KR101889331B1

KR101889331B1 - A composition having anti-oxidation, anti-inflammation or anti-bacterial activity comprising Acanthopanax koreanum Nakai stem extracts, fractions thereof or compounds isolated therefrom as an active ingredient

Info

Publication number: KR101889331B1
Application number: KR1020160012350A
Authority: KR
Inventors: 이남호; 문승리
Original assignee: 제주대학교 산학협력단
Priority date: 2016-02-01
Filing date: 2016-02-01
Publication date: 2018-08-17
Also published as: KR20170091391A

Abstract

The present invention relates to a method for producing an Acanthopanax koreanum Nakai) stem extract, a fraction thereof, or a compound isolated therefrom as an active ingredient, to a cosmetic composition, a food composition and a pharmaceutical composition for antioxidant, anti-inflammation or antimicrobial use.
Island of the present invention wood Acanthopanax (Acanthopanax The anti-oxidative, anti-inflammatory or antimicrobial composition comprising koreanum Nakai stem extract, fractions thereof, or a compound isolated therefrom as an active ingredient is excellent in activity of scavenging DPPH free radicals in a concentration-dependent manner, The present invention is not only excellent in the activity of inhibiting the production of nitric oxide (NO), but also has an excellent growth inhibitory activity against inflammatory skin microbes. Therefore, it is possible to prevent oxidative, inflammatory and microbial diseases, And can be usefully used for cosmetics, foods and medicines for improvement or treatment.

Description

TECHNICAL FIELD [0001] The present invention relates to an antioxidant, anti-inflammatory or antimicrobial composition containing an extract of an insect trilobal stem, a fraction thereof or a compound isolated therefrom as an active ingredient. , fractions thereof or compounds isolated therefrom as an active ingredient}

The present invention relates to a method for producing an Acanthopanax koreanum Nakai) stem extract, a fraction thereof, or a compound isolated therefrom as an active ingredient, to a cosmetic composition, a food composition and a pharmaceutical composition for antioxidant, anti-inflammation or antimicrobial use.

Oxygen is essential for many metabolic reactions for life support and it is necessary for the detoxification of toxic substances in the human body. However, not only oxygen is beneficial to human body, but also oxygen, reducing metabolism, chemical, , Environmental factors, etc., it is converted into a highly reactive free radical such as superoxide radical, hydroxyl radical, hydrogen peroxide, singlet oxygen There is a bi-aspect that causes lethal oxygen toxicity in living organisms ( Korea J. Biotechnol . Bioeng . , 2001, 16 (6), 592-602). BHT (butylated hydroxy toluene) and BHA (butylated hydroxy anisole), which are synthetic antioxidants for the removal of active oxygen, have been widely used because of their excellent antioxidative and economic properties. However, hepatic hypertrophy, And the amount of food and the amount of food to be used are strictly restricted. In addition, natural antioxidants such as tocopherol and ascorbic acid are high in safety, but they are disadvantageous in that they are low in chain reaction inhibiting ability and expensive. For this reason, researches for isolating and using more safe, economical and effective antioxidants from natural products have been actively conducted recently. In particular, plant-derived secondary metabolites inhibit the production of free radicals and free radicals since removing to prevent oxidation, many studies have been made about this (Korean Journal of Plant Res. , 2011, 24 (1), 30-39; J. Korean Soc . Food Sci . Nutr . , 2010, 39 (9), 1249-1256).

The color of skin is determined by three kinds of melanin, hemoglobin and carotene. Melanin is the most important role. Melanocytes are leaf-shaped dendritic cells that produce dark brown melanin in the basal layer of the epidermis. The main role of melanin is to remove active oxygen and free radicals from the skin and to protect the inside of the skin by blocking the penetration of ultraviolet rays. The starting material for making melanin is tyrosine, a kind of amino acid normally present in the human body. Tyrosine is oxidized by tyrosinase in melanocytes to become dopa (DOPA), and dopa is further oxidized into dopaquinone (DOPA quinone). Dopaquinone then undergoes autoxidation and ultimately produces dark brown eumelanin. On the other hand, when dopaquinone encounters cysteine, cysteinyl DOPA is formed, resulting in reddish brown pheomelanin. The resulting melanin is then transferred to the keratinocyte on the melanosome. However, when melanin is overproduced, pigmentation such as spots, freckles, dots, and black spots occurs, leading to skin aging and damage, and it is also known to be involved in the induction of skin cancer ( J. Korean Ind . Eng . Chem . 2005, 16 (3), 348-353). Studies on the recent whitening effects are proceeding with a comprehensive concept including skin whitening effect, antioxidant action, and prevention of skin aging. Currently known whitening studies include studies on UV blocking materials, studies to control the action of the cytokine, a signaling substance that commands the synthesis of melanin in melanocytes, suppression of gene expression, inhibition of tyrosinase action , Active oxygen scavenging material, pigment reduction, research to promote stratum corneum removal, and so on. Of these, the mechanism by which the activity of tyrosinase is inhibited and the initial production of melanin is reduced is the most studied.

Inflammation is a vital reaction caused by wound, infection, or autoimmune mechanism. When inflammation occurs, immune cells penetrate into the inflammation site and these cells are exposed to various kinds of

Chemical secretion, and cytokine production to produce bio-defense and inflammatory responses ( J. Korean Soc . Food Sci . Nutr . , 2010, 39 (7), 980-985). Lipopolysaccharide (LPS), also known as endotoxin, is present in the extracellular membrane of Gram-negative bacteria, and is expressed in macrophages or monocytes, such as RAW 264.7, by proinflammatory factors such as TNF-α, IL-6, It is known to increase pro-inflammatory cytokines. In addition, the formation of such inflammatory mediators leads to the process of converting arachidonic acid into prostaglandin and the formation of nitric oxide (NO) due to the activity of phospholipase A ₂ (phospholipase A ₂ ) . In vivo inflammatory process is formed by the excessive amount of NO and PGE ₂ (prostaglandin E ₂₎ inflammatory markers are iNOS (inducible NO synthase) and such as COX-2 (cyclooxygenase-2) . Although normal NO formation plays an important role in killing bacteria or eliminating tumors, NO produced by iNOS in the inflammatory state not only promotes inflammatory responses such as vascular permeability and swelling but also stimulates inflammation mediator biosynthesis, ( Korean Soc . Of Food Sci . Techn . , 2007, 39 (4), 464-469). There are numerous causes of inflammation, but one of them is biological causes such as bacteria, fungi, and viruses. Skin involved in causing the overburden skin inflammation of the bacterium Staphylococcus epidermidis (Staphylococcus epidermidis), PROFIBUS sludge tumefaciens arc Ness (Propionibacterium acnes), Stephen Pyrococcus Ou Russ (Staphylococcus aureus) and Rouge Labor (Pseudomonas Pseudomonas aeruginosa , and the like. Bacteria such as S. epidermidis , P. acnes , S. aureus , and P. aeruginosa are causative agents of purulent diseases such as boils, acne and otitis media in the skin. Because these fungi play a major role in inducing inflammatory responses, various antibiotics have been used to treat inflammatory skin diseases. Among the currently used antibiotics are erythromycin, tetracycline, clindamycin, macrolide, azelaic acid, retinoid, benzoyl peroxide and the like, , Benzoyl peroxide causes skin dryness and hypersensitivity, and erythromycin, tetracycline, clindamycin, and macrolide are resistant to antibiotics and are difficult to continue to use, have severe toxicity, and may have opportunistic infections such as candidiasis . Therefore, in order to solve these problems, we are trying to develop a therapeutic agent for skin irritation which has antimicrobial effect and no side effects using safe natural materials. In the recent inflammatory field research, it is necessary to find a natural antiinflammatory substance Is due to the high incidence of adverse side effects of conventional anti-inflammatory agents such as cortisol synthetic agents ( Korean J. Medicinal Crop Sci. , 2010, 18 (2), 105-112).

Under these circumstances, the present inventors have made intensive studies to find natural products having antioxidative, anti-inflammatory, or antimicrobial effects and less side effects, and as a result, (1, 1-diphenyl-2-picryl-hydrazyl) free radical scavenging effect of ethyl acetate fraction of Acanthopanax koreanum Nakai, ) Production inhibitory effect, an inflammatory cytokine production inhibitory effect and an inhibitory effect on the growth of the skin uptake bacteria as well as an excellent melanogenesis and intracellular tyrosinase inhibitory activity, .

Korea J. Biotechnol. Bioeng., 2001, 16 (6), 592-602 Korean Journal of Plant Res., 2011, 24 (1), 30-39 J. Korean Soc. Food Sci. Nutr., 2010, 39 (9), 1249-1256 J. Korean Ind. Eng. Chem., 2005, 16 (3), 348-353 Korean Soc. of Food Sci. Techn., 2007, 39 (4), 464-469 Korean J. Medicinal Crop Sci., 2010, 18 (2), 105-112

It is an object of the present invention to provide a method for treating acanthopanax koreanum Nakai) extract as an active ingredient. The present invention also provides a composition for antioxidant, anti-inflammation or antibacterial activity.

Another object of the present invention is to provide a cosmetic composition comprising the antioxidant, anti-inflammatory or antimicrobial composition.

It is another object of the present invention to provide a food composition comprising the antioxidant, anti-inflammatory or antimicrobial composition.

It is still another object of the present invention to provide a pharmaceutical composition comprising the antioxidant, anti-inflammatory or antibacterial composition.

In one aspect of the present invention, the present invention provides a composition for antioxidant, anti-inflammation or antimicrobial containing an ethyl acetate fraction of Acanthopanax koreanum Nakai stem extract as an active ingredient.

Terms of this invention "island wood Acanthopanax (Acanthopanax koreanum Nakai "is a deciduous shrub with many branches of Araliaceae. It is a rare species of Jeju native species that grows rarely in valleys or forests from the coast of Jeju Island to 1,400 m above sea level. It is also known as Tamala Ogapi. The flower has a mountain-shaped inflorescence, bloomed in July-August, and the fruit of about 7 mm is black in October. The stem has a broad base, a strong hook-like thorn, and thick leaf. In the case of seed propagation, it is produced as immature seed, it takes more than 2 years to germinate, the germination rate is low, and the reproduction rate to plant is low due to low rooting power even in the case of propagation. Since the contents of physiologically active substances vary depending on the season and the area, when cultivated for the purpose of producing physiologically active substances, To verify that it is known to have been used and the higher the reported physiologically active substance content, as a folk medicine for example from a medicinal plant for treatment of neuralgia, osteoarthritis.

The above-mentioned Acanthopanax The ethylacetate fraction of koreanum Nakai stem extract is preferably but not limited to be produced by a manufacturing method comprising the following steps:

1) Acanthopanax koreanum Nakai) by adding an extraction solvent to the stem;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and then drying it to prepare an extract of Trichomones truncalis; And

4) Extracting the extract of the trichomonads trichomes in step 3) with an organic solvent to prepare truncated trichomes.

In the above method, the Acanthopanax ( Acanthopanax < RTI ID = 0.0 > koreanum Nakai) Stem can be used without restrictions such as cultivated or marketed. In addition, although not limited thereto, in step 1), it may be a method including extracting all the leaves or roots in addition to the trichomere trunks.

In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent of the step 1). As the alcohol, a C ₁ to C ₄ lower alcohol is preferably used, and as the lower alcohol, ethanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable that the extraction solvent is added by 1 to 10 times the amount of dried tricholinergic trunks. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. The number of times of extraction is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but is not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In the method, the organic solvent of step 4) is n-hexane (n -Hexane), one preferably in ethyl acetate (EtOAc) or butanol (BuOH), but not always limited thereto. The fraction was suspended island Acanthopanax extract a tree trunk in water n-hexane (n -Hexane), ethyl acetate (EtOAc), n-butanol fraction to the system sequentially (n -BuOH) and water (H ₂ O) But it is preferably one of the obtained n-hexane fraction, ethyl acetate fraction, n-butanol fraction or water fraction, more preferably ethyl acetate fraction, but not limited thereto. The fraction may be obtained by repeating the fractionation process 1 to 5 times, preferably 3 times, from the islet trunk extract, followed by fractionation and concentration under reduced pressure. However, the fraction is not limited thereto.

The EtOAc fraction may include, but is not limited to, 3,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid (3,4-dihydroxybenzoic acid) methyl ester), cafe acid (caffeic acid) and the chloro transgenic acid methyl ester (chlorogenic acid methyl ester) may be one containing the compound, the n-hexane (n -Hexane) fraction has claws come acid (coriolic acid), 16 alpha-hydroxy-17-child Nassau going to yloxy-ent-Koran -19- O acid (16α-hydroxy-17-isovaleroyloxy- ent -kauran-19-oic acid), 16 alpha-hydro-17-child Nassau reoro yloxy - may be one containing the Koran -19- O acid (16α-hydro-17-isovaleroyloxy- ent- kauran-19-oic acid) and no collection acid (kaurenoic acid) - ent.

The term " antioxidant " of the present invention means an antioxidant action. The human body has a balance between prooxidant and antioxidant, but due to various factors, And leaning towards oxidative stimulation leads to oxidative stress leading to potential cellular damage and pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is unstable and highly reactive. It reacts easily with various bio-materials, attacks irreversible damage to cells and tissues, Cytotoxicity and carcinogenesis. The reactive nitrogen species (RNS) such as NO, HNO2, and ONOO- are produced in large quantities by the immune response of macrophage neutrophils and other immune cells during the inflammatory reaction. Such active oxygen oxidizes and destroys cells in the body, thereby exposing them to various diseases. Therefore, the antioxidant effect of the extract of the present invention, especially the ethyl acetate fraction of the above-mentioned stem extract, is included in cosmetics, foods or medicines, thereby contributing to anti-aging and health promotion.

In a specific example of the present invention, the DPPH free radical scavenging activity was measured in order to confirm the antioxidative effect of the extract of Trichomonas sieboldii or its fractions. As a result, it was found that the extract of Tricholoma edulis or its fraction, especially the ethyl acetate fraction, Dependent activity of DPPH free radicals (Table 1 and Figure 2). Therefore, the antioxidant effect of the present invention may be achieved by free radical scavenging activity.

The term " anti-inflammatory " of the present invention means an action to suppress inflammation. It is known that the control of the inflammatory reaction is extremely complicated, which is known to reduce the enhancement and damage of the in vivo restoration system. However, if the inflammatory response is sustained by repeated tissue damage or regeneration, ROS and RNS are overproduced in inflammation-related cells, resulting in permanent gene deformation. Thus, ROS and RNS are deeply involved in the inflammatory response that regulates the action of various cells in vivo. During inflammation, large amounts of pro-inflammatory cytokines, nitric oxide (NO) and prostaglandin (prostaglandin E ₂ , PGE ₂ ) induce nitric oxide synthase (iNOS) And cyclooxygenase-2 (COX-2). The inflammation is a cause of various inflammatory diseases. The composition of the present invention comprising the extract of Trichophytonthymophilus, especially the fraction containing ethyl acetate of the Trichothecificus extract, has anti-inflammatory action to prevent and improve various inflammatory diseases have.

In a specific example of the present invention, inhibition of nitric oxide (NO) production using RAW 264.7 murine macrophage cells (RAW 264.7 murine macrophage cells) was performed to examine the anti- As a result of measurement of IL-6 (interleukin-6) production inhibitory activity as an active and inflammatory cytokine, it was found that the ethanol extract or its fractions, especially ethyl acetate fraction, (Table 2, Table 3, Table 4, Fig. 3, Fig. 4 and Fig. 5). Thus, the anti-inflammatory effect of the present invention may be achieved by inhibiting NO production and inflammatory cytokine production inhibition.

The term " antibacterial " of the present invention means an action of inhibiting or controlling the growth and proliferation of microorganisms such as bacteria or fungi. The microorganism of the present invention is not limited to this, S. epidermidis or P. acnes . Many kinds of skin diseases are mainly caused by skin fungi, and acne bacteria and dandruff are typical. The onset of acne has been reported to be the main cause of inflammation reaction of acne bacteria, and dandruid is ideally proliferating on skin such as back and neck, causing seborrheic dermatitis. The cause of atopic dermatitis has not been elucidated yet, but it has been reported as staphylococcus as a cause of atopic dermatitis. Preservatives and antimicrobial agents are essential to reduce the incidence of skin diseases caused by such skin diseases and other bacteria, and to protect the skin. However, synthetic substances used in the past may cause allergic reactions to the skin. It is important to do. Therefore, when the extract of the present invention is incorporated into cosmetics, foods or medicines, it can contribute to prevention and improvement of skin diseases by achieving microorganisms, especially skin microbial growth inhibitory effect.

In a specific example of the present invention, in order to confirm the antimicrobial effect of the extract of Trichomonas sieboldii or its fractions, a skin disc diffusion method, Staphylococcus epidermidis ( Staphylococcus epidermidis) ; CCARM 3709, 3710, 3711), Propionibacterium acnes ; CCARM 9009, 9010, 0081), Staphylococcus aureus ; KCCM 11335) and Pseudomonas aeruginosa ( KCCM 11802) were measured for antimicrobial activity against eight strains. The results showed that the diameter of the circular growth zone was smaller than that of all eight strains in the n - hexane fraction, The ethyl acetate fraction showed excellent growth inhibitory activity against the four strains (Table 5). Therefore, the antimicrobial effect of the present invention can be achieved by the activity of inhibiting growth of the skin microorganism.

In another aspect, the present invention relates to a method of treating acanthopanax The present invention provides a cosmetic composition comprising an antioxidant, an anti-inflammatory or antimicrobial composition containing an ethyl acetate fraction of Korean napa Nakai stem extract as an active ingredient.

The ethylacetate fraction of the extract of Tricholoma japonica extract of the present invention has an antioxidative effect, an anti-inflammatory effect and an antimicrobial effect, and thus the antioxidative, anti-inflammatory or antibacterial May be added to the cosmetic composition.

The cosmetic composition can prevent or ameliorate any one of skin diseases selected from the group including acne, atopy, athlete's foot, psoriasis, eczema and dermatitis, not only has a skin whitening effect, but also ages skin, wrinkles, Prevention or amelioration.

In a particular embodiment of the invention, the effect of the island Acanthopanax ethyl acetate fraction of a tree trunk extract Staphylococcus epidermidis (S. epidermidis) and propionyl sludge inhibiting the growth of tumefaciens arc Ness (P. acnes) strains (Table 5), and it was confirmed that melanin contents and melanin contents decreased and the activity of intracellular tyrosinase was decreased, thereby proving that there was also a whitening effect (Table 15, Table 16, Table 19 , Figs. 16, 17, and 20A). In addition, excellent antioxidative effects were also confirmed (Table 1 and Fig. 2).

The present invention relates to a method for preventing acne bacteria from acne-causing bacteria such as Propionibacterium acnes, which is an acne causative agent, as well as a second antibacterial agent such as Staphylococcus epidermidis after acne infection, And it has excellent anti-inflammatory effect which inhibits inflammation-related cytokine IL-6 in macrophages and inhibits NO production secreted by macrophages after LPS stimulation. Antibiotics such as tetracycline have limitations in use such as side effects and the appearance of resistant bacteria, while the ethyl acetate fraction of the extract of the extract of the trilobite extract according to the present invention is safe and suitable as a cosmetic composition because it has no side effects.

The cosmetic composition of the present invention can be produced in the form of a general emulsified formulation and a solubilized formulation. Examples of the emulsified formulations include nutritive lotions, creams, essences, and the like, and the solubilization formulations include softening longevity. Suitable formulations include, but are not limited to, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) A cream, a skin, a lotion, a powder, an ointment, a spray, or a conical stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, surfactants, water, ionic or nonionic emulsifiers, Auxiliaries such as ionic sequestering agents, chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetic compositions &Lt; / RTI >

In yet another aspect, the present invention provides a method of treating acanthopanax The present invention provides a food composition comprising an antioxidant, anti-inflammatory or antimicrobial composition containing, as an active ingredient, an ethyl acetate fraction of Korean napa Nakai stem extract.

When the composition of the present invention is used as a food additive, the fraction of ethyl acetate in the extract of Trichophyton trunks may be added as it is or may be added to other foods or food ingredients Can be used together, and can be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment), and may further include a food-acceptable food-aid additive. Since the composition of the present invention contains an extract derived from a natural product and a fraction thereof as an active ingredient, there is no problem in terms of stability and there is no great limitation on the amount of the mixture.

The food composition of the present invention may include all foods having a conventional meaning, and may be mixed with terms known in the art such as functional foods, health functional foods, and the like.

The term " functional food " in the present invention means a food prepared and processed by using a raw material or ingredient having functionality useful to the human body according to Law No. 6727 on health functional foods, and " And function of the nutrient for the purpose of obtaining a beneficial effect in health use such as controlling the nutrient or physiological action.

The term " health functional food " of the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, mixing, or the like a specific ingredient contained in a food raw material, Refers to a food which is designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease and the like can be sufficiently exhibited to the living body by the above components. Recovery, and so on.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition comprising the ethyl acetate fraction of the extract of Tricholoma matsutakeii as an active ingredient of the present invention can be prepared by mixing other suitable auxiliary ingredients that may be contained in foods and known additives in accordance with the selection of a person skilled in the art. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention and its fractions as a main ingredient.

Examples of foods that can be used in the present invention include special nutritional foods such as crude oil, milk, baby food, meat products, fish meat products, tofu, mushrooms, noodles such as ramen noodles, (Such as soy sauce, doenjang, kochujang, mixed potatoes), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickles ), Beverages (such as fruit, vegetable beverages, beverages, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a drink, it may contain various sweeteners, flavors, or natural carbohydrates as an additional ingredient such as ordinary beverages. In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

In yet another aspect, the present invention provides a method of treating acanthopanax The present invention provides a pharmaceutical composition comprising an antioxidant, an anti-inflammatory or antimicrobial composition containing an ethyl acetate fraction of a Korean nacre ( Nakai) stem extract as an active ingredient.

The extract of the present invention and its fractions have antioxidative, antiinflammatory and antimicrobial effects against skin sclerosing bacteria, as described above for the ethyl acetate fraction of the extract of Trichomonas sieboldii. It can be used as a pharmaceutical composition to be included in medicines for preventing, improving and treating diseases caused by active oxygen, inflammatory diseases and skin diseases caused by skin diseases.

The above-mentioned antioxidation is as described above, and thus can be included in a pharmaceutical composition for the purpose of antioxidation, and can have a preventive or therapeutic effect on a disease caused by active oxygen. The diseases caused by the active oxygen include, but are not limited to, degenerative neurological diseases including arteriosclerosis, Lou Gehrig's disease, Parkinson's disease, Alzheimer's disease, proximal axillary sclerosis and Huntington's disease, myocardial infarction, angina pectoris, coronary artery disease, May include various diseases such as cardiovascular diseases including diseases, ischemic brain diseases including stroke, digestive system diseases including diabetes, gastritis and gastric cancer, cancer, leukemia, aging, rheumatoid arthritis, hepatitis, atopic dermatitis, Preferably aging caused by active oxygen.

In addition, the anti-inflammatory activity is as described above, and thus can be included in a pharmaceutical composition for the prophylaxis or treatment of anti-inflammatory, i.e., inflammatory diseases. The inflammatory diseases include allergic diseases including allergic asthma, allergic rhinitis, allergic mucositis, urticaria, and anaphylax, although it is not limited thereto. Arthritis, atopic dermatitis, psoriasis, asthma, multiple sclerosis, ssRNA and dsRNA viral infection, sepsis, multiple chondritis, scleroderma, including systemic sclerosis, dermatomyositis and inclusion body myositis , Eczema, gout, periodontal disease, Behcet's syndrome, edema, vasculitis, Kawasaki disease, diabetic retinitis, autoimmune pancreatitis, vasculitis, glomerulonephritis, acute and chronic bronchitis, and influenza infection.

In addition, the above-mentioned antimicrobial activities are as described above and can be included in a pharmaceutical composition for antibacterial purposes. Staphylococcus ( Staphylococcus epidermidis) epidermidis ; CCARM 3709, 3710, 3711), Propionibacterium < RTI ID = 0.0 > acnes ; CCARM 9009, 9010, 0081), Staphylococcus aureus ; KCCM 11335), and Pseudomonas aeruginosa ( KCCM 11802). The present invention also provides a method for preventing or treating a skin disease caused by a fungus such as Pseudomonas aeruginosa ( KCCM 11802). Diseases caused by the above-mentioned skin-bearing bacteria include, but are not limited to, acne, atopy, athlete's foot, psoriasis, eczema and dermatitis.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term " pharmaceutically acceptable " of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants.

In addition, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, spray agents, patches, creams, powders, suspensions, gels or gels. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term " administering " of the present invention means introducing a predetermined substance into an individual by an appropriate method, and the administration route of the composition can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal.

The term " individual " refers to all animals, including humans, including mice, mice, and livestock. Preferably, it may be a mammal, including a human.

The term " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. The administration may be carried out once per day, or divided into several doses.

Since the ethyl acetate fraction of the extract of Tricholoma japonica extract of the present invention is a natural medicinal plant raw material, it can be used safely and usefully when it is used as a cosmetic composition, a food composition or a pharmaceutical composition because it has less side effects than a general synthetic compound have.

Island of the present invention wood Acanthopanax (Acanthopanax The anti-oxidative, anti-inflammatory or antimicrobial composition comprising koreanum Nakai stem extract, fractions thereof, or a compound isolated therefrom as an active ingredient is excellent in activity of scavenging DPPH free radicals in a concentration-dependent manner, The present invention is not only excellent in the activity of inhibiting the production of nitric oxide (NO), but also has an excellent growth inhibitory activity against inflammatory skin microbes. Therefore, it is possible to prevent oxidative, inflammatory and microbial diseases, And can be usefully used for cosmetics, foods and medicines for improvement or treatment.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram schematically illustrating a method for producing an ethanol extract of the trichomonads trichomes and fractions thereof according to the present invention. FIG.
FIG. 2 is a graph showing the concentration-dependent DPPH free radical scavenging effect of the ethanol extract of the trichomonads of the present invention and the fraction of the solvent by fraction.
FIG. 3 shows the results of measuring the inhibitory activity of NO production by the ethanol extract of the trichomonads of the present invention against the production of NO induced by LPS in RAW 264.7 cells and their solvent fractions (A) and the effect on the cell viability And a result (B).
Figure 4 shows inhibition of NO production according to the concentration of the n -hexane ( n- Hex) fraction and ethyl acetate (EtOAc) fraction of the ethanol extract of the trichomonads of the present invention against LPS-induced NO production in RAW 264.7 cells (A) and the effect on cell survival rate (B).
Figure 5 shows the IL-6 production of IL-6 according to the concentration of the n -hexane ( n- Hex) fraction and the ethyl acetate (EtOAc) fraction of the ethanol extract of the trichomonads of the present invention against LPS- -6 production inhibitory activity.
FIG. 6 is a graph showing the results obtained when the ethyl acetate (EtOAc) fraction of the ethanol extract of the trichomonads of the present invention was treated with 3,4-dihydroxybenzoic acid (Compound 1), caffeic acid (Compound 3) And a chlorogenic acid methyl ester (Compound 4) are separated from each other.
7 is a schematic diagram illustrating the process of separating 3,4-dihydroxybenzoic acid methyl ester (Compound 2) from the ethyl acetate (EtOAc) fraction of the ethanol extract of the trichomonads of the present invention Fig.
8 is n of the ethanol extract of Acanthopanax island tree of the present invention stem - from hexane (n -Hex) fraction claws come acid (coriolic acid; Compound 5), 16-alpha-hydroxy-17-to-yloxy when multiple child Nassau - ent-Koran -19- O acid (16α-hydroxy-17-isovaleroyloxy- ent -kauran-19-oic acid; compound 6), 16-alpha-yloxy when a dihydro-17-child Nassau multiple-ent-Koran -19- A process for separating 16α-hydro-17-isovaleroyloxy- ent- kauran-19-oic acid (compound 7) and kaurenoic acid (compound 8).
9 is a graph showing the effect of the 3,4-dihydroxybenzoic acid (Compound 1) and caffeic acid (Compound 1) isolated from the ethyl acetate (EtOAc) 3). &Lt; tb >< TABLE >
10 is a graph showing the activity of the 3,4-dihydroxybenzoic acid (Compound 1) and caffeic acid (Compound 1) isolated from the ethyl acetate (EtOAc) 3). &Lt; tb >< TABLE >
11 is a graph showing the concentration-dependent DPPH free radical scavenging effect of the compounds of the present invention isolated from the fractions of ethyl acetate (EtOAc) fraction and n -hexane ( n- Hex) fraction.
Figure 12 shows the NO production by the total eight compounds isolated from the fractions of the trichotomous trusses ethyl acetate (EtOAc) and n -hexane ( n- Hex) of the present invention against LPS-induced NO production in RAW 264.7 cells (A) and the effect on cell survival rate (B).
Figure 13 shows Compounds 2, 4 and 6 (SEQ ID NOs: 1, 2, 3, 4 and 5) isolated from the trichotomous trusses ethyl acetate (EtOAc) fraction and the n -hexane ( n- Hex) fraction of the present invention for the production of NO induced by LPS in RAW 264.7 cells (A) and the effect (B) on the cell survival rate according to the concentration of Compound (7).
14 shows the results of measurement of inhibitory activity of NO production according to the concentrations of Compound 1 and Compound 3 isolated from the fraction of the trichomonas trichoderma (EtOAc) fractions of the present invention against LPS-induced NO production in RAW 264.7 cells (A) And cell survival rate (B).
Figure 15 shows a comparison of the compound of the present invention against the compound of the present invention, i.e., the compound of formula (I), which is isolated from the extract of the present invention's trichotomous trine ethyl acetate (EtOAc) fraction and the n -hexane ( n- Hex) fraction for the production of TNF-α and IL-6 induced by LPS in RAW 264.7 cells , Compound 4, Compound 6, and Compound 7 according to the present invention.
16 is a graph showing the results (A) and (B) of the results of measurement of inhibitory activity on melanogenesis by the ethanol extract of the trichomonoides of the present invention and their solvent fractions against the production of melanin pigment induced by? -MSH in B16F10 cells (B). Fig.
FIG. 17 is a graph showing the effect of the ethanol extract of the present invention on the production of melanin pigment induced by? -MSH in B16F10 cells according to the concentration of n -hexane ( n- Hex) fraction and ethyl acetate (EtOAc) (A) and the effect (B) on the cell survival rate of melanogenesis inhibitory activity.
FIG. 18 is a graph showing the effect of the total of eight compounds isolated from the extract of the present invention on the production of melanin pigment induced by? -MSH in B16F10 cells from the extract of Trichoderma herbicides ethyl acetate (EtOAc) and the fraction of n -hexane ( n- Hex) (A) and (B) the results of measuring the effect on melanogenesis inhibitory activity (A) and cell survival rate.
FIG. 19 shows the effect of melanin on the production of melanin pigment induced by? -MSH in B16F10 cells according to the concentration of Compound 6, Compound 7 and Compound 8 isolated from the trichomonas trichomes n -hexane ( n- Hex) fraction of the present invention (A) and the effect (B) on the cell survival rate of melanogenesis inhibitory activity.
Figure 20 is n the ethanol extract of Acanthopanax island tree of the present invention stem-hexane (n -Hex) fraction and ethyl acetate (EtOAc) fraction (A), and the n-hexane is separated from (n -Hex) fractions, Compound 6 , Compound 7, and Compound 8 (B).

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: preparation of experimental material

Example 1-1: Reagents And devices

In the present invention, the solvents used for the extraction of the sample, the separation of the solvent fractions and the analysis were the products of Merck and Junsei. Silica gel (95-110 ㎛, Merck Co.) for normal column chromatography and Sephadex LH-20 (Merck Co.) for gel filtration chromatography were used for vacuum liquid chromatography (VLC) (0.1-0.025 mm) was used. Also, MPLC (medium pressure liquid chromatography, KP-C ₁₈ -HS, Biotage Co.) was used for separation. The thin-layer chromatography (TLC) used in the separation process was a precoated silica gel aluminum sheet (silica gel 60 F ₂₅₄ , 2.0 mm, Merck Co.). In order to identify the materials separated on TLC, a UV lamp (254 nm) was used or a visualizing agent was immersed and dried using a heat-gun. KMnO ₄ aqueous solution (3% KMnO ₄ , 20% K ₂ CO ₃ , 0.25% NaOH) and 1% anisaldehyde-MeOH were used as visualizing agents as needed.

For the measurement of the physiological activity of the separated compounds, Sunrise ^™ (Tecan Co.) or Biochrom Libra S22 (Biochrom Co.) was used for absorbance measurement.

The nuclear magnetic resonance spectrometer used in the structural analysis was JNM-ECX 400 (FT-NMR system, JEOL) and AVANCE III (FT-NMR, Bruker Co.) CD ₃ OD and CDCl ₃ were used.

For HPLC analysis, HPLC (Waters Co. 600 Pump) was used. The column was an INNO column (250 × 4.6 mm, 5 μm) and a detector was a UV-Visible Detector (Waters Co. 966 PDA 254.0 nm).

Example 1-2: Experimental sample

The samples used in the present invention, Acanthopanax koreanum Nakai) Stem is collected from Goseong-ri, Ayeol-eup, Jeju Island in December 2013 through the "Halla San Ogapy", a food processing company in Jeju Island that cultivates, processes and sells island oak tree in a direct farm. Respectively.

Example 2: Island oak tree Stem ethanol extract and its solvent Fraction Produce

1 kg of dried and crushed islet trunks were placed in 20 L of 70% (v / v) ethanol (EtOH) and stirred at room temperature for 24 hours. The leached samples were filtered using a filtration apparatus under reduced pressure, and the residue thus separated was further subjected to the same filtration twice. The filtrate obtained by filtration was concentrated on a rotary evaporator in a water bath at 40 ° C or lower to obtain 100.6 g of a 70% ethanol extract. The 70% ethanol extracts obtained above were suspended in distilled water 1 L, and sequentially fractionated by the polarity order by using a separatory funnel n - hexane (n-hexane, n -Hex) , ethyl acetate (ethyl acetate, EtOAc), n A total of four solvent fraction layers were obtained in the order of -butanol ( n -butanol, n- BuOH) and water (water, H ₂ O) (FIG. The resulting fractions were each dried under reduced pressure to prepare a fraction of the dried island-like organs.

Example 3: Island oak tree Stem ethanol extract and its solvent Fraction Antioxidant effect analysis

The Blois method (Blois, MS, Nature , 1958, 181) was used to measure the free radical scavenging activity of the 70% ethanol (EtOH) extract and the four solvent fractions obtained in Example 2 above. DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging activity experiment was performed. At this time, DPPH was dissolved in ethanol so as to have a concentration of 0.2 mM.

Specifically, the extracts of the extracts of Ishigaki japonica and its n -hexane ( n- Hex) fraction, ethyl acetate (EtOAc) fraction, n -butanol ( n- BuOH) fraction and water (H ₂ O) ml, 6.25 ㎍ / mL, 12.5 ㎍ / mL, 25 ㎍ / mL, 50 ㎍ / mL and 100 ㎍ / mL. Subsequently, 180 μl of 0.2 mM DPPH solution and 20 μl of each of the diluted sample solutions were mixed in a 96-well plate and allowed to react at room temperature for 10 minutes. Then, using a spectrophotometer, 515 nm The absorbance was measured. The scavenging rate (%) was calculated by the following equation (1), and the concentration (SC ₅₀ ; scavenging concentration of 50%) when each sample inhibited DPPH by 50% was determined. Each sample was subjected to three repeated experiments to obtain the average value. Vitamin C was used as an antioxidant to compare the activity.

Island Acanthopanax tree trunk 70% ethanol (EtOH) extract and its four kinds of solvent fraction in n - hexane (n -Hex) fraction, ethyl acetate (EtOAc) fraction, n - butanol (n -BuOH) fraction, water (H ₂ O The free radical scavenging activity was found in both the extracts and fractions. In particular, the ethyl acetate (EtOAc) fraction had a DPPH SC ₅₀ value of 34.6 ㎍ / mL, indicating free radical scavenging activity (Table 1 and Fig. 2).

sample SC ₅₀ (쨉 g / mL) EtOH > 100 n- Hex > 100 EtOAc 34.6 n- BuOH 88.7 H ₂ O > 100 Vitamin C 9.1

Example 4: Island oak tree Stem ethanol extract and its solvent Fraction Analysis of anti-inflammatory effect

( N- Hex) fraction, ethyl acetate (EtOAc) fraction, n -butanol ( n- BuOH) fraction, and n -hexane fraction obtained in Example 2, and the four solvent fractions thereof, In order to confirm the antiinflammatory effect of water (H ₂ O) fractions, RAW 264.7 murine macrophage cells were used to inhibit nitric oxide (NO) production and IL-6, an inflammatory cytokine, (interleukin-6) production inhibitory activity. In order to evaluate the anti-inflammatory effect, MTT (dimethylthiazolyl diphenyl tetrazolium salt) assay was performed to evaluate the cytotoxicity of the treated samples.

Example 4-1: Cell culture

RAW 264.7 RAW 264.7 murine macrophage cells were purchased from Korean Cell Line Bank (KCLB; Seoul, Korea) and cultured with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) Were cultured at 37 ° C in a 5% CO ₂ incubator using DMEM medium (Dulbecco's modified eagle's medium, GIBCO, Grand Island, YY, USA) and subcultured at 2-day intervals.

Example 4-2: Island oak tree Stem ethanol extract and its solvent Fraction Measurement of NO production inhibitory activity and evaluation of cytotoxicity

RAW 264.7 cells cultured in Example 4-1 were dispensed in a 24-well plate at a cell number of 2 x 10 ⁵ cells / mL and cultured for 18 hours at 37 ° C under 5% CO ₂ incubator conditions. After the culture, the medium was removed, washed with phosphate buffered saline (PBS), and then replaced with a medium containing 1 μg / mL LPS (lipopolysaccharides). After replacing the medium with LPS, the ethanol extract of Ishigaki japonica and its four solvent fractions were treated at a concentration of 100 ㎍ / mL and cultured for 24 hours. After 24 hours of incubation, the amount of NO produced was measured in the form of NO ₂ ^- present in cell culture medium using Greiss reagent (1% sulfanilamide, 0.1% naphthylethylenediamine in 2.5% phosphoric acid). 100 μl of the cell culture solution cultured in the 96-well plate for 24 hours and 100 μl of the Griess reagent were mixed and reacted for 10 minutes, and the absorbance was measured at 540 nm using a microplate reader. The amount of NO produced was calculated using a sodium nitrite (NaNO ₂ ) calibration curve. At this time, 2-amino-4-picoline (10 μM) was used as an anti-inflammatory agent to compare the activity.

Meanwhile, MTT reagent at a concentration of 500 / / mL was added to 900 쨉 l of the remaining cell culture used for the NO production inhibitory activity, and the reaction was allowed to proceed for 3 hours. The supernatant was completely removed, and the formazan DMSO and cytotoxicity was assessed by measuring the absorbance at 570 nm using a microplate reader. Cytotoxicity was calculated by the following equation (2).

Island Acanthopanax tree trunk 70% ethanol (EtOH) extract and its four kinds of solvent fraction in n - hexane (n -Hex) fraction, ethyl acetate (EtOAc) fraction, n - butanol (n -BuOH) fraction, water (H ₂ O ) Fraction showed NO activity inhibition activity and cytotoxicity, and 89.2% and 87.7% of n -hexane ( n- Hex) fraction and ethyl acetate (EtOAc) fraction, respectively, (Table 2 and Fig. 3).

sample Treatment concentration NO Inhibition ( % ) Cell survival rate % ) Control (LPS processing) - 0 100.0 2-Amino-4-picoline 10 uM 84.8 97.3 EtOH 100 / / mL 34.5 109.5 n- Hex 100 / / mL 89.2 103.0 EtOAc 100 / / mL 87.7 104.6 n- BuOH 100 / / mL 13.6 110.5 H ₂ O 100 / / mL N.D. 104.6

(N.D. *: Not Detected)

Example 4-3: Island oak tree Stem of ethanol extract n - Hexane ( n -Hex) Fraction And ethyl acetate (EtOAc) Fraction Measurement of inhibitory activity of NO production by concentration and evaluation of cytotoxicity

4 kinds of the solvent fraction, n in Example 4-2 island Acanthopanax tree trunk ethanol extract in-check-hexane (n -Hex) fraction and ethyl acetate (EtOAc) fraction is produced with no NO cytotoxicity inhibiting activity is excellent and, In addition, the NO production inhibitory activity and cytotoxicity of the two fraction fractions were measured at concentrations of 25 / / mL, 50 / / mL, 100 / / mL and 200 / / mL, respectively. At this time, the NO production inhibitory activity and the cytotoxicity measurement method were performed in the same manner as in Example 4-2.

Island Acanthopanax tree trunk 70% ethanol (EtOH) an extract of n - hexane (n -Hex) fraction and ethyl acetate (EtOAc) results confirmed the NO production-inhibiting activity and cytotoxicity of the concentration of the fraction, n - hexane (n -Hex ) Fractions and ethylacetate (EtOAc) fractions were highly effective inhibition of NO production in a concentration-dependent manner without cytotoxicity (Table 3 and Fig. 4).

sample Treatment concentration NO Inhibition ( % ) Cell survival rate % ) Control (LPS processing) - 0 100.0 2-Amino-4-picoline 10 uM 77.6 113.1 n- Hex 25 / / mL 39.3 117.9 50 / / mL 72.7 110.4 100 / / mL 88.4 103.8 200 [mu] g / mL 95.1 95.4 EtOAc 25 / / mL 31.3 120.8 50 / / mL 65.9 110.6 100 / / mL 90.6 105.1 200 [mu] g / mL 93.8 100.4

Example 4-4: Island oak tree Stem of ethanol extract n - Hexane ( n -Hex) Fraction And ethyl acetate (EtOAc) Fraction Measurement of inhibitory activity of IL-6 production by concentration

RAW 264.7 cells cultured in Example 4-1 were dispensed in a 24-well plate at a cell number of 2 x 10 ⁵ cells / mL and cultured for 18 hours at 37 ° C under 5% CO ₂ incubator conditions. After the culture, the medium was removed, washed with phosphate buffered saline (PBS), and then replaced with a medium containing 1 μg / mL LPS (lipopolysaccharides). After exchange with a medium containing LPS, Acanthopanax island of a tree trunk ethanol extract n - hexane 25 ㎍ / mL to (n -Hex) fraction and ethyl acetate (EtOAc), each fraction sample, 50 ㎍ / mL, 100 ㎍ / mL And 200 [mu] g / mL and cultured for 24 hours. The IL-6 content was measured using the supernatant of the culture medium obtained after culturing for 24 hours. At this time, the content of IL-6 was measured using an ELISA kit (Life Technologies Co. invitrogen) according to the manufacturer's manual, and the absorbance was measured and quantified at 450 nm using a microplate reader. The inhibitory activity of IL-6, an inflammatory cytokine, was analyzed in comparison with the LPS-treated control group.

The inhibitory activity of IL-6 production by the concentration of n -hexane ( n- Hex) fraction and ethyl acetate (EtOAc) fraction of 70% ethanol extracts 6-induced IL-6 production was significantly reduced in a concentration-dependent manner (Table 4 And Fig. 5).

sample Treatment concentration IL-6 Inhibition ( % ) Control (LPS processing) - 0 n- Hex 25 / / mL 36.4 50 / / mL 44.6 100 / / mL 61.3 200 [mu] g / mL 71.5 EtOAc 25 / / mL 37.8 50 / / mL 48.4 100 / / mL 74.4 200 [mu] g / mL 84.1

Example 5: Island oak tree Stem ethanol extract and its solvent Fraction Antibacterial effect analysis

( N- Hex) fraction, ethyl acetate (EtOAc) fraction, n -butanol ( n- BuOH) fraction, and n -hexane fraction obtained in Example 2, and the four solvent fractions thereof, In order to confirm the antimicrobial effect of the water (H ₂ O) fraction, antimicrobial activity was measured using a paper disc diffusion method.

Example 5-1: Culture of strain

Staphylococcus epidermidis ( CCARM 3709, 3710, 3711), which is a skin antibiotic, was used to measure the antimicrobial activity of the extract of Tricholinus edulis and the four solvent fractions thereof obtained in Example 2, , Propionibacterium ( Propionibacterium < RTI ID = 0.0 > acnes ; CCM 9009, 9010, 0081), Staphylococcus aureus ( KCCM 11335) and Pseudomonas aeruginosa ( KCCM 11802) were purchased from the Korean Microorganism Conservation Center. In the case of S. epidermidis , S. aureus and P. aeruginosa , 3 passages were cultured using a TSB (tryptic soy broth) culture medium, and P. acnes was cultured using GAM (gifu anaerobic medium) And then used for the antimicrobial activity test.

Example 5-2: Paper disc diffusion method

(EtOH) extract of the present invention and its four solvent fractions, n -hexane fraction ( n- Hex) fraction, ethyl acetate ( The growth inhibitory activity was measured using the paper disc diffusion method to measure the antibacterial activity of the fractions of EtOAc, n -BuOH ( n- BuOH) and water (H ₂ O) fractions.

Specifically, P. acnes (CCARM 9009, 9010, 0081) was adjusted to 1.5 × 10 ⁶ CFU / mL by controlling the turbidity with a 0.5 McFarland standard, and then placed in GAM medium containing 0.8% agar containing 1.5% agar GAM medium. When the medium was hardened, the ethanol extract of the trilobalis truncatus and its four solvent fractions were anaerobically cultured at 37 ° C for 48 hours on an 8 mm paper disc having a diameter of 100 ㎎ / mL. The diameter of the circular growth zone formed after incubation was measured. In addition, the turbidity of S. epidermidis (CCARM 3709, 3710, 3711), S. aureus (KCCM 11335) and P. aeruginosa (KCCM 11802) was adjusted to 0.5 × 10 ⁶ CFU / mL with 0.5 McFarland standard, % Agar in TSB medium and poured onto TSB medium containing 1.5% agar. When the medium was hardened, an 8 mm paper disc having a diameter of 100 ㎎ / mL was extruded on the culture medium and cultured at 37 ° C. for 24 hours. The diameter of the circular growth zone formed after incubation was measured. At this time, erythromycin (1 mg / mL) was used as an active substance to be compared.

Staphylococcus epidermidis ( CCARM 3709, 3710, 3711), Propionibacterium acnes ( Staphylococcus epidermidis , acnes ; CCARM 9009, 9010, 0081), Staphylococcus aureus ( KCCM 11335) and Pseudomonas aeruginosa ; KCCM 11802) 70% islands Acanthopanax tree trunk for a total of 8 strains of ethanol (EtOH) extract and four solvent fraction of its n-hexane (n -Hex) fraction, ethyl acetate (EtOAc) fraction, n-butanol (n - (H ₂ O) fractions were measured and the results showed that the diameter of the circular zone of cleavage was smaller than that of all eight strains of ethyl acetate (EtOAc) in the n - hexane fraction ( n - Hex) ) Fractions showed excellent growth inhibitory activity against the four strains (Table 5).

Bacterial density
(1.5 x 10 < ⁶ > CFU / mL) Extracts and fractions (treatment concentration: 100 mg / mL) Erythromycin
(Treatment concentration:
1 mg / mL)
Extract n- Hex EtOAc n- BuOH H ₂ O Clear zone (mm) S. epidermidis (CCARM 3709) N.D. 12 18 N.D. N.D. 30 S. epidermidis (CCARM 3710) N.D. 10 14 N.D. N.D. N.D. S. epidermidis (CCARM 3711) N.D. 9 10 N.D. N.D. 26 P. acnes
(CCARM 0081) N.D. 10 N.D. N.D. N.D. N.D. P. acnes
(CCARM 9009) N.D. 12 12 N.D. N.D. 30 P. acnes
(CCARM 9010) N.D. 10 N.D. N.D. N.D. 28 S. aureus
(KCCM 11335) N.D. 15 N.D. N.D. N.D. 28 P. aeruginosa
(KCCM 11802) N.D. 14 N.D. N.D. N.D. 30

(N.D. *: Not Detected)

Example 6: Island oak tree Solvent of Stem Ethanol Extract From the fraction Isolation and structure identification of physiologically active compounds

Based on the results of Examples 3 to 5, n -hexane ( n -hexane) having a physiologically active function was obtained in order to isolate the physiologically active compound from the fractions obtained in Example 2, Fractions and ethylacetate (EtOAc) fractions were selected and the physiologically active compounds were isolated and their structures were identified.

Example 6-1: Ethyl acetate ( EtOAc ) Fraction Isolation and structure identification of active ingredients

Medium pressure liquid chromatography (MPLC) was carried out to sequentially fractionate 5.0 g of the ethyl acetate (EtOAc) fraction obtained in Example 2 according to the polarity. The ethyl acetate fraction (5.0 g) was dissolved in methanol (MeOH) (8 mL), filtered through a 0.45 ㎛ PVDF filter, and injected. Reverse column silica gel (C ₁₈ ) was used as a column. The polarity ratios were sequentially lowered with solvent conditions of methanol (MeOH): water (H ₂ O) (10 → 90%, 80 min) and methanol (90 → 100%, 10 min) mL to obtain a total of 43 fractions (Fr., Fr.). Was from a fraction of the MPLC Fr.7-9 scored compound 1 (196.0 mg), Fr.11-12 (100.0 mg) of chloroform (CHCl _3): methanol (MeOH) = 3: Sephadex the solvent conditions of 1 Compound 4 (11.5 mg) was obtained by performing LH-20 column chromatography (Sephadex LH-20 column chromatograpy). Also, compound 3 (100.8 mg) was obtained from Fr.13-15 of the MPLC fractions (Fig. 6).

On the other hand, to fractionate 4.8 g of the ethyl acetate (EtOAc) fraction obtained in Example 2 in order according to polarity, a glass column filled with normal phased silica gel for vacuum liquid chromatography (VLC) ) Was used to perform VLC. The polarity ratios were determined by gradient elution using solvent conditions of n -hexane ( n- Hex): ethyl acetate (EtOAc) (0 → 100%), ethyl acetate (EtOAc): methanol (MeOH) 5, 10, or 20%, respectively, and eluted with 300 mL each, resulting in a total of 27 fractions (Fr. Of VLC fraction Fr.2-4 (150.0 mg) a n - hexane (n -Hex): ethyl acetate (EtOAc) = 1: 1 solvent conditions with normal phase silica gel column chromatography (normal phased silica gel column chromatograpy) To give compound 2 (11.5 mg) (Figure 7).

A total of four active compounds were separated from the ethyl acetate (EtOAc) fraction as described above, and ¹ H-nuclear magnetic resonance spectrum ( ¹ H-NMR spectrum) and ¹³ C-nuclear magnetic resonance spectrum ( ¹³ C-NMR spectrum) As a result, compound 1 was 3,4-dihydroxybenzoic acid, compound 2 was 3,4-dihydroxybenzoic acid methyl ester, Compound 3 was identified as caffeic acid and compound 4 was identified as chlorogenic acid methyl ester (Table 6).

Example 6-2: n - Hexane ( n -Hex) Fraction Isolation and structure identification of active ingredients

To fractionate 4.0 g of the n -hexane ( n- Hex) fraction obtained in Example 2 in order according to the polarity, a glass column filled with normal phased silica gel for vacuum liquid chromatography (VLC) column) was used to perform VLC. Using a gradient elution method n - hexane (n -Hex): ethyl acetate (EtOAc) while increasing the ratio of polar solvent to the conditions of (0 → 100%) at 5 or 10% total of 15 fractions were eluted by 300 mL each (fraction, Fr.) was obtained. It was obtained the compound 8 (222.2 mg) from the fractions of the VLC Fr.2, Fr.3 (50.0 mg) in chloroform (CHCl ₃₎ to 100% solvent criteria of Sephadex LH-20 column chromatography (Sephadex LH-20 column chromatograpy) to obtain compound 7 (17.0 mg). Compound 5 (18.3 mg) and compound 6 (31.4 mg) were obtained from Fr.6 (180.0 mg) by the same method as that for obtaining compound 7 (Fig. 8).

The total of four active compounds were separated from the n -hexane ( n- Hex) fraction as described above and analyzed by ¹ H-nuclear magnetic resonance spectrum ( ¹ H-NMR spectrum) and ¹³ C-nuclear magnetic resonance spectrum ( ¹³ C- The compound 5 was identified as coriolic acid and the compound 6 as 16 alpha -hydroxy-17-isovalerolyloxy- ent -koran-19-oxic acid (16? hydroxy-17-isovaleroyloxy- ent -kauran- 19-oic acid) with, compound 7 is 16 alpha-yloxy when a dihydro-17-child Nassau multiple-ent-Koran -19- O acid (16α-hydro-17-isovaleroyloxy - ent- kauran-19-oic acid, and compound 8 was identified as kaurenoic acid (Table 7).

Example 7: Island oak tree Ethyl Acetate of Stem Ethanol Extract ( EtOAc ) Fraction Qualitative and quantitative analysis of active compounds isolated from water

3,4-dihydroxybenzoic acid (Compound 1), which is present in a relatively large amount among the compounds isolated from the fractions obtained from Example 6-1 and the fractions isolated from the fractions of the fractions of the fractions of ethyl acetate (EtOAc) High performance liquid chromatography (HPLC) analytical tests were performed for qualitative and quantitative analysis of caffeic acid (Compound 3).

Specifically, for the qualitative analysis of the compound, Compound 1 and Compound 3 dissolved in 10 mg / mL were added, respectively, and filtered using a 0.45 쨉 m PVDF filter. The filtrate was analyzed, and when the compounds 1 and 3 were separately added, And the retention time of each compound was determined. In addition, quantitative analysis of the compound described above was conducted using 3,4-dihydroxybenzoic acid (Cas: 99-50-3, Sigma-Aldrich Co.) and caffeic acid (caffeic acid, Cas: 331-39-5, Sigma-aldrich Co.) standard solution was used to prepare a calibration curve for quantification of each compound with the area value of the obtained peak. To prepare a calibration curve, 1,000 mg of each of 3,4-dihydroxybenzoic acid and caffeic acid standard was dissolved in 1 liter methanol (MeOH) to prepare a stock solution at 1,000 ppm, and each of 25, 50, and 100 And 200 ppm to prepare a calibration curve.

The column and detector used were HPLC column (250 × 4.6 mm, 5 μm) and UV-Visible Detector (Waters Co., 966 PDA 254.0 nm). The eluent was gradient eluted with acetonitrile / 0.1% acetic acid under the conditions of Table 8 below and analyzed at a flow rate of 1.0 mL / min (min) for 60 minutes (Table 8).

Time (min) Flow (mL / min) 0.1% acetic acid (%) Acetonitrile (%) 0 1.0 90 10 15 1.0 60 40 30 1.0 30 70 42 1.0 5 95 45 1.0 90 10

3,4-dihydroxybenzoic acid (Compound 1), which is present in a comparatively excessive amount among the compounds isolated from the fraction of the trichloroacetic acid ethyl acetate (EtOAc) fraction using the reversed phase column (C ₁₈ ) Qualitative and quantitative analysis of caffeic acid (Compound 3) revealed that 7 minutes of the retention time of the ethyl acetate (EtOAc) fraction peak in the HPLC qualitative analysis was Compound 1 and 18 minutes was Compound 3 9).

In addition, a calibration curve using standard solutions of 3,4-dihydroxybenzoic acid and caffeic acid was prepared from the results of HPLC quantitative analysis, and the resultant was subjected to an external standard method using ethyl acetate (EtOAc) as a result of measuring the content of the compound 1 and compound 3 isolated from the fractions, compound 1 and compound 3 Ethyl acetate (EtOAc) fractions were present in fractions of 4.18% and 2.30%, respectively (FIG. 10).

Example 8: Island oak tree Ethyl Acetate of Stem Ethanol Extract ( EtOAc ) Fraction And n - Hexane ( n -Hex) From the fraction Antioxidative, anti-inflammatory and antibacterial effects of isolated active compounds

Antioxidant, anti-inflammatory and antimicrobial agents against the eight compounds isolated and identified in Example 6 above In order to confirm the effect, in the same manner as in Example 3, Example 4 and Example 5 DPPH free radical scavenging activity, nitric oxide (NO) production inhibitory activity, and growth inhibitory activity against skin fungi were measured.

Example 8-1: Island oak tree Stem ethyl acetate ( EtOAc ) Fraction And n - Hexane ( n Antioxidative Effect of Active Compounds Isolated from Fractions

In order to examine the antioxidant effect of eight active compounds isolated from the trichloroacetic acid ethyl acetate (EtOAc) fraction and the n -hexane ( n- Hex) fraction, DPPH free radical scavenging activity was measured in the same manner as in Example 3 Respectively. At this time, DPPH assay was performed at a concentration of 25 μM, 50 μM, 100 μM, 300 μM and 500 μM, respectively.

The DPPH free radical scavenging activity of the compounds isolated from the trichloroacetic acid ethyl acetate (EtOAc) fraction and the n -hexane ( n- Hex) fraction was measured to find that Compound 1, Compound 2 , Compound 3 and Compound 4 showed excellent free radical scavenging activity at 361.8 [mu] M, 275.8 [mu] M, 276.0 [mu] M and 165.8 [mu] M, respectively (Table 9 and Fig. 11).

compound
(compound) Name of compound SC ₅₀
( μM ) Cpd1 3,4-dihydroxybenzoic acid
(3,4-dihydroxybenzoic acid) 361.8 Cpd2 3,4-dihydroxybenzoic acid methyl ester
(3,4-dihydroxybenzoic acid methyl ester) 275.8 Cpd3 Caffeic acid 276.0 Cpd4 Chlorogenic acid methyl ester
(chlorogenic acid methyl ester) 165.8 Cpd5 Coriolic acid > 100 Cpd6 16 alpha -hydroxy-17-isovalerroyloxy- endo -koran-19-aoxic acid
(16α-hydroxy-17-isovaleroyloxy- ent- kauran-19-oic acid) > 100 Cpd7 16 alpha -hydro-17-isovalerroyloxy- endo -koran-19-
(16α-hydro-17-isovaleroyloxy- ent- kauran-19-oic acid) > 100 Cpd8 Kaurenoic acid > 100 Vit.C Vitamin C (vitamin C) 44.6

Example 8-2: Island oak tree Stem ethyl acetate ( EtOAc ) Fraction And n - Hexane ( n -Hex) From the fraction Analysis of anti-inflammatory effects of isolated active compounds

In order to confirm the antiinflammatory effect of a total of eight active compounds isolated from the trichloroacetic acid ethyl acetate (EtOAc) fraction and the n -hexane ( n- Hex) fraction, RAW 264.7 murine TNF-α, which is an inflammatory cytokine, and TNF-α and TNF-.alpha. Were measured using an ELISA Kit (Life Technologies co. Invitrogen) in the same manner as in Example 4-4, IL-6 (interleukin-6) production inhibitory activity was measured. MTT assay was also performed to assess the cytotoxicity of the treated compounds to confirm the anti-inflammatory effect.

Island Acanthopanax tree trunk ethyl acetate (EtOAc) fraction and n - hexane (n -Hex) at a concentration of 100 μM of the compounds isolated from the fractions, each process in the murine macrophage RAW 264.7 and verified against NO production inhibitory activity and cytotoxicity results, All compounds showed NO production inhibitory activity without cytotoxicity. It was confirmed that Compound 6 and Compound 8 significantly inhibited the production of NO (Table 10 and Fig. 12).

sample Treatment concentration ( uM ) NO Inhibition ( % ) Cell survival rate % ) Control (LPS processing) - 0 100.0 2-Amino-4-picoline 10 83.8 118.3 Compound 1 (Cpd1) 100 9.9 104.8 Compound 2 (Cpd2) 100 38.6 112.5 Compound 3 (Cpd3) 100 19.6 104.6 Compound 4 (Cpd4) 100 19.9 109.2 Compound 5 (Cpd5) 100 49.4 115.6 Compound 6 (Cpd6) 100 61.7 103.4 Compound 7 (Cpd7) 100 29.7 100.6 Compound 8 (Cpd8) 100 59.9 105.2

Of the total of eight active compounds isolated from the fractions of the tricholinergic trichloroacetate (EtOAc) and n -hexane ( n- Hex) fractions, the compounds 2, 4, 6 and 7 were diluted to 25 μM and 50 μM And 100 μM of RAW 264.7 murine macrophages. As a result of confirming the NO production inhibitory activity and cytotoxicity according to the concentrations of the four compounds, it was confirmed that NO production was inhibited in a concentration-dependent manner without cytotoxicity And Fig. 13).

sample Treatment concentration ( uM ) NO Inhibition ( % ) Cell survival rate % ) Control (LPS processing) - 0 100.0 2-Amino-4-picoline 10 77.9 108.2 Compound 2 (Cpd2)

25 0.7 112.3 50 22.4 117.6 100 36.7 105.9 Compound 4 (Cpd4)

25 N.D. 100.9 50 4.7 106.2 100 18.3 105.7 Compound 6 (Cpd6)

25 5.7 102.5 50 21.6 102.2 100 61.7 96.2 Compound 7 (Cpd7)

25 0.7 101.5 50 14.7 100.4 100 26.6 94.2

(N.D. *: Not Detected)

Compounds 1 and 3, which were present in a relatively large amount and were subjected to qualitative and quantitative analysis by HPLC, were dissolved in RAW 264.7 murine macrophages at concentrations of 100 μM and 200 μM, respectively, in the active compounds isolated from the truncated ethyl acetate (EtOAc) mu] M and 400 [mu] M, and the NO production inhibitory activity and cytotoxicity according to the concentrations of the two compounds were examined. As a result, it was confirmed that NO production was inhibited in a concentration-dependent manner without cytotoxicity (Table 12 and Fig.

sample Treatment concentration ( uM ) NO Inhibition ( % ) Cell survival rate % ) Control (LPS processing) - 0 100.2 2-Amino-4-picoline 10 77.7 108.2 Compound 1 (Cpd1)

100 10.4 111.3 200 19.5 108.2 400 28.7 106.3 Compound 3 (Cpd3)

100 16.5 109.6 200 25.6 111.3 400 46.8 110.3

Among the total of 8 active compounds isolated from the fractions of the trichotomous truss ethyl acetate (EtOAc) and the n -hexane ( n- Hex), Compound 2, Compound 4, Compound 6 and Compound 7 were dissolved in 25 μM and 50 μM And RAW 264.7 murine macrophages at a concentration of 100 [mu] M, and TNF- [alpha] and IL-6 production inhibitory activities were determined according to the concentrations of the above four compounds. As a result, It was confirmed that the amount of TNF-α, which is an inflammatory mediator, decreased slightly, and the amount of IL-6 produced was also significantly decreased in a concentration-dependent manner (Table 13 and FIG. 15).

sample Treatment concentration ( uM ) TNF -α inhibition rate ( % ) IL-6 Inhibition ( % ) Control (LPS processing) - 0 0 Compound 2 (Cpd2)

25 N.D. 10.6 50 9.6 37.1 100 25.0 71.8 Compound 4 (Cpd4)

25 4.5 40.2 50 10.6 57.2 100 19.7 71.5 Compound 6 (Cpd6)

25 N.D. 46.3 50 17.6 64.4 100 20.1 77.2 Compound 7 (Cpd7)

25 N.D. 40.3 50 16.9 57.2 100 23.3 67.6

(N.D. *: Not Detected)

Example 8-3: Island oak tree Stem ethyl acetate ( EtOAc ) Fraction And n - Hexane ( n -Hex) From the fraction Analysis of antibacterial effect of isolated active compounds

Island Acanthopanax tree trunk ethyl acetate (EtOAc) fraction and n - hexane (n -Hex) to confirm that the antimicrobial activity of the total of eight active compounds isolated from the fractions, for a total of 8 strains in the same manner as Example 5 Growth inhibitory activity was measured.

Staphylococcus epidermidis ( CCARM 3709, 3710, 3711), Propionibacterium acnes ( Staphylococcus epidermidis , acnes ; (EtOAc) fractions and n ( n- hexane) acetate fractions for eight strains of Staphylococcus aureus ( KCCM 11335) and Pseudomonas aeruginosa ( KCCM 11802) -Hexane ( n- Hex) fractions, the antimicrobial activities of eight compounds isolated from the fraction of n -hexane ( n- Hex) were measured. As shown in Table 14, the diameters of the circular zone- It was confirmed that there were excellent growth inhibitory activities against three strains in compound 3, three strains in compound 5, five strains in compound 6, and one strain in compound 8 (Table 14).

Bacterial density
(1.5 x 10 < ⁶ > CFU / mL) Compound (treatment concentration: 100 mM) Erythromycin
(Treatment concentration:
1 mM)
Cpd1 Cpd2 Cpd3 Cpd4 Cpd5 Cpd6 Cpd7 Cpd8 Clear zone (mm) S. epidermidis (CCARM 3709) N.D. N.D. 14 N.D. N.D. N.D. N.D. N.D. 22 S. epidermidis (CCARM 3710) N.D. N.D. 10 N.D. N.D. N.D. N.D. N.D. N.D. S. epidermidis (CCARM 3711) N.D. N.D. 9 N.D. N.D. 10 N.D. N.D. 19 P. acnes
(CCARM 0081) N.D. N.D. N.D. N.D. 9 12 N.D. 9 N.D. P. acnes
(CCARM 9009) N.D. 12 N.D. N.D. 9 14 N.D. N.D. 24 P. acnes
(CCARM 9010) N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 22 S. aureus
(KCCM 11335) N.D. N.D. N.D. N.D. 10 12 N.D. N.D. 22 P. aeruginosa
(KCCM 11802) N.D. N.D. N.D. N.D. N.D. 11 N.D. N.D. 22

(N.D. *: Not Detected)

Example 9: Island oak tree Stem ethanol extract, its solvent Fraction And From the fraction Analysis of whitening effect of isolated compounds

Example 2 island Acanthopanax tree trunk obtained in ethanol (EtOH) extract, four solvent fraction of its n-hexane (n -Hex) fraction, ethyl acetate (EtOAc) fraction, n-Butanol (n -BuOH) fraction, To confirm the whitening effect of the water (H ₂ O) fractions and the compounds isolated from the fractions, the melanogenesis inhibitory activity was measured using B16F10 murine melanoma cell (B16F10 murine melanoma cell). In order to evaluate the whitening effect, MTT (dimethylthiazolyl diphenyl tetrazolium salt) assay was performed to evaluate the cytotoxicity of the treated samples.

Example 9-1: Cell culture

B16F10 murine melanoma cells were cultured in a cell line of Korean Cell Line (Bacillus subtilis) in order to measure the whitening activity of the extracts of the extracts of Tricholinus edulis, the four solvent fractions thereof and the compounds separated from the fractions obtained in Example 2 above, (Dulbecco's modified eagle's medium, GIBCO, Grand Island, NY) containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) YY, USA) at 37 ° C in a 5% CO ₂ incubator and subcultured at 3-day intervals.

Example 9-2: Island oak tree Stem ethanol extract and its solvent Fraction Measurement of melanogenesis inhibitory activity and evaluation of cytotoxicity

B16F10 cells cultured in Example 9-1 were dispensed in a 6-well plate to a cell number of 5 x 10 ⁴ cells / mL and cultured at 37 ° C under a 5% CO ₂ incubator for 24 hours. After the culture, the medium was removed, washed with phosphate buffered saline (PBS), and then replaced with a medium containing 100 nM of α-MSH (α-melanocyte stimulating hormone). After exchanging with a medium containing α-MSH, the ethanol extract of Ishigaki japonica and its four solvent fraction samples were treated at a concentration of 100 μg / mL and cultured for 72 hours. After culturing for 72 hours, the cells were washed with PBS and treated with trypsin-EDTA to recover the cells. The recovered cells were centrifuged and only the supernatant-removed cell pellet was taken. Sonication buffer (6.7 mM sodium phosphate buffer containing 1% triton X-100, 0.2 mM PMSF) was added to the cell pellet for sonication and then centrifuged again. The separated supernatant was reacted with L-DOPA and the intracellular tyrosinase inhibitory activity was measured at 475 nm using a microplate reader. The pellet was washed with 1 N sodium hydroxide (NaOH), and the absorbance was measured at 405 nm to confirm melanin contents. Melasolv (20 μM) was used as a whitening agent to compare the activity.

On the other hand, MTT reagent at a concentration of 500 / / mL was added to 900 세포 of the cell culture which had been cultured under the same conditions as the cells used for the melanin formation inhibitory activity, and the reaction was allowed to proceed for 3 hours. The supernatant was completely removed, formazan) was dissolved in DMSO and the cytotoxicity was evaluated by measuring the absorbance at 570 nm using a microplate reader. Cytotoxicity was calculated by the above formula (2).

Island Acanthopanax tree trunk 70% ethanol (EtOH) extract and its four kinds of solvent fraction in n - hexane (n -Hex) fraction, ethyl acetate (EtOAc) fraction, n - butanol (n -BuOH) fraction, water (H ₂ O ) confirmed the formation of melanin (melanogenesis) inhibitory activity and cytotoxicity of the resulting fraction, n - hexane (n -Hex) fraction and ethyl acetate (EtOAc), respectively the melanin content was decreased by 79.7% and 63.7% in fraction, cytotoxic (Table 15 and Fig. 16).

sample Treatment concentration Inhibition rate of melanin formation ( % ) Cell survival rate % ) Control (α-MSH treatment) - 0 100.0 Melasolv 20 uM 74.7 111.4 EtOH 100 / / mL N.D. 111.7 n- Hex 100 / / mL 79.7 73.0 EtOAc 100 / / mL 63.7 70.3 n- BuOH 100 / / mL N.D. 100.4 H ₂ O 100 / / mL N.D. 99.3

(N.D. *: Not Detected)

Example 9-3: Island oak tree Stem of ethanol extract n - Hexane ( n -Hex) Fraction And ethyl acetate (EtOAc) Fraction Melanin formation by concentration ( melanogenesis ) Inhibitory activity measurement and cytotoxicity evaluation

The n -hexane ( n- Hex) fraction and the ethyl acetate (EtOAc) fraction, which showed cytotoxicity but decreased the melanin contents in the four solvent fractions of the ethanol extract of the trichomonads, Melanogenesis inhibition activity and cytotoxicity were measured by concentrations of 25 ㎍ / mL, 50 ㎍ / mL and 100 ㎍ / mL, respectively. At this time, the melanin formation inhibitory activity and cytotoxicity were measured in the same manner as in Example 9-2.

Island Acanthopanax tree trunk 70% ethanol (EtOH) an extract of n - hexane (n -Hex) fraction and ethyl acetate (EtOAc) results confirmed the melanin formation inhibitory activity and cytotoxicity of the concentration of the fraction, n - hexane (n -Hex ) Fractions and ethylacetate (EtOAc) fractions reduced the melanin content in a concentration-dependent manner without lethal cytotoxicity at concentrations below 100 [mu] g / mL (Table 16 and Figure 17).

sample Treatment concentration Inhibition rate of melanin formation ( % ) Cell survival rate % ) Control (α-MSH treatment) - 0 100.0 Melasolv 20 uM 70.7 110.4 n- Hex 25 / / mL N.D. 121.2 50 / / mL 29.8 102.3 100 / / mL 83.4 78.5 EtOAc 25 / / mL N.D. 115.4 50 / / mL 22.7 95.4 100 / / mL 68.8 71.2

(N.D. *: Not Detected)

Example 9-4: Island oak tree Ethyl Acetate of Stem Ethanol Extract ( EtOAc ) minute And n - Hexane ( n -Hex) From the fraction Measurement of melanogenesis inhibitory activity and cytotoxicity evaluation of isolated active compounds

To confirm the melanogenesis inhibitory activity and cytotoxicity of the eight compounds isolated and identified in Example 6, the same procedure as in Example 9-2 was conducted Melanin contents (melanin contents) and cell viability were confirmed.

Island Acanthopanax tree trunk ethyl acetate (EtOAc) fraction and n - hexane (n -Hex) at a concentration of 100 μM of the compounds isolated from the fractions, each processing a B16F10 cell, check the melanin formation inhibitory activity and cytotoxicity results, n - hexane 52.6%, and 42.7%, respectively, in Melanin contents without lethal cytotoxicity in Compound 6, Compound 7 and Compound 8 isolated from the ( n- Hex) fraction ).

sample Treatment concentration (uM) Inhibition rate of melanin formation ( % ) Cell survival rate (%) Control (α-MSH treatment) - 0 100.0 Melasolv 20 68.8 116.2 Compound 1 (Cpd1) 100 6.7 102.2 Compound 2 (Cpd2) 100 76.6 63.4 Compound 3 (Cpd3) 100 19.3 105.4 Compound 4 (Cpd4) 100 41.8 75.2 Compound 5 (Cpd5) 100 13.5 103.4 Compound 6 (Cpd6) 100 73.7 82.2 Compound 7 (Cpd7) 100 52.6 90.4 Compound 8 (Cpd8) 100 42.7 91.2

Compounds 6, 7, and 8, in which the melanin pigment content was decreased without lethal cytotoxicity, of the compounds isolated from the truncated trichomonas n -hexane ( n- Hex) fraction were measured at concentrations of 25 μM, 50 μM and 100 μM And the melanin formation inhibitory activity and cytotoxicity were examined according to the concentration of the above three compounds. As a result, the melanin content was decreased in a concentration-dependent manner without lethal cytotoxicity at a concentration of 100 μM or less (Table 18 and Fig. 19).

sample Treatment concentration (uM) Inhibition rate of melanin formation ( % ) Cell survival rate (%) Control (α-MSH treatment) - 0 100.0 Melasolv 20 67.9 110.3 Compound 6 (Cpd6) 25 18.5 94.6 50 57.9 85.4 100 70.9 79.2 Compound 7 (Cpd7) 25 N.D. 106.4 50 4.7 100.1 100 46.7 90.2 Compound 8 (Cpd8) 25 N.D. 103.2 50 4.7 94.3 100 36.7 91.3

(N.D. *: Not Detected)

Example 9-5: Island oak tree stem n - Hexane ( n -Hex) Fraction And ethyl acetate (EtOAc) Fraction , remind n - Hexane ( n -Hex) From the fraction Measurement of intracellular tyrosinase inhibitory activity according to the concentration of isolated compounds

Example 9-3 and Example 9-4 island proceeds melanin production inhibition experiment at different concentrations in Acanthopanax tree trunk n - hexane (n -Hex) fraction and ethyl acetate (EtOAc) and the fractions, n - hexane (n - Hex) fractions, Compound 6, Compound 7 and Compound 8 were assayed for intracellular tyrosinase inhibitory activity by the method of Example 9-2. As a result, as shown in the following Table 19 and FIG. 20, the n -hexane ( n- Hex) fraction and the ethyl acetate (EtOAc) fraction and the compounds 6, 7 and 8 showed a concentration-dependent intracellular tyrosinase activity (Table 19 and Fig. 20).

sample Treatment concentration Intracellular Tyrosinase activity inhibition rate ( % ) Control (α-MSH treatment) - 0 n- Hex 25 / / mL 1.8 50 / / mL 26.7 100 / / mL 65.5 EtOAc 25 / / mL N.D. 50 / / mL 27.9 100 / / mL 58.8 Compound 6 (Cpd6) 25 uM 10.4 50 uM 49.6 100 uM 64.7 Compound 7 (Cpd7) 25 uM 3.7 50 uM 5.7 100 uM 37.8 Compound 8 (Cpd8) 25 uM 3.7 50 uM 6.9 100 uM 28.4

(N.D. *: Not Detected)

Manufacturing example One: Island oak tree Ethyl acetate of the stem extract The fraction Antioxidant, anti-inflammatory or antibacterial active ingredient Cosmetic Produce

Manufacturing example 1-1: Manufacture of flexible lotion

The emollient lotion containing the ethyl acetate fraction of the extract of Trichomonas silybacoli as an active ingredient was prepared as shown in Table 20 below.

Raw material content( Weight portion ) The ethyl acetate fraction of the extract of Tricholoma japonica extract of the present invention 10.00 1,3-butylene glycol 1.00 Disodium iodide 0.05 Allantoin 0.10 Dipotassium glycyrrhizate 0.05 Citric acid 0.01 Sodium citrate 0.02 Glycereth-26 1.00 Arbutin 2.00 Hydrogenated castor oil 1.00 ethanol 30.00 Preservative a very small amount coloring agent a very small amount Flavoring agent a very small amount Purified water Balance

Manufacturing example 1-2: Manufacture of nutrition cream

The soft nutritive cream containing the ethyl acetate fraction of the extract of Trichomonas sieboldii as an active ingredient was prepared as shown in the following Table 21.

Raw material content( Weight portion ) The ethyl acetate fraction of the extract of Tricholoma japonica extract of the present invention 10.0 1,3-butylene glycol 7.0 glycerin 1.0 D-Panthenol 0.1 Plant extract 3.2 Magnesium aluminum silicate 0.3 PEG-40 stearate 1.2 Stearic acid 2.0 Polysorbate 60 1.5 Chin type glyceryl stearate 2.0 Sorbitan sesquioleate 1.5 Cetearyl alcohol 3.0 Mineral oil 4.0 Squalane 3.8 Carlyric / capric triglyceride 2.8 vegetable oil 1.8 Dimethicone 0.4 Dipotassium glycyrrhizate a very small amount Alantouille a very small amount Sodium hyaruronate a very small amount Tocopheryl acetate Suitable amount Triethanolamine Suitable amount Preservative Suitable amount Flavoring agent Suitable amount Purified water Balance

Manufacturing example 2: Island oak tree Ethyl acetate of the stem extract The fraction Production of antioxidant, anti-inflammatory or antibacterial food containing active ingredient

Manufacturing example 2-1: Manufacture of Flour Food

0.5 to 5.0 parts by weight of the ethyl acetate fraction of the extract of the present invention was added to wheat flour and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

Manufacturing example 2-2: soup And juicy ( gravies )

0.1 to 5.0 parts by weight of the ethyl acetate fraction of the extract of the present invention was added to the soup and the juice to prepare health promotion meat products, noodle soup and juice.

Manufacturing example 2-3: Ground Beef Produce

A ground beef for health promotion was prepared by adding 10 parts by weight of the ethyl acetate fraction of the extract of the present invention to the ground beef.

Manufacturing example 2-4: Dairy products ( dairy products )

5 to 10 parts by weight of the ethyl acetate fraction of the extract of the present invention was added to milk and various dairy products such as butter and ice cream were prepared using the milk.

Manufacturing example 2-5: Solar Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The ethylacetate fraction of the extract of Trichotomous stem extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and dried in a hot air drier, and pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The above-prepared cereals, seeds, and ethyl acetate fraction of the extract of Tricholoma japonica were prepared in the following proportions:

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley), seeds (7 parts by weight of perilla seeds, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds), ethyl acetate fraction (3 parts by weight), Ganoderma lucidum (0.5 parts by weight) and Rhizophorus (0.5 part by weight).

Manufacturing example 2-6: Health drink Produce

5 g of the ethyl acetate fraction of the extract of Tricholoma matsutake extract of the present invention was added to a homogeneous mixture such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5% And sterilized at the moment, and then packaged in small containers such as glass bottles and plastic bottles.

Manufacturing example 2-7: Manufacture of vegetable juice

Vegetable juice was prepared by adding 5 g of the ethyl acetate fraction of the extract of the present invention to 1,000 ml of tomato or carrot juice.

Manufacturing example 2-8: Manufacture of fruit juice

A fruit juice was prepared by adding 1 g of the ethyl acetate fraction of the extract of the present invention to 1,000 ml of apple or grape juice.

Manufacturing example 3: Island oak tree Ethyl acetate of the stem extract The fraction Preparation of a pharmaceutical composition for antioxidant, anti-inflammation or antibacterial activity containing as an active ingredient

Manufacturing example 3-1: Sanje Produce

1 g of lactose was mixed with 2 g of the ethyl acetate fraction of the extract of the present invention, and the mixture was packed in an airtight container to prepare a powder.

Manufacturing example 3-2: Preparation of tablets

100 mg of the ethyl acetate fraction, 100 mg of the corn starch, 100 mg of the lactose, and 2 mg of the magnesium stearate were mixed with the extract of the present invention, and the tablets were prepared by tableting according to the conventional preparation method.

Manufacturing example 3-3: Preparation of capsules

100 mg of the ethyl acetate fraction, 100 mg of the corn starch, 100 mg of the lactose and 2 mg of the magnesium stearate were mixed with the gelatin capsule according to the conventional preparation method of the capsules to obtain capsules .

Manufacturing example 3-4: Manufacture of rings

1 g of the ethyl acetate fraction, 1.5 g of lactose, 1 g of glycerin and 0.5 g of xylitol were mixed with each other to prepare 4 g per one ring according to a conventional method.

Manufacturing example 3-5: Preparation of granules

150 mg of the ethyl acetate fraction of the present invention, 50 mg of soybean extract, 200 mg of glucose and 600 mg of starch were mixed with 100 mg of 30% ethanol and dried at 60 ° C to form granules And filled in foams.

Claims

3,4-dihydroxybenzoic acid (3,4-dihydroxybenzoic acid), 3,4-dihydroxybenzoic acid (3,4-dihydroxybenzoic acid) isolated from the ethyl acetate fraction of the Acanthopanax koreanum Nakai stem extract acid methyl ester, caffeic acid and chlorogenic acid methyl ester as an active ingredient.

The cosmetic composition for antimicrobial use according to claim 1, wherein the islet extract is extracted with water, a C ₁ -C ₄ lower alcohol or a mixed solvent thereof.

The antimicrobial cosmetic composition according to claim 2, wherein the lower alcohol is ethanol.

delete

The method according to claim 1, wherein the antimicrobial effect is inhibited by at least one microorganism selected from the group consisting of S. epidermidis , P. aeruginosa , P. acnes , Wherein the antimicrobial cosmetic composition is an antimicrobial cosmetic composition.

delete

The antimicrobial cosmetic composition according to claim 1, wherein the cosmetic composition has a whitening effect.

3,4-dihydroxybenzoic acid (3,4-dihydroxybenzoic acid), 3,4-dihydroxybenzoic acid (3,4-dihydroxybenzoic acid) isolated from the ethyl acetate fraction of the Acanthopanax koreanum Nakai stem extract acid methyl ester, caffeic acid and chlorogenic acid methyl ester as effective ingredients.

delete

12. The method of claim 11, wherein the antimicrobial effect is a growth inhibitory activity against at least one microorganism selected from the group consisting of S. epidermidis , P. aeruginosa , P. aeruginosa , P. aeruginosa , P. aeruginosa , P. aeruginosa , &Lt; / RTI >