CN102813687A - Use of ligustilide in preparing anti-thrombotic medicines - Google Patents

Abstract

The present invention relates to the use of ligustilide in the preparation of antithrombotic drugs. Specifically, the present invention relates to a Chuanxiong extract, which contains 30-90% ligustilide. The extract is prepared by the following method: crush the Rhizoma Chuanxiong medicinal material, soak it with 70%~99% ethanol (for example, 75%~98%) and extract it under reflux for 1-4 times, combine the extracts, concentrate, and use petroleum ether Extract, recover the solvent from the combined extract to dryness, and obtain petroleum ether extraction extract; take petroleum ether extraction part of the extract, add normal phase silica gel to mix the sample, and then pass through a normal phase silica gel column, with a volume ratio of 50:0.5~5( For example, 50:0.5~3) petroleum ether and ethyl acetate solution are used as eluent for elution, the eluate is collected, concentrated, and obtained.

Description

Use of Ligustilide in the Preparation of Antithrombotic Drugs

technical field

The invention belongs to the field of traditional Chinese medicine chemistry, and specifically relates to an effective component extracted from Rhizoma Chuanxiong and its preparation method, as well as the use of the component in the preparation of antithrombotic drugs.

Background technique

Thrombosis refers to the process in which blood coagulates or certain formed components in blood adhere to each other to form solid masses in the heart and blood vessel cavity. Under pathological conditions, the vascular lumen obstruction and other effects caused by thrombus will cause serious or even fatal harm to the body, and become an important pathogenic factor of cardiovascular and cerebrovascular diseases such as myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism.

Vascular intimal injury is one of the important causes of thrombus formation. Vascular endothelial cells are a single-cell membrane that separates subendothelial connective tissue from coagulation factors and platelets in blood, and have the functions of anticoagulation and anti-platelet adhesion. However, when the endothelial cells are damaged, the basement membrane collagen, fibrillar collagen, and elastin in the subendothelial connective tissue all have a strong procoagulant effect, leading to platelet aggregation to form a thrombus. Central to triggering the coagulation process is the activation of platelets. Substances that can activate platelets include collagen, thrombin, ADP, etc. After endothelial injury, the first thing that activates platelets is the collagen in contact with the platelets, and then the coagulation chain reaction is initiated to generate thrombin, and the platelets continue to be activated ADP and thromboxane A2 are continuously released, and platelets are continuously activated locally along with the blood flow. Therefore, blocking platelet activation and preventing endothelial damage are effective ways to prevent and treat thrombotic diseases.

Antithrombotic therapy has always been an effective means for the prevention and treatment of stroke, myocardial infarction caused by coronary artery thrombosis, thromboangiitis obliterans and other diseases. Commonly used antithrombotic drugs mainly include three categories, namely anticoagulants, antiplatelet drugs and thrombolytic drugs, represented by heparin, aspirin and urokinase respectively. Modern pharmacological studies have shown that some traditional Chinese medicines for promoting blood circulation and removing blood stasis, such as Xuefu Zhuyu Decoction, can improve hemorheological properties, reduce the adhesion and aggregation of platelets, reduce the surface activity of platelets, and at the same time protect damaged endothelial cells by inhibiting platelet aggregation. and other ways to prevent and treat thrombotic diseases. Therefore, finding effective components with anti-platelet aggregation and endothelial protection activities from traditional Chinese medicine has become a new source for the development of antithrombotic drugs.

Chuanxiong is the dry rhizome of Umbelliferae Ligusticum chuanxiong Hort. Its nature and flavor are pungent and warm. Return liver, gallbladder, pericardium channel. It has a wide range of functions of promoting blood circulation and removing blood stasis, suitable for stagnation of various diseases; dispelling wind and relieving pain, has very good effects, and can cure head wind, headache, rheumatic arthralgia and other diseases. Modern pharmacological studies have shown that Chuanxiong extract has a dose-dependent inhibitory effect on isolated guinea pig hearts, and has a significant effect on increasing coronary artery flow in isolated rat or guinea pig hearts. Its main active ingredient, ligustrazine, can expand the pulmonary blood vessels of rats. , inhibits the hypoxic pulmonary vasoconstrictor response and right ventricular hypertrophy. In addition, Ligusticum Chuanxiong also has the inhibitory effect on the central nervous system, which has an inhibitory effect on the activity of the animal brain, and has an excitatory effect on the medullary respiratory center, vasomotor center and spinal cord reflex center. Ligustrazine, the main compound in Chuanxiong, can inhibit the synthesis of thromboxane A2 (TXA ₂ ), and its effect is similar to that of TXA ₂ synthetase inhibitors. However, most of the current research focuses on ligustrazine and ferulic acid, and it is not clear whether ligustrazine has antithrombotic activity and its mechanism of action.

Contents of the invention

The object of the present invention is to provide an effective component of Rhizoma Chuanxiong, which mainly contains ligustilide with a content of 30-90%.

Another object of the present invention is to provide the preparation method of the effective components of Rhizoma Chuanxiong, which is realized by the following steps: add 20%~90% ethanol to soak for 5~12 hours after the Rhizoma Chuanxiong medicinal material is pulverized, heat and reflux for 0.8~1.2 hours, extract 1 to 3 times, combine the filtrates to obtain the extract, extract the extract with petroleum ether, concentrate the extract and purify it with a normal-phase silica gel column, using petroleum ether and ethyl acetate with a volume ratio of 50:1 as the washing Remove the agent, collect the eluate and concentrate to obtain the effective components.

Another object of the present invention is to provide the application of the effective components of Chuanxiong Rhizoma in the preparation of antithrombotic drugs.

The first aspect of the present invention provides a Chuanxiong extract, which contains 30-90% ligustilide.

The Chuanxiong extract according to the first aspect of the present invention contains 80-97% (eg 85%-95%) ligustilide.

The Rhizoma Chuanxiong extract according to the first aspect of the present invention is prepared by the following method: after pulverizing the medicinal material of Rhizoma Chuanxiong, soak it with 70%~99% ethanol (such as 75%~98%) and reflux extract it for 1-4 times, Combine the extracts, concentrate, extract with petroleum ether, recycle the combined extracts to dryness, and obtain petroleum ether extracts; take part of the extracts extracted with petroleum ether, add normal phase silica gel to mix the sample, and pass through a normal phase silica gel column to obtain Petroleum ether and ethyl acetate solution with a volume ratio of 50:0.5~5 (for example, 50:0.5~3) are used as the eluent for elution, the eluate is collected, concentrated, and obtained.

The Rhizoma Chuanxiong extract according to the first aspect of the present invention is prepared by the following method: crush the Rhizoma Chuanxiong medicinal material, add 20%~90% ethanol to soak for 5~12 hours, heat and reflux for 0.8~1.2 hours, extract for 1~ 3 times, the filtrate was combined to obtain the extract, the extract was extracted with petroleum ether, and the extract was concentrated and then purified with a normal phase silica gel column, using petroleum ether and ethyl acetate at a volume ratio of 50:1 as the eluent , the eluate was collected and concentrated to obtain the extract.

The Rhizoma Chuanxiong extract according to the first aspect of the present invention is prepared by the following method: After pulverizing 1000g of Rhizoma Chuanxiong medicinal material, add 4000ml of 95% ethanol to soak overnight, reflux extraction for 2 hours, then add 4000ml and heat reflux with 95% ethanol to extract 3 times, 2 hours each time, combine the extracts and concentrate to a density of 1.08; add the concentrate to 500ml water to disperse into a suspension, add 500ml petroleum ether to extract 3 times, combine the extracts, and recover the solvent to dryness. Take petroleum ether to extract part of the extract, add normal phase silica gel to mix the sample, pass through normal phase silica gel column, use petroleum ether and ethyl acetate solution with a volume ratio of 50:1 as the eluent to elute, collect the eluate, and pass through the normal phase silica gel column. Available after concentration.

The second aspect of the present invention provides a method for preparing the extract of Rhizoma Chuanxiong, which includes the following steps: after crushing the medicinal material of Rhizoma Chuanxiong, soak it with 70%~99% ethanol (for example, 75%~98%) and extract it by refluxing 1-4 times, combine The extract is concentrated, extracted with petroleum ether, and the combined extract is recovered to dryness to obtain a petroleum ether extract; take a part of the extract extracted with petroleum ether, add normal phase silica gel to mix the sample, and then pass it through a normal phase silica gel column. Petroleum ether and ethyl acetate solution with a ratio of 50:0.5~5 (for example, 50:0.5~3) are used as the eluent for elution, the eluate is collected, concentrated, and obtained.

According to the method of the second aspect of the present invention, it comprises the following steps: adding 20% to 90% ethanol to soak for 5 to 12 hours after crushing the Rhizoma Chuanxiong medicinal material, heating and refluxing for 0.8 to 1.2 hours, extracting for 1 to 3 times, and combining the filtrates to obtain Extract, extract the extract with petroleum ether, concentrate the extract and purify it with a normal phase silica gel column, use petroleum ether and ethyl acetate at a volume ratio of 50:1 as the eluent, collect the eluate and concentrate After obtaining the extract.

According to the method of the second aspect of the present invention, it comprises the following steps: After pulverizing 1000g of Rhizoma Chuanxiong medicinal material, add 4000ml 95% ethanol to soak overnight, reflux extraction for 2 hours, then add 4000ml and heat reflux extraction with 95% ethanol for 3 times, each time 2h , combine the extracts, concentrate to a density of 1.08; add the concentrate to 500ml of water to disperse into a suspension, add 500ml of petroleum ether to extract 3 times, combine the extracts, and recover the solvent to dryness. Take petroleum ether to extract part of the extract, add normal phase silica gel to mix the sample, pass through normal phase silica gel column, use petroleum ether and ethyl acetate solution with a volume ratio of 50:1 as the eluent to elute, collect the eluate, and pass through the normal phase silica gel column. Available after concentration.

According to the method of the second aspect of the present invention, the obtained Rhizoma Chuanxiong extract contains 30-90% ligustilide.

According to the method of the second aspect of the present invention, the resulting Chuanxiong extract contains 80-97% (eg 85%-95%) ligustilide.

The third aspect of the present invention provides the application of the Rhizoma Chuanxiong extract of the first aspect of the present invention in the preparation of antithrombotic drugs.

The fourth aspect of the present invention provides a pharmaceutical composition, which includes the Rhizoma Chuanxiong extract of the first aspect of the present invention and a pharmaceutically acceptable carrier or excipient.

The effective components of Rhizoma Chuanxiong of the present invention can be used as the active part, added with pharmaceutical excipients or carriers acceptable in pharmacy, and prepared according to the method recorded in pharmacy.

The preparation forms of the medicine include liquid preparations, solid preparations, capsules and capsules. Including injections, drips, powder injections, granules, tablets, granules, powders, oral liquids, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, Buccal preparations, granules, pills, ointments, elixirs, sprays, dropping pills, disintegrants, orally disintegrating tablets, pellets, etc.

In the present invention, the term "Chuanxiong effective components" can also be called "Chuanxiong components", "Chuanxiong extract", "Chuanxiong lactone", etc., and these terms can be used interchangeably.

The beneficial effects of the present invention are:

1. The extraction and separation process of the present invention is simple, and effective components can be obtained quickly and accurately.

2. The active components of Rhizoma Chuanxiong provided by the present invention have simple and clear chemical components, which makes it easier to clarify its mechanism of action in pharmacological research and easier to control the quality of drugs in production.

3. For the first time, the present invention evaluates the effect of chuanxiong lactone extract on anti-platelet aggregation and protection of endothelial cells from inflammatory damage on the model, and obtains better pharmacological activity, which provides a basis for its use in the preparation of antithrombotic drugs .

Description of drawings

Fig. 1. is the high-efficiency liquid chromatographic analysis chart of the Rhizoma Chuanxiong extract of the present invention.

Figure 2. Effects of different concentrations of Chuanxiong extract on arachidonic acid-induced platelet aggregation.

Figure 3. The effect of different concentrations of Chuanxiong extract on ADP-induced platelet aggregation.

Figure 4. The effect of different concentrations of Chuanxiong extract on collagen-induced platelet aggregation.

Figure 5. Effects of different concentrations of Chuanxiong extract on adhesion factors secreted by TNF-α-injured endothelial cells.

Figure 6. The effect of different concentrations of Chuanxiong extract on the expression of nuclear factor NF-κB protein in TNF-α-injured endothelial cells.

Detailed ways

The present invention further describes the essence of the present invention in detail in conjunction with the accompanying drawings and the following embodiments, which are only used to illustrate the present invention rather than limit the present invention.

Embodiment 1 The preparation of effective components of Rhizoma Chuanxiong

After crushing 1000g of Rhizoma Chuanxiong medicinal material, add 4000ml of 95% ethanol to soak overnight, reflux extraction for 2 hours, then add 4000ml of 95% ethanol to heat and reflux for extraction for 3 times, each time for 2h, combine the extracts, and concentrate to a density of 1.08. Add the concentrated solution to 500ml of water to disperse into a suspension, add 500ml of petroleum ether to extract three times, combine the extracts, and recover the solvent to dryness. Take petroleum ether to extract part of the extract, add normal phase silica gel to mix the sample, pass through normal phase silica gel column, use petroleum ether and ethyl acetate solution with a volume ratio of 50:1 as the eluent for elution, collect the eluent, and pass through the normal phase silica gel column. After concentration, 11.3 g of light yellow liquid was obtained, which was the Rhizoma Chuanxiong extract of the present invention, which can also be referred to as Chuanxiong lactone in the present invention, and was used in the following biological test examples.

Embodiment two The analysis of the effective components of Rhizoma Chuanxiong

Weigh and dissolve the Rhizoma Chuanxiong extract as shown in Example 1, and transfer an appropriate amount to a brown liquid phase sampling bottle for liquid chromatography analysis.

The analysis conditions are as follows: Agilent 1100 liquid chromatograph, injection volume 10 μl; flow rate 1ml/min, column temperature 30°C. The mobile phase gradient for chromatographic analysis is: 0~40min: water (0.1% formic acid): 70%~20%, acetonitrile: 30%~80%; 40-50min: water (0.1% formic acid): 20%~5%, Acetonitrile: 80%~95%; 50~55min: water (0.1% formic acid): 5%, acetonitrile: 95%; detection wavelength 271nm. The analytical spectrogram is shown in Figure 1 (the abscissa is minutes). Liquid chromatographic analysis showed that the extract of Chuanxiong Rhizoma had an obvious chromatographic peak at 26.5min, and the compound represented by this peak was identified as ligustilide after comparison with the standard. The analysis results showed that the main component of ligustilide extract was ligustilide, and its content was about 90%. Surprisingly, it is found that in Example 1, if the concentration is lower than 70% for ethanol extraction, or petroleum ether is an eluent, or the ethyl acetate portion is greater than 5 in the eluent used, that is, 50 parts of petroleum ether and 5 When the ethyl acetate mixed solution of more than one part is eluent, the obtained amount of ligustilide is more than 30% lower than that obtained in Example 1. When extracting with more than 75% ethanol, or using a mixture of petroleum ether: ethyl acetate at a ratio of 50:0.5~3 as the eluent, the amount of ligustilide obtained is 90%~115% of that obtained in Example 1. %, and the content of ligustilide in the extract is 80~97%.

Embodiment three Rhizoma Chuanxiong effective component preparation

Take 0.5 g of the effective components of Ligusticum chuanxiong obtained in Example 1 and mix evenly with 10.5 g of polyethylene glycol-6000, heat and melt, and after the chemical material is moved to drip irrigation, the drug solution is dropped into 6 ~ 8 ℃ of liquid paraffin, and the Oil, made 400 drop pills.

Embodiment four Rhizoma Chuanxiong effective component preparation

Take 0.5 g of the effective components of Ligusticum chuanxiong obtained in Example 1, grind into fine powder, mix with about 1/3 of the starch, add starch slurry and mix to make a soft material, granulate with a 16-mesh sieve, dry at 70°C, and dry the granules Pass through a 14-mesh sieve for granulation, add the remaining starch (pre-dried at 100-105 ℃) and talcum powder adsorbed with liquid paraffin (spray light liquid paraffin into the talcum powder and mix), then pass through a 14-mesh sieve, and press into tablets , that is.

Example 5 Evaluation of Anti-platelet Aggregation Activity of Chuanxionglide Extract

Platelets can be activated by various substances, but mainly through the following pathways: ADP pathway, AA cyclooxygenase pathway and PAF (platelet activating factor) pathway. These three pathways ultimately activate platelets by increasing the Ca ²⁺ concentration in platelets. Exogenous collagen mainly through the PAF pathway. In this example, the effects of the ligusticolidinide extract obtained in Example 1 on platelet aggregation induced by arachidonic acid (AA), adenosine diphosphate (ADP) and collagen (COLL) were investigated.

New Zealand big-eared white rabbits were anesthetized locally with lidocaine, and the left common carotid artery was isolated. Blood was collected from the common carotid artery and anticoagulated with 3.8% sodium citrate solution. Centrifuge at 1000rmp for 12min, take the supernatant part that is platelet-rich plasma (PRP), and centrifuge the remaining part at 3000rmp for 10min, take the supernatant part that is platelet-poor plasma (PPP). Platelets were counted with an automatic hematology analyzer, and PRP was adjusted with PPP so that the platelet count was (3.0-5.0)×10 ⁹ /L. After inducing coagulation with different types of inducers ADP, AA and COLL, measure the anti-platelet aggregation activity of Ligustin extract (gained in Example 1) at 0.05, 0.1, 0.2, 0.4, 0.6, 0.8mg/ml concentration, respectively 0.05mg/ml aspirin and 0.2mg/ml clopidogrel were used as positive drugs, and platelet aggregation rate was measured with 570-VS platelet aggregation analyzer.

The results showed that after being induced by AA, they aggregated rapidly, and the average maximum aggregation rate was 68.04. Under the intervention of the positive drug Aspirin, the maximum aggregation rate decreased significantly ( p< 0.01). The chuanxiong lactone extract obtained in Example 1 has no significant difference between the 0.05 mg/ml and 0.1 mg/ml dosage groups and the blank control group, and the aggregation rate in each dosage group of 0.2-0.8 mg/ml is significantly decreased, compared with the blank control group The difference was significant ( p< 0.01), and there was a dose-dependent relationship, as shown in Figure 2 (control in the figure represents the control group, and aspirin represents the aspirin group).

Platelet-rich plasma aggregated rapidly after being induced by ADP, and the average maximum aggregation rate was 48.15. Under the intervention of the positive drug clopidogrel, the maximum aggregation rate decreased significantly ( p< 0.01). The chuanxiong lactone extract obtained in Example 1 significantly decreased the platelet aggregation rate induced by ADP in the concentration range of 0.6-1.4mg/ml, which was significantly different from that of the blank control group ( p< 0.01), and was in a dose-dependent manner, as shown in the attached As shown in Fig. 3 (in the figure contr ... represents the control group, clopi ... represents the clopidogrel group).

Platelet-rich plasma aggregated rapidly after being induced by collagen, and the average maximum aggregation rate was 70.77. After aspirin and clopidogrel intervention, the maximum aggregation rate decreased significantly ( p< 0.01). Ligustilide 0.05 mg/ml, 0.1 mg/ml dose group had no significant difference compared with the blank control group, and the aggregation rate of each dose group of 0.2-0.8 mg/ml decreased significantly, which was significantly different from the blank control group ( p< 0.01), And there is a dose-dependent relationship, as shown in Figure 4 (control in the figure represents the control group, aspirin represents the aspirin group, clopido... represents the clopidogrel group).

Example 6 Evaluation of anti-endothelial cell injury activity of chuanxiong lactone extract

The protective effect of chuanxiong lactone extract obtained in Example 1 on inflammatory injury endothelial cells was investigated. Human umbilical vein endothelial cells were isolated and cultured in vitro, TNF-α injury model was established, and different concentrations (6.25, 12.5, 25ug/ml) of ligusticum lactone extracts were added for co-incubation for 24 hours, and the enzyme-linked immunoassay (Elisa method) was used to detect the cell The content of adhesion molecules sICAM-1 and sVCAM-1 in the culture medium, and the expression of NF-κB protein in the nucleus were determined by Western blot. The results showed that MTT results showed that TNF-α10ng/ml could obviously cause cell damage, and chuanxiong lactone dose-dependently protected endothelial cells from TNF-α damage; TNF-α significantly increased endothelial cell adhesion molecules ICAM-1 and The expression of sVCAM-1 (p<0.01), high dose of chuanxionglide can significantly reduce its expression; at the same time, Western blot results showed that chuanxionglide can reduce the protein expression of NF-kB in human umbilical vein endothelial cells in a dose-dependent manner. Chuanxiong lactone extract can protect endothelial cells from TNF-α damage, and its mechanism may be related to reducing the content of endothelial cell adhesion molecule ICAM-1 by inhibiting the protein expression of NF-κB. The results are shown in Figure 5, which shows the effect of different concentrations of Chuanxiong extract on the secretion of adhesion factors from TNF-α-injured endothelial cells. The vertical axis represents the concentration of human sICAM-1 and sVCAM-1 (ug/ml). In addition, Figure 6 shows the effect of different concentrations of Chuanxiong extract on the expression of nuclear factor NF-κB protein in endothelial cells injured by TNF-α, and the ordinate in the figure indicates the expression of NF-κB (compared with the control).

Claims (10)

1. a Rhizoma Chuanxiong extract wherein contains 30 ~ 90% ligustilide.

2. according to the Rhizoma Chuanxiong extract of claim 1, wherein contain 80 ~ 97% ligustilide.

3. according to the Rhizoma Chuanxiong extract of claim 1 or 2; It prepares through following method: after the Rhizoma Chuanxiong pulverizing medicinal materials, soak and reflux, extract, 1-4 time merge extractive liquid, with 70% ~ 99% ethanol (for example 75% ~ 98%); Concentrate; Use petroleum ether extraction, the extract that merges is reclaimed solvent to doing, get petroleum ether extraction extractum; Getting petroleum ether extraction part extractum, add and cross normal phase silicagel column after purification on normal-phase silica gel is mixed appearance, is that the petroleum ether and the ethyl acetate solution of 50:0.5 ~ 5 (for example 50:0.5 ~ 3) is that eluant carries out eluting with the volume ratio, collects eluent, concentrates, and promptly gets.

4. according to the Rhizoma Chuanxiong extract of claim 1 or 2, it prepares through following method: with adding 20% ~ 90% soak with ethanol after the Rhizoma Chuanxiong pulverizing medicinal materials 5 ~ 12 hours, heating and refluxing extraction 0.8 ~ 1.2 hour; Extract 1 ~ 3 time; Merging filtrate gets extracting solution, and extracting solution is used petroleum ether extraction, extract is concentrated the back with normal phase silicagel column it is carried out purification; The petroleum ether and the ethyl acetate that with the volume ratio are 50:1 are eluant, collect to obtain extract after eluent concentrates.

5. according to the Rhizoma Chuanxiong extract of claim 1 or 2; It prepares through following method: after 1000g Rhizoma Chuanxiong pulverizing medicinal materials, add 4000ml 95% soak with ethanol and spend the night reflux, extract, 2 hours; Adding 4000ml again extracts 3 times with 95% alcohol heating reflux; Each 2h, merge extractive liquid, is concentrated into density 1.08; Concentrated solution added in the 500ml water be dispersed into suspension, add the 500ml petroleum ether extraction 3 times, combining extraction liquid reclaims solvent to doing; Getting petroleum ether extraction part extractum, add and cross normal phase silicagel column after purification on normal-phase silica gel is mixed appearance, is that petroleum ether and the ethyl acetate solution of 50:1 is that eluant carries out eluting with the volume ratio, collects eluent, after concentrating, promptly gets.

6. prepare the method for Rhizoma Chuanxiong extract, it may further comprise the steps: after the Rhizoma Chuanxiong pulverizing medicinal materials, soak and reflux, extract, 1-4 time with 70% ~ 99% ethanol (for example 75% ~ 98%); Merge extractive liquid, concentrates, and uses petroleum ether extraction; The extract that merges is reclaimed solvent to doing, get petroleum ether extraction extractum; Getting petroleum ether extraction part extractum, add and cross normal phase silicagel column after purification on normal-phase silica gel is mixed appearance, is that the petroleum ether and the ethyl acetate solution of 50:0.5 ~ 5 (for example 50:0.5 ~ 3) is that eluant carries out eluting with the volume ratio, collects eluent, concentrates, and promptly gets.

7. according to the method for claim 6, it may further comprise the steps: with adding 20% ~ 90% soak with ethanol after the Rhizoma Chuanxiong pulverizing medicinal materials 5 ~ 12 hours, heating and refluxing extraction 0.8 ~ 1.2 hour; Extract 1 ~ 3 time; Merging filtrate gets extracting solution, and extracting solution is used petroleum ether extraction, extract is concentrated the back with normal phase silicagel column it is carried out purification; The petroleum ether and the ethyl acetate that with the volume ratio are 50:1 are eluant, collect to obtain extract after eluent concentrates.

8. according to the method for claim 6, it may further comprise the steps: after 1000g Rhizoma Chuanxiong pulverizing medicinal materials, add 4000ml 95% soak with ethanol and spend the night; Reflux, extract, 2 hours adds 4000ml again and extracts 3 times with 95% alcohol heating reflux, each 2h; Merge extractive liquid, is concentrated into density 1.08; Concentrated solution added in the 500ml water be dispersed into suspension, add the 500ml petroleum ether extraction 3 times, combining extraction liquid reclaims solvent to doing; Getting petroleum ether extraction part extractum, add and cross normal phase silicagel column after purification on normal-phase silica gel is mixed appearance, is that petroleum ether and the ethyl acetate solution of 50:1 is that eluant carries out eluting with the volume ratio, collects eluent, after concentrating, promptly gets.

9. the application of the Rhizoma Chuanxiong extract of claim 1-6 in the preparation antithrombotic reagent.

10. pharmaceutical composition is comprising Rhizoma Chuanxiong extract and pharmaceutically acceptable carrier or the excipient of claim 1-6.

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