KR101641498B1

KR101641498B1 - Complex formulation comprising natural antimicrobial compound and process for producing thereof

Info

Publication number: KR101641498B1
Application number: KR1020150105016A
Authority: KR
Inventors: 전용진; 황근익
Original assignee: 청운대학교 인천캠퍼스 산학협력단
Priority date: 2015-07-24
Filing date: 2015-07-24
Publication date: 2016-07-21

Abstract

The present invention relates to a nature-derived complex composition having low capacity, and highly efficacious antibiotic and anti-inflammatory effects. More particularly, the present invention relates to a natural antibiotic complex medicine comprising caprylhydroxamic acid (CHA), a Boswellia serrata resin extract, and an oriental medicine extract as effective components. The natural antibiotic complex medicine has preservative power and efficacy to alleviate stimuli, and is composed of polymeric compounds forming a thin protection layer on the skin to protect the skin from penetration of substances inducing stimuli which may become a secondary stimuli source.

Description

TECHNICAL FIELD The present invention relates to a natural antimicrobial compound preparation composition and a method for producing the same,

The present invention relates to a low-dose, high-efficacy composite preparation of a natural antimicrobial agent and a method of preparing the same, and more particularly, to a composition comprising a moisturizing agent or a solvent; Caprylhydroxamic Acid (CHA); Cationic polymer compounds; Boswellia serrata resin extract; And a herbal extract. When the composition is applied to the skin, a thin protective film is formed on the skin to protect the skin from penetration of the stimulant, thereby providing the stimulant reduction effect and the repelling force.

Preservatives are essential to maintain the quality of various products such as cosmetics, medicines, daily necessities and foods for a long time.

Preservatives are especially essential for cosmetics containing glycerin, sorbitol, etc., which are relatively long in distribution period and composed mainly of oil or water, and are carbon sources of microorganisms, and amino acid derivatives and proteins that are nitrogen sources.

It is therefore common to add chemical preservatives in order to prevent contamination by fingers during use, as well as cosmetic manufacturing processes, or secondary contamination due to carelessness in storage.

Features required for preservatives and preservatives are high preservative effects, broad spectrum of antimicrobial activity, long-term stability of preservatives and preservatives itself, and other effects on the base should be small and not be inactivated as a base component. It also has high safety in keeping with low cost.

In recent years, the demand for cosmetics and quasi-drugs of hypoallergenicity and hypoallergeness has been increasing due to the improvement in consciousness of consumers' safety. As a result, cosmetics companies are trying to minimize prescription of parabens, Is required.

Preservatives of parabens, which are chemical preservatives commonly used in cosmetics and pharmaceuticals, have been shown to be effective in the treatment of skin allergies (Andera Countti et al., Contact Dermatitis, 1997, 37; 35-36) and potential as environmental hormones (Edwin et al., Toxicology and Applied Pharmacology , 1998, 153; 12-19.), And there is a problem of inducing resistant bacteria.

Cosmetics used as daily necessities have been reported to be the main cause of accumulation and allergic reactions in the body. Several synthetic chemicals, such as parabens, have been found to be debilitating for estrogen from studies by Brunek University in the UK. There is growing concern that this will have a negative impact on the endocrine system of all living things, including humans. In particular, the problem is related to the breast cancer of women.

Preservatives that can be incorporated into cosmetics are limited to those described in the cosmetics standards, but not only those preservatives that have been developed or utilized but also silver-copper zeolite, polyaminopropyl biguanide, iodopropynyl butyl Newly formulated preservatives such as Iodopropynyl Butyl-carbamate have been developed.

In addition, components such as alcohols and polyhydric alcohols that support a preservative effect having a certain repelling force and components not incorporated in cosmetics for the purpose of inhibiting the growth of microorganisms in cosmetics are not classified as preservatives, to be.

A number of natural antimicrobial substances have been reported from spices, foods, essential oils and herbal medicines. They have been found to be effective in the production of various antibacterial substances such as phenolic, polyphenol, quinine, flavone, flavonoid, antimicrobial substances are classified into flavonol, tannin, coumarin, terpenoid, alkaloid, lectin, polypeptide and the like.

Although the mechanism of action against the antimicrobial activity of plant-derived natural substances has not been elucidated, terpenoids and phenolics have been reported to have antimicrobial action through the mechanism of destroying the cell membranes of microorganisms. Phenol and flavonoids are substances essential for microbial metabolism To inhibit the growth of microorganisms.

In addition, coumarin and alkanoides are known to inhibit the growth of microorganisms at the genetic level. Phenolic compounds have a high antimicrobial activity against hydroxyl groups despite their low water solubility, and monoterpenes (Mihee Cho et al., Food Science and Industry, 2005, Vol. 38-2). The microbial cell walls and membranes are highly permeable to microbial cell walls and cell membranes.

In recent years, studies on the search for natural antimicrobial substances derived from microorganisms have been actively conducted. Among them, antimicrobial peptides or bacteriocins, which are produced by lactic acid bacteria, are the most representative ones. , It does not affect the beneficial bacteria in the intestines and has no spot in the intestines. Therefore, it is popular as a natural preservative such as foods.

However, most of the natural preservatives may deteriorate the stability of the raw material itself, and the viscosity of the cosmetic formulations may deteriorate and the emulsion stability may be deteriorated. And bacteriocin is limited in its use as a natural preservative because of its narrow range of antibacterial activity.

Due to such problems, only a small part of natural preservatives such as Hinokitiol, Magnoliae extract, Megnonol, and grapefruit seed extract DF-100 are commercially available.

Therefore, it is urgent to develop an effective preservative that has a wide range of antimicrobial action, exerts excellent effects, and is free from the safety of formulation, bio-toxicity, and skin irritation.

Korean Registered Patent No. 10-0615892 (registered date 2006.08.18) Korean Registered Patent No. 10-1249920 (Registration date 2013.03.27) Korean Registered Patent No. 10-1427462 (Registered on July 31, 2014) Korean Registered Patent No. 10-1367512 (Registration date 2014.02.19) Korean Registered Patent No. 10-0782599 (Registered on November 29, 2007) Korean Registered Patent No. 10-1532005 (Registered on June 22, 2015)

As described above, the present invention recognizes limitations such as limited antimicrobial effect, safety, and stability of synthetic preservatives including parabens and natural preservatives, and provides a safe, effective, and non-irritating, It is an object of the present invention to provide a composite preparation of a natural antimicrobial substance and a method for producing the same.

In order to achieve the above object,

The present invention relates to a composition comprising 60 to 90 wt% of a moisturizing agent or a solvent;

5 to 30 wt% of caprylhydroxamic acid (CHA);

0.1 to 5 wt% of a cationic polymer compound selected from a polymer of vinylpyrrolidone and an alpha-olefin or polyquaternium-10;

1 to 15 wt% of Boswellia serrata resin extract;

1 to 15 wt% of a herbal extract comprising 20 to 50 wt% of an alabaster, 20 to 50 wt% of an omelet, and 20 to 50 wt% of a gold powder.

Also disclosed is a method for producing a composition for a combination of natural antimicrobial substances,

Adding a caprylhydroxamic acid (CHA) to the solvent to dissolve the solvent,

Introducing a cationic polymer into the solvent through the dissolution step to swell the polymer;

And cooling the solvent after the swelling step, and then adding Boswellia serrata resin extract and one-sided extract to the mixture and stirring the mixture.

The composition of the present invention can be applied to various cosmetics and can be effectively used even in a small amount because the antimicrobial effect of the conventional preservative, the limited antibacterial pH range, and safety and stability limitations are overcome. .

Even when applied to skin, there is no usability stimulus such as itching and erythema, maintains its effectiveness even in a wide pH range, and has an advantage of having a low capacity and high efficacy.

And has high economical efficiency due to low manufacturing cost.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the function of Caprylhydroxamic Acid (CHA) and Boswellia serrata resin extract as components constituting a complex composition of a natural antimicrobial agent according to the present invention; .
2 is a view showing a test procedure for skin stability evaluation according to Experimental Example 1 of the present invention.
FIG. 3 is a graph showing an evaluation value of antimicrobial activity against a microorganism according to a concentration change of a natural antimicrobial compound preparation composition according to the present invention, and a survival value (OD) of each antimony compound according to concentration of a natural antimicrobial compound preparation composition.
FIG. 4 is a graph showing the results of a flotation test of the composition of a natural antimicrobial compound preparation according to the present invention and its numerical values. FIG.
FIG. 5 is a graph showing the cytotoxicity test results of a combination product of a natural antimicrobial substance according to the present invention. FIG.
FIG. 6 is a graph showing the anti-inflammatory activity of TNF-.alpha.
FIG. 7 is a graph showing the anti-inflammatory efficacy evaluation using the test compound inhibiting IL-6 production of the natural antimicrobial compound preparation composition according to the present invention.

The cell absorbs the protonated acid, which receives hydrogen ions through the cell membrane. In the plasma of the cell, the acid dissociates and changes the pH of the cell.

At this point, the bacteria continue to release hydrogen ions (protons) out of the cell while sodium ions are absorbed into the cells to keep the physiological pH of the cells constant.

This decrease in the pH value of the cell due to the energy-consuming process leads to a decrease in the breeding rate of the bacteria.

This process also lowers the extracellular pH of the cell to maintain proper acidic levels in the cell, allowing the cell to pass through acidic substances bound to the active species, hydrogen ions, Which in turn promotes acidification and makes the situation worse. This process eventually leads to the death of microorganisms.

The character of the acid to obtain an effective active substance is very important. Suitable materials as active substances ensure biological effectiveness and can penetrate cell membranes and dissociate hydrogen ions within cells. If sufficient organic acids are sufficient, sufficient solubility in the water phase, efficacy as a protonated form at a given pH of the formulation (pH), structure capable of passing through the cell membrane Dissociation is possible) to effectively inhibit the growth of bacteria.

Organic acids are safe ingredients in toxicity and are a preservative with a broad spectrum of antimicrobial activity.

Each composition of the antimicrobial composition according to the present invention is evaluated for cytotoxicity through separate experiments and confirmed the anti-inflammatory effect through the antimicrobial effect and the expression level of TNF-α and IL-6 stimulating factor The content was limited and validated.

The antimicrobial composition of the present invention is an antibacterial composition containing an organic acid having an antibacterial effect and a boswellia extract having a function of reducing the burning sensation appearing in antiseptics such as anti-inflammation and anti-inflammation, and a thin protective film on the skin to form an antimicrobial substance It consists of a polymer compound that blocks skin from penetration and prevents further stimulation.

More specifically, 60 to 90 wt% of a moisturizing agent or a solvent; 5 to 30 wt% of caprylhydroxamic acid (CHA) which is an organic acid having an antibacterial effect; Polymers of vinylpyrrolidone and alpha-olefin or poly (vinylpyrrolidone) as a polymeric substance that protects the skin from oil, moisture and stimulation sources from the outside when forming the viscosity of the formulation or by forming a film 0.1 to 5 wt% of a cationic polymer compound selected from quaternium-10; 1 to 15 wt% of Boswellia serrata resin extract; It is composed of 1 ~ 15wt% of Oriental herb extract which is composed of 20 ~ 50wt% of alfalfa, 20 ~ 50wt% of omija, and 20 ~ 50wt% of gold and has excellent antibacterial and anti - inflammatory effect and minimizes skin irritation Based on the total weight of the composition.

On the other hand, according to the regulations on the safety standards of cosmetics (notified by the Food and Drug Administration), the pH range of the cosmetics should be in the range of 3.0 to 9.0 in the cosmetics except for the washable cosmetics. Within this range, the cosmetics are developed through various formulations . An acidic condition with a pH range of 3.0 or less or a basic condition of 9.0 or more gives an environmental condition that can inhibit the growth of some microorganisms, so that a special environment that does not require prescription of a preservative can be provided, It is true that this pH range is not free from pathogenic bacteria and fungi because it is suitable for the growth conditions of pathogens or microorganisms that can act as cosmetics and toxic to skin. Therefore, in order to be utilized as a general-purpose preservative, it is desirable to have an effective preservation spectrum in a wide range of pH range.

Therefore, it has been found that the use of six organic acids [caprylhydroxamic acid (CHA), caprylohydroxamic acid (Caprylohydroxamic Acid), benzoic acid benzoic acid, sorbic acid and salicylic acid] were selected to screen for components with an antimicrobial spectrum in a wide pH range. Among these candidate groups, caprylic hydroxamic acid (CHA) It is confirmed that the antimicrobial activity against fungi is effective in the pH range and the cost is reduced.

pH A. fungi (A. Niger ) For Buoyancy Comparative Test [Unit: fungus (A. Niger )% Growth inhibition rate] pH3 pH4 pH5 pH6 pH7 Caprylohydroxamic Acid 100 100 100 100 100 Dehydroacetic Acid 100 95 65 16 2 Benzoic Acid 94 61 13 1.5 0 Sorbic Acid 98 85 37 5.5 0 Salicylic Acid 48 9 One 0 0

Hereinafter, the technical composition will be described in more detail.

As described above, the combined preparation for natural antimicrobial agent for cosmetics according to the present invention comprises 60 to 90 wt% of a humectant or a solvent;

5 to 30 wt% of caprylhydroxamic acid (CHA);

1 to 15 wt% of Boswellia serrata resin extract;

1 to 15 wt% of herbal extract composed of a mixture of 20 to 50 wt% of corn syrup, 20 to 50 wt% of omija and 20 to 50 wt% of gold.

Specific examples of the moisturizing agent include polyhydric alcohols conventionally used in the art such as glycerin, butylene glycol, dipropylene glycol, and propylene glycol. Preferably, the moisturizers include glycerin, butylenol Use one or more of the licenses.

The solvent may be pure water, purified water, various antibacterial activity, and water-extracting water having skin moisturizing / antioxidant ability, and any one or more selected from these may be used.

If the amount of the humectant or solvent used is less than 60 wt%, there is a problem of precipitation of caprylic hydroxamic acid (CHA) due to a decrease in solubility at a low temperature. When the amount of the humectant or solvent exceeds 90 wt% The use feeling of the moisturizing agent or the solvent is preferably limited within a range of 60 to 90 wt% with respect to the total weight of the antimicrobial composite preparation.

When the amount of caprylhydroxamic acid (CHA) is less than 5 wt%, it is difficult to have antibacterial effectiveness as an organic acid. When the amount of caprylhydroxamic acid is more than 30 wt%, factors causing stimulation and the acidity of the formulation The amount of the caprylhydroxamic acid (CHA) used is limited within the range of 5 to 30 wt% with respect to the total weight of the antimicrobial composite preparation. Therefore, the amount of the caprylhydroxamic acid (CHA) .

The cationic polymer compound is a polymeric substance that protects the skin from external secondary stimulation sources upon application of the skin. When the amount of the cationic polymer compound is less than 0.1 wt%, there is a problem that the effectiveness of stimulation reduction is poor. When the amount exceeds 5 wt% Or more. The amount of the cationic polymer compound used is preferably limited within a range of 0.1 to 5 wt% with respect to the total weight of the antimicrobial compound preparation.

The Boswellia serrata resin extract has a function of reducing the burning sensation appearing in preservatives when applying cosmetics containing a complexing agent to the skin.

If the amount is less than 1 wt%, the effectiveness of the stimulation reduction is limited. If the amount is more than 15 wt%, there is a problem in usability that the product tends to stick and absorb in the application. Therefore, the Boswellia serrata resin extract is preferably limited within a range of 0.1 to 5 wt% with respect to the total weight of the antimicrobial composite preparation.

The herbal extract is added to enhance antimicrobial activity. When the amount of the herbal extract is less than 1 wt%, the improvement in antibacterial activity is insignificant. When the content exceeds 15 wt%, the antibacterial activity does not change, Is preferably limited to a range of 0.1 to 5 wt% with respect to the total weight of the material combination preparation.

The herbal extract may be a mixture of 20 to 50 wt% of an alfalfa extract, 20 to 50 wt% of Omija, and 20 to 50 wt% of gold.

The above-mentioned extracts of alfalfa, omija and gold are obtained by hot water extraction or Ethyl acetate extraction.

The hot-water extract was prepared by pulverizing a sample selected from the group consisting of phaeophyceae, omija and gold with a pulverizer, heating the mixture for 2 to 4 hours using a reflux heater at a ratio of 1: 8 to 10: And the extracted extract is prepared by centrifugation, filtering the supernatant using Whatman No. 2 filter paper, and filtering with sterile filter paper.

The ethyl acetate extract is prepared by pulverizing a sample selected from the group consisting of alfalfa, omija, and gold with a pulverizer, and then extracting the mixture at a ratio of 1: 8 to 10 for 65 to 75 hours. Thereafter, the extract is centrifuged at 6,000 to 9,000 rpm for 10 to 30 minutes, and the supernatant is filtered using a Whatman No.2 filter paper, followed by filtering with sterile filter paper.

(Example, Comparative Examples 1 to 4) [0097] The antibacterial composition of the present invention was prepared in the same manner as in Example 1,

Composition ratio of natural antimicrobial compound preparation composition ingredient Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 1,3-BG 74.50 85.00 70.00 80.00 75.00 Caprylic hydroxamic
Acid
15.00 15.00 30.00 15.00 15.00 Bosswellia resin extract 8.00 - - 50.00 10.00 Polyquaternium-10 0.500 - - - - Herbal extracts * 2.00 - - - -

* Oriental herb extract: Morus alba extract: Omiza extract: Golden extract = 1: 1: 1 weight ratio

The preparation process of the combination product of natural antimicrobial substances according to the compounding ratio is as follows.

(S1) adding capryloyloxamic acid to a solvent (1,3-butyleneglycol) and dissolving at 60 to 70 ° C by heating;

(S2) slowly adding a polymer compound (polyquaternium-10) to swell,

(S3), the mixture is cooled to 45 캜, and boswellia resin extract and oriental herb extract are added.

The mixing of S1) and S2) is carried out in a vacuum emulsification tank / stirrer capable of temperature control and stirring by a conventional method, and stirring and heating are applied as necessary.

Hereinafter, the skin safety evaluation and antibacterial effect of Example 1 will be described.

[ Test Example One]

[Evaluation of Skin Safety of Combination of Natural Antimicrobial Substance]

1. Antimicrobial Substance The antimicrobial compound preparation of various composition ratio was tested in a single human body skin test as follows to confirm the optimal composition ratio safe from skin irritation. The antimicrobial compound preparation was diluted to 2.0%, which is 10 times the antimicrobial effective content of 0.20%, with each sample being 100%.

2. Test method

Skin irritation was observed by attaching the patches to the inner part of the upper arm of the subject for 48 hours. After 24 hours and 48 hours, the skin irritation was visualized.

3. Evaluation Items

The results were as follows: negative (-), positive (+), weak (+), strongly positive (++)

In order to evaluate the stimulation abatement efficacy of the compositions of Example 1 and Comparative Examples 1 to 4 in Table 2, the following test was conducted.

A total of 12 subjects were recruited and 10 subjects completed the trial. The average age of the subjects was 23.5 years and the age distribution was 20-24 years.

The composition of the antimicrobial compound preparation composition was visually inspected after 24 and 48 hours of skin irritation by attaching a sample to the inside of the upper arm of the subject's arm, respectively.

Read criterion and score Positive reaction sign division Score No reaction at all - voice 0 Faint erythema ± Positive 0.5 Pronounced erythema + positivity One edema ++ Yangyang castle 2 Minority, necrosis +++ River breeding 4

Chemical Cosmetics Stimulation of the composition Abatement Efficacy evaluation

Referring to Table 4, in Comparative Examples 1 and 2 comprising only 1,3-BG and caprylhydroamic acid according to the present invention, erythema and irritation are largely exhibited, but in Example 1 containing boswellia resin extract, 3 and 4, it was confirmed that the effect of inhibiting erythema formation was increased as the content of boswellia resin extract increased.

In Comparative Example 3 containing 5% or more of boswellia resin extract, erythema and irritation were significantly reduced at 48 h, and no stimulation was observed in a combination of boswellia resin extract and a cationic polymer compound. Therefore, it was confirmed that the boswellia extract and the polymer compound have the effect of reducing the skin irritation.

[ Test Example 2]

[Antimicrobial Effect of Combination of Natural Antimicrobial Substance]

The antimicrobial effect of the natural antimicrobial compound preparation composition of Example 1, which is the result of the present invention, was confirmed as follows.

A disk agar plate diffusion method was used to measure the antimicrobial activity against the antimicrobial complex composition. The obtained results are shown in FIG. 3 and Table 5. [Korean J. Plant Res. 25 (1): 80-88 (2012)]

The test microorganisms were cultured at a concentration of 107 CFU / mL in a medium suitable for each test microorganism. The soft agar was dispensed into a petri dish, cooled to 25 ° C, and sterilized. Each sample extract was inoculated into a filter paper disk in an amount of 40 μL, incubated at 37 ° C for 24 hours in an incubator, and the formation of inhibitory rings around the filter paper disk was observed.

FIG. 3 is a graph and a table showing the evaluation of the antibacterial activity against each microorganism according to the concentration change of the complex of natural antimicrobial agent. The detailed test results are shown in Table 5 below.

Detailed test results ( MIC Value measurement) Strain classification Strain name Inoculation number Use badge MIC Value Gram
positive bacteria S. aureus 3.31X106cfu / Well Tryptic Soy Agar 0.031
Gram
negative bacteria
P. aeruginosa 3.23X106cfu / Well Tryptic Soy Agar 0.031 E. coli 3.56X106cfu / Well Tryptic Soy Agar 0.016 Yeast C. albicans 3.45X106cfu / Well Sabouraud
Dextrose Agar 0.063 to 0.031

As a result of the test, it was confirmed that the growth of microorganisms was inhibited at a concentration of about 0.03% or more in the case of bacteria and about 0.06% or more in the case of fungi.

Therefore, the results of the comparative tests of synthetic preservatives and non - preservatives showed that they have excellent antibacterial effect to replace conventional preservatives in various bacteria and fungi. In particular, it was confirmed that the minimum effective concentration (MIC) was 1000 ppm (0.1%) or less.

[In the formulation Buoyancy evaluation]

The antimicrobial effect (USP25 / NF20) in the formulation was evaluated by blending the combination preparation of the natural antimicrobial compound according to Example 1 at a concentration of 0.20, 0.50, and 1.00 in a conventional general lotion, and the results are shown in FIG. 4 .

FIG. 4 shows the results of the flotation test of the composite preparation of natural antimicrobial agent according to Example 1. As a result, it was confirmed that the antibacterial composition showed high antiseptic effect against each fungus and bacteria (effective concentration: 0.20% or more) All of them were killed.

[ Test Example 3]

[ Stimulation Reduction Validation test]

In order to confirm the effectiveness of the stimulation reduction, the expression levels of TNF-α and IL-6, which are stimulating-inducing factors, were tested in the following amounts of no toxicity of the cells.

end. Cell culture

(100 IU / ml), streptomycin (100 μg / ml) and streptomycin (100 μg / ml) were inoculated on the bottom of the culture dish. Dulbecco's Modified Eagle's Medium (DMEM, Welgene, Korea) containing 10% fetal bovine serum (FBS) was added and incubated at 37 ° C in an incubator containing 5% carbon dioxide.

I. Preparation of test solution

The test solution used in this test was prepared by diluting a combination of Antibiotic Compound 1 and extracts of Matsuchi, known to be effective for stimulation reduction, at a suitable concentration in the cell culture medium. In addition, preliminary experiments were carried out using the WST-1 assay, and then the concentration of the cells was determined to not exhibit cytotoxicity, and the effect of the salt was evaluated. Positive controls were also diluted with cell culture medium.

All. Protein amount measurement

The protein was measured using the Bio-rad DC Protein Kit based on the Lowry method.

1) Add 5 μl of each cell lysate into a 96-well plate.

2) Preparation of protein standard: BSA (Bovine Serum Albumine, Bio-Rad, 500-0007) at a concentration of 4.5mg / ml is diluted to 8 concentrations in cell lysis buffer and 5μl is added to a 96-well plate.

3) Mix reagent A and reagent S in a ratio of 40: 1 and add 25 μl to each well.

4) Add 200 μl of the reagent B contained in the kit.

5) After reacting at room temperature for 15 minutes, absorbance is measured at 750 nm.

6) Calculate the total protein content of the cells according to the BSA calibration curve.

la. statistics

All data were expressed as mean ± SD. Student's t-test was used for statistical analysis. The significance was P <0.005, and the two-sided test was used.

1) Cytotoxicity test (MTT) test

To confirm the toxicity of the test sample, cytotoxicity was measured in raw 264.7 cells. Cells were seeded in 96-well plates at an appropriate concentration and cultured in cell culture conditions for 24 hours. After the medium was removed and washed with PBS, the sample solution and fresh medium (Serum-free medium) were added and cultured for 24 hours. After 24 hours, the test solution and medium were removed and washed with PBS. To measure cell viability, the WST-1 reaction solution was diluted 1/10 in serum-free medium and treated with 100 μl of each solution for 1 hour After the reaction, absorbance was measured at 450 nm.

The in vitro cytotoxicity of the natural antimicrobial compound composition according to Example 1 and the marginal toxicity was measured by the WST-1 reaction solution which can determine the survival of cells depending on the mitochondrial activity of the cells. Cell viability was assessed in raw 264.7 cells and expressed as a percentage of control (control) except for the sample.

FIG. 5 is a graph showing the cytotoxicity evaluation of antimicrobial compound preparation and mash extract according to the concentration of the test substance. Referring to FIG. 5, it can be seen that the combined preparation of the antimicrobial substance has no cytotoxicity even when it is used up to the level of 0.5%.

Cell Viability Assay (n = 5) sample density(%) % control Contro 100.00 5.992

Antibiotic compound combination 1
0.1 108.32 + - 4.152 0.5 121.643 + 4.940 1.0 67.734 +/- 2.413 5.0 16.099 + - 0.403 10.0 17.091. + -. 0.882

Marchia extract

0.1 100.94 + 5.826 0.5 101.58 ± 8.714 1.0 99.00 + - 6.467 5.0 85.38 ± 6.180 10.0 39.29 ± 3.831

2) Anti-inflammatory test (TNF-α synthesis inhibition test)

This study was conducted to evaluate the anti-inflammatory activity of TNF-α in murine macrophages by ELISA. For this purpose, raw 264.7 cells were plated at 2 × 10 5 cells / well on the plate and cultured for 24 hours. After 24 hours, the test substance was diluted in DMEM (serum free medium), which was a cell culture medium, and the cells were treated for each concentration and cultured for 24 hours. The medium of the cultured cells was collected and the amount of TNF-α was measured using a TNF-α ELISA (R & D system, DY410). On the other hand, the cells attached to the bottom were washed with PBS, lysed with 1N NaOH, and the total amount of protein was measured to calculate the amount of TNF-α synthesis per a certain protein (FIG. 6)

Evaluation of anti-inflammatory efficacy by inhibition of TNF- α production (n = 3) sample density % of LPS-

LPS
1 ppm

- 100.00 ± 22.041 + 3438.0 ± 280.576 Dexa 100ppm 1998.3 ± 121.968
Example 1 0.05% 2943.1 ± 205.103 0.10% 1597.5 ± 223.921 0.50% 1055.7 ± 191.553
Marchia extract
0.50% 3520.3 ± 126.537 1.00% 2186.9 ± 210.055 5.00% 1622.1 + - 134.817

<TNF-α synthesis inhibition test result>

Macrophages involved in inflammation are produced by stimulation with lipopolysaccharide (LPS), such as TNF-α. Therefore, this test also induced TNF-α synthesis inhibition by inducing LPS-induced inflammation in raw 264.7 cells, macrophages. The concentrations used in the tests were evaluated by setting the concentrations without cytotoxicity.

As a result, when treated with 1 μg / ml of LPS, 348.0% of TNF-α was produced in the LPS alone group, indicating that the raw 264.7 cells were induced by LPS, and Dexamethasone (Dexa) Was 100 ppm, the TNF-α production was decreased significantly compared with the LPS alone treatment group.

The antimicrobial complex substance 1 was treated with 0.05%, 0.10%, 0.50% and 0.5%, 1.00%, and 5.00% of the mashin and then measured with a TNF-α ELISA kit (R & D system, DY410) As shown in FIG. 6, TNF-α was measured at 2943.1%, 1597.5%, and 1055.7%, respectively, in the antimicrobial compound preparation group, compared to the LPS alone treatment group. Respectively.

Especially, 0.10% and 0.50% were statistically significant, indicating that the anti - inflammatory effect is excellent.

3) Anti-inflammatory test (IL-6 synthesis inhibition test)

This test was conducted to evaluate the effect of the sample on the synthesis of IL-6 in mouse macrophages by ELISA. For this purpose, raw 264.7 cells were plated at 2 × 10 5 cells / well on the plate and cultured for 24 hours. After 24 hours, the test substance was diluted in DMEM (serum-free medium) as a cell culture medium, treated with cells at different concentrations, and cultured for 24 hours. The culture medium was then harvested and the amount of IL-6 was measured using an IL-6 ELISA (R & D system, DY406). On the other hand, the cells on the bottom were washed with PBS, lysed with 1N NaOH, and the total amount of protein was measured to calculate the amount of IL-6 synthesis per a certain protein (FIG. 7).

Evaluation of anti-inflammatory efficacy by inhibition of TNF- α production (n = 3) sample density % of LPS-

LPS
1 ppm

- 100.00 + - 9.616 + 1368.46 ± 19.093 Dexa 100ppm 913.96 ± 55.169
Example 1 0.05% 1235.16 ± 92.946 0.10% 574.87 + - 84.969 0.50% 257.80 ± 10.379
Marchia extract
0.50% 1572.23 + - 61.815 1.00% 1404.50 ± 82.772 5.00% 969.20 ± 81.744

The inflammatory macrophages are stimulated by lipopolysaccharide (LPS) to produce IL-6. Therefore, this test also induced the inflammation of raw macrophage RAW 264.7 cells with LPS and evaluated the ability of the sample to inhibit IL-6 synthesis. The concentrations used in the tests were evaluated by setting the concentrations without cytotoxicity.

As a result, IL-6 was found to be 1368.4% of IL-6 treated with 1 μg / ml of LPS, indicating that the raw 264.7 cells were induced by LPS, and Dexamethasone (Dexa) Was 100 ppm, the TNF-α production was decreased significantly compared with the LPS alone treatment group.

On the other hand, IL-6 ELISA kit (R & D system, DY410) was used after 0.05%, 0.10% and 0.50% antimicrobial combination treatment and 0.5%, 1.00% and 5.00% , As shown in FIG. 7, IL-6 was measured at 1235.1%, 574.8%, and 257.8%, respectively, in the antimicrobial compound preparation group compared to the LPS alone treatment group. Respectively.

INDUSTRIAL APPLICABILITY The complex composition of a natural antimicrobial agent according to the present invention is excellent in antimicrobial and anti-inflammatory properties of hypoallergenic and can be applied to various cosmetics.

Claims

60 to 90 wt% of 1,3-butylene glycol;
5 to 30 wt% of caprylhydroxamic acid (CHA);
0.1 to 5 wt% of cationic polymer compound having polyquaternium-10;
1 to 15 wt% of Boswellia serrata resin extract;
Wherein the composition comprises 1 to 15 wt% of an extract of Oriental herbarium extract: Omiza extract: golden extract, and a herbal extract prepared by mixing 1: 1: 1 by weight of the extract.

Adding 1,3-butylene glycol to caprylhydroxamic acid (CHA) and dissolving the same,
Introducing a cationic polymer compound such as polyquaternium-10 into 1,3-butylene glycol, which has been subjected to the above steps,
After cooling the 1,3-butylene glycol by the swelling step, Boswellia serrata resin extract,
Wherein the extract is prepared by mixing an extract of Allium cepa L.: Omija extract: golden extract at a weight ratio of 1: 1: 1, and stirring the mixture.