KR20210018408A - Composition containing grape leaf extract and centella asiatica extract for improvement of venous circulation disorder - Google Patents

Abstract

The present invention relates to a health functional food composition, a food composition, a pharmaceutical composition and a cosmetic composition, for alleviating venous circulation disorders, each comprising a grape leaf extract and a Centella asiatica extract as active ingredients.

Description

A composition for improving venous circulation disorders comprising grape leaf extract and centella asiatica extract {COMPOSITION CONTAINING GRAPE LEAF EXTRACT AND CENTELLA ASIATICA EXTRACT FOR IMPROVEMENT OF VENOUS CIRCULATION DISORDER}

The present invention relates to a health functional food composition for improving venous circulation disorders, a food composition, a pharmaceutical composition and a cosmetic composition comprising grape leaf extract and centella asiatica extract as active ingredients.

Veins are divided into superficial veins, which are shallow veins located very close to the skin, and deep veins, which are deep veins located between or below the muscles under the skin. The veins connecting the shallow and deep veins are called penetrating veins. There is a valve inside the vein to keep the blood flow constantly toward the heart.If the pressure in the vein increases, such as standing or sitting for a long time, the wall of the vein becomes weak and the valve is damaged, causing the blood to flow back to the heart. Veins become visible outside the skin, which is called varicose veins. Varicose veins are a type of venous circulation disorder, and if not treated early, symptoms worsen and the skin turns black, and it can progress to increased venous pressure, inflammation, valve failure (defect), microvascular hyperpermeability, and skin ulcers. In addition, when blood and body fluids are stored in the vein and cannot move toward the heart, various venous circulation disorders occur, and when venous circulation is not smooth, the legs become swollen or numb, and symptoms appear.

If venous circulatory disorder persists, blood circulation to the heart is not smooth, making systemic blood circulation difficult. Pain, heaviness, swelling, night muscle spasms, itching, etc. may appear in the lower extremities. If left untreated, the blood vessels in the legs appear uneven. It may cause ulcers. These venous circulation disorders can be treated through surgery, but the possibility of recurrence is high due to the absence of a fundamental treatment, so it is important to prevent and manage venous circulation disorders.

The prevalence of varicose veins, which is a representative venous circulation disorder, is reported to be the highest among women in their 50s who are interested in health, and the number of patients and medical expenses continue to increase in 2012-2016. In addition, the production performance of pharmaceuticals for phlebitis and varicose veins is also continuously increasing, but there are no products targeting venous circulation disorders among domestic health functional foods. Korean Patent Laid-Open Publication No. 10-2018-0076262 discloses a fermentation method of black currant having an effect of strengthening capillaries and antioxidant, and improving blood circulation of a composition containing grape seed extract in Korean Patent No. 10-1469801 Although the effects and antioxidant effects of grape seed extract have been disclosed, studies on natural products that can effectively improve venous circulation disorders have not been conducted yet.

In the present invention, a natural product-derived composition effective in preventing inflammation and improving swelling that may occur in vein valves and vein walls by combining grape leaves having antioxidant, anti-inflammatory and edema inhibiting effects and centella asiatica having wound healing and anti-inflammatory effects Was intended to provide.

To this end, we confirmed the pharmacological efficacy of Centella asiatica extract and grape leaf extract through in vitro venous endothelial cell experiments and in vivo venous permeability experiments related to venous circulation, and furthermore, the synergistic effect of the combination of the two extracts. I tried to check the effect. That is, in the present invention, a complex of centella asiatica extract and grape leaf extract was selected as a candidate for a functional raw material capable of managing venous circulation disorders, and to prove the synergistic pharmacological efficacy of the complex for improving venous circulation.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

As a technical means for achieving the above technical problem, the first aspect of the present invention can provide a health functional food composition for preventing or improving venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

A second aspect of the present invention can provide a food composition for preventing or improving venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

A third aspect of the present invention can provide a pharmaceutical composition for preventing or treating venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

A fourth aspect of the present invention can provide a cosmetic composition for preventing or improving venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

The above-described problem solving means are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, there may be additional embodiments and embodiments described in the drawings and detailed description of the invention.

The complex of centella asiatica extract and grape leaf extract of the present invention does not exhibit cytotoxicity even at high concentrations, so it is safe for the living body, and exhibits a synergistic effect on the inhibition of oxidative damage and inflammatory factors of vascular endothelial cells that cause venous circulation disorders. Therefore, it is possible to effectively prevent, improve and treat venous circulation disorders such as varicose veins.

1 is a graph showing by measuring the antioxidant power of centella extract and grape leaf extract complex according to an embodiment of the present invention.
Figure 2 is a graph showing the inhibitory effect of active oxygen (ROS) in blood vessel cells of centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
3 is a graph showing the effect of releasing nitrogen monoxide (NO) in vascular cells of a centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
4 is a graph showing the effect of restoring electrical resistance of a cell monolayer in blood vessel cells of a centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
5 is a graph showing the effect of inhibiting cell permeability in vascular cells induced oxidative stress of centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
6 is a graph showing the effect of inhibiting cell permeability in vascular cells induced an inflammatory response of centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
7 is a graph showing the results of measuring cytotoxicity by concentration of centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
8 is an experimental result showing the effect of centella extract and grape leaf extract complex according to an embodiment of the present invention on the expression of inflammatory factors in macrophages.
9 is a result of a mouse intraperitoneal vascular permeability test measured after oral administration of 400 mg per kg of mouse body weight by varying the mixing ratio of centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
FIG. 10 is a result of an intraperitoneal vascular permeability experiment in mice in which the mixing ratio and oral dose of centella extract and grape leaf extract complex were varied according to an embodiment of the present invention.
FIG. 11 is a photograph of a result of a mouse bivein vascular permeability animal experiment using a centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention, and a graph quantifying it.
12 is a photograph of the results of an animal test of carrageenan-induced mouse paw edema using a centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
13 is a graph showing the measurement of the thickness of the foot edema according to the animal test of carrageenan-induced mouse foot edema using a centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
14 is a graph showing the measurement of the volume of paw edema according to an animal test of carrageenan-induced mouse paw edema using a centella asiatica extract and grape leaf extract complex according to an embodiment of the present invention.
15 is a photograph of foot pad edema according to an animal test of carrageenan-induced mouse foot edema in which oral doses of centella extract and grape leaf extract complex according to an embodiment of the present invention are varied.
16 is a side photograph of the sole of the foot according to an animal test of carrageenan-induced mouse foot edema in which oral doses of centella extract and grape leaf extract complex according to an embodiment of the present invention are varied.
FIG. 17 is a graph showing the measurement of the volume of paw edema according to an animal experiment in carrageenan-induced mouse paw edema in which oral doses of centella extract and grape leaf extract complex were varied according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Throughout this specification, when a certain part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

Throughout this specification, the term "combination of these" included in the expression of the Makushi form means one or more mixtures or combinations selected from the group consisting of the constituent elements described in the expression of the Makushi form, and the constituent elements It means to include one or more selected from the group consisting of.

Throughout the present specification, the term "prevention" refers to any action that inhibits or delays the onset by administration of the composition, and "treatment" or "improvement" refers to symptomatic improvement or beneficial change by administration of the composition. It means all actions.

Hereinafter, a health functional food composition, a food composition, a pharmaceutical composition, and a cosmetic composition comprising the grape leaf extract and centella asiatica extract of the present invention will be described in detail with reference to embodiments and examples and the drawings. However, the present invention is not limited to these embodiments and examples and drawings.

A first aspect of the present invention can provide a health functional food composition for preventing or improving venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

Grape leaf extract and centella asiatica extract contained in the health functional food composition of the present invention reduce oxidative stress, vascular permeability and inflammatory response of vascular cells, inhibit NO production, and reduce edema in vivo, thereby impairing venous circulation Can prevent and/or improve. In particular, the health functional food composition of the present invention may exhibit a synergistic effect compared to the case of using the grape leaf extract alone or centella asiatica extract of the same dose by including the grape leaf extract and centella asiatica extract together.

The composition of the present invention may contain a grape leaf extract and centella asiatica extract in a weight ratio of 5:1 to 5:1, specifically 3:1 to 1:3, more specifically 2:1 to 3:1 It may be contained in a weight ratio, but may not be limited thereto. For example, the composition may contain a grape leaf extract and centella asiatica extract in a weight ratio of 1:1, 2:1, 3:1, 4:1 or 5:1, specifically 2:1, 2.5: It may be contained in a weight ratio of 1 or 3:1, but may not be limited thereto. When the composition contains the grape leaf extract and centella asiatica extract in a weight ratio of 2:1 to 3:1, the effect of preventing and/or improving venous circulation disorders may be particularly excellent.

The composition of the present invention may be for ingestion in a dose of 1 mg to 100 mg per kg of body weight of the subject, but may not be limited thereto. Specifically, the composition may be for ingestion at a dose of 2 mg to 50 mg, 3 mg to 30 mg, 4 mg to 20 mg, or 5 mg to 10 mg per kg of the subject's body weight, but may not be limited thereto. .

According to one embodiment of the present invention, the venous circulation disorder may be varicose veins, specifically varicose veins, or venous insufficiency, but may not be limited thereto, and any disorder or disease caused by poor venous circulation is limited. Without it can be prevented and / or improved by the health functional food composition of the present invention.

The composition of the present invention can prevent and/or ameliorate one or more symptoms selected from symptoms associated with venous circulation disorders, such as lower extremity pain, swelling, nocturnal muscle spasms, itching, heaviness in the lower extremities, increased venous pressure, and valve failure. However, it may not be limited thereto.

Grape leaf extract and centella asiatica included in the composition of the present invention may be, for example, pulverized grape leaves, extracted using an extraction solvent, filtered and concentrated, and dried to form a powder, but is limited thereto. May not be.

The extraction solvent may be selected from water, an alcohol having 1 to 5 carbon atoms, and a combination thereof, for example, methanol, ethanol, propanol, butanol, pentanol, a mixture of the alcohol and water, or hot water. May not be limited.

Foods that may contain the grape leaf extract and centella asiatica extract of the present invention as an active ingredient, for example, various foods, powders, granules, tablets, capsules, syrup, beverages, gum, tea, vitamin complex, health functional foods And the like, but may not be limited thereto, and may also be added to food or beverage. For example, the composition of the present invention may be a formulation selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids, but may not be limited thereto.

At this time, the total amount of the grape leaf extract and centella asiatica extract in food or beverage may be added in an amount of about 0.01 to 15% by weight of the total food weight, and the health beverage composition may be added in an amount of about 0.01 to 10g based on 100 ml. However, it may not be limited thereto.

When the food composition of the present invention is a beverage composition, there is no particular limitation on other ingredients other than containing the above extract as an essential ingredient in the indicated ratio, and various flavoring agents or natural carbohydrates, etc., as an ordinary beverage, may be contained as additional ingredients. I can. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used, but are not limited thereto. The ratio of the natural carbohydrate may generally be in the range of about 1 to 20 g per 100 ml of the composition of the present invention, but may not be limited thereto.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. may be included, but may not be limited thereto.

In addition, the composition of the present invention may contain natural fruit juice and flesh for the production of fruit juice beverages and vegetable beverages, and these components may be used independently or in combination. The ratio of these additives is not important, but may be selected from about 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention, but may not be limited thereto.

A second aspect of the present invention can provide a food composition for preventing or improving venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

Detailed descriptions of parts overlapping with other aspects of the present invention have been omitted, but descriptions of other aspects of the present invention may be equally applied even if the description is omitted in the second aspect of the present invention.

For example, the food composition may be a formulation selected from the group consisting of beverages, bread, fats and oils, ice cream, confectionery, gum, rice cakes, snacks, alcoholic beverages, seasonings, and retort foods, including general food forms, but may not be limited thereto. have.

A third aspect of the present invention can provide a pharmaceutical composition for preventing or treating venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

Detailed descriptions of parts overlapping with other aspects of the present invention have been omitted, but descriptions of other aspects of the present invention may be equally applied even if the description is omitted in the third aspect of the present invention.

Grape leaf extract and centella asiatica extract contained in the pharmaceutical composition of the present invention reduce the oxidative stress, vascular permeability and inflammatory response of vascular cells, inhibit NO production, and reduce edema in vivo, thereby reducing venous circulation disorders. It can be prevented and/or treated. In particular, the pharmaceutical composition of the present invention may exhibit a synergistic effect compared to the case of using the grape leaf extract alone or Centella asiatica extract of the same dose by including the grape leaf extract and centella asiatica extract together.

For example, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and/or diluent.

Here, “carrier” may be defined as a compound that facilitates the addition of the target active ingredient into cells or tissues, and is a commonly used carrier that facilitates the introduction of many organic compounds into cells or tissues of an organism. It can be selected and used without limitation.

Herein, “diluent” may be defined as a compound that is diluted in a solvent that will dissolve the active ingredient as well as stabilize the biologically active form of the target active ingredient. The salt dissolved in the buffer solution is used as a diluent in the art, and the buffer solution commonly used is a phosphate buffered saline solution that mimics the salt state of a human solution, but may not be limited thereto. Because the buffer salt controls the pH of the solution at low concentrations, the buffer diluent may not modify the biological activity of the compound.

The pharmaceutical composition of the present invention is formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. I can. Carriers, excipients, and diluents that can be included in the composition containing natural extracts, such as the pharmaceutical composition of the present invention, include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. , May not be limited thereto. In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, such as starch, calcium carbonate, sucrose ) Or lactose (lactose), gelatin, etc. can be prepared by mixing. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by a person skilled in the art.

The pharmaceutical composition of the present invention may be administered in a dose of 1 mg to 100 mg per kg of body weight of the subject, but may not be limited thereto. Specifically, the pharmaceutical composition may be for administration in a dose of 2 mg to 50 mg, 3 mg to 30 mg, 4 mg to 20 mg, or 5 mg to 10 mg per kg of body weight of the subject, but is not limited thereto. I can. For example, the dosage may be a daily dosage.

Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or by intracerebroventricular injection.

A fourth aspect of the present invention may provide a cosmetic composition for preventing or improving venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

Detailed descriptions of parts overlapping with other aspects of the present invention have been omitted, but descriptions of other aspects of the present invention may be equally applied even if the description is omitted in the fourth aspect of the present invention.

The cosmetic composition of the present invention includes, for example, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack , Soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, press powder, and loose powder, but limited thereto. May not be.

Further, the ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to grape leaf extract and centella as an active ingredient, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. It may include a conventional adjuvant, such as, and a carrier.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation, and spray, etc., but are not limited thereto, and in addition, flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing It can be made of foam, cleansing water, pack, spray or powder.

If the formulation of the cosmetic composition is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide are used as carrier components. Can be. Or when the formulation of the cosmetic composition is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan can be used. When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used. When the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether. When the formulation of the cosmetic composition is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

The present invention is to be described in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Example]

A. Study on the efficacy of grape leaf extract, centella asiatica extract, and complexes thereof in vascular cells

1. Purpose of the experiment

The purpose of this study was to evaluate the efficacy and synergistic effects of centella, grape leaves, and their complex extracts, which are candidate materials for improving venous circulation, using vascular cells.

2. Raw material manufacturing

1) Preparation of Centella asiatica extract

For the preparation of centella asiatica extract, centella asiatica leaves were pulverized with a grinder and then extracted using alcohol (Daehan Alcohol Life Co., Ltd., Korea) to the raw material. The extract was filtered under reduced pressure with a paper filter (ADVANTEC 2, 280 mm, China), and the filtrate was concentrated with a rotary vacuum concentrator (Rotavapor® R-220SE, BUCHI, switzerland), and then in a vacuum dryer (Daeil engineering, Korea). After drying, an extract in powder form was obtained.

2) Preparation of grape leaf extract

For the preparation of grape leaf extract, grape leaves were pulverized with a grinder, and then extracted using 40% alcohol (Daehan Alcohol Life Co., Ltd., Korea) in the raw material. The extract was filtered under reduced pressure with a paper filter (ADVANTEC 2, 280 mm, China), and the filtrate was concentrated with a rotary vacuum concentrator (Rotavapor® R-220SE, BUCHI, switzerland), and then in a vacuum dryer (Daeil engineering, Korea). After drying, an extract in powder form was obtained.

3) Preparation of Grape Centella asiatica extract complex

The grape leaf extract and centella asiatica extract prepared by the above method were mixed at a specific mixing ratio and used in the experiment.

3. Experimental method

1) Analysis of Oxygen Radical Antioxidant Capacity (ORAC)

Experiments were performed according to the protocol in the OxiSelect™ ORAC Activity Assay Kit (CELL BIOLABS, INC.). First, 25 µl of each sample and Trolox were added to a 96-well plate, 150 µl of Fluorescein Solution dissolved in a diluent buffer was added and reacted at 37° C. for 30 minutes. Then, 25 µl of a free radical inhibitor solution was added to each well, and then mixed well. The plate was placed in a VICTOR3 multilabel counter (PerkinElmer, USA) and measured at 37°C for 1 hour at ex/em 485/530 nm at 5 minute intervals, which were converted to the relative equivalents of Trolox and calculated. Marked.

2) Intracellular ROS production

HUVEC cells were aliquoted into a 96-well plate at 3×10 ⁴ cells/well and cultured for 24 hours. After removing the medium, it was washed once with an assay buffer, and treated with 2', 7'-dichlorofluorcein (DCFDA) 25 μM, and reacted in an incubator at 37° C. for 45 minutes. After the reaction, the sample was pretreated for 30 minutes after washing once with an analysis buffer. H ₂ O ₂ 500 μM was treated and reacted for 1 hour and then measured at ex/em 485/530 nm.

3) Nitric oxide (NO) emission

HUVEC cells were aliquoted into a 96-well plate at 3×10 ⁴ cells/well and cultured for 24 hours. After removing the medium, the sample was pretreated for 30 minutes, and reacted with 500 μM of H ₂ O ₂ for 4 hours. After sampling the culture medium, total NO was measured with a NO assay kit (R&D systems).

4) TEER (Transepithelial Electrical Resistance) measurement

HUVEC cells were dispensed into 12-well transwells at 4×10 ⁵ cells/well and cultured for 24 hours. After removal of the medium, the sample was pretreated for 30 minutes and reacted with 500 μM of H ₂ O ₂ for 4 hours. Then, the cell monolayer gap was measured using a Millicell ERS (Millipore) machine.

5) Permeability measurement

HUVEC cells were aliquoted into 24-well transwells at 2×10 ⁵ cells/well and cultured for 24 hours. After removal of the medium, the sample was pretreated for 30 minutes, and reacted with 500 μM of H ₂ O ₂ for 4 hours or TNF-α 50 ng/ml for 6 hours. After 20 minutes reaction by treatment with FITC-Dextran working solution, 100 μl of the medium in the insert well was sampled. Subsequently, fluorescence was measured at ex/em 485/530 nm.

4. Experiment result

1) ORAC analysis

As a result of ORAC analysis to evaluate the antioxidant activity of centella asiatica extract and grape leaf extract individual raw materials and grape centella asiatica (grape leaf + centella asiatica) complex, all three samples showed antioxidant activity, especially grape leaf and centella asiatica 3: It was confirmed that the composite sample combined with 1 showed higher antioxidant power than each individual sample (FIG. 1).

2) Production of active oxygen in cells

Reactive oxygen (ROS) is a substance that induces an inflammatory reaction by inducing oxidative damage to vascular endothelial cells, and ultimately causes dysfunction of the vascular wall. In this example, after inducing oxidative stress by treating cells with H ₂ O ₂ , the efficacy of the candidate material for inhibiting ROS production was evaluated.

As a result of the experiment by subdividing the grape vinegar complex sample and individual samples into four concentrations, the ROS production increased by H ₂ O ₂ in a concentration-dependent manner decreased (FIG. 2). When comparing individual samples, Centella asiatica showed higher efficacy than grapes. In order to confirm the synergistic (synergistic) effect of the composite sample, the expected inhibitory effect from the composite was calculated using the Colby equation (Colby, SR Calculation of the synergistic and antagonistic response of herbicide combinations. Weeds 1967, 15, 20-22) . The Colby equation is as follows.

Colby estimate = A + B-(A x B/100)

A = observed efficacy of active ingredient A at the same concentration as used in the mixture;

B = observed efficacy of active ingredient B at the same concentration as used in the mixture.

As a result of the calculation, it was confirmed that the ROS inhibitory effect of Grape Centella asiatica complex was higher than Colby's expected value at all concentrations, so that the Grape Centella asiatica complex sample had a synergistic effect on ROS inhibition compared to grape or Centella asiatic alone sample (Table 1).

[Table 1]

3) nitrogen monoxide release

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) in vascular endothelial cells is an active substance that plays a key role in maintaining the homeostasis of endothelial cells such as vascular relaxation and permeability. Unlike NO secreted by macrophages, NO produced by vascular endothelial cells, in addition to vascular relaxation, interferes with the expression of inflammatory cytokines, chemokines, and adhesion molecules, thereby blocking recruitment of leukocytes into blood vessels, proliferation of vascular smooth muscle, and platelet aggregation It plays a positive role in regulating the function of vascular endothelial cells by inhibiting the production of tissue factors involved in thrombus formation. In order to confirm the production of NO, the cells were treated with H ₂ O ₂ to induce oxidative stress, and then the NO release action of the candidate material was measured. When the cells were treated with H ₂ O ₂ , the concentration of NO was decreased compared to the control, but it was confirmed that the concentration-dependently decreased NO was recovered when treated with the grape plant complex (FIG. 3 ).

4) TEER measurement

In order to confirm the cell permeability inhibitory action of the candidate material, the cells were treated with H ₂ O ₂ to induce oxidative stress, and then the TEER value was measured. When H ₂ O ₂ treatment, the TEER value was decreased due to the weakening of the cell monolayer, but it was observed that the TEER value was recovered in a concentration-dependent manner when the vinegar plant complex was treated (FIG. 4).

5) Permeability measurement (oxidation stress condition)

In order to confirm the inhibitory effect of the candidate material on cell permeability, the cells were treated with H2O2 to induce oxidative stress, and then a permeability test was performed. When the vinegar plant complex was tested for each concentration, it showed the effect of inhibiting the increased cell permeability by H ₂ O ₂ in a concentration-dependent manner (FIG. 5).

In addition, when a single sample of Centella asiatica and grapes were treated at the concentration contained in 50 μg/ml of the Grape Centella asiatica complex, and their efficacy was compared with the complex sample, the Colby equation was substituted and Colby's expected value was 74.8%, whereas the actual observed value was Since it was 77.8%, it was confirmed that the grape centella asiatica complex sample had a synergistic effect on cell permeability inhibition compared to the grape or centella asiatica alone sample (Table 2).

[Table 2]

6) Permeability measurement (inflammation induction condition)

After inducing an inflammatory response by treating cells with TNF-α, the effect of inhibiting permeability by the sample was evaluated. As a result of treatment of the grape vinegar complex sample by concentration, it was confirmed that the increased permeability was suppressed by TNF-α in a concentration-dependent manner (FIG. 6).

5. Conclusion

In this example, the efficacy of the combination of grape leaf extract and centella asiatica extract with a mixing ratio of 3:1 (grape plantain complex) was evaluated at the in vitro level. As a result of confirming the antioxidant activity, which is an important mechanism for improving venous circulation, by ORAC analysis, the Grape Centella asiatica complex showed superior antioxidant capacity than the individual extracts of Grape and Centella asiatica. In the experiment using HUVEC cells, which are human venous endothelial cells, it was confirmed that the grape plantain complex exhibited a synergistic effect compared to individual raw materials in inhibiting ROS production, and had a synergistic effect compared to individual extracts even under hydrogen peroxide induction conditions. In addition, the grape vinegar complex showed an increase in NO production in cells, and in the TEER measurement and permeability analysis to confirm the inhibitory effect of cell membrane permeability, the vinegar plant complex significantly increased cell membrane permeability due to oxidative stress and inflammation. It was confirmed to suppress. Accordingly, it is analyzed that the vascular permeability inhibitory effect of the candidate material identified through animal experiments is mediated by the inhibition of inflammation induction and vascular protection by the antioxidant action of the Grapevine.

B. Study on the efficacy of grape leaf extract, centella asiatica extract, and complexes thereof in inflammatory cell and vascular permeation animal models

1. Summary

In order to test the vascular permeability pharmacological efficacy of the test substances, grape leaf extract, centella asiatica extract, and their complexes, anti-inflammatory efficacy was tested in Raw264.7 cells stimulated with lipopolysaccharide (LPS). In ICR mice, vascular permeability and A paw edema test was conducted. In the mouse animal test, the vascular permeability was tested after oral administration of the test substance for 4 to 7 days, and the vascular permeability test of all animal tests was conducted on the last day 1 hour after administration of the test substance.

2. Test substances and excipients

(1) Test substance: same as Example A

(2) Excipient: sterile distilled water

3. Materials and Methods

(1) Test system

a) Species and strains: Specific pathogen-free (SPF) ICR mice

b) Source: Korea Animal Technology (KOATECH, Korea)

c) Week age: male, 5 weeks old upon receipt

(2) breeding environment

a) environmental conditions

The environmental conditions of this test are: temperature 23±3℃, relative humidity 50±10%, ventilation frequency 10 to 20 times/hour, lighting time 12 hours (08:00 lit up to 20:00 off) and illuminance 150 to 300 lux. It was carried out in the established laboratory. This test was conducted in compliance with all regulations applicable to the management and use of experimental animals.

b) breeding box, breeding density and identification of breeding box

Animals were kept in individual ventilated stainless steel mesh cages (200W×320L×145H mm) for acclimatization, administration, and observation periods, with no more than 4 animals per cage. An individual identification card with the test number and animal number was attached to the breeding box.

c) feed and water

㉠ Feed feeding method and confirmation of pollutants

For feed, solid feed for laboratory animals (Lab Diet® #5002, PMI Nutrition International, 505 North 4th Street Richmond, IN 47374, USA) was irradiated [12.6~32.0 kGy (Lot No.: APR 15 052A, JUN 02 053A) It was sterilized with] and ingested freely.

㉡ How to supply water and check pollutants

Water was freely ingested after sterilizing tap water with a microfilter and UV sterilizer.

(3) Administration of test substance

After weighing the test substance, the test substance was suspended and prepared using sterile distilled water at a concentration of 200 to 600 mg/kg. The test substance was orally administered to the test animal after aspirating the test substance with a feeding needle and a syringe for oral administration to the animal.

(4) Observation and inspection items

1) Observation of general symptoms and dead animals

During the administration and observation period, all animals were observed for general symptoms and death once a day.

2) autopsy

A mixture of zoletil and lumpen (3:1) was diluted 10 times and injected at 0.3 cc per mouse, anesthetized during the vascular permeability and foot edema test, and then cervical spine dislocation was performed at the end of the test.

(5) Statistical method

For statistical analysis of the obtained data, Student's t-test was performed.

4. Cell test results

(1) Cell viability analysis using Raw264.7 cells-Cell viability in Raw264.7 cell line treated with Centella asiatica extract, grape leaf extract, and grape Centella asiatica complex

Raw264.7 cells were dispensed into 96 wells with 1×10 ⁴ cells per well, and after 24 hours, various concentrations of Centella asiatica extract, grape leaf extract, and grape Centella asiatica complex (0~400 μg/ml) were added and treated for 24 hours. As a result of measuring the cell viability, most of the extracts did not show cytotoxicity and showed a cell viability of 80% or more even at the highest concentration of 400 μg/ml (FIG. 7). Therefore, centella asiatica extract, grape leaf extract, and grape centella asiatica complex were evaluated to have no cytotoxicity at concentrations of 400 μg or less per ml, the highest concentration in this test.

(2) Changes in the expression of inflammatory factors in LPS-stimulated Raw264.7 cells according to the pretreatment of the Grapefruit complex (initial inflammatory cytokines TNF-α, IL-1β and IL-6, iNOS and COX-2)

Raw264.7 cell line was pretreated with the extract of grapefruit for 1 to 4 hours, and then LPS (0.5 μg/ml) was reacted for 24 hours, and the expression of inflammatory targets was evaluated. As a result, it was found that the expression of IL-1β, IL-6, iNOS and COX-2 was inhibited in a concentration-dependent manner (FIG. 8).

5. Animal test results

(1) Mouse intraperitoneal vascular permeability animal test

1) 5-week-old ICR mice were purified in the animal room for 1 week, and then Sencia or Anti-Starx as a positive control material was administered at 400 mg per kg for 1 week. The complex (1: 1) and grape vinegar complex (3: 1) were administered orally at 400 mg per kg (Table 3).

[Table 3]

One hour after the last day of administration, 0.1 cc of 2% Evans blue was administered causally, stabilized for 15 minutes, and then 0.1 cc of 0.78% acetic acid per body weight was injected into the abdominal cavity, and 10 ml of physiological saline was added into the abdominal cavity after 20 minutes. After retrieval, the OD value was measured at 620 nm to test intraperitoneal hemopermeability.

As a result of animal experiments, the result of decreased vascular permeability in the Sencia-administered group was derived, and it was evaluated that the administration group of the vinegar plant complex showed similar efficacy to the Sencia-administered group. In addition, it was evaluated to exhibit similar efficacy between the complex administration groups by ratio (Fig. 9).

2) Secondary mouse intraperitoneal vascular permeability animal test

In the second mouse intraperitoneal vascular permeability animal experiment, animal experiments were carried out with the Grape Centella asiatica complex at 2:1 ratio and 3:1 ratio in order to finally set the clinical dose. In addition, by incorporating the preliminary results, the vinegar plant complex was divided into 200 mg and 400 mg per kg of body weight and orally administered to mice for 4 days by mixing ratio (Table 4). The difference from the previous intraperitoneal vascular permeability animal experiments was to test the efficacy by lowering the concentration of acetic acid administration from 0.78% to 0.5% in order to capture the subtle difference between the mixture extract 2:1 and 3:1 ratio.

[Table 4]

In the second mouse intraperitoneal vascular permeability animal experiment, vascular permeability was inhibited in all groups administered with the vinegar plant complex compared to the trigger group with increased vascular permeability, and also showed a tendency to be inhibited in a concentration-dependent manner (FIG. 10). In particular, it was evaluated that the 400 mg administration group with a ratio of 3:1 ratio of the grape plantain plant exhibits excellent vascular permeability inhibitory effect compared to the administration group with a 2:1 ratio (FIG. 10).

(2) Mouse bivenous vascular permeability animal test

Five-week-old ICR mice were purified in an animal room for one week, and then each test substance was orally administered for one week (Table 5). 1 hour after administration on the last day, 0.1 cc of 2% Evans Blue was administered causally and stabilized for 15 minutes. 15 minutes after Evans Blue administration, 20 µl of 20% mustard oil was evenly applied to the ear area using a cotton swab, and a picture was taken 20 minutes later and observed. After that, the ear part was cut and dried at 60°C for one day. Then, after immersing in 1 ml of formamide at 56°C to elute Evans Blue staining solution for 48 hours, the OD value was measured at 620 nm to confirm the bivenous vascular permeability. Before measuring the OD value, a standard curve was made for the Evans Blue solution and the relative ratio of the Evans Blue eluate in the animal test group was calculated, and the results were summarized.

[Table 5]

In the mouse bivenous vascular permeability animal experiment, it was observed that the bivenous vascular permeability was suppressed in the group administered with Sencia, antistarx, and various ratios of the vinegar plant complexes compared to the triggered control group (Fig. 11).

In order to quantify the vascular permeability of the ear veins in mice, the same part of the ear veins was punched out and eluted in formamide. It was evaluated that the vascular permeability of the vein was inhibited (Fig. 11). There was no significant difference between the composites.

(3) Carrageenan-induced mouse paw edema animal test

1) Primary carrageenan-induced mouse paw edema animal experiment

5-week-old ICR mice were acclimatized in an animal room for 1 week, and then each test substance was orally administered for 4 days. The test substance was orally administered 1 hour before the last day of administration. After that, 50 µl of 1% carrageenan was administered intradermally to the sole of the foot, and at 1, 3, or 5 hours, the thickness and volume of the sole of the foot were swelled using a digital caliper and a digital plethysmometer (LE-7500). Was measured.

Mice were administered with a normal control group, an induction control group, dexamethasone, and a variety of mixed ratios of grape vinegar complex at 400 mg per kg body weight (Table 6). Dexamethasone was used as a positive control by administering 2 mg per kg of body weight. The test results were evaluated through a photograph of the foot edema, the thickness of the foot, and the volume of the foot.

[Table 6]

In the carrageenan-induced mouse paw edema test, paw edema was strongly suppressed in the dexamethasone-administered group, which is a positive control, compared to the inducing control, and it was observed that paw edema was decreased compared to the inducing control group in the complex administration group of various mixing ratios (Figs. 12 to 14). It was evaluated that the foot edema was also reduced in the group administered with the vinegar plant complex.

2) Secondary carrageenan-induced mouse paw edema animal experiment

In the second carrageenan-induced mouse paw edema, in an animal experiment, to determine the clinically applied dose of Grape Centella asiatica complex, grape leaf extract and centella asiatica extract were mixed at a ratio of 2:1 and administered orally at 300 mg and 600 mg per kg body weight, respectively. Was (Table 7). Dexamethasone was used as a positive control by administering 2 mg per kg of body weight.

[Table 7]

The test results were evaluated through a photograph of the foot edema, the thickness of the foot, and the volume of the foot. On the plantar edema photographs, swelling and redness were the least observed in the Grape Centella asiatica complex administration groups (300 mg and 600 mg) (Fig. 15), and the edema was the most in the Grape Centella asiatica complex administration groups (300 mg and 600 mg) on the plantar lateral picture. It was observed to be weak (Figure 16). In addition, it was evaluated that the volume of foot edema decreased in the groups administered with the vinegar plant complex in terms of the volume change of foot edema (Fig. 17).

6. Test results and discussion

Changes in venous permeability lead to a continuous increase in venous pressure, consequent venous dilation, and functional abnormalities of venous valves. In particular, when the venous permeability changes chronically, the morphology and functional reorganization of the veins accompanied by inflammatory reactions of the venous vascular wall and vascular endothelium appear. The cause of the change in venous permeability is not yet well known, but it is predicted to be caused by abnormalities in the circulatory system that cause an increase in venous pressure. In particular, it is reported that chronic venous diseases are increased by westernized eating habits, and it is reported that there is a higher possibility of developing an obesity predisposition.

On the other hand, a lot of investments are being made in the development of protection and therapeutic agents through the study of the mechanism of such vascular disease, and in particular, due to the nature of vascular disease, many studies on new natural drugs with little side effects even when taken for a long time are being conducted. As a representative natural product, centella asiatica and grape leaf extract are reported to have vascular protection function. In particular, asiatic acid, known as an active ingredient of Centella asiatica (L.) Urban, is known to have wound healing, neuroprotection, and blood lipid reduction effects, and is known to have efficacy in several clinical studies on atherosclerosis. The total triterpenic fraction of is reported to inhibit the formation of atherosclerotic plaques when administered in combination with pycnogenol, an active ingredient of pine bark extract. In addition, it has been reported that the total triterphenyl fraction of Centella asiatica has improved capillary permeation pressure and alleviating symptoms of microcirculatory disorders in hypertensive patients. In addition, asiatic acid is known to have anti-inflammatory, anti-angiogenic, anti-oxidative, plasma glucose reduction and metabolic effects.

Grape leaf extract is known to be effective for mucous membrane protection, defecation relief, antipyretic action, gastrointestinal protection, anti-diuretic, chronic bronchitis, gout and heart disease, but not as much research as grape fruit and grape seed.

Therefore, in this Example, in order to examine the effect of the combination of grape leaf extract and centella asiatica extract on anti-inflammatory and vascular permeability inhibition, anti-inflammatory effects on various inflammatory factors were tested in Raw264.7 cells stimulated with LPS, and ICR Using mice, vascular permeability animal tests and carrageenan-induced paw edema animal tests in the abdominal cavity and bile vein were performed. Also, based on these results, we tried to establish the clinical trial concentration.

Since the grape vinegar complexes did not show cytotoxicity up to 400 μg/ml concentration in Raw264.7 cells, it was evaluated that there was no cytotoxicity even at very high concentrations. As a result of testing the anti-inflammatory effect of the Grapefruit complex against inflammatory cytokines, which are factors affecting vascular permeability, TNF-α, IL-1β, and IL-6, which were mainly increased by LPS, were found in the pretreatment of Grapevine complex. It has been shown to be strongly inhibited by. In addition, it was found that the expression of these inflammatory targets was strongly suppressed in the test to evaluate the mRNA and protein expression levels of the inflammatory targets iNOS and COX-2. Therefore, it was analyzed that the vinegar plant complex has an inhibitory effect on vascular permeability by inhibiting inflammatory cytokines and inflammatory target proteins.

In various animal tests related to vascular permeability, the vinegar plant complex was evaluated to inhibit the vascular permeability caused by acetic acid, mustard oil and carrageenan increased intraperitoneal, intravenous, and foot edema, respectively. These complexes were evaluated to exhibit particularly excellent efficacy at a concentration of 300 mg or more per kg body weight, and were evaluated to have efficacy in both 2:1 and 3:1 in the mixing ratio.

Increased vascular permeability in the intraperitoneal cavity by intraperitoneal administration of acetic acid is due to direct damage to vascular endothelial cells by acetic acid, and it was analyzed that the vinegar plant complex reduces vascular permeability by protecting such vascular endothelial cell damage. In addition, the change in vascular permeability of the bivein due to mustard oil is also directly stimulated by the blood vessels, thereby increasing vascular permeability.In a mouse test of bivenous vascular permeability, it was confirmed that the vascular permeability was reduced by the vasoprotective action of the vinegar plant complex. . In the carrageenan-induced mouse paw edema test, it was confirmed that the increased paw edema due to inflammation was reduced by the anti-inflammatory and vasoprotective effects of the Grapevine complex.

That is, when the experimental results of Examples are summarized, when the Grape Centella asiatica complex is administered at a dose of 300 mg or more per kg of body weight, particularly excellent vascular permeability inhibitory effect appears, and in the mixing ratio, the grape leaf extract and Centella asiatica extract were 2:1 or 3 It was found to have excellent efficacy in a composite mixed in a :1 ratio, particularly a composite having a ratio of 3:1.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are illustrative in all respects and should be understood as non-limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later rather than the detailed description, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention. do.

Claims (1)

Health functional food composition for preventing or improving venous circulation disorders, comprising grape leaf extract and centella asiatica extract as active ingredients.

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