阅读说明：本技术 一种葛根异黄酮提取方法及其应用 (Extraction method and application of pueraria isoflavone ) 是由 向维 于 2021-09-06 设计创作，主要内容包括：本发明提供一种葛根异黄酮提取方法及其应用：具体步骤如下：氯化胆碱/乙二醇的制备：将139.6g的氯化胆碱与111.5-223.0mL的乙二醇混合(即摩尔比控制在1：2-4),在80℃的水浴锅中磁力搅拌2-3h,直到变成透明溶液,然后在60℃真空干燥箱中干燥36h,即得深度共熔溶剂氯化胆碱/乙二醇,保藏在干燥器中,待用,本申请通过利用深度共熔溶剂氯化胆碱/乙二醇对植物细胞壁具有溶解破坏作用,以及与黄酮类化合物形成氢键促进其溶解等特性,同时辅助超声波破碎大大提高了葛根异黄酮的提取率并具备抗氧化性。(The invention provides a method for extracting pueraria isoflavone and application thereof: the method comprises the following specific steps: preparation of choline chloride/ethylene glycol: 139.6g of choline chloride and 111.5-223.0mL of ethylene glycol are mixed (namely the molar ratio is controlled to be 1: 2-4), the mixture is magnetically stirred in a water bath kettle at 80 ℃ for 2-3 hours until the mixture becomes transparent solution, then the transparent solution is dried in a vacuum drying oven at 60 ℃ for 36 hours, and the deep eutectic solvent choline chloride/ethylene glycol is obtained and stored in a dryer for later use.)

1. A method for extracting pueraria isoflavone and application thereof are characterized in that: the method comprises the following specific steps:

(1) preparation of choline chloride/ethylene glycol: mixing 139.6g of choline chloride with 111.5-223.0mL of ethylene glycol (i.e. the molar ratio is controlled at 1: 2-4), magnetically stirring for 2-3h in a water bath kettle at 80 ℃ until the choline chloride becomes a transparent solution, then drying for 36h in a vacuum drying oven at 60 ℃ to obtain a deep eutectic solvent of choline chloride/ethylene glycol, and storing in a dryer for later use;

(2) preparing 10-40% choline chloride/ethylene glycol aqueous solution with distilled water, adding into 60 mesh radix Puerariae powder, stirring, extracting under ultrasonic wave-assisted heating to obtain radix Puerariae isoflavone crude extract, and separating and purifying to obtain radix Puerariae isoflavone;

(3) the feed-liquid ratio of the kudzu root powder to the choline chloride/ethylene glycol is 1: 10-25;

(4) in the extraction process, the power of ultrasonic wave is 300-600W, the temperature is 60-80 ℃, and the extraction time is 30-90 min;

(5) after extraction, removing pueraria residue by a high-speed centrifugation mode to obtain a crude extract, wherein the content of pueraria isoflavone in the crude extract is 1.19-1.54mg/mL, and the extraction rate is 3.58-4.62%;

(6) the separation and purification uses AB-8 type macroporous resin as a filler, and the purification is realized through the separation and purification of a chromatographic column. The separation and purification process comprises the following steps: loading on column by wet method, loading the crude extract, eluting with ethanol solution, collecting eluate, concentrating the eluate by rotary evaporation, adjusting pH, cooling to separate out precipitate, filtering to obtain crude radix Puerariae isoflavone precipitate, dissolving the precipitate with lime water, adjusting pH, cooling to separate out precipitate, filtering, and freeze drying to obtain refined radix Puerariae isoflavone.

2. The method for extracting pueraria isoflavone and the application thereof according to claim 1, wherein the method comprises the following steps: the AB-8 resin was loaded into a chromatography column (15mm by 500mm) with a column volume of 50mL by wet packing.

3. The method for extracting pueraria isoflavone and the application thereof according to claim 1, wherein the method comprises the following steps: the loading volume of the crude extract is 15 mL.

4. The method for extracting pueraria isoflavone and the application thereof according to claim 1, wherein the method comprises the following steps: the eluate was collected at 70% ethanol concentration.

5. The method for extracting pueraria isoflavone and the application thereof according to claim 1, wherein the method comprises the following steps: weighing the pueraria isoflavone powder, wherein the extraction rate is 21.48-27.23mg/g (calculated according to the pueraria raw material).

6. The method for extracting pueraria isoflavone and the application thereof according to claim 1, wherein the method comprises the following steps: and (3) determining the hydroxyl free radical clearance rate, DPPH clearance rate and ABTS clearance rate of the pueraria isoflavone.

Technical Field

The invention relates to the technical field, in particular to a method for extracting pueraria isoflavone and application thereof, belonging to the field of food processing.

Background

The root of kudzu vine is root tuber of Pueraria plant of Leguminosae, and is originally recorded in Shen nong's herbal Jing of Han Dynasty of China; the kudzu root is rich in starch and flavonoid substances (namely daidzin, pueraria flavonol and puerarin), and has high edible value and medical health care value; the department of health of China in 1988 publishes that the pueraria lobata is a plant which is both edible and medicinal, the growing area of the pueraria lobata is fast in recent years, but the processing and utilization of the pueraria lobata are still in the primary stage, the processing technology of the pueraria lobata in the prior art emphasizes on the extraction of the pueraria lobata powder or emphasizes on the extraction of the pueraria lobata total isoflavone, the technology capable of simultaneously extracting the pueraria lobata powder and the pueraria lobata total isoflavone is lacked, and the extraction rate of the pueraria lobata isoflavone is low.

Summary of the invention

In the method, the deep eutectic solvent choline chloride/ethylene glycol is utilized to have the characteristics of dissolving and destroying the plant cell wall, forming a hydrogen bond with the flavonoid compound to promote the dissolution of the flavonoid compound and the like, and the auxiliary ultrasonic crushing greatly improves the extraction rate of the pueraria isoflavone and has oxidation resistance.

The invention can achieve the purpose by the following scheme, including the following steps, but not limited to:

(1) preparation of choline chloride/ethylene glycol: mixing 139.6g of choline chloride with 111.5-223.0mL of ethylene glycol (i.e. the molar ratio is controlled at 1: 2-4), magnetically stirring for 2-3h in a water bath kettle at 80 ℃ until the choline chloride becomes a transparent solution, then drying for 36h in a vacuum drying oven at 60 ℃ to obtain a deep eutectic solvent of choline chloride/ethylene glycol, and storing in a dryer for later use;

(2) preparing 10-40% choline chloride/ethylene glycol aqueous solution with distilled water, adding into 60 mesh radix Puerariae powder, stirring, extracting under ultrasonic wave-assisted heating to obtain radix Puerariae isoflavone crude extract, and separating and purifying to obtain radix Puerariae isoflavone;

(3) the feed-liquid ratio of the kudzu root powder to the choline chloride/ethylene glycol is 1: 10-25;

(4) in the extraction process, the power of ultrasonic wave is 300-600W, the temperature is 60-80 ℃, and the extraction time is 30-90 min;

(5) after extraction, removing pueraria residue by a high-speed centrifugation mode to obtain a crude extract, wherein the content of pueraria isoflavone in the crude extract is 1.19-1.54mg/mL, and the extraction rate is 3.58-4.62%;

(6) the separation and purification uses AB-8 type macroporous resin as a filler, and the purification is realized through the separation and purification of a chromatographic column. The separation and purification process comprises the following steps: loading the crude extract into a column by a wet method, eluting the crude extract by an ethanol solution, collecting the eluent, concentrating the eluent by rotary evaporation, adjusting the pH, cooling to separate out a precipitate, filtering to obtain a crude pueraria isoflavone precipitate, dissolving the precipitate by lime water, adjusting the pH, cooling to separate out the precipitate, filtering, and freeze-drying to obtain refined pueraria isoflavone;

(7) loading AB-8 resin into a chromatography column (15mm x 500mm) by a wet method, wherein the volume of the column is 50 mL;

(8) the sample volume of the crude extract is 15 mL;

(9) collecting the eluent with 70% ethanol concentration;

(10) adjusting the pH of the concentrated eluate and the redissolved solution to pH3.0 with saline;

(11) weighing the pueraria isoflavone powder with the extraction rate of 21.48-27.23mg/g (calculated according to the pueraria raw material);

(12) and (3) determining the hydroxyl free radical clearance rate, DPPH clearance rate and ABTS clearance rate of the pueraria isoflavone.

Advantageous effects

According to the method, the choline chloride/ethylene glycol serving as the deep eutectic solvent has the characteristics of dissolving and destroying the plant cell wall, forming a hydrogen bond with a flavonoid compound to promote the dissolution of the flavonoid compound and the like, and meanwhile, the extraction rate of the pueraria isoflavone is greatly improved by assisting ultrasonic crushing, and the pueraria isoflavone has oxidation resistance.

Detailed Description

The first embodiment is as follows:

(1) and (3) preparing a deep eutectic solution. Drying choline chloride with the purity of 99% in a vacuum drying oven at the temperature of 60 ℃ for 24 hours, and then carrying out vacuum drying according to the weight ratio of 1:2, 1:3,1: 4, filling the mixture into a 50mL round bottom flask, plugging the flask with a plug, and stirring and heating the mixture for about 2 to 3 hours at the rotating speed of 300rpm in an oil bath kettle at the temperature of 100 ℃ until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. Adding appropriate amount of distilled water to adjust to 20% before use.

(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.

(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 20mL of DES (20% strength) is added and stirred uniformly. Then extracting for 1h at 60 ℃ under the assistance of 500W ultrasound. The mixture was centrifuged at 12000g for 10min using a tabletop high speed centrifuge to separate the solid particles and obtain a crude extract.

(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.

Molar ratio of	1：2	1：3	1：4
				Content of radix Puerariae isoflavone (mg/mL) in the crude extractive solution	1.27	1.35	1.19
Extraction ratio (%) of isoflavone in the crude extract	3.82	4.06	3.58
				Extraction rate (mg/g) of refined rutin	22.95	24.27	21.48

Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed.

Example two:

(1) and (3) preparing a deep eutectic solution. Drying choline chloride with the purity of 99% in a vacuum drying oven at 60 ℃ for 24 hours, mixing the choline chloride with ethylene glycol according to the molar ratio of 1:3, filling the mixture into a 50mL round-bottom flask, plugging the flask, stirring and heating the mixture in an oil bath kettle at 100 ℃ and the rotating speed of 300rpm for about 2 to 3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. Before use, appropriate amount of distilled water is added to adjust the concentration to 10,20, 30, 40%.

(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.

(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 20mL of DES (20% strength) is added and stirred uniformly. Then extracting for 1h at 60 ℃ under the assistance of 500W ultrasound. The mixture was centrifuged at 12000g for 10min using a tabletop high speed centrifuge to separate the solid particles and obtain a crude extract.

(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.

DES concentration%)	10	20	30	40
					Content of radix Puerariae isoflavone (mg/mL) in the crude extractive solution	1.21	1.35	1.31	1.21
Extraction ratio (%) of isoflavone in the crude extract	3.64	4.06	3.94	3.62
					Extraction rate (mg/g) of refined rutin	21.85	24.47	23.24	21.72

Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed.

Example three:

(1) and (3) preparing a deep eutectic solution. Drying choline chloride with the purity of 99% in a vacuum drying oven at 60 ℃ for 24 hours, mixing the choline chloride with ethylene glycol according to the molar ratio of 1:3, filling the mixture into a 50mL round-bottom flask, plugging the flask, stirring and heating the mixture in an oil bath kettle at 100 ℃ and the rotating speed of 300rpm for about 2 to 3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. Adding appropriate amount of distilled water to adjust to 20% before use.

(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.

(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 10,20 and 25mL of DES with the concentration of 20 percent is added and stirred evenly. Then extracting for 1h at 60 ℃ under the assistance of 500W ultrasound. The mixture was centrifuged at 12000g for 10min using a tabletop high speed centrifuge to separate the solid particles and obtain a crude extract.

(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.

Ratio of material to liquid	10	20	25
				Content of radix Puerariae isoflavone (mg/mL) in the crude extractive solution	1.22	1.35	1.44
Extraction ratio (%) of isoflavone in the crude extract	3.67	4.06	4.32
				Extraction rate (mg/g) of refined rutin	22.01	24.47	25.81

Example four:

(1) preparing a deep eutectic solution. Drying choline chloride with the purity of 99% in a vacuum drying oven at 60 ℃ for 24 hours, mixing the choline chloride with ethylene glycol according to the molar ratio of 1:3, filling the mixture into a 50mL round-bottom flask, plugging the flask, stirring and heating the mixture in an oil bath kettle at 100 ℃ and the rotating speed of 300rpm for about 2 to 3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. Adding appropriate amount of distilled water to adjust to 20% before use.

(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.

(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 20% DES was added to 25mL and stirred well. Then extracting for 1h at 60 ℃ under the assistance of 300, 500 and 600W ultrasound. The mixture was centrifuged at 12000g for 10min using a tabletop high speed centrifuge to separate the solid particles and obtain a crude extract.

(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.

Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed.

Example five:

(1) preparing a deep eutectic solution. Drying choline chloride with the purity of 99% in a vacuum drying oven at 60 ℃ for 24 hours, mixing the choline chloride with ethylene glycol according to the molar ratio of 1:3, filling the mixture into a 50mL round-bottom flask, plugging the flask, stirring and heating the mixture in an oil bath kettle at 100 ℃ and the rotating speed of 300rpm for about 2 to 3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. Adding appropriate amount of distilled water to adjust to 20% before use.

(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.

(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 20% DES was added to 25mL and stirred well. Then extracting for 1h at 60, 70 and 80 ℃ under the assistance of 300W ultrasound. The mixture was centrifuged at 12000g for 10min using a tabletop high speed centrifuge to separate the solid particles and obtain a crude extract.

(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.

Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed.

Example six:

(1) preparing a deep eutectic solution. Drying choline chloride with the purity of 99% in a vacuum drying oven at 60 ℃ for 24 hours, mixing the choline chloride with ethylene glycol according to the molar ratio of 1:3, filling the mixture into a 50mL round-bottom flask, plugging the flask, stirring and heating the mixture in an oil bath kettle at 100 ℃ and the rotating speed of 300rpm for about 2 to 3 hours until a uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 60 deg.C for 36h, transferring to a dryer, and sealing for storage. Adding appropriate amount of distilled water to adjust to 20% before use.

(2) And (4) pretreating the kudzuvine root. Drying the purchased radix Puerariae in the sun, drying for 12h, slicing, grinding with a traditional Chinese medicine grinder, sieving with a 60-mesh sieve, bagging the fine powder in a room temperature dryer, and storing.

(3) Extracting radix Puerariae isoflavone. Weighing fine powder according to a material-liquid ratio of 1 g: 20% DES was added to 25mL and stirred well. Then extracting at 70 deg.C for 30, 60, 90min under 300W ultrasound assistance. The mixture was centrifuged at 12000g for 10min using a tabletop high speed centrifuge to separate the solid particles and obtain a crude extract.

(4) Separating and purifying rutin. 15mL of the crude extract was slowly added to the column, followed by elution of other impurities with 600mL of deionized water, followed by 70% ethanol solution, and collection of the ethanol eluate. The eluate was then concentrated to 10mL using a rotary evaporator and the pH was adjusted to 3.0 with dilute hydrochloric acid. Placing the concentrated solution in 4 deg.C environment, standing for 24 hr to separate out radix Puerariae isoflavone crystal, filtering, and washing with cold water to obtain crude radix Puerariae isoflavone. Dissolving crude radix Puerariae isoflavone in lime water under heating, filtering with 0.22um membrane, adjusting pH of the filtrate to 3.0 with saline water, cooling, standing, recrystallizing to precipitate, filtering, washing with cold water, and lyophilizing to obtain refined radix Puerariae isoflavone.

Note that: the ratio of the content of the pueraria isoflavone in the crude extract to the extraction rate of the isoflavone in the crude extract is fixed.

Example six:

oxidation resistance