j. Cosmet. Sci.

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

New cosmetic agentsfor skinwhitening from Anelicadahurica

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. dahzrica. 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-fLro[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. dahzrica, 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

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JOURNAL OF COSMETIC SCIENCE

Concerns of changes in skin colorare frequentlyraisedfor medicalor cosmetic reasons. Hyperpigmentation disorders are often treatedwith hydroquinones, retinoids,arbutin, kojic acid, ascorbic acid, and tyrosinase inhibitors,but resultsof suchtreatments are sometimes disappointing (7). Nowadays, tyrosinase inhibitorsfrom naturalplants,such Morusalba L. (8), Glycyrrhiza g/abraL. (9), and greentea (10), have beenstudiedin consideration of safety.To developplant materialsprotectinghyperpigmentation, we havechecked the effectof 20 medicinalplant extracts on the inhibition of melanogenesis,the inhibition of tyrosinase synthesis, and the activityof tyrosinase in B16 melanoma cells,respectively. From the resultsof thesescreening procedures, we found that Angelica dahurica exerteda strongmelanogenic inhibitoryeffecton B16 mouse melanomacells.A. dahurica hasbeenusedin Korea,Japan,and China as a folk medicineto treat menstrualdisorder,abdominalpain, hysteria,bleeding,headache, and excessive leukorrhea (11). We alsoidentifiedthe activecompounds in the extractof A. dahurica. To elucidatethe action mechanism of the active compounds of A. dahurica, we investigated the changes in the mRNA level of tyrosinase using the reverse transcriptionpolymerase chain reaction(RT-PCR) technique.

EXPERIMENTAL
MATERIALS

Medicinal plants were purchased from a local market (Kyeong-Dongmarket, Korea). The plants were extractedin 70% aqueous ethanolunder reflux for four hours.The extractswere filtered and concentrated in vacuo. For evaluation,the plant extractswere dissolved in dimethylsulfoxide (DMSO).

The melting pointsweretakenon a Mel-Temp II (Laboratory Devices, USA) melting-

point-determining apparatus and uncorrected. H- and3C-NMR spectra were recorded

with a Unity Inova 500 (Varian, USA) and a DPX 300 (Bruker, Germany).Chemical shiftsweregivenin 8 (ppm)from TMS. IR, ELMS,and UV spectra weremeasured on an FT/IR-5300 (Jasco, Japan),a JMS 700 (Jeol,Japan),and a Cary 1E (Varian, Australia), respectively. The purity of the compounds isolatedwas identified with highperformance liquid chromatography (WatersAlliance2695-996 PDA detector, Waters, USA). Thin-layer chromatography (TLC) wasperformed onpre-coated Kieselgel 60 F254 (Art. 1.05554 and 1.13895)and RP-18 F254(Art. 1.05559)plates(Merck,Germany). Silica gel 60 was purchased from Merck. The organic solventsand chemicalswere obtained from Sigma(USA), Bio Whittaker (USA), andGibcoBRL (USA), andpurified by the appropriate methods beforeuse.
CELL VIABILITY ASSAY

Cell survivalwas measured by the level of mitochondrialrespirationin cells after treatingthe samples, which wasdetermined by the reduction of the tetrazoliumsalt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), into a blue

formazan precipitate. Thecells were plated at a density of approximately 1 x 104

cells/wellin a 96-well microplate. After sample treatmentfor 24 hours,the cellswere incubatedin MTT solution (0.5 mg/ml) for four hoursat 37C. The blue formazan produced wassolubilized in 0.4 ml of acid-isopropanol (0.04 N HC1in isopropanol), and

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13

the optical densitywas read at 565 nm. Only cells with functionalmitochondriaare capable of cleavingMTT to generate the dark purple formazan.The resultswere expressed in percentages relativeto the control (12).
EFFECT OF A. DAHURICA EXTRACT ON MELANOGENESiS iN B16 MELANOMA CELLS

The A. dahurica extractwasquantitatively evaluated regarding its inhibitoryactivityon melanogenesis in B16 melanoma cells.The inhibitory activity on melanogenesis of A. dahurica extractwasdemonstrated according to the procedure of Imokawaetal. (13) with minor modification.B16 melanomacells were seededinto a 12-well microplate at a

density of8 x 105cells, and incubated in 37C in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% fetal calf serum(FCS).After 24 hours,the medium wasexchanged with DMEM supplemented with 2% FCSandthe plant extractat various
concentrations, and cultured at 37C. The intracellular melanin content and the cell
number were measured after 72 hours. The melanin content of B16 melanoma cells was

quantifiedby the followingprocedure. The cellswere washed with phosphate buffer saline(PBS)andthen collected by trypsinization andcentrifugation. The cellswerelysed with phosphate buffer(50 mM, pH 6.8) containing1% Triton-X, 2 mM phenylmethyl sulfonyl fluoride(PMSF)and collected by centrifugation. The pelletsof the cellswere solubilizedin iN NaOH containing 10% DMSO to determinethe melanin content. The melanincontentwasquantifiedwith an absorbance at 405 nm. A standard curvefor melanindetermination wasprepared using syntheticmelanin (SigmaChemicalCo., St.
Louis, MO). The cell number was determined with a Coulter counter.

EFFECT

OF A. 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 37C for 24 hours.After washingwith PBS twice, the cellswere lysedfor 30 minutesat 37C with 50 pl of phosphate buffer(50 mM, pH 6.8) containing 1% Triton-X, 2 mM PMSF.Then 50 pl of phosphate buffer(50 mM, pH 6.8) containing 0.1% L-3, 4-dihydroxyphenylanine (DOPA) and 50 pl ofA. dahurica extract was addedto the lysateand incubatedat 37C for three hours.When DOPA was used asa substrate of tyrosinase, melaninwasyieldedasa final product.Therefore, to evaluate tyrosinase activity, melanin content was determined by the method describedin the previous section. A proteinin the cellswasquantified with the Bio-Radproteinassay kit (PierceCo., Rockford,Illinois) according to the supplier's instruction.Tyrosinase activity was expressed as pg melanin/pg protein.
EFFECT OF A. DAHURICA EXTRACT ON THE SYNTHESIS OF TYROSINASE iN B16 MELANOMA CELLS

The synthesis of tyrosinase was assa4Y, ed by enzyme-linked immunosorbent assay (ELISA)

by Fulleret al. (14). Cells(5 x 10 ) wereseeded into a 96-well microplate in DMEM
supplemented with 5% FCS and culturedat 37C for 24 hours.The cellswere cultured in DMEM supplemented with A. dahurica extractfor 24 hours. After washing with PBS, the cellswerelysed at 37Cfor 30 minutes with 75 pl of phosphate buffer(50 raM, pH 6.8) containing1% Triton-X and 2 mM PMSF. Then the supernatants weretransferred

into a 96-wellmicroplate andthe coating buffer(Na2CO 3 0.159%, NaHCO3 0.293%,

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JOURNAL OF COSMETIC SCIENCE

NaN 0.02%, pH 9.6) wasadded1:1 (v/v) and incubated overnight at 4C. 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 37C. After

washing three times with PBS-T, 150 pl of 1:2000 diluted primary antibody (antityrosinase) in PBS-T wasaddedto each well andincubated for onehourand 30 minutes. After washing the wellswith PBS-T threetimes, 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.After washingwith PBS-T five times, 150 pl of 5 mg/ml 0-phenylenediamine (OPD) in OPD solution wasaddedand incubated for 40 minutes.The opticaldensitywasmeasured at 490 nm.

EFFECT OF A. 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 37C for 24 hours.The cellswere then cultured in DMEM supplementedwith A. dahzzrica extractfor 24 hours.Total cellular RNA was prepared using the UltraspecII RNA isolationsystem (BiotecxLab., Houston,Texas)according to the supplier's instructions. Primersusedfor RT-PCR analysis in this studywereasfollows;

tyrosinase (716 bp), 5'-ACGCCCGAGGGACCTTTACGGCGTAATCCT-3' (5' primer),5'-TTATAAATGGCTCTGATACAAGCTGTGGTAA-3' (3' primer);[3-actin (400 bp), 5'-CGAGCTGCCTGACGGCCAGG-3' (5' primer) 5'-ATTTGCGGTGGACGATGGAG-3' (3' primer).These primersweresynthesized by Bioneer Corporation
(Korea). The extractedRNA was reverse-transcribed and amplified using an Access RT-PCR system kit (Promega, USA) on a thermalcycler(PCR system PC801, ASTEC,

Japan). The PCR cycleconditions were:meltingfor 30 seconds at 94C,annealing for 30 seconds at 65C, and extension for 90 seconds at 68C for 28 cycles. PCR products were resolved on 2% agarose gel and visualized by ethidium bromide(EtBr) staining, photographed, and analyzed with a FluoroS multi-image analyzer (Bio-Rad, USA).

ISOLATION

AND

IDENTIFICATION

OF ACTIVE

COMPOUNDS

FROM

ANGELICA

DAHURICA

Active compounds from A. dahzzrica were roughly separated using silica gel column chromatography. The dried roots(100 g) of A. dahrica Benthet Hook (Umbelliferae) wererefluxedwith 70% aqueous ethanol,and the extractwasevaporated to afford30 g
of the residue. The residue was dissolved in MeOH and then divided into a MeOH

soluble fraction (14 g) anda MeOH unsoluble fraction (9.5 g), respectively. The MeOH soluble fraction(14 g) waschromatographed on a silicagel column(250 g, 230-400 mesh, 15 x 50 cm) using stepwise gradientelution with the solvents CH2C12and
MeOH (50:1, 1:1, v/v) to divide the fraction into eleven subfractions (Fr. 1 - Fr. 11).

The inhibitoryeffectof eachelutionon melanogenesis wasexamined to select the elution containingactivecompounds. Further,the fractionthat possessed the greatereffect, subfraction 3, wasrechromatographed on a silicagel column (70 g, 230-400 mesh, 2x 50 cm) usingstepwise gradientelutionwith the solvents hexane and EtOAc (5:1, 3:1, v/v) to divide the fraction into four subfractions (Fr. I - Fr. IV). The inhibitory effects of fourfractions on melanogenesis wereexamined, and thencompound 1 (114 mg) and

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15

compound 2 (94 mg) wereobtainedby recrystallization in MeOH from subfractions 2 and 4, respectively.
COMPOUND 1 (ISOIMPERATORIN)

Amorphous white powder;mp: 101-103C; UVmax: 220, 249, 309 nm; IRma x (KBr)

cm-:2950(C-H),1720(C -- O);ElMSm/z: 270[M+]; H-NMR (500MHz, CDC13)

8H:8.15 (1H, d,J -- 9.76 Hz, H-4), 7.60 (1H, d,J = 2.45 Hz, H-2'), 7.16 (1H, s,H-8),
6.96 (1H, d,J = 2.45 Hz, H-3'), 6.27 (1H, d,J = 9.76 Hz, H-3), 5.54 (1H, m, H-2"),

4.92(2H, d,J = 6.99Hz, H-l"), 1.81(3H, s,H-5"),1.71(3H, s,H-4"));3C-NMR(75

MHz, CDC13) 8c (TableI).
COMPOUND 2 (IMPERATORIN)

Amorphous white powder;mp ' 95-97C; UVma x ' 217, 247, 298 nm; IRma x (KBr) -1

cm ' 2950(C-H),1720 (C -- O);ElMS m/z270[M+]; H-NMR(500MHz,CDC13)

8 H 7.76 (1H, d,J -- 9.60 Hz, H-4), 7.69 (1H, d,J -- 2.21 Hz, H-2'), 7.36 (1H, s, H-5), 6.81 (1H, d,J = 2.21 Hz, H-3'), 6.36 (1H, d,J = 9.60 Hz, H-3), 5.62 (1H, m, H-2"), 5.01 (2H, d, J = 7.16 Hz, H-i"), 1.75 (3H, s, H-5"), 1.72 (3H, s, H-4");

3C-NMR (75MHz, CDC13) 8 c(Table I).

STATISTICAL ANALYSIS

The results wereexpressed asthe averages + S.D. of threeindependent experiments. The

Student's t-test was used to evaluate the differences of the means between the control and

the samples. P valuesof <0.05 were taken to be significant.

Table 1

' C-NMRDataof Compounds 1 and2 Isolated from Angelica dahurica*

'BC(8) in CDC1B
No. 1 2

161.26 112.55

160.50

114.68

4 5
6

139.55 148.95
114.20 158.12

144.31 113.12
125.84

148.61

8 9
10

94.21 152.66
107.52

131.67 143.82
116.47

2'

144.87

146.60

3' 1" 2" 3" 4" 5"

105.02 69.74 119.09 139.80 18.20 25.79

106.69 70.14 119.76 139.71 18.09 25.79

* TMS wasusedasinternalstandard; the datafor compounds 1 and 2 wereobtained

at 75 MHz. CDCI wasusedasthe solvent.

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RESULTS AND

JOURNAL OF COSMETIC SCIENCE

DISCUSSION

EFFECT

OF A. DAHURICA

EXTRACT

ON

MELANOGENESIS

IN

B16 MELANOMA

CELLS

We quantitativelyexaminedthe effect of A. dahurica extract on melanogenesis. The resultsare shownin Figure 1. A. dahurica extractdecreased the intracellularmelanin content at all testing concentrations. At a concentration of 100 pg/ml, A. dahurica extractdecreased the melanincontentto 68 +_ 2.5% compared to thoseof controlcells. Theseare very significant decreases in melanincontentcompared to other melanogenic inhibitorssuchasarbutin. In theseexperimental conditions, A. dahurica extractdid not haveany cytotoxiceffects.

EFFECT

OF A. DAHURICA

EXTRACT

ON THE

ACTIVITY

OF TYROSINASE

IN B16 MELANOMA

CELLS

Tyrosinase is the rate-limiting enzymein melaninsynthesis, and somemelaninproduction-inhibiting agentssuchas arbutin and kojic acid are known to inhibit tyrosinase activity (15,16). To clarify the inhibitory mechanism of A. dahurica extracton melanogenesis, we examinedthe effect of A. dahurica extract on tyrosinase activity in B16 melanoma cells.The resultsare shownin Figure 2. At all concentrations, A. dahurica extractexhibitedno influence on tyrosinase activity. From theseresults,it wasindicated that A. dahurica extractdid not inhibit tyrosinase activity in B16 melanoma cells.

EFFECT

OF A. DAHURICA

EXTRACT

ON THE

SYNTHESIS

OF TYROSINASE

IN B16 MELANOMA

CELLS

Tyrosinase synthesis wasexamined by an enzyme-linked immunosorbent assay (ELISA). The resultsare shownin Figure 3. A. dahurica extractat concentrations of 50, 100, and 1000 pg/ml reducedtyrosinase synthesis to 56 _+1.5%, 43 _+1.5%, and 18 +_
120

11o

lOO

:::.

.,.

...:.

70

.:.

ii; :.::'

": '::
Ctrl

:.iiii. i::.:.:.
1 lO 100 ArbulJn

Conc.ofA. dahurica(pglmL)

Figure 1. Inhibitory effectsof A. dahurica extract on melanin synthesis in B16 melanomacells. B16 melanomacells were cultured in the presence of A. dahurica extract at concentrations of 1, 10, and 100 pg/ml for 72 hours.The concentration of arbutinwas2 mg/ml. The determination of melanincontent(I) wasmeasured asdescribed in Materials andMethods. Cell cytotoxity wasmeasured by an MTT assay ( ). The viability of cellswasexpressed asa percentage. Results are the averages of threeindependent experiments _+SD. *p < 0.05 comparedto the control.

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17

lOO

75

Ctrl

10

1O0

Conc.ofA. dahurica (pglmL)

Figure 2. Effects of A. dahuritextract on the activityof tyrosinase. The lysates of B16 melanoma cells containing tyrosinase wereincubated withA. dahurica extract andDOPA forthreehours. Tyrosinase activity
was measuredas describedin Materials and Methods. Results are the averages of three independent experiments_+SD.

lOO

50

25

Ctd

50

100

1000

Conc.ofA. dahurica(FglrnL)

Figure 3. Inhibitory effects of A. dahuritextract on tyrosinase synthesis in B16 melanoma cells.B16

melanoma cellswerecultured in the presence ofA. dahuritextractat concentrations of 50, 100, and 1000 Fg/mlfor 24 hours. The tyrosinase synthesis was examined by ELISAasdescribed in Materials andMethods. Resultsare the averages of threeindependent experiments _+ SD. *p < 0.05 compared to the control.

1.5% of the controlvalue,respectively. Theseresults suggested the possibility that A. dahurica extractinhibitedtyrosinase synthesis in B16 melanoma cells.
EFFECT OF A. DAHURICA
MELANOMA CELLS

EXTRACT

ON EXPRESSION

OF THE TYROSINASE

GENE IN B16

To elucidate the action mechanismof A. dahuricaextract, we investigatedthe changes

in themRNA levelof tyrosinase usingtheRT-PCR technique. B16 melanoma cells were treated with 20 and 100 pg/ml ofA. dahurica extract for 48 hours, respectively, andthen eachmRNA levelwasexamined. The resultsare shownin Figure4. When normalized with the mRNA levelof -actin, the mRNA levelof tyrosinase wasdecreased by 75% at 100 pg/ml of the untreated controlvalue.Theseresultssuggest that A. dahurica extract might act on the common upstream eventthat controls the transcription of the
tyrosinase gene.

18
(A)

JOURNAL OF COSMETIC SCIENCE

:Tyresinase

(B)

120

100

o (.3

80

"' o

60

,--

40

20

Ctrl

20

Conc.ofA. dahurica (p./mL)

Figure 4. Inhibitoryeffects of A. dahurica extracton expression of the tyrosinase genein B16 melanoma

cells. B16mouse melanoma cells were seeded intoa T-75 flask at a density of l x 10 6 cells perflask and
treatedwith or without A. dahurica extractfor 48 hours.(A) is the result of gel electrophoresis of the RT-PCR products. Lane1 shows the tyrosinase (716 bp) and [3-actin gene(400 bp) of the controlcell. Lane 2 shows those of cellstreatedwith 20 []g/ml of A. dahurica extract.Lane3 shows those of cellstreatedwith 100 [g/ml of A. dahurica extract.(B) shows the decreased mRNA level of cellstreatedwith A. dahurica extractexpressed as% inhibitionof the control.Results are the averages of threeindependent experiments _+SD. *p < 0.05 comparedto the control.
ISOLATION AND IDENTIFICATION OF ACTIVE COMPOUNDS FROM A. DAHIIRICA

The ethanolicextractof A. dahzzrica decreased significantintracellularmelanin content andtyrosinase biosynthesis. Thus,a laboratory investigation wasperformed on the active ethanolic extract.Activity-guidedfractionation led to the isolation of compounds 1 and 2 as active compounds.

Compound 1 wasobtained asan amorphous white powder,and showed [M] + at m/z 270 in the ElMS spectrum. The UV spectrum exhibitedtypical bandsof the 5-substituted

linear furanocoumarin ringat 220,249,and 309nm(17,18). In the]H-NMR spectrum,

two doubletsignals (1H,J = 9.76 Hz) at 8 6.27 and 8.15 wereassignable to the ortho coupled protons of the pyronering, and anothertwo doubletsignals (1H,J = 2.45 Hz) weredetected at 6.96 and 7.60 ppm, attributableto the protons of the furan ring. A peakof the aromatic protonwasdetected asa singletsignal(1H, H-8) at 7.16 ppm, and the signalfor H-4 wasobserved at a ratherlower field (8 8.15) to indicatethe absence of protonat C-5 (17,18). Therefore,a sidechainwassuggested to substituteat the C-5

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EXTRACTS

19

position. A doublet signal (2H,J = 6.99 Hz, H-I") anda multipletsignal (1H, m, H-2") weredetected at 4.92 and 5.54 ppm, respectively, andprotons of methylgroups were

detected at 1.71and1.81ppmassinglet signals. In the3C-NMR spectrum, carbonyl carbon was shown at 161.26 ppm, along withtwelve sp 2carbons (8 94.12to 158.12) and three sp 3carbons (8 18.20 to69.74). From these data, compound 1 was postulated tobe
isoimperatorin, and the structurewasverified by the reportedNMR data (18,19).

Compound 2 wasobtained asan amorphous white powder,and [M] + at m/z 270 in the

ElMSspectrum. In the H- and3C-NMR spectrum, most of thespectral aspect of

compound2 was similar to that of compound1, but the proton signal for H-4 was

detected at a higherfield (8 7.76) thanthat of compound 1, andtwo carbon signals for

C-5 and 8 werechanged. From thesedata,compound 2 wasassumed to be the position isomerof compound 1, and identifiedas imperatorin. This was finally confirmedby comparingits NMR data with thosein the reportedreferences (18,19). The structures

of compounds 1 and2 arepresented in Figure 5 and3C-NMRdata arelisted in Table

I. We investigated the inhibitoryeffects of thesecompounds on melanogenesis in B16 melanoma cells.Compounds 1 and 2 at a concentration of 80 pM and 100 pM reduced the melanincontentby 50% compared with non-treated controlcells,respectively. Thesecompounds inhibitedmelanogenesis of B16 melanoma cellsin a dose-dependent
manner (data not shown).

We furtherstudiedby ELISA the inhibitoryeffects of thesecompounds on tyrosinase

expression in B16 melanomacells. 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 < 0.05) at 10 pM; 40+ 4.5% (P < 0.05) at 50 pM; and60+ 5.6% (p < 0.05) at 100 pM of compound 1; 35+ 5% (p < 0.05) at 50 pM; 50+ 3.4% (p < 0.05) at 100 pM; and 75+ 6.5% (p < 0.05) at 200 pM of

R
4"

R2

Isoimperatorin

0 2' 1'
H

5"

4"

Imperatorin

Figure 5. Chemical structure of compounds 1 and 2 isolated from the root of Angelica dahurica.

20

JOURNAL OF COSMETIC SCIENCE

compound2, compared with non-treatedcontrol cells, respectively (data not shown). Therefore,in this study, we investigated in B16 melanomacellswhetherthesecompoundsmodulatethe expression of tyrosinase steady-state mRNA levels.Thesecompounds significantly reduced tyrosinase production at the mRNA level(datanot shown). These resultssuggestthat thesecompounds suppress the tyrosinse productionat the protein and mRNA levels.

CONCLUSIONS

We foundthat A. dahz/rica extracthada stronginhibitoryactivityagainstmelanogenesis and exerted its melanogenic inhibitoryeffectthroughthe modulation of mRNA levels

of tyrosinase. Also,we isolated isoimperatorin andimperatorin, newcosmetic agents for skinwhitening,fromAngelica dahz/rica. Theseresults suggest that the activecompounds, in their actionmechanism, are novelwhiteningagents differentfrom otherthosebeing
used in the cosmeticindustry.

REFERENCES

(1) H. Y. Park,J. M. Perez,R. Laursen, M. Hara, and B. A. Gilchrest,ProteinkinaseC-[3 activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain, J. Bid. Chem., 274(23),
16470-16478 (1999).

(2) A.M. KornerandJ. Pawelek, Dopachrome conversion: A possible controlpoint in melaninbiosynthesis,J. Invet.Dermatol., 75, 192-195 (1980). (3) V.J. Hearingand M. Jimenez, Analysis of mammalian pigmentation at the molecular level,Pigment
Ce//Res., 2, 75-85 (1989).

(4) T. Kobayashi, K. Urabe,A. Winder, C. Jimenez-Cervantes, G. Imokawa; T. Brewington, F. Solano, J. C. Garcia-Borron, and . J. Hearing,Tyrosinase relatedprotein 1 (TRP1) functions asa DHICA oxidase in melaninbiosynthesis. EMBOJ., 13, 5818-5825 (1994). (5) K. Yokoyama, H. Suzuki,K. Yasumoto, Y. Tomita, and S. Shibahara, Molecular cloningand functional analysis of a cDNA codingfor humanDOPAchrometautomerase/tyrosinase-related protein-2, Biochim. Biophys. Acta., 1217, 317-321 (1994). (6) V. J. Hearing and K. Tsukamoto, Enzymaticcontrolof pigmentation in mammals, FASEBJ., 5,
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