Persulfide-containing liquid composition

Abstract

The present invention addresses the problem of providing: a liquid composition which contains a high concentration of an oxidized glutathione persulfide and can be used as an external or oral composition; and a method for producing the same. The present invention also addresses the problem of providing a method for increasing the concentration of oxidized glutathione persulfide in a liquid composition. The present invention solves said problems by preparing a liquid composition containing at least 1.5 wt% of an oxidized glutathione persulfide by suspending a persulfide of oxidized glutathione in a polyol and then heating the same.

Description

Persulfide-containing liquid composition

[0001] The present invention relates to a liquid composition containing a high concentration of persulfide and a method for producing the same.

[0002] In recent years, persulfides have been discovered as glutathione-related substances, and their high physiological activity has attracted attention. However, persulfides of oxidized glutathione are known to be highly reactive, i.e., less stable. Furthermore, their lower solubility in water compared with reduced glutathione (GSH) and oxidized glutathione (GSSG) has also been problematic. Oxidized glutathione trisulfide dihydrate crystals obtained by purifying oxidized glutathione trisulfide, a type of oxidized glutathione persulfide, have been reported (Patent Document 1). However, due to their low solubility, they are thought to precipitate as persulfide hydrate crystals over time. In fact, when oxidized glutathione trisulfide dihydrate crystals are dissolved in water at room temperature and then left standing at room temperature for a long period of time, they recrystallize and precipitate; therefore, the concentration of the aqueous solution at which they do not form crystals is approximately 0.1%. Although persulfides of oxidized glutathione dissolve in strong acids such as hydrochloric acid to form highly concentrated solutions, low pH solutions may be irritating to the skin and mucous membranes when used in topical or oral compositions. Therefore, they must be adjusted to a usable pH after dissolution or used as a diluted solution at a low concentration. On the other hand, although some solubility can be improved by adding basic solvents or compounds to raise the pH, it is known that persulfides decompose under basic conditions.

[0003] On the other hand, a method using a 1,2-diol compound having a cyclohexane ring and an alkylene group is known as a method for dissolving poorly water-soluble compounds (Patent Document 2). However, because this method uses a chemically synthesized compound, it does not meet the needs of consumers who prefer natural products.

[0004] Therefore, there has been a need for a liquid composition containing a high concentration of persulfides of oxidized glutathione that can be used as a topical or oral composition and that uses compounds derived from natural products or that are readily available.

[0005] WO2018 / 117186 Pamphlet Japanese Patent Application Laid-Open No. 2022-031761

[0006] Br J Pharmacol., 176(4):671-683 (2019)

[0007] The present invention aims to provide a liquid composition containing a high concentration of oxidized glutathione persulfide that can be used as an external or oral composition, a method for producing the same, and a method for increasing the concentration of oxidized glutathione persulfide in the liquid composition.

[0008] As a result of intensive research into solving the above problems, the present inventors discovered that a liquid composition containing 1.5 wt % or more of oxidized glutathione persulfide can be prepared by suspending oxidized glutathione persulfide in a polyol and then heating the suspension, and further, that no precipitation of oxidized glutathione persulfide is observed even when the composition is stored in a refrigerator, thereby completing the present invention.

[0009] That is, the present invention relates to the following (1) to (8). (1) A liquid composition containing 1.5 wt% or more of glutathione persulfide or persulfide of a glutathione analog. (2) The composition according to (1), wherein the solvent of the composition is a polyol having a water content of 30% or less. (3) A method for producing the liquid composition according to (1) or (2), comprising the steps of suspending glutathione persulfide or persulfide of a glutathione analog in a polyol and heating the resulting mixture. (4) A method for producing the liquid composition according to (3), wherein the water content of the polyol is 30% or less. (5) A method for increasing the concentration of glutathione persulfide or persulfide of a glutathione analog, comprising suspending glutathione persulfide or persulfide of a glutathione analog in a polyol and heating the resulting mixture. (6) A method for inhibiting precipitation of glutathione persulfide or persulfide of a glutathione analog, comprising suspending glutathione persulfide or persulfide of a glutathione analog in a polyol and dissolving the suspension by heating. (7) A topical or oral composition comprising the liquid composition according to (1). (8) A method for producing a topical or oral composition, comprising adding the liquid composition according to (1).

[0010] According to the present invention, a liquid composition containing a high concentration of persulfide that can be used as an external or oral composition can be provided by suspending glutathione persulfide or glutathione analog persulfide in a liquid polyol and heating the resulting suspension.

[0011] Liquid composition using glycerin or water as a solvent Presence or absence of insoluble components in liquid composition using glycerin or water as a solvent

[0012] The liquid composition of the present invention is a liquid composition containing 1.5 wt. % or more of glutathione persulfide or glutathione analog persulfide (hereinafter, sometimes collectively referred to as "persulfide") and a polyol. Here, the glutathione analog in the present invention refers to reduced or oxidized γ-glutamylcysteine ​​or reduced or oxidized cysteinylglycine. Furthermore, the persulfide in the present invention 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 the molecule of the typical glutathione analog, reduced glutathione, or oxidized glutathione (e.g., RS-SH or RSSS-R'; R and R' are any functional groups). Any compound in which one or more sulfur atoms are directly bonded to a sulfur atom in the molecule of glutathione analog, reduced glutathione, or oxidized glutathione 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 liquid composition of the present invention is preferably an oxidized type, more preferably an oxidized glutathione persulfide, and even more preferably an oxidized glutathione persulfide containing three sulfur atoms per molecule (oxidized glutathione trisulfide; hereinafter sometimes referred to as GSSSG).

[0013] Persulfides can be obtained using commonly available glutathione analogs, reduced glutathione, or oxidized glutathione as raw materials. For example, 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 substances, solutions, suspensions, or the like. For the liquid composition of the present invention, crystals or amorphous substances are preferably used, with amorphous substances being more preferred.

[0014] Glutathione persulfide and glutathione analog persulfide are prone to precipitation. For example, oxidized glutathione trisulfide dihydrate crystals temporarily dissolve in water at room temperature, but precipitate over time, resulting in a solution with a concentration of approximately 0.1% by weight, which is low enough to prevent precipitation. Furthermore, their solubility in organic solvents is even lower, being almost insoluble in ethanol, methanol, acetone, acetonitrile, hexane, etc., even when heated. They dissolve in strong acids such as hydrochloric acid and sulfuric acid at pH 1 or below at room temperature.

[0015] The liquid composition of the present invention contains 1.5% by weight or more, preferably 2% by weight, of glutathione persulfide or glutathione analog persulfide. Furthermore, because precipitation of glutathione persulfide or glutathione analog persulfide is inhibited in the liquid composition of the present invention, the liquid composition contains glutathione persulfide or glutathione analog persulfide at the aforementioned concentration as a solution even when stored under refrigerated conditions. The persulfide content of the composition of the present invention can be determined by a common method using HPLC.

[0016] The liquid composition of the present invention contains a polyol. In the present invention, a polyol refers to an aliphatic compound having two or more hydroxy groups, or a carbohydrate in which at least some of the carbonyl groups in the molecule have been reduced to hydroxy groups. The polyol used in the present invention can be a commonly available polyol, such as glycerol, erythritol, arabitol, xylitol, mannitol, maltitol, 1,3-butylene glycol, propylene glycol, 3-methyl-1,3-butanediol, 1,3-propanediol, 2-methyl-1,3-propanediol, trimethylolpropane, pentaerythritol, sorbitol, pentylene glycol, hexylene glycol, 1,2-hexanediol, caprylyl glycol, diglycerin, polyglycerin, diethylene glycol, polyethylene glycol, dipropylene glycol, polypropylene glycol, and reduced starch saccharification products. The polyol used in the liquid composition of the present invention may be any of the aforementioned polyols, or a combination of two or more thereof. The polyol used in the liquid composition of the present invention is preferably one having a low water content, preferably 30% or less.

[0017] Furthermore, since the liquid composition of the present invention is used as an external composition that can be applied to the skin or mucous membranes or as an oral composition that can be orally ingested, the pH of the composition is preferably 2 to 7, more preferably 2.5 to 6.5, and even more preferably 3 to 6.

[0018] Furthermore, the composition of the present invention is less likely to produce precipitation of glutathione persulfides or glutathione analog persulfides even when left standing under refrigerated conditions, and when left standing under refrigerated conditions such as 4°C for 12 hours or more, the composition exhibits a concentration of dissolved persulfides of 1.5 wt% or more.

[0019] Whether or not the persulfide is dissolved in the liquid composition of the present invention can be determined by visual inspection. The liquid composition of the present invention is clear because 1.5 wt % or more of the persulfide is dissolved therein, but if the persulfide is not dissolved or precipitated, the liquid composition becomes a white suspension due to the presence of persulfide particles, and the precipitated particles can be visually confirmed as pellets by a general method such as centrifugation.

[0020] The solubility of persulfide can be determined by quantitatively analyzing the amount of persulfide added as a solute, the amount of dissolved persulfide, and the amount of undissolved persulfide by a general method using HPLC.

[0021] The liquid composition of the present invention can be produced by heating the polyol and persulfide in a coexistent state. For example, it can be produced by suspending the persulfide in the polyol and then heating. Heating may be performed with stirring. Heating conditions are preferably 55°C or higher, more preferably 60°C or higher, and even more preferably 70°C or higher. Furthermore, since high-temperature conditions may cause decomposition of the persulfide, heating temperatures are preferably 100°C or lower, more preferably 90°C or lower, and even more preferably 85°C or lower. The heating time can be adjusted as long as the conditions allow the persulfide to dissolve in the polyol. However, since prolonged heating may cause decomposition of the persulfide, heating times are preferably 1 to 60 minutes, more preferably 3 to 30 minutes, and even more preferably 5 to 20 minutes.

[0022] The liquid composition of the present invention thus obtained can be used directly as a topical composition that can be applied to the skin or mucous membranes, or as an oral composition that can be orally ingested. Furthermore, as long as the effects of the present invention are not impaired, it may be prepared as a mixture with other ingredients. Examples of other ingredients include thickeners, pH adjusters, flavorings, excipients, and bases for topical preparations, which are commonly used in topical and oral preparations. Furthermore, the composition can be prepared in the form of a solution, dispersion, powder, granules, capsules, tablets, paste, gel, or sheet according to conventional methods using solvents and bases commonly used in foods, supplements, cosmetic compositions, pharmaceutical compositions, and topical skin preparations. In this way, topical or oral compositions containing the liquid composition of the present invention can be obtained according to conventional methods.

[0023] 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.

[0024] The present invention also relates to a method for increasing the concentration of persulfides in a liquid composition. The persulfides in the liquid composition can be increased in concentration by suspending the persulfides in a polyol and heating the resulting mixture. The polyols that can be used include those described in the preceding paragraphs. The conditions for heating and other processes are not particularly limited as long as they can increase the concentration of persulfides in the composition, and can be set appropriately. This method allows the concentration of persulfides to be increased to 1.5% to 20% by weight, compared to a solution of about 0.1% by weight in water.

[0025] The present invention also relates to a method for inhibiting the precipitation of persulfides in a liquid composition. The precipitation of persulfides in a liquid composition can be confirmed, for example, by preparing a liquid composition containing a persulfide and then allowing it to stand overnight at 4°C. The method for inhibiting the precipitation of persulfides of the present invention can be achieved by suspending a persulfide in a polyol and heating the resulting mixture. The polyols that can be used may be those described in the preceding paragraphs. The conditions for heating, etc., are not particularly limited as long as they can increase the concentration of persulfides in the composition, and can be set appropriately. The liquid composition containing a persulfide prepared in this manner has inhibited persulfide precipitation, and can be handled while maintaining a high persulfide concentration. By diluting the liquid composition as needed, the composition can be made into a topical composition that can be applied to the skin or mucous membranes or an oral composition that can be ingested.

[0026] The present invention will be specifically explained below using examples, but the present invention is not limited to these examples.

[0027] The persulfide was produced by the following method based on a previously published paper (Non-Patent Document 1). Oxidized glutathione (GSSG), available commercially 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 the mixture was 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 recovered precipitate was dried under reduced pressure for 1 hour. 2.5 mL of ultrapure water was added to the resulting dried product, which was then dissolved. 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 obtained oxidized glutathione persulfide was purified using a synthetic adsorption resin, concentrated, and then freeze-dried to obtain an amorphous powder of oxidized glutathione trisulfide (GSSSG) (Sample 1). Furthermore, crystals of oxidized glutathione trisulfide dihydrate (Sample 2) were obtained according to the description in the Examples of Patent Document 1.

[0028] Preparation of Liquid Compositions and Confirmation of Persulfide Solubility Using water or glycerin, a representative polyol, as a solvent, suspensions of Sample 1 or Sample 2 were prepared at 10 mg / mL or 200 mg / mL. Each suspension was heated at 60°C or 80°C for 10 minutes with stirring to prepare a liquid composition. After heating, the suspensions were centrifuged, and the amount of GSSSG in the supernatant was quantified using HPLC, and the concentration of GSSSG in the liquid composition was calculated. The pH of the 1 wt% aqueous solution at this time was 2.7. Furthermore, for the test sections of Sample 1 (water or glycerin) heated at 80°C, the liquid composition was photographed after standing overnight at 4°C to confirm the presence or absence of persulfide precipitation.

[0029] The concentrations of GSSSG in the liquid compositions immediately after preparation, calculated from the results of HPLC analysis, are shown in Table 1. Figures 1 and 2 also show the test sections (water or glycerin) of Sample 1 heated at 80°C and then left overnight at 4°C. Table 1 shows that when glycerin was used as the solvent, Sample 1 heated at 60°C yielded a 2 wt% liquid composition, and when heated at 80°C, a 20 wt% liquid composition (pH 3.6) was obtained. On the other hand, when the solvent was water, both Sample 1 and Sample 2 exhibited low solubility regardless of temperature, with a maximum concentration of approximately 1 wt% immediately after preparation. Furthermore, Sample 2, which is crystalline, did not become a highly concentrated liquid composition in the presence of glycerin, and exhibited a solubility similar to that of water. Furthermore, Figures 1 and 2 (1: glycerin, 2: water) show that the test section in which sample 1 was heated in glycerin remained clear even after standing overnight at 4°C, despite being 20% ​​by weight. However, the test section in which sample 1 was heated in water became cloudy after standing, and GSSSG precipitation was observed. A 1% by weight aqueous solution of sample 2 (pH 2.7 immediately after dissolution) prepared in a similar manner showed precipitation within approximately 2 hours of standing at room temperature. Furthermore, when GSSSG solutions of the same concentration were prepared by changing the pH of the solvent, no precipitation was observed at pH 1.3, but precipitation of GSSSG was observed in solutions prepared at pH 1.7, 1.8, 2.0, 2.4, or 2.5. These results suggest that heating GSSSG in glycerin can achieve a concentration of GSSSG at least twice that of water. Furthermore, no precipitation was observed even when left standing at 4°C, suggesting that the precipitation and recrystallization of GSSSG was inhibited.

[0030]

[0031] <Effect of Water Content of Glycerin on Liquid Compositions> Sample 1 (amorphous GSSSG) was used to examine the effect of water content of glycerin on solubility and precipitation. Glycerin was mixed with water to give 25%, 50%, 75%, 80%, 85%, 90%, or 95% glycerin. Tests were also conducted using water (0% glycerin) and 100% glycerin. Sample 1 was added to each solvent to a concentration of 200 mg / mL and heated to 80°C. The solution was then left to stand overnight at 4°C, and the concentration of GSSSG in the solution was measured by HPLC.

[0032] The results are shown in Table 2. A liquid composition containing 2.41 wt% oxidized glutathione trisulfide was obtained in the 75% glycerin (25% water) group. It was also revealed that the concentration of oxidized glutathione trisulfide could be increased up to approximately 20 wt% by further reducing the water content. Furthermore, in the test groups containing 95% or more glycerin, no GSSSG precipitation was observed even when left refrigerated, suggesting that not only was the GSSSG concentration increased, but precipitation was also suppressed. These results demonstrate that the use of glycerin allows for the production of a high-concentration liquid composition containing approximately 20 wt% oxidized glutathione trisulfide, and that a water content of 25% or less results in a solution with a concentration more than twice that of water.

[0033]

[0034] <Preparation of Liquid Compositions Using Reduced Starch Saccharides and Sugar Alcohols> As polyols other than glycerin, "Sorbit D-70 (sorbitol, manufactured by Mitsubishi Corporation Life Sciences)" and "PO-20 (reduced starch saccharides, manufactured by Mitsubishi Corporation Life Sciences)" were used. Both polyols contain 30% water. "PO-20" is a reduced starch saccharide with a sugar composition of 2-5% monosaccharides, 9-14% disaccharides, 11-16% trisaccharides, and 67-76% tetrasaccharides or more. Sample 1 (amorphous GSSSG) was suspended in each polyol and heated at 80°C for 10 minutes to prepare liquid compositions. A liquid composition containing 1.5 wt% GSSSG was prepared in the sorbitol group. Furthermore, a liquid composition containing 1.7 wt% GSSSG was prepared in the reduced starch saccharide group. Both liquid compositions contained 1.5 wt% or more GSSSG. Furthermore, these liquid compositions were allowed to stand at 4°C for 12 hours or more, but no precipitation was observed.

[0035] <Effect of Water Content of Reduced Starch Saccharification Products and Sugar Alcohols on Liquid Compositions> As in the previous test, "Sorbit D-70 (sorbitol, manufactured by Mitsubishi Corporation Life Sciences)" and "PO-20 (reduced starch saccharification products, manufactured by Mitsubishi Corporation Life Sciences)" were used as polyols other than glycerin. Because both polyols contain 30% water, powdered sorbitol "LTS-P (manufactured by Mitsubishi Corporation Life Sciences)" was added to adjust the water content, and solvents with polyol contents of 20%, 50%, 65%, and 77.5% were prepared. Sample 1 (amorphous GSSSG) was suspended in each solvent and heated at 80°C for 10 minutes to prepare liquid compositions. The solutions were then allowed to stand overnight at 4°C, and the GSSSG concentrations in the solutions were analyzed by HPLC.

[0036] The results are shown in Tables 3 and 4. In all test plots, the concentration of oxidized glutathione trisulfide reached 1.5 wt% or higher when the moisture content was 30% or less. In test plots with even lower moisture contents, the concentration was 8.92 wt% in the "Sorbit D-70" test plot and 1.85 wt% in the "PO-20" test plot. Furthermore, when a similar test was conducted using liquid maltitol "Amalti Syrup (Mitsubishi Corporation Life Sciences)" with a moisture content of 25% and powdered sorbitol "LTS-P (Mitsubishi Corporation Life Sciences)" to achieve a moisture content of 18.75%, a liquid composition with a concentration of 4.12 wt% was obtained. These results indicate that, as long as the moisture content is at least 30% or less, a liquid composition with a higher concentration can be obtained compared to a solution of oxidized glutathione trisulfide using water as the solvent. Furthermore, since no precipitation was observed even when the high-concentration liquid composition was stored under refrigerated conditions, it became clear that precipitation of persulfides was also suppressed when polyols other than glycerin were used.

[0037]

[0038]

[0039] From the above, it was revealed that a high-concentration liquid composition can be prepared by suspending oxidized glutathione persulfide in a polyol and heating it. The same is thought to be true for persulfides of glutathione analogs with similar chemical structures. Furthermore, since no precipitation was observed even when a high-concentration liquid composition of persulfide was prepared and left to stand at 4°C, it is thought that the precipitation of persulfide is suppressed by the polyol.

Claims

1. A liquid composition containing 1.5% by weight or more of glutathione persulfide or glutathione analog persulfide.

2. The composition of claim 1, wherein the solvent of said composition is a polyol having a water content of 30% or less.

3. A method for producing the liquid composition according to claim 1 or 2, comprising the steps of suspending glutathione persulfide or persulfide of a glutathione analogue in a polyol and heating the mixture.

4. The method for producing a liquid composition according to claim 3, wherein the water content of the polyol is 30% or less.

5. A method for increasing the concentration of glutathione persulfide or glutathione analog persulfide, which comprises suspending glutathione persulfide or glutathione analog persulfide in a polyol and heating the suspension.

6. A method for inhibiting the precipitation of glutathione persulfide or glutathione analog persulfide, which comprises suspending glutathione persulfide or glutathione analog persulfide in a polyol and dissolving the suspension by heating.

7. A topical or oral composition comprising the liquid composition of claim 1.

8. A method for producing a composition for external use or an oral composition, comprising the step of adding the liquid composition according to claim 1.

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