CN112300234A

CN112300234A - Method for extracting isoflavone, isoflavone extracted by method and application of isoflavone

Info

Publication number: CN112300234A
Application number: CN202011158670.8A
Authority: CN
Inventors: 刘青; 孔令姗; 黄芳; 周利丹; 卢伊娜; 田军
Original assignee: Shanghai Jiakai Biological Technology Co ltd
Current assignee: Shanghai Jiakai Biological Technology Co ltd
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2021-02-02

Abstract

The invention provides an extraction method of isoflavone, isoflavone prepared by the extraction method and application of the isoflavone. The extraction method of isoflavone comprises the following steps: (1) crushing plants, and then extracting in a mixed solvent consisting of an acidic eutectic solvent, alcohol and water to obtain a crude extraction liquid; (2) and (2) sequentially concentrating, filtering and washing the crude extract obtained in the step (1) to obtain the isoflavone. The extraction method can efficiently extract isoflavone from plants, namely the content of the total isoflavone is high, and the ratio of the isoflavone in an aglycone form to the ratio is ensured. The isoflavone provided by the invention has a remarkable oil control effect, and can be applied to products for preventing alopecia, controlling oil of skin and inhibiting acne.

Description

Method for extracting isoflavone, isoflavone extracted by method and application of isoflavone

Technical Field

The invention belongs to the field of extraction of medicinal chemical components from plants, and particularly relates to an extraction method of isoflavone, isoflavone obtained by extraction and application of the isoflavone.

Background

Isoflavones are a class of phytochemicals with simple diphenyl ring structures that are found primarily in leguminous plants. In plants, isoflavones occur predominantly in several forms: (1) the basic aglycone form; (2) glycosides that bind to a sugar component such as glucose through a beta-glycosidic bond (glycosidic form); (3) glycoside form + malonyl moiety; (4) glycoside form + acetyl moiety. Wherein, in plant cells, β -glucosidase cleaves the sugar moiety to produce an aglycone, an aglycone form of isoflavone, which is more biologically active but substantially insoluble in water. And researches show that the isoflavone aglycone is easier to be absorbed into a human body than isoflavone glycoside, the human body has higher absorption rate on the isoflavone aglycone, the isoflavone aglycone can keep stable concentration for a longer time, and the isoflavone aglycone shows more obvious antioxidant activity and oil control capability, which indicates that the isoflavone aglycone has higher bioavailability than the glycoside thereof.

At present, the existing extraction process of isoflavone utilizes alcohol solution to reflux and extract, and the extract is subjected to column chromatography or organic solvent extraction to obtain total flavone part; or hydrolyzing with strong alkali and strong acid to obtain flavonoid aglycone. The defects of the prior art are as follows: firstly, isoflavone aglycone cannot be extracted in a targeted manner, so that the obtained isoflavone extract has insufficient oil control effect; secondly, the solubility of the isoflavone compound in water and alcohol is poor, and the extraction rate is not high; thirdly, the hydrolysis process of strong base and strong acid is not beneficial to environmental protection and industrial production.

CN110183541A discloses a preparation method of red clover polysaccharide and total isoflavone, which comprises the following steps: (1) crushing the dried red clover raw material; (2) adding water into the crushed red clover raw material, stirring and extracting, and filtering to obtain a water extracting solution and extracting residues; (3) sequentially passing the obtained water extract through anion and cation exchange resin columns, concentrating under reduced pressure, adding active carbon, heating and stirring, filtering, drying, and pulverizing to obtain Trifolium pratense polysaccharide; (4) adding alkali liquor into the obtained extraction residue, stirring, extracting, and filtering to obtain alkali liquor extract; (5) adding aluminum oxide into the obtained alkaline water extract, stirring, nano-filtering, adjusting pH of the obtained permeate with acid solution, standing for crystallization, centrifuging, washing the obtained filter cake with water, vacuum drying, and pulverizing to obtain herba Trifolii Pratentis total isoflavone. The total isoflavone of red clover finally obtained by the extraction method still contains the form of glycoside, so the oil control effect is poor.

CN108642102A discloses a method for extracting soybean isoflavone aglycone produced by microorganisms by microwave extraction, which comprises inoculating Aspergillus niger strains in a CYM culture medium for fermentation culture; transferring the aspergillus niger strain subjected to fermentation culture and a culture medium into a triangular flask filled with sterile water and glass beads together to obtain a mixed solution mixed with thalli hyphae; then inoculating the soybean meal into a soybean meal culture medium for fermentation; filtering the fermentation liquid, taking the crude enzyme liquid, mixing the crude enzyme liquid with the ethanol solution to obtain a mixed solution, heating and extracting in microwave, and repeating the operation to obtain the soybean isoflavone aglycone. The invention adopts the processes of mould fermentation culture and microwave extraction to extract the soybean isoflavone aglycone, but the extraction rate is still to be improved, and the mould fermentation method has complex procedure and is difficult to apply in industrial production.

Therefore, the development of an extraction method capable of effectively increasing the yield of isoflavone in aglycone form is the key point of research in the field.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an isoflavone extraction method, isoflavone obtained by extraction and application. The extraction method can efficiently extract isoflavone from plants, namely the total isoflavone content is high, and the yield of isoflavone in an aglycone form is high.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for extracting isoflavones, the method comprising the steps of:

(1) crushing plants, and then extracting in a mixed solvent consisting of an acidic eutectic solvent, alcohol and water to obtain a crude extraction liquid;

(2) and (2) sequentially concentrating, filtering and washing the crude extract obtained in the step (1) to obtain the isoflavone.

In the invention, the acid eutectic solvent, alcohol and water are matched to extract the plants in the step (1), the extraction rate of isoflavone can be increased during reflux extraction, and isoflavone glycoside can be hydrolyzed into isoflavone in an aglycone form, so that the yield of isoflavone aglycone is effectively improved. In the invention, the concentration in the step (2) is carried out, and the isoflavone aglycone can be separated out from the water; filtering, wherein the crude product is residue, and water-soluble impurities such as saccharides, eutectic solvent, phenolic acid and the like are removed along with the filtrate; and finally, washing the solid obtained by filtering to remove alcohol-soluble and fat-soluble impurities such as coumarin, sterol, fatty acid and the like.

Preferably, the plant in step (1) is selected from any one or a combination of at least two of chickpea, soybean, formononetin, white lupin, sophora flower rice, kudzu, mulberry leaf, genistein or red clover, preferably red clover and/or chickpea.

Preferably, the particle size of the plant powder obtained by the pulverization in the step (1) is 10 to 100 meshes, and may be, for example, 10 meshes, 20 meshes, 30 meshes, 40 meshes, 50 meshes, 60 meshes, 70 meshes, 80 meshes, 90 meshes, 100 meshes, or the like.

Preferably, the mass ratio of the acidic eutectic solvent, the alcohol and the water in the step (1) is (0.5-2): (4-6): (3-5), preferably 1:5: 4;

wherein "0.5-2" can be 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, etc.;

wherein "4-6" can be 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, etc.;

wherein "3-5" can be 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, etc.

Preferably, the acidic eutectic solvent in step (1) consists of a quaternary ammonium salt and an organic acid.

Preferably, the organic acid is selected from any one of malic acid, citric acid, malonic acid or succinic acid or a combination of at least two of them.

Preferably, the quaternary ammonium salt is preferably choline chloride or betaine.

Preferably, the molar ratio of the quaternary ammonium salt to the organic acid is (0.5-2): (2-4), preferably 1: 3.

Preferably, the alcohol in step (1) is selected from any one of methanol, ethanol, n-propanol, isopropanol or tert-butanol or a combination of at least two thereof.

Preferably, the number of times of the extraction in step (1) is 1 to 3, and may be, for example, 1, 2, or 3.

Preferably, the temperature for each extraction is 70-90 deg.C, such as 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, etc., and the time for each extraction is 1-3h, such as 1h, 1.5h, 2h, 2.5h, 3h, etc.

Preferably, the concentration in step (2) is specifically: concentration is carried out until the alcohol content in the crude extract is 5 wt% or less, and may be, for example, 0 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, or the like.

Preferably, the concentration in step (2) is carried out at a temperature of 40 to 60 ℃, for example, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ or the like, and the degree of vacuum of the concentration is-0.05 MPa or less, for example, -0.05MPa, -0.06MPa, -0.07MPa, -0.08MPa, -0.09MPa, -0.1MPa, -0.2MPa or the like.

Preferably, the filtration in step (2) is performed by using filter paper and/or polypropylene membrane;

preferably, the pore size of the polypropylene membrane is 0.8-5 μm, and may be, for example, 0.8 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, or the like.

Preferably, the washing in step (2) is specifically: washing the solid obtained by filtering with water, ethanol and dichloromethane in sequence; the three solvents are adopted to wash the solid obtained by filtering in sequence, so that residual water-soluble impurities and alcohol-soluble and fat-soluble impurities such as coumarin, sterol, fatty acid and the like can be better removed.

Preferably, the mass ratio of the solid obtained by filtering, water, ethanol and dichloromethane is 1 (4-6) to (2-4).

Wherein "4-6" can be 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, etc.;

wherein two "2-4" each independently can be 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, etc.

Preferably, the mass concentration of ethanol is 40-60 wt%, and may be, for example, 40 wt%, 42 wt%, 44 wt%, 46 wt%, 48 wt%, 50 wt%, 52 wt%, 54 wt%, 56 wt%, 58 wt%, 60 wt%, etc.

Preferably, the method for extracting isoflavones comprises the following steps:

(1) crushing plants to 10-100 meshes, putting the crushed plants into an acidic eutectic solvent, alcohol and water in a mass ratio of (0.5-2): 4-6): 3-5, and extracting for 1-3 times at the temperature of 70-90 ℃ for each time for 1-3 hours to obtain a crude extract;

(2) concentrating the crude extraction liquid obtained in the step (1) at 40-60 ℃ under the vacuum degree of-0.05 MPa until the alcohol content in the crude extraction liquid is below 5 wt%, filtering, and washing the solid obtained by filtering with water, ethanol with the mass concentration of 40-60 wt% and dichloromethane in sequence to obtain the isoflavone.

In a second aspect, the present invention provides an isoflavone extracted by the extraction method of the first aspect.

The Molish reaction of the total isoflavone extracted by the method is negative, namely the Molish reaction can not detect sugar and carbohydrate functional groups, the isoflavone is in an aglycone form, and the oil control effect is obviously superior to that of the total isoflavone obtained by the conventional extraction method.

In a third aspect, the present invention provides the use of an isoflavone as described in the second aspect for inhibiting the secretion of lipids;

preferably, the isoflavone is used for preparing products for preventing alopecia, controlling oil of skin or inhibiting pox.

Compared with the prior art, the invention has the following beneficial effects:

(1) the extraction method can efficiently extract isoflavone from plants, namely the extraction rate of the total isoflavone is high, the extraction rate is 30-45% higher than that of the conventional extraction method, and the purity of the total isoflavone is higher.

(2) The Molish reaction of the total isoflavone extracted by the method is negative, namely the Molish reaction can not detect sugar and carbohydrate functional groups, the isoflavone is in an aglycone form, and the oil control effect is obviously superior to that of the total isoflavone obtained by the conventional extraction method.

(3) The extraction method disclosed by the invention is environment-friendly, simple in process and convenient for industrialization.

Drawings

FIG. 1 is a schematic view under a cell microscope of a model group seb-E6E7 to which a sample to be tested is not added;

FIG. 2 is a schematic representation under a cell microscope of seb-E6E7 after the addition of isoflavones obtained by extraction in example 1;

FIG. 3 is a schematic representation under a cell microscope of seb-E6E7 after the addition of the isoflavone obtained by extraction in comparative example 7.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

This example provides a method for extracting isoflavones, which comprises the following steps:

(1) crushing 100g of red clover into 50 meshes of particle size, placing the crushed red clover into a mixed solvent of 50g of acid eutectic solvent, 250g of ethanol and 200g of water, and extracting for 3 times, wherein the temperature of each extraction is 70 ℃, and the time of each extraction is 1h, so as to obtain 1.2kg of crude extract; wherein the acidic eutectic solvent is a mixture of choline chloride and malic acid with a molar ratio of 1: 3;

(2) concentrating 1.2kg of crude extract obtained in step (1) at 40 deg.C under vacuum degree of-0.08 MPa or below until the alcohol content in the crude extract is 0.5 wt%, filtering to obtain 5.1g of solid (wet weight), and washing the filtered solid with 20.4g of water, 10.2g of 50% ethanol and 10.2g of dichloromethane in sequence to obtain 1.2g of isoflavone extract (dry weight).

Example 2

(1) crushing 100g of red clover to 80 meshes, putting the crushed red clover into a mixed solvent of 100g of acidic eutectic solvent, 500g of ethanol and 400g of water, and extracting for 1 time at the extraction temperature of 90 ℃ for 3 hours to obtain 710g of crude extract; wherein the acidic eutectic solvent is a mixture of choline chloride and citric acid with a molar ratio of 1: 3;

(2) concentrating 710g of crude extract obtained in step (1) at 60 deg.C under vacuum degree of-0.09 MPa or less until the alcohol content in the crude extract is 5 wt%, filtering to obtain 4.5g of solid (wet weight), and washing the solid obtained by filtering with 27g of water, 18g of 50% ethanol and 18g of dichloromethane in sequence to obtain 0.9g of isoflavone extract (dry weight).

Example 3

(1) crushing 100g of red clover into 50 meshes, putting the crushed red clover into a mixed solvent of 80g of acidic eutectic solvent, 400g of ethanol and 320g of water, and extracting for 2 times, wherein the temperature of each extraction is 80 ℃, and the extraction time is 2 hours, so as to obtain 1.1kg of crude extraction liquid; wherein the acidic eutectic solvent is a mixture of betaine and malic acid with a molar ratio of 1: 3;

(2) concentrating 1.1kg of crude extract obtained in step (1) at 50 deg.C under vacuum degree of-0.08 MPa or below until the alcohol content in the crude extract is 2 wt%, filtering to obtain 4.8g of solid (wet weight), and washing the solid obtained by filtering with 21g of water, 14g of 50% ethanol and 14g of dichloromethane in sequence to obtain 1.1g of isoflavone extract (dry weight).

Example 4

This example provides a method for extracting isoflavones, which is different from example 1 only in that red clover is replaced with chickpea bean sprouts, and the other extraction steps are the same as example 1.

Comparative example 1

This comparative example provides a method for extracting isoflavones, which is different from example 1 only in that the mass of the acidic eutectic solvent is reduced to 25g, the ethanol content is increased to 275g, and the other extraction steps are the same as example 1.

Comparative example 2

This comparative example provides a process for the extraction of isoflavones, which differs from example 1 only in that the mass of the acidic eutectic solvent is increased to 100g, the ethanol content is reduced to 200g, and the other extraction steps are the same as in example 1.

Comparative example 3

This comparative example provides a method for extracting isoflavones, which differs from example 1 only in that the acidic eutectic solvent is a mixture of choline chloride and malic acid in a molar ratio of 1:0.5, and the other extraction steps are the same as in example 1.

Comparative example 4

This comparative example provides a process for the extraction of isoflavones, which differs from example 1 only in that the acidic eutectic solvent is a mixture of choline chloride and malic acid in a molar ratio of 1:5, and the other extraction steps are the same as in example 1.

Comparative example 5

This comparative example provides a conventional extraction method of isoflavones, which differs from example 1 only in that the extraction solvent is 50% ethanol, and does not contain an acidic eutectic solvent.

Comparative example 6

This comparative example provides a conventional extraction method of isoflavone, which is different from example 1 only in that 50g of the acidic eutectic solvent is replaced with 50g of 10 wt% hydrochloric acid and the other extraction steps are the same as example 1.

Comparative example 7

This comparative example provides a conventional extraction method of isoflavone, which is different from example 1 in that 50g of the acidic eutectic solvent is replaced with 50g of 10 wt% aqueous NaOH solution, pH is adjusted to 6.0 before concentration of the crude extract, and the other steps are the same as example 1.

Comparative example 8

This comparative example provides a process for the extraction of isoflavones, which differs from example 1 only in that an alkaline eutectic solvent is used for the extraction, said alkaline eutectic solvent being a mixture of choline chloride and urea in a molar ratio of 1:3, and the other extraction steps are the same as in example 1.

Test example 1

The absorbance of the isoflavone extracts obtained in examples 1-4 and comparative examples 1-8 at 263nm was measured by ultraviolet spectrophotometry using biochanin a as a control, and the content and yield of total isoflavones were calculated, wherein the yield of total isoflavones is (the mass of extracted total isoflavones/the mass of raw materials) × 100%.

The Molish reaction was used to identify whether the total isoflavone extracts obtained in examples 1-4 and comparative examples 1-8 contained carbohydrate groups: preparing the total isoflavone extract into a 10mg/mL solution by DMSO and ethanol (1:1), adding 2-3mL solution into a test tube, dropwise adding 2 drops of a 5 wt% alpha-naphthol ethanol solution, shaking uniformly, adding 2 drops of concentrated sulfuric acid along the wall of the test tube, and observing whether purple exists between the two liquid surfaces. The experimental results are expressed as follows:

the more obvious purple color was marked as positive and indicated by "+" indicating that the product contained more saccharide groups

The appearance of faint purple color was marked as weak positive and indicated by "+ -", indicating that the product contained a small amount of carbohydrate groups

The absence of purple color was marked as negative and is indicated by "-" indicating that no saccharide groups were detected by this method

The specific test results are shown in table 1:

TABLE 1

As can be seen from Table 1, the isoflavone extract obtained by the extraction method of the present invention was negative to Molish reaction, and had higher total isoflavone content and higher extraction rate than the conventional extraction method (comparative examples 5, 6 and 7) by 30-45%. The method adopts the cooperation of the acid eutectic solvent, alcohol and water to extract the plants, can increase the extraction rate of isoflavone during reflux extraction, and can hydrolyze the isoflavone glycoside into isoflavone in aglycone form, thereby effectively improving the yield of isoflavone aglycone. The plants are extracted by matching of alkaline eutectic solvent, alcohol and water (comparative example 8), the extraction rate of total isoflavone is close to that of the method, but the Molish reaction is positive, and the yield of aglycone is low.

Test example 2

Test samples: isoflavone extracts obtained by extraction of example 1 and comparative example 7;

subject: seb-E6E7 sebaceous gland cells;

the experimental method comprises the following steps: seb-E6E7 cells were trypsinized and counted, and then they were plated at 10000/well in a 96-well plate, the next day cells were stimulated with linoleic acid as a stimulant at a final concentration of 0.0125%, and isoflavone extracts of example 1 and comparative example 7 (both of which had the same final concentration of total isoflavones in the 96-well plate and were 20. mu.g/mL) were added, and after culturing for 48 hours, they were stained with oil red O to observe the oil secretion of the cells.

FIG. 1 is a schematic diagram of a model group seb-E6E7 cell microscope without a test sample added thereto, and a fat liquid is clearly seen. FIG. 2 is a schematic representation under a cell microscope of seb-E6E7 after the addition of isoflavones obtained by extraction in example 1; FIG. 3 is a schematic representation under a cell microscope of seb-E6E7 after the addition of the isoflavone obtained by extraction in comparative example 7. As shown in figures 1-3, the isoflavone obtained by the extraction method can obviously reduce the secretion of fat droplets, has an oil control effect obviously superior to that of isoflavone prepared by the conventional extraction method, and can be used for the development of products for preventing alopecia, controlling oil of skin and inhibiting acne.

The applicant states that the present invention is illustrated by the above examples to show the extraction method of isoflavone, the isoflavone obtained by extraction and the application of the isoflavone, but the present invention is not limited by the above examples, i.e. the present invention is not limited by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. The method for extracting the isoflavone is characterized by comprising the following steps of:

2. The method for extracting isoflavone according to claim 1, wherein the plant in step (1) is selected from any one or a combination of at least two of chickpea, soybean, formononetin, lupin, sophora japonica, kudzu, mulberry leaf, genistein or red clover, preferably red clover and/or chickpea;

preferably, the particle size of the plant powder obtained by the pulverization in the step (1) is 10-100 meshes.

3. The method for extracting isoflavone according to claim 1 or 2, wherein the mass ratio of the acidic eutectic solvent, the alcohol and the water in the step (1) is (0.5-2): (4-6): (3-5), preferably 1:5: 4.

4. The method for extracting isoflavone according to any one of claims 1 to 3, wherein the acidic eutectic solvent in the step (1) consists of a quaternary ammonium salt and an organic acid;

preferably, the molar ratio of the quaternary ammonium salt to the organic acid is (0.5-2) to (2-4), preferably 1: 3;

preferably, the quaternary ammonium salt is preferably choline chloride and/or betaine;

preferably, the organic acid is selected from any one of malic acid, citric acid, malonic acid or succinic acid or a combination of at least two of the two;

5. The method for extracting isoflavone according to any one of claims 1 to 4, wherein the number of times of the extraction in the step (1) is 1 to 3;

preferably, the temperature of each extraction is 70-90 deg.C, and the time of each extraction is 1-3 h.

6. The method for extracting isoflavone according to any one of claims 1 to 5, wherein the concentration in the step (2) is specifically: concentrating until the alcohol content in the crude extract is below 5 wt%;

preferably, the concentration temperature in the step (2) is 40-60 ℃, and the vacuum degree of the concentration is below-0.05 MPa.

7. The method for extracting isoflavone according to any of claims 1 to 6, wherein the filtration in the step (2) is performed by using filter paper and/or polypropylene membrane;

preferably, the pore size of the polypropylene membrane is 0.8-5 μm.

8. The method for extracting isoflavone according to any one of claims 1 to 7, wherein the washing in the step (2) is specifically: washing the solid obtained by filtering with water, ethanol and dichloromethane in sequence;

preferably, the mass ratio of the solid obtained by filtering, water, ethanol and dichloromethane is 1 (4-6) to (2-4);

preferably, the mass concentration of the ethanol is 40-60 wt%.

9. An isoflavone which is extracted by the extraction method according to any one of claims 1 to 8;

preferably, the isoflavones are in the aglycon form.

10. Use of isoflavones according to claim 9 for inhibiting lipid secretion.