阅读说明：本技术 一种低共熔溶剂提取结合大孔树脂纯化银杏叶中黄酮类化合物的方法 (Method for extracting flavonoid compounds from ginkgo leaves by combining eutectic solvent with macroporous resin purification ) 是由 金君素 李吉超 于 2019-08-31 设计创作，主要内容包括：本发明提供了一种利用低共熔溶剂结合大孔树脂高效提取纯化银杏叶中黄酮类化合物的方法。针对目前工业上多采用60％乙醇提取银杏叶中黄酮工艺中存在的提取率低、流程繁琐、有机溶剂损失严重、溶剂易燃易爆等问题,开发了一种以胆碱类低共熔溶剂水溶液为提取剂,在一定的超声条件下从银杏叶中高效提取黄酮类化合物,结合大孔树脂纯化的方法,最后制备含量为24％以上的黄酮类化合物,本发明的优点是：与传统提取剂相比,低共熔溶剂不挥发、不易燃、损失少,该方法操作简便、提取率高、具有广泛的工业化应用前景。(The invention provides a method for efficiently extracting and purifying flavonoid compounds in ginkgo leaves by combining a eutectic solvent with macroporous resin. Aiming at the problems of low extraction rate, complicated flow, serious loss of organic solvent, inflammable and explosive solvent and the like existing in the process of extracting flavone from ginkgo leaves by adopting 60% ethanol in the prior industry, the invention develops a method for efficiently extracting flavonoid compounds from ginkgo leaves by taking choline eutectic solvent aqueous solution as an extracting agent under certain ultrasonic conditions and finally preparing the flavonoid compounds with the content of more than 24% by combining a macroporous resin purification method, and the method has the advantages that: compared with the traditional extractant, the eutectic solvent is non-volatile, non-flammable and less in loss, and the method is simple and convenient to operate, high in extraction rate and wide in industrial application prospect.)

1. A method for extracting flavonoid compounds in ginkgo leaves by combining a eutectic solvent with macroporous resin purification is characterized by comprising the following steps:

(1) preparation of eutectic solvent: choline chloride and polyol are mixed according to a certain molar ratio and heated at 60 ℃ to synthesize uniform and stable liquid.

(2) Extraction: the flavonoid compounds in the ginkgo leaves are intensively extracted by using the eutectic solvent water solution under certain conditions.

(3) And (3) purification: adsorbing the extract with macroporous resin, eluting with low-concentration ethanol solution, eluting with high-concentration ethanol solution, and drying to obtain the final product.

2. The eutectic solvent according to claim 1, wherein the eutectic solvent is a choline-based eutectic solvent, and is synthesized from choline chloride and ethylene glycol or one of 1, 2-propylene glycol, glycerol, 1, 3-butylene glycol, 1, 4-butylene glycol, 1, 2-pentanediol, 1, 5-pentanediol, diethylene glycol, and triethylene glycol.

3. The molar ratio of choline chloride to polyol according to claim 1 is from 1:1 to 1:6, preferably from 1:2 to 1: 4.

4. The volume ratio of water in the eutectic solvent aqueous solution according to claim 1 is 10% to 60%, preferably 20% to 40%.

5. The extraction method according to claim 1, wherein: the dosage ratio of the dried ginkgo leaf powder to the eutectic solvent aqueous solution is 1g:5mL-40mL, preferably 1g:10mL-30mL, the extraction temperature is 40-70 ℃, preferably 50-65 ℃, the extraction time is 10min-40min, preferably 20min-35min, and the ultrasonic power in the extraction method according to claim 1 is 250W.

6. The macroporous resin used according to claim 1 is one of NKA-9, D101, AB-8, DM 130.

7. The extract according to claim 1, wherein the volume of the extractive solution is 0.5BV-3BV, the concentration of the low concentration ethanol solution is 5% -30%, the volume is 0.5BV-2BV, the concentration of the high concentration ethanol solution is 60% -95%, and the volume is 0.5BV-2 BV.

Technical Field

The invention relates to the field of traditional Chinese medicine extraction, in particular to a method for extracting flavonoids compounds in ginkgo leaves by combining eutectic solvent extraction with macroporous resin purification.

Background

The use of ginkgo in medicine can be traced back to Bencao gang mu written by Li Shizhen in 1596 for the earliest, and ginkgo leaves are considered to have the curative effects of promoting blood circulation to remove blood stasis and dredging collaterals in traditional Chinese medicine, and are mainly applied to treating cardiovascular and cerebrovascular diseases in modern medical treatment, the extraction of effective components from ginkgo leaves starts in the 20 th century, and the product EGB761 developed by Germany Schwabe company and the standard preparation method thereof continue to the present, at present, more than 140 chemical components are separated from ginkgo leaves, wherein the flavonoid compounds have obvious physiological activity as the main components in the flavonoid compounds, so how to improve the extraction efficiency of the flavonoid compounds in the ginkgo leaves is the key of the ginkgo industry.

The polarity of various substances in ginkgo leaves is different, soThere is a certain difference in solubility in the solution. The method for extracting flavonoids from folium Ginkgo can be divided into organic solvent method and supercritical (subcritical) CO method according to the kind of the extractant₂Methods and enzyme extraction methods. In order to remove fat-soluble impurities such as alkylphenol and chlorophyll from ginkgo leaves and extract ginkgetin with the greatest possibility, some solvents with higher polarity such as water, methanol, ethanol, acetone or their mixed solvents are often used for extraction, and the method is also an extraction technology widely used in the industry at present, for example, plum shin et al use ethanol water solution as the best extractant for extracting flavonoid compounds in ginkgo leaves, see: lixinzhou et al, chemical and biological engineering, 2014,31(03):31-34. in CN106822194A, 60% ethanol solution is used as extractant and Soxhlet extraction process is combined to obtain extraction rate of only 75% under optimal conditions; the organic solvent extraction method is easy to industrialize, but the used solvent is high in volatility, inflammable and explosive, has certain potential safety hazard, is relatively low in extraction rate, is generally extracted for 2-4 times industrially, and is complex to operate. The supercritical (sub-) critical fluid technology is widely applied to the extraction of plant components at present, for example, the supercritical CO is determined by Qi Zhao et al under the condition of 35MPa₂The method for extracting flavonoids from ginkgo leaves is as follows: 27-28 of 2009,36(03) in Zhao Qi Jun, Jiangxi forestry science and technology, and the solvent used by the method is green and safe but has high requirements on equipment. The enzyme extraction method utilizes cellulase to decompose cellulose and hemicellulose of ginkgo leaves under certain conditions, destroys cell structures and dissolves out effective components, for example, Zhangqing researches the process conditions of extracting the active component ginkgo flavone in ginkgo leaves by combining enzyme method and solvent extraction, see: zhang Xiao Qing, applied to chemical industry, 2005,34(10):48-49, the main defect of the method is that the enzyme is easy to inactivate and difficult to recover in the extraction process, and the cost is high. The eutectic solvent is a mixture composed of a hydrogen bond donor and a hydrogen bond acceptor, and has strong dissolving capacity for effective components in natural products, so in recent years, researches for extracting bioactive components by combining the eutectic solvent with other methods for assistance are gradually developed, for example, in patent CN108619202A, extraction of the eutectic solvent and separation and purification of burdock leaves by macroporous resin are reportedThe method for preparing the middle flavonoid compound can separate and enrich effective components; in patent CN104887723A, a method for extracting total flavonoids in ginkgo leaves by combining a ternary eutectic solvent choline chloride-ethylene glycol-oxalic acid with ultrasonic assistance is reported; the use of a hydrophilic eutectic solvent system is reported in patent CN 107789376A: a method for simultaneously extracting multiple effective components from folium Ginkgo by choline chloride-levulinic acid-malonic acid and a hydrophobic eutectic solvent system of methyl trioctyl ammonium chloride-octanol-octanoic acid has a total flavone extraction rate of 77.72%; patent CN107312047A reports that salidroside and rosavin can be synchronously separated and prepared from rhodiola rosea by using a method of extracting with an alcohol aqueous solution containing a eutectic solvent and then performing gradient elution by using mixed macroporous resin.

In conclusion, compared with the traditional organic solvent, the eutectic solvent is used as a novel extracting agent and has a good extracting effect on flavonoid compounds in the Chinese herbal medicines, and the method for extracting the flavonoid compounds in the ginkgo leaves by combining the eutectic solvent with macroporous resin can fully extract the flavonoid compounds in the ginkgo leaves, separate and purify the flavonoid compounds, and realize the effective utilization of resources.

Disclosure of Invention

Aiming at the problems existing at present, the invention develops a green chemical process for purifying flavonoid compounds in ginkgo leaves by combining eutectic solvent extraction with macroporous resin, the used extracting agent is non-volatile and non-flammable, the regeneration is easy, the extraction rate is higher than that of the traditional organic solvent, and the whole process is simple and convenient to operate, short in period and easy to industrialize.

The technical scheme of the invention is as follows: a method for extracting flavonoids compounds from ginkgo leaves by using a eutectic solvent and combining macroporous resin purification is characterized in that ginkgo leaf powder is used as a raw material, a eutectic solvent aqueous solution is added, and the flavonoids compounds are extracted in an enhanced manner under an ultrasonic condition, and comprises the following steps:

1) synthesizing the choline chloride and the polyol according to the molar ratio of 1:1-1:6 at the condition of heating at 60 ℃ to obtain the colorless and uniform choline eutectic solvent.

2) Pulverizing dried folium Ginkgo, sieving with 60 mesh sieve, extracting with eutectic solvent water solution with water volume of 10-60%, mixing folium Ginkgo powder and eutectic solvent water solution at a ratio of 1g: 5-40 mL, extracting under ultrasonic power of 250W and temperature of 50-90 deg.C for 10-40 min, and centrifuging to obtain upper layer ginkgetin crude extract. Hydrolyzing the extractive solution with hydrochloric acid, filtering with 0.45 μm microporous membrane, measuring peak areas of quercetin, kaempferol, and isorhamnetin with high performance liquid chromatograph, and calculating total flavone extraction rate with reference to 2015 version of Chinese pharmacopoeia.

3) Adsorbing the 0.5-3 BV extractive solution with resin column, eluting with 0.5-2 BV 5-30% ethanol solution to remove impurities, eluting with 0.5-2 BV 60-95% ethanol solution to obtain flavonoid solution, and drying to obtain flavonoid.

The polyalcohol used for synthesizing the choline eutectic solvent in the step 1) is one of ethylene glycol, 1, 2-propylene glycol, glycerol, 1, 3-butanediol, 1, 4-butanediol, 1, 2-pentanediol, 1, 5-pentanediol, diethylene glycol and triethylene glycol.

The invention has the advantages that: the used extractant is non-volatile, non-flammable, low in loss, easy to regenerate and simple and convenient to operate, the one-time extraction rate reaches more than 95% under the optimal condition, the one-time extraction rate of a 60% ethanol solvent commonly used in industry is 75% -90%, the extraction rate of a eutectic solvent is higher than that of a traditional organic solvent, the operation is simple, the industrialization is easy, the extractant can replace the traditional organic solvent to be used for high-efficiency extraction of ginkgo flavonoid compounds, the content of the flavonoid compounds obtained after the extracting solution is purified by macroporous resin is higher than 24%, and the requirement of 2015 edition of Chinese pharmacopoeia on the content of the flavonoid compounds in the ginkgo leaf extract is met.

Detailed Description

The present invention is further illustrated with reference to the following specific examples, which are not intended to limit the scope of the invention.

Example 1:

a method for extracting flavonoids compounds in ginkgo leaves by combining a eutectic solvent with macroporous resin purification comprises the following steps:

1) mixing a certain mass of choline chloride and 1, 3-butanediol according to a molar ratio of 1:3, and heating at 60 ℃ for 1h to synthesize a eutectic solvent for later use.

2) Taking 1g of ginkgo leaf powder which is sieved by a 60-mesh sieve, adding 6.4mL of prepared eutectic solvent and 1.6mL of water, uniformly mixing, carrying out ultrasonic extraction for 20min at the power of 250W and the temperature of 60 ℃, and carrying out centrifugal separation on the extracting solution for 10min at 4000rpm to obtain the ginkgo flavone extracting solution. Hydrolyzing the extractive solution with hydrochloric acid, filtering with 0.45 μm microporous membrane, measuring the peak areas of quercetin, kaempferol, and isorhamnetin with high performance liquid chromatograph, and calculating to obtain total flavone extraction rate of 71.3% with reference to 2015 edition of Chinese pharmacopoeia.

3) Loading AB-8 macroporous resin with the dry weight of 5g into a column, adding 2BV of the extracting solution in the step 2) for dynamic adsorption at 0.5 mL/min, firstly washing the adsorbed resin with excessive water to remove the eutectic solvent attached to the resin, eluting with an ethanol solution with the mass fraction of 2BV of 20% to remove impurities, eluting the ginkgetin with the mass fraction of 2.5BV of 60% to obtain a ginkgetin eluent, drying the ginkgetin eluent to obtain flavonoid compound powder 59.1mg, hydrolyzing with hydrochloric acid, passing through a 0.45-mu m microporous membrane, and calculating the content of the flavonoid compound by using a high performance liquid chromatograph according to the 'Chinese pharmacopoeia' 2015 edition.

Example 2:

a method for extracting flavonoids compounds in ginkgo leaves by combining a eutectic solvent with macroporous resin purification comprises the following steps:

1) mixing a certain mass of choline chloride and ethylene glycol according to a molar ratio of 1:4, and heating at 60 ℃ for 1h to synthesize a eutectic solvent for later use.

2) Taking 1g of ginkgo leaf powder which is sieved by a 60-mesh sieve, adding 16mL of prepared eutectic solvent and 4mL of water, uniformly mixing, carrying out ultrasonic extraction for 20min at the power of 250W and the temperature of 60 ℃, and carrying out centrifugal separation on the extracting solution for 10min at 4000rpm to obtain the ginkgo flavone extracting solution. Hydrolyzing the extractive solution with hydrochloric acid, filtering with 0.45 μm microporous membrane, measuring the peak areas of quercetin, kaempferol, and isorhamnetin with high performance liquid chromatograph, and calculating to obtain total flavone extraction rate of 93.3% with reference to 2015 edition of Chinese pharmacopoeia.

3) Loading AB-8 macroporous resin with the dry weight of 5g into a column, adding 2.5BV of the extracting solution in the step 2) for dynamic adsorption at the speed of 0.5mL per minute, firstly washing the adsorbed resin with excessive water to remove the eutectic solvent attached to the resin, eluting with 2BV of ethanol solution with the mass fraction of 20% to remove impurities, eluting the ginkgetin with 3BV of ethanol solution with the mass fraction of 90%, drying the obtained ginkgetin eluent to obtain flavonoid compound powder 82.7mg, hydrolyzing with hydrochloric acid, and then passing through a 0.45-mu m microporous membrane to obtain the content of 28.04% by calculation of a high performance liquid chromatograph according to the Chinese pharmacopoeia of the 2015 edition.

Example 3:

a method for extracting flavonoids compounds in ginkgo leaves by combining a eutectic solvent with macroporous resin purification comprises the following steps:

1) mixing a certain mass of choline chloride and ethylene glycol according to a molar ratio of 1:4, and heating at 60 ℃ for 1h to synthesize a eutectic solvent for later use.

2) Taking 1g of ginkgo leaf powder which is sieved by a 60-mesh sieve, adding 12mL of prepared eutectic solvent and 3mL of water, uniformly mixing, carrying out ultrasonic extraction for 30min at the power of 250W and the temperature of 60 ℃, and carrying out centrifugal separation on the extracting solution for 10min at 4000rpm to obtain the ginkgo flavone extracting solution. Hydrolyzing the extractive solution with hydrochloric acid, filtering with 0.45 μm microporous membrane, measuring the peak areas of quercetin, kaempferol, and isorhamnetin with high performance liquid chromatograph, and calculating to obtain total flavone extraction rate of 94.7% with reference to 2015 edition of Chinese pharmacopoeia.

3) Packing a D101 macroporous resin with the dry weight of 5g into a column, adding 2BV of the extracting solution in the step 2) for dynamic adsorption at 0.5 mL/min, firstly washing the adsorbed resin with excessive water to remove a eutectic solvent attached to the resin, eluting with an ethanol solution with the mass fraction of 2BV of 20% to remove impurities, eluting the ginkgetin with an ethanol solution with the mass fraction of 2.5BV of 60%, drying the obtained ginkgetin eluent to obtain flavonoid compound powder of 76.8mg, hydrolyzing with hydrochloric acid, passing through a 0.45-mu m microporous membrane, and calculating the content of the flavonoid compound by using a high performance liquid chromatograph according to the Chinese pharmacopoeia of the 2015 edition.