% EB dye (2 mL / kg) was injected through the tail vein. Before sacrifice, the mice were heart perfused with 0.9% saline to remove the residual dye in the blood vessels, and then the brain tissue was collected for imaging analysis. The brain tissue was frozen in a -80°C refrigerator, and after weighing, the brain tissue was homogenized with a 50% trichloroacetic acid solution at a ratio of 1:3 (weight mg / volume mm 3 ) and centrifuged at 12000 x g for 30 min. The supernatant was taken for EB colorimetric determination, and the absorbance was detected at 620 nm wavelength using a microplate reader.

[0063] Brain water content: After the mice with photothrombotic brain injury were sacrificed, the brain tissue was taken out, and the weight of the right cerebral hemisphere was recorded as the wet weight. Then the brain tissue was placed in a 60°C oven for drying for 3 days, and then weighed again and recorded as the dry weight. The brain water content percentage was calculated by the following formula: brain water content (%) = (wet weight - dry weight) / wet weight x 100%.

[0064] Brain hemorrhage evaluation: After the mice with photothrombotic brain injury were heart perfused with saline, the brain tissue was immediately taken out and stored at -80°C. The brain tissue was homogenized in a 2wt% ethylenediaminetetraacetic acid (EDTA) solution, and then centrifuged at 12000 rpm for 15 min (4°C) to separate. The hemoglobin content was determined by colorimetry using a kit, and the experimental operation strictly followed the requirements of the reagent instruction.

[0065] Figure 3 The pharmacodynamic comparison chart of EA extract and MeOH extract in the photothrombotic model, where A is the neurological score, B is the cerebral arterial blood flow, C is the Evan’s Blue leakage, D is the brain water content, and E is the hemoglobin content. The experimental results show that DBE, MeOH extract and EA extract can significantly reduce the neurological score of mice with cerebral thrombosis, increase the cerebral arterial blood flow after thrombosis. In addition, DBE and EA extract can significantly improve the Evans’ blue leakage and reduce the brain water content. MeOH extract can significantly increase the hemoglobin content and increase the risk of cerebral hemorrhage. Therefore, the effective components of EA extract are further separated and identified.

[0066] Example 4 Screening of anti-platelet aggregation effect of 25 monomer compounds A total of 25 monomer compounds were isolated from the EA extract, and the anti-platelet aggregation effect of the 25 monomer compounds was preliminarily screened in vitro at a concentration of 10 μM. The specific steps are as follows: Blood samples were collected from the abdominal aorta of SD rats into acidic citrate-glucose (ACD) anticoagulant tubes. Platelet-rich plasma (PRP) was obtained by centrifugation (800 rpm, 10 min). The PRP was then centrifuged at high speed (3000 rpm, 10 min) to separate and purify platelets. Platelet counts were performed to calculate the platelet concentration, which was then adjusted to 4.8 × 10⁻⁶. 8 Platelet aggregation was assessed using an AggRAM analyzer (Helena Laboratories, USA). After instrument calibration, 200 μL of platelet plasma at a concentration of 4.8 × 10⁻⁶ was collected. 8 Platelet samples of 10 μM / mL were incubated with 25 μL of the test compound solution and the positive control ticagrelor (Tigl) under static conditions (37℃, 5 min). Then, 25 μL of the agonist adenosine diphosphate (ADP) solution was added for stimulation, and its antiplatelet aggregation effect was detected.

[0067] Figure 4 The image shows the screening results of antiplatelet aggregation activity of 25 monomeric compounds isolated from EA extract (concentration 10 μM, n=3). The results show that a total of 6 compounds can significantly reduce platelet aggregation rate, namely compound 1, compound 2, compound 7, compound 12, compound 17 and compound 24, and their effects are comparable to those of the positive control ticagrelor.

[0068] Example 5: Antiplatelet IC50 of 6 monomeric compounds 50 Detection IC testing of 6 monomeric compounds 50 The value is determined by the following steps: Six monomeric compounds (compounds 1, 2, 7, 12, 17, and 24) were prepared into test compound solutions with different concentration gradients (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM). Blood samples were collected from the abdominal aorta of SD rats into acidic citrate-glucose (ACD) anticoagulant tubes. Platelet-rich plasma (PRP) was obtained by centrifugation (800 rpm, 10 min), and the PRP was further purified by high-speed centrifugation (3000 rpm, 10 min). Platelet counts were performed, and the platelet concentration was calculated and adjusted to 4.8 × 10⁻⁶. 8 Platelet aggregation was assessed using an AggRAM analyzer (Helena Laboratories, USA). After instrument calibration, 200 μL of platelet plasma at a concentration of 4.8 × 10⁻⁶ was collected. 8Platelet sample of 200 μL / mL was added with 25 μL of the compound to be tested and positive control product Ticagrelor (Tigl) under static conditions (37℃, 5 min), followed by the addition of 25 μL of 3 μM concentration of agonist adenosine diphosphate (ADP) solution for stimulation, and the anti-platelet aggregation effect was detected.

[0069] Figure 5 The anti-platelet half-inhibition concentration (IC 50 ) determination results of 6 monomer compounds are shown in the figure (n=4). The results show that the anti-platelet IC 50 of compound 1, compound 2, compound 7, compound 12, compound 17 and compound 24 are respectively: 4.31 μM, 9.75 μM, 8.04 μM, 7.96 μM, 4.21 μM, 8.93 μM.

[0070] Example 6 Brain protection effect of Loureirin D on cerebral infarction rats caused by middle cerebral artery embolism Permanent middle cerebral artery occlusion (pMCAO) of rats: male SD rats weighing 280-300 g were selected, and permanent occlusion of the middle cerebral artery of the rats was caused by operation to establish an animal model similar to the pathological state of ischemic stroke. Minocycline was used as a positive drug, and 5 groups of Sham, Model (CMC-Na), Loureirin D (12 mg / kg), Loureirin D (24 mg / kg), and Minocycline (5 mg / kg) were set. The samples were taken after 3 days of gavage administration. Experimental parameters: after 3% isoflurane induction, maintain the anesthesia state with 2.5% concentration, make a midline incision on the neck to expose the right common carotid artery, bluntly dissect the subcutaneous tissue and the right common carotid artery (CCA), bluntly dissect the muscles covering the CCA, dissect the vagus nerve accompanying the CCA, and dissect the CCA about 1-1.5 cm. At this time, the external carotid artery (ECA), the internal carotid artery (ICA) and the CCA are in a "Y shape", and two independent arteries (ECA and ICA) are separated. After the blood vessels are separated, the proximal end of the ECA and the distal end of the CCA are ligated with 4-0 silk, and the proximal end of the CCA is ligated. A 45° small opening is cut between the proximal and distal ends of the CCA ligation line with an ophthalmic scissors, and a wire plug is carefully inserted. Push the wire plug into the ICA and insert it into the MCA, stop pushing when a significant resistance is felt (about 1.6 cm black mark), and fix the wire plug and CCA ligation.

[0071] Bederson neurological score: suspension test posture abnormalities: 0 points: no abnormalities; 1 point: when lifting the tail, the rat's forelimbs are flexed (pointing to the chest), and the body is twisted; 2 points: ipsilateral (affected side) forelimbs are flexed (pointing to the chest), and the contralateral forelimbs are normal; 3 points: ipsilateral forelimbs are flexed (pointing to the chest), and the contralateral forelimbs are also flexed (pointing to the chest). Forelimb extension ability: 0 points: both forelimbs can be extended symmetrically and grasp the ground; 1 point: ipsilateral (affected side) forelimb extension is impaired (cannot be extended or weakly extended), and cannot grasp the ground. Walking ability (circling behavior): 0 points: can walk straight on open ground; 1 point: walks in a circle to the affected side (circling behavior). Total score calculation: add the scores of the above three items. 0 points: completely normal, no neurological deficits; 1-3 points: mild neurological deficits; 4-6 points: moderate to severe neurological deficits.

[0072] Rotarod fatigue test: Before the experiment, prepare four identical white boards at the bottom of the experimental instrument to set the rotation speed, with a range of 4-40 r / min. Place the experimental animals in the instrument for the experiment. Once the experimental animals fall, record the time and stop the data recording.

[0073] Open field test: The motor function of pMCAO rats was detected, and the average speed and total distance of movement were defined as the autonomous movement ability. The experimental process was as follows: the rats were placed in a quiet laboratory environment for a period of time to adapt to the environment, thereby reducing the interference of the external environment on the experimental results. During the formal test, the rats were placed gently in the center of the open field box (100x100x40cm) with their backs to the experimenter to avoid interfering with the behavior of the rats. The video monitoring system (Xinruan VisuTrack2.0) was started, and the movement trajectory, residence time, distance, and other behavioral indicators of the rats in the open field were recorded. The experimental time was 5 minutes. Each rat was tested once, and the equipment was wiped with 75% alcohol during the test to remove the odor and feces left by the previous rat. After the alcohol completely evaporated, the next rat was placed in the equipment. The total distance of movement and average speed of the rats were analyzed.

[0074] Evans blue leakage: Evan's Blue (EB) dye method was used to determine the permeability of the blood-brain barrier (BBB). 90 minutes before the experiment, 2% EB dye (2 mL / kg) was injected through the tail vein. Before sacrifice, the rats were perfused with 0.9% saline through the heart to remove residual dye in the blood vessels, and then the brain tissue was collected for imaging analysis. The brain tissue was frozen in a -80°C refrigerator, and after weighing, a 50% trichloroacetic acid solution was added at a ratio of 1:3 (weight mg / volume mm 3 ) to homogenize, and centrifuged at 12000xg for 30 minutes. The supernatant was taken for EB colorimetric determination, and the absorbance was detected at 620 nm wavelength using a microplate reader.

[0075] Brain tissue water content: After the rats were sacrificed, the brain tissue was taken out, and the weight of the left cerebral hemisphere was recorded as the wet weight. Then the brain tissue was placed in a 60℃ oven for drying for 3 days, and weighed again and recorded as the dry weight. The percentage of brain water content was calculated by the following formula: brain water content (%) = (wet weight-dry weight) / wet weight x 100%.

[0076] Figure 6 The blood-brain barrier pharmacodynamic comparison chart of Loureirin D in the pMCAO cerebral infarction model, wherein A is Evan's Blue leakage, B is Evan's Blue leakage quantification, C is brain water content, D is nerve score, E is open field test schematic diagram, F is open field total distance statistics, G is average speed statistics, and H is rotarod test statistics. The experimental results show that Loureirin D can significantly reduce the blood-brain barrier leakage and brain water content of pMCAO rats, has a blood-brain barrier protection effect, and is comparable to the positive drug minocycline. In addition, Loureirin D can reduce the nerve score after cerebral infarction, and according to the open field test and rotarod test, it is found that Loureirin D can significantly improve the motor dysfunction of cerebral infarction rats.

[0077] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of dragon's blood or a phenolic extract of dragon's blood in the preparation of an anti-thrombus drug, wherein the phenolic extract of dragon's blood comprises one or more of a total phenolic extract of dragon's blood, an ethyl acetate phenolic extract of dragon's blood, and a methanol phenolic extract of dragon's blood.

2. Use of a phenolic extract of dragon's blood or a monomeric compound in the preparation of an anti-thrombus drug or an anti-platelet aggregation drug, wherein the phenolic extract of dragon's blood comprises one or more of a total phenolic extract of dragon's blood, an ethyl acetate phenolic extract of dragon's blood, and a methanol phenolic extract of dragon's blood, and the monomeric compound is any one of the compounds having the structures of Formulae I to VI: Formula I; Formula II; Formula III; Formula IV; Formula V; Formula VI.

3. Use according to claim 1 or 2, characterized in that, the thrombus comprises an arterial thrombus.

4. Use according to claim 3, characterized in that, the arterial thrombus comprises a common carotid artery thrombus or a middle cerebral artery thrombus.

5. Use according to claim 4, characterized in that, the common carotid artery thrombus comprises a FeCl3-induced common carotid artery thrombus.

6. Use according to claim 4, characterized in that, the middle cerebral artery thrombus comprises a photochemical-induced middle cerebral artery thrombus.

7. Use according to claim 2, characterized in that, the platelet aggregation comprises an adenosine diphosphate-induced platelet aggregation.

8. Use of a monomeric compound in the preparation of a cerebral infarction protective drug, wherein the monomeric compound is loureirin D having the structure of Formula V: Formula V.

9. Use according to claim 8, characterized in that, the cerebral infarction comprises a cerebral infarction caused by middle cerebral artery embolism.

10. Use of a phenolic extract of dragon's blood or an ethyl acetate phenolic extract of dragon's blood in the preparation of an Evans' blue leakage improving drug, a blood-brain barrier protective drug, a cerebral water content reducing drug, or a cerebral hemorrhage reducing drug.

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