WO2018145622A1 - Method for preparing salvianolic acid a salt - Google Patents

Abstract

Provided in the present invention is a method for preparing a salvianolic acid A salt, the preparation method being: dissolving salvianolic acid A in water, then adjusting the pH value to be 1.2-5.5 by using an aqueous solution of a basic substance, and collecting a precipitate, i.e., a salvianolic acid A salt; the present invention uses a method of adding a basic solution to a salvianolic acid A aqueous solution to increase the pH value and precipitate a salvianolic acid A salt, thereby preparing and obtaining a salvianolic acid A salt product which has strong stability, cannot be easily oxidized, has high purity and reduced impurities; said preparation method is efficient, simple and easy to implement, while being more suitable for industrial mass production, and being beneficial to the development and application of salvianolic acid A salt.

Description

Preparation method of salvianolic acid A salt (1) Technical field

The invention relates to a preparation method of salvianolic acid A salt.

(2) Background technology

Salvianic acid A (Salvianic acid A) is a water-soluble phenolic acid compound contained in Salvia miltiorrhiza. It was first isolated from Salvia miltiorrhiza in 1984 by Professor Li Lian Niang (LiLian-Niang, et al. Planta Medica. 1984, 50: 227). Salvianolic acid A has strong pharmacological activities such as myocardial ischemia protection, antithrombotic, anti-fibrosis and anti-oxidation, and has potential therapeutic value for cardiovascular and cerebrovascular diseases.

The presence of salvianolic acid A is easily oxidized and has poor stability, and it is difficult to ensure long-term stability in various dosage forms. CN105523926 A discloses a method for extracting and separating and purifying salvianolic acid A and a method for preparing salvianolic acid A salt, wherein a magnesium salt of salvianolic acid A is used, but the obtained method comprises sodium chloride or potassium chloride. Magnesium salt complex. CN102432467 B discloses a preparation method of salvianolic acid A magnesium salt, which has the advantages of complicated process flow, high production cost, and is not suitable for industrial production or preparation.

(3) Invention content

The object of the present invention is to provide a preparation method of salvianolic acid A salt which has high stability, is not easily oxidized, and has high purity. The preparation method has the advantages of good processability, simple operation, low production cost, and is suitable for industrial production or preparation. specialty.

The invention adopts the following technical solutions:

A method for preparing salvianolic acid A salt, wherein the preparation method is:

The salvianolic acid A is dissolved in water, and the pH is adjusted to a pH of 1.2 to 5.5 (preferably 3.2 to 4.2) with an aqueous solution of an alkaline substance, and the precipitate is collected to be the salvianolic acid A salt.

In the preparation method of the present invention, the mass ratio of the salicylic acid A to water is 1:0.25 to 4, preferably 1:0.67 to 1.5.

The concentration of the aqueous solution of the alkaline substance is 1% to 15%, preferably 4% to 11%; the alkaline substance includes a strong base, a weak acid strong base salt, specifically, for example, sodium hydroxide, sodium hydrogencarbonate, carbonic acid Sodium, potassium hydroxide, potassium hydrogencarbonate, potassium carbonate, magnesium hydroxide, magnesium hydrogencarbonate, magnesium carbonate, calcium hydroxide, calcium hydrogencarbonate, calcium carbonate, sodium citrate, sodium tartrate, potassium citrate, potassium tartrate, lemon Magnesium or magnesium tartrate.

The salvianolic acid A salt obtained by the present invention is a sodium salt, a potassium salt, a magnesium salt or a calcium salt of salvianolic acid A.

In the preparation method, the collected precipitates are washed with water and dried, and then directly used in the preparation or used together with a human acceptable pharmaceutical excipient for the preparation.

The salvianolic acid A of the invention is obtained by extracting raw material Danshen by water or ethanol aqueous solution, separating by macroporous resin column chromatography and separating by polyamide chromatography column. For the preparation method, for example, the method disclosed in Chinese Patent No. CN101361728 B can be referred to.

Specifically, the preparation method of the salvianolic acid A is generally:

(1) Mixing the raw material Danshen according to the mass ratio of the liquid: 1:6~12 with water or volume fraction of 10%-90% ethanol solution, extracting at 70-100 °C for 1-3 hours, collecting the filtrate by filtration, and extracting the residue. ~3 times, combining the filtrates obtained each time to obtain the extract of Salvia miltiorrhiza;

(2) taking the salvia miltiorrhiza extract obtained in the step (1), concentrating (usually concentrated to 2 to 4 times the mass of the raw salvia miltiorrhiza), and adjusting the pH to 3.5 to 6.0 (normal operation, adjustment with an aqueous solution of a mineral acid, the inorganic Acids such as hydrochloric acid, sulfuric acid, nitric acid, etc., are heated to 110-130 ° C for 1 to 6 hours, then cooled to room temperature (20 ~ 30 ° C), filtered, and the filtrate is separated by macroporous resin column chromatography, first with water, volume fraction 10 After elution with %~30% ethanol aqueous solution, elute with 30%-70% ethanol aqueous solution, collect eluate containing salvianolic acid A, concentrate (to no alcohol taste) and continue polyamide chromatography Separation of the column, first elution with water, volume fraction of 20% ~ 50% ethanol aqueous solution, and then eluted with a volume fraction of 50% ~ 95% ethanol aqueous solution, collecting eluate containing salvianolic acid A, concentrated (to alcohol-free Taste) adjust the pH to 2 to 5 (conventional operation, adjusted with an aqueous solution of a basic substance, the basic substance is as defined above), extracted with an organic solvent, and the extract is concentrated and dried to obtain Salvianolic acid A;

The raw material Salvia miltiorrhiza is the dried root and rhizome of Salvia miltiorrhiza Bge., which is commercially available on the market;

The macroporous resin is HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101, 1300-I, 1400 or AB-8;

The organic solvent used for the extraction is ethyl acetate, propyl acetate, butyl acetate, n-butanol or isopropanol;

The obtained salvianolic acid A has a purity of 50% to 100%, and can be directly used for the preparation of the subsequent salvianolic acid A salt.

The invention has the beneficial effects that the invention adopts a method for increasing the pH and extracting the salvianolic acid A salt by adding an alkali solution in the aqueous solution of salvianolic acid A, and preparing a method which has strong stability, is not easily oxidized, has high purity, and has reduced impurities. Phenolic acid A salt product. The preparation method is simple and more suitable for industrial large production. Therefore, the present invention provides an efficient and simple method for the preparation of salvianolic acid A salt, which is beneficial to the development and application of salvianolic acid A salt.

(4) Description of the drawings

Figure 1: High resolution mass spectrum of salvianolic acid A sodium salt;

Figure 2: ¹ H NMR spectrum of salvianolic acid A sodium salt;

Figure 3: ¹³ C NMR spectrum of salvianolic acid A sodium salt;

Figure 4: HMQC spectrum of salvianolic acid A sodium salt.

(5) Specific implementation methods

The present invention will be further illustrated by the following specific examples, but the scope of the present invention is not limited thereto.

Examples 1 to 9 are preparation examples of salvianolic acid A, and refer to the method disclosed in CN 101361728 B. Raw material Danshen was purchased from Shanghai Huayu Pharmaceutical Co., Ltd.

Example 1

The raw material Danshen 200kg and the volume fraction 50% ethanol aqueous solution 2000L were mixed, extracted at 82 ° C for 2h, the filtrate was collected by filtration, the filter residue was re-extracted 3 times, and the obtained filtrate was combined to obtain Salvia miltiorrhiza extract; the obtained Salvia miltiorrhiza extract was concentrated to 600L, adjust the pH to 4.5 with hydrochloric acid, heat to 120 ° C for 4h, then cool to room temperature, filter, filtrate for HPD-100 macroporous resin column chromatography, first elute with water, volume fraction of 20% ethanol solution Then, elute with a 50% aqueous solution of ethanol, collect the eluate containing salvianolic acid A, concentrate to an alcohol-free taste, and continue to separate the polyamide column, first elute with water and a volume fraction of 35% ethanol solution. Miscellaneous, and eluted with 75% aqueous solution of ethanol, collected the eluate containing salvianolic acid A, concentrated to an alcohol-free taste, adjusted to pH 3.0 with potassium bicarbonate solution, extracted with ethyl acetate, extract After concentration and drying, salvianolic acid A 816g was obtained in an amount of 93.20%.

Example 2

The raw material Danshen 200kg and the volume fraction 50% ethanol aqueous solution 2000L were mixed, extracted at 82 ° C for 2h, the filtrate was collected by filtration, the filter residue was double-recovered twice, and the obtained filtrate was combined to obtain the Salvia miltiorrhiza extract; the obtained Salvia miltiorrhiza extract was concentrated to 400L, adjust the pH to 4.4 with hydrochloric acid solution, heat to 120 ° C for 2.5h, then cool to room temperature, filter, filtrate for HPD-300 macroporous resin column chromatography, first elute with water, volume fraction of 20% ethanol solution Miscellaneous, and then eluted with a 50% aqueous solution of ethanol, collected eluate containing salvianolic acid A, concentrated to an alcohol-free taste, and then separated on a polyamide column, first eluted with water, a volume fraction of 35% ethanol After removing impurities, elute with a 75% aqueous solution of ethanol, collect the eluate containing salvianolic acid A, concentrate to an alcohol-free taste, adjust the pH to 3.0 with potassium carbonate solution, extract with propyl acetate, extract After concentration and drying, salvianolic acid A 808 g was obtained in an amount of 93.80%.

Example 3

The raw material Danshen 200kg and the volume fraction 50% ethanol aqueous solution 2000L were mixed, extracted at 82 ° C for 2h, the filtrate was collected by filtration, the filter residue was double-recovered twice, and the obtained filtrate was combined to obtain the Salvia miltiorrhiza extract; the obtained Salvia miltiorrhiza extract was concentrated to 400L, adjust the pH to 4.6 with hydrochloric acid solution, heat to 120 ° C for 3h, then cool to room temperature, filter, the filtrate is separated by 1400 macroporous resin column chromatography, first elute with water, volume fraction of 20% ethanol solution, and then remove impurities. The eluate containing salvianolic acid A was collected by elution with a volume fraction of 50% aqueous ethanol solution, concentrated to an alcohol-free taste, and then separated by a polyamide chromatography column, and then eluted with water and a 35% aqueous solution of ethanol. After eluting with a 75% aqueous solution of ethanol, the eluate containing salvianolic acid A was collected, concentrated to an alcohol-free taste, adjusted to pH 3.0 with magnesium hydrogencarbonate solution, extracted with butyl acetate, and concentrated. , dried, to obtain salvianolic acid A 797g, the content of 93.50%.

Example 4

200 kg of raw material Danshen and 1600 L of 70% ethanol solution of ethanol were mixed, and extracted at 80 ° C for 2 h. The filtrate was collected by filtration, and the residue was re-extracted 3 times. The obtained filtrate was combined to obtain Salvia miltiorrhiza extract. The obtained Salvia miltiorrhiza extract was concentrated to 600L, adjust the pH to 3.5 with hydrochloric acid, heat to 120 ° C for 3.5h, then cool to room temperature, filter, the filtrate is separated by 1400 macroporous resin column chromatography, first elution with water, volume fraction of 20% ethanol solution, The eluate containing salvianolic acid A was collected and eluted with a 50% aqueous solution of ethanol. The mixture was concentrated to an alcohol-free atmosphere and then separated on a polyamide column. The mixture was eluted with water and a 35% aqueous solution of ethanol. Then, eluting with a 70% aqueous solution of ethanol, collecting the eluate containing salvianolic acid A, concentrating to an alcohol-free taste, adjusting the pH to 3.0 with sodium hydrogen carbonate solution, extracting with ethyl acetate, and extracting the solution Concentrated and dried to obtain 903 g of salvianolic acid A with a content of 91%.

Example 5

200 kg of raw material Salvia miltiorrhiza and 2400 L of a 90% ethanol aqueous solution were mixed, and extracted at 78 ° C for 3 h. The filtrate was collected by filtration, and the residue was twice extracted. The obtained filtrate was combined to obtain Salvia miltiorrhiza extract. The obtained Salvia miltiorrhiza extract was concentrated to 800L, adjust the pH to 4.5 with nitric acid solution, heat to 130 ° C for 1 h, then cool to room temperature, filter, filtrate and 1300-I macroporous resin column chromatography, first elute with water, volume fraction of 30% ethanol solution Then, elute with a 70% aqueous solution of ethanol, collect the eluate containing salvianolic acid A, concentrate to an alcohol-free taste, and continue to separate the polyamide column, first elute with water, volume fraction of 50% ethanol solution. Miscellaneous, and then eluted with 95% aqueous solution of ethanol, collected eluate containing salvianolic acid A, concentrated to an alcohol-free taste, adjusted to pH 2.0 with sodium carbonate solution, extracted with butyl acetate, the extract was Concentrated and dried to obtain salvianolic acid A 711 g, content 84%.

Example 6

The raw material Danshen 200kg and water 1200L were mixed, extracted at 100 ° C for 1 h, the filtrate was collected by filtration, and the filter residue was re-extracted once, and the obtained filtrate was combined to obtain Salvia miltiorrhiza extract; the obtained Salvia miltiorrhiza extract was concentrated to 400 L, and adjusted with nitric acid solution. pH to 6, warmed to 110 ° C for 6h, then cooled to room temperature, filtered, the filtrate was separated by AB-8 macroporous resin column chromatography, first eluted with water, volume fraction of 10% ethanol solution, and then used to volume fraction 30 After elution with % ethanol solution, the eluate containing salvianolic acid A was collected, concentrated to an alcohol-free taste, and then separated by polyamide chromatography column, first eluted with water, volume fraction of 20% aqueous ethanol solution, and then used for volume fraction. After elution with 50% ethanol aqueous solution, the eluate containing salvianolic acid A was collected, concentrated to an alcohol-free taste, adjusted to pH 5.0 with sodium hydroxide solution, extracted with propyl acetate, and the extract was concentrated and dried to obtain Salvianolic acid A 495g, content 63%.

Example 7

250 kg of raw salvia miltiorrhiza and 2000 L of aqueous solution of 60% by volume were mixed and extracted at 81 ° C for 3 h. The filtrate was collected by filtration, and the residue was twice extracted. The obtained filtrate was combined to obtain Salvia miltiorrhiza extract. The obtained Salvia miltiorrhiza extract was concentrated to 645L, adjust the pH to 4.5 with nitric acid solution, heat to 120 ° C for 3h, then cool to room temperature, filter, the filtrate is separated by 1400 macroporous resin column chromatography, first elute with water, volume fraction of 20% ethanol solution, and then remove impurities. The eluate containing salvianolic acid A was collected by elution with a volume fraction of 40% aqueous solution of ethanol, concentrated to an alcohol-free taste, and then separated by a polyamide chromatography column, and then eluted with water and a 40% aqueous solution of ethanol. The eluate containing salvianolic acid A was collected by elution with a volume fraction of 80% aqueous ethanol solution, concentrated to an alcohol-free taste, adjusted to pH 3.2 with sodium bicarbonate solution, extracted with ethyl acetate, and concentrated. , dried, to obtain salvianolic acid A 1260g, content 95.8%.

Example 8

250 kg of raw salvia miltiorrhiza and 2000 L of aqueous solution of 60% by volume were mixed and extracted at 81 ° C for 3 h. The filtrate was collected by filtration, and the residue was twice extracted. The obtained filtrate was combined to obtain Salvia miltiorrhiza extract. The obtained Salvia miltiorrhiza extract was concentrated to 638L, adjust the pH to 4.6 with nitric acid solution, heat to 120 ° C for 3h, then cool to room temperature, filter, the filtrate is separated by 1400 macroporous resin column chromatography, first elute with water, volume fraction of 20% ethanol solution, and then remove impurities. The eluate containing salvianolic acid A was collected by elution with a volume fraction of 40% aqueous solution of ethanol, concentrated to an alcohol-free taste, and then separated by a polyamide chromatography column, and then eluted with water and a 40% aqueous solution of ethanol. The eluate containing salvianolic acid A was collected by elution with a volume fraction of 80% aqueous ethanol solution, concentrated to an alcohol-free taste, adjusted to pH 3.2 with sodium bicarbonate solution, extracted with ethyl acetate, and concentrated. , dried, to obtain salvianolic acid A 1600g, the content of 98.2%.

Example 9

250 kg of raw salvia miltiorrhiza and 2000 L of aqueous solution of 60% by volume were mixed and extracted at 81 ° C for 3 h. The filtrate was collected by filtration, and the residue was twice extracted. The obtained filtrate was combined to obtain Salvia miltiorrhiza extract. The obtained Salvia miltiorrhiza extract was concentrated to 635L, adjust the pH to 4.6 with nitric acid solution, heat to 120 ° C for 3h, then cool to room temperature, filter, the filtrate is separated by 1400 macroporous resin column chromatography, first elute with water, volume fraction of 20% ethanol solution, and then remove impurities. The eluate containing salvianolic acid A was collected by elution with a volume fraction of 40% aqueous solution of ethanol, concentrated to an alcohol-free taste, and then separated by a polyamide chromatography column, and then eluted with water and a 40% aqueous solution of ethanol. The eluate containing salvianolic acid A was collected by elution with a volume fraction of 80% aqueous ethanol solution, concentrated to an alcohol-free taste, adjusted to pH 3.2 with sodium bicarbonate solution, extracted with ethyl acetate, and concentrated. , dried, to obtain salvianolic acid A 1554g, the content of 98.5%.

Examples 10 to 24 are preparation examples of salvianolic acid A salt.

Example 10

The 600 mg of salvianolic acid A prepared in Example 1 was dissolved in 2400 mL of water at a weight ratio of 2:8, filtered, and the pH was adjusted to 1.2 by adding 5% sodium carbonate solution, and the precipitate was collected and dried under normal reduced pressure. The salvianolic acid A sodium salt was 336 g, and the purity was 99.90%.

Example 11

Take 600 mg of salvianolic acid A prepared in Example 2, dissolve it in 1400 mL of water at a weight ratio of 3:7, filter, add 15% sodium hydroxide solution to adjust the pH to 1.8, treat with activated carbon, filter, collect precipitates, reduce Press dry to obtain 343 g of salvianolic acid A sodium salt with a purity of 99.50%.

Example 12

The 600 mg of salvianolic acid A prepared in Example 3 was dissolved in 900 mL of water at a weight ratio of 4:6, and the pH was adjusted to 1.5 by adding 1% sodium tartrate solution, activated carbon, filtered, and the precipitate was collected and dried under reduced pressure. The salvianolic acid A sodium salt was 343 g, and the purity was 99.50%.

Example 13

100 g of salvianolic acid A prepared in Example 6 was dissolved in 100 mL of water by weight ratio of 5:5, and the pH was adjusted to 1.2 by adding 5% sodium carbonate solution, and the precipitate was collected by filtration and dried under reduced pressure to obtain dansyl alcohol. Acid A sodium salt 34g, purity 91.10%.

Example 14

100 g of salvianolic acid A prepared in Example 5 was dissolved in 100 mL of water at a weight ratio of 5:5, and the pH was adjusted to 1.2 by adding 5% sodium carbonate solution, and the precipitate was collected by filtration and dried under reduced pressure to obtain dansyl alcohol. Acid A sodium salt 47g, purity 97.70%.

Example 15

100 g of salvianolic acid A prepared in Example 4 was dissolved in 100 mL of water by weight ratio of 5:5, and the pH was adjusted to 1.2 by adding 5% sodium carbonate solution, and the precipitate was collected by filtration and dried under reduced pressure to obtain dansyl alcohol. Acid A sodium salt 57g, purity 98.30%.

Example 16

100 g of salvianolic acid A prepared in Example 6 was dissolved in 100 mL of water at a weight ratio of 5:5, and the pH was adjusted to 3.5 by adding 5% magnesium hydrogencarbonate solution, and the precipitate was collected by filtration, and dried under reduced pressure. Phenolic acid A magnesium salt 36g, purity 92.10%.

Example 17

100 g of salvianolic acid A prepared in Example 5 was dissolved in 100 mL of water at a weight ratio of 5:5, and the pH was adjusted to 3.5 by adding 5% magnesium hydrogencarbonate solution, and the precipitate was collected by filtration and dried under reduced pressure. The phenolic acid A magnesium salt was 51 g, and the purity was 97.20%.

Example 18

100 g of salvianolic acid A prepared in Example 4 was dissolved in 100 mL of water by weight ratio of 5:5, and the pH was adjusted to 3.5 by adding 5% magnesium hydrogencarbonate solution, and the precipitate was collected by filtration and dried under reduced pressure. Phenolic acid A magnesium salt 58g, purity 98.60%.

Example 19

100 g of salvianolic acid A prepared in Example 6 was dissolved in 100 mL of water at a weight ratio of 5:5, and the pH was adjusted to 5.5 by adding 5% potassium hydrogencarbonate solution, and the precipitate was collected by filtration, and dried under reduced pressure. Phenolic acid A potassium salt 39g, purity 90.40%.

Example 20

100 g of salvianolic acid A prepared in Example 5 was dissolved in 100 mL of water at a weight ratio of 5:5, and the pH was adjusted to 5.5 by adding 5% potassium hydrogencarbonate solution, and the precipitate was collected by filtration, and dried under reduced pressure. Phenolic acid A potassium salt 52g, purity 96.80%.

Example 21

100 g of salvianolic acid A prepared in Example 4 was dissolved in 100 mL of water at a weight ratio of 5:5, and the pH was adjusted to 5.5 by adding 5% potassium hydrogencarbonate solution, and the precipitate was collected by filtration, and dried under reduced pressure. Phenolic acid A potassium salt 58g, purity 97.50%.

Example 22

Take 1260 g of salvianolic acid A prepared in Example 7, dissolved in 1030 mL of water at a weight ratio of 6:4, adjust the pH to 3.5 by adding 5% sodium hydroxide solution, filter, collect the precipitate, and dry it under reduced pressure. The phenolic acid A sodium salt was 812 g, and the purity was 99.80%.

Example 23

Take 1600 g of salvianolic acid A prepared in Example 8, dissolved in 1310 mL of water at a weight ratio of 6:4, adjust the pH to 3.5 by adding 5% sodium hydroxide solution, filter, collect the precipitate, and dry it under reduced pressure. Phenolic acid A sodium salt 948g, purity 99.80%.

Example 24

1554 g of salvianolic acid A prepared in Example 9 was dissolved in 1269 mL of water at a weight ratio of 6:4, and the pH was adjusted to 3.4 by adding 5% sodium hydroxide solution, and the precipitate was collected by filtration, and dried under reduced pressure. Phenolic acid A sodium salt 915 g, purity 99.80%.

The following is an analysis of the results of the technical solution of the present invention:

The purity of salvianolic acid A salt was obtained at different pH values for different purity raw materials, as shown in Table 1.

The different purity raw materials, salvianolic acid A, are dissolved in water, and an alkali solution is added to adjust different pH values, and the precipitates are collected to obtain salicylic acid A salt.

Table 1

The yields were dissolved in different weight ratios as shown in Table 2.

The raw material salvianolic acid A is dissolved in different proportions of water, and the pH is adjusted to 3.6 by adding an alkali solution, and the precipitate is dried to obtain salicylic acid A salt.

Table 2

The stability of salvianolic acid A before and after salt formation is shown in Table 3.

table 3

Conclusion: The salicylic acid A salt of Example 10 was prepared from Example 1 and the salicylic acid A salt of Example 11 was prepared from Example 2 salvianolic acid A, and was prepared from Example 3 salvianolic acid A. Example 12 salicylic acid A salt; from the data in the table, the content of salvianolic acid A decreased from about 93% to about 75% after one year of storage, and the impurity increased from 4% to 9%, and the salvianolic acid A was prepared. After the salt, there is no change before and after the placement, so it is quite stable after the salt is formed.

High temperature, high humidity, strong light test

Table 4 High temperature inspection

Table 5 High humidity inspection

Table 6 Light inspection

Conclusion: The salicylic acid A salt of Example 10 was prepared from the salicylic acid A of Example 1. From the experimental data of high temperature, high humidity and strong light, it was found that the salt was more stable after salt formation.

The solubility of salicylate A before and after salt formation was compared as shown in Table 7.

Table 7

Salvianolic acid A example	Salvianolic acid A	Water soluble	Salvianolic acid A salt example	Salvianolic acid A sodium salt	Water soluble
6	63%	Very soluble	16	92.1%	Slightly soluble
5	84%	Very soluble	17	97.2%	Slightly soluble
4	91%	Very soluble	18	98.6%	Slightly soluble

1 to 4 are respectively high-resolution mass spectrometry, ¹ H NMR spectrum, ¹³ C NMR spectrum, and HMQC spectrum of salvianolic acid A sodium salt prepared by the present invention.

High resolution mass spectrometry

(1) Test equipment: Qstar Elite (Applietl Biosyhtems/MBS Sclex)

Determination of elements: C, H, O, Na

(2) The measurement results are shown in Table 8 below:

Table 8 High Resolution Mass Spectrometry Analysis Results Data Sheet

High resolution mass spectrometry data analysis:

High-resolution mass spectrometry uses the ESI ⁺ ion mode, so the high-resolution mass spectrometry results in a molecular formula that is one more hydrogen than the sample formula. The results of high-resolution mass spectrometry showed that there was a sodium ion in the molecule, indicating that salvianolic acid A exists in the form of sodium salt, and the salvianolic acid A and sodium ion in this product are 1:1 salt.

¹ H-NMR nuclear magnetic resonance spectroscopy

(1) Instrument: MP-400

Solvent: DMSO-d ₆ , internal standard: TMS

(2) ¹ H NMR nuclear magnetic resonance spectrum, see Figure 2

(3) The measured data is shown in Table 9 below:

Table 9 ¹ H NMR nuclear magnetic resonance measurement data sheet

Atomic number	chemical shift	Peak shape	Number of protons	Corresponding proton	J (Hz)
A-5	6.80	d	1	Φ-H	8.4
A-6	7.16	d	1	Φ-H	8.4
A-7	7.88	d	1	=C-H	15.6
A-8	6.29	d	1	=C-H	15.6
B-2	6.75	d	1	Φ-H	8.4
B-3	6.47	d	1	Φ-H	8.4
B-6	6.67	s	1	Φ-H
B-7α	2.81	m	1	-CH 2
B-7β	3.01	d	1	-CH 2
B-8	4.98	Dd	1	-OCH
C-2	7.01	s	1	Φ-H
C-5	6.60	d	1	Φ-H
C-6	6.79	d	1	Φ-H
C-7	6.53	d	1	=C-H	16.8
C-8	7.04	d	1	=C-H	16.2

¹ H NMR nuclear magnetic resonance data analysis:

(1) The ¹ H-NMR spectrum showed a peak of δ 2.5, and a broad peak appeared at δ 3.2-4.8, indicating that a water molecule was present in the molecule, which was consistent with the TGA data of the sample.

(2) ¹ H-NMR spectrum showed a broad peak of δ 9.03, containing about 6 protons, and should be 6 phenolic hydroxyl hydrogen protons contained in the molecule.

(3) High field, δ2.81, multiple peaks, containing one hydrogen proton; δ3.01, doublet, containing one hydrogen proton, respectively two hydrogen protons on methylene B7, subject to ortho chirality The effects of carbon produce different chemical shifts. δ 4.98 is the hydrogen proton signal on the methine carbon B8 attached to the carboxyl group and the ester group.

(4) The low field region, δ7.88 and δ6.29, each containing 1 proton, double peak, coupling constant is 15.6, which is trans-hydrogen A7, hydrogen proton on A8, δ7.04 and δ6.53, each Containing 1 proton, bimodal, coupling constant of about 16.2, is a hydrogen proton on trans-olefinic hydrogen C7, C8, indicating that there are two sets of trans double bond hydrogen atoms in the molecule.

(5) The low field region, δ6.2～7.9, contains a total of 12 protons, 4 of which are hydrogen protons on the trans-olefin hydrogen, and the remaining 8 are aromatic ring hydrogen protons, which are completely consistent with the molecular structure.

The nuclear magnetic resonance spectrum data showed that the nuclear magnetic resonance spectrum of salvianolic acid A sodium was basically consistent with the nuclear magnetic resonance spectrum of salvianolic acid A, and the displacement of some hydrogen protons was slightly shifted.

¹³ C-NMR nuclear magnetic resonance spectroscopy

(1) Instrument: ARX400

Solvent: DMSO-d6, internal standard: TMS

(2) ¹³ C NMR nuclear magnetic resonance spectrum, see Figure 3

C, H related two-dimensional nuclear magnetic resonance (HMQC) spectrum see Figure 4

(3) The measured data is shown in Table 10 below:

Table 10 ¹³ CNMR NMR data sheet

¹³ C-NMR nuclear magnetic resonance spectroscopy data analysis:

(1) ¹³ CNMR spectra give a total of 25 carbon atom signal peaks, of which δ119.24 contains 2 carbon protons, indicating 26 carbons in the molecule.

(2) In the high field, δ36.85 is methylene carbon, which is related to two hydrogens in the HMQC diagram, which is B7 carbon; δ74.58 is methine carbon, which is related to one hydrogen in the HMQC diagram. For B8 carbon. Compared with the carbon spectrum of salvianolic acid A, it was found that the displacement of B7 carbon shifted slightly to the high field, and the displacement of B8 carbon shifted slightly to the lower field due to the influence of sodium salt formation.

(3) The low field region, δ172.31, is the carbonyl carbon at the B9 position in the carboxylate; δ166.43, which is the carbonyl carbon at the A9 position in the ester group.

(4) A carbon atom having a chemical shift at δ 113 to 148 is an aromatic ring carbon atom or an olefin carbon atom. The chemical shift of the unsubstituted carbon atom on the benzene ring is δ 113-121, and the carbon atom having a chemical shift of δ 143-147 is a carbon atom connected to the hydroxyl group on the benzene ring. δ 119.24, containing 2 carbon atoms, is associated with 2 aromatic hydrogens in the HMQC correlation diagram.

The nuclear magnetic carbon spectrum data showed that the carbon spectrum of salvianolic acid A sodium salt compared with the carbon spectrum of salvianolic acid A, the chemical shift of carbonyl carbon B9 and its attached B8 carbon shifted slightly to the low field, and the displacement of other carbon atoms was basically the same.

Claims (9)

1. A method for preparing salvianolic acid A salt, characterized in that the preparation method is as follows:

   The salvianolic acid A is dissolved in water, and the pH is adjusted to 1.2 to 5.5 with an aqueous solution of an alkaline substance, and the precipitate is collected to be the salvianolic acid A salt.
2. The method for producing salvianolic acid A salt according to claim 1, wherein the pH is adjusted to 3.2 to 4.2.
3. The method for producing salvianolic acid A salt according to claim 1, wherein the mass ratio of the salvianolic acid A to water is 1:0.25-4.
4. The method for producing a salvianolic acid A salt according to claim 3, wherein a mass ratio of the salvianolic acid A to water is 1:0.67 to 1.5.
5. The method for producing a salvianolic acid A salt according to claim 1, wherein the aqueous solution of the basic substance has a concentration of from 1% to 15%.
6. The method for producing a salvianolic acid A salt according to claim 5, wherein the aqueous solution of the basic substance has a concentration of 4% to 11%.
7. The method for preparing a salvianolic acid A salt according to claim 1, wherein the aqueous solution of the alkaline substance is prepared by dissolving the following basic substance in water: sodium hydroxide, sodium hydrogencarbonate, sodium carbonate, Potassium hydroxide, potassium hydrogencarbonate, potassium carbonate, magnesium hydroxide, magnesium hydrogencarbonate, magnesium carbonate, calcium hydroxide, calcium hydrogencarbonate, calcium carbonate, sodium citrate, sodium tartrate, potassium citrate, potassium tartrate, magnesium citrate Or magnesium tartrate.
8. The method for preparing salvianolic acid A salt according to claim 1, wherein the salvianolic acid A is extracted from the raw material Danshen by water or ethanol aqueous solution, separated by macroporous resin column chromatography, and the polyamide chromatography column is used. Prepared by separation.
9. The method for preparing salvianolic acid A salt according to claim 8, wherein the salvianolic acid A is prepared as follows:

   (1) Mixing the raw material Danshen according to the mass ratio of the liquid: 1:6~12 with water or volume fraction of 10%-90% ethanol solution, extracting at 70-100 °C for 1-3 hours, collecting the filtrate by filtration, and extracting the residue. ~3 times, combining the filtrates obtained each time to obtain the extract of Salvia miltiorrhiza;

   (2) taking the salvia miltiorrhiza extract obtained in the step (1), concentrating, adjusting the pH to 3.5 to 6.0, heating to 110 to 130 ° C for 1 to 6 hours, then cooling to room temperature, filtering, and separating the filtrate by macroporous resin column chromatography. First elute with water, volume fraction 10% ~ 30% ethanol solution, and then elute with 30% ~ 70% ethanol aqueous solution, collect eluate containing salvianolic acid A, concentrate and continue polyamide chromatography Separation of the column, first elution with water, volume fraction 20% ~ 50% ethanol aqueous solution, and then eluted with 50% ~ 95% ethanol aqueous solution, collect eluate containing salvianolic acid A, concentrate and adjust the pH value To 2 to 5, extracted with an organic solvent, the extract is concentrated and dried to obtain the salvianolic acid A;

   The macroporous resin is HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101, 1300-I, 1400 or AB-8;

   The organic solvent used for the extraction is ethyl acetate, propyl acetate, butyl acetate, n-butanol or isopropanol.

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