阅读说明：本技术 一种分离同分异构体齐墩果酸和熊果酸的方法 (Method for separating isomers of oleanolic acid and ursolic acid ) 是由 童胜强 王超越 王香 于 2019-08-30 设计创作，主要内容包括：一种分离同分异构体齐墩果酸和熊果酸的方法,所述方法为：在分液漏斗中加入羟丙基-β-环糊精水溶液、正己烷和乙酸乙酯组成的混合溶剂体系,充分振摇后静置分层,取上相作为固定相,下相作为流动相；取齐墩果酸和熊果酸混合物用固定相和流动相的混合溶液溶解,得到样品溶液；取固定相注满逆流色谱仪的分离柱,开启速度控制器,使分离柱正转,注入流动相,等到柱尾端流出流动相时,表明两相已达到平衡,这时将样品溶液注入分离柱,紫外检测器检测,用自动部分收集器收集洗脱液,分别合并含目标物质的洗脱液,经后处理得到齐墩果酸、熊果酸；本发明操作简便,高效快捷,溶剂消耗少,齐墩果酸与熊果酸纯度高,且分离过程中样品回收率高。(A method of separating isomers of oleanolic acid and ursolic acid, said method comprising: adding a mixed solvent system consisting of a hydroxypropyl-beta-cyclodextrin aqueous solution, normal hexane and ethyl acetate into a separating funnel, fully shaking, standing for layering, taking an upper phase as a stationary phase, and taking a lower phase as a mobile phase; dissolving a mixture of oleanolic acid and ursolic acid by using a mixed solution of a stationary phase and a mobile phase to obtain a sample solution; filling a separation column of a counter-current chromatograph with a stationary phase, starting a speed controller to enable the separation column to rotate forwards, injecting a mobile phase, and when the mobile phase flows out from the tail end of the column, indicating that the two phases are balanced, injecting a sample solution into the separation column, detecting by an ultraviolet detector, collecting eluent by an automatic part collector, respectively combining the eluents containing target substances, and performing post-treatment to obtain oleanolic acid and ursolic acid; the method has the advantages of simple and convenient operation, high efficiency, rapidness, less solvent consumption, high purity of the oleanolic acid and the ursolic acid and high sample recovery rate in the separation process.)

1. A method for separating isomers of oleanolic acid and ursolic acid, comprising:

(1) adding a mixed solvent system consisting of a hydroxypropyl-beta-cyclodextrin aqueous solution, normal hexane and ethyl acetate into a separating funnel, fully shaking, standing for layering, taking an upper phase as a stationary phase of high-speed countercurrent chromatography, and adjusting the pH of a lower phase to 2-8 by using a disodium hydrogen phosphate-phosphoric acid buffer solution to be used as a mobile phase;

The concentration of the hydroxypropyl-beta-cyclodextrin aqueous solution is 0.05-0.30 mol/L;

In the mixed solvent system, the volume parts of the hydroxypropyl-beta-cyclodextrin aqueous solution, the normal hexane and the ethyl acetate are respectively 10 parts, 8.5-9.5 parts and 0.5-1.5 parts;

(2) Dissolving a mixture of oleanolic acid and ursolic acid by using a mixed solution of a stationary phase and a mobile phase to obtain a sample solution;

(3) Filling a separation column of a high-speed counter-current chromatograph with a stationary phase, starting a speed controller to enable the separation column to rotate forwards, injecting a mobile phase at the speed of 500-1000 r/min at the flow rate of 0.5-5 mL/min, when the mobile phase flows out of the tail end of the column, indicating that the two phases are balanced, injecting a sample solution into the separation column, detecting by using an ultraviolet detector with the wavelength of 200-254 nm, collecting eluent by using an automatic part collector, respectively combining the eluents containing target substances oleanolic acid and ursolic acid, acidifying the eluent by using 10 wt% of HCl to the pH value of 2-3, extracting by using ethyl acetate, drying an ethyl acetate layer by using anhydrous sodium sulfate, filtering, and evaporating the solvent under reduced pressure to obtain oleanolic acid and ursolic acid.

2. The method for separating oleanolic acid and ursolic acid as claimed in claim 1, wherein in the step (1), the concentration of said hydroxypropyl- β -cyclodextrin aqueous solution is 0.1 to 0.15 mol/L.

3. the method for separating oleanolic acid and ursolic acid as claimed in claim 1, wherein in the step (1), the volume parts of the hydroxypropyl- β -cyclodextrin aqueous solution, n-hexane and ethyl acetate in the mixed solvent system are 10 parts, 9 parts and 1 part respectively.

4. the method for separating the isomers of oleanolic acid and ursolic acid as claimed in claim 1, wherein in the step (2), the volume ratio of the stationary phase to the mobile phase in the mixed solution of the stationary phase and the mobile phase is 1: 1.

5. The method for separating the isomers of oleanolic acid and ursolic acid as claimed in claim 1, wherein in the step (2), the volume usage of the mixed solution of the stationary phase and the mobile phase is 0.3-0.7 mL/mg based on the mass of the mixture of oleanolic acid and ursolic acid.

6. The method for separating oleanolic acid and ursolic acid as claimed in claim 1, wherein in the step (3), said sample solution is injected through a sample injection loop or pumped into a counter current chromatograph through a pump.

7. the method for separating oleanolic acid and ursolic acid as claimed in claim 1, wherein in the step (3), the sample solution is injected in a volume of less than 30% of the column volume of the separation column.

8. the method for separating the isomers of oleanolic acid and ursolic acid as claimed in claim 1, wherein in step (3), said target substance is detected by thin layer chromatography or high performance liquid chromatography, and combined with oleanolic acid and ursolic acid standard R, respectively_feluent containing oleanolic acid and ursolic acid with the same value or retention time;

the developing agent for the thin layer chromatography is cyclohexane, ethyl acetate and glacial acetic acid in a volume ratio of 10:3:0.5 of a mixed solvent;

the detection conditions of the high performance liquid chromatography are as follows: agilent 5HC-C₁₈(2) column; the column temperature is 25 ℃; the mobile phase is acetonitrile-methanol-0.5% ammonium acetate solution with volume ratio of 67: 12: 21, isocratic elutionA mode; flow rate: 1 mL/min; detection wavelength: 210 nm; sample introduction amount: 20 μ L.

(I) technical field

the invention relates to a method for separating isomers of oleanolic acid and ursolic acid

(II) background of the invention

Oleanolic acid (oleanolic acid) and ursolic acid (ursolic acid) are pentacyclic triterpenoid compounds, are widely distributed in nature, are isomers with each other, and have a molecular formula of C₃₀H₄₈O₃Only one methyl group is in different position and is not easy to separate. They are widely present in plants and are always present in the same species at the same time. Based on the close chemical structures of oleanolic acid and ursolic acid, both generally exhibit the same or similar therapeutic and biological effects. They are well known to have significant hepatoprotective effects against chemical carcinogen-induced acute liver injury, chronic liver fibrosis and cirrhosis. Numerous other pharmacological actions of oleanolic acid and ursolic acid have been reported, such as antioxidant, anti-inflammatory, anti-tumor, anti-HIV, antibacterial, stomach-protective and potential anti-obesity actions. In view of the characteristics of low natural abundance and no or extremely low ultraviolet absorption of triterpenic acid compounds in Chinese herbal medicines, researches on oleanolic acid and ursolic acid have attracted extensive attention of researchers at home and abroad.

counter current chromatography (counter current chromatography) is a continuous modern chromatographic separation technology without a solid carrier, can avoid pollution and sample loss caused by solid phase adsorption compared with high performance liquid chromatography, has large sample feeding amount and low operation cost, and is an ideal preparation chromatographic technology for separating isomers.

Disclosure of the invention

The invention aims to provide a method for separating isomers of oleanolic acid and ursolic acid by using high-speed counter-current chromatography, which utilizes cyclodextrin to align different recognition effects of oleanolic acid and ursolic acid so as to separate the oleanolic acid and the ursolic acid quickly, and has stronger purposiveness, quickness, high efficiency and simple process.

The technical scheme of the invention is as follows:

A method of separating isomers of oleanolic acid and ursolic acid, said method comprising:

(1) Adding a mixed solvent system consisting of a hydroxypropyl-beta-cyclodextrin aqueous solution, normal hexane and ethyl acetate into a separating funnel, fully shaking, standing for layering, taking an upper phase as a stationary phase of high-speed countercurrent chromatography, and adjusting the pH of a lower phase to 2-8 (preferably 6-7) by using a disodium hydrogen phosphate-phosphoric acid buffer solution to be used as a mobile phase;

The concentration of the hydroxypropyl-beta-cyclodextrin aqueous solution is 0.05-0.30 mol/L, preferably 0.1-0.15 mol/L;

In the mixed solvent system, the volume parts of the hydroxypropyl-beta-cyclodextrin aqueous solution, the normal hexane and the ethyl acetate are respectively 10 parts, 8.5-9.5 parts and 0.5-1.5 parts, preferably 10 parts, 9 parts and 1 part;

(2) Dissolving a mixture of oleanolic acid and ursolic acid by using a mixed solution of a stationary phase and a mobile phase to obtain a sample solution;

The volume ratio of the stationary phase to the mobile phase is 1: 1;

The volume dosage of the mixed solution of the stationary phase and the mobile phase is 0.3-0.7 mL/mg by mass of the mixture of oleanolic acid and ursolic acid;

(3) Filling a separation column of a high-speed counter-current chromatograph with a stationary phase, starting a speed controller to enable the separation column to rotate forwards, injecting a mobile phase at a flow rate of 0.5-5 mL/min at a rotation speed of 500-1000 r/min, and when the mobile phase flows out from the tail end of the column, indicating that the two phases are balanced, injecting a sample solution into the separation column, detecting by using an ultraviolet detector with a wavelength of 200-254 nm, collecting eluent by using an automatic part collector, respectively combining the eluents containing target substances oleanolic acid and ursolic acid, acidifying the eluent by using 10 wt% of HCl to a pH value of 2-3, extracting by using ethyl acetate, drying an ethyl acetate layer by using anhydrous sodium sulfate, filtering, and evaporating the solvent under reduced pressure to obtain oleanolic acid and ursolic acid;

The sample solution can be injected through a sample injection ring or pumped into a counter-current chromatograph through a pump;

The sample injection volume of the sample solution is generally less than 30% of the volume of the separation column, and preferably the sample injection volume is 5% -10% of the volume of the separation column;

The target substanceCan be monitored by thin layer chromatography or high performance liquid chromatography, and combined with oleanolic acid and ursolic acid standard substance R_feluent containing oleanolic acid and ursolic acid with the same value or retention time;

The developing agent for the thin layer chromatography is cyclohexane, ethyl acetate and glacial acetic acid in a volume ratio of 10:3:0.5 of a mixed solvent;

the detection conditions of the high performance liquid chromatography are as follows: agilent 5HC-C₁₈(2) column (4.6X 250mm, 5 μm); the column temperature is 25 ℃; the mobile phase is acetonitrile-methanol-0.5% ammonium acetate solution (67: 12: 21, v/v/v), isocratic elution mode; flow rate: 1 mL/min; detection wavelength: 210 nm; sample introduction amount: 20 μ L.

The invention has the beneficial effects that: the method is simple and convenient to operate, efficient and rapid, the solvent consumption is low, and the sample is not lost; the hydroxypropyl-beta-cyclodextrin aligned oleanolic acid and ursolic acid have strong recognition effect, strong purpose and obviously better separation effect than the traditional separation method.

(IV) description of the drawings

FIG. 1: high-speed countercurrent chromatography (hscc) profile of example 1;

FIG. 2: high-speed countercurrent chromatography (hscc) profile for example 2;

FIG. 3: high-speed countercurrent chromatography (hscc) profile for example 3;

FIG. 4: high-speed countercurrent chromatography (hscc) profile for example 4;

in FIGS. 1 to 4, peak 1 represents oleanolic acid, and peak 2 represents ursolic acid;

FIG. 5: a High Performance Liquid Chromatography (HPLC) chart of oleanolic acid and ursolic acid mixture; b High Performance Liquid Chromatography (HPLC) plot of peak number 1 in FIGS. 1-4; c High Performance Liquid Chromatography (HPLC) of peak No. 2 in FIGS. 1 to 4.

(V) detailed description of the preferred embodiments

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

In the embodiment of the invention, an analytical countercurrent chromatograph and a semi-preparative countercurrent chromatograph are adopted, and a separation system of the countercurrent chromatograph comprises a constant flow pump, an ultraviolet detector, a recorder and the like.