JPH07179489A - New polyphenol glycoside - Google Patents

Abstract

PURPOSE:To obtain a new polyphenol glycoside having excellent stability to light, pH and heat and good taste and useful for foods, cosmetics, pharmaceuticals, etc., by reacting a polyphenol mixture and starch, etc., with a cyclodextrin glucanotransferase. CONSTITUTION:This new polyphenol glycoside having excellent stability to light, pH and heat and good taste and expressed by formula (X is D- glucopyranose residue or a maltooligosaccharide residue having a degree of polymerization of 2-10; R1 to R5 each is H or same as X; Y is R4 or gallic acid group; Z is H or OR5) is produced by reacting a material selected from gallocatechin, catechin-3-O-gallate, gallocatechin-3-O-gallate and a polyphenol mixture containing at least two kinds of the above compounds and a material selected from dextrin, cyclodextrin, starch and a mixture containing at least two kinds of the above compounds with a cyclodextrin glucanotransferase originated from Bacillus thermophilus.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a polyphenol glycoside, and more specifically to a novel polyphenol glycoside having improved stability and taste by glycosylation, a method for producing the same, and a polyphenol glycoside. It relates to a composition.

[0002]

2. Description of the Related Art Polyphenols have an inhibitory effect on cholesterol elevation (Japanese Patent Publication No. 2-44449), an antibacterial action (Japanese Patent Application Laid-Open No. 2-276562), an antioxidant action (Japanese Patent Publication No. 1-44434), and antitumor. Action (JP-A-60-
No. 190719), an inhibitory effect on blood pressure increase and an enzyme activity inhibitory effect (JP-A-3-133928), and the active ingredients thereof are gallocatechin, catechin-3-O-gallate and gallocatechin-3-O. −
It is known to be one of gallates or a mixture thereof.

However, these substances are unstable to temperature and light, and not only have the property of changing while coloring, but are also astringent components having strong astringent properties. Taste is significantly impaired, which is a major drawback in considering its use in various fields including food. Furthermore, these polyphenols act as inhibitors of phenol oxidase, which is involved in the production of pigments and browning of foods such as vegetables and fruit juices, but on the other hand, the polyphenols themselves become substrates for these enzymes and become colored. Has the drawback.

Recently, a method has been developed in which catechin glycoside is used as an external agent for the skin as an inhibitor of tyrosinase involved in the formation of melanin pigment (JP-A-4-273890). However, catechin is converted into gallocatechin, catechin-3-O-gallate or gallocatechin-3-O-gallate in the physiologically active actions such as the above-mentioned cholesterol elevation inhibitory action, antibacterial action, antioxidant action, antitumor action and blood pressure elevation inhibitory action. On the other hand, its physiological activity is not so satisfactory that it is extremely weak and, in some cases, it has no activity.

Therefore, it is not possible to expect strong physiological activity in catechin glycosides. Further, a method for producing a catechin glycoside having easily soluble property and color stability by allowing sucrose phosphorylase to act on a mixed solution of catechins and glucose-1-phosphate or sucrose (Japanese Patent Laid-Open No. HEI-5 (1999)). No. 176786) was developed, and (−)-epicatechin gallate, (−)-epigallocatechin and (−)-epigallocatechin gallate are also exemplified. However, in this method, 1
Since only the glucose residue of the molecule is bound, it is hard to say that the strong astringency and astringency of these substances have been sufficiently improved, and because the binding position of this glucose residue is at the 3'position, In particular, it is doubtful whether the stability can be sufficiently improved for epigallocatechin and epigallocatechin gallate type substances.

[0006]

The object of the present invention is to improve the light, pH or thermal stability of polyphenols having a strong physiological activity and prevent the coloring (change to other compounds) by phenol oxidase, In addition, by improving the taste, it is possible to fully utilize the original functionality of these substances in a wide range of fields such as foods, cosmetics, and pharmaceuticals, and to more effectively demonstrate the inhibitory activity against phenol oxidase. Another object of the present invention is to provide a novel polyphenol compound which is effective for the purpose.

[0007]

Means for Solving the Problems The present inventors have glycosylated the 4′-position of polyphenols having a strong physiological activity, are excellent in light, pH and heat stability and have sufficient taste. As a result of earnest research on a method for improving and more effectively exhibiting the inhibitory ability against phenol oxidase, these polyphenols and dextrin, cyclodextrin, starch or a mixture thereof were derived from Bacillus stearothermophilus. It was found that glycosylation can be carried out specifically at the 4'-position of polyphenols by the action of cyclodextrin glucanotransferase. The present invention has been completed based on such findings.

That is, the present invention uses the general formula 1

[0009]

[Chemical 5]

(In the formula, X represents a D-glucopyranose residue or a maltooligosaccharide residue having a degree of polymerization of 2 to 10, and Y represents R.
₄ or gallic acid group, Z is hydrogen or OR ₅
, R _{1 to} R ₅ are each independently hydrogen, D-
A glucopyranose residue or a maltooligosaccharide residue having a degree of polymerization of 2 to 10 is shown. ) A novel polyphenol glycoside and (A) gallocatechin, catechin-3-O-
Any one of a gallate, a gallocatechin-3-O-gallate and a polyphenol mixture containing at least two kinds of these compounds, and (B) a dextrin, a cyclodextrin, a starch and a mixture containing at least two kinds of these compounds. And a method for producing the novel polyphenol glycoside characterized by obtaining a polyphenol glycoside by reacting a cyclodextrin glucanotransferase derived from Bacillus stearothermophilus with the novel polyphenol glycoside The present invention provides a composition having a phenol oxidase activity-inhibiting activity containing a body and a composition containing the novel polyphenol glycoside and having excellent stability and taste.

The basic skeleton of the polyphenol in the present invention is shown in the general formula 5, and in this structural formula, the 2-position and 3-position of the benzopyran ring may be either R-configuration or S-configuration. That is, in gallocatechin, (+)
Alternatively, for (-)-gallocatechin and (+) or (-)-epigallocatechin, catechin gallate, (+) or (-)-catechin gallate and (+)
Alternatively, for (−)-epicatechin gallate and gallocatechin gallate, (+) or (−)-gallocatechin gallate and (+) or (−)-epigallocatechin gallate can be mentioned.

[0012]

[Chemical 6]

The method for producing the polyphenol glycoside represented by the general formula 1 is not particularly limited,
Chemical synthesis methods, plant tissue culture cell methods, methods using microbial cells, etc. are conceivable, but in order to significantly improve the stability and taste of polyphenols, one or more of these polyphenols are used. Dextrin, cyclodextrin, starch, or a mixture thereof is added to the mixture of Bacillus stearothermophilus (Baci
(llus stearothermophilus) -derived cyclodextrin glucanotransferase is preferable.

As the enzyme reaction conditions, the reaction pH is 3 to
8, preferably pH 4.5-6.0, the reaction temperature is 2
0 to 70 ° C., preferably 40 to 65 ° C.,
0.1-20 w / w polyphenols as substrate concentration
% (Hereinafter, w / w% is simply abbreviated as%) unless otherwise specified), preferably 2 to 10%, and dextrin, cyclodextrin, starch or a mixture thereof is 1 to 40%, preferably It is preferable to use a reaction solution containing 10 to 30%. The amount of enzyme and the reaction time can be optimally set according to the above reaction conditions.

The reaction solution thus produced with the polyphenol glycoside can be used as it is, but it can also be converted into a glucosyl sugar compound by further performing a glucoamylase treatment. These reaction solutions can be used after syrup or pulverization, if necessary. When a higher-purity polyphenol glycoside is desired, a porous synthetic adsorbent such as DIAION HP manufactured by Mitsubishi Kasei Kogyo Co., Ltd.
-10, Diaion HP-20, Diaion HP-
40 and Rohm & Haas brand names, Amberlite XAD-1, Amberlite XA
D-4, Amberlite X AD-7, Amberlite X
It may be purified using AD-8 or the like by utilizing the difference in adsorptivity between the polyphenol glycoside and the contaminant. For example, when it is necessary to separate free saccharides from polyphenol glycosides or polyphenols, the reaction solution is passed through a column filled with a porous synthetic adsorbent to adsorb polyphenol compounds and free saccharides. Elute without. Then, the adsorbed polyphenols such as polyphenol glycosides are eluted with a lower alcohol solution, for example, 50 v / v% ethanol aqueous solution, and the eluate is concentrated to form a syrup, and further dried and powdered for collection. . Furthermore, when it is necessary to separate polyphenol glycosides and unreacted polyphenols, the unreacted polyphenols are transferred to an organic solvent phase such as ethyl acetate by utilizing the difference in polarities of these compounds. It can also be removed. Furthermore, if necessary, a specific fraction of the polyphenol glycoside can be collected and used by a method such as chromatography.

Unlike the conventional polyphenols, the polyphenol glycoside of the present invention obtained as described above has little bitterness, astringency, harshness such as acridity and astringency, and light, pH and It is also excellent in heat stability and can be freely used in a wide range of fields such as foods, quasi-drugs, cosmetics, and pharmaceuticals, irrespective of its degree of purification or purity, or as it is contained together with other materials.

The polyphenol glycoside of the present invention is
Since it easily returns to the original polyphenols by the action of α-amylase, α-glucosidase, etc. in the body, without worrying about its drug efficacy, the original polyphenols,
For example, because it can exert physiologically active functions such as cholesterol elevation inhibitory effect and in vivo antioxidant effect,
It can be advantageously used as a health maintenance food and a health recovery food.

When using the polyphenol glycoside of the present invention, for example, seasonings, Japanese confectionery, Western confectionery, ice confectionery,
Syrups, processed fruits, processed vegetables, pickles, meat products, fish products, delicacies, cans / bottles, alcoholic beverages, soft drinks, foods such as instant food and drink, tobacco, toothpaste, lipstick, lip Cream, oral medication, troche, liver oil drop, mouthwash, mouthwash, mouthwash, various solid forms,
It is desirable that 0.01% or more, and preferably 0.1% or more, of the composition such as pasty or liquid luxury products, cosmetics, and pharmaceuticals is contained.

[0019]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples. Example 1 100 g of α-cyclodextrin (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) and 5 g of (−)-epigallocatechin (manufactured by Mitsui Norin Co., Ltd.) were mixed with 10 mM calcium chloride.
After dissolving in 500 ml of 1 M acetate buffer (pH 5.5), 1000 units of cyclodextrin glucanotransferase (manufactured by Hayashibara Biochemical Laboratories, Inc.) derived from Bacillus stearothermophilus was added per α-cyclodextrin glam, 50 The reaction was performed at 24 ° C. for 24 hours.

Then, after removing the enzyme by UF membrane filtration,
Add 100 M of 0.1 M acetate buffer (pH 4.5) to the reaction solution.
0 ml and Rhizopus nibeus-derived glucoamylase (manufactured by Seikagaku Corporation) were added in an amount of 50 units per gram of solid matter, and the reaction was carried out at 40 ° C for 20 hours. After removing the enzyme using a UF membrane, the resulting reaction solution was equilibrated with water, Diaion HP-10 (manufactured by Mitsubishi Kasei Co., Ltd.).
It was adsorbed on a column filled with 1000 ml. After washing the column with 3000 ml of deionized water, the column was eluted with 1000 ml of 50 v / v% methanol aqueous solution and then 2000 ml of 100 v / v% methanol to collect the adsorbed fraction.
The eluate is concentrated under reduced pressure to remove methanol and
Dissolved in distilled water. 500 ml of ethyl acetate in this solution
After washing 7 times with, the unreacted (−)-epigallocatechin was removed. Then, this aqueous solution fraction was added to Diaion HP-
20 (Mitsubishi Chemical Industries Co., Ltd.) SS column 2500m
The mixture was developed to 1 and thoroughly washed with distilled water, and then eluted with 8000 ml of 25 v / v% methanol aqueous solution to fractionate 2000 ml each. Furthermore, 5 v of 50 v / v% methanol solution
Eluted at. These fractions were analyzed by HPLC to obtain fractions A and B containing epigallocatechin glycoside.

Preparative HPLC for these fractions
Separation was carried out, and from the A fraction, epigallocatechin glycoside 1,
Epigallocatechin glycoside 2 was obtained from the B fraction. These glycosides showed a single peak in HPLC analysis.

HPLC condition for analysis Column: Capsule pack AG-120 ODS S-5 (4.6 × 2)
Mobile phase: Acetonitrile: Ethyl acetate: 0.05 v / v
% Phosphoric acid water = 12: 1: 87 Flow rate: 0.7 ml / min Detection: UV 280 nm (40 ° C.) Preparative HPLC condition Column: Capsule pack ODS AG-120 S-5 (20 × 25
Mobile phase: Fraction A; 10v containing 0.05v / v% phosphoric acid water
/ V% methanol aqueous solution B fraction; 15 v / v% methanol aqueous solution containing 0.05 v / v% phosphoric acid water Flow rate: 8 ml / min Detection: UV 280 nm (room temperature)

The yield of each epigallocatechin glycoside was 282 mg for 1 and 590 mg for 2, respectively. As a result of analyzing these glycosides 1 and 2 by instrumental analysis by NMR and MS, it was determined to have the following structure.
The results of instrumental analysis are also listed. For reference, the ¹ H-NMR spectrum, ¹³ C-NMR spectrum and mass spectrum of epigallocatechin glycoside 2 are shown in FIGS. 1, 2 and 3, respectively.

[0024]

[Chemical 7]

(-)-Epigallocatechin-7,4'-O
-Di-α-D-glucopyranoside (epigallocatechin glycoside 1) ¹ H-NMR (ppm, methanol (d ₄ ) -D ₂ O) 2.80 (1H, dd, H-4a), 2.
87 (1H, dd, H-4b), 3.45-3.92 (12H, ring H, Glc × 2), 4.21
(1H, m, H-3), 4.83 (1H, s, H-2), 5.36 (1H, d, H-1 ''', J
_{H-1 ''',H-2'''} = 4 Hz), 5.41 (1H, d, H-1 ", J
_{H-1 ''',H-2'''} = 4 Hz), 6.26 (1H, d, H-8), 6.31 (1H, d, H-
6), 6.76 (1H, d, H-6 '), 6.98 (1H, d, H-2') ¹³ C-NMR (ppm, methanol (d ₄ ) -D ₂ O) 28.5 (C-4), 60.5 (C-
6 ", C-6 '''), 64.5 (C-3), 69.8 (C-4", C-4'''), 71.6 (C-
2 "), 72.0 (C-2 '''), 73.0 (C-3"), 73.1 (C-3'''), 73.5
(C-5 "), 73.6 (C-5 '''), 78.2 (C-2), 95.6 (C-8), 96.7 (C
-6), 98.0 (C-1 "), 100.6 (C-1 '''), 101.5 (C-4a), 107.9
(C-2 '), 109.8 (C-6'), 129.6 (C-1 '), 134.9 (C-4'), 14
5.1 (C-3 '), 145.3 (C-5'), 155.6 (C-8a), 156.3 (C-7), 1
56.5 (C-5) FAB-MS (pos.) M / z = 631 = (M + H) ⁺ , molecular weight 630

[0026]

[Chemical 8]

(-)-Epigallocatechin-4'-O-α
-D-glucopyranoside (epigallocatechin glycoside 2) ¹ H-NMR (ppm, methanol (d ₄ ) -D ₂ O) 2.73 (1H, dd, H-4a), 2.
85 (1H, dd, H-4b), 3.45 (1H, dd, H-4 "), 3.57 (1H, dd, H-
2 "), 3.78 (3H, m, H-3", 6 "), 3.88 (1H, m, H-5"), 4.18 (1H,
m, H-3), 4.79 (1H, s, H-2), 5.35 (1H, d, H-1 ", J
_{H-1 ''',H-2'''} = 3.2 Hz), 5.91 (1H, d, H-8), 5.93 (1H, d,
H-6), 6.75 (1H, d, H-6 '), 6.95 (1H, d, H-2') ¹³ C-NMR (ppm, methanol (d ₄ ) -D ₂ O) 29.3 (C-4 ), 62.4 (C-
6 "), 67.4 (C-3), 71.4 (C-4"), 73.5 (C-2 "), 74.4 (C-
3 "), 74.9 (C-5"), 79.3 (C-2), 95.9 (C-8), 96.4 (C-6),
100.0 (C-4a), 101.2 (C-1 "), 108.9 (C-2 '), 110.4 (C-
6 '), 131.6 (C-1'), 135.9 (C-4 '), 146.7 (C-3', 5 '), 15
7.2 (C-8a), 157.6 (C-7), 158.0 (C-5) FAB-MS (pos.) M / z = 469 = (M + H) ⁺ , molecular weight 468

Example 2 (-)-Epicatechin gallate (manufactured by Mitsui Norin Co., Ltd.)
The enzyme reaction was carried out according to the method shown in Example 1 using, and unreacted sugar was removed using Diaion HP-10 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.). The adsorbed fraction is eluted with an aqueous methanol solution, concentrated under reduced pressure, and then collected by HPL.
It was subjected to C, and epicatechin gallate glycoside 1 (1.14
g) was obtained. This glycoside showed a single peak in the HPLC analysis under the above conditions (Example 1).

Preparative HPLC conditions Column: Capsule pack AG-120 ODS S-5 (50 × 50
Mobile phase: 20 v / v% aqueous methanol solution containing 0.05 v / v% phosphoric acid water; 40 min 23 v / v% aqueous methanol solution containing 0.05 v / v% phosphoric acid water; 90 min Flow rate: 120 ml / min Detection: UV 280nm (room temperature)

As a result of analyzing this glycoside by instrumental analysis by NMR and MS, it was determined to have the following structure. The results of instrumental analysis are also listed.

[0031]

[Chemical 9]

(-)-Epicatechin gallate-4'-O
-Α-D-glucopyranoside (epicatechin gallate glycoside 1) ¹ H-NMR (ppm, DMSO-d ₆ ) 2.71 (1H, dd, H-4a), 2.94 (1H, dd,
H-4b), 3.21-3.61 (6H, ring H, Glc), 5.02 (1H, d, H-1 ''',
J _{H-1 ''',H-2'''} = 3.8 Hz), 5.07 (1H, s, H-2), 5.34 (1H,
m, H-3), 5.83 (1H, d, H-8), 5.94 (1H, d, H-6), 6.74 (1H, d,
H-5 '), 6.84 (2H, s, H-2 ", 6"), 7.00 (1H, dd, H-6'), 7.37
(1H, d, H-2 ') ¹³ C-NMR (ppm, DMSO-d ₆ ) 25.5 (C-4), 60.2 (C-6'''), 68.
0 (C-3), 69.4 (C-4 '''), 71.9 (C-2'''), 73.0 (C-5 '''), 7
3.2 (C-3 '''), 76.3 (C-2), 94.3 (C-8), 95.5 (C-6), 97.0
(C-4a), 100.0 (C-1 '''), 108.5 (C-2 ", 6"), 115.1 (C-
5 '), 116.0 (C-2'), 118.9 (C-1 "), 121.1 (C-6 '), 129.5 (C
-1 '), 138.5 (C-4 "), 144.5 (C-4'), 145.3 (C-3", 5 "), 14
6.6 (C-3 '), 155.4 (C-8a), 156.4 (C-7), 156.4 (C-5), 16
5.0 (C-7 ") FAB-MS (pos.) M / z = 605 = (M + H) ⁺ , molecular weight 604

Example 3 (−)-Epigallocatechin gallate (manufactured by Mitsui Norin Co., Ltd.) was used to carry out the same procedure as in Example 2 to give epigallocatechin gallate glycosides 1 (375 mg) and 2
(960 mg) was obtained. As a result of analyzing these glycosides by instrumental analysis by NMR and MS, it was determined to have the following structure. The results of instrumental analysis are also listed.

[0034]

[Chemical 10]

(-)-Epigallocatechin gallate-7,
4′-O-di-α-D-glucopyranoside (epigallocatechin gallate glycoside 1) ¹ H-NMR (ppm, methanol (d ₄ ) -D ₂ O) 2.94 (1H, dd, H-4a), 3.
08 (1H, dd, H-4b), 3.43-3.92 (12H, ring H, Glc × 2), 4.87
(1H, d, H-1 "", J _{H-1 ""} , _{H-2 ""} = 4 Hz), 5.06 (1H, s, H-
2), 5.50 (1H, d, H-1 '", J _{H-1''', H-2 '''} = 3.5 Hz), 5.51
(1H, m, H-3), 6.38 (1H, d, H-8), 6.38 (1H, d, H-6), 6.71 (1
H, d, H-6 '), 7.02 (2H, s, H-2 ", 6"), 7.13 (1H, d, H-2') ¹³ C-NMR (ppm, methanol (d ₄ ) -D ₂ O) 27.7 (C-4), 62.7 (C-
6`` '), 63.1 (C-6 ""), 70.5 (C-3), 71.7 (C-4'''), 72.3
(C-4 ""), 74.1 (C-2 '''), 74.2 (C-2 ""), 74.7 (C-3'''),
75.0 (C-3 ""), 75.7 (C-5 '''), 75.7 (C-5 ""), 79.6 (C-2),
98.4 (C-8), 99.0 (C-6), 99.9 (C-4a), 101.9 (C-1 '''),
102.9 (C-1 ""), 109.5 (C-2 '), 111.1 (C-6'), 111.2 (C-2 ",
6 "), 122.0 (C-1"), 131.4 (C-1 '), 136.7 (C-4 "), 140.8
(C-4 '), 147.1 (C-3 ", 5"), 147.3 (C-3'), 147.8 (C-5 '),
157.9 (C-8a), 158.5 (C-7), 158.9 (C-5), 168.1 (C-7 ") FAB-MS (pos.) M / z = 783 = (M + H) ⁺ , molecular weight 782

[0036]

[Chemical 11]

(-)-Epigallocatechin gallate-4 '
-O-α-D-glucopyranoside (epigallocatechinga
Rate glycoside 2)¹ H-NMR (ppm, methanol (d_Four) -D₂O) 2.87 (1H, dd, H-4a), 2.
99 (1H, dd, H-4b), 3.42 (1H, dd, H-4 '' '), 3.51 (1H, t, H-
2 '' '), 3.68 (1H, dt, H-5' ''), 3.81 (1H, t, H-3 '' '), 3.85
(2H, m, H-6 '' '), 4.90 (1H, d, H-1' '', J_{H-1 ''',H-2'''}= 4
 Hz), 5.94 (1H, d, H-8), 5.95 (1H, d, H-6), 6.64 (1H, d, H-
6 '), 7.00 (2H, s, H-2 ", 6"), 7.11 (1H, d, H-2') ¹³ C-NMR (ppm, methanol (d_Four) -D₂O) 27.8 (C-4), 63.0 (C-
6 '' '), 70.2 (C-3), 71.0 (C-4' ''), 74.2 (C-2 '' '), 74.7
(C-3 '' '), 75.5 (C-5' ''), 80.1 (C-2), 98.6 (C-8), 99.3 (C
-6), 100.2 (C-4a), 101.8 (C-1 '' '), 109.8 (C-2'), 111.3
(C-6 '), 111.5 (C-2 ", 6"), 122.1 (C-1 "), 131.5 (C-1'),
136.5 (C-4 "), 141.0 (C-4 '), 147.1 (C-3", 5 "), 147.1 (C-
3 '), 147.5 (C-5'), 158.0 (C-8a), 158.5 (C-7), 158.6 (C
-5), 168.0 (C-7 ") FAB-MS (pos.) M / z = 621 = (M + H)⁺, Molecular weight 620

Example 4 The inhibitory effect of the polyphenol glycosides obtained in the above examples on phenol oxidase (manufactured by Sigma) was examined. That is, phenol oxidase (170,000 units / 8.4 ml) 0.06 was added to 0.84 ml of a citrate phosphate buffer solution containing 1.0 mM (+)-catechin and 0.1 mM polyphenol or a glycoside thereof.
ml was added and the reaction was carried out at 30 ° C. For the measurement of the enzyme activity, the increase in absorbance at 400 nm was followed over time, and the enzyme activity was determined from the initial rate. Further, as a control, (+)-catechin glycoside 1 ((+)-catechin-3 ′
-O-α-D-glucopyranoside) and (+)-catechin glycoside 2 ((+)-catechin-4′-O-α-D-
Glucopyranoside) was used. In addition, (+)-catechin glycoside 1 was prepared according to the method disclosed in JP-A-4-273890, and (+)-catechin glycoside 2 was prepared by using (+)-catechin. Each was prepared according to the method of 1.

The results are shown in Table 1. As is clear from the table, the polyphenol glycoside of the present invention shows almost the same inhibitory effect on the enzyme as the conventional polyphenols, and the two types of (+)-catechin glycosides used as controls were used. It had an excellent inhibitory ability compared to.

[0040]

[Table 1]

Example 5 The stability of the polyphenol glycosides obtained in the above Examples to light was examined. That is, the sample was dissolved in distilled water so as to have a concentration of 1 mM, and 5 ml thereof was placed in a test tube with a cap. This was set in a UV carbon arc light resistance tester (manufactured by Suga Seisakusho) and irradiated with light at 40 ° C for 10 hours (main wavelength: 3
(80 nm), the spectrum of the sample solution was measured with a spectrophotometer to evaluate its coloring degree. The sample solution before light irradiation was used as a control. FIG. 4 shows the result of evaluation of the degree of coloring using the integrated value of the absorbance in the visible region absorption region (400 to 700 nm). From these results, it was revealed that the polyphenol glycoside has excellent photostability as compared with the non-glycosylated polyphenol glycoside. As described above, the polyphenol glycoside of the present invention has excellent stability even in a solution state, and thus can be used for various purposes.

Example 6 Cyclodextrin glucanotransferase was applied to a mixed solution of (−)-epigallocatechin or (−)-epigallocatechin gallate and dextrin (trade name: Paindex # 1, manufactured by Matsutani Chemical Co., Ltd.). The reaction was carried out according to the method shown in Example 1. The reaction solution is Diaion H
P-10 (manufactured by Mitsubishi Kasei Co., Ltd.) was thoroughly washed with distilled water to remove unreacted sugars, buffer solution and enzyme, and then the glycoside was eluted with 50-100 V / V% ethanol aqueous solution. . Next, ethanol was removed by concentration under reduced pressure, and water was added to make 200 ml. This solution was washed 5 times with an equal volume of ethyl acetate to remove unreacted polyphenol. In addition, it was confirmed by HPLC analysis that unreacted polyphenol was removed.

The yield of the glycoside thus obtained is
(−)-Epigallocatechin 5 g to 23.4 g, (−)
-Epigallocatechin gallate from 5 g to 14.8 g. From the measurement result of ultraviolet absorption (molecular extinction coefficient), it was considered that 6 to 8 glucoses were bonded to these polyphenols on average per molecule.

Next, the astringency of these glycosides was examined by a sensory test by 15 panel members. The results are shown in Table 2. As is clear from the table, (-)-epigallocatechin and (-)-epigallocatechin gallate begin to feel astringency at 100 to 200 ppm, whereas glycosides do not feel astringency even at 1000 ppm, and 200
At 0 ppm, a slight astringency began to be felt.
As described above, the polyphenol glycoside is a substance whose taste is greatly improved as compared with conventional polyphenols. Therefore, the polyphenol glycoside of the present invention can be applied to all articles that can enjoy the taste regardless of foods, luxury items, cosmetics, pharmaceuticals and the like.

[0045]

[Table 2]

Example 7 Jelly Confectionery (−)-epigallocatechin or (−)-epigallocatechin gallate 20 g and dextrin (trade name: Paindex # 1, Matsutani Chemical Co., Ltd.) 200 g 1 mM
After dissolving in 1000 ml of water containing calcium chloride, cyclodextrin glucanotransferase was allowed to act according to the method of Example 1. After heat inactivating the enzyme,
Insoluble matter was removed by filtration. The filtrate was dried by a spray dryer to obtain 210 g of powder (yield about 95%).

126 g of coupling sugar (registered trademark, manufactured by Hayashibara Biochemical Laboratory Co., Ltd.), Oligomate 50
(Product name, Yakult Pharmaceutical Co., Ltd.) 136 g, lactose 6 g, aspartame (Product name, Ajinomoto Co., Inc.)
0.3 g, 10 g of (-)-epigallocatechin gallate glycoside-containing powder prepared by the above method and 150 g of water were mixed, and then dissolved by heating while stirring. Pectin in this solution
After gradually adding and dissolving 4.5 g, 50% citric acid dissolution 3.
3 g, 1/5 concentrated lemon juice 6 g, natural pigment 0.1 g and lemon flavor 0.2 g were added and mixed thoroughly,
This solution was poured into a mold and allowed to cool at room temperature for 12 hours to solidify to prepare pectin jelly. This product is a jelly confectionery with excellent flavor without coloring or astringency peculiar to polyphenols. It is also suitable as a jelly confectionery having the functionality of polyphenols.

Example 8 Powdered lactic acid foamed beverage 2.7 g of sugar, 0.6 g of vitamin C, 0.6 g of foaming agent (baking soda), 0.6 g of acidulant (citric acid) per cup,
Powdered fermented milk 0.5g, powdered flavor (grapefruit)
0.2 g and (-)-epigallocatechin gallate glycoside-containing powder prepared in Example 7 (0.6 g) were uniformly mixed to prepare a powdered lactose sparkling beverage. 180 ml of this product in cold water
When I drank it in, it was a drink with a refreshing taste and aroma that was easy to drink. In addition, this product is an easy-to-drink powdered lactic acid effervescent beverage having the original functions of polyphenols, such as cholesterol elevation inhibitory action, blood glucose elevation inhibitory action, and antioxidant action.

Example 9 Cosmetic Cream Squalane 23 g, stearic acid 5 g, behenyl alcohol 0.8 g, polyethylene glycol monostearate 2 g, self-emulsifying glyceryl monostearate 2.5.
g, titanium oxide 2.5 g and methyl paraoxybenzoate 0.05 g were dissolved under heating to 80 ° C., and allantoin 0.1 g, 1,3-butylene glycol 2 g, methyl paraoxybenzoate 0.15 g, in Example 7. The prepared (-)-epigallocatechin glycoside-containing powder (1 g) and purified water which had been heated to 80 ° C. were added, and the mixture was emulsified. After cooling to 30 ° C. with good stirring, the product was filled in a container. This product is a cosmetic cream that does not have the coloring peculiar to polyphenols and is easy to adapt to the skin. In addition, it is a cosmetic cream having the original functions of polyphenols, such as an antioxidant effect and an ultraviolet absorbing effect.

Example 10 Tablets Lactose 77 g, (-)-epigallocatechin glycoside-containing powder 25 g prepared in Example 7, aspartame 0.5 g,
Sugar ester 0.7g, 1/5 concentrated lemon juice 2
g, Vitamin C 5 g, Vitamin E powder 0.2 g, β-carotene powder 0.2 g and powdered lemon flavor 1 g were uniformly mixed, and then tableted to a tablet of 500 mg, a diameter of 10 mm, and a tablet thickness of 3.2 mm. This product is a tablet with no astringency and a good flavor. In addition, the original polyphenols, for example, cholesterol elevation inhibitory action, blood glucose elevation inhibitory action,
It is an easy-to-drink tablet that has functionality such as anti-oxidant effect.

[0051]

The polyphenol glycoside of the present invention is
Not only it has excellent stability against pH and heat, but it can also effectively improve the strong astringency and astringency of conventional polyphenols, and has sufficient inhibitory effect on phenol oxidase. Therefore, it is a novel compound that can be applied to various compositions such as foods, luxury items, cosmetics, quasi drugs, and pharmaceuticals.

[Brief description of drawings]

FIG. 1 ¹ H-NM of (−)-epigallocatechin glycoside 2.
The R spectrum is shown.

FIG. 2 shows ¹³ C-N of (−)-epigallocatechin glycoside 2.
An MR spectrum is shown.

FIG. 3 shows a mass spectrum of (−)-epigallocatechin glycoside 2.

FIG. 4 is a graph showing the degree of coloring of polyphenol glycosides.

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[Procedure amendment]

[Submission date] December 28, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

(-)-Epigallocatechin-7,4'-O
-Di-α-D-glucopyranoside (epigallocatechin glycoside 1) ¹ H-NMR (ppm, methanol (d ₄ ) -D ₂ O) 2.79 (1H, dd, H-4a), 2.
87 (1H, dd, H-4b), 3.45-3.92 ( 11 H, ring H, Glc × 2), 4.20
(1H, m, H-3) , 4.26 (1H, m, H-5 "') , 4.83 (1H, s, H-2), 5.05
(1H, d, H-1 "', J _{H-1"', H-2 "'=} 4 Hz ), 5.41 (1H, d, H-1", J
_{H-1 "} , _H-2" = 4Hz), 6.26 (1H, d, H-8), 6.31 (1H, d, H-6),
6.60 (2H, d, H-2 ', 6') ¹³ C-NMR (ppm, methanol (d ₄ ) -D ₂ O) 28.5 (C-4), 60.5 (C-
6 '" ), 62.3 (C-6"), 64.5 (C-3), 68.1 (C-4 ") , 69.8 (C-
4 '" ), 71.6 (C-2"), 72.0 (C-2'"), 73.1 (C-3 '"), 73.6 (C
-5 "'), 75.1 (C-5") , 78.2 (C-2), 95.6 (C-8), 96.7 (C-
6), 98.0 (C-1 " ), 101.5 (C-4a), 105.6 (C-1 '") , 107.9 (C
-2 ' , 6'), 137.9 (C-1 ' ), 134.9 (C-4' ), 150.8 (C-3 ',
5 ' ), 155.6 (C-8a), 156.3 (C- 5) , 156.5 (C- 7) FAB-MS (pos.) M / z = 631 = (M + H) ⁺ , molecular weight 630

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0027

[Correction method] Change

[Correction content]

(-)-Epigallocatechin-4'-O-α
-D-glucopyranoside (epigallocatechin glycoside 2) ¹ H-NMR (ppm, methanol (d ₄ ) -D ₂ O) 2.72 (1H, dd, H-4a), 2.
87 (1H, dd, H-4b), 3.44-3.93 (5H, ring H Glc), 4.22 (1
H, m, H-3), 4.25 (1H, m, H-5 "), 4.80 (1H, s, H-2), 5.08 (1
H, d, H-1 ", J _{H-1", H-2 "} = 4.1Hz), 5.96 (1H, d, H-8), 5.9
8 (1H, d, H-6), 6.58 (2H, d, H-2 ', 6') ¹³ C-NMR (ppm, methanol (d ₄ ) -D ₂ O) 29.9 ( C-4), 62.8 (C-
6 "), 65.0 (C-3), 68.0 (C-4"), 71.6 (C-2 "), 74.0 (C-
3 "), 75.6 (C-5"), 80.3 (C-2), 96.9 (C-8), 97.2 (C-6),
100.9 (C-4a), 106.0 (C-1 "), 108.0 (C-2 ', 6'), 135.5 (C-
4 '), 138.5 (C-1'), 152.0 (C-3 ', 5'), 157.7 (C-8a), 15
8.1 (C-7), 158.5 (C-5) FAB-MS (pos.) M / z = 469 = (M + H) ⁺ , molecular weight 468

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

(-)-Epicatechin gallate-4'-O
-Α-D-glucopyranoside (epicatechin gallate glycoside 1) ¹ H-NMR (ppm, DMSO-d ₆ ) 2.71 (1H, dd, H-4a), 2.94 (1H, dd,
H-4b), 3.21-3.61 (6H, ring H, Glc), 5.02 (1H, d, H-1 ''',
J _{H-1 ''',H-2'''} = 3.8 Hz), 5.07 (1H, s, H-2), 5.34 (1H,
m, H-3), 5.83 (1H, d, H-8), 5.94 (1H, d, H-6), 6.72 (1H, d
d, H-6 '), 6.77 (1H, d, H-2') , 6.84 (2H, s, H-2 ", 6"), 7.14
(1H, d, H-5 ' ) ¹³ C-NMR (ppm, DMSO-d ₆ ) 25.5 (C-4), 60.2 (C-6'''), 68.
0 (C-3), 69.4 (C-4 '''), 71.9 (C-2'''), 73.0 (C-5 '''), 7
3.2 (C-3 '''), 76.3 (C-2), 94.3 (C-8), 95.5 (C-6), 97.0
(C-4a), 100.0 (C-1 '''), 108.5 (C-2 ", 6"), 115.1 (C-
5 '), 116.0 (C-2'), 118.9 (C-1 "), 121.1 (C-6 '), 129.5 (C
-1 '), 138.5 (C-4 "), 144.5 (C-4'), 145.3 (C-3", 5 "), 14
6.6 (C-3 '), 155.4 (C-8a), 156.4 (C-7), 156.4 (C-5), 16
5.0 (C-7 ") FAB-MS (pos.) M / z = 605 = (M + H) ⁺ , molecular weight 604

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction content]

(-)-Epigallocatechin gallate-7,
4′-O-di-α-D-glucopyranoside (epigallocatechin gallate glycoside 1) ¹ H-NMR (ppm, methanol (d ₄ ) -D ₂ O) 2.94 (1H, dd, H-4a), 3.
08 (1H, dd, H-4b), 3.43-3.92 (11H , ring H, Glc × 2), 4.17
(1H, m, H-5 "'), 5.02 (1H, d, H-1"', J _{H-1 "', H-2"'} = 4Hz),
5.06 (1H, s, H-2), 5.50 (1H, d , H-1 "", J _{H-1 "", H-2 ""} = 3.
5Hz), 5.51 (1H, m, H-3), 6.38 (1H, d, H-8) , 6.38 (1H, d, H
-6), 6.61 (2H, d, H-2 ', 6') , 7.02 (2H, s, H- 2 ", 6") ¹³ C-NMR (ppm, methanol (d ₄ ) -D ₂ O) 27.7 (C-4), 62.7 (C-
6 '"), 63.1 (C-6""), 70.5 (C-3), 71.7 (C-4'"), 72.3 (C-
4 ""), 74.1 (C-2 '"), 74.2 (C-2""), 74.7 (C-3'"), 75.9 (C
-5 '"), 75.7 (C-3"") , 75.7 (C-5""), 79.6 (C-2), 98.4 (C
-8), 99.1 (C-6) , 99.9 (C-4a), 106.3 (C-1 '") , 102.9 (C-
1 ""), 108.0 (C-2 '), 111.1 (C-6'), 111.2 (C-2 ", 6"), 12
2.0 (C-1 "), 135.4 (C-1 '), 135.8 (C-4'), 136.7 (C-4") ,
147.1 (C-3 ", 5") , 152.0 (C-3 ', 5'), 157.9 (C-8a), 158.5
(C -5 ), 158.9 (C -7 ), 168.1 (C-7 ") FAB-MS (pos.) M / z = 783 = (M + H) ⁺ , molecular weight 782

[Procedure Amendment 5]

[Document name to be amended] Statement

[Name of item to be corrected] 0037

[Correction method] Change

[Correction content]

(-)-Epigallocatechin gallate-4 '
-O-α-D-glucopyranoside (epigallocatechin gallate glycoside 2) ¹ H-NMR (ppm, methanol (d ₄ ) -D ₂ O) 2.83 (1H, dd, H-4a), 2.
99 (1H, dd, H-4b), 3.30-3.86 (5H, ring H, Glc), 4.14 (1H,
m, H-5 "'), 4.99 (1H, d, H-1'", J _{H-1 "', H-2"'} = 3.8Hz),
5.00 (1H, s, H-2), 5.53 (1H, m, H-3), 5.95 (2H, s, H-6,8),
6.56 (2H, s, H-2 ', 6'), 6.93 (2H, s, H-2 ", 6") ¹³ C-NMR (ppm, methanol (d ₄ ) -D ₂ O) 27.7 ( C- 4), 63.0 (C-
6 '"), 70.6 (C-3), 71.8 (C-4'"), 74.1 (C-2 '"), 75.7 (C-
3 '"), 76.0 (C-5'"), 79.2 (C-2), 96.7 (C-8), 97.4 (C-
6), 100.2 (C-4a) , 106.4 (C-1 '"), 107.8 (C-2', 6 '), 11
1.0 (C- 2 ", 6"), 122.1 (C-1 ") , 135.9 (C-4 '), 138.0 (C-
1 '), 140.7 (C-4 "), 147.1 (C-3", 5 " ), 152.3 (C-3', 5 '),
157.8 (C-8a), 158.7 (C-5,7), 168.4 (C-7 ") FAB-MS (pos.) M / z = 621 = (M + H) ⁺ , molecular weight 620

[Procedure correction 6]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

[Figure 1]

[Procedure Amendment 7]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 2

[Correction method] Change

[Correction content]

[Fig. 2]

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Takashi Shibuya 318 Shimohara, Soja City, Okayama Prefecture

Claims (8)

[Claims] 1. The following general formula 1 (In the formula, X represents a D-glucopyranose residue or a maltooligosaccharide residue having a degree of polymerization of 2 to 10, Y represents R ₄ or a gallic acid group, Z represents hydrogen or OR ₅ , and R _{1 to} R ₅ is a hydrogen atom, D-glucopyranose residue or a maltooligosaccharide residue having a degree of polymerization of 2 to 10). 2. A novel polyphenol glycoside is represented by the following general formula 2 (In the formula, X represents a D-glucopyranose residue or a maltooligosaccharide residue having a degree of polymerization of 2 to 10, and R _{1 to} R ₅ each independently represent hydrogen, a D-glucopyranose residue or a degree of polymerization of 2 to 10). A novel polyphenol glycoside according to claim 1, which is a gallocatechin glycoside represented by a malto-oligosaccharide residue. 3. A novel polyphenol glycoside is represented by the following general formula 3: (In the formula, X represents a D-glucopyranose residue or a maltooligosaccharide residue having a degree of polymerization of 2 to 10, and R _{1 to} R ₃ each independently represent hydrogen, a D-glucopyranose residue or a degree of polymerization of 2 to 10. A catechin-3-O-gallate glycoside represented by (1) represents a malto-oligosaccharide residue.
The novel polyphenol glycoside described. 4. A novel polyphenol glycoside is represented by the following general formula 4 (In the formula, X represents a D-glucopyranose residue or a maltooligosaccharide residue having a degree of polymerization of 2 to 10, and R _{1 to} R ₃ and R ₅ each independently represent hydrogen, a D-glucopyranose residue or a degree of polymerization of 2). 10. The novel polyphenol glycoside according to claim 1, which is a gallocatechin-3-O-gallate glycoside represented by the following formula 10). 5. (A) Gallocatechin, catechin-3-O
-Gallate, gallocatechin-3-O-gallate, and any one of a mixture of polyphenols containing at least two kinds of these compounds, and (B) dextrin, cyclodextrin, starch and any of a mixture containing at least two kinds of these compounds. Crab Bacillus
The method for producing a novel polyphenol glycoside according to claim 1, wherein the polyphenol glycoside is obtained by reacting a cyclodextrin glucanotransferase derived from stearothermophilus. 6. A composition having a phenol oxidase activity inhibitory activity, which comprises the polyphenol glycoside according to claim 2. 7. A composition having excellent stability and taste, which comprises the polyphenol glycoside according to claim 2. 8. The composition according to claim 6 or 7, which is used as a food or drink, a luxury item, a cosmetic, a quasi drug, a drug or a raw material thereof.