Preparation method of total lignans of Penthorum chinense Pursh and application thereof in preparing antithrombotic drugs

Abstract

The present invention discloses a preparation method of total lignans from Penthorum chinense Pursh, belonging to the field of pharmaceuticals. First, an extract of Penthorum chinense Pursh containing total lignans is obtained by using the combined method of enzymatic extraction - supercritical fluid extraction, and then macroporous adsorption resin purification is used to further improve the product purity. This method has the advantages of high extraction efficiency and high product purity. The present invention also discloses a new pharmaceutical use of total lignans from Penthorum chinense Pursh, especially its new use in the preparation of antithrombotic drugs, providing a new treatment means and drug option for the clinical treatment of thrombosis.

Description

Technical Field

[0001] The present invention belongs to the pharmaceutical field, and relates to an extraction and purification method of total lignans from Penthorum chinense Pursh. The present invention also relates to a new pharmaceutical use of total lignans from Penthorum chinense Pursh, especially a new use in the preparation of antithrombotic drugs. Background Art

[0002] Thrombotic diseases are different diseases caused by the continuous accumulation of fibrin in the blood, resulting in stenosis and occlusion of blood vessels, and causing ischemia and infarction in the body, leading to dysfunction, such as cardiovascular and cerebrovascular diseases. Fibrinogen is the precursor of fibrin. Under the action of thrombin, fibrinogen is converted into fibrin, and then forms a fibrin clot through polymerization. In the human body, fibrin clots and platelets together form a clot that can stop bleeding, and at the same time, pathological vascular embolisms can also be generated. The fibrin coagulation and dissolution systems in the body are coordinated with each other to maintain the balance between thrombus formation and dissolution. Once the balance is broken, pathological conditions will occur. At present, the incidence of thromboembolic diseases ranks first among cardiovascular and cerebrovascular diseases, and the fatality rate and recurrence rate are extremely high. As China enters the aging stage, thrombotic diseases will pose a greater threat to our health.

[0003] According to the thrombus formation process, current antithrombotic drugs are mainly divided into three categories: antiplatelet drugs, anticoagulant drugs, and fibrinolytic (thrombolytic) drugs, such as clopidogrel, heparin, urokinase, etc. Thrombolytic therapy is a commonly used method for treating thrombotic diseases at present. Although it has good effects, it has side effects such as bleeding, gastrointestinal irritation, allergy, etc., and even serious adverse reactions such as granulocytopenia and thrombotic thrombocytopenic purpura (TIP), and the cost is high. Therefore, finding new thrombolytic drugs is a current research hotspot. Food-derived natural plant extracts not only have efficacy, but also can improve safety and reduce side effects, and at the same time, the treatment cost is lower, which is a very good choice.

[0004] Penthorum chinense Pursh, also known as water eupatorium and water willow, is the dried aerial part of Penthorum chinense Pursh, a plant of the genus Penthorum. It is mainly distributed in North China, East China, Central South, Shaanxi, Sichuan, Guizhou and other places in China, and is mainly produced in Gulin, Sichuan. Penthorum chinense Pursh is a traditional medicine of the Miao nationality. The tender seedlings can be used as vegetables, and the whole herb is used as medicine in folk, with the effects of removing dampness and promoting diuresis, removing stasis and relieving pain, soothing the liver and strengthening the spleen, etc. In recent years, great progress has been made in the research on the chemical constituents of Penthorum chinense Pursh, and it is found that it mainly contains flavonoids, organic acids and glycosides, esters, lignans and other components. Among them, penthorin A, B, C, D and other penthorins are rare 2,4`-epoxy-8,5`-neolignans. The biosynthetic precursors of these special lignans are all 8,3`-neolignans, but further transformation occurs in the biosynthetic process of secondary metabolites of Penthorum chinense Pursh, thus forming unique and specific lignans.

[0005] The extraction methods of natural products mainly include solvent method, distillation method, enzymatic hydrolysis method, supercritical fluid extraction method, physical field-assisted extraction method, etc. Among them, the enzymatic hydrolysis method decomposes and transforms plant tissues and chemical components by using enzymatic hydrolysis reactions under mild conditions, but there are problems such as low yield and long time consumption; supercritical fluid extraction uses CO2 in the supercritical state as the extraction solvent, and a higher extraction rate can be obtained by changing the extraction conditions. However, large consumption of CO2, high operating pressure, and long extraction time are common problems in supercritical fluid extraction. Therefore, the present invention combines the enzymatic hydrolysis extraction method with the supercritical fluid extraction technology in order to solve the main problems existing in the traditional process.

[0006] In 2020, the National Health Commission listed Penthorum chinense Pursh as a new food raw material, indicating that Penthorum chinense Pursh, as a medicine and food homologous substance with high safety, has great research value. At present, the research on the pharmacological effects of Penthorum chinense Pursh mainly focuses on aspects such as antioxidant, liver protection, and antiviral, etc., and there is no report on the antithrombotic effect of the lignan components in Penthorum chinense Pursh. Summary of the Invention

[0007] The first object of the present invention is to provide a preparation method of total lignans from Penthorum chinense Pursh, which for the first time realizes the extraction of total lignans from Penthorum chinense Pursh with relatively high purity. The second object of the present invention is to provide a new use of total lignans from Penthorum chinense Pursh in the preparation of antithrombotic drugs. The experimental results show that total lignans from Penthorum chinense Pursh play an antithrombotic effect by two ways of degrading fibrin and improving vascular endothelial cell injury. Specifically as follows:

[0008] One, the preparation method provided by the present invention mainly includes the following two steps:

[0009] Step one, use the combined technology of enzyme-supercritical CO2 extraction to extract total lignans

[0010] After pulverizing the Penthorum chinense Pursh medicinal materials, add the enzymatic hydrolysis solution, carry out enzymatic hydrolysis at a temperature of 30 - 60 °C for 0.5 - 2 hours, then add the entrainer, and carry out CO2 supercritical fluid extraction at a temperature of 30 - 60 °C and a pressure of 5 - 30 Mpa. The extraction solution is centrifuged, and the supernatant is concentrated and dried to obtain the Penthorum chinense Pursh extract containing total lignans.

[0011] Among them, the enzymolysis solution has two functions. On the one hand, it can break the cell walls of plant cells to accelerate the dissolution of active ingredients. On the other hand, since a part of the lignans in plants are combined with glycosides and exist in the form of glycosides or other derivatives, the enzymolysis solution can also cause the glycosidic bonds or other binding sites to break and release lignans, thereby improving the extraction efficiency and purity of lignans. The free lignans in Penthorum chinense Pursh have very low polarity and strong lipophilicity and can be dissolved in low-polarity solvents such as fluid CO2. According to this characteristic, the present invention adopts a combined technology of enzymatic method - supercritical CO2 extraction. First, hydrolytic enzymes are used to pretreat Penthorum chinense Pursh, and then the enzymolysis product is extracted by fluid CO2 supercritical extraction to enhance the solubility and selectivity of CO2 in the extraction process. This method combines the advantages of the enzymatic method and supercritical fluid extraction, saves extraction time, reduces the consumption of CO2, lowers the extraction cost, and the entire extraction process is environmentally friendly and can efficiently extract most of the lignans in Penthorum chinense Pursh.

[0012] Step 2. Purify the total lignans using macroporous adsorption resin

[0013] The extract is adsorbed by macroporous resin, then eluted with water and ethanol in sequence, the ethanol eluate is collected, concentrated and dried to obtain the total lignans of Penthorum chinense Pursh with high purity.

[0014] First of all, according to the van der Waals force and hydrogen bond binding ability between the total lignans of Penthorum chinense Pursh and the macroporous adsorption resin, the present invention realizes the selective adsorption of the substances to be separated on the macroporous resin; then, according to the solubility and polarity of the substances to be separated, corresponding organic solvents are used for selective elution, and finally, the effective separation of the total lignans of Penthorum chinense Pursh from other components is realized and the product purity is improved.

[0015] Among them, the enzymolysis solution contains one or more of cellulase, pectinase, laccase, protease, α-amylase;

[0016] Among them, the entrainer is one or more of ethanol, methanol, n-butanol, acetone, ethyl acetate.

[0017] Further preferably, the enzymolysis solution contains pectinase, and the mass of the pectinase accounts for 0.8 - 1.5% of the mass of Penthorum chinense Pursh medicinal materials.

[0018] Further preferably, the temperature of the enzymolysis is 45 - 55 °C and the time is 1 - 1.5 hours.

[0019] Preferably, the entrainer is a mixed solution of ethanol and ethyl acetate, and the volume ratio of ethanol to ethyl acetate is 1:2 - 5.

[0020] Further preferably, the volume ratio of the entrainer to the enzymolysis solution is 1:3 - 5.

[0021] Preferably, the temperature of the CO2 supercritical fluid extraction is 50 - 55 °C, and the pressure is 25 - 30 Mpa.

[0022] Preferably, the CO of the supercritical fluid extraction 2 flow rate is 30 - 50 / L·h -1 , and the extraction time is 1 - 3 h.

[0023] Preferably, the type of the macroporous resin is AB - 8 type, and the ethanol is 95% ethanol.

[0024] According to an embodiment of the present invention, a preferred preparation method of the total lignans of Penthorum chinense Pursh is as follows:

[0025] 1) After pulverizing the Penthorum chinense Pursh medicinal materials, add an enzymatic hydrolysis solution containing 1.4% pectinase by mass of the Penthorum chinense Pursh medicinal materials, perform enzymatic hydrolysis at 50 °C for 75 min, then add an entrainer, the entrainer is a mixed solution of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 1:3, the volume ratio of the entrainer to the enzymatic hydrolysis solution is 1:4, perform CO2 supercritical fluid extraction at 55 °C and 30 Mpa, control the CO 2 flow rate to be 40 L·h -1 , after extraction for 2 h, centrifuge, and after concentrating and drying the supernatant, obtain the Penthorum chinense Pursh extract containing total lignans;

[0026] 2) After redissolving the extract with ethanol, adsorb it on AB - 8 type macroporous resin, the sample loading concentration is 2 mg / mL, the sample loading volume is 7 times the column volume, after adsorption equilibrium, first wash with 5 times the column volume of water, then elute with 11 times the column volume of 95% ethanol, the elution flow rate is 1 ml / min, collect the ethanol eluate, concentrate and dry to obtain.

[0027] This preferred technical solution can further improve the extraction efficiency of the total lignans of Penthorum chinense Pursh, and has a relatively higher product yield and purity.

[0028] Second, the present invention provides a new pharmaceutical use of the total lignans of Penthorum chinense Pursh, specifically, a new use in the preparation of antithrombotic drugs.

[0029] To achieve the above - mentioned purpose, the applicant first investigated the activity of the total lignans of Penthorum chinense Pursh in dissolving fibrin through an in vitro thrombolysis experiment. Then, the applicant constructed a mouse thrombus model by intraperitoneal injection of carrageenan and investigated the antithrombotic efficacy of the total lignans of Penthorum chinense Pursh. The results showed that the total lignans of Penthorum chinense Pursh not only had plasmin - like activity and was positively correlated with the purity of the total lignans, but also could significantly reduce the formation of tail thrombus in model mice, and also had the effect of improving vascular endothelial cell injury.

[0030] Based on the above test results, it is speculated that the total lignans of Penthorum chinense Pursh play an anti-thrombotic effect by degrading fibrin and improving vascular endothelial cell injury, and further it is expected to prevent and treat vascular embolism diseases such as varicose veins, acute myocardial infarction, ischemic stroke, atherosclerosis and other diseases.

[0031] When preparing these drugs, the total lignans of Penthorum chinense Pursh can be used alone as the active ingredient, or it can be combined with other anti-thrombotic drugs and used together as the active ingredient. Further, these active ingredients can be combined with a pharmaceutical carrier and made into various pharmaceutical preparations suitable for clinical use according to the well-known pharmaceutical methods in the art. In addition, the extract of the total lignans of Penthorum chinense Pursh can also be used as a food homologous substance for health foods or functional foods.

[0032] The present invention realizes the preparation of the total lignans of Penthorum chinense Pursh for the first time. The method uses the combined extraction of enzymatic method-supercritical fluid extraction and macroporous resin purification, making it have the advantages of high extraction efficiency and high product purity. At the same time, it also provides a new treatment means and drug choice for the clinical treatment of anti-thrombosis. The present invention is derived from natural plants and has the advantages of high safety and low cost, which is beneficial to reducing the burden on doctors and patients and better meeting the clinical drug demand. Description of the Drawings

[0033] Figure 1 : Influence of the type of hydrolase on the extraction rate of total lignans.

[0034] Figure 2 : Influence of the dosage of pectinase on the extraction rate of total lignans.

[0035] Figure 3 : Influence of the enzymatic hydrolysis temperature on the extraction rate of total lignans.

[0036] Figure 4 : Influence of the enzymatic hydrolysis time on the extraction rate of total lignans.

[0037] Figure 5 : Influence of the type of entrainer on the extraction rate of total lignans.

[0038] Figure 6 : Influence of the dosage of entrainer on the extraction rate of total lignans.

[0039] Figure 7 : Influence of the CO2 flow rate on the extraction rate of total lignans.

[0040] Figure 8 : Influence of the extraction pressure on the extraction rate of total lignans.

[0041] Figure 9 : Influence of the extraction temperature on the extraction rate of total lignans.

[0042] Figure 10 : Influence of extraction time on the extraction rate of total lignans.

[0043] Figure 11 : Influence of total lignans from Penthorum chinense Pursh on endothelin-1 in thrombus mice. Compared with the normal group, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01. Specific implementation manners

[0044] The present invention will be described in detail below through specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention rather than to limit it. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

[0045] The experimental methods not described in detail in the embodiments can be conventional operations in the art or implemented according to reference books such as "Medicinal Botany" and "Pharmacological Experiment Methodology".

[0046] The percentages involved in the following embodiments, unless otherwise specified, all refer to volume percentages. For example, 95% ethanol means that there are 95 ml of ethanol in every 100 ml of ethanol aqueous solution.

[0047] The method for determining the content of total lignans involved in the following embodiments is as follows:

[0048] 1) Preparation of test sample

[0049] Take 1 g of the extract of Penthorum chinense Pursh, accurately weigh it, place it in a 50 mL Erlenmeyer flask, accurately add 50 mL of 60% ethanol, weigh it, ultrasonically treat it for 30 min, take it out and let it stand at room temperature, make up the lost weight with 60% ethanol, shake well, filter, take 2.5 ml of the continued filtrate and place it in a 10 ml volumetric flask, dilute it to the mark with 60% ethanol, shake well, and use it as the test sample solution.

[0050] 2) Preparation of standard

[0051] Accurately weigh 7.78 mg of the syringaresinol reference substance, place it in a 10 ml volumetric flask, add 60% ethanol to the mark, shake well, and obtain a reference standard solution with a concentration of 0.778 mg / ml. Respectively pipette 0, 2, 4, 6, and 8 ml of the syringaresinol standard solution into 10 mL stoppered test tubes, make up to 10 ml with 60% ethanol for later use.

[0052] 3) Determination

[0053] Precisely pipette 0.5 ml of the test solution and the syringaresinol reference solutions of each concentration into a 10 mL stoppered test tube, evaporate to dryness, successively add 1 mL of 10% chromotropic acid solution and 6 mL of concentrated H2SO4, shake well, then add 3 mL of distilled water, seal the test tube mouth, shake well, place in a boiling water bath and heat for 30 min, take out and quickly cool with running water, shake well, zero with the blank reagent, and measure at 573 nm.

[0054] 4) Calculation method

[0055] Taking the absorbance value as the abscissa and the standard product concentration (mg / ml) as the ordinate, the standard curve Y = 252.3X - 0.0108 (R 2 = 0.998) is obtained. Substitute the absorbance value measured for the sample into it, calculate the total lignan concentration, and calculate the total lignan extraction rate and purity according to the formula.

[0056]

[0057]

[0058] Example 1 Preparation and Process Research of Total Lignans from Penthorum chinense Pursh

[0059] Step 1: Combined extraction of enzymatic method - supercritical CO2 extraction

[0060] Supercritical fluid extraction is a commonly used method for extracting active ingredients from traditional Chinese medicines developed in recent years. It uses CO2 in the supercritical state as the extraction solvent and obtains a higher extraction rate by changing the extraction conditions. However, large amounts of CO2, high operating pressure, and long extraction time are common problems in supercritical fluid extraction. Therefore, in this invention, before supercritical CO2 extraction, Penthorum chinense Pursh is pretreated with the enzymatic method to improve the content of lignans in Penthorum chinense Pursh.

[0061] The specific process flow is: Penthorum chinense Pursh powder → add enzymatic hydrolysis solution → control temperature for enzymatic hydrolysis → add entrainer → supercritical fluid extraction → centrifugation → concentration → drying.

[0062] Air-dry the washed Penthorum chinense Pursh, pulverize it through a 60-mesh sieve, then add 2 times the mass of the enzymatic hydrolysis solution, carry out enzymatic hydrolysis at the optimal temperature of the enzyme for a certain time, then add a certain amount of entrainer according to the volume ratio of the enzymatic hydrolysis solution, and carry out extraction with CO2 supercritical fluid at a certain temperature and pressure. The extract is centrifuged, and the supernatant is concentrated and dried to obtain the Penthorum chinense Pursh extract containing total lignans.

[0063] Preparation of the enzymatic hydrolysis solution: Weigh 100 g of distilled water, heat it in a water bath to the optimal temperature of the enzyme, adjust the pH to the optimal pH of the enzyme with 0.1 mol / L HCl or 0.1 mol / L NaOH, weigh a certain amount of the enzyme and add it to the solution with the adjusted pH, and stir until completely dissolved for standby.

[0064] 1. Single - factor experimental design

[0065] The factors affecting the enzymatic action effect are mainly the type of enzyme, the dosage of enzyme, the enzymatic hydrolysis temperature, and the enzymatic hydrolysis time, etc. According to the type and characteristics of the enzyme (Table 1), a single - factor experiment was designed (Table 2).

[0066] Table 1 Types and characteristics of enzymes

[0067]

[0068] The initial conditions of the experiment were that the enzyme dosage was 0.8%, enzymatic hydrolysis was carried out at 50 °C for 30 min, the dosage of the entrainer was 1:2, the extraction pressure was 15 MPa, the extraction temperature was 50 °C, the CO2 flow rate was 35 L / h, and the extraction time was 1 h. Fixing other factors, according to Table 2, the influence of a single factor on the total lignan content of Penthorum chinense Pursh was investigated.

[0069] Table 2 Single - factor experimental design

[0070]

[0071]

[0072] 2. Results of single - factor experiments

[0073] From Figure 1 it can be seen that when pectinase is selected, the extraction rate of lignan is higher. Therefore, pectinase was selected subsequently to investigate the influence of the enzyme dosage, enzymatic hydrolysis temperature, and enzymatic hydrolysis time on the total lignan extraction rate. The results are shown in Figures 2 - 4 , and it was found that when the enzyme dosage was 1.2%, the enzymatic hydrolysis temperature was 50 °C, and the enzymatic hydrolysis time was 75 min, the extraction rate was relatively high.

[0074] A suitable entrainer is helpful for the extraction of lignan. From Figure 5 it can be seen that when ethanol and ethyl acetate are mixed in a volume ratio of 1:3 as the entrainer, it has an enhancing effect on the lignan extraction rate. Therefore, a mixed solution of ethanol and ethyl acetate at 1:3 was selected as the entrainer for subsequent single - factor experiments.

[0075] From Figure 6 it can be seen that when a mixed solution of ethanol and ethyl acetate at 1:3 is used as the entrainer, the extraction rate of lignan is 1 - 3 times that without using an entrainer, and the extraction rate is relatively high when the dosage of the entrainer is 1:3 (the volume ratio of the entrainer to the enzymatic hydrolysate).

[0076] CO 2 The flow rate, extraction pressure, extraction temperature, and extraction time all have an impact on the extraction rate. From Figures 7 - 10 it can be seen that the CO 2 flow rate is 40 L·h -1When the extraction pressure is 25 MPa, the extraction temperature is 55 °C, and the extraction time is 2 h, the extraction rate of total lignans is relatively high.

[0077] 3. Optimization of the extraction process by orthogonal experiment

[0078] According to the results of the single-factor experiment, four factors with a greater impact on the extraction rate were selected. When the enzyme was determined to be pectinase, the enzymatic hydrolysis temperature was 50 °C, the enzymatic hydrolysis time was 75 min, the entrainer was a mixed solution of ethanol and ethyl acetate (volume ratio 1:3), the extraction temperature was 55 °C, and the CO 2 flow rate was 40 L·h -1 an orthogonal experiment was carried out under the conditions of, and the factor level design is shown in Table 3 to further optimize the extraction process.

[0079] Table 3 Factor design table for orthogonal experiment

[0080]

[0081] As can be seen from Table 4, the order of the influence of each factor on the extraction rate of total lignans is A > B > D > C. The optimal process obtained by orthogonal experiment optimization is that the enzyme dosage is 1.4%, the entrainer dosage is 1:4, the extraction pressure is 30 MPa, and the extraction time is 2 h.

[0082] Table 4 Results of orthogonal experiment

[0083]

[0084] 4. Verification test of process parameters

[0085] According to the optimal process parameters obtained from the orthogonal experiment, the verification test was repeated 3 times, and the average extraction rate of total lignans was 26.2%. It shows that when pectinase is selected, the enzyme dosage accounts for 1.4% of the mass of the Herba Senecionis Scandentis, the enzymatic hydrolysis temperature is 50 °C, the enzymatic hydrolysis time is 75 min, the entrainer is a mixed solution of ethanol and ethyl acetate (volume ratio 1:3), the volume ratio of the entrainer to the enzymatic hydrolysate is 1:4, the extraction temperature is 55 °C, the extraction pressure is 30 MPa, and the CO 2 flow rate is 40 L·h -1 , under the condition of an extraction time of 2 h, the extraction rate of total lignans is relatively high, and its purity is 26.25%.

[0086] Step 2. Purification by macroporous resin

[0087] Before the purification by macroporous adsorption resin, the types of 5 macroporous adsorption resins, namely AB-8, NKA-2, S-8, HPD100, and DM301, were screened through the static adsorption rate and desorption rate. The specific method is as follows:

[0088] 1. Investigation of static adsorption rate: Weigh 2 g of each of the five treated wet resins and place them in 250-ml conical flasks respectively. Then inject 40 ml of the total lignans extract of Penthorum chinense Pursh into each flask. Cover the flask with a stopper and shake it in a constant temperature shaker at 100 r / min and 25 °C for 2 h. Let it stand for 24 h, filter, and calculate the total lignans content according to the above measurement method. The static adsorption rates of the 5 resins are 85.3%, 76.8%, 62.7%, 71.7% and 79.5% respectively.

[0089] 2. Investigation of static desorption rate: Put the macroporous resin adsorbed with the medicinal liquid into 250-ml conical flasks, add 100 ml of 95% ethanol solution to each flask. Cover the flask with a stopper and shake it in a constant temperature shaker at 100 r / min and 25 °C for 2 h. Let it stand for 24 h for static elution, filter, and calculate the total lignans content according to the above measurement method. The static desorption rates of the 5 resins are 90.3%, 80.8%, 70.1%, 73.9% and 85.6% respectively.

[0090] Based on the above experiments, AB-8 resin was used as the best adsorption resin in this experiment. The specific purification operation is as follows: Redissolve the extract of Penthorum chinense Pursh with 30% ethanol and load it onto AB-8 macroporous adsorption resin. The loading concentration is 2 mg / mL, and the loading volume is 7 times the column volume. Pass through the macroporous resin at a flow rate of 1.5 BV / h. After complete adsorption, first wash with 5 BV of distilled water to remove impurities, and then elute with 11 BV of 95% ethanol at a flow rate of 1 ml / min. Collect the ethanol eluate, concentrate and dry it, and the purity of the total lignans detected is 69.38%, which is 2.64 times that before purification.

[0091] Example 2 In vitro thrombolysis experiment of total lignans from Penthorum chinense Pursh

[0092] ① Test method

[0093] Using the fibrin plate method, observe the activities of the crude extract, extract and purified product in dissolving fibrin. The fibrin plate method is currently the standard method for the Ministry of Health of China to determine the activities of thrombolytic enzymes such as urokinase and lumbrokinase, and it is also the most commonly used method for measuring the thrombolytic activity of plasmin. This method is simple and easy to operate, the area of the dissolution circle is visually visible, and multiple samples can be measured simultaneously.

[0094] Mix the 1% agarose solution, 0.3% fibrinogen stock solution, and 1 BP / ml thrombin solution that have been incubated in a 50°C water bath in proportion and pour them into a plate to prepare an artificial thrombus plate. After solidification, punch holes (3 mm in diameter), add 10 μL of sample to each hole (the concentrations of the 3 extracts are all 10 mg / mL), then place it in an incubator at 37°C for 18 h. Use a caliper to measure the two perpendicular diameters of the clear zone respectively to calculate the relative activity of each part of the sample. Use standard urokinase as a positive control, prepare urokinase with different concentrations (31, 62, 124, 248, 496 IU / mL), take the logarithm of the fibrinolytic activity of urokinase as the abscissa, and the logarithm of the product of the perpendicular diameters of the clear zone as the ordinate, perform a correlation regression analysis, and draw the urokinase standard curve.

[0095] ② Test results

[0096] On the fibrin plate, there are obvious clear zones around the holes with the 3 extracts added and the holes with urokinase added, indicating that these 3 extracts of Penthorum chinense Pursh all have thrombolytic activity. According to the urokinase thrombolytic activity standard curve y = 0.2439x + 2.4024 (R 2 = 0.996), calculate the fibrinolytic enzyme activities of the 3 extracts, and the results are shown in Table 5.

[0097] Table 5 Fibrinolytic enzyme activities of different extracts

[0098] Group Crude extract Extract Purified product Fibrinolytic enzyme activity (IU / mL) 11.59 156.80 657.32

[0099] Note: The crude extract is the product obtained by extracting Penthorum chinense Pursh by the conventional water decoction method, and the purity of total lignans is 1.15%; the extract is the product obtained by extracting by the combined method of enzyme - supercritical CO2 extraction optimized in Example 1, and the purity of total lignans is 26.25%; the purified product is the product obtained by the purification method optimized in Example 1, and the purity of total lignans is 69.38%.

[0100] It can be seen from Table 5 that the fibrinolytic enzyme activity increases with the increase of the purity of total lignans. Thus, it is confirmed that lignans are the key active ingredients in the extracts of Penthorum chinense Pursh that exert the anti - thrombus effect.

[0101] Example 3 Evaluation of the in - vivo anti - thrombus effect of total lignans from Penthorum chinense Pursh

[0102] We used the purified product prepared in Example 1 as the raw material for the efficacy investigation and detected the effect of total lignans from Penthorum chinense Pursh on carrageenan - induced thrombosis in mice.

[0103] Since a mouse's tail has only one caudal artery and is a typical single-artery circulatory blood supply, once embolism occurs, collateral circulation cannot be carried out, leading to dry necrosis at the thrombus site. Injecting carrageenan into the abdominal cavity of mice to simulate a clinical thrombus model causes little trauma to the animals, and the thrombus appears in the tail, making it easy to observe and measure. It is currently widely used in the evaluation of the antithrombotic efficacy of drugs.

[0104] Forty SPF-grade healthy Kunming mice, with 20 males and 20 females, weighing 20 ± 2 g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal license number was SCXK(Jing)2016-0006. The 40 mice were randomly divided into 4 groups, with 10 mice in each group. Normal group and model group: gavaged with normal saline; low-dose group: 50 mg / kg·BW of the total lignans of Penthorum chinense Pursh; high-dose group: 200 mg / kg·BW of the total lignans of Penthorum chinense Pursh. Gavage was performed once a day for 7 consecutive days. One hour after the last administration, except for the normal group, the other 3 groups of mice were intraperitoneally injected with 1% carrageenan at 50 mg / kg. A dark red thrombus formation area appeared at the tip of the mouse tail 4 - 24 h after injection, gradually expanding towards the tail root to a certain extent. After 48 - 72 h, it changed from purple to black, with a clear demarcation from the normal tail. After measuring the black tail rate of the mice (black tail rate % = number of mice with black tails / total number of mice * 100) and the black tail length 72 h later, the mice were anesthetized with 10% chloral hydrate, and blood was collected from the orbital cavity. The blood of each group was allowed to clot naturally at room temperature for 20 min, centrifuged at 3000 r / min for 20 min, and the supernatant was collected. The content of endothelin-1 (ET-1) in each group was measured according to the kit method.

[0105] As can be seen from Table 6, except for the normal group, different degrees of dark red thrombi appeared in the tails of the mice in the other groups. Compared with the normal group, the black tail rate of the mice in the model group reached 80%, and the tail thrombus accounted for about 37% of the total tail length, with a statistically significant difference (P < 0.01), indicating that the model was successfully established. Compared with the model group, the black tail rate and the length of the tail thrombus in the low-dose and high-dose administration groups of mice were significantly reduced, indicating that the total lignans of Penthorum chinense Pursh have an antithrombotic effect.

[0106] Table 6 Effects of Penthorum chinense Pursh extract on mouse tail thrombus formation

[0107] Group Black tail rate at 72 h / % Black tail length at 72 h / cm Normal group 0 0 Model group 80 3.71±1.69** Low - dose administration group 50 1.60±0.81# High - dose administration group 30 0.86±0.24##

[0108] Compared with the normal group, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01

[0109] Serum endothelin (ET) is a bioactive polypeptide synthesized and secreted by vascular endothelial cells and is the most potent vasoconstrictor factor discovered in current research. As one of the ET isomers, ET-1 is the most important vasoconstrictor factor, which can regulate the function of vascular endothelial cells, is highly expressed in damaged blood vessels, and can cause vascular damage.

[0110] According to Figure 11 As shown, compared with the normal group, the serum endothelin-1 (ET-1) in the model group was significantly increased, indicating that the vascular endothelial cells of the thrombus mice in the model group were damaged. After administration of the total lignans of Penthorum chinense, the content of ET-1 was significantly decreased (P < 0.01), indicating that the antithrombotic effect of the extract of Penthorum chinense may be related to the improvement of vascular endothelial cell injury.

Claims

1. A preparation method of total lignans from Penthorum chinense Pursh for anti - thrombosis, characterized in that it comprises the following steps: 1) After pulverizing the Patrinia scabra Buch.-Ham. ex D. Don medicinal material, add an enzymatic hydrolysate, enzymatically hydrolyze at a temperature of 30 - 60 °C for 0.5 - 2 hours, then add an entrainer, and carry out CO 2 supercritical fluid extraction at a temperature of 30 - 60 °C and a pressure of 5 - 30 Mpa. Centrifuge the extract, and after concentrating and drying the supernatant, obtain a Patrinia scabra Buch.-Ham. ex D. Don extract containing total lignans; 2) Adsorb the extract with AB - 8 macroporous resin, then elute successively with water and 95% ethanol, collect the ethanol eluate, concentrate and dry to obtain the product. The enzymatic hydrolysate contains pectinase, and the mass of the pectinase accounts for 0.8 - 1.5% of the mass of Penthorum chinense Pursh medicinal materials; The entrainer is a mixed solution of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 1:2 - 5, and the volume ratio of the entrainer to the enzymatic hydrolysate is 1:3 - 5.

2. The preparation method of total lignans from Penthorum chinense Pursh according to claim 1, characterized in that: The temperature of the enzymatic hydrolysis is 45 - 55 °C and the time is 1 - 1.5 hours.

3. The preparation method of total lignans from Penthorum chinense Pursh according to claim 1, characterized in that: The CO 2 The temperature of the supercritical fluid extraction is 50-55 °C and the pressure is 25-30 Mpa.

4. The preparation method of total lignans from Penthorum chinense Pursh according to claim 1, characterized in that: The CO of the supercritical fluid extraction 2 flow rate is 30 - 50 / L·h -1 , and the extraction time is 1 - 3 h.

5. A total lignans from Penthorum chinense Pursh for anti - thrombosis, which is prepared by the method according to any one of claims 1 - 4.

6. Use of the total lignans from Penthorum chinense Pursh according to claim 5 in the preparation of anti - thrombosis drugs.

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