JP5124712B2 - Method for producing polyphenol glycoside - Google Patents

Description

  The present invention relates to a method for producing a polyphenol glycoside. In particular, the present invention relates to a method for producing a polyphenol glycoside in which a plurality of glucoses are bonded to the phenolic hydroxyl group of a polyphenol compound.

  Polyphenol is a general term for compounds having a plurality of phenolic hydroxyl groups in the same molecule, and is known to be contained in plants and the like. Many polyphenols have physiologically active functions, but are often poorly soluble in water, and are used for various applications by inclusion or glycosylation of polyphenols using cyclodextrins or the like. .

  For example, JP-A-9-3089 (Patent Document 1) describes a glycoside of tea polyphenols and a method for producing the same. Specifically, polyphenols are distributed by allowing cyclomaltodextrin glucanotransferase derived from Bacillus stearothermophilus to act on tea polyphenols and dextrin, cyclodextrin, starch or a mixture thereof. It is described that it can be saccharified. According to this, unlike conventional polyphenols, there is little bitterness such as bitterness, astringency, sashimi and astringency, and it can be contained as it is or with other materials regardless of the degree and purity of its purification. It can be used in a wide range of fields such as foods, quasi drugs, cosmetics, and pharmaceuticals. However, there are many cases where glycosides cannot always be obtained efficiently, and improvements have been desired.

  Japanese Patent Application Laid-Open No. 2005-41817 (Patent Document 2) describes a curcuminoid glycoside and a method for producing the same. Specifically, by condensing a glycosylated aromatic aldehyde compound as a starting material with an acetylacetone-boron oxide complex to form a curcuminoid glycoside, and removing the boron by heating the reaction product with a lower alcohol, It is said that a wide range of curcuminoid glycosides can be produced efficiently. However, there are many cases where glycosides cannot always be efficiently obtained by such a chemical synthesis method, and improvements have been desired.

Japanese Patent Laid-Open No. 9-3089 JP 2005-41817 A

  The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a method for producing a polyphenol glycoside capable of efficiently obtaining a polyphenol glycoside having a plurality of glucose bonded thereto. .

  The above-mentioned problem is that a γ-cyclodextrin glucanotransferase is allowed to act on the compound (B) in which one molecule of glucose is bonded to the phenolic hydroxyl group of the polyphenol compound (A) and the sugar donor (C). This is solved by providing a method for manufacturing the body (D).

  At this time, it is preferable that the polyphenol compound (A) is curcumin, and the sugar donor (C) is at least one selected from the group consisting of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin. It is preferable that It is also preferable that the γ-cyclodextrin glucanotransferase is an enzyme derived from Bacillus macerans.

  According to the method for producing a polyphenol glycoside of the present invention, a polyphenol glycoside having a plurality of glucose bonds can be obtained efficiently. Since the polyphenol glycoside thus obtained has a unit in which a plurality of glucoses are bonded in the molecule, it has good solubility and stability against light, heat, pH and the like. In addition, when administered in vivo, the bond between the polyphenol compound and glucose is easily cleaved, so that the physiologically active function of the polyphenol compound is not reduced. Therefore, it can be suitably used as food, pharmaceuticals, quasi drugs, cosmetics and the like.

  In the method for producing a polyphenol glycoside of the present invention, the compound (B) in which one molecule of glucose is bonded to the phenolic hydroxyl group of the polyphenol compound (A) and the sugar donor (C) are converted to γ-cyclodextrin glucanotransferase (hereinafter “ It may be abbreviated as “γ-CGTase”).

  The polyphenol compound (A) used in the present invention is not particularly limited as long as it is a natural compound or a derivative thereof having two or more phenolic hydroxyl groups in the same molecule. Examples of the polyphenol compound (A) include flavone, flavonol, flavanone, flavanonol, isoflavone, flavan, flavanol (catechin), flavan-3,4-diol, chalcone, aurone, anthocyanidin, lignan, coumarin, curcumin, ellagic acid, Examples include chlorogenic acid, among which curcumin is preferably used.

  Polyphenol compounds are known to have antioxidative action, antimutagenicity, anticancer action, blood pressure rise inhibiting action, blood sugar rise inhibiting action, antiallergic action, antibacterial action, antiviral action and the like. In particular, since polyphenol compounds have a plurality of hydroxyl groups, they are more easily oxidized than other coexisting compounds, and are considered to have an excellent antioxidant effect.

  The method for obtaining the polyphenol compound (A) used in the present invention is not particularly limited, and the polyphenol compound (A) can be obtained by extraction from a plant or the like using an organic solvent or supercritical carbon dioxide, or chemical synthesis. For example, as a method for obtaining curcumin, a method of extracting from a plant containing a large amount of curcumin such as turmeric can be mentioned.

  The method for producing a polyphenol glycoside (D) of the present invention uses a compound (B) obtained by binding only one molecule of glucose to one phenolic hydroxyl group of the polyphenol compound (A). . Such a compound (B) is not particularly limited, and may be one in which glucose is bonded to a plurality of positions of the phenolic hydroxyl group of the compound (B), or only one position of the phenolic hydroxyl group. In addition, one molecule of glucose may be bonded. At that time, the bond between the phenolic hydroxyl group and glucose may be an α bond or a β bond. From the viewpoint that the activity such as antioxidant action is better when having as many phenolic hydroxyl groups as possible, the compound (B) is preferably one in which one molecule of glucose is bonded to only one position of the phenolic hydroxyl group. .

The method for obtaining such a compound (B) is not particularly limited, but can be preferably obtained by chemical synthesis. For example, when the polyphenol compound (A) is curcumin, curcumin and tetra-O-acetyl-α-D-glucosyl bromide (hereinafter may be abbreviated as “TAGB”) in the presence of Ag ₂ CO ₃ . By reacting and then deacetylating, a curcumin derivative which is a compound (B) in which one molecule of glucose is bonded can be obtained.

  The present invention is characterized in that γ-CGTase is allowed to act on the compound (B) and the sugar donor (C). By such a method, polyphenol glycoside (D) in which a plurality of glucoses are bonded to compound (B) can be obtained. The present inventors have confirmed that when γ-CGTase is allowed to act using curcumin instead of compound (B), it is difficult to obtain a polyphenol glycoside having a plurality of glucose bound thereto. . Therefore, in the present invention, it is important to use the compound (B) in which one molecule of glucose is bonded to the phenolic hydroxyl group. Since the polyphenol glycoside (D) obtained by the present invention has a unit in which a plurality of glucoses are bonded in the molecule, the solubility is good and the stability to light, heat, pH, etc. is good. Have advantages.

  The sugar donor (C) used in the present invention is not particularly limited, but is abbreviated as α-cyclodextrin (hereinafter sometimes abbreviated as “α-CD”), β-cyclodextrin and abbreviated as “β-CD”. And at least one selected from the group consisting of γ-cyclodextrin (hereinafter sometimes abbreviated as “γ-CD”). The α-CD, β-CD and γ-CD may be used alone or as a mixture.

  In the present invention, the number of glucose bonds in the resulting polyphenol glycoside (D) varies depending on the mode of use of the sugar donor (C). Actually, as is apparent from Examples 1, 2, and 3 in Examples using α-CD, β-CD, and γ-CD, which will be described later, polyphenol glycosides having different glucose binding numbers depending on the reaction time. Body (D) was obtained. Although it may vary depending on the type of polyphenol compound (A) used, it is preferable to use α-CD from the viewpoint of obtaining a polyphenol glycoside (D) having as many glucose bonds as possible in a short reaction time. From the viewpoint of having advantages in terms of surface, it is preferable to use β-CD.

  In the present invention, γ-CGTase is allowed to act on the compound (B) in which one molecule of glucose is bonded to the phenolic hydroxyl group of the polyphenol compound (A). Therefore, the number of glucose bonds in the polyphenol glycoside (D) is 2 or more. is there. From the viewpoint of improving the solubility by increasing the sugar chain, those having a glucose bond number of 7 or more are preferably 5 mol% or more, and those having 7 or more are 10 mol% or more. More preferably, 7 or more is more preferably 20 mol% or more. Here, the said mol% was calculated | required from the peak area at the time of performing LC-MS measurement with the light absorbency of 430 nm. The absorbance 430 nm is an absorption wavelength mainly derived from the curcumin skeleton. In addition, the number of glucose bonds is usually 30 or less.

［式中、ｎは１以上の正の整数である。］ The polyphenol glycoside (D) obtained by the present invention is represented by the following chemical formula (1) or (2), for example, when the polyphenol compound (A) used is curcumin.

[Wherein n is a positive integer of 1 or more. ]

［式中、ｎは１以上の正の整数である。］

[Wherein n is a positive integer of 1 or more. ]

  In the above chemical formulas (1) and (2), the bond between glucose directly bound to the phenolic hydroxyl group derived from curcumin is a β bond, but the bond may be an α bond. From the viewpoint of having better activity such as antioxidant action when having as many phenolic hydroxyl groups as possible, the polyphenol glycoside (D) is one place of the curcumin-derived phenolic hydroxyl group as shown in the above chemical formula (1). It is preferable that only a large number of glucoses are bonded to each other.

  Γ-CGTase used in the present invention refers to an enzyme that mainly produces γ-CD among α-CD, β-CD, and γ-CD cyclodextrins when acting on starch. The γ-CGTase used in the present invention is not particularly limited. However, Bacillus macerans, Bacillus stearothermophilus, Bacillus circulans, and those produced by microorganisms such as Bacillus polymyxa, Klebsiella pneumoniae, and the like. Among these, from the viewpoint of utilization of food, it is preferably derived from Bacillus macerans.

  In the present invention, the molar ratio of sugar donor (C) in terms of glucose to compound (B) (sugar donor (C) / compound (B)) is preferably 10 to 10,000. When the molar ratio is less than 10, the decomposition of cyclodextrin may be suppressed and the reaction may not proceed, and is more preferably 100 or more. On the other hand, when the molar ratio exceeds 10,000, the cyclodextrin is decomposed quickly, and there is a possibility that sugars may bond (polymerize) to form a high molecular weight dextran (starch), more preferably 2000 or less. .

  The addition amount of (gamma) -CGTase used by this invention is not specifically limited, It is preferable that it is 300-1000 units per 1 mol of compound (B), and it is more preferable that it is 400-800 units.

  Moreover, the reaction temperature at the time of reacting by adding γ-CGTase is not particularly limited, and the reaction temperature is 20 to 70 ° C. in consideration of obtaining the desired polyphenol glycoside (D) having a glucose bond number. Preferably there is. When reaction temperature is less than 20 degreeC, there exists a possibility that progress of reaction may become difficult, More preferably, it is 35 degreeC or more, More preferably, it is 45 degreeC or more. On the other hand, when the reaction temperature exceeds 70 ° C., γ-CGTase may be deactivated, more preferably 60 ° C. or less.

  Moreover, the pH at the time of reacting by adding γ-CGTase is not particularly limited, and can be determined by considering the optimum pH of the enzyme. The pH is preferably from 3 to 11, and more preferably from 4 to 8.

  The reaction time after adding γ-CGTase is not particularly limited, and a polyphenol glycoside (D) having a different number of glucose bonds depending on the sugar donor (C) used is obtained. In the present invention, there is a tendency that the number of glucose bonds decreases as the reaction time increases. The reaction time is usually 1 minute to 100 hours, but a polyphenol glycoside (D) having a desired number of glucose bonds can be obtained by appropriately adjusting the reaction time. In the present invention, the preferred reaction time when α-CD is used is 1 minute to 6 hours, the preferred reaction time when β-CD is used is 1 hour to 12 hours, and γ-CD is The preferred reaction time when used is 3 to 24 hours.

  When the polyphenol glycoside (D) obtained by the present invention is administered in vivo, the binding between the polyphenol compound (A) and glucose is caused by the action of α-amylase, α-glucosidase, etc. present in the living body. Since it is easily cleaved, the antioxidant action and the like possessed by the polyphenol compound (A) will be exhibited. Therefore, the polyphenol glycoside (D) obtained by the present invention does not reduce the physiologically active function of the polyphenol compound (A), and can be suitably used as food, pharmaceuticals, quasi drugs, cosmetics, and the like. it can.

  Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1
By reacting 20 g of penta-O-acetyl-β-D-glucose with 100 g of 25% hydrogen bromide-acetic acid solution at 25 ° C. for 8 hours, tetra-O-acetyl-α-D-glucosyl bromide (TAGB) Got. The obtained TAGB was reacted with 0.4 g of Ag ₂ CO ₃ in acetone in the presence of molecular sieves 4A (MS-4A), whereby 3.2 g of curcumin-O-acetyl-β-D-glucopyranoside was obtained. Obtained. Next, the obtained curcumin-O-acetyl-β-D-glucopyranoside was hydrolyzed in methanol using 1 g of K ₂ CO ₃ , isolated and purified using open column chromatography, and curcumin-O 100 mg of -β-D-glucopyranoside was obtained. The chemical reaction formula is shown below.

  Weigh out 3 mmol of curcumin-O-β-D-glucopyranoside obtained above, 0.5 mmol of α-CD (manufactured by Wako Pure Chemical Industries, Ltd.) and 1 mg of calcium chloride, and add 50 mM sodium citrate buffer (pH 5.5). 3 ml) and 600 units of γ-CGTase (manufactured by Amano Co., Ltd.) derived from Bucillus macerans were added and stirred at 55 ° C. for 5 minutes, 1 hour, 6 hours, 12 hours, 24 hours. And 48 hours reaction, respectively. After the reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 1 hour, and passed through a membrane filter (pore size 0.45 μm) to obtain curcumin glycosides. When the obtained curcumin glycoside was subjected to LC-MS measurement at an absorbance of 430 nm, peaks up to 15 glycosides were confirmed. The obtained chart is shown in FIG.

Example 2
In Example 1, instead of using α-CD, 0.5 mmol of β-CD (manufactured by Wako Pure Chemical Industries, Ltd.) was used for 5 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours. The reaction was performed in the same manner as in Example 1 except that each reaction was performed.

Example 3
In Example 1, instead of using α-CD, 0.5 mmol of γ-CD (manufactured by Wako Pure Chemical Industries, Ltd.) was used for 5 minutes, 1 hour, 6 hours, 12 hours, 24 hours and 48 hours, respectively. The reaction was performed in the same manner as in Example 1 except that the reaction was performed.

Example 4
In Example 1, instead of using α-CD, the reaction was performed in the same manner as in Example 1 except that the reaction was performed using 600 mg of soluble starch for 24 hours.

  In Example 1 using α-CD, Example 2 using β-CD, and Example 3 using γ-CD, the number of glucose bonds is relatively higher than that of glycoside having a small number of glucose bonds. Glycosides with a high content were mainly obtained. On the other hand, in Example 4 using soluble starch, many glycosides with a small number of glucose bonds were mainly observed.

  In the case of Example 1 using α-CD, a large number of glycosides having 7 or more sugars were confirmed when the reaction time was 5 minutes, and as the reaction time progressed, there was a tendency for the number of glucose bonds to decrease due to hydrolysis. It was seen. In addition, in the case of Example 2 using β-CD, when the reaction time is 3 hours, in the case of Example 3 using γ-CD, a glycoside having 7 or more sugars when the reaction time is 6 hours. Many were confirmed. In the case of using β-CD and γ-CD, as in the case of using α-CD, when the reaction time became too long, the number of glucose bonds tended to decrease.

It is LC-MS analysis chart of the polyphenol glycoside (D) obtained by making it react for 5 minutes, 1 hour, 6 hours, 12 hours, 24 hours, and 48 hours using (alpha) -CD. It is LC-MS analysis chart of the polyphenol glycoside (D) obtained by making it react for 5 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours using β-CD. It is LC-MS analysis chart of the polyphenol glycoside (D) obtained by making it react for 5 minutes, 1 hour, 6 hours, 12 hours, 24 hours, and 48 hours using (gamma) -CD. It is LC-MS measurement chart of the polyphenol glycoside obtained by making it react for 24 hours using soluble starch.

Claims (4)

  Polyphenol glycoside (D) characterized in that γ-cyclodextrin glucanotransferase is allowed to act on compound (B) in which one molecule of glucose is bonded to the phenolic hydroxyl group of polyphenol compound (A) and sugar donor (C) Manufacturing method.   The method for producing a polyphenol glycoside (D) according to claim 1, wherein the polyphenol compound (A) is curcumin.   The production of polyphenol glycoside (D) according to claim 1 or 2, wherein the sugar donor (C) is at least one selected from the group consisting of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin. Method.   The method for producing a polyphenol glycoside (D) according to any one of claims 1 to 3, wherein the γ-cyclodextrin glucanotransferase is an enzyme derived from Bacillus macerans.

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