Syringic acid as well as preparation, pharmaceutical composition and medical application thereof

Abstract

The invention provides syringic acid as well as preparation, a pharmaceutical composition and medical application thereof. The purity of the syringic acid is 95% or above, and the syringic acid can be extracted from motherwort. The syringic acid disclosed by the invention has a good dissolving effect on fibrin in thrombus formation, is relatively high in blood-brain barrier transmittance, has thrombus dissolving and antithrombotic effects, and can be used for preparing medicines for treating thrombotic diseases. The syringic acid 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 injection freeze-dried powder injections, and external preparations such as ointments or emulsifiable paste according to a conventional production process of pharmaceutics, and is used for treating thrombotic diseases, especially cerebral thrombosis diseases.

Description

Technical Field

[0001] The present invention relates to syringic acid, its preparation, pharmaceutical composition and medical use, belonging to the field of medical technology. Background Art

[0002] Thrombotic diseases are diseases caused by two pathological processes: thrombosis formation and thromboembolism. Thrombosis formation refers to the pathological process in which blood formed elements form emboli in blood vessels (mostly small blood vessels) under certain conditions, causing partial or complete blockage of blood vessels and blood supply disorders in the corresponding parts. Thromboembolism is the pathological process in which a thrombus detaches from the formation site and partially or completely blocks some blood vessels during the process of moving with the blood flow, causing ischemia, hypoxia, necrosis (arterial thrombus) and congestion, edema (venous thrombus) in the corresponding tissues and (or) organs. Thrombotic diseases seriously threaten human life and health, with their incidence ranking first among various diseases and showing an increasing trend in recent years. They are one of the key points and hotspots in contemporary medical research.

[0003] Syringic acid is a kind of phenolic acid, a natural plant compound with various biological activities, and is often used in the fields of medicine and health products. Syringic acid is a potent natural antibacterial agent that can kill a variety of bacteria and inhibit the growth and reproduction of bacteria; it can inhibit the activation and secretion of inflammatory cells and reduce the release of inflammatory mediators, thus alleviating the inflammatory response; it has good antioxidant capacity, can neutralize free radicals and remove harmful substances in the body; it can reduce blood lipid and cholesterol levels and prevent the occurrence of cardiovascular diseases; it can regulate the function of the nervous system, reduce pain signal conduction and the activation of pain receptors, thus reducing pain and discomfort; in addition, syringic acid can also promote blood circulation and metabolism, and accelerate tissue repair and recovery. However, the prior art has not disclosed that syringic acid has the effect of dissolving thrombus or anti-thrombosis. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a syringic acid, its preparation, pharmaceutical composition and medical use. The syringic acid of the present invention, with a purity of more than 95%, can be extracted from Leonurus japonicus. The syringic acid of the present invention has a good dissolving effect on fibrin in thrombosis formation, and has a relatively high transmittance through the blood-brain barrier, with the effects of dissolving thrombus and anti-thrombosis, and can be used to prepare drugs for treating thrombotic diseases. The syringic acid of the present invention can be processed into oral preparations such as tablets, capsules, granules, dripping pills and other common preparations or sustained-release preparations according to the conventional pharmaceutical production process, injection preparations such as freeze-dried powder for injection, and external preparations such as ointments or creams for treating thrombotic diseases, especially for the treatment of cerebral thrombosis.

[0005] The technical solution of the present invention is as follows:

[0006] A syringic acid, with a purity greater than or equal to 95%, has the following structure:

[0007]

[0008] In the present invention, the source of syringic acid includes but is not limited to being isolated and prepared from Leonurus japonicus, and also includes being isolated and prepared from other plants containing syringic acid, and being prepared by total chemical synthesis or semi-synthesis, or can also be obtained commercially.

[0009] The preferred preparation method of syringic acid in the present invention includes the steps of: using the aerial stems and leaves of Leonurus japonicus as raw materials, and obtaining syringic acid through extraction, enrichment, separation, and purification.

[0010] The extraction method is water extraction method, alcohol-water extraction method or alkaline water extraction method; the enrichment method is extraction method, alkaline water kneading and dissolving 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; the purification method is high performance liquid chromatography (HPLC) method.

[0011] According to the preference of the present invention, the water extraction method or alcohol-water extraction method includes the steps of: crushing the aerial stems and leaves of Leonurus japonicus into coarse powder, then immersing it in water or alcohol-water solvent, and obtaining the Leonurus japonicus extract through extraction and filtration; or,

[0012] Or, crushing the aerial stems and leaves of Leonurus japonicus into coarse powder, wetting it in an aqueous NaOH solution, adjusting the pH value to neutral with a 1-5wt% aqueous hydrochloric acid solution, and filtering; then immersing it in water or alcohol-water solvent, and obtaining the Leonurus japonicus extract through extraction and filtration.

[0013] Preferably, the alcohol-water solvent is a 50wt% - 100wt% methanol solution or a 50wt% - 95wt% ethanol aqueous solution; the weight ratio of water or alcohol-water solvent to the coarse powder is 5:1 - 20:1.

[0014] Preferably, the concentration of the aqueous NaOH solution is 0.5 - 2wt%, the wetting temperature is room temperature, and the wetting time is 2 - 8h.

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

[0016] Preferably, the Leonurus japonicus extract can be further concentrated under normal pressure or reduced pressure to obtain the Leonurus japonicus extract paste.

[0017] Preferably according to the present invention, the alkaline water extraction method comprises the steps of: pulverizing the aerial stems and leaves of Leonurus japonicus into coarse powder, impregnating and extracting in an NaOH aqueous solution, and filtering to obtain an alkaline water extract of Leonurus japonicus.

[0018] Preferably, the concentration of the NaOH aqueous solution is 0.5 wt% to 1.5 wt%; the mass ratio of the NaOH aqueous solution to the coarse powder is (5 to 12):1.

[0019] Preferably, the number of impregnation and extraction times is 1 to 5 times; the method for each impregnation and extraction is: standing at room temperature for 2 to 12 h, or ultrasonically treating at room temperature for 15 to 30 minutes, or standing at room temperature for 2 to 12 h and then ultrasonically treating at room temperature for 15 to 30 minutes.

[0020] Preferably, after the filtering step, the pH of the alkaline water extract needs to be adjusted to 5 to 7 using a hydrochloric acid aqueous solution.

[0021] Preferably according to the present invention, the extraction method comprises the steps of: diluting the extract obtained from the above extraction 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 vacuum 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 the extraction agent, and subjecting the ethyl acetate extract 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 Leonurus japonicus phenolic acid enriched product.

[0022] Preferably, in the dilution of the extract with water, the volume of water is 3 to 6 times the volume of the extract; during the sequential extraction with petroleum ether, dichloromethane, and ethyl acetate as extraction agents, extraction is performed 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; during the suspension of the dichloromethane extract (A) in water, the volume of water is the same as the volume of the dichloromethane extract (A); during the extraction of the suspension with ethyl acetate as the extraction agent, the number of extraction times is 2 to 5 times, and the volume of ethyl acetate used each time is the same as the volume of the suspension.

[0023] Preferably according to the present invention, the alkaline water kneading method comprises the steps of: adding an NaOH aqueous solution to the extract obtained from the above extraction, kneading and filtering; adding the filter residue to an NaOH aqueous solution, kneading 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 Leonurus japonicus; using this alkaline aqueous solution of Leonurus japonicus, or adjusting the pH value to neutral with a hydrochloric acid aqueous solution to obtain a feed liquid, subjecting the feed liquid to vacuum concentration to obtain a Leonurus japonicus phenolic acid enriched product, or directly treating it by a strong basic anion exchange resin method to obtain a Leonurus japonicus phenolic acid enriched product.

[0024] Preferably, the concentration of the NaOH aqueous solution is 1 wt% to 5 wt%; the mass of the NaOH aqueous solution is 2 to 5 times the mass of the extract; the pH value of the obtained aqueous solution of motherwort alkali is adjusted to neutral with a hydrochloric acid aqueous solution having a concentration of 4 - 8N.

[0025] Preferably according to the present invention, the macroporous adsorption resin column chromatography includes the steps of: adjusting the pH value of the obtained extract to 2 - 7, loading it onto the macroporous adsorption resin column bed, and then successively 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 enrichment.

[0026] Preferably, a hydrochloric acid aqueous solution with a concentration of 4 - 6N is used to adjust the pH value to 2 - 7; when loading, the flow rate of the liquid flowing out from the bottom end of the macroporous adsorption resin column bed is 0.1 - 0.3 BV / h; when the eluent is eluting, the flow rate of the liquid flowing out from the bottom end of the column 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% ethanol aqueous solution and the 95 wt% ethanol aqueous solution are respectively eluted for 6 - 20 retention volumes (BV).

[0027] Preferably according to the present invention, the strong basic anion exchange resin method includes the steps of: directly injecting the motherwort alkali water extract obtained by the above alkali water extraction method or the motherwort alkali aqueous solution obtained by the alkali water kneading and dissolving method into a strong basic anion exchange resin column, and then successively using water and an acid aqueous solution as eluents for elution, adjusting the pH value of the eluate obtained with the acid aqueous solution as the eluent to 5 - 7, and concentrating under reduced pressure to obtain a concentrated solution; extracting with ethyl acetate and concentrating to obtain a motherwort phenolic acid enrichment.

[0028] Preferably, when injecting the motherwort alkali water extract or the motherwort alkali aqueous solution, the flow rate of the liquid flowing out from the bottom end of the column is 0.1 - 0.3 BV / h; when the eluent is eluting, the flow rate of the liquid flowing out from the bottom end of the column is 0.3 - 1 BV / h; the eluent water is eluted for 2 - 5 BV, and the eluent acid aqueous solution is eluted for 4 - 10 BV; the concentration of the eluent acid aqueous solution is 0.5 - 2N, and the acid is hydrochloric acid, sulfuric acid or phosphoric acid; the pH value is adjusted to 5 - 7 with a 4 - 6N NaOH aqueous solution; concentrating under reduced pressure to 1 - 3 times the mass of the motherwort alkali water extract or the motherwort alkali aqueous solution; the number of extraction times is 2 - 6 times, and the volume ratio of ethyl acetate to the concentrated solution used for each extraction is 1:1.

[0029] Preferably according to the present invention, the silica gel column chromatography method includes the steps of: dissolving the enriched leonuritic acid enriched product in methanol, mixing the sample with silica gel of 100-200 mesh, and drying at room temperature to obtain a silica gel sample mixture; suspending silica gel of 200-300 mesh with water-saturated dichloromethane to pack the column; then loading the silica gel sample mixture 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 sequentially 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 eluents 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 leonuritic acid mixture. 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.

[0030] Preferably according to the present invention, the ODS silica gel column chromatography method includes the steps of: dissolving the enriched leonuritic acid enriched product in methanol, loading the sample onto the top of 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 sequentially eluting with a 50 wt% methanol aqueous solution, a 70 wt% methanol aqueous solution, and 100% methanol for 2-10 BV; combining the eluents of the 50 wt% methanol aqueous solution and the 70 wt% methanol aqueous solution, and concentrating under reduced pressure to obtain a leonuritic acid mixture. Preferably, the mass ratio of the leonuritic acid enriched product to methanol is 1:1-3.

[0031] Preferably according to the present invention, the purification method comprises the steps of: using high performance liquid chromatography (HPLC), through two preparations: for the first preparation, dissolving the mixture of phenolic acids of Leonurus japonicus obtained by the above separation in methanol, injecting the sample into a preparative or semi-preparative C18 high performance liquid chromatography column of HPLC, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid water (v / v), gradient elution, and the elution program is: 0 - 10 min, the volume ratio of mobile phase A is 10%, the volume ratio of mobile phase B is 90%; 10 - 15 min, the volume ratio of mobile phase A is 10% → 20%, the volume ratio of mobile phase B is 90% → 80%; 15 - 30 min, the volume ratio of mobile phase A is 20% → 50%, the volume ratio of mobile phase B is 80% → 50%; collecting the eluate of the chromatographic peak with a retention time of 21.5 min, concentrating to obtain crude syringic acid; for the second preparation, dissolving the crude syringic acid in methanol, injecting the sample into a preparative or semi-preparative C18 high performance liquid chromatography column of HPLC, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid water (v / v), gradient elution, and the elution program is: 0 - 5 min, the volume ratio of mobile phase A is 0% → 30%, the volume ratio of mobile phase B is 100% → 70%; 5 - 20 min, the volume ratio of mobile phase A is 30%, the volume ratio of mobile phase B is 70%; collecting the eluate of the chromatographic peak with a retention time of 12.8 min, concentrating to obtain pure syringic acid. The meaning of the above arrows used is linear change.

[0032] The present invention adopts the internationally and domestically common fibrin plate method, and through fibrinolytic activity tracking, for the first time discovers the active sites ACT1 (see Test Example 1) and ACT2 (see Test Example 2) with fibrinolytic activity in Leonurus japonicus. Through component separation and structure identification of the active site ACT2, it is determined that its main component structures are respectively: vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethyl gallic acid (see Test Example 3), and the composition and content of the main active components in the fibrinolytic active site of Leonurus japonicus are analyzed and determined, among which the content of syringic acid is the highest (see Test Example 4).

[0033] The commonly used thrombolytic drug urokinase in current clinical practice was used as the positive control (see Test Example 5), and a solution of 40 U / ml was prepared with physiological saline. Using the internationally and domestically common fibrin plate method, with physiological saline as the solvent, 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 were respectively dissolved in 100 μL of chromatographic methanol to make their concentration 20 mg / mL, and then the diameter φ (mm) of the fibrinolytic circle on the fibrin plate was measured. The results showed that the five single compounds, namely vanillic acid, syringic acid, trans-ferulic acid, 3,4,5-trimethylgallic acid, and cis-ferulic acid (a mixture containing a small amount of trans-ferulic acid), all had strong protein fibrinolytic activity, among which syringic acid and vanillic acid had the strongest fibrinolytic activity (see Test Example 6).

[0034] Using the internationally and domestically common Chandler loop experiment method, with the blood of male Wistar rats as the research object and aspirin as the positive control drug, the experimental results are shown in Test Example 7. Compared with the blank group, in the high, medium, 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 anti-thrombosis, laying a foundation for further development into a drug for treating thrombotic diseases.

[0035] For the in vitro blood-brain barrier permeability experiment, the measured values and experimental values of the literature theoretical permeability values of four commercially available drugs (estradiol, progesterone, alprazolam, oxazepam) were used to make a standard curve for subsequent correction, and a good linear correlation equation was 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 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 that the compound has poor blood-brain barrier permeability. The experimental results found that among the 5 single compounds isolated from the active part 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-trimethylgallic acid were 7.1×10 -6 cm / s, 9.2×10 -6 cm / s, and 8.1×10 -6 cm / s respectively, all of which were greater than 5.1×10 -6cm / s, indicating that they all have good blood-brain barrier permeability, and syringic acid has the strongest permeation activity, similar to the penetration ability of the control drug oxazepam, and has the potential value of being developed into a drug for treating cerebral thrombosis.

[0036] The above-mentioned activity test results show that the present invention innovatively discovers that syringic acid has relatively good fibrinolytic activity and good permeability through the blood-brain barrier. Therefore, syringic acid can be used to develop into a therapeutic drug for treating thrombotic diseases, especially cerebral thrombosis.

[0037] The above application of syringic acid is used to prepare a drug with thrombolytic or antithrombotic effects.

[0038] Preferably according to the present invention, the thrombus is cerebral thrombosis.

[0039] The syringic acid or a pharmaceutically acceptable salt thereof of the present invention is combined with pharmaceutical excipients to form pharmaceutical preparations of different dosage forms for treating thrombotic diseases, especially cerebral thrombosis.

[0040] A drug composition with thrombolytic effect includes the syringic acid or a pharmaceutically acceptable salt thereof of the present invention, one or more pharmaceutically acceptable carriers or excipients, and an antioxidant.

[0041] Preferably according to the present invention, the pharmaceutically acceptable salt is prepared by dissolving syringic acid 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 a sodium salt, potassium salt or magnesium salt. Preferably, the alkalizing reagent is NaHCO3 or KHCO3.

[0042] Preferably according to the present invention, the antioxidant is selected from sodium ascorbate, ascorbic acid, citric acid or sodium metabisulfite.

[0043] Preferably according to the present invention, the drug composition is one of tablets, capsules, granules, freeze-dried injection preparations, powder injections, pellets, ointments or creams, and can be prepared according to the conventional production processes of pharmacy.

[0044] The pharmaceutical preparations of different dosage forms of the drug composition are described in detail as follows:

[0045] (1) Preparation of granules, capsules, enteric-coated capsules, tablets or enteric-coated tablets:

[0046] Take the syringic acid described above. Based on the weight of the syringic acid as 1 weight multiple, add 1 - 4 weight multiples of diluent I, 2 - 8 wt% of wetting agent, and 3 - 20 wt% of disintegrant, and granulate by the conventional wet granulation method, then dry, size the granules, and bag them to obtain the granule agent; or add 0.2 - 10 wt% of lubricant to the sized granules, mix well, fill them into the capsule shell to obtain the capsule agent, or press them into tablets, or fill them into the enteric capsule shell to obtain the enteric capsule, or coat the tablets with enteric coating after pressing to obtain the enteric-coated tablets.

[0047] The above-mentioned diluent I is selected from starch, powdered sugar, dextrin, microcrystalline cellulose or hydroxypropyl cellulose;

[0048] The above-mentioned wetting agent is selected from water or ethanol;

[0049] The above-mentioned disintegrant is sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose;

[0050] The above-mentioned lubricant is magnesium stearate or talc powder.

[0051] (2) Preparation of pellets, pellet capsules, enteric-coated pellet capsules, enteric-coated pellet tablets, enteric-coated pellet sustained-release capsules or tablets

[0052] Take the syringic acid described above. Prepare a film agent with 1.2 wt% hydroxypropyl methylcellulose aqueous solution and polysorbate - 80, add talc powder, and mix well; add blank pellets to the fluidized bed, and spray the drug solution from the bottom to obtain the pellet cores. Coating the pellet cores, coating the isolation layer, and drying to obtain the pellets; or coating the enteric coating with polyacrylic resin, polyvinyl acetate phthalate and / or diethyl phthalate / enteric type Opadry, and drying to obtain the enteric-coated pellets, or after coating the isolation layer on the pellet cores, spraying the aqueous solution of enteric type Opadry coating premix to coat the enteric coating to obtain the enteric-coated pellets. Fill the pellets or enteric-coated pellets into capsules to obtain pellet capsules or enteric-coated pellet capsules; press 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 the sustained-release enteric-coated pellets, filling them into ordinary capsule shells to obtain the sustained-release enteric-coated pellet capsules, or pressing them into tablets to obtain the sustained-release enteric-coated pellet tablets.

[0053] Preferably, the excipient for the above-mentioned isolation layer is hydroxypropyl methylcellulose;

[0054] Preferably, the excipients 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;

[0055] Preferably, the above-mentioned sustained-release layer is Eudragit NE30D, DrugCoat L30D coating materials, and more preferably Eudragit NE30D.

[0056] (3) Preparation of Soft Capsules and Enteric-coated Soft Capsules

[0057] Take the said syringic acid, add a diluent, mix evenly, then add a suspending agent, stir well, and let stand at room temperature to obtain the liquid medicine for the content of soft capsules of syringic acid; press it together with the soft capsule shell material prepared in advance with gelatin, glycerin, and preservatives through a mold to form soft capsules. The soft capsules prepared according to the above method are then coated with a separating 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, and enteric-coated soft capsules are obtained.

[0058] The above-mentioned diluent is selected from vegetable oils;

[0059] The above-mentioned suspending agent is selected from beeswax, chitin methylcellulose or agar.

[0060] (4) Preparation of Freeze-dried Injectable Preparation of Syringic Acid (Salt)

[0061] Take the said syringic acid, 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 injectable preparation of syringic acid. When in use, dissolve it with an equivalent amount of injectable alkaline reagent to form a solution.

[0062] Or, dissolve the said syringic acid in distilled water, adjust the pH to 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 vials under aseptic conditions, freeze-dry, and then seal under nitrogen to obtain it.

[0063] The above-mentioned antioxidant is sodium ascorbate or ascorbic acid.

[0064] The above-mentioned alkalizing reagent is an aqueous solution of NaHCO3 or KHCO3.

[0065] The above-mentioned injectable alkaline reagent is an aqueous solution of NaOH, NaHCO3 or KHCO3.

[0066] (5) Preparation of Powder for Injectable of Syringate

[0067] Take the said syringic acid, 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, freeze-dry under aseptic conditions, grind and mix the obtained pharmaceutical composition evenly, dispense into vials, and seal under nitrogen to obtain it.

[0068] The antioxidant described above is sodium ascorbate or ascorbic acid.

[0069] The alkalizing agent described above is an aqueous solution of NaHCO3 or KHCO3.

[0070] (6) Preparation of syringic acid ointment

[0071] Dissolve azone or triethanolamine, glycerol, sodium lauryl sulfate, and methylparaben in an appropriate amount of distilled water at 85 °C, add syringic acid, 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, stir rapidly in the same direction to fully mix and emulsify. After cooling, it is obtained. (7) Preparation of syringic acid cream

[0072] Take the syringic acid described above, moisten it with an appropriate amount of propylene glycol, and grind it until no visible particles remain. Take glyceryl monostearate, dimethicone, 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, which is 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 it.

[0073] The application of a pharmaceutical composition with thrombolytic effect of the present invention is used for the treatment of thrombotic diseases, especially cerebral thrombosis, to dissolve its thrombus, and can improve or eliminate the blood circulation disorder caused by thrombus.

[0074] The technical features and beneficial effects of the present invention are as follows:

[0075] The present invention preferably extracts syringic acid from Leonurus japonicus. The method of the present invention can effectively obtain high-purity syringic acid, and the purity of the obtained syringic acid is greater than or equal to 95%.

[0076] The present invention obtains a variety of different chemical parts through different extraction, separation and other methods, combines activity evaluation, and conducts step-by-step experimental tests. After adopting different separation methods, evaluating the activities of the obtained different chemical parts, and repeated experiments, the chemical part with the optimal fibrinolytic activity in Leonurus japonicus is obtained.

[0077] There are many methods for separating the chemical components of traditional Chinese medicine. Different methods will result in different separation and purification results of the chemical components, and there will be differences in the composition of the components. On the basis of not knowing the structure of the fibrinolytic active components in Leonurus japonicus, during the early stage of tracking the 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 parts either had no fibrinolytic activity or had no difference in fibrinolytic activity, and the active part of Leonurus japonicus could not be determined. It was not until the method of the present invention was adopted that an ideal fibrinolytic active part was obtained, and the component with the best performance was determined to be syringic acid. Then, further purification was carried out to obtain high-purity syringic acid.

[0078] The present invention designs a more suitable extraction, separation and purification method according to the physical and chemical properties of syringic acid components.

[0079] The present invention adopts the commonly used in vitro fibrin plate assay method at home and abroad. Through fibrinolytic activity tracking, the active part with fibrinolytic activity in Leonurus japonicus was first discovered (see Test Examples 1 and 2), and the main components of the active part in Test Example 2 were separated and structurally identified, and it was determined that its main components were vanillic acid, syringic acid, trans-ferulic acid, cis-ferulic acid and 3,4,5-trimethylgallate (see Test Example 3). The content of syringic acid in the active part was the highest (see Test Example 4).

[0080] The present invention first discovered that syringic acid has good fibrinolytic activity, that is, it has a relatively good dissolving effect on fibrin in thrombus formation, thereby exerting its good thrombolytic effect. Using the commonly used fibrin plate method at home and abroad, with the currently clinically used thrombolytic drug urokinase as a positive control, the fibrinolytic activities of the fibrinolytic active part obtained from Leonurus japonicus and the five single compounds separated from this part were measured. The results showed that the active part and the five single compounds vanillic acid, syringic acid, trans-ferulic acid, 3,4,5-trimethylgallate and cis-ferulic acid (a mixture containing a small amount of trans-ferulic acid) obtained from it all had strong protein fibrinolytic activity, and among them, the fibrinolytic activities of syringic acid and vanillic acid were the strongest (see Test Example 6), clarifying the chemical part with fibrinolytic effect in Leonurus japonicus and its fibrinolytic active components, and in vitro experiments showed that syringic acid has significant anti-thrombotic activity, indicating that syringic acid has good potential application value for anti-thrombosis, laying a foundation for further development into a drug for treating thrombotic diseases.

[0081] The present invention first discovered that syringic acid has good blood-brain barrier permeability. Through in vitro blood-brain barrier permeability experiments, it was found that among the four components obtained from the fibrinolytic active part of Leonurus japonicus, except for trans-ferulic acid (Pe value is 1.2×10 -6In addition to (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 5.1×10 -6 cm / s, indicating that they all have good blood-brain barrier permeability. Moreover, syringic acid has the strongest permeability activity and is similar to the penetration ability of the control drug oxazepam, suggesting that syringic acid also has the potential value to be developed into a drug for treating cerebral thrombosis.

[0082] Among the five fibrinolytic active components, 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) obtained from Leonurus japonicus in the present invention, the content of syringic acid is the largest. Its fibrinolytic activity is superior to that of trans-ferulic acid, 3,4,5-trimethyl gallic acid, and cis-ferulic acid (a mixture containing a small amount of trans-ferulic acid), and its blood-brain barrier permeability rate is better than that of vanillic acid. Therefore, syringic acid has the most important value among these five components to be developed into drugs for thrombotic diseases, especially cerebral thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 It is the HPLC fingerprint of the active site ACT1 in Test Example 1.

[0084] Figure 2 It is the HPLC fingerprint of the active site ACT2 in Test Example 2.

[0085] Figure 3 It is the standard curve for the determination of the contents of vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethyl gallic acid in Test Example 4.

[0086] Figure 4 It is the HPLC content determination chromatogram of the phenolic acid extract ACT2 (A) of Leonurus japonicus and the reference substance (B) in Test Example 4.

[0087] Figure 5 It is the HPLC chromatogram of syringic acid prepared by the method of Example 1 in Test Example 4.

[0088] Figure 6 It is the standard curve of the fibrinolytic activity of different doses of urokinase with the diameter of the fibrinolytic circle as the index in Test Example 5.

[0089] Figure 7 It is the calibration curve of the measured Pe value and the literature penetration value Pe of four commercially available drugs (estradiol, progesterone, alprazolam, and oxazepam) in Test Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0090] The present invention will be further described below in conjunction with embodiments, but not limited thereto.

[0091] Example 1

[0092] A preparation method of syringic acid with fibrinolytic effect, comprising the steps:

[0093] After the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, distilled water is used as the extraction solvent for heating and 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, followed by filtration; 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 removed by filtration, and the filtrates are combined. It is concentrated to a paste to obtain the Leonurus japonicus extraction extract.

[0094] Distilled water is added to the Leonurus japonicus extraction extract for dilution to obtain a mixed solution. The amount of water added is 6 times the weight of the Leonurus japonicus extraction extract. It is successively extracted with petroleum ether, dichloromethane, and ethyl acetate, and each phase is extracted 5 times repeatedly. 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 liquid A and the ethyl acetate phase extraction liquid B are collected. The extraction liquid A is concentrated under reduced pressure to a paste at 50 °C, and water with the same weight as the paste is added for suspension to obtain a suspension. It 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. The solvent is evaporated under reduced pressure to obtain a solid C. The extraction liquid B is evaporated under reduced pressure to remove the solvent at 50 °C to obtain a solid B. The solid C and B are combined to obtain the crude extract of Leonurus japonicus phenolic acid.

[0095] After dissolving the crude extract of Leonurus japonicus phenolic acid in methanol (the mass ratio of the crude extract of Leonurus japonicus 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 Leonurus japonicus phenolic acid is 3:1). It is air-dried at room temperature to obtain a silica gel sample mixture, and silica gel partition chromatography column separation and purification are carried out. The column is filled with silica gel suspended in dichloromethane saturated with water with a mesh size of 200 - 300. The above silica gel sample mixture is added to the top of the silica gel column bed. First, the lower phase of dichloromethane - water with a volume ratio of 100:5 (dichloromethane phase saturated with water) is used for elution of 2 BV, then the lower phase of dichloromethane - methanol - water with a volume ratio of 100:1:5 (dichloromethane phase saturated with water and methanol) is used for elution of 4 BV, then the lower phase of dichloromethane - methanol - water with a volume ratio of 100:2:5 (dichloromethane phase saturated with water and methanol) is used for elution of 8 retention volumes, then the lower phase of dichloromethane - methanol - water with a volume ratio of 100:4:5 (dichloromethane phase saturated with water and methanol) is used for elution of 6 retention volumes, and then the lower phase of dichloromethane - methanol - water with a volume ratio of 100:10:8 (dichloromethane phase saturated with water and methanol) is used for elution of 4 BV; the eluates of the lower phases of dichloromethane - methanol - water with volume ratios of 100:2:5 and 100:4:5 are combined, and the solvent is evaporated under reduced pressure at 50 °C to obtain a solid, namely, the Leonurus japonicus phenolic acid mixture.

[0096] Syringic acid was prepared by high performance liquid chromatography (HPLC). The above-mentioned phenolic acid mixture of Leonurus artemisia was dissolved in methanol (mass ratio 1:100), and 0.5 mL of the sample solution was loaded onto a semi-preparative C18 high performance liquid chromatography column of HPLC. Mobile phase A was acetonitrile, and mobile phase B was 0.1% formic acid in water (v / v). Gradient elution was performed, and the elution program was as follows: from 0 to 10 min, the volume percentage of mobile phase A was 10%, and the volume percentage of mobile phase B was 90%; from 10 to 15 min, the volume percentage of mobile phase A was 10% → 20%, and the volume percentage of mobile phase B was 90% → 80%; from 15 to 30 min, the volume percentage of mobile phase A was 20% → 50%, and the volume percentage of mobile phase B was 80% → 50%; The eluate of the chromatographic peak with a retention time of 21.5 min was collected and concentrated to obtain the crude syringic acid; for the second preparation, the obtained crude syringic acid was dissolved in methanol (mass ratio 1:100), and injected into a semi-preparative C18 high performance liquid chromatography column of HPLC. Mobile phase A was acetonitrile, and mobile phase B was 0.1% formic acid in water (v / v). Gradient elution was performed, and the elution program was as follows: from 0 to 5 min, the volume percentage of mobile phase A was 0% → 30%, and the volume percentage of mobile phase B was 100% → 70%; from 5 to 20 min, the volume percentage of mobile phase A was 30%, and the volume percentage of mobile phase B was 70%; The eluate of the chromatographic peak with a retention time of 12.8 min was collected and concentrated to obtain the pure syringic acid. The meaning of the above-mentioned arrows is linear change; for example, from 10 to 15 min, the volume percentage of mobile phase A was 10% → 20%, which means that within 10 to 15 min, the volume percentage of mobile phase A linearly increased from 10% to 20%; and so on for the others.

[0097] 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 was as follows: from 0 to 10 min, 10% methanol aqueous solution; from 10 to 15 min, 10% → 20% methanol aqueous solution; from 15 to 25 min, 20% → 40% methanol aqueous solution; from 25 to 40 min, 40% → 80% methanol aqueous solution. Column temperature: 30 °C, flow rate: 0.3 mL / min, injection volume: 10 μL.

[0098] After HPLC analysis and determination, the purity of the obtained syringic acid was 97.2%.

[0099] Example 2

[0100] A preparation method of syringic acid with fibrinolytic activity is as described in Example 1, except that: the extraction method is different, specifically as follows:

[0101] After the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, 70wt% ethanol aqueous solution is used as the extraction solvent for reflux extraction. For the first time, 12 times the amount (i.e., 12 times the mass of the coarse powder) of 70wt% 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 70wt% ethanol aqueous solution is repeatedly added to the filter residue, and each 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 is removed, and an extract of Leonurus japonicus is obtained.

[0102] Other steps and conditions are the same as in Example 1, and syringic acid is prepared.

[0103] Determined by HPLC analysis, the purity of the obtained syringic acid is 96.8%.

[0104] Example 3

[0105] A preparation method of syringic acid with fibrinolytic activity is as described in Example 1, the difference is: the extraction method is different, specifically as follows:

[0106] 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, 13 times the amount (i.e., 13 times the mass of the coarse powder) of the solvent is added, and reflux extraction is carried out for 2h, followed by filtration; for the second and third extractions, 10 times the amount of the solvent is repeatedly added to the filter residue, and reflux extraction is carried out for 1h. The filter residue is filtered off, and the filtrates are combined. Under the condition of 50 °C, it is concentrated under reduced pressure until the alcohol is removed. An extract of Leonurus japonicus is obtained.

[0107] Other steps and conditions are the same as in Example 1, and syringic acid is prepared.

[0108] Determined by HPLC analysis, the purity of the obtained syringic acid is 96.2%.

[0109] Example 4

[0110] A preparation method of syringic acid with fibrinolytic activity is as described in Example 1, the difference is: the extraction method is different, specifically as follows:

[0111] After the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, 2 times the weight of its powder of 1wt% NaOH aqueous solution is added and moistened at room temperature for 6 hours, then the pH is adjusted to neutral with 1wt% hydrochloric acid aqueous solution, and the filter residue is obtained by filtration; then for the first time, 10 times the weight of its coarse powder of 95wt% ethanol aqueous solution is added to the filter residue for reflux extraction for 2h, and the filter residue is obtained by filtration; for the second and third extractions, 1 times the weight of the coarse powder of 70wt% ethanol aqueous solution is repeatedly added to the filter residue, and reflux extraction is carried out for 1h. The medicinal residues are removed, the filtrates are combined, and under the condition of 50 °C, it is concentrated under reduced pressure until the alcohol is removed. An extract of Leonurus japonicus is obtained.

[0112] Other steps and conditions are the same as in Example 1, and syringic acid is prepared.

[0113] HPLC analysis showed that the purity of the obtained syringic acid was 97.3%.

[0114] Example 5

[0115] A method for preparing syringic acid with fibrinolytic activity is as follows:

[0116] The above-ground stems and leaves of Leonurus japonicus were ground into a coarse powder. Ultrasonic extraction was then performed at 50°C for 30 minutes using a 70wt% ethanol aqueous solution (8 times the amount of the coarse powder) as the extraction solvent. The extraction was repeated five times. The residue was removed, and the filtrate was combined. The extract was then concentrated under reduced pressure at 50°C to a paste to obtain a Leonurus japonicus extract.

[0117] To the motherwort extract, 3 times its weight was added a 1 wt% aqueous solution of NaOH, and the mixture was kneaded and filtered. The filter residue was then kneaded with 2 times the weight of the motherwort extract in a 1 wt% aqueous solution of NaOH, and filtered. This step was repeated twice. The combined kneading solutions were neutralized with 8N aqueous hydrochloric acid to neutrality, and concentrated to a paste to obtain a crude extract of motherwort phenolic acid.

[0118] The crude extract of leonuronic acid was dissolved in methanol (the mass ratio of leonuronic acid mixture to methanol was 1:2) and loaded onto C 18 At the top of the reversed-phase silica gel (ODS) column, 3 BV of the solution was first eluted with a 10 wt% aqueous methanol solution, followed by 6 BV, 10 BV, and 4 BV of the solution using 50 wt% aqueous methanol, 70 wt% aqueous methanol, and 100 wt% methanol, respectively. The 50% and 70% aqueous methanol eluates were combined and concentrated under reduced pressure to obtain a mixture of lemurocanic acids.

[0119] Then, HPLC was used to separate and prepare syringic acid from the crude syringic acid product in the same manner as in Example 1.

[0120] HPLC analysis showed that the purity of the obtained syringic acid was 98.2%.

[0121] Example 6

[0122] A method for preparing syringic acid with fibrinolytic activity comprises the steps of:

[0123] The above-ground stems and leaves of Leonurus japonicus are ground into coarse powder, and then immersed in a 1wt% NaOH aqueous solution at room temperature for 8 hours. The mixture is then ultrasonically extracted at room temperature for 30 minutes and filtered. The medicinal residue is then subjected to a second and third extraction, each of which is repeated with a 1wt% NaOH aqueous solution at room temperature for 30 minutes. The extraction is then filtered and the filtrates are combined. The pH of the filtrate is adjusted to 5 with a 5N hydrochloric acid aqueous solution to obtain an alkaline water extract of Leonurus japonicus.

[0124] The alkaline aqueous extract of Leonurus japonicus Houttuyn is directly loaded onto the upper end of the macroporous adsorption resin column bed, and the solution is allowed to flow through the macroporous adsorption resin column bed at a flow rate of 0.2 BV / h. Then, it is 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. The eluates of the 50 wt% ethanol aqueous solution and the 95 wt% ethanol aqueous solution are combined and concentrated until the alcohol smell disappears to obtain the crude extract of Leonurus japonicus phenolic acid.

[0125] Successively adopt silica gel column chromatography for distribution and HPLC method to separate and prepare syringic acid from the phenolic acid extract of Leonurus japonicus, and the method is the same as that in Example 1.

[0126] Determined by HPLC analysis, the purity of the obtained syringic acid is 97.1%.

[0127] Example 7

[0128] A preparation method of syringic acid with fibrinolytic activity, including the steps:

[0129] After the aerial stems and leaves of Leonurus japonicus are crushed into coarse powder, add 10 times the weight of the coarse powder of 1 wt% NaOH aqueous solution and soak at room temperature for 12 hours, then perform ultrasonic extraction at room temperature for 30 minutes and filter; the medicinal residues are extracted for the second and third times, each time adding 10 times the weight of the coarse powder of 1 wt% NaOH aqueous solution, and each time performing ultrasonic extraction at room temperature for 30 minutes and filtering. The filtrates are combined to obtain the alkaline aqueous extract of Leonurus japonicus.

[0130] The alkaline aqueous extract of Leonurus japonicus is directly loaded onto the upper end of the strong alkaline anion exchange resin column bed, and it is allowed to flow through the column bed of the anion exchange resin at a flow rate of 0.2 BV / h, eluted with distilled water for 4 BV, and then eluted with 1N hydrochloric acid aqueous solution for 8 BV, and the flow rate of the eluent is 1.0 BV / h. The acid water eluate is combined, adjusted to pH 5 with 5N NaOH aqueous solution, concentrated under reduced pressure to 1.25 times the mass of the alkaline aqueous extract of Leonurus japonicus to obtain the 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. The ethyl acetate extraction solutions are combined and concentrated to obtain the crude extract of Leonurus japonicus phenolic acid.

[0131] Successively adopt silica gel column chromatography for distribution and HPLC method to separate and prepare syringic acid from the crude extract of Leonurus japonicus phenolic acid, and the method is the same as that in Example 1.

[0132] Determined by HPLC analysis, the purity of the prepared syringic acid is 97.9%.

[0133] Example 8: Preparation of ordinary oral preparations (capsules, granules, tablets)

[0134] 15 g of syringic acid obtained in Example 1 was added with 15 g of starch, 5 g of dextrin, and 3 g of microcrystalline cellulose. All the above solid substances were ground through an 80-mesh sieve respectively, then 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 syringic acid granules.

[0135] 1.5 g of talc was added to the above granules and mixed evenly, then filled into ordinary capsule shells to obtain the syringic acid capsules. 1.5 g of talc was added to the above syringic acid granules, mixed evenly, and tabletted to obtain the syringic acid tablets.

[0136] Example 9: Preparation of enteric-coated oral preparations (enteric-coated capsules, enteric-coated tablets)

[0137] 20 g of syringic acid obtained in Example 2 was taken and added with 20 g of pregelatinized starch, 5 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 syringic acid capsules.

[0138] The above-prepared granules were tabletted to obtain the tablet cores, then an isolation layer was coated with hydroxypropylcellulose 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 syringic acid tablets.

[0139] Example 10: Preparation of pellet oral preparations (pellets, pellet capsules, enteric-coated pellet tablets)

[0140] 20 g of syringic acid obtained in Example 1 was taken and added to an aqueous solution of hydroxypropylmethylcellulose with a mass fraction of 3%, then 2.4 g of polysorbate 80 was added and stirred evenly; 0.8 g of talc was added and mixed evenly. Preparation of pellet cores: Coating was carried out by the bottom spraying process in a fluidized bed. The fluidized bed was preheated, 20 g of starch blank pellets were added, first sprayed with an aqueous solution of hydroxypropylmethylcellulose with a mass fraction of 3% to moisten the fluidized bed, and then sprayed with the 3% hydroxypropylmethylcellulose aqueous solution containing the drug, and the drug was sprayed onto the bottom to obtain the pellet cores. Then an isolation layer was coated: First, an aqueous solution of hydroxypropylcellulose with a mass fraction of 5% was prepared, 0.7 g of magnesium stearate and 8.3 g of talc were weighed respectively, poured into the hydroxypropylmethylcellulose E50 solution, stirred evenly and sieved for standby; in the fluidized bed, the above-prepared aqueous solution containing hydroxypropylcellulose, magnesium stearate and talc was sprayed onto the bottom to complete the isolation layer coating and obtain the syringic acid pellets. The pellets were filled into capsules to obtain the syringic acid pellet capsules; an appropriate amount of 10% starch paste was added as a binder, 1.2 g of talc was added, and tabletted to obtain the syringic acid pellet tablets.

[0141] Example 11: Preparation of enteric-coated pellets oral preparations (enteric-coated pellets, enteric-coated pellet capsules, enteric-coated pellet tablets)

[0142] The pellets prepared in Example 10 above were further coated with an enteric layer. 2.5 g of glyceryl monostearate was dissolved in pure water, and 1.6 g of Tween-80 and 3.5 g of triethyl citrate were added successively, stirred and dissolved to obtain a suspension, and then it was slowly poured into 24 g of enteric Opadry, filtered to obtain an enteric layer coating solution. Then the pellets prepared above were placed in a fluidized bed and coated by bottom spraying to obtain syringic acid enteric-coated pellets containing an enteric coating. They were filled into ordinary capsule shells to obtain syringic acid enteric-coated pellet capsules. The above pellets were added with an appropriate amount of 10% starch paste as a binder, 1.2 g of magnesium stearate was added, and pressed into tablets to obtain syringic acid enteric-coated pellet tablets.

[0143] Example 12: Preparation of sustained-release enteric-coated pellets oral preparations (sustained-release enteric-coated pellets, sustained-release enteric-coated pellet capsules, sustained-release enteric-coated pellet tablets)

[0144] The enteric-coated pellets prepared in Example 11 above were further coated with a sustained-release layer. 20 g of talc powder and 20 g of Eudragit NE30D solids content (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 an enteric layer prepared above were placed in a fluidized bed and coated by bottom spraying to obtain syringic acid sustained-release enteric-coated pellets. They were filled into ordinary capsule shells to obtain syringic acid sustained-release enteric-coated pellet capsules. The above pellets were added with an appropriate amount of 10% starch paste as a binder, 1.2 g of magnesium stearate was added, and pressed into tablets to obtain syringic acid enteric-coated and sustained-release pellet tablets.

[0145] Example 13: Preparation of soft capsules of syringic acid

[0146] 20 g of syringic acid 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 soft capsule content; by weight, gelatin: glycerol: water: sorbic acid was proportioned according to a 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 holding tank at 70 °C to prepare the rubber sheet of the soft capsule, and then the prepared soft capsule content was injected, and rolled and pressed into soft capsules, dried, washed with pills, and dried again to obtain the soft capsule preparation of syringic acid.

[0147] The soft capsules are prepared according to the above method, and then Nutrilis, a commercial product of cellulose derivatives recognized as GRAS (Generally Recognized as Safe), or a commercial product based on shellac through special processes and chemical modifications is used as the enteric coating material to coat the enteric coating, and the syringic acid enteric soft capsule preparation is obtained.

[0148] Example 14: Freeze-dried preparation of syringic acid for injection

[0149] Take 20 g of syringic acid prepared in Example 1, add 15 g of sodium ascorbate and 12 g of mannitol, dissolve in 250 ml of distilled water, make up the volume to 300 ml, filter and sterilize, and dispense into 100 vials under aseptic conditions, then freeze-dry and seal under nitrogen to obtain the product.

[0150] Example 15: Freeze-dried preparation of syringate for injection

[0151] Take 10 g of syringic acid prepared in Example 2, dissolve it in 250 ml of distilled water, adjust the pH value to 7.0 with 10% Na2CO3 aqueous 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, and dispense into 100 vials under aseptic conditions, then freeze-dry and seal under nitrogen to obtain the product.

[0152] Example 16: Freeze-dried powder for injection of syringic acid

[0153] Take 10 g of syringic acid prepared in Example 4, dissolve it in 250 ml of distilled water, adjust the pH value to 7.0 with 10% NaOH aqueous 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, and dispense into 100 vials under aseptic conditions, then freeze-dry and seal under nitrogen to obtain the product.

[0154] Example 17: Freeze-dried powder for injection of syringate

[0155] Take 20 g of syringic acid prepared 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.

[0156] Example 18: Preparation of syringic acid ointment

[0157] Take 10 g of syringic acid prepared by the method of Example 1, and add it to an 85°C mixed aqueous phase containing 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 to obtain the aqueous 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 to obtain the oil phase. Then slowly add the aqueous phase to the oil phase, while rapidly stirring in the same direction to fully mix and emulsify, and cool to obtain the product.

[0158] Example 19: Preparation of Syringic Acid Emulsion

[0159] Take 10 g of syringic acid prepared by the method of Example 1, moisten it with 15 ml of propylene glycol, and grind 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 base. When the temperature reaches about 60°C, add the mixture of syringic acid and propylene glycol, fully mix, and condense to obtain the product.

[0160] Test Example 1. Tracking of Fibrinolytic Active Components in Leonurus japonicus Houtt. 1 (Water Extraction - Alcohol Precipitation - Macroporous Adsorption Resin - Ion Exchange Resin - Silica Gel Partition Column Chromatography Separation / Fibrinolytic Activity Evaluation)

[0161] Tracking of fibrinolytic active components: During the extraction and preliminary separation of the chemical components in Leonurus japonicus Houtt., the fibrinolytic activity of each obtained chemical fraction was continuously evaluated by the fibrin plate assay method, and the chemical fractions with better activity were further separated and their fibrinolytic activities were evaluated until the fibrinolytic active components were clarified. The specific experimental steps are as follows.

[0162] 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 liquids, filter, and concentrate the filtrate to a small volume. Precipitate the concentrated liquid twice with 95% ethanol, stir continuously when adding ethanol, control the alcohol content in the solution after adding ethanol, so that the alcohol content reaches 70% for the first time and 83% for the second time. After the second alcohol precipitation, concentrate the filtrate until it has no alcohol smell to obtain the Leonurus japonicus Houtt. extract.

[0163] Macroporous adsorption resin: The above-mentioned Leonurus japonicus extract was diluted with an appropriate amount of distilled water, filtered, and the filtrate was directly loaded onto a macroporous adsorption resin column. First, it was eluted with distilled water for 5 retention volumes, and then eluted with 50wt% ethanol aqueous solution and 95wt% ethanol aqueous solution for 5 retention volumes respectively. Each eluate was concentrated to obtain the water-eluted fraction, 50% ethanol-eluted fraction, and 95% ethanol-eluted fraction.

[0164] Ion exchange resin column chromatography separation and purification: The water-eluted fraction of the above-mentioned macroporous adsorption resin column was dissolved with an appropriate amount of distilled water and loaded onto an ion exchange resin column, and eluted with distilled water and 3N HCl aqueous solution respectively. The water eluate was concentrated to obtain the water-soluble non-alkaloid fraction; the HCl aqueous solution eluted part 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, ultrasonicated for 1 h, filtered, and this process was repeated twice. The two filtrates were combined. The solvent was evaporated to dryness, dissolved with methanol, filtered, and this process was repeated five times until no more NaCl solid was precipitated to obtain the alkaloid fraction.

[0165] On the basis of not knowing the structure of the fibrinolytic active ingredient 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, the following mainly used different component separation methods, combined with the fibrin plate assay, to trace its fibrinolytic active ingredient.

[0166] In the early stage of the fibrinolytic activity tracking process 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 tracking, so it will not be elaborated here.

[0167] 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. The sample was loaded, and eluted successively with organic phases of different ratios of dichloromethane: methanol: water (25:3:1, 10:3:1, 10:5:1.3, 10:7:2.5, lower phase) as eluents. The fractions eluted with each solvent gradient were divided into several components to obtain the eluted fractions Fr.A1.1 - Fr.A4.6, as shown in Table 1.

[0168] Table 1 Silica Gel Distribution Column Chromatography Eluent and Components

[0169]

[0170] 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 physiological saline. Using the internationally and domestically common fibrin plate method, with physiological 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.

[0171] 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 the macroporous adsorption resin column and cation exchange resin column was determined by the above fibrin plate method.

[0172] 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 a 50 mg / mL methanol aqueous solution; weigh an appropriate amount 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 a 50 mg / mL aqueous solution. 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 alkaloid obtained from Leonurus japonicus and the 50% ethanol part of macroporous resin have no fibrinolytic effect.

[0173] Table 2 Effects of Extractive and Isolated Parts of Leonurus japonicus on Fibrinolysis by External Administration

[0174]

[0175]

[0176] Quantitative-effect relationship of fibrinolytic activity of water-soluble non-alkaloids: Using the above fibrin plate method, aqueous solutions of water-soluble non-alkaloid fractions at different concentrations were measured. The results showed that the diameters of the fibrinolytic zones of water-soluble non-alkaloid fractions at different concentrations in the fibrin plate were different, with good dose-dependence and a good quantitative-effect relationship, indicating that this water-soluble non-alkaloid fraction has good protein fibrinolytic activity, which was named active fraction 1 (ACT1).

[0177] Fibrinolytic activity of the sample purified by silica gel partition column chromatography: Using the above fibrin plate method, the fibrinolytic activity of the fractions obtained by further separating active fraction 1 (ACT1) using 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, samples numbered 1 to 5 were the fractions eluted with dichloromethane: methanol: water (25:3:1; lower phase) as the mobile phase. The experimental results showed that silica gel partition column chromatography could further separate and purify active fraction 1 (ACT1).

[0178] Table 3 Effects of in vitro administration of each eluted fraction Fr.A1.1~Fr.A2.1 on fibrinolysis

[0179]

[0180] HPLC analysis of active fraction 1 (ACT1):

[0181] 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.

[0182] Using the above HPLC conditions to analyze the components of the obtained active fraction 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 with a retention time of about 3 min and the chromatographic peak with a retention time of about 20 min. From this chromatogram, it can be seen that the composition of active fraction 1 (ACT1) is relatively complex, consisting of the above two parts of components, and further separation and purification are required. The results show that the eluent ratio of the silica gel column chromatography in this part is not the best.

[0183] Experimental Example 2, Tracking of the Fibrinolytic Active Ingredients in Leonurus japonicus Houtt. 2 (Water Extraction - Extraction - Partition Column Chromatography Separation / Fibrinolytic Activity Evaluation)

[0184] Extraction and Extraction: The aerial stems and leaves of Leonurus japonicus Houtt. were crushed into coarse powder. Then, 200 g of the powder was extracted by decoction with water twice. For the first time, 2.6 L of water was added, and it was refluxed at 100 °C for 2 hours using 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 water 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 water solution after extraction (YMC - H2O - 1, 14.65 g).

[0185] 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 filled 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 successively 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).

[0186] Table 4 Eluents and Each Elution Component of Silica Gel Partition Column Chromatography

[0187]

[0188] 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, dissolved in methanol, and prepared into 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 fibrinolytic activities of each extraction part YMC-PE-1, YMC-DCM-1, YMC-EA-1, and YMC-H2O-1 obtained in the active ingredient tracking 2 of this test example were determined by the fibrin plate method in Test Example 1. The measurement results are shown in Table 5. Among them, YMC-DCM-1 and YMC-EA-1 have fibrinolytic activity, the former has weak activity, and the latter has strong activity; while YMC-PE-1 and YMC-H2O-1 have no activity. 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 weak.

[0189] Table 5 Effects of each extraction part obtained in the active ingredient tracking 2 on fibrinolytic

[0190]

[0191] 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, dissolved in methanol, and prepared into a methanol solution with a concentration of 20 mg / mL. The fibrinolytic activity was determined by the fibrin plate method in Test Example 1, 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 activity. Among them, the sample Fr.B1004 numbered 4 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, named active part 2 (ACT2).

[0192] Table 6 Fibrinolytic effects of samples after separation and purification by silica gel partition column chromatography

[0193]

[0194] HPLC analysis of active part 2 (ACT2): Weigh 1 mg of the sample Fr.B1004 (ACT2) separated by silica gel partition column chromatography in Test Example 2, dissolve it in 1 mL of chromatographically pure methanol, and prepare a 1 mg / mL methanol solution. Using the HPLC analysis conditions in Test Example 1, the ACT2 in Test Example 2 was analyzed, and its HPLC fingerprint is shown inFigure 2 There are 5 main chromatographic peaks between the retention times of 18 - 28 min in its fingerprint spectrum 2, indicating that its main components are concentrated within this retention time range.

[0195] Separation and Structure Identification of the Components in the Fibrinolytic Active Site (ACT2) of Leonurus japonicus Houtt.

[0196] Using the extraction and extraction method of the active site ACT2 in Test Example 2, the ethyl acetate part after extraction was obtained. Using the silica gel partition column chromatography method in Test Example 2, the ethyl acetate extraction part was separated (the volume ratios of the eluents dichloromethane:methanol:water were 100:0:5, 100:1:5, 100:2:5, 100:3:5, 100:4:5, 100:10:8 respectively). Its elution fractions were identified and combined by thin layer chromatography, and then its fibrinolytic activity was measured to obtain its fibrinolytic active site components Fr.3 - 1 to Fr.3 - 4. Using a method combining TLC and HPLC, the separation and preparation of single compounds were carried out respectively.

[0197] Compound 1 (50.02 mg) was obtained by repeatedly separating and purifying the above Fr.3 - 2 part 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.

[0198] Compound 2 (36.87 mg) was first prepared by TLC from the ethyl acetate extraction part of the Leonurus japonicus Houtt. extract. The developing agent was dichloromethane:methanol (13:1), and the R f was about 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.

[0199] 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. The mobile phase for the second preparation 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.

[0200] Compound 5 (47.8 mg) was obtained by HPLC preparation of Fr.3 - 1. The mobile phase was Phase A: water, Phase B: methanol, 0 - 15 min, 40% A → 25% A, 15 - 25 min, 25% A → 0% A, and the retention time was 15.6 min.

[0201] Spectral data for structural identification:

[0202] Spectral data of compound 1: 1 H-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 C-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: 167.0.13410[M-H] － 。 Its structure was determined to be vanillic acid.

[0203] Spectral data of compound 2: 1 H-NMR(400 MHz, CD3OD) δ 7.33(2H, s, H-2, H-6), 3.88(6H, s, H-8, H-9). 13 C-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.

[0204] Spectral data of compound 3: 1 H-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). 13 C-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.

[0205] Spectral data of compound 4: 1H-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.

[0206] 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.

[0207] Among them, Compound 4 (cis-ferulic acid) has poor stability. After standing, part of it is converted into Compound 3 (trans-ferulic acid).

[0208] Experimental Example 4 HPLC Content Determination of Leonurine Acid Extract and Prepared Syringic Acid

[0209] 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: 0 - 10 min, 10% methanol solution; 10 - 15 min, 10% → 20% A; 15 - 25 min, 20% → 40% A; 25 - 40 min, 40% → 80% A. Column temperature: 30 °C, flow rate: 0.3 mL / min, injection volume: 10 μL.

[0210] Preparation of the extract solution of phenolic acids from Leonurus japonicus Houtt.: Weigh accurately 10 mg of the extract of phenolic acids from Leonurus japonicus Houtt. (active fraction ACT2) prepared in Test Example 2, place it in a 10 mL volumetric flask, dissolve it with methanol and dilute it to the mark, shake well, filter, and set aside for use.

[0211] Preparation of the solution of syringic acid for the sample to be measured: Weigh accurately 10 mg of syringic acid prepared in Example 1, place it in a 10 mL volumetric flask, dissolve it with chromatographic pure methanol and dilute it to the mark, shake well, filter, and set aside for use.

[0212] Preparation of the reference solution: Weigh accurately 10.00 mg each of the reference substances vanillic acid, syringic acid, trans-ferulic acid, and 3,4,5-trimethylgallic acid, dissolve them separately in 5 mL volumetric flasks with chromatographic methanol, make up the volume to obtain a reference stock solution with a concentration 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.

[0213] Establishment of the standard curve: Using the above HPLC determination conditions, inject the above-prepared reference and sample solutions to be measured respectively for determination, count the peak areas of the samples at each concentration of each compound, and establish the HPLC standard curves 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.

[0214] Determination results of the contents of each component in the extract ACT2 of phenolic acids from Leonurus japonicus Houtt. and the purity of the prepared syringic acid: According to the above-established standard curve ( Figure 3 ) and the HPLC chromatogram of the reference substance ( Figure 4 B), calculate the HPLC chromatogram of the extract of phenolic acids from Leonurus japonicus Houtt. ( Figure 4A) The percentage contents of the compounds of chromatographic peaks F1 (vanillic acid), F2 (syringic acid), F3 (trans-ferulic acid), and F5 (3,4,5-trimethylgallic acid) are shown in Table 7. The contents of the four main compounds in ACT2 are 10.4%, 54.0%, 23.4%, and 7.3% respectively. The total content of these four compounds is 95.1% (see Table 7), and the content of syringic acid is the highest (54.0%). Similarly, according to Figure 3 the syringic acid standard curve in Figure 5 , the content of syringic acid in the syringic acid chromatogram prepared in Example 1 (see

[0215] ) was calculated, and its purity was found to be 97.2%.

[0216]

[0217] Test Example 5 Dose-effect relationship of the fibrinolytic circle diameter of urokinase

[0218] 2.8 mg of urokinase at 50 U / mg was dissolved in 1 mL of distilled water and then diluted with distilled water to obtain urokinase aqueous solutions at 20, 40, 60, 80, 100, 120, and 140 U / mL. The fibrinolytic circles of urokinase at different dose concentrations were measured on fibrin plates, and the results are shown in Table 8. The fibrinolytic circle diameters of urokinase at different dose concentrations were different, and as the urokinase dose increased, the diameter of its fibrinolytic circle also increased, showing a good dose-effect relationship. The fibrinolytic circle diameters at each concentration were statistically analyzed to establish a dose-effect relationship standard curve, as shown in Figure 6 . The regression equation was calculated as: y = 0.083x + 13.857, R 2 = 0.9714. The experimental results show that it is feasible to use the fibrin plate assay method to evaluate the fibrinolytic effect of compounds. The diameter (φ) of the fibrinolytic circle is proportional to the strength of fibrinolytic activity, indicating that the size of the fibrinolytic circle 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.

[0219] Table 8 Fibrinolytic activities of urokinase at different doses

[0220]

[0221]

[0222] Test Example 6 Determination of the fibrinolytic activities of individual phenolic acid compounds in the phenolic acid extract of Leonurus japonicus Houtt.

[0223] Using the commonly used thrombolytic drug urokinase in current clinical practice as a positive control, a solution of 40 U / ml was prepared with normal saline. The internationally and domestically common fibrin plate method was adopted. Using normal saline as a solvent, fibrinogen solutions of 10 mg / mL, thrombin solutions of 1 U / mL, and agarose solutions of 10 mg / mL were prepared. 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. Use a 7 mm punch to punch holes, 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.

[0224] 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, and the incubation time at 37 °C is 2 h. Measure the diameter φ (mm) of each fibrinolytic circle in the fibrin plate to evaluate the strength of their fibrinolytic activity.

[0225] The fibrinolytic experiment results of each single compound and mixture are shown in Table 9. The five single compounds vanillic acid, syringic acid, trans-ferulic acid, 3,4,5-trimethylgallic acid, and the mixture of cis-ferulic acid and trans-ferulic acid all have strong protein fibrinolytic activity, among which syringic acid and vanillic acid have the strongest fibrinolytic activity.

[0226] Table 9 Protein fibrinolytic activity of each monomeric phenolic acid compound in the fibrinolytic active site

[0227]

[0228] Test Example 7 Evaluation of the in vitro antithrombotic formation activity of syringic acid

[0229] The internationally and domestically common Chandler loop test method was adopted in this experiment, 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 (10 μmol / L, 25 μmol / L, 50 μmol / L), and a syringic acid (prepared by the method of Example Ⅰ) group (10 μmol / L, 25 μmol / L, 50 μmol / L).

[0230] Male Wistar rats, weighing approximately 180 - 300 g, were provided by Jinan Pengyue Laboratory Animal Breeding Co., Ltd. Anesthetize by intraperitoneal injection of 0.2 mL of 1.5% pentobarbital solution per 100 g of body weight, fix in the supine position, open the abdominal cavity, and collect blood from the abdominal aorta using a common negative pressure blood collection tube without anticoagulant. Add 10 μL of the sample to be tested into the plastic hose for thrombus formation in advance. After blood collection, quickly inject 1 mL of blood sample along the inner wall of one end of the plastic hose, avoiding generating air bubbles during injection. Then connect the plastic tubes into a ring and install them into the turntable (preheated to 37 °C in advance) of the automatic thrombus formation instrument. Try to control the process from blood collection to installation within 1 min. Let the turntable rotate at a speed of (20 ± 2) rpm for 10 min, then take out the plastic ring, remove the thrombus, measure and record the wet weight of the thrombus. Then place it in an oven at 60 °C for 30 min and take it out, and record the dry weight of the thrombus.

[0231] Experimental results: The results of the in vitro anti-thrombosis experiment of the syringic acid group, each dose group of the positive drug aspirin, and the blank control group are shown in Table 10. Compared with the blank group, in the high, medium, and low dose syringic acid administration groups and the positive drug aspirin administration groups, there were significant differences in the average wet weight and average dry weight of the thrombus, indicating that syringic acid obtained from Leonurus japonicus has a significant anti-thrombosis effect. Among them, the high dose syringic acid group showed a better anti-thrombosis effect than the high dose aspirin group, indicating its potential application prospect in anti-thrombosis.

[0232] Table 10 Evaluation of the in vitro anti-thrombosis activity of syringic acid

[0233]

[0234] Note: Compared with the blank group, *P < 0.05, **P < 0.01, ***P < 0.001

[0235] Test Example 8: Determination of the in vitro blood-brain barrier permeability rate of syringic acid and other monomeric phenolic acid compounds

[0236] The pig polar brain extract was prepared into a 20 mg / mL solution with dodecane as the solvent for standby. Four commercially available drugs (estradiol, progesterone, alprazolam, oxazepam) and commercially available phenolic acid compounds (vanillic acid, syringic acid, trans-ferulic acid, 3,4,5-trimethyl gallic acid) were preliminarily dissolved with a small amount of DMSO solution respectively, and then diluted into a 25 μg / mL solution with a buffer of PBS:EtOH = 7:3 for standby. 4 μL of the pig polar brain extract was pipetted and dropped onto the PVDF membrane of the receptor microplate to simulate the human biological membrane. Then, 300 μL of the test compound solution was added to the donor well, and 200 μL of the PBS:EtOH = 7:3 buffer was added to the receptor well, so that the compound solution just contacted the biological membrane. The receptor well was placed on the donor well to form a sandwich-like structure of donor well / biological membrane / receptor well. The drug to be tested diffused from the donor well through the artificial simulated biological membrane into the receptor well in the form of passive diffusion. The whole microplate was left standing at room temperature for 16 h, then the receptor microplate and the donor microplate were separated, and the liquids in their donor wells and receptor wells were taken out respectively. The OD values were measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Five replicate experiments were set for each compound to be tested.

[0237] Calculate the effective permeability Pe of the compound according to the following formula:

[0238] Pe (cm·s -1 ): Pe = -VdVa / [(Vd + Va)At]ln(1 – OD drugacceptor / OD drugreceiver )

[0239] where 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.

[0240] Experimental results: Through the above experimental method, we first obtained the experimental permeation values of 4 commercially available drugs. By consulting relevant materials, the theoretical permeation values of these 4 drugs were determined. All the results are shown in Table 11. A standard curve was made based on the theoretical permeation values and the experimental permeation values for subsequent correction ( Figure 7 ), and a good linear correlation equation was obtained: y = 1.9489x + 2.559, R 2 = 0.9616.

[0241] According to this equation and considering the limit 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 that the compound has poor blood-brain barrier permeability. According to the experimental results (see Table 12), except for trans-ferulic acid (Pe value is 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. This indicates that vanillic acid, syringic acid, and 3,4,5-trimethyl gallic acid all have a certain ability to penetrate the blood-brain barrier. Among them, syringic acid has the largest permeability, which is similar to the penetration ability of the control drug oxazepam, showing good blood-brain barrier penetration ability and good application value.

[0242] Table 11 Measured values and literature penetration values of blood-brain barrier permeability of four drugs

[0243]

[0244] Table 12 Results of blood-brain barrier permeability of monomeric phenolic acid compounds

[0245]

[0246]

Claims

1. A syringic acid, characterized in that, The purity of the syringic acid is greater than or equal to 95%, and it has the structure as shown below:

2. The preparation method of the syringic acid according to claim 1, comprising the steps of: using the aerial stems and leaves of Leonurus japonicus as raw materials, and obtaining syringic acid through extraction, enrichment, separation and purification; The extraction method is water extraction method, 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; the purification method is high performance liquid chromatography (HPLC) method.

3. The preparation method of syringic acid according to claim 2, characterized in that, The water extraction method or alcohol-water extraction method comprises the steps of: pulverizing the aerial stems and leaves of Leonurus japonicus into coarse powder, then immersing it in water or alcohol-water solvent, and obtaining the Leonurus japonicus extract through extraction and filtration; Alternatively, pulverize the aerial stems and leaves of Leonurus japonicus into coarse powder, soak it in NaOH aqueous solution, adjust the pH value to neutral with 1-5wt% hydrochloric acid aqueous solution, and filter; then immerse it in water or alcohol-water solvent, and obtain the Leonurus japonicus extract through extraction and filtration.

4. The preparation method of syringic acid according to claim 3, characterized in that, Comprising one or more of the following conditions: i. The alcohol-water solvent is 50wt% - 100wt% methanol solution or 50wt% - 95wt% ethanol aqueous solution; the weight ratio of water or alcohol-water solvent to the coarse powder is 5:1 - 20:1; ii. The concentration of the NaOH aqueous solution is 0.5 - 2wt%, the soaking temperature is room temperature, and the soaking time is 2 - 8h; iii. The extraction method is heating reflux extraction method, ultrasonic extraction method, percolation method or maceration method; more preferably, the extraction method is heating reflux extraction method or ultrasonic extraction method; the heating reflux extraction method has an extraction time of 1 - 2h each time, the ultrasonic temperature of the ultrasonic extraction method is 40 - 60°C, and the ultrasonic extraction time each time is 20 - 40min; the number of extractions is 1 - 5 times; iv. The Leonurus japonicus extract can be further concentrated into Leonurus japonicus extract paste under normal pressure or reduced pressure conditions.

5. The preparation method of syringic acid according to claim 2, wherein, The alkaline water extraction method comprises the steps of: pulverizing the aerial stems and leaves of Leonurus japonicus into coarse powder, impregnating and extracting it in NaOH aqueous solution, and filtering to obtain the Leonurus japonicus alkaline water extract; Preferably, it comprises one or more of the following conditions: i. The concentration of the NaOH aqueous solution is 0.5wt% - 1.5wt%; the mass ratio of the NaOH aqueous solution to the coarse powder is (5 - 12):1; ii. The number of impregnation extractions is 1 - 5 times; the impregnation extraction method each time is: standing at room temperature for 2 - 12h, or, ultrasonic at room temperature for 15 - 30 minutes, or, standing at room temperature for 2 - 12h and then ultrasonic at room temperature for 15 - 30 minutes; iii. After the filtration step, the pH of the alkaline water extract needs to be adjusted to 5 - 7 with hydrochloric acid aqueous solution.

6. The preparation method of syringic acid according to claim 2, characterized in that, Comprising one or more of the following conditions: i. The extraction method includes the steps of: diluting the extract obtained by extraction 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 subjecting the dichloromethane extract and the ethyl acetate extract to vacuum concentration respectively to obtain dichloromethane extract (A) and ethyl acetate extract (B); suspending the dichloromethane extract (A) in water to obtain a suspension, extracting with ethyl acetate as the extraction agent, and subjecting the ethyl acetate extract to vacuum concentration to obtain ethyl acetate extract (C); combining the ethyl acetate extract (B) and the ethyl acetate extract (C) to obtain the leonuritic 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 carried out 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 the extraction agent, the extraction is carried out 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 and dissolving method includes the steps of: adding an NaOH aqueous solution to the extract obtained by extraction, kneading and dissolving, and filtering; adding the filter residue to the NaOH aqueous solution, kneading and dissolving, and filtering, and repeating this step 2 - 5 times for the obtained filter residue, combining the filtrates to obtain the leonurine aqueous solution; adjusting the pH value of the leonurine aqueous solution to neutral with a hydrochloric acid aqueous solution to obtain a feed solution, and subjecting the feed solution to vacuum concentration to obtain the leonuritic acid enriched product, or directly treating it by the strong basic anion exchange resin method to obtain the leonuritic acid enriched product; Preferably, the concentration of the NaOH aqueous solution is 1wt% - 5wt%; the mass of the NaOH aqueous solution is 2 - 5 times the mass of the extract; the pH value of the obtained leonurine aqueous solution is adjusted to neutral with a hydrochloric acid aqueous solution with a concentration of 4 - 8N; iii. The macroporous adsorption resin column chromatography method includes the steps of: adjusting the pH value of the extract obtained by extraction to 2 - 7, loading it onto the macroporous adsorption resin column bed, and then sequentially using water, 50wt% ethanol aqueous solution, and 95wt% ethanol aqueous solution as eluents for elution, combining the eluates of the 50wt% ethanol aqueous solution and the 95wt% ethanol aqueous solution, and concentrating until the alcohol smell disappears to obtain the leonuritic acid enriched product; Preferably, the pH value is adjusted to 2 - 7 with a 4 - 6N hydrochloric acid aqueous solution; the flow rate of the liquid flowing out from the bottom end of the macroporous adsorption resin column bed during loading is 0.1 - 0.3BV / h; the flow rate of the liquid flowing out from the bottom end of the column during elution with the eluent is 0.4 - 0.6BV / h; the eluent water is eluted for 2 - 6 retention volumes (BV), and the eluents of 50wt% ethanol aqueous solution and 95wt% ethanol aqueous solution are respectively eluted for 6 - 20 retention volumes (BV); iv. The strong basic anion exchange resin method includes the steps of: directly injecting the leonurine aqueous extract obtained by the alkaline water extraction method or the leonurine aqueous solution obtained by the alkaline water kneading and dissolving method into a strong basic anion exchange resin column, then sequentially eluting with water and an acidic aqueous solution as the eluents, adjusting the pH of the eluate obtained with the acidic aqueous solution as the eluent to 5 - 7, and concentrating under reduced pressure to obtain a concentrated solution; Extracting with ethyl acetate and concentrating to obtain a leonurine phenolic acid enriched product; Preferably, when injecting the leonurine aqueous extract or the leonurine aqueous solution, the flow rate of the effluent at the bottom of the column is 0.1 - 0.3 BV / h; when eluting with the eluents, the flow rate of the effluent at the bottom of the column is 0.3 - 1 BV / h; the eluent water elutes 2 - 5 BV, and the eluent acidic aqueous solution elutes 4 - 10 BV; the concentration of the eluent acidic aqueous solution is 0.5 - 2 N, and the acid is hydrochloric acid, sulfuric acid or phosphoric acid; adjusting the pH to 5 - 7 with a 4 - 6 N NaOH aqueous solution; concentrating under reduced pressure to 1 - 3 times the mass of the leonurine aqueous extract or the leonurine aqueous solution; the number of extraction times is 2 - 6 times, and the volume ratio of ethyl acetate to the concentrated solution used for each extraction is 1:

1.

7. The preparation method of syringic acid according to claim 2, characterized in that, Including one or more of the following conditions: i. The silica gel partition column chromatography method includes the steps of: dissolving the obtained leonurine phenolic 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; then 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 sequentially 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 leonurine phenolic acid mixture; preferably, the mass ratio of the leonurine phenolic acid enriched product to methanol is 1:2 - 3, and the mass ratio of silica gel to the leonurine phenolic acid enriched product is 2 - 4:1; ii. The ODS silica gel column chromatography method includes the steps of: dissolving the obtained leonurine phenolic acid enriched product in methanol, loading onto the top of the ODS silica gel column bed by wet method, first eluting with a 10 wt% methanol aqueous solution for 2 - 4 BV, and then sequentially eluting with a 50 wt% methanol aqueous solution, a 70 wt% methanol aqueous solution, and pure methanol for 2 - 10 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 leonurine phenolic acid mixture; preferably, the mass ratio of the leonurine phenolic acid enriched product to methanol is 1:1 - 3.

8. The preparation method of syringic acid according to claim 2, characterized in that, The purification method comprises the steps of: using high performance liquid chromatography (HPLC) method, with two preparations: for the first preparation, dissolve the separated phenolic acid mixture of Leonurus japonicus in methanol, inject it into a preparative or semi-preparative C18 high performance liquid chromatography column of HPLC, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid water (v / v), gradient elution, and the elution program is: 0 - 10 min, the volume ratio of mobile phase A is 10%, and the volume ratio of mobile phase B is 90%; 10 - 15 min, the volume ratio of mobile phase A is 10% → 20%, and the volume ratio of mobile phase B is 90% → 80%; 15 - 30 min, the volume ratio of mobile phase A is 20% → 50%, and the volume ratio of mobile phase B is 80% → 50%; collect the eluate of the chromatographic peak with a retention time of 21.5 min, concentrate it to obtain the crude syringic acid; for the second preparation, dissolve the crude syringic acid in methanol, inject it into a preparative or semi-preparative C18 high performance liquid chromatography column of HPLC, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid water (v / v), gradient elution, and the elution program is: 0 - 5 min, the volume ratio of mobile phase A is 0% → 30%, and the volume ratio of mobile phase B is 100% → 70%; 5 - 20 min, the volume ratio of mobile phase A is 30%, and the volume ratio of mobile phase B is 70%; collect the eluate of the chromatographic peak with a retention time of 12.8 min, concentrate it to obtain the pure syringic acid.

9. The application of syringic acid according to claim 1, characterized in that, For preparing a drug having a thrombolytic or antithrombotic effect; Preferably, the thrombus is a cerebral thrombus.

10. A pharmaceutical composition with thrombolytic effect, characterized in that, Comprising the syringic acid or a pharmaceutically acceptable salt thereof as claimed in claim 1, one or more pharmaceutically acceptable carriers or excipients, and an antioxidant; Preferably, the pharmaceutical composition is one of tablets, capsules, granules, freeze-dried injection preparations, powder injections, pellets, ointments or creams, and can be prepared according to the conventional pharmaceutical production process.

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