阅读说明：本技术 一种从丹参中制备丹酚酸b、隐丹参酮、丹参酮i和丹参酮iia的方法 (A method for preparing salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA from Saviae Miltiorrhizae radix ) 是由 唐万 徐剑 杨跃军 石万银 江丹 巫岳 向诗银 于 2020-06-15 设计创作，主要内容包括：本发明公开了一种从丹参中制备丹酚酸B、隐丹参酮、丹参酮I和丹参酮IIA的方法,具体为获得丹参提取物后用一定量有机试剂溶解做前处理,然后上样进制备液相,在一个程序下可以同时将丹酚酸B、隐丹参酮、丹参酮I和丹参酮IIA四种单体成分分离收集。本发明具有操作便利、重现性好、周期短、同时制备4种单体成分等特点,制备的丹酚酸B、隐丹参酮、丹参酮I和丹参酮IIA产品具有纯度高的特点。(The invention discloses a method for preparing salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA from salvia miltiorrhiza, which is characterized by dissolving a certain amount of organic reagent for pretreatment after a salvia miltiorrhiza extract is obtained, then loading the solution into a preparation liquid phase, and separating and collecting four monomer components of salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA simultaneously under one program. The method has the characteristics of convenient operation, good reproducibility, short period, simultaneous preparation of 4 monomer components and the like, and the prepared products of the salvianolic acid B, the cryptotanshinone, the tanshinone I and the tanshinone IIA have the characteristic of high purity.)

1. A method for preparing salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA from Salvia miltiorrhiza Bunge is characterized by comprising the following steps:

(1) single medicine extraction: crushing a salvia miltiorrhiza medicinal material, adding 8-10 times of 65-75% ethanol solution, heating and refluxing for extraction for 2-3 times at 55-70 ℃ for 50-70 min each time, roughly filtering after extraction is finished, and combining filtrates to obtain a salvia miltiorrhiza extracting solution;

(2) concentrating and drying: vacuum concentrating the filtered extractive solution under reduced pressure, and drying;

(3) purification and preparation: adding 100-200 times of 60-70% ethanol into the dried extract, ultrasonically dissolving, filtering with a 0.22 μm microporous filter membrane, and preparing a liquid phase with a chromatographic column: c18 reverse phase chromatography column;

(4) and (3) fraction collection: collecting the fractions of salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA.

(5) Concentrating and drying: and (3) carrying out vacuum concentration and drying on the collected fractions at the temperature of 50-60 ℃.

2. The method according to claim 1, wherein the specific chromatographic conditions in step (3) are: the mobile phase is methanol: and (3) carrying out gradient elution on 0.1-0.2% formic acid according to the following table, wherein the flow rate is 10-20 mL/min, the detection wavelengths are 285-290 nm and 268-275 nm, and the column temperature is 25-35 ℃.

3. The method according to claim 1, wherein the conditions of gradient elution in step (3) are as follows:

time (min) Acetonitrile (%) 0.1 to 0.2% formic acid (%) 0 24～26 76～74 12 24～26 76～74 15 60～65 40～35 30 60-65 40～35 35 89～93 11～7 45 89-93 11-7

4. The method according to claim 1, wherein the temperature in the step (2) is 50 to 60 ℃ and the vacuum degree is 0.05 to 0.08 MPa.

Technical Field

The invention relates to the technical field of biological medicine extraction, and particularly relates to a method for preparing salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA.

Background

Salvia miltiorrhiza, which is the dried root and rhizome of Salvia miltiorrhiza bge, is listed as the superior product in Shen nong's herbal Jing. All the herbs in all the generations are recorded and collected, and are common Chinese herbal medicines collected in Chinese pharmacopoeia. Collected in spring and autumn, removed of silt and dried. This herb is bitter in flavor and slightly cold in nature. Enters heart and liver meridians, and has the effects of promoting blood circulation, removing blood stasis, dredging meridians, relieving pain, clearing away heart-fire, relieving restlessness, cooling blood and resolving carbuncle. Can be used for treating thoracic obstruction, heart pain, abdominal pain, hypochondriac pain, abdominal mass, pain due to pyretic arthralgia, vexation, insomnia, menoxenia, dysmenorrhea, amenorrhea, and pyocutaneous disease with swelling and pain.

Research shows that the salvia miltiorrhiza mainly contains chemical components such as salvianolic acids, tanshinone, volatile oils and inorganic elements, and has wide pharmacological activity, wherein the phenolic acids and the tanshinone are the main material basis of the salvia miltiorrhiza playing pharmacological action, have obvious curative effects on treating cardiovascular diseases, liver cirrhosis, ulcer, lymphoma, neonatal hypoxic encephalopathy and the like, have good curative effects on the cardiovascular and cerebrovascular diseases, and also have the drug effects of resisting bacteria, resisting inflammation, resisting tumors, protecting organs and the like.

Salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA are several main functional components in Saviae Miltiorrhizae radix. According to pharmacological research, the salvianolic acid B has important pharmacological action on organs such as heart, brain, liver, kidney and the like, and has better effects on oxidation resistance, heart protection, brain protection, tumor resistance and the like; cryptotanshinone has antioxidant and antiaging effects, and can be used for treating coronary heart disease, angina pectoris, and myocardial damage; cryptotanshinone has antibacterial effect, 1mg/ml has inhibiting effect on Staphylococcus aureus and Pseudomonas aeruginosa, and also has certain inhibiting effect on hemolytic streptococcus; research proves that tanshinone I has certain antiandrogen effect; tanshinone IIA has effects of improving coronary artery blood circulation, repairing vascular endothelial cells, resisting Atherosclerosis (AS) formation, resisting myocardial hypertrophy, and preventing and treating diabetic complications.

At present, the method for obtaining the functional components is mainly a method for extracting and separating the salvia miltiorrhiza from the salvia miltiorrhiza: generally, ultrasonic extraction, countercurrent extraction, extraction and the like are adopted, or target components are modified and modified in a certain mode and then are further separated, and separation and purification are further carried out in the later stage by means of a chromatographic column and a medium-low pressure preparation combined mode, and monomer compounds with higher purity are obtained by means of separation and purification in supercritical fluid extraction and other modes.

Disclosure of Invention

The invention aims to provide a convenient, efficient, simple-process and easy-to-popularize method for separating and preparing high-purity salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA monomers from salvia miltiorrhiza.

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

a method for preparing salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA from Saviae Miltiorrhizae radix comprises the following steps:

(1) single medicine extraction: crushing a salvia miltiorrhiza medicinal material, adding 8-10 times of 65-75% ethanol solution, heating and refluxing for extraction for 2-3 times at 55-70 ℃ for 50-70 min each time, roughly filtering after extraction is finished, and combining filtrates to obtain a salvia miltiorrhiza extracting solution;

(2) concentrating and drying: vacuum concentrating the filtered extractive solution under reduced pressure, and drying;

(3) purification and preparation: adding 100-200 times of 60-70% ethanol into the dried extract, ultrasonically dissolving, filtering with a 0.22 μm microporous filter membrane, and preparing a liquid phase with a chromatographic column: c18 reverse phase chromatography column;

(4) and (3) fraction collection: collecting the fractions of salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA;

(5) concentrating and drying: and (3) carrying out vacuum concentration and drying on the collected fractions at the temperature of 50-60 ℃.

Further, the specific chromatographic conditions in step (3) are as follows: the mobile phase is methanol: and (3) carrying out gradient elution on 0.1-0.2% formic acid according to the following table, wherein the flow rate is 10-20 mL/min, the detection wavelengths are 285-290 nm and 268-275 nm, and the column temperature is 25-35 ℃.

Further, the conditions of gradient elution in step (3) are as follows:

time (min)	Acetonitrile (%)	0.1 to 0.2% formic acid (%)
			0	24～26	76～74
12	24～26	76～74
			15	60～65	40～35
30	60-65	40～35
			35	89～93	11～7
45	89-93	11-7

Further, in the step (2), the temperature is 50-60 ℃, and the vacuum degree is 0.05-0.08 MPa.

Compared with the prior art, the invention has the following advantages:

firstly, the process is convenient and fast: the preparation method of most effective components of radix salviae miltiorrhizae mainly comprises the steps of carrying out adsorption and impurity removal in a resin chromatographic column mode after solvent countercurrent extraction, or purifying step by means of other solvents so as to achieve the purpose of separating and purifying the effective components in the radix salviae miltiorrhizae; the invention dries the concentrated solution after extracting the salvia miltiorrhiza, then dissolves and passes through a membrane by virtue of a mobile phase, and then directly separates and purifies the solution by a prepared liquid phase, thereby obtaining high-purity salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA.

Secondly, separation is efficient: compared with other methods for preparing the salvia monomer, the preparation method has the advantages that the salvia extract is dissolved by virtue of a mobile phase and then passes through a membrane, detection and separation are carried out on a prepared liquid phase, 4 monomer components can be sequentially collected after being separated by virtue of a fraction collector under one detection and separation procedure according to peak output information of an absorption peak of a reference substance, the separation efficiency is high, the effect of separating four monomers at one time can be achieved, and compared with methods such as repeated resin passing and reagent purification in other methods, the separation efficiency is greatly improved by the method.

Thirdly, the product purity is high: after pretreatment, the salvia miltiorrhiza extract is separated and purified by a preparative liquid chromatograph, so that monomer components with the product purity of more than 98 percent can be obtained, the impurities are less, and the product purity is high.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.