KR100396184B1 - Natural antibiotic composition - Google Patents

Abstract

The present invention relates to a natural antimicrobial agent, and more specifically, as a bio-new material, having antiseptic and antioxidant and anti-aging functions in foods, medicines, cosmetics, textiles, household goods, etc. to increase the safety and stability of quality, and in mammals including humans. The present invention relates to a natural antimicrobial agent that has the effect of treating and preventing infectious diseases because it is safe and does not induce resistance and has adequate antioxidant and anti-aging functions.

Description

Natural antibiotic composition

The present invention relates to a natural antimicrobial agent having an antibacterial activity while having an antioxidant function and an anti-aging activity. More specifically, the present invention relates to D. TF (dimethoxy tetrahydroxy flavone) obtained from a golden extract as a main active ingredient. It has anti-aging activity, high antibacterial activity, and antibacterial spectrum relates to a wide range of multifunctional natural antibacterial agents.

Many products such as food, medicine, and cosmetics are essential to prevent decay by microorganisms in order to maintain the quality for a long time, but in the case of cosmetics with a long shelf life and a lot of microbial nutrients due to the characteristics of the product.

However, even parabens preservatives, which are the safest as conventional preservatives and are widely used in cosmetics and medicines, are allergic to skin allergies (Andrea Counti et al., Contact Dermatitis, 1997, 37; 35-36.) And their potential as environmental hormones (Edwin). Et al., Toxicology and Applied Pharmacology, 1998, 153; 12-19.) And resistant bacteria. In addition, the use of food preservatives in accepted standards is distrusted, and new problems such as acute, chronic toxicity, and mutagenesis due to continuous accumulation in the body are emerging (Xindonghua, Food Science and Industry, 1990, 23 (4)). 68-72).

Due to these problems, research on natural preservatives having excellent safety and economical efficiency of products is ongoing, and as a result, many natural antimicrobial substances have been reported from foods such as spices, milk and fish, essential oils, and herbal medicines.

Alkaloids (alkaloids), flavonoids (phytoalexin), antimicrobial peptides, and the like are known as natural antimicrobial substances, and antimicrobial agents such as organic acids and fatty acids are also known. Most of them are thought to be the main mechanisms of acid pH and chelate effects (EI-shenawy, MA et al., J. Food Protec. 1989,52 (11): 771-778) (Bizri, JN et al. , J Food Science, 1994, 59 (1), 130-135).

However, most of the reported natural antimicrobial substances have not been commercialized due to color odor, poor stability, narrow antimicrobial spectrum, problems with formulation, and the like. Henokithiol, which is a protein extract, and Magnool, which is a magnolia extract, Only a few, such as grapefruit seed extract DF-100, are commercially available.

In the case of DF-100, which is the most developed product, antibacterial activity is known to be due to organic acid and synthetic preservative benzethonium chloride contained in DF-100, and other natural antimicrobial agents have low economical or narrow antibacterial spectrum or physical properties. Due to the problem of limitation of use range, there is an urgent need for natural antimicrobial agents that can be more advanced and commercialized.

Meanwhile, antimicrobial agents currently used in general products are not intended to maintain product quality, but synthetic antibiotics are widely used to prevent and treat disease in humans and livestock caused by microorganisms. In the hospital, a vicious cycle that requires the use of a high level of antibiotics to treat infectious diseases is being formed, and the public health problems such as susceptibility of causative bacteria to the livestock and residual antibiotics are emerging.

In particular, two or more antibiotics may be used in combination or vancomycin to combat Methicillin Resistant Staphylococcus Aureus (MRSA) (Voss, A et al., Int J. Antimicrob.Agents, 1995,5: 101-106). Vancomycin is used, but resistant strains such as Vancomycin-resistant enterococci (Billot-klein D, Antimicrob. Agents. Chemother, 1992, 36: 1487-1490) are resistant to vancomycin.

Accordingly, in recent years, it has been reported that natural products are used together with flavonoids to increase the sensitivity of antibiotics (IAIN X, LIU et al., J. Pharm.Pharmacol, 2000, 52: 361-366), antimicrobial peptides ( Giacometti A et al., J. Antimicrob. Chemother 2000 Nov; 46 (5): 807-811), although many studies have been conducted as an alternative for the combined effect, antimicrobial peptides are limited to show antimicrobial properties and have problems with economic mass production methods. Lee JH et al., Protein. Expr. Purif, 1998 Feb; 12 (1), 53-60).

On the other hand, free radicals generated by biochemical oxidation reactions in vitro or in vivo are causing various diseases such as rheumatoid arthritis, heart disease, circulatory disorders, and cancer. Free radicals cause diseases by damaging factors of the immune system such as proteins, DNA and immune cells, attacking unsaturated fatty acids on cell membranes, accumulating lipid peroxide, which leads to deterioration of biological function, aging and adult disease. have.

In addition, deterioration of quality due to rancidity of foods, cosmetics, medicines, etc. can not only cause economic loss, but also affect the people who consume and apply it. It is preferable to add the substance present.

Infections caused by microorganisms and antioxidants are inseparably related to the expression of free radical scavengers such as superoxide dismutase, which is induced by Gram-negative endotoxins and free radicals are inherent defense factors. It is also known that this decrease (Leach M et al., Br J. Pharmacol, 1998, oct; 125 (4): 817-825).

In addition, free radicals produced by polymorphonuclear leukocytes activated by the interaction of bacteria and foods can disrupt the balance of oxidoreductant activity, induce the destruction of the periodontal tissue, and further promote inflammation (Waddington RJ et al., Oral. Dis, 2000, May; 6 (3): 138-151. In this context, vitamin C has been reported to alleviate symptoms associated with endotoxin shock as one of the important water soluble antioxidants (Victor VV et al., Immunopharmacology, 2000 Jan; 46 (1): 89-101). Rarely do antioxidants be used in combination for proper treatment.

Since ancient times, antioxidant and non-enzyme-based natural antioxidants have been used a lot, but one of the most practical tocopherols (tocopherol) has a relatively low antioxidant effect. It is known to cause disease. Therefore, there is a need for safe and effective natural antioxidants.

The present inventors continued to study the natural antimicrobial agent that exhibits excellent antibacterial activity with antioxidant function, and as a result, as a part of the technology regarding natural antimicrobial agent in the patent application No. 10-2001-0006372 and the gold extract Disclosed is a technique related to a natural antimicrobial agent.

However, the natural antimicrobial agent presented by the present inventors, as well as the existing natural antimicrobial agent is lowered in stability, and has a narrow antimicrobial spectrum, so there is a restriction in the range of use and has a lot of difficulties in commercialization due to formulation problems.

Thus, the present invention is separated and purified from the anti-gold extract D-Tif (dimethoxy tetrahydroxy flavone) showing the antimicrobial component to utilize it as an antimicrobial composition, not only very excellent antimicrobial power but also has a broad spectrum of appropriate antioxidant function and aging Its purpose is to provide a multifunctional natural antibacterial agent that has an inhibitory function.

In addition, the present invention is another object to provide a natural antimicrobial agent containing a substance to increase the antimicrobial activity of DTF with DTF in order to maximize the antibacterial activity.

1 to 4 are photographs showing the results of aging inhibitory activity of the cell line,

1 is a photograph showing the cell state of the untreated control group,

Figure 2 is a photograph showing the cell state after treatment for 12 hours using 4-HNE as an oxidizing agent,

Figure 3 is a photograph showing the cell state after 12 hours treatment using an antimicrobial agent containing 0.001% by weight DTI as an antimicrobial agent,

Figure 4 is a photograph showing the cell state after 12 hours treatment using an antimicrobial agent containing 1.0% by weight of DTF as an antimicrobial agent.

5 is a photograph showing the results of the anti-aging activity test.

In the present invention to achieve the above object, in the natural antimicrobial agent containing di-Tef (dimethoxy tetrahydroxy flavone) separated and purified from the golden extract as the main active ingredient, the content of the D-Tif is 0.001 to 1.0% by weight It provides a natural antibacterial agent characterized.

In another aspect, the present invention provides a natural antimicrobial agent, which comprises D-IF as the main active ingredient, and contains Baikalin purified from the anti-gold extract to increase the antimicrobial activity of the D-TF.

Hereinafter, the present invention will be described in more detail.

The present invention provides a natural antimicrobial agent comprising diTF (dimethoxy tetrahydroxyflavone) separated and purified from golden extract as a main active ingredient.

Golden extract is obtained by using dried gold ( Scutellaria baicalensis ) and is known to be effective for antipyretic, bile secretion, gastric juice secretion, prevention of atherosclerosis, antibacterial and antifungal and antiviral. In addition, anti-oxidation, anti-aging (Shieh DE, Anticancer Res, 2000 Sep-Oct; 20 (5A): 2861-2865), anti-inflammatory and anti-allergic effects (Williamson EM, etc., Potter's New Cyclpaedia of Botanical Drug and Preparations, revised edn) , 1988, p362, The CW Daniel Co., Ltd, UK).

The golden extract can be obtained by a variety of methods commonly used to extract the active ingredient from the plant.

For example, 10 times (by weight) of a pulverized product, a polar solvent such as ethanol and methanol, or a mixed solution of purified water is added to a golden dry matter, and the extract is heated at a temperature of about 80 ° C. for 24 hours in an extractor equipped with a cooling capacitor. Obtained and concentrated under reduced pressure by filtration to obtain a concentrated extract. Purified water corresponding to 2 times (weight ratio) of the obtained extract was added, followed by 4 times (weight ratio) of non-polar solvents such as ethyl acetate and methylene chloride, and vigorously shaken to separate the non-polar solvent layer, which was concentrated under reduced pressure. You can get golden extract.

As another example, two times (weight ratio) of purified water was added to a golden dry matter, and 20 parts by weight of a polar solvent such as ethanol and methanol or a mixed solution of them and purified water was added to 100 parts by weight of the ground product to which a cooling capacitor was attached. The extract was heated at 60 ° C. for 10 hours in an extractor, and the extract was filtered and concentrated under reduced pressure to obtain a concentrated extract. A polar extract, such as ethanol and methanol, or a mixed solution of purified water and 10 times the weight of the extract is added, followed by strong shaking extraction, followed by filtration to remove solids and filtration to obtain a golden extract.

As another example, the main antimicrobial active ingredient in the golden extract can be obtained by the following extraction method using supercritical fluid extraction technology. In other words, after filling the golden dry matter with a supercritical extractor, the extract was first extracted with carbon dioxide at a pressure of 4,000 to 6,000 PSI at 70 to 90 ° C., and then 2 to 20 (v / v)% of diethylamine or tree to 100 parts by volume of carbon dioxide. One to 20 parts by volume of one or more alkaline cosolvents selected from methanol, ethanol, water, or a mixed solvent of ethylamine in which they are dissolved can be extracted by mixing in a reactor, and then a selective fraction of golden extract can be obtained using column chromatography. .

In addition to the above-mentioned methods, a golden extract can be obtained through various methods, and the obtained golden extract includes baicalein, which is the main ingredient, and includes baicalin, DTF, wogonin, 7- It contains flavonoids such as 7-methoxy baicalein, oroxylin-A and skullcapflavone I and II, and also beta-sitosterol and campestrol ( sterols such as campesterol), sugars such as sucrose and D-glucose, essential oils and various other resins.

It is known that the yield obtained by the conventional extraction method is about 2 to 3% by weight of Baikallein and Baikal relative to the weight of the raw material in the dry state and less than 1% by weight of Ogonin, D.T. The yield of each active ingredient thus varies.

As described above, the golden extract is mainly composed of baicalein (baicalein), but has a limited antimicrobial activity, while the D-Tif contained in the golden extract, although its content is extremely small, but excellent antibacterial activity.

Therefore, in the present invention, separated and purified DTIF from the golden extract was used as a natural antimicrobial agent, and the separated and purified DTIFF from the golden extract can be easily separated and purified by conventional column chromatography.

In the present invention, the golden extract was mixed with a solvent selected from ethanol, methanol, ethyl acetate or methylene chloride using a silica gel column to fractionate in a concentration gradient method to obtain a D.T.

The obtained DITF itself has a high antibacterial activity, a broad spectrum of antimicrobial spectrum, high antioxidant activity, and anti-aging activity, which is selected from propylene glycol, glycerin, 1,3-butylene glycol, water or alcohol. It can be dissolved in a solvent to be used as an antimicrobial agent or added to various other items to have antimicrobial activity.

In the process of using DTIFF, if its content exceeds 1% by weight, the antibacterial activity does not increase any more, so it should be added within 1% by weight. If the amount is less than 0.001% by weight, DTIFF cannot be obtained. It is preferable to be contained -1.0 weight%.

According to the present invention, the addition of bicalin separated and purified from anti-gold extract to DTF increases the antimicrobial activity of DTF, thereby obtaining more maximized antimicrobial activity, compared to when the content of the bicalin is 0.1 to 5.0% by weight. Maximized synergy can be obtained. That is, in the case of a natural antimicrobial agent containing 0.001 to 1.0% by weight of DTF and 0.1 to 5.0% by weight of Baikal, the antimicrobial activity of DTF is increased by Baikal to obtain more excellent antibacterial activity. At this time, if the content of Baikal is less than 0.1% by weight can not sufficiently obtain the synergistic effect of DTIFF antibacterial activity, even if the content exceeds 5% by weight, the synergistic effect of antimicrobial activity does not increase proportionally. Therefore, it is preferable to contain 0.1-5.0 weight% of bicalin .

The Baikal can be easily obtained by mixing the golden extract, like the DITF, using a silica gel column, a solvent selected from ethanol, methanol, ethyl acetate or methylene chloride, fractionation by concentration gradient method.

Natural antimicrobial agents, including D.T. and Baikallin, to contain at least 10% by weight of at least one herbal extract selected from high ginseng extract, sterling silver extract, rhubarb extract, fragrance extract, sesame bark extract, gallola extract, garlic extract or licorice extract When added, it has high anti-oxidation and anti-aging activity, high antimicrobial activity, and antibacterial spectrum gives a wide range of multifunctional natural antimicrobial agents.

At this time, the high ginseng extract, sterling silver extract, rhubarb extract, fragrance extract, velvet bark extract, gall bladder extract, garlic extract or licorice extract can be extracted and used in the same manner as the conventional extraction method to obtain the golden extract. In addition, tannin can be removed in advance using sodium chloride for effective extraction, and the extraction conditions can be deposited for 2 to 7 days at room temperature instead of heating.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples, which are only presented to aid the understanding of the present invention, but the present invention is not limited thereto.

<Gold extract extract example>

2 times (weight ratio) of purified water was added to the golden dried pulverized product, and 20 parts by weight of a polar solvent such as ethanol and methanol or a mixed solution of them and purified water was added to 100 parts by weight of the pulverized powder, and the extractor was attached to a cold condenser. The extract was heated at a temperature of 10 ° C. for 10 hours, filtered and concentrated under reduced pressure to obtain a concentrated extract. Polar solvents, such as ethanol and methanol, or mixed solutions of purified water and 10 times the weight of the extract were added thereto, followed by strong shaking extraction, followed by filtration to remove solids, filtration, and drying to obtain a powdery golden extract.

<Activation ingredient fraction and qualitative identification>

The extract obtained in the golden extract preparation example was fractionated with a silica gel column to obtain anti-microbial activity of Baikal, D.T., and Ogonin. The composition of the obtained Baikal, D.T., and Ogonin was confirmed by high pressure liquid chromatography (column; Microbondapak C18 column, mobile phase; acetonitrile: 0.5% phosphoric acid was mixed at 27:73 (v / v) solvent, flow rate; 2ml per minute, injection amount; 10ul, detection wavelength 280nm. As a result, the peak was detected in the 16-minute baikal, 18-minute gonnin, and 17-minute DTIFF, which is consistent with the detection time range of each standard. In addition, the results of ¹ H NMR or ¹³ CNMR of each component was as follows, which was found to be the same ¹ H NMR or ¹³ C NMR results as the standard material.

DTI: ¹ H NMR (DMSO-d6) δ: 3.74,3.77 (3H each, s, -OMe), 6.30,6.35 (1H each, s), 6.61,6.94 (1H each, d, 9.0, aromatic H) , 9.0, 9.40, 10.72 (1H ech, br s, -OH), 12.56 (1H, s, OH5)

¹³ C NMR (DMSO-d6) δ: 161.7 (s, C2), 111.1 (d), 181.7 (s), 157.0 (s), 99.0 (d), 150.4 (s), 127.6 (s), 156.3 (s ), 103.7 (s), 114.8 (s, C1 '), 148.2 (s), 111.6 (d), 119.8 (d), 142.4 (s), 146.0 (s), 60.8 (q, -OCH3), 60.4 ( q, -OCH3)

Baikal: ¹ H NMR (DMSO-d6) δ: 7.01 (1H, s, H3 or 8), 7.07 (1H, s, H8 or 3), 7.40-7.70 (3H, m, H3 ', 4', 5 '), 7.90-8.20 (2H, m, H2', 6 '), 12.59 (1H, br.s, OH5), 5.11 (1H, d, 8.0, nanomeric H)

¹³ C NMR (DMSO-d6) δ: 163.7, 104.8, 182.8, 146.7, 131.0, 151.8, 94.3, 149.4, 106.2, 130.8, 126.5, 129.3, 132.2, 129.3, 126.5, 101.1, 73.2, 75.9, 69.8, 77.4, 60.7

Ogonin: ¹³ C NMR (DMSO-d6) δ: 162.9, 105.1, 182.0, 157.3, 99.2, 149.6, 127.8, 156.2, 103.8, 130.9, 126.2, 129.1, 131.9, 129.1, 126.2, 61.0

As described above, the following experimental examples were performed to determine the antibacterial activity and characteristics of Baikallin, D.T., Ogonin and powdered golden extract obtained by fractionation from the golden extract.

Experimental Example 1

In order to measure the antimicrobial activity of Baikallin, D.T., Ogonin and powdered golden extract obtained by fractionation from the golden extract, the strain was cultured using the Agar Serial Dilution Method, and the bacteria were mueller. Hinton broth) was used to inoculate the culture medium and incubated for 20 hours in a 37 ℃ incubator. For fungal culture, Sabouraud dextrose broth was used, inoculated with the culture medium, and incubated for 7 days in a 25 ° C incubator. Potato dex for the plant pathogen Fusarium solani Incubated for 7 days at 25 ℃ using a rose medium was used. Positive bacteria to the strain used is gram as Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538P, gram-negative bacteria as Escherichia coli ATCC 10536, Pseudomonas aeroginosa ATCC 1636, the yeast Candida albicans ATCC 10231, fungi Aspergillus niger ATCC 9029, Trichophyton mentagrophytes KCTC 6077, Fusarium solani was used.

For more specific antimicrobial test, first inoculate the bacteria in Mullertonton broth (2 × 10 ⁶ CFU / ml) and pre-incubate at 37 ℃ for 24 hours. Meanwhile, 2 ml of antimicrobial solution (5% DMSO; Dimethylsulfoxide physiological saline solution) of 3μg / ml concentration was added to the sterilized petri dish twice, and the control group was added 2ml of sterile distilled water, and the control group was added Amocla Put 2ml. After sterilization in each Petri dish, add 18 ml of Muller-Hinterton fusion media cooled to 50 ℃ and rotate the bottom of the Petri dish to mix well. Afterwards, pre-cultivated bacteria are plated about 5mm using platinum. Bacteria were incubated for 24 hours at 37 ℃, fungi and yeasts were incubated for 6 days at 22 ℃ incubator, and then colony formation was observed in each compartment to minimize the minimum antimicrobial concentration of the plate where growth was not recognized (MIC, Minimum inhibitory). concentration).

division B.subtilis S.aureus M.luteus MRSA E.coli E.coliO157 P.aeruginosa K.pneumoniae C.albicans A.niger T.mentagrophytes F.solani Baikalin 250 250 125 125 500 500 500 250 63 500 1000 1000 Dtyev 125 125 32 63 125 125 250 125 16 63 125 63 Ogonin 500 250 250 1000 500 1000 1000 500 125 1000 2000 1000 Golden extract 500 250 250 500 500 1000 1000 500 125 500 1000 1000

As shown in Table 1, DTF obtained from the golden extract was found to have the highest antibacterial activity with a minimum inhibitory concentration of 16 ppm to 250 ppm, but in the case of ongonin, the minimum inhibitory concentration was 125 ppm to 2,000 ppm, and the golden extract had a minimum It can be seen that the inhibitory concentration shows low antibacterial activity from 125ppm to 1,000ppm. Therefore, it can be seen that DTF alone can be sufficiently used as an antimicrobial agent.

Experimental Example 2

To determine the bactericidal activity according to the content of Baikallin, D.T. and O.gonin, which was obtained by fractionation from the golden extract, the Bacalin, O. ni .., and D.T. should be in the weight ratio shown in Table 2 below, and the remaining amount was mixed to be glycerin. A natural antimicrobial agent was prepared, and a paper disk agar assay method was used to measure the antimicrobial activity of the natural antimicrobial agent. In other words, each of the bacteria prepared in sterile saline is prepared at 10 ⁸ cfu / ml, sterilized Hangar agar medium, the culture medium for flat plate production sterilized at 121 ℃ for 15 minutes and added to the medium cooled to 45 ~ 50 ℃ 10 ⁶ cfu / Use a solid medium prepared in ml ml concentration. Place a 6-millimeter diameter paper disk on the Hinterton agar bacteria medium, add 25ul of Baikallin, Ogonin, and D-Tif at 0.0001 to 10% by weight, incubate for 24 hours at 37 ℃, and measure the diameter of the growth inhibitory ring. The optimum antibacterial activity range was evaluated.

Bacterial mussels (Mueller Hinton broth) was used and inoculated with the culture medium incubated for 20 hours in a 37 ℃ incubator. For fungal culture, Sabouraud dextrose broth was used, inoculated with the culture medium, and cultured for 6 days in a 22 ° C incubator. Potato dextrose for the plant pathogen Fusarium solani Medium was used.

Staphylococcus aureus ATCC 6538P was used as Gram-positive bacteria, Escherichia coli ATCC 10536 as Gram-negative bacteria, Candida albicans ATCC 10231 as yeast, and Aspergillus niger ATCC 9029 as fungi.

division Strain Antimicrobial activity (mm) 0.0001% 0.001% 0.01% 0.1% 1.0% 2.0% 5.0% 10% Baikalin S. aureus ND TR TR 14 18 22 27 27 E. coli ND ND ND 11 15 18 22 22 C. albicans ND TR TR 16 21 26 27 28 A. niger ND ND TR 10 13 17 19 20 Ogonin S. aureus ND ND ND 8 10 14 15 15 E. coli ND ND ND 7 8 11 11 12 C. albicans ND ND TR 8 11 15 16 16 A. niger ND ND TR 9 11 14 14 15 Dtyev S. aureus TR 11 15 18 23 26 26 27 E. coli ND 10 14 17 21 21 22 21 C. albicans 8 12 17 23 26 28 28 28 A. niger TR 10 13 17 19 20 20 20

As a result of Table 2, it can be seen that the range of 0.1 to 5.0% by weight of bicalin is an effective effective concentration having antimicrobial activity against bacteria, yeasts and fungi. It can be confirmed that it has a proper effective antibacterial activity at 0.001 to 1.0% by weight.

<Examples 1 to 54>

D.T.F., Baikalin, Ogonin and golden herb extracts obtained from golden extracts (Ginseng extract, Geummisu extract, Rhubarb extract, Ductus extract, Bark extract, Garlic extract, Garlic extract, Licorice extract ) To the weight ratio shown in Table 3 below, the remaining amount was mixed to become glycerin to prepare a natural antibacterial agent.

division DTF (%) Baikalin (%) Ogonin (%) Herbal extract / content (%) Example 1 0.0005 0 0 0 Example 2 0.001 0 0 0 Example 3 0.001 0.05 0 0 Example 4 0.001 0.1 0 0 Example 5 0.001 1.0 0 0 Example 6 0.001 5.0 0 0 Example 7 0.001 7.0 0 0 Example 8 0.001 0 0.05 0 Example 9 0.001 0 0.1 0 Example 10 0.001 0 0.5 0 Example 11 0.001 0 2.0 0 Example 12 0.001 0 5.0 0 Example 13 0.01 0 0 0 Example 14 0.01 0.05 0 0 Example 15 0.01 0.1 0 0 Example 16 0.01 1.0 0 0 Example 17 0.01 5.0 0 0 Example 18 0.01 7.0 0 0 Example 19 0.01 0 0.05 0 Example 20 0.01 0 0.1 0 Example 21 0.01 0 0.5 0 Example 22 0.01 0 2.0 0 Example 23 0.01 0 5.0 0 Example 24 0.1 0 0 0 Example 25 0.1 0.05 0 0 Example 26 0.1 0.1 0 0 Example 27 0.1 1.0 0 0 Example 28 0.1 5.0 0 0 Example 29 0.1 7.0 0 0

<Continued Table 3>

division DTF (%) Baikalin (%) Ogonin (%) Herbal extract / content (%) Example 30 0.1 0 0.05 0 Example 31 0.1 0 0.1 0 Example 32 0.1 0 0.5 0 Example 33 0.1 0 2.0 0 Example 34 0.1 0 5.0 0 Example 35 1.0 0 0 0 Example 36 1.0 0.05 0 0 Example 37 1.0 0.1 0 0 Example 38 1.0 1.0 0 0 Example 39 1.0 5.0 0 0 Example 40 1.0 7.0 0 0 Example 41 1.0 0 0.05 0 Example 42 1.0 0 0.1 0 Example 43 1.0 0 0.5 0 Example 44 1.0 0 2.0 0 Example 45 1.0 0 5.0 0 Example 46 2.0 0 0 0 Example 47 0.001 5.0 0 Red ginseng extract (10%) Example 48 0.001 5.0 0 Gold and Silver Extract (10%) Example 49 0.001 5.0 0 Huang lotus extract (10%) Example 50 0.001 5.0 0 Soft extract (10%) Example 51 0.001 5.0 0 Bark Extract (10%) Example 52 0.001 5.0 0 Schisandra chinensis extract (10%) Example 53 0.001 5.0 0 Garlic Extract (10%) Example 54 0.001 5.0 0 Licorice Extract (10%)

Experimental Example 3

Using the natural antibacterial agent prepared in Examples 1 to 54 was carried out in the same manner as in Experiment 1 to determine the minimum inhibitory concentration. At this time, a positive bacteria to the strain used is gram Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538P, Micrococcus luteus ATCC 9341, Methicillin resistant staphylococcus aureus (MRSA) C2207, the Gram-negative bacteria Escherichia coli ATCC 10536, Escherichia coli 0157: K88ac, Pseudomonas aeroginosa ATCC 1636, Klebsiella pneumoniae ATCC 10031, Candida albicans ATCC 10231 as yeast, Aspergillus niger ATCC 9029, Trichophyton mentagrophytes KCTC 6077, Fusarium solani as fungi.

division B.subtilis S.aureus M.luteus MRSA E.coli E.coli0157: K88ac P.aerosinosa K.pneumonia C.albicans A.niger T.mentagr-ophyte F.solani Example 1 1000 2000 1000 2000 1000 2000 2000 1000 500 500 500 1000 Example 2 1000 1000 500 2000 1000 2000 1000 1000 500 500 500 500 Example 3 500 1000 500 1000 1000 1000 500 1000 250 500 250 500 Example 4 500 1000 500 1000 1000 1000 500 1000 250 500 250 500 Example 5 500 500 250 500 1000 1000 500 250 63 250 125 125 Example 6 250 250 125 250 500 500 125 125 32 125 63 63 Example 7 250 125 125 250 500 500 125 125 32 125 63 63 Example 8 2000 2000 2000 2000 2000 2000 2000 1000 1000 2000 1000 1000 Example 9 1000 1000 1000 1000 2000 2000 1000 1000 1000 1000 500 500 Example 10 1000 1000 1000 1000 500 1000 500 1000 500 500 500 250 Example 11 500 500 1000 1000 500 1000 500 1000 250 500 250 500 Example 12 500 500 500 1000 500 1000 500 1000 250 500 250 500 Example 13 500 250 250 500 500 500 500 1000 500 500 250 250 Example 14 250 250 250 500 250 500 500 500 250 250 250 125 Example 15 125 250 250 500 250 500 250 500 125 125 125 63 Example 16 63 125 63 250 250 250 125 250 125 63 63 32 Example 17 32 63 63 125 125 250 125 250 63 63 63 32 Example 18 32 32 63 125 125 250 125 250 63 63 63 32 Example 19 1000 1000 1000 500 500 500 250 250 250 125 250 250 Example 20 500 500 500 500 250 500 250 250 125 125 250 125 Example 21 500 250 250 250 125 250 125 125 125 125 250 125 Example 22 250 125 250 250 125 125 125 63 125 63 125 125 Example 23 250 125 125 250 125 125 125 63 125 63 125 125 Example 24 125 250 250 500 500 250 500 250 125 250 125 125 Example 25 125 250 125 500 250 250 250 125 125 125 125 63 Example 26 63 125 125 250 250 250 125 125 63 63 125 63 Example 27 125 125 63 63 125 125 63 63 32 32 63 32 Example 28 63 63 32 32 63 63 32 16 8 16 32 16 Example 29 63 63 32 32 63 32 32 16 8 16 32 16 Example 30 500 500 250 500 250 500 250 125 250 250 250 250 Example 31 250 250 125 500 250 250 125 125 125 250 125 125 Example 32 250 250 125 125 500 125 125 63 63 125 63 63 Example 33 125 125 63 125 250 125 63 63 32 32 16 16 Example 34 125 125 63 125 125 125 63 63 32 32 16 16 Example 35 63 125 63 125 125 250 125 250 125 63 63 63 Example 36 63 63 32 63 125 125 63 63 63 32 32 63 Example 37 32 32 16 32 63 63 32 32 16 16 8 8 Example 38 32 16 16 32 32 32 16 8 8 4 4 4 Example 39 16 8 16 16 16 16 8 8 4 4 2 2 Example 40 16 8 16 16 16 8 8 8 4 4 2 2 Example 41 125 250 63 125 125 63 63 32 63 32 63 32 Example 42 125 125 63 63 125 63 63 32 63 16 63 16 Example 43 125 63 63 63 125 63 32 16 32 16 16 16 Example 44 63 32 16 32 32 32 16 16 16 16 16 16 Example 45 63 32 16 32 16 32 16 16 16 16 8 16

<Continued Table 4>

division B.subtilis S.aureus M.luteus MRSA E.coli E.coli0157: K88ac P.aerosinosa K.pneumonia C.albicans A.niger T.mentagr-ophyte F.solani Example 46 63 63 32 363 125 125 63 125 125 63 32 63 Example 47 125 250 125 250 250 125 125 125 63 63 63 32 Example 48 125 250 125 250 250 250 125 125 32 63 125 63 Example 49 250 500 125 250 500 250 250 125 63 250 125 125 Example 50 125 250 125 250 250 500 250 125 63 125 250 125 Example 51 125 250 250 250 125 250 125 250 125 63 125 250 Example 52 125 125 250 125 63 125 125 63 63 125 63 63 Example 53 250 125 250 250 250 250 125 250 125 125 250 125 Example 54 125 63 125 63 125 125 63 125 63 63 125 63

As shown in Tables 3 and 4, it can be seen that the antimicrobial activity is very high when DTI is used within the preferred range of the present invention, but when added to include Baikal within the range of 0.1 to 5.0% by weight, It can be seen that it brings about an antimicrobial activity. In addition, in the case of the embodiment in which the herbal extracts were additionally added to DTF and Baikal, it can be seen that the antimicrobial activity is improved more than the embodiment without the addition.

Experimental Example 4

Examples 4 to 6, Examples 9 to 11, Examples 15 to 17, Examples 20 to 22, and Examples 26 to 28, which prepared an antimicrobial agent within the preferred range of the present invention based on the results of Experimental Example 3 Induction resistance was confirmed using the antibacterial agents prepared in Examples 31 to 33, Examples 37 to 39, and Examples 42 to 44. In addition, erythromycin, a commonly used antibiotic for comparison, was confirmed to be resistant. The strain used in the experiment was Escherichia coli 0157: K88ac, inoculated in lactose broth (0.5% yeast extract, tryptone, 1% sodium chloride), incubated at 37 ° C and 150rpm for 12 hours, and centrifuged (500g, 4 minutes) to isolate bacteria. Then, the bacterial count was adjusted by diluting with phosphate buffer solution (0.1M, pH 7.0), and 0.05 ppm of the antimicrobial agent of Example 16 was added to 20 ml of lactose broth culture and inoculated so that the concentration of the bacterium was 2 × 10 ⁶ . The erythromycin treatment group was incubated with erythromycin at 0.05 ppm for 12 hours, followed by centrifugation of the bacteria in the same manner, and then inoculated to the same concentration in the culture medium containing the same concentration of antimicrobial agent. After repeating the above procedure five times, the antimicrobial activity of the antimicrobial agent in the initial state and the antimicrobial activity of the six times incubation were compared. After 12 hours, the culture solution was aliquoted into 10ul aliquots and placed in a test tube containing 990ul of phosphate buffer solution.

division Subculture After six subcultures AI Example 4 1000 1000 1.0 Example 5 500 500 1.0 Example 6 250 250 1.0 Example 9 125 125 1.0 Example 10 1000 1000 1.0 Example 11 250 250 1.0 Example 15 125 125 1.0 Example 16 125 125 1.0 Example 17 250 250 1.0 Example 20 250 250 1.0 Example 21 125 125 1.0 Example 22 63 63 1.0 Example 26 125 125 1.0 Example 27 63 63 1.0 Example 28 63 125 1.0 Example 31 250 250 1.0 Example 32 125 125 1.0 Example 33 125 125 1.0 Example 37 63 63 1.0 Example 38 32 32 1.0 Example 39 32 32 1.0 Example 42 125 125 1.0 Example 43 125 125 1.0 Example 44 63 63 1.0 Erythromycin 150 1300 8.7

In Table 5, AI (Adaptation Index) = MIC after subculture and MIC before subculture. As shown in Table 5, the adaptation index (AI), that is, the resistance-induced resistance to natural antimicrobial agents prepared according to the present invention, did not cause resistance at all without changing the minimum inhibitory concentration even after 6 subcultures. In the case of mycin, the initial minimum inhibitory concentration was increased from 150ppm to 1,300ppm after the 6th passage, and the adaptation index was 8.7, indicating that the resistance-induced resistance was high.

Experimental Example 5

Examples 4 to 6, Examples 9 to 11, Examples 15 to 17, Examples 20 to 22, and Examples 26 to 28, which prepared an antimicrobial agent within the preferred range of the present invention based on the results of Experimental Example 3 Using the antimicrobial agents prepared in Examples 31 to 33, Examples 37 to 39, and Examples 42 to 44, the antioxidant effect was confirmed as follows, and the results are shown in Table 6 below.

First, blood collected from rabbits was centrifuged (8,000 rpm, 5 minutes), washed, and the obtained red blood cells were diluted with physiological saline to prepare a red blood cell suspension (60 million red blood cells / 4 ml). Nine 10 ml Pyrex test tubes with a diameter of 1.0 cm are prepared, and 4 ml of red blood cell suspension is added to each. One of the nine test tubes was added 50 μl of ethanol as a control and the remaining eight were added 50 μl of the antimicrobial sample prepared in Example as a treatment group and fused in the dark for 30 minutes. After the completion of the melting, add 50 μl of an aqueous solution of Hematoporphyrin (80 μM) as a photosensitizer, seal the inlet with parafilm, and install a 20 W fluorescent lamp in a box of 50 cm × 20 cm × 25 cm rectangular black painted inside. The tubes were arranged at a distance of 5 cm from the fluorescent tube and irradiated with light for 15 minutes. After light irradiation, the absorbance was measured at 700 nanometers at 15 minute intervals while leaving the test tubes in the dark. The increase in the transmittance of the red blood cell suspension at this wavelength is proportional to the hemolysis of the red blood cells. All experiments were carried out in a 27 ℃ constant temperature room, the activity of the sample to protect the cells from free radicals was defined as the time (minutes) for 50% of the red blood cells added under the above measurement conditions for photohemolysis.

Natural Antibacterial Antioxidant effect (min) Tocopherol 2.0% Ascorbic acid 3% 60 Natural Antibacterial Exclusion 35 Example 4 40 Example 5 50 Example 6 78 Example 9 125 Example 10 70 Example 11 90 Example 15 115 Example 16 135 Example 17 125 Example 20 140 Example 21 165 Example 22 130 Example 26 200 Example 27 160 Example 28 125 Example 31 150 Example 32 163 Example 33 170 Example 37 192 Example 38 200 Example 39 220 Example 42 200 Example 43 210 Example 44 220

In Table 6, the antioxidant effect is represented by the time required for each sample to hemolyze 50% of red blood cells (minutes), and as shown in Table 6, the natural antimicrobial agent prepared within the preferred range of the present invention is conventional 3 It can be seen that% ascorbic acid, 2% tocopherol and the residual amount have an antioxidant power of up to three times or more compared to the antioxidant consisting of active water.

Experimental Example 6

In order to determine the anti-aging activity of the antioxidant cell line of the present invention was carried out as follows and the results are shown in Figures 1 to 4. The cell lines used in the experiments were Bovine aortic endothelial cells (BAEC) and Bovine pulmonary artery endothelial cells (CPAE). Cells were cultured in a 5% CO ₂ incubator at 37 ° C. using Dubelcos Modify Eagle Medium (DMEM, GIBCO BRL, Grand Island, NY) containing 10% Petalcalp Serum (FCS, GIBCO BRL). Cell lines incubated for 24 hours were treated with 0.2 μM 4-hydroxy 2,3-nonenal (4-HNE) as an oxidant and cultured for 12 hours. After washing the cells well, the cells were cultured by removing the oxidant. It was again collected after 12 hours after 24 hours by adding 10 μM of the antioxidant according to the present invention (Example 4, Example 39). Afterwards, the cells were observed using a fluorescence microscope, and the use of the fluorescence microscope was performed using 100 ㎍ / ml acridine orange and 100 ㎍ / ml ethidium bromide as fluorescent dye binding dyes. Acridine orange stains the cytoplasm and is inserted into DNA, which is observed in green in living cells, and ethidium bromide appears in orange in dead cells. Stained cells were observed with a specular reflector microscope (Olympus, Japan), Figure 1 shows the cell state of the untreated control, Figure 2 shows the cell state after treatment for 12 hours using 4-HNE as the oxidant. 3 shows the cell state after 12 hours of treatment using an antimicrobial agent (Example 4) containing 0.001% by weight of DTI as an antimicrobial agent, and FIG. 4 shows an antimicrobial agent containing 1.0% by weight of DTIF as an antimicrobial agent (execution). Example 39) was used to show the cell state after 12 hours of treatment.

As shown in FIGS. 1 to 4, the cells treated with the oxidant 4-HNE show signs of apoptosis due to apoptosis in the cytoplasm. The apoptosis-inducing cells were treated with the anti-regulator according to the present invention. Results The cytoplasm was observed green, as was the case with the oxidant-free group. This shows aging inhibitory activity as a result showing that the dead cells killed by the oxidant has the activity of restoring cellular function by reactivating the antimicrobial agent according to the present invention.

Experimental Example 7

In order to confirm the anti-aging activity of the antimicrobial agent according to the present invention was carried out as follows and the results are shown in FIG. Western blotting was used to analyze bcl-2 proteins involved in aging. As a cell line, Bovine pulmonary artery endothelial cells (CPAE) were used, and the antioxidant according to the present invention after exposure to 4-hydroxy 2,3-nonenyl for 6 hours (Example 4, Example 39) Proteins were collected from cells by treatment with 10 μM and confirmed by Western blotting analysis. The cultured cells were collected and centrifuged and the protein was dissolved buffer solution (1% Triton X-100.50 mM TrisciCl, 150 mM sodium chloride, containing 1 μg / mL aprotinin and 100 μg / mL phenylmethyl sulfonyl fluoride (PMSF)). 0.2% sodium azide). Cell lysis was collected by centrifugation at 12,000 rpm for 10 minutes, and protein concentration was measured by a BS assay. Cellular proteins were separated by SD polyacrylamide electrophoresis (SDS-PAGE) and transferred to nitrocellulose transfer membrane. Schkim milk was added to Tris buffer solution at 4 ° C. to block non-specific binding by blocking the nitrocellulose membrane. The antibody was used by diluting Bcl-2 at 1: 1000 for the antibody. The secondary antibody was an anti-mouse IgG antibody. (1: 1000 dilution) was used. The immunocomplex thus formed was confirmed by an enhanced chemiluminescence search kit and the results are shown in FIG. 5. 5A shows the cell state of the untreated control group, B of FIG. 5 shows the cell state after treatment for 12 hours using 4-HNE as an oxidizing agent, and C of FIG. The cell state after treatment for 12 hours using an antimicrobial agent containing 4% by weight (Example 4) was shown, and D of FIG. The cell state after the treatment is shown.

As shown in FIG. 5, P21 protein related to aging was expressed in Bovine pulmonary artery endothelial cells (CPAE) treated with oxidizing agent 4-HNE (FIG. 5B). When treated with an antimicrobial agent according to the present invention it can be seen that the P21 protein expression is inhibited and also has anti-aging activity.

Experimental Example 8

Bacillus subtilis ATCC 6633, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, Aspergillus niger ATCC 9029, and Fusarium solani were cultured in the same manner as in Experimental Example 1, and then inoculated in a nutrient lotion formulation having the composition shown in Table 7 X10 ⁶ CFU / g. Since the increase and decrease of the number of bacteria was observed over time to examine the antiseptic power in the product, the results are shown in Table 8.

Comparative Example 1

The experiment was carried out in the same manner as in Experimental Example 8, but the antimicrobial agent was used as a mixed antibacterial agent (methyl paraben 2.0%, propyl paraben 1.0%, Germal115 2.0%) to examine the antiseptic force and the results are shown in Table 8.

Comparative Example 2

The experiment was carried out in the same manner as in Experiment 8, but the antiseptic power was examined without adding an antimicrobial agent and the results are shown in Table 8 below.

ingredient Composition ratio (%) Glyceryl stearate 1.50 Squalane 7.00 Mineral oil 7.00 Cetearyl alcohol 1.20 Sorbitan stearate 0.30 Polysorbate 60 1.00 Carbomer 0.12 Glycerin 10.00 Natural Antibacterial Dtyev 1.0 Baikalin 5.0 Deionized water Remaining amount

Natural Antibacterial At the time of inoculation 1 day later 3 days later 1 week later 2 weeks later 3 weeks later 4 weeks later Experimental Example 8 2.0 * 10 ⁶ 45 10 0 0 0 0 Comparative Experimental Example 1 2.0 * 10 ⁶ 3.0 * 10 ⁴ 5.0 * 10 ² 1.0 * 10 ² 10 0 0 Comparative Experiment 2 2.0 * 10 ⁶ 2.5 * 10 ⁶ 4.5 * 10 ⁶ 5.5 * 10 ⁶ 1.4 * 10 ⁷ 3.5 * 10 ⁷ 6.8 * 10 ⁷

As shown in Table 8, in the case of the natural antimicrobial agent according to the present invention, it can be confirmed that the antiseptic property is very excellent.

As described above, the present invention shows a wide range of antimicrobial spectrum, showing high antibacterial activity against resistant bacteria and fungi without inducing resistance even though natural antibacterial agent is produced from natural products, and not only suitable antioxidant function but also anti-aging activity It can be seen that it has a natural material. Therefore, it is expected to be widely applied as a multifunctional preservative in food, medicine, cosmetics and household goods and as a multifunctional natural antibacterial agent of anti-aging activity in mammals including humans.

As described above, the multifunctional natural antimicrobial agent according to the present invention is a bio-new material, and has antiseptic and antioxidant and anti-aging functions in foods, medicines, cosmetics, textiles, household goods, etc., thereby enhancing the safety and stability of quality and in mammals including humans. It is safe and does not induce resistance, and has adequate antioxidant and anti-aging functions, thereby treating and preventing infectious diseases.

Claims (5)

delete A natural antimicrobial agent comprising dithief (dimethoxy tetrahydroxy flavone) separated and purified from golden extract as a main active ingredient, wherein the content of the dithief is 0.001 to 1.0% by weight. The natural antimicrobial agent of claim 2 comprising 0.1 to 5.0% by weight of bicalin. The natural antimicrobial agent of claim 3 comprising a solvent selected from propylene glycol, glycerin, 1,3-butylene glycol, water, and alcohol. The natural antimicrobial agent according to claim 4, comprising 10% by weight of one kind of herbal extract selected from high ginseng extract, sterling silver extract, rhubarb extract, fragrance extract, sesame seed extract, gallola extract, garlic extract or licorice extract.