阅读说明：本技术 一种深共熔溶剂及采用其提取鹰嘴豆中异黄酮的方法 (Deep eutectic solvent and method for extracting isoflavone from chickpea by adopting same ) 是由 谭家能 尚宪超 张忠锋 窦玉青 刘新民 杜咏梅 李冉 于 2018-07-09 设计创作，主要内容包括：本发明提出了一种深共熔溶剂,包括氯化胆碱、1,2-丙二醇和水,且氯化胆碱和1,2-丙二醇的摩尔比为1:1,含水量为0-50％(V/V)。一种采用该深共熔溶剂提取鹰嘴豆中异黄酮的方法,包括如下步骤：(a)鹰嘴豆处理；(b)异黄酮提取；(c)异黄酮分析；(d)取精制提取液用大孔树脂吸附,然后纯水-乙腈洗脱,分别收集洗脱液,合并相同组分,浓缩结晶,将结晶产物45℃真空烘干后得到含有鹰嘴豆4种异黄酮单体的提取物。本发明提供一种深共熔溶剂及采用其提取鹰嘴豆中异黄酮的方法,采用该提取方法可以有效解决现有鹰嘴豆异黄酮提取技术使用有机溶剂存在的污染问题。(The invention provides a deep eutectic solvent which comprises choline chloride, 1, 2-propylene glycol and water, wherein the molar ratio of the choline chloride to the 1, 2-propylene glycol is 1:1, and the water content is 0-50% (V/V). A method for extracting isoflavone from chickpea by adopting the deep eutectic solvent comprises the following steps: (a) processing chickpeas; (b) extracting isoflavone; (c) analyzing isoflavone; (d) adsorbing the refined extractive solution with macroporous resin, eluting with pure water-acetonitrile, respectively collecting eluates, mixing the same components, concentrating for crystallization, and vacuum drying at 45 deg.C to obtain extract containing 4 isoflavone monomers of semen Ciceris Arietini. The invention provides a deep eutectic solvent and a method for extracting isoflavone in chickpea by using the same, and the extraction method can effectively solve the pollution problem caused by using an organic solvent in the existing chickpea isoflavone extraction technology.)

1. A deep eutectic solvent, characterized in that: comprises choline chloride, 1, 2-propylene glycol and water, wherein the mol ratio of the choline chloride to the 1, 2-propylene glycol is 1:1, and the water content is 0-50% (V/V).

2. The deep eutectic solvent as claimed in claim 1, wherein the molar ratio of choline chloride to 1, 2-propanediol in the deep eutectic solvent is 1:1, and the water content is 30% (V/V).

3. A method for extracting isoflavone from chickpeas using the deep eutectic solvent of claim 1 or 2, characterized in that it comprises the following steps:

(a) treating chickpeas: placing semen Ciceris Arietini in constant temperature and humidity incubator, and allowing germination for 3-7 days until the sprout length is 2-3 cm. Freeze drying germinated semen Ciceris Arietini at-80 deg.C for 48 hr, grinding, and sieving with 40 mesh sieve;

(b) and (3) isoflavone extraction: adding the deep eutectic solvent into a chickpea sample, wherein the dosage ratio of the chickpea sample to the deep eutectic solvent is 10-100mg/mL, the mixture of the chickpea sample and the deep eutectic solvent is subjected to ultrasonic extraction for 10-50min under the conditions that the ultrasonic power is 100W, the ultrasonic frequency is 25kHz and the temperature is 30-80 ℃, and then the mixture is centrifuged at 5000r/min for liquid-solid separation to obtain a crude extract of isoflavone;

(c) and (3) isoflavone analysis: filtering the crude extractive solution with 0.45 μm filter membrane, removing impurities to obtain refined extractive solution of isoflavone, and performing component analysis by ultra high performance liquid chromatography;

(d) adsorbing the refined extract with macroporous resin, eluting with pure water-acetonitrile, collecting eluates, mixing the same components, concentrating, crystallizing, and vacuum drying at 45 deg.C to obtain extract containing 4 isoflavone monomers of semen Ciceris Arietini.

4. The method for extracting isoflavone from chickpea by using the deep eutectic solvent as claimed in claim 3, characterized in that: in the step (b), the dosage ratio of the chickpea sample to the deep eutectic solvent is 40mg/mL, and the mixture of the chickpea sample and the deep eutectic solvent is subjected to ultrasonic extraction for 30min under the conditions that the ultrasonic power is 100W, the ultrasonic frequency is 25kHz and the temperature is 50 ℃.

5. The method for extracting isoflavone from chickpea by using the deep eutectic solvent according to claim 3, characterized in that: adsorbing the refined extracting solution in the step (d) by using DM130 macroporous resin, eluting by using pure water until the refined extracting solution does not contain a deep eutectic solvent, and then taking 90% acetonitrile as an eluent and 1 BV.h^-1And (3) elution at a high rate.

Technical Field

The invention relates to a biochemical engineering technology, in particular to a deep eutectic solvent and a method for extracting isoflavone in chickpea by adopting the deep eutectic solvent.

Background

The isoflavone compound is a bioactive substance widely existing in leguminous plants such as chickpea, has wide physiological activity, and has various beneficial effects on human and animal health. Research shows that isoflavone has the functions of resisting oxidation and hemolysis and preventing and treating cardiovascular diseases by reducing blood fat and plasma cholesterol. Cicer arietinum L, a family of vetch of Leguminosae, is the second largest consumption bean in the world, and has various pharmacological activities of reducing blood sugar, reducing blood fat, resisting tumor, resisting oxidation, protecting heart and blood vessels, and the like, and the efficacies are related to isoflavone which is an active ingredient of the chickpea.

The extraction method of isoflavone compounds in chickpea mainly comprises a heating extraction method, a microwave extraction method, an ultrasonic extraction method, a supercritical CO2 extraction method and the like. Most of the methods use methanol and the like as solvents, have great pollution to the environment and limit large-scale industrial production. Although the supercritical CO2 extraction is more environment-friendly and efficient, the supercritical CO2 extraction has high requirements on industrial equipment and high cost and is difficult to be commercialized in a large scale.

In 2003, Abbott et al first discovered a Deep Eutectic Solvent (DES), a good physicochemical solvent, formed from a quaternary ammonium salt and an amide compound. The solvent is non-toxic and biodegradable, and the atom utilization rate in the synthesis process reaches 100 percent, so the solvent is a novel green solvent. With the continuous and intensive research on the DES properties, the DES shows strong superiority in the separation and extraction process, particularly in the extraction process of natural products such as anthocyanin, chlorogenic acid, alkaloid and the like, but the DESS has not been used for extracting the isoflavone of the chickpea.

Disclosure of Invention

Based on the above problems, the present invention aims to provide a deep eutectic solvent and a method for extracting isoflavone from chickpea by using the same, wherein the method can effectively solve the problem of pollution caused by using organic solvents in the existing chickpea isoflavone extraction technology.

Aiming at the problems, the following technical scheme is provided: a deep eutectic solvent, characterized in that: comprises choline chloride, 1, 2-propylene glycol and water, wherein the mol ratio of the choline chloride to the 1, 2-propylene glycol is 1:1, and the water content is 0-50% (V/V).

The invention is further provided that the molar ratio of choline chloride and 1, 2-propanediol in the deep eutectic solvent is 1:1, and the water content is 30% (V/V).

The method for extracting isoflavone from chickpea by adopting the deep eutectic solvent is characterized by comprising the following steps:

(a) treating chickpeas: placing semen Ciceris Arietini in constant temperature and humidity incubator, and allowing germination for 3-7 days until the sprout length is 2-3 cm. Freeze drying germinated semen Ciceris Arietini at-80 deg.C for 48 hr, grinding, and sieving with 40 mesh sieve;

(b) and (3) isoflavone extraction: adding the deep eutectic solvent into a chickpea sample, wherein the dosage ratio of the chickpea sample to the deep eutectic solvent is 10-100mg/mL, the mixture of the chickpea sample and the deep eutectic solvent is subjected to ultrasonic extraction for 10-50min under the conditions that the ultrasonic power is 100W, the ultrasonic frequency is 25kHz and the temperature is 30-80 ℃, and then the mixture is centrifuged at 5000r/min for liquid-solid separation to obtain a crude extract of isoflavone;

(c) and (3) isoflavone analysis: filtering the crude extractive solution with 0.45 μm filter membrane, removing impurities to obtain refined extractive solution of isoflavone, and performing component analysis by ultra high performance liquid chromatography;

(d) adsorbing the refined extract with macroporous resin, eluting with pure water-acetonitrile, collecting eluates, mixing the same components, concentrating, crystallizing, and vacuum drying at 45 deg.C to obtain extract containing 4 isoflavone monomers of semen Ciceris Arietini.

Further setting that the dosage ratio of the chickpea sample to the deep eutectic solvent in the step (b) is 40mg/mL, and the ultrasonic extraction of the mixture of the chickpea sample and the deep eutectic solvent is carried out for 30min under the conditions that the ultrasonic power is 100W, the ultrasonic frequency is 25kHz and the temperature is 50 ℃.

Further setting that the refined extract in the step (d) is absorbed by DM130 macroporous resin, pure water is eluted until the extract does not contain deep eutectic solvent, and then 90 percent acetonitrile is used as eluent, and 1 BV.h^-1And (3) elution at a high rate.

The invention has the beneficial effects that: the scheme provides a deep eutectic solvent which is non-toxic and biodegradable, has an atom utilization rate of 100% in the synthesis process, and is a novel green solvent; meanwhile, the extraction method has the characteristics of non-volatilization of an extracting agent, no pollution, high efficiency, simple and convenient operation, industrial production and the like, and can be applied to efficient green extraction of isoflavone compounds and comprehensive utilization of chickpea resources.

Drawings

FIG. 1 is a high performance liquid chromatogram of 4 isoflavones in a chickpea extract in an embodiment of the invention;

FIG. 2 is a schematic diagram illustrating the effect of a deep eutectic solvent system on the extraction rate of isoflavone from chickpeas in an embodiment of the present invention;

FIG. 3 is a schematic diagram illustrating the effect of water content in the deep eutectic solvent on the extraction rate of isoflavone from chickpeas in an embodiment of the present disclosure;

FIG. 4 is a schematic diagram illustrating the effect of extraction temperature on the extraction rate of isoflavone from chickpeas in the example of the present invention;

FIG. 5 is a schematic diagram illustrating the effect of extraction time on the extraction rate of isoflavone from chickpeas in the example of the present invention;

FIG. 6 is a schematic diagram showing the influence of solid-liquid ratio on the extraction rate of isoflavone from chickpea in the example of the present invention.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

1. Materials and reagents

Chickpeas were purchased from Xinjiang province;

23 different deep eutectic solvent system components: choline chloride, glucose, fructose, sucrose, xylitol, urea, citric acid, malic acid, lactic acid, malonic acid, ethylene glycol, 1, 4-butanediol, 1, 2-propanediol, glycerol, betaine, proline (analytical purity, alatin reagent, china);

chickpea isoflavones (analytically pure, Shanghai-derived leaf Biotech limited, China);

methanol, acetonitrile (chromatographically pure, MERCK, germany).

2. Laboratory apparatus

Waters UPLC H-CLASS ultra high performance liquid chromatograph (Waters corporation, USA); BSA124S-CW electronic balance (sensitivity 0.0001g, Sidorist scientific instruments Beijing, Inc., China); IKA magnetic stirrer (IKA corporation, germany); KQ-500GVDV type double-spliced constant temperature numerical control ultrasonic generator (Kunshan ultrasonic instruments Co., Ltd., China).

3. Experimental methods

3.1 sample treatment and analysis

Germinating 500g chickpeas under appropriate temperature and humidity conditions, freeze drying at-80 deg.C to obtain 40 mesh powder, and storing in 4 deg.C refrigerator as raw material for extraction;

accurately weighing 100mg of sample powder, placing the sample powder in an extractor, adding 2mL of deep eutectic solvent, fully and uniformly mixing, and placing the mixture in a constant-temperature ultrasonic extractor, wherein the ultrasonic power is 100W, and the ultrasonic frequency is 25kHz for extraction;

after the extraction was completed, the volume was adjusted to 10mL with methanol, and the mixture was filtered through a 0.45 μm filter, and 1mL of the filtrate was subjected to UPLC/UV analysis.

3.2 high Performance liquid chromatography conditions

The mobile phase A is acetonitrile, the mobile phase B is pure water, and the gradient elution procedure is that the time is 0-8min, 50% of A and 50% of B; column Acquity UPLC BEH C18column (50mm × 2.1mm,1.7 μm); the flow rate is 0.3 mL/min; the sample introduction rate is 5 mu L; the temperature of the chromatographic column is 40 ℃; the detection wavelength is 260 nm.

3.3 Standard solution preparation

Weighing formononetin (ON), biochanin (SI), Formononetin (FO) and biochanin glycoside (BI) with certain mass, dissolving and diluting with absolute ethyl alcohol to prepare chickpea isoflavone mixed standard solution, and refrigerating and storing the chickpea isoflavone standard working solution at 4 ℃.

4. Optimization of experimental conditions

4.1 deep eutectic solvent System optimization

The extraction rates of the four isoflavones by the 23 different deep eutectic solvent systems are compared, and the best deep eutectic solvent system is screened out, as shown in table 1.

TABLE 1 composition of deep eutectic solvents

4.2 deep eutectic solvent System Water content optimization

The method comprises the steps of firstly screening the water content of a deep eutectic solvent system, taking the total extraction rate of four isoflavone compounds as an index, and screening the optimum water content of the deep eutectic solvent by adopting a principal component analysis method.

4.3 optimization of the extraction Process conditions for the deep eutectic solvent

And carrying out single-factor experimental design of the deep eutectic solvent extraction process, wherein the single-factor experimental design comprises extraction time, extraction temperature and solid-to-liquid ratio, and obtaining the optimal extraction process conditions for extracting the chick pea isoflavone by using the deep eutectic solvent.

4.4 separation and purification

The chickpea crude extract is obtained by an optimal extraction process, and the optimal separation macroporous resin and the composition of eluent are obtained by screening the type, the sample loading amount, the eluent and the like of the macroporous resin.

5. Results of the experiment

5.1 Standard Curve for four isoflavones

TABLE 1 Standard curves for the four isoflavones (see FIG. 1)

5.2 optimization of deep eutectic solvent System

The results are shown in fig. 2, and the best deep eutectic solvent system is selected to be choline chloride and 1, 2-propylene glycol with a molar ratio of 1: 1.

5.3 optimization of Water content in deep eutectic solvent

Under the extraction conditions of extraction temperature of 40 deg.C, extraction time of 20min and solid-to-liquid ratio of 50mg/mL, as shown in FIG. 3, when DES water content is increased within the range of 0% -50%, the extraction rate of chickpea isoflavone tends to increase first and then decrease, and when 30%, the extraction rate reaches the highest value. Therefore, the optimum deep eutectic solvent water content was selected to be 30%.

5.4 optimization of extraction temperature

Under the extraction conditions of DES water content of 25%, extraction time of 20min, and solid-to-liquid ratio of 50mg/mL, as shown in FIG. 4, when the extraction temperature is increased within the range of 30 deg.C-50 deg.C, the extraction rate of chickpea isoflavone increases, and reaches the maximum value at 50 deg.C. While the extraction rate slightly increased with the subsequent increase in temperature, the difference was not significant (p > 0.05). Therefore, the optimum extraction temperature was selected to be 50 ℃.

5.5 optimization of extraction time

Under the extraction conditions of DES water content of 25%, extraction temperature of 40 deg.C, and solid-to-liquid ratio of 50mg/mL, as shown in FIG. 5, when the extraction time is increased within 10-30min, the extraction rate of semen Ciceris Arietini isoflavone increases, and reaches maximum value at 30 min. The extraction rate slightly increased with time after that, but the difference was not significant (p > 0.05). Therefore, the extraction time 30min was selected as the optimum extraction time.

5.6 optimization of solid-to-liquid ratio

Under the extraction conditions of DES water content of 25%, extraction temperature of 40 deg.C, and extraction time of 20min, as shown in FIG. 6, when the solid-to-liquid ratio is increased in the range of 40-100mg/mL, the extraction rate of semen Ciceris Arietini isoflavone is decreased, and when the solid-to-liquid ratio is in the range of 10-40mg/mL, the extraction rate increase effect is not significant (p > 0.05). And finally selecting the solid-liquid ratio to be 40mg/mL from the aspects of saving the solvent, controlling the extraction cost and the like.

Through a single factor test, the optimal extraction conditions are determined as follows: the water content in the deep eutectic solvent is 30%, the dosage ratio of the chickpea sample to the deep eutectic solvent is 40mg/mL, the temperature is 50 ℃, and the time is 30 min.

5.7 separation and purification of four isoflavones

The result shows that the DM130 macroporous resin has the best separation and purification effect on formononetin, biochanin, formononetin and biochanin glycoside, and has higher recovery rates of 88.08%, 89.51%, 94.96%, 91.83% and 88.35% respectively. The purity of the 4 purified isoflavones was greater than 92.5% by HPLC.

In conclusion, the extraction solvent adopted by the invention is a deep eutectic solvent, replaces toxic organic solvents such as methanol and the like extracted traditionally, reduces the environmental pollution in the extraction process, is green and environment-friendly, greatly reduces the extraction cost, and is suitable for industrial mass production.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and those modifications and variations assumed in the above are also considered to be within the protective scope of the present invention.