Method for producing physiologically active high-molecular-weight polyphenols

Abstract

This invention provides a novel method for producing bioactive high-molecular-weight polyphenols from fermented tea. [Solution] In reverse-phase high-performance liquid chromatography performed on an extract of fermented tea under conditions in which polyphenols and caffeine are separated and eluted, a group of compounds derived from the portion of the broad peak detected that has a longer retention time than the point at which epicatechin gallate is detected is purified from the water or water-containing organic solvent extract of the fermented tea using preparative chromatography, etc., which uses at least an aqueous solution containing an alcohol with 1 to 2 carbon atoms as the eluent, thereby obtaining the high molecular weight polyphenols. This makes it possible to produce a functional agent selected from the group consisting of a mitochondrial activator, a blood glucose level elevation inhibitor, an inhibitor of the formation of abnormal prion proteins, and a preventive and / or therapeutic agent for fatty liver.

Description

Technical Field

[0001] The present disclosure relates to a method for producing a physiologically active polymer polyphenol. The present disclosure also relates to a method for producing a functional agent selected from the group consisting of a mitochondrial activator, a blood glucose level increase inhibitor, an abnormal prion protein formation inhibitor, and a preventive and / or therapeutic agent for fatty liver.

Background Art

[0002] Polyphenols are a general term for various compounds having a plurality of phenolic hydroxyl groups in the same molecule. To date, the discovery, extraction, and pharmacological actions of various polyphenols have been reported.

[0003] Patent Document 1 discloses a polymer polyphenol extracted from fermented tea, having a number average molecular weight of 9,000 to 18,000, including a procyanidin structure in which catechins and / or their gallic acid esters are polymerized in a partial structure, and a structure in which B rings of catechins and / or their gallic acid esters are bonded to each other. And it is disclosed that this polymer polyphenol has a mitochondrial activation action and a blood glucose level increase inhibitory action.

[0004] Patent Document 2 discloses that a polymer polyphenol extracted and purified from fermented tea, similar to Patent Document 1, has an action of preventing and / or treating fatty liver.

[0005] Patent Document 3 discloses that a polymer polyphenol extracted and purified from fermented tea, similar to Patent Document 1, has an effect of suppressing the formation of abnormal prion proteins.

[0006] Patent Document 4 discloses that a polymer polyphenol extracted and purified from fermented tea, similar to Patent Document 1, has an action of promoting the slow muscle transformation of muscles and is effective for enhancing endurance and recovering fatigue.

Prior Art Documents

Patent Documents

[0007] [Patent Document 1] International Publication No. 2006 / 049258 [Patent Document 2] Japanese Patent Publication No. 2007-320958 [Patent Document 3] Japanese Patent Publication No. 2009-29752 [Patent Document 4] Japanese Patent Publication No. 2010-37323 [Overview of the project] [Problems that the invention aims to solve]

[0008] Further investigation into the preparation methods for the physiologically active polymeric polyphenols disclosed in Patent Documents 1 to 4 is significant for the production of pharmaceuticals containing the above polymeric polyphenols and for the manufacture of food and beverages utilizing the above polymeric polyphenols. The object of this disclosure is to provide a novel method for producing bioactive high-molecular-weight polyphenols, mainly from fermented tea. [Means for solving the problem]

[0009] The inventors of this invention have conducted extensive research on high-molecular-weight polyphenols extracted from fermented tea, obtained new insights, and completed the present invention based on these substitutions. Typical aspects of this disclosure include the following:

[0010] <1> A method for producing physiologically active high-molecular-weight polyphenols, A method for producing fermented tea, comprising purifying a group of compounds derived from portions of a broad range of peaks detected in reverse-phase high-performance liquid chromatography, performed under conditions in which polyphenols and caffeine are separated and eluted from the fermented tea extract, where the retention time is longer than the point at which epicatechin gallate is detected, from an extract of the fermented tea with water or a water-containing organic solvent. <2> The above purification includes performing preparative chromatography. The above preparative chromatography uses an aqueous solution containing at least one or two carbon atoms of alcohol as the eluent. <1> The manufacturing method described above. <3> The above purification process The method involves loading a solution containing the above-mentioned fermented tea with water or an extract of water-containing organic solvent onto a column made of polystyrene gel, octadecyl silica gel, dextran gel, or hydrophilic vinyl polymer gel and performing preparative chromatography. <2> The manufacturing method described above.

[0011] <4> A method for producing physiologically active high-molecular-weight polyphenols, The above manufacturing method is The method includes loading a solution containing water or an extract of fermented tea with an organic solvent onto a column made of polystyrene gel, octadecyl silica gel, dextran gel, or hydrophilic vinyl polymer gel and performing preparative chromatography. The above preparative chromatography uses an aqueous solution containing at least one or two carbon atoms of alcohol as the eluent. A method for producing the above-mentioned high-molecular-weight polyphenol, comprising obtaining the above-mentioned high-molecular-weight polyphenol from the fraction with a longer retention time in the above-mentioned preparative chromatography. <5> The above preparative chromatography further uses an aqueous acetone solution as the eluent. <2> ~ <4> A manufacturing method described in any of the following. <6> The above column is a dextran-based gel column. <5> The manufacturing method described above. <7> The process involves subjecting the fraction eluted with the above-mentioned aqueous acetone solution to a liquid-liquid separation treatment with an organic solvent and water, thereby obtaining the above-mentioned high-molecular-weight polyphenol from the aqueous layer. <6> The manufacturing method described above. <8> The above dextran-based gel column does not require chromatography or liquid-liquid treatment before preparative chromatography. <7> The manufacturing method described above. <9> A solution containing an extract of fermented tea with water or a water-containing organic solvent is loaded onto a dextran-based gel column and preparative chromatography is performed to obtain a fraction eluted with an aqueous acetone solution containing urea and acid, and The production method according to any one of <1> to <8>, which comprises subjecting the fraction obtained above to acetone distillation, then loading it onto a column of a polystyrene-based gel and performing preparative chromatography using an aqueous solution containing an alcohol having 1 to 2 carbon atoms as an eluent. <10> The production method according to any one of <1> to <9>, wherein the physiological activity is selected from the group consisting of a mitochondrial activation action, a blood glucose level increase inhibitory action, an abnormal prion protein formation inhibitory action, and an action of preventing and / or treating fatty liver. <11> A production method of a functional agent containing the above polymer polyphenol as an active ingredient, which comprises obtaining the above polymer polyphenol by the production method according to any one of <1> to <9>, The production method, wherein the functional agent is selected from the group consisting of a mitochondrial activator, a blood glucose level increase inhibitor, an abnormal prion protein formation inhibitor, and a preventive and / or therapeutic agent for fatty liver.

Advantages of the Invention

[0012] The present disclosure provides a new method for producing a polymer polyphenol having physiological activity from fermented tea.

Brief Description of the Drawings

[0013] [Figure 1] Shows the elution pattern of the reverse-phase HPLC of the black tea extract. [Figure 2] Shows the elution pattern of the reverse-phase HPLC of the powder obtained in Example 1. [Figure 3] Shows the elution pattern of the reverse-phase HPLC of the powder obtained in Reference Example 1. [Figure 4] Shows the 13C-NMR spectra of the powders obtained in Example 1 and Reference Example 1.

Modes for Carrying Out the Invention

[0014] Hereinafter, representative embodiments for carrying out the present invention will be described in detail with reference to the drawings. Note that the embodiments described below are for illustrative purposes only, and the following description in these embodiments should not be construed as limiting the language of the claims. In this specification, a numerical range represented by "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively. In this specification, "%" means "mass%" unless otherwise specified. In this specification, "fermented tea" refers to tea that includes a stage in which enzymes contained in the tea leaves oxidize the components within the tea leaves. Examples of fermented teas include oolong tea and black tea.

[0015] Patent Document 1 discloses that, among the extracts obtained by fractionating the butanol-eluting components extracted from fermented tea leaves with ethyl acetate, extracting the ethyl acetate-non-eluting components that were not extracted by the ethyl acetate extraction with butanol, and fractionating the butanol-eluting components extracted by the butanol extraction using column chromatography with an aqueous acetone solvent, the fraction that elutes at an acetone concentration of 35-50% and contains polyphenols with a number-average molecular weight in the range of 9,000-18,000 is a fraction that exhibits a high degree of mitochondrial membrane potential elevation. In this specification, this fraction may be referred to as the "MAF fraction" as a fraction containing mitochondrial activation factors (MAF). This fraction can be used as a mitochondrial membrane potential raising agent and, consequently, as a mitochondrial activator.

[0016] This disclosure primarily relates to a method for producing a bioactive high-molecular-weight polyphenol that corresponds to the MAF fraction. Specifically, the bioactive high-molecular-weight polyphenol refers to a high-molecular-weight polyphenol obtained from the group of high-molecular-weight polyphenols extracted from fermented tea leaves, as described in Patent Document 1, which has a higher degree of mitochondrial membrane potential elevation than other parts. This high-molecular-weight polyphenol is a mixture of multiple polyphenols with different molecular weights and structures.

[0017] In Patent Document 1, and in subsequent research, it has been found that the MAF fraction possesses the physiological activity described above and can be used as an active ingredient in functional agents for mitochondrial activators, blood glucose elevation inhibitors, abnormal prion protein formation inhibitors, and preventive and / or therapeutic agents for fatty liver. In this specification, "functional agent" refers to a compound(s) or composition that exhibits useful effects as an active ingredient in a pharmaceutical or health food based on the unique functions of the MAF fraction, and includes pharmaceuticals.

[0018] Mitochondrial activators can be used as pharmaceuticals to prevent or treat mitochondrial diseases. Mitochondria are major intracellular organelles whose primary role is ATP synthesis, and they are responsible for energy production, which is fundamental to cellular activity. Therefore, activating mitochondria can lead to cell activation and cell membrane stabilization. Consequently, using mitochondrial activators can provide anti-aging effects, skin beautifying effects, energy metabolism promotion effects, and anti-obesity effects. In addition to being applicable as pharmaceuticals, mitochondrial activators can also be used in cosmetics. Furthermore, mitochondrial activators may be incorporated into food and beverages as health foods.

[0019] Furthermore, since sperm motility largely depends on the ATP synthesis capacity of mitochondria in flagella, mitochondrial activators can be applied to the treatment of infertility in men (or males) and to improve fertilization rates in artificial insemination of humans, cattle, and other animals.

[0020] Furthermore, since ciliary motility is highly dependent on mitochondrial ATP synthesis, mitochondrial activators can enhance expectorant effects and ciliary motility in the fallopian tubes. Therefore, mitochondrial activators can be applied as expectorants and as infertility treatments for women (or females).

[0021] Furthermore, Patent Document 1 specifically demonstrates that the MAF fraction has an effect of suppressing the rise in blood glucose levels.

[0022] Patent Document 2 specifically discloses that the MAF fraction can be used as a preventive and / or therapeutic agent for fatty liver disease. Furthermore, Patent Document 3 specifically discloses that the MAF fraction can be used as an inhibitor of the formation of abnormal prion proteins.

[0023] Furthermore, Patent Document 4 (Japanese Patent Publication No. 2010-37323) discloses that the MAF fraction can be used as a muscle slow-twitch muscle development promoter, which is thought to be based on the mitochondrial activating effect of the MAF fraction.

[0024] On the other hand, it has long been said that the polyphenols extracted from fermented tea, especially black tea, are mainly theaflavins and thearubigins, of which thearubigin is a complex mixture of catechin enzyme oxidation products with a relatively large molecular weight. Many aspects of thearubigin's chemical composition remain unclear, and neither its structure nor its production mechanism has been elucidated. Recently, Tanaka et al. proposed a thearubigin production mechanism in which a quinone, produced by the oxidation of the catechin B ring by enzyme oxidation, binds to the catechin A ring in a reaction that is repeated several times [Hashiguchi, K.; Teramoto, S.; Katayama, K.; Matsuo, Y.; Saito, Y.; Tanaka, T. Journal of Agricultural and Food Chemistry 2023, 71 (41), 15319-15330.]. This explains why thearubigin is a mixture that is difficult to separate. Thus, it is impossible and meaningless to attribute the physiological activity of thearubidin to the specific structure that constitutes thearubidin; therefore, it had to be discussed as a mixture.

[0025] In the aforementioned paper by Tanaka et al., reverse-phase HPLC was performed on a black tea extract under specific conditions. While the detected sharp peaks were attributed to various polyphenols, thearubigin was detected as a broad peak spanning the time points where these sharp peaks were detected. In this specification, a "broad peak" refers to a portion of the HPLC elution pattern that is detected as a continuous rise from the baseline for a long retention time (e.g., 10 minutes or more). Compounds detected as broad peaks indicate a mixture that is difficult to separate.

[0026] However, the inventors have gained new insights into this difficult-to-separate mixture. As shown in Figure 1, it was found that among the broad peaks mentioned above, the portion with a longer retention time than the point at which epicatechin gallate is detected corresponds to the MAF fraction. In other words, it was found that the MAF fraction corresponds to the hydrophobic group of thearubidin compounds disclosed in the above-mentioned literature, and it was revealed for the first time that the hydrophobic group of thearubidin compounds corresponds to a physiologically active high-molecular-weight polyphenol, and that thearubidin has specific physiological activity. Furthermore, this discovery revealed that the method for preparing the hydrophobic group of thearubidin compounds can be applied as a method for producing physiologically active high-molecular-weight polyphenols, and that the method for preparing the hydrophobic group of thearubidin compounds can be applied as a method for producing a functional agent selected from the group consisting of mitochondrial activators, blood glucose elevation inhibitors, abnormal prion protein formation inhibitors, and preventive and / or therapeutic agents for fatty liver.

[0027] Previous methods for purifying thearubigin included fractionation using Sephadex LH-20 (dextran gel) of the extract or a fraction obtained by solvent partitioning [Cattell DJ, Nursten HE, Phytochemistry, 15, 1967-1970 (1976); Kusano R., Andou H., Fujieda M., Tanaka T., Matsuo Y., Kouno I., Chem. Pharm. Bull., 56, 266-272 (2008).], octadecyl silica gel [Bailey R., Nursten H., McDowell I., J. Chromatogr. A, 542, 115-128 (1991); Bailey R., Nursten H., McDowell I., J. Chromatogr. A, 662, 101-112 (1994).], and the acrylic resin Amberlite. Methods such as column chromatography using XAD-7 [Stodt UW, Stark J., Engelhardt UH, J. Food Compos. Anal., 43, 160-168 (2015).], polyvinyl gel (Toyopearl HW40F) [Japanese Patent Publication No. 2012-5413], or high-speed countercurrent chromatography [Degenhard A., Engelhardt UH, Wendt AS, Winterhalter P., J. Agric. Food Chem., 48, 5200-5205 (2000).] have been reported. In addition, a method is known in which thearubigin is obtained by applying the above chromatography to the precipitate formed by adding an excess amount of caffeine to a black tea extract [Powell C., Clifford MN, Opie SC, Ford MA, Robertson A., Gibson CL, J. Sci. Food Agric., 63, 77-86 (1992).].

[0028] However, these methods have not achieved the separation of hydrophobic and hydrophilic thearubidin. Furthermore, thearubidin obtained by these methods often contains small molecule compounds such as catechins and catechin dimers, and highly purified methods involve complex procedures. In addition, none of the above-mentioned literature describes the physiological activity of thearubidin, such as its ability to activate mitochondria.

[0029] The aforementioned paper by Tanaka et al. also does not contain any description of the physiological activity of thearubidin. Our discovery that the hydrophobic compounds of thearubidin constitute the MAF fraction has revealed that a method for preparing the hydrophobic fraction of thearubidin can be used as a method for producing physiologically active high-molecular-weight polyphenols.

[0030] The method for producing physiologically active high-molecular-weight polyphenols according to this disclosure comprises separating and purifying physiologically active high-molecular-weight polyphenols from fermented tea or an extract of fermented tea. As the extract of fermented tea, an extract of fermented tea using water or a water-containing organic solvent is preferred.

[0031] The physiologically active high molecular weight polyphenols are a group of compounds derived from the more hydrophobic portion, specifically the portion with a longer retention time than the point at which epicatechin gallate is detected, among the broad peaks derived from thearubidin detected when reverse-phase high-performance liquid chromatography is performed on an extract of fermented tea under conditions in which polyphenols such as theasinensins, epicatechins, theaflavins, and caffeine are separated and eluted. The production method of this disclosure includes purifying the above group of compounds from fermented tea or an extract of fermented tea. The conditions under which polyphenols and caffeine are separated and eluted do not need to be conditions under which all polyphenols are separated and eluted, but it is preferable that at least epicatechin and epigallocatechin gallate are separated.

[0032] The purification of the above-mentioned physiologically active high-molecular-weight polyphenols can be carried out, for example, by methods using adsorbents such as various resin gels, or by preparative chromatography. In this case, the point at which epicatechin gallate is detected in the reversed-phase HPLC described above should be used as a reference, and conditions should be selected to obtain a purified product in which a broad range of peaks, including those with longer retention times than the point at which epicatechin gallate is detected, are detected. Furthermore, it is preferable to select conditions such that when the above-mentioned reversed-phase HPLC is performed on the purified product, peaks derived from various polyphenols and caffeine, for example as shown in Figure 1, are not detected.

[0033] In one embodiment, the method for producing the physiologically active polymeric polyphenol of the present disclosure includes loading a solution containing an extract of fermented tea with water or a water-containing organic solvent onto a column of polystyrene gel, octadecyl silica gel, dextran gel, or hydrophilic vinyl polymer gel and performing chromatography. The chromatography is preferably performed using an aqueous solution containing at least one to two carbon atoms as the eluent. The physiologically active polymeric polyphenol can be obtained from the fraction with a longer retention time in the chromatography.

[0034] A solution containing an extract of fermented tea with water or a water-containing organic solvent includes the extract itself, a concentrated or diluted version of the extract, the extract from which insoluble matter has been removed, the extract subjected to chromatography or liquid-liquid separation, and a powder obtained by removing the solvent from the extract and drying it, which is then redissolved in water or a water-containing organic solvent.

[0035] Examples of organic solvents in water or water-containing organic solvents include acetone, methanol, and ethanol. The concentration of the organic solvent in water-containing organic solvents is not particularly limited; for example, it may be 10-90%, preferably 30-80%, and more preferably 50-70%. Preferred water or water-containing organic solvents are water, a 60% aqueous acetone solution, and a 60% aqueous ethanol solution. When performing extraction with water, it is preferable to use water at 60°C or higher, preferably water at 90°C or higher, and more preferably water at 100°C (boiling water, hot water, or steam).

[0036] Examples of columns that can be used include polystyrene gels, octadecyl silica gels, dextran gels, hydrophilic vinyl polymer gels, or methacrylate ester gels. An example of a polystyrene gel is Diaion HP20SS. Octadecyl silica gel is a silica gel column to which octadecyl groups (ODS) are bound, and any commercially available column can be used. An example of a dextran gel is Sephadex LH-20. Examples of hydrophilic vinyl polymer gels are TOYOPEARL HW40 (Tosoh Corporation) and TOYOPEARL HW-50 (Tosoh Corporation).

[0037] Elution with an aqueous solution containing an alcohol with 1 to 2 carbon atoms is preferably carried out by gradually increasing the amount of alcohol in the aqueous solution. For example, the amount of alcohol can be increased by 10% or 20% at a time. By gradually increasing the amount of alcohol during elution, low molecular weight compounds such as sugars in the fermented tea extract can be eluted with lower alcohol concentrations, and high molecular weight polyphenols corresponding to the MAF fraction, which are eluted as fractions with longer retention times with higher alcohol concentrations or more hydrophobic eluents, can be separated. Elution with water may be included before elution with an aqueous solution containing an alcohol with 1 to 2 carbon atoms, and elution with an alcohol with 1 to 2 carbon atoms may be included afterward. Note that elution with water or elution with an aqueous solution containing an alcohol with 1 to 2 carbon atoms may also be called washing. Alcohols with 1 to 2 carbon atoms are, in other words, methanol or ethanol.

[0038] The above chromatography preferably includes elution with an aqueous solution containing methanol followed by elution with an aqueous solution containing acetone. For example, depending on the type of column, particularly the gel (resin) used in the column, if the MAF fraction does not elute with methanol, a methanol-water-acetone mixture, or even an aqueous acetone solution (water-acetone mixture), can be used. The acetone concentration in the aqueous acetone solution can be appropriately set depending on the type of gel (resin) used in the column, but is typically 40% to 70%, preferably 50% to 60%.

[0039] The fractions with longer retention times vary depending on the type of column, but are, for example, fractions eluted with aqueous solutions containing 80% or more or 90% or more methanol, or with solutions containing methanol or acetone. The MAF fraction can be identified as a broad peak that rises from approximately the point at which epicatechin gallate is detected in the reverse-phase HPLC described above. The MAF fraction can also be identified using silica gel TLC. In silica gel TLC, the MAF fraction is detected at the origin.

[0040] The fraction eluted as a fraction with a longer retention time is preferably subjected to liquid-liquid separation of an organic solvent and water, either as is, or after removal of the solvent or dilution with a solvent as necessary. The MAF fraction can be obtained from the aqueous layer. Ethyl acetate can be used as the organic solvent. The aqueous layer can be concentrated and dried as is to obtain a powder of physiologically active high molecular weight polyphenol, but additional chromatography or liquid-liquid separation may be performed as necessary.

[0041] In a preferred embodiment of the manufacturing method of this disclosure, a dextran-based gel column is used as the column. Using this column, elution is performed with an aqueous solution containing a carbon-1 to carbon-2 alcohol, followed by elution with an aqueous acetone solution. The fraction eluted with the aqueous acetone solution is subjected to liquid-liquid separation with an organic solvent (e.g., ethyl acetate) and water to obtain the MAF fraction from the aqueous layer. In this method, chromatography using the dextran-based gel column can be performed by loading the fermented tea extract as is, or a solution that has undergone only simple operations such as concentration, dilution, or filtration, and does not require chromatography or liquid-liquid separation as a pretreatment. The MAF fraction can be obtained with high purity even without such pretreatment. Furthermore, sufficient purity can be obtained without further purification in post-treatment. However, post-treatment may be performed as needed. For example, a polystyrene-based gel column may be loaded with water or a low-concentration methanol aqueous solution (e.g., 20% to 60%) and dissolved with a higher-concentration methanol aqueous solution (e.g., 80% or more, or 60%) or methanol.

[0042] In another preferred embodiment of the manufacturing method of the present disclosure, a solution containing an extract of fermented tea with water or a water-containing organic solvent is loaded onto a dextran-based gel column to obtain a fraction eluted with an aqueous acetone solution containing urea and acid. After removing the acetone from this fraction, the solution is loaded onto a polystyrene-based gel column and chromatography is performed using an aqueous solution containing a carbon-1 to carbon-2 alcohol as the eluent. In this method, chromatography using the dextran-based gel column can be performed using the fermented tea extract as is, or a solution that has undergone only simple operations such as concentration, dilution, or filtration, and does not require chromatography or liquid-liquid extraction as a pretreatment.

[0043] As the aqueous acetone solution containing urea and acid, for example, a urea aqueous solution of about 3 to 10 M, preferably 5 to 8 M, can be mixed with acetone in a volume ratio of about 1:3 to 3:1. As the acid, hydrochloric acid, trifluoroacetic acid, formic acid, acetic acid, trichloroacetic acid, perchloric acid, etc., can be used. For example, concentrated hydrochloric acid may be mixed in at a concentration of about 0.1 mL to 10 mL. In this step using the aqueous acetone solution containing urea and acid, polyphenols with small molecular weights (for example, catechins and theaflavins) can be removed.

[0044] Elution of polystyrene gel columns with an aqueous solution containing a carbon-1 to carbon-2 alcohol is preferably carried out by gradually increasing the amount of alcohol in the aqueous solution, as described above. For example, the amount of alcohol can be increased by 10% or 20% at a time. When methanol is used as the alcohol, the MAF fraction can be obtained from the fraction eluted with 40% to 80% methanol.

[0045] Furthermore, this disclosure clarifies the relationship between thearubigin and the MAF fraction, demonstrating that the MAF fraction in tea (including fermented tea) can be quantified using a simple reversed-phase HPLC method. By comparing the portion of the broad range of peaks detected in the aforementioned reversed-phase HPLC that retains longer than the point at which epicatechin gallate is eluted, the amount of the MAF fraction can be compared. This makes it possible to produce fermented tea containing a larger amount of physiologically active high-molecular-weight polyphenols.

[0046] A functional agent containing a high molecular weight polyphenol as an active ingredient can be produced using the manufacturing method of this disclosure. The functional agent is not particularly limited, but examples include functional agents selected from the group consisting of mitochondrial activators, blood glucose level elevation inhibitors, abnormal prion protein formation inhibitors, and agents for the prevention and / or treatment of fatty liver.

[0047] The polymer polyphenol obtained by the production method of the present disclosure can be formulated by known methods. Examples of the formulation include oral preparations such as granules, tablets, and capsules, and parenteral preparations such as injections. By administering this formulation to mammals including humans, known effects (for example, the effects exerted by the MAF fraction described in Patent Documents 1 to 4) can be obtained based on the physiological activity of the polymer polyphenol. The dosage of the formulation can be appropriately determined according to the age, sex, weight, physical condition, degree of fatty liver, etc. of the application target. In addition, an effective amount showing physiological activity can be added to various forms of food and drink (including supplements) containing the polymer polyphenol obtained by the production method of the present disclosure to produce a food with functional claims.

Examples

[0048] The present invention will be described more specifically with reference to the following examples. The materials, reagents, amounts of substances and their ratios, operations, etc. shown in the following examples can be appropriately changed as long as they do not deviate from the gist of the present invention. Therefore, the scope of the present invention is not limited to the following examples.

[0049] <Preparation of polymer polyphenol corresponding to MAF fraction> (Reference Example 1) The neutral component extracted from black tea with butanol described in Example 1 of Patent Document 1 (International Publication No. 2006 / 049258) was fractionated by column chromatography according to the procedure described in Patent Document 1, and a fraction eluted with an aqueous acetone solvent having an acetone concentration of 35% to 50% was obtained. As disclosed in Patent Document 1, this fraction is a fraction having a high degree of increase in mitochondrial membrane potential among the fractions containing polymer polyphenols extracted from black tea, and has a higher mitochondrial activation ability. This fraction was concentrated under reduced pressure and freeze-dried to obtain a reddish-brown powder.

[0050] (Example 1) 20g of commercially available Kenyan black tea was extracted with a 60% acetone aqueous solution, and the organic solvent was removed by vacuum distillation. The precipitate remaining in the aqueous solution was dissolved with a small amount of methanol. The aqueous solution was loaded onto a Diaion HP20SS column (3cm diameter x 18cm length), and sugars and other components were eluted with water. Then, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol (the remaining component when not 100% methanol is water; the same applies below) were sequentially passed through the column to obtain fractions eluted with 70% and 80% methanol. These fractions were attached to a Sephadex LH-20 (4cm diameter x 45cm length) and eluted with a 7M urea-acetone mixture (2:3, volume ratio) containing HCl (5mL / L). After removing acetone from the fraction containing only high molecular weight polyphenols detected at the origin using silica gel TLC, the column was loaded onto a Diaion HP2-SS column (2 cm in diameter × 15 cm in length) and eluted with water and methanol (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%). The fraction eluted with 40-80% methanol was concentrated under reduced pressure and freeze-dried to obtain a reddish-brown powder.

[0051] (Example 2) 1.91 kg of commercially available black tea (Assam CTC) was extracted with 6 L of an acetone-water mixture (6:1) and filtered. The residue on the filter paper was extracted twice more in the same manner, and the three extracts were combined and the acetone was removed by distillation using a rotary evaporator to obtain an aqueous solution. Insoluble matter formed in the aqueous solution was removed by decantation, and the supernatant was attached to a Sephadex LH-20 column (10 cm diameter × 35 cm length) that had been replaced with water. The eluents were, first, water, then 20%, 40%, 60%, 70%, 80%, 90%, and 100% methanol, then methanol-water-acetone mixtures (90:5:5, 80:10:10, 70:15:15, 60:20:20; 1 L each), and finally 4 L of an aqueous acetone solution (1:1). The eluates were monitored for components by normal-phase silica gel thin-layer chromatography and reverse-phase HPLC and fractionated into 1 to 10 fractions. The last eluted fraction (33.4 g) was dissolved in 200 mL of acetone-aqueous mixture (1:1), and ethyl acetate (500 mL) and water (500 mL) were added. The mixture was then transferred to a separatory funnel and the solvent was partitioned. The aqueous layer was further partitioned three times with ethyl acetate (500 mL) (ethyl acetate layer: 19.5 g). The aqueous layer was concentrated and freeze-dried to obtain 13.8 g of reddish-brown powder.

[0052] <Reference Example 1, Comparison of powders obtained in Example 1 and Example 2> The powders obtained in Reference Example 1, Example 1, and Example 2 were subjected to reversed-phase high-performance liquid chromatography (reverse-phase HPLC) under the same conditions as described in the aforementioned paper by Tanaka et al. (Journal of Agricultural and Food Chemistry 2023, 71 (41), 15319-15330). The results for Example 1 are shown in Figure 2, and the results for Reference Example 1 are shown in Figure 3.

[0053] Column: Cosmosil 5C18-ARII (250×4.6 mm id, 5μm) (Nacalai Tesque, Kyoto, Japan) Mobile phase: The concentration of CH3CN in a 50 mMH3PO4 aqueous solution is increased from 4% to 30% between 0 and 39 minutes, then from 30% to 75% between 39 and 54 minutes, and after maintaining 75% for 5 minutes, it is returned to the initial concentration. Flow rate: 0.8mL / min Column temperature: 35℃ Detector: Ultraviolet-Visible detector UV: Max Absorbance (Traces the wavelength with the highest ultraviolet-visible absorption during detection) (For quantitative analysis, it is necessary to fix the wavelength to a specific one, e.g., 275 nm or 280 nm) Injection volume: 5mg / mL 60%CH3CN solution 5μL

[0054] Furthermore, the powders obtained in Reference Example 1 and Example 1 were analyzed using a Varian NMR System 500PS SN spectrometer (126MHz) manufactured by Varian. 13 ¹³C-NMR nuclear magnetic resonance (C-NMR) spectroscopy measurements were performed. The results of comparing the two methods are shown in Figure 4. The top row shows the results for Reference Example 1, and the bottom row shows the results for Example 1.

[0055] Figures 2, 3, and 4 clearly show that the powder of Reference Example 1 is the same as the powder of Example 1. Furthermore, the same chromatography results as in Figures 2 and 3 were obtained for the powders obtained using the method of Example 2. In other words, the powders obtained using the methods of Example 1 and Example 2 are high molecular weight polyphenols with the same physiological activity as the powder obtained using the method of Reference Example 1 (MAF fraction), and can similarly be used as active ingredients in functional agents selected from the group consisting of mitochondrial activators, blood glucose elevation inhibitors, abnormal prion protein formation inhibitors, and preventive and / or therapeutic agents for fatty liver.

Claims

1. A method for producing physiologically active high-molecular-weight polyphenols, A method for producing fermented tea, comprising purifying a group of compounds derived from portions of a broad range of peaks detected in reverse-phase high-performance liquid chromatography, performed under conditions in which polyphenols and caffeine are separated and eluted from the extract of fermented tea, where the retention time is longer than the time at which epicatechin gallate is detected, from an extract of the fermented tea with water or a water-containing organic solvent.

2. The purification includes performing preparative chromatography. The manufacturing method according to claim 1, wherein the preparative chromatography uses an aqueous solution containing at least one or two carbon atoms of alcohol as the eluent.

3. The aforementioned purification process The production method according to claim 2, comprising loading a solution containing the fermented tea with water or an extract of water-containing organic solvent onto a column of polystyrene gel, octadecyl silica gel, dextran gel, or hydrophilic vinyl polymer gel and performing preparative chromatography.

4. A method for producing physiologically active high-molecular-weight polyphenols, The aforementioned manufacturing method is The method includes loading a solution containing water or an extract of fermented tea with an organic solvent onto a column made of polystyrene gel, octadecyl silica gel, dextran gel, or hydrophilic vinyl polymer gel and performing preparative chromatography. The aforementioned preparative chromatography uses an aqueous solution containing at least one or two carbon atoms of alcohol as the eluent. A method for producing the high molecular weight polyphenol, comprising obtaining the high molecular weight polyphenol from the fraction with a longer retention time in the aforementioned preparative chromatography.

5. The manufacturing method according to claim 4, wherein the preparative chromatography further uses an aqueous acetone solution as the eluent.

6. The manufacturing method according to claim 5, wherein the column is a dextran-based gel column.

7. The manufacturing method according to claim 6, comprising subjecting the fraction eluted with the aqueous acetone solution to a liquid-liquid separatory treatment with an organic solvent and water, and obtaining the high-molecular-weight polyphenol from the aqueous layer.

8. The manufacturing method according to claim 7, wherein chromatography or liquid-liquid treatment is not performed before the preparative chromatography of the dextran-based gel column.

9. A solution containing an extract of fermented tea with water or a water-containing organic solvent is loaded onto a dextran-based gel column and preparative chromatography is performed to obtain a fraction eluted with an aqueous acetone solution containing urea and acid, and The manufacturing method according to claim 4, comprising removing acetone from the fraction by distillation, loading a polystyrene gel column, and performing preparative chromatography using an aqueous solution containing an alcohol having 1 to 2 carbon atoms as the eluent.

10. The manufacturing method according to any one of claims 1 to 9, wherein the physiological activity is selected from the group consisting of mitochondrial activation, blood glucose elevation suppression, abnormal prion protein formation suppression, and fatty liver prevention and / or treatment.

11. A method for producing a functional agent containing the polymer polyphenol as an active ingredient, comprising obtaining the polymer polyphenol by the manufacturing method described in any one of claims 1 to 9, A method for producing the functional agent selected from the group consisting of mitochondrial activators, blood glucose level elevation inhibitors, abnormal prion protein formation inhibitors, and fatty liver preventive and / or therapeutic agents.

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