WO2004096171A1

WO2004096171A1 - Skin whitening cosmetic composition comprising the extract of machilus thunbergii and the compounds isolated therefrom

Info

Publication number: WO2004096171A1
Application number: PCT/KR2003/001636
Authority: WO
Inventors: Jong-Keun Son; Seung-Ho Lee; Hyeun-Wook Chang
Original assignee: Yeungnam Educational Foundation
Priority date: 2003-04-29
Filing date: 2003-08-13
Publication date: 2004-11-11
Also published as: AU2003251200A1

Abstract

The present invention is related to a cosmetic composition comprising the extract of Machilus thunbergii and lignan compounds isolated therefrom having potent inhibitory effect on melanin synthesis. Inventive composition shows the excellent inhibitory activity on cultured B-16 melanoma cell therefore, it can be useful in skin whitening cosmetic composition as an cream, skin, lotion, pack and the like.

Description

SKIN WHITENING COSMETIC COMPOSITION COMPRISING THE EXTRACT OF MACHILUS THUNBERGII AND THE COMPOUNDS ISOLATED THEREFROM

TECHNICAL FIELD

The present invention is related to skin whitening cosmetic composition comprising the extract of Machilus Thunbergii and the compounds isolated therefrom.

BACKGROUND ART

Skin hyper-pigmentation comes of various origins such as the hormonal disorder followed by the inflammatory response of skin, genetic disease and ultraviolet irradiation, mainly the synthetic disorder and distribution disorder of melanin pigment. The main function of melanin is to scavenge oxygen radical, which can protect skin from the injury. Therefore, it has been known that the plenty of melanin shows potent response on skin system for protecting skin from physical or chemical toxic substance. Melanin is formed by serial step i.e., converting tyrosine to dopaquinone by tyrosinase enzyme followed by further enzymatic reaction and spontaneous oxidative reaction and so on. Therefore, the inhibiting methods of melanin biosynthesis for protecting skin tanning are classified by follows: (1) UV protecting method to get rid of the main cause of melanin formation, which is expected to give satisfactory results (2) Inhibiting method of core carbohydrate biosynthesis necessary to tyrosinase activity (3) Inhibiting method of the function of tyrosinase enzyme participating in melanin formation using kojic acid or arbutin (4) Inhibiting method of cell differentiation using hydroquinone which has specific toxicity on melanocyte, melanin forming cell, (5) Decolorizing method by reducing melanin formation.

In order to inhibit melanin biosynthesis, ascorbic acid, kojic acid, arbutin, hydroquinone and the extract of natural herb etc have been used till now. However, kojic acid or arbutin, representative skin whitening material, has several problems such as product safety in spite of their strong skin whitening effect.

Recently, the extract of natural herb has been highlighted as skin whitening material due to its safety on human body.

Therefore, there has been needed to study skin whitening material using the extract of natural herb which is safe in human body.

Machilus cortex, a cortex of Machilus thunbergii SIEB. et ZUCC. has been reported to contain lignan, lignan glycoside, alkaloid, flavonoid and essential oil, in particular, 0.48% tannin, 12.38% resin, 0.688% caoutchouc and the plenty of mucus in cortex (B. S. Chung and M. K. Shin; Dohaehyangyak Dictionary, pp458-459, Youngrim Press, 1998).

Korean Patent Publication No. 207958 discloses the preparation method of licarin compounds specifically inhibiting the activity of ACAT (Acyl-CoA: cholesterol Acyltransferase) isolated from the leaves of Machilus thunbergii and the composition containing the same for prevention and treatment of cardiovascular disease.

Korean Patent Publication No. 78252 discloses the skin cosmetics comprising the extract of Chinese Galls (Galla Rhois), Sinapsis semen or Machilus cortex, which have preventing and protecting activity of skin aging by blocking skin cell injury from harmful active oxygen.

Korean Patent Publication No. 352269 discloses pharmaceutical composition comprising novel lignan compound showing anti-oxidative activity for LDL (low- density lipoprotein) for the prevention and treatment of circulatory disease such as artheriosclerosis.

Korean Patent Publication No. 345825 discloses the extraction method of serotonin, lignan and flavonoid isolated from Carthamus tinctorius L stimulating bone formation.

Korean Patent Publication No. 321313 discloses the extract of Magnolia flos and lignan compounds isolated therefrom having inhibiting activity of leucotriene reproduction.

Korean Patent Publication No. 263439 discloses novel lignan compound isolated from Magnolia flos acting as a PAF (Platelet Activating Factor) antagonist.

However, there have been no disclosure or suggestion that the extract of Machilus cortex and lignan compounds isolated therefrom have inhibiting activity of melanin biosynthesis in above disclosed prior references.

Therefore, the present inventors have endeavored to find active substance from lots of plant extract group which can protect from skin pigmentation.

Finally, the present inventors have found that the extract of Machilus Thunbergii and the compounds isolated therefrom have potent inhibiting activity of melanin biosynthesis.

DISCLOSURE OF THE INVENTION

Accordingly, it is an object of the present invention to provide a skin cosmetic composition comprising crude extract of Machilus thunbergii.

Above described crude extract can be soluble in water, lower alcohol such as methanol, ethanol etc or the solvent mixture thereof, preferably, 70% methanol. It is another object of the present invention to provide a skin cosmetic composition comprising a non-polar solvent soluble extract of Machilus thunbergii.

Above described non-polar solvent soluble extract can be soluble in hexane, carbon tetrachloride, chloroform, methylene chloride, ether or ethylacetate, preferably, methylene chloride.

It is another object of the present invention to provide a skin cosmetic composition comprising polar solvent soluble extract of Machilus thunbergii.

Above described polar solvent extract can be soluble in water, lower alcohol such as methanol, ethanol etc or the solvent mixture thereof, preferably, water and obtained by removing non-polar soluble extract from above described crude extract.

The present invention also provides a skin cosmetic composition comprising lignan compounds expressed as following general formula (I), which can be isolated from Machilus thunbergii or synthesized by general procedure well known in the art:

Wherein R¹, R², R³, R⁴, R⁵ and R⁶ are independently hydrogen atom, hydroxyl group, Cl to C3 lower alkyl or Cl to C3 lower alkoxy group,

A and A' are independently hydrogen, Cl to C3 lower alkyl or five -member ring fused with oxygen or sulfur atom each other.

Preferable compound is the group of which R¹, R⁵ and R⁶ is methoxyl group, R² is hydroxyl group, R³ and R is hydrogen atom, A and A' are hydrogen atoms respectively, i.e., mesO-monomethyl dihydroguaiaretic acid, wherein R , R and R is methoxyl group, R¹ is hydroxyl group, R³ and R⁴ is hydrogen atom, A and A' are fused with oxygen atom to form furan ring, i.e., nectandrin A, and wherein R and R is methoxyl group, R¹ and R⁵ is hydroxyl group, R³ and R⁴ is hydrogen atom, A and A' are fused with oxygen atom to form furan ring, i.e., nectandrin B.

The inventive extract and the compounds isolated therefrom of the present invention may be prepared in accordance with the following preferred embodiment.

For the present invention, the bark of Machilus thunbergii is dried at room temperature and cut into small pieces. The each pieces is mixed with 1 to 20-fold, preferably, 2 to 5 -fold volume of water, alcohols such as methanol, ethanol, butanol and the like, or the mixtures thereof, preferably, the mixture of 70% methanol; and is heated at the temperature ranging from 20 to 100°C, preferably from 50 to 90°C, for the period ranging from 1 to 48 hours, preferably 2 to 24 hours, with 1 to 10 times, preferably 2 to 5 times, by hot-water, sonication, reflux or conventional extraction to obtain an aqueous crude extract. The crude extract is added with 1 to 100-fold, preferably, 1 to 5-fold volume of non-polar solvent such as hexane, carbon tetrachloride, chloroform, methylene chloride or ethylacetate and extracted with the non-polar solvent at 1 to 10 times, preferably 2 to 5 times to obtain non-polar solvent soluble fraction. The residue obtained from above fractionation, is collected to obtain polar solvent soluble fraction and to obtain purified fraction thereof and additional conventional fractionation procedure can be subjected in the literature (Harborne J. B. Phytochemical methods: A guide to modern technique of plant analysis. 3^rd Ed., pp6-7, 1998).

For example, dried bark of Machilus thunbergii is subjected to reflux extraction with to 3-fold volume of 70% methanol at the temperature ranging from 50 to 70°C, for the period ranging from 10 to 18 hours, once and similar procedure to above extraction method is repeated with 90% methanol at 2 times to collect and obtain crude extract thereof. The optimum amount of distilled water and methylene chloride are added thereto, extracted and subjected to fractionation with separatory funnel three times to obtain water soluble layer and methylene chloride soluble layers. The purified fraction and preferable compounds, i.e., røeso-monomethyl dihydroguaiaretic acid, nectandrin A, B, which show more effective activity than crude extract can be obtained by further Silica gel column chromatographic purification and thin layer column chromatographic isolation procedure from above described dimethylene chloride soluble fraction well known in the art.

Accordingly, present invention also presents the process for the preparation of the extract and compounds isolated therefrom having inhibiting activity of melanin biosynthesis obtained from above described method.

Additionally, present invention presents cosmetic composition comprising crude extract and non-polar solvent soluble extract of Machilus thunbergii prepared by above- described procedure. Present invention also presents cosmetic composition comprising the compounds expressed as above described general formula (I), which can be isolated from the extract of Machilus thunbergii and synthesized by synthetic methods well known in the art. It is confirmed that present composition showed potent inhibiting activity of melanin bio-synthesis by following experiments for determining the inhibition of melanin bio-synthesis. Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

It is preferable that the present cosmetic composition contains 0.001-40%, more preferably, 0.01- 10% by the weight of the inventive composition based on the total weight of the composition. The other components may be a mixture of the ingredients of a conventional cosmetic composition well known in the art.

Cosmetic formulations containing above composition may be prepared in any form such as skin, astringent, lotion, nutrient face lotion, cream, nutrient cream, massage cream, essence, pack, skin adhesive patch and skin adhesive gel or cleansing agent, cleansing toner, gel, balm, spray solution and the like.

The cosmetic composition of the present invention can comprises additional additives selected from the group consisting of water soluble vitamin, lipid soluble vitamin, peptide polymer, polysaccharide polymer, sphingolipid and sea-weed extract. Preferable water soluble vitamins are any one which can be mixed with cosmetic, however, various vitamin such as vitamin B_ls B₂, B₆, pyridoxine, pyridoxine HC1, vitamin B₁₂, pantothenic acid, nicotinic acid, nicotiamide, folic acid, vitamin C, vitamin H etc, the salt thereof such as thiamin HC1 salt, ascorbic acid Na salt etc or their derivatives such as ascorbic acid-2-phosphonic acid Na salt, ascorbic acid-2-phosphonic acid Mg salt are preferable and those can be obtained by conventional method such as microbial conversion method, purification method from the microbial cultivates, enzymatic method or chemical synthetic method.

Preferable lipid soluble vitamins are any one which can be mixed with cosmetic, however, various vitamin such as vitamin A, D₂, D₃, E (dl-α-tocopherol, d-α-tocopherol, d-δ-tocopherol) and their derivatives such as palmitic acid ascorbate, stearic acid ascorbate, dipalmitic acid ascorbate, acetic acid- dl-α-tocopherol, nicotinic acid dl-α- tocopherol vitamin E, dl-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethylether etc. including the lipid soluble vitamin used in examples of present invention are preferable and those can be obtained by conventional method such as microbial conversion method, purification method from the microbial cultivates, enzymatic method or chemical synthetic method.

Preferable peptide polymers are any one which can be mixed with cosmetic, however, collagen, hydrolysable collagen, gelatin, elastin, hydrolysable gelatin, keratin etc. including the peptide polymer used in examples of present invention are preferable.

Preferable polysaccharide polymers are any one which can be mixed with cosmetic, however, hydroxy ethyl cellulose, xanthin gum, hyaluronic acid Na, chondroitin sulfate or their salt (Na salt etc) and the like are preferable. For example, chondroitin sulfate or the salt thereof etc can be used by being purified from mammal or fishes ordinarily.

Preferable sphingolipid are any one which can be mixed with cosmetic, however, ceramide, pit-sphingosin, sphingo-lipopolysaccharide and the like are preferable. Sphingo-lipid can be obtained by being purified from mammal, fish, shellfish, yeast or plant etc in conventional method.

Preferable seaweed extract is any one which can be mixed with cosmetic, however, the extract of brown algae, red algae, green algae and the like or the purified carrageenan, alginic acid, arginic acid Na, K isolated therefrom are preferable. Algae extract can be obtained by being purified from seaweed in conventional method.

The cosmetic composition of the present invention may combine with other ingredients used in conventional cosmetic composition, if necessary, together with above described essential ingredient.

Preferable above described other ingredients may comprise oil ingredient, humectants, emollients, surfactants, organic or inorganic dye, organic powder, ultraviolet ray absorbing agent, preservatives, antiseptics, antioxidants, plant extract, pH controller, alcohol, pigments, perfumes, refrigerants, blood circulator, antihidrotic, distilled water etc.

Preferable oil ingredients may comprise ester oil, hydrocarbon oil, silicone oil, fluoride oil, animal oil, plant oil and so on.

Preferable ester oil described above may comprise glyceryl tri-2-ethyl hexanoic acid, cetyl 2-ethyl hexanoic acid, isopropyl myristic acid, butyl myristic acid, isopropyl palmitic acid, ethyl stearic acid, octyl palmitic acid, isocetyl isostearic acid, butyl stearic acid, ethyl linoleic acid, isopropyl linoleic acid, ethyl oleic acid, isocetyl myristic acid, isostearyl myristic acid, isostearyl palmitic acid, octyldodecyl myristic acid, isocetyl isostearic acid, diethyl sebasic acid, isopropyl adipic acid, isoalkyl neopetanoic acid, glyceryl tri(capryl, capric acid), trimethylopropane tri-2-ethyl hexanoic acid, trimethylopropane triisostearic acid, pentaerythritol tetra-2 ethyl hexanoic acid, cetyl caprylic acid, decyl lauric acid, hexyl lauric acid, decyl myristic acid, myristyl myristic acid, cetyl myristic acid, stearyl stearic acid, decyl oleic acid, cetyl licinoleic acid, isostearyl lauric acid, isotridecyl myristic acid, isocetyl palmitic acid, octyl stearic acid, isocetyl stearic acid, isodecyl oleic acid, octyldodecyl oleic acid, octyldodecyl linoleic acid, isopropyl isostearic acid, cetostearyl 2-ethyl hexanoic acid, stearyl 2-ethyl hexanoic acid, hexyl isostearic acid, ethylene glycol dioctanoic acid, ethylene glycol dioleic acid, propylene glycol dicapric acid, propylene glycol di(capryl, capric acid), propylene glycol dicaprylic acid, neopentylglycol dicapric acid, neopentylglycol dioctanoic acid, glyceryl tricaprylic acid, glyceryl triundecylic acid, glyceryl triisopalmitic acid, glyceryl triisostearic acid, octyldodecyl neopentanoic acid, isostearyl octanoic acid, octyl isononanoic acid, hexyldecyl neodecanoic acid, octyldodecyl neodecanoic acid, isocetyl isostearic acid, isostearyl isostearic acid, octyldecyl isostearic acid, polyglycerin oleanoic acid ester, polyglycerin isostearic acid ester, triisocetyl citric acid, triisoalkyl citric acid, triisooctyl citric acid, lauryl lactic acid, myristyl lactic acid, cetyl lactic acid, octyldecyl lactic acid, triethyl citric acid, acetyltriethyl citric acid, acetyl tributyl citric acid, trioctyl citric acid, diisostearyl maleic acid, di 2-ethylhexyl hydroxy stearic acid, 2-ethyl hexyl succinic acid, diisobutyl adipic acid, diisopropyl sebasinic acid, dioctyl sebacinic acid, cholesteryl stearic acid, cholesteryl isostearic acid, cholesteryl hydroxy stearic acid, cholesteryl hydroxy stearic acid, cholesteryl oleic acid, dihydrocholesteryl oleic acid, pitsteryl isostearic acid, pitsteryl oleic acid, isocetyl 12- stealoyl hydroxy stearic acid, stearyl 12-stealoyl hydroxy stearic acid, isostearyl 12- stealoyl hydroxy stearic acid.

Preferable hydrocarbon oil described above may comprise squalene, liquid paraffin, α-olefm oligomer, isoparaffin, ceresm, paraffin, liquid isoparaffin, polybuden, microcrystalline wax, vaselin and the like.

Preferable silicone oil may comprise polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethyl siloxane-methyl cetyloxysiloxan copolymer, dimethyl siloxane-methyl stealoxysiloxane copolymer, alkyl modified silicone oil, amino modified silicone oil and the like.

Preferable fluoride oil can comprise perfluoropolyether and the like. Preferable animal or plant oil can comprise avocado oil, almond oil, olive oil, sesame oil, rice husk oil, safflower oil, soy-bean oil, corn oil, rape oil, amygdalin oil, palm kernel oil, palm oil, pimaja oil, sunflower oil, finite seed oil, cotton seed oil, coconut palm oil cucui nut oil, wheat embryo bud oil, rice embryo bud oil, sia butter, evening-primrose oil, marker daymia nut oil, medo home oil, egg yolk oil, lanolin, hempseed oil, mink oil, orange ruppy oil, hohoba oil, carnawa wax, liquid lanolin, solid pimaja wax and the like. Preferable humectants can comprise water-soluble low molecular humectants, lipophilic low molecular humectants, water-soluble polymer and lipid soluble polymer. Specifically, preferable water soluble low molecular humectants can comprise cerin, glutamine, sorbitol, mannitol, pyrrolidone-carboxylic acid Na, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol (polymerization index.

>2), polypropylene glycol (polymerization index >2), lactic acid, lactate salt and the like.

Preferable lipid soluble low molecular humectants can comprise cholesterol, cholesteryl ester and the like.

Preferable water soluble polymer can comprise carboxy vinyl polymer, poly asparaginic acid salt, tragacanth, xanthin gum, HMC (hydroxy methyl celluose), HEC (hydroxy ethyl celluose), HPC (hydroxy propyl celluose), carboxymethylcellulose, water soluble chitin, chitosan, dextrin and the like. Preferable lipid soluble polymer can comprise polyvinylpyrrolidone-eicocene copolymer, polyvinylpyrrolidone-hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, silicone polymer and the like.

Preferable emollients can comprise long chain acyl glutamic acid cholesteryl ester, cholesteryl hydroxy stearic acid, 12-hydroxy stearic acid, rogic acid, lanolin fatty acid cholesteryl ester and the like.

Preferable surfactant can comprise nonionic surfactants, anionic surfactants, cationic surfactants, ambivalent surfactants and the like.

Specifically, preferable non-ionic surfactants can comprise self-emulsified monostearic acid glycerin, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, polyoxyethylene (POE) sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE solid pimaja oil, POE pimaja oil, POE-POP copolymer, POE-POP alkyl ether, polyether modified silicone, lauric acid alkanol amide, alkyl amine oxide, hydrogen addition soybean phospholipid and the like. Preferable anionic surfactants can comprise fatty acid soap, α-acyl sulfonic acid salt, alkyl sulfonic acid salt, alkyl ally sulfonic acid, alkyl naphthalene sulfonic acid salt, alkyl sulfonic acid salt, POE alkylether sulfate salt, alkyl amide sulfate salt, alkyl phosphate salt, POE alkyl phosphate salt, alkylamide phosphate salt, alkyloylalkyl taurine salt, N-acyl-amino acid salt, POE alkyl ether carboxylic acid salt, alkyl sulfo succinic aid salt, alkyl sulfo-acetic acid salt, acylated hydrolysable collagen peptide salt, perfluoro alkyl phosphate ester and the like.

Preferable cationic surfactant can comprise alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, setostearyltrimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, vehenyltrimethyl ammonium bromide, benzalkonium chloride, diethylamino ethyl amide stearic acid, dimethylaminopropyl amide stearic acid, lanolin derivatives quaternary ammonium and the like. Preferable ambivalent surfactants can comprise carboxy betaine type, amide betaine type, hydroxy sulfo betaine type, phosphobetaine type, aminocarboxylic acid, imidazoline derivatives type, amide amine type and the like.

Preferable organic and inorganic dyes can comprise silicic acid, anhydrous silicic acid, magnesium silicic acid, talc, ceracyte, mica, caolin, bengala, clay, bentonite, titan film mica, oxy chlorine bismuth, zirconium oxide, magnesium oxide, zinc oxide, titan oxide, aluminium oxide, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, ferrous oxide, chromium oxide, chromium hydroxide, calamine, carbon black and the complex thereof as an inorganic dyes; polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluoride resin, silicone resin, acryl resin, melamine resin, epoxy resin, polycarbonated resin, divinyl benzene-styrene copolymer, silk powder, cellulose, Cl pigment yellow, Cl pigment orange as an organic dyes; and their complex etc.

Preferable organic powder can comprise metal soap such as calcium stearate; alkyl phosphonate metal salt such as sodium zinc cetylic acid, zinc laurylic acid, calcium laurylic acid; acylamino acid polyvalent metal salt such as calcium N-lauroyl- β-alanine, zinc N-lauroyl-β-alanine, calcium N-lauroyl-glycine etc.; amide sulfonic acid polyvalent metal salt such as calcium N-lauroyl-taurine, calcium N-palmitoyl-taurine; N-acyl basic amino acid such as Nε-lauroyl-L-lysine, Nε-palmitoyl-lysine, Nα- palmitoyl ornitine, Nα-lauroly arginine, hardened lanolin fatty acid acyl arginine and the like; N-acylpolypeptide such as N-lauroylglycyl glycine; α-amino fatty acid such as α-amino caprylic acid, α-amino lauric acid and the like; polyethylene, polypropylene, nylon, polymethylmetacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride and so on. Preferable ultraviolet absorbing agents can comprise paraaminobenzoic acid, paraamonoethyl benzoate, paraamino amyl benzoate, paraamino octyl benzoate, ethyleneglycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylate, benzyl cinnamic acid, paramethoxy 2- ethoxy ethyl cinnamic acid, paramethoxy octyl cinnamic acid, diparamethoxy mono-2- ethylhexane glyceryl cinnamic acid, paramethoxy isopropyl cinnamic acid, diisopropyl- diisopropyl cinnamate ester mixture, urokanic acid, ethyl urokanic acid, hydroxy methoxy benzophenone, hydroxymethoxy benzophenone sulfonic acid and their salt, dihydroxy methoxy benzophenone, dihydroxy methoxy benzophenone disulfonate Na, dihydroxy benzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4'- methoxydibenzoylmethane, 2,4,6-triatu ino-/?-(carbo-2'-ethylhexyl-l '-oxy)-l,3,5- triazine, 2-(2-hydroxy-5-methylphenyl) benzotriazole and the like.

Preferable preservatives can comprise hinokitiol, trichloric acid, trichlorohydroxydiphenylether, chlorohexidine glucuronate, phenoxyethanol, resorcine, isopropylmethylphenol, azulene, salicylic acid, zinc pilithione, bezalconium HC1, photosensitizer 301, mononitroguaiacol Na, undecylenic acid etc.

Preferable antioxidants can comprise butylhydroxyanisole, propyl gallate, ellisorbate and the like.

Preferable pH controller can comprise citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumaric acid, succinic acid, sodium succinic acid, sodium hydroxide, sodium hydrogen phosphate and the like. Preferable alcohol can comprise cetyl alcohol etc. Furthermore, other ingredient addable to above described component and the amount thereof is not limited within the scope of the purpose and effect of the present invention, however, it is preferable that the amount of the other ingredients ranges from 0.01 to 5%, more preferably, 0.01 to 3% in that of total composition.

The cosmetic composition of the present invention can be modified as a solution, emulsion, cohesive mixture etc.

Above described ingredients such as water-soluble vitamin, lipid soluble vitamin, peptide polymer, polysaccharide polymer, sphingolipid, sea weed extract and addable ingredients which can be added other than above described ingredients if necessary, can be obtained by conventional methods disclosed in the literature (Matsumoto Mithio; Manual for the development of transdermal applied preparation. Seisi Press, 1^st Ed., 1985).

Inventive extract and compounds of the present invention have no toxicity and adverse effect therefore, they can be used with safe.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

BEST MODE FOR CARRING OUT THE INVENTION

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention. The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

EXAMPLES

The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Reference Example 1. Apparatus and reagents Melting point was determined by Fischer-Johns melting point apparatus and optical rotation was determined by JASCO DIP-1000 model (Japan). ¹H-NMR and ¹³C-NMR was determined by Bruker 250MHz spectrometer (DMX 250) and Mass spectometer was FAB-MS (VR TRIO 2A mass spectrometer). All solvent was used the highest grade reagents.

Example 1. The crude extract of Machilus thunbergii

The bark of dried Machilus thunbergii (10kg) purchased from Daegu Oriental Drug Market located in Daegu was crushed, refluxed 1 times in 20-2 of 70% methanol at 60 ^°C for 12 hours and further extracted 2 times in 20# of 90% methanol at 60 ^°C for 12 hours. The residue was filtered with filter paper (Whatman), concentrated in vacuo with rotary evaporator to remove solvent at 65 ^°C and dried to obtain 1.45kg of crude extract.

Example 2. The preparation of non-polar solvent extract (1)

The crude extract (1.45kg) in Example 1 was suspended with 2 I of distilled water and 2 t of methylene chloride was added thereto. The solution was subjected to fractionation 3 times, concentrated in vacuo to obtain 1102.0 g of water soluble fraction and 193.86g of methylene chloride soluble fraction, respectively.

Example 3. The preparation of non-polar solvent extract (2) The crude extract (1.45kg) in Example 1 was suspended with 2 I of distilled water and 2 I of ethylacetate was added thereto. The solution was subjected to fractionation 3 times, concentrated in vacuo to obtain 1102.0 g of water soluble fraction and 390g of ethylacetate fraction, respectively.

Example 4. Isolation of active compounds

The methylene chloride fraction (120g) obtained in the Example 2 was purified by flash column chromatography (10x100cm) on silica gel (Merck, 230-400 mesh, No. 9385) at the rate 2000m#/min with hexane/ethyl acetate (100:1— »1:1) as an eluant to give 31 numbers of fractions (MAM-1 to 31) and for each fractions, reverse phase column chromatographic isolation was subjected repeatedly to obtain following three compounds which are belonged to the group expressed as general formula 1 (See Table

1).

[Table 1]

Compound 1 : eso-monomethyl dihydroguaiaretic acid

Fraction MAM-13(2.5g) was loaded to reverse phase column chromatography (3x30cm, LiChroprep RP-18) at the rate 150m^/min with methanol/water (1:9— 1:100) as an eluant to give 19 number of fractions(MAM 13-1 to 19). Among those fractions, the 9th fraction (MAM13-9, 680g) was loaded to reverse phase column chromatography (2x60cm, LiChroprep RP-18) at the rate 50m /min with methanol/water (3:7-»l:100) as an eluent to give 12 number of fractions(MAM 13-9-1 to 12). From 6^th fraction (MAM 13-9-6), 24mg of colorless oil merø-monomethyl dihydroguaiaretic acid (compound 1) having following physicochemical property was obtained;

[α]²⁵ _D + 1.3° (c 0.1, CHCl₃);

1H (CD₃OD, 250 MHz) 56.71 (IH, d, J= 8.5 Hz, H-5), 6.59 (IH, d, J= 8.0 Hz, H-5'), 6.58 (IH, dd J= 8.5, 1.8 Hz, H-6), 6.48 (2H, br , H-2, 2'), 6.51 (IH, dd, J= 8.0, 1.8 Hz, H-6'), 3.7 (6H, s, 4', 4-OMe), 3.6 (3H, s, 3'-OMe), 2.62 (IH, dd, J= 13.4, 5.3 Hz, H-7b), 2.58 (IH, dd, J = 13.4, 5.8 Hz, H- 7'b), 2.16 (IH, dd, J - 13.4, 9.2 Hz, H-7a), 2.15 (IH, dd,.J= 13.4, 9.2 Hz, H-7'a), 1.62 (2H, m, H-8, 8'), 0.72 (3H, d, J= 6.6 Hz, H-

9'), 0.71 (3H, d, J= 6.6 Hz, H-9);

¹³C NMR (CD₃OD, 62.9 MHz) δ 150.2 (C-3'), 148.7 (C-4'), 148.4 (C-3), 145.4 (C-4), 136.1 (C-r), 134.5 (C-l), 122.5 (C-6), 122.4 (C-6') , 115.8 (C-5), 113.8 (C-5'), 113.5 (C-2), 112.8 (C-2'), 56.5 (3'-OMe), 56.3 (4', 4-OMe), 40.2 (C-8), 40.0 (C-8'), 39.7 (C-7'), 39.4 (C-7), 16.6 (C-9')₅ 16.5 (C-9); positive FAB-MS m/z : 345 [M+H]⁺.

Compound 2: Nectandrin A Fraction MAM-16(1.5g) was loaded to reverse phase column chromatography (2x60cm, LiChroprep RP-18) at the rate 50m£/min with methanol/water (3:7-»l:100) as an eluant to give 12 number of fractions(MAM 13-9-1 to 12). From 10^th fraction (MAM 13-9-10), 250mg of colorless oil nectandrin A (compound 2) having following physicochemical property was obtained;

[α]²⁵ _D -3.78°(c 0.1, CHCl₃);

1H (CDC1₃, 250 MHz) δ 6.81 6.96 (6H, m, Ar-H), 4.47 (2H, m, H-7 and -7'), 3.85 (9H, s, 3x-OCH3), 2.30 (2H, m, H-8, 8'), 1.02 (3H, d, J = 6.6 Hz, H-9 or H-9'), 1.00 (3H, d, J= 6.6 Hz, H-9 or H-9');

¹³C NMR (CDC1₃, 62.9 MHz) δ 148.8 (C-3*), 148.3 (C-4'), 146.4 (C-3), 144.9 (C-4), 134.7 (C-l'), 134.0 (C-l), 119.1 (C-6), 118.5 (C-6'), 114.0 (C-5), 110.8 (C-5'), 109.6 (C-2'), 109.0 (C-2), 87.3 (C-7), 87.1 (C-7'), 55.8, 55.7, 55.7 (3x-OCH3), 44.3 (C- 8), 44.2 (C-8'), 12.9 (C-9), 12.8 (C-9'); positive FAB-MS m/z : 359 [M+H]⁺. Compound 3: Nectandrin B

Fraction MAM-18(0.7g) was loaded to reverse phase column chromatography (2x60cm, LiChroprep RP-18) at the rate 50m£/mrn with methanol water (3:7-»l:100) as an eluant to give 12 number of fractions(MAM 13-9-1 to 12). From 6^th fraction (MAM 18-6), 15mg of colorless oil nectandrin B (compound 3) having following physicochemical property was obtained;

[α]²⁵ _D 0°(c 0.41, CHC1₃);

1H (CDCI₃, 250 MHz) δ6.89 (6H, m, Ar-H), 4.47 (2H, d, J = 6.4 Hz, H-7, 7), 3.85 (6H, s, 3, 3'-OMe)2.30 (2H, m, H-8, 8'), 1.02 (6H, d, J= 6.4 Hz, H-9, 9');

¹³C NMR (CDC1₃, 62.9 MHz) δl46.4 (C-3, C-3'), 144.9 (C-4, C-4'), 134.1 (C-l, C-l'), 119.2 (C-6, C-6'), 114.0 (C-5, C-5'), 109.1 (C-2, C-2'), 87.2 (C-7, C-7'), 55.8 (3, 3'-OCH3), 44.2 (C-8, C-8'), 12.9 (C-9, C-9'); positive FAB-MS m/z : 345 [M+H]⁺.

Experimental Example 1. Inhibitory effect on melanin synthesis in Cultured B-16 Mouse Melanoma Cells.

In order to confirm the inhibition effect of the extracts prepared in Example 1 to 2 and the compounds prepared in Example 3 on melanin biosynthesis, the experiment was performed with the procedure described in the literature (Curto E. et al.; Biochem. Pharmacol, 57, ρp663-672, 1999).

Mouse melanoma B-16 cells (2xl0⁴ cells/200/ β) were subcultured in Eagle's minimum essential medium using a 96-well plate.

The cells were grown in a 5% CO₂-95% air atmosphere at 37 ^°C, for 1 day, and the samples dissolved in DMSO was added to the medium at the concentration of 25 βg /ml As a negative control, only DMSO solvent was added thereto without sample. As positive controls, 150 βglmi ofkojic acid (Sigma Co.) and 25 μg/ml of arbutin (Sigma Co.) were added thereto. After incubation for another 2 days, the adherent cells were washed with 200/z£ of PBS (phosphate buffered saline, pH 7.2), and 200/^ of IN NaOH was added. The cells were homogenized. Optical density (OD) value was measured at 405nm by an enzyme-linked immunosorbent assay (ELISA) reader, and the inhibitory activity as calculated from the change from the initial rate of OD.

As shown in Table 2, the extracts showed potent inhibitory activity on B-16 melanoma cell. In particular, the crude extract of Example 1 and methylene chloride soluble extract exhibit 11.3% and 42.9% inhibition on cultured B-16 mouse melanoma cells.

[Table 2]

As shown in Table 3, the IC₅₀ values of compound 1, 2 and 3 on melanin biosynthesis were 15.1, 19.4 and 37.8μM, respectively, which is about 2-6-fold more potent than arbutin used as a positive control.

[Table 3]

Experimental Example 2. Cytotoxicity Test

In order to examine the cytotoxicity of inventive extracts and compounds of the present invention prepared in the Example 1 to 3, the experiment was performed as follows.

Mouse Melanoma B-16 cells (2xl0⁴ cells/200 ^) were subcultured in Eagle's minimum essential medium using a 96-well plate.

The cells were grown in a 5% CO₂-95% air atmosphere at 37 ^°C, for 1 day, and the samples dissolved in DMSO at the concentration ranging from 5μM to 500 uM, was added to the medium. After incubation for another 2 days, the cytotoxicity of the extracts and compounds was measured by tetrazolium-based colorimetric assay (MTT assay) according to the method disclosed in the literature (Rubinstein L. V., et al: J. Nat.

Cancer Inst., 82, pplll3-1118, 1990). OD value was measured at 540nm by an ELISA reader and the result (LD₅₀) was shown in Table 4. As shown in Table 4, compound 1 showed hardly toxic, therefore, it is safe and useful as a cosmetic composition.

[Table 4]

Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

Preparation of skin lotion

Compound 1 1.00(%)

Glycerol 3.00

Ethanol 1.00

Propylene glycol 0.10

Flavor trace amount Distilled water made to 100%

Skin preparation was prepared by dissolving active component according to conventional lotion preparation method.

Preparation of lotion

Compound 1 3.00(%)

L-ascorbic acid-2-magnesium phosphate 1.00

Soluble collagen (1 % solution) 1.00 Sodium citric acid 0.10

Citric acid 0.05

1 ,3 -butylene glycol 3.00

Distilled water made to 100%

Lotion preparation was prepared by dissolving active component according to conventional lotion preparation method.

Preparation of cream

Compound 1 3.00(%)

Polyethyleneglycolmonosterate 2.00

Monostearate glycerin 1.00

Cetyl alcohol 4.00

Squalene 6.00

Tri 2- glyceryl ethylhexanoate 6.00

Sphingo-glycolipid 1.00

1,3 -butylene glycol 7.00

Distilled water made to 100%

Preparation of pack

Crude extract in Example 1 5.00(%)

Polyvinyl alcohol 13.00

L-ascorbic acid-2-magnesium phosphate 1.00

Lauroylhydroxyproline 1.00

Soluble collagen (1% solution) 2.00

1,3 -butylene glycol 3.00

Ethanol 5.00

Distilled water made to 100% Pack preparation was prepared by dissolving active component according to conventional pack preparation method.

Preparation of beauty solution

Methylene Chloride extract in Example 2 2.00(%)

Hydroxyethylenecellulose (2% solution) 12.00

Xanthin gum (2% solution) 2.00

1,3-butylene glycol 3.00 Cone. Glycerin 4.00

Sodium hyaluronate 5.00

Distilled water made to 100%

Beauty solution preparation was prepared by dissolving active component according to conventional beauty solution preparation method.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

As described in the present invention, the extracts of Machilus thunbergii and the compound isolated therefrom have potent inhibitory effect on melanin synthesis and low toxicity, the cosmetic composition comprising above extract or compounds can be useful in skin whitening cosmetic composition as an cream, skin, lotion, pack and the like.

Claims

What is claimed is;

1. A skin cosmetic composition comprising crude extract of Machilus thunbergii.

2. The cosmetic composition according to claim 1, wherein said crude extract is soluble in water, lower alcohol such as methanol, ethanol etc or the solvent mixture thereof.

3. The skin cosmetic composition comprising non-polar solvent soluble extract of Machilus thunbergii.

4. The cosmetic composition according to claim 3, wherein said non-polar solvent is at least one selected from the group consisting of hexane, carbon tetrachloride, chloroform, methylene chloride, ethyl ether and ethylacetate.

5. The cosmetic composition according to claim 4, wherein said nonpolar solvent is methylene chloride.

6. A skin cosmetic composition comprising lignan compounds expressed as following general formula (I):

7. The cosmetic composition according to claim 6, wherein said lignan compounds are 7røeso-monomethyl dihydroguaiaretic acid, nectandrin A and nectandrin B.

8. The cosmetic composition according to claim 6, wherein said cosmetic composition comprises 0.001 to 40 w/w% of compound as set forth in claim 6.

9. The cosmetic composition according to any of claims 1 to 8, wherein said cosmetic composition is selected from skin, astringent, nutrient face lotion, nutrient cream, massage cream, essence, pack, skin adhesive patch and skin adhesive gel or cleansing agent.