JPH02233795A - Antioxidant - Google Patents

Abstract

PURPOSE:To obtain an antioxidant effective at a small amount in preventing the oxidation reaction catalyzed by oxidase, having chemical stability and useful in the fields of foods, cosmetics, pharmaceuticals, etc., by using glabridin as an active component. CONSTITUTION:Glabridin is extracted from glabridin-containing licorice or its water-extraction residue by using a lower aliphatic alcohol, lower aliphatic ether, aliphatic or aromatic hydrocarbon, halogenated hydrocarbon, lower aliphatic ester, etc., as an extraction solvent. The obtained crude extract liquid is purified several times by column chromatography using silica gel, reversed phase silica gel, polyamide, porous polymer or weakly basic ion exchange resin as a carrier to obtain glabridin having high purity. The objective antioxidant is produced by using the glabridin as an active component.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an antioxidant that is effective in preventing oxidation reactions catalyzed by oxidases.

[Conventional technology]

The tissues of animals and plants contain various heptenolic components such as catechin, chlorogenic acid, anthocyanide, and dopa, which are oxidized by oxygen and turn into colored substances, or conversely, fade. This causes blackening of the epidermis in animals, and browning or discoloration in vegetables, fruits, and flower petals. Such discoloration is often considered an undesirable phenomenon, and efforts are being made to prevent it.

The above-mentioned oxidation reaction is influenced by temperature and metal ions, but it proceeds particularly quickly when an oxidation reaction catalytic enzyme is present. Catechol oxidase, polyphenol oxidase, and the like are known as enzymes that catalyze the oxidation of phenolic components.

As a means to prevent oxidation reactions involving oxidases,
Conventionally, there have been methods of blocking oxygen and methods of using antioxidants. Oxygen-blocking methods include packaging with an oxygen-blocking material using an oxygen absorber and vacuum packaging, but these methods cannot be used when oxygen-blocking packaging is not possible. However, it is not common because it loses its effectiveness once the package is opened.

Antioxidants that slow down oxidation reactions caused by oxidizing enzymes and prevent the accumulation of oxides have traditionally been available in synthetic and natural products, but in recent years there has been a trend among users to prefer natural products. As antioxidant activity has become stronger, the search for animal and plant extracts that have antioxidant effects is being widely conducted.

As a result, it has been found that mulberry bark extract, rhododendron extract, quercetin, rutin, kojic acid, oryzanol, etc. are effective. However, these natural products have problems in terms of efficacy, color, taste, odor, stability over time, etc., and very few of them can actually be used.

[Problem to be solved by the invention]

Therefore, an object of the present invention is to provide a more practical natural antioxidant.

[Means to solve the problem]

The present inventors conducted repeated studies on many animal and plant components in search of substances that can effectively inhibit oxidation reactions involving oxidases, and found that a specific species of licorice (plants of the genus Glyeyrhix) contains strong oxidants. It was found that it has a preventive effect. We then proceeded with research to confirm its active ingredients.
As a result, they found out that it was glabridin, and completed the present invention.

That is, the antioxidant provided by the present invention contains glabridin as an active ingredient or is composed of a licorice extract containing glabridin.

Glabridin has the following chemical structure and is an isoflavan, which is extremely rare among natural products.

Glabridin is classified into 1labrs species (GIycyrrl+it* (li
Br.

Also, Glyeyrrhixi 1l*
The content of glabridin has not been confirmed in species other than brs. Therefore, when extracting glabridin from licorice, care must be taken in selecting the raw material licorice.

Glabridin is extracted from licorice containing it or its water-extracted residue using lower aliphatic alcohols, lower aliphatic ethers, aliphatic or aromatic hydrocarbons, halogenated hydrocarbons, lower aliphatic esters, etc. as extraction solvents. be able to. Glabridin cannot be extracted with water or weakly alkaline water.

The crude extract containing glabridin can be used as an antioxidant either in the form of a concentrate or as a crude extract obtained by drying and powdering it, but it is necessary to purify it to remove coloring substances and odor components. By increasing the content, it becomes easier to use and more effective. Examples of purification methods for obtaining high purity glabridin include purification several times using column chromatography using silica gel, reversed-phase silica gel, polyamide, porous polymer, or weakly basic ion exchange resin as a carrier;
There is a method of fractionating while confirming the target component using thin layer chromatography or high performance liquid chromatography.

Incidentally, when natural antioxidant substances are used in combination with reducing substances such as ascorbic acid, synergistic effects often give good results (Japanese Patent Application Laid-open No. 63-269942, etc.), and in the case of glabridin, it can be used alone as well. In addition, even better results can be obtained by using it in combination with reducing substances such as ascorbic acid, phytic acid, and phosphates.

[Action/Effect]

Glabridin has far superior antioxidant effects than previously known natural antioxidants, is not only effective in small amounts, but also has no undesirable physiological side effects.
Since it is a chemically stable compound, the antioxidant of the present invention containing it as an active ingredient can be used in foods, cosmetics, pharmaceuticals, etc.
It is an excellent product that can be used in a wide range of fields.

(Example〕 Hereinafter, the present invention will be explained with reference to Examples.

Example 1 (Production Example of Purified Glabridin) 2 kg of crushed roots and rhizomes of G41sbra containing glabridin were mixed with 101 dichloromethane.
Heat under reflux for a period of time to extract dichloromethane-soluble components. The extract was separated and the same operation was repeated on the extraction residue to obtain a total of 301 extracts. The solvent of this extract was distilled off, and the extract was further dried under reduced pressure to obtain 54 g of a dark brown solid.

30 g of this extract was dissolved in chloroform and sprinkled on silica gel (Wako Gel C-300, Wako Pure Chemical Industries, Ltd.), dried, and then packed in layers on a column filled with silica gel. Next, elution treatment with a chloroform/methanol mixture (40/1) was performed to obtain a fraction containing high glabridin content. Elution of the target product was confirmed by thin layer chromatography (developing solvent: chloroform/methanol; carrier: Merck & Co. silica gel 60F; detection method: heating after spraying with 19% sulfuric acid).

The solvent of the glabridin-rich fraction was distilled off under reduced pressure,
15 g of the obtained solid was dissolved in methanol, and the solution was sprinkled on reverse-phase silica gel (30 to 50 mesh, manufactured by ODSG-131 Mito Chemical Technology Institute) and dried.

This reversed phase silica gel was stacked and packed on a column previously filled with reversed phase silica gel, separated and eluted with 2% acetic acid/acetonitrile (40/60), and a glabridin fraction was collected. This glabridin fraction was concentrated under reduced pressure, chloroform was added to the concentrated solution, liquid-liquid partition extraction was performed, and the chloroform layer was separated. In this chloroform layer,
After dehydration by adding anhydrous sodium sulfate, dissolving in benzene, gradually adding n-hexane, heating when it becomes cloudy, making it clear again, and standing still to obtain 300 m of pale yellow needle crystals of glabridin ( I got it.

Example 2 Regarding glabridin and other known antioxidants,
The ability to prevent oxidation reactions involving catechol oxidase was investigated using the following method.

Reagent A solution: Catechol oxidase solution (pH 6.8
; Using l/Is potassium triphosphate buffer; 200
and/ml) 1 ml B solution: Catechol solution (p
Hs. s; I/15M-potassium phosphate buffer for stool; concentration 0.6m! /+I) lml (0.0 0
Solution C: 1/15M potassium phosphate buffer (contains 6H ascorbic acid).
8) A lm test solution sample was dissolved in 11 ml of ethanol and diluted with l/IsM-potassium phosphate buffer (pH 6.8) to make a 100 ml test solution. Use l1 once.

Procedure ① Add Solution A to the test solution and incubate grains at 25°C for 10 minutes. Then add Solution B and incubate for 10 minutes, then measure the absorbance A at 440 nm.

(2) Add liquid C instead of the test solution and proceed in the same manner as (2) to measure the absorbance A at 440 am.

■ Add liquid C instead of liquid B and operate in the same way as ■ 4.
Measure the absorbance at 40 am.

(2) Perform the above measurements on sample solutions of various concentrations, and add the sample amount (amount in 3 liters of reaction test solution) I Cs at which the antioxidant rate becomes 50% according to the following formula. is determined by interpolation.

Antioxidant rate - p- b - (A-p-゜) x 10
0 (%) A, the measurement results are as shown in Table 1.

Table 1 Sample ■CS. (a+S) Glabridin 0.0053 Licorice G. ++rslu+sis methylene chloride extract (
(Does not contain glabridin) 0.22 Rutin
Greening (ineffective) Ebigaro Tekin Gallate Browning (ineffective) Kojic acid
0.055 Example 3 G. The ability of the extracts obtained by extracting G. glxbra and other various licorice with a hexane/ethanol (10/1) mixed solvent to prevent oxidation reactions involving polyphenol okindase was investigated using the method described below.

Reagent A solution: polyphenol oxidase (mushroom) solution (
pH 6.8 potassium phosphate buffer for stool: 600 units/
m1) 1+sl Solution B: caffeic acid solution (pH 6.8 potassium phosphate buffer for stool; concentration 0.5 mg/ml) lml (0
．． 0 0 5m (contains L-ascorbic acid) Solution C: pH 6. 8 potassium phosphate buffer 1ml
The test solution sample was dissolved in lml of ethanol and diluted with l/1 5M potassium phosphate buffer (pH 6.8) to 100ml.
1 test solution. Use l@lml.

Perform the same operation as in Operation Example 2, and add the sample amount (amount in reaction test liquid 31) IC, which will give an antioxidant rate of 50%. is determined by interpolation. However, the absorbance measurement is performed at 4 7 5 am
I did it.

The measurement results are shown in Table 2.

Table 2 G. ur*lemsis extract* 0
．． 24G. imflatx extract I
O. 36G. ecb:n*ts extract
Invalid G. psllidiflor*extract back
None of them contain glabridin.Example 4 The ability of glabridin and other antioxidants to prevent oxidation reactions involving tyrosinase was investigated by the following method.I;o K'' Solution A: Tyrosinase (mushroom) solution (pH 6.8
Using potassium phosphate buffer; 110 units/1) lm
Solution IB: Tyrosine solution (pH 6.8 potassium phosphate buffer; concentration 0.3 u/ml) 1 ml
Solution C: Potassium phosphate buffer with p86.8 1 test solution sample was dissolved in 1 liter of ethanol and diluted with I/ISM-potassium phosphate buffer (pH 6.8) to make 1001 test solution. . Use 1ml at a time.

Example 2 except that the operation absorbance was measured at 475 nm.
Perform the same operation as above, and add the sample amount (amount in reaction test liquid 31) IC, which makes the antioxidant rate 50%. is determined by interpolation.

The measurement results are shown in Table 3.

Table 3 Sample x c So (+g) Glabridin 0.00028 (without glabridin) 0.04G. i
IIflits methylene chloride extract (without glabridin) 0.032 glabrene
o. oos Rikokumaron
0. Ol licochalcone A
O. 07 Kenorecetin 0
．． 015 Kojic acid 0.02
2. Shrimp Gallocatechin Ineffective Example 5 Glabridin was dissolved in a small amount of ethanol and added to tap water to make a 20 pp hazy solution. For comparison, L-ascorbic acid was dissolved in tap water and 2000 ppm
A solution was prepared.

Apples that had been peeled and sliced into 1-2cm thick pieces were immersed in these solutions for a few seconds, and after thoroughly draining the water, they were placed in a container and left at 20°C to observe the degree of discoloration due to oxidation. .

The results are shown in Table 4.

Table 4 Example 6 A test solution was prepared in the same manner as in Example 5. Peeled and sliced potatoes with a thickness of 1 to 2 mm are immersed in this test liquid for a few seconds, and after draining well, they are placed in an open container and left at 20°C to determine the degree of discoloration due to oxidation. Observed. The results of Part 4 are shown in Table 5.

Table 5

Claims (2)

[Claims] (1) An antioxidant characterized by containing glabridin as an active ingredient. (2) An antioxidant consisting of a licorice extract containing glabridin.