WO2005070443A1 - Timber bark extract - Google Patents

Abstract

A timber bark extract obtained by extracting the bark of timber less than 100 years of age with the use of at least one solvent selected from the group consisting of water, polar solvents and mixed solvents thereof. In particular, it is preferred that the timber be one 20 to 80 years of age. This timber bark extract contains OPC at a high ratio.

Description

 Bark extract

Technical field

 The present invention relates to bark extracts, and more particularly to bark extracts obtained from the bark of trees less than 100 years old.

 Akita

Background art

 Polyphenols, especially proanthocyanidins, have a strong antioxidant action to scavenge active oxygen. Proanthocyanidins are compounds that generate anthocyanidins when subjected to chemical treatment (for example, acid treatment), and are a group of compounds composed of polycondensates having a flavan-1-ol derivative as a constituent unit.

 It is known that such proanthocyanins are contained, for example, in various plants. Therefore, a method for producing an extract containing a large amount of proanthocyanidins has been studied (for example, Japanese Patent Application Laid-Open Nos. Sho 644-42479 and Hei 11-180148). ). However, the effectiveness of physiological activities such as antioxidant activity of these plant extracts is not determined solely by the proanthocyanidin content, but rather by the structure of the proanthocyanidin and the plant. Various factors are involved, such as inhibitory or synergistic effects with other components contained therein.

Japanese Patent Application Laid-Open Publication No. 2001-1588739 discloses that an inorganic salt is added to a hot water extract of a plant in order to obtain an extract having a high content of a biologically active oligomeric porifenol. The tannin is precipitated, the precipitate is filtered off, the resulting filtrate is treated with ethyl acetate, concentrated, replaced with water, washed with solvent, the solvent is removed, and the aqueous phase concentrate is dried. Preparation methods are described. However, such as antioxidant action Examination for obtaining a plant extract having excellent physiological activity is still not sufficient. In addition, the bark extract has low solubility in water, which causes problems such as precipitation or cloudiness when the bark extract is used as a liquid cosmetic or beverage material. Such problems are undesirable in the appearance of the product or impede the absorption of components such as proanthocyanidins into the body which cause precipitation or cloudiness.

 Various methods have been studied to enhance the solubility of bark extracts. For example, Japanese Patent Application Laid-Open No. 2003-284549 discloses a polyphenol by adding a skin extract having a high polyphenol content and a predetermined amount of a saccharide to an aqueous solution. It describes a method for improving the solubility of bark extract in water, and consequently the solubility of bark extract in water. However, in the above method, since it is essential to add a saccharide, a step of dissolving the saccharide in water is required in advance. The addition of this step complicates the manufacturing process, and the addition of carbohydrates is not preferable because it leads to an increase in calorie intake, especially when used in foods (drinks). Disclosure of the invention

 An object of the present invention is to provide a plant extract having an excellent antioxidant effect. The present inventors have conducted intensive studies on plant extracts having excellent antioxidant activity. As a result, the bark extract obtained from the bark of a tree less than 100 years old was obtained from oligomeric proanthocyanidin (hereinafter referred to as OPC). ) At a high ratio and have excellent antiacid activity. Furthermore, it has been found that by extracting this specific bark with water, a polar solvent, or a mixed solvent thereof, a bark extract having excellent antioxidant activity can be obtained in a high yield, and the present invention has been completed. Was.

That is, the bark extract of the present invention is obtained from bark of a tree less than 100 years old, It is obtained by extraction with at least one solvent selected from the group consisting of polar solvents and mixed solvents thereof.

 In a preferred embodiment, the tree is a tree of 20 to 80 years of age.

 The present invention also provides a bark extraction method comprising the step of extracting from the bark of a tree younger than 100 years with at least one solvent selected from the group consisting of water, a polar solvent and a mixed solvent thereof. Provided is a method for manufacturing a product.

 By using the bark of a tree less than 100 years old, the solid concentration in the bark extract is about 1.5 to 2 times or more than when using the bark of a tree older than 100 years. A bark extract is obtained that is high and has an increased absolute amount of proanthocyanin containing OPC in the solid content. Further, by purifying the skin extract, OPC, which is a dimer to tetramer compound of a flapan-13-ol derivative, and a pentamer or higher compound which is a flavan-13-ol derivative, are obtained. A bark extract containing the molecule proanthocyanidin at a high concentration is obtained. That is, a bark extract in which 25% by mass or more of all proanthocyanidins is OPC can be obtained. Since the bark extract has excellent solubility in water, it does not cause turbidity and precipitation in aqueous solution, is quickly absorbed into the body, and has excellent antioxidant activity (particularly, superoxide dismutase activity). Having. Therefore, it is easy to use the bark extract for beverages, liquid cosmetics, and the like. Brief Description of Drawings

 FIG. 1 is a diagram showing pine bark extract A separated into various fractions.

FIG. 2 is a graph showing the change over time in the rate of increase in SOD activity when a test solution was administered to rats. BEST MODE FOR CARRYING OUT THE INVENTION

 The bark extract of the present invention is obtained by a method comprising a step of extracting from the bark of a tree less than 100 years old with at least one water-polar solvent. The tree that is the raw material of the bark extract of the present invention is not particularly limited as long as it is less than 100 years old, but is preferably 80 years or less, more preferably 20 to 80 years, and still more preferably. It is a tree from 20 to 60 years. Trees over 100 years old have a thick bark, and it is possible to obtain a large amount of bark as a raw material from a single tree.However, the amount of polyphenols such as proanthocyanin contained in the tree is poor. Unfavorable due to low c content. In the present specification, the anthocyanidins are classified according to the degree of polymerization. That is, a dimer to tetramer compound of a flavan-13-ole derivative is defined as OPC, and a pentamer or higher compound is defined as high molecular weight proanthocyanidin. A flavan-13-ol derivative is a compound having a flavan-13-ol as a basic skeleton. Examples of the flavan-13-ol include a flavan-13-ol, a flavan-13,4-diolone, a flavan-1,3,4-diolone derivative, Catechins (generic term for polyhydroxyflapan-1-ol). OPC has particularly excellent bioactivity among proanthocyanidins. The age of the 齢 tree is measured, for example, by a radiocarbon measurement method or by counting annual rings after falling.

 The tree may be, for example, either a conifer or a hardwood. Specifically, there are trees belonging to the order Pinaceae, oaks, yamazaki, morisima acacia, acacia mangium, trees belonging to the order willow, hiba, radia-tapain, and the like. These barks generally contain many proanthocyanidins. The bark of a tree belonging to the order Poniferae is preferred because of its high OPC content.

Trees belonging to the order Pinaceae include French pine (Pinus Martima), Japanese pine, Japanese pine, Japanese larch, Japanese larch, Japanese black pine, Japanese pine, Japanese pine, Japanese pine, Korean pine, Nojimatsu, Ryukyu pine, Peck Shimatsu, Daiomatsu, Shiromatsu, and Aneda in Quebec, Canada. French coastal pine, bay pine, northern ezo pine, new Zealand pine, and akamatsu are preferred! / ヽ.

The bark extract of the present invention contains the oligomeric proanthocyanin (OPC) in an amount of 25% by mass or more, preferably 30 mass%, based on the solid content in the bark extract. / ₀ or more, more preferably ₃₅ mass. / ₀ or more, more preferably 40% by mass or more, most preferably 40% by mass to 60% by mass. /. contains. The higher the OPC content, the greater the water solubility of the bark extract tends to be. The bark extract has high solubility in water. Therefore, the generation of cloudiness and precipitation of the aqueous solution is suppressed. Specifically, the dissolution rate of the bark extract is 90% or more, preferably 95% or more, more preferably 99% or more, still more preferably 99.5% or more, and most preferably 99.7% or more. % Or more. The “dissolution rate” means that the bark extract was suspended in distilled water so as to have a concentration of 1% by mass, Z, and stirred with a vortex, then allowed to stand at room temperature (about 25 ° C) for 60 minutes. In this case, it refers to the percentage of solids that are not detected as filter cake, and is expressed by the following equation 1. r ヽ

 Dissolution rate (%) = f] ζin solution _ ;: | dry mass of residue (g).

 1 X I 00 Formula 1 Dry mass of bark extract added (g)

(Method for producing bark extract)

To obtain the bark extract of the present invention, first, a step of extracting the bark of a tree less than 100 years old with at least one solvent of water and a polar solvent is included. Preferably, a method including (a) a crude extraction step and a purification step is employed. The purification step is, specifically, (b) a step of reducing high molecular weight proanthocyanidin (high molecular weight PC) and (c) a step of enriching proanthocyanidin. (B) of The high molecular weight PC reduction step is a step of reducing high molecular weight PC from the processed product (for example, crude extract) obtained in the previous step, and the proanthocyanin concentration step (c) is performed in the previous step. This is a step of removing contaminants such as saccharides, organic acids, and fat-soluble components from the obtained processed product (for example, a high-molecular PC reduced product) and reducing the solvent. The order of the (b) polymer PC reduction step and (c) proanthocyanidin concentration step may be reversed. In the present specification, first, the production method (the above steps (a) to (c)) for obtaining the bark extract of the present invention will be described, and then the bark extract of the present invention will be described. The configuration described below does not limit the present invention, and it is apparent to those skilled in the art that various modifications can be made within the scope of the present invention.

(a) Crude extraction process

 The rough extraction step is, specifically, by immersing the bark of a tree less than 100 years old in at least one solvent of water and a polar solvent, and maintaining it at a predetermined temperature as necessary. Done. .

 The bark is preferably crushed to an appropriate size in order to increase the surface area per volume, from the viewpoint of extraction efficiency. For the crushing, for example, a cutter, a slicer, a mixer, a juicer, a blender, and a masco mouth lider are used. The size of the bark crushed product is not particularly limited, but is preferably 0.1 to 10 cm, more preferably 0.1 to 5 cm. At the time of crushing, water or a polar solvent such as ethanol, methanol, or ethyl acetate, or a mixed solvent of water and a polar solvent may be added to increase the crushing efficiency.

The solvent used to obtain the bark extract of the present invention is water, a polar solvent, or a mixed solvent thereof. Polar solvents include, for example, methanol, ethanol, 1-propanol, 2-propanol. 1-butanol, 2-butanol. Nol, Acetone, Hexane, Hexane Hexane, Propylene Glyconore, Hydrous Ethanol, Hydrous Propylene Glycol, Ethyl Methyl Ketone, Glycerin, Methynolacetate, Etinole Acetate, Getinoreether, Dichloromethane, Edible Oil, 1, 1,1,2-Tetrafluoroethane and 1,1,2-trichloroethane. Examples of the mixed solvent include a solvent obtained by mixing one or more of the above polar solvents and water, and a solvent obtained by mixing two or more of the above polar solvents.

 The selection of the solvent may be determined based on, for example, the following items. First, it is preferable to use water from the viewpoint of waste liquid treatment during production or from the viewpoint of adding a metal salt or the like (described later). When concentration is performed at a relatively low temperature in a short time, a polar solvent having a lower boiling point than water (eg, ethanol) or a mixed solvent of a polar solvent having a lower boiling point than water and water may be used. Such a polar solvent can be easily removed at the time of concentration, and a bark extract can be efficiently obtained. When the bark extract of the present invention is used as food or medicine, it is preferable to use ethanol or a mixed solvent of ethanol and water in consideration of the safety of its use.

 The amount of the solvent may be set in consideration of the concentration of proanthocyanidin in the bark extract or the extraction efficiency. For example, when water is used as the extraction solvent, the ratio of bark to water is 1: 5 to 1: 100, preferably 1:10 to 1:50 by mass. When crushing by adding water and / or a polar solvent, the amount of extraction solvent to be added may be adjusted in consideration of the amount used for crushing.

The extraction temperature is preferably set to a higher temperature in consideration of extraction efficiency. For example, when water is used, hot water extraction is performed at 50 to 120 ° C, preferably at 70 to LOO. In this case, hot water may be added to the bark (or bark crushed product), or water may be added to the bark (or bark broken frame) and then heated. However, to prevent thermal denaturation of proanthocyanidins, extract with relatively low temperature water. It is preferable to do.

 The extraction time may be determined in consideration of the extraction conditions such as the size of the bark (or bark rubbish) to be used and the extraction temperature, and the extraction efficiency. In general, the extraction time is 10 minutes to 24 hours. is there.

 Next, a specific extraction method will be described. As an extraction method, a warm extraction method or a supercritical fluid extraction method can be used.

 As the warm extraction method, a method of adding a heated solvent to bark, or a method of adding bark to a solvent and heating the solvent is used. For example, using a mixed solvent of water and ethanol having a mass ratio of water to ethanol of 1: 1 to: L: 9 and pulverized leather, the mixture is refluxed at 70 to 75 ° C. to obtain a mixture. A method of stirring for 5 to 6 hours can be used. When this method is used, the amount of the solvent may be 1 to 20 times the amount of the bark.

 By the way, a process is adopted in which the solvent is once extracted without heating to the temperature at which the solvent is refluxed, the supernatant is collected by filtration, etc., and the solvent is again added to the remaining residue, followed by similar heating and extraction. As a result, high extraction efficiency can be obtained. When a polar solvent is used, the extraction temperature must be set to a value equal to or lower than the boiling point of the polar 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 a substance's gas-liquid. As the supercritical fluid, carbon dioxide, ethylene, propane, nitrous oxide (laugh gas) and the like are used, and dioxide carbon 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 the supercritical fluid. In the separation step, either extraction separation by pressure change, extraction separation by temperature change, or extraction separation using an adsorbent or absorbent may be performed.

Further, supercritical fluid extraction by an entrainer addition method may be performed. this The method is as follows. For example, ethanol, propanol, n-hexane, acetone, toluene, other aliphatic lower alcohols, aliphatic hydrocarbons, aromatic hydrocarbons, or ketones are added to a supercritical fluid. 0 By adding WZV% and adding supercritical fluid with the obtained extraction fluid, the solubility of OPC, catechins (described below), etc., in the target extraction target fluid in the extraction fluid is dramatically increased. This is a method to increase the selectivity of separation and to extract proanthocyanin efficiently.

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

 The obtained crude extract can be used as it is as a bark extract, but is further purified. By purifying, it is possible to obtain a bark extract that is more excellent in antioxidant action. Specifically, it is subjected to (b) a step of reducing high molecular weight PC or (c) a step of concentrating proanthocyanidins.

Prior to subjecting the crude extract to (b) the step of reducing high molecular PC or (c) the step of concentrating proanthocyanidin, it is preferable to carry out a concentration treatment for reducing the solvent from the crude extract in advance. By reducing the solvent from the crude extract, advantageous effects can be obtained in the subsequent steps, especially in the step (b) for reducing the high molecular weight PC. For example, (b) when a salt treatment is performed as the polymer PC reduction step, the amount of salt used can be reduced. As a result, it is possible to efficiently remove proanthocyanidins (polymer PC) having a high degree of polymerization. When the crude extract contains an organic solvent, the organic solvent can be removed by concentration and replaced with water. By substituting with water, the ionization of the salt in the solution is increased, and the salt and the high molecular weight PC are combined to easily precipitate. Therefore, polymer PC can be removed efficiently. The amount of water to be replaced can be adjusted so that the volume of the concentrate after the replacement is at most twice the volume of the crude extract before the concentration. When performing the concentration treatment, it is preferable to remove insolubles by filtration in advance. Yes. If such a crude extract is used, since the insoluble matter is removed, the concentration can be performed uniformly, and the concentration ratio can be easily adjusted.

 The concentration method is not particularly limited as long as it is a concentration method usually used by those skilled in the art. For example, heating concentration, reduced-pressure concentration, freeze concentration, concentration using an ultrafiltration membrane, concentration using a dialysis membrane, concentration using an adsorbent and the like can be mentioned. Freeze-concentration is a method in which a crude extract is frozen, and the water is sublimated by reducing the pressure below the water vapor pressure of the frozen product to remove it. Preferably, it is concentration under reduced pressure and freeze concentration in which the heat denaturation of proanthocyanin is small, and more preferably, concentration under reduced pressure. Concentration under reduced pressure can minimize denaturation of proanthocyanidins.

 The concentration method may be performed by only one method, or may be performed by combining a plurality of methods. In addition, when performing heat concentration, it is preferable to perform the heat concentration at a temperature of 40 ° C. to 100 ° C. in order to prevent thermal denaturation of proanthocyanidin by heating.

 The concentration rate of the obtained concentrate is not particularly limited. Generally, the volume of the concentrate is 1/2 to 1 Z 100 volumes, preferably 1/5 to 170 volumes, more preferably the volume of the crude extract before concentration. Is concentrated to a volume of 10 to 50 volumes.

 The above-mentioned concentration treatment is useful in terms of cost reduction and environmental conservation, since the amount of salt used is reduced when the salt treatment is carried out as the subsequent (b) polymer PC reduction step. Specifically, the amount of salt used can be reduced to about 1/2 to about 1-100, preferably about 1/5 to about 1/50, as compared to the case where the crude extract is directly salted. .

(b) Polymer PC reduction process

Next, the polymer PC reduction step will be described. In this step, the polymer proanthocyanin in the crude extract (or concentrate) obtained in the above (a) crude extraction step is used. This is done to reduce gin (polymer PC). Alternatively, (a) crude extraction is performed after the (c) proanthocyanidin enrichment step described below, with the purpose of reducing high molecular weight PC. Examples of the polymer PC reduction step include (b-1) salt treatment and (b-2) adsorbent treatment. By this high-molecular-weight PC reduction step, high-molecular-weight PC can be reduced, and as a result, the OPC content in the obtained high-molecular-weight PC reduction product can be relatively increased.

(b-1) Salt treatment

 The salt treatment is carried out by adding a salt to a crude extract (or concentrate) or a proanthocyanidin concentrate obtained in the (c) proanthocyanidin concentration step described below, whereby polymer PC, for example, a precipitate is added. This is a process that removes this insoluble matter by generating it as insoluble matter.

 The salt used in the salt treatment may be any one that is ionized in the solution. Examples of the salt include a monovalent metal salt, a divalent metal salt, and a nonmetallic salt.

 Monovalent metal salts include salts of alkali metals such as lithium, sodium, potassium, rubidium, cesium, and francium, for example, halide salts (chloride salts, bromide salts, etc.), phosphates, carbonates, and organic acids. Salts (carboxylates such as acetate, sulfonates, etc.). Specific examples include sodium salt sodium, sodium sulfate, sodium citrate, potassium chloride, sodium phosphate, potassium phosphate, sodium acetate and the like. Particularly preferred are sodium sulfate, potassium phosphate, sodium citrate, and sodium chloride, which are preferably used for salt prayer.

Examples of divalent metal salts include beryllium, magnesium, and alkaline earth metals (calcium, strontium, barium, and radium). The divalent metal salt is a metal used as an oxidizing agent (for example, copper Care must be taken when using a salt containing) because proanthocyanin may be oxidized. Since the divalent metal salt has a strong adsorptive power particularly to proanthocyanidin, the polymer PC can be made insoluble with a small amount of salt. As the nonmetal salt, for example, ammonium sulfate (ammonium sulfate) is preferably used.

 Among the above salts, a monovalent metal salt or ammonium sulfate is preferred from the viewpoint of minimizing the bond between OPC and the salt and increasing the purification efficiency of OPC.

The amount of the salt to be added is not particularly limited, but is 0.1% by mass to 50% by mass relative to the total mass of the crude extract (or concentrate) or the proanthocyanin concentrate. / ₀ , preferably 3% to 50% by mass, more preferably 5% by mass. /. It can be added so as to be ~ 45 mass%. In particular, 10 to 75% by mass, preferably 20 to 60% by mass of the saturated amount of salt with respect to water is added to the crude extract (or concentrate) or the proanthocyanidin concentrate. Is preferred. When a divalent metal salt such as a calcium salt or a magnesium salt is used as the salt, it is preferable to add the salt so as to be 0.1% by mass to 30% by mass in the concentrate. /. In the salt treatment, particularly the treatment with a metal salt, it is preferable to perform the treatment on the acidic side. If the alkalinity is weak to strong, the stability of proanthocyanidins may be degraded and may be decomposed. Therefore, the pH in the solution is preferably adjusted to be less than 7.5, more preferably less than 6, and even more preferably 5.5 or less. In particular, care should be taken when adding a divalent metal salt, because the pH in the solution is easily increased. The pH is adjusted by using, for example, an auxiliary agent for stabilizing proanthocyanidin (pH adjusting agent such as ascorbic acid). Specifically, a metal salt solution is prepared in advance so that the metal salt concentration becomes 2 to 10 times the final concentration at the time of salt treatment, and this solution is further adjusted to have a pH of 4 to 6, preferably 4 to 5. 5, more preferably 4 to 5. This solution is used as a crude extract (or concentrate) or By adding to the cyanidin concentrate, proanthocyanin degradation can be avoided.

 In the salt treatment step, the treatment temperature is not particularly limited. Preferably it is 1 to 40 ° C. There is no particular limitation on the processing time, and it can be set as appropriate according to the processing temperature. For example, after addition of the salt, the mixture is allowed to stand at 1 ° C. to 40 ° C. for 30 minutes to 48 hours to sufficiently generate an insoluble matter such as a precipitate. The standing time may be 48 hours or more, but it is preferable to move to the next step before the OPC is automatically oxidized and the reddish brown turns deep brown.

 In the next step, the insoluble matter such as the generated precipitate is removed. As a method for removing insolubles, a method commonly used by those skilled in the art, for example, filtration or centrifugation may be used. Filtration is preferably used in terms of processing time. Filtration may preferably be carried out at 1-4 ° C. The lower the temperature, the more polymer PC can be removed, and the reaction is preferably performed at 30 ° C. or lower, more preferably at 25 ° C. or lower. This filtration treatment may be performed before the addition of the salt, but must be performed after the addition of the salt in order to remove insoluble matters such as precipitates. In the filtration, it is preferable to repeatedly wash the residue remaining after the filtration with an aqueous solution of a salt having a similar saturation concentration in order to minimize the loss of OPC.

 The amount of high molecular weight proanthocyanidin contained in the solution after salt treatment (high-molecular weight PC-reduced product) is 12 or less compared to the crude extract (or concentrate) or the salt of broanthocyanidin before salt treatment. , Preferably 1 Z 3 or less, more preferably 15 or less, and even more preferably 1/6 or less.

If the (c) proanthocyanin enrichment step described below is adopted after the salt treatment step (polymer PC reduction step), the salt used in the salt treatment is removed in the adsorbent treatment step. There is no need to provide a step for removing salts, which is efficient. (b-2) Adsorbent treatment

 (b) As the adsorbent used in the polymer PC reduction step, an adsorbent that can effectively adsorb OPC and does not easily adsorb polymer PC (hereinafter referred to as the first adsorbent) should be used. Is preferred. The first adsorbent is brought into contact with (a) the crude extract (or concentrate) obtained in the crude extraction step or (c) the proanthocyanin concentrate obtained in the (c) proanthocyanidin concentration step described below. By recovering the adsorbate with a predetermined solvent, the non-adsorbed polymer PC can be removed. Details of the adsorbent treatment will be described in the section of (ii) Adsorbent treatment described later.

(c) Proanthocyanin concentration process

 Next, the proanthocyanidin concentration step will be described. In this step, the crude extract of bark (or its concentrate) or the high molecular weight PC-reduced material obtained above is treated with a predetermined adsorbent to obtain saccharides, organic acids, and fats from the treated material. This is a step of removing contaminants such as soluble components. Specifically, a crude extract of bark (or its concentrate) or a high-molecular-weight PC-treated product is brought into contact with a synthetic resin-based adsorbent, and proanthocyanidin containing OPC and high-molecular-weight PC is adsorbed. After being adsorbed on the surface, the adsorbent is washed and eluted with a predetermined solvent to obtain a proanthocyanidin concentrate (or bark extract). The adsorbent used in this treatment is called a second adsorbent. The proanthocyanidin concentrate (or bark extract) thus obtained contains more proanthocyanidins (such as OPC) as a result of removing contaminants. The details of the adsorbent treatment will be described later in the section of (ii) Adsorbent treatment.

If the polymer PC reduction step of (b) is performed after the proanthocyanidin concentration step of (c), reduce the solvent such as concentration under reduced pressure between the above steps (c) and (b). May be performed. By this concentration process, For example, as described above, in the subsequent salt treatment as the (b) polymer PC reduction step, effects such as a reduction in the amount of salt used can be obtained. In this case,

(c) In the proanthocyanidin concentration step, the solvent used for the crude extraction can be replaced with a highly volatile organic solvent. Substitution with such a highly volatile organic solvent facilitates concentration compared to a crude extract using water as an extraction solvent. Specifically, after contacting the crude extract with water as the extraction solvent and the adsorbent, when desorbing the components adsorbed on the adsorbent, an organic solvent (for example, anhydrous ethanol) is used. Elute all of the adsorbate from the column. This ethanol eluate is easily concentrated by vacuum concentration or the like. Hereinafter, (i) the adsorbent and (ii) the treatment with the adsorbent will be described.

(i) Adsorbent

 The first adsorbent and the second adsorbent used in the (b) polymer PC reduction step and (c) the proanthocyanidin enrichment step behave differently with respect to proanthocyanidin. It is thought to be due to the material of the adsorbent, pore radius, specific surface area, molecular weight fractionation range, etc. Therefore, first, adsorbents generally used for adsorption of proanthocyanidins will be described.

Preferred properties of each of the first adsorbent and the second adsorbent will be described. The individual adsorbents of the first adsorbent and the second adsorbent are not particularly limited as long as they are generally used for column chromatography. Examples of the adsorbent used in the present invention include a synthetic adsorbent, a cation-exchange resin-based adsorbent, an anion-exchange resin-based adsorbent, a crosslinked dextran derivative-based adsorbent, and an adsorbent composed of a polyester resin. Agarose derivative-based adsorbent and cellose derivative-based adsorbent. Here, a synthetic adsorbent is a material that has no functional groups such as ion-exchange groups, is porous and has fine continuous pores (pores). For example, van der Waals Can be absorbed by force. Synthetic adsorbents are further classified according to their materials, and are classified into synthetic adsorbents such as aromatic synthetic adsorbents, substituted aromatic synthetic adsorbents, and ataryl synthetic adsorbents. These synthetic adsorbents have different degrees of hydrophilicity and hydrophobicity depending on the material. From the viewpoint of the stability of the synthetic adsorbent, the adsorption efficiency of proanthocyanidin (OPC), and the separation and fractionation ability, an aromatic synthetic adsorbent is preferred.

 As the aromatic synthetic adsorbent, an adsorbent composed of a porous resin such as a crosslinked styrene resin is preferable. Commercially available adsorbents include, for example, DIAION (registered trademark) HP-10, HP-20, HP-21, HP-30, HP-40, and HP-50 (all manufactured by Mitsubishi Chemical Corporation). ); Amberlight® XAD-4, XAD-16, XAD-1180, and XAD

-2000 (all manufactured by Organo Co., Ltd.); and Sepabeads (registered trademark) SP-825, SP-800, SP-850, and SP-875 (all manufactured by Mitsubishi Chemical Corporation).

 Examples of the substituted aromatic synthetic adsorbent include an adsorbent made of a strongly hydrophobic resin in which a bromine atom or the like is bonded to an aromatic nucleus of an aromatic polymer. Examples of commercially available adsorbents include Sepabeads (registered trademark) SP-205, SP-206, and SP-207 (all manufactured by Mitsubishi Chemical Corporation).

 As the ataryl-based synthetic adsorbent, for example, an adsorbent made of a highly hydrophilic resin having a skeleton of a methacrylic acid ester polymer or the like can be used. Commercially available adsorbents include, for example, Diaion (registered trademark) HP 1MG and HP 2MG (all manufactured by Mitsubishi Chemical Corporation); and Amperlite (registered trademark) X AD-7 (produced by Organo Corporation). Can be

Since the synthetic adsorbent is porous having fine continuous pores (pores) as described above, the target solute present in the solution can be separated by the effect of a molecular sieve. That is, when the synthetic adsorbent is brought into contact with the solution, The porous molecules permeate and diffuse through the pores to the inside of the synthetic adsorbent and are adsorbed. On the other hand, molecules larger than this pore size cannot diffuse into the synthetic adsorbent and are not adsorbed.

 Examples of the cation exchange resin-based adsorbent include resins having a sulfonate group as a functional group, such as Amberlite (registered trademark) CG-4000, CG-5000, CG-6000, CG-8000, IR-1. 16, IR-118, IR-120B, IR-122, IR-124, XT-1007, XT-1009, XT-1002 (all manufactured by Organo Co., Ltd.).

 Examples of anion exchange resin-based adsorbents include OPT I PORE-XUS 402 85.00 and OPT I PORE-XUS 40390.00 (which are weakly basic anion exchange resins having a quaternary amine as a functional group). And Dow Chemical Co., Ltd.).

 Examples of the cross-linked dextran derivative-based adsorbent include SEPHADEX (registered trademark) LH20 and LH60 (all manufactured by Amersham Bioscience Co., Ltd.).

 Examples of the adsorbent composed of a polybutyl-based resin (gel) include Toyopearl HW-40 and 50 (Toyo Soda Kogyo Co., Ltd.).

 Examples of the agarose derivative-based adsorbent include Sepharose CL, 4B and 6B (Amersham Biosciences, Inc.) and Bio-Gel A (Biorad Inc.).

 Cellulose derivative-based adsorbents include Cellguchi Fine CL-90, GCL-300, GCL-1000 (all of which are manufactured by Seikagaku Corporation).

Among the above adsorbents, it is particularly preferable that the adsorbent is porous and has a network-like molecular structure. For example, Diaion HP-20, Amberlite XA Particularly preferred are synthetic adsorbents such as D-4 and crosslinked dextran derivative-based adsorbents such as Sephadex LH20 and LH60.

 Hereinafter, preferable properties of the first adsorbent and the second adsorbent will be described.

 When the first adsorbent and the second adsorbent are selected based on the material, the first adsorbent is preferably a synthetic adsorbent, more preferably an aromatic synthetic adsorbent. As the second adsorbent, a synthetic adsorbent or a crosslinked dextran derivative is preferably selected, and an aromatic synthetic adsorbent or a bridged dextran derivative is particularly preferably selected.

 When selecting based on the pore radius, the fact that the size of the molecule to be adsorbed or the adsorptive power to the molecule differs depending on the pore radius is used.

 The first adsorbent preferably has a pore radius of 90 A or less, more preferably 20 to 90 A, still more preferably 3 to 8 O A (A =

^{1 X 1 0- 1 D m)} . The smaller the pore radius, the higher the adsorption capacity for low molecules (molecular weight of several thousand or less) than for high molecules. Therefore, it is possible to adsorb low-molecular-weight compounds and remove proanthocyanidins without adsorbing pentamers or higher pentamers having relatively high molecular weights. As such adsorbents, for example, aromatic synthetic adsorbents Sepabeads SP-825, Sepabeads SP-850 Amperlite XAD-4 and XAD-200 are preferable. .

 The second adsorbent is preferably 100 A or more, more preferably 100 A to 5

It is preferred to have a pore radius of 100 A, more preferably 100 A to 30 OA. The larger the pore radius is, the more efficiently it absorbs a wide range of molecular weight of anthocyanidin having a molecular weight of several thousand to several tens of thousands.However, since the ability to adsorb impurities is weak, it is not necessary to adsorb the impurities. This can be easily removed. Such adsorbents include, for example, aromatic synthetic adsorbents such as Diaion HP-20, Diaion HP-21, and Amperlite XAD-16, or Is preferably Sephadex LH20 which is a crosslinked dextran derivative-based adsorbent.

When the first adsorbent and the second adsorbent are selected based on the specific surface area, from the viewpoint of the adsorbing power, the first adsorbent and the second adsorbent each have a specific surface area of 50%.

It is preferable that it is above. Further, the first adsorbent preferably has a specific surface area of 70 Om ² ^ or more, because it adsorbs OPC efficiently. The specific surface area of the adsorbent varies depending on the size of the individual adsorbent or, in the case of porous adsorbent, on the size and number of pores. It is possible to choose.

 It is also possible to fractionate by molecular weight using an adsorbent such as a cross-linked dextran derivative or polybier resin (using a gel). The molecular weight fraction range is not particularly limited, but the first adsorbent preferably has a molecular weight fraction range of 100 to 20,000, preferably 100 to 5,000. The second adsorbent preferably has a molecular weight fractionation range of 100 to 20,000, preferably 100 to 10,000. An adsorbent having such a molecular weight fraction range can adsorb proanthocyanidins and remove contaminants. Furthermore, it is possible to elute and separate the OPCs in the adsorbed proanthocyanidins. Among them, SEPHADEX LH-20 and SEPHADEX LH-60 are preferred.

 Among the above various adsorbents, the first adsorbent is porous,

Amberlite XAD-4, which is an adsorbent composed of an aromatic synthetic adsorbent with a pore radius of not more than A and a specific surface area of 700 m ² / g or more, and Sepabeads SP 825 and SP 850 are particularly suitable. is there. (ii) Treatment with adsorbent

The amount of adsorbent depends on the substance to be treated (crude extract or its concentrate, polymer The amount may be appropriately determined depending on the amount of solids contained in the chemical substance or the proanthocyanidin concentrate), the type of solvent (described later), the type of adsorbent, and the like. For example, it is preferable to use 0.1 to 100 parts by mass, preferably 0.1 to 50 parts by mass of the adsorbent with respect to 1 part by mass of the dry mass of the solid content contained in the substance to be treated. . If the amount is less than 0.01 part by mass, the recovery of proanthocyanidin decreases, and if the amount exceeds 100 parts by mass, sufficient adsorption is possible, but the recovery from the adsorbent becomes poor.

 In the first adsorbent, especially when an adsorbent having a high OPc adsorption efficiency is used, the bark before the treatment is measured without measuring the dry mass in the object to be operated more easily. The amount of the adsorbent may be set based on the mass. For example, when using an aromatic synthetic adsorbent such as Diaion HP-20, Sepabeads SP-825, Amberlite XAD-16, etc., 1 part by mass of dry mass of bark before treatment is By using the adsorbent in a dry mass of 0.1 to 10 parts by mass, preferably 0.2 to 5 parts by mass, the object to be treated and the adsorbent are sufficiently contacted, and the adsorbent can be efficiently adsorbed.

 Contact between the object to be treated and the adsorbent may be performed by any method. For example, as a simple method, a column method in which an adsorbent is packed in a column and the substance is passed through the column, or a batch method in which the adsorbent is added to the substance to be treated and the adsorbent is removed after a certain period of time And the like.

To perform treatment using the column method, the solvent of the substance to be treated is replaced with a solvent suitable for contact with the adsorbent, if necessary. This substitution can be performed by a method including a step commonly used by those skilled in the art, such as heat drying, freeze drying, reduced pressure concentrating to dryness, and dialysis. For example, when using an aromatic synthetic adsorbent such as Diaion HP-20, the solvent is replaced with water. When a crosslinked dextran derivative-based adsorbent such as Sephadex LH20 is used, the solvent is replaced with ethanol. Next, for example, a second adsorbent (for example, a synthetic resin-based adsorbent) is packed in the column, and The material to be treated is passed through the column (1), and water, for example, 5 to 10 times the volume of the adsorbent is passed through. As a result, saccharides and organic acids as contaminants are removed. Then, by using an appropriate solvent, proanthocyanidins, particularly proanthocyanidins with a high OPC content, can be eluted. Various conditions in the column method may be appropriately determined depending on the adsorbent to be used.For example, when an adsorbent made of an ion exchange resin is used, the column temperature is set at 10 ° C to 120 ° C. However, it is preferable that the inside of the column is set to a normal pressure or a pressurized state.

 To perform treatment using the patch method, an adsorbent (for example, a synthetic lipophilic adsorbent) having the same mass ratio as the above column method is added to the material to be treated, and the mixture is contacted for 1 to 3 hours with stirring. The adsorbent is recovered by filtration or centrifugation. For example, when the second adsorbent is used as the adsorbent, impurities can be removed by this operation. Next, the second adsorbent to which proanthocyanidin has been adsorbed is further stirred with an appropriate solvent (described later) for 1 to 3 hours to elute proanthocyanidin, and then filtered or centrifuged to separate the supernatant. By recovering the extract, a bark extract containing proanthocyanidin or proanthocyanidin containing more OPC can be obtained.

The elution solvent may be appropriately selected depending on the type of the adsorbent and the type of the substance to be adsorbed or eluted. For example, the elution solvent when the first adsorbent is used includes water, methanol, ethanol, ethyl acetate, chloroform, and a mixed solvent thereof. From the viewpoint of safety, a mixed solvent of water and ethanol is preferably used. For example, when using an aromatic synthetic adsorbent for removing high molecular weight PC such as Amberlite XAD-4, Amberlite XAD-200, Sepabeads SP825, Sepabeads SP850, water The mixing ratio between ethanol and ethanol is a relatively high concentration of 10% by volume or more, preferably 30% by volume or more, and more preferably 50% by volume or more because the polymer PC is removed. Using ethanol aqueous solution to elute adsorbed OPC It is preferable from the viewpoint of increasing the yield.

Solvents that can be used to elute the proanthocyanidin-rich component when using the second adsorbent include, for example, water, methanol, ethanol, ethyl acetate, cro-form, and mixtures thereof. No. Among them, a mixed solvent of water and ethanol is preferably used from the viewpoint of the safety of the skin extract. In the adsorbent treatment step, it is preferable to select a solvent capable of selectively eluting OPC and separating and fractionating from the components adsorbed on the adsorbent. Therefore, it is preferable to use a mixed solvent of water and ethanol (aqueous ethanol solution). For example, when an aromatic synthetic adsorbent such as Diaion HP-20, HP-21, XAD-16 is used, the capacity is 10 volumes. /. ~ 50% by volume, preferably 10% by volume. /. ~ 30 capacity. A / ₀ aqueous ethanol solution is preferred. In the case of a crosslinked dextran derivative-based adsorbent such as Sephadex LH-20 or Sephadex LH-60, an ethanol aqueous solution of 70% by volume or more, preferably 80% by volume or more is preferable. When an ion-exchange resin-based adsorbent is used, water is preferably used as an elution solvent. (A) crude extraction step, (b) polymer PC reduction step, and (c) proanthocyanidin concentration step, or (a) crude extraction step, (c) proanthocyanidin concentration step, and (B) By the high-molecular PC reduction step, a bark extract with an extremely high OPC content is obtained, and the yield is high. In the above bark extract, the amount of high molecular weight proanthocyanidins is reduced, and the fat-soluble component contained in the bark extract is also reduced, so that the solubility of the bark extract in water is further improved and However, the occurrence of cloudiness when the bark extract is mixed with water can be significantly reduced. (Bark extract)

The bark extract purified by the method including the above (a) to (c) is as described above. In addition, OPCs, which are dimers to tetramers, are abundant, and the proportion of OPCs in the total proanthocyanidins contained in the bark extract is 45% by mass or more, preferably 50% by mass or more. Such a percutaneous extract containing proanthocyanidins not only has excellent solubility in water, but also has high physiological activity.

 The bark extract of the present invention preferably further contains phytotechins. As described above, catechins are a general term for polyhydroxyflavan-1-ol. As catechins, (+)-force techin, (1) eppicatechin,

(+)-Gallocatechin, (_) -epigallocatechin, epigallocatechin gallate, eppicatechin gallate and the like. In addition, it includes afzelequin derived from natural products, as well as the (+) 3-gallonole derivative of one-pot techin or gallocatechin. Catechins have carcinogenesis-preventing action, arteriosclerosis-preventing action, fat metabolic disorder-suppressing action, blood pressure elevation-inhibiting action, platelet aggregation-inhibiting action, antiallergic action, antiviral action, antibacterial action, caries prevention action, bad breath prevention action, It is known to have 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. Coexistence of catechins and OPC increases the water solubility of catechins. Therefore, it is considered that absorption of catechins into the body can be facilitated, and the above-mentioned action of catechins is activated. Therefore, a bark extract containing OPC and catechins is particularly useful because it can maintain the high solubility in water and can further exert an effect on the organism of activated catechins.

The content of the catechins is not particularly limited, but is preferably 5 mass with respect to the solid content in the bark extract. / ₀ or more, more preferably 5 to 15% by mass.

The bark extract produced by the above method can be concentrated or diluted by a method commonly used by those skilled in the art and adjusted as appropriate. For example, various methods such as membrane concentration, heat concentration, vacuum (reduced pressure) concentration, and freeze concentration can be used for concentration. Yes. Further, if necessary, the bark extract may be sterilized and stored. Sterilization may be performed by a method commonly used by those skilled in the art, such as air sterilization, high-pressure sterilization, and heat sterilization.

 The bark extract of the present invention has excellent solubility in water. Specifically, the dissolution rate of the bark extract is 90% or more, preferably 95% or more, more preferably 99% or more, still more preferably 99.5% or more, and most preferably 99.7%. That is all. An aqueous solution containing such a bark extract has little precipitation and cloudiness. Therefore, when the bark extract containing proanthocyanin is used in beverages, liquid cosmetics, etc., the appearance of the product is excellent and the absorption of polyphenols in the bark extract, especially proanthocyanidin, into the body is promoted. Therefore, it can exert excellent physiological effects immediately after ingestion, for example, an antioxidant effect, in particular, a superoxide dismutase activity (SOD activity). The antioxidant action, particularly the SOD activity, of the bark extract of the present invention is more persistent than the action of ascorbic acid.

 The bark extract of the present invention may be liquid or dry (powder). The dried bark extract (powder) of the present invention can be obtained by removing the solvent (eg, water, ethanol, etc.) contained in the bark extract. Drying may be performed by a method commonly used by those skilled in the art, and among them, drying by freeze drying, vacuum drying, and air drying is preferred.

The above bark extract (especially dried bark extract) can be used not only for eating and drinking but also for excipients, bulking agents, binders, thickeners, emulsifiers, fragrances, food additives, seasonings, etc. They may be mixed and formed into granules, tablets and the like depending on the application. For example, it may be mixed with royal jelly, vitamins, protein, calcium, chitosan, lecithin, caffeine and the like, and further seasoned with seasonings. Further, capsules such as hard capsules and soft capsules, pills, or tea bags may be used. These are Depending on their shape or preference, they may be eaten as they are, or may be dissolved in water, hot water, milk, etc. for drinking. In the case of tea bags, etc., the ingredients may be leached before drinking. As described above, the bark extract and the dried bark extract of the present invention can be widely used as raw materials for food (drinks), cosmetics, quasi-drugs, and pharmaceuticals.

 Since the bark extract of the present invention contains a large amount of proanthocyanidins, particularly OPC, it has an excellent antioxidant effect. Furthermore, it has high solubility in water and is quickly absorbed into the body. It has excellent effects of improving hyperlipidemia, suppressing blood sugar rise, preventing hypertension, improving blood flow, and improving beauty (beautiful skin). It is thought that the improvement effect is exhibited.

 Note that the present invention is not limited to the above embodiment, and various applications are possible within the scope of the present invention. Example

 Hereinafter, examples of the present invention will be described. The following examples do not limit the present invention. In the following examples, the units (V / V) shown in the examples represent (capacity Z capacity), (w / w) represents (mass / mass), and (vzw) represents (capacity / mass). (Example 1: Production of bark extract)

25 kg of pine bark (Pine bark of the French coast) with 1 kg of 5.4 L of 80 νΖ ^ ν% aqueous solution of ethanol, crushed by a Waring Blender. The mixture was heated and extracted under reflux at 24 ° C for 24 hours. Then, the mixture was immediately filtered, and the insoluble material after the filtration was washed with 1.6 V of an 80 V / W% aqueous ethanol solution. The filtrate and the washing solution were combined to obtain 7 L of a crude extract A. The crude extract (100 mL) was freeze-dried to obtain 700 mg of a dry powder. From this value, The amount of dry powder in 7 L was determined to be 49 g.

 Next, in order to examine the content of each component contained in this powder, the pine bark crude extract A was fractionated into the following fractions. Figure 1 shows the fractionation process of pine bark crude extract A.

 First, 25 mL of Sefadex LH-20 (manufactured by Amersham Biotech) swollen with water was packed in a 15 × 30 Omm column, and washed with 50 mL of ethanol. Next, 100 mg of the dry powder of the crude extract A was dissolved in 2 mL of ethanol, and the solution was passed through a column to be adsorbed. After that, gradient elution was carried out with a mixed solvent of 100-80% (V / V) ethanol / water, and 10 mL each was collected.

 At the time of fractionation, the presence or absence of OPC in each fraction was detected by silica gel thin-layer chromatography (TLC), using a dimer to tetramer OPC sample as an index. Then, it was separated into an OPC-free fraction (fraction 1) and an OPC-containing fraction (fraction 2) that did not contain OPC and flowed before OPC.

In the above TLC, proanthocyanin B—2 (R f value: 0.6) was used as a sample of dimeric OPC, and proanthocyanidin C—1 was used as a sample of trimeric OPC. (R f value: 0.4), and cinnam tannin A ₂ (R f value: 0.2) was used as a sample of tetramer OPC. TLC was performed under the following conditions.

 (TLC conditions)

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

 • Developing solvent: benzene / ethyl formate Z formic acid (2/7/1)

 • Detection reagent: sulfuric acid

 • Sample volume: 10 L each

Next, when OPC is no longer detected, 300 mL of a 50% (V / V) water-acetone mixed solvent is passed through, and the remaining adsorbed on the column (resin) is removed. Of the adsorbed product was eluted and fractionated in 10 mL portions. This was designated as OPC-free fraction (resin adsorbed fraction: fraction 3).

 This fraction 3 was further subjected to TLC to separate it into a high molecular weight PC fraction (fraction 3a) and another fraction (fraction 3b). The TLC development conditions and detection method were performed in the same manner as above, and the fraction that developed color with an R f value smaller than the tetramer sample (R f value: 0.2) was converted to the polymer PC fraction (fraction 3a). ), And the non-colored fraction was designated as the other fraction (fraction 3b).

 Next, for the OPC-free fraction (fraction 1), which bleeds ahead of OPC and does not contain OPC, the phytotechin fraction (fraction la) and other It was separated into fractions (fraction lb). First, fraction 1 was freeze-dried to obtain a powder. This powder was dissolved in 3 mL of water and passed through a column (15 x 300 mm) packed with 20 mL of MC I gel (manufactured by Mitsubishi Chemical Corporation) swelled with water to separate the components. Adsorbed. Next, the column was washed with water, and then eluted with a mixed solvent of 10% to 100% (V / V) ethanol / aqueous solution, and fractionated in 7 mL portions. After completion of the elution, catechins in each fraction were detected by TLC using catechin as an index, and the fraction was divided into a motive force fraction (fraction la) and other fractions (fraction 1b).

 Fractions 1 to 3 obtained as described above were each powdered by freeze-drying, and the dry mass was measured. The sum of fractions 1 to 3 was 99.9 mg per 10 Omg of the dry powder of the bark extract, indicating that almost the entire amount was recovered. Furthermore, the content of each component contained in the dry powder of the bark extract was calculated from the dry mass of each fraction. The results are shown in Table 1.

(Example 2)

Same as Example 1 except that the bark of a 50-year-old tree (New Zealand pine) was used instead of the 25-year-old pine bark (Pine bark of the French coast). Thus, a dry powder of the crude extract (crude extract B) was obtained. The content of each component contained in this powder was measured in the same manner as in Example 1, and the content was determined. The results are shown in Table 1. ·

(Example 3)

 Crude extraction was performed in the same manner as in Example 1, except that the bark of an 80-year-old tree (North-Ezo pine) was used instead of the 25-year-old pine bark (French coastal pine bark). A liquid (crude extract C) was obtained. The content of each component contained in the powder was measured in the same manner as in Example 1, and the content was determined. The results are shown in Table 1.

(Comparative Example 1)

 The same procedure as in Example 1 was carried out except that the bark of a tree over 100 years old (Pine pine) was used instead of the bark of a 25-year-old pine tree (Pine bark of the French coast). Thus, a crude extract (crude extract D) was obtained. The content of each component contained in this powder was measured in the same manner as in Example 1, and the content was determined. The results are shown in Table 1. table 1

PC… Pro-Antian From the results in Table 1, the crude extract obtained from the bark of the 25-year-old, 50-year-old, 80-year-old trees of Examples 1 to 3 It can be seen that the OPC content is higher than the crude extract obtained from the skin. This indicates that the crude extract obtained from the bark of a tree less than 100 years old has a higher OPC content than the extract obtained from the bark of a tree older than 100 years. In particular, the crude extract obtained from the bark of the 25-year-old and 50-year-old trees of Examples 1 and 2 shows the proportion of OPC in the proanthocyanidins contained in the crude extract (OPCZ total Anthocyanin) was 10% by mass or more, and had a high OPC content. In addition, a large difference was found in the amount of extracted solid substances. That is, when bark of a tree less than 100 years old is used, the bark extract has a solid concentration of about 1.5 to 2 times higher than that of a bark of a tree older than 100 years. It can be seen that the proanthocyanidin content, especially the OPC content, was high. (Example 4)

 1 L of crude extract A obtained in Example 1 was concentrated under reduced pressure to completely remove ethanol. Thereafter, purified water was added to adjust the volume to 5 OmL to obtain a concentrate A corresponding to 1/20 volume of the extract. To 5 OmL of the concentrated solution A1, sodium chloride (about 3 g) was added and stirred well. After the solution was allowed to stand at 4 ° C. for 24 hours, the precipitated insolubles were removed by filtration to obtain 51 mL of filtrate A.

Next, the filtrate A1 was passed through a 30 X 300 mm column filled with 100 mL of an aromatic synthetic resin swollen with water (Diaion HP-20: manufactured by Mitsubishi Chemical Corporation). The proanthocyanidin in 1 was adsorbed on the column. This column was washed with 1 L of purified water to remove saccharides, organic acids, and the like remaining on the column. Then, apply 20% (VV) ethanol / water mixed solvent to the column. Then, proanthocyanidins were eluted to obtain 20 O rn L of pine bark extract A1. Next, the pine bark extract A was freeze-dried to obtain a dry powder (pine bark extract A). In the same manner as in Example 1, the content of each component contained in the pine bark extract A was measured, and the content was determined. Table 2 shows the results.

(Example 5)

 A dry powder (pine bark extract B) of pine bark extract B was obtained in the same manner as in Example 4 except that crude extract B of Example 2 was used instead of crude extract A. The content of each component contained in was measured and the content was determined. Table 2 shows the results.

(Example 6)

 A dry powder (pine bark extract C) of pine bark extract C was obtained in the same manner as in Example 4, except that crude extract C of Example 3 was used instead of crude extract A. The content of each component contained in was measured and the content was determined. Table 2 shows the results.

(Comparative Example 2)

A dry powder (pine bark extract D) of pine bark extract D was obtained in the same manner as in Example 4, except that crude extract D of Comparative Example 1 was used instead of crude extract A. The content of each component contained in was measured and the content was determined. Table 2 shows the results.

Table 2

From the results in Table 2, the crude extract obtained from the bark of the trees of Examples 1 to 3 at the ages of 25 years, 50 years, and 80 years was the same as that of the bark of the tree of Comparative Example 1 over the age of 100 years. It can be seen that the OPC content is higher than that of the obtained extract. It can be seen that the yield (solid content) of the extracts of Examples 4 to 6 obtained by further purification is high. This means that extracts obtained from the bark of trees less than 100 years old have a higher OPC content than extracts obtained from the bark of trees older than 100 years, but have higher recovery rates. Indicates that In particular, the extract obtained from the bark of the 25-year-old and 50-year-old trees of Examples 1 and 2 showed that the proportion of OPC in the proanthocyanidin contained in the extract (OPC, total proanthocyanidin) was 50%. At over 50%, OPC accounted for more than half of proanthocyanidins, and yields also increased significantly.

(Example 7: Production of bark extract)

 Pine bark (French coastal pine bark, less than 100 years old) 1 kg, purified water 5

Add 4L, crush with Waring Blender and add 10L The mixture was heated and extracted while refluxing at 0 ° C for 24 hours. Then, the mixture was immediately filtered, and the insoluble matter after the filtration was washed with 1.6 L of purified water, and the filtrate and the washing liquid were combined to obtain a 7 L crude extract. When 1 OmL of the crude extract was freeze-dried, the dry mass was 70 mg. The extract was allowed to cool to 25 ° C.

Add 1 L of the above extract (7 g dry weight of extract powder) to specific surface area y O

An aromatic synthetic adsorbent with a pore radius of 8 OA or less (Amperlite (registered trademark) X AD-4 (manufactured by Organo Corporation)) packed in a column of 5 cm in diameter packed with 30 OmL (equivalent to about 200 g) The liquid was passed. Next, the column was washed with 60 OmL of purified water, and 80 OmL of an 80% (V / V) aqueous ethanol solution was passed through the column to elute the adsorbed material. The components adsorbed on the adsorbent were eluted by passing the ethanol through. The eluate was concentrated under reduced pressure to remove ethanol, and water was added to adjust the volume to 50 OmL.

Next, the eluate 5 0 OML, further specific surface area 6 0 0 m ² Zg, DIAION (registered trademark) is aromatic synthetic adsorbents Hosoanahan diameter 1 00~1 2 OA HP-20 (Mitsubishi The solution was passed through a 5 cm diameter column packed with 20 OmL (equivalent to about 140 g). The column was washed with 60 OmL of purified water, and then eluted by passing a 20% (V / V) aqueous ethanol solution (70 OmL) to obtain pine husk extract E.

 Next, in order to examine the content of each component in the obtained pine bark extract E, pine bark extract E was separated in the same manner as in Example 1.

As in Example 1, the total of fractions 1 to 3 was 99.9 mg with respect to 10 Omg of the dry powder of the bark extract. Therefore, it was found that almost the entire amount had been recovered. When the content of each component was calculated from the mass of each component of the bark extract, the OPC fraction (fraction 2) was 45.2%, and the high-molecular PC fraction (fraction 3a) was 21%. The fraction of catechins (fraction 1a) was 14.9% and that of the other fractions (fraction lb + fraction 3b) was 18.7%. The OPC ratio (image The fraction 2 / fraction 3 a) was 2.13.

(Example 8: Production of bark extract)

 Pine bark (under 100 years old) To 900 g, 80 (V / V)% ethanol aqueous solution 7.2, after crushing it with a Waring Blender, 70. The mixture was extracted with heating while refluxing with C for 1 hour. Then, the mixture was immediately filtered, and the insoluble material after the filtration was washed with 1.8 L of an 80 (V_V)% ethanol aqueous solution. The combined filtrate and washings were combined with 9 L of pine bark aqueous ethanol-free crude extract F. Got. One liter of this ethanol extract (dry mass of extract powder 1 Og) was allowed to cool to 25 ° C and concentrated to completely remove ethanol. Thereafter, pine bark extract F of 20 OmL was obtained in the same manner as in Example 4. Next, the pine peel extract F was freeze-dried to obtain a dry powder. This was used as a dry powder of pine bark extract F. When the content of each component in the pine bark extract F was measured in the same manner as in Example 7, OPC was 43.2%, polymer PC was 36.4%, and other components were 20. 4%. The OPC ratio (fraction 2Z fraction 3a) was 1.19.

(Example 9: Production of bark extract)

 In the same manner as in Example 8, a crude extract G of hydrated ethanol containing pine bark (tree age less than 100 years) was obtained and further concentrated to completely remove ethanol. Thereafter, purified water was added to adjust the volume to 10 OmL to obtain a concentrate B2 corresponding to 1 to 10 volumes of the extract.

 Next, sodium chloride (15 g of yarn) was added to the concentrated solution G of 1 O OmL, and the mixture was stirred well. After the solution was allowed to stand at 4 ° C for 24 hours, the precipitated insolubles were removed by filtration to obtain 96 mL of filtrate G.

Subsequent operations were performed in the same manner as in Example 8 to obtain pine bark extract G, which was dried. The dried powder of the pine bark extract G was obtained by drying. The content of each component in the pine bark extract G was determined in the same manner as in Example 7, with 0% being 39.3%, polymer PC being 39.9%, and other components being 20. 8%. The OPC ratio (fraction 2 fraction 3a) was 0.98.

(Comparative Example 3: Component analysis of commercially available pine bark extract)

 The content of each component in the extract of a commercially available pine bark extract powder (pine bark extract H) was measured in the same manner as in Example 7 to find that OPC was 19.3% and polymer PC 37.1%, the catechin fraction was 10.8%, and the other components were 32.8%. In addition, OP Ci: dani (fraction 2 fraction 3a) was 0.52.

(Comparative Example 4: Component analysis of commercially available pine peel extract)

 The content of each component in the extract of a commercially available pine bark extract powder (pine bark extract I) was measured in the same manner as in Example 7, and found that OPC was 15.9% and polymer PC was 39.6%, catechin fraction was 12.9%, and other components were 31.6%. In addition, OPCJ: dani (fraction 2 fraction 3a) was 0.40. (Example 10: solubility)

 Dry powders of the pine bark extracts obtained in Examples 7 to 9 (bark extracts E, F, and G), and commercially available pine bark extracts of Comparative Examples 3 and 4 (4 bark extracts H and I) ) Was used to conduct a water solubility test.

Each pine bark extract was suspended in distilled water so as to be 1% by mass and stirred well with Vortex. Then, it was left still at room temperature (about 25 ° C). After 60 minutes of standing, the precipitate and turbidity in the solution were visually checked and evaluated according to the following criteria. It was. Table 3 shows the results.

 Hidden)

 +: A large amount of precipitation is observed.

 Soil: Some precipitation is observed.

 1: No precipitation is observed.

 (Turbidity)

 +: The cloudiness is remarkable.

 Shi: Some clouding is observed.

 1: No clouding is observed.

 Further, after the visual confirmation, the entire amount of each of the aqueous solutions was filtered using a glass fiber filter paper. The dry mass of the obtained filter cake was measured, and the dissolution rate was determined from the above formula 1. The results are shown in Table 3. Table 3

From the results in Table 3, it can be seen that the pine bark extracts (E, F, and G) of Examples 7 to 9 did not show any visible precipitation and no cloudiness. one On the other hand, with respect to the commercially available pine bark extracts (H and I) of Comparative Examples 3 and 4, a slight amount of precipitation was observed, and it was cloudy. Further, the dissolution rates of the pine bark extracts (E, F, and G) of Examples 7 to 9 were all 99.7%, whereas the commercially available pine bark extracts of Comparative Examples 3 and 4 were all soluble. The dissolution rates of the products (H and I) were 85.7% and 89.7%. These results indicate that the pine bark extracts (E, F, and G) of Examples 7 to 9 are more soluble in water than the commercially available pine bark extracts of Comparative Examples 3 and 4 (Η and I). Is higher.

 As described above, the pine bark extracts of Examples 7 to 9 did not show sedimentation and cloudiness, and had high dissolution rates. This means that the OPC content of the bark extract is at least 25% by mass, preferably at least 30% by mass, more preferably at least 35% by mass, even more preferably at least 40% by mass, even more preferably at least 45% by mass. It shows that the solubility of the bark extract in water is dramatically increased when it is contained as described above. If the OPC ratio is 0.9 or more, it indicates that the solubility in water is increased.

 On the other hand, the powders H and I of the commercially available pine bark extracts of Comparative Examples 3 and 4 showed slight precipitation, became cloudy, and had a low dissolution rate. This indicates that when the content of OPC in the bark extract is small, the solubility of the bark extract in water is small. Also, when the polymer proanthocyanidin content in the bark extract is considerably larger than the OPC content, the solubility of the bark extract in water tends to decrease.

(Example 11: Antioxidant activity (SOD activity))

 (1) Preparation of test rats

After purchasing a 5-week-old male SD rat, it was pre-bred until the age of 6 weeks. During the pre-breeding period, rats were observed and rats showing abnormalities such as abnormal weight gain were removed. During the preliminary breeding period, rat solid feed MF (Orient Tal Yeast Co., Ltd.) and tap water were allowed to freely ingest.

 The 6-week-old rat was fasted for 12 hours, and immediately thereafter, blood was collected from the orbital vein of the rat. This blood was centrifuged at 3000 rpm (1700 XG) for 20 minutes to obtain serum. Then, the serum SOD activity was measured using a kit (SOD Test Co., Ltd. (Code 435-70601): Wako Pure Chemical Industries, Ltd.) for NB.

It was measured by the T reduction method. After the measurement, the animals were divided into 5 animals per group so that the average value of the SOD activities was uniform.

(2) Preparation of test solution

 The dried powder (pine bark extract G) of the pine bark extract G obtained in Example 9 was dissolved in distilled water so that the final concentration was 1 Omg / mL (referred to as test solution 1). Instead of the pine bark extract G, any of the pine bark extract of Comparative Example 3 (pine bark extract H), commercially available (I) catechin, commercially available vitamin C (ascorbic acid), and commercially available grape seed polyphenol Test solutions were prepared in the same manner as above, except that was used (test solutions 2 to 5 respectively). As a control, distilled water was used as test solution 6.

(3) Measurement of SOD activity

 Each test solution (containing 1 OmgZmL of each component) was orally administered to the above-mentioned five rats per group using a sonde so that the ratio of each component was 10 mkg body weight (10 OmgZkg body weight as a component). After oral administration, rats were bred in an environment where tap water was freely available, and blood was collected again from the orbital vein 45 minutes and 90 minutes after oral administration, and SOD activity was measured in the same manner as described above. Then, when the measured value of the SOD activity before administration was set to 1, the relative value of the SOD activity after administration was determined. The result is shown in figure 2:

From the results in Fig. 2, pine bark extract G containing at least 25% by mass of OPC is included Test solution 1 was composed of a test solution containing pine husk extract H containing 19.3% OPC and a test solution 2 containing other antioxidants, or a test solution containing other antioxidants. It can be seen that there is a tendency for

In particular, it can be seen that the SOD activity 45 minutes after administration of Test Solutions 1 and 4 was higher than the SOD activity 45 minutes after administration of Test Solutions 2, 3, and 5. This means that pine bark extract G (test solution 1) and ascorbic acid (test solution 4) containing at least 25% by mass of OPC S, OPC 19.3. / ₀ containing that pine bark extract H (test solution 2), catechin (test solution 3), compared to Pudou seeds polyphenylene Nord (test solution 5), are absorbed rapidly in vivo, to have a high SOD activity Show.

 Furthermore, in FIG. 2, the increase (slope) of the SOD activity from 45 minutes to 90 minutes after administration of pine bark extract G (test solution 1) was due to the difference between 45 minutes and 90 minutes after administration of ascorbic acid (test solution 4). It can be seen that it is higher than the increase in SOD activity after one minute. This indicates that pine bark extract G containing 25% by mass or more of OPC is superior in persistence of SOD activity as compared with ascorbic acid. Ascorbic acid had a low sustained activity despite a rapid increase in SOD activity. Ascorbic acid had poor persistence of SOD activity as compared with pine peel extract H containing 19.3% OPC (test solution 2).

 Finally, the SOD activity was highest at 90 minutes after administration of Test Solution 1. This is considered to be because the pine bark extract G containing 25% by mass or more of OPC contained in the test solution 1 has high solubility in water and is quickly absorbed into the living body. Thus, it can be seen that the bark extract of the present invention tends to exhibit an excellent antioxidant action, particularly an SOD activity, relatively quickly and in vivo compared to other antioxidants.

 From the above, pine bark extract G containing 25% by mass or more of OPC

It has both the immediate effect of rapidly increasing OD activity and excellent sustainability You can see that. This effect is an effect not seen in the conventional antioxidant. Thus, it can be seen that pine husk extract G is highly water-soluble and can be used as an easily-absorbable antioxidant (easy-absorbable SOD activator) that is easily absorbed into a living body. Industrial Available Individuals

 The bark extract of the present invention can be obtained by extracting from the bark of a tree with an age of less than 0 years L0 with at least one of water and a polar solvent. A bark extract containing proanthocyanin can be obtained in a higher yield than bark over 100 years of age, and the proportion of OPC in the total proanthocyanin is high. This skin extract has excellent solubility in water, so there is little turbidity and precipitation in aqueous solution, it is quickly absorbed into the body, and it has excellent antioxidation activity (particularly superoxide dismutase activity). ). Therefore, the bark extract can be easily used in beverages, liquid cosmetics, quasi-drugs, pharmaceuticals, foods, beverages, and the like.

Claims

The scope of the claims

1. A bark extract obtained by extracting the bark of a tree less than 100 years old with at least one solvent selected from the group consisting of water, a polar solvent and a mixed solvent thereof.

2. The bark extract according to claim 1, wherein the tree is a tree of 20 to 80 years of age.

3. A method for producing a bark extract, comprising the step of extracting from the bark of a tree less than 100 years old with at least one solvent selected from the group consisting of water, a polar solvent and a mixed solvent thereof.

1/2

Figure 1

Pine bark crude extract A

OPC not · | ί fraction Fraction containing OPC opc N Γ fraction

 h) (Fraction 2) (Fraction of resin absorption)

 (Drawings, 3)

Force fraction Other fraction High polymer PC fraction Other fraction

(Fraction 1a) (fraction lb) (fraction 3a) (fraction 3b)

2/2

 20 40 60 80 100

 Hours (minutes)

 Test liquid 1 (pine bark G, OPC39.3 mass%) Test liquid 2 (pine bark! ■), OPC19.3 mass W

H ~ Test solution 3 (catechin) ❖ --Test solution 4 (Vitamin C)

 D-Test solution 5 (Podo seed refill) Test solution 6 (Distilled water)