CN115768739B - Aqueous solution containing stilbene compound and superoxide generation inhibitor containing stilbene compound - Google Patents

Aqueous solution containing stilbene compound and superoxide generation inhibitor containing stilbene compound Download PDF

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CN115768739B
CN115768739B CN202180044521.5A CN202180044521A CN115768739B CN 115768739 B CN115768739 B CN 115768739B CN 202180044521 A CN202180044521 A CN 202180044521A CN 115768739 B CN115768739 B CN 115768739B
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aqueous solution
stilbene compound
resveratrol
compound
stilbene
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CN115768739A (en
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益冈典芳
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Maseki Noriyoshi
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Maseki Noriyoshi
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
    • C07C37/72—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by liquid-liquid treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/205—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing only six-membered aromatic rings as cyclic parts with unsaturation outside the rings
    • C07C39/21—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing only six-membered aromatic rings as cyclic parts with unsaturation outside the rings with at least one hydroxy group on a non-condensed ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D475/00—Heterocyclic compounds containing pteridine ring systems
    • C07D475/12—Heterocyclic compounds containing pteridine ring systems containing pteridine ring systems condensed with carbocyclic rings or ring systems
    • C07D475/14—Benz [g] pteridines, e.g. riboflavin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide

Abstract

The present invention provides an aqueous solution containing 0.3 to 200mM of a stilbene compound (A) and 0.3 to 200mM of a flavin derivative (B) selected from riboflavin, FMN and FAD, wherein the stilbene compound (A) is a compound represented by the following formula (1) or a glycoside thereof. Thus, an aqueous solution containing a stilbene compound at a high concentration can be provided. In addition, inhibitors of superoxide production catalyzed by xanthine oxidase can be provided. In the formula (1), R 1 、R 2 And R is 3 Each independently represents a hydrogen atom or a methyl group, and X represents a hydrogen atom, a hydroxyl group or a methoxy group.

Description

Aqueous solution containing stilbene compound and superoxide generation inhibitor containing stilbene compound
Technical Field
The present invention relates to aqueous solutions comprising stilbene compounds and to water solubility enhancers for stilbene compounds. Further, the present invention relates to a composition containing a stilbene compound having excellent water solubility and a method for producing the same. In addition, it relates to an efficient extraction method of stilbene compounds. Furthermore, superoxide generation inhibitors comprising stilbene compounds are described.
Background
Grape juice is known to contain polyphenols as an active ingredient. Among the functional compounds contained in the wine named in French Parado are resveratrol (resveratrol). Resveratrol has long life, anti-aging, antihyperglycemic, antihyperlocytic, hyperLDL, antihypertensive, antiinflammatory, obesity and diabetes improving effects, and also has been reported to prevent Alzheimer disease (see non-patent documents 1 to 4). Grape peel contains resveratrol glycoside (Piceid), but grape juice contains only very small amounts of Piceid, so that it is difficult to achieve a large supply of glycoside from the juice. For example, an example of the composition of a commercially available grape concentrate reduced 100% juice contains only 0.02mM of picrin as shown below.
Water: 86.9+ -2.5 g/dL;
glucose: 5.87+ -0.21 g/dL;
fructose: 5.76.+ -. 0.05g/dL;
picea new glycoside: 0.00068g/dL (0.02 mM);
polyphenols (in terms of gallic acid): 0.088g/dL.
As a source of resveratrol, resveratrol contained in extracts of leaves, stems and seeds of grape can be used. However, resveratrol is very poorly soluble in water because it is hydrophobic, and its solubility in water at 20 ℃ is only 0.19mM (see table 2 of the present specification). In addition, in the above-mentioned non-patent documents 1 to 4, in which physiological activities of resveratrol are reported, in order to exhibit their physiological activities, a considerable amount (50 to 3000 mg/day) of resveratrol is taken in. Therefore, a large amount of intake of 0.2mmol or more is required for 1 day in order to sufficiently exhibit the effect. For example, in the case of use as a supplement, it is necessary to prepare a beverage containing resveratrol in a high concentration for efficient ingestion.
In order to produce a beverage containing resveratrol which is well absorbed by living bodies, a method of converting the resveratrol into a glycoside, clathrating the glycoside with cyclodextrin, adding an alcohol to an aqueous solution, and the like have been attempted (see non-patent documents 5 and 6). However, efficient glycosylation of resveratrol using biocatalysis has not been reported. In addition, a method for obtaining a high-concentration aqueous solution by clathration of cyclodextrins with resveratrol has not been reported yet. In addition, the solubility of resveratrol in an aqueous solution containing ethanol is not so great in the case of a small ethanol content (see fig. 3 of the present specification). Therefore, many resveratrol cannot be dissolved in alcohol-containing drinks, and if health is considered, it is desirable to dissolve resveratrol in alcohol-free drinks.
Xanthine Oxidase (XO) is an enzyme that catalyzes a reaction of oxidizing hypoxanthine to produce xanthine and a reaction of oxidizing xanthine to produce uric acid in an organism. Thus, inhibition of xanthine oxidase is effective in the treatment of hyperuricemia or gout. For example, gallic acid alkyl ester is effective as a xanthine oxidase inhibitor as described in non-patent document 7. Reaction from hypoxanthine to xanthine, uric acid, and oxygen (O) 2 ) Is reduced to superoxide (O) 2 － Hydrogen peroxide (H) 2 O 2 ) The reaction of (2) is carried out in a so-called ping-pong mechanism. The excessive production of reactive oxygen species (ROS: reactive Oxygen Species) such as superoxide and hydrogen peroxide may cause oxidative stress including the formation of lipid peroxide, and may adversely affect health and food industry (see non-patent document 8).
Prior art literature
Non-patent literature
Non-patent document 1: szkudielski T, szkudielski K.resuratrol and diabetes from animal to human publications.Biochim Biophys acta.2015;1852 (6) 1145-1154. Non-patent document 2: 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.biochem Biophys acta.2015;1852 (6):1202-1208.
Non-patent document 3: khodabandhoo H, seyyedebrahimi S, esfahani EN, razi F, meshkani r.resuratrol supplementation decreases blood glucose without changing the circulating CD14+cd16+ monocytes and inflammatory cytokines in patients with type 2diabetes:a randomized,double-blind, placebo-controlled student.nutr.res, 2018;54:40-51.
Non-patent document 4: pennyroyal Tian Wuyan, xiaoxiyou flower, dan Yuanhao two, yigang classical aryl, isomerization of stilbene compound and stilbene Naturalistae 2009 contained in polygonum cuspidatum (Polygonum cuspidatum); 14:17-21.
Non-patent document 5: 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.
Non-patent document 6: szkudielska K, deniziak M, ros P, gwozoz K, szkudielski t.resuveratrol alleviates ethanol-induced hormonal and metabolic disturbances in the rate.physiol.res.2017; 66:135-145.
Non-patent document 7: masaoka N, nihei K, kubo I, xanthine oxidase inhibitory activity of alkyl gallates, mol. 50,725-731 non-patent document 8: vine Tian Zhi, reactive oxygen species, peroxidized lipids, mechanisms of free radical generation and scavenging, their biological effects, YAKUGAKU ZASSHI 2002;122 (3):203-218
Disclosure of Invention
Technical problem to be solved by the invention
The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide an aqueous solution containing a stilbene compound at a high concentration. It is also an object of the present invention to provide a water-solubility enhancer capable of enhancing the solubility of a stilbene compound having low solubility in water. It is also an object of the present invention to provide a composition comprising a stilbene compound and having good water solubility and a suitable method for the production thereof. It is also an object of the present invention to provide a process for efficiently extracting stilbene compounds using water. Furthermore, it is an object of the present invention to provide inhibitors of xanthine oxidase-catalyzed superoxide generation comprising the above stilbene compounds or aqueous solutions and compositions containing the above stilbene compounds.
Technical scheme for solving technical problems
The above technical problems are solved by providing an aqueous solution in which 0.3 to 200mM of a stilbene compound (A) which is a compound represented by the following formula (1) or a glycoside or polymer thereof and 0.3 to 200mM of a flavin derivative (B) which is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof are dissolved.
[ in formula (1), R 1 、R 2 And R is 3 Each independently represents a hydrogen atom or a methyl group, and X represents a hydrogen atom, a hydroxyl group or a methoxy group. ]
In the 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). ]
In this case, the stilbene compound (A) is preferably at least 1 selected from resveratrol, piceatannol (piceatannol), rhapontigenin (rhapontigenin), isorhapontigenin (isorhapontigenin), pterostilbene (pterostilbene) and pinostilbene (pinostilbene), and glycosides and polymers thereof. It is also preferable that the molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) is from 0.5 to 50. The pH of the aqueous solution is preferably 1.5 to 6. In addition, it is also preferable that the concentration of ethanol in the solvent is less than 20% by volume.
The aqueous solution containing the pH adjuster (C) selected from carboxylic acids having 2 to 6 carbon atoms, ascorbic acid and its glycoside is a preferred embodiment. A preferred embodiment of the present invention is an inhibitor of the formation of superoxide catalyzed by xanthine oxidase comprising the aqueous solution described above. In addition, a preferred embodiment of the present invention is a beverage or cosmetic comprising the above aqueous solution.
The above-mentioned problems can also be solved by providing a water-solubility enhancer containing a flavin derivative (B) for enhancing the solubility of a stilbene compound (a) in water, wherein the stilbene compound (a) is a compound represented by the above-mentioned formula (1) or a glycoside or polymer thereof, and the flavin derivative (B) is a compound represented by the above-mentioned formula (2) or a pharmaceutically acceptable salt thereof.
The above technical problems can also be solved by providing a composition in a form selected from the group consisting of powder, granules, tablets and ointments, which comprises a stilbene compound (A) as a compound represented by the above formula (1) or a glycoside or polymer thereof, and a flavin derivative (B) as a compound represented by the above 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. In this case, the pH adjuster (C) is preferably selected from carboxylic acids having 2 to 6 carbon atoms, ascorbic acid and its glycoside.
In addition, the above-mentioned method for producing a composition is preferable, in which the water is removed after the aqueous solution in which the stilbene compound (A) and the flavin derivative (B) are dissolved is prepared. A preferred embodiment of the present invention is an inhibitor of the formation of superoxide catalyzed by xanthine oxidase comprising the above composition.
The above-mentioned problems can also be solved by providing a method for extracting a stilbene compound (a) which comprises contacting a plant material containing a stilbene compound (a) or a crude extract obtained by extracting the plant material with an organic solvent with an aqueous solution in which a flavin derivative (B) is dissolved, and extracting the stilbene compound (a) into the aqueous solution from the plant material or the crude extract, wherein the stilbene compound (a) is a compound represented by the above formula (1) or a glycoside or a polymer thereof, and the flavin derivative (B) is a compound represented by the above formula (2) or a pharmaceutically acceptable salt thereof.
In addition, the above technical problems can be solved by providing an inhibitor of superoxide generation catalyzed by xanthine oxidase, the inhibitor of superoxide generation comprising stilbeneThe stilbene compound (A) is a compound represented by the above formula (1) or a glycoside or polymer thereof as an active ingredient. In a preferred embodiment, in formula (1), R 1 、R 2 、R 3 And X is a hydrogen atom. In another preferred embodiment, R in formula (1) 1 And R is 2 Is a hydrogen atom, R 3 Is hydrogen atom or methyl, X is hydroxy.
ADVANTAGEOUS EFFECTS OF INVENTION
The stilbene compound such as resveratrol in the aqueous solution of the present invention is dissolved and contained in a high concentration, and the stilbene compound can be efficiently taken up only by drinking a small amount. Since it is dissolved in water, the absorption performance in a living body after intake is also excellent. The aqueous solution of the present invention can dissolve a stilbene compound at a high concentration without adding an organic solvent such as ethanol. In addition, the flavin derivative contained in the aqueous solution of the present invention can also be used as vitamin B 2 And metabolites thereof exert useful effects on organisms. Therefore, the aqueous solution of the present invention is useful as a beverage or a cosmetic. In addition, the use of the water-solubility enhancer of the present invention can greatly enhance the solubility of the stilbene compound in water.
The composition of the present invention has a form of powder, granule, tablet or paste, and is excellent in solubility in water and absorbability in living bodies. Therefore, the composition of the present invention is useful as a supplement or the like. In addition, according to the extraction method of the present invention, even without using an organic solvent, stilbene compounds can be extracted efficiently by water.
In addition, the stilbene compound, the aqueous solution containing the stilbene compound, and the composition containing the stilbene compound can inhibit the formation of superoxide catalyzed by xanthine oxidase.
Drawings
Fig. 1 is a graph showing the change in water solubility of stilbene compounds when FMN concentration is changed in example 1.
Fig. 2 is a graph showing the change in the solubility of resveratrol in an aqueous solution when the concentrations of FMN and FAD in the aqueous solution were changed in example 2.
Fig. 3 is a graph showing a change 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. 4 is a graph comparing the absorption spectrum (dotted line) of an aqueous solution containing 10mM piceatannol and 10mM FMN with the absorption spectrum (solid line) of an aqueous solution containing only 10mM FMN in example 5.
FIG. 5 is a graph comparing the absorption spectrum (dotted line) of an aqueous solution containing 0.01mM piceatannol and 0.01mM FMN with the absorption spectrum (solid line) of an aqueous solution containing only 0.01mM FMN in example 5.
Fig. 6 is a graph showing pH when the acetic acid concentration of an aqueous solution containing resveratrol, FMN and acetic acid was changed in example 8.
Fig. 7 is a graph showing the pH when the concentration ratio of FMN and various acids was changed in reference example 2.
Fig. 8 is a graph in which a part of the horizontal axis of fig. 7 is enlarged.
Fig. 9 is a graph showing the dissolution rate in water of a mixed powder 1 obtained by mixing resveratrol powder and FMN powder and a mixed powder 2 obtained by drying a mixed aqueous solution of resveratrol and FMN in comparative example 9.
FIG. 10 is a graph showing the change in superoxide scavenging activity when the concentration of stilbene compound was changed in example 14.
Detailed Description
The present invention provides an aqueous solution in which 0.3 to 200mM of a stilbene compound (A) or a glycoside or polymer thereof represented by the following formula (1) and 0.3 to 200mM of a flavin derivative (B) or a pharmaceutically acceptable salt thereof are dissolved. The stilbene compound (a) is hydrophobic, so that the solubility in water is small, but the solubility of the stilbene compound (a) can be greatly improved by dissolving together with the flavin derivative (B).
First, the stilbene compound (a) will be described. The stilbene compound (A) is a compound represented by the following formula (1) or a glycoside or polymer thereof.
R in formula (1) 1 、R 2 And R is 3 Each independently is a hydrogen atom or a methyl group. From the viewpoint of water solubility, R is preferable 1 、R 2 And R is 3 More preferably, 1 or more of them are hydrogen atoms, more preferably 2 or more thereof, and even more preferably, 3 or more thereof are all hydrogen atoms. X in the formula (1) is a hydrogen atom, a hydroxyl group or a methoxy group.
The stilbene compound (A) may be a glycoside of a compound represented by the formula (1). In this case, the sugar chain is bonded to the hydroxyl group contained in the compound represented by formula (1). The sugar chain may be a monosaccharide or a polysaccharide. In the case of the sugar chain length, the water solubility of the stilbene compound (A) is improved, and the stilbene compound (A) has sufficient water solubility even in the case where the flavin derivative (B) is not used in many cases. Therefore, the meaning of the present invention is great in the case where the sugar chain is a monosaccharide or a disaccharide, particularly in the case of a monosaccharide. The sugar forming the sugar chain is not particularly limited, and glucose, β -rutin sugar and the like can be exemplified, but glucose is preferable. Preferred are glucosides conjugated with 1 molecule of glycol. In addition, a part of the hydroxyl groups of the stilbene compound (a) may form a salt, but it is preferable that the pH of the aqueous solution is kept to a small amount within a range of less than 7. Preferred specific examples of the stilbene compound (A) are shown in Table 1 together with the structures thereof.
TABLE 1
Stilbene compounds (A) R 1 R 2 R 3 X
Resveratrol H H H H
Novel glycosides of spruce H Glc H H
Resveratrol-4' -glucoside H H Glc H
Piceatannol H H H OH
Rhein of Danye H H CH 3 OH
Isodon Okamek (Roxb.) kuntze H H H OCH 3
Pine stilbene H CH 3 H H
Pterostilbene (Pterocarpus gracilis) CH 3 CH 3 H H
Resveratrol is a stilbene compound having 3 hydroxyl groups, and is an important compound in terms of physiological activity. The resveratrol has 1 hydroxyl group combined with glucose (Glc) to obtain piceid (resveratrol-3-glucoside) and resveratrol-4' -glucoside. X is hydroxyl and piceatannol having 4 hydroxyl groups in total. The DANSAIHUANG and MIDANSAIHUANG are substances obtained by converting 1 of 4 hydroxyl groups of piceatannol into methoxy groups, and 1 or 2 of hydroxyl groups of resveratrol are substances obtained by converting 1 or 2 hydroxyl groups into methoxy groups, such as stilbene and pterostilbene. These compounds are reported to have physiological activities, respectively.
The stilbene compound (A) may be a polymer of the compound represented by the formula (1). The polymer may be a polymer in which molecules of the compound represented by the formula (1) are simply bonded to each other, a polymer in which the molecules are dehydrogenated to each other, or a polymer in which the molecules are dehydrated to each other. Particularly preferred are polymers obtained by dehydrogenation-bonding. The polymer may be a dimer or a polymer of a trimer or more, but is preferably a dimer. Examples of the resveratrol polymer include dehydrogenated dimers of epsilon-glucitol (formula (4) below), delta-glucitol, and Gnetrin C, dehydrogenated trimers of alpha-glucitol, and dehydrogenated tetramers of Hopeaphenol.
Next, the flavin derivative (B) will be described. The flavin derivative (B) is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof.
In the 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 B) 2 ). The compound when n is 1 and Y is a hydrogen atom is Flavin Mononucleotide (FMN). In addition, the compound in which 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 is desirably ingested continuously in order to maintain health. Further, since there is substantially no problem of excessive intake, a compound which is dissolved in the aqueous solution of the present invention is preferable. FMN and FAD are both metabolites from riboflavin, as are useful and safe compounds.
The flavin derivative (B) may also be a pharmaceutically acceptable salt. The type of salt is not particularly limited as long as it is a pharmaceutically acceptable salt. Among them, it is desirable that at least a part of the phosphoric acid units contained in the compound represented by the formula (2) form a salt with cations when n is 1 or 2. The cation species in this case is not particularly limited, and examples thereof include sodium, potassium, magnesium, calcium, zinc, ammonium, and the like.
The flavin derivative (B) used in the present invention may be riboflavin (vitamin B) 2 ) Any of Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD), but is effective in increasing stilbene compoundsFrom the viewpoint of solubility of the substance (a), FMN and FAD are preferable. As shown in the example (fig. 2), the same number of moles of FMN and FAD are capable of dissolving the stilbene compound (a) in the same amount. Therefore, if the molecular weights of FMN and FAD are compared, FMN capable of improving solubility with less mass is more preferable.
The concentration of the stilbene compound (A) dissolved in the aqueous solution of the present invention is 0.3 to 200mM. The concentration of the stilbene compound (A) is preferably 1mM or more, more preferably 2mM or more, still more preferably 5mM or more, particularly preferably 10mM or more. Dissolving more stilbene compound (a) can effectively take up stilbene compound (a) by drinking a smaller amount of aqueous solution. In addition, the cosmetic composition can be absorbed from the skin more effectively even when used as a cosmetic. Depending on the type of the stilbene compound (A), it may not be possible to dissolve up to 200mM, and in such a case, the saturation concentration in the aqueous solution may be an upper limit.
The concentration of the flavin derivative (B) dissolved in the aqueous solution of the present invention is 0.3 to 200mM. The concentration of the flavin derivative (B) is preferably 2mM or more, more preferably 5mM or more, still more preferably 10mM or more, particularly preferably 15mM or more. More of the flavin derivative (B) is dissolved, and more of the stilbene compound (A) can be dissolved. Depending on the type of the flavin derivative (B), the concentration may not be as high as 200mM, and in such a case, the saturation concentration may be an upper limit.
As shown in example 1 (FIG. 1), the solubility of stilbene compound (A) increases substantially in proportion to the concentration of flavin derivative (B). The number of molecules of the flavin derivative (B) required for dissolving 1 molecule of the stilbene compound (a) varies depending on the kind of the stilbene compound (a). The more water-soluble stilbene compound (A) is, the more soluble the stilbene compound (A) is in addition to the polymer, the more soluble the stilbene compound (A) can be by a smaller number of molecules of the flavin derivative (B). The number of molecules of the flavin derivative (B) required for dissolving the stilbene compound (A) 1 molecule is less than 1 molecule, piceatannol, danshinin, isodanshinin are about 1 molecule, resveratrol is about 4 molecules, and pterostilbene is about 6 molecules. Although the original water solubility of epsilon-viniferin, which is a dehydrogenated dimer of resveratrol, is not so high, the water solubility is greatly improved by a small amount of the flavin derivative (B).
As shown in example 5, in the absorption spectrum of stilbene compound in aqueous solution, dark shift occurs for each absorption maxima by addition of FMN. Thus, in the aqueous solution of the present invention, it is considered that the stilbene compound (a) molecule and the flavin derivative (B) molecule are associated at a prescribed ratio. Further, this association disappeared by dilution of the aqueous solution, which was confirmed by example 5 (comparison of fig. 4 and 5). Thus, the association of the aromatic ring of the stilbene compound (A) with the aromatic ring of the flavin derivative (B) by pi-pi interaction is an association by a relatively weak force, and is considered to be 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 from 0.2 to 50. The molar ratio (B/A) is more preferably 0.5 or more, and still more preferably 0.8 or more. In the case where 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) hardly has a problem due to excessive intake, it is irrelevant that the flavin derivative (B) having a larger number of molecules than that required for association is dissolved.
The pH of the aqueous solution of the present invention is preferably 1.5 to 6. As shown in example 7 (table 4), when an aqueous solution having a pH of 7 or more was prepared, the amount of stilbene compound (a) was reduced and easily changed to other compounds. The pH is more preferably 5.5 or less, and still more preferably 5 or less. On the other hand, if the pH is too low, the sour taste is too strong when used as a beverage and the skin irritation is too strong when used as a cosmetic, so the pH is more preferably 2.5 or more, and still more preferably 3 or more. In addition, if a product which is diluted with water and used as a beverage or a cosmetic by a consumer is considered, the pH of the aqueous solution of the present invention is more preferably 1.5 or more, and still more preferably 2 or more.
The method for adjusting the pH of the aqueous solution of the present invention to 1.5 to 6 is not particularly limited. Preferably, a pharmaceutically acceptable acid is added as pH adjuster (C). Suitable acids for use as the pH adjuster (C) include carboxylic acids having 2 to 6 carbon atoms, ascorbic acid and its glycosides. Examples of the carboxylic acid 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 view of taste. In addition, ascorbic acid and its glycoside are preferable because they can also provide an antioxidant effect. As the glycoside of ascorbic acid, ascorbic acid 2-glucoside and the like can be exemplified.
The molar ratio (C/B) of the pH regulator (C) to the flavin derivative (B) is preferably 0.2 to 50. If the molar ratio (C/B) is too small, the pH does not sufficiently decrease. Therefore, the molar ratio (C/B) is more preferably 0.3 or more, and still more preferably 0.5 or more. On the other hand, if the molar ratio (C/B) is too large, the sour taste becomes strong and the skin irritation becomes strong. Therefore, the molar ratio (C/B) is more preferably 10 or less, still 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 components used in beverages and cosmetics can be blended as needed. Among these, the concentration of ethanol in the solvent is preferably less than 20% by volume. As shown in reference example 1 (fig. 3), the solubility of the stilbene compound (a) when a mixed solvent of water and ethanol is used increases greatly after the ethanol content exceeds 20% by volume. Therefore, when the concentration of ethanol in the solvent is less than 20% by volume, the incorporation of the flavin derivative (B) is significant. The concentration of ethanol in the solvent is more preferably 5% by volume or less, still more preferably 1% by volume or less, and particularly preferably 0.05% by volume or less. If the aqueous solution of the present invention is used, an alcohol-free beverage can be provided.
The use of the aqueous solution of the present invention is not particularly limited, and beverages and cosmetics are suitable uses. Since a large amount of the stilbene compound (a) is dissolved in water, the stilbene compound (a) is excellent in the absorbability in an organism. In the case of beverages, they are absorbed rapidly from the intestinal tract, and in the case of cosmetics, they are easily absorbed from the skin. As the beverage, not only alcohol-free beverage but also alcohol beverage with low alcohol content of less than 20% can be prepared. Is particularly suitable for use as a supplement beverage. As a cosmetic, it can be formulated into a astringent or an emulsion. In addition, it can also be made into medicinal products such as drop.
As shown in example 6 and example 7 (table 4), the aqueous solutions of the present invention containing the stilbene compound (a) and the flavin derivative (B) sometimes decrease in the amount of the stilbene compound (a) by irradiation with light. In contrast, when the stilbene compound (a) is contained alone without the flavin derivative (B), the decrease in the amount of the stilbene compound (a) due to light irradiation can be suppressed. It is therefore considered that the photostability of the stilbene compound (A) is lowered by associating with the flavin derivative (B). Therefore, the aqueous solution of the present invention is preferably preserved in the dark. As described above, the aqueous solution of the present invention is preferably maintained at a pH of 1.5 to 6 in terms of stability. Therefore, in the case of storing the aqueous solution of the present invention, it is desirable to store the aqueous solution in a dark place at a pH of 1.5 to 6, and a package contained in a container that can be stored in a dark place is a preferred embodiment.
As described above, by compounding the flavin derivative (B), the solubility of the stilbene compound (a) in water is improved. Accordingly, the technical problem of the present invention can also be solved by providing a water solubility enhancer comprising a flavin derivative (B) for enhancing the solubility of a stilbene compound (a) in water.
In another aspect of the present invention, the composition is a composition in a form selected from a powder, a granule, a tablet, and a paste, and the composition comprises a stilbene compound (a) and a flavin derivative (B), and the molar ratio (B/a) of the flavin derivative (B) to the stilbene compound (a) is 0.5 to 50. Such a composition is useful as a raw material for obtaining the above aqueous solution, and even when it is directly taken up or applied, the stilbene compound (a) is easily absorbed by living bodies. In this case, the preferable molar ratio (B/A) is as described in the above description of the aqueous solution. The pH adjuster (C) which further contains a carboxylic acid having 2 to 6 carbon atoms, ascorbic acid and its glycoside is also preferably the same as in the case of the above aqueous solution. The preferred molar ratio (C/B) is as described in the description of the aqueous solution above.
The composition of the present invention may be in a form substantially free of moisture, such as powder, granules or tablets, or may be in a form in which the stilbene compound (a) is not completely dissolved in water and dispersed as granules in a high-viscosity liquid, such as a paste. These compositions may contain a stilbene compound (A), a flavin derivative (B), a pH adjustor (C) and components other than water as required. The stilbene compound (A) was reduced only a little by light irradiation even in the state where water was hardly present as shown in example 6. Therefore, the composition of the present invention is also preferably stored in the same light-shielding manner as the aqueous solution of the present invention. In addition, a package in which the composition is contained in a light-shielding container is a preferred embodiment. The use of the composition of the present invention is not particularly limited, and the composition can be used as a health food or a supplement. In addition, it is also possible to prepare 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 stilbene compound (a) powder and the flavin derivative (B) powder may be simply mixed with other components as needed. However, from the viewpoint of the dissolution rate in water, it is preferable to prepare an aqueous solution in which the stilbene compound (a) and the flavin derivative (B) are dissolved, and then remove the water contained therein to produce the composition of the present invention. As shown in example 9 (fig. 9), the powder of the composition obtained by forming the aggregate of the stilbene compound (a) and the flavin derivative (B) in advance and then drying the same was particularly high in dissolution rate in water as compared with the powder obtained by simply mixing the powder of the stilbene compound (a) and the powder of the flavin derivative (B). Therefore, the time required for dissolving the composition in water to prepare an aqueous solution can be shortened, and thus the consumer can easily prepare an aqueous solution. In addition, the absorption rate and the absorption rate of the composition when the composition is directly taken into the body can be improved.
Another aspect of the present invention is a method for extracting a stilbene compound (a), comprising contacting a plant material containing a stilbene compound (a) or a crude extract obtained by extracting the plant material with an organic solvent with an aqueous solution in which a flavin derivative (B) is dissolved, and extracting the stilbene compound (a) from the plant material or the crude extract to the aqueous solution. Since the aqueous solution in which the flavin derivative (B) is dissolved can dissolve a larger amount of the stilbene compound (a) than water, the stilbene compound (a) can be efficiently extracted using the aqueous solution. The concentration of the flavin derivative (B) dissolved in the aqueous solution is 0.3 to 200mM. The concentration of the flavin derivative (B) is preferably 2mM or more, more preferably 5mM or more, still more preferably 10mM or more, particularly preferably 15mM or more.
In extracting hydrophobic active ingredients from plant materials, organic solvents are often used as extraction solvents. However, from the viewpoint of environmental protection, it is desirable to use no organic solvent as the extraction solvent in order not to volatilize the organic solvent to the surroundings. In addition, even in the case of use in beverages or cosmetics, it is desirable that no organic solvent be used 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. Further, since only the stilbene compound (a) which can be associated with the flavin derivative (B) can be selectively extracted, the stilbene compound (a) having high purity can be obtained. The aqueous solution containing the stilbene compound (A) and the flavin derivative (B) thus obtained can be used as a raw material for beverages or cosmetics. Further, the stilbene compound (a) having a high purity can be obtained by extracting a crude extract component obtained by extracting a plant material in advance with an organic solvent or the like with an aqueous solution containing the flavin derivative (B).
Further, an inhibitor of the production of superoxide catalyzed by xanthine oxidase, which comprises as an active ingredient the stilbene compound (a) which is a compound represented by the above formula (1) or a glycoside or polymer thereof, is also an embodiment of the present invention. These compounds inhibit the reaction of forming superoxide more strongly than inhibit the reaction of forming uric acid from xanthine with xanthine oxidase as a catalyst. This was the first time a compound was reported that specifically inhibited the production of superoxide catalyzed by xanthine oxidase.
In this case, in the formula (1), R is preferably 1 、R 2 、R 3 And X is a hydrogen atom. That is, a preferred embodiment is that the stilbene compound (a) is resveratrol or a glycoside or polymer thereof. Resveratrol and its derivatives are considered to be particularly useful as physiologically active substances.
In this case, R in the above formula (1) is also preferable 1 And R is 2 Is a hydrogen atom, R 3 Is a hydrogen atom or a methyl group, X is a hydroxyl group. That is, stilbene compound (A) is chrysin or piceatannol, or a glycoside or polymer thereof, as a preferred embodiment. These compounds strongly inhibit both the reaction of producing uric acid from xanthine using xanthine oxidase as a catalyst and the reaction of producing superoxide. Rhein inhibits particularly strongly the reaction of xanthine to uric acid from xanthine with xanthine oxidase as catalyst. On the other hand, piceatannol inhibits the formation of superoxide more strongly than rhein by accelerating the reaction of hydrogen peroxide from oxygen in addition to the reaction of uric acid from xanthine with xanthine oxidase as a catalyst.
In addition, an inhibitor of the formation of superoxide catalyzed by xanthine oxidase, comprising the above aqueous solution in which stilbene compound (a) and flavin derivative (B) are dissolved, is a preferred embodiment. Furthermore, an inhibitor of the formation of superoxide catalyzed by xanthine oxidase, comprising a composition comprising stilbene compound (a) and flavin derivative (B) in a form selected from the group consisting of powder, granules, tablets or paste, is also a preferred embodiment.
Examples
Example 1 (solubility in aqueous FMN solution)
Flavin Mononucleotide (FMN) was dissolved in pure water at 20℃to prepare aqueous solutions at concentrations of 2.5, 5, 10, 20, 25, 50 and 100mM (mmol/L), respectively. The FMN used herein is a sodium salt of a compound in which n is 1 and Y is a hydrogen atom in the compound of formula (2), and 1 mole of phosphate groups contained in FMN are neutralized with about 1 mole of sodium ions. The pH of the 20mM aqueous solution of FMN was 5.8. Further, the concentration of the saturated aqueous solution of FMN at 20℃was 173mM. The same applies to FMN used in other examples described below.
Resveratrol powder manufactured by tokyo chemical industry co was added to pure water and FMN aqueous solutions of various concentrations, and the mixture was dissolved by irradiation with ultrasonic waves for 30 minutes. After standing for 1 hour, the undissolved powder was centrifuged to remove the precipitate, and the supernatant was separated to determine the resveratrol concentration. In the measurement, the above supernatant was diluted with 0.1M sodium citrate buffer (pH 4.0), the difference spectrum against the same concentration of FMN aqueous solution was measured, and the solubility (mM) of resveratrol in the aqueous solution was calculated from the absorbance of the absorption peak (molar absorption coefficient. Epsilon.: 29,000) at 317 nm. The resulting solubilities are summarized in table 2 and in the graph shown in fig. 1.
The absorbance was measured at each wavelength as in the case of piceatannol (LKT lab, absorption maxima 319nm, epsilon=30,500), piceatannol (BLD pharm, absorption maxima 324nm, epsilon= 27,200), danshinolic rheum officinale (tokyo chemical industry Co., ltd., absorption maxima 324nm, epsilon= 28,900), isodanshinolic rheum officinale (tokyo chemical industry Co., ltd., absorption maxima 324nm, epsilon= 29,300), pterostilbene (BLD pharm, absorption maxima 317nm, epsilon= 29,200), and epsilon-grape (Fuji film and light purity chemical Co., ltd., absorption maxima 322nm, epsilon=25,500), and the solubility was obtained. The measurement results are summarized in table 2, and the graphs shown in fig. 1 are summarized.
TABLE 2
As is clear from Table 2 and FIG. 1, the solubility of picroside, rhein and isorhein in pure water was 1.19 to 2.73mM, but the solubility was far higher than that in pure water by dissolution in FMN aqueous solution. Since the solubility of these stilbene compounds increases substantially linearly at 1:1 in proportion to the concentration of FMN, it is known that 1 molecule of FMN is required to dissolve these stilbene compounds. Thus, it is speculated that these stilbene compounds associate with FMN in a 1 to 1 ratio in aqueous solution.
On the other hand, when resveratrol having a solubility of 0.19mM in pure water is dissolved in an aqueous FMN solution, the solubility of resveratrol increases substantially linearly in proportion to the concentration of FMN, but in order to dissolve resveratrol 1 molecule, about 4 molecules of FMN are required. Therefore, resveratrol is presumed to be surrounded by about 4 molecules of FMN in water and dissolved.
In the case of pterostilbene (0.09 mM) which has low solubility in pure water, more FMN molecules than resveratrol are required for dissolving 1 molecule. On the other hand, in the case of piceatannol (8.34 mM) and epsilon-glucinol (3.83 mM) having high solubility in pure water, FMN required for dissolving 1 molecule was 1 molecule or less.
Example 2 (solubility in aqueous FAD solution)
Flavin Adenine Dinucleotide (FAD) was dissolved in pure water at 20℃to prepare aqueous solutions at concentrations of 5, 10 and 20mM, respectively. The FAD used herein is a sodium salt of a compound in which n is 2 and Y is a group represented by formula (3) in the compound of formula (2), and the diphosphate groups contained in the FAD are neutralized with about 2 moles of sodium ions. In addition, the concentration of the saturated aqueous solution of FMN at 20℃was 324mM.
The solubility of resveratrol was measured by dissolving resveratrol powder in pure water and FAD aqueous solutions of respective concentrations in the same manner as in example 1. The solubility of resveratrol in the resulting FAD aqueous solution is summarized in table 3 and fig. 2 together with the solubility of resveratrol in the FMN aqueous solution obtained in example 1.
TABLE 3
As can be seen from table 3 and fig. 2, resveratrol having the same number of moles can be dissolved in the same molar concentration in both the FMN aqueous solution and the FAD aqueous solution. Thus, both FMN and FAD can be considered to act on resveratrol molecules in the same way, contributing to their water solubility.
Example 3 (solubility in aqueous Riboflavin solution)
A saturated aqueous solution of riboflavin was prepared, having a solubility of 0.43mM. The riboflavin is a compound in which n is 0 and Y is a hydrogen atom in the compound of formula (2). The saturated aqueous solution of riboflavin was dissolved in resveratrol powder in the same manner as in example 1, and the solubility of resveratrol was measured. As a result, 0.45mM resveratrol was dissolved, and the solubility was doubled or more compared with the solubility in pure water (0.19 mM).
Reference example 1 (solubility in aqueous ethanol)
Ethanol was added to pure water to prepare aqueous ethanol solutions having concentrations of 5, 10, 20, 30 and 40 vol%, respectively. The solubility of resveratrol was measured by dissolving resveratrol powder in pure water and ethanol aqueous solutions of respective concentrations in the same manner as in example 1. The solubility (mM) of resveratrol at each ethanol concentration was 0.19.+ -. 0.03 (pure water), 0.25.+ -. 0.02 (5 vol%), 0.37.+ -. 0.02 (10 vol%), 1.30.+ -. 0.30 (20 vol%), 7.38.+ -. 1.10 (30 vol%), 32.9.+ -. 3.0 (40 vol%). The solubility with respect to the ethanol concentration is shown in the graph of fig. 3.
As can be seen from fig. 3, the solubility of resveratrol increases as the ethanol concentration increases. However, the solubility of resveratrol, which has an ethanol concentration of about 20 vol%, is not so high, and the solubility increases sharply as it becomes 30 vol% and 40 vol%. As the polarity of the solvent changes, the solubility changes, and the solubility of the stilbene compound increases in linearity in proportion to the concentration of the flavin derivative.
Example 4 (preparation of a Mixed powder of stilbene Compound and FMN)
An aqueous solution comprising stilbene compounds (resveratrol, piceatannol or piceatannol) and FMN was prepared. At this time, referring to the results of example 1 (Table 2), FMN was added in an amount required to make each stilbene compound completely soluble in water. That is, FMN was added in a number of moles that was 4 times that of resveratrol, 1.5 times that of piceatannol, and 1 time that of piceatannol.
Specifically, 1mmol (228 mg) of resveratrol was completely dissolved in an aqueous solution obtained by dissolving 4mmol (1913 mg) of FMN in 200mL of water. In addition, 2mmol (780 mg) of picroside was completely dissolved in an aqueous solution obtained by dissolving 3mmol (1436 mg) of FMN in 150mL of water. Further, 1mmol (244 mg) of piceatannol was completely dissolved in an aqueous solution obtained by dissolving 1mmol (479 mg) of FMN in 100mL of water. The aqueous solution thus obtained was dried under reduced pressure to obtain a mixed powder containing resveratrol and FMN in a molar ratio of 1:4, a mixed powder containing piceatannol and FMN in a molar ratio of 2:3, and a mixed powder containing piceatannol and FMN in a molar ratio of 1:1, respectively. The mass of each mixed powder containing 1 mol of FMN was 535g, 738g, 723g, respectively.
Example 5 (spectra of a Mixed aqueous solution of stilbene Compound and FMN)
When the mixed powder of the stilbene compound obtained in example 4 and FMN was dissolved in water, both of them exhibited a reddish orange color. Since the same concentration of FMN aqueous solution containing no stilbene compound exhibited yellow color, it was found that the color tone was changed by containing the stilbene compound. In order to quantitatively grasp this phenomenon, the absorbance 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 7.23mg/mL to prepare an aqueous solution containing piceatannol and FMN at 10 mM. The visible light absorption spectrum of the obtained aqueous solution was measured with a glass cuvette having an optical path length of 0.1mm, to obtain a spectrum of an aqueous solution of a mixture of piceatannol and FMN (dotted line of fig. 4). The spectra of aqueous solutions containing only 10mM FMN are also shown in solid lines in FIG. 4. The absorbance epsilon of each absorption maximum of the aqueous mixture was 27,800 (331 nm), 6,490 (377 nm), 8,330 (449 nm) and 2,020 (510 nm). It is presumed that the occurrence of weak absorption around 510nm occurs in a change in hue from yellow to red-orange. The absorption band decreases if diluted with water. The above mixed aqueous solution was diluted 100 times with pure water, and the FMN concentration was set to 0.01mM, and the spectrum (dotted line in FIG. 5) was measured in a cuvette having an optical path length of 10mM and was substantially the same as the sum of the spectrum of piceatannol alone and the spectrum of FMN alone.
The mixed powder of picroside and FMN obtained in example 4 was dissolved in pure water at a concentration of 7.38mg/mL to prepare an aqueous solution containing 6.7mM of picroside and 10mM of FMN. As a result of the spectroscopic measurement in the same manner as described above, the absorbance epsilon of each absorbance maximum was 21,100 (328 nm), 6,900 (380 nm), 8,730 (449 nm) and 1,700 (510 nm), respectively. In addition, the mixed powder of resveratrol and FMN obtained in example 4 was dissolved in pure water at a concentration of 5.35mg/mL to prepare an aqueous solution containing 2.5mM resveratrol and 10mM FMN. As a result of spectral measurement, the absorbance epsilon was 10,100 (336 nm), 8,070 (376 nm), 9,920 (448 nm) and 730 (510 nm) at each absorption maximum.
It is known that in the absorption spectrum of stilbene compounds, dark shifts occur in the absorption maxima by addition of FMN. In addition, in an aqueous solution containing a stilbene compound and FMN, absorption at 510nm was observed, and thus it is considered that a complex of both compounds was formed in the aqueous solution. Since the complex produced in a high concentration aqueous solution is reduced by dilution, it is presumed that stilbene compound and FMN associate in the aqueous solution through a comparatively weak pi-pi interaction produced between the two.
Example 6 (stability of 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 ℃) for 30 days under a fluorescent lamp, and then the absorbance was measured, whereby the amount of decrease in resveratrol contained in the mixed powder was only 3.6±1.5%. The mixed powder was dissolved in pure water at a concentration of 5.35mg/mL to prepare an aqueous solution containing 2.5mM resveratrol and 10mM FMN. The resulting aqueous solution was placed in a transparent glass bottle, and stored at room temperature (20 ℃) for 5 days in a fluorescent lamp room, and a 25.6% decrease was confirmed by measuring the amount of resveratrol. On the other hand, if the brown glass bottle is stored under the same conditions instead of the transparent glass bottle, the decrease in resveratrol is only 3.4% even after 17 days. Further, the above aqueous solution was diluted 100 times with pure water to prepare an aqueous solution containing resveratrol 0.025mM and FMN 0.1 mM. The diluted aqueous solution was allowed to stand at 20℃for 1 day without being protected from light, and as a result, the absorbance (317 nm) of resveratrol was significantly reduced, and the amount of resveratrol remaining was half or less. If light is shielded, the decrease in absorbance of resveratrol is suppressed.
Example 7 (Effect of light and pH on the aqueous Mixed solution of resveratrol and FMN)
100mM sodium citrate buffer as a buffer at pH4, 100mM sodium phosphate buffer as a buffer at pH6 and pH7, and 40mM sodium phosphate buffer as a buffer at pH8 were prepared, respectively. The buffers of the above-mentioned pH4, 6, 7 and 8 were added to 0.6mL of a saturated (0.19 mM) resveratrol aqueous solution, and the total amount was 3.00mL and mixed to obtain 4 aqueous solutions. The above buffers were added to 0.03mL of an aqueous solution containing 2.5mM resveratrol and 10mM FMN, and the total amount was 3.00mL and mixed to obtain 4 aqueous solutions. The aqueous solution thus obtained was placed in a cuvette for measurement made of quartz glass. The absorbance at 317nm of each of the obtained aqueous solutions was measured by irradiation with light at 20℃or by standing for 1 hour in the absence of light. In this case, the light source was a fluorescent lamp, and light was irradiated through the vessel at a position of about 2m just below 2 fluorescent lamps of 2250 lumens. The reduction rate (%) of resveratrol amount was calculated from the absorbance thus obtained. The results are summarized in Table 4.
TABLE 4
As shown in table 4, it was found that resveratrol in a thin aqueous solution prepared by dissolving a mixed powder of resveratrol and FMN in water was unstable to light. This is presumed to be due to inclusion of FMN. In addition, it is also known that if the pH is increased, the pH becomes unstable gradually. Therefore, it is found that when an aqueous solution containing resveratrol and FMN is stored, it is desirable to keep the aqueous solution acidic and light-proof.
Example 8 (pH of aqueous solution comprising resveratrol, FMN and acetic acid)
To an aqueous solution containing 5mM resveratrol and 20mM FMN, acetic acid was added so that the ratio of the number of moles of acetic acid to the number of moles of FMN (acetic acid/FMN) became 0, 0.25, 0.5, 1, 2, 5, 10, 25, 50 and 100, to prepare an aqueous solution, and their pH was measured. In addition, aqueous solutions were prepared from which resveratrol was removed from the respective aqueous solutions, and their pH was also measured. The results are shown in fig. 6. The pH of an aqueous solution containing resveratrol and FMN can be adjusted to a desired value by adjusting the amount of acetic acid to be incorporated. The presence or absence of resveratrol does not have a large influence on the pH of the aqueous solution, but it is considered that this is because the pKa value of the stilbene compound (a) is large. In this way, the stability of the stilbene compound (a) is ensured by lowering the pH by adding an appropriate amount of acid, and a mixture of moderate sourness can be obtained.
Reference example 2 (pH of aqueous solution comprising FMN and acid)
To a 20mM aqueous FMN solution, an acid (acetic acid, citric acid, lactic acid, ascorbic acid) was added so that the ratio of the number of moles of the acid to the number of moles of FMN (acid/FMN) became 0, 0.25, 0.5, 1, 2, 5, 10, 25, 50 and 100 to prepare an aqueous solution, and the pH thereof was measured. The results are shown in fig. 7 and 8. By adjusting the type and the mixing ratio of the acid, the pH of the aqueous solution can be adjusted to a desired value of 1.5 to 6.
Example 9 (dissolution rate of Mixed powder in Water)
A mixed powder 1 was prepared by mixing 114mg (0.5 mmol) of resveratrol powder and 957mg (2 mmol) of FMN powder and mixing the mixture with an agate mortar. On the other hand, a mixed powder 2 obtained by drying a mixed aqueous solution of resveratrol and FMN in example 4 was prepared. To 3mL of 0.1M citric acid buffer (pH 4.0) was added about 0.2mg of any of the mixed powder 1 and mixed powder 2, and the mixture was stirred slowly immediately, absorbance at 317, 374 and 445nm was measured every 3 minutes, and stirring and measurement were repeated until after 33 minutes. 317nm is the absorption peak from resveratrol, and 374nm and 445nm are the absorption peaks from FMN. The amount of resveratrol dissolved was calculated from the absorbance at 317nm and is shown in FIG. 9. As shown in fig. 9, not just mixing the powder, but a method of previously preparing a mixed aqueous solution and then drying it can obtain a powder having a higher dissolution rate. In addition, the taste of the mixture powder of resveratrol, FMN and citric acid has a moderate sour taste, and the mixture powder of resveratrol and FMN is substantially odorless.
Example 10 (extraction of resveratrol)
Commercial products of crude extract powder obtained by extraction from grapes and the like with an organic solvent and drying are used. The crude extract powder contained resveratrol about 10 mass%. To 100mL of the crude extract powder, 10mL of the aqueous solution or pure water shown below was added, and the extract was obtained by irradiating with ultrasonic waves for 30 minutes. After standing, insoluble components were centrifuged, and the supernatant was used as an extract. The solubility of resveratrol was calculated from the absorbance at 317nm obtained by diluting the obtained extract with 0.1M citric acid buffer (pH 4.0) and measuring. The recovery rate of resveratrol thus obtained was 9.2% when pure water was used as the extract, 84% when 25mM FMN aqueous solution was used, and 96% when 50% by volume ethanol aqueous solution was used. From this, it is found that the use of an aqueous FMN solution can efficiently extract the FMN as an aqueous solution even without using an organic solvent.
Example 11 (test for uric acid production)
Xanthine Oxidase (XO) enzyme solution (EC1.1.3.22, gradeIV) was purchased from Sigma-Aldrich. Stilbene compounds were dissolved in DMSO to prepare 10mM sample solutions. A DMSO solution (0-0.03 mL) of xanthine and stilbene compound (0-200. Mu.M) was added to a 40mM sodium carbonate buffer (pH 10) containing EDTA (0.1 mM), and a mixed solution (2.88 mL) was prepared. To this mixture at 25℃was added 0.12mL of XO enzyme solution (0.04 units), and the reaction was started, and the absorbance at 293nm (ultraviolet absorbance maximum of uric acid) was measured for 60 seconds. The control experiment was an assay with the sample solution replaced with DMSO. The reaction rate was obtained from a linear increase in absorbance. Determination of inhibition constant Ki (μM) of uric acid production reaction by stilbene Compound and 50% inhibition concentration IC at xanthine concentration of 200 μM 50 The results are shown in Table 5. In order to confirm absorption by the stilbene compound at 293nm, uric acid can be quantified only when the concentration of the stilbene compound is lower than 0.1 mM.
Each measurement was performed 3 times or more in each experiment. Analysis was performed with Sigma plot 2001 (SPSS inc., chicago, IL). Inhibition pattern and reaction rate theory were analyzed using Enzyme Kinetics Module 1.1.1 (SPSS inc.) attached to Sigma plot 2001. These methods are also used in examples 12 to 14 below.
TABLE 5
Stilbene compounds Ki(μM) IC 50 (μM)
Resveratrol 7.7±0.7 34±4
Resveratrol-3-glucoside － >50
Resveratrol-4' -glucoside 7.0±0.8 25±4
Resveratrol-3-oligosaccharin － >50
Pterostilbene (Pterocarpus gracilis) － >50
Piceatannol 6.7±1.6 15±5
Rhein of Danye 3.5±0.2 7.9±0.5
Isodon Okamek (Roxb.) kuntze 96±2 161±11
As shown in table 5, resveratrol-4' -glucoside, piceatannol, rhein, and isorhein calculated only the inhibition constants Ki and not others, and thus analysis inhibited uric acid production by competitive inhibition. This competitive inhibition is expected from the slow rate of oxidation of xanthine and the fast rate of reduction of oxygen. Slave IC 50 Among these, rhein has particularly strong uric acid production inhibitory activity.
Example 12 (test for superoxide production)
A sample solution (2.88 mL) prepared by dissolving 0.06mL of xanthine (0.200. Mu.M), 0.03mL of 0.5% bovine serum albumin, 0.03mL of 2.5mM of azulene tetrazolium, and 10mM of stilbene compound in DMSO was added to a 40mM sodium carbonate buffer (pH 10) containing 0.1mM of EDTA. To this mixture at 25℃was added 0.12mL of XO enzyme solution (0.04 units) to start the reaction, and the absorbance at 560nm was measured for 60 seconds. The control experiment was an assay with the sample solution replaced with DMSO. The reaction rate was obtained from the linear increase in absorbance. The above reaction was carried out in order to detect unstable superoxide (O 2 － And. Cndot.) is carried out at pH 10. The superoxide reduces and converts the nitrogen blue tetrazolium into blue formazan (absorption maximum value is 560 nm), so that quantitative detection is carried out on the nitrogen blue tetrazolium. When the concentration of xanthine is varied from 0 to 200. Mu.M, the formation of superoxide is inhibited by stilbene compounds. The results are summarized in Table 6.
TABLE 6
Stilbene compounds Ki(μM) K'i(μM) IC 50 (μM)
Resveratrol 7.1±1.0 － 21±3
Resveratrol-3-glucoside 19±2 － 49±1
Resveratrol-4' -glucoside 5.0±0.1 － 11±2
Resveratrol-3-oligosaccharin 121±19 － 323±20
Pterostilbene (Pterocarpus gracilis) 43±5 － 124±16
Piceatannol 2.2±0.1 21.1±1.0 4.5±0.4
Rhein of Danye 2.4±0.2 － 5.0±0.2
Isodon Okamek (Roxb.) kuntze 36±3 － 99±8
As shown in table 6, since stilbene compounds other than piceatannol calculated only the inhibition constant Ki and not other inhibition constants, the analysis inhibited superoxide production by competitive inhibition. On the other hand, the analysis further calculated that piceatannol with inhibition constant K' i was inhibited by mixed (mixed) to inhibit superoxide generation. Slave IC 50 Among these, piceatannol has particularly strong superoxide generation inhibitory activity. These stilbene compounds strongly inhibit superoxide formation through uric acid formation, thus indicating that stilbene compounds bind to FAD sites within Xanthine Oxidase (XO).
Example 13 (DPPH scavenger Activity)
The method of scavenging DPPH (2, 2-diphenyl-1-picrylhydrazine) as a stable free radical is the method described in Blois, M.S. (1958) Antioxidant determinations by the use of astable free radical Nature,181, 1199-1200. In addition, DPPH activity shows properties of electron donating or hydrogen atoms (Zhang, r., kang, k.a., piao, m.j., lee, k.h., jang, h.s., park, m.j., kim, b.j., kim, j.s., kim, y.s., ryu, s.y., hyun, j.w. (2007)). After adding 1.0mL of 100mM acetic buffer (pH 5.5), 1.87mL of ethanol and 0.1mL of ethanol solution of DPPH of 3mM to a test tube, 0.03mL of a sample solution obtained by dissolving stilbene compound in DMSO was added, and the reaction was carried out at 25℃for 20 minutes. Meanwhile, absorbance at 517nm (DPPH, ε=8.32X10) 3 ). Control experiments were assayed by replacing the sample solution with DMSO. The decrease in absorbance was measured for 20 minutes, and the number of DPPH molecules for scavenging 1 molecule of stilbene compound was calculated as the scavenging activity. Reduction of initial rate of the scavenging reaction from absorbance after sample additionThe values were obtained (Masuoka, N., isobe, T., kubo, I. (2006) Antioxidants from Rabdosia japonica. The results are summarized in Table 7.
TABLE 7
As shown in table 7, resveratrol and its glycoside, and pterostilbene showed as weak a scavenging activity as alkenyl phenol. On the other hand, piceatannol, rhein and isorhein show the same degree of strong activity. However, if the initial rate of the scavenging activity of these compounds is determined, the initial rate of piceatannol is faster than the other 2 compounds. This suggests that Pi Shachun is capable of reducing XO molecules (see 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 compositions. Phytochemistry (2018) 155, 100-106). Wherein the DPPH scavenging activity of dodecyl gallate of food additive with antioxidant effect is 7.32+ -0.04.
Example 14 (superoxide scavenging Activity)
Superoxide (O) 2 － In the PMS-NADH system (Nishikimi, M., rao, N.A., yagi, K. (1972) The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxgen. Biochem. Biophys. Res. Commun.46, 849-854). A mixed solution (2.97 mL) was prepared by dissolving 0.1mM EDTA in 2.82mL of 40mM sodium carbonate buffer (pH 10), 0.03mL of 0.5% bovine serum albumin aqueous solution, 0.03mL of 2.5mM nitroblue tetrazolium aqueous solution, 0.06mL of a sample solution of stilbene compound 10mM in DMSO, and 0.03mL of 7.8mM NADH aqueous solution. To this mixture at 25℃was added 0.03mL of a 115. Mu.M aqueous solution of PMS (phenazine methosulfate), and the reaction was started, and the absorbance at 560nm was measured for 60 seconds. The superoxide is scavenged (eliminated) in the reaction with stilbene compounds. Unreacted superoxide radical and nitro blueTetrazolium reacts to become blue (formazan), and is therefore quantified. Control experiments were assayed by replacing the sample solution with 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 figure 10.
Clearance activity (%) = [1- (sample speed)/(control speed) ]. Times.100
In the radical scavenging reaction, the superoxide (O 2 － And stilbene compounds in a molar ratio of 1:1. Among these stilbene compounds, piceatannol is exceptionally high in activity. Even with caffeic acid (IC) 50 =51 μm) or gallic acid (IC 50 =29 μm), piceatannol IC 50 The value (9.0.+ -. 2.0. Mu.M) is also significantly smaller, indicating that piceatannol is a strong scavenger. This scavenging activity may be associated with a stronger stabilization of piceatannol radicals by the long conjugated ene glycol structure of piceatannol.

Claims (13)

1. An aqueous solution characterized in that:
0.3 to 200mM of stilbene compound (A) which is a compound represented by the following formula (1) or a glycoside or polymer thereof and 0.3 to 200mM of flavin derivative (B) which is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof are dissolved,
the glycoside is a glycoside having 1 molecule of diol bound thereto, and the polymer is epsilon-glucagons, delta-glucagons or GmbH C as dehydrogenated dimers of resveratrol,
in the formula (1), R 1 、R 2 And R is 3 Each independently is a hydrogen atom or a methyl group, X is a hydrogen atom, a hydroxyl group or a methoxy group,
In the formula (2), when n is 1 or 2, and n is 1, Y is a hydrogen atom, and when n is 2, Y is a group represented by the following formula (3),
2. the aqueous solution of claim 1, wherein:
the stilbene compound (A) is at least 1 selected from resveratrol, piceatannol, rheum officinale, isorhapontin, pterostilbene and pinostilbene and their glycosides.
3. The aqueous solution according to claim 1 or 2, characterized in that:
the molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) is 0.5 to 50.
4. The aqueous solution according to claim 1 or 2, characterized in that:
the pH is 1.5-6.
5. The aqueous solution according to claim 1 or 2, characterized in that:
further comprises a pH regulator (C) selected from carboxylic acids having 2 to 6 carbon atoms, ascorbic acid and its glycosides.
6. The aqueous solution according to claim 1 or 2, characterized in that:
the concentration of ethanol in the solvent is less than 20% by volume.
7. A beverage, characterized in that:
an aqueous solution comprising any one of claims 1 to 6.
8. A water solubility enhancer characterized by:
for increasing the solubility of stilbene compounds (A) in water, the water-solubility-increasing agent comprising a flavin derivative (B),
Stilbene compound (A) is a compound represented by the following formula (1) or a glycoside thereof, which is a glucoside to which 1 molecule of diol is bound, or a polymer thereof, which is epsilon-glucagons, delta-glucagons or GmbH C as a dehydrogenated dimer of resveratrol, and,
the flavin derivative (B) is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof,
in the formula (1), R 1 、R 2 And R is 3 Each independently is a hydrogen atom or a methyl group, X is a hydrogen atom, a hydroxyl group or a methoxy group,
in the formula (2), when n is 1 or 2, and n is 1, Y is a hydrogen atom, and when n is 2, Y is a group represented by the following formula (3),
9. a composition in a form selected from the group consisting of powder, granules, tablets, and paste, characterized in that:
comprising a stilbene compound (A) which is a compound represented by the following formula (1) or a glycoside or a polymer thereof, a flavin derivative (B) which is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof, a glycoside which is a glucoside to which 1 molecule of glycol is bonded, a polymer which is epsilon-glucagons, delta-glucagons or Gnetin C which are dehydrogenated dimers of resveratrol, a molar ratio (B/A) of the flavin derivative (B) to the stilbene compound (A) being from 0.5 to 50,
In the formula (1), R 1 、R 2 And R is 3 Each independently is a hydrogen atom or a methyl group, X is a hydrogen atom, a hydroxyl group or a methoxy group,
in the formula (2), when n is 1 or 2, and n is 1, Y is a hydrogen atom, and when n is 2, Y is a group represented by the following formula (3),
10. the composition of claim 9, wherein:
further comprises a pH regulator (C) selected from carboxylic acids having 2 to 6 carbon atoms, ascorbic acid and its glycosides.
11. An inhibitor of superoxide generation catalyzed by xanthine oxidase, characterized by:
a composition comprising the composition of claim 9 or 10.
12. A method for producing the composition according to any one of claims 9 to 11, characterized in that:
after preparing an aqueous solution in which stilbene compound (A) and flavin derivative (B) are dissolved, water is removed.
13. A method for extracting stilbene compound (A) is characterized in that:
contacting a plant material comprising a stilbene compound (A) or a crude extract obtained by extracting the plant material with an organic solvent with an aqueous solution in which a flavin derivative (B) is dissolved, extracting the stilbene compound (A) into the aqueous solution from the plant material or the crude extract, wherein the stilbene compound (A) is a compound represented by the following formula (1) or a glycoside or a polymer thereof, the flavin derivative (B) is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof, the glycoside is a glucoside to which 1 molecule of glycol is bound, and the polymer is epsilon-glucopyranoside, delta-glucopyranoside or Gnetin C which is a dehydrogenated dimer of resveratrol,
In the formula (1), R 1 、R 2 And R is 3 Each independently is a hydrogen atom or a methyl group, X is a hydrogen atom, a hydroxyl group or a methoxy group,
in the formula (2), when n is 1 or 2, and n is 1, Y is a hydrogen atom, and when n is 2, Y is a group represented by the following formula (3),
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