ArticlePDF Available

In vivo evaluation of piperine and synthetic analogues as potential treatments for vitiligo using a sparsely pigmented mouse model


Abstract and Figures

Piperine and its analogues have been reported to stimulate melanocyte replication in vitro and may be useful in treating the depigmenting disease, vitiligo. To investigate the ability of piperine (PIP) and three analogues to stimulate pigmentation in a strain of sparsely pigmented mice. The test compounds were PIP [5-(3,4-methylenedioxyphenyl)-2,4-pentadienoylpiperidine], tetrahydropiperine [THP, 5-(3,4-methylenedioxyphenyl)-pentanoylpiperidine], a cyclohexyl analogue of piperine [CHP, 5-(3,4-methylenedioxyphenyl)-2,4-pentadienoylcyclohexylamine], and reduced CHP [rCHP, 5-(3,4-methylenedioxyphenyl)-2,4-pentanoylcyclohexylamine]. Sparsely pigmented, HRA/Skh-II mice were randomized to receive topical treatment with test compounds or vehicle twice a day for five days a week, with or without ultraviolet (UV) irradiation on 3 days a week. Treatment was either continuous or interrupted to evaluate fading and repigmentation. Skin inflammation and pigmentation were evaluated regularly during treatment. DOPA+ melanocytes were determined histologically at the termination of treatment. Four weeks of treatment with one of the compounds PIP, THP or rCHP, but not CHP, induced greater pigmentation than vehicle with low levels of inflammation. Additional exposure to UVR led to darker pigmentation than did the compound or UVR alone, and greater numbers of DOPA+ melanocytes were found. The combination produced an even pigmentation pattern, contrasting with the speckled, perifollicular pattern produced by UVR alone. Treatment interruption led to a decrease in pigmentation but not its loss. Repigmentation was achieved by administering one of the compounds, UVR or both, and occurred faster than in naïve mice. Treatment with PIP, THP or rCHP and UVR induced a marked pigmentation response in HRA/Skh-II mice, with clinically better results than UVR alone. This result supports the potential use of these compounds in treating vitiligo.
Content may be subject to copyright.
In vivo evaluation of piperine and synthetic analogues
as potential treatments for vitiligo using a sparsely
pigmented mouse model
L. Faas,*,R. Venkatasamy,* R.C. Hider,* A.R. Young,# and A. Soumyanath*,,#
*Department of Pharmacy King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, U.K.
St John’s Institute of Dermatology, Division of Genetics and Molecular Medicine, King’s College School of Medicine, King’s College London, Guy’s Hospital,
London SE1 9RT, U.K.
#Equal last authors.
Present address: Department of Biology, University of York, Heslington, York YO10 5DD, U.K.
Present address: Department of Neurology, Oregon Health & Science University, Portland, Oregon 97239, U.S.A.
A. Soumyanath.
Accepted for publication
4 September 2007
Key words
HRA Skh-II, melanocyte, pigmentation, piperine,
tetrahydropiperine, vitiligo
Conflicts of interest
None declared.
Background Piperine and its analogues have been reported to stimulate melano-
cyte replication in vitro and may be useful in treating the depigmenting disease,
Objective To investigate the ability of piperine (PIP) and three analogues to stimu-
late pigmentation in a strain of sparsely pigmented mice.
Methods The test compounds were PIP [5-(3,4-methylenedioxyphenyl)-2,4-penta-
dienoylpiperidine], tetrahydropiperine [THP, 5-(3,4-methylenedioxyphenyl)-
pentanoylpiperidine], a cyclohexyl analogue of piperine [CHP, 5-(3,4-methyl-
enedioxyphenyl)-2,4-pentadienoylcyclohexylamine], and reduced CHP [rCHP,
5-(3,4-methylenedioxyphenyl)-2,4-pentanoylcyclohexylamine]. Sparsely pigmen-
ted, HRA Skh-II mice were randomized to receive topical treatment with test
compounds or vehicle twice a day for five days a week, with or without ultra-
violet (UV) irradiation on 3 days a week. Treatment was either continuous
or interrupted to evaluate fading and repigmentation. Skin inflammation and
pigmentation were evaluated regularly during treatment. DOPA+ melanocytes
were determined histologically at the termination of treatment.
Results Four weeks of treatment with one of the compounds PIP, THP or rCHP,
but not CHP, induced greater pigmentation than vehicle with low levels of
inflammation. Additional exposure to UVR led to darker pigmentation than did
the compound or UVR alone, and greater numbers of DOPA+ melanocytes were
found. The combination produced an even pigmentation pattern, contrasting
with the speckled, perifollicular pattern produced by UVR alone. Treatment inter-
ruption led to a decrease in pigmentation but not its loss. Repigmentation was
achieved by administering one of the compounds, UVR or both, and occurred
faster than in naı
¨ve mice.
Conclusions Treatment with PIP, THP or rCHP and UVR induced a marked pigmenta-
tion response in HRA Skh-II mice, with clinically better results than UVR alone.
This result supports the potential use of these compounds in treating vitiligo.
The skin disorder vitiligo is the most common acquired hypo-
melanosis, affecting approximately 1% of the world’s popula-
tion, with serious cosmetic and psychological effects.
characteristic depigmentation can be restricted to a limited
skin area (segmental vitiligo) or generalized in symmetrical
patches (nonsegmental vitiligo). In most cases, loss of skin
colour corresponds with melanocyte loss, first in the epider-
mal compartment, and later in the follicular reservoir where
most melanocytic stem cells are probably situated.
Treatment of vitiligo is often difficult and disappointing.
This is most probably because the aetiopathogenesis is
unknown, and a treatment directed to the cause has not
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950 941
been established. Several treatment modalities, such as PUVA
[psoralen + UVA (320–400 nm) radiation], broad-band (280–
320 nm) and narrow-band (311 nm) UVB, and local corticos-
teroids are currently used. However, it has been reported that
these standard treatments result in limited success; less than
25% of patients responded successfully to topical corticoster-
Moreover, corticosteroids applied either systemically or
topically carry the risk of significant side effects in long-term
Alternatively, PUVA therapy seldom achieves extensive
repigmentation that is cosmetically acceptable, and treatment
response is often followed by relapse.
A recent Cochrane
highlights the lack of research on current treatments
as well as the need to identify novel clinical approaches for
Several clinical studies
strongly suggest that reservoirs in
hair follicles are the source of melanocytes in skin repigmented
by standard therapies. Small circular areas of repigmentation
centred around hair follicles enlarge and eventually coalesce.
Consequently, the identification of stimuli that activate outer
root sheath melanocytes is a prospective means of developing
new treatments for vitiligo.
Recent evidence from our laboratory indicates that piperine
[5-(3,4-methylenedioxyphenyl)-2,4-pentadienoylpiperidine; PIP]
has a potent stimulatory effect on mouse melanocytes in vitro.
Culture media supplemented with Piper nigrum (black pepper)
fruit extract or its main alkaloid, PIP, induced nearly 300%
stimulation of melan-a mouse melanocyte proliferation after
8 days of treatment in vitro.
The increase of growth was
effectively inhibited by RO-31-8220, a broad-spectrum pro-
tein-kinase C (PKC) inhibitor, suggesting that PKC signalling
is involved in its activity. Both Piper nigrum extract and PIP also
induced an increase in the number and length of cell den-
Melanin synthesis, however, was not stimulated. We
have also shown that several synthetic derivatives of piperine
share these in vitro effects.
The aim of the present study was to evaluate the melano-
cyte-stimulatory activity of PIP and three of its synthetic deriv-
atives (Fig. 1) in vivo as a putative new chemical group for the
treatment of vitiligo, alone or in association with UVR. Studies
were performed in HRA.HRII-c +Skh mice, a hairless, spar-
sely-pigmented mouse model
that has white skin except for
the ears and tails. This line, congenic with albino inbred
HRA Skh mice, segregates into albino and pigmented pheno-
types and was developed by Dr P. Forbes, Temple University
Centre for Photobiology, Philadelphia, PA, U.S.A. These mice
have melanocytes in the epidermal layer (as in human skin),
whereas in many other pigmented mouse strains, melanocytes
are found only in the dermis. The numbers of epidermal mel-
anocytes in this model are small – two or three DOPA+ mela-
nocytes mm
However, unlike albino mice, pigmentation
in HRA.HRII-c +Skh mice is inducible
with melanocyte
numbers reaching close to 600 mm
. As with vitiligo, peri-
follicular pigmentation is evident after exposure to UVR with
and without photosensitizers.
We therefore advocate the
induction of pigmentation in this strain as an in vivo model for
repigmentation in vitiligo.
Materials and methods
Male and female inbred HRA.HRII-c +Skh hairless pigmented
mice, age-matched (8–16 weeks old), were used. Animals
were bred by the Biological Services Division, KCL, University
of London, U.K. and the Rayne Institute, St Thomas’s Hospital,
London, U.K. Animals were killed by cervical dislocation and
skin samples removed surgically when required.
PIP [5-(3,4-methylenedioxyphenyl)-2,4-pentadienoylpiperi-
dine] was purchased from Sigma-Aldrich Ltd (Dorset, U.K.).
PIP derivatives, i.e. tetrahydropiperine [5-(3,4-methylenedi-
oxyphenyl)-pentanoylpiperidine; THP], a cyclohexyl analogue
of piperine [5-(3,4-methylenedioxyphenyl)-2,4-pentadienoyl-
cyclohexylamine; CHP] and reduced CHP [5-(3,4-methyl-
enedioxyphenyl)-2,4-pentanoylcyclohexylamine; rCHP] were
synthesised in our laboratory.
Selection of vehicles by ex vivo skin assays
To determine the optimum vehicle for delivery of test agents,
ex vivo permeation studies were conducted with vertical Franz
diffusion cells using a modification of reported methods.
5-(3,4-methylenediox y phe ny l)-2,4-pentadieno yl pi peridine (PIP)
5- (3,4-methylenedioxypheny l)-pentano yl piperidine (THP)
5- (3,4-methylenedioxypheny l)-2,4-pentadienoylc yc lohexy lamine (CHP)
5- (3,4-methy le nedioxy pheny l)-2,4-pentano yl cy clohexylamine (rCHP)
Fig 1. Piperine (PIP) and structural analogues tetrahydropiperine
(THP), a cyclohexyl analogue of piperine (CHP) and reduced CHP
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
942 Evaluation of piperine and analogues for vitiligo, L. Faas et al.
Each cell consists of a chamber with upper (donor) and lower
(receiver) compartments divided by the mounted skin sample.
The skin acts as a seal between the two half-cells when they
are clamped together. The upper, stratum corneum side is
filled with the drug formulation and the lower one (dermal
side) with receiving fluid. Samples of the receiving fluid are
taken at intervals to quantify the amount of the drug passing
through the skin. The cells used in this study had a 10 mL
capacity receptor compartment and a 1Æ75 cm
diffusion area.
A circular piece of full-thickness dorsal skin from HRA.HRII-
c+ Skh mice was carefully mounted onto the receiver com-
partment of the diffusion cells with the stratum corneum
facing the donor compartment. The receptor compartment
was filled with phosphate buffered saline; PBS, pH 7Æ4) which
was continuously stirred with a magnetic bar. Test solutions
[175 mmol L
PIP in ethanol, diethylene glycol monoethyl
ether (Transcutol
, Gattefosse
´, Saint-Priest Cedex, France),
dimethyl sulfoxide (DMSO), polyethylene glycol (PEG) or 5%
oleic acid (OA) in PEG] were added into the donor compart-
ment of each cell (n= 4 for each formulation). Samples of
fluid from the receiver cell were taken at 3, 19 and 22 h and
the concentration of PIP was determined by high performance
liquid chromatography (HPLC) using a model 3100 pump
(LDC Analytical, Riviera Beach, FL, U.S.A.) with a Spectro-
monitor 3100 UV detector (LDC Analytical) and Hewlett Pack-
ard 3390 A integrator. A 4Æ6·25 cm, 10 lm, C18 Econosil
reverse phased column (Alltech U.K., Stamford, U.K.) was
used, eluting with methanol : water (60 : 40; HPLC grade,
1 mL min
). The detector wavelength was set at 348 nm.
Under these conditions, PIP eluted at 10Æ59 min. Results were
expressed as mg mL
according to a previously determined
calibration curve (0Æ003–0Æ1mgmL
PIP in PBS).
Topical application of test compounds in vivo
Test agents were dissolved in vehicle (either OA PEG or in
DMSO) to a final concentration of 175 mmol L
and 100 lL
(17Æ5lmoles) applied with a micropipette on the central area
of mouse dorsal skin (2–3 cm
), twice a day (weekdays only)
with an interval of 5–6 h between applications. In protocols
with UVR exposure, the irradiations were carried out every
Monday, Wednesday and Friday immediately prior to the first
daily application, to avoid a possible photosensitizing effect
and or photodamage
to the test compound.
UV irradiation and dosimetry
The UVR source was a bank of eight Bellarium SA-1-12-100W
fluorescent tubes (Wolff, Erlangen, Germany), the emission
spectrum of which has been published.
This UVR source emits
4Æ1% UVB (280–320 nm) and 95Æ8% UVA (320–400 nm), but
the UVB accounts for the 71Æ5% erythemally effective energy
when biologically weighted with the human erythema spec-
Given that tanning and erythema action spectra are very
it is probable that the small UVB component accounts
for most of the tanning effect. Irradiations were carried out in a
purpose-built unit with ventilation, temperature and humidity
controls. The irradiance was monitored daily immediately
before irradiation with an International Light radiometer (IL
422A; Newburyport, MA, U.S.A.) equipped with UVR sensors.
The radiometer was calibrated for the source, as described
Irradiance measured at mouse level was typically about
0Æ16 mW cm
. Animals were irradiated unrestrained in metal
cages with a dose of 354 mJ cm
confirmed to be sub-
inflammatory from a single exposure (increase in skin fold
thickness (SFT) < 10%; data not shown). Irradiations lasted for
a maximum of 1 h. The position of cages was systematically
rotated to ensure even UVR exposure.
Experimental groups
In initial experiments, animals were treated topically with PIP,
THP and CHP dissolved in either OA PEG or in DMSO or with
vehicle alone for 9 weeks with concomitant exposure to UVR
during weeks 5–9. Further experiments (summarized in
Fig. 2) were conducted using compounds dissolved in DMSO,
with DMSO as control. For continuous treatment, animals
(n4) were treated topically with PIP, THP, rCHP or DMSO
for up to 13 weeks (Fig. 2, Group A). A second group
(Fig. 2, Group B) received the same treatment, but was add-
itionally exposed to UVR from week 5 to 13. For studies on
discontinuous treatment, animals were treated as in Group B
up to week 7. All treatment was then suspended for 3 weeks
(weeks 8–10) and re-started as topical application only
(Fig. 2, Group C), UVR only (Fig. 2, Group D), or topical
application plus UVR (Fig. 2, Group E) for weeks 11–13.
Mice exposed only to UVR (i.e. no vehicle treatment) from
week 5 onwards (Fig. 2, Group F) were used as controls for
all groups treated with UVR.
Group Week
No treatment Compound UVR
Fig 2. Treatments. Mice were treated for 13 weeks with topical
compounds alone (A) or with additional ultraviolet radiation (UVR)
exposure (B). For other groups, treatment was interrupted and
restarted as compounds only (C), UVR only (D), or compounds +
UVR (E). Group F received UVR alone.
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
Evaluation of piperine and analogues for vitiligo, L. Faas et al. 943
Assessment of inflammation and pigmentation
Dorsal SFT was recorded to evaluate potential inflammatory
effects of treatments, Measurements were taken every day dur-
ing the first week of treatment and twice a week thereafter
with a spring-loaded micrometer (Mitutoyo, Kawasaki, Japan).
Pigmentation was assessed independently by two investigators
and the average score calculated. The first type of assessment
was conducted visually, every day, and pigmentation scored
from 0 to 5 according to the following scheme: 0 = no
pigmentation; 1 = first signs of pigmentation (freckles);
2 = light brown; 3 = medium brown; 4 = dark brown;
5 = black. Pigmentation was also assessed histologically by
DOPA staining at the end of the experiment. Animals were
killed and skin samples from representative dorsal areas
(1 cm
) were removed surgically and incubated in 2 mol L
NaBr in PBS for 2 h at 37 C. The epidermis was carefully
removed with tweezers and further incubated in 0Æ1% L-DOPA
in PBS (pH 7Æ2) for 4 h at 37 C. The DOPA solution was
changed periodically to prevent auto-oxidation. Finally, epi-
dermal sheets were fixed in 4% paraformaldehyde in PBS (pH
7Æ4) for 15 min, dehydrated through a graded series of alco-
hol concentrations and mounted on glass microscope slides
for examination. The number of DOPA+ cells per mm
calculated from at least 30 fields per sample (n= 4 animals).
DOPA+ cells were also classified as highly or poorly melanized
according to their melanin granule content. The percentage of
cells in each category per mm
was calculated.
Selection of vehicles based on skin penetration
measured with Franz cells
Skin penetration of PIP when dissolved in five vehicles was
compared using Franz cells. At 22 h after application, the con-
centration of PIP in the receiver compartment was highest
with DMSO followed by OA PEG in both male and female
skin (Fig. 3). PIP was undetectable when delivered in other
vehicles (ethanol, diethylene glycol monoethyl ether and
PEG). No PIP was detected at shorter time periods (3 h and
19 h) with any vehicle. DMSO and OA PEG were therefore
chosen as vehicles for the in vivo studies.
Inflammatory and irritant effects
Differences in inflammatory response were seen depending on
the vehicle, test compound and sex of animal. OA PEG based
formulations induced stronger adverse effects than those with
DMSO in both male and female mice (Figs 4 and 5). However,
in males, PIP and THP solutions in OA PEG induced a stronger
inflammatory response (more than 30% increase in SFT;
Fig. 4a), than in females where THP had only a mild
inflammatory effect (20% increase in SFT, Fig. 5a). The
inflammatory effect of CHP was comparable to vehicle alone in
both males and females (around a 20% increase in SFT, Figs 4a
and 5a). The inflammatory response induced by formulations
PIP Concentration (mg mL–1)
Fig 3. DMSO is the best of five skin penetration enhancers for
piperine (PIP). Penetrance of PIP dissolved in ethanol (EtOH), DMSO,
diethylene glycol monoethyl ether (Transc), polyethylene glycol (PEG)
and 5% oleic acid in PEG (OA PEG) through HRA.HRII-c+ Skh
mouse dorsal skin was tested using Franz cells.
Increase in SFT (%)Increase in SFT (%)
Fig 4. Inflammation in male mice treated with piperine (PIP),
tetrahydropiperine (THP) or cyclohexyl analogue of piperine (CHP) in
5% oleic acid in polyethylene glycol (OA PEG) (a) or DMSO (b).
Inflammation was assessed as percentage increase in skin fold
thickness (SFT). Only mean values (n= 5) are given for clarity (% CV
not more than 30%). V, vehicle; UV, ultraviolet radiation.
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
944 Evaluation of piperine and analogues for vitiligo, L. Faas et al.
in DMSO was observed to be milder than with OA PEG. DMSO
alone had a mild inflammatory effect (up to 20% increase in
SFT; Figs 4b and 5b). Both vehicles and test solutions had a
transient irritant effect, producing redness and desquamation
of treated areas during the first 10 days of treatment (data not
shown). Irritancy induced by DMSO (and DMSO-based solu-
tions) was milder than with OA PEG. Redness decreased after
an hour of topical application, and desquamation was less
severe than with OA PEG. PIP and THP in OA PEG showed the
most powerful irritant effects, in agreement with the strong in-
flammatory response observed. The irritant effects were over-
come by week 2 of topical treatment. UVR alone had lower
inflammatory effects than any of the topical treatments.
Four weeks of topical treatment with PIP or THP, in either
DMSO (Fig. 6a) or OA PEG (not shown), induced a light,
even pigmentation of the treated area compared with vehicle
control whereas CHP had virtually no effect. The vehicles used
also showed some effect, as previously reported for DMSO
and OA.
For PIP (Fig. 6b) and THP, subsequent subery-
themal exposure to two UVR exposures alone significantly
enhanced pigmentation induced by the test compounds com-
pared with controls treated with vehicle and UVR, or UVR
alone. Pigmentation was observed as a dark, even pattern after
6–8 exposures (Fig. 6c,d). The pigmentation induced by topi-
cal treatment with vehicle was lighter and uneven (DMSO,
Fig. 6a–d). The pigmentation induced by UVR alone (Fig. 6c)
was observed to be perifollicular and therefore speckled, in
contrast to the even pigmentation of PIP and THP alone
(Fig. 6a), or in combination with UVR (Fig. 6b–d).
Different pigmentation responses observed in vivo corre-
sponded with changes in the number of DOPA+ cells mm
in the skin (Fig. 7). Pigmentation responses were slower and
less evident in females (scores not shown) than in males. In
male mice, treatment with PIP and THP in either DMSO or
OA PEG significantly (P<0Æ05) increased the number of
DOPA+ cells compared with vehicles. The lower pigmenta-
tion responses in female mice corresponded with a smaller
mean number of DOPA+ cells mm
under all treatment
conditions compared with males receiving equivalent treat-
ments (Fig. 7). In females the stimulatory effects of THP and
PIP on pigmentation reached statistical significance only with
PIP in OA PEG and THP in DMSO (Fig. 7) although a trend
towards an increase was apparent with both compounds in
either vehicle. CHP, in contrast, did not show any effect on
the number of DOPA+ cells compared with vehicles in either
males and females, in agreement with the low pigmentation
levels observed on visual examination of the animals
(Fig. 6).
Further experiments were carried out in order to determine
the persistence of the pigmentation effect after the cessation of
treatment and the stimuli needed to restore pigmentation if
lost, according to protocols summarized in Fig. 2. PIP, THP
and a novel compound, the reduced form of CHP (rCHP)
were tested. rCHP was chosen because of its high stimulatory
activity on melanocyte proliferation in vitro.
All solutions
were prepared in DMSO because of its better performance as a
penetration enhancer (Fig. 3) and milder inflammatory effects
than OA PEG (Figs 4 and 5). The results of this experiment
are shown in Fig. 8. The application of the compounds alone
for 13 weeks (Group A) stimulated pigmentation up to a
maximum of level 2 (light brown). The first change was
observed at 4 weeks and maximum pigmentation was reached
by week 6. The application of concomitant UVR (Group B)
significantly enhanced pigmentation reaching up to level 5
(black) by week 7. This was greater than the highest mean
scores obtained with either compound alone (Group A; score
2, light brown) or UVR alone (Group F; score 3, medium
brown). In mice treated topically with a compound, four
exposures of suberythemal doses of UVR were sufficient to
induce a pigmentation score of 3 (Group B), in contrast with
the greater number of UVR exposures (more than 10) needed
Increase in SFT (%)
Increase in SFT (%)
Fig 5. Inflammation in female mice treated with piperine (PIP),
tetrahydropiperine (THP) and cyclohexyl analogue of piperine (CHP)
in 5% oleic acid in polyethylene glycol (OA PEG) (a) or DMSO (b).
Inflammation was assessed as percentage increase in skin fold
thickness (SFT). Only mean values (n= 5) are given for clarity (% CV
not more than 20%). V, vehicle; UV, ultraviolet radiation.
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
Evaluation of piperine and analogues for vitiligo, L. Faas et al. 945
to obtain a similar, but less even, response in naı
¨ve mice
(Group F). This data clearly shows a combined pigmentation
enhancing effect of PIP, THP and rCHP with UVR. Pigmenta-
tion in Group B was maintained up to week 13 with contin-
ued treatment with the compounds plus UVR.
After three weeks without treatment (Week 8–10), the
degree of pigmentation decreased in animals treated with a
compound and UVR (Fig. 8, Groups C, D and E) compared
with week 7 pigmentation levels, but did not disappear
completely. By contrast, there was no remaining detectable
pigmentation after week 9 in animals treated with only
DMSO and UVR in both male (Fig. 8, Group C, D and E)
and female (data not shown) mice. Retreatment with topical
solutions, UVR, or a combination of both, all resulted in re-
pigmentation after 3 weeks (Week 11–13; Fig. 8, Group C,
D and E). The rate of increase in pigmentation was faster than
the initial pigmentary response (weeks 1–4), reaching scores
of 2 or more within 2 weeks of retreatment (Week 12;
Group C, D and E). Retreatment with the compounds alone
(Fig. 8, Group C) increased pigmentation to levels comparable
with those obtained by continuous topical treatment alone
(Fig. 8, Group A). Retreatment with UVR alone (Fig. 8,
Group D) or combined topical applications plus UVR (Fig. 8,
Group E) resulted in higher pigmentation levels (score 3)
than after retreatment with the compounds alone (score about
2), but comparable with UVR alone (Fig. 8, Group F). The
pigmentation patterns resulting from a compound alone
(Group C) or a compound plus UVR (Group E) were both
even (Fig 6c and data not shown). In contrast, pigmentation
induced by retreatment with UVR (Fig. 8, Group D) resem-
bled the spotted pattern obtained with continuous UVR
exposure (Fig. 6c).
Histological analysis of skin melanocyte numbers (Fig. 9)
again showed a good correlation with visually observable dif-
ferences in pigmentation for both male and female mice.
Group B animals (compound plus UVR) showed significantly
more melanocytes mm
than those receiving compounds
(Group A) or UVR (Group F) alone. Based on the fading of
pigmentation, treatment withdrawal (Groups C–E, weeks
8–10) is assumed to have caused a decrease in the activity of
melanocytes. In males (Fig. 9a), retreatment with UVR alone
(Group D) or with topical compounds plus UVR (Group E)
increased the number of DOPA+ cells mm
at the end of
Week 13 to levels comparable with those in Group B which
Fig 6. Pigmentation induced in male mice by
piperine (PIP) and derivatives applied in
DMSO. (a) PIP and tetrahydropiperine (THP)
[but not cyclohexyl analogue of piperine
(CHP)] applied for 4 weeks induce greater
pigmentation than DMSO. After two
ultraviolet radiation (UVR) exposures (b) or
eight UVR exposures (c,d), pigmentation is
darker in mice treated with PIP or THP (but
not CHP) than in mice treated with DMSO or
previously untreated (UVR) mice.
Compounds produce an even pigmentation
compared with the speckles (c, arrows)
caused by UVR alone.
Fig 7. Increase of DOPA+ cell number after continuous treatment
with piperine (PIP), tetrahydropiperine (THP) or cyclohexyl analogue
of piperine (CHP) in DMSO or 5% oleic acid in polyethylene glycol
(OA PEG) for 9 weeks, with exposure to ultraviolet radiation (UVR)
from week 5 to 9 (mean ± SD; n= 4). (M), male; (F), female.
*P<0Æ05 compared with vehicle (Student’s t-test). Untr, untreated
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
946 Evaluation of piperine and analogues for vitiligo, L. Faas et al.
had received continuous treatment with UVR and a com-
pound. The number of cells mm
in Group D animals was
significantly higher than in the group that received continuous
UVR (Group F) or continuous topical treatment (Group A).
Retreatment with a compound alone significantly increased
the number of DOPA+ cells mm
compared with vehicle
control (Group C), reaching comparable levels with those in
animals treated continuously with a compound alone (Group
A). However, the number of cells mm
was lower than for
groups that were retreated with UVR alone (Group D) or with
a compound and UVR (Group E). The results obtained in
female mice (Fig. 9b) showed the same trends as in males,
except that in animals retreated with UVR alone (Group D) or
UVR with a compound (Group E) no significant differences
were observed compared with vehicle controls.
To investigate whether the differences in pigmentation
observed were due to an increase in melanocyte number or in
melanin production, DOPA+ cells were classified as highly or
poorly melanized according to the content of pigment gran-
ules, and the percentage of DOPA+ cells in each category per
of skin was calculated for each experimental group. As
expected, UVR exposure considerably increased the degree of
melanization of DOPA+ cells (Fig. 10c and Groups B, D, E
and F in Fig. 11a,b) compared with mice treated with a
compound alone (Fig. 10b or Groups A and C in Fig. 11a,b),
where poorly melanized cells were predominant.
4 5 6 7 8 9 10 11 12 13
7 8 9 10111213
Group A: Compound only 13 weeks Group B: As group A, plus UV on weeks 5–13
Group C: Interrupt treatment,
then compound only Group D: Interrupt treatment, then UV only
Group E: Interrupt treatment,
then compound + UV
4 5 6 7 8 9 10 11 12 13
** ** ** ** ** **
Group F: Treat with UV only
Fig 8. Pigmentation response of male mouse skin (n= 4) to continuous (Groups A, B, F) or discontinuous (Groups C–E) treatment as
summarized in Figure 2. Mice were treated for 13 weeks with topical compounds alone (A) or with additional ultraviolet radiation (UVR)
exposure (B). For other groups, treatment was interrupted and restarted as compounds only (C), UVR only (D), or compounds + UVR (E).
Group F received UVR alone. Pigmentation scores range from 1 (freckles) to 5 (black). *P<0Æ05 compared with vehicle; **P<0Æ05 compared
with Group B (Mann–Whitney U-test).
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
Evaluation of piperine and analogues for vitiligo, L. Faas et al. 947
Topical treatment of HRA Skh-II mice with PIP, or two of its
synthetic derivatives, THP and rCHP, stimulates the develop-
ment of even skin pigmentation in vivo after four or more
weeks of continuous topical application. The darkening of skin
in treated areas corresponds with an increase in the number
of DOPA+ melanocytes. This in vivo finding correlates well
with our previous studies showing the stimulation of in vitro
melanocyte proliferation by PIP and chemically related com-
Animals treated with PIP or analogues before
UVR exposure showed more rapid and darker pigmentation
than those treated with UVR exposure or a compound alone
(Fig. 8). These findings highlight the potential of these com-
pounds as novel treatments for vitiligo. Notably, supplement-
ing UVR with these compounds may offer a means of
reducing UV exposure in vitiligo therapy, thereby reducing
the risk of developing skin cancer.
The degree of skin pigmentation is a consequence of both
number of melanocytes and their degree of melanization.
UVR, for example, stimulates both melanocyte proliferation
DOPA+ cells/mm2
DOPA+ cells/mm2
A B C D E F Untr
A B C D E F Untr
Fig 9. DOPA+ cell numbers (mean ± SD) in male (a) and female (b)
mouse skin (n= 4) after continuous and discontinuous treatment as
in Fig. 2, Groups A–F). Cell numbers correlate well with visually
determined pigmentation scores. *Significant increase compared with
vehicle; **Significant decrease compared with groups B and D
(P<0Æ05; Student’s t-test).
(a) (b) (c)
Fig 10. Histology of DOPA+ melanocytes in skin from animals treated with (a) vehicle, (b) piperine (PIP) and (c) PIP + ultraviolet radiation
(UVR). The ratio of highly (arrowheads) to poorly (arrows) pigmented melanocytes increases in the order a < b < c. Original magnification
Fig 11. Percentage of highly (black bars) vs. poorly (grey bars)
melanized melanocytes in male (a) and female (b) mice (mean ± SD;
n= 4). Groups receiving ultraviolet radiation (UVR) (B, D, E and F)
show greater melanization than those receiving a compound alone
(A, C) prior to histology. Treatment groups as shown in Figure 2.
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
948 Evaluation of piperine and analogues for vitiligo, L. Faas et al.
and melanin synthesis.
The relatively low pigmentation
scores in the absence of UVR (Fig. 8) and the low degree of
melanization of DOPA+ cells observed in skin treated with a
compound alone (Fig. 11) suggests that these compounds
stimulate melanocyte proliferation rather than melanin synthe-
sis. This is in good agreement with in vitro data showing that
PIP derivatives do not stimulate melanin production although
they stimulate melanocyte proliferation.
Retreatment with
a compound alone induced a higher difference in DOPA+ cell
numbers between compound and vehicle (Fig. 9a,b, Group C)
than did retreatment with UVR alone (Fig. 9a,b, Group D),
This again suggests that the primary effect of piperine is to
stimulate rapid melanocyte proliferation and population of
epidermal areas. This phenomenon, as well as effects on mela-
nocyte differentiation by PIP analogues could be further exam-
ined through bromodeoxyuridine incorporation experiments
and immunohistochemical determination of specific markers
such as Kit, Mitf, TRP-1 and TRP-2, indicative of different
developmental stages of melanocytes.
A gender difference in induced pigmentation was observed
in these studies, with males showing a greater response than
female mice. However, skin penetration of PIP was the same
in both sexes using a Franz cell model (Fig. 3), suggesting
equal bioavailability in both sexes. However, the mild inflam-
matory and irritant effects seen with PIP and its analogues
(Figs 4 and 5) may be significant, in explaining the activity of
the compounds per se, as well as the differences in pigmentary
response of male and female animals. Females showed a lower
inflammatory response than males. Gender differences in sen-
sitivity to UVR have also been observed in humans with males
showing a greater sensitivity and lower MED.
An important feature of treatment with PIP and its analogues
is the even pigmentation pattern that is obtained with or with-
out additional UVR (Fig. 6). This correlates well with the find-
ing that DOPA+ melanocytes in treated skin (Fig. 10) were
distributed in interfollicular areas rather than associated with
hair follicles, and suggests an active epidermal distribution of
melanocytes after treatment with PIP or its analogues. An exam-
ination of the in vivo cutaneous absorption and distribution of
PIP and its analogues, particularly the relative roles of the
stratum corneum and hair follicles, would be of interest in
determining their site of action and understanding the repig-
mentation patterns seen. Hair follicles are known to play a sig-
nificant role in the percutaneous absorption of many drugs.
The use of PIP and its analogues in vitiligo clearly offers po-
tential cosmetic advantages over the use of PUVA or UVR alone
(common current treatments for vitiligo) if an even pattern can
be obtained in humans. PUVA repigmentation, when successful,
progresses from a perifollicular pattern in early stages of ther-
apy, with the circular patches of pigment coalescing after fur-
ther treatment to a more even pattern in humans
and in
A similar progression has been observed using therapies
based on UVR.
Mice treated with UVR alone in the present
study also showed this speckled pattern (Fig. 6c).
Continuous treatment appears to be needed to maintain
pigmentation as shown by the gradual, though not complete,
loss of pigmentation when treatment is suspended. Retreat-
ment with either UVR alone, topical compounds alone or the
combination of both, restored pigmentation over a shorter
period of time than in naı
¨ve mice. This indicates the possible
presence of poorly melanized melanocytes but in greater num-
bers than in naı
¨ve skin. Consistent with our previous observa-
tions, the resulting pigmentation after retreatment with UVR
often showed darker perifollicular areas, in contrast to the
even pattern produced by retreatment with a compound alone
or a compound plus UVR.
Although our results suggest that the melanocyte is the main
target for these compounds, no known melanocytic receptor
for PIP or its derivatives has been identified to date. Interest-
ingly, the presence of one of the subtypes of vanilloid receptor,
the receptor for PIP and PIP-related molecules, has recently
been shown in keratinocytes.
In this respect, it is well known
that melanocytes and keratinocytes exhibit a close functional
relationship. Keratinocytes are known to produce several fac-
tors that regulate melanocyte activity and survival, such as
nerve growth factor, granulocyte-monocyte colony stimulating
factor, basic fibroblast growth factor, endothelin-1, stem cell
factor and other cytokines.
It has recently been shown
that some of these molecules are imbalanced in vitiligo skin,
suggesting that the deregulation of the melanocyte microenvi-
ronment could be involved in the selective destruction of mel-
anocytes in vitiligo. Indeed, an impairment of keratinocyte
function is observed in perilesional skin.
It is reasonable to
speculate that PIP and PIP analogues could have an effect on
modulating cytokine production by keratinocytes in vivo, con-
sequently stimulating melanocyte replication or activity, which
could result in an increase in pigmentation. Nevertheless, we
have observed an effect on PKC activation by PIP in vitro that is
suggestive of a direct effect on melanocytes.
In summary, we have shown that topical treatment with
PIP, and two of its synthetic analogues, THP and rCHP, stimu-
lates even pigmentation in mice. Topical treatment in combin-
ation with low dose UVR significantly enhances the
pigmentation response with results that are cosmetically better
compared with conventional vitiligo therapies when applied
to mice. Although fading may occur when the treatment is
interrupted, a good pigmentation response is readily achieved
again after short periods of retreatment. Side effects, such as
irritation and inflammation, were transient and tolerable.
These data provide strong support for the future clinical evalu-
ation of PIP and its derivatives as novel treatments for vitiligo.
This work was funded by BTG International Ltd and by an
Overseas Research Student Award to RV. We thank Dr Marc
Brown and Richard Harper of the Pharmacy Department,
King’s College London for, respectively, guidance on the Franz
cell assay and photography of the mice. At St John’s Institute
of Dermatology, we acknowledge the technical support in
histology provided by Guy Orchard and thank Dr Susan
Walker for helpful discussions and critical reading of the
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
Evaluation of piperine and analogues for vitiligo, L. Faas et al. 949
manuscript. Dr A Soumyanath and Professor AR Young held
joint responsibility for the supervision of this work.
1 Agarwal G. Vitiligo: an under-estimated problem. Fam Pract 1998;
2 Taieb A. Intrinsic and extrinsic pathomechanisms in vitiligo. Pigment
Cell Res 2000; 13 (Suppl. 8):41–7.
3 Njoo MD, Bossuyt PM, Westerhof W. Management of vitiligo.
Results of a questionnaire among dermatologists in The Nether-
lands. Int J Dermatol 1999; 38:866–72.
4 Nordlund JJ, Ortonne JP. Genetic hypomelanoses: acquired
de-pigmentation. In: The Pigmentary System: Physiology and Pathophysiology,
2nd Edition (Nordlund JJ, Boissy RE, Hearing VJ et al., eds).
Oxford: Blackwell Publishing, 2006; 551–98.
5 Kwok YK, Anstey AV, Hawk JL. Psoralen photochemotherapy
(PUVA) is only moderately effective in widespread vitiligo: a
10-year retrospective study. Clin Exp Dermatol 2002; 27:104–10.
6 Whitton ME, Ashcroft DM, Barrett CW, Gonzalez U. Interventions
for vitiligo. Cochrane Database Syst Rev 2006; 1:1–37.
7 Arrunategui A, Arroyo C, Garcia L et al. Melanocyte reservoir in
vitiligo. Int J Dermatol 1994; 33:484–7.
8 Cui J, Shen L, Wang G. Role of hair follicles in the re-pigmenta-
tion of vitiligo. J Invest Dermatol 1991; 97:410–16.
9 Jimbow K. Vitiligo. Therapeutic advances. Dermatol Clin 1998;
10 Ortonne JP, Schmitt D, Thivolet J. PUVA-induced re-pigmentation
of vitiligo: scanning electron microscopy of hair follicles. J Invest
Dermatol 1980; 74:40–2.
11 Lin Z, Hoult RSJ, Bennett DC, Raman A. Stimulation of mouse
melanocyte proliferation by Piper nigrum fruit extract and its main
alkaloid, piperine. Planta Med 1999; 65:600–3.
12 Venkatasamy R, Faas L, Young AR et al. Effects of piperine analogs
on stimulation of melanocyte proliferation and melanocyte differ-
entiation. Bioorg Med Chem 2004; 12:1905–20.
13 Kipp C, Lewis EJ, Young AR. Furocumarin-induced epidermal
melanogenesis does not protect against skin photocarcinogenesis in
mice. Photochem Photobiol 1998; 67:126–1.
14 Welsh BM, Mason RS, Halliday GM. Topical all-trans retinoic acid
augments ultraviolet radiation-induced increases in activated mela-
nocyte numbers in mice. J Invest Dermatol 1999; 112:271–8.
15 Kinley JS, Brunborg G, Moan J, Young AR. Photoprotection by
furocoumarin-induced melanogenesis against DNA photodamage
in mouse epidermis in vivo.Photochem Photobiol 1997; 65:486–91.
16 Akomeah FK, Martin GP, Brown MB. Variability in human skin
permeability in vitro: comparing penetrants with different physico-
chemical properties. J Pharm Sci 2007; 96:824–34.
17 Soumyanath A, Venkatasamy R, Joshi M et al. UV irradiation affects
melanocyte stimulatory activity and protein binding of piperine.
Photochem Photobiol 2006; 82:1541–8.
18 McKinlay AF, Diffey BL. A reference action spectrum for ultraviolet
induced erythema in human skin. CIE J 1987; 66:17–22.
19 Parrish JA, Jaenicke KF, Anderson RR. Erythema and melanogenesis
action spectra of normal human skin. Photochem Photobiol 1982;
20 Morison WL, Hood AF, Sayre RM, Agin PP. A novel model for
testing enhancers of pigmentation. Photodermatol 1987; 4:32–5.
21 Nordlund JJ, Ackles AE, Traynor FF. The proliferative and toxic
effects of ultraviolet light and inflammation on epidermal pigment
cells. J Invest Dermatol 1981; 77:361–8.
22 Lashmar UT, Hadgraft J, Thomas N. Topical application of penetra-
tion enhancers to the skin of nude mice: a histopathological study.
J Pharm Pharmacol 1989; 41:118–22.
23 Louw L. Keloids in rural black South Africans. Part 3: a lipid model
for the prevention and treatment of keloid formations. Prostaglandins
Leukot Essent Fatty Acids 2000; 63:255–62.
24 Tanojo H, Boelsma E, Junginger HE et al. In vivo human skin per-
meability enhancement by oleic acid: a laser Doppler velocimetry
study. J Control Release 1999; 58:97–104.
25 Carsberg CJ, Warenius HM, Friedmann PS. Ultraviolet radiation-
induced melanogenesis in human melanocytes. Effects of modulat-
ing protein kinase C. J Cell Sci 1994; 107:2591–7.
26 Kawaguchi Y, Mori N, Nakayama A. Kit+ melanocytes seem to
contribute to melanocyte proliferation after UV exposure as precur-
sor cells. J Invest Dermatol 2001; 116:920–5.
27 Broekmans WMR, Vink AA, Boelsma E et al. Determinants of
skin sensitivity to solar irradiation. Eur J Clin Nutr 2003; 57:1222–
28 Meidan VM, Bonner MC, Michniak BB. Transfollicular drug deliv-
ery – is it a reality? Int J Pharm 2005; 306:1–14.
29 Mofty ME, Zaher H, Esmat S et al. PUVA and PUVB in vitiligo – are
they equally effective? Photodermatol Photoimmunol Photomed 2001;
30 Inoue K, Koizumi S, Fuziwara S et al. Functional vanilloid receptors
in cultured normal human epidermal keratinocytes. Biochem Biophys
Res Commun 2002; 291:124–9.
31 Abdel-Malek Z, Swope VB, Pallas J et al. Mitogenic, melano-
genic, and cAMP responses of cultured neonatal human melano-
cytes to commonly used mitogens. J Cell Physiol 1992; 150:
32 Gordon PR, Mansur CP, Gilchrest BA. Regulation of human mela-
nocyte growth, dendricity, and melanization by keratinocyte
derived factors. J Invest Dermatol 1989; 92:565–72.
33 Grichnik JM, Burch JA, Burchette J, Shea CR. The SCF KIT pathway
plays a critical role in the control of normal human melanocyte
homeostasis. J Invest Dermatol 1998; 111:233–8.
34 Halaban R, Langdon R, Birchall N et al. Paracrine stimulation
of melanocytes by keratinocytes through basic fibroblast growth
factor. Ann N Y Acad Sci 1988; 548:180–90.
35 Imokawa G, Yada Y, Morisaki N, Kimura M. Biological character-
ization of human fibroblast-derived mitogenic factors for human
melanocytes. Biochem J 1998; 15:330.
36 Imokawa G. Autocrine and paracrine regulation of melanocytes in
human skin and in pigmentary disorders. Pigment Cell Res 2004;
37 Yaar M, Eller MS, DiBenedetto P et al. The trk family of receptors
mediates nerve growth factor and neurotrophin-3 effects in mela-
nocytes. J Clin Invest 1994; 94:1550–62.
38 Moretti S, Spallanzani A, Amato L et al. New insights into the path-
ogenesis of vitiligo: imbalance of epidermal cytokines at sites of
lesions. Pigment Cell Res 2002; 15:87–92.
39 Moellmann G, Klein-Angerer S, Scollay DA et al. Extracellular gran-
ular material and degeneration of keratinocytes in the normally
pigmented epidermis of patients with vitiligo. J Invest Dermatol 1982;
2008 The Authors
Journal Compilation 2008 British Association of Dermatologists British Journal of Dermatology 2008 158, pp941–950
950 Evaluation of piperine and analogues for vitiligo, L. Faas et al.
... This bothersome disease begins on average at the age of 20 but is most common between the ages of 10 and 30 [5]. This apparent disorder can lead to many psychological, social and physiological problems in individuals [6]. ...
... Despite the excellent therapeutic properties of piperine, it has a low aqueous solubility (40 mg/L at 18 C) [6] which leads to poor skin penetration and poor topical skin bioavailability. In addition, piperine has shown excessive degradation in acid hydrolysis, base hydrolysis, and under oxidative stress [17], limiting its effective use in conventional dosage forms. ...
... One of these herbal components, piperine, is an alkaloid found in several species of piper, mainly P. nigrum and P. longum, among other species [5]. Studies have shown that piperine can induce skin pigmentation even when used alone, thanks to its melanocyte proliferation potential [3,6]. In addition, topical treatment with piperine may play a role in protecting melanocytes from being destroyed by reactive oxygen species, thanks to its antioxidant capacity [7]. ...
Full-text available
Background: Eplerenone is a member of antihypertensives used individually or in combination with other medicines. Eplerenone exhibits poor solubility and is considered a class II drug. Objective: Increasing the solubility of eplerenone by using both liquid and solid self-emulsifying drug delivery system as an alternative to its marketed tablet product. Methods: Solubility studies of eplerenone were done with different oils, surfactants, and co-surfactants to determine which one has the highest solubility for eplerenone and determine the preference in the formulations of liquid self-emulsifying drug delivery system. The solidification process was carried out with the adsorption to solid carrier method. Optimal ratios of components were specified with pseudo-ternary phase diagram technique. Self-emulsifying drug delivery system formulations were characterized in terms of chemical interaction, droplet size/distribution, crystallization behaviors, and rheological evaluation. In vitro drug release studies were conducted and compared to pure drug and marketed product. Results: The solubility screening results showed high solubility of EPL in triacetin (11.99 mg/mL) as oil, Kolliphor®EL (≈ 2.65 mg/mL), and Tween®80 (≈ 1.91 mg/mL) as surfactant and polyethylene glycol 200 (PEG200) (≈ 8.50 mg/mL), dimethyl sulfoxide (≈ 7.57 mg/mL), Transcutol®P (≈ 6.03 mg/mL) as co-surfactant, respectively. Rheology studies revealed that liquid self-emulsifying drug delivery formulations exhibited non-Newtonian pseudoplastic flow. Conclusion: Solid self-emulsifying drug delivery systems prepared with Aerosil and Neusilin have shown tremendous improvement in terms of eplerenone dissolution by releasing the entire dose with boosted effect within 5 and 30 minutes respectively compared to the marketed product and pure eplerenone (p < 0.05).
... Anderson 17 initially used alkalis to hydrolyze piperine into a base and an acid, which were subsequently given the names 18 piperidine and piperic acid. The piperoyl chloride's reaction with piperidine produced the alkaloid 19 . . Following the administration of carbamazepine and carbamazepine combined with piperine, the analysis of pharmacokinetic parameters from blood samples taken at regular intervals revealed that piperine considerably raised the mean plasma concentrations of carbamazepine in both dose groups. ...
Full-text available
Drug oral absorption is a crucial concern, particularly when the medication is costly, poorly bioavailable, and administered for extended periods of time. Chemical substances known as "bioenhancers" are those that, when combined with pharmaceuticals, increase their bioavailability without having a synergistic impact on the drug itself. Toxicity, expense, poor bioavailability, and long-term medication administration all contribute to the need for bioenhancers, which aid in solving the majority of these issues. Piperine, also known as 1-peperoyl piperidine, is an aromatic alkaloid produced by the Piper species. Piperine alters the lipid milieu and membrane dynamics at the site of absorption to improve permeability. The molecular nature of piperine makes it appropriate for inhibiting enzymes. By blocking several metabolising enzymes, it increases the bioavailability of many medications, including carbamazepine, curcumin, ciprofloxacin, ampicillin, metronidazole, oxytetracycline, and many more. As a result, piperine, a potent inhibitor of medication metabolism, effectively increases absorption. The mechanism, metabolic inhibition, influence of structural alterations on activity, and medications that are bioenhanced by piperine are all explored in the review that follows. It offers insight into the use of piperine as a useful bioenhancer and the advantages of a bioenhanced drug formulation over one without one. Bioavailability enhancers are typically plant-based molecules that support the biological activity, bioavailability, or uptake of drugs in combination therapy. This review article finishes with discussing piperine's capacity to increase bioavailability. Keywords: Bioenhancers, Piperine, Oral absorption, Alkaloid.
... de dicho informe sugieren que la combinación de piperina y sus derivados sintéticos aumentaba significativamente la respuesta a la pigmentación con la posible reducción del cáncer de piel. Además, la piperina inducía a una estimulación de casi el 300% de la proliferación de melanocitos en modelos murinos después de 8 días de tratamiento in vitro."(Faas et al., 2008) Por otra parte, en otro estudio se evaluó la eficacia de la piperina combinada con dosis de radiación ultravioleta en el cual 63 paciente participaron, haciendo estudios de casos y controles tratados un grupo con piperina al 1% y placebo, adicionalmente ambos grupos recibieron fototerapia.En el grupo de pacientes que recibieron la piper ...
El vitíligo es una enfermedad autoinmune que da como resultado la despigmentación de la piel, generando un importante impacto en el estilo de vida y la autoestima de quienes lo padecen. Por estas razones muchos pacientes en su afán de ver resultados permanentes optan por la aplicación de tratamientos con pocas o ninguna base científica, siendo víctimas de la propaganda engañosa o promesas milagrosas de productos de dudosa procedencia. El presentar artículo de revisión bibliográfica busca resumir los datos más importantes en torno a la enfermedad, desde su epidemiologia, fisiopatología y clínica hasta profundizar en las opciones terapéuticas mas importantes.
... 77 Herbal Taraxaci, Herba Agrimoniae and Cortex Phellodendri et al have remarkable anti-mites potential along with human skin safety versus the Demodex folliculorum. 78,79 Based on in vivo melanocyte proliferation potential of Piper nigrum extract (containing piperine) 80 there has been a clinical human study on few patients using the piperine extract, piperine alone or piperine extract associated with prostaglandins. Pigmentation effects were reported with such therapies and latter combination speeded up the pigmentation with changed pigmentation pattern. ...
Full-text available
Introduction: Vitiligo is disfiguring and devastating condition that can humans feel stigmatic and devalued. Melasma is a general condition of hyperpigmentation particularly involving the face. The pigmentation disorders of vitiligo (hypopigmentation or de-pigmentation) and melasma (Hypermelanosis) are common among the world's population (around 1% for vitiligo). Objective: The identification of medicinal plants used in the treatment of vitiligo and hypermelanosis. A systematic literature review on harms associated with the medicinal plants used in the treatment of vitiligo and hypermelanosis. To review and summarize information on reported adverse drug reactions (ADRs) associated with these medicinal plants contained in (where access is available) national and global individual case safety report databases. Methods: A systematic review of the literature with special reference to all types of clinical trial and case reports using biomedical databases including Medline, EMBASE, Scopus, International Pharmaceutical Abstracts and so forth to identify medicinal plants alone or as an adjuvant with other treatments and their safety/tolerability in the treatment of vitiligo and Hypermelanosis. Other sources of this search were medicinal plants text books, pharmacopoeias and authentic websites discussing possible treatments for vitiligo/hypermelanosis. It also included databases such as VigiAccess containing data from spontaneous reporting schemes for ADRs. Results: A total of 55 articles (47 clinical trials and 8 case reports) met the inclusion criteria. Some trials did not reported safety information, some did report, but not very well. Reports of blistering, erythema, acute hepatitis and mutagenesis with Psoralea corylifolia. Adverse effects of erythema (mild to severe), phototoxic reactions, mild raise in liver transaminases, gastrointestinal disturbances, burns, itching, scaling, depigmented macules, pruritis, and giddiness with the use of psoralens. Khellin-related erythema, perilesional hyperpigmentation, gastrointestinal disturbances, mild raise in liver transaminases and orthostatic complaints. Infrequent side effects with Ginkgo biloba. Lower grade of erythema and edema reported with the use of Polypodium leucotomos. Conclusion: Primarily the retrieved clinical studies were efficacy oriented and safety parameters were secondary in priority whilst the general protocol of clinical trials requires the screening of drugs/medicinal plants on the basis of safety studies before testing the clinical aspects of efficacy. Thereby it is recommended that efficacy studies may be followed once the safety has been established for a particular medicinal plant in treating vitiligo and hypermelanosis.
Piperine (a naturally occurring alkaloid), is a biologically active natural product belonging to the alkaloid series of compounds. In this study, the spectral characterization of piperine isolated from black pepper has been performed using FT-IR, 1H-NMR, and 13C-NMR. To explain spectral features, we have calculated the vibrational frequencies of the optimized structure of piperine using the B3LYP/6-311 + G(d,p) level of theory and NMR spectra using the GIAO method. All vibrational modes of the title molecule have been assigned based on potential energy distribution. The calculated scaled wavenumbers and NMR chemical shifts show good agreement with the experimental FT-IR and NMR data, respectively. To assess the bioactivity of piperine, we have first performed molecular docking using the ITK receptor and subsequently, the molecular dynamics simulation of the resulting complex for 100 ns. The results suggest the potential of piperine for possible anti-inflammatory action.
Vitiligo is a chronic disease of unknown etiology that causes progressive cutaneous depigmentation. Current pharmacological treatments have limited success and present significant risks. Many efforts have been made in recent years to explore new anti-vitiligo therapeutic strategies, including herbal-based therapies. The objective of the present review is to provide an updated overview on the most frequently used medicinal plants in the treatment of vitiligo. A bibliographical search was carried out in scientific databases Pubmed, Scifinder, Scopus, Google Scholar, and Medline up to October 2021 using the descriptors vitiligo, herbal, medicinal plants, and alternative therapies. In our search, the highest number of published studies comprise plants commonly used in traditional herbal medicine, highlighting the usefulness of ethnopharmacology in the discovery of new therapeutic agents. The review outlines current understanding and provides an insight into the role of psoralens and khellin (photosensitizing agents obtained from plants such as Cullen corylifolium or Ammi visnaga). The paper also describes other traditional herbs such as Ginkgo biloba, Phlebodium aureum, Piper nigrum, Picrorhiza kurroa, and Baccharoides anthelmintica that can likewise act as potential therapeutical agents. Based on our findings, photosensitizing agents in combination with phototherapy, the association of oral Phebodium aureum with phototherapies as well as oral G. biloba in monotherapy showed greater scientific evidence as therapeutic options. The research results emphasize that further investigation in this area is merited. More long-term follow up clinical trials and higher quality randomized trials are needed.
In this study, it was aimed to develop a topical piperine nanoemulsion (P-NE) using an ultrasonic emulsification process to find an alternative treatment option for some hypopigmentation disorders such as vitiligo.Results showed that 150 mg piperine loaded NE with 1:2 oil phase to Smix ratio and manufactured with 20 minutes ultrasonication duration with pre-emulsification step was the most durable formulation with a mean globule size of 216.00 ± 2.65, a PdI value of 0.094 ± 0.02 and a zeta potential value of -27.50 ± 2.48 mV.After three months of storage, the selected P-NE (coded as F3P2) remained kinetically stable without visual changes. This formulation displayed a sustained release pattern with a release of 81.92% ± 3.04% piperine after 72 hours. According to our in vitro activity experiments, it was determined that the P-NE had no toxic effect including dose of 5 mg/mL, and the highest P-NE formulation dose of 5 mg/mL increased tyrosinase activity by 32.77% ± 9.09% and melanogenesis activity by 34.90% ± 0.73%.In conclusion, it was demonstrated that the P-NE formulation may serve as a promising therapy for efficient treatment of vitiligo. Moreover, P-NE formulation may also help in preventing irregular pigmentation and skin cancer, associated with the conventional treatment methods.
Full-text available
Background Piperine is a type of amide alkaloid that exhibits pleiotropic properties like antioxidant, anticancer, anti-inflammatory, antihypertensive, hepatoprotective, neuroprotective and enhancing bioavailability and fertility-related activities. Piperine has the ability to alter gastrointestinal disorders, drug-metabolizing enzymes, and bioavailability of several drugs. The present review explores the available clinical and preclinical data, nanoformulations, extraction process, structure–activity relationships, molecular docking, bioavailability enhancement of phytochemicals and drugs, and brain penetration properties of piperine in the prevention, management, and treatment of various diseases and disorders. Main body Piperine provides therapeutic benefits in patients suffering from diabetes, obesity, arthritis, oral cancer, breast cancer, multiple myeloma, metabolic syndrome, hypertension, Parkinson's disease, Alzheimer’s disease, cerebral stroke, cardiovascular diseases, kidney diseases, inflammatory diseases, and rhinopharyngitis. The molecular basis for the pleiotropic activities of piperine is based on its ability to regulate multiple signaling molecules such as cell cycle proteins, anti-apoptotic proteins, P-glycoprotein, cytochrome P450 3A4, multidrug resistance protein 1, breast cancer resistance protein, transient receptor potential vanilloid 1 proinflammatory cytokine, nuclear factor-κB, c-Fos, cAMP response element-binding protein, activation transcription factor-2, peroxisome proliferator-activated receptor-gamma, Human G-quadruplex DNA, Cyclooxygenase-2, Nitric oxide synthases-2, MicroRNA, and coronaviruses. Piperine also regulates multiple signaling pathways such as Akt/mTOR/MMP-9, 5′-AMP-activated protein kinase-activated NLR family pyrin domain containing-3 inflammasome, voltage-gated K+ current, PKCα/ERK1/2, NF-κB/AP-1/MMP-9, Wnt/β-catenin, JNK/P38 MAPK, and gut microbiota. Short conclusion Based on the current evidence, piperine can be the potential molecule for treatment of disease, and its significance of this molecule in the clinic is discussed. Graphical abstract
Full-text available
Ethnopharmacological relevance Anethum sowa Roxb. ex Fleming (Syn. Peucedanum sowa Roxb. ex Fleming, Family: Apiaceae) is a pharmacologically important as aromatic and medicinal plant. Various parts of this plant are used in traditional medicine systems for carminative, uterine and colic pain, digestion disorder, flatulence in babies, appetite-stimulating agent and used to treat mild flue and cough. The essential oil is used for aromatherapy. It is also used as a spice for food flavouring and culinary preparations in many Asian and European countries. Aim of the review This review aims to provide a comprehensive and critical assessment from the reported traditional and pharmaceutical uses and pharmacological activities of the extracts, essential oil and phytoconstituents with emphasis on its therapeutic potential as well as toxicological evaluation of A. sowa. Materials and methods Online search engines such as SciFinder®, GoogleScholar®, ResearchGate®, Web of Science®, Scopus®, PubMed and additional data from books, proceedings and local prints were searched using relevant keywords and terminologies related to A. sowa for critical analyses. Results The literature studies demonstrated that A. sowa possesses several ethnopharmacological activities, including pharmaceutical prescriptions, traditional applications, and spice in food preparations. The phytochemical investigation conducted on crude extracts has been characterized and identified various classes of compounds, including coumarins, anthraquinone, terpenoids, alkaloid, benzodioxoles, phenolics, polyphenols, phenolic and polyphenols, fatty acids, phthalides and carotenoids. The extracts and compounds from the different parts of A. sowa showed diverse in vitro and in vivo biological activities including antioxidant, antiviral, antibacterial, analgesic and anti-inflammatory, Alzheimer associating neuromodulatory, cytotoxic, anticancer, antidiabetes, insecticidal and larvicidal. Conclusion A. sowa is a valuable medicinal plant which is especially used in food flavouring and culinary preparations. This review summarized the pertinent information on A. sowa and its traditional and culinary uses, as well as potential pharmacological properties of essential oils, extracts and isolated compounds. The traditional uses of A. sowa are supported by in vitro/vivo pharmacological studies; however, further investigation on A. sowa should be focused on isolation and identification of more active compounds and establish the links between the traditional uses and reported pharmacological activities with active compounds, as well as structure-activity relationship and in vivo mechanistic studies before integrated into the medicine. The toxicological report confirmed its safety. Nonetheless, pharmacokinetic evaluation tests to validate its bioavailability should be encouraged.
Vitiligo is an autoimmune disease of the skin which causes loss of melanocytes from the epidermis. Recently, it is demonstrated that oxidative stress (OS) plays a significant role in the immuno-pathogenesis of vitiligo. A major mechanism in the cellular defense against OS is activation of the nuclear factor erythroid2-related factor (Nrf2)-Kelch-like ECH-associated protein 1(Keap1)-antioxidant responsive element (ARE) signaling pathway. Recently it has been shown that vitiligo melanocytes have impaired Nrf2-ARE signaling. A number of drugs including those known as Nrf2 activators and those known to possess effects to activate Nrf2, have been used in treating vitiligo with certain therapeutic effects. Also, studies have shown that a number of compounds can protect melanocytes against OS via activating Nrf2. These compounds may be considered as candidates for developing new drugs for vitiligo in the future. Nrf2 can be considered as a potential therapeutic target for vitiligo.
The most comprehensive and integrated book on pigmentation. The Pigmentary System, Second Edition, gathers into one convenient, all-inclusive volume a wealth of information about the science of pigmentation and all the common and rare clinical disorders that affect skin color. The two parts, physiology (science) and pathophysiology (clinical disorders), are complementary and annotated so that those reading one part can easily refer to relevant sections in the other. For the clinician interested in common or rare pigment disorders or the principles of teaching about such disorders, this book provides an immediate and complete resource on the biologic bases for these disorders. For the scientist studying the biology of melanocyte function, the book provides a list of disorders that are related to basic biological functions of melanocytes. New features of this Second Edition include: Completely new section on the basic science of pigmentation - explaining the integration of melanocyte functions with other epidermal cells and with various organ systems like the immune system. New chapters on pigmentary disorders related to intestinal diseases, the malignant melanocyte, benign proliferations of melanocytes (nevi) and phototherapy with narrow band UV. All clinical chapters include the latest genetic findings and advances in therapy. More than 400 color images of virtually all clinical disorders. The book is ideal for all dermatologists and especially those interested in disorders of pigmentation. It is of particular use for pediatric dermatologists and medical geneticists caring for patients with congenital and genetic pigmentary disorders. This authoritative volume will fill the gap for dermatology training programs that do not have local experts on pigmentation. Basic and cosmetic scientists studying pigmentation and melanocytes will find the science and clinical correlations very useful in showing human significance and relevance to the results of their studies.
Erythemal action spectra have been the subject of experimental and theoretical interest for over 50 years. Despite all of these studies, or perhaps because of the diversification of the published data resulting from them, no one action spectrum has been universally adopted. In response to a request by Division 6 of the Commission on Illumination (CIE) for the formulation of a reference action spectrum for adoption by the CIE, members of Technical Committee 2 propose the following: 1. The reference action spectrum for erythema should be that shown in figure 1 (proposed action spectrum). 2. The data contained in the action spectrum should be used only as spectral weighting factors to indicate the approximate relative erythemal efficacy of a light source (between 250 and 400 nm).
Vitiligo is the most commonly acquired hypomelanosis, and is restricted to a limited cutaneous territory (focal/segmental vitiligo [SV]) or generalized in symmetric patches (nonsegmental vitiligo [NSV]). In the majority of cases, vitiligo corresponds to a loss of melanocytes, first in the epidermal compartment, and later in the follicular reservoir where most melanocytic stem cells are probably situated. There are many data currently supporting an impaired redox status of the epidermal melanin unit as a primary defect leading to inappropriate immune responses in NSV. SV is probably a mosaic developmental predisposition to melanocytic loss, with similar mechanisms at work on a limited scale, as suggested by its cutaneous distribution and success of autografting. In NSV, engraftment of autologous melanocytes is less durable, especially in areas prone to repeated trauma or pressure. Although melanocytes are the obvious target of the disease, keratinocytes, as providers of antioxidant molecules to melanocytes as well as cofactors in the synthesis of melanin, are probably involved. The production of autoantibodies and specific cytotoxic T cells is not surprising in the context of the massive uptake of melanocytic antigens by Langerhans cells in unstable vitiligo vulgaris, thereby allowing the self-perpetuation of lesions. This article reviews the recent data on the pathophysiology of vitiligo, on the basis of clinical classification and the intrinsic/extrinsic nature of proposed pathomechanisms. Unfortunately, basic issues like pathological staging, clinical scoring, and eliciting factors have not yet been fully resolved. More genetic studies in vitiligo-prone families and in specific genetic disorders associated with vitiligo are also needed.
Background Several therapeutic options are available for the treatment of vitiligo. Concern exists that there is no uniform approach towards the management of vitiligo among Dutch dermatologists. Methods A written survey concerning the management of vitiligo was sent to 332 dermatologists in The Netherlands. Results The response rate was 86%. “Giving information and reassurance concerning the nature of disease” was regarded by most dermatologists (68%) as being the most important goal in the management of vitiligo. Only 16% of the dermatologists aimed for active treatment in vitiligo. The reported therapy choices in children resembled those of adults, except that slightly more dermatologists did not prescribe active therapy in children. Nine different therapeutic modalities were mentioned as first choice therapies. Topical corticosteroids were indicated by most dermatologists as first choice therapy (241 out of 266, i.e. 91%); however, only 2% indicated that 50% or more of the patients achieved a successful treatment; 66% found that less than 25% of the patients were successfully treated with topical corticosteroids. Only 15% of the respondents reported that 50% or more of the patients were treated successfully with narrow-band UVB. The observed response profile to broad-band UVB therapy was found to be comparable with that of narrow-band UVB. The classical therapy with oral psoralen plus UVA (PUVA) was prescribed as first choice therapy by only 12% (32 out of 266) of the dermatologists. Only 6% of these respondents observed that 50% or more of the patients achieved successful therapy using oral PUVA. The recommended maximum treatment duration for topical corticosteroids, oral PUVA, and UVB therapy was found to vary from 3 to 12 months. Conclusions Most dermatologists in The Netherlands do not offer active treatment in vitiligo, probably because the estimated effectiveness of (nonsurgical) repigmentation therapy is low. In cases where treatment is prescribed, there appears to be no consensus on the choice of therapies and treatment strategies. The development of practice guidelines may be helpful in reducing inappropriate care and improving treatment outcome.
The following studies have been undertaken to compare and correlate the effects of 12-O-tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration. NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10(-4) M IBMX. TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly. Human recombinant (hr) bFGF could replace TPA in the NHM growth medium. Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10(-4) M IBMX. The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX. TPA alone in the absence of CT and IBMX caused a dose-dependent stimulation of tyrosinase activity. Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10(-4) M IBMX. Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity. In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone. Neither TPA nor hrbFGF alone could increase intracellular cAMP levels. The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF. Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose-dependent increase in cAMP concentration. Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.
Vitiligo is a common pigment disease that is difficult to treat. The mechanism of repigmentation is not known. We combined Dopa-Toluidine Blue complex stain, hair follicle split-Dopa stain, and hair follicle split-scanning electron microscope (SEM) to observe the changes of melanocytes in 23 normal, 24 vitiliginous, and 36 repigmented skin specimens. We found that only active (Dopa-positive) melanocytes existed in the epidermis of normal skin. There were some inactive (Dopa-negative) melanocytes in the outer root sheaths of normal hair follicles, which form the melanocyte reservoir in human skin. In the patients with vitiligo the active melanocytes in the epidermis were totally missing, whereas the inactive melanocytes in the outer root sheaths of hair follicles were not affected. Treatment stimulated the inactive melanocytes in the middle and/or lower parts of the outer root sheaths of hair follicles to divide, proliferate, and migrate upward along the surface of the outer root sheath to the nearby epidermis, where the melanocytes continued to migrate radially to form the pigmented island visible clinically in repigmented vitiligo lesions. During the migration to the epidermis, the melanocytes matured gradually from an inactive phase to an active condition. In conclusion, the existence of these inactive melanocytes provided the melanocyte sources for repigmentation of vitiligo.
Epidermal pigmentation involves the synthesis of melanin in melanocytes and its transfer to surrounding keratinocytes, where it functions in photoprotection. To investigate the possible role of the keratinocyte in regulating pigmentation, human keratinocytes were incubated for 24 h in a defined culture medium, which was then transferred to pure human melanocyte cultures. After 1 week, the conditioned medium produced a fourfold increase in melanocyte yield and a seven-fold increase in total melanin. Increased melanocyte dendricity was clearly visible within 24 h as well. Ultrafiltration of the keratinocyte-conditioned medium suggested approximately one-half of the growth promoting activity as well as most of the dendricity and melanization stimulating activities were of low molecular weight (less than 500 Da). High molecular weight fractions stimulated only melanocyte growth. Of the several known keratinocyte-derived factors tested, none could be implicated as a mediator of the observed effects. Basic fibroblast growth factor, known to stimulate melanocyte growth in some culture systems, failed to stimulate growth, dendricity, or melanin content when added to the complete non-conditioned medium. Interleukin-1 alpha, interleukin-1 beta, 12-hydroxyeicosatetraenoic acid, prostaglandin E2, leukotriene B4, and adenosine 3',5'-cyclic monophosphate analogues also had no effect. These studies demonstrate that keratinocytes in vitro release factors that modulate melanocyte behavior and expand our understanding of controls for human epidermal pigmentation.