KR101070805B1 - A composition for prevention or treatment of atopic dermatitis - Google Patents

Abstract

The present invention relates to a composition for preventing or treating atopic dermatitis, and more specifically, including 80 to 90% by volume of deep seawater, 5 to 15% by volume of honey, 0.4 to 0.6% by volume of vinegar, and 0.5 to 1.0% by volume of chitosan. As a composition consisting of, it exhibits inhibitory activity against bacteria and fungi involved in secondary skin infection, and exhibits atopic dermatitis effect on atopic dermatitis mice, and can be usefully used for preventing or treating skin diseases such as atopic dermatitis.

Deep sea water, honey, vinegar, chitosan, antibacterial, antifungal, atopy, skin diseases

Description

A composition for prevention or treatment of atopic dermatitis

The present invention relates to a composition for preventing or treating atopic dermatitis, and more particularly, showing inhibitory activity against bacteria and fungi involved in secondary skin infection, including deep seawater, honey, vinegar and chitosan, and inducing atopic dermatitis. The present invention relates to a composition exhibiting an atopy-releasing effect on mice.

Bacterial infections are one of the most common and fatal causes of human disease. Unfortunately, the abuse of antibiotics has resulted in antibiotic resistance of bacteria. Indeed, the rate at which bacteria are resistant to new antibiotics is much faster than the rate at which new antibiotic analogs are developed. For example, which may pose a threat to life, Enterococcus faecalis kusu nose (Enterococcus faecalis), Mycobacterium-to M. tuberculosis (Mycobacterium tuberculosis) and Pseudomonas her rugi bacterial species such as labor (Pseudomonas aeruginosa) have raised resistance to all antibiotics known to date.

Tolerance to antibiotics is a distinct phenomenon from resistance to antibiotics. In the 1970s, Pneumococcus sp . Was first discovered and provided important clues about the mechanism of action of penicillin. Tolerant species stop growing in the presence of the usual concentration of antibiotics but do not die as a result. Resistance is due to the absence of the activity of bacterial autolytic enzymes such as autolysin when antibiotics inhibit cell wall synthase, which in the case of penicillin activates endogenous hydrolytic enzymes. This kills the bacteria, but also inhibits the activity of the enzyme, resulting in survival during antibiotic treatment.

It is clinically very important for bacteria to be resistant to antibiotics, because the inability to eradicate resistant bacteria makes antibiotic treatment less effective in clinical infections. In addition, resistance is regarded as a prerequisite for bacterial resistance to antibiotics, because strains survive even antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow even in the presence of antibiotics. Indeed, all bacteria that are resistant to antibiotics are known to be resistant to antibiotics, and therefore, new antibiotics that can kill antibiotic resistant bacteria are needed.

On the other hand, atopic dermatitis is a recurrent chronic dermatitis with severe itching, and about 10-15% of children have atopic dermatitis, and 90% have reported spontaneously improving within 5 years. Such atopic dermatitis generally improves in adulthood, and about 30-40% of the dermatitis does not reappear in appearance, but the rest is known to cause severe dermatitis such as skin dryness, skin irritation caused by irritant substances, and housewife eczema. This ratio is increasing with the progress of industrialization and the increase of environmental pollution.

Dry and sensitive skin has low water retention and recovery ability, and thus, skin keratinization, itching, etc. tend to appear, and in severe cases, dermatitis such as atopic dermatitis develops. Several compositions have been proposed as a method for treating skin dryness and itching, such as atopic dermatitis, but all of these compositions are pharmaceutical compositions and most of them are applied to the skin for a long time since they contain steroid preparations or antibiotics as active ingredients. May cause serious side effects such as capillary dilator and / or thickening of the stratum corneum, or dilating. The gamma-linolenic acid (Korean Patent Laid-Open Publication No. 2000-0046633), which is widely used to alleviate atopic dermatitis, is the main ingredient. Since the composition is easily oxidized, the stability is inferior, and the skin irritation is relatively strong, making it difficult to use for sensitive skin.

In recent years, attempts have been made to treat and prevent atopic dermatitis from natural materials.A representative conventional techniques include atopic dermatitis cosmetic composition (Korean Patent Registration No. 10-0377262), herbal medicine for atopic dermatitis. Cosmetic composition (Korean Patent Registration No. 10-0517465), cosmetic composition for treating atopic dermatitis (Korean Patent Registration No. 10-0451444), functional food for treating atopic dermatitis (Korean Patent Registration No. 10-0454752), There was a composition for preventing or treating atopic dermatitis, a method for producing the same (Korea Patent Registration No. 10-0483539), a therapeutic agent for atopic skin disease containing herbs and a method for manufacturing the same (Korea Patent Registration No. 10-0597997), and the like. .

The inventors have been paying attention to deep sea water, honey, vinegar and chitosan while searching for natural materials as described above.

Deep sea water is pure water in the deep sea that is not exposed to sunlight. In general, more than 200 meters deep is divided into surface waters and less than deep seas. Deep sea water exists only in the deep sea because of the sedimentation between temperature and salinity. Deep sea water is known to cycle through the Atlantic, Indian and Pacific oceans every 4,000 years. It is characteristic of deep sea water because it is difficult to grow or reproduce microorganisms because it is pulled up from low temperature to ultra high pressure and rich in minerals. There are several steps involved in making deep ocean water human drinking water. The deep ocean water withdrawn is first subjected to a water quality analysis room that examines the components and bacteria. Afterwards, the sterilization chamber equipped with a 15-20μ air circulation system that does not pass through germs passes through UV sterilization to become drinking water. It removes inedible substances such as sodium and is processed into water that leaves only the minerals needed by humans.

In particular, experts believe that the deep seawater in the East Sea has better water quality than that of other oceans. For example, the deep sea water temperature of the East Sea is 0.2 ° C on average, which is lower than the deep sea water of 2 ° C in other oceans. The core of the development technology is the 'membrane' technology called membrane. It is a high-tech technology that leaves only minerals that are beneficial to the body, excluding unnecessary components such as salt in deep sea water. As much as it is difficult, only five countries have succeeded in developing deep sea water, including Korea, the United States, Japan, Norway and Taiwan.

Deep sea water is not only used as drinking water but also as a raw material in various fields such as medicine, cosmetics and household goods. Deep seawater contains little pathogens, maintains stable low temperatures throughout the year, and is a seawater resource that has been aged for a long time. That is, deep ocean water is very useful because of its cleanliness, low temperature stability, and rich nutrition. "The US FDA's stability tests have shown that deep sea water is not harmful to the body. Rather, experiments have shown benefits. Animal testing has shown that deep sea water calms atopy. Japan has published the same findings. In addition, experimental results showing that deep sea water is effective in preventing cancer such as breast cancer, liver cancer and rectal cancer. Of course, the hardness should be about 700 mg / L.

Hardness is a quantified amount of magnesium and calcium contained in 1 liter of water. Normal hardness is less than 100 mg / L is called soft water or sweet water. If it is 1,100-1,300 mg / L, it is called hard water, and if it is 310 mg / L or more, it is called hard water or hard water. Higher hardness means more minerals. However, the higher the hardness, the bitter taste of magnesium. The most delicious water has a hardness of 50mg / L, which is about this, and groundwater is 60-100mg / L. The depth of deep sea water is 6,000 ㎎ / L, so it cannot be drunk immediately. Desalting must be done to remove the salt. At this time, the mineral component is also removed. The salt-free minerals are then injected back into deep sea water. In the process, the hardness of the water is lowered to 150 mg / L, which makes it possible for humans to drink.

Indigenous honey is literally honey made by the native wild bees of Korea freely flying in the mountains and making honeycombs between stones or trees. Native honey, which obtains honey in one place, may be unable to drop even one year due to bad weather.

Indigenous bees are pure Korean native bees that have been domesticated in oriental traditions, and differ from bee bees strictly in the West. Jirisan native honey is pulverized only once a year after the first frost, and is contained in naturally occurring native bees that eat herbs and wildflowers that bloom in spring in the pollution-free area of the highlands over 1,800 meters above sea level. It contains a lot of royal jelly, propolis, pollen and beeswax, which is effective in preventing various adult diseases and treating atopic dermatitis, and is an essential health nutrition food for modern people.

In order to collect 1kg of honey, bees have to make 5.6 million wings, collect the collected native honeycomb honey and drop it back to the traditional method to extract moisture and concentrate to produce pure honey. The color is dark chestnut, slightly different from the color of honeycomb honey, because beeswax honey removes beeswax and moisture.

Since the main components of honey are absorbed into glucose and fructose without digestion, chronic gastrointestinal disease, relieving asthma, bronchitis, sputum, neuron gastrointestinal disease, hepatitis, nervous breakdown, pulmonary tuberculosis, constipation, forgetfulness (presented by the Seoul National University Institute of Medicine), If you continue to eat honey, your face will look like a flower, and the longer you eat, the better, "Nothing in the duodenum". It is well suited to the weak constitution, it is known to grow strong children and good for high blood pressure.

Chitosan is a β-1,4 conjugated pyranose unit of glucoseamine (glucosamine), and is a natural polymer having a molecular weight of 1 million or more in which 5,000 or more glucoseamine residues are bound. This chitosan can be extracted from aquatic systems including crustaceans such as crab shells and shrimps and squids, and its molecular structure is similar to that of cellulose, a kind of polysaccharides. Since it does not occur, it has been applied to the pharmaceutical industry, and recently certified as a food by the US FDA, chitosan has been applied as an important bioindustry and biomedical material of the 21st century.

In particular, the "water-soluble free amine chitosan" disclosed in Korean Patent Registration No. 10-0441270 may be usefully used for a functional product using a strong positive charge of the free amine of chitosan itself.

On the other hand, vinegar is generally known to be good for fatigue recovery, to maintain the blood alkaline to cause the action to clear the blood, as well as effective in skin care. Such vinegar is a fermented food that has been used for a long time regardless of the East and West, and the consumption of vinegar using various fruits and grains is increasing rapidly. Moreover, in recent years, health promotion effects, including regulation of metabolism in the body, have been reported, and vinegar diversification and high quality are apparent.

Brown rice is rich in dietary fiber, calcium, iron, and vitamins such as thiamin and riboflavin, and is widely used as a health food to prevent adult diseases such as arteriosclerosis and diabetes.

In addition, brown rice vinegar fermented brown rice is a typical traditional fermented food that has been used in cooking by manufacturing a variety of vinegar from the traditional method in each family in Korea.

Some of the ingredients are known to be effective in atopic dermatological diseases, but there is no known atopy product having a synergistic effect by mixing them all in an appropriate ratio.

Accordingly, the inventors conducted experiments on the natural substances, and found that a mixed composition of marine deep water, honey, vinegar, and low molecular weight water-soluble free amine chitosan is a pathogenic bacterium that causes secondary skin infection in atopic patients. The present invention was completed by confirming that it has an antibiotic effect against fungi and shows a therapeutic effect in atopic dermatitis-induced rats.

An object of the present invention to provide a composition for preventing or treating atopic dermatitis.

In order to achieve the above object, the present invention provides a composition for preventing or treating atopic dermatitis comprising 80 to 90% by volume of deep sea water, 5 to 15% by volume of honey, 0.4 to 0.6% by volume of vinegar, and 0.5 to 1.0% by volume of chitosan. to provide.

The present invention relates to a composition for preventing or treating atopic dermatitis comprising 80 to 90% by volume of deep sea water, 5 to 15% by volume of honey, 0.4 to 0.6% by volume of vinegar, and 0.5 to 1.0% by volume of chitosan. It exhibits inhibitory activity against bacteria and fungi involved in secondary skin infections, and exhibits atopic dermatitis effect on atopic dermatitis mice, and can be usefully used for preventing or treating skin diseases such as atopic dermatitis.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing or treating atopic dermatitis, comprising 80 to 90% by volume of deep sea water, 5 to 15% by volume of honey, 0.4 to 0.6% by volume of vinegar, and 0.5 to 1.0% by volume of chitosan. .

The deep sea water in the composition of the present invention is water collected at a depth of 200 meters or less, which means that the purified water is filtered 2-3 times. Although commercial deep sea water may be used, it is preferable to use deep water having a hardness of 50 to 300 mg / L.

Honey of the composition of the present invention can be used as commercial honey, rapeseed honey, acacia honey, chestnut honey, miscellaneous honey, honey honey, etc., preferably native honey.

The vinegar in the composition of the present invention may be any of synthetic vinegar, alcohol vinegar, natural vinegar, etc., but it is most preferable to use brown rice vinegar. The brown rice vinegar contains 5 to 10% by weight of brown rice, it is preferable to use a total acid content of 6.0 to 7.0% (w / v).

Chitosan in the composition of the present invention can be used commercially available chitosan products, preferably it is preferred to use a low molecular weight water-soluble free amine chitosan (LMWSC) prepared by the manufacturing method disclosed in Korea Patent Registration No. 10-0441270. .

The low molecular weight water soluble free amine chitosan is prepared by the following method.

That is, 1) treating a solution of an organic or inorganic acid salt of chitosan oligosaccharide with trialkyl amine which is the base;

2) recovering the chitosan oligosaccharide from which the organic or inorganic acid bound to the chitosan oligosaccharide is removed in the form of the trialkyl amine salt by adding an organic solvent to the solution;

3) The acid-free chitosan oligosaccharide solution from which the acid is removed can be purified by an activated carbon / ion exchange resin column to prepare a water-soluble free amine chitosan having a molecular weight of 1,000 to 100,000 Daltons. have.

Looking at the manufacturing method in more detail,

Insoluble chitosan extracted from chitin is dissolved in the form of a salt using an organic acid including lactic acid, acetic acid, propionic acid, formic acid, ascorbic acid, and tartaric acid and an inorganic acid including hydrochloric acid, nitric acid and sulfuric acid. The chitosan solution is enzymatically digested to obtain chitosan oligosaccharides.

The acid solution of the chitosan oligosaccharide is a solvent used to prepare a solution of an organic acid salt including lactic acid, acetic acid, propionic acid, formic acid, ascorbic acid, and tartaric acid and an inorganic acid salt including hydrochloric acid, nitric acid, and sulfuric acid. ) Is selected from groups 7.0, 7.2 and 7.4 to dissolve.

In order to effectively remove the form of the organic or inorganic acid salt contained in the chitosan oligosaccharide, trialkyl amine is added in a ratio of 2 to 3 equivalents to 1 equivalent of the amine group of the chitosan oligosaccharide, preferably 2 equivalents.

The quantitative ratio of the trialkyl amine added above is that the organic or inorganic acid in the amine group at the C-2 position in one unit of the chitosan oligosaccharide is removed in the form of the trialkyl amine salt, and protects the -CH 2 OH group at the C-6 position. It is necessary for the role. To be more specific, in the case of the process to dissolve the water-insoluble chitosan in organic or inorganic acid the C-2 position lactates -NH ₃ + and CH ₃ for CHOHCOO-, acetates -NH ₃ + and CH ₃ COO-, if acid salt -NH ₃ + and CH ₃ CH ₂ COO-, -NH ₃ + in the case of formic acid and HCOO-, for ascorbic acid and -NH ₃ + CO (COH) ₃ CHOHCH ₂ O-, and if the tartrate -NH ₃ + and HOOC (CHOH ₂₎ COO-, and in the case of a mineral acid hydrochloride -NH ₃ + and Cl-, if the sulfate -NH ₃ + and HSO ₄ - in, and -NH ₃ + and nitrate NO ₃ - are in the form . At this time, when trialkyl amine is added, trialkyl amine attracts H + from the amine group of strongly acidic chitosan oligosaccharide, and electrostatic interaction between CH ₃ CHOHCOO- or CH ₃ COO-, Cl- and trialkyl amine By binding to and removing the C-2 position to obtain the free amine form.

Said tree is an alkylamine, preferably a tree, -C _{1 ~4} alkylamine, characterized in that the specifically selected from the group consisting of trimethylamine, triethylamine, tripropylamine, tri-isopropyl ethyl amine, and tributyl amine More preferably, triethyl amine is used.

The reaction product is reacted at room temperature for 2 hours, and then stirred by adding an organic solvent selected from acetone, methanol, chloroform and dichloromethane group, followed by centrifugation.

After the above process, -CH ₂ OH group in the C-6 position protected by trialkyl amine is removed by treatment with 0.0005 ~ 0.010 N inorganic acid, the inorganic acid can be selected from the inorganic acid consisting of hydrochloric acid, nitric acid or sulfuric acid can be used. have. Preferably, 0.001 N hydrochloric acid is used, which is removed in the form of (C ₂ H ₅ ) ₃ NH + .Cl- salt. The present invention also purifies a solution of chitosan oligosaccharides from which organic or inorganic acids have been removed with an activated carbon / ion exchange resin column to produce pure water soluble free amine chitosan.

In the composition of the present invention, the deep ocean water is preferably used in an amount of 80 to 90% by volume, and has a disadvantage in that the effect of improving atopy is lowered at 80% by volume or less.

In addition, honey is preferably used in the range of 5 to 15% by volume, there is a disadvantage that the activity is lowered when the volume is less than 5% by volume, there is almost no change in activity when more than 15% by volume.

The composition of the present invention prevents or treats atopic dermatitis by exhibiting antimicrobial or antifungal activity.

The antimicrobial activity of Staphylococcus aureus (Staphylococcus aureus ), Bacillus subtilis subtilis ), Pseudomonas aeruginosa ) and E. coli ( Esherichia coli ) is an inhibitory activity against any one selected from the group consisting of, the antifungal activity is Candida albicans ( Candida albicans ) or Trichosporon beigelli .

The composition of the present invention showed a minimum growth inhibitory concentration (MIC) of 1.25 to 5.0 μg / mL against bacteria, showed excellent activity compared to cepypyramid (see Table 2), and 5 to 10 μg / mL against fungi By indicating the minimum growth inhibitory concentration of (see Table 3), it can be used to treat skin diseases such as atopy, eczema, vaginitis, dermatitis, dandruff or athlete's foot.

In addition, as a result of in - vivo experiments, the atopy-induced mice were treated with the mixed composition of the present invention to obtain a sensory evaluation result close to that of normal mice (see FIG. 10), and showed an effect of reducing the content of IgE (FIG. 15). As a result of histology, it has been shown to alleviate inflammation (see FIGS. 16 and 17), which can be used very effectively to treat or ameliorate atopic dermatitis.

The composition of the present invention may be used in various administration methods from the viewpoint of those skilled in the art, but most preferably, the composition may be used as an external agent for applying to the affected part of the skin.

Hereinafter, the present invention will be described in detail with reference to specific embodiments, but the scope of the present invention is not limited to these embodiments.

Example  1: Preparation of skin external composition

To prepare the external skin composition, deep sea water, native honey, brown rice vinegar and low molecular weight low molecular water-soluble chitosan (LMWSC) were selected. Deep sea water with a hardness of 300 mg / L was purchased from Chito Life Co., Ltd., and native honey is a home-made product. Its main components are water 19.0 ~ 20.5%, fructose 36.0 ~ 41.5%, glucose 26.0 ~ 38%, water 3.0-7.0% cross-acid, 0.1-0.2% organic acid and 0.13-0.45% ash, with vitamin B1 (1.56 mg), B2 (1.33 mg), nicotinic acid 11.6 mg, pantothenic acid 1.73 mg, B6 (0.88 mg), biotin 0.6 Honey containing mg and folic acid 1.56 mg was used. Brown rice vinegar is purchased from Chito Life Co., Ltd. and contains total acid content of 6.0 ~ 7.0% (w / v) and brown rice 8% by weight. Other ingredients include alcohol, maltose, oligosaccharide, citric acid. Brown rice vinegar was used.

On the other hand, the low molecular weight water-soluble free amine chitosan (LMWSC) used a low molecular weight water-soluble free amine chitosan prepared by the method of the present inventors registered patent (No. 10-0441270). The components were mixed in the following amounts to prepare a skin external blend composition.

Composition ratio of external skin composition ingredient Content (% by volume) Usable content range Deep ocean water 88.65 80-90 Native Honey 10 5-15 Brown Rice Vinegar 0.55 0.4-0.6 LMWSC 0.8 0.5-1.0

Experimental Example  1: Determination of antimicrobial activity of the external composition for skin of the present invention

The antibacterial activity of the composition for external application of skin prepared in Example 1 was measured.

Fungus, preparation of antibiotics

Bacteria to be used for antimicrobial activity are Gram-positive bacteria, Staphylococcus aureus KCTC 1621 and Bacillus subtilis , which are involved in secondary infections of the skin such as atopy and eczema and in respiratory diseases such as bronchus. KCTC 1918) was selected as Pseudomonas aeruginosa KCTC 1637 and Escherichia coli KCTC 1682, which are involved in urinary tract infections, enterotoxicity and dermatitis as gram-negative bacteria, and Korea Research Institute of Bioscience and Biotechnology (KCTC). Received from. Among the bacteria, Escherichia coli and Bacillus subtilis were cultured in LB medium, Pseudomonas aeruginosa in NB + 0.5% NaCl medium, and Staphylococcus aureus in TSB medium until the mid-log phase. It was.

As a positive control, cefpiramide, which is widely used as a cephalosporin-based third generation antibiotic, was used.

Concentration and Dilution of Mixed Compositions

After filtering the external composition of Example 1 with a 0.22 μm filter, Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem. 72. 248-254) was used to measure the concentration. Specifically, 800 μL of sterilized tertiary distilled water and 200 μL of a Bradford solution (1 liter reference content: Coomassie brilliant blue G250 0.1 g / 50 mL ethanol, phosphoric acid 100 mL, tertiary distilled water 850 ml) were added, and then 10 μL of the mixed composition of the present invention was added. Added. The control group was prepared by adding only 800 μL of distilled water and 200 μL of the Bradford solution without adding the mixed composition of the present invention. The OD value was measured at a wavelength of 595 nm using a spectrometer, and the quantification was calculated using the following formula.

When OD ₅₉₅ <0.241, Quantitative Concentration (μg / mL) = (2.8136 ＊ OD ₅₉₅ ) -0.0146

OD ₅₉₅ 0.241, quantitative concentration (μg / mL) = (11.702 * OD ₅₉₅ ) -2.1208

Based on the measured results, the mixed composition of the present invention was diluted stepwise in a 96-well microtiter plate (Nunc) using 1% Bactopeptone.

Antimicrobial Activity

The antimicrobial activity of the mixed composition of the present invention was measured as follows. In 96-well plates, the mixed composition of the present invention and the ceftyramide antibiotic, a positive control, were diluted by concentration. 100 μL (number of bacteria per well: 2 × 10 ⁵ CFU / mL) was added to each well of a medium containing bacteria to measure antimicrobial activity, and then left at 37 ° C. overnight. Negative control was performed in the same manner without adding the mixed composition or antibiotic of the present invention. E. coli and Bacillus subtilis cultured overnight were plated in LB agar medium, Pseudomonas aeruginosa in NA + 0.5% NaCl agar medium, and Staphylococcus aureus were incubated for 12 hours to confirm growth. It was.

Measurement result

Measurement results are shown in FIGS. 1 to 4.

Figure 1 shows the antimicrobial results for Gram-positive bacteria Staphylococcus aureus, A is a negative control, B is added to the mixed composition of the present invention at 5 μg / mL, C is a mixed composition of the present invention Was added at 5 μg / mL, D represents 5 μg / mL of ceftyramide which is a positive control, and E shows 2.5 μg / mL of ceftyramide. As a result, it was found that Staphylococcus aureus inhibited growth when 5 μg / mL of the mixed composition was added, and 5 μg / mL of Sefpyramid, a positive control, was inhibited.

Figure 2 shows the antimicrobial results for gram positive bacteria Bacillus subtilis, A to E are the same as shown in Figure 1 above. As a result, Bacillus subtilis inhibited growth when 5 μg / mL and 2.5 μg / mL were added to the mixed composition, and growth was inhibited when 5 μg / mL and 2.5 μg / mL were added to ceftyramide, a positive control. Could.

Figure 3 shows the antimicrobial results for E. coli, Gram-negative bacteria, A to E are the same as shown in FIG. As a result, E. coli inhibited growth when 5 μg / mL and 2.5 μg / mL were added to the mixed composition, and growth was inhibited when 5 μg / mL and 2.5 μg / mL were added to the ceftyramide, a positive control. .

Figure 4 shows the antimicrobial results for gram negative bacteria Pseudomonas aeruginosa, A to E are the same as shown in Figure 1 above. As a result, Pseudomonas aeruginosa inhibited growth when 5 μg / mL and 2.5 μg / mL were added to the mixed composition, and 5 μg / mL was inhibited when cephipyamide, a positive control, was added.

From the above results, it was possible to determine the minimum growth inhibitory concentration (MIC) for various bacteria of the external composition of the present invention and antibiotics, summarized in Table 2 below.

Minimum Growth Inhibition Concentration (MIC) against Bacteria of the Mixed Composition and Antibiotic (positive Control) of the Present Invention Mixed composition (μg / mL) Ceftyramide (μg / mL) S. aureus 5 5 B. subtilis 1.25 2.5 E. coli 2.5 2.5 P. aeruginosa 2.5 5

Taken together, the external composition for skin of the present invention exhibits antimicrobial activity similar to that of the positive control (antibiotic) against bacteria causing skin secondary infection, thereby preventing skin diseases such as atopy and eczema. Or may be usefully used for treatment.

Experimental Example  2: Determination of antifungal activity of the mixed composition of the present invention

The antifungal activity of the mixed composition prepared in Example 1 was measured.

Fungus, preparation of antibiotics

Fungi for antifungal activity include Candida albicans KCTC 7121, which causes secondary infections in women's vaginitis and dermatitis, and Trichosporon beigelli KCTC 7707, which causes dandruff and athlete's foot, called seborrheic dermatitis. ) Was selected by the Korea Biotechnology Research Institute (KCTC). As a fungal culture medium, YPD medium was used. As a positive control, clotrimazole, an antifungal antibiotic used as an ointment for fungal skin infections, was used.

Antifungal Activity Measurement

Using the YPD medium, 50 μL of the mixed composition and the antibiotics were added to each well and diluted in stages.

The fungal water was dispensed into 50 μL of YPD medium containing fungi so as to be 2 × 10 ⁴ cells per well on a 96-well microtiter plate, and left in a 28 ° C. incubator for 24 hours. Then, YPD agar was plated on the medium, and growth was measured.

Measurement result

The measurement results are shown in FIGS. 5 to 6.

Figure 5 shows the antifungal results for Candida albicans, A is a negative control, B is added 10 μg / mL of the mixed composition, C is a 5 μg / mL of the mixed composition, D is a negative control The addition of DMSO to phosphorus fungi shows the result of the addition of 0.63 μg / mL of clotrimazole, a positive control. As a result, the growth of Candida albicans was inhibited by the addition of 10 μg / mL and 5 μg / mL of the mixed composition, and the addition of 0.63 μg / mL was inhibited by the positive control clotrimazole.

Figure 6 shows the antifungal results for tricosphorone Beigelli, A to E are the same as shown in FIG. As a result, it was found that the growth of tricosphorone Beige was slightly inhibited when the mixed composition of 10 μg / mL concentration was added, and that 0.63 μg / mL was inhibited when the positive control clotrimazole was added.

From the above results, it was possible to determine the minimum growth inhibitory concentration (MIC) for the various molds of the mixed composition and antibiotic of the present invention, summarized in Table 3 below.

Minimum growth inhibitory concentration (MIC) against fungi of the mixed composition and antibiotics (positive control) of the present invention Mixed composition (μg / mL) Clotrimazole C. albicans 5 <0.63 T. beigelli 10 <0.63

Taken together, the mixed composition of the present invention exhibits antifungal activity similar to that of a positive control (antibiotic) against fungi causing skin secondary infection, such as vaginitis and dermatitis, dandruff or athlete's foot. It may be used to prevent or treat a disease.

Experimental Example  3: Measurement of cytotoxicity of the mixed composition of the present invention

Cytotoxicity of the mixed composition of the present invention was measured using HaCaT cells, which are human skin cells, human keratinocyte cells. The HaCaT cells were distributed from Seoul National University Cell Bank.

Cultured in RPMI-1640 culture medium containing the cells, 10% FBS (fetal bovine serum), and insert the herb honey and antibiotics was stepwise diluted with a 96-well plate, and thus the cells per well each 2 × 10 ³ Dogs were mixed and incubated at 37 ° C for 24 hours. 20 μL of MTT solution (5 mg / mL MTT in phosphate-buffered saline) was added thereto, followed by incubation at 37 ° C. for 4 hours, and then DMSO solution was added to dissolve the resulting MTT-formazan product. The absorbance was then measured at 570 nm using an ELISA reader.

The measurement results are shown in FIG. 7. A is the cytotoxicity rate (%) according to the concentration of the mixed composition of antibiotics clotrimazole and the present invention, B is the cytotoxicity of clotrimazole by MTT solution, C is the cytotoxicity of the mixed composition by MTT solution It is shown. As shown in FIG. 7, the positive control of clotrimazole showed high cytotoxicity while the mixed composition of the present invention showed no cytotoxicity at all. From the above results, the mixed composition of the present invention turned out to be a very safe material.

Experimental Example  4: Analysis of the effect of the mixed composition of the present invention on bacterial and fungal cells

After each bacteria and fungi were grown to a log-phase in the medium, the cells were resuspended at 2 × 10 ⁵ CFU / mL for fungi and 2 × 10 ⁴ CFU / mL for fungi, and then the mixed composition of the present invention was used at 37 ° C. for 12 hours at MIC concentration. The reaction is carried out for 24 hours at 28 ℃ in the case of fungi. The control was reacted without the composition of the present invention. Then fixed with 2% glutaraldehyde (glutaraldehyde) at 4 ℃ 12 hours. The reaction control group and the experimental group were dehydrated using 50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol. After dehydration, the mixture was washed with 1% osmium tetroxide containing 5% sucrose and freeze-dried to coat with gold. Each sample was then analyzed using a scanning electron microscope (HITHACHI S-2400 SEM). The analysis results are shown in FIGS. 8 and 9.

8A shows E. coli itself without treatment of the mixed composition of the present invention, FIG. 8B shows the result of treating the mixed composition of the present invention with E. coli, and FIG. 8C shows Staphylococcus aureus without the treatment of the mixed composition of the present invention. 8D itself shows the result of treating Staphylococcus aureus with the mixed composition of the present invention. As a result, the gram-positive bacteria S. aureus and the gram-negative bacteria E. coli were found to be distorted or destroyed when compared to the negative control.

9A shows Candida albicans itself without treatment of the mixed composition of the present invention, and FIG. 9B shows the result of treating Candida albicans with the mixed composition of the present invention. As a result, the treatment group treated with the mixed composition of the present invention showed that more fungal cells were destroyed or distorted than the negative control group.

Experimental Example  5: mixed composition of the present invention in - vivo  Atopic dermatitis treatment effect verification (1)

Induced atopic dermatitis and treatment of the mixed composition of the present invention

In order to demonstrate the atopic dermatitis treatment effect of the mixed composition of the present invention was applied to the atopic dermatitis induction model was compared by applying the external composition of the skin of the present invention.

The experimental animals were purchased from NC / Nga mice (5 weeks old, 30 males) from the central experimental animals and acclimatized under conditions of 23 ± 3 ° C. and 55 ± 15% for one week. Feed was freely fed Feedlab Formula M-07 (Central Experimental Animal), and drinking water was also freely consumed using a water bottle. One week later, the NC mice were depilated and treated with 1% DNCB (dinitrochlorobenzene; 150 μL / dorsal area in AOO) (where AOO is acetone: olive oil = 3: 1). Thereafter, 0.2 to 0.4% DNCB (150 μL / dorsal area in AOO) was treated for 5 weeks to induce atopic dermatitis. The DNCB treatment was discontinued on the first day of week 7, and then the mixed composition of the present invention was applied twice a day for 5 days to the back of the mouse. As a drug control, 1% pimerochlorimus cream (Novatis) was applied in the same manner.

To summarize the experimental group and the control group, a control drug pimecrolimus 1% cream was added to the negative control (negative control), atopic dermatitis-induced positive control, atopic dermatitis-induced positive control that did not induce atopic dermatitis The applied drug control (drug control), atopic dermatitis-positive control group is divided into experimental groups coated with the mixed composition of the present invention.

Sensory evaluation results

The control group and the experimental group were evaluated using a clinical visual evaluation method commonly used. The visual evaluation results are expressed as the total sum of the scores of the five items in the table below, and the dermatitis score was determined by consultation with two or more experts.

Dermatitis Score none weakness Severity Severe Redness, bleeding 0 One 2 3 Formation, Dry 0 One 2 3 edema 0 One 2 3 Tissue damage 0 One 2 3 Tissue damage 0 One 2 3

The score can be from the lowest 0 to 15 points, the comparison between the control group and the experimental group on the obtained data was tested for the difference between the groups using the student's t- test .

Atopic scores for each of the control and experimental groups as a result of sensory evaluation are shown in FIG. 10, and specific pictures are shown in FIGS. 11 to 14. FIG. 11 shows a negative control, FIG. 12 shows a positive control, FIG. 13 shows a drug control, and FIG. 14 shows an experimental group.

Referring to the graph of FIG. 10, the atopic dermatitis-induced positive control group had an atopy score of 11.00, whereas the control group applied the control drug was confirmed that the atopic was reduced to 4.70. Furthermore, in the case of the mixed composition of the present invention, an atopy score of 2.50 was found to have a much better effect than the control drug. Since the atopy score of the negative control group that did not induce atopy was 2.10, it was confirmed that the treatment of the mixed composition of the present invention was almost close to the normal rat, from which the composition of the present invention was determined to be very effective for the treatment of atopy.

Experimental Example  6: mixed composition of the present invention in - vivo  Atopic dermatitis treatment effect verification (2)

IgE is an antibody that induces an allergic reaction by binding to a receptor on the surface of mast cells or white blood cells (basophil) .IgE secretes histamine when cells are activated due to the binding of IgE. The reaction will take place. People with atopic dermatitis usually have high serum IgE levels (ng / mL) and are sensitive to various antigens in their surroundings.The more severe the skin symptoms, the higher the levels of IgE (ng / mL) and relief of skin symptoms. Is associated with a decrease in IgE levels.

In this experiment, the effect of the mixed composition of the present invention on serum IgE levels (ng / mL) was examined to examine the effect of atopic dermatitis.

Serum was collected from each of the control and experimental groups of Experimental Example 5, and then the IgE content was measured, and the measurement results are shown in FIG. 15. As can be seen in Figure 15, there was no value close to the negative control, of which the mixed composition of the present invention was found to be effective in reducing the IgE content showing the lowest value. Furthermore, the compounds of the present invention were superior to the drug control. From the above results, it was confirmed that the compound of the present invention acts to alleviate atopic dermatitis.

Experimental Example  7: Mixed composition of the present invention in - vivo  Validation of atopic dermatitis treatment effect (3)

The histological examination was performed on the dorsal tissue of the control group and the experimental group of Experimental Example 5. Each result is shown in FIGS. 16 and 17.

As shown in FIG. 16, in the atopic dermatitis-induced positive control group, atopic symptoms were reduced in skin lesions (mild spongiosis) and inflammation along with epidermal hyperplasia and parakeratosis. It was accompanied by infiltration of cells (inffiltration with leukocytes) and decreased in the case of drug control group, but showed inflammation and thick stratum corneum.

In FIG. 17, although some inflammation remains in the mixed composition of the present invention, this degree of inflammation is a level of inflammation that appears in a negative control group (normal group), and the mixed composition of the present invention has an effect of alleviating dermatitis. It could be confirmed.

As can be seen from the experimental examples, the mixed composition of the present invention showed a dermatitis-improving effect on the positive control in appearance evaluation, IgE levels and histology, and in particular, it was much superior to the control drug. In addition, it can be very useful for treating or improving atopic dermatitis.

1 is a plate photograph showing the effect of the mixed composition of the present invention on the growth of Gram-positive bacteria Staphylococcus aureus.

Figure 2 is a plate photograph showing the effect of the mixed composition of the present invention on the growth of gram positive bacteria Bacillus subtilis.

Figure 3 is a plate photograph showing the effect of the mixed composition of the present invention on the growth of E. coli, Gram-negative bacteria.

Figure 4 is a plate photograph showing the effect of the mixed composition of the present invention on the growth of Pseudomonas aeruginosa Gram-negative bacteria.

Figure 5 is a plate photograph showing the effect of the mixed composition of the present invention on the growth of the fungus Candida albicans.

Figure 6 is a plate photograph showing the effect of the mixed composition of the present invention on the growth of the fungus Tricosphorone bergelli.

Figure 7 shows the cytotoxicity and cytotoxicity results according to the concentration of the mixed composition and positive control (clotrimazole) of the present invention.

8 is a photograph of the change in appearance of the bacteria according to the addition of the mixed composition of the present invention.

Figure 9 is a photograph of the change in appearance of the fungi according to the addition of the mixed composition of the present invention.

10 is a graph showing the results of sensory evaluation according to the application of the mixed composition of the present invention in atopic dermatitis induced mice.

11 is a photograph of the appearance of a normal mouse not causing atopy.

12 is a photograph of the appearance of the atopy-induced mouse (positive control).

Figure 13 is a photograph of the appearance of the mouse changed by applying a control drug pimecrolimus cream to atopy-induced mice.

Figure 14 is a photograph of the appearance of the mouse changed by applying the mixed composition of the present invention to atopy-induced mice.

15 is a graph showing the level of IgE, an atopic related antibody in serum, according to the application of the mixed composition of the present invention in atopic dermatitis-inducing mice.

Figure 16 is a histological picture of normal mice (negative control) and atopy-induced mice (positive control) and mice to which the control drug pimecrolimus cream was applied (drug control).

Figure 17 is a histological picture of normal mice (negative control) and atopy-induced mice (positive control) and the mouse (drug control) to which the mixed composition of the present invention is applied.

Claims (8)

Deep sea water 80-90% by volume, honey 5-15% by volume, vinegar 0.4-0.6% by volume and chitosan 0.5-1.0% by volume, characterized in that Bacteria or Candida albicans or Tricosphoron Bay, including Staphylococcus aureus , Bacillus subtilis , Pseudomonas aeruginosa , or Escherichia coli Antimicrobial or antifungal composition for the prevention or treatment of atopic secondary skin infections caused by fungi, including Jelly ( Trichosporon beigelli ). The antimicrobial or antifungal composition of claim 1, wherein the deep sea water has a hardness of 50 to 300 mg / L. According to claim 1, wherein the vinegar contains 5 to 10% by weight of brown rice, the total acid content is 6.0 to 7.0% (w / v) antimicrobial or antifungal composition for preventing or treating atopic secondary skin infections . The method of claim 1, wherein the chitosan is 1) treating a solution of an organic or inorganic acid salt of chitosan oligosaccharide with trialkyl amine which is a base, 2) recovering the chitosan oligosaccharide in which the organic or inorganic acid bound to the chitosan oligosaccharide is removed in the form of a trialkyl amine salt by adding an organic solvent to the solution, 3) Water-soluble free amine chitosan having a molecular weight of 1,000 to 100,000 Daltons prepared by purifying an acid-free chitosan oligosaccharide solution after inorganic acid treatment and then using an activated carbon / ion exchange resin column. Antimicrobial or antifungal composition for preventing or treating atopic dermatitis, characterized in that the secondary skin infection. delete delete delete The antimicrobial or antifungal composition for preventing or treating atopic dermatitis, wherein the composition is an external preparation applied to the affected part of the skin.

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