JP2011256165A - Novel methylated theaflavin, and composition containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel methylated theaflavin which has one or two or more hydroxyl groups having a methylated galloyl group, and has excellent stability and functionality.SOLUTION: The methylated theaflavin which has one or two or more hydroxyl groups having a methylated galloyl group is a compound represented by one formula of formulae (I) to (III) wherein in the formula (I), Rto Rdenote each hydrogen or methyl, and at least two of Rto Rare methyl; in the formula (II), Rto Rdenote each hydrogen or methyl, and at least one of Rto Ris methyl; and in the formula (III), Rto Rdenote each hydrogen or methyl, and at least one of Rto Ris methyl, excluding that Ronly is methyl.

Description

  The present invention relates to a novel methylated theaflavin having a galloyl group in which one or more hydroxyl groups are methylated.

  One of the components mainly contained in plant fruits and leaves is polyphenol. Examples of polyphenols include flavones, flavonols, flavanones, isoflavones, anthocyanins, flavanols, ellagitannins, phenylpropanoids, anthocyanidins, proanthocyanidins, chalcones, aurones, phenylethanoids, etc. Is mentioned. More specifically, gallic acid, ellagic acid, hydroxytyrosol, epigallocatechin-3-O-gallate (EGCG), epicatechin-3-O-gallate (ECG), catechin-3-O-gallate (CG) ), Gallocatechin-3-O-gallate (GCG), epicatechin (EC), catechin (C), epigallocatechin (EGC), gallocatechin (GC), chromanin, delphinidin, delphinidin 3-O-glucoside, procyanidin B2 ( PB2), caffeic acid, chlorogenic acid, rosmarinic acid, caffeic acid phenethyl ester, strictinin, quercetin, isoquercitrin, rutin, myricetin, butein, sulfretin, luteolin, eriodictyol and the like. These polyphenols are characterized by having multiple phenolic hydroxyl groups in their structure, and have antioxidant activity, blood pressure rise inhibition, blood sugar level rise inhibition, internal fat accumulation inhibition, arteriosclerosis inhibition, anti-inflammatory action, anticancer activity Various functions such as allergy suppression, anti-cariogenic activity, deodorant activity, antibacterial activity, etc. have been studied and reported. In addition to these polyphenols, various functionalities have been studied and reported for plant-derived components.

  Among these polyphenols, there are many reports that methylated polyphenols having a hydroxyl group modified with a methyl group are superior in absorbability, metabolic stability, functionality, etc. compared to polyphenols not modified with a methyl group. ing. For example, epigallocatechin-3-O- (3-O-methyl) gallate (EGCG3) is used for tea varieties such as “Aoshin Daipan”, “Benihomare”, “Benifuuji”, “Benifuuki”, etc. Me) and epigallocatechin-3-O- (4-O-methyl) gallate (EGCG4 "Me), but when these methylated catechins were orally administered to mice, 60 minutes after administration It has been reported that the blood methylated catechin concentration is about 9 times higher in the free form compared to epigallocatechin-3-O-gallate (EGCG) (see, for example, Non-Patent Document 1). ). In addition, when 7-hydroxyflavone, 7,4′-dihydroxyflavone, chrysin, and apigenin were compared with these methylated products, the methylated product had higher absorption in the human intestinal absorption model test using Caco-2 cells. It has also been reported that it exhibits good results in metabolic stability tests using human liver tissue-derived S9 fraction (see, for example, Non-Patent Document 2). Further, regarding the histamine release inhibition test using mouse mast cells, EGCG3 "Me, epigallocatechin-3-O- (3,5-O-dimethyl) gallate, and epi (3-O-methyl) gallocatechin-3 It has also been reported that -O- (3,5-O-dimethyl) gallate showed a high antiallergic action as compared with EGCG and showed a tendency that the action became stronger as the methyl group increased ( For example, see Patent Document 1.)

  Theaflavin, a kind of black tea polyphenol, has also been reported to have various functionalities such as fat absorption inhibitory action, antioxidant action, blood sugar level increase inhibitory action, and antitumor action. In order to effectively exert these functions in vivo, it is considered important to take a sufficient amount of theaflavin. However, the theaflavin content in black tea is low and it is easily degraded by oxidation. For this reason, even if tea is eaten or consumed as it is, it is difficult to take a large amount of theaflavin stably.

It is considered that the metabolic stability and functionality of theaflavin can be improved by methylation in the same manner as catechin. Actually, it has been reported that black tea contains methylated theaflavin, which is a kind of methylated polyphenol, but its content is about 0.05% in dry tea leaves, which is even lower than normal theaflavin. (For example, refer nonpatent literature 3.).
For this reason, it is difficult to obtain a large amount of methylated theaflavin from black tea.

As a method for artificially obtaining methylated theaflavin, a method in which methylated catechin such as epigallocatechin 3-O- (3-O-methyl) gallate, epicatechin, and epicatechin gallate are used as raw materials and polymerized by oxidase (For example, refer nonpatent literature 3).
However, in such a method using a naturally rare methylated catechin as a raw material, it is difficult to obtain the raw material in the first place, and the synthesized methylated theaflavin is classified into the type of methylated catechin used as a raw material. Dependent. For this reason, only theaflavin 3-O- (3-O-methyl) gallate, theaflavin 3-O- (3-O-methyl) gallate, and 3′-O-gallate have been obtained so far. In Non-Patent Document 3, the functionality of methylated theaflavin is not clarified.

JP 2008-189628 A

Sano, 3 others, FRAGRANCE JOURNAL, 2000, No. 4, pp. 46-52. Wen, 1 other, Drug Metabolism and Disposition, 2006, Vol. 34, No. 10, pp. 1786-1792. Nishimura, 5 others, Journal of Agricultural and Food Chemistry 2007, 55, 7252-7257.

  Polyphenols such as theaflavin have a very large number of functions, but these functions may vary depending on the type of polyphenol. Similarly, in methylated polyphenols, there is a high possibility that the action effect and activity intensity differ depending on which hydroxyl group in the polyphenol is methylated. For this reason, it is expected that methylated theaflavins with more excellent metabolic stability and functionality can be obtained by methylating various theaflavins.

  The present invention has been made in view of the above problems, and provides a novel methylated theaflavin having a galloyl group in which one or more hydroxyl groups are methylated and having excellent stability and functionality. With the goal.

  As a result of intensive studies to solve the above problems, the present inventors have obtained a novel methyltransferase capable of efficiently methylating hydroxyl groups of polyphenols such as theaflavin, and using the enzyme as a substrate, theaflavin is used as a substrate. The present invention was completed by synthesizing a novel methylated theaflavin by performing the enzymatic reaction method described above.

That is, the present invention
(1) A methylated theaflavin having a galloyl group in which one or two or more hydroxyl groups are methylated, and is represented by any one of the following general formulas (I) to (III) Compound,
[In formula (I), R _{1 to} R ₃ represent a hydrogen atom or a methyl group, and at least two of R _{1 to} R ₃ are methyl groups. ]
[In Formula (II), R _{4 to} R ₆ represent a hydrogen atom or a methyl group, and at least one of R _{4 to} R ₆ is a methyl group. ]
[In Formula (III), R _{7 to} R ₁₂ represent a hydrogen atom or a methyl group, and at least one of R _{7 to} R ₁₂ is a methyl group. However, the case where only R ₇ is a methyl group is excluded. ]

(2) Said (1) characterized by being represented by one formula selected from the group consisting of the following formulas (I) -1, (II) -1 to 4, and (III) -1 to 3 ),

(3) A composition comprising the compound according to (1) or (2),
(4) A fat accumulation inhibitor comprising the compound according to (1) or (2), (5) The compound is a compound represented by the formula (I) -1. The fat accumulation inhibitor according to (4),
(6) An antioxidant comprising the compound according to (1) or (2),
(7) One type in which the compound is selected from the compounds represented by the formulas (II) -1, (II) -3, (III) -1, (III) -2, and (III) -3 The antioxidant according to (6) above, wherein
(8) A food or drink comprising the compound according to (1) or (2),
(9) A pharmaceutical product or quasi-drug comprising the compound according to (1) or (2),
(10) A cosmetic comprising the compound according to (1) or (2),
(11) 1 selected from the group consisting of a compound represented by the following formula (I) -0, a compound represented by the following formula (II) -0, and a compound represented by the following formula (III) -0 By reacting a methyltransferase enzyme using a compound of more than one species as a substrate, the compound represented by the general formula (I), the compound represented by the general formula (II), and the general formula (III) A method for producing methylated theaflavins, comprising synthesizing one or more compounds selected from the group consisting of:
(12) The method for producing a methylated theaflavin according to (11), wherein the methyltransferase enzyme is an enzyme derived from Enokitake (Flamulinavelipepes),
Is to provide.

  The methylated theaflavin of the present invention has a physiological function similar to that of the unmethylated theaflavin, and also has very good pH stability and metabolic stability. For this reason, by applying the methylated theaflavin of the present invention to various products such as foods and drinks, pharmaceuticals, quasi-drugs, and cosmetics, it is superior in functionality and economy than using unmethylated products. Product development is possible.

It is the figure which showed the base sequence of the methyl transferase gene derived from Enokitake, and the corresponding amino acid sequence. FIG. 2 (A) is a result of comparing the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequences of the above-mentioned two types of basidiomycetes, and the top four genes having high homology as a result of homology inspection. is there. FIG. 2 (B) is a diagram showing a sequence presumed to be a consensus sequence of an O-methyltransferase enzyme from the region indicated by 1-4 in FIG. 2 (A). In Reference Example 1, it is a two-dimensional electrophoresis image of the active fraction having methyltransferase enzyme activity fractionated from the enokitake mycelia culture. In Reference Example 2, E. coli lysate before IPTG treatment (lane 1), E. coli lysate after IPTG treatment (lane 2), and purified product obtained from IPTG-treated E. coli lysate using histidine tag (lane 3). ) SDS-PAGE electrophoresis image. In the reference example 5, it is the figure which showed the EGCG methylated body density | concentration in the enzyme reaction liquid fractionated after each reaction time. FIG. 5A shows the result of the natural enzyme, and FIG. 5B shows the result of the recombinant enzyme. In the reference example 6, it is the figure which showed the EGCG methylated body density | concentration in the enzyme reaction liquid of each pH. In the reference example 7, it is the figure which showed the EGCG methylated body density | concentration in the enzyme reaction liquid of each temperature. In Example 2, it is the figure which showed the result of having measured the composition of theaflavin and methylated theaflavin in the manufacturing process of methylated theaflavin containing black tea polyphenol with time. In Example 3, it is the figure which showed the result of having measured the residual rate of various theaflavins and methylated theaflavins 2 hours after the reaction start. FIG. 9A shows the result at pH 5.0, and FIG. 9B shows the result at pH 7.5. In Example 4, it is the figure which showed the result of having measured the residual rate of various theaflavins and methylated theaflavin 60 minutes after the reaction start. In Example 5, it is the figure which showed the measurement result of the fat accumulation amount of the cell group which performed each process. FIG. 11A shows the result when each test material is added to the medium so as to be 1 μM, and FIG. 11B shows the result when it is added to the medium so as to be 3 μM. In Example 6, it is the figure which showed the measurement result of HepG2 cell viability. FIG. 12A shows the measurement results when TF3′G and its methylated product are added, and FIG. 12B shows the measurement results when TF3, 3′G and its methylated product are added.

Abbreviations of each compound used in the present invention and the present specification are shown below.
TF: theaflavin TF3G: theaflavin 3-O-gallate 3MeTF3G: theaflavin 3-O- (3-O-methyl) gallate 3,5diMeTF3G: theaflavin 3-O- (3,5-O-dimethyl) gallate TF3′G: theaflavin 3′-O-gallate 3MeTF3′G: theaflavin 3′-O- (3-O-methyl) gallate 4MeTF3′G: theaflavin 3′-O- (4-O-methyl) gallate 3,5diMeTF3′G: theaflavin 3 '-O- (3,5-O-dimethyl) gallate 3,4diMeTF3'G: theaflavin 3'-O- (3,4-O-dimethyl) gallate TF3,3'G: theaflavin 3,3'-di- O-gallate 3MeTF3,3′G: theaflavin 3-O-gallate, 3′-O- (3-O-methyl) gallate
4MeTF3,3′G: theaflavin 3-O-gallate, 3′-O- (4-O-methyl) gallate
3,5diMeTF3,3′G: theaflavin 3-O-gallate, 3′-O- (3,5-O-dimethyl) gallate
Tea extract: black tea extract, black tea extract

In the present invention and the present specification, theaflavin refers to a compound represented by the following formula (I) -0 (TF3G), a compound represented by the following formula (II) -0 (TF3′G), or the following formula ( III) Compound (TF3,3′G) represented by −0 is shown. The compounds represented by these formulas have a plurality of isomers, and these isomers are also included in theaflavins.
In the present invention and the present specification, methylated theaflavin means a compound in which at least one hydroxyl group in the galloyl group of theaflavin is methylated.

<New methyltransferase>
In order to synthesize a novel methylated theaflavin, the present inventors have isolated and identified a novel methyltransferase derived from Enokitake (Flamulina velutipes) as shown in Reference Example 1 and the like described later.
FIG. 1 shows the base sequence (SEQ ID NO: 1) of the gene encoding the enokitake mushroom-derived methyltransferase and the corresponding amino acid sequence (SEQ ID NO: 2). The 45th to 734th positions of the base sequence represented by SEQ ID NO: 1 are coding regions.

  As a result of performing a homology search on the nucleotide sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2 using a publicly available NCBI (National Center for Biotechnology Information) database, The highly homologous gene is the foxtail mushroom-derived gene S238N-H82 (accession number: XM — 001878803), which had a homology of about 63.6% in terms of base sequence and about 61.6% in terms of amino acid sequence.

  Similarly, homology search was performed using a database of model mushrooms, basidiomycetes for which the entire genome sequence was known (http://genome.jgi-psf.org/Phchr1/Phchrl.home.html). As a result, the most homologous gene was the O-methyltransferase gene (Protein ID: 127115), which had a homology of about 63.9% in terms of base sequence and about 56.8% in terms of amino acid sequence.

  FIG. 2 (A) is a result of comparing the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequences of the above-mentioned two types of basidiomycetes, and the top four genes having high homology as a result of homology inspection. is there. In FIG. 2A, “F. velipepes” represents the amino acid sequence represented by SEQ ID NO: 2. In addition, “L. bicolor S238N-H82” is a foxtail bamboo-derived gene S238N-H82, “P. chrysosporium” is a model mushroom-derived O-methyltransferase gene, and “B. indicica subsp.” Is Beijerindicia subsp. O-methyltransferase family protein gene (accession number: YP — 001832797), “B. ambifaria AMMD” is the Burkholderia ambifaria AMMD-derived O-methyltransferase family protein gene (accession number: YP — 777310), “B. MSMB43-derived O-methyltransferase family protein gene (accession number: ZP — 0246606609), and “E. sakazakii” is Enterobacter sakazakii ATCC BAA-894-derived gene (accession number: YP — 001437785) Represents the deduced amino acid sequence.

  The present inventors have found from the alignment that the regions shown in 1 to 4 in FIG. 2A are regions having high conservation in all seven types of genes. In the amino acid sequence represented by SEQ ID NO: 2, region 1 is the 70th to 88th region, region 2 is the 139th to 148th region, region 3 is the 171st to 175th region, region Reference numeral 4 denotes the 215th to 223rd areas. Furthermore, the present inventors deduced a consensus sequence of the O-methyltransferase enzyme from the amino acid sequences of these four regions. FIG. 2 (B) represents a sequence estimated from the region indicated by 1-4 in FIG. 2 (A) as a consensus sequence for the O-methyltransferase enzyme. In FIG. 2B, “x” means any amino acid residue, and “(/)” means any amino acid residue in parentheses. .

  Furthermore, as a result of homology search with a plant-derived methyltransferase enzyme using the NCBI database for the amino acid sequence represented by SEQ ID NO: 2, the gene derived from soybean (Zea mays) (accession number: AY279004) The homology was 53.9% for the nucleotide sequence and 30.4% for the amino acid sequence. Similarly, rice (Olysatativa) -derived gene (accession number: AY279004), 50.7% (base sequence) and 30.0% (amino acid sequence), Arabidopsis thaliana-derived gene (accession number: MN_116065) 52. The homology was about 3% (base sequence) and 30.8% (amino acid sequence). Further, as a result of comparing EGCG isolated from tea described in Patent Document 4 with a methyltransferase enzyme capable of being methylated, the homology was about 53.9% in the base sequence and about 30.4% in the amino acid sequence. It was.

  The present inventors obtained methylated theaflavin by an enzyme reaction with the identified enokitake-derived methyltransferase using theaflavin as a substrate. As a result, in addition to 3MeTF3G, which is a known methylated theaflavin, many novel methylated theaflavins were obtained.

  That is, the methylated theaflavin of the present invention is represented by any one of the following general formulas (I) to (III).

[In formula (I), R _{1 to} R ₃ represent a hydrogen atom or a methyl group, and at least two of R _{1 to} R ₃ are methyl groups. ]

[In Formula (II), R _{4 to} R ₆ represent a hydrogen atom or a methyl group, and at least one of R _{4 to} R ₆ is a methyl group. ]

[In Formula (III), R _{7 to} R ₁₂ represent a hydrogen atom or a methyl group, and at least one of R _{7 to} R ₁₂ is a methyl group. However, the case where only R ₇ is a methyl group is excluded. ]

In the general formula _(I), R 1 ~R ₃ represents a hydrogen atom or a methyl group, _and at least two R 1 to R ₃ is a methyl group.
In general formula (II), R _{4 to} R ₆ represent a hydrogen atom or a methyl group, and at least one of R _{4 to} R ₆ is a methyl group.
In the general formula _{(III), R} 7 ~R ₁₂ represents a hydrogen atom or a methyl group, and at _least one or more R 7 _{to R 12} is a methyl group. However, the case where only R ₇ is a methyl group is excluded.
In addition, although the compounds represented by these general formulas have a plurality of isomers, any isomer is included in the methylated theaflavin of the present invention as long as it is a compound represented by these general formulas. It is.

  As the methylated theaflavin of the present invention, in particular, a compound represented by formula (I) -1 (3,5diMeTF3G), a compound represented by formula (II) -1 (3MeTF3′G), formula (II) — 2, a compound represented by formula (II) -3 (3,5diMeTF3′G), a compound represented by formula (II) -4 (3,4 diMeTF3′G), Compound represented by formula (III) -1 (3MeTF3, 3′G), compound represented by formula (III) -2 (4MeTF3, 3′G), and compound represented by formula (III) -3 (3,5diMeTF3,3′G) is preferred.

  The methylated theaflavin of the present invention is synthesized for the first time by an enzymatic reaction using enokitake mushroom-derived methyltransferase. Black tea contains theaflavin, and a methyltransferase derived from fresh tea leaves is also known (for example, JP-A-2007-306806). Nonetheless, it is not clear why these methylated theaflavins have not been obtained so far, but there is a possibility that methyltransferase from fresh tea leaves has high specificity for catechins and not enough specificity for theaflavins In addition, the enzyme activity of enokitake mushroom-derived methyltransferase may be higher than that of known methyltransferases such as fresh tea leaf-derived methyltransferase.

  The methylated theaflavin of the present invention can be produced by methylating theaflavin with methyltransferase. More specifically, for example, one or more methyltransferases are suspended in a buffer solution having a pH of 5 to 12, preferably 6 to 9, more preferably 6.5 to 8.5, and further containing a theaflavin. And a methyl group donor are added and reacted at 0 to 80 ° C., preferably 10 to 50 ° C., more preferably 30 to 42 ° C., whereby a methylated product can be obtained in the enzyme reaction solution. As the theaflavin-containing material, black tea, black tea extract, black tea polyphenol, standard theaflavin, and the like can be used. The donor for the methyl group can be appropriately selected from substances generally used for methylation of phenolic hydroxyl groups, such as S-adenosyl-L-methionine (SAM). .

  The methyltransferase used for producing the methylated theaflavin of the present invention is not particularly limited as long as it is an enzyme capable of methylating a hydroxyl group in the galloyl group of theaflavin, and methylation of a phenolic hydroxyl group is possible. It can be used by appropriately selecting from possible general methyltransferases. Examples of such methyltransferases include microorganism-derived or plant-derived methyltransferases, catechol methyltransferases derived from rats, pigs, and cattle liver.

  In order to produce the methylated theaflavin of the present invention, it is preferable to use a methyltransferase obtained from the fruiting body or mycelium of a basidiomycete, which is a methyltransferase derived from Enokitake or a homolog derived from another basidiomycete of the enzyme It is more preferable, and enokitake mushroom-derived methyltransferase is particularly preferable.

  Specifically, homologs derived from other basidiomycetes derived from enokitake mushroom transferase are 40% or more, preferably 50% or more, more preferably 55% or more, and still more preferably 62, with the amino acid sequence represented by SEQ ID NO: 2. % And a polypeptide having methyltransferase enzyme activity.

  In the case of performing homology search between a base sequence and an amino acid sequence, generally, a method of first searching for a core portion of homology without considering GAP and performing alignment processing therefrom (Homology Search), There is a method (Maximum Matching) in which rearrangement or deletion is considered so that the number of base (amino acid residue) pairs that match between base (amino acid) sequences is maximized. The value of “homology” in the present invention and the specification of the present application indicates a value obtained by analysis using Maximum Matching. The homology search by Maximum Matching can be performed using general-purpose analysis software such as GENETYX Version 6.1.0 (manufactured by GENETYX).

  The methyltransferase used for the production of methylated theaflavin may be a natural enzyme or a recombinant enzyme produced using a known gene recombination technique or the like. The recombinant enzyme may have the same amino acid sequence as that of a natural enzyme, consists of an amino acid sequence in which one or more amino acids are substituted, deleted or added to the natural enzyme, and is a methyltransferase enzyme It may be a variant that retains activity. Moreover, as these methyltransferases, commercially available products may be used.

  Natural methyltransferase can be obtained by extracting and purifying from an organism containing the enzyme. A purification method from a living organism is not particularly limited, and can be appropriately selected from publicly known known purification methods. Moreover, what is necessary is just to refine | purify until the state which methyltransferase enzyme activity is exhibited, and a refinement | purification degree is not specifically limited, Crude purification may be sufficient.

  For example, in the case of basidiomycetous fruit bodies or mycelium-derived methyltransferases, specifically, basidiomycetous fruit bodies or mycelium containing the gene of the present invention were disrupted by sonication or the like in an appropriate buffer solution. Thereafter, the recovered supernatant can be used as a crude enzyme. In addition, the supernatant of the basidiomycete fruit body or mycelium may be collected after homogenizing in an appropriate buffer. As the buffer solution for obtaining the crude enzyme, for example, a Tris buffer solution or a phosphate buffer solution having a pH of 6.5 to 8.5 can be used.

  Further, the basidiomycetes from which the enzyme is extracted may be those immediately after collection, may be commercially available, or may be a culture. The mycelium of basidiomycetes can be cultured by a conventional method.

  The crude enzyme thus obtained can be used for the enzymatic reaction as it is. Furthermore, a purified product can be obtained from the crude enzyme by combining centrifugation, ultrafiltration membrane, salting out, and various types of chromatography.

  The recombinant enzyme for methyltransferase may be produced by any known technique. Specifically, a gene encoding methyltransferase is incorporated into an expression vector to prepare a recombinant expression vector, and this recombinant expression vector is introduced into an appropriate organism (host) such as a microorganism or cultured cell, A transformant containing the recombinant expression vector is produced. By culturing the transformant thus obtained under the optimum conditions of each organism, a recombinant enzyme of methyltransferase is produced in the transformant. Therefore, a methyltransferase recombinant enzyme can be obtained by extraction from a culture of the transformant and purification.

  The methylated theaflavin obtained by the enzyme reaction can be extracted from the enzyme reaction solution by using a solvent or the like that can extract methylated theaflavin. In addition, you may use an enzyme reaction liquid for extraction, after concentrating. In addition, the methylated theaflavin can also be obtained by subjecting the enzyme reaction solution to a column filled with a synthetic adsorbent to adsorb the methylated theaflavin to the synthetic adsorbent, and after washing and eluting with an appropriate solvent. It is possible to obtain Further, it is possible to produce a novel methylated theaflavin with high purity by purification using HPLC (High Performance Liquid Chromatography).

  The methylated theaflavin of the present invention can be used in various products including foods and drinks, pharmaceuticals, quasi drugs and cosmetics. The methylated theaflavin of the present invention may be used as a raw material for these products as it is, or a composition obtained by premixing with other components may be used as a raw material.

  The methylated theaflavin of the present invention has better pH stability and metabolic stability than unmethylated products. For this reason, when the methylated theaflavin of the present invention is used in various products, the same function can be exerted with a smaller amount of addition than in the case of using an unmethylated product.

  In addition, in this invention and this-application specification, pH stability means the difficulty to receive decomposition | disassembly (stability) when it preserve | saves in the solution of pH near neutrality. Metabolic stability refers to the difficulty of being decomposed when stored in a living body or in an environment similar thereto.

  Moreover, the methylated theaflavin of the present invention has a fat accumulation suppressing action and an antioxidant action. For this reason, a fat accumulation inhibitor and an antioxidant can be produced by using the methylated theaflavin of the present invention or a composition containing the same as an active ingredient. In addition, the methylated theaflavin contained in the fat accumulation inhibitor and the antioxidant may be one kind, or may be contained in combination of two or more kinds.

  As the fat accumulation inhibitor, it is particularly preferable to contain 3,5diMeTF3G represented by the formula (I) -1 as an active ingredient. This is because 3,5diMeTF3G has a very good fat accumulation inhibitory action compared to TF3G which is an unmethylated form. The amount of methylated theaflavin contained in the fat accumulation inhibitor is not particularly limited as long as it is a concentration at which the ability to inhibit fat accumulation is exhibited. Considering the type of methylated theaflavin, the desired strength of inhibiting fat accumulation, etc. And can be set as appropriate. For example, when 3,5diMeTF3G is used, it can be 0.5 to 10 μM, preferably 0.5 to 5 μM, more preferably 1 to 3 μM.

  Antioxidants include 3MeTF3′G represented by formula (II) -1, 3,5diMeTF3′G represented by formula (II) -3, and 3MeTF3,3 represented by formula (III) -1. It is preferable that at least one of 'G, 4MeTF3, 3'G represented by the formula (III) -2, or 3,5diMeTF3, 3'G represented by the formula (III) -3 is included as an active ingredient. This is because these methylated theaflavins have a very good antioxidant action compared to the corresponding unmethylated form. The amount of methylated theaflavin to be included in the antioxidant is not particularly limited as long as the antioxidant ability is exerted, considering the kind of methylated theaflavin, the strength of the desired antioxidant ability, etc. It can be set appropriately. For example, when 3MeTF3′G, 3,5diMeTF3′G, 3MeTF3, 3′G, 4MeTF3,3′G, or 3,5diMeTF3,3′G is used, 0.1 μM or more, preferably 0.1 The amount can be 50 μM, more preferably 0.1 to 20 μM, and still more preferably 0.2 to 2 μM.

  When used for foods and drinks, pharmaceuticals, quasi drugs, and cosmetics, it is preferable to use methylated theaflavins produced using methyltransferase enzymes recovered from edible basidiomycetes such as enokitake. Methyltransferase enzymes recovered from edible basidiomycetes such as enokitake are highly safe and suitable for industrial production of products that require sufficient safety such as foods, beverages, pharmaceuticals, and cosmetics. In addition, basidiomycetes can be easily cultivated (cultivated) in large quantities.

  Examples of foods and beverages to which the methylated theaflavin or the composition thereof is added as a raw material include soft drinks, carbonated drinks, nutrition drinks, fruit drinks, lactic acid drinks and other drinks (including concentrated concentrates and adjustment powders of these drinks); Ice cream, ice sherbet, shaved ice and other frozen desserts; buckwheat noodles, udon, harusame, gyoza skin, Shumai skin, Chinese noodles, instant noodles and other noodles; rice cakes, candy, gum, chocolate, tablets, snacks, biscuits, jelly , Jams, creams, baked confectionery, etc .; kamaboko, chikuwa, ham, sausage and other acid and livestock processed foods; dairy products such as processed milk and fermented milk; margarine, mayonnaise, shortening, whipped cream, dressing, etc. Fat and oil processed foods; seasonings such as sauces and sauces; soups, stews, salads, prepared dishes, pickles; other species Health foods, dietary supplements in the form, can be cited for specified health foods.

Examples of dosage forms of pharmaceuticals and quasi drugs to which the methylated theaflavin of the present invention or a composition thereof is added as a raw material include tablets, liquids, capsules, drinks, troches and the like.
Cosmetics include basic cosmetics such as face-wash creams, lotions, packs, and serums; makeup cosmetics such as foundations, lipsticks, and eye colors; nail enamels, soaps, bathing agents, sunscreen agents, deodorant sprays, etc. Body cosmetics; hair cosmetics such as shampoos, rinses, hair treatments, hair mousses; hair cosmetics such as hair restorers, hair nourishing agents, hair tonics; and fragrance cosmetics such as perfumes and eau de cologne.

  In producing these, the methylated theaflavin or the composition thereof of the present invention can be added together with auxiliary materials and additives that are usually used. Examples of such raw materials and additives include glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene Glycol, glycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin C, vitamin B group, vitamin E, nicotinamide, calcium pantothenate, amino acids, calcium Salts, surfactants, pigments, fragrances, preservatives and the like can be mentioned.

  Next, although a reference example and an example are shown and the present invention is explained still in detail, the present invention is not limited to the following reference examples and examples.

[Reference Example 1]
We isolated and identified the methyltransferase gene from Enokitake.
<Screening of methyltransferase enzyme activity>
Japanese shiitake, shimeji, maitake, enokitake, beech shimeji, oyster mushroom, oyster mushroom, tamogitake, eringi, and abalone are commercially available for edible use.
The stem portion was cut using these fruit bodies, soaked in 0.5% hypochlorous acid, and then washed with sterilized water. About 5 mm was cut out from the inside of the handle and cultured on a Difco Potato Dextrose Ager medium at 25 ° C. to isolate the mycelium. The obtained mycelium was added to a liquid medium for mycelium culture (0.02% glucose, 0.01% peptone, 0.002% Yeast Extract, 0.002% KH ₂ PO ₄ , 0.001% MgSO ₄ .7H ₂ O) and swirl culture at 28 ° C.

The obtained mycelium culture solution is filtered to collect mycelia, and after adding a crude enzyme solution (20 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA, 10% glycerol) and ultrasonically crushing it. The supernatant was collected by centrifugation. This supernatant was used as a crude enzyme solution for enzyme activity measurement.
In the enzyme activity measurement, EGCG was used as a substrate, and the amount of methylated product produced was used as an index.
The composition of the enzyme reaction solution was 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl ₂ , 0.25 mM EGCG, 0.5 mM SAM, and 50% crude enzyme solution, and a total volume of 3 ml was reacted at 37 ° C. for 16 hours. I let you.
After the reaction, 70 μl of 1N HCl and 5 ml of ethyl acetate were added to 3 ml of the enzyme reaction solution, and the mixture was stirred and centrifuged to recover the organic layer. The organic layer was dried with nitrogen, dissolved in 30% aqueous methanol containing 1% (w / v) ascorbic acid, and measured using HPLC. The HPLC conditions are shown below.

Column: Wakopak Navi C18-5 (4.6 × 150 mm) and C18-5 (4.6 × 10 mm) (manufactured by Wako Pure Chemical Industries, Ltd.)
Mobile phase A: Solution water phase in which distilled water, acetonitrile and phosphoric acid were mixed at 400: 10: 1 (v / v) Mobile phase B: Solution gradient condition in which mobile phase A and methanol were mixed at 2: 1 (v / v) : 20% solution B (2 minutes) → 80% solution B (25 minutes) → 80% solution B (10 minutes), linear concentration gradient flow rate: 1 ml / min
Detection: UV280nm

As a result, when the crude enzyme extracted from the enokitake mushroom mycelium was used, epigallocatechin-3-O- (3-O-methyl) gallate, epigallocatechin-3-O- (4-O-methyl) Galate, epigallocatechin-3-O- (3,4-O-dimethyl) gallate, epigallocatechin-3-O- (3,5-O-dimethyl) gallate, epi (4-O-methyl) gallocatechin- Formation of 3-O- (3,5-O-dimethyl) gallate was confirmed. In addition, epigallocatechin-3-O- (3,4-O-dimethyl) gallate, epigallocatechin-3-O- (3,5-O-dimethyl) gallate, epi (4-O-methyl) gallocatechin- Regarding 3-O- (3,5-O-dimethyl) gallate, the peaks were collected and the structure was confirmed using TOF-MS and NMR. In addition, when the crude enzyme solution was boiled and then added to the enzyme reaction solution, or when EGCG or SAM was not added to the enzyme reaction solution, formation of these methylated products was not observed.
From the above results, it was confirmed that the crude enzyme extracted from the enokitake mushroom culture mycelium has methyltransferase enzyme activity.

<Confirmation of various enokitake enzyme activities>
The presence or absence of methyltransferase enzyme activity due to differences in the location of Enokitake was collected. As the mycelium of enokitake mushroom, one purchased from Agricultural Biological Resources Genebank was used and cultured at 25 ° C. on Difco Potato Dextrose Ager medium. The mycelial liquid culture and enzyme activity measurement were performed in the same manner as in the above <Screening of methyltransferase enzyme activity>. MAFF numbers 430204, 430205, 430206, 430207, 430209, 435210, 430212, 430214, 430224, 4308585, 430211, 430213, 435121, 440110, 440111, and 440118 were used for the test. As a result, the enzyme activity of methyltransferase was confirmed in all mycelia although each mycelium had strong and weak enzyme activity.

<Identification of Enokitake Methyltransferase>
The enokitake mycelium was liquid-cultured in the same manner as described above, and the mycelium culture solution obtained was filtered to recover the mycelium and freeze-dried. About 12 g of the lyophilized product was crushed in a mortar and then suspended in 600 ml of a crude enzyme solution. The suspension was sonicated, centrifuged (10,000 rpm × 10 min, 4 ° C.), and the collected supernatant was centrifuged again (30,000 rpm × 30 min, 4 ° C.) to recover the supernatant. . To this supernatant, ammonium sulfate was added so as to be 60% saturated, stirred and centrifuged, and the supernatant was collected. Furthermore, ammonium sulfate was added to the collected supernatant so as to be 80% saturation, and the mixture was stirred and centrifuged to obtain a precipitate. The obtained precipitate was dissolved in 40 ml of a crude enzyme solution, and then desalted using PD-10 (manufactured by GE Healthcare Bioscience). The desalted sample was adsorbed on an anion exchange column (HiPrep 16/10 DEAE FF, manufactured by GE Healthcare Bioscience) equilibrated with Tris buffer (20 mM Tris-HCl (pH 7.5), 1 mM DTT). The fraction in which methyltransferase enzyme activity was confirmed was eluted using a linear concentration gradient of 0-500 mM NaCl solution adjusted with the above Tris buffer, and fractionated as an active fraction. The methyltransferase enzyme activity was confirmed in the same manner as in <Screening of methyltransferase enzyme activity>.

  The obtained active fraction was desalted and concentrated with an ultrafiltration column, and then an anion exchange column equilibrated with the above Tris buffer (HiLoad 26/10 Q-sepharose HP, manufactured by GE Healthcare Biosciences) The active fraction was fractionated by elution using a linear concentration gradient of 0-500 mM NaCl solution adjusted with the above Tris buffer. The obtained active fraction was desalted and concentrated with an ultrafiltration column, adsorbed again on the same anion exchange column, and the active fraction was fractionated with the preparative fraction more detailed than the previous conditions. The solution was desalted and concentrated using an ultrafiltration column.

  The concentrated fraction obtained was equilibrated with Tris buffer (20 mM Tris-HCl (pH 7.5), 1 mM DTT, 150 mM NaCl) containing 150 mM NaCl, and a gel filtration column (HiLoad 16/60 Superdex 200 prep grade, GE). Fractionation was performed using Healthcare Bioscience. When each fractionated fraction was subjected to SDS-PAGE electrophoresis and the obtained protein-stained image was compared with the methyltransferase enzyme activity of each fraction, a band correlated with the enzyme activity was located at a position of about 24 to 25 kDa. Was confirmed. The fraction in which the enzyme activity was confirmed was further adsorbed to an anion exchange column (TSK-GEL BIASSIST Q, manufactured by Tosoh Corporation) equilibrated with the above-described Tris buffer, and 0 to 500 mM NaCl adjusted with the above-described Tris buffer. The active fraction was fractionated by elution using a linear concentration gradient of the solution. As a result of subjecting the active fraction to SDS-PAGE electrophoresis again, a specific band was confirmed again at a position of about 24 to 25 kDa. In order to clarify the internal sequence of this enzyme protein, the sample purified by the above anion exchange column (HiLoad 26/10 Q-sepharose HP, manufactured by GE Healthcare Bioscience) was subjected to SDS-PAGE electrophoresis, and the band was gelled. Recovered from.

  The recovered band was subjected to in-gel trypsin digestion according to a conventional method, and the internal sequence of the protein was analyzed using LC / MS / MS (manufactured by Thermo Fisher Scientific). Furthermore, the same active fraction was electrophoresed by two-dimensional electrophoresis (first dimension: isoelectric focusing at pH 3-10, second dimension: polyacrylamide gel with a 4-20% gradient), molecular weight of about 24-25 kDa, A spot near pH 5 was recovered from the gel. FIG. 3 is a two-dimensional electrophoresis image. In the figure, the circled spot was collected as the target enzyme protein.

  The collected spot was digested with trypsin in gel according to a conventional method, and the internal sequence of the protein present in the spot was confirmed using LC / MS / MS (manufactured by Thermo Fisher Scientific). It was. As a result, internal sequences RVLEVGTLGGYSTTTWARA (SEQ ID NO: 3) and TGGIIIVDNVVR (SEQ ID NO: 4) were obtained. As a result of homology search using NCBI database based on this amino acid sequence, it showed high homology with the internal sequence of known O-methyltransferase.

<Isolation and Identification of Enokitake Methyltransferase Gene>
A degenerate primer was designed based on the sequence information of the internal sequence, and a methyltransferase gene derived from Enokitake was isolated and identified from total-RNA recovered from Enokitake.
Specifically, first, total RNA was recovered from about 1 g of enokitake mushroom mycelium crushed under liquid nitrogen using a mortar using TRI Reagent (manufactured by Sigma). From about 4 μg of the obtained total-RNA, cDNA was synthesized by reacting at 55 ° C. for 50 minutes using a thermoscript RT-PCR system (manufactured by Invitrogen). Using this cDNA as a template, PCR was performed using degenerate primers (FVOMT-F and FVOMT-R) designed based on the sequence information of the internal sequence, and the Enokitake mushroom-derived methyltransferase gene was isolated. The nucleotide sequence of the designed degenerate primer and the PCR conditions are described below. In the nucleotide sequence, S is guanine or cytosine, M is adenine or cytosine, Y is thymine or cytosine, D is adenine, guanine, or thymine, K is guanine or thymine, V is adenine, guanine, Or cytosine, and R represents guanine or adenine, respectively.

FVOMT-F: GAGGTSGGMACYYTDGGMGSTA
FVOMT-R: GCKSACVACRTTRTCMAC
PCR conditions: 94 ° C., 5 min → (94 ° C., 1 min → 55 ° C., 1 min → 72 ° C., 1 min) × 40 cycles → 72 ° C., 7 min

  As a result of subjecting the PCR product to agarose gel electrophoresis, an amplification band of about 400 bp was confirmed. The band was cut out from the agarose gel, and the PCR product was collected using QIAquick Gel Extraction Kit (Qiagen) and cloned into the pGEM-T vector (Promega), and then Escherichia coli JM-109 (Takara). Transformed into The obtained transformant was cultured with shaking in LB medium at 37 ° C. overnight, and a plasmid was extracted from the obtained culture using QIAprep Spin Miniprep Kit (manufactured by Qiagen). The base sequence of the extracted plasmid insert was confirmed by Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and ABI PRISM 3100-AVANT Genetic Analyzer (Applied Biosystems).

  Based on the confirmed partial base sequence of the gene, specific primers (FVOMT-5′GSP1, FVOMT-5′GSP2, FVOMT-3′GSP1, FVOMT-3′GSP2) are designed, and 5 ′ and 3 The RACE-PCR method was performed to obtain the full length on the 'side. The nucleotide sequence of the designed specific primer is shown below.

FVOMT-5'GSP1: AGCCTCTTAGCCCTCAACAAAGTA
FVOMT-5'GSP2: TCTTCGAGCTCGAAGGTGAT
FVOMT-3'GSP1: ATCACCTCTCGAGCTCGAAGA
FVOMT-3'GSP2: TACTTTGTTGAGGCTAAGAGGCT

In 5′RACE-PCR, first, 2.5 μg of isolated total-RNA was reacted at 55 ° C. for 50 minutes using FVOMT-5′GSP1 primer and using Thermoscript RT-PCR system (manufactured by Invitrogen). CDNA was synthesized. Subsequently, from the obtained cDNA, 5′RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (manufactured by Invitrogen) was used, and the FVOMT-5′GSP1 primer and the FVOMT-5′GSP2 primer were used on the 5 ′ side. Was isolated and the base sequence was confirmed.
Similarly, 3′RACE-PCR is performed by synthesizing cDNA using a thermoscript RT-PCR system (manufactured by Invitrogen), and 3′RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (manufactured by Invitrogen). Using the obtained cDNA, 3′-side isolation was performed using the FVOMT-3′GSP1 primer and the FVOMT-3′GSP2 primer, and the nucleotide sequence was confirmed.

  Based on the confirmed 5'-end and 3'-end base sequences, the following FVOMT-5'NdeI primer and FVOMT-3'BamHI primer were designed and RT-PCR was performed. The obtained PCR product was subjected to agarose gel electrophoresis, and the amplified band was recovered. The collected PCR product was cloned into the pGEM-T vector and transformed into Escherichia coli JM109 strain, and then the base sequence of the insert was confirmed. As a result, the entire gene of Enokitake mushroom methyltransferase enzyme was isolated. The base sequence of the identified enokitake mushroom-derived methyltransferase gene is the base sequence represented by SEQ ID NO: 1 and encodes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.

FVOMT-5'NdeI: TACATATGTCCAACCCCGACAAGCATACT
FVOMT-3'BamHI: TAGGATCCCAAGTTTGATAGCGTACAAGAATCC

[Reference Example 2]
<Production of recombinant enokitake methyltransferase enzyme>
A recombinant expression vector incorporating the enokitake mushroom-derived methyltransferase gene isolated in Reference Example 1 was prepared, and this recombinant expression vector was introduced into Escherichia coli to recover the enokitake mushroom-derived methyltransferase enzyme expressed in E. coli. .
Specifically, first, the enokitake mushroom-derived methyltransferase gene-containing pGEM-T vector prepared in Reference Example 1 was cleaved with restriction enzymes NdeI and BamHI, and the insert was recovered by agarose electrophoresis. A recombinant expression vector was prepared by cloning this insert into the NdeI and BamHI sites of the pET28a (+) vector (Novagen). This recombinant expression vector was introduced into Escherichia coli BL21 (DE3) strain (Stratagene) to obtain a transformant containing the recombinant expression vector.

  The obtained transformant was cultured with shaking in an LB medium at 37 ° C. overnight, and a part thereof was added again to a new LB medium and cultured. O. D. After culturing so that 600 = 0.6, IPTG was added to a final concentration of 1 mM, and further cultured with shaking at 28 ° C. to induce expression of histidine-tagged enzyme protein. The expression-induced E. coli was centrifuged, and the precipitate was suspended in the above Tris buffer. This E. coli suspension was sonicated and centrifuged again. The obtained supernatant (lysate) was electrophoresed with 12% SDS-PAGE. As a result, compared to the case where E. coli before IPTG induction was treated in the same manner and electrophoresed, about 29 kDa was obtained by IPTG treatment. Protein bands in which expression was induced were confirmed.

  FIG. 4 shows lysate of E. coli before IPTG treatment (lane 1), lysate of E. coli after IPTG treatment (lane 2), and purified product obtained from lysate of E. coli after IPTG treatment using histidine tag (lane 3). It is an SDS-PAGE electrophoresis image of. In the figure, “M” is a lane in which a molecular weight marker is passed. The band (about 29 kDa) at the position of the arrow in the figure was subjected to image analysis, and the amount of protein was quantified and compared. For the image analysis, Photoshop (manufactured by Adobe Systems) and Scion Image (manufactured by Scion Corporation) software were used. As a result, the amount of the protein of about 29 kDa is 8.4% after IPTG treatment and 25.1% in the purified product after treatment when IPTG treatment is 0%. The protein was confirmed to have a histidine tag whose expression was induced.

  When cloned into the NdeI and BamHI sites of the pET28a (+) vector, a vector-derived expressed protein (including a histidine tag) of about 5 kDa is added. The theoretical molecular weight of the enzyme protein calculated from the amino acid sequence of SEQ ID NO: 2 is 24.7 kDa. That is, it was found that the protein of about 29 kDa indicated by the arrow in FIG. 4 matches the estimated molecular weight obtained from the amino acid sequence introduced by the recombinant expression vector, and is a histidine-tagged enokitake mushroom-derived methyltransferase enzyme.

[Reference Example 3]
<Enzyme activity of recombinant enokitake methyltransferase enzyme>
The IPTG-treated Escherichia coli lysate obtained in Reference Example 2 was used as a crude enzyme solution of a recombinant enzyme of enokitake mushroom-derived methyltransferase enzyme, and the methyltransferase enzyme activity of this crude enzyme solution was examined.
Specifically, 3 ml of an enzyme reaction solution was prepared so as to be 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl ₂ , 0.25 mM EGCG, 0.5 mM SAM, and 50% crude enzyme solution, This enzyme reaction solution was incubated at 37 ° C. for 16 hours to be reacted. The enzyme reaction solution after the reaction was treated and analyzed in the same manner as in <Screening of methyltransferase enzyme activity> in Reference Example 1, and as a result, epigallocatechin-3-O- (3-O-methyl) gallate, epigallocatechin -3-O- (4-O-methyl) gallate, epigallocatechin-3-O- (3,4-O-dimethyl) gallate, epigallocatechin-3-O- (3,5-O-dimethyl) Formation of gallate and epi (4-O-methyl) gallocatechin-3-O- (3,5-O-dimethyl) gallate could be confirmed.
From these results, it was confirmed that the recombinant enzyme expressed in E. coli in Reference Example 2 had methyltransferase enzyme activity.

[Reference Example 4]
<Production of methylated product using mycelium extraction crude enzyme solution>
Extracted from the mycelium of the enokitake mushroom mycelium culture solution in the same manner as in Reference Example 1, and the 60% to 80% ammonium sulfate fraction of the obtained extract was desalted, and the natural form of enokitake mushroom-derived methyltransferase enzyme A crude enzyme solution of the enzyme was used.
Specifically, first, the enokitake mycelium culture solution was filtered to recover the mycelium and lyophilized. The obtained lyophilized product was crushed in a mortar, suspended in the crude enzyme solution, sonicated, and centrifuged to collect the supernatant. To this supernatant, ammonium sulfate was added so as to be 60% saturated, stirred and centrifuged, and the supernatant was collected. Furthermore, ammonium sulfate was added to the collected supernatant so as to be 80% saturation, and the mixture was stirred and centrifuged to obtain a precipitate. The obtained precipitate was dissolved in a crude enzyme solution, and then desalted using PD-10 (manufactured by GE Healthcare Bioscience). This desalted sample was used as a crude enzyme solution of a natural enzyme of enokitake mushroom-derived methyltransferase enzyme.
The methyltransferase activity for various substrates of the obtained crude enzyme solution was confirmed. The composition of the enzyme reaction solution was 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl ₂ , 0.05 mM substrate, 0.5 mM SAM, 0.04% (w / v) ascorbic acid, and crude enzyme solution ( The total amount of 3 ml was reacted at 37 ° C. for 6 hours. Substrates include epicatechin-3-O-gallate (ECG), catechin-3-O-gallate (CG), gallocatechin-3-O-gallate (GCG), caffeic acid, chlorogenic acid, ellagic acid, butein, sulfretin , Luteolin, myricetin, or rosmarinic acid, respectively.
The enzyme reaction solution after the reaction was treated in the same manner as in Reference Example 1 <Screening of methyltransferase enzyme activity>, and the methylated product in the enzyme reaction solution was analyzed using LC-MS under the following conditions. Identifications were also made for samples that could be compared with the standard. The HPLC conditions and MS conditions are shown below.

HPLC condition column: Inertsil ODS-3, 2.1 × 150 mm (manufactured by GL Science)
Mobile phase A: 0.1 (v / v)% formic acid aqueous solution Mobile phase B: acetonitrile gradient containing 0.1 (v / v)% formic acid (i): 8% solution B (20 minutes) → 25% B Liquid (88 minutes)
Gradient condition (ii): 8% solution B (10 minutes) → 50% solution B (31 minutes)
Flow rate: 0.2 ml / min
Detection: UV280nm

MS condition detector: API 3000 (Applied Biosystems)
Ion source: ESI (negative)
Curtain gas: 10
Nebulizer gas: 14
Turbo gas: 6 l / min
Ion spray voltage: -4000V
Ion spray temperature: 500 degrees Cone voltage: -41V

The results of LC-MS analysis are shown in Table 1. As a result, the formation of methylated products was confirmed for all the substrates used. From these results, it was revealed that the enzyme of the present invention can modify a methyl group using various compounds having a hydroxyl group as a substrate.
Moreover, in the compound having a plurality of hydroxyl groups in the compound like ECG, the generation of a plurality of types of methylated products was confirmed, and the dimethylated product was also confirmed. From these results, it was also confirmed that the enzyme of the present invention can independently methylate each of a plurality of hydroxyl groups in a substrate compound.

[Reference Example 5]
<Examination of reaction time>
A crude enzyme solution of a natural enzyme of enokitake mushroom-derived methyltransferase used in Reference Example 4 (hereinafter referred to as a natural enzyme) and a recombinant enzyme solution purified with a histidine tag of an enokitake mushroom-derived methyltransferase enzyme used in Reference Example 2 (hereinafter referred to as “enzyme-take”) , The reaction time of the enzyme reaction was examined.
The composition of the enzyme reaction solution was 20 mM phosphate buffer (pH 7.0), 2.5 mM MgCl ₂ , 0.05 mM EGCG, 0.5 mM SAM, 0.04% (w / v) ascorbic acid, and each crude enzyme. A liquid (2.5 ml with respect to the total volume of 30 ml) was reacted at a temperature of 37 ° C. 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours from the start of the reaction After 24 hours, 1 ml of the enzyme reaction solution was collected. These enzyme reaction solutions were treated in the same manner as in Reference Example 1 <Screening of methyltransferase enzyme activity>, and methylated products in the enzyme reaction solution were analyzed under the following HPLC conditions.

Column: Inertsil ODS-3, 2.1 × 150 mm (manufactured by GL Science)
Mobile phase A: 0.1 (v / v)% formic acid aqueous solution Mobile phase B: acetonitrile gradient containing 0.1 (v / v)% formic acid Conditions: 10% solution B (10 minutes) → 16% solution B (50 Minutes)
Flow rate: 0.2 ml / min
Detection: UV280nm

  FIG. 5 shows the concentration of EGCG methylated product in the enzyme reaction solution collected after each reaction time. FIG. 5A shows the result of the natural enzyme, and FIG. 5B shows the result of the recombinant enzyme. As a result, the production of EGCG methylated product showed the highest value 8 hours after the start of the reaction with the natural enzyme and 20 minutes after the recombinant enzyme.

[Reference Example 6]
<Examination of optimum pH>
The optimum pH in the enzyme reaction of the natural enzyme and the recombinant enzyme used in Reference Example 5 was examined.
The composition of the enzyme reaction solution was as follows: 20 mM various buffers, 2.5 mM MgCl ₂ , 0.05 mM EGCG, 0.5 mM SAM, 0.04% (w / v) ascorbic acid, and each crude enzyme solution (total volume 3 ml) To 0.25 ml). The buffers used were acetate buffer (pH 3.0 to 5.5), phosphate buffer (pH 6.0 to 7.0), and Tris-HCl (pH 7.5 to 10). A total amount of 3 ml of the prepared enzyme reaction solution was reacted at 37 ° C. The reaction time was 6 hours for the natural enzyme and 10 minutes for the recombinant enzyme. These enzyme reaction solutions were treated in the same manner as in Reference Example 1 <Screening for methyltransferase enzyme activity>, and methylated products in the enzyme reaction solution were analyzed.
FIG. 6 shows the EGCG methylated product concentration in the enzyme reaction solution at each pH. As a result, the production of EGCG methylated product was confirmed at pH 6.5 to 8.5 for the natural enzyme and at pH 6 or more for the recombinant enzyme.

[Reference Example 7]
<Examination of optimum temperature>
The optimum temperature in the enzyme reaction of the natural enzyme and the recombinant enzyme used in Reference Example 5 was examined.
The composition of the enzyme reaction solution was 20 mM phosphate buffer (pH 7.0), 2.5 mM MgCl ₂ , 0.05 mM EGCG, 0.5 mM SAM, 0.04% (w / v) ascorbic acid, and each crude enzyme. The liquid (0.25 ml with respect to the total volume of 3 ml) was reacted with the total volume of 3 ml at 4, 10, 20, 30, 37, 42, 50, or 60 ° C., respectively. The reaction time was 6 hours for the natural enzyme and 10 minutes for the recombinant enzyme. These enzyme reaction solutions were treated in the same manner as in Reference Example 1 <Screening for methyltransferase enzyme activity>, and methylated products in the enzyme reaction solution were analyzed.
FIG. 7 shows the concentration of EGCG methylated product in the enzyme reaction solution at each temperature. As a result, synthesis of EGCG methylated product was confirmed at 10 to 50 ° C. with the natural enzyme and at all temperatures performed with the recombinant enzyme. Among them, good enzyme activity was confirmed at 30 to 42 ° C. for both enzymes.

[Reference Example 8]
<Preparation of standard theaflavin as substrate>
Shimane Prefecture Izumo black tea (trade name: Izumo domestic black tea (Leaf), manufactured by Nishi Seisho) is crushed, 40 times 2% (w / v) ascorbic acid containing 50% (v / v) EtOH aqueous solution Using, the room temperature extraction for 20 minutes was performed 3 times. The extract was centrifuged to remove tea leaves from the extract. After the organic solvent was removed by concentration under reduced pressure, the extract was subjected to Sep-Pak Vac C18 (manufactured by Waters) to perform 15%, 40%, 100% EtOH stepwise elution. The 40% EtOH fraction containing theaflavin was freeze-dried, redissolved in 40% EtOH as appropriate, and theaflavin was fractionated under the following conditions. The collected theaflavins were deacidified using Sep-Pak Vac C18 to prepare freeze-dried products. Thereby, TF3G standard product, TF3′G standard product, TF3, 3′G standard product were obtained.

Sorting conditions;
Column: Inertsil ODS-3 (20 × 250 mm, manufactured by GL Science) Mobile phase A: 0.05 (v / v)% phosphoric acid aqueous solution mobile phase B: 12.5 (v / v) acetonitrile containing ethyl acetate Gradient condition: 24% solution B (50 minutes) → 50% solution B (0.1 minute) → 50% solution B (10 minutes)
Flow rate: 18 ml / min
Column temperature: 30 ° C
Detection: UV280nm

[Example 1]
<Production of methylated theaflavin>
Using the recombinant enzyme produced in Reference Example 2, methylated theaflavin was produced. As substrates, the TF3G standard product, the TF3′G standard product, and the TF3,3′G standard product obtained in Reference Example 8 were used.
The composition of the enzyme reaction solution was 20 mM phosphate buffer (pH 6.5), 2.5 mM MgCl ₂ , 0.025% (w / v) theaflavin (substrate), 0.5 mM SAM, 0.04% (w / v v) Ascorbic acid and methyltransferase (33.3 ml with respect to a total volume of 1000 ml) were reacted at 37 ° C. for 6 hours. Thereafter, 24 ml of 1N HCl was added to 1000 ml of the enzyme reaction solution to terminate the enzyme reaction. An equal amount of ethyl acetate was added to the enzyme reaction solution, stirred vigorously, and allowed to stand, and then the ethyl acetate layer was recovered. The same operation was performed again on the remaining aqueous layer. The collected ethyl acetate layer was concentrated under reduced pressure, and methylated theaflavin was fractionated under the same conditions as in Reference Example 8. The fractionated methylated theaflavin was deacidified using Sep-Pak C18 to prepare a freeze-dried product. The obtained methylated theaflavin freeze-dried product was subjected to TOF-MS analysis under the following conditions, and the molecular formula was calculated from the obtained precise molecular weight. In addition, NMR (H-NMR, C-NMR, HSQC, HMBC, COSY) was measured under the following conditions to identify the structure.

TOF-MS conditions;
Detector: QSTAR ELITE (Applied Biosystems)
Ion source: ESI (negative mode)
Curtain gas: 30
Ion source gas 1:50
Ion source gas 2:50
Ion spray voltage: -4500V
Ion spray temperature: 450 ° C
Declustering potential: -30V
Focus potential: -250
Declustering potential: -15V

NMR conditions;
Detector: XWIN-NMR AV600 (Bruker Biospin)
Probe temperature: 300K
Solvent: acetone-d6

  The results of TOF-MS and NMR analysis are shown in Tables 2-4. In addition to the known compound 3MeTF3G, eight new compounds were obtained. Each of the novel compounds was converted into 3,5diMeTF3G (chemical formula (I) -1), 3MeTF3′G (chemical formula (II) -1), 4MeTF3′G (chemical formula (II) -2), 3,5diMeTF3′G (chemical ( II) -3), 3,4diMeTF3′G (formula (II) -4), 3MeTF3,3′G (chemical formula (III) -1), 4MeTF3,3′G (chemical formula (III) -2), 3, 5diMeTF3,3′G (chemical formula (III) -3) was determined.

[Example 2]
<Production of methylated theaflavin-containing black tea polyphenol and reaction efficiency>
The enzyme reaction was carried out in the same manner as in Example 1 using the lyophilized product of the 40% EtOH fraction prepared in Reference Example 8 instead of the standard product as a substrate. The enzyme reaction solution was divided into 0.5 ml over time, treated in the same manner as in Example 1, and then analyzed under the following conditions to examine the compositions of theaflavin and methylated theaflavin.

Analysis conditions;
Column: Inertsil ODS-3 (4.6 × 250 mm, manufactured by GL Science) Mobile phase A: 0.05 (v / v)% phosphoric acid aqueous solution mobile phase B: 12.5 (v / v)% ethyl acetate Containing acetonitrile gradient: 24% solution B (50 minutes) → 50% solution B (0.1 minute) → 50% solution B (10 minutes)
Flow rate: 1 ml / min
Column temperature: 50 ° C
Detection: UV375nm

  FIG. 8 shows the results of examining the transition of the composition of theaflavin and methylated theaflavin over time in the production process of methylated theaflavin-containing black tea polyphenol. As a result, it was clarified that the amount of methylated theaflavin produced increased with the passage of the reaction time, and the amount produced became maximum after 24 hours. From this result, it became clear that the methylated theaflavin content in black tea polyphenols can be increased by enzyme treatment using methyltransferase.

[Example 3]
<Evaluation of pH stability of methylated theaflavin>
The theaflavin standard used in Example 1 and the methylated theaflavin obtained in Example 1 were mixed with 40% (v / v) ethanol-containing 0.3 M acetate buffer (pH 5.0) or 40 so as to be 400 μM. % (V / v) ethanol-containing 0.3 M Tris-HCl buffer solution (pH 7.5) was dissolved, and the pH stability at room temperature was examined. Samples of 50 μl were taken at 0, 1, and 2 hours after the start of the reaction, and the decomposition reaction was stopped by adding an equal amount of 1% (w / v) ascorbic acid aqueous solution to 40% (v / v). EtOH was added and the remaining various theaflavins were analyzed by the method described in Example 2.

  The residual rate of methylated theaflavin 2 hours after the start of the reaction is shown in FIG. FIG. 9A shows the result at pH 5.0, and FIG. 9B shows the result at pH 7.5. As a result, almost 100% of all the compounds remained at pH 5.0 where the polyphenol was stabilized. On the other hand, at pH 7.5 where polyphenols are unstable, methylated theaflavin had better pH stability than theaflavin. Moreover, it became clear that pH stability improved, so that the number of methyl groups increased in all the evaluated compounds.

[Example 4]
<Metabolic stability test of methylated theaflavin using human liver tissue-derived S9 fraction>
The metabolic stability of the theaflavin standard product used in Example 1 and the methylated theaflavin obtained in Example 1 was evaluated using the S9 fraction derived from human liver tissue (manufactured by Sigma Aldrich).
The reaction composition was 50 mM Tris-HCl buffer (pH 7.5), 0.1 mM 3′-phosphoadenosine-5′-phosphosulfate, 1 mM uridine-5 ′ diphosphate-α-D-glucuronic acid, 1 mM nicotinamide adenine. Prepare 250 μl of a reaction solution consisting of dinucleotide phosphate, 10 mM MgCl ₂ , 0.063% bovine serum albumin, 8 mM DTT, human liver tissue-derived S9 fraction (final amount 50 μg protein), 5 μM various theaflavins or methylated theaflavins, 37 Incubated at 0 ° C. 50 μl was taken at 0, 15, 30, and 60 minutes after the start of the reaction, 45 μl of ice-cooled methanol and 5 μl of 1% (w / v) ascorbic acid aqueous solution were added and stirred. Then, it centrifuged at 14,000g for 3 minutes, 90 microliters was collect | recovered from the obtained supernatant, and various remaining theaflavins were analyzed by the method as described in Example 2.

  FIG. 10 shows various theaflavin residual rates 60 minutes after the start of the reaction. As a result, all the methylated theaflavins had a high residual rate compared with the corresponding theaflavins and showed good metabolic stability. It was also found that the metabolic stability of all the evaluated compounds increased as the methyl group increased.

[Example 5]
<Evaluation of fat accumulation inhibitory effect of methylated theaflavin>
The effect of TF3G methylation on fat accumulation was examined. Tea extract (extracted liquid before being used for Sep-Pak Vac C18 in Reference Example 8), TF3G (standard product) used in Example 1, 3MeTF3G and 3,5diMeTF3G obtained in Example 1 were used as test materials. did.

First, human organ fat-derived preadipocytes (Lot. No. 8F3482 manufactured by Sanko Junyaku Co., Ltd.) were cultured in a growth medium for preadipocytes at 37 ° C. in a 5% CO ₂ incubator and proliferated. Thereafter, the preadipocytes were seeded in 96-well plate (manufactured by Sumitomo Bakelite Co., Ltd.) so as to be 1.5 × 10 ⁴ cells / well and cultured for 24 hours. Differentiation was induced. Simultaneously with the differentiation induction, the final concentration was dissolved in DMSO (dimethyl sulfoxide) so that the final extract was 10 μg / ml, and each test material was 1 or 3 μM. Thereafter, every other day, the medium was replaced with a differentiation medium for preadipocytes so that Tea extract was 10 μg / ml and each test material was 1 or 3 μM, and cultured for 10 days. After completion of the culture, the amount of accumulated fat in the cells was evaluated by fluorescence intensity using AdipoRedassay reagent (Lonza Walkersville) (Ex: 485 nm, Em: 572 nm).

FIG. 11 shows the measurement results of the fat accumulation amount of the cells subjected to each treatment. FIG. 11 (A) shows the result when each test material is added to the medium so as to be 1 μM, and FIG. 11 (B) shows the result when it is added to the medium so as to be 3 μM. As a result, it was clarified that when TF3G, 3MeTF3G, or 3,5diMeTF3G was added, the numerical value was lower than when no addition or Tea extract was added, and fat accumulation was suppressed. In addition, 3MeTF3G and 3,5diMeTF3G have a stronger fat accumulation-inhibiting action than TF3G, which is an unmethylated form. Among them, it is clear that 3,5diMeTF3G has a fat accumulation-inhibiting action superior to 3MeTF3G, which is a known compound. It became.
From these results, it became clear that, in at least TF3G among the theaflavins, the action of inhibiting fat accumulation increases as the number of hydroxylated hydroxyl groups increases.

[Example 6]
<Evaluation of antioxidant activity of methylated theaflavin>
The inhibitory effect of methylated theaflavin on the oxidative stress caused by aqueous hydrogen peroxide on the human liver cancer cell line HepG2 was evaluated. Tea extract (extracted solution in Reference Example 8 before being subjected to Sep-Pak Vac C18), TF3′G and TF3,3′G (standard product) used in Example 1, and 3MeTF3 obtained in Example 1 'G, 3,5diMeTF3'G, 3MeTF3,3'G, 4MeTF3,3'G, and 3,5diMeTF3,3'G were used as test materials. These test materials were previously dissolved in DMSO.

HepG2 cells were subcultured in D-MEM medium containing 10% inactivated FBS (fetal bovine serum), 100 units / ml penicillin, 100 μg / ml streptomycin in a 37 ° C., 5% CO ₂ incubator. Then, HepG2 cells were seeded on 96-well plate (manufactured by Sumitomo Bakelite Co., Ltd.) so as to be 4 × 10 ⁴ cells / well. The cells were cultured for 23 hours at 37 ° C. in a 5% CO ₂ incubator in a D-MEM medium dissolved in DMSO so as to be 2, 2 or 20 μM. After removing the medium, 150 μl of D-MEM medium containing 12 mM hydrogen peroxide solution was added to each well, and the cells were exposed to hydrogen peroxide solution for 2 hours. Thereafter, 200 μl of 10% Alamar Blue-containing D-MEM medium was added to each well from which the supernatant was removed, and the fluorescence intensity was measured after culturing for 3 hours (Ex: 544 nm, Em: 590 nm).

  The HepG2 cell viability when no hydrogen peroxide solution was added was taken as 100%, and the HepG2 cell viability when each concentration of the test material was added was determined. The measurement results of the HepG2 cell viability are shown in FIG. FIG. 12A shows the measurement results when TF3′G and its methylated product are added, and FIG. 12B shows the measurement results when TF3, 3′G and its methylated product are added. In addition, the Tukey method was used for the test of the significant difference.

As a result of comparison with the case where only hydrogen peroxide solution was added or the case where Tea extract was added, the difference was not confirmed when theaflavin was added, but the cell viability was almost 100% when methylated theaflavin was added. It was found that oxidative stress was significantly suppressed. In particular, in TF3′G and TF3,3′G, it was confirmed that methylated theaflavin has a higher antioxidant effect than an unmethylated product.
In addition, when each test material was added so that it might become 2 or 20 micromol, the significant difference was not obtained between a non-methylated body and various methylated bodies. This is presumably because the effect of the antioxidant action was necessary and sufficient depending on the processing conditions such as the concentration of hydrogen peroxide solution.

In addition, the purchase place of the reagent used in Reference Examples 1-8 and Examples 1-6 is as follows.
Difco Potato Dextrose Ager: Nippon Becton Dickinson Peptone: Nippon Becton Dickinson Yeast Extract: Japanese Becton Dickinson Preadipocyte Growth Medium: Sanko Pure Drug D-MEM Medium: Wako Pure Drug Differentiation Medium: Sanko Pure Drug Alamar Blue Invitrogen FCS: JRH Bioscience Penicillin: GIBCO
Streptomycin: GIBCO
EGCG: Theavigo, DSM Nutrition Japan

Epicatechin-3-O-gallate (ECG): Funakashicatechin-3-O-gallate (CG): Funakoshigallocatechin-3-O-gallate (GCG): Funakoshicaffeic acid: Sigmamacrogogenic acid: Funakoshiellagic acid: Funakoshi Butein: Funakoshisulfuretin: Funacocilteolin: Funakoshimiricetin: Funakoshirosmarinic acid: Funakoshi

  Since the novel methylated theaflavin of the present invention is excellent in stability and functionality, it can be used in the production field of various products such as foods and drinks, pharmaceuticals, quasi drugs, and cosmetics.

Claims (12)

A methylated theaflavin having a galloyl group in which one or two or more hydroxyl groups are methylated, and represented by any one of the following general formulas (I) to (III):
[In formula (I), R _{1 to} R ₃ represent a hydrogen atom or a methyl group, and at least two of R _{1 to} R ₃ are methyl groups. ]
[In Formula (II), R _{4 to} R ₆ represent a hydrogen atom or a methyl group, and at least one of R _{4 to} R ₆ is a methyl group. ]
[In Formula (III), R _{7 to} R ₁₂ represent a hydrogen atom or a methyl group, and at least one of R _{7 to} R ₁₂ is a methyl group. However, the case where only R ₇ is a methyl group is excluded. ] It is represented by the formula of 1 selected from the group which consists of following formula (I) -1, (II) -1-4, and (III) -1-3, The Claim 1 characterized by the above-mentioned. Compound.
  A composition comprising the compound according to claim 1 or 2.   A fat accumulation inhibitor comprising the compound according to claim 1 or 2.   The fat accumulation inhibitor according to claim 4, wherein the compound is a compound represented by the formula (I) -1.   An antioxidant comprising the compound according to claim 1 or 2.   The compound is at least one selected from compounds represented by formulas (II) -1, (II) -3, (III) -1, (III) -2, and (III) -3 The antioxidant according to claim 6.   A food or drink comprising the compound according to claim 1 or 2.   A pharmaceutical product or quasi-drug comprising the compound according to claim 1 or 2.   A cosmetic comprising the compound according to claim 1 or 2. One or more selected from the group consisting of a compound represented by the following formula (I) -0, a compound represented by the following formula (II) -0, and a compound represented by the following formula (III) -0 By reacting a methyltransferase enzyme with the compound as a substrate, a compound represented by the following general formula (I), a compound represented by the following general formula (II), and a compound represented by the following general formula (III) A method for producing methylated theaflavin, comprising synthesizing one or more compounds selected from the group consisting of:
[In formula (I), R _{1 to} R ₃ represent a hydrogen atom or a methyl group, and at least two of R _{1 to} R ₃ are methyl groups. ]
[In Formula (II), R _{4 to} R ₆ represent a hydrogen atom or a methyl group, and at least one of R _{4 to} R ₆ is a methyl group. ]
[In Formula (III), R _{7 to} R ₁₂ represent a hydrogen atom or a methyl group, and at least one of R _{7 to} R ₁₂ is a methyl group. However, the case where only R ₇ is a methyl group is excluded. ]   The method for producing a methylated theaflavin according to claim 11, wherein the methyltransferase enzyme is an enzyme derived from Enokitake (Flamulina velutipes).