j. Cosmet.

Sci., 53, 237-240 (July/August2002)

A test for antioxidantactivityin cosmeticformulations

E. PELLE, T. MAMMONE, K. MARENUS, D. DICANIO, and
D. MAES, EsteeLauderResearch
Laboratories,
125 Pindawn Road,
Me/ville, NY l 1747.

Accepted
for publication
March15, 2002.

Synopsis

The aim of this studywas to developa techniqueto assayfor the activity of antioxidantsin a finished
cosmeticproduct.This wasaccomplished
by adaptingthe RandoxAssayfor Total AntioxidantStatuskit
so that diluted samplescould be evaluatedby kinetic as well as end-pointdeterminations.
Using this
technique,we foundthat a finishedproducthad an IC5oof 0.07 gm of productand a relativeantioxidant
activity concentrationof 52.7 nmoles/mg.

INTRODUCTION

Environmentalinsultto humanskinby ultraviolet(UV) radiation,aswell asby cigarette

smokeandair pollution,generates
reactiveoxygenintermediates
that contributeto both
acuteand chronicskin damage(1,2). For example,immediatelyafter overexposure
to
sunlight,an erythemalresponse
is inducedthat is associated
with epidermalinflammatory oxidativereactions.Moreover,in terms of chronicexposure,the involvementof
oxygenfreeradicalshasalsobeenimplicatedin actinicskindamagethat manifestsitself
in elastosis,
collagendisorganization,
andmostnotablyin the appearance
of wrinkles(3).
Due to increasedoutdoor leisure activities, these visible signs of photodamageand
prematureaging havebecomewidespreadin our society.To addressthis problem,the
cosmeticsindustryhasdevotedmuch researchtoward the developmentof variousskin
care products.Although protectivesunscreen
productsthat absorbUV and diffuse
photonicenergyare widely used,cosmeticproductsthat containantioxidants,which
scavengedeleteriousreactive oxygen speciesproduced in skin after environmental
trauma, havealsobecomestandardfor a healthyskin careregimen.

Although analyticaltechniquesare availableto measurethe level of antioxidantsin

cosmeticproducts,in general, they do not provide any information regarding their
potentialactivity.Further,due to the complexnatureof cosmeticformulations,extracting and determiningbiochemicalactivity in a finishedproductcan be a challenging
task. Previously,we evaluatedthe antioxidantpotentialof certaincosmeticingredients
(4) and alsothe antioxidantefficacyof finishedproductson skin (5). In this study, we
237

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JOURNAL OF COSMETIC SCIENCE

now report on a noveltechniqueto measureantioxidantactivity directly in a complete

cosmeticproduct.

MATERIALS
SAMPLE

AND

METHODS

PREPARATION

A typicalcosmeticformulationcontaininga blendof emulsifiers

waspreparedaseither
a controlwith no antioxidantsor asa completeformulawith a mixture of antioxidants.
The following antioxidantswere used in the formulation:2.0% tocopherylacetate
(Hoffman-LaRoche,
Parsippany,NJ), 0.1% butylatedhydroxytoluene(Rhone-Poulenc,
Cranbury,NJ), 1.0% magnesiumascorbylphosphate(Barnet,Englewood,NJ), 0.1%
ubiquinone50 and 0.5% N-acetyl-L-cysteine(Seizer,Carlsbad,CA), 0.1% rosemary
(Robertet,Oakland,CA), and 0.1% tocopherolcysteamine
(Mercier,S. Plainfield,NJ).
ASSAY

The RandoxAssayfor Total AntioxidantStatuskit (Randox,Antrim, UK) wasadapted

for usein cosmeticproductsby diluting the formulationsto be testedto 1% in isopropyl
alcohol.At 1% in isopropylalcohol,the samplesare sufficientlyclarified and the
antioxidantssolubilizedto allow the reactionto proceedwithout interference.
Briefly,
2,2'-azino-di-(3-ethylbenzthiazoline
sulphonate)(ATBS) is reactedwith a peroxidase
andH20 2 to convertATBS into a radicalcation.In this state,ATBS formsa chromogen
that canbemeasured
spectrophotometrically
at 600 nm. In the presence
of antioxidants,
this colorformationis inhibited. Typically, 50-100 pl of the 1% sampleis diluted in
water up to 250 pl. Then, 1.5 ml of chromogensolutionis added,followedby the
addition of 0.3 ml of substratesolution.The absorbance
(A) of the samplesis then
measuredimmediatelyin a BeckmanDU-7500 spectrophotometer
usingthe kinetics/
time program.

CALCULATIONS

Percent
inhibition
wascalculated
as(dAvehicle-dAproduct/dA,ehicle)
x 100andusedto
quantitateanIC5ovalue.Also,a rangeof 15 to 85 nanomoles
of anantioxidant
standard
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid)was usedto determinethe
relativeactivity of a product.

RESULTS

One hundredmicroliters(1 mg) of a 1% cosmeticsampledilution wasassayed,

and a
typicalthree-minutekinetic plot of the data is shownin Figure 1. The samplewithout
antioxidantshad a dA/min of 0.126 whereasthe samplecontainingantioxidantshad a
dA/min of 0.011. Thus, there was an 86.8% decrease
from the controlsample.From
similarkineticplots,the averagevaluethat wouldinhibit the reactionby 50% wasthen
determinedto be 0.7 mg of the sample.Sincethe samplewas a 1% dilution of the

TEST FOR ANTIOXIDANT

Zoom

ZoomOut

Trace

fluLoscale

flnnotate

ACTIVITY

239

Print

8.58088

[Abs]

...........................

:...........................
:

8.88888

: ..........................

: ...........................

sec

8.8888

..........................

188.88

Figure 1. Kineticplot of the increase

in absorbance
at 600 nm overtime. (-I-): Vehicleformulationwas
0.126 dA/min. (-(2)-): Antioxidant formulation was 0.011 dA/min.

product,0.7 mg is multiplied by 100 in orderto determinethat 0.07 gm of the cosmetic

productis equivalentto 50% inhibition in this assay.In this way, relativemeasurements
of effectiveness
can be calculatedand usedfor comparisonto other products.
Basedon regression
analysisfrom the standardcurveand end-pointassayanalysis,the
relativeantioxidantactivityconcentration
that wasdeterminedfor this productwas52.7
nmoles/mg(+ SD 1.7) of material.In contrastthe placebocontainedonly 11.4 nmoles/
mg (+ SD 1.2), although when comparedto a blank, there appearedto be some
backgroundantioxidantactivityby the formulationitself.Thesedataaresummarizedin
Figure 2.

40

20

lO

blank

vehicle

vehicle + antioxidants

Figure 2. Increasein the amountof nmoles/mgof antioxidantactivity in cosmeticformulationsasdetermined by standardcurveand end-pointassaymeasurements.

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JOURNAL OF COSMETIC SCIENCE

DISCUSSION

Antioxidantsin skin careproductshavebeenfoundto be effectiveprotectantsagainst

freeradical-mediated
oxidativedamagein skin.Also,dueto the risein photodamage
in
an aging population,the topical applicationof cosmeticproductsthat containantioxidantshasbecomean importantareaof researchin skin careproducts.Thus, the needto
measurethe activityof antioxidantsin a finishedproductis of criticalimportance.In this
reportwe describea novelandsimpletechniqueto quicklyassess
the relativeantioxidant
potential of whole productformulations.Additionally, it can be utilized for stability
studiesand, sincethe chromogendevelopsin the visibleregion,perhapsevena qualitative resultcanbe obtainedby workersin the field who lack spectrophotometers
but
needto assaythe antioxidantactivity of a product.

REFERENCES

(1) B. A. Gilchrest,SkinandAgingProcesses
(CRC Press,BocaRaton,FL, 1989), pp. 97-116.
(2) A. V. Benedetto,The environmentand skin aging, Clin. Dermatol.,16, 129-139 (1998).
(3) J. Fuchsand L. Packer,Eds.,OxidativeStress
in Dermatology
(MarcelDekker, New York, 1993).
(4) E. Pelle, D. Maes, G.A. Padulo, E-K. Kim, and W. P. Smith, An in vitro model to test relative
antioxidantpotential:Ultraviolet-inducedlipid peroxidationin liposomes,Arch. Blochem.
Biophys.,
283, 234-24O (1990).
(5) E. Pelle,N. Muizzuddin,T. Mammone,K. Marenus,andD. Maes,Protectionagainstendogenous
and
UVB-induced oxidativedamagein stratumcorneumlipids by an antioxidant-containing
cosmetic
formulation, Photodermatol.
Photoimmnol.
Photoreed.,
15, 115-119 (1999).