Anti-aging composition comprising persulfide and polyphenol

Abstract

To provide a composition having anti-aging ability such as excellent melanogenesis inhibitory effect and protective effect against oxidative stress, and usable for the purpose of whitening and skin beautification.SOLUTION: By using a polyphenol and a persulfide in combination, it is possible to obtain an external composition or an oral composition having an excellent melanin production inhibitory effect and an excellent protective effect against oxidative stress, that is, having an excellent anti-aging ability. In addition, by using the polyphenol and the persulfide in combination, the anti-aging ability of the persulfide can be improved.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an anti-aging composition containing a persulfide and a polyphenol, which is suitable for use in whitening and beautifying skin, a method for producing the composition, and a method for inhibiting aging. [Background technology]

[0002] Aging is known to be driven by genetic and environmental factors. The same is true for skin aging, which is characterized by pigmentation, age spots and freckles, wrinkles, and sagging skin. While the detailed mechanisms of skin pigmentation, age spots, and freckles remain unclear, one possible cause is thought to be excessive melanin production and deposition in the skin due to hormonal abnormalities and excessive or chronic exposure to ultraviolet light. Additionally, a decline in skin cell function caused by oxidative stress is thought to be one of the causes of increased wrinkles and loss of skin firmness and luster.

[0003] To date, ascorbic acid, kojic acid, arbutin, reduced glutathione (GSH), and other active ingredients have been found in melanin production inhibitors and skin protectors that protect skin cells from oxidative stress, and are used in foods, beverages, and cosmetics for whitening and skin beautification. However, these ingredients may not be effective enough when used alone, and multiple compounds have often been used in combination to achieve greater melanin production inhibitory effects, antioxidant effects, and other anti-aging effects.

[0004] In recent years, persulfides (also referred to as active sulfur compounds, supersulfur compounds, or supersulfur molecules) have attracted attention as glutathione-related substances, and an intraocular irrigation solution (Patent Document 1) and a neuroprotective method (Patent Document 2) have been reported. However, no compounds that exhibit combined effects with persulfides have been reported.

[0005] Therefore, there has been a demand for compositions containing persulfides that exhibit even better melanin production inhibitory effects and protective effects against oxidative stress, i.e., anti-aging properties. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2017 / 057768 Brochure [Patent Document 2] WO2021 / 231476 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Bianco CL., et al. (2019) “The reaction of hydrogen sulfide with disulfides: formation of a stable trisulfide and implications for biological systems”, British Journal of Pharmacology, 176, 671-683 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a composition containing a persulfide that has an excellent effect of inhibiting melanin production and a protective effect against oxidative stress, i.e., an anti-aging effect, and can be used for whitening and skin beautifying. [Means for solving the problem]

[0009] As a result of intensive research into solving the above problems, the inventors discovered that the combined use of polyphenols and persulfides exhibits a synergistic effect in inhibiting melanin production and protecting against oxidative stress, and thus completed the present invention.

[0010] That is, the present invention relates to the following (1) to (7). (1) An anti-aging composition containing a persulfide of a glutathione analog or a persulfide of a cysteine ​​analog and a polyphenol. (2) The anti-aging composition according to (1), wherein the polyphenol is contained in an amount of 0.01 to 100 parts by weight per part by weight of the persulfide of a glutathione analog or the persulfide of a cysteine ​​analog. (3) The anti-aging composition according to (1) or (2), wherein the polyphenol is a polyphenol having a galloyl group or a resorcinol group. (4) A composition for external use comprising the composition according to (1) or (2). (5) An oral composition comprising the composition according to (1) or (2). (6) A method for inhibiting aging, characterized by using the anti-aging composition according to (1) or (2) as an oral or external preparation. (7) A method for improving the anti-aging activity of a persulfide of a glutathione analog or a persulfide of a cysteine ​​analog, characterized by using a polyphenol in combination with a persulfide of a glutathione analog or a persulfide of a cysteine ​​analog. [Effects of the Invention]

[0011] According to the present invention, a composition containing a glutathione analog persulfide or a cysteine ​​analog persulfide and a polyphenol exhibits anti-aging effects such as improving skin wrinkles, inhibiting the occurrence and deposition of blemishes, and improving the skin's moisture retention ability. In particular, the composition exhibits effects that are greater than the sum of the individual effects, particularly in inhibiting melanin production and protecting against oxidative stress, making it possible to obtain an anti-aging composition with excellent whitening and skin-beautifying effects. [Brief explanation of the drawings]

[0012] [Figure 1] Inhibitory effect of melanin production by combined use of GSSSG and polyphenols [Figure 2] Antioxidant effect of combined use of GSSSG and polyphenols DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be specifically described below. The compositions of the present invention contain glutathione analog persulfides or cysteine ​​analog persulfides (hereinafter, sometimes collectively referred to as persulfides). Here, glutathione analogs in the present invention refer to reduced or oxidized glutathione, reduced or oxidized γ-glutamylcysteine, reduced or oxidized cysteinylglycine, and compounds in which one or more amino or carboxyl groups in these structures are modified with a linear or branched alkyl group having 1 to 24 carbon atoms, as well as compounds esterified with an organic or inorganic acid. Furthermore, cysteine ​​analogs in the present invention refer to cysteine, cystine, reduced or oxidized N-acetylcysteine ​​(hereinafter, sometimes referred to as NAC), and compounds in which one or more amino or carboxyl groups in these structures are modified with a linear or branched alkyl group having 1 to 24 carbon atoms, as well as compounds esterified with an organic or inorganic acid. The term "persulfide" as used herein refers to a molecule having a structure in which one or more sulfur atoms are directly bonded to a sulfur atom constituting a thiol group or disulfide bond present in a molecule of the aforementioned gluthione analog or cysteine ​​analog (e.g., RS-SH or RSSS-R'; R and R' are any functional groups). Any glutathione analog or cysteine ​​analog in which one or more sulfur atoms are directly bonded to a sulfur atom in the molecule can be used in the present invention. A mixture of multiple compounds with different numbers of sulfur atoms in the molecule may also be used. The persulfide contained in the composition of the present invention is preferably an oxidized type, more preferably oxidized glutathione persulfide or oxidized NAC persulfide, and even more preferably oxidized glutathione persulfide (oxidized glutathione trisulfide) or oxidized NAC persulfide (oxidized NAC trisulfide), which contain three sulfur atoms per molecule. Furthermore, the composition of the present invention is not particularly limited as long as it contains each of the ingredients, such as yeast extract, a mixture of purified or crudely purified products, or all of them.

[0014] Persulfides can be obtained using publicly available glutathione analogs or cysteine ​​analogs as raw materials according to methods published in papers such as Non-Patent Document 1. Specifically, oxidized glutathione and peracetic acid are reacted on ice for 30 minutes, followed by the addition of a solvent consisting of equal parts ethanol and tetrahydrofuran (THF). The precipitate obtained by centrifugation is then further reacted with sodium hydrogen sulfide at room temperature to obtain oxidized glutathione persulfide. By purifying this oxidized glutathione persulfide, oxidized glutathione trisulfide can be produced, and it can be obtained in the form of crystals, amorphous matter, solution, suspension, or the like. Oxidized NAC persulfide can be obtained by a similar process using oxidized N-acetylcysteine ​​as a raw material.

[0015] The composition of the present invention contains the persulfide and a polyphenol. The polyphenol in the present invention refers to a compound having two or more phenolic hydroxyl groups in an aromatic ring contained in one molecule. Among polyphenols, those having a galloyl group or a resorcinol group and a molecular weight of 120 to 2000 are particularly preferred. Examples of such polyphenols include pyrogallol (CAS No.: 87-66-1), hydroxyquinol (CAS No.: 533-73-3), galacetophenone (CAS No.: 528-21-2), gallic acid (CAS No.: 149-91-7), methyl gallate (CAS No.: 99-24-1), ethyl gallate (CAS No.: 831-61-8), propyl gallate (CAS No.: 121-79-9), octyl gallate (CAS No.: 1034-01-1), and dodecyl gallate. Examples of suitable polyphenols include decyl catechin (CAS No. 1166-52-5), tannic acid (CAS No. 1401-55-4), quercetin (CAS No. 117-39-5), epigallocatechin gallate (CAS No. 989-51-5), hesperidin (CAS No. 520-26-3), ellagic acid (CAS No. 476-66-4), and resveratrol (CAS No. 501-36-0). Pyrogallol, propyl gallate, tannic acid, and quercetin are particularly preferred. Glycosides in which sugars are bound to the polyphenols may also be used. Each polyphenol can be quantified by standard analytical methods. For example, the amount of polyphenols contained in an analytical sample can be determined by a calibration curve prepared by HPLC using a standard sample of known concentration.

[0016] The composition of the present invention may be used in combination with other materials, as long as they do not impair the effects of the present invention. These materials may include melanin production inhibitors other than the persulfides and polyphenols, antioxidants, thickeners, pH adjusters, fragrances, bases for topical preparations, excipients, etc.

[0017] The method for making the persulfide and polyphenol coexist is not particularly limited, and they may be mixed together with other raw materials when preparing the composition, or they may be mixed at a different time from the other raw materials. The form of the composition is not limited as long as it can be taken orally or applied to the skin, and can be prepared in the form of a solution, dispersion, powder, granules, capsules, tablets, paste, gel, or sheet according to a conventional method using solvents or bases used in general foods, supplements, cosmetic compositions, pharmaceutical compositions, and topical skin preparations.

[0018] The content of each component in the composition of the present invention can be appropriately adjusted by adding excipients and the like that are generally used in the above forms.

[0019] By the method described in the preceding paragraphs, a topical or oral composition containing the composition of the present invention can be produced.

[0020] The content of persulfides in the composition of the present invention can be determined by a common method using HPLC. The content of persulfides in the composition of the present invention can be adjusted as appropriate, and the composition typically contains 0.001 to 0.7 wt %, preferably 0.0012 to 0.5 wt % of persulfides. The amount of polyphenols contained in the composition of the present invention is preferably 0.01 to 100 parts by weight, more preferably 0.02 to 50 parts by weight, more preferably 0.04 to 10 parts by weight, and even more preferably 0.1 to 5 parts by weight, per part by weight of persulfides.

[0021] In the present invention, aging refers to increased wrinkles, loss of firmness and luster, decreased skin moisture retention, and the development of age spots and pigmentation. Furthermore, the anti-aging effect in the present invention refers to the improvement of wrinkles, firmness, luster, or brightness of the skin, improved skin moisture retention (moisturizing), and inhibition of pigmentation. The antioxidant activity of the composition of the present invention refers to the ability to exhibit the above-mentioned anti-aging effect. The development of age spots and pigmentation are thought to be related to melanin production, while hyaluronic acid and collagen are thought to be involved in the development of wrinkles. The degree of aging can be determined from any of the commonly used indicators of aging, such as decreased cell proliferation, hyaluronic acid production, and melanin production in cultured cells, or increased wrinkles, loss of firmness and luster, decreased skin moisture retention, or the development of age spots and pigmentation in human skin.

[0022] The composition of the present invention exhibits anti-aging properties, and therefore, aging can be inhibited by using the composition as an oral or topical preparation. That is, the anti-aging method of the present invention can be achieved by using the composition of the present invention as an oral or topical preparation. When used as an oral or topical preparation, there is no limit to the amount used, as long as the anti-aging properties are exhibited. When used as an oral preparation for humans, the dosage and frequency of administration vary depending on the administration form, age, and body weight, but is typically administered once or several times a day so that an adult receives 50 mg to 30 g, preferably 100 mg to 10 g, and particularly preferably 200 mg to 3 g of persulfide per day.

[0023] The composition of the present invention exhibits anti-aging properties and can inhibit melanin production. The amount of melanin in cultured cells can be quantified by a common method, such as measuring absorbance at 405 nm. The effect of inhibiting melanin production can be determined by a decrease in the amount of melanin.

[0024] In addition, the composition of the present invention has anti-aging ability, and can protect cells from oxidative stress.Anti-oxidative ability can be determined by the proliferation ability of fibroblasts that are continuously exposed to hydrogen peroxide, and can be evaluated by a general cell proliferation test system such as MTT assay.It is known that fibroblasts are generally involved in the production of collagen and hyaluronic acid, so it is believed that by improving the survival rate and proliferation ability of fibroblasts, the amount of collagen and hyaluronic acid can increase, and this can be effective in improving skin elasticity and wrinkles.

[0025] A method for enhancing the anti-aging activity, particularly the melanin production inhibitory activity or antioxidant activity, of a persulfide of a glutathione analog or a persulfide of a cysteine ​​analog is to make the persulfide of a glutathione analog or the persulfide of a cysteine ​​analog coexist with a polyphenol. By checking the amount of melanin produced and the cell viability using the methods described above, it can be confirmed that a synergistic effect can be obtained compared to when either persulfides or polyphenols are used alone.

[0026] The method for coexisting a glutathione analog persulfide or a cysteine ​​analog persulfide with a polyphenol is not particularly limited, and the polyphenol may be added to a composition containing the glutathione analog persulfide or the cysteine ​​analog persulfide, or the glutathione analog persulfide or the cysteine ​​analog persulfide may be added to a composition containing the polyphenol.Furthermore, the glutathione analog or the cysteine ​​analog persulfide may be prepared in the coexistence of the glutathione analog or the cysteine ​​analog with the polyphenol. [Example]

[0027] The present invention will be specifically explained below using examples, but the present invention is not limited to these examples.

[0028] Persulfide was produced as follows. Commercially available oxidized glutathione as a reagent was dissolved in ultrapure water to prepare a 170 mM oxidized glutathione aqueous solution. 4 mL of 32 wt% peracetic acid solution was added to 2.5 mL of this oxidized glutathione aqueous solution and allowed to react on ice for 30 minutes. 30 mL of a solvent consisting of an equal mixture of ethanol and tetrahydrofuran was added to the resulting reaction solution, which was then centrifuged at 10,000 rpm at 4°C for 10 minutes. The collected precipitate was dried under reduced pressure for 1 hour. 2.5 mL of ultrapure water was added to dissolve the resulting dried product. An equal volume of sodium hydrogen sulfide solution adjusted to 60 mM with 0.3 M sodium hydroxide was then added, and the mixture was allowed to react at room temperature for 1 hour to obtain oxidized glutathione persulfide. The resulting oxidized glutathione persulfide was purified using a synthetic adsorption resin, concentrated, and dried to obtain GSSSG powder.

[0029] (Test to confirm the melanin production inhibitory effect of the combined use of oxidized glutathione trisulfide and polyphenols) B16 mouse melanoma cells were seeded in 6-well plates (2.0 x 10^4 cells / well) and cultured for 24 hours in D-MEM medium supplemented with 5% fetal bovine serum (5% CO2, 37°C). Then, α-MSH (final concentration 200 nM) was added to serum-free D-MEM medium and cultured for 24 hours to induce melanin. After melanin induction, the cells were washed with PBS(-) and harvested by treatment with 0.25% trypsin (containing EDTA). Then, the cells were centrifuged at 10,000 rpm for 3 minutes to pellet the cells. The resulting pellet was washed twice with PBS(-) and dissolved in 150 μL of 1 M sodium hydroxide solution at 100°C for 10 minutes. This was used as the sample for Reference Example 1. Furthermore, a test group to which 7.5 μg / mL of oxidized glutathione trisulfide and 3.75 μg / mL of tannic acid were added during melanin induction was designated as the sample of Example 1. Similarly, a test group to which 7.5 μg / mL of oxidized glutathione trisulfide and 15 μg / mL of tannic acid were added during melanin induction was designated as the sample of Example 2. In addition, test groups containing 7.5 μg / mL of oxidized glutathione trisulfide alone, 3.75 μg / mL of tannic acid alone, or 15 μg / mL of tannic acid alone were also set up. Samples from each test group were added to a 96-well plate, and the absorbance at 405 nm was measured using a microplate reader (n=3). The melanin production amount (%) was calculated as a relative value for each test group, with the value for Reference Example 1 set at 100. The melanin production amount for each test group was then subtracted from 100 to obtain a value that was used as the improvement rate of melanin production for each test group.

[0030] The calculated improvement rates (calculated values) and actual improvement rates (measured values) are shown in Figure 1. When the amount of melanin produced in the test group where only melanin induction was performed (Reference Example 1) was set to 100, the amount of melanin produced in the test group containing 7.5 μg / mL of GSSSG alone was 95.3 (improvement rate: 4.7%), the amount of melanin produced in the test group containing 3.75 μg / mL of tannic acid alone was 82.1 (improvement rate: 17.9%), and the amount of melanin produced in the test group containing 15 μg / mL of tannic acid alone was 80.2 (improvement rate: 19.8%). In other words, the sum of the improvement rates for the test group to which 7.5 μg / mL of oxidized glutathione trisulfide was added and the test group to which 3.75 μg / mL of tannic acid was added was 22.6%, and the sum of the improvement rates for the test group to which 7.5 μg / mL of oxidized glutathione trisulfide was added and the test group to which 15 μg / mL of tannic acid was added was 24.5%. On the other hand, the melanin production amount in the test group (Example 1) to which 7.5 μg / mL of oxidized glutathione trisulfide and 3.75 μg / mL of tannic acid were added was 75.9 (improvement rate: 24.1%), and the melanin production amount in the test group (Example 1) to which 7.5 μg / mL of oxidized glutathione trisulfide and 15 μg / mL of tannic acid were added was 75.9 (improvement rate: 24.1%). As described above, the measured melanin production inhibition rates of Examples 1 and 2 were higher than the calculated values. In other words, it was revealed that the combination of GSSSG with polyphenols showed even greater effects than the use of GSSSG alone. Furthermore, although results are not shown, a synergistic melanin production inhibition effect was observed when combined with propyl gallate, suggesting that similar effects can be expected with polyphenols other than tannic acid. These results suggest that the composition of the present invention is effective against pigmentation in the skin and exhibits a synergistic anti-aging effect.

[0031] (Antioxidant effect confirmation test of the combined use of oxidized glutathione trisulfide and polyphenols) Normal human fibroblasts were seeded in 96-well plates (3.0 × 10^3 cells / well, N = 3) and pre-cultured for 24 hours at 5% CO2 and 37°C. MEM-α nucleosides medium (Gibco) containing fetal bovine serum (10% exogenous fetal bovine serum) was used. After washing the wells once with PBS(-), the medium was replaced with MEM-α nucleosides medium containing fetal bovine serum (5% exogenous fetal bovine serum) containing GSSSG and / or propyl gallate and incubated for another 30 minutes. Oxidative stress was then induced by treatment with hydrogen peroxide at a final concentration of 0.5 mmol / L. Subsequently, MTT assays were performed according to standard methods, and fibroblast viability was calculated relative to the viability of cells not treated with oxidized glutathione trisulfide or oxidative stress induction. In addition, the survival rate of cells in which oxidative stress was only induced without adding either oxidized glutathione or polyphenols was calculated. Here, the test groups to which GSSSG and polyphenols were added were a test group using 60 μg / mL GSSSG and 60 μg / mL propyl gallate in combination (Example 3), and a test group using 60 μg / mL GSSSG and 120 μg / mL propyl gallate in combination (Example 4). The difference in cell viability between these groups and cells subjected to oxidative stress induction alone was calculated to calculate the recovery rate, which indicates the degree to which cell viability in the test group using these two groups improved compared to the test group subjected to oxidative stress induction alone. Furthermore, the recovery rate for cells to which either GSSSG or polyphenols were added was calculated, and the total recovery rate when each was used alone was compared with the recovery rate when both were used in combination.

[0032] The recovery rates in the test plots where GSSSG and propyl gallate were used together are shown in Figure 2. The calculated values ​​in the figure are the sum of the recovery rates obtained when GSSSG or propyl gallate was tested alone. The cell viability in the test group where only oxidative stress was induced was 52.1%. The cell viability in the test group where 60 μg / mL of GSSSG was administered alone was 64.7%, representing a recovery rate of 12.6 points. The cell viability in the test group where 60 μg / mL of propyl gallate was administered alone was 102.2%, and the cell viability in the test group where 120 μg / mL of propyl gallate was administered alone was 135.6%, representing recovery rates of 50.1 and 83.5 points, respectively. On the other hand, the actual measured survival rate in Example 3 was 119.2%, resulting in a recovery rate of 67.1 points, and the actual measured survival rate in Example 4 was 157.5%, resulting in a recovery rate of 105.4 points, both of which were higher than the calculated values. In other words, it was revealed that the combined use of GSSSG and polyphenols exhibited a synergistic antioxidant effect compared to GSSSG alone. Furthermore, since a synergistic effect on the inhibition of melanin production was observed between propyl gallate and tannic acid, a similar antioxidant effect is expected in the test area where tannic acid was also used. Fibroblasts are generally known to be involved in the production of collagen and hyaluronic acid in the skin. Therefore, improving the survival rate of fibroblasts is thought to improve the production of hyaluronic acid, collagen, and other substances in the skin, thereby improving wrinkles and improving elasticity in the skin, thereby providing an anti-aging effect.

[0033] These results demonstrate that the combined use of glutathione persulfide and polyphenols can synergistically suppress melanin production in melanin-induced cultured cells. Furthermore, a synergistic cytoprotective effect was observed in fibroblasts induced with oxidative stress. Therefore, the composition of the present invention was shown to have excellent anti-aging properties. Furthermore, since the polyphenols used in the present invention can be used in cosmetics and foods, the composition of the present invention can be used in general foods and beverages, cosmetics, pharmaceuticals, etc.

Claims

1. An anti-aging composition comprising a persulfide of a glutathione analog or a persulfide of a cysteine ​​analog, and a polyphenol.

2. 2. The anti-aging composition according to claim 1, wherein the polyphenol is present in an amount of 0.01 to 100 parts by weight per part by weight of the persulfide of a glutathione analog or the persulfide of a cysteine ​​analog.

3. 3. The anti-aging composition according to claim 1, wherein the polyphenol is a polyphenol having a galloyl group or a resorcinol group.

4. A composition for external use comprising the composition according to claim 1 or 2.

5. An oral composition comprising the composition of claim 1 or 2.

6. A method for inhibiting aging, comprising administering the anti-aging composition according to claim 1 or 2 as an oral or external preparation.

7. A method for improving the anti-aging activity of a persulfide of a glutathione analog or a cysteine ​​analog, characterized by using a persulfide of a glutathione analog or a persulfide of a cysteine ​​analog in combination with a polyphenol.

Images