j. Cosmet.

sci., 58, 35-44 (January/February

2007)

High-performance
liquid chromatographic
determinationof
arbutinin skin-whiteningcreamsand medicinal
plant extracts
WISANU

THONGCHAI,

BOONSOM LIAWRUANGRATH,

and SAISUNEE LIAWRUANGRATH, Department

of
Pharmaceutical
Sciences,
Facultyof Pharmacy
(W.T., B.L.), and
Department
of Chemistry,
FacultyofScience
(S.L.), Chiang
University,
ChiangMai, 50200, Thailand.

Accepted
for publication
November
1, 2006.
Synopsis

A high-performance
liquid chromatographic
methodwasdeveloped
for quantitativeanalysis
of arbutin.The
arbutinwasseparated
on an ODS Hypersil %8 columnwith a mobilephaseof water:methanol:0.1
M
hydrochloric
acid(89:10:1,v/v/v).The levelof arbutinwasmeasured
by meansof UV detectionat 222 nm.
The optimum conditionsfor arbutin quantitativeanalysiswere investigated.The calibrationcurvewas

foundto be linearup to 1,000[tg/ml- of arbutinconcentration,

andtheworkingcalibration
curvefor
arbutindetermination
overthe range0.5-30.0 lxg/ml
- of arbutin(r2 = 0.9999)wasestablished.
The
relativestandarddeviationsfor intradayand interdaywerefoundto be 0.98% and 1.15%, respectively.
A

detection
limit (3cr)andquantitation
limit(10cr)of0.02pg/ml- and0.212g/ml
-, respectively,
anda mean
percentage
recovery
of the spikedarbutinof 99.88 -+1.12% wereobtained.The proposed
methodhasbeen
appliedto the determination
of arbutinin commercial
skin-whitening
creams(Arbuwhite
cream,Super

Whitening
cream,andShiseido
cream)with average
contents
of 7.60, 5.30,and57.90mg/g-, respectively. It wasalsoappliedto the determinationof arbutin in medicinalplant extractsfrom Betu/aa/noides
Buch. Ham., Clerodendrum
petasites
S. Moore, Curculigo
latifo/ia Dryand. Var. latifolia, and HeJperethusa

crenulata
(Roxb.)Roem,levels
ofwhichwerefoundto be3.50,1.50,1.10,and0.12ing/g
- , respectively
(no
articlereportedin the literatureaboutarbutinanalysis).
The proposed
HPLC methodis rapid,simple,and
selectivefor routineanalysis.

INTRODUCTION

Skin-whiteningproductshavebecomeincreasingly
in demandin the pastfew years.The
main purposefor skin-lighteningproductsis to lighten the skin aswell as to evenout
skin toneor to treatpigmentationdisorders
suchasfreckles,melasma,pregnancymarks,
and age spots(1). The most successful
recentand natural skin-whiteningagentsare
arbutin, vitamin C, kojic acid, licoriceextract,burnet root extract,scutellariaextract,

Addressall correspondence
to BoonsomLiawruangrath.
35

36

JOURNAL OF COSMETIC SCIENCE

andmulberry.Theseagentsareall tyrosinase
inhibitors,whichinactivatetyrosinase
(the
enzymeresponsible
for skin pigmentation)by chelatingwith its vital copperion and
suppressing
tautomerizationfrom dopachrome
to DHICA. Normal skin coloris formed
by melanin,a naturalpigment that alsodetermineshair and eyecolor.In the skin,the
enzymetyrosinase
biochemicallyconvertsthe aminoacidtyrosineinto melanin.Hyperpigmentationoccurswhentoomuchmelaninis producedandformsdepositsin the skin.
Hyperpigmentationis not a medicallyharmfulcondition.However,it is alwaysadvisableto havenewbrownspotscheckedby a dermatologist
to makesurethey arenot skin
cancers(2). Arbutin is a naturallyoccurringglycosideof hydroquinone(Figure 1).
Arbutin is foundin the barkandleavesof variousplants,usuallyoccurringtogetherwith
methylarbutin.Naturally occurringarbutinwasfirst characterized
by Kawalier(3), who
obtainedit from bearberryleaves.It is alsofoundin the leavesof blueberry,cranberry,
and pear. Syntheticarbutin was first reportedby Mannich (4), and later by others.
Commercialarbutin is almostalwayssyntheticin origin. Becauseof its antibacterial
properties,arbutinis a constituentof the traditionalmedicineva rsi, and it is widely
usedin a varietyof formulations(5). The ability of arbutin to inhibit humanmelanin
synthesishas given rise to its wide use in many cosmeticformulations(6). Arbutin
protectsthe skinagainstdamagecausedby freeradicals.It is a skin-whiteningagentthat
is verypopularin JapanandAsiancountriesfor skin depigmentation.
Arbutin inhibits
the formationof melaninpigmentby inhibitingtyrosinase
activity(7). Backin the 18th
centuryarbutinwasfirst usedin medicalareasasan anti-inflammatory
andantibacterial
agent.It wasusedparticularlyfor cystiris,urethritis,andpyelitis.Theseuseshavebeen
applied until today, when natural medicineusesonly natural ingredientsto treat any
disease.It may be usedto repressthe virulenceof bacterialpathogensand to repress
contaminatingbacteria.It is alsousedfor treatingallergicinflammationof the skin.It
can be usedto whiten the skin, to prevent liver spotsand freckles,to treat sunburn
marks,and to regulatemelanogenesis
(8).
Arbutin is a very safe skin-whiteningagent for external use, which doesnot have
toxicity,a stimulatingeffect,an unpleasant
odor,or sideeffectssuchas hydroquinone
(Figure la). Hydrophilicarbutin can be incorporatedin lipophilic media by encapsulation. Arbutin hasthreemain properties:a whiteningeffect,an anti-agingeffect,and
a UVB/UVC filter (9). Arbutin is determinedby manymethods:the spectrophotometric
method (10-12), capillary zone electrophoresis
(13), and thin-layer chromatographydensitometry(14). The proposedHPLC method for determiningarbutin in skinwhitening cosmeticproductsand somemedicinalplants is more sensitive,precise,and
lesstime-consumingthan the previousHPLC methodsdescribedin the literature (15OH

OH

Hydroquinone
(a)

CH20
H
OH

Arbutin
(b)

Figure 1. Chemicalstructuresof hydroquinone

(a) and arbutin (b).

HPLC

DETERMINATION

OF ARBUTIN

37

18). Furthermore,there is no data in the literature about the isolationand quantitative

analysisof arbutin in BetMaalnoides

Buch.Ham., Clerodendrz/m
petasites
S. Moore,CrcMigolatifoliaDryand.Var. latifolia,andHesperethzsa
crenzdata
(Roxb.)Roem.Therefore,
it is interestingto investigatethe arbutin content in thesemedicinalplants, because
naturallyoccurringarbutin is very safeskin-whiteningagent.

EXPERIMENTAL
APPARATUS

HPLC analyseswere carriedout with Hewlett PackardModel 1100 liquid chromatograph with autosampler,thermostaticcolumn compartment,online degasser,and a
UV-visibledetectormodelG 1313 A. The columnusedwasODS Hypersil Cs (125

mm x 4 mm, 5.0 tm [Chromtech,

Stockholm,
Sweden])with a Lichrosphere
100
RP-18 (4 mm x 4 mm, 5.0 tm) guard column. The mobile phasewas a mixture
containingvaryingratiosof methanol,water, and 0.1 M hydrochloricacid, vacuumfiltered through 0.45-tm nylon membranes(Germany)beforeuse.The following instrumentswerealsoused:a simultaneous
spectrophotometer
(Spekol1200), a pH-meter
(Model pH 900, Precisa,Switzerland),a water bath and shaker(Model SB-200-10,
Thailand), an ultrasonicator(Model 889, Cole Parmer,USA), and a rotary evaporator
(EYELA N-N series).

REAGENTS

The followingstandardreagents
wereused:arbutinHPLC grade98% (Sigma,St Louis,
MO) and resorcinol98% (Fluka). The following reagentswere used:hydrochloricacid

(AR) (FarmitaliaCarlo Erba, Italy), glacial aceticacid (AR) (FarmitaliaCarlo Erba),

acetonitrile(HPLC grade) (Lab-ScanAnalytical Sciences,Ireland), ethyl acetate(AR)
(BDH laboratorysupplies,England),methanol(HPLC grade)(Lab-Scan
AnalyticalSciences),and ether (AR) (Lab-ScanAnalytical Sciences).
De-ionizeddistilled water was
usedthroughout.

SAMPLES

Skin whitening
prodz/cts.
Arbuwhite cream(Nature Best Health ProductCo., Ltd.,
Thailand),SuperWhitening cream(Aunyamanee
Herbs,Thailand),and Shiseido

cream(ShiseidoCo., Ltd., Tokyo,Japan)were used.

Plant materialand location.The bark of BetMaalnoideswas collectedat Bah Bae Village,

Mae Dtang District, ChiangMai Province,Thailand,in October2003 and wasidentified by W. Thongchai1. Voucherspecimens
havebeendepositedat CMU Herbarium,
ChiangMai University,ChiangMai, Thailand. The rootsof Clerodendrz/m
petasites
S.
Moorewerecollectedfrom ChiangDow, ChaingMai, Thailand.The tubersof Curculigo
latij$liaDryand.Var. latifoliawerecollectedfrom Papae,Maetang,ChiangMai, Thailand. The trunk of HeJ;Oerethma
cremdata(Roxb.) Roere. was collectedfrom Mae Sai,
Chiang Rai, Thailand.

38

JOURNAL OF COSMETIC SCIENCE

PROCEDURES

Samplepreparation
(skin-whitening
cream).About 0.5 g of each whitening cream was
accuratelyweighed and transferredinto three separate25-ml volumetricflasksand

dissolved
in methanol.
To eachflask50 tg/ml- of resorcinol
wasaddedasaninternal
standard.
The solutionwassonicated
vigorouslyfor 30 min, centrifugedat 4000 rpm for
30 min, and filtered on a Millipore membrane(0.45 tm) to obtain a transparent
solution.The supernatant
liquid wasusedfor chromatographic
analysis.
Extraction
of medicinal
plants.The dried medicinalplantswerepowdered.Then 6 kg of
the powderwasextractedwith two successive
portionsof 5.0 1 of de-ionizedwater and
methanol.They wereshakenin a wrist-actionshakerfor five hoursandfiltered.Then the
solventof the filtrate couldbe removedeither by usinga spray-driedtechnique(tem-

perature
100Candflowrate1.0 ml/min-) to givea brownpowder,
or by usinga
rotatoryevaporatorto give a dark browncruderesidue.

Preparation
ofstandard
soluions.
A 1,000tag/ml
- stocksolution
ofarbutinstandard
was
preparedin methanol.A seriesof eachstandardsolutioncontaining0.5, 1.0, 3.0, 5.0,

10.0,and30.0lag/ml
- wasprepared
fromthestockstandard
solution.
Preparation
of sample
soluions.
Three setsof medicinalcrudeextracts(5 g) and cosmetic
samples(0.5 g) of eachset were extractedunderrefluxwith 100 ml of 75% methanol
for 30 min andfiltered.The flitrate wasevaporated
to about12 ml andtransferred
into
a 250-ml separatorfollowedby addition of 50 ml of water. The mixture was then
extractedwith ether(2 x 30 ml). The combinedaqueouslayerwasextractedwith ethyl
acetate(3 x 50 ml). The combinedethyl acetateextractwasthen evaporated
to dryness
and dissolved in 10 ml of methanol.

Preliminaryinvestigation.
A preliminary investigationwas carriedout to separatesome
chemicalconstituentsby TLC. The crude extract was extractedwith 75% methanol
underrefluxfor 30 min andthen filtered.The flitrate wasevaporated
to about12 ml and
transferredto a 250-ml separatingfunnel togetherwith 50 ml of water.This solution
wasextractedthree times with 50 ml of ethyl acetate,and the combinedethyl acetate
extractswere evaporatedto drynessand the residuesdissolvedin 10% methanol.The
samplesolutionandthe standardsolutionswereseparated
on a silicagel GF254(20 x 20
cm) glassplate, using ethyl acetate:methanol
(9:1) as a developingsolvent.The crude
extractgavefive well-definedspots.The Rf valueof eachspotwasexactlythe sameas
that obtainedfrom eachspotof standard.

Optimization
of experimental
conditions
for RP-HPLC. RP-HPLC was performedunder
isocraticconditions.All experimentalconditionswereoptimizedby meansof a univariate method as follows:

Analyticalwavelength.Optimum absorbance
of eachstandardsolutionwasdetermined

byinjection
ofthesame
amount
ofmixedstandard
solutions
(5.0lag/ml
-) at different
wavelengths
from 200 nm to 400 nm. The mobilephasewasa mixtureconsisting
of

water:methanol
(80:20v/v)witha flowrateof 1.0ml/min-.Astheoptimum
to obtain
the bestsensitivity,maxwaschosen.
Mobilephase.Varioussolventsystems
weretestedasthe mobilephasefor the separation
of arbutinin the samples,
e.g.,water:acetonitrile:0.1
M hydrochloric
acid(94:5:1,v/v/v),
water:methanol:0.1M hydrochloricacid (89:10:1, v/v/v), and methanol:100mM phosphatebuffer,pH 2.1 (10:90 v/v).

HPLC DETERMINATION

OF ARBUTIN

39

Mobile phaseflow rate.The optimum flow rate of the mobilephaseshouldprovidegood

separation,high sensitivity,and short analysistime. In this work, after the optimal
wavelengthwasselected,optimizationof the flow rate wascarriedout by injecting the

same
concentration
ofmixedstandard
solutions
atvarying
flowratesfrom0.5ml/min
to 1.0 ml/min -.

Recommended
RP-HPLC procedure.
A sampleand/orstandardsolutioncontainingarbutin

wasseparated
ona reverse-phase
ODSHypersil
C8column(125 mm x 4 mm, 5.0 pm)
and detectedat 222 nm. An aliquot of 100 pl of a seriesof arbutin standardsolutions
and 100 pl of sampleextractwasinjectedinto the LC systemand elutedwith the mobile
phase,water:methanol:0.1M hydrochloricacid (89:10:1, v/v/v) (flow rate = 1.0 ml/

min ). Theareaof thearbutinpeakwasmeasured.

Arbutinconcentration
in theplant
extractwas determinedby referenceto the calibrationcurvepreparedunder identical
experimentalconditions.

RESULTS

AND

DISCUSSION

A high-performance
liquid chromatographic
methodfor the determinationof arbutinin
skin-whiteningcreamsand medicinalplant extractscontainingarbutin wasdeveloped.
The experimentalconditionswere investigatedand the proposedmethodwasalsovalidated.

OPTIMIZATION

OF RP-HPLC

CONDITIONS

The optimal conditionsof HPLC for determiningarbutin were carriedout under isocratic conditions.Variousmobile phasesystemswith differentcompositions
were investigated.First, the optimal wavelengthfor the detectionof arbutin and other compounds,as mentionedearlier,wasinvestigated,and the UV spectrumof eachstandard
compoundshowedthe absorptionmaximaat 222 nm. A wavelengthof 222 nm showed
the highestsensitivityfor arbutin. Second,amongthe mobile phasesstudied,a mixture
consistingof water:methanol:0.1
M hydrochloricacid (89:10:1, v/v/v) wasusedas the
mobilephase,and it wasfoundthat this mobile phasewasthe most suitablebecauseit
resultedin goodretentiontimes, resolution,and satisfactory
peak profiles(Figure2).

Finally,theoptimumflowratewas1.0 ml/min-, asit gavea goodresolution,

high
sensitivity,a shortanalysistime, etc. In the RP-HPLC analysis,arbutin and resorcinol
(internal standard)showedsinglesymmetricalpeaks(retentiontime 5.7 min and 10.7
min), respectively.

METHOD

VALIDATION

The proposedmethod wasvalidated accordingto U.S. Pharmacopoeia;

USP (19).

Sensitivity.
The sensitivityof the assaywasdeterminedin termsof the detectionlimit
(LOD) and the quantitationlimit (LOQ). Detection limits and quantitationlimits were
estimatedfor eachof the examinedcompounds.The valueswere calculatedfrom the
standarddeviation(S.D.) of response
and the slopeof the curve(S) by meansof the
equations:LOD = 3.3 (S.D./S)and LOQ = 10 (S.D./S).Low LOD and LOQ valueswere

40

JOURNAL OF COSMETIC SCIENCE

mAU

Resorcinol

2o.i
Arbutm

8O

4O

10

12

14

Figure2. HPLCchromatogram
of thearbutinstandard
(5 pg/ml ) andtheresorcinol
internalstandard
(5
pg/ml ).

obtainedas shownin Table I, which indicatesthe goodsensitivityof the proposedLC

method.

Li,earity. To determinelinearity, five different concentrations

of the arbutin standard

wereusedin a workingrangeof0.5-30.0Hg/ml- . Thelinearregression

equations
and
thecorrelation
coefficient
(r2) values
forarbutinandtheinternalstandard
aregivenin
TableI. Ther2 values
showgoodlinearityin theexamined
concentration
range.
PRECISION

AND

ACCURACY

The intradayreproducibilitystudywasperformedduring a period of three days.The

resultsobtainedshowedthat the arbutin peak areavariabilitiesfor standardsolutions
werewithin 0.00-0.02% R.S.D. For interdayreproducibility,five replicateinjectionsof
variousconcentrations
of arbutin weremadewithin a day. The resultsobtainedshowed
that the arbutin peak area variabilities for the standardsolutionswere within 0.000.02% R.S.D. The resultsare shownin Table II. The R.S.D. valuesdemonstrated
good
precision.
Table

LinearRegression
Analysis(n = 3) and Limits of Detection(S/N = 3) and Quantitation(S/N = 10)

Standard

Linearityrange
(Hg/ml
-)

Arbutin

0.5-30

Resorcinol*

0.5-30

* Internal

standard.

Equation
Y = SX + C
Y
Y
+

r2
Mean(+S.D.)

= 132.84 (_+0.36)X
0.9999
9.32 (+1.23)
(-+5.77x 10 5)
= 257.03 (_+0.91)X
0.9999
7.34 (_+0.04)
(+1.49 x 10-s)

LOD
(Hg/ml1)

LOQ
(Hg/ml-)

5.07 x 10 .3

1.01 x 10 2

5.04 x 10

1.00 x 10 2

HPLC DETERMINATION

OF ARBUTIN

Table

41

II

Intraday and Interday PrecisionAnalysisof Arbutin StandardSolutions

Concentration

Within-day variability (n - 5)

Between-dayvariability (n = 5)

(concentration found)

(concentrationfound)

added

(pg/ml)

Mean S.D.(pg/ml)

%R.S.D.

Mean_+S.D.(pg/ml)

%R.S.D.

1.00

0.99

+ 0.02

0.02

1.01 _+0.02

5.00

5.00 + 0.02

0.00

5.00 _+0.02

0.02
0.00

10.00

10.09 + 0.07

0.01

10.06 _+0.11

0.01

15.00

15.02

_+0.08

0.00

15.02

_+0.08

0.00

20.00

19.88 + 0.28

0.01

19.88 _+0.28

0.01

The accuracyof the proposedmethod was determinedby analyzingsampleextract

solutionsspiked with three different concentrationsof standardarbutin solution (3.0,

5.0,and10.0pg/ml-, respectively),
usingtheproposed
LCmethod.
Therecoveries
of
the arbutinstandardsolutionswerealsoanalyzed.The resultsarepresentedin Table III.
The excellentrecoveries
of the standardadditionamountssuggestthat the method is
accurate.

APPLICATION

TO COSMETIC

AND

MEDICINAL

PLANTS

The proposedRP-HPLC methodwasappliedto the determinationof arbutin in whitenlug creamsand medicinal plant extractsafter TLC extraction.The sampleswere
separated
on a silicagel GF254(20 cm x 20 cm) glassplate usingethyl acetate:methanol:water(85:17:13, v/v/v) as mobilephase.After visualization,eachsampleshoweda
blue spotwith an Rf valueof 0.36, whichcorresponded
to that of the standard.Arbutin
in eachsamplewas then determinedby the proposedRP-HPLC method(Figure 3). A
comparativedeterminationof arbutin in the originalskin-whiteningcreamsampleswas
also carried out using the UV spectrophotometric
method. The resultsare shown in
TableIV. It is indicatedthat the amountsof arbutinfoundby both methodsarequite
identical.Excellentcorrelationbetweenthe two methodswasobtained.The performance
of the methodwasassessed
by calculationof the Studentt-test comparedwith the UV
spectrophotometricmethod. It was evident that the t-value for arbutin determination,
comparedto the resultsobtained by the UV spectrophotometric
method, was 1.38,
which did not exceedthe theoreticalvalue (2.44) at the 95% confidencelimit for the six
degreesof freedom.Theseresultsindicatedthat the 95% confidencelimit betweenthe

proposedHPLC methodand the UV spectrophotometric

method for the arbutin assay
doesnot differ significantly.Fourmedicinalplant sampleswerecollected:BetzdaM, oides
Buch. Ham, Clerodendrm
petasites
S. Moore., Czrcligo
latifolia Dryand. Var. latifolia,
Table

III

Accuracyof the ProposedLC Method (n = 7)

Concentration

Standard

added

Concentration

found:

(pg/ml
-)

mean+_S.D.(pg/ml1)

Arbutin

3.00

2.96 _+0.006

a .[....:

5.00

q 2

Arbutin

10.00

10.15 + 0.009

Recovery
(%)

Relative
error(%)

98.71 + 0.22

0.04

/,

101.53 _+0.09

r rv

0.15

42

JOURNAL OF COSMETIC SCIENCE

mAU

_L
Figure 3. HPLC chromatogram
of a samplecontainingarbutinfrom skin-whiteningcream.
Table

IV

Comparisonof Two Methodsfor MeasuringArbutin in Samplesof CommercialWhitening Cream

Amountsof arbutin found(g%)

Cosmeticsample
(Arbuwhite cream)

Average

RP-HPLC method

Spectrophotometric
method (20)

0.75

0.77

0.75

0.78

0.77

0.80

0.76
0.76

0.78
0.79

0.75

0.78

0.77

0.80

0.76

0.79

Calculatedt-values

1.38

a Each value is the averageof 7 determinations.

s Calculated
t-valueforp = 005 andsixdegrees
of freedom
is 2.44.

and HeJperethusa
crenu/ata
(Roxb.) M. Roem.The sampleswereextractedwith water and
methanol. Then the amounts of arbutin in the sampleswere determined by the
RP-HPLC method. The amountsof arbutin found in the aqueousextract were 3.50,

1.50,1.10,and0.12pg/g-,respectively.
Theamounts
ofarbutinfoundin themethanol
extractwere0.77,0.0002,and0.0012pg/g-,respectively.
Themethod
wassuccessfully applied to the determination of arbutin in three commercialskin-whitening

creams.The amountsof arbutin found in the samples(Arbuwhite cream,Super

Whitening
cream,andShiseido
cream)were0.76, 0.58, and5.79 mg/g-, respectively.
CONCLUSION

The reversed-phase
high-performance
liquid chromatographic
procedurehas beendevelopedfor determiningarbutin in commercialskin-whiteningcreamsand someme-

HPLC DETERMINATION

OF ARBUTIN

43

dicinalplant extracts,respectively.
The methodwasalsovalidatedfor limit of detection,
limit of quantitation,repeatability,reproducibility,and accuracy.The optimum conditionsand analyticalcharacteristics
for RP-HPLC determinationof arbutin exhibited
goodresolution,shortanalysistime, andratherhigh sensitivity.In the proposed
method
for determiningarbutin in skin-whiteningcreams,the workingcalibrationcurvesover

theranges
of 0.5-30.0pg/ml- wereestablished.
Themethod
wassuccessfully
applied
to the determinationof arbutinin threecommercialskin-whiteningcreams.The content

of arbutinfoundin the samples

(Arbuwhite
cream,SuperWhitening cream,and

Shiseido
cream)
were0.76,0.58,and5.79mg/g-, respectively.
Themethod
wasalso
appliedto the determinationof arbutin in somemedicinalplant extracts.The amounts
of arbutin in Betla alnoidesBuch. Ham., Clerodendrm
petasites
S. Moore., Crc/igo
latifoliaDryand.Var. latifolia,andHesperethma
crenlata(Roxb.).Roemin the aqueous

extracts
were3.50, 1.50, 1.10,and 0.12 lg/g-, respectively.
Arbutinfromthese
medicinalplants can be used for the productionof skin-whiteningcosmetics.The
benefitsof the proposedmethodare simplicity, convenience,
rapidity, sensitivity,good
precision,and accuracy.
The methodis suitablefor routineanalysisof arbutinin commercial cosmeticsand raw plant materials.

ACKNOWLEDGMENTS

The authorsexpresstheir sincerethanksto the GraduateSchooland the Facultyof

Pharmacy, Chiang Mai University, for financial and chemical support. Saisunee
Liawruangrathexpresses
her sincerethanksto the Postgraduate
Educationand Research
Programin Chemistry(PERCH) for partial support.

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44

JOURNAL OF COSMETIC SCIENCE

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