JP2004305027A - Method for producing soluble flavonoids - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To directly produce soluble flavonoids from a plant or its part by using a glycosyltransferase. <P>SOLUTION: Glycosides of the soluble flavonoids are directly produced from the plant or its part as follows. An alkali liquid and cyclodextrin, cycloamylose, dextrin or starch are added to the plant or its part to carry out a transglycosylation with the glycosyltransferase in an alkaline region. The resultant product is then separated and purified. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
INDUSTRIAL APPLICABILITY The present invention comprises adding an alkaline liquid and cyclodextrin, cycloamylose, dextrin, soluble starch or starch to a plant or a part thereof, and subjecting it to a glycosyltransferase using a glycosyltransferase in an alkaline region, The present invention relates to a method for producing soluble flavonoids directly from plants by separating and purifying the produced glycosyl transfer compound.
[0002]
Prior art and problems to be solved by the invention Since flavonoids such as hesperidin and rutin, which are conventionally known as vitamin P, have a pharmacological action of suppressing an increase in vascular permeability, antihypertensive agents It is used as an agent for preventing bleeding disorders. Moreover, since most flavonoids have an ultraviolet-absorbing action and a radical scavenger action, these flavonoids are expected to be used as functional additives in cosmetics and foods.
Currently, about 3,000 kinds of flavonoids are known, and since they are present in a large amount in citrus fruits, their use is easy despite being readily available at low cost. Very limited. That is because most of the flavonoids are slightly soluble in water or not at all.
As a method for solving these problems, soluble flavonoid glycosides are prepared from flavonoids using glycosyltransferases in JP-A-48-44260, JP-A-3-7593 or JP-A-11-346792. It shows how to do it. However, in these methods, it is necessary to purify flavonoids from plants, and purified flavonoids are also on the market. However, since these are originally expensive, the production cost of glycosides is naturally high.
[0005] Methods for producing hesperidin, a flavonoid, from citrus juice lees are reported in Eclipse Journal, 37 , 631-636 (1990) and Eclipse Journal, 40 , 833-840 (1993). ing. The former method is relatively simple and includes only pH adjustment, centrifugation, and drying steps, but the yield is as low as about 25%. On the other hand, in the latter case, the yield is improved to 70%, but complicated treatment such as heat treatment, enzyme treatment, pH adjustment, centrifugation, and drying process is required. Next, in order to obtain a glycoside, it is necessary to redissolve this sparingly soluble hesperidin, and then it is produced by a transglycosylation reaction and must be prepared in a two-step complicated process.
As described above, there is no method for efficiently producing flavonoid glycosides directly from plants.
[0007]
The method for producing a soluble flavonoid glycoside according to the present invention is a method for producing a flavonoid glycoside directly from a plant, wherein the flavonoid contained in the plant is an alkaline solution having a pH of 8 or more. And a step of solubilizing cyclodextrin, dextrin, soluble starch or starch, and a step of glycosyltransferase with a glycosyltransferase in an alkaline region, whereby the above object is achieved.
Flavonoids are a general term for a group of substances in which two phenyl groups are bonded via a pyran ring or three carbon atoms having a structure close thereto.
Any plant may be used in the present invention, but fruits are particularly preferred, and citrus fruits such as mandarin oranges, oranges, lemons, grapefruits, apples, grapes, peaches, pears, pineapples, bananas, Fruits such as melon and strawberry are good. Moreover, in the plant which can be used by this invention, especially fruit, you may use parts, such as not only the whole fruit but a skin, a streak, a seed, fruit juice, fruit meat, a sardine, and a juice waste juice. Further, these dry products may be used.
The flavonoids contained in plants and used as glycosides in the present invention are not particularly limited, and known flavonoids can be used. Examples include flavanones, flavones, flavonols, flavonols, flavanols, anthocyans, isoflavones, and chalcones.
Flavanones include eriodictyol contained in lemon and the like, eriodictin, liquiritin contained in licorice and the like, naringenin contained in liquiritigenin and peach Examples include Naringin, Neohesperidin, Hesperetin, Hesperidin, Hesperidin contained in citrus, etc., and Prune contained in cherries.
Examples of flavones include apigenin contained in dahlia and the like, apiin contained in parsley and the like, baicalein contained in seaweed, etc., synaroside contained in honeysuckle, lemon and the like. Include diosmethin, diosmin, galteolin contained in horsetail, etc., luteolin contained in all plant species, and nobiletin contained in hassaku and the like.
Examples of flavonols include astragalin contained in tea and the like, fisetin contained in saikachi and the like, isorhamnetin contained in almond and the like, kaempferitrin contained in crow uri and the like Kaempferol contained in citrus, Morin contained in mulberry, Myricetin contained in bayberry etc., Myricitrin, Quercitrin contained in onions etc., Quercitriline (Quercitrin) Rhamnazin, a quaternine, quercetin, kisstus, etc. Rhamnetin contained in police, etc. (Rhamnetin), and rutin (rutin) and the like contained in buckwheat and the like.
Examples of the flavonols include aromadendrin contained in grapes and the like, pinobanksin contained in pine species, and taxifolin.
Flavanols include epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, catechin, catechin, and catechin, which are contained in tea and the like. (Catechin gallate), gallocatechin (Gallocatechin), gallocatechin gallate, and the like.
Examples of the anthocyans include apigenindin contained in sorghum and the like, cyanidin contained in grapes and the like, delphinidin contained in blueberries and the like, delphinin contained in delphinium and delphin (Delphine), luteolinidin contained in sorghum and the like, luvininidin contained in the moss, malvin, malonylshisonin contained in red perilla, etc. ), Peranin contained in purple potatoes, etc., petanin, and Dutch Pelargonidin (pelargonidin) and the like contained in the like.
Examples of isoflavones include genistin, genistin, daidzin, daidzein and the like contained in soybeans and the like.
Examples of the chalcones include carthamine contained in safflower and the like, and isoliquiritigenin contained in beans and the like.
In the present invention, as a first step of the method for dissolving flavonoids, the flavonoids are improved in solubility in water in the alkaline region, and when cyclodextrin (hereinafter referred to as “CD”) is added, It can be solubilized by forming a contact compound.
In the first stage, when a dissolution process using an alkaline solution and CD is used, the alkaline solution and then CD can be added first, but each step can also proceed simultaneously.
The flavonoids contained in the plant and used as glycosides in the present invention can be dissolved in an alkaline solution using sodium hydroxide, ammonia, potassium hydroxide or the like. The pH at this time may be 8 or more. The flavonoids are dissolved even at a pH lower than 8, but the type of flavonoids to be dissolved is limited, and it is difficult to prepare a high-concentration solution of flavonoids. Preferably, it is the range of pH 8-11, More preferably, it is pH 9-10.
For solubilization of flavonoids in plants, an alkaline solution is added to the plant or a part thereof, and if necessary, extraction is carried out using a combination of a stirrer, squeezer, homogenizer, filter separator, centrifuge, etc. It is obtained by preparing a liquid. At this time, the temperature can be selected from room temperature to 100 ° C, but is preferably 20 to 80 ° C. In addition, since pH may fall to 8 or less during preparation, it is good to add alkaline liquid suitably and hold | maintain pH to 8 or more.
CD forms a clathrate with flavonoids to solubilize flavonoids and become a sugar donor in the production of flavonoid glycosides. A commercially available CD can be used, but cyclodextrin synthase (1,4-α-D-Glucan: 4-α-D- (1,4-glucano) -transferase (EC) is used for starch. 4.1.19), hereinafter referred to as “CGTase”). The CD produced here is a substance in which 6 to 8 glucoses are bound cyclically by α-1,4 bonds, and those having 6, 7, 8 glucoses are α-CD, β-CD, γ, respectively. -It is called CD.
In addition to CD, cycloamylose or cluster dextrin described in JP-A-8-311103 and JP-A-8-134104 can also be used as a donor. Furthermore, various types of dextrin, soluble starch, starch and the like that are commercially available can also be used as donors.
CD, cycloamylose, dextrin, soluble starch, starch can be added directly to the alkaline solution as long as it can be easily dissolved in an alkaline solution at room temperature, but it must be gelatinized and dissolved by heating. The solution after heating is preferably added to the alkaline solution.
The concentration of the donor is 0.1 to 30% by weight, preferably 1 to 20% by weight.
In the second step, glycosyltransferase is used to produce solubilized flavonoids that do not cause precipitation even after neutralization by glycosylating flavonoids.
The flavonoid glycoside according to the present invention is produced by utilizing a transfer reaction by glycosyltransferase.
The enzyme used in the present invention can be any glycosyltransferase. For example, CGTase, α-amylase (EC 3.2.2.1), α-glucosidase (EC 3.2.2.1.20), and the like. However, since the present invention has a reaction in the alkaline region, the yield of flavonoid glycosides is remarkably reduced when an unstable one is used in the alkaline region. Therefore, it is preferable to use an alkali-resistant enzyme. Particularly preferred is alkali-resistant CGTase produced by alkalophilic Bacillus genus cells in JP-A-7-107972.
The transfer reaction by glycosyltransferase is appropriately selected depending on the substrate and the amount of enzyme, pH, reaction temperature, etc. The conditions for the sugar transfer to flavonoids are the same as in the conventional method except that the transfer is carried out in an alkaline region. It ’s not different. When the transfer reaction is performed in a neutral region or an acidic region, the solubilized flavonoids may be insolubilized, and efficient glycoside formation cannot be performed. Therefore, the transfer reaction is preferably performed in an alkaline region. The reaction temperature is preferably 20 to 75 ° C.
In the case of using alkali-resistant CGTase in JP-A-7-107972, the enzyme concentration is preferably 0.1-100 units / ml. The longer the reaction time, the better the enzyme activity lasts. The reaction is stopped by heating at 100 ° C. for 5 minutes.
The produced flavonoid glycosides are soluble flavonoids which do not cause precipitation even after neutralization and / or CD removal or even in the acidic region because the solubility is remarkably improved.
Since the flavonoid glycoside obtained from a plant by glycosyltransferase is a hybrid composed of a plurality of flavonoids, ion exchange, gel filtration or the like can be used alone or in combination as required. It may be separated and purified.
[0034]
【Example】
Example 1 50 g of orange skin was dried at 60 ° C. overnight, and powdered with a pulverizer. To swell the powder while further suspending it as a strong alkaline solution (pH 11.8) by adding 285 mL of 33 mM potassium monohydrogen phosphate solution to 3 g of this orange peel dry powder and then 15 mL of 1N sodium hydroxide. Heated temporarily in 80 ° C. water for 5 minutes. After cooling to room temperature, 5 g of β-CD was added thereto, and then adjusted to pH 7.0, 8.0, 9.0, 10.0 or 11.0 by adding 1N hydrochloric acid. To these, 1500 units of alkali-resistant CGTase produced by alkaliphilic Bacillus genus cells in JP-A-7-107972 was added, the pH was finely adjusted, and the mixture was placed in a thermostatic bath set at 40 ° C. Was started. After the reaction for 16 hours, the reaction was stopped by heating at 80 ° C. for 5 minutes.
When each of these solutions was analyzed by HPLC (ODS), there was no difference in the amount of dissolved flavonoids before reaction (here, hesperidin) at each pH as shown in FIG. The amount of hesperidin dissolved from the orange skin dry powder was 17 to 18 mg per 1 g of skin dry powder. The amount of hesperidin in 1 g of orange dry skin is Agric. Food. Chem. 47 , 4391-4397 (1999), it was 12-31 mg, and it was revealed that the method in this example was dissolved in an appropriate amount.
Furthermore, the amount of hesperidin glycoside produced by the enzyme reaction is large in the alkaline region at pH 8, as shown in FIG. There was found. Moreover, the dissolution amount of hesperidin at pH 8.0 was 18 mg in 100 ml of the reaction solution, the amount of produced glycoside was 15 mg, and the yield exceeded 80%. These facts indicate that the hesperidin in the orange peel is efficiently glycosylated according to this example.
The analytical conditions of the HPLC (ODS) were as follows: column: ODS eluent: AcCN / water = 20/80 flow rate: 0.5 ml / min column temperature: 40 ° C. detection method: UV280.
Example 2 50 g of orange skin was dried overnight at 60 ° C. and pulverized with a pulverizer. 2 g of this dry powder was added to the next 300 mL of each buffer containing 5 g of β-CD. That is, 0.5 M potassium phosphate buffer (pH 7.0), 0.5 M Tris-aminomethane-hydrochloric acid buffer (pH 8.0), 0.5 M Tris-aminomethane-hydrochloric acid buffer (pH 9. 0), 0.5 M sodium carbonate-sodium bicarbonate buffer (pH 10.0), and potassium monohydrogen phosphate-sodium hydroxide buffer (pH 11.0). While suspended, they were heated in water at 80 ° C. for 5 minutes. Subsequently, the solution was placed in a thermostat set at 40 ° C., and after reaching 40 ° C., the pH of each buffer solution was finely adjusted. In this solution, alkali-resistant CGTase produced by alkalophilic Bacillus genus cells was added to 1500. The unit was added to start the reaction. After the reaction for 16 hours, the reaction was stopped by heating at 80 ° C. for 5 minutes.
When each of these solutions was analyzed by HPLC (ODS), the amount of hesperidin dissolved before the reaction at each pH as shown in FIG. 2 was large in the alkaline region at pH 8 or higher. In addition, the amount of hesperidin glycoside produced by the enzyme reaction was large in the alkaline region from pH 8 to 9, especially pH 9.
Example 3
Add 150 mL of 0.5 M Tris-aminomethane-hydrochloric acid buffer (pH 8.0) or 150 mL of 0.5 M Tris-aminomethane-hydrochloric acid buffer (pH 9.0) to 1 g of orange peel dry powder, For 5 minutes. While suspended in this, 3 g of α-CD, β-CD, γ-CD, or soluble starch was added, and after dissolution, it was cooled to room temperature. To these, 800 units of alkali-resistant CGTase produced by alkalophilic Bacillus cells were added and placed in a thermostat set at 40 ° C. to initiate the reaction. After the reaction for 16 hours, the reaction was stopped by heating at 80 ° C. for 5 minutes.
When each of these solutions was analyzed by HPLC (ODS), as shown in FIG. 3 (pH 8) and FIG. 4 (pH 9), the addition of β-CD or γ-CD produced hesperidin glycosides in a particularly high yield. .
[0039]
[Brief description of the drawings]
FIG. 1 shows the results of HPLC (ODS) analysis of the dissolved amount of flavonoids before reaction (here, hesperidin) dissolved in an alkaline solution and adjusted to each pH and the amount of glycoside produced by the reaction. (Example 1)
FIG. 2 shows the result of HPLC (ODS) analysis of the dissolved amount of hesperidin before the reaction after dissolving in a buffer solution at each pH and the amount of glycoside produced by the reaction. (Example 2)
FIG. 3 is a result of yield analysis of hesperidin glycoside added with α-CD, β-CD, γ-CD, or soluble starch at pH 8. (Example 3)
FIG. 4 is a result of yield analysis of hesperidin glycoside added with α-CD, β-CD, γ-CD, or soluble starch at pH 9. (Example 3)

Claims (3)

A method for producing a soluble flavonoid glycoside comprising: adding an alkaline solution having a pH of 8 or higher and β-cyclodextrin or γ-cyclodextrin to a plant or a part thereof to solubilize flavonoids; and the alkaline region And a step of transglycosylation with a glycosyltransferase. A method for producing a soluble flavonoid glycoside, comprising: adding an alkaline solution having a pH of 8 or higher and α-cyclodextrin, dextrin, soluble starch or starch to a plant or a part thereof to solubilize flavonoids; and A process for transglycosylation with a glycosyltransferase in an alkaline region. A method for producing a soluble flavonoid glycoside comprising: adding an alkaline solution having a pH of 8 or higher and cycloamylose or cluster dextrin to a plant or a part thereof to solubilize flavonoids; and sugar transfer in the alkaline region A process for transglycosylation with an enzyme.

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