KR20190084887A - Composition containing bee venom for improving skin inflammation - Google Patents

Abstract

The present invention relates to a therapeutic composition for alleviating inflammation caused by pathogenic fungi containing bee venom (BV). A composition for alleviating inflammation caused by pathogenic fungi according to the present invention can alleviate inflammation without side effects since purified bee venom derived from natural products is used within a concentration range without side effects.

Description

[0001] The present invention relates to a composition for controlling skin inflammation containing bee venom as an active ingredient,

The present invention relates to a composition for controlling skin inflammation containing bee venom (BV) as an active ingredient. More particularly, the present invention relates to therapeutic compositions containing bee venom (BV) for treating skin inflammatory diseases caused by pathogenic fungi, pathogenic bacteria and antibiotic-resistant bacteria causing skin inflammation.

Fungi are fungi, yeasts, and mushrooms, which contain microorganisms, which have nuclear membranes, and belong to eukaryotes that are different from bacteria or viruses. Some fungi live in the form of parasitic or symbiotic. When infected with weak immunity, they cause various skin diseases. The majority of the cells in the outer layers of the skin are composed of keratinocytes. These skin cells are the first defenses against the invasion of external pathogens through the skin and are associated with congenital immunity. When fungi such as Candida albicans and Malassezia furfur penetrate into the skin, they react with various TLR receptors. When skin cells are damaged, histamine, prostaglandin, and other substances are released, expanding blood vessels, collecting phagocytes, and releasing cytokines.

Recently, amphotericin B and azole-based drugs such as fluconazole and itraconazole have been used to alleviate such inflammatory reactions. However, these drugs are known to have serious side effects such as thrombocytopenia, renal dysfunction, and liver toxicity (Rafael Laniado-Laborin R., Cabrales-Vargas MN, 2009. Amphotericin B: side effects and toxicity. Rev. Iberoam. Micor, 26 (4), 223-227; Martin M, 1999. The use of fluconazole and itraconazole in the treatment of Candida albicans infections: a review, J. Antimicrob. Chemother. 44 (4), 429-437). Therefore, there is a restriction in its use, and development of an antifungal agent of a natural material which minimizes toxicity to the human body is required.

On the other hand, bee venom, also called honey bee, is a substance composed of about 40 components contained in the bee venom of a bee, mainly including a polypeptide, an enzyme, and a compound having a small molecular weight. In bee venom collected from honey bees, melittin, which is the main component, is contained in an amount of 40-50%, and peptides such as apamin, MCD peptide, adolapin, protease inhibitor), phospholipase and hyaluronidase are contained in an amount of 10 ~ 12% and 1.5 ~ 2%, respectively, and they are known to exhibit anti-inflammatory and anticancer effects (Son DJ et al., Pharmacology & Therapeutics 2007, 115, 246-270). In addition, components contained in less than 1% are known to be the components of bee venom, such as histamine, dopamine, and norepinephrine.

Bee venom has been reported to induce allergies when it is used at high concentration, but it has been used traditionally as a folk remedy and as an important material of oriental medicine because it shows various therapeutic effects when the appropriate concentration is used. The major effects are known to have analgesic effects and have been used in traditional therapies such as degenerative arthritis and acute arthritis. In recent years, there has been a growing interest in the treatment of rheumatoid arthritis (Seo BK et al., Korean Acupuncture Society 2005, 22, 19-32), improvement of Parkinson's disease (Doo AR et al., Brain Research 2012 1429 106-115; Alvarez- D., PLoS One. 2013, 8, e61700) and anti-dementia, but also to treat skin whitening (Patent Document 1) and skin regeneration (Patent Document 2) . However, studies on the mitigation of skin inflammation by fungi have not been conducted.

Korean Patent Publication No. KR 10-2012-0003178 Korean Patent Publication No. KR 10-2011-0139486

It is an object of the present invention to develop an inflammation-improving agent for improving inflammation caused by pathogenic fungi using a substance derived from a natural substance with a low side effect. In particular, by verifying the effect of using bee venom to alleviate inflammation caused by pathogenic fungi, it is possible to solve various problems of currently used chemicals and to develop new substances that achieve skin inflammation mitigation effect.

In order to solve the above problems, a composition for improving inflammation or treating skin diseases, which inhibits inflammation caused by pathogenic fungi or bacteria according to one aspect of the present invention, comprises bee venom or a pharmacologically acceptable bee venom composition as an active ingredient .

The present invention provides a pharmaceutical composition for preventing or treating skin diseases, comprising bee venom as an active ingredient.

The active ingredient may be included in the composition as 32 to 256 [mu] g / ml. When the concentration is less than 32 μg / ml, the efficacy is low. When the concentration exceeds 256 μg / ml, the efficacy is not greatly increased, resulting in cytotoxicity, which is inefficient.

The skin diseases are selected from the group consisting of skin inflammation, acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simplex poisoning, biliary cirrhosis, deprived dermatitis, urticaria, psoriasis, sunshine dermatitis and acne But is not limited thereto.

The skin disease may be caused by fungal infection or bacterial infection.

The fungi may be selected from the group consisting of Malassezia spp., Microsporum spp., Trychophyton spp., Penicillium spp., Cladosporium spp., Malassezia spp. furfur, Malassezia nana, Malassezia pachydermatis, and Malassezia sympodialis, Candida albicans, Candida lutaniae, Candida albicans, Candida parasilosis, Candidatropicalis, Candidaguilliermondii, Epidermophyton floccosum, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida albicans, , Trichophyton rubrum, Trichophytonmentagrophytes, Trichophyton rubrum, Cryptococcus neoform The present invention relates to a method for the production of microorganisms such as Cryptococcus neoformans, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Epidermophyton flocosomes, Histoplasmacapsulatum, for example, blastomyces, Cryptococcus neoformans, Pneumocystis jiroveci, Cocidioides mimitis, Aspergillus fumigatus and Aspergillus niger, niger). < / RTI >

The fungus may be an antifungal-resistant fungus. The antifungal drug resistance may be selected from the group consisting of terbinafine, amphotericin B, candicidin, filipin, hamycin, nystatin, rimocidin, , Bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, ruliconazole laciconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, , Fluconazole, isavuconazole, itraconazole, psoaconazole, ravuconazole, terconazole, voriconazole, aba-phanzine, abafungin, amorolfin, butenafine, naftifine, Aniline, anidulafungin, caspofungin, micafungin, ciclopirox, flucytosine, haloprogin, griseofulvin, and tolnaftate ), ≪ / RTI > and one or more antifungal agents selected from the group consisting of < RTI ID = 0.0 >

The bacterium may be a bacterium that causes acne, and may be preferably Propionibacterium acnes.

The bacterium may be an antibacterial-resistant bacterium, and the antibacterial agent may be clindamycin, but is not limited thereto.

In the present invention, "treatment" means treating or ameliorating an inflammatory skin disease by administering the composition of the present invention to an individual.

The subject includes, without limitation, mammals including rats, livestock, humans, and the like.

"Prevention" in the present invention means all actions of inhibiting inflammation skin disease or delaying the onset of the disease by administering the composition of the present invention to an individual.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration may include tablet pills, powders, granules, capsules and the like, which may contain one or more excipients, such as starch, calcium carbonate, sucrose or lactose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may also be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solutions, suspensions, emulsions, ≪ / RTI > and suppositories.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dosage may be determined depending on the condition of the patient The severity of the disease, the form of the drug, the route of administration, and the time, but may be suitably selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and excretion, the type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight may be administered daily or every other day, or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect, the present invention provides a cosmetic composition for improving skin diseases containing bee venom as an active ingredient.

The active ingredient may be included in the composition as 32 to 256 [mu] g / ml. When the concentration is less than 32 μg / ml, the efficacy is low. When the concentration exceeds 256 μg / ml, the efficacy is not greatly increased, resulting in cytotoxicity, which is inefficient.

The skin disease may be caused by fungal infection or bacterial infection.

The fungus may be a fungus resistant to an antifungal agent, Types of antifungal agents and types of skin diseases are as described above.

The bacterium may be a bacterium that causes acne, and may be preferably Propionibacterium acnes.

The bacterium may be an antibacterial-resistant bacterium, and the antibacterial agent may be clindamycin, but is not limited thereto.

The cosmetic composition may be selected from the group consisting of lotion, essence, lotion, cream, pack, foundation, gel, ointment or spray. But the present invention is not limited thereto.

The cosmetic composition may further contain, in addition to the bee venom as an active ingredient according to the present invention, a lipid, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an anti-aging agent, a suspending agent, a stabilizer, a foaming agent, Surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, lipophilic or lipophilic active agents, lipid vesicles or cosmetics Such as any other ingredients conventionally used in cosmetics.

The present invention can prevent and / or ameliorate inflammatory skin diseases by applying a cosmetic composition containing bee venom as an active ingredient to the skin of an individual.

The term "application" in the present invention means to contact the composition according to the present invention with the skin of the individual by any suitable method, thereby aiming to absorb the composition into the skin.

In the present invention, the term "improvement" refers to all the actions of the composition according to the present invention in which the function of the skin barrier is strengthened and the skin inflammation caused by the fungus is improved or the inflammation does not occur easily.

The composition for improving inflammation caused by pathogenic fungus according to the present invention can improve inflammation without side effects because it uses the conventionally used purified bee venom derived from natural products within a concentration range without side effects. In particular, bee venom can attain sufficient inflammation-improving effect by skin application without oral administration, and thus can prevent the adverse effects of conventional antifungal agents and induce the expression of inflammatory cytokines which are increased by inflammation induced by fungi without stimulation of skin cells It is possible to achieve an effect of decreasing the secretion amount, increasing the expression amount of a growth factor that assists skin growth, and alleviating fungal-induced apoptosis. In addition, it is possible to further diversify a substance having a skin inflammation relieving effect derived from a natural product.

1 is bee venom at various concentrations to the skin keratinocyte (HaCaT keratinocytes) (1, 10 , 50, 100, 200 ng / ㎖) C. albicans (10 3, 10 and ^{4, 10 5, 10 6,} 10 7 cells / Lt; / RTI > cells), and then subjected to toxicity test on cells.
FIG. 2 shows the effect of reducing the inflammatory cytokine gene expression and the expression of the skin growth factor in various concentrations of bee venom (1, 10, 100 ng / ml) after inducing inflammation with C. albicans .
FIG. 3 shows the effect of reducing the amount of inflammatory cytokine protein expression when various concentrations of bee venom (1, 10, 100 ng / ml) were treated after inducing inflammation with C. albicans .
FIG. 4 shows the effect of suppressing apoptosis when various concentrations of bee venom (1, 10, 100 ng / ml) were treated after inducing inflammation with C. albicans .
FIG. 5 is a graph showing the effect of bee venom (2.5, 5, 10 and 20 ng / ml) and M. furfur (10 ⁵ , 10 ⁶ , 10 ⁷ , 5 × 10 ⁷ and 10 ⁸ cells / Ml), and then the toxicity to the cells was confirmed.
FIG. 6 shows the effect of reducing the expression level of inflammatory cytokines when various concentrations of bee venom (2.5, 5, 10 ng / ml) were treated after inducing inflammation with M. furfur .
FIG. 7 shows the effect of reducing the amount of inflammatory cytokine protein expressed when M. furfur induced inflammation and then treated with various concentrations of bee venom (2.5, 5, 10 ng / ml).
FIG. 8 shows the effect of suppressing apoptosis when various concentrations of bee venom (2.5, 5, 10 ng / ml) were treated after inducing inflammation with M. furfur .
Fig. 9 shows the result of measurement of the antibacterial activity according to the use amount of bee venom of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example

Example 1. Preparation of materials

Before confirming whether bee venom helps to relieve inflammation caused by pathogenic fungi, toxicity to cells was performed, and inflammatory fungi were administered to HaCaT keratinocytes and human dermal papilla cells After the inflammation was induced, bee venom was administered to perform an inflammation relieving test.

1.1 Manufacture and purification of bee venom

The non-purified bee venom collected from Apis mellifera species was dissolved in boiled and chilled purified water at 2 ~ 3% concentration and stirred at 800 rpm for 30 minutes. The dissolved bee venom solution was centrifuged at 4 DEG C and 4000 rpm for 10 minutes. The centrifuged supernatant was filtered through a paper filter, and the filtrate after the first filtration was secondary filtered with a microfilter. The filtered solution was lyophilized for 96-120 hours to form a solid lump, which was pulverized to be a plate-like powder.

1.2 Strain preparation

Malassezia furfur was purchased from KCCM (Korea Microorganisms Conservation Center) and Candida albicans (ATCC 90028 ™) from ATCC. Human dermal papilla cells (hDPc) and HaCaT cell (human keratinocyte) were distributed from the Korean Cell Line Bank (Seoul, Korea). Malassezia furfur was incubated in Sabouraud dextrose agar containing 1% peptone, 4% glucose, 10% olive oil, and 0.2% Tween 80 for 4 days in an incubator (37 ° C). M. furfur cultured for 4 days was washed twice with physiological saline, and then subjected to heat bacterium treatment at 80 ° C for 30 minutes. C. albicans were cultured in SDB (Sabouraud Dextrose Broth, BD) for 4 days in an incubator (37 ° C, 5% CO ₂ ). The cultured bacteria were washed twice with physiological saline, and then subjected to heat treatment at 80 ° C for 30 minutes.

1.3 Data Statistics Processing

Statistical analysis was performed using SigmaPlot Version 10.0 and SPSS version 21. One-way analysis of variation (ANOVA) was performed. When significance was observed, Tukey HSD was performed (* p <0.05) to identify test groups that were significantly different from the positive control group.

Example 2. Culture and differentiation of HaCaT cells

HaCaT cells were cultured in Dulbecco's modied Eagle's medium (DMEM) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin at 37 ° C and 5% CO ₂ . All experiments were carried out 24 hours after cell culture. When the experiment was conducted to examine the effect of bee venom, a medium without FBS was used.

Example 3. C. albicans  Induction of inflammation by treatment

To induce inflammation in HaCaT cells, the cells cultured by the method of Example 1 were treated with 10 ⁶ cells / ml of C. albicans and cultured together for 24 hours.

Example 4. Culture and differentiation of human dermal papilla cell (hDPc)

hDPc was cultured in Dulbecco's modied Eagle's medium (DMEM) supplemented with 10% FBS, 1% penicillin / streptomycin at 37 ° C and 5% CO ₂ . All experiments were carried out 24 hours after cell culture. When the experiment was conducted to examine the effect of bee venom, a medium without FBS was used.

Example 5. M. furfur  Induction of inflammation by treatment

To induce inflammation in hDPc, M. furfur at a concentration of 10 ⁷ cells / ml was added to the cells cultured by the method of Example 3 and cultured together for 24 hours.

Example 6. Measurement of gene expression associated with inflammation by fungi

Total RNA was isolated using AccuZol ™ Total RNA Extraction Reagent (Bioneer, Seoul, Korea) for quantitative real-time polymerase chain reaction (qPCR) and RevertAid First Strand cDNA kit (Fermentas, Burlington, Ontario, Canada) .

The qPCR was performed using a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers used in qPCR are shown in Table 1 below. The PCR reaction was carried out at 95 ° C for 10 minutes, followed by 40 cycles of reaction at 95 ° C for 20 seconds, 60 ° C for 20 seconds, and 72 ° C for 30 seconds. The GAPDH gene was used as an endogenous control, and the amount of gene expression in the negative control sample was 1 (relative to 2 ^-ΔΔCt method).

Gene Primers GAPDH Forward 5'-ACC CAC TCC TCC ACC TTT GA-3 '
Reverse 5'-CTG TTG CTG TAG CCA AAT TCG T-3 ' TNF-a Forward 5'-CAG AGG GCC TGT ACC TCA TC-3 '
Reverse 5'-GGA AGA CCC CTC CCA GAT AG-3 ' IL-6 Forward 5'-GGA GCC CAG CTA TGA ACT CC-3 '
Reverse 5'-GGT CAG GGG TGG TTA TTGCA-3 ' IL-1? Forward: 5'- AAC AGG CTG CTC TGG GAT TC-3 '
Reverse: 5'-TAT CCT GTC CCT GGA GGT GG-3 ' IGF-1 Forward: 5'-CAT TTC ACC TCC ACC ACC AC-3 '
Reverse: 5'-AGG CAT CCT GCC CAT CAT AC-3 ' FGF-2 Forward: 5'- GGT GAA ACC CCG TCT CTA CA-3 '
Reverse: 5'-TCT GTT GCC TAG GCT GGA CT-3 '

Example 7. Test for antibacterial activity

The minimum inhibitory concentration (MIC) was tested using microdilution method to determine the effect of bee venom on the growth of antibiotic resistant bacteria. The strains used in the experiments were Propionibacterium acnes (KCTC3314) and CCARM 9010 (Clindamycin-resistant strain) and cultured in GAM (Gifu Anaerobic Medium) broth at 37 ° C and 5% CO ₂ . The bacterium that had undergone the second activation process was adjusted to the turbidity of 0.5 McFarland solution using 0.85% NaCl solution, which was again diluted 1/1000 and used as inoculum. Bee venom was prepared at 0 - 500 ㎍ / ㎖ concentration. 100 μl of bacterial solution and 100 μl of bee venom at each concentration were applied to a 96-well plate. After incubation at 37 ° C and 5% CO ₂ for 48 hours, the concentration at which the growth of the bacteria was inhibited by 80% or more was judged as the minimum inhibitory concentration.

Experimental Example

Experimental Example 1: C. albicans Of cytotoxicity

Example 1 Method bee venom at various concentrations to the incubation of the cells with (1, 10, 50, 100 , 200 ng / ㎖) C. albicans (10 3, 10 and ^{4, 10 5, 10 6,} 10 7 cells / ㎖ MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to determine the cytotoxicity observed when the cells were treated. As a negative control group, samples treated with the same amount of physiological saline were used.

As a result, C. albicans did not show cytotoxicity at a concentration of 10 ⁶ cells / ml or less and induction of inflammation was carried out at a concentration of 10 ⁶ cells / ml of C. albicans (FIG. 1A). Bee venom showed a slight concentration-dependent proliferation effect but did not show cytotoxicity up to 200 ng / ㎖, and cell proliferation increased significantly at 200 ng / ㎖ treatment group (Fig. 1B).

Experimental Example 2. Decrease of inflammatory cytokine expression and increase of gene expression of growth factors by bee venom in keratinocytes

When the bee venom (1, 10, 100 ng / ㎖) was added to HaCaT cells induced by C. albicans , the expression levels of inflammation-inducible cytokines and growth factors were quantitated using a quantitative real-time polymerase chain reaction ). Cells cultured by the method of Example 1 were cultured in the same manner as in Example 2, except that the cells were treated with bee venom at a concentration of 1, 10, and 100 ng / ml, a positive control group without bee venom treatment, And the negative control group treated with PBS only.

Total RNA was isolated using AccuZol ™ Total RNA Extraction Reagent (Bioneer, Seoul, Korea) for qPCR and converted to cDNA using RevertAid First Strand cDNA kit (Fermentas, Burlington, Ontario, Canada).

The analysis included inflammation-inducible cytokines IL-1β (interleukin-1β), tumor necrosis factor-α (TNF-α) and insulin-like growth factor-1 (IGF-1) and fibroblast growth factor -2) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

As a result, the positive control group without bee venom treatment showed an increase in the amount of cytokine expression and a decrease in the expression level of the growth factor compared to the negative control group. In the bee venom treatment, cytokine expression was decreased in a concentration-dependent manner (FIGS. 2A and 2B) , And expression of the growth factor was increased (Fig. 2C and Fig. 2D).

Experimental Example 3: Reduction of protein expression of inflammatory cytokine by bee venom in keratinocytes

When the bee venom (1, 10, 100 ng / ㎖) was added to HaCaT cells induced by C. albicans , secretion of inflammation-inducible cytokines was measured by enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences; Franklin Lakes, SD, USA). Cells cultured by the method of Example 1 were cultured in the same manner as in Example 2, except that the cells were treated with bee venom at a concentration of 1, 10, and 100 ng / ml, a positive control group without bee venom treatment, And the negative control group treated only with physiological saline. Cell culture medium was used as an analytical sample and used without dilution. The protein expression levels of inflammatory cytokines IL-1β and TNF-α were analyzed.

As a result, C. albicans induced inflammation In the positive control group without bee venom treatment, the amount of cytokine secretion was increased compared with the negative control group. C. albicans induced inflammation In the bee venom treatment, it was confirmed that the secretion of cytokine decreased in a concentration-dependent manner (Fig. 3).

Experimental Example 4. Confirmation of the effect of bee venom mitigating apoptosis in keratinocytes

(BD Biosciences; Franklin Lakes, SD, USA) in the negative control group treated with saline only, the positive control group treated with C. albicans alone, and the test group treated with C. albicans and bee venom Respectively. The recovered cells were stained with Ezway Annexin V-FITC Apoptosis Detection Kit (KOMA BIOTECH, Seoul, Korea) and analyzed by flow cytometry.

As a result of the experiment, it was confirmed that the death rate was significantly increased in the group that only induced inflammation of the bacterium as compared with the negative control group, and that the death rate was significantly decreased in the bee venom-treated group (FIG. 4).

Experimental Example 5: M. furfur Of cytotoxicity

Example 3 How the various concentrations of bee venom in cultured cells of (2.5, 5, 10, 20 ng / ㎖) and ^{M. furfur (10 5, 10 6} , 10 7, 5 × 10 7, 10 8 cells / ㎖ ), MTT assay was performed to confirm cytotoxicity. As a negative control group, samples treated with the same amount of physiological saline were used.

As a result, no cytotoxicity was observed according to the concentration of bee venom, as shown in FIG. 5 (FIG. 5A). M. furfur did not show cytotoxicity at 10 ⁵ , 10 ⁶ , or 10 ⁷ cells / ml, but showed significant cytotoxicity compared to negative control at a concentration of 5 × 10 ⁷ cells / ml (FIG. 5B). Therefore, the inflammation relaxation experiment was carried out at a concentration of 10 ⁶ cells / ml.

Experimental Example 6. Decrease of inflammatory cytokine gene expression and increase of gene expression of growth factor by bee venom in dermal papilla cells

When bee venom (2.5, 5, 10 ng / ㎖) was added to inflamed hDPc through M. furfur , the expression level of inflammation - inducible cytokine was confirmed by qPCR. Cells cultured by the method of Example 3 were cultured in the same manner as in Example 4 except that bee venom was treated at a concentration of 2.5, 5, or 10 ng / ml, a positive control group without inflammatory induction, And the negative control group treated only with physiological saline.

Total RNA was isolated using AccuZol ™ Total RNA Extraction Reagent (Bioneer, Seoul, Korea) for qPCR and converted to cDNA using RevertAid First Strand cDNA kit (Fermentas, Burlington, Ontario, Canada).

The analysis items were inflammation-inducible cytokines IL-6 and TNF-α and analyzed based on the expression level of GAPDH.

As a result of the experiment, it was confirmed that the positive control group not treated with bee venom increased the expression level of cytokine compared with the negative control group, and the cytokine expression was decreased in a concentration-dependent manner when bee venom was treated (FIG. 6).

Experimental Example 7. Reduction of inflammatory cytokine protein expression by bee venom in dermal papilla cells

When the addition of bee venom (2.5, 5, 10 ng / ml) to hDPc induced inflammation through M. furfur , protein expression of inflammation-inducible cytokines was measured by enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences; Franklin Lakes, SD, USA). Cells cultured by the method of Example 3 were cultured in the same manner as in Example 4 except that bee venom was treated at a concentration of 2.5, 5, or 10 ng / ml, a positive control group without inflammatory induction, And the negative control group treated only with physiological saline. Cell culture medium was used as an analytical sample and used without dilution. The analysis items were inflammatory cytokines IL-6 and TNF-α.

As a result, the positive expression of cytokine was increased in the positive control group not treated with bee venom, and the cytokine expression was decreased in a concentration-dependent manner when the bee venom was treated (FIG. 7).

Experimental Example 8. Confirmation of effect of bee venom mitigating apoptosis in dermal papilla cells

Confocal laser scanning biological microscope was used to observe cell death in the negative control group treated with saline only, the positive control group treated with M. furfur alone, and the test group treated with M. furfur and bee venom. Cells cultured by the method of Example 3 were cultured in the same manner as in Example 4 except that bee venom was treated at a concentration of 2.5, 5, or 10 ng / ml, a positive control group without inflammatory induction, And the negative control group treated only with physiological saline. The cells were stained with Ezway Annexin V-FITC Apoptosis Detection Kit (KOMA BIOTECH, Seoul, Korea) and the degree of apoptosis was observed immediately through a confocal microscope.

As a result, it was found that the cell death was significantly increased in the positive control group as compared with the negative control group, and the cell death was reduced in the bee venom treated group (FIG. 8). Especially, late apoptosis was confirmed to be alleviated in bee venom treated group.

Experimental Example 9. Confirmation of antibacterial activity of bee venom

As a result of observation after 48 hours of incubation according to Example 7, bacteria were grown in the group treated with bee venom at a concentration of 0 to 16 / / ml. However, there was no bacterial proliferation in the group treated with bee venom at a concentration of 32 to 256 占 퐂 / ml (see FIG. 9). Therefore, the minimum inhibitory concentration (MIC) inhibiting the growth of P. acnes KCTC3314 and CCARM 9010 (Clindamycin-resistant strain) was 32 ㎍ / ㎖, indicating that bee venom was also effective against clindamycin-resistant bacteria See Table 2).

Strain MIC ([mu] g / ml) A common strain Propionibacterium acnes
(Propionibacterium acnes, KCTC3314) 32 Resistant strain Clindamycin-resistant strain
(Clindamycin-resistant strain, CCARM 9010) 32

Manufacturing example

Production Example 1. Preparation of cosmetic composition

The cosmetic composition was prepared under the conditions shown in Table 3 below (unit, weight%).

Raw material name Content (% by weight) Purified water Balance Glycerin 5.000 Sodium hyaluronate 0.010 hydrogel PFKC 1.500 Dissolvine NA2-S 0.020 Betaine 1.500 carbopol 980 0.250 1.3 ㆍ BG 1,000 Keltrol F 0.060 CS-165 1.200 ARLACEL 60 0.800 TWEEN 60 0.800 M.C.T. 5.000 Lanetto O 1.500 Lipex shea soft 0.100 Tocopheryl acetate 0.100 Bees wax 0.500 Dimethicone 6CS 0.300 TEA 0.250 SEPPIPLUS 400 0.150 Spices a very small amount The bee venom of the present invention 7.000 Phenoxyethanol 0.300 1,2-HEXANEDIOL 0.400

Preparation Example 2. Preparation of ointment in external preparation for skin

The ointments were prepared according to the conventional ointment preparation methods in the amounts shown in Table 4 below and uniformly mixed.

Compounding ingredient Content (% by weight) The bee venom of the present invention 1.0 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 1.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 Wax 4.0 Preservatives, coloring and flavoring Proper amount Purified water Balance (up to 100% by weight)

Production Example 3: Preparation of nutritional cream

Nutritive creams were prepared according to the compositions shown in Table 5 in a conventional manner.

Compounding ingredient Content (% by weight) The bee venom of the present invention 0.2 glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 7.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 Triethanolamine 0.1 Preservatives, coloring and flavoring Proper amount Purified water Balance (up to 100% by weight)

Production Example 4. Preparation of pharmaceutical composition

The pharmaceutical composition was prepared under the conditions shown in Table 6 below.

Constituent Content (% by weight) The bee venom of the present invention 2 saccharin 0.8 Party 25.4 glycerin 8 Spices 0.04 ethanol 4 Sorbic acid 0.4 Distilled water 59.36

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (16)

A pharmaceutical composition for preventing or treating skin diseases, comprising bee venom (BV) as an active ingredient.
2. The pharmaceutical composition according to claim 1, wherein the active ingredient is contained in the composition as 32 to 256 占 퐂 / ml.
The method according to claim 1, wherein the skin disease is selected from the group consisting of skin inflammation, acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simplex poisoning, biliary cirrhosis, deprived dermatitis, urticaria, psoriasis, Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &gt;
The pharmaceutical composition according to claim 1, wherein the skin disease is caused by fungal infection or bacterial infection.
The method of claim 4, wherein the fungi are selected from the group consisting of Malassezia, Microsporum, Trychophyton, Penicillium, Cladosporium, Malassezia furfur, Malassezia nana, Malassezia pachydermatis, and Malassezia sympodialis, Candida albicans, Candida albicans, Candida species such as Candida species, Candida species, Candida species, Candida species, Candida species, Candida species, Candida parapsilosis, Candidatropicalis, Epidermophyton floccosum, Trichophyton rubrum, Trichophytonmentagrophytes, Trichophyton rubrum, Cryptococcus, Such as Cryptococcus neoformans, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Epidermophyton flocosum, Histoplasmacapsulatum, For example, blastomyces, Cryptococcus neoformans, Pneumocystis jiroveci, Cocidioidesimmitis, Aspergillus fumigatus and Aspergillus, Aspergillus niger, &lt; / RTI &gt; and the like.
5. The pharmaceutical composition according to claim 4, wherein the fungus is an antifungal drug resistant fungus.
7. The method of claim 6, wherein the antifungal drug resistance is selected from the group consisting of terbinafine, amphotericin B, candicidin, filipin, hamycin, nystatin, But are not limited to, rimocidin, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, ), Luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, It has been reported that albaconazole, fluconazole, isavuconazole, itraconazole, psoaconazole, ravuconazole, terconazole, voriconazole, abafungin, amorolfin, butenafine, napthin (na) anhydroglucosidase inhibitors, such as fuftine, anidulafungin, caspofungin, micafungin, ciclopirox, flucytosine, haloprogin, griseofulvin, Wherein the composition is resistant to at least one antifungal agent selected from the group consisting of tolnaftate.
5. The method of claim 4,
Wherein the bacterium is Propionibacterium acnes.
The pharmaceutical composition according to claim 4, wherein the bacterium is a bacterium resistant to an antimicrobial agent.
10. The pharmaceutical composition according to claim 9, wherein the antibacterial agent is Clindamycin.
A cosmetic composition for the improvement of skin diseases containing bee venom as an active ingredient.
12. The cosmetic composition according to claim 11, wherein the active ingredient is contained in the composition as 32 to 256 占 퐂 / ml.
12. The cosmetic composition according to claim 11, wherein the skin disease is caused by fungal infection or bacterial infection.
14. The method of claim 13,
Wherein the bacterium is Propionibacterium acnes.
14. The cosmetic composition according to claim 13, wherein the bacterium is an antibacterial-resistant bacterium.
16. The cosmetic composition according to claim 15, wherein the antibacterial agent is Clindamycin.

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