JP5382676B2 - Method for producing anthocyanidins - Google Patents

Description

  The present invention relates to a method for producing anthocyanidins.

  Anthocyanins are flavonoid glycosides with anthocyanidins as aglycones. Anthocyanins are widely present in the plant kingdom and are useful for expressing the color of flowers and fruits. Using this property, anthocyanins are used as coloring agents. Anthocyanins are attracting attention as polyphenols having various physiological functions. For example, since anthocyanin has antioxidant properties as a kind of polyphenol, it is expected to be used as a protective factor against in vivo oxidative stress. In addition, it has been reported that anthocyanins have functions such as capillary permeability inhibitory action, visual improvement action, and antidiabetic action.

  Many anthocyanin supplies rely on extraction from plants that contain anthocyanins. However, in this method, a plant containing anthocyanins must be cultivated, and there is a problem from the viewpoint of stable supply. In addition, when a plant containing anthocyanins is cultivated, it takes time and labor, and there is a problem that production efficiency is poor. Therefore, development of a production method by a synthetic reaction is desired as a method for stably and efficiently obtaining anthocyanins.

  As a method of obtaining anthocyanins by a synthetic reaction, a method of reducing glycosylated flavonols obtained in large quantities at a lower cost than plants is known. For example, Non-Patent Documents 1 and 2 and Patent Documents 1 and 2 below describe methods for obtaining anthocyanins by reducing glycosylated flavonols with metals such as magnesium under acidic conditions. Non-patent documents 3 and 4 listed below describe methods for obtaining anthocyanins by electrolysis of glycosylated flavonols. Further, Patent Documents 3 and 4 below describe methods for obtaining anthocyanins using a hydride reagent.

J. Am. Chem. Soc. (1919) 41, 208-20 Tetrahedron Letters, (1995) 36 (26), 4611-14 Collection of Czechoslovak Chemical Communication, (1950) 15, 433-6 Bulletin de Liaison-Groupe Polyphenols, (1990) 15, 33-6 JP 51-121034 A Japanese Patent Laid-Open No. 54-134736 Japanese Patent Laid-Open No. 55-111418 European Patent Publication No. 490615

  However, the reduction reaction with metal has a problem that the yield is about 20 to 30% and the yield is low. In addition, in the said nonpatent literature 2, although the yield is described as 60%, the molar extinction coefficient used as a basis is wrong and the actual yield is about 30%. In addition, the yield is low even in the electrolytic reaction, and the product becomes a mixture having various structures. On the other hand, in the method using a hydride reagent, the yield is described as about 60 to 70%, but the reagent is expensive and is not suitable for production on a large scale.

  An object of the present invention is to provide a method for producing anthocyanidins by a synthetic reaction at a lower cost and higher efficiency than conventional methods. Another object of the present invention is to provide a flavenol derivative that can be used in a method for producing anthocyanidins.

（式中、Ｒ^３は−ＯＲで表されるアルコキシ基（Ｒは置換基を有していない炭素数１〜６のアルキル基）又は−ＯＧ基である。Ｒ^５は水素原子又は水酸基である。Ｒ^１、Ｒ^２、Ｒ^４及びＲ^６はそれぞれ独立に水素原子、水酸基、アルコキシ基、又は−ＯＧ基である。尚、Ｇは糖鎖である。）

（式中、Ｒ^３は−ＯＲで表されるアルコキシ基（Ｒは置換基を有していない炭素数１〜６のアルキル基）又は−ＯＧ基である。Ｒ^１、Ｒ^４、及びＲ^６はそれぞれ独立に水素原子、水酸基、又は−ＯＧ基である。尚、Ｇは糖鎖である。） Anthocyanidins production method of the present invention, (1) in an oxygen-free atmosphere, acid selected from hydrogen chloride and sulfuric acid in a methanol solvent, as well as powdered or granular single metal selected from zinc and magnesium A step of reducing a flavonol derivative represented by the following general formula (A) in the presence to obtain a reduction product;
(2) Next, a step of oxidizing the reduction product in a state not containing the metal,
Have
A method for producing anthocyanidins, wherein in the step (1), the flavonol derivative represented by the following general formula (B1) or (B2) is generated by reducing the flavonol derivative.

(Wherein R ³ is an alkoxy group represented by —OR (R is an alkyl group having 1 to 6 carbon atoms which does not have a substituent) or —OG group, and R ⁵ is a hydrogen atom or a hydroxyl group. R ¹ , R ² , R ⁴ and R ⁶ are each independently a hydrogen atom, a hydroxyl group, an alkoxy group, or an —OG group, where G is a sugar chain.

(In the formula, R ³ is an alkoxy group represented by —OR (R is an alkyl group having 1 to 6 carbon atoms which does not have a substituent) or —OG group. R ¹ , R ⁴ , and R ^6. Are each independently a hydrogen atom, a hydroxyl group, or an -OG group, where G is a sugar chain.)

  According to the present invention, anthocyanidins can be produced by a synthetic reaction with higher efficiency than conventional methods. In addition, anthocyanidins can be produced by a cheaper and easier method than conventional methods.

(1) Method for producing anthocyanidins (A) Step (1)
In the production method of the present invention, the flavonol derivative is a compound represented by the following general formula (A) . In the present invention, various types and structures of flavonol derivatives can be used depending on the anthocyanidins to be synthesized.

（式中、Ｒ^３は−ＯＲで表されるアルコキシ基（Ｒは置換基を有していない炭素数１〜６のアルキル基）又は−ＯＧ基である。Ｒ^５は水素原子又は水酸基である。Ｒ^１、Ｒ^２、Ｒ^４及びＲ^６はそれぞれ独立に水素原子、水酸基、アルコキシ基、又は−ＯＧ基である。尚、Ｇは糖鎖である。）

(Wherein R ³ is an alkoxy group represented by —OR (R is an alkyl group having 1 to 6 carbon atoms which does not have a substituent) or —OG group, and R ⁵ is a hydrogen atom or a hydroxyl group. R ¹ , R ² , R ⁴ and R ⁶ are each independently a hydrogen atom, a hydroxyl group, an alkoxy group, or an —OG group, where G is a sugar chain.

  In the general formula (A), the type and structure of the alkoxy group (—OR, R: hydrocarbon group) are not limited. The hydrocarbon group contained in the alkoxy group may be linear or branched. The alkyl group or the like may have a chain structure or a cyclic structure (a cycloalkyl group, a cycloalkenyl group, or a cycloalkynyl group). Further, the hydrocarbon group may be a saturated hydrocarbon group or an unsaturated hydrocarbon group. Examples of the hydrocarbon group include an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an arylalkyl group, an arylalkenyl group, and an arylalkynyl group. In the general formula (A), when two or more alkoxy groups are present, each alkoxy group may be the same alkoxy group or an alkoxy group having a different structure. Moreover, the aromatic ring contained in the aryl group, arylalkyl group, arylalkenyl group, and arylalkynyl group may contain other substituents.

The number of carbon atoms of the hydrocarbon group is not particularly limited. Carbon number of the said hydrocarbon group is 1-10 normally, Preferably it is 1-6, More preferably, it is 1-4. Furthermore, the hydrocarbon group may be composed of only carbon atoms and hydrogen atoms, or may contain one or more atoms other than carbon atoms and hydrogen atoms in the structure. For example, the hydrocarbon group may have one or more substituents containing atoms other than carbon atoms and hydrogen atoms as substituents. Moreover, the said alkyl group etc. may contain 1 or 2 or more atoms other than a carbon atom and a hydrogen atom in a chain structure or a cyclic structure. Specific examples of the alkoxy group include a methoxy group and an ethoxy group.
However, the alkoxy group (—OR, R: hydrocarbon group) in R ³ of the general formula (A) is an alkyl group having 1 to 6 carbon atoms in which R constituting the alkoxy group does not have a substituent. It is.

  In the above general formula (A), G is a sugar chain. The sugar chain is glycosidic bonded through an oxygen atom. There is no particular limitation on the structure of the sugar chain and the type of sugar constituting the sugar chain. The number of sugars constituting the sugar chain is not particularly limited, but is usually 1 to 4, preferably 1 to 3, and more preferably 1 or 2. The sugar constituting the sugar chain may be a pentose sugar or a hexose sugar. Furthermore, the hydroxyl group contained in the sugar chain may be modified with other groups. For example, the hydroxyl group may be esterified as described later or may be alkoxylated. The glycosidic bond is usually a β-bond, but may be an α-bond.

Specific examples of the sugar include, for example, α-D-ribose, β-D-glucose, β-D-mannose, β-D-galactose, α-L-rhamnose, β-D-xylose, β-D- arabinose, beta-D-glucuronic acid, rutinose, sophorose, gentiobiose, Zanbubiosu, Rachirosu, lamina Livio North, gentiotriose, Robinobiosu, 2 G ^- glucosyl Ruchino scan, and 2 ^G - is xylosyl Ruchino scan the like.

  The sugar may be a sugar (organic acid-bonded sugar) in which an organic acid is singly or plurally esterified. There is no particular limitation on the bonding position of the organic acid. Specific examples of the binding position of the organic acid include one or more of the 2-position, 3-position, and 6-position of the sugar. Usually, the binding position of the organic acid is the 6th position of the sugar.

  There is no limitation in particular in the kind and structure of the said organic acid. The organic acid may be an aromatic organic acid or an aliphatic organic acid. Examples of the aromatic organic acid include cinnamic acids (E-form or Z-form) and benzoic acids. Examples of the cinnamic acids and benzoic acids include hydroxycinnamic acids (E-form or Z-form) and hydroxybenzoic acids. Specific examples of the cinnamic acid include cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, and sinapinic acid. Specific examples of the benzoic acids include benzoic acid, p-hydroxybenzoic acid, and gallic acid. The aliphatic organic acid may be a monocarboxylic acid or a dicarboxylic acid. Specific examples of the aliphatic organic acid include acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, and malic acid.

In the general formula (A), the number and position of the -OG group are not particularly limited. The number of said -OG groups is 1-5 normally, Preferably it is 1-4, More preferably, it is 1-3, More preferably, it is 1-2. Also, the position of the -OG group is usually the 3-position ^(R 3), 3 'position ^(R 1), 5-position ^(R 4), 5' position ^(R 2), and 7 out of ^{(R 6)} 1 or 2 or more. Specific examples of the flavonol derivative include, for example, 3,5, 3,7, 3,3 ′, 3,5,7, 3,5,3 ′, and 3,7,3 ′. Are -OG group flavonol glycosides. The flavonol derivative is preferably a flavonol glycoside in which the 3rd or 5th position is an -OG group.

In the general formula (A), the number and position of the hydroxyl groups are not particularly limited. The number of the hydroxyl groups is usually 1 to 5, preferably 1 to 4, more preferably 1 to 3, and more preferably 1 to 2. Further, the position of the hydroxyl group is usually one or more of the 3 ′ position (R ¹ ), the 5 position (R ⁴ ), the 5 ′ position (R ² ), and the 7 position (R ⁶ ). Specific examples of the flavonol derivative include flavonol derivatives having hydroxyl groups at the 5,7 and 5,7,3 ′ positions.

  More specifically, examples of the flavonol derivative include a flavonol derivative represented by the following general formula (A1). Specific examples of the flavonol derivative represented by the general formula (A1) include quercetin glycosides (rutin, quercetin-3-glucoside, quercetin-3-robinobioside, etc.) and kaempferol glycosides (kaempferol- 3-glucoside and kaempferol-3-robinobioside).

（式中、Ｒ^１は水素原子、水酸基、アルコキシ基、又は−ＯＧ基である。Ｒ^３はアルコキシ基又は−ＯＧ基である。Ｒ^４及びＲ^６はそれぞれ独立に水酸基、アルコキシ基、又は−ＯＧ基である。）

(In the formula, R ¹ is a hydrogen atom, a hydroxyl group, an alkoxy group, or an —OG group. R ³ is an alkoxy group or an —OG group. R ⁴ and R ⁶ are each independently a hydroxyl group, an alkoxy group, or — OG group.)

  Step (1) is performed in an oxygen-free atmosphere. The specific content of the “oxygen-free atmosphere” is not particularly limited as long as the effects of the present invention are not impaired. The “oxygen-free atmosphere” may include only one kind of gas or may contain two or more kinds of gases. Specific examples of the gas constituting the “oxygen-free atmosphere” include one or more of nitrogen gas, rare gas (helium gas, neon gas, argon gas), hydrogen gas, and carbon dioxide gas. . The “oxygen-free atmosphere” usually does not contain any oxygen in the atmosphere, but may contain oxygen to the extent that it does not impair the effects of the present invention. The oxygen concentration in the “oxygen-free atmosphere” is usually 0.1% by volume or less (for example, 0 to 0.1% by volume), preferably 0.05% by volume or less, more preferably 0.01% by volume or less ( For example, 0.001 to 0.01% by volume).

  The acid may be either hydrogen chloride or sulfuric acid, or both. The acid concentration is not particularly limited. The concentration of the acid is usually 1 to 20% by volume, preferably 3 to 20% by volume, more preferably 3 to 15% by volume, and more preferably 5 to 12% by volume.

The metal is a powdery or granular simple metal selected from Zn and Mg. In the present invention, only one of Zn and Mg may be used, or both may be used.

  In step (1), methanol is used as a solvent. In step (1), if water is not present in the solvent, the progress of the reaction can be promoted. Therefore, the methanol is preferably anhydrous methanol. However, water may be contained in the solvent as long as the effect of the present invention is not impaired. The content of water in the solvent is usually 1% by volume or less, preferably 0.5% by volume or less, more preferably 0.1% by volume or less (for example, 0.01 to 0.1% by volume).

  In the present invention, the method for adding the flavonol derivative, the acid and the metal is not particularly limited. For example, in the present invention, the flavonol derivative, the acid, and the metal can be added to the solvent. In addition, a solution prepared by dissolving or suspending the flavonol derivative, the acid, and a part of the metal in a solvent and a solution obtained by dissolving or suspending the rest in a solvent are prepared separately, and then the above solutions are mixed May be.

  There are no particular limitations on the reaction conditions of step (1). Step (1) of the present invention usually proceeds in a short time. Therefore, the reaction time is usually 1 to 10 minutes, preferably 2 to 8 minutes. Moreover, reaction temperature is -10-50 degreeC normally, Preferably it is 0-30 degreeC. The flavonol derivative may be completely dissolved or in a suspended state. Further, in the above step (1), other operations such as stirring and ultrasonic irradiation can be appropriately performed. For example, the reaction solution can be irradiated with ultrasonic waves for the purpose of dissolving the flavonol derivative.

In step (1), the flavonol derivative is reduced to produce a reduced product. Usually, as the reduction product, a Furabenoru derivative represented by the general formula (B1) or (B2). The description of R ¹ , R ³ , R ⁴ , and R ⁶ in the general formula (A1) is valid for R ¹ , R ³ , R ⁴ , and R ^{6 in} the general formulas (B1) and (B2). Specific examples of the flavenol derivative represented by the general formula (B1) or (B2) include flavenol derivatives represented by the following general formula (B1 ′) or (B2 ′). In addition, in the manufacturing method of this invention, another reduction | restoration product may produce | generate from the flavenol derivative represented by general formula (B1) or (B2).

（式中、Ｒ^１は水素原子、水酸基、アルコキシ基、又は−ＯＧ基である。Ｇは糖鎖である。）

(In the formula, R ¹ is a hydrogen atom, a hydroxyl group, an alkoxy group, or an —OG group. G is a sugar chain.)

  In the production method of the present invention, step (2) may be subsequently performed without isolating the reduction product (flavenol derivative represented by the general formula (B1) or (B2)) from the reaction solution. In the production method of the present invention, the reaction product is passed through an appropriate column to adsorb the reduction product onto the column, and then the reduction product is eluted with an appropriate eluent, thereby reducing the reduction product. It can be isolated. Therefore, in the production method of the present invention, the reduction product may be isolated from the reaction solution and added to another solvent to perform step (2).

(B) Step (2)
In the step (2), the reduction product is oxidized without containing the metal. Thereby, the yield of anthocyanidins can be increased. This effect is considered as follows. That is, as will be described later, in the present invention, the reaction from a flavonol derivative to an anthocyanidin is considered to consist of a reduction reaction from a flavonol derivative to a flavenol derivative and an oxidation reaction from the subsequent flavenol derivative to an anthocyanidin. In the latter oxidation reaction, when a metal such as Zn is present, the produced anthocyanidins are reduced or decomposed, and as a result, the yield of the anthocyanidins is considered to be reduced. It is not an explanation of the purpose of limiting the present invention, nor an explanation of the purpose of defining the present invention.)

  In the step (2), there is no particular limitation on the means for realizing the “state not containing the metal”. As described above, powder metal is used as the metal. Therefore, in this invention, the said metal is removed by filtering the reaction liquid of a process (1), and then a process (2) can be performed. In addition, the “state not containing the metal” includes not only the case where the metal is removed from the reaction solution in the step (1) but also the isolation of the reduction product from the reaction solution in the step (1). . That is, “oxidize the reduction product in a state not containing the metal” in the step (2) means that the reduction product is isolated from the reaction solution in the step (1), and then the isolated reduction product. The case where the product is added to another solvent not containing the metal to oxidize the reduction product is also included.

  There is no particular limitation on the method for oxidizing the reduction product. The reduction product can be oxidized by adding an appropriate oxidizing agent. The reduction product can be oxidized by bringing the reaction solution into contact with an oxygen-containing gas. More specifically, examples of this method include a method of supplying an oxygen-containing gas into the reaction solution, or a method of bringing the reaction solution into contact with the oxygen-containing gas by appropriately stirring the reaction solution. The reduction product can be oxidized, for example, by removing the metal from the reaction solution obtained in step (1) and then appropriately stirring the reaction solution in the atmosphere. There is no particular limitation on the type of the oxygen-containing gas. The oxygen-containing gas may be oxygen itself or a mixed gas of oxygen and another gas. Examples of the oxygen-containing gas include air.

  Step (2) is usually performed under acidic conditions. It is preferable to perform the step (2) under acidic conditions because decomposition of the produced anthocyanins can be suppressed. More specifically, in the step (2), the pH of the reaction solution can be adjusted to 4 or less, preferably 3 or less. There is no particular limitation on the lower limit of pH. The lower limit value may be an appropriate value such as −2, −1, 0, 1 or the like. Examples of the method of performing the step (2) under acidic conditions include a method of adding an appropriate acid (hydrogen chloride or the like) to the reaction solution.

  Step (2) is usually performed in an oxygen atmosphere such as air. However, step (2) may be performed in an “oxygen-free atmosphere” as in step (1). For example, the step (2) is performed by performing the step (1) in an “oxygen-free atmosphere” and subsequently adding an oxidizing agent to the reaction solution in the “oxygen-free atmosphere” or blowing an oxygen-containing gas into the reaction solution. It can be performed.

  There is no limitation in particular in the reaction conditions of a process (2). The reaction time in step (2) is usually 1 minute to 30 hours, preferably 1 to 24 hours. Moreover, reaction temperature is -10-50 degreeC normally, Preferably it is 0-30 degreeC.

(C) Others The present invention may include other steps as necessary. For example, after the completion of the step (2), the target anthocyanidins can be recovered and purified by a known method, for example, a method such as distillation, adsorption, extraction, and recrystallization, or a combination of these methods.

In the present specification, “anthocyanidins” means anthocyanidins and anthocyanins (glycosides containing anthocyanidins as aglycones). Examples of the anthocyanidins produced according to the present invention include anthocyanidins represented by the following general formula (C). Incidentally, in the general formula ^(C), and the content of R 1 to R ⁶ is an explanatory of ^R 1 to R ⁶ in the general formula (A) are valid.

（式中、Ｒ^３はアルコキシ基又は−ＯＧ基である。Ｒ^５は水素原子又は水酸基である。Ｒ^１、Ｒ^２、Ｒ^４及びＲ^６はそれぞれ独立に水素原子、水酸基、アルコキシ基、又は−ＯＧ基である。尚、Ｇは糖鎖である。）

(In the formula, R ³ is an alkoxy group or —OG group. R ⁵ is a hydrogen atom or a hydroxyl group. R ¹ , R ² , R ⁴ and R ⁶ are each independently a hydrogen atom, a hydroxyl group, an alkoxy group, or -OG group, where G is a sugar chain.)

Specific examples of the anthocyanidins include anthocyanins represented by the following general formula (C1). The anthocyanins represented by the general formula (C1) are preferably anthocyanins in which either one or both of R ⁴ and R ⁶ are —OG groups or hydroxyl groups. In the general formula (C1), the description of R ¹ , R ⁴ and R ⁶ in the general formula (A1) is appropriate for the contents of R ¹ , R ⁴ and R ⁶ . More specifically, as anthocyanins represented by the general formula (C1), for example, cyanidin-3-glucoside, cyanidin-3-rutinoside, cyanidin-3,5-diglucoside, pelargonidin-3-glucoside, pelargonidin-3-rutinoside, And pelargonidin-3,5-diglucoside.

（式中、Ｒ^１、Ｒ^４及びＲ^６はそれぞれ独立に水素原子、水酸基、アルコキシ基、又は−ＯＧ基である。尚、Ｇは糖鎖である。）

(Wherein R ¹ , R ⁴ and R ⁶ are each independently a hydrogen atom, a hydroxyl group, an alkoxy group, or an —OG group, where G is a sugar chain.)

  Hereinafter, the present invention will be described specifically by way of examples. In addition, this invention is not restricted to the form shown in the Example. The embodiment of the present invention can be variously modified within the scope of the present invention depending on the purpose and application.

(1) Example 1
The reaction mechanism is shown below.

  Rutin (1.00 g, 1.64 mmol) and Zn powder (10.0 g) were dried under vacuum and placed in a reactor. Subsequently, argon (Ar) gas was introduce | transduced in the reactor, and the inside of a reactor was made into Ar gas atmosphere. Then, anhydrous methanol (60 ml) was added to the reactor, and ultrasonic waves (40 KHz) were irradiated from the outside for 5 minutes, whereby rutin was completely dissolved in anhydrous methanol. The temperature of this solution was adjusted to 5 ° C., and a 7% hydrogen chloride-anhydrous methanol solution (60 ml) was added under ultrasonic irradiation. After performing ultrasonic irradiation and stirring for 5 minutes, the reaction solution was filtered under dry conditions to remove Zn powder, and a filtrate was obtained.

  When the filtrate was brought into contact with dry air at room temperature with vigorous stirring, the color of the reaction solution changed from light red to dark red. Three hours after the start of stirring, the reaction solution was poured into water (2.5 L) and then filtered. The obtained filtrate is poured onto a column (inner diameter 5 cm, height 50 cm) packed with “Amberlite XAD-7 gel” (substituted with 0.5% trifluoroacetic acid (TFA) aqueous solution) to adsorb the dye molecules to the column. I let you. The column was then washed with 0.5% aqueous TFA (2 L). Thereafter, 90% acetonitrile aqueous solution containing 0.5% TFA was added to elute the dye adsorbed on the column. This dye fraction was concentrated to dryness at 40 ° C. under reduced pressure to obtain 1.00 g (yield 86%) of cyanidin-3-rutinoside.

The purity of the dye fraction was measured by high performance liquid chromatography (HPLC) analysis using an ODS column (see FIG. 1). The HPLC analysis conditions are as follows. As a result, the purity of the dye fraction was 90% or more.
Column: Develosil “ODS-HG-5” (2.0 mm × 250 mm)
Solvent A: 0.5% TFA-10% CH ₃ CN aqueous solution Solvent B: 0.5% TFA-10% CH ₃ CN aqueous solution Elution condition: Linear gradient elution (0-15 minutes; Solvent A 100% -80%, 15 To 25 minutes; solvent A 80% to 50%, 25 to 35 minutes; solvent 50% to 0%)
Flow rate: 0.2 ml / min
Analysis temperature: 40 ° C
Detection temperature: 280 nm

(2) Example 2
Rutin (1.00 g, 1.64 mmol) and Zn powder (10.0 g) were reacted in the same manner as in Example 1 to obtain filtrate 1. The filtrate 1 was poured into 3 L of water, and then poured onto a column (inner diameter 5 cm, height 50 cm) packed with Amberlite XAD-7 gel (replaced with water) to adsorb the reaction molecules. Next, 2 L of water was flowed to wash the column, and after confirming that chlorine ions were no longer detected, the reaction molecules were eluted with 90% acetonitrile aqueous solution (1 L). The effluent was concentrated to dryness under reduced pressure to obtain a mixture 1 of four kinds of reaction products.

  Using preparative ODS-HPLC, the mixture 1 was adsorbed on an ODS column, and then the elution was performed by gradually increasing the acetonitrile concentration of the acetonitrile aqueous solution from 5% to 50%. As a result, two types of 3,4-flavenol compound (1,2), 2,3-flavenol compound (3), and cyanidin-3-rutinoside (4) were respectively obtained at 215 mg (yield 22%), 464 mg (yield). Yield 47%), 100 mg (yield 10%), and 178 mg (yield 15%). The HPLC chromatogram of mixture 1 is shown in FIG. The HPLC analysis conditions are the same as in Example 1.

The structures of the flavenol bodies 1 to 3 were determined by nuclear magnetic resonance spectrum analysis ( ¹ H-NMR and ¹³ C-NMR) and mass spectrometry (FAB-MASS). Table 1 shows the ¹ H-NMR analysis results of the flavenol bodies 1 to 3. Moreover, the spectrum chart of ¹ H-NMR and ¹³ C-NMR of the flavenol bodies 1 to 3 is shown in FIGS. The results of mass spectrometry of the flavenol bodies 1 to 3 are as follows. All mass spectrometry was measured as proton adducts.
Flabenol body 1 (C ₂₇ H ₃₃ O ₁₅ ):
Actual value: 597.1839, calculated value: 597.1819
Flabenol body 2 (C ₂₇ H ₃₃ O ₁₅ );
Actual value: 597.1839, calculated value: 597.1819
Flabenol body 3 (C ₂₇ H ₃₃ O ₁₅ );
Actual value: 597.817, calculated value: 597.1819

(3) Example 3
Mixture 1 (10.1 mg) obtained in Example 2 was dissolved in anhydrous methanol (0.5 ml). A 7% hydrogen chloride-anhydrous methanol solution (0.5 ml) was added to the solution, and the mixture was stirred under dry air for 12 hours. The dye fraction was analyzed by HPLC analysis using an ODS column in the same manner as in Example 1. As a result, it was confirmed that the flavenol compound contained in the mixture 1 was converted into a single cyanidin-3-rutinoside (see FIG. 9).

(4) Example 4
Rutin (103 mg) and Zn powder (1.07 g) were dried under vacuum and placed in a reactor. Subsequently, Ar gas was introduce | transduced in the reactor and Ar inside was made into Ar gas atmosphere. Then, anhydrous methanol (5.0 ml) was added to the reactor, and ultrasonic waves (40 KHz) were irradiated from the outside for 15 minutes to completely dissolve rutin in anhydrous methanol. A 10% concentrated sulfuric acid-methanol solution (5.0 ml) was added to this solution with stirring at room temperature. After stirring for 2 hours, the reaction solution was filtered to remove Zn powder. The filtration residue was washed with methanol (6 ml) and this methanol was also added to the filtrate.

  The filtrate was stirred at room temperature for 23 hours and contacted with dry air to oxidize. Water (300 ml) was added to the reaction solution and immediately poured onto a column (inner diameter 2 cm, height 25 cm) packed with “Amberlite XAD-7 gel” (substituted with 0.5% TFA aqueous solution) to adsorb the dye molecules. . The column was then washed with 0.5% aqueous TFA (200 ml). Thereafter, a 90% acetonitrile aqueous solution containing 0.5% TFA was added to elute the dye molecules adsorbed on the column. This dye fraction was concentrated to dryness under reduced pressure at 40 ° C. to obtain 106 mg of the dye (yield 89%). The method for measuring the purity of the dye fraction is the same as in Example 1. As a result, the purity of the dye fraction was 90% or more (see FIG. 10).

(5) Example 5
The reaction mechanism is shown below.

  Quercetin-3-glucoside (102 mg, 0.22 mmol) and Zn powder (1.25 g) were dried under vacuum and placed in a reactor. Subsequently, Ar gas was introduce | transduced in the reactor and Ar inside was made into Ar gas atmosphere. And anhydrous methanol was added in the reactor, and the quercetin-3-glucoside was completely dissolved in anhydrous methanol by irradiating ultrasonic waves (40 KHz) from the outside for 5 minutes. An anhydrous methanol solution (10 ml) containing 10.5% hydrogen chloride was added to this solution at room temperature under ultrasonic irradiation. After performing ultrasonic irradiation and stirring for 3 hours and 30 minutes, the reaction solution was filtered under dry conditions to remove Zn powder, and a filtrate was obtained.

  When the filtrate was stirred for 20 hours and contacted with dry air at room temperature, the color of the reaction solution changed from light red to dark red. Then, water (400 ml) was added to the reaction solution and immediately poured onto a column (inner diameter 2 cm, height 25 cm) packed with “Amberlite XAD-7 gel” (substituted with 0.5% TFA aqueous solution) to adsorb the dye molecules. I let you. The column was then washed with 0.5% aqueous TFA (500 ml). Thereafter, a 90% acetonitrile aqueous solution containing 0.5% TFA was added to elute the dye molecules adsorbed on the column. This dye fraction was concentrated to dryness under reduced pressure at 40 ° C. to obtain 113 mg of cyanidin-3-glucoside (yield 91%). The method for measuring the purity of the dye fraction is the same as in Example 1. As a result, the purity of the dye fraction was 95% or more (see FIG. 11).

(6) Experimental example 6
Rutin (102 mg, 0.167 mmol) and granular Mg (326 mg) were dried under vacuum and these were placed in the reactor. Subsequently, Ar gas was introduce | transduced in the reactor and Ar inside was made into Ar gas atmosphere. And anhydrous methanol (2 ml) was added in the reactor, and rutin was dissolved in anhydrous methanol. The solution was cooled to 0 ° C. and 10% hydrogen chloride-anhydrous methanol solution (6 ml) was added. The reaction solution was allowed to reach room temperature and stirred for 30 minutes, and the reaction solution was filtered under dry conditions to remove particulate Mg and obtain a filtrate.

  While stirring the above filtrate in dry air, 10% hydrogen chloride-anhydrous methanol solution (2 ml) was added, and stirring was continued at room temperature for 16 hours. Then, water (300 ml) was added to the reaction solution, and immediately poured onto a column (inner diameter 5 cm, height 25 cm) packed with “Amberlite XAD-7 gel” (substituted with 0.5% TFA aqueous solution) to adsorb the dye molecules. I let you. The column was then washed with 0.5% aqueous TFA (2 L). Thereafter, 90% acetonitrile aqueous solution containing 0.5% TFA was added to elute the dye adsorbed on the column. When the dye fraction was analyzed by HPLC analysis using an ODS column in the same manner as in Example 1, almost a single cyanidin-3-rutinoside peak was detected.

(5) Example 7
The reaction mechanism is shown below.

  Kaempferol-3- (6-O-acetyl) glucoside (9.75 mg, 0.020 mmol) and Zn powder (164 mg) were dried under vacuum and these were placed in a reactor. Subsequently, Ar gas was introduce | transduced in the reactor and the inside of a reactor was made into Ar gas atmosphere. Then, a 3.5% hydrogen chloride-anhydrous methanol solution (1.2 ml) was added at 0 ° C. under ultrasonic irradiation. After 7 minutes of ultrasonic irradiation and strong stirring, the reaction solution was filtered under dry conditions to remove Zn powder, and a filtrate was obtained.

  When the filtrate was brought into contact with dry air at room temperature with vigorous stirring and reacted for 3.5 hours, the color of the reaction solution changed from light red to dark red. The reaction solution was poured into water (30 ml) and then poured onto a column (inner diameter 1.5 cm, height 17 cm) packed with “Amberlite XAD-7 gel” (substituted with 0.5% TFA aqueous solution) Molecules were adsorbed onto the column. The column was then washed with 0.5% aqueous TFA (250 ml). Thereafter, 90% acetonitrile aqueous solution containing 0.5% TFA was added to elute the dye adsorbed on the column. The dye fraction was purified by preparative ODS-HPLC. As a result of HPLC analysis using an ODS column, the dye fraction contained two types of anthocyanins (see FIG. 12). The obtained pigment fraction was concentrated to dryness at 40 ° C. under reduced pressure to obtain 5.8 mg of pelargonidin-3- (6-O-acetyl) glucoside (yield 50%) and 5.4 mg of pelargonidin-3-glucoside. (Yield 50%) was obtained. The method for measuring the purity of the dye fraction is the same as in Example 1. As a result, the purity of the dye fraction was 95% or more for all the dyes (see FIG. 12).

2 is an HPLC chromatogram of a dye fraction of Example 1. 2 is an HPLC chromatogram of the mixture 1 of Example 2. 1 is a ¹ H-NMR chart of flavenol body 1. It is a ¹³ C-NMR chart of flavenol body 1. ¹ is a ¹ H-NMR chart of flavenol body 2. It is a ¹³ C-NMR chart of flavenol body 2. ¹ is a ¹ H-NMR chart of a flavenol body 3. 3 is a ¹³ C-NMR chart of flavenol body 3. 3 is an HPLC chromatogram of a dye fraction of Example 3. 2 is an HPLC chromatogram of a dye fraction of Example 4. 6 is an HPLC chromatogram of a dye fraction of Example 5. 2 is an HPLC chromatogram of a dye fraction of Example 7.

Claims (6)

Under (1) an oxygen-free atmosphere, acid selected from hydrogen chloride and sulfuric acid in a methanol solvent, and the presence of single metal powder or granular selected from zinc and magnesium, the following general formula (A) Reducing the represented flavonol derivative to obtain a reduced product;
(2) Next, a step of oxidizing the reduction product in a state not containing the metal,
Have
A method for producing anthocyanidins, wherein in the step (1), the flavonol derivative represented by the following general formula (B1) or (B2) is generated by reducing the flavonol derivative.

(Wherein R ³ is an alkoxy group represented by —OR (R is an alkyl group having 1 to 6 carbon atoms which does not have a substituent) or —OG group, and R ⁵ is a hydrogen atom or a hydroxyl group. R ¹ , R ² , R ⁴ and R ⁶ are each independently a hydrogen atom, a hydroxyl group, an alkoxy group, or an —OG group, where G is a sugar chain.

(In the formula, R ³ is an alkoxy group represented by —OR (R is an alkyl group having 1 to 6 carbon atoms which does not have a substituent) or —OG group. R ¹ , R ⁴ , and R ^6. Are each independently a hydrogen atom, a hydroxyl group, or an -OG group, where G is a sugar chain.)   The method for producing anthocyanidins according to claim 1, wherein the reduction product is a flavenol derivative represented by the general formula (B1) or (B2).   The method for producing anthocyanidins according to claim 1 or 2, wherein, in the step (2), the metal is removed from the reaction solution in the step (1), and then the reduction product is oxidized.   The method for producing anthocyanidins according to any one of claims 1 to 3, wherein the oxidation is performed by bringing a reaction solution into contact with an oxygen-containing gas.   The method for producing anthocyanidins according to any one of claims 1 to 4, wherein in the step (2), the pH of the reaction solution is 4 or less.   The method for producing anthocyanidins according to any one of claims 1 to 5, wherein the sugar constituting the G sugar chain in the general formula (A) and the general formulas (B1) and (B2) is rutinose.

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