WO2010092941A1

WO2010092941A1 - Composition having vasodilation activity, process for producing same, and use of same

Info

Publication number: WO2010092941A1
Application number: PCT/JP2010/051851
Authority: WO
Inventors: 平山　匡男; 賀代子中山; 悠介渡辺; 彩子明田; 忠幸横山; 充南場; 亮古内
Original assignee: 株式会社ブルボン
Priority date: 2009-02-16
Filing date: 2010-02-09
Publication date: 2010-08-19
Also published as: JPWO2010092941A1

Abstract

Disclosed are: a composition which has a vasodilation activity and therefore has an alleviation and regression activity on various conditions associated with blood circulation, such as an alleviation activity on blood circulation, an anti-hypertensive activity, and an alleviation or regression activity on shoulder stiffness, head ache, excessive sensitivity to cold and cerebral circulation; a process for producing the composition; and a food, a beverage or a pharmaceutical preparation each having the above-mentioned functions. Specifically disclosed are: a composition having a synergistically improved vasodilation activity, which comprises a fused tannin oligomer component and an organic acid component; a process for producing the composition; and a food, a beverage or a pharmaceutical preparation each having the above-mentioned function.

Description

Composition having vasodilatory effect, production method and use

The present invention relates to a composition having a vasodilating action and a composition for alleviating or alleviating a disease ameliorated by vasodilation, more specifically, at least one condensed tannin oligomer component of dimer to 13mer, and at least one The present invention relates to a composition, a production method and a use for synergistically improving a vasodilatory action comprising an organic acid component as an active ingredient.

In recent years, with the diversification and aging of the living environment, the risk of lifestyle-related diseases has increased. For prevention and risk reduction, it has become clear that eating a food with bioregulatory functions is important, and research and development of functional food ingredients has been energetically conducted. ing. For example, there is a circulatory function as an important function to maintain a healthy body. By taking foods that maintain and improve the circulatory function on a daily basis, various symptoms associated with the deterioration of blood circulation, that is, only blood flow It is thought that it will be possible to improve blood pressure, stiff shoulders, headache, coldness, cerebral circulation, and the like. Therefore, the search and evaluation of food ingredients that improve blood flow are one target for functional food development. Vegetables and fruits we eat on a daily basis contain a wide variety of polyphenols, and research on their health functions has been rapidly expanding in recent years. Among the polyphenols, flavonoid components such as soybean isoflavone, tea catechin, onion quercetin, and berry anthocyanins, which are relatively easy to prepare, have been scientifically clarified to have their respective healthy functions. It has been used as a useful food ingredient in health foods and supplements. On the other hand, among polyphenols, a condensed tannin oligomer component having a particularly complicated molecular structure has been left as an unexamined area because it has been difficult to purify and produce the conventional technology. Recently, however, research has been advanced to extract condensed tannin oligomer components by applying the latest advanced technology and to elucidate their health functions.

Condensed tannin is a structural unit of catechin, epicatechin, gallocatechin, epigallocatechin and their 3-O-gallate well known as green tea catechins, ie, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate (Monomer) is a general term for a group of polymerized substances. Depending on the degree of polymerization, a low polymerization component (approximately from a dimer to a 20-mer) may be referred to as a condensed tannin oligomer. Further, when condensed tannins are classified according to their structural units, those having catechin, epicatechin and / or their gallate as the structural unit as procyanidins, and at least one of gallocatechin, epigallocatechin and / or their gallate as the structural unit. There are many reports that prodelphinidin is added. For example, condensed tannin obtained from cacao has only epicatechin as a constituent unit and is classified as procyanidin. On the other hand, the condensed tannin contained in persimmon fruit has epigallocatechin and epicatechin gallate added to epicatechin and epicatechin gallate which are constituent units of procyanidins, and belongs to prodelphinidin.

Among the condensed tannin oligomers, the procyanidin oligomer component has a relatively simple molecular structure because its constituent units are limited to catechin, epicatechin and / or gallate thereof, and plant varieties with a high content have been found. As a result, there are several research examples. Therefore, it can be said that the research on the condensed tannin oligomer started from the procyanidin oligomer component. For example, research on procyanidin oligomer components contained in apples, grape seeds, cacao beans, pine bark and the like is ongoing, and their health functions include antioxidant activity and therapeutic agents for autoimmune diseases (Patent Document 1). : WO 2005/030200), blood flow improving action (Patent Document 2: Patent Publication 2003-128560 and Patent Document 3: Patent Publication 2003-530410, etc.) have been reported.

Prodelphinidin oligomer components in which gallocatechin, epigallocatechin and / or their gallates are added to the constituent units in addition to catechin and epicatechin are found to be contained in strawberry fruits and sea buckthorn. ing. Prodelphinidin oligomer components, which have a more complicated structure than procyanidin oligomer components, have not yet been found in a way to extract and purify them in large quantities, and there are still very few scientific studies on their health functions. There have been reports of ingesting strawberry fruits containing a prodelphinidin oligomer component, but experiments using freeze-dried products of the entire strawberry fruits without extracting the prodelphinidin component have been conducted (Non-Patent Document 1: Hibino). G et al., Reguration of the peripheral body temperature by foods: a temperature decrease induced by the Japanese persimmon (kaki, Diospyros kaki) Biosci. Biotechnol. Biochem., 67, 23-28 (2003)). In addition, the results show an action of increasing blood pressure and suppressing blood flow, suggesting a vasoconstrictive action opposite to the vasodilatory action.

This result is the result when the whole persimmon fruit was ingested, and it was not clear whether it was the action of prodelphinidin in the persimmon fruit or the other ingredients. Therefore, in order to evaluate the effect of true prodelphinidin oligomer components on blood vessels, it was necessary to obtain and evaluate a purified sample, but no efficient extraction method or purification method has been found. . As described above, the evaluation of the vasodilatory effect of the prodelphinidin oligomer component among the condensed tannins has not yet been clarified.

In this way, the vasodilatory effect of condensed tannin oligomer components is limited to procyanidin oligomer components contained in apples, grape seeds, cacao beans, pine bark, etc., and new active ingredients including prodelphinidin oligomers It has been desired to develop a component having a better action by searching and evaluating. Furthermore, since the condensed tannin oligomer components of the procyanidin oligomer and the prodelphinidin oligomer have a low content in the plant, there is a disadvantage that much labor is required for the extraction and production. Accordingly, it has been desired to search for an enhancing component that efficiently exerts the vasodilatory effect with a smaller amount of use, and to develop a composition and a production method thereof.

WO2005 / 030200 JP 2003-128560 A Japanese Patent Publication No. 2003-530410

The object of the present invention is to find a new composition that synergistically improves the excellent health function exhibited by the condensed tannin oligomer component, in particular, the vasodilatory effect, and to provide the composition, production method, and use thereof.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the vasodilatory action of procyanidin oligomer components, which has been conventionally known, is at least of dimers to 13-mers including prodelphinidin oligomer components. This is an action commonly exhibited by one condensed tannin oligomer component, and more surprisingly, a composition in which at least one organic acid component coexists with at least one condensed tannin oligomer component of dimer to 13-mer. As a result, the inventors have found a synergistic effect that dramatically improves the vasodilatory effect, thereby completing the present invention. More specifically, the present invention has the following features.
1. A vasodilator comprising as an active ingredient at least one condensed tannin oligomer component of dimer to 13-mer derived from berry fruit or cacao bean or persimmon fruit or persimmon leaf, and at least one organic acid component, The vasodilator described above, wherein the condensed tannin oligomer component is a procyanidin oligomer and / or a prodelphinidin oligomer having at least one of catechin, epicatechin, gallocatechin, epigallocatechin and / or their gallate as a constituent unit.
2. The vasodilator according to claim 1, wherein the berry fruit is Boysenberry, Natsuhaze, Cassis, Blueberry, Cranberry, Strawberry or Grape.
3. The vasodilator according to claim 1 or 2, wherein the organic acid component is citric acid, succinic acid, malic acid, acetic acid, phytic acid and / or lactic acid or a salt thereof.
4). The vasodilator according to claim 3, wherein the organic acid component is citric acid or a salt thereof.
5. The vasodilator according to any one of claims 1 to 4, which has a blood flow improving action and / or a blood pressure rise inhibiting action.
6). A pharmaceutical product or food comprising the vasodilator according to any one of claims 1 to 5.
7). A berry fruit or cacao bean or persimmon fruit or persimmon leaf containing at least one condensed tannin oligomer component and a sugar component, or an organic acid component is produced or increased by microbial fermentation of an extract thereof. The method for producing a vasodilator according to claim 1.
8). The production method according to claim 7, wherein the berry fruit or an extract thereof is Boysenberry, Natsuhaze, Cassis, Blueberry, Cranberry, Strawberry or Grape.
9. The production method according to

claim

7 or 8, wherein the organic acid component is citric acid, succinic acid, malic acid, acetic acid, phytic acid and / or lactic acid or a salt thereof.
10. The production method according to claim 9, wherein the organic acid component is citric acid or a salt thereof.

A composition comprising at least one condensed tannin oligomer component of dimer to 13-mer derived from berry fruit or cacao bean or persimmon fruit or persimmon leaf and at least one organic acid component of the present invention, It has an excellent vasodilatory action due to its synergistic action.

A chromatogram obtained by analyzing a reaction product obtained by thiol decomposition of a bamboo leaf extract by high performance liquid chromatography (ODS column, 280 nm UV detector). This indicates that the koji leaf extract is procyanidin composed of catechin (CA), epicatechin (EC), epicatechin gallate (ECg), epigallocatechin (EGC) and epigallocatechin gallate (EGCg) . The figure which shows the composition of the structural unit corresponding to the chromatogram of the polymerization degree distribution obtained by carrying out the MALD-TOF-MS analysis of a bamboo leaf extract, and a measured polymerization degree peak. From this chromatogram, by analyzing the combination of epicatechin or catechin unit (EC), epigallocatechin or gallocatechin unit (EGC), and gallate (gal), the degree of polymerization of condensed tannin and the constituent unit ratio can be calculated. . The numbers indicate the number of the existing units, and the parentheses indicate the degree of polymerization. Grape seed extract (4-8-linked procyanidin dimer structural isomers (molecular weight 577/289, B1, B2, B3, B4) analyzed by high performance liquid chromatography / mass spectrometry (LC / MS) ( 3a), Boysenberry extract (3b), cacao bean extract (3c), bamboo leaf extract (3d). From this chromatogram, it can be seen that 4-8-linked procyanidin dimer structural isomers are included depending on the plant species. The chromatogram which quantifies the condensed tannin oligomer derived from a berry fruit or cacao bean or a persimmon fruit or a persimmon leaf. Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr) (4a), Boysenberry fruit condensed tannin oligomer extract-2 (BBF-XAD-LH20 fr) (4b), and epicatechin standard ( 4c) is illustrated. Boysenberry fruit condensed tannin oligomer extract-2 (BBP-XAD-LH20 fr, 1μg / mL) alone (5a) and its blood expansion effect of citric acid (100 μg / mL) composition (5b) . When BBP-XAD-LH20 fr alone was added (5a arrow), the vascular dilation indicated by the change in the broken line was hardly observed, and after addition of the citric acid (100 μg / mL) composition (5b arrow) It is a graph in which vasodilation is recognized as a large change in. The measurement graph which shows the blood expansion effect of persimmon fruit condensed tannin oligomer extract (PAF-XAD / EtOH fr, 1 μg / mL) alone (6a) and its citric acid (100 μg / mL) composition (6b). Compared to the vasodilation after the addition of PAF-XAD / EtOH） fr alone (arrow 6a) (changes within the dashed line 6a), larger blood vessels than when the citric acid (100 µg / mL) composition was added (arrow 6b) It is a graph by which expansion (change in a 6b broken line) is recognized. The graph which shows the blood flow change of the tail vein of the mouse | mouth from the 15th to 70 minutes after sample administration by the laser Doppler blood flow measuring device. It is a graph which shows that the blood flow of the citric acid composition is synergistically improved compared with BBF-XAD / EtOH fr alone intake. The graph which shows the time-dependent change of the blood pressure of the SHR rat which administered the composition (solid line) and distilled water (dashed line) of BBP * XAD / EtOH fr, and a citric acid. The blood pressure of rats administered with the composition of BBP XAD / EtOH fr and citric acid decreased after administration, particularly significantly after 1 hour and 6 hours compared to the control group. This result indicates that the composition of BBP XAD / EtOH fr and citric acid has a blood pressure lowering effect.

The vasodilatory action in the present invention has an action of dilating blood vessels, and various symptoms related to vascular circulation function, improvement of blood flow, reduction of blood pressure increase, shoulder stiffness, headache, coldness, cerebral circulation, etc. Refers to action.
At least one condensed tannin oligomer component of dimer to 13-mer derived from berry fruit or cacao bean or persimmon fruit or persimmon leaves is at least one of catechin, epicatechin, gallocatechin, epigallocatechin and / or gallate thereof. It is a procyanidin oligomer and / or prodelphinidin oligomer having one structural unit. Adjacent structural units are bonded at the 4th and 8th positions, and the steric structure at the 4th position is not particularly limited, and can be arranged in both 4α- or 4β-. Taking the procyanidin dimer as the simplest example, the structural components of catechin (CA) and epicatechin (EC) are CA8-4βEC (generic name procyanidin B1), EC8-4βEC (generic name). There are four types of procyanidins B2), CA8-4αCA (generic name procyanidin B3), and EC8-4αCA (generic name procyanidin B4). Further, considering the gallated structure, there are one (mono-) and two (di-) for each of the four types of non-gallate structural components, so eight types are added. Therefore, 12 possible structures exist only with the structural component of procyanidin dimer having 4-8 bonds. Thus, the component constituting the procyanidin oligomer and / or prodelphinidin oligomer having at least one of catechin, epicatechin, gallocatechin, epigallocatechin and / or their gallate as a structural unit is a structural unit and its sequence order, Many components exist depending on the degree of polymerization, the steric structure of adjacent bonds, and the ratio of gallation.

As the condensed tannin oligomer component which is an active ingredient of the present invention, extracts and concentrates such as fruits, leaves and bark of plants containing them can be used. The plant species is not particularly limited, but fruits and leaf extracts having food experience can be preferably used for pharmaceuticals and foods and drinks. For example, berry fruits and cacao beans (Theobroma cacao) often containing procyanidin oligomer components, Diospyros kaki and koji leaves containing prodelphinidin oligomer components can be preferably used. Specific examples of berry fruits include Boysenberry (Rubus sp. Hydrid “Boysen”), Natsuhaze (Vaccinium oldhamii), Cassis (Ribes nigrum), Blueberry (Vaccinium angustifolium, V. ashei, V. corymbosum ), Strawberry (Fragari sp.), Grapes (Vitis vinifera), Bilberry (Vaccinium myrtillus), Cowberry (Vaccinium vits-idea), Blackberry (Rubus fruticosus), Elderberry (Sambucus nigra), Yama grape (Vitis coigia) Examples include berry cocoons (Photinia melanocarpa) cocoons, and fruits include flesh, pericarp, and seeds. Specific examples of persimmon fruit and persimmon leaves can be used as sweet persimmon or persimmon, but preferably persimmon contains a large amount of condensed tannin oligomer components.

When a specific structural component is a main component as the condensed tannin oligomer component, the content can be increased by appropriately selecting the fruit, leaf, bark, etc. of the plant as the raw material. For example, when a procyanidin oligomer component is used as a main component, components prepared from cacao beans, grape seeds, boysenberry, blueberry, cranberry, bilberry and the like can be blended and used. In order to increase the blending ratio of the prodelphinidin oligomer component, it is possible to add a large amount of components prepared from persimmon fruits and persimmon leaves.

The condensed tannin oligomer component derived from plants can take the form of an extract or a concentrate, but can be prepared by selecting and combining juice, extraction, fractionation, concentration, and the like. For example, the fruit juice obtained by pressing the fruit can be used as such. Furthermore, fruit juice is concentrated by membrane or reduced pressure, such as Amberlite XAD-4 and XAD-7 (trademark, manufactured by Organo Corporation, the same applies hereinafter), Diaion HP-10 and HP-20 (trademark, Synthetic adsorption resin from Mitsubishi Chemical Corporation, the same shall apply hereinafter), Sephadex LH-20 (trademark, manufactured by GE Healthcare Biosciences, the same shall apply hereinafter), Toyopearl HW-40 and HW-50 (trademark, It can be used as a concentrate concentrated and fractionated by a gel filtration carrier such as (made by Tosoh Corporation). There are also berry varieties containing both effective components of condensed tannin oligomer component and organic acid component in the juice of juice, so that a composition utilizing the organic acid can also be obtained. Moreover, the condensed tannin oligomer component extracted with the hydrophilic solvent from the peel and seed in the pressing residue can also be used. Examples of the hydrophilic solvent include methanol, ethanol, isopropanol, ethylene glycol, acetone, acetonitrile, dimethyl sulfoxide and the like, and these can be used alone or as a mixed solvent of plural kinds including water. . Among these solvents, ethanol and hydrous ethanol can be preferably used in consideration of the use for food and drink. Similar to fruit juice, the extract can be led to a concentrate by combining a concentration step and a fractionation step.

The organic acid component constituting the composition of the present invention is a vasodilator by interaction with at least one condensed tannin oligomer component of a dimer to a 13-mer derived from berry fruits or cacao beans or persimmon fruits or persimmon leaves. It can be an active ingredient of a composition that synergistically improves the action.
The organic acid component is not particularly limited, but preferably citric acid, succinic acid, malic acid, acetic acid, phytic acid, lactic acid, or a salt thereof. These may be used singly or as a mixture of two or more. Specific examples of the salt include sodium, potassium, calcium, magnesium, ammonium and the like. The composition can be prepared by adding a predetermined amount of an organic acid component to an extract or concentrated solution containing a condensed tannin oligomer component. As a form of the organic acid component to be used, raw materials as food and drink, food additives, and citrus and fermented products containing the organic acid component can also be used.

The content of the condensed tannin oligomer component in the composition that synergistically improves the vasodilatory effect of the present invention is not limited by procyanidin oligomer and prodelphinidin oligomer, but generally 0.00004 to 90 wt. %, Preferably 0.00004 to 40% by weight, more preferably 0.004 to 40% by weight. The condensed tannin oligomer component and the organic acid are contained in a weight ratio of 1: 1 to 70000, preferably 1: 2 to 200.

On the other hand, if a sugar component in a plant containing a condensed tannin oligomer component is processed and converted to produce an organic acid and a desired composition can be obtained, it is a great advantage in producing the composition of the present invention. It becomes. The present inventors produce or increase an organic acid component to a predetermined amount from a plant containing a condensed tannin oligomer component and a sugar component or an extract thereof by subjecting the sugar component to microbial fermentation without decomposing the condensed tannin oligomer component. As a result of intensive studies on the method of making the composition, a new production method for obtaining a desired composition has been established, and the method has been completed as an efficient production method. Specifically, a microorganism that performs organic acid fermentation is allowed to act on a plant or its extract to increase the amount of the organic acid without damaging the condensed tannin oligomer component, so that the condensed tannin oligomer component and the organic acid component are effective ingredients. A composition that synergistically improves the vasodilatory action.

The plant containing the condensed tannin oligomer component to be subjected to organic acid fermentation and the sugar component or the extract thereof is not particularly limited, but a berry fruit or an extract thereof, or an astringent fruit or an extract thereof can be preferably used. Specific examples include Boysenberry (Rubus sp. Hydrid “Boysen”), Natsuhaze (Vaccinium oldhamii), Cassis (Ribes nigrum), Blueberry (Vaccinium angustifolium, V. ashei, V. corymbosum), Cranberry (Vaccponium cro (Fragari sp.), Grape (Vitis vinifera), bilberry (Vaccinium myrtillus), cowberry (Vaccinium vits-idea), blackberry (Rubus fruticosus), elderberry (Sambucus nigra), wild grape (Vitis coignetiae), chalkberry melanocarpa), Tone Sayabu Shibutsu, Hirakuen Shibutoshi and other fruits or extracts thereof. The microorganism to be acted on is not particularly limited as long as it is a microorganism that performs organic acid fermentation without impairing the condensed tannin oligomer component, and microorganisms that perform citric acid fermentation or acetic acid fermentation can be suitably used. Specific examples thereof include organic acid fermentation in which black mold (Aspergillus niger), Bacillus bacterium licheniformis, yeast (Saccharomyces cerevisiae), acetic acid bacterium (Acetobactor aceti), lactic acid bacterium (Lactobacillus sp.) And the like alone or in combination.

A composition containing a condensed tannin oligomer component and an organic acid component that synergistically improve the vasodilatory effect can be applied to pharmaceuticals or foods and drinks in various forms. When the composition of the present invention is used as a pharmaceutical product, for example, oral preparations such as powders, granules, capsules, pills, tablets and other solid preparations, liquids such as liquids, suspensions and emulsions are exemplified. In addition to the above-mentioned composition, this oral administration agent includes excipients, disintegrating agents, binders, lubricants, surfactants, alcohols, water, water-soluble polymers generally used according to the form of the oral administration agent. It can be produced by adding sweeteners, flavoring agents, acidulants and the like.

When the composition of the present invention is used as a food or drink, it exerts a vasodilatory effect and can be used alone or in combination with other drinks and food materials that can be eaten or consumed. Specific drinks include fruit juice drinks, fruit drinks, vegetable drinks, carbonated drinks, tea drinks, sports drinks, milk drinks and other bakery foods, sauces, soups, dressings, etc. Seasonings, dairy products such as milk and yogurt, confectionery such as chocolate and candy, supplements such as capsules, tablets, powders and granules, but are not limited thereto.

When the composition of the present invention is ingested as a food or drink, the amount of intake varies depending on the degree of purification, narrow components, age, weight, sex, form of intake, etc., but the condensed tannin oligomer which is an active ingredient Ingredient intake is calculated based on the amount of condensed tannin oligomer contained in the range of usually 10 to 5000 g, preferably 100 to 1000 g per day per adult, in terms of the weight of the berry fruit contained. An effective dose is easy and preferable. However, in the case of food and drink, unlike pharmaceuticals, in the case of considering the purpose of maintaining health functions, taste, and palatability, it is not limited to the above range.
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

(Reference Example 1: Specification of the target component of the present invention by measuring the structure of the condensed tannin oligomer component)
The dimer to 13-mer condensed tannin oligomer component in the present invention is measured by measuring the structural unit and arrangement order, the degree of polymerization, the steric structure of adjacent bonds, the gallation ratio, and measuring the characteristics of the structural component. It is possible to identify the corresponding plant.

1) Identification by measurement of structural units, sequence order and gallation ratio: Guyot S et al.'S method in which condensed tannin component by thiol decomposition reaction is subjected to thiol decomposition reaction with toluene thiol in 3% hydrochloric acid-containing methanol solution (J Agric. Food Chem. 49, 14-20, 2001). For example, when the reaction product obtained by thiol decomposition of a bamboo leaf extract was analyzed by liquid chromatography (ODS column, 280 nm UV detector), the spectrum of FIG. 1 was obtained. The observed peaks (retention time) could be assigned as follows: catechin (12.1 minutes), epigallocatechin-4β-sulfide (17.9 minutes), epigallocatechin gallate-4β-sulfide (20.2 minutes), epicatechin -4β-sulfide (22.3 min), epicatechin gallate-4β-sulfide (24.4 min). This result is prodelphinidin in which the coconut leaf tannin component has catechin as a terminal constitutional unit and 4-4 linked epigallocatechin, epigallocatechin gallate, epicatechin, and epicatechin gallate as extension constitutional units. It showed that. Further, from the respective peak areas and relative sensitivities, it was possible to calculate that the gallated ratio was 43%. Similarly, the target substances used in the examples were measured, and the measurement results of the structural units, arrangement order and gallation ratio are shown in Table 1. Has been.

2) Identification by measurement of degree of polymerization: Based on MALD-TOF MS analysis The above-mentioned cocoon leaf condensed tannins were analyzed with MALD-TOF MS (equipment, Bruker Reflex III TOF / TOF; mode, linear positive, matrix, dihydroxybenzoic acid). Upon analysis, the spectrum shown in FIG. 2 was obtained. Also shown in FIG. 2 is a corresponding peak obtained by examining the measured molecular weight based on the combination of constituent units of condensed tannin and the degree of polymerization. From this MALD-TOF MS result, it was found that the Kashiwa condensate tannin contains oligomers having a degree of polymerization of up to 12, and each component having a polymerization degree is composed of components having the same or different molecular weight. As a specific example, when a structural unit having a polymerization degree of 12 (molecular weight around 4064) is analyzed, a component (molecular weight 4090) in which epicatechin (EC) is composed of 1 unit and 11 units of epigallocatechin, of which 3 units are gallated. ) And epicatechin (EC) 10 units and epigallocatechin 3 units, of which 5 units were gallated (molecular weight 4090). Thus, as a result of measuring the degree of polymerization of the target substance used in the examples, it became clear that it had a specific molecular weight distribution and structural unit depending on the raw material and the preparation method. The results are also shown in Table 1.

3) Identification of condensed tannin dimer by steric structure: 4-8-linked procyanidin dimers (molecular weight: 578) containing no gallate by high performance liquid chromatography / mass spectrometry (LC / MS) B2, B3, B4) exist, and it is a structural isomer that has been confirmed even to the three-dimensional structure. As a result of the content ratio of these isomers being different depending on the raw material and the preparation method, measurement of the constituent components of the procyanidin dimer helps to identify the origin of the condensed tannin oligomer. Specific examples are shown below. Kashiwa tannin was analyzed by high performance liquid chromatography / mass spectrometry (LC / MS) (equipment, Shimadzu Prominence (Shimadzu), equipped with ODS column, gradient elution with formic acid / methanol / water); MS / MS, API 3200 (Applied) Using a negative-ion mode (manufactured by Biosystems), the molecular weight of 577/289 was measured, and the spectrum shown in FIG. 3d was obtained. Similarly, when grape seed preparations were measured, the four stereoisomers (B1, B2, B3, J. Chromatography A, 1177, 114-125, 2008) reported by Kohler N et al. Four peaks consistent with B4,) were observed (FIG. 3a). Although the procyanidin dimer derived from cocoa beans is known to contain B2 as a main component, the results of the measurement of the target substance of the present invention were in good agreement (FIG. 3c). In addition, the Boysenberry fruit extract showed a spectrum different from the above three types (3b). By comparing these measured values, it is clear that procyanidin dimer constituents have B1 as the main component in the bamboo leaf extract and B4 and B3 as the main components in the Boysenberry fruit extract. It was. In addition to the molecular weight of 577, the protephinidin dimer having molecular weights of 593 and 609 having epigallocatechin as the structural unit was confirmed as the dimer of kashiwa tannin.

Specific examples in which the plant species contained by the above three measurement methods and the structural characteristics of the condensed tannin oligomer component used in the following examples were measured are shown in Table 1. As is clear from this table, the measured values of the condensed tannin oligomers obtained from the raw material plants have different parts, and the substances derived from them can be specified.

(Reference Example 2: Quantification of condensed tannin oligomers derived from berry fruits, cacao beans, persimmon fruits or persimmon leaves)
Liquid chromatography (HPLC) profiles of the condensed tannin oligomer extract of the present invention, for example, BBF-XAD / EtOH fr, BBF-XAD-LH20 fr obtained from Boysenberry, and epicatechin standards, respectively, (4a), Shown in (4b) and (4c). HPLC measurement conditions: column, Inertsil ODS-3 (250 x 4.6 mm); mobile phase, A (0.5% trifluoroacetic acid aqueous solution), B (0.5% trifluoroacetic acid methanol solution); gradient, 0-5 minutes (A / B = 90/10), 5-15 minutes (A / B = 10/90-25/75), 15-35 minutes (A / B = 25/75-35/65), 35-50 minutes (A / B = 35/65-50/50), 50-60 minutes (A / B = 0/100), 60-75 minutes (A / B = 90/10); flow rate, 1.0 mL / min; detection method, UV (280 & 520 nm). Under the measurement conditions, the procyanidin oligomer component and the prodelphinidin oligomer component were observed as a peak having an absorption of 280 nm during a retention time of 15 minutes to 32 minutes. Dimer and trimer components are liquid chromatography / mass mass (LC / MSMS)
The analysis confirmed that it was in the same range. The content of the condensed tannin oligomer component was quantified as an epicatechin equivalent amount using an epicatechin calibration curve. By adding these contents to the mass balance in the extraction and fractionation operations, the amount of each component contained in the raw materials used and the intermediate preparation was calculated. As a result, the content of condensed tannin oligomer component in Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr) and Boysenberry fruit condensed tannin oligomer extract-2 (BBF-XAD-LH20 fr) is Calculated as 1.6 and 3.2%, respectively. The content of the condensed tannin oligomer component of the present invention measured in the same way is shown in the corresponding examples.

(Examples 1 and 2: Vasodilatory effect of the composition of Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr) and citric acid)
(1) Preparation of Boysenberry fruit condensed tannin oligomer extract Boysenberry juice concentrate (100 g) obtained by squeezing and concentrating Boysenberry fruit is passed through an Amberlite XAD-7 column and eluted. Boysenberry syrup containing organic acids and saccharides as adsorbing components. The concentrate of the reddish brown fraction eluted with ethanol from the adsorbed polyphenol component was Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr). Furthermore, the ethanol solution of BBF-XAD / EtOH fr was loaded onto a Sephadex LH-20 column, passed through methanol, and then the concentrate eluted with 70% acetone aqueous solution was extracted with Boysenberry fruit condensed tannin oligomer. It was set to thing-2 (BBF-XAD-LH20 fr).

(2) Vasodilation measurement method Magnus experimental device (AP-5 type, sardine and Kishimoto Medical Industry, which cuts the thoracic aorta of Wistar rats aged 8-11 weeks to 2-3 mm and filled with 20 mL of phosphate buffer ( And a tension of 300 mg was applied. After equilibration and stabilization of the baseline baseline, 0.2 mL of 10 μM norepinephrine (NE) was added as a contracting agent (final concentration, NE 0.1 μM). After the tension increased to about 3000 mg and became constant, the sample solution was added to a predetermined concentration. The amount of decrease in tension due to the addition of the sample was shown as an average value ± standard deviation for four specimens, with the percentage when the value without adding each sample as 100 was the vasodilation rate.

(3) Vasodilation effect of a composition comprising Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr) and citric acid Condensed tannin oligomer extract-1 prepared in (1) (BBF-XAD / EtOH fr) and citric acid were mixed to the final concentrations shown in Table 2 as sample solutions, and the vasodilation rate was measured by the method (3) to give Examples 1 and 2. . Further, as the activity of the single component, the results of the same measurement at the concentrations of BBF-XAD / EtOH fr and citric acid alone at 100 and 4 μg / mL were set as Comparative Examples 1, 1a and 2a, and the results are shown in Table 2. The vasodilatory effect of the composition of BBF-XAD / EtOH fr and citric acid of Examples 1 and 2 was improved by 8.7 to 7.2 times compared to the vasodilatory effect of a single component.

Examples 3, 4, 5 and 6: Vasodilation of a composition comprising Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr) and succinic acid, phytic acid, malic acid, acetic acid or lactic acid Action)
Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr) prepared in Example 1 and (1) and succinic acid, phytic acid, malic acid, acetic acid, or lactic acid are shown in Table 3. The composition mixed so as to have the final concentration shown was used as a sample solution, and the vasodilation rate was measured according to the method of Example 1 and 2 (2) to give Examples 3, 4, 5, 6 and 7. Moreover, the result of having measured only the organic acid similarly as activity of a single component was shown in Table 3 as Comparative Examples 3, 4, 5, 6, and 7. The vasodilatory effect of the composition comprising BBF-XAD / EtOH fr and organic acid in Examples 3, 4, 5, 6 and 7 is 9.2 compared to the vasodilatory effect of BBF-XAD / EtOH fr or organic acid alone. Improved by 3.0 times. This result shows that vasodilatory action is improved by the synergistic action of the composition consisting of condensed tannin oligomer extract, BBF-XAD / EtOH fr and succinic acid, phytic acid, malic acid, acetic acid, or lactic acid. It is shown that.

(Examples 8, 9, 10 and 11: Vasodilatory effect of a composition comprising Boysenberry fruit condensed tannin oligomer extract-2 (BBP-XAD-LH20 fr) and citric acid)
A composition prepared by mixing condensed tannin oligomer extract-2 (BBF-XAD-LH20 fr) prepared in Example 1 and 2 (1) and citric acid so as to have final concentrations shown in Table 4, respectively. As the sample solution, the vasodilation rate was measured according to the method of Example 1 and 2 (2) to give Examples 8, 9, 10 and 11. In addition, as the activity of the single component, the results of measuring the sample of only BBF-XAD-LH20 fr in the same manner are referred to as Comparative Examples 8, 9, 10 and 11, respectively, and the results of the same measurement of the sample of citric acid alone are the comparative examples. It is shown in Table 4 as 1a. Compared to the administration of Boysenberry fruit condensed tannin oligomer extract-2 (BBP-XAD-LH20 fr, 1.0 μg / mL) alone in Comparative Example 11, Boysenberry fruit condensed tannin oligomer extract-2 (BBP -XAD-LH20 fr, 1.0 μg / mL) and a measurement graph showing the synergistic vasodilatory effect of a composition consisting of citric acid (100 μg / mL) are illustrated in FIG. The vasodilatory effect of the composition of Boysenberry fruit condensed tannin oligomer extract-2 (BBF-XAD-LH20 fr) and citric acid in Examples 8, 9, 10 and 11 is the Boysenberry fruit condensed tannin oligomer extract- 2 (BBF-XAD-LH20 fr) was improved by 3.6 to 14.5 times compared with the vasodilatory effect alone. Furthermore, the magnitude of the expansion rate was positively correlated with the concentration of Boysenberry fruit condensed tannin oligomer extract-2 (BBF-XAD-LH20 fr). These results indicate that the vasodilatory action is improved by the synergistic action of the composition comprising Boysenberry fruit condensed tannin oligomer extract-2 (BBP-XAD-LH20 fr) and citric acid.

Examples 12, 13, 14 and 15: Vasodilatory effect of a composition comprising a condensed tannin oligomer extract derived from berry fruits and citric acid)
(1) Preparation of condensed tannin oligomer extract derived from berry fruit 70% acetone was added to the pressed residue obtained by pressing the natsuhaze fruit and stirred to extract the natsuhaze condensed tannin oligomer component. The concentrated solution obtained by distilling off acetone from this extract under reduced pressure was loaded onto an Amberlite XAD-7 column, and water was passed through it. Then, the adsorbed Natsuhaze fruit condensed tannin oligomer component was methanol, followed by 60% acetone aqueous solution. It eluted with. Both eluates were combined and concentrated to obtain reddish brown NHF-XAD fr. Furthermore, an ethanol solution of BBF-XAD fr was loaded onto a Sephadex LH-20 column, passed through ethanol, and the concentrate eluted with a 60% acetone aqueous solution was extracted with a nuthaze fruit condensed tannin oligomer extract (NHF -XAD-LH20 fr). Cassis fruit, bilberry fruit and grape seed are treated in the same manner, and cassis fruit condensed tannin oligomer extract (CAF-XAD-LH20 fr), bilberry fruit condensed tannin oligomer extract (BIF-XAD-LH20 fr) and grape seed, respectively. A condensed tannin oligomer extract (GRS-XAD-LH20 fr) was obtained. Nutzeze fruit condensed tannin oligomer extract (NHF-XAD-LH20 fr), Bilberry fruit condensed tannin oligomer extract (BIF-XAD-LH20 fr) and grape seed condensed tannin oligomer extract (GRS-XAD-LH20 fr) are procyanidin oligomers The Cassis fruit condensed tannin oligomer extract (CAF-XAD-LH20 fr) was a prodelphinidin oligomer.

(2) Vasodilatory action of berry fruit-derived condensed tannin oligomer extract and citric acid composition Natsuhaze condensed tannin oligomer extract (NHF-XAD-LH20 fr) prepared in (1), Cassis fruit condensed tannin oligomer extract (CAF-XAD-LH20 fr), bilberry fruit condensed tannin oligomer extract (BIF-XAD-LH20 fr), grape seed condensed tannin oligomer extract (GRS-XAD-LH20 fr) and citric acid are shown in Table 5, respectively. Using the composition mixed to the final concentration as a sample solution, the vasodilatation rate was measured according to the method of Example 1 and 2 (2) to give Examples 12, 13, 14, and 15. Moreover, the result of having measured only each single condensed tannin oligomer extract and citric acid in the same manner is shown in Table 5 as Comparative Examples 12, 13, 14, 15 and 1a as the activity of the single component. The vasodilatory effect of the composition comprising the condensed tannin oligomer extract derived from the berry fruits of Examples 12, 13, 14 and 15 and citric acid was compared with the vasodilatory effect of each condensed tannin oligomer extract alone or citric acid alone. Improved by 3.1 to 1.6 times. This result shows that the vasodilatory action is improved by the synergistic action of the composition comprising the condensed tannin oligomer extract of berry fruits and citric acid.

(Examples 16, 17 and 18: Cocoa bean condensed tannin oligomer extract (CCP-XAD-LH20 fr), persimmon fruit condensed tannin oligomer extract (PAF-XAD / EtOH fr) and persimmon leaf condensed tannin oligomer extract (PAL) -XAD / EtOH fr) and vasodilatory effect of a composition comprising citric acid)
(1) Preparation of condensed tannin oligomer extract A cocoa bean condensed tannin oligomer extract (CCP-XAD-LH20 fr) was prepared from cocoa beans according to the method described in Example 12 (1). Astringent fruit (plain nucleus seedless) was strip-shaped, and 70% acetone was added and stirred to extract the condensed fruit tannin oligomer component. The concentrated solution obtained by distilling off acetone from the extract under reduced pressure was loaded onto an Amberlite XAD-7 column and passed through water. The adsorbed persimmon fruit polyphenol component was eluted with ethanol and concentrated to obtain a light brown persimmon fruit condensed tannin oligomer extract (PAF-XAD / EtOH fr). In addition, 70% acetone was added to Shibuya leaves (Tone seedlings) and stirred to extract the Kashiwa leaf polyphenol component. A concentrated aqueous solution obtained by evaporating acetone from the extract under reduced pressure was extracted with ethyl acetate to remove fat-soluble components, and then loaded onto an Amberlite XAD-7 column and passed through water. The adsorbed components were eluted with ethanol and concentrated to obtain a light brown maple leaf condensed tannin oligomer extract (PAL-XAD / EtOH fr). Cocoa bean condensed tannin oligomer extract (CCP-XAD-LH20 fr) is procyanidin, and persimmon fruit and persimmon leaf condensed tannin oligomer extract has a high epigallocatechin structural unit ratio of 65 and 45% and is also gallated. 62 and 43% were characteristic procyanidins.

(2) Vasodilatory action Cocoa bean condensed tannin oligomer extract (CCP-XAD-LH20 fr), persimmon fruit condensed tannin oligomer extract (PAF-XAD / EtOH fr) and persimmon leaf condensed tannin oligomer extraction prepared in (1) Vasodilatation rate according to the method of Example 1 and 2 (2), using as a sample solution a composition in which the product (PAL-XAD / EtOH fr) and citric acid were mixed to the final concentrations shown in Table 6 respectively. Are shown in Table 6 as Examples 16, 17 and 18. In addition, as the activity of the single component, the results of measuring each of the single condensed tannin oligomer extract and citric acid in the same manner are shown in Table 6 as Comparative Examples 16, 17, 18 and 1a. Compared with the single administration of persimmon fruit condensed tannin oligomer extract (PAF-XAD / EtOH fr, 1.0 μg / mL) of Comparative Example 17, persimmon fruit condensed tannin oligomer extract (PAF-XAD / EtOH fr, Example 17), A measurement graph showing the synergistic vasodilatory effect of a composition comprising 1 μg / mL) and citric acid (100 μg / mL) is shown in FIG. The vasodilatory action of the composition comprising CCP-XAD-LH20 fr, PAF-XAD / EtOH fr and PAL-XAD / EtOH fr and citric acid in Examples 16, 17 and 18 is a blood vessel of each alone or only citric acid only. Compared with the expansion effect, it improved by 1.4 to 1.7 times. This result shows that the vasodilatory action is improved by the synergistic action of the composition comprising the condensed tannin oligomer component and the citric acid in the cacao bean, persimmon fruit or persimmon leaf extract.

(Example 19: Method for producing Boysenberry juice acetic acid fermentation liquid composition)
Water and alcohol were added to Boysenberry concentrated fruit juice obtained by pressing and concentrating Boysenberry fruit to prepare a charging solution. A culture solution containing acetic acid bacteria (Acetobactor aceti) was added thereto, and acetic acid fermentation was performed at 28 ° C. to 32 ° C. for about 5 days. Water and alcohol vinegar were added to the resulting Boysenberry juice-containing raw vinegar (fruit juice 60%), and after adjusting the juice content and acidity, sterile filtration was performed to obtain a Boysenberry juice 30% acetic acid fermentation broth composition. The organic acid content of this composition by LC / MSMS analysis is 49.2 μg / mL (acetic acid 45 μg / mL, citric acid 3.7 μg / mL, malic acid 0.5 μg / mL), and the content of Boysenberry juice before fermentation Although the amount was increased 14 times from the amount of 3.5 μg / mL (acetic acid 0 μg / mL, citric acid 3.1 μg / mL, malic acid 0.4 μg / mL), the composition and content of the condensed tannin oligomer component were not changed.

(Example 20: Vasodilatory effect of Boysenberry juice acetic acid fermentation liquid composition)
The Boysenberry juice acetic acid fermentation broth composition prepared by the production method of Example 19 was used as a sample solution with a final concentration of 10 mg / mL (condensed tannin oligomer concentration of 8 ng / mL, organic acid concentration of 0.49 μg / mL). And when the vasodilatation rate was measured according to the method of (2) of 2 and 2, it was 26.7 ± 8.3%. As Comparative Example 19, when the vasodilatation rate of Boysenberry juice condensed tannin oligomer extract (BBF-XAD-LH20 fr, 0.27 μg / mL) alone was measured, it was 1.3 ± 0.7%, the former being 21 times higher in activity. showed that. This activity improvement was due to the result of conversion of Boysenberry juice into a composition containing a condensed tannin oligomer having an organic acid content enhanced by acetic acid fermentation and an organic acid.

(Example 21: Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr) and citric acid improve blood flow of composition beverage)
(1) Animals used, administration sample, administration method and dosage 7 weeks old male ICR mice (average body weight 40 g) were divided into 2 groups (4 groups for each of the test group and the control group). Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr, 10 mg / mL) and citric acid (20 mg / mL) composition drink 30 μL (condensed tannin oligomer component 15 μg, citric acid 400 μg) Orally administered. Each mouse in the control group was orally administered 30 μL of beverage (condensed tannin oligomer component 0.5 μg) of Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr, 10 mg / mL) alone. Five minutes after administration, a pentobarbital solution (6.48 mg / mL) was intraperitoneally administered (10 mL / kg). When the amount of Boysenberry fruit condensed tannin oligomer component in this administration sample is converted to a human intake of 60 kg body weight, it corresponds to 22.5 mg. This amount corresponds to the amount of condensed tannin oligomer component contained in 480 mL as Boysenberry juice, and is in the range of daily intake as juice.

(2) Test method and results A probe is mounted on the tail vein of a mouse, and blood flow from 15 to 70 minutes after sample administration using a laser Doppler blood flow measuring device (ALF21, manufactured by Advance Co., Ltd.) Changes were measured over time. As shown in FIG. 7, the blood flow of the test group administered with the composition beverage consisting of Boysenberry fruit condensed tannin oligomer extract and citric acid was 6-9 mL / min / 100 g. On the other hand, in the control group to which only the Boysenberry fruit condensed tannin oligomer extract was administered, it was 5-7 mL / min / 100 g, which was always lower than the value of the test group during the measurement period. This result has shown that the blood flow of a mouse | mouth is synergistically improved by administering the citric acid (20 mg / mL) composition rather than a Boysenberry fruit condensed tannin oligomer extract single administration.

(Example 22: Boysenberry fruit condensed tannin oligomer extract-1 (BBF-XAD / EtOH fr) and citric acid)
Spontaneous hypertensive rats (SHR / Izm) pre-bred from 12 weeks of age with solid feed and free water were divided into 2 groups. After 12 hours fasting, the control group (6 animals) was deionized water, the test group (5 animals) was BBP XAD / EtOH fr 200 mg / kg and citric acid monohydrate 500 mg / kg. Was forcibly orally administered to the stomach with a sonde. At 0, 1, 2, 4, and 8 hours after administration, the blood pressure of SHR rats was fixed on a warmer preliminarily kept at 37 ° C., and the blood pressure was measured three times per tail by the tail-cuff method. FIG. 8 shows a temporal change in blood pressure with the measured values obtained by averaging three measured values. As shown in FIG. 8, the blood pressure of the rats that received the composition of BBP XAD / EtOH fr and citric acid decreased, and showed a significant decrease compared to the control group at 1 hour and 6 hours after the intake. This result shows that the composition of BBP XAD / EtOH fr and citric acid has a blood pressure lowering effect.

Claims

A vasodilator comprising as an active ingredient at least one condensed tannin oligomer component of dimer to 13-mer derived from berry fruit or cacao bean or persimmon fruit or persimmon leaf, and at least one organic acid component,
The vasodilator described above, wherein the condensed tannin oligomer component is a procyanidin oligomer and / or a prodelphinidin oligomer having at least one of catechin, epicatechin, gallocatechin, epigallocatechin and / or their gallate as a constituent unit.
The vasodilator according to claim 1, wherein the berry fruit is Boysenberry, Natsuhaze, Cassis, Blueberry, Cranberry, Strawberry or Grape.
The vasodilator according to claim 1 or 2, wherein the organic acid component is citric acid, succinic acid, malic acid, acetic acid, phytic acid and / or lactic acid or a salt thereof.
4. The vasodilator according to claim 3, wherein the organic acid component is citric acid or a salt thereof.
The vasodilator according to any one of claims 1 to 4, which has an effect of improving blood flow and / or suppressing an increase in blood pressure.
A pharmaceutical or food comprising the vasodilator according to any one of claims 1 to 5.
A berry fruit or cacao bean or persimmon fruit or persimmon leaf containing at least one condensed tannin oligomer component and a sugar component, or an organic acid component is produced or increased by microbial fermentation of an extract thereof. The method for producing a vasodilator according to claim 1.
The production method according to claim 7, wherein the berry fruit or an extract thereof is Boysenberry, Natsuhaze, Cassis, Blueberry, Cranberry, Strawberry or Grape.
The production method according to claim 7 or 8, wherein the organic acid component is citric acid, succinic acid, malic acid, acetic acid, phytic acid and / or lactic acid or a salt thereof.
10. The production method according to claim 9, wherein the organic acid component is citric acid or a salt thereof.