KR100855314B1 - Antimicrobial composition comprising natural plant extract - Google Patents

Abstract

An antimicrobial composition comprising natural plant extracts is provided to improve abtimicrobial effects by widening the range of antimicrobial spectrum, suppress oxidation, and inhibit toxicity and stimulation to human and animal. An antimicrobial composition comprises 1-30 wt.% of Kalopanax pictus extract and Siegesbeckia glabrescens extract in a weight ratio of 1:0.5-1:2, which are prepared by extracting Kalopanax pictus and Siegesbeckia glabrescens with water, organic solvent including methanol, ethanol, isopropanol, butanol, ethylene, acetone, ether, hexane, chloroform, ethylacetate, N-N-dimethylformamide and dimethylsulfoxide, or a mixture thereof, and is a cosmetic, pharmaceutical or food composition.

Description

Antimicrobial composition comprising natural plant extracts

The present invention relates to an antimicrobial composition comprising a natural plant extract, and more particularly, to an antimicrobial composition comprising an extract of a plant of Mt.

Nowadays, with the development of transportation, global movement is active, and accordingly, various infectious diseases and new diseases are spreading rapidly according to the movement of people. In the meantime, the use of indiscriminate antimicrobial materials makes various microorganisms resistant and threaten humanity.

Pesticides can be broadly divided into fungicides, insecticides, herbicides and growth regulators. In the case of crop damage caused by viruses or microorganisms, pesticides are controlled using fungicides. Inexpensive and powerful artificially synthesized antimicrobials in agriculture have threatened our table. Various antibacterial materials are also used in household goods that are applied directly to the human body, and most of them are inexpensive chemical components. Unfortunately, many of these antimicrobials are scientifically found to be harmful to humans over long-term use.

Many products such as food, medicine, daily necessities, and cosmetics widely used in our daily life contain antiseptics, which are antimicrobial substances licensed for human use to prevent changes in internal properties and to preserve for a certain period of time. As the material used as the preservative, there are some materials produced in nature, but almost all chemically synthesized artificial materials are used. Even parabens preservatives that are the safest as conventional preservatives and are widely used in daily necessities, cosmetics and medicines, are potential for skin allergies (Andrea Counti et al., Contact Dermatitis, 1997, 37; 35-36) and environmental hormones. (Edwin et al., Toxicology and applied pharmacology, 1998, 153; 12-19) and has a problem of inducing resistant bacteria.

Most of the active substances used as preservatives are not commercialized due to color, deterioration of stability, narrow antimicrobial spectrum, formulation problems, and the like.Hinokitiol, a white powder extract, magnool, a magnolia extract, Only a portion of the grapefruit extract, such as DF-100 is being commercialized.

Antibiotics in medicine are used to prevent infections in surgical sites or to treat various infectious diseases. Most of these antimicrobial agents for medical purposes have been obtained by culturing actinomycetes.

In order to prevent contamination of pathogenic microorganisms, sterilization and antibacterial substances for environmental spraying are widely used. In fact, it is used to prevent contamination of hospitals or to prevent contamination of ports and airports when cleaning toilets, including livestock raising. However, most of them are synthetic compounds that can be harmful to livestock or humans after long-term overdose.

Therefore, the necessity of a natural antimicrobial substance that can replace the existing synthetic chemical antimicrobial substance is maximized. In particular, there is a need for the development of natural antimicrobial materials that reduce side effects, which are the biggest disadvantages of antimicrobials, and at the same time possess a broad antimicrobial spectrum, formulation stability, and the like. The present inventors performed antimicrobial screening on plant species native to Jeju, to develop a natural antimicrobial agent having excellent antimicrobial activity, broad antimicrobial spectrum, and safe for human body among these plant species.

An object of the present invention is to provide an excellent antimicrobial activity, a broad antimicrobial spectrum, antioxidant efficacy, a natural antibacterial agent that is safe for human body, which can be applied to various fields such as cosmetics, pharmaceuticals, pesticides, food, household goods.

The present invention provides an antimicrobial composition comprising a natural plant extract.

The antimicrobial composition of the present invention includes an extract of a plant of Mt.

Kalopanax pictus is a deciduous broad-leaved arboreous tree that grows all over the country. It is not a big tree growing up to 25m in height and 1m in diameter.It grows at an altitude of 100-1,800m and is generally around 400-500m above sea level. . The bark and root bark of Yin-tree are used as health food additives, and the stem, branch, and root bark are used for medicinal purposes. It is applied to expectoration, antipyretic and analgesia. It is used to take bruises, rheumatic myalgia and arthritis in 11 ~ 19g. It is powdered and attached to the sore spot.

Siegesbeckia glabrescens is an annual herb belonging to the genus Asteraceae (Compositae). When used for daily treatment, it eliminates moist heat and treats muscle pain or numbness caused by dampness. If you put it in the Hwangju steaming customs (風濕) while removing the liver (肝) and god (腎) is effective to strengthen the bones and muscles. It is mainly used for paralysis of the limbs, pain in the knees, ache, swelling, and half body incontinence, and to treat nasal congestion and inflammation of the nose, and recent research has shown that it is effective in treating hypertension and heart disease. In addition, there are records that it has been used for skin diseases such as eczema and tumors, and it is also used as a detoxifying effect for snake and insect bites.

The plant extract included in the composition of the present invention may extract each plant using water, an organic solvent, or a mixed solvent thereof. All of these components may be mixed and extracted at the same time, or individual components may be extracted and mixed under optimum conditions. In the case of hot water extraction, for example, the components of the composition of the present invention are added to water and heated at a temperature of at least 70 ° C, preferably at least 80 ° C, more preferably at least 90 ° C, for at least 2 hours, preferably at least 3 hours, The heated mixture can be prepared by filtration. When heating at a high temperature, the time is reduced, but the active ingredient is destroyed and can be lost. When heating at a temperature lower than the temperature, it is important to control the temperature and time properly because the active ingredient of the mixture is not completely extracted. This extract can be dried or used as it is. When extracted using an organic solvent, alcohols such as methanol, ethanol, isoprotanol, butanol, ethylene, acetone, ether, hexane, chloroform, ethyl acetate, DMF (N, N-dimethylformamide), DMSO (dimethyl sulfoxide) Side), mixtures thereof and the like can be extracted with an organic solvent at room temperature or by heating.

The extract extracted by heat extraction or cold extraction is filtered to remove suspended solid particles, for example, by filtering the particles using nylon or the like, or filtering using a freeze filtration method, and then using them as they are or using lyophilization, vacuum drying, and hot air. Drying, spray drying, etc. may be used for drying.

The present invention relates to an antimicrobial composition comprising an extract of a plant of Mt. Each natural extract in this antimicrobial composition is preferably included in approximately equal proportions.

The plant extract in the antimicrobial composition of the present invention may be included in an amount of 1 to 30% by weight, preferably 1 to 20% by weight, more preferably 1 to 10% by weight of each plant extract relative to the total weight of the composition. have.

The antimicrobial compositions of the present invention exhibit broad antimicrobial activity against a variety of microorganisms, in particular bacteria (Table 7), safe for human skin (Table 9) without cytotoxicity (Table 8) and do not exhibit acute oral toxicity.

In specific embodiments, the compositions of the present invention are Gram-positive bacteria, Gram-negative bacteria, in particular Gram-positive bacteria, for example Bacillus subtilis ATCC 6633 ( B. subtilis ), Staphylococcus aureus ATCC 6538P ( S. aureus ), Micrococcus luteus ATCC 9341 ( M. luteus ), Methicillin resistant staphylococcus aureus (MRSA) C2207, Gram-negative bacteria, for example Escherichia coli (E. coli) ATCC 10536, Escherichia coli (E. coli ) 0157: K88ac, Pseudomonas It showed antimicrobial activity against aeroginosa (P. aeroginosa ) ATCC 1636, Klebsiella pneumoniae (K. pneumoniae ) ATCC 10031.

The antimicrobial composition of the present invention may contain additional ingredients in addition to the above-described plant extracts as long as it does not inhibit its antimicrobial activity.

As described above, the antimicrobial composition according to the present invention having a broad antimicrobial spectrum may be widely used to achieve the purpose of antibacterial, sterilization, disinfection, antiseptic, etc. in pesticides, medicine, cosmetics, household goods, food, and the like. Specifically, for the purpose of antibacterial, disinfecting and disinfecting in agriculture, for antibiotics and antifouling agents in medicine, for antiseptic and antibacterial purposes in foods, for preventing dandruff in cosmetics and household goods, for preventing athlete's foot, for armpits. It may be used in products directly related to microorganisms such as anti-acne and anti acne, or for preservative, antibacterial or sterilizing purposes such as cleaning detergents or dish washing detergents, but is not limited thereto.

Thus, in another aspect, the present invention provides cosmetics, foods, pharmaceuticals, pesticides, household goods processed with the antimicrobial composition described above.

As a specific embodiment of the processed article, the antimicrobial composition of the present invention is added to cosmetics such as various creams, lotions, skins, etc., without the addition of artificial antibacterial agents (preservatives) to shampoos, rinses, cleansing, face washes, soaps, treatments, cosmetics, etc. Provide cosmetics that provide antimicrobial effects.

Cosmetics of the present invention include aqueous vitamins, oily vitamins, polymer peptides, polymer polysaccharides, sphingolipids and the like.

The aqueous vitamin may be any compound that can be incorporated into cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like. Their salts (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, etc.) may also be used in the water-soluble vitamins that can be used in the present invention. Included.

The oil-based vitamin may be any compound that can be formulated in cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-α-tocopherol, d-α-tocopherol, d-δ-tocopherol) and the like. Derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic palmitate, dl-α-tocopherol acetate, dl-α-tocopherolvitamin E nicotinic acid, DL-pantothenyl alcohol, D-pantothenyl alcohol , Pantotenylethyl ether, etc.) are also included in the oil-soluble vitamins used in the present invention.

The polymer peptide may be any compound as long as it can be incorporated into cosmetics. Preferably, collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, and the like can be given.

The polymer polysaccharide may be any compound as long as it can be blended into cosmetics. Preferably, hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.) may be mentioned. For example, chondroitin sulfate or its salt can be normally purified from a mammal or fish.

The sphingolipid may be any compound as long as it can be blended into cosmetics. Preferably, ceramide, pit spingosine, sphingolipid lipid and the like can be given.

In addition to the said component, you may mix | blend the cosmetics of this invention with the other components normally mix | blended with cosmetics as needed.

Examples of the blending component that may be added include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavors, and the like. .

Examples of the fat or oil component include ester fats, hydrocarbon fats, silicone fats, fluorine fats, animal fats, and vegetable fats and oils.

Examples of the moisturizing agent include a water-soluble low molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

Examples of the emollient include long-chain acyl glutamic acid cholesteryl esters, hydroxy stearic acid cholesterol, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl esters, and the like.

As surfactant, nonionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, etc. are mentioned.

Nonionic surfactants include self-emulsifying glycerin monostearate, propylene glycol fatty acid esters, glycerin fatty acid esters, polyglycerol fatty acid esters, sorbitan fatty acid esters, POE (polyoxyethylene) sorbitan fatty acid esters, POE sorbitan fatty acid esters, POE Glycerin Fatty Acid Ester, POE Alkyl Ether, POE Fatty Acid Ester, POE Cured Castor Oil, POE Castor Oil, POE · POP (PolyoxyethylenePolyoxypropylene) Copolymer, POEPOP Alkyl Ether, Polyether Modified Silicone, Laurin Acid Alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc. are mentioned.

As anionic surfactant, fatty acid soap, (alpha)-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl phosphorus salt, alkylamide Phosphates, alkyloylalkyltaurine salts, N-acylamino acid salts, POE alkyl ether carboxylate salts, alkyl sulfosuccinate salts, sodium alkyl sulfo acetates, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphate esters, and the like. have.

As cationic surfactant, alkyl trimethylammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium bromide, behenyl trimethyl ammonium chloride, chloride Benzalkonium, stearic acid diethylaminoethylamide, stearic acid dimethylaminopropylamide, lanolin

Derivative quaternary ammonium salts; and the like.

Examples of amphoteric surfactants include carboxybetaine type, amidebetaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type and amideamine type. An amphoteric surfactant etc. are mentioned.

Organic and inorganic pigments include silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium film mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine, carbon black and composites thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene, styrene copolymer, Organic pigments such as silk powder, cellulose, CI pigment yellow, CI pigment orange, and composite pigments of these inorganic pigments and organic pigments;

As organic powder, Metal soaps, such as a calcium stearate; Alkyl phosphate metal salts such as sodium cetylinate, zinc lauryl acid and calcium laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-β-alanine calcium, N-lauroyl-β-alanine zinc, and N-lauroylglycine calcium; Amide sulfonic acid polyvalent metal salts, such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N-acyl basic amino acids, such as N (epsilon) -lauroyl-L- lysine, N (epsilon) -palmitolysine, N (alpha)-partoylol nitin, N (alpha)-lauroyl arginine, and N (alpha) -cured fatty acid acyl arginine; N-acyl polypeptides, such as N-lauroyl glycyl glycine; α-amino fatty acids such as α-aminocaprylic acid and α-aminolauric acid; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride and the like.

Examples of the ultraviolet absorber include paraaminobenzoic acid, ethyl paraaminobenzoate, amyl paraaminobenzoic acid, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylate and cinnamic acid benzyl , Paramethoxy cinnamic acid 2-ethoxyethyl, paramethoxy cinnamic acid octyl, diparamethoxy cinnamic acid mono 2-ethylhexane glyceryl, paramethoxy cinnamic acid isopropyl, diisopropyl diisopropyl cinnamic acid ester mixture, urokanoic acid Ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenonedisulfonate, dihydroxybenzophenone, tetra Hydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p- (carbo-2'-ethylhexyl-1'-oxy) -1,3 , 5-triazine, 2- (2-hydric When the like can be mentioned 5-methylphenyl) benzotriazole.

Examples of the alcohol include higher alcohols such as cetyl alcohol.

Specific examples of the cosmetic of the present invention face wash, face wash foam, cleansing cream, cleansing milk, cleansing lotion, massage cream, cold cream, moisturizing cream, latex, lotion, pack, aftershave cream, sunburn prevention cream, suntan oil, soap , Body shampoo, hair shampoo, hair rinse, hair treatment, wool, hair care, hair cream, hair liquid, set lotion, hair spray, hair bridge, coloring, color spray, permanent wave liquid, press powder, loose powder Eye shadows, hand creams, lipsticks, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are only intended to illustrate the present invention in more detail and are not intended to limit the present invention thereto.

[ Example  1] Preparation of Plant Extracts

Yin ( Kalopanax 1 kg of stem (Nanji Agricultural Research Institute) of pictus ) was chopped with a mill, and 100 kg of 70% ethanol was added and extracted by heating at 100 ° C. for 4 hours using a device equipped with a reflux device. The extract was filtered using a filter aid, left to mature in a cold place for 7 to 10 days, and the precipitate was filtered, followed by adding 70% ethanol to obtain a 100 kg product.

Squirrel ( Siegesbeckia 1 kg of leaves and stems (Nanji Agricultural Research Institute ) of the glabrescens were chopped with a mill, and 100 kg of 10% ethanol was added and extracted by heating at 100 ° C. for 4 hours. The extracted solution was filtered using a filter aid, an additional 10% ethanol was added to obtain a 100 kg product.

[ Example  2] Antimicrobial efficacy evaluation

Staphylococcus using the extract obtained in Example 1 aureus and Propionibacterium The antimicrobial effect on acnes was measured.

Antimicrobial activity was measured by agar diffusion method, which was placed on a solid agar medium coated with test strains with antibiotic solution of known concentration on several discs, and then measured the size of growth inhibition halo around the disc. Table 1 shows the results obtained by limiting the antibacterial test concentration to 100 ppm (0.01%). The results for the minimum concentrations are shown in Table 2.

Extract Type S. aureus inhibitory activity P. acnes inhibitory ability Yin ++ ++ Rind ++ ++

+: Transparent band size is less than 2 mm, ++: transparent band size is less than 2 to 5 mm, +++: transparent band size is 5 mm or more.

Microbial strain Extract concentration (ppm) Yin Squirt P. acnes 10 9 S. aureus 8 8

Example 3 Preparation of Natural Antibacterial Formulation

A natural antimicrobial formulation was prepared by mixing the Yin-wood extract and Jindukchal extract extract prepared in Example 1 in various component ratios, respectively. Component ratios were combined with each plant extract to make a total of three component ratios (1: 1, 1: 2, 2: 1). As a result of various investigations on the compounding ratio of each component, the combination of 1: 1 showed excellent antibacterial effect. Antimicrobial activity was measured for Staphylococcus aureus using agar diffusion.

Kinds Staphylococcus Antimicrobial efficacy against aureus Yin-tree (15ug / ml) + squirting (15ug / ml) ++++ Yin-tree (20ug / ml) + squirting (10ug / ml) +++ Yin-tree (10ug / ml) + squirting (20ug / ml) +++ Yin extract (30ug / ml) ++ Ginseng Extract Extract (30ug / ml) ++

+: Transparent band size of less than 2 mm, ++: Transparent band size of less than 2 to 4 mm, +++: Transparent band size of 4 to 8 mm, ++++: 8 mm or more

Table 4 shows the compounding ratio of the plant extract showing the best antimicrobial activity.

Raw material name Yin extract Ginseng Extract Purified water or solvent content(%) 1.0 1.0 to 100

Example 4 Antibacterial Activity of a Natural Antimicrobial Formulation

The antimicrobial activity of natural antimicrobial formulations was investigated using agar diffusion method. Experiments were performed with concentrations of natural antimicrobial formulations (1: 1) at 0.25%, 0.5%, 1.0%, 2.0% and the like. The microbial strains were Gram-positive bacteria Staphylococcus aureus , Gram-negative bacteria Escherichia coli , and yeast Candida albicans .

Antimicrobial solutions of known concentrations were applied to several discs, placed on solid agar plates coated with test strains, and cultured, and then the size of growth inhibition halo around the discs was measured. As shown in Table 5, it showed high antibacterial activity against Staphylococcus aureus and Candida albicans , whereas Escherchia Coli showed relatively low antimicrobial activity.

Size measurement of the transparent zone around the disc. Microbial strain E. coli S. aureus C. albicans Concentration (%) ^a 0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0 Diameter of Suppression Zone (mm) ^b 10.5 11.0 12 16 13.5 15.5 18 19 13 14.5 16.5 19

a: Amount (%) of natural antimicrobial formulation per disc

b: average of three observations, transparent zone contains disk diameter (10 mm)

Example 5 Determination of Minimum Inhibitory Concentration (MIC) of a Natural Antimicrobial Formulation

Minimum Inhibition Concentration (MIC) of the natural antimicrobial formulation was measured. Staphylococcus aureus , Candida albicans and Escherichia coli , which were incubated for 24 hours, were placed in 3 ml brain heart infusion broth. At the same time, natural antimicrobial formulations were added at different concentrations, followed by incubation at 37 ° C for 24 hours. . A 2-fold dilution method was used to determine the MIC of the natural antimicrobial formulation. As shown in Table 6, the MIC for Escherichia coli was 320 ug / ml, the MIC for Staphylococcus aureus was 20 ug / ml, and the MIC for Candida albicans was 80 ug / ml. The antimicrobial activity of natural antimicrobial formulations showed the highest antimicrobial activity against Staphylococcus aureus , a Gram-positive bacterium.

Strain MIC (ug / ml) E. coli 320 S. aureus 20 C. albicans 80

Example 6 Antimicrobial Activity Spectrum of Natural Antimicrobial Combinations

Various kinds of microorganisms were used to examine the antimicrobial activity spectrum of the natural antimicrobial formulations prepared in Example 3. Bacillus subtilis ATCC 6633 ( B. subtilis ), Staphylococcus aureus ATCC 6538P ( S. aureus ), Micrococcus luteus ATCC 9341 ( M. luteus ), Methicillin resistant staphylococcus aureus (MRSA) C2207, Escherichia coli (E. coli) ATCC 10536, Escherichia coli (E. coli) 0157: K88ac, Pseudomonas aeroginosa (P. aeroginosa ) ATCC 1636, Klebsiella pneumoniae (K. pneumoniae) ATCC 10031, yeast Candida albicans (C. albicans) ATCC 10231, fungi The antibacterial activity of Aspergillus niger (A. niger) ATCC 9029, Trichophyton mentagrophytes (T. mentagrophytes) KCTC 6077, Fusarium solani (F. solani) was measured by agar diffusion method. The results are shown in Table 7.

Microbial strain B. subtilis S. aureus M. luteus MRSA E. coli E. coli 0157: K88ac P. aerosinosa K. pneumonia C. albicans A. niger T. mentagrophyte F. solani Antibacterial Activity of Natural Antimicrobial Blends (400 ug / ml ) +++ +++ ++ ++ ++ ++ +++ ++ +++ ++ ++ ++ Antimicrobial Activity of Yard Trees (400 ug / ml ) + ++ + + + + + + ++ + + + Antimicrobial Activity of Jindukchal (400 ug / ml ) + ++ + + + + + + ++ + + +

+: Weak (transparent stand size: less than 2 mm), ++: strong (transparent stand size: less than 2 to 5 mm), +++: very strong (transparent stand size: 5 mm or more)

Example 7 Cytotoxicity Evaluation

When used as an antimicrobial agent, it can be expanded its use only if it is not toxic to the human body. In this toxicity test, the simplest and most obvious toxicity test is the cytotoxicity test using human cells. In general, the absence of toxicity in cytotoxicity is considered to be very unlikely to occur at the individual level.

MTT experiments were performed using HaCaT (Korean Cell Line Bank) and human normal fibroblasts (Pacific), a human keratinocyte line, for cytotoxic effects of natural antimicrobial formulations. For human normal fibroblasts and HaCaT, the natural antimicrobial formulation did not show cytotoxicity at concentrations below 500 ug / ml.

Natural Antibiotic Concentration (ug / ml) 0 100 200 300 400 500 Cell survival rate 100 102 99.3 99.3 99.0 89.7

Example 8 Safety Confirmation Experiment on Human Skin of Natural Antibacterial Formulation

In order to confirm whether the natural antimicrobial formulations that were found to be excellent in antimicrobial activity as safe as in the above examples were also safe to human skin, skin safety verification experiments were performed. The cumulative skin irritation test was used as a test method.

Squalene-based formulations containing 400 ug / ml, 600 ug / ml, 800 ug / ml, and 1 mg / ml of natural antimicrobial formulations, respectively, were used to prepare the upper forearm area for 30 healthy adults. A total of nine 24-hour cumulative patches were carried out every other day to determine whether the natural antimicrobial agent irritated the skin.

Fin chamber (Finn chamber, Epitest Ltd, Finland) was used. 15ul of each said skin external preparation was dripped at the chamber, and the patch was performed. The degree of response to the skin each time was scored using Equation 2 below, and the results are shown in Table 9 below.

Average responsiveness = [[response index x responsiveness / total number of subjects x highest score (4 points)] x 100] ÷ number of tests (9 times)

Test substance Number of subjects with response Average reactivity 1 week 2 weeks 3 weeks Primary Secondary 3rd 4th 5th 6th 7th 8th 9th ± + + + ± + + + ± + + + ± + + + ± + + + ± + + + ± + + + ± + + + ± + + + Control group (squalene) 2 - - --- --- --- --- --- --- --- --- 0.18 Natural Antibiotic Blend (400 ug / ml) [Test Group 1] 2 - - 0 - - --- --- --- --- --- --- --- 0.18 Natural Antibiotic Blend (600 ug / ml) [Test Group 2] 3-- One - - --- --- --- --- --- --- --- 0.37 Natural Antibiotic Blend (800 ug / ml) [Test Group 3] 2 - - 0 - - --- --- --- --- --- --- --- 0.18 Natural Antibiotic Blend (1 mg / ml) [Test Group 4] 2 - - 0 - - --- --- --- --- --- --- --- 0.18 Inspector 30 30 30 30 30 30 30 30 30

In the reactivity, ± gives 1 point, + gives 2 points, and ++ gives 4 points, and when the average reactivity is less than 3, it is determined as a safe composition.

In Table 8, the test group 1 [natural antimicrobial formulation (400 ug / ml)], the number of people corresponding to ±, +, ++ was 2, 0, 0, respectively, the rest of the response was Did not appear. The average reactivity of test group 1 to test group 4 was 0.18, 0.37, 0.18, respectively. As 0.18 was all 3 or less, it was judged as a safe composition.

Example 9 Acute Oral Toxicity Test

An acute oral toxicity test was conducted to determine whether the natural antimicrobial formulation could be safely applied to food. Twenty SPF SD rats, 5 to 6 weeks old, were used as experimental animals and subjected to an acute toxicity test under the following conditions.

Temperature and humidity conditions: temperature 22 ± 2 ℃. Relative Humidity 50 ± 10%

ㆍ Contrast cycle: Fluorescent light (8 o'clock ~ 20 o'clock)

Roughness: 200 to 300 Lux

ㆍ Free drinking water treated with ultraviolet rays

ㆍ Dilute the test substance in sterile distilled water to prepare a sample for administration and add the same amount using sterile distilled water as a control.

On the day of administration, the general condition is observed every hour up to 4 hours, and once a day from the day after the administration, the general condition changes, poisoning symptoms, mobility, appearance, autonomic nerves, and the presence of dead animals are carefully observed. Pathological anatomical verification was performed to determine the toxicity. LD ₅₀ was 2500 mg / kg BW and no toxicity was determined.

Plant-derived natural antimicrobial agent according to the present invention exhibits a broad spectrum of antimicrobial activity against a variety of bacteria, do not cause any toxicity or irritation in animal and human safety tests, so it is a safe substance for human use, cosmetics, medicine, household goods, It can be usefully used for sterilization, antibacterial and antiseptic purposes in various fields such as pesticides.

Claims (9)

Antimicrobial composition comprising an extract of Kalopanax pictus and Siegesbeckia glabrescens as an active ingredient, wherein the extract and the extract of Jinkuk are included in the composition in a weight ratio of 1: 0.5-1: 2. delete delete delete The antimicrobial composition of claim 1, wherein the composition has antimicrobial activity against bacteria, fungi and yeast. The antimicrobial composition of claim 1, wherein the yin-tree extract and Jinchuk-ri extract are included in an amount of 1-30% by weight based on the total weight of the composition. The composition according to any one of claims 1, 5 and 6, wherein the composition is a cosmetic composition. 7. A composition according to any one of claims 1, 5 and 6, wherein said composition is a pharmaceutical composition. 7. A composition according to any one of claims 1, 5 and 6, wherein said composition is a food composition.