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[0001] The present invention relates to a composition for retarding skin ageing and, more particularly, to a composition comprising a certain specific herb medicine or medicines selected from among various plant extracts having melanine formation-inhibiting, elastase-inhibiting, hyaluronidase-inhibiting, active oxygen-eliminating and/or radical-capturing type antioxidant activities. The composition, when incorporated in certain preparations, is useful in promoting the so-called whitening effects, namely skin whitening effects, maintaining the tension and elasticity of the skin, facilitating skin moistening and, further, providing the skin with anti-inflammatory and/or anti-allergic properties.
[0002] As for the inhibition of melanine formation, certain whitening agents such as ascorbic acid or derivatives thereof, placenta extracts, kojic acids, glutathion, hydroquinone and a derivative thereof (arbutin), and plant extracts have so far been incorporated in dermatologic preparations for skin whitening for the purpose of preventing such phenomena as sunburn-induced skin darkening or inflammation and pigmentation-due formation of spots and freckles (Fragrance Journal, Vol. 18, No. 6, 1990, pp. 47-58).
[0003] Meanwhile, elastase is a protease specifically acting on elastin, which is concerned with the tension and elasticity of the skin. This participates in decreasing the quantity of elastin, leading to particularly grave results among the skin aging phenomena. Further, ultraviolet rays activate elastase, with the results that the skin loses it tension and elasticity. The effect of elastase increases with the advance of age, bringing about the same results. Therefore, if the excessive effect of elastase can be lessened, it will become possible to prevent the skin aging due to ultraviolet rays and/or aging. From such viewpoint, substances having elastase inhibiting activity have been searched for. As a result, it has been demonstrated that extracts from Schima species, eucalyptus, and Meniran (
[0004] As for hyaluronidase, its functions in connective tissu s are considered to retain water intercellular spaces, form a j lly-like matrix in th tissu to maintain cells, maintain skin lubricity and flexibility, and protect th skin against external forces (mechanical lesions) and bacterial infection. It is said that hyaluronic acid in the skin decreases with aging, with the result that such aging phenomena as fine wrinkles formation and skin roughening. Therefore, it is considered that inhibition of the activity of hyaluronidase, which decomposes such hyaluronic acid, may contribute to the stabilization of skin hyaluronic acid and will be also effective in improving the barrier function of the skin by preventing the same from decreasing. Presumably, this also contribute to stabilization of hyaluronic acid used in preparations for the treatment of arthritis, among others, and further to stabilization of hyaluronic acid in preparations applied to the skin for improving the skin function.
[0005] Hyaluronidase is also known as an inflammatory enzyme, and it is known that inhibition of its activity is effective in reducing inflammation and that it suppresses allergy as well. Several substances capable of inhibiting hyaluronidase have been reported. Hyaluronidase inhibiting activity has been confirmed in extracts of peony root, coptis rhizome (JP-A-1-128933), ziyu (
[0006] Active oxygen eliminators having SOD activity are widely used in the treatment and/or alleviation, for instance, of various diseases caused by active oxygen (O
[0007] More specifically, there are known cosmetics in which various characteristics of SOD are utilized (e.g. JP-B59-010324, JP-A-55-087712) and dermatologic preparation compositions comprising a placenta or liver extract having stable SOD activity (JP-A61-277626). Further known as plant extracts having SOD activity are plant flavonoid-containing xtracts of
[0008] Referring to antioxidants, sebum is secreted on the human skin to protect the same. When exposed to ultraviolet rays, for instance, this sebum is oxidized to give lipid peroxides, which irritate the skin. These lipid peroxides attack the cell membrane, damaging or adversely affecting the same in various ways, and these damages, in turn, are said to be involved in human skin aging. Therefore, it is considered effective not only in preventing the skin condition from worsening but also in preventing the living body from aging to inhibit the formation of such lipid peroxides by means of antioxidants.
[0009] Among specific antioxidants, there have been reported plants extracts such as extracts from birch (
[0010] As regards the melanine formation, however, some dermatologic preparations containing these whitening agents are unsatisfactory in whitening effec, and others are effective in inhibiting melanine formation but still have problems from the safety viewpoint. In many instances, the whitening agents are denatured in the preparations, for instance, hence the desired effects are not obtained. Improvements in this respect have thus been desired.
[0011] As regards the elastase activity inhibition, if the excess elastase activity can be inhibited, it may become possible to prevent skin aging. However, the elastase inhibitors so far reported are not fully satisfactory from the viewpoint of stability, effect, and feel upon application.
[0012] As for the hyaluronidase activity inhibition, in spite of various investigations in search of cosmetic ingredients having antinflammatory or antiallergic activity, any safe and promising one has not yet been obtained. Thus, substances have been desired which are natural products and have been eaten by men for long and whose safety has thus been established and which have strong hyaluronidase inhibiting activity and further have some other effect on the skin.
[0013] The in vivo enzyme superoxide dismutase (SOD) is known as an active oxygen eliminator. However, this is difficult to purify, thermally unstable and readily deactivated, hence it is very expensive. Therefore, the advent of SOD substitutes (substances having SOD-like activity) which show active oxygen eliminating activity, like SOD and are highly stable and can be obtained at low cost has been awaited.
[0014] As for the antioxidants so far used, cosmetics and dermatologic preparations containing BHT, BHA and the like tend to cause contact dermatitis and their safety is questionable. On the other hand, gallic acid derivatives and vitamin E species are insufficient in antioxidant activity and thus fail to effectively prevent fat/oil-containing products from becoming rancid. Even if the effect of preventing skin aging due to peroxidation of skin lipids, in particular, is expected of the dermatologic preparations containing them, no satisfactory effects can be obtained.
[0015] Further, referring to the whitening effect, the direct inhibitory effect on melanine-synthesizing melanocytes as well as the effect of suppressing the production of melanocyte-stimulating agents (MSH, endotehlin, NO, histamine, PGE2, etc.) r I ased by melanocyte-surrounding cells is an important factor. Therefore, the antinflammatory action inhibiting these stimulators from being released by keratinocyt s is also considered to be effective in promoting whitening.
[0016] Furthermor, ascorbic acid d rivatives and arbutin, which are whitening agents in conventional use, have themselves antioxidant activity, and it Is considered that their active oxygen- and free radical-quenching activity is party involved in the mechanisms of whitening. Therefore, herb extracts having such whitening and antioxidant activities together with antiinflammatory and other activities will be more effective. However, no investigations have so far been made in search of such herb medicine extracts having such a plurality of activities.
[0017] It is an object of the present invention to provide a composition for retarding skin aging which can promote whitening, allow the skin to retain its tension and elasticity, facilitate skin moistening, and exert antiinflammatory and antiallergic effects on the skin.
[0018] In an aspect of the invention, the skin age-retarding composition comprises, together with a base and/or additive medicinally acceptable for external dermal application, one or more plant extracts selected from the group consisting of extracts of Suei-Ding Hsiang (
[0019] In another aspect of the invention, the skin age-retarding composition comprises, together with a base and/or additive medicinally acceptable for external dermal application, one or more plant extracts having, in particular, melanine formation-inhibiting activity as the aging retarding effect as selected from the group consisting of extracts of Yui-Jen-Sau (
[0020] In a further aspect of the invention, the skin age-retarding composition comprises, together with a base and/or additive medicinally acceptable for external dermal application, one or more plant extracts having elastase-inhibiting activity as the aging retarding effect as selected from the group consisting of extracts of YehSia-Chu (
[0021] In a still further aspect of the invention, the skin age-retarding composition comprises, together with a base and/or additive medicinally acceptable for external dermal application, one or more plant extracts having hyaluronidase-inhibiting activity as the aging retarding effect as selected from the group consisting of extracts of Huang-Gin-Guei (
[0022] In a still further aspect of the invention, the skin age-retarding composition comprises, together with a base and/or additive medicinally acceptable for external dermal application, one or more plant extracts having active oxygen eliminating activity as the aging retarding effect as selected from the group consisting of extracts of Suei-Ding Hsiang (
[0023] In a still further aspect of the invention, the skin age-retarding composition comprises, together with a base and/or additive medicinally acceptable for external dermal application, one or more plant extracts having antioxidant activity as the aging retarding effect as selected from the group consisting of extracts of SueiDing Hsiang (
[0024] The present invention also provides a cosmetic composition and a dermatologic preparation each containing the skin age-retarding composition mentioned above.
[0025] The medicinal herbs to be used in accordance with the invention respectively have the common names and scientific names listed below in Table 1. Various parts (whole herb, flower, caput, floral spike, female floral spike, fruit spike, corpus glandulae, leaf, branch, leaf-bearing branch, rhizome, velamen, root, seed, etc.) of each plant may be extracted either as such or after grinding to give extracts ready for use.
TABLE 1 No. Name of herb Botanical name 1 Suei-Ding Hsiang 2 Yui-Jen-Sau 3 Ban-Ji-Lien 4 Ban-Bien-Lien 5 Chi-Mu-Sau 6 Ma-Bien-Sau 7 Tu-Chi-Chi 8 Ma-Ti-Gin 9 Yeh-Sia-Chu 10 Ku-Chi 11 Bai-Hua-Tsai 12 San-Jei-Tsai 13 Pien-Di-Gin 14 Sarng-Han-Sau 15 Gin-New-Kou 16 Mei-Zen-Giau 17 Ma-An-Tung 18 Hua-Shi-Su 19 Gin-Di-Lo 20 Gin-Hsien-Lien 21 Nan-Ling-Yia-Hua 22 Bai-Yeh-Chi-Ken 23 Yui-Yeh-Gin-Hua 24 Hsien-Sau 25 Taiwan Ma-Dou-Ling 26 Shi-Chung-Pu 27 Huang-Gin-Guei 28 Chou-Mor-Li 29 Da-Ching 30 Bai-Lorng-Chun-Hua 31 Mu-Chu-Ma 32 Ya-Jou 33 San-Yeh-Pu-Tao 34 Hsia-Yeh-Pu-Tao 35 Gi-Hua-Mu 36 Gin-Shi-Liu 37 Ha-Sai-Hua 38 Shi-Sharng-Bor 39 Hu-Ji-Chi 40 Ci-Yeh-Suei-Ding-Hsiang 41 Chein-Gin-Tung 42 Wu-Jei 43 Tie-Hsien-Jei 44 San-Hsiang 45 Gi-Yien-Sau 46 Hai-Chou-Chung-San 47 Huang-Ging 48 Sarng-Er 49 Hsien-Fong-Sau 50 Su-Chi-Sau 51 Tsu-Bei-Sau 52 Harn-Hsiu-Sau 53 Zu-Chi-Sau 54 Cher-Chien-Sau 55 Dau-Di-Ling 56 Bai-Hua-Tung 57 Ding-Di-Wu-Gong 58 Da-Gia-Sau 59 Gi-Yeh-Tsu-Hua-Di-Ding 60 Taiwan Her-Shou-Wu 61 New-Zu-Zong 62 Bi-Yeh-Dung-Lorng-Sau 63 Karng-Hsiang-Tung 64 Taiwan Kou-Tung 65 Lo-Chi-Yien-Fu-Mu 66 Gin-Gien-Sau 67 Horng-Hua-Su-Wei-Sau 68 Tu-Chi-Tsai 69 Dau-Sharng-Sau 70 Ku-Larn-Parn 71 Tou-Hua-Hsiang-Ku-Sau 72 Du-Horng-Hua 73 Yeh-Hu 74 Torng-Chuei-Yui-Di-Sau 75 San-Yeh-Wu-Gia
[0026] The method of producing such medicinal herb extracts is not particularly restricted but may be any of the methods in conventional use. The following method, for example, may be preferably employed. Thus, any of the starting herb materials mentioned above is finely ground, 5 to 10 weights of an extracting solvent (water, alcohol or organic solvent, or mixture thereof) is added, the mixture is allowed to stand at room temperature for at least one week and then filtered, the filtrate is lyophilized, and the lyophilization product is used as a medicinal herb extract. Alternatively, an extracting solvent (water or organic solvent) is added to the herb material and, after a certain period of digestion or heating at the refluxing temperature of the solvent, the mixture is filtered, and the filtrate is concentrated to dryness.
[0027] Usable as specific extracting solvents are aqueous solvents (e.g. water, acidic or basic aqueous solvents, etc.), alcohols (e.g. lower alcohol such as methanol, absolute ethanol, ethanol, or polyhydric alcohols such as propylene glycol, 1,3-butylene glycol, etc.), ketones such as acetone, diethyl ether, dioxane, acetonitrile, esters such as ethyl acetate, and organic solvents such as xylene, benzene and chloroform. These may be used either singly or in the form of a mixture of two or more. In particular, water, methanol, ethanol, and 50% ethanol are preferred. Two or more solvent extracts may be used in combination.
[0028] In the practice of the invention, the plant extract(s) mentioned above may be used as such or may be admixed with per se known bases or additives medicinally acceptable for dermal application, including, as cosmetics components, for instance, water, alcohols, oleaginous substances, surfactants, preservatives, perfumes, colorants, humectants, thickening agents, water-soluble polymers, antioxidants, chelating agents, pH adjusting agents, foaming agents, (pigments, ultraviolet absorbing/scattering agents, powders), vitamins, amino acids, antimicrobial agents, algae extracts, various drugs, additives, and so forth.
[0029] More specifically, the oleaginous substances include vegetable oils and fats such as macadamia nut oil, castor oil, olive oil, cacao oil, tsubaki oil, coconut oil, Japan wax, jojoba oil, grape seed oil, and avocado oil, animal oils and fats such as mink oil and yolk oil, waxes such as beeswax, spermaceti, lanolin, carnauba wax, and candelilla wax, hydrocarbons such as liquid paraffin, squalane, microcrytalline wax, ceresin wax, paraffin wax, and Vaseline, natural or synthetic fatty acids such as capric acid, myristic acid, palmitic acid, stearic acid, behenic acid, lanolin fatty acids, linolic acid, linolenic acid, lauric acid, myristic acid, oleic acid, and isostearic acid, natural and synthetic higher alcohols such as cetanol, stearyl alcohol, hexyldecanol, octyldodecanol, lauryl alcohol, capryl alcohol, myristyl alcohol, cetyl alcohol, cholesterol, and phytosterol, and esters such as isopropyl myristate, isopropyl palmitate, octyldodecyl myristate, octyidodecyl oleate, and cholesterol oleate, among others.
[0030] Examples of the above-mentioned surfactants are nonionic surfactants such as sorbitan monolaurate, sorbitan monoplamitate, sorbitan sesquioleate, sorbitan trioleate, polyoxyethylenesorbitan monolaurate, polyoxyethylenesorbitan monostearate, polyethylene glycol monooleate, polyethylene glycol alkylates, polyoxyethylene alkyl ethers, polyglycol diethers, lauroyldiethanolamide, fatty acid isopropanolamides, maltitol hydroxyfatty acid ethers, alkylated polysaccharides, alkyl glucosides, and sugar esters; nonionic surfactants such as lipophilic glycerol monostearate, self-emulsificable glycerol monostearate, polyglycerol monostearate, polyglycerol alkylates, sorbitan monooleate, polyethylene glycol monostearate, polyoxyethylenesorbitan monooleate, polyoxyethylene cetyl ether, polyoxyethyienateu sterol, polyoxyethylenated lanolin, polyoxyethylenated beeswax, and polyoxyethylenated hardened castor oil; anionic surfactants such as sodium stearate, potassium palmitate, sodium cetyl sulfate, sodium lauryl phosphate, palmitic acid triethanolamide, sodium polyoxyethylenelauryl phosphate, sodium N-acylglutamate, sodium palmitate, sodium laurate, sodium lauryl, potassium lauryl sulfate, alkyl sulfate triethanolamine ethers, Turkey red oil, linear dodecylbenzenesulfuric acid, polyoxyethylene-hardened castor oil maleic acid, and acylmethyltaurine; cationic surfactants such as stearyidimethylbenzylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium chlrodie, benzalkonium chloride, and laurylamine oxide; and amphoteric surfactants such as alkylaminoethylglycine hydrochloride solutions, and lecithin.
[0031] Examples of the above preservatives are benzoic acid salts, salicylic acid salts, sorbic acid salts, dehydroacetic acid salts, parahydroxybenzoic acid esters, 2,4,4′-tri-chloro-2′-hydroxydiphenyl ether, 3,4,4′-trichlorocarbanilide, benzalkonium chloride, hinokitiol, resorcinol, and ethanol.
[0032] The above humectants include polyhydric alcohols such as glycerol, erythritol, xylitol, maltitol glycerol, propylene glycol, 1,3-butylene glycol, sorbitol, polyglycerol, polyethylene glycol, dipropylene glycol, 1,2-pentanediol, and isoprene glycol; NMF components such as amino acids, sodium lactate, and sodium pyrrolidonecarboxylate; water-soluble polymer substances such as xyloglucan, quince seeds, carrageenan, pectin, mannan, curdlan, galactan, dermatan sulfate, glycogen, keratan sulfate, chondroitin, mucoitin sulfate, keratosulfate, locust bean gum, succinoglucan, charonic acid, hyaluronic acid, heparan sulfate, sodium hyaluronate, collagen, mucopolysaccharides, and chondroitin sulfate; silicones such as dimethylpolysiloxane and methylphenylsiloxane; and culture supernatants such as of lactic acid bacteria/bifidus bacilli, among others.
[0033] The above thickeners include, among others, natural polymer substances such as sodium alginate, xanthan gum, aluminum silicate, marmelo seed extracts, gum arabic, hydroxyethyl-guar gum, carboxymethyl-guar gum, guar gum, dextran, tragacanth gum, starch, chitin, chitosan, carboxymethylchitin, and agar; semisynthetic polymer substances such as cellulose, hydroxypropylcellulose, methylhydroxypropylcellulose, methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, soluble starch, and cationized cellulose; and synthetic polymer substances such as carboxyvinyl polymers, polyvinyl alcohol, polyvinylpyrrolidone, and vinyl alcohol-vinyl acetate copolymers.
[0034] The above antioxidants include, among others, dibutylhydoxytoluene, butylhydroxyanisole, propyi gaiiate, and ascorbic acid; the chelating agents include, among others, disodium edetate, ethylenediaminetetraacetic acid salts, pyrophosphoric acid salts, hexametaphosphoric acid salts, citric acid, tartaric acid, and gluconic acid; the pH adjusting agents include, among others, sodium hydroxide, triethanolamine, citric acid, sodium citrate, boric acid, borax, and potassium hydrogen phosphate.
[0035] The above ultraviolet absorbing/scattering agents include, among others, paraaminobenzoic acid type ultraviolet absorbers, anthranilic acid type ultraviolet absorbers, salicylic acid type ultraviolet absorbers, cinnamic acid type ultraviolet absorbers, benzophenone type ultraviolet absorbers, sugar type ultraviolet absorbers, 3-(4′-methylbenzylidene)-d-camphor, 3-benzylidene-d, I-camphor, urocanic acid, ethyl urocanate, 2-phenyl-5-methylbenzoxazole, 2,2′-hydroxy-5-methylphenylbenzotriazole, 2-(2′-hydroxy-5′-t-octylphenyl)benzotriazole, 2-(2′-hydroxy-5′-methylphenyl)benzotriazole, dibenzalazine, dianisoylmethane, 4-methoxy-4′-t-butylbenzophenone, 5-(3,3-dimethyl-2-norbornylidene)-3-pentan-2-one, 2-hydroxy-4-methoxybenzophenone, octyidiemthyl paraaminobenzoate, ethylhexyl paramethoxycinnamate, titanium oxide, kaolin, and talc.
[0036] The above vitamins include, among others, vitamin A and derivative thereof, vitamin B species such as vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 and derivatives thereof, vitamin B12, vitamin B15 and derivatives thereof, ascorbic acid, ascorbic acid sulfate and salts thereof, ascorbic acid phosphate and salts thereof, ascorbic acid dipalmitates, ascorbic acid glucosides, acylascorbic acid glucosides, vitamin E species such as α-tocopherol, α-tocopherol, β-tocopherol, and vitamin E acetate, vitamin D species, vitamin H, pantothenic acid, pantethine, vitamin F, vitamin K, vitamin P, vitamin U, carnitine, ferulic acid, γ-oryzanol, α-lipoic acid, orotic acid and derivatives thereof.
[0037] The above amino acids include glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, asparagine, glutamine, taurine, tryptophan, cystine, cysteine, methionine, proline, hydroxyproline, aspartic acid, glutamic acid, arginine, histidine, lysine, and derivatives thereof.
[0038] The above antimicrobial agents include benzoic acid, salicylic acid, parahydroxybenzoic acid alkyl esters, and hexchlorophene, among others.
[0039] The above algae extracts include extracts from brown algae, red algae, green algae, blue algae and so on. More specific examples are extracts from tangle, Japanese tangle, hijiki, agar-agar, coralline, Palmara, carrageen moss, lav r, sea lettuce, holey sea lettuce, Ascopifyfllum, wrack mozuku, Okinawa mozuku, Himantada, etc.
[0040] Specific examples of the various drugs mentioned above are nictlnamiide, benzyl nicotinate, γ-oryzanol, allantoin, glycyrrhizic acid (salts), glycyrrhetic acid and derivatives thereof, hinokitiol, bisabolol, eucalyptone, thymol, inositol, saponins such as psychosaponin, carrot saponin, loofah saponin and soapberry saponin, pantothenyl ethyl ether, ethynylestradiol, tranexamic acid, arbutin, capharanthine, and placenta extract.
[0041] In the practice of the invention, the level of incorporation of the medicinal herb extract is not particularly restricted but may be adequately selected according to the intensity (IC
[0042] The cosmetics, dermatologic preparations, quasi drugs and so forth, which contain the skin age-retarding composition of the invention, can be produced in the conventional manner. They may have various forms, for example, basic cosmetic forms such as skin lotions, milky lotions, moisturizing creams, deansing creams, massage creams, facial cleansing creams, packs, and essence, hair care products such as shampoos and rinses, bath products such as bath salt compositions, makeup cosmetics such as foundations, and special cosmetics such as anti-suntan compositions.
[0043] The following test examples and working examples illustrate the invention in further detail. They are, however, by no means limitative of the scope of the invention.
[0044] To 282.3 g of Nan-Ling-Yia-Hua was added 1.8 L of 100% methanol, and extraction was effected at room temperature for 1 week, followed by filtration. The filtrate obtained was evaporated to dryness. The dry weight was 21.49 g (yield 7.6%).
[0045] For the medicinal herbs listed in Table 1, 1 to 10 L of 100% methanol was added to about 300 g of each bulk herb medicine, and extraction was effected in the same manner to give an extract. The following Table 2 shows yields of respective xtracts in 100% methanol. Also shown in Table 2 are th pH values found when 0.5% solutions were prepared from th dried products, th states of dissolution and th colors as well as the absorbance values (As) at 312 nm in 50% thanol solutions at a solid matter concentration of 0.5%.
TABLE 2 Yield Dissolution state Color* pH As No. Herb name (%) (0.5%/50% ethanol) (ditto) (ditto) (312 nm) 1 Suei-Ding Hsiang 2.1 Partly insoluble YB 3.9 0.542 2 Yui-Jen-Sau 10.6 Precipitation DB 4.6 1.563 3 Ban-Ji-Lien 14.5 Precipitation DG 4.7 1.199 4 Ban-Bien-Lien 9.7 Mostly dissolved YB 3.9 0.268 5 Chi-Mu-Sau 3.1 Mostly dissolved LYB 4.2 1.128 6 Ma-Bien-Sau 9.0 Precipitation DB 4.3 0.993 7 Tu-Chi-Chi 16.9 Partly insoluble YB 4.5 1.225 8 Ma-Ti-Gin 6.9 Oily DB 4.1 0.765 9 Yeh-Sia-Chu 6.9 Precipitation DB 3.8 1.604 10 Ku-Chi 6.2 Precipitation DB 4.8 0.427 11 Bai-Hua-Tsai 5.0 Precipitation DB 4.2 0.567 12 San-Jei-Tsai 4.2 Oily DDB 3.7 0.528 13 Pien-Di-Gin 7.0 Partly insoluble DB 4.3 1.130 14 Sarng-Han-Sau 5.0 Oily YB 4.8 1.536 15 Gin-New-Kou 8.5 Oily YE 4.2 0.663 16 Mei-Zen-Giau 1.6 Mostly dissolved LYB 4.3 0.550 17 Ma-An-Tung 4.5 Emulsified LYB 4.0 1.933 18 Hua-Shi-Su 4.7 Oily DG 4.2 0.757 19 Gin-Di-Lo 3.2 Partly insoluble BR 3.9 2.101 20 Gin-Hsien-Lien 6.5 Oily DG 4.9 0.938 21 Nan-Ling-Yia-Hua 7.6 Mostly dissolved LYB 4.9 2.496 22 Bai-Yeh-Chi-Ken 3.9 Dissolved RB 4.5 1.145 23 Yui-Yeh-Gin-Hua 3.7 Mostly dissolved LY 4.4 1.038 24 Hsien-Sau 3.4 Partly insoluble LDG 5.0 0.736 25 Taiwan Ma-Dou-Ling 5.3 Oily RB 3.8 0.992 26 Shi-Chung-Pu 7.3 Oily YB 3.8 2.494 27 Huang-Gin-Guei 7.0 Mostly dissolved RB 4.9 6.078 28 Chou-Mor-Li 3.8 Mostly dissolved YE 4.7 0.425 29 Da-Ching 3.8 Mostly dissolved YB 3.8 1.209 30 Bai-Lorng-Chun-Hua 5.8 Mostly dissolved YE 4.9 0.335 31 Mu-Chu-Ma 2.6 Mostly dissolved RB 4.8 0.588 32 Ya-Jou 1.2 Mostly dissolved YE 4.5 1.206 33 San-Yeh-Pu-Tao 2.3 Partly insoluble YB 4.6 0.879 34 Hsia-Yeh-Pu-Tao 6.8 Dissolved RB 4.3 2.739 35 Gi-Hua-Mu 12.6 Dissolved LRB 4.8 0.961 36 Gin-Shi-Liu 3.7 Precipitation DG 4.2 1.044 37 Ha-Sai-Hua 8.4 Partly insoluble BDB 4.6 1.302 38 Shi-Sharng-Bor 2.1 Partly insoluble DG 5.9 1.017 39 Hu-Ji-Chi 1.9 Precipitation YB 4.5 1.338 40 Ci-Yeh-Suei-Ding-Hsiang 2.4 Mostly dissolved YB 3.7 0.618 41 Chein-Gin-Tung 4.9 Precipitation BR 4.0 1.030 42 Wu-Jei 4.7 Precipitation DG 7.2 0.847 43 Tie-Hsien-Jei 4.3 Precipitation YE 4.0 0.700 44 San-Hsiang 1.6 Precipitation YE 4.6 0.965 45 Gi-Yien-Sau 5.3 Oily DG 5.1 0.910 46 Hai-Chou-Chung-San 2.2 Dissolved YE 5.0 1.396 47 Huang-Ging 1.8 Dissolved YB 4.7 >4.5 48 Sarng-Er 1.9 Mostly dissolve LY 4.3 0.257 49 Hsien-Fong-Sau 6.8 Precipitation YE 4.9 0.857 50 Su-Chi-Sau 4.4 Suspended matter YE 4.2 1.166 51 Tsu-Bei-Sau 2.5 Precipitation YE 4.1 0.500 52 Harn-Hsiu-Sau 3.1 Mostly dissolved DB 3.7 0.324 53 Zu-Chi-Sau 8.0 Mostly dissolved YE 4.0 0.570 54 Cher-Chien-Sau 5.8 Suspended matter YE 3.6 0.693 55 Dau-Di-Ling 2.1 Suspended matter YE 4.8 0.783 56 Bai-Hua-Tung 4.3 Mostly dissolved DB 3.9 0.400 57 Ding-Di-Wu-Gong 2.0 Precipitation YE 6.5 2.698 58 Da-Gia-Sau 6.3 Suspended matter LY 4.1 0.618 59 Gi-Yeh-Tsu-Hua-Di-Ding 4.6 Precipitation OC 4.8 0.700 60 Taiwan Her-Shou-Wu 4.5 Suspended matter YB 4.3 0.468 61 New-Zu-Zong 2.9 Mostly dissolved LY 4.5 0.436 62 Bi-Yeh-Dung-Lorng-Sau 6.3 Suspended matter OC 4.7 0.258 63 Karng-Hsiang-Tung 5.3 Mostly dissolved LYB 4.7 1.024 64 Taiwan Kou-Tung 9.0 Dissolved DB 4.6 0.418 65 Lo-Chi-Yien-Fu-Mu 3.4 Mostly dissolved YE 4.2 1.314 66 Gin-Gien-Sau 1.5 Precipitation YE 4.6 0.506 67 Horng-Hua-Su-Wei-Sau 2.6 Mostly dissolved OC 6.2 0.499 68 Tu-Chi-Tsai 4.0 Precipitation YE 4.7 0.544 69 Dau-Sharng-Sau 3.6 Dissolved LY 4.7 0.379 70 Ku-Larn-Parn 3.7 Dissolved YE 4.5 0.445 71 Tou-Hua-Hsiang-Ku-Sau 3.8 Suspended matter LY 4.7 0.642 72 Du-Horng-Hua 3.3 Dissolved LY 4.1 1.029 73 Yeh-Hu 1.0 Mostly dissolved YB 6.0 1.491 74 Torng-Chuei-Yui-Di-Sau 10.5 Suspended matter LOC 4.6 0.428 75 San-Yeh-Wu-Gia 5.0 Suspended matter LY 4.5 1.445
[0046] Measurements were made by the 96-well plate method using Bi 6 mouse melanoma cells (B16-F1 cells, ATCC No. CRL-6323) purchased from Dainippon Pharmaceutical. The cells (1×10
[0047] As a result, as is evident from Table 3, 31 herb extracts were found to show an inhibition percentage of 20% or higher at the test concentration of 0.001% and, in particular, 18 items, namely Ku-Chi, Bi-Yeh-Dung-Lorng-Sau, Nan-Ling-Yia-Hua, Shi-Chung-Pu, Ding-Di-Wu-Gong, Yui-Jen-Sau, Sarng-Er, Bai-Hua-Tung, Gi-Hua-Mu, Taiwan Ma-Dou-Ling, Hsien-Fong-Sau, Huang-Gin-Guei, San-Jei-Tsai, Ma-Ti-Gin, New-Zu-Zong, Shi-Sharng-Bor, Horng-Hua-Su-Wei-Sau, Gi-Yeh-Tsu-Hua-Di-Ding, showed an IC50 value (i.e., the concentration of the test substance for reducing the amount of extracellular melanine of the controls used) not higher than 10 μg/mL and were found to be more active than arbutin or kojic acid used as the positive contols (Table 3). These active herb extracts were low in toxicity to cells, namely no cytotoxicity was observed at the test concentration of 0.001%.
TABLE 3 Melanine formation inhibition % No. Herb name Inhibition at 0.001% IC 10 Ku-Chi 104.6 1.9 62 Bi-Yeh-Dung-Lorng-Sau 94.3 1.9 21 Nan-Ling-Yia-Hua 93.5 0.015 26 Shi-Chung-Pu 93.1 2.7 57 Ding-Di-Wu-Gong 79.5 4.5 2 Yui-Jen-Sau 77.9 3.0 48 Sarng-Er 72.7 3.1 56 Bai-Hua-Tung 70.1 3.9 35 Gi-Hua-Mu 67.3 4.6 25 Taiwan Ma-Dou-Ling 65.5 4.6 49 Hsien-Fong-Sau 64.9 3.1 27 Huang-Gin-Guei 64.7 3.8 12 San-Jei-Tsai 53.3 8.7 8 Ma-Ti-Gin 52.8 8.6 61 New-Zu-Zong 51.6 7.5 38 Shi-Sharng-Bor 39.4 8.8 67 Horng-Hua-Su-Wei-Sau 34.5 9.9 66 Gin-Gien-Sau 34.5 10.9 51 Tsu-Bei-Sau 29.8 10.9 59 Gi-Yeh-Tsu-Hua-Di-Ding 29.1 10.0 15 Gin-New-Kou 26.6 — 29 Da-Ching 26.4 — 11 Bai-Hua-Tsai 24.6 — 39 Hu-Ji-Chi 22.7 — 40 Ci-Yeh-Suei-Ding-Hsiang 22.5 — 13 Pien-Di-Gin 22.4 — 5 Chi-Mu-Sau 22.0 — 20 Gin-Hsien-Lien 22.0 — 16 Mei-Zen-Giau 21.9 — 7 Tu-Chi-Chi 21.6 — 54 Cher-Chien-Sau 20.2 — Mulberry bark 61.9 3.8 Arbutin 32.4 12.1 Kojic acid 25.7 12.9
[0048] The synthetic substrate, methoxysuccinyl-Ala-Ala-Ala-Pro-p-nitroanilide (8.0 mM, 25 μL), was reacted with 25 μL of 0.1 M Hepes buffer (pH 7.4, containing 0.5 M NaCl) containing 5 μg/mL human neutrophilic leukocyte-derived elastase and 50 AL of the test sample at 37° C. for 30 minutes and, then, the yield of the decomposition product 4-nitroaniline was determined by measuring the absorbance at 405 nm using the microplate reader EL-340 (Bio-Tek Instrument). The enzyme inhibition rate was calculated using the following formula:
[0049] where A: absorbance without addition of sample, B: absorbance with addition of sample.
[0050] The results are shown below in Table 4.
TABLE 4 Elastase inhibition % No. Herb name Inhibition at 0.025% IC 65 Lo-Chi-Yien-Fu-Mu 66.6 1.4 34 Hsia-Yeh-Pu-Tao 66.5 9.0 52 Harn-Hsiu-Sau 63.8 46.0 35 Gi-Hua-Mu 61.4 10.5 57 Ding-Di-Wu-Gong 60.6 54.8 56 Bai-Hua-Tung 58.3 — 60 Taiwan Her-Shou-Wu 56.4 63.8 33 San-Yeh-Pu-Tao 56.2 22.8 31 Mu-Chu-Ma 52.1 50.9 39 Hu-Ji-Chi 49.6 — 9 Yeh-Sia-Chu 35.4 — 64 Taiwan Kou-Tung 34.8 — 21 Nan-Ling-Yia-Hua 34.3 — 55 Dau-Di-Ling 32.0 — 27 Huang-Gin-Guei 30.4 — 40 Ci-Yeh-Suei-Ding-Hsiang 28.6 — 43 Tie-Hsien-Jei 28.4 — 36 Gin-Shi-Liu 24.5 — ELHIBIN 29.3
[0051] As shown in Table 4, 18 herb medicines were found to have inhibitory activity of 20% or more at the concentration of 0.025% and, in particular, Lo-Chi-Yien-Fu-Mu, Hsia-Yeh-Pu-Tao, Gi-Hua-Mu, and San-Yeh-Pu-Tao showed a lower lC50 value as compared with the positive control substance ELHIBIN (soybean extract, product of PentaPharm) and thus found to have strong elastase-inhibiting activity.
[0052] The sample (100 μL) was added to 50 μL of 0.1 M acetate buffer solution (pH 4.0) containing hyaluronidase (type IV-S from bovine testis, 500 NF units/mL), and the mixture was incubated at 37° C. for 20 minutes. Then, 100 μL of a 0.1 mg/mL solution of the enzyme activator, Compound 48/80, was added, and incubation was further continued at 37° C. for 20 minutes. Thereto was added 250 AL of hyaluronic acid (from rooster comb, 0.5 mg/mL) and after allowing the reaction to proceed at 37° C. for 40 minutes, the reaction was terminated with 100 μL of 0.4 N-NaOH. Then, for assaying N-acetylhexosamine, 120 μL of 0.8 M potassium borate was added to the enzyme reaction mixture, the mixture was heated at 100° C. for 3 minutes and then cooled to room temperature. A 30-ItL portion of the mixture was taken out, 180 AL of 1% p-dimethylaminobenzaldehyde in acetic acid was added, and the reaction was allowed to proceed at 37° C. for 20 minutes, After cooling to room temperature, the OD at 590 nm was measured using the microplate reader EL-340 (Bio-Tek Instrument). The inhibition rate (%) was calculated according to the following formula:
[0053] where A: absorbance without addition of sample, B: absorbance with addition of sample.
[0054] The results are shown below in Table 5.
TABLE 5 Hyaluronidase inhibition No. Herb name % Inhibition at 0.02% IC 34 Hsia-Yeh-Pu-Tao 85.9 0.0048 27 Huang-Gin-Guei 80.1 0.0077 35 Gi-Hua-Mu 41.7 0.0122 Glycyrrhizin — 0.0054
[0055] As a result, when the test was performed at a concentration of 0.02%, an inhibitory activity higher than 40% was observed with three items, namely Hsia-Yeh-Pu-Tao, Huang-Gin-Guei, and Gi-Hua-Mu. Hsia-Yeh-Pu-Tao, in particular, gave a lower IC
[0056] TEST EXAMPLE 5
[0057] The test was performed by the 96-well microplate method using the kit “SOD Test Wako” (Wako Pure Chemical Industries) based on the principle of Nitro Blue Tetrazolium (NBT) reduction.
[0058] The sample (10 μL) was added to 95 μL of the color reagent solution containing 0.24 M NBT and 0.4 M xanthine. Further, 95 μL of the enzyme solution (xanthine oxidase) was added, and the mixture was incubated at 37° C. for 30 minutes. Thereafter, the enzyme reaction was terminated with 100 μL of the reaction terminating solution (69 mM sodium dodecyl sulfate), and the OD 562 nm was measured on the microplate reader EL-340 (Bio-Tek Instrument). A sample prepared by adding purified water in lieu of the enzyme was used as a blank, and a sample prepared by adding the solvent in lieu of the sample was used as a control. The superoxide anion (02-) elimination rate was calculated by the following formula:
[0059] where A: absorbance of control, B: absorbance with addition of sample.
[0060] IC
[0061] As a result, 57 herb medicines were found to have SOD-like activity (30% or higher) at the concentration of 0.025%. Huang-Gin-Guei, Lo-Chi-Yien-Fu-Mu, and Yeh-Sia-Chu gave a lower IC
TABLE 6 SOD-like activity % No. Herb name Elimination at 0.025% IC 27 Huang-Gin-Guei 100.8 34.5 35 Gi-Hua-Mu 100.2 71.6 65 Lo-Chi-Yien-Fu-Mu 99.5 53.8 34 Hsia-Yeh-Pu-Tao 95.4 77.3 9 Yeh-Sia-Chu 90.9 54.5 22 Bai-Yeh-Chi-Ken 79.7 82.3 19 Gin-Di-Lo 79.6 61.9 60 Taiwan Her-Shou-Wu 78.2 88.3 32 Ya-Jou 74.4 86.1 44 San-Hsiang 72.1 85.0 64 Taiwan Kou-Tung 70.6 90.0 50 Su-Chi-Sau 69.0 — 36 Gin-Shi-Liu 65.3 — 46 Hai-Chou-Chung-San 65.0 — 58 Da-Gia-Sau 61.5 — 37 Ha-Sai-Hua 60.9 — 31 Mu-Chu-Ma 59.7 — 73 Yeh-Hu 59.1 — 2 Yui-Jen-Sau 58.1 — 29 Da-Ching 58 — 14 Sarng-Han-Sau 56.5 — 6 Ma-Bien-Sau 56.2 — 55 Dau-Di-Ling 56.2 — 69 Dau-Sharng-Sau 54.5 — 26 Shi-Chung-Pu 53.0 — 3 Ban-Ji-Lien 52.3 — 72 Du-Horng-Hua 52.3 — 63 Karng-Hsiang-Tung 52.1 — 53 Zu-Chi-Sau 51.4 — 40 Ci-Yeh-Suei-Ding-Hsiang 50.8 — 71 Tou-Hua-Hsiang-Ku-Sau 50.0 — 18 Hua-Shi-Su 49.8 — 54 Cher-Chien-Sau 49.0 — 66 Gin-Gien-Sau 47.3 — 39 Hu-Ji-Chi 47.0 — 75 San-Yeh-Wu-Gia 46.1 — 24 Hsien-Sau 45.3 — 17 Ma-An-Tung 45.2 — 70 Ku-Larn-Parn 43.6 — 47 Huang-Ging 42.8 — 21 Nan-Ling-Yia-Hua 42.6 — 4 Ban-Bien-Lien 41.8 — 7 Tu-Chi-Chi 41.6 — 74 Torng-Chuei-Yui-Di-Sau 41.4 — 68 Tu-Chi-Tsai 41.0 — 23 Yui-Yeh-Gin-Hua 40.9 — 1 Suei-Ding Hsiang 40.0 — 57 Ding-Di-Wu-Gong 39.6 — 16 Mei-Zen-Giau 39.4 — 41 Chein-Gin-Tung 39.2 — 33 San-Yeh-Pu-Tao 37.2 — 28 Chou-Mor-Li 37 — 11 Bai-Hua-Tsai 32.3 — 52 Harn-Hsiu-Sau 31.9 — 10 Ku-Chi 31.2 — 30 Bai-Lorng-Chun-Hua 31.1 — 43 Tie-Hsien-Jei 30.3 — Mellisa — 50.5 Gambir — 55.0
[0062]
(1) 0.2 M Acetate buffer (pH 5.5) 50 μL (2) 250 μM DPPH/ethanol 100 μL (3) Sample/50% ethanol 100 μL
[0063] (1) to (3) were placed in each well of a 96-well micrpalte (product of NUNC) and, after 30 minutes of incubation at 30 C, the absorbance at the wavelength 515 nm was measured using the microplate reader EL-340 (BIO-TEK INSTRUMENT). The extent of activity was expressed in terms of DPPH Radial Scavenging (%) calculated according to the following formula:
[0064] where A: absorbance without addition of sample, B: absorbance with addition of sample.
[0065] IC
TABLE 7 DPPH radical scavenging % No. Herb name Inhibition at 0.002% IC 60 Taiwan Her-Shou-Wu 83.5 9.3 65 Lo-Chi-Yien-Fu-Mu 82.2 8.2 35 Gi-Hua-Mu 77.5 2.6 22 Bai-Yeh-Chi-Ken 76.4 5.1 32 Ya-Jou 75.7 7.2 9 Yeh-Sia-Chu 75.6 5.3 27 Huang-Gin-Guei 75.3 11.4 31 Mu-Chu-Ma 74.9 6.4 19 Gin-Di-Lo 74.5 10.4 36 Gin-Shi-Liu 73.2 14.6 64 Taiwan Kou-Tung 69.4 9.6 33 San-Yeh-Pu-Tao 68.5 11.1 46 Hai-Chou-Chung-San 62.8 26.0 2 Yui-Jen-Sau 62.3 16.5 29 Da-Ching 59.6 — 37 Ha-Sai-Hua 57.8 — 34 Hsia-Yeh-Pu-Tao 57.1 — 23 Yui-Yeh-Gin-Hua 54.4 — 17 Ma-An-Tung 50.5 — 40 Ci-Yeh-Suei-Ding-Hsiang 49.9 — 1 Suei-Ding Hsiang 49.2 — 63 Karng-Hsiang-Tung 45.9 — 44 San-Hsiang 44.2 — 6 Ma-Bien-Sau 42.7 — 43 Tie-Hsien-Jei 38.4 — 45 Gi-Yien-Sau 37.7 — 24 Hsien-Sau 37.6 — 61 New-Zu-Zong 37.2 — 14 Sarng-Han-Sau 36.2 — 47 Huang-Ging 35.5 — 58 Da-Gia-Sau 34.7 — 57 Ding-Di-Wu-Gong 33.8 — 42 Wu-Jei 33.3 — 39 Hu-Ji-Chi 32.4 — 53 Zu-Chi-Sau 32.2 — 3 Ban-Ji-Lien 31.8 — 21 Nan-Ling-Yia-Hua 30.8 — Ascorbic acid — 4.1 Glutathione — 13.5
[0066] As a result, 37 herb medicines were found to have 30% or higher DPPH radical scavenging activity at the concentration of 0.002%. In particular, Gi-Hua-Mu gave an IC
[0067] The medicinal herb extracts from Gi-Hua-Mu and Huang-Gin-Guei were found to have all the above-mentioned activities, namely melanine formation-inhibiting, elastase-inhibiting, hyaluronidase-inhibiting, SOD-like, and DPPH radical-capturing activities. The herb extracts from Nan-Ling-Yia-Hua, Hu-Ji-Chi, Ci-Yeh-Suei-Ding-Hsiang, and Ding-Di-Wu-Gong were found to have melanine formation-inhibiting, eiastase-inhibiting, SOD-like, and DPPH radical-capturing activities simultaneously. The extract from Hsia-Yeh-Pu-Tao was a herb medicine having melanine formation-inhibiting, hyaluronidase-inhibiting, SOD-like and DPPH radical-capturing activities simultaneously. Thus were found a number of herb medicines each having a combination of whitening-anti-inflammatory-antioxidant, whitening-antioxidant, whitening-anti-inflammatory, or antioxidant-anti-inflammatory activities.
[0068] The use of such herb medicines having a plurality of activities as medicinal herb extracts to be incorporated into anti-aging compositions can be expected to produce diversified and efficient effects with a smaller number of herb medicines. Further, their effects will be produced synergistically, making it possible to establish formulations with enhanced anti-aging effects.
[0069] In the following, typical examples of different preparation forms containing the aging-retarding composition of the invention are described.
[0070] Face lotions having the respective formulations shown below in Table 8 were prepared by using the extracts of the medicinal herbs Gi-Hua-Mu, Huang-Gin-Guei, Lo-Chi-Yien-Fu-Mu, Hsia-Yeh-Pu-Tao, and Nan-Ling-Yia-Hua.