KR101557788B1 - Cosmetic Composition and Topical Composition for Anti-inflammation Containing Peptide derived from Harmonia axyridis larva - Google Patents

Abstract

The present invention relates to an anti-inflammatory cosmetic composition and a composition for external application for skin containing a novel peptide derived from a ladybug larva, and more particularly to an anti-inflammatory cosmetic composition containing a novel peptide derived from a ladybug larva to prevent and improve inflammation- A cosmetic composition and a composition for external application for skin.
The anti-inflammatory cosmetic composition and the composition for external application for skin according to the present invention are characterized by containing a novel peptide HaGF derived from the sequence consisting of the amino acid sequence shown in SEQ ID NO: 1.
The anti-inflammatory cosmetic composition and the composition for external application for skin are effective for prevention and improvement of skin diseases caused by inflammation by inducing the production and suppression of pro-inflammatory mediator.

Description

TECHNICAL FIELD The present invention relates to an anti-inflammatory cosmetic composition containing a novel peptide derived from a ladybug larva and a composition for topical application to skin,

The present invention relates to an anti-inflammatory cosmetic composition and a composition for external application for skin containing a novel peptide derived from a ladybug larva, and more particularly to an anti-inflammatory cosmetic composition containing a novel peptide derived from a ladybug larva to prevent and improve inflammation- A cosmetic composition and a composition for external application for skin.

The inflammatory reaction is a mechanism to repair and regenerate the injured area when an invasion that brings about physical changes such as physical action, chemical substance, bacterial infection, etc. is applied to the living body or tissue. As the mediator that induces the inflammation reaction, radicals, nitric oxide (NO), and prostaglandin.

Once the stimulation is applied, the vasoactive material such as inflammatory component is released locally to increase the vascular permeability, causing the inflammation. However, the continuous inflammation reaction promotes the mucosal injury, and as a result, do.

Macrophages are known to be involved in various host responses such as acquired immunity as well as innate immunity, and they are known to be involved in homeostasis. In the inflammatory reaction, they produce nitric oxide (NO) and cytokine and play an important role in biological defense in the early stage of infection. It is known that iNOS is induced by inflammatory stimuli such as TNF-α, IL-1β and IFN-γ in macrophages regardless of the concentration of calcium. Especially, It is known to be generated in large quantities.

TNF-α is an important mediator of inflammation and immune responses and is known to regulate the growth and differentiation of various cells. It is also known to cause toxicity to cells, promote angiogenesis, bone resorption, thrombus formation and inhibit lipogenetic metabolism (Aggarwal, BB, Nat. Rev. Immunol. 3,745-756, 2003).

On the other hand, from ancient times, insects have been widely used for medicinal / edible purposes in the Orient. According to the records of the ancient times, about 95 kinds of medicinal insects are recorded in Donguibogam, and 106 kinds of medicinal insects are recorded in the main river water.

Recent advances in biotechnology have diversified the use of insect resources, the most notable of which is industrial use. The exoskeleton (extraction of chitin) of silkworms (silk, silkworm), insects (honey, royal jelly, propolis) insects producing various useful substances and the like are good examples. In addition, insect extracts have been used as traditional herbal medicines. Recently, studies on the efficacy and effective ingredient analysis have been conducted in recent years. In addition, insect resources are useful biological resources, and they are widely used and developed widely in various fields worldwide today.

In particular, the ladybug ( Harmonia axyridis ) belongs to the beetle, which is about 7 mm in body length, hemispherical, and the back of the head except for the side eye varies from yellow to black. Adults and larvae of ladybirds have been used for a long time to control the aphid which inhabits the habitat while living in the backside of the host plant, net, etc., and has been used in civilian to soothe the cough .

Korean Patent No. 0665000 discloses a ladybug extract having antiinflammatory activity and a composition containing the same, but the ladybug was extracted with a solvent and it was not confirmed what active body exhibiting anti-inflammatory effect.

Accordingly, the present inventors have made intensive efforts to solve the above problems. As a result, the present inventors have found that a novel peptide derived from a larva of a ladybug larva is a nitric oxide (NO), an inducible nitric oxide synthase (iNOS), a cyclooxygenase Pro-inflammatory factors such as IL-2 (Cyclooxygenase-2: COX-2), TNF-alpha (tumor necrosis factor-?), IL- inflammatory mediator by inhibiting the production and suppression of inflammation, and thus it can be used as an anti-inflammatory cosmetic and skin external preparation for prevention and improvement of skin diseases caused by inflammation, and completed the present invention .

It is an object of the present invention to provide an anti-inflammatory cosmetic and dermatological composition which is effective for prevention and improvement of skin diseases caused by inflammation by inducing the generation and suppression of pro-inflammatory mediator with little irritation to the skin .

In order to achieve the above object, the anti-inflammatory cosmetic composition of the present invention is characterized by containing a novel peptide HaGF derived from a larva of a ladybug larva consisting of the amino acid sequence shown in SEQ ID NO: 1.

The novel peptide HaGF derived from the ladybug larva is contained in an amount of 0.005 to 5% by weight, preferably 0.025 to 5% by weight, based on the total weight of the anti-inflammatory cosmetic composition.

The novel peptide HaGF derived from the ladybug larva has an anti-inflammatory effect by inducing the production and suppression of pro-inflammatory mediator.

The pro-inflammatory mediator may be selected from the group consisting of nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2 ), TNF-alpha (tumor necrosis factor-alpha), IL-1 beta (interleukin-1 beta), and IL-6 (interleukin-6).

The composition for external application for skin disease prevention and improvement of the present invention is characterized by containing a novel peptide HaGF derived from a larva of a ladybug larva having an amino acid sequence represented by SEQ ID NO: 1.

The new peptide HaGF derived from the ladybug larva is contained in an amount of 0.005 to 5% by weight, preferably 0.025 to 5% by weight, based on the total weight of the skin external composition for prevention and improvement of skin diseases caused by oxidative stress and inflammation .

The novel peptide HaGF derived from the ladybug larva has an anti-inflammatory effect by inducing the production and suppression of pro-inflammatory mediator.

The pro-inflammatory mediator may be selected from the group consisting of nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2 ), TNF-alpha (tumor necrosis factor-alpha), IL-1 beta (interleukin-1 beta), and IL-6 (interleukin-6).

The skin disease is characterized by being a skin disease caused by oxidative stress and inflammation, and is characterized in that it is selected from the group consisting of allergic dermatitis, atopic dermatitis, contact dermatitis and eczema.

The anti-inflammatory cosmetic composition and composition for external application for skin according to the present invention are effective for prevention and improvement of skin diseases caused by inflammation by inducing the production and suppression of pro-inflammatory mediator.

FIG. 1 is a graph showing cytotoxicity against mouse macrophages (Raw 264.7) of a novel peptide HaGF derived from a ladybug larva.
FIG. 2 is a graph showing the inhibitory effect of nitric oxide (NO) on the generation of a novel peptide HaGF derived from a ladybug larva to mouse macrophages (Raw 264.7) (Nor: LPS untreated group, Con: LPS treated group).
FIG. 3 is a graph showing the inhibition of protein expression of iNOS and COX-2 in mouse macrophages (Raw 264.7) of a novel peptide HaGF derived from a ladybug larva (A: iNOS, B: COX-2; Nor: Con: LPS treatment group).
FIG. 4 is a graph showing the inhibitory effect on generation of new peptide HaGF derived from ladybug larvae to cytokines stimulated by LPS (A: TNF-α, B: IL-6, C: IL-1β; Nor: Group, Con: LPS treatment group).

In the present invention, HaGF (GEILHRFRRSFCDY-NH ₂ ) represented by SEQ ID NO: 1, which is a partial region of the peptide gene Harmoniasin isolated from a ladybug larva, was synthesized and the synthesized ladybug larva-derived new peptide HaGF was cytotoxic And had an anti - inflammatory effect.

In the present invention, evaluation of cytotoxicity and inhibition of pro-inflammatory mediator generation of a novel peptide HaGF derived from a ladybug larva was carried out on mouse macrophages (Raw 264.7). As a result, the novel peptide HaGF derived from the ladybug larvae has no cytotoxicity against mouse macrophages (Raw 264.7), and has no nitric oxide (NO), inducible nitric oxide synthase (iNOS) Early-inflammatory < / RTI > cells, such as cyclooxygenase-2 (COX-2), tumor necrosis factor-a (TNF-alpha), interleukin- Inflammatory effect by inducing the production and suppression of pro-inflammatory mediator, and thus it can be used as an anti-inflammatory cosmetic and skin external preparation for prevention and improvement of skin diseases caused by inflammation.

Thus, in one aspect, the present invention relates to an anti-inflammatory cosmetic composition containing a novel peptide HaGF derived from a ladybug larva consisting of the amino acid sequence shown in SEQ ID NO: 1.

At this time, the novel peptide HaGF derived from the ladybug larva is contained in an amount of 0.005 to 5 wt%, preferably 0.025 to 5 wt%, based on the total weight of the anti-inflammatory cosmetic composition. If it is contained in an amount less than 0.005% by weight, it is difficult to exhibit the desired anti-inflammatory effect due to the ability to induce the production and suppression of pro-inflammatory mediator, Due to the excessive amount of new peptide HaGF, the stability as an emulsified product is broken and it is a problem to use as a safe cosmetic product for human body.

In the present invention, the novel peptide HaGF derived from the ladybug larva having the amino acid sequence shown in SEQ ID NO: 1 can be synthesized by a conventional peptide synthesis method.

In the present invention, the novel peptide HaGF derived from the ladybug larva having the amino acid sequence represented by SEQ ID NO: 1 has an anti-inflammatory effect by inducing the production and suppression of pro-inflammatory mediator .

The pro-inflammatory mediator may be selected from the group consisting of nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2 ), TNF-alpha (tumor necrosis factor-alpha), IL-1 beta (interleukin-1 beta), and IL-6 (interleukin-6).

Nitric oxide (NO) has various physiological functions such as defense function in the body, signal transduction function, neurotoxicity and vasodilation. Three types of NOS (neuronal NO synthase (nNOS), endothelial NO synthase (eNOS) inducible NO synthase (iNOS). Among these NOS, NO production by iNOS is absolutely abundant, which is known to play an important pathological role. INOS expressed by LPS (lipopolysaccharide) stimulation produces a large amount of NO, which is known to be involved in inflammatory reactions, cell mutations and tumorigenesis. It has been reported that the expression of NO and iNOS is increased in tissue injury associated with inflammatory reaction.

Inducible nitric oxide synthase (iNOS) is not normally present in the cell, but once induced, it produces a large amount of NO over a long period of time. The resulting NO promotes inflammatory responses such as vascular permeability and edema But it is known to promote inflammation by promoting the biosynthesis of inflammatory mediators.

NO synthase (NOS) acts on the production of NO. In the case of iNOS induced by constitutive NO synthase (cNOS) and iNOS, iNOS produces a large amount of NO over a long period of time. Resulting in cytotoxicity. Therefore, anti-inflammatory effect can be expected by confirming the decrease of protein level of iNOS in NO-induced RAW 264.7 cell. In addition, it is expected that anti-inflammatory effect can be expected by increasing the proinflammatory cytokine such as TNF-α and IL-6 in the monocyte Anti-inflammatory effects can be expected by inducing a decrease in the protein level of one of COX-2.

Macrophages are the immune cells distributed in all tissues of animals and secrete inflammatory mediators such as IL-1β, IL-6 and TNF-α in the course of phagocytosis of bacteria and foreign substances and play a major role in the initial inflammatory response . In particular, TNF-α plays an important role in the inflammatory response and is secreted by macrophage and mast cell, and plays an important role in endogenous immunity as a main mediator of LPS response and also in chronic inflammatory response. IL-1β activates T-cell activation, B-cell maturation, and NK cell activation, IL-6 activates lymphocytes to increase antibody production, and IL-6 levels are always elevated in inflammatory lesions (Delgado, AV et al., P. Neuro. 37, 355-361, 2003).

In the present invention, the anti-inflammatory cosmetic composition may be exemplified by lotions, skins, lotions, nutritional lotions, nutritional creams, massage creams, essences, packs and the like, but is not limited thereto.

In another aspect, the present invention relates to a composition for external application for skin for preventing and improving skin diseases, which contains a novel peptide HaGF derived from a larva of a ladybug larva having an amino acid sequence represented by SEQ ID NO: 1.

At this time, the new peptide HaGF derived from the ladybug larva is contained in an amount of 0.005 to 5% by weight, preferably 0.025 to 5% by weight, based on the total weight of the composition for external application for skin diseases prevention and improvement. If it is contained in an amount less than 0.005% by weight, it is difficult to use as an external preparation for skin because it is difficult to exhibit the desired anti-inflammatory effect due to its ability to inhibit the production and expression of pro-inflammatory mediators, , There is a problem in using as an external preparation for skin which is safe for human body due to an excessive amount of HaGF, a novel peptide derived from a ladybug larva.

In the present invention, the skin disease is a skin disease caused by oxidative stress and inflammation. Examples of the skin disease include allergic dermatitis, atopic dermatitis, contact dermatitis, eczema, and the like.

In the present invention, the external preparation for skin diseases prevention and improvement may be a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta or a cataplasma But is not limited thereto.

The anti-inflammatory cosmetic composition of the present invention or the composition for external skin application for prevention and improvement of skin diseases caused by oxidative stress and inflammation contains a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymeric polysaccharides, sphingolipids and seaweed extract . ≪ / RTI >

The water-soluble vitamin may be any compound that can be compounded in cosmetics, but preferably vitamin B 1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, And their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate etc.) are also included in the water-soluble vitamins usable in the present invention do. The water-soluble vitamin can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzymatic method, or a chemical synthesis method.

Usable vitamins include vitamins such as vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol) , Derivatives thereof (such as palmitic acid ascorbin, stearic acid ascorbic acid, dipalmitic acid ascorbin, dl-alpha tocopherol acetic acid, dl-alpha tocopherol nicotinic acid vitamin E, dl-pantothenyl alcohol, D-pantothenyl alcohol, Ether, etc.) are also included in the usable vitamins used in the present invention. Usability Vitamins can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzyme or a chemical synthesis method.

The polymeric peptide may be any compound as long as it can be compounded in cosmetics, and examples thereof include collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. The polymeric peptide can be obtained by a conventional method such as purification from a culture broth of a microorganism, an enzymatic method, or a chemical synthesis method, or it can be purified from natural products such as ducks such as pigs and cows and silk fiber of silkworms.

The polymeric polysaccharide may be any compound as long as it can be incorporated in cosmetics, and examples thereof include hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.). For example, chondroitin sulfate or a salt thereof can be usually purified from mammals or fish.

Sphingo lipids may be any as long as they can be incorporated into cosmetics, and preferable examples thereof include ceramides, phytosphingosine and sphingoglycolipids. Sphingoid lipids can be purified from ordinary mammals, fish, shellfish, yeast or plants by conventional methods or can be obtained by chemical synthesis.

The seaweed extract may be any of those which can be compounded in cosmetics. Preferably, the seaweed extract is selected from the group consisting of algae extract, red pepper extract, green algae extract, and the like. Potassium alginate and the like are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained from seaweed by a conventional method.

The anti-inflammatory cosmetic composition of the present invention or the composition for external application for skin disease prevention and improvement caused by oxidative stress and inflammation may be blended with other essential ingredients, if necessary, in combination with a cosmetic or an external preparation for skin.

Examples of the compounding ingredients that may be added include organic solvents such as a preservative component, a moisturizer, an emollient, a surfactant, an organic and inorganic pigment, an organic powder, an ultraviolet absorbent, a preservative, a bactericide, an antioxidant, a plant extract, a pH adjuster, A blood circulation accelerator, a cold agent, an antiperspirant agent, and purified water.

Examples of the oil retaining component include ester-based oil retaining, hydrocarbon-based oil retaining, silicone-based oil retaining, fluoric oil retaining, animal retention and plant retention.

Examples of ester-based fats include glyceryl tri-2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isostearyl isostearate, Butyl isopropyl myristate, isopropyl myristate, isopropyl myristate, isopropyl myristate, isopropyl myristate, isopropyl myristate, butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isosilyl myristate, isostearic acid isostearyl, isostearyl palmitate, octyldodecyl myristate, Trimethylol propane, triisostearic acid trimethylol propane, tetra 2-ethylhexanoic acid pentaerythritol tetra (2-ethylhexanoate) , Decyl caprylate, decyl laurate, hexyl laurate, myristate decyl, myristyl myristate, myristine monoethyl stearate, stearyl stearate, decyl oleate, ricinoleic acid tri , Isostearyl stearate, isostearyl stearate, isodecyl stearate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, isopropyl stearate, isopropyl stearate, isopropyl stearate, -Hexyl stearate, stearyl ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprate, di (capryl, capric acid) propylene glycol, Propyleneglycol propionate, propyleneglycol propionate, dicaproic acid neopentyl glycol, dioctanoic acid neopentyl glycol, tricarboxylic acid glyceryl, triunsaturated glyceryl, triisopalmitic acid glyceryl, triisostearic acid glyceryl, neopentanoic acid octyldodecyl Octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, Octyldecyl lactate, octyldecyl lactate, octyldecyl lactate, polyglycerin oleic acid ester, polyglycerin isostearic acid ester, triisocetyl citrate, triisobutyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, But are not limited to, ethyl, acetyltriethyl citrate, acetyltributyl citrate, trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di-2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, But are not limited to, dioctyl sebacate, stearic acid cholesteryl, isostearic acid cholesteryl, hydroxystearic acid cholesteryl, oleic acid cholesteryl, oleic acid dihydrocholesteryl, isostearic acid pitostearyl, Stearoyl hydroxystearic acid isostearyl, 12-stearoyl stearyl hydroxystearate, 12-stearo And monohydroxystearic acid and esters such as sostearyl.

Examples of the hydrocarbon hydrocarbon-based fats include hydrocarbon fats and oils such as squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, floating isoparaffin, polybutene, microcrystalline wax and vaseline.

Examples of silicone based oils include polymethyl silicone, methylphenyl silicone, methyl cyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane-methylcetyloxysiloxane copolymer, dimethylsiloxane-methylstarchoxysiloxane copolymer, alkyl Modified silicone oils, and amino-modified silicone oils.

Examples of the fluorine-based oil include perfluoropolyether and the like.

Examples of animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, new flower oil, soybean oil, corn oil, rape oil, apricot kernel oil, palm kernel oil, palm oil, castor oil, , Corn oil, palm oil, palm oil, cucumber nut oil, wheat germ oil, rice germ oil, shea butter, coltsfoot colostrum, marker daisy nut oil, mead home oil, egg oil, , Canned wax, carnauba wax, liquid lanolin, hardened castor oil, and the like.

Examples of the moisturizing agent include water-soluble low-molecular moisturizing agents, oil-soluble molecular moisturizing agents, water-soluble polymers, and oil-soluble polymers.

Examples of the water-soluble low-molecular moisturizing agent include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B Glycol (polymerization degree n = 2 or more), polyglycerin B (polymerization degree n = 2 or more), lactic acid, lactic acid salt and the like.

Examples of the lipid-soluble low-molecular moisturizing agent include cholesterol and cholesterol ester.

Examples of the water-soluble polymer include carboxyvinyl polymer, polyaspartic acid, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, dextrin, etc. .

Examples of the oil-soluble polymer include polyvinylpyrrolidone / eicosene copolymer, polyvinylpyrrolidone / hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymer silicone.

Examples of the emollients include long chain acyl glutamic acid cholesteryl ester, hydroxystearic acid cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid and lanolin fatty acid cholesteryl ester.

Examples of the surfactant include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

Examples of the nonionic surfactant include self emulsifying monostearate glycerin, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbit fatty acid ester, POE (Polyoxyethylene / polyoxypropylene) copolymer, POE.POP alkyl ether, polyether-modified silicone, polyether-modified silicone, polyoxyethylene-polyoxypropylene (POE) Alkanolamides, alkylamine oxides, hydrogenated soybean phospholipids, and the like.

Examples of the anionic surfactant include fatty acid soap, alpha-acylsulfonate, alkylsulfonate, alkylarylsulfonate, alkylnaphthalenesulfonate, alkylsulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl ginseng salt, Alkylsulfosuccinic acid salts, acylated hydrolyzed collagen peptide salts, and perfluoroalkyl phosphoric acid esters, and the like can be mentioned. have.

Examples of the cationic surfactant include alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, behenyl trimethyl ammonium chloride, Benzalkonium, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, quaternary ammonium salts of lanolin derivatives, and the like.

Examples of the amphoteric surfactant include carboxybetaine type, amide betaine type, sulfobetaine type, hydroxysulfobetaine type, amidosulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type and amide amine type Amphoteric surfactants and the like.

Examples of the organic and inorganic pigments include inorganic pigments such as silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, Bengala, clay, bentonite, titanium mica, titanium oxide, bismuth chloride, zirconium oxide, magnesium oxide, Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, chromium oxide, chromium oxide, chromium hydroxide, But are not limited to, polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluororesin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, Silk powder, cellulose, CI Pigment Yellow, CI Pigment Orange, and composite pigments of inorganic pigments and organic pigments thereof.

As the organic powder, metallic soap such as calcium stearate; Metal salts of alkyl phosphates such as sodium zinc cetylate, zinc laurylate and calcium lauryl laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc and N-lauroylglycine calcium; Amidosulfonic acid multivalent metal salts such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; Such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoylidene, N-alpha-paratyylnitine, N-alpha-lauroyl arginine, Acyl basic amino acids; N-acylpolypeptides such as N-lauroylglycylglycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid, alpha-aminoaurauric acid, and the like; Polyethylene, polypropylene, nylon, polymethylmethacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride, and the like.

Examples of ultraviolet absorbers include paraaminobenzoic acid, ethyl parnamobenzoate, amyl paranobenzoate, octyl paranobenzoate, ethyleneglycol salicylate, phenyl salicylate, benzyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, benzyl cinnamate , Octyl methoxycinnamate, dioctyl methoxycinnamate, mono-2-ethylhexane glyceryl dipyrromethoxycinnamate, isopropyl paratumoxycinnamate, diisopropyl-diisopropyl cinnamate ester mixture, Carninoic acid, ethyl urocanoate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and salts thereof, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone disulfonate, dihydroxybenzophenone , Tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianylino-p- (carbo-2'-ethylhexyl- , 3,5-triazine, 2- (2- When the like can be mentioned 5-methylphenyl) benzotriazole.

Examples of the disinfectant include hinokitiol, trichloroacid, trichlorohydroxydiphenyl ether, crohexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zinc filitione, benzalkonium chloride, No. 301, mononitro and eicol sodium, and undecylenic acid.

Examples of the antioxidant include butylhydroxyanisole, gallic acid propyl, and eicosorbic acid.

Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogenphosphate and the like.

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

In addition, any of the above-mentioned components may be blended within the range not to impair the objects and effects of the present invention, but it is preferably 0.01 to 5% by weight based on the total weight, More preferably from 0.01 to 3% by weight.

The anti-inflammatory cosmetic composition of the present invention or the composition for external skin application for prevention and improvement of skin diseases caused by oxidative stress and inflammation can take the form of solution, emulsion, and a viscous mixture.

The cosmetic composition of the present invention can be prepared into any formulation conventionally produced in the art, and examples thereof include emulsions, creams, lotions, packs, foundations, lotions, essences, and hair cosmetics.

Specifically, the cosmetic composition of the present invention can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, Packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

[Example]

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 1: Synthesis of a novel peptide HaGF derived from a larva of a ladybug and cell culture

1-1: Synthesis of novel peptide HaGF derived from ladybug larvae

A novel peptide HaGF derived from a ladybug larva consisting of the amino acid sequence shown in SEQ ID NO: 1 was synthesized at a purity of 98% or more with the aid of AnyGen (Gwang-ju, Korea) and dissolved in 0.01% acetic acid.

[SEQ ID NO: 1]: GEILHRFRRSFCDY-NH ₂

1-2: Mouse macrophage (Raw 264.7) culture

Mouse macrophage Raw 264.7 (available from Korean Cell Line Bank) was inoculated into DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal Bovine Serum) (Invitrogen) and 1% penicillin- streptomycin (Invitrogen) The cells were cultured at 37 ° C under 5% CO ₂ .

Example 2: Cytotoxicity test

Cytotoxicity measurement of the novel peptide HaGF derived from the larva of the ladybug larva consisting of the amino acid sequence shown in SEQ ID NO: 1 prepared in Example 1 was analyzed by the MTT method. Mouse macrophages, Raw 264.7, were seeded in 96-well plates at a concentration of 5 × 10 ⁴ cells / ml and treated with HaGF at concentrations of 5, 10, 25, 50, 100 and 500 μg / ml for 24 h . 20 μl of MTT solution per well was added and reacted for 4 hours at 37 ° C. in a 5% CO ₂ incubator. The absorbance was measured at 540 nm using a microplate reader (Beckman, USA) The cell viability is shown in Fig.

From FIG. 1, it was confirmed that the cell viability was reduced by 95% at a concentration of 500 / / ml although the toxicity did not appear up to a concentration of 100 / / ml of HaGF.

Therefore, HaGF has low cytotoxicity at a concentration of 100 μg / ml or less and does not affect cell viability. The anti-inflammatory effect of HaGF does not inhibit the production of cellular inflammatory mediators by simple cell death, Effect.

Example 3: Evaluation of inducibility of early-inflammatory factor production and suppression of expression of a novel peptide HaGF derived from a ladybug larva

3-1: Assessment of nitric oxide (NO) formation inhibition

Mouse macrophage Raw 264.7 was dispensed into 96-well plates at a concentration of 5 × 10 ⁵ cells / ml and cultured in a 5% CO ₂ incubator for 24 hours. After culturing, Raw 264.7 cells were treated with 1 μg / ml of LPS (lipopolysaccharide) and treated with HaGF at 1, 5, 10, 25, 50, 100 μg / ml for 1 hour and then cultured for 24 hours. Next, the supernatant of the culture was reacted with the same amount of griess reagent (Sigma), and the absorbance at 540 nm was measured with an ELISA reader and the percentage of nitric oxide production was shown in FIG. 2 as a percentage.

2, after the treatment with LPS (lipopolysaccharide), the amount of nitric oxide production was increased about 4 times as compared with that of normal cells (Nor), and it was confirmed that inhibition of nitric oxide formation was caused by 0% or more at a concentration of HaGF 100 μg / ml Could.

3-2: Evaluation of inhibitory effect of iNOS and COX-2 on expression

Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was measured by Western blotting to confirm the inhibition mechanism of NO production by HaGF .

Mouse macrophage Raw 264.7 was dispensed into a 100 mm cell culture dish at a concentration of 5 × 10 ⁵ cells / ml and cultured in a 5% CO ₂ incubator for 24 hours. After culturing, Raw 264.7 cells were treated with 1 μg / ml of LPS (lipopolysaccharide) and treated with HaGF at 1, 5, 25, 50, 100 μg / ml for 1 hour and then cultured for 24 hours. Next, the cells were washed twice with PBS (Phosphate Buffered Saline), proteins were extracted using a lysis buffer, and the supernatant was recovered by centrifugation.

The supernatant was quantitated by Bradford assay, 10% SDS-PAGE was performed, and the developed protein was transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skim milk for 1 hour.

Rabbit COX-2 (1: 1000) (cayman) was used as an antibody to examine the expression level of COX-2 and anti-mouse iNOS (BD bioscienc) ) Was diluted in TBST solution, reacted overnight at 4 ° C, and then washed three times with TBST solution. Anti-rabbit IgG (Santa Cruz) conjugated with horse radish peroxidase (HRP) was diluted 1: 1000 as a secondary antibody, reacted for 2 hours at room temperature, and washed three times with TBST solution to obtain ECL substrate (Amersham Co. And the reaction was performed for 1 to 3 minutes. Then, development and quantification were performed using a LAS 4000 chemiluminescence detection system (Fuji, Tokyo, Japan), and the results are shown in FIG.

FIG. 3 shows that the expression level of HaGF induced by lipopolysaccharide (LPS) increased in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) Concentration-dependent decrease in the number of the cells. That is, up to 49% inhibition of iNOS protein expression and up to 51% inhibition of protein expression of cyclooxygenase-2 (COX-2) according to the band density ratio of β-Actin Respectively.

These results indicate that iNOS and COX-2 expressed by LPS stimulate a large amount of NO, and that HaGF inhibits NO production as a result.

3-3: Inhibition of TNF-α, IL-6 and IL-1β production

In order to confirm the inhibitory effect of HaGF on TNF-α, IL-6, and IL-1β (interleukin-1β) R & D systems).

Mouse macrophages, Raw 264.7, were plated at a density of 5 × 10 ⁵ cells / ml in a 96-well plate, washed with 5% CO ₂ And incubated in an incubator for 24 hours. After culturing, Raw 264.7 cells were treated with 1 μg / ml of LPS (lipopolysaccharide) and treated with HaGF at 1, 5, 25, 50, 100 μg / ml for 1 hour and then cultured for 24 hours.

After culturing, TNF-α, IL-6 and IL-1β produced from the medium were measured, and the results are shown in FIG.

For reference, after the incubation, 50 μl Assay Diluent was added to the culture medium in accordance with the protocol of the ELISA Kit for measurement of TNF-α, IL-6 and IL-1β produced from the culture medium, and the sample was placed and left at room temperature for 2 hours. After washing the wells 5 times, 100 의 of conjugate was added, incubated at room temperature for 2 hours, and then washed 5 times. Subsequently, 100 μl of the substrate solution was added, and the solution was allowed to stand in a dark place for 30 minutes, followed by the addition of 100 μl of stop solution, followed by measurement at 450 nm.

4, LPS (lipopolysaccharide) increased the production of TNF-α, IL-6 and IL-1β, but when HaGF was treated at a concentration of 100 μg / ml, IL- 14%. ≪ / RTI >

Thus, in general, LPS acts on macrophages to induce inflammatory responses by promoting the secretion of TNF-α, IL-6 and IL-1β, but HaGF inhibits IL-6 among these three cytokines Showed the best efficacy, and therefore, is expected to be effective in the treatment of inflammatory diseases.

For reference, all experiments in the examples were repeated three times, and the results were expressed as means ± standard deviation. Statistical analysis was performed using the SPSS 10.0 program with significance ( ^* p <0.05, ^** p <0.01 ) Were analyzed by variance analysis (ANOVA) followed by multiple comparisons with tukey test.

Preparation Example 1: Preparation of various formulations containing novel peptide HaGF derived from ladybug larvae

1-1: Preparation of skin lotion

Skin lotion in the cosmetic containing HaGF of Example 1-1 was prepared as follows.

ingredient weight(%) HaGF
glycerin
Carboxyvinyl polymer
Alcohol
Tea trio day
Phenoxyethanol
Small polyphonic
Methyl paraoxybenzoate
Citric acid
Sodium City Trade
Spices
Purified water 1.0
1.0
0.2
5.0
0.02
0.3
0.2
0.15
0.04
0.
0.05
Balance system 100.0

1-2: Manufacture of Lotion

A lotion in the cosmetic containing HaGF of Example 1 was prepared as follows.

ingredient weight(%) HaGF
glycerin
Carboxyvinyl polymer
Beads wax
Decaglyceryl myristate
Squalane
Methyl polysiloxane
Non-sabo roll
Phenoxyethanol
Butyl p-hydroxybenzoate
Spices
Purified water 1.0
7.0
0.6
1.8
2.0
6.0
2.0
0.1
0.7
0.1
0.1
Balance system 100.0

1-3: Manufacture of serum

A serum in cosmetics containing HaGF of Example 1 was prepared as follows.

ingredient weight(%) HaGF
Amino coat
Carboxyvinyl polymer
Arbutin
ethanol
1,3-butylene glycol
Triethanolamine
Methyl paraoxybenzoate
Spices
Purified water 1.0
7.0
0.6
1.8
6.0
2.0
0.1
0.1
0.1
Balance system 100.0

1-4: Manufacture of Cream

A cream in a cosmetic containing HaGF of Example 1 was prepared as follows.

ingredient weight(%) HaGF
Stearic acid
Cetanol
glycerin
Sorbitol rebate
Methyl polysiloxane
Phenoxyethanol
Beta Glucan
Methyl paraoxybenzoate
Non-sabo roll
Spices
Purified water 1.0
0.5
1.5
7.0
0.3
0.3
0.7
3.0
0.2
0.05
0.1
Balance system 100.0

EXPERIMENTAL EXAMPLE 1: Performance Evaluation of Formulation

In order to confirm whether skin lotions, lotions, creams and creams manufactured in Production Examples 1-1 to 1-4 were caused to develop dermatitis, a test for the patches was conducted on human arms and the like in preliminary experiments.

Patch test Waggoner etc. (Waggoner WC et al., Marcel Dekker. New York 105, 1990), Matsumura , etc. (Mastsumura H. et al., Contact Dermatitis. 33, 231-235, 1995), Aberer etc. (Aberer W. et al ., Contact Dermatitis 28 , 1-2, 1993), Kim (Kim, JI, Journal of the Society of Cosmetic Scientists of Korea 23 , 159-184, 1997), the formulation containing HaGF stored at room temperature for 30 days was applied to the skin of the inside of the arm with a Finn chamber on a Scanpor tape for 24 hours, It was visually confirmed. This experiment was carried out on 10 persons in each of male and female, and the results are shown in Table 5 below.

Formulation Subjects who responded after 24 hours (persons) Number of subjects who responded after 48 hours (persons) 0 One 2 3 4 0 One 2 3 4 Skin Lotion 20 0 0 0 0 20 0 0 0 0 Lotion 20 0 0 0 0 20 0 0 0 0 Serum 20 0 0 0 0 20 0 0 0 0 cream 20 0 0 0 0 20 0 0 0 0

As shown in Table 5, the skin lotions, lotions, serum, and creams manufactured in Production Examples 1-1 to 1-4 were found to be in a non-stimulated range and thus safe for human body.

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. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Cosmetic Composition and Topical Composition for          Anti-inflammation Containing Peptide derived from Harmonia          axyridis larva <130> P2012-0197 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> HaGF <400> 1 Gly Glu Ile Leu His Arg Phe Arg Arg Ser Phe Cys Asp Tyr   1 5 10

Claims (12)

A novel peptide HaGF derived from a ladybug larva consisting of the amino acid sequence shown in SEQ ID NO:
Anti-inflammatory cosmetic composition.
The method according to claim 1,
The new peptide HaGF derived from the ladybug larva is contained in an amount of 0.005 to 5% by weight based on the total weight of the anti-inflammatory cosmetic composition.
Anti-inflammatory cosmetic composition.
3. The method of claim 2,
Wherein the new peptide HaGF derived from the ladybug larva is contained in an amount of 0.025 to 5% by weight based on the total weight of the anti-inflammatory cosmetic composition,
Anti-inflammatory cosmetic composition.
4. The method according to any one of claims 1 to 3,
Wherein the new peptide HaGF derived from the ladybug larva has an anti-inflammatory effect by inducing the production and suppression of pro-inflammatory mediator.
Anti-inflammatory cosmetic composition.
5. The method of claim 4,
The pro-inflammatory mediator may be selected from the group consisting of nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2 ), TNF-alpha (tumor necrosis factor-alpha), IL-1 beta (interleukin-1 beta), and IL-6 (interleukin-6)
Anti-inflammatory cosmetic composition.
A novel peptide HaGF derived from a ladybug larva consisting of the amino acid sequence shown in SEQ ID NO:
A composition for external application for skin for preventing and improving skin diseases.
The method according to claim 6,
The new peptide HaGF derived from the ladybug larva is contained in an amount of 0.005 to 5% by weight based on the total weight of the composition for external application for skin disease prevention and improvement,
A composition for external application for skin for preventing and improving skin diseases.
8. The method of claim 7,
Wherein the new peptide HaGF derived from the ladybug larva is contained in an amount of 0.025 to 5% by weight based on the total weight of the skin external composition for skin disease prevention and improvement,
A composition for external application for skin for preventing and improving skin diseases.
The method according to claim 6,
Wherein the new peptide HaGF derived from the ladybug larva has an anti-inflammatory effect by inducing the production and suppression of pro-inflammatory mediator.
A composition for external application for skin for preventing and improving skin diseases.
10. The method of claim 9,
The pro-inflammatory mediator may be selected from the group consisting of nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2 ), TNF-alpha (tumor necrosis factor-alpha), IL-1 beta (interleukin-1 beta), and IL-6 (interleukin-6)
A composition for external application for skin for preventing and improving skin diseases.
11. The method according to any one of claims 6 to 10,
The skin disease is a skin disease caused by oxidative stress and inflammation,
A composition for external application for skin for preventing and improving skin diseases.
12. The method of claim 11,
Wherein the skin disease is selected from the group consisting of allergic dermatitis, atopic dermatitis, contact dermatitis and eczema.
A composition for external application for skin for preventing and improving skin diseases.

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