JPH093089A - New polyphenol glycoside, its production and use thereof - Google Patents

Abstract

PURPOSE: To obtain the subject new glycoside, capable of improving astringency and stypticity and excellent in taste and useful for foods, favorite foods, cosmetics and medicines, etc., by reacting a mixture of a tea polyphenol, such as epigallocatechin, and dextrin, etc., with an enzyme. CONSTITUTION: This new polyphenol glycoside is expressed by the formula [R1 and R2 are each OH or a maltooligosaccharide residue having 2-10 polymerization degree; X is H or OH; Y is OH or galoyl; (n) is an integer of 1-9] [e.g. (-)-epigallocatechin 3',7-di-O-α-D-glucopyranoside]. The glycoside is improved in astringency and stypticity of tea polyphenols and excellent in taste and useful in wide fields such as foods, favorite foods, medicines, etc. The polyphenol glycoside is obtained by mixing a tea polyphenol such as (epi)gallocatechin(3- O-gallate) with dextrin, cyclodextrin, starch, etc., and reacting the mixture with cyclomaltodextringlucanotransferase.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a glycoside of tea polyphenols, a method for producing the same, and a use thereof. More specifically, the present invention relates to a polyphenol glycoside having a novel structure with improved taste and a method for producing the same. Regarding its use.

[0002]

2. Description of the Related Art Tea polyphenols have an inhibitory effect on cholesterol elevation (Japanese Patent Publication No. 2-44449) and an antibacterial effect (JP-A-2-27).
No. 6562), antioxidative effect (Japanese Patent Publication No. 1-44234)
No.), an antitumor effect (JP-A-60-190719), a blood pressure elevation inhibitory effect and an enzyme activity inhibitory effect (JP-A-3-133928), and the active ingredient thereof is epigalo. Catechin, epicatechin 3
-O-gallate and epigallocatechin 3-O-gallate are known. However, since these substances are astringent components having a strong astringent property, their taste properties are significantly impaired, which is a major drawback in considering their use in various fields including foods.

At present, for (+)-catechin having a structure similar to that of tea polyphenol, sucrose phosphorylase is allowed to act on a mixed solution of (+)-catechin and glucose-1-phosphate or sucrose. A method for producing a (+)-catechin glycoside to which easy solubility and color stability are imparted (Japanese Patent Laid-Open No. 176786/1993), and a (+)-catechin glycoside of tyrosinase involved in the formation of a melanin pigment. A method (Japanese Patent Application Laid-Open No. 4-273890) has been developed in which it is used as an inhibitor in a skin external preparation. However, (+)-catechin is equivalent to epigallocatechin, epigallocatechin 3-O-gallate, and epigallocatechin in physiologically active actions such as cholesterol elevation inhibitory action, antibacterial action, antioxidant action, antitumor action and blood pressure elevation inhibitory action. Compared with catechin 3-O-gallate, its physiological activity is extremely weak, and in some cases, it has no activity. Therefore, regarding glycosides, the glycosides of epigallocatechin, epigallocatechin 3-O-gallate and epicatechin 3-O-gallate are expected to have more physiological activity than the (+)-catechin glycoside. be able to.

On the other hand, tea polyphenols (-)-epicatechin, (-)-epicatechin 3-O-gallate,
Regarding the glycosides of (−)-epigallocatechin and (−)-epigallocatechin 3-O-gallate, there is no report that the structure shown in the present invention has been clarified so far.
Therefore, the characteristics and functionality of the novel polyphenol glycoside of the present invention have not been clarified.

[0005]

[Means for Solving the Problems] The present inventors acted a cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus on the above tea polyphenols and dextrin, cyclodextrin, starch or a mixture thereof. By letting
It has been found that polyphenols can be glycosylated. Further, the obtained polyphenol glycoside was isolated to clarify the structure of a novel compound in which the 3'position, 3'and 5 position and 3'and 7 position of tea polyphenols were glycosylated. Also,
The novel polyphenol glycoside of the present invention was confirmed to have almost no astringent, astringent and excellent taste properties, and the present invention was completed.

That is, the present invention relates to the general formula 1 (wherein R ₁ ,
R ₂ is independently a hydroxyl group or a polymerization degree of 2 to 10
Malto-oligosaccharide residue, X is a hydroxyl group or hydrogen, Y is a hydroxyl group or a galloyl group, and n is an integer from 1 to 9. ) Provides a polyphenol glycoside represented by
Furthermore, epigallocatechin, gallocatechin, epigallocatechin 3-O-gallate, gallocatechin 3-O-gallate
, Epicatechin, epicatechin 3-O-gallate, catechin 3-O-gallate or a mixture of polyphenols containing two or more of these with dextrin, cyclodextrin, starch or a mixture thereof with cyclomaltodextrin glucano It is intended to provide a method for producing the above polyphenol glycoside, which is characterized by causing a transferase to act, and a composition containing the above polyphenol glycoside.

[0007]

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Hereinafter, the present invention will be described in detail. The basic skeleton of the polyphenol used in the present invention is represented by the following general formula 5.

[0009]

[Chemical 6]

In this structural formula, 2 of the benzopyran ring is
Positions 3 and 4 may each be in the R or S configuration. For example, (+) or (−)-gallocatechin, (+) or (−)-epigallocatechin, (+) or (−) catechin 3-O-gallate, (+) or ( Examples include-)-epicatechin 3-O-gallate, (+) or (-)-gallocatechin 3-O-gallate, and (+) or (-)-epigallocatechin 3-O-gallate.

The method for producing the polyphenol glycoside represented by the above general formula 1 is not particularly limited, and chemical synthesis method, plant tissue culture cell method, method using microbial cells and the like can be considered. In order to improve the stability and taste of these polyphenols, dextrin, cyclodextrin, starch or a mixture thereof is added to one or a mixture of two or more of the polyphenols, and cyclomaltodextrin is added thereto. A method of acting glucanotransferase can be used. As the cyclomaltodextrin glucanotransferase, those derived from the genus Bacillus are used, and particularly Bacillus stearothermophilus (Bacillus
The enzyme derived from stearothermophilus) has a high ability to glycosylate polyphenols and can be advantageously used.

The condition of the enzyme reaction is that the pH of the reaction is 3
-9, preferably pH 5-8, reaction temperature 20-80
C., preferably 30 to 70.degree. C., and a substrate concentration of polyphenols of 0.1 to 20% (w / v), preferably 0.1.
5-10% (w / v), dextrin, cyclodextrin, starch or a mixture of these 1-40% (w / v).
/ V), preferably a reaction solution containing 2 to 30% (w / v). Further, the amount of enzyme and the reaction time can be optimally set according to the above reaction conditions.

Thus, the polyphenol glycoside represented by the general formula 1 is obtained. As the polyphenol glycoside, for example, when gallocatechin is used as the polyphenol, the polyphenol glycoside represented by the general formula 2 is obtained, and when gallocatechin 3-O-gallate is used, the polyphenol glycoside is represented by the general formula 3. The polyphenol glycoside is obtained, and when catechin 3-O-gallate is used, the polyphenol glycoside represented by the general formula 4 is obtained.

[0014]

[Chemical 7]

[0015]

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[0016]

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The reaction solution containing the polyphenol glycoside thus obtained can be used as it is for various purposes, but can also be used after forming the reaction solution into a syrup or a powder, if necessary. . Further, it is also possible to use a product purified as described below.

In the case of collecting high-purity polyphenol glycoside, a porous synthetic adsorbent such as DIAION HP-10, DIAION HP-10 manufactured by Mitsubishi Chemical Co., Ltd.
P-20, Diaion HP-40, Rohm & Haas
Using the trade name of Amberlite XAD-1, Amberlite XAD-4, Amberlite XAD-7, Amberlite XAD-8, etc., the difference in the adsorption between polyphenol glycosides and impurities is used. Can be purified.

For example, when separating polyphenol glycosides, polyphenols and free saccharides, the reaction solution is passed through a column packed with a porous synthetic adsorbent and free saccharides are not adsorbed on the column. Elute, but the polyphenol compound is adsorbed. Then, the adsorbed polyphenol compound such as polyphenol glycoside is eluted with a lower alcohol solution, for example, a 50% (v / v) ethanol aqueous solution, and the eluate is concentrated to form a syrup, and further dried and powdered. Can be collected. further,
When it is necessary to separate polyphenol glycosides and unreacted polyphenols, polyphenols unreacted in an organic solvent phase such as ethyl acetate can be used by utilizing the difference in polarities of these compounds by liquid separation. Can also be removed by shifting. It is also possible to collect and use a specific fraction of the polyphenol glycoside by a method such as chromatography.

Unlike the conventional polyphenols, the polyphenol glycoside of the present invention collected as described above has little bitterness, astringency, harshness such as acridness and astringency, and its degree of purification. It can be used in a wide range of fields such as foods, quasi-drugs, cosmetics, and pharmaceuticals, as it is or together with other materials, regardless of its purity.

Further, the polyphenol glycoside of the present invention is
It has been confirmed that the original polyphenol is released by being decomposed by α-glucosidase or glucoamylase. Therefore, when the polyphenol glycoside of the present invention is administered to the human body, the polyphenols are easily released by the action of α-amylase or α-glucosidase in the body, and the physiological activity functions inherent to the polyphenols, for example, cholesterol elevation suppression. It exhibits an action, an antibacterial action, an antioxidant action, an antitumor action, a blood pressure increase suppressing action, and the like. Therefore, the polyphenol glycoside of the present invention can be used in a wide range of fields such as foods, quasi-drugs, cosmetics, and pharmaceuticals without reducing the physiological activity of polyphenols. In particular, it can be advantageously used for health promotion food, health maintenance food, health recovery food, and the like.

The fields of application of the polyphenol glycoside of the present invention are listed as seasonings, Japanese confectionery, Western confectionery, ice confectionery, syrups, fruit processed products, vegetable processed products, pickles, livestock meat products,
Fish products, delicacies, canned / bottled products, alcoholic beverages, soft drinks,
Foods such as instant food and drink, tobacco, toothpaste, lipstick, lip balm, internal medicine, troches, liver oil drops, mouth fresheners, oral pastilles, mouthwash, various solids, pastes, liquids, cosmetics, Pharmaceuticals and raw materials for these.

[0023]

EXAMPLES Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. Example 1 100 g of dextrin (trade name: Paindex # 1, manufactured by Matsutani Chemical Co., Ltd.) and 5 g of (−)-epigallocatechin (manufactured by Mitsui Norin Co., Ltd.) in 0.1 M acetate buffer (pH 5) containing 10 mM calcium chloride. .5) After dissolving in 500 ml, add 1000 units of Bacillus stearothermophilus-derived cyclomaltodextrin glucanotransferase (manufactured by Hayashibara Biochemical Research Institute) per 1 g of dextrin solid content, and react at 50 ° C. for 24 hours Let Then, after removing the enzyme by UF membrane filtration, 1000 ml of 0.1 M acetate buffer (pH 4.5) was added to the reaction solution.
And 50 units of glucoamylase derived from Rhizopus niveus (manufactured by Seikagaku Corporation) were added per 1 gram of solid matter, and the reaction was carried out at 40 ° C. for 20 hours. After the reaction was completed, the enzyme was removed using a UF membrane, and the resulting reaction solution was adsorbed on a column packed with 1000 ml of Diaion HP-10 (manufactured by Mitsubishi Chemical Corporation) equilibrated with water.

After washing the column with 3000 ml of deionized water, 1000 ml of 50% (v / v) aqueous methanol solution,
Then, the mixture was eluted with 2000 ml of 100% (v / v) methanol to collect the adsorbed fraction. The eluate was concentrated under reduced pressure to remove methanol and then dissolved in 500 ml of distilled water. This solution was washed 5 times with 500 ml of ethyl acetate to remove unreacted (−)-epigallocatechin. Next, this aqueous solution fraction was developed on 2500 ml of Diaion HP-20SS column, washed thoroughly with distilled water, and
Elution was performed with 8000 ml of a 5% (v / v) methanol aqueous solution, and 2000 ml fractions were fractionated. Furthermore, 50% (v /
v) Elution with 5 liters of methanol solution. These fractions were analyzed by HPLC to obtain fractions A and B containing epigallocatechin glycoside. These fractions were separated by preparative HPLC, and from the A fraction, epigallocatechin glycoside 1 (127 mg) and epigallocatechin glycoside 2 (85 mg), and from the B fraction, epigallocatechin glycoside 3 (327 mg) was obtained. These glycosides are HPLC
Analysis showed a single peak.

Analytical HPLC conditions are as follows. Column: Shiseido Capsule Pack AG-120 ODS (4.6 x
250 mm) Mobile phase: acetonitrile: ethyl acetate: 0.05% (v
/ V) Phosphoric acid water = 12: 0.6: 90 Flow rate: 1.0 ml / min Detection: UV 280 nm (40 ° C.) Preparative HPLC conditions are as follows. Column: Capsule pack ODS AG-120 S-5 (50 × 500 mm) Mobile phase: A fraction: 10% (v / v) aqueous methanol solution containing 0.05% (v / v) phosphoric acid aqueous solution B fraction : 15% (v / v) methanol aqueous solution containing 0.05% (v / v) phosphoric acid water Flow rate: 120 ml / min Detection: UV 280 nm (room temperature)

As a result of analyzing these glycosides 1, 2 and 3 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, ¹ H-NMR, ¹³ C-NMR spectrum and mass spectrum of (-)-epigallocatechin glycoside 3 are shown in FIGS. 1, 2 and 3, respectively.

(-)-Epigallocatechin glycoside 1 (-)-Epigallocatechin 3 ', 7-di-O-α-D-
Glucopyranoside 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, Glcx2), 4.21 (1
H, m, H-3), 4.83 (1H, s, H-2), 5.36 (1H, d, H-1 '", JH-1'", H-
2 '"= 4 Hz), 5.41 (1H, d, H-1", JH-1 ", H-2" = 4 Hz), 6.26 (1
H, d, H-8), 6.31 (1H, d, H-6), 6.76 (1H, d, H-6 '), 6.98 (1
H, 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'), 1
09.8 (C-6 '), 129.6 (C-1'), 134.9 (C-4 '), 145.1 (C-
3 '), 145.3 (C-5'), 155.6 (C-8a), 156.3 (C-7), 156.5 (C
-5) FAB-MS (pos.) M / z = 631 = (M + H) ⁺ , molecular weight 630

(−)-Epigallocatechin glycoside 2 (−)-Epigallocatechin 3 ′, 5-di-O-α-D-
Glucopyranoside ¹ H-NMR (ppm, methanol (d ₄ )) 2.79 (1H, dd, H-4a), 3.03 (1
H, dd, H-4b), 3.43-3.92 (12H, ring H, Glcx2), 4.20 (1H, m,
H-3), 4.83 (1H, s, H-2), 5.37 (1H, d, H-1 '", JH-1'", H-
2 '"= 3.5 Hz), 5.52 (1H, d, H-1", JH-1 ", H-2" = 3.5 Hz),
6.09 (1H, d, H-8), 6.33 (1H, d, H-6), 6.74 (1H, d, H-6 '),
6.97 (1H, d, H-2 ') FAB-MS (pos.) M / z = 631 = (M + H) ⁺ , molecular weight 630

(−)-Epigallocatechin glycoside 3 (−)-Epigallocatechin 3′-O-α-D-glucopyranoside 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 ", JH-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), 1
00.0 (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'), 157.2
(C-8a), 157.6 (C-7), 158.0 (C-5) FAB-MS (pos.) M / z = 469 = (M + H) ⁺ , molecular weight 468

Example 2 After the cyclomaltodextrin glucanotransferase reaction and the glucoamylase reaction were carried out in the same manner as in Example 1 using 5 g of (−)-epigallocatechin 3-O-gallate (manufactured by Mitsui Norin Co., Ltd.). , Diaion HP-1
0 was used to remove unreacted sugar. The adsorbed fraction was eluted with an aqueous methanol solution, concentrated under reduced pressure, and then subjected to preparative HPLC to obtain epigallocatechin gallate glycoside 1 (315 m
g) and epigallocatechin gallate glycoside 2 (780
mg). This glycoside showed a single peak in the HPLC analysis under the above conditions (Example 1).

Preparative HPLC conditions are as follows. Column: Capsule pack AG-120 ODS S-5 (50 x 50
Mobile phase: 0.05% (v / v) 20% (v) containing phosphoric acid water
/ V) Methanol aqueous solution; 40 min Flow rate: 120 ml / min Detection: UV 280 nm (room temperature)

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. (-)-Epigallocatechin gallate glycoside 1 (-)-Epigallocatechin gallate 3 ', 7-di-O-
α-D-glucopyranoside 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, Glcx2), 4.87 (1
H, d, H-1 "", JH-1 "", H-2 "" = 4 Hz), 5.06 (1H, s, H-2), 5.5
0 (1H, d, H-1 '", JH-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 (1H, 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 '), 15
7.9 (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

(-)-Epigallocatechin gallate glycoside 2 (-)-Epigallocatechin gallate 3'-O-α-D-
Glucopyranoside ¹ H-NMR (ppm, methanol (d ₄ ) -D ₂ O) 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 (2
H, m, H-6 '"), 4.90 (1H, d, H-1'", JH-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 ₄ ) -D ₂ O) 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'), 13
6.5 (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 3 (−)-Galocatechin (manufactured by Mitsui Norin Co., Ltd.) was subjected to cyclomaltodextrin glucanotransferase reaction and glucoamylase reaction in the same manner as in Example 1, and then DIAION HP-10 was added. Used to remove unreacted sugar. The adsorbed fraction was eluted with an aqueous methanol solution, concentrated under reduced pressure, and then subjected to solvent fractionation with ethyl acetate and water.
The aqueous layer was concentrated under reduced pressure and then subjected to preparative HPLC to obtain gallocatechin glycoside 1 (750 mg). This glycoside showed a single peak in the HPLC analysis according to Example 1.

Preparative HPLC conditions are as follows. Column: Capsule pack AG-120 ODS S-5 (50 x 50
0 mm) Mobile phase: 10% (v / v) acetonitrile aqueous solution; 40
min Flow rate: 100 ml / min Detection: UV 280 nm (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. (−)-Gallocatechin glycoside 1 (−)-Gallocatechin 3′-O-α-D-glucopyranoside ¹ H-NMR (ppm, methanol-d ₄ ) 2.73 (1H, dd, H-4a), 2.87 (1
H, dd, H-4b), 3.42-3.94 (6H, ring H, Glc), 4.00 (1H, ddd,
H-3), 5.39 (1H, d, H-1 ", JH-1", H-2 "= 4.1 Hz), 5.55 (1H,
d, H-2) 5.88 (1H, d, H-8), 5.96 (1H, d, H-6), 6.63 (1H, d,
H-6 '), 6.89 (1H, d, H-2') FAB-MS (pos.) M / z = 455 = (M + H) ⁺ , molecular weight 454

Example 4 The cyclomaltodextrin glucanotransferase reaction and the glucoamylase reaction were carried out in the same manner as in Example 1 using 5 g of (-)-epicatechin 3-O-gallate (manufactured by Mitsui Norin Co., Ltd.). Unreacted sugar was removed using Diaion HP-10. The adsorbed fraction was eluted with an aqueous methanol solution, concentrated under reduced pressure, and then subjected to preparative HPLC to obtain epicatechin gallate glycoside 1 (923 mg). This glycoside showed a single peak in the HPLC analysis according to Example 1.

Preparative HPLC conditions are as follows. Column: Capsule pack AG-120 ODS S-5 (50 x 50
Mobile phase: 0.05% (v / v) 20% (v) containing phosphoric acid water
/ V) Methanol aqueous solution; 40 min Flow rate: 120 ml / min Detection: UV 280 nm (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. (−)-Epicatechin 3-O-gallate glycoside 1 (−)-Epicatechin gallate 3′-O-α-D-glucopyranoside ¹ H-NMR (ppm, methanol-d ₄ ) 2.86 (1H, dd, H-4a), 3.01 (1
H, dd, H-4b), 3.30-3.87 (6H, ring H, Glc), 4.93 (1H, d, H-
1 '", JH-1'", H-2 '"= 3.8 Hz), 5.05 (1H, s, H-2), 5.48 (1H,
m, H-3), 5.95 (1H, d, H-8), 5.97 (1H, d, H-6), 6.77 (1H, d,
H-5 '), 7.00 (1H, dd, H-6'), 7.01 (2H, s, H-2 ", 6"), 7.57
(1H, d, H-2 ') ¹³ C-NMR (ppm, methanol-d ₄ ) 26.9 (C-4), 61.8 (C-6'"),
70.0 (C-3), 70.7 (C-4 '"), 73.4 (C-2'"), 73.9 (C-5 '"),
74.9 (C-3 '"), 78.6 (C-2), 95.9 (C-8), 96.6 (C-6), 99.2
(C-4a), 101.1 (C-1 '"), 110.4 (C-2", 6 "), 116.6 (C-5'),
117.3 (C-2 '), 121.2 (C-1 "), 123.0 (C-6'), 131.6 (C-
1 '), 139.9 (C-4 "), 146.3 (C-4'), 146.3 (C-3", 5 "), 14
8.0 (C-3 '), 157.2 (C-8a), 157.8 (C-7), 157.8 (C-5), 16
7.3 (C-7 ") FAB-MS (pos.) M / z = 605 = (M + H) ⁺ , molecular weight 604

Example 5 100 g of dextrin (trade name: Paindex # 1, manufactured by Matsutani Chemical Co., Ltd.) and (−)-epigallocatechin,
(-)-Epigallocatechin 3-O-gallate, (-)
-Epicatechin 3-O-gallate (all manufactured by Mitsui Norin Co., Ltd.) was dissolved separately in 500 ml of a 0.1 M acetate buffer (pH 5.5) containing 10 mM calcium chloride. Cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus (Hayashibara Biochemical Laboratories, Inc.) was added to each mixed solution in an amount of 1000 units per 1 g of dextrin solid content, and 5
The reaction was performed at 0 ° C. for 24 hours. Then, the reaction solution is at 100 ° C
The enzyme was inactivated by heating at 30 minutes.

Each of the obtained three kinds of reaction solutions was adsorbed on a column filled with 1000 ml of Diaion HP-10 (manufactured by Mitsubishi Chemical Corporation) equilibrated with water. After washing the column with 3000 ml of deionized water, 70% (v /
v) The adsorbed fraction was collected by elution with 1000 ml of an aqueous ethanol solution. The eluate was concentrated under reduced pressure to remove ethanol and dissolved in 500 ml of distilled water. This solution was washed 5 times with 500 ml of ethyl acetate to remove unreacted polyphenols. It was confirmed by the HPLC method of Example 1 that the unreacted polyphenol was removed. The reaction solutions washed with ethyl acetate were lyophilized to obtain polyphenol glycosides. The yields of the glycosides thus obtained were 21.4 g from epigallocatechin, 13.8 g from epigallocatechin 3-O-gallate, and 16.2 g from epicatechin gallate. Each of the obtained glycosides was dissolved in distilled water and the absorption of ultraviolet rays was measured. From the molecular extinction coefficient, it was considered that an average of 6 to 8 glucoses were bound to each polyphenol molecule.

Example 6 100 mg of each of the three kinds of polyphenol glycosides obtained in Example 5 were weighed and dissolved in 1 ml of distilled water. Glucoamylase (Gluczyme AF6, manufactured by Amano Pharmaceutical Co., Ltd.) (1.6 mg) and α-glucosidase (manufactured by Sigma) (0.23 mg) were added to the dissolved liquids, and the mixture was stirred well. The mixture was incubated at 37 ° C for 3 hours. The reaction solution was analyzed by the following HPLC method.

The HPLC conditions are as follows. Column: Shiseido Capsule Pack AG-120 ODS (4.6 x
250 mm) Mobile phase: acetonitrile: ethyl acetate: 0.05% (v
/ V) Phosphoric acid water = 12: 0.6: 90 Flow rate: 1.0 ml / min Detection: Photodiode array detector

From the reaction solution of epigallocatechin glycoside, epigallocatechin, and from the reaction solution of epigallocatechin 3-O-gallate glycoside, epigallocatechin 3-O-gallate, epicatechin 3-O. -Epicatechin 3-O-gallate was detected in each reaction liquid of gallate glycoside.

The above results show that the polyphenols are actually glycosylated as they are by the glycosylation reaction carried out in Example 4. Furthermore, since the polyphenol glycoside is hydrolyzed by α-glucosidase or glucoamylase to release polyphenols, it can be easily hydrolyzed in vivo by enzymes such as α-glucosidase and α-amylase. It is considered that the polyphenols having the physiologically active function are released to exhibit the original physiologically active function of the polyphenols.

Example 7 The astringency of the glycoside obtained in Example 5 was examined by a sensory test on 20 panelists. The results are shown in Table 1.

[0047]

[Table 1]

As is clear from Table 1, polyphenols begin to feel astringency at 100 to 200 ppm, whereas polyphenol glycosides do not feel astringency at 1000 ppm, and slightly astringent at 2000 ppm. I started to feel it. As described above, polyphenol glycosides are substances having improved taste properties 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, and pharmaceuticals.

Example 8 Jelly Confectionary Epigallocatechin 3-O-obtained in the same manner as in Example 5.
Galate glycosides 10 g, coupling sugar (registered trademark, manufactured by Hayashibara Co., Ltd.) 126 g, Oligomate 50 (trade name: Yakult Pharmaceutical Co., Ltd.) 136 g, lactose 6 g, aspartame (trade name, manufactured by Ajinomoto Co., Inc.) To 3 g, 150 g of water was added and mixed, and then dissolved while stirring. 4.5g of pectin was gradually added to this solution to dissolve it, then 50%
(W / v) 3.3 g of citric acid solution, 6 g of 1/5 concentrated lemon juice, 0.1 g of natural pigment, and lemon flavor of 0.
Add 2 g and mix well, pour this solution into a mold,
A pectin jelly was prepared by allowing it to cool at room temperature for 12 hours for solidification. This product is a jelly confectionery that has no astringency peculiar to polyphenols and has an excellent flavor. It is also suitable as a jelly confectionery having the functionality of polyphenols.

Example 9 Powdered lactic acid effervescent beverage 0.6 g of epigallocatechin 3-O-gallate glycoside and 2.7 g of sugar per cup were obtained by the same method as in Example 5.
Vitamin C 0.6 g, effervescent agent (baking soda) 0.6 g, acidulant (citric acid) 0.6 g, powdered fermented milk 0.5 g, and powdered flavor (strawberry) 0.2 g are uniformly mixed to prepare a powdered lactic acid effervescent beverage. I made a prototype. When this product was dissolved in 180 ml of cold water and drunk, it was a drink with a refreshing taste and aroma that was easy to drink. In addition, this product is an original polyphenol, for example, cholesterol elevation inhibitory action, blood glucose elevation inhibitory action,
It is an easy-to-drink powdered lactic acid foamed beverage that has antioxidant and other functionalities.

Example 10 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, and Example 6 were added. In a similar manner, 1 g of epigallocatechin gallate glycoside and purified water that had been heated and dissolved at 80 ° C. were added, and mixed and emulsified. After cooling to 30 ° C. with good stirring, the product was filled in a container. This product is a cosmetic cream that is unique to polyphenols and has functionality such as antioxidation and ultraviolet absorption.

[0052]

EFFECTS OF THE INVENTION The polyphenol glycoside of the present invention effectively improves the strong astringency and astringency of conventional polyphenols and is not only excellent in taste but also polyphenols in vivo. Since it can be expected to be released, it is a novel compound that can be applied to foods, luxury items, cosmetics, quasi drugs, pharmaceuticals, and the like.

[Brief description of drawings]

FIG. 1 (−)-epigallocatechin 3′-O-α-D
- it is a ¹ H-NMR spectrum of glucopyranoside.

FIG. 2 (−)-epigallocatechin 3′-O-α-D
-Is a ¹³ C-NMR spectrum of glucopyranoside.

FIG. 3 (−)-epigallocatechin 3′-O-α-D
-Mass spectrum of glucopyranoside.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C08B 37/00 C08B 37/00 G C12P 19/44 C12P 19/44 (72) Inventor Takahiko Bandai Okayama 1428 Masatsu, Okayama City, Japan (72) Inventor Takashi Shibuya 318 Shimohara, Soja City, Okayama Prefecture

Claims (9)

[Claims] 1. General formula 1 (wherein R ₁ and R ₂ are each independently a hydroxyl group or a maltooligosaccharide residue having a degree of polymerization of 2 to 10, X is a hydroxyl group or hydrogen, Y is a hydroxyl group or a galloyl group, and n is A polyphenol glycoside represented by the formula 1) to 9). Embedded image 2. The polyphenol glycoside has the formula 2 (wherein R ₁ and R ₂ are each independently a hydroxyl group or a polymerization degree 2).
To 10 of maltooligosaccharide residues, and n represents an integer of 1 to 9. The polyphenol glycoside according to claim 1, which is a gallocatechin glycoside represented by Embedded image 3. The polyphenol glycoside has the formula 3 (wherein R ₁ and R ₂ are each independently a hydroxyl group or a polymerization degree 2).
To 10 of maltooligosaccharide residues, and n represents an integer of 1 to 9. ) Gallocatechin 3-O-galle represented by
The polyphenol glycoside according to claim 1, which is a glycoside. Embedded image 4. The polyphenol glycoside has the formula 4 (wherein R ₁ and R ₂ are independently a hydroxyl group or a polymerization degree 2).
To 10 of maltooligosaccharide residues, and n represents an integer of 1 to 9. The polyphenol glycoside according to claim 1, which is a catechin 3-O-gallate glycoside represented by (4). Embedded image 5. The polyphenol glycoside according to claim 1, which has reduced astringency. 6. Epigallocatechin, gallocatechin, epigallocatechin 3-O-gallate, gallocatechin 3-O-
Galate, epicatechin, epicatechin 3-O-gallate
A cyclomaltodextrin glucanotransferase is allowed to act on dextrin, cyclodextrin, starch, or a mixture of polyphenols, catechin 3-O-gallate, or a polyphenol mixture containing two or more kinds of these, dextrin, cyclodextrin, starch, or a mixture thereof. A method for producing a polyphenol glycoside. 7. The method for producing a polyphenol glycoside according to claim 6, wherein the cyclomaltodextrin glucanotransferase is derived from Bacillus stearothermophilus. 8. A composition comprising the polyphenol glycoside according to claim 1. 9. The composition according to claim 8, wherein the composition is any one of food and drink, a luxury item, a cosmetic, a quasi drug, a drug, and a raw material thereof.