A preparation method of salvianolic acid A

Abstract

The present invention relates to a method for preparing salvianolic acid A, comprising the following steps: purifying salvianolic acid A purified and extracted using medium- and low-pressure resins using normal-phase silica gel, crystallizing, extracting, and freeze-drying. This method is characterized by low cost, simple separation and purification, ease of operation, and susceptibility to industrial production, ultimately yielding high-content, high-purity salvianolic acid A.

Description

Technical Field

[0001] The present invention relates to the field of drug preparation, and in particular to a method for preparing salvianolic acid A. Background Art

[0002] Salvianolic acid A is a water-soluble phenolic acid compound contained in the medicinal material of Salvia miltiorrhiza, a plant of the Lamiaceae family. Its content in the medicinal material is extremely low. It was first isolated from Salvia miltiorrhiza by Professor Li Lian-Niang in 1984 (Li Lian-Niang, et al. Planta Medica, 1984, 50: 227). Salvianolic acid A is one of the strongest antioxidant compounds known to date and has a wide range of pharmacological activities in antioxidant, myocardial ischemia protection, anti-thrombotic, neuroprotective, anti-liver fibrosis, and prevention and treatment of diabetes (Du Guanhua, Qiu Yue, Zhang Juntian. Protective effect of salvianolic acid A on myocardial ischemia-reperfusion injury in rats [J]. 1995, (10): 731; Ren Zihua. Research progress on the pharmacological effects of salvianolic acid A [J]. Medical Information, 2013 (1): 322-322, etc.). Its structural formula is as follows:

[0003]

[0004] Numerous patents and literature exist regarding methods for preparing salvianolic acid A, such as: 1) CN101041620A discloses a method for preparing salvianolic acid A from salvia miltiorrhiza. The salvianolic acid A is extracted from medicinal herbs and then directly purified. However, due to the extremely low salvianolic acid A content in the medicinal herbs, the final product produced by this method has low purity and high production costs. Furthermore, the extraction solvents used are often Class II solvents, which pose safety risks. These factors are all detrimental to industrial production and preparation. 2) CN103570548A discloses a method for preparing salvianolic acid A. This process addresses the source of salvianolic acid A, but because the extract is directly heated to react without purification and impurity removal through alcohol precipitation, there are the following disadvantages: the extract contains substances such as proteins, colloids, and polysaccharides, which react with salvianolic acid A during the heating reaction, increasing the difficulty of subsequent separation and reducing the service life of the chromatographic filler, thereby increasing production costs. 3) The method for preparing salvianolic acid A disclosed in CN103242161A lacks in-depth research into the pathway and conditions for producing salvianolic acid A through a heating reaction. Furthermore, the purity of the salvianolic acid A produced is only 90%, far below the current requirement for APIs in new drug research. 4) In the method disclosed in CN103044251A, a catalyst (such as zinc chloride) is added during the conversion process. The salvianolic acid A is dried in a microwave vacuum and purified using polyamide, a polymer compound that is often in powder form. The resulting particles are uneven in size, requiring filtration to remove fine powder and activation treatment to remove residual impurities before use. This complicates the process and is not conducive to industrial production.

[0005] In summary, although the extraction and yield of salvianolic acid A have been improved to a certain extent in the existing technology, there are still problems such as difficulty in separation and purification, cumbersome operation, unfavorable for industrial / industrial production, high cost, and low purity and content of the final product. Summary of the Invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides a preparation method for salvianolic acid A with low cost, simple separation and purification, easy operation, and conducive to industrial production, thereby ultimately obtaining high-content and high-purity salvianolic acid A.

[0007] The present invention provides a method for preparing high-purity and high-content salvianolic acid A, comprising the following steps:

[0008] (1) Medium and low pressure resin purification step: Take the converted solution of salvianolic acid A and -1 ~3CV·h -1 The flow rate passes through the qualified styrene type macroporous adsorption resin column, and the resin dosage is M 丹酚酸A :M 大孔吸附树脂 =1:35~1:60, diameter-to-height ratio =1:5~1:10. -1 ~4CV·h -1 flow rate, eluting with water for 3-5CV, 15-30% acidic ethanol (pH = 2.5-3.5) for 8-12CV, and 35-55% acidic ethanol (pH = 2.5-3.5) for 4-8CV, collecting the 35-55% acidic ethanol eluate in fractions, and detecting the purity of each fraction by HPLC. The fractions with a purity of ≥80% were combined to obtain the purified solution of the macroporous adsorption resin;

[0009] (2) Salvianolic acid A extraction step: taking the macroporous adsorption resin purified solution from step (1), concentrating under reduced pressure, and extracting with tert-butyl methyl ether or ethyl acetate to obtain an extract;

[0010] (3) Normal phase silica gel purification: The extract of step (2) was concentrated to a concentration of 150 mg / ml to 250 mg / ml of salvianolic acid A, mixed with silica gel, and loaded onto a silica gel column for chromatography. A cyclohexane: tert-butyl methyl ether mixed solvent was used for gradient elution to obtain a normal phase silica gel purified solution;

[0011] (4) Crystallization: The normal phase silica gel purified solution of step (3) was concentrated, dissolved in water, and concentrated to a concentration of 100 mg / ml to 300 mg / ml of salvianolic acid A in the solution. The pH value was adjusted to 3.5 to 3.8. The sample was placed in an environment (0 to 15°C), allowed to stand for crystallization, and filtered to obtain a crude primary crystal;

[0012] (5) Recrystallization: The crude product of the primary crystallization of step (4) is recrystallized and purified:

[0013] (6) Extraction, concentration, and drying: Dissolve the crystals obtained in step (5) in water, add 10% to 20% acid solution dropwise while stirring, and adjust the pH to 1.5 to 3.0; extract with tert-butyl methyl ether several times (for example, 3 times), combine the extracts to obtain a crystalline extract; concentrate under reduced pressure, and freeze-dry in a vacuum.

[0014] Preferably, the salvianolic acid A conversion liquid is obtained by extracting salvianolic acid B, precipitating with alcohol, and converting salvianolic acid B;

[0015] More preferably, wherein:

[0016] (a) Extraction: Crush and sieve Danshen medicinal materials, soak in water at 75-85°C, and extract 2-4 times (e.g., 3 times with 6-10 times the amount of water) to obtain a salvianolic acid B extract; or crush and sieve Danshen medicinal materials, and extract under reflux with 20-40% ethanol to obtain a salvianolic acid B extract;

[0017] and / or, (b) alcohol precipitation: taking the extract from step (1), concentrating it under reduced pressure at T≤65°C (e.g., 50°C-60°C) to a relative density of 1.10-1.15 (preferably 1.10-1.15), combining the concentrates; adding 90-95% ethanol to adjust the ethanol concentration in the solution to 70%-75%, allowing it to stand at 10°C-35°C for more than 24 hours, filtering, and concentrating the filtrate under reduced pressure to a ethanol concentration in the solution of ≤5%, thereby obtaining an alcohol precipitation concentrate;

[0018] And / or, (c) conversion step: take the alcohol precipitation concentrate from step (2), add purified water to dilute to a salvianolic acid B concentration of about 5-30 mg / ml (preferably 5-15 mg / ml), adjust the pH to 3.5-4.0, react at 110-130° C. for 2.5-4.5 hours, cool, and centrifuge to obtain a salvianolic acid A conversion solution.

[0019] Preferably, in step (1), the styrene-type macroporous adsorption resin is HPD-100S, LX-1180, D101B, HPD-300, D101-I or HPD-600, preferably the filler particle size is 0.30 to 1.25 mm, and the pressure resistance is 2 to 10 bar (preferably 2 to 5 bar);

[0020] Preferably, in step (1), the concentration of the salvianolic acid A conversion solution is 1.59 mg / ml to 6.56 mg / ml;

[0021] Preferably, in step (1), the loading flow rate of the salvianolic acid A conversion solution is 1CV. -1 ~2CV·h -1 ;

[0022] Preferably, in step (1), the eluent flow rate is 2CV flow rate -1 ~3CV·h -1 ;

[0023] Preferably, in step (2), the purified solution of salvianolic acid A macroporous adsorption resin is concentrated under reduced pressure at T≤85°C to a solution ethanol concentration of ≤15%, extracted multiple times with tert-butyl methyl ether, and the extracts are combined and dehydrated;

[0024] More preferably, the mixture is extracted three times with tert-butyl methyl ether, and / or anhydrous sodium sulfate is added and allowed to stand for more than 2 hours to remove water.

[0025] Preferably, in step (3), the silica gel used for purification is 300-400 mesh, with a particle size of 40-150 μm (preferably 40-60 μm, more preferably 40-45 μm), and a pressure of 500-1500 bar (preferably 800-1200 bar);

[0026] Preferably, in step (3), the amount of silica gel used for mixing the sample satisfies: M 丹酚酸A :M 硅胶 The ratio is 1:2.0 to 1:3.0 (preferably 1:2.0 to 1:2.5), and the amount of silica gel used for column chromatography meets the following requirements: 丹酚酸A :M 硅胶 1:10-1:50, preferably 1:15-1:20;

[0027] Preferably, in step (3), the volume ratio of cyclohexane: tert-butyl methyl ether mixed solvent is 6:4 to 2:8, preferably 5:5 to 3:7;

[0028] Preferably, in step (3), the gradient elution is: eluting with cyclohexane: tert-butyl methyl ether (5:5-4:6) for 8-12CV, eluting with cyclohexane: tert-butyl methyl ether (4:6-3:7) for 4-8CV, with a flow rate of 2.5CV·h -1 ~7.5CV·h -1 , and collect the eluate of cyclohexane: tert-butyl methyl ether (4:6~3:7) in fractions.

[0029] Preferably, step (3) is: the extract of step (2) is concentrated under reduced pressure at T≤50°C to a concentration of salvianolic acid A of 130mg / ml to 250mg / ml (preferably 150-210mg / ml), and 丹酚酸A :M 硅胶(300-400目) =1:2~1:3.0 (preferably 1:2~1:2.5) and silica gel for sample mixing, take the sample silica gel column, silica gel column chromatography dosage M 丹酚酸A :M 硅胶(300-400目) The mixture was eluted with cyclohexane: tert-butyl methyl ether (5:5-4:6) for 8-12CV and cyclohexane: tert-butyl methyl ether (4:6-3:7) for 4-8CV, with a flow rate of 2.5CV·h -1 ~7.5CV·h -1, collecting the cyclohexane: tert-butyl methyl ether (4:6-3:7) eluate in fractions, detecting the purity of each fraction by HPLC, and combining the salvianolic acid A with a purity of ≥90% to obtain salvianolic acid A purified by normal phase silica gel;

[0030] Preferably, in step (4), the concentration of salvianolic acid A is 100 mg / ml to 300 mg / ml, preferably 150 mg / ml to 250 mg / ml;

[0031] Preferably, in step (4), the pH regulator is one or more selected from meglumine, sodium bicarbonate, sodium carbonate, potassium bicarbonate, magnesium bicarbonate, magnesium carbonate, calcium hydroxide, calcium bicarbonate, and calcium carbonate, preferably sodium bicarbonate;

[0032] Preferably, in step (4), the crystallization is allowed to stand for more than 12 hours;

[0033] Preferably, step (4) comprises: concentrating the normal phase silica gel purified solution of step (3) to dryness under reduced pressure at T≤50°C, adding purified water or water for injection to dissolve the solution to a concentration of about 80 mg / ml of salvianolic acid A, concentrating the solution under reduced pressure at T≤65°C to a concentration of 150 mg / ml to 250 mg / ml of salvianolic acid A, adjusting the pH to 3.5 to 3.8, placing the sample in an environment (0 to 15°C), standing for crystallization for more than 12 hours, and filtering to obtain a crude primary crystal.

[0034] Preferably, step (5) is:

[0035] The crude product of the primary crystallization in step (4) is dissolved in purified water at a temperature of 50-65°C, filtered while hot, and the volume is adjusted to a concentration of 200-250 mg / ml of salvianolic acid A. The sample is placed in an environment of 0-15°C, stirred and crystallized for more than 24 hours, the crystals are filtered, and dried.

[0036] Preferably, in step (6), the water is purified water or water for injection;

[0037] Preferably, in step (6), the concentration of salvianolic acid A in the solution is 25 mg / ml to 50 mg / ml;

[0038] Preferably, in step (6), the acid solution is hydrochloric acid or phosphoric acid solution;

[0039] Preferably, in step (6), the pH is adjusted to 1.5-2.0;

[0040] Preferably, the reduced pressure concentration and freeze-vacuum drying steps are as follows: the recrystallized extract is concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A is above 200 mg / ml, the solution is diluted to a concentration of salvianolic acid A of 150 mg / ml to 200 mg / ml, and freeze-vacuum drying is performed;

[0041] Preferably, step (6) comprises: adding purified water or water for injection to the crystals obtained in step (5) to dissolve the solution containing salvianolic acid A at a concentration of 25 mg / ml to 50 mg / ml, adding 10% to 20% acid solution dropwise while stirring, and adjusting the pH value to 1.5 to 2.0; extracting with tert-butyl methyl ether several times (for example, 3 times), combining the extracts to obtain a crystallization extract; concentrating the recrystallized extract under reduced pressure at T≤65°C until the salvianolic acid A concentration is above 200 mg / ml, diluting the solution to a salvianolic acid A concentration of 150 mg / ml to 200 mg / ml, and freeze-drying in a vacuum.

[0042] Preferably, the freeze-vacuum drying conditions in step (6) are:

[0043]

[0044] Beneficial technical effects of the present invention:

[0045] 1. Use specific processes and solvents to obtain high-purity and high-content products:

[0046] (1) The present invention uses an industrial medium- and high-pressure chromatography system with normal-phase silica gel to purify salvianolic acid A. Silica gel with a particle size of 300-400 mesh is used as a filler. Gradient elution is performed using a mixed solvent of cyclohexane and tert-butyl methyl ether in different volume ratios within the range of 6:4 to 2:8 (e.g., 6:4, 5:5, and 3:7). This effectively removes pigments, pyrogenic, and allergenic impurities; further removes colloidal impurities and phenolic acid compounds, removes impurities adjacent to salvianolic acid A, and enriches and purifies salvianolic acid A. Purification on normal-phase silica gel allows the purity of salvianolic acid A to reach over 95%, and the content to reach over 90%.

[0047] (2) In order to further improve the content, purity and stability of the raw material of salvianolic acid A, a crystallization purification method was used to purify salvianolic acid A. According to the structural characteristics of salvianolic acid A, salvianolic acid A belongs to the polyphenolic acid class of compounds, and can be precipitated by changing the solubility by adjusting the pH value of the sample solution. An alkaline substance (such as a saturated sodium bicarbonate aqueous solution) was creatively used as a pH regulator to neutralize the carboxyl group in the structure of salvianolic acid A, so that salvianolic acid A crystallized and precipitated in the form of ions to obtain salvianolic acid A salt. It was then dissolved in water, and the pH was adjusted to 1.5-2.0 with a dilute acidic solution to free salvianolic acid A in the form of molecules. It was then extracted with a solvent (such as tert-butyl methyl ether or ethyl acetate). The purity of salvianolic acid A was above 98% and the yield was above 80%. This shows that the process parameters for purifying salvianolic acid A by crystallization are scientific and reasonable, the operation process is simple, and it is conducive to industrial production. The production cost is low, and the purity and content of the final product meet the expected goals.

[0048] (3) In order to further improve the purity of the final salvianolic acid A and control the impurity limit, further recrystallization can be performed according to product requirements. The crystals are dissolved in water and recrystallized to obtain a higher purity salvianolic acid A salt. The salvianolic acid A salt is dissolved and dispersed with purified water. The pH of the sample solution is adjusted with dilute hydrochloric acid solution to free salvianolic acid A in molecular form. The salvianolic acid A in the sample solution is extracted with tert-butyl methyl ether. After concentration and freeze-vacuum drying, a high-purity (99.5% or more) and high-content (99.5% or more) salvianolic acid A raw material is obtained. The raw material has few impurities and can be used as a chemical raw material. The freeze-vacuum drying is beneficial to ensure the quality of the product.

[0049] 2. Produces significantly better results than existing technologies:

[0050] (1) Compared with the method in CN103044252A, the present invention adopts an industrial medium- and high-pressure chromatography system for purification, shortening the separation and purification time to 1 / 3 to 1 / 2 of the original time, reducing labor intensity, and ensuring that the yield of the product in each process is increased by more than 10% compared with the atmospheric pressure column.

[0051] (2) CN103044251A uses polyamide purification. Polyamide filler is a high-value-added fine polymer filler, mostly in powder form with uneven particle size. Before use, it is necessary to filter out the fine powder and perform activation treatment to remove residual impurities in the product itself. The pretreatment process is relatively complicated and is not conducive to industrial production. Catalysts are used, and microwave drying easily changes the material structure, which is not conducive to industrial production. The present invention does not use the polyamide filler purification method commonly used in the prior art, but instead uses a double extraction combined with a crystallization method, which is simple to operate and low in cost. No catalyst is used, and the production process is highly controllable, which is conducive to industrial production.

[0052] (3) The solvent used in CN103044251A is n-pentane-tert-butyl methyl ether. N-pentane is highly flammable, and its vapor can form an explosive mixture with steam. It is very easy to burn and explode when exposed to open flames and high heat. It reacts violently with oxidants and may even cause combustion, which poses a high risk in production operations. High concentrations can cause mild irritation to the eyes and respiratory mucosa and anesthesia, or even loss of consciousness. Chronic effects are mild irritation to the eyes and respiratory tract, which can cause mild dermatitis, requiring the adoption of higher-level effective labor protection measures. The solvents used in the process of the present invention (cyclohexane, tert-butyl methyl ether or ethyl acetate) are safe, reusable, and environmentally friendly.

[0053] 3. The salvianolic acid A obtained in the present invention has high purity (above 99.5%), high content (above 99.5%), and few impurities, and can be used as a chemical raw material. The preparation method of the present invention has a scientific and reasonable process route design, fully considering both pharmaceutical economics and practical production feasibility, and has good process reproducibility, which can achieve continuous large-scale production. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0055] The medicinal material is derived from the dried root and rhizome of Salvia miltiorrhiza Bge. of the Labiatae family. The medicinal material and the solvents and reagents mentioned in the following examples can all be directly purchased from the market.

[0056] Example 1-4: Water extraction and alcohol precipitation of salvianolic acid B

[0057] Example 1

[0058] Take 4 portions of Danshen medicinal material (decoction pieces, thick slices), 100g each, crush them, pass through a four-mesh sieve, and extract them three times with water at 78°C ± 2°C (10-fold, 8-fold, and 6-fold volume), each extraction for 1 hour. The extracts are concentrated under reduced pressure at 60°C to a relative density of 1.15. The concentrates are combined and adjusted to an ethanol concentration of 70% with 95% ethanol. The solution is allowed to settle at 25°C ± 2°C for 36 hours, filtered, and the filtrate is concentrated under reduced pressure at T ≤ 65°C (60°C) to an ethanol concentration of ≤ 5% (4%) to obtain an alcohol-precipitated concentrate.

[0059] Example 2

[0060] Take 4 portions of Danshen medicinal material (short segment), 100g each, crush, pass through a four-mesh sieve, add water and soak at 75℃±2℃ for three times (10 times the amount, 8 times the amount, and 6 times the amount), each extraction time for 1.5 hours. The extract is concentrated under reduced pressure at 50℃ to a relative density of 1.05. The concentrates are combined and adjusted to an ethanol concentration of 75% by adding 90% ethanol. The solution is allowed to stand at 35℃±2℃ for 24 hours to allow alcohol precipitation. Filter, and the filtrate is concentrated under reduced pressure at T≤65℃ (50℃) to an ethanol concentration of ≤5% (5%) to obtain an alcohol precipitation concentrate.

[0061] Example 3

[0062] Take 4 portions of Danshen medicinal material (long section), 100g each, crush them, pass through a four-mesh sieve, and extract them three times with water at 85℃±2℃ (10 times the volume, 8 times the volume, and 6 times the volume), each extraction time for 2 hours. The extracts are concentrated under reduced pressure at 55℃ to a relative density of 1.10. The concentrates are combined and adjusted to an ethanol concentration of 72% by adding 92% ethanol. The solution is allowed to settle at 10℃±2℃ for 25 hours, filtered, and the filtrate is concentrated under reduced pressure at T≤65℃ (55℃) to an ethanol concentration of ≤5% (3%) to obtain an alcohol-precipitated concentrate.

[0063] Example 4

[0064] Take 4 portions of Danshen medicinal material (decoction pieces, thick slices), 100g each, crush them, pass through a four-mesh sieve, and add water to soak at 82℃±2℃ for three extractions (10 times the amount, 8 times the amount, and 6 times the amount), each extraction for 1.5 hours. The extract is concentrated under reduced pressure at 50℃ to a relative density of 1.15. The concentrates are combined and adjusted to an ethanol concentration of 75% by adding 95% ethanol. The solution is allowed to stand at 20℃±2℃ for 48 hours for alcohol precipitation. Filter the filtrate and concentrate under reduced pressure at T≤65℃ (55℃) to an ethanol concentration of ≤5% (4%) to obtain an alcohol precipitation concentrate.

[0065] Examples 5-8: Conversion Steps

[0066] Example 5

[0067] The alcohol precipitate concentrate of Example 1 was diluted with purified water to a salvianolic acid B concentration of about 10 mg / ml, the pH was adjusted to 3.8, the mixture was reacted at 120° C. for 3.5 h, cooled, and centrifuged to obtain a salvianolic acid A conversion solution.

[0068] Example 6

[0069] The alcohol precipitate concentrate of Example 2 was diluted with purified water to a salvianolic acid B concentration of about 30 mg / ml, the pH was adjusted to 4.0, and the mixture was reacted at 110° C. for 4.5 h, cooled, and centrifuged to obtain a salvianolic acid A conversion solution.

[0070] Example 7

[0071] The alcohol precipitate concentrate of Example 3 was diluted with purified water to a salvianolic acid B concentration of about 5 mg / ml, the pH was adjusted to 3.5, and the mixture was reacted at 130° C. for 2.5 h, cooled, and centrifuged to obtain a salvianolic acid A conversion solution.

[0072] Example 8

[0073] The alcohol precipitate concentrate of Example 4 was diluted with purified water to a salvianolic acid B concentration of about 15 mg / ml, the pH was adjusted to 4.0, and the mixture was reacted at 115° C. for 3 h, cooled, and centrifuged to obtain a salvianolic acid A conversion solution.

[0074] Examples 9-12: Purification of Salvianolic Acid A by Low-Pressure Resin

[0075] Example 9

[0076] Take the converted solution of salvianolic acid A in Example 5, where the concentration of salvianolic acid A is 3.00 mg / ml, and heat it at 1.5 CV·h -1 The flow rate passes through the qualified styrene type macroporous adsorption resin column, the macroporous adsorption resin is HPD-100S, the filler particle size is 0.3~1.25mm, the pressure is 2bar, M 丹酚酸A :M 大孔吸附树脂 =1:35, diameter-to-height ratio = 1:5; 3CV·h -1 The product was eluted with purified water for 4CV, 20% acidic ethanol (pH = 3.0) for 9CV, three wastes were treated, and 45% acidic ethanol (pH = 3.0) was used for 5CV. The 45% acidic ethanol eluate was collected in fractions, and the purity of each fraction was detected by HPLC. The purity of the combined salvianolic acid A was ≥80%, and the purified salvianolic acid A macroporous adsorption resin solution was obtained.

[0077] Example 10

[0078] Take the converted solution of salvianolic acid A in Example 6, the concentration of salvianolic acid A is 1.59 mg / ml, and -1 The flow rate passes through the qualified styrene type macroporous adsorption resin column, the macroporous adsorption resin is HPD-100S, the filler particle size is 0.3~1.25mm, the pressure is 5bar, M 丹酚酸A :M 大孔吸附树脂 =1:50, diameter-to-height ratio = 1:10; 2CV·h -1 The product was eluted with purified water for 3CV, 30% acidic ethanol (pH = 3.5) for 8CV, three wastes were treated, and 55% acidic ethanol (pH = 3.5) was used for 4CV. The 55% acidic ethanol eluate was collected in fractions, and the purity of each fraction was detected by HPLC. The purity of the combined salvianolic acid A was ≥80%, and the purified salvianolic acid A macroporous adsorption resin solution was obtained.

[0079] Example 11

[0080] Take the converted solution of salvianolic acid A in Example 7, the concentration of salvianolic acid A is controlled at 6.56 mg / ml, and the mixture is heated at 1 CV·h -1 The flow rate passes through the qualified styrene type macroporous adsorption resin column, the macroporous adsorption resin is LX-1180, the filler particle size is 0.3~1.25mm, the pressure is 10bar, M 丹酚酸A :M 大孔吸附树脂 =1:40, diameter-to-height ratio = 1:8; at 2.5CV·h -1The product was eluted with purified water for 5CV, 25% acidic ethanol (pH = 2.5) for 12CV, three wastes were treated, and 40% acidic ethanol (pH = 2.5) was used for 6CV. The 40% acidic ethanol eluate was collected in fractions, and the purity of each fraction was detected by HPLC. The purity of the combined salvianolic acid A was ≥80%, and the purified salvianolic acid A macroporous adsorption resin solution was obtained.

[0081] Example 12

[0082] Take the converted solution of salvianolic acid A in Example 8, the concentration of salvianolic acid A is controlled at 5.00 mg / ml, and -1 The flow rate passes through the qualified styrene type macroporous adsorption resin column, the macroporous adsorption resin is LX-1180, the filler particle size is 0.3~1.25mm, the pressure is 0bar, M 丹酚酸A :M 大孔吸附树脂 =1:60, diameter-to-height ratio = 1:10; 4CV·h -1 The product was eluted with purified water for 3CV, 15% acidic ethanol (pH = 3.5) for 10CV, three wastes were treated, and 35% acidic ethanol (pH = 3.5) was used for 8CV. The 35% acidic ethanol eluate was collected in fractions, and the purity of each fraction was detected by HPLC. The purity of the combined salvianolic acid A was ≥80%, and the salvianolic acid A macroporous adsorption resin purified solution was obtained.

[0083] Examples 13-16: Single extraction of salvianolic acid A

[0084] Example 13

[0085] The purified solution of salvianolic acid A obtained by macroporous adsorption resin in Example 9 was concentrated under reduced pressure at T ≤ 85° C. (70° C.) to a solution ethanol concentration of ≤ 15% (10%). The solution was extracted three times with tert-butyl methyl ether, with the amount of tert-butyl methyl ether used each time being 1 / 2, 1 / 3, and 1 / 3 of the concentrated solution, respectively. The extracts were combined.

[0086] Example 14

[0087] The purified solution of salvianolic acid A obtained by macroporous adsorption resin in Example 10 was concentrated under reduced pressure at T ≤ 85° C. (60° C.) to a solution ethanol concentration of ≤ 15% (15%), and extracted three times with ethyl acetate, using 1 / 2, 1 / 3, and 1 / 3 of the concentrated solution each time, respectively. The extracts were combined.

[0088] Example 15

[0089] The purified solution of salvianolic acid A prepared by macroporous adsorption resin in Example 11 was concentrated under reduced pressure at T≤85°C (65°C) to a solution ethanol concentration of ≤15% (12%), and extracted with tert-butyl methyl ether to obtain an extract.

[0090] Example 16

[0091] The purified solution of salvianolic acid A prepared by macroporous adsorption resin in Example 12 was concentrated under reduced pressure at T≤85°C (50°C) to a solution ethanol concentration of ≤15% (6%), and extracted with tert-butyl methyl ether to obtain an extract.

[0092] Examples 17-20: Purification of Salvianolic A by High Pressure Chromatography

[0093] Example 17

[0094] The extract of Example 13 was added with anhydrous sodium sulfate and allowed to stand for more than 2 hours (2.5 hours) to remove water. The extract was concentrated under reduced pressure at T≤50°C (40°C) to a concentration of 200 mg / ml of salvianolic acid A. The silica gel used in the sample and silica gel column was 300-400 mesh, with a particle size of 60 μm, and the pressure was 800 bar. 丹酚酸A :M 硅胶 =1:2 sample mixing, silica gel column chromatography dosage M 丹酚酸A :M 硅胶 The mixture was eluted with cyclohexane: tert-butyl methyl ether (4:6) for 10CV and cyclohexane: tert-butyl methyl ether (3:7) for 4CV, with a flow rate of 4.0CV·h -1 The eluent of cyclohexane: tert-butyl methyl ether (3:7) was collected in fractions, and the purity of each fraction was detected by HPLC. The fractions with purity ≥ 90% were combined to obtain normal phase silica gel purified solution of salvianolic acid A. The yield of salvianolic acid A was 81.23%. Further liquid phase detection showed that the content of salvianolic acid A was 93.02% and the purity was 98.51%.

[0095] Example 18

[0096] The extract of Example 14 was added with anhydrous sodium sulfate and allowed to stand for more than 2 hours (3.5 hours) to remove water. The extract was concentrated under reduced pressure at T ≤ 50 ° C (30 ° C) to a concentration of salvianolic acid A of 150 mg / ml. The silica gel used in the sample and silica gel column was: 300-400 mesh, particle size 120 μm, and pressure 1200 bar; M 丹酚酸A :M 硅胶 =1:2.5, mix the sample, silica gel column chromatography dosage M 丹酚酸A :M 硅胶 The dilution ratio was 1:15, and 12CV was used for elution with cyclohexane: tert-butyl methyl ether (5:5), and 8CV was used for elution with cyclohexane: tert-butyl methyl ether (4:6), with a flow rate of 7.5CV·h -1 The cyclohexane: tert-butyl methyl ether (4:6) eluate was collected in fractions, and the purity of each fraction was detected by HPLC. The fractions with purity ≥ 90% were combined to obtain a normal phase silica gel purified solution of salvianolic acid A. The yield of salvianolic acid A was 80.29%. Further liquid phase detection showed that the content of salvianolic acid A was 92.39% and the purity was 96.67%.

[0097] Example 19

[0098] The extract of Example 15 was added with anhydrous sodium sulfate and allowed to stand for more than 2 hours (3 hours) to remove water. The extract was concentrated under reduced pressure at T ≤ 50 ° C (40 ° C) to a concentration of 250 mg / ml of salvianolic acid A. The silica gel used in the sample and silica gel column was: 300-400 mesh, particle size 150 μm, and pressure 1500 bar; M 丹酚酸A :M 硅胶 =1:2, silica gel 300-400 mesh, mix sample, silica gel column chromatography dosage M 丹酚酸A :M 硅胶 The dilution ratio was 1:18, and 8CV was eluted with cyclohexane: tert-butyl methyl ether (4:6), and 5CV was eluted with cyclohexane: tert-butyl methyl ether (3:7), with a flow rate of 2.5CV·h -1 The cyclohexane: tert-butyl methyl ether (3:7) eluate was collected in fractions, and the purity of each fraction was detected by HPLC. The fractions with purity ≥ 90% were combined to obtain a normal phase silica gel purified solution of salvianolic acid A. The yield of salvianolic acid A was 84.45%. Further liquid phase detection showed that the content of salvianolic acid A was 92.58% and the purity was 96.92%.

[0099] Example 20

[0100] The extract of Example 16 was added with anhydrous sodium sulfate and allowed to stand for more than 2 hours (2.5 hours) to remove water. The extract was concentrated under reduced pressure at T ≤ 50 ° C (25 ° C) to a concentration of 180 mg / ml of salvianolic acid A. The silica gel used in the sample and silica gel column was: 300-400 mesh, particle size 40 μm, pressure 500 bar; M 丹酚酸A :M 硅胶 =1:2.5, silica gel 300-400 mesh for sample mixing, silica gel column chromatography dosage M 丹酚酸A :M 硅胶 The dilution ratio was 1:16, and 12CV was used for elution with cyclohexane: tert-butyl methyl ether (5:5), and 6CV was used for elution with cyclohexane: tert-butyl methyl ether (3:7), with a flow rate of 6.0CV·h -1 The eluent of cyclohexane: tert-butyl methyl ether (3:7) was collected in fractions, and the purity of each fraction was detected by HPLC. The fractions with purity ≥ 90% were combined to obtain normal phase silica gel purified solution of salvianolic acid A. The yield of salvianolic acid A was 83.82%. Further liquid phase detection showed that the content of salvianolic acid A was 92.98% and the purity was 95.31%.

[0101] Examples 21-24: Crystallization and Recrystallization

[0102] Example 21

[0103] The salvianolic acid A purified solution of normal phase silica gel in Example 17 was concentrated to dryness under reduced pressure at T≤50°C (40°C), dissolved in purified water to a concentration of about 80 mg / ml of salvianolic acid A, and concentrated under reduced pressure at T≤65°C to a concentration of 150 mg / ml of salvianolic acid A in the solution. Saturated sodium bicarbonate solution was added dropwise with stirring, and the pH was adjusted to 3.5. The solution was allowed to stand at 0-15°C for 25 h to allow crystallization. The crystals were filtered to obtain crude primary crystals of salvianolic acid A with a purity of 99.04% and a yield of 81.21%.

[0104] The crude product of the primary crystal of salvianolic acid A can be further recrystallized by adding purified water at 50°C, stirring and dissolving the product, filtering the product while hot, and adjusting the volume to a concentration of 240 mg / ml of salvianolic acid A. The product is stirred and crystallized at 0-15°C for 25 hours. The crystals are filtered and dried to obtain recrystallized salvianolic acid A with a purity of 99.74% and a yield of 86.54%.

[0105] Example 22

[0106] The salvianolic acid A purified solution of normal phase silica gel in Example 18 was concentrated to dryness under reduced pressure at T≤50°C (45°C), dissolved in purified water or water for injection until the concentration of salvianolic acid A was about 80 mg / ml, and concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A in the solution was 200 mg / ml. Saturated sodium bicarbonate solution was added dropwise with stirring, and the pH was adjusted to 3.8. The solution was allowed to stand at 0-15°C for 30 h to crystallize. The crystals were filtered to obtain crude primary crystals of salvianolic acid A with a purity of 99.19% and a yield of 84.11%.

[0107] The method can be further recrystallized by adding purified water or water for injection to the crude primary crystal of salvianolic acid A at 60°C, stirring and dissolving the crude product, filtering the product while hot, and adjusting the volume to a concentration of 250 mg / ml of salvianolic acid A. The product is stirred and crystallized at 0-15°C for 30 hours. The crystals are filtered and dried to obtain recrystallized salvianolic acid A with a purity of 99.66% and a yield of 83.28%.

[0108] Example 23

[0109] The salvianolic acid A purified solution of normal phase silica gel in Example 19 was concentrated to dryness under reduced pressure at T≤50°C (45°C), dissolved in purified water to a concentration of about 80 mg / ml of salvianolic acid A, and concentrated under reduced pressure at T≤65°C to a concentration of 200 mg / ml of salvianolic acid A in the solution. Saturated potassium bicarbonate solution was added dropwise while stirring, and the pH was adjusted to 3.7. The solution was allowed to stand at 0-15°C for 25 h to crystallize. The crystals were filtered to obtain crude primary crystals of salvianolic acid A with a purity of 99.03% and a yield of 84.05%.

[0110] The crude product of the first crystal of salvianolic acid A can be further recrystallized by adding purified water at 55°C, stirring and dissolving the product, filtering the product while hot, and adjusting the volume to a concentration of 220 mg / ml of salvianolic acid A. The product is stirred and crystallized at 0-15°C for 28 hours. The crystals are filtered and dried to obtain recrystallized salvianolic acid A with a purity of 99.69% and a yield of 84.29%.

[0111] Example 24

[0112] The salvianolic acid A purified solution of normal phase silica gel in Example 20 was concentrated to dryness under reduced pressure at T≤50°C (40°C), dissolved in purified water to a concentration of about 80 mg / ml of salvianolic acid A, and concentrated under reduced pressure at T≤65°C to a concentration of 250 mg / ml of salvianolic acid A in the solution. The solution was then added dropwise with meglumine solution while stirring, and the pH was adjusted to 3.5. The solution was allowed to stand at 0-15°C for 25 h to crystallize. The crystals were filtered to obtain crude primary crystals of salvianolic acid A with a purity of 99.36% and a yield of 82.29%.

[0113] The crude product of the primary crystal of salvianolic acid A can be further recrystallized by adding purified water at 65°C, stirring and dissolving the product, filtering the product while hot, and adjusting the volume to a concentration of 200 mg / ml of salvianolic acid A. The product is stirred and crystallized at 0-15°C for 26 hours. The crystals are filtered and dried to obtain recrystallized salvianolic acid A with a purity of 99.67% and a yield of 81.87%.

[0114] Examples 25-29: Secondary extraction of salvianolic acid A

[0115] Example 25

[0116] The crude recrystallized product of salvianolic acid A from Example 21 was dissolved in purified water with stirring to form a 30 mg / ml solution. 10% hydrochloric acid solution was added dropwise while stirring to adjust the pH to 2.0. The product was extracted three times with tert-butyl methyl ether, using 1 / 2, 1 / 3, and 1 / 3 of the solution, respectively. The extracts were combined.

[0117] Example 26

[0118] The crude recrystallized product of salvianolic acid A from Example 22 was dissolved in purified water with stirring to form a 40 mg / ml solution containing salvianolic acid A. 20% hydrochloric acid solution was added dropwise while stirring to adjust the pH to 1.5. The product was extracted three times with tert-butyl methyl ether, using 1 / 2, 1 / 3, and 1 / 3 of the solution, respectively. The extracts were combined.

[0119] Example 27

[0120] The crude recrystallized product of salvianolic acid A from Example 23 was dissolved in purified water with stirring to form a 50 mg / ml solution containing salvianolic acid A. 10% phosphoric acid solution was added dropwise while stirring to adjust the pH to 2.0. The product was extracted three times with tert-butyl methyl ether, using 1 / 2, 1 / 3, and 1 / 3 of the solution, respectively. The extracts were combined.

[0121] Example 28

[0122] The crude recrystallized product of salvianolic acid A from Example 24 was dissolved in purified water with stirring to form a 25 mg / ml solution containing salvianolic acid A. 10% hydrochloric acid solution was added dropwise while stirring to adjust the pH to 3.0. The product was extracted three times with tert-butyl methyl ether, using 1 / 2, 1 / 3, and 1 / 3 of the solution, respectively. The extracts were combined.

[0123] Example 29

[0124] Take the crude crystalline product of salvianolic acid A from Example 21 and add an appropriate amount of purified water with stirring to dissolve it into a solution containing 25 mg / ml of salvianolic acid A. While stirring, add 20% phosphoric acid solution dropwise to adjust the pH of the solution to 2.0. Extract with ethyl acetate three times, using 1 / 2, 1 / 3, and 1 / 3 of the solution volume, respectively. Combine the extracts.

[0125] Examples 30-34: Concentration and drying

[0126] Example 30

[0127] The extract obtained in Example 25 was concentrated to dryness under reduced pressure at T≤50°C (40°C), dissolved in purified water until the concentration of salvianolic acid A was about 100 mg / ml, concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A in the solution was above 200 mg / ml (220 mg / ml), diluted to a concentration of 160 mg / ml, and freeze-dried under vacuum under the following conditions:

[0128]

[0129] HPLC detection showed that the content of salvianolic acid A in the dried product was 99.3%, the purity was 99.70%, and the residual solvents were: methyl tert-butyl ether was 0.002%, cyclohexane was not detected, and ethanol was not detected.

[0130] Example 31

[0131] The extract obtained in Example 26 was concentrated to dryness under reduced pressure at T≤50°C (30°C), dissolved in purified water until the concentration of salvianolic acid A was about 100 mg / ml, concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A in the solution was above 200 mg / ml (200 mg / ml), and the solution was diluted to a concentration of 150 mg / ml of salvianolic acid A and freeze-dried under vacuum under the following conditions:

[0132]

[0133] HPLC detection showed that the content of salvianolic acid A in the dried product was 99.9%, the purity was 99.57%, and the residual solvents were: methyl tert-butyl ether was 0.008%, cyclohexane was not detected, and ethanol was 0.007%.

[0134] Example 32

[0135] The extract obtained in Example 27 was concentrated to dryness under reduced pressure at T≤50°C (35°C), dissolved in purified water until the concentration of salvianolic acid A was about 100 mg / ml, concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A in the solution was above 200 mg / ml (250 mg / ml), diluted to a concentration of 200 mg / ml, and freeze-dried under vacuum under the following conditions:

[0136]

[0137] HPLC detection showed that the content of salvianolic acid A in the dried product was 100.0%, the purity was 99.53%, and the residual solvents were: methyl tert-butyl ether was 0.004%, cyclohexane was not detected, and ethanol was 0.015%.

[0138] Example 33

[0139] The extract obtained in Example 28 was concentrated to dryness under reduced pressure at T≤50°C (30°C), dissolved in purified water until the concentration of salvianolic acid A was about 100 mg / ml, concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A in the solution was above 200 mg / ml (250 mg / ml), diluted to a concentration of 180 mg / ml, and freeze-dried under vacuum under the following conditions:

[0140]

[0141] HPLC detection showed that the content of salvianolic acid A in the dried product was 99.9%, the purity was 99.54%, and the residual solvents were: no methyl tert-butyl ether was detected, no cyclohexane was detected, and 0.006% ethanol.

[0142] Example 34

[0143] The extract obtained in Example 29 was concentrated to dryness under reduced pressure at T≤50°C (50°C), dissolved in purified water until the concentration of salvianolic acid A was about 100 mg / ml, concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A in the solution was above 200 mg / ml (250 mg / ml), diluted to a concentration of 200 mg / ml, and freeze-dried under vacuum under the following conditions:

[0144]

[0145] HPLC detection showed that the content of salvianolic acid A in the dried product was 99.7%, the purity was 99.72%, and the residual solvents were: methyl tert-butyl ether was 0.002%, cyclohexane was not detected, ethyl acetate was not detected, and ethanol was not detected.

[0146] Comparative experiment

[0147] Comparative Example Group 1 Example of CN103044251A

[0148] The method comprises the following steps: taking Danshen medicinal material, cutting it into slices, adding 15 times the amount of water to decoct and extracting, extracting for a total of 3 times, each time for 4 hours; concentrating the extract under reduced pressure to a relative density of 1.20 (60° C.), adding ethanol to make the alcohol content 65%, letting it stand, filtering, recovering the ethanol from the filtrate under reduced pressure and concentrating it until there is no alcohol taste; diluting the Danshen extract with water to contain 30 mg of salvianolic acid B per 1 ml, adjusting the pH of the aqueous solution to 4.5 with alkali, adding zinc chloride with a molar percentage of 0.3% of salvianolic acid B as a catalyst, and heating at 140° C. for 6 hours; adjusting the pH of the conversion solution to 3.5 with acid, letting it stand, centrifuging, concentrating the supernatant under reduced pressure to contain 10 mg of salvianolic acid A per 1 ml, and separating the mixture by chromatography on an HPD-100 macroporous resin column, with a salvianolic acid A loading ratio of 1:50 and a resin column diameter-height ratio of 1:10, respectively, with 8-fold column chromatography. The solution was washed with 10 times the column volume of water and 30% ethanol to remove impurities, and then eluted with 10 times the column volume of 50% ethanol. After HPLC detection, the 50% ethanol elution fraction containing salvianolic acid A was collected, the ethanol was recovered under reduced pressure and concentrated until there was no alcohol smell; the aqueous solution was concentrated to a solution containing 10 mg of salvianolic acid A per 1 ml, and separated by polyamide chromatography column, the ratio of salvianolic acid A loading amount to polyamide was 1:20, the resin column diameter-to-height ratio was 1:10, and 10 times the column volume of water and 10 times the column volume were used respectively. The elution was performed with 40% ethanol solution to remove impurities, and then eluted with 80% ethanol solution for 10 times the column volume. The 80% ethanol solution containing salvianolic acid A was collected, the ethanol was recovered under reduced pressure and concentrated to an alcohol-free aqueous solution. The pH was adjusted to 4.0, and the aqueous solution was extracted with tert-butyl methyl ether in an amount of 8 times that of the aqueous solution for 3 times. The organic layer was separated and the tert-butyl methyl ether was recovered under reduced pressure to prepare an extract containing 10 g of salvianolic acid A per 1 ml. 3 times the amount of silica gel was added, stirred, and evaporated to dryness. The stirred silica gel was added to the packed silica gel. The product was purified by gradient elution using 20 times the column volume of n-pentane-tert-butyl methyl ether (4:6) and 20 times the column volume of n-pentane-tert-butyl methyl ether (6:4), respectively. The eluent was recovered under reduced pressure, and the recovered salvianolic acid A was dissolved in 10 times the amount of water and dried under microwave vacuum to obtain salvianolic acid A. HPLC analysis showed a content of 95.7% and a purity of 98.58%.

[0149] Investigation of parameters of normal phase silica gel purification step in comparative example group 2

[0150] Comparative Example 2-1 Investigation of the concentration degree of the extract

[0151] 2000 ml of the salvianolic acid A extract was taken, and the concentration of salvianolic acid A in the extract was determined by HPLC. The amount of salvianolic acid A was calculated. The extract was concentrated under reduced pressure at T ≤ 50°C to different concentrations (about 150 mg / ml for Comparative Example 2-1-1 group, about 200 mg / ml for Comparative Example 2-1-2 group, and about 250 mg / ml for Comparative Example 2-1-3 group). The dissolution and fluidity of the samples at different concentrations were observed. The results are shown in Table 1.

[0152] Table 1: Results of investigation on different concentration levels of extracts

[0153]

[0154] Data Analysis and Conclusions:

[0155] The results of the sample dissolution and fluidity tests at different concentrations in Table 1 show that when the salvianolic acid A extract was concentrated to a concentration of 150 mg / ml to 250 mg / ml, the sample dissolved and exhibited good fluidity, facilitating silica gel mixing. Subsequent silica gel mixing was performed with the salvianolic acid A extract concentrated to a concentration of 150 mg / ml to 250 mg / ml.

[0156] Comparative Example 2-2 Investigation of elution solvent and elution gradient

[0157] Based on the properties of salvianolic acid A, thin-layer chromatography was used to initially explore column chromatography elution solvent conditions. Low-polarity solvents suitable for silica gel chromatography purification of salvianolic acid A include n-hexane, n-pentane, cyclohexane, and petroleum ether, while high-polarity solvents include methanol, ethanol, isopropanol, ethyl acetate, and tert-butyl methyl ether. Based on the solvent's inherent properties (safety, quality controllability, boiling point, etc.), solvent usage principles, and solvent price, four solvent systems were selected for purification of salvianolic acid A: n-hexane / tert-butyl methyl ether, n-hexane / ethyl acetate, cyclohexane / tert-butyl methyl ether, and cyclohexane / ethyl acetate. Based on the results of preliminary experiments, column chromatography elution solvent systems and elution gradients were explored, and these elution solvent systems and elution gradients were then confirmed by column chromatography.

[0158] Use a spotting capillary to take a small amount of sample and reference substance and spot them on a GF254 silica gel plate (0.5cm to 1.0cm from the bottom of the plate). Place the sample-spotted thin layer plate into a developing tank with different systems and different proportions of developing agents. The depth of immersion in the developing agent should be 2 to 3mm from the origin (do not allow the sample to penetrate the developing agent). Seal the top cover and wait until the solvent front reaches the specified development distance. Remove the thin layer plate, dry it, and observe it under ultraviolet light (254nm). Calculate the corresponding Rf value (Rf is the ratio of the migration value: the distance from the origin to the center of the component point / the distance from the origin to the flow front). Use the developing system with an Rf of about 0.2 to 0.3 at the first point of thin layer chromatography as the first elution gradient of column chromatography. The results are shown in Tables 2 and 3:

[0159] Table 2: Results of investigation on elution solvent and elution gradient (R of the first impurity at the front) f value)

[0160]

[0161] Table 3: Results of investigation on elution solvent and elution gradient (R f value)

[0162]

[0163] Data Analysis and Conclusions:

[0164] According to the results of thin-layer chromatography experiments and the safety of the solvent, the normal-phase silica gel chromatography purification of salvianolic acid A is planned to use a cyclohexane / tert-butyl methyl ether system (Class II solvent) for elution. The elution gradient is preliminarily judged by thin-layer chromatography and then confirmed by column chromatography.

[0165] Comparative Example 2-3 Elution Conditions

[0166] Comparative Example 2-3-1: Take an appropriate amount of silica gel mixed with a sample, containing about 10g of salvianolic acid A, and fill it into a chromatography column filled with 200g of silica gel (300-400 mesh) for column chromatography (M 丹酚酸A With M 层析硅胶(300-400目) A dynamic axial compression column was used (with a ratio of 1:20), using cyclohexane:tert-butyl methyl ether (4:6) as the eluent. The eluent volume was 20 CV, the elution rate was 40 ml / min, and 350 ml fractions were collected. The concentration and purity of salvianolic acid A in the fractions were determined by HPLC. Fractions with a purity greater than 90% were combined, and the amount and yield of salvianolic acid A were calculated. The results are shown in Table 4:

[0167] Comparative Example 2-3-2: Take an appropriate amount of silica gel mixed with a sample, containing about 10g of salvianolic acid A, and fill it into a chromatography column filled with 200g of silica gel (300-400 mesh) for column chromatography (M 丹酚酸A With M 层析硅胶(300-400目) A dynamic axial compression column was used (with a ratio of 1:20), using cyclohexane:tert-butyl methyl ether (3:7) as the eluent. The eluent dosage was 10 CV, the elution rate was 40 ml / min, and 350 ml fractions were collected. The concentration and purity of salvianolic acid A in the fractions were determined by HPLC. Fractions with a purity greater than 90% were combined, and the amount and yield of salvianolic acid A were calculated. The results are shown in Table 4:

[0168] The present invention: take an appropriate amount of silica gel that has been mixed evenly, containing about 10g of salvianolic acid A, and fill it into a chromatography column filled with 200g of silica gel (300-400 mesh) for column chromatography (M 丹酚酸A With M 层析硅胶(300-400目)The elution was performed using a dynamic axial compression column with varying gradients of cyclohexane:tert-butyl methyl ether (5:5 to 4:6) for 10 CV and cyclohexane:tert-butyl methyl ether (4:6 to 3:7) for 6 CV. The elution flow rate was 40 ml / min, and 350 ml fractions were collected (with a UV detector). The concentration and purity of salvianolic acid A in the fractions were determined by HPLC. Fractions with a purity greater than 90% were combined, and the amount and yield of salvianolic acid A were calculated. The results are shown in Table 4.

[0169] Table 4: Experimental data of elution conditions

[0170]

[0171] Data Analysis and Conclusions:

[0172] (1) When elution was performed using cyclohexane: tert-butyl methyl ether (4:6) in Comparative Example 2-3-1, impurities were removed significantly, but the target compound, salvianolic acid A, was eluted slowly. Until the end, salvianolic acid A was not completely eluted. Although the sample quality obtained under this elution condition met the requirements, the yield was low, the material was wasted, the time wasted, and the production cost was high.

[0173] (2) When elution was performed using cyclohexane: tert-butyl methyl ether (3:7) as in Comparative Example 2-3-2, the elution fraction was small, but the target product, salvianolic acid A, and impurities could not be effectively separated, and the sample yield was low, and the process was uncontrollable.

[0174] (3) Using cyclohexane: tert-butyl methyl ether gradient elution, cyclohexane: tert-butyl methyl ether 5:5 to 4:6 elution 10CV can remove most impurities. Cyclohexane: tert-butyl methyl ether 4:6 to 3:7 elution can obtain salvianolic acid A with a purity of more than 90%, and the salvianolic acid A sample is concentrated, with the highest yield. In order to effectively remove impurities and improve the purity and yield of the target product salvianolic acid A, cyclohexane: tert-butyl methyl ether 5:5 to 4:6 was selected as the eluent for eluting impurities, and cyclohexane: tert-butyl methyl ether (4:6 to 3:7) was then used to elute high-purity salvianolic acid A.

[0175] The experimental results show that the separation and purification effect of salvianolic acid A is good when using a cyclohexane: tert-butyl methyl ether solvent system for normal phase silica gel purification, such as a gradient elution of 5:5 to 3:7, and the yield and purity of salvianolic acid A can meet the requirements of process research.

[0176] Investigation of crystallization conditions of comparative group 3

[0177] Comparative Example 3-1 Crystallization Solution pH Investigation

[0178] Take the crude product of salvianolic acid A purified on normal phase silica gel, containing approximately 15g of salvianolic acid A, and dissolve it in purified water by ultrasonication (containing a salvianolic acid A concentration of approximately 80mg / ml). Concentrate under reduced pressure at T≤65°C until the salvianolic acid A concentration in the solution is 150-250mg / ml. Add saturated sodium bicarbonate solution dropwise while stirring, and adjust the pH of the sample solution to 3.0, 3.5, 4.0, and 4.5, respectively. Refrigerate the sample (2-8°C) and allow it to stand for 24 hours to crystallize. Observe the precipitation of the sample. Filter the crystals, vacuum dry the sample (T≤45°C), weigh it, and calculate the crystal yield. Analyze the purity of the crystals by HPLC. The results are shown in Table 5:

[0179] Table 5: Crystallization solution pH investigation results

[0180]

[0181] Note: SAA salt yield (%) = dry weight of crystals (g) / amount of SAA before crystallization (g) × 100%. " / " indicates no crystals precipitated under these conditions. The English abbreviation for salvianolic acid A is: SAA.

[0182] Data Analysis and Conclusions:

[0183] Experimental observations and test results indicate that by adjusting the solution pH, salvianolic acid A can be crystallized. At a pH of 3.0-4.0, the purity reaches over 99%. The optimal pH of 3.5 achieves the highest sample yield, increasing the purity of salvianolic acid A from 95% to over 99%, demonstrating significant impurity removal.

[0184] Comparative Example 3-2 Crystallization Solution pH Optimization

[0185] Take the crude product of salvianolic acid A purified on normal phase silica gel, containing approximately 10g of salvianolic acid A, add purified water and ultrasonically dissolve it (containing a salvianolic acid A concentration of approximately 80mg / ml). Concentrate under reduced pressure at T≤65°C until the salvianolic acid A concentration in the solution is 150mg / ml to 250mg / ml. Add saturated sodium bicarbonate solution dropwise while stirring, and adjust the pH to 3.4, 3.5, 3.6, 3.7, 3.8, and 3.9. Place the sample in an environment (0-15°C) and let it stand for crystallization for more than 24 hours. Filter the crystals. Place the sample in a vacuum drying oven (T≤45°C) and vacuum dry it for 12 hours. Weigh it and calculate the crystal yield. Detect the crystal purity by HPLC. The results are shown in Table 6:

[0186] Table 6: Crystallization solution pH optimization results

[0187]

[0188] Data Analysis and Conclusions:

[0189] Experimental phenomena and test results show that after the pH value of the crystallization solution was adjusted to 3.5, 3.6, 3.7, and 3.8, the crystallization yield was above 80% and the crystal purity was above 99%. Therefore, after optimization research, the pH value of the sample solution before crystallization was adjusted from 3.5 to 3.5-3.8.

[0190] Investigation of drying method of comparative group 4

[0191] Comparative Example 4-1 Microwave Drying

[0192] Microwave drying utilizes the thermal effect generated by the interaction between material molecules and high-frequency microwaves to evaporate the moisture in the material to achieve drying, and in the process the material structure changes.

[0193] Comparative Example 4-2 Vacuum drying

[0194] Take the extract after crystallization, extraction, and purification of salvianolic acid A (containing approximately 10g of salvianolic acid A). Sample 1: Concentrate to dryness at T≤50°C; Sample 2: Concentrate to dryness at T≤50°C, dissolve in purified water (containing approximately 100mg / ml of salvianolic acid A), and continue concentrating to dryness at T≤65°C. Samples 1 and 2 are vacuum-dried in a vacuum drying oven (T≤45°C) for 12 hours and weighed. The yield of salvianolic acid A, purity of related substances, content, moisture content, and residual solvents are examined. The results are shown in Table 7:

[0195] Table 7: Results of investigation on sample treatment methods before drying

[0196]

[0197] Note: “-” in Table 7 indicates that the purity of salvianolic acid A decreases before and after drying.

[0198] Data Analysis and Conclusions:

[0199] Comprehensive analysis of the purity, content, moisture content, and residual solvent data for salvianolic acid A revealed that the quality of the final products after vacuum drying using various pretreatment methods was substandard. Given the thermal instability of salvianolic acid A, freeze-vacuum drying was preferred to achieve quality standards for purity, content, moisture content, and residual solvent content.

Claims

1. A method for preparing salvianolic acid A, comprising the following steps: (1) Medium and low pressure resin purification step: Take the converted solution of salvianolic acid A and -1 ~3CV·h -1 The flow rate passes through the qualified styrene type macroporous adsorption resin column, and the resin dosage is M 丹酚酸A :M 大孔吸附树脂 =1:35~1:60, diameter-to-height ratio =1:5~1:10, 2CV·h -1 ~4CV·h -1 flow rate, eluting with water for 3CV to 5CV, 15% to 30% acidic ethanol for 8CV to 12CV, and 35% to 55% acidic ethanol for 4CV to 8CV, collecting the 35% to 55% acidic ethanol eluate in fractions, detecting the purity of each fraction by HPLC, and combining fractions with a purity of ≥80% to obtain a purified solution of the macroporous adsorption resin; the pH of the acidic ethanol is 2.5 to 3.5; (2) Salvianolic acid A extraction step: taking the macroporous adsorption resin purified solution from step (1), concentrating under reduced pressure, and extracting with tert-butyl methyl ether or ethyl acetate to obtain an extract; (3) Normal phase silica gel purification: The extract of step (2) was concentrated to a concentration of 150 mg / mL to 250 mg / mL of salvianolic acid A, mixed with silica gel, and loaded onto a silica gel column for chromatography. A cyclohexane: tert-butyl methyl ether mixed solvent was used for gradient elution to obtain a normal phase silica gel purified solution. The gradient elution is as follows: 8CV to 12CV of cyclohexane: tert-butyl methyl ether with a volume ratio of 5:5 to 4:6, and 4CV to 8CV of cyclohexane: tert-butyl methyl ether with a volume ratio of 4:6 to 3:7, with a flow rate of 2.5CV·h -1 ~7.5CV·h -1 , the fractions were collected with a cyclohexane: tert-butyl methyl ether eluate having a volume ratio of 4:6 to 3:7; (4) Crystallization: The normal phase silica gel purified solution of step (3) was concentrated, dissolved in water, and concentrated to a concentration of 100 mg / mL to 300 mg / mL of salvianolic acid A in the solution. The pH value was adjusted to 3.5 to 3.

8. The sample was placed in an environment of 0 to 15°C, allowed to stand for crystallization, and filtered to obtain a crude primary crystal. (5) Recrystallization: Recrystallize and purify the crude product of the primary crystallization in step (4); (6) Extraction, concentration, and drying: Dissolve the crystals obtained in step (5) in water, add 10% to 20% acid solution dropwise while stirring, and adjust the pH to 1.5 to 3.0; extract with tert-butyl methyl ether three times, combine the extracts to obtain a crystalline extract; concentrate under reduced pressure, and freeze-dry in a vacuum.

2. The preparation method according to claim 1, wherein: The salvianolic acid A conversion liquid is prepared by the steps of salvianolic acid B extraction, alcohol precipitation, and salvianolic acid B conversion, wherein: (a) Extraction: Take the medicinal material of Salvia miltiorrhiza, crush it, sieve it, add water at 75-85°C, and extract it 2-4 times to obtain the salvianolic acid B extract; or take the medicinal material of Salvia miltiorrhiza, crush it, sieve it, and add 20-40% ethanol to reflux and extract it to obtain the salvianolic acid B extract; (b) Alcohol precipitation: The extract from step (a) was concentrated under reduced pressure at a temperature of ≤ 65°C to a relative density of 1.05 to 1.15, and the concentrates were combined; 90% to 95% ethanol was added to adjust the ethanol concentration of the solution to 70% to 75%, and the solution was allowed to settle under alcohol precipitation at a temperature of 10°C to 35°C for more than 24 hours, filtered, and the filtrate was concentrated under reduced pressure at a temperature of ≤ 65°C to a ethanol concentration of ≤ 5% to obtain an alcohol-precipitated concentrate; (c) Conversion step: taking the alcohol precipitation concentrate from step (b), diluting it with purified water to a salvianolic acid B concentration of 5 mg / mL to 10 mg / mL, adjusting the pH to 3.5 to 4.0, reacting at 110°C to 130°C for 2.5 hours to 4.5 hours, cooling, and centrifuging to obtain a salvianolic acid A conversion solution; and / or, the concentration of salvianolic acid A in the salvianolic acid A conversion solution in step (1) is 1.59 mg / mL to 6.56 mg / mL; And / or, the styrene-type macroporous adsorption resin in step (1) is HPD-100S, LX-1180, D101B, HPD-300, D101-I or HPD-600; the filler particle size is 0.30mm to 1.25mm; the pressure resistance is 2bar to 10bar; And / or, the acid solution in step (6) is hydrochloric acid or phosphoric acid solution.

3. The preparation method according to claim 1 or 2, characterized in that In step (1), the styrene-type macroporous adsorption resin is HPD-100S or LX-1180; and the pressure resistance is 2 bar to 5 bar.

4. The preparation method according to claim 1 or 2, characterized in that In step (2), the purified solution of salvianolic acid A macroporous adsorption resin is concentrated under reduced pressure at T≤85°C to a solution ethanol concentration of ≤15%, extracted three times with tert-butyl methyl ether, the extracts are combined, anhydrous sodium sulfate is added, and the mixture is allowed to stand for more than 2 hours to remove water.

5. The preparation method according to claim 1 or 2, characterized in that In step (3), the silica gel used for purification is 300-400 mesh, with a particle size of 40 μm-150 μm, and a pressure of 500 bar-1500 bar; The amount of silica gel used for mixing samples should meet the following requirements: M 丹酚酸A :M 硅胶 1:2.0~1:3.0; The amount of silica gel used for column chromatography is as follows: M 丹酚酸A :M 硅胶 It is 1:10 to 1:

50.

6. The preparation method according to claim 5, characterized in that In step (3), the silica gel particle size used for purification is 40 μm to 60 μm, and the pressure is 800 bar to 1200 bar; The amount of silica gel used for mixing samples should meet the following requirements: M 丹酚酸A :M 硅胶 1:2.0~1:2.5; The amount of silica gel used for column chromatography is as follows: M 丹酚酸A :M 硅胶 1:15~1:

20.

7. The preparation method according to claim 1 or 2, characterized in that Step (3) is specifically as follows: the extract of step (2) is concentrated under reduced pressure at T≤50°C to a concentration of salvianolic acid A of 150mg / mL to 250mg / mL, and 丹酚酸A :M 硅胶 =1:2~1:3.0 to mix with silica gel, take the mixed sample and pack it into the silica gel column, the amount of silica gel column chromatography is M 丹酚酸A :M 硅胶 The flow rate was 2.5 CV h, and the volume ratio of cyclohexane to tert-butyl methyl ether was 1:15 to 1:20, and 8 CV to 12 CV was eluted with cyclohexane to tert-butyl methyl ether in a volume ratio of 5:5 to 4:6, and 4 CV to 8 CV was eluted with cyclohexane to tert-butyl methyl ether in a volume ratio of 4:6 to 3:

7. -1 ~7.5CV·h -1 , the fractions were collected with a volume ratio of 4:6 to 3:7 of cyclohexane: tert-butyl methyl ether, the purity of each fraction was detected by HPLC, and the purity of salvianolic acid A was ≥90% to obtain salvianolic acid A normal phase silica gel purified solution; wherein the silica gel was 300-400 mesh.

8. The preparation method according to claim 1 or 2, characterized in that In step (4), the concentration of salvianolic acid A is 150 mg / mL to 250 mg / mL; The pH regulator for adjusting the pH value is selected from one or more of meglumine, sodium bicarbonate, sodium carbonate, potassium bicarbonate, magnesium bicarbonate, magnesium carbonate, calcium hydroxide, calcium bicarbonate, and calcium carbonate; Let it stand for more than 12 hours to crystallize.

9. The preparation method according to claim 8, characterized in that In step (4), the pH regulator for adjusting the pH value is selected from sodium bicarbonate.

10. The preparation method according to claim 1 or 2, characterized in that: Step (4) is specifically as follows: the normal phase silica gel purified solution of step (3) is concentrated to dryness under reduced pressure at T≤50°C, purified water or water for injection is added to dissolve it into a solution containing 80 mg / mL of salvianolic acid A, and concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A in the solution is 150 mg / mL to 250 mg / mL, the pH value is adjusted to 3.5 to 3.8, the sample is placed in an environment of 0°C to 15°C, and allowed to stand for crystallization for more than 12 hours, and filtered to obtain a crude primary crystal.

11. The preparation method according to claim 1 or 2, characterized in that: Specifically, step (5) comprises adding purified water to the crude primary crystallization product of step (4) at a temperature of 50°C to 65°C, stirring and dissolving the product, filtering the product while hot, and adjusting the volume to a concentration of 200 mg / mL to 250 mg / mL of salvianolic acid A. The sample is placed in an environment of 0°C to 15°C, stirring and crystallizing the product for more than 12 hours, filtering the crystals, and draining the product.

12. The preparation method according to claim 1 or 2, characterized in that: In step (6), The water is purified water or water for injection; The concentration of salvianolic acid A in the solution is 25 mg / mL to 50 mg / mL; Adjust pH to 1.5-2.0; The reduced pressure concentration and freeze vacuum drying steps are as follows: after the recrystallization extract is concentrated under reduced pressure at T≤65°C until the concentration of salvianolic acid A is above 200 mg / mL, the solution is diluted to a concentration of salvianolic acid A of 150 mg / mL to 200 mg / mL, and freeze vacuum drying is performed.

13. The preparation method according to claim 1 or 2, characterized in that: Step (6) is specifically as follows: adding purified water or water for injection to the crystals obtained in step (5) to dissolve the crystals into a solution containing 25 mg / mL to 50 mg / mL of salvianolic acid A, adding 10% to 20% of an acid solution dropwise while stirring, and adjusting the pH value to 1.5 to 2.0; extracting with tert-butyl methyl ether three times, combining the extracts to obtain a crystallization extract; concentrating the recrystallized extract under reduced pressure at T≤65°C until the salvianolic acid A concentration is above 200 mg / mL, diluting the solution to a salvianolic acid A concentration of 150 mg / mL to 200 mg / mL, and freeze-drying in a vacuum.

14. The preparation method according to claim 1 or 2, characterized in that: The freeze vacuum drying conditions in step (6) are:

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