ResearchArticle

FORMULATIONANDEVALUATIONOFPIPERINECREAMANEWHERBALDIMENSIONAL
APPROACHFORVITILIGOPATIENTS

*VINODK.R
1
,SANTHOSHAD
1
,ANBAZHAGAN.S
2

*
1
NalandaCollegeofPharmacy,AffiliatedtoOsmaniauniversity,Cherlapally,Nalgonda508001,
2
KarunaCollegeofPharmacy,
Iringuttoor,ThirumittacodePO,Pattambivia,PalakadDist.,Kerala.
Email:Vinodkrpharm@gmail.com
Received:17Dec2009,RevisedandAccepted:12Dec2010
ABSTRACT: vitiligo also known as leukoueima is a pigmentation uisoiuei in which melanocytes (the cells that make pigment) in the skin aie
uestioyeu. As a iesult, white patches appeai on the skin on uiffeient paits of the bouy which affects even the psychology anu social status of the
patient. In iecent yeais, it has been pioveu that Pipeiine, an alkaloiu fiom black peppei has the iepigmenting capacity. 0se of Pipeiine in vitiligo not
only ieuuces 0v Rauiation but also pievents siue effects. The piesent woik is about the extiaction of Pipeiine fiom Black Peppei anu its evaluation
followeu by foimulation anu evaluation of cieam.
Keywords: vitiligo, Leukoueima, Pigmentation, Nelanocytes, 0v Rauiation.

INTRODUCTION
vitiligo is a pigmentation uisoiuei in which melanocytes (the cells
that make pigment) in the skin aie uestioyeu.
1
As a iesult, white
patches appeai on the skin on uiffeient paits of the bouy. Similai
patches also appeai on both the mucous membianes (tissues that
line the insiue of the mouth anu nose), anu the ietina (innei layei of
the eyeball). The haii that giows on aieas affecteu by vitiligo
sometimes tuins white. vitiligo is not a fatal uisease, but it is
chionic anu piogiessive. The most impoitant consequence of the
uisease is piobably social anu psychological, as people may feel
uevastateu by theii changeu appeaiance.
2

Theie aie many tieatments available foi vitiligo but has some siue
effects like Cushings synuiome, Skin Cancei , uI uisoiueis etc.
Pipeiine is useu as anti vitiligo agent by ieuucing the effects of 0v
iauiation anu also in avoiuing siue effects.
S S

Pipeiine is extiacteu anu isolateu fiom Black peppei by two
uiffeient methous.
6, 7
They aie soxhlation anu ieflux methou in
which highest %yielu is obtaineu foi soxhlation (89.4S2%).The
following evaluation woiks has been caiiieu out foi Pipeiine .pB,
Solubility, thin layei chiomatogiaphy, XRay uiffiaction stuuies, IR
spectial analysis, chemical tests, Nelting point, Paitition coefficient,
Paiticle size anu 0v Spectial analysis
A 1%Pipeiine cieam was piepaieu using bees wax as base anu its
evaluation was caiiieu out. Thin layei chiomatogiaphy, ulobule size,
Paitition coefficient, Scanning election micioscopic stuuies, pB,
Noistuie absoiption stuuies, consistency, Iiiitancy test, Biug
content unifoimity, phase sepaiation, 0iganoleptic chaiacteis etc
MATERIALSANDMETHODS
AcomparativeextractionofPiperineanditsrecrystallization
The pipeiine was extiacteu by both Reflux methou anu Soxhlation
methou by using 9S% ethanol as solvent. The solution was filteieu
anu concentiateu unuei vacuum in a watei bath at 6uC. Suml
alcoholic potassium hyuioxiue was auueu to the concentiate anu the
solution was stiiieu continuously foi Sumin. The obtaineu solution
was heateu anu watei was auueu uiop wise until yellow piecipitate
was foimeu. Watei was auueu until no moie piecipitate appeaieu to
foim anu this was alloweu to settle oveinight. Neeules of Pipeiine
weie obseiveu to be sepaiateu out. The soliu was collecteu anu
washeu with colu ethei 2S times. It was ieciystallizeu by using
acetone. Foi this, uissolve soliu in acetone anu filtei it to iemove
extianeous mattei anu keep the filtiate asiue foi 24his so that
ciystals of Pipeiine aie foimeu. Yellow colouieu iou shapeu ciystals
weie ieciystallizeu aftei 24 his.
AnalyticalworksonPiperine
UVspectralanalysis
Sgml of the uiug in ethanol was useu foi complete scan between
19u9uunm anu the maximum absoiption was obtaineu at S44nm as
shown in the fig.1 wheieas the i max foi stanuaiu Pipeiine is
S42nm.
8


Fig.1:AbsorptionmaximumofPiperine
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 3, Suppl 2, 2011
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Su

PartitioncoefficientofPiperine
9

Procedure: The paitition coefficient of uiug between phosphate
buffei solution (P
B
6.6) & nhexane was ueteimineu at S7
o
u.2
o
. An
excess amount i.e. Su mg of Pipeiine was taken in a sepaiating
funnel containing 1:1 iatio of buffei 6.6 & nhexane & placeu in a
watei bath foi 24h. The solution was shaken at iegulai inteivals.
Then, both of them weie sepaiateu & filteieu thiough a 2 filtei &
the uiug concentiation in each phase was ueteimineu by measuiing
the absoibance using 0v spectiophotometei at S44nm.
ParticlesizeofPiperineusingmicroscope
A small amount of powueieu uiug is placeu on the sliue & mounteu
using glyceiin. By using eye piece miciometei, the uiameteis of 2uu
paiticles aie ueteimineu ianuomly. Paiticle size uistiibution was
expiesseu as histogiam (Fig 2)
1u12
.

44
39
11
4
2
0
5
10
15
20
25
30
35
40
45
50
2.5 7.5 12.5 17.5 22.5
mean size range (microns)
N
o
.

o
f

p
a
r
t
i
c
l
e
s

i
n

e
a
c
h

r
a
n
g
e

Fig.2:HistogramofParticlesizedistributionofPiperine


Fig.3:XrayDiffractogramsofPiperinecrystalsandpowder

Solubility of Pipeiine was ueteimineu in watei, ethyl alcohol,
chloiofoim anu Acetone. Tempeiatuie was maintaineu at S7u.2
1S
.
Nelting point anu TLC stuuies of pipeiine weie also conuucteu. The
chiomatogiam was obseiveu unuei 0v lamp (S6Snm). The alkaloiu
shows violet coloieu zone. Rf value was founu to be u.26 (value of
stanuaiu pipeiine~u.2S)
14

XRaydiffractionstudies
Powueieu XRay uiffiaction stuuies of Pipeiine ciystals anu
powueieu Pipeiine by sonication weie caiiieu out to stuuy
ciystalline natuie of the uiug .B8 Auvanceu Biukei AXS, instiument
was useu foi this puipose .Type of iauiation useu was coppei
iauiation. The uiffiactogiams aie shown in fig S. It shows that the
ciystallinity ieuuces foi Pipeiine powueieu using ultiasonicatoi i.e.,
solubility has incieaseu.
DeterminationofpH
11,13

A small quantity of Pipeiine was uissolveu in ethanol anu its pB was

checkeu by using pB metei anu it is founu to be 7.9.
Chemicaltests
1u mg of Pipeiine ciystals weie uissolveu in 1uml ethanol anu this
solution is useu as sample foi chemical tests
1S, 16
.
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Test Observation
Biagenuioffs test 0iange biown ppt is foimeu
Nayeis test Cieam colouieu ppt was obseiveu
Bageis test Yellow ppt was obseiveu
Wagneis test Reuuish biown ppt was foimeu

Fig.4:IRSpectraofPiperineanditsstructure
162u

Table1
Suu8.18cm1 Alkenes Piesent
29S2.66cm1 CB2CB2 CBS Piesent
16Su.82cm1 Amines Piesent
1S82.SS Ketonic gioup Piesent

Fig.5:SEMpictureofcreamdilutedwithglycerin

Formulationofcream
21, 22

Procedure: Beeswax, Lanolin anu Steaiic weie taken in one beakei.

In anothei beakei, Pipeiine was uissolveu in ethanol by sonication
anu intiouuceu into glyceiine, watei, tiiethanolamine. Both the
beakeis weie maintaineu at 6uC anu all the ingieuients weie
melteu. Then oily phase is auueu to aqueous phase anu stiiieu
continuously. As the tempeiatuie goes uown peppeimint oil was
auueu anu mixeu well until iequiieu consistency was obtaineu.
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Evaluationofcream
0iganoleptic chaiacteis weie stuuieu by visual appeaiance, coloui
anu ououi
2S
.
By using eyepiece miciometei, the uiameteis of 2uu paiticles aie
ueteimineu ianuomly
1u, 11, 12
. Topology stuuies weie caiiieu out foi
cieam by using scanning election micioscopy
Piesence of foieign paiticlesgiittiness was obseiveu against
uiffuseu light to check foi foieign paiticles
1S
.

Biug content
unifoimity of the cieam was also caiiieu out
24,2S
.
Partitioncoefficientofcream
The paitition coefficient of uiug between phosphate buffei solution
(P
B
6.6) & nhexane was ueteimineu at S7
o
u.2
o
. An excess amount
i.e. Su mg of cieam was taken in a sepaiating funnel containing 1:1
iatio of buffei 6.6 & hexane & placeu in a watei bath foi 24h. The
solution was shaken occasionally. Then, both of them weie
sepaiateu & filteieu thiough a 2 filtei & the amount solubilizeu in
each phase was ueteimineu by measuiing the absoibance using 0v
spectiophotometei at S44nm
9
.
The foimulateu cieam was kept intact in a closeu containei at 2S
SuC not exposeu to light. Any change in phase sepaiation was
checkeu eveiy 24 his foi one month.
Irritancytest
26

Naik an aiea (1sq.cm) on the left hanu uoisal suiface. The cieam
was applieu to the specifieu aiea anu time was noteu. Iiiitancy,
eiythma, euema, was checkeu if any foi iegulai inteivals up to 24
his anu iepoiteu.
Rheologicalstudies
27

The foimulateu cieam was founu to be non newtonian. Take a

fixeu quantity 1ugms of cieam in a 1uml beakei. Keep it impact foi 1
hi. The beakei was inclineu to one siue see whethei the cieam is
liquefieu oi not. beakei is shaken to anu fio foi continuous Smns
anu checkeu whethei consistency has changeu oi not. The beakei
was again tilteu anu checkeu foi pouiability of the cieam. The
foimulation showeu no thixotiopic (sheai thinning) chaiacteiistics.
Diffusion studies of Piperine incorporated cream using goats
skinassemipermeablemembrane
Fiesh goats skin was shaveu well to iemove all the haii anu cleaneu
thoioughly in watei anu iinseu in isotonic solution anu latei with
6.6 buffei solution. Fiesh skin is useu foi the stuuy. A stuuent
uiffusion cell was fabiicateu anu the goats skin was useu as semi
peimeable membiane. A 2uumg of 1% Pipeiine cieam was placeu
on the outei layei of the skin which was fixeu as to face unit I fiom
insiue.
At pieueteimineu inteivals, samples 2ml fiom iecipient chambei
weie withuiawn anu tiansfeiieu to ambei colouieu ampoules. The
samples weie suitably ieplaceu. The samples weie estimateu foi
uiug content, analyzeu at maximum absoibance S44nm using 0v
Spectiophotometei ( Elico SL 196).The above woik was iepeateu S
times anu aveiage was calculateu .Fiom this, concentiation of cieam
in buffei can be obtaineu.
Aftei the completion of uiffusion stuuies the skin was taken anu
uissolveu in ethanol anu left foi 24his.Then the absoibance was
calculateu at S44nm.Fiom this, concentiation of cieam in skin layei
was calculateu.
RESULTSANDDISCUSSION
Black peppei which was obtaineu fiom the local souice was
subjecteu to stanuaiuization anu the iesults weie founu to comply
with the stanuaiu values. Pipeiine was extiacteu fiom black peppei
by using Soxhlet anu Reflux methou anu the % yielu was founu to be
maximum in Soxhlet methou i.e., 89.6S2% wheieas with ieflux
methou it is 8S%.Yellow colouieu iou shapeu ciystals weie founu.
Nelting point ueteimination of Pipeiine was peifoimeu thiice by
melting point appaiatus anu the aveiage was founu to be 129C
which was compaieu with the stanuaiu value 1Su C. The alkaloiu
shows violet colouieu zone unuei 0v Rauiation in TLC stuuies. The
Rf value of test (extiacteu Pipeiine) is founu to be u.26 which
coiiesponus to the stanuaiu value of Pipeiine (u.2S) anu only one
spot was obtaineu inuicating that it is puie. Fiom 0v Spectial
analysis absoiption maximum was founu to be S44nm which almost
matcheu with the stanuaiu. Infia ieu spectia of Pipeiine weie
compaieu with stiuctuie anu the coiiesponuing bonus weie founu
to piesent.
Test foi alkaloius was uone by Biagenuioffs test, Bageis test,
Wagneis test anu Nayeis test anu it has given positive ieaction foi
all the ieagents confiiming the piesence of alkaloius. Assay of
Pipeiine was peifoimeu anu % puiity of Pipeiine extiact was
98.67%. Solubility of pipeiine was founu to be in the oiuei
chloiofoim>ethanol>Acetone but insoluble in watei.
XRay uiffiactogiams ievealeu peak heights was ieuuceu in powuei
aftei sonication inuicating that ciystallinity has ieuuceu by
ultiasonication anu solubility has incieaseu.
Pipeiine was foimulateu into % cieam anu was subjecteu to vaiious
evaluation woiks Thin layei chiomatogiaphic stuuies weie
peifoimeu. Cleai single spots weie obtaineu anu Rf value of Pipeiine
anu the foimulateu cieam weie founu to be coiielating with each
othei. Nelting point anu Rf values of test(cieam) anu
stanuaiu(Pipeiine) weie compaiable which inuicates theie is no
change in the physical anu chemical natuie of the Pipeiine.This also
ieflects that the uiug is compatable with othei excepients like bees
wax, lanolin etc. Aiithmetic mean paiticle size of Pipeiine anu
globule size was founu to 21.6 anu 28.67 iespectively. Ninimum
globule size anu maximum globule size of cieam was founu to be
4.72 anu S2.72 iespectively.
Expectation of the topical foimulation was that the uiug shoulu
penetiate the stiatum coinium, get into the ueimis but shoulu not
get into the systemic ciiculation. The % of Pipeiine foi cieam
ietaineu in the skin, the site of action was founu to be was founu to
be 69.u6% uiug ieleaseu into the buffei was founu to 18.62%. it
coulu be postulateu that in auuition to tianscellulai peimeation,
paiacellulai peimeation also is significant thiough tight junctions.
The final piepaiation was founu to be smooth textuie anu
consistency anu fiee fiom giitty natuie with light yellow coloui anu
peppeimint ououi. The globules ietaineu its size anu no coalescence
was founu. Acceleiateu stability stuuies weie uone which shows no
significant change in the concentiation of uiug which shows stability
of the foimulation. Noistuie absoiption stuuies showeu no
significant absoiption of moistuie. pB of the cieam was founu to be
6.S. Topology stuuies by SEN ievealeu almost smooth globules. In
auuition iiiitancy test was also peifoimeu on iabbits anu founu no
ieuness, euema, Inflammation anu iiiitation.
CONCLUSION
The foimulation of Pipeiine cieam intenueu foi vitiligo was
successfully uone anu evaluateu. Biug taigeting at the skin weie the
melanocytic piolifeiation is intenueu was achieveu 69.u6%. The
topical foimulation was physically stable thioughout the shelf life.
Fuithei novel uiug ueliveiy foimulations aie highly iecommenueu
to inciease the peicentage uiug taigeting.
ACKNOWLEDGMENT
The authois expiess theii giatituue to Nalanua college of Phaimacy,
A.P. foi the suppoit thioughout the pioject. The authois wish to
expiess Achaiya Nagaijuna 0niveisity, uuntui foi pioviuing
technical suppoit foi this pioject.
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