MX2007010344A - Method of producing proanthocyanidin oligomer. - Google Patents

Abstract

A composition containing, as the main component, a proanthocyanidin oligomer which is obtained by heating a plant material containing a proanthocyanidin polymer or its extract together with a substance having a phloroglucinol ring or resorcinol ring structure in an acidic solution, carries the substance having the fluoro/resorcinol ring structure attached thereto and has been degraded; a method of producing the same; and use of the composition in health foods, drugs and so on. Thus, the proanthocyanidin oligomer having a biological activity, carrying the substance having a phloroglucinol ring or resorcinol ring structure attached to the terminus thereof and having been degraded to a level allowing the absorption <i>in vivo, </i>which can be hardly obtained from a plant material at a high yield, can be efficiently and easily obtained.

Description

METHOD OF PRODUCTION OF OLIGOMERO DE LA PROANTOCIANIDINE Technical Field The invention relates to a method for producing an oligomer of proanthocyanidin, which reduces a molecular weight of the proanthocyanidin polymer in plants to a level that can be absorbed (easily by the gastrointestinal tract) in a living body. . More specifically, the invention relates to a composition containing the oligomer of proanthocyanidin, which has a degree of polymerization of 2 to 4 and has a ring structure of the phloroglucinol or ring structure of the resorcinol attached at its terminus, which is obtains by heating a material containing the proanthocyanidin polymer together with a substance having a ring structure of the phloroglucinol or ring structure of the resorcinol in an acid solution, its method of production, the uses of the composition and a novel oligomer of the proanthocyanidin having a ring structure of the phloroglucinol or ring structure of the resorcinol. The composition containing the oligomer of the, according to the invention, can be used in food products, health food products, specific foods for use in health and medical products. Especially, the composition is useful as a composition for food products for the health and medical products for the prevention of diseases related to a lifestyle, caused by the generation of reactive oxygen species, prevention and treatment of brain diseases or the prevention of aging.

Previous Technique Due to the excessive admission of fat due to changes in our eating habits, increased exposure to UV rays due to the change in the environment, ozone depletion and others, the increase in environmental and similar pollutants, the incidence regimes of so-called lifestyle-related diseases, such as hyperlipidemia, hypercholesterolemia, high blood pressure, diabetes and cancers, are increasing, and the number of patients with allergies or with brain diseases, such as dementia, is also increasing. There is a problem that the number of patients with dementia or Alzheimer's syndromes will be increased in the future, with rapid aging of society. The complication of reactive oxygen species, generated in vivo, has been pointed out in the factors that contribute to these diseases. (Biorganic &Medicinal Chemistry, Vol. 10 (2002, pp 2497-2509; Document 1, unpatented) However, since the perfect technology to suppress or control the generation of reactive oxygen species has not been developed, a sufficiently safe medical technique, useful for the treatment and prevent diseases related to lifestyle, brain diseases and the like. Recently, natural substances present in plants and exhibiting physiological activities, especially polyphenol compounds that have attracted attention. Polyphenols, which are generally contained in tea, vegetables, fruits, herbs and the like, can be expected to be ingested as food and drinks for a long period of time and serve as treatment / prevention agents, free from side effects. Polyphenol compounds, secondary plant metabolites, which exist universally in the plant world in a large quantity, are known to exhibit various physiological activities, attracted attention in the pharmaceutical and phytochemical fields, in earlier times, and have recently called attention in the field of health foods. For example, tea polyphenols, especially catechins, are known to have a wide range of physiological activities, such as antibiotic properties, antiviral action, antimutagenic property. , antioxidant effect, suppression of blood pressure increase, property of reducing blood cholesterol, property against decay, antiallergic activity, improvement of enteric flora, odor elimination activity, and the like. Among the polyphenols, proanthocyanidins are contained in a wide variety of plants, in order that these Proanthocyanidins exhibit several physiological activities, the proanthocyanidin compound needs to be absorbed into the living body by the gastrointestinal tract. However, the molecular weights of proanthocyanidins are generally said to be in the order of several thousand to several hundred thousand. The substance, which has such a large molecular weight, is difficult to absorb by gastrointestinal touch and, in many cases, even if it is ingested, it is not absorbed in the living body and is not used as a nutrient. The term "proanthocyanidins" is a generic name of procyanidin, prodelfinidin, propelargonidin and the like of polymers of dimers, trimers, tetramers, decamers or higher oligomers, having as a constituent unit flavan-3-ol also referred to as catechins) and those with the gallic acid esterified there, and their stereoisomers, which are polyphenol compounds, which generalize antrocyanidins through acid treatment. The constituent units are linked together through a carbon to carbon bond between position 4 and position 8 of the carbon skeleton or between position 4 and position 6, or sometimes through ether bonds between the position 2 and position 7, in addition to the carbon to carbon bond.

Proanthocyanidin has an excellent antioxidant effect (Arch Biochem, Biophys., Vol. 374, p.p. 347-355, 2000, Document 2, not patented) and also, since it has other effects, such as improved blood flow, action against stress, antihypertensive efficacy, antibiotic effect, effect against tumors, activity against cataracts and antidiarrheal effect, has been used as a naturally occurring substance, which has a maintenance effect of health. Proanthocyanidins are isolated as a mixture of pineapple bark, unripe apple fruit, grape seeds and the like and are now mixtures in beverages, confectionery, health foods, cosmetic products, hair growth drugs and the like , which are commercially available. In many proanthocyanidin-containing plants, several proanthocyanidins from those with a low degree of polymerization to those with a high molecular weight are contained as mixtures, and many of them are plants that contain mainly high-polymerization-grade proanthocyanidin, such as the persimmon, banana and Chinese quince. However, among the proanthocyanidins, a proanthocyanidin polymer, which has a high degree of polymerization, is said to be lower in the pharmacological activities than proanthocyanidin oligomers having a degree of polymerization of 2 to 4, due to its poor capacity of absorption in the intestine. Likewise, it is preferable that such a proanthocyanidin polymer, which has a strong astringency and poor water solubility, be eliminated when the plant is used in food products (Free Radical Res., Vol. 29, p.p. 351-358, 1998, Document 3, not patented). Based on these facts, the oligomer of proanthocyanidin, which has a degree of polymerization of 2 a, has attracted attention as having an excellent effect in the maintenance of health and those derived from the bark of pinto are used in beverages and in food products. In order to obtain only proanthocyanidin from plant extracts, the absorption method (see, for example, H06 49053, Patent Document 1) and the like, is employed. But it is difficult to isolate these different degrees of polymerization. In order to obtain only oligomers of proanthocyanidin with polymerization degrees from 2 to 4, the solvent division method, which uses ethyl acetate, the solid phase extraction method with methyl acetate and the chromatography method (PCT Publication WO00 / 64883, Patent Document 2), the chitin absorption method (PCT Publication WO03 / 091237, Patent Document 3) and the like, are used to isolate only the low molecular weight proanthocyanidins, through of the extraction. However, in these methods, a large number of high molecular weight proanthocyanidin polymers are discarded, which is disadvantageous in terms of yield. As an alternative method, which replaces the methods that isolate proanthocyanidin oligomers of polymerization degree 2 to 4 from plants containing the polymers of the proanthocyanidin, the present inventors previously proposed a method of reacting the material containing the proanthocyanidin polymer with an SH-containing compound, such as cysteine, in an acid solution, to reduce the molecular weight of the oligomer of proanthocyanidin (PCT Publication). WO2004 / 103988, Patent Document 4), according to this method, the oligomer of proanthocyanidin, which has the cysteine bound to thereby reduce the molecular weight and have an excellent systemic absorption, can be obtained. It has been confirmed that the oligomer of proanthocyanidin has no toxicity and can be used safely. However, currently, in some countries (including Japan) there is a problem that strict procedures are in place to obtain approval and use of health food products, which contain the proanthocyanidin that has cysteine attached, which is a substance non-natural chemistry [Patent Document 1] Japanese Patent Application, Open to the Public, No. H06-49053; [Patent Document 2] PCT Publication No.

WO00 / 64883; [Patent Document 3] PCT Publication No. WO003 / 091237; [Patent Document 4] PCT Publication No. WO2004 / 103988 [Non-Patent Document 1] Bioorganic & Medicinal Chemistry, Vol. 10 (2002), p. P.2497-2509 [Non-patent document 2] Arch. Biochem. Biophys., Vol. 374, p.p.347-355, 2000 [Non-patent document 3 [Free Radial Res., Vol. 29, p.p. 351-358, 1998.

DESCRIPTION OF THE INVENTION Problems to be solved by the invention The object of the invention is to provide a convenient and efficient method for reducing the molecular weight of the oligomer of proanthocyanidin. widely distributed in nature as a proanthocyanidin polymer, but limited in naturally occurring materials as an oligomer, by use, as a starting material of the proanthocyanidin polymer, plants containing the proanthocyanidin polymer or its extract and binding the material to a substance that has a ring structure of phloroglucinol or ring structure of resorcinol. Means to solve the problem In order to achieve the above object, the inventors have done intensive studies and as a result, have found that the Proanthocyanidin can be fractionated and its molecular weight reduced and, at the same time, it can be converted into the oligomer of proanthocyanidin which has the catechin bound in the terminal by moderately boiling fruits, fruit skin, bark, leaves or its extract, which contains the polymer of proanthocyanidin, such as plum, banana, grapes, pineapple, Chamaecyparis obtuse, camphor tree, wax myrtle, Chinese quince, litche, Myrica rubra and cinnamon bark, together with green tea leaves or fresh tea, containing a large amount of low molecular weight catechins, in an acid solution, for 2 to 3 hours. Also, the inventors have found that proanthocyanidin can be fractionated, reduced in its molecular weight and converted into the oligomer of proanthocyanidin, having a substance with a ring structure of the phloroglucinol or a ring structure of the resorcinol attached thereto, by the use of the substance having the ring structure of the floroglucinol or the structure of the ring of resorcinol and other plant materials (such as the seeds of grapes and skin of ace) containing such substance instead of the leaves of green tea or of fresh tea and have carried out the invention based on these findings. That is, the invention is referred to in the following paragraphs 1 to 14, to a composition containing as its main component the oligomer of proanthocyanidin at the terminal of the which substance having a ring structure of phloroglucinol or a ring structure of resorcinol, is bound by heating to the plant containing the proanthocyanidin polymer or its extract, with the leaves of green tea or fresh tea in a solution acid (1 to 5). Its production method (6 to 9) uses the composition (10 to 12) a novel oligomer of proanthocyanidin. 1. A composition that contains, as its main component, the proanthocyanidin oligomer in whose terminal a substance having the ring structure of floroglucinol or the ring structure of resorcinol, has been bound and reduced in molecular weight, which was obtained by heating plant materials containing the proanthocyanidin polymer or its extract, with a substance having the ring structure of the phloroglucinol or the ring structure of the resorcinol, the plants containing such substance or its extract in an aqueous acidic solution. 2. The composition containing as its main component the oligomer of proanthocyanidin, described in paragraph 1, in which the plant containing the proanthocyanidin polymer is at least one class selected from the group consisting of grapes, pineapple. Chamaecyparis obtuse, camphor tree, myrtle of wax, cocoa, plum season, banana, chinese quince, hawthorn apple, litche, Myrica rubra and cinnamon bark. The composition containing as its main component the oligomer of proanthocyanidin, described in paragraph 1, in which the substance having a ring structure of the phloroglucinol or the ring structure of the resorcinol, is at least one class selected from the group consists of resveratrol, phloroglucinol, flavonoid and flavannoid (Galoyl ester of the catechin). The composition containing as its main component the oligomer of proanthocyanidin, described in paragraph 1, in which the substance having the structure of the floroglucinol ring or the ring structure of the resorcinol, is at least one class selected from the group consisting of of green tea leaves, fresh tea leaves, grape seeds, lining of cubic gambir grape seeds, red algae and their extracts; The composition containing as its main component the oligomer of proanthocyanidin, described in paragraph 1, having a degree of polymerization of 2 to 4; A method for producing a composition containing, as its main component, the oligomer of proanthocyanidin. described in any of paragraphs 1 to 5, which comprises the step of heating the materials of plants containing the proanthocyanidin polymer or its extract, with plants having a ring structure of the floruglucinol or ring structure of the resorcinol, or its extract, in an aqueous acidic solution, and a step of concentrating the reaction solution, containing the oligomer of proanthocyanidin, with a ring structure of the floroglucinol or ring structure of the resorcinol, attached to the terminals, and reducing the molecular weight and drying the solution; A method for producing the composition containing as its main component the proanthocyanidin described in any of paragraphs 1 to 5, comprising the step of concentrating the reaction solution containing the oligomer of proanthocyanidin with a substance having a ring structure from phloroglucinol or ring structure of resorcinol attached to the terminals, and reduced in molecular weight,, and a step of subjecting the concentrated solution to the fractionation treatment; The method of producing a composition containing, as its main component, the oligomer of the proanthocyanidin described in paragraphs 6 or 7, in which the acid condition is prepared using an inorganic acid, an organic acid or both of them.

The method for producing a composition containing as its main component the oligomer of proanthocyanidin, described in paragraph 8, in which at least one class selected from the group consisting of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, citric acid , ascorbic acid and malic acid. The composition containing the oligomer of proanthocyanidin, described in any of paragraphs 1 to 5, used in health food products for the treatment / prevention of lifestyle-related diseases, caused by the generation of oxygen species reagent and diseases of the brain or prevention of aging. The composition containing the oligomer of proanthocyanidin, described in paragraph 10, used in pharmaceutical products for the treatment / prevention of lifestyle-related diseases, caused by the generation of reactive oxygen species and diseases of the brain or for the prevention of aging. The composition containing the oligomer of proanthocyanidin, described in paragraph 10, used in cosmetic products for the prevention of aging, caused by the generation of reactive oxygen species.

An oligomer of proanthocyanidin, represented by the following formula (1): A proanthocyanidin oligomer, represented by the following formula (2): [in the formula, n is O or an integer of 1 or 3) Effect of the Invention The invention provides a method for producing a composition containing, as its main component, the oligomer of proanthocyanidin to which a substance having a ring structure of the floroglucinol or resorcinol ring structure has been attached, in the terminals and where it has been reduced in molecular weight, this composition is obtained by concentrating and drying a reaction solution obtained by heating plant materials containing the proanthocyanidin polymer or its extract, with a substance or plants having a ring structure of floroglucinol or the ring structure of resorcinol, or its extract, in an aqueous acidic solution. According to the invention, the oligomer of proanthocyanidin to which a substance having the ring structure of the floroglucinol or ring structure of the resorcinol has been attached at the terminals and which has been reduced in molecular weight and which is useful as a composition for salid food products, and pharmaceutical products for treatment / prevention of reactive oxygen species and diseases of the brain or for the prevention of aging, can be produced efficiently from materials containing the proanthocyanidin polymer.

The Best Mode for Carrying Out the Invention In the following, the invention is described in greater detail. The raw materials used to produce the oligomer of proanthocyanidin, according to the method of the invention, are plants that contain the proanthocyanidin polymer (such as a fruit, fruit peel, bark and leaves) or their extracts. Here, examples of plants containing the proanthocyanidin polymer include fruits, vegetables, such as astringent persimmon, banana, apple, pear, grape, strawberries, raspberry, hawthorn, lotus root, buckwheat, litchee and Myrica rubra, herbs, spices, wood, cinnamon bark and pineapple tenderloin. Among these, the astringent persimmon, banana, grape, pineapple, Chamaecyparis obtusa, Camphor tree, wax myrtle, Chinese quince, litchee and Myrica rubra are preferably used. In the invention, these plants containing the proanthocyanidin polymer are cut or ground and then used and the extracts obtained by heating and concentrating / drying these materials, in an aqueous solvent, are used. There is no particular limitation in the substances having the ring structure of the phloroglucinol or the ring structure of resorcinol, used in the reaction of the present invention, as long as the substance is a plant containing resverstrol, phloroglucinol, flavonoid and flavanoid ( galloyl ester or catechin) p its extracts. Examples include green tea leaves, fresh tea, grape seed, grape seed coating, bucket gambir, red algae and their extracts. Among them, in consideration that the main uses of the oligomer of proanthocyanidin produced in the present invention, is for health food products, ingredients for food for the specified use in health, cosmetics and pharmaceutical products, especially specified foods for the use in health and pharmaceutical products, the seeds of grapes, coating of grape seeds, vede tea leaves, fresh tea, and their extracts, which have been conventionally applied to beverages and the safety of which has been confirmed, they are preferred.

The proportion of the materials containing the proanthocyanidin polymer and the substrate having the ring structure of the phloroglucinol or the resorcinol ring used in the reaction is arbitrarily selected. and it prefers that the amount of the latter be sufficiently large to bind to the fragments of the proanthocyanidin polymer having a reduced molecular weight. If the amount of the substance having the ring structure of the chlorchloroglycin or the structure of the ring of resorcinol, is too small, proanthocyanidin that has a high molecular weight can remain unreacted, and in that case, the remaining proanthocyanidin has a high molecular weight that can be easily removed by column chromatography. The reaction between the plants containing the proanthocyanidins or the proanthocyanidins contained in their extract, and the substance having the ring structure of the floroglucinol or the ring structure of the resorcinol (hereinafter sometimes referred to simply as "substance containing the floro ring / resorcinol ", is conducted in a solvent, by heating.As the reaction solvent, water, methanol, ethanol and a mixture of two or more of them are used. Food and pharmaceuticals, water and ethanol are preferred.

It is preferred that the reaction be carried out under an acidic condition. An acid appropriately selected from inorganic acids, such as hydrochloric acid, sulfuric acid and nitric acid, and organic acids, such as acetic acid, citric acid, ascorbic acid, and malic acid, are used in a concentration of 0.1 to 1.0 N , preferably in about 0.5 N The reaction between the plant or plant extract containing the proanthocyanidins and the substance containing the floro / resorcinol ring, is carried out at a temperature from room temperature to 100 ° C, for 0.5 hours to 1 week, preferably at 90 to 100 ° C, for 1 to 4 hours. The reaction solution, after the reaction is subjected to filtration or a similar treatment, to remove the solids content and isolate the liquid. The resulting extract (liquid) containing the oligomer of proanthocyanidin can be used in various forms, such as a liquid, powder, gel, solid molded product or the like, after condensing and drying. The reaction solution, after the reaction, can be concentrated and dried or concentrated and fractionated. By these methods, the objective substance is separated from the reaction solution, concentrated and stored by a conventional method. For example, the waste is separated by filtration and after the filtrate is concentrated, the concentrated liquid can be purified by subjecting the extract to a film treatment (such as ultrafiltration and reverse osmosis treatment) or a treatment with an adsorbent, or a similar treatment to concentrate and isolate the target substance. Examples of adsorbents include the styrene-divinylbenzene adsorbent, methacrylic acid adsorbent, hydrophilic vinyl polymer, modified dextran gel, polyarylamide gel, reverse phase silica gel, and ion exchange resin. In a case where such an adsorbent is used, proanthocyanidin oligomers that have been reduced in molecular weight through reaction with the substance containing the floro / resorcinol ring, are contained in adsorbed fractions to the adsorbent (hereinafter referred to as "fractions) adsorbed"). By diluting the adsorbed fraction with the hydro-alcohol, alcohol, acetone or the like, the components having various molecular weights can be obtained. On this occasion, isolating the oligomer of the target proanthocyanidin, which has a degree of polymerization d 2 to 4 from the reaction solution through column chromatography, using a synthetic adsorbent based on an aromatic compound, the proanthocyanidin having a high molecular weight, it can be sieved and separated and the concentration of the eluate is easy, which is preferred. Preferred examples of synthetic adsorbents based on aromatic compounds include the crosslinked styrene-based porous polymers, such as SEPABEADS.

From the results of the measurements, using 1H-NMR, UV, HPLC, GPC and TLC, it was confirmed that the proanthocyanidin oligomer obtained contains tetramer dimers of proanthocyanidin, whose typical chemical structure is represented by the formulas (1) and (2): (in the formula, Ri represents a hydrogen atom or a hydroxyl group, and R 2 represents a hydrogen atom or a hydroxyl group, R 2 represents a hydrogen atom or a group of galloyl. and n represents O to an integer of 1 or 2). Although in formulas (1) and 2), only structures that have junctions between position 4 and position 8 are shown, those that have links between position 4 and position 6 or links of 2-0-7, also exist. Through the reaction between the proanthocyanidin polymer and the substance containing the floro / resorcinol ring, according to the invention, the proanthocyanidin polymer having a high molecular weight (5 in terms of the degree of polymerization) and can not be absorbed in the living body, it can be easily fragmented in order to supply the polymer with a substance of low molecular weight that can be absorbed and at the same time, a composition containing mainly oligomers of the proanthocyanidin from the dimer to the tetramer, can be obtained. The proanthocyanidin oligomer, having a substance containing the ring structure of the floro / resorcmol, attached to its terminal, obtained according to the invention, which exhibits a strong antioxidant activity in the anti-oxidation tests of DPPH, TEAC and FRAP It has a high antioxidant function, compared to other materials of polyphenol. In addition, in experiments with animals, with respect to diseases related to lifestyle and in surveillance tests, the Human Anti-oxidation Index, data have been obtained that show that the effect can be judged based on antioxidant activity. Therefore, the products containing the proanthocyanidin oligomer of the invention, as the active ingredient, have not only the function of suppressing lipid peroxide generation in vivo, but also effects on diseases caused by induced oxidation. to active oxygen. Therefore, the products, which have effects in preventing various organ failures, caused by the generation of lipid peroxide or active oxygen and also prevent aging, are effective in preventing and treating various diseases caused by such organ failures and the aging. Likewise, the products can be considered effective in the suprssion / prevention / treatment of cerebral dysfunctions,. such as dementia, presumably caused by aging of the brain. At the same time, along with the improvement in brain functions, the learning function increases, presumably the easy irritation, relief of insomnia, easy deconcentration state and similar effects, can be expected. Thus, the products containing the proanthocyanidin oligomer of the invention, as an active ingredient, can be used in avalanche food products, pharmaceuticals, cosmetics and the like. No toxicity was observed with the products containing the proanthocyanidin oligomer of the invention, as an act ingredient and the products can be used safely. These products are used orally or parenterally. The amount of dose, in the case of oral use, differs, depending on age, weight, symptoms, objective therapeutic effect, method of administration and the like. Generally, it is in the range of 50 to 1000 mg per adult dose. In the case of oral administration, the products of the invention are used in the form of tablets, pills, capsules, powder, granulated powder, syrup or the like. In case of parenteral administration, they are used in the form of an injectable solution, coating agent and the like. In the case of preparing granulated products, the products of tablets or syrup products, suitable auxiliary agents (such as starches, dextrin, sweetening agents, colorants and flavoring agents) can be used.

EXAMPLES Hereinafter, the invention is specifically described with reference to the Examples. The invention is in no way limited by these Examples.

Example 1 Procyanidin B 3-0-gallate (+) - catechin (4-β-8) - -} -epigalocatechin + + 3-0-epigallocatechin (-) epicatechin gallate Procyanidin B4 (100 mg) and epigallocatechin 3-O-galeate (100 mg) were dissolved in 40 ml of a 2% citric acid solution and heated at 100 ° C for 2 hours. The solution was subjected to column chromatography of M20-gel P20P (aqueous methanol) and then to column chromatography of Sephadex LH-20 (60% methanol), to recover procyanidin B4 (10 mg) as a starting material and 3-0-gallate of epigallocatechin (25.4 mg) and 3-0-gallate of (+) - catechin (4ß-8) - (-) -3-0-epigallocatechin gallate (17.3 mg). By comparing the generated catechin and proanthocyanidin with compositions of the prior art in the J H-NMR spectrum, the identification of the obtained compositions. (See the previous structural formulas).

Example 2 Pofoaan'dina polymer extracted nutty and escara years + i + (+) - catechin 3-0-gallate of (-) - epigallocatechin 0. 5 g of the procyanidin polymer extracted from the beetle nut and the 3-0-gallate of (-) - epigallocatechin (0.5 g) were dissolved in 200 ml of a 2% citric acid solution and heated to 95 ° C. during 3 hours. The reaction solution was subjected to column chromatography in the same manner as in Example 1, 3-0-epigallocatechin gallate (403 mg). (+) Recently generated catechin (48.6 mg) and 3-0-gallate (-) -epicatechin (148.3 mg) which is the proanthocyanidin were obtained (see above structural formulas.

Example 3 5 g of Japanese cypress bark, containing a mixture of catechin and procyanidin and 1 g of green tea, were dissolved in 100 ml of a 2% citric acid solution and heated at 95 ° C for 3 hrs. . After cooling, 100 ml of ethanol was added to the solution and the solution subjected to suction filtration. The filtrate was analyzed by HPLC chromatography. The conditions for the HPLC analysis are as follows: Column: Cosmosil SC18 ARII (4.6 x 250 mm), Column temperature: 36 ° Mobile phase: A; 50 mM phosphoric acid, 3CN, B from 4 to 30% (for 39 minutes) from 30 to 75% (for 15 minutes) flow rate: 0.8 ml / min.

Detection: Detection of the photodiode array 5 g of a Japanese cypress bark and 1 g of green tea, were heated separately in a citric acid solution under the same conditions and set were subjected to a treatment of extraction. The HPLC analysis was conducted in the same manner (see HPLC in Figure 1). In the solution of the extract obtained by the treatment of the mixture of the Japanese cypress bark and green tea in the citric acid solution, the subtractions derived from many new compounds, which are not found in the cases of treating the bark of the cypress Japanese and green tea separately, were detected. It is presumed by comparison in the spectrum of ultraviolet absorption of the crests between those obtained in this Example and those obtained in Examples and 2, that all the compounds were proanthocyanidins. The liquid extract, obtained in Example 3, by heating the bark of the Japanese cypress and the green tea in the citric acid solution, was concentrated and then subjected to the division of solvent with 50 ml of water and 50 ml of acetate of ethyl for 5 times. The obtained ethyl acetate layer was collected and also concentrated to obtain 0.67 g of the product extracted from ethyl acetate. Extracts obtained from the bark of Japanese cypress and green tea, respectively, were treated separately, subjected to solvent division in the same way to obtain 0.30 g of the product extracted from ethyl acetate and Japanese cypress bark. and 0.37 g of the product extracted from ethyl acetate of green tea. Thus, the extracts obtained from ethyl acetate were analyzed by TLC chromatography. The conditions of the FTA are as follows: Silica gel 60, Development solvent: benzene / ethyl formate / formic acid (1: 7: 1, volume / volume). Color reagent: Vanilla hydrochloric acid reagent (see TLC in Figure 2). The vanilla hydrochloric acid reagent, which is a stopping reagent for catechins and proanthocyanidins, takes on a characteristic red color when these substances are present. With respect to the extract obtained through the treatment of the Japanese cypress bark and green tea, areas derived from the dimer and trimer of proanthocyanidin are recognized. In the remaining water layer, after the division of ethyl acetate in Example 3, the proanthocyanidin having a molecular weight greater than the molecular weight of those transferred to the ethyl acetate layer remains. Then, the molecular weights of the acetylated compounds of the proanthocyanidins contained in the water layer were compared by the analysis of gel permeation chromatography. The water layer, after the Japanese cypress bark and green tea were treated with the citric acid solution and the water layer of the Japanese cypress bark, were concentrated and dried to a solid. After dissolving in acetic anhydride - pyridine, the solution was left stand at room temperature for 8 hrs. The reaction solutions were respectively drained in ice water and the insoluble matter deposited was separated through filtration and dried under vacuum. The obtained acetylated body was analyzed under column conditions TSK-GEL 0G4000H6, tetrahydrofuran solvent and detection with UV absorption of 254 nm. According to the molecular weight of the products obtained estimated based on the calibration curves, prepared using benzene and polystyrenes, which have molecular weights of 4000, 25000 and 50000. The crest of the proanthocyanidin contained in the water layer, after of dealing with the bark of Japanese cypress and green tea and with citric acid, was around 1300, while the upper part of the crest of the proanthocyanidin contained in the water layer of the Japanese cypress bark was around 2000. It was found that by adding green tea in the citric acid treatment, the weight of the molecule was reduced.

Example 4 100 g of fresh banana peel was sprayed together with 300 ml of a solution of a mixture of acetone and water (4: 1, vol / vol), using a swirl mixer and subjecting to a suction filtration. The acetone was distilled off from the filtrate using an evaporator to thereby prepare an aqueous solution, the insoluble matter was separated by filtration and added water to the solution to obtain a total amount of 200 ml. Separately, 3 g of green tea leaves were boiled out in 300 ml of water and then the resulting product was subjected to suction filtration, the water was added to obtain a total sanity of 300 ml. 100 ml of the banana extract (corresponding to 50 g of banana peel) and 100 ml of green tea extract (corresponding to 1 g of green tea) were mixed together and 2 g of citric acid was dissolved in the mixture. Then the mixture was heated at 96 ° C for 3 hours. After cooling, the solution was extracted with ethyl acetate 3 times to obtain 0.312 g of the product extracted from ethyl acetate. In the same manner, the banana extract liquid and the green tea extract liquid were separated separately with ethyl acetate, to obtain 0.015 g and 0.18 g of ethyl acetate extracts, respectively. The extracts obtained from ethyl acetate were analyzed by TLC chromatography (see TLC in Figure 3). Due to the high molecular weight of proanthocyanidin in the banana skin extract, the extract is positive for the vanilla hydrochloric acid reagent only at the point of origin in the TLC analysis, while in the product obtained by treating the extract of banana peel and green tea extract with citric acid, derived areas that did not exist in the original extracts were observed before treatment.

Example 5 The astringent component contained in a large amount in astringent persimmon is the proanthocyanidin which has a very high molecular weight and is constituted by four kinds of tea catechins (Tanaka et al., J. Chem. Soc. Perkin Trans 1, 1013-1022, 1994). 100 g of fresh immature persimmon fruit were sprayed together with 500 ml of a 1% citric acid solution, using a whirlpool mixer and in addition 500 ml of a 1% citric acid solution and 20 g of green tea were mixed and the mixture was boiled moderately for 3 hours. The reaction solution was subjected to suction filtration while it was hot, to thereby obtain 950 ml of filtrate. Half of the filtrate, 475 ml, was partitioned with ethyl acetate 4 times to obtain 2.58 g of the ethyl acetate layer. The remaining half, 475 ml, was allowed to pass through a column of Sepabeads SP850 beads (200 ml) and after washing with water to remove the sugar, the adsorbed polyphenols were eluted and separated with 40-60% ethanol. The eluate was concentrated to obtain 3.26 g of the fraction containing the catechin and proanthocyanidin. On the other hand, 200 g of the unripe persimmon fruit was sprayed together with 900 ml of a mixed solution of acetone-water (4: 1, vol / vol) and subjected to an extraction. The acetone was distilled and completely separated from the filtrate and the extract solution obtained was allowed to pass through a column of pearls Spabeads SP 850 (200 ml). As a result, most of the di-kaki proanthocyanidins (persimmon tannin) were not adsorbed but eluted with only water. The amount of the polyphenol fraction adsorbed to the column and eluted with aqueous alcohol was as small as 0.76 g. The molecular weight of proanthocyanidin in the persimmon was assumed to be approximately 1.38 c 104 (Mastuo, T. et al., Agic, Biol. Chem, 42, 1537-1643, 1978). too large to enter the pores of the Sepabeads pearls. On the other hand, the persimmon proanthocyanidin treated with green tea, fractionated and reduced in molecular weight, could enter the pores of Sepabeads to be adsorbed. For the present Example, it was found that Sepabeads beads can be separated by screening the proanthocyanidins, which have large molecular weights. The product extracted from ethyl acetate, obtained by treating the persimmon with green tea and citric acid, and the substance adsorbed to Sepabeads SP825 were compared with the product extracted from ethyl acetate from green tea and the aqueous acetone extract from the immature persimmon, using TLC chromatography (see TLC in Figure 4). With respect to the portion adsorbed to Sepabeads SP725 beads, normal phase HPLC analysis was conducted and the comparison was made with the polyphenol fraction (containing the monomer of the catechin, procyanidin, trimer and tetramer dimer and procyanidin having a higher molecular weight) obtained by separating the extracted product with water hot from Japanese cypress with DIAION HP20SS column chromatography (see HPLC in Figure 5). The conditions of the normal phase HPLC analysis are as follows: Column LiChroCART Superspher Si 60 (4.6x250 mm) column temperature: 28 ° C mobile phase, hexane: methanol: tetrahydrofuran: trifluoroacetate (45: 40: 13.5: 1.5) Flow rate: 1.0 ml / min. Detection: 143 nm. As controls, (-) epicatechin (monomer), procyanidin B4 (dimer), procyanidin Cl (trimer) and dynamatanine A2 (tetramer) were used. These controls were separated from the medlar seeds and identified by comparison of the 1-ti-NMR spectrum with the values described in the references. In HPLC chromatography, although a ridge was observed for caffeine and it was further confirmed that polymers of up to four monomer molecules were present, they imply less crest of the ridge, next to compare with proanthocyanidin from Japanese cypress.

Example 6 1.0 kg of the fresh immature persimmon fruit were sprayed together with 2 liters of water and the resulting product was mixed with 200 g of green tea and 80 g of citric acid. Added water to obtain the total amount of 8 liters and then the mixture was boiled moderately for 3 hours. After heating, a filtration was conducted while it was hot. The obtained filtrate was cooled, allowed to pass through a Sepabeads SP825 column and washed with water. The adsorbed portion was separated by elution with concentrated aqueous ethanol and then dried by freezing to obtain 59.0 g of a mixture of catechin and proanthocyanidin. 6 g of the obtained mixture was left for through a Sephadex LD-20 column to divide it into 8 fractions (Fr.) (See Figure 6). The amount obtained from Fr 1, which mainly contained the epicatechin and epigallocatechin, was 0.79 g. The amount obtained from Fr 2, which mainly contained the 3-0-epicatechin gallate, was 0.15 g. The amount obtained from Fr 3, which mainly contained the 3-0-epigallocatechin gallate, was 0.87 g. The amount obtained from Fr 4, which was a mixture of the 3-0-gallate of the epigallocatechin and the dimer of proanthocyanidin, was 0.2 g. The amounts obtained from Fr 5 and Fr 6, which both contained the dimer of proanthocyanidin, were 0.25 g and 0.51 g, respectively. The amount obtained from Fr 7, which mainly contained the trimer of proanthocyanidin, was 0.88 g. The sanity obtained from Fr 8, which contained the same trimer as Fr 7, or the proanthocyanidins that have greater molecular weights thereof, was 1.63 g. Among these reactions, the Fr 5 and Fr 6 were purified with column chromatography on MLC-CHP20P gel column chromatography of Chromatorex ODS (both using aqueous methanol as solvent), to thereby obtain 101.6 mg of the (-) epicatechin (4ß-8) - (- ) epigallo-catechin-3-0-gallate, 121 mg of epigallocatechin (4ß-8) - (-) -epigalocateuin-3-0-gallate and 24.3 mg of the (-) epicatechin (4ß-8) - (-) epigallo-catechin-3-0-gallate, 121 mg epigallocatechin (4ß-8) - (-) -epigalocateuin-3-0-gallate (See the following structural formulas). In addition, although many classes of proanthocyanidin v-dimers were present, the inventors succeeded in pure separation of the three above types, which identify their structures by comparison of the * H-NMR spectrum. 3-0-gallate (-) - catechin (4ß-8) - (-) - β-pigalocatechin 3-0-gallate (-) - catechin (4ß-8) - (-) - epigallocatechin 3-0-gallate (-) - catequin (4ß-8) - (-) -epigalocatechin Example 7 - Example of production of the compound bound to resveratrol. 100 mg of polyphenol powder from grape seeds (Grape Speed PE, products of Guilin Layn Natural Ingredients Corp., proanthocyanidin content of 95% or more) and 120 mg of resveratrol, together with 100 mg of citric acid, dissolved in 10 ml of water were placed in water for 3 hours (87 to 93 ° C) to allow the reaction to proceed, and the resulting product was allowed to stand to cool to room temperature. With this liquid, a column of Sephadex LH-20 (internal diameter of 2 cm, length of 15 cm, about 50 ml of 70% methanol) were charged. 40 ml of 70% methanol and then 50 ml of 100% methanol were drained in sequence to pass through the column, and thus concentrate the fraction of the target substance. After the freeze-dried treatment, 6.0 mg of a powder (hereinafter abbreviated as Substance A of the Invention) was obtained. The dry powder was dissolved in 1 ml of 70% methanol and with this solution, a column of MCl-gel CHP-20 (internal diameter 2 cm, length 15 cm, about 50 ml) was loaded. 50 ml of the same solvent was allowed to pass through the column to concentrate the fraction of the target substance. After the freeze-drying treatment, 2.4 mg of a powder was obtained. The powder obtained was subjected to HR-FAB-MS (Rapid Atom Bombardment Mass Spectrum, High Resolution) and the nuclear magnetic resonance spectrum 1-H-NMR, See Table 1). The [M] + of the compound of the obtained fraction contained mainly was observed in m / z: 516, 1404 in HR-FAB-MS, which coincided with the value of 516. 1420 corresponding to a molecular ula of C2gH25? G, with an error of 3.1 ppm. There, the fraction was assumed to contain a compound represented by the molecular ula C2gH25? 9, which led to a chemical structure, where the resveratrol bound to the catechin or epicatechin through the carbon-to-carbon bond was considered.

In the spectrum * H-NMR (see Table 1) of the compound, in addition to the five proton signals in the aromatic ring, which are common in the catechin and epicatechin, a group of signals that have oxygen atoms in the bases they were observed ad 5.04 (1H, br, s, 2-H) and d 4.01 (1H, br, s.3-H), which suggests the presence of the epicatechin spherical configuration. The signal at d 4.64 (1H, br, s) was attributed to position 4a of the epicatechin part and the respostrol part was assumed to be placed in position 4b. In addition, an envelope in 2H, which has the same configuration as that of the signal observed at 66.55 in the floroglucinoal was displaced at 0.56 ppm less, which shows that the newly ed C-bond had a similar steric environment as well, in addition, the AB system in the conjugated double bond of type E derived from a trans-stilbene structure of resveratrol (56.79, 5.97, each 1H, J = 16.5 Hz, and the A2B2 signal in the other aromatic ring (6.807.36 each 2H J = 8.6 Hz) were observed Based on the ination, the chemical structure of the compound was assumed to be 4b- (4-resveratroyl) - (-) -apicatechin, shown as follows: Table 1: Assignment of the 'H-NMR Signal of Substance A of the Invention (d, in ppm) HNb. 2-H 5.01 (br.s) 3-H 3.96 (br.s) 4-H 4.53 (br.s) 6-H 6.01 (d, * = 2.2 W) 8-H 5.98. { d, J! = 2.2 H?) ff-H 6.74 (d, ^ 8.3 H?) t-H 6.67 (dd, -> g = 8.3, 1.7 Hr) R? 4-H 5.99 (envelope) 6-H 5.99. { envel ope) Note 1) The substance of the invention was measured in acetone dß-D2O EXAMPLE 8 Production example of the compound bound to phloroglucinol Each 1.00 g of polyphenol from grape seeds and phloroglucinol were dissolved together with 500 mg of citric acid in 50 ml of water and the mixture was placed in water (87 to 93 ° C). ) during 3 hours. After the reaction, the resulting product was allowed to stand cooling to room temperature. A column of DIAION HP20 (internal diameter of 3 cm, length of 14 cm, about 100 ml) was then charged with this solution, and the wash was conducted with about 300 ml of water. Elution was then carried out with 200 ml of methanol and the objective fraction was concentrated and freeze-dried to obtain 1.24 g of powder (hereinafter abbreviated as Substance B of the invention). Substance B of the invention was dissolved in 5 ml of 70% methanol and a column of Sephadex 1H-20 (internal diameter 3 cm, length 25 cm, about 180 ml) was charged with the solution. 500 ml of the same solvent was allowed to pass through the column, and the objective fraction was concentrated and freeze dried to obtain 62.2 mg of powder. The powder obtained was subjected to the analysis HR-FAB-MS and 1H-NMR With respect to the compound, the reaction obtained contained mainly. [M] + was observed in m / z 414.0941 in HR-FAB-MS 'which coincided with the value of the calculation 424.0960, which corresponds to the molecular ula C2? H18? 9, with a margin of error of 2.appm ( an error of 10 ppm or less is permitted) . Therefore, it was assumed that the compound had a molecular formula of C2iH? 809, which suggests a chemical structure where phloroglucinol is linked to the catechin or epicatechin through the carbon-to-carbon bond. In the XH-NMR spectrum of the compound (see Table 2) in addition to the five proton signals in the aromatic ring common to the catechin and epicatechin, a group of signals having oxygen atoms in the foot was observed in 5.01 (1H, br.s, 2-H) and 3.96 (1H, br.s. 3-H), which suggests that the compound has a spherical configuration of the epicatechin. It was assumed that a proton signal of d4.5 (Ih, br s) was attributable to position 4 of the epicatechin part and that the phloroglucinol part was located in position 4. In addition, a signal that can be attributed at c-4 and c-6 derivatives of phloroglucinol at 2H, was observed at 55.99 as the equivalent envelope, which suggests that the newly formed -C-bond was generated by a rotation barrier. Based on this information, the chemical structure of the compound was assumed to be 4- (2-phloroglucinol) - (-) -epicatechin, as shown in the following formula: 13C-NMR (Carbon-Nuclear Magnetic Resonance Spectrum 13) , see Table 3). in the compound supported by the chemical structure: carbon-13 Table 2: H- NMR Sgnal assignment of Invention Substance B (d i n pprr) H ND. 2-H 5.04 (br. S) 3-H 4.01 (br.s) 4-H 4.64 (br.s) 6-H 6.00 (d, J = 2.2 l-fc) 8-H 6.02 (d, J = 2.2 l-fc) 2'-H 6.97 (d, J = 1.7 te) 5'-H 6.74 (d, = 8.3 Hz) 6'-H 6.67 (dd, J = 8.3.1.7 l-fc) Ftes 2 and 6- H 6.55 (envel ope) to -H 6.79 (d, ^ = 16.5 hfc) a) ß -H 6.97 (d, .M6.5 hfe) a) 2"and 6 '- H 6.80 (d, -« = 8.6 l-fc) c) 3 'and 5' - H 7.36 (d, J = 8.6 hk) c) Nbte 1) Invention Substance B \? TS rreasured in acet one- - RP 2) If the same letter is appended on right shoulders, the assignrrant nay be exchanged vi th each ot her Table 3] Table 3: Assignment of Signal C1 -NMR of Invention Substance B (d, in ppm) C D. C-2 76.8 - 3 72.3 - 4 36.7 - 5 157.7 a) - 6 95.4 - 7 158.4 b) - 8 96 - 9 158.5 b) -10 100.3 - r 132.2 - 2 115 - 3 '145.0C) - 4 * 145.3 c) - 5"115.1 - 6 '119 Fh - 1 157.78) - 2 100.3 - 3 157.5 a) - 4 106.6 - 5 157.7 a) - 6 106.6 Notes 1) Substance B of the invention was measured in acetone dß-D20 2) If the same letter is attached to the supports on the right, the assignment can be exchanged with each other. / Example 9: Example of production of the compound qpae has epigallocatechin gallate linked to grape seeds. 5.00 g of vanilla polyphenol powder (Grape Seed P.E. 15., 95% proanthocyanidin content, Table 2: Assignment of the 1 H-NMR Signal of the Invention Substance B (d, ßn ppm) HN). 2-H 5.04. { br.s) 3-H 4.01 (br.s) 4-H 4.64 (br.s) 6-H 6.00 (d, J2.2 l-fc) 8-H 6.02 (á, 2.2 H?) ZH 6.97 ( d, J =? 7 H?) 5 * -H 6.74 (d, J = 8.3 H?) 6 * -H 6.67 (dd, = 8.3, 1.7 B?) Fes 2 and 6-H 6.55 (envelop?) aH 6.79 (d, J = 16.5 B?) a > ß -H 6.97 (d, ^ = 16.5 Hr) a > Zand 6"-H 6.80 (d, ^ = 8.6 Hz) c> 3 'and 5' - H 7.36 { D, 8.6 H?) C) Notes 1) Substance B of the invention was measured in acetone d? -D20 2) If the same letter is attached to the supports on the right, the assignment can be exchanged between itself Example 9 -: Example of production of the compound having the epigallocatechin gallate, derived from the seeds of grapes, together with 5.00 g of the polyphenol powder of grape seeds (Grape Seed PE LAYN, 95% proanthocyanidin content) they were dispersed and dissolved in about 100 ml of water and then the solution was emptied onto a SEPABEADS SP 850 column (internal diameter 3.8 cm, length 20 cm, about 230 ml) and eluted with water. The fraction obtained was concentrated and dried by freezing to obtain 2.44 g of a polymer powder (48.8%). Each 1.00 of the polyphenol powder of the obtained grape seeds and the EGCG were dissolved together with 500 g of citric acid in 50 ml of water and the container containing the mixture was placed in water (87 to 93 ° C) for three hours . After the reaction, it was left to rest to cool it to room temperature. A column of internal diameter of 3 cm, length of 14 cm, about 100 ml) was charged with this liquid and washed with about 300 ml of water. The fraction obtained by eluting with about 200 ml of methanol was concentrated and freeze-dried to thereby obtain 1.85 g of a powder (hereinafter abbreviated as the substance of the invention).

Example 10: Production Example of the compound that has the polyphenol of EGCG of the cortex Myricae Cortex (polymer EGCG = attached .. A piece of myrtle of wax (Myricaceae), a piece of bark of (Myricaceae), that is to say "Myricae Cortex" "Bark of Myrica rubra ie Myricae ortex", were submerged in cold in 50% acetone, in an amount of 5 to 10 times (weight / volume) of the piece, for 3 to 7 days, in order to obtain a liquid of The extract obtained was concentrated and the liquid crystals of myricitin were filtered off. (mirecetin 3-0-L-rhamnopyranoside)) repeatedly using filter paper. The filtrate obtained, after further concentration, was dried by freezing to obtain a dark brown powder with a yield of 14% from the resin part. 14.0 g of this powder were dissolved in about 70 ml of 50% methanol and a column of Sephadex LH-20 (internal diameter of 5 cm, length of 20 cm, about 400 ml) were loaded with the solution. Then, 1.5 liters of the same solvent and then 0.7 liter of 70% methanol were allowed to pass through the column. 1 liter of 70% acetone was allowed to pass through it to recover the fraction of the EGCG polymer. The fraction obtained was concentrated and freeze-dried to obtain 9.37 g of a 66.9% EGCG polymer powder). each 1 g of the EGCG polymer derived from the bark Myricae ortex and the EGCG was dissolved with 500 mg of citric acid in 50 ml of water and the vessel containing the mixture was placed in hot water (87 to 93 ° C) for 3 hours. hours. After the reaction, the resulting product was allowed to stand to cool it to room temperature. A column of HP20 (internal diameter of 3 cm, length of 14 cm, about 100 ml) was charged with this liquid, and the substance D of the invention was conducted ") 1.0 g of dry powder of persimmon shell and 300 mg of tea extract (PF-TP 90) manufactured by Pharma Foods International Co., Ltd., containing polyphenol tea: 90% or more, total catechin content: 80% or more (with EGC content of 50% or more ) were dissolved with 500 mg of citric acid in 50 ml of water and the vessel containing the mixture was placed in hot water (87 to 93 ° C) for 3 hours. After the reaction, the resulting product was allowed to stand to cool it to ambient temperature (Then a column of SEPABEAD SP 850 (internal diameter: 3 cm, length 14 cm, about 100 ml with this liquid, washing was conducted with 300 ml of water and elution with about 200 ml of methanol was performed.After, a column of DIADION HP20 (internal diameter of 3 cm, length of 14 cm, about 100 ml) was loaded with the fraction obtained, washing with about 300 ml of water was conducted, then the fraction obtained by elution with about 200 ml of methanol was concentrated and freeze dried to thereby obtain 464 mg of a powder (hereinafter referred to as "Substance E of the Invention"). ") Example 12: Example of production of the compound having polyphenol epigallocatechin gallate (EGCG), litchee derivative 5.00 g of litchee polyphenol powder (Litch) PE, product of Guilin Latn Natural Ingredients, Corp.,) content of proanthocyanidin: 90% or more) were dispersed and dissolved in about 100 ml of water and a column of SEPABEADS SP 850 (internal diameter of 3.8 cm, length of 20 cm, about 230 ml) was charged with the mixture. The fraction obtained by eluting with water was concentrated and freeze dried to obtain 3.02 g of a polymer powder 60.4%).

Each 1.00 g of the powder the obtained polyphenol nut polymer and the EGCG were dissolved with 500 mg of citric acid in 50 ml of water and the vessel containing the mixture was placed in hot water (87 to 93 ° C) during 3 hours. After the reaction, the resulting product was allowed to stand to cool it to room temperature. Then a column of DIAION HP20 (internal diameter: 3 cm, length 14 cm, about 100 ml with this liquid, washing was carried out with about 300 ml of water, the fraction obtained by elution with 200 ml of water was concentrated and dried by freezing to obtain 1.80 g of a powder ( hereinafter referred to as Substance F of the Invention). Test Example 1: With respect to the invention, substances A through E, obtained in Examples 7 to 11, respectively, were evaluated in the antioxidant properties by measuring the radical clearance activity of 1, 1-diphenyl- 2-picrylhydrazil (DPPH) and the TEAC (Trolox Equivalent Antioxidant Capacity) method. DPPH Assay Procedures: With respect to each sample, the purifying activity of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was evaluated as follows. In a 96-well microplate, 100 μl of the DPPH solution (60 μl of ethanol solution from the test sample or 100 μl of ethanol as control, and the mixture was mixed moderately and allowed to stand at room temperature for 30 minutes. Then, the absorbance at 520 nm was measured. The depuration activity of DPPH radical was calculated by the following formula and the effective concentration (EC50) of the value of the DPPH radical purifying activity of the test sample, gradually diluted, and its concentration was calculated. Formula 1 Radical clearance activity D (%) = (1-absorbance of the test sample / control absorbency per 100. As the substance to be compared with the invention, Substance A, epicatechin (EP) and the resveratrol (RS) were used.As the substance to be compared with the invention, Substance B, epicatechin (EP) and phloroglucinol were used.As the substance to be compared with the invention, Substances C to E , the polyphenol d (GP) seed polymer of grapes was used, the results of the measurements are shown in Figures 7 to 9, together with the data of the compared substances, in the epicatechin, the D activity of 48.7. % was recognized, while in resveratrol, activity was only 23.1% in Substance A of the invention, where epicatechin and resveratrol were combined with each other, 1 activity was so high as of 76.1%, which was a significantly excellent result (Figure 7). In the epicatechin, the DPPH activity of 43.0% was observed, while in the phloroglucinol, little of this activity was observed. Substance F of the invention, where phloroglucinol was combined with epicatechin, showed a significantly high purifying activity, as compared to both of the substances (Figure 8). Likewise, the Substances of the Invention C (41.3%) and D (35.1%) showed higher purification activities than the compared substances (26.9%) The purifying activity of the Substance E of the Invention (26.8%) showed was equivalent to that of the compared substances (Figure 9) (TEAC). Method: The TEAC (Trolox Equivalent Antioxidant Capacty) method is a method of relatively assessing the antioxidant strength by converting the antioxidant activity of a compound into the antioxidant activity of Trolox, which is a derivative of α-tocopherol and the method is widely employed as An index of antioxidant activity: 36 μl of a 70 μM metamyoglobin solution, 300 μl of an ABTS solution and 487 μM -L? of phosphate buffered saline were added in. Subsequently, the sample solution or 1.25 mM of the Yrolox solution was added there and mixed moderately, for 5 minutes at 0 ° C. After 167 μl of a 450 μM hydrogen peroxide solution was added there and mixed for 10 seconds, the reaction was allowed to proceed for 5 minutes at room temperature. The absorbance at 734 nm was measured and the absorbance ratio between the sample and the Trolox solution was calculated to serve as the REAC value of the sample against 1.00 mM Trolox. As in the DPPH assay, described above, as the substance to be compared with Substance A of the invention, epicatechin and resveratrol were used and as the substance to be compared with Substance B of the invention, the Epicatechin and phloroglucinol were used. As the substance to be compared with Substances C to E of the Invention, the polyphenol polymer from grape seeds was used. The results of the measurements are shown in Figures 10 to 13, together with the data of the substances compared. In the epicatechin. the TEAC activity of 1.2 mM was observed, while in resveratrol, the activity was only 1.1 mM. In Substance A of the Invention, where the epicatechin and the reseratrol were combined together, the activity was 1.33 mM, which is significantly high (Figure 10). In phloroglucinol, the activity of TEAC was as low as 0.52 mM, while Substance B of the invention, where epicatechin was combined with phloroglucinol, activity was 1.44 mM, which is significantly greater than that of the two when treated alone (Figure 11). Substances 1.11), DI.11) and E (0.97) of the invention showed high TEAC values, compared to the compared substance (0.73) (Figure 12). Test Example 2 As substances to be compared with Substance F of the Invention, obtained in Example 12, the polyphenol (LP) of litchee nut and the tea extract were used to conduct the comparative experiment.

Test in vi tro Test Method: N1H373 cells were seeded in a 96-well plate and cultured overnight at 37 ° C. The next day, the medium was changed to a culture medium free of its serum and the substance to be tested was added there and the subsequent culture was conducted for one hour. Then, UV light was irradiated for 20 minutes. The medium was changed to a medium containing serum and the culture was conducted overnight at 37 ° C. Cell viability was evaluated by the MTT method. The cells in the medium, without the addition of the substance to be tested, were used as the control group ©. The results are shown in Figure 13. In the control group, the cell viability against UV irradiation was about 20%, while in Substance F of the invention, the viability was the highest, which showed that Substance F of the invention, significantly reduced the number of cell deaths compared to the compared substance, had an effect of protection of UV light. In vivo Test Test Method: Male Sic: ddy mice of 9 weeks of age, substance F of the invention, LP or SE are administered orally in a forced manner, respectively, each in an amount of 50 mg / kg of body weight, every day for 3 weeks. To a mouse control group, the same amount of water was administered. Two hours after the administration of the test substance on the final day, the antioxidant activity of TEAC and the sanity of the lipid peroxide in the serum were measured by collecting blood from the heart, under ether anesthesia. The measurement of TEAC was conducted in the manner described above. The amount of lipid peroxide was measured using commercially available equipment (lipid peroxide - Wako Test, manufactured by Wako Puré Chemical Industries, Ltd.) and measuring the fluorescence at the excitation wavelength of 515 nm and the wavelength Fluorescent of 553 nm, in the reaction between the precipitation of the lipid peroxide and the reagent of 2-thiobarbituric acid in the solution of phosphotungstic acid under the acidic condition of sulfuric acid.

The results are shown in Figures 14 and 15. Substance F of the invention showed a significantly high antioxidant activity compared to the compared substance and the control group. Likewise, the amount of the lipid peroxide in the serum was significantly low compared to the case using the compared substance. In vivo test Test method: Male Sic: ddy mice 6 weeks of age, Substance F of the invention, LP and TE, were orally administered in a forced manner, respectively, to each in an amount of 50 mg / kg of body weight every day for 3 weeks. To a mouse control group, the same amount of water was administered. On the day before the dissection, 2-NP (70 mg / kg body weight) was administered intraperitoneally and 24 hours later, heart blood collection was conducted under anesthesia with ether, to measure the GT and GPT in the serum. Also, the liver was isolated to measure the amount of lipid peroxide in the organ. The results are shown in Figures 16 and 17. Due to the administration of 2-NP, a disorder in the liver was caused, which resulted in an increase in the concentrations of GOT and GPT. However, Substance F of the invention significantly reduced such increases compared to the group of control and the compared substance. Also, the amount of lipid peroxide in the liver was significantly low compared to the case of using the compared substance.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (A) is an HPLC chromatogram in Example 3, which treats Japanese cypress bark and green tea with heat, under an acidic condition. Figures 1 (B) and (C) are HPLC chromatograms in Example 3, which treat the bark of Japanese cypress (Figure 1 (B)) or green tea (Figure 1 (C)), each independently, with heat, under an acid condition, respectively. Figure 2 is a TLC chromatogram showing that the Japanese cypress bark, as raw material, and green tea, are generated by heat treating the materials in Example 4. In the Figure, A is the result in the layer of ethyl acetate of green tea, after the treatment, B is the result in the ethyl acetate layer of the Japanese cypress bark, after the treatment, and is the result in the ethyl acetate layer of the cypress bark Japanese, and green tea, after the treatment. The M zone was derived from the non-galloylated monomer, MG was derived from the monomer of the galloylated proanthocyanidin, the D-dimer was derived mainly from the dimer of proanthocyanidin galloilated and T is derived mainly from the trimer of galloated proanthocyanidin. Figure 3 is a photograph of TLC showing that the new proanthocyanidins, which do not occur in banana skin extract as a raw material and green tea, were generated by heat treatment of the materials, in Example 6. In Figure A is the result in the lime acetate layer of green tea only after the treatment, B is the result in the ethyl acetate layer of the banana skin extract only after the treatment, and C is the result in the ethyl acetate layer of the banana peel extract and the green tea, after the treatment. Figure 4 is a photograph of TLC, which shows that the new proanthocyanidins, which were not present in the unripe persimmon fruit of the raw material and green tea, were generated by the heat treatment of the materials in Example 5. In the Figure, A is the result in the ethyl acetate layer of the green tea only after the treatment, B is the result in the ethyl acetate layer of the immature persimmon fruit alone after the treatment, is the result in the ethyl acetate layer of the unripe persimmon fruit and green tea after the treatment, and D is the result of the product obtained by allowing the resulting product (liquid extract) obtained by treating the unripe fruit of persimmon and green tea by passing through Sepalée 825 pearls and eluting the adsorbed portion with ethanol-water. The zones M, MG, D and T are the same as in Figure 2. Figure 5a shows the results of the analysis of the normal phase HLPC in the product obtained after allowing the resulting product (liquid extract) obtained by treat the unripe persimmon fruit and green tea pass through Sepabead 826 beads and then elute the adsorbed portion with water-ethanol in Example 5. Figure 5 (B) shows the result of normal phase HPLC analysis in proanthocyanidin of Japanese cypress used as a comparative example. Figure 6 shows the result of the TLC analysis in fractions (Fr 1 to Fr 8) obtained by treating the unripe persimmon fruit and green tea with heat, under an acidic condition, and then allowing the obtained catechins and the proanthocyanidins to pass through a chromatographic column of Sephadex LH-20 and in a mixture (E) before separation in Example 6. Figure 7 shows the purifying activity of the DPPH radical of Substance A of the Invention, obtained in Example 7 Figure 8 shows the purifying activity of the radical DPPH of Substance B of the Invention, obtained in Example 9.

Figure 9 shows the purifying activity of the DPPH radical of Substances C to E of the Invention, obtained in Examples 9 to 11. Figure 10 shows the results of the evaluation by the TEAC method on the antioxidant ability of Substance A of the invention, obtained in Example 7. Figure 12 shows the results of the evaluation by the TEAC method in the antioxidant ability of Substance B of the Invention, obtained in Example 8. Figure 12 shows the results of the evaluation by the method TEAC in the antioxidant ability of Substances C to E of the invention, obtained in Examples 9 to 11. Figure 13 shows the results of the UV protection effect test in substance F of the invention, obtained in Example 12. Figure 14 shows the results of the evaluation by the TEAC method in the antioxidant ability of Substance F of the invention, obtained in Example 12. Figure 15 shows the results of measurement of the level of LPO in the serum in the antioxidant skill test in Substance F of the Invention, obtained in Example 12. Figure 16 shows the results of measuring the levels of GOT and GT in the serum in the skill test Antioxidant in Substance F of the Invention, obtained in Example 12. Figure 17 shows the results of measurement of the level of LPO in the liver in the antioxidant skill test in Substance F of the Invention, obtained in Example 12.

Claims (14)

CLAIMS A composition containing as its main component the oligomer of proanthocyanidin to the terminal from which a substance having a ring structure of floroglucinol or resorcinol ring structure has been attached, and with reduced molecular weight, this composition has been obtained by heating plant materials containing the proanthocyanidin polymer or its extract, with a substance having a ring structure of the floroglucinoal or ring structure of the resorcinol, said plant contains such substance or its extract in an aqueous acidic solution.
1. The composition containing as its main component the oligomer of proanthocyanidin, described in claim 1, wherein the plant containing the proanthocyanidin polymer is at least one class selected from the group consisting of grapes, pineapple, Chamaecyparis obtuse, Camphor tree of bayberry wax, cocoa, seasonal plum, Myrica rubra and Cortex cinnamon (cinnamon bark).
2. The composition containing as its main component the oligomer of proanthocyanidin, described in claim 1, in which the substance having a
3. Ring structure of the floroglucinoal or resorcinol ring structure is at least one class selected from the group consisting of resveratrol, floroglucinol, flavonoid and flavanoid (catechin galloyl ester).
4. The composition containing as its main component the oligomer of proanthocyanidin, described in claim 1, wherein the substance having the ring structure of floroglucinol or resorcinol ring structure is at least one class selected from the group consisting of green tea, fresh tea leaves, grape seeds, grape seed coating, cubic gambir, red algae and their extracts.
5. The composition containing as its main component the oligomer of proanthocyanidin, described in claim 1, having a degree of polymerization of 2 to 4.
6. A method for the production of the composition containing as its main component the oligomer of proanthocyanidin, described in any of claims 1 to 5, comprising the step of heating plant materials containing the proanthocyanidin polymer or its extract, with plants that have a ring structure of phloroglucinol or structure resorcinol ring or its extracts, in an acidic aqueous solution, and a step of concentrating the reaction solution containing the oligomer of proanthocyanidin, having a ring structure of floroglucinol or resorcinol ring structure attached to the terminals, and a reduced molecular weight, and then dry the solution.
7. A method for the production of the composition containing as its main component the oligomer of proanthocyanidin, described in any of claims 1 to 5, comprising the step of concentrating the reaction solution containing the oligomer of proanthocyanidin, which has a substance with a phloroglucinol ring structure or a resorcinol ring structure attached to the terminals and reduced in molecular terms, and a step of subjecting the concentrated solution to a fractionation treatment.
8. A method for the production of the composition containing as its main component the oligomer of proanthocyanidin, described in claims 6 or 7, wherein the acid condition is prepared when an inorganic acid, organic acid or both.
9. A method for the production of the composition containing as its main component the oligomer of the proanthocyanidin, described in claim 9, wherein at least one class selected from the group consisting of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, citric acid, ascorbic acid and malic acid.
10. 10. A method for the production of the composition containing as its main component the oligomer of proanthocyanidin, described in any of claims 1 to 5, used in health food products, for the treatment / prevention of diseases related to the style of life by the generation of reactive oxygen species and diseases of the brain or for the prevention of aging.
11. 11. A method for the production of the composition containing as its main component the proanthocyanidin oligomer, described in claim 10, used in pharmaceutical products for the treatment / prevention of diseases related to lifestyle, caused by the generation of the species of reactive oxygen and diseases of the brain or for the prevention of aging.
12. 12. A method for the production of the composition containing as its main component the oligomer of proanthocyanidin, described in claim 10, used in Cosmetic products for the prevention of aging caused by the generation of reactive oxygen species.
13. 13. An oligomer of proanthocyanidin, represented by the following formula: In the formula, n is 0 or an integer of 1 or 2 '
14. 14. An oligomer of proanthocyanidin, represented by the following formula (2): In the formula, n is O or an integer of 1 or 2)