CN112755079A

CN112755079A - Medicine for preventing thrombosis or dissolving formed thrombus and preparation method thereof

Info

Publication number: CN112755079A
Application number: CN202110062225.XA
Authority: CN
Inventors: 李明; 梅丽萍
Original assignee: Hangzhou Kebei'an Biopharmaceutical Co ltd
Current assignee: Hangzhou Kebei'an Biopharmaceutical Co ltd
Priority date: 2021-01-18
Filing date: 2021-01-18
Publication date: 2021-05-07

Abstract

The invention discloses a medicine for preventing thrombosis or dissolving formed thrombus and a preparation method thereof, belonging to the field of medicines. The medicament comprises a combination of orthocyanobacteria active compounds, which is isolated from an orthocyanobacteria extraction, from which potentially hepatotoxic compounds are removed, and which comprises polyphenolic compounds and/or pentacyclic triterpenoids. The administration of the drug to a subject mammal can achieve the prevention of thrombosis in the blood of the mammal and the dissolution of the formed thrombus. The medicine can inhibit thrombosis in blood and dissolve thrombosis through synergistic effect with blood coagulation factor/fibrinolytic factor, so as to prevent and treat different diseases or disease states caused by thrombosis and thromboembolism in different tissues or organs.

Description

Medicine for preventing thrombosis or dissolving formed thrombus and preparation method thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a medicine for preventing thrombosis or dissolving formed thrombus and a preparation method thereof.

Background

Thromboembolic disorders include thrombosis and embolism, and can occur in any artery, capillary, or vein of the blood circulation. If any local blood clots in the blood circulation form a blood clot called thrombosis; the thrombus formed is removed from its original position and plugs the other part of the distal end along the blood flow.

Vascular endothelial injury, platelet activation and aggregation and other factors can lead to increased blood coagulability, even initiation of the coagulation process, and promotion of thrombosis. The decrease of fibrinolytic activity of human body, such as abnormal plasminogen-emia, can lead to decrease of fibrin-removing ability of human body, and is beneficial to thrombosis and expansion. The blood flow stasis and slowness of the whole body or the local blood flow caused by various reasons are important factors of the thrombosis, such as high-viscosity syndrome caused by high fibrinogen blood disease, hyperlipidemia, dehydration and erythrocytosis, circulatory disturbance and the like, such as high aggregative property of erythrocytes, and red thrombosis is easily caused. The damage to the vascular endothelium causes the adhesion and aggregation of platelets to the vascular endothelium, and fibrin deposition, which leads to white thrombosis, usually occurring in arteries.

Yet another clinically common thrombotic disorder is microcirculation thrombosis and microthrombosis in the blood. Thrombi occurring in the small veins, microcirculation and capillaries of the microcirculation can only be detected under microscope, so they are called microthrombus. For example, DIC activates the coagulation system and microvascular microthrombus is widely formed, resulting in ischemic organ dysfunction. Microvascular thrombosis can cause intravascular coagulation due to microcirculatory disturbance; small blood vessels can also be occluded by sloughed emboli or microvascular endothelial cells can be directly damaged by certain factors resulting in the deposition of platelets and fibrin. In coronary arteries, autopsy data indicate that the incidence of thrombosis in the blocked coronary arteries is 15% -95%. Microthrombosis are quite common in autopsy, and can reach 37.6%, and are commonly seen in lung, liver, kidney and brain.

Diseases or disease states associated with thrombotic and embolic diseases are one of the most harmful causes of human health, disability and mortality, such as myocardial infarction due to coronary thrombosis or embolism, cerebral thrombosis, cerebral embolism, deep vein thrombosis, pulmonary embolism, limb embolism, and the like. The clinical treatment method mainly comprises the prevention of thrombosis and the dissolution of the formed thrombus. The thrombolytic therapy plays an extremely important role in thrombosis and thromboembolism of important organs, and aims to dissolve thrombus of blood vessels of the important organs in vivo as soon as possible, promote blood vessel recanalization, recover blood supply of the important ischemic organs as soon as possible, and reduce disability rate and lethality rate of diseases.

However. The thrombolytic therapy has a certain window period, for example, the myocardial infarction thrombolysis is preferably performed within 3h, which is the best thrombolysis time. If 3h are passed, it is also possible to be within 6 h. Usually, the thrombolytic therapy is not suitable for thrombolysis after 12 hours, and the thrombolytic therapy is not suitable for thrombolysis after 24 hours. Bleeding complications of thrombolytic therapy, especially cerebral hemorrhage, are important reasons limiting thrombolytic therapy.

The key to the prevention of thrombosis or embolic diseases is to improve the hypercoagulable state of blood and to recanalize or reestablish blood flow paths to prevent ischemia or avascular necrosis of the relevant tissues or organs. The preventive measures mainly include anticoagulation therapy, thrombolytic therapy, antiplatelet therapy and the like. The prognosis of thrombotic or embolic disease depends largely on the size of the infarct size, the timely establishment of collateral circulation, and whether the treatment is timely.

Clinically, the anticoagulant therapy is mainly used for preventing and treating thrombosis or embolic diseases. The main principle of prevention and treatment is to prevent the conversion of fibrinogen into fibrin by using an anticoagulant, thereby preventing the final formation and development of thrombus. The conventional measures for preventing thrombosis basically employ heparin, low molecular heparin, aspirin, hirudin, etc., which are commonly used drugs for anticoagulant therapy, and they can reduce the viscosity of plasma and inhibit the thrombosis. Heparin has no thrombolytic effect and thus no therapeutic effect on the already formed thrombus.

In addition to anticoagulation therapy, dissolving already formed thrombi is also an important treatment. Thrombolytic agents are generally plasminogen activators that activate the fibrinolytic reaction in the blood to dissolve the thrombus that has formed. The thrombolytic agent mainly includes streptokinase, urokinase, tissue plasminogen activator (t-PA, serine protein kinase), staphylokinase, etc.

Statistics show that the number of people suffering from various thrombotic and embolic disorders is surprisingly high and shows a continuously rising trend with the aging society of the human society. While there are many of the above-mentioned treatment regimens for thrombotic disorders currently available, these platelet inhibitors, anticoagulation and fibrinolytic activation and thus thrombolytic treatment regimens have major side effects, especially the most significant complication of thrombolytic therapy is bleeding. Special precautions are taken against gastrointestinal tract, intracranial bleeding, etc. Therefore, it is imperative to develop new therapeutic strategies that are effective in preventing thrombosis and dissolving already formed thrombus without causing bleeding tendency side effects.

Disclosure of Invention

In order to solve at least one of the above technical problems, the present invention aims to provide a medicament for preventing thrombosis and/or dissolving a formed thrombus in blood of a mammal including a human and a method for preparing the same.

To this end, the invention provides in a first aspect a medicament for the prevention of thrombosis and/or the lysis of a thrombosis in a mammal, said medicament comprising a combination of orthocyanobacterial active compounds isolated from an orthocyanobacterial extraction, depleted of potentially hepatotoxic compounds, and comprising polyphenolic compounds and/or pentacyclic triterpenoids.

Optionally, the polyphenolic compound comprises one or more of Gemin a, ellagic acid and gallic acid. In some embodiments of the invention, the polyphenolic compounds include Gemin a, ellagic acid, and gallic acid.

Optionally, the pentacyclic triterpenoid comprises one or more of rubusoside F1, rubusoside F1 aglycone, potentilla discolor glycoside a and tormentic acid. In some embodiments of the invention, the pentacyclic triterpenoid includes picraspberrin F1, picraspberrin F1 aglycone, potentilla discolor a and tormentic acid.

Optionally, the medicament is an oral formulation. In use, the combination of compounds is administered orally to an individual to prevent thrombosis in the blood of a mammal or to dissolve an established thrombus.

Optionally, the medicament is an injectable formulation. Further, the medicament is a subcutaneous injection preparation, an intramuscular injection preparation or an intravenous injection preparation. In use, the combination of compounds for positive blue tooth activity is administered to an individual by subcutaneous injection, intramuscular injection or intravenous infusion to prevent thrombosis in the blood or to dissolve a thrombus already formed.

In some embodiments of the invention, the agent is administered in an amount of 0.01mg to 5g/kg of mammal body weight per day.

In the present invention, the prevention of thrombus formation in mammalian blood, the dissolution of a formed thrombus include thrombus formation in an artery, vein or capillary vessel of a tissue or organ, ischemia of a tissue or organ or interruption of blood supply caused by thrombus formation or embolism in blood.

In the present invention, the thrombus is a venous thrombus, an arterial thrombus or a microthrombus.

In the present invention, the mammal includes, but is not limited to, a human, preferably, a human.

In a second aspect, the present invention provides a process for the preparation of a medicament according to the first aspect of the present invention for the prevention of thrombosis and/or for the lysis of a thrombosis in a mammal, comprising the steps of:

s1, cleaning the whole herb of bluecloth, drying in the shade, and cutting into pieces;

s2, refluxing and extracting the fragments for 6-8 hours by using 6-8 times of petroleum ether or chloroform;

s3, extracting the plant material extracted by petroleum ether or chloroform with 10 times of 60% ethanol under reflux, concentrating the ethanol extractive solution, and spray drying to obtain solid powder;

s4, dissolving and suspending the ethanol extract in water, and extracting with ethyl acetate;

s5, concentrating the ethanol extract extracted by ethyl acetate, standing at low temperature for 24 hours, filtering, concentrating, and drying under reduced pressure to obtain the blue-bluecloth positive active compound combination.

In step S2, the ineffective and hepatotoxic compounds mainly containing fat-soluble components can be removed by extraction with petroleum ether or chloroform.

In step S4, ethyl acetate is used to remove chlorophyll and less polar compounds, and also relatively fat soluble hepatotoxic compounds.

In some embodiments of the invention, in step S5, the low temperature placement refers to a 4 ℃ placement.

Further, the preparation method comprises the step of separating the monomeric polyphenol compounds and the pentacyclic triterpenoid compounds by a reverse column chromatography method.

In a third aspect, the present invention provides the use of a combination of orthocyanobacteria active compounds isolated from orthocyanobacteria extraction, which has been depleted of potentially hepatotoxic compounds and comprises polyphenolic compounds and/or pentacyclic triterpenoids, for the preparation of a medicament for the prevention of thrombosis and/or the lysis of a thrombosis in a mammal.

In the present invention, the drug is an oral preparation or an injection preparation. Further, the medicament is a subcutaneous injection preparation, an intramuscular injection preparation or an intravenous injection preparation.

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial technical effects:

the medicine of the present invention includes active compound composition extracted and separated from blue cloth as Chinese medicine. The administration of the drug to a subject mammal can achieve the prevention of thrombosis in the blood of the mammal and the dissolution of the formed thrombus. The medicine can inhibit thrombosis in blood and dissolve thrombosis through synergistic effect with blood coagulation factor/fibrinolytic factor, so as to prevent and treat different diseases or disease states caused by thrombosis and thromboembolism in different tissues or organs.

Drawings

Figure 1 shows the in vitro lysis activity (serum free) of a whole blood clot with the compound combination.

Figure 2 shows the activity of the compound combinations to lyse whole blood thrombi in vitro (with serum).

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

EXAMPLE 1 extraction and isolation of Compound combinations

A process for preparing a medicament for preventing thrombosis in the blood of a mammal and for dissolving thrombosis formed in the blood, comprising the steps of:

1. cleaning whole herb of Bluecloth Zhengcao collected from autonomous level of 1000-1400 m of Bluey of Guizhou province in 9 months in 2020, drying in shade, and cutting into pieces.

2. Extracting dried piece (2 kg) of herba Bluellae Doederlensis with 8 times of petroleum ether under reflux for 8h, recovering petroleum ether, and discarding extract mainly containing liposoluble extract which has no thrombosis preventing activity and no thrombosis dissolving activity in blood and hepatotoxic compounds;

3. extracting the above blue cloth raw material with 60% ethanol 10 times the concentration for 8 hr under reflux, concentrating the ethanol extractive solution, and spray drying to obtain solid powder;

4. dissolving the ethanol extract in 10 times of water, extracting with ethyl acetate to remove chlorophyll and less polar compounds, recovering ethyl acetate, and discarding the extract containing relatively liposoluble chlorophyll and residual hepatotoxic compounds;

5. extracting the above with ethyl acetate, concentrating the ethanol extractive solution to remove hepatotoxic compounds, standing at low temperature (4 deg.C) for 24 hr, filtering, concentrating, and drying under reduced pressure to obtain the blue-cloth positive active compound composition.

6. The obtained blue-bunsen compound combination can be further separated into monomer polyphenol compounds and pentacyclic triterpenoid compounds by a reverse column chromatography method. Through mass spectrum and nuclear magnetic resonance analysis, the main components are polyphenol compounds: such as Gemin a, Ellagic acid (Ellagic acid), Gallic acid (galic acid), and pentacyclic triterpenoids: such as picrorhiza glycoside F1(niga-ichigoside F1) and its aglycone, potentioglifloside A, Tormentic acid (Tormentic acid), etc.

The following examples all use this combination of compounds as experimental agents.

EXAMPLE 2 Effect of Compound combinations on Clotting Time (CT) in mice

Male experimental mice with the basic physiological blood coagulation time of 40-160S are selected for experiments, and are grouped based on the uniform distribution of the blood coagulation time. Randomly dividing into;

group 1 (n-15) normal group, 0.5% sodium carboxymethylcellulose (CMC-Na) solution;

2 (n-15) positive control group, 20mg/kg aspirin;

and 3 groups, administration groups respectively give active combinations of 200, 400 and 600mg/kg body weight. Each group was orally administered 1 time a day. The number of animals in each of the three dose groups of the administration group was 15.

The three groups of animals are continuously treated by oral administration for 3 days, after 1 hour of the last treatment, a glass capillary with the inner diameter of 1mm is inserted into the inner canthus of the mouse to take blood from the venous plexus, the timing is started from the blood inflow tube, the capillary is taken out after the blood is filled, the capillary is placed in a clean and disinfected dish, the capillary at two ends is broken at intervals of 30S, the broken capillary is slowly pulled left and right, whether the blood coagulation silks appear at the broken part is observed, the timing is stopped until the blood coagulation silks appear at the broken part, and the obtained time is CT.

The results show that the blood coagulation time of the experimental animals in the normal group is not obviously changed, and the blood coagulation time of the experimental animals in the high, medium and low dose groups combined by the positive drug and the compound is obviously prolonged. The clotting time of the low, medium and high dose groups of the compound combination was significantly prolonged compared to the normal group (P <0.05, 0.01), suggesting that the compound combination may affect the clotting time of mice, and the results are shown in table 1.

TABLE 1 Effect of Activity combinations on Clotting Time (CT) in mice

Group of	Dosage to be administered	Coagulation time/s before administration	Coagulation time/s after administration
				Normal control group	0.5％CMC—Na	86.12±35.43	99.45±33.13
Positive control group	20mg/kg Aspirin	87.25±38.65	136.23±38.53*++
				Administration set (Low)	200mg/kg of active combination	88.00±32.12	139.25±39.76*++
Administration set (middle)	400mg/kg active combination	86.56±36.32	158.35±41.23**++
				Administration set (high)	600mg/kg active combination	88.08±37.78	162.78±43.63**++

Comparison with the normal group: p <0.05, P <0.01

Comparison with this group before dosing: + P <0.01

EXAMPLE 3 Effect of Compound combinations on bleeding time in Experimental animals

Kunming male mice, randomly divided into 3 groups.

Group 1 (n-8) normal group, 0.5% sodium carboxymethylcellulose (CMC-Na) solution;

2 (n-8) positive control group, 20mg/kg aspirin;

and 3 groups, administration groups respectively give 200, 400 and 600mg/kg of active combination. Each group was orally administered 1 time a day. The number of animals in the three dose groups of the administration group was 8.

Each group of experimental animals is orally administered 1 time a day for 3 days, and the mice are taken 1h after the last administration and placed in a fixer, the tail part of the mice is vertical, the mice are cut off at a position 1.5mm away from the tail tip, and the blood is sucked by filter paper until the mice can not bleed. And recording the time from the broken tail tip to the blood sucking failure as the tail bleeding time. .

The results showed that the tail bleeding time was significantly longer in both aspirin and active combination dose groups than in the normal group (P <0.01, 0.05, 0.01) (table 2).

TABLE 2 Effect of the combination of activities on bleeding time in laboratory animals

Group of	Dosage to be administered	Time/s of tail bleeding
			Normal control group	0.5％CMC—Na	196.22±65.54
Positive control group	20mg/kg Aspirin	363.15±103.16**
			Administration set (Low)	200mg/kg of active combination	279.00±92.34*
Administration set (middle)	400mg/kg active combination	381.65±113.37**
			Administration set (high)	600mg/kg active combination	378.15±121.37**

Comparison with the normal group: p <0.05, P <0.01

EXAMPLE 4 Effect of Compound combinations on thrombosis in vitro in Experimental animals

SD rats (250 g) were randomly assigned to 3 groups.

2 (n-8) positive control group, 10mg/kg aspirin;

the administration groups were given 200, 400, and 600mg/kg of the active combination, respectively, and the number of animals in each of the three dose groups was 8. Each group was orally administered 1 time a day for 5 days. After the last administration for 1h, the siliconized plastic tube is taken out and rotated into a ring (the diameter is 4mm), 1mL of blood is taken out from the orbit of the rat, the blood is rapidly injected into the thrombus ring, the plastic tube is connected into a ring, the ring is rapidly placed into a groove of a metal turntable of the thrombus instrument, and the timing is immediately carried out. Starting the turntable, rotating for 15min at 37 ℃ and rotating at the speed of 16 r/min. After completion, the rotation was stopped, the thrombus was taken out, the blood was blotted with filter paper, and the wet weight of the thrombus was weighed. The thrombus was placed at 60 ℃ and baked for 24 hours, and then the dry weight of the thrombus was weighed.

The results showed that both wet and dry thrombus mass were significantly lighter (P <0.05, 0.01) in the aspirin group and the compound combination in each dose group compared to the normal group of rats (table 3).

TABLE 3 Effect of Compound combinations on thrombosis in vitro in laboratory animals

Comparison with the normal group: p <0.05, P <0.01

EXAMPLE 5 Effect of Compound combinations on rat inferior vena cava thrombosis

SD rats (250 g) were randomly assigned to 3 groups.

2 (n-8) positive control group, 10mg/kg aspirin;

the administration groups were given 200, 400, and 600mg/kg of the active combination, respectively, and the number of animals in each of the three dose groups was 8. Each group was orally administered 1 time a day for 5 days. Venous thrombosis model is made after 1h of last administration. Rats were anesthetized with 3% sodium pentobarbital 0.1ml/100g intraperitoneal injection. After the anesthesia is effective, the abdominal cavity is opened along the midline of the abdomen of the rat, the inferior vena cava is separated, the inferior vena cava is ligated at the level below the left renal vein, and the abdominal wall is sutured. After 3h, the abdominal cavity was reopened and the vessel was occluded 2cm below the original ligation site. The lumen of the inferior vena cava between the two ligations was then cut longitudinally, and the intravascular blood was removed with a filter paper strip and observed for the presence of thrombosis. If a thrombus had formed, the thrombus was carefully removed and immediately weighed on a scale as the wet mass of the thrombus. Then dried at 60 ℃ for 24h and weighed as the dry mass of thrombus. The thrombus inhibition rate was then calculated.

The thrombus inhibition rate is (thrombus quality of normal group, thrombus quality of administered group)/Zhengzheng

Quality of thrombus in common group is multiplied by 100

The results show that the active combination can significantly inhibit the thrombosis of the inferior vena cava at high, medium and low doses (P <0.01, 0.01) (Table 4).

TABLE 4 Effect of the combination of activities on the thrombosis of the inferior vena cava in rats

Comparison with the normal group: p <0.01

EXAMPLE 6 Activity of Compound combinations to lyse thrombi in whole blood in vitro

Taking venous blood of healthy people in a polyvinyl chloride tube (diameter of 0.34cm), placing the tube in an inclined position, allowing the tube to coagulate at room temperature, pushing out the blood clot with a syringe after 60 minutes, washing the blood clot with 10ml of 0.9% sodium chloride normal saline to remove residual serum, and cutting the blood clot into 0.15cm³The size 24 blocks are reserved. The spare blood clots were dispensed into 24 tubes. The method comprises the following groups:

1. for control (n ═ 6), three test tubes were each charged with 5ml of 0.9% sodium chloride physiological saline;

2. high dose (n ═ 6) three tubes were each filled with 5ml of 0.9% sodium chloride in physiological saline containing the active composition (100 mg);

3. medium dose (n ═ 6), three tubes were each charged with 5ml of 0.9% sodium chloride physiological saline containing the active composition (50 mg);

4. at low dose (n ═ 6), three tubes were each charged with 5ml of 0.9% sodium chloride saline containing the active composition (25 mg).

All the samples were placed in a 37 ℃ water bath. After 24 hours of incubation, the thrombus in each tube was removed, the weight thereof was weighed, and the thrombolysis rate was calculated for each group for different treatments:

[ (starting Wet weight average of thrombus-Wet weight average of thrombus after 24h incubation for each treatment group)/starting Wet weight average of thrombus ]. times.100%

Then, the thrombi were replaced into the respective tubes to which 1ml of human serum was added, and then the tubes were placed in a 37 ℃ water bath and incubated for 24 hours. The thrombus (if any) in each tube was then removed, weighed, and then divided by the initial wet weight of thrombus in each different tube prior to addition of serum.

The experimental results show that no thrombolysis was found after 24h incubation under serum-free conditions, no significant difference in thrombus weight was observed between groups (1-4) regardless of whether the thrombus was treated with high, medium or low dose combination of compounds (fig. 1). It is worth exciting that after adding 1ml serum in vitro, the high, medium or low dose of the active combination treatment of the thrombocytes, after 24h incubation, a significant thrombolysis effect was found (P <0.01, 0.01) compared to the control saline group (fig. 2). Wherein the thrombolysis rate of the high dose reaches 80%, the thrombolysis rate of the medium dose reaches 87%, and the thrombolysis rate of the low dose reaches 58%. These results suggest that the compound combination itself does not have a complete thrombolytic effect, since the compound combination was not found to have any significant thrombolytic effect before the serum was added to the test tube, whereas the compound combination exhibited a dose-dependent thrombolytic effect after the serum was added.

EXAMPLE 7 Effect of the active combination on the lysis of Microthromboses in human blood

Four patients found massive microthrombosis (clear thrombi) in peripheral blood smears at the cardiovascular department of the first subsidiary hospital at the university of Hebei medical sciences. The examination also revealed that the blood cells in the blood of these patients also agglutinated and adhered to each other in a cluster, string, or dendritic form. However, after one week of oral treatment of patients with the combination of compounds (1g each, 2 times a day), the massive amount of microthrombosis that had been found before treatment in peripheral blood smears of all four patients disappeared and the severely agglutinated blood cells also significantly improved. After two weeks of treatment, peripheral blood smears of all four patients did not find any microthrombus, the agglutination state of blood cells was completely restored to normal, and the morphology of blood cells was also restored to normal and well separated from each other.

EXAMPLE 8 Effect of Compound combinations on dissolving arterial and venous thrombi

A male patient (65 years old) admitted to the first subsidiary hospital of Hebei medical university suffered from acute myocardial infarction of the anterior wall of the heart 2 months before visiting the clinic, and the B-mode echocardiography of the heart revealed a thrombus with a diameter of about 1.5cm inside the transmural infarct site of the anterior wall of the left ventricle.

The patient took the combination orally (1 g/dose, 2 times a day) before meals. After the compound combination is continuously taken for one week, the echocardiogram is used for rechecking the heart to find that the volume of the thrombus is obviously reduced, and after the compound combination is continuously taken for one week, the echocardiogram is used for rechecking the heart to find that the volume of the thrombus is further obviously reduced. After one month of continuous compound combination, the heart was examined by echocardiography to find complete disappearance of the thrombus inside the left ventricular anterior transmural infarct site.

The deep vein thrombosis model of the rat is established by adopting an inferior vena cava ligation method. The experiment was divided into three groups: treatment group (n ═ 6, 400mg compound combination/kg body weight, gavage in dissolved water, once a day); control group (n-6, equal amount of saline gavage, once daily). Mice were sacrificed on the seventh day after molding, the inferior vena cava thrombus formation section separated the vein wall from the thrombus, and the thrombus was weighed. Peripheral venous blood was drawn from each mouse before sacrifice and used for four examinations of clotting.

The results show that on day seven, the weight of the rat deep vein thrombus with the combination of gavage compounds (0.008 + -0.002 g vs 0.065 + -0.017 g) was significantly lower (P <0.01) than the average thrombus weight in the control group. However, the compound combination gavage for seven consecutive days did not have a statistically different effect on the thrombin time, prothrombin time and activated partial thrombin time in the experimental animals.

Thus, the compound combination not only can remarkably enhance the dissolution of deep vein thrombosis of rats, but also can not increase the bleeding tendency. This thrombolytic property of the combination of compounds may require co-action with active factors in the fibrovascular system of the blood, since the combination of compounds loses its thrombolytic effect upon in vitro serum removal.

Patients had a single deep vein thrombosis in the right lower limb (2 cases) for men, a single deep vein thrombosis in the left lower limb and the right lower limb (2 cases) for women, and the patients had swollen, painful and locally painful muscles, with the course of the disease being longer than one month. The ultrasonic examination of the blood vessel B shows that the deep vein lumen is filled with low echo, so that the lumen is completely or incompletely occluded. 4 patients were given the oral combination of compounds (1.5 g/time, 2 times per day) for one month. After the medicine is taken for about 2 weeks, the patient is rechecked to find that the swelling and pain symptoms of the affected limb of all four cases are obviously reduced or completely disappear, and the local muscle tenderness is also obviously reduced. After taking the medicine for four weeks, the symptoms of swelling, pain and local muscle tenderness of the affected limb disappear completely. Doppler ultrasound examination shows that the venous thrombus image disappears, the blood flow is smooth, and the mural thrombus symptom is not found.

In conclusion, the invention provides a medicament for preventing thrombosis in blood of mammals or dissolving formed venous thrombosis, arterial thrombosis and microthrombosis and a preparation method thereof. Wherein the agent for preventing thrombosis and dissolving thrombosis comprises a combination of compounds extracted and separated from blue cloth; the compound combination mainly comprises polyphenol compounds: such as Gemin a, Ellagic acid (Ellagic acid), Gallic acid (galic acid), and pentacyclic triterpenoids: such as picrorhiza glycoside F1(niga-ichigoside F1) and its aglycone, potentioglifloside A, Tormentic acid (Tormentic acid), or at least one of them. The medicine of the invention comprises a compound combination extracted and separated from blue cloth, and the medicine is administered to human bodies, so that the prevention of individual thrombosis can be realized, or even the formed thrombosis can be dissolved. The compound combination mainly inhibits the formation of thrombus in blood and dissolves the formed thrombus through the synergistic action with blood coagulation factors/fibrinolytic factors in blood, thereby achieving the purposes of preventing and treating different diseases or disease states caused by the thrombus formation and the thromboembolism in different tissues or organs.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A medicament for preventing thrombosis and/or thrombolysis in a mammal, said medicament comprising a combination of orthocoestive active compounds isolated from orthocoestive extract, depleted of potentially hepatotoxic compounds, and comprising polyphenolic compounds and/or pentacyclic triterpenoids.

2. The medicament of claim 1, wherein the polyphenolic compound comprises one or more of Gemin A, ellagic acid, and gallic acid.

3. The medicament of claim 1, wherein the pentacyclic triterpenoid comprises one or more of rubusoside F1, rubusoside F1 aglycone, potentilla discolor glycoside A and tormentic acid.

4. The medicament according to any one of claims 1 to 3, wherein the medicament is an oral preparation or an injectable preparation.

5. The medicament of claim 4, wherein the medicament is a subcutaneous, intramuscular, or intravenous formulation.

6. A medicament as claimed in any one of claims 1 to 3, wherein the thrombus is a venous thrombus, an arterial thrombus or a microthrombus.

7. The medicament of any one of claims 1 to 3, wherein the mammal is a human.

8. A process for preparing a medicament for preventing thrombosis and/or dissolving thrombosis in a mammal according to claim 1, comprising the steps of:

s2, refluxing and extracting the fragments for 6 hours by using 6 times of petroleum ether or trichloromethane;

s5, concentrating the ethanol extract which is extracted by ethyl acetate and is used for removing hepatotoxic compounds, standing at low temperature for 24 hours, filtering, concentrating, and drying under reduced pressure to obtain the blue-cloth positive active compound combination.

9. The method of claim 8, further comprising the step of separating the monomeric polyphenolic compound and the pentacyclic triterpenoid compound by reverse phase column chromatography.

10. Use of a combination of bunkan active compounds for the preparation of a medicament for preventing thrombosis and/or dissolving a thrombosis in a mammal, wherein the combination of bunkan active compounds is isolated from a bunkan extract, removes potentially hepatotoxic compounds, and comprises polyphenolic compounds and/or pentacyclic triterpenoids.