CN116730958A - A kind of salvianolic acid conversion product that is converted into salvianolic acid A in proportion, preparation method, process parameter determination method and its use - Google Patents

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

A salvianolic acid conversion product converted into salvianolic acid A in proportion, a preparation method, a method for determining process parameters and its use

Technical Field

The invention relates to a salvianolic acid conversion product converted into salvianolic acid A in proportion, a preparation method, a process parameter determination method and use thereof, and belongs to the technical field of medicine.

Background Art

Salvia miltiorrhiza is a plant of the Lamiaceae family. Its medicinal parts are its dried roots and rhizomes. It is a commonly used drug in my country for cardiovascular diseases such as coronary heart disease. It has the effects of promoting blood circulation and removing blood stasis, and treating chest pain and heartache. The effective ingredients of Salvia miltiorrhiza are mainly water-soluble components and fat-soluble components. Among them, the water-soluble components are mainly salvianolic acid B, while the content of salvianolic acid A is very low. Studies have shown that salvianolic acid B and salvianolic acid A have a relatively good therapeutic effect on cardiovascular diseases such as coronary heart disease and angina pectoris. In clinical practice, there are already injections of salvianolic acid salts with salvianolic acid B as the main active ingredient, which are used to treat cardiovascular diseases such as coronary heart disease and angina pectoris. Tannic acid A also has a strong antioxidant effect, which can effectively inhibit oxidative stress damage to cell membranes and reduce myocardial ischemia-reperfusion injury; it has a good anti-platelet aggregation effect, which can effectively reduce blood viscosity, significantly increase the cAMP content in platelets, inhibit platelet adhesion on the collagen coating surface, and inhibit the binding of platelets to fibrinogen; it also has pharmacological activity against liver fibrosis and prevention of diabetic complications.

Salvianolic acid B and salvianolic acid A have synergistic effects, and their combination has better efficacy. Different ratios of salvianolic acid A and salvianolic acid B (4:1, 2:1, 1:1, 1:2, 1:4) can significantly reduce the size of myocardial infarction caused by myocardial ischemia-reperfusion injury in rats, and the effect of salvianolic acid A or salvianolic acid B alone is not as obvious as the combination of the two. Among all the ratio groups, the ratio of salvianolic acid A: salvianolic acid B of 2:1 has the most significant effect (Wang Guozhen et al., Protective effects of different ratios of salvianolic acid A/salvianolic acid B on myocardial ischemia-reperfusion injury in rats, Journal of Hebei Traditional Chinese Medicine, 2006, 21(2): 4-5, 12).

However, the content of salvianolic acid A in plants of the same genus such as Salvia miltiorrhiza or Salvia miltiorrhiza miltiorrhiza is not high, which is difficult to meet the requirements of industrial production, limiting the application of salvianolic acid A. At present, there are two main ways to convert salvianolic acid B into salvianolic acid A: (1) adjusting the pH value of the aqueous solution of salvianolic acid B to a certain value, and converting it into salvianolic acid A under high temperature and high pressure conditions (Wang Ying et al., Study on the process of synthesizing salvianolic acid A by high temperature and high pressure conversion, Journal of Dalian Polytechnic University, 2011, 30(6): 412-415; Jiang Feng et al., Method for converting salvianolic acid A in Salvia miltiorrhiza, Chinese Patent Medicine, 2018, 40(9): 2091-2096); (2) adding acid or alkali solution to the powder of medicinal materials of the same genus such as Salvia miltiorrhiza or Salvia miltiorrhiza miltiorrhiza, adjusting the pH value to a certain value, and converting salvianolic acid B into salvianolic acid A in plant tissues under high temperature and high pressure conditions (Sun Longru et al., A medicinal material with high content of salvianolic acid A, preparation method and use, ZL 2012 1 0334295.7). However, the above two methods still have some shortcomings: in method (1) (the former), both salvianolic acid B and the converted salvianolic acid A are in aqueous solution. Since the temperature of the aqueous solution is relatively high, salvianolic acid B and salvianolic acid A have poor stability in aqueous solution (especially at high temperature), which affects their yields; in addition, the optimal concentration of salvianolic acid B in aqueous solution is 5 mg/mL, which limits the feed amount of salvianolic acid A for each conversion. Method (2) (the latter): Although the conversion of salvianolic acid B to salvianolic acid A is carried out in the tissue of medicinal plants and has good stability, the volume of medicinal materials is relatively large, and the amount of medicinal materials processed in the actual operation process is limited, which affects the application of this method in industrial production processes.

Summary of the invention

In order to overcome the shortcomings of the prior art, the present invention provides a salvianolic acid conversion product converted into salvianolic acid A in proportion, a preparation method, a process parameter determination method and its use. The present invention extracts salvianolic acid B from salvia miltiorrhiza or other Salvia medicinal materials used as salvia miltiorrhiza, adsorbs it on a macroporous adsorption resin, and then converts salvianolic acid B into salvianolic acid A on the macroporous adsorption resin to prepare a salvianolic acid conversion product containing salvianolic acid A and salvianolic acid B; the method is simple, low in cost, has good product stability, low loss, and is suitable for industrial production.

According to the specific ratio requirements of salvianolic acid A and salvianolic acid B, the process parameters of the required conversion products can be optimized efficiently and accurately according to the process parameter determination method of the present invention. The method is simple, the number of tests is small, the result is highly accurate, and the feasibility is strong.

The salvianolic acid conversion product of the present invention can be used for the preparation of antithrombotic drugs and anti-atherosclerotic drugs.

The technical solution of the present invention is as follows:

A salvianolic acid conversion product converted into salvianolic acid A in proportion, the salvianolic acid conversion product comprising: salvianolic acid B, salvianolic acid A, danshensu, protocatechuic aldehyde, salvianolic acid D, rosmarinic acid, and the total content of the six components in the salvianolic acid conversion product is not less than 60wt%; wherein salvianolic acid B, salvianolic acid A, and danshensu are main components, accounting for more than 80wt% of the total amount of the six components; the content ratio of salvianolic acid B to salvianolic acid A can be set to any ratio as required; the salvianolic acid conversion product is prepared from a Salvia plant medicinal material as a raw material, and salvianolic acid B is extracted, adsorbed on a macroporous adsorption resin, salvianolic acid B is converted into salvianolic acid A in proportion as required, and then separated to obtain the salvianolic acid conversion product.

The method for preparing the above-mentioned salvianolic acid conversion product converted into salvianolic acid A in proportion comprises the steps of:

(1) Extraction of Salvianolic Acid B

The method uses water as solvent and ultrasonic extraction method: the medicinal materials of the genus Salvia are crushed and passed through a 40-mesh sieve; distilled water is added, ultrasonic extraction is performed at 40-80° C. for 20-60 minutes, and a filtrate and a medicinal residue are obtained by filtering; the obtained medicinal residue is subjected to the above ultrasonic extraction steps for 1-5 times; and the filtrate is combined to obtain a salvianolic acid B extract.

(2) Preparation of macroporous adsorption resin adsorbing salvianolic acid B

The whole of the salvianolic acid B extract is loaded onto a macroporous adsorption resin column at a flow rate of 0.5 to 1 mL per minute. After the filtrate has flowed out, it is eluted with 1 to 5 times the retention volume of distilled water at a flow rate of 0.5 to 2 mL per minute to obtain a 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 products

The macroporous adsorption resin adsorbed with salvianolic acid B is moistened with an acidic solution of pH=1.0-5.0, placed in a high-temperature and high-pressure steam sterilizer, and treated at 105-125°C for 1-5 hours to obtain a macroporous adsorption resin adsorbed with the conversion product of salvianolic acid; the acid is hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, formic acid or acetic acid.

(4) Separation of salvianolic acid conversion products

The macroporous adsorption resin adsorbed with the salvianolic acid conversion product is loaded into a column for elution, or the macroporous adsorption resin adsorbed with the salvianolic acid conversion product is subjected to ultrasonic extraction; then the salvianolic acid conversion product is obtained by vacuum concentration, or vacuum concentration and freeze drying.

Preferably according to the present invention, in step (1), the sage plant is salvia miltiorrhiza, white salvia miltiorrhiza, yunnan salvia miltiorrhiza, southern salvia miltiorrhiza, gansu salvia miltiorrhiza, brown-haired salvia miltiorrhiza, wild salvia miltiorrhiza, white-backed salvia miltiorrhiza, three-pair-leaf salvia miltiorrhiza or small salvia miltiorrhiza.

Preferably, according to the present invention, in step (1), the mass of distilled water is 5 to 40 times the mass of the medicinal material powder; preferably, the mass of distilled water is 20 to 40 times the mass of the medicinal material powder; more preferably, it is 30 times.

Preferably according to the present invention, in step (1), the extraction temperature is 60-80°C, preferably 73°C.

Preferably according to the present invention, in step (1), the extraction time is 30 to 50 minutes, preferably 41 minutes.

Preferably according to the present invention, in step (1), the ultrasonic extraction step is repeated 2 to 4 times, preferably 2 times.

Preferably, in step (1), the liquid-to-solid ratio, extraction time and extraction temperature are carefully investigated by central composite design-response surface methodology to obtain the optimal liquid-to-solid ratio, extraction time and extraction temperature process parameters of the salvianolic acid B extraction process.

Preferably, in step (2) of the present invention, the pretreatment method of the macroporous adsorption resin column is as follows: the macroporous adsorption resin is soaked in 90-100wt% ethanol for 20-30h, suspended in 90-100wt% ethanol, and wet column loading is performed; the 90-100wt% ethanol is used to flush the effluent until it does not become turbid after adding an equal amount of water; the column is flushed with distilled water until the effluent has no alcohol taste; the column is flushed with 3 to 4 times the retention volume (BV) of 0.5wt% HCl aqueous solution and soaked for 2 to 4h; the column is flushed with distilled water until the pH of the effluent is neutral; the column is flushed with 3 to 4 times the retention volume (BV) of 2wt% NaOH aqueous solution and soaked for 2 to 4h; the pretreatment of the macroporous adsorption resin column is completed.

Preferably according to the present invention, in step (2), the macroporous adsorption resin is HPD450.

Preferably, according to the present invention, in step (2), the mass ratio of the amount (dry weight) of the macroporous adsorption resin filler in the macroporous adsorption resin column to the medicinal material powder used for extracting salvianolic acid B is 1:(0.5-3).

Preferably according to the present invention, in step (2), the retention volume of distilled water elution is 1 to 4 times, preferably 4 times.

Preferably according to the present invention, in step (3), the acid is hydrochloric acid or sulfuric acid, preferably hydrochloric acid.

Preferably, according to the present invention, in step (3), the ratio of the acidic solution to the dry weight of the macroporous adsorption resin adsorbed with salvianolic acid B is (1-2):1 (v/w, mL/g), preferably 1:1 (v/w, mL/g).

Preferably according to the present invention, in step (3), the pH of the acidic solution is 2.0 to 5.0, preferably the pH is 3.0.

Preferably according to the present invention, in step (3), the reaction temperature is 115 to 125°C, preferably 118.5 to 125°C.

Preferably according to the present invention, in step (3), the reaction time is 2 to 5 hours, preferably 2 to 4 hours.

Preferably, in step (3), the process parameters of pH, conversion time and conversion temperature of the acidic solution can 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, according to the present invention, in step (4), the elution solvent or extraction solvent is water, 10wt% to 90wt% ethanol aqueous solution, pure methanol or a methanol-water mixed solution in any proportion; preferably, the elution solvent or extraction solvent is 30wt% to 60wt% ethanol aqueous solution or pure methanol; more preferably, the elution solvent or extraction solvent is 40wt% to 50wt% ethanol aqueous solution or pure methanol.

According to the preferred embodiment of the present invention, in step (4), the volume of the elution solvent is 2 to 8 retention volumes; preferably, the volume of the elution solvent is 2 to 6 retention volumes; more preferably, the volume of the elution solvent is 5 to 6 retention volumes.

Preferably, according to the present invention, in step (4), the reduced pressure concentration temperature is 40-60°C; preferably, the reduced pressure concentration temperature is 40-50°C; more preferably, the reduced pressure concentration temperature is 40-45°C.

Preferably, according to the present invention, in step (4), when the elution solvent or the extraction solvent is pure methanol, the obtained eluate or extract is concentrated under reduced pressure to a solvent-free state to obtain the salvianolic acid conversion product; or, when the elution solvent or the extraction solvent is an ethanol-water solution or a methanol-water mixed solution, the eluate or the extract is concentrated under reduced pressure to a small volume and freeze-dried to obtain the salvianolic acid conversion product.

The method for determining the conversion process parameters of the salvianolic acid conversion product converted into salvianolic acid A in proportion comprises the steps of:

(1) The pH value, conversion time and conversion temperature of the acidic solution were used as the investigation factors. The central point design with three factors and five levels and the effect surface method were used to determine the mass content of salvianolic acid A and salvianolic acid B in each group of conversion products. The mass content of salvianolic acid A and salvianolic acid B was used as the response value. The data were processed by Design Expert 8.0 software. The fitting equations of the mass content of salvianolic acid A (Y ₁ ) and salvianolic acid B (Y ₂ ) and the investigation factors were established 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), and c is the conversion temperature (°C).

(2) According to the desired mass content ratio of salvianolic acid B to salvianolic acid A in the transformation product, the Design Expert 8.0 software was used for analysis using the fitting equation _Y1 and the fitting equation _Y2 to predict the optimal process parameters corresponding to the mass content ratio of salvianolic acid B to salvianolic acid A in the transformation product;

(3) Determine the final optimal process parameters based on the optimal process parameters predicted in step (2).

Preferably, according to the present invention, in step (1), the mass content of salvianolic acid A and salvianolic acid B in each group of conversion products is determined, and the determination experiment can be carried out according to the existing method.

Preferably, in step (2), the method of using Design Expert 8.0 software for analysis is as follows: in the data set corresponding to the fitting equation _Y1 , determine the data set I corresponding to the mass content of salvianolic acid A in the range of 37.1 to 701.5 μg/mL; in the data set corresponding to the fitting equation _Y2 , determine the data set II corresponding to the mass content of salvianolic acid B in the range of 191.2 to 1662.5 μg/mL; according to the mass content ratio of salvianolic acid B to salvianolic acid A in the desired conversion product, compare and analyze the data set I and the data set II, and the two sets of values corresponding to the closest values of the factors under investigation in the data set I and the data set II are the predicted optimal process parameters.

According to the preferred embodiment of the present invention, in step (3), the optimal process parameters predicted in step (2) are used as the base point, and further optimization experiments are performed to obtain the final optimal process parameters. The predicted optimal process parameters are used as the base point, and the process parameters are fine-tuned near the base point, and further small-scale optimization experiments are performed to determine the final optimal process parameters; the optimization experiment can be performed according to the existing method.

The use of the salvianolic acid conversion product of the present invention can be used to prepare antithrombotic drugs, especially for preparing drugs 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 a pharmaceutically acceptable salt thereof of the present invention is combined with pharmaceutical excipients to prepare pharmaceutical preparations of different dosage forms for anti-thrombosis.

A pharmaceutical composition of an anti-thrombotic salvianolic acid conversion product comprises the salvianolic acid conversion product of the present invention or a pharmaceutically acceptable salt thereof, one or more pharmaceutically acceptable carriers or excipients, and an antioxidant.

Preferably according to the present invention, the pharmaceutically acceptable salt of the salvianolic acid conversion product is prepared by dissolving the salvianolic acid conversion product in distilled water, adjusting the pH to 6.8-8.0 with one of the alkalizing agents selected from the group consisting of NaOH, KOH, Na ₂ CO ₃ , K ₂ CO ₃ , NaHCO ₃ , KHCO ₃ , MgHPO ₃ , and Mg(H ₂ PO ₃ ) ₂ to form a sodium salt, a potassium salt, or a magnesium salt; the preferred alkalizing agent is NaHCO ₃ , KHCO ₃ , or MgH ₂ PO ₃ .

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

Preferably, according to the present invention, the pharmaceutical composition is one of tablets, capsules, granules and freeze-dried preparations for injection of the conversion product of salvianolic acid, which can be prepared according to conventional pharmaceutical production processes.

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

(1) In the preparation of tablets, capsules or granules of salvianolic acid conversion products, the following raw materials are used in amounts expressed as a percentage of the weight of the salvianolic acid conversion products:

Take the phenolic acid conversion product, add 2 to 4 times the weight of diluent I, 5wt% to 10wt% of a wetting agent, 4wt% to 15wt% of a disintegrant, granulate according to a conventional wet method, dry, granulate, and bag to obtain an granule; or add 0.5wt% to 3wt% of a lubricant to the granules after granulation, mix well, or compress to obtain tablets, or put into capsule shells to obtain capsules.

The diluent I is selected from starch, powdered sugar, dextrin or microcrystalline cellulose;

The wetting agent is selected from water or ethanol;

The above disintegrant is sodium carboxymethyl starch;

The lubricant is magnesium stearate or talc.

(2) In the preparation of enteric-coated tablets and capsules of salvianolic acid conversion products, the following raw materials are used in amounts expressed as a percentage by weight of the salvianolic acid conversion products:

Take the phenolic acid conversion product of salvianolic acid, add 3 to 5 times by weight of pregelatinized starch, 1 to 2 times by weight of polyvinyl pyrrolidone or hydroxypropyl cellulose, and appropriate amounts of microcrystalline cellulose, cross-linked sodium carboxymethyl cellulose, and talcum powder, mix well, make a soft material with a cross-linked polyvinyl pyrrolidone aqueous solution, granulate, dry, granulate, or press to obtain tablets, or put into enteric capsule shells to obtain enteric capsules.

(3) In the preparation of enteric-coated sustained-release tablets of salvianolic acid conversion products, the following raw materials are used in amounts expressed as a percentage by weight of the salvianolic acid conversion products:

Take the product of salvianolic acid conversion, add 1 to 2 times by weight of hydroxypropyl methylcellulose, 3 to 5 times by weight of microcrystalline cellulose, appropriate amounts of starch (or dextrin) and sodium carboxymethyl cellulose, spray with 10wt% polyvinyl pyrrolidone anhydrous ethanol to make a soft material, granulate, dry, granulate, add appropriate amount of magnesium stearate, mix, and tablet to obtain a sustained-release tablet core; dissolve 1 to 2 times by weight of hydroxypropyl methylcellulose phthalate or acrylic resin L-100 in ethanol, add appropriate amount of talcum powder, homogenize, and use it to coat the sustained-release tablet core to obtain the enteric-coated sustained-release tablet.

(4) Preparation of freeze-dried preparations for injection of salvianolic acid conversion products

Take the phenolic acid conversion product of salvia miltiorrhiza, add an appropriate amount of antioxidant, add 5% (w/v) mannitol to the total volume, dissolve in distilled water, fix the volume, filter and sterilize, package under aseptic conditions, freeze-dry, and then seal under nitrogen to obtain. When used, dissolve it in sterile water for injection or sodium bicarbonate water for injection.

(5) Preparation of freeze-dried preparation of salvianolic acid conversion product salt for injection

The product of the transformation of salvianolic acid is dissolved in distilled water, an appropriate amount of antioxidant is added, 5% (w/v) of mannitol is added, the pH is adjusted to 6.8-8.0 with an alkalinizing reagent KHCO ₃ aqueous solution, insoluble matter is removed by filtration, sterilized by filtration, dispensed into vials under sterile conditions, freeze-dried, and then sealed under nitrogen to obtain the product.

The salvianolic acid conversion product and the pharmaceutical composition preparation thereof of the present invention are used for the preparation of antithrombotic drugs, especially for preventing thrombosis in the treatment of atherosclerosis, and have a certain auxiliary therapeutic effect on atherosclerosis.

The preparation of the salvianolic acid conversion product of the present invention is carried out through systematic process parameter screening and optimization. First, a simple and easy aqueous solution ultrasonic extraction method is adopted, and the main factors affecting the extraction of salvianolic acid B, such as liquid-to-solid ratio, extraction time and extraction temperature, are carefully investigated through the star point design-effect surface method, and the process parameters such as liquid-to-solid ratio, extraction time and extraction temperature of the optimal extraction process are obtained. Secondly, considering the poor stability of salvianolic acid B and salvianolic acid A in hot water, salvianolic acid B in the water extract was adsorbed on a macroporous adsorption resin. After water elution to remove water-soluble impurities such as polysaccharides in the water extract, the macroporous adsorption resin adsorbed with a large amount of salvianolic acid B was directly placed under high temperature, high pressure and high humidity conditions to convert the adsorbed salvianolic acid B into phenolic acid components such as salvianolic acid A. The design-response surface method was used to process the experimental data using the content ratio of its main components salvianolic acid B and salvianolic acid A as an indicator, and the software Design Expert 8.0 was used to process the experimental data to obtain the fitting equations of salvianolic acid B and salvianolic acid A, respectively. Based on these two fitting equations, the conversion process parameters of different ratios of salvianolic acid B and salvianolic acid A in the desired salvianolic acid conversion product can be optimized. After a small-scale optimization experiment, the expected goal was achieved. Finally, the elution and drying methods of the treated macroporous adsorption resin were investigated. Through the above research, a preparation process of a salvianolic acid conversion product of the present invention is obtained; the present invention can arbitrarily optimize the conversion process parameters containing different content ratios of salvianolic acid B and salvianolic acid A, so as to obtain the desired conversion product. The invention meets the needs of conversion products of salvianolic acid B and salvianolic acid A in various different ratios, has a simple method, a simple process, low experimental cost, and is suitable for industrial production.

The salvianolic acid conversion product of the present invention has the effect of treating and preventing thrombosis, and is particularly suitable for the prevention and treatment of atherosclerosis.

Compared with the prior art, the advantages of the present invention are as follows:

(1) The present invention provides a method for efficiently and accurately optimizing the process parameters of the desired conversion product according to the requirements. According to the fitting equation of the present invention, combined with the ratio of different salvianolic acid B and salvianolic acid A in the desired salvianolic acid conversion product, the corresponding conversion process parameters are predicted by using Design Expert 8.0 software, and the optimal process parameters can be determined by further small amount of optimization experiments based on the predicted process parameters. The method is simple, greatly reduces the number of experiments, has high accuracy of the prediction results, and is highly feasible, achieving the purpose of controlling the content ratio of salvianolic acid A and salvianolic acid B in the product.

(2) The pre-conversion sample treatment of the present invention uses water as the extraction solvent and performs extraction under heating and ultrasonic conditions. The obtained water extract is directly loaded onto a macroporous adsorption resin, which has the advantages of simple operation and cost saving.

(3) Salvianolic acid B is extracted from medicinal materials, adsorbed on a macroporous adsorption resin, and then converted into salvianolic acid A under high temperature, high pressure and high humidity conditions. This has the following advantages: ① It avoids the problem of large volume caused by direct conversion using medicinal material powder; ② The converted salvianolic acid A and the unconverted salvianolic acid B are adsorbed on the macroporous resin, which is easier to cool and has better stability and less loss than if they were dissolved in a larger volume of aqueous solution.

(4) The conversion raw materials and conversion products used in the present invention are adsorbed on a solid macroporous adsorption resin, and the extraction and conversion process is also carried out on the macroporous adsorption resin, so that the entire production process such as loading, conversion, elution, and drying is easy to operate. The method is simple to operate and has low experimental cost. It is suitable for industrial production and lays a foundation for the large-scale preparation of salvianolic acid A and the development and utilization of salvianolic acid conversion products.

(5) The salvianolic acid conversion product prepared by the present invention mainly contains salvianolic acid A, salvianolic acid B, danshensu, protocatechuic aldehyde, salvianolic acid D, and rosmarinic acid. The salvianolic acid conversion product of the present invention can be used for the preparation of antithrombotic drugs and anti-atherosclerotic drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of the solid-liquid ratio on the extraction amount of salvianolic acid B in Experimental Example 1.

FIG. 2 shows the effect of extraction time on the extraction amount of salvianolic acid B in Experimental Example 1.

FIG. 3 shows the effect of extraction temperature on the extraction amount of salvianolic acid B in Experimental Example 1.

FIG. 4 is a response surface diagram of the extraction factors to the extraction amount of salvianolic acid B in Experimental Example 1.

FIG. 5 shows the effect of pH on the conversion of salvianolic acid B to salvianolic acid A in Experimental Example 2.

FIG. 6 shows the effect of conversion time on the conversion of salvianolic acid B to salvianolic acid A in Experimental Example 2.

FIG. 7 shows the effect of conversion temperature on the conversion of salvianolic acid B to salvianolic acid A in Experimental Example 2.

FIG8 is a response surface diagram of the conversion factors to the content of salvianolic acid A in Experimental Example 2.

FIG. 9 is a response surface diagram of the conversion factors to the content of salvianolic acid B in Experimental Example 2.

FIG. 10 is a gradient elution curve of salvianolic acid A in Experimental Example 3.

FIG. 11 is a methanol elution curve of salvianolic acid A in Experimental Example 3.

FIG. 12 is a comparison chart of thrombus weight in the inferior vena cava thrombosis model in Experimental Example 7.

FIG. 13 is a comparison diagram of thrombus areas in the inferior vena cava thrombosis model in Experimental Example 7. FIG.

FIG. 14 is a HPLC spectrum of the extract obtained by ultrasonic extraction of salvianolic acid B in Example 1.

FIG. 15 is a HPLC spectrum of the salvianolic acid conversion product obtained in Example 1.

FIG. 16 is a HPLC spectrum of the phenolic acid conversion product obtained in Example 2.

FIG. 17 is a HPLC spectrum of the phenolic acid conversion product obtained in Example 3.

FIG. 18 is a HPLC spectrum of the phenolic acid conversion product obtained in Example 4.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with the embodiments and test examples, but is not limited thereto. The raw materials and equipment used in the embodiments and test examples are all existing technologies and commercially available products.

Experimental Example 1: Study on the Optimal Extraction Process Parameters of Salvianolic Acid B from Salvia miltiorrhiza

The extraction method is as follows: using water as solvent and ultrasonic extraction method: crushing white salvia miltiorrhiza and passing it through a 40-mesh sieve; adding distilled water, then ultrasonic extraction, filtering to obtain a filtrate and a medicinal residue; repeating the above ultrasonic extraction steps (i.e. adding distilled water, then ultrasonic extraction, and filtering) twice on the obtained medicinal residue; combining the filtrates to obtain a salvianolic acid B extract.

In the experiment, the liquid-to-solid ratio (a), extraction time (b), and extraction temperature (c) were investigated factors, the number of extractions was 3, and a three-factor five-level central-point design-effect surface method was adopted. The content of salvianolic acid B in each group of Salvia miltiorrhiza extract was determined by conventional HPLC, and the content of salvianolic acid B was used as the response value. The data was processed by Design Expert 8.0 software to optimize and determine the optimal extraction process.

(1) Single factor study

The main factors affecting the extraction of salvianolic acid B are liquid-to-solid ratio, extraction time and extraction temperature. Two of them are fixed and the influence of the other phase is investigated. The results are shown in Figures 1, 2 and 3 (see attached drawings). The optimal extraction process parameters are: liquid-to-solid ratio of 1:30, extraction time of 40 min and temperature of 70°C.

(2) Experimental design

The three parameters of solid-liquid ratio of 1:30, extraction time of 40 min, and temperature of 70°C were determined as 0 level. The coding of each factor and level is shown in Table 1. Each factor and level was tested in a certain combination (see Table 2), and the content of salvianolic acid B in the salvia miltiorrhiza extract was determined. The results are shown in Table 2.

Table 1 Experimental factors and level coding table

Table 2 Central combination experimental design and results

Design-Expert software was used to perform regression model analysis on the experimental results. The results are shown in Table 3. The fitted binary multinomial regression equation was: Y=12.44+0.17a+0.18b+1.66c-0.059ab-0.12ac-0.076bc-0.56a ² -0.55b ² -0.64c ² , with P<0.01 and R ² =0.7682, indicating that the experimental design was reliable and the regression model established with the extraction amount of salvianolic acid B as the response value was significant.

Table 3 Variance analysis of regression model for the content of salvianolic acid B in salvia miltiorrhiza extract

Note: ^* P＜0.05, ^** P＜0.01, ^*** P＜0.001

The response surface diagram was established based on the above results, as shown in Figure 4. When the temperature was 60.00-77.32°C, the extraction time was 31.34-48.66 min, and the solid-liquid ratio was 21.34-38.66, the extraction amount of salvianolic acid B was the highest. After analysis by Design-Expert software, the optimal extraction process was selected as 30.08 times the extraction solvent, 72.86°C, and ultrasonic 40.71 min. Considering practical and industrial factors, the optimal extraction process can be changed to 30 times the extraction solvent, 73°C, ultrasonic 41 min, and extraction 3 times.

Experimental Example 2: Study on the optimal process parameters for converting salvianolic acid B into salvianolic acid A

The preparation method is as follows: according to the optimal extraction process parameters of Experimental Example 1, 8g of Salvia miltiorrhiza powder is used to extract the salvianolic acid B water extract, and the amount (dry weight) of the macroporous adsorption resin HPD450 filler is 10g. After pre-treatment of the macroporous adsorption resin, the salvianolic acid B water extract is all loaded onto the macroporous adsorption resin column at a flow rate of 0.5mL per minute, and after the filtrate is exhausted, it is eluted with 4 times the retention volume of distilled water at a flow rate of 1mL per minute to obtain a macroporous adsorption resin adsorbed with salvianolic acid B. Most of the water is removed from the macroporous adsorption resin adsorbed with salvianolic acid B, and it is moistened with 10mL of hydrochloric acid aqueous solution, and then the moistened macroporous adsorption resin is placed in a high-temperature and high-pressure steam sterilizer for reaction to obtain a macroporous adsorption resin adsorbed with salvianolic acid conversion products.

The pretreatment method of the macroporous adsorption resin column is as follows: the macroporous adsorption resin (HPD450) is soaked in 95wt% ethanol for 24h, suspended in 95wt% ethanol, and wet-packed; the column is flushed with 95wt% ethanol until the effluent does not become turbid when an equal amount of water is added; the column is flushed with distilled water until the effluent has no alcohol taste; the column is flushed with 0.5wt% HCl aqueous solution with 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral; the column is flushed with 2wt% NaOH aqueous solution with 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral, thus completing the preparation of the macroporous adsorption resin column.

The experiment used the pH, conversion time and conversion temperature of the acidic solution as the investigation factors, determined the pH to be 2, the conversion time to be 3 hours, and the conversion temperature to be 120°C as the 0 level, adopted the three-factor five-level central point design-effect surface method, and used the conventional HPLC determination method to determine the content of salvianolic acid A (SAA) and salvianolic acid B (SAB) in each group of conversion products, and used the content of salvianolic acid A and salvianolic acid B as the response value to conduct the following experiment. Among them, the preparation method of the methanol solution for HPLC determination of the conversion product is: placing the macroporous adsorption resin adsorbed with the conversion product of salvianolic acid in a conical flask, adding 50mL of methanol, ultrasonically extracting at room temperature for 30 minutes, filtering, transferring the filtrate to a 50mL volumetric flask, and fixing the volume with methanol.

(1) Single factor study

The main factors affecting the conversion of phenolic acid components are pH, conversion time, and conversion temperature. Two of them are fixed and the influence of the other phase is investigated. The results are shown in Figures 5, 6, and 7. In the figure, SAD is salvianolic acid D. When pH is 2, the conversion time is 3 hours, and the conversion temperature is 120°C, the conversion of salvianolic acid B to salvianolic acid A has the greatest impact. These three parameters are determined as 0 level. The coding of each factor and level is shown in Table 4. The experiment was carried out with each factor and level in a certain combination (see Table 5), and the content of salvianolic acid A and salvianolic acid B in the conversion product was determined. The results are shown in Table 5.

Table 4 Experimental factors and level coding table

Table 5 Central combination experimental design and results

Design Expert 8.0 software was used for data processing, and regression analysis was performed on the data of salvianolic acid A in the above experimental results. The results are shown in Table 6. The fitting equation Y ₁ of salvianolic acid A was obtained, the correlation coefficient R ² =0.9514, and the adjusted determination coefficient R ² _Adj =0.9076, indicating that the model can better reflect the true relationship between each factor and the content of salvianolic acid A, and its response surface diagram was established (Figure 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)

Design Expert 8.0 software was used for data processing, and regression analysis was performed on the data of salvianolic acid B in the above experimental results. The results are shown in Table 7. The fitting equation Y ₂ of salvianolic acid B was obtained, the correlation coefficient R ² =0.922, and the adjusted determination coefficient R ² _Adj =0.8518, indicating that the model can better reflect the true relationship between each factor and the content of salvianolic acid B, and its response surface diagram was established (Figure 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) Table 6 Variance analysis of regression model of salvianolic acid A

Note: ^* P＜0.05, ^** P＜0.01, ^*** P＜0.001

Table 7 Analysis of variance of regression model of salvianolic acid B

Note: ^* 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 desired transformation product, the Design Expert 8.0 software was used for analysis using the fitting equation Y ₁ and the fitting equation Y ₂ to predict the optimal process parameters corresponding to the mass content ratio of salvianolic acid B and salvianolic acid A in the desired transformation product. The method of using the Design Expert 8.0 software for analysis is as follows: in the data set corresponding to the fitting equation Y ₁ , the data set I corresponding to the mass content of salvianolic acid A in the range of 39.1 to 701.5 μg/mL was determined; in the data set corresponding to the fitting equation Y ₂ , the data set II corresponding to the mass content of salvianolic acid B in the range of 191.2 to 1662.5 μg/mL was determined; according to the mass content ratio of salvianolic acid B and salvianolic acid A in the desired transformation product, the data set I and the data set II were compared and analyzed, and the two sets of values corresponding to the closest values of the factors in the data set I and the data set II were the predicted optimal process parameters.

The final optimal process parameters are determined based on the predicted optimal process parameters. The predicted optimal process parameters are used as the base point, the process parameters are fine-tuned near the base point, and the final optimal process parameters are determined through further small-scale optimization tests according to the existing optimization experimental method.

The analysis results of Design Expert 8.0 software show that:

(1) When the content of salvianolic acid B in the desired conversion product is 2:1 (B2A1), according to Y _1, a conversion condition for salvianolic acid A is pH = 2.95, the conversion time is 3.96 hours, and the conversion temperature is 117.04°C; and according to Y _2, a conversion condition for salvianolic acid B is pH = 2.96, the conversion time is 3.99 hours, and the conversion temperature is 118.02°C. Taking the above-predicted optimal process parameters as the base point, the process parameters are fine-tuned near the base point. After further small-scale optimization tests, the optimal conversion conditions for B2A1 are optimized as follows: pH = 3, the conversion time is 4 hours, and the conversion temperature is 118.5°C.

(2) When the content of salvianolic acid B in the desired conversion product is 1:1 (B1A1), according to Y ₁ , a conversion condition for salvianolic acid A is pH = 2.19, the conversion time is 3.55 hours, and the conversion temperature is 122.69°C; and according to Y _2, a conversion condition for salvianolic acid B is pH = 2.17, the conversion time is 3.51 hours, and the conversion temperature is 123.75°C. Taking the above-predicted optimal process parameters as the base point, the process parameters are fine-tuned near the base point. After further small-scale optimization tests, the optimal conversion conditions for B1A1 are optimized as follows: pH = 3, the conversion time is 3.55 hours, and the conversion temperature is 123°C.

(3) When the content of salvianolic acid B in the desired conversion product is 1:2 (B1A2), according to Y _1, a conversion condition for salvianolic acid A is pH = 2.94, the conversion time is 3.98 hours, and the conversion temperature is 124.88°C; and according to Y ₂ , a conversion condition for salvianolic acid B is pH = 3, the conversion time is 3.99 hours, and the conversion temperature is 124.92°C. Taking the above-predicted optimal process parameters as the base point, the process parameters are fine-tuned near the base point. After further small-scale optimization tests, the optimal conversion conditions of B1A2 are optimized as follows: pH = 3, the conversion time is 4 hours, and the conversion temperature is 124°C.

Test Example 3: Investigation of elution solvent concentration

The macroporous adsorption resin adsorbed with the conversion product of salvianolic acid was loaded into a column, and eluted with 5 retention volumes of 10wt%, 20wt%, 30wt%, 40wt%, 50wt%, 60wt%, 70wt%, 80wt%, and 90wt% ethanol aqueous solution, respectively. The results are shown in Figure 10. The elution effect of 40wt% and 50wt% ethanol aqueous solution was the best. In order to obtain elution conditions with higher elution rates of salvianolic acid A, salvianolic acid B, and danshensu, 40wt%, 50wt% ethanol aqueous solution and pure methanol were further used as elution solvents to investigate the elution again. The amount of elution solvent used was 5 retention volumes. The results are shown in Table 8. The results show that the elution efficiency of salvianolic acid A, salvianolic acid B, and danshensu is higher when pure methanol is used for elution with 5 retention volumes. When methanol was used for elution, an eluent was collected for each retention volume, and the methanol elution curve of salvianolic acid A was drawn. The result is shown in Figure 11. The volume of methanol eluent was 6 retention volumes, and almost all the conversion products of salvianolic acid were eluted. Therefore, the elution effect of 40% to 50% ethanol or methanol was the best.

Table 8 Investigation of different elution solvents

Test Example 4: Determination of in vivo antiplatelet aggregation rate

Male Wistar rats, weighing approximately 220-250 g, were housed for one week at a temperature of 25 ± 1 °C. Wistar rats were randomly divided into 10 groups, 6 rats in each group, and the grouping was as follows: blank control group: 0.5% sodium carboxymethylcellulose (CMC-Na); B2A1 (content of salvianolic acid B: content of salvianolic acid A = 2:1) low-dose group: 50 mg·kg ^-1 ; B2A1 medium-dose group: 100 mg·kg ^-1 ; B2A1 high-dose group: 150 mg·kg ^-1 ; B1A1 (content of salvianolic acid B: content of salvianolic acid A = 1:1) low-dose group: 50 mg·kg ^-1 ; B1A1 medium-dose group: 100 mg·kg ^-1 ; B1A1 high-dose group: 150 mg·kg ^-1 ; B1A2 (content of salvianolic acid B: content of salvianolic acid A = 1:2) low-dose group: 50 mg·kg ^-1 ; B1A2 medium-dose group: 100 mg·kg ^-1 ; B1A2 high-dose group: 150 mg kg ^-1 ; once a day, for seven consecutive days, blood was collected 5 hours after the last administration. Rats were anesthetized with 10% chloral hydrate, and blood was collected from the abdominal aorta into a negative pressure blood collection tube with 3.8% sodium citrate 1:9 anticoagulation. Rat plasma was centrifuged at 1000 rpm for 10 minutes to prepare platelet-rich plasma (PRP), and the remaining plasma was centrifuged at 3000 rpm for 10 minutes to prepare platelet-poor plasma (PPP).

The platelet aggregation rate of the experimental rat plasma collected above was determined by turbidimetry at 37°C. PPP was used to adjust to zero, PRP was incubated for 15 minutes, and the inducer ADP was added to determine the inhibitory effect of each group of plasma on ADP-induced platelet aggregation. The experimental results are shown in Table 9.

The test results showed that after adding various compounds at different concentrations, they could significantly inhibit ADP-induced platelet aggregation compared with the blank group. Specifically, compound B1A2 could significantly reduce ADP-induced platelet aggregation rate at a concentration of 50 mg kg ^-1 . The antiplatelet aggregation rate of the B2A1, B1A1 and B1A2 groups showed a good dose-effect relationship, and the antiplatelet aggregation effect gradually increased with the increase of the dosage. The maximum inhibition rates of the three groups were 61%, 59% and 47%, respectively, among which the B1A2 group had the best effect.

Table 9 Effects of conversion products containing different ratios of salvianolic acid B and salvianolic acid A on platelet aggregation rate

^* P＜0.05, ^** P＜0.01, ^*** P＜0.001 compared with blank control group.

Test Example 5: In vivo anticoagulation test

The method of administration to rats and the method of preparing plasma were the same as those in Experimental Example 4.

The prepared reagents were placed on a set semi-automatic coagulation analyzer for preheating. The platelet-poor plasma (PPP) prepared above was taken and the prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) and thrombin time (TT) were detected according to the instrument operating instructions. The results are shown in Table 10.

The results showed that the three doses of B2A1, B1A1 and B1A2 had no significant effect on PT and TT, but could significantly reduce APTT. Among them, the B1A2 150mg·kg ^-1 dose group had the best effect.

Table 10 Anticoagulant effect of conversion products containing different ratios of salvianolic acid B and salvianolic acid A

^* P＜0.05, ^** P＜0.01, ^*** P＜0.001 compared with blank control group.

Test Example 6: Test on Inhibition of Macrophage Foam Formation

Human acute monocytic leukemia THP-1 cells were inoculated in a six-well plate. THP-1 cells grew in suspension and were cultured in a 1640 medium containing 15% FBS and 1% penicillin-streptomycin mixture. They were usually passaged once every four days. THP-1 cells with good growth were inoculated in a culture plate. PMA (100 ng/mL) was added to the 1640 medium containing 3% FBS for 24 hours. Fresh 1640 medium containing 3% FBS was replaced. ox-LDL (80 μg/mL) was added for 48 hours of induction. The medium was discarded, washed once with PBS, and replaced with a fresh 1640 medium containing 3% FBS. Different concentrations (10, 20, 30 μg/mL) of salvianolic acid conversion product aqueous solution (content of salvianolic acid B: content of salvianolic acid A = 1:2) were added for 24 hours. The culture medium was discarded, the cells were washed twice with PBS, and the cell suspension was collected with a cell scraper; the supernatant was discarded by centrifugation (1000 rpm, 10 min), isopropanol was added, and the cells were ultrasonically disrupted under ice bath conditions; the supernatant was collected by centrifugation, and the intracellular TC, TG, and FC contents were determined according to the instructions of the kit, and CE=TC-FC was further obtained from TC and FC. The experimental results are shown in Table 11.

The effect of salvianolic acid conversion products on the lipid content in foam cells was determined by a kit, with atorvastatin as a positive control. The results are shown in Table 11. After the addition of ox-LDL, the contents of TC, TG, FC, and CE in the cells were significantly increased, with significant differences compared with the blank group (P < 0.001). The salvianolic acid conversion products significantly reduced the contents of TC, TG, and CE in the cells at concentrations of 10, 20, and 30 μg/mL, indicating that the salvianolic acid conversion products have a significant inhibitory effect on the formation of THP-1 macrophage foam cells and can be used to treat atherosclerosis.

Table 11 Anti-macrophage foaming effect of salvianolic acid transformation products

Note: ^b cells were treated with PMA (100 ng/mL) for 24 h and then incubated with ox-LDL (80 μg/mL) for 48 h.

^* P＜0.05, ^** P＜0.01, ^*** P＜0.001 compared with the ox-LDL (80 μg/mL) group.

^### P＜0.001 compared with the blank group.

Experimental Example 7: Inferior vena cava thrombosis model

Wistar rats, male, 220-250g, were randomly divided into 5 groups, 6 rats in each group. The grouping was as follows: blank control group: 0.5% sodium carboxymethylcellulose aqueous solution; aspirin group (25mg/kg); salvianolic acid conversion product (salvianolic acid B content: salvianolic acid A content = 1:2) low-dose group (12.5mg/kg), medium-dose group (25mg/kg), and high-dose group (50mg/kg). All drugs were administered orally once a day for seven consecutive days. One hour after the last administration, 10% chloral hydrate anesthesia, supine fixation, abdominal wall cut open, inferior vena cava separated, inferior vena cava ligated at the intersection of left renal vein and inferior vena cava, abdominal wall sutured, abdominal cavity reopened 6 hours later, blood vessels were ligated again 2cm below the ligation, emboli were removed, residual blood was absorbed with filter paper, and then weighed with an electronic balance and data were recorded. The thrombus tissue specimen of the inferior vena cava was taken out, fixed with 4% paraformaldehyde for 1 hour, frozen and sectioned, stained with HE, and observed under a microscope. The test results are shown in Figures 12 and 13.

Figure 12 is a comparison of thrombus weight in the inferior vena cava thrombosis model, and Figure 13 is a comparison of thrombus area in the inferior vena cava thrombosis model. As shown in the figure, compared with the model group, the thrombus weight and thrombus area in the aspirin group and the three groups of low, medium and high salvianolic acid conversion products were significantly reduced, and with the increase in the dosage of salvianolic acid conversion products, the thrombus weight and thrombus area gradually decreased, indicating that salvianolic acid conversion products can significantly inhibit the formation of inferior vena cava thrombosis and show a significant anti-thrombotic effect.

Example 1: Preparation of a salvianolic acid conversion product having a content ratio of salvianolic acid B to salvianolic acid A of 2:1

The Danshen medicinal material was crushed so that the particle size of the particles could pass through a 40-mesh sieve to obtain Danshen powder. 8 g of Danshen powder was taken, and 30 times the amount of distilled water was added, and ultrasonic extraction was performed at 73°C for 41 minutes, and the filtrate and the residue were filtered; the obtained residue was subjected to the above ultrasonic extraction steps twice; the filtrate was combined to obtain the salvianolic acid B extract, and the HPLC content analysis was performed under conventional determination conditions. The determination results are shown in Figure 14.

The preparation method of the macroporous adsorption resin column is as follows: the macroporous adsorption resin (HPD300) is soaked in 95wt% ethanol for 24h, suspended in 95wt% ethanol, and wet-packed, the mass ratio of the macroporous adsorption resin filler (dry weight) to the medicinal powder used for extracting salvianolic acid B is 1:1; the column is flushed with 95wt% ethanol until the effluent does not become turbid when adding an equal amount of water; the column is flushed with distilled water until the effluent has no alcohol taste; the column is flushed with 0.5wt% HCl aqueous solution with 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral; the column is flushed with 2wt% NaOH aqueous solution with 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral, and the preparation of the macroporous adsorption resin column is completed.

All of the above-mentioned salvianolic acid B extract was loaded onto a macroporous adsorption resin column at a flow rate of about 1 mL per minute. After the filtrate was exhausted, it was eluted with distilled water of 4 times the retention volume at a flow rate of about 1 mL per minute. After the loading was completed, the solvent in the macroporous adsorption resin column was drained to obtain a macroporous adsorption resin adsorbed with salvianolic acid B extract.

The content ratio of salvianolic acid B to salvianolic acid A in the conversion product was set to 2:1. The fitting equation _Y1 and the fitting equation Y2 in the conversion process parameters of Experimental Example ₂ were used for analysis, and the optimal conversion process parameters with a content ratio of salvianolic acid B to salvianolic acid A of 2:1 in the conversion process were selected: infiltration with a hydrochloric acid aqueous solution with a pH of 3.0 and treatment at 118.5°C for 4 hours.

The conversion process using the above process parameters is as follows: remove most of the water from the macroporous adsorption resin adsorbed with the salvianolic acid B extract, place it in an evaporating dish, moisten it with a hydrochloric acid aqueous solution with a pH of 3.0 equal to the macroporous adsorption resin filler (dry weight) (v/w), and then place it in a high-pressure steam sterilizer, treat it at 118.5°C for 4 hours to obtain a treated macroporous adsorption resin, which adsorbs the converted salvianolic acid conversion product. Then, the treated macroporous adsorption resin is loaded into a glass column, eluted with 40wt% ethanol water for 5 retention volumes, concentrated under reduced pressure at 40-45°C until there is no alcohol taste, and freeze-dried to obtain the converted salvianolic acid conversion product. The content of salvianolic acid B and salvianolic acid A is determined by HPLC chromatography. The content of salvianolic acid B and salvianolic acid A in the product is about 2:1 (see Figure 15). The total content of six components, namely, salvianolic acid B, salvianolic acid A, danshensu, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid in the conversion product is 60.69wt%, of which the content of salvianolic acid A, salvianolic acid B and danshensu accounts for 96wt% of the total content of the six components.

From the above, the method for determining the conversion process parameters of the present invention can efficiently and accurately determine the optimal process parameters according to the desired conversion products.

Example 2: Preparation of a salvianolic acid conversion product having a content ratio of salvianolic acid B to salvianolic acid A of 1:1

The Danshen medicinal material is crushed so that the particle size can pass through a 40-mesh sieve to obtain Danshen powder. Take 10g of Danshen powder, add 30 times the amount of distilled water, ultrasonically extract at 70°C for 40 minutes, filter to obtain a filtrate and a medicinal residue; repeat the ultrasonic extraction steps twice with the obtained medicinal residue; combine the filtrate to obtain a salvianolic acid B extract.

The preparation method of the macroporous adsorption resin column is as follows: the macroporous adsorption resin (D101) is soaked in 95wt% ethanol for 24h, suspended in 95wt% ethanol, and wet-packed, the mass ratio of the amount of macroporous adsorption resin filler (dry weight) to the mass ratio of the medicinal powder used for extracting salvianolic acid B is 5:4; the column is flushed with 95wt% ethanol until the effluent does not become turbid when adding an equal amount of water; the column is flushed with distilled water until the effluent has no alcohol taste; the column is flushed with 0.5wt% HCl aqueous solution with 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral; the column is flushed with 2wt% NaOH aqueous solution with 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral, and the preparation of the macroporous adsorption resin column is completed.

All of the above-mentioned salvianolic acid B extract was loaded onto a macroporous adsorption resin column at a flow rate of about 0.5 mL per minute. After the filtrate was exhausted, it was eluted with 1 retention volume of distilled water at a flow rate of about 0.5 mL per minute. After the loading was completed, the solvent in the macroporous adsorption resin column was drained to obtain a macroporous adsorption resin adsorbed with salvianolic acid B extract.

The content ratio of salvianolic acid B to salvianolic acid A in the conversion product was set to 1:1. The fitting equation _Y1 and the fitting equation Y2 in the conversion process parameters of Experimental Example ₂ were used for analysis, and the optimal conversion process parameters with a content ratio of salvianolic acid B to salvianolic acid A of 1:1 in the conversion process were selected: infiltration with a hydrochloric acid aqueous solution with a pH of 3.0 and treatment at 123°C for 3.55 hours.

The conversion process using the above process parameters is as follows: remove most of the water from the above macroporous adsorption resin adsorbed with the salvianolic acid B extract, place it in an evaporating dish, moisten it with a pH=3.0 hydrochloric acid aqueous solution equal to the macroporous adsorption resin filler (dry weight) (v/w), and then place it in a high-pressure steam sterilizer, treat it at 123°C for 3.55 hours to obtain a treated macroporous adsorption resin, which adsorbs the converted salvianolic acid conversion product. Then, the treated macroporous adsorption resin is loaded into a glass column, eluted with 50wt% methanol water for 4 retention volumes, concentrated under reduced pressure at 40-45°C until there is no alcohol taste, and freeze-dried to obtain the converted salvianolic acid conversion product. The content of salvianolic acid A and salvianolic acid B is determined by HPLC chromatography. The content of salvianolic acid B and salvianolic acid A in the product is about 1:1 (see Figure 16). The total content of six components, namely, salvianolic acid B, salvianolic acid A, danshensu, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid in the conversion product is 60.41wt%, of which the content of salvianolic acid A, salvianolic acid B and danshensu accounts for 93wt% of the total content of the six components.

From the above, the method for determining the conversion process parameters of the present invention can efficiently and accurately determine the optimal process parameters according to the desired conversion products.

Example 3: Preparation of a salvianolic acid conversion product having a content ratio of salvianolic acid B to salvianolic acid A of about 1:2

The Danshen medicinal material is crushed so that the particle size can pass through a 40-mesh sieve to obtain Danshen powder. Take 10g of Danshen powder, add 30 times the amount of distilled water, ultrasonically extract at 70°C for 40 minutes, filter to obtain a filtrate and a medicinal residue; repeat the ultrasonic extraction steps twice with the obtained medicinal residue; combine the filtrate to obtain a salvianolic acid B extract.

The preparation method of the macroporous adsorption resin column is as follows: the macroporous adsorption resin (HPD450) is soaked in 95wt% ethanol for 24h, suspended in 95wt% ethanol, and wet-packed, the mass ratio of the amount of macroporous adsorption resin filler (dry weight) to the mass ratio of the medicinal powder used for extracting salvianolic acid B is 5:4; the column is flushed with 95wt% ethanol until the effluent does not become turbid when adding an equal amount of water; the column is flushed with distilled water until the effluent has no alcohol taste; the column is flushed with 0.5wt% HCl aqueous solution of 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral; the column is flushed with 2wt% NaOH aqueous solution of 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral, and the preparation of the macroporous adsorption resin column is completed.

All of the above-mentioned salvianolic acid B extract was loaded onto a macroporous adsorption resin column at a flow rate of about 0.5 mL per minute. After the filtrate was exhausted, it was eluted with 3 times the retention volume of triple distilled water at a flow rate of about 0.5 mL per minute. After the loading was completed, the solvent in the macroporous adsorption resin column was drained to obtain a macroporous adsorption resin adsorbed with salvianolic acid B extract.

The content ratio of salvianolic acid B to salvianolic acid A in the conversion product was set to 1:2. The fitting equation _Y1 and the fitting equation Y2 in the conversion process parameters of Experimental Example ₂ were used for analysis, and the optimal conversion process parameters with a content ratio of salvianolic acid B to salvianolic acid A of 1:2 in the conversion process were selected: infiltration with a hydrochloric acid aqueous solution with a pH of 3.0 and treatment at 124°C for 4 hours.

The conversion process using the above process parameters is as follows: remove most of the water from the above macroporous adsorption resin adsorbed with the extract of salvianolic acid B, place it in a crucible, moisten it with an equal amount (v/w) of hydrochloric acid aqueous solution with pH=3.0 to the macroporous adsorption resin filler (dry weight), and then transfer it to a high-temperature and high-pressure steam sterilizer to react for 4 hours at 124°C. After the reaction is completed, take out the crucible containing the macroporous resin, place it on ice for cooling, and then put the macroporous adsorption resin into a glass column, elute it with pure methanol, and elute 6 retention volumes. The methanol eluate is concentrated and evaporated to dryness under reduced pressure at 40-45°C to obtain the converted salvianolic acid conversion product. The content of salvianolic acid B and salvianolic acid A is determined by HPLC chromatography. The content ratio of salvianolic acid B to salvianolic acid A in the product is about 1:2 (see Figure 17). The total content of six components, namely, salvianolic acid B, salvianolic acid A, danshensu, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid in the conversion product is 64.85wt%, of which the content of salvianolic acid A, salvianolic acid B and danshensu accounts for 94wt% of the total content of the six components.

From the above, the method for determining the conversion process parameters of the present invention can efficiently and accurately determine the optimal process parameters according to the desired conversion products.

Example 4: Preparation of a salvianolic acid conversion product having a content ratio of salvianolic acid B to salvianolic acid A of about 1:3.5

The Danshen medicinal material is crushed so that the particle size can pass through a 40-mesh sieve to obtain Danshen powder. Take 10g of Danshen powder, add 30 times the amount of distilled water, ultrasonically extract at 70°C for 40 minutes, filter to obtain a filtrate and a medicinal residue; repeat the ultrasonic extraction steps twice with the obtained medicinal residue; combine the filtrate to obtain a salvianolic acid B extract.

The preparation method of the macroporous adsorption resin column is as follows: the macroporous adsorption resin (HPD600) is soaked in 95wt% ethanol for 24h, suspended in 95wt% ethanol, and wet-packed, the mass ratio of the amount of macroporous adsorption resin filler (dry weight) to the mass ratio of the medicinal powder used for extracting salvianolic acid B is 1:1; the column is flushed with 95wt% ethanol until the effluent does not become turbid when adding an equal amount of water; the column is flushed with distilled water until the effluent has no alcohol taste; the column is flushed with 0.5wt% HCl aqueous solution with 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral; the column is flushed with 2wt% NaOH aqueous solution with 4 times the retention volume (BV), soaked for 3h, and flushed with distilled water until the pH of the effluent is neutral, and the preparation of the macroporous adsorption resin column is completed.

All of the above-mentioned salvianolic acid B extract was loaded onto a macroporous adsorption resin column at a flow rate of about 1 mL per minute. After the filtrate was exhausted, it was eluted with 2 retention volumes of distilled water at a flow rate of about 0.5 mL per minute. After the loading was completed, the solvent in the macroporous adsorption resin column was drained to obtain a macroporous adsorption resin adsorbed with salvianolic acid B extract.

The content ratio of salvianolic acid B to salvianolic acid A in the conversion product was set to 1:3.5. The fitting equation _Y1 and the fitting equation Y2 in the conversion process parameters of Experimental Example ₂ were used for analysis, and the optimal conversion process parameters with a content ratio of salvianolic acid B to salvianolic acid A of 1:3.5 in the conversion process were selected: infiltration with a hydrochloric acid aqueous solution with a pH of 3.5 and treatment at 125°C for 4 hours.

The conversion process using the above process parameters is as follows: remove most of the water from the macroporous adsorption resin adsorbed with the salvianolic acid B extract, place it in an evaporating dish, moisten it with a hydrochloric acid aqueous solution with a pH of 3.5 equal to the macroporous adsorption resin filler (dry weight) (v/w), and then transfer the evaporating dish to a high-temperature and high-pressure steam sterilizer, and react for 4 hours at 125°C. After the reaction is completed, take out the evaporating dish containing the macroporous resin, transfer the resin to a conical flask, and then cool it on ice, then add pure methanol to ultrasonically extract at room temperature for 30 minutes, filter it, and repeat the ultrasonic twice. The methanol extract is concentrated and evaporated to dryness under reduced pressure at 40-45°C to obtain the converted salvianolic acid conversion product. The content of salvianolic acid B and salvianolic acid A is determined by HPLC chromatography. The content ratio of salvianolic acid B to salvianolic acid A in the product is about 1:3.5 (see Figure 18). The total content of six components, namely, salvianolic acid B, salvianolic acid A, danshensu, protocatechuic aldehyde, salvianolic acid D and rosmarinic acid in the conversion product is 63.62wt%, of which the content of salvianolic acid A, salvianolic acid B and danshensu accounts for 88wt% of the total content of the six components.

From the above, the method for determining the conversion process parameters of the present invention can efficiently and accurately determine the optimal process parameters according to the desired conversion products.

Claims (10)

1. A salvianolic acid conversion product that is converted into salvianolic acid A in proportion, characterized in that the salvianolic acid conversion product includes: salvianolic acid B, salvianolic acid A, salvianoside, and protocatechuic aldehyde , salvianolic acid D, rosmarinic acid, and the total content of the six components in the salvianolic acid conversion product is not less than 60wt%; among them, salvianolic acid B, salvianolic acid A, and salvianoside are the main components, accounting for six More than 80wt% of the total amount of these ingredients; the content ratio of salvianolic acid B and salvianolic acid A can be set to any ratio as needed; the salvianolic acid conversion product is made from Salvia plant medicinal materials as raw materials, and is extracted Salvianolic acid B, adsorb salvianolic acid B on macroporous adsorption resin, convert salvianolic acid B into salvianolic acid A in proportion as needed, and then separate and obtain the salvianolic acid conversion product. 2. The preparation method of the salvianolic acid conversion product converted into salvianolic acid A in proportion as claimed in claim 1, comprising the steps: (1) Extraction of salvianolic acid B Using water as the solvent, use the ultrasonic extraction method: crush the medicinal materials of the genus Sage and pass them through a 40-mesh sieve; add distilled water, conduct ultrasonic extraction at 40 to 80°C for 20 to 60 minutes, and filter to obtain the filtrate and medicinal residue; the resulting medicine Repeat the above ultrasonic extraction step 1 to 5 times with the residue; combine the filtrate to obtain salvianolic acid B extract; (2) Preparation of macroporous adsorption resin adsorbed with salvianolic acid B Load all the salvianolic acid B extract onto the macroporous adsorption resin column at a flow rate of 0.5 to 1 mL per minute. When the filtrate is exhausted, elute with distilled water 1 to 5 times the retention volume at a flow rate of 0.5 to 2 mL per minute. , obtain a 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 products The macroporous adsorption resin adsorbed with salvianolic acid B is moistened with an acidic solution of pH=1.0~5.0, placed in a high-temperature and high-pressure steam sterilization pot, and treated at 105~125°C for 1~5 hours to obtain the adsorbed salvia miltiorrhiza. Macroporous adsorption resin for phenolic acid conversion products; the acid is hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, formic acid or acetic acid; (4) Isolation of salvianolic acid conversion products The macroporous adsorption resin adsorbed with the salvianolic acid conversion product is loaded into a column for elution, or the macroporous adsorption resin adsorbed with the salvianolic acid conversion product is subjected to ultrasonic extraction; and then concentrated under reduced pressure, or concentrated under reduced pressure, Freeze-drying to obtain salvianolic acid conversion product. 3. The preparation method of salvianolic acid conversion product proportionally converted into salvianolic acid A according to claim 2, characterized in that, in step (1), one or more of the following conditions are included: i. Plants of the genus Salvia include Salvia miltiorrhiza, Salvia miltiorrhiza, Yunnan Salvia miltiorrhiza, South Salvia miltiorrhiza, Gansu Salvia miltiorrhiza, Salvia miltiorrhiza, Salvia miltiorrhiza, Salvia miltiorrhiza, Salvia miltiorrhiza or Salvia miltiorrhiza; ii. The quality of distilled water is 5 to 40 times the mass of medicinal powder; preferably, the quality of distilled water is 20 to 40 times the mass of medicinal powder; more preferably, it is 30 times; iii. The extraction temperature is 60-80°C, preferably 73°C; iv. The extraction time is 30 to 50 minutes, preferably 41 minutes; v. Repeat the ultrasonic extraction step 2 to 4 times, preferably 2 times; vi. Through the star point design-effect surface method, the liquid-to-material ratio, extraction time and extraction temperature were carefully investigated, and the optimal liquid-to-material ratio, extraction time and extraction temperature process parameters of the salvianolic acid B extraction process were obtained. 4. The method for preparing the salvianolic acid conversion product proportionally converted into salvianolic acid A according to claim 2, characterized in that, in step (2), one or more of the following conditions are included: i. The pretreatment method for the macroporous adsorption resin column is as follows: soak the macroporous adsorption resin in 90-100wt% ethanol for 20-30h, suspend it in 90-100wt% ethanol, and perform wet column packing; use 90-100wt% Flush the column with ethanol until the effluent remains turbid by adding an equal amount of water; flush the column with distilled water until the effluent has no alcohol smell; flush the column with 0.5wt% HCl aqueous solution 3 to 4 times the retention volume (BV), and soak for 2 to 4 hours; distilled water Flush until the pH of the effluent is neutral; flush the column with 2wt% NaOH aqueous solution 3 to 4 times the retention volume (BV), and soak for 2 to 4 hours; flush with distilled water until the pH of the effluent is neutral, that is, the macroporous adsorption resin column is completed. preprocessing; ii. The macroporous adsorption resin is HPD450; iii. The mass ratio of the amount of macroporous adsorption resin filler in the macroporous adsorption resin column (dry weight) to the medicinal powder used to extract 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 for preparing a salvianolic acid conversion product that is proportionally converted into salvianolic acid A according to claim 2, characterized in that step (3) includes one or more of the following conditions: 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 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 to 5.0, preferably pH 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 for preparing a salvianolic acid conversion product that is proportionally converted into salvianolic acid A according to claim 2, characterized in that in step (3), the pH, conversion time, and conversion temperature process parameters of the acidic solution can be based on The mass content ratio of salvianolic acid B and salvianolic acid A in the required salvianolic acid conversion product is determined according to the conversion process parameter determination method of the present invention. 7. The method for preparing the salvianolic acid conversion product proportionally converted into salvianolic acid A according to claim 2, characterized in that step (4) includes one or more of the following conditions: i. The elution solvent or extraction solvent is water, 10wt% ~ 90wt% ethanol aqueous solution, pure methanol or a methanol-water mixed solution in any proportion; preferably, the elution solvent or extraction solvent is 30wt% ~ 60wt% ethanol aqueous solution or pure Methanol; more preferably, the elution solvent or extraction solvent is 40wt% to 50wt% ethanol aqueous solution or pure methanol; ii. 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; iii. The vacuum concentration temperature is 40-60℃; preferably, the vacuum concentration temperature is 40-50℃; more preferably, the vacuum concentration temperature is 40-45℃; iv. When the elution solvent or extraction solvent is pure methanol, the resulting eluate or extraction solution is concentrated under reduced pressure until it is solvent-free to obtain the salvianolic acid conversion product; or, the elution solvent or extraction solvent is an ethanol aqueous solution or a methanol-water mixed solution. When the eluate or extract is concentrated under reduced pressure to a small volume, the salvianolic acid conversion product is obtained by freeze-drying. 8. The method for determining the conversion process parameters of the salvianolic acid conversion product converted into salvianolic acid A in proportion as claimed in claim 1, comprising the steps: (1) Using the pH, transformation time, and transformation temperature of the acidic solution as investigation factors, a three-factor five-level star point design-effect surface method was used to determine the mass content of salvianolic acid A and salvianolic acid B in each group of transformation products. , and using the mass content of salvianolic acid A and salvianolic acid B as the response value, the data was processed through Design Expert8.0 software to establish the mass of salvianolic acid A (Y ₁ ) and salvianolic acid B (Y ₂ ) respectively. The fitting equation of content and investigation factors is as follows: Fit 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) Fit 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) Among them: a is the pH of the acidic solution, b is the conversion time (hours), c is the conversion temperature (℃); (2) According to the mass content ratio of salvianolic acid B and salvianolic acid A in the required transformation products, use the fitting equation Y ₁ and the fitting equation Y ₂ to analyze and predict the required transformation products using Design Expert 8.0 software The optimal process parameters corresponding to the mass content ratio of salvianolic acid B and salvianolic acid A; (3) Determine the final optimal process parameters based on the optimal process parameters predicted in step (2). 9. The method for determining the conversion process parameters of the salvianolic acid conversion product that is proportionally converted into salvianolic acid A according to claim 8, characterized in that it includes one or more of the following conditions: i. In step (2), the analysis method using Design Expert 8.0 software is as follows: In the data set corresponding to the fitting equation Y ₁ , it is determined that the mass content of salvianolic acid A is in the range of 37.1 to 701.5 μg/mL. Data set I; in the data set corresponding to the fitting equation Y ₂ , it is determined that the mass content of salvianolic acid B is in the range of 191.2 ~ 1662.5 μg/mL and the corresponding data set II; according to the required salvianolic acid in the conversion product B. Salvianolic acid A mass content ratio, comparative analysis of data set I and data set II. The two sets of values corresponding to the closest values of the investigated factors in data set I and data set II are the predicted optimal process parameters; ii. In step (3), based on the optimal process parameters predicted in step (2), the final optimal process parameters are obtained through further optimization experiments. 10. The use of the salvianolic acid conversion product proportionally converted into salvianolic acid A according to claim 1, which is characterized in that it can be used to prepare anti-thrombotic drugs, especially for the preparation of preventive and therapeutic treatments for atherosclerosis. A drug for thrombosis; preferably, in the salvianolic acid conversion product, the mass content ratio of salvianolic acid B and salvianolic acid A is 1:2.