JP2805325B2 - Antioxidant - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an antioxidant effective for preventing an oxidation reaction catalyzed by an oxidase.

[Conventional technology]

The tissues of animals and plants contain various phenolic components such as catechin, chlorogenic acid, anthocyan, dopa, and the like, which are oxidized by oxygen to change into coloring substances, or discolor. As a result, epidermal blackening occurs in animals, and browning or fading occurs in vegetables, fruits, and flowers. Such discoloration is often considered an undesirable phenomenon, and efforts are being made to prevent it.

The oxidation reaction described above is affected by temperature and metal ions, but proceeds particularly quickly when an oxidation reaction catalytic enzyme is present. As enzymes that catalyze the oxidation of phenolic components, catechol oxidase, polyphenol oxidase, and the like are known.

As means for preventing an oxidation reaction involving an oxidase, there have conventionally been a method of blocking oxygen and a method of using an antioxidant. The oxygen barrier method includes a method of packaging with an oxygen absorber using an oxygen barrier packaging material and a method of vacuum packaging.These methods can be adopted when oxygen barrier packaging cannot be performed. It is not common because it loses its effect after opening.

Antioxidants that delay the oxidation reaction by oxidases and prevent the accumulation of oxides have been classified into synthetic products and natural products in the past. Therefore, the search for animal and plant extracts having an antioxidant effect has been widely conducted. as a result,
It has been found that mulberry bark extract, mushroom extract, quercetin, rutin, kojic acid, oryzanol and the like are effective. However, these natural products have problems in potency, color, taste, odor, stability over time, and the like, and there are few practically usable ones.

[Problems to be solved by the invention]

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

[Means for Solving the Problems] As a result of repeated studies on many animal and plant components in search of a substance capable of effectively inhibiting an oxidation reaction involving an oxidase, the present inventors found that licorice (Glycyrrhiza plant) It has been found that the components contained in certain species have a strong antioxidant effect.
Then, as a result of conducting research for confirming the active ingredient, they found that it was glabridine, and completed the present invention.

That is, the antioxidant provided by the present invention contains glabridine as an active ingredient.

Gravlidine has the following chemical structure and belongs to isoflavane, which is very rare in natural products.

Grabradine is a component peculiar to the glabra species (Glycyrrhiza glabra Linn) of leguminous licorice plants in the botanical classification, and is contained in roots and rhizomes.
Even ra species may not contain this compound. Also,
Even in licorice plants, the content of glabridine has not been confirmed in species other than Glycyrrhiza glabra. Therefore, when extracting glabridine from licorice, care must be taken in selecting the raw material licorice.

Gravlidine extracts liquorice containing it or its aqueous extraction residue, using lower aliphatic alcohols, lower aliphatic ethers, aliphatic or aromatic hydrocarbons, halogenated hydrocarbons, lower aliphatic esters, etc. as extraction solvents. be able to. With water or weakly alkaline water, glabridine is not extracted.

The crude extract containing grabradine can be used as an antioxidant in the form of a concentrate or a crude extract obtained by simply drying and powdering the concentrate, but it can be purified to remove coloring substances and odorous components, and it can be used as an antioxidant. When the content is high, the composition is easy to use and the efficacy is further improved. Examples of the purification method for obtaining high-purity glabridine, silica gel, reverse-phase silica gel, polyamide, a porous polymer or a weakly basic ion-exchange resin and the like by performing purification several times by column chromatography, as a carrier,
There is a method of performing fractionation while confirming the target component by thin-layer chromatography or high-performance liquid chromatography.

In addition, when a natural product-based antioxidant is used in combination with a reducing substance such as ascorbic acid, good results are often obtained due to a synergistic effect (JP-A-63-269942, etc.). In addition, more excellent results can be obtained by using in combination with reducing substances such as ascorbic acid, phytic acid and phosphates.

[Action / Effect]

Gravlidine has a much better antioxidant effect than previously known natural antioxidants, and is effective not only in small amounts, but also has no undesirable physiological side effects and is chemically stable. Therefore, the antioxidant of the present invention containing this as an active ingredient is an excellent one that can be used in a wide range of fields such as foods, cosmetics, and pharmaceuticals.

〔Example〕

 Hereinafter, the present invention will be described with reference to examples.

Example 1 (Production example of purified glabridine) 2 kg of the ground and rhizome parts of G. glabra containing grabradine were heated under reflux with 10 dichloromethane for 2 hours to extract dichloromethane-soluble components. The extract was separated, and the same operation was repeated for the extraction residue to obtain a total of 30 extracts. The solvent of this extract was distilled off and further dried under reduced pressure to obtain 54 g of a brown solid.

30 g of this extract was dissolved in chloroform, sprayed on silica gel (Wakogel C-300, Wako Pure Chemical Industries, Ltd.), dried, and then layered and packed on a column filled with silica gel. Subsequently, elution treatment was performed with a mixed solution of chloroform / methanol (40/1) to obtain a glabridine-rich fraction. The elution of the target product was confirmed by thin-layer chromatography (developing solvent: chloroform / methanol; carrier: silica gel 60F, Merck Co .; detection method: heating after spraying 19% sulfuric acid).

The solvent of the glabridine-rich fraction is distilled off under reduced pressure, 15 g of the obtained solid is dissolved in methanol, and the solution is reversed-phase silica gel (30 to 50 mesh; ODSG-3; manufactured by Mito Chemical Research Laboratory) Dried and dried. This reverse-phase silica gel was stacked and packed on a column previously filled with reverse-phase silica gel, separated and eluted with 2% acetic acid / acetonitrile (40/60), and a glabridine fraction was collected. The glabridine fraction was concentrated under reduced pressure, chloroform was added to the concentrate, liquid-liquid partition extraction was performed, and the chloroform layer was separated. Anhydrous sodium sulfate was added to the chloroform layer to dehydrate it. After dissolving in benzene, n-hexane was gradually added. When cloudiness occurred, the mixture was heated, clarified again, and allowed to stand. 300 mg of needle-like crystals were obtained.

Example 2 Gravlidine and other known antioxidants were tested for their ability to prevent catechol oxidase-related oxidation reactions by the following method.

Reagent A: 1 ml of catechol oxidase solution (pH 6.8; using 1/15 M potassium phosphate buffer; 200 unit / ml) Solution B: Catechol solution (pH 6.8; using 1/15 M potassium phosphate buffer; concentration) 0.6 mg / ml) 1 ml (containing 0.006 mg L-ascorbic acid) Solution C: 1/15 M-potassium phosphate buffer (pH 6.8) 1 ml test solution Dissolve the sample in 1 ml of ethanol, and add 1/15 M- It was diluted with potassium phosphate buffer (pH 6.8) to obtain 100 ml of a test solution. Use 1 ml at a time.

Operation test solution was added solution A was preincubated for 10 minutes at 25 ° C. to then solution B was added and after incubation for 10 minutes, measuring the 440nm absorbance A _t.

C was added instead of the test solution, and operating similarly to measure the 440nm absorbance A _b.

Add liquid C instead of liquid B and operate in the same manner as
Measuring the 440nm absorbance A _o.

The above measurements were carried out for the sample solution at various concentrations to determine the sample amount (reaction test solution amount in 3 ml) IC ₅₀ for preventing oxidation rate by the following equation becomes 50% interpolation.

Table 1 shows the measurement results.

Example 3 With respect to the extract obtained by extracting G. glabra and various other licorices containing glabridine with a mixed solvent of hexane / ethanol (10/1), the ability to prevent an oxidation reaction involving polyphenol oxidase was determined as follows. Investigated by method.

Reagent A: 1 ml of polyphenol oxidase (mushroom) solution (using a potassium phosphate buffer of pH 6.8; 600 units / ml) Solution B: Caffeic acid solution (using a potassium phosphate buffer of pH 6.8; concentration: 0.5 mg) 1 ml (containing 0.005 mg of L-ascorbic acid) Solution C: 1 ml of potassium phosphate buffer solution of pH 6.8 Test solution The sample was dissolved in 1 ml of ethanol, and 1/15 M potassium phosphate buffer solution (pH 6 .8) to make a 100 ml test solution. Use 1 ml at a time.

Operation performed in the same manner as in Example 2, obtaining a sample amount of antioxidant content became 50% IC ₅₀ (the amount in the reaction test solution 3ml) at interpolation. However, the absorbance was measured at 475 nm.

 The measurement results were as shown in Table 2.

Example 4 Gravlidine and other antioxidants were tested for their ability to prevent oxidative reactions involving tyrosinase by the following method.

Reagent A: 1 ml of tyrosinase (mushroom) solution (pH 6.8 potassium phosphate buffer; 110 units / ml) Solution B: Tyrosine solution (pH 6.8 potassium phosphate buffer; concentration 0.3 mg / ml) 1) C solution: 1 ml of potassium phosphate buffer solution of pH 6.8 1 ml test solution A sample was dissolved in 1 ml of ethanol, and diluted with 1 / 15M-potassium phosphate buffer solution (pH 6.8) to obtain 100 ml of test solution. . Use 1 ml at a time.

Except for measuring the operation absorbance at 475nm following the procedure of Example 2, obtaining a sample amount of antioxidant content became 50% IC ₅₀ (the amount in the reaction test solution 3ml) at interpolation.

 The measurement results were as shown in Table 3.

Example 5 Gravlidine was dissolved in a small amount of ethanol and added to tap water to form a 20 ppm solution. For comparison, L-ascorbic acid was dissolved in tap water to prepare a 2000 ppm solution.

Apples, peeled and sliced to a thickness of 1 to 2 mm, were immersed in these solutions for several seconds, drained well, placed in a container, and left at 20 ° C. to observe the degree of discoloration due to oxidation.

 Table 4 shows the results.

Example 6 A test solution was prepared in the same manner as in Example 5. In this test solution, immerse the potatoes peeled and sliced to a thickness of 1 to 2 mm for several seconds, drain the water well, put them in a container without a lid and leave them at 20 ° C to measure the degree of discoloration due to oxidation. Observed. Table 5 shows the results.

Claims (1)

(57) [Claims] 1. An antioxidant comprising glabridine as an active ingredient.