JPH07173148A - Production of genistein - Google Patents

Abstract

PURPOSE:To separate and purify high-purity genistein by a simple operation. CONSTITUTION:High-purity isoflavone is separated by bringing an isoflavone- containing mixture into contact with a solvent represented by the formula CHnCl4-n (n is 0, 1 or 2) to extract genistein and subsequently concentrating the solvent layer. Since a complicated operation such as column chromatography or thin layer chromatography is not required, the time cost or the economical cost can be reduced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for obtaining highly pure genistein from an isoflavone mixture by a simple operation.

[0002]

BACKGROUND OF THE INVENTION Many plants such as legumes, Asteraceae and Iridaceae contain daidzein, genistein and isoflavones (isoflavone aglycones and their glycosides) such as daidzin and genistin which are glycosides thereof. It has been confirmed that these have many pharmacological effects including estrogen action, antibacterial action, antioxidant action, and carcinogenic action. Isoflavones in plants are
Although 95% or more of them exist as glycosides, aglycones such as daidzein and genistein have stronger pharmacological effects such as carcinogenicity than glycosides.

Conventionally, a method for obtaining an isoflavone solution containing genistein from a plant raw material is known (Japanese Patent Publication No.
4-21670, Japanese Examined Patent Publication No. 4-34526), these are methods for obtaining a mixture of several isoflavones, not for separating genistein.

A method for isolating genistein from an isoflavone mixture is also known (Japanese Patent Laid-Open No. 61-247396),
This is a very complicated method in which column chromatography and thin layer chromatography are repeated, and there is a problem that time and cost are high. Therefore, it is further expected to establish a method for separating highly pure genistein from an isoflavone mixture by a simple operation.

[0005]

[Means for Solving the Problems] In view of such a current situation,
The present inventors have investigated a method for separating genistein from an isoflavone mixture by a simple operation, and as a result, of the isoflavones, only genistein has the general formula CH _n Cl.
_The present invention was completed by discovering that it is easily soluble in a solvent represented by _4-n (n = 0, 1, 2). That is, the present invention provides a mixture of isoflavones of the general formula CH _n Cl _4-n (n = 0,1,
The method for producing genistein is characterized by performing extraction treatment with a solvent represented by 2).

The present invention will be described in detail below. The starting material for obtaining the isoflavone mixture may be any plant extract containing isoflavone, but it is particularly preferable to use soybean containing a large amount of isoflavone. As the soybean extract, whole soybean, dehulled soybean, defatted soybean, soybean powder, soybean germ and the like extracted with water, an organic solvent or a mixed solvent of water and an organic solvent, or tofu, miso, soy sauce, isolated soybean protein, etc. Examples include "boiled soup", "yu", "whey" and the like produced in the process of producing. It is also possible to use soy sauce cake extracted with alcohol such as methanol or ethanol, or hydrous alcohol. Furthermore, caustic soda solution may be added to soy sauce, heated to saponify unnecessary oil, and isoflavone aglycone, which is an unsaponifiable product, may be extracted with ethyl ether.

A method for obtaining an isoflavone mixture using a soybean soaking solution as a starting material will be described below with reference to specific examples. 1 10 times the amount of dehulled soybeans in warm water at 50 ° C
Immerse in H9) for 2 hours to separate the immersion liquid. 2 Use this immersion liquid in a synthetic resin (for example, Diaion HP-2
0, YMC-GEL ODS-A type 60-01) to adsorb isoflavone in the immersion liquid on the synthetic resin. For the contact between the immersion liquid and the synthetic resin, either a batch method or a column method may be used. 3 Desorb and elute isoflavone adsorbed on the synthetic resin using an alkaline aqueous solution, alcohol, or hydrous alcohol, and collect as a resin adsorbate. 4 The obtained resin adsorbate is concentrated under reduced pressure to obtain a syrupy concentrate. In addition to isoflavone glycosides, this concentrate contains multiple fat-soluble components such as chlorophyll, carotenoids and phospholipids. Therefore, add organic solvent (such as hexane) to this concentrate to extract the fat-soluble components. After removing
The extraction residue is collected. The main components of the extraction residue are isoflavone glycosides, specifically daidzin and genistin,
Genistin is a glycoside of genistein targeted by the present invention.

The present invention can be carried out by using this extraction residue, but in order to increase the yield of genistein,
It is preferable to use isoflavone aglycone as the glycoside component in the extraction residue by hydrolysis. 5 Hydrochloric acid acidic alcohol is added to the extraction residue obtained by the above method, and hydrolysis is carried out at 100 ° C. for 6 hours while refluxing. By this treatment, daidzin and genistin, which are the main components of the isoflavone glycoside, are hydrolyzed into genistein and daidzein.

The hydrolysis treatment can also be carried out using an enzyme (β-glucosidase). 6. After concentrating the hydrolyzate under reduced pressure, it is thoroughly washed with distilled water and adjusted to a neutral region (pH 5-7). After washing, the residue is collected and dried. This residue (isoflavone mixture)
The main component of isoflavone aglycone, specifically daidzein and genistein.

In the present invention, an isoflavone mixture prepared as described above is converted into a compound of the general formula CH _n Cl _4-n (n = 0,1,2).
The genistein is separated by extraction treatment with a solvent represented by, specifically, chloroform, dichloromethane and carbon tetrachloride. The main component of the isoflavone mixture obtained by the above method is isoflavone aglycone, specifically genistein and daidzein, of which only genistein is readily soluble in the above solvent. Therefore, when the isoflavone mixture is contacted with these solvents, only genistein is extracted in the solvent layer. Of the solvents used in the present invention,
In particular, chloroform has an excellent effect of specifically extracting genistein.

A reflux extraction method is preferably used for contacting the isoflavone mixture with the above solvent. The amount of the solvent is 30 to 500 times the amount of the isoflavone mixture, the extraction time is about 3 hours, and this is preferably repeated several times. The target genistein can be separated by subjecting the isoflavone mixture to extraction treatment with a solvent and then concentrating the solvent layer, for example, under reduced pressure. The genistein obtained by the method of the present invention may be further purified using alumina, silica gel or the like, if necessary.

The effects of the present invention will be described below with reference to experimental examples. Experimental Example 1 (Method for preparing isoflavone mixture) Dehulled soybeans are immersed in 10 times the amount of warm water at 50 ° C (adjusted to pH 9 with alkali) for 2 hours to separate the immersion liquid. Next, a synthetic resin (Diaion HP-20) is added to the immersion liquid by the batch method and stirred for 1 hour. After thoroughly washing the synthetic resin with water, 1N-ammonia water is added to elute the resin adsorbate. After performing the same elution operation twice, the eluates are combined and concentrated under reduced pressure to obtain a syrup-like concentrate. Chloroform was added to this concentrate and reflux extraction was carried out 3 times for 3 hours. Then, the extraction residue was recovered, 4% hydrochloric acid methanol solution was added to this extraction residue, and the mixture was hydrolyzed at 100 ° C. for 6 hours under reflux. The hydrolyzate was concentrated under reduced pressure, washed thoroughly with distilled water, and the residue after washing was dried to obtain a brown solid. When this solid was analyzed by HPLC (Waters 209D type),
This solid was an isoflavone mixture containing daidzein and genistein as main components. The amount ratio of daidzein and genistein was about 1: 1.

(Extraction of genistein) To the isoflavone mixture prepared by the above method, the solvent shown in Table 1 was added, and the mixture was subjected to reflux extraction for 3 hours 3 times, then the solvent layer was filtered with a membrane filter, and the filtrate was obtained. When analyzed by HPLC under the same conditions as above, the components were genistein and daidzein. The results are shown in Table 1.

[0014]

As is apparent from this experimental example, the isoflavone mixture is subjected to extraction treatment with a solvent represented by the general formula CH _n Cl _4-n (n = 0,1,2), that is, chloroform, dichloromethane and carbon tetrachloride. Thus, high-purity genistein can be separated by a simple operation. In particular, it can be seen that chloroform has an excellent effect of specifically extracting genistein.

[0016]

According to the present invention, highly pure genistein can be separated and purified from a soybean isoflavone mixture by a simple operation. Therefore, complicated operations such as column chromatography and thin layer chromatography are unnecessary,
Time and economic costs can be reduced.

[0017]

EXAMPLES Examples will be shown below. Example 1 2 kg of dehulled soybeans was adjusted to pH 9 with alkali at 50 ° C.
It was immersed in 20 liters of water for 2 hours to separate the immersion liquid. Then, by a batch method, the dipping solution was made into synthetic resin (Diaion HP-2
0) 500 g was added and stirred for 1 hour. The synthetic resin was thoroughly washed with water, and 700 ml of 1N-ammonia water was added to elute the resin adsorbate. After performing the same elution operation twice, the eluates were combined and concentrated under reduced pressure to obtain 1.57 g of a syrupy concentrate. Hexane 100 is added to this concentrate.
ml was added and reflux extraction was performed 3 times for 3 hours. Next, the extraction residue was recovered, 150 ml of a 4% hydrochloric acid methanol solution was added to the extraction residue, and the mixture was hydrolyzed under reflux for 3 hours. After concentrating the hydrolyzate under reduced pressure, the hydrolyzate was thoroughly washed with distilled water, and the residue after washing was dried to obtain 367 mg of a brown solid. After adding 50 ml of chloroform to the solid and performing reflux extraction for 3 hours three times,
The solvent layer was concentrated under reduced pressure to obtain highly pure genistein 152 mg.
Got

Example 2 To 50 g of soy sauce meal, which is a by-product of soy sauce produced from defatted soybeans, 1500 ml of methanol was added and reflux extraction was repeated 3 times for 3 hours, and then the extracts were combined and decompressed. Concentration gave 4.0 g of a syrupy concentrate. 200 ml of hexane was added to the concentrate, and the mixture was subjected to reflux extraction for 3 hours 3 times. Then, the extraction residue is collected and thoroughly washed with distilled water, and then the residue is dried to give a brown solid 1.
450 mg was obtained. The main component of this solid was isoflavone aglycone, specifically daidzein and genistein.

Since the isoflavone glycosides in the soy sauce cake are hydrolyzed by β-glucosidase into aglycones during the soy sauce production process, there is no need to perform a hydrolysis operation. Chloroform (500 ml) was added to the above residue, reflux extraction was carried out in the same manner as in Example 1, and the solvent layer was concentrated under reduced pressure to obtain 769 mg of highly pure genistein.

Claims (1)

[Claims] 1. An isoflavone mixture having the general formula CH _n Cl
_A method for producing genistein, which comprises extracting with a solvent represented by _4-n (n = 0, 1, 2).