O Artemisia capillaris Thunberg and Its In Vivo

Research ArticleIsolation of 45-O-Dicaffeoylquinic Acid as a PigmentationInhibitor Occurring in Artemisia capillaris Thunberg and ItsValidation In Vivo

Nadia Tabassum1 Ji-Hyung Lee1 Soon-Ho Yim2 Galzad Javzan Batkhuu3

Da-Woon Jung1 and Darren R Williams1

1New Drug Targets Laboratory School of Life Sciences Gwangju Institute of Science and TechnologyGwangju 500-712 Republic of Korea2Department of Pharmaceutical Engineering Dongshin University 185 Geonjaero Naju Jeonnam 520-714 Republic of Korea3School of Engineering and Applied Sciences National University of Mongolia 14200 Ulaanbaatar Mongolia

Correspondence should be addressed to Da-Woon Jung junggistackr and Darren R Williams darrengistackr

Received 7 February 2016 Revised 5 June 2016 Accepted 23 June 2016

Academic Editor Thierry Hennebelle

Copyright copy 2016 Nadia Tabassum et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

There is a continual need to develop novel and effectivemelanogenesis inhibitors for the prevention of hyperpigmentation disordersThe plant Artemisia capillarisThunberg (Oriental Wormwood) was screened for antipigmentation activity using murine culturedcells (B16-F10 malignant melanocytes) Activity-based fractionation using HPLC and NMR analyses identified the compound 45-119874-dicaffeoylquinic acid as an active component in this plant 45-119874-Dicaffeoylquinic acid significantly reduced melanin synthesisand tyrosinase activity in a dose-dependentmanner in themelanocytes In addition 45-119874-dicaffeoylquinic acid treatment reducedthe expression of tyrosinase-related protein-1 Significantly we could validate the antipigmentation activity of this compound invivo using a zebrafish model Moreover 45-119874-dicaffeoylquinic acid did not show toxicity in this animal model Our discovery of45-119874-dicaffeoylquinic acid as an inhibitor of pigmentation that is active in vivo shows that this compound can be developed as anactive component for formulations to treat pigmentation disorders

1 Introduction

The visible color of the mammalian skin hair and eyesresults from the quantity quality and epidermal distributionof the melanosomes These are organelles produced byspecialized dendritic cells called melanocytes [1] Melaninpigment is synthesized in the melanosomes which is trans-ported to keratinocytes by melanocyte dendrites Thus thedistribution pattern of melanin determines skin color [2]Melanin provides broad wavelength protection from solarUV radiation and absorbs free radicals generated in theskin [3] Melanogenesis is regulated by the expression ofenzymes involved in melanin formation More than 100proteins are involved in regulating pigmentation [2 4] Forexample melanogenesis is initiated with tyrosine oxidationto dopaquinone which is catalyzed by the key regulatory

enzyme tyrosinase Dopaquinone is further converted toeumelanin by intramolecular cyclization and polymeriza-tions reactions [5] Dysregulation in melanogenesis mayresult in the accumulation of excessive levels of pigmentationproducing disorders such as melasma age spots and sitesof solar keratosis Changes in skin color are also desiredfor cosmetic reasons which has produced a significantglobal market for skin lightening products [6] Lighteningproducts and therapeutics include hydroquinones retinoidsand tyrosinase inhibitors However these treatments maycause problems including mutations toxicity and ochrono-sis (blue-black hyperpigmentation of skin) [7]

In this study we used in vitro melanocyte-based screen-ing to screen extracts from the plant Artemisia capillarisThunberg (A capillaris Oriental Wormwood) to discovernovel melanin regulatory compounds A capillaris has been

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7823541 11 pageshttpdxdoiorg10115520167823541

2 Evidence-Based Complementary and Alternative Medicine

traditionally used as a herbal medicine in Korea and Chinasince ancient times [8] Extractspreparations from this plantexhibit various pharmacological activities such as antiviralinfection [9] antioxidant effects [10] hepatoprotective prop-erties [11] and anti-inflammatory effects [11 12] Numerousactive compounds have been extracted from A capillarissuch as phenolic compounds flavonoids flavonoid glyco-sides and coumarins [11 13]We isolated an antipigmentationcompound from the methanol extract of A capillaris whichwas identified as 45-119874-dicaffeoylquinic acid (45-diCQA)45-diCQAwas shown to be a pigmentation inhibitor in bothmelanocytes and the zebrafish vertebratemodel Significantlyno toxicity was observed in our analysis indicating that 45-diCQA is an attractive candidate for further development asa pharmaceutical or cosmetic depigmenting agent

2 Material and Methods

21 Chemicals PTU NaOH DMSO (dimethyl sulfoxide)L-DOPA (-34-dihydroxyphenylalanine) CellLytic buffermushroom tyrosinase MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide) and tricaine methanesul-fonate solution were purchased from Sigma (St Louis MOUSA) L-tyrosine was purchased from Duchefa Biochemie(Haarlem Netherland) All test compounds were dissolved inDMSO and protected from light at minus20∘C until use HPLCgrade solvents acetonitrile and methanol were obtainedfromMerck (Darmstadt Germany)

22 Plant Material The leaves and stems of Artemisia cap-illaris Thunberg were provided by Professor Soon-Ho YimDongshin University Naju Republic of Korea

23 Cell Culture Murine melanoma B16-F10 cells were ob-tained from the American Type Culture Collection (ATCCManassas VA) and cultured in DMEM supplemented with10 FBS 1 penicillin-streptomycin mixture (Gibco USA)Cultured cells were maintained in a 37∘C humidified incuba-tor with 5 CO

2

24 Determination of Melanin Content in B16-F10 Melan-ocytes Melanocytes were rinsed with phosphate bufferedsaline (PBS) and lysed with CellLytic buffer at 4∘C Cellextracts were spun at 13000 rpm for 10min at 4∘C Theremaining pellet was assayed for melanin by rinsing twicewith ethanol ether (1 1) and dissolving in 200120583L of 1NNaOH in 10 DMSO at 80∘C A 100 120583L aliquot of theresulting solution was then measured for absorbance at400 nm using microplate reader (VersaMax MolecularDevices Corporation California USA) [14]

25 Determination of In Vitro Tyrosinase Activity Tyrosinaseactivity was determined as described previously [15] BrieflyB16-F10 melanocytes were seeded in a 6-well plate at adensity of 2 times 105 cellswell The melanocytes were treatedwith compound for 48 hr Melanocytes were lysed using lysisbuffer and centrifuged at 13000 rpm for 10min 100 120583L ofeach lysate containing an equal amount of protein (250120583g)was placed into a 96-well plate and 100 120583L of 5mM L-DOPA

was added to each well After incubation at 37∘C for 60mindopachrome formation was measured at 475 nm using amicroplate reader

26 HPLC-Based Activity Profiling of Artemisia capillarisExtract The dried powder of the leaves and stems fromArtemisia capillaris was extracted with 100 MeOH andconcentrated in vacuo to yield a MeOH extract (6 g) HPLCwas performed on an Agilent HP1100 series comprised of adegasser a binary mixing pump a column oven and a DADdetector using YMC-PAC Pro C18 (10mm 250mm 5120583m)columns in conjunction with a gradient system of MeCNand H

2O containing 01 HCOOH For activity profiling

a portion (6 g) of the MeOH extract was fractionated bysemipreparative HPLC (Agilent 1100 Series USA) usingthe gradient eluent system with acetonitrile (MeCN) andwater containing 01 formic acid that is 20 MeCN to60 MeCN for 50min The mobile phase was delivered atthe flow rate of 60mLmin and detection of eluate wascarried out at 280 nm A total of 23 fractions were collectedconcentrated and their biological activities were evaluatedFraction ACMF09 was selected on the basis of its inhibitionof melanogenesis to obtain potentially bioactive compoundsThe ACMF09 fraction of MeOH extract of A capillaris wasseparated on a RP-18 column with a gradient of H

2O-MeOH

started at 60 40 (v v) and was kept constant for 50minThegradient system was then decreased to 0 100 and was keptconstant for 20min to yield a purified compound (72mg)

27 45-O-Dicaffeoylquinic Acid 1H-NMR (in CD3OD

600MHz) 120575 759 (1H d 119869 = 159Hz H-71015840 or H-710158401015840) 751 (1Hd 119869 = 159Hz H-71015840 or H-710158401015840) 702 (1H d 119869 = 18Hz H-21015840 orH-210158401015840) 700 (1H d 119869 = 18Hz H-21015840 or H-210158401015840) 692 (1H dd119869 = 81 18Hz H-61015840 or H-610158401015840) 690 (1H dd 119869 = 81 18HzH-61015840 or H-610158401015840) 675 (1H d 119869 = 81Hz H-51015840 or H-510158401015840) 674(1H d 119869 = 81Hz H-51015840 or H-510158401015840) 628 (1H d 119869 = 159Hz H-81015840 or H-810158401015840) 619 (1H d 119869 = 159Hz H-81015840 or H-810158401015840) 562 (1Hbr s H-5) 511 (1H br s H-4) 437 (1H br s H-3) 240 (2HH-6) 199 (2H m H-2) 13C-NMR (in CD

3OD 125MHz) 120575

1687 (C-91015840 or 910158401015840) 1684 (C-91015840 or 910158401015840) 1498 (C-41015840 or 410158401015840) 1478(C-71015840 or 710158401015840) 1477 (C-71015840 or 710158401015840) 1469 (C-31015840 or 310158401015840) 1468 (C-31015840or 310158401015840) 1278 (C-11015840 or 110158401015840) 1277 (C-11015840 or 110158401015840) 1233 (C-61015840 or 610158401015840)1166 (C-51015840 or 510158401015840) 1153 (C-21015840 or 210158401015840) 1152 (C-21015840 or 210158401015840) 1148(C-81015840 or 810158401015840) 1177 (C-81015840 or 810158401015840) 758 (C-1) 757 (C-2) 698(C-3) 686 (C-5) 397 (C-6) 385 (C-14) ESI-MS mz 5151[MndashH]minus (C

25H24O12)

28MushroomTyrosinase Assay Theeffect of the samples onmushroom tyrosinase activity was investigated according tothe method of Zhang et al [16] with minor modifications Inbrief mushroom tyrosinase enzyme was dissolved in 50mMpotassium phosphate buffer (pH 65) at a concentration of500 unitsmL 550 120583L of 50mM potassium phosphate and50 120583L of tyrosinase solution were mixed with an appropriatevolume of test samples in a microfuge tube and incubated for5min at room temperature 100 120583L of 15mM L-tyrosine wasadded to the solution and loaded into a 96-well plate Theamount of dopachrome formed in the reaction mixture was

Evidence-Based Complementary and Alternative Medicine 3

determined by measuring the absorbance at 490 nm using amicroplate reader

29 Quantitative Real-Time PCR Analysis B16-F10 melan-ocytes were treated with test samples for 48 hr Total RNAwas extracted using the TRI-Solution according to themanufacturerrsquos instructions and quantified using a Nan-oDrop 2000 spectrophotometer (NanoDrop Technologies)cDNA synthesis was carried out from 1120583g RNA usingAccuPower PCR PreMix (Bioneer) following the manufac-turerrsquos recommendation mRNA expressions of the MITFgene tyrosinase gene and TRP-1 were quantified using aPower SYBT Green PCR Master Mix (Applied Biosystems)mRNA levels were normalized with 120573-actin and fold changeof expression was calculated with the ΔΔCT method Theprimer sequences were as follows mouse tyrosinase for-ward 51015840-TACTTGGAACAAGCCAGTCGTATC-31015840 reverse51015840-ATAGCCTACTGCTAAGCC CAGAGA-31015840 mouse TRP-1forward 51015840- AAACCCATTTGTCTCCCAA -TGA-31015840 reverse51015840-CGTTTTCCAACGG -GAAGGT A-31015840 mouse MITF for-ward 51015840-GGACTTTCCCTTATCCCATCCA-31015840 reverse 51015840-GCCGAGGTTGTTGGTAAAG -GT-31015840The PCR conditionswere 95∘C for 2min followed by 40 cycles of 95∘C for 30 s60∘C for 1min and 72∘C for 1min followed by a final 30 secextension at 72∘C Data were analyzed using the Steponesoftware v23 (Applied Biosystems)

210 Origin and Maintenance of Parental Zebrafish Adultzebrafish were obtained from a commercial dealer and 10ndash15 fishes were kept in 5 L acrylic tanks under the fol-lowing conditions 285∘C with a 1410 hr lightdark cycleZebrafish were fed two times a day 7 dweek with livebrine shrimps (Artemia salina) Embryos were obtained fromnatural spawning that was induced at the morning around930 AM by turning on the light Collection of the embryoswas completed within 30min

211 Phenotype-Based Evaluation of Test Compounds UsingZebrafish Zebrafish embryos were maintained in 100mm2petri dishes in embryo media at a density of 70ndash80 embryosper dish Synchronized embryos were collected and arrayedby pipette at three embryos per well in 96-well plates con-taining 200120583L embryo medium (5mM NaCl 017mM KCl033mM CaCl

2sdot2H2O 033mM MgSO

4sdot7H2O to provide

a 60x stock solution) Test extracts were dissolved in 01DMSO and added to the embryo medium from 9 to 72 hpf(63 hr exposure) Occasional stirring and replacement of themedium were done every 24 hr to ensure even distributionof the test compounds In all experiments 75120583M PTU wasused to generate transparent zebrafish without interferingwith developmental process [17] Phenotype-based evalu-ations of body pigmentation were carried out at 72 hpfEmbryos were dechorionated using forceps anesthetizedwith tricaine methanesulfonate solution and mounted in 3methyl celluloseThe effects on the pigmentation of zebrafishwere observed using stereomicroscopy (LEICA DFC425 C)Melanocyte area was calculated using the Image J program(National Institutes of Health USA) as previously described[18]

212 Melanin Content and Tyrosinase Activity Determinationin Zebrafish Tyrosinase activity and melanin content wasdetermined as described previously [19] About 40 zebrafishembryos were treated with melanogenic modulators from 9to 48 hpf and sonicated in CellLytic buffer Optical densityof the supernatant was measured at 400 nm to measuremelanin level To determine tyrosinase activity 250120583g of totalprotein in 100 120583L of lysis buffer was transferred into a 96-well plate and 100 120583L of 5mML-34-dihydroxyphenylalanine(L-DOPA) was added Control wells contained 100 120583L lysisbuffer and 100 120583L 5mM L-DOPA After incubation for60min at 37∘C absorbance was measured at 475 nm usinga microplate reader The blank was removed from eachabsorbance value and the final activity was expressed as apercentage of the water control PTU-treated embryos wereused as a positive control

213 Melanocyte Counting Assay Embryos were assessed formelanocyte cell number as previously [20] Embryos werefirst exposed to light to contract the melanin within themelanocytes followed by imaging using stereomicroscopyMelanocytes were counted within a defined head region inthe micrographs

214 Measurement of Embryo Heart Rate The heart rate ofboth the atrium and ventricle was measured at 48 hpf todetermine compound toxicity Counting and recording ofatrial and ventricular contraction were performed for 3minusing stereomicroscopy and results were represented as theaverage heart rate per min [18]

215 MTT Assay for Cell Viability Cell viabilities wereassessed using the MTT assay as previously described [20]B16-F10 melanocytes were seeded into a 96-well plate at thedensity of 5 times 103 cellswell for 12 hours Cells were treatedwith compound or extract for 48 hr

216 Statistical Analysis Data were evaluated statisticallyusing Studentrsquos 119905-test Statistical significance was set at 119875 lt005 The data are shown as the mean plusmn SEM of threeindependent experiments

3 Results

31 Screening of A capillaris Extract for Pigmentation Reg-ulatory Activity Using a Melanocyte-Based Screening SystemA methanol extract of the aerial parts of A capillaris wasscreened to investigate its potential melanogenesis regulatoryactivity using murine melanocytes Melanocytes were treatedwith two different concentrations 25 and 50120583gmL of theextract for 48 hr When B16-F10 melanocytes were treatedwith A capillaris the cells became visibly less dark comparedto untreated cells indicating reduced cellular melanogenesis(Figure 1(a))Thiswas confirmed by observation of the lightlycolored crude extract of the treated cell pellet (Figure 1(b))Treatment with 50 or 25120583gmL A capillaris reduced melaninproduction to 9192plusmn888 and 8581plusmn1012 compared tountreated melanocytes (Figure 1(c)) To assess the effect of A


Control A capillaris (

45-diCQA (50120583M)ACMF09 (50120583gmL)

50120583gmL)AZ (5mM)

(a)

Control 45-diCQA (50120583M)A capillaris (50120583gmL)AZ (5mM) ACMF09 (50120583gmL)

(b)

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120

Con

trol

A capillaris ACMF09 45-diCQA

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

lowastlowastlowast

lowast

(c)

Tyro

sinas

e act

ivity

( o

f con

trol)

020406080

100120

Con

trol


50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

lowastlowast lowast lowast

lowast

lowast

(d)

Figure 1 Inhibitory effect of the A capillaris extract active fraction ACMF09 and 45-diCQA onmelanogenesis in B16-F10 melanocytes (a)Phase-contrast microscopy of melanocytes showing less pigmented cells after exposure to test samples for 48 hr Scale bar = 20120583m (b) Grossappearance of the cell pellets from treated melanocytes (c) Melanin content in B16-F10 melanocytes treated with the A capillaris extractactive fraction ACMF09 and 45-diCQA (d) Tyrosinase activity in melanocytes after treatment with the indicated concentrations of testsamples Azelaic acid (AZ) was used as a positive control The results are expressed as percentages of the untreated control and the data aremean plusmn SEM of three independent experiments lowast119875 lt 005 compared to the untreated control


HOOC

OH OH OH

OH

OH

OH

OO

OO

12 3

45

6

7998400

7998400

1998400

1998400

3998400

3998400

4998400

4998400

8998400

8998400

9998400

9998400

(a)

70057

82738

83681

60

50

40

30

20

10

0

0 10 20 30 40 50 60 70 80

(min)

Nor

m

(b)

Figure 2 HPLC chromatogram of purified compound 45-diCQA (a) Chemical structure of 45-diCQA (b) 45-diCQA was isolated fromA capillaris by HPLC as described in Section 2

capillaris on melanin content and tyrosinase activity azelaicacid (AZ) a known tyrosinase inhibitor was used as a positivecontrol Although previous studies reported that azelaic acidwas effective at concentrations of 40 and 20mM [21] weobserved that treatment with these concentrations for 48 hrcaused immediate detachment of more than 50 of the B16-F10 melanocytes (data not shown) We observed that 5mMAZ treatment decreased melanin content by 15ndash20 withoutproducing noticeable toxicity in the melanocytes (Figures1(a)ndash1(c)) Subsequently tyrosinase inhibition was assessedand it was observed that tyrosinase activity was partiallyinhibited by treatmentwith 25 or 50120583gmL of theA capillarisextract for 48 h 8782 plusmn 0065 and 7568 plusmn 068 comparedto untreated respectively (Figure 1(d))

32 Isolation and Characterization of an AntimelanogenesisCompound from the A capillaris Extract High performanceliquid chromatography (HPLC) was performed to isolateantimelanogenesis compounds (the chromatogram is shownin Supplementary Figure S1 in Supplementary Materialavailable online at httpdxdoiorg10115520167823541)23 fractions of varying weight were isolated from the Acapillaris plant extract via activity-guided separation (thefraction weights are shown in Supplementary Table 1) Thefractions were collected dried and dissolved in DMSO andevaluated for their antimelanogenic activity at the sameconcentration used for the plant extract (25120583gmL) Fourfractions (ACMF09 ACMF13 ACMF14 and ACMF23) wereactive and their antipigmentation activity was confirmedin vivo using zebrafish embryos (Supplementary FigureS2) Fraction ACMF09 was found to produce the great-est antimelanogenesis effect and selected for analysis inmammalian melanocytes ACMF09 reduced pigmentationin the melanocytes (Figures 1(a) and 1(b)) Moreover thisfraction also significantly reduced melanin production andtyrosinase activity (Figures 1(c) and 1(d)) To isolate potentialmelanogenic regulatory compound(s) the ACMF09 wasfractionated on a silica gel column and a reversed-phaseHPLC column A linear gradient solvent condition appliedto HPLC separation was H

2O-methanol and started at 60 40

(v v) and kept constant for 50min The gradient system wasthen decreased to 0 100 and kept constant for 20min The

mobile phase was delivered at the flow rate of 60mLmin anddetection of the eluate was carried out at 280 nmWe isolatedand purified a compound identified as 45-O-dicaffeoylquinicacid (45-diCQA) on the basis of 1H-NMR and ESI-MSdetector analysis (Figures 2(a) and 2(b))This compound wasa yellow amorphous powder One spot was detected underUV at 280 nm 1H- and 13C-NMR spectra of compoundshowed the existence of two caffeoyl moieties six aromaticprotons [120575H 702 (1H d 119869 = 18Hz H-21015840 or H-210158401015840) 700(1H d 119869 = 18Hz H-21015840 or H-210158401015840) 692 (1H dd 119869 = 8118Hz H-61015840 or H-610158401015840) 690 (1H dd 119869 = 81 18Hz H-61015840 orH-610158401015840) 675 (1H d 119869 = 81Hz H-51015840 or H-510158401015840) 674 (1H d119869 = 81Hz H-51015840 or H-510158401015840)] and trans doublets [120575H 759 (1Hd 119869 = 159Hz H-71015840 or H-710158401015840) 751 (1H d119869 = 159Hz H-71015840 or H-710158401015840) 628 (1H d 119869 = 159Hz H-81015840 or H-810158401015840) 619(1H d 119869 = 159Hz H-81015840 or H-810158401015840)] and two carboxyl groups(120575C 1687 and 1684) and three hydroxyl carbons (120575C 14981469 and 1468) 1H- and 13C-NMR spectra of the compoundalso showed the existence of a quinic acid moiety [120575H 562(1H br s H-5) 511 (1H br s H-4) 437 (1H br s H-3) 240(2H H-6) 199 (2H m H-2)] The LCUVMS profile of thecompound displayed UV absorption bands at 328 292 and245 nm and ESI-MS [MndashH]minus peak atmz 51511 On the basisof these results the structure of compound was elucidated as45-O-dicaffeoylquinic acid [22] 45-diCQA was examinedfor its effects on pigmentation in mammalian melanocytesMicroscopic inspection indicated that melanocyte pigmenta-tion decreased after treatment with 45-diCQA (Figure 1(a))The cell pellet was markedly lighter in color compared tocontrol cells (Figure 1(b)) Quantitative analysis confirmedthat 45-diCQA treatment reduced melanin level in themelanocytes (Figure 1(c)) In addition 45-diCQA reducedtyrosinase activity in the melanocytes at the 50120583M treatmentconcentration (Figure 1(d))

33 Effects of Fraction ACMF09 and 45-diCQA onMushroomTyrosinase Activity in a Cell-Free System To investigatewhether 45-diCQA directly inhibits the enzymatic activity oftyrosinase the in vitro cell-free mushroom tyrosinase assaywas used It was observed that 45-diCQA inhibited mush-room tyrosinase in dose-dependent manner (Figure 3) Atthe 50 120583M concentration 45-diCQA produced 32 enzyme


ACMF09

Con

trol

45-diCQA

50120583

M

25120583

m

120583M

125

625120583

M

50120583

gm

L

25120583

gm

L

125120583

gm

L

625120583

gm

L

AZ

(5m

M)0

20406080

100120

Tyro

sinas

e act

ivity

in a

cell

free

syste

m (

of c

ontro

l)

lowast

lowast lowast lowastlowast

lowast

Figure 3 Inhibitory effect of the active fraction ACMF09 and 45-diCQA on tyrosinase activity The inhibition of cell-free tyrosinaseactivity was determined using mushroom tyrosinase Azelaic acid(AZ) was used as a positive control The results are expressed aspercentages of the control and the data are mean plusmn SEM of threeindependent experiments lowast119875 lt 005 as compared to the untreatedcontrol

MITF Tyrosinase TRP-1

mRN

A ex

pres

sion

( o

f con

trol)

Control

0

20

40

60

80

100

120

140lowast lowast lowast

AZ (5mM)45-diCQA (25120583M)

Figure 4 Quantitative real-time PCR analysis of the effect of 45-diCQA on the expression of the melanogenesis-related genes MITFtyrosinase and TRP-1 in B16-F10 melanocytes mRNA signal wasnormalized to actin mRNA expression Azelaic acid (AZ) was usedas a positive control The results are expressed as percentages ofthe control and the data are mean plusmn SEM of three independentexperiments lowast119875 lt 005 as compared to the untreated control

inhibition compared to untreatedThepositive control (5mMAZ) produced a 77 inhibition of enzyme activity

34 Effect of 45-diCQA on the Expression of MITF Tyrosi-nase and TRP-1 To explore the possible mechanism ofthe antipigmentation effects of 45-diCQA the expressionof levels of three key regulatory genes for melanogene-sis microphthalmia-associated transcription factor (MITF)tyrosinase-related protein-1 (TRP-1) and tyrosinase wasexamined using quantitative real-time PCR As shown inFigure 4 the mRNA level of tyrosinase was unchanged bytreatmentwith 45-diCQA In contrast themRNAexpressionof TRP-1 was significantly reduced

35 A capillaris Extract ACMF09 and 45-diCQA InhibitPigmentation in the Zebrafish Vertebrate Model SystemZebrafish is an emerging animal model in pigmentationresearch [23] We tested the A capillaries methanol extractand ACMF09 active fraction in the zebrafish larvae-based invivo system for comparison with the in vitro data A well-known pigmentation inhibitor 1-phenyl 2-thiourea (PTU)which reduces tyrosinase activity was used as a positivecontrol [24ndash26] However it has been reported that at the 28-somite stage PTUmay cause delayed hatching and mortalityby 120 hpf [17] Thus for this study we tested different con-centrations of PTU (25 50 75 100 and 200120583M) on zebrafishpigmentation It was observed that the 75 120583M dose of PTUreduced pigmentation in the zebrafish without significantlyaffecting mortality or producing teratogenic effects (data notshown)

The methanol extract of A capillaris fraction ACMF09and 45-diCQA inhibited pigmentation in the zebrafish(Figure 5(a)) Interestingly depigmentation was observed tobe caused by shrinkage of the melanocytes in the head regionof the embryos (Figure 5(a)) In addition it was observedthat the A capillaris extract fraction ACMF09 and 45-diCQA decreased melanin synthesis in zebrafish embryos ina dose-dependent manner (Figure 5(b)) Moreover partialinhibition of tyrosinase activity was observed (Figure 5(c))indicating that the reduction of melanogenesis in zebrafish isdue to the partial inhibition of cellular tyrosinase activity

36 Effect of 45-diCQA on Melanocyte Survival in the Zebra-fish Larval Head Portion Melanocytes in zebrafish embryobegin to produce melanin at around 24 hpf By 60 hpf thereare approximately 460 melanocytes in the head body tailand yolk sac that form the pattern of pigmentation [27]In our study we imaged melanocytes in the head region ofthe whole embryo When 9 hpf larvae were incubated with25 120583M 45-diCQA the number of melanocytes was slightlyless than in untreated the embryos (Figure 6(a)) 45-diCQAdid not affect melanocyte cell number at the 125 120583M doseHowever melanocyte area was significantly decreased at alltested doses of 45-diCQA For example the 25 120583M dosereducedmelanocyte area by approximately 4 times comparedto untreated embryos (Figure 6(b))

37 Determination of the Toxic Effects of A capillaris ExtractACMF09 and 45-diCQA In Vitro and In Vivo To confirmthe effect of 45-diCQA on melanogenesis the cytotoxi-city of different concentrations of 45-diCQA on B16-F10melanocytes was evaluated by MTT assay As shown inFigure 7 cell viability did not change in the presence of 45-diCQA in all treatment groups compared to the control indi-cating that the isolated compound is not cytotoxic to B16-F10melanocytes A very useful feature of zebrafish-based analysisis that the toxicity of candidate drugs can be readily testedin the developing larvae [28] To evaluate the A capillarisextract ACMF09 and 45-diCQA for their potential toxiceffects in zebrafish we observed treated embryos at 24 hpf 48and 72 hpf for any morphological malformations embryonicmortality and heartbeat disturbances We did not observeany adverse effects on zebrafish morphology and physiology


Control A capillaris

45-diCQA (25120583M)ACMF09 (25120583gmL)

PTU (75120583M) (25120583gmL)

(a)

A capillaris ACMF09

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120140

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowast lowast

(b)

020406080

100120140

Tyro

sinas

e act

ivity

( o

f con

trol)

A capillaris ACMF09

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowastlowast

lowast

(c)Figure 5 Inhibitory effect of the A capillaris methanol extract active fraction ACMF09 and 45-diCQA on pigmentation in developingzebrafish embryos (a) Zebrafishwas treatedwith test samples from9 hfp to 72 hpf Treatmentwith test samples at the indicated concentrationsresulted in decreased pigmentation as indicated by imaging the dorsal view of live embryos and the head portion Scale bar = 250 120583m (b)Melanin content in zebrafish embryos treated with test samples from 9 hfp to 48 hpf (c) Tyrosinase activity in the treated zebrafish PTU wasused as positive control Results are expressed as percentages of the control and the data are mean plusmn SEM of three independent experimentslowast

119875 lt 005 compared to the untreated control

The zebrafish heart at 72 hpf is identical to that of a humanembryo at three weeksrsquo gestation stage and can be used as atoxic test for compoundcompounds [29] We observed thatthe average heart rate of treated embryos was not significantlydifferent compared to untreated embryos We also notedthat the observed heart rates of treated embryos were notsignificantly different compared to untreated embryos Wealso noted that the observed heart rates of treated embryoswere similar to heart rates reported in previous studies [30ndash32]

4 Discussion

There is a research and therapeutic need to develop com-pounds that effectively regulate melanin synthesis [14]

Commonly utilized pigmentation inhibitors such as cor-ticosteroids or tyrosinase inhibitors are effective but mayproduce toxic side effects [33] In recent years there hasbeen renewed research in developing depigmenting productsfrom natural sources because there is greater potentialto avoid safety issues [34] Interestingly the utilization ofnatural products to treat pigmentation has a long historyIn Ancient China it was common practice to use herbs toproduce hypopigmentation [35 36] In our study we usedcell-based screening to examine the effect of A capillarison melanogenesis To our knowledge no study has beenpublished concerning the melanogenic regulatory activity ofthis plant In our study we have demonstrated that an Acapillaris extract produces pronounced inhibitory effects onpigmentation in B16-F10 melanocytes in a dose-dependent


Con

trol

Mela

nocy

te ce

ll nu

mbe

r

20253035404550556065

45

-diC

QA

(25120583

M)

45

-diC

QA

(125120583

M)

45

-diC

QA

(625120583

M)

PTU

(75120583

M)

(a)

Spot

area

( o

f con

trol)

Con

trol

PTU

(75120583

M)

45-diCQA

25120583

M

120583M

125

625120583

M

lowast

lowast

lowast

lowast

0

20

40

60

80

100

120

140

(b)

Figure 6 The effect of 45-diCQA on melanocyte cell number and spot area in zebrafish embryos (a) Box and whisker plots of melanocytesnumber in the head region of 45-diCQA-treated embryos indicated no major difference compared with untreated embryos The standardmean is indicated outliers are represented by an asterisk (b) Surface area of the melanocytes in zebrafish embryos PTU was used as positivecontrol Azelaic acid (AZ) was used as a positive control The results are expressed as percentages of control and the data are the means plusmnSEM of three independent experiments lowast119875 lt 005 as compared to the untreated control

Con

trol


Cel

l via

bilit

y (

of c

ontro

l)

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

020406080

100120

Figure 7 Effect of theA capillaris extract active fraction ACMF09and 45-diCQA on melanocyte cell viability B16-F10 melanocyteswere treated with test samples at the indicated concentrations for48 h Cell viability was determined using the MTT assay Azelaicacid (AZ) was used as a positive control The results are expressedas percentages of the control and the data are mean plusmn SEM of threeindependent experiments

manner Our data showed that fraction ACMF09 displayedthe antityrosinase activity in the melanocytes and zebrafishsystem We isolated the potential pigmentation inhibitorcompound from ACMF09 using HPLC and identified it as45-diCQA This compound has been shown to have manypharmacological properties [37ndash41] Our study is the first toshow that 45-diCQA can be isolated from A capillaries andcan suppress melanin biosynthesis in B16-F10 melanocytesvia partial inhibition of tyrosinase activity 45-diCQA hasalso been extracted from other plant sources for examplegreen coffee beans [42] and Gnaphalium affineD DON [43]These previous studies also showed that 45-diCQA inhibitedtyrosinase activity and produced antioxidant effects How-ever these previous studies only assessed 45-diCQA using

enzyme-based cell-free systems there was no assessment onpigmentation in cells or animal models

Our real-time PCR analysis of tyrosinase indicated that45-diCQA does not affect gene expressionThis is consistentwith our finding that 45-diCQA partially inhibits tyrosi-nase enzyme activity Moreover our finding that 45-diCQAshowed significant inhibitory effects on TRP-1 expressionindicates that this compound may also inhibit pigmentationby targeting TRP-1 (TRP-1 has been demonstrated to activatethe tyrosinase and enhance its stability and thus inducemelanin synthesis [44]) Elucidating exactly how 45-diCQAdownregulates both TRP-1 and its effect on pigmentation rel-ative to tyrosinase enzyme inhibition could be an interestingavenue for further research

The in vivo imaging data of zebrafish melanocytes indi-cated that 45-diCQA caused shrinkage of these cells Toour knowledge melanocyte shrinkage without cytotoxicityis not a common feature of depigmentation compounds(our MTT data suggests that 45-diCQA is not cytotoxicfor melanocytes even though some studies demonstrate thatphenolic compounds producemelanocyte toxicity) [27 45] Ithas been shown that cytochalasin B (amycotoxin that inhibitsactin filament formation) or dysregulation of melanocytefunction by T-helper cell 17-related cytokines can inducemelanocyte shrinkage [46 47]

Our compound also caused a slight reduction inmelanocyte numbers in the zebrafish skin This decreasein melanocyte numbers could be due to factors such asmelanocyte cell death clustering of melanocytes to makethem indistinguishable as separate units or the inhibition ofmelanoblast proliferation [45] Assessing the precise mech-anism of 45-diCQA on melanocyte morphology in vivoshould be an interesting area for future investigation Manycandidate drugs have been shown to induce toxic effects bytargeting the circulatory system [48] It has been demon-strated that the pharmacological responses of zebrafish to


various classes of drugs and the development of the cardio-vascular system are markedly similar to humans [49 50]Our results revealed that 45-diCQA produced antipigmen-tation effects in vivo with no obvious developmental defectsor effect on heart rate

In summary in this workwe employedmelanocyte-basedscreening for the activity-guided fractionation of ArtemisiacapillarisThunberg to identify pigmentation regulatory com-poundsThe cell-based screeningwas coupledwith validationin the zebrafish animal model Using this approach weidentified 45-diCQA as a depigmenting compound thatinhibits tyrosinase activity and is effective in vivo Our resultsfurther support the zebrafish as a valuable model for drugdiscovery which has also been demonstrated in other diseasecontexts such as diabetes and cancer [51 52] The discoveryof 45-diCQA as a nontoxic cosmetic and pharmaceuticaldepigmenting should be of interest to companies developingskin whitening products in addition to researchers studyingmelanogenesis

Abbreviations13C-NMR Carbon 13 nuclear magnetic resonance1H-NMR Proton nuclear magnetic resonance45-diCQA 45-O-Dicaffeoylquinic acidA capillaris Artemisia capillarisACMF09 Artemisia capillarisMeOH Fraction

number 09AZ Azelaic acidDMSO Dimethyl sulfoxideHpf Hours postfertilizationHPLC High performance liquid chromatographyL-DOPA L-34-DihydroxyphenylalanineMeCN AcetonitrileMeOH MethanolMITF Microphthalmia-associated transcription

factorNaOH Sodium hydroxidePTU 1-Phenyl 2-thioureaTRP-1 Tyrosinase-related protein-1

Competing Interests

The authors declare no conflict of interests

Authorsrsquo Contributions

Nadia Tabassum Ji-Hyung Lee and Soon-HoYimhave equalcontribution

Acknowledgments

This research was supported by the following grants (1) BasicScience Research Program through the NRF funded by theKorean government MSIP (NRF-2015R1A2A2A11001597)and (2) A grant from the Integrative Aging ResearchCenter of the Gwangju Institute of Science and Technol-ogy

References

[1] H-YThong S-H Jee C-C Sun andR E Boissy ldquoThepatternsof melanosome distribution in keratinocytes of human skinas one determining factor of skin colourrdquo British Journal ofDermatology vol 149 no 3 pp 498ndash505 2003

[2] V J Hearing and K Tsukamoto ldquoEnzymatic control of pigmen-tation in mammalsrdquo FASEB Journal vol 5 no 14 pp 2902ndash2909 1991

[3] D A Brown ldquoSkin pigmentation enhancersrdquo Journal of Photo-chemistry and Photobiology B Biology vol 63 no 1ndash3 pp 148ndash161 2001

[4] D C Bennett and M L Lamoreux ldquoThe color loci of micemdashagenetic centuryrdquo Pigment Cell Research vol 16 no 4 pp 333ndash344 2003

[5] C Olivares C Jimenez-Cervantes J A Lozano F Solano and JC Garcıa-Borron ldquoThe 56-dihydroxyindole-2-carboxylic acid(DHICA) oxidase activity of human tyrosinaserdquo BiochemicalJournal vol 354 no 1 pp 131ndash139 2001

[6] E V Curto C Kwong H Hermersdorfer et al ldquoInhibitors ofmammalian melanocyte tyrosinase in vitro comparisons ofalkyl esters of gentisic acid with other putative inhibitorsrdquoBiochemical Pharmacology vol 57 no 6 pp 663ndash672 1999

[7] O Nerya J Vaya R Musa S Izrael R Ben-Arie and S TamirldquoGlabrene and isoliquiritigenin as tyrosinase inhibitors fromlicorice rootsrdquo Journal of Agricultural and Food Chemistry vol51 no 5 pp 1201ndash1207 2003

[8] K S Seo H J Jeong and KW Yun ldquoAntimicrobial activity andchemical components of two plants Artemisia capillaris andArtemisia iwayomogi used as Korean herbal Injinrdquo Journal ofEcology and Field Biology vol 33 no 2 pp 141ndash147 2010

[9] K S Seo and KW Yun ldquoAntioxidant activities of extracts fromArtemisia capillaris Thunb and Artemisia iwayomogi Kitamused as Injinrdquo Korean Journal of Plant Resources vol 21 no 1pp 292ndash298 2008

[10] J-H Choi D-W Kim N Yun et al ldquoProtective effects ofhyperoside against carbon tetrachloride-induced liver damagein micerdquo Journal of Natural Products vol 74 no 5 pp 1055ndash1060 2011

[11] I J Seon Y-J Kim W-Y Lee et al ldquoScoparone from Artemisiacapillaris inhibits the release of inflammatory mediators inRAW 2647 cells upon stimulation cells by interferon-120574 plusLPSrdquo Archives of Pharmacal Research vol 28 no 2 pp 203ndash208 2005

[12] T-Y Shin J-S Park and S-H Kim ldquoArtemisia iwayomogiinhibits immediate-type allergic reaction and inflammatorycytokine secretionrdquo Immunopharmacology and Immunotoxicol-ogy vol 28 no 3 pp 421ndash430 2006

[13] A Mase B Makino N Tsuchiya et al ldquoActive ingredients oftraditional Japanese (kampo) medicine inchinkoto in murineconcanavalin A-induced hepatitisrdquo Journal of Ethnopharmacol-ogy vol 127 no 3 pp 742ndash749 2010

[14] L Ni-Komatsu and S J Orlow ldquoIdentification of novel pigmen-tation modulators by chemical genetic screeningrdquo Journal ofInvestigative Dermatology vol 127 no 7 pp 1585ndash1592 2007

[15] K J S Kumar J-C Yang F-H Chu S-T Chang and S-YWang ldquoLucidone a novel melanin inhibitor from the fruit ofLindera erythrocarpa Makinordquo Phytotherapy Research vol 24no 8 pp 1158ndash1165 2010

[16] X Zhang X Hu A Hou and H Wang ldquoInhibitory effectof 242101584041015840-tetrahydroxy-3-(3-methyl-2-butenyl)-chalcone on


tyrosinase activity and melanin biosynthesisrdquo Biological andPharmaceutical Bulletin vol 32 no 1 pp 86ndash90 2009

[17] J Karlsson J VonHofsten and P-E Olsson ldquoGenerating trans-parent zebrafish a refinedmethod to improve detection of geneexpression during embryonic developmentrdquoMarine Biotechnol-ogy vol 3 no 6 pp 522ndash527 2001

[18] T-Y Choi J-H Kim D H Ko et al ldquoZebrafish as a newmodelfor phenotype-based screening ofmelanogenic regulatory com-poundsrdquo Pigment Cell Research vol 20 no 2 pp 120ndash127 2007

[19] R Busca C Bertolotto J-P Ortonne and R Ballotti ldquoInhibi-tion of the phosphatidylinositol 3-kinasep70S6-kinase pathwayinduces B16 melanoma cell differentiationrdquo The Journal ofBiological Chemistry vol 271 no 50 pp 31824ndash31830 1996

[20] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[21] J S Yu and A K Kim ldquoEffect of combination of taurine andazelaic acid on antimelanogenesis in murine melanoma cellsrdquoJournal of Biomedical Science vol 17 supplement 1 article S452010

[22] E J Lee J S KimH P Kim J-H Lee and S S Kang ldquoPhenolicconstituents from the flower buds of Lonicera japonica and their5-lipoxygenase inhibitory activitiesrdquo Food Chemistry vol 120no 1 pp 134ndash139 2010

[23] R N Kelsh M L Harris S Colanesi and C A EricksonldquoStripes andbelly-spots-a reviewof pigment cellmorphogenesisin vertebratesrdquo Seminars in Cell and Developmental Biology vol20 no 1 pp 90ndash104 2009

[24] B L Bohnsack D Gallina and A Kahana ldquoPhenothioureasensitizes zebrafish cranial neural crest and extraocular muscledevelopment to changes in retinoic acid and IGF signalingrdquoPLoS ONE vol 6 no 8 Article ID e22991 2011

[25] M P Craig S D Gilday and J R Hove ldquoDose-dependenteffects of chemical immobilization on the heart rate of embry-onic zebrafishrdquo Laboratory Animals vol 35 no 9 pp 41ndash472006

[26] R N Kelsh M Brand Y-J Jiang et al ldquoZebrafish pigmentationmutations and the processes of neural crest developmentrdquoDevelopment vol 123 pp 369ndash389 1996

[27] C Yang and S L Johnson ldquoSmall molecule-induced ablationand subsequent regeneration of larval zebrafish melanocytesrdquoDevelopment vol 133 no 22 pp 3563ndash3573 2006

[28] N S Sipes S Padilla and T B Knudsen ldquoZebrafish-As anintegrativemodel for twenty-first century toxicity testingrdquoBirthDefects Research Part CmdashEmbryo Today Reviews vol 93 no 3pp 256ndash267 2011

[29] S-K Ko H J Jin D-W Jung X Tian and I Shin ldquoCardio-sulfa a small molecule that induces abnormal heart develop-ment in zebrafish and its biological implicationsrdquo AngewandteChemiemdashInternational Edition vol 48 no 42 pp 7809ndash78122009

[30] C-Y Chen and C-Y Chen ldquoInfluences of textured substrateson the heart rate of developing zebrafish embryosrdquo Nanotech-nology vol 24 no 26 Article ID 265101 2013

[31] J R Guyon A N Mosley Y Zhou et al ldquoThe dystrophinassociated protein complex in zebrafishrdquo Human MolecularGenetics vol 12 no 6 pp 601ndash615 2003

[32] W R Barrionuevo and W W Burggren ldquoO2

consumptionand heart rate in developing zebrafish (Danio rerio) influenceof temperature and ambient O

2

rdquo The American PhysiologicalSociety vol 276 no 2 part 2 pp R505ndashR513 1999

[33] J H Lee H Chen V Kolev et al ldquoHigh-throughput high-content screening for novel pigmentation regulators usinga keratinocytemelanocyte co-culture systemrdquo ExperimentalDermatology vol 23 no 2 pp 125ndash129 2014

[34] S Zhong Y Wu A Soo-Mi et al ldquoDepigmentation of mel-anocytes by the treatment of extracts from traditional Chineseherbs a cell culture assayrdquo Biological and PharmaceuticalBulletin vol 29 no 9 pp 1947ndash1951 2006

[35] H-Y Ding T-S Chang H-C Shen and S S-K Tai ldquoMurinetyrosinase inhibitors from Cynanchum bungei and evaluationof in vitro and in vivo depigmenting activityrdquo ExperimentalDermatology vol 20 no 9 pp 720ndash724 2011

[36] H Park K H Song P M Jung et al ldquoInhibitory effect ofarctigenin from fructus arctii extract on melanin synthesis viarepression of tyrosinase expressionrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 96531210 pages 2013

[37] P Basnet K Matsushige K Hase S Kadota and T NambaldquoPotent antihepatotoxic activity of dicaffeoyl quinic acids frompropolisrdquo Biological and Pharmaceutical Bulletin vol 19 no 4pp 655ndash657 1996

[38] T Nagaoka A H Banskota Q Xiong Y Tezuka and S KadotaldquoSynthesis and antihepatotoxic and antiproliferative activitiesof di- and tri-O-caffeoylquinic acid derivativesrdquo Journal ofTraditional Chinese Medicine vol 18 no 5 pp 183ndash190 2015

[39] Y-J Chen M-S Shiao M-L Hsu T-H Tsai and S-Y WangldquoEffect of caffeic acid phenethyl ester an antioxidant frompropolis on inducing apoptosis in human leukemic HL-60cellsrdquo Journal of Agricultural and Food Chemistry vol 49 no11 pp 5615ndash5619 2001

[40] K Zhu M L Cordeiro J Atienza W Edward Robinson Jrand S A Chow ldquoIrreversible inhibition of human immunode-ficiency virus type 1 integrase by dicaffeoylquinic acidsrdquo Journalof Virology vol 73 no 4 pp 3309ndash3316 1999

[41] B McDougall P J King B W Wu Z Hostomsky M GReinecke and W E Robinson Jr ldquoDicaffeoylquinic and dicaf-feoyltartaric acids are selective inhibitors of human immun-odeficiency virus type 1 integraserdquo Antimicrobial Agents andChemotherapy vol 42 no 1 pp 140ndash146 1998

[42] K Iwai N Kishimoto Y Kakino K Mochida and T FujitaldquoIn vitro antioxidative effects and tyrosinase inhibitory activitiesof seven hydroxycinnamoyl derivatives in green coffee beansrdquoJournal of Agricultural and Food Chemistry vol 52 no 15 pp4893ndash4898 2004

[43] N R ImH S Kim J HHa G Y Noh and S N Park ldquoAntioxi-dant and tyrosinase inhibitory activities of dicaffeoylquinic acidderivatives isolated from Gnaphalium affine D Donrdquo AppliedChemistry for Engineering vol 26 no 4 pp 470ndash476 2015

[44] M-H Jeong K-M Yang J-K Kim et al ldquoInhibitory effects ofAsterina pectinifera extracts on melanin biosynthesis throughtyrosinase activityrdquo International Journal ofMolecularMedicinevol 31 no 1 pp 205ndash212 2013

[45] S Colanesi K L Taylor N D Temperley et al ldquoSmallmolecule screening identifies targetable zebrafish pigmentationpathwaysrdquo Pigment Cell and Melanoma Research vol 25 no 2pp 131ndash143 2012

[46] Y Kotobuki A Tanemura L Yang et al ldquoDysregulation ofmelanocyte function by Th17-related cytokines significance ofTh17 cell infiltration in autoimmune vitiligo vulgarisrdquo PigmentCell amp Melanoma Research vol 25 no 2 pp 219ndash230 2012


[47] MAWikswo andG Szabo ldquoEffects of cytochalasin B onmam-malian melanocytes and keratinocytesrdquo Journal of InvestigativeDermatology vol 59 no 2 pp 163ndash169 1972

[48] B-L Ma and Y-M Ma ldquoPharmacokinetic properties potentialherb-drug interactions and acute toxicity of oral Rhizomacoptidis alkaloidsrdquo Expert Opinion on Drug Metabolism andToxicology vol 9 no 1 pp 51ndash61 2013

[49] P K Chan C C Lin and S H Cheng ldquoNoninvasive techniquefor measurement of heartbeat regularity in zebrafish (Daniorerio) embryosrdquo BMC Biotechnology vol 9 article 11 2009

[50] D Raldua and B Pina ldquoIn vivo zebrafish assays for analyzingdrug toxicityrdquo Expert Opinion on Drug Metabolism and Toxicol-ogy vol 10 no 5 pp 685ndash697 2014

[51] P Gut B Baeza-Raja O Andersson et al ldquoWhole-organismscreening for gluconeogenesis identifies activators of fastingmetabolismrdquo Nature Chemical Biology vol 9 no 2 pp 97ndash1042013

[52] D-W Jung W-H Kim S Seo et al ldquoChemical targeting ofGAPDH moonlighting function in cancer cells reveals its rolein tubulin regulationrdquo Chemistry and Biology vol 21 no 11 pp1533ndash1545 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


traditionally used as a herbal medicine in Korea and Chinasince ancient times [8] Extractspreparations from this plantexhibit various pharmacological activities such as antiviralinfection [9] antioxidant effects [10] hepatoprotective prop-erties [11] and anti-inflammatory effects [11 12] Numerousactive compounds have been extracted from A capillarissuch as phenolic compounds flavonoids flavonoid glyco-sides and coumarins [11 13]We isolated an antipigmentationcompound from the methanol extract of A capillaris whichwas identified as 45-119874-dicaffeoylquinic acid (45-diCQA)45-diCQAwas shown to be a pigmentation inhibitor in bothmelanocytes and the zebrafish vertebratemodel Significantlyno toxicity was observed in our analysis indicating that 45-diCQA is an attractive candidate for further development asa pharmaceutical or cosmetic depigmenting agent

2 Material and Methods

21 Chemicals PTU NaOH DMSO (dimethyl sulfoxide)L-DOPA (-34-dihydroxyphenylalanine) CellLytic buffermushroom tyrosinase MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide) and tricaine methanesul-fonate solution were purchased from Sigma (St Louis MOUSA) L-tyrosine was purchased from Duchefa Biochemie(Haarlem Netherland) All test compounds were dissolved inDMSO and protected from light at minus20∘C until use HPLCgrade solvents acetonitrile and methanol were obtainedfromMerck (Darmstadt Germany)

22 Plant Material The leaves and stems of Artemisia cap-illaris Thunberg were provided by Professor Soon-Ho YimDongshin University Naju Republic of Korea

23 Cell Culture Murine melanoma B16-F10 cells were ob-tained from the American Type Culture Collection (ATCCManassas VA) and cultured in DMEM supplemented with10 FBS 1 penicillin-streptomycin mixture (Gibco USA)Cultured cells were maintained in a 37∘C humidified incuba-tor with 5 CO

2

24 Determination of Melanin Content in B16-F10 Melan-ocytes Melanocytes were rinsed with phosphate bufferedsaline (PBS) and lysed with CellLytic buffer at 4∘C Cellextracts were spun at 13000 rpm for 10min at 4∘C Theremaining pellet was assayed for melanin by rinsing twicewith ethanol ether (1 1) and dissolving in 200120583L of 1NNaOH in 10 DMSO at 80∘C A 100 120583L aliquot of theresulting solution was then measured for absorbance at400 nm using microplate reader (VersaMax MolecularDevices Corporation California USA) [14]

25 Determination of In Vitro Tyrosinase Activity Tyrosinaseactivity was determined as described previously [15] BrieflyB16-F10 melanocytes were seeded in a 6-well plate at adensity of 2 times 105 cellswell The melanocytes were treatedwith compound for 48 hr Melanocytes were lysed using lysisbuffer and centrifuged at 13000 rpm for 10min 100 120583L ofeach lysate containing an equal amount of protein (250120583g)was placed into a 96-well plate and 100 120583L of 5mM L-DOPA

was added to each well After incubation at 37∘C for 60mindopachrome formation was measured at 475 nm using amicroplate reader

26 HPLC-Based Activity Profiling of Artemisia capillarisExtract The dried powder of the leaves and stems fromArtemisia capillaris was extracted with 100 MeOH andconcentrated in vacuo to yield a MeOH extract (6 g) HPLCwas performed on an Agilent HP1100 series comprised of adegasser a binary mixing pump a column oven and a DADdetector using YMC-PAC Pro C18 (10mm 250mm 5120583m)columns in conjunction with a gradient system of MeCNand H

2O containing 01 HCOOH For activity profiling

a portion (6 g) of the MeOH extract was fractionated bysemipreparative HPLC (Agilent 1100 Series USA) usingthe gradient eluent system with acetonitrile (MeCN) andwater containing 01 formic acid that is 20 MeCN to60 MeCN for 50min The mobile phase was delivered atthe flow rate of 60mLmin and detection of eluate wascarried out at 280 nm A total of 23 fractions were collectedconcentrated and their biological activities were evaluatedFraction ACMF09 was selected on the basis of its inhibitionof melanogenesis to obtain potentially bioactive compoundsThe ACMF09 fraction of MeOH extract of A capillaris wasseparated on a RP-18 column with a gradient of H

2O-MeOH

started at 60 40 (v v) and was kept constant for 50minThegradient system was then decreased to 0 100 and was keptconstant for 20min to yield a purified compound (72mg)

27 45-O-Dicaffeoylquinic Acid 1H-NMR (in CD3OD

600MHz) 120575 759 (1H d 119869 = 159Hz H-71015840 or H-710158401015840) 751 (1Hd 119869 = 159Hz H-71015840 or H-710158401015840) 702 (1H d 119869 = 18Hz H-21015840 orH-210158401015840) 700 (1H d 119869 = 18Hz H-21015840 or H-210158401015840) 692 (1H dd119869 = 81 18Hz H-61015840 or H-610158401015840) 690 (1H dd 119869 = 81 18HzH-61015840 or H-610158401015840) 675 (1H d 119869 = 81Hz H-51015840 or H-510158401015840) 674(1H d 119869 = 81Hz H-51015840 or H-510158401015840) 628 (1H d 119869 = 159Hz H-81015840 or H-810158401015840) 619 (1H d 119869 = 159Hz H-81015840 or H-810158401015840) 562 (1Hbr s H-5) 511 (1H br s H-4) 437 (1H br s H-3) 240 (2HH-6) 199 (2H m H-2) 13C-NMR (in CD

3OD 125MHz) 120575

1687 (C-91015840 or 910158401015840) 1684 (C-91015840 or 910158401015840) 1498 (C-41015840 or 410158401015840) 1478(C-71015840 or 710158401015840) 1477 (C-71015840 or 710158401015840) 1469 (C-31015840 or 310158401015840) 1468 (C-31015840or 310158401015840) 1278 (C-11015840 or 110158401015840) 1277 (C-11015840 or 110158401015840) 1233 (C-61015840 or 610158401015840)1166 (C-51015840 or 510158401015840) 1153 (C-21015840 or 210158401015840) 1152 (C-21015840 or 210158401015840) 1148(C-81015840 or 810158401015840) 1177 (C-81015840 or 810158401015840) 758 (C-1) 757 (C-2) 698(C-3) 686 (C-5) 397 (C-6) 385 (C-14) ESI-MS mz 5151[MndashH]minus (C

25H24O12)

28MushroomTyrosinase Assay Theeffect of the samples onmushroom tyrosinase activity was investigated according tothe method of Zhang et al [16] with minor modifications Inbrief mushroom tyrosinase enzyme was dissolved in 50mMpotassium phosphate buffer (pH 65) at a concentration of500 unitsmL 550 120583L of 50mM potassium phosphate and50 120583L of tyrosinase solution were mixed with an appropriatevolume of test samples in a microfuge tube and incubated for5min at room temperature 100 120583L of 15mM L-tyrosine wasadded to the solution and loaded into a 96-well plate Theamount of dopachrome formed in the reaction mixture was














3 Results




45-diCQA (50120583M)ACMF09 (50120583gmL)

50120583gmL)AZ (5mM)

(a)


(b)

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120

Con

trol


50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

lowastlowastlowast

lowast

(c)

Tyro

sinas

e act

ivity

( o

f con

trol)

020406080

100120

Con

trol


50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)


lowast

lowast

(d)



HOOC

OH OH OH

OH

OH

OH

OO

OO

12 3

45

6

7998400

7998400

1998400

1998400

3998400

3998400

4998400

4998400

8998400

8998400

9998400

9998400

(a)

70057

82738

83681

60

50

40

30

20

10

0

0 10 20 30 40 50 60 70 80

(min)

Nor

m

(b)









ACMF09

Con

trol

45-diCQA

50120583

M

25120583

m

120583M

125

625120583

M

50120583

gm

L

25120583

gm

L

125120583

gm

L

625120583

gm

L

AZ

(5m

M)0

20406080

100120

Tyro

sinas

e act

ivity

in a

cell

free

syste

m (

of c

ontro

l)

lowast


lowast



mRN

A ex

pres

sion

( o

f con

trol)

Control

0

20

40

60

80

100

120


AZ (5mM)45-diCQA (25120583M)










45-diCQA (25120583M)ACMF09 (25120583gmL)

PTU (75120583M) (25120583gmL)

(a)

A capillaris ACMF09

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120140

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowast lowast

(b)

020406080

100120140

Tyro

sinas

e act

ivity

( o

f con

trol)

A capillaris ACMF09

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowastlowast

lowast




4 Discussion




Con

trol

Mela

nocy

te ce

ll nu

mbe

r

20253035404550556065

45

-diC

QA

(25120583

M)

45

-diC

QA

(125120583

M)

45

-diC

QA

(625120583

M)

PTU

(75120583

M)

(a)

Spot

area

( o

f con

trol)

Con

trol

PTU

(75120583

M)

45-diCQA

25120583

M

120583M

125

625120583

M

lowast

lowast

lowast

lowast

0

20

40

60

80

100

120

140

(b)


Con

trol


Cel

l via

bilit

y (

of c

ontro

l)

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

020406080

100120













Competing Interests




Acknowledgments


References




































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results




45-diCQA (50120583M)ACMF09 (50120583gmL)

50120583gmL)AZ (5mM)

(a)


(b)

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120

Con

trol


50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

lowastlowastlowast

lowast

(c)

Tyro

sinas

e act

ivity

( o

f con

trol)

020406080

100120

Con

trol


50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)


lowast

lowast

(d)



HOOC

OH OH OH

OH

OH

OH

OO

OO

12 3

45

6

7998400

7998400

1998400

1998400

3998400

3998400

4998400

4998400

8998400

8998400

9998400

9998400

(a)

70057

82738

83681

60

50

40

30

20

10

0

0 10 20 30 40 50 60 70 80

(min)

Nor

m

(b)









ACMF09

Con

trol

45-diCQA

50120583

M

25120583

m

120583M

125

625120583

M

50120583

gm

L

25120583

gm

L

125120583

gm

L

625120583

gm

L

AZ

(5m

M)0

20406080

100120

Tyro

sinas

e act

ivity

in a

cell

free

syste

m (

of c

ontro

l)

lowast


lowast



mRN

A ex

pres

sion

( o

f con

trol)

Control

0

20

40

60

80

100

120


AZ (5mM)45-diCQA (25120583M)










45-diCQA (25120583M)ACMF09 (25120583gmL)

PTU (75120583M) (25120583gmL)

(a)

A capillaris ACMF09

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120140

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowast lowast

(b)

020406080

100120140

Tyro

sinas

e act

ivity

( o

f con

trol)

A capillaris ACMF09

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowastlowast

lowast




4 Discussion




Con

trol

Mela

nocy

te ce

ll nu

mbe

r

20253035404550556065

45

-diC

QA

(25120583

M)

45

-diC

QA

(125120583

M)

45

-diC

QA

(625120583

M)

PTU

(75120583

M)

(a)

Spot

area

( o

f con

trol)

Con

trol

PTU

(75120583

M)

45-diCQA

25120583

M

120583M

125

625120583

M

lowast

lowast

lowast

lowast

0

20

40

60

80

100

120

140

(b)


Con

trol


Cel

l via

bilit

y (

of c

ontro

l)

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

020406080

100120













Competing Interests




Acknowledgments


References




































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















45-diCQA (50120583M)ACMF09 (50120583gmL)

50120583gmL)AZ (5mM)

(a)


(b)

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120

Con

trol


50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

lowastlowastlowast

lowast

(c)

Tyro

sinas

e act

ivity

( o

f con

trol)

020406080

100120

Con

trol


50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)


lowast

lowast

(d)



HOOC

OH OH OH

OH

OH

OH

OO

OO

12 3

45

6

7998400

7998400

1998400

1998400

3998400

3998400

4998400

4998400

8998400

8998400

9998400

9998400

(a)

70057

82738

83681

60

50

40

30

20

10

0

0 10 20 30 40 50 60 70 80

(min)

Nor

m

(b)









ACMF09

Con

trol

45-diCQA

50120583

M

25120583

m

120583M

125

625120583

M

50120583

gm

L

25120583

gm

L

125120583

gm

L

625120583

gm

L

AZ

(5m

M)0

20406080

100120

Tyro

sinas

e act

ivity

in a

cell

free

syste

m (

of c

ontro

l)

lowast


lowast



mRN

A ex

pres

sion

( o

f con

trol)

Control

0

20

40

60

80

100

120


AZ (5mM)45-diCQA (25120583M)










45-diCQA (25120583M)ACMF09 (25120583gmL)

PTU (75120583M) (25120583gmL)

(a)

A capillaris ACMF09

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120140

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowast lowast

(b)

020406080

100120140

Tyro

sinas

e act

ivity

( o

f con

trol)

A capillaris ACMF09

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowastlowast

lowast




4 Discussion




Con

trol

Mela

nocy

te ce

ll nu

mbe

r

20253035404550556065

45

-diC

QA

(25120583

M)

45

-diC

QA

(125120583

M)

45

-diC

QA

(625120583

M)

PTU

(75120583

M)

(a)

Spot

area

( o

f con

trol)

Con

trol

PTU

(75120583

M)

45-diCQA

25120583

M

120583M

125

625120583

M

lowast

lowast

lowast

lowast

0

20

40

60

80

100

120

140

(b)


Con

trol


Cel

l via

bilit

y (

of c

ontro

l)

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

020406080

100120













Competing Interests




Acknowledgments


References




































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HOOC

OH OH OH

OH

OH

OH

OO

OO

12 3

45

6

7998400

7998400

1998400

1998400

3998400

3998400

4998400

4998400

8998400

8998400

9998400

9998400

(a)

70057

82738

83681

60

50

40

30

20

10

0

0 10 20 30 40 50 60 70 80

(min)

Nor

m

(b)









ACMF09

Con

trol

45-diCQA

50120583

M

25120583

m

120583M

125

625120583

M

50120583

gm

L

25120583

gm

L

125120583

gm

L

625120583

gm

L

AZ

(5m

M)0

20406080

100120

Tyro

sinas

e act

ivity

in a

cell

free

syste

m (

of c

ontro

l)

lowast


lowast



mRN

A ex

pres

sion

( o

f con

trol)

Control

0

20

40

60

80

100

120


AZ (5mM)45-diCQA (25120583M)










45-diCQA (25120583M)ACMF09 (25120583gmL)

PTU (75120583M) (25120583gmL)

(a)

A capillaris ACMF09

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120140

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowast lowast

(b)

020406080

100120140

Tyro

sinas

e act

ivity

( o

f con

trol)

A capillaris ACMF09

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowastlowast

lowast




4 Discussion




Con

trol

Mela

nocy

te ce

ll nu

mbe

r

20253035404550556065

45

-diC

QA

(25120583

M)

45

-diC

QA

(125120583

M)

45

-diC

QA

(625120583

M)

PTU

(75120583

M)

(a)

Spot

area

( o

f con

trol)

Con

trol

PTU

(75120583

M)

45-diCQA

25120583

M

120583M

125

625120583

M

lowast

lowast

lowast

lowast

0

20

40

60

80

100

120

140

(b)


Con

trol


Cel

l via

bilit

y (

of c

ontro

l)

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

020406080

100120













Competing Interests




Acknowledgments


References




































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ACMF09

Con

trol

45-diCQA

50120583

M

25120583

m

120583M

125

625120583

M

50120583

gm

L

25120583

gm

L

125120583

gm

L

625120583

gm

L

AZ

(5m

M)0

20406080

100120

Tyro

sinas

e act

ivity

in a

cell

free

syste

m (

of c

ontro

l)

lowast


lowast



mRN

A ex

pres

sion

( o

f con

trol)

Control

0

20

40

60

80

100

120


AZ (5mM)45-diCQA (25120583M)










45-diCQA (25120583M)ACMF09 (25120583gmL)

PTU (75120583M) (25120583gmL)

(a)

A capillaris ACMF09

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120140

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowast lowast

(b)

020406080

100120140

Tyro

sinas

e act

ivity

( o

f con

trol)

A capillaris ACMF09

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowastlowast

lowast




4 Discussion




Con

trol

Mela

nocy

te ce

ll nu

mbe

r

20253035404550556065

45

-diC

QA

(25120583

M)

45

-diC

QA

(125120583

M)

45

-diC

QA

(625120583

M)

PTU

(75120583

M)

(a)

Spot

area

( o

f con

trol)

Con

trol

PTU

(75120583

M)

45-diCQA

25120583

M

120583M

125

625120583

M

lowast

lowast

lowast

lowast

0

20

40

60

80

100

120

140

(b)


Con

trol


Cel

l via

bilit

y (

of c

ontro

l)

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

020406080

100120













Competing Interests




Acknowledgments


References




































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















45-diCQA (25120583M)ACMF09 (25120583gmL)

PTU (75120583M) (25120583gmL)

(a)

A capillaris ACMF09

Mela

nin

cont

ent (

o

f con

trol)

020406080

100120140

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowast lowast

(b)

020406080

100120140

Tyro

sinas

e act

ivity

( o

f con

trol)

A capillaris ACMF09

Con

trol

45-diCQA

25120583

M

120583M

125

625120583

M

50120583

gm

L

75120583

gm

L

25120583

gm

L

25120583

gm

L

PTU

(75120583

M)

lowast

lowast

lowast

lowast

lowastlowast

lowast




4 Discussion




Con

trol

Mela

nocy

te ce

ll nu

mbe

r

20253035404550556065

45

-diC

QA

(25120583

M)

45

-diC

QA

(125120583

M)

45

-diC

QA

(625120583

M)

PTU

(75120583

M)

(a)

Spot

area

( o

f con

trol)

Con

trol

PTU

(75120583

M)

45-diCQA

25120583

M

120583M

125

625120583

M

lowast

lowast

lowast

lowast

0

20

40

60

80

100

120

140

(b)


Con

trol


Cel

l via

bilit

y (

of c

ontro

l)

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

020406080

100120













Competing Interests




Acknowledgments


References




































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

Mela

nocy

te ce

ll nu

mbe

r

20253035404550556065

45

-diC

QA

(25120583

M)

45

-diC

QA

(125120583

M)

45

-diC

QA

(625120583

M)

PTU

(75120583

M)

(a)

Spot

area

( o

f con

trol)

Con

trol

PTU

(75120583

M)

45-diCQA

25120583

M

120583M

125

625120583

M

lowast

lowast

lowast

lowast

0

20

40

60

80

100

120

140

(b)


Con

trol


Cel

l via

bilit

y (

of c

ontro

l)

50120583

M

25120583

M

50120583

gm

L

25120583

gm

L

50120583

gm

L

25120583

gm

L

AZ

(5m

M)

020406080

100120













Competing Interests




Acknowledgments


References




































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Competing Interests




Acknowledgments


References




































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































2




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















O Artemisia capillaris Thunberg and Its In Vivo

Documents

Transcript of O Artemisia capillaris Thunberg and Its In Vivo