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j. Cosmet. ci., 58, 35-44 (January/February 007)

High-performance iquid chromatographic etermination farbutin n skin-whitening reams and medicinalplant extracts

WISANU THONGCHAI, BOONSOM LIAWRUANGRATH,

and SAISUNEE LIAWRUANGRATH, Department fPharmaceutical ciences, aculty f Pharmacy W. T., B.L.), andDepartment f Chemistry, aculty f Science S.L.), ChiangUniversity, hiang Mai, 50200, Thailand.

Acceptedor publication ovember , 2006.

Synopsis

A high-performance iquid chromatographic ethod was developed or quantitative nalysis f arbutin. The

arbutin was separated n an ODS Hypersil %8 column with a mobile phase of water:methanol:0.1hydrochloric cid 89:10:1, v/v/v). The level of arbutin was measured y means f UV detection t 222 nm.The optimum conditions or arbutin quantitative nalysis were investigated. he calibration urve wasfound o be linear up to 1,000 tg/ml of arbutin oncentration, nd he working alibration urve orarbutin determination ver he range .5-30.0 lxg/ml of arbutin r 2 = 0.9999) was established. herelative standard eviations or intraday and interday were found o be 0.98% and 1.15%, respectively.detection imit (3cr) nd quantitation imit (10cr) f 0.02 pg/ml- and 0.2 12g/ml, respectively, nda meanpercentage ecovery f the spiked rbutin of 99.88 -+ 1.12% were obtained. he proposed ethod has beenapplied o the determination f arbutin n commercial kin-whitening reams Arbuwhite cream, SuperWhitening cream, nd Shiseido cream) ith average ontents f 7.60, 5.30, and 57.90 mg/g-, respec-tively. It was also applied o the determination f arbutin in medicinal plant extracts rom Betu/a a/noidesBuch. Ham., Clerodendrum etasites . Moore, Curculigo atifo/ia Dryand. Var. latifolia, and HeJperethusa

crenulataRoxb.) oem, evels f whichwere ound o be 3.50, 1.50, 1.10,and 0.12 ng/g , respectively noarticle eported n the literature bout arbutin analysis). he proposed PLC method s rapid, simple, andselective or routine analysis.

INTRODUCTION

Skin-whitening roducts ave become ncreasingly n demand n the past ew years. hemain purpose or skin-lightening products s to lighten the skin as well as to even outskin tone or to treat pigmentation isorders uch as reckles, melasma, regnancy marks,and age spots 1). The most successful ecent and natural skin-whitening agents are

arbutin, vitamin C, kojic acid, licorice extract, burnet root extract, scutellaria xtract,

Address ll correspondence o Boonsom iawruangrath.

35

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36 JOURNAL OF COSMETIC SCIENCE

and mulberry. These agents re all tyrosinase nhibitors, which nactivate yrosinase theenzyme esponsible or skin pigmentation) by chelating with its vital copper on andsuppressing automerization rom dopachrome o DHICA. Normal skin color s formed

by melanin, a natural pigment that also determines air and eye color. n the skin, theenzyme yrosinase iochemically onverts he amino acid tyrosine nto melanin. Hyper-pigmentation ccurs hen oo much melanin s produced nd forms deposits n the skin.Hyperpigmentation s not a medically harmful condition. However, t is always dvis-able to have new brown spots hecked y a dermatologist o make sure hey are not skincancers 2). Arbutin is a naturally occurring glycoside f hydroquinone Figure 1).

Arbutin is found n the bark and eaves f various lants, usually occurring ogether withmethylarbutin. Naturally occurring rbutin was irst characterized y Kawalier 3), whoobtained t from bearberry eaves. t is also ound n the leaves f blueberry, ranberry,and pear. Synthetic arbutin was first reported by Mannich (4), and later by others.

Commercial arbutin is almost always synthetic n origin. Because f its antibacterialproperties, rbutin s a constituent f the traditional medicine va rsi, and it is widelyused n a variety of formulations 5). The ability of arbutin to inhibit human melaninsynthesis as given rise to its wide use in many cosmetic ormulations 6). Arbutinprotects he skin against damage aused y free adicals. t is a skin-whitening gent hatis very popular n Japan and Asian countries or skin depigmentation. rbutin inhibitsthe formation of melanin pigment by inhibiting yrosinase ctivity 7). Back n the 18thcentury rbutin was irst used n medical reas s an anti-inflammatory nd antibacterialagent. t was used particularly or cystiris, urethritis, and pyelitis. These uses have beenapplied until today, when natural medicine uses only natural ingredients o treat anydisease. t may be used o repress he virulence of bacterial pathogens nd to represscontaminating acteria. t is also used or treating allergic nflammation of the skin. tcan be used to whiten the skin, to prevent liver spots and freckles, o treat sunburnmarks, and to regulate melanogenesis 8).

Arbutin is a very safe skin-whitening agent for external use, which does not havetoxicity, a stimulating effect, an unpleasant dor, or side effects uch as hydroquinone(Figure la). Hydrophilic arbutin can be incorporated n lipophilic media by encapsu-lation. Arbutin has hree main properties: whitening effect, an anti-aging effect, anda UVB/UVC filter (9). Arbutin is determined y many methods: he spectrophotometricmethod (10-12), capillary zone electrophoresis 13), and thin-layer chromatography-

densitometry 14). The proposed HPLC method for determining arbutin in skin-whitening cosmetic products and some medicinal plants is more sensitive, precise, andless ime-consuming han the previous HPLC methods described n the literature (15-

OH

CH20H OH

Hydroquinone Arbutin(a) (b)

Figure 1. Chemical structures f hydroquinone a) and arbutin (b).

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HPLC DETERMINATION OF ARBUTIN 37

18). Furthermore, here is no data in the literature about the isolation and quantitativeanalysis f arbutin n BetMa alnoides uch. Ham., Clerodendrz/m etasites . Moore, Cr-cMigoatifolia Dryand. Var. latifolia, and Hesperethzsarenzdata Roxb.) Roem. Therefore,

it is interesting o investigate he arbutin content n these medicinal plants, becausenaturally occurring rbutin s very safe skin-whitening gent.

EXPERIMENTAL

APPARATUS

HPLC analyses ere carried out with Hewlett Packard Model 1100 liquid chromato-graph with autosampler, hermostatic olumn compartment, nline degasser, nd a

UV-visible detector model G 1313 A. The column used was ODS Hypersil Cs (125mm x 4 mm, 5.0 tm [Chromtech, tockholm, weden]) ith a Lichrosphere 100RP-18 (4 mm x 4 mm, 5.0 tm) guard column. The mobile phase was a mixturecontaining arying ratios of methanol, water, and 0.1 M hydrochloric cid, vacuum-filtered through 0.45-tm nylon membranes Germany) before use. The following in-struments were also used: simultaneous pectrophotometer Spekol 1200), a pH-meter(Model pH 900, Precisa, Switzerland), 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 following standard eagents ere used: rbutin HPLC grade 98% (Sigma, St Louis,MO) and resorcinol 8% (Fluka). The following reagents were used: hydrochloric cid(AR) (Farmitalia Carlo Erba, Italy), glacial acetic acid (AR) (Farmitalia Carlo Erba),acetonitrile HPLC grade) (Lab-Scan Analytical Sciences, reland), ethyl acetate AR)(BDH laboratory upplies, ngland), methanol HPLC grade) Lab-Scan nalytical Sci-ences), nd ether (AR) (Lab-Scan Analytical Sciences). e-ionized distilled water wasused hroughout.

SAMPLES

Skin whitening rodz/cts. rbuwhite cream Nature Best Health Product Co., Ltd.,Thailand), Super Whitening cream Aunyamanee erbs, Thailand), and Shiseidocream Shiseido Co., Ltd., Tokyo, Japan) were used.

Plant material and location. he bark of BetMa alnoides as collected at Bah Bae Village,Mae Dtang District, Chiang Mai Province, hailand, n October 2003 and was den-tified by W. Thongchai 1. Voucher specimens ave been deposited t CMU Herbarium,Chiang Mai University, Chiang Mai, Thailand. The roots of Clerodendrz/m etasites .

Moore were collected rom Chiang Dow, Chaing Mai, Thailand. The tubers of Curculigolatij lia Dryand. Var. latifolia were collected rom Papae, Maetang, Chiang Mai, Thai-land. The trunk of HeJ;Oerethma remdata Roxb.) Roere. was collected rom Mae Sai,Chiang Rai, Thailand.

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Mobile phase low rate. The optimum flow rate of the mobile phase hould provide goodseparation, igh sensitivity, and short analysis ime. In this work, after the optimalwavelength was selected, ptimization of the flow rate was carried out by injecting the

same oncentration fmixed tandard olutions t varying low ates rom .5 ml/min to 1.0 ml/min -.

Recommended P-HPLC procedure. sample and/or standard olution containing rbutinwas eparated na reverse-phase DSHypersil C8 column 125 mm x 4 mm, 5.0 pm)and detected at 222 nm. An aliquot of 100 pl of a series of arbutin standard solutionsand 100 pl of sample extract was njected nto the LC system nd eluted with the mobilephase, water:methanol:0.1 M hydrochloric acid (89:10:1, v/v/v) (flow rate = 1.0 ml/min ). The area f the arbutin eak wasmeasured. rbutin oncentration n the plantextract was determined by reference o the calibration curve prepared under identicalexperimental conditions.

RESULTS AND DISCUSSION

A high-performance iquid chromatographic ethod or the determination f arbutin nskin-whitening reams nd medicinal plant extracts ontaining rbutin was developed.The experimental onditions were nvestigated nd the proposed method was also vali-dated.

OPTIMIZATION OF RP-HPLC CONDITIONS

The optimal conditions of HPLC for determining arbutin were carried out under iso-cratic conditions. Various mobile phase systems with different compositions ere in-vestigated. First, the optimal wavelength or the detection of arbutin and other com-pounds, as mentioned earlier, was nvestigated, nd the UV spectrum of each standardcompound howed he absorption maxima at 222 nm. A wavelength f 222 nm showedthe highest sensitivity or arbutin. Second, mong he mobile phases tudied, a mixtureconsisting f water:methanol:0.1 hydrochloric cid (89:10:1, v/v/v) was used as themobile phase, and it was ound that this mobile phase was the most suitable because tresulted n good retention imes, resolution, nd satisfactory eak profiles Figure 2).

Finally, he optimum low ate was 1.0 ml/min , as t gave good esolution, ighsensitivity, a short analysis ime, etc. In the RP-HPLC analysis, rbutin and resorcinol(internal standard) howed ingle symmetrical peaks retention ime 5.7 min and 10.7min), respectively.

METHOD VALIDATION

The proposed method was validated according o U.S. Pharmacopoeia; SP (19).

Sensitivity. he sensitivity of the assay was determined n terms of the detection imit

(LOD) and the quantitation imit (LOQ). Detection imits and quantitation imits wereestimated or each of the examined compounds. he values were calculated rom thestandard eviation S.D.) of response nd the slope of the curve S) by means of theequations: OD = 3.3 (S.D./S) and LOQ = 10 (S.D./S). Low LOD and LOQ values were

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mAU

2o.i

8O

4O

Resorcinol

Arbutm

2 4 6 8 10 12 14

Figure 2. HPLC chromatogram f the arbutin tandard 5 pg/ml ) and he esorcinol nternal tandard 5pg/ml ).

obtained as shown n Table I, which indicates he good sensitivity of the proposed Cmethod.

Li,earity. To determine inearity, five different concentrations f the arbutin standardwere sed n a working ange f 0.5-30.0 Hg/ml . The inear egression quations ndthe correlation oefficientr2) values or arbutin nd he nternal tandard regiven nTable . The r2 values how ood inearity n the examined oncentration ange.

PRECISION AND ACCURACY

The intraday reproducibility tudy was performed during a period of three days. Theresults obtained showed hat the arbutin peak area variabilities or standard olutionswere within 0.00-0.02% R.S.D. For nterday eproducibility, ive replicate njections fvarious concentrations f arbutin were made within a day. The results obtained showed

that the arbutin peak area variabilities for the standard solutions were within 0.00-0.02% R.S.D. The results are shown n Table II. The R.S.D. values demonstrated oodprecision.

Table I

Linear Regression nalysis n = 3) and Limits of Detection S/N = 3) and Quantitation S/N = 10)

Linearity ange Equation r 2 LOD LOQStandard (Hg/ml ) Y = SX + C Mean +S.D.) (Hg/ml 1) (Hg/ml-)

Arbutin 0.5-30 Y = 132.84 (_+0.36)X 0.9999 5.07 x 10 .3 1.01 x 10 2- 9.32 (+1.23) (-+5.77 x 10 5)

Resorcinol* 0.5-30 Y = 257.03 (_+0.91)X 0.9999 5.04 x 10 1.00 x 10 2+ 7.34 (_+0.04) (+1.49 x 10 s)

* Internal standard.

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mAU

_LFigure 3. HPLC chromatogram f a sample containing arbutin from skin-whitening ream.

Table IV

Comparison f Two Methods or Measuring Arbutin in Samples f Commercial Whitening Cream

Amounts of arbutin found g%)

Cosmetic sample Spectrophotometric(Arbuwhite cream) RP-HPLC method method (20) Calculated -value s

Average

0.75 0.77

0.75 0.780.77 0.80

0.76 0.780.76 0.790.75 0.78

0.77 0.800.76 0.79

1.38

a Each value is the average of 7 determinations.s Calculated -value orp = 005 and six degrees f freedom s 2.44.

and HeJperethusa renu/ata Roxb.) M. Roem. The samples were extracted with water andmethanol. Then the amounts of arbutin in the samples were determined by theRP-HPLC method. The amounts of arbutin found in the aqueous xtract were 3.50,1.50, 1.10,and 0.12 pg/g-, espectively. heamounts farbutin ound n the methanolextract ere .77, 0.0002, and 0.0012 pg/g-, respectively. he method as uccess-fully applied to the determination of arbutin in three commercial skin-whiteningcreams. The amounts of arbutin found in the samples Arbuwhite cream, SuperWhitening cream, nd Shiseido cream) ere 0.76, 0.58, and 5.79 mg/g , respec-tively.

CONCLUSION

The reversed-phase igh-performance iquid chromatographic rocedure as been de-veloped or determining arbutin in commercial kin-whitening creams and some me-

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dicinal plant extracts, espectively. he method was also validated or limit of detection,limit of quantitation, repeatability, eproducibility, and accuracy. he optimum con-ditions and analytical haracteristics or RP-HPLC determination f arbutin exhibitedgood esolution, hort analysis ime, and rather high sensitivity. n the proposed methodfor determining arbutin in skin-whitening reams, he working calibration curves verthe anges f 0.5-30.0 pg/ml were stablished. he method as uccessfully ppliedto the determination f arbutin n three commercial kin-whitening creams. he contentof arbutin ound n the samples Arbuwhite cream, Super Whitening cream, ndShiseido cream) ere .76, 0.58, and 5.79 mg/g , respectively. he method as lsoapplied o the determination f arbutin in some medicinal plant extracts. he amountsof arbutin in Betla alnoides uch. Ham., Clerodendrm etasites . Moore., Crc/igolatifolia Dryand. Var. latifolia, and Hesperethma renlata Roxb.). Roem n the aqueousextracts ere 3.50, 1.50, 1.10, and 0.12 lg/g , respectively. rbutin rom hesemedicinal plants can be used for the production of skin-whitening cosmetics. hebenefits of the proposed method are simplicity, convenience, apidity, sensitivity, goodprecision, nd accuracy. he method s suitable or routine analysis f arbutin n com-mercial cosmetics nd raw plant materials.

ACKNOWLEDGMENTS

The authors express heir sincere hanks o the Graduate School and the Faculty ofPharmacy, Chiang Mai University, for financial and chemical support. SaisuneeLiawruangrath xpresses er sincere hanks o the Postgraduate ducation and ResearchProgram n Chemistry PERCH) for partial support.

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