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1 26 3 ( ) J Korean Oriental Med 2005;26(3):55-65 The Effcects of Psoraleae fructus Extract on Melanin Synthesis of B16 Melanoma Cells Jae-Ho Chung, Hyung-Sik Seo Dept. of Ophthamology, Otolaryngology & Dermatology, College of Oriental Medicine, Sangji University Objectives : This study was carried out for the development of medicine for vitiligo treatment and focused on the effect of Psoraleae fructus extract on melanin synthesis of B16 melanoma cells. Methods : Activity of tyrosinase playing a vital role in the synthesis and quantity of melanin, which is the final product in cultured B16 melanoma cells, the effects of Psoraleae fructus extract were measured. Results : The results indicated that Psoraleae fructus extract increased both the amount of melanin and the activity of tyrosinase according to concentration, also supported by western blot analysis. Conclusions : The results suggest that Psoraleae fructus extract has an advantageous effect on the promotion of melanin synthesis and will contribute to the development of vitiligo treatment through further related studies. Key Words: Psoraleae fructus, melanogenesis, tyrosinase, vitiligo. (vitiligo), : : : :. (Tel : , aran99@sangji.ac.kr) 1 3% 1-4). L-Tyrosine tyrosinasel-tyrosine dihydroxyl-l-phenylalanine(l-dopa), L-DOPA dopaquinone, dopachrome, indole-5,6-quinone 5). tyrosinase 6).,,, 55

2 (514) 26 3 ( ), 7-9),. 9), 7), 10), 11), 12), 13).,, ) 20),. B16 melanoma tyrosinase western blot. 1). 100g (50 % ) PBS(phosphate buffered saline; Sigma Co. U.S.A.). 2) B16 melanoma B16 melanoma RPMI-1640(Gibco BRL Co, Gaithersburg, MD) 5 % FBS(fetal bovine serum; Hyclones Co.) Fungizone 37, 5 % CO2. 1) crystal violet. 24-well culture plate well cells PBS 0.5 % crystal violet(in 20 % methanol) 300 /well 5 tap water. lysis buffer(10 mm Tris HCl, ph7.6, 150 mm NaCl, 1 % SDS) nm(reference 450 nm). 2) Tyrosinase Tyrosinase Martinez-Esparza 21). PBS 100 Buffer(1% Triton X-100, 10 mm Sodium phosphate, 0.1mM PMSF).. 100mM Sodium phosphate(ph 7.0) mM catechol nm. 3) Hosoi 22). PBS 10% DMSO(dimethyl sulfoxide) 1N NaOH nm ) Western bolt (1) % SDS

3 (515) Fig. 1. The survival of B16 melanoma Cells treated with Psoraleae fructus extract in kao-liang liquor. Fig. 2. The microscopic images of B16 melanoma cells. lysis. 10, (2) BCA(bicinchoninic acid, Sigma, U.S.A.) BSA(bovine serum albumin, Sigma, U.S.A.). 96-well plate BSA(1 ) 0, 1, 2, 4, 8, 16 BCA nm. 2 BCA nm. (3) Western blot % SDS-polyacrylamide gel nitrocellulose membrane transfer. Membrane 5 % milk PBS-Tween(0.01 %) 1 hybridization. membrane tyrosinase(santa Cruz, U.S.A.) 1 shaking hybridization PBS-Tween 20, membrane horseradish peroxidase 57

4 (516) 26 3 ( ) conjugated antimouse IgG antirabbit IgG 1 hybridization. Membrane PBS-Tween chemiluminescence (DuPont, NEM) Fugi X-ray film. 5) SigmaStat(ver 2.1) one way ANOVA test,, p<0.05. B16 melanoma crystal violet , 10 / 64.3% (p<0.05)(fig.1), (Fig.2). 10 / 0.5 /, 1 /, 2 /, 3 /, 4 /, 5 /. tyrosine dopa, L-dopaquinone /, 1 /, 2 /, 3 / 3 / %, % 5 / % 4 (p<0.05)(fig. 3). Tyrosinase. tyrosinase B16 melanoma tyrosinase Fig. 3. The melanin content of B16 melanoma cells treated with Psoraleae fructus extract in kao-liang liquor. 58

5 (517) Fig. 4. The tyrosinase activity of B16 melanoma cells treated with Psoraleae fructus extract in kao-liang liquor.. Tyrosinase 0.5, 1, 2, 3 3 / %, % % 4 (p<0.05)(fig. 4). - western blot 0, 1, 3, actin tyrosinase (Fig. 5). B16 melanoma tyrosinase %, 23). 1-4)., 7). Fig. 5. Western blot analysis. 59

6 (518) 26 3 ( ). tyrosine dopa dopaquinone, indoleindole 2 24). tyrosinase..,,, 4 1,2,7)., 2).,,, tyrosinase. 1).,,,, 1) % 1%., 9). 1 3% 20 30% 4,25).. posralen,, 1-4)., psoralen 8- methoxypsoralen trimethylpsoralen (UVA) (PUVA) psoralen (15 25 ) 100 1,9). 9).,,,,, 26). 1,3,4)..,,,, 1,2,4). covermark, 1,27). 60

7 (519),,,, 7-9). <> 28),.,,,,,,,,,,,,,, 7).,,,,.,,,, 9)., 7). 11) B16 melanoma, 12), 13), 29), 30), 6), 31). 8). 32). 33), 34), 35).,.. (Leguminosae) Psoralea corylifolia L.,,,,,,,,, 14-19)., melanin 15-18) < > 36) < > 37)., coumarin psoralen, isopsoralen, angelicin, psoralidin, isopsoralidin, corylidin, 8-methoxypsoralen, flavon bavachalcone, isobavachalcone, 61

8 (520) 26 3 ( ) corylifolinin, bavachin, coryfolin, bavachinin, isobavachinin, psoralenol,,,, bakuchiol, stigmasterol, - sitosterol-d-glucoside, triacontane, psoraldehyde, saponin 38,39). psoralen (PUVA) B.C. 2000, 1948 El-Mofty 40) 1974 Parrish psoralen UVA (PUVA) 25).,, psoralen 41). B16 melanoma Crystal violet % 0.5, 1, 2, 3, 4, 5., 4. tyrosinase rate-limiting step, tyrosine dopa tyrosinase hydroxlase, dopa dopachrome dopa oxidase melanin polymer 48). Tyrosinase, 4. tyrosinase, tyrosinase, 48). tyrosinase,. tyrosinase. western blot actin tyrosinase tyrosinase. B16 melanoma tyrosinase,. psolaren. 62

9 (521). psoralen,. tyrosinase % , 1, 2, 3, 5 tyrosinase 3, , 1, 2, 3, 5 3, Western blot tyrosinase protein level, tyrosinase. B16 melanoma tyrosinase,., :. 2001:3, 8, : 2003:3, 4, ,,.. : 1993: :. 1987: ,,,. L :34(1): ,,,, :17(2): :16(2): , :8(2): ,, :4(1): ,., :3(1): ,,,,,. B16 melanoma 63

10 (522) 26 3 ( ) :14(1): ,, :16(2): ,,,,,, :13(2): :. 1995: ,. (). :. 1997: ,,,. 4. :. 1997: :. 1994: ,.. :. 2004: :. 2003: ,,,,,,,. 6. :. 2001: Martinez-Esparza M, Jimenez-Cervantes C, Solano F, Lozano JA, Garcia-Borron JC. Mechanisms of melanogenesis inhibition by tumor necrosis factor-alpha in B16/F10 mouse melanoma cells. Eur J Biochem. 1998:255: Hosoi J, Abe E, Suda T, Kuroki T. Regulation of melanin synthesis of B16 mouse melanoma cells by 1 alpha, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res. 1985:45: ,,, :28(2): Pawelek J, Korner A, Bergstrom A, et al. New regulation of melanin biosynthesis and autodestruction of melanoma cells. Nature. 1980:286: Thomas P. Habif. Clinical Dermatology a color guide to Diagnosis and Therapy. St. Louis : The C.V. Mosby Company: 1985: ,,,. PUVA :37(12): pp :. 1996: , :. 1965: ,,,,. B16/F :15(6): ,,,, ;16(1): ,,,,. B16/F10 melanoma ;16(4): , ;10(1): ,,, ;12(2):

11 (523) 36.,. ;. 1986: :. 1982: (). :. 1984: :. 1983: El-Mofty, A.M.. Vitiligo and psoralen. oxford: Pergamon Press Ltd. 1963:

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical