阅读说明：本技术 一种甘草地上部分总黄酮的提取及其脂质体的制备方法 (Method for extracting total flavonoids from aerial parts of liquorice and preparing liposome of total flavonoids ) 是由 宋吉明 董庄庄 朱泽华 方晶 于 2019-09-11 设计创作，主要内容包括：本发明公开了一种甘草地上部分总黄酮的提取及其脂质体的制备方法,属于中医药技术领域。其包括以下步骤：将甘草地上部分茎叶烘干、粉粹,过筛,按一定料液比加入乙醇溶液进行加热回流提取,取滤液进行减压浓缩至提取液的1/10,在浓缩液中加入乙酸乙酯溶液,取上清液过滤、减压浓缩、烘干,得甘草地上部分粗黄酮。本发明的有效结果是：甘草地上部分利用度高,操作简单,成本低,黄酮提取率高,可达85.5%。本发明同时着眼于甘草黄酮新型纳米给药系统的构建,采用薄层分散法制备黄酮脂质体。经本发明制备得到的黄酮脂质体颜色为乳黄色,呈圆形或类圆形,平均粒径为157.8 nm,分布均匀,脂质体的包封率为72.33%,该方法简单、原料易得、稳定性好,具有良好的生物利用度。(The invention discloses an extraction method of total flavonoids in aerial parts of liquorice and a preparation method of liposome of the total flavonoids, and belongs to the technical field of traditional Chinese medicines. Which comprises the following steps: drying, crushing and sieving the overground part stems and leaves of the liquorice, adding an ethanol solution according to a certain material-liquid ratio, heating and refluxing for extraction, taking the filtrate, concentrating under reduced pressure to 1/10 of the extracting solution, adding an ethyl acetate solution into the concentrated solution, taking the supernatant, filtering, concentrating under reduced pressure, and drying to obtain the overground part crude flavone of the liquorice. The effective results of the invention are: the utilization rate of the aerial part of the liquorice is high, the operation is simple, the cost is low, and the extraction rate of the flavone is high and can reach 85.5 percent. The invention simultaneously focuses on the construction of a novel liquorice flavone nano drug delivery system, and the thin-layer dispersion method is adopted to prepare the flavone liposome. The flavone liposome prepared by the method is creamy yellow, is round or quasi-round, has the average grain diameter of 157.8 nm, is uniformly distributed, has the entrapment rate of 72.33 percent, and has the advantages of simple method, easily obtained raw materials, good stability and good bioavailability.)

1. A method for extracting the total flavone of the overground part of liquorice and preparing liposome thereof is characterized by comprising the following steps:

pulverizing Glycyrrhrizae radix stem with pulverizer, sieving with 60 mesh sieve, adding ethanol, extracting with condensing reflux device at 65-85 deg.C for 1-3 hr with a material-liquid ratio of 1:10-1: 20;

vacuum filtering the above extractive solution, concentrating, removing alcohol, and extracting with ethyl acetate;

taking the upper layer chromatography liquid in the step (2), concentrating to remove ethyl acetate to 1/10 of the original volume, and drying;

dissolving the concentrate obtained in the step (3) with ethanol, filtering, passing the filtrate through macroporous resin, leaving the flavonoid compounds on the macroporous resin, and allowing other compounds to flow out along with tail liquid; eluting macroporous resin with ethanol solution, and concentrating the eluate to dryness to obtain Glycyrrhrizae radix total flavone;

accurately weighing 100-600 mg of lecithin and 50-100 mg of cholesterol in the same pear-shaped bottle, adding 5-30 ml of absolute ethyl alcohol or 95% ethyl alcohol, and carrying out ultrasonic treatment until the lecithin and the cholesterol are completely dissolved; dissolving the licoflavone obtained in step (4), transferring 1-5 ml into a pear-shaped bottle, and shaking uniformly until the solution is yellow transparent mixed solution;

removing the reaction solvent in the mixed solution under reduced pressure on a rotary evaporator, preferably at 35-75 deg.C, to form a uniform film on the container wall;

adding 10-50 ml mixed phosphate buffer solution into pear-shaped bottle, rotary steaming at 35-75 deg.C water bath for 10-30 min to dissolve membrane, standing for 2-6 hr for sufficient hydration, and filtering the suspension with microporous membrane to obtain Glycyrrhrizae radix total flavone liposome emulsion by thin film dispersion method.

2. The method for extracting the total flavonoids from the aerial parts of the liquorice and the preparation method of the liposome thereof according to claim 1 are characterized in that the concentration of ethanol extracted by refluxing is 65 to 85 percent.

3. A method for extracting the total flavone from the aerial parts of liquorice root and preparing its liposome as claimed in claim 1, is characterized by concentrating to 1/10 of its original volume and adding ethyl acetate three times the volume of the concentrated solution.

4. The method for extracting the total flavone from the overground part of the liquorice and preparing the liposome thereof according to claim 1 is characterized in that the total flavone is separated from other components by adopting macroporous resin, and the eluent is 60-95% ethanol.

5. The method for preparing licorice total flavone liposome according to claim 1, wherein the pH value of the PBS solution is 6-8.

6. A method for extracting total flavonoids from aerial parts of Glycyrrhrizae radix and preparing liposome thereof according to any one of claims 1-5.

Technical Field

The invention relates to a method for extracting active ingredients from overground parts of liquorice, in particular to a method for extracting total flavonoids from the overground parts of the liquorice and a method for preparing liposome thereof.

Background

The aerial part of licorice is rich in flavonoid compounds, and licorice flavone has obvious antioxidation, can prevent tumor, has female hormone-like effect, and has obvious pharmacological action on gastric ulcer, liver damage, spasmolysis, pathogenic microorganism, enzyme, inflammation resistance, allergic reaction resistance and the like. However, in the traditional application method of liquorice, only liquorice root is taken, but overground parts are not reasonably utilized, and the development and clinical application of related preparations are limited due to the properties of low solubility, low stability, poor absorption, low bioavailability and the like of many flavonoid active ingredients. In the literature (Kangxuefeng, etc., the research on the antioxidant activity of the overground part fat-soluble general flavone of liquorice, 2016, 9 (05): 567-. Therefore, the development and research of the aerial parts of the liquorice are necessary.

At present, few literature reports are reported on the development process of the overground part of liquorice, and the literature (Korean male, etc., research on the dynamic accumulation rule of total flavonoids and total polysaccharides in different organs of wild and cultivated liquorice, the university of Beijing traditional Chinese medicine, 2014, 5: 344 plus 348) respectively researches the total flavonoids and the total polysaccharides in roots, stems and leaves of the liquorice at different periods, and the result shows that the content of the total flavonoids in the liquorice stems is 1.6-1.8% in the whole growth period. The literature (Zhang et al, HPLC-MS analysis of flavonoids in aerial parts of licorice, university of Liaoning traditional Chinese medicine, 2018, 20 (05): 48-51) researches the analysis of flavonoids in stems and leaves of aerial parts of licorice, provides a more reliable basis for the analysis of chemical components of aerial parts of licorice, but does not separate and purify the active ingredients. Patent CN102836202A (bolatide macarabi, a method for comprehensively developing and utilizing aerial parts of licorice) discloses the development and utilization of aerial effective components of licorice, but the extraction rate of flavone products obtained by separating various biological activities is low, and compared with the method of the present invention, the method of the present invention has the advantages of simple preparation method and high extraction rate.

In order to fully utilize the liquorice resources and avoid excessive waste of the liquorice overground part resources, the invention determines the optimal extraction process condition by researching various factors influencing the reflux extraction effect of the liquorice overground part total flavonoids, extracts, separates and purifies the liquorice overground part total flavonoids, prepares the flavonoid liposome to obtain the flavonoid extraction rate under the optimal extraction condition, and simultaneously achieves the purposes of increasing the solubility of the liquorice flavonoids and improving the bioavailability of the liquorice flavonoids.

Disclosure of Invention

Aiming at the prior art, the invention aims to solve the technical problems of the waste of the overground part of the liquorice, the low bioavailability of the liquorice total flavonoids and the incapability of well absorbing the active ingredients by the organism. Provides a method for extracting and separating the total flavonoids of the overground part of liquorice and a method for preparing the flavonoid liposome, and prepares the liquorice total flavonoid liposome capable of improving water solubility and improving bioavailability on the basis of extracting, separating and purifying the liquorice total flavonoids.

In order to achieve the above purpose, the technical scheme adopted by the invention is to provide a liquorice total flavone liposome and a preparation method thereof, wherein the liquorice total flavone liposome comprises the following steps:

an extraction of the total flavone component of the aerial parts of liquorice and the preparation of liposome, which comprises the following steps:

1. a method for extracting total flavonoids from aerial parts of liquorice comprises the following steps:

(1) preparing a coarse extract of the overground part of liquorice:

cutting the aerial parts of the liquorice into small sections, placing the small sections in an oven at 50-70 ℃ for drying overnight, crushing the small sections by using a crusher, sieving the small sections by using a 60-mesh sieve, precisely weighing 100 g of aerial part powder of the liquorice, adding 10-20 times of 80% ethanol solution, refluxing the mixture for 1-3 h at 65-85 ℃, performing vacuum filtration to separate filtrate and filter residue, repeatedly extracting the filter residue for 1-3 times, and combining supernate to obtain crude extract of the aerial parts of the liquorice.

(2) Separation of flavone component from aerial part extract of licorice

Concentrating the crude extract of aerial part of Glycyrrhrizae radix obtained in step (1) under reduced pressure to 1/10-1/5 of the original volume, adding 1-3 times of ethyl acetate solution for extraction, collecting upper layer chromatography liquid, repeating for 1-3 times, mixing the upper layer chromatography liquid, concentrating under reduced pressure to remove ethyl acetate, and oven drying to constant weight.

(3) Purification of flavone component from aerial part extract of licorice

Soaking the macroporous resin in ethanol for 12-24 h for pretreatment to fully swell the macroporous resin, dissolving the crude extract in the step (2) with ethanol, filtering the solution through a 0.45-micron filter membrane, passing the filtrate through AB-8 macroporous resin firstly, adsorbing saturated licorice flavonoids compounds, remaining the licorice flavonoids compounds on the resin, remaining the other compounds in the tail solution, eluting with ethanol solution, finally obtaining eluent, and concentrating to dryness to obtain the licorice overground part total flavonoids.

2. The method for preparing the liquorice overground part total flavone liposome by the thin film dispersion method is implemented specifically as follows:

(1) accurately weighing 600 mg of lecithin and 50-100 mg of cholesterol in the same pear-shaped bottle, adding 5-30 ml of absolute ethyl alcohol or 95% ethyl alcohol, carrying out ultrasonic treatment until the solutions are completely dissolved, then adding 1-10 ml of 1 mg/ml of liquorice overground part total flavone solution, and shaking uniformly until the solution is yellow and transparent mixed solution.

(2) The reaction solvent in the mixture is removed under reduced pressure on a rotary evaporator, preferably at a temperature of 35-75 deg.C, to form a uniform thin film on the container wall.

(3) Taking 10-50 ml of mixed Phosphate (PBS) buffer solution with pH of 6-8 to put in a pear-shaped bottle, carrying out rotary evaporation for 10-30 min under the condition of 35-75 ℃ water bath to dissolve the membrane, standing for 2-6 h for full hydration, and filtering the suspension with a microporous filter membrane to obtain the licorice total flavone liposome emulsion prepared by the thin film dispersion method.

The invention obtains the general flavone of the overground part of the liquorice and the flavone liposome thereof by the preparation method, and the optimal extraction process obtained by orthogonal experiments comprises the following steps: the extraction rate of the total flavonoids in the overground part of the liquorice is 1.54 percent and reaches 85.6 percent of the total flavonoids in the liquorice stems, and the process has the advantages of high extraction rate, simple process and no toxicity, wherein the mass concentration of the ethanol is 80 percent, the reflux time is 2 hours, the extraction temperature is 85 ℃, and the material-to-liquid ratio is 1:20 g/ml. Meanwhile, the prepared flavone liposome is creamy yellow, is round or round-like in appearance, has the average particle size of 174.5 nm and is uniformly distributed, the entrapment rate of the liposome is 72.33 percent, and the prepared liposome has the advantages of good shape, stable dispersion system, uniform distribution and high entrapment rate. The preparation method is simple, the raw materials are easy to obtain, and the prepared licorice total flavone liposome has good bioavailability.

Drawings

FIG. 1 is a Scanning Electron Microscope (SEM) image of Licorice root total flavonoids liposome drug-loaded particles obtained in example 2.

FIG. 2 is a Transmission Electron Microscope (TEM) image of Licorice root total flavonoids liposome drug-loaded particles obtained in example 2.

FIG. 3 is a distribution diagram of the particle size of the Licorice root Total Flavonoids liposome drug-loaded particles obtained in example 2.

Detailed Description

The present invention will be described in detail with reference to specific examples.