JPH06153976A - Production of phenol glycoside - Google Patents

Abstract

PURPOSE:To obtain a phenol glycoside having excellent physiological activities such as antioxidation activity and antibacterial activity and low toxicity and useful as food additive, etc., in high efficiency by treating a phenolic compound with sucrose phosphorylase in the presence of a sugar donor. CONSTITUTION:Various phenolic compounds and a sugar donor (e.g. sucrose) are mixed to a buffering solution (pH 7.5) containing sucrose phosphorylase and made to react at 42 deg.C for 15hr to obtain the objective phenol glycoside in high efficiency without deteriorating the characteristic physiological activity of the phenolic compound. The glycoside is free from the problems such as side effect and the solubility in water, has excellent physiological activities such as antioxidation action and antibacterial action and low toxicity and is useful as a food, food material, food additive, medicine, intermediate for medicine, organic synthetic compound, intermediate for organic synthetic compound, cosmetics and chemicals. The phenolic compound is preferably phenol, hydroxybenzenes, hydroxybenzoic acids, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a phenol glycoside, which can be expected to have various physiological activities such as an antioxidant effect and an antibacterial effect. More specifically, sucrose phosphorylase is used to efficiently produce conjugates of various phenol compounds having a phenolic hydroxyl group with the phenolic hydroxyl group and sugars such as glucose (hereinafter referred to as phenol glycoside). On how to do.

[0002]

2. Description of the Related Art Conventionally, phenol glycosides are widely present in plants, albeit in small amounts, and have low toxicity despite having excellent physiological activity. Therefore, they are food materials, food additives, pharmaceuticals, chemical products, etc. It is getting more and more attention as a target for development. For example, the Arctostap
hylos uva-ursi), cowberry (Vacc
Hydroquinone β-D-glucopyranoside (one of the hydroquinone glycosides belonging to hydroxybenzenes, also known as arbutin) contained in plants such as inium vitis-idea) (hereinafter referred to as arbutin) is an antioxidant. Action (Biosci. Bio
tech. Biochem. Volume 56, 1658-16
59, 1992, see), anti-allergic action (Pharmaceutical magazine 110, 56-67, 68-76, 1
990, reference) and melanin pigment suppression effect (Pro
c. Jpn. Soc. Invest. Dermato
l. 12 (pp. 138-139, 1988)). Further, arbutin is known to be used as an active ingredient for highly safe external preparations for skin, especially skin lightening cosmetics (see JP-A-61-227516), and also has an effect of maintaining a good scent balance. Therefore, it is also known that it can be used as a perfume base component of cosmetics (see JP-A-62-84010).

[0003] Further, glycosides of catechol and resorcinol, which belong to hydroxybenzenes, are effective for the prevention and treatment of skin pigmentation, and therefore they should be used as a component of an external preparation for skin (see JP-A-4-1115). It is also known to be used as a component of hair cosmetics (see JP-A-4-5218) because it has a remarkable effect of preventing the occurrence of dandruff on the scalp and imparting moisturizing and suppleness to the hair. There is.

As described above, the glycosides of hydroquinone, catechol, and resorcinol have excellent pharmacological activity as described above, while the non-sugar portion (also called aglycone) of these glycosides, For example, hydroquinone, which is an aglycone of arbutin, is also known to have an excellent pharmacological effect such as a melanin pigment suppressing effect. However, its effect is low, so that when it is used as a whitening cosmetic, it is inevitable to use it at a high concentration, and when it is used for a long period of time, it causes a new problem that it causes side effects such as white spots on the skin. In addition, ellagic acid (aglycone), which is a dimer of gallic acid, also has excellent pharmacological effects such as melanin pigment suppression effect including antioxidant effect, and thus is effective as an antioxidant in foods and in whitening cosmetics. Although it is expected to be used as a component, it has a problem that it is highly reactive and easily oxidized, its structure is unstable, and it is inconvenient to handle because its solubility in water is very low. Have Thus, the phenolic compound (aglycone) is a useful substance having various physiological activities, and by glycosylating this, side effects and solubility in water can be prevented without impairing its physiological activity. It is expected to improve such problems. However, the development of an effective technique for converting these phenolic compounds into glycosides has not been developed yet, which is an obstacle to their use.

[0005]

Conventionally, as a method for producing a phenol glycoside, a method of extracting and purifying from a contained plant or the like, or a method of binding a phenol moiety of an aglycone and a sugar by a chemical reaction by a method of organic synthesis, Alternatively, a method of binding a phenol moiety and a sugar by a reaction of a sugar-related enzyme is known, but none of them is a satisfactory method.
That is, in the method of extracting and purifying from a plant, the content of phenol glycosides is small and there are many extracted contaminants, which requires a complicated operation and a large cost, and the recovery rate is low. For example, a method of tissue culture of Catharanthus roseus in the presence of hydroquinone to generate and accumulate arbutin in the culture solution and obtain arbutin from this culture solution (see JP-A-61-124391) is Conversion rate of 60%
Although it is quite high as above, it is very difficult to set the culture conditions,
The addition amount of hydroquinone is as low as 2-5 mM,
It has low productivity and has a problem in industrial implementation. As a method of obtaining a phenol glycoside by a chemical reaction, acetylation of sugar, substitution with bromine, binding with phenol in the presence of silver salt such as silver carbonate, and deacetylation to obtain the desired glycoside. Although a method for obtaining the same is known, this method requires very careful handling because the steps are very complicated and a highly toxic Brom reagent is used. Also, as a phenol glycoside synthase, Bacillus mac
Cyclodextrins synthase from erans, Ba
α-amylase derived from cillus (See the 1992 Annual Meeting of the Japanese Society of Agricultural Chemistry, pp. 248-249)
Alternatively, a galactanase from Penicillium citrinum and several β-galactosidases (Denpun Kagaku 39, 1-6, 19)
The method for obtaining a phenol glycoside by using a sugar-related enzyme (see 1992) has a merit that it can be used for various phenol compounds because of its relatively wide substrate specificity. Has a non-industrial problem due to its low transfer rate.

[0006]

Therefore, the present inventors have conducted various studies to develop a new effective technique for glycosylating a phenol compound without the above-mentioned drawbacks, and as a result,
Finally, the present invention has been completed. That is, the present invention is a method for producing a phenol glycoside, which comprises allowing a sucrose phosphorylase to act on a phenol compound in the presence of a sugar donor.

The present invention will be described in detail below. The phenol compound used as the starting material of the present invention,
Phenol; hydroxybenzenes such as catechol, resorcinol and hydroquinone; trihydroxybenzenes such as pyrogallol; hydroxybenzoic acids such as salicylic acid; dihydroxybenzoic acids such as catechol-o-carboxylic acid, .beta.-resorcinic acid and protocatechuic acid; pyrogallol Examples include trihydroxybenzoic acids such as -4-carboxylic acid and pyrologlucin carboxylic acid; and hydroxybenzyl alcohols such as salicyl alcohol.

Examples of the sugar donor used in the present invention include those capable of transferring at least one molecule of a glucosyl group to a phenol compound, such as sucrose and glucose-1-phosphate. These may be used together if necessary.

Next, the sucrose phosphorylase used in the present invention is an enzyme that acts on sucrose in the presence of inorganic phosphorus to produce glucose-1-phosphate and fructose, or catalyzes the reverse reaction. , A known enzyme, for example, can be easily produced by using a microorganism described in the following literature. That is, for example, Leuconostoc Mecenteroides (Leuconos)
tocmesenteroides, Pseudomonas sacc
harophila), Pseudomonas putrefaci
ens), Clostridium pastillianum (Cl
ostridium pasteurianum), Acetobacter xylinum (Acetobacterium)
xylinum), pullularia pullulans (Pull)
ularia pullulans, etc. [Biotechnology and Baneo Engineering (Biote
chnol. Bioeng. ,) Vol. 29th, 8th
Page 15, 1987], inoculating a nutrient medium with a microorganism capable of producing sucrose phosphorylase.
It can be obtained by culturing to allow the enzyme to be produced and accumulated, and then separating the enzyme by a conventional method. As the sucrose phosphorylase, any of crude enzyme, purified enzyme, commercially available enzyme (eg, manufactured by Kikkoman, sold by Funakoshi, sucrose phosphorylase) and the like may be used.

To carry out the present invention, first, a phenol compound and a sugar donor are dissolved in water to prepare a mixed solution.
The addition amount of the above-mentioned two components to water is 5 to 80%, preferably 20 to 60% in terms of concentration by weight as a whole.

The addition amount of sucrose phosphorylase to the above mixture is 1 unit or more, preferably 50 to 200 units, per 1 gram of the total weight of the phenol compound and the sugar donor. It should be noted that 1 unit means Japanese Patent Laid-Open No. 3-
4785 means the amount determined according to the method described in "Production method of sucrose phosphorylase", that is, the amount of enzyme that converts 1 µmol of sucrose into glucose-1-phosphate per minute at 25 ° C.

The pH of the enzyme reaction is 5.0 to 8.5,
It is preferably 7.0 to 8.0 and the temperature is 20 to 5
0 ° C, preferably 35 to 45 ° C, and the time is 1 to
It is 48 hours, preferably 5 to 40 hours.

In this way, the reaction-terminated liquid containing the phenol glycoside is obtained. Separation of the desired phenol glycoside from the obtained reaction-terminated liquid is carried out as it is, or after pretreatment as described below, followed by isolation and purification by a liquid chromatography method, an organic solvent extraction method or the like.

As a specific method of the pretreatment, the obtained enzyme reaction-terminated liquid is passed through a column packed with an adsorption resin (for example, activated carbon or reverse phase silica gel), and the desired phenol distribution is applied to the resin. Glucose and unreacted phenol compounds are adsorbed, and then distilled water is passed through to elute unreacted sugar donors, sucrose phosphorylase (protein), etc., and then ethyl alcohol when activated carbon is used as the adsorption resin. In aqueous solution, or when using reversed-phase silica gel as the adsorption resin, pass an aqueous acetonitrile solution to elute and collect unreacted phenol compounds and phenol glycosides. The eluate containing the saccharide may be removed by a method such as evaporation to remove ethyl alcohol or acetonitrile and then concentrated. That.

As a method for separating the desired phenol glycoside from the reaction-terminated liquid obtained without or with the above-mentioned pretreatment, in the case of liquid chromatography, for example, strong acidic ion exchange, Reverse-phase partition mode ODS column treatment, or normal-phase partition mode amino column treatment (treatment to an amino column in which polyamine is chemically bonded to a hard polymer and separation and elution of a phenol glycoside fraction with an acetonitrile solvent solution, etc.) Or, based on a partition mode based on a molecular sieve in a polar organic solvent or a partition mode showing a gel-solute interaction effect, for example, a Sephadex LH-20 column treatment is performed to obtain a desired phenol glycoside fraction. The product is obtained by concentration under reduced pressure, freeze-drying and the like. In the case of using the organic solvent extraction method, the reaction-terminated liquid may be mixed with an organic solvent such as methyl alcohol, chloroform, acetic acid or ethyl acetate, and the extraction may be performed by a conventional method. The above-mentioned liquid chromatography method and organic solvent extraction method,
You may isolate and purify each individually or in combination.

[0016]

EFFECTS OF THE INVENTION According to the present invention, a phenol glycoside can be efficiently obtained by allowing sucrose phosphorylase to act on a phenol compound in the presence of a sugar donor. Then, it is possible to obtain a phenol glycoside having no side effects and problems such as solubility in water without impairing the physiological activity which the phenol compound originally has. The phenol glycoside obtained in the present invention has low toxicity in spite of having excellent physiological activity, therefore, food, food material, food additive, drug, pharmaceutical intermediate, organic synthetic compound, organic synthesis. It can be suitably used for compound intermediates, cosmetics, and chemical products. Further, among the phenol glycosides obtained in the present invention, α-arbutin, that is, hydroquinone α-D-glucopyranoside, is a highly safe external preparation for skin, particularly as an active ingredient of skin whitening cosmetics, and as a fragrance for cosmetics. It can be used as a base component. When it is used as a whitening cosmetic material, it has a very high melanin pigment inhibitory effect, and thus it has the characteristics that it achieves the purpose with a small amount and has no side effects even when used for a long period of time. Further, the catechol and the glycoside of resorcinol obtained in the present invention are effective in the prevention and treatment of skin pigmentation disease, as a component of an external preparation for the skin, prevent the occurrence of dandruff on the scalp, and moisturize and supple the hair. It can be used as a component of hair cosmetics that gives strength. In addition, the ellagic acid glycoside obtained in the present invention, that is, ellagic acid 4-O-α-D-glucopyranoside is itself very stable against oxidation, and has high solubility in water, and thus is easy to handle. Moreover, since it has an excellent pharmacological effect such as an antioxidant effect and a melanin pigment suppressing effect, it can be used as an antioxidant in foods and as an active ingredient in whitening cosmetics and the like.

Hereinafter, the present invention will be described more specifically by showing examples.

Example 1 Sucrose phosphorylase (manufactured by Kikkoman Co., sold by Funakoshi Co., Ltd.) was prepared by using 60 mg of sucrose and 2 mg of each phenol compound shown in Table 1.
0.2 ml of 100 mM HEPES containing 5.0 units
(PH 7.5) mixed with a buffer solution and reacted at 42 ° C. for 15 hours, and this reaction solution was subjected to thin layer chromatography (TLC).
Was analyzed. The chromatogram thus obtained was compared with that of a control to which no phenol compound was added to confirm the presence or absence of metastases. The results are shown in Table 1. As a result, the enzyme has a wide range of receptor specificity that can accept various phenolic compounds, that is, phenol, hydroxybenzenes, trihydroxybenzenes, hydroxybenzoic acids, trihydroxybenzoic acids, ellagic acid, and hydroxybenzyl alcohols. It has become clear that it has a sex.

[Conditions for analysis by thin layer chromatogram] Plate: Silica gel 60 (Merck) Mobile phase: Ethyl acid acid / acetic acid / water = 3/1/1 Detection: Sulfuric acid / methyl alcohol = 1/1 on the plate Spray and heat. Table 1 Types of phenolic compounds (receptors) Presence or absence of transfer products 1.1,2-DHB (catechol) Yes 2.1,3-DHB (resorcinol) Yes 3.1,4-DHB (hydroquinone) Yes 4.1 , 2,3-THB (pyrogallol) Yes 5.1,2,4-THB (1,2,4-trihydroxybenzene) No 6.1,3,5-THB (Ploroglucinol) No 7. HB (phenol) Yes 8. No BAD (benzoic acid) No 9.2-HBAD (salicylic acid) Yes 10.3-HBAD (3-hydroxybenzoic acid) Yes 11.4-HBAD (4-hydroxybenzoic acid) Yes 12.2,3-DHBAD (catechol) -O-carboxylic acid) Yes 13.2,4-DHBAD (β-resorcinic acid) Yes 14.2,5-DHBAD (gentisic acid) No 15.2,6-DHBAD (γ-resorcinic acid) No 16.3 , 4-DHBAD (protocatechuic acid) Yes 17.3,5-DHBAD (3,5-dihydroxybenzoic acid) No 18.2,3,4-THBAD (pyrogallol-4-carboxylic acid) Yes 19.2,4 , 6-THBAD (pyrrologlycine carboxylic acid) Yes 20.3,4,5-THBAD (gallic acid) No 21. EA (ellagic acid) Yes 22. BAL (benzyl alcohol) Yes 23.2-HBAL (salicyl alcohol) Yes 24.3-HBAL (3-hydroxybenzyl alcohol) Yes 25.4-HBAL (4-hydroxybenzyl alcohol) Yes

[0019]

Example 2 As a phenol compound, 2 g of hydroquinone (hereinafter sometimes referred to as HQ) and 30 g of sucrose were dissolved in 100 ml of 100 mM HEPES (pH 7.5) buffer. Sucrose phosphorylase (manufactured by Kikkoman, sold by Funakoshi)
Add 4,000 units, react at 42 ° C for 14 hours,
A sugar compound formation reaction was performed to obtain an enzyme reaction completed liquid. The result of the HPLC analysis of this reaction completed liquid is shown in FIG.

From the results shown in FIG. 1, it can be confirmed that the reaction-terminated liquid contains hydroquinone and hydroquinone glycoside. As shown in FIG. 1, the peak fraction eluted first is hydroquinone glycoside, and the peak fraction eluted later is hydroquinone.

FIG. 2 shows the time course of the hydroquinone glycoside transfer rate in the enzymatic reaction by HPLC analysis. The transfer rate shown on the vertical axis of FIG. 2 is the number of moles of hydroquinone glycoside produced relative to the number of moles of hydroquinone used in the reaction (calculated from the peak area of HPLC).
Expressed as a percentage of.

[HPLC by High Performance Liquid Chromatogram]
Analysis Conditions] Column; CAPCELL PAK C18 SG12
0, inner diameter, 4.6 mm, length, 250 mm Flow rate; 1 ml / min Mobile phase; 0-50% methyl alcohol concentration gradient (30
Min), containing 0.1% trifluoroacetic acid. Detection wavelength: 270 nm

[0023]

[Example 3] Enzyme reaction completed liquid 1 obtained in Example 2 above
00 ml of activated carbon column (inner diameter 40 mm, length 350 m
After passing through m), unreacted sugar and enzyme were eluted with 5% ethyl alcohol aqueous solution, and then hydroquinone and hydroquinone glycoside fraction were eluted with 20% ethyl alcohol aqueous solution. The solution containing the unreacted hydroquinone and the desired hydroquinone glycoside thus obtained was concentrated by a rotary evaporator to 1/40 volume. Then, the concentrated solution was mixed with O in a reverse phase distribution mode.
CAPCELL PAK C18 SG which is a DS column
120 (made by Shiseido) column (inner diameter 20 mm, length 25
(0 mm), and a 7.5% methyl alcohol aqueous solution was passed through to obtain the desired hydroquinone glycoside, which was concentrated under reduced pressure by a rotary evaporator and freeze-dried. About 2.3 g of odorless powder which is considered to be the target hydroquinone glycoside was obtained. The above powder under the above conditions,
The purity of the hydroquinone glycoside was measured by high performance liquid chromatography and found to be 95%.

Next, the structure of the above hydroquinone glycoside was analyzed by the following nuclear magnetic resonance spectrum, and it was confirmed that this compound was hydroquinone O-α-D-glucopyranoside. That is, according to a conventional method the powder of the compound was dissolved in deuterated DMSO, ¹ H- nuclear magnetic resonance spectrum and ¹³ C- was measured by a nuclear magnetic resonance spectrum, Fig. 3 was the analysis of the structure ⁽¹ H-NMR
(200MH _Z)) and FIG. 4 ⁽¹³ C-NMR (50M
H _Z )) was obtained, and it was found from these figures that the constituents were 1 molecule of glucose and 1 molecule of hydroquinone, and the 1-position of glucose and the 4-position of hydroquinone were α-bonded.

Next, the UV and IR spectra of the compound obtained by the method of the present invention were investigated by a conventional method. As a result, FIGS. 5 and 6 were obtained, respectively. The UV spectrum was measured by dissolving the sample in distilled water to a concentration of 0.02 mg / ml, and the IR spectrum was measured by the KBr method. From this result, it was confirmed that the obtained sugar compound was hydroquinone O-α-D-glucopyranoside (α-arbutin) (hereinafter abbreviated as HQG).

[0026]

[Application Example 1] "Color stability stability test of HQG in aqueous solution" Example 3 above
HQG obtained in step 1, hydroquinone as a starting material thereof, and arbutin (manufactured by Nacalai Tesque, Inc.) were each dissolved in distilled water to prepare a 1000 ppm aqueous solution,
A pseudo-sunlight irradiation device (made by Irie Seisakusho Co., Ltd.) was maintained at a temperature of 30 ° C. for 8 hours, and a part of the aqueous solution was taken in a 10 mm cell over time to measure the increase in absorbance at 460 nm, which reflects the coloring degree. did. The result is shown in FIG. 7.

From the results shown in FIG. 7, the absorbance of the hydroquinone category gradually increased and increased to 0.4 or more after 8 hours, whereas the HQG category was similar to the arbutin category.
The increase in absorbance was about 0.1 even after 8 hours. From this, HQ obtained by the method of the present invention
It can be seen that G has a very stable structure that is hardly oxidized and has excellent color stability.

[0028]

[Application Example 2] "Tyrosinase activity inhibitory effect of HQG" The tyrosinase activity inhibitory effect of HQG (α-arbutin) obtained in Example 3 above was examined in-house (in vitro). The test method was basically carried out according to JP-A-4-45791, and dopachrome (maximum absorption wavelength 475 nm) produced from L-tyrosine by tyrosinase was measured.

(1) Reaction system test solution L-tyrosine solution: 0.05% (W / V) aqueous solution of L-tyrosine (manufactured by Wako Pure Chemical Industries, Ltd.) Sample solution: HQG to a concentration of 5 nM / ml It was prepared by dissolving in 50 mM acetate buffer (pH 6.8). Tyrosinase solution: Tyrosinase (manufactured by Sigma, mushroom origin, 2000 units / mg) 0.7 mg 50
It was prepared by dissolving in 1 ml of mM acetate buffer (pH 6.8). The above reaction system test solution was chilled and stored in ice until measurement.

(2) Measurement of tyrosinase activity inhibition rate Preparation of reaction solution In measuring the tyrosinase activity inhibition rate, the reaction system test solutions were mixed so as to have the composition shown in Table 2, and C1 and C were added.
Four reaction solutions of 2, T1, and T2 were prepared. Next, 12 test tubes were divided into 4 groups of 3 tubes each, and the above reaction solution was put into each section and allowed to stand at room temperature for 5 minutes or more. Then, the procedure was shifted to the next measurement operation and measurement was carried out at 37 ° C. Table 2 (Composition of reaction system test solution) Unit (ml) Reaction system test solution C1 C2 T1 T2 L-tyrosine solution 1.0 0 1.00 Test solution 0 0 1.0 1.0 1.0 Acetate buffer 1.9 2.9 0.9 1.9 Total 2.9 2.9 2.9 2.9 Measurement When 0.05 ml of tyrosinase solution was added to each reaction solution of C1, C2, T1 and T2 prepared above, tyrosinase was added. L-tyrosine decreases and dopachrome increases on the contrary, and the reaction solution gradually changes to reddish orange. Therefore, the absorbance at 475 nm is measured every 1 minute by a spectrophotometer manufactured by Guilford (cell temperature 3
It was measured at 7 ° C. For C1-C2 (control) and T1-T2 (invention) of HQG, the change in the increased absorbance was examined. The result is shown in FIG.

FIG. 8 shows that the lower the value compared with the control, the greater the inhibition of tyrosinase activity. Calculation of tyrosinase activity inhibition rate From the absorbance 5 minutes after the addition of the tyrosinase solution, C1
Since three data are obtained for each of C2, T1, and T2, an average value is obtained for each, and C1,
The tyrosinase activity inhibition rate was determined according to the following formula, where C2, T1 and T2 were used. Formula; inhibition rate (%) = [1- (T1-T2) / (C1-C)
2)] x 100

From the results of FIG. 8, in the control section not containing HQG, the action of tyrosinase causes a decrease in L-tyrosine and conversely an increase in dopachrome so that the absorbance increases with the passage of time. Since the presence of the substance inhibits the action of tyrosinase, the increase in absorbance derived from the reaction product dopachrome is suppressed to a low level, and the category of the present invention containing HQG has a tyrosinase activity inhibition rate of 81.1. %, Which was a very high value. Further, HQG (α-obtained by the method of the present invention
It is understood that arbutin) has a tyrosinase activity inhibitory action and can be used as an effective component of a whitening cosmetic composition.

[0033]

Example 4 1 g of ellagic acid was added as a phenol compound.
In order to accelerate the dissolution of this ellagic acid, 1.28 g of arginine was added to dissolve the ellagic acid, and 23.8 g of HEPES and 400 g of sucrose as a sugar donor were dissolved therein. To this, 20,000 units of sucrose phosphorylase (manufactured by Kikkoman, sold by Funakoshi) were added, and the total amount was 1000 ml.
Was allowed to react at 42 ° C. for 14 hours to carry out a sugar compound-forming reaction to obtain an enzyme reaction completed liquid. HPL of this reaction completed liquid
The result of C analysis is shown in FIG.

From the results shown in FIG. 9, it can be confirmed that the reaction completed liquid contains ellagic acid and ellagic acid glycoside.
As shown in FIG. 9, the peak fraction eluted first is ellagic acid glycoside, and the peak fraction eluted later is ellagic acid.

FIG. 10 shows the change over time in the ellagic acid glycoside transfer rate in the enzymatic reaction by HPLC analysis. The transfer rate shown on the vertical axis of FIG. 10 is represented by% of the number of moles of ellagic acid glycoside produced (calculated from the peak area of HPLC) with respect to the number of moles of ellagic acid used in the reaction.

[HPLC by High Performance Liquid Chromatogram]
Analysis Conditions] Column; CAPCELL PAK C18 SG12
0, inner diameter, 4.6 mm, length, 250 mm Flow rate; 1 ml / min Mobile phase; 5 mM KH ₂ PO ₄ (pH 2.5) / methyl alcohol = 55/45 Detection wavelength; 254 nm

[0037]

[Example 5] Enzyme reaction completion liquid 1 obtained in Example 4 above
000 ml was passed through an ODS-AQ (YMC) column (inner diameter 45 mm, length 150 mm) to elute unreacted sugar and enzyme with water, and then ellagic acid and ellagic acid glycoside were added with 50% acetonitrile solution. The body fraction was eluted. The solution containing the unreacted ellagic acid and the desired ellagic acid glycoside was thus obtained by a rotary evaporator.
Concentrated to a volume of / 40. Then, this concentrated solution was applied to Sephadex LH-20 (inner diameter 50 mm, length 7
20 mm), and a 0.05 N KOH solution is passed through to elute unreacted ellagic acid first, and then a fraction containing the target ellagic acid glycoside is obtained, to which concentrated hydrochloric acid is added. The target ellagic acid glycoside was separated by acid precipitation. Then, the mixture was centrifuged by a conventional method, washed with a small amount of water, and dried in a vacuum to obtain about 80 mg of an odorless powder which is considered to be the target ellagic acid glycoside. The purity of the ellagic acid glycoside was 95% when the above powder was measured by high performance liquid chromatography under the above-mentioned conditions. When IR spectrum of 1 mg of the ellagic acid glycoside was examined by the KBr method, FIG. 11 was obtained, and ellagic acid 4-O-α-D was obtained.
-It was confirmed to be glucopyranoside.

[Brief description of drawings]

FIG. 1 shows the results of HPLC analysis of an enzyme reaction completed solution containing HQ and HQG obtained in Example 2.

FIG. 2 shows the results of changes over time in the HQG transfer rate in the enzymatic reaction of Example 2 determined by HPLC analysis.

FIG. 3 ¹ H-nuclear magnetic resonance spectrum of HQG (200
MH _Z) indicating the (heavy DMSO).

FIG. 4 shows the ¹³ C-nuclear magnetic resonance spectrum (50
MH _Z) indicating the (heavy DMSO).

FIG. 5 shows a UV spectrum when the same compound was dissolved in distilled water.

FIG. 6 shows an IR spectrum of the same compound measured by a KBr method.

FIG. 7 shows color stability over time of the same compound, hydroquinone, and arbutin in an aqueous solution.

FIG. 8 shows time-course changes of the same compound regarding inhibition of tyrosinase activity.

FIG. 9 shows the results of HPLC analysis of the enzyme reaction completed liquid containing ellagic acid and ellagic acid glycoside obtained in Example 4.

FIG. 10 shows the results of changes over time in the transfer rate of ellagic acid glycosides in the enzymatic reaction of Example 4 determined by HPLC analysis.

FIG. 11 shows an IR spectrum of ellagic acid glycoside by the KBr method.

Claims (2)

[Claims] 1. A phenol compound in the presence of a sugar donor,
A method for producing a phenol glycoside, which comprises reacting sucrose phosphorylase. 2. The phenol compound is one selected from the group consisting of phenol, hydroxybenzenes, trihydroxybenzenes, hydroxybenzoic acids, trihydroxybenzoic acids, ellagic acid and hydroxybenzyl alcohols. Method for the production of phenol glycosides.