CN116730958A - Salvianolic acid conversion product converted into salvianolic acid A in proportion, preparation method, process parameter determination method and application thereof - Google Patents
Salvianolic acid conversion product converted into salvianolic acid A in proportion, preparation method, process parameter determination method and application thereof Download PDFInfo
- Publication number
- CN116730958A CN116730958A CN202210210071.9A CN202210210071A CN116730958A CN 116730958 A CN116730958 A CN 116730958A CN 202210210071 A CN202210210071 A CN 202210210071A CN 116730958 A CN116730958 A CN 116730958A
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- Prior art keywords
- salvianolic acid
- acid
- conversion
- adsorption resin
- macroporous adsorption
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- YMGFTDKNIWPMGF-AGYDPFETSA-N 3-(3,4-dihydroxyphenyl)-2-[(e)-3-[2-[(e)-2-(3,4-dihydroxyphenyl)ethenyl]-3,4-dihydroxyphenyl]prop-2-enoyl]oxypropanoic acid Chemical compound C=1C=C(O)C(O)=C(\C=C\C=2C=C(O)C(O)=CC=2)C=1/C=C/C(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 YMGFTDKNIWPMGF-AGYDPFETSA-N 0.000 title claims abstract description 143
- 238000000034 method Methods 0.000 title claims abstract description 132
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- 229930183842 salvianolic acid Natural products 0.000 title claims abstract description 99
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- SNKFFCBZYFGCQN-UHFFFAOYSA-N 2-[3-[3-[1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyloxy]-3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)C(C(=O)OC(CC=3C=C(O)C(O)=CC=3)C(O)=O)C=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-UHFFFAOYSA-N 0.000 claims abstract description 185
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/86—Benzo [b] furans; Hydrogenated benzo [b] furans with an oxygen atom directly attached in position 7
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
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- C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
- C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
- C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
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Abstract
The invention provides a salvianolic acid conversion product which is converted into salvianolic acid A in proportion, a preparation method, a process parameter determination method and application thereof. The invention extracts salvianolic acid B from Salvia Miltiorrhiza or other Salvia medicinal materials used as Salvia Miltiorrhiza, makes the salvianolic acid B adsorbed on macroporous adsorption resin, and then converts the salvianolic acid B into salvianolic acid A on macroporous adsorption resin to prepare salvianolic acid conversion products containing the salvianolic acid A and the salvianolic acid B; the method is simple, low in cost, good in product stability, low in loss and suitable for industrial production. According to the invention, the process parameters of the required conversion products can be efficiently and accurately optimized according to the specific proportion requirements of the salvianolic acid A and the salvianolic acid B, the method is simple and convenient, the test times are few, the accuracy of the results is high, and the feasibility is strong. The salvianolic acid conversion product of the invention can be used for preparing antithrombotic and anti-atherosclerosis medicines.
Description
Technical Field
The invention relates to a salvianolic acid conversion product which is converted into salvianolic acid A in proportion, a preparation method, a process parameter determination method and application thereof, and belongs to the technical field of medicines.
Background
The red sage root is a Labiatae plant, and the medicinal parts are dry roots and rhizomes, which are commonly used in cardiovascular diseases such as coronary heart disease in China, and have the effects of promoting blood circulation to remove blood stasis and treating chest stuffiness and pains. The effective components of Saviae Miltiorrhizae radix mainly comprise water-soluble component and fat-soluble component. Wherein the water-soluble component is mainly salvianolic acid B, and the content of salvianolic acid A is very low. Studies show that the salvianolic acid B and the salvianolic acid A have better treatment effects on cardiovascular diseases such as coronary heart disease, angina and the like. An injection salvianolic acid salt with salvianolic acid B as a main active ingredient is clinically used for treating cardiovascular diseases such as coronary heart disease, angina pectoris and the like. The salvianolic acid A also has strong antioxidation effect, can effectively inhibit oxidative stress injury suffered by cell membranes and relieve myocardial ischemia reperfusion injury; has good platelet aggregation resisting effect, can effectively reduce blood viscosity, remarkably increase cAMP content in platelets, inhibit adhesion of platelets on the surface of a collagen coating, and inhibit combination of platelets and fibrinogen; also has pharmacological activity of resisting hepatic fibrosis and preventing diabetic complications.
The salvianolic acid B and the salvianolic acid A have synergistic effect and better curative effect when being combined. The different proportions (4:1, 2:1,1:1,1:2, 1:4) of the salvianolic acid A and the salvianolic acid B can obviously reduce the range of myocardial infarction of rats caused by myocardial ischemia reperfusion injury, and the effects of the salvianolic acid A or the salvianolic acid B used alone are not obvious as those of the two compatibility groups, and the salvianolic acid A is used in each proportion group: the ratio of the salvianolic acid B to the total weight of the traditional Chinese medicine is 2:1, the most obvious effect is achieved (Wangguozhen, etc., the different ratios of the salvianolic acid A/the salvianolic acid B have the protection effect on the myocardial ischemia reperfusion injury of rats, and the Hebei traditional Chinese medicine report 2006,21 (2): 4-5, 12).
However, because the content of salvianolic acid A in the salvia miltiorrhiza or the salvia miltiorrhiza bunge and other plants belonging to the genus Salvia Miltiorrhiza is not high, the industrial production is difficult to meet, and the application of the salvianolic acid A is limited. At present, two main modes for converting salvianolic acid B into salvianolic acid A are available: (1) The method comprises the steps of adjusting a certain pH value of an aqueous solution of salvianolic acid B, converting the aqueous solution into salvianolic acid A under high temperature and high pressure (Wang Ying and the like), carrying out technical research on the high temperature and high pressure conversion synthesis of salvianolic acid A, university of Daian university, 2011,30 (6): 412-415; jiang Feng and the like, and carrying out a salvianolic acid A conversion method in red sage root, wherein the method comprises the steps of (2018,40) (9): 2091-2096); (2) Adding acid or alkali solution into powder of plant belonging to Salvia Miltiorrhiza or Salvia alba, adjusting pH, and converting salvianolic acid B into salvianolic acid A (Sun Longru, ZL 2012 1 0334295.7) under high temperature and high pressure. However, the two methods still have the defects: in the method (1) (the former), the salvianolic acid B and the salvianolic acid A produced by conversion are both in aqueous solution, and the stability of the salvianolic acid B and the salvianolic acid A in the aqueous solution (especially when the temperature is higher) is poor due to the higher temperature of the aqueous solution, so that the yield is influenced; in addition, the optimal concentration of salvianolic acid B in the aqueous solution is 5mg/mL, so that the feeding amount of salvianolic acid A converted each time is limited. Method (2) (the latter): although the conversion of the salvianolic acid B into the salvianolic acid A is performed in the plant tissues of the medicinal materials, the stability is good, and the volume ratio of the medicinal materials is large, so that the quantity of the medicinal materials treated in the actual operation process is limited, and the application of the method in the industrial production process is influenced.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a salvianolic acid conversion product which is converted into salvianolic acid A in proportion, a preparation method, a process parameter determination method and application thereof. The invention extracts salvianolic acid B from Salvia Miltiorrhiza or other Salvia medicinal materials used as Salvia Miltiorrhiza, makes the salvianolic acid B adsorbed on macroporous adsorption resin, and then converts the salvianolic acid B into salvianolic acid A on macroporous adsorption resin to prepare salvianolic acid conversion products containing the salvianolic acid A and the salvianolic acid B; the method is simple, low in cost, good in product stability, low in loss and suitable for industrial production.
According to the invention, the process parameters of the required conversion products can be efficiently and accurately optimized according to the specific proportion requirements of the salvianolic acid A and the salvianolic acid B, the method is simple and convenient, the test times are few, the accuracy of the results is high, and the feasibility is strong.
The salvianolic acid conversion product of the invention can be used for preparing antithrombotic and anti-atherosclerosis medicines.
The technical scheme of the invention is as follows:
a salvianolic acid conversion product which is proportionally converted into salvianolic acid a, wherein the salvianolic acid conversion product comprises: the total content of the six components in the salvianolic acid B, salvianolic acid A, salvianic acid A, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid is not less than 60wt%; wherein, salvianolic acid B, salvianolic acid A and salvianic acid A are used as main components, and account for more than 80wt% of the total of the six components; the content ratio of the salvianolic acid B to the salvianolic acid A can be set to be any ratio according to the requirement; the salvianolic acid conversion product is obtained by taking a sage plant medicinal material as a raw material, extracting salvianolic acid B, adsorbing the salvianolic acid B on macroporous adsorption resin, converting the salvianolic acid B into salvianolic acid A according to the proportion as required, and separating.
The preparation method of the salvianolic acid conversion product which is converted into salvianolic acid A according to the proportion comprises the following steps:
(1) Extraction of salvianolic acid B
Water is used as solvent, and an ultrasonic extraction method is adopted: pulverizing Salvia plant, and sieving with 40 mesh sieve; adding distilled water, ultrasonically extracting for 20-60 minutes at 40-80 ℃, and filtering to obtain filtrate and dregs; repeating the ultrasonic extraction step for 1-5 times; mixing the filtrates to obtain salvianolic acid B extractive solution.
(2) Preparation of macroporous adsorption resin with adsorbed salvianolic acid B
Loading all the salvianolic acid B extract onto a macroporous adsorption resin column at a flow rate of 0.5-1 mL per minute, eluting with distilled water with a retention volume of 1-5 times when the filtrate is completely discharged, and at a flow rate of 0.5-2 mL per minute to obtain macroporous adsorption resin adsorbed with salvianolic acid B; the macroporous adsorbent resin is D101, HPD100, HPD300, HPD450, HPD600 or HPD700.
(3) Conversion of salvianolic acid conversion product
Wetting the macroporous adsorption resin adsorbed with salvianolic acid B with acidic solution with pH=1.0-5.0, placing the wet macroporous adsorption resin into a high-temperature high-pressure steam sterilization pot, and treating the wet macroporous adsorption resin for 1-5 hours at 105-125 ℃ to obtain macroporous adsorption resin adsorbed with salvianolic acid conversion products; the acid is hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, formic acid or acetic acid.
(4) Separation of salvianolic acid conversion products
Loading the macroporous adsorption resin adsorbed with the salvianolic acid conversion product into a column for eluting, or carrying out ultrasonic extraction on the macroporous adsorption resin adsorbed with the salvianolic acid conversion product; then concentrating under reduced pressure or concentrating under reduced pressure and lyophilizing to obtain salvianolic acid conversion product.
According to the preferred embodiment of the present invention, in step (1), the Salvia plant is selected from the group consisting of Salvia Miltiorrhiza, salvia alba, salvia yunnanensis, salvia Miltiorrhiza, salvia Gansu, salvia fusca, salvia Miltiorrhiza, salvia alba, salvia pria ternate, and Salvia xiaoda.
According to the invention, in the step (1), the mass of distilled water is 5-40 times of the mass of the medicinal material powder; preferably, the mass of distilled water is 20-40 times of the mass of the medicinal material powder; more preferably 30 times.
According to a preferred embodiment of the invention, in step (1), the extraction temperature is 60 to 80 ℃, preferably 73 ℃.
According to a preferred embodiment of the present invention, in step (1), the extraction time is 30 to 50 minutes, preferably 41 minutes.
According to a preferred embodiment of the invention, in step (1), the ultrasonic extraction step is repeated 2 to 4 times, preferably 2 times.
According to the invention, in the step (1), the liquid-material ratio, the extraction time and the extraction temperature are carefully examined by a star point design-effect surface method to obtain the optimal liquid-material ratio, the extraction time and the extraction temperature technological parameters of the salvianolic acid B extraction technology.
According to the invention, in the step (2), the pretreatment method of the macroporous adsorption resin column is as follows: soaking macroporous adsorption resin in 90-100deg.C ethanol for 20-30 hr, suspending with 90-100deg.C ethanol, and wet packing; washing the effluent with 90-100wt% ethanol until the effluent is not turbid after adding water with equal amount; flushing the column with distilled water until the effluent liquid has no alcohol smell; 3-4 times of retention volume (BV) of 0.5wt% HCl aqueous solution is used for flushing a column, and soaking is carried out for 2-4 hours; distilled water is washed until the pH of effluent liquid is neutral; 3-4 times of retention volume (BV) of 2wt% NaOH aqueous solution is used for flushing a column, and soaking is carried out for 2-4 hours; and (5) flushing distilled water until the pH of the effluent is neutral, thus finishing the pretreatment of the macroporous adsorption resin column.
According to a preferred embodiment of the present invention, in the step (2), the macroporous adsorbent resin is HPD450.
According to the invention, in the step (2), the mass ratio of the dosage (dry weight) of the macroporous adsorption resin filler in the macroporous adsorption resin column to the medicine powder used for extracting the salvianolic acid B is 1 (0.5-3).
According to a preferred embodiment of the invention, in step (2), the retention volume of the distilled water elution is 1 to 4 times, preferably 4 times.
According to a preferred embodiment of the invention, in step (3), the acid is hydrochloric acid or sulfuric acid, preferably hydrochloric acid.
According to a preferred embodiment of the invention, in step (3), the acidic solution: the dry weight of the macroporous adsorption resin adsorbed with the salvianolic acid B is (1-2) 1 (v/w, mL/g), preferably 1:1 (v/w, mL/g).
According to a preferred embodiment of the invention, in step (3), the pH of the acidic solution is between 2.0 and 5.0, preferably pH 3.0.
According to a preferred embodiment of the invention, in step (3), the reaction temperature is 115 to 125 ℃, preferably 118.5 to 125 ℃.
According to the invention, in step (3), the reaction time is preferably 2 to 5 hours, more preferably 2 to 4 hours.
According to the preferred embodiment of the present invention, in the step (3), the pH, the conversion time and the conversion temperature of the acidic solution may be determined according to the mass content ratio of salvianolic acid B to salvianolic acid a in the desired salvianolic acid conversion product according to the conversion process parameter determination method of the present invention.
Preferably, in the step (4), the eluting solvent or the extracting solvent is water, 10-90 wt% ethanol water solution, pure methanol or methanol-water mixed solution with any proportion; preferably, the eluting solvent or the extracting solvent is 30-60 wt% ethanol water solution or pure methanol; more preferably, the eluting solvent or extracting solvent is 40wt% to 50wt% aqueous ethanol or pure methanol.
Preferably according to the invention, in step (4), the elution solvent volume is 2 to 8 retention volumes; preferably, the elution solvent volume is 2 to 6 retention volumes; more preferably, the elution solvent volume is 5 to 6 retention volumes.
According to the invention, in the step (4), the reduced pressure concentration temperature is 40-60 ℃; preferably, the reduced pressure concentration temperature is 40-50 ℃; more preferably, the reduced pressure concentration temperature is 40-45 ℃.
According to the invention, in the step (4), when the eluting solvent or the extracting solvent is pure methanol, the obtained eluent or extracting solution is concentrated under reduced pressure until no solvent exists, and the salvianolic acid conversion product is obtained; or, when the eluting solvent or the extracting solvent is ethanol water solution or methanol-water mixed solution, concentrating the eluent or the extracting solution under reduced pressure to small volume, and lyophilizing to obtain salvianolic acid conversion product.
The method for determining the transformation technological parameters of the salvianolic acid transformation product which is transformed into salvianolic acid A according to the proportion comprises the following steps:
(1) The pH value, the conversion time and the conversion temperature of an acid solution are taken as investigation factors, a three-factor five-level star point Design-effect curved surface method is adopted to measure the mass contents of salvianolic acid A and salvianolic acid B in each group of conversion products, the mass contents of the salvianolic acid A and the salvianolic acid B are taken as response values, data processing is carried out through Design expert8.0 software, and the salvianolic acid A (Y 1 ) Salvianolic acid B (Y) 2 ) The fit equation of the mass content of (c) and the investigation factor is as follows:
fitting equation Y 1 ：
Y 1 ＝270.35+66a+106.7b+126.25c+2.7ab+26.72ac+48.03bc-27.31a 2 +29.97b 2 +15.35c 2
(R 2 ＝0.9514，P＜0.001)
Fitting equation Y 2 ：
Y 2 ＝886.66-57.79a-141.41b-329.34c-154.25ab-39.3ac+134.42bc+2.99a 2 +86.55b 2 -25.2c 2
(R 2 ＝0.9220，P＜0.001)
Wherein: a is the pH of the acidic solution, b is the conversion time (hours), c is the conversion temperature (DEG C).
(2) According to the mass content ratio of salvianolic acid B and salvianolic acid A in the required conversion products, a fitting equation Y is utilized 1 And fitting equation Y 2 Analyzing by using Design Expert 8.0 software, and predicting the optimal technological parameters corresponding to the mass content ratio of salvianolic acid B to salvianolic acid A in the required conversion products;
(3) Determining final optimal process parameters based on the predicted optimal process parameters of step (2).
According to the invention, in the step (1), the mass content of the salvianolic acid A and the salvianolic acid B in each group of transformation products is determined, and the determination experiment is carried out according to the prior method.
In the step (2), the analysis method using Design Expert 8.0 software is preferably as follows: fitting equation Y 1 Determining a data set I corresponding to the mass content of the salvianolic acid A within the range of 37.1-701.5 mug/mL from the corresponding data set; in fitting equation Y 2 Determining a data set II corresponding to the mass content of the salvianolic acid B in the range of 191.2-1662.5 mug/mL in the corresponding data set; and comparing and analyzing the data set I and the data set II according to the mass content ratio of the salvianolic acid B and the salvianolic acid A in the required conversion products, wherein two groups of values, which are closest to the corresponding values of the examination factors in the data set I and the data set II, are the predicted optimal process parameters.
According to the invention, in the step (3), the optimal process parameters predicted in the step (2) are taken as base points, and the final optimal process parameters are obtained through further optimization experiments. Taking the predicted optimal technological parameter as a base point, finely adjusting the technological parameter near the base point, and determining the final optimal technological parameter through further small-range optimization experiments; the optimization experiment is carried out according to the prior method.
The application of the salvianolic acid conversion product can be used for preparing antithrombotic medicines, in particular medicines for preventing thrombosis in the treatment of atherosclerosis. Preferably, in the salvianolic acid conversion product, the mass content ratio of salvianolic acid B to salvianolic acid A is 1:2.
The salvianolic acid conversion product or the pharmaceutically acceptable salt thereof and the pharmaceutical excipients are combined to prepare pharmaceutical preparations with different dosage forms for resisting thrombus.
A pharmaceutical composition of antithrombotic salvianolic acid conversion product comprises salvianolic acid conversion product or pharmaceutically acceptable salt thereof, one or more pharmaceutically acceptable carriers or excipients and an antioxidant.
According to the preferred embodiment of the present invention, the pharmaceutically acceptable salt of salvianolic acid is prepared by dissolving salvianolic acid in distilled water, and alkalizing the salt with an alkalizing agent NaOH, KOH, na 2 CO 3 、K 2 CO 3 、NaHCO 3 、KHCO 3 、MgHPO 3 、Mg(H 2 PO 3 ) 2 The pH value of the mixture is adjusted to be between 6.8 and 8.0, so that sodium salt, potassium salt or magnesium salt is formed; preferred alkalizing agents are NaHCO 3 、KHCO 3 Or MgH 2 PO 3 。
Preferably, according to the invention, the antioxidant is selected from sodium ascorbate, ascorbic acid, citric acid or sodium metabisulfite.
According to the invention, the pharmaceutical composition is preferably one of tablets, capsules, medicinal granules and freeze-dried preparations for injection of salvianolic acid conversion products, and can be prepared according to a conventional production process of pharmacy.
The pharmaceutical preparations of the different dosage forms of the pharmaceutical composition are described in detail as follows:
(1) The preparation of the salvianolic acid conversion product tablet, capsule or granule comprises the following raw materials in percentage by weight:
taking salvianolic acid conversion products, adding 2-4 times of diluent I, 5-10 wt% of wetting agent and 4-15 wt% of disintegrating agent, granulating by a conventional wet method, drying, granulating, and bagging to obtain medicinal granules; or adding 0.5-3wt% of lubricant into the granule after finishing, mixing, or tabletting to obtain tablet, or encapsulating to obtain capsule.
The diluent I is selected from starch, sugar powder, dextrin or microcrystalline fiber;
The above-mentioned wetting agent is selected from water or ethyl alcohol;
the disintegrating agent is sodium carboxymethyl starch;
the lubricant is magnesium stearate or talcum powder.
(2) The preparation of the salvianolic acid conversion product enteric-coated tablet and the enteric-coated capsule comprises the following raw materials in percentage by weight:
taking salvianolic acid conversion products, adding 3-5 times of pregelatinized starch, 1-2 times of polyvinylpyrrolidone or hydroxypropyl cellulose, and a proper amount of microcrystalline cellulose, crosslinked sodium carboxymethyl cellulose and talcum powder, uniformly mixing, preparing soft materials by using crosslinked polyvinylpyrrolidone aqueous solution, granulating, drying, finishing, or tabletting to obtain tablets, or filling into enteric capsule shells to obtain enteric capsules.
(3) The preparation of the salvianolic acid conversion product enteric-coated sustained-release tablet comprises the following raw materials in percentage by weight:
taking salvianolic acid conversion products, adding 1-2 times by weight of hydroxypropyl methylcellulose and 3-5 times by weight of microcrystalline cellulose, spraying a proper amount of starch (or dextrin) and sodium carboxymethyl cellulose with 10wt% of polyvinylpyrrolidone absolute ethyl alcohol to prepare a soft material, granulating, drying, finishing, adding a proper amount of magnesium stearate, uniformly mixing, tabletting and obtaining a sustained release tablet core; dissolving 1-2 times of hydroxypropyl methyl cellulose phthalate or acrylic resin L-100 in ethanol, adding a proper amount of talcum powder, homogenizing, and coating a slow-release tablet core to obtain the enteric slow-release tablet.
(4) Preparation of freeze-dried preparation for injection of salvianolic acid conversion product
Taking salvianolic acid conversion product, adding appropriate amount of antioxidant, adding mannitol accounting for 5% (w/v) of total volume, dissolving in distilled water, fixing volume, filtering, sterilizing, packaging under aseptic condition, freeze drying, and sealing under nitrogen. The preparation method comprises dissolving with sterile water for injection or sodium bicarbonate water for injection.
(5) Preparation of freeze-dried preparation for injection of salvianolic acid conversion product salt
Dissolving salvianolic acid conversion product in distilled water, adding antioxidant, adding mannitol 5% (w/v) of total volume, and alkalizing with KHCO 3 Adjusting pH of the aqueous solution to be 6.8-8.0, filtering to remove insoluble substances, filtering to remove bacteria, sub-packaging into penicillin bottles under aseptic condition, freeze-drying, and sealing under nitrogen.
The salvianolic acid conversion product and the pharmaceutical composition preparation thereof are used for preparing antithrombotic medicaments, particularly for preventing thrombosis in the treatment of atherosclerosis, and have a certain auxiliary treatment effect on the atherosclerosis.
The preparation of the salvianolic acid conversion product is screened and optimized by systematic technological parameters. Firstly, a simple and easy aqueous solution ultrasonic extraction method is adopted, and the main factors influencing the extraction of the salvianolic acid B, such as the liquid-material ratio, the extraction time, the extraction temperature and the like, are carefully examined through a star point design-effect curved surface method, so that the technological parameters of the optimal extraction process, such as the liquid-material ratio, the extraction time, the extraction temperature and the like, are obtained. Secondly, taking the factors of poor stability of the salvianolic acid B and the salvianolic acid A in hot water into consideration, adsorbing the salvianolic acid B in the water extract on macroporous adsorption resin, eluting water to remove water-soluble impurities such as polysaccharide and the like in the water extract, directly treating the macroporous adsorption resin adsorbed with a large amount of salvianolic acid B under high-temperature high-pressure high-humidity conditions, and converting the adsorbed salvianolic acid B into phenolic acid components such as salvianolic acid A and the like; the star point Design-effect surface method is adopted, the content ratio of the main components of the salvianolic acid B and the salvianolic acid A is used as an index, design expert8.0 software is used for processing experimental data to respectively obtain fitting equations of the salvianolic acid B and the salvianolic acid A, conversion process parameters of the salvianolic acid B and the salvianolic acid A in different proportions in the required salvianolic acid conversion products can be optimized according to the two fitting equations, and the expected target is achieved through a small-range optimization experiment. Finally, the elution and drying method of the treated macroporous adsorption resin are examined. Through the research, the preparation process of the salvianolic acid conversion product is obtained; the invention can arbitrarily select conversion process parameters containing different content ratios of salvianolic acid B and salvianolic acid A, thereby obtaining the required conversion product. The invention meets the requirements of various salvianolic acid B and salvianolic acid A conversion products with different proportions, has simple method, simple process and low experimental cost, and is suitable for industrial production.
The salvianolic acid conversion product has the effects of treating and preventing thrombosis, and is particularly suitable for preventing and treating atherosclerosis.
Compared with the prior art, the invention has the following advantages:
(1) The invention provides a method for efficiently and accurately optimizing the technological parameters of the required conversion products according to requirements. According to the fitting equation, the corresponding conversion process parameters are predicted by combining the proportions of different salvianolic acids B and salvianolic acids A in the required salvianolic acid conversion products and applying Design expert8.0 software, and the optimal process parameters can be determined by further few optimization experiments according to the predicted process parameters.
(2) The sample treatment before conversion of the invention uses water as an extraction solvent, extraction is carried out under the condition of heating and ultrasonic, and the obtained water extract is directly loaded on macroporous adsorption resin, thus having the advantages of simple operation, cost saving and the like.
(3) The salvianolic acid B is extracted from medicinal materials, adsorbed on macroporous adsorption resin, and then converted into salvianolic acid A under the conditions of high temperature, high pressure and high humidity, and has the following advantages: (1) the problem of large volume caused by direct use of drug powder conversion is avoided; (2) the converted salvianolic acid A and the unconverted salvianolic acid B are adsorbed on macroporous resin, and compared with the macroporous resin which is dissolved in a water solution with a larger volume, the salvianolic acid A and the salvianolic acid B are easy to cool, have better stability and lower loss.
(4) The conversion raw materials and conversion products used in the invention are adsorbed on the solid macroporous adsorption resin, and the extraction and conversion process is also carried out on the macroporous adsorption resin, so that the whole production processes of loading, conversion, elution, drying and the like are easy to operate, the method is simple to operate, the experimental cost is low, the method is suitable for industrial production, and a foundation is laid for the mass preparation of salvianolic acid A and the development and utilization of salvianolic acid conversion products.
(5) The salvianolic acid conversion product prepared by the invention mainly contains salvianolic acid A, salvianolic acid B, salvianolic acid A, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid. The salvianolic acid conversion product can be used for preparing antithrombotic and anti-atherosclerosis medicines.
Drawings
FIG. 1 shows the effect of the feed-liquid ratio on the extraction amount of salvianolic acid B in test example 1.
FIG. 2 shows the effect of the extraction time on the extraction amount of salvianolic acid B in test example 1.
FIG. 3 shows the effect of the extraction temperature on the extraction amount of salvianolic acid B in test example 1.
FIG. 4 is a graph showing the response of the extraction factor to the amount of salvianolic acid B extracted in test example 1.
FIG. 5 is a graph showing the effect of pH on the conversion of salvianolic acid B to salvianolic acid A in test example 2.
FIG. 6 is a graph showing the effect of conversion time on the conversion of salvianolic acid B to salvianolic acid A in test example 2.
FIG. 7 is a graph showing the effect of conversion temperature on the conversion of salvianolic acid B to salvianolic acid A in test example 2.
FIG. 8 is a graph showing the response of the conversion factor to the content of salvianolic acid A in test example 2.
FIG. 9 is a graph showing the response of the conversion factor to the content of salvianolic acid B in test example 2.
FIG. 10 is a gradient elution profile of salvianolic acid A in test example 3.
FIG. 11 is a methanol elution profile of salvianolic acid A in test example 3.
FIG. 12 is a graph showing comparison of thrombus weights in the inferior vena cava thrombus model in test example 7.
FIG. 13 is a graph showing a comparison of thrombus areas in the inferior vena cava thrombus model in test example 7.
FIG. 14 is an HPLC chart of an extract obtained by ultrasonic extraction of salvianolic acid B in example 1.
FIG. 15 is an HPLC chart of a salvianolic acid conversion product obtained in example 1.
FIG. 16 is an HPLC chart of a salvianolic acid conversion product obtained in example 2.
FIG. 17 is an HPLC chart of a salvianolic acid conversion product obtained in example 3.
FIG. 18 is an HPLC chart of a salvianolic acid conversion product obtained in example 4.
Detailed Description
The present invention will be further described with reference to examples and test examples, but is not limited thereto. The raw materials and equipment used in the examples and the test examples are all products in the prior art and the market.
Test example 1: optimum extraction process parameter research of salvianolic acid B in salvia miltiorrhiza bunge
The extraction method comprises the following steps: water is used as solvent, and an ultrasonic extraction method is adopted: pulverizing Saviae Miltiorrhizae radix, and sieving with 40 mesh sieve; adding distilled water, performing ultrasonic extraction, and filtering to obtain filtrate and residue; repeating the above steps (i.e. adding distilled water, then performing ultrasonic extraction, and filtering) for 2 times; mixing the filtrates to obtain salvianolic acid B extractive solution.
In the test, the liquid-material ratio (a), the extraction time (B) and the extraction temperature (c) are taken as investigation factors, the extraction times are 3 times, a three-factor five-level star point Design-effect curved surface method is adopted, the content of salvianolic acid B in each group of red sage root extracting solutions is measured by adopting a conventional HPLC measuring method, the content of salvianolic acid B is taken as a response value, data processing is carried out by Design expert8.0 software, and the optimal extraction process is optimized and determined.
(1) Single factor investigation
The main factors influencing the extraction of the salvianolic acid B include liquid-material ratio, extraction time and extraction temperature, two of the two factors are fixed, the influence degree of the other phase change is inspected, and the results are shown in figures 1, 2 and 3 (see the drawings), wherein the optimal extraction process parameters are as follows: the liquid-material ratio is 1:30, the extraction time is 40min, and the temperature is 70 ℃.
(2) Test design
The three parameters of feed liquid ratio of 1:30, extraction time of 40min and temperature of 70 ℃ are respectively determined to be 0 level. The coding of each factor and level is shown in table 1. The factors and the levels were tested in a certain combination (see table 2), and the content of salvianolic acid B in the extract of red sage root was measured, and the results are shown in table 2.
Table 1 experimental factors and horizontal encoding table
Table 2 center combined experimental design and results
Regression model analysis is carried out on the experimental results by adopting Design-Expert software, the results are shown in table 3, and a binary polynomial regression equation is obtained through fitting: y=12.44+0.17a+0.18b+1.66c-0.059 ab-0.12ac-0.076bc-0.56a 2 -0.55b 2 -0.64c 2 Its P<0.01，R 2 The experimental design was reliable, and the regression model established with the salvianolic acid B extraction as the response value was remarkable as demonstrated by = 0.7682.
TABLE 3 analysis of variance of regression model of salvianolic acid B content in Saviae Miltiorrhizae radix extract
Note that: * P＜0.05， ** P＜0.01， *** P＜0.001
from the above results, a response curve diagram is created, and as shown in FIG. 4, the extraction amount of salvianolic acid B is highest when the temperature is 60.00-77.32 ℃, the extraction time is 31.34-48.66 min, and the feed-liquid ratio is 21.34-38.66. Through analysis of Design-Expert software, the optimal extraction process is preferably 30.08 times of extraction solvent, the temperature is 72.86 ℃, the ultrasonic treatment is carried out for 40.71 minutes, the optimal extraction process is changed into 30 times of extraction solvent, the temperature is 73 ℃, the ultrasonic treatment is carried out for 41 minutes, and the extraction is carried out for 3 times by taking actual factors and industrial factors into consideration.
Test example 2: optimum technological parameter research for converting salvianolic acid B into salvianolic acid A
The preparation method comprises the following steps: according to the optimal extraction process parameters of test example 1, 8g of red sage root powder is used for extracting salvianolic acid B water extract, and the dosage (dry weight) of macroporous adsorption resin HPD450 filler is 10g. And (3) after pretreatment of the macroporous adsorption resin, loading all the salvianolic acid B water extract on a macroporous adsorption resin column, wherein the flow rate is 0.5mL per minute, eluting with distilled water with the retention volume of 4 times when the filtrate is completely discharged, and the flow rate is 1mL per minute, so as to obtain the macroporous adsorption resin with the salvianolic acid B adsorbed. Removing most of water from the macroporous adsorption resin adsorbed with the salvianolic acid B, wetting with 10mL of hydrochloric acid aqueous solution, and then placing the wetted macroporous adsorption resin into a high-temperature high-pressure steam sterilization pot for reaction to obtain the macroporous adsorption resin adsorbed with the salvianolic acid conversion product.
The pretreatment method of the macroporous adsorption resin column comprises the following steps: soaking macroporous adsorbent resin (HPD 450) in 95wt% ethanol for 24 hr, suspending with 95wt% ethanol, and wet packing; washing the column with 95wt% ethanol until the effluent liquid is not turbid after adding an equal amount of water; washing the column with distilled water until the effluent liquid has no alcohol smell; 4 times of retention volume (BV) of 0.5wt% HCl aqueous solution is used for flushing, soaking is carried out for 3 hours, and distilled water is used for flushing until the pH of effluent liquid is neutral; 4 times of retention volume (BV) of 2wt% NaOH aqueous solution is used for flushing a column, soaking is carried out for 3 hours, distilled water is used for flushing until the pH value of effluent liquid is neutral, and then the preparation of the macroporous adsorption resin column is completed.
The test uses pH, conversion time and conversion temperature of an acid solution as investigation factors, the pH is determined to be 2, the conversion time is 3 hours, the conversion temperature is 120 ℃ to be 0 level, a three-factor five-level star point design-effect curved surface method is adopted, the content of Salvianolic Acid A (SAA) and Salvianolic Acid B (SAB) in each group of conversion products is measured by adopting a conventional HPLC measuring method, and the following test is carried out by taking the content of the salvianolic acid A and the content of the salvianolic acid B as response values. The preparation method of the methanol solution for determining the conversion product by HPLC comprises the following steps: placing macroporous adsorbent resin with salvianolic acid conversion product in conical flask, adding 50mL of methanol, ultrasonic extracting at room temperature for 30 min, filtering, transferring filtrate into 50mL volumetric flask, and fixing volume with methanol.
(1) Single factor investigation
The main factors influencing the conversion of phenolic acid components include pH, conversion time and conversion temperature, two of the factors are fixed, and the influence degree of the other phase change is examined, so that the results are shown in figures 5, 6 and 7, wherein SAD is salvianolic acid D. At pH 2, conversion time of 3 hours and conversion temperature of 120 ℃, the influence on the conversion of salvianolic acid B into salvianolic acid A was the greatest, and the three parameters were respectively determined to be 0 level. The coding of the factors and levels is shown in table 4. The factors and levels were combined in a certain amount (see Table 5), and the contents of salvianolic acid A and salvianolic acid B in the converted products were measured, and the results are shown in Table 5.
Table 4 experiment factors and horizontal coding table
Table 5 center combined experimental design and results
Carrying out data processing by adopting Design expert8.0 software, carrying out regression analysis on the data of the salvianolic acid A in the experimental results, and obtaining a fitting equation Y of the salvianolic acid A, wherein the result is shown in Table 6 1 Correlation coefficient R 2 ＝0.9514，Correction of the determination coefficient R 2 Adj 0.9076, the model is described as reflecting the real relationship between each factor and the content of salvianolic acid a, and a response curve graph is established (fig. 8).
Y 1 ＝270.35+66a+106.7b+126.25c+2.7ab+26.72ac+48.03bc-27.31a 2 +29.97b 2 +15.35c 2 (P＜0.001)
Carrying out data processing by adopting Design expert8.0 software, carrying out regression analysis on the data of the salvianolic acid B in the experimental results, and obtaining a fitting equation Y of the salvianolic acid B, wherein the result is shown in table 7 2 Correlation coefficient R 2 =0.922, correct the decision coefficient R 2 Adj 0.8518, the model is described as reflecting the real relationship between each factor and the content of salvianolic acid B, and a response curve graph is established (fig. 9).
Y 2 ＝886.66-57.79a-141.41b-329.34c-154.25ab-39.3ac+134.42bc+2.99a 2 +86.55b 2 -25.2c 2 (P < 0.001) analysis of variance of regression model of salvianolic acid A of Table 6
Note that: * P＜0.05， ** P＜0.01， *** P＜0.001
TABLE 7 analysis of variance of regression model of salvianolic acid B
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Note that: * P＜0.05， ** P＜0.01， *** P＜0.001
according to the mass content ratio of salvianolic acid B and salvianolic acid A in the required conversion products, a fitting equation Y is utilized 1 And fitting equation Y 2 By application of Design Expert 8.0.0 software is used for analysis, and the optimal technological parameters corresponding to the mass content ratio of salvianolic acid B and salvianolic acid A in the required conversion products are predicted. The analysis method using Design Expert 8.0 software is as follows: fitting equation Y 1 Determining a data set I corresponding to the mass content of the salvianolic acid A within the range of 39.1-701.5 mug/mL in the corresponding data set; in fitting equation Y 2 Determining a data set II corresponding to the mass content of the salvianolic acid B in the range of 191.2-1662.5 mug/mL in the corresponding data set; and comparing and analyzing the data set I and the data set II according to the mass content ratio of the salvianolic acid B and the salvianolic acid A in the required conversion products, wherein two groups of values, which are closest to the corresponding values of the examination factors in the data set I and the data set II, are the predicted optimal process parameters.
And determining final optimal process parameters according to the predicted optimal process parameters. And (3) taking the predicted optimal process parameters as base points, fine-tuning the process parameters near the base points, and determining final optimal process parameters through further small-range optimization experiments according to the existing optimization experiment method.
Analysis results by Design Expert 8.0 software show that:
(1) When the content of salvianolic acid B in the required conversion product is: when the content of salvianolic acid a=2:1 (B2 A1), according to Y 1 One transformation condition of the available salvianolic acid A is pH=2.95, the transformation time is 3.96 hours, and the transformation temperature is 117.04 ℃; according to Y 2 One conversion condition for the available salvianolic acid B was ph=2.96, conversion time was 3.99 hours, and conversion temperature was 118.02 ℃. Taking the predicted optimal process parameters as base points, fine-tuning the process parameters near the base points, and optimizing the optimal conversion conditions of the B2A1 by further small-range optimization tests: ph=3, conversion time 4 hours, conversion temperature 118.5 ℃.
(2) When the content of salvianolic acid B in the required conversion product is: when the content of salvianolic acid a=1:1 (B1 A1), according to Y 1 One transformation condition of the available salvianolic acid a is ph=2.19, the transformation time is 3.55 hours, and the transformation temperature is 122.69 ℃; according to Y 2 One transformation condition of the available salvianolic acid B is pH=2.17, the transformation time is 3.51 hours, and the transformation is carried outThe temperature was 123.75 ℃. Taking the predicted optimal process parameters as base points, fine-tuning the process parameters near the base points, and optimizing the optimal conversion conditions of the B1A1 by further small-range optimization tests: ph=3, conversion time of 3.55 hours, conversion temperature of 123 ℃.
(3) When the content of salvianolic acid B in the required conversion product is: when the content of salvianolic acid a=1:2 (B1 A2), according to Y 1 One transformation condition of the available salvianolic acid A is pH=2.94, the transformation time is 3.98 hours, and the transformation temperature is 124.88 ℃; according to Y 2 One transformation condition for the available salvianolic acid B was ph=3, transformation time was 3.99 hours, and transformation temperature was 124.92 ℃. Taking the predicted optimal process parameters as base points, fine-tuning the process parameters near the base points, and optimizing the optimal conversion conditions of the B1A2 by further small-range optimization tests: ph=3, conversion time 4 hours, conversion temperature 124 ℃.
Test example 3 concentration investigation of elution solvent
The macroporous adsorption resin with the salvianolic acid conversion product adsorbed thereon is loaded into a column, and is eluted by using 5 retention volumes of 10wt%, 20wt%, 30wt%, 40wt%, 50wt%, 60wt%, 70wt%, 80wt% and 90wt% ethanol water solution respectively, and the results show in figure 10, 40wt% and 50wt% ethanol water solution have the best eluting effect. In order to obtain the eluting conditions with higher eluting rate of salvianolic acid A, salvianolic acid B and salvianic acid A, 40wt% ethanol aqueous solution and pure methanol are further adopted as eluting solvents, the eluting conditions are further examined, the using amount of the eluting solvents is 5 reserved volumes, the results are shown in table 8, and the eluting efficiency of the eluting of the salvianolic acid A, the salvianolic acid B and the salvianic acid A by using the pure methanol is higher. When methanol is used for elution, one eluent is collected for each retention volume, and a methanol elution curve of salvianolic acid A is drawn, and the result is shown in figure 11, wherein the volume of the methanol eluent is 6 retention volumes, and almost all salvianolic acid conversion products are eluted. Therefore, the eluting effect is best by adopting 40 to 50 percent of ethanol or methanol.
TABLE 8 investigation of different elution solvents
Test example 4 measurement of in vivo anti-platelet aggregation Rate
Male Wistar rats weighing about 220-250 g and were kept for one week with an adaptive temperature of 25+ -1deg.C. Wistar rats were randomly divided into 10 groups of 6 animals each, with the following groupings: blank control group: 0.5% sodium carboxymethyl cellulose (CMC-Na); b2A1 (content of salvianolic acid B: content of salvianolic acid a = 2:1) low dose group: 50 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the Dose group in B2 A1: 100 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the B2A1 high dose group: 150 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the B1A1 (content of salvianolic acid B: content of salvianolic acid a=1:1) low dose group: 50 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the Dose group in B1 A1: 100 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the B1A1 high dose group: 150 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the B1A2 (content of salvianolic acid B: content of salvianolic acid a=1:2) low dose group: 50 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the Dose group in B1 A2: 100 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the B1A2 high dose group: 150 mg/kg -1 The method comprises the steps of carrying out a first treatment on the surface of the Once daily, the stomach is continuously irrigated for seven days, and blood is taken 5h after the last administration. 10% chloral hydrate anesthetized rats, and abdominal aorta was bled to 3.8% sodium citrate 1:9 anticoagulated negative pressure blood collection tube. Platelet Rich Plasma (PRP) was prepared by centrifugation of rat plasma at 1000rpm for 10min, and Platelet Poor Plasma (PPP) was prepared by centrifugation of the remaining plasma at 3000rpm for 10 min.
The platelet aggregation rate of the above-collected experimental rat plasma was measured by a turbidimetry method at 37 ℃. Zeroing by PPP, incubating PRP for 15min, adding inducer ADP, and determining the inhibition of platelet aggregation induced by ADP by each group of plasma. The experimental results are shown in table 9.
Test results show that platelet aggregation induced by ADP can be obviously inhibited compared with a blank group after various compounds with different concentrations are added. Specifically, the concentration of Compound B1A2 was 50 mg/kg -1 The platelet aggregation rate induced by ADP can be obviously reduced. The anti-platelet aggregation rates of the groups B2A1, B1A1 and B1A2 show good dose-effect relationship, and the anti-platelet aggregation effect is gradually enhanced along with the increase of the administration dosage. The maximum inhibition rates of the three groups were 61%, 59% and 47%, respectively, with the B1A2 group being the most effective.
TABLE 9 influence of the conversion products of salvianolic acid B and salvianolic acid A in different ratios on the platelet aggregation rate
* P＜0.05， ** P＜0.01， *** P < 0.001 was compared to the blank.
Test example 5 in vivo anticoagulation test
Rat administration method and plasma preparation method are the same as in test example 4.
The prepared reagents were placed on a set-up semi-automatic coagulation analyzer and preheated. Platelet Poor Plasma (PPP) prepared as described above was assayed for Prothrombin Time (PT), activated Partial Thromboplastin Time (APTT), fibrinogen (FIB) and Thrombin Time (TT) as indicated in Table 10.
The test results show that the doses of the three groups B2A1, B1A1 and B1A2 have no obvious effect on PT and TT, but can obviously reduce APTT. Wherein B1A2 group is 150mg.kg -1 The dose group effect is optimal.
Table 10 contains anticoagulant effect of the conversion products of salvianolic acid B and salvianolic acid A in different proportions
* P＜0.05， ** P＜0.01， *** P < 0.001 was compared to the blank.
Test example 6 test for inhibition of macrophage foam formation
Six-hole plate inoculated human acute monocytic leukemia cells THP-1, THP-1 in suspension state growth, cultured in 1640 culture medium containing 15% FBS and 1% blue chain mycin mixed solution, and generally passed once every four days. THP-1 cells with good growth state are inoculated into a culture plate, PMA (100 ng/mL) is added into 1640 culture medium of 3% FBS to induce for 24 hours, fresh 1640 culture medium containing 3% FBS is replaced, ox-LDL (80 mug/mL) is added to induce for 48 hours, the culture medium is discarded, PBS is washed once, fresh 1640 culture medium of 3% FBS is replaced, and salvianolic acid conversion product aqueous solutions with different concentrations (10, 20 and 30 mug/mL) are added (the content of salvianolic acid B: the content of salvianolic acid A=1:2) to treat for 24 hours. Sucking and discarding the culture medium, washing twice by PBS, and collecting cell suspension by using a cell scraper; centrifuging, removing supernatant (1000 rpm,10 min), adding isopropanol, and ultrasonically breaking cells under ice bath condition; the supernatant was centrifuged, and the intracellular TC, TG and FC contents were measured according to the kit instructions, and CE=TC-FC was further obtained from TC and FC, and the experimental results are shown in Table 11.
The effect of salvianolic acid conversion products on the lipid content in foam cells was measured by using a kit, and the results are shown in Table 11, wherein the content of TC, TG, FC, CE in cells was significantly increased after ox-LDL was added, and the difference was significantly greater than that in the blank cells (P < 0.001). The salvianolic acid conversion products have remarkable reducing effect on the content of TC, TG and CE in cells when the concentration is 10, 20 and 30 mug/mL, which indicates that the salvianolic acid conversion products have remarkable inhibiting effect on the foam formation of THP-1 macrophages, and can be used for treating atherosclerosis.
TABLE 11 anti-macrophage foam action of salvianolic acid conversion products
Note that: b cells were treated with PMA (100 ng/mL) for 24 hours and then incubated with ox-LDL (80. Mu.g/mL) for 48 hours.
* P＜0.05， ** P＜0.01， *** P < 0.001 was compared with ox-LDL (80. Mu.g/mL) group.
### P < 0.001 was compared to the blank.
Test example 7, inferior vena cava Thrombus model
Wistar rats, male, 220-250 g, were randomly divided into 5 groups of 6 animals each. The grouping is as follows, blank group: 0.5% sodium carboxymethyl cellulose aqueous solution; aspirin group (25 mg/kg); the salvianolic acid conversion product (salvianolic acid B content: salvianolic acid A content=1:2) was used in the low dose group (12.5 mg/kg), the medium dose group (25 mg/kg), and the high dose group (50 mg/kg). All drugs were administered orally once a day for seven consecutive days. After 1h from the last administration, 10% chloral hydrate was anesthetized, fixed in the supine position, the abdominal wall was dissected, the inferior vena cava was isolated, the inferior vena cava was ligated at the intersection of the left renal vein and the inferior vena cava, the abdominal wall was sutured, the abdominal cavity was reopened after 6h, the vessel was again ligated at 2cm below the ligation, the embolus was removed, residual blood was sucked with filter paper, and the data was weighed with an electronic balance and recorded. The vena cava thrombotic tissue specimens were removed, fixed with 4% paraformaldehyde for 1 hour, frozen, and HE stained, and observed with a microscope, and the test results are shown in FIG. 12 and FIG. 13.
Fig. 12 is a graph showing a comparison of thrombus weights in the inferior vena cava thrombus model, and fig. 13 is a graph showing a comparison of thrombus areas in the inferior vena cava thrombus model. Compared with the model group, the weight of thrombus in the aspirin group and the salvianolic acid conversion products in the low, medium and high groups is obviously reduced, the thrombus area is obviously reduced, and the thrombus weight and the thrombus area are gradually reduced along with the increase of the administration dosage of the salvianolic acid conversion products, so that the salvianolic acid conversion products can obviously inhibit the formation of inferior vena cava thrombus and show obvious antithrombotic effect.
Example 1: the content ratio of the salvianolic acid B to the salvianolic acid A is 2:1, preparation of salvianolic acid conversion product
Pulverizing Saviae Miltiorrhizae radix, and sieving with 40 mesh sieve to obtain Saviae Miltiorrhizae radix powder. Taking 8g of red sage root powder, adding 30 times of distilled water, ultrasonically extracting at 73 ℃ for 41 minutes, and filtering to obtain filtrate and dregs; repeating the ultrasonic extraction step for 2 times; combining the filtrates to obtain salvianolic acid B extract, and performing HPLC content analysis under conventional measurement conditions, wherein the measurement result is shown in figure 14.
The preparation method of the macroporous adsorption resin column comprises the following steps: soaking macroporous adsorption resin (HPD 300) in 95wt% ethanol for 24h, suspending with 95wt% ethanol, and carrying out wet column packing, wherein the mass ratio of the dosage (dry weight) of macroporous adsorption resin filler to the medicine powder used for extracting salvianolic acid B is 1:1; washing the column with 95wt% ethanol until the effluent liquid is not turbid after adding an equal amount of water; washing the column with distilled water until the effluent liquid has no alcohol smell; 4 times of retention volume (BV) of 0.5wt% HCl aqueous solution is used for flushing, soaking is carried out for 3 hours, and distilled water is used for flushing until the pH of effluent liquid is neutral; 4 times of retention volume (BV) of 2wt% NaOH aqueous solution is used for flushing a column, soaking is carried out for 3 hours, distilled water is used for flushing until the pH value of effluent liquid is neutral, and then the preparation of the macroporous adsorption resin column is completed.
And (3) loading all the salvianolic acid B extract onto a macroporous adsorption resin column at a flow rate of about 1mL per minute, eluting with distilled water with a retention volume of 4 times when the filtrate is completely discharged, and drying the solvent in the macroporous adsorption resin column after the loading is completed at a flow rate of about 1mL per minute to obtain the macroporous adsorption resin with the salvianolic acid B extract adsorbed.
Setting the content ratio of salvianolic acid B and salvianolic acid A in the conversion product to be 2:1, and utilizing a fitting equation Y in the conversion process parameters of test example 2 1 And fitting equation Y 2 Analysis is carried out by using Design Expert 8.0 software, and the content ratio of salvianolic acid B to salvianolic acid A in the conversion process is preferably 2:1, optimal conversion process parameters: aqueous hydrochloric acid at ph=3.0, and treated at 118.5 ℃ for 4 hours.
The conversion process using the above process parameters is as follows: removing most of water from the macroporous adsorption resin adsorbed with the salvianolic acid B extract, placing the macroporous adsorption resin in an evaporation dish, wetting the macroporous adsorption resin with an equal amount (v/w) of (dry weight) hydrochloric acid aqueous solution with pH=3.0, placing the macroporous adsorption resin in a high-pressure steam sterilization pot, and treating the macroporous adsorption resin at 118.5 ℃ for 4 hours to obtain the treated macroporous adsorption resin, wherein the treated macroporous adsorption resin is adsorbed with converted salvianolic acid conversion products. And loading the treated macroporous adsorption resin into a glass column, eluting with 40wt% ethanol water for 5 retention volumes, concentrating under reduced pressure at 40-45 ℃ until no ethanol smell exists, and freeze-drying to obtain a converted salvianolic acid conversion product. The content of salvianolic acid B and salvianolic acid A in the product is about 2:1 (see figure 15) by HPLC chromatography. The total content of salvianolic acid B, salvianolic acid A, salvianic acid A, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid in the converted product is 60.69wt%, wherein the content of salvianolic acid A, salvianolic acid B and salvianic acid A accounts for 96wt% of the total amount of the six components.
Therefore, the conversion process parameter determination method can efficiently and accurately determine the optimal process parameters according to the required conversion products.
Example 2: the content ratio of the salvianolic acid B to the salvianolic acid A is 1:1, preparation of salvianolic acid conversion product
Pulverizing Saviae Miltiorrhizae radix, and sieving with 40 mesh sieve to obtain Saviae Miltiorrhizae radix powder. Taking 10g of red sage root powder, adding 30 times of distilled water, carrying out ultrasonic extraction at 70 ℃ for 40 minutes, and filtering to obtain filtrate and dregs; repeating the ultrasonic extraction step for 2 times; mixing the filtrates to obtain salvianolic acid B extractive solution.
The preparation method of the macroporous adsorption resin column comprises the following steps: soaking macroporous adsorption resin (D101) in 95wt% ethanol for 24h, suspending with 95wt% ethanol, and carrying out wet column packing, wherein the mass ratio of the dosage (dry weight) of macroporous adsorption resin filler to the medicine powder used for extracting salvianolic acid B is 5:4; washing the column with 95wt% ethanol until the effluent liquid is not turbid after adding an equal amount of water; washing the column with distilled water until the effluent liquid has no alcohol smell; 4 times of retention volume (BV) of 0.5wt% HCl aqueous solution is used for flushing, soaking is carried out for 3 hours, and distilled water is used for flushing until the pH of effluent liquid is neutral; 4 times of retention volume (BV) of 2wt% NaOH aqueous solution is used for flushing a column, soaking is carried out for 3 hours, distilled water is used for flushing until the pH value of effluent liquid is neutral, and then the preparation of the macroporous adsorption resin column is completed.
And (3) loading all the salvianolic acid B extract onto a macroporous adsorption resin column, wherein the flow rate is about 0.5mL per minute, eluting with distilled water with 1 retention volume after the filtrate is completely discharged, and drying the solvent in the macroporous adsorption resin column after the loading is completed, thereby obtaining the macroporous adsorption resin adsorbed with the salvianolic acid B extract.
Setting the content ratio of salvianolic acid B and salvianolic acid A in the conversion product to be 1:1, and utilizing a fitting equation Y in the conversion process parameters of test example 2 1 And fitting equation Y 2 Analysis is carried out by using Design Expert 8.0 software, and the content ratio of salvianolic acid B to salvianolic acid A in the conversion process is preferably 1:1, optimal conversion process parameters: ph=3.0 aqueous hydrochloric acid, and treated at 123 ℃ for 3.55 hours.
The conversion process using the above process parameters is as follows: removing most of water from the macroporous adsorption resin adsorbed with the salvianolic acid B extract, placing the macroporous adsorption resin in an evaporation dish, wetting the macroporous adsorption resin with an equal amount (v/w) of (dry weight) hydrochloric acid aqueous solution with pH=3.0, placing the macroporous adsorption resin in a high-pressure steam sterilization pot, and treating the macroporous adsorption resin for 3.55 hours at 123 ℃ to obtain the treated macroporous adsorption resin, wherein the treated macroporous adsorption resin is adsorbed with converted salvianolic acid conversion products. And loading the treated macroporous adsorption resin into a glass column, eluting with 50wt% methanol water for 4 retention volumes, concentrating under reduced pressure at 40-45 ℃ until no alcohol smell exists, and freeze-drying to obtain a converted salvianolic acid conversion product. The content of salvianolic acid A and salvianolic acid B in the product is about 1:1 (see figure 16) as determined by HPLC chromatography. The total content of salvianolic acid B, salvianolic acid A, salvianic acid A, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid in the converted product is 60.41wt%, wherein the content of salvianolic acid A, salvianolic acid B and salvianic acid A accounts for 93wt% of the total content of the six components.
Therefore, the conversion process parameter determination method can efficiently and accurately determine the optimal process parameters according to the required conversion products.
Example 3: preparation of salvianolic acid conversion product with salvianolic acid B and salvianolic acid A content ratio of about 1:2
Pulverizing Saviae Miltiorrhizae radix, and sieving with 40 mesh sieve to obtain Saviae Miltiorrhizae radix powder. Taking 10g of red sage root powder, adding 30 times of distilled water, carrying out ultrasonic extraction at 70 ℃ for 40 minutes, and filtering to obtain filtrate and dregs; repeating the ultrasonic extraction step for 2 times; mixing the filtrates to obtain salvianolic acid B extractive solution.
The preparation method of the macroporous adsorption resin column comprises the following steps: soaking macroporous adsorption resin (HPD 450) in 95wt% ethanol for 24h, suspending with 95wt% ethanol, and carrying out wet column packing, wherein the mass ratio of the dosage (dry weight) of macroporous adsorption resin filler to the medicine powder used for extracting salvianolic acid B is 5:4; washing the column with 95wt% ethanol until the effluent liquid is not turbid after adding an equal amount of water; washing the column with distilled water until the effluent liquid has no alcohol smell; 4 times of retention volume (BV) of 0.5wt% HCl aqueous solution is used for flushing, soaking is carried out for 3 hours, and distilled water is used for flushing until the pH of effluent liquid is neutral; 4 times of retention volume (BV) of 2wt% NaOH aqueous solution is used for flushing a column, soaking is carried out for 3 hours, distilled water is used for flushing until the pH value of effluent liquid is neutral, and then the preparation of the macroporous adsorption resin column is completed.
And (3) loading all the salvianolic acid B extract onto a macroporous adsorption resin column, wherein the flow rate is about 0.5mL per minute, eluting with three distilled water with 3 times of the retention volume after the filtrate is completely discharged, and drying the solvent in the macroporous adsorption resin column after the loading is completed, thereby obtaining the macroporous adsorption resin adsorbed with the salvianolic acid B extract.
Setting the content ratio of salvianolic acid B and salvianolic acid A in the conversion product to be 1:2, and utilizing a fitting equation Y in the conversion process parameters of test example 2 1 And fitting equation Y 2 Analysis is carried out by using Design Expert 8.0 software, and the content ratio of salvianolic acid B to salvianolic acid A in the conversion process is preferably 1:2, optimal conversion process parameters: ph=3.0 aqueous hydrochloric acid, and treated at 124 ℃ for 4 hours.
The conversion process using the above process parameters is as follows: the macroporous adsorption resin absorbed with the salvianolic acid B extract is dehydrated to a large extent, placed in a crucible, wetted by a hydrochloric acid aqueous solution with the pH of 3.0 and equal (v/w) amount (dry weight) of macroporous adsorption resin filler, transferred into a high-temperature high-pressure steam sterilization pot, and reacted for 4 hours at the temperature of 124 ℃. And after the reaction is finished, taking out the crucible filled with macroporous resin, placing the crucible on ice for cooling, loading the macroporous resin into a glass column, eluting with pure methanol, eluting for 6 retention volumes, concentrating methanol eluent under reduced pressure at 40-45 ℃ and evaporating to dryness to obtain a converted salvianolic acid conversion product. The content of the salvianolic acid B and the salvianolic acid A is measured by an HPLC chromatography, and the content ratio of the salvianolic acid B to the salvianolic acid A in the product is about 1:2 (see fig. 17). The total content of salvianolic acid B, salvianolic acid A, salvianic acid A, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid in the converted product is 64.85wt%, wherein the content of salvianolic acid A, salvianolic acid B and salvianic acid A accounts for 94wt% of the total amount of the six components.
Therefore, the conversion process parameter determination method can efficiently and accurately determine the optimal process parameters according to the required conversion products.
Example 4: preparation of salvianolic acid conversion product with salvianolic acid B and salvianolic acid A content ratio of about 1:3.5
Pulverizing Saviae Miltiorrhizae radix, and sieving with 40 mesh sieve to obtain Saviae Miltiorrhizae radix powder. Taking 10g of red sage root powder, adding 30 times of distilled water, carrying out ultrasonic extraction at 70 ℃ for 40 minutes, and filtering to obtain filtrate and dregs; repeating the ultrasonic extraction step for 2 times; mixing the filtrates to obtain salvianolic acid B extractive solution.
The preparation method of the macroporous adsorption resin column comprises the following steps: soaking macroporous adsorption resin (HPD 600) in 95wt% ethanol for 24h, suspending with 95wt% ethanol, and carrying out wet column packing, wherein the mass ratio of the dosage (dry weight) of macroporous adsorption resin filler to the medicine powder used for extracting salvianolic acid B is 1:1; washing the column with 95wt% ethanol until the effluent liquid is not turbid after adding an equal amount of water; washing the column with distilled water until the effluent liquid has no alcohol smell; 4 times of retention volume (BV) of 0.5wt% HCl aqueous solution is used for flushing, soaking is carried out for 3 hours, and distilled water is used for flushing until the pH of effluent liquid is neutral; 4 times of retention volume (BV) of 2wt% NaOH aqueous solution is used for flushing a column, soaking is carried out for 3 hours, distilled water is used for flushing until the pH value of effluent liquid is neutral, and then the preparation of the macroporous adsorption resin column is completed.
And (3) loading all the salvianolic acid B extract onto a macroporous adsorption resin column at a flow rate of about 1mL per minute, eluting with 2 distilled water with a retention volume after the filtrate is completely discharged, at a flow rate of about 0.5mL per minute, and drying the solvent in the macroporous adsorption resin column after loading, thereby obtaining the macroporous adsorption resin adsorbed with the salvianolic acid B extract.
Setting the content ratio of salvianolic acid B and salvianolic acid A in the conversion product to be 1:3.5, and utilizing a fitting equation Y in the conversion process parameters of test example 2 1 And fitting equation Y 2 Analysis is carried out by using Design Expert 8.0 software, and the content ratio of salvianolic acid B to salvianolic acid A in the conversion process is preferably 1:3.5 optimal conversion process parameters: ph=3.5 aqueous hydrochloric acid, and treated at 125 ℃ for 4 hours.
The conversion process using the above process parameters is as follows: the macroporous adsorption resin absorbed with the salvianolic acid B extract is dehydrated to a large extent, placed in an evaporation dish, wetted by a hydrochloric acid aqueous solution with the pH of 3.5 and equal (v/w) amount (dry weight) of macroporous adsorption resin filler, and then transferred into a high-temperature high-pressure steam sterilization pot for reaction for 4 hours at the temperature of 125 ℃. After the reaction is completed, the evaporating dish filled with macroporous resin is taken out, the resin is transferred into a conical flask, then the conical flask is placed on ice for cooling, then pure methanol is added for ultrasonic extraction for 30 minutes at normal temperature, and after filtration, ultrasonic is repeated for 2 times. Concentrating the methanol extract under reduced pressure at 40-45 ℃ and evaporating to dryness to obtain a salvianolic acid conversion product after conversion. The content of the salvianolic acid B and the salvianolic acid A is measured by an HPLC chromatography, and the content ratio of the salvianolic acid B to the salvianolic acid A in the product is about 1:3.5 (see FIG. 18). The total content of salvianolic acid B, salvianolic acid A, salvianic acid A, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid in the converted product is 63.62wt%, wherein the content of salvianolic acid A, salvianolic acid B and salvianic acid A accounts for 88wt% of the total amount of the six components.
Therefore, the conversion process parameter determination method can efficiently and accurately determine the optimal process parameters according to the required conversion products.
Claims (10)
1. A salvianolic acid conversion product which is converted into salvianolic acid a in proportion, wherein the salvianolic acid conversion product comprises: the total content of the six components in the salvianolic acid B, salvianolic acid A, salvianic acid A, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid is not less than 60wt%; wherein, salvianolic acid B, salvianolic acid A and salvianic acid A are used as main components, and account for more than 80wt% of the total of the six components; the content ratio of the salvianolic acid B to the salvianolic acid A can be set to be any ratio according to the requirement; the salvianolic acid conversion product is obtained by taking a sage plant medicinal material as a raw material, extracting salvianolic acid B, adsorbing the salvianolic acid B on macroporous adsorption resin, converting the salvianolic acid B into salvianolic acid A according to the proportion as required, and separating.
2. The method for preparing salvianolic acid conversion product proportionally converted into salvianolic acid A as claimed in claim 1, comprising the steps of:
(1) Extraction of salvianolic acid B
Water is used as solvent, and an ultrasonic extraction method is adopted: pulverizing Salvia plant, and sieving with 40 mesh sieve; adding distilled water, ultrasonically extracting for 20-60 minutes at 40-80 ℃, and filtering to obtain filtrate and dregs; repeating the ultrasonic extraction step for 1-5 times; mixing the filtrates to obtain salvianolic acid B extract;
(2) Preparation of macroporous adsorption resin with adsorbed salvianolic acid B
Loading all the salvianolic acid B extract onto a macroporous adsorption resin column at a flow rate of 0.5-1 mL per minute, eluting with distilled water with a retention volume of 1-5 times when the filtrate is completely discharged, and at a flow rate of 0.5-2 mL per minute to obtain macroporous adsorption resin adsorbed with salvianolic acid B; the macroporous adsorption resin is D101, HPD100, HPD300, HPD450, HPD600 or HPD700;
(3) Conversion of salvianolic acid conversion product
Wetting the macroporous adsorption resin adsorbed with salvianolic acid B with acidic solution with pH=1.0-5.0, placing the wet macroporous adsorption resin into a high-temperature high-pressure steam sterilization pot, and treating the wet macroporous adsorption resin for 1-5 hours at 105-125 ℃ to obtain macroporous adsorption resin adsorbed with salvianolic acid conversion products; the acid is hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, formic acid or acetic acid;
(4) Separation of salvianolic acid conversion products
Loading the macroporous adsorption resin adsorbed with the salvianolic acid conversion product into a column for eluting, or carrying out ultrasonic extraction on the macroporous adsorption resin adsorbed with the salvianolic acid conversion product; then concentrating under reduced pressure or concentrating under reduced pressure and lyophilizing to obtain salvianolic acid conversion product.
3. The method of claim 2, wherein the step (1) comprises one or more of the following conditions:
i. The Salvia plant is Saviae Miltiorrhizae radix, white flower Saviae Miltiorrhizae radix, yunnan Saviae Miltiorrhizae radix, radix Adenophorae, gansu Saviae Miltiorrhizae radix, brown Saviae Miltiorrhizae radix, radix Salviae Miltiorrhizae, and radix Salviae Miltiorrhizae;
ii. The mass of distilled water is 5-40 times of the mass of the medicinal material powder; preferably, the mass of distilled water is 20-40 times of the mass of the medicinal material powder; more preferably 30 times;
iii, the extraction temperature is 60-80 ℃, preferably 73 ℃;
iv, the extraction time is 30-50 minutes, preferably 41 minutes;
v, repeating the ultrasonic extraction step for 2-4 times, preferably 2 times;
vi, carefully examining the liquid-material ratio, the extraction time and the extraction temperature by a star point design-effect surface method to obtain the optimal liquid-material ratio, the extraction time and the extraction temperature technological parameters of the salvianolic acid B extraction technology.
4. The method of preparing a salvianolic acid conversion product which is proportionally converted into salvianolic acid a according to claim 2, wherein in the step (2), one or more of the following conditions are included:
i. the pretreatment method of the macroporous adsorption resin column comprises the following steps: soaking macroporous adsorption resin in 90-100deg.C ethanol for 20-30 hr, suspending with 90-100deg.C ethanol, and wet packing; washing the effluent with 90-100wt% ethanol until the effluent is not turbid after adding water with equal amount; flushing the column with distilled water until the effluent liquid has no alcohol smell; 3-4 times of retention volume (BV) of 0.5wt% HCl aqueous solution is used for flushing a column, and soaking is carried out for 2-4 hours; distilled water is washed until the pH of effluent liquid is neutral; 3-4 times of retention volume (BV) of 2wt% NaOH aqueous solution is used for flushing a column, and soaking is carried out for 2-4 hours; distilled water is flushed until the pH value of effluent liquid is neutral, thus finishing the pretreatment of the macroporous adsorption resin column;
ii. The macroporous adsorption resin is HPD450;
iii, the mass ratio of the dosage (dry weight) of the macroporous adsorption resin filler in the macroporous adsorption resin column to the medicine powder used for extracting the salvianolic acid B is 1 (0.5-3);
iv the retention volume of distilled water elution is 1 to 4 times, preferably 4 times.
5. The method of preparing a salvianolic acid conversion product which is proportionally converted into salvianolic acid a according to claim 2, wherein in the step (3), one or more of the following conditions are included:
i. the acid is hydrochloric acid or sulfuric acid, preferably hydrochloric acid;
ii. Acidic solution: the dry weight of the macroporous adsorption resin adsorbed with the salvianolic acid B is (1-2) 1 (v/w, mL/g), preferably 1:1 (v/w, mL/g);
iii, the pH of the acidic solution is 2.0-5.0, preferably the pH is 3.0;
iv, the reaction temperature is 115-125 ℃, preferably 118.5-125 ℃;
v, the reaction time is 2 to 5 hours, preferably 2 to 4 hours.
6. The method according to claim 2, wherein in the step (3), the pH, the conversion time and the conversion temperature of the acidic solution are determined according to the mass content ratio of salvianolic acid B to salvianolic acid a in the desired salvianolic acid conversion product according to the conversion process parameter determination method.
7. The method of preparing a salvianolic acid conversion product which is proportionally converted into salvianolic acid a according to claim 2, wherein in the step (4), one or more of the following conditions are included:
i. the eluting solvent or the extracting solvent is water, 10-90 wt% ethanol water solution, pure methanol or methanol-water mixed solution with any proportion; preferably, the eluting solvent or the extracting solvent is 30-60 wt% ethanol water solution or pure methanol; more preferably, the eluting solvent or the extracting solvent is 40wt% to 50wt% ethanol water solution or pure methanol;
ii. The volume of the eluting solvent is 2-8 reserved volumes; preferably, the elution solvent volume is 2 to 6 retention volumes; more preferably, the elution solvent volume is from 5 to 6 retention volumes;
iii, the reduced pressure concentration temperature is 40-60 ℃; preferably, the reduced pressure concentration temperature is 40-50 ℃; more preferably, the reduced pressure concentration temperature is 40-45 ℃;
iv, when the eluting solvent or the extracting solvent is pure methanol, concentrating the obtained eluent or extracting solution under reduced pressure until no solvent exists, and obtaining a salvianolic acid conversion product; or, when the eluting solvent or the extracting solvent is ethanol water solution or methanol-water mixed solution, concentrating the eluent or the extracting solution under reduced pressure to small volume, and lyophilizing to obtain salvianolic acid conversion product.
8. The method for determining transformation process parameters of the salvianolic acid transformation product proportionally transformed into salvianolic acid A according to claim 1, comprising the steps of:
(1) The pH value, the conversion time and the conversion temperature of an acid solution are taken as investigation factors, a three-factor five-level star point Design-effect curved surface method is adopted to measure the mass contents of salvianolic acid A and salvianolic acid B in each group of conversion products, the mass contents of the salvianolic acid A and the salvianolic acid B are taken as response values, data processing is carried out through Design expert8.0 software, and the salvianolic acid A (Y 1 ) Salvianolic acid B (Y) 2 ) The fit equation of the mass content of (c) and the investigation factor is as follows:
fitting equation Y 1 ：
Y 1 ＝270.35+66a+106.7b+126.25c+2.7ab+26.72ac+48.03bc-27.31a 2 +29.97b 2 +15.35c 2
(R 2 ＝0.9514，P＜0.001)
Fitting equation Y 2 ：
Y 2 ＝886.66-57.79a-141.41b-329.34c-154.25ab-39.3ac+134.42bc+2.99a 2 +86.55b 2 -25.2c 2
(R 2 ＝0.9220，P＜0.001)
Wherein: a is the pH of the acidic solution, b is the conversion time (hours), c is the conversion temperature (DEG C);
(2) According to the mass content ratio of salvianolic acid B and salvianolic acid A in the required conversion products, a fitting equation Y is utilized 1 And fitting equation Y 2 Analyzing by using Design Expert8.0 software, and predicting the optimal technological parameters corresponding to the mass content ratio of salvianolic acid B to salvianolic acid A in the required conversion products;
(3) Determining final optimal process parameters based on the predicted optimal process parameters of step (2).
9. The method for determining the transformation process parameters of the salvianolic acid transformation product which is proportionally transformed into salvianolic acid A according to claim 8, wherein the method comprises one or more of the following conditions:
i. In the step (2), the analysis method using Design Expert 8.0 software is as follows: fitting equation Y 1 Determining a data set I corresponding to the mass content of the salvianolic acid A within the range of 37.1-701.5 mug/mL from the corresponding data set; in fitting equation Y 2 Determining a data set II corresponding to the mass content of the salvianolic acid B in the range of 191.2-1662.5 mug/mL in the corresponding data set; according to the mass content ratio of salvianolic acid B and salvianolic acid A in the required conversion products, comparing and analyzing a data set I and a data set II, wherein two groups of values, which are closest to the corresponding values of the examination factors in the data set I and the data set II, are predicted optimal process parameters;
ii. In the step (3), the optimal technological parameters predicted in the step (2) are taken as base points, and the final optimal technological parameters are obtained through further optimization experiments.
10. Use of the salvianolic acid conversion product, which is proportionally converted into salvianolic acid a, according to claim 1, for the preparation of antithrombotic agents, in particular for the preparation of agents for the prevention of thrombosis in the treatment of atherosclerosis; preferably, in the salvianolic acid conversion product, the mass content ratio of salvianolic acid B to salvianolic acid A is 1:2.
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