JPH1077472A - Natural type antioxidant and production thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antioxidant which not only shows a significant oxidation-resistant effect, but also is of high safety, and further provide a method which enables the production of the antioxidant on a large scale and with high efficiency. SOLUTION: This antioxidant comprises, as an active component, 4- deoxygadusol. This method for producing 4-deoxygadusol comprises culturing Pseudoalteromonas genus strain which is capable of converting amino acids analogous to mycosporin to 4-deoxygadusol in a medium containing amino acids analogous to mycosporin, to thereby accumulate 4-deoxygadusol in the medium, and separating thus produced 4-deoxygadusol from this culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a natural antioxidant, and more particularly to an antioxidant containing 4-deoxygadosol as an active ingredient. The present invention also relates to a method for producing the same using microorganisms.

[0002]

2. Description of the Related Art In the fields of foods and cosmetics, various antioxidants are used mainly for the purpose of improving storage stability or maintaining quality. Recently, tocopherol (vitamin E), ascorbic acid, erythorbic acid,
Natural product antioxidants such as polyphenols derived from various plants are widely used.

[0003] Antioxidants used in foods and cosmetics are preferable not only from the viewpoint of improving storage stability or maintaining quality as described above, but also from the viewpoint of promoting health. This is because the antioxidant has an effect of inactivating active oxygen, which causes aging, carcinogenesis, cardiovascular disease and the like. Especially,
It has been shown that active oxygen is likely to be generated in the environment and lifestyle of modern society, so by adding safe antioxidants to foods and cosmetics, the oxidation of biological components is suppressed and the health promotion effect is improved. Is expected to get.

[0004] Under the circumstances described above, a natural antioxidant having excellent antioxidant activity and high safety has been desired. Such an antioxidant is useful not only as a preservative for foods and cosmetics, but also in the field of health foods.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an antioxidant having a remarkable antioxidant effect and high safety. Another object of the present invention is to provide a method which enables the above-mentioned antioxidant to be produced in a large amount and efficiently.

[0006]

Means for Solving the Problems The present inventors have widely searched for a natural substance having an excellent antioxidant action, and as a result, it has been found that a known compound, 4-deoxygadosol, has a strong antioxidant activity. This has led to the present invention.

[0007] Pseudoaltomonas (Pseudoalt)
It has been found that a bacterium belonging to the genus eromonas) efficiently produces 4-deoxygadosol from a precursor of 4-deoxygadosol, and has established a production method according to the present invention.

[0008] That is, the antioxidant according to the present invention is characterized by containing 4-deoxygadosol as an active ingredient. Further, the method for producing an antioxidant according to the present invention comprises culturing a strain of the genus Pseudoalteromonas having the ability to convert a mycosporin-like amino acid to 4-deoxygadsol in a medium containing a mycosporin-like amino acid. And a step of accumulating 4-deoxygadosol in the medium, and a step of separating 4-deoxygadosol from the medium.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION

<Antioxidant> The main component of the antioxidant according to the present invention, namely, 4
-Deoxygadazole has been found in trace organisms in very limited living organisms such as eggs of soda bonito (Bulletin Societes Chimiques Belges, 89, 11).
01 (1980)). However, the biological function of this compound is not completely clear, and of course its antioxidant activity was not known. The antioxidant effect of 4-deoxygadazole was first discovered by the present inventors. Therefore, the antioxidants of the present invention are based on this finding.
-Use invention of deoxygadosol. 4-deoxygadosol is a compound represented by the following formula.

[0010]

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The chemical name of this compound is 1,5-dihydroxy-5-hydroxymethyl-2-methoxy-cyclohex-1-en-3-one (Biochemical Journal
nal, vol. 199, 741-747 (1981)). However, in the present specification, the common name of 4-deoxygadosol is used (Comparative Biochemistry and Physiology,
vol.80B, No.4, 755-759 (1985)). In addition, IUPAC
According to the nomenclature, the name of the compound is 3,5-dihydroxy-5-hydroxymethyl-2-methoxycyclohex-2-enone.

The above-mentioned 4-deoxygadosol has an excellent antioxidant action, as described in the examples below. Therefore, by adding it as an antioxidant to various products such as foods and cosmetics, the preservability of the product can be enhanced and the quality can be maintained. Further, since this compound is a natural product, there is no problem in terms of safety even when applied to products used directly for the human body such as food and cosmetics.

In addition, since 4-deoxygadosol is present in marine organisms, it can have antioxidant activity in vivo. As a result, when this is taken into the body, a health promotion effect by reducing active oxygen in the living body is expected. Therefore, when the antioxidant of the present invention is added to food, not only effects of improving storage stability and maintaining quality are obtained, but the food itself has utility as a health food. Similar utility is also expected when applied to cosmetics.

When 4-deoxygadosol is used as an antioxidant, an appropriate amount thereof may be added to a product such as food or cosmetics alone or in a mixture with an appropriate carrier. The amount used can be easily determined by those skilled in the art depending on the product to be applied.

<Production method of antioxidant> By the way, in order to industrially implement the antioxidant of the present invention, it is indispensable to produce a large amount and efficiently of 4-deoxygadosol which is an active ingredient. It is. In order to achieve this object, the inventors have developed a method using microorganisms.

As mentioned above, 4-deoxygadosol is found in marine organisms. Therefore, it has been noted that among marine microorganisms, there is a high possibility that there is a strain that efficiently produces 4-deoxygadosol.
In addition, the inventors focused on the fact that the structure of 4-deoxygadosol is similar to the structure of mycosporin-like amino acids.
From these two viewpoints, marine microorganisms having the ability to convert these mycosporin-like amino acids to 4-deoxygadsol were searched for by screening marine bacteria isolated from seawater in various places. As a result, they have found that bacteria of the genus Pseudoalteromonas have desired properties, and have completed the present invention.

Mycosporine-like amino acids are a general term for compounds having an aminocyclohexenone structure found in fungi and the like and compounds having an aminocyclohexenimine structure found in marine organisms, and include, for example, the following compound groups.

[0018]

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In the present invention, the mycosporine-like amino acid used in the microorganism medium is not particularly limited. Also, 4
-Any one containing a mycosporin-like amino acid which is a precursor of deoxygadulsol may be used. For example, an extract of a red algae such as a red alga, Akahaginnansou, Fudaraku, Kalexa, Aedes pine, Obakusa, Ezotsunomata, Tengusa and Nori can be mentioned. These red algae contain mycosporin-like amino acids such as sinorin, parisin, and porphyra-334. Therefore, when these red algae are immersed in a hydrated lower alcohol, an extract containing a mycosporin-like amino acid can be obtained. This is concentrated under reduced pressure and redissolved in seawater, which can be suitably used as a medium for culturing Pseudoalteromonas spp.

It has been found that the microorganism used for the production of 4-deoxygadosol is not particularly limited as long as it is a strain belonging to the genus Pseudoalteromonas. Examples include the AR0307 and FUO605 strains isolated from seawater by the present inventors, as well as Pseudoalteromonas haloplanktis : I
AM 12918) and Pseudoalteromonasatlantica (IAM 12927). Among them, AR0307 strain and FU06
The detailed microbiological properties of the 05 strain are as follows.

[FU0605] [AR0307] Gram staining negative negative form Bacillus bacillus pigmentation Red orange Not producing Motile Yes Yes Flagella polar flagella polar flagellum halophilic Viable area 0.5-10% 0.5-10% Optimal growth area 2.5- 4.0% 1.7 ~ 4.0% Na Requirement Requirement Requirement OF Test Oxidized Oxidized DNase Activity Positive Positive Gelatin Decomposition Positive Oxidase Activity Positive Positive Arginine Degradation Negative Negative STD Negative Negative Ornithine Degradation Negative Negative Lysine Degradation Negative Negative Gas production from glucose Negative Negative GC content 40.05% 41.35% Quinone analysis Q-8 type Q-8 According to the above mycological properties, strains AR0307 and FU
It was revealed that each of the 0605 strains belongs to the genus Pseudoalteromonas. However, as described above, the present invention is not limited to these two strains, and can be carried out with other strains of the genus Pseudoalteromonas.

In carrying out the production method according to the present invention,
For example, the above strain may be inoculated into a medium containing a mycosporin-like amino acid obtained from a red algae extract and cultured for a certain period of time. For the purpose of increasing production efficiency, various commonly used nutrient sources may be added.

When the AR0307 strain is cultured using a solution obtained by re-dissolving a red algae extract in seawater as a medium, usually 1
By culturing for ４4 days, the desired 4-deoxygadosol is accumulated in the culture solution. At that time, a part of the culture solution was taken out with time and subjected to high performance liquid chromatography (column: YMC-Pack.NH
₂ , 250 × 4.6mm ID, S-5 μm 120A, eluent: 40 mM ammonium acetate, 17.5 mM acetic acid / 80% methanol, flow rate:
1 ml / min, detector: Waters 991J photodiode array detector, detection wavelength: 200 to 400 n
m) can be used to determine the amount of 4-deoxygadosol and of the precursor mycosporin-like amino acid over time. In this analysis, the maximum wavelength of the precursor mycosporin-like amino acid is 320-335 nm and the elution time is about 10 minutes to about 15 minutes. On the other hand, since the maximum wavelength of 4-deoxygadazole is 294 nm and the elution time is about 8.5 minutes, the precursor and the target product are detected as clearly distinguishable peaks. Therefore, based on the measurement results of the high performance liquid chromatography analysis, the amount of 4-deoxygadosol reaches the maximum,
The culture may be stopped when the precursor has disappeared.

Next, 4-deoxygadosol is isolated from the above-mentioned culture using a conventional separation and purification method. For example, the cultured cells are removed by centrifugation or filtration, and several times the amount of methanol or ethanol is added to the obtained culture supernatant, and the mixture is concentrated under reduced pressure, and isolated and purified using various preparative chromatography. I do.

The above method has made it possible to obtain 4-deoxygadosol in large quantities and to have a detailed study of its properties and its biological role. As an example, the inventors have found that 4-deoxygadazole has very strong antioxidant activity and can prevent oxidation of lipids and biological membranes. That is, the antioxidant of the present invention described above has been made possible for the first time by the above production method. The production method of the present invention strongly backs up the further study of 4-deoxygadazole and is expected to make a great contribution to the pharmacology and biology of the compound.

Hereinafter, the present invention will be described in more detail with reference to Examples. Example 1: 4 by marine bacteria strain AR0307
-Production of deoxygadulsol 850 ml of ethanol was added to 250 g (wet weight, 85% moisture) of Asakusanori and immersed for several days to extract mycosporin-like amino acids. The obtained green extract was dried under reduced pressure, redissolved in 170 ml of seawater, and filtered to remove insolubles. Further, the pH is adjusted to 7.6 to 7.
After adjustment to about 8, the mixture was injected through a 0.45 μm sterile filter into an Erlenmeyer flask that had been autoclaved in advance.

In this flask, Zobell 2216
Only 850 μl of a culture solution of the AR0307 strain cultured in a liquid medium was added. While the culture was continued, a culture solution was collected over time and analyzed by high performance liquid chromatography. 82.5 hours after the start of the culture according to the present invention, the peak of Porphyra 334 disappeared, and the peak value of 294 nm indicating the amount of 4-deoxygadosol reached the maximum value. The entire amount was recovered.

After removing the cells by centrifuging the culture, ethanol three times the amount of the culture was added. After insoluble salts were removed by filtration, the mixture was concentrated under reduced pressure. This is 10
The residue was dissolved in ml of distilled water, and subjected to adsorption chromatography (35 mm × 360 mm) of Toyopearl HW-40S, and purified by elution with distilled water. 294 nm of the eluted fraction
The fractions having the maximum absorption were collected, concentrated three times the amount of ethanol, concentrated under reduced pressure, and redissolved in 5 ml of distilled water. Then, preparative high performance liquid chromatography (column: YMC D-ODS-5-ST, S-5 μm 120A,
Mobile phase: 2.5% methanol containing 0.2% acetic acid, flow rate: 4
(ml / min), and the operation of collecting peaks eluted at a retention time of about 20 minutes was repeated.

The procedure of adding three times the amount of ethanol to the obtained fraction and repeating concentration under reduced pressure by an evaporator was repeated three times, and it was confirmed that the sample had no acetic acid odor. By this operation, 18.8 mg of the target substance was obtained.

Example 2: Confirmation of the chemical structure of 4-deoxygadosol In order to confirm that the substance obtained in Example 1 was 4-deoxygadosol, a mass spectrum analysis was performed. . FIG. 1 shows the result. The result of this analysis revealed that the molecular weight was 188.

The substance obtained in the above example (Example product)
The ^13C -NMR analysis was performed, and the obtained chemical shift value was compared with the literature value of 4-deoxygadosol. As a result, the following results were obtained. Reference 1 in the following table refers to Tetrahedron Lette
rs) and reference 2 is Bulletin Societes Chimiques Belges
It is.

Chemical shift value of each carbon indicated by carbon No. (No. 1) (No. 2) (No. 3) (No. 4) (No. 5) (No. 6) (N0.7) ( No. 8) Reference 1 181.0 134.7 181.0 41.4 73.1 41.4 68.5 60.9 Reference 2 180.0 134.5 180.0 41.0 72.8 40.9 68.3 60.5 Example product 179.4 134.3 179.4 41.4 73.3 41.4 68.5 60.6 As described above, the chemical shift value of the example product was previously reported. It can be said that it almost matches the literature value. From these results, the substance having a molecular weight of 188 obtained in Example 1 was
It was confirmed to be 4-deoxygadsol.

In addition, FU06 was used instead of the AR0307 strain.
In the same manner as described above, it was confirmed that this was 4-deoxygadosol in the same manner as described above for the substance obtained by using the strain No. 05 and otherwise conducting the same procedure as in Example 1.

Example 3 Prevention of Lipid Oxidation by 4-Deoxygadosol The antioxidant activity of the purified 4-deoxygadosol obtained in Example 1 was measured using the oxidation of methyl linoleate as an index.

The amounts of each component added to the measuring system are as shown in the following table. Here, with respect to 4-deoxygadazole, its 10 mM ethanol solution was appropriately diluted and added as a sample solution. As a control when no sample solution was added, the same amount of ethanol as the sample solution was added. For the purpose of initiating and promoting the oxidation reaction of methyl linoleate, 2,2-azobis (2-amidinopropane) dihydrochloride (abbreviation: AAP) which is a radical generator
Note that H) was added.

<Component><Amount> 129 mM NaCl 560 μl 10 mM EDTA 10 μl 100 mM Triton X 80 μl Methyl linoleate 40 μl Sample solution 10 μl EtOH (control) 10 μl 200 mM AAPH 100 μl Total 800 μl The above components were added sequentially. Stir to form an emulsion. While shaking this emulsion in a thermostat at 37 ° C., 10 μl was taken out every 30 minutes, and the amount of peroxide (HPO: hydroperoxide) generated by oxidation of methyl linoleate was analyzed by high performance liquid chromatography. . FIG. 2 shows the results when the final concentrations of 4-deoxygadazole in the measurement system were 50 μM and 125 μM. In the figure, black filled squares indicate the results for the control solution using EtOH, Δ indicates the results for the sample solution (final concentration of 4-deoxygadsol 50 μM), and ○ indicates the sample solution (4-deoxygadazole). (Final concentration of Dosole 125 μM). The results indicated that 4-deoxygadazole significantly suppressed lipid oxidation.

For comparison, an aqueous solution of ascorbic acid was added to a final concentration of 125 μM instead of the 4-deoxygadazole sample solution, and the same analysis as above was performed. 2 in FIG. 2 shows the result of the comparative example using the ascorbic acid aqueous solution. From these results, it was found that the lipid oxidation inhibitory effect of 4-deoxygadazole was equivalent to that of ascorbic acid.

Example 4 Inhibition of Oxidation of Liposomal Membrane by 4-Deoxygadosol In general, cells are formed by a lipid bilayer composed of phospholipids. Thus, the effect of 4-deoxygadosol on its oxidation was examined using a liposome formed of phospholipid as a model of a biological membrane. As a method for this, Yamamoto et al. (Bulletin of the Chemical Society of Japan, 5
7, 1260 (1984); Analytical Biochemistry, 160, 7 (1
987)).

A 40 mg / ml methanol solution of the purified soybean phosphatidylcholine (phospholipid) was prepared. 0.4 ml of this solution was placed in an eggplant-shaped flask and dried at room temperature under reduced pressure. Then, 0.1 M NaCl containing 100 μM EDTA
4 ml of the aqueous solution was added, and the mixture was slowly stirred with a vortex mixer to form liposomes.

Each of the liposome emulsions was 1.5 ml
Each into two glass screw-lid test tubes, 100 mM A
APH was added in 15 μl portions. This was stirred in a water bath at 37 ° C. to start an oxidation reaction. While stirring in a water bath to continue the oxidation reaction, a part (30 μl) of the sample was taken out every 15 minutes,
The peroxides of the phospholipids generated by the oxidation reaction were analyzed. This analysis was performed by high performance liquid chromatography (column: silica LC-S, 4.6 × 250 mm ID, manufactured by Spelco, mobile phase: 40 mM NaH ₂ PO ₄ /90% methanol, flow rate:
1.0 ml / min). Ninety minutes after the start of the reaction, one test tube contained 10 mM of 4-deoxygadazole.
5 μl of the ethanol solution was added, and 5 μl of ethanol was added to the other test tube. Thereafter, the amount of peroxide (PC-OOH; phosphatidylcholine hydroperoxide) was analyzed while continuing the oxidation reaction.

FIG. 3 shows the results of the above-mentioned peroxide analysis.
The arrow in the figure indicates 10 mM of 4-deoxygadazole.
This shows the time when the ethanol solution was added. As is clear from the figure, it was found that the peroxidation reaction of the liposome membrane which had been progressed by AAPH was remarkably suppressed by 4-deoxygadosol. This indicates that in cell biological membranes, 4-deoxygadosol can stop the chain reaction of radical-induced lipid peroxidation.

[0042]

As described in detail above, according to the present invention,
It is possible to provide a safe natural antioxidant capable of preventing quality deterioration due to oxidation of foods, cosmetics and the like. Moreover,
Since 4-deoxygadosol, the active ingredient of the antioxidant of the present invention, suppresses the production of active oxygen and free radicals in cells when taken into the body, the antioxidant of the present invention was added. Foods are also useful as health foods. Also,
According to the present invention, it is possible to provide a method capable of producing a large amount of 4-deoxygadosol, which is essential for industrially implementing the antioxidant of the present invention.

[Brief description of the drawings]

FIG. 1 is a diagram showing a mass spectrum of a substance obtained by a production method of the present invention. FIG. 3A shows [M−H] ^−.
FIG. 3B shows a [M + H] ⁺ spectrum.

FIG. 2 is a graph showing the effect of 4-deoxygadosol on the oxidation of methyl linoleate.

FIG. 3 is a graph showing the effect of 4-deoxygadosol on the oxidation reaction of a liposome membrane.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:01) (72) Inventor Junhiro Yamamoto 3-34-38-107 Motomanuma, Suginami-ku, Tokyo (72) Inventor Yukio Karube 1-3-16 Higashi-Arima, Miyamae-ku, Kawasaki-shi, Kanagawa (72) Inventor Randy Magdalen Larsen Australia, 2617 AC, Evatt, Carlyle Street 117

Claims (2)

[Claims] 1. An antioxidant comprising 4-deoxygadosol as an active ingredient. 2. A strain of the genus Pseudo-Alteromonas having the ability to convert a mycosporin-like amino acid to 4-deoxygadosol is cultured in a medium containing a mycosporine-like amino acid, and A method for producing an antioxidant, comprising: a step of accumulating gadusol; and a step of separating 4-deoxygadusol from the medium.