Antioxidant composition

Abstract

[Problem] To provide a composition that can be administered orally or topically and that has excellent anti-oxidative capacity, in particular, excellent free radical scavenging effect. [Solution] The present invention involves combinational use of γ-Glu-Cys-Gly (GSH), γ-Glu-Cys (γ-GC), and Cys-Gly (CG) which are cysteine-including peptides.

Description

Antioxidant composition

[0001] The present invention relates to a composition having antioxidant ability that can be used orally or externally.

[0002] Free radicals are known to be generated in vivo by exposure to ultraviolet rays and lipid peroxides in food. In vivo, free radicals at appropriate concentrations play an important role in biological defense, but it has been clarified that excessive free radicals have various adverse effects on the living body. For example, it is known that the generation of free radicals causes aging of cells, decline of liver function, appearance of spots and freckles, and aging of the skin such as loss of skin elasticity.

[0003] In order to remove or eliminate free radicals that are excessively generated in vivo as described above, substances having an antioxidant action are known to be useful. For example, as having excellent antioxidant action, there is a fermentation broth obtained by fermenting plants of the Artemisia genus and Taraxacum officinale with Aspergillus oryzae (Prior art 1). In addition, a rice fermented product fermented by Lactobacillus curvatus AOK L 2296 strain (NITE P-03596) is known (Prior art 2). However, since natural plants are used as raw materials, there is a problem that the components contained vary depending on the season.

[0004] In addition, a glutathione-containing antioxidant composition containing glutathione and a general antioxidant substance is known (Prior art 3). However, most of the general antioxidant substances exhibit an action mechanism different from that of glutathione, and a synergistic effect by a combination with an antioxidant substance exhibiting an action mechanism similar to that of glutathione, for example, free radical scavenging ability, has not been reported.

[0005] Japanese Patent Application Laid-Open No. 2023-171950, Japanese Patent Application Laid-Open No. 2024-006803, WO2015 / 163165 pamphlet

[0006] An object of the present invention is to provide a composition having excellent antioxidant ability, particularly excellent free radical scavenging ability.

[0007] As a result of diligent research to solve the above problems, the present inventors have found that combining glutathione (GSH), a cysteine-containing peptide, with γ-Glu-Cys (γ-GC) and Cys-Gly (CG) exhibits excellent antioxidant activity.

[0008] In other words, the present invention relates to the following (1) to (7): (1) An antioxidant composition comprising GSH, γ-GC, and Cys-Gly. (2) An antioxidant composition comprising GSH, γ-GC, and Cys-Gly, wherein the composition ratio (mass ratio) of GSH, γ-GC, and Cys-Gly is 1:0.01 to 0.1:0.005 to 0.50. (3) A method for inhibiting oxidation, characterized by using the composition described in (1) or (2) or applying it orally or topically. (4) The method for inhibiting oxidation according to (3), wherein the method for inhibiting oxidation is free radical scavenging. (5) A food or beverage for improving skin comprising the composition of (1) or (2). (6) A cosmetic for improving skin comprising the composition of (1) or (2). (7) A pharmaceutical for improving skin comprising the composition of (1) or (2).

[0009] In this invention, by using glutathione (GSH), γ-glutamylcysteine ​​(γ-Glu-Cys: γ-GC), and cysteinylglycine (Cys-Gly: CG), which are cysteine-containing peptides, in combination, it exhibits a superior synergistic effect rather than simply an additive effect of adding up their respective antioxidant capacities, particularly their free radical scavenging effects. As a result, it provides a high level of protection against oxidative stress and yields excellent whitening and skin beautifying effects. Furthermore, since GSH, γ-GC, and CG are naturally occurring components in food and living organisms, they are also highly safe for the body.

[0010] Radical scavenging ability of GSH, γ-GC, or CG alone, and the results of radical scavenging tests in combined groups of yeast extracts from multiple lots, as well as the results of radical scavenging tests in combined groups of GSH, γ-GC, and CG.

[0011] The present invention will be described in detail below. In the present invention, the cysteine-containing peptide refers to glutathione (GSH), γ-glutamylcysteine ​​(γ-Glu-Cys: γ-GC), and cysteinylglycine (Cys-Gly: CG). The composition of the present invention is not particularly limited as long as it contains GSH, γ-GC, and CG, or a mixture of refined or crude products of the cysteine-containing peptides GSH, γ-GC, and CG, or all of them, as well as food materials such as yeast extract. Whether a composition contains GSH, γ-GC, and CG can be confirmed by the examples described herein.

[0012] The composition of the present invention uses GSH, γ-GC, and CG in combination, and the composition ratio (mass ratio) of each is arbitrary, but it is preferable that the composition ratio (mass ratio) of GSH, γ-GC, and CG is 1:0.01 to 1:0.005 to 0.50, preferably 1:0.05 to 0.50:0.01 to 0.30, and more preferably 1:0.10 to 0.21:0.03 to 0.13. These composition ratios are measured by the method described in the examples of this specification.

[0013] The GSH, γ-GC, and CG in the composition of the present invention are optional, but since the composition of the present invention is used by adding it to food products, it is preferable that the composition contains 5% by mass or more of GSH. More preferably, the composition contains 15% by mass or more. The content of γ-GC and CG may be as long as they are present in the mass ratios mentioned above.

[0014] Other free radical scavengers, such as ascorbic acid, kojic acid, arbutin, edaravone, α-tocopherol, hydroquinone, 3,4-dibutyl-4-hydroxytoluene, butylhydroxyanisole, and derivatives thereof, may be added to the composition of the present invention, to the extent that they do not inhibit the effects of the present invention.

[0015] In addition to the substances mentioned above, the composition of the present invention may contain various food ingredients, food additives, cosmetic additives, and excipients used in pharmaceuticals. For example, various amino acids, peptides, proteins, nucleic acids, sugars, oils, etc., can be mixed in without limitation, as long as they do not inhibit the effects of this product.

[0016] The composition of the present invention can be added to various foods and beverages, cosmetics, oral medications, topical skin preparations, and other quasi-drugs, including pharmaceuticals. When added to foods and beverages, it can also be used in functional foods and foods for specified health uses. Since the composition of the present invention can be used as a whitening composition and a skin beautifying composition, foods and beverages, cosmetics, topical skin preparations, and pharmaceuticals to which the composition of the present invention has been added can be suitably used as foods and beverages for skin improvement, such as skin beautification or whitening. There are no particular restrictions on the content of the composition of the present invention, but it is preferable to contain about 0.0001 to 10% by mass in products such as foods and beverages.

[0017] The composition of the present invention possesses antioxidant activity, and in particular, excellent free radical scavenging activity. The free radical scavenging activity of the composition of the present invention can be evaluated by its scavenging activity against 2,2-diphenyl-1-picryldihydrazyl (DPPH), which is commonly used in free radical scavenging tests. When DPPH is dissolved in a solvent, it turns purple, and as the radicals are scavenged by the antioxidant, the purple color fades. Therefore, the free radical scavenging activity of the antioxidant can be determined from the difference in absorbance between the unadded and added antioxidant groups. The free radical scavenging activity can be compared with other substances by converting it to the amount of Trolox (μmol), a type of antioxidant.

[0018] <Method for Measuring Cysteine-Containing Peptides> The cysteine-containing peptides contained in the composition of the present invention were measured using the DTNB-HPLC method. Under the following conditions, 50 μL of 0.5 mol / L phosphate buffer (pH 8.0) and 200 μL of 0.2 mol DTNB solution were added to 2 mL of standard aqueous solutions of GSH, γ-GC, and CG with known concentrations, as well as to 2 mL of the sample aqueous solution, to derivatize the thiol groups of each component. By HPLC analysis, the cysteine-containing peptides in the composition were identified and their content calculated from the peak retention times and areas of the cysteine-containing peptides and standard solutions, respectively. Mobile phase: 0.025 mol / L ammonium formate (pH 4.0) and methanol mixture (9:1 V / V) Column: Nucleosil 10C18 Φ4.6 mm × 250 mm Column No. H3A31 Column temperature: 40°C Flow rate: 0.7 mL / min Injection volume: 10 μL Detector: PDA (UV) Detection wavelength: 280 nm Analysis time: 25 min

[0019] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

[0020] <Confirmation Test of Free Radical Scavenging Ability> The GSH, γ-GC, and CG used as samples were generally available purified products. The tablets containing yeast extract with cysteine-containing peptides (hereinafter referred to as Tablet A) were manufactured by Mitsubishi Corporation Life Sciences. Three Tablet A tablets were dissolved in 100 mL of water and analyzed. The cysteine-containing peptides contained in Tablet A were 42 mg for GSH, 6 mg for γ-GC, and 1.5 mg for CG. First, the following five types of solutions were prepared. Sample solution: The samples shown in Comparative Examples 1-3 and Examples 1 and 2 were dissolved in water. DPPH solution: 7.89 mg of DPPH reagent was dissolved in 100 mL of ethanol. Trolox solution: Dissolved and prepared in ethanol to concentrations of 40, 60, 80, and 100 μg / mL. Assay buffer: 0.1 M Tris-HCl buffer (pH 7.4) Next, as a preliminary study, a concentration range was set for each sample in which linearity was confirmed including a DPPH radical scavenging rate of 50%. For measurement, 80 μL of assay buffer was added to 20 μL of each sample solution or Trolox solution within the set concentration range, and 100 μL of DPPH solution was added. After incubation at 25°C for 30 minutes under light-shielding conditions, the absorbance at 517 nm was measured. The measurement value without the sample and DPPH solution was denoted as Ac, and the measurement value with Trolox solution was denoted as AR, and the radical scavenging rate (%) of Trolox was calculated using Equation 1 below. Furthermore, the radical scavenging rate (%) of the sample was calculated using Equation 2 below, with the difference between the measurement value with the sample solvent added instead of the sample and the measurement value with ethanol added instead of DPPH solution being denoted as Acs, and the difference between the measurement value of the sample and the measurement value without the sample and DPPH solution being denoted as As. The radical scavenging rate of the samples was converted to an equivalent Trolox concentration, and the relative radical scavenging ability was evaluated.

[0021] <Equation 1> Radical scavenging rate of Trolox (%) = (Ac - AR) / Ac × 100 <Equation 2> Radical scavenging rate of the sample (%) = (Acs - As) / Acs × 100

[0022] The following samples were used as comparative examples: <Comparative Example 1> A purified product of GSH was used. <Comparative Example 2> A purified product of γ-GC was used. <Comparative Example 3> A purified product of CG was used.

[0023] The following samples were used as examples. <Example 1> The purified products of each cysteine-containing peptide were mixed and used in a composition ratio of GSH:γ-GC:CG = 1:0.143:0.036. <Example 2> Tablet A was dissolved in water to prepare the solution.

[0024] <Results> Figure 1 shows the ratio of radical scavenging ability when the sum of Comparative Examples 1-3 (numbered 4 in the figure) is set to 1, based on the measured values ​​of DPPH radical scavenging ability converted to Trolox concentration for Comparative Examples 1-4 and Examples 1 and 2. The numbers in the figure correspond to the respective samples as follows. 1: Radical scavenging ability of Comparative Example 1 (GSH) 2: Radical scavenging ability of Comparative Example 2 (γ-GC) 3: Radical scavenging ability of Comparative Example 3 (CG) 4: Sum of radical scavenging abilities of Comparative Examples 1 to 3 5: Radical scavenging ability of Example 1 (combination of GSH, γ-GC, and CG) 6: Radical scavenging ability of Example 2 (cysteine-containing peptide) Although the concentrations of each cysteine-containing peptide contained in Example 1 were the same as those of Comparative Examples 1 to 3, the DPPH radical scavenging ability of Examples 1 and 2 showed higher values ​​than the theoretical sum, indicating that a synergistic effect is achieved by using GSH, γ-GC, and CG in combination.

[0025] Furthermore, similar studies were conducted on tablets A from a different lot, using yeast extract containing cysteine-containing peptides in the ratio GSH:γ-GC:CG = 1:0.125:0.063 and yeast extract containing cysteine-containing peptides in the ratio GSH:γ-GC:CG = 1:0.139:0.035. The results of the free radical scavenging ability confirmation test are shown in Figure 2. When comparing the theoretical sum of the radical scavenging ability of the test group using GSH, γ-GC, and CG in combination with the test group using each of them individually (number 10 in the figure; sum of numbers 7-9), it became clear that the radical scavenging ability in the combined group (numbers 11 and 12) was higher than the sum of the radical scavenging ability values ​​of each individual test group, indicating a synergistic antioxidant capacity. The numbers correspond to the respective samples as follows. 7: Radical scavenging ability of GSH 8: Radical scavenging ability of γ-GC 9: Radical scavenging ability of CG 10: Combined value of radical scavenging abilities of GSH, γ-GC, and CG 11: Radical scavenging ability of test groups using GSH, γ-GC, and CG in combination 12: Radical scavenging ability of cysteine-containing peptides

[0026] The free radical scavenging ability of the present invention, achieved by using the cysteine-containing peptides GSH, γ-GC, and CG in combination, exhibits a superior synergistic effect rather than simply an additive effect from the sum of their individual free radical scavenging abilities. This results in a high protective effect against oxidative stress and excellent whitening and skin beautifying effects. In other words, the composition of the present invention possesses excellent antioxidant capacity. Furthermore, since GSH, γ-GC, and CG are naturally present in food and living organisms, they are highly safe for the body and can be used in food, cosmetics, and pharmaceuticals.

Claims

1. An antioxidant composition comprising GSH, γ-GC, and Cys-Gly.

2. An antioxidant composition comprising GSH, γ-GC, and Cys-Gly, wherein the composition ratio (mass ratio) of GSH, γ-GC, and Cys-Gly is 1:0.01 to 0.1:0.005 to 0.

50.

3. A method for inhibiting oxidation, characterized by using the composition described in claim 1 or 2 orally or topically.

4. The oxidation suppression method according to claim 3, wherein the oxidation suppression method is free radical scavenging.

5. A food or beverage for improving skin containing the composition of claim 1 or 2.

6. A skin-improving cosmetic comprising the composition of claim 1 or 2.

7. A pharmaceutical product for improving skin comprising the composition of claim 1 or 2.

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