WO2003091237A1

WO2003091237A1 - Process for producing proanthocyanidine-rich material

Info

Publication number: WO2003091237A1
Application number: PCT/JP2003/002001
Authority: WO
Inventors: Isao Kouno; Takashi Tanaka; Gen-Ichiro Nonaka; Kinya Takagaki
Original assignee: Toyo Shinyaku Co., Ltd.
Priority date: 2002-04-24
Filing date: 2003-02-24
Publication date: 2003-11-06
Also published as: AU2003211279A1; AU2002311198A1; WO2003090770A1; JPWO2003091237A1; JP3595816B2; JPWO2003090770A1

Abstract

A material containing proanthocyanidine, which is rich in OPC having a high physiological activity, can be obtained by extracting proanthocyanidine from a plant, then bringing the obtained extract into contact with a definite amount of chitin, chitosan or a derivative thereof and recovering the unadsorbed matters. By further treating this proanthocyanidine-containing material with a synthetic resin adsorbent, a proanthocyanidine-containing material with an extremely high OPC content can be obtained.

Description

Description Method for producing high content of proanthocyanidins

The present invention relates to a method for efficiently producing a proanthocyanidin-containing substance containing a large amount of OPC having high physiological activity. Background art

Proanthocyanidins are polymerized or condensed tannins present in various plants. They are formed by condensation or polymerization of flavan-1-ol or flavan-1,4-diol as a structural unit (hereinafter referred to as condensation polymerization). , Are the combined compounds. These are given their names because they produce anthocyanidins such as cyanidin, delphinidin, and pelargonidin by acid treatment.

Among proanthocyanidins, proanthocyanidins having a low polymerization degree are suitably used. A polycondensate having a degree of polymerization of 2 to 30 (2 to 30 mer) is preferable, a polycondensate having a degree of polymerization of 2 to 10 (2 to 10 mer) is more preferable, and a degree of polymerization of 2 to 30 is preferred. Condensed polymers of (4) to (4 to 4) are more preferably used.

In the present specification, among the condensed polymers of proanthocyanidins, the condensed polymers having a degree of polymerization of 2 to 4 and containing flavan-13-ol-Z or flavan-3,4-diol as a structural unit are referred to as OPCs. (Oligomeric 'proanthocyanidin; oligomeric proanthocyanidm;

Proanthocyanidins, a type of polyphenol, are potent antioxidants produced by plants and are concentrated in plant leaves, bark, fruit bark or seed parts. Proanthocyanidins, especially OPC, are specifically It is found in dough seeds, pine bark, peanut skin, ginkgo biloba, false acacia fruit, and lingonberry fruit. It is also known that cola nuts in West Africa, the roots of ratania in Peru, and green tea in Japan also contain OPC. OPC is a substance that cannot be produced in the human body.

In addition, OPC has the power of antioxidant action, the effect of suppressing bacterial growth in the oral cavity and reducing plaque (gum); the effect of restoring the elasticity of blood vessels; Prevents damage, prevents damaged fat from aggregating on the inner wall of blood vessels, and adhering cholesterol; regenerating vitamin E decomposed by active oxygen; as an enhancer of vitamin E It is known to have the effect of

In addition, tannin, which is a polymer of flavan-3-ol and / or flavan-1,4-diol, and chitin, chitosan, and derivatives thereof are useful for staphylococcus aureus, salmonella, typhoid, cholera, and the like. Its antibacterial and bactericidal effects are remarkable. In particular, it has been reported that MRSA (methicillin-resistant staphylococci), which has been talked about because most antibiotics do not work, is also effective.

Proanthocyanidins are generally obtained by extraction from plants. Examples of the solvent used for the extraction include water; organic solvents such as methanol, ethanol, acetone, hexane, and ethyl acetate; and mixtures thereof (Japanese Patent Application Laid-Open No. 11-801488). However, simply extracting with a solvent results in low recovery and low purity of oral anthocyanidins. Therefore, in order to use it as a raw material for health foods, cosmetics, and pharmaceuticals, further concentration and purification processes are required to increase the purity, which requires a long time.

Japanese Patent Application Laid-open No. Hei 5-279694 and Japanese Patent Application Laid-Open No. Hei 6-56689 describe a method for recovering polyphenols containing proanthocyanidins. In this method, polyphenols are adsorbed to chitin, and polyphenols are The chitin with adsorbed phenols is used as a polyphenol product. However, the adsorbed polyphenols are polyphenols having a high degree of polymerization, and the content of the above-mentioned highly active dimer to tetramer OPC is extremely low, so that the physiological activity such as antioxidant action is low. Low.

Japanese Patent Application Laid-Open Nos. Hei 4 _ 1 0 7 7 4, Japanese Patent Laid-Open No. 10-218 769, Japanese Patent Laid-Open No. 2001-131 0 27, and Eberhard Scholz et al., Proanthocyani dins From Krameria triandra Root, Planta Medica, 55 (1989), pp. 379-384, describe a method for extracting OPC from a plant and a method for synthesizing Z or OPC. In order to obtain OPC, a process of adsorbing and eluting the plant extract on a synthetic resin-based adsorbent, collecting the obtained fraction, and repeating this process are necessary. And not efficient. The synthesis method also has problems such as a large number of steps, cost and time. Disclosure of the invention

The present inventors diligently studied a method for efficiently obtaining a proanthocyanidin-containing substance having a useful OPC content and having a useful physiological activity. As a result, proanthocyanidins are extracted from the plant, and the resulting extract is brought into contact with a predetermined amount of chitin, chitosan, or a derivative thereof, and non-adsorbed substances are recovered, thereby achieving high bioactivity. It has been found that a proanthocyanidin-containing substance rich in OPC can be obtained efficiently. The present inventors have found that a proanthocyanidin-containing material having a higher OPC content can be obtained by further treating the proanthocyanidin-containing material with a synthetic resin-based adsorbent, thereby completing the present invention.

The present invention provides a method for producing a proanthocyanidin-containing substance, the method comprising: (a) a step of extracting or squeezing from a plant by extraction or squeezing; and (b) the extract or squeezed juice. The polyphenol content of the extract or juice And contacting it with 2 to 10 times the dry weight of chitin, chitosan, or a derivative thereof to recover non-adsorbed substances.

In a preferred embodiment, the extraction is a hot water extraction.

In a further preferred embodiment, the method further includes the step of (c) contacting the non-adsorbed substance obtained in the step (b) with the synthetic resin-based adsorbent.

The present invention also provides a proanthocyanidin-containing substance containing an oligomeric proanthocyanidin and a catechin, wherein the proanthocyanidin-containing substance is contained in the proanthocyanidin-containing substance in terms of dry weight in terms of oligomeric proanthocyanin. It contains 30% by weight or more of nisin and 5 to 15% by weight of techin.

BEST MODE FOR CARRYING OUT THE INVENTION

The plant used in the present invention is a plant containing proanthocyanidin, and its type is not particularly limited. Bark of cedar, cypress, pine, etc., which contains a large amount of proanthocyanin; grape, blueberry, strawberry, apogado, fruit of Nisekasia, lingonberry, elderberry, peel or seed; barley; wheat; soybean; black soybean; cacao; red bean Conker shell; peanut shell; ginkgo biloba; tea leaves and tea extract; sorghum cane; apple fruit; kumazasa; fucoidan; yacon leaves; cola nuts (eg, West African cola nuts); ratania (eg, Peru) Ratania) root; and the like. Of these, pine bark, grape seeds and pericarp, peanut thin skin, and the like are particularly preferably used.

Hereinafter, the method for producing the proanthocyanidin-containing material of the present invention will be described. First, proanthocyanidins are extracted from the plant, or juice is obtained from the plant. In the case of performing extraction, the plant is preferably crushed to an appropriate size from the viewpoint of extraction efficiency to increase the surface area per volume. The method of crushing is not particularly limited. For example, crushed material processed by cutter, slicer, etc .; mixer 1. A crushed product treated with a juicer, blender, or masco mouth lider may be used. The size of the crushed or crushed material is 0.1 to 0.1 cm, preferably 0.1 to 5 cm. To increase the crushing efficiency, water or an organic solvent such as ethanol, methanol or ethyl acetate may be added at the time of crushing.

Water or an organic solvent is used as the extraction solvent. Examples of the organic solvent include methanol, ethanol, 1-propanol, 2_propanol Λ ^, 1-butanol, 2-butanol, acetone, hexane, cyclohexane, propylene glycolone, hydrous ethanolanol, hydrous propylene glycol, and ethynolemethinole. Ketones, glycerin, methyl acetate, ethyl acetate, ethynoleether, dichloromethane, edible fats and oils, 1,1,1,2-tetrafluoroethane, and 1,1,2-trichloroethene. Further, a mixed solvent of water and an organic solvent is also preferably used. These organic solvents may be used alone or in combination. From the viewpoint of waste liquid treatment during production, water is preferably used.

The amount of the extraction solvent to be added to the plant can be set in consideration of the target proanthocyanidin concentration and the extraction efficiency. For example, when water is used as the extraction solvent, the weight ratio of plant: water is 1: 5 to 1:50, preferably 1:10 to 1:20. When water and / or an organic solvent are added and crushed, the amount of extraction solvent to be added may be adjusted in consideration of the amount used for crushing.

The higher the extraction temperature is, the higher the extraction efficiency is. When water is used, extraction is performed with hot water at 50 to 120 ° C, preferably 70 to 100 ° C. Hot water may be added to the plant, or water may be added to the plant and then heated. The extraction time is generally 10 minutes to 24 hours, but can be appropriately determined depending on the extraction temperature.

Further, as an extraction method using an organic solvent, extraction by warming or supercritical fluid extraction may be performed. As the warm extraction method using an organic solvent, A method of adding a heated solvent to the body or a method of adding a solvent to the plant and heating is used. For example, a water-ethanol mixed solvent with a weight ratio of water to ethanol of 1: 1 is used as an extraction solvent in a 5-fold amount with respect to the crushed plant, and the mixture is refluxed at 70 to 75 ° C. While stirring for 3 hours. When an organic solvent is used, the extraction temperature must be set to a value equal to or lower than the boiling point of the organic solvent.

The supercritical fluid extraction method is a method of extracting a target component using a supercritical fluid, which is a fluid that has exceeded the critical point (critical temperature, critical pressure) of the gas-liquid of a substance. As the supercritical fluid, carbon dioxide, ethylene, propane, nitrous oxide (laugh gas) and the like are used, and carbon dioxide is preferably used. The supercritical fluid extraction method includes an extraction step of extracting a target component with a supercritical fluid and a separation step of separating the target component from a supercritical fluid. In the separation process, either extraction separation by pressure change, extraction separation by temperature change, or extraction separation using adsorbent / absorbent may be performed.

Further, supercritical fluid extraction by an entrainer addition method may be performed. In this method, for example, ethanol, propanol, n-hexane, acetone, toluene, other aliphatic lower alcohols, aliphatic hydrocarbons, aromatic hydrocarbons, or ketones are added to a supercritical fluid. By adding about 20 W / V% and performing supercritical fluid extraction with the resulting extraction fluid, the solubility of the target extract, such as OPCs and catechins (described below), in the extraction fluid is dramatically improved. It increases or increases the selectivity of the separation and allows for efficient extraction of proanthocyanidins.

For the extraction, for example, any of a batch type, a semi-continuous type, and a continuous type may be used.

When juice is obtained from a plant, the plant is directly squeezed or, after being appropriately cut or crushed, squeezed. This method uses plants with a high moisture content. It is suitably adopted when For example, in the case of grape berries, squeezing produces squeezed juice containing pulp anthocyanidins. A crushed plant containing solids derived from the plant obtained by crushing the plant (eg, the crushed grape fruit) is also used in the same manner as squeezed juice. The crushed plant containing the solid content of is also included in the juice.

Next, the obtained extract or squeezed juice (hereinafter referred to as extract, etc.) and a predetermined amount of chitin, chitosan, or a derivative thereof (hereinafter, chitin, chitosan, and at least one of these derivatives) (Referred to as chitins). Hereinafter, this step is referred to as a contact step.

The chitin used in the contacting step may be in the form of a powder, or may be in the form of a fiber, fibril, film, porous body, microbead, or the like. Alternatively, it may be bonded to a carrier such as a resin.

The predetermined amount of chitin means 2 to 10 times, preferably 3 to 8 times, more preferably 4 to 7 times the amount of chitin by dry weight with respect to the polyphenol content in the extract and the like. Kind. When the chitin is bound to a carrier such as a resin, the weight excluding the weight of the carrier portion is defined as the weight of the chitin.

The polyphenol content in extracts and the like can be determined, for example, by fractionating a part (about 1 to 5 mL) of the extract, powdering by hot air drying, freeze drying, vacuum drying, and vacuum concentration. It can be calculated by measuring the weight of the powder of extract, etc. (hereinafter referred to as extract powder), and measuring the polyphenol content (%) in the extract powder by the Fo1 in method. it can.

The measurement of the polyphenol content (%) in the extract powder by the F o 1 in method is performed, for example, as follows. First, catechin is dissolved in purified water as a polyphenol standard substance, and a standard specimen is prepared stepwise within a range of 0.05 to 0.05% by weight / volume. Next, the extract powder is appropriately dissolved in purified water within the range of 0.01 to 0.05 weight / volume% to prepare a specimen. Standard samples Each sample and 2.5 ml of Folin reagent (2.5 g of sodium tungstate, 0.5 g of phosphomolybdic acid, and 1.25 ml of an 85% by weight phosphoric acid solution in 10 O ml) Aqueous solution) and stirred for 3 minutes, then add 3 mL of 10% by volume sodium carbonate solution, and leave for 1 hour. After standing, collect the supernatant by centrifugation, and measure the absorbance of this supernatant at 70 nm. A calibration curve is created from the absorbances of the standard samples at each concentration, and the concentration of polyphenol contained in the sample is obtained using this calibration curve. The polyphenol content (%) in the extract powder can be calculated from the obtained polyphenol concentration and the sample concentration.

In the contacting step, the contact may be performed by any method as long as the extract or the like contacts the chitin. For example, as a simple method, a chitin resin in which chitin is bound to a synthetic resin carrier is filled in a column, a column method in which an extract is passed, a chitin is added to the extract, etc., and after a certain time, the chitin is removed. There is a batch method for removal. When an organic solvent is contained in the extract or the like, it is necessary to dilute the extract or the like to such an extent that chitins do not dissolve in the organic solvent or to remove the organic solvent.

In the column method, it is preferable to remove insolubles contained in the extract and the like in advance in consideration of clogging of the column and the like. Methods for removing insolubles include methods commonly used by those skilled in the art, for example, filtration and centrifugation. Filtration is preferably used in terms of processing time. Filtration may preferably be performed at 50 to 100 ° C. The higher the temperature, the more proanthocyanidin can be recovered, and it is preferably carried out at 50 ° C or higher, more preferably at 70 ° C or higher. More proanthocyanidins can be recovered by washing the insolubles removed by filtration with hot water or a heated solvent at 70 ° C or more.

After removing insolubles, the resulting extract is taken into account in consideration of the efficiency of adsorption to chitins. Is preferably cooled. Preferably, the extract is cooled to 4 to 30 ° C. Examples of the cooling include natural cooling by standing to cool; forced cooling using water, ice, or a cooling device such as a freezer or a refrigerator.

After cooling, the extract is passed through a column packed with chitins. For example, when a proanthocyanin-containing substance is obtained from pine bark using chitin derived from Rikiji, the polyphenol content in the hot water extract of pine bark is measured, and then the content of the polyphenol is determined. The pine bark extract is passed through a column packed with 2 to 10 times, more preferably 3 to 8 times, the amount of chitin by dry weight. Thereafter, the column is washed with about 2 to 3 times the volume of the column with water, and the resulting protean cyanidin-containing substance can be obtained by collecting the column passing liquid and the washing liquid. Here, the proanthocyanidin-containing substance also includes a concentrate, a diluent, a powder, and the like obtained by a method usually used by those skilled in the art.

In the batch method, the treatment with chitins may be performed, for example, by adding 2 to 10 times, preferably 3 to 8 times the dry weight of chitin relative to the dry weight of polyphenol in the hot water extract of pine bark. It is preferable to carry out the reaction for 1 to 3 hours with stirring.

After the treatment, the chitins are removed by filtration or centrifugation to obtain a broantocyanidin-containing substance.

Through the above contacting step, a proanthocyanidin-containing substance having a high OPC content (hereinafter referred to as proanthocyanidin-containing substance I) can be easily obtained. Pro Ant Xia two gin inclusions I are dry weight in OPC (Origome Rick Pro Ant Xia two gin) 3 0-9 0 weight ^_0/0, and more preferably contains 4 0 wt% to 7 0% by weight.

The resulting pro Ant Xia two gin inclusions I is more preferably a catechin in terms of dry weight 5-1 5 wt%, more preferably 1 0-1 5 weight ^_0/0 can contain. Catechins are poorly water-soluble by themselves, and their physiological activities OPC has the property of increasing the water solubility of catechins and, at the same time, activating the action of catechins when coexisting with catechins. Therefore, the proanthocyanidin-containing substance I containing OPC and catechins is particularly useful.

Catechins are a general term for polyhydroxyflavan-1-ol. Examples of power techins include (+)-catechin, (1) i-epicatechin, (+)-gallocatechin, (1) i-epigallocatechin, epigallocatechin gallate, and epeki tekin gallate. In addition, it includes afzelequin, which is derived from natural products, and 3- (galloyl) derivatives of (+)-forcechin or gallocatechin. The effects of catechins include carcinogenesis inhibitory action, arteriosclerosis preventive action, fat metabolic disorder inhibitory action, blood pressure increase inhibitory action, platelet aggregation inhibitory action, antiallergic action, antiviral action, antibacterial action, caries prevention action, bad breath It has a protective effect, a normalizing effect on the intestinal flora, a scavenging effect of active oxygen and free radicals, an antioxidant effect, and an antidiabetic effect for suppressing an increase in blood sugar.

The resulting proanthocyanidin-containing substance I is further treated with a synthetic resin-based adsorbent to remove impurities such as saccharides and organic acids, and further purified. That is, the proanthocyanidin-containing substance I is brought into contact with a synthetic resin-based adsorbent, the proanthocyanidin is adsorbed on the synthetic resin-based adsorbent, and then eluted with a predetermined solvent, whereby the OPC content is reduced. An extremely high proanthocyanidin content (hereinafter referred to as proanthocyanidin content II) can be obtained. Examples of the synthetic resin-based adsorbent used for the above purification include an organic resin, an ion exchange resin, silica gel, and reversed-phase silica gel.

As the organic resin, for example, an aromatic resin such as a styrene-dibutylbenzene-based resin, an atalylic acid-based methacrylic resin, and an atarilonitrile-based aliphatic resin can be used. Preferably, it is an aromatic resin. As the aromatic resin, an aromatic resin having a hydrophobic substituent, an unsubstituted aromatic resin, An aromatic resin obtained by subjecting a substituted group to a special treatment is exemplified, and an aromatic resin obtained by subjecting an unsubstituted group to a special treatment is more preferable. These resins are preferably porous. Such synthetic resins are commercially available and include, for example, the following: Acrylic methacrylic resins include DIAION (registered trademark) HP 1MG and HP 2MG (all manufactured by Mitsubishi Chemical Corporation); As the aromatic resin, SP-900 (manufactured by Mitsubishi Chemical Corporation), Amberlite (registered trademark) XAD-2, XAD-4, XAD-16, XAD-2000 (all manufactured by Organo Corporation); hydrophobic Examples of the aromatic resin having a hydrophilic substituent include Diaion (registered trademark) SP-205, SP-206, SP-207, HP-1MG, EX-0021 (all manufactured by Mitsubishi Chemical Corporation), Amberley G (registered trademark) XAD-7, XAD-8 (all manufactured by Organo Co., Ltd.); Unsubstituted aromatic resins include DIAION (registered trademark) HP-10, HP-20, HP-2 1, HP-30, HP-40, HP-50 (or more, manufactured by Mitsubishi Chemical Corporation) Aromatic resins obtained by specially treating unsubstituted groups include SP-825, SP-800, SP-850, and SP-875 (all manufactured by Mitsubishi Chemical Corporation); derivatives of cross-linked dextran Examples include SEPHADEX (registered trademark) LH20 and LH60 (all manufactured by Pharmacia Biotech Co., Ltd.). Among them, Diaion HP-20 or Sephadex LH20 is preferable.

As the ion exchange resin, any of a cation exchange resin and an anion exchange resin can be used. Commercially available cation exchange resins include, for example, resins having a sulfonate group as a functional group, such as Amberlite (registered trademark) CG-4000, CG-5000, CG_6000, CG-8000, IR- 116, IR-118, IR-120B, IR-122, IR-124, XT-1007, XT-1009, XT-1002 (all manufactured by Olgano Corporation). As a weakly basic anion exchange resin, for example, OPT I PORE—XUS 40285.00, OPT I PORE—XUS 40390.00 (both manufactured by Da Chemical Co., Ltd.), which are resins having a quaternary amine as a functional group. The solvent to be dissolved when using the ion exchange resin is preferably water, and the column temperature is 50 to 120 ° (:, preferably, the column is under normal pressure or pressurized state.

The amount of the synthetic resin-based adsorbent may be appropriately set depending on the type of the solvent, the type of the synthetic resin-based adsorbent, and the like. For example, it is preferable to use 0.01 to 5 times the weight of the synthetic resin-based adsorbent with respect to the dry weight of the proanthocyanidin-containing substance I.

In order to purify the proanthocyanidin-containing substance I using a synthetic resin-based adsorbent, for example, first, a synthetic resin-based adsorbent is packed in a column, and the proanthocyanidin-containing substance I is passed through the column. Pass water 5 to 10 times the weight of the synthetic resin adsorbent. Thereby, saccharides and organic acids as impurities are removed. Then elute proanthocyanidin with a suitable solvent. Examples of the solvent include water, methanol, ethanol, ethyl acetate, chloroform, and a mixed solvent thereof. Preferably, a mixed solvent of water and ethanol is used. The mixing ratio of water and ethanol depends on the synthetic resin adsorbent used. For example, in the case of Diaion HP-20, the ratio of water: ethanol is 1: 1 to 4: 1 by volume. In this way, a proanthocyanidin-containing substance II having an extremely high OPC content is obtained.

The proanthocyanidin-containing substance II obtained through the above purification step may contain 0 to 80% by weight or more, preferably 95% by weight or more in terms of dry weight. This proanthocyanidin-containing substance II contains an extremely large amount of OPC, but hardly contains phytotechins.

Using the proanthocyanidin-containing substance II obtained through the above purification step, a proanthocyanidin-containing substance containing 5 to 15% by weight of phytotechins Object I can also be. For example, it can be obtained by adding high-purity tetekins to proanthocyanin-containing substance II.

The proanthocyanidin-containing substance I or II produced by the above method can be concentrated to produce a food material. For the concentration, various methods such as membrane concentration, heat concentration, vacuum (reduced pressure) concentration, and freeze concentration are used.

If necessary, these proanthocyanidin-containing substances I or II are sterilized and stored. Sterilization is performed by a method used by those skilled in the art, such as airflow sterilization, high-pressure sterilization, and heat sterilization.

Further, these proanthocyanidin-containing substances I or II may be concentrated, dried, or powdered after sterilization. Drying is performed by a method commonly used by those skilled in the art. Among them, freeze drying, vacuum drying, and spray drying are preferably used.

The proanthocyanidin-containing substance I or II thus obtained can be used as a raw material for foods, cosmetics, and pharmaceuticals.

The resulting proanthocyanidin-containing substance I or II can also be used as a drink, a gelled food or the like. In addition, proanthocyanidin-containing substances are not only used for eating and drinking as they are, but also mixed with excipients, bulking agents, binders, thickeners, emulsifiers, flavors, food additives, seasonings, etc., depending on the application. In addition, it can be formed into the form of granules, tablets, and the like. For example, it can be mixed with royal jelly, vitamins, protein, calcium, chitosan, lecithin, caffeine, etc., and taste can be adjusted with a sugar solution and a seasoning. Further, they are formed into capsules such as hard capsules and soft capsules, pills, or tea bags. These may be eaten as they are, or may be dissolved in water, hot water, milk, or the like, depending on their shape or preference. In the case of tea bags, etc., the ingredients may be leached before drinking. Example

Hereinafter, the present invention will be described with reference to examples, but the examples do not limit the present invention. The unit (V / V) shown in the examples represents (capacity / capacity), and (W / W) represents (weight / weight).

(Example 1)

2.4 L of purified water was added to 300 g of pine bark, crushed with a blender (Waring Blender), and heated at 100 ° C for 10 minutes. Then, the mixture was immediately filtered, and the insoluble material after the filtration was washed with 60 OmL of purified water, and the filtrate and the washing liquid were combined to obtain a 3 L extract.

A part (5 mL) of this extract was concentrated under reduced pressure, and the weight of the obtained extract powder was measured (4 Omg). Next, the polyphenol content in the extract powder was measured by the Fo1 in method and found to be 15.78% by weight. Therefore, it was found that the dry weight of 1 mL of this extract was 8 mg, and the polyphenol content in the extract was 1.26 mg.

40 OmL of the above extract (3.2 g of extract powder and 0.5 g of polyphenol content) was allowed to cool to 25 ° C, and it was cooled to 3 times the polyphenol content (1.5 g). The solution was passed through a column filled with chitin (manufactured by Nakarai Tester Co., Ltd.). Next, this column was washed with 4 OmL of purified water, and the passing solution and the washing solution were combined to obtain 44 OmL of the proanthocyanidin-containing solution Ia.

Half of the proanthocyanidin-containing solution Ia (22 OmL) was concentrated under reduced pressure, and the weight of the obtained dry powder was measured. The weight of the dry powder was 574 mg, and it was proved that the dry weight of the total amount of the liquid Ia containing oral anthocyanidin was 1148 mg.

Next, the following operation was performed in order to measure the content and the content of OPC and catechin in the proanthocyanidin-containing solution Ia in terms of dry weight. The dry powder of the proanthocyanidin-containing solution Ia obtained as described above is mixed with a component including OPC, a component including force techins, and other components including a polymer (hereinafter, referred to as other components). Then, they were separated as follows. First, 25 mL of Sefadex LH-20 (manufactured by Pharmacia Biotech Co., Ltd.) swollen with water was packed in a 15 × 30 OmL column, and washed with 25 mL of ethanol. Dissolve 10 Omg of the above dry powder in 2 mL of ethanol, pass it through the column and allow it to adsorb, then perform gradient elution with 100-80% (V / V) ethanol-aqueous mixed solvent. OmL was dispensed. At the same time as the fractionation, a dimer to tetramer of OPC standard (dimer: proanthocyanidin B-2 (R f value: 0.6), trimer: proanthocyanidin C- Using 1 (R f value: 0.4) and tetramer: cinnamtannin A ₂ (R f value: 0.2)) as the index, the OPC in each fraction was analyzed by silica gel thin-layer chromatography ( TLC).

The TLC development conditions and detection method are as follows.

TLC: Silica gel plate (Merck & CO., Inc.)

Developing solvent: benzene Z-ethyl formate Z-formic acid (27/1)

Detection reagents: sulfuric acid and anisaldehyde sulphate

Sample volume: 10 μL each

The elution fractions confirmed to contain OPC by TLC detection were combined to obtain an OPC fraction.

Then, when OPC was no longer detected, the remaining components adsorbed on the column were eluted by passing lOOmL of a 50% (V / V) water-acetone mixed solvent.

Next, the fractions other than the OPC fraction were subjected to TLC using catechin as an index (R f value: 0.8), and separated into a fraction containing phytotechins and a fraction of other components. The TLC development conditions and detection method were the same as described above. The fraction containing phytotechins was further separated into catechins and components other than phytotechins as follows. First, the fraction containing force techins was freeze-dried, the obtained powder was dissolved in 3 mL of water, and this was swollen with water and 2 OmL of MCI gel (manufactured by Mitsubishi Chemical Corporation) was applied to 15X. The liquid was adsorbed by passing through a column packed in a 30 OmL column. After the column was washed with water, it was eluted with a gradient of 10 to 100% (V / V) ethanol-water mixed solvent, and fractionated in 10 mL portions. After completion of elution, catechins in each fraction were detected by TLC using catechin as an index, and separated into tetekins fractions and fractions of components other than tetekins.

The OPC fraction, the hapticins fraction, the fractions other than the catechins, and the other component fractions obtained as described above were each powdered by freeze-drying, and the dry weight was measured.

The obtained OPC is a dry powder of the proanthocyanin-containing solution Ia 10

Omg was 30.2 mg. That is, the content of OPC in proanthocyanidin-containing liquid Ia is 30.2% by weight in terms of dry weight, and 347 mg of OPC should be contained in the total amount of oral anthocyanidin-containing liquid Ia. I was strong.

The resulting tetekins were 10.1 mg. That is, the content of catechins in the proanthocyanidin-containing solution Ia was 10.1% by weight in terms of dry weight, and the amount of catechins in the total amount of the proanthocyanidin-containing solution Ia was 1 16 It was found to contain mg. Note 99, OPC fraction, force catechins fraction, fraction of components other than the force text emissions such, and the sum of the fractions of the other components, the dry powder 10 Omg of Puroantoshi Anijin containing liquid I _a. It was 2 mg, and almost all of it had been recovered.

Table 1 shows the dry weight of the proanthocyanidin-containing liquid Ia, and the contents and contents of OPC and kytechins in terms of dry weight. (Examples 2 and 3)

Example 1 was repeated except that the amount of chitin packed in the column was 2.5 g, equivalent to 5 times the dry weight of polyphenol in the extract, and 3.5 g, equivalent to 7 times the dry weight of the polyphenol in the extract. In the same manner as in the above, 44 OmL of proanthocyanidin-containing solutions Ib and Ic were obtained. The proanthocyanidin-containing solutions Ib and Ic were concentrated under reduced pressure at a volume of 22 OmL, and the weight of each of the obtained dry powders was measured.The entire amount of each proanthocyanidin-containing solution Ib and Ic was dried. It was converted to weight. The dry weight of the proanthocyanidin-containing solution Ib was 1024 mg, and the dry weight of the proanthocyanidin-containing solution Ic was 632 mg. Next, using 100 mg of each of the proanthocyanidin-containing solutions Ib and Ic, the content and content of OPC and catechins in terms of dry weight were determined in the same manner as in Example 1. The results are shown in Table 1. The total dry weight of the obtained fractions was 98.9 mg and 99.5 mg, respectively, for 10 Omg of dry powder of proanthocyanidin-containing solutions Ib and Ic, respectively. Had been recovered.

(Example 4)

Further purification was performed using 22 OmL of the half amount of the proanthocyanidin-containing solution Ib obtained in Example 2. First, the proanthocyanidin-containing solution Ib was passed through a column filled with 5 g of an aromatic synthetic resin (Diaion HP-20: manufactured by Mitsubishi Chemical Corporation), and the proanthocyanidin was adsorbed on the column. Was. The column was washed with 2 OmL of purified water, and then the column was eluted with a 20 to 40% (V / V) mixed solvent of ethanol and water to obtain 40 mL of proanthocyanidin-containing solution IIb.

Dry powder obtained by concentrating this proanthocyanidin-containing solution IIb under reduced pressure Was 143 mg, and was found to be 286 mg in terms of the dry weight of the entire proanthocyanidin-containing solution IIb. In the same manner as in Example 1, the content and the content in terms of dry weight of OPC and force techins were determined using 10 Omg of the dry powder of the anthocyanidin-containing liquid IIb. The results are shown in Table 1.

(Comparative Examples 1 and 2)

Example 1 Example 1 was repeated except that the amount of chitin packed in the column was 0.5 g, which was equivalent to 1 times the dry weight of polyphenol in the extract, and 6.5 g, which was equivalent to 13 times the dry weight of polyphenol in the extract. In the same manner as in, 44 OmL of proanthocyanidin-containing solutions Id and Ie were obtained. 22 OmL of each half of the proanthocyanidin-containing solutions Id and Ie were concentrated under reduced pressure, and the weight of each of the obtained dry powders was measured.The total amount of each of the oral anthocyanidin-containing solutions Id and Ie was determined. It was converted to dry weight. The dry weight of the proanthocyanidin-containing solution Id was 2288 mg, and the dry weight of the proanthocyanidin-containing solution Ie was 472 mg. Next, using 10 Omg of each of the dry powders of the proanthocyanidin-containing liquids Id and Ie, the content and the content in terms of dry weight of the OPC and force techins were measured in the same manner as in Example 1. The results are shown in Table 1. The total dry weight of the obtained fractions was 99.3 mg and 98.8 mg, respectively, for 10 Omg of dry powder of proanthocyanidin-containing solutions Id and Ie, respectively, and almost all of them were Had been recovered. Example Comparative example

1 2 3 4 1 2 Name la lb Ic lib Id Ie

1148 1024 632 286 2288 472 Proant "flM (mg) 347 463 349 231 394 50

Inclusion Content (%) 30.2 45.2 55.2 80.8 17.2 10.5

Content (mg) 116 152 171 n.d. 166 18 Catechin

Content (%) 10.1 14.8 13.5 n.d.7.3 3.8 Chitin (g)

Column 3 5

E 7 1. Riff I Nord (g) 13

nd; not detected As can be seen from Table 1, proanthocyanidin-containing solutions Ia to I obtained by treating with 3 to 7 times the amount of chitin relative to the polyphenol content in the extract. Contains 30% by weight or more of OPC on a dry weight basis. These O PC content corresponds to about 2 times the polyphenol contained in extracts powder (15.78 wt. / _0). Particularly Puroantoshia - Gin containing liquid I b and I c are the OPC on a dry weight amount conversion also contains 40 weight ^_0/0 above. On the other hand, in proanthocyanidin-containing solutions Id and Ie obtained by treating with 1 or 13 times the amount of chitin relative to the dry weight of polyphenol in the extract, OPC was calculated to be 17.2 in terms of dry weight. % And 10.5% by weight, which were lower than those treated with 3 to 7 times the amount of chitin. From these results, it is shown that by treating a plant extract or the like with a predetermined amount of chitin, a proanthocyanidin, particularly a brothocyanidin-containing substance containing a high proportion of OPC can be obtained. Furthermore, pro-Ant Xia two Jin-containing solution I to I c is, 10. a force enemy emissions such in terms of dry weight from 1 to 14.8 wt ^_0/0 T while containing, Puroantoshia two Jin-containing solution I The dtekines contained in d and Ie were 7.3% by weight and 3.8% by weight, respectively. This is because treatment of plant extracts and the like with a predetermined amount of chitin allows not only OPCs but also high-technologies to be more expensive. Fig. 7 shows that a proanthocyanin-containing material can be obtained.

Furthermore, the proanthocyanin-containing solution I Ib contained 80% by weight or more of OPC in terms of dry weight. Thus, it was found that a proanthocyanidin-containing substance containing OPC at an extremely high ratio can be easily obtained only by the step of passing the extract through two types of columns.

(Example 5)

1 L of purified water was added to 100 g of pine bark, crushed, and heated at 100 ° C for 10 minutes. Then, the mixture was immediately filtered, and the insoluble material after the filtration was washed with 20 OmL of purified water. The filtrate and the washing liquid were combined to obtain 1.2 L of an extract. A part (5 mL) of this extract was concentrated under reduced pressure, and the weight of the obtained extract powder was measured (39 mg). Further, the content of polyphenol in the extract powder was measured by the Fo1 in method, and was found to be 15.55% by weight. The polyphenol content in the total amount of the extract was 1.46 g.

Next, the above extract powder was added to the remaining extract, adjusted to a total volume of 1.2 L with hot water, allowed to cool to 25 ° C, and chitin in a dry weight of about 7 times the polyphenol content. 10 g was added, and the mixture was stirred for 3 hours. After stirring, the mixture was filtered to obtain 1.2 L of a proanthocyanidin-containing solution If.

Next, the proanthocyanidin-containing solution If was concentrated under reduced pressure to obtain 3.36 g of a dry powder of the proanthocyanidin-containing solution If. l The OPC and catechin contents were measured in the same manner as in Example 1 using the dry powder of the proanthocyanidin-containing solution If of O Omg, and it was found that OPC was 40% by weight or more on a dry weight basis. It was found to contain 12.1% by weight of force techins. Furthermore, 3.26 g of the proanthocyanidin-containing solution If dry powder was dissolved in 2 L of methanol and passed through a column packed with 50 g of Diaion HP 20 as in Example 1. The proanthocyanidins were adsorbed. Purify this column After washing with water, the column was eluted with a 20-40% (V / V) ethanol-aqueous solvent mixture to obtain 40 OmL of proanthocyanidin-containing solution IIf. The proanthocyanidin-containing solution IIf was concentrated under reduced pressure to obtain 2.55 g of a dry powder of the proanthocyanidin-containing solution IIf. When the content of OPC in the proanthocyanidin-containing liquid IIf was measured in the same manner as in Example 1, it was found that the content of OPC was 80% by weight or more in terms of dry weight. Industrial applicability

According to the method of the present invention, a proanthocyanidin-containing substance containing OPC having a high physiological activity in a high ratio can be easily obtained. The proanthocyanidin-containing material having a high OPC content also contains phytotechins. Therefore, OPC has a synergistic effect of activating the action of catechins. By simply treating the proanthocyanidin-containing substance with a synthetic resin-based adsorbent, a proanthocyanidin-containing substance having an extremely high OPC content can be provided. Since the proanthocyanidin-containing substance having an extremely high OPC content contains almost no catechins, it can be used as a more purified OPC. These proanthocyanidin-containing substances are effective in improving vascular enhancement, hypertension, coldness, etc., and are very useful as raw materials for producing foods, cosmetics, and pharmaceuticals.

Claims

The scope of the claims

1. (a) obtaining an extract or juice from a plant by extraction or pressing; and (b) converting the extract or juice to the polyphenol content in the extract or juice. A method for producing a proanthocyanidin-containing substance, which comprises a step of bringing non-adsorbed substances into contact with chitin, chitosan, or a derivative thereof in an amount of 2 to 10 times by dry weight.

2. The method of claim 1, wherein the extraction is a hot water extraction.

3. The method according to claim 1, further comprising: (c) contacting the non-adsorbed substance obtained in the step (b) with a synthetic resin-based adsorbent.

4. Oligomeric 'proanthocyanidin-containing material containing proanthocyanidins and catechins, wherein the proanthocyanidin-containing material contains 30% of oligomeric' proanthocyanidin in terms of dry weight. weight ^_0/0 or more, and the force catechins in a proportion of 5 to 1 5% by weight, pro Ant Xia guanidinium down inclusions.