JP7095861B2 - Functional composition - Google Patents

Description

The present invention relates to a functional composition containing a plant-derived ingredient as an active ingredient, and more particularly, a composition for preventing or ameliorating oxidative stress, which comprises an active ingredient derived from asitaba and olive, and protecting or damaging the vascular endothelium. The present invention relates to a composition for promoting healing, a composition for scavenging or detoxifying active oxygen, and a composition for activating endothelial nitric oxide synthase.

Ashitaba (Ashitaba, scientific name: Angelica keiskei), a plant of the Umbelliferae family, contains xanthangelol and 4-hydroxydelicin, which belong to the chalcones, as the main functional components. In addition, olive (scientific name: Olea europaea), which is a plant of the family Oleaceae, contains oleuropein in its leaves and hydroxytyrosol in its fruits as its main functional components. Oleuropein has a hydroxytyrosol structure in the molecule, and it is known that oleuropein changes to hydroxytyrosol as the fruit matures. In addition, it is known that oleuropein ingested in vivo is partially converted to hydroxytyrosol.

According to the research by the present inventors on the functionality of the above-mentioned plant-derived components, it has been clarified that oleuropein and hydroxytyrosol have an action effect of improving ovulation disorder (Patent Document 1). Further, it has been clarified that oleuropein and hydroxytyrosol have an action and effect of promoting bone formation (Patent Document 2). Further, it has been clarified that the Angelica keiskei extract has an effect of improving ovulation disorder (Patent Document 3). Further, it has been clarified that Ashitaba extract and its functional components such as xanthangelol and 4-hydroxydelicin have an action effect of improving sperm function deterioration due to heat stress (Patent Document 4). ). Further, it has been clarified that the Angelica keiskei extract has an action and effect of preventing or ameliorating mastitis in livestock (Patent Document 5).

Japanese Unexamined Patent Publication No. 2009-191012 Japanese Unexamined Patent Publication No. 2009-2271616 Japanese Unexamined Patent Publication No. 2012-102055 Japanese Unexamined Patent Publication No. 2016-033131 JP-A-2017-071564

However, there has been no previous report on the simultaneous action of ashitaba-derived components and olive-derived components, which are known to have various functions.

An object of the present invention is to provide a technique for synergistically exerting its functionality by allowing ashitaba-derived components and olive-derived components to act simultaneously.

In order to achieve the above object, as a result of diligent research by the present inventors, various uses are used in combination with xanthangelol or 4-hydroxydelicin, which is a component derived from Ashitaba, and hydroxytyrosol, which is a component derived from olive. We have found that the effect is synergistically enhanced in the functionality of Ashitaba, and have completed the present invention.

That is, the present invention first comprises (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein simultaneously or separately as active ingredients. It provides a characteristic functional composition.

Secondly, the present invention comprises (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein simultaneously or separately as active ingredients. It is intended to provide a composition for preventing or ameliorating characteristic oxidative stress.

The present invention also includes, thirdly, (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein simultaneously or separately as active ingredients. It provides a composition for protecting the vascular endothelium or promoting injury healing.

Fourth, the present invention comprises (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein simultaneously or separately as active ingredients. It is intended to provide a composition for scavenging or detoxifying active oxygen, which is a characteristic.

Fifth, the present invention also comprises (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein simultaneously or separately as active ingredients. It is an object of the present invention to provide a composition for activating an endothelial nitric oxide synthase.

In the composition according to the present invention, the xanthangelol and / or the 4-hydroxydelicin is derived from Ashitaba, and / or the hydroxytyrosol and / or the oleuropein is derived from olive. Is preferable.

Further, in the above composition according to the present invention, the composition is preferably in the form of food, pharmaceutical, quasi-drug, cosmetic, supplement, veterinary drug or feed.

According to the present invention, xanthangelol and / or 4-hydroxydelicin, which are components derived from Ashitaba, and oleuropein and / or hydroxytyrosol, which are components derived from olive, are used in combination, and thus, in various functionalities. , The effects of these ingredients are synergistically enhanced. Therefore, utilizing this, a composition for preventing or improving oxidative stress, a composition for protecting or promoting damage healing of vascular endothelium, a composition for scavenging or detoxifying active oxygen, and activity of endothelial nitrogen monoxide synthase. Various functional compositions such as a chemical composition can be provided. In addition, it is possible to provide foods, pharmaceuticals, quasi-drugs, cosmetics, supplements, veterinary drugs, feeds and the like with enhanced functionality.

In Test Example 1, it is a chart which shows the result of having evaluated the influence of the test substance on the oxidative stress resistance of vascular endothelial cells by the measurement of the cell viability by trypan blue staining. In Test Example 2, it is a chart which shows the result of having evaluated the influence which a test substance has on the wound healing promotion effect of a vascular endothelial cell by observation under a microscope. FIG. 3 is a chart showing the results of evaluation of the effect of a test substance on the glutathione peroxidase activity of vascular endothelial cells in Test Example 3 by in vitro enzyme activity measurement using a total protein extract of vascular endothelial cells. In Test Example 4, it is a chart which shows the result of having evaluated the influence of the test substance on the activation of the endothelial type nitrogen monoxide synthase of the vascular endothelial cell by the western blot analysis using the total protein extract of the vascular endothelial cell. FIG. 4 (a) is a photograph of a band corresponding to p-eNOS (active eNOS) or α-tubulin detected on a western blot membrane by developing a total protein extract of vascular endothelial cells by SDS gel electrophoresis. 4 (b) is a graph in which the relative expression level is obtained from the band intensity and plotted.

In the present invention, as the first active ingredient, xanthoangelol represented by the following chemical formula (1) and / or 4-hydroxyderricin represented by the following chemical formula (2) are used. .. As the first active ingredient, either of these may be used alone, or both may be used in combination.

In the present invention, hydroxytyrosol represented by the following chemical formula (3) and / or oleuropein represented by the following chemical formula (4) are used as the second active ingredient. As the second active ingredient, either of these may be used alone, or both may be used in combination. It is known that oleuropein has a hydroxytyrosol structure in the molecule, and oleuropein is partially converted to hydroxytyrosol in vivo.

As the above compound, a chemically synthesized product may be used, but from the viewpoint of availability and safety when administered to a living body, it is preferable to use a compound derived from a natural product.

Ashitaba (Ashitaba, scientific name Angelica keiskei), a plant of the Umbelliferae family, has been edible for a long time and is rich in xanthangelol and / or 4-hydroxydelicin. Therefore, it is preferable to use such a plant-derived material as the first active ingredient used in the present invention. However, the base may be any plant containing these compounds other than Angelica keiskei, and is not particularly limited.

There are no particular restrictions on the varieties used and the cultivation area of Ashitaba, but if a specific variety is mentioned as the base, for example, there is "Rie" of Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 14641. According to this, it is possible to obtain a material containing xanthangelol and / or 4-hydroxydelicin having stable quality. The site of Ashitaba to be used is not particularly limited as long as the object of the present invention is not impaired, and examples thereof include whole plants, or sites such as leaves, stems, rhizomes, inflorescences, fruits, and seeds. These may be one kind or two or more kinds of selected sites, and may be used in combination as appropriate. Of these, leaves, stems and rhizomes are particularly preferable from the viewpoint of the content and yield of the target component. Although it depends on the variety used and the harvest time, for example, Ashitaba leaves usually contain about 0.02% by mass to 0.20% by mass of xanthangelol in terms of dry weight. Further, 4-hydroxydelicin is contained in an amount of about 0.01% by mass to 0.10% by mass. Ashitaba stalks usually contain about 0.05% by mass to 0.40% by mass of xanthangelol in dry weight. Further, 4-hydroxydelicin is contained in an amount of about 0.05% by mass to 0.35% by mass. Ashitaba root usually contains about 0.10% by mass to 0.30% by mass of xanthangelol in dry weight. Further, 4-hydroxydelicin is contained in an amount of about 0.10% by mass to 0.35% by mass.

As the first active ingredient used in the present invention, the plant body of the active ingredient such as Angelica keiskei may be used as it is, for example, in the form of dried powder of a part such as a leaf, a stem, and / or a rhizome, or It may be used in the form of a squeezed juice obtained by pressing or a processed product obtained by concentrating, drying, powdering or the like. However, from the viewpoint of increasing the content of xanthangelol and / or 4-hydroxydelicin, the extract thereof is more preferably used.

Extraction can be performed by a conventionally known method. For example, the whole plant of Ashitaba or one or more kinds of parts of Ashitaba as a basic plant, or a squeezed solution thereof, or a processed product obtained by concentrating, drying, powdering, etc., is placed at a low temperature in an appropriate solvent. Alternatively, it can be carried out by immersing in a warm state for a predetermined time or heating and refluxing. The obtained primary extract may be subjected to filtration, concentration, purification / separation using an ion exchange resin, liquid chromatography, or the like, freeze-drying, or the like, if necessary. According to these means such as fractionation / purification, the content of xanthangelol and / or 4-hydroxydelicin can be appropriately used as an index to increase the content thereof.

The solvent used for extraction is not particularly limited, and one type or two or more types of solvents usually used for plant extraction can be selected and used. For example, water, alcohols, glycols, ketones, esters, ethers, halogenated carbons and the like can be mentioned. Examples of alcohols include ethanol, methanol, propanol and the like. Examples of glycols include ethylene glycol, diethylene glycol, butylene glycol, propylene glycol, glycerin and the like. Examples of the ketone include acetone, methyl ethyl ketone and the like. Examples of the esters include ethyl acetate, propyl acetate, ethyl formate and the like. Examples of the ketone include acetone, methyl ethyl ketone and the like. Examples of the esters include ethyl acetate, propyl acetate, ethyl formate and the like. Examples of ethers include diethyl ether and the like. Examples of the halogenated carbons include chloroform, dichloromethane and the like.

The obtained extract may be prepared and used in any form such as liquid (juice), paste, gel, oil, and emulsion. Alternatively, it may be dried to a solid state, or may be dried by freeze-drying, spray-drying, or the like to form a powder. In this case, a drying aid such as dextrin or an emulsifier such as sucrose fatty acid ester may be added.

As the first active ingredient used in the present invention, a material containing xanthangelol and / or 4-hydroxydelicin in an amount of 0.01% by mass to 100% by mass in terms of dry solid content is used. Can be done. For example, in the case of a primary extract from a plant, ashitaba leaf powder itself, etc., the upper limit may be typically 20% by mass or less, but more typically 10% by mass or less. It may be present, and more typically, it may be 5% by mass or less. The lower limit is typically 0.01% by mass or more, more typically 0.1% by mass or more, and even more typically 0.2% by mass or more. May be good. Further, for example, in the case of a material obtained by further refining from a primary extract from a plant or ashitaba leaf powder itself, the upper limit thereof may be typically 100% by mass or less. However, it may be more typically 90% by mass or less, and even more typically 80% by mass or less. The lower limit may be typically 50% by mass or more, more typically 60% by mass or more, and even more typically 70% by mass or more.

On the other hand, olive (scientific name: Olea europaea), which is a plant of the family Oleaceae, has been edible for a long time and is rich in hydroxytyrosol and / or oleuropein. In particular, the leaves contain oleuropein and the fruits contain hydroxytyrosol. Therefore, it is preferable to use such a plant-derived material as the second active ingredient used in the present invention. However, since hydroxytyrosol and oleuropein are contained in Enju, Ligustrum obtusa, etc. in addition to olive, those derived from these may be used as the base, and there is no particular limitation.

As for olives, there are no particular restrictions on the varieties and cultivation areas used. The part of the olive to be used is not particularly limited as long as the object of the present invention is not impaired, and examples thereof include parts such as fruits, leaves, flowers, stems and roots. These may be one kind or two or more kinds of selected sites, and may be used in combination as appropriate. Of these, fruits and leaves are particularly preferable from the viewpoint of the content and yield of the target component. Although it depends on the variety used and the harvest time, for example, olive oil usually contains about 5 mg / kg to 150 mg / kg of hydroxytyrosol. Further, for example, olive leaves usually contain about 5% by mass to 20% by mass of oleuropein in terms of dry weight.

As the second active ingredient used in the present invention, the plant body of the original plant such as olive may be used as it is, for example, in the form of dried powder of parts such as fruits and / or leaves, or may be pressed. The juice may be used in the form of a processed product obtained by concentrating, drying, powdering or the like. However, from the viewpoint of increasing the content of hydroxytyrosol and / or oleuropein, the extract thereof is more preferably used. Here, in the normal olive oil production process, a large amount of vegetable water, treated water and squeezed residue are generated when olive oil is squeezed from olive fruits, and hydroxytyrosol and / or squeezed residue is generated from such by-products. You can also extract olive oil. Further, since hydroxytyrosol and / or oleuropein, particularly hydroxytyrosol, is also contained in olive oil, it may be extracted from olive oil and, in some cases, olive oil itself may be used.

Extraction can be performed by a conventionally known method. For example, one or more parts of olive as a basic plant, or a juice squeezed thereof, or a processed product obtained by concentrating, drying, powdering, etc., is subjected to low temperature or heating in an appropriate solvent. It can be done by immersing underneath for a predetermined time or heating and refluxing. The obtained primary extract may be subjected to filtration, concentration, purification / separation using an ion exchange resin, liquid chromatography, or the like, freeze-drying, or the like, if necessary. According to these means such as fractionation / purification, the content of hydroxytyrosol and / or oleuropein can be appropriately used as an index to increase the content thereof.

The solvent used for extraction is not particularly limited, and one type or two or more types of solvents usually used for plant extraction can be selected and used. For example, water, alcohols, glycols, ketones, esters, ethers, halogenated carbons and the like can be mentioned. Examples of alcohols include ethanol, methanol, propanol and the like. Examples of glycols include ethylene glycol, diethylene glycol, butylene glycol, propylene glycol, glycerin and the like. Examples of the ketone include acetone, methyl ethyl ketone and the like. Examples of the esters include ethyl acetate, propyl acetate, ethyl formate and the like. Examples of the ketone include acetone, methyl ethyl ketone and the like. Examples of the esters include ethyl acetate, propyl acetate, ethyl formate and the like. Examples of ethers include diethyl ether and the like. Examples of the halogenated carbons include chloroform, dichloromethane and the like.

The obtained extract may be prepared and used in any form such as liquid (juice), paste, gel, oil, and emulsion. Alternatively, it may be dried to a solid state, or may be dried by freeze-drying, spray-drying, or the like to form a powder. In this case, a drying aid such as dextrin or an emulsifier such as sucrose fatty acid ester may be added.

As the second active ingredient used in the present invention, a material containing hydroxytyrosol and / or oleuropein in an amount of 1% by mass to 100% by mass in terms of dry solid content can be used. For example, in the case of a primary extract from a plant, olive oil itself, etc., the upper limit may be typically 40% by mass or less, but more typically 30% by mass or less. It may be more preferably 20% by mass or less. The lower limit may be typically 0.1% by mass or more, more typically 5% by mass or more, and even more typically 7% by mass or more. Further, for example, in the case of a material obtained by further refining from a primary extract from a plant body or olive oil itself, the upper limit thereof may be typically 100% by mass or less. , More typically 90% by mass or less, and even more typically 80% by mass or less. The lower limit may be typically 50% by mass or more, more typically 60% by mass or more, and even more typically 70% by mass or more.

The functional composition according to the present invention contains (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein as active ingredients simultaneously or separately. .. Here, the "functional composition" means that it is intended to act on a living body of a human or an animal. Further, "contains simultaneously or separately" may mean that the above (1) and (2) are contained in the same form as an active ingredient, and that the above (1) and (2) are contained in different forms as an active ingredient. The meaning is to include the case where the action is to be made to act on the living body at the same time, without a short time, or in sequence at an arbitrary time.

For example, in the present invention, when the above (1) and (2) are contained in the same form as active ingredients, xanthangelol and / or 4-hydroxydelicin as the above (1) in the total amount thereof. It may be in the form of containing 0.01% by mass to 99% by mass in terms of dry solid content. The upper limit may be 99% by mass, but typically 95% by mass or less, more typically 93% by mass or less, and even more typically 80% by mass or less. In particular, it may be 70% by mass or less, and most typically 60% by mass or less. The lower limit may be 0.01% by mass, but typically 0.1% by mass or more, more typically 0.2% by mass or more, and even more typical. It may be 5% by mass or more, particularly typically 10% by mass or more, and most typically 20% by mass or more. Further, as the above (2), hydroxytyrosol and / or oleuropein may be contained in an amount of 1% by mass to 99.99% by mass in terms of dry solid content in the total amount. The upper limit may be 99.99% by mass, but typically 90% by mass or less, more typically 80% by mass or less, and even more typically 70% by mass. % Or less, particularly typically 60% by mass or less, and most typically 50% by mass or less. The lower limit may be 1% by mass, but typically 5% by mass or more, more typically 7% by mass or more, and particularly typically 20% by mass or more. It may be present, and most typically, it may be 30% by mass or more. In addition, as the compounding ratio of the above (1) and the above (2), the dry solid content equivalent amount of the total amount of xanthangelol and / or 4-hydroxydelicin and the total amount of hydroxytyrosol and / or oleuropein. The dry solid content conversion amount can be contained in a ratio of 100: 1 to 1: 100, more typically in a ratio of 20: 1 to 1:20, and even more typically. Can be contained in a ratio of 5: 1 to 1: 5.

For example, in the present invention, when the above (1) and the above (2) are contained in separate forms as active ingredients, the first form thereof includes xanthangelol and / or 4- or 4- as the above (1). It may be in the form of containing hydroxydelicin in an amount of 0.01% by mass to 100% by mass in terms of dry solid content in the total amount. The upper limit may be 100% by mass, but typically 90% by mass or less, more typically 80% by mass or less, and even more typically 70% by mass or less. It may be 60% by mass or less, and most typically 50% by mass or less. The lower limit may be 0.01% by mass, but typically 0.1% by mass or more, more typically 0.2% by mass or more, and even more typical. It may be 5% by mass or more, particularly typically 10% by mass or more, and most typically 20% by mass or more. In addition, the second form may contain hydroxytyrosol and / or oleuropein as the above (2) in an amount of 1% by mass to 100% by mass in terms of dry solid content. The upper limit may be 100% by mass, but typically 90% by mass or less, and more typically 80% by mass or less. Furthermore, it may be more typically 70% by mass or less, particularly typically 60% by mass or less, and most typically 50% by mass or less. The lower limit may be 1% by mass, but typically 5% by mass or more, more typically 7% by mass or more, and particularly typically 20% by mass or more. It may be present, and most typically, it may be 30% by mass or more.

In the functional composition according to the present invention, the above (1) xanthangelol and / or 4-hydroxydelicin and the above (2) hydroxytyrosol and / or oleuropein are allowed to act on a living body of a human or an animal. And use it like this. In that case, the above (1) and the above (2) may be contained in the same form as an active ingredient and administered to humans or animals, or the above (1) and the above (2) may be administered. ) And the active ingredient may be contained in separate forms, which may be administered to humans or animals at the same time, at short intervals, or at arbitrary intervals in sequence. From the viewpoint of ease of daily administration, it is more preferable to include the above (1) and (2) as active ingredients in the same form and administer them to humans or animals.

The administration form is not particularly limited, and any administration form such as oral, intravenous, enteral, nasal, abdominal cavity, transdermal, transpulmonary, oral cavity, and external use may be used. In this case, a dosage form prepared to contain the above-mentioned active ingredient may be formed by a well-known pharmaceutical means together with an appropriate pharmaceutical substrate and the like so as to be suitable for the various administration forms. For example, in dosage forms such as powders, granules, soft capsules, tablets, granules, powders, liquids, hard capsules, jelly-like agents, troches, oral disintegrants, injections, inhalants, suppositories, coatings, etc. In addition, there is no particular limitation on the use of the above active ingredient contained therein. Further, the above (1) and the above (2) may be administered in the same administration form, or may be administered in different administration forms.

From the viewpoint of ease of daily administration, the functional composition according to the present invention is preferably used so as to be taken orally. Therefore, if necessary, powders, granules, soft capsules, tablets, granules, powders, liquids, hard capsules, jelly-like agents, troches, orally disintegrating agents are used using substrates and carriers that are acceptable for oral ingestion. Etc. can be in the form of a composition for oral ingestion.

The dose may be appropriately set according to the desired type of functionality and conditions such as the age, gender, and health condition of the ingestor, and although it is not unconditional, in the case of oral administration, for example, an adult adult. Xanthangelol and / or 4-hydroxydelicin as described in (1) above can be administered in the range of 1 mg to 1000 mg in terms of the total amount thereof, and more typically in the range of 1 mg to 100 mg per day. It can be administered, and more typically in the range of 1 mg to 10 mg. In addition, hydroxytyrosol and / or oleuropein can be administered in the range of 1 mg to 1000 mg in terms of the total amount, and more typically in the range of 1 mg to 100 mg, as described in (2) above. And even more typically in the range of 1 mg to 10 mg. In addition, as the administration ratio between the above (1) and the above (2), the dry solid content equivalent amount of the total amount of xanthangelol and / or 4-hydroxydelicin and the total amount of hydroxytyrosol and / or oleuropein. The dry solid content equivalent can be administered at a ratio of 100: 1 to 1: 100, more typically at a ratio of 20: 1 to 1:20, and even more typically. Can be administered at a ratio of 5: 1 to 1: 5.

According to the results of Examples described later, the functional composition according to the present invention is, in particular, a composition for preventing or ameliorating oxidative stress, a composition for protecting or promoting injury healing of vascular endothelium, scavenging of active oxygen or It is more preferable to use it as a detoxification composition, an activation composition for endothelial nitrogen monoxide synthase, and the like.

Here, "prevention or improvement of oxidative stress" means that in the living body, various stresses such as smoking, excessive exercise, mental stress, lack of sleep, and aging are converted into oxidative stress in the body, and often. Since this causes damage to health, it means to prevent or reduce the degree of stress. In livestock, summer heat stress is converted into oxidative stress in the body and causes reproductive disorders in males and females, and prevention or reduction thereof is also included. The same is true for stress-dependent reproductive disorders in humans.

In addition, "protection of vascular endothelium or promotion of injury healing" means that in the living body, various blood vessels such as hypertension, hypertension, and high cholesterol plasma are loaded, and this is often atherosclerosis and angina. , It causes various diseases such as myocardial infarction and stroke, and causes damage to health, so it is meant to include prevention or reduction of the degree thereof.

In addition, "elimination or detoxification of active oxygen" means that various active oxygen species such as superoxide, hydroxy radicals, hydrogen peroxide, and singlet oxygen are generated on a daily basis in living organisms, which often impair health. Since it causes a cause, it means to include preventing or reducing the degree of it. Specifically, the scavenging effect of the compound eliminates or detoxifies active oxygen species, or efficiently eliminates or detoxifies active oxygen species through increased expression of antioxidant enzymes, and suppresses the production of active oxygen species. Etc.

In addition, "activation of endothelial nitric oxide synthase" means that in living organisms, especially in tissues such as vascular endothelium, endothelial nitric oxide synthase (eNOS) is used on a daily basis. Nitric oxide is produced to protect blood vessels from damage caused by oxidative stress, excessive contraction, inflammatory cytokines, etc. and maintain homeostasis, but it is often healthy if it is inactive or its production is low. Since it causes harm to the disease, it means to prevent it or reduce its degree.

The functional composition according to the present invention is particularly preferably applied to a healthy human or animal.

The form of use of the above composition is not particularly limited as long as its action and effect are not impaired. For example, it can be used in various forms such as foods, pharmaceuticals, quasi-drugs, cosmetics, supplements, veterinary drugs, feeds, etc., or in combination with these products. Here, the food includes a nutritional supplement, a food with a functional claim, a food for specified health use, or a food additive for those foods, and the feed also includes a feed additive and the like.

Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

(material)
Xanthoangelol (hereinafter abbreviated as "XA") and 4-Hydroxyderricin (hereinafter abbreviated as "4HD") are Japan Biological Research Institute Co., Ltd. After extraction with 80% Methanol (methanol) from "Tomorrow's leaf polyphenol CHALSAP" obtained from Mfg. Co., Ltd., HPLC (High Performance Liquid Chromatography) was repeated twice to purify the product to a purity of 95% or higher.

Here, the HPLC conditions are as follows.
<Purification conditions by HPLC>
Column: Intersil OSD-4 (10.0 x 250 mm)
Mobile phase: 80% Methanol
Flow rate: 4.0 ml / min
Solvent: Methanol
Detection: 280 nm
Injection amount: 0.75 ml

Hydroxytyrosol (Hydroxytyrosol) Hereinafter, it is abbreviated as "HT". ) Was obtained from Cayman.

Vascular endothelial cells were collected from the thoracic thoracic aorta of pigs purchased from the Tsuchiura Meat Cooperative, isolated and cultured. Specifically, the thoracic aorta of a pig purchased from the Tsuchiura Meat Cooperative was washed with PBS (-) and then immersed in PBS (-) containing an antibiotic for 30 minutes or more for sterilization. The sterilized blood vessels were cut open with scissors, and cells on the surface of the intima of the blood vessels were collected with a scalpel and suspended in DMEM medium. Then, the cells were collected by centrifugation at 1,500 rpm for 5 minutes, the cells were seeded in a 6 cm dish, and cultured at 37 ° C. in a 5% CO ₂ environment. For culturing, DMEM medium containing 10% FBS (Fetal Bovine Serum), 1% P / S (penicillin-streptomycin solution), and glucose at a concentration of 1.0 g / L was used. In the experiment, cells with passages from 7 to 10 generations were used.

Reduced glutathione (GSH: glutathione) was obtained from Wako Pure Chemical Industries, Ltd.

Glutathione reductase (GR: Glutathione reductase) was obtained from Sigma-Aldrich.

tert-Butyl hydroperoxide (t-BuOOH) was obtained from Sigma-Aldrich.

Specific antibodies against phosphorylated eNOS (p-eNOS), which is an active form of endothelial nitrogen monoxide synthase (eNOS) for Western blot analysis, were obtained from Sigma-Aldrich.

Specific antibodies against α-tubulin for Western blot analysis were obtained from Calbiochem.

<Test Example 1>
The effect of the test substance on the oxidative stress resistance of vascular endothelial cells was evaluated by measuring the cell viability by trypan blue staining. Specifically, vascular endothelial cells were seeded on a 24-well plate at 5 × 10 ⁴ cells / well, adhered for 7 hours, and then subjected to synchronized culture for 17 hours in DMEM medium containing 1% FBS (Fetal Bovine Serum). After that, each test substance was added and cultured for 24 hours. After washing twice with PBS (-) to remove each test substance, DMEM medium containing 1% FBS (Fetal Bovine Serum) and _H202 at a _final concentration of 800 μM was added, and the cells were cultured for 24 hours. Cells are stripped from the dish using trypsin EDTA solution, harvested, mixed with 0.5% trypan blue stain, and cells using the automated cell counter "Countess® 11FL Automated Cell Counter (Thermo Fisher Scientific). Survival was measured.

The test substances to be added to the vascular endothelial cells were added to the medium so that the final concentration of HT was 2 μM and that of XA and 4HD was 0.3 μM. _In addition, as a control, a reference control in which _H202 was not added and a comparative control in which _H202 was added but the test substance was not added _were carried out. The test was conducted in 4 cases for each test substance and control, and the relative survival rate when the cell survival rate of the reference control was 100% was determined. The results are shown by mean ± SE (Standard Error). The comparison of each population was performed by Tukey-Kramer test or t-test, and it was judged to be significant when p <0.05. The results are shown in FIG.

As shown in FIG. 1 (a), the cell viability of the vascular endothelial cells of the comparative control treated with _H202 at a _final concentration of 800 μM decreased to _{approximately} 57%, and that of the reference control not treated with _H202 . Compared with vascular endothelial cells, cell damage due to oxidative stress was observed. On the other hand, when vascular endothelial cells were treated alone with HT having a final concentration of ₂ μM or XA having a final concentration of 0.3 μM and then treated with _H202 , the cell viability was about 63% and 59%, respectively. Yes, there was not much difference from the result of the comparative control (in FIG. 1 (a), "b" indicates that no significant difference from the comparative control was observed). On the other hand, the cell viability when the vascular endothelial cells were treated in combination with HT having a final concentration of ₂ μM and XA having a final concentration of 0.3 μM and then treated with _H202 was about 90%, which is a comparative control. (In FIG. 1 (a), "a" indicates that a significant difference from the comparative control was observed at p <0.05).

FIG. 1B shows the result of comparing the sum of the increase in the cell viability due to the single treatment of HT or XA and the increase in the cell viability due to the combined treatment of HT and XA. From this FIG. 1 (b), it can be seen that HT and XA act synergistically to significantly increase the oxidative stress resistance of vascular endothelial cells.

Further, as shown in FIG. 1 (c), the cell viability of the vascular endothelial cells of the comparative control obtained by treating the vascular endothelial cells with _H202 at a _final concentration of 800 μM decreased to about 49%, and the final concentration was previously reached. The cell viability when treated alone with ₂ μM HT and then treated with _H202 was approximately 60% (as shown in FIG. 1A), whereas the final concentration was 0.3 μM in advance. The cell viability was about 65% when treated alone with _4HD and then treated with _H202 . Therefore, similarly, there was not much difference from the result of the comparative control (in FIG. 1 (c), "b" indicates that no significant difference from the comparative control was observed). On the other hand, the cell viability when the vascular endothelial cells _were treated in combination with HT having a final concentration of ₂ μM and 4HD having a final concentration of 0.3 μM and then treated with H202 was about 88%, which is a comparative control. (In FIG. 1 (c), "a" indicates that a significant difference from the comparative control was observed at p <0.05).

FIG. 1 (d) shows the result of comparing the sum of the increase in the cell viability due to the single treatment of HT or 4HD with the increase in the cell viability due to the combined treatment of HT and 4HD. From this FIG. 1 (d), it can be seen that HT and 4HD acted synergistically to significantly increase the oxidative stress resistance of vascular endothelial cells.

<Test Example 2>
The effect of the test substance on the wound healing promoting action of vascular endothelial cells was evaluated by observation under a microscope. Specifically, vascular endothelial cells were seeded in a 3.5 cm dish at 3 × 10 ⁵ cells / dish, adhered for 7 hours, and then subjected to synchronized culture for 17 hours in DMEM medium containing 1% FBS (Fetal Bovine Serum). And each test substance was added. After the addition of each test substance, a pseudo-scratch was made on the cells adhered to the dish surface almost confluently using the tip of a 200 μL chip, and the cells were observed and photographed under a microscope. The image was taken again 24 hours later, and the degree of wound repair was evaluated by counting the number of cells existing within the range of the wound initially created.

The test substances to be added to the vascular endothelial cells were added to the medium so that the final concentration of HT was 20 μM and that of XA and 4HD was 0.3 μM. In addition, as a control, a comparative control without adding the test substance was provided. The test was conducted in 3 cases for each test substance and control. In each test, imaging was performed for each of the test substances and controls for 3 fields of view per dish, and each field of view was relative to the number of cells existing within the range of the wound observed in the case of comparative control as 100%. Wound healing rate was determined. The results are shown by mean ± SE (Standard Error). Comparison of each population was performed by Dunnett's test and judged to be significant when p <0.05. The results are shown in FIG.

As shown in FIG. 2A, the wound healing rates when vascular endothelial cells were treated alone with HT at a final concentration of 20 μM or XA at a final concentration of 0.3 μM were approximately 110% and 118%, respectively. There was not much difference from the result of the comparative control (in FIG. 2 (a), "a" indicates that no significant difference from the comparative control was observed). On the other hand, the wound healing rate when the vascular endothelial cells were treated in combination with HT having a final concentration of 2 μM and XA having a final concentration of 0.3 μM was about 167%, and the wound healing was remarkable as compared with the comparative control. (In FIG. 2A, "b" indicates that a significant difference from the comparative control was observed at p <0.05).

FIG. 2B shows the result of comparing the sum of the increase in the wound healing rate by the single treatment of HT or XA and the increase in the wound healing rate by the combined treatment of HT and XA. From this FIG. 2 (b), it can be seen that HT and XA act synergistically to significantly enhance the wound healing promoting effect of vascular endothelial cells.

Further, as shown in FIG. 2 (c), the wound healing rate when the vascular endothelial cells were treated alone with HT having a final concentration of 20 μM was about 103% (FIG. 2 (a)). (As shown), the wound healing rate was about 145% when treated alone with 4HD having a final concentration of 0.3 μM in advance, and the wound healing promoting effect was enhanced as compared with the comparative control (in FIG. 2 (c), No significant difference from the comparative control was observed as shown by "a"). On the other hand, the wound healing rate when the vascular endothelial cells were treated in combination with HT having a final concentration of 20 μM and 4HD having a final concentration of 0.3 μM was about 242%, and the wound healing promoting effect was even more remarkable as compared with the comparative control. (In FIG. 2 (c), "b" indicates that a significant difference from the comparative control was observed at p <0.05).

FIG. 2D shows the result of comparing the sum of the increase in the wound healing rate by the single treatment of HT or 4HD and the increase in the wound healing rate by the combined treatment of HT and 4HD. From this FIG. 2 (d), it can be seen that HT and 4HD act synergistically to significantly enhance the wound healing promoting effect of vascular endothelial cells.

<Test Example 3>
The effect of the test substance on the activity of glutathione peroxidase (hereinafter abbreviated as "GPx") of vascular endothelial cells was evaluated by in vitro enzyme activity measurement using a total protein extract of vascular endothelial cells. .. Specifically, vascular endothelial cells were seeded in a 3.5 cm dish at 3 × 10 ⁵ cells / dish, adhered for 7 hours, and then subjected to synchronized culture for 17 hours in DMEM medium containing 1% FBS (Fetal Bovine Serum). After that, each test substance was added and cultured for 24 hours. After washing twice with PBS (-) to remove each test substance, the substance was immediately frozen and stored at -80 ° C. Then, in each dish, elution buffer (50mM HEPES, pH7.5, 50mM NaC1, 1mM EDTA, 50% Glycero1, 100mM NaF, 10mM Sodium pyrophosphate, 1% TritonX-100, 1mM NaVO ₄ , 1mM PMSF, 10pg / mL Antipain, (10 μg / mL Leupeptin, 10 pg / mL Aprotinin) was added at 150 μL / dish, and the cells were allowed to stand for 5 minutes, and then the cells were separated and collected with a scraper. After allowing to stand on ice for 15 minutes, centrifugation was performed at 4 ° C. and 14,000 rpm for 15 minutes, and the collected supernatant was used as the final sample.

The test substances to be added to the vascular endothelial cells were added to the medium so that the final concentration of HT was 2 μM and the final concentration of XA was 0.3 μM. In addition, as a control, a comparative control without adding the test substance was carried out. The test was carried out in 4 cases for each test substance and control, and the relative activity increase ratio when the GPx activity of the comparative control was 1 times was determined. The results are shown by mean ± SE (Standard Error). The comparison of each population was judged to be significant when p <0.05 by performing Games Howell or t-test.

The measurement of GPx activity was performed as follows. That is, 50 μL of 1 × TE (Tris buffer, pH 8.0) was added to Eppen to be measured, 290 μL of double-distilled water was added, and 10 μL of 0.1 M reduced glutathione (GSH: glutathione) was added to 10 U / mL glutathione reductase (10 U / mL glutathione reductase). GR: Glutathione reductase) was added in an amount of 50 μL, 2 mM NADPH was added in an amount of 50 μL, and the mixture was placed at 37 ° C. for 2 minutes. .. The measurement was performed every 30 seconds at a setting of 300 seconds, and the change in absorbance over time was followed. For the measurement blank, 1 × TE (Tris buffer, pH 8.0), 0.1 M reduced glutathione (GSH: glutathione), 10 U / mL glutathione reductase (GR: Glutathione reductase), and 50 μL of double-distilled water, respectively. A combination of 10 μL, 50 μL and 390 μL was used. After the measurement, the value obtained by dividing the amount of change in the absorption value in each sample by the amount of protein was defined as the enzyme activity. The results are shown in FIG.

As shown in FIG. 3 (a), the GPx activity increase ratios when the vascular endothelial cells were treated alone with HT having a final concentration of 2 μM or XA having a final concentration of 0.3 μM were approximately 1.03 times and approximately 1.03 times, respectively. It was 1.10 times, and there was not much difference from the result of the comparative control (in FIG. 3 (a), "a" indicates that no significant difference from the comparative control was observed). On the other hand, when the vascular endothelial cells were treated in combination with HT having a final concentration of 2 μM and XA having a final concentration of 0.3 μM, the GPx activity increase rate was about 2.32 times, which was remarkable as compared with the comparative control. A significant increase was observed (in FIG. 3A, "b" indicates that a significant difference from the comparative control was observed at p <0.05).

FIG. 3B shows the result of comparing the sum of the increase in the GPx activity increase rate due to the single treatment of HT or XA and the increase in the GPx activity increase rate due to the combined treatment of HT and XA. From this FIG. 3 (b), it can be seen that HT and XA acted synergistically to significantly increase the GPx activity of vascular endothelial cells.

<Test Example 4>
The effect of the test substance on the activation of endothelial nitric oxide synthase (hereinafter abbreviated as "eNOS") of vascular endothelial cells was examined using a total protein extract of vascular endothelial cells. It was evaluated by Western blot analysis. Specifically, a total protein extract was prepared in the same manner as in Test Example 3 except that the final concentration of HT added to the vascular endothelial cells was 20 μM and the culture time after adding the test substance was 12 hours. , This is subjected to SDS gel electrophoresis according to a conventional method, and Western blot analysis using a specific antibody against phosphorylated eNOS (hereinafter abbreviated as “p-eNOS”), which is an active form of eNOS, is performed. The expression level was measured. In addition, α-Tubulin was used as an internal standard for the amount of test protein in Western blot analysis, and the strength of the obtained band was measured by Image Studio Software, and the relative expression level was obtained. Asked. The test was carried out in 3 cases for each test substance and control, and the relative expression level increase rate was determined when the p-eNOS amount of the comparative control was set to 1 time. The results are shown by mean ± SE (Standard Error). Comparison of each population was performed by Dunnett's test and judged to be significant when p <0.05. The results are shown in FIG.

As shown in FIGS. 4 (a) and 4 (b), the p-eNOS production increase ratio when the vascular endothelial cells were treated alone with HT having a final concentration of 20 μM or XA having a final concentration of 0.3 μM, respectively. It was about 1.22 times and 1.60 times, and only a slight increase was observed from the comparative control. On the other hand, when the vascular endothelial cells were treated in combination with HT having a final concentration of 20 μM and XA having a final concentration of 0.3 μM, the increase ratio of p-eNOS production was about 2.27 times, which was p compared to the comparative control. -A significant increase in eNOS production was observed.

Claims (12)

The active ingredients include (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein, which is converted to the hydroxytyrosol in vivo, simultaneously or separately. A composition for preventing or ameliorating oxidative stress. The active ingredients include (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein, which is converted to the hydroxytyrosol in vivo, simultaneously or separately. A composition for protecting vascular endothelium or promoting injury healing. The active ingredients include (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein, which is converted to the hydroxytyrosol in vivo, simultaneously or separately. A composition for scavenging or detoxifying active oxygen. The active ingredients include (1) xanthangelol and / or 4-hydroxydelicin and (2) hydroxytyrosol and / or oleuropein, which is converted to the hydroxytyrosol in vivo, simultaneously or separately. A composition for activating endothelial nitric oxide synthase. The oxidation according to claim 1 , wherein the xanthangelol and / or the 4-hydroxydelicin is derived from Ashitaba, and / or the hydroxytyrosol and / or the oleuropein is derived from olive. A composition for preventing or ameliorating stress. The blood vessel according to claim 2 , wherein the xanthangelol and / or the 4-hydroxydelicin is derived from Ashitaba, and / or the hydroxytyrosol and / or the oleuropein is derived from olive. A composition for protecting the endothelium or promoting damage healing. The activity according to claim 3 , wherein the xanthangelol and / or the 4-hydroxydelicin is derived from Ashitaba, and / or the hydroxytyrosol and / or the oleuropein is derived from olive. A composition for scavenging or detoxifying oxygen. The endothelium according to claim 4 , wherein the xanthangelol and / or the 4-hydroxydelicin is derived from Ashitaba, and / or the hydroxytyrosol and / or the oleuropein is derived from olive. A composition for activating type nitric oxide synthase. The composition for preventing or ameliorating oxidative stress according to claim 1 or 5 , which is in the form of foods, pharmaceuticals, quasi-drugs, cosmetics, supplements, veterinary drugs or feeds. The composition for protecting the vascular endothelium or promoting damage healing according to claim 2 or 6 , which is in the form of a food, a drug, a quasi drug, a cosmetic, a supplement, an animal drug or a feed. The composition for eliminating or detoxifying active oxygen according to claim 3 or 7 , which is in the form of foods, pharmaceuticals, quasi-drugs, cosmetics, supplements, veterinary drugs or feeds. The composition for activating an endothelial nitrogen monoxide synthase according to claim 4 or 8 , which is in the form of foods, pharmaceuticals, quasi-drugs, cosmetics, supplements, veterinary drugs or feeds.

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