Leonurus phenolic acid extract with thrombolysis effect as well as preparation method and application thereof

Abstract

The invention provides a motherwort phenolic acid extract with a thrombolysis effect as well as a preparation method and application of the motherwort phenolic acid extract. The method provided by the invention can effectively extract the effective part with fibrinolysis effect in motherwort; the herba leonuri phenolic acid extract is mainly prepared from vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3, 4, 5-trimethyl gallic acid, and the total content of the herba leonuri phenolic acid extract is not lower than 70%. The herba leonuri phenolic acid extract has a good dissolving effect on fibrin in thrombosis, has thrombus dissolving and antithrombotic effects, and can be processed into various common preparations or sustained release preparations such as oral preparations such as tablets, capsules, granules and dropping pills according to a conventional production process of pharmaceutics; injection preparations such as injection freeze-dried powder injections and external preparations such as ointment and emulsifiable paste are used for treating thrombotic diseases (including cerebral thrombosis and subcutaneous blood stasis).

Description

Technical Field

[0001] The present invention relates to an extract of the traditional Chinese medicine Leonurus japonicus Houtt., and more particularly to an extract of phenolic acids from Leonurus japonicus Houtt. with thrombolytic effect, its preparation method and application. Background Art

[0002] Thrombotic diseases refer to vascular diseases in which, under certain conditions, blood components form emboli in blood vessels, causing partial or complete blockage of blood vessels and blood supply disorders in the corresponding parts. When a thrombus detaches from its formation site and flows with the blood, thromboembolism can cause partial or complete blockage of blood vessels, leading to ischemia, infarction and dysfunction of major organs, thus triggering thrombotic diseases. Among them, cardiovascular and cerebrovascular diseases such as cerebral infarction, myocardial infarction, coronary heart disease, and atherosclerosis cause 12 million deaths globally every year. At the same time, cardiovascular and cerebrovascular diseases can occur at all ages, and the incidence of thrombotic diseases ranks first among various diseases. Its high disability rate and mortality rate make it one of the most fatal health threats faced by humans.

[0003] Leonurus japonicus Houtt. is the fresh or dried aerial part of the plant Leonurus japonicus Houtt. in the family Lamiaceae, which is produced throughout the country and has the effects of activating blood circulation and regulating menstruation. Clinically, it is used in thrombotic diseases and has achieved satisfactory curative effects. Duan Chenglin of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine treated 24 patients with cerebral thrombosis with single-flavor Leonurus japonicus Houtt., and after treatment, 16 cases were cured and 6 cases improved, with a total effective rate of 92% (Duan Chenglin et al., Chinese Folk Remedies, 1997, (04): 39-40).

[0004] Pharmacological experimental studies have shown that Leonurus japonicus Houtt. has a strong antithrombotic effect. Xie Wenguang et al. studied the effects of more than 20 commonly used drugs for activating blood circulation and removing blood stasis on fibrinolytic activity, and found that the fibrinolytic activity of the 56°C water bath extract of Leonurus japonicus Houtt. was the strongest (Xie Wenguang et al., Acta Chinese Medicine and Pharmacology, 1996, 11(6): 18-21). Leonurus japonicus Houtt. injection has a significant inhibitory effect on ADP-induced platelet aggregation (Zhao Xiaomei et al., Chinese Journal of Experimental Traditional Medical Formulae, 2014, 20(4): 128-130). Oral administration of the water decoction of Leonurus japonicus Houtt. can significantly prolong the clotting time of mice and show a dose-effect relationship (Liu Weiguo et al., Contemporary Animal Husbandry, 2018(9): 44-45).

[0005] The chemical components in Leonurus japonicus Houtt. are relatively complex, mainly including alkaloids (such as stachydrine, leonurine, leonuridine, etc.), flavonoids (such as kaempferol, quercetin, wogonin, etc.), terpenoids (such as preleonurine, leonurine, preleonurine B, etc.), coumarins (such as xanthotoxin, isoimperatorin, etc.) and phenethyl alcohols (such as leonurinoside C, isomucronulatol glycoside, etc.).

[0006] So far, the components with fibrinolytic (hemolytic) effects in Leonurus japonicus Houtt. are not clear. Therefore, studying and effectively extracting the fibrinolytic active components in Leonurus japonicus Houtt. to obtain the chemical parts with fibrinolytic effects is of great significance for the efficient utilization of Leonurus japonicus Houtt. and the development of anti-thrombotic drugs. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technology, the present invention provides a phenolic acid extract of Leonurus japonicus Houtt. with thrombolytic effects, its preparation method and application. The method of the present invention can effectively extract the chemical parts with fibrinolytic effects in Leonurus japonicus Houtt.; the obtained phenolic acid extract of Leonurus japonicus Houtt. is mainly composed of vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethyl gallic acid, and its total content is not less than 70%. The phenolic acid extract of Leonurus japonicus Houtt. of the present invention has a good dissolving effect on fibrin in thrombus formation, and can be processed into oral preparations such as tablets, capsules, granules, dripping pills and other common preparations or sustained-release preparations, injection preparations such as freeze-dried powder for injection, and external preparations such as ointments, creams, etc. according to the conventional pharmaceutical production process for the treatment of thrombotic diseases.

[0008] The technical solution of the present invention is as follows:

[0009] A phenolic acid extract of Leonurus japonicus Houtt. with thrombolytic effects, which is mainly composed of vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethyl gallic acid, and the total mass content of vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethyl gallic acid is greater than or equal to 70%; preferably, in the phenolic acid extract of Leonurus japonicus Houtt., the total mass content of vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethyl gallic acid is greater than or equal to 80%.

[0010] The preparation method of the above-mentioned phenolic acid extract of Leonurus japonicus Houtt. with thrombolytic effects includes the steps of: using the aerial stems and leaves of Leonurus japonicus Houtt. as raw materials, and preparing the phenolic acid extract of Leonurus japonicus Houtt. with thrombolytic effects through extraction, enrichment, separation and purification;

[0011] The extraction method is an alcohol-water extraction method or an alkali-water extraction method; the enrichment method is an extraction method, an alkali-water kneading and dissolving method, a macroporous adsorption resin column chromatography method or a strongly basic anion exchange resin method; the separation method is a silica gel partition column chromatography method or an ODS silica gel column chromatography method.

[0012] According to the preference of the present invention, the alcohol-water extraction method includes the steps of: crushing the aerial stems and leaves of Leonurus japonicus Houtt. into coarse powder, then immersing it in a solvent, and obtaining the Leonurus japonicus Houtt. extract through extraction and filtration;

[0013] Alternatively, the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, soaked in an aqueous NaOH solution, the pH value is adjusted to neutral, and then filtered; then it is immersed in a solvent, extracted, and filtered to obtain an extract of Leonurus japonicus.

[0014] Preferably, the solvent is water, a 50 wt% - 100 wt% methanol solution, or a 50 wt% - 95 wt% ethanol aqueous solution; the weight ratio of the solvent to the coarse powder is 5:1 - 20:1.

[0015] Preferably, in the alcohol-water extraction method, the extraction method is heating reflux extraction, ultrasonic extraction, percolation, or maceration; more preferably, the extraction method is heating reflux extraction or ultrasonic extraction; the extraction time for heating reflux extraction is 1 - 2 h each time, the ultrasonic temperature for ultrasonic extraction is 40 - 60 °C, and the ultrasonic extraction time each time is 20 - 40 min; the number of extractions is 1 - 5 times.

[0016] Preferably, the concentration of the aqueous NaOH solution is 0.5 - 2 wt%, the soaking temperature is room temperature, and the soaking time is 2 - 12 h; a 1 - 5 wt% hydrochloric acid aqueous solution is used to adjust the pH.

[0017] Preferably, the extract of Leonurus japonicus can be further concentrated under normal pressure or reduced pressure to obtain an extract paste of Leonurus japonicus.

[0018] According to the preference of the present invention, the alkaline water extraction method includes the steps: the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, soaked and extracted in an aqueous NaOH solution, and filtered to obtain an alkaline water extract of Leonurus japonicus.

[0019] Preferably, before the soaking and extraction step, it further includes a soaking step in water; the mass ratio of water to the coarse powder is 1 - 5:1; the soaking temperature is room temperature, and the soaking time is 2 - 8 h.

[0020] Preferably, the concentration of the aqueous NaOH solution is 0.5 wt% - 1.5 wt%; the mass ratio of the aqueous NaOH solution to the coarse powder is 5 - 12:1.

[0021] Preferably, the number of soaking and extraction times is 1 - 5 times; the method for each soaking and extraction is: standing at room temperature for 2 - 12 h, or ultrasonic treatment at room temperature for 15 - 30 minutes, or standing at room temperature for 2 - 12 h and then ultrasonic treatment at room temperature for 15 - 30 minutes.

[0022] Preferably, after the filtration step, it may further include a step of adjusting the pH, and a hydrochloric acid aqueous solution is used to adjust the pH to 5 - 7.

[0023] Preferably, the alkaline water extract of Leonurus japonicus can be further concentrated under normal pressure or reduced pressure to obtain an extract paste of Leonurus japonicus.

[0024] Preferably according to the present invention, the extraction method comprises the steps of: diluting the obtained extract with water to obtain a mixed solution; sequentially extracting with petroleum ether, dichloromethane, and ethyl acetate as extraction agents, discarding the petroleum ether extraction solution, and subjecting the dichloromethane extraction solution and the ethyl acetate extraction solution to vacuum concentration respectively to obtain a dichloromethane extract (A) and an ethyl acetate extract (B); suspending the dichloromethane extract (A) in water to obtain a suspension, extracting with ethyl acetate as an extraction agent, and subjecting the ethyl acetate extraction solution to vacuum concentration to obtain an ethyl acetate extract (C); combining the ethyl acetate extract (B) and the ethyl acetate extract (C) to obtain a motherwort phenolic acid enriched product.

[0025] Preferably, in the dilution of the extract with water, the volume of water is 3 to 6 times the volume of the extract; in the sequential extraction with petroleum ether, dichloromethane, and ethyl acetate as extraction agents, extraction is carried out 2 to 5 times under each extraction agent, and the volume of the extraction agent used each time is the same as the volume of the mixed solution; in the process of suspending the dichloromethane extract (A) in water, the volume of water is the same as the volume of the dichloromethane extract (A); in the process of extracting the suspension with ethyl acetate as an extraction agent, the number of extractions is 2 to 5 times, and the volume of ethyl acetate used each time is the same as the volume of the suspension.

[0026] Preferably according to the present invention, the alkaline water kneading and dissolving method comprises the steps of: adding an NaOH aqueous solution to the obtained extract, kneading and dissolving, and filtering; adding the filter residue to the NaOH aqueous solution, kneading and dissolving, and filtering, and repeating this step 2 to 5 times for the obtained filter residue, combining the filtrates to obtain an alkaline aqueous solution of motherwort; adjusting the pH value to neutral to obtain a feed liquid; subjecting the feed liquid to vacuum concentration to obtain a motherwort phenolic acid enriched product.

[0027] Preferably, the concentration of the NaOH aqueous solution is 1 - 5 wt%; the mass of the NaOH aqueous solution is 2 to 5 times the mass of the extract; the obtained alkaline aqueous solution of motherwort is adjusted to neutral with a hydrochloric acid aqueous solution having a concentration of 4 - 8 N.

[0028] Preferably according to the present invention, the macroporous adsorption resin column chromatography method comprises the steps of: adjusting the pH value of the obtained extract to 2 - 7, injecting it into a macroporous adsorption resin column bed, and then sequentially using water, a 50 wt% ethanol aqueous solution, and a 95 wt% ethanol aqueous solution as eluents for elution, combining the eluates of the 50 wt% ethanol aqueous solution and the 95 wt% ethanol aqueous solution, and concentrating until there is no alcohol smell to obtain a motherwort phenolic acid enriched product.

[0029] Preferably, a 4 - 6 N hydrochloric acid aqueous solution is used to adjust the pH value to 2 - 7; the flow rate of injecting into the macroporous adsorption resin column bed is 0.1 - 0.3 BV / h; the flow rate of the eluent is 0.4 - 0.6 BV / h; the eluent is washed with water for 2 - 6 retention volumes (BV), and the 50 wt% ethanol aqueous solution and the 95 wt% ethanol aqueous solution of the eluent are respectively washed for 6 - 20 retention volumes (BV).

[0030] Preferably according to the present invention, the strong basic anion exchange resin method comprises the steps of: injecting the obtained extract into a strong basic anion exchange resin column, and then eluting successively with water and an acid aqueous solution as the eluent, adjusting the pH of the eluate obtained with the acid aqueous solution as the eluent to 3-7, and concentrating under reduced pressure to obtain a concentrated solution; extracting with ethyl acetate and concentrating to obtain a leonuritic acid enriched product.

[0031] Preferably, the injection flow rate of the extract is 0.1-0.3 BV / h; the flow rate of the eluent is 0.3-0.8 BV / h; the eluent is washed with water for 2-5 BV, and the eluent is washed with the acid aqueous solution for 4-10 BV; the concentration of the acid aqueous solution of the eluent is 0.5-2 N, and the acid is hydrochloric acid, sulfuric acid or phosphoric acid; the pH is adjusted to 3-7 with a 4-6 N NaOH aqueous solution; the extract is concentrated under reduced pressure to 2-6 times the mass of the extract; the extraction times are 2-6 times, and the volume ratio of ethyl acetate to the concentrated solution used for each extraction is 1:1.

[0032] Preferably according to the present invention, the silica gel partition column chromatography method comprises the steps of: dissolving the leonuritic acid enriched product in methanol, mixing with silica gel of 100-200 mesh, and drying at room temperature to obtain a silica gel mixed sample; suspending silica gel of 200-300 mesh with water-saturated dichloromethane to pack a column, loading the silica gel mixed sample onto the top of the silica gel column bed, first eluting with the lower phase (water-saturated dichloromethane phase) of dichloromethane-water with a volume ratio of 100:5 for 1-3 BV, then eluting with the lower phase (dichloromethane phase saturated with water and methanol) of dichloromethane-methanol-water with a volume ratio of 100:1:5 for 2-5 BV, then successively eluting with the lower phase (dichloromethane phase saturated with water and methanol) of dichloromethane-methanol-water with volume ratios of 100:2:5 and 100:4:5 for 3-8 BV respectively, and then eluting with the lower phase (dichloromethane phase saturated with water and methanol) of dichloromethane-methanol-water with a volume ratio of 100:10:8 for 2-5 BV; combining the eluates of the lower phases of dichloromethane-methanol-water with volume ratios of 100:2:5 and 100:4:5, and concentrating under reduced pressure to obtain a crude leonuritic acid product. Preferably, the mass ratio of the leonuritic acid enriched product to methanol is 1:2-3, and the mass ratio of silica gel to the leonuritic acid enriched product is 2-4:1.

[0033] Preferably according to the present invention, the ODS silica gel column chromatography method comprises the steps of: dissolving the leonuritic acid enriched product in methanol, loading the sample onto the ODS silica gel column bed by the wet method, first eluting with a 10 wt% methanol aqueous solution for 2-4 BV, and then successively eluting with 50 wt% methanol aqueous solution, 70 wt% methanol aqueous solution, 90 wt% methanol aqueous solution, and 100 wt% methanol for 2-8 BV; combining the eluates of the 50 wt% methanol aqueous solution and the 70 wt% methanol aqueous solution, and concentrating under reduced pressure to obtain a crude leonuritic acid product. Preferably, the mass ratio of the leonuritic acid enriched product to methanol is 1:1-3.

[0034] Preferably according to the present invention, the purification method comprises the steps of: dissolving the above-mentioned crude phenolic acid of Leonurus japonicus in methanol, loading it onto the bed of an MCI chromatographic column, first eluting with an 80 wt% methanol aqueous solution for 5 - 10 BV, and then eluting with 100% methanol for 3 - 6 BV; taking the eluate of the 80 wt% methanol aqueous solution, concentrating it under reduced pressure to obtain the phenolic acid extract of Leonurus japonicus. Preferably, the mass ratio of the crude phenolic acid of Leonurus japonicus to methanol is 1:1 - 3.

[0035] The present invention adopts the internationally and domestically common fibrin plate method. Through fibrinolysis activity tracking, the active sites ACT1 and ACT2 with fibrinolysis activity in Leonurus japonicus were first discovered (see Test Examples 1 and 2). Through component separation and structure identification of the active site ACT2, its main components were determined to be: vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid, and 3,4,5-trimethyl gallic acid (see Test Example 3). The composition and content of the main active components in the fibrinolysis active site of Leonurus japonicus were analyzed and determined, and the content of syringic acid was the highest among them (see Test Example 4).

[0036] Adopting the internationally and domestically common fibrin plate method, using the clinically commonly used thrombolytic drug urokinase as a positive control, preparing a urokinase solution with a concentration of 40 U / ml with physiological saline. The fibrinolysis active site obtained from Leonurus japonicus and the single compounds vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid (containing a small amount of trans-ferulic acid), and 3,4,5-trimethyl gallic acid obtained in Test Example 3 were respectively dissolved in 100 μL of chromatographic methanol to make their concentration 20 mg / mL, and then the diameter φ (mm) of the fibrinolysis circle on the fibrin plate was measured. The results showed that the five single compounds vanillic acid, syringic acid, trans-ferulic acid, 3,4,5-trimethyl gallic acid, and cis-ferulic acid (a mixture containing a small amount of trans-ferulic acid) all had strong protein fibrinolysis activity, and among them, the fibrinolysis activities of syringic acid and vanillic acid were the strongest; the phenolic acid extracts ACT2 and ACT3 of Leonurus japonicus (Example 1) both had relatively large fibrinolysis circle diameters (see Test Examples 5, 6, and 7).

[0037] Adopting the internationally and domestically common in vitro thrombus formation experimental method (Chandler loop method), using the blood of male Wistar rats as the research object, using the blank group as the negative control and aspirin as the positive control, the experimental results are shown in Test Example 8. Compared with the blank group, in the high-dose, medium-dose, and low-dose syringic acid administration groups and the positive drug aspirin administration group, there were significant differences in the average wet weight and average dry weight of the thrombus, indicating that syringic acid has a significant anti-thrombotic effect. Among them, the high-dose syringic acid group showed a better anti-thrombotic effect than the high-dose aspirin group, indicating its potential application value in good anti-thrombotic formation, laying a foundation for further development into a drug for treating thrombotic diseases.

[0038] The present invention adopts the Chandler loop experiment method, uses the blood of male Wistar rats as the research object, takes the blank group as the negative control, and takes urokinase as the positive control. The experimental results are shown in Test Example 9. After the high-dose group and the medium-dose group of each administration group were co-incubated with the newly formed thrombus for 4 hours, the thrombolysis rates of the high-dose group and the medium-dose group were significantly higher than or equivalent to those of the high-dose group and the medium-dose group of urokinase, indicating that the fibrinolytic active sites obtained from Leonurus japonicus have extremely significant thrombolytic effects and can be used for the treatment of thrombotic diseases.

[0039] The present invention adopts an in vitro blood-brain barrier penetration experiment. The measured values and experimental penetration values of the literature theoretical penetration values of four commercially available drugs (estradiol, progesterone, alprazolam, oxazepam) are used to make a standard curve for subsequent correction, and a good linear correlation equation is obtained: y = 1.9489x + 2.559, R 2 = 0.9616. According to this equation and considering the limits established by Di et al., the following criteria are established: A Pe value above 5.1×10 -6 cm / s indicates that the compound has good blood-brain barrier permeability, and a Pe value less than 5.1×10 -6 cm / s indicates that the compound has poor blood-brain barrier permeability. The measured Pe values of the phenolic acid extracts ACT2 and ACT3 of Leonurus japonicus are 6.1×10 -6 cm / s and 6.4×10 -6 cm / s respectively, which are greater than the Pe value of 5.1×10 -6 cm / s established by Di et al.; among the 4 single compounds separated from the phenolic acid extract of Leonurus japonicus, except for trans-ferulic acid (Pe value of 1.2×10 -6 cm / s), the Pe values of vanillic acid, syringic acid, and 3,4,5-trimethyl gallic acid are 7.1×10 -6 cm / s, 9.2×10 -6 cm / s, and 8.1×10 - 6 cm / s respectively, all of which are greater than the Pe value of 5.1×10 -6 cm / s, indicating that the phenolic acid extracts ACT2 and ACT3 of Leonurus japonicus and the single compounds vanillic acid, syringic acid, and 3,4,5-trimethyl gallic acid all have good blood-brain barrier penetration ability, and the permeability of syringic acid is the largest, which is similar to the penetration ability of the control drug oxazepam. Therefore, the phenolic acid extracts ACT2 and ACT3 of Leonurus japonicus and the vanillic acid, syringic acid, and 3,4,5-trimethyl gallic acid separated therefrom all have good blood-brain barrier penetration ability and can be used for the treatment of cerebral thrombosis diseases (see Test Example 12).

[0040] In addition, through experimental studies, it has been found that the phenolic acid extract of Leonurus japonicus Houtt. has certain anticoagulant and antiplatelet aggregation effects (see Test Examples 10 and 11).

[0041] The above experimental studies show that the phenolic acid extract of Leonurus japonicus Houtt. in the present invention has good fibrinolytic activity, significant thrombolytic effect, and high blood-brain barrier permeability, and can be used for the treatment of thrombotic diseases, especially cerebral thrombosis.

[0042] The application of the above-mentioned phenolic acid extract of Leonurus japonicus Houtt. with thrombolytic effect is used for preparing drugs with thrombolytic or antithrombotic effects.

[0043] The phenolic acid extract of Leonurus japonicus Houtt. of the present invention or its pharmaceutically acceptable salts are combined with pharmaceutical excipients to form pharmaceutical preparations of different dosage forms for the treatment of thrombotic diseases.

[0044] A drug composition with thrombolytic effect, comprising the phenolic acid extract of Leonurus japonicus Houtt. of the present invention or its pharmaceutically acceptable salts, one or more pharmaceutically acceptable carriers or excipients, and antioxidants.

[0045] Preferably according to the present invention, the pharmaceutically acceptable salt is prepared by dissolving the phenolic acid extract of Leonurus japonicus Houtt. in distilled water and adjusting the pH to 6.8 - 8.0 with one of the alkalizing reagents NaOH, KOH, Na2CO3, K2CO3, NaHCO3, KHCO3, MgHPO3, Mg(H2PO3)2 to form sodium salt, potassium salt or magnesium salt. Preferably, the alkalizing reagent is NaHCO3 or KHCO3.

[0046] Preferably according to the present invention, the antioxidant is selected from sodium ascorbate, ascorbic acid, citric acid or sodium metabisulfite.

[0047] Preferably according to the present invention, the drug composition is one of tablets, capsules, granules, freeze-dried injection preparations, powder injections, pellets, ointments, and can be prepared according to the conventional production processes of pharmacy.

[0048] The detailed descriptions of the pharmaceutical preparations of different dosage forms of the drug composition are as follows:

[0049] (1) Preparation of granules, capsules, enteric-coated capsules, tablets or enteric-coated tablets:

[0050] Take the phenolic acid extract of Leonurus japonicus Houtt., and based on the weight of the phenolic acid extract of Leonurus japonicus Houtt. being 1 weight times, add 1 - 4 weight times of diluent I, 2 - 8 wt% of wetting agent, and 3 - 20 wt% of disintegrant, granulate by the conventional wet granulation method, dry, size the granules, and bag them to obtain a granule preparation; or add 0.2 - 10 wt% of lubricant to the sized granules, mix evenly, fill them into capsule shells to obtain a capsule preparation, or compress them into tablets, or fill them into enteric capsule shells to obtain enteric capsules, or apply an enteric coating after compression to obtain enteric tablets.

[0051] The above-mentioned diluent I is selected from starch, sugar powder, dextrin, microcrystalline cellulose or hydroxypropyl cellulose;

[0052] The above-mentioned wetting agent is selected from water or ethanol;

[0053] The above-mentioned disintegrant is sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose;

[0054] The above-mentioned lubricant is magnesium stearate or talc powder.

[0055] (2) Preparation of pellets, pellet capsules, enteric-coated pellet capsules, enteric-coated pellet tablets, enteric-coated pellet sustained-release capsules or tablets

[0056] Take the above-mentioned phenolic acid extract of Leonurus japonicus Houtt., prepare a film coating agent with 1.2 wt% hydroxypropyl methylcellulose aqueous solution and polysorbate - 80, add talc powder, and mix evenly; add blank pellets into a fluidized bed, spray the medicine from the bottom to obtain pellet cores. Coating the pellet cores, coating the isolation layer, and drying to obtain pellets; or coating an enteric coating with polyacrylic resin, polyvinyl acetate phthalate and / or diethyl phthalate / enteric-type Opadry, and drying to obtain enteric-coated pellets, or after coating the isolation layer on the pellet cores, spraying an aqueous solution of enteric-type Opadry coating premix to coat the enteric coating to obtain enteric-coated pellets. Fill the pellets or enteric-coated pellets into capsules to obtain pellet capsules or enteric-coated pellet capsules; compress the pellets or enteric-coated pellets together with binders such as povidone and microcrystalline cellulose into tablets to obtain pellet tablets or enteric-coated pellet tablets. Coating the above-mentioned enteric-coated pellets with a sustained-release layer to obtain sustained-release enteric-coated pellets, filling them into ordinary capsule shells to obtain sustained-release enteric-coated pellet capsules, or compressing them into tablets to obtain sustained-release enteric-coated pellet tablets.

[0057] Preferably, the auxiliary material for the above-mentioned isolation layer is hydroxypropyl methylcellulose;

[0058] Preferably, the auxiliary materials for the above-mentioned enteric coating are polyacrylic resin, polyvinyl acetate phthalate, diethyl phthalate or enteric-type Opadry, and more preferably the enteric-type Opadry coating system;

[0059] Preferably, the above-mentioned sustained-release layer is a coating material such as Eudragit NE30D and DrugCoat L30D, and more preferably Eudragit NE30D.

[0060] (3) Preparation of soft capsules and enteric-coated soft capsules

[0061] Take the prepared phenolic acid extract of Leonurus japonicus, add a diluent, mix well, then add a suspending agent, stir evenly, and let stand at room temperature to obtain the liquid medicine for the content of the soft capsules of phenolic acid of Leonurus japonicus; press it together with the soft capsule shell material prepared in advance with gelatin, glycerol, and preservatives through a mold to form soft capsules. The soft capsules prepared according to the above method are then coated with an isolating layer with hydroxypropyl methylcellulose phthalate, and then coated with an enteric coating with cellulose acetate phthalate or polyvinyl acetate phthalate as the enteric coating material; or the soft capsules prepared by the above method are sprayed with an aqueous solution of an enteric-type Opadry coating premix to coat the enteric coating to obtain enteric-coated soft capsules.

[0062] The above diluent is selected from vegetable oils;

[0063] The above suspending agent is selected from beeswax, chitin methylcellulose or agar.

[0064] (4) Preparation of freeze-dried preparation for injection of phenolic acid (salt) of Leonurus japonicus

[0065] Take the prepared phenolic acid extract of Leonurus japonicus, add an antioxidant, add mannitol accounting for 5% (w / v) of the total volume, dissolve it in distilled water, make up the volume, filter and sterilize, dispense under aseptic conditions, freeze-dry, and then seal under nitrogen to obtain the freeze-dried preparation for injection of phenolic acid of Leonurus japonicus. When in use, dissolve it with an equivalent amount of basic reagent for injection to form a solution.

[0066] Or, dissolve the prepared phenolic acid extract of Leonurus japonicus in distilled water, adjust the pH = 6.8 - 8.0 with an aqueous solution of an alkalizing reagent, then add an antioxidant, add mannitol accounting for 5% (w / v) of the total volume, filter to remove insoluble substances, filter and sterilize, dispense into ampoules under aseptic conditions, freeze-dry, and then seal under nitrogen to obtain it.

[0067] The above antioxidant is sodium ascorbate or ascorbic acid.

[0068] The above alkalizing reagent is an aqueous solution of NaHCO3 or KHCO3.

[0069] The above basic reagent for injection is an aqueous solution of NaOH, NaHCO3 or KHCO3.

[0070] (5) Preparation of powder for injection of phenolic acid salt of Leonurus japonicus

[0071] Take the phenolic acid extract of Leonurus japonicus, dissolve it in distilled water, add an antioxidant, add mannitol accounting for 5% (w / v) of the total volume, adjust the pH to 6.8 - 8.0 with an aqueous solution of an alkalizing reagent, stir, filter to remove insoluble substances, then filter and sterilize, and freeze-dry under aseptic conditions. Grind and mix the prepared pharmaceutical composition, and dispense it into ampoules, and seal it under nitrogen to obtain the product.

[0072] The above-mentioned antioxidant is sodium ascorbate or ascorbic acid.

[0073] The above-mentioned alkalizing reagent is an aqueous solution of NaHCO3 or KHCO3.

[0074] (6) Preparation of Leonurus japonicus phenolic acid ointment

[0075] Dissolve azone or triethanolamine, glycerol, sodium lauryl sulfate, and methylparaben in an appropriate amount of distilled water at 85°C, add the phenolic acid extract of Leonurus japonicus, and mix evenly to obtain the aqueous phase; melt vaseline, lanolin, liquid paraffin, and stearic acid at 85°C to obtain the oil phase. Then slowly add the aqueous phase to the oil phase, and at the same time, quickly stir in the same direction to make it fully mixed and emulsified, and obtain the Leonurus japonicus extract ointment after cooling.

[0076] (7) Preparation of Leonurus japonicus phenolic acid cream

[0077] Take the phenolic acid extract of Leonurus japonicus, moisten it with an appropriate amount of propylene glycol, and grind it until no visible particles remain. Take glyceryl monostearate, dimethyl silicone oil, white vaseline, cetyl alcohol, and ethylparaben, heat to 80°C, and keep warm to obtain the oil phase; take glycerol, citric acid, sodium citrate, Tween-80, Peregal A-20, and distilled water, heat to 80°C to obtain the aqueous phase. Slowly add the oil phase to the aqueous phase, stir while adding, and perform ultrasonic treatment to obtain the cream matrix. When it reaches about 60°C, add the mixture of syringic acid and propylene glycol, mix well, and condense to obtain the product. The application of a pharmaceutical composition with thrombolytic effect of the present invention is used for the treatment of thrombotic diseases, dissolving thrombus, and can improve or eliminate blood circulation disorders caused by thrombus.

[0078] The technical features and beneficial effects of the present invention are as follows:

[0079] The method of the present invention can effectively extract the chemical part with fibrinolytic effect from Leonurus japonicus; the obtained phenolic acid extract of Leonurus japonicus is mainly composed of vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid, and 3,4,5-trimethyl gallic acid, and its total content is not less than 70%.

[0080] Through different extraction, separation and other methods, various different chemical fractions are obtained in the present invention. By combining activity evaluation and conducting step-by-step experimental tests, through the adoption of different separation methods, activity evaluation of the obtained different chemical fractions, and repeated experiments, the chemical fraction with fibrinolytic activity in Leonurus japonicus Houtt. is obtained.

[0081] There are many kinds of separation methods for traditional Chinese medicine chemical components. Different methods are adopted, and the results of separation and purification of their chemical components will be different, and the composition of their components will be different. On the basis of not knowing the structure of the fibrinolytic active components in Leonurus japonicus Houtt., in the early stage of the process of tracing its fibrinolytic active components, separation methods such as adsorption silica gel column chromatography, reverse-phase silica gel column chromatography, polyamide column chromatography, Sephadex LH-20 gel column chromatography, and MCI column chromatography were used. As a result, the separated chemical fractions either have no fibrinolytic activity or have no difference in fibrinolytic activity, and the active site of Leonurus japonicus Houtt. cannot be determined. It was not until the method of the present invention was adopted that an ideal fibrinolytic active site was obtained. After obtaining the fibrinolytic active site, methods such as MCI column chromatography can be used for further purification to increase the content of active ingredients in the active site.

[0082] In view of the physicochemical properties of phenolic acid components, the present invention designs more suitable extraction, separation and purification methods. For example, the method of heating and reflux extraction with solvent water is adopted. First, the medicinal material powder is moistened with an alkaline solution and left standing (the syringic acid group contained in components such as leonurine can be released by alkaline hydrolysis), and the obtained extract is kneaded and dissolved with an alkaline solution, so as to extract or dissolve its phenolic acid components more effectively.

[0083] The present invention uses modern separation techniques and methods such as nuclear magnetic resonance spectroscopy determination to separate and identify the structure of its active site, and determines that the main components contained therein are vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethylgallic acid. By using high performance liquid chromatography and combining corresponding reference substances, the composition components of its active site are analyzed, further proving that its active site is mainly vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethylgallic acid, and the contents of its main components are determined.

[0084] The present invention discovers for the first time the fibrinolytic activity of the phenolic acid extract of Leonurus japonicus Houtt. and its contained main monomer compounds. Its fibrinolytic active fractions ACT2 and ACT3 and the single compounds vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethylgallic acid obtained therefrom all have good fibrinolytic activity, thus indicating that these several compounds and the phenolic acid extract of Leonurus japonicus Houtt. all have good thrombolytic effects, and these several compounds are the main effective components of the phenolic acid extract of Leonurus japonicus Houtt.

[0085] The present invention for the first time discovers that the phenolic acid extract of Leonurus japonicus has good thrombolytic effect in vitro. Taking urokinase as the positive control, the thrombolysis rates of its high-dose group and medium-dose group are significantly higher than or equivalent to those of the high-dose group and medium-dose group of urokinase, indicating that the fibrinolytic active parts obtained from Leonurus japonicus all have extremely significant thrombolytic effects and can be used for the treatment of thrombotic diseases.

[0086] The present invention adopts the blood-brain barrier penetration experiment research and discovers that the phenolic acid extract of Leonurus japonicus and the single compounds vanillic acid, syringic acid, 3,4,5-trimethyl gallic acid (except trans-ferulic acid) isolated therefrom all have good blood-brain barrier penetration ability and can be used for the treatment of cerebral thrombosis diseases.

[0087] In addition, the present invention also discovers that the phenolic acid extract of Leonurus japonicus has good anticoagulant, antiplatelet aggregation effects, and good in vitro antithrombotic formation effect. It indicates that it can be used for preventing the formation of thrombus, thus playing a synergistic role together with its thrombolytic effect, which is more beneficial to the treatment of thrombotic diseases, including cerebral thrombosis. Description of the Drawings

[0088] Figure 1 It is the HPLC fingerprint of ACT1 in Test Example 1.

[0089] Figure 2 It is the HPLC fingerprint of ACT2 in Test Example 2.

[0090] Figure 3 It is the content determination standard curve of vanillic acid, syringic acid, trans-ferulic acid, 3,4,5-trimethyl gallic acid in Test Example 4.

[0091] Figure 4 It is the HPLC content determination chromatogram of ACT2 (A) and ACT3 (B) and reference substance (C) in Test Example 4.

[0092] Figure 5 It is the standard curve of fibrinolytic activity of different doses of urokinase with the fibrinolytic circle diameter as the index in Test Example 5.

[0093] Figure 6 It is the in vitro antithrombotic formation activity of ACT2 and ACT3 in Test Example 8; wherein, A is the blank control; B is each dose group of aspirin; C is each dose group of ACT2; D is each dose group of ACT3.

[0094] Figure 7 It is the thrombolytic activity of ACT2 and ACT3 in vitro on newly formed thrombus in Test Example 9; wherein, A is the blank control; B is each dose group of urokinase; C is each dose group of ACT2; D is each dose group of ACT3.

[0095] Figure 8It is the effects of aspirin (ASP), ACT2, and ACT3 on platelet aggregation in Experimental Example 11.

[0096] Figure 9 It is the calibration curve of the measured values and literature penetration values of four commercially available drugs (estradiol, progesterone, alprazolam, oxazepam) in Experimental Example 12. Specific implementation manners

[0097] The present invention will be further described below in conjunction with embodiments, but is not limited thereto.

[0098] Embodiment 1

[0099] A preparation method of a phenolic acid extract of Leonurus japonicus with thrombolytic effect, comprising the steps of:

[0100] The aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, and 60wt% ethanol aqueous solution is used as the extraction solvent for heating under reflux for extraction. For the first time, 12 times the amount (i.e., 12 times the mass of the coarse powder) of 60wt% ethanol aqueous solution is added, and reflux extraction is carried out for 2h, followed by filtration; for the second and third extractions, 10 times the amount of 60wt% ethanol aqueous solution is repeatedly added to the filter residue, and reflux extraction is carried out for 1h. The medicinal residues are filtered off, and the filtrates are combined. Under the condition of 50°C, it is concentrated under reduced pressure until the alcohol smell disappears to obtain the Leonurus japonicus extraction paste.

[0101] Distilled water is added to the Leonurus japonicus extraction paste to obtain a mixed solution. The amount of water added is 6 times the weight of the Leonurus japonicus extraction paste. It is successively extracted with petroleum ether, dichloromethane, and ethyl acetate, and each phase is repeatedly extracted 5 times. The volume of the extraction solvent used each time is the same as the volume of the mixed solution. The petroleum ether layer is discarded, and the dichloromethane phase extraction solution A and the ethyl acetate phase extraction solution B are collected. The extraction solution A is concentrated under reduced pressure to a paste at 50°C, and 5 times the weight of water of the paste is added to suspend it to obtain a suspension, which is extracted with ethyl acetate, and the extraction is repeated 4 times. The volume of the extraction solvent used each time is the same as the volume of the suspension, and the solvent is evaporated under reduced pressure to obtain a solid C. The extraction solution B is evaporated under reduced pressure at 50°C to obtain a solid B. The solids C and B are combined to obtain the crude phenolic acid extract of Leonurus japonicus.

[0102] After dissolving the crude extract of leonurine phenolic acid in methanol (the mass ratio of the crude extract of leonurine phenolic acid to methanol is 1:2.5), silica gel with a mesh size of 100 - 200 is added for sample mixing (the mass ratio of silica gel to the crude extract of leonurine phenolic acid is 3:1). After air-drying at room temperature, the sample-mixed silica gel is obtained, and then subjected to silica gel partition chromatography column separation and purification. Silica gel with a mesh size of 200 - 300 is suspended in water-saturated dichloromethane to pack the column, and the sample-mixed silica gel is loaded onto the top of the silica gel column bed. First, elute with the lower phase (water-saturated dichloromethane phase) of dichloromethane - water with a volume ratio of 100:5 for 2 bed volumes (BV), then elute with the lower phase (dichloromethane phase saturated with water and methanol) of dichloromethane - methanol - water with a volume ratio of 100:1:5 for 4 BV, then elute with the lower phase (dichloromethane phase saturated with water and methanol) of dichloromethane - methanol - water with a volume ratio of 100:2:5 for 8 BV, then elute with the lower phase (dichloromethane phase saturated with water and methanol) of dichloromethane - methanol - water with a volume ratio of 100:4:5 for 6 bed volumes, and then elute with the lower phase (dichloromethane phase saturated with water and methanol) of dichloromethane - methanol - water with a volume ratio of 100:10:8 for 4 BV; combine the eluents of the lower phases of dichloromethane - methanol - water with volume ratios of 100:2:5 and 100:4:5, and evaporate the solvent under reduced pressure at 50 °C to obtain a solid, that is, the crude product of leonurine phenolic acid.

[0103] Dissolve the crude product of leonurine phenolic acid in methanol (the mass ratio of the crude product of leonurine phenolic acid to methanol is 1:2.5), directly load the sample solution onto the top of the MCI chromatographic column bed, first elute with 80 wt% methanol aqueous solution for 8 BV, and then elute with 100% methanol for 3 BV; take the eluate of 80 wt% methanol aqueous solution, concentrate it under reduced pressure to obtain the leonurine phenolic acid extract (named active fraction 3, marked as ACT3).

[0104] HPLC determination conditions: Use a Thermo 25002 - 101030 (1 mm * 100 mm, 1.9 μm) chromatographic column; use methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, and the gradient elution time program is: 0 - 10 min, 10% methanol aqueous solution; 10 - 15 min, 10% → 20% methanol aqueous solution; 15 - 25 min, 20% → 40% methanol aqueous solution; 25 - 40 min, 40% → 80% methanol aqueous solution. Column temperature: 30 °C, flow rate: 0.3 mL / min, injection volume: 10 μL.

[0105] After HPLC analysis and determination, the total content of the main components vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethyl gallic acid in the leonurine phenolic acid extract ACT3 is 87.4%.

[0106] Example 2

[0107] A preparation method of a phenolic acid extract of Leonurus japonicus with thrombolytic effect, as described in Example 1, the difference is: the extraction method is different, specifically as follows:

[0108] After the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, distilled water is used as the extraction solvent for reflux extraction. For the first time, 12 times the amount of water (i.e., 12 times the mass of the coarse powder) is added, and extraction is carried out at 100 °C for 2 h, and then filtered; for the second and third extractions, 10 times the amount of water is repeatedly added to the filter residue, and extraction is carried out at 100 °C for 1 h. The filter residue is filtered off, and the filtrates are combined. It is concentrated to a paste to obtain an extract of Leonurus japonicus.

[0109] Other steps and conditions are the same as in Example 1 to obtain a phenolic acid extract of Leonurus japonicus.

[0110] Determined by HPLC analysis, the total content of the main components vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid in the phenolic acid extract of Leonurus japonicus is 94.6%.

[0111] Example 3

[0112] A preparation method of a phenolic acid extract of Leonurus japonicus with thrombolytic effect, as described in Example 1, the difference is: the extraction method is different, specifically as follows:

[0113] After the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, 100% methanol is used as the extraction solvent for reflux extraction. For the first time, 12 times the amount of solvent (i.e., 12 times the mass of the coarse powder) is added, and reflux extraction is carried out for 2 h, and then filtered; for the second and third extractions, 10 times the amount of solvent is repeatedly added to the filter residue, and reflux extraction is carried out for 1 h. The filter residue is filtered off, and the filtrates are combined. It is concentrated under reduced pressure at 50 °C until alcohol-free. An extract of Leonurus japonicus is obtained.

[0114] Other steps and conditions are the same as in Example 1 to obtain a phenolic acid extract of Leonurus japonicus.

[0115] Determined by HPLC analysis, the total content of the main components vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid in the phenolic acid extract of Leonurus japonicus is 87.2%.

[0116] Example 4

[0117] A preparation method of a phenolic acid extract of Leonurus japonicus with thrombolytic effect, as described in Example 1, the difference is: the extraction method is different, specifically as follows:

[0118] After the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, an aqueous solution of 1 wt% NaOH twice the weight of the powder is added and moistened at room temperature for 6 hours. Then, the pH is adjusted to neutral with an aqueous solution of 1 wt% hydrochloric acid, and the filter residue is obtained by filtration. Then, an aqueous solution of 95 wt% ethanol ten times the weight of the coarse powder is added to the filter residue for the first time and refluxed for 2 hours, and the filter residue is filtered. For the second and third extractions, an aqueous solution of 70 wt% ethanol ten times the weight of the coarse powder is repeatedly added to the filter residue and refluxed for 1 hour. The medicinal residues are removed, and the filtrates are combined and concentrated under reduced pressure at 50 °C until the ethanol is removed completely, obtaining the Leonurus japonicus extraction paste.

[0119] Other steps and conditions are the same as in Example 1, obtaining the phenolic acid extract of Leonurus japonicus.

[0120] Determined by HPLC analysis, the total content of the main components vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid in the phenolic acid extract of Leonurus japonicus is 91.8%.

[0121] Example 5

[0122] A preparation method of a phenolic acid extract of Leonurus japonicus with thrombolytic effect, as described in Example 1, the difference is: the extraction method is different, specifically as follows:

[0123] After the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, an aqueous solution of distilled water 1.5 times the weight of the coarse powder is added and moistened at room temperature for 5 hours. Then, an aqueous solution of 1 wt% NaOH eight times the weight of the coarse powder is added and impregnated at room temperature for 8 hours, and then filtered. The medicinal residues are subjected to the second and third extractions, and an aqueous solution of 1 wt% NaOH eight times the weight of the coarse powder is repeatedly added for ultrasonic extraction at room temperature for 30 minutes, and the filtrates are combined after filtration. The alkaline aqueous extract is neutralized to neutral with an 8N aqueous solution of hydrochloric acid and concentrated under reduced pressure to a paste, obtaining the Leonurus japonicus extraction paste.

[0124] Other steps and conditions are the same as in Example 1, obtaining the phenolic acid extract of Leonurus japonicus.

[0125] Determined by HPLC analysis, the total content of the main components vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid in the phenolic acid extract of Leonurus japonicus is 88.2%.

[0126] Example 6

[0127] A preparation method of a phenolic acid extract of Leonurus japonicus with thrombolytic effect, as described in Example 1, the difference is: the extraction method and the enrichment method are different, specifically as follows:

[0128] After the aerial stems and leaves of Leonurus japonicus were crushed into coarse powder, 70 wt% methanol aqueous solution eight times the amount of the coarse powder was used as the extraction solvent, and ultrasonic extraction was carried out at 50 °C for 30 minutes, and the extraction was repeated 5 times. The filter residue was removed, and the filtrates were combined. Under the condition of 50 °C, it was concentrated under reduced pressure to a paste to obtain the Leonurus japonicus extraction extract.

[0129] To the Leonurus japonicus extraction extract was added 1 wt% NaOH aqueous solution three times its weight and kneaded and dissolved, and then filtered; the filter residue was kneaded and dissolved again with 1 wt% NaOH aqueous solution twice its weight of the Leonurus japonicus extraction extract, filtered, and this step was continued to be repeated 2 times; the solutions obtained after kneading and dissolving were combined, neutralized to neutral with 8N hydrochloric acid aqueous solution, and concentrated to a paste to obtain the crude extract of Leonurus japonicus phenolic acids.

[0130] Other steps and conditions were the same as in Example 1 to obtain the Leonurus japonicus phenolic acid extract.

[0131] Determined by HPLC analysis, the total content of the main components vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid in the Leonurus japonicus phenolic acid extract was 92.5%.

[0132] Example 7

[0133] A preparation method of a Leonurus japonicus phenolic acid extract with thrombolytic effect, as described in Example 1, the difference is: the extraction method and the enrichment method are different, specifically as follows:

[0134] After the aerial stems and leaves of Leonurus japonicus were crushed into coarse powder, 1 wt% NaOH aqueous solution eight times the weight of the coarse powder was added and impregnated at room temperature for 8 hours, then ultrasonic extraction was carried out at room temperature for 30 minutes, and filtered; the medicinal residues were subjected to the second and third extractions, and 1 wt% NaOH aqueous solution eight times the weight of the coarse powder was repeatedly added to each filter residue, and ultrasonic extraction was carried out at room temperature for 30 minutes each time, and filtered, and the filtrates were combined. The pH value of the filtrate was adjusted to 5 with 5N hydrochloric acid aqueous solution to obtain the Leonurus japonicus alkaloid aqueous extract.

[0135] The Leonurus japonicus alkaloid aqueous extract was directly loaded onto the upper end of the macroporous adsorption resin column bed, and the solution was allowed to flow through the macroporous adsorption resin column bed at a flow rate of 0.2 BV / h, and then eluted with distilled water at a flow rate of 0.5 BV / h for 4 retention volumes (BV), and then successively eluted with 50 wt% and 95 wt% ethanol aqueous solutions at a flow rate of 0.5 BV / h for 8 BV each, and the eluates of 50 wt% ethanol aqueous solution and 95 wt% ethanol aqueous solution were combined and concentrated to no alcohol smell to obtain the crude extract of Leonurus japonicus phenolic acids.

[0136] Other steps and conditions were the same as in Example 1 to obtain the Leonurus japonicus phenolic acid extract.

[0137] Determined by HPLC analysis, the total content of the main components vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid in the Leonurus japonicus phenolic acid extract was 92%.

[0138] Example 8

[0139] A preparation method of a phenolic acid extract of Leonurus japonicus with thrombolytic effect is as described in Example 1, except that the extraction method, enrichment method and separation method are different, specifically as follows:

[0140] The aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, then added with 10 times the weight of 1wt% NaOH aqueous solution based on the coarse powder and impregnated at room temperature for 12 hours, then ultrasonically extracted at room temperature for 30 minutes and filtered; the residue is extracted for the second and third times, and 10 times the weight of 1wt% NaOH aqueous solution based on the coarse powder is added to the filter residue each time, ultrasonically extracted at room temperature for 30 minutes each time and filtered, and the filtrates are combined to obtain an aqueous extract of Leonurine.

[0141] The aqueous extract of Leonurine is directly loaded onto the upper end of the column bed of a strongly basic anion exchange resin column, and allowed to flow through the column bed of the anion exchange resin column at a flow rate of 0.2BV / h, eluted with distilled water for 4BV, and then eluted with 1N hydrochloric acid aqueous solution for 8BV, and the flow rate of the eluent is 0.5BV / h. The acid-washed eluate is combined, adjusted to pH 5 with 5N NaOH aqueous solution, concentrated under reduced pressure to 5 times the mass of the aqueous extract of Leonurine to obtain a concentrated solution, and then extracted with ethyl acetate 5 times, and the volume of ethyl acetate used for each extraction is the same as that of the concentrated solution, and the ethyl acetate extracts are combined and concentrated to obtain a crude extract of phenolic acid of Leonurus japonicus.

[0142] Then the crude extract of phenolic acid of Leonurus japonicus is dissolved in a small amount of methanol (the mass ratio of the crude product of phenolic acid of Leonurus japonicus to methanol is 1:2), and loaded onto the upper end of the column bed of an ODS silica gel column by wet method. First, elute with 10wt% methanol aqueous solution for 3BV, and then elute with 50wt% methanol aqueous solution, 70wt% methanol aqueous solution, 90wt% methanol aqueous solution, 100wt% methanol in turn for 6BV, 8BV, 8BV, 4BV. The 50% and 70% methanol aqueous eluates are combined and concentrated under reduced pressure to obtain a crude product of phenolic acid of Leonurus japonicus.

[0143] Other steps and conditions are the same as in Example 1 to obtain an extract of phenolic acid of Leonurus japonicus.

[0144] Determined by HPLC analysis, the total content of the main components vanillic acid, syringic acid, trans-ferulic acid, 3,4,5-trimethylgallic acid in the extract of phenolic acid of Leonurus japonicus is 92.7%.

[0145] Example 9: Preparation of ordinary oral preparations (capsules, granules, tablets)

[0146] 15 g of the phenolic acid extract of Leonurus japonicus Houttuyn obtained in Example 1 was added with 15 g of starch, 5 g of dextrin, and 3 g of microcrystalline cellulose. The above solid substances were each ground through an 80-mesh sieve, and 2.5 g of sodium carboxymethyl starch was added and mixed evenly. An appropriate amount of 10 wt% starch aqueous solution was added as a binder to make wet granules, which were dried at 60 °C, sized, and bagged to obtain the granule preparation of the phenolic acid extract of Leonurus japonicus Houttuyn.

[0147] 1.5 g of talc was added to the above granules and mixed evenly, and then filled into ordinary capsule shells to obtain the capsule preparation of the phenolic acid extract of Leonurus japonicus Houttuyn.

[0148] 1.5 g of talc was added to the above granule preparation of the phenolic acid extract of Leonurus japonicus Houttuyn, mixed evenly, and then tabletted to obtain the tablet preparation of the phenolic acid extract of Leonurus japonicus Houttuyn.

[0149] Example 10: Preparation of enteric-coated oral preparations (enteric-coated capsules, enteric-coated tablets)

[0150] 20 g of the phenolic acid extract of Leonurus japonicus Houttuyn obtained in Example 2 was taken and added with 20 g of pregelatinized starch, 6 g of polyvinylpyrrolidone, 1.5 g of microcrystalline cellulose, 1.2 g of cross-linked carboxymethylcellulose sodium, and 0.8 g of talc. After mixing evenly, a soft material was made with 6 g of 5 wt% cross-linked polyvinylpyrrolidone aqueous solution, granulated, dried, and sized. The obtained granules were filled into enteric capsule shells to obtain the enteric-coated capsule of the phenolic acid extract of Leonurus japonicus Houttuyn.

[0151] The above-obtained granules were tabletted to obtain a tablet core, then an isolation layer was coated with hydroxypropyl cellulose phthalate, and then an enteric coating was coated with cellulose acetate phthalate and diethyl phthalate (the mass ratio of the two is 2:1) to obtain the tablet preparation of the phenolic acid extract of Leonurus japonicus Houttuyn.

[0152] Example 11: Preparation of pellet oral preparations (pellets, pellet capsules, enteric-coated pellet tablets)

[0153] Take 20 g of the leonurine acid extract obtained in Example 2, add it to an aqueous solution of hydroxypropyl methylcellulose with a mass fraction of 3%, then add 2.4 g of polysorbate 80, and stir to mix evenly; add 0.8 g of talc powder and mix well. Preparation of pellet cores: Use the bottom spraying process of a fluidized bed for coating. Preheat the fluidized bed, add 20 g of starch blank pellet cores, first spray an aqueous solution of hydroxypropyl methylcellulose with a mass fraction of 3% to moisten the fluidized bed, and then spray the 3% aqueous solution of hydroxypropyl methylcellulose containing the drug, and spray the drug from the bottom to obtain pellet cores. Then coat the isolation layer: First prepare an aqueous solution of hydroxypropyl cellulose with a mass fraction of 5%. Weigh 0.7 g of magnesium stearate and 8.3 g of talc powder respectively, pour them into the hydroxypropyl methylcellulose E50 solution, stir to mix evenly and sieve, and set aside; in the fluidized bed, spray the above-prepared aqueous solution containing hydroxypropyl cellulose, magnesium stearate and talc powder from the bottom to complete the coating of the isolation layer, and obtain the leonurine acid extract pellets. Divide the pellets into capsules to obtain the leonurine acid extract pellet capsules; add an appropriate amount of 10% starch paste as a binder, add 1.2 g of talc powder, and press tablets to obtain the leonurine acid extract pellet tablets.

[0154] Example 12: Preparation of enteric-coated pellet oral preparations (enteric-coated pellets, enteric-coated pellet capsules, enteric-coated pellet tablets)

[0155] Coat the pellets prepared in Example 11 above with an enteric layer. Dissolve 2.5 g of glyceryl monostearate in pure water, sequentially add 1.6 g of Tween 80 and 3.5 g of triethyl citrate, stir to dissolve to obtain a suspension, and then slowly pour it into 24 g of enteric Opadry, filter to obtain the enteric layer coating solution. Then place the above-prepared pellets in a fluidized bed and spray the coating from the bottom to obtain the enteric-coated leonurine acid extract enteric-coated pellets. Put them into ordinary capsule shells to obtain the leonurine acid extract enteric-coated pellet capsules. Add an appropriate amount of 10% starch paste as a binder to the above pellets, add 1.2 g of magnesium stearate, and press tablets to obtain the leonurine acid extract enteric-coated pellet tablets.

[0156] Example 13: Preparation of sustained-release enteric-coated pellet oral preparations (sustained-release enteric-coated pellets, sustained-release enteric-coated pellet capsules, sustained-release enteric-coated pellet tablets)

[0157] The enteric-coated pellets prepared in Example 12 above were further coated with a sustained-release layer. 20 g of talc powder and 20 g of the solids content of Eudragit NE30D (i.e., the dry weight of the coating material) were added to purified water to prepare a coating suspension with a total solids content of 25%. The pellets containing the enteric layer prepared above were placed in a fluidized bed and coated by bottom spraying to obtain the sustained-release enteric-coated pellets of the motherwort phenolic acid extract. They were filled into ordinary capsule shells to obtain the capsule of the sustained-release enteric-coated pellets of the motherwort phenolic acid extract. An appropriate amount of 10% starch paste was used as a binder for the above pellets, 1.2 g of magnesium stearate was added, and then tablet pressing was carried out to obtain the enteric-sustained-release enteric-coated pellets of the motherwort phenolic acid extract tablets.

[0158] Example 14: Preparation of Soft Capsule of Motherwort Phenolic Acid Extract

[0159] 20 g of the motherwort phenolic acid extract obtained in Example 3 was taken, ground finely, passed through a 40-mesh sieve, added to 5 g of polyethylene glycol-400, heated and stirred, then diluted with 160 g of peanut oil, 4 g of white beeswax was added, and heated to about 38 °C while stirring to obtain the content of the soft capsule; by weight, gelatin: glycerol: water: sorbic acid were proportioned according to the mass ratio of 1.0:0.25:0.80:0.15. First, a certain amount of glycerol and water were put into a gelatinizing tank and heated to 65 °C, then medicinal gelatin and sorbitol were put into the gelatinizing tank, stirred, and heated to 65 °C to completely dissolve the medicinal gelatin to obtain the capsule shell material; then, using a soft capsule machine, the prepared capsule shell material was placed in a gelatin heat preservation barrel at 70 °C to make the rubber sheet of the soft capsule, and then the prepared content of the soft capsule was injected, and it was roll-molded into soft capsules, dried, washed with pills, and dried again to obtain the soft capsule preparation of the motherwort phenolic acid extract.

[0160] The soft capsules were prepared according to the above method, and then a commercial product Nutrilys of cellulose derivatives recognized as GRAS (Generally Recognized as Safe) Or a commercial product based on shellac after special processes and chemical modifications was used as an enteric coating material to coat the enteric coating to obtain the enteric-coated soft capsule preparation of the motherwort phenolic acid extract.

[0161] Example 15: Freeze-dried Preparation for Injection of Motherwort Phenolate

[0162] 20 g of the motherwort phenolic acid extract prepared in Example 2 was taken, 15 g of sodium ascorbate and 12 g of mannitol were added, dissolved in 250 ml of distilled water, made up to 300 ml, filtered and sterilized, aseptically dispensed into 100 vials, freeze-dried, and then sealed under nitrogen to obtain.

[0163] Example 16: Freeze-dried Preparation for Injection of Motherwort Phenolate

[0164] Take 10 g of the phenolic acid extract of Leonurus japonicus Houttuyn obtained in Example 2, dissolve it in 250 ml of distilled water, adjust the pH value to 7.0 with 10% aqueous Na2CO3 solution, add 20 g of sodium ascorbate and 10 g of mannitol, add distilled water to make up the volume to 300 ml, filter and sterilize, dispense into 100 vials under aseptic conditions, freeze-dry, and then seal under nitrogen to obtain the product.

[0165] Example 17: Freeze-dried powder for injection of phenolic acid of Leonurus japonicus Houttuyn

[0166] Take 10 g of the phenolic acid extract of Leonurus japonicus Houttuyn obtained in Example 4, dissolve it in 250 ml of distilled water, adjust the pH value to 7.0 with 10% aqueous NaOH solution, add 15 g of sodium ascorbate and 12 g of mannitol, add distilled water to make up the volume to 300 ml, filter and sterilize, dispense into 100 vials under aseptic conditions, freeze-dry, and then seal under nitrogen to obtain the product.

[0167] Example 18: Freeze-dried powder for injection of phenolic acid salt of Leonurus japonicus Houttuyn

[0168] Take 20 g of the phenolic acid extract of Leonurus japonicus Houttuyn obtained in Example 6, dissolve it in 250 ml of distilled water, add 20 g of sodium ascorbate, 10 g of anhydrous magnesium hydrogen phosphate, and 12.5 g of mannitol, stir at room temperature for half an hour, filter to remove insoluble substances, then filter and sterilize, freeze-dry under aseptic conditions, grind and mix the prepared pharmaceutical composition, dispense into 100 vials, and seal under nitrogen to obtain the product.

[0169] Example 19: Preparation of ointment of phenolic acid of Leonurus japonicus Houttuyn

[0170] Take 10 g of the phenolic acid extract of Leonurus japonicus Houttuyn obtained by the method of Example 1, add it to the 85°C mixed water phase of 15 ml of glycerol, 0.3 g of sodium dodecyl sulfate, 0.2 g of methylparaben, 5 ml of azone and 30 ml of distilled water as the water phase; separately melt 15 g of petrolatum, 8 g of lanolin, 10 ml of liquid paraffin and 8 g of stearic acid in an 85°C water bath as the oil phase. Then slowly add the water phase to the oil phase while performing rapid stirring in the same direction to fully mix and emulsify, and cool to obtain the product.

[0171] Example 20: Preparation of emulsion of phenolic acid of Leonurus japonicus Houttuyn

[0172] Take 10 g of the phenolic acid extract of Leonurus japonicus Houtt. prepared by the method of Example 1, moisten it with 15 ml of propylene glycol, and grind it until no visible particles remain. Take 8 g of glyceryl monostearate, 20 ml of dimethicone, 10 g of white petrolatum, 3 g of cetyl alcohol, and 0.3 g of ethylparaben, heat to 80 °C, and keep warm to obtain the oil phase; take 15 ml of glycerol, 0.5 g of citric acid, 0.5 g of sodium citrate, 5 ml of polysorbate 80, 6 g of Peregal A-20, and 30 ml of distilled water, heat to 80 °C to obtain the aqueous phase. Slowly add the oil phase to the aqueous phase, stir while adding, and perform ultrasonic treatment to obtain the cream matrix. When the temperature reaches about 60 °C, add the mixture of syringic acid and propylene glycol, mix well, and condense to obtain the product.

[0173] Test Example 1. Tracking of fibrinolytic active components 1 (Water extraction - Alcohol precipitation - Macroporous adsorption resin - Ion exchange resin - Silica gel partition column chromatography separation / Fibrinolytic activity evaluation)

[0174] Tracking of fibrinolytic active components: During the extraction and preliminary separation of the chemical components in Leonurus japonicus Houtt., the fibrin plate assay was used to continuously evaluate the fibrinolytic activities of the obtained chemical fractions, and the chemical fractions with better activities were further separated and evaluated for fibrinolytic activity until the fibrinolytic active components were clarified. The specific experimental steps are as follows.

[0175] Water extraction and alcohol precipitation: Crush the dried aerial parts of Leonurus japonicus Houtt., take 300 g of the powder, add 3900 mL of water for the first time, heat with an electric heating mantle at 100 °C and reflux for 2 h; add 3000 mL of water for the second time, heat at 100 °C and reflux for 1 h. Combine the two extraction solutions, filter, and concentrate the filtrate to a small volume. Precipitate the concentrated solution twice with 95% ethanol, stir continuously when adding ethanol, and control the ethanol content in the solution after adding ethanol so that the ethanol content reaches 70% for the first time and 83% for the second time. After the second alcohol precipitation, concentrate the filtrate until there is no alcohol smell to obtain the extract of Leonurus japonicus Houtt.

[0176] Macroporous adsorption resin: Dilute the above-mentioned extract of Leonurus japonicus Houtt. with an appropriate amount of distilled water, filter, directly load the filtrate onto a macroporous adsorption resin column, first elute with distilled water for 5 retention volumes, and then elute with 50 wt% ethanol aqueous solution and 95 wt% ethanol aqueous solution for 5 retention volumes respectively, and concentrate them respectively to obtain the water elution fraction, 50% ethanol elution fraction, and 95% ethanol elution fraction.

[0177] Separation and purification by cation exchange resin column chromatography: The water elution fraction of the above macroporous adsorption resin column was dissolved in an appropriate amount of distilled water, loaded onto a cation exchange resin column, and eluted with distilled water and 3N HCl aqueous solution respectively. After concentration of the water eluate, a water-soluble non-alkaloid fraction was obtained; the HCl aqueous solution elution fraction was adjusted to neutral pH with 20% NaOH aqueous solution, evaporated to dryness, and a large amount of solid was precipitated. 95wt% ethanol was added thereto, sonicated for 1 h, filtered, and this process was repeated twice. The two filtrates were combined. The solvent was evaporated to dryness, dissolved in methanol, filtered, and this process was repeated five times until no more NaCl solid was precipitated, and the alkaloid fraction was obtained.

[0178] On the basis of not knowing the structure of the fibrinolytic active components of Leonurus japonicus, combined with the following fibrin plate assay, it was found that the water-soluble non-alkaloid fraction obtained from the above cation exchange resin column had relatively strong fibrinolytic activity. Therefore, different component separation methods were mainly used below, combined with the fibrin plate assay, to trace its fibrinolytic active components.

[0179] In the early stage of the tracing process of the fibrinolytic activity of Leonurus japonicus, separation methods such as adsorption silica gel column chromatography, reverse-phase silica gel column chromatography, polyamide column chromatography, Sephadex LH-20 gel column chromatography, and MCI column chromatography were used to obtain many chemical fractions composed of different components. Combined with the fibrin plate assay, the results showed that the separated chemical fractions either had no fibrinolytic activity or had no difference in fibrinolytic activity, and the active fraction of Leonurus japonicus could not be determined. Because the experimental time of this part of the research work was relatively long and there were also a lot of experimental data, but the experimental results basically did not achieve the goal of fibrinolytic activity tracing, so it will not be elaborated here.

[0180] Subsequently, silica gel partition column chromatography was used for separation. 31.3 g of the water-soluble non-alkaloid fraction was dissolved in methanol-water, mixed with 70 g of silica gel (100 - 200 mesh), and 200 g of silica gel (200 - 300 mesh) was suspended in dichloromethane saturated with water to pack the column. After loading the sample, organic phases with different ratios of dichloromethane: methanol: water (25:3:1, 10:3:1, 10:5:1.3, 10:7:2.5, lower phase) were used as eluents for elution. The fractions eluted with each solvent gradient were divided into several components to obtain elution fractions Fr.A1.1 - Fr.A4.6, as shown in Table 1.

[0181] Table 1 Eluents and components of silica gel partition column chromatography

[0182]

[0183] Fibrin plate assay for each component: Using urokinase, a commonly used thrombolytic drug in current clinical practice, as a positive control, a solution of 40 U / ml was prepared with normal saline. Using the internationally and domestically common fibrin plate method, with normal saline as the solvent, a fibrinogen solution of 10 mg / mL, a thrombin solution of 1 U / mL, and an agarose solution of 10 mg / mL were prepared. Take 8 mL of the agarose solution and heat it until completely dissolved. When the temperature drops to 50 - 60 °C, add 1 mL of the fibrinogen solution and 1 mL of the thrombin solution, stir evenly, pour it into a glass petri dish with a diameter of 9 cm, and place it horizontally at room temperature for about 15 minutes until the fibrin plate completely solidifies. Punch holes with a 7 mm puncher, add 30 μL of the methanol solution of the sample to be tested (concentration 20 mg / mL) to each hole, place it in an incubator at 37 °C for 16 h, and measure the diameter φ (mm) of the fibrinolytic circle. For each experimental group, each concentration was repeated three times, and the average value of the diameter was taken.

[0184] Evaluation of fibrinolytic activity of each chemical part of macroporous adsorption resin column and cation exchange resin column: The fibrinolytic activity of several chemical parts obtained from each chemical part of macroporous adsorption resin column and cation exchange resin column was determined by the above fibrin plate method.

[0185] Weigh the 50% ethanol part and 95% ethanol part obtained from the above macroporous adsorption resin column and dissolve them in 80% methanol respectively to prepare 50 mg / mL methanol aqueous solutions; weigh appropriate amounts of the alkaloid part and water-soluble non-alkaloid part obtained from the above cation exchange resin column and dissolve them in distilled water respectively to prepare 50 mg / mL aqueous solutions. Using the above fibrin plate method, add 30 μL of the sample, place it in an incubator at 37 °C for 16 h, and the experimental results are shown in Table 2. Among them, urokinase, water-soluble non-alkaloid part, and 95% ethanol part of macroporous resin all have good fibrinolytic activity. Among them, the fibrinolytic effect of the water-soluble non-alkaloid part is better than that of urokinase. However, the alkaloids obtained from Leonurus japonicus and the 50% ethanol part of macroporous resin have no fibrinolytic effect.

[0186] Table 2 Effects of different extraction and separation parts of Leonurus japonicus on fibrinolytic activity after external administration

[0187]

[0188] Dose-effect relationship of fibrinolytic activity of water-soluble non-alkaloids: Using the above fibrin plate method, aqueous solutions of water-soluble non-alkaloid parts with different concentrations were measured. The results show that the diameters of the fibrinolytic circles of water-soluble non-alkaloid parts with different concentrations in the fibrin plate are different, showing good dose dependence and dose-effect relationship, indicating that this water-soluble non-alkaloid part has good protein fibrinolytic activity, and it is named active part 1 (ACT1).

[0189] Fibrinolytic activity of the sample purified by silica gel partition column chromatography: Using the above fibrin plate method, the fibrinolytic activity of the components obtained by further separating the active site 1 (ACT1) with silica gel partition column chromatography was evaluated. The experimental results are shown in Table 3. Samples numbered 1 to 5 had fibrinolytic activity, while samples numbered 6 and 7 had no fibrinolytic activity. Combining the analysis of the separation and elution conditions of the above silica gel partition column chromatography, the samples numbered 1 to 5 were the components eluted with dichloromethane: methanol: water (25:3:1; lower phase) as the mobile phase. The experimental results show that silica gel partition column chromatography can further separate and purify the active site 1 (ACT1).

[0190] Table 3 Effects of in vitro administration of each eluted component Fr.A1.1 - Fr.A2.1 on fibrin lysis

[0191]

[0192]

[0193] HPLC analysis of the active site 1 (ACT1):

[0194] HPLC analysis conditions. Chromatographic column: COSMOSIL5 C18-MS-II (4.6×250mm, 5μm). The mobile phase was a gradient elution of acetonitrile (A) - 0.1% formic acid in water (B) (0 - 10 min, 10% A; 10 - 15 min, 10% → 20% A; 15 - 30 min, 20% → 50% A; 30 - 55 min, 50% → 75% A; 55 - 60 min, 75% → 10% A). Injection volume: 10 μL, flow rate 1 mL / min, detection wavelength: 254 nm.

[0195] Using the above HPLC conditions to analyze the components of the obtained active site 1 (ACT1), its HPLC fingerprint is shown in Figure 1 . From Figure 1 , it can be seen that its main components are two parts: the chromatographic peak at about 3 min retention time and the chromatographic peak at about 20 min retention time. From this chromatogram, it can be seen that the components in the active site 1 (ACT1) are relatively complex, consisting of the above two parts of components, and need to be further separated and purified. The results show that the eluent ratio of the silica gel column chromatography in this part is not the best.

[0196] Test Example 2, Tracking of Fibrinolytic Active Components 2 (Water Extraction - Extraction - Partition Column Chromatography Separation / Fibrinolytic Activity Evaluation)

[0197] Extraction and Extraction: After the aerial stems and leaves of Leonurus japonicus were crushed into coarse powder, 200 g of the powder was taken and extracted twice by boiling with water. For the first time, 2.6 L of water was added, and it was refluxed at 100 °C for 2 hours with an electric heating mantle; for the second time, 2 L of water was added, and it was refluxed at 100 °C for 1 h. The two extraction solutions were combined and filtered. It was concentrated to about 200 mL, and the ethanol precipitation process was repeated twice (the first ethanol precipitation concentration was 70%, and the second ethanol precipitation concentration was 83%). The second ethanol precipitation solution was filtered, and the filtrate was rotary evaporated until there was no alcohol smell, and then diluted with water to 450 mL. It was extracted twice with equal amounts of petroleum ether, dichloromethane, and ethyl acetate in turn. The extraction solutions were combined and concentrated respectively, and then the aqueous solution after extraction was concentrated to obtain four chemical parts, namely the petroleum ether extraction part (YMC-PE-1, 39.61 mg), the dichloromethane extraction part (YMC-DCM-1, 424.4 mg), the ethyl acetate extraction part (YMC-EA-1, 362.9 mg), and the concentrate of the aqueous solution after extraction (YMC-H2O-1, 14.65 g).

[0198] Silica gel partition column chromatography separation: The above-mentioned ethyl acetate extraction part was separated by silica gel partition column chromatography. The sample was mixed with silica gel of 100 - 200 mesh, and the column was packed with silica gel of 200 - 300 mesh suspended in dichloromethane saturated with water. First, it was eluted with dichloromethane saturated with water, and then gradient elution was carried out in turn with different ratios of dichloromethane: methanol: water (100:1:5, 100:2:5, 100:4:5, 100:10:8, 100:20:10, 100:40:10, 100:60:17, lower phase) to obtain several different elution fractions (see Table 4).

[0199] Table 4 Eluents and Each Elution Component of Silica Gel Partition Column Chromatography

[0200]

[0201]

[0202] Evaluation of fibrinolytic activity of components obtained by extraction method: Appropriate amounts of each extraction chemical part YMC-PE-1, YMC-DCM-1, YMC-EA-1 in Test Example 2 were weighed separately and dissolved in methanol to prepare a methanol solution with a concentration of 20 mg / mL; while YMC-H2O-1 was dissolved in distilled water to prepare a 20 mg / mL aqueous solution. The fibrin plate method in Test Example 1 was used to determine the fibrinolytic activities of each extraction part YMC-PE-1, YMC-DCM-1, YMC-EA-1 and YMC-H2O-1 obtained in the tracking of active ingredients 2 in this test example. The measurement results are shown in Table 5. Among them, YMC-DCM-1 and YMC-EA-1 have fibrinolytic activities, the former has a weak activity, and the latter has a strong activity; while YMC-PE-1 and YMC-H2O-1 have no activities. Therefore, the results show that the fibrinolytic active components are mainly concentrated in the YMC-EA-1 part, and the content in YMC-DCM-1 is less or the component activity is weaker.

[0203] Table 5 Effects of each extraction part obtained in the second active ingredient tracking on fibrin dissolution

[0204]

[0205] Evaluation of fibrinolytic activity of samples after separation and purification by silica gel partition column chromatography: Appropriate amounts of each chemical part obtained by separation and purification of silica gel partition column chromatography in Test Example 2: Fr.B1000, Fr.B1001, Fr.B1002, Fr.B1004, Fr.B10010, Fr.B10020, Fr.B10040-1 to Fr.B10040-5, Fr.B10060 were weighed separately and dissolved in methanol to prepare a methanol solution with a concentration of 20 mg / mL. The fibrin plate method in Test Example 1 was used to determine their fibrinolytic activities, and the measurement results are shown in Table 6. From these results, it can be seen that the samples numbered from 2 to 12 have different degrees of fibrinolytic activities. Among them, the sample numbered 4, Fr.B1004, has the strongest fibrinolytic activity (φ34 mm), indicating that the fibrinolytic active components are mainly concentrated in the Fr.B1004 part. This part was determined as the fibrinolytic active part in Leonurus japonicus Houtt. and named Active Part 2 (ACT2).

[0206] Table 6 Effects of samples after separation and purification by silica gel partition column chromatography on fibrin dissolution

[0207]

[0208]

[0209] HPLC Analysis of Active Site 2 (ACT2): Weigh 1 mg of the sample Fr.B1004 (ACT2) obtained from the silica gel partition column chromatography in Test Example 2 and dissolve it in 1 mL of chromatographically pure methanol to prepare a methanol solution with a concentration of 1 mg / mL. Using the HPLC analysis conditions in Test Example 1, analyze ACT2 in Test Example 2. The HPLC fingerprint is shown in Figure 2 . There are 5 main chromatographic peaks between the retention times of 18 - 28 min in Fingerprint 2, indicating that its main components are concentrated within this retention time range.

[0210] Test Example 3. Separation and Structure Identification of the Fibrinolytic Active Site (ACT2) of Leonurus japonicus

[0211] Using the extraction and extraction method of the active site ACT2 in Test Example 2, obtain the ethyl acetate fraction after extraction. Using the silica gel partition column chromatography method in Test Example 2, separate this ethyl acetate extraction fraction (the volume ratios of the eluents dichloromethane:methanol:water are 100:0:5, 100:1:5, 100:2:5, 100:3:5, 100:4:5, 100:10:8 respectively). The elution fractions are identified and combined by thin layer chromatography, and then their fibrinolytic activities are measured to obtain the fibrinolytic active site components Fr.3 - 1 to Fr.3 - 4. Use a method combining TLC and HPLC to separate and prepare single compounds from them respectively.

[0212] Compound 1 (50.02 mg) was obtained by repeatedly separating and purifying the above Fr.3 - 2 fraction by HPLC. The HPLC conditions were: Phase A: water, Phase B: methanol, 0 - 15 min: 70% A → 40% A, 15 - 25 min: 40% A → 0. The retention time of Compound 1 was 18.1 min.

[0213] Compound 2 (36.87 mg) was first prepared by TLC from the ethyl acetate extraction fraction of the Leonurus japonicus extract. The developing agent was dichloromethane:methanol (13:1), and the R f was approximately 0.5. Then it was obtained by HPLC preparation. The mobile phase was Phase A: water, Phase B: methanol, 0 - 15 min: 70% A → 40% A. The retention time was 8 min.

[0214] Compounds 3 and 4 (23.46 mg) were obtained by repeatedly preparing Fr.3 - 2 by HPLC. The mobile phase was Phase A: water, Phase B: methanol, 0 - 15 min: 40% A → 25% A, 15 - 25 min: 25% A → 10% A. Compounds 3 and 4 were the same peak, and the retention time was 13 min. For the second preparation, the mobile phase was Phase A: 0.1% formic acid water, Phase B: acetonitrile, and it was prepared with 80% A. The retention time of Compound 3 was 12.5 min, and the retention time of Compound 4 was 13.1 min.

[0215] Compound 5 (47.8 mg) was prepared from Fr. 3-1 by HPLC. The mobile phase was phase A: water, phase B: methanol. From 0 to 15 min, 40% A → 25% A, from 15 to 25 min, 25% A → 0% A, and the retention time was 15.6 min.

[0216] Spectral data for structure identification:

[0217] Spectral data for compound 1: 1 1H-NMR (CD3OD, 400 MHz) δ 7.57 (2H, m, H-2, H-6), 6.85 (1H, d, H-5), 3.91 (3H, s, H-8). 13 13C-NMR (CD3OD, 100 MHz) δ 121.8 (C-1), 112.4 (C-2), 147.3 (C-3), 151.2 (C-4), 114.4 (C-5), 123.9 (C-6), 168.7 (C-7), 55.0 (C-8). HR-ESI-MS m / z: 167.013410 [M-H] － Its structure was determined to be vanillic acid.

[0218] Spectral data for compound 2: 1 1H-NMR (400 MHz, CD3OD) δ 7.33 (2H, s, H-2, H-6), 3.88 (6H, s, H-8, H-9). 13 13C-NMR (100 MHz, CD3OD) 120.5 (C-1), 106.9 (C-2, C-6), 147.4 (C-3, C-5), 140.3 (C-4), 168.6 (C-7), 55.4 (C-8, C-9). HR-ESI-MS m / z: 197.04396 [M-H] － Its structure was determined to be syringic acid.

[0219] Spectral data for compound 3: 1 1H-NMR (400 MHz, CD3OD) δ 7.62 (1H, d, H-7), 7.20 (1H, d, H-2), 7.08 (1H, dd, H-6), 6.83 (1H, d, H-5), 6.33 (1H, d, H-8), 3.91 (3H, s, H-10). 13C-NMR (100 MHz, CD3OD) δ 126.4 (C-1), 110.3 (C-2), 149.1 (C-3), 148.0 (C-4), 114.5 (C-5), 122.6 (C-6), 145.5 (C-7), 115.1 (C-8), 169.6 (C-9), 55.0 (C-10). HR-ESI-MS m / z: 193.04906 [M-H] － Its structure was determined to be trans-ferulic acid.

[0220] Spectral data of compound 4: 1 H-NMR (400 MHz, CD3OD) δ 7.90 (1H, d, H-2), 7.21 (1H, dd, H-6), 6.75 (1H, d, H-7), 6.81 (1H, d, H-5), 5.83 (1H, d, H-8), 3.80 (3H, s, H-10). 13 C-NMR (100 MHz, CD3OD) δ 127.2 (C-1), 114.0 (C-2), 146.8 (C-3), 141.3 (C-4), 114.3 (C-5), 125.3 (C-6), 141.3 (C-7), 117.8 (C-8), 168.2 (C-9), 55.4 (C-10). HR-ESI-MS m / z: 193.04901 [M-H] － Its structure was determined to be cis-ferulic acid.

[0221] Spectral data of compound 5: 1 H-NMR (400 MHz, CD3OD) δ 7.34 (2H, s, H-2, H-6), 3.89 (6H, s, H-8, H-9), 3.84 (3H, s, H-10). 13 C-NMR (100 MHz, CD3OD) δ 125.8 (C-1), 106.7 (C-2, C-6), 152.8 (C-3), 141.6 (C-5), 168.0 (C-7), 59.7 (C-10), 55.2 (C-8, C-9). HR-ESI-MS m / z: 211.06979 [M-H] － Its structure was determined to be 3,4,5-trimethylgallic acid.

[0222] Among them, compound 4 (cis-ferulic acid) has poor stability. After standing, part of it is converted into compound 3 (trans-ferulic acid).

[0223] Test Example 4, HPLC Component Analysis and Content Determination of Leonurine Acid Extract

[0224] HPLC determination conditions: Use a Thermo 25002-101030 (1 mm * 100 mm, 1.9 μm) chromatographic column; use methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B. The gradient elution time program is as follows: 0 - 10 min, 10% methanol aqueous solution; 10 - 15 min, 10% → 20% methanol aqueous solution; 15 - 25 min, 20% → 40% methanol aqueous solution; 25 - 40 min, 40% → 80% methanol aqueous solution. Column temperature: 30 °C, flow rate: 0.3 mL / min, injection volume: 10 μL.

[0225] Preparation of the phenolic acid extract solution of Leonurus japonicus: Take 10 mg each of the phenolic acid extract of Leonurus japonicus (active part ACT2) prepared in Test Example 2 and the phenolic acid extract of Leonurus japonicus (active part ACT3) prepared in Example 1, accurately weigh, place in a 10 mL volumetric flask, dissolve with methanol and dilute to the mark, shake well, filter, and set aside for use.

[0226] Preparation of the reference solution: Take 10.00 mg each of the reference substances vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid reference substances, dissolve them separately in 5 mL volumetric flasks with chromatographic methanol, make up the volume to obtain a reference stock solution of 2.00 mg / mL. Then take appropriate amounts of these reference stock solutions and dilute them with chromatographic pure methanol to obtain reference solutions with concentrations of 1000, 800, 600, 400, 200, and 100 μg / mL respectively, and set aside for use.

[0227] Establishment of the standard curve: Using the above HPLC determination conditions, inject the above-prepared reference substances and the sample solution to be measured for determination respectively, count the peak areas of the samples of each compound at each concentration, and establish the HPLC standard curve of each compound, as Figure 3 shown. The regression equation of vanillic acid was calculated as: y = 12.788x + 1332.4, R 2 = 0.9991; the regression equation of syringic acid was: y = 13.639x - 91.274, R 2 = 0.9994; the regression equation of trans-ferulic acid was: y = 7.2717x + 127.59, R 2 = 0.9993; the regression equation of 3,4,5-trimethylgallic acid was: y = 16.661x + 433.95, R 2 = 0.9997. The results show that vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid have good linear relationships within their respective ranges.

[0228] HPLC analysis results of the components of the phenolic acid extract of Leonurus japonicus: The determination results of the phenolic acid extract ACT2 of Leonurus japonicus, the phenolic acid extract ACT3 of Leonurus japonicus, and the four reference substances are shown respectively inFigure 4 . Figure 4 A and Figure 4 In B, the sample of Leonurus phenolic acid extract mainly has five chromatographic peaks F1, F2, F3, F4, and F5. Figure 4 In C, the retention times of the chromatographic peaks of reference substances vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid are the same as the retention times of the main chromatographic peaks F1, F2, F3, and F5 in samples ACT2 and ACT3, respectively, indicating that the chromatographic peaks F1, F2, F3, and F5 in leonuri phenolic acid ACT2 and ACT3 are vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid, respectively.

[0229] Determination results of the content of each component: According to the standard curve established above ( Figure 3 ) and the HPLC chromatogram of the reference substance ( Figure 4 C), calculate separately Figure 4 A and Figure 4 The percentage contents of the compounds of chromatographic peaks F1, F2, F3, and F5 in B are shown in Tables 7 and 8. In ACT2, the contents of the four main compounds are 10.4%, 54.0%, 23.4%, and 7.3%, respectively, and their total content is 95.1%; in ACT3, the contents of the four main compounds are 8.4%, 52.6%, 24.1%, and 2.3%, respectively, and their total content is 87.4%.

[0230] Table 7 The percentage of each main component in the motherwort phenolic acid extract ACT2

[0231]

[0232] Table 8 The percentage of each main component in the motherwort phenolic acid extract ACT3

[0233]

[0234]

[0235] Experimental Example 5: Dose-effect relationship of urokinase fibrinolytic zone diameter

[0236] Take 2.8 mg of 50 U / mg urokinase and dissolve it in 1 mL of distilled water, and then dilute it with distilled water to obtain 20, 40, 60, 80, 100, 120, and 140 U / mL urokinase aqueous solutions. The fibrinolysis zone of urokinase with different dose concentrations on the fibrin plate was measured, and the results are shown in Table 9. The diameter of the fibrinolysis zone of urokinase with different dose concentrations on the fibrin plate is different, and as the dose of urokinase increases, the diameter of the fibrinolysis zone also increases, and a good dose-effect relationship is shown. The diameter of the fibrinolysis zone at each concentration is counted to establish a dose-effect relationship standard curve, such as Figure 5。The regression equation calculated is: y = 0.083x + 13.857, R 2 = 0.9714. The experimental results show that it is feasible to use the fibrin plate assay to evaluate the fibrinolytic effect of compounds. The diameter (φ) of the fibrinolytic zone is proportional to the strength of fibrinolytic activity, indicating that the diameter of the fibrinolytic zone can be directly used to represent the strength of the fibrinolytic activity of the test component in the fibrin plate assay, providing a reliable method for the evaluation and tracking of fibrinolytic active components in Leonurus japonicus Houtt.

[0237] Table 9 Fibrinolytic activities of different doses of urokinase

[0238]

[0239] Test Example 6: Determination of fibrinolytic activity of monomeric phenolic acids of Leonurus japonicus Houtt

[0240] Using urokinase, a commonly used thrombolytic drug in clinical practice, as a positive control, a solution of 40 U / ml was prepared with normal saline. Using the internationally and domestically common fibrin plate method, a fibrinogen solution of 10 mg / mL, a thrombin solution of 1 U / mL, and an agarose solution of 10 mg / mL were prepared with normal saline as the solvent. Take 8 mL of the agarose solution and heat it until it is completely dissolved. When the temperature drops to 50 - 60 °C, add 1 mL of the fibrinogen solution and 1 mL of the thrombin solution, stir evenly, pour it into a glass petri dish with a diameter of 9 cm, and place it horizontally at room temperature for about 15 minutes until the fibrin plate is completely solidified. Punch holes with a 7 mm puncher, add 30 μL of the methanol solution of the test sample (concentration 20 mg / mL) to each hole, place it in an incubator at 37 °C for 16 h, and measure the diameter φ (mm) of the fibrinolytic zone.

[0241] Weigh 2 mg each of the single compounds vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid (containing a small amount of trans-ferulic acid), and 3,4,5-trimethylgallic acid obtained in Test Example 3, dissolve them in 100 μL of chromatographic methanol to make their concentration 20 mg / mL, incubate at 37 °C for 2 h, and measure the diameter φ (mm) of each fibrinolytic zone in the fibrin plate to evaluate the strength of their fibrinolytic activity.

[0242] The fibrinolytic experimental results of each single compound and mixture are shown in Table 10. All five single compounds have significant protein fibrinolytic activity. Among them, urokinase has the best fibrinolytic effect; the single compounds vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid all have strong protein fibrinolytic activity. The fibrinolytic activity of the cis- and trans-ferulic acid mixture is equivalent to that of trans-ferulic acid, indicating that the protein fibrinolytic activities of cis-ferulic acid and trans-ferulic acid are equivalent and there is no synergistic effect after their mixing.

[0243] Table 10 Protein fibrinolytic activities of monomeric compounds in fibrinolytic active sites

[0244]

[0245] Test Example 7 Fibrin plate test of phenolic acid extract from Leonurus japonicus

[0246] Using the fibrin plate method as in Test Example 6, with urokinase as the positive control, the fibrinolytic activity of the fibrinolytic active site of phenolic acid from Leonurus japonicus was determined. The phenolic acid extract ACT2 prepared in Test Example 2 and the phenolic acid extract ACT3 prepared by the method of Example 1 were formulated into methanol solutions with a concentration of 20 mg / mL, and urokinase was formulated into an aqueous solution with a concentration of 40 U / mL. 30 μL of the methanol solution of the sample to be tested and 30 μL of the aqueous solution of urokinase were added to each well of the fibrin plate, and the diameter φ of the fibrinolytic circle was measured. The measurement was repeated three times, and the average value of the diameters of the fibrinolytic circles was taken.

[0247] The experimental results of the fibrin plate test are shown in Table 11. In the fibrin plate experiment, both the phenolic acid extract from Leonurus japonicus (ACT2) and the phenolic acid extract from Leonurus japonicus (ACT3) had relatively large fibrinolytic circle diameters, and the fibrinolytic circle diameter of ACT2 was larger than that of ACT3, indicating that both ACT2 and ACT3 had relatively good fibrinolytic activities.

[0248] Table 11 Dissolution effect of phenolic acid from Leonurus japonicus on fibrin

[0249]

[0250] Test Example 8 In vitro antithrombotic activity experiment of phenolic acid extract from Leonurus japonicus

[0251] This experiment used the internationally and domestically common Chandler loop method, referring to the literature method Chandler A, Laboratory Investigation, 1958, (7): 110. This experiment was divided into a blank group (normal saline), a positive drug aspirin group (2 mg / mL, 5 mg / mL, 10 mg / mL), and high, medium, and low dose groups (2 mg / mL, 5 mg / mL, 10 mg / mL) of the phenolic acid extract ACT2 prepared in Test Example 2 and the phenolic acid extract ACT3 prepared in Example 1.

[0252] Male Wistar rats, weighing approximately 180 - 300 g, were provided by Jinan Pengyue Laboratory Animal Breeding Co., Ltd. They were anesthetized by intraperitoneal injection of 0.2 mL of 1.5% pentobarbital solution per 100 g of body weight, fixed in the supine position, and the abdominal cavity was opened. Blood was collected from the abdominal aorta using an ordinary negative pressure blood collection tube without anticoagulant. 10 μL of the test samples ACT2 and ACT3 were added to the plastic hoses for thrombus formation in advance. After blood collection, 1 mL of blood sample was quickly injected into the tube along the inner wall of one end of the plastic hose, avoiding the generation of air bubbles during injection. Then, the plastic tubes were connected into a ring and installed in the turntable (preheated to 37 °C in advance) of an automatic thrombus formation instrument. The process from blood collection to installation was controlled within 1 min as much as possible. After the turntable rotated at a speed of 20 ± 2 rpm for 10 min, the plastic ring was taken out, the thrombus was removed, and the wet weight and length of the thrombus were measured and recorded. Then, it was placed in an oven at 60 °C for 30 min and taken out, and the dry weight of the thrombus was recorded.

[0253] Experimental results: The results of the in vitro anti - thrombosis experiments of each dose group of ACT2 and ACT3, each dose group of the positive drug aspirin, and the blank control group are shown in Table 12 and Figure 6 . Compared with the blank group, in the high, medium, and low dose groups of phenolic acid from Leonurus japonicus and the positive drug aspirin administration groups, there were significant differences in the average thrombus length, average wet weight of the thrombus, and average dry weight of the thrombus, indicating that the fibrinolytic active sites ACT2 and ACT3 obtained from Leonurus japonicus both have significant anti - thrombosis effects. Among them, the medium dose groups of ACT2 and ACT3 showed anti - thrombosis effects equivalent to those of the positive drug aspirin.

[0254] Table 12 In vitro anti - thrombosis activities of ACT2 and ACT3

[0255]

[0256] Note: Compared with the blank group, *P < 0.05, **P < 0.01, ***P < 0.001

[0257] Test Example 9: In vitro thrombolytic activity experiment of phenolic acid extract from Leonurus japonicus

[0258] Refer to the general in vitro thrombus formation experimental methods at home and abroad (Chandler loop experiment method, Chandler A, Laboratory Investigation, 1958, (7): 110). This experiment was divided into a blank group (normal saline), high, medium, and low-dose groups of the positive drug urokinase (urokinase solutions of 10 U / mL, 20 U / mL, and 40 U / mL), high, medium, and low-dose groups of the phenolic acid extract ACT2 from Leonurus japonicus prepared in Test Example 2 (2 mg / mL, 5 mg / mL, and 10 mg / mL), and high, medium, and low-dose groups of the phenolic acid extract ACT3 from Leonurus japonicus prepared in Example 1 (2 mg / mL, 5 mg / mL, and 10 mg / mL).

[0259] Sixty male Wistar rats, weighing 180 - 200 g, were provided by Jinan Pengyue Laboratory Animal Breeding Co., Ltd. Blood was collected from the abdominal aorta of the rats. 1 mL of blood sample was injected into a plastic hose, connected into a loop, and installed into the turntable of a thrombus generator. It was rotated at a speed of 20 ± 2 rpm for 10 min. Thrombus was formed in the plastic hose. Then, 10 μL of the test samples ACT2 and ACT3 solutions were respectively added to the plastic hose, and it was continuously rotated in the turntable at a speed of 20 ± 2 rpm for 4 h. Then the thrombus was taken out, and the wet weight and length of the thrombus were measured and recorded. After that, it was taken out from the 60 °C oven after 30 min, and the dry weight of the thrombus was recorded.

[0260] Calculation method of thrombolysis rate:

[0261]

[0262] Experimental results: The in vitro thrombolysis experimental results of the blank control group, each dose group of the positive drug urokinase, and each dose group of ACT2 and ACT3 on newly formed thrombus are shown in Table 13 and Figure 7 . After each administration group was co-incubated with the newly formed thrombus for 4 hours, compared with the blank group, there were significant differences in the average length, average wet weight, and average dry weight of the thrombus in the high, medium, and low doses of ACT2, ACT3, and urokinase compared with the blank group, indicating that they could significantly dissolve the newly formed thrombus. In particular, the thrombolysis rates of the medium and high dose groups of ACT2 and ACT3 (ACT2: 70.9%, 79.1%; ACT3: 71.6%, 80.2%) were significantly higher than or equivalent to the thrombolysis rates of the medium and high dose groups of urokinase (54.2%, 78.6%), indicating that the fibrinolytic active sites ACT2 and ACT3 obtained from Leonurus japonicus both had extremely significant thrombolytic effects.

[0263] Table 13 Thrombolytic effects of ACT2 and ACT3 on newly formed thrombus in vitro

[0264]

[0265] Note: Compared with the blank group, *P<0.05, **P<0.01, ***P<0.001

[0266] The above experimental results show that the phenolic acids of Leonurus japonicus in the present invention have the effects of dissolving fibrin activity, anti-thrombosis formation, and dissolving thrombus, and can be used for the treatment of thrombotic diseases, with the characteristics of good curative effect and convenient use.

[0267] Compared with urokinase currently used clinically for the treatment of thrombotic diseases, the thrombolysis rates of the medium and high dose groups of phenolic acids of Leonurus japonicus are higher than or equivalent to those of the urokinase control group, indicating that the Leonurus japonicus extract can be applied to the thrombolysis treatment of thrombotic diseases clinically. This Leonurus japonicus extract can be processed into various dosage forms according to the conventional pharmaceutical production process, such as ordinary or enteric-coated tablets, capsules, soft capsules, dripping pills, and granules, as well as injection preparations.

[0268] Test Example 10: Anticoagulation experiment of phenolic acid extract of Leonurus japonicus

[0269] This experiment was divided into a blank group (normal saline), high, medium, and low dose groups of positive drug aspirin (ASP) (ASP solutions of 1 μM, 50 μM, 100 μM, 200 μM), high, medium, and low dose groups of phenolic acid extract ACT2 prepared in Test Example 2 (3.3 μg / mL, 165 μg / mL, 330 μg / mL, 660 μg / mL), and high, medium, and low dose groups of phenolic acid extract ACT3 prepared in Example 1 (3.3 μg / mL, 165 μg / mL, 330 μg / mL, 660 μg / mL).

[0270] Male Wistar rats, weighing about 250 - 300 g, at room temperature of 25 ± 1°C. The rats were anesthetized with 3% pentobarbital, and the administration dose was 3.5 mL / kg. The rats were fixed in the supine position, the abdominal cavity was opened, and blood was collected with a negative pressure blood collection tube anticoagulated with 3.8% sodium citrate at a ratio of 1:9 at the position of the abdominal aorta. The rat plasma in the blood collection tube was centrifuged at 1000 rpm for 10 min, and the upper plasma was taken out with a pipette to obtain PRP. The remaining plasma in the blood collection tube was centrifuged again at 3000 rpm for 10 min, and the upper plasma PPP was taken out with a pipette. Place the prepared various reagents on a semi-automatic coagulation analyzer set up for preheating. Mix PPP with the drug to be tested, and detect PT, APTT, and TT according to the instrument operation instructions. The experiment should be completed within 2 hours.

[0271] Experimental results: The in vitro anticoagulant activity test results of the blank control group, each dose group of the positive drug ASP, and each dose group of ACT2 and ACT3 are shown in Table 14. Compared with the blank group, most of the high-dose groups in ASP, ACT2, and ACT3 showed significant differences, indicating that they could significantly prolong PT, APTT, and TT, showing obvious anticoagulant activity. Among them, the low, medium, and high-dose groups of ACT2 and ACT3 were significantly better than the positive drug ASP in prolonging TT, indicating that they could significantly reduce the generation of thrombin and had strong anticoagulant activity.

[0272] Table 14 Evaluation of the in vitro anticoagulant activity of ACT2 and ACT3

[0273]

[0274] Note: Compared with the blank group, *P<0.05, **P<0.01, ***P<0.001

[0275] Test Example 11 Antiplatelet aggregation experiment of phenolic acid extract from Leonurus japonicus

[0276] Using aspirin (ASP) as the positive control, the phenolic acid extracts ACT2 and ACT3 from Leonurus japonicus prepared in Test Example 2 and Example 1 were dissolved in 1% sodium bicarbonate aqueous solution, and three concentrations of high, low, and medium were set to make the final concentrations in the plasma measurement system 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, and 20 mg / mL, and the effects of the phenolic acid extracts ACT2 and ACT3 from Leonurus japonicus on platelet aggregation were measured.

[0277] After preheating the platelet aggregometer for 30 min, the stir bar was placed in the reaction cup. 250 μL of PRP prepared in Test Example 5 and 10 μL of the test drug were aspirated into the reaction cup with a pipette gun, and the reaction cup was placed in the slot of the instrument and incubated at 37 °C for 20 minutes. After adjusting the baseline to zero with PPP prepared in Test Example 5, 10 μL of ADP was added to induce platelet aggregation, and the maximum aggregation rate of the compound within 6 minutes was recorded.

[0278] Experimental results: The results of the inhibition of ADP-induced platelet aggregation by the positive drug ASP, the phenolic acid extract ACT2, and ACT3 from Leonurus japonicus are shown in Figure 8 . Both ACT2 and ACT3 of phenolic acid from Leonurus japonicus had good effects on inhibiting platelet aggregation, and with the increase of concentration, the inhibitory effect on platelet aggregation showed an increasing trend. Among them, the inhibitory effect of ACT3 of phenolic acid from Leonurus japonicus on platelet aggregation was close to that of the positive drug ASP, and at a larger dose of 50 mg / mL, the inhibitory effect of ACT3 on platelets was better than that of the positive drug ASP. The phenolic acid extracts ACT2 and ACT3 from Leonurus japonicus had good antiplatelet aggregation effects and had potential application prospects.

[0279] Experimental Example 12: Experiment on the Permeation of Leonurine Acid Extract through the Blood-Brain Barrier

[0280] Using dodecane as a solvent, prepare a 20 mg / mL solution of porcine polar brain extract for later use. Dissolve four commercially available drugs (estradiol, progesterone, alprazolam, oxazepam), the leonurine acid extracts (ACT2, ACT3) prepared in Experimental Example 2 and Example 1, and commercially available phenolic acid compounds (vanillic acid, syringic acid, trans-ferulic acid, 3,4,5-trimethylgallate) separately with a small amount of DMSO solution, and then dilute them with a buffer solution of PBS:EtOH = 7:3 to a 25 μg / mL solution for later use. Use a pipette to take 4 μL of porcine polar brain extract and drop it on the PVDF membrane of the receptor microplate to simulate the human biological membrane. Then add 300 μL of the test compound solution to the donor well and 200 μL of the PBS:EtOH = 7:3 buffer solution to the receptor well, so that the compound solution just touches the biological membrane. Place the receptor well on the donor well to form a sandwich-like structure of donor well / biological membrane / receptor well. The drug to be tested diffuses from the donor well through the artificial simulated biological membrane to the receptor well in the form of passive diffusion. The entire microplate is left standing at room temperature for 16 h, then separate the receptor microplate and the donor microplate, and then take out the liquids in their donor wells and receptor wells respectively, and measure their OD values with an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm. Set 5 sets of repeated experiments for each compound to be tested.

[0281] Calculate the effective permeability Pe of the compound according to the following formula:

[0282] Pe (cm·s -1 ): Pe = -VdVa / [(Vd + Va)At]ln(1 – OD drugacceptor / OD drugreceiver )

[0283] Wherein, Vd is the volume of the donor well, Va is the volume of the receptor well, A is the area of the artificial phospholipid membrane, t represents the permeation time, OD drugacceptor is the absorbance of the liquid in the donor well, OD drugreceiver is the absorbance of the liquid in the receptor well, and the results are expressed as the mean “±” standard error.

[0284] Experimental results: Through the above experimental method, we first obtained the experimental permeation values of 4 commercially available drugs. By referring to relevant data, the theoretical permeation values of these 4 drugs were determined. All the results are shown in Table 15. A standard curve was made based on the theoretical permeation values and the experimental permeation values for subsequent correction ( Figure 9 ), and a good linear correlation equation was obtained: y = 1.9489x + 2.559, R 2 = 0.9616.

[0285] Based on this equation and considering the limits established by Di et al., the following criteria were established: A Pe value above 5.1×10 -6 cm / s indicates that the compound has good blood-brain barrier permeability, and a Pe value less than 5.1×10 -6 cm / s indicates poor blood-brain barrier permeability of the compound. According to the experimental results (Table 16), the measured Pe values of leonuritic acid ACT2 and ACT3 were 6.1×10 -6 cm / s and 6.4×10 -6 cm / s respectively, both greater than the Pe value of 5.1×10 -6 cm / s; except for trans-ferulic acid (Pe value of 1.2×10 -6 cm / s), the Pe values of vanillic acid, syringic acid, and 3,4,5-trimethylgallic acid were 7.1×10 -6 cm / s, 9.2×10 -6 cm / s, and 8.1×10 -6 cm / s respectively, all greater than the Pe value of 5.1×10 -6 cm / s, indicating that the leonuritic acid extracts ACT2, ACT3 and the single compounds vanillic acid, syringic acid, and 3,4,5-trimethylgallic acid all have good blood-brain barrier permeability, and syringic acid has the largest permeability, which is similar to the penetration ability of the control drug oxazepam. Therefore, the leonuritic acid extract has good blood-brain barrier permeability and has the potential value to become a drug for treating cerebral thrombosis.

[0286] Table 15 Measured values and literature penetration values of commercially available drugs

[0287]

[0288] Table 16 Blood-brain barrier permeability results of leonuritic acid ACT2 and ACT3

[0289]

Claims

1. An extract of phenolic acid from Leonurus japonicus with thrombolytic effect, characterized in that, It is mainly composed of vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethyl gallic acid, and the total mass content of vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethyl gallic acid is greater than or equal to 70%; preferably, in the leonurine acid extract, the total mass content of vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethyl gallic acid is greater than or equal to 80%.

2. The preparation method of the leonurine acid extract with thrombolytic effect as described in claim 1, comprising the steps of: using the aerial stems and leaves of leonurus artemisia as raw materials, and preparing the leonurine acid extract with thrombolytic effect through extraction, enrichment, separation and purification; the extraction method is alcohol-water extraction method or alkaline water extraction method; the enrichment method is extraction method, alkaline water kneading dissolution method, macroporous adsorption resin column chromatography method or strongly basic anion exchange resin method; the separation method is silica gel partition column chromatography method or ODS silica gel column chromatography method.

3. The preparation method of the phenolic acid extract of Leonurus artemisia with thrombolytic effect according to claim 2, characterized in that, It includes one or more of the following conditions: i. The alcohol-water extraction method includes the steps of: crushing the aerial stems and leaves of leonurus artemisia into coarse powder, then immersing it in a solvent, extracting, and filtering to obtain the leonurus artemisia extract; Or, crushing the aerial stems and leaves of leonurus artemisia into coarse powder, moistening it in an aqueous NaOH solution, adjusting the pH value to neutral, filtering; then immersing it in a solvent, extracting, and filtering to obtain the leonurus artemisia extract; Preferably, the solvent is water, 50wt% - 100wt% methanol solution or 50wt% - 95wt% ethanol aqueous solution; the weight ratio of the solvent to the coarse powder is 5:1 - 20:1; Preferably, in the alcohol-water extraction method, the extraction methods are heating reflux extraction method, ultrasonic extraction method, percolation method, maceration method; further preferably, the extraction method is heating reflux extraction method or ultrasonic extraction method; the extraction time for the heating reflux extraction method is 1 - 2h each time, the ultrasonic temperature for the ultrasonic extraction method is 40 - 60°C, and the ultrasonic extraction time each time is 20 - 40min; the extraction times are 1 - 5 times; Preferably, the concentration of the aqueous NaOH solution is 0.5 - 2wt%, the moistening temperature is room temperature, and the moistening time is 2 - 12h; use 1 - 5wt% hydrochloric acid aqueous solution to adjust the pH; Preferably, the leonurus artemisia extract can be further concentrated under normal pressure or reduced pressure to obtain the leonurus artemisia extract paste; ii. The alkaline water extraction method includes the steps of: crushing the aerial stems and leaves of leonurus artemisia into coarse powder, soaking and extracting it in an aqueous NaOH solution, and filtering to obtain the leonurus artemisia alkaline water extract; Preferably, before the soaking and extraction step, it further includes a moistening step in water; the mass ratio of water to the coarse powder is 1 - 5:1; the moistening temperature is room temperature, and the moistening time is 2 - 8h; Preferably, the concentration of the aqueous NaOH solution is 0.5wt% - 1.5wt%; the mass ratio of the aqueous NaOH solution to the coarse powder is 5 - 12:1; Preferably, the number of soaking and extraction times is 1 - 5 times; the method of each soaking and extraction is: standing at room temperature for 2 - 12h, or, ultrasonicating at room temperature for 15 - 30 minutes, or, standing at room temperature for 2 - 12h and then ultrasonicating at room temperature for 15 - 30 minutes; Preferably, after the filtration step, a step of adjusting the pH may further be included, and the pH is adjusted to 5-7 using an aqueous hydrochloric acid solution; Preferably, the leonurine aqueous extract may be further concentrated under normal pressure or reduced pressure to obtain a leonurus extract paste.

4. The preparation method of the salvianolic acid extract of Leonurus japonicus with thrombolytic effect according to claim 2, characterized in that, including one or more of the following conditions: i. The extraction method includes the steps of: diluting the obtained extract with water to obtain a mixed solution; sequentially extracting with petroleum ether, dichloromethane, and ethyl acetate as extraction agents, discarding the petroleum ether extract, and separately subjecting the dichloromethane extract and the ethyl acetate extract to reduced pressure concentration to obtain a dichloromethane extract (A) and an ethyl acetate extract (B); suspending the dichloromethane extract (A) in water to obtain a suspension, extracting with ethyl acetate as an extraction agent, and subjecting the ethyl acetate extract to reduced pressure concentration to obtain an ethyl acetate extract (C); combining the ethyl acetate extract (B) and the ethyl acetate extract (C) to obtain a leonurus phenolic acid enriched product; Preferably, in the dilution of the extract with water, the volume of water is 3-6 times the volume of the extract; in the sequential extraction with petroleum ether, dichloromethane, and ethyl acetate as extraction agents, the extraction is performed 2-5 times under each extraction agent, and the volume of the extraction agent used each time is the same as the volume of the mixed solution; in the process of suspending the dichloromethane extract (A) in water, the volume of water is the same as the volume of the dichloromethane extract (A); in the process of extracting the suspension with ethyl acetate as an extraction agent, the extraction is performed 2-5 times, and the volume of ethyl acetate used each time is the same as the volume of the suspension; ii. The alkaline water kneading method includes the steps of: adding an aqueous NaOH solution to the obtained extract, kneading and dissolving, and filtering; adding the filter residue to the aqueous NaOH solution, kneading and dissolving, and filtering, and the obtained filter residue continues to repeat this step 2-5 times, combining the filtrates to obtain an alkaline aqueous solution of leonurus; adjusting the pH value to neutral to obtain a feed liquid; subjecting the feed liquid to reduced pressure concentration to obtain a leonurus phenolic acid enriched product; Preferably, the concentration of the aqueous NaOH solution is 1-5 wt%; the mass of the aqueous NaOH solution is 2-5 times the mass of the extract; the obtained alkaline aqueous solution of leonurus is adjusted to neutral with an aqueous hydrochloric acid solution with a concentration of 4-8 N; iii. The macroporous adsorption resin column chromatography method includes the steps of: adjusting the pH value of the obtained extract to 2-7, injecting it into the macroporous adsorption resin column bed, and then sequentially using water, a 50 wt% aqueous ethanol solution, and a 95 wt% aqueous ethanol solution as eluents for elution, combining the eluates of the 50 wt% aqueous ethanol solution and the 95 wt% aqueous ethanol solution, and concentrating until there is no alcohol smell to obtain a leonurus phenolic acid enriched product; Preferably, the pH value is adjusted to 2-7 using a 4-6 N aqueous hydrochloric acid solution; the flow rate of injecting into the macroporous adsorption resin column bed is 0.1-0.3 BV / h; the flow rate of the eluent is 0.4-0.6 BV / h; the eluent water is eluted for 2-6 retention volumes (BV), and the eluents of the 50 wt% aqueous ethanol solution and the 95 wt% aqueous ethanol solution are respectively eluted for 6-20 retention volumes (BV); iv. The strong basic anion exchange resin method comprises the steps of: injecting the obtained extract into a strong basic anion exchange resin column, and then eluting successively with water and an acid aqueous solution as the eluent. The pH of the eluate obtained with the acid aqueous solution as the eluent is adjusted to 3 - 7, and it is concentrated under reduced pressure to obtain a concentrated solution; extracting with ethyl acetate and concentrating to obtain a leonuritic acid enriched product; Preferably, the injection flow rate of the extract is 0.1 - 0.3 BV / h; the flow rate of the eluent is 0.3 - 0.8 BV / h; the eluent is washed with water for 2 - 5 BV and with the acid aqueous solution for 4 - 10 BV; the concentration of the acid aqueous solution of the eluent is 0.5 - 2 N, and the acid is hydrochloric acid, sulfuric acid or phosphoric acid; the pH is adjusted to 3 - 7 with a 4 - 6 N NaOH aqueous solution; it is concentrated under reduced pressure to 2 - 6 times the mass of the extract; the extraction times are 2 - 6 times, and the volume ratio of ethyl acetate to the concentrated solution used for each extraction is 1:

1.

5. The preparation method of the salvianolic acid extract of Leonurus japonicus Houtt. with thrombolytic effect according to claim 2, wherein, including one or more of the following conditions: i. The silica gel partition column chromatography method comprises the steps of: dissolving the obtained leonuritic acid enriched product in methanol, mixing with silica gel of 100 - 200 mesh, and drying at room temperature to obtain a silica gel mixed sample; suspending silica gel of 200 - 300 mesh with water - saturated dichloromethane to pack the column, loading the silica gel mixed sample onto the top of the silica gel column bed, first eluting with the lower phase (water - saturated dichloromethane phase) of dichloromethane - water with a volume ratio of 100:5 for 1 - 3 BV, then eluting with the lower phase (water - and methanol - saturated dichloromethane phase) of dichloromethane - methanol - water with a volume ratio of 100:1:5 for 2 - 5 BV, then eluting successively with the lower phase (water - and methanol - saturated dichloromethane phase) of dichloromethane - methanol - water with volume ratios of 100:2:5 and 100:4:5 for 3 - 8 BV respectively, and then eluting with the lower phase (water - and methanol - saturated dichloromethane phase) of dichloromethane - methanol - water with a volume ratio of 100:10:8 for 2 - 5 BV; combining the eluates of the lower phases of dichloromethane - methanol - water with volume ratios of 100:2:5 and 100:4:5, and concentrating under reduced pressure to obtain crude leonuritic acid; preferably, the mass ratio of the leonuritic acid enriched product to methanol is 1:2 - 3, and the mass ratio of silica gel to the leonuritic acid enriched product is 2 - 4:1; ii. The ODS silica gel column chromatography method comprises the steps of: dissolving the obtained leonuritic acid enriched product in methanol, loading it onto the ODS silica gel column bed by wet method, first eluting with a 10 wt% methanol aqueous solution for 2 - 4 BV, and then eluting successively with 50 wt% methanol aqueous solution, 70 wt% methanol aqueous solution, 90 wt% methanol aqueous solution, and 100 wt% methanol for 2 - 8 BV; combining the eluates of the 50 wt% methanol aqueous solution and the 70 wt% methanol aqueous solution, and concentrating under reduced pressure to obtain crude leonuritic acid; preferably, the mass ratio of the leonuritic acid enriched product to methanol is 1:1 - 3.

6. The preparation method of the phenolic acid extract of Leonurus artemisia with thrombolytic effect according to claim 2, characterized in that, The purification method comprises the steps of dissolving the crude extract of leonuritic acid obtained by separation in methanol, loading it onto the bed of an MCI chromatographic column, first eluting with an 80 wt% aqueous methanol solution for 5 - 10 bed volumes (BV), and then eluting with 100% methanol for 3 - 6 BV; taking the eluate of the 80 wt% aqueous methanol solution, concentrating it under reduced pressure to obtain the leonuritic acid extract; preferably, the mass ratio of the crude extract of leonuritic acid to methanol is 1:1 - 3.

7. Use of the phenolic acid extract of Leonurus japonicus with thrombolytic effect according to claim 1, characterized in that, It is used for preparing a drug for treating thrombotic diseases.

8. Use of the phenolic acid extract of Leonurus japonicus with thrombolytic effect as described in claim 1, characterized in that, The leonuritic acid extract is used for preparing a drug having the effect of dissolving thrombus or anti - thrombosis.

9. A pharmaceutical composition having a thrombolytic effect, characterized in that, It comprises the leonuritic acid extract or its pharmaceutically acceptable salt as claimed in claim 1, one or more pharmaceutically acceptable carriers or excipients, and an antioxidant.

10. The pharmaceutical composition with thrombolytic effect according to claim 9, characterized in that, The pharmaceutical composition is one of tablets, capsules, granules, freeze - dried injection preparations, powder injections, pellets, and ointments.

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