JP2005139124A - Silsesquioxane-polyphenol hybrid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a new polyphenol-polymer hybrid to expand polyphenol applications in view of the fact that polyphenols have been hopeful of application to the biomedical field through utilizing the effects such as anticancer effects and anti-inflammatory effects inherent thereto, and if such polyphenols are bound to organic polymers, the residence time of the resulting hybrids is estimated to be longer than that of low-molecular polyphenols, therefore the resulting hybrid polymers are hopeful of the increase in biochemical activity than low-molecular polyphenols, and technological development for the polymeric modification of low-molecular polyphenols including catechin, epigallocatechin gallate and rutin by hybridizing them with existing organic polymers has been desired. <P>SOLUTION: A silsesquioxane-polyphenol hybrid is provided. This hybrid is obtained from an amino group-bearing silsesquioxane derivative and a polyphenol. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a polyphenol-binding polymer useful for an antioxidant, a biocompatible material, a drug carrier, an antibacterial material, a deodorant and the like, and a method for producing the same.

  It has been conventionally known that polyphenols such as catechin, egalocatechin gallate, quercetin, hesperidin, and rutin contained in plants have antioxidant and bactericidal action. Furthermore, these include anti-cancer action, anti-inflammatory action, ultraviolet absorption action, strengthening of capillaries, suppression of blood pressure rise, stress suppression, female hormone balance adjustment, anti-tumor action, mutation suppression, blood cholesterol suppression, intestinal action It is also known to have such effects, and application to the biomedical field is expected. If such a polyphenol is combined with a high molecular organic polymer, it is estimated that the residence time in the cell is longer than that of the low molecular weight polyphenol. Therefore, the polyphenol-bonded polymer thus obtained is more vigorous than the low molecular weight polyphenol. Increased chemical activity is expected.

  For example, a polymer in which polyphenol such as catechin is bonded to polyacrylamide or polymethacrylamide and a production method thereof are known (Patent Document 1). However, the method of Patent Document 1 is limited to polyacrylamide and polymethacrylamide, and it is necessary to modify the polymer or monomer in order to bind the polyphenol. Naturally-occurring polyphenols are known that are water-soluble and have a molecular weight of 10,000 or more, but their structure is not clear. Therefore, it has been desired to develop a technology for polymerizing low molecular weight polyphenols, such as catechin, epigallocatechin gallate, and rutin, whose biochemical activities have been investigated in detail, by combining them with a wide range of existing high molecular organic polymers.

In order to solve such problems, a method for producing a polyamine-polyphenol hybrid by introducing polyphenol into a polyamine through a covalent bond by reacting polyamine and polyphenol using an oxidase catalyst has been found. It was. And it became clear that the obtained polyamine-polyphenol hybrid has high superoxide elimination ability (patent document 2). However, since the use of polyphenols is diverse as described above, the polyamine-polyphenol hybrid disclosed in Patent Document 2 cannot be used for all uses. The polyamine-polyphenol hybrid means a conjugate of polyamine and polyphenol.
JP-A-8-27226 JP2003-137925

  The present invention aims to develop new polyphenol-polymer conjugates and to expand the use of polyphenols.

The present invention has the following configuration. The term “silsesquioxane-polyphenol hybrid” used in the present invention means a silsesquioxane-polyphenol conjugate.
[1] A silsesquioxane-polyphenol hybrid obtained from a silsesquioxane derivative having an amino group and a polyphenol.

ここに、ｎは３〜１００の整数であり；ｍは０または１であり；Ｒ^２は反応性を有しない１価の有機基であり、同じであって異なっていてもよく；そして、Ｒ^１の少なくとも１つはアミノ基を有する基であり、Ｒ^１の残りはＲ^２と同様に定義される基である： [2] The silsesquioxane-polyphenol hybrid according to item [1], wherein the silsesquioxane derivative having an amino group is a compound represented by the formula (1):

Where n is an integer from 3 to 100; m is 0 or 1; R ² is a monovalent organic group having no reactivity and may be the same and different; ^At least one of 1 is a group having an amino group, and the rest of R ¹ is a group defined similarly to R ² :

  [3] The silsesquioxane-polyphenol hybrid according to item [2], wherein n is an integer of 4 to 12.

ここに、Ｒ^３の少なくとも１つは式（３）で表される基であり、Ｒ^３の残りは式（４）で表される基である：

ここに、Ｒ^２は式（１）におけるＲ^２と同一の意味を有し；Ｒ^４はアミノ基を有する基であり；そして、ｍは０または１である。 [4] The silsesquioxane-polyphenol hybrid according to item [2], wherein the silsesquioxane derivative having an amino group is a compound represented by formula (2):

Here, at least one of R ³ is a group represented by formula (3), and the rest of R ³ is a group represented by formula (4):

Here, ^{R 2} has the same meaning as ^{R 2} in Formula (1); ^{R 4} is a group having an amino group; and, m is 0 or 1.

ここに、Ｒ^２は反応性を有しない１価の有機基であり、同じであっても異なっていてもよく；Ｒ^５はＲ^２またはＲ^４であり；そして、Ｒ^４はアミノ基を有する基である： [5] The silsesquioxane-polyphenol hybrid according to item [1], wherein the silsesquioxane derivative having an amino group is a compound represented by formula (5):

Where R ² is a monovalent organic group having no reactivity and may be the same or different; R ⁵ is R ² or R ⁴ ; and R ⁴ has an amino group The group is:

ここに、Ｒ^２は反応性を有しない１価の有機基であり、同じであっても異なっていてもよく；Ｒ^５はＲ^２またはＲ^４であり；そして、Ｒ^４はアミノ基を有する基である： [6] The silsesquioxane-polyphenol hybrid according to item [1], wherein the silsesquioxane derivative having an amino group is a compound represented by formula (6):

Where R ² is a monovalent organic group having no reactivity and may be the same or different; R ⁵ is R ² or R ⁴ ; and R ⁴ has an amino group The group is:

ここに、Ｒ^２は反応性を有しない１価の有機基であり、同じであっても異なっていてもよく；Ｒ^５はＲ^２またはＲ^４であり；そして、Ｒ^４はアミノ基を有する基である： [7] The silsesquioxane-polyphenol hybrid according to item [1], wherein the silsesquioxane derivative having an amino group is a compound represented by formula (7):

Where R ² is a monovalent organic group having no reactivity and may be the same or different; R ⁵ is R ² or R ⁴ ; and R ⁴ has an amino group The group is:

  [8] The polyphenol is selected from catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, quercetin, hesperidin, tannic acid, theaflavin, procyanidin, leucoanthocyanidin and rutin The silsesquioxane-polyphenol hybrid according to any one of [1] to [7], which is at least one.

  [9] The silsesquioxane-polyphenol hybrid according to any one of [1] to [7], wherein the polyphenol is catechin.

  [10] The silsesquioxane-polyphenol hybrid according to item [4], wherein the silsesquioxane derivative having an amino group is a compound represented by formula (2), and the polyphenol is catechin.

ここに、Ｒ^６は３−アミノプロピル基である。 [11] The silsesquioxane-polyphenol hybrid according to item [4], wherein the silsesquioxane derivative having an amino group is a compound represented by formula (8), and the polyphenol is catechin.

Here, R ⁶ is a 3-aminopropyl group.

  [12] The process for producing a silsesquioxane-polyphenol hybrid according to item [1], wherein a polyphenol is reacted with a silsesquioxane derivative having an amino group in the presence of an enzyme catalyst.

  The present invention provides a silsesquioxane-polyphenol hybrid having high antioxidant properties and enzyme inhibitory ability.

First, terms used in the present invention will be described.
“Silsesquioxane” is a class name indicating a compound in which each silicon atom is bonded to three oxygen atoms, and each oxygen atom is bonded to two silicon atoms. This term is used as a general term for an oxane structure and a silsesquioxane-like structure in which a part thereof is deformed. In any case, the alkyl and alkylene in the present invention may be a straight-chain group or a branched group. This is the same when any hydrogen in these groups is replaced with a halogen or a cyclic group, or when any —CH ₂ — is replaced with —O—, cycloalkylene, or the like. “Arbitrary” indicates that not only the position but also the number is arbitrary. Then, a plurality of hydrogen or -CH 2 _- when is replaced by other groups may be replaced by different groups. Note that the description that any —CH ₂ — is replaced by —O— does not include the replacement of a plurality of consecutive —CH ₂ — with —O—.

  The silsesquioxane-polyphenol hybrid of the present invention can be obtained by reacting a silsesquioxane derivative having an amino group with polyphenol. The silsesquioxane having an amino group is not particularly limited. However, when controlling the molecular weight and shape, a silsesquioxane having a cage-type or incomplete cage-type structure is preferable.

式（１）において、ｎは３〜１００の整数であり、４〜１２が好ましい。ｍは０または１である。Ｒ^２は反応性を有しない１価の有機基であり、同じであっても異なっていてもよい。そして、Ｒ^１の少なくとも１つはアミノ基を有する基であり、Ｒ^１の残りはＲ^２と同様に定義される基である： The first preferred example of such a silsesquioxane derivative is a compound represented by the formula (1). In the following description, the compound represented by formula (1) may be referred to as compound (1). The compounds represented by other formulas may be represented according to the same notation as in the case of the compound represented by formula (1).

In Formula (1), n is an integer of 3-100, and 4-12 are preferable. m is 0 or 1. R ² is a monovalent organic group having no reactivity and may be the same or different. And at least one of R ¹ is a group having an amino group, and the rest of R ¹ is a group defined similarly to R ² :

One preferred example of R ² is alkyl having 1 to 10 carbon atoms. Any hydrogen in the alkyl may be replaced with fluorine, and any —CH ₂ — may be replaced with —O—. Specific examples of unsubstituted alkyl among such alkyl include methyl, ethyl, propyl, 1-methylethyl, butyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, hexyl, 1,1,2- Trimethylpropyl, heptyl, octyl, 2,4,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, docosyl, methyloxypropyl, ethyloxypropyl, propyloxypropyl, methyloxybutyl, Ethyloxybutyl, propyloxybutyl, methyloxyisobutyl, ethyloxyisobutyl, propyloxyisobutyl, trifluoromethyl, 2-fluoroethyl, 2,2-difluoroethyl, 3,3,3-trifluoropropyl, hexaf Oropropyl, tridecafluoro-1,1,2,2-tetrahydrooctyl, nonafluoro-1,1,2,2-tetrahydrohexyl, heptadecafluoro-1,1,2,2-tetrahydrodecyl, 2-fluoroethyloxy Propyl, 2,2,2-trifluoroethyloxypropyl, 2-fluoro-1-fluoromethylethyloxypropyl, 2,2,3,3-tetrafluoropropyloxypropyl, 2,2,3,3,3- Pentafluoropropyloxypropyl, hexafluoroisopropyloxypropyl, hexafluorobutyloxypropyl, heptafluorobutyloxypropyl, octafluoroisobutyloxypropyl, octafluoropentyloxypropyl, 2-fluoroethyloxybutyl, 2,2,2-tri Fluoroethyloxybutyl, 2-fluoro-1-fluoromethylethyloxybutyl, 2,2,3,3-tetrafluoropropyloxybutyl, 2,2,3,3,3-pentafluoropropyloxybutyl, hexafluoro Isopropyloxybutyl, hexafluorobutyloxybutyl, heptafluorobutyloxybutyl, octafluoroisobutyloxybutyl, octafluoropentyloxybutyl, 2-fluoroethyloxyisobutyl, 2,2,2-trifluoroethyloxyisobutyl, 2-fluoro -1-fluoromethylethyloxyisobutyl, 2,2,3,3-tetrafluoropropyloxyisobutyl, 2,2,3,3,3-pentafluoropropyloxyisobutyl, hexafluoroisopropyloxyisobutyl Hexafluorobutyloxyisobutyl, heptafluorobutyloxyisobutyl, octafluoroisobutyloxyisobutyl, and octafluoropentyloxyisobutyl. Of these groups, more preferred examples are methyl, ethyl, isopropyl, tert-butyl and 3,3,3-trifluoropropyl.

Another preferred example of R ² is substituted or unsubstituted phenyl. Preferred examples of substituted phenyl are phenyl in which any hydrogen is replaced with halogen, methyl or methoxy. In phenyl having a plurality of substituents, these substituents may be the same group or may be composed of different groups. Preferred examples of substituted or unsubstituted phenyl include phenyl, pentafluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-methylphenyl, 4-methoxyphenyl, 3-chloro-4-methylphenyl, and 2,5-dichloro. -4-methylphenyl, 3,5-dichloro-4-methylphenyl, 2,3,5-trichloro-4-methylphenyl, 2,3,6-trichloro-4-methylphenyl, 3-bromo-4-methyl Phenyl, 2,5-dibromo-4-methylphenyl, 3,5-dibromo-4-methylphenyl, 2,3-difluoro-4-methylphenyl, 3-chloro-4-methoxyphenyl, 3-bromo-4- Methoxyphenyl, 3,5-dibromo-4-methoxyphenyl, and 2,3-difluoro-4-methoxyphenyl. The most preferred example of substituted or unsubstituted phenyl is phenyl.

Yet another preferred example of R ² is cycloalkyl having 4 to 8 carbon atoms. In this cycloalkyl, two non-adjacent carbon atoms may be bridged. Specific examples of such cycloalkyl are cyclobutyl, cyclopentyl, cyclohexyl, 2-bicycloheptyl, and cyclooctyl. More preferred examples of cycloalkyl are cyclopentyl and cyclohexyl.
And more preferred examples of R ² are methyl, tert-butyl and phenyl.

One preferred example of a group having an amino group is an aminoalkyl having 1 to 10 carbon atoms. In this aminoalkyl, arbitrary —CH ₂ — may be replaced by —NH—. Specific examples of such aminoalkyl include aminomethyl, 3-aminopropyl, 4-aminobutyl, N- (2-aminoethyl) aminomethyl, N- (2-aminoethyl) -3-aminopropyl, N- (2-aminoethyl) -3-aminoisobutyl, and N- (6-aminohexyl) -3-aminopropyl. Another preferred example of a group having an amino group is aminophenyl. Specific examples of aminophenyl are 3-aminophenyl and 4-aminophenyl.

The silsesquioxane derivative represented by the formula (1) is obtained by hydrolyzing a trialkoxysilane represented by R ¹ Si (OR ⁷ ) ₃ . R ⁷ is alkyl, preferably methyl or ethyl. When trialkoxysilane having an amino functional group and R ² Si (OR ⁷ ) ₃ are mixed and hydrolyzed, a silsesquioxane derivative in which a part of R ¹ is R ² is obtained.

In the silsesquioxane derivative in which m is 1 in the formula (1), for example, octakis (tetramethylammonium) octasilsesquioxane is obtained by reacting tetraethoxysilane with tetramethylammonium hydroxide in methanol. The compound obtained by synthesis is reacted with H—SiR ² ₂ —Cl to obtain a Si—H functional silsesquioxane derivative, which is obtained by hydrosilylation reaction of a compound having an amino group and an alkenyl group. It is done.

式（２）において、Ｒ^３の少なくとも１つは式（３）で表される基であり、Ｒ^３の残りは式（４）で表される基である：

式（３）および式（４）において、Ｒ^２は式（１）におけるＲ^２と同様に定義される基であり、Ｒ^４はアミノ基を有する基であり、そしてｍは０または１である。アミノ基を有する基の例は前記の通りである。 A preferred example of the silsesquioxane derivative represented by the formula (1) is a compound represented by the formula (2).

In formula (2), at least one of R ³ is a group represented by formula (3), and the rest of R ³ is a group represented by formula (4):

In the formula (3) and (4), R ² represents a group defined as for R ² in Formula (1), R ⁴ is a group having an amino group, and m is 0 or 1 . Examples of the group having an amino group are as described above.

式（５）において、Ｒ^２は式（１）におけるＲ^２と同様に定義される基であり、同じであっても異なっていてもよく、Ｒ^５はＲ^２またはＲ^４であり、そしてＲ^４は式（３）におけるＲ^４と同様に定義される基である。 A second preferred example of the silsesquioxane derivative having an amino group is a compound represented by the formula (5).

In the formula (5), ^{R 2} is a group defined as for ^{R 2} in Formula (1) or different be the same, ^{R 5} is ^{R 2} or ^{R 4,} and R ⁴ is a group defined in the same manner as R ⁴ in formula (3).

式（Ａ）において、Ｒ^２は式（１）におけるＲ^２と同様に定義される基であり、Ｍは１価のアルカリ金属原子である。 Compound (5) can be obtained by reacting compound (A) with a compound represented by R ⁴ R ⁵ SiCl ₂ . At this time, the amino group in R ⁴ R ⁵ SiCl ₂ needs to be protected with trimethylsilyl or the like. A dichlorosilane compound having a cyano functional group or a nitro functional group may be used in place of the amino group, and the cyano group or nitro group may be converted into an amino group by hydrogen reduction after the reaction.

In formula (A), R ² represents a group defined as for R ² in formula (1), M is a monovalent alkali metal atom.

The compound (A) is obtained by hydrolyzing R ² —SiR ⁸ ₃ with a monovalent alkali metal hydroxide and water in the presence of alcohols and condensing them. R ^{8 in} this formula is a hydrolyzable group, and alkoxy having 1 to 4 carbon atoms is preferable. Examples of alkali metal hydroxides are sodium hydroxide and potassium hydroxide.

式（６）におけるＲ^２、Ｒ^４およびＲ^５は、式（５）におけるこれらの記号と同様に定義される基である。化合物（６）は、前記の化合物（Ａ）とＲ^４Ｒ^５ _２ＳｉＣｌで表される化合物を反応させることによって得られる。 A third preferred example of the silsesquioxane derivative having an amino group is a compound represented by the formula (6).

R ² , R ⁴ and R ⁵ in the formula (6) are groups defined similarly to these symbols in the formula (5). Compound (6) can be obtained by reacting the compound (A) with a compound represented by R ⁴ R ⁵ ₂ SiCl.

式（７）におけるＲ^２、Ｒ^４およびＲ^５は、式（５）におけるこれらの記号と同様に定義される基である。 A fourth preferred example of the silsesquioxane derivative having an amino group is a compound represented by the formula (7).

R ² , R ⁴ and R ⁵ in the formula (7) are groups defined in the same manner as those symbols in the formula (5).

式（Ｂ）において、Ｒ^２は式（１）におけるＲ^２と同様に定義される基であり、Ｍは１価のアルカリ金属原子である。 Compound (7) is obtained by reacting compound (B) with a compound represented by R ⁴ R ⁵ ₂ SiCl. At this time, the amino group in R ⁴ R ⁵ ₂ SiCl needs to be protected with trimethylsilyl or the like. A chlorosilane compound having a cyano functional group or a nitro functional group may be used in place of the amino group, and the cyano group or nitro group may be converted into an amino group by hydrogen reduction after the reaction.

In the formula (B), R ² represents a group defined as for R ² in formula (1), M is a monovalent alkali metal atom.

Similarly to the compound (A), the compound (B) can also be obtained by hydrolyzing and condensing R ² —SiR ⁸ ₃ with a monovalent alkali metal hydroxide and water in the presence of an alcohol. . The only difference from the case of the compound (A) is the molar ratio of the alkali metal hydroxide to R ² —SiR ⁸ ₃ . The most preferred value of this molar ratio in the production of compound (B) is 1/2. In the case of the compound (A), the most preferable molar ratio is 2/3.

  Of compound (1), compound (5), compound (6) and compound (7), compound (1) is most preferred in that more polyphenols can be added in one molecule.

  The polyphenol used in the present invention is preferably water-soluble. And when high antioxidant provision is required, the polyphenol which has a flavonoid skeleton is preferable. Examples of such polyphenols are catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, quercetin, hesperidin, tannic acid, theaflavin, procyanidins, leucoanthocyanidins and rutin It is. These can be used alone or as a mixture. The preferred use amount of the polyphenol is in the range of 1 mg to 10 g with respect to 1 g of the amino-functional silsesquioxane derivative.

  The silsesquioxane-polyphenol hybrid of the present invention is obtained by reacting an amino group of a silsesquioxane derivative with a polyphenol in the presence of an enzyme catalyst. Examples of enzymes that can be used as enzyme catalysts are oxidases, hydrolases, transferases, desorption enzymes and isomerases. From the viewpoint of the oxidation catalyst function of polyphenols, an oxidase is preferable.

  Examples of oxidase are tyrosinase (EC 1.14.18.1), phenolase, laccase and bilirubin oxidase, but are not particularly limited as long as they have an oxidizing ability sufficient to cause oxidation of polyphenols. . And the usage-amount of an enzyme is 1-1 million units with respect to 1g of silsesquioxane derivatives which have an amino group. A preferable range of the amount used is 3 units to 500000 units, and a more preferable range is 5 units to 200000 units.

  A solvent is preferably used for the reaction between the silsesquioxane derivative having an amino group and polyphenol. As the solvent, those that dissolve both the silsesquioxane derivative having an amino group, the polyphenol, and the enzyme catalyst are preferable. When the silsesquioxane derivative having an amino group is water-soluble, water or a mixture of an organic solvent and water can be used. The water may be distilled water or deionized water, but a buffer may be used instead of water. When using a buffer solution, the range of pH 2 to pH 12 is preferable.

  Examples of organic solvents that can be used by mixing with water are methanol, ethanol, 2,2,2-trifluoroethanol, N, N-dimethylformamide, N, N-dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone. , Nitromethane, nitrobenzene, pyridine, 1,4-dioxane, acetone and methyl ethyl ketone. These can also be used alone. What is necessary is just to adjust the mixing ratio of an organic solvent and water so that the silsesquioxane derivative which has an amino group, polyphenol, and an enzyme catalyst may melt | dissolve together.

  The reaction temperature is preferably a temperature at which the enzyme catalyst is not inactivated. Preferably it is the range of -20-100 degreeC, More preferably, it is the range of 0-60 degreeC. When the reaction temperature is high, the enzyme is generally deactivated, but depending on the type of solvent, the reaction can be performed at a higher temperature.

  The amount of polyphenol bonds in the silsesquioxane-polyphenol hybrid of the present invention is in the range of 0.01 mol% to 100 mol% with respect to the amino group in the silsesquioxane derivative. A preferable range of this value is 0.05 mol% to 100 mol%, and a more preferable range is 0.1 to 100 mol%.

これらの式におけるＱは、ポリフェノールを示す式からジヒドロフェニル基を除いた残基である。 In addition, when the polyphenol is represented by Formula (C), it is estimated that the coupling | bonding mode of the polyphenol in the silsesquioxane-polyphenol hybrid of this invention will be either of the following, when patent document 2 is referred.

Q in these formulas is a residue obtained by removing a dihydrophenyl group from formulas representing polyphenols.

式（９）におけるＲ^９は３−アミノプロピル基である。 <Synthesis of Silsesquioxane Derivative Having Amino Group>
According to Frank J. Feher, Chem. Commun., 1998, 323, a silsesquioxane derivative having an amino group was synthesized by the following procedure. 3-Aminopropyltriethoxysilane (150 ml) and concentrated hydrochloric acid (200 ml) were dissolved in 3.6 liters of methanol and allowed to stand at room temperature for 6 weeks to obtain fine crystals. Fine crystals obtained by removing the solvent were washed with methanol and dried at room temperature under a pressure of 1.33 Pa to obtain a compound represented by the formula (9). The yield was 30%.

R ⁹ in Formula (9) is a 3-aminopropyl group.

<Synthesis of Silsesquioxane-Catechin Hybrid>
An aqueous solution (150 μl) containing compound (9) (0.4 mmol), catechin (1.5 mmol), and laccase derived from Myceliophthora (150 units) was added to a water / methanol mixed solvent (mixing ratio 5/1; 12 ml). Thereafter, the pH was adjusted to 7 using 6N hydrochloric acid. The resulting solution was allowed to stand at room temperature for 24 hours with gentle stirring, and then unreacted substances were removed by ultrafiltration using a cutoff membrane having a molecular weight of 500. Thereafter, the obtained reaction solution was lyophilized to isolate the polymer, and a silsesquioxane-catechin hybrid was obtained (hereinafter, this compound may be abbreviated as SC-L). The yield was 23%. As a result of measuring the visible / ultraviolet absorption spectrum of SC-L, it was found that absorption specific to catechin disappeared as shown in FIG. 1, and absorption of silsesquioxane-catechin hybrid was newly generated.
The catechin content determined by elemental analysis was 43 mol% with respect to the amino group in the silsesquioxane derivative.

<Synthesis of Silsesquioxane-Catechin Hybrid>
Compound (9) (0.4 mmol), catechin (1.5 mmol) and horseradish peroxidase (5 mg) were added to a water / methanol mixed solvent (mixing ratio 5/1; 12 ml), and 6N hydrochloric acid was used. The pH was adjusted to 7. While gently stirring the resulting solution, 51 μl of 30% aqueous hydrogen peroxide was added to this four times every 5 minutes. Then, after standing at room temperature for 1 hour, unreacted substances were removed by ultrafiltration using a cutoff membrane having a molecular weight of 500. Thereafter, the resulting reaction solution was lyophilized to isolate the polymer, thereby obtaining a silsesquioxane-catechin hybrid (hereinafter, this compound may be abbreviated as SC-H). The yield was 43%. As a result of measuring the visible-ultraviolet absorption spectrum of SC-H, it was found that the absorption specific to catechin disappeared in the same manner as in Example 2, and the absorption of the silsesquioxane-catechin hybrid was newly generated.
The catechin content determined by elemental analysis was 25 mol% with respect to the amino group in the silsesquioxane derivative.

<Inspection of superoxide anion scavenging ability>
The superoxide anion was generated in the xanthine / xanthine oxidase system, and the superoxide anion scavenging ability of the silsesquioxane-catechin hybrid and catechin produced in Examples 2 and 3 was examined by chemiluminescence method.
(1) Preparation of reagents
Buffer: A 100 mM aqueous solution of KH ₂ PO ₄ is prepared. To 200 ml of this aqueous solution, 2.9 mg (0.01 mmol) of EDTA was added.
XO solution: 30 mg of Xanthine oxidase (from Butter milk, 0.25 U / mg, Wako Pure Chemical Industries) was dissolved in 50 ml of the previously prepared Buffer. This was stored at room temperature for use.
Xathine solution: 7.5 mg (0.05 mmol) of Xanthine was dissolved in 600 μl of 1N aqueous sodium hydroxide solution. After adding 7.5 ml of distilled water, 16.9 ml of Buffer was further added to make the total volume 25 ml. This was stored and used in the refrigerator.
MPEC solution: 2.8 mg of MPEC (2-methyl-p-methoxyphenylethynylimidazopyrazinon) was dissolved in 10 ml of methanol. This was diluted 3 times with water and used.
(2) Preparation of sample solution 6 mM DMSO solutions of catechin, SC-L prepared in Example 2 and SC-H prepared in Example 3 were prepared. Each solution was appropriately diluted with Buffer, and a sample solution was prepared so that the concentration shown in FIG.
(3) Measurement First, an Xanthine solution is introduced into a luminescence measuring device (photon counter MTP-700CL (manufactured by Corona Electric Co., Ltd.)). A 96-well microplate is mixed with 10 μl of sample solution, 170 μl of Buffer, 60 μl of XO solution, and 10 μl of MPEC solution. The amount of luminescence immediately after 50 μl of the Xanthine solution is dispensed into each well of the microplate is measured for 30 seconds. The erasure rate was calculated by the following formula using this light emission amount.
SOD-like activity (%)
= 100 × (emission level (control) −emission level (sample)) / emission level (control)
The erasure rate at each concentration is shown in FIG. As a result, it was found that by using a silsesquioxane-catechin hybrid, the SDO-like activity was greatly improved over catechin itself.

<Examination of xanthine oxidase enzyme inhibition>
Instead of measuring the chemiluminescence intensity using MPEC, it was found by measuring the absorption intensity of uric acid that it has the ability to inhibit xanthine oxidase enzyme that was not expressed by catechin alone as shown in FIG.
(1) Preparation of reagents
Buffer: A 100 mM aqueous solution of KH ₂ PO ₄ is prepared. To 200 ml of Buffer, 2.9 mg (0.01 mmol) of EDTA was added.
XO solution: 30 mg of Xanthine oxidase (from Butter milk, 0.25 U / mg, Wako Pure Chemical Industries) was dissolved in 50 ml of the buffer prepared above. This was stored at room temperature for use.
Xanthine solution: 7.5 mg (0.05 mmol) of Xanthine was dissolved in 600 μl of 1N aqueous sodium hydroxide solution. After adding 7.5 ml of distilled water, 16.9 ml of Buffer was further added to make the total volume 25 ml. This was stored and used in the refrigerator.
(2) Preparation of sample solution Catechin, SC-L prepared in Example 2 and SC-H prepared in Example 3 were dissolved in DMSO, and the sample solution was prepared so as to have the concentration shown in FIG. did.
(3) Measurement In a 1.5 ml quartz cell for UV measurement, 740 μl of Buffer, 20 μl of sample solution and 240 μl of XO solution are mixed and pre-incubated at 37 ° C. for 5 minutes. The absorbance at 295 nm is measured for 5 minutes immediately after adding 200 μl of the Xanthine solution, and the increase in absorbance is read. For the control, use only the solvent used to dissolve the sample instead of the sample solution. Using this absorbance, the inhibition rate was determined from the following formula.
Inhibitory ability (%) ＝
100 × (absorbance (control) −absorbance (sample)) / absorbance (control)
The inhibitory ability at each concentration is shown in FIG. As can be seen from FIG. 3, catechin alone showed almost no inhibitory ability, but it was found that the inhibitory ability was expressed by using a silsesquioxane-catechin hybrid.

<Measurement of collagenase enzyme inhibition>
Enzchek Gelatinase / Collagenase Assay Kit (Molecular Probes) is used.
(1) Preparation of reagents 1X Buffer: Diluted by adding distilled water (8 ml) to 10X Buffer (2 ml) attached to the above kit.
DQ gelatin solution: NaN ₃ solution was prepared by adding 10 ml of distilled water to 1.3 mg of NaN ₃ . One ml of the attached DQ gelatin was added with 1 ml of NaN ₃ solution and sonicated at 50 ° C. for 5 minutes to completely dissolve. This was diluted 2-fold with 1X Buffer.
Collagenase solution: 0.5 ml of distilled water is added to one attached Collagenase (Type IV from Clostridium histolyticum 500 U). Dilute 100 times with 1X Buffer. This was stored in the refrigerator.
At the time of use, it was further diluted 50 times with 1 × Buffer (it becomes 0.2 U / ml).
(2) Preparation of sample solution A predetermined amount of catechin and silsesquioxane-catechin hybrid (pH 8) were dissolved in distilled water, and a sample solution was prepared so as to have a concentration shown in FIG.
(3) Measurement In a 96-well microplate, 60 μl of 1 × Buffer, 20 μl of sample solution having different concentrations and 20 μl of DQ gelatin solution are mixed. Finally, incubate in the dark at room temperature for 30 minutes after adding 100 μl of collagenase solution. The fluorescence intensity after 30 minutes is measured. (Absorption maximum: 495 nm, emission maximum: 515 nm) For the control, a solution containing only the solvent used to dissolve the sample is used instead of the sample solution. Using this fluorescence intensity, the inhibition ability was determined from the following formula.
Inhibitory capacity (%)
= 100 × (fluorescence intensity (control) −fluorescence intensity (sample)) / fluorescence intensity (control)
The inhibitory ability at each concentration is shown in FIG.
It was found that the collagenase / collagen system has the ability to inhibit the collagenase enzyme that was hardly expressed by catechin alone.

  The silsesquioxane-polyphenol hybrid of the present invention is obtained by introducing a polyphenol having a known biochemical function into a polymer, and an effect of enhancing the activity of the polyphenol and an effect of developing a novel action such as inhibition of enzyme activity. There is. And since the residence time in a living body can be extended, it can be used for various uses, such as an antioxidant, a biocompatible material, a drug carrier, an antibacterial agent, and a cosmetic material.

The absorption spectrum of catechin and silsesquioxane-catechin hybrid is shown. The superoxide anion scavenging ability of catechin and silsesquioxane-catechin hybrid is shown. The ability of catechin and silsesquioxane-catechin hybrids to inhibit xanthine oxidase is shown. Fig. 3 shows the ability of catechin and silsesquioxane-catechin hybrids to inhibit collagenase.

Explanation of symbols

SC-L: Silsesquioxane-catechin hybrid synthesized in Example 2 SC-H: Silsesquioxane-catechin hybrid synthesized in Example 3

Claims (12)

  A silsesquioxane-polyphenol hybrid obtained from a silsesquioxane derivative having an amino group and a polyphenol. The silsesquioxane-polyphenol hybrid according to claim 1, wherein the silsesquioxane derivative having an amino group is a compound represented by the formula (1):

Where n is an integer from 3 to 100; m is 0 or 1; R ² is a monovalent organic group having no reactivity and may be the same and different; ^At least one of 1 is a group having an amino group, and the rest of R ¹ is a group defined similarly to R ² :   The silsesquioxane-polyphenol hybrid according to claim 2, wherein n is an integer of 4 to 12. The silsesquioxane-polyphenol hybrid according to claim 2, wherein the silsesquioxane derivative having an amino group is a compound represented by the formula (2):

Here, at least one of R ³ is a group represented by formula (3), and the rest of R ³ is a group represented by formula (4):

Here, ^{R 2} has the same meaning as ^{R 2} in Formula (1); ^{R 4} is a group having an amino group; and, m is 0 or 1. The silsesquioxane-polyphenol hybrid according to claim 1, wherein the silsesquioxane derivative having an amino group is a compound represented by the formula (5):

Where R ² is a monovalent organic group having no reactivity and may be the same or different; R ⁵ is R ² or R ⁴ ; and R ⁴ has an amino group The group is: The silsesquioxane-polyphenol hybrid according to claim 1, wherein the silsesquioxane derivative having an amino group is a compound represented by formula (6):

Where R ² is a monovalent organic group having no reactivity and may be the same or different; R ⁵ is R ² or R ⁴ ; and R ⁴ has an amino group The group is: The silsesquioxane-polyphenol hybrid according to claim 1, wherein the silsesquioxane derivative having an amino group is a compound represented by formula (7):

Where R ² is a monovalent organic group having no reactivity and may be the same or different; R ⁵ is R ² or R ⁴ ; and R ⁴ has an amino group The group is:   At least one selected from polycatechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, quercetin, hesperidin, tannic acid, theaflavin, procyanidin, leucoanthocyanidin and rutin The silsesquioxane-polyphenol hybrid according to any one of claims 1 to 7.   The silsesquioxane-polyphenol hybrid according to any one of claims 1 to 7, wherein the polyphenol is catechin.   The silsesquioxane-polyphenol hybrid according to claim 4, wherein the silsesquioxane derivative having an amino group is a compound represented by the formula (2), and the polyphenol is catechin. The silsesquioxane-polyphenol hybrid according to claim 4, wherein the silsesquioxane derivative having an amino group is a compound represented by formula (8), and the polyphenol is catechin.

Here, R ⁶ is a 3-aminopropyl group. The method for producing a silsesquioxane-polyphenol hybrid according to claim 1, wherein a polyphenol is reacted with a silsesquioxane derivative having an amino group in the presence of an enzyme catalyst.

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