the inhibition of 1 on cell - free melan - a tyrosinase 
 was demonstrated by a lineweaver  burk plot . the results shown 
 in figure 1 revealed both 1 and 
 resveratrol to be noncompetitive inhibitors of tyrosinase , with kojic 
 acid being a competitive inhibitor . thus , the addition of 1 to the reaction mixture led to a reduction of the maximal velocity 
 ( vmax = 4.7  10 ) and km value ( 2.4 mm l - dopa ) 
 of tyrosinase activity . the vmax value 
 decreased in a dose - dependent manner from 3.1  10 to 2.4  10 and 2.0  10 in the presence of 60 , 120 , and 240 m of 1 , 
 respectively . the ki value also decreased 
 from 3.1 to 2.8 and 2.5 mm , respectively . resveratrol , a stilbenoid 
 of the same oxidative level as 1 , exhibited comparable ki and vmax values 
 at 120 m ( table 1 ) . these results are 
 in agreement with a previous observation that the tyrosinase inhibitory 
 activity of stilbenes varies with the extent of hydroxylation , having 
 more potent inhibitory activity with a higher degree of hydroxylation . a pretreatment experiment was also performed 
 in which tyrosinase was mixed with 1 for 24 h prior to 
 kinetic analysis . the ki and vmax values of pretreated samples ( data not shown ) were 
 similar to those without the pretreatment . this finding suggests that 
 the binding between 1 and tyrosinase is likely reversible . taken together , the available evidence indicated 1 to 
 be a reversible noncompetitive inhibitor of melan - a tyrosinase showing 
 a higher potency than kojic acid ( table 1 ) . inhibitory 
 effects of 60 , 120 , and 240 m 1 , 
 120 m kojic acid , or 120 m resveratrol on tyrosinase 
 activity in murine melan - a cells . the vmax and km values in the absence ( control ) 
 or presence of 1 with l - dopa as the substrate 
 are analyzed using lineweaver the kinetic parameters are obtained 
 with l - dopa as a substrate using the lineweaver km ( michaelis constant ) and ki ( inhibition constant ) values are represented 
 as molar concentration , and vmax ( maximum 
 velocity ) values are expressed as the change of absorbance at 490 
 nm per second . forskolin 
 and 12-o - tetradecanoylphorbol-13-acetate 
 ( tpa ) are inducers of melanogenesis via the pka and pkc pathways , 
 respectively . the present results showed 
 that 1 reduces tyrosinase activity in melan - a cells in 
 a dose - dependent manner in the presence or absence of these melanogenesis 
 inducers ( figure 2a ) . in the presence of forskolin , similarly , the enzyme activity was reduced from 100% to 
 65% in the presence of tpa . these results suggest that 1 is a more potent blocker of the pka melanogenic pathway than the 
 pkc pathway . possible inhibitory mechanisms of 1 include 
 the inhibition of melanocortin-1 receptor from forskolin binding , 
 inhibition of camp synthesis , or inactivation of camp responding element ( cre ) binding protein . further study 
 is warranted to elucidate the inhibitory mechanism of 1 on the pka pathway . cells were incubated with or without 1 in the medium 
 containing 20 nm 12-o - tetradecanoylphorbol-13-acetate 
 ( tpa ) , 1 m forskolin , or vehicle alone for 3 days . tyrosinase 
 activity was measured by l - dopa oxidation ( a ) , and cell viability 
 was determined by the sulforhodamine b assay ( b ) . the values are denoted 
 as mean  sd of triplicate experiments . among stilbene compounds , resveratrol and oxyresveratrol the number and location of the hydroxy group(s ) , as well as the trans - olefin structure of the stilbene skeleton , have been 
 shown to be associated with the inhibitory properties on murine tyrosinase . on the other hand , glycosylation of the hydroxy 
 group did not show a significant effect . in our hands , 1 was the least cytotoxic compound among 
 the hydroxystilbenes tested including resveratrol , oxyresveratrol , 
 and picetanneol ( supporting information , figure s1 ) . moreover , it did not exert any cytotoxic effect in 
 samples with or without forskolin ( figure 2 ) . the nascent tyrosinase core is a polypeptide of 60 kda that is n - glycosylated to become a 70 kda folding intermediate and 
 further post - translationally modified to a mature form of 80 kda in 
 the golgi apparatus . in cases where the 
 polypeptide is misfolded or not properly glycosylated , it will remain 
 bound to calnexin in the er until a quality glycoprotein intermediate 
 is formed . if there is misfolding or aberration trafficking from the 
 late endosome to melanosome , the enzyme will likely be trapped in 
 the lysosome . consistent with the observations 
 made by others , functional tyrosinase colocalizes 
 with trp-1 ( figure 3a and b ) , but not with 
 lamp-1 of the lysosome ( figure 3c and d ) or 
 calnexin of the er ( figure 3f and g ) , regardless 
 of the presence or absence of 1 . these findings suggest 
 that no aberrant tyrosinase is trapped in the er and lysosome in 1-treated cells and tyrosinase is properly transported outside 
 to the melanosomes where it forms a complex with trp-1 . as a positive 
 control , calnexin and tyrosinase clearly colocalize to the er in resveratrol - treated 
 cells ( figure 3h ) , because resveratrol is capable 
 of retarding the post - translational - modified tyronsinase in the er . colocalization of both tyrosinase and lamp-1 
 were also observed ( figure 3e ) in progesterone - treated 
 cells in which misfolded tyrosinase was induced and retarded in the 
 lysosomes . confocal immunofluorescence 
 microscopy of melan - a melanocytes incubated 
 in the presence of 1 , progesterone , or resveratrol . the 
 locations of trp-1 ( red , a , b ) , lamp-1 ( red , c  e ) , or calnexin 
 ( cnx , red , f overlay 
 of the red and green images resulted in a yellow fluorescence , indicating 
 strong colocalization of the targeted proteins . cells were treated 
 with vehicle alone ( a , c , f ) , or 1 ( 60 m , b , d , 
 g ) for 3 days , progesterone ( 15 m ) for 4 days ( e ) , or resveratrol 
 ( 85 m ) for 1 day ( h ) . glycosylation analysis further confirmed the normal trafficking 
 of tyrosinase in both untreated and 1-treated cells ( figure 4 ) . the immature oligosaccharide added to tyrosinase 
 in the er can be digested only with endoglycosidase h ( endo h ) , whereas 
 both mature and immature oligosaccharide added to tyrosinase in the 
 golgi apparatus can be removed by peptide - n - glycosidase 
 f ( pngase f ) . results of western blotting 
 revealed two bands : an upper band at 80 kda for the mature and endo 
 h - resistant form of tyrosinase and a lower band at 60 kda for the 
 immature and endo h - sensitive form ( figure 4 ) . no immature endo h - sensitive form of tyrosinase was present in 
 both the untreated control and 1-treated murine cells 
 after being digested with endo h. these findings agree with the immunofluorescence 
 results to indicate that 1 does not interfere with tyrosinase 
 maturation in the er , golgi apparatus , and protein trafficking to 
 the melanosome . the deglycosylated tyrosinase digested by pngase f 
 ( 60 kda ) represented the total amount of tyrosinase . protein lysates 
 were obtained from 1-treated melan - a cells in forskolin - supplemented 
 medium . the lysates 
 were incubated with either peptide - n - glycosidase 
 f ( pngase f ) or endoglycosidase h ( endo h ) following the manufacturer s 
 instruction . the western blot was analyzed for the amounts of glycosylated 
 or deglycosylated tyrosinase . the upper band at 80 kda and the lower 
 band at 60 kda represent the mature endo h - resistant form and immature 
 endo h - sensitive form of tyrosinase , respectively . stilbene derivatives such as resveratrol and 2,2-dihydroxy-5,5-dipropylbiphenyl 
 have been associated with the unfolded protein response that triggers 
 er stress and subsequent apoptosis , but 1 seems to induce neither er stress nor lysosomal storage 
 stress as suggested by the above results . despite the structural similarity 
 between 1 and resveratrol , they do not share the same 
 canonical maturation retardation and apoptotic properties . being a weak acid as conferred from its phenolic 
 groups , and unlike basic compounds such as quinolines that would alter 
 the lysosomal ph and retard lysosomal function,1 appears not to alter the acidic lysosomal 
 ph , or otherwise it would have resulted in lysosomal storage stress . melanogenesis is controlled by mitf , a key regulator of a number 
 of melanogenic proteins such as tyrosinase , trp-1 , and trp-2 . since 
 melanogenesis is significantly increased through the forskolin pka 
 pathway ( figure 2 ) , the inhibitory action of 1 on these proteins was analyzed further in forskolin - supplemented 
 melan - a cells . it was found that 1 suppressed the expression 
 of mitf and tyrosinase in a dose - dependent manner , but the reduction 
 of trp-1 expression was not prominent ( figure 5 ) . such a down - regulation of mitf and tyrosinase protein could explain , 
 at least partially , the reduction of l - dopa oxidation with 
 increasing doses observed in figure 2a . expression 
 of melanogenesis - related proteins in melan - a melanocytes 
 after 1 treatment for 1 day in fsk - supplemented medium . the expressions of mitf ( a ) , tyrosinase ( b ) , 
 and trp-1 ( c ) were analyzed by western blotting , and the expression 
 of melanogenic proteins relative to actin is shown in panel ( d ) . trp-1 influences tyrosinase activity 
 by stabilizing and forming 
 complexes with tyrosinase . without stabilization from trp-1 during 
 the processing and intracellular trafficking , tyrosinase will be prompted 
 to degrade in the proteasome , leading to a reduction of pigment formation . this phenomenon has been demonstrated in mutant 
 brown melan - b melanocytes in which the trp-1 locus is mutated . co - immunoprecipitation using antibodies against trp-1 resulted in 
 a reduction of immunoprecipitated tyrosinase after treatment with 1 ( figure 6b ) , whereas trp-2 was not 
 pulled down together with tyrosinase ( figure 6c ) . these results are in agreement with the findings of hearing and 
 associates that trp-1 forms a complex with tyrosinase but not with 
 trp-2 . therefore , reduction of tyrosinase 
 activity after treatment with 1 was not only caused by 
 the down - regulation of tyrosinase expression but also affected by 
 the reduction of tyrosinase / trp-1 complex formation . association of tyrosinase 
 and trp-1 in melan - a melanocytes after 
 treatment with 1 for 3 days in forskolin - supplemented 
 medium . co - immunoprecipitation with antibody against trp-1 proteins 
 showed that trp-1 ( a ) and tyrosinase ( b ) , but not trp-2 ( c ) , proteins 
 were co - immunoprecipitated . in conclusion , compound 1 displayed antimelanogenic 
 activity in murine melanocytes . it noncompetitively inhibited the 
 enzyme activity of tyrosinase , down - regulated the expression of melanogenic 
 proteins , and reduced the tyrosinase / trp-1 complex formation . studies 
 of the antimelanogenic properties of 1 and its analogues 
 in the future using human melanocytes are needed to confirm these 
 activities . 2,3,5,4-tetrahydroxystilbene-2-o--d - glucopyranoside ( 1 ) was 
 isolated from polygonum multiflorum , and a larger 
 quantity was obtained from the hong kong jockey club institute of 
 chinese medicine ; the purity was determined to be > 98% by hplc . kojic 
 acid , forskolin , 12-o - tetradecanoylphorbol-13-acetate , 
 progesterone , and resveratrol were purchased from sigma - aldrich co. 
 ( st louis , mo , usa ) . all antibodies were obtained from santa cruz 
 biotechnology ( dallas , tx , usa ) , unless otherwise specified . mouse melan - a melanocytes derived from 
 c57bl/6 mice were a gift from professor d. c. bennett ( st george s 
 hospital medical school , london , uk ) . cells were cultured in rpmi-1640 
 medium supplemented with 5% fbs , 100 iu / ml penicillin ( sigma ) , 100 
 g / ml streptomycin sulfate ( sigma ) , and 200 nm tpa ( sigma ) for 
 cell attachment and proliferation . during treatment , cells were cultured 
 in the absence of tpa unless specified . cells used in the experiments 
 were restricted to passages of < 30 as recommended by the donor . the sulforhodamine 
 b ( srb , sigma ) colorimetric cytotoxicity assay was adopted in this 
 study . briefly , melan - a melanocytes ( 20  000 cells/0.1 ml / well ) 
 were treated with the test compounds in 96-well corning costar plates 
 for 72 h. the reaction was stopped and subjected to srb cytotoxicity 
 assays as described previously . the 3,4-dihydroxy - l - phenylalanine ( l - dopa ) ( sigma ) conversion assay is 
 a colorimetric assay in which the oxidation rate of l - dopa 
 specifically reflects the tyrosinase activity . tyrosinase inhibitory 
 property of 1 in the presence of various melanogenic 
 inducers was investigated under the same conditions . after treatment , 
 one volume cell lysate in 20 mm tris/0.1% triton x-100 ( ph 6 ) reacted 
 with one volume 1 g / ml l - dopa . data represent the mean values and 
 standard deviations of triplicate assays in at least three separate 
 experiments . the melan - a 
 cells were rinsed with phosphate buffer saline twice , then lysed with 
 20 mm tris/0.1% triton x-100 ( ph 7 ) . the protein lysate ( 50 g ) 
 was preincubated with the test compounds , i.e. , 1 , resveratrol , 
 or kojic acid , for 10 min at room temperature before reacting with l - dopa ( ph 6 ) . the rate of dopachrome formation 
 was measured at 490 nm per 20 s for a period of 10 min at 37 c 
 using a fluostar microplate reader ( bmg labtech , ortenberg , germany ) . the tyrosinase activity was determined based on the rate of l - dopa oxidation ( absorbance at 490 nm per second ) ; michaelis menten 
 constants ( km ) and maximal velocity ( vmax ) were calculated from the lineweaver for colocalization of 
 tyrosinase and trp-1 , cells cultured on coverslips were fixed in 100% 
 methanol , permeablized with 0.2% triton x-100 , and blocked in 2% bovine 
 serum albumin before subjected to confocal immunofluorescence microscopy . the procedures for costaining tyrosinase with organelle marker proteins , 
 i.e. , lamp-1 and calnexin ( sigma ) for lysosome and endoplasmic reticulum , 
 respectively , have been described in a previous paper . cells cotreated 
 with 1 m 
 forskolin with or without 1 before total proteins were 
 extracted with phosphosafe extraction buffer ( novagen , darmstadt , 
 germany ) . the proteins were resolved on a 10% sds - page gel and transferred 
 onto a pvdf membrane using a semidry transfer cell ( bio - rad , hercules , 
 ca , usa ) . the blot was blocked with 3% bovine serum albumin before 
 incubation with primary antibodies against tyrosinase , trp-1 ( generous 
 gift from dr . hearing , nih , bethesda , md , usa ) , trp-2 , mitf ( thermo 
 fisher scientific , pittsburgh , pa , usa ) , or -actin . signals 
 were detected by incubating the blot for 1 h at room temperature with 
 secondary antibodies , horseradish peroxidase - conjugated goat anti - mouse , 
 or donkey anti - goat , or goat anti - rabbit ( dako cytomation , glostrup , 
 denmark ) , and visualized using an enhanced chemiluminescence system 
 ( ecl , ge healthcare , lower chalfont , uk ) . for reprobing with other 
 primary antibodies , previous antibodies were stripped away using the 
 reblot plus western blot recycling kit ( millipore , temecula , ca , usa ) . protein lysates ( 10 g ) 
 obtained in the immunoblotting analyses were digested with either 
 1000 units of endoglycosidase h ( new england biolabs , beverly , ma , 
 usa ) or 1000 units of peptide - n - glycosidase f ( new 
 england biolabs ) according to the manufacturer s instruction . 
 in brief , the protein lysates were subjected to denaturation by treating 
 with glycoprotein denaturing buffer at 100 c for 10 min . the 
 denatured lysates were incubated with endo h or pngase f at 37 c 
 for 1 h and then subjected to western blotting analysis using anti - tyr 
 antibody . tyrosinase and trp-1 proteins 
 were co - immunoprecipitated using a dynabeads co - immunoprecipitation 
 kit with minor modifications ( invitrogen , grand island , ny , usa ) . briefly , harvested cell pellets were lysed with immunoprecipitation 
 lysis buffer supplemented with 0.1 m nacl , 0.5 mm pmsf , and 10 g / ml 
 apotinin on ice for 30 min . the extracted proteins of the same amount 
 from 1-treated or untreated samples ( 1 mg each ) were 
 reacted with antibody - coupled dynabeads overnight and separated by 
 sds - page analysis . following protein transfer on pvdf blot , melanogenic 
 proteins were detected by corresponding antibodies using ecl plus 
 ( ge healthcare ) or supersignal west dura chemiluminescent substrate 
 ( thermo fisher scientific ) .