阅读说明：本技术 一种利用天然低共熔溶剂催化转化丹酚酸b的方法 (Method for catalytic conversion of salvianolic acid B by using natural eutectic solvent ) 是由 李畅 万浩芳 万海同 何新平 于 2020-08-12 设计创作，主要内容包括：本发明属于药物化学领域,具体涉及一种以天然低共熔溶剂作为溶剂兼催化剂催化转化丹酚酸B生成其他丹酚酸类化合物的方法。该方法利用天然低共熔溶剂(NaDES)作为溶剂兼催化剂,将丹参中含量丰富的丹酚酸B催化转化为丹酚酸A、紫草酸、丹参素、原儿茶醛等天然丹参中含量较低的活性成分。主要步骤包括：(1)将丹参根茎粉碎,加水后超声辅助提取,大孔树脂分离纯化丹酚酸B；(2)将纯度大于90％的丹酚酸B加入到不同组成的NaDES水溶液中,加热反应催化丹酚酸B为活性成分混合物,该混合物利用柱层析分离,得到丹酚酸A、紫草酸、丹参素、原儿茶醛等活性成分。本发明方法具有操作简单、转化率高、绿色环保等优势,有望用于工业化生产。(The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a method for catalytically converting salvianolic acid B into other salvianolic acid compounds by taking a natural eutectic solvent as a solvent and a catalyst. The method uses natural eutectic solvent (NaDES) as solvent and catalyst to catalytically convert salvianolic acid B rich in Saviae Miltiorrhizae radix into lower active components such as salvianolic acid A, lithospermic acid, tanshinol, protocatechualdehyde, etc. The method mainly comprises the following steps: (1) pulverizing Saviae Miltiorrhizae radix, adding water, extracting with ultrasonic wave, and separating and purifying salvianolic acid B with macroporous resin; (2) adding salvianolic acid B with purity of more than 90% into NaDES aqueous solution of different compositions, heating to react and catalyze salvianolic acid B to obtain active ingredient mixture, and separating the mixture by column chromatography to obtain salvianolic acid A, lithospermic acid, tanshinol, protocatechualdehyde, etc. The method has the advantages of simple operation, high conversion rate, environmental protection and the like, and is expected to be used for industrial production.)

1. A method for catalytically converting salvianolic acid B by using a natural eutectic solvent is characterized in that the method takes the natural eutectic solvent as a solvent and a catalyst, and the salvianolic acid B is catalytically converted into other salvianolic acid compounds under normal pressure, and the specific method comprises the following steps:

(1) extracting salvianolic acid B: extracting Saviae Miltiorrhizae radix with water as solvent under heating and ultrasonic conditions, and separating and purifying with macroporous resin to obtain salvianolic acid B;

(2) preparing a natural eutectic solvent: mixing a hydrogen bond donor with a hydrogen bond acceptor and/or deionized water according to a certain proportion, and heating and stirring to prepare a natural eutectic solvent;

(3) catalytic conversion of salvianolic acid B: adding salvianolic acid B into the eutectic solvent, and heating for reacting for a certain time to obtain salvianolic acid B conversion solution;

(4) separation and purification: and (4) performing column chromatography separation and purification on the salvianolic acid B conversion liquid obtained in the step (3) to obtain the salvianolic acid compound.

2. The method for catalytic conversion of salvianolic acid B based on natural eutectic solvent in claim 1, wherein the hydrogen bond donor in step (2) comprises: glycerol, lactic acid, urea; hydrogen bond acceptors include: choline chloride, betaine and L-proline.

3. The method for catalytic conversion of salvianolic acid B by using the natural eutectic solvent as claimed in claim 1, wherein the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor in the step (2) is 1: 1-2.

4. The method for catalytic conversion of salvianolic acid B by using the natural eutectic solvent as claimed in claim 1, wherein the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor to the deionized water in step (2) is 1:1-2: 1-5.

5. The method for catalytic conversion of salvianolic acid B based on natural eutectic solvent as claimed in claim 1, wherein the heating temperature in step (2) is 80-100 ℃ and the stirring time is 0.5-2 h.

6. The method for catalytic conversion of salvianolic acid B by using natural eutectic solvent as claimed in claim 1, wherein the heating temperature in step (2) is 80 ℃ and the stirring time is 1 h.

7. The method for catalytic conversion of salvianolic acid B by using the natural eutectic solvent as claimed in claim 1, wherein the volume fraction of the eutectic solvent in the step (3) is 75-100%.

8. The method for catalytic conversion of salvianolic acid B based on natural eutectic solvent in claim 1, wherein the charge amount of salvianolic acid B in step (3) is 2mg/mL of eutectic solvent.

9. The method for catalytic conversion of salvianolic acid B based on natural eutectic solvent as claimed in claim 1, wherein the reaction temperature in step (3) is 80-140 ℃ and the reaction time is 2-48 h.

10. The method for catalytically converting salvianolic acid B by using the natural eutectic solvent as claimed in claim 1, wherein the salvianolic acid compounds are salvianolic acid A, lithospermic acid, tanshinol and protocatechuic aldehyde, and the reaction conditions for converting salvianolic acid B into various substances are as follows:

a. the optimal reaction conditions for converting the salvianolic acid B into the salvianolic acid A are as follows: the betaine-lactic acid eutectic solvent with 25 percent of water (volume fraction) is reacted for 6 hours at 120 ℃;

b. the optimal reaction conditions for converting the salvianolic acid B into the lithospermic acid are as follows: the betaine-lactic acid eutectic solvent with 25 percent of water (volume fraction) is reacted for 36 hours at 80 ℃;

c. the optimal reaction conditions for converting the salvianolic acid B into the danshensu and the protocatechuic aldehyde are as follows: betaine-urea eutectic solvent containing 25% (volume fraction) of water is reacted for 2h at 140 ℃.

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a method for generating other salvianolic acid compounds by catalytic conversion of salvianolic acid B by taking a natural eutectic solvent as a solvent and a catalyst.

Background

Salvia miltiorrhiza (Salvia miliiorhiza Bunge) is a widely used traditional Chinese medicine, has certain curative effects on cerebrovascular diseases, metabolic disorders, hepatitis and cancers, and has been widely accepted in clinical curative effect. The main effective component of Saviae Miltiorrhizae radix is salvianolic acid compound, wherein salvianolic acid B has the highest content. The content of other salvianolic acids such as salvianolic acid A, lithospermic acid, tanshinol and protocatechualdehyde is very low. The research shows that the salvianolic acid A has excellent biological activity of resisting oxidation, eliminating free radicals, protecting nerves, preventing and treating cardiovascular diseases and the like, and is superior to the salvianolic acid B in certain aspects. The salvianolic acid A can obviously improve the myocardial ischemia range of experimental animals by both intravenous administration and oral administration, and obviously inhibit the activity rise of serum lactate dehydrogenase LDH and creatine kinase CK caused by myocardial infarction (Leilei, tension phar, Linzhirong, Stapf, Malayan and Liujian. different administration routes of the salvianolic acid A have the influence on the acute myocardial ischemia of dogs [ J ] Chinese traditional medicine journal, 2016,41(05): 910-. The model of platelet aggregation induced by adenosine diphosphate, arachidonic acid and thrombin, as compared with aspirin, by Wanghanggang et al, it was found that salvianolic acid A not only has a comprehensive mild inhibitory effect on platelet aggregation, but also has a significant protective effect on cerebral ischemia caused by thrombus (Wanghanggang, Koranei, Kirui, Chenyanxia, Yangren, Zhao Xiao, Zhou enmeng, Duguanhua. the comparative study on antithrombotic effect of salvianolic acid A and aspirin [ J ] pharmaceutics, 2019,54(02): 301-. The results of rat myocardial ischemia models of Wangshoubao et al show that salvianolic acid A is more effective than salvianolic acid B10 mg/kg when the dosage is 5 and 10mg/kg, and further that the therapeutic effect is equivalent to that of salvianolic acid B10 mg/kg when the dosage is 2.5 mg/kg. (Wang S B, Tian S, Yang F, et. Cardioprotective effect of salvianoic acid A on isoprotenol-induced myocardial attack in rates. Eur J Pharm,2009,615: 125).

However, the chemical component research of the salvia miltiorrhiza medicinal material shows that: the main component of Salvia miltiorrhiza is salvianolic acid B, and the contents of other salvianolic acids such as tanshinol, protocatechuic aldehyde, lithospermic acid and salvianolic acid A are extremely low, so that the salvianolic acid compounds can not be obtained by direct extraction. There are several prior art methods that attempt to convert salvianolic acid B to salvianolic acid A, etc., by chemical reaction, such as: salvianolic acid B can be converted to other salvianolic acids by high temperature and pressure and adjusting the pH of the solvent, such as salvianolic acid B which produces large amounts of salvianolic acid A at pH 1 and large amounts of salvianic acid B at pH 12 (Xia H, Sun L, Lou H, Rahman M. conversion of salvianic acid B into salvianic acid A in properties of Radix salviae miltiorrhizae using high temperature, high pressure and high purity. photomedicine, 2014,21,906 and 911). Chinese patent with application number CN201310487751.6 discloses a method for preparing salvianolic acid A, which comprises adding an extractant into a salvia miltiorrhiza medicinal material, heating and extracting, filtering and concentrating the extract to obtain salvia miltiorrhiza extract; adjusting pH of the Saviae Miltiorrhizae radix extractive solution to 4-8, concentrating under reduced pressure, heating the concentrated solution at 70-100 deg.C for reaction for 5-40h to obtain crude salvianolic acid A solution; the pH value of the crude salvianolic acid A solution is adjusted to 6-8, and the pure salvianolic acid A is obtained by adsorption and resolution of reversed-phase resin. Chinese patent application No. CN201610806533.8 discloses another preparation method of salvianolic acid A, which comprises preparing salvianolic acid B into 35-45mg/mL solution with NaOH or NaHCO3 water with pH of 3.5-4.5, placing the solution in a subcritical water reaction kettle, placing the heating furnace in a heating furnace after the heating furnace reaches 170-190 ℃ and is stable, placing the reaction kettle in the heating furnace for 50-70 min, taking out the reaction kettle, placing the reaction kettle in an ice water bath or cold water bath for cooling, taking out the liquid, freeze-drying to obtain a crude product rich in salvianolic acid A, and separating and purifying the crude product of salvianolic acid A by using high-efficiency countercurrent chromatography to obtain salvianolic acid A with purity of more than 98%. In addition, patent No. CN201310192210.0 discloses that transformation of salvianolic acid B under normal pressure is achieved by using high boiling point organic solvents such as ethylene glycol, dimethyl sulfoxide, and dimethylformamide to convert the extract of salvia miltiorrhiza, but the use of a large amount of organic solvents increases the process components and has high toxicity.

In addition to the above problems, the salvianolic acid B conversion experiment also has the problems of low reaction efficiency, complex products and the like, and at present, a large number of patents only make adjustments on adjusting the pH value, changing the reaction temperature, changing the reaction solvent or the additive, but no deep new methods are available, and chinese patent application No. CN201210487598.2 discloses that the salvianolic acid B is catalytically converted by using ferric chloride, ruthenium trichloride, zinc chloride, palladium chloride and the like as catalysts, and besides most of the catalysts are expensive, the catalytic effects of the catalysts are not obviously different. Although the method improves the efficiency of converting the salvianolic acid B into the salvianolic acid A, the conversion rate of the salvianolic acid B is only about 63 percent, and the yield of the salvianolic acid A is about 42 percent. The patent application No. CN201410200983.3 suggests that air isolation during the transformation of salvianolic acid a can not only ensure that salvianolic acid B is damaged as little as possible to transform salvianolic acid a as much as possible, but also prevent the generated salvianolic acid a from being damaged by oxidation. Therefore, the reaction is carried out under the condition of air isolation by using nitrogen or argon as a protective gas, and the best experimental result is as follows: concentrating the extractive solution to 400mL, placing in a high pressure reaction kettle, reacting at 135 deg.C and 0.31M protocatechuic aldehyde for 2h under nitrogen protection, and stopping reaction. The content of salvianolic acid B in the extract was determined to be 0.95%, the content of salvianolic acid A in the conversion solution was determined to be 0.47%, and the conversion rate was determined to be 71.9%. Although the above method increases the conversion rate to about 72%, the participation of nitrogen or argon not only greatly increases the cost, but also makes the air removal operation complicated and requires expensive sealing equipment, which is not favorable for large-scale industrial production.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide a method for selectively preparing other salvianolic acid compounds by using a natural eutectic solvent as a solvent and a catalyst and using salvianolic acid B as a raw material, which is green and efficient.

In order to achieve the above object, the present invention provides the following technical solutions:

a method for catalytically converting salvianolic acid B by using a natural eutectic solvent is characterized in that the natural eutectic solvent is used as a solvent and a catalyst, and the salvianolic acid B is catalytically converted into other salvianolic acid compounds under normal pressure, and the specific method comprises the following steps:

(1) extracting salvianolic acid B: extracting Saviae Miltiorrhizae radix with water as solvent under heating and ultrasonic conditions, filtering the extractive solution, centrifuging, collecting supernatant, distilling under reduced pressure to obtain paste, and separating and purifying with macroporous resin to obtain salvianolic acid B;

(2) synthesizing natural eutectic solvent: mixing a hydrogen bond donor HBD and a hydrogen bond acceptor HBA and/or deionized water in a certain proportion, heating and stirring to obtain a transparent clear solution, namely a natural eutectic solvent;

(3) catalytic conversion of salvianolic acid B: adding salvianolic acid B into the eutectic solvent, and heating for reacting for a certain time to obtain salvianolic acid B conversion solution;

(4) separation and purification: and (4) performing column chromatography separation and purification on the salvianolic acid B conversion liquid obtained in the step (3) to obtain the salvianolic acid compound with the purity of 95%.

Preferably, the specific operation of extracting salvianolic acid B in the step (1) is as follows: adding water into Saviae Miltiorrhizae radix powder, ultrasonic extracting at 50 deg.C for 2 hr, filtering with gauze to obtain extractive solution, centrifuging at 7000G for 10min, collecting supernatant, repeatedly adding water into residue, ultrasonic extracting for 3 times, mixing supernatants, concentrating at 50 deg.C to obtain paste, purifying by AB-8 macroporous resin column chromatography, eluting with water and 75% ethanol, collecting 75% ethanol eluate, and spin drying at 50 deg.C to obtain salvianolic acid B.

Preferably, the hydrogen bond donor in step (2) comprises: glycerol (Gly), lactic acid (Lac), urea (Ur); hydrogen bond acceptors include: choline chloride (ChCl), betaine (Bet), L-proline (L-Pro).

Preferably, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor in the step (2) is 1: 1-2.

Preferably, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor to the deionized water in the step (2) is 1:1-2: 1-5.

Preferably, the heating temperature in the step (2) is 80-100 ℃, and the stirring time is 0.5-2 h; more preferably, the temperature is 80 ℃ and the time is 1 h.

Preferably, the volume fraction of the low eutectic solvent in the step (3) is 75-100%.

Preferably, the charging amount of the salvianolic acid B in the step (3) is 2mg/mL of eutectic solvent.

Preferably, the reaction temperature in the step (3) is 80-140 ℃, and the reaction time is 2-48 h.

Preferably, the separation and purification in the step (4) comprises the following specific steps: extracting salvianolic acid B conversion solution with ethyl acetate, rotary evaporating organic phase, and separating and purifying the product by column chromatography, wherein the eluent is petroleum ether, ethyl acetate and methanol.

Preferably, the salvianolic acid compounds are salvianolic acid A, lithospermic acid, tanshinol and protocatechualdehyde, and the reaction conditions for converting salvianolic acid B into various substances are as follows:

a. the reaction conditions for converting the salvianolic acid B into the salvianolic acid A are as follows: betaine (Bet) -lactic acid (Lac) -deionized water (the molar ratio is 1:1:1) with the volume fraction of 75% is taken as a eutectic solvent, and the reaction is carried out for 6 hours at the temperature of 120 ℃, so that the conversion rate of salvianolic acid B is 89.8%, and the generation rate of salvianolic acid A is 72.4%;

b. the reaction conditions for converting the salvianolic acid B into the lithospermic acid are as follows: betaine (Bet) -lactic acid (Lac) -deionized water (the molar ratio is 1:1:1) with the volume fraction of 75% is taken as a eutectic solvent, and the reaction is carried out for 36 hours at the temperature of 80 ℃, so that the conversion rate of salvianolic acid B is 67.6%, and the generation rate of alkannic acid is 31.9%;

c. the reaction conditions for converting the salvianolic acid B into the danshensu and the protocatechuic aldehyde are as follows: betaine (Bet) -urea (Ur) -deionized water (molar ratio of 1:1:2) with volume fraction of 75% is used as eutectic solvent, and the reaction is carried out at 140 ℃ for 2h, so that the salvianolic acid B is completely converted, and the yields of tanshinol and protocatechuic aldehyde are respectively 0.77 and 0.56mol/mol SAB (calculated by the amount of the substance relative to salvianolic acid B).

The method for catalytically converting the salvianolic acid B by utilizing the natural eutectic solvent can realize the conversion selectivity of the salvianolic acid B by selecting different natural eutectic solvents:

(1) using 75% betaine (Bet) -lactic acid (Lac) as a eutectic solvent, reacting for 6h at 120 ℃, wherein the conversion rate of salvianolic acid B is 89.8%, and the generation rate of salvianolic acid A is 72.4%;

(2) using 75% betaine (Bet) -lactic acid (Lac) as a eutectic solvent, reacting at 80 ℃ for 48h, wherein the conversion rate of the salvianolic acid B is 71.4%, and lithospermic acid (yield is 29.0%) and salvianolic acid A (yield is 31.9%) are generated;

(3) using 75% betaine (Bet) -urea (Ur) as eutectic solvent, reacting at 140 deg.C for 2h to convert salvianolic acid B completely, and generating large amount of danshensu (0.77mol/mol SAB) and protocatechuic aldehyde (0.56mol/mol SAB).

Compared with the prior art, the invention has the following beneficial effects: the invention provides a high-efficiency, green and simple method for converting salvianolic acid B into active ingredients with lower content in salvia miltiorrhiza, such as salvianolic acid A, lithospermic acid, tanshinol, protocatechuic aldehyde and the like by utilizing a eutectic solvent, and the method selectively prepares a plurality of salvianolic acid compounds with lower Natural content by introducing a Natural Deep eutectic solvent (NaDES) as a solvent and a catalyst, wherein the conversion rate of salvianolic acid B reaches 89.8-100%, and the generation rate of salvianolic acid A also reaches 72.44%. Compared with the previously reported method, the method can be carried out under normal pressure, does not need to repeatedly adjust the pH value, does not have the defects of flammability and explosiveness of an organic solvent, has high conversion efficiency, has different products along with different types of natural eutectic solvents, has the advantages of greenness, high efficiency, simple operation, safety and the like, and is very suitable for industrial production. The experiment introduces the natural eutectic solvent into the preparation of the traditional Chinese medicine components, and provides a new research and development idea for the reaction preparation of the traditional Chinese medicine components.

Drawings

FIG. 1 is a high performance liquid chromatogram of salvianolic acid B;

FIG. 2 is a high performance liquid chromatogram of salvianolic acid A obtained by catalytic conversion of salvianolic acid B with betaine-lactic acid (4mg salvianolic acid B/2mL 75% DES-1, 120 deg.C, 2 h);

FIG. 3 is a high performance liquid chromatogram of salvianolic acid A and lithospermic acid obtained by catalytic conversion of salvianolic acid B with betaine-lactic acid (4mg salvianolic acid B/2mL 75% DES-1, 80 deg.C, 36 h);

FIG. 4 is a high performance liquid chromatogram of the betaine-urea catalyzed conversion of salvianolic acid B to tanshinol and protocatechualdehyde (4mg salvianolic acid B/2mL 75% DES-2, 120 deg.C, 2 h);

FIG. 5 is a control HPLC chromatogram (120 ℃ C., 2h) of salvianolic acid B converted by heating with water.

Detailed Description

The technical solutions of the present invention are further described in detail with reference to the following embodiments and the accompanying drawings to help those skilled in the art to more completely, accurately and deeply understand the inventive concept and technical solutions of the present invention, but it should be noted that the scope of the present invention is not limited thereto.