UV irradiation affects melanocyte stimulatory activity and protein binding of piperine

Article abstract of DOI:10.1562/2006-04-21-RA-882

Piperine, the major alkaloid of black pepper (Piper nigrum L.; Piperaceae), stimulates melanocyte proliferation and dendrite formation in vitro. This property renders it a potential treatment for the skin depigmentation disorder vitiligo. However, piperine does not stimulate melanin synthesis in vitro, and treatments based on this compound may therefore be more effective with concomitant exposure of the skin to ultraviolet (UV) radiation or sunlight. The present study investigated the effect of UVA and simulated solar radiation (SSR) on the chemical stability of piperine, its melanocyte stimulatory effects and its ability to bind protein and DNA. Chromatographic and spectroscopic analysis confirmed the anticipated photoisomerization of irradiated piperine and showed the absence of any hydrolysis to piperinic acid. Isomerization resulted in the loss of ability to stimulate proliferation of a mouse melanocyte cell line, and to bind to human serum albumin. There was no evidence of DNA binding by piperine either before or after irradiation, showing the absence of photoadduct formation by either piperine or its geometric isomers. This is unlike the situation with psoralens, which form DNA adducts when administered with UVA in treating skin diseases. The present study suggests that exposure to bright sunlight should be avoided both during active application of piperine to the skin and in the storage of piperine products. If UVA radiation is used with piperine in the treatment of vitiligo, application of the compound and irradiation should be staggered to minimize photoisomerization. This approach is shown to effectively induce pigmentation in a sparsely pigmented mouse strain.

Full text of DOI:10.1562/2006-04-21-RA-882

UV Irradiation Affects Melanocyte Stimulatory Activity and Protein Binding of
Piperine
Author(s): Amala Soumyanath, Radhakrishnan Venkatasamy, Meghna Joshi, Laura Faas, Bimpe
Adejuyigbe, Alex F. Drake, Robert C. Hider, and Antony R. Young
Source: Photochemistry and Photobiology, 82(6):1541-1548.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/2006-04-21-RA-882
URL: http://www.bioone.org/doi/full/10.1562/2006-04-21-RA-882
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Photochemistry and Photobiology, 2006, 82: 1541–1548
UV Irradiation Affects Melanocyte Stimulatory Activity and
Protein Binding of Piperine
Amala Soumyanath*1y, Radhakrishnan Venkatasamy1, Meghna Joshi1, Laura Faas2z
Bimpe Adejuyigbe1, Alex F. Drake1, Robert C. Hider1 and Antony R. Young2
,
1Department of Pharmacy, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, UK
2St. John’s Institute of Dermatology, Division of Genetics and Molecular Medicine, King’s College London,
St. Thomas’ Hospital, London SE1 7EH, UK
Received 14 April 2006; accepted 4 July 2006; published online 14 July 2006 DOI: 10.1562/2006-04-21-RA-882
Melanocytes are pigment-producing dendritic cells located within
ABSTRACT
the basal layer of the epidermis and in the matrix of hair follicles.
Piperine, the major alkaloid of black pepper (Piper nigrum L.;
Piperaceae), stimulates melanocyte proliferation and dendrite
formation in vitro. This property renders it a potential
treatment for the skin depigmentation disorder vitiligo.
However, piperine does not stimulate melanin synthesis in vitro,
and treatments based on this compound may therefore be more
effective with concomitant exposure of the skin to ultraviolet
(UV) radiation or sunlight. The present study investigated the
effect of UVA and simulated solar radiation (SSR) on the
chemical stability of piperine, its melanocyte stimulatory effects
and its ability to bind protein and DNA. Chromatographic and
spectroscopic analysis confirmed the anticipated photoisomer-
ization of irradiated piperine and showed the absence of any
hydrolysis to piperinic acid. Isomerization resulted in the loss of
ability to stimulate proliferation of a mouse melanocyte cell line,
and to bind to human serum albumin. There was no evidence
of DNA binding by piperine either before or after irradiation,
showing the absence of photoadduct formation by either
piperine or its geometric isomers. This is unlike the situation
with psoralens, which form DNA adducts when administered
with UVA in treating skin diseases. The present study suggests
that exposure to bright sunlight should be avoided both during
active application of piperine to the skin and in the storage of
piperine products. If UVA radiation is used with piperine in
the treatment of vitiligo, application of the compound and
irradiation should be staggered to minimize photoisomeriza-
tion. This approach is shown to effectively induce pigmentation
in a sparsely pigmented mouse strain.
Melanin is synthesized within organelles known as melanosomes,
which are transferred through melanocytic dendrites to epidermal
keratinocytes (4), resulting in the observed pigmentation of mam-
malian skin. The stimulatory effects of piperine on melanocyte
proliferation and dendricity render it a potential treatment for
vitiligo, a skin disorder characterized by depigmented lesions (4).
Melanocytes have been shown to be absent, or present in very
small numbers, in vitiligo lesions apart from reservoirs found in the
hair follicles (5–7). Piperine is expected to cause the repopulation
of vitiligo patches through a stimulatory effect on perilesional and
follicular melanocytes. However, piperine does not stimulate mel-
anin synthesis in melanocytes in vitro (2,3). A potential treatment
option is to expose the skin to ultraviolet radiation (UVR) along
with piperine in order to induce pigmentation in the new mela-
nocytes. UVR can act directly on melanocytes to increase skin
pigmentation or indirectly through the release of keratinocyte-
derived factors such as cytokines, eicosanoids, growth factors,
nitric oxide or melanotropic hormones (8–10).
UVA irradiation (320–400 nm) is already used in the treatment
of vitiligo, in conjunction with orally or topically administered
psoralens (PUVA therapy—psoralens plus UVA) (11). Psoralens
form monofunctional and bifunctional photoadducts with cellular
DNA upon exposure to UVA; this process stimulates melanocyte
replication and melanogenesis (12,13). Solar simulated radiation
(SSR) plus 5-methoxypsoralen (5-MOP) has been used to induce
human pigmentation (12). 8-MOP, which is more commonly used
in clinical practice, has been shown to induce epidermal mela-
nogenesis in human (14) and murine (15) skin when administered
with UVA.
INTRODUCTION
Unlike psoralens (12–15), piperine is able to induce melanocyte
proliferation in vitro even in the absence of UVR, possibly by a
mechanism involving stimulation of protein kinase C (2). A
potential complicating factor in the concomitant use of UVR
to induce melanogenesis in these cells is that piperine is known
to undergo photoisomerization at the double bonds on exposure to
UVR (350 nm) (16) and sunlight (17). Although a number of
structural analogs of piperine have been found to share its
melanocyte stimulatory effects (3), the activity of its geometric
isomers has not previously been examined.
We have previously reported that piperine (Fig. 1), the major
alkaloid found in the fruit of black pepper (Piper nigrum L.;
Piperaceae) (1), stimulates the replication of melanocytes and
induces the formation of melanocytic dendrites in vitro (2,3).
*Corresponding author email: soumyana@ohsu.edu (Amala Soumyanath)
Current address: Department of Neurology, Oregon Health & Science
University, Portland, OR 97239.
àCurrent address: Department of Biology, University of York, Heslington,
York YO10 5DD, UK.
The present study investigates the effect of UVR on the in vitro
and in vivo stimulatory effects of piperine on melanocytes and on
Ó 2006 American Society for Photobiology 0031-8655/06
1541

1542 Amala Soumyanath et al.
accounting for 8.5% of total output) at 11 cm, which was the distance to the
test sample. The emission spectrum of this source has been previously
reported (25).
Broad-spectrum UVA. Broad spectrum UVA-I (340–400 nm) was ob-
tained from a UVASUN2000 (Mutzhas, Munich, Germany), which was
housed on a custom-built trolley allowing irradiation from above. Two
fans blew across the irradiation area to provide cooling. Routine irradiance
was measured with an IL 442 radiometer (International Light, Newburyport,
MA) and calibrated in the same way as the thermopile used for SSR.
Irradiance was 74 mW cmꢀ2 (over the spectral range 340–400 nm) at the test
surface (a distance of 10 cm); of the total output, 98.88% was UVA-I
(340–400 nm), 1.11% was UVA-II (320–340 nm) and 0.01% was UVB
(280–320 nm). The emission spectrum of this source has been previously
reported (25).
Irradiation of piperine solutions. Piperine was dissolved in CD3OD
(40 mg/mL). This solution (2 mL) was carefully pipetted into petri dishes
placed on ice to minimize evaporation. Petri-dish lids were removed prior
to irradiation with either UVA or SSR. After irradiation, solutions were
collected and any volume lost by evaporation made up to 2 mL with
CD3OD. The control solutions (nonirradiated) were covered with aluminum
foil and kept at 48C.
Analysis of irradiated solutions. 1H NMR was recorded using a Bruker
(360 MHz) NMR spectrometer. Reversed-phase HPLC analysis of irra-
diated and unexposed piperine was performed with the use of an Alltech
Adsorbosphere 4.6 mm 3 25 cm RP-C18 (10 lm) column with a 10 mm
C18 guard column with a LDC Analytical 3100 pump and a HP 3390A
Integrator connected to a spectromonitor 3100 UV detector (LDC Ana-
lytical, Riviera Beach, FL). Analyses were performed isocratically with
a mobile phase containing 60% water, 40% acetonitrile; 1 mL/min. An
injection volume of 10 lL was used; the eluent was monitored at 348 nm.
The retention time of piperine in this system was 12.7 min. LC-MS analysis
was undertaken with the use of an Alltech Adsorbosphere 4.6 mm 3 25 cm
RP-C18 (10 lm) column in a ThermoFinnigan Surveyor LC system directly
coupled to ThermoFinnigan LCQ DECA XP ion-trap mass spectrometer
operating in the electrospray positive mode. UV spectra were obtained from
the ThermoFinnigan Surveyor Photo Diode Array (PDA) detector
associated with the LC-MS system. Analyses were performed isocratically
with a mobile phase comprising 85% acetonitrile, 15% water. An injection
volume of 10 lL was used; the samples were eluted at a flow rate of 0.2
mL/ min and monitored at 348 nm on the PDA for piperine. The retention
time of piperine in this system was 11.7 min.
Cell culture experiments
Stock cultures. Melan-a cells are an immortal pigmented mouse cell line,
cultured from epidermal melanoblasts from embryos of inbred C57BL mice
(26). Subconfluent to nearly confluent melan-a cultures (passage number
26–31) were used in this study. Cell cultures were maintained in culture
flasks in supplemented RPMI 1640 growth medium with 200 nM TPA,
trypsinized, harvested and resuspended for experiments in the same me-
dium without TPA, as described earlier (2,3).
Figure 1. Molecular structure of piperine (1-2E,4E-piperinoyl-piperidine)
and its three geometric isomers.
its ability to interact with proteins and DNA. The involvement of
DNA binding either before or after irradiation in the action of
piperine has not previously been investigated. These studies are
relevant to the design of preclinical and clinical studies on the
efficacy of piperine with UVR as a stimulant for skin repigmenta-
tion in vitiligo.
Preparation of microplates with melan-a cell suspension. Melan-a cells
were inoculated (100 lL; 63103 cells per well) with a repeater pipetter into
96-well microtiter plates (Nunc, Cambridge) and incubated at 378C in a
10% CO2, 90% air humidified atmosphere for 4 h. Piperine was dissolved in
methanol and the solutions were sterilized by filtration (pore size 0.2 lm)
and then diluted with the cell culture medium to give a final stock solution
of 30 lM piperine and a nontoxic concentration of methanol. Each plate
was subdivided into sections each consisting of two adjacent columns of
6 wells each for piperine (10 lM; 50 lL of stock solution) or control (50 lL
medium only), with a gap of 2 empty columns in between each section.
Irradiation and culture of melan-a cells. The plates, placed on ice, were
positioned under the UVA and SSR irradiation sources. In order to maintain
sterility of the cell cultures, irradiations needed to be carried out with the
microplate lid in place. The lid reduced the UVB and UVA irradiances of
the SSR source by about 45 and 30%, respectively, and the UVA irradiance
of the UVA source by about 20%. The doses given below and in Figs. 6 and
7 are not corrected for the effects of the lid, so that comparisons can be
made with Fig. 3B. Microplates were irradiated with UVA doses ranging
from 0 to 124 J cmꢀ2 with the use of the Mutzhas UVASUN2000
broadband UVA source. This represents a dose of less than 2 minimal
erythema doses (MED) (27) for sun-sensitive skin Type II after correction
for absorption by the 96-well-plate lid. Different doses were achieved by
exposing particular sections of the plate (each consisting of one row of
MATERIALS AND METHODS
Chemicals. All chemicals and reagents were purchased from Sigma-Aldrich
Co. Ltd. (Irvine, UK) unless stated otherwise. Piperinic acid was prepared
in our laboratory by hydrolysis of piperine (24).
Radiation sources and dosimetry for in vitro experiment simulated solar
radiation (SSR). SSR was obtained from a Solar Simulator (Oriel, Stratford,
CT) with the use of a 1-kW xenon arc lamp (ORC Lighting Products,
Azusa, CA) in conjunction with a quartz collimator, quartz lens and
a Schott WG320 filter (1 mm thick). Irradiance was routinely measured
with
a wideband thermopile radiometer (Medical Physics, Dryburn
Hospital, Durham, UK) calibrated with a double-monochromator spectro-
radiometer (DM150, Bentham, Reading, UK) which had been calibrated
against a UK National Physics Laboratory (NPL) standard lamp. Irradiance
was 12 mW cmꢀ2 scanned between 280 and 400 nm (UVB 280–320 nm

Photochemistry and Photobiology, 2006, 82 1543
piperine exposed cells and one row of control cells) for different time
periods (0, 7, 13, 22 or 28 minutes; n 5 6). Unexposed areas were covered
with a piece of cardboard. Another set of microplates was irradiated with
SSR doses ranging from 0 to 15 J cmꢀ2 with the use of the solar simulator
for 0, 5, 7, 15 or 21 min (n 5 6). This represents doses of about 1.5 MEDs
for skin Type II (filter 2 data [27]) after corrections for the effect of the
microplate lid. All plates were incubated at 378C in a 10% CO2, 90% air
humidified atmosphere incubator for 4 days.
Measurement of cell proliferation. The basic protocol was based on the
assay developed by Skehan et al. (28) with modifications (29). Briefly, after
4 days incubation cells were fixed with the use of cold trichloroacetic acid
solution, incubating at 48C for 1 h. After washing with tap water to remove
acid, medium and dead cells, plates were dried in air and SRB dye was
added. At the end of the staining period (30 min) unbound SRB was re-
moved by washing with acetic acid and air drying. Cell-bound dye was
solubilized in Tris [tris(hydroxymethyl)aminomethane] base and absor-
bance was read at 550 nm in a microplate spectrophotometer (Spectromax
190 Molecular Devices, Softmax Pro Version 2.2.1, 1998).
Figure 2. Ultraviolet absorption spectrum of piperine (0.06 mM) in
methanol.
Statistical analysis. Statistical significance of differences between the
number of melanocytes in control and test incubations was determined with
the use of one-way ANOVA followed by a Dunnett’s t test.
following scoring system: 0 5 no pigmentation; 1 5 first signs of
pigmentation (spots); 2 5 light brown; 3 5 medium brown; 4 5 dark
brown; 5 5 black. Scores obtained at the end of each week (Friday) are
shown in Fig. 11.
DNA and human serum albumin (HSA) binding assay
DNA and HSA binding of piperine was monitored using a 5 : 1 DNA base pair:
drug or protein: drug molar ratio, using piperine (100 lM) and DNA or HSA
(500 lM), all made up in 1% methanolic phosphate buffer (pH 7.4). Mixtures
were made of piperine with buffer, DNA solution, or HSA solution. Pure
solutions of piperine, DNA and HSA and mixtures were divided into three sets
with one set left unexposed, the second set exposed to UVA for 5 min (22 J
cmꢀ2) and the third set exposed to SSR for 21 min (15 J cmꢀ2). These solutions
were then stored in a refrigerator for 3 days prior to circular dichroism, CD
(Jasco J-600 spectropolarimeter) and linear dichroism, LD (Jasco J-720
spectropolarimeter) analysis. The irradiated samples were analyzed by UV
spectroscopy (Perkin-Elmer Lambda-2 UV/VIS spectrometer) immediately
after irradiation and after 3 days’ storage in a refrigerator. No changes were
found to have occurred during the storage period (results not shown). CD
spectroscopy was performed using a CD cell of 1 cm path length. LD analysis
was performed to validate CD experiments on piperine binding to DNA.
Statistical analysis. Differences between treatment groups across the
entire treatment period were compared by the Mann–Whitney U-test.
RESULTS AND DISCUSSION
Piperine is 1-2E,4E-piperinoyl-piperidine (Fig. 1) with two trans
double bonds in the chain connecting the methylenedioxyphenyl
and piperidine groups. Figure 2 shows the UV spectrum of this
compound. Possible geometric isomers (Fig. 1) are chavicine
(2Z, 4Z; cis-cis), isopiperine (2Z, 4E; cis-trans) and isochavicine
(2E, 4Z; trans-cis). Conversion of piperine to these geometric
isomers has been reported following exposure to UVR (kmax 350
nm [16,18] or 366 nm [19]) and sunlight (17). In early work (16),
chavicine, and later isochavicine, were noted as being the major
product of piperine’s photoisomerization. However, later studies
(17–19) have shown that all three isomers form from piperine, their
ratio being dependent on the length of exposure to irradiation.
Chavicine appears to be the last isomer to be produced (17,18) and
was the dominant isomer after 24 h exposure to sunlight (17).
In the present study, irradiated and unirradiated solutions of
piperine in methanol were compared by high-performance liquid
chromatography (HPLC), liquid chromatography coupled to mass
spectrometry (LC-MS) and UV and 1H NMR (nuclear magnetic
resonance) spectroscopy. An HPLC chromatogram of piperine
irradiated with UVA (124 J cmꢀ2) is shown in Fig. 3A. The main
peak at Rt 12.7 min corresponds to piperine, whereas the peak
eluting just before piperine (Rt 12.0 min) represents one or more
photoproducts of piperine. Figure 3B shows that the relative area of
the photoproduct(s) at 12.0 min increased with increasing UVR
dose. No peak for piperinic acid (retention time of standard 5 6.3
min) was observed in HPLC analysis, showing that no hydrolysis
had taken place at the amide function. Individual isomers were not
resolved on this HPLC system, but methods for their baseline
separation have been reported elsewhere (17).
In vivo evaluation of pigmentation induced by piperine and /or UVR
Animals. Male inbred HRA.HRII-c/þ/Skh hairless pigmented mice, age-
matched (8–16 weeks old), were used in each study. This line, congenic with
albino inbred HRA/Skh mice, segregates into albino and pigmented phe-
notypes and was developed by Dr. P. Forbes, Temple University Centre for
Photobiology (bred by the Biological Services Division, KCL, University of
London, and the Rayne Institute, St. Thomas’s Hospital, London).
Treatment groups. Mice (n 5 4 per group) were treated with (A)
dimethylsulfoxide (DMSO) for 9 weeks, (B) piperine (175 mM) dissolved
in DMSO for 9 weeks, (C) piperine in DMSO for 9 weeks with UVR from
Weeks 5–9, or (D) UVR only for 5 weeks. Piperine solution and DMSO
were applied with a micropipette (100 lL) on dorsal skin twice a day
(weekdays) with an interval of 5–6 h between applications. UVR was
administered as described below. For group (C) the irradiations were carried
out every Monday, Wednesday and Friday immediately prior to the first
daily application of piperine to avoid photodegradation of piperine.
UV irradiation and dosimetry. The UVR source was a bank of eight
Bellarium SA-1-12-100 W fluorescent tubes (Wolff, Erlangen, Germany).
This UVR source emits 4.1% UVB (280–320 nm) and 95.8% UVA, but the
UVB accounts for the 71.5% erythemally effective energy when bio-
logically weighted with the human erythema spectrum (30,31). Irradiations
were carried out in a custom-built unit with ventilation, temperature and
humidity controls. The irradiance was monitored daily immediately before
irradiations with International Light radiometer (IL 422A; Newburyport,
MA) equipped with UVR sensors. The radiometer was calibrated for the
source, as described before (30). Irradiance at mouse level was typically
about 0.16 mW/cm2. Animals were unrestrained in metal cages and irra-
diated with a dose of 354 mW cmꢀ2 (30) that was further confirmed to be
subinflammatory from a single exposure (increase in skin folding thickness
,10%; data not shown). Irradiations lasted for a maximum of 1 h. The
position of cages was systematically rotated to ensure even UVR exposure.
Assessment of pigmentation. Pigmentation was assessed visually by an
investigator blinded to the treatment that the animals had received, with the
Tandem LC-MS (LC-MSn) analysis of the two HPLC peaks
revealed that both were comprised of substances with identical
mass spectra. ESI-MS m/z in positive-ion mode for the piperine
peak gave a quasimolecular ion at m/z 286 (100%) [M þ H]þ.
Further fragmentation (MS2) of this ion gave m/z 201, which was
then further fragmented (MS3) to give m/z 173, 171, 143 and 115.
The photoproduct HPLC peak gave virtually identical ions in terms

1544 Amala Soumyanath et al.
Figure 3. (A) Partial HPLC chromatogram of a methanolic solution of
piperine after exposure to UVA (124 J cmꢀ2). The peak at 12.7 min
corresponds to that obtained with unexposed piperine, whereas that at 12.0
min is present only in irradiated samples. HPLC conditions as in the text.
(B) Relative areas of the HPLC peaks seen at 12.7 min (piperine) and 12.0
min (product) when piperine is exposed to different levels of radiation. The
two peaks were not completely resolved, and are not composed purely
of piperine or its photoproducts. Areas were obtained by integrating the
peak areas on either side of a vertical line dropped from the valley be-
tween the two peaks.
of both m/z value and relative abundance. Mass spectra are
reported to be identical for the four isomeric compounds (19,20).
The MS fragments obtained in this study were in excellent
agreement with the literature for piperine and its isomers (19).
Figure 5. 1H-NMR spectra of piperine (A), piperine after exposure to
SSR at 22 J cmꢀ2 (B) and piperine after exposure to UVA at 112 J cmꢀ2
(C). Spectra were recorded in CD3OD.
LC-MS data therefore confirm that the photomodified product
consists of one or more geometric isomers of piperine.
Piperine isomers vary in their UV absorption kmax values.
Reported values (17,19,20) range as follows: piperine 340–343
nm, isochavicine 330–336 nm, isopiperine 332–335 nm and
chavicine 317–321 nm. UV analysis of the peaks observed in
HPLC, using diode array detection coupled to the LC-MS
instrument, showed a kmax 5 326 nm for the photomodified
product peak as compared to 340 nm for piperine (Fig. 4). This
clearly shows the presence of chavicine, which is the only isomer
with a kmax value lower than 330 nm. The presence of other
isomers is likely to have caused the kmax to shift to a slightly higher
wavelength than the range reported for chavicine (317–321 nm).
The 1H-NMR spectra of piperine in CD3OD (deuterated
methanol) before and after irradiation are shown in Fig. 5A–C.
Figure 4. UV spectra of HPLC peaks corresponding to piperine (A) and
photoproduct (B) after exposure to UVA (124 J cmꢀ2). Spectra were
obtained using a photodiode array detector attached to the HPLC system.

Photochemistry and Photobiology, 2006, 82 1545
Figure 7. Effect of SSR irradiation on cell growth in the presence or
absence (control) of 10 lM piperine. Cell growth is expressed as
a percentage of cell growth in cultures unexposed to radiation or piperine.
**P , 0.01 when compared to control incubation at the same radiation
dose (one-way ANOVA followed by Dunnett’s t-test). Note that doses are
not corrected for the absorption of the 96-well-plate lid, which absorbs
about 30% of the UVA and 45% of the UVB.
Figure 6. Effect of UVA irradiation on cell growth in the presence or
absence (control) of 10 lM piperine. Cell growth is expressed as
a percentage of cell growth in cultures unexposed to radiation or piperine.
**P , 0.01 when compared to control incubation at the same radiation
dose (one-way ANOVA followed by Dunnett’s t-test). Note that doses are
not corrected for the absorption of the 96-well-plate lid, which absorbs
about 20% of the UVA.
The 1H NMR (CD3OD, 360MHz) signals for piperine are at d
7.31(1H, ddd, J3,2 5 14.7 Hz, J3,4 5 8.2 Hz, J3,5 5 2.0 Hz, H-3),
7.07 (1H, d, J7,11 5 1.6 Hz, H-7), 6.95 (1H, dd, J11,10 5 8.0 Hz,
J11,7 5 1.6 Hz, H-11), 6.88 (1H, dd, J4,5 5 15.2Hz, J4,3 5 8.2 Hz,
H-4), 6.80 (1H, d, J5,4 5 15.2 Hz, H-5), 6.78 (1H, d, J10,11 5 8.0
Hz, H-10), 6.60 (1H, d, J2,3 5 14.7 Hz, H-2), 5.95 (2H, s,
OCH2O), 4.90 (solvent), 3.60 (4H, br, H-19, H-59), 1.70 (2H, m,
H-39), 1.60 (4H, m, H-2949). This spectrum is in agreement with
the literature (17,19,20), minor differences in d values being due
to the use of CDCl3 (deuterated chloroform) as solvent in the
earlier studies.
signal at d 7.75 (1.08 SSR; 0.42 UVR) with the value obtained for
the six piperidine protons at H-29, 39, 49 appearing at d , 2.0
(29.84 SSR; 23.09 UVR). Interestingly, there is less isochavicine
in the UVR-treated (11%) than the SSR-treated (22%) sample.
In cell cultures unexposed to irradiation (Figs. 6 and 7), piperine
stimulated melanocyte proliferation and dendrite formation (Fig. 8)
as expected from earlier studies (2,3). However this effect was lost
on exposure to UVA (Fig. 6) or SSR (Fig. 7). For SSR, the effect
The 1H-NMR spectra of irradiated piperine solutions (Fig. 5B,C)
showed significant differences from that of piperine, which were
more pronounced in the UVA- than in the SSR-irradiated sample.
The spectra were very similar to that previously reported for
piperine samples exposed to sunlight (17). In both cases (Fig.
5B,C), the most significant changes are observed in the 6–8 ppm
region, representing the double-bond and phenyl protons, indic-
ative of changes in the configuration of the double bond. In both
irradiated samples, the strong signal at about 5.98 ppm is still
apparent, indicating that the methylenedioxyphenyl group is intact,
although there is now some overlap with the double-bond signals.
Three previous studies (17,19,20) discuss comparative 1H-NMR
spectra of the four isomers. A doublet assignable to H-2 is found at
around d 6.0 in the 1H-NMR spectra of isopiperine and chavicine,
both of which have a cis arrangement of the H-2, H-3 double
bond), whereas this proton is seen at around d 6.5 for piperine or
isochavicine. Isochavicine is the only isomer reported to have
a signal (H-3) downfield of d 7.5 (15,17,18), and the H-4 signal
appears uniquely in the d 6.3–6.4 region for this isomer. New
signals around d 6 were seen in the two irradiated samples of
piperine (Fig. 5B,C), showing that either chavicine or isopiperine,
or both, are present in the mixture. The irradiated solutions also
show a new double doublet at d 6.4 and multiplet at around d 7.75,
confirming the presence of isochavicine. 1H NMR analysis
therefore confirms that UVA and, to a lesser extent, SSR cause
the photoisomerization of piperine in the present study. The
proportion of isochavicine in the mixture can be obtained from
a comparison of the integration value for the isochavicine H-3
Figure 8. Melan-a cells grown in control culture medium show a bipolar
morphology (A). Exposure to piperine (10 lM) induces dendricity in
these cells (B).

1546 Amala Soumyanath et al.
the structural requirements for activity may not be very stringent.
However, the present study shows that the configuration around the
double bonds in the 1-piperinoyl-piperidine molecule may have
a profound effect on activity. The isomerization induced by UVA
and SSR radiation results in a loss of activity of the molecule in
proportion to the radiation dose received. A change from a trans,
trans configuration to one involving cis bonds would lead to
a considerable alteration in the overall shape of the molecule.
Piperine would be expected to have a relatively linear structure,
whereas the other three isomers, each containing at least one cis
bond, are more folded (Fig. 1). It is of note that tetrahydropiperine,
in which the two double bonds are replaced by a saturated 4-carbon
aliphatic chain is also active (3), presumably because it can adopt
a linear conformation similar to piperine.
Potential binding of piperine to protein and DNA before and
after irradiation was investigated using circular dichroism (CD).
Piperine is not optically active, but if bound to a chiral substance
such as human serum albumin (HSA) or DNA, optical activity is
induced and a signal will be observed in the CD spectrum asso-
ciated with piperine’s UV absorption kmax (about 340 nm).
Piperine was found to bind to HSA (Fig. 9A) but on exposure
to UVA (Fig. 9B) or SSR (Fig. 9C), this was abolished, sug-
gesting that the structural changes induced by radiation were
detrimental to protein binding of the molecule. No binding to DNA
was observed by CD either before or after irradiation (Fig. 10A–C);
the increase in absorbance at 275nm (Fig. 10B,C) is due to
a concentration effect (solvent evaporation). Absence of binding to
DNA was confirmed with the use of linear dichroism (data not
shown). This suggests that unlike the situation with psoralens (13),
piperine and its isomers do not bind DNA and would not form
photoadducts in vivo.
The present results show that UVR-induced photoisomerization
of the piperine molecule results in the loss of its protein binding
and melanocyte stimulatory activity. These results do not, how-
ever, preclude the use of UVR in conjunction with piperine in the
treatment of vitiligo. We have conducted in vivo experiments with
a hairless, sparsely pigmented mouse strain (HRA.HRII-cþ/Skh)
in which piperine solution was applied topically twice every
weekday for 9 weeks and UV irradiation was administered three
times a week from Weeks 5–9, just prior to application of piperine.
Using this protocol, the melanocyte stimulatory effect of piperine
was retained. Pigmentation was better in mice receiving both pip-
erine and UVR than in mice treated with either agent alone
(Fig. 11), verifying the usefulness of their concomitant use. It
is important that administration of UV irradiation to patients
receiving piperine must be appropriately timed and that piperine-
containing preparations are protected from light.
Figure 9. CD study of piperine’s interaction with HSA, before exposure to
irradiation (A), after exposure to UVA (22 J cmꢀ2) (B), and after exposure
to SSR (15 J cmꢀ2) (C).
was radiation dose dependent, probably due to lower levels of
isomerization with this form of radiation as compared to the doses
used with UVA alone. UVA has previously been shown to cause
DNA damage in cultured human melanocytes in vitro (21–23).
However, in the present study, this does not account for the
observed decline in cell growth in irradiated, piperine-treated
cultures. Control cells exposed to SSR radiation at all test doses
grew as well as unexposed cells (Fig. 7), whereas UVA-exposed
cells showed a small decline in growth only at radiation doses of 98
J cmꢀ2 and above, whereas the stimulatory effect of piperine was
lost even at 31 J cmꢀ2 of UVA (Fig. 6).
CONCLUSIONS
When exposed to physiologically relevant doses of UVA and SSR,
piperine photoisomerizes at the conjugated double bond to give
1
a mixture of isomeric products. UV and H NMR data provided
good evidence for the presence of chavicine and isochavicine in the
mixture, although the presence of isopiperine could not be ruled
out. Piperinic acid, a potential hydrolysis product of piperine, was
not detected. Conversion to its geometric isomers led to a loss of
piperine’s ability to stimulate melanocyte proliferation and to bind
to HSA in vitro. No in vitro binding to DNA was observed either
before or after irradiation. These results are not surprising, given
We have previously reported that a large number of analogues of
piperine can stimulate melanocyte proliferation (3), implying that

Photochemistry and Photobiology, 2006, 82 1547
Figure 11. Pigmentation scores in mice (n 5 4 per group) treated with (A)
topical DMSO (vehicle) for 9 weeks, (B) topical piperine in DMSO for 9
weeks, (C) topical piperine in DMSO for 9 weeks with UVR from Weeks
5–9, and (D) UVR alone from Weeks 5–9. Piperine treatment (B) gave
significantly (P , 0.05) higher scores than vehicle alone (A). Treatment
with piperine and UVR (C) gave significantly (P , 0.05) better pigmen-
tation scores than either piperine alone (B) or UVR alone (D). Groups
were compared with the use of the Mann–Whitney U-test.
Acknowledgements—Dr. R. Venkatasamy was funded by Overseas Re-
search Students Award Scheme (ORS) and the project was funded by
British Technology Group (BTG), UK. The authors thank Professor
D. C. Bennett of St. Georges Medical School, UK for the gift of
the melan-a cell line and Mr. Graham Harrison for assistance with the
UV radiations and dosimetry. Some data reported here were presented in
posters at the following conferences: 42nd Annual Meeting of the Ameri-
can Society of Pharmacognosy, Oaxaca, Mexico, July 14–18, 2001; Inter-
national Symposium of the Phytochemical Society of Europe (PSE),
2001, Lausanne, Switzerland, September 12–14, 2001; British Pharma-
ceutical Conference, 2001, Glasgow, Scotland, September 23–26, 2001,
and in one published abstract (32).
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