1. A method for efficiently preparing salvianolic acid B and lithospermic acid is characterized in that a pH zone countercurrent chromatography is adopted to separate and purify a sample containing salvianolic acid B and lithospermic acid; the solvent system of the pH zone countercurrent chromatography is composed of tert-butyl methyl ether and water, the volume ratio of tert-butyl methyl ether to water is 1: 0.9-1.1, an upper phase solution of the solvent system is used as a stationary phase, a lower phase solution of the solvent system is used as a mobile phase, acid is added into the stationary phase, and alkali is added into the mobile phase.

2. The method according to claim 1, wherein the acid concentration in the stationary phase is 4.5 to 5.5mM, and the alkali concentration in the mobile phase is 59 to 61 mM.

3. The method according to claim 1, wherein the acid is trifluoroacetic acid and the base is aqueous ammonia.

4. Use of the method of any one of claims 1 to 3 for extracting salvianolic acid B and lithospermic acid from Salvia miltiorrhiza Bunge.

5. A method for extracting salvianolic acid B and lithospermic acid from Salvia miltiorrhiza Bunge is characterized by comprising the following steps:

(1) extracting total phenolic acid of Yunnan red sage root from Yunnan red sage root;

(2) separating and purifying the total salvianolic acid extract by pH zone countercurrent chromatography to obtain fraction containing salvianolic acid B and lithospermic acid;

(3) separating and purifying the fraction containing salvianolic acid B and lithospermic acid by pH zone countercurrent chromatography;

wherein the solvent system of the pH zone countercurrent chromatography in the step (3) consists of tert-butyl methyl ether and water, the volume ratio of tert-butyl methyl ether to water is 1: 0.9-1.1, the upper phase solution of the solvent system is used as a stationary phase, the lower phase solution of the solvent system is used as a mobile phase, acid is added into the stationary phase, and alkali is added into the mobile phase.

6. The extraction process according to claim 5, wherein in the step (3), the concentration of the acid in the stationary phase is 4.5 to 5.5mM, and the concentration of the base in the mobile phase is 59 to 61 mM;

or, in the step (3), the acid concentration in the stationary phase is 4.5-5.5 mM, and the alkali concentration in the mobile phase is 59-61 mM;

or, in the step (3), the acid is trifluoroacetic acid, and the base is ammonia water.

7. The extraction method according to claim 5, wherein in the step (1), the process for extracting the total phenolic acid extract of the salvia yunnanensis from the salvia yunnanensis comprises the following steps: extracting the salvia miltiorrhiza bunge by adopting an ethanol extraction method, carrying out primary extraction on the extracted liquid by adopting petroleum ether, adjusting the pH of the extracted water phase to 1.5-2.5, then adding ethyl acetate for secondary extraction, and concentrating the ethyl acetate phase obtained by the secondary extraction to obtain the total phenolic acid extract of the salvia miltiorrhiza bunge.

8. The extraction method as claimed in claim 7, wherein the Salvia yunnanensis is added into ethanol water solution, reflux extraction is carried out at 65-75 ℃, the extract is concentrated and then added with water for redissolution, and then petroleum ether is adopted for primary extraction; preferably, the concentration of the ethanol water solution is 55-65% by volume fraction; preferably, the material-liquid ratio of the salvia yunnanensis to the ethanol water solution is 1: 7.5-8.5; preferably, the reflux extraction times are 2-4 times, and the time of each reflux extraction is 1.5-2.5 h.

9. The extraction method as claimed in claim 5, wherein the pH zone countercurrent chromatographic separation extraction process comprises: conveying the stationary phase to a separation column, injecting the solution of the total phenolic acid extract of the Yunnan red sage root into the separation column, and conveying the mobile phase to the separation column continuously for separation and extraction; preferably, the flow rate of conveying the stationary phase to the separation column is 25-35 mL/min; preferably, the flow rate of the mobile phase is 1.5-2.5 mL/min; preferably, in the pH zone countercurrent chromatographic separation and extraction, the detection wavelength of the ultraviolet detector is 252-256 nm.

10. The extraction method as claimed in claim 5, wherein in the step (2), the solvent system of pH zone countercurrent chromatography comprises petroleum ether, ethyl acetate, acetonitrile and water, wherein the volume ratio of the petroleum ether to the ethyl acetate to the acetonitrile to the water is 1.46-1.54: 2.46-2.54: 1: 4.6-5.4; preferably, the concentration of trifluoroacetic acid in the stationary phase is 9-11 mM, and the concentration of ammonia in the mobile phase is 29-31 mM.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Salvia yunnanensis Radix (Salvia yunnanensis Radix) is dry root of Salvia yunnanensis C.H. Wright (Salvia yunnanensis C.H. Wright) of Labiatae, also called as Salvia miltiorrhiza Radix, has a long medicinal history in Yunnan and Guizhou areas, and is recorded in Yunnan Bencao in Ming Dynasty. The activity of the Yunnan red sage root is similar to that of the red sage root (Salvia milithiorrhizibge), and the biological activity of the Yunnan red sage root is mainly the influence on a circulatory system, such as improving microcirculation and blood circulation functions, improving coronary blood flow of heart, repairing heart function, reducing blood fat, preventing atherosclerosis, protecting liver, treating gastric ulcer, easing pain, resisting oxidation, resisting bacteria, resisting inflammation, resisting tumors and the like. In addition, researches show that the salvia miltiorrhiza bunge has obvious effect of inhibiting platelet aggregation, and the inhibition effect is obviously better than that of the salvia miltiorrhiza bunge. Phenolic acid compounds in the salvia yunnanensis mainly play roles in resisting inflammation, oxidation and thrombus and protecting cardiovascular, and are represented by salvianolic acid A-K, wherein the content of salvianolic acid B is highest. However, the inventors have found that the structure of salvianolic acid B is similar to that of lithospermic acid, and it is difficult to obtain high-purity salvianolic acid B and lithospermic acid simultaneously by common chromatography.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a method for efficiently preparing salvianolic acid B and lithospermic acid, which can simultaneously obtain the salvianolic acid B and the lithospermic acid with the purity of over 95 percent.

The pH zone countercurrent chromatography is a liquid-liquid chromatography technology, realizes the separation of components according to the difference of pKa and polarity of a compound, and has the advantages of high separation efficiency, good reproducibility, large sample injection amount and the like. The invention firstly adopts various solvent systems (such as petroleum ether-ethyl acetate-methanol-water, ethyl acetate-n-butanol-water, ethyl acetate-water and chloroform-methanol-water), but the separation and extraction of the salvianolic acid B and the lithospermic acid are difficult to realize, and when tert-butyl methyl ether-acetonitrile-water (4:1:5, v/v) is adopted as a solvent system, the result shows that the solvent system can realize the separation and extraction of the salvianolic acid B and the lithospermic acid, but the separation and extraction time is longer, 9 hours are needed only to elute the salvianolic acid B, and the separation and extraction of the salvianolic acid B and the lithospermic acid by pH zone countercurrent chromatography is difficult due to the longer separation and extraction time.

In order to achieve the purpose, the technical scheme of the invention is as follows:

on the one hand, the method for efficiently preparing the salvianolic acid B and the lithospermic acid adopts pH zone countercurrent chromatography to separate and purify a sample containing the salvianolic acid B and the lithospermic acid; the solvent system of the pH zone countercurrent chromatography is composed of tert-butyl methyl ether and water, the volume ratio of tert-butyl methyl ether to water is 1: 0.9-1.1, an upper phase solution of the solvent system is used as a stationary phase, a lower phase solution of the solvent system is used as a mobile phase, acid is added into the stationary phase, and alkali is added into the mobile phase.

According to the invention, through research, when tert-butyl methyl ether-acetonitrile-water (4:1:5, v/v/v) is used as a solvent system, the solubility of a sample in acetonitrile is better than that in water and tert-butyl methyl ether, and the affinity of acetonitrile to tert-butyl methyl ether is better, so that the retention time of the sample is longer. Therefore, the solvent system is adjusted again, and experiments show that the salvianolic acid B and the lithospermic acid can be separated within 13 hours by adopting the solvent system, so that the separation time of the salvianolic acid B and the lithospermic acid is greatly reduced.

Meanwhile, further research shows that the acid concentration of the stationary phase and the alkali concentration of the mobile phase can also reduce the separation time of the salvianolic acid B and the lithospermic acid, and when the acid concentration of the stationary phase is 4.5-5.5 mM and the alkali concentration of the mobile phase is 59-61 mM, the separation time of the salvianolic acid B and the lithospermic acid can be shortened to 9 hours.

On the other hand, the application of the method in extracting salvianolic acid B and lithospermic acid from the salvia miltiorrhiza bunge is provided.

Because the content of the salvianolic acid B in the salvia miltiorrhiza bunge is high and the lithospermic acid exists, the method is used for extracting the salvia miltiorrhiza bunge, and the salvianolic acid B and the lithospermic acid in the salvia miltiorrhiza bunge can be effectively extracted simultaneously.

In a third aspect, a method for extracting salvianolic acid B and lithospermic acid from Salvia yunnanensis comprises the following steps:

(1) extracting total phenolic acid of Yunnan red sage root from Yunnan red sage root;

(2) separating and purifying the total salvianolic acid extract by pH zone countercurrent chromatography to obtain fraction containing salvianolic acid B and lithospermic acid;

(3) separating and purifying the fraction containing salvianolic acid B and lithospermic acid by pH zone countercurrent chromatography;

wherein the solvent system of the pH zone countercurrent chromatography in the step (3) consists of tert-butyl methyl ether and water, the volume ratio of tert-butyl methyl ether to water is 1: 0.9-1.1, the upper phase solution of the solvent system is used as a stationary phase, the lower phase solution of the solvent system is used as a mobile phase, acid is added into the stationary phase, and alkali is added into the mobile phase.

When phenolic acid compounds in the salvia yunnanensis are extracted, the salvianolic acid B and the lithospermic acid in the salvia yunnanensis are difficult to separate by adopting one-time pH-zone countercurrent chromatography, so that the salvianolic acid B and the lithospermic acid in the salvia yunnanensis are difficult to separate by adopting one-time pH-zone countercurrent chromatography, the separation and purification of the salvianolic acid B and the lithospermic acid are further carried out on the basis of one-time pH-zone countercurrent chromatography, and the separation of the salvianolic acid B and the lithospermic acid can be realized only by using a tert-butyl methyl ether-acetonitrile-water (4:1:5, v/v/v/v) solvent system through the selection of various solvent systems, but the separation time is extremely long, and experiments show that the salvianolic acid B and the lithospermic acid can be completely separated by adopting the tert-butyl methyl ether and the water as the solvent systems, and the separation time is greatly shortened.

The invention has the beneficial effects that:

1. according to the invention, tert-butyl methyl ether and water are used as a solvent system of the pH zone countercurrent chromatography, so that the salvianolic acid B and the lithospermic acid can be completely separated, the separation rate of the salvianolic acid B and the lithospermic acid can be increased, and the separation time of the salvianolic acid B and the lithospermic acid can be shortened.

2. The method adopts pH zone countercurrent chromatography to separate the salvianolic acid B and the lithospermic acid, not only can extract a large amount of salvianolic acid B and lithospermic acid from the salvia miltiorrhiza bunge, but also has higher purity of the obtained salvianolic acid B and lithospermic acid, and can reach more than 95 percent.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a High Performance Liquid Chromatography (HPLC) analysis chart of the total phenolic acid extract of Salvia yunnanensis with pure components separated by pH-ZRCCC according to the present invention;

FIG. 2 is a graph of the separation and enrichment of pH-ZRCCC in an example of the present invention;

FIG. 3 is a graph showing the separation of pH-ZRCCC in the example of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The chemical structure of the salvianolic acid B is

The chemical structure of alkannic acid is

In view of the fact that a solvent system of a pH zone countercurrent chromatography has a large influence on the separation effect and the separation efficiency of the salvianolic acid B and the lithospermic acid, the invention provides a method for efficiently preparing the salvianolic acid B and the lithospermic acid.

The invention provides a typical embodiment of a method for efficiently preparing salvianolic acid B and lithospermic acid, which adopts pH zone countercurrent chromatography to separate and purify a sample containing salvianolic acid B and lithospermic acid; the solvent system of the pH zone countercurrent chromatography is composed of tert-butyl methyl ether and water, the volume ratio of tert-butyl methyl ether to water is 1: 0.9-1.1, an upper phase solution of the solvent system is used as a stationary phase, a lower phase solution of the solvent system is used as a mobile phase, acid is added into the stationary phase, and alkali is added into the mobile phase.

According to the invention, through research, when tert-butyl methyl ether-acetonitrile-water (4:1:5, v/v/v) is used as a solvent system, the solubility of a sample in acetonitrile is better than that in water and tert-butyl methyl ether, and the affinity of acetonitrile to tert-butyl methyl ether is better, so that the retention time of the sample is longer. Therefore, the solvent system is adjusted again, and experiments show that the salvianolic acid B and the lithospermic acid can be separated within 13 hours by adopting the solvent system, so that the separation time of the salvianolic acid B and the lithospermic acid is greatly reduced.

And (3) uniformly mixing tert-butyl methyl ether and water, standing, separating liquid, wherein the upper phase after liquid separation is a stationary phase, and the lower phase after liquid separation is a mobile phase.

In some embodiments, the acid concentration in the stationary phase is 4.5 to 5.5mM and the base concentration in the mobile phase is 59 to 61 mM. Experiments show that under the condition, the efficiency of separating the salvianolic acid B and the lithospermic acid is further improved, so that the separation time of the salvianolic acid B and the lithospermic acid is further shortened.

In some embodiments, the acid is trifluoroacetic acid and the base is aqueous ammonia.

The invention also provides an application of the method in extracting salvianolic acid B and lithospermic acid from the salvia miltiorrhiza bunge.

The third embodiment of the invention provides a method for extracting salvianolic acid B and lithospermic acid from Yunnan red sage root, which comprises the following steps:

(1) extracting total phenolic acid of Yunnan red sage root from Yunnan red sage root;

(2) separating and purifying the total salvianolic acid extract by pH zone countercurrent chromatography to obtain fraction containing salvianolic acid B and lithospermic acid;

(3) separating and purifying the fraction containing salvianolic acid B and lithospermic acid by pH zone countercurrent chromatography;

wherein the solvent system of the pH zone countercurrent chromatography in the step (3) consists of tert-butyl methyl ether and water, the volume ratio of tert-butyl methyl ether to water is 1: 0.9-1.1, the upper phase solution of the solvent system is used as a stationary phase, the lower phase solution of the solvent system is used as a mobile phase, acid is added into the stationary phase, and alkali is added into the mobile phase.

In some embodiments, in step (3), the acid concentration in the stationary phase is 4.5 to 5.5mM, and the base concentration in the mobile phase is 59 to 61 mM. Experiments show that under the condition, the efficiency of separating the salvianolic acid B and the lithospermic acid is further improved, so that the separation time of the salvianolic acid B and the lithospermic acid is further shortened.

In some embodiments, in step (3), the acid is trifluoroacetic acid and the base is aqueous ammonia.

In some embodiments, the process for obtaining the total phenolic acid extract of the salvia yunnanensis from the salvia yunnanensis comprises the following steps: extracting the salvia miltiorrhiza bunge by adopting an ethanol extraction method, carrying out primary extraction on the extracted liquid by adopting petroleum ether, adjusting the pH of the extracted water phase to 1.5-2.5, then adding ethyl acetate for secondary extraction, and concentrating the ethyl acetate phase obtained by the secondary extraction to obtain the total phenolic acid extract of the salvia miltiorrhiza bunge.

The 'primary' and 'secondary' in the primary extraction and the secondary extraction only indicate different extraction processes, and are not limited to the extraction times.

In one or more embodiments, the salvia yunnanensis is added into an ethanol water solution, reflux extraction is carried out at 65-75 ℃, an extracting solution is concentrated, water is added for redissolution, and then petroleum ether is adopted for primary extraction. Wherein the concentration of the ethanol water solution is 55-65% by volume fraction. The material-liquid ratio of the salvia yunnanensis to the ethanol water solution is 1: 7.5-8.5, kg: and L. The reflux extraction times are 2-4 times, and the time of each reflux extraction is 1.5-2.5 h.

In one or more embodiments, the number of secondary extractions is 3-7.

The process of pH zone countercurrent chromatographic separation and extraction comprises the following steps: conveying the stationary phase to a separation column, injecting the solution of the total phenolic acid extract of the Yunnan red sage root into the separation column, and then conveying the mobile phase to the separation column continuously for separation and extraction. The flow rate of the stationary phase conveyed to the separation column is 25-35 mL/min. The flow rate of the mobile phase is 1.5-2.5 mL/min. The time to collect the fractions was 6min per collection (vial). In the pH zone countercurrent chromatographic separation and extraction, the detection wavelength of an ultraviolet detector is 252-256 nm.

In some embodiments, in step (2), the solvent system comprises petroleum ether, ethyl acetate, acetonitrile and water, wherein the volume ratio of petroleum ether, ethyl acetate, acetonitrile and water is 1.46-1.54: 2.46-2.54: 1: 4.6-5.4. Mixing petroleum ether, ethyl acetate, acetonitrile and water uniformly, standing, separating liquid, wherein the upper phase after liquid separation is a stationary phase, and the lower phase after liquid separation is a mobile phase. When the concentration of trifluoroacetic acid in the stationary phase is 9-11 mM and the concentration of ammonia water in the mobile phase is 29-31 mM, the separation and extraction effect is more excellent.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

The reagents used in the following examples are all commercially available unless otherwise specified. The Yunnan Danshen root is purchased from Xinping county of Yunnan province.

Examples

Extracting and preparing a sample:

weighing 3kg of Yunnan red sage root, crushing, and performing reflux extraction for 3 times at 70 ℃ by using 60% ethanol, wherein the material-liquid ratio is 1:8(w/v) and each time is 2 hours. The extract was filtered, concentrated to about 800mL at 50 ℃ under reduced pressure, and reconstituted to 1000mL with water. Extracting the extractive solution with petroleum ether for three times, removing the upper layer, adding hydrochloric acid into the water phase to adjust the pH of the extractive solution to 2, and extracting with ethyl acetate for 5 times. Mixing ethyl acetate phases, and concentrating under reduced pressure to obtain 136g of total phenolic acid extract of Saviae Miltiorrhizae radix.

Preparation of two-phase solvent system and sample solution:

according to a two-phase solvent system, the solvents are respectively weighed according to a proportion and are sequentially added into a separating funnel. After shaking, the mixture was allowed to stand for 12 hours, and the upper and lower phases were separated. Trifluoroacetic acid with a certain concentration is added into the upper phase as a stationary phase, and ammonia water with a certain concentration is added into the lower phase as a mobile phase. Ultrasonic degassing the upper and lower phases for 2min to obtain solvent system for pH-ZRCCC separation.

And (3) putting the sample into a test tube, and ultrasonically dissolving the sample by using the acidified upper phase solution and the equivalent non-alkalized lower phase solution, wherein the total volume is not more than 20mL, so as to prepare a sample solution for separating the pH-ZRCCC.

Separation by pH-zone countercurrent chromatography

Pumping the upper phase solution at the flow rate of 30mL/min until the separation column is filled, injecting the sample solution into the separation column through a sample injection ring, adjusting the separation column to rotate at the positive rotation speed of 800rpm, and simultaneously pumping the lower phase solution at the flow rate of 2 mL/min. The detection wavelength of the ultraviolet detector was adjusted to 254nm, fractions were collected at 6 min/vial intervals, and the pH of each vial fraction was measured using a pH meter. After the separation process is finished, the residual liquid in the column is blown into a measuring cylinder by using an air compressor, and the retention rate of the stationary phase is calculated. And finally, sequentially cleaning the separation column by using water and ethanol.

HPLC analysis and structural identification

HPLC analysis conditions of the total phenolic acid extract of Salvia yunnanensis and the pure components separated by pH-ZRCCC are Compass C18 chromatographic column (250X 4.6mm,5 μm), and the mobile phase is acetonitrile (A) -0.1% formic acid water solution (B): 0-10 min, 14% A; 10-20 min, 14% -17% A; 20-35 min, 17% -30% A. The flow rate is 1.0mL/min, the detection wavelength is 280nm, and the sample injection amount is 10 mu L.

The pure fraction isolated from pH-ZRCCC was purified using HPLC-ESI-QTOF-MS and¹³C-NMR、¹H-NMR structural identification was carried out on the pure components.

Results of HPLC analysis

FIG. 1 shows HPLC analysis chart of total salvianolic acid extract and pure component separated by pH-ZRCCC, and the peak area ratios of salvianolic acid B and lithospermic acid in total phenolic acid crude extract are 32.12% and 5.52% respectively, when calculated by peak area normalization at 280 nm.

Optimization of two-phase solvent systems

First, using petroleum ether-ethyl acetate-acetonitrile-water (1.5:2.5:1:5, v/v/v/v) as a separation solvent system, aqueous ammonia (30mM) was added to the lower phase, trifluoroacetic acid (10mM) was added to the upper phase, and 2g of Salvia yunnanensis was separated. In combination with HPLC analysis, as shown in FIG. 2, since the chromatographic peaks of salvianolic acid B and lithospermic acid in the system have large overlap and no obvious separation tendency, the fractions of the two compounds are combined, concentrated under reduced pressure and freeze-dried to obtain 1.30g of sample 1 for secondary separation of pH-ZRCCC.

For the pH-ZRCCC separation of sample 1, a series of attempts were first made using systems such as petroleum ether-ethyl acetate-methanol-water, ethyl acetate-n-butanol-water, ethyl acetate-water, chloroform-methanol-water, and the like, and it was difficult to separate compounds IV and V. Sample 1 was isolated according to the solvent system proposed by Ito, again trying a t-butyl methyl ether-acetonitrile-water (4:1:5, v/v/v) solvent system with trifluoroacetic acid (10mM) added to the upper phase and aqueous ammonia (10mM) added to the lower phase. As a result, the solvent system can realize the separation of two compounds, but the elution time is longer, and the elution of only the first compound V lasts for 9 hours, so that how to shorten the elution time becomes a key problem. It was found experimentally that the solubility of the sample in acetonitrile was better than that in both water and tert-butyl methyl ether, whereas in the tert-butyl methyl ether-acetonitrile-water (4:1:5, v/v/v) system the affinity of acetonitrile for tert-butyl methyl ether was better. Therefore, the presence of acetonitrile in the above solvent system makes the sample distributed in the stationary phase in a large amount, which is a major factor in the long retention time of the sample. Thus, the experiment removed the acetonitrile in the solvent system, allowed the sample to dissolve more in the lower phase, and then sample 1 was separated using a t-butyl methyl ether-water (1:1, v/v) system with trifluoroacetic acid (10mM) added to the upper phase and aqueous ammonia (10mM) added to the lower phase, resulting in complete separation of the two compounds over 13 h. Based on the above experiments, this example performed a limiting adjustment of the concentrations of the retained acid and the elution base in the solvent system, reducing the retained acid concentration to 5mM, increasing the elution base concentration to 60mM, and again using a t-butyl methyl ether-water (1:1, v/v) solvent system to separate 1.30g of sample 1, the pH-ZRCCC separation scheme is shown in FIG. 3. The salvianolic acid B and lithospermic acid are successfully separated within 9h, the platform limit of the two zones is obvious, and the purity is 95.6 percent and 95.0 percent respectively. After freeze-drying, 259.9mg of salvianolic acid B and 28.75mg of lithospermic acid are obtained.

Structural identification of compounds

According to ESI-MS,¹H-NMR and¹³C-NMR carries out structural identification on the compounds I to III, and specific data are as follows:

salvianolic acid B: the peak of the excimer ion is M/z 717M-H]^–A relative molecular weight of 718 was suggested, and the fragment of the second-order mass spectrum was M/z 519[ M-salvanic acid A ]]^–。¹H-NMR(DMSO-d₆,400MHz)δ(ppm):7.14(1H,s,H-5),6.82(1H,s,H-6),6.19(1H,s,H-8),6.23(1H,d,H-2’),6.80-6.51(8H,H-5’,6’,2”,5”,6”,2”’,5”’,6”’),5.77(1H,s,7”’),4.22(1H,s,H-8”),4.83(2H,s,H-8’,8”’).¹³C-NMR(DMSO-d₆,100MHz)δ(ppm):125.6(C-1),125.6(C-2),146.1(C-3),147.9(C-4),119.8(C-5),121.9(C-6),144.0(C-7),117.3(C-8),166.5(C-9),129.6(C-1’),116.8(C-2’),146.1(C-3’),145.9(C-4’),116.0(C-5’),123.3(C-6’),37.7(C-7’),76.6(C-8’),173.1(C-9’),132.2(C-1”),113.2(C-2”),145.5(C-3”),145.5(C-4”),115.6(C-5”),117.0(C-6”),86.6(C-7”),56.7(C-8”),172.4(C-9”),130.0(C-1”’),116.3(C-2”’),144.3(C-3”’),144.1(C-4”’),119.8(C-5”’),123.3(C-6”’),37.3(C-7”’),77.5(C-8”’),170.9(C-9”’)。

Alkannic acid: the peak of the excimer ion is M/z 537[ M-H ]]^–The suggested relative molecular weight is 538 and the fragment of the secondary mass spectrum is M/z 493[ M-H-CO ]₂]^–And M/z 295[ M-H-CO ]₂–salvianic acid A]^–。¹H-NMR(DMSO-d₆,400MHz)δ(ppm):7.13(1H,d,J＝8.0Hz,H-6),8.00(1H,d,J＝16.0Hz,H-7),6.19(1H,d,J＝16.0Hz,H-8),6.75-6.66(5H,m,H-5,2’,2”,5”,6”),6.51(1H,d,J＝8.0Hz,H-5’),6.37(1H,d,J＝8.0Hz,H-6’),2.88(1H,d,J＝12.0Hz,H-7’a),2.72(1H,d,J＝12.0Hz,H-7’b),4.58(1H,t,J＝24.0,12.0Hz,H-8’),5.82(1H,d,J＝4.0Hz,H-7”),4.05(1H,d,J＝4.0Hz,H-8”).¹³C-NMR(DMSO-d₆,100MHz)δ(ppm):123.1(C-1),129.7(C-2),147.2(C-3),143.7(C-4),118.6(C-5),119.5(C-6),142.0(C-7),116.4(C-8),166.3(C-9),131.0(C-1’),117.4(C-2’),145.8(C-3’),144.2(C-4’),116.4(C-5’),119.5(C-6’),37.8(C-7’),76.7(C-8’),173.1(C-9’),133.5(C-1”),113.6(C-2”),146.3(C-3”),145.7(C-4”),118.5(C-5”),115.9(C-6”),88.7(C-7”),60.1(C-8”),174.5(C-9”)。

The invention uses a solvent system of petroleum ether-ethyl acetate-acetonitrile-water (1.5:2.5:1:5, v/v/v/v) (trifluoroacetic acid (10mM) is added on the upper phase, and ammonia water (30mM) is added on the lower phase) to obtain the fractions of salvianolic acid B and lithospermic acid from the crude extract of total salvianolic acid in Yunnan, and then uses a solvent system of tert-butyl methyl ether-water (1:1, v/v) to obtain 259.9mg of salvianolic acid B and 28.75mg of lithospermic acid, wherein the purity is higher than 95%.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.