JP3024848B2 - Method for producing catechin glycosides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a catechin glycoside having a catechin that is easily soluble and has good color stability without substantially impairing the excellent physiological activity of the catechin. A catechin glycoside having the above characteristics in which glucose and catechins are combined from glucose-1-phosphate or sucrose and catechins by utilizing sucrose phosphorylase, particularly using sucrose phosphorylase. On how to get.

[0002]

2. Description of the Related Art Conventionally, catechins have an antioxidant effect on edible oils and fats, an antibacterial effect on food poisoning bacteria, a blood cholesterol level increase inhibitory action, angiotensin converting enzyme inhibitory action and blood pressure increase inhibitory action, α-amylase inhibitory action. It is an important chemical substance in the food and pharmaceutical industries because it has excellent physiological activities such as an anti-hyperglycemic effect, an anti-mutation in microorganisms and an anti-tumor effect in mice or rats.

[0003]

However, catechins have low solubility in water and very poor stability against photo-oxidation, which limits their use in the food and pharmaceutical industries. Has problems.

[0004]

The present inventors have made various studies to solve such problems, and as a result,
When sucrose phosphorylase is allowed to act on a mixed solution of catechins and glucose-1-phosphate or sucrose, the solubility in water is high without substantially impairing the excellent physiological activity of the catechins, It has been found that catechin glycosides having extremely high stability against irozawa can be obtained, and the present invention has been completed based on this finding.

That is, the present invention relates to catechins and glucose-
A method for producing a catechin glycoside characterized by allowing sucrose phosphorylase to act on a mixed solution with 1-phosphate or sucrose.

Hereinafter, the present invention will be described in detail. The catechins used in the present invention include (+)-catechin,
(-)-Epicatechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epigallocatechin gallate, and the like.

Since the catechins generally have very low solubility in water (1 to 2.5 mg / ml), the catechins are previously dissolved in a polar solvent such as methanol or ethanol (100 to 100 mg / ml).
(400 mg / ml) and increasing the concentration of catechins as much as possible is preferred in order to efficiently obtain catechin glycosides.

Next, sucrose phosphorylase used in the present invention is an enzyme which acts on sucrose in the presence of inorganic phosphate to produce glucose-1-phosphate and fructose, or catalyzes the reverse reaction. is there.

[0009] The origin is, for example, Leuconostoc mesenteroides (Leuconostoc)
mesenteroides), Pseudomonas saccharophila (Pseudomonas saccha)
rophila), Pseudomonas putrefacien (Pseudomonas putrefacien)
s), Clostridium pastirianum (Clos)
tridium pasteurianum), Acetobacter xylinum
linum), Pullulia pullulans (Pululul)
aria pullulans) [Biotechnology and Bioengineering (Biotechnol. Bioeng.,) Vo]
l. 29, Pp8-15, 1987], but are not limited thereto.

Next, in order to carry out the present invention, first, glucose-1-phosphate or sucrose and the above catechins are dissolved in water to prepare a mixed solution. The addition amount of the above two components to water is 5 to 5% by weight as a whole.
It is 100%, more preferably 20 to 60%.

The amount of sucrose phosphorylase to be added to the above mixture is 1 unit or more, preferably 50 to 100 units, per gram of the total weight of glucose-1-phosphate or sucrose and catechins.

One unit is determined according to the method described in JP-A-3-4785, "Method for producing sucrose phosphorylase".

The pH of the enzymatic reaction is from 5 to 8.5, preferably from 7 to 8, the temperature is from 20 to 50 ° C, preferably from 35 to 45 ° C, and the time is from 1 to 24 hours, preferably from 1 to 24 hours. Is 8 to 15 hours.

For separation of the target catechin glycoside from the reaction solution thus obtained, a conventional method for isolating a catechin compound may be employed. That is, a chromatography method using a dextran derivative such as Sephadex LH-20 as a carrier [R. S. Tompson et al.
Chem. Soc. Perkin I, No. 11,1
387 (1972)], a column chromatography method using polyamide as a carrier [J. P. Van Buren
Etc. J. Food Sci. , Vol. 31.964
(1966)], a liquid chromatography method using silica gel [C. J. William Glennie et al. Agric. FoodChem. , Vol. 2
9.965-968 (1981)], a method by countercurrent distribution between water and ethyl acetate [Andrew G., et al. H. Lea
Author, J. et al. Sci. FdAgric. , Vol. 29.
471-477 (1978)], a polystyrene resin,
For example, Diaion HP20, HP21, SP206,
SP207, CHP3C, CHP5C, CHP20P
(All are manufactured by Mitsubishi Kasei Kogyo Co., Ltd.), Amberlite XA
Chromatographic method using D-1, XAD-2, and XAD-4 (all of which are manufactured by Organo Corporation)
62685], or a method of fractionation using an ultrafiltration membrane or a reverse osmosis membrane [JP-A-63-267774]. These can be used alone or in combination to separate the desired catechin glycosides. For example, the solution is passed through a column filled with a filtration resin (for example, Sephadex LH-20 manufactured by Pharmacia), and then water is passed through to remove unreacted sugars, enzymes (proteins), and the like. The solution was passed through the column to elute unreacted catechins and catechin glycosides, which were subjected to liquid chromatography using silica gel (for example, TSOSOH, TS
(K-gel ODS-80TM), and the target catechin glycoside fraction is separated by a methanol concentration gradient.

[0015]

Conventionally, it has been known that starch is added to catechins to react with cyclodextrin synthase to produce catechins glucoside (Japanese Society of Agricultural Chemistry, Vol. 5 pages, 2 Ap14, 1991
In this method, a plurality of catechin glucosides having different degrees of glucose polymerization are formed in the reaction solution, and thus, in order to collect a target catechin monoglucoside from the reaction solution, Furthermore, the reaction solution has to be treated with glucoamylase, which has a problem that the operation is complicated. On the other hand, according to the present invention, by the above-mentioned extremely simple operation of adding a sugar-phosphorylating enzyme sucrose phosphorylase to a solution containing glucose-1-phosphate or sucrose and catechins, A single, desired catechin glycoside, for example, (+)-catechin 3′-O-α-D-glucopyranoside can be obtained in good yield. That is, the operation of isolating and purifying the target catechin glycoside compound from the reaction solution is very simple.

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

Example 1 600 mg of (+)-catechin was dissolved in 2 ml of methanol, and this was mixed with 100 ml of a sucrose solution dissolved at a concentration of 400 mg / ml in 100 mM MES (pH 7.5) buffer, and sucrose was added thereto. Add 4500 units of phosphorylase (manufactured by Seishin Pharmaceutical Co., Ltd.)
After reacting for 5 hours, a sugar compound formation reaction was performed.
Heat treatment was performed at 00 ° C. for 5 minutes to stop the enzyme reaction, thereby obtaining an enzyme reaction-treated solution. Next, the obtained reaction solution was passed through a Sephadex LH-20 column (inner diameter 3 cm, length 30 cm), and water was passed through to wash off unreacted sugars and proteins (enzymes). The solution was passed through a 50% solution of methanol to obtain a solution containing unreacted (+)-catechin and the target catechin glucoside.

The thus obtained unreacted (+)-
The solution containing catechin and the desired catechin glucoside is then subjected to a reverse phase distribution adsorption chromatogram, T.
The solution is applied to an SK-gel ODS-80TM column (inner diameter 2 cm, length 25 cm) and passed through a 15% methanol solution to obtain a solution containing the desired catechin glucoside, which is concentrated under reduced pressure by a rotary evaporator. , Followed by freeze-drying to obtain 410 mg of a powder that was considered to be catechin glucoside.

The powder was analyzed by high performance liquid chromatography (TSK-gel ODS-80TM) to determine the purity of catechin glucoside, which was 98%.

The structure was measured by the following nuclear magnetic resonance spectrum and mass spectrometry analysis methods, and the obtained compound was (+)-catechin 3'-
It was confirmed to be O-α-D-glucopyranoside. That is, according to a conventional method the powder was dissolved in deuterated acetone, ¹ H- nuclear magnetic resonances determined by spectrum and ¹³ C- nuclear magnetic resonance spectrum by analytical methods, views, respectively were analyzed structure 1 ⁽¹ H- NMR (200M
_{H Z) (Acetone-d 6} )) and FIG. 2 ⁽¹³ C-NM
_{R (50MH Z) (Acetone-} d 6)) is obtained,
From these figures, the constituent components are one molecule of glucose and (+)-
One molecule of catechin, and it was found that the 1-position of glucose and the 3′-position of (+)-catechins were α-bonded.

FIG. 3 shows the result of mass spectrometry analysis according to a conventional method. From the results, it was confirmed that the molecular weight of the compound obtained by the above method was 452.

From the above results, it was confirmed that the obtained sugar compound was (+)-catechin 3'-O-α-D-glucopyranoside.

[0022]

Example 2 600 mg of (+)-catechin was dissolved in 2 ml of methanol, and this was mixed with 100 ml of a glucose-1-phosphate solution dissolved at a concentration of 200 mg / ml in 100 mM MES (pH 7.5) buffer. Add 4500 units of sucrose phosphorylase (Seishin Pharmaceutical Co., Ltd.) to this,
The mixture was reacted at 42 ° C. for 15 hours to perform a saccharide compound generation reaction, and then heated at 100 ° C. for 5 minutes to stop the enzyme reaction, thereby obtaining an enzyme reaction-treated solution. Hereinafter, 370 mg of powder of (+)-catechin 3′-O-α-D-glucopyranoside was obtained in exactly the same manner as in Example 1 above.

[0023]

[Application Example 1] “Anti-photooxidative test of (+)-catechin 3′-O-α-D-glucopyranoside” 20 ppm of riboflavin in pure water
Dissolve so that it becomes 1
In the first section, (+)-catechin 3′-O-α-D-glucopyranoside is dissolved to a concentration of 2500 ppm, and in the second section, (+)-catechin is dissolved to a concentration of 2500 ppm. , Without adding anything to the third section,
Each of the samples was irradiated with a pseudo-sunlight irradiation device (manufactured by Irie Seisakusho Co., Ltd., precision sunlight bath) under the conditions of visible light of 14600 lux and ultraviolet intensity (310 to 400 nm) of 0.12 mW / cm ^2. Non-degraded riboflavin was quantified by high-performance liquid chromatogram.

Conditions for High Performance Liquid Chromatogram TSK-gel ODS-80TM (inner diameter 4.6 mm,
Flow length; 1 ml / min. Mobile phase; methanol: 10 mM NaH ₂ PO ₄ (pH 5.
5) = 35: 65 detection; 266 nm The results above are shown in FIG.

From the results shown in FIG. 4, in the case of category 3, where nothing is added, riboflavin is rapidly decomposed and disappears, but together with the category 2 to which (+)-catechin is added, (+)-catechin 3 '.
Category 1 to which -O-α-D-glucopyranoside was added,
It can be seen that riboflavin is hardly decomposed and is stable. That is, it can be seen that (+)-catechin 3′-O-α-D-glucopyranoside has no inferiority to (+)-catechin in anti-photooxidative property.

[0026]

[Application Example 2] “Color stability test of (+)-catechin 3′-O-α-D-glucopyranoside” Two sections of pure water were prepared in 3 ml portions, and (+)-catechin 3′- was assigned to the first section. O-α-D-
Glucopyranoside is dissolved at 1000 ppm, and (+)-catechin is dissolved at a concentration of 1000 ppm in the second section, and visible light is illuminated by a pseudo-sunlight irradiation device (a precision sunlight thermostat manufactured by Irie Seisakusho Co., Ltd.). 14600 lux, UV intensity (310-400 nm)
Irradiation was performed under the condition of 0.12 mW / cm ² , a part of the sample was collected over time, and the degree of coloring was measured by increasing the absorbance at 460 nm. The result is shown in FIG.

From the results shown in FIG. 5, it is found that Category 2 in which (+)-catechin was dissolved was colored brown with the passage of time, and (+)
-Catechin has very poor color stability, whereas Category 1 to which (+)-catechin 3'-O-α-D-glucopyranoside is added is almost colorless and transparent even after 7 hours, and It can be seen that the stability of is very good.

[0028]

[Application Example 3] "Water solubility test of (+)-catechin 3'-O-α-D-glucopyranoside" (+)-catechin and (+)-
Catechin 3′-O-α-D-glucopyranoside was dissolved in pure water so as to have respective concentrations shown in Table 1, and this was dissolved in 1
It was left for 15 hours in a constant temperature room at 5 ° C., and the presence or absence of precipitation was examined. Table 1 shows the results.

From the results in Table 1, (+)-catechin 3 '
It can be seen that -O-α-D-glucopyranoside has a solubility that is at least 10 times higher than that of (+)-catechin and is extremely convenient for practical use.

[0030] Note 1. In the table,-indicates that no precipitate is formed, + indicates that a precipitate is formed, and ++ indicates that a precipitate is significantly formed.

[Brief description of the drawings]

FIG. 1: ¹ H-nuclear magnetic resonance spectrum (200 M) of (+)-catechin 3′-O-α-D-glucopyranoside
H _Z) indicating the (Acetone-d _6).

FIG. 2: ¹³ C-nuclear magnetic resonance spectrum (50 M) of (+)-catechin 3′-O-α-D-glucopyranoside
H _Z) indicating the (Acetone-d _6).

FIG. 3 shows a mass spectrum of (+)-catechin 3′-O-α-D-glucopyranoside.

FIG. 4 shows the results of an anti-photooxidative test of (+)-catechin 3′-O-α-D-glucopyranoside.

FIG. 5 shows (+)-catechin and (+) obtained by the present invention.
-The catechin 3'-O- (alpha) -D-glucopyranoside shows the graph which compared the color stability with the color stability.

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 19/00-19/64 C07H 17/075 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A method for producing a catechin glycoside, which comprises reacting sucrose phosphorylase with a mixture of catechins and glucose-1-phosphate or sucrose.