KR20090069732A - SKIN WHITENING COMPOSITION COMPRISING ARBUTIN beta-DERIVATIVE - Google Patents

Abstract

A skin whitening composition containing albutin beta derivative is provided to suppress tyrosinase activation and over synthesis of melanin and have excellent safety. A composition for skin whitening comprises a beta-D-glucosyl-(1->3)-arbutin which is an albutin beta derivative of the chemical formula 1. The albutin beta derivative is synthesized through the transglycosylation of thermotoga neapolitana beta-glucosidase. The composition is used in a form of oral preparation, cosmetic product, parenteral formulation of external skin preparation, and medicinal product. The cosmetic product is used in a form of ointment, solution, cream, emulsion, lotion, gel, essence, foundation, pack mask, rouge, stick, and bath product. The composition further comprises 0.0001-10 weight% of whitening agent.

Description

Skin whitening composition comprising arbutin β-derivative}

The present invention relates to a skin whitening agent, and more particularly, to a composition for skin whitening having a whitening function and a skin protection function by inhibiting tyrosinase activity using an arbutin beta derivative to inhibit melanin oversynthesis.

In general, when the skin is exposed to ultraviolet light, the epidermis becomes black, because the melanin, a black pigment, is synthesized and released from melanocytes. Tyrosinase of melanocytes synthesizes dopaquinone from tyrosine, and dopaquinone undergoes spontaneous oxidation to form melanin. Melanin absorbs excess light in vivo, but it is concentrated in specific areas to produce spots, blotch, etc., and may cause abnormal skin discoloration due to pregnancy blockage or skin pathology. In addition, many cosmetic compositions that are commercially available because of a strong desire to whiten the skin also contains a whitening agent component.

As such, melanin is a human skin pigment that plays a primary role in photoprotection (Riley 2003; Ahn et al. 2006) and is secreted by melanocytes distributed throughout the basal dermis (Spritz). et al. 1994). One of these melanin's roles is to protect the skin and subcutaneous tissue from UV-induced skin damage, but overproduction of melanin in the skin has a negative hyperpigmentation effect, causing spots, freckles and blotch (Ha et al. 2005) Min et al. 2004; Unver et al. 2006), and hyperpigmentation in the epidermis and dermis also depends on an increase in the number of melanocytes or an increase in the activity of melanogenesis enzymes (Griffiths et al. 1993; Kanwar et al. 1994).

In addition, tyrosinase (EC 1.14.18.1) is one of the important enzymes in melanin synthesis (Kubo et al. 2000; Perez-Gilabert and Garcia-Carmona, 2001) and dopa (3), the first two steps in melanin synthesis. It acts as a catalyst for the hydroxylation of-(3,4-dihydroxyphenyl) -alanine, DOPA) and the oxidation of dopa to dopaquinone (Shin et al. 1998). Many tyrosinase inhibitors are also obtained by natural and synthetic, and only a few of them are utilized as skin lightening agents, mainly due to various safety considerations. For example, linoleic acid, hinokitol, kojic acid, naturally occurring hydroquinone and catechol are all reported to inhibit enzymatic activity, but deleterious side effects have been demonstrated (Seo et al. 2003), among which 4-hydroxyphenyl β -D-glucopyranoside (arbutin) shows significant tyrosinase inhibitory activity.

In previous studies, 4-hydroxyphenyl α-D-glucopyranoside (α-arbutin) and α-arbutin-α-glycosides were enzymatically synthesized (Sugimoto et al. 2003; Sugimoto et al. 2005), and their inhibitory effects on melanin formation The inhibitory activity of α-arbutin and α-arbutin-α-glycosides against several tyrosinase from different sources was also compared. α-arbutin and α-arbutin-α-glycosides showed higher inhibitory activity than that of arbutin against mammalian tyrosinase (Sugimoto et al. 2005). In other words, tyrosinase inhibition studies with arbutin derivatives show that α-glycoside association in hydroquinone-glycosides will play an important role in tyrosinase inhibition in humans.

As described above, the melanin pigment produced in the melanocytes of the body skin is a phenolic polymer having a complex form of black protein, and plays an important role in inhibiting skin damage caused by ultraviolet rays irradiated from the sun, and melanin for melanin biosynthesis Since the action of tyrosinase present in cells is important, most of the studies for skin whitening and skin cancer prevention through melanin overproduction and deposition inhibition have focused on the inhibition of tyrosinase activity.

In addition, inhibitors of tyrosinase activity developed to date include chelate-forming substances for copper ions of tyrosinase active sites, reducing agents such as vitamin C (ascorbic acid) or kojic acid, which have reduced quinones to phenols, and tyrosinase itself. Modified bisulfite agents and the like are known. In addition, 4-hydroxyanisole (4-hydroxyanisole), hydroquinone (hydroquinone), retinol (retinol) or hydrocortisole (hydrocortisole), etc. are known as substances that exhibit a whitening effect by inhibiting tyrosinase activity. Of these, kojic acid and hydroquinone are known as representative materials having a whitening effect. Kojic acid is a component obtained from yeast mold fermentation broth and is mainly used as a raw material for whitening cosmetics because it inhibits melanin production, and hydroquinone is used in more than 80% of the whitening agent currently produced and sold (Ardnt K. A. et al. 1965).

Hydroquinone, however, has a strong inhibitory effect on tyrosinase, but not only causes skin irritation or dermatitis caused by cytotoxicity such as degeneration or lethality of melanocytes, but also acts as a strong mutagen for mammalian cells. It is known.

Therefore, as part of efforts to find low cytotoxic tyrosinase inhibitors, studies on alternatives to hydroquinone such as arbutin have been conducted, but no effective tyrosinase inhibitors have been developed. Thermotoga in the previous study of this study Arbutin beta derivatives were produced by transglycosylation of beta-glycoside derived from neapolitana , and arbutin and cellobiose were used as acceptors and donors, respectively. Arbutin derivative production was confirmed using TLC and HPAEC. As a result, compounds 1, 2, and 3 were formed, and NMR analysis showed β-D-glucosyl- (1 → 6) -arbutin (compound 1), β-D-glucosyl- (1 → 4) -arbutin (compound 2) and β-D-glucosyl- (1 → 3) -arbutin (compound 3).

Accordingly, the present inventors have led to the present invention by studying the tyrosinase inhibitory effect using the arbutin and arbutin beta derivatives.

references

1.Ahn SJ, Koketsu M, Ishihara H, Lee SM, Ha SK, Lee KH, Kang TH, Kima SY (2006) Regulation of melanin synthesis by selenium-containing carbohydrates. Chem Pharm Bull 54: 281-286

2. Griffiths CE, Finkel LJ, Ditre CM, Hamilton TA, Ellis CN, Voorhees JJ (1993) Topical tretinoin (retinoic acid) improves melasma. A vehicle-controlled, clinical trial. Br J Dermatol 129: 415-421

3.Ha SK, Koketsu M, Lee K, Choi SY, Park JH, Ishihara H, Kim SY (2005) Inhibition of tyrosinase activity by N, N-unsubstituted selenourea derivatives. Biol Pharm Bull 28: 838-840

Kanwar AJ, Dhar S, Kaur S (1994) Treatment of melasma with potent topical corticosteroids. Dermatology 1

5. Kubo I, Kinst-Hori I, Chaudhuri SK, Kubo Y, Sanchez Y, Ogura T (2000) Flavonols from Heterotheca inuloides: tyrosinase inhibitory activity and structural criteria. Bioorg Med Chem 8: 1749-1755

6.Maeda K, Fukuda M (1991) In vitro effectiveness of several whitening cosmetic components in human melanocytes. J. Soc. Cosmet. Chem. 42: 361-368

7.Min KR, Kim KS, Ro JS, Lee SH, Kim JA, Son JK, Kim Y (2004) Piperlonguminine from Piper longum with inhibitory effects on alpha-melanocyte-stimulating hormone-induced melanogenesis in melanoma B16 cells. Planta Med 70: 1115-1118.

8.Miyazawa M, Tamura N (2007) Inhibitory compound of tyrosinase activity from the sprout of Polygonum hydropiper L. (Benitade). Biol Pharm Bull 30: 595-597

9.Park TH, Choi KW, Park CS, Lee SB, Kang HY, Shon KJ, Park JS, Cha J (2005) Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana . Appl Microbiol Biotechnol 69: 411-422

10. Perez-Gilabert M, Garcia-Carmona F (2001) Dimethyl sulfide, a volatile flavor constituent, is a slow-binding inhibitor of tyrosinase. Biochem Biophys Res Commun 285: 257-261

11. Riley PA (2003) Melanogenesis and melanoma pigment. Cell Res 16: 548-552

12. Seo SY, Sharma VK, Sharma N (2003) Mushroom tyrosinase: recent prospects. J Agric Food Chem 51: 2837-2853

13. Shin NH. Ryu SY, Choi EJ, Kang SH, Chang IM, Min K, Kim Y (1998) Oxyresveratrol as the potent inhibitor on dopa oxidase activity of mushroom tyrosinase. Biochem Biophys Res Commun 243: 801-803

14. Spritz RA, Hearing VJ Jr (1994) Genetic disorders of pigmentation. Adv Hum Genet 22: 1-45

15. Sugimoto K, Nishimura T, Nomura K, Sugimoto K, Kuriki T (2003) Syntheses of arbutin-α-glycosides and a comparison of their inhibitory effects with those of α-arbutin and arbutin on human tyrosinase. Chem Pharm Bull 51: 798-801

Sugimoto K, Nishimura T, Nomura K, Sugimoto K, Kuriki T (2004) Inhibitory effects of α-arbutin on melanin synthesis in cultured human melanoma cells and a three-dimensional human skin model. Biol Pharm Bull 27: 510-514

17. Sugimoto K, Nomura K, Nishimura T, Kiso T, Sugimoto K, Kuriki T (2005) Syntheses of α-arbutin-alpha-glycosides and their inhibitory effects on human tyrosinase. J Biosci Bioeng 99: 272-276

18.Unver N, Freyschmidt-Paul P, HorsterS, Wenck H, St Mu F, Blatt T, Elsasser HP (2006) Alterations in the epidermal-dermal melanin axis and factor XIIIa melanophages in senile lentigo and ageing skin. BrJ Dermatol 155: 119-128

An object of the present invention is to provide a skin lightening agent of arbutin beta derivatives having tyrosinase inhibitory effect and not toxic to skin.

In order to solve the above object, the present invention provides an arbutin beta derivative which inhibits melanin production by inhibiting tyrosinase activity and has no toxicity to skin.

More specifically, the present invention provides a composition for skin whitening containing β-D-glucosyl- (1 → 3) -arbutin, which is an arbutinbeta derivative, having the following chemical structure represented by the formula (1).

The present invention also relates to a cosmetic composition for skin whitening, wherein the composition is a cosmetic.

The present invention also relates to a pharmaceutical composition for skin whitening, wherein the composition is a medicament.

Preferably the present invention relates to a composition for skin whitening further comprising a whitening component other than the arbutin beta derivative.

The composition for skin whitening of the present invention suppresses melanin production in melanocytes, and prevents and improves pigmentation of skin such as whitening of sunburned skin after ultraviolet irradiation, and blotch, freckles, and spots caused by sunburn. And treatment.

The composition of the present invention may take the form of oral (content) or parenteral (external). In the case of oral use, the composition of this invention can be prepared, for example in the form of a medicine or food. In the case of parenteral use, it can be prepared in the form of cosmetics, quasi-drugs, medicines, skin external preparations and the like.

In the composition containing the arbutin beta derivative in the present invention, that is, the composition such as the whitening composition, the whitening medicine, the whitening skin external preparation, and the like, the whitening agent component other than the arbutin beta derivative (hereinafter also simply referred to as the whitening component) It may further contain, by the combination of the arbutin beta derivative and the whitening agent components, it is possible to synergistically improve the melanin production inhibitory effect and thereby the whitening effect.

As said whitening agent component, it does not specifically limit, For example, ascorbic acid, its derivative (s), and its salts (for example, ascorbic acid, ascorbic acid-2-glucoside, ascorbic acid phosphate ester magnesium salt) Sodium ascorbate, stearic acid ascorbyl, palmitic acid ascorbyl, dipalmitic acid ascorbyl, tetraisopalmitate ascorbyl, magnesium ascorbate, chitosan ascorbate, ascorbyl methyl silanolfecti Nate, Ascorbic Acid Polypeptide, Diazelaic Acid Ascorbyl, Ascorbic Acid Polyphosphate Ester, Ascorbic Acid Polyoxyethylene Derivatives, Ricinol Acid Ascorbyl, Ascorbic Acid-2-Sulfate Ester Salt, etc.), hydroquinone And derivatives thereof and salts thereof (e.g. arbutin), cysteine and derivatives thereof and salts thereof (e.g. L-cysteine, N, N'-diacetyl Cystinedimethyl, etc.), placenta extract, kojic acid and its derivatives, rucinol, errac acid and its derivatives, glucosamine and its derivatives, azeline and its derivatives, hydroxycinnamic acid and its derivatives, glutathione, plant extracts (camomile extract, arnica Extract, Ogon Extract, Cheongung Extract, Mulberry Leaf Extract, Psycho Extract, Windproof or Water Extract, Gaetung Extract, Gymnema Extract, Basswood Extract, Peach Leaf Extract, Red Ginseng Extract, Chew Extract, Toki Extract, Yulmu Rice Extract, Persimmon Leaf Extract, Rhubarb Extract, Peony Extract, Hamamelis Extract, Horse Chestnut Extract, Red Pepper Extract, Clown Extract, Licorice Extract, Capillary Extract, Cinnamon Extract, Mountain Head Extract, Ibukitorano bistorta Linn) extract, red ginseng extract, Sanjay extract, lily extract, hop extract, brier extract, etc.) Dean, there may be mentioned Gras Brenner, François Come Tin, isopropyl leak Come Augustine, hydrophobic licorice flavonoid, Ricoh chalcone A, such as placenta extract. 1 type (s) or 2 or more types are arbitrarily selected and can be mix | blended with the whitening agent component. As for the compounding quantity (content) of the whitening agent component at the time of mix | blending the whitening agent component, 0.0001-10 weight% is preferable in the said whole composition, More preferably, it is 0.001-5 weight%. In addition, in the case of an extract, it is the quantity converted into the dry weight which removed the solvent from the extract.

Hereinafter, specific compositions of the present invention containing arbutin beta derivatives will be described in more detail by use.

The arbutin first use cosmetic of the present invention may be used as an external preparation for skin such as an ointment, a solution, a cream, an emulsion, a lotion, a lotion, a gel, an essence (a cosmetic), a foundation, a pack mask, a rouge, a stick, a bathing agent, and the like. Applicable in a wide range including quasi drugs.

In addition, the formulations of cosmetics, solution, solubilizing, emulsifying, powder, powder dispersion, emulsion, gel, ointment, aerosol, water-oil two-layer, water-oil- three-layer, etc. Formulations may be employed. As for the compounding quantity of the arbutin beta derivative to the cosmetics of this invention, 0.001-20 weight% is preferable in cosmetics whole quantity, More preferably, it is 0.01-16 weight%, More preferably, it is 0.1-12 weight%. Most preferred is 1 to 10% by weight. The cosmetic of the present invention has a melanin production inhibitory effect and a whitening effect, and thus is used as a melanin production inhibitory cosmetic and a whitening cosmetic. Moreover, since the cosmetics of this invention can be preferably applied especially to the use as a skin external preparation, it is used as a skin external preparation for melanin production suppression, and a whitening skin external preparation.

The pharmaceutical of the second use of the present invention can be adopted by any method of oral administration, parenteral administration, and can be in the form of a pharmaceutical formulation suitable for each. Examples of pharmaceutical preparations include liquid preparations such as liquids, syrups, injections, inhalants, and emulsions, tablets, powders, granules, capsules, solid preparations such as inhalants, and external skin preparations such as ointments and suppositories. External preparations; and the like.

As for the compounding quantity of the arbutin beta derivative to a pharmaceutical agent, 0.001-30 weight% is preferable in the whole pharmaceutical preparation. More preferably, it is 0.01-20 weight%, Most preferably, it is 0.1-10 weight%. The dosage is appropriately changed based on the age and weight of the patient, the route of application, the progression of the disease, and the treatment being performed in parallel, but is not particularly limited, but is generally about 4 to 10 ml per day, Although administration can be carried out once per day or divided into 2-3 times, it is not limited to this. Since the medicine of this invention has a melanin production inhibitory effect and a whitening effect, it is used as a melanin production inhibitory medicine and a whitening medicine. In addition, the medicament of the present invention can be suitably applied to the use as a skin external preparation, and therefore can be used as a skin external preparation for inhibiting melanin production and a skin external preparation for whitening.

In addition to the arbutin beta derivatives described above, other components usually used in cosmetics, medicines, external preparations for skin and the like can be blended into the compositions such as cosmetics and medicines of the present invention in the range that does not impair the effects of the present invention.

For example, cosmetics include oils, powders, surfactants, humectants, thickeners, lower alcohols, coatings, ultraviolet absorbers, metal ion sequestrants, organic amines, pH adjusters, active ingredients, sugars, preservatives, vitamins, antioxidants, Fragrance, water, etc. are mentioned.

Examples of the oil include jojoba oil, olive oil, avocado oil, castor oil, palm oil, tallow, hydrogenated oil, natural oils and derivatives thereof such as liquid lanolin, lead such as carnauba wax, beeswax and lanolin, liquid paraffin, micro crystalline wax , Hydrocarbons such as squalene and petrolatum, higher fatty acids such as stearic acid, higher alcohols such as cetyl alcohol and stearyl alcohol, glycerin monostearic acid ester, glyceryl triglycerate, glycerin monooleate and isopropyl myristin Esters such as diisostearyl malic acid, glycerin di2-heptyl undecanoate, trimethylolpropane tri-ethylhexyl trimetholpropane, trimethylolpropane trioctanoate, di2-ethylhexyl sebacic acid, peppermint oil, rose oil, Essential oils such as citroneral, silicone oils such as dimethylpolysiloxane, decamethylcyclopentasiloxane, and the like. Although the compounding quantity of an oil component is suitably selected according to the form, formulation, etc. of cosmetics, Usually, it can be 0.1-95 weight% in cosmetics whole quantity.

Examples of the powder include talc, mica, kaolin, silica, galvanized, mica titanium, titanium oxide, iron oxide, nylon powder, and the like.

Examples of the surfactant include polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene hardened castor oil, polyoxyethylene sorbitol fatty acid ester, polyoxyalkylene-modified polysiloxane, and the like. Nonionic surfactants, anionic surfactants such as sodium palmitate, cationic surfactants such as stearyltrimethylammonium chloride, and amphoteric surfactants such as betaine, amidebetaine, sulfobetaine, and imidazolinium. Can be.

Examples of the moisturizing agent include glycerin, 1,3-butylene glycol, polyethylene glycol, dipropylene glycol, sorbitol, and the like.

Examples of the thickener include water-soluble polymers such as carboxyvinyl polymer, carboxymethyl cellulose and polyvinyl alcohol, and clay minerals such as bentonite.

Examples of ultraviolet absorbers include paraamino benzoic acid (abbreviated as PABA), glyceryl PABA, ethyldihydroxypropyl PABA, octylmethoxycinnamate, 2-ethoxyethyl-p-methoxycinnamate, 2,4- Dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4-methylbenzophenone, 2-hydroxy-4-methoxy-4-methylbenzophenonesul Fonates, urokanoic acid ethyl esters, 2-phenyl-5-methylbenzokizazole, 4-methoxy-4-t-butyldibenzoylmethane, paramethoxycinnamic acid ethylhexyl, and the like.

Next, as a medicament, various additives such as excipients, stabilizers, moisturizers, emulsifiers, absorption accelerators, pH adjusters, surfactants, diluents, and carriers can be blended. Specific examples of these additives include starch, sugars such as lactose, cellulose derivatives such as magnesium sulfate, talc, gelatin, hydroxypropyl cellulose, vegetable oils such as soybean oil, sesame oil, animal oils or synthetic oils, rubber, physiological Water such as saline, alcohols such as ethanol, 1,3-butylene glycol, polyalkylene glycol, and the like. In addition, it can also select and mix | blend among the components which can be mix | blended with the said cosmetics and the foodstuff mentioned later.

The said composition of this invention can mix 1 type (s) or 2 or more types of the essential component of this invention and the said arbitrary compounding components, and can be prepared in arbitrary forms and formulations by a conventional method.

Β-D-glucosyl- (1 → 3) -arbutin, which is newly synthesized according to the present invention, is a novel skin lightening agent, has melanogenesis inhibitory activity, is more effective in whitening than arbutin, a melanogenesis inhibitor, and cytotoxicity is greater than arbutin. It is effective in protecting skin.

Hereinafter, the present invention will be described in detail with reference to Examples, but the content of the present invention is not limited to the following Examples.

<Example 1>

1-1 materials

α-Melanocyte Stimulating Hormone (MSH), Arbutin, Cellobiose, Mushroom Extract Tyrosinase and L-Tyrosine were obtained from Sigma Chemical Co. (St. Louis, USA). α-D-glucosyl- (1 → 4) -arbutin was supplied by Professor Park Cheon-seok of Kyung Hee University.

Preparation and Purification of 1-2 Arbutin β-Glycoside

Arbutin β-glycosides were prepared by the method described in Park et al. (Reference 9). Thermotoga A 5 ml reaction mixture comprising 17.5 μg of neapolitana β -glucosidase and 15% cellobis and 15% arbutin in 100 mM sodium phosphate buffer (pH 7.0) was incubated at 80 ° C. for 12 hours. The transferred product was isolated by recycling preparative high-performance liquid chromatography. About 3 ml of the sugar transition product was applied to a JAIGEL W-251 (JAI Korea) column (2 × 50 cm) and eluted with demineralized water at a flow rate of 3 ml / min.

Fractions comprising arbutin β-glycoside were collected and lyophilized. The purity of each of the isolated arbutin β-glycosides was confirmed by thin layer chromatography (TLC) and high performance anion exchange chromatography (HPAEC).

<Example 2>

Enzymatic assay of tyrosinase

Tyrosinase activity was analyzed according to a method slightly modified from the method disclosed in Miyazawa et al. (Reference 8). That is, the reaction mixture containing 100 µl of 0.03% L-tyrosine, 640 µl of 67 mM phosphate buffer (pH 7.0), and 50 µl of the test sample solution was preincubated at 37 ° C for 10 minutes. Then 10 ml of 60 U mushroom tyrosinase solution dissolved in 67 mM phosphate buffer was added and the reaction mixture was incubated at 37 ° C. for 30 minutes in a 96-well plate.

Tyrosinase activity was determined by the following equation.

Tyrosinase Activity (%) = [( AB ) / (Cp-Cn)] × 100

Where A is the absorbance of the test sample (67 mM phosphate buffer, L-tyrosine, sample solution and tyrosinase) and B is the absorbance of the unknown sample (67 mM phosphate buffer, distilled water, sample solution and tyrosinase). The absorbance of the control group (67 mM phosphate buffer, L-tyrosine and tyrosinase), and Cn is the absorbance of the negative control group (67 mM phosphate buffer, L-tyrosine and tyrosinase).

As shown in FIG. 2, β-D-glucosyl- (1 → 3) -arbutin showed the highest inhibitory effect among the arbutin derivatives. The IC ₅₀ value of β-D-glucosyl- (1 → 3) -arbutin was 5 mM, whereas the IC ₅₀ value of arbutin was 6 mM (Table 1). Here, IC (Inhibitory concentration) ₅₀ is a criterion for evaluating the inhibitory concentration of a substance, and if it is 100% inhibition when no inhibitor is present, the concentration value corresponds to inhibiting about 50% of inhibitory activity. Therefore, it can be seen that the tyrosine inhibitory activity of β-D-glucosyl- (1 → 3) -arbutin and α-D-glucosyl- (1 → 4) -arbutin is similar. This suggests that regioselectivity of the glycosidic bonds of arbutin glycosides is involved in tyrosinase inhibition.

IC ₅₀ Value of Arbutin for Tyrosinase Extracted from Mushrooms Compound Inhibition at 10 mM (%) IC ₅₀ (mM) Arbutin 66 6 β-D-glucopyranosyl- (1 → 3) -arbutin 74 5 β-D-glucopyranosyl- (1 → 4) -arbutin 69 5

<Example 3>

Inhibition of melanin production in B16F10 melanoma cells

B16F10 mouse melanoma cell lines were obtained from the Korea Cell Line Bank (KCLB, Seoul, South Korea), and DMEM (Dulbecco's modified Eagle's medium) medium (BioWhittaker, Walkersville, MD, USA supplemented with 10% fetal bovine serum (FBS) (BioWhittaker) ) And incubated at 37 ° C. in an atmosphere containing 5% CO ₂ .

B16F10 cells were cultured in 60 mm tissue culture dishes (Iwaki, Japan) and kept 2 ml of DMEM containing 10% FBS. After 48 hours the cells were washed twice with phosphate-buffered saline (PBS) (BioWhittaker), then 2 ml of fresh medium was fed and treated with α-MSH at a final concentration of 200 nM. Samples were dispensed into dishes in a dilution series (final concentrations: 1, 5, 10, 20 mM). Cells were harvested after 48 hours and washed twice with 2 ml of PBS. Cells were collected by centrifugation (1700 × g) for 5 minutes at 4 ° C. and the supernatant was removed. 200 μl of 1 N NaOH (10% DMSO) was added to the cells and incubated at 80 ° C. for 1 hour. After melanin was dissolved by vortex, the melanin content was determined at 405 nm with Synergy HT Multi-Detection Microplate Reader (Biotek Instruments, Inc, Winooski, VT, USA).

As shown in FIG. 3, the degree of inhibition of B16F10 melanoma cell pigment as a result of melanin synthesis after reacting with B16F10 melanoma cells was 1-20 in β-D-glucosyl- (1 → 3) -arbutin. In the range of mM showed the highest inhibitory effect, especially melanin synthesis was significantly inhibited at 5 mM, about 70% less than untreated control cells. This demonstrates that β-D-glucosyl- (1 → 3) -arbutin effectively inhibits melanin production.

<Example 4>

Toxicity Analysis

B16F10 cells were cultured in 96-well tissue culture plates (Nunc, USA) and maintained in 200 μl of DMEM containing 10% FBS. After 48 hours, cells were washed twice with PBS, 200 μl of fresh medium was supplied, and treated with α-MSH at a final concentration of 200 nm. Samples were dissolved in PBS and then dispensed onto plates in dilution series (final concentrations: 1, 5, 10, 20 mM). After 48 hours the cells were washed twice with PBS and then replaced with fresh medium. 20 μl of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide (MTT) solution (5 mg / ml in PBS) was added to each well, followed by 37 ° C. for 12 hours. It was further cultured in. After media removal, cells were lysed with 100 μl of DMSO and conversion to MTT's formazan via absorbance measurement at 570 nm using Synergy HT Multi-Detection Microplate Reader (Biotek Instruments, Inc., Winooski, VT, USA). Quantification.

As a result, as shown in FIG. 4, β-D-glucosyl- (1 → 3) -albutin was reacted with B16F10 melanoma cells and reacted with B16F10 melanoma cell pigment as a result of melanin synthesis. Cell viability up to mM concentration was confirmed that little change.

<Results and Discussions>

Thermotoga The glycosyltransferase reaction of β-glycosidase from neapolitana was successfully used in the production of arbutin β-glycoside in this study, and arbutin and cellobiose were used as acceptor and donor molecules, respectively. Synthesis of arbutin β-glycoside was confirmed using TLC and HPAEC. Purification via repeated preparation HPLC yielded Compounds 1, 2 and 3 (8.1 mg, 8.0 mg, and 5.3 mg) of white crystals with a total probability of 2.84% based on the added donor. NMR analysis of three purified arbutin β-glycosides showed that the structures of the three compounds were β-D-glucosyl- (1 → 6) -arbutin (compound 1) and β-D-glucosyl- (1 → 4), respectively. -Arbutin (compound 2), and β-D-glucosyl- (1 → 3) -arbutin (compound 3) (Park et al. 2005) (FIG. 1).

In order to investigate the tyrosine inhibitory effect of the purified arbutin β-glycoside of the present invention, IC ₅₀ values of three arbutin β-glycosides against tyrosinase extracted from mushrooms were evaluated using L-tyrosine as a substrate. The highest inhibitory effect on mushroom tyrosinase was demonstrated between the evaluated arbutin glycosides and β-D-glucosyl- (1 → 3) -arbutin (see FIG. 2). The IC ₅₀ value of β-D-glucosyl- (1 → 3) -arbutin was 5 mM, while the value of arbutin was 6 mM (Table 1). IC ₅₀ values of β-D-glucosyl- (1 → 4) -arbutin (β-D-glucosyl- (1 → 4) -arbutin) and β-D-glucosyl- (1 → 6) -arbutin Compared with arbutin and α-D-glucosyl- (1 → 4) -arbutin used as a control, mushroom extracts were higher than those observed for arbutin and α-D-glucosyl- (1 → 4) -arbutin. The inhibitory effect on tyrosinase has been demonstrated to be less.

Inhibitory effects of α-arbutin and arbutin α-glycoside on human tyrosinase have been studied (Sugimoto et al. 2003; Sugimoto et al. 2005). Previous studies have shown that α-glycoside association of hydroquinone-glycosides is essential for inhibitory effects on human tyrosinase, and that α-arbutin and arbutin α-glycosides demonstrate stronger inhibitory activity compared to arbutin. The calculation of the concentration functional theory of arbutin derivatives on the effect of molecular size and the electrostatic potential around the benzene ring of the inhibitor is also proposed (Sugimoto et al. 2005).

As a result of tyrosinase inhibition assay according to the present invention, the inhibitory activity of α-D-glucosyl- (1 → 4) -arbutin, in which β-D-glucosyl- (1 → 3) -albutin has α-glucoside association, It proved to have very similar inhibitory activity. While the other two arbutin β-glycosides, β-D-glucosyl- (1 → 4) -arbutin and β-D-glucosyl- (1 → 6) -arbutin, proved to have weaker inhibitory activity . Therefore, it is assumed that the region selectivity of the glycoside association of arbutin glycoside is also related to the inhibitory effect of tyrosinase inhibitors.

Preferred skin lightening compositions inhibit melanin synthesis in melanocytes, show low cytotoxicity and should be nonmutagenic, in particular by reducing the synthesis or activity of tyrosinase. Melanoma cells were used to evaluate melanin synthesis and inhibition of cytotoxicity. In the present invention, arbutin and arbutin β-glycoside were fed to cultured B16F10 melanoma cells for 2 days. As a result of melanin synthesis, inhibition of pigmentation in cultured B16F10 melanoma cells was evaluated and compared with cytotoxic effects. This suppression of melanin synthesis is shown in FIG. The cell content of melanin was reduced through the addition of arbutin β-glycoside to the medium in a dose dependent manner, with the exception of β-D-glucosyl- (1 → 4) -albutin.

Of the four tested arbutin derivatives, β-D-glucosyl- (1 → 3) -arbutin showed the best inhibitory effect at concentrations ranging from 1-20 mM. Melanin synthesis was particularly inhibited at 5 mM. At this concentration, the melanin content was reduced by 70% compared to that observed in the untreated cells. To exclude the possibility that the above inhibitory effect of β-D-glucosyl- (1 → 3) -albutin on melanin formation may occur as a result of inhibition of cell growth, the number of cell growth of these compounds was compared. As shown in FIG. 4, the compound did not show inhibition of cell viability up to 20 mM concentration. On the other hand, β-D-glucosyl- (1D → 6) -arbutin showed high cytotoxicity at the 5 mM concentration. In this study, we compared β-D-glucosyl- (1 → 3) -albutin as the most effective melanogenesis inhibitor by comparing the inhibitory activity of tyrosinase extracted from mushrooms with the inhibition of melanin production in melanoma cell lines. It became.

In addition, although the present study on the inhibitory activity of tyrosinase and the inhibition of melanin synthesis in melanoma cells using arbutin α-glycoside derivatives (Sugimoto et al. 2004), in melanoma cells using arbutin β-glycoside Since the study of melanin synthesis has not been carried out before, arbutin β-glycoside together with arbutin α-glycoside would be of value as a potent melanin synthesis inhibitor. The development of these enzymes, which only generate β-D-glucosyl- (1 → 3) -arbutin via enzymatic reactions, is necessary to promote the industrialization potential of these compounds as bleaching compositions. We have recently succeeded in producing mutants that produce β-D-glucosyl- (1 → 3) -arbutin, particularly through site-induced mutations. Mutants increased the production of this compound by 3-folding compared to wild type enzymes. Research is currently underway aiming to optimize the enzymatic reaction conditions for the maximum yield of this compound.

1 is β-D-glucosyl- (1 → 6) -arbutin (compound 1) and β-D-glucosyl- (1 → 4) which are arbutin beta derivatives synthesized using beta-glucosidase, a carbohydrate enzyme. ) -Arbutin (Compound 2), and β-D-glucosyl- (1 → 3) -Arbutin (Compound 3).

FIG. 2 is a graph showing the inhibitory effect of arbutin derivatives on tyrosinase activity on L-tyrosine substrates compared to the inhibitory effect of arbutin known as a potent tyrosinase inhibitor.

3 is a graph showing the melanogenesis inhibitory effect using melanogenesis cells compared with the melanogenesis inhibitory effect of arbutin known as melanogenesis inhibitors.

4 is a graph showing the cytotoxic effect of arbutin derivatives compared to arbutin.

However, in Fig. 2 to Fig. 4, (?) Arbutin, (?)? -D-glucopyranosyl- (1 → 3) -arbutin, and (?)? -D-glucopyranosyl- (1 → 4) -arbutin.

Claims (4)

A skin whitening composition containing β-D-glucosyl- (1 → 3) -arbutin, which is an arbutinbeta derivative represented by the following formula (1).

The cosmetic composition for skin whitening according to claim 1, wherein the composition is a cosmetic. The pharmaceutical composition for skin whitening according to claim 1, wherein the composition is a medicament. The method of claim 1, Skin whitening composition, characterized in that it further comprises a whitening component other than the arbutin beta derivative.

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