j. Cosmet. Sci.

, 57, 11-21 (January/February 2006)

New cosmetic agentsfor skinwhitening from Anõelicadahurica
Y. H. CHO, J. H. KIM, S. M. PARK, B. C. LEE, H. B. PYO, and H. D. PARK, R&D Center, Hanbz//Cosmetic Corporation, 72-7

Yongszmg-ri, Samsz/ng-myzm, Umsz/ng-kz/n, Chz/ngbz//e 369-834 (Y.H.C., J.H.K., S.M.P., B.C.L., H.B.P.), and Department of Biotechnology, College of Engineering, Daegz/ University, Gyeongsan-si, GyeongbM 712-714 (H.D.P.) Korea. Accepted for pz/blication onAz/gz/st 9, 2005.
Synopsis

To develop a new whitening agentfor cosmetics from naturalproducts, Angelica dahz/rica wasselected for its inhibitory effecton melanogenesis in B16 melanoma cells.Fromthe mechanism study,it wasclarified that the ethanolic extracts of this plant showed the suppression of tyrosinase synthesis but no inlnibition of tyrosinase activity. In orderto find the activeconstituents from this plant, the ethanolextracts were chromatographed repeatedly with silicagel. Two coumarin compounds wereisolated fromA. dahz•rica. Their structures were identifiedby physicoclnemical and spectral data suchas UV, IR, NMR, and MS. It was shown that the activesubstance wasisoimperatorin (10-[(3-methyl-2-butenyl)oxy]-7H-furo[3,2-g][1] benzopyran-7-one) and imperatorin(9-[(3-methyl-2-butenyl)oxy]-7H-f•Lro[3,2-g][1] benzopyran-7-one). They
significantly inhibited tyrosinase synthesis in B l 6 melanoma cells.To elucidate the actionmechanism of the activecompounds of A. dahz•rica, we investigated the changes in the mRNA level of tyrosinase usingtlne RT-PCR technique. As a result,the mRNA levelof tyrosinase wasmarkedly reduced by activecompounds of A. dahz/rica. Fromthese results, we suggest tinatthese extracts might be useful asa newwhitening agent in cosmetics, but the in vitrofindingsmust be verifiedin in vivoskin-lighteningstudies.

INTRODUCTION

Melaninproduction is principallyresponsible for skin colorandplaysan importantrole in theprevention of sun-induced skininjury(1). However, abnormal hyperpigmentation suchasfreckles, chloasma, lentigines, and otherformsof melaninhyperpigmentation could be a serious aesthetic problem(2). In mammalianmelanocytes, melaninsare synthesized within melanosomes that contain tyrosinase, which plays a key role in melanogenesis, as it catalyzes the rate-limiting reactionof the melanogenic process (3-5). Accordingly, melaninproduction is mainly controlled by the expression and
activationof tyrosinase (6).

Address all correspondence to Y. H. Cho.
11

05559)plates(Merck. USA) and a DPX 300 (Bruker. After sample treatmentfor 24 hours.5-diphenyltetrazolium bromide (MTT). and greentea (10).Japan). 1.04 N HC1in isopropanol). The purity of the compounds isolatedwas identified with highperformance liquid chromatography (WatersAlliance2695-996 PDA detector.such Morusalba L. The organic solventsand chemicalswere obtained from Sigma(USA). USA). We alsoidentifiedthe activecompounds in the extractof A. Germany). have beenstudiedin consideration of safety. andGibcoBRL (USA). Thecells were plated at a density of approximately 1 x 104 cells/wellin a 96-well microplate.respectively.but resultsof suchtreatments are sometimes disappointing (7). Australia).12 JOURNAL OF COSMETIC SCIENCE Concerns of changes in skin colorare frequentlyraisedfor medicalor cosmetic reasons. dahurica. Hyperpigmentation disorders are often treatedwith hydroquinones. kojic acid. Waters. and excessive leukorrhea (11). and the activityof tyrosinase in B16 melanoma cells. The melting pointsweretakenon a Mel-Temp II (Laboratory Devices.the cellswere incubatedin MTT solution (0. retinoids.the plant extractswere dissolved in dimethylsulfoxide (DMSO). Japan).Germany). (9). 1.To developplant materialsprotectinghyperpigmentation. andpurified by the appropriate methods beforeuse.13895)and RP-18 F254(Art. Thin-layer chromatography (TLC) wasperformed onpre-coated Kieselgel 60 F254 (Art. into a blue formazan precipitate. •H. we investigated the changes in the mRNA level of tyrosinase using the reverse transcriptionpolymerase chain reaction(RT-PCR) technique. ELMS.5-dimethylthiazol-2-yl)-2.and•3C-NMR spectra were recorded with a Unity Inova 500 (Varian. hysteria. USA) melting- point-determining apparatus and uncorrected. To elucidatethe action mechanism of the active compounds of A. Nowadays. IR.4 ml of acid-isopropanol (0.A. Glycyrrhiza g/abraL.abdominalpain.and China as a folk medicineto treat menstrualdisorder. Silica gel 60 was purchased from Merck. we found that Angelica dahurica exerteda strongmelanogenic inhibitoryeffecton B16 mouse melanomacells. dahurica. Bio Whittaker (USA). 3-(4. dahurica hasbeenusedin Korea.5 mg/ml) for four hoursat 37øC.Chemical shiftsweregivenin 8 (ppm)from TMS. tyrosinase inhibitorsfrom naturalplants.arbutin. EXPERIMENTAL MATERIALS Medicinal plants were purchased from a local market (Kyeong-Dongmarket.headache. we havechecked the effectof 20 medicinalplant extracts on the inhibition of melanogenesis.05554 and 1.a JMS 700 (Jeol. From the resultsof thesescreening procedures. ascorbic acid.and UV spectra weremeasured on an FT/IR-5300 (Jasco. The plants were extractedin 70% aqueous ethanolunder reflux for four hours. For evaluation. and tyrosinase inhibitors. CELL VIABILITY ASSAY Cell survivalwas measured by the level of mitochondrialrespirationin cells after treatingthe samples.the inhibition of tyrosinase synthesis.Japan.bleeding. (8). The blue formazan produced wassolubilized in 0.and a Cary 1E (Varian. which wasdetermined by the reduction of the tetrazoliumsalt. Korea). and . respectively.The extractswere filtered and concentrated in vacuo.

the cellswere lysedfor 30 minutesat 37øC with 50 pl of phosphate buffer(50 mM.The inhibitory activity on melanogenesis of A.. 2 mM phenylmethyl sulfonyl fluoride(PMSF)and collected by centrifugation.293%. pH 6. DAHURICA EXTRACT ON THE SYNTHESIS OF TYROSINASE iN B16 MELANOMA CELLS The synthesis of tyrosinase was ass•a4Y.8) containing1% Triton-X. The cellswerelysed with phosphate buffer(50 mM. After washing with PBS. The melanin content of B16 melanoma cells was quantifiedby the followingprocedure.Tyrosinase activity was expressed as pg melanin/pg protein. A proteinin the cellswasquantified with the Bio-Radproteinassay kit (PierceCo. melaninwasyieldedasa final product. Rockford. The cell number was determined with a Coulter counter. NaHCO3 0. EFFECT OF A.The cellswere cultured in DMEM supplemented with A. The pelletsof the cellswere solubilizedin iN NaOH containing 10% DMSO to determinethe melanin content.B16 melanomacells were seededinto a 12-well microplate at a density of8 x 105cells. and cultured at 37øC. St. ed by enzyme-linked immunosorbent assay (ELISA) by Fulleret al. melanin content was determined by the method describedin the previous section. Then the supernatants weretransferred into a 96-wellmicroplate andthe coating buffer(Na2CO 3 0. (14).Therefore. DAHURICA EXTRACTS 13 the optical densitywas read at 565 nm. The cellswere washed with phosphate buffer saline(PBS)andthen collected by trypsinization andcentrifugation. The intracellular melanin content and the cell number were measured after 72 hours. The melanincontentwasquantifiedwith an absorbance at 405 nm.8) containing1% Triton-X and 2 mM PMSF.After washingwith PBS twice.1% L-3. EFFECT OF A. EFFECT OF A.159%. pH 6. pH 6. DAHURICA EXTRACT ON MELANOGENESiS iN B16 MELANOMA CELLS The A. the cellswerelysed at 37øCfor 30 minutes with 75 pl of phosphate buffer(50 raM. to evaluate tyrosinase activity. Only cells with functionalmitochondriaare capable of cleavingMTT to generate the dark purple formazan. dahurica extractwasdemonstrated according to the procedure of Imokawaetal. dahurica extractwasquantitatively evaluated regarding its inhibitoryactivityon melanogenesis in B16 melanoma cells. and incubated in 37øC in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum(FCS).8) containing 1% Triton-X. .Illinois) according to the supplier's instruction.After 24 hours. MO).Then 50 pl of phosphate buffer(50 mM. Cells(5 x 10 ) wereseeded into a 96-well microplate in DMEM supplemented with 5% FCS and culturedat 37øC for 24 hours.8) containing 0.When DOPA was used asa substrate of tyrosinase. A standard curvefor melanindetermination wasprepared using syntheticmelanin (SigmaChemicalCo. (13) with minor modification.the medium wasexchanged with DMEM supplemented with 2% FCSandthe plant extractat various concentrations. 2 mM PMSF. 4-dihydroxyphenylanine (DOPA) and 50 pl ofA..The resultswere expressed in percentages relativeto the control (12).SKIN WHITENING BY A. pH 6. Louis. DAHURICA EXTRACT ON THE ACTIVITY OF TYROSINASE IN BI 6 MELANOMA CELLS Cells (5 x 104) were seeded into a96-well microplate inDMEMsupplemented with5% FCS and culturedat 37øC for 24 hours. dahurica extract was addedto the lysateand incubatedat 37øC for three hours. dahurica extractfor 24 hours.

230-400 mesh. The dried roots(100 g) of A.02%. Primersusedfor RT-PCR analysis in this studywereasfollows. EFFECT OF A.The opticaldensitywasmeasured at 490 nm. The MeOH soluble fraction(14 g) waschromatographed on a silicagel column(250 g. Further. 1:1. v/v) to divide the fraction into eleven subfractions (Fr.6) wasadded1:1 (v/v) and incubated overnight at 4øC. respectively.the fractionthat possessed the greatereffect. wasrechromatographed on a silicagel column (70 g. photographed. Houston. 1 . I . 150 pl of 1:3000 diluted secondary Ab (Goat anti-rabbit IgG conjugated horse-radish peroxidase) in PBS-T was addedand incubated for onehour and 30 minutes. 5'-CGAGCTGCCTGACGGCCAGG-3' (5' primer) 5'-ATTTGCGGTGGACGATGGAG-3' (3' primer). dahzzrica were roughly separated using silica gel column chromatography. After washing three times with PBS-T. 150 pl of 5 mg/ml 0-phenylenediamine (OPD) in OPD solution wasaddedand incubated for 40 minutes. IV).[3-actin (400 bp).5'-TTATAAATGGCTCTGATACAAGCTGTGGTAA-3' (3' primer). 3:1. Japan). The inhibitory effects of fourfractions on melanogenesis wereexamined.The cellswere then cultured in DMEM supplementedwith A. USA). dah•rica Benthet Hook (Umbelliferae) wererefluxedwith 70% aqueous ethanol.14 JOURNAL OF COSMETIC SCIENCE NaN• 0. tyrosinase (716 bp).Total cellular RNA was prepared using the UltraspecII RNA isolationsystem (BiotecxLab. v/v) to divide the fraction into four subfractions (Fr. and analyzed with a FluoroS multi-image analyzer (Bio-Rad. The inhibitoryeffectof eachelutionon melanogenesis wasexamined to select the elution containingactivecompounds.Fr.After washingwith PBS-T five times.annealing for 30 seconds at 65øC.Fr. USA) on a thermalcycler(PCR system PC801. 230-400 mesh. ISOLATION AND IDENTIFICATION OF ACTIVE COMPOUNDS FROM ANGELICA DAHURICA Active compounds from A. and extension for 90 seconds at 68øC for 28 cycles.5 g).. 11). pH 9. 150 pl of 1:2000 diluted primary antibody (antityrosinase) in PBS-T wasaddedto each well andincubated for onehourand 30 minutes. 15 x 50 cm) using stepwise gradientelution with the solvents CH2C12and MeOH (50:1. DAHURICA MELANOMA CELLS EXTRACT ON EXPRESSION OF THE TYROSINASE GENE IN B16 Cells (1 x 106) were seeded intoaT-75flask in DMEMsupplemented with5%FCS and then culturedat 37øC for 24 hours. 5'-ACGCCCGAGGGACCTTTACGGCGTAATCCT-3' (5' primer). and thencompound 1 (114 mg) and .These primersweresynthesized by Bioneer Corporation (Korea). The PCR cycleconditions were:meltingfor 30 seconds at 94øC. 2x 50 cm) usingstepwise gradientelutionwith the solvents hexane and EtOAc (5:1. ASTEC.Texas)according to the supplier's instructions. PCR products were resolved on 2% agarose gel and visualized by ethidium bromide(EtBr) staining. After washing the wellswith PBS-T threetimes. The residue was dissolved in MeOH and then divided into a MeOH soluble fraction (14 g) anda MeOH unsoluble fraction (9. The supernatants wereremoved and the coated well waswashed with PBS-T threetimesand blocked with 3% bovine serum albumin (BSA) in PBS-T for two hours at 37øC. subfraction 3. The extractedRNA was reverse-transcribed and amplified using an Access RT-PCR system kit (Promega. dahzzrica extractfor 24 hours.and the extractwasevaporated to afford30 g of the residue.

01 (2H. d. H-4"). H-i").76 (1H.1720 (C -. H-l").O). s. m.55 160.27 (1H. •H-NMR (500MHz.60 3' 1" 2" 3" 4" 5" 105.J -.74 119. H-5). H-3). 5. 7. CDC13) 8 c(Table I). CDC13) 8H:8.20 25.71 18.81(3H.71(3H.45 Hz. d.26 112.96 (1H.62 (1H. 6.09 25. COMPOUND 2 (IMPERATORIN) Amorphous white powder. 5.80 18.69 70. s. UVmax: 220.ElMS m/zß270[M+]. d. 249. s.J -.J = 9.50 114.mp: 101-103øC. d. •H-NMR(500MHz.72 (3H. d.1720(C -.21 Hz. 7.36 (1H. 1. H-2").79 106.69 (1H.67 143.15 (1H.52 131.H-8). 1. UVma x ' 217. 6. STATISTICAL ANALYSIS The results wereexpressed asthe averages + S. •3C-NMR (75MHz.12 125.21 Hz. H-3).1. 7.95 114.16 (1H. 247.02 69. P valuesof <0.CDC13) 8 H ß7. s.12 144.60 Hz. d. 309 nm. 298 nm. H-2'). 5. CDCI• wasusedasthe solvent. 6.87 146.•3C-NMR(75 MHz. H-2").47 2' 144. s. d.J = 2. The Student's t-test was used to evaluate the differences of the means between the control and the samples.76 Hz.45 Hz.76 Hz. .2. respectively. 1 2 2 3 161.55 148.79 * TMS wasusedasinternalstandard. 4.68 4 5 6 139.H-5").09 139. 1. H-3'). H-3').82 116.J = 2.99Hz. 7. H-5").60 (1H. DAHURICA EXTRACTS 15 compound 2 (94 mg) wereobtainedby recrystallization in MeOH from subfractions 2 and 4.81 (1H. d.SKIN WHITENING BY A. s.14 119.J -.J = 2. m.9. of threeindependent experiments.31 113.mp ' 95-97øC.84 7 148.20 158.O).54 (1H. Table 1 ' •C-NMRDataof Compounds 1 and2 Isolated from Angelica dahurica* 'BC(8) in CDC1B No. IRma x (KBr) -1 cm ' 2950(C-H). H-4). 6. J = 7.66 107.75 (3H.61 8 9 10 94.76 139.H-4")).92(2H.16 Hz. IRma x (KBr) cm-•:2950(C-H).J = 9.ElMSm/z: 270[M+]. H-2').9.36 (1H. the datafor compounds 1 and 2 wereobtained at 75 MHz. d. H-4). CDC13) 8c (TableI).60 Hz.J = 6.05 were taken to be significant. d.21 152.D. COMPOUND 1 (ISOIMPERATORIN) Amorphous white powder.

:. dahurica extract on melanogenesis. From theseresults. . 70 . A. and 18 +_ 120 11o lOO . •i•i.. dahurica extract on tyrosinase activity in B16 melanoma cells. dahurica extracton melanogenesis.16 RESULTS AND JOURNAL OF COSMETIC SCIENCE DISCUSSION EFFECT OF A. To clarify the inhibitory mechanism of A. . Inhibitory effectsof A.The resultsare shownin Figure 2.:. i::. 1 lO 100 ArbulJn Conc. DAHURICA EXTRACT ON MELANOGENESIS IN B16 MELANOMA CELLS We quantitativelyexaminedthe effect of A. and 100 pg/ml for 72 hours.. dahurica extractdecreased the melanincontentto 68 +_ 2. Cell cytotoxity wasmeasured by an MTT assay ( ß ).:. dahurica extractat concentrations of 50. and somemelaninproduction-inhibiting agentssuchas arbutin and kojic acid are known to inhibit tyrosinase activity (15. A. :::. In theseexperimental conditions. 43 _+1. A. Theseare very significant decreases in melanincontentcompared to other melanogenic inhibitorssuchasarbutin. dahurica extractdecreased the intracellularmelanin content at all testing concentrations. :. EFFECT OF A. The determination of melanincontent(I) wasmeasured asdescribed in Materials andMethods. 10. we examinedthe effect of A. dahurica extract on melanin synthesis in B16 melanomacells.:.it wasindicated that A. dahurica extractdid not haveany cytotoxiceffects. At all concentrations. DAHURICA EXTRACT ON THE SYNTHESIS OF TYROSINASE IN B16 MELANOMA CELLS Tyrosinase synthesis wasexamined by an enzyme-linked immunosorbent assay (ELISA). . A. and 1000 pg/ml reducedtyrosinase synthesis to 56 _+1.05 comparedto the control.The concentration of arbutinwas2 mg/ml.5%. At a concentration of 100 pg/ml. The resultsare shownin Figure 3. EFFECT OF A. dahurica(pglmL) Figure 1.. 100. dahurica extractdid not inhibit tyrosinase activity in B16 melanoma cells.16). DAHURICA EXTRACT ON THE ACTIVITY OF TYROSINASE IN B16 MELANOMA CELLS Tyrosinase is the rate-limiting enzymein melaninsynthesis. *p < 0.5% compared to thoseof controlcells. The viability of cellswasexpressed asa percentage. dahurica extract at concentrations of 1. Results are the averages of threeindependent experiments _+SD. The resultsare shownin Figure 1..::' ": '::• Ctrl :.ofA. A. B16 melanomacells were cultured in the presence of A.5%.iiii. dahurica extractexhibitedno influence on tyrosinase activity.

Effects of A. Results are the averages of three independent experiments_+SD. The resultsare shownin Figure4. Inhibitory effects of A. we investigatedthe changes in themRNA levelof tyrosinase usingtheRT-PCR technique. dahurica (pglmL) Figure 2. Tyrosinase activity was measuredas describedin Materials and Methods.ofA. The lysates of B16 melanoma cells containing tyrosinase wereincubated withA. dahurica(FglrnL) Figure 3. 1. the mRNA levelof tyrosinase wasdecreased by 75% at 100 pg/ml of the untreated controlvalue. Theseresults suggested the possibility that A. lOO • • 50 25 Ctd 50 100 1000 Conc. dahuricaextract. Resultsare the averages of threeindependent experiments _+ SD. DAHURICA MELANOMA CELLS EXTRACT ON EXPRESSION OF THE TYROSINASE GENE IN B16 To elucidate the action mechanismof A. dahurica extractinhibitedtyrosinase synthesis in B16 melanoma cells.5% of the controlvalue. and 1000 Fg/mlfor 24 hours. *p < 0. respectively.B16 melanoma cellswerecultured in the presence ofA.ofA. dahurit•extractat concentrations of 50.SKIN WHITENING BY A. 100.05 compared to the control. EFFECT OF A. dahurit•extract on tyrosinase synthesis in B16 melanoma cells.• • •o Ctrl 1 10 1O0 Conc. dahurica extract might act on the common upstream eventthat controls the transcription of the tyrosinase gene. dahurit•extract on the activityof tyrosinase. . DAHURICA EXTRACTS 17 lOO I 75 .Theseresultssuggest that A. When normalized with the mRNA levelof •-actin. andthen eachmRNA levelwasexamined. B16 melanoma cells were treated with 20 and 100 pg/ml ofA.respectively. The tyrosinase synthesis was examined by ELISAasdescribed in Materials andMethods. dahurica extract for 48 hours. dahurica extract andDOPA forthreehours.

DAHIIRICA The ethanolicextractof A.a laboratory investigation wasperformed on the active ethanolic extract. dahurica extract. dahurica extractexpressed as% inhibitionof the control.60 ppm. dahurica extracton expression of the tyrosinase genein B16 melanoma cells.ofA. Lane 2 shows those of cellstreatedwith 20 []g/ml of A.õ/mL) Figure 4. dahurica extractfor 48 hours.96 and 7. dahzzrica decreased significantintracellularmelanin content andtyrosinase biosynthesis.18).Lane3 shows those of cellstreatedwith 100 [•g/ml of A.Activity-guidedfractionation led to the isolation of compounds 1 and 2 as active compounds. Lane1 shows the tyrosinase (716 bp) and [3-actin gene(400 bp) of the controlcell. In the]H-NMR spectrum.-- 40 20 Ctrl 20 Conc.(B) shows the decreased mRNA level of cellstreatedwith A.05 comparedto the control.and showed [M] + at m/z 270 in the ElMS spectrum.15) to indicatethe absence of protonat C-5 (17.J = 2.(A) is the result of gel electrophoresis of the RT-PCR products.16 ppm. A peakof the aromatic protonwasdetected asa singletsignal(1H.J = 9. *p < 0. two doubletsignals (1H. The UV spectrum exhibitedtypical bandsof the 5-substituted linear furanocoumarin ringat 220.76 Hz) at 8 6. B16mouse melanoma cells were seeded intoa T-75 flask at a density of l x 10 6 cells perflask and treatedwith or without A.45 Hz) weredetected at 6. ISOLATION AND IDENTIFICATION OF ACTIVE COMPOUNDS FROM A.249.Results are the averages of threeindependent experiments _+SD. attributableto the protons of the furan ring. dahurica extract.and 309nm(17.3 80 "' o 60 .18).27 and 8. Thus.a sidechainwassuggested to substituteat the C-5 . H-8) at 7. and the signalfor H-4 wasobserved at a ratherlower field (8 8. dahurica (p. Inhibitoryeffects of A. Compound 1 wasobtained asan amorphous white powder.18 (A) JOURNAL OF COSMETIC SCIENCE :Tyresinase (B) 120 100 o (. and anothertwo doubletsignals (1H.15 wereassignable to the ortho coupled protons of the pyronering. Therefore.

SKIN WHITENING BY A. respectively.05) at 100 pM of compound 1. and the structurewasverified by the reportedNMR data (18.compound 2 wasassumed to be the position isomerof compound 1. and60+ 5. 50+ 3.05) at 100 pM. A doublet signal (2H. H-2") weredetected at 4.6% (p < 0. From thesedata.19). H-I") anda multipletsignal (1H.05) at 50 pM. andtwo carbon signals for C-5 and 8 werechanged.05) at 50 pM. In the•3C-NMR spectrum.54 ppm. . and 75+ 6. We investigated the inhibitoryeffects of thesecompounds on melanogenesis in B16 melanoma cells.92 and 5. The structures of compounds 1 and2 arepresented in Figure 5 and•3C-NMRdata arelisted in Table I. most of thespectral aspect of compound2 was similar to that of compound1. and identifiedas imperatorin.Compounds 1 and 2 at a concentration of 80 pM and 100 pM reduced the melanincontentby 50% compared with non-treated controlcells. The treatment of B16 melanomacells with these compounds significantly suppressed tyrosinase productionat the protein levelsin a dose-dependent manner: by an average of 20+ 5% (P < 0.81ppmassinglet signals. carbonyl carbon was shown at 161. 35+ 5% (p < 0.05) at 200 pM of 4 R• 4" R2 Isoimperatorin 0 2'•• 1' H 5" 4" Imperatorin Figure 5.26 ppm.74). From these data. 40+ 4. along withtwelve sp 2carbons (8 94.05) at 10 pM.12) and three sp 3carbons (8 18.99 Hz. DAHURICA EXTRACTS 19 position.J = 6.respectively. m.12to 158.76) thanthat of compound 1.20 to69. andprotons of methylgroups were detected at 1. This was finally confirmedby comparingits NMR data with thosein the reportedreferences (18. compound 1 was postulated tobe isoimperatorin. Compound 2 wasobtained asan amorphous white powder.and•3C-NMR spectrum.5% (P < 0. We furtherstudiedby ELISA the inhibitoryeffects of thesecompounds on tyrosinase expression in B16 melanomacells.and [M] + at m/z 270 in the ElMSspectrum. but the proton signal for H-4 was detected at a higherfield (8 7. In the •H.19). Thesecompounds inhibitedmelanogenesis of B16 melanoma cellsin a dose-dependent manner (data not shown).71and1.5% (p < 0. Chemical structure of compounds 1 and 2 isolated from the root of Angelica dahurica.4% (p < 0.

A. 274(23). in their actionmechanism.J. Y. Molecular cloningand functional analysis of a cDNA codingfor humanDOPAchrometautomerase/tyrosinase-related protein-2. (13) G.. Jung. Therefore. 75-85 (1989). (11) T. Mishima. Bid. Kim. and S.J. Mosmann. D. (3) V.T. (8) S. Inhibitionof tyrosinase by greentea components. B. Tsukamoto. G. Irare/tool. Solano.Dermatol. Y. Singapore. J. are novelwhiteningagents differentfrom otherthosebeing used in the cosmeticindustry. Inve•t. K.Cancer Res.Inhibitoryeffectof arbutinon melano- . Hwang. (7) S. Mol. B. Sung. Gilchrest.K. H. Perez. Song. Akiu. J. 177-186 (1996). Iman. FASEBJ. Y. (15) S.. 117-118. 55-63 (1983). pp. B.Pigment Ce//Res. 134(1). E. CONCLUSIONS We foundthat A. dahz/rica extracthada stronginhibitoryactivityagainstmelanogenesis and exerted its melanogenic inhibitoryeffectthroughthe modulation of mRNA levels of tyrosinase. Suzuki. Lunsford.Exp. andH. Asahara. Jimenez-Cervantes. H.we isolated isoimperatorin andimperatorin. Gao. J.J.in this study. M. Y..Tyrosinase relatedprotein 1 (TRP1) functions asa DHICA oxidase in melaninbiosynthesis. Y. Methods. Stimulation of melanogenesis by glycyrrhizin in B16 melanomacells. Urabe. (6) V. Dopachrome conversion: A possible controlpoint in melaninbiosynthesis. compared with non-treatedcontrol cells.J.Y.International Collation of Traditional andFolk Medicine. 65(1-2). and B. Theseresults suggest that the activecompounds. Rapidcolorimetric assay for cellular growthandsurvival: Application to proliferation and cytotoxicity assays. H. and C. Y. Fuller.M. P. we investigated in B16 melanomacellswhetherthesecompoundsmodulatethe expression of tyrosinase steady-state mRNA levels. Rhee.J. 10451048 (2002). Choi. These resultssuggestthat thesecompounds suppress the tyrosinse productionat the protein and mRNA levels. Imokawa. 99(6).ProteinkinaseC-[3 activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain. Lee. Hearing. EMBOJ. Analysis of mammalian pigmentation at the molecular level. Park. Yokozawa. (14) B. 33(2).Loss of melanogenic properties in tyrosinases induced by glycosylation inhibitorswithin malignantmelanoma cells.. 1994-2002 (1982).A. Yasumoto. D. PL241-246 (1999). No. 13.. Yokoyama. M. Pawelek. Brewington. Y. KornerandJ. REFERENCES (1) H. Acta. andM.J... K. 75. M. S. H. J. Y.20 JOURNAL OF COSMETIC SCIENCE compound2. Fukuda. newcosmetic agents for skinwhitening..J. G. Biochim. 149-154 (1988). Y. Cell. (2) A. Jimenez. (10) J.Comparison of tyrosinase levelsin amelanotic and melanotic melanoma cell cultures by a competitive enzyme-linked immunoadsorbent assay and by immunotitration analysis.. But.Commondisorders of pigmentation: When aremorethan cosmetic cover-ups required?. H. and J. J. 2. Chung. 2902-2909 (1991). Pharm. Postgrad. 25(8). J. S.fromAngelica dahz/rica.. S. D. J. K. Imokawaand Y. Y. Yang. J. Hearingand M. Biol. Winder. and ¾.65(21). and S. S.andJ. Hacker. Lij• Sci. Shim. 131-135 (2001). 317-321 (1994). J. Kimura. C. Shibahara. Par.Jun. Laursen. Lee. Part 1 (World Scientific. Enzymaticcontrolof pigmentation in mammals. 1217.R.T. W. Also. Med. Garcia-Borron.. K. (9) G. (12) T. (5) K. S. Soung. 16470-16478 (1999). Chem. Kim. Fujinuma. 42. Suzuki. X. Biophys. Guo. Hara.Thesecompounds significantly reduced tyrosinase production at the mRNA level(datanot shown). Kobayashi.Physiol. S. C. 5. Kim. T. 192-195 (1980).Bull. Hearing and K. (4) T. 5818-5825 (1994). Mulverroside F isolated from the leaves of Morus alba inhibits melaninbiosynthesis. H. 1996).. P. Tomita. Med. F. respectively (data not shown).

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