JP2022117993A - Aqueous solution containing stilbene compound, and superoxide production inhibitor containing stilbene compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an aqueous solution containing high-concentration stilbene compounds, and an inhibitor of superoxide production catalyzed by xanthine oxidase.

SOLUTION: The present invention discloses an aqueous solution in which 0.3 to 200 mM of a stilbene compound (A) being a compound expressed by the following formula (1) or a glycoside or multimer thereof, and 0.3 to 200 mM of a flavin derivative (B) selected from riboflavin, FMN or FAD are dissolved. Further, provided is an inhibitor of superoxide production catalyzed by xanthine oxidase, where the inhibitor contains the stilbene compound (A) as an active ingredient. In the formula (1), R¹, R² and R³ denote each independently a hydrogen atom or a methyl group, and X denotes a hydrogen atom, a hydroxy group or a methoxy group.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2022,JPO&INPIT

Description

TECHNICAL FIELD The present invention relates to an aqueous solution containing a stilbene compound and a water-solubility improver for the stilbene compound. The present invention also relates to a composition containing a highly water-soluble stilbene compound and a method for producing the same. It also relates to an efficient method for extracting stilbene compounds. Furthermore, it relates to a superoxide production inhibitor containing a stilbene compound.

Grape juice is known to contain polyphenols as active ingredients. Among them, resveratrol is a functional compound contained in wine, which is famous for the French paradox. Resveratrol has longevity, anti-aging, anti-hyperglycemia, anti-hyperuricemia, hyper-LDL, anti-hypertension, anti-inflammatory effects, improves obesity, diabetes, etc., and is also reported to be effective in preventing the onset of Alzheimer's disease. (See Non-Patent Documents 1 to 4). Grape skins contain resveratrol glycosides (piceide), but grape juice contains only a very small amount of piceide, so it is difficult to supply a large amount of glycosides from the juice. For example, an example of the component composition of commercially available 100% concentrated reduced grape juice is as follows, and contains only 0.02 mM of piceide.
Water: 86.9±2.5g/dL
Glucose: 5.87±0.21 g/dL
Fructose: 5.76±0.05 g/dL
Piceid: 0.00068 g/dL (0.02 mM)
Polyphenol (as gallic acid): 0.088 g/dL

Resveratrol contained in grape leaf, stem and seed extracts can be used as a source of resveratrol. However, due to its hydrophobicity, resveratrol is very sparingly soluble in water, with a solubility of only 0.19 mM in water at 20° C. (see Table 2 herein). In addition, in Non-Patent Documents 1 to 4, which report on the physiological activity of resveratrol, a fairly large amount (50 to 3000 mg/day) of resveratrol is ingested in order to express their physiological activity. there is Therefore, in order to sufficiently develop the effect, a large amount of intake such as 0.2 mmol or more per day is required. For example, when used as a supplement, preparation of a beverage containing high-concentration resveratrol is desired for efficient ingestion.

In order to produce a beverage containing resveratrol that is well absorbed by the body, techniques such as conversion to glycosides, clathration with cyclodextrin, and addition of alcohol to aqueous solutions have been attempted (non-patent literature 5, 6). However, efficient biocatalytic glycosylation of resveratrol has not yet been reported. Moreover, a method for obtaining a highly concentrated aqueous solution by inclusion of cyclodextrins and resveratrol has not yet been reported. Furthermore, the solubility of resveratrol in ethanol-containing aqueous solutions is not as great at low ethanol contents (see FIG. 3 herein). Therefore, it is not possible to dissolve a large amount of resveratrol in sake with a low alcohol content, and it is desirable to dissolve resveratrol in non-alcoholic beverages in consideration of health.

Xanthine oxidase (XO) is an enzyme that catalyzes the reaction of oxidizing hypoxanthine to produce xanthine in vivo and further catalyzes the reaction of oxidizing xanthine to produce uric acid. Therefore, inhibition of the action of xanthine oxidase is effective in treating hyperuricemia and gout. For example, Non-Patent Document 7 describes that gallic acid alkyl esters are effective as xanthine oxidase inhibitors. A reaction in which hypoxanthine is oxidized to xanthine and uric acid, and a reaction in which oxygen (O ₂ ) is reduced to superoxide (O ₂ ^- .) and hydrogen peroxide (H ₂ O ₂ ) form the so-called ping-pong mechanism. to proceed. Excessive production of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide causes oxidative stress, including lipid peroxide formation, and may adversely affect health and the food industry (Non-Patent Document 8. reference).

Szkudelski T, Szkudelska K. Resveratrol and diabetes: from animal to human studies. Biochim Biophys Acta. 2015; 1852(6):1145-1154. Pasinetti GM, Wang J, Ho L, Zhao W, Dubner L. Roles of resveratrol and other grape-derived polyphenols in Alzheimer's disease prevention and treatment. Biochim Biophys Acta. 2015; 1852(6):1202-1208. Khodabandehloo H, Seyyedebrahimi S, Esfahani EN, Razi F, Meshkani R. Resveratrol supplementation decreases blood glucose without changing the circulating CD14+CD16+ monocytes and inflammatory cytokines in patients with type 2 diabetes: a randomized, double-blind, placebo-controlled study. Nutr. Res, 2018; 54:40-51. Okada, T., Konishi, Y., Ishihara, K., Masuoka, N., Isomerization of stilbene compounds and stilbene in Polygonum cuspidatum. Naturalistae. 2009; 14: 17-21. Shimoda K, Kubota N, Hamada H, Hamada H. Synthesis of resveratrol glycosides by plant glucosyltransferase and cyclodextrin glucanotransferase and their neuroprotective activity. Nat. Prod. Commun. 2015; 10(6): 995-6. Szkudelska K, Deniziak M, Ros P, Gwozodz K, Szkudelski T. Resveratrol alleviates ethanol-induced hormonal and metabolic disturbances in the rat. Physiol. Res. 2017; 66: 135-145. Masuoka N, Nihei K, Kubo I, Xanthine oxidase inhibitory activity of alkyl gallates, Mol. Nutr. Food Res. 2006; 50, 725-731 Sunao Fujita, Generation and elimination mechanisms of active oxygen, lipid peroxides and free radicals and their biological actions, YAKUGAKU ZASSHI 2002; 122(3): 203-218

The present invention has been made to solve the above problems, and an object of the present invention is to provide an aqueous solution containing a stilbene compound at a high concentration. Another object of the present invention is to provide a water solubility improver capable of improving the solubility of a stilbene compound having low solubility in water. It is also an object of the present invention to provide a composition containing a stilbene compound and having good water solubility and a suitable method for producing the same. It is also an object of the present invention to provide a method for efficiently extracting stilbene compounds using water. It is also an object of the present invention to provide an inhibitor of superoxide formation catalyzed by xanthine oxidase, comprising the stilbene compound or an aqueous solution or composition containing the same.

The above problem is a compound represented by the following formula (1) or a stilbene compound (A) which is a glycoside or multimer thereof, and 0.3 to 200 mM of a compound represented by the following formula (2) or a pharmaceutical It is solved by providing an aqueous solution in which 0.3 to 200 mM of the flavin derivative (B), which is an acceptable salt for

[In formula (1), R ¹ , R ² and R ³ are each independently a hydrogen atom or a methyl group, and X is a hydrogen atom, a hydroxy group or a methoxy group. ]

[In formula (2), n is 0, 1 or 2. When n is 0 or 1, Y is a hydrogen atom. When n is 2, Y is a group represented by the following formula (3). ]

At this time, the stilbene compound (A) is at least one selected from the group consisting of resveratrol, piceatannol, rhapontigenin, isolapontigenin, pterostilbene and pinostilbene, and their glycosides and polymers. Preferably. It is also preferred that the molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) is 0.5-50. It is also preferable that the aqueous solution has a pH of 1.5-6. It is also preferred that the concentration of ethanol in the solvent is less than 20% by volume.

Furthermore, the aqueous solution containing a pH adjuster (C) selected from carboxylic acids having 2 to 6 carbon atoms and ascorbic acid and its glycosides is a preferred embodiment. A preferred embodiment of the present invention is an inhibitor of superoxide formation catalyzed by xanthine oxidase, comprising said aqueous solution. A preferred embodiment of the present invention is a beverage or cosmetic comprising the aqueous solution.

The subject is a water-solubility improver comprising a flavin derivative (B) for improving the water solubility of the stilbene compound (A); the stilbene compound (A) is represented by the formula (1) By providing a water-solubility-improving agent which is a compound or a glycoside or multimer thereof, and wherein the flavin derivative (B) is the compound represented by the above formula (2) or a pharmaceutically acceptable salt thereof is also resolved.

The subject is the compound represented by the formula (1) or a stilbene compound (A) which is a glycoside or polymer thereof, and the compound represented by the formula (2) or a pharmaceutically acceptable salt thereof. wherein the molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) is 0.5 to 50, and is selected from powders, granules, tablets or pastes It is also solved by providing a composition of the form. At this time, it is preferable to further contain a pH adjuster (C) selected from carboxylic acids having 2 to 6 carbon atoms, ascorbic acid and glycosides thereof.

Moreover, the method for producing the composition is preferable, in which an aqueous solution in which the stilbene compound (A) and the flavin derivative (B) are dissolved is prepared, and then water is removed. A preferred embodiment of the present invention is an inhibitor of superoxide formation catalyzed by xanthine oxidase, comprising said composition.

The object is to provide a plant raw material containing the compound represented by the formula (1) or a stilbene compound (A) which is a glycoside or multimer thereof, or a crude extract extracted from the plant raw material with an organic solvent, The compound represented by the formula (2) or a flavin derivative (B), which is a pharmaceutically acceptable salt thereof, is brought into contact with an aqueous solution in which the stilbene compound ( It is also solved by providing a method for extracting the stilbene compound (A), which extracts A).

Further, the object is to provide an inhibitor of superoxide formation catalyzed by xanthine oxidase, which contains, as an active ingredient, a stilbene compound (A) which is the compound represented by the formula (1) or its glycoside or multimer. can also be solved by In a preferred embodiment, R ¹ , R ² , R ³ and X in formula (1) are hydrogen atoms. In another preferred embodiment, in formula (1), R ¹ and R ² are hydrogen atoms, R ³ is a hydrogen atom or a methyl group, and X is a hydroxy group.

The aqueous solution of the present invention contains a stilbene compound such as resveratrol dissolved at a high concentration, and the stilbene compound can be ingested efficiently only by drinking a small amount. Since it is dissolved in water, it has excellent bioavailability after ingestion. Moreover, the aqueous solution of the present invention can dissolve a stilbene compound at a high concentration without adding an organic solvent such as ethanol. Furthermore, the flavin derivative contained in the aqueous solution of the present invention can also exert useful effects _on the living body as vitamin B2 and its metabolites. Therefore, the aqueous solution of the present invention is useful as beverages and cosmetics. Further, by using the water-solubility improver of the present invention, the solubility of the stilbene compound in water can be greatly improved.

The composition of the present invention is in the form of powder, granules, tablets, or paste, and has excellent water solubility and bioabsorbability. Therefore, the composition of the present invention is useful as a supplement or the like. Moreover, according to the extraction method of the present invention, the stilbene compound can be efficiently extracted with water without using an organic solvent.

Further, the stilbene compounds, the aqueous solutions containing the same, and the compositions containing the same are capable of inhibiting superoxide production catalyzed by xanthine oxidase.

1 is a graph showing changes in water solubility of stilbene compounds when the concentration of FMN is changed in Example 1. FIG. 2 is a graph showing changes in the solubility of resveratrol in an aqueous solution when the concentrations of FMN and FAD in the aqueous solution are changed in Example 2. FIG. 1 is a graph showing changes in the solubility of resveratrol in an aqueous solution when the concentration of ethanol in the aqueous solution is changed in Reference Example 1. FIG. In Example 5, the absorption spectrum of an aqueous solution containing 10 mM piceatannol and 10 mM FMN (dotted line) is compared with the absorption spectrum of an aqueous solution containing only 10 mM FMN (solid line). In Example 5, the absorption spectrum (dotted line) of an aqueous solution containing 0.01 mM piceatannol and 0.01 mM FMN was compared with the absorption spectrum (solid line) of an aqueous solution containing only 0.01 mM FMN. be. 10 is a graph showing the pH of an aqueous solution containing resveratrol, FMN and acetic acid when the acetic acid concentration is changed in Example 8. FIG. 4 is a graph showing pH when the concentration ratios of FMN and various acids are changed in Reference Example 2. FIG. 8 is a graph in which a portion of the horizontal axis of FIG. 7 is enlarged; Dissolution rate in water of mixed powder 1 obtained by mixing resveratrol powder and FMN powder and mixed powder 2 obtained by drying a mixed aqueous solution of resveratrol and FMN in Example 9. is a graph comparing 10 is a graph showing changes in superoxide scavenging activity when the concentration of the stilbene compound was varied in Example 14. FIG.

The present invention provides a compound represented by the following formula (1) or a stilbene compound (A) which is a glycoside or multimer thereof, and a compound represented by the following formula (2) or a pharmaceutical compound thereof. It is an aqueous solution in which 0.3 to 200 mM of a flavin derivative (B), which is an acceptable salt for , is dissolved. Although the stilbene compound (A) is hydrophobic and has low solubility in water, the solubility of the stilbene compound (A) can be greatly increased by dissolving it together with the flavin derivative (B).

First, the stilbene compound (A) will be explained. The stilbene compound (A) is a compound represented by the following formula (1) or a glycoside or polymer thereof.

R ¹ , R ² and R ³ in formula (1) are each independently a hydrogen atom or a methyl group. From the viewpoint of water solubility, one or more of R ¹ , R ² and R ³ are preferably hydrogen atoms, more preferably two or more are hydrogen atoms, and all three are hydrogen atoms. is more preferred. X in formula (1) is a hydrogen atom, a hydroxy group or a methoxy group.

The stilbene compound (A) may be a glycoside of the compound represented by formula (1). In that case, the sugar chain is bound to the hydroxyl group contained in the compound represented by formula (1). The sugar chain may be monosaccharide or polysaccharide. When the sugar chain is long, the water solubility of the stilbene compound (A) is improved, and the stilbene compound (A) often has sufficient water solubility without using the flavin derivative (B). Therefore, when the sugar chain is a monosaccharide or a disaccharide, especially when it is a monosaccharide, the adoption of the present invention is of great significance. The sugar that forms the sugar chain is not particularly limited, and examples thereof include glucose and β-rutinose, but glucose is preferred. Preferably, it is a glucoside in which one molecule of glucose is bound. Also, a part of the hydroxyl groups of the stilbene compound (A) may form a salt, but the amount is preferably such that the pH of the aqueous solution is kept below 7. Preferred specific examples of the stilbene compound (A) are shown in Table 1 together with their structures.

Resveratrol is a stilbene compound having three hydroxyl groups and is an important compound in terms of physiological activity. Glucose (Glc) is bound to one of the hydroxyl groups of resveratrol in piceide (resveratrol-3-glucoside) and resveratrol-4'-glucoside. Piceatannol has a total of four hydroxyl groups with X being a hydroxyl group. Rhapontigenin and isorapontigenin are those in which one of the four hydroxyl groups of piceatannol has been changed to a methoxy group, and pinostilbene in which one or two of the hydroxyl groups of resveratrol have been changed to methoxy groups. and pterostilbene. Each of these compounds has been reported to have bioactivity.

Furthermore, the stilbene compound (A) may be a polymer of the compound represented by formula (1). The multimer may be one in which the molecules of the compound represented by formula (1) are simply bound to each other, may be one in which the molecules are dehydrogenated and bound, or one in which the molecules are dehydrated and bound. There may be. In particular, those bound by dehydrogenation are preferred. Further, it may be a dimer or a polymer of trimers or higher, but a dimer is preferred. For example, resveratrol multimers include ε-viniferin (formula (4) below), δ-viniferin, dehydrogenated dimers such as gnetin C, dehydrogenated trimers such as α-viniferin, hopeaphenol Dehydrogenated tetramers such as are exemplified.

Next, the flavin derivative (B) will be described. Flavin derivative (B) is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof.

In formula (2), n is 0, 1 or 2, Y is a hydrogen atom when n is 0 or 1, and Y is a group represented by the following formula (3) when n is 2. The compound when n is 0 and Y is a hydrogen atom is riboflavin ₍ vitamin B2). Compounds where n is 1 and Y is a hydrogen atom are flavin mononucleotides (FMN). Further, the compound when n is 2 and Y is a group represented by the following formula (3) is flavin adenine dinucleotide (FAD). Riboflavin is a water-soluble vitamin, and continuous intake is desired for maintenance of health. Moreover, since there is almost no problem of overdose, it is a desirable compound to be dissolved in the aqueous solution of the present invention. Both FMN and FAD are metabolites from riboflavin and are useful and safe compounds like riboflavin.

Flavin derivative (B) may be a pharmaceutically acceptable salt. The type of salt is not particularly limited as long as it is pharmaceutically acceptable. In particular, when n is 1 or 2, it is desirable that at least part of the phosphate units contained in the compound represented by formula (2) form a salt with a cation. The cationic species at this time is not particularly limited, but sodium, potassium, magnesium, calcium, zinc, ammonium and the like are exemplified.

The flavin derivative (B) used in the present invention may be any of riboflavin (vitamin B ₂ ), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). FMN and FAD are preferred from the viewpoint of effectively improving the As shown in the Examples (FIG. 2), the same molar number of FMN and FAD can dissolve similar amounts of the stilbene compound (A). Therefore, comparing the molecular weights of FMN and FAD, FMN is more preferable since it can improve the solubility with a smaller mass.

The concentration of the stilbene compound (A) dissolved in the aqueous solution of the present invention is 0.3-200 mM. The concentration of the stilbene compound (A) is preferably 1 mM or higher, more preferably 2 mM or higher, even more preferably 5 mM or higher, and particularly preferably 10 mM or higher. The more the stilbene compound (A) is dissolved, the more effectively the stilbene compound (A) can be ingested by drinking a small amount of the aqueous solution. Also, when used as a cosmetic, it can be more effectively absorbed through the skin. Some types of stilbene compound (A) cannot be dissolved at 200 mM, and in such cases, the saturated concentration in the aqueous solution is the upper limit.

The concentration of the flavin derivative (B) dissolved in the aqueous solution of the present invention is 0.3-200 mM. The concentration of the flavin derivative (B) is preferably 2 mM or higher, more preferably 5 mM or higher, even more preferably 10 mM or higher, and particularly preferably 15 mM or higher. The more flavin derivative (B) is dissolved, the more stilbene compound (A) can be dissolved. Some types of flavin derivative (B) cannot be dissolved at 200 mM, and in such cases, the upper limit is the saturating concentration.

As shown in Example 1 (FIG. 1), the solubility of the stilbene compound (A) increases approximately proportionally with the concentration of the flavin derivative (B). The number of molecules of the flavin derivative (B) required to dissolve one molecule of the stilbene compound (A) varies depending on the type of the stilbene compound (A). Excluding multimers, the stilbene compound (A) originally having higher water solubility can be dissolved by a smaller number of flavin derivative (B) molecules. The number of molecules of the flavin derivative (B) required to dissolve one molecule of the stilbene compound (A) is less than 1 molecule of piceatannol, about 1 molecule of piceid, rapontigenin, and isolapontigenin, and about 4 molecules of resveratrol. , pterostilbene is about 6 molecules. ε-viniferin, which is a dehydrogenated dimer of resveratrol, is originally not very water-soluble, but the water-solubility is greatly improved with a small amount of the flavin derivative (B).

As shown in Example 5, in the absorption spectra of stilbene compounds in aqueous solution, the addition of FMN bathochromically shifts each absorption maximum. Therefore, in the aqueous solution of the present invention, the stilbene compound (A) molecules and the flavin derivative (B) molecules are thought to associate at a predetermined ratio. It is confirmed in Example 5 (comparison between FIGS. 4 and 5) that this association disappears by diluting the aqueous solution. Therefore, it is considered that the association of the aromatic ring of the stilbene compound (A) and the aromatic ring of the flavin derivative (B) due to the π-π interaction is due to a relatively weak force and is dissociated by dilution.

In the aqueous solution of the present invention, the molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) is preferably 0.2-50. The molar ratio (B/A) is more preferably 0.5 or more, even more preferably 0.8 or more. When the stilbene compound (A) is resveratrol, the molar ratio (B/A) is preferably 2 or more, more preferably 3 or more. Since the flavin derivative (B) has almost no problems due to overdose, it does not matter if the flavin derivative (B) having a number of molecules larger than the number of molecules required for association is dissolved.

The pH of the aqueous solution of the present invention is preferably 1.5-6. As shown in Example 7 (Table 4), when an aqueous solution having a pH of 7 or higher is used, the amount of the stilbene compound (A) is reduced and easily changed to other compounds. The pH is more preferably 5.5 or less, more preferably 5 or less. On the other hand, if the pH is too low, the acidity is too strong when used as a beverage, or the skin is too irritated when used as a cosmetic. It is more preferable that it is above. In addition, considering that there are products that consumers dilute with water and use as beverages and cosmetics, the pH of the aqueous solution of the present invention is more preferably 1.5 or higher, more preferably 2 or higher. preferable.

The method for adjusting the pH of the aqueous solution of the present invention to 1.5-6 is not particularly limited. It is preferred to add a pharmaceutically acceptable acid as pH adjuster (C). Acids suitable for use as the pH adjuster (C) are carboxylic acids having 2 to 6 carbon atoms, ascorbic acid and glycosides thereof. Examples of carboxylic acids having 2 to 6 carbon atoms include acetic acid, citric acid, lactic acid, tartaric acid, succinic acid, malic acid, glutamic acid and gluconic acid. Among them, citric acid and acetic acid are preferable in consideration of taste. In addition, ascorbic acid and its glycosides are preferable because they also provide an antioxidant effect. Examples of glycosides of ascorbic acid include ascorbic acid 2-glucoside.

The molar ratio (C/B) of the pH adjuster (C) to the flavin derivative (B) is preferably 0.2-50. If the molar ratio (C/B) is too small, the pH will not drop sufficiently. Therefore, the molar ratio (C/B) is more preferably 0.3 or more, more preferably 0.5 or more. On the other hand, if the molar ratio (C/B) is too large, the sour taste will become strong and the irritation to the skin will also become strong. Therefore, the molar ratio (C/B) is more preferably 10 or less, even more preferably 5 or less, and particularly preferably 2 or less.

The aqueous solution of the present invention may contain components other than the stilbene compound (A), the flavin derivative (B) and the pH adjuster (C). Various ingredients used in beverages and cosmetics can be blended as needed. However, it is preferred that the concentration of ethanol in the solvent is less than 20% by volume. As shown in Reference Example 1 (FIG. 3), the solubility of the stilbene compound (A) when using a mixed solvent of water and ethanol greatly increases when the ethanol content exceeds 20% by volume. Therefore, when the concentration of ethanol in the solvent is less than 20% by volume, the addition of the flavin derivative (B) is significant. The concentration of ethanol in the solvent is more preferably 5% by volume or less, even more preferably 1% by volume or less, and particularly preferably 0.05% by volume or less. Alcohol-free beverages can be provided using the aqueous solution of the present invention.

Applications of the aqueous solution of the present invention are not particularly limited, but beverages and cosmetics are suitable applications. Since a large amount of the stilbene compound (A) is dissolved in water, the stilbene compound (A) is highly bioabsorbable. Beverages are rapidly absorbed through the intestinal tract, and cosmetics are easily absorbed through the skin. Beverages include not only non-alcoholic beverages, but also alcoholic beverages with a low alcohol content of less than 20%. It is particularly suitable for use as a supplement drink. As cosmetics, lotions and milky lotions can be used. It can also be used as a medicine such as a drip solution.

As shown in Examples 6 and 7 (Table 4), the amount of the stilbene compound (A) in the aqueous solution of the present invention containing the stilbene compound (A) and the flavin derivative (B) is reduced by irradiation with light. Sometimes. On the other hand, when the flavin derivative (B) is not included and only the stilbene compound (A) is included, the decrease in the amount of the stilbene compound (A) due to light irradiation is suppressed. Therefore, it is considered that the photostability of the stilbene compound (A) is reduced by associating with the flavin derivative (B). Therefore, it is preferable to store the aqueous solution of the present invention in the dark. Further, as described above, it is desirable to maintain the pH of the aqueous solution of the present invention at 1.5 to 6 from the standpoint of stability. Therefore, when the aqueous solution of the present invention is to be stored, it is desirable to keep the aqueous solution at a pH of 1.5 to 6 and to store it from light.

As described above, the addition of the flavin derivative (B) improves the solubility of the stilbene compound (A) in water. Therefore, the object of the present invention is also solved by providing a water-solubility improver comprising a flavin derivative (B) for improving the water-solubility of the stilbene compound (A).

Another aspect of the present invention comprises a stilbene compound (A) and a flavin derivative (B), wherein the molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) is 0.5 to 50. It is a composition in a form selected from powders, granules, tablets or pastes. Such a composition is useful as a raw material for obtaining the aqueous solution, and the stilbene compound (A) is easily absorbed by the body even when it is directly ingested or applied. At this time, a suitable molar ratio (B/A) is as described in the description of the aqueous solution. It is also preferable to further contain a pH adjuster (C) selected from carboxylic acids having 2 to 6 carbon atoms, ascorbic acid and glycosides thereof, as in the case of the aqueous solution. A suitable molar ratio (C/B) is as described in the description of the aqueous solution.

The composition of the present invention may be in a form containing almost no water such as powder, granules and tablets, or may be in a form such as a paste in which the stilbene compound (A) is not completely dissolved in water and contains a high amount of water. It may be in the form of particles dispersed in a viscous liquid. These compositions may optionally contain components other than the stilbene compound (A), the flavin derivative (B), the pH adjuster (C) and water. In the composition of the present invention, as shown in Example 6, the stilbene compound (A) is slightly reduced by light irradiation even in the absence of water. Therefore, it is preferable to store the composition of the present invention in the same way as the aqueous solution of the present invention. A preferred embodiment is a package in which the composition is contained in a light-shielding container. Although the use of the composition of the present invention is not particularly limited, it can be used as health foods and supplements. It can also be made into pharmaceuticals in the form of capsules, tablets, and the like.

The method for producing the composition of the present invention is not particularly limited, and the powder of the stilbene compound (A) and the powder of the flavin derivative (B) may be simply mixed together with other ingredients as necessary. I do not care. However, from the viewpoint of dissolution rate in water, the composition of the present invention can be produced by previously preparing an aqueous solution in which the stilbene compound (A) and the flavin derivative (B) are dissolved, and then removing the contained water. is preferred. As shown in Example 9 (FIG. 9), the powder of the composition obtained by preliminarily forming an association between the stilbene compound (A) and the flavin derivative (B) and then drying the stilbene compound (A ) and the powder of the flavin derivative (B) are simply mixed, the rate of dissolution in water is much faster. Therefore, the time required for dissolving the composition in water to prepare an aqueous solution can be shortened, so that consumers can easily prepare an aqueous solution. In addition, it is possible to improve the absorption speed and absorption rate when the composition is directly ingested into the body.

Furthermore, in another aspect of the present invention, a plant material containing a stilbene compound (A) or a crude extract extracted from the plant material with an organic solvent is brought into contact with an aqueous solution in which the flavin derivative (B) is dissolved, A method for extracting a stilbene compound (A), wherein the stilbene compound (A) is extracted from the plant raw material or crude extract into the aqueous solution. An aqueous solution in which the flavin derivative (B) is dissolved can dissolve a large amount of the stilbene compound (A) as compared with water, so the stilbene compound (A) can be efficiently extracted using this aqueous solution. . The concentration of the flavin derivative (B) dissolved in the aqueous solution is 0.3-200 mM. The concentration of the flavin derivative (B) is preferably 2 mM or higher, more preferably 5 mM or higher, even more preferably 10 mM or higher, and particularly preferably 15 mM or higher.

Organic solvents are often used as extraction solvents when extracting hydrophobic active ingredients from plant raw materials. However, from the viewpoint of environmental protection, it is desirable not to use an organic solvent as an extraction solvent in order not to volatilize the organic solvent to the surroundings. Also, when used in beverages and cosmetics, it is desirable not to use an organic solvent in the extraction process. In contrast, if an aqueous solution in which the flavin derivative (B) is dissolved is used, the stilbene compound (A) can be efficiently extracted without using an organic solvent. Moreover, since only the stilbene compound (A) that can associate with the flavin derivative (B) can be selectively extracted, the stilbene compound (A) with high purity can be obtained. Moreover, the obtained aqueous solution containing the stilbene compound (A) and the flavin derivative (B) can be used as it is as a raw material for beverages and cosmetics. Furthermore, when extracting with an aqueous solution containing the flavin derivative (B) from a crude extract obtained in advance from a plant raw material with an organic solvent or the like, a highly pure stilbene compound (A) can be obtained.

In addition, an inhibitor of superoxide formation catalyzed by xanthine oxidase, which contains as an active ingredient a stilbene compound (A), which is the compound represented by the above formula (1) or its glycoside or multimer, is also of the present invention. It is an embodiment. These compounds inhibit the reaction of producing superoxide more strongly than the reaction of producing uric acid from xanthine catalyzed by xanthine oxidase. This is the first time that compounds that specifically inhibit superoxide formation catalyzed by xanthine oxidase have been reported.

At this time, in formula (1), R ¹ , R ² , R ³ and X are preferably hydrogen atoms. That is, it is a preferred embodiment that the stilbene compound (A) is resveratrol or its glycoside or multimer. Resveratrol and its derivatives are said to be particularly useful as bioactive substances.

At this time, in the formula (1), it is also preferable that R ¹ and R ² are hydrogen atoms, R ³ is a hydrogen atom or a methyl group, and X is a hydroxy group. That is, it is a preferred embodiment that the stilbene compound (A) is rhapontigenin, piceatannol, or a glycoside or multimer thereof. These compounds strongly inhibit the reaction of producing uric acid from xanthine catalyzed by xanthine oxidase, and further inhibit the reaction of producing superoxide. Rhapontigenin particularly strongly inhibits the reaction of producing uric acid from xanthine catalyzed by xanthine oxidase. On the other hand, piceatannol inhibits the production of uric acid from xanthine using xanthine oxidase as a catalyst, and also accelerates the reaction of producing hydrogen peroxide from oxygen, thereby inhibiting the production of superoxide more strongly than rapontigenin. do.

A preferred embodiment is an inhibitor of superoxide formation catalyzed by xanthine oxidase, which comprises the aqueous solution in which the stilbene compound (A) and the flavin derivative (B) are dissolved. Also suitable is an inhibitor of superoxide formation catalyzed by xanthine oxidase, comprising a composition comprising a stilbene compound (A) and a flavin derivative (B) and in a form selected from powder, granules, tablets or pastes. It is an embodiment.

Example 1 (Solubility in FMN aqueous solution)
Flavin mononucleotide (FMN) was dissolved in pure water at 20° C. to prepare aqueous solutions with concentrations of 2.5, 5, 10, 20, 25, 50 and 100 mM (mmol/L). FMN used here is a sodium salt of a compound of formula (2) in which n is 1 and Y is a hydrogen atom, and 1 mol of a phosphate group contained in FMN is equivalent to about 1 mol of sodium ion. is neutralized by The pH of a 20 mM aqueous solution of the FMN is 5.8. Also, the concentration of the saturated aqueous solution of FMN at 20° C. was 173 mM. The FMN used in other examples described below is the same.

Resveratrol powder manufactured by Tokyo Chemical Industry Co., Ltd. was added to pure water and FMN aqueous solutions of various concentrations, and dissolved by irradiation with ultrasonic waves for 30 minutes. After allowing to stand for 1 hour, undissolved powder was centrifuged to remove the precipitate, and after separating the supernatant, the resveratrol concentration was measured. For the measurement, the supernatant was diluted with 0.1 M sodium citrate buffer (pH 4.0), the difference spectrum was measured against an FMN aqueous solution of the same concentration, and the absorption peak at 317 nm (molar extinction coefficient ε : 29,000), the solubility (mM) of resveratrol in the aqueous solution was calculated. The obtained solubilities are summarized in Table 2 and shown in the graph of FIG.

Piceido (manufactured by LKT lab: absorption maximum 319 nm, ε = 30,500), piceatannol (manufactured by BLD pharm: absorption maximum 324 nm, ε = 27,200), Rhapontigenin (manufactured by Tokyo Chemical Industry Co., Ltd.: absorption maximum 324 nm, ε = 28,900), isorapontigenin (manufactured by Tokyo Chemical Industry Co., Ltd.: absorption maximum 324 nm, ε = 29,300), pterostilbene (manufactured by BLD pharm: absorption maximum 317 nm, ε = 29,200) and ε-viniferin ( FUJIFILM Wako Pure Chemical Industries, Ltd.: absorption maximum 322 nm, ε = 25,500) was also measured for absorbance at each wavelength in the same manner as for resveratrol, and the solubility was obtained. The measurement results are summarized in Table 2 and shown in the graph of FIG.

As can be seen from Table 2 and FIG. 1, the solubility of piceide, rapontigenin, and isolapontigenin in pure water is 1.19 to 2.73 mM, but by dissolving in the FMN aqueous solution, the solubility in pure water is much higher than that in pure water. High solubility was obtained. Since the solubility of these stilbene compounds increases approximately 1:1 linearly with the concentration of FMN, it is estimated that approximately one molecule of FMN is required to dissolve one molecule of these stilbene compounds. all right. Therefore, it is presumed that these stilbene compounds associate with FMN on a one-to-one basis in aqueous solutions.

On the other hand, even when resveratrol with a solubility in pure water of 0.19 mM is dissolved in an aqueous FMN solution, the solubility of resveratrol increases almost linearly in proportion to the concentration of FMN. About 4 molecules of FMN were required to dissolve one molecule. Therefore, it is estimated that resveratrol is dissolved in water surrounded by about 4 molecules of FMN.

Pterostilbene (0.09 mM), which has lower solubility in pure water, requires more FMN molecules than resveratrol to dissolve one molecule. On the other hand, piceatannol (8.34 mM) and ε-viniferin (3.83 mM), which have higher solubility in pure water, require less than one FMN molecule to dissolve one molecule.

Example 2 (solubility in FAD aqueous solution)
Flavin adenine dimononucleotide (FAD) was dissolved in pure water at 20° C. to prepare aqueous solutions with concentrations of 5, 10 and 20 mM, respectively. FAD used here is a sodium salt of a compound of formula (2) in which n is 2 and Y is a group represented by formula (3), and the diphosphate group contained in FAD is It is neutralized with about 2 moles of sodium ions. Also, the concentration of the saturated aqueous solution of FMN at 20° C. was 324 mM.

In the same manner as in Example 1, resveratrol powder was dissolved in pure water and FAD aqueous solutions of various concentrations, and the solubility of resveratrol was measured. The solubility of resveratrol in the obtained aqueous FAD solution is summarized in Table 3 and FIG.

As can be seen from Table 3 and FIG. 2, both the FMN aqueous solution and the FAD aqueous solution can dissolve the same number of moles of resveratrol at the same molar concentration. Therefore, it is believed that both FMN and FAD act similarly on the resveratrol molecule to aid its water solubility.

Example 3 (solubility in riboflavin aqueous solution)
A saturated aqueous solution of riboflavin was prepared and its solubility was 0.43 mM. The riboflavin is a compound of formula (2) in which n is 0 and Y is a hydrogen atom. Resveratrol powder was dissolved in the saturated aqueous solution of riboflavin in the same manner as in Example 1, and the solubility of resveratrol was measured. As a result, 0.45 mM of resveratrol was dissolved, which was more than double the solubility in pure water (0.19 mM).

Reference Example 1 (solubility in aqueous ethanol solution)
Ethanol was added to pure water to prepare ethanol aqueous solutions with concentrations of 5, 10, 20, 30 and 40% by volume, respectively. In the same manner as in Example 1, resveratrol powder was dissolved in pure water and aqueous ethanol solutions of various concentrations, and the solubility of resveratrol was measured. The solubility (mM) of resveratrol for each ethanol concentration was 0.19 ± 0.03 (pure water), 0.25 ± 0.02 (5% by volume), 0.37 ± 0.02 (10% by volume ), 1.30±0.30 (20% by volume), 7.38±1.10 (30% by volume), 32.9±3.0 (40% by volume). The solubility versus ethanol concentration is shown in the graph of FIG.

As can be seen from Figure 3, the solubility of resveratrol increases as the ethanol concentration increases. However, the solubility of resveratrol up to an ethanol concentration of about 20% by volume is not so high, and the solubility rises sharply at 30% by volume and 40% by volume. It seems that the solubility changes with the change in the polarity of the solvent, which is very different from the linear increase in the solubility of the stilbene compound in proportion to the concentration of the flavin derivative.

Example 4 (Preparation of mixed powder of stilbene compound and FMN)
An aqueous solution containing a stilbene compound (resveratrol, piceid or piceatannol) and FMN was prepared. At this time, referring to the results of Example 1 (Table 2), FMN was added in an amount necessary for each stilbene compound to dissolve completely in water. That is, 4 times the moles of resveratrol, 1.5 times the moles of piceide, and 1 times the moles of piceatannol were added to FMN.

Specifically, 1 mmol (228 mg) of resveratrol was completely dissolved in an aqueous solution prepared by dissolving 4 mmol (1913 mg) of FMN in 200 mL of water. Also, 2 mmol (780 mg) of piceide was completely dissolved in an aqueous solution prepared by dissolving 3 mmol (1436 mg) of FMN in 150 mL of water. Furthermore, 1 mmol (244 mg) of piceatannol was completely dissolved in an aqueous solution of 1 mmol (479 mg) of FMN dissolved in 100 mL of water. The aqueous solution thus obtained is dried under reduced pressure to form a mixed powder containing resveratrol and FMN at a molar ratio of 1:4, a mixed powder containing piceide and FMN at a molar ratio of 2:3, and piceatannol. Mixed powders containing FMN at a molar ratio of 1:1 were respectively obtained. Here, the mass of each mixed powder containing 1 mol of FMN is 535 g, 738 g, and 723 g, respectively.

Example 5 (Spectrum of mixed aqueous solution of stilbene compound and FMN)
When the mixed powder of the stilbene compound and FMN obtained in Example 4 was dissolved in water, all exhibited a reddish orange color. Since the FMN aqueous solution of the same concentration without containing the stilbene compound exhibited a yellow color, it was found that the color tone changed due to the inclusion of the stilbene compound. In order to quantitatively grasp this phenomenon, the absorption spectrum of each aqueous solution was measured.

The mixed powder of piceatannol and FMN obtained in Example 4 was dissolved in pure water at a concentration of 7.23 mg/mL to prepare an aqueous solution containing 10 mM of both piceatannol and FMN. The visible light absorption spectrum of the resulting aqueous solution was measured with a glass cuvette having an optical path length of 0.1 mm to obtain the spectrum of the aqueous solution of the mixture of piceatannol and FMN (dotted line in FIG. 4). FIG. 4 also shows the spectrum of an aqueous solution containing only 10 mM FMN as a solid line. The extinction coefficient ε at each absorption maximum of the mixed aqueous solution was 27,800 (331 nm), 6,490 (377 nm), 8,330 (449 nm), and 2,020 (510 nm), respectively. It is presumed that the appearance of weak absorption around 510 nm causes the color change from yellow to reddish orange. This absorption band decreased upon dilution with water. The mixed aqueous solution was diluted 100-fold with pure water to make the FMN concentration 0.01 mM, and the measurement was performed using a cuvette with an optical path length of 10 mm. is almost the same as the sum of the spectra of

The mixed powder of piceide and FMN obtained in Example 4 was dissolved in pure water at a concentration of 7.38 mg/mL to prepare an aqueous solution containing 6.7 mM piceide and 10 mM FMN. When the spectrum was measured in the same manner as above, the extinction coefficient ε at each absorption maximum was 21,100 (328 nm), 6,900 (380 nm), 8,730 (449 nm), and 1,700 (510 nm), respectively. there were. Further, the mixed powder of resveratrol and FMN obtained in Example 4 was dissolved in pure water at a concentration of 5.35 mg/mL to prepare an aqueous solution containing 2.5 mM of resveratrol and 10 mM of FMN. . Upon spectral measurement of this, the extinction coefficients .epsilon.

It was found that each absorption maximum in the absorption spectrum of the stilbene compound undergoes a bathochromic shift by adding FMN. In addition, since an aqueous solution containing a stilbene compound and FMN shows absorption at 510 nm, it is considered that a complex of both compounds is formed in the aqueous solution. Since the complex formed in a high-concentration aqueous solution is reduced by dilution, it is assumed that the stilbene compound and FMN are associated in the aqueous solution by a relatively weak π-π interaction occurring between them. Presumed.

Example 6 (Stability of a mixture of resveratrol and FMN)
The mixed powder of resveratrol and FMN obtained in Example 4 was placed in a transparent glass bottle and stored at room temperature (20 ° C.) for 30 days in a room under a fluorescent lamp. The amount of resveratrol contained in the powder was reduced by only 3.6±1.5%. The mixed powder was dissolved in pure water at a concentration of 5.35 mg/mL to prepare an aqueous solution containing 2.5 mM resveratrol and 10 mM FMN. The resulting aqueous solution was placed in a transparent glass bottle and stored at room temperature (20° C.) for 5 days in a room under a fluorescent light. On the other hand, when it was placed in a brown glass bottle instead of a clear glass bottle and stored under the same conditions, the amount of resveratrol decreased by only 3.4% even after 17 days. Further, the aqueous solution was diluted 100-fold with pure water to prepare an aqueous solution containing 0.025 mM resveratrol and 0.1 mM FMN. When this diluted aqueous solution was allowed to stand at 20° C. for 1 day without shielding from light, the absorbance (317 nm) of resveratrol decreased significantly, and the amount of residual resveratrol decreased to less than half. Shielding from light prevented the decrease in absorbance of resveratrol.

Example 7 (Effect of light and pH on a mixed aqueous solution of resveratrol and FMN)
A 100 mM sodium citrate buffer was prepared as a pH 4 buffer, a 100 mM sodium phosphate buffer as a pH 6 and pH 7 buffer, and a 40 mM sodium phosphate buffer as a pH 8 buffer. To 0.6 mL of saturated (0.19 mM) resveratrol aqueous solution, each of the above pH 4, 6, 7 and 8 buffer solutions was added to make a total volume of 3.00 mL and mixed to obtain 4 kinds of aqueous solutions. In addition, 0.03 mL of an aqueous solution containing 2.5 mM of resveratrol and 10 mM of FMN was added with each of the above buffer solutions to make a total volume of 3.00 mL and mixed to obtain four kinds of aqueous solutions. The aqueous solution thus obtained was placed in a measuring cuvette made of quartz glass. The absorbance at 317 nm of each of the obtained aqueous solutions was measured at 20° C. after being allowed to stand for 1 hour while irradiating with light or under light shielding. The light source at this time was a fluorescent lamp, and the light was irradiated through the container at a position about 2 m directly below the two 2250 lumen fluorescent lamps. The reduction rate (%) of the amount of resveratrol was calculated from the absorbance thus obtained. The results are summarized in Table 4.

As shown in Table 4, resveratrol in a dilute aqueous solution prepared by dissolving a mixed powder of resveratrol and FMN in water is unstable to light. This is presumed to be due to the inclusion of FMN. In addition, it can be seen that the higher the pH, the more unstable it gradually becomes. Therefore, it was found that when storing an aqueous solution containing resveratrol and FMN, it is desirable to keep it acidic and protect it from light.

Example 8 (pH of aqueous solution containing resveratrol, FMN and acetic acid)
For an aqueous solution containing 5 mM resveratrol and 20 mM FMN, the ratio of moles of acetic acid to moles of FMN (acetic acid/FMN) is 0, 0.25, 0.5, 1, 2, 5, 10. , 25, 50 and 100 to prepare aqueous solutions and measure their pH. In addition, aqueous solutions were prepared by removing resveratrol from each aqueous solution, and their pH was also measured. The results are shown in FIG. By adjusting the amount of acetic acid added to the aqueous solution containing resveratrol and FMN, the pH of the aqueous solution could be adjusted to a desired value. The presence or absence of resveratrol did not significantly affect the pH of the aqueous solution, presumably because the pKa value of the stilbene compound (A) is large. Thus, by adding an appropriate amount of acid, a moderately sour mixture can be obtained while lowering the pH and ensuring the stability of the stilbene compound (A).

Reference Example 2 (pH of aqueous solution containing FMN and acid)
An acid (acetic acid, citric acid, lactic acid, ascorbic acid) was added to a 20 mM FMN aqueous solution, and the ratio of the number of moles of the acid to the number of moles of FMN (acid/FMN) was 0, 0.25, 0.5, 1. , 2, 5, 10, 25, 50 and 100 were prepared and the pH was measured. The results are summarized in FIGS. 7 and 8. FIG. The pH of the aqueous solution can be adjusted to a desired value between 1.5 and 6 by adjusting the type and blending ratio of the acid.

Example 9 (Dissolution rate of mixed powder in water)
Mixed powder 1 was prepared by mixing 114 mg (0.5 mmol) of resveratrol powder and 957 mg (2 mmol) of FMN powder and mixing them in an agate mortar. On the other hand, mixed powder 2 obtained by drying the mixed aqueous solution of resveratrol and FMN in Example 4 was prepared. About 0.2 mg of powder of either mixed powder 1 or mixed powder 2 is added to 3 mL of 0.1 M citrate buffer (pH 4.0), and immediately gently stirred and stirred every 3 minutes. Absorbance at 445 nm was measured, and stirring and measurement were repeated until 33 minutes later. 317 nm is the absorption peak derived from resveratrol, and 374 nm and 445 nm are the absorption peaks derived from FMN. The dissolved amount of resveratrol was calculated from the absorbance at 317 nm and shown in FIG. As shown in FIG. 9, a powder having a higher dissolution rate could be obtained not only by simply mixing powders but also by preparing a mixed aqueous solution in advance and then drying it. The powder mixture of resveratrol, FMN and citric acid had a moderately sour taste, and the powder mixture of resveratrol and FMN was almost tasteless.

Example 10 (Extraction of resveratrol)
A commercially available crude extract powder obtained by extracting from grapes or the like with an organic solvent and drying was used. The crude extract powder contains about 10% resveratrol by weight. 10 mL of the following aqueous solution or pure water was added to 100 mL of the crude extract powder, and extracted by irradiating ultrasonic waves for 30 minutes. After allowing to stand, the insoluble matter was centrifuged, and the supernatant was used as an extract. The solubility of resveratrol was calculated from the absorbance at 317 nm measured by diluting the obtained extract with 0.1 M citrate buffer (pH 4.0). The resveratrol recovery rate thus obtained was 9.2% when pure water was used as the extract, 84% when 25 mM FMN aqueous solution was used, and 96% when 50% by volume ethanol aqueous solution was used. . From this, it was clarified that the use of the FMN aqueous solution enables highly efficient extraction as an aqueous solution without using an organic solvent.

Example 11 (Assay for uric acid production)
Xanthine oxidase (XO) enzyme solution (EC 1.1.3.22, Grade IV) was purchased from Sigma-Aldrich. A 10 mM sample solution was prepared by dissolving the stilbene compound in DMSO. A mixed solution (2.88 mL) was prepared by adding 0 to 200 μM xanthine and a DMSO solution (0 to 0.03 mL) of a stilbene compound to 40 mM sodium carbonate buffer (pH 10) containing 0.1 mM EDTA. 0.12 mL of XO enzyme solution (0.04 units) was added to the mixed solution at 25° C. to initiate the reaction, and absorbance at 293 nm (ultraviolet absorption maximum of uric acid) was measured for 60 seconds. Control experiments were performed by replacing the sample solution with DMSO. Reaction rate was determined from the linear increase in absorbance. The inhibition constant Ki (μM) of the uric acid production reaction by the stilbene compound and the 50% inhibition concentration IC ₅₀ (μM) at a xanthine concentration of 200 μM were determined, and the results are shown in Table 5. Since absorption by the stilbene compound was observed at 293 nm, uric acid could be quantified only when the concentration of the stilbene compound was less than 0.1 mM.

Each measurement was measured 3 or more times in separate experiments. Analysis was performed with Sigma plot 2001 (SPSS Inc., Chicago, Ill.). Inhibition modes and kinetics were analyzed with Enzyme Kinetics Module 1.1 (SPSS Inc.) attached to Sigma plot 2001. These methods were similarly employed in Examples 12-14 below.

As shown in Table 5, for resveratrol, resveratrol-4'-glycoside, piceatannol, rapontigenin and isolapontigenin, only the inhibition constant Ki was calculated and no other inhibition constants were calculated. , analyzed to inhibit uric acid production by competitive inhibition. This competitive inhibition is expected due to the slow rate of xanthine oxidation and the fast rate of oxygen reduction. Judging from the _IC50 value, it was found that among these, rapontigenin has a particularly strong uric acid formation inhibitory activity.

Example 12 (Assay for superoxide production)
0-200 μM xanthine, 0.03 mL of 0.5% bovine serum albumin, 0.03 mL of 2.5 mM nitro blue tetrazolium and 10 mM of stilbene compound in DMSO. A mixture (2.88 mL) was prepared by adding sodium carbonate buffer (pH 10). 0.12 mL of XO enzyme solution (0.04 units) was added to the mixture at 25° C. to initiate the reaction, and the absorbance at 560 nm was measured for 60 seconds. Control experiments were performed by replacing the sample solution with DMSO. Reaction rate was determined from the linear increase in absorbance. The reaction was carried out at pH 10 in order to detect unstable superoxide (O ₂ ^- .). Since superoxide reduces nitroblue tetrazonium and converts it to blue formazan (absorption maximum at 560 nm), this was quantitatively detected. When the concentration of xanthine was varied from 0 to 200 μM, the formation of superoxide was inhibited by stilbene compounds. The results are summarized in Table 6.

As shown in Table 6, the stilbene compounds excluding piceatannol were analyzed to inhibit superoxide production by competitive inhibition, since only the inhibition constant Ki was calculated and no other inhibition constants were calculated. . On the other hand, it is analyzed that piceatannol, whose inhibition constant K'i was calculated, inhibited superoxide production by mixed inhibition. Judging from the _IC50 value, it was found that piceatannol among these has a particularly strong superoxide formation inhibitory activity. These stilbene compounds inhibit superoxide production more strongly than uric acid production, suggesting that the stilbene compounds bind to the FAD site within xanthine oxidase (XO).

Example 13 (DPPH scavenging activity)
A method for scavenging the stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) is described in Blois, MS (1958). Antioxidant determinations by the use of a stable free radical. 1200. In addition, DPPH activity indicates the property of donating electrons or hydrogen atoms (Zhang, R., Kang, KA, Piao, MJ, Lee, KH, Jang, HS, Park, MJ, Kim, BJ, Kim, JS, Kim, YS, Ryu, SY, Hyun, JW (2007)). In a test tube, 1.0 mL of 100 mM acetate buffer (pH 5.5), 1.87 mL ethanol and 0.1 mL of ethanol solution of 3 mM DPPH were added, and then 0.03 mL of sample solution in which 10 mM of stilbene compound was dissolved in DMSO. was added and reacted at 25° C. for 20 minutes. Meanwhile, the absorbance at 517 nm (DPPH, ε=8.32×10 ³ ) was measured. Control experiments were performed by changing the sample solution to DMSO. The decrease in absorbance for 20 minutes was measured, and the number of DPPH molecules scavenging by one molecule of the stilbene compound was calculated and defined as the scavenging activity. The initial rate of the scavenging reaction was determined from the decrease in absorbance after addition of the sample (Masuoka, N., Isobe, T., Kubo, I. (2006). Antioxidants from Rabdosia japonica. Phytother. Res., 30(3 ), 206-213). The results are summarized in Table 7.

As shown in Table 7, resveratrol and its glycosides, and pterostilbene showed weak scavenging activity like alkenylphenols. On the other hand, piceatannol, rapontigenin and isolapontigenin showed similar strong activity. However, when measuring the initial rate of scavenging activity of these compounds, the initial rate of piceatannol was faster than the other two compounds. This suggests that piceatannol can reduce the XO molecule (Hille, R., & Massey, V. (1981) Studies on the oxidative half-reaction of xanthine oxidase. J. Biol. Chem. 256 , 9090-9095 and Masuoka, N., Kubo, I (2018) Characterization of the xanthine oxidase inhibitory activity of alk(en)yl phenols and related compounds. Phytochemistry (2018) 155, 100-106). The DPPH scavenging activity of dodecyl gallate, a food additive having an antioxidant effect, is 7.32±0.04.

Example 14 (superoxide scavenging activity)
Superoxide (O ₂ ⁻ ·) is produced in the PMS-NADH system (Nishikimi, M., Rao, NA, Yagi, K. (1972). The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys). Res. Commun. 46, 849-854). 2.82 mL of 40 mM sodium carbonate buffer (pH 10) containing 0.1 mM EDTA, 0.03 mL of 0.5% bovine serum albumin aqueous solution, 0.03 mL of 2.5 mM nitro blue tetrazolium aqueous solution, and 10 mM of a stilbene compound were dissolved in DMSO. A mixture (2.97 mL) of 0.06 mL of the sample solution and 0.03 mL of 7.8 mM NADH aqueous solution was prepared. 0.03 mL of a 115 μM aqueous solution of PMS (phenazine methosulfate) was added to the mixture at 25° C. to initiate the reaction, and the absorbance at 560 nm was measured for 60 seconds. Superoxide is scavenged (erased) by reaction with stilbene compounds. Unreacted superoxide radicals were quantified as they reacted with nitroblue tetrazolium to give a blue color (formazan). Control experiments were performed by changing the sample solution to DMSO. The reaction rate was determined from the linear increase in absorbance, and the scavenging activity was determined from the following equation. The scavenging activity of stilbene compounds is shown in FIG.
Scavenging activity (%) = [1 - (rate of sample) / (rate of control)] X 100

In this radical scavenging reaction, the superoxide (O ₂ ⁻ .) and the stilbene compound react in a molar ratio of 1:1. Among these stilbene compounds, piceatannol is unusually active. Compared to caffeic acid (IC ₅₀ =51 μM) and gallic acid (IC ₅₀ =29 μM), the IC ₅₀ value of piceatannol (9.0±2.0 μM) is clearly smaller, suggesting that piceatannol is a potent scavenger. It was shown that there is This scavenging activity may be related to stronger stabilization of the piceatannol radical by the long conjugated enediol structure of piceatannol.

Claims (17)

0.3 to 200 mM of a stilbene compound (A) which is a compound represented by the following formula (1) or a glycoside or multimer thereof, and a compound represented by the following formula (2) or a pharmaceutically acceptable compound thereof An aqueous solution in which 0.3 to 200 mM of the flavin derivative (B), which is a salt, is dissolved.

[In formula (1), R ¹ , R ² and R ³ are each independently a hydrogen atom or a methyl group, and X is a hydrogen atom, a hydroxy group or a methoxy group. ]

[In formula (2), n is 0, 1 or 2. When n is 0 or 1, Y is a hydrogen atom. When n is 2, Y is a group represented by the following formula (3). ]

The stilbene compound (A) is at least one selected from the group consisting of resveratrol, piceatannol, rhapontigenin, isolapontigenin, pterostilbene and pinostilbene, and glycosides and multimers thereof. Item 1. The aqueous solution according to item 1. 3. The aqueous solution according to claim 1, wherein the molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) is 0.5-50. The aqueous solution according to any one of claims 1 to 3, which has a pH of 1.5 to 6. The aqueous solution according to any one of claims 1 to 4, further comprising a pH adjuster (C) selected from carboxylic acids having 2 to 6 carbon atoms and ascorbic acid and glycosides thereof. Aqueous solution according to any one of claims 1 to 5, wherein the concentration of ethanol in the solvent is less than 20% by volume. An inhibitor of superoxide formation catalyzed by xanthine oxidase, comprising an aqueous solution according to any one of claims 1-6. A beverage or cosmetic comprising the aqueous solution according to any one of claims 1 to 6. A water solubility improver comprising a flavin derivative (B) for improving the water solubility of a stilbene compound (A);
The stilbene compound (A) is a compound represented by the following formula (1) or a glycoside or multimer thereof,
A water-solubility improver, wherein the flavin derivative (B) is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof.

[In formula (1), R ¹ , R ² and R ³ are each independently a hydrogen atom or a methyl group, and X is a hydrogen atom, a hydroxy group or a methoxy group. ]

[In formula (2), n is 0, 1 or 2. When n is 0 or 1, Y is a hydrogen atom. When n is 2, Y is a group represented by the following formula (3). ]

A stilbene compound (A) which is a compound represented by the following formula (1) or a glycoside or multimer thereof, and a compound represented by the following formula (2) or a flavin derivative which is a pharmaceutically acceptable salt thereof (B), wherein the molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) is 0.5 to 50, and the composition is in a form selected from powder, granules, tablets and pastes. .

[In formula (1), R ¹ , R ² and R ³ are each independently a hydrogen atom or a methyl group, and X is a hydrogen atom, a hydroxy group or a methoxy group. ]

[In formula (2), n is 0, 1 or 2. When n is 0 or 1, Y is a hydrogen atom. When n is 2, Y is a group represented by the following formula (3). ]

11. The composition according to claim 10, further comprising a pH adjuster (C) selected from carboxylic acids having 2 to 6 carbon atoms and ascorbic acid and glycosides thereof. 12. An inhibitor of superoxide formation catalyzed by xanthine oxidase, comprising a composition according to claim 10 or 11. 13. The method for producing the composition according to any one of claims 10 to 12, wherein an aqueous solution in which the stilbene compound (A) and the flavin derivative (B) are dissolved is prepared and then water is removed. A plant raw material containing a compound represented by the following formula (1) or a stilbene compound (A) which is a glycoside or multimer thereof, or a crude extract extracted from the plant raw material with an organic solvent; ) or a pharmaceutically acceptable salt thereof, which is a flavin derivative (B), is brought into contact with an aqueous solution in which the stilbene compound (A) is extracted from the plant raw material or crude extract into the aqueous solution. A method for extracting a stilbene compound (A).

[In formula (1), R ¹ , R ² and R ³ are each independently a hydrogen atom or a methyl group, and X is a hydrogen atom, a hydroxy group or a methoxy group. ]

[In formula (2), n is 0, 1 or 2. When n is 0 or 1, Y is a hydrogen atom. When n is 2, Y is a group represented by the following formula (3). ]

An inhibitor of superoxide formation catalyzed by xanthine oxidase, comprising a compound represented by the following formula (1) or a stilbene compound (A) which is a glycoside or multimer thereof as an active ingredient.

[In formula (1), R ¹ , R ² and R ³ are each independently a hydrogen atom or a methyl group, and X is a hydrogen atom, a hydroxy group or a methoxy group. ] 16. The inhibitor according to claim 15, wherein ^R1 , ^{R2 ,} R3 and X in formula (1) are hydrogen atoms. 16. The inhibitor according to claim 15, wherein in formula (1), ^R1 and ^{R2 are} hydrogen atoms, R3 is a hydrogen atom or a methyl group, and X is a hydroxy group.

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