j. Cosmet.

sci., 50, 221-229 (July/August 1999)

Comparisonof antioxidantactivityof extractfrom seedsof

white pepper(Pipernigrurn,Linn.)to commercial
antioxidantsin 2% hydroquinone
cream
ARANYA

MANOSROI,

MASAHIKO

ABE, and

JIRADEJ MANOSROI, Pharmaceutical-Cosmetic

Raw Materialsand
Natural Products
Research
and Development
Center(PCRNC), Natural
Products
for Thai TraditionalMedicines
Research
Unit, Department
of
Pharmaceutical
Technology,
Facultyof Pharmacy,
ChiangMai
University,
ChiangMai 50200, Thailand(A.M., J.M.); The
Cosmetic
QualityControlSubcommittee,
FDA, Ministryof Health,
Nontaburi,Thailand (A.M.); Facultyof Science
and Technology,
Science
University
of Tokyo,Yamazaki2641, Noda,Chiba278, Japan
(M.A.); and Instituteof Colloidand Interface
Science,
Science
University
of Tokyo,1-3 Kagurazaka,Shinjuku,Tokyo162, Japan (M.A.).

Accepted
for publication
June30, 1999.
Synopsis

Powdereddry fruits of white pepper(Pipernigrum,Linn.) were extractedwith dichloromethane

and fractionarealinto variousportions.The powderobtainedfrom the aqueousweaklyacidicfraction,which gave
a yield of 1.09% w/w of crudepepper,wastestedfor antioxidativeactivityin comparison
with commercial
antioxidants(sodiummetabisulfiteand BHT) at 0.1%, 0.5%, and 1.0% w/w in 2% w/w hydroquinone
cream.The systemswere incubatedin a dark room at 24 + 1.0C for two weeksand at 45 + 0.5C for
threemonths.The physicalstabilityand the percentages
of hydroquinoneremainingafter two weeksand
one, two, and threemonthsweredeterminedby UV spectrophotometry
at 294 nm accordingto official
standardprocedures.
The experimentrevealedthat oxidationdegradationof hydroquinonewasaccelerated
by heat evenwith the existenceof antioxidants.The higher percentages
of remaininghydroquinonewere
observedfor higher antioxidantconcentration
but showedlower physicalstability of the formulation,
especially
in the cases
of 1% BHT and 1% extract.In two monthsat 45 + 0.5C,the extractdemonstrated
the sameactivity asthe other two commercialantioxidantsat all concentrations,
with about 20-30% more
hydroquinoneremainingthan in the controlsystem.The creamscontaining0.1% and 0.5% extractgave
goodphysicalformulationstability,with about66% and72% of hydroquinone
remaining.This suggested
the possibilityof usingan extractfrom white pepperat 0.1% and 0.5% asa water-solubleantioxidantfor
substances
that are oxidation-susceptible.

INTRODUCTION

One of the mostimportantcharacteristics

of many cosmeticproductsis stability.Hydroquinone,a hypopigmentingagent employedpercutaneously
to lighten localized
221

222

JOURNAL OF COSMETIC SCIENCE

areasof hyperpigmented
skinsuchasblemishes,
lentigo,melasma,chloasma,
andfreckles,is knownfor its high oxidativereactivity.It is oneof the chemicals
that aredifficult
to stabilize.It becomesbrown in air due to oxidation.One gram of hydroquinoneis
solublein 17 ml of waterandfreelysolublein alcohol(1,2). In Thailand,hydroquinone
is classifiedin a speciallycontrolledcosmeticcategoryaccordingto the FDA-Cosmetic
Act BE 1992. In antifreckleandhair dyeingcosmeticproducts,its contentis limited by
law to 2% w/w. Most hydroquinoneproductsusedin creams,gels,lotions,and ointmentsare locally manufacturedin Thailand.

Beinglocatedin a tropicalarea,mostThai peoplesufferfromsolareffectson their skin.

Frecklesare one of theseeffects.Hydroquinoneis one of the cheapesthypopigmenting
agentsavailablein the Thai market that mostmiddle and lowerclassThai peoplehave
preferredto usealongwith sunscreening
products.However,with low stabilityandthe
sideeffectsof allergyand irritation by hydroquinone
for manyconsumers,
the Cosmetic
Control Division, FDA, of Thailand has specialconcernsfor theseproducts.
In 1995, the FDA and the ToxicologyDivision of the Departmentof Medical Science,
Ministry of Health, in Thailand reportedthat six out of 20 samplescollectedfrom the
market containedmore hydroquinonethan the allowedamount (3). This was excused
becauseof the high instabilityof hydroquinone,and most manufacturers
had put the
excesshydroquinone
in their productsin orderto keep the constantamountof 2.0%
during storage.

Recently,many researchers
have been involvedin finding meansto preventor delay
deteriorationby oxidativereactionsin cosmeticpreparations.
A varietyof antioxidants
both from naturalsources
and syntheticprocesses
are availablein the market. In Thai
markets,antioxidantsthat are usuallyincorporatedin hydroquinoneformulationsare
sodiummetabisulfite,BHT (butylatedhydroxytoluene),BHA (butylatedhydroxyanisole),ascorbicacid, vitamin E, citric acid, and/or combinationsof thesechemicals.Their

shelf-lifeis about one year. Hydroquinoneitself has been usedas an antioxidantin

combinationwith othersin the concentration
of 0.05-0.1% (4). Variousdisadvantages
of someof theseantioxidantshavecausedconcern.For example,an applicationof BHA
andBHT is nowrestrictedin manycountries,sinceundesirable
effectsof theseadditives
on the enzymes
of the liverandlungscanoccur(5). Occasionally,
the antioxidantability
of vitamin E is lessactive(6). More recently,research
hasfocusedon developingsaferand
more effectiveantioxidantsfrom natural sources,suchas Rosmarinus
officinalisL. (7,8)
and Piper spp.(8-10). Chemicalconstituents
of white pepper(Pipernigrum,Linn.) and
their applicationto foodpreservation
havebeenevaluated(6). However,a studyof the
antioxidativeactivity of the extractfrom white pepperhasneverbeenperformedfor
cosmeticpreparations.
Hydroquinone,which is knownfor its high sensitivityto oxidation, hasbeenchosenas an indicatorfor comparison
of the antioxidativeactivity of
white pepperextractto commercialantioxidantsin the form of 2% w/w hydroquinone
cream.

MATERIALS
EXTRACTION

AND
AND

METHODS

FRACTIONATION

OF PEPPER

SEEDS

An amountof 729.56 g of the powdereddry fruits of white pepper(P. nigrum,Linn.)

ANTIOXIDANT

ACTIVITY

OF P. NIGRUM

223

wassoakedin 600 ml of dichloromethane

(Merck, Germany)for 24 hrs.The mixture was
filtered and the flitrate waswashedwith 25 ml of distilled water (Figure 1) threetimes.
The organiclayer(dichloromethane)
wasextractedwith 25 ml of 2N-HCL solutionthree

times.The organiclayerwasfurtherextracted
with 25 ml of saturated
NaHCO3 solution
threetimes. The resultingorganiclayerwasextractedwith 25 ml of 1N-NaOH solution
three times, and the aqueouslayerwasacidified(pH = 1) with concentratedHCL. The
resultingsolutionwasextractedwith 25 ml of dichloromethane
threetimes.The organic
layerwasdiscarded,and the aqueousweaklyacidicfractionwasfinally evaporatedand
the white powder of the extractobtained.The percentageyield of this fractionwas
determined.

PREPARATION

OF TEST

SAMPLES

Hydroquinonecreamwasfreshlypreparedaccordingto the formula in Table I. Light

mineral oil, isopropylmyristate,and glyceryl monostearate
were addedto the melted
cetostearylalcoholin a water bath at 70C. Separately,propyleneglycol and paraben
solutionwere added to a sodium lauryl sulfate solution in a water bath at 73C. The
formermixture wasaddedto the latter and stirredconstantlyuntil an emulsionformed.
The 2% hydroquinonesolutionin 95% ethyl alcoholwasaddedto the creamat 40C,
and the resultingmixture was stirred while coolingto room temperature.The incorporationof pepperextractor commercialantioxidantsto the fomulationduring preparationdependson their solubilityproperties.The extract(0.1%, 0.5%, and 1.0% w/w)
andsodiummetabisulfite(0.1%, 0.5%, and 1.0% w/w) werein the waterphase,whereas
BHT (0.1%, 0.5%, and 1.0% w/w) was in the oil phase.The control was 2% hydroquinone cream without the extract or any commercialantioxidants.

ANTIOXIDATIVE

ACTIVITY

STUDY

A 10-g samplewas put in a 20-ml, tightly screw-capped,

test tube. One set of test
sampleswasincubatedat 45 + 0.5Cin an incubator(MemmertIncubator,Germany)
Table

Formulationof 2% w/w HydroquinoneCream

% w/w

Hydroquinone
95% ethyl alcohol
Light mineraloil
Cetostearylalcohol
Isopropylmyristate
Glyceryl monostearate
Sodium lauryl sulfate
Propyleneglycol
Paraben solution 20% w/w

Extract or antioxidants

Functions

Humectant

Merck, Germany
Merck, Germany
Honghuad,Bangkok
Honghuad,Bangkok
Honghuad,Bangkok
Chemikit, Bangkok
Sunnyworld,Bangkok
Vithayasom,Bangkok

Preservative

Kech's, USA

2
8
6
5

Active ingredient
Solventfor hydroquinone

Emollient

10
5
5

Emollient

qs

Emollient

Emollient

stabilizer

Emulsifier

Antioxidant

BHT

Sunnyworld,Bangkok
May & Baker,England

Sodium metabisulfite

Distilled water qs to

Sources

100

Solventfor waterphase

224

JOURNAL OF COSMETIC SCIENCE

Groundpeperseeds

Soaked
indichloroTethane
for
24
hrs.
Extracted
dfiltered

Exacts

Refidue

Washed
with
water
and
extractedDiscard
with2N-HC1
Aqueouslayer
Discarded

Organiclayer

Extracted
with
saturated
NaHCCh
solutio
AqueOus
layer Organi
Ilayer
Discarded Extracted
wi 1N-NaOH

Aqueo
layer Organi
Ilayer

Addified
and
extracd
with
CH2C12
Discarded

Aqueouslayer
Organi
layer

(weakly addic fracUon)

Discarded

Evaporateon water bath

I

Dried powder
Figure 1. Extractionand fractionationof aqueous
weaklyacidicfractionfrom white pepperseeds.

for threemonthsto evaluateformulationstability.Anothersetwaskept in a dark room

at 24 + 1.0Cfor two weeks.Samplesat eachconcentration
of the extractand commercialantioxidantsweredonein triplicate.Physicalstabilitybehaviors,
i.e., changesin
colorand separationof emulsion,were observedopticallyeveryweek.
For the determination
of the averagepercentages
of hydroquinone
remainingaftertwo
weeksin the darkroomandfor formulationstabilitystudiesat 45 + 0.5Cin twoweeks
and one, two, and three months,one gram of the testedsampleswas extractedwith
methanoland the amountof hydroquinone
wasmeasured
by an UV spectrophotometer
(Spectronic301, Milton Roy Company,USA) at 293 nm accordingto the official
standardhydroquinoneassay(11).
RESULTS

AND

DISCUSSION

The white powderwasobtainedwhenthe aqueous

weaklyacidicfractionwasevaporated,

ANTIOXIDANT

ACTIVITY

Table

OF P. NIGR URI

225

II

--

Comparison
of AveragePercentages
(X _+SD) of HydroquinoneRemainingin 2% HydroquinoneCream
ContainingWhite PepperExtractand CommercialAntioxidantsAfter Incubationin a Dark Room at
24 +_ 1.0C and at 45 +_0.5C for 2 Weeks

Systems*
CB
CB
CB
CB
CB
CB
CB
CB
CB
CB

+
+
+
+
+
+
+
+
+
+

HY
HY
HY
HY
HY
HY
HY
HY
HY
HY

+
+
+
+
+
+
+
+
+

Dark roomat 24 +_1.0C

66.76
85.13
86.26
89.63
78.00
88.65
96.60
90.42
91.85
93.19

SM 0.1%
SM 0.5%
SM 1.0%
BHT 0.l%
BHT 0.5%
BHT 1.0%
EXT 0.1%
EXT 0.5%
EXT 1.0%

Dark room at 45 +_0.5C

_+1.38
+_1.89
_+1.65
_+1.73
_+2.76
_+1.35
: 1.44
+_0.47
_+3.75
-+ 2.90

61.33
73.49
79.27
82.11
75.35
87.52
85.53
86.46
84.68
88.70

+_4.22
_+4.50
_+4.92
+_4.67
+_4.51
+_5.00
+_4.13
+_4.17
+_1.88
+_1.67

* CB, creambase;HY, hydroquinone,

SM, sodiummetabisulfite;BHT, butylatedhydroxytoluene;EXT,
white pepper seedextract.
100

7o+

45o.5 c

50-

ao80

10

Figure 2. Comparison
of averagepercentages
of hydroquinone
remainingincubatedat 24 _+1.0Cand45 o
+_0.5C for two weeks.A, CB + HY; B, CB + HY + SM (0.1%); C, CB + HY + SM (0.5%); D, CB + HY
+ SM (1.0%); E, CB + HY + BHT (0.1%); F, CB + HY + BHT (0.5%); G, CB + HY + BHT (1.0%); H,
CB + HY + EXT (0.1%); I, CB + HY + EXT (0.5%); J, CB + HY + EXT (1.0%).

andthe yield was1.09% w/w of the dry white pepperseeds.The powderappearedto be

water-soluble
and a 1% solutiongavea pH of 6.75 + 0.25. It hasbeenpreviously
revealedthat the structureof the major compoundsof this acidicfractionare phenolic
amides,which areN-trans-feruloyltyramine,coumaperine,
N-trans-feruloylpiperidine,

226

JOURNAL OF COSMETIC SCIENCE

Table

--

III

Comparisonof AveragePercentages
(X + SD) of HydroquinoneRemainingin 2% W/W Hydroquinone
CreamContainingWhite PepperExtractandCommercialAntioxidantsKept at 45 _+0.5Cfor
Three Months

Incubationperiods(weeks)
4

Systems*
CB

CB
CB
CB
CB
CB
CB
CB
CB
CB
CB

+
+
+
+
+
+
+
+
+
+

HY
HY
HY
HY
HY
HY
HY
HY
HY
HY

+
+
+
+
+
+
+
+
+

SM 0.1%
SM 0.5%
SM 1.0%
BHT 0.1%
BHT 0.5%
BHT 1.0%
EXT 0.1%
EXT 0.5%
EXT 1.0%

(1 month)

0.00

_+0.00

0.00

61.33
73.49
79.27
82.11
75.35
84.70
85.53
81.86
84.68
88.70

-+4.22
-+4.50
-+4.92
_+4.67
+ 4.51
5.00
4.13
_+4.17
1.88
_+1.67

59.32
65.93
70.85
74.94
65.11
72.19
73.29
74.37
75.97
80.05

_+ 0.00

-+ 2.66
-+0.39
+ 1.45
+ 2.74
2.17
-+4.18
2.73
+ 2.30
_+1.48
_+2.11

(2 months)
0.00

41.21
62.47
65.53
74.14
67.56
73.01
74.04
66.35
72.36
74.96

_+0.00

+ 2.47
_+2.37
-+ 1.48
+ 2.01
_+4.06
+ 2.04
+ 1.94
2.17
+ 2.52
_+3.83

12

(3 months)
0.00

_+0.00

14.55
16.15
23.65
39.22
25.62
30.80
35.05
17.94
20.84
33.12

_+4.52
+ 3.54
_+3.93
_+3.96
_+3.65
+ 4.06
+ 4.27
2.59
+ 3.12
2.93

* CB, creambase;HY, hydroquinone;

SM, sodiummetabisulfite;
BHT, butylatedhydroxytoluene;EXT,
white pepper seedextract.

feruperine,and N-5-(4-hydroxy-3-methoxy
phenyl)-2E-pentenoyl
piperidine(6). All
phenolicamideshad shownsignificantactivity,moreeffectivethan ot-tocopherol
at the
sameconcentration(0.01%). Somewere as highly effectiveas thoseof the synthetic
antioxidantsBHA and BHT at the samelevel (6). Our extractwasusedin this study
insteadof individualpure isolatedphenolicamidessinceit hasbeenour purposeto test
the antioxidativeactivity of the wholegroupof thesecompounds
derivedfrom pepper.
In addition, this extractcan be interpretedasan antioxidantfrom naturalsources.The
antioxidativeactivity of separatephenolicamidesmay not be as effectiveaswhen they
are combinedasa wholein naturalpepperseeds.This studyhasbeendesignedto be as
closeto natureas possible.
Our powderextractshowednoskinirritationby a patchteston the backof rabbits.After
storageat roomtemperature
(25-30C)for two years,thepowdershowedgoodphysical
stability (data not shown).
From physicalobservation
(datanot shown),the colorof all samplesystemswasdarker
startingfrom the first week, with the darkestintensityin creamwithout any antioxidants(CB + HY) and the leastdarknessin the systemsof 1.0% BHT and 1.0% extract.
However,thesetwo systems
showedemulsioninstabilityearlierthan othersystems.
The
1.0% extract cream started to crack from the first month, whereas 1.0% BHT cream

startedat the secondmonth and continued.This may be due to the high concentrations
of both antioxidantscomparedwith the other test systems.BHT is a phenolicantioxidant for fatty acid and vegetableoil. Usually it is usedat a level of 0.01-0.1% in
cosmetics
containingunsaturated
materials(4). The emulsionsystems
with all concentrations (1.0%, 0.5%, and 0.1%) of the extract were crackedat three months. Incompatibility may occurin thesesystemssincethe extractscontainmore than one single
phenolicconstituentwith acidicproperties.Incubationfor a longer period at high
temperature(45 + 0.5C)may accelerate
this incompatibilityreaction.

ANTIOXIDANT

ACTIVITY

OF P. NIGRUM

227

1oo

90

7o

(50-

CB+HY+BHT

0. I ,

CB+HY+BHT

0.5

C'B+HY+BXT

1.0

50'

30-

We

6
e

ks

10

12

Weeks

8c

lOO

10 12
1oo

90

9o

o_8o

7o
'

6O

60

--'-CB+HY
CB+H
--e:CB+HY+EXT0.1

so

CB+HY+SM
0.1%
I
CB+HY+BHT
0.

--a--CB+HY+EXT0.5

_ 4o

--t--CB+HY+EXT1.0

30

30

CB+HY+EXT
0.1

20-

lO
0

2
2

10

10

12

Weeks

Weeks

100

12

3f

lOO

90

90

----CB+HY
370 CB+
-----CB+HY+SM
0560
--CB+HY+BHT0.5
50
o.s t4o

o
o

CB+HY+SM
1.0%
I
CB+HY+BHT
1.0]

3o

CB+HY+BXT
1.0

20-

10-

lO

10

10

12

12
Weeks

Weeks

Figure3. Formulation
stability
studyof2%w/whydroquinone
cream
containing
whitepepper
extract
and
commercialantioxidantsincubatedat 45 _+0.5C for threemonths.3a, systemswith SM at 0.1%, 0.5%,

and1.0%;3b, systems
with BHT at 0.1%,0.5%,and1.0%;3c,systems
with EXT at 0.1%, 0.5% and
1.0%;3d,systems
withSM/BHT/EXTat 0.1%;3e,systems
withSM/BHT/EXTat 0.5%;3f, systems
with
SM/BHT/EXT

at 1.0%.

228

JOURNAL OF COSMETIC SCIENCE

When comparingthe percentages

of hydroquinone
remainingafter incubationat differenttemperatures
Fortwo weeks,the sodiummetabisulfite,BHT, andpepperextract
systemsin all concentrations
in the dark room at 24C showedhigher remaining
hydroquinone
thanthoseincubatedat 45C.This indicatedthe effectsof temperaturein
acceleratingoxidativedegradationof hydroquinone(Table II, Figure 2). Table III and
Figure 3 comparesthe averagepercentages
of hydroquinoneremaining in 2% w/w
hydroquinonecreamcontainingwhite pepperextractand the commercialantioxidants
kept at 45C for threemonths.Both water-soluble
antioxidant(sodiummetabisulfite)
and oil-soluble antioxidant (BHT), as well as the extract at all concentrations,showed

about 10-20% more hydroquinoneremainingthan in the controlsystem.More than

60% of hydroquinoneremainingin all systemsat two monthswas observed,whereas
only 25-30% oFhydroquinone
wasleft at threemonths.A shelflife of oneyearandfour
monthsat roomtemperaturewith 60% hydroquinone
remainingcanbe anticipatedfor
all thesesystems.
The systemcontainingpepperextractat all concentrations
appearedto showthe same
activity as sodiummetabisulfiteand BHT. Both water-solubleand oil-solubleantioxidantsand the water-soluble
pepperextractin our studyshowedsomeprotectionfrom
oxidativedegradationin hydroquinone(but not 100% protection)after two months.
Accordingto ourprocedure,by incorporating
hydroquinone
afterthe creamwasformed,
and togetherwith the solubilitypropertyof hydroquinonethat is freely solublein
alcoholand slightly solublein oil and water, we had expectedthat hydroquinonehad
beenabsorbed
in both the oil and the waterphaseof the formulation.It is alsoevident
that hydroquinone
canbe incorporated
in the formulationin differentways.Besidesour
procedure,hydroquinone
wasusedin the oil phasewith laevo-ascorbic
acidasantioxidants(12,13) and in the water phasewith sodiummetabisulfiteand ascorbicand citric
acidsasantioxidants(14). This meantthat the typesof antioxidantsselectedfor usein
the formulationdependedon the methodof the incorporationof hydroquinone
into the
system.In our hydroquinonesystem,combinations
of both water- and oil-solubleantioxidantsshouldbe usedin orderto achievecompleteoxidativedegradationprotection
for the formulation.

In conclusion,the pepperextractat 0.1% and 0.5% can be usedas a water-soluble

antioxidantwith an expectedshelflife oFone year and four months,having 66% and
72% hydroquinoneremaining,respectively,
at room temperature.The resultsindicate
that the extractgives the sameeffectiveness
as sodiummetabisulfiteand BHT at the
sameconcentrations.
Thus,compounds
presentin the extractmaybe usedassubstitutes
for water-soluble

antioxidants

in oxidation-sensitive

formulations.

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ANTIOXIDANT

ACTIVITY

OF P. NIGRUM

229

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