JP7784718B2 - Composition for improving blood vessel flexibility - Google Patents

Description

The present invention relates to a composition for improving vascular flexibility and a composition for improving vascular endothelial function, which contain pine bark and at least one member selected from the group consisting of ginger, elastin, and vitamin E. The present invention also relates to an oral composition containing pine bark, ginger, and elastin.

In recent years, lifestyle-related diseases have been increasing due to changes in diet and lifestyle. Lifestyle-related diseases are a general term that includes not only diseases such as hypertension, hyperlipidemia, and diabetes, but also diseases such as angina pectoris, myocardial infarction, and cerebral circulatory disorders, the onset of which is thought to be preventable by improving lifestyle habits. It has been revealed that reduced vascular flexibility and reduced vascular endothelial function are involved as factors in these diseases. Furthermore, because lifestyle-related diseases are chronic, their prevention and treatment require a long period of time. Therefore, there is a need for the development of foods that can safely and effectively improve vascular flexibility and vascular endothelial function over the long term. Hops and chlorogenic acid, for example, are known as food ingredients that have such effects (Patent Documents 1 and 2).

JP 2005-104951 JP 2003-261444

However, according to the inventors' research, the effects of hops and chlorogenic acid on improving vascular flexibility and vascular endothelial function are not sufficient.

The inventors therefore conducted various studies with the objective of providing a composition that contains ingredients that can be safely ingested over a long period of time and that exhibits excellent effects of improving vascular flexibility and vascular endothelial function.

The inventors of the present invention conducted extensive research to solve the above-mentioned problems and surprisingly discovered that combining pine bark with at least one selected from the group consisting of ginger, elastin, and vitamin E exerts an excellent effect of improving vascular flexibility and vascular endothelial function. The present invention was completed based on this finding.

The outline of the present invention is as follows.
[1] A composition for improving vascular flexibility, comprising pine bark and at least one selected from the group consisting of ginger, elastin, and vitamin E.
[2] A composition for improving vascular endothelial function, comprising pine bark and at least one selected from the group consisting of ginger, elastin, and vitamin E.
[3] An oral composition comprising pine bark, ginger, and elastin.
[4] An oral composition comprising pine bark, ginger, elastin, and vitamin E.
[5] A composition for improving vascular flexibility, comprising pine bark and at least two or more members selected from the group consisting of ginger, elastin, and vitamin E.
[6] A composition for improving vascular endothelial function, comprising pine bark and at least two or more members selected from the group consisting of ginger, elastin, and vitamin E.
[7] A composition for improving vascular flexibility, comprising pine bark, ginger, elastin, and vitamin E.
[8] A composition for improving vascular endothelial function, comprising pine bark, ginger, elastin, and vitamin E.

Function of the invention

According to the present invention, by containing at least one selected from the group consisting of pine bark, ginger, elastin, and vitamin E, a composition can be provided that has excellent effects of improving vascular flexibility and vascular endothelial function.

FIG. 1 is a diagram showing the expression levels of NO synthase genes in Examples and Comparative Examples.

The present invention will be described in detail below. Note that the present invention is not limited to the following embodiments.

The composition of the present invention is characterized by containing pine bark and at least one component selected from ginger, elastin, and vitamin E. The component selected from ginger, elastin, and vitamin E used together with pine bark may be used alone or in combination of two or three components. However, from the standpoint of improving vascular flexibility and endothelial function, it is preferable to use two or three components in combination, and it is particularly preferable to use three components in combination.

Each component contained in the composition of the present invention will be described below.
[Pine bark]
The present invention is characterized by the use of pine bark. Examples of pine species from which pine bark is obtained include, but are not limited to, French maritime pine (Pinus Martima), larch, black pine, Japanese red pine, Pinus densiflora, Pinus sieboldii, Pinus sieboldii, Pinus sieboldii, Pinus sieboldii, Pinus palustris ...

In the present invention, pine bark can be processed and used. Examples of processed pine bark products include chips, crushed material, squeezed product, extract, and dried powder thereof. Considering formulation feasibility, the pine bark used in the present invention is preferably crushed, squeezed product, extract, or dried powder thereof, as these are easy to apply. Extract or dried powder thereof is more preferred in terms of improving vascular flexibility and vascular endothelial function. Pine bark processed products may be produced by methods commonly known to those skilled in the art, or may be commercially available products. For example, pine bark extract manufactured by Toyo Shinyaku Co., Ltd. can be used.

Extraction solvents used to obtain pine bark extract include, for example, water, organic solvents, and aqueous organic solvents (aqueous alcohols such as aqueous ethanol). Examples of organic solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, butane, acetone, hexane, cyclohexane, propylene glycol, aqueous ethanol, aqueous propylene glycol, ethyl methyl ketone, glycerin, methyl acetate, ethyl acetate, diethyl ether, dichloromethane, edible oils and fats, 1,1,1,2-tetrafluoroethane, and 1,1,2-trichloroethene. These water and organic solvents may be used alone or in combination. Water is preferably used as the extraction solvent. The temperature of the solvent used during extraction is not limited, as long as it is below the boiling point of the solvent used.

The method for obtaining pine bark extract is not particularly limited, but examples include heat extraction, supercritical fluid extraction, liquid carbon dioxide batch extraction, liquid carbon dioxide reflux, and supercritical carbon dioxide reflux. Multiple extraction methods may also be combined. By combining multiple extraction methods, it is possible to obtain pine bark extracts with various compositions.

Supercritical fluid extraction is a method of extraction using a supercritical fluid, which is a fluid in a state above the gas-liquid critical point (critical temperature, critical pressure) of a substance. Examples of supercritical fluids that can be used include carbon dioxide, ethylene, propane, and nitrous oxide (laughing gas), with carbon dioxide being preferred.

Supercritical fluid extraction involves an extraction process in which the target component is extracted using a supercritical fluid, and a separation process in which the target component is separated from the supercritical fluid. The separation process may involve extraction and separation due to pressure changes, temperature changes, or the use of adsorbents or absorbents.

Supercritical fluid extraction may also be performed using the entrainer addition method. In this method, approximately 2-20 wt./vol. % of ethanol, propanol, n-hexane, acetone, toluene, or other aliphatic lower alcohols, aliphatic hydrocarbons, aromatic hydrocarbons, or ketones is added to the extraction fluid, and supercritical fluid extraction is performed using this fluid. This method dramatically increases the solubility of the target extract, such as OPCs (oligomeric proanthocyanidins) and catechins, in the extraction solvent or enhances separation selectivity, resulting in an efficient method for obtaining pine bark extracts.

Supercritical fluid extraction has the advantages of being able to operate at relatively low temperatures, making it applicable to substances that change or decompose at high temperatures, leaving no residual extracted fluid, and allowing the solvent to be recycled, eliminating the need for desolvation steps and simplifying the process.

From a safety standpoint, it is preferable to purify the pine bark extract obtained by the above extraction using a column method or batch method. Examples of column methods include purification methods using adsorptive carriers such as Diaion HP-20, Sephadex-LH20, and chitin.

The pine bark extract used in the present invention contains proanthocyanidins as one of its main components. Proanthocyanidins are a group of compounds consisting of condensation polymers with a degree of polymerization of 2 or more, whose constituent units are flavan-3-ol and/or flavan-3,4-diol.

The pine bark extract used in the present invention preferably contains condensation polymers with a degree of polymerization of 2 or more as proanthocyanidins. Proanthocyanidins containing a large amount of condensation polymers with a low degree of polymerization are particularly preferred. Examples of condensation polymers with a low degree of polymerization include condensation polymers with a degree of polymerization of 2 to 30 (dimers to tridecamers). From the standpoint of improving vascular flexibility and vascular endothelial function, condensation polymers with a degree of polymerization of 2 to 10 (dimers to decamers) are preferred, and condensation polymers with a degree of polymerization of 2 to 4 (dimers to tetramers) are particularly preferred. In this specification, polymers with a degree of polymerization of 2 to 4 are referred to as OPCs (oligomeric proanthocyanidins).

There are no particular restrictions on the proportion of pine bark contained in the composition of the present invention, and the proportion can be appropriately set within a wide range depending on various conditions, such as the purpose, shape, and intended use. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the proportion is preferably 0.001 to 50% by mass, and from the standpoint of improving vascular flexibility and vascular endothelial function, 0.01 to 40% by mass is more preferable, and 0.1 to 30% by mass is particularly preferable. Furthermore, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the proportion is preferably 0.0001 to 30% by mass, more preferably 0.001 to 20% by mass, and particularly preferably 0.01 to 10% by mass.

[Ginger]
In the present invention, ginger can be used together with pine bark. The ginger used in the present invention is not particularly limited as long as it is ginger (Zingiber officinale), a perennial plant of the Zingiberaceae family. Specific ginger varieties include Sanshu ginger, Omi ginger, Yanaka ginger, Kintoki ginger, Shizuoka No. 4, yellow ginger, earth ginger, and Otafuku ginger. However, Kintoki ginger is preferred in terms of its effects of improving vascular flexibility and vascular endothelial function.

The form of ginger used in the present invention is not particularly limited, and in addition to using the rhizome as is, ginger can also be processed and used. Examples of processed ginger products include chips, pulverized material, squeezed product, extract, and dried powder thereof. Considering formulation ease, the ginger used in the present invention is preferably pulverized, squeezed, extract, or dried powder thereof, and is more preferably pulverized, extract, or dried powder thereof, from the standpoint of improving vascular flexibility and vascular endothelial function, with pulverized material or dried powder thereof being particularly preferred. The processed ginger product may be one produced by a method commonly known to those skilled in the art, or it may be one that is commercially available.

Extraction solvents used to obtain ginger extract include, for example, water, organic solvents, and aqueous organic solvents (aqueous alcohols such as aqueous ethanol). Examples of organic solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, butane, acetone, hexane, cyclohexane, propylene glycol, aqueous ethanol, aqueous propylene glycol, ethyl methyl ketone, glycerin, methyl acetate, ethyl acetate, diethyl ether, dichloromethane, edible oils and fats, 1,1,1,2-tetrafluoroethane, and 1,1,2-trichloroethene. These water and organic solvents may be used alone or in combination. From the standpoint of improving vascular flexibility and vascular endothelial function, it is preferable to use water, ethanol, or aqueous ethanol as the extraction solvent, with water being particularly preferred. The temperature of the solvent used during extraction is not limited, as long as it is below the boiling point of the solvent used.

There are no particular restrictions on the proportion of ginger to be incorporated into the composition of the present invention, and the proportion can be appropriately set within a wide range depending on various conditions, such as the purpose, shape, and target of use. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the proportion is preferably 0.0001 to 20% by mass, and from the standpoint of improving vascular flexibility and vascular endothelial function, 0.001 to 10% by mass is more preferred, and 0.01 to 5% by mass is particularly preferred. Furthermore, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the proportion is preferably 0.00001 to 10% by mass, and from the standpoint of improving vascular flexibility and vascular endothelial function, 0.0001 to 5% by mass is more preferred, and 0.001 to 1% by mass is particularly preferred.

[Elastin]
The present invention can use elastin together with pine bark. Elastin is a major elastic protein present, along with collagen, in mammals, birds, fish, etc., particularly in the aorta, ligamentum nuchale, ligamentum flavum, lung, skin, uterus, elastic cartilage, etc. of mammals, and is also known as elastic fiber. The elastin used in the present invention includes, in addition to water-soluble elastin, hydrolyzed elastin, α-elastin, κ-elastin, elastin peptides, etc.

Elastin can be extracted from animal biological tissues, such as the connective tissue of mammals such as pigs, cows, horses, and sheep, and fish such as bonito, tuna, yellowtail, and salmon, by hydrolyzing the elastin with acid or alkali, or by enzymatic treatment. The elastin used in the present invention can be naturally derived or synthetic, and is not particularly limited. However, from the standpoint of improving vascular flexibility and vascular endothelial function, elastin derived from fish is preferred, and of fish, elastin derived from bonito or tuna is more preferred, with bonito being particularly preferred.

The proportion of elastin contained in the composition of the present invention is not particularly limited, and can be appropriately set within a wide range depending on various conditions such as the purpose, shape, and intended use. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the proportion is preferably 0.0001 to 20% by mass, and from the standpoint of improving vascular flexibility and vascular endothelial function, 0.001 to 10% by mass is more preferable, and 0.01 to 5% by mass is particularly preferable. Furthermore, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the proportion is preferably 0.00001 to 10% by mass, and from the standpoint of improving vascular flexibility and vascular endothelial function, 0.0001 to 5% by mass is more preferable, and 0.001 to 1% by mass is particularly preferable.

[Vitamin E]
The present invention can use vitamin E together with pine bark. Vitamin E used in the present invention refers to vitamin E (tocopherol, tocotrienol) and its derivatives. The type of vitamin E used in the present invention is not particularly limited. For example, tocopherol and tocotrienol may be any of α-, β-, γ-, and δ-tocopherols, and may be either the d- or dl-isomer. Examples of vitamin E derivatives include organic tocopherol esters such as tocopherol acetate, tocopherol succinate, tocopherol linoleate, tocopherol linolenate, tocopherol nicotinate, and tocopherol (linoleate/oleate). From the viewpoint of improving vascular flexibility and vascular endothelial function, α-tocopherol and γ-tocopherol are preferred, with α-tocopherol being more preferred. Among α-tocopherols, the d-isomer is particularly preferred.

The proportion of vitamin E contained in the composition of the present invention is not particularly limited, and can be appropriately set within a wide range depending on various conditions such as the purpose, shape, and target of use. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the proportion is preferably 0.0001 to 20% by mass, and from the standpoint of improving vascular flexibility and vascular endothelial function, 0.001 to 10% by mass is more preferable, and 0.01 to 5% by mass is particularly preferable. Furthermore, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the proportion is preferably 0.00001 to 10% by mass, and from the standpoint of improving vascular flexibility and vascular endothelial function, 0.0001 to 5% by mass is more preferable, and 0.001 to 1% by mass is particularly preferable.

The total proportion of pine bark, ginger, elastin, and vitamin E contained in the composition of the present invention is not particularly limited, and can be appropriately set within a wide range depending on various conditions, such as the purpose, form, and intended use. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the proportion is preferably 0.02 to 100% by mass. From the viewpoint of improving vascular flexibility and vascular endothelial function, the proportion is more preferably 0.1 to 70% by mass, and particularly preferably 1.0 to 45% by mass. Furthermore, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the proportion is preferably 0.0001 to 30% by mass. From the viewpoint of improving vascular flexibility and vascular endothelial function, the proportion is more preferably 0.001 to 20% by mass, and particularly preferably 0.01 to 10% by mass.

In the composition of the present invention, the ratio of ginger to pine bark is not particularly limited, but from the standpoint of improving vascular flexibility and vascular endothelial function, the ratio of ginger to pine bark is preferably 0.0001 to 50 times, more preferably 0.0005 to 20 times, and particularly preferably 0.001 to 10 times, the mass of pine bark per 1.

In the composition of the present invention, the ratio of elastin to pine bark is not particularly limited, but from the standpoint of improving vascular flexibility and vascular endothelial function, the ratio of elastin to pine bark is preferably 0.0001 to 50 times, more preferably 0.0005 to 20 times, and particularly preferably 0.001 to 10 times, the mass of which is 1 part pine bark.

In the composition of the present invention, the ratio of vitamin E to pine bark is not particularly limited, but from the standpoint of improving vascular flexibility and vascular endothelial function, the ratio of vitamin E to pine bark is preferably 0.0001 to 50 times, more preferably 0.0005 to 20 times, and particularly preferably 0.001 to 10 times, the mass of pine bark per 1 mass of pine bark.

In the composition of the present invention, the ratio of the total amount of ginger, elastin, and vitamin E to the pine bark is not particularly limited, but from the standpoint of improving vascular flexibility and vascular endothelial function, the ratio of the total amount of ginger, elastin, and vitamin E to the pine bark is preferably 0.0001 to 150 times, more preferably 0.0005 to 60 times, and particularly preferably 0.001 to 30 times, the mass of pine bark per 1 mass of pine bark.

The daily intake amount per body weight of the composition of the present invention is not particularly limited and can be set appropriately depending on the mode of use and the user's intended use. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the intake amount is preferably 1 to 1,000 mg/kg, more preferably 3 to 200 mg/kg, based on the user's body weight. In terms of the effects of improving vascular flexibility and vascular endothelial function, the intake amount is particularly preferably 5 to 100 mg/kg. Furthermore, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the intake amount is preferably 200 to 20,000 mg/kg, more preferably 600 to 15,000 mg/kg, based on the user's body weight. In terms of the effects of improving vascular flexibility and vascular endothelial function, the intake amount is particularly preferably 1,000 to 10,000 mg/kg.

Similarly, the single intake amount of the composition of the present invention per body weight is not particularly limited. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the amount is preferably 0.3 to 1,000 mg/kg, more preferably 1.0 to 200 mg/kg, based on the user's body weight. In terms of the effects of improving vascular flexibility and vascular endothelial function, 1.6 to 100 mg/kg is particularly preferred. Furthermore, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the amount is preferably 66 to 20,000 mg/kg, more preferably 200 to 15,000 mg/kg, based on the user's body weight. In terms of the effects of improving vascular flexibility and vascular endothelial function, 333 to 10,000 mg/kg is particularly preferred.

Furthermore, the daily intake of the composition of the present invention is not particularly limited. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the daily intake is preferably 0.05 to 50 g, more preferably 0.1 to 10 g, and from the viewpoint of improving vascular flexibility and vascular endothelial function, it is particularly preferably 0.2 to 5 g. Furthermore, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the daily intake is preferably 10 to 1000 g, more preferably 30 to 750 g, and from the viewpoint of improving vascular flexibility and vascular endothelial function, it is particularly preferably 50 to 500 g.

The amount of the composition of the present invention to be taken at one time is not particularly limited. For example, when the composition of the present invention is a solid formulation (powder, granules, tablets, capsules, chewable tablets, etc.), the amount is preferably 0.01 to 50 g, more preferably 0.03 to 10 g, and from the viewpoint of improving vascular flexibility and vascular endothelial function, particularly preferably 0.06 to 5 g. Also, when the composition of the present invention is a liquid formulation (liquid, gel, paste, etc.), the amount is preferably 3.3 to 1000 g, more preferably 10 to 750 g, and from the viewpoint of improving vascular flexibility and vascular endothelial function, particularly preferably 16 to 500 g.

The composition of the present invention may contain only components selected from the group consisting of pine bark, ginger, elastin, and vitamin E, or may contain other components in addition to these. Examples of such other components include proteins, dietary fiber such as soluble dietary fiber and insoluble dietary fiber, vitamins other than vitamin E, minerals, plants or plant products other than pine bark and ginger, algae, lactic acid bacteria, yeast, and other microorganisms. Furthermore, as needed, ingredients commonly used in the food industry, such as sugars (dextrin, starch, etc.), oligosaccharides, sweeteners, acidulants, colorants, thickeners, glazing agents, excipients, nutritional supplements, binders, lubricants, stabilizers, diluents, bulking agents, emulsifiers, food additives, and seasonings, may also be included. The content of these other components can be appropriately selected depending on the form of the composition of the present invention.

The composition of the present invention can be used, for example, as a pharmaceutical product (including quasi-drugs), a so-called health food such as a food for specified health uses, a food with nutrient function claims, or a food with functional claims whose efficacy has been approved by a designated organization, or as a general food, food additive, feed, etc. The composition of the present invention may be taken before meals, between meals, after meals, or simultaneously with meals. However, in terms of its effects of improving vascular flexibility and vascular endothelial function, it is preferable to take it before meals, after meals, or simultaneously with meals, and more preferably before meals.

The composition of the present invention is not particularly limited as long as it can be distinguished from other products in that it is used to improve vascular flexibility or vascular endothelial function. For example, the scope of the present invention includes products that display the function of improving vascular endothelial function on the product itself, packaging, instructions, or promotional materials. Examples of such products include so-called health foods, such as pharmaceuticals (including quasi-drugs), functional foods such as specified health foods, nutritional functional foods, and functional food products whose efficacy has been approved by a designated organization, and animal feed. Examples of so-called health foods include those that "help maintain vascular flexibility (vasodilation after vasoconstriction) that declines with age," "enhance vascular function," "maintain vascular function," "enhance vascular endothelial function," "maintain vascular endothelial function," "enhance vascular endothelial function of arteries that declines with age," "maintain vascular endothelial function of arteries that declines with age," "enhance vasoconstriction after vasoconstriction," "maintain vasoconstriction after vasoconstriction," "enhance vascular flexibility," "maintain vascular flexibility," "enhance vascular flexibility," and "maintain vascular flexibility."

The composition of the present invention may be in any form. Examples include forms suitable for oral administration, such as powder, granules, tablets, liquids, gels, pastes, capsules (e.g., hard capsules and soft capsules), caplets, tablets, gels, jellies, gummies, wafers, biscuits, cookies, cakes, chewable tablets, syrups, and sticks. Depending on the intended form, various excipients, binders, lubricants, stabilizers, diluents, bulking agents, thickeners, gelling agents, emulsifiers, colorants, flavors, sweeteners, and additives may be added. From the viewpoint of the inhibitory effect on postprandial uric acid level elevation, the composition of the present invention is preferably in granular, tablet, capsule, or liquid form, with granular, tablet, and capsule forms being more preferred. Here, granular refers to a composition obtained by granulating powder. It may be consumed directly or dissolved in a liquid such as water and consumed. Additionally, gel refers to a composition that contains water and a gelling agent and has viscosity or elasticity.

The packaging form of the composition of the present invention is not particularly limited and can be selected appropriately depending on the dosage form, etc., but examples include blister packs such as PTPs, strip packaging, heat seals, aluminum pouches, film packaging using plastic or synthetic resin, glass containers such as vials, and plastic containers such as ampoules.

The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be solved.

<Tests> Evaluation of the effects of improving vascular flexibility and vascular endothelial function
In this test, the expression level of NO synthase gene was evaluated. When NO produced by NO synthase (eNOS) in the vascular endothelium diffuses and reaches the vascular smooth muscle, the muscle relaxes and the blood vessels dilate. When the expression level of NO synthase gene increases, the amount of NO synthase increases and the amount of NO produced increases, which can be expected to improve vascular flexibility and vascular endothelial function. From the above, by evaluating the expression level of NO synthase gene, the effect of improving vascular flexibility and vascular endothelial function can be evaluated.

(1) A hot water extract of pine bark containing OPCs (proanthocyanidins with a degree of polymerization of 2 to 4) was used as the test substance pine bark, crushed Kintoki ginger was used as the ginger, elastin derived from bonito was used as the elastin, and d-α-tocopherol was used as the vitamin E. Chlorogenic acid was also used as a food ingredient known to have the effects of improving vascular flexibility and vascular endothelial function. Commercially available pine bark, ginger, elastin, vitamin E, and chlorogenic acid were all used. These components were prepared in EGM-2 medium (manufactured by Lonza) in the proportions shown in Table 1 to prepare test substance-containing media for the Examples and Comparative Examples.

(2) Cell Culture Human umbilical vein endothelial cells (Lonza) were cultured in EGM-2 medium (Lonza). The cells were seeded onto a collagen-coated 96-well plate at 100 μL/well to give a density of 1 × 10 ⁴ cells/well, and pre-cultured for 24 hours in a 37°C, 5% CO ₂ incubator. After removing the medium from each well, 100 μL/well of medium containing a test substance listed in Table 1 was added, and the cells were cultured for 24 hours.

(3) Measurement of NO synthase gene expression level After removing the medium from each well, the wells were washed once with PBS and RNA was recovered using RNeasy Mini Kit (QIAGEN). One-step real-time PCR was performed on the obtained RNA using One Step TB Green (registered trademark) PrimeScript RT-PCR Kit II (Perfect Real Time). The gene expression level of NOS3 (eNOS) was measured using NOS3 (eNOS) primers (Takara). As an endogenous control, the gene expression level of GAPDH was measured using GAPDH primers (QIAGEN).

(4) Evaluation of NO synthase gene expression level The expression level of the NO synthase gene was calculated relative to the expression level of the group not administered the test substance (control) set at 1. The results are shown in Figure 1.

In the groups administered with pine bark and at least one selected from the group consisting of ginger, elastin, and vitamin E (Examples 1-7), an increase in NO synthase gene expression was observed compared to the group administered with pine bark alone (Comparative Example 1). No increase in NO synthase gene expression was observed in the group administered with ginger alone (Comparative Example 2), the group administered with elastin alone (Comparative Example 3), or the group administered with vitamin E alone (Comparative Example 4). Therefore, it was found that the intake of a composition containing at least one selected from the group consisting of pine bark, ginger, elastin, and vitamin E increases NO synthase gene expression, thereby improving vascular flexibility and vascular endothelial function. In particular, the groups administered with two or more ingredients selected from the group consisting of pine bark, ginger, elastin, and vitamin E (Examples 4 to 7) showed a more excellent increase in NO synthase gene expression, and the group administered with pine bark, ginger, elastin, and vitamin E (Example 7) showed a significantly excellent increase in NO synthase gene expression. These results demonstrate that the composition of the present invention, containing at least one ingredient selected from the group consisting of pine bark, ginger, elastin, and vitamin E, exhibits excellent vascular flexibility-improving effects and vascular endothelial function-improving effects, and that the composition containing two or more ingredients selected from the group consisting of pine bark, ginger, elastin, and vitamin E exhibits more significant vascular flexibility-improving effects and vascular endothelial function-improving effects, and that the composition containing pine bark, ginger, elastin, and vitamin E exhibits extremely significant vascular flexibility-improving effects and vascular endothelial function-improving effects. On the other hand, although the group administered only chlorogenic acid (Comparative Example 5) showed an increase in NO synthase gene expression compared to the control, the group administered pine bark and chlorogenic acid (Comparative Example 6) did not show an increase in NO synthase gene expression compared to the group administered only pine bark (Comparative Example 1) or the group administered only chlorogenic acid (Comparative Example 5), indicating that the combination of pine bark and chlorogenic acid does not have the effect of improving vascular flexibility or vascular endothelial function.

(Manufacturing example)
Based on the results of the Examples, the following Production Examples of the present invention are given below.

[Production Example 1-4: Granules]
Pine bark, at least one selected from the group consisting of ginger, elastin, and vitamin E, and other ingredients were mixed according to the formulation in Table 2, and then fluidized bed granulation was carried out using a granulator to produce the granules described in Production Examples 1-4. The granules described in Production Examples 1-4 only need to be taken at a dose of 3 g per day, and may be taken by dissolving in 100 ml of a solvent such as water, or may be taken as is without dissolving. All of the granules in Production Examples 1-4 are effective in improving vascular flexibility and vascular endothelial function.

[Production Example 5-8: Tablets]
As shown in Table 3, pine bark, at least one selected from the group consisting of ginger, elastin, and vitamin E, and other ingredients were mixed, and then tableted using a rotary tablet press to produce tablets of Production Examples 5-8. The tablets were produced with a tablet diameter of 8 mmφ, a tablet thickness of 4.5 mm, a weight of 250 mg, and a hardness of 5 kgf or more. One to two tablets of Production Examples 5-8 can be taken per day, and can be taken with 100 ml of water or the like. All tablets of Production Examples 5-8 are effective in improving vascular flexibility and vascular endothelial function.

[Production Example 9-12: Hard Capsule]
As shown in Table 4, pine bark, at least one selected from the group consisting of ginger, elastin, and vitamin E, and other ingredients were mixed, and the mixture was encapsulated with a coating containing gelatin or hydroxypropyl cellulose to produce hard capsules. Each hard capsule was produced at 300 mg. One to two capsules per day could be taken with 100 ml of water, for example. All of the hard capsules of Production Examples 9-12 were effective in improving vascular flexibility and vascular endothelial function.

[Manufacture example 13-16: PET beverage]
As shown in Table 5, a liquid preparation containing pine bark, at least one selected from the group consisting of ginger, elastin, and vitamin E, and other ingredients was mixed and packed into a PET container to produce a PET beverage. Each PET beverage was produced in a 500 ml bottle. One bottle should be consumed per day. Furthermore, all of the PET beverages of Production Examples 13-16 are effective in improving vascular flexibility and vascular endothelial function.

The composition of the present invention exhibits excellent effects of improving vascular flexibility and vascular endothelial function, and is therefore highly useful industrially.

Claims (3)

An oral composition for improving blood vessel flexibility, which contains pine bark as an active ingredient for improving blood vessel flexibility, and further contains elastin and vitamin E. An oral composition for improving vascular endothelial function, which contains pine bark as an active ingredient for improving vascular endothelial function, and further contains elastin and vitamin E. An oral composition for improving blood vessel flexibility, which contains pine bark as an active ingredient that helps maintain the flexibility of blood vessels, which decreases with age, and further contains elastin and vitamin E.