JPH05176785A - Production of arbutin - Google Patents

Abstract

PURPOSE:To readily and efficiently obtain arbutin by reacting glucose with hydroquinone in the presence of a beta-glucosidase. CONSTITUTION:Glucose is blended with hydroquinone so as to provide preferably (20:1) to (3:1) weight ratio of the glucose to the hydroquinone and an organic solvent such as preferably ethanol is then added thereto. A beta-glucosidase is added into the obtained mixture and enzymic reaction is carried out to afford the objective arbutin.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing arbutin which is a glycoside of hydroquinone. More specifically, it relates to an efficient method for producing arbutin from glucose and hydroquinone using an enzymatic reaction.

Arbutin (p-hydroxyphenyl-
β-D-glucopyranoside) has long been known as a glycoside having a bitter taste that is widely contained in azalea plants and rhododendron plants such as thornbill and cowberry, and has bactericidal ability (Cesk. Farm., 34 , 174, 1985), recorded in the Japanese Pharmacopoeia and used as a diuretic and urinary tract disinfectant. In addition, it has recently been used as a stabilizer for color photography (U.S. Pat. No. 3,287,126) and has been applied to the field of cosmetics because of its strong inhibitory effect on melanin-producing cells, that is, a whitening effect. Sho 60-16906, 6
0-56912 etc.).

[0003]

2. Description of the Related Art As a method for producing arbutin, in addition to an extraction method from an early natural product, a chemical synthesis method is conventionally known. In the chemical synthesis method, β-pentaacetylglucose obtained by acetylating glucose and hydroquinone are converted to tetraacetylarbutin using an appropriate catalyst, and arbutin is obtained by alkaline hydrolysis of the tetraacetylarbutin. There are several methods depending on the solvent system used. For example, a method using benzene [Doklady Akad. Nauk. SSSR, 8
6 , 333, (1952)], a method using xylene (ML Wolfro
m. A. Thompson, "Methods in Carbohydrate.", Vol.2,
p211, 1963) and a method using polyethylene glycol dialkyl ether (JP-A-62-263194).

Recently, arbutin is produced using plant callus cells (Phytochemistry, 15 , 12).
25, 1976). For example, a method in which hydroquinone and uridine diphosphate (UDP) -glucose are bound by an enzyme (glucose transferase) reaction to produce arbutin by utilizing the glycosidation ability (glycosyl transfer reaction) of cultured cells of Catharanthus roseus or Datsura (JP-A-62-181795, 6)
3-251092).

However, these methods have various drawbacks. That is, the former chemical synthesis method has a wide variety of reactions, is difficult to control, and requires a plurality of isolation and purification steps, resulting in a low total yield. On the other hand, the latter production method using plant cells has a drawback that it takes a long time to cultivate plant callus, resulting in high production cost. Therefore, an object of the present invention is to easily provide arbutin, which is an industrially useful hydroquinone glycoside,
And to provide a method for efficient production.

[0006]

Means for Solving the Problems The present inventors have conducted extensive studies to obtain arbutin in a relatively simple system consisting of glucose and hydroquinone by utilizing an enzymatic reaction, and as a result, use of β-glucosidase. As a result, they found that arbutin can be easily obtained, and completed the present invention. That is, the present invention relates to a method for producing arbutin, which comprises reacting glucose and hydroquinone in the presence of β-glucosidase and then collecting and collecting arbutin.

In the method for producing arbutin of the present invention, first, a reaction solution containing the substrates glucose and hydroquinone is prepared. The concentration of the substrate in the reaction solution is preferably within a concentration range in which each substrate can be dissolved, but even if the substrate is not dissolved and exists as a precipitate in the reaction solution, it may be used for arbutin production. The maximum dissolved concentration of substrate is about 91% glucose by weight percent and about 7% hydroquinone (25 ° C). It is also possible to increase the concentration of hydroquinone above the solubility in water to produce arbutin by adding an organic solvent such as dioxane or ethanol to the reaction system.

The amount of the organic solvent used is not particularly limited,
The enzyme used in the present invention is preferably used within a range that does not inactivate the enzyme or significantly reduce the solubility of the substrate. Regarding the composition of the substrate in the reaction solution, a relatively high glucose concentration is preferable because the stability of the enzyme in the reaction solution is increased and the productivity of arbutin is increased. For example, the weight ratio of glucose to hydroquinone is about 90: 1 to 1:10,
More preferably, it is in the range of about 20: 1 to 3: 1.

Next, β-glucosidase is allowed to act on the reaction solution as an enzyme. The origin of β-glucosidase used here is not particularly limited, and examples thereof include seeds of higher plants such as apricot and almond, filamentous fungi such as Aspergillus niger, and yeast. β-glucosidase need not be a pure enzyme, a culture of β-glucosidase-producing bacteria, a production strain collected by a method such as centrifugation from the culture,
It is also possible to use a dried bacterial cell or a treated bacterial cell obtained by pulverizing, autolyzing, or sonicating the bacterial cell, an extract from these bacterial cells, and an enzyme composition obtained from the extract.

The method of allowing β-glucosidase to act on the reaction system can be roughly classified into two. One is a batch system in which an enzyme or a microorganism having an enzyme is directly added to a reaction solution, and the other is an enzyme or a microorganism having an enzyme immobilized on which the reaction solution is applied. It is a method to let. The amount of enzyme added to the reaction solution can increase the amount of arbutin produced in a relatively short time as the amount increases, but is not particularly limited as long as it is a sufficient amount for the reaction of glucose and hydroquinone to proceed at a practically sufficient rate. Not done.

Generally, in the enzymatic reaction, reaction conditions such as pH and temperature are important factors, but in the method of the present invention,
The pH and temperature may be set within a range in which the enzyme used retains its activity, and it is particularly preferable to select the optimum temperature and optimum pH of the enzyme used. Also, the reaction p
H can usually be set using an appropriate buffer. The type and concentration of the buffer used here are not particularly limited as long as they do not significantly inhibit the enzyme reaction.

The reaction time according to the method of the present invention is not particularly limited, but it is preferable that the reaction time is such that glucose and hydroquinone do not actually increase. Arbutin obtained according to the method of the present invention is separated from the starting materials glucose and hydroquinone by a conventional method, after removing the enzyme as necessary, and by utilizing various chromatography, solvent extraction, recrystallization, etc. By doing so, arbutin can be easily and efficiently recovered.

[0013]

The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Glucose 30% (W / V), Hydroquinone 10% (W / V)
50 ml of the reaction solution (pH 5) consisting of was placed in a 100 ml container and set in a constant temperature bath previously set at 40 ° C. Next, β-glucosidase (derived from almond, manufactured by Sigma) was added to this reaction solution at a protein concentration of 10 mg / ml.
Then, the reaction was started, sampling was performed at regular intervals, and the enzyme in the sampling solution was removed using a membrane filter to stop the enzymatic reaction. The arbutin produced was analyzed by high performance liquid chromatography (HPL
It was analyzed in C). That is, using a silica-based partition adsorption chromatography column, Shodex F-511A, eluting with 10% methanol (pH 2.5) and using a commercially available arbutin (manufactured by Wako Pure Chemical Industries) as a standard substance, a UV detector (wavelength 29
1 nm). As a result, the amount of arbutin produced was 0.06 wt% in 24 hours after the reaction started, and was 0.11 in 72 hours.
It was wt%.

Examples 2 to 3 50 ml of a reaction solution (pH 5) consisting of 50% glucose and 5% hydroquinone was placed in a 100 ml container,
It was set in a constant temperature bath previously set to 40 ° C. Then, β-glucosidase (derived from almond, manufactured by Sigma) was added to this reaction solution so that the protein concentrations were 10 mg / ml and 50 mg / ml to start the reaction, and 24 hours after the same as in Example 1. The amount of arbutin produced was measured. The amount produced is 0.1 when the enzyme concentration is 10 mg / ml.
It was 0.08 wt% when the enzyme concentration was 05 wt% and the enzyme concentration was 50 mg / ml.

Examples 4 to 6 Glucose 20% and hydroquinone 5% (Example 4) Glucose 50% and hydroquinone 5%
(Example 5), and a reaction solution (pH 5) consisting of 90% glucose and 5% hydroquinone (Example 6).
Put 50 ml in each 100 ml container,
It was set in a constant temperature bath which had been set to ° C. Next, β-glucosidase (derived from almond, manufactured by Sigma) was added to each reaction solution so that the protein concentration was 10 mg / ml to start the reaction, and sampling was performed at regular intervals in the same manner as in Example 1 to generate. The amount of arbutin was measured. As a result, the amount of arbutin produced 24 hours after the start of the reaction was 0.03 wt% at a glucose concentration of 20% and 50% at a glucose concentration of 50%.
It was 0.05 wt% and 0.18 wt% at a glucose concentration of 90%.
The amount of arbutin produced 72 hours after the start of the reaction was 0.03 wt% at a glucose concentration of 20% and 50% at a glucose concentration of 50%.
It was 0.08 wt% and 0.17 wt% at a glucose concentration of 90%.

Example 7 50 ml of a reaction solution (pH 5) consisting of 30% glucose, 10% hydroquinone and 50% dioxane was used.
It was placed in a 0 ml container and set in a constant temperature bath previously set at 40 ° C. Next, β-glucosidase (derived from almond, manufactured by Sigma) was added to this reaction solution at a protein concentration of 10
When the amount of arbutin after 24 hours was quantified in the same manner as in Example 1, the reaction amount was 0.04 wt%.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Gen Sato 2-24-25 Tamagawa, Ota-ku, Tokyo Showa Denko Co., Ltd.

Claims (1)

[Claims] 1. A method for producing arbutin, which comprises reacting glucose and hydroquinone in the presence of β-glucosidase, and then collecting and collecting arbutin.