JPH11172246A - Anti-oxidant and manufacture thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anti-oxidant having a preventive effect on an oxidation of food and a sufficient suppressive effect on an oxidative damage in a living matter due to an active oxygen by extracting a plant having a suppressive effect on a damage of a protein by peroxy nitrite with an aqueous alcohol solution of a specified concentration. SOLUTION: A gluten meal(GM) derived from a corn is extracted with a 50-80% aqueous ethyl alcohol. A coffee bean is extracted with a 60-99% aqueous ethyl alcohol. A grape is extracted with a 10-50% aqueous ethyl alcohol. These extracts contain an anti-oxidant represented by formula I (wherein R1 is H, OH or OCH3 , R2 is OH and R3 is H; R1 is OH, R2 is OCH3 and R3 is H; or R1 and R3 are each OCH3 and R2 is OH), that which is represented by formula II (wherein R1 and R2 are each a methoxyl group and R3 is H or the like), and the same represented by formula III (wherein R1 and R3 are each OH and R2 is H or the like).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to a plant-derived antioxidant having a biological injury suppressing action and a method for producing the same.

[0003]

2. Description of the Related Art

Heretofore, some naturally occurring substances have been reported as antioxidants, but the purpose of utilizing these naturally occurring antioxidants was mainly to prevent oxidation of foods and cosmetics.

[0005] In recent years, it has been suggested that oxidative damage in living bodies is involved in various diseases, and it has been reported that antioxidants are effective in preventing diseases.

However, many plant-derived antioxidants which have been found so far, for example, Japanese Patent Application Laid-Open No. 2-193
No. 930 and JP-A-6-128138 have a remarkable antioxidant activity as compared with conventionally used tocopherol and ascorbic acid in terms of antioxidation of foods and whitening action of cosmetics. However, due to various problems, it has not yet been sufficiently commercialized.

[0007] Therefore, like tocopherol and ascorbic acid, which have been conventionally used as antioxidants, they have a satisfactory effect on the prevention of food oxidation and the whitening effect of cosmetics, and furthermore, the oxidation by active oxygen in living organisms. What also has the sufficient inhibitory effect also with respect to the injury was desired.

[0008]

[Means for Solving the Problems]

The present inventors have conducted intensive studies to overcome the above-mentioned problems, and have recently found that 3-nitrotyrosine selectively produced by peroxynitrite is present in various lesions. We focused on peroxynitrite as a new peroxide in living organisms.

As a result of further studies by the present inventors, the inhibitory effect of peroxynitrite on the oxidative damage of collagen is hardly found in conventional antioxidants such as tocopherol, ascorbic acid and BHA. In contrast, we found that it was found in some plant-derived antioxidants.

Further, as a result of a test using LDL, similar results were obtained, and the present invention was completed.

That is, means for solving the problems of the present invention are as follows.

First, a plant-derived antioxidant having a peroxynitrite inhibitory action on proteins.
Second, a corn-derived antioxidant having a peroxynitrite inhibitory effect on protein damage. Third
And an antioxidant derived from coffee beans, which has an action of inhibiting protein damage caused by peroxynitrite. Fourth, a grape-derived antioxidant having a protein damage inhibitory action by peroxynitrite.

Fifthly, a plant-derived antioxidant represented by the following formula and having a protein damage inhibitory action by peroxynitrite.

[0015]

Embedded image

[0016] (wherein, R ₁ represents a hydrogen atom, a hydroxyl group or a methoxyl group, and R ₂ represents a hydroxyl group, and either R ₃ represents a hydrogen atom, or, R ₁ is a hydroxyl group, and R ₂ Represents a methoxyl group and R ₃ represents a hydrogen atom, or R ₁ and R ₃ represent a methoxyl group and R ₂ represents a hydroxyl group.)

That is, the substances represented by the above formula are summarized in Table 1 below.

[0018]

[Table 1]

Sixth, a plant-derived antioxidant represented by the following formula and having an inhibitory action on protein damage by peroxynitrite.

[0020]

Embedded image

Wherein R ₁ and R ₃ represent a methoxyl group and R ₂ represents a hydroxyl group, or R ₁ represents a hydrogen atom or a methoxyl group, and R ₂ represents a hydroxyl group;
And R ₃ represents a hydrogen atom, or R ₁ represents a hydroxyl group, R ₂ represents a methoxyl group, and R ₃ represents a hydrogen atom. )

That is, the substances represented by the above formula are summarized as shown in Table 2.

[0023]

[Table 2]

Seventh, a plant-derived antioxidant represented by the following formula and having an inhibitory action on protein damage by peroxynitrite.

[0025]

Embedded image

Wherein R ₁ and R ₃ represent a hydroxyl group and R ₂ represents a hydrogen atom, or R ₁ represents a hydroxyl group and R ₂ represents a methoxyl group, and R ₃ represents a carboxyl group. Represents a group.)

That is, the substances represented by the above formula are summarized as shown in Table 3.

[0028]

[Table 3]

Eighth, a method for producing a plant-derived antioxidant, which comprises producing a plant having an inhibitory effect on protein damage by peroxynitrite by extracting it with an aqueous solution of 10-99% alcohol. Ninth, a method for producing a corn-derived antioxidant, which comprises producing corn-derived gluten meal (GM) having a protein damage inhibitory action by peroxynitrite by extracting it with a 50 to 80% aqueous alcohol solution. Tenth, a method for producing an antioxidant derived from coffee beans, which comprises producing coffee beans having a protein-injury inhibitory action by peroxynitrite with a 60-99% alcohol aqueous solution. First
1. A method for producing a grape-derived antioxidant, which comprises producing a grape having a protein damage inhibitory action by peroxynitrite by extracting it with a 10 to 50% alcohol aqueous solution.

[0030]

Embodiment 1

Corn steep liquor (CS) produced as a by-product in the production of corn starch from corn
L) 100 g was diluted with a three-fold amount of saturated saline (300 ml), stirred with 100 ml of ethyl acetate, centrifuged (3500 rpm, 15 min.), And liquid-liquid distribution was repeated three times. Thus, an ethyl acetate extract was obtained.

The ethyl acetate extract was extracted with 100 ml of saturated sodium hydrogen carbonate solution, acidified with 1N hydrochloric acid, and then extracted three times with 100 ml of ethyl acetate to obtain a fraction soluble in ethyl acetate.

After the ethyl acetate-soluble fraction was washed lightly with water and saturated saline, anhydrous sodium sulfate was added thereto, followed by dehydration, filtration and evaporation under reduced pressure to obtain 280 mg of a crude extract. Was.

This crude extract was prepared by the method described in Osawa et al.
ric. Food. Chem. , 35, 808 (198
Using a rabbit erythrocyte membrane ghost developed by 7)) as an index, a silica gel column chromatograph (id 20 × 250 mm, eluen) is used according to a conventional method.
t; n-Hex. : EtOAc = 1: 2), preparative HPL
C (column Wakosil-II5C18 i.
d. 20 x 250 mm, eluent; 30% MeO
H, 0.1% TFA, flow; 5.0 ml / mi
n. , Detc; UV254 nm) to obtain 0.9 mg of the active substance.

The active substance was analyzed by ¹ H-NMR, EI-MS
When structural analysis was performed by instrumental analysis including
It was completely consistent with the spectrum of sinapinic acid.

In addition, from the crude extract obtained above,
Silica gel column chromatography, preparative TLC, preparative H
Purification, isolation, and structural analysis using a technique such as PLC revealed that the following substances were contained together with sinapic acid.

[0037]

Embedded image

The substances represented by the above formula are as shown in Table 1 above.

[0039]

Embedded image

The substances represented by the above formula are as shown in Table 2 above.

[0041]

Embedded image

The substances represented by the above formulas are as shown in Table 3 above.

[0043]

[Test Example 1]

For each substance purified in Example 1 above,
Similar to tocopherol and ascorbic acid, which have been conventionally used as antioxidants, whether or not they have a satisfactory effect on the prevention of food oxidation and the whitening effect of cosmetics, Osawa et al. (J. Agric. Food. Ch.
em. , 35, 808 (1987)), the antioxidant activity was determined by determining the oxidation inhibition rate using an antioxidant test using a rabbit erythrocyte membrane ghost.

Further, in order to examine whether or not each substance purified in Example 1 has a sufficient inhibitory effect on oxidative damage caused by active oxygen in a living body, Kato et al. (J. Agric. Food) Chem., 45,
3007 (1997))
A peroxynitrite injury inhibition test was performed on the protein using the above method.

(1) Antioxidant test

The antioxidant test using a rabbit erythrocyte membrane ghost is basically an antioxidant test using a rabbit erythrocyte membrane ghost developed by Osawa et al. (J. Agric. Food. C).
hem. , 35, 808 (1987)) using each substance and tocopherol for comparison as follows. First, 100 μl of a methanol solution (1.0 mg / ml) of a sample of each substance was added to 0.5 ml of erythrocyte membrane ghost obtained from stored rabbit blood, and the mixture was gently stirred, and then 24 mM t-butylylhyd.
Add 50 μl of droperoxide solution, and add
For 20 minutes. After the reaction, 2.0M
3 ml of a 1: 2 mixed solution of trichloroacetic acid-1.7M hydrochloric acid solution and 0.67% thiobarbituric acid was added, stirred, and heated for 15 minutes in boiling water to develop color. After ice cooling, 3
Centrifuge at 500 rpm, and discard the supernatant at 532 nm.
The oxidation inhibition rate was determined by measuring the absorbance at. The result is shown in FIG. According to FIG.
It was confirmed that among the substances obtained in the above, some of the substances showed stronger activity than tocopherol.

(2) Injury suppression test

The injury inhibition test was carried out using a peroxynitrite injury inhibition test (J. Agric. Food. Che) using ELISA developed by Kato et al.
m. , 45, 3007 (1997)). In the injury suppression test, collagen which plays a role of moisturizing and the like in the lower part of the skin as a substrate protein and LDL which is pointed out to be associated with arteriosclerosis were used, but there is no particular limitation as long as it is a protein. First, a 0.1 M phosphate buffer (pH 7.4) 90
Collagen was dissolved in μl to a final concentration of 0.5 mg / ml. Next, 10 μl of a DMSO solution of various antioxidants at an equal concentration (mol%) was added, and a final concentration of 1.0 m
M amount of peroxynitrite was reacted, and the generated nitrotyrosine site was measured using ELISA. As a result, as shown in FIG. 2, tocopherol, ascorbic acid, and BHA showed almost no activity, whereas the majority of the substances contained in the CSL showed remarkable activity. Similarly, when LDL 1.0 mg / ml, which has been similarly linked to arteriosclerosis, was used as a substrate, similar results were obtained as shown in FIG.

[0050]

Embodiment 2

Instead of using CSL in the first embodiment,
An antioxidant was produced under the same conditions except that gluten meal (GM) was used, except that the conditions for extraction were changed as follows. First, when an extract using water, a water-ethyl alcohol solution and hexane was evaluated using a rabbit erythrocyte membrane ghost, the most active was found in the 50-80%, preferably 50% ethyl alcohol aqueous solution extract. Was.
Therefore, 150 ml of 50, 70, and 80% ethyl alcohol was placed in a 500 ml Erlenmeyer flask with respect to 50 g of GM, extracted at room temperature for 2 hours using a shaker, and then filtered and the solvent was distilled off under reduced pressure. Was adjusted to 10 and 100 mg / ml using the extraction solvent. The obtained preparation was evaluated using a rabbit erythrocyte membrane ghost.
Strong activity was seen in the% and 70% EtOH extracts.

[0052]

[Table 4]

[0053]

Embodiment 3

The dried product of the GM 50% EtOH extract was
The solution was dissolved using 5% EtOH to a concentration of 50.0 mg / ml. A column (id 19 mm × 2) containing 2.0 ml of this solution and containing a synthetic adsorbent (HP20) in which the solvent was replaced with water was used.
Placed in 50mm), H ₂ O, 20 and 50% EtOH
Elution was performed sequentially at 300 ml each, and the weight and activity of each fraction were measured. As shown in Table 5, strong activity was observed in the 50% EtOH elution fraction.

[0055]

[Table 5]

[0056]

Embodiment 4

1.0 g of coffee extract cake is placed in a test tube with a lid, and 5.0 ml of each of five kinds of solvents of H ₂ O, 30%, 60%, 99% ethyl alcohol and n-hexane are added, and the mixture is shaken with a shaker. Extraction was performed at room temperature for 3 hours. The supernatant was used for evaluation using a rabbit erythrocyte membrane, and 2.0 ml of the supernatant was concentrated and dried to measure the weight. As a result, a strong activity was found in the 60% and 99% ethyl alcohol extracts. Admitted. Table 6 shows the results.

[0058]

[Table 6]

Then, 60, 70, 80, 9
When the activity per unit weight was compared using 0,99% ethyl alcohol, as shown in FIG.
Almost equally strong activity was seen in the 0% ethyl alcohol extract.

[0060]

Embodiment 5

The present inventors have also studied grape, which is a raw material of red wine, which has recently attracted attention as an antioxidant food material. That is, as a result of examining the activity of the grape extract, a strong activity was found in the 30-50% ethyl alcohol aqueous solution extract. Table 7 shows the results.

[0062]

[Table 7]

The same concentration of the GM 50% EtOH extract, coffee extract cake 80% EtOH extract, and grape 40% EtOH extract at the same concentration (1
0 mg / ml) was compared to tocopherol. The result is shown in FIG.

[0064]

【The invention's effect】

According to the antioxidant according to the present invention, as with tocopherol and ascorbic acid which have been conventionally used as antioxidants, the antioxidant has a satisfactory effect on the prevention of oxidation of foods and the like, It also has a sufficient inhibitory effect on oxidative damage caused by active oxygen.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of an antioxidant test.

FIG. 2 is a graph showing the results of an injury suppression test using collagen as a substrate.

FIG. 3 is a graph showing the results of an injury suppression test using LDL as a substrate.

FIG. 4 is a graph showing the activity when coffee extract cake is extracted with ethyl alcohol solvents at various concentrations.

FIG. 5 is a graph showing the inhibition rates of peroxynitrite injury suppression and rabbit erythrocyte membrane ghost injury suppression.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C09K 15/06 C09K 15/06 15/08 15/08 // A23L 3/3508 A23L 3/3508

Claims (11)

[Claims] 1. A plant-derived antioxidant which has an inhibitory action on protein damage by peroxynitrite. 2. An antioxidant derived from corn, which has an inhibitory effect on protein damage by peroxynitrite. 3. An antioxidant derived from coffee beans, which has an effect of inhibiting protein damage caused by peroxynitrite. 4. A grape-derived antioxidant having an inhibitory effect on protein damage by peroxynitrite. 5. A plant-derived antioxidant represented by the following formula and having an inhibitory action on protein damage by peroxynitrite. Embedded image (Wherein, R ₁ represents a hydrogen atom, a hydroxyl group or a methoxyl group, and R ₂ represents a hydroxyl group and R ₃ represents a hydrogen atom, or R ₁ represents a hydroxyl group, and R ₂ represents a methoxyl group. And R ₃ represents a hydrogen atom, or
R ₁ and R ₃ represent a methoxyl group, and R ₂ represents a hydroxyl group. ) 6. A plant-derived antioxidant represented by the following formula and having an inhibitory action on protein damage by peroxynitrite. Embedded image (Wherein R ₁ and R ₃ represent a methoxyl group and R ₂ represents a hydroxyl group, or R ₁ represents a hydrogen atom or a methoxyl group, and R ₂ represents a hydroxyl group, and R ₃ represents a hydrogen group. Represents an atom, or R ₁ represents a hydroxyl group and R ₂
Represents a methoxyl group, and R ₃ represents a hydrogen atom. ) 7. A plant-derived antioxidant represented by the following formula and having an inhibitory action on protein damage by peroxynitrite. Embedded image (Wherein R ₁ and R ₃ represent a hydroxyl group and R ₂ represents a hydrogen atom, or R ₁ represents a hydroxyl group and R ₂ represents a methoxyl group, and R ₃ represents a carboxyl group. .) 8. A method for producing a plant-derived antioxidant, which comprises producing a plant having an inhibitory action on protein damage by peroxynitrite by extracting the plant with a 10-99% aqueous alcohol solution. 9. A method for producing a corn-derived antioxidant, which comprises producing corn-derived gluten meal (GM) having a protein damage inhibitory action by peroxynitrite by extracting it with a 50-80% aqueous alcohol solution. 10. A method for producing an antioxidant derived from coffee beans, comprising extracting coffee beans having a protein damage inhibitory action by peroxynitrite with a 60-99% aqueous alcohol solution. 11. A method for producing a grape-derived antioxidant, which comprises producing a grape having a protein damage inhibitory action by peroxynitrite by extracting it with a 10 to 50% alcohol aqueous solution.