CN1994997B - A method for separating and purifying 6-gingerol - Google Patents

Abstract

The invention discloses a separating method of 6-gingerol, which is characterized by the following: adopting multiple chromatographic ways to separate effective component 6-gingerol in the traditional Chinese medicine ginger; accelerating the separating course; simplifying the operation; improving the quality and production.

Description

A method for separating and purifying 6-gingerol

technical field

The invention relates to the field of natural medicines, in particular to a method for separating the active ingredient 6-gingerol, that is, a method for separating the active ingredient 6-gingerol in the traditional Chinese medicine ginger by using multiple chromatographic means in orderly combination.

Background technique

Ginger and dried ginger are the fresh rhizome and dried rhizome of Zingiber officinale Rosc., which are commonly used in traditional Chinese medicine. The main components in ginger are volatile oil components, gingerol components and diphenylheptane components. Among them, gingerol has a large content of components, has obvious spicy taste and biological activity, and has been confirmed by modern research to have various pharmacological activities, which can be used as the research basis for the pharmacological activity and quality control of ginger and dried ginger. There are many kinds of gingerol components, among which 6-gingerol has the highest content, and its Chinese names include 6-gingerol, 6-gingerol, etc. There is some confusion in naming, so this patent uses its English name as the standard. Therefore, the separation and purification of 6-gingerol has obvious significance in promoting the pharmacological research and quality control of ginger traditional Chinese medicine.

6-gingerol has unstable physical and chemical properties because of its 8-hydroxyketone structure in its molecule, and it coexists with a variety of chemical components with similar structures, making it difficult to separate. Some foreign companies have commercialized 6-gingerol reference substance supply, but the price is expensive, the unit price per 20mg is about 200 US dollars. However, existing domestic separation methods often use some methods with high separation ability but low output, such as thin-layer scraper method and analytical HPLC method.

CN1616391A discloses a method for separating 6-gingerol from ginger. It first obtains ginger oleoresin from the acetone extract of ginger, and then separates through silica gel column chromatography and HPLC. The raw material is ginger oleoresin soaked in acetone, the yield is low, and the components are relatively complex, which is not conducive to subsequent separation. When it is separated on silica gel in the first step, the eluent is a mixed solution of n-hexane and ether, which is expensive and extremely volatile, and the ratio is prone to change during the chromatographic process. In the HPLC separation step, the mixed mobile phase of water-methanol-glacial acetic acid is used, which leads to cumbersome post-processing steps, reduces the yield and increases the cost.

There are also bibliographical reports (preparation and identification of crystalline gingerol, "Food and Industrial Fermentation" 2005 volume 31, the 10th phase: p48-50), its first and second steps are the same as CN1313391A, and it uses dry method column chromatography, As stated in the literature, 700g of silica gel can only load 20g of sample, which is not conducive to large-scale applications. In addition, the literature uses preparative thin layer and Sephadex LH 20 column chromatography to refine the 6-gingerol crude product obtained in the first step of silica gel column chromatography. The preparative thin-layer method has less sample load and limited separation ability, so it is only suitable for a small amount of preparation in the laboratory. The filler used in Sephadex LH 20 column chromatography is expensive, the operation time is long, and the separation efficiency is low, so it is not suitable for large-scale applications. In addition, the literature does not specifically describe the final n-hexane recrystallization step. It has been proved by experiments that 6-gingerol does not dissolve well in n-hexane under the condition of only heating.

Obviously, due to the constraints of cost and output, it is still difficult to realize the pharmacological research and quality control of ginger traditional Chinese medicine based on 6-gingerol.

my country's supercritical fluid extraction research began in the early 1980s, and has entered the stage of commercial application. There is already a mature commodity ginger supercritical fluid extract available on the market. Due to the low temperature and airtight system, the supercritical fluid extraction technology can preserve a large amount of thermally unstable and easily oxidized 6-gingerol, creating a good prerequisite for the separation and purification of 6-gingerol. Therefore, how to use ginger supercritical fluid extract as the basis, properly apply the combination of multiple chromatographic means, and study a method for preparing 6-gingerol that is fast, easy to operate, good in finished product quality and has a large output is a challenge faced by this technical field. main problem.

Contents of the invention

In order to overcome the inherent deficiencies of the prior art, the present invention discloses a method for preparing 6-gingerol, the main active ingredient in ginger, which is fast, easy to operate, good in finished product quality and relatively large in yield.

The present invention separates and purifies the method for 6-gingerol from ginger supercritical fluid extract, comprises the following steps successively:

a, silica gel column chromatographic separation;

b. HPLC purification;

c, recrystallization,

Wherein the eluent of gradient elution in a step is the mixed solution of sherwood oil and ethyl acetate.

The inventor found in the research that using the diethyl ether:n-hexane mixed solvent reported in the literature as the eluent is expensive and extremely volatile, and the ratio is prone to change during the chromatographic process. The inventor has also tried many eluents, and finally found that using a mixed solution of petroleum ether and ethyl acetate as the eluent is not only cheap, but also has less compositional change during the chromatographic process. The yield of this step can reach 4-7%, which is much higher than the yield of elution with diethyl ether: n-hexane mixed solvent.

Since 6-gingerol is an oily liquid at room temperature, it is easily soluble in various medium and high polar organic solvents, but hardly soluble in water and n-hexane. The inventors have found that 6-gingerol can be dissolved in boiling n-hexane under the condition of ultrasound as an auxiliary means, and after standing in the refrigerator, 6-gingerol is separated out in a white yellow-white crystal state, and the product purity can be further improved, and The product looks good. Therefore, the solvent used for the recrystallization of the present invention is n-hexane, and boiling water and ultrasound are used to aid in dissolution. Experiments have proved that about 1g of 6-gingerol can be dissolved per 100ml of n-hexane, which is suitable for batch preparation.

In the present invention, the mobile phase used in HPLC purification is preferably methanol. The present invention uses a single methanol mobile phase, the post-treatment is simple, and the recovered methanol can be considered to be reused, which has certain environmental protection factors.

In the above-mentioned preparation method, after the step a, a silica gel column chromatography refining step may be further included, and the eluent in the refining step is a mixed solution of petroleum ether and acetone. The refining step can make the purity of the final product reach more than 98%, while the final purity of the finished product without the refining process is more than 95%.

In the above preparation method, the volume ratio of petroleum ether and ethyl acetate is preferably 5-9:1.

In the above-mentioned preparation method, the volume ratio of sherwood oil and acetone is preferably 4～8: 1

The more preferred preparation method of the present invention comprises the steps in turn:

a. Silica gel column chromatographic separation

Using supercritical fluid extract of ginger as raw material, mixing samples, using silica gel as stationary phase for chromatography, gradient elution, eluent is a mixed solution of petroleum ether and ethyl acetate, collecting the part containing 6-gingerol, and setting rotary evaporation Evaporate to dryness on the instrument to obtain the crude product of 6-gingerol;

b. Refined by silica gel column chromatography

Mix the crude product of 6-gingerol obtained in step a, use silica gel for chromatography as a stationary phase, and the eluent is a mixed solution of sherwood oil and acetone, collect the main color bands therein, and evaporate to dryness on a rotary evaporator, i.e. Obtain 6-gingerol refined product;

c. HPLC purification

The 6-gingerol refined product obtained in step b is dissolved in methanol to prepare HPLC purification, the mobile phase is pure methanol, the main chromatographic peaks are collected, and evaporated to dryness on a rotary evaporator to obtain pure 6-gingerol;

d. Recrystallization

The pure product of 6-gingerol obtained in step c is recrystallized with n-hexane, and when dissolving 6-gingerol with n-hexane, two means of boiling water and ultrasound are used for dissolution aid.

The preparation method of the invention is easy to operate, and the finished product has good quality and high output. In particular, the purity of the 6-gingerol prepared by the product of the refining step is over 98%. The product yield of the present invention is far higher than the yield in the prior art. The yields in each step are as follows:

Steps Coarse fractionation of silica gel Purification of silica gel Preparative HPLC Recrystallization

Yield 4-7% About 30% 30-50% 50-60%

Detailed ways

Example 1

(1) Preliminary separation by silica gel column chromatography

Use 300g of commercial ginger supercritical fluid extract as raw material, mix the sample with 200g of 100-200 mesh column chromatography silica gel, use 100-200 mesh column chromatography with 2kg of silica gel as the stationary phase, gradient elution, and the polarity of the eluents in sequence Petroleum ether: ethyl acetate = 12: 1, petroleum ether: ethyl acetate = 9: 1 and petroleum ether: ethyl acetate = 6: 1, collected petroleum ether: ethyl acetate = 6: 1 fraction, TLC test combined , and evaporated to dryness on a rotary evaporator to obtain 16.5 g of crude 6-gingerol, with a yield of 5.5%. Its purity is above 65% (HPLC test, area normalization method).

(2) Refined by silica gel column chromatography

Mix the 6-gingerol crude product obtained in the previous step, use 200-300 mesh chromatography silica gel as the stationary phase, the amount of silica gel used is 20 times that of the 6-gingerol crude product used, and the eluent is petroleum ether: acetone=4: 1. Collect the main color bands, combine them for TLC inspection, and evaporate to dryness on a rotary evaporator to obtain 6.5 g of 6-gingerol refined product, with a yield of 39%. Its purity is above 80% (HPLC test, area normalization method).

(3) Preparative HPLC purification

Dissolve the refined product of 6-gingerol obtained in the previous step with an appropriate amount of methanol, filter it through a 0.45 μm microporous membrane, _and purify it by HPLC using Angilent1100 series. , the detection wavelength was 254nm, the main chromatographic peaks were collected, and evaporated to dryness on a rotary evaporator to obtain 2 g of pure 6-gingerol, with a yield of 30%. Its purity is above 96% (HPLC test, area normalization method).

(4) Recrystallization

The pure 6-gingerol obtained in the previous step was recrystallized with n-hexane.

The recrystallization steps are as follows: adding boiling water into an ultrasonic cleaner, adding 100 mL of n-hexane to the pure 6-gingerol, and dissolving under ultrasonic conditions. After it is completely dissolved, immediately place it in the freezer of the refrigerator (about -10°C) and let it stand overnight, then yellowish white to white 6-gingerol crystals can be seen to precipitate. About 1.2g, yield 60%. Its purity is above 98% (HPLC test, area normalization method).

Example 2

(1) Preliminary separation by silica gel column chromatography

Using 250g of commercial ginger supercritical fluid extract as raw material, 200g of 100-200 mesh column chromatography silica gel as a sample, 1.5kg of 100-200 mesh column chromatography silica gel as a stationary phase, gradient elution, and the polarity of the eluent Petroleum ether: ethyl acetate = 12:1, petroleum ether: ethyl acetate = 9:1 and petroleum ether: ethyl acetate = 6:1, collected petroleum ether: ethyl acetate = 6:1, TLC inspection Combined and evaporated to dryness on a rotary evaporator to obtain about 12 g of crude 6-gingerol with a yield of 4.8%. Its purity is above 65% (HPLC test, area normalization method).

(2) Refined by silica gel column chromatography

Mix the 6-gingerol crude product obtained in the previous step, use 200-300 mesh chromatography silica gel as the stationary phase, the amount of silica gel used is 15 times that of the 6-gingerol crude product used, and the eluent is petroleum ether: acetone=5: 1. Collect the main color bands, combine them for TLC inspection, and evaporate to dryness on a rotary evaporator to obtain about 4 g of 6-gingerol refined product, with a yield of 33%. Its purity is above 80% (HPLC test, area normalization method).

(3) Preparative HPLC purification

Dissolve the refined product of 6-gingerol obtained in the previous step with an appropriate amount of methanol, filter it through a 0.45 μm microporous membrane, _and purify it by HPLC using Angilent1100 series. , the detection wavelength was 254nm, the main chromatographic peaks were collected, and evaporated to dryness on a rotary evaporator to obtain about 2 g of pure 6-gingerol, with a yield of 50%. Its purity is above 95% (HPLC test, area normalization method).

(4) Recrystallization

The pure 6-gingerol obtained in the previous step was recrystallized with n-hexane.

The recrystallization steps are as follows: adding boiling water into an ultrasonic cleaner, adding 100 mL of n-hexane to the pure 6-gingerol, and dissolving under ultrasonic conditions. After it is completely dissolved, immediately place it in the freezer of the refrigerator (about -10°C) and let it stand overnight, then yellowish white to white 6-gingerol crystals can be seen to precipitate. About 1.3g, yield 65%. Its purity is above 98% (HPLC test, area normalization method).

Example 3

(1) Preliminary separation by silica gel column chromatography

Use commercial ginger supercritical fluid extract 100g as raw material, mix sample with 60g silica gel for 100-200 mesh column chromatography, use 1.0kg silica gel for column chromatography with 100-200 mesh as stationary phase, gradient elution, eluent polarity Petroleum ether: ethyl acetate = 12:1, petroleum ether: ethyl acetate = 9:1 and petroleum ether: ethyl acetate = 6:1, collected petroleum ether: ethyl acetate = 6:1, TLC inspection Combined and evaporated to dryness on a rotary evaporator to obtain the crude product of 6-gingerol. It is about 6.5g, the yield is 6.5%, and its purity is above 65% (HPLC test, area normalization method).

(2) Preparative HPLC purification

Dissolve the crude 6-gingerol obtained in the previous step with an appropriate amount of methanol, filter through a 0.45 μm microporous membrane, and purify it by HPLC using Angilent 1100 series. The mobile phase is pure methanol, the stationary phase is C ₁₈ , and the flow rate is 10 mL/min. The detection wavelength is 254nm, and the main chromatographic peaks are collected and evaporated to dryness on a rotary evaporator to obtain the pure product of 6-gingerol. It is about 2 g, the yield is 31%, and its purity is above 85% (HPLC test, area normalization method).

(4) Recrystallization

The pure 6-gingerol obtained in the previous step was recrystallized with n-hexane.

The recrystallization steps are as follows: adding boiling water into an ultrasonic cleaner, adding 100 mL of n-hexane to the pure 6-gingerol, and dissolving under ultrasonic conditions. After it is completely dissolved, immediately place it in the freezer of the refrigerator (about -10°C) and let it stand overnight, then yellowish white to white 6-gingerol crystals can be seen to precipitate. Finally, 1.2 g of the finished product was obtained with a yield of 60% and a purity of over 95% (HPLC test, area normalization method).

Claims (6)

1. a method for separating and purifying 6-gingerol from ginger supercritical fluid extract, comprising the following steps successively: a, silica gel column chromatographic separation; b. HPLC purification; c, recrystallization, It is characterized in that: the eluent for gradient elution in step a is a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether and ethyl acetate is 5-9:1. 2. The method of claim 1, wherein the solvent used for recrystallization is n-hexane, and boiling water and ultrasonic waves are used to aid dissolution. 3. the method for claim 1, also comprise silica gel column chromatography refining step after a step, the eluent of refining step is the mixed solvent of sherwood oil and acetone. 4. The method of claim 3, wherein the volume ratio of sherwood oil and acetone is 4 to 8:1. 5. The method of claim 1, wherein the mobile phase used during HPLC purification is methanol. 6. The method of any one of claims 1 to 5, comprising the steps of: a. Silica gel column chromatographic separation Using ginger supercritical fluid extract as raw material, mixing samples, using silica gel as a stationary phase for chromatography, and gradient elution, the eluent is a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether and ethyl acetate is 5 ~9:1, collect the part containing 6-gingerol, and evaporate to dryness on a rotary evaporator to obtain the crude product of 6-gingerol; b. Refined by silica gel column chromatography Mix the crude product of 6-gingerol obtained in step a, use silica gel for chromatography as a stationary phase, and the eluent is a mixed solvent of sherwood oil and acetone, collect the main color bands therein, and evaporate to dryness on a rotary evaporator, i.e. Obtain 6-gingerol refined product; c. HPLC purification The 6-gingerol refined product obtained in step b is dissolved in methanol to prepare HPLC purification, the mobile phase is pure methanol, the main chromatographic peaks are collected, and evaporated to dryness on a rotary evaporator to obtain pure 6-gingerol; d. Recrystallization The pure product of 6-gingerol obtained in step c is recrystallized with n-hexane, and when dissolving 6-gingerol with n-hexane, two means of boiling water and ultrasound are used for dissolution aid.