Liquid composition containing persulfide and amino acid

Abstract

[Problem] The present invention addresses the problem of providing a liquid composition that contains a high concentration of oxidized glutathione persulfide and can be used as a pharmaceutical, a food or beverage, or a cosmetic, and a method for producing the liquid composition. The present invention also addresses the problem of providing a method for increasing the concentration of oxidized glutathione persulfide in a liquid composition and a method for suppressing precipitation of oxidized glutathione persulfide. [Solution] A liquid composition containing at least 0.3 wt% of oxidized glutathione persulfide can be prepared by suspending oxidized glutathione persulfide and an amino acid in a solvent and then heating the suspension.

Description

Liquid composition containing persulfides and amino acids

[0001] The present invention relates to a liquid composition containing persulfide and amino acids, a method for increasing the concentration of persulfide, and a method for suppressing precipitation.

[0002] In recent years, supersulfur molecules (also known as active sulfur compounds, supersulfur compounds, persulfides, sulfen sulfur, persulfide, polysulfide, and supersulfide) have attracted attention as glutathione-related and cysteine-related substances, and their high reactivity and physiological activity are anticipated. However, persulfides of oxidized glutathione are known to be highly reactive, meaning they have low stability, and their low solubility in water compared to reduced glutathione (GSH) and oxidized glutathione (GSSG) has also been a problem.

[0003] To date, oxidized glutathione trisulfide dihydrate crystals, obtained by purifying oxidized glutathione trisulfide, a type of oxidized glutathione persulfide, have been reported (Patent Document 1). However, this is thought to be due to precipitation as persulfide hydrate crystals over time because of low solubility. In fact, when oxidized glutathione trisulfide dihydrate crystals are dissolved in water at room temperature and left to stand at room temperature for a long time, they precipitate by recrystallization, resulting in an aqueous solution concentration of about 0.1% that does not produce crystals. Although oxidized glutathione persulfides dissolve in strong acids such as hydrochloric acid to form high-concentration solutions, if intended for use in topical or oral compositions, low-pH solutions may be irritating to the skin and mucous membranes. Therefore, it is necessary to adjust the pH to a usable level after dissolution or to use a low-concentration diluted solution. On the other hand, although some improvement in solubility is observed by raising the pH with the addition of basic solvents or compounds, it has been known that decomposition of persulfides progresses under basic conditions.

[0004] As described above, persulfides have been problematic due to their low stability resulting from high reactivity and their low solubility. For this reason, various studies have been conducted to obtain stably handleable liquid compositions containing oxidized glutathione persulfide, and for example, a method for stabilizing persulfides by using a novel tyrosine derivative has been reported (Patent Document 2). In addition, a method for stabilizing persulfides using a hydroxy compound is known (Patent Document 3).

[0005] On the other hand, as a method for dissolving poorly water-soluble compounds, a method using a 1,2-diol compound having a cyclohexane ring and an alkylene group is known (Patent Document 4). However, since it uses chemically synthesized compounds, it did not satisfy the needs of consumers who prefer products derived from natural products.

[0006] For this reason, there has been a demand for a liquid composition containing a high concentration of oxidized glutathione persulfide, which uses a compound derived from a natural product or a readily available compound.

[0007] WO 2018 / 117186 pamphlet, WO 2021 / 172523 pamphlet, Japanese Unexamined Patent Application Publication No. 2021-66722, Japanese Unexamined Patent Application Publication No. 2022-031761

[0008] Br J Pharmacol., 176(4):671-683 (2019)

[0009] An object of the present invention is to provide a liquid composition containing a high concentration of persulfide of oxidized glutathione or persulfide of an oxidized glutathione analog, which can be used as a pharmaceutical product, food and drink, or cosmetic product, and a method for producing the same. Another object of the present invention is to provide a method for increasing the concentration of oxidized glutathione persulfide in a liquid composition and suppressing the precipitation thereof.

[0010] As a result of intensive studies on the above problems, the present inventors found that by suspending oxidized glutathione persulfide and an amino acid in a solvent and then heating the mixture, a liquid composition containing 0.3% by weight or more of oxidized glutathione persulfide can be prepared, and further that precipitation of oxidized glutathione persulfide is not observed even after refrigerated storage, and thus completed the present invention.

[0011] In other words, the present invention relates to the following (1) to (8). (1) A liquid composition characterized by containing 0.3% by weight or more of glutathione persulfide or glutathione analog persulfide and an amino acid. (2) The liquid composition according to (1), wherein the amino acid is one or more selected from the group consisting of glycine, alanine, valine, serine, threonine, homoserine and salts thereof. (3) A method for producing the liquid composition according to (1) or (2), comprising the steps of suspending glutathione persulfide or glutathione analog persulfide and an amino acid in an aqueous solvent and heating the suspension. (4) A method for increasing the concentration of glutathione persulfide or glutathione analog persulfide, characterized by suspending glutathione persulfide or glutathione analog persulfide and an amino acid in an aqueous solvent and dissolving them by heating. (5) A method for suppressing the precipitation of glutathione persulfide or glutathione analog persulfide, characterized by suspending glutathione persulfide or glutathione analog persulfide and an amino acid in an aqueous solvent and dissolving them by heating. (6) A food or beverage containing the liquid composition described in (1) or (2). (7) A pharmaceutical product containing the liquid composition described in (1) or (2). (8) A cosmetic product containing the liquid composition described in (1) or (2).

[0012] According to the present invention, by coexisting glutathione analog persulfides and amino acids in a liquid composition, it is possible not only to increase the concentration of glutathione analog persulfides but also to suppress the precipitation of glutathione analog persulfides. Furthermore, it is possible to provide pharmaceuticals, food and beverages, and cosmetics containing a liquid composition comprising glutathione analog persulfides and amino acids.

[0013] The liquid composition of the present invention is a liquid composition comprising 0.3% by weight or more of glutathione persulfide or glutathione analog persulfide (hereinafter sometimes collectively referred to as persulfide) and an amino acid. Here, glutathione analog in the present invention refers to reduced or oxidized γ-glutamylcysteine ​​or reduced or oxidized cysteinylglycine. Furthermore, persulfide in the present invention refers to a molecule having a structure in which one or more sulfur atoms are directly bonded to a thiol group or sulfur atom constituting a disulfide bond present in the molecules of the usual glutathione analog, reduced glutathione, and oxidized glutathione (for example, R-S-SH or R-S-S-S-R'; R and R' are arbitrary functional groups). Any compound in which one or more sulfur atoms are directly bonded to a sulfur atom in the molecule of the glutathione analog, reduced glutathione, or oxidized glutathione can be used in the present invention, and it may also be used as a mixture of multiple types of compounds with different numbers of sulfur atoms in the molecule. The persulfide contained in the liquid composition of the present invention is preferably of the oxidized form, more preferably of oxidized glutathione persulfide, and even more preferably of oxidized glutathione persulfide containing three sulfur atoms per molecule (oxidized glutathione trisulfide; hereinafter sometimes referred to as GSSSG).

[0014] The method for obtaining the aforementioned persulfide can be based on previously published papers using the glutathione analogs that are generally available as raw materials (Non-Patent Literature 1). For example, oxidized glutathione persulfide can be obtained by reacting oxidized glutathione with peracetic acid on ice for 30 minutes, adding a solvent of equal parts ethanol and THF (tetrahydrofuran), and then centrifuging the resulting precipitate, which is then reacted with sodium hydrogen sulfide at room temperature. By purifying this oxidized glutathione persulfide, oxidized glutathione trisulfide can be produced, and it can be obtained in crystalline, amorphous, solution, or suspension forms. It is preferable to use crystalline or amorphous forms in the liquid composition of the present invention, and amorphous forms are more preferable.

[0015] Glutathione persulfides or glutathione analog persulfides are prone to precipitation. For example, dihydrate crystals of oxidized glutathione trisulfide temporarily dissolve in water at room temperature but precipitate over time, so a solution of about 0.1% by weight is preferred to prevent precipitation. Furthermore, their solubility in organic solvents is even lower; they are almost insoluble in ethanol, methanol, acetone, acetonitrile, and hexane, and this remains true even when heated. They dissolve at room temperature in strong acids such as hydrochloric acid and sulfuric acid with a pH of 1 or lower.

[0016] The liquid composition of the present invention contains 0.3% by weight or more, preferably 0.5% by weight, of glutathione persulfide or glutathione analog persulfide. Furthermore, because the precipitation of glutathione persulfide or glutathione analog persulfide is suppressed in the liquid composition of the present invention, even when left standing under refrigerated conditions, the solution contains the aforementioned concentration of glutathione persulfide or glutathione analog persulfide. The persulfide content in the composition of the present invention can be determined by a general method using HPLC.

[0017] The liquid composition of the present invention comprises the persulfide and an amino acid. In the present invention, an amino acid refers to a compound containing one or more amino groups and one or more carboxyl groups in a single molecule. Examples include glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, asparagine, glutamine, proline, phenylalanine, tryptophan, lysine, cysteine, methionine, tyrosine, arginine, histidine, serine, threonine, homoserine, and salts thereof. The amino acids contained in the liquid composition of the present invention can be those that are generally available. Furthermore, a combination of multiple amino acids may be used, either the L-form or the D-form may be used, or a mixture of both may be used. Glycine, alanine, valine, serine, threonine, and homoserine are preferred as amino acids that can be used in the present invention, and glycine, alanine, and serine are more preferred. Each amino acid can be quantified by analyzing it using general methods. For example, the amount of amino acids in the analytical sample can be determined from a calibration curve created using HPLC from standards of known concentration.

[0018] The persulfide and amino acids contained in the liquid composition of the present invention preferably consist of 3.3 parts by weight or more, and more preferably 3.5 parts by weight or more, of amino acids per 1 part by weight of persulfide. Furthermore, considering the solubility of amino acids in the liquid composition, it is preferable that the amount of amino acids be 10 parts by weight or less, and more preferably 9 parts by weight or less, of amino acids per 1 part by weight of persulfide.

[0019] An aqueous solvent can be used as the solvent for the liquid composition of the present invention. In the present invention, an aqueous solvent refers to water, a solvent that mixes with water in any ratio, such as ethanol, which is used in food and beverages, pharmaceuticals, and cosmetics, and a buffer solution, with water being a preferred solvent.

[0020] Furthermore, since the liquid composition of the present invention is used as a topical composition applicable to the skin and mucous membranes, or as an orally ingestible pharmaceutical, food, beverage, or cosmetic, the pH of the composition is preferably 2 to 7, more preferably 2.5 to 6.5, and even more preferably 3 to 6. The pH of the liquid composition may be adjusted by general methods.

[0021] Furthermore, the composition of the present invention is less prone to precipitation of glutathione persulfide or glutathione analog persulfide even when left standing under refrigerated conditions. For example, when left standing for 12 hours or more under refrigerated conditions such as 4°C, the dissolved persulfide shows a concentration of 0.3% by weight or more, preferably 0.5% by weight or more.

[0022] In the liquid composition of the present invention, it is possible to visually determine whether or not persulfides are dissolved. The liquid composition of the present invention is clear because the persulfides are dissolved, but if they do not dissolve or precipitate, the presence of persulfide particles results in a white suspension, which can be visually confirmed. In addition, the precipitated particles can be visually confirmed as pellets by common methods such as centrifugation.

[0023] Furthermore, the solubility of persulfides can be determined by quantitatively analyzing the amount of persulfide added as a solute, the amount of persulfide dissolved, or the amount of persulfide remaining undissolved, using a general method involving HPLC.

[0024] The liquid composition of the present invention can be produced by heating the persulfide and amino acids suspended in a solvent. The heating may be done while stirring. The heating temperature is preferably 55°C or higher, more preferably 60°C or higher, and even more preferably 70°C or higher. However, since high temperatures may cause the persulfide to decompose, the temperature is 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 appropriate to ensure the persulfide dissolves, but since prolonged heating may cause the persulfide to decompose, the heating time is preferably 1 to 60 minutes, more preferably 3 to 30 minutes, and even more preferably 5 to 20 minutes.

[0025] The liquid composition of the present invention obtained in this manner can be used as a pharmaceutical, food, beverage, or cosmetic, such as a topical composition that can be applied directly to the skin or mucous membranes, or an oral composition that can be ingested orally. Furthermore, as long as the effects of the present invention are not inhibited, a mixture prepared with other materials may also be used. Other materials include thickeners, pH adjusters, fragrances, excipients, and bases for topical preparations—materials commonly used in topical and oral preparations. Additionally, it can be prepared in the form of a solution, dispersion, powder, granules, capsule, tablet, paste, gel, or sheet using solvents and bases commonly used in foods, supplements, cosmetic compositions, pharmaceutical compositions, and topical skin preparations, according to conventional methods. In this way, pharmaceuticals, food, beverages, and cosmetics containing the liquid composition of the present invention can be obtained by following conventional methods.

[0026] The content of each component in the composition of the present invention can be appropriately adjusted by adding excipients and the like that commonly used in the above-described forms.

[0027] The present invention also relates to a method for increasing the concentration of persulfides in a liquid composition. The concentration of persulfides in a liquid composition can be increased by suspending persulfides and amino acids in a solvent and heating the mixture. The amino acids that can be used are those described above. The heating conditions are not particularly limited as long as they can increase the concentration of persulfides in the composition and can be set as appropriate. By this method, while a solution of about 0.1% by weight is obtained with water, the concentration of persulfides can be increased to 0.3% by weight or more.

[0028] The present invention also relates to a method for suppressing 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 persulfides and letting it stand overnight at 4°C. The method for suppressing persulfide precipitation of the present invention can be achieved by suspending persulfides and amino acids in a solvent and heating it. The amino acids that can be used are those described above. The heating conditions are not particularly limited as long as the concentration of persulfides in the composition can be increased, and can be set as appropriate. Since the precipitation of persulfides is suppressed in the liquid composition containing persulfides prepared in this way, it can be handled while maintaining a high concentration of persulfides, and by diluting it as necessary, it can be used as a topical composition that can be applied to the skin or mucous membranes, or an oral composition that can be taken orally, making it a pharmaceutical, food or beverage, or cosmetic.

[0029] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0030] The persulfide was prepared by the following method: Commercially available oxidized glutathione (GSSG) was dissolved in ultrapure water to prepare a 170 mM aqueous solution of oxidized glutathione. 2.5 mL of this aqueous solution was mixed with 4 mL of 32% by weight peracetic acid solution and reacted on ice for 30 minutes. 30 mL of a solvent mixture of equal parts ethanol and tetrahydrofuran was added to the resulting reaction mixture, and the mixture was 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 dried product and dissolved. An equal volume of sodium hydrogen sulfide solution, prepared to 60 mM with 0.3 M aqueous sodium hydroxide solution, was added, and the mixture was reacted 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 freeze-dried to obtain oxidized glutathione trisulfide (GSSG) powder.

[0031] (Preparation of liquid composition and confirmation test 1 of the effect of one type of amino acid on suppressing GSSSG precipitation) The precipitation suppression effect was confirmed using the GSSSG powder obtained by the method described above and a commercially available reagent-grade amino acid by the method described below. The GSSSG powder and amino acid were suspended in water, heated and stirred at 95°C for 5-10 minutes to completely dissolve, and then processed through a 0.2 μm filter to prepare a liquid composition. The obtained liquid composition was left to stand overnight at 4°C, a condition in which precipitation is likely to occur, and the presence or absence of persulfide precipitation was visually confirmed. At this time, the liquid composition was prepared so that the GSSSG content was 0.5% by weight. In addition, the test group to which amino acids were added was prepared by dissolving one of the following amino acids together with GSSSG to a concentration of 5% by weight. The amino acids used were glycine (Gly), alanine (Ala), valine (Val), serine (Ser), threonine (Thr), and homoserine (Hse). Hydroxyproline (Hyp) was also used as a comparison.

[0032] (Results of Test 1 to Confirm the Effect of One Amino Acid on Inhibiting GSSSG Precipitation) The results are shown in Table 1, with ○ indicating no GSSSG precipitation and × indicating precipitation. In the test group with GSSSG alone, GSSSG precipitation was observed after standing overnight under refrigerated conditions. On the other hand, when GSSSG was present with one of the following amino acids: glycine, alanine, valine, serine, threonine, or homoserine, no GSSSG precipitation was observed even after standing overnight under refrigerated conditions. In addition, although not shown in Table 1, as an additional study, the concentration of glycine, alanine, or serine, which showed a precipitation inhibitory effect at 5% by weight, was set to 2% by weight, and a precipitation inhibitory effect was observed in the test group with 0.5% by weight of GSSSG. Furthermore, a precipitation inhibitory effect was also observed when the concentration of GSSSG was 1% by weight and the amino acid concentration was 5% by weight.

[0033]

[0034] (Preparation of Liquid Composition and Confirmation Test 2 of the GSSSG Precipitation Inhibition Effect by One Amino Acid) The precipitation inhibitory effect in a 2 wt% GSSSG solution was confirmed using the same method as in the previous confirmation test 1 of the GSSSG precipitation inhibitory effect by one amino acid. The amino acid used was glycine, alanine, or serine, as examples of amino acids that showed a precipitation inhibitory effect in the previous step. The GSSSG concentration was also prepared to 3 wt%, 4 wt%, or 5 wt%, and glycine was used as a representative example of an amino acid that can be used in the present invention to obtain the liquid composition of the present invention. Each of the obtained liquid compositions was left to stand overnight at 4°C, a condition in which precipitation is likely to occur, and then the presence or absence of persulfide precipitation was checked visually.

[0035] (Results of Test 2 to Confirm the Effect of One Amino Acid on Inhibiting GSSSG Precipitation) The results are shown in Table 2, with ○ indicating no GSSSG precipitation and × indicating precipitation. In the test group with GSSSG alone, GSSSG precipitation was observed after standing overnight under refrigerated conditions. On the other hand, no precipitation was observed in any of the test groups with 16% by weight of glycine, alanine, or serine and a GSSSG concentration of 2% by weight. Furthermore, an inhibitory effect on precipitation was observed in the test group with 16% by weight of glycine and a GSSSG concentration of 3% or 4% by weight, but precipitation was observed when the GSSSG concentration reached 5% by weight, indicating that a liquid composition with a GSSSG concentration of less than 5% by weight can be obtained. From the above, it has become clear that glycine, alanine, and serine exhibit an inhibitory effect on precipitation of high concentrations of GSSSG. Threonine and homoserine are expected to have similar effects.

[0036]

[0037] (Confirmation test of high concentration and precipitation inhibition effect of GSSSG using multiple amino acids) Using the same method as in the previous confirmation test of the precipitation inhibition effect of GSSSG using one type of amino acid, the precipitation inhibition effect of combinations of two or more amino acids was confirmed. The same GSSSG and amino acids used in the previous tests were used. The presence or absence of persulfide precipitation was checked under the same conditions except for the use of two or more types of amino acids.

[0038] (Results of the test to confirm the effect of multiple amino acids on increasing the concentration of GSSSG and suppressing precipitation) The results are shown in Table 3. It was shown that by combining two types of amino acids from glycine, alanine, and serine, the precipitation of GSSSG can be suppressed in liquid compositions containing at least 2% by weight of GSSSG. In addition, although not shown in the table, in the test group in which 5% by weight of each of the three types of amino acids were used in combination, no precipitation of GSSSG was observed even when a 2% by weight solution of GSSSG was left standing at 4°C for 4 days. From the above, it was shown that it is possible to achieve a high concentration of GSSSG and further suppress precipitation by combining multiple amino acids. It was also suggested that the liquid composition can be stored under refrigeration for a long period of time.

[0039]

[0040] From the above, it has become clear that a liquid composition containing a high concentration of persulfide can be prepared by suspending glutathione persulfide and amino acids in a solvent and heating it. It is thought that the same can be done with glutathione analogues persulfides that have similar chemical structures. Furthermore, after preparing a liquid composition with a high concentration of persulfide, no precipitation was observed even when left to stand at 4°C, a condition where precipitation is likely to occur, suggesting that the amino acids suppress the precipitation of persulfide. Since the precipitation of persulfide is suppressed in the composition of the present invention, it can be used in general foods and beverages, cosmetics, pharmaceuticals, etc.

Claims

1. A liquid composition characterized by containing 0.3% by weight or more of glutathione persulfide or glutathione analog persulfide, and also containing amino acids.

2. The liquid composition according to claim 1, wherein the amino acid is one or more selected from the group consisting of glycine, alanine, valine, serine, threonine, homoserine, and salts thereof.

3. A method for producing a liquid composition according to claim 1 or 2, comprising the steps of suspending glutathione persulfide or glutathione analog persulfide and an amino acid in an aqueous solvent, and heating the suspension.

4. A method for increasing the concentration of glutathione persulfide or glutathione analog persulfide, characterized by suspending glutathione persulfide or glutathione analog persulfide and an amino acid in an aqueous solvent and dissolving them by heating.

5. A method for suppressing the precipitation of glutathione persulfide or glutathione analog persulfide, characterized by suspending glutathione persulfide or glutathione analog persulfide and an amino acid in an aqueous solvent and dissolving them by heating.

6. Food or beverage comprising the liquid composition according to claim 1 or 2.

7. A pharmaceutical product comprising the liquid composition according to claim 1 or 2.

8. A cosmetic comprising the liquid composition according to claim 1 or 2.

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