JP2015040230A - Antioxidant composition and cosmetic containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antioxidant composition which has higher stability than ascorbic acid/tocopherol-based antioxidants, can exhibit an excellent antioxidant effect equal to or higher than that of the ascorbic acid/tocopherol-based antioxidants and is suitable for the recovery or improvement of various inflammations or diseases, and to provide a cosmetic preparation containing the same.SOLUTION: There are provided: an antioxidant composition composed mainly of a mixture of flavonoids and gallic acid; and a cosmetic preparation containing the antioxidant composition.

Description

  The present invention relates to an antioxidant composition and a cosmetic preparation containing the same, and more particularly to an antioxidant composition suitable for the recovery and improvement of various inflammations and diseases and a cosmetic preparation containing the same.

In recent years, the amount of ultraviolet rays falling on the ground due to the decrease in the ozone layer has been increasing year by year, and there is an increasing interest in ultraviolet ray damage. Various reactive oxygen species (ROS) are generated in the skin that is directly affected by ultraviolet rays, and these are considered to be involved in skin inflammation, disease, and aging. The deep ROS associated with skin disorders, superoxide anion radicals _{(O 2} ^- ·), hydroxyl radical (· OH), hydrogen peroxide _(H 2 _{O 2),} singlet oxygen ⁽¹ _{O 2)} is known In addition, lipid radicals (see Non-Patent Document 1) have been reported. ROS generated in the living body are antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase, catalase and thioredoxin reductase, antioxidants such as glutathione, thioredoxin and vitamins, or ceruloplasmin, transferrin, ferritin, It is usually quickly cleared by various proteins having antioxidative action such as albumin.

However, out of balance and erase generation of ROS in vivo, at the oxidative stress conditions, superoxide anion radical _- hydroxy radical derived from ^{(O 2 ·) (· OH} ) and hydrogen peroxide (H ₂ O ₂ More reactive radical species such as) and singlet oxygen ( ¹ O ₂ ) are produced excessively, causing not only serious diseases such as cancer, but also the aforementioned skin inflammations, diseases and aging. Therefore, the development of an excellent antioxidant composition has been strongly demanded in the industrial field using external preparations such as cosmetics and pharmaceuticals, and many antioxidants have been developed and reported. For example, dibutylhydroxytoluene (BHT), butylhydroxyanisole (BHA), ascorbic acid (vitamin C), tocopherol (vitamin E) and the like are widely used.

H. Masaki and H. Sakurarai, J. Dermatol. Sci., 14,207-216 (1997) E. Niki, Annals New York Academy of Science, 498, 186-199 (1987)

  Among the above antioxidants, BHT and BHA, which are one of the synthetic antioxidants, are recognized as mutagenic in BHT and carcinogenic in BHA. The safety to living bodies including the skin has been questioned, and there is a problem that there is a limit to the amount to be added in the Pharmaceutical Affairs Law.

  Among the above-mentioned antioxidants, ascorbic acid and tocopherol, which are the other naturally occurring antioxidants, for example, tocopherol, which is an important antioxidant component of biological membranes, has a synergistic effect with ascorbic acid (so-called sparing action). ), It is reported that a high and sustained antioxidant effect is realized (see Non-Patent Document 2), and exhibits a high safety and an excellent antioxidant effect. However, in these naturally-occurring antioxidants, for example, in the case of cosmetics or pharmaceutical preparations, ascorbic acid is extremely vulnerable to heat and oxidation in a coexisting system with water, and is susceptible to inactivation and decomposition. There was a problem of poor stability.

  Therefore, in reality, it is extremely difficult to design a cosmetic preparation having an antioxidant composition that is highly safe and stable.

  Under such circumstances, there is a strong demand for an antioxidant composition that is a naturally-occurring antioxidant and has a high safety and at the same time an excellent anti-oxidant effect, such as an ascorbic acid / tocopherol system.

  The present invention has been made in view of these points, and unlike the ascorbic acid / tocopherol system, the present invention has high stability, and can exhibit an antioxidant effect equivalent to or better than those, and various inflammations. An object of the present invention is to provide an antioxidant composition suitable for recovery and improvement of diseases and cosmetic preparations containing the same.

In order to solve the above problems, the present inventors have obtained an antioxidant composition that is highly stable and can exhibit an excellent antioxidant effect equivalent to or higher than that of an ascorbic acid / tocopherol system. intensive result of extensive research, superoxide in petal extracts of Western NatsuYukikusa (Filipendula Ulmaria Maximowicz), which is a kind of European production herbs anion radicals (O ₂ ^- ·), hydroxyl radical (· OH), hydrogen peroxide ( It has been found that there is an excellent erasing activity of H ₂ O ₂ ) and singlet oxygen ( ¹ O ₂ ).

  The petal extract of Western summer snow grass was obtained as follows. A petal of 5 kg of dried Western summer snow grass was pulverized with a blender, and 5 kg of 50% aqueous ethanol was added to the petal, followed by heating and extraction while refluxing. The filtrate obtained by filtering the extract was concentrated under reduced pressure to obtain a petal extract. As a result of analyzing the petal extract by high performance liquid chromatography (HPLC) and gas chromatography (GC-MS), the main component of the fraction having the above excellent antioxidant effect is flavonoid quercetin (hereinafter referred to as Quercetin). It was found that it was “QT” as required) and gallic acid of polyphenols (hereinafter referred to as “GA” if necessary). Therefore, it was considered that an antioxidant composition capable of exhibiting an excellent antioxidant effect can be obtained by combining various naturally derived flavonoids with GA, and further studies were made to solve the problem. As a result, it was found that the mixed system of QT and GA, and the mixed system of myricetin (hereinafter referred to as “MT” if necessary) and GA have extremely excellent antioxidant effect.

  More specifically, QT is a kind of flavonoid and is contained in many plants including citrus fruits, onions and buckwheat in a glycoside (rutin, quercitrin, etc.) or in a free form. QT is a part of a vitamin-like substance (vitamin P) together with rutin and hesperidin, and its glycoside is known to exhibit various pharmacological actions and is also known to exhibit a strong antioxidant action. .

  Furthermore, MT is a kind of flavonoid and is contained in many plants such as grapes, berries, fruits, vegetables and herbs. MT studies have shown that there is a correlation between high myricetin intake and a reduction in the incidence of prostate cancer, and it is also known to have high antioxidant activity.

  GA is distributed in the leaves, stems, roots, gallic and pentaploids of plants, and is distributed as a constituent component of tannin, and is known to exhibit an antioxidant action as a kind of polyphenol.

  Thus, it is known that QT and MT each independently exhibit an antioxidant effect, but the levels thereof are other flavonoids (isoflavone, galangin, fisetin, kaempferol, rutin, taxifolin, catechin, epigallocatechin). Compared to luteolin, etc.), the number of OH groups bonded to each other is considered to be excellent in terms of structure, but the level was not sufficient. Moreover, although it is known that GA also exhibits an antioxidant action alone, its level is lower than that of flavonoids.

  However, as a result of the diligent research by the present inventors, in a mixed system of flavonoids and gallic acid, specifically, a mixed system of QT and GA, and a mixed system of MT and GA, each of QT, MT, and GA is independent. It was found that the level was significantly increased compared to the antioxidant capacity of, and that it was possible to construct an antioxidant system that has never existed before. In addition, these mixed systems, unlike ascorbic acid / tocopherol-based ascorbic acid, are all chemically stable in an aqueous system and have also been found to have an excellent level of antioxidant ability. Therefore, by using a mixed system of flavonoids and gallic acid, specifically, a mixed system of QT and GA, a mixed system of MT and GA, each flavonoid alone, each polyphenol alone, and further ascorbic acid / tocopherol It was possible to obtain an antioxidant composition having an antioxidant ability superior to that of the system and having excellent stability and safety, thereby completing the present invention.

Further, by utilizing the antioxidant composition of the present invention, superoxide anion radical (O ₂ ⁻ ·), hydroxy radical (· OH), hydrogen peroxide (H ₂ O ₂ ), singlet oxygen ( ¹ O ₂ It was possible to provide a cosmetic preparation excellent in erasing activity), and the present invention was completed.

  The antioxidant composition of the first aspect of the present invention thus completed is characterized by comprising a mixture of flavonoids and gallic acid as a main component.

  By using a mixed system of flavonoids and gallic acid, which are naturally derived substances, with the antioxidant composition of the first aspect, each flavonoid alone, each polyphenol alone, and further, ascorbic acid / tocopherol system It is possible to provide an antioxidant composition that can exhibit a superior antioxidant effect and is excellent in stability and safety.

  The antioxidant composition according to the second aspect of the present invention is characterized in that, in the first aspect, the flavonoid is composed of at least one of quercetin and myricetin.

  With the antioxidant composition of the second aspect, quercetin and myricetin, which are particularly structurally excellent in flavonoids, are used, so that a more excellent antioxidant effect can be exhibited.

  The antioxidant composition according to the third aspect of the present invention is the antioxidant composition according to the first or second aspect, wherein the flavonoids and gallic acid are in a weight ratio of 4/1 to 1/4, preferably 3/1. 1/3.

  With the antioxidant composition of the third aspect, it is possible to provide an antioxidant composition that exhibits an excellent antioxidant effect by selecting various mixing ratios of flavonoids and gallic acid.

  Furthermore, the cosmetic preparation according to the first aspect of the present invention is characterized by including the antioxidant composition according to any one of the first to third aspects.

The cosmetic preparation according to the first aspect has a scavenging activity of superoxide anion radical (O ₂ ⁻ ·), hydroxy radical (· OH), hydrogen peroxide (H ₂ O ₂ ), and singlet oxygen ( ¹ O ₂ ). An excellent cosmetic preparation can be provided.

  The cosmetic preparation according to the second aspect of the present invention is the cosmetic preparation according to the first aspect, wherein the antioxidant composition is 0.000001 wt% to 5.0 wt%, preferably 0.00001 wt% to 2 wt%, more preferably 0. .0001 wt% to 0.5 wt%.

The cosmetic preparation of this second aspect allows superoxide anion radical (O ₂ ⁻ ·), hydroxy radical (· OH), hydrogen peroxide (H ₂ O ₂ ), singlet by addition of a small amount of the antioxidant composition of the present invention. It is possible to provide a cosmetic preparation excellent in elimination activity of term oxygen ( ¹ O ₂ ).

  Thus, since the present invention is constructed and acts, unlike the ascorbic acid / tocopherol system, it is highly stable and can exhibit an excellent antioxidant effect equivalent to or higher than those, and various inflammations It is possible to provide an antioxidant composition suitable for recovery and improvement of diseases and cosmetic preparations containing the same.

  Hereinafter, embodiments of the present invention will be described in detail.

<Antioxidant composition>
First, the antioxidant composition of the present invention will be described.

  The antioxidant composition of the present invention mainly comprises a mixture of flavonoids and gallic acid.

  In addition to these main components, the antioxidant composition of the present invention can contain components that promote antioxidants and other components that cannot be avoided.

  The flavonoids that can be used in the present invention are naturally derived substances, and may be selected from quercetin, myricetin, isoflavone, galangin, fisetin, kaempferol, rutin, taxifolin, catechin, epigallocatechin, luteolin and the like.

  As a selection criterion, it is preferable that the number of bonded OH groups is large. For example, quercetin (see the following formula (1)) and myricetin (see the following formula (2)) may be used.

  The gallic acid used in the present invention is a naturally derived substance, a kind of polyphenol, and is represented by the following formula (3).

  Since the antioxidant composition of the present embodiment is formed as a mixed system of flavonoids and gallic acid, which are naturally derived substances, each flavonoid alone, each polyphenol alone, and further, ascorbic acid / tocopherol system In addition to exhibiting an excellent antioxidant effect, it is excellent in stability and safety.

  Further, when the flavonoids in the antioxidant composition of the present embodiment are formed of at least one of quercetin and myricetin, the use of quercetin and myricetin that are particularly excellent in structure in the flavonoids, a more excellent antioxidant effect Can be demonstrated.

  Furthermore, in the antioxidant composition of the present embodiment, flavonoids and gallic acid may be mixed in a weight ratio of 4/1 to 1/4, preferably 3/1 to 1/3. Thereby, the antioxidant composition which exhibits the outstanding antioxidant effect by selecting various mixing ratios of flavonoids and gallic acid can be provided.

<Cosmetic preparation>
The cosmetic preparation of the present invention will be described.

  The cosmetic preparation of this embodiment is characterized by including the antioxidant composition of this embodiment, that is, a composition comprising a mixture of flavonoids and gallic acid as a main component.

  The dosage form of the cosmetic preparation of this embodiment is arbitrary, for example, an aqueous solution type, a solubilized type, an aqueous gel type, an emulsion type, a liposome type, a freeze-dried type, a powder type by spray drying, or a dispersed type. It is good to choose from according to the intended use.

When the cosmetic preparation of this embodiment is used, an excellent antioxidant effect is exhibited by a mixture of flavonoids and gallic acid, specifically, a mixture of at least one of quercetin and myricetin and gallic acid. There superoxide in the applied site anion radicals (O ₂ ^- ·), hydroxyl radical (· OH), hydrogen peroxide (H ₂ O _2), scavenging activity of singlet oxygen ⁽¹ O ₂₎ is exhibited, various Recovery and improvement of inflammation and disease will be made.

  Furthermore, the addition amount of the antioxidant composition in the cosmetic preparation is, for example, 0.000001 wt% to 5.0 wt%, preferably 0.00001 wt% to 2 wt%, more preferably 0.0001 wt% to 0.5 wt%. Good.

In the cosmetic preparation of the present embodiment, superoxide anion radical (O ₂ ⁻ ·), hydroxy radical (· OH), hydrogen peroxide (H ₂ O ₂ ), singlet oxygen ( ¹ ) are added by adding a small amount of an antioxidant composition. It is possible to provide a cosmetic preparation excellent in O ₂ ) elimination activity.

  Furthermore, as the cosmetic preparation of this embodiment, in addition to the above-mentioned essential ingredients, ingredients blended as a base material for ordinary cosmetic preparations, such as oils and fats, hydrocarbons, waxes, fatty acids, synthetic esters, alcohols, etc. Book, powder, surfactant, thickener (polymer compound, gelling agent), moisturizer, UV absorber, UV scattering agent, antioxidant, dye, pigment, preservative, fragrance or water It is good to select and use suitably in the range which does not impair the effect of invention.

  The main components among these components will be further described.

  Examples of the fats and oils include jojoba oil, castor oil, olive oil, soybean oil, coconut oil, palm oil, cacao oil, mink oil, turtle oil and the like.

  Examples of hydrocarbons include liquid paraffin, petrolatum, microcrystalline wax, squalane and the like.

  Examples of waxes include beeswax, lanolin, carnauba wax, and candelilla wax.

  Examples of fatty acids include myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, lauric acid and the like.

  Synthetic esters include isopropyl myristate, isopropyl palmitate, cetyl palmitate, ethyl oleate, decyl oleate, myristyl myristate, octyldodecyl myristate, diisostearyl malate, ethyl linoleate, 2-ethylhexanoic acid Examples thereof include cetyl and glyceryl tri-2-ethylhexanoate.

  Oils and fats, hydrocarbons, waxes, fatty acids, and synthetic esters are usually combined in a proportion of 0 to 50 wt% per skin cosmetic.

  Examples of alcohols include ethanol, 1,3-butylene glycol, propylene glycol, pentylene glycol, isoprene glycol, lauryl alcohol, cetanol, stearyl alcohol, and oleyl alcohol. Alcohols are usually blended at a rate of 0 to 40 wt% per skin cosmetic.

  Surfactants include polyglycerin fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, coconut oil fatty acid monoethanolamide, polyoxyethylene cured Castor oil, sodium lauryl sulfate, polyglutethylene isostearate polyoxyethylene glyceryl, sodium alkylbenzenesulfonate, polyoxyethylene stearyl ether, dialkylsulfosuccinic acid, cetylpyridinium bromide, -n-octadecyltrimethylammonium chloride, monoalkyl phosphate, dialkyl Phosphoric acid, N-acyl glutamine, sucrose fatty acid ester, polyoxyethylene sorbitan monostearate, polyoxyethylene Emissions lauryl ether sodium sulfate, polyoxyethylene reduced lanolin, alkyl glucosides, propylene glycol monostearate, glyceryl monostearate, and the like distearyl dimethyl ammonium chloride and the like. Surfactant is normally mix | blended in the ratio of 0-20 wt% per skin cosmetics.

  Examples of the thickener (polymer compound, gelling agent) include carboxyvinyl polymer, methylpolysiloxane, dextran, carboxymethylcellulose, carrageenan, hydroxypropylmethylcellulose, hydroxypropylcellulose and the like. A thickener is normally mix | blended in the ratio of 0-5 wt% per skin cosmetics.

  As humectants, glycerin, trehalose, 1,3-butylene glycol, propylene glycol, dipropylene glycol, arginine, hydroxyproline, acetylhydroxyproline, pyroglutamic acid, acetylglutamic acid, sodium hyaluronate, atelocollagen, hydrolyzed collagen, hydrolyzed Examples include elastin, hydrolyzed hyaluronic acid, and phospholipid. A moisturizer is normally mix | blended in the ratio of 0-40 wt% per skin cosmetics.

  Examples of the pigment include iron oxide, titanium dioxide, zinc oxide, kaolin and talc. A pigment is normally mix | blended in the ratio of 0-30 wt% per skin cosmetics.

  Examples of the preservative include benzoic acid, salicylic acid, dehydroacetic acid or salts thereof, paraoxybenzoic acid esters, phenols of phenoxyethanol, chlorphenesin, and the like. A preservative is normally mix | blended in the ratio of 0-1.0 wt% per skin cosmetics.

  Any fragrance may be used as long as it is used in ordinary cosmetic preparations.

  Examples of water include tap water, purified water, mineral water, brine, seawater, deep ocean water, ultrapure water, polar ice-derived water, mineral-containing water, and the like. Water is blended in an arbitrary ratio.

  Examples of the present invention will be described in detail below.

  Examples 1 to 5 show the antioxidant composition of the present invention, and Example 6 shows the cosmetic preparation of the present invention.

  Note that the present invention is not limited to the embodiments.

[Example 1] Antioxidant activity by TBA method <Experimental procedure>
In Example 1, the antioxidant activity of the antioxidant composition of the present invention and the comparative example was determined by measuring by the TBA method.
The TBA method (Kenji Fukuzawa, Junji Terao, “Lipid Peroxidation Experimental Method” Yodogawa Shoten (1990)) reacts 2-thiobarbituric acid (TBA) with lipid peroxide and converts the resulting red dye to a fluorescence spectrum [excitation In this method, the degree of lipid peroxidation is determined by quantification at a wavelength of 515 nm and a fluorescence wavelength of 551 nm. As the substance to be oxidized, linoleic acid contained in the stratum corneum intercellular lipid was used.

The experimental procedure is shown below.
(1) 0.32 mL of a test sample (antioxidant (0.05 w%) ethylene glycol solution) containing 0.03 mL of linoleic acid was placed in a 25 mL volumetric flask and diluted with acetone.
As an antioxidant, control (no antioxidant added), QT alone, MT alone, GA alone, QT / GA (1/1 weight ratio), MT / GA (1/1 weight ratio), ascorbic acid alone, tocopherol A simple substance, ascorbic acid / tocopherol (1/1 weight ratio) was prepared.
(2) 100 μL of (1) was taken and transferred to a 10 mL screw mouth bottle and dried.
(3) UV irradiation was performed for 2 hours with the cap removed. As the UV lamp, a 32 W low-pressure mercury lamp (32-LB: Riko Kagaku Sangyo Co., Ltd.) was used.
(4) Add 0.3 mL of 0.8% (w / w) BHT (dibutylhydroxytoluene) (in CH ₃ COOH) to ( ₃ ), then add 8.1% (w / w) SDS solution to 0. 4 mL was added, and 3.0 mL of 10% (w / w) CH ₃ COOH buffer solution (pH 4.0) was further added.
(5) 4.3 mL of 0.56% (w / w) TBA solution was added to (4) and mixed well.
(6) (5) was heated in a boiling water bath at 95 ° C. for 2 hours with the lid closed.
(7) (6) was allowed to cool to room temperature, and then transferred to a centrifuge tube (3 mL).
(8) After 5 mL of n-butanol was added to (7) and mixed, the mixture was centrifuged at 3000 rpm for 10 minutes at room temperature.
(9) 1 mL of upper butanol was filtered through a 0.50 μm membrane filter.
(10) After adding 7 mL of n-butanol to (9) and mixing, 3 mL was transferred to a quartz cell, and the fluorescence spectrum was measured. [Excitation wavelength: 515 nm, fluorescence wavelength: 551 nm]

(result)
The results are as shown in Tables 1 and 2 below.

  From the results in Table 1, the control without addition of the antioxidant showed high fluorescence intensity and the progress of oxidation of linoleic acid was recognized, but the progress of the oxidation was suppressed with QT alone, MT alone and GA alone. It was found that the fluorescence intensity was lower in the / GA mixed system and the MT / GA mixed system, indicating strong antioxidant ability. Further, comparing the results of Table 1 and Table 2, the fluorescence intensity of the ascorbic acid / tocopherol mixed system is 145, whereas the fluorescence intensity of one QT / GA mixed system is 60, which is 1/2 or less. The fluorescence intensity of the other MT / GA mixed system is 40, which is 1/3 or less, and the QT / GA mixed system and the MT / GA mixed system of this example are clearly higher than the ascorbic acid / tocopherol mixed system. It was also observed to exhibit antioxidant capacity.

  Furthermore, the QT / GA mixed system, MT / GA mixed system and ascorbic acid / tocopherol mixed system were heated at 90 ° C. for 5 hours, and thereafter each antioxidant ability was measured by the same method. As a result, the fluorescence intensities of the QT / GA mixed system and the MT / GA mixed system were almost the same as the results of Table 1 at 65 and 42, respectively, but the fluorescence intensity of the ascorbic acid / tocopherol mixed system was 165 at Table 2. Compared with the results, the level of antioxidant capacity decreased. From this, it was confirmed that the QT / GA mixed system and the MT / GA mixed system are clearly superior to the ascorbic acid / tocopherol mixed system also from the viewpoint of thermal stability.

Example 2 O ₂ ⁻ · Erasing Activity <Experimental Procedure>
In Example 2, the O ₂ ⁻ · erasing activity of the antioxidant composition of the present invention and the comparative example was determined.
O 2 _- ^· is a precursor of the active oxygen species, O _{2 -} O 2 ^· since it is possible to suppress the generation of additional reactive oxygen species by deleting, for each antioxidant ^- - it is very important to consider the elimination activity.

The experimental procedure is shown below.
(1) To 25 mL (sample A) of a test sample (antioxidant in dimethyl sulfoxide (DMSO) solution) containing 0.05 wt% of antioxidant, 6.4 mg of potassium superoxide (KO ₂ ) and 18-crown-6 15.8 mg of ether was added and stirred for 15 minutes to generate O ₂ ⁻ . (Sample B).
As an antioxidant, a control (no antioxidant added), QT alone, MT alone, GA alone, QT / GA (1/1 weight ratio), MT / GA (1/1 weight ratio) were prepared.
Ascorbic acid alone, tocopherol alone, and ascorbic acid / tocopherol (1/1 weight ratio) were found to have low antioxidant ability in Example 1, and therefore it was determined that O ₂ ⁻ · erasing activity was also low. It was decided not to measure.
(2) 0.1 ml of sample A was dispersed in 4.6 ml of 2 mM nitro blue tetrazolium (NBT) solution.
(3) 0.3 mL of sample B was added to (2), and 8 mL of chloroform was added to the resulting blue precipitate (blue formazan). As a result, the sample solution was separated into two layers, an unreacted NBT layer and a generated blue formazan layer.
(4) 0.1 mL of blue formazan layer was collected from (3), diluted with 0.9 mL of chloroform, and the absorbance was measured. Subsequently, the peak intensity appearing in the vicinity of 530-570 nm was compared with the control (antioxidant-free test sample) to quantify O ₂ ⁻ .

(result)
The results are as shown in Table 3 below.

From the results in Table 3, the absorbance ratios of the test samples to which each antioxidant was added were lower than those of the control without addition of the antioxidant, and they had a high scavenging ability against O ₂ ⁻ . I understand. In particular, it was confirmed that the QT / GA mixed system and the MT / GA mixed system of this example had a lower absorbance ratio and exhibited a strong O ₂ ⁻ .erasing ability.

[Example 3] H ₂ O ₂ scavenging activity <Experimental procedure>
In Example 3, the H ₂ O ₂ scavenging activity of the antioxidant composition of the present invention and the comparative example was determined.
It is known that H ₂ O ₂ is an active oxygen produced when O ₂ ⁻ is decomposed by SOD, and is relatively stable, so that it is diffused through a cell membrane when generated in vivo. In addition, H ₂ O ₂ itself is not highly reactive, but it is easily decomposed by the presence of metal ions to generate OH. Therefore, it is rich in reactivity. It is important in considering oxidation damage as a precursor of OH. is there.
H ₂ O ₂ was quantified using a hydrogen peroxide quantification kit (BIOXYTECH H ₂ O ₂ Assay Kit [funakoshi]).
The experimental procedure is shown below.
(1) A 10 ⁻⁵ mol H ₂ O ₂ aqueous solution is prepared, and a test sample (antioxidant in ethylene glycol) containing 0.05 wt% antioxidant is added thereto.
As an antioxidant, a control (no antioxidant added), QT alone, MT alone, GA alone, QT / GA (1/1 weight ratio), MT / GA (1/1 weight ratio) were prepared.
Ascorbic acid alone, tocopherol alone, and ascorbic acid / tocopherol (1/1 weight ratio) were found to have low antioxidant ability in Example 1, and therefore judged to have low H ₂ O ₂ scavenging activity. It was decided not to measure.
(2) After leaving the solution of (1) for 10 minutes, H ₂ O ₂ was quantified from the absorbance at 560 nm using the above-described hydrogen peroxide quantification kit.

(result)
The results are as shown in Table 4 below.

From the results shown in Table 4, the absorbance ratios of the test samples to which each antioxidant was added were lower than those in the case where no antioxidant was added, and the scavenging ability against H ₂ O ₂ was high. I understand. In particular, it was confirmed that the QT / GA mixed system and the MT / GA mixed system of this example had a lower absorbance ratio and exhibited a strong H ₂ O ₂ erasing ability.

[Example 4] OH scavenging activity <Experimental procedure>
In Example 4, the .OH elimination activity of the antioxidant composition of the present invention and the comparative example was determined.
-OH has the highest reactivity and strongest oxidizing power among ROS. In addition, it reacts with all biological substances such as carbohydrates, proteins and lipids. This .OH is generated by irradiating H ₂ O ₂ with ultraviolet rays or by a method of catalytically reacting H ₂ O ₂ and a divalent iron compound under acidic conditions (Fenton reaction). Therefore, it is very important to examine the scavenging activity of .OH in terms of biological defense including skin against ultraviolet rays.

The experimental procedure is shown below.
(1) An antioxidant 0.05 wt% was dissolved in tert-butyl alcohol (sample A).
As an antioxidant, a control (no antioxidant added), QT alone, MT alone, GA alone, QT / GA (1/1 weight ratio), MT / GA (1/1 weight ratio) were prepared.
Ascorbic acid alone, tocopherol alone, and ascorbic acid / tocopherol (1/1 weight ratio) were found to have low antioxidant ability in Example 1. It was decided.
(2) OH was generated by adding phenazine methyl sulfate to NADH (reduced form of nicotinamide adenine dinucleotide) (sample B).
(3) Sample B was added to Sample A, and a tert-butyl alcohol solution of p-nitrosodimethylaniline was added thereto.
(4) The absorbance of the solution of (3) was measured at a wavelength of 440 nm, and OH was quantified by comparing with the control with no antioxidant added. This is a measurement method utilizing the fact that p-nitrosodimethylaniline changes from yellow to colorless by reacting with .OH.

(result)
The results are shown in Table 5 below.

  From the results in Table 5, it can be seen that the test samples to which each antioxidant was added had a lower absorbance ratio than the control without addition of an antioxidant, and had a high erasability against OH. . In particular, it was confirmed that the QT / GA mixed system and the MT / GA mixed system of this example had a lower absorbance ratio and exhibited strong OH erasing ability.

[Example 5] ¹ O ₂ scavenging activity <Experimental procedure>
In Example 5, the ¹ O ₂ scavenging activity of the antioxidant composition of the present invention and the comparative example was determined.
¹ O ₂ is one of the most reducible ROS and is considered to be continuously generated by UV exposure in daily life (Tomio Okada: wrinkles, active oxygen / free radicals that cause sagging, skin aging) And active oxygen / free radical, Fragrance Journal, Tokyo, 1996, pp. 69-88). ¹ O ₂ generated in the skin surface and tissues quickly peroxidizes squalene and unsaturated fatty acids in sebum and denatures proteins such as collagen in the dermis (Gilchrest, BA, et al, Photochem. Photobiol. , 63, 1-10 (1996)). However, unlike SOD for O ₂ ^−. , There is no function for efficiently eliminating ¹ O ₂ in the living body, and it is necessary to protect it by the action of an external antioxidant. Therefore, an antioxidant system that efficiently eliminates ¹ O ₂ is very important in preventing oxidative damage in the skin.
^{The 1} O ₂ erasing activity changes the mixing ratio of MT and GA with respect to the MT / GA mixed system and changes the mixing ratio of QT and GA with respect to the QT / GA mixed system as follows. Measured according to the procedure.

The experimental procedure is shown below.
(1) Mukai et al. Measured singlet oxygen ( ¹ O ₂ ) scavenging activity using test samples (antioxidant ethylene glycol solution) with MT / GA total concentration and QT / GA total concentration of 0.05 wt%. (Kazuo Mukai, et al., J. Org. Chem. 56 , 4188-4192 (1991)).
Here, the mixing ratio of each flavonoid (MT and QT) and GA is 100/0, 80/20, 75/25, 50/50, 25/75, 20/80, 0/100 in weight ratio. Changed.
In the evaluation reaction system, 3- (1,4-epidioxy-4-methyl-1,4-dihydro-1-naphtyl) propionic acid (EP) was used as a singlet oxygen donor, and 2,5-diphenyl-3,4- Designed to accept benzofuran (DPBF) as an acceptor. EP generates a sustained ¹ O ₂ at 40 ° ^C., 1 if _{the O 2} scavengers are not co-exist in the reaction, reacts rapidly with DPBFs, over time reduces the absorbance at 415nm of DPBFs. ^When a component having ¹ O ₂ scavenging activity is added to the reaction solution, the digestion rate of DPBF decreases, and the decrease in the digestion rate and the concentration of the ¹ O ₂ scavenger in the reaction system are in a relative relationship. Therefore, in this evaluation condition, the value obtained by subtracting the natural decrease rate of DPBF from the decrease rate of the blank measured using only the solvent without MT and GA is set to 100, and 10% of this decrease rate is reduced. The required amount of ¹ O ₂ erasing agent in the evaluation reaction solution is indicated as EC10. The smaller this value, the higher the ¹ O ₂ erasing activity value.
(3) Add 50 μL of EP solution (2 mg / mL) and 50 μL of DPBF solution (0.1 mg / mL) to 100 μL of the test solution corresponding to each mixing ratio of MT / GA mixed system and QT / GA mixed system, Warmed at 40 ° C.
(4) ¹ O ₂ scavenging activity was calculated by measuring the rate of decrease in DPBF absorption (415 nm) with a spectrophotometer.

(result)
The results are as shown in Table 6 (MT / GA mixed system) and Table 7 (QT / GA mixed system) below.

From the results of Tables 6 and 7, it can be seen that the value of EC10 varies depending on the mixing ratio of MT and GA and the mixing ratio of QT and GA. That is, when MT, QT, and GA are each independent, they exhibit ¹ O ₂ scavenging activity equivalent to or slightly higher than catechin, ascorbic acid, tocopherol, and ascorbic acid / tocopherol mixed system, It became clear that extremely high ¹ O ₂ erasing activity was exhibited by combining QT and GA. In particular, MT / GA (1/1 weight ratio) is 0.00007, QT / GA (1/1 weight ratio) is 0.00010, and both are 0 of ascorbic acid / tocopherol (1/1 weight ratio). It was revealed that it exhibits extremely high ¹ O ₂ erasing activity of 20 to 15 times that of .00147.

  Furthermore, in the antioxidant composition of the present invention from this example, when the flavonoids and gallic acid are in a weight ratio of 4/1 to 1/4, preferably 3/1 to 1/3, an excellent antioxidant effect is obtained. I found it to work.

[Example 6] Inhibitory effect on human skin oxidation reaction by UV irradiation <Experimental procedure>
In Example 6, the inhibitory effect on the human skin oxidation reaction by UV irradiation between the cosmetic preparation of the present invention and the comparative example was determined.
In this example, an experiment was conducted on MT / GA (1/1 weight ratio) in the antioxidant composition of the present invention as a cosmetic preparation. This is because other substances in the antioxidant composition of the present invention, such as QT / GA (1/1 weight ratio), are found to exhibit the same inhibitory effect. In this example, healthy men ( Using the back of 3 persons), the inhibitory effect of the MT / GA (1/1) system of the present invention on the oxidation reaction generated by UV irradiation was evaluated.

The experimental procedure is shown below.
(1) 10 μg of linoleic acid was applied to the test area (10 cm ² ) on the back of the subject.
(2) A test sample cosmetic solution (see Table 8 below for components) having a total concentration of 50 μL of MT / GA of 0.05 wt% was applied to the test area (10 cm ² ) on the back of the subject.

(3) The area was irradiated with UV (60 mJ / cm ² , 285 nm).
(4) Four hours after the irradiation, the oxide in the area was extracted by a cup method with 30 ml of an acetone: diethyl ether (50:50) solution.
(5) The oxide amount of (4) was measured by the TBA method.

(result)
As a result, the measured values of the amount of oxide in the extract of the back portion of the subject irradiated with UV (60 mJ / cm ² , 285 nm, 1 MED) not coated with the antioxidant were 2.14, 2.26. 2.20 (average 2.20 nmol MDA (malondialdehyde) / mL). The measured value of the amount of oxide in the extract of the UV non-irradiated section was 1.05, 1.20, 1.14 (average 1.13 nmol MDA / mL). Therefore, about twice as much lipid peroxide was generated as compared with the portion not irradiated by UV irradiation. In contrast, the measured values of oxide in the extract of the UV-irradiated section after applying the MT / GA (1/1) test sample of the present invention are 1.18, 1.25, 1.19 (average 1. 21 nmol MDA / mL) showed a value almost the same as that of the UV non-irradiated section. On the other hand, the measured values of the oxide amount in the extract of the section irradiated with UV after applying the test sample of 0.05% MT and GA alone were 1.57, 1.90, 1.69 (average) in MT. 1.72 nmol MDA / mL), 1.98, 2.01, and 1.83 (average 1.94 nmol MDA / mL) for GA, and the MT / GA (1/1) system and UV irradiation without antioxidants. An intermediate value was shown. Table 9 summarizes the results.

  From the results in Table 9, it was found that the MT / GA (1/1) system can effectively suppress the actual human skin peroxidation reaction caused by UV irradiation. ROS generated in the skin tissue by UV irradiation radicalizes lipid components having unsaturated bonds such as squalene and linoleic acid present in sebum and stratum corneum lipids, and these radical components are further lipid components. It is thought to induce peroxidation and increase the surface peroxide (Tomio Okada: Wrinkles, active oxygen / free radicals causing sagging, skin aging and active oxygen / free radicals, fragrance journal Co., Tokyo, 1996, pp. 69-88), the MT / GA (1/1) system of the present invention effectively eliminates ROS, which is a temporary reaction product in this series of oxidation reactions, so Peroxidation is suppressed. In this way, the MT / GA (1/1) system effectively suppresses skin oxidation, thus preventing disturbance of skin surface lipids and stratum corneum lipid structures caused by oxidation of lipids present in the skin. It is considered that the barrier function and moisture retention function of the entire stratum corneum including the surface can be protected.

With the cosmetic preparation of the present invention shown in this example, superoxide anion radical (O ₂ ⁻ ·), hydroxy radical (· OH), hydrogen peroxide (H ₂ O ₂ ) and singlet oxygen ( ¹ O ₂ ) are eliminated. It was found that a cosmetic preparation excellent in activity can be provided, and further, the addition amount of the antioxidant composition of the present invention in the cosmetic preparation can be adjusted as necessary.

Claims (5)

An antioxidant composition comprising a mixture of flavonoids and gallic acid as a main component. The antioxidant composition according to claim 1, wherein the flavonoid is composed of at least one of quercetin and myricetin. The antioxidant composition according to claim 1 or 2, wherein the flavonoids and the gallic are in a weight ratio of 4/1 to 1/4, preferably 3/1 to 1/3. A cosmetic preparation comprising the antioxidant composition according to any one of claims 1 to 3. The antioxidant composition according to claim 4, comprising 0.000001 wt% to 5.0 wt%, preferably 0.00001 wt% to 2 wt%, more preferably 0.0001 wt% to 0.5 wt%. Cosmetic preparation.