JPH09249688A - Chromanol glycoside, its production and antioxidant using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a new chromanol glycoside, excellent in solubility in water and useful as an antioxidant, etc., for cosmetics, medicines, foods, etc., by reacting a β-galactosidase with a solution containing chromanol and a β- galactosyl saccharide compound. SOLUTION: This new chromanol glycoside is represented by formula I [R<1> to R<4> are each H or a lower alkyl; R<5> is H, a lower alkyl or a lower acyl; H atoms of the hydroxyl groups in the saccharide residue may be substituted with a lower alkyl group or a lower acyl group; (n) is an integer of 0-4] and is excellent in solubility in water and useful as an antioxidant, etc., for foods, cosmetics, medicines, etc. The glycoside is obtained by reacting a β-galactosidase with a solution containing a 2-substituted alcohol represented by formula II and a β-galactosylsaccharide compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel chromanol glycoside, a method for producing the same, and an antioxidant using the same. More specifically, the general formula (2) known as a known antioxidant

[0002]

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(Wherein R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atom or lower alkyl group, R ⁵ represents hydrogen atom, lower alkyl group or lower acyl group, and n is an integer of 0 to 4) A novel water-soluble compound having excellent chemical stability obtained by binding a sugar to a 2-substituted alcohol (hereinafter, also simply referred to as “2-substituted alcohol”) represented by And a method for producing the same, and a water-soluble antioxidant using the same.

[0004]

2. Description of the Related Art In recent years, it has become increasingly clear that active oxygen and free radicals damage the living body, which leads to various diseases, cancer, and even aging. Therefore, the development of antioxidants that can efficiently protect against damages due to active oxygen and free radicals has attracted attention in many fields such as cosmetics, pharmaceuticals, and foods.

Although many antioxidants are known at present,
In particular, Vitamin E is widely used in the fields of foods, cosmetics and pharmaceuticals because of its excellent antioxidant activity. However, since vitamin E is a viscous oily substance that is insoluble in water, it must be solubilized with a surfactant or the like before it can be used as an injection or solution, resulting in the incorporation of a large amount of originally unwanted substances. Occurs. Further, it is impossible to make a high-concentration aqueous solution of vitamin E with a surfactant or the like. That is, there is a need for a water-soluble vitamin E analog compound that retains the excellent antioxidant activity of vitamin E.

In Japanese Patent Laid-Open No. 7-118287,
Vitamin E-like compounds (chromanol glycosides) with excellent water solubility and their production by enzymatic methods have been shown. In the production method of the above publication, although a simple description is given in the case of binding galactose to a 2-substituted alcohol by a β bond, the physical properties of the product in Examples are
No manufacturing method or antioxidant activity is shown. In addition, when administered to a living body, 2-
Glucose α-bonded to a substituted alcohol (hereinafter,
Glucose-type glycosides and galactose-type glycosides (hereinafter referred to as galactose-type glycosides) have different glycoside glycosides due to differences in the location and activity of glycolytic enzymes in vivo. It is common knowledge that the stability of the bond is different, and also because of the problem of the receptor of the biological tissue, the way in which both are taken up by the biological tissue is different.

[0007]

Therefore, an object of the present invention is to provide a novel galactose-type compound which exhibits different stability in vivo and how it is taken up by living tissues than glucose-type glycosides known in vivo. It is also to provide a glycoside, a method for producing the same, and a water-soluble antioxidant using the same.

[0008]

Means for Solving the Problems The above-mentioned object is (A) general formula (1)

[0009]

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(Wherein R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms or lower alkyl groups, R ⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group, and The hydrogen atom of the hydroxyl group in the residue may be substituted with a lower alkyl group or a lower acyl group, and n is an integer of 0 to 4).

Further, the above object is (B) general formula (2)

[0012]

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(Wherein R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atom or lower alkyl group, R ⁵ represents hydrogen atom, lower alkyl group or lower acyl group, and n represents an integer of 0 to 4), a solution containing a 2-substituted alcohol represented by β-galactosyl sugar compound and β-galactosidase (EC
It can also be achieved by a method for producing a chromanol glycoside represented by the general formula (1) shown in (A) above, which is characterized by causing 3.2.1.23) to act.

Further, the above object is (C) above (A)
This is achieved by an antioxidant characterized in that the chromanol glycoside represented by the general formula (1) shown below is used as an active ingredient.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION An enzyme used for the enzymatic synthesis of the chromanol glycoside according to the present invention (the above general formula (1)) includes β-galactosidase.
As the β-galactosidase, those derived from almost all sources can be used, and examples thereof include those manufactured by Toyobo Co., Ltd., Oriental Yeast Co., Ltd., and Sigma (SIGMA) Escherichia coli.
li) -derived β-galactosidase, manufactured by Toyobo Co., Ltd. (Aspergillus s)
p. ) -Derived β-galactosidase, SIGMA (SIGM
A) bovine liver, bovine testes, Aspergillus niger, Aspergillus oryzae
ryzae), Saccharomyces fragilis (Sacc
and .beta.-galactosidase derived from jack bean. The amount of enzyme added is, for example,
Escherichia coli (E manufactured by Toyobo Co., Ltd.
5 to 70 when β-galactosidase derived from Scherichia coli) is added to 3 ml of the reaction solution.
U, preferably 10 to 40 U. When the amount of the enzyme is less than 5 U, the catalytic action by the enzyme is small, and when the amount of the enzyme exceeds 70 U, the effect commensurate with the excessive addition cannot be obtained, which is uneconomical. Aspergillus genus (Aspergillus) manufactured by Toyobo Co., Ltd.
p. ) -Derived β-galactosidase is added to 3 ml of the reaction solution, 0.1-20 U, preferably 0.5-15
U. When the amount of the enzyme is less than 0.1 U, the catalytic action by the enzyme is small, and when the amount of the enzyme exceeds 20 U, the effect commensurate with the excessive addition cannot be obtained, which is uneconomical. It should be noted that 1 U of β-galactosidase derived from Escherichia coli mentioned above herein uses o-nitrophenyl-β-D-galactopyranoside as a substrate, and
7.3, which is the amount of enzyme that produces 1 μmol of o-nitrophenol in 1 minute at 37 ° C., 1 U of β-galactosidase derived from the genus Aspergillus described above is used as a substrate for o-nitrophenyl-β-D-galactopyra. 1 μmol / min at 37 ° C and pH 5.0
Is the amount of enzyme that produces o-nitrophenol.

Next, as a condition for synthesizing the chromanol glycoside according to the present invention (the above general formula (1)) by an enzymatic method, first, a 2-substituted alcohol (the above general formula (2)) is β A galactosyl sugar compound (herein,
It is desirable to dissolve it in a solution (also simply referred to as “sugar”), and for that purpose, it is necessary to add an organic solvent capable of dissolving a 2-substituted alcohol having a very low solubility in water. Furthermore, the organic solvent that can be added must be one that can efficiently express the transfer activity of β-galactosidase, and examples thereof include dimethyl sulfoxide, N, N-dimethylformamide, methanol, ethanol, acetone, and acetonitrile. And the like.In order to enhance the transfer activity of β-galactosidase derived from Escherichia coli, dimethyl sulfoxide is preferable, and in order to enhance the transfer activity of β-galactosidase derived from Aspergillus, dimethyl sulfoxide, N, N. -Dimethylformamide, methanol, ethanol and acetonitrile are preferred. When the above-mentioned two types of β-galactosidase are used, the concentration of the organic solvent to be added is 1 to 50 (v / v)%, preferably 5 to 40 (v / v)% in view of reaction efficiency.
When the organic solvent concentration is less than 1 (v / v)%, it is difficult to dissolve the desired 2-substituted alcohol (the above general formula (2)) in the sugar solution, and 50 (v / v) is required. %
If it exceeds, the stability of the enzyme is lowered and the transfer efficiency is deteriorated, which is not preferable.

A 2-substituted alcohol (general formula (2)) used as a starting material when the chromanol glycoside according to the present invention (general formula (1) above) is synthesized by an enzymatic method.
Is a known substance, and can be obtained by methods such as Japanese Examined Patent Publication No. 1-43755 and Japanese Examined Patent Publication No. 1-49135.
Further, for example, in the general formula (2), R ¹ = R ² = R ^{3
_{= R 4 = CH 3, R}} 5 = H, 1 or 2-substituted alcohol with n = 1, the Trolox the (Trolox) such as by heating under reflux with diethyl ether in the presence of lithium aluminum hydride Can be easily obtained. 2-substituted alcohol (the above general formula (2))
It is desirable that the concentration of is a saturated concentration or a concentration close to it in the reaction solution. This is because the maximum yield can be obtained practically (and theoretically).

Furthermore, the types of β-galactosyl sugar compounds used when synthesizing the chromanol glycoside according to the present invention (the above general formula (1)) are lactose (lactose) and o-nitrophenyl- Oligos having sugar residues 2 to 5 with a structure in which β-D-galactopyranoside, p-nitrophenyl-β-D-galactopyranoside and galactose are linked by β-1,4-glycosidic bonds Lactose is preferable, and lactose is preferable in view of price. The concentration of lactose is 5 to 50 (w / v)%, preferably 10 to 50 (w / v)%. Lactose concentration is 5
If it is less than (w / v)%, a high transfer rate from the 2-substituted alcohol to the chromanol glycoside cannot be obtained due to lack of sugar. From the aspect of lactose solubility, 50 (w /
v) It is difficult to prepare a lactose solution in excess of%.

Furthermore, the pH of the reaction system when synthesizing the chromanol glycoside according to the present invention (the above general formula (1)) by the enzymatic method is β-derived from Escherichia coli as described above.
When using galactosidase, it is 5.0 to 7.5, preferably 5.5 to 7.0, and when using β-galactosidase derived from the genus Aspergillus described above, 3.0 to 7.
5, preferably 3.5 to 7.0. When the pH is out of the above range, the above-mentioned enzyme is inactivated, which is not preferable.

Furthermore, the reaction temperature for synthesizing the chromanol glycoside according to the present invention (the above general formula (1)) by an enzymatic method is 20 to 60 ° C. when the above-mentioned β-galactosidase derived from Escherichia coli is used. The temperature is preferably 30 to 50 ° C, and when the β-galactosidase derived from the genus Aspergillus is used, the temperature is 20 to 70 ° C, preferably 30 to 60 ° C. When the reaction temperature is lower than the above range, the activity of the above-mentioned enzyme decreases, and it takes a long time to secure a sufficient transfer rate, which is not economical.
Further, if the reaction temperature exceeds the above range, the above-mentioned enzyme is deactivated, which is not preferable.

The reaction time for synthesizing the chromanol glycoside according to the present invention (the above general formula (1)) by an enzymatic method is 2 to 40 hours when the above-mentioned two types of β-galactosidase are used, It is preferably 10 to 30 hours. When the reaction time is less than 2 hours, the reaction does not reach near equilibrium, and thus it is not possible to obtain a sufficient transfer rate, and when it exceeds 40 hours, further effects commensurate with this cannot be expected. .

However, such conditions in the above-mentioned enzymatic synthesis are slightly affected by the amount of enzyme used.

After completion of the reaction, the reaction solution is subjected to column chromatography using XAD (manufactured by Organo Corporation) as a carrier to obtain a high-purity chromanol glycoside (general formula (1)). .

In the above general formulas (1) and (2), R ¹ , R ² , R ³ and R ⁴ in the formula are the same or different hydrogen atoms or lower alkyl groups, of which lower ones are lower. The alkyl group preferably has 1 carbon atom.
~ 8 alkyl groups, most preferably 1-6 carbon atoms
An alkyl group of, specifically, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a pentyl group, an isopentyl group, a hexyl group, a heptyl group, and an octyl group. Out of
A methyl group and an ethyl group are preferred. Similarly, R ⁵ is a hydrogen atom, a lower alkyl group or a lower acyl group. Among them, the lower alkyl group is preferably an alkyl group having 1 to 8 carbon atoms, and most preferably 1 carbon atom.
To 6 alkyl groups, and specific examples thereof include the same ones as described above. Further, the lower acyl group is an acyl group having 1 to 10 carbon atoms, most preferably an acyl group having 1 to 7 carbon atoms, and specifically, formyl group, acetyl group, propionyl group, butyryl group. Group, valeryl group, caproyl group, and benzoyl group (C ₆ H ₅ CO
-) And the like, and of these, a formyl group, an acetyl group, a propionyl group and a butyryl group are preferable. Further, similarly, the hydrogen atom of the hydroxyl group of the sugar residue may be substituted with a lower alkyl group or a lower acyl group, and the lower alkyl group preferably has 1 to 10 carbon atoms.
An alkyl group having 8 carbon atoms, most preferably an alkyl group having 1 to 6 carbon atoms, and the lower acyl group includes an acyl group having 1 to 10 carbon atoms, and most preferably 1 carbon atom.
~ 7, and specific examples of the alkyl group and the acyl group include the same ones as described above. Furthermore, n
Is an integer of 0 to 4, preferably 1 to 3.

The solubility of the chromanol glycoside (general formula (1)) thus obtained in water is 6-hydroxy-2,5,7 in which the isoprenoid side chain at the 2-position of tocopherol is substituted with a carboxyl group. , 8-Tetramethylchroman-2-carboxylic acid (hereinafter, also simply referred to as Trolox) and 2-substituted alcohol are significantly higher.

Further, the obtained chromanol glycoside (general formula (1)) is equivalent to tocopherol, as shown by the measurement of lipid peroxidation rate of highly unsaturated fatty acid methyl ester in a hexane-isopropyl alcohol solution. It has antioxidant activity. In addition, chromanol glycoside (general formula (1)) has a remarkable inhibitory effect on the oxidation rate as compared with ascorbic acid which is known as a water-soluble antioxidant. For example, when a liposome containing a fat-soluble radical generator inside is prepared by using a biological membrane as a model and the oxidation reaction is promoted, the chromanol glycoside (general formula (1)) is allowed to exist in the aqueous phase. As a result, the oxidation reaction can be suppressed significantly more than ascorbic acid or the like. In addition, chromanol glycosides (general formula (1))
Since it has a chroman ring, it also has the ability to eliminate various active oxygen such as singlet oxygen.

The resulting chromanol glycoside (general formula (1)) is an injectable agent effective for various diseases associated with active oxygen due to its high solubility (water solubility) and excellent antioxidant activity. Alternatively, it can be used as an active ingredient of an antioxidant such as a solubilizer such as an internal medicine, and in this case, an aqueous solution of the chromanol glycoside (general formula (1)) can be directly used as an antioxidant. The solution may contain other active ingredients which are not reactive with each other in an appropriate amount.

[0028]

EXAMPLES Next, the present invention will be explained with reference to Examples and the like.
It goes without saying that the scope of the present invention is not limited by these.

Reference Example 1 (1) Activity measurement of β-galactosidase derived from Escherichia coli 2.5 ml of 100 mM phosphate buffer (pH 7.3),
3.36M mercaptoethanol solution 0.1 ml, 3
0.1 ml of 0 mM MgCl ₂ solution (pH 7.3), 3
0.2 ml of 4 mM o-nitrophenyl-β-D-galactopyranoside solution was placed in a cuvette (d = 1.0 cm) and set in a spectrophotometer controlled at 37 ° C. 5
After incubating for a minute, 0.1 ml of the enzyme solution was added and the change in absorbance at 410 nm was determined. Note that 1 U was the amount of enzyme that produced 1 μmol of o-nitrophenol per minute under the above conditions.

(2) Activity measurement of β-galactosidase derived from Aspergillus genus 2 in 1.0 ml of 100 mM acetate buffer (pH 5.0)
0.2 ml of 0 mM o-nitrophenyl-β-D-galactopyranoside solution was added, and the mixture was incubated at 37 ° C for 5 minutes, then 0.5 ml of the enzyme solution was added, and incubated at the same temperature for 15 minutes. And 0.2
The reaction was stopped by adding 2.0 ml of M Na ₂ CO ₃ solution, and the absorbance at 410 nm was measured. Note that 1 U was the amount of enzyme that produced 1 μmol of o-nitrophenol per minute under the above conditions.

Example 1 40 prepared with 50 mM phosphate buffer (pH 6.5)
Formula (3) of 5 (w / v)% prepared with dimethyl sulfoxide to 160 ml of (w / v)% lactose solution

[0032]

[Chemical 6]

32 m of 2-substituted alcohol solution represented by
1 and 1600 U of β-galactosidase derived from Escherichia coli manufactured by Toyobo Co., Ltd. were added and reacted at 40 ° C. for 20 hours. At this time, the conversion rate of the 2-substituted alcohol to the chromanol glycoside was about 20% in terms of molar ratio.

This reaction solution was applied to a column of XAD-4 (manufactured by Organo Co.) equilibrated with a 30% methanol solution, and the non-adsorbed substance was eluted with 30% methanol and then 80%.
The chromanol glycoside was eluted with methanol. Next, the obtained chromanol glycoside fraction was subjected to silica gel column chromatography (ethyl acetate: methanol = 5: 1, v
/ V) The formula (4), which is a highly pure chromanol glycoside by treatment

[0035]

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2- (α-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman represented by
6-ol [2- (α-D-glucopyranosyl) methyl-2,5,7,8-
tetramethylchroman-6-ol] (n = 1 in the general formula (1), R
¹ = R ² = R ³ = R ⁴ = CH ₃ , R ⁵ = H) was obtained (about 300 mg).

The infrared absorption spectrum of the obtained chromanol glycoside represented by the above formula (4) is shown in FIG.

The results of ¹ H-NMR, ¹³ C-NMR, mass spectrometry and optical rotation of the chromanol glycoside represented by the above formula (4) are as follows.

¹ H-NMR δ (270 MHz, DM
SO-d ₆₎ 1.21 and 1.24 (s, 3H),
1.67 to 1.73 (m, 1H), 1.90 to 1.
93 (m, 1H), 1.98 (s, 3H), 2.02
(S, 3H), 2.05 (s, 3H), 2.50 (br
oad t, 2H) 3.17 to 4.78 (m, 13
H), 7.38 (s, 1H) ¹³ C-NMR δ (67.8 MHz, DMSO-d ₆ ,
Proton decoupling spectrum) 11.7, 1
1.8, 12.6, 19.7 and 19.8, 22.2
And 22.5, 28.0 and 28.1, 60.3 and 60.4, 68.1, 70.5, 73.1 and 7
3.2, 73.4, 73.9 and 74.0, 75.1
And 75.2, 104.0 and 104.2, 11
6.8, 120.2, 120.8, 122.5, 14
4.2, 145.2 Mass spectrum (FAB) m / z 398 (Molecular ion peak)

[0040]

[Outside 1]

Example 2 40 prepared with 50 mM acetate buffer (pH 4.5)
Formula (3) of 5 (w / v)% prepared with dimethyl sulfoxide to 160 ml of (w / v)% lactose solution

[0042]

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32 m of 2-substituted alcohol solution represented by
1- and 1000 U β-derived from Aspergillus sp. manufactured by Toyobo Co., Ltd.
Galactosidase was added and reacted at 50 ° C. for 20 hours. At this time, the conversion rate of 2-substituted alcohol to chromanol glycoside was about 7% in terms of molar ratio.

This reaction solution was applied to an XAD-4 (manufactured by Organo Co.) column equilibrated with a 30% methanol solution, and the non-adsorbed material was eluted with 30% methanol, and then 80%.
The chromanol glycoside was eluted with methanol. Next, the obtained chromanol glycoside fraction was subjected to silica gel column chromatography (ethyl acetate: methanol = 5: 1, v
/ V) which is a high-purity chromanol glycoside represented by the formula (5)

[0045]

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2- (α-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman represented by
6-ol [2- (α-D-glucopyranosyl) methyl-2,5,7,8-
tetramethylchroman-6-ol] (n = 1 in the general formula (1), R
¹ = R ² = R ³ = R ⁴ = CH ₃ and R ⁵ = H)
About 90 mg was obtained.

The infrared absorption spectrum of the obtained chromanol glycoside represented by the above formula (5) is shown in FIG.

The results of ¹ H-NMR, ¹³ C-NMR, mass spectrometry and optical rotation of the chromanol glycoside represented by the above formula (5) are as follows.

¹ H-NMR δ (270 MHz, DM
SO-d ₆₎ 1.21 and 1.24 (s, 3H),
1.67 to 1.73 (m, 1H), 1.90 to 1.
93 (m, 1H), 1.98 (s, 3H), 2.02
(S, 3H), 2.05 (s, 3H), 2.50 (br
oad t, 2H) 3.17 to 4.78 (m, 13
H), 7.38 (s, 1H) ¹³ C-NMR δ (67.8 MHz, DMSO-d ₆ ,
Proton decoupling spectrum) 11.7, 1
1.8, 12.6, 19.7 and 19.8, 22.2
And 22.5, 28.0 and 28.1, 60.3 and 60.4, 68.1, 70.5, 73.1 and 7
3.2, 73.4, 73.9 and 74.0, 75.1
And 75.2, 104.0 and 104.2, 11
6.8, 120.2, 120.8, 122.5, 14
4.2, 145.2 Mass spectrum (FAB) m / z 398 (Molecular ion peak)

[0050]

[Outside 2]

Example 3 The antioxidative activity of the chromanol glycosides represented by the formulas (4) and (5) obtained in Examples 1 and 2 was suppressed by suppressing the radical chain autooxidation of methyl linoleate. evaluated. 132 μmol of methyl linoleate, 1
6.5 μmol of fat-soluble radical generator (2,2′-azobis (2,4-dimethylvaleronitrile)), 0.1
Incubate 1.1 ml of a hexane / isopropyl alcohol (1: 1, v / v) solution consisting of μmol of the chromanol glycoside represented by the formula (4) or the formula (5), butylhydroxytoluene or α-tocopherol at 37 ° C. Then, it was sampled with time, and the amount of methyl linoleate hydroperoxide produced was measured by high performance liquid chromatography. As shown in FIG. 3, the antioxidant activity of the chromanol glycosides represented by formulas (4) and (5) is higher than that of butylhydroxytoluene and is equivalent to that of α-tocopherol.

Example 4 The antioxidative activity of the chromanol glycosides represented by the formulas (4) and (5) obtained in Examples 1 and 2 was suppressed by suppressing the radical chain autoxidation reaction of multilamellar liposomes. evaluated. 5.5 μmol egg yolk phosphatidylcholine, 1.1 μmol fat-soluble radical generator (2,2 ′)
-Azobis (2,4-dimethylvaleronitrile)
5 mM diethylenetriamine-N, N, N ', N ",
N "-pentaacetic acid (diethylethylene
-N, N, N ', N ", N" -pentaacetic
acid-containing 10 mM Tris-HCl buffer (p
H7.4) was suspended in 1 ml to prepare a multilamellar liposome, to which 0.1 ml of 100 μM of the chromanol glycoside represented by the formula (4) or the formula (5) or ascorbic acid was added. It was Then, the mixture was incubated at 50 ° C., sampled over time, and the amount of phosphatidylcholine hydroperoxide produced was measured by high performance liquid chromatography.

As shown in FIG. 4, the chromanol glycosides represented by the formulas (4) and (5) are clearly more effective antioxidants than ascorbic acid.

Example 5 The solubility of the chromanol glycosides represented by the formulas (4) and (5) obtained in Examples 1 and 2 in water was evaluated. An excess amount of the chromanol glycoside represented by the formula (4) or the formula (5) is added to 1 ml of water, incubated at 25 ° C., stirred for 20 hours (200 rpm),
Transfer the solution to Sunprep C02-LG and centrifuge (41
(00 × g, 10 minutes, 20 ° C.) to remove insoluble matter, and the amount of chromanol glycoside dissolved in the aqueous solution was measured by high performance liquid chromatography. For comparison, Trolox (Trolox, manufactured by Aldrich Chemical Company, Inc.) and 2-substituted alcohol represented by the formula (3) were also evaluated in the same manner.

[0055]

[Table 1]

As shown in Table 1, equations (4) and (5)
It was clarified that the chromanol glycoside shown in Fig. 3 has an excellent solubility in water.

Example 6 Mouse T lymphoma strain EL-4 cells were transferred to RPMI-164.
0 + 10% fetal calf serum + HEDES buffer (25 mM)
37 in a system culture solution (hereinafter abbreviated as "complete culture solution").
C., subculture in 5% CO ₂ atmosphere, cell density 2 ×
It was adjusted to 10 ⁵ cells / ml. The supernatant of the EL-4 cell culture broth thus cultivated was removed, and the chromanol glycoside (general formula (4)) solution prepared in the same manner as in Example 1 was added to the final solution in the complete culture broth. The cell culture was performed under the same conditions as above for 30 minutes until irradiation with X-rays, in which the concentration was adjusted to 1 mM. After culturing in a culture solution containing chromanol glycoside for a predetermined time, it was irradiated with 3 Gy of radiation at a dose rate of 0.92 Gy / min. Immediately after the completion of irradiation, the cells are spun down (400 g × 5 minutes), and R
The cells were washed twice with PMI-1640, resuspended in complete culture medium, and cultured. To this, a solution of cytochalasin B in DMSO (concentration of 2 mg / ml) was added so that the final concentration was 3 μg / ml, and after 20 hours of culture, the frequency of micronucleus-bearing cells in the binuclear cells (micronucleus induction frequency) was measured. It was measured and used as a scale representing the frequency of radiation damage to cells. In addition, the micronucleus induction inhibitory rate was calculated from the following formula based on the micronucleus induction frequency (Comparative Example 1) obtained by repeating the above operation except that the treatment concentration of the chromanol glycoside was 0 mM. On this occasion,
The above cells were subjected to radiation irradiation experiments in groups of 4 to 5 and the results were expressed as the average value of these. Table 2 shows the results.

[0058]

[Equation 1]

[0059]

[Table 2]

As shown in Table 2, the micronucleus induction frequency was not treated when treated with the chromanol glycoside represented by the general formula (4) (Example 6) (Comparative Example 1).
Compared with the case, it was significantly smaller, which indicates that the chromanol glycoside of the present invention has an excellent radioprotective effect.

[0061]

INDUSTRIAL APPLICABILITY The chromanol glycoside of the present invention (general formula (1)) is novel and exhibits a different stability in vivo than that of a known glucose-type glycoside in vivo and a way of being taken up by living tissues. Galactose type glycoside,
It has significantly higher solubility (about 1000 times or more) than Trolox and 2-substituted alcohols, and exhibits an antioxidant activity effect superior to that of vitamin C.

Next, the galactose type glycoside of the present invention is
The enzyme can be efficiently synthesized by using an enzyme that is stable even in the presence of a high concentration of organic solvent and has a high transfer activity.

Further, by utilizing the excellent characteristics of galactose-type glycosides which are inexpensively supplied by the production method of the present invention, they can be used as active ingredients of water-soluble antioxidants applicable to foods, cosmetics, pharmaceuticals and the like. Can be used.

Furthermore, the galactose-type glycoside of the present invention has an excellent radioprotective action.

[Brief description of drawings]

1 is an infrared absorption spectrum of the chromanol glycoside represented by the formula (4) obtained in Example 1. FIG.

FIG. 2 is an infrared absorption spectrum of the chromanol glycoside represented by the formula (5) obtained in Example 2.

FIG. 3 shows the antioxidant activity of the chromanol glycosides represented by formulas (4) and (5), butylhydroxytoluene, and α-tocopherol obtained in Examples 1 and 2 as a radical chain of methyl linoleate. It is a graph which shows the result of having measured the production amount of methyl linoleic acid hydroperoxide over time so that it may evaluate by suppression of an autoxidation reaction.

FIG. 4 shows that the antioxidant activity of the chromanol glycosides and ascorbic acid represented by the formulas (4) and (5) obtained in Examples 1 and 2 is suppressed by the radical chain autoxidation reaction of multilamellar liposomes. 5 is a graph showing the results of measuring the amount of phosphatidylcholine hydroperoxide produced over time for evaluation by.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12P 19/60 C12P 19/60 // A23L 3/3562 A23L 3/3562

Claims (3)

[Claims] 1. A compound of the general formula (1) (In the formula, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atom or lower alkyl group,
⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group, the hydrogen atom of the hydroxyl group in the sugar residue may be substituted with a lower alkyl group or a lower acyl group, and n is an integer of 0 to 4 ) A chromanol glycoside represented by 2. A compound of the general formula (2) (In the formula, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atom or lower alkyl group,
⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group, and n is an integer of 0 to 4) 2
-Β-galactosidase (EC 3.2.1.) In a solution containing a substituted alcohol and a β-galactosyl sugar compound.
23) is acted on, the method for producing a chromanol glycoside represented by the general formula (1) according to claim 1. 3. An antioxidant comprising the chromanol glycoside represented by the general formula (1) according to claim 1 as an active ingredient.