JP2619491B2 - Astaxanthin-containing composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an antioxidant containing astaxanthin and / or its ester as a natural product as an active ingredient,
The present invention relates to a medicine for preventing oxidative tissue damage in a living body and an anti-inflammatory agent. An antioxidant containing astaxanthin and / or its ester as a natural active ingredient is mainly used as an antioxidant for foods, cosmetics, and pharmaceutically active substances due to its safety and antioxidant ability, as well as oxidative tissue damage in living bodies Can be used as a pharmaceutically active ingredient to protect against. In addition, anti-inflammatory agents containing astaxanthin and / or an ester thereof as a natural active ingredient can be used mainly as an anti-inflammatory component in functional foods, cosmetics, and pharmaceuticals due to their safety and strong anti-inflammatory effect. It is effective against various symptoms such as inflammation caused by vitamin E deficiency. Further, an anti-mutagenic agent containing astaxanthin and / or its ester as a natural active ingredient can be mainly used as an anti-mutagenic component in functional foods, cosmetics, and pharmaceuticals due to its safety and anti-mutagenic effect. .

[Conventional technology and problems]

Many antioxidants have been devised and manufactured to prevent the oxidation of foods, cosmetics, medicines, and fats and oils, but among these antioxidants, butylhydroxyanisole (hereinafter abbreviated as BHA) is mainly used. I have been. However, in recent years, due to safety issues, administrative measures have been taken to prohibit use in foods. Therefore, the development of a safe antioxidant having the same antioxidant capacity as BHA has been desired.

As an antioxidant that can be used in place of BHA, a natural product is most preferred in view of its safety and the like, and a method using tocopherol which is a natural antioxidant (for example, JP-A 61-28)
9835). As one of such attempts, a product which overcomes this drawback has been developed by converting an oil-in-water (O / W) emulsion of fat-soluble tocopherol, which is hardly dispersed in foods containing multi-water molecules. However, this product is inferior to BHA in terms of antioxidant power, and its use is greatly limited in that a large amount of product is required to exhibit a certain antioxidant power. Therefore, attempts have been made to add L-ascorbic acid, citric acid, gallic acid and the like as synergists (synergistic factors) to increase the antioxidant power by a synergistic effect with tocopherol, and in particular, tocopherol, L-ascorbic acid, and gallic Emulsion antioxidants containing acids have been developed, but they are still inferior to BHA in antioxidant power.

On the other hand, in the living body, active oxygen is produced enzymatically or non-enzymatically for various reasons. This active oxygen attacks biological membrane systems and causes dysfunction by peroxidizing the constituent polyunsaturated fatty acids. For example, hyperbaric oxygen therapy, high-concentration oxygen absorption, and resumption of circulation increase the production of active oxygen due to an increase in intracellular phosphorus dioxide, resulting in oxygen poisoning, retinopathy of prematurity, encephalopathy, and the like. Insufficiency of oxygen induces the emission of electrons from the electron transport system to produce active oxygen, and induces shock, myocardial infarction, cerebral infarction and the like. Drugs that stimulate the production of active oxygen, such as paraquat and adriamycin, also induce lung damage. Insufficiency of vitamins E and A also promotes intracellular production of active oxygen and causes various disease states. Furthermore, when active oxygen produced by polynuclear leukocytes and macrophages is released outside the cells, inflammation (acute and chronic) is induced as a typical pathological condition (the above-mentioned medical history, Vol. 129, p. 1214, 1984). I know that.

In addition, tocopherol has a number of beneficial effects in vivo as vitamin E, and even when used externally, it has the effect of alleviating inflammation and the like caused by trauma and burns, hastening its healing and preventing deep scars from remaining. Industrial applications are immeasurable if there is a safe compound that shows strong antioxidant ability and antimutagenic effect, and also has effects for internal use and external use.

[Means for solving the problem]

The present inventors have intensively searched for compounds having a high antioxidant activity and high safety from nature, and as a result, they were mainly used as additives to fish and livestock feed (for example, Japanese Patent Application Laid-Open No.
206342 or JP-A-60-54647) It has been discovered that astaxanthin and its esters, which are often used and are known as a kind of carotenoid, show very strong antioxidant activity. And it was confirmed that this antioxidant activity can be used not only as an antioxidant component for foods, cosmetics and pharmaceutically active substances, but also as a pharmaceutically active ingredient for preventing oxidative tissue damage in living organisms.

In addition, they have found that astaxanthin and its ester have an anti-inflammatory effect in a very small amount, and thus are effective as anti-inflammatory agents.

Accordingly, the present invention provides an antioxidant containing astaxanthin or an ester thereof or both thereof as an active ingredient, a medicament for preventing oxidative tissue damage in a living body, and an anti-inflammatory agent.

[Specific explanation]

According to the present invention, there are provided an antioxidant, an anti-inflammatory agent and an anti-mutagenic agent containing astaxanthin and / or an ester thereof as an active ingredient.

Astaxanthin and its esters are used in shrimp eggs (Ku
hn et al., Angew. Chem. 51 , 465 (1938) or Ber. 71 , 1879.
(1938)], organs of animals [Kuhn et al., Ber. 72 , 1688 (193)
9)], plants [Tischer et al., Z. Physiol. Chem. 267 , 281 (19
41), petals of Fukusou and Kimbo (Seybold et al., Nature 184 ,
1714, (1959)], red feathers of birds [Z. Physiol. Chem. 288 ,
20 (1951)] and its structure has been determined [Grangaud, Comt. Rend. 242 , 1767, (1956) or Andrew et al., Acta. Chem. Scand. B28 , 730 (1974)]. Synthetic methods have been established (Cooper et al., J. Chem. Soc. Perkin
Trans. I 1975 , 2195, Kienzl et al., Helv. Chim. Acta 61 , 2609.
(1978), Widmer et al., Helv. Chim. Acta 64 , 2405 (1981),
Mayer et al., Helv. Chim, Acta 64 , 2419 (1981)], and can be easily obtained as a chemically synthesized product. The active ingredient in the present invention may be chemically synthesized astaxanthin, or an extract from red yeast, tigliopath (red flea) or krill containing astaxanthin and its ester, particularly an organic solvent, preferably ethanol or acetone. It may be in the form of an extract, or may be used after being appropriately purified if necessary. For example,
Phaffia yeast (Phaffia rhodoz) as described below
yma Miller) is cultured in an appropriate medium, and the culture is separated and purified.

Astaxanthin esters include, for example, oleic acid esters, palmitic acid esters, stearic acid esters and the like.

The medium used for producing astaxanthin and its ester using Phaffia rhodozyma Miller may be liquid or solid. Usually, shaking culture or aeration and stirring culture using a liquid medium is convenient. The medium may be any medium as long as the red yeast grows and accumulates astaxanthin and its ester in the cells. That is, as a carbon source, for example, glucose, lactose, glycerin, starch, sucrose, dextrin, molasses, organic acids, etc., and as a nitrogen source, for example, peptone, protein hydrolyzate such as casamino acid, meat extract, yeast Extracts, soybean meal, corn steep liquor, amino acids, ammonium salts, nitrates and other various organic or inorganic nitrogen compounds are used.
As the inorganic salts, various phosphates, magnesium sulfate, and sodium chloride may be added, and vitamins, nucleic acid-related compounds, and the like may be added for the purpose of promoting the growth of bacteria. In some cases, the addition of an antifoaming agent such as silicon, a polypropylene glycol derivative, or soybean oil to the medium may be effective in increasing the accumulated amount of the substance of the present invention.

In culturing, it is desirable to inoculate a medium with a culture obtained by conducting a small-scale preculture in advance rather than suddenly performing main culture. Conditions such as culture temperature, culture period, and liquid properties of the culture are appropriately selected and adjusted so as to maximize the accumulation amount of the substance of the present invention, but in many cases, 15 ° C under aerobic conditions.
The culture can be carried out at ~ 27 ° C for 3-7 days.
Good to keep at ~ 9.5.

By culturing in this way, astaxanthin and its ester are produced and accumulated in the cells. In the case of culturing using a liquid medium, the target substance is accumulated mainly in the cell portion. Therefore, the culture is once collected by filtration or centrifugation, and then washed once or several times with water. The cells thus obtained are crushed using a physical method and then dried, or directly dried without crushing. Various means based on the chemical properties of astaxanthin and its esters are employed for the separation and purification of the target substance from the dried cells or their crushed products. That is, for example, n-
Examples include extraction with an organic solvent which is not arbitrarily mixed with water such as butanol and can dissolve the substance of the present invention, and dissolution in a highly polar solvent such as methanol, ethanol or acetone. Particularly preferred extraction methods are organic solvents,
For example, cell extraction using methanol, ethanol or acetone. The extraction solvent is usually used at a ratio of 3 to 5 times the weight of the dried cells or the crushed product, and it is sufficient to repeat the extraction by stirring at room temperature for half a day to one day two to three times. Then, the whole extract is concentrated under reduced pressure at 40 ° C. or lower and dried to obtain an oily crude extract containing astaxanthin and its ester. The content of astaxanthin and its ester in the crude extract varies depending on the extraction conditions used, but is usually about 5 to 10%. Furthermore, to purify the target substance from this crude extract, removal of impurities by treating with hexane etc. depending on the application, gel filtration with Sephadex, ion exchange resins, ion exchange cellulose, various ion exchangers such as ion exchange Sephadex Ion chromatography, alumina, silica gel, cyanopropyl, octadecyl silica gel, adsorption chromatography using an adsorbent such as Amberlite-XAD-1,2, etc. are effectively used, and these means should be used in an appropriate combination. By this, the substance of the present invention is isolated. However, any other method can be used as long as it effectively utilizes the properties of the substance of the present invention. Particularly preferred adsorbents include
Examples include Diaion HP-20, Sephadex LH-20, Cosmosil 10C18, silica gel for Volem dry column, Dupont Zorbax-CN, or Elma FRC-CN.

For use of the astaxanthin of the present invention as an antioxidant or as an anti-inflammatory agent, the above-mentioned crude extract or purified astaxanthin may be used. When a crude extract or purified astaxanthin is used, the active ingredient itself is oily, so that the active ingredient can be made into a floating agent or a compound that becomes a synergist can be made into a floating agent by a conventional method.

The antioxidant activity of astaxanthin is 200 times or more higher in any antioxidant test as compared to tocopherol at a molar concentration ratio.
It showed antioxidant power more than 10 times.

In addition, mice bred on a diet containing no vitamin E (tocopherol) show vitamin E deficiency in 8 weeks, but adding astaxanthin to a diet containing no vitamin E at 1/100 of the required amount of vitamin E results in 8 weeks Even afterwards, there were few symptoms of membrane function associated with vitamin E deficiency. In addition, astaxanthin also showed an effect of restoring mice showing vitamin E deficiency to a normal state.

 Next, the composition and the preparation of the present invention will be described.

The astaxanthin used as the active ingredient of the antioxidant in the present invention may be chemically synthesized astaxanthin or natural astaxanthin and its crude ester extract, and these can be used alone or in an appropriate combination. Astaxanthin and its crude extract can be directly used after dissolving in ethanol and diluting with water, but an emulsion can be prepared as needed. In preparing an emulsion preparation, gallic acid, L-ascorbic acid (or an ester or salt thereof), gum (eg, locust bean gum, guar gum, gelatin, etc.), and vitamin P (eg, hesperidin, rutin) are added to the aqueous phase. Flavonoids such as quercetin, catechin, thianidine, eriodictin or a mixture thereof), and astaxanthin or an astaxanthin-inhibiting extract or a mixture thereof in the oil phase, and glycerin fatty acid esters or fats such as seed oils, Soybean oil,
It can be easily prepared by adding and emulsifying a normal liquid oil such as corn oil. The emulsification may be performed by mixing and emulsifying using a high-speed stirrer, a homogenizer or the like.

Also, as the medicament of the present invention, solid dosage forms such as tablets and powders, or liquid dosage forms such as elixirs, syrups and suspensions are orally administered. It is also used parenterally, for example, as injections, ointments, and suppositories. As an active ingredient of these medicines, astaxanthin may be chemically synthesized astaxanthin or natural astaxanthin and its crude ester extract, and these can be used alone or in an appropriate combination. Auxiliaries for oral administration contained in the pharmaceutical composition include, for example, solid powder carriers, lactose, saccharose, dextrose, mannitol, sorbitol, cellulose, glycine and the like. Examples of the lubricant include silicon dioxide, talc, magnesium stearate, and polyethylene glycol. Examples of the binder include starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. Disintegrators include starch and agar. The dose of the active ingredient in the medicament of the present invention is usually 10 to 4000 mg, preferably 100 to 1000 mg orally, or 1 to 2000 mg, preferably 50 to 500 mg per day for adults. Administer parenteral doses. The dose is
It is clear that the conditions vary depending on the type of disease to be administered, the age, weight, degree of symptoms, and administration form of the patient.

 The toxicity of the active ingredient of the medicament of the present invention is extremely low.

Hereinafter, the present invention will be described in detail with reference to Examples and Reference Examples, but it goes without saying that the present invention is not limited to these.

Reference Example 1. Phaffia rhodozyma Miller
Production of astaxanthin and its crude ester extract by cultivation of yeast. Liquid synthetic medium consisting of 0.3% yeast extract, 0.5% polypeptone, and 1.0% glucose is dispensed into five Sakaguchi flasks each in 100 ml volumes, and is autoclaved at 120 ° C. for 20 minutes at 120 ° C. Heat sterilized. This is followed by Phaffia Rhodjima Miller shares (AT
CC-24201 strain) was inoculated with a loopful of pure platinum at 25 ° C, 7
The cells were cultured for 2 hours using a reciprocating shaker. Then 0.3% yeast extract,
A liquid synthetic medium 25 composed of 0.5% of polypeptone and 1.0% of glucose is placed in a 50 jar fermenter and placed in a 120 jar fermenter.
The solution was heat-sterilized in an autoclave at 20 ° C. for 20 minutes. This was inoculated with 500 ml of the preculture, and cultured at 25 ° C. for 48 hours with aeration and stirring at 12 / min. Thereafter, glucose for 1% was further added, and the cells were further cultured for 48 hours. About 500 g of cells by wet weight could be obtained from the obtained culture solution by centrifugation. After the cells thus obtained were dried by a freeze-drying method, 5-fold weight of acetone was added, and extraction was performed while mechanically crushing the cells in a mortar. The residue was removed by a filtration method, and the residue was further extracted by adding 500 ml of acetone, and this operation was repeated two to three times. The resulting red acetone extraction solution was concentrated under reduced pressure at 40 ° C. or lower to obtain a crude extract of astaxanthin and its ester.

The crude extract was first subjected to column chromatography using silica gel for a dry column as a carrier and 10% ethyl acetate-containing dichloromethane as a developing solvent to separate a red coloring pigment as a main component. In addition, this component can be used with Elma ERC
-Astaxanthin can be separated and purified by high-performance liquid chromatography using CN as a carrier and performing a gradient elution from hexane / dichloromethane (70:30) to hexane / dichloromethane / ethanol (70:30:30). did it. The yield was about 1 mg / g of Phaffia dry matter. FIG. 1 shows the elution pattern of astaxanthin in high performance liquid chromatography.

Example 1. Prevention of oxidative tissue damage in living body (1) Inhibitory effect of astaxanthin on lipid peroxidation reaction of normal rat erythrocytes caused by dialial acid and ferric iron (antioxidant effect) Dialial acid and ferrous iron cause How astaxanthin prevented the peroxidation of normal rat erythrocytes was measured as follows.

First, erythrocytes of normal rats were collected and analyzed by KRP (Krebs
After washing three times with a buffer solution (Ringer / phosphate), an appropriate amount of astaxanthin was added, or an aqueous solution of a ferric salt (100 μM) and an aqueous solution of dialial acid (1 mM) were added to the final concentration, respectively. . In this state, a lipid peroxidation reaction was performed at 37 ° C. for 20 minutes. A 40% aqueous solution of trichloroacetic acid (0.5 ml) was added to the reaction solution (2 ml), 5N hydrochloric acid (0.25 ml),
And 2% thiobarbituric acid aqueous solution (0.5 ml)
Boil at ℃ for 15 minutes, TBA reaction (thiobarbituric acid reaction)
Was done. Centrifuge the reaction solution (3000 rpm, 10 minutes),
The supernatant was assayed for lipid peroxide at an absorbance of 535 nm to determine the inhibitory effect of astaxanthin on lipid peroxidation (see
Figure) was examined. Astaxanthin inhibited lipid peroxidation by 50% or more at a low concentration of 2 μM or less.

Example 2. Prevention of oxidative tissue damage in living body (2) Inhibitory effect of astaxanthin on lipid peroxidation reaction of mitochondria in normal rat liver induced by ferrous iron (Mohr salt) (antioxidant effect) Divalent iron (Mohr salt) ) Caused by peroxidation of mitochondria in normal rat liver was measured as follows.

First, liver mitochondria of normal rats were separated by the method of Hodziboom et al., And washed three times with a buffer solution of potassium chloride (150 mM) -tris-hydrochloric acid (10 mM) (pH 7.4) (8000 g, 10 mM).
Minutes). Next, 2 mg / ml protein mitochondria with or without an appropriate amount of astaxanthin added to potassium chloride (150 mM) -tris-hydrochloric acid (10 mM) (pH 7.4) buffer was added to adjust the final ferric iron concentration to 50 μM. (Mall salt)
Was added. In this state, a lipid peroxidation reaction was performed at 37 ° C. for 60 minutes. To this reaction solution (2 ml), a 40% aqueous solution of trichloroacetic acid (0.5 ml), 5N hydrochloric acid (0.25 ml), and a 2% aqueous solution of thiobarbituric acid (0.5 ml) were added, and the mixture was boiled at 100 ° C. for 15 minutes. Uric acid reaction). The reaction solution was centrifuged (3000 rpm, 10 minutes), the supernatant was quantified for lipid peroxide by absorbance at 535 nm, and the inhibitory effect of astaxanthin on the lipid peroxidation reaction was examined (FIG. 3). Astaxanthin inhibited lipid peroxidation by 50% or more at a low concentration of 400 mM or less.

Example 3 Protection of Oxidative Tissue Damage in Living Body (3) Comparison of Inhibitory Effect of Astaxanthin and Vitamin E on Lipid Peroxidation of Normal Rat Liver Mitochondria Induced by Ferrous Iron (Mole's Salt) (Antioxidant Action and Vitamin E) E Inhibitory Effect) The inhibitory effect of ferrous iron (Mohr's salt) on the peroxidation of mitochondria in normal rat liver caused by astaxanthin and vitamin E was compared as follows.

First, liver mitochondria of normal rats were separated by the method of Hodziboom et al., And washed three times with a buffer solution of potassium chloride (150 mM) -tris-hydrochloric acid (10 mM) (pH 7.4) (8000 g, 10 mM).
Minutes). Next, to a potassium chloride (150 mM) -tris-hydrochloric acid (10 mM) (pH 7.4) buffer, 2 mg / ml protein mitochondria with or without the addition of an appropriate amount of astaxanthin or vitamin E was added to a final concentration of 100 μM. Was added with ferrous iron (mole salt). In this state, a lipid peroxidation reaction was performed at 37 ° C. for 60 minutes. 40% in this reaction solution (2 ml)
Trichloroacetic acid aqueous solution (0.5ml), 5N hydrochloric acid (0.25m
l) and a 2% aqueous solution of thiobarbituric acid (0.5 ml) were added and boiled at 100 ° C for 15 minutes to give TBA (thiobarbituric acid reaction).
The reaction was performed. The reaction solution was centrifuged (3000 rpm, 10 minutes), and the supernatant was examined for residual lipid peroxide (FIG. 4) at an absorbance of 535 nm. Compared with vitamin E, astaxanthin inhibited the peroxidation of mitochondrial lipids in rat liver at a low concentration of about 1/1000 or more.

Example 4. Protection of oxidative tissue damage in living body (4) Superoxide anion radicals in erythrocyte ghosts of vitamin E-deficient rats and rats fed vitamin E-deficient but supplemented with astaxanthin
Comparison of lipid peroxidation reaction by ferric iron (antioxidant effect and vitamin E substitute effect) Vitamin E deficient and astaxanthin substitute rat are vitamin E deficient feed and vitamin E deficient astaxanthin substitute feed (feed containing 4 mg / 160 g astaxanthin) Red blood cells were collected using each of them for 8 weeks,
Wash 3 times with calcium-free KPP buffer (1300rpm,
10 minutes). This was added to a 5 mM phosphate buffer (pH 8.0) at 0 ° C30.
The cells were lysed for 1 minute, centrifuged (16000 rpm, 10 minutes), and a ghost (Dodge method) was obtained as a sediment. Next, Tris
Trivalent iron (50 μM) and xanthine (200 μM) were added to a hydrochloric acid buffer (20 mM, pH 7.5) to each concentration, and xanthine oxidase (Boehringer diluted 16 times, 10 μ / ml), and Ghost (1mg / m
l) was added and reacted at 37 ° C. This reaction is a peroxidation reaction of [superoxide anion radical-trivalent iron] with a hydroxy radical, and is a superoxide anion radical-dependent reaction. 40 ml of this reaction solution (2 ml)
% Trichloroacetic acid aqueous solution (0.5ml), 5N hydrochloric acid (0.25m
l) and a 2% aqueous solution of thiobarbituric acid (0.5 ml) were added and boiled at 100 ° C for 15 minutes to give TBA (thiobarbituric acid reaction).
Was done. Centrifuge the reaction solution (3000 rpm, 10 minutes),
The supernatant was quantified for lipid peroxide (malondialdehyde) at an absorbance of 535 nm, and its reaction time dependence (FIG. 5) was examined. As a result, a strong peroxidation reaction was observed in the vitamin E-deficient rats, despite that no peroxidation reaction was observed in the ghost of normal rats. Also vitamin E
Despite the deficiency, it was found that the amount of lipid peroxide generated was significantly suppressed in rats to which astaxanthin was administered.

Example 5. Prevention of oxidative tissue damage (5) Lipid peroxidation reaction by ferrous iron in rat mitochondria deficient in vitamin E deficient rat and accompanying reduction in respiratory control ability, and the effect of astaxanthin on it (antioxidant action and vitamin E (Substitution effect) Vitamin E-deficient rats were bred on a vitamin E-deficient diet for 8 weeks, liver mitochondria were separated by the method of Hodziboom et al., And potassium chloride (150 mM) -magnesium chloride (3 mM) -phosphate buffer (5 mM, (pH 7.4) three times. The liver mitochondria were reacted at 25 ° C. in the same buffer as described above with or without the addition of an appropriate concentration of ferrous iron (Mohr's salt). Thereafter, succinic acid and adenosine diphosphate were sequentially added, and oxygen consumption was measured with an oximeter. RCI (respiratory control ability) was determined from the time-dependent ratio of oxygen consumption after the addition of succinic acid (state 4) and after the addition of adenosine diphosphate (state 3) (state 3 / state 4: chance state). Further, the inhibitory effect of astaxanthin on the above-mentioned peroxidation reaction was determined by adding astaxanthin before adding ferrous iron and measuring oxygen consumption by the same method to determine RCI.

As a result, rat mitochondria to which no ferrous ion was added showed RCI = 4.00, but their respiratory control ability decreased as the ferrous ion concentration increased (Table 1). At this time, the consumption of oxygen was observed by the addition of ferrous ion, but this was not due to respiration but due to a peroxidation reaction, and the consumption of oxygen was dependent on the concentration of ferrous ion. In addition, the effect of astaxanthin on the above-mentioned peroxidation reaction is that if astaxanthin is added before the addition of ferrous ion, oxygen consumption by ferrous iron is inhibited depending on the concentration of astaxanthin added, and the reduced RCI also recovers (Table 2).

Example 6 Inhibitory Effect of Astaxanthin on Carrageenan-Induced Foot Edema [Anti-inflammatory Effect] Under ether anesthesia, Wistar male rats (200 g) received 0.2 ml of physiological saline in the left leg and 10 mg / ml of carrageenin in the right leg. The same amount of physiological saline was administered subcutaneously to cause carrageenan-induced paw edema, and the change in paw volume was measured over time. The control group received 1 ml of 1% carboxymethylcellulose (CMC) solution, and the experimental group received 1 mg of α-tocopherol or the same amount of CMC solution containing 1 mg of astaxanthin.
The measurement was performed using a rat administered intraperitoneally 30 minutes before.

As a result, α-tocopherol showed the same results as the control, and no inhibitory effect on carrageenin-induced foot edema was observed, whereas astaxanthin showed a significant inhibitory effect on foot edema (FIG. 6). .

Example 7 Antioxidant Effect Inhibitory Effect of Astaxanthin on Generation of Singlet Oxygen by Photoreaction of Methylene Blue (Antioxidant Effect) In a test tube, one of the dyes, methylene blue (1.0
μg) as a singlet oxygen source, an ethanol solution (1.0 ml) containing 1.0% (volume ratio) linoleic acid was dissolved in 1.0 ml of water, and various carotenoids were added to each (100 μl).
g) Added. Irradiate this solution with 1800 lux white light,
Over time, the peroxidation of linoleic acid by singlet oxygen generated by the photoreaction of methylene blue was quantified as follows. A 20% aqueous solution of trichloroacetic acid was added to the reaction solution (2 ml),
And 0.67% thiobarbituric acid aqueous solution (0.5 ml) to add 1
The mixture was boiled at 00 ° C. for 15 minutes to perform a TBA reaction (thiobarbituric acid reaction). After the reaction, the mixture is washed with ether, and the aqueous layer is separated by 530 n.
The inhibitory effect of astaxanthin on lipid peroxidation was compared with that of control, α-tocopherol, diester of astaxanthin and the like (FIG. 8).

As a result, in the system to which astaxanthin was added, the generation of lipid peroxide was inhibited even when the light irradiation was continued for a long time, whereas in the system to which α-tocopherol was added, the generation of lipid peroxide with time was similar to the control. It was observed to occur. The same applies to the diester of astaxanthin.

Reference Example 2. Antimutagenicity test of astaxanthin (antimutagenic effect accompanying antioxidant action) The antimutagenic effect of astaxanthin was examined by Ames / Salmonella test (preincubation method).
The strain used was Salmonella typhimurium.
la · typhimurium) TA102 strain, which is highly sensitive to mutagens that produce free radicals such as bleomycin and aldehydes. Mitomycin C (MMC) was used as a positive control. Dimethyl sulfoxide (DMSO) was used as a solvent control. Samples were of two types: astaxanthin alone and astaxanthin plus MMC. For each sample, the concentration of astaxanthin was set to a maximum of 5 mg / plate, and five concentrations were examined.

First, a DMSO solution of astaxanthin is dispensed into a sterile small test tube (100 μm for a sample containing only astaxanthin, and 50 μm for the other sample). For samples other than astaxanthin alone, add MMC to a final amount of 0.5 μ / plate.
μ was added. Next, 0.5 ml of a sodium-phosphate buffer (pH 7.4) was added to each. 0.1 ml of pre-cultured bacterial suspension
Was added. Pre-incubate them with shaking at 37 ° C for 20 minutes in a shaking culture high temperature bath, add 2.5 ml of soft tangerine, mix carefully not to form bubbles, pour on minimal glucose agar medium, and uniformly spread on a plate. Spread out. This is 37
After incubation at 2 ° C. for 2 days, the presence or absence of growth inhibition of the test strain on the plate was examined using a stereoscopic microscope, and the number of colonies generated by reversion (FIG. 7) was counted.

As a result, no mutagenicity was observed in astaxanthin itself. However, anti-mutagenicity against MMC suppressed mutagenicity by 34.5% at the maximum concentration of 5 mg / plate, confirming its effect.

Formulation Example 1. Antioxidant or topical agent Oil phase (wt%) Astaxanthin 1.0% Rapeseed oil 39.0% Glyceride succinate 2.0% Water phase L-ascorbic acid 2.0% Gallic acid 1.0% Quercetin 1.0% Locust bean gum 0.1% Water 53.9% Water containing locust bean gum dissolved therein is heated to 65 ° C, and then gallic acid, L-ascorbic acid and quercetin are mixed, and the oil phase previously mixed and dissolved at 65 ° C is mixed.
After stirring, the mixture was passed through a homogenizer, homogenized, and cooled to 10 ° C. to obtain an emulsion preparation having the above composition.

Formulation Example 2. Antioxidant or topical agent Oil phase (% by weight) Astaxanthin 1.0% Rapeseed oil 38.0% Citric acid monoglyceride 2.0% Water phase L-ascorbic acid 2.0% Gallic acid 1.0% Hesperidin 1.0% Locust bean gum 0.05% Water Heat the water in which 54.95% locust bean gum is dissolved to 65 ° C, then mix gallic acid, L-ascorbic acid and hesperidin, mix the oil phase previously mixed and dissolved at 65 ° C, mix and stir After passing through a homogenizer, the mixture was homogenized and cooled to 10 ° C. to obtain an emulsion having the above composition.

Formulation Example 3. Tablets and capsules (% by weight) Astaxanthin 10% Lactose 75% Heavy magnesium oxide 15% were uniformly mixed to give tablets or capsules.

Formulation Example 4. Powders and granules (% by weight) Astaxanthin 45% Starch 15% Lactose 40% were uniformly mixed to give powders and granules.

Formulation Example 5. Injection (wt%) Astaxanthin 1% Solubilizer 5% Physiological saline 94% was heated and mixed, and sterilized to give an injection.

Formulation Example 6 Ointment Astaxanthin 10 g White Vaseline qs Amount of fragrance qs 100 g [Effect of the Invention] Astaxanthin represented by the present invention and its formulation have a low concentration of lipid peroxidation due to various oxidation conditions as described in Examples. Food, cosmetics,
And an effect as an antioxidant for pharmaceuticals and the like can be expected.
In addition, since it has an inhibitory effect on inflammation such as tissue destruction, tissue damage, mutagenicity or drug-induced edema due to various oxidizing conditions in biological systems, it is used as a composition for functional foods, cosmetics, pharmaceuticals, etc. The law can be expected. Furthermore, astaxanthin and its preparations are vitamin E
Prevents various symptoms caused by deficiency,
Alternatively, it can be expected to be used as a substitute for vitamin E because of its relaxing effect. In addition, since it is recognized that various phenomena derived from peroxidation reactions which could not be prevented by vitamin E were prevented, a new method of using the material as a composition for functional foods, cosmetics, pharmaceuticals, and the like can be expected.

[Brief description of the drawings]

FIG. 1 shows an elution pattern of astaxanthin by high performance liquid chromatography. FIG. 2 shows the lipid peroxidation reaction of normal rat erythrocytes by dialial acid and iron (III), and the inhibitory effect of astaxanthin on it. FIG. 3 shows the lipid peroxidation reaction of normal rat liver mitochondria by ferrous iron and the inhibitory effect of astaxanthin on it. FIG. 4 shows a comparison of the lipid peroxidation reaction of normal rat liver mitochondria with ferrous iron and the inhibitory effect thereof against astaxanthin and vitamin E. FIG. 5 shows a comparison of lipid peroxidation by superoxide anion radicals and ferric iron in erythrocyte ghosts of rats fed a vitamin E-deficient diet and a diet supplemented with vitamin E-deficient diet and astaxanthin. . FIG. 6 compares the inhibitory effect of astaxanthin on carrageenin-induced foot edema with α-tocopherol. FIG. 7 shows how astaxanthin suppresses the mutagenicity induced by mitomycin C in a mutagenicity test using Salmonella. FIG. 8 compares the inhibitory effect of astaxanthin on the generation of singlet oxygen by the photoreaction of methylene blue with α-tocopherol and astaxanthin diester.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location A61K 31/12 ADS A61K 31/12 ADS

Claims (3)

(57) [Claims] An antioxidant comprising astaxanthin or an ester thereof or both as an active ingredient. 2. A medicament comprising astaxanthin or an ester thereof or both thereof as an active ingredient for preventing oxidative tissue damage in a living body. 3. An anti-inflammatory agent comprising astaxanthin or its ester or both as an active ingredient.