CN110613739A - Method for separating flavonoid compounds in cotton rose based on high-speed countercurrent chromatography - Google Patents

Abstract

The purpose of the present invention is to provide a method for rapidly separating anti-inflammatory components from Hibiscus hibiscus, and significantly speed up the preparation process of flavonoids in Hibiscus hibiscus. In this method, the crude extract only needs to be refluxed with ethanol and extracted with petroleum ether and ethyl acetate. The pH adjustment is directly completed in the solvent of high-speed countercurrent chromatography, without the need for excessive macroporous resin, and high-speed countercurrent chromatography is directly used to extract flavonoids from the crude extract. The steps are simple, the operation is convenient, and the recovery rate is high.

Description

A method for separation of flavonoids from Hibiscus hibiscus based on high-speed countercurrent chromatography

technical field

The invention relates to a method for rapidly separating anti-inflammatory components from Hibiscus hibiscus by high-speed countercurrent chromatography

Background technique

Hibiscus mutabilis Linn. (Hibiscus mutabilis Linn.) is a plant of the genus Hibiscus in the Malvaceae family. It is distributed in all parts of my country except the northeast and northwest regions. Its flowers, leaves and roots can be used as medicine. Muhi Rong has the functions of clearing away heat and detoxifying, reducing swelling and discharging pus, cooling blood and stopping bleeding. It has been recorded in the literature that the drug has good anti-inflammatory and detumescent effects when used externally and orally. It can treat appendicitis, mumps, gouty arthritis, erysipelas and burns.

The anti-inflammatory active ingredients of Hibiscus hibiscus are mainly flavonoids. At present, the extraction of flavonoids from Hibiscus hibiscus leaves is mainly through ethanol heating and reflux method, alkali extraction and acid precipitation method and microwave-assisted extraction method. Ethanol heating and refluxing costs high, the residual amount of organic solvent in the product exceeds the standard, the alkali extraction and acid precipitation method takes a long time, the strong acid and alkali are highly corrosive to the equipment, and the product quality is poor, which is not suitable for industrial production.

High speed countercurrent chromatography is a liquid-liquid chromatography separation technology. Its stationary phase and mobile phase are both liquids, without irreversible adsorption, and have no sample loss, no pollution, high efficiency, rapid and large-scale separation. Etc. Because HSCCC has obvious advantages compared with traditional separation and purification methods, this technology has been widely used in the separation of traditional Chinese medicine components, health food, biochemistry, bioengineering, natural product chemistry, organic synthesis, environmental analysis and other fields.

CN101716199A discloses a method for extracting flavonoids from Hibiscus hibiscus leaves. The Hibiscus Hibiscus leaves are mixed with a solid phase reagent in a mass ratio of 1:0.05-2, and the powder is ground until the D90 of the obtained powder particle size analysis result is 10-200 μm, and then water is fully added. Stirring, centrifuging, taking the supernatant and adding acid to adjust the pH to 1.0-6.0, then concentrating to the crude drug content of 0.1-1.0 g Hibiscus hibiscus leaves/mL, super-macroporous resin, washing with water, eluting with 75wt% ethanol aqueous solution, collecting ethanol Eluent, ethanol eluent is concentrated and dried to obtain hibiscus leaf flavonoid extract; the yield and content of flavonoids extracted by this method are significantly improved, but the steps are longer, the number of extraction steps is many, and the pH needs to be adjusted, and each method is used. types of macroporous resins.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for rapidly separating anti-inflammatory components from Hibiscus hibiscus, and significantly speed up the preparation process of flavonoids in Hibiscus hibiscus. In this method, the crude extract only needs to be refluxed with ethanol and extracted with petroleum ether and ethyl acetate. The pH adjustment is directly completed in the solvent of high-speed countercurrent chromatography, without the need for excessive macroporous resin, and high-speed countercurrent chromatography is directly used to extract flavonoids from the crude extract. The steps are simple, the operation is convenient, and the recovery rate is high.

The technical scheme adopted in the present invention is:

A method for separating flavonoids in Hibiscus hibiscus based on high-speed countercurrent chromatography, the method comprises the steps:

1) Take the dried Hibiscus hibiscus, pulverize and extract with ethanol for 1-4 times, each time for 1-2 hours; combine the extracts, concentrate and recover ethanol to obtain an ethanol extract, and extract the ethanol extract with petroleum ether in turn to obtain the petroleum ether layer , and then extracted with ethyl acetate, the ethyl acetate layer was taken, the solvent was distilled off under reduced pressure, and dried in vacuo to obtain the crude extract of Hibiscus hibiscus

2) Take the mixed solution of n-hexane-ethyl acetate-ethanol-water-glacial acetic acid as the two-phase solvent system, fully mix and then stand for layering, ultrasonically degas after the two-phase separation, the upper phase is used as the stationary phase, and the lower phase is used as the stationary phase. For the mobile phase, the stationary phase is filled with the column of the high-speed countercurrent chromatograph, and the high-speed countercurrent chromatograph is set. The flow rate was injected into the mobile phase until the two-phase solvent reached an equilibrium state in the column, and the crude extract of Hibiscus hibiscus was dissolved in a mixed solvent with a volume ratio of the upper phase and the lower phase, and the prepared sample solution was injected, and detected with a UV detector with a wavelength of 254 nm. , collect the effluent according to the peak shape of the UV detector spectrum, concentrate and freeze-dry to obtain the target flavonoid product.

In some embodiments of the present invention, the ethanol reflux extraction of Hibiscus hibiscus is 90% ethanol.

In some embodiments of the present invention, the hibiscus is extracted with ethanol under reflux for 3 times, 2 hours each time.

In some embodiments of the present invention, the ethanolic extract is extracted three times with petroleum ether and ethyl acetate.

In some embodiments of the present invention, the two-phase solvent body is n-hexane-ethyl acetate-ethanol-water-glacial acetic acid in a volume ratio of 1:1:1:1:0.05.

In some embodiments of the present invention, the high-speed countercurrent chromatography water bath temperature is 25°C.

In some embodiments of the present invention, the amount of the crude extract of Hibiscus hibiscus is 10 mg-20 mg/mL based on the volume of the mixed solvent.

In some embodiments of the present invention, the loading amount of the crude extract of Hibiscus hibiscus is 100 mg-200 mg.

In some embodiments of the invention, the mobile phase flow rate is 2 mL/min.

In some embodiments of the present invention, the rotational speed of the high-speed countercurrent chromatograph is 850 r/min.

The invention uses Hibiscus as a raw material, ethanol reflux extraction, petroleum ether ethyl acetate extraction, concentrated to obtain a crude extract of Hibiscus, and high-speed countercurrent chromatography is used to separate and obtain flavonoid anti-inflammatory components in Hibiscus. The method is simple and has good reproducibility. Can be used for the large-scale preparation of compounds.

Detailed ways

The solution of the present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited to this.

Example 1

Take 500 g of dried hibiscus, pulverize and extract with 90% ethanol for 3 times, 2 hours each time. Combine the extracts, concentrate and recover ethanol to obtain an ethanol extract, extract the ethanol extract 3 times with petroleum ether in turn, take the petroleum ether layer, and then extract 3 times with ethyl acetate, take the ethyl acetate layer, and remove the solvent by distillation under reduced pressure , and dried in vacuo to obtain 10.8 g of the crude extract of Hibiscus hibiscus.

The mixed solution of n-hexane-ethyl acetate-ethanol-water-glacial acetic acid (1:1:1:1:0.05, v/v) was used as a two-phase solvent system. Ultrasonic degassing, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase. The stationary phase is filled with the chromatographic column of the high-speed countercurrent chromatograph. The mobile phase was injected at a flow rate of 2 mL/min until the two-phase solvent reached an equilibrium state in the column, and 200 mg of the crude extract of Hibiscus hibiscus was dissolved in a mixed solvent with a volume ratio of the upper phase and the lower phase, and the prepared sample solution was injected with a wavelength of 254nm ultraviolet detector detects, collects the effluent according to the peak shape of the ultraviolet detector spectrum, concentrates and freeze-dries to obtain 9.6 mg of the target anti-inflammatory flavonoid product.

Example 2

Take 100 g of dried Hibiscus, crushed and extracted with 90% ethanol under reflux for 3 times, 2 hours each time. Combine the extracts, concentrate and recover ethanol to obtain an ethanol extract, extract the ethanol extract 3 times with petroleum ether in turn, take the petroleum ether layer, and then extract 3 times with ethyl acetate, take the ethyl acetate layer, and remove the solvent by distillation under reduced pressure , and vacuum-dried to obtain 2.7 g of the crude extract of Hibiscus hibiscus.

The mixed solution of n-hexane-ethyl acetate-ethanol-water-glacial acetic acid (1:1:1:1:0.05, v/v) was used as a two-phase solvent system. Ultrasonic degassing, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase. The stationary phase is filled with the chromatographic column of the high-speed countercurrent chromatograph. The mobile phase was injected at a flow rate of 2 mL/min until the two-phase solvent reached equilibrium in the column, and 100 mg of the crude extract of Hibiscus hibiscus was dissolved in a mixed solvent with a volume ratio of the upper phase and the lower phase, and the prepared sample solution was injected with a wavelength of 254nm UV detector detects, collects the effluent according to the peak shape of the UV detector spectrum, concentrates and freeze-dries to obtain 5.1 mg of the target anti-inflammatory flavonoid product.

Example 3

Take 1000 g of dried Hibiscus, crushed and extracted with 90% ethanol under reflux for 3 times, 2 hours each time. Combine the extracts, concentrate and recover ethanol to obtain an ethanol extract, extract the ethanol extract 3 times with petroleum ether in turn, take the petroleum ether layer, and then extract 3 times with ethyl acetate, take the ethyl acetate layer, and remove the solvent by distillation under reduced pressure , and vacuum-dried to obtain 21.1 g of the crude extract of Hibiscus hibiscus.

The mixed solution of n-hexane-ethyl acetate-ethanol-water-glacial acetic acid (1:1:1:1:0.05, v/v) was used as a two-phase solvent system. Ultrasonic degassing, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase. The stationary phase is filled with the column of the high-speed countercurrent chromatograph, and the high-speed countercurrent chromatograph is set. The mobile phase was injected at a flow rate of 2 mL/min until the two-phase solvent reached an equilibrium state in the column, and 200 mg of the crude extract of Hibiscus hibiscus was dissolved in a mixed solvent with a volume ratio of the upper phase and the lower phase, and the prepared sample solution was injected with a wavelength of 254nm UV detector detects, collects the effluent according to the peak shape of the UV detector spectrum, concentrates and freeze-dries to obtain 8.9 mg of the target anti-inflammatory flavonoid product.

Example 4

Take 1000 g of dried Hibiscus, crushed and extracted with 90% ethanol under reflux for 3 times, 2 hours each time. Combine the extracts, concentrate and recover ethanol to obtain an ethanol extract, extract the ethanol extract 3 times with petroleum ether in turn, take the petroleum ether layer, and then extract 3 times with ethyl acetate, take the ethyl acetate layer, and remove the solvent by distillation under reduced pressure , and vacuum-dried to obtain 21.1 g of the crude extract of Hibiscus hibiscus.

The mixed solution of n-hexane-ethyl acetate-ethanol-water-glacial acetic acid (1:1:1:1:0.05, v/v) was used as a two-phase solvent system. Ultrasonic degassing, the upper phase is used as the stationary phase, and the lower phase is used as the mobile phase. The stationary phase is filled with the column of the high-speed countercurrent chromatograph, and the high-speed countercurrent chromatograph is set. The mobile phase was injected at a flow rate of 2 mL/min until the two-phase solvent reached an equilibrium state in the column, and 200 mg of the crude extract of Hibiscus hibiscus was dissolved in a mixed solvent with a volume ratio of the upper phase and the lower phase, and the prepared sample solution was injected with a wavelength of 254nm ultraviolet detector detects, collects the effluent according to the peak shape of the ultraviolet detector spectrum, concentrates and freeze-dries to obtain 9.3 mg of the target anti-inflammatory flavonoid product.

Claims (10)

1. the method for separating flavonoids in Hibiscus hibiscus based on high-speed countercurrent chromatography, is characterized in that, described method comprises the steps: 1) Take the dried Hibiscus hibiscus, pulverize and extract with ethanol for 1-4 times, each time for 1-2 hours; combine the extracts, concentrate and recover ethanol to obtain an ethanol extract, and extract the ethanol extract with petroleum ether in turn to obtain the petroleum ether layer , and then extracted with ethyl acetate, the ethyl acetate layer was taken, the solvent was removed by distillation under reduced pressure, and dried in vacuo to obtain the crude extract of Hibiscus hibiscus; 2) Take the mixed solution of n-hexane-ethyl acetate-ethanol-water-glacial acetic acid as the two-phase solvent system, fully mix and then stand for layering, ultrasonically degas after the two-phase separation, the upper phase is used as the stationary phase, and the lower phase is used as the stationary phase. For the mobile phase, the stationary phase is filled with the column of the high-speed countercurrent chromatograph, and the high-speed countercurrent chromatograph is set. The flow rate was injected into the mobile phase until the two-phase solvent reached an equilibrium state in the column, and the crude extract of Hibiscus hibiscus was dissolved in a mixed solvent with a volume ratio of the upper phase and the lower phase, and the prepared sample solution was injected, and detected with a UV detector with a wavelength of 254 nm. , collect the effluent according to the peak shape of the UV detector spectrum, concentrate and freeze-dry to obtain the target flavonoid product. 2. method as claimed in claim 1 is characterized in that: Hibiscus hibiscus ethanol reflux extraction is 90% ethanol. 3. method as claimed in claim 1 is characterized in that: hibiscus ethanol reflux extraction 3 times, each 2h. 4. The method of claim 1, wherein the ethanolic extract is extracted 3 times with petroleum ether ethyl acetate. 5. method as claimed in claim 1 is characterized in that: two-phase solvent body is that the volume ratio of n-hexane-ethyl acetate-ethanol-water-glacial acetic acid is 1:1:1:1:0.05. 6. The method of claim 1, wherein the temperature of the high-speed countercurrent chromatography water bath is 25°C. 7. The method of claim 1, wherein the amount of the crude extract of Hibiscus hibiscus is 10 mg-20 mg/mL in terms of the volume of the mixed solvent. 8. The method of claim 1, wherein the loading amount of the crude extract of Hibiscus hibiscus is 100mg-200mg. 9. The method of claim 1, wherein the flow rate of the mobile phase is 2 mL/min. 10. The method of claim 1, wherein the rotating speed of the high-speed countercurrent chromatograph is 850 r/min.