KR100761331B1 - A topical composition comprising the extract, the fraction and the isolated compounds from aerial part of platycodon grandiflorum - Google Patents

Abstract

A composition comprising an extract of an aerial part of Platycodon grandiflorum, a polyphenol fraction thereof or a compound isolated therefrom is provided to inhibit collagenase and active oxygen species, thereby being applied as a pharmaceutical and a cosmetic composition for preventing skin aging and improving wrinkle. A composition for preventing and treating wrinkles caused by skin aging comprises an extract of Platycodon grandiflorum, which is prepared by extracting an aerial part of the Platycodon grandiflorum with methanol, or luteolin-7-P-glucoside isolated therefrom as an effective ingredient. The composition is in the formulation of cream, gel, patch, sprayer, ointment, adhesive plaster, lotion, liniment, paste type or cataplasma.

Description

A topical composition comprising the extract, the fraction and the isolated compounds from aerial part of Platycodon grandiflorum}

1 is a diagram showing the HPLC chromatogram of the bellflower ground portion extracted with methanol solvent,

FIG. 2 shows luteolin-7-O-glucoside (125 MHz, DMSO) in the ¹³ C-NMR spectrum.

     3 is a diagram illustrating luteolin-7-O-glucoside in the DEPT 90 NMR spectrum,

4 is a diagram showing luteolin-7-O-glucoside in the DEPT 135 NMR spectrum,

5 is a diagram showing luteolin-7-O-glucoside (500 MHz, DMSO) in the ¹ H-NMR spectrum,

6 is a diagram showing luteolin-7-O-glucoside in ¹³ C- ¹ H COSY spectrum,

7 is a diagram showing a luteolin -7-O- glucoside in the ¹ H- ¹ H COSY spectrum,

8 shows luteolin-7-O-glucoside in the ¹³ C- ¹ H HMBC spectrum,

        9 is a diagram showing the type 1 collagenase inhibitory effect of the bellflower ground extract,

        10 is a diagram showing the inhibitory effect of type 3 collagenase of the bellflower ground extract,

        11 is a diagram showing the collagenase-related gene expression inhibitory effect of the bellflower ground extract,

        12 is a diagram showing the inhibitory effect of the active oxygen species of the bellflower ground extract,

        Figure 13 is a diagram showing the inhibitory effect of AP-1 (activator protein-1) activity of bellflower ground extract,

  14 is a diagram showing the AP-1-luciferase vector map of the bellflower ground extract.

     The present invention relates to a composition comprising a bellflower ground extract, fractions thereof, or a compound separated therefrom that exhibits anti-aging and anti-wrinkle effect.

Human skin is constantly changing, the most representative of which is deterioration of the skin function due to aging and reduction of visual beauty. Skin aging is classified into internal aging due to genetic factors and external aging due to external environmental factors such as sunlight. Such external aging may cause wrinkles on the skin, and representative factors of wrinkle formation include active oxygen, dispatch ultraviolet rays, and decreased biosynthesis of collagen (Danielle, A study in the epidermiology of crow's feet., Ann . Intern . Med . , 75 , pp. 873-880, 1971; Grove et al., Optical profilometry; an objective method for quantification of facial wrinkles., J. Am . Acad . Dermatol ., 21 , pp. 631-637, 1989). In the case of internal aging of the skin, artificial control is impossible, but in the case of external aging, it is easier to control than internal factors such as removal of free radicals, growth of fibroblasts and promotion of collagen biosynthesis (Schwartz et al., Topical all-trans retinoic). acid stimulates collagen synthesis in vivo., J. Invest. Dermatol., 96 (6), pp.975-978, 1991).

As human skin ages, a great deal of change occurs both in quantity and quality. In addition to this natural aging is stimulated by various external environments. That is, a series of changes are caused by molecular biological changes or external physical and chemical factors. In particular, damage caused by ultraviolet rays (ultraviolet ray) included in sunlight is called photoaging, and research on this has been actively conducted. In general, ultraviolet light is classified into three types according to its wavelength: ultraviolet A (320-400 nm), ultraviolet B (280-320 nm), and ultraviolet C (190-280 nm). Among the ultraviolet rays reaching the surface from sunlight, ultraviolet C is absorbed, scattered and filtered in the ozone layer in the upper part of the atmosphere, and thus does not affect the natural photochemical reaction. Ultraviolet A is primarily emitted from the sun but also by artificial lamps and penetrates deeply into the epidermis and dermis of the skin. Therefore, UV A is currently regarded as the most influential factor on the skin. However, ultraviolet B irradiation is more dangerous than ultraviolet A. In other words, UV B is shorter in wavelength than UV A, so it does not penetrate deep into the skin. However, UV B has much stronger energy than UV A, causing sharp erythema on the surface of the skin and promoting photoaging of the skin. One of the causes of skin damage caused by ultraviolet rays is MMP (matrix), an enzyme that breaks down collagen or gelatin, which is a component of the extracellular matrix (ECM), of free radicals formed by UV rays, signaling to cellular signaling systems. By synthesizing metalloproteinases, collagen and gelatin are broken down to form wrinkles on the skin. Many functional cosmetics have been developed so far to suppress photoaging, and most of them use vitamin A-based retinol and vitamin C-based ascorbic acid. However, these two components are unstable at room temperature, so if the cosmetics are stored for a long time, they are easily degraded and lose their efficacy. In order to prevent the deactivation of these two components, various methods have been mobilized, such as binding fatty acids to these substances, or collecting them into liposomes.

Collagen is a major matrix protein produced by skin fibroblasts and is present in extracellular epilepsy. It is an important protein that accounts for 30% of the total weight of biological proteins and has a solid triple helix structure. Its main functions are known as mechanical firmness of skin, resistance of connective tissue and binding of tissue, support of cell adhesion, induction of cell division and differentiation. Collagen is also destroyed by ultraviolet light exposure, which is an external cause of skin aging, and the change by ultraviolet light is known to be proportional to the accumulation of time exposed to ultraviolet light. Ultraviolet rays accumulate elastic fibrous material in the dermis of the skin and denature collagen fibers, causing wrinkles on the skin and reducing elasticity.

Meanwhile, matrix metalloproteinases (MMPs) are calcium and zinc-dependent endopeptidase secreted from cells such as polymorphonuclear neutrophils, macrophages, fibroblasts, bone cells, and the like. Endopeptidase) works at neutral pH and uses various extracellular matrix as substrate. These proteolytic enzymes have various pathological processes such as embryonic development, tissue formation, cancer metastasis, periodontal disease, rheumatoid arthritis, inflammation, diabetes, corneal ulcer, osteoporosis, gastric ulcer, trauma, skin aging and wrinkles, burns and wounds It is known to be involved in the disease. As research for the prevention and treatment of wrinkles has progressed, important functions of collagen have been revealed in the skin. From these studies, if collagen metabolism is activated by promoting the synthesis of collagen in the skin, the components of dermal matrix are increased, which improves wrinkles and elasticity. It has been found to have effects such as enhancement and skin strengthening. Retinoids (RE36068), TGF-β (trans-forming growth factor), animal placental proteins (JP Pyung 08-2312709 (A)), betulinic acid (JP Pyung) 08-208424 (A)) and chlorella extracts (JP-P09-09-0405239, JP-P10-0362839 (A)) are known, but retinoic acid is unstable and its use is limited due to stability problems such as irritation and redness when applying skin. There are chlorella extracts, such as the effect is insignificant and can not be expected to substantially improve the skin function due to the promotion of collagen synthesis of the skin. Recently, new wrinkle treatments include ultrasound therapy and skin scaling, laser dermabrasion, botulinum toxin injection, restilene injection, etc., but they do not show significant effects in terms of cost and persistence. Therefore, there is an urgent need for development and discovery of a promoter having excellent collagen synthesis promoting effect in vivo.

Bellflower (Gilyeong, Platycodon grandiflorum A. DC) has been used in respiratory diseases such as drainage, expectoration, bronchitis, and asthma, and has various pharmacological effects, with 273 cases of oriental medicine prescription with bellflower and 49 cases of chemotherapy. It is known to have.

The main pharmacological component of the ground top of the bellflower is terpene saponin. The pharmacological component is antitussive, expectorant, central nervous suppression (sedative, analgesic, antipyretic effect), anti-inflammatory action against acute inflammation, and anti-ulcer through animal experiments. And gastric secretion inhibition, anticholinergic action to lower blood pressure by dilation of blood vessels, hypoglycemic activity and cholesterol metabolism improvement (Tada A. et al., Chem . Pharm . Bull ., 23 , pp2965, 1975 ).

In addition, the hot water and ethanol extract of bellflower inhibit the fungal aflatoxin, and the inulin fraction is known to have phagocytosis, potent anti-tumor effect against solid and ascites, and 40% bellflower extract is known to inhibit alcohol absorption. JP Small 60-0894279 (A)).

      The inventors of the present invention are looking for an agent that inhibits skin aging against UV rays from safe and non-toxic natural products, while useful physiologically active substances in the ground area of the bellflower promote collagenase synthesis and inhibit collagenase enzyme activity and gene expression. It was confirmed that the inhibition of the present invention was completed.

An object of the present invention is to provide a skin external pharmaceutical composition and cosmetic composition containing a bellflower ground extract, fractions thereof, or a compound separated therefrom showing anti-aging and anti-wrinkle effect.

      In order to achieve the above object, the present invention provides a skin external pharmaceutical composition for preventing skin aging and wrinkle improvement containing a bellflower ground extract or polyphenol fraction as an active ingredient.

     In another aspect, the present invention provides a skin external pharmaceutical composition for preventing skin aging and wrinkle improvement containing luteolin-7-O-glucoside, which is separable from the above-ground extract of bellflower.

     Extracts as defined herein include extracts available in water, including purified water, lower alcohols having 1 to 4 carbon atoms or at least one solvent selected from the group consisting of propylene glycol, ethyl acetate, butylene glycol, ether, chloroform.

       Hereinafter, the present invention will be described in detail.

      The extract of the present invention is dried on the ground portion of the bellflower, preferably the leaves and stems of the bellflower so that the water content is 5% or less and pulverized, water, lower alcohol or propylene glycol having 1 to 4 carbon atoms, acetic acid as an extraction solvent Ethyl, butylene glycol, ether or chloroform, preferably water or ethanol, 0.5 to 30 times, preferably 5 to 15 times the volume of the bellflower ground powder, 10 to 100 ℃, preferably 40 to 95 ℃ Extraction methods such as cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, heating extraction for 0.5 to 48 hours, preferably 2 to 10 hours at the extraction temperature, preferably 1 to 7 times by hot water extraction, preferably After extracting 1 to 3 times, filtered and freeze-dried can be obtained the bellflower extract of the present invention.

      In addition, the present invention can purify and isolate the polyphenol fraction and luteolin-7-O-glucoside which is the main bioactive substance having skin aging prevention and anti-wrinkle effect from the bellflower ground extract obtained from the manufacturing method,

       Specifically, the polyphenol fraction of the present invention and the compound to be separated therefrom are subjected to HPLC of the bellflower ground extract obtained in the above manufacturing process under specific conditions as follows, and the polyphenol fraction and luteolin-7 containing a large amount of polyphenol components. -O-glucoside can be purified and separated.

       [HPLC Terms}

      Column: Lichrospher RP-18 column (250 × 4 mm I.D. Merck),

      Mobile phase: D.W. (solvent A) with 0.1% acetic acid and methanol (solvent B) with 0.1% acetic acid,

      -20 μL sample addition

       Flow rate 1 mL / min,

      UV meters: 254 and 270 nm

      In addition, the present invention can prepare a polyphenol fraction having a skin aging prevention and anti-wrinkle effect from the bellflower ground polyphenol extract obtained from the manufacturing method,

       Specifically, the polyphenol fraction of the present invention can purify the polyphenol fraction containing a large amount of polyphenol components by performing HPLC under specific conditions on the bellflower ground extract obtained in the manufacturing process. As a result of confirming the collagenase inhibitory effect and the active algae inhibitory effect of the bellflower ground extract obtained by the above extraction method, it was confirmed that the excellent effect.

The present invention provides a skin external pharmaceutical composition for preventing skin aging and improving wrinkles, which contains a bellflower ground extract obtained by the above method as an active ingredient.

As defined herein, a "flower field" includes the leaves, stems, of the bellflower.

The pharmaceutical composition for preventing skin aging and improving wrinkles of the present invention comprises 0.1 to 50% by weight of the extract or compound based on the total weight of the composition.

      Pharmaceutical compositions comprising the extract or compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

     Pharmaceutical compositions comprising extracts or compounds according to the invention can be used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, respectively, according to conventional methods. And, preferably, in the form of an external preparation for the skin of a cream, gel, patch, spray, ointment, warning, lotion, linen, pasta or cataplasma. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The external skin pharmaceutical composition for skin aging and wrinkle improvement containing bellflower extract or compound of the present invention is an external skin formulation that can be applied to the skin, such as creams, gels, patches, sprays, ointments, warning agents, lotions, and linings. Preparation, or may be used as a pharmaceutical composition in the form of an external preparation for skin of pasta or cataplasma, but is not limited thereto.

     Preferred dosages of the extracts or compounds of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In another aspect, the present invention provides a cosmetic composition having a skin aging prevention and anti-wrinkle effect containing bellflower extract or polyphenol fraction as an active ingredient.

In another aspect, the present invention provides a cosmetic composition having a skin aging prevention and anti-wrinkle effect containing luteolin-7-O-glucoside as an active ingredient that is separable from the extract of the bellflower.

The cosmetic composition of the present invention comprises 0.01 to 10% by weight, preferably 0.1 to 8% by weight, most preferably 0.5 to 5% by weight, based on the total weight of the composition.

     The composition containing the bellflower ground extract or compound of the present invention may be used in various ways, such as cosmetics and face wash for the anti-aging and wrinkle improvement effect of the skin. Examples of products to which the present composition can be added include cosmetics such as various creams, lotions, skins, and the like, cleansing agents, face washes, soaps, treatments, and essences.

    Cosmetics of the present invention comprises a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract.

    The water-soluble vitamins may be any compound that can be incorporated into cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like. Their salts (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, etc.) may also be used in the water-soluble vitamins that can be used in the present invention. Included. The water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microorganism culture, enzyme or chemical synthesis.

    The oil-soluble vitamin may be any compound that can be incorporated into cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), and the like. And derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherolvitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl) Ethers, etc.) are also included in the oil-soluble vitamins used in the present invention. Oil-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification of microorganism culture, enzyme or chemical synthesis.

     The polymer peptide may be any compound as long as it can be incorporated into cosmetics. Preferably, collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, and the like can be given. Polymeric peptides can be purified and obtained by conventional methods such as purification from microbial cultures, enzymatic methods or chemical synthesis methods, or can be purified and used from natural products such as dermis and pig silk such as pigs and cattle.

      The polymer polysaccharide may be any compound as long as it can be blended into cosmetics. Preferably, hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.) may be mentioned. For example, chondroitin sulfate or its salt can be normally purified from a mammal or fish.

     The sphingolipid may be any compound as long as it can be blended into cosmetics. Preferably, ceramide, phytosphingosine, sphingosaccharide lipid, etc. may be mentioned. Sphingolipids can usually be purified from mammals, fish, shellfish, yeasts or plants by conventional methods or obtained by chemical synthesis.

     The seaweed extract may be any compound as long as it can be blended into cosmetics. Preferably, the seaweed extract may include brown algae extract, red algae extract, green algae extract, and the like. Also, calginine, arginic acid, sodium arginate, Potassium arginate and the like are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purification from seaweed by conventional methods.

In addition to the said essential component, you may mix | blend the cosmetics of this invention with the other components normally mix | blended with cosmetics as needed.

Other components that may be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, Blood circulation accelerators, cooling agents, restriction agents, purified water and the like.

    Examples of the fat or oil component include ester fats, hydrocarbon fats, silicone fats, fluorine fats, animal fats, and vegetable fats and oils.

As ester fats and oils, glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl mystinate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, and stearic acid Butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl acid isocetyl, isostyl acid isostearyl, isostaryl palmitate, octylate acid octyldodecyl, isostearic acid isetyl, diethyl sebacate, adipine Sandiisopropyl, isoalkyl neopentane, tri (capryl, capric acid) glyceryl, trimethyl ethyl trimethylolpropane, trimethyl stearate trimethylol propane, tetra 2-ethylhexanoic penta erythritol Cetyl caprylate, lauric acid decyl, hexyl laurate, decyl myristin, myristin acid myristyl, myritic acid cetyl, stearyl stearate, decyl oleate, rininooleic acid Teyl, isostearyl laurate, isotridecyl myristin, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyl dodecyl oleate, octyl dodecyl linoleate, isopropyl isopropyl acid, 2-ethylhexanoate cetostearyl, 2-ethylhexanoate stearyl, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicapric acid, propylene glycol di (capryl, capric acid), Dicaprylic acid propylene glycol, dicapric acid neopentyl glycol, dioctanoate neopentyl glycol, tricaprylate glyceryl, triundecyl glyceryl, triisopalmitinate glyceryl, triisostearate glyceryl, octylate octadodode Sil, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isstearyl isostearate, Sothete octylate, polyglycerol oleate, polyglycerine isostearate, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myritic lactate, octyl lactate, octyl lactate Ethyl, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malic acid, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipicate, diisopropyl sebacinate, Dioctyl sebacate, cholesteryl stearate, cholesteryl isostearic acid, cholesteryl hydroxystearate, cholesteryl oleate, oleic acid dihydrocholesteryl, physosterate isostearic acid, phytic oleate , 12-stealoylhydroxystearate isocetyl, 12-stealoylhydroxystearate stearyl, 12-steal One hydroxy stearic acid and the like esters such as cetearyl source.

Hydrocarbon-based fats and oils, such as squalene, a liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, a liquid isoparaffin, polybutene, microcrystal wax, and a vaseline, etc. are mentioned as a hydrocarbon-type fats and oils.

      Examples of the silicone-based oils and fats include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane, methylcetyloxysiloxane copolymer, dimethylsiloxane and methylsteoxysiloxane copolymer, and alkyl. Modified silicone oil, amino modified silicone oil and the like.

Perfluoro polyether etc. are mentioned as fluorine-based fats and oils.

Animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, soybean oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil. , Cottonseed oil, Palm oil, Cucumber nut oil, Wheat germ oil, Rice germ oil, Shea butter, Walnut colostrum oil, Marker demia nut oil, Meadow home oil, Egg yolk oil, Uji, Horse oil, Mink oil, Orange rape oil, Jojoba oil And animal or plant fats and oils such as candeler wax, carnava wax, liquid lanolin and hardened castor oil.

Examples of the moisturizing agent include a water-soluble low molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

     As the water-soluble low molecular moisturizer, serine, glutamine, sorbitol, mannitol, pyrrolidone-sodium carbonate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or more), polypropylene Glycol (polymerization degree n = 2 or more), polyglycerol B (polymerization degree n = 2 or more), lactic acid, lactic acid salt, etc. are mentioned.

    Examples of the fat-soluble low molecular humectants include cholesterol and cholesterol esters.

     Examples of the water-soluble polymer include carboxyvinyl polymer, polyasparaginate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water soluble chitin, chitosan, and dextrin. Can be.

     Examples of the fat-soluble polymers include polyvinylpyrrolidone-eicosene copolymers, polyvinylpyrrolidone-hexadecene copolymers, nitrocellulose, dextrin fatty acid esters, polymer silicones, and the like.

     Examples of the emollient include long-chain acyl glutamic acid cholesteryl esters, hydroxy stearic acid cholesterol, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl esters, and the like.

     As surfactant, nonionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, etc. are mentioned.

      Examples of nonionic surfactants include self-emulsifying monoglycerate, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE castor oil, POE-POP (polyoxyethylene polyoxypropylene) copolymer, POE-POP alkyl ether, polyether modified silicone, laurin Acid alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc. are mentioned.

     Anionic surfactants include fatty acid soaps, alpha-acyl sulfonates, alkyl sulfonates, alkyl allyl sulfonates, alkyl naphthalene sulfonates, alkyl sulfates, POE alkyl ether sulfates, alkylamide sulfates, alkyl phosphates, POE alkyl phosphates, and alkylamide phosphates. , Alkyloylalkyl taurine salt, N-acylamino acid salt, POE alkyl ether carboxylate, alkyl sulfosuccinate, sodium alkyl sulfo acetate, acyl hydrolyzed collagen peptide salt, perfluoroalkyl phosphate ester, etc. are mentioned. .

     As cationic surfactant, alkyl trimethylammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium bromide, behenyl trimethyl ammonium chloride, chloride Benzalkonium, diethylaminoethyl stearate, dimethylaminopropyl stearate, lanolin derivatives, quaternary ammonium salts, and the like.

     Examples of amphoteric surfactants include the carboxybetaine type, the amide betain type, the sulfobetain type, the hydroxysulfobetain type, the amide sulfobetain type, the phosphobetaine type, the aminocarboxylate type, the imidazoline derivative type, and the amideamine type. An amphoteric surfactant etc. are mentioned.

As organic and inorganic pigments, silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengal, clay, bentonite, titanium film mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine and composites thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene, styrene copolymer, Organic pigments such as silk powder, cellulose, CI pigment yellow, CI pigment orange, and composite pigments of these inorganic pigments and organic pigments;

     As organic powder, Metal soaps, such as a calcium stearate; Alkyl phosphate metal salts such as sodium cetylinate, zinc lauryl acid and calcium laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroylglycine calcium; Amide sulfonic acid polyvalent metal salts, such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N-epsilon-lauroyl-L-lysine, N-epsilon-palmitolyzine, N-alpha-paratoylol nitin, N-alpha-lauroyl arginine, N-alpha-cured fatty acid acyl arginine -Acyl basic amino acid; N-acyl polypeptides, such as N-lauroyl glycyl glycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride and the like.

Examples of the ultraviolet absorber include paraaminobenzoic acid, ethyl paraaminobenzoate, amyl paraaminobenzoic acid, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylic acid and benzyl cinnamic acid. , Paramethoxy cinnamic acid-2-ethoxyethyl, paramethoxy cinnamic acid octyl, diparamethoxy cinnamic acid mono-2-ethylhexane glyceryl, paramethoxy cinnamic acid isopropyl, diisopropyl diisopropyl cinnamic acid ester mixture, wuro Canonic acid, ethyl urokanoate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone sodium sulfonate, dihydroxybenzophenone , Tetrahydroxybenzophenone, 4- tert -butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino- p- (carbo-2'-ethylhexyl-1'-oxy) -1 , 3,5-triazine, 2- (2-hi And the like can be mentioned hydroxy-5-methylphenyl) benzotriazole.

      As fungicides, hinokithiol, trichloric acid, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, ginxitlionone, benzalkonium chloride, photosensitive Sodium No. 301, sodium mononitroguicol, undecylenic acid, and the like.

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

      Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, fmaric acid, sodium pmarate, succinic acid, sodium succinate, sodium hydroxide, sodium dihydrogen phosphate, and the like.

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

     In addition, the compounding component which may be added other than this is not limited to this, Moreover, Although all said components can be mix | blended within the range which does not impair the objective and effect of this invention, Preferably it is 0.01-5% weight percentage with respect to a total weight, More preferably 0.01 to 3% by weight.

    The cosmetic of the present invention may take the form of a solution, an emulsion, a viscous mixture, or the like.

Ingredients included in the cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the extract as an active ingredient, for example, conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments and flavorings. And carriers.

    Functional cosmetic composition exhibiting the anti-aging and anti-wrinkle effect of the present invention can be prepared in any formulation commonly prepared in the art, for example, emulsion, cream, lotion, pack, foundation, lotion, essence, hair Cosmetics; and the like.

    Specifically, the cosmetic composition of the present invention skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, Formulations of 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 carriers, vegetable fibers, waxes, paraffins, starches, tracantes, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide, etc. may be used as carrier components. Can be.

     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, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

     When the formulation of the present invention is a solution or emulsion, a solvent, solvating or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

     When the dosage form of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

     When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

       Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example  1.Material production of bellflower ground

 1-1. Extraction Separation from Bellflower Ground

The bellflower ground was harvested in August 2005 at Hadong, Gyeongnam. ¹ H-NMR, ¹³ C-NMR and 2D-NMR spectra were measured using a Bruker AW-500 spectrophotometer for structural characterization of the isolated compounds.

      Methanol (1 L) was added to 100 g of the bellflower grounds (leaves and stems), and extracted three times by reflux cooling at 70 ° C. for 2 hours, followed by filtration using a vacuum filter. The filtrate was concentrated under reduced pressure using a rotary vacuum evaporator (EYELA N-1000, Japan) at 30 ° C. or lower to obtain 12.15 g of a deep green methanol extract.

1-2. Isolation of Polyphenol-Containing Fractions from Bellflower Grounds

      12.15 g of the extract was qualitatively and quantitatively analyzed using a HPLC (Agilent 1100) method.

      Licrospher RP-18 column (250 × 4 mm ID Merck) for separating polyphenol components using HPLC, mobile phase is DW (A solvent) with 0.1% acetic acid and 0.1% acet Acid-containing methanol (solvent B) was used to analyze the proportion of solvent A gradually decreasing from 100% to 0%. A sample addition amount of 20 μL, flow rate of 1 mL / min, and a measuring instrument were separated at 254 and 270 nm by UV to obtain 8.429 g of polyphenol-containing fractions and 100 g of bellflower ground portion.

1-3. Isolation of Luteolin-7-O-Glucoside Compounds

The compound separated as described above was subjected to structural identification through a device such as NMR. The total polyphenol compound content was extracted by reflux cooling for 2 hours at 70 ° C. by adding 100 ml of 50% methanol to 10 g of the ground part, and 3 ml of distilled water and 0.016 M potassium ferricyanide (K ₃ Fe) (CN) ₆ ) Add 1 mL of 0.01 M ferric trichloride (FeCl ₃ /0.1N HCl) solution, mix, and leave for 15 minutes. Stabilizer (H ₂ O: 1% Arabian gum: 85% phosphoric acid = 3: 1 : 1, v / v / v) After adding 5 mL, absorbance was measured at 700 nm, and the content was converted to a calibration curve prepared with gallic acid.

As a result, the main useful bioactive substance was determined by HPLC quantifying It was confirmed that luteolin-7-O-glucoside having the following physical properties.

Luteolin-7-O-glucoside

Molecular Formula: C ₂₁ H ₁₆ O ₁₁

_- yellow amorphous powder form

¹ H-NMR (500 MHz, DMSO- d ₆ ) δ 12.97 (1H, OH), 6.93 (1H, d, J = 2.0, H-2 ′), 6.80 (1h, d, J = 8.0 and 2.0 , H-6 '), 6.80 (1H, d, J = 8.0, H-5'), 6.72 (1H, d, J = 1.8, H-8), 6.47 (1H, d, J = 2.0, H- 6), 5.09 (1H, d, J = 7.2, anomeric H of Glu), 4.09 (1H, s), 2.52 (3H, d, J = 6.2, Glu-CH ₂ );

¹³ C-NMR (125 MHz, DMSO- d ₆ ) δ; 182.16 (C-4), 164.76 (C-7), 163.24 (C-2), 161.54 (C-5), 157.31 (C-9), 149.96 (C-4 '), 145.89 (C-3') , 121.84 (C-1 '), 119.41 (C-6'), 116.17 (C-5 '), 113.69 (C-2'), 105.66 (C-10), 103.50 (C-3), 99.76 (C -6), 95.05 (C-8), 100.26 (Glu-1), 73.35 (Glu-2), 77.41 (Glu-3), 69.78 (Glu-4), 73.35 (Glu-5), 60.85 (Glu- 6).

Reference Example  1. Preparation of Reagents and Cell Lines

       Human fibroblast line, CCD986SK, was purchased from ATCC (Accession No .: CRL-1947), Matrigel, Hematoxylin, Eosin, Gelatin, IMEM Culture, Trypsin, Fetal Serum (Fetal) bovine serum was purchased from Sigma (Sigma, USA). Activator-protein-1 (AP-1) luciferase vector was purchased from Invitrogen Corporation, and an enzo-lyte kit for measuring collagenase activity was purchased from Anaspec. Ltd. .

Experimental Example  One. Collagenase ( collagenase ) Determination of activity inhibitory effect

After passage of CCD986SK cells in 48 well plates, the bellflower ground extract obtained in Example 1 was treated with PBS for 12 hours, washed once with PBS, and irradiated with ultraviolet A (UVA). After 24 hours, only supernatant was taken and 10,000 rpm. Centrifuge for 5 minutes at. 50 μl of the supernatant and a final concentration of 1 mM 4-aminophenylmercuric acetate (APMA) were added to the 96 well plate and mixed well. The MMP-1 enzyme activity was reacted at 37 ° C. for 6 hours and the MMP-13 enzyme activity was 37 It reacted at 40 degreeC for 40 minutes. After each time, MMP-1 and MMP-13 substrate solutions were added and mixed, followed by fluorescence measurement at 5 minutes intervals for 1 hour at an excitation wavelength of 340 nm and an emission wavelength of 490 nm. Type collagenase) and MMP-13 (type 3 collagenase) enzyme activity inhibitory effects were measured.

     Experimental results, as shown in Figures 9 and 10, the bellflower ground extract (methanol extract fraction), polyphenol fraction, and luteolin-7-O-glucoside were all concentration-dependently dependent on the activity of MMP-1 and MMP-13 enzymes. Inhibition was confirmed.

Experimental Example  2. Collagenase  ( collagenase ) Gene expression inhibitory effect

    To investigate the gene expression inhibitory effect of MMP-1 and MMP-13, the CCD986SK cells were pretreated with the bellflower ground extract obtained in Example 1 for 24 hours, washed once with washing buffer (PBS), and irradiated with UVA for 24 hours. Gene expression of MMP-1 and MMP-13 was measured using RT-PCR.

    As shown in FIG. 11, the expression of MMP-1 and MMP-13 genes was reduced by the polyphenol fraction, luteolin-7-O-glucoside, which is a useful bioactive substance of the bellflower ground (methanol extract fraction). Could confirm.

Experimental Example  3. Free radical species ( ROS ) Inhibition effect measurement

    In view of the fact that UVA increases free radical species in skin epithelial cells and increased free radical species increases the expression of collagen degrading enzyme collagenase (matrix metalloproteinases-MMPs), Experiments were conducted to see if they inhibit the formation of.

The CCD986SK cells were passaged in a 48 well plate for 24 hours with 0.1% serum, and then changed to a medium containing 0.1% serum. Then, the bellflower ground extract obtained in Example 1 was treated for 24 hours. After 24 hours, 10 uM of 2'.7-dichlorofluorescein diacetate (2 ', 7'-Dichlorofluorescein diacetate; DCF-DA) was dispensed into each well for color reaction for 30 minutes. After 30 minutes, the medium was removed, replaced with medium containing 0.1% of serum, and irradiated with UVA (200 mJ / cm ² ), followed by fluorescence measurement at an excitation wavelength of 495 nm and an emission wavelength of 520 nm every 5 minutes for 1 hour. It was.

    As shown in FIG. 12, it was confirmed that the bellflower ground extract (methanol fraction), the polyphenol fraction, and the luteolin-7-O-glucoside all reduced the formation of increased intracellular reactive oxygen species by TPA. .

Experimental Example  4. AP -One( activator protein -1) activity inhibition effect

     UVA increases free radical species in skin epithelial cells, and the increased free radical species increases the activity of activator protein-1 (AP-1), an intracellular transcription factor. Focusing on increasing the expression of, experiments were conducted to confirm the transcriptional activity inhibitory effect of the transcription factor AP-1 by the treatment of bellflower ground extract.

Bellflower ground extract obtained in Example 1 by transfection of a vector containing AP-1-luciferase reporter gene (AP-1-luciferase reporter gene-invitrogen corporation, model number: K1118) into CCD986SK cells. After treatment with TPA to increase AP-1 transcriptional activity, an experiment (AP-1-luciferase reporter gene assay) was performed. As a result, as shown in Figure 13, the bellflower ground extract (methanol extract fraction), polyphenol fraction, luteolin-7-O-glucoside reduced the transcriptional activity of AP-1 increased by TPA. Could.

Formulation examples of the cosmetic composition comprising the extract of the present invention will be described, but the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation example  1. Flexible Lotion

number Raw material Content (% by weight)  One Bellflower ground extract of Example 1  0.5  2 Butylene Glycol  4.0  3 glycerin  8.0  4 Carboxy Vinyl Polymer  0.06  5 Disodium ethylenediamineteracetic acid  0.01  6 ethanol  7.0  7 Triethanolamine  0.2  8 Sorbitan monostearate  0.8  9 Hydrogenated Castor Oil  0.4  10 Phenyltrimethicone  3.5  11 Paraoxybenzoic acid propyl  0.2  12 Methyl paraoxybenzoate  0.2  13 Purified water  Remaining amount  system  100.0

Formulation example  2. nutrition lotion

number Raw material Content (% by weight)  One Bellflower ground extract of Example 1  0.5  2 Stearic acid  1.0  3 Cetyl alcohol  1.0  4 Glyceryl Monostearate  1.0  5 Polyoxyethylene sorbitan monostearate  1.0  6 Sorbitan sesquioleate  1.0  7 Liquid paraffin  4.0  8 Carlylic / Capric Triglycerides  4.0  9 Squalane  3.0  10 Carboxy Vinyl Polymer  0.1  11 Butylene glycol  5.0  12 Triethanolamine  0.2  13 Paraoxybenzoic acid propyl  0.2  14 Methyl paraoxybenzoate  0.2  15 Purified water  Remaining amount  system  100.0

Formulation example  3. Nutrition Cream

number Raw material Content (% by weight)  One Bellflower ground extract of Example 1  0.5  2 Stearic acid  2.0  3 Cetyl alcohol  2.0  4 Glyceryl Monostearate  2.0  5 Polyoxyethylene sorbitan monostearate  0.5  6 Sorbitan sesquioleate  0.5  7 Glyceryl Monostearate / Glyceryl Stearate / Polyoxyethylene Stearate  1.0  8 Wax  1.0  9 Liquid paraffin  4.0  10 Carlylic / Capric Triglycerides  4.0  11 Squalane  4.0  12 Carboxy Vinyl Polymer  0.3  13 Butylene glycol  5.0  14 glycerin  3.0  15 Triethanolamine  0.5  16 Paraoxybenzoic acid propyl  0.2  17 Methyl paraoxybenzoate  0.2  18 Purified water  Remaining amount  system  100.0

Formulation example  4. Essence

number Raw material Content (% by weight)  One Bellflower ground extract of Example 1 0.5  2 Cytostolol 1.70  3 Polyglyceryl 2-oleate 1.50  4 Ceramide 0.7  5 Ceteares-4 1.2  6 cholesterol 1.5  7 Dicetylphosphate 0.4  8 Concentrated glycerin 5.0  9 Sunflower oil 15.0  10 Carboxy Vinyl Polymer 0.2  11 Xanthan Gum 0.2  12 antiseptic a very small amount  13 Spices a very small amount  14 Purified water Remaining amount  system 100.0

Production Example  5. Preparation of Hydrophilic Ointment

number Raw material Content (% by weight) Content (% by weight) One   Bellflower ground extract of Example 1 0.5 - 2   Bellflower ground extract of Example 1 - 0.5 3   White vaseline 250 250 4   Stearyl alcohol 220 220 5   Ethyl (or methyl) p-oxybenzoate 0.25 0.25 6   Propylene glycol 120 120 7   Sodium lauryl sulfate 15 15 8   Propyl p-oxybenzoate 0.15 0.15

As described above, the bellflower ground extract, polyphenol fraction and the compound separated therefrom of the present invention show collagenase inhibitory effect and inhibitory effect of reactive oxygen species, and the skin external pharmaceutical composition for skin aging and wrinkle improvement and It can be used as a cosmetic composition.

Claims (8)

      Skin external pharmaceutical composition for the prevention and treatment of wrinkles caused by skin aging containing methanol extract of the bellflower grounds as an active ingredient. delete      A skin external pharmaceutical composition for the prevention and treatment of wrinkles caused by skin aging containing Luteolin-7-O-glucoside isolated from the above-ground extract of bellflower.       The external dermal pharmaceutical composition according to claim 1 or 3, wherein the composition is a cream, gel, patch, spray, ointment, warning, lotion, linen, pasta or cataplasma formulation.        Cosmetic composition for the prevention and improvement of wrinkles caused by skin aging containing methanol extract of the bellflower grounds as an active ingredient. delete        Cosmetic composition for the prevention and improvement of wrinkles caused by skin aging containing Luteolin-7-O-glucoside (Luteolin-7-O-glucoside) isolated from the bellflower ground extract. delete

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