Scientific Studies Pigmerise™ · (360 MHz) NMR spectrometer. Reversed-phase HPLC analysis of irra- diated and unexposed piperine was performed with the use of an Alltech Adsorbosphere

Scientific StudiesPigmerise™

Photochemistry and Photobiology, 2006, 82: 1541-1 548

UV Irradiation Affects Melanocyte Stimulatory Activity and Protein Binding of Piperine

Amala Soumyanath*’+, Radhakrishnan Venkatasamy‘, Meghna Joshi’, Laura Faas’*, Bimpe Adejuyigbe’, Alex F. Drake’, Robert C. Hider’ and Antony R. Young’ ’Department 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 SEl 7EH, UK

Received 14 April 2006; accepted 4 July 2006; published online 14 July 2006 DOI: 10.1562/2006-04-21-RA-882

ABSTRACT

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.

INTRODUCTION We have previously reported that piperine (Fig. l), the major alkaloid found in the fruit of black pepper (Piper nigrum L.; Piperaceae) ( I ) , stimulates the replication of melanocytes and induces the formation of melanocytic dendrites in vitro (2,3).

*Corresponding author email: [email protected] (Amala Soumyanath) ?Current address: Department of Neurology, Oregon Health & Science

$Current address: Department of Biology, University of York, Heslington,

0 2006 American Society for Phutobiology 003 I -8655/06

University, Portland, OR 97239.

York YO10 SDD, UK.

Melanocytes are pigment-producing dendritic cells located within the basal layer of the epidermis and in the matrix of hair follicles. 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 melanin 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 melanocytes. 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) ( I I). 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 melanogenesis in human (14) and murine (15) skin when administered with UVA.

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.

The present study investigates the effect of UVR on the in vitro and in vivo stimulatory effects of piperine on melanocytes and on

1541

1542 Arnala Soumyanath et a/.

H H O

10 4' 10 4'

Piperine [E,E-(trans-trans)-piperine]

H H

5 ' U Y 4'

Isopiperine [Z,E-(cis-trans)-piperine]

H

4'

Chavicine [Z~-(cis-cis)-piperinel

H

S U 3'

Isochavicine [E,Z-(h.ans-cis)-piperine]

Figure 1. Molecular structure of piperine (1-2E,4E-piperinoyl-piperidine) and its three geometric isomers.

its ability to interact with proteins and DNA. T h e 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 repigmentation in vitiligo.

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) calibrited 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 cm? scanned between 280 and 400 nm (UVB 280-320 nm

accounting for 8.5% of total output) at I 1 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 oh- tained from a UVASUN2000 (Mutzhas, Munich, Gemiany), which was housed on a custom-built trolley allowing irradiation from above. Two fans blew across the irradiation area to provide cooling. Routine irrddiance 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 cm-' (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-I1 (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 CD30D (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 CD30D. The control solutions (nonirradiated) were covered with aluminum foil and kept at 4°C.

Analysis of irradiated solutions. 'H NMR was recorded using a Bruker (360 MHz) NMR spectrometer. Reversed-phase HPLC analysis of irradiated and unexposed piperine was performed with the use of an Alltech Adsorbosphere 4.6 mm X 25 cm RP-C18 (10 pm) 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 pL 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 X 25 cm RP-C18 (10 pm) 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 ii?jection volume of 10 pL 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 medium without TPA, as described earlier (23).

Preparation of microplates with melan-a cell suspension. Melan-a cclls were inoculated (100 pk 6 X 10' cells per well) with a repeater pipetter into 96-well microtiter plates (Nunc, Cambridge) and incubated at 37OC in a 10% COz, 90% air humidified atmosphere for 4 h. Piperine was dissolved in methanol and the solutions were sterilized by filtration (pore size 0.2 pm) and then diluted with the cell culture medium to give a final stock solution of 30 pM 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 pM; 50 pL of stock solution) or control (50 pL 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 cm-' 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 11 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

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 = 6). Unexposed areas were covered with a piece of cardboard. Another set of microplates was irradiated with SSR doses ranging from 0 to IS J cm-2 with the use of the solar simulator for 0, 5 , 7, IS or 21 min (n = 6). This represents doses of about 1.5 MEDs for skin Type I1 (filter 2 data [27]) after corrections for the effect of the microplate lid. All plates were incubated at 37°C in a 10% CO1, 90% air humidified atmosphere incubator for 4 days.

Measurement of cell proliferation. The basic protocol was based on the assay developed by Skehan er al. (28) with modifications (29). Briefly, after 4 days incubation cells were fixed with the use of cold trichloroacetic acid solution, incubating at 4°C 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 removed by washing with acetic acid and air drying. Cell-bound dye was solubilized in Tris [ms(hydroxymethyl)aminomethane] base and absorbance was read at 550 nm in a microplate spectrophotometer (Spectromax 190 Molecular Devices, Softmax Pro Version 2.2.1, 1998).

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.

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 pM) and DNA or HSA (SO0 pM), 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 rnin (22 J cm-*) and the third set exposed to SSR for 2 1 rnin (1 5 J crn-'). 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 UVNIS 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 perfoimed using a CD cell of 1 cm path length. LD analysis was performed to validate CD experiments on piperine binding to DNA.

In vivo evaluation of pigmentation induced by piperine and /or UVR

Animals. Male inbred HRA.HRI1-e/+/Skh hairless pigmented mice, age- matched (8-16 weeks old), were used in each study. This line, congenic with albino inbred HRAISkh 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 = 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 pL) 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% U V B (280-320 nm) and 95.8% UVA, but the UVB accounts for the 715% erythemally effective energy when biologically weighted with the human erythema spectrum (30,3 1). 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). Irradianee at mouse level was typically about 0.16 mW/cm2. Animals were unrestrained in metal cages and i ra- diated with a dose of 354 mW cm-2 (30) that was further confirmed to be subinflammatory from a single exposure (increase in skin folding thickness <lo%; 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

200 300 400 500

Wavelength (nm)

Figure 2. Ultraviolet absorption spectrum of piperine (0.06 mM) in methanol.

following scoring system: 0 = no pigmentation; I = first signs of pigmentation (spots); 2 = light brown; 3 = medium brown; 4 = dark brown; 5 = black. Scores obtained at the end of each week (Friday) are shown in Fig. 11.

Statistical analysis. Differences between treatment groups across the entire treatment period were compared by the Mann-Whitney I/-test.

RESULTS AND DISCUSSION Piperine is 1-2E,4E-pipennoyl-piperidine (Fig. I ) 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 (22, 42; cis-cis), isopiperine (22, 4E; cis-trans) and isochavicine (2E. 4Z; trans-cis). Conversion of piperine to these geometric isomers has been reported following exposure to UVR (h,,, 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 'H NMR (nuclear magnetic resonance) spectroscopy. An HPLC chromatogram of piperine irradiated with UVA (124 J crnp2) is shown in Fig. 3A. The main peak at R, 12.7 min corresponds to piperine, whereas the peak eluting just before piperine (R, 12.0 min) represents one or more photoproducts of piperine. Figure 3B shows that the relative area of the photoproduct(s) at 12.0 rnin increased with increasing UVR dose. No peak for piperinic acid (retention time of standard = 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).

Tandem LC-MS (LC-MS") analysis of the two HPLC peaks revealed that both were comprised of substances with identical mass spectra. ESI-MS mlz in positive-ion mode for the piperine peak gave a quasimolecular ion at mlz 286 (100%) [M + HI ' . Further fragmentation (MS2) of this ion gave nzlz 201, which was then further fragmented (MS3) to give mlz 173, 171, 143 and 115. The photoproduct HPLC peak gave virtually identical ions in terms

1544 Amala Soumyanath et a/.

A

p p m l l 10 9 8 7 6 5 4 3 2 I

B

C Figure 3. (A) Partial HPLC chromatogram of a methanolic solution of piperine after exposure to UVA (124 J c K 2 ) . The peak at 12.7 rnin corresponds to that obtained with unexposed piperim, whereas that at 12.0 min is prescnt 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 between the twn 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 4. UV \pectra of HPLC peaks corresponding to iperine (A) and photoproduct (B) after exposure to UVA (124 J cm-5,. Spectra were obtained using a photodiode anay detector attached to the HPLC system.

i 1

~ - ~ . ~ - ~ ~ - - - ~ , ~ , ~ - , ~ - ~ ~ ~ - ~ ~ , ~ - r-lTn I T . 1

ppm11 10 9 8 7 6 5 4 3 2 I

Figure 5. 'H-NMR spectra of piperine (A), piperine after exposure to SSR at 22 J cm-* (B) and piperine after exposure to UVA at I12 J cm-* (C). Spectra were recorded in CDIOD.

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 h,,,, values. Reported values (17,19,20) range as follows: piperine 34c-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 h,,, = 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 h,,, value lower than 330 nm. The presence of other isomers is likely to have caused the h,,, to shift to a slightly higher wavelength than the range reported for chavicine (317-321 nm).

The 'H-NMR spectra of piperine in CD,OD (deuterated methanol) before and after irradiation are shown in Fig. SA-C.


**

150 I\ t control t-Piperine ( IOpM)

0 I 0 25 50 75 100 125 150

UVA DOSE (J cm‘)

Figure 6. Effect of UVA irradiation on cell growth in the presence or ab\ence (control) of 10 pM 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 ‘H NMR (CD30D, 360MHz) signals for piperine are at 6 7.31(1H, ddd, J3.2 = 14.7 Hz, J3.4 8.2 Hz, J3,5 = 2.0 Hz, H-3), 7.07 (IH, d, J7,11 = 1.6 Hz, H-7), 6.95 (IH, dd, J l l , l o = 8.0 Hz, J11,7 = 1.6 Hz, H-11), 6.88 (IH, dd, J4,5 = 15.2Hz,J4,3 = 8.2 Hz, H-4), 6.80 (IH, d, J5,4 = 15.2 Hz, H-5), 6.78 (lH, d, J,o,ll = 8.0 Hz, H-lo), 6.60 (IH, d, J2.3 = 14.7 Hz, H-2), 5.95 (2H, S,

OCH20), 4.90 (solvent), 3.60 (4H, br, H-l’ , H-5’), 1.70 (2H, m, H-3’), 1.60 (4H, m, H-2’4’). This spectrum is in agreement with the literature (17,19,20), minor differences in 6 values being due to the use of CDCI, (deuterated chloroform) as solvent in the earlier studies.

The ‘H-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, indicative 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 ‘H-NMR spectra of the four isomers. A doublet assignable to H-2 is found at around 6 6.0 in the ‘H-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 6 6.5 for piperine or isochavicine. lsochavicine is the only isomer reported to have a signal (H-3) downfield of 6 7.5 (15,17,18), and the H-4 signal appears uniquely in the 6 6.3-6.4 region for this isomer. New signals around 6 6 were seen in the two irradiated samples of piperine (Fig. SB,C), showing that either chavicine or isopiperine, or both, are present in the mixture. The irradiated solutions also show a new double doublet at 6 6.4 and multiplet at around 6 7.75, confirming the presence of isochavicine. ‘H NMR analysis therefore confirms that UVA and, to a lesser extent, SSR cause the photoisonierization 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

I t Control

,I 0 6 10 15 20

SSR DOSE (J cm-*)

Figure 7. Effect of SSR irradiation on cell growth in the presence or absence (control) of 10 pM 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.

signal at 6 7.75 (1.08 SSR; 0.42 UVR) with the value obtained for the six piperidine protons at H-2’, 3’, 4’ appearing at 6 < 2.0 (29.84 SSR, 23.09 UVR). Interestingly, there is less isochavicine in the UVR-treated ( 1 1 %) 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

Figure 8. Melan-a cells grown in control culture medium show a bipolar morphology (A). Exposure to piperine (10 pM) induces dendricity in these cells (B).

1546 Amala Soumyanath et a/.

Figure 9. CD study of piperine’s interaction with HSA, before exposure to irradiation (A), after exposure to UVA (22 J cm-2) (B), and after exposure to SSR (15 J cm?) (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 cm-* and above, whereas the stimulatory effect of piperine was lost even at 31 J cmp2 of UVA (Fig. 6).

We have previously reported that a large number of analogues of piperine can stimulate melanocyte proliferation (3), implying that

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 associated with piperine’s UV absorption h,,, (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, suggesting 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, however, 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.HRI1-c+/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 piperine and UVR than in mice treated with either agent alone (Fig. ll), 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.

CONCLUSIONS When exposed to physiologically relevant doses of UVA and SSR, piperine photoisomerizes at the conjugated double bond to give 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


Figure 10. CD study of piperine’s interaction with DNA, before exposure to irradiation (A), after ex owre to UVA (22 J cm-’) (B), and after exposure to SSR (15 J cm ) (C). 8 ‘

that piperine absorbs UVR across the whole solar range (Fig. 2) . Thus both UVB and UVA are likely to have contributed to the photoisomerization and loss of melanocyte stimulatory activity observed here with environmentally and physiologically relevant doses of SSR. If UVR is used with piperine in the treatment of vitiligo, application of the compound and irradiation should take place at different times, to minimize photoisomerization of piperine. This protocol was more effective than piperine or radiation alone in inducing pigmentation in a sparsely pigmented mouse strain. Bright sunlight should be avoided both during active application of piperine to the skin and in the storage of piperine products.

Figure 11. Pigmentation scores in mice ( n = 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 pigmentation 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. Venkatasarny 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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Parmar, V., S. Jain, K. Bisht, R. Jain, P. Taneja, A. Jha, 0. Tyagi, A. Rasad, J. Wengel, C. Olsen and P. Boll (1997) Phytochemistry of the genus Piper. Phytochem. 46, 597-673. Lin, 2. X., .I. R. S. Hoult, D. C. Bennett and A. Raman (1999) Stimulation of mouse melanocyte proliferation by Piper nigrum fruit extract and its main alkaloid, piperine. Plunta Med. 65, 6 0 M 0 3 . Venkatasamy, R., L. Faas, A. R. Young, A. Raman and R. C. Hider (2004) Effects of piperine analogues on stimulation of melanocyte proliferation and melanocyte differentiation. Bioorg. Med. Chern. 12, 1905-1 920. Ortonne, J. P., D. Mosher and T. B. Fitzpatrick (1983) Hypomelanotic disorders. In Vitiligo and olher hypomelanoses of hair and skin, (Edited by J. P. Ortonne J. D. Mosher and T. B. Fitzpatrick), pp. 129- 310. Plenum, New York. Hu, F., R. P. Fosnaugh ,and P. F. Lesney (1959) In vitro studies on vitiligo. 1. Invest. Dermatol. 33, 267-280. Le Poole, I. C., R. M. J. G. Vandenwijngaard, W. Westerhof, R. P. Dutrieux and P. K. Das (1993) Presence or absence of melanocytes in vitiligo lesions-An immunohistochemical investigation. J . Invest. Dermatol. 100, 816-822. Tobin, D. J., N. N. Swanson, M. R. Pittelkow, E. M. Peters and K. U. Schallreuter (2000) Melanocytes are not absent in lesional skin of long duration vitiligo. J . Pathot. 191, 407416. Abdel-Malek, Z., V. B. Swope, I. Suzuki, C. Akcali, M. D. Harriger, S. T. Boyce, K. Urabe and V. J. Hearing (1995) Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides. Proc. Natl. Acad. Sci. USA 92, 1789-1793. Aberdam, E., P. Auberger, J. P. Ortonne and R. Ballotti (2000) Neprilysin, a novel target for ultraviolet B regulation of melanogenesis via melanocortins. J . Invest. Dermatol. 115, 381-387. Romero-Graillet, C., E. Aberdam, M. Clement, J. P. Ortonne and R. Ballotti (1997) Nitric oxide produced by ultraviolet-irradiated keratinocytes stimulates melanogenesis. .I. Clin. Invest. 99, 635-642.

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11. Whitton M. E., D. M. Ashcroft, C. W. Barrett and U. Gonzalez (2006) Cochruni, Database Syst. Rev. 1, 1-37.

12. Grimes, P. E. (1997) Psoralen photochemotherapy for vitiligo. Clin. Dermurol. 15, 921-926.

13. Gange, R. W., A. D. Blackett, E. A. Matzinger, B. M. Sutherland and 1. E. Kochevar (1985) Comparative protection efficiency of UVA- induced and UVB-induced tans against erythema and formation of endonuclease-sensitive sites in DNA by UVB in human-skin. J . Invest. Derniutol. 85, 362-364.

14. Kwok, Y. K., A. V. Anstey and J. L. Hawk (2002) Psoralen photochemotherapy (PUVA) is only moderately effective in widespread vitiligo: A 10-year retrospective study. Clin. Exp. Dermatol. 27, 104-1 10.

15. Kinley, J., S . J. Moan, F. Dall’Aqua and A. R. Young (1994) Quantitative assessment of epidermal melanogenesis in C3HDif hr/hr mice treated wilh topical furocoumarins and UVA radiation. J . Invest. Dcrmutol. 103, 97-103.

16. De Cleyn, R. and M. Verzele (1972) Constituents of peppers. I. Qualitative analysis of piperine isomers. Chromutographiu 5, 346-350.

17. Hashimoto K., T. Yaoi, H. Koshiba, T. Yoshida, T. Maoka, Y. Fujiwara, Y. Yamamoto and K. Mori (1996) Photochemical isomer- isation of piperine, a pungent constituent in pepper. Food Sci. Technol. Inr. 2( I), 24-29.

18. Verzele, M., P. Mussche and S. A. Qureshi (1979) High performance liquid chromatographic analysis of the pungent principles of pepper and pepper extracts. J . Chromatogr. 172,493497.

19. Temes, W. and E. L. Krause (2002) Characterization and determination of piperine and piperine isomers in eggs. Anal. Biounal. Chem. 364, 155-160.

20. De Cleyn, R. and M. Verzele (1975) Constituents of peppers. Part VII. Spectroscopic structure elucidation of piperine and its isomers. Bull. SOC. Chinz. Belg. 84, 435438.

21. Kobayashi, N., S. Katsumi, K. Imoto, A. Nakagawa, S. Miyagawa, M. Furumura and T. Mori (2001) Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: Applica- tion to pigment cell biology. Pigm. Cell. Res. 14, 94-102.

22. Yohn, J. J., M. B. Lyons and D. A. Noms (1992) Cultured human melanocytes from black-and-white donors have different sunlight and ultraviolet-A radiation sensitivities. J . Invest. Dermatul. 99, 454459.

23. Kadekaro, A. L., R. J. Kavanagh, K. Wakamatsu, S . Ito, M. A. Pipitone and Z. A. Abdel-Malek (2003) Cutaneous photobiology. The melanocyte vs. the sun: Who will win the final round? Pigment. Cell. Res. 16, 434447.

24. Chatterjee, A. and C. Dutta (1967) Alkaloids of Piper longum Linn.- I. Structure and synthesis of piperlongumine and piperlonguminine. Tetruhedron, 23, 1769-1781.

25. Harrison, G. I., A. R. Young and S. B. McMahon (2004) Ultraviolet radiation-induced inflammation as a model for cutaneous hyperalgesia. J . Invest. Dermatul. 122, 183-189.

26. Bennett, D. C., P. J. Cooper and I. R. Hart (1987) A line of non- turnorigenic mouse melanocytes, syngeneic with the B-I6 melanoma and requiring a tumor promoter for growth. Int. J . Cancer. 39, 414-418.

27. Harrison, G. I. and A. R. Young (2002) Ultraviolet radiation-induced erythema in human skin. Methods 28, 14-19.

28. Skehan, P., R. Storeng, D. Scuderio, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H. Bokesch, S. Kenney and M. R. Boyd (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J . Nutl. Cancer. Insr. 82, 1107-1 112.

29. Lin, Z. X., J. R. S. Hoult and A. Raman (1999) Sulphorhodamine B assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evaluation of crude drugs used in the treatment of vitiligo. J Ethnopharrnucol. 66, 141-150.

30. Kipp, C., E. J. Lewis and A. R. Young (1998) Furocumarin-induced epidermal melanogenesis does not protect against skin photocarcinogenesis in mice. Photochem. Photobiol. 67(1), 126-131.

3 I . McKinlay, A. F. and B. L. Diffey (1987) A reference action spectrum for ultraviolet induced erythema in human skin. CIE J , 66, 17-22.

32. Joshi, M., R. Venkatasamy, B. Adejuyigbe, F. Drake, R. C. Hider and A. Raman (2001) Effect of UVA on piperine’s ability to bind DNA and protein and stimulate melanocyte proliferation in vitro. J . Pharm. Phurmacol. 53 (Suppl.), 175.

CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2008.08464.x

In vivo evaluation of piperine and synthetic analoguesas potential treatments for vitiligo using a sparselypigmented mouse modelL. 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.

CorrespondenceA. Soumyanath.

E-mail: [email protected]

Accepted for publication4 September 2007

Key wordsHRA ⁄Skh-II, melanocyte, pigmentation, piperine,

tetrahydropiperine, vitiligo

Conflicts of interestNone declared.

Summary

Background Piperine and its analogues have been reported to stimulate melano-cyte replication in vitro and may be useful in treating the depigmenting disease,vitiligo.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 testcompounds 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 continuousor interrupted to evaluate fading and repigmentation. Skin inflammation andpigmentation were evaluated regularly during treatment. DOPA+ melanocyteswere 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 ofinflammation. Additional exposure to UVR led to darker pigmentation than didthe compound or UVR alone, and greater numbers of DOPA+ melanocytes werefound. The combination produced an even pigmentation pattern, contrastingwith the speckled, perifollicular pattern produced by UVR alone. Treatment inter-ruption led to a decrease in pigmentation but not its loss. Repigmentation wasachieved by administering one of the compounds, UVR or both, and occurredfaster than in naıve 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.1 The

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.2

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-

oids.3 Moreover, corticosteroids applied either systemically or

topically carry the risk of significant side effects in long-term

therapy.4 Alternatively, PUVA therapy seldom achieves extensive

repigmentation that is cosmetically acceptable, and treatment

response is often followed by relapse.5 A recent Cochrane

review6 highlights the lack of research on current treatments

as well as the need to identify novel clinical approaches for

vitiligo.

Several clinical studies7–10 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.11 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-

drites.11 Melanin synthesis, however, was not stimulated. We

have also shown that several synthetic derivatives of piperine

share these in vitro effects.12

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 model13 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)2.14 However, unlike albino mice, pigmentation

in HRA.HRII-c ⁄+ ⁄Skh mice is inducible13,14 with melanocyte

numbers reaching close to 600 mm)2. As with vitiligo, peri-

follicular pigmentation is evident after exposure to UVR with

and without photosensitizers.13–15 We therefore advocate the

induction of pigmentation in this strain as an in vivo model for

repigmentation in vitiligo.

Materials and methods

Animals

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.

Chemicals

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.12

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.16

O

N

O

O 1'

2'

3' 4'

5'

1 2

3

4

5 6

7 8

9 10

11

5-(3,4-methylenediox y phe ny l)-2,4-pentadieno yl pi peridine (PIP)

O

N

O

O 1'

2'

3' 4'

5'

1 2

3

4

5 6

7 8

9 10

11

5- (3,4-methylenedioxypheny l)-pentano yl piperidine (THP)

1'

2' 3'

4'

5' 1 2

3

4

5 6

7 8

9 10

11

O

NH

O

O 6'

5- (3,4-methylenedioxypheny l)-2,4-pentadienoylc yc lohexy lamine (CHP)

1'

2' 3'

4'

5' 1 2

3

4

5 6

7 8

9 10

11

O

NH

O

O 6'

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

(rCHP).


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 cm2 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)1 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)1). The detector wavelength was set at 348 nm.

Under these conditions, PIP eluted at 10Æ59 min. Results were

expressed as mg mL)1 according to a previously determined

calibration curve (0Æ003–0Æ1 mg mL)1 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)1 and 100 lL

(17Æ5 lmoles) applied with a micropipette on the central area

of mouse dorsal skin (2–3 cm2), 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 photodamage17 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.13 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-

trum.18 Given that tanning and erythema action spectra are very

similar19 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

before.13 Irradiance measured at mouse level was typically about

0Æ16 mW cm)2. Animals were irradiated unrestrained in metal

cages with a dose of 354 mJ cm)2,13 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

(n ‡ 4) 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 Week1 2 3 4 5 6 7 8 9 10 11 12 13

A

B

C

D

E

F

Untr

No treatment Compound UVR

3

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.



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 cm2) were removed surgically and incubated in 2 mol L)1

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 mm2 was

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 mm2 was calculated.

Results

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

0

0·001

0·002

0·003

0·004

0·005

0·006

0·007

0·008

DMSO EtOH Transc OA/PEG PEG

PIP

Co

nce

ntr

atio

n (

mg

mL

–1)

MalesFemales

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+ ⁄Skhmouse dorsal skin was tested using Franz cells.

–10

10

30

50

70In

crea

se in

SF

T (

%)

Incr

ease

in S

FT

(%

)

1 2 3 4 5 6 7 8 9Weeks

1 2 3 4 5 6 7 8 9Weeks

PIP

THP

CHP

(a)

(b)

V

UV

.

–10

10

30

50

70

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.




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.

Pigmentation

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 DMSO20,21

and OA.22–24 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)2

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)2 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.12 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

–10

10

30

50

70

Incr

ease

in S

FT

(%

)

1 2 3 4 5 6 7 8 9Weeks

–10

10

30

50

70In

crea

se in

SF

T (

%)

PIP

THP

CHP

V

UV

1 2 3 4 5 6 7 8 9Weeks

(a)

(b)

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.




to obtain a similar, but less even, response in naıve 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)2 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)2 at the end of

Week 13 to levels comparable with those in Group B which

(a)

(c)

(b)

(d)

DMSOPIP

CHP

THP

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.

0

100

200

300

400

500

600

700

Cel

ls/m

m2

M F M F M F M F M F M F M F M F M FPIP+

M FPIP+ THP+ THP+ CHP+ CHP+ UVR Untr

DMSO DMSODMSO

DMSOOA OA OAOA

**

*

*

* *

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

(naıve).




had received continuous treatment with UVR and a com-

pound. The number of cells mm)2 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)2 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)2 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

mm2 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 13Weeks

0

1

2

3

4

5

6

Pig

men

tati

on

DMSO

PIP

THP

rCHP

7 8 9 10 11 12 13Weeks

0

1

2

3

4

5

6

Pig

men

tati

on

* * * *

7 8 9 10 11 12 13Weeks

0

1

2

3

4

5

6

Pig

men

tati

on

**

* *

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

0

1

2

3

4

5

6

Pig

men

tati

on

0

1

2

3

4

5

6

Pig

men

tati

on

4 5 6 7 8 9 10 11 12 13Weeks

* ****

7 8 9 10 11 12 13Weeks

**

0

1

2

3

4

5

6

Pig

men

tati

on

5 6 7 8 9 10 11Weeks

** ** **** ** **

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).




Discussion

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-

pounds.11,12 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

0

100

200

300

400

500

600

700

800

900

DO

PA

+ ce

lls/m

m2

DO

PA

+ ce

lls/m

m2

A B C D E F Untr

A B C D E F Untr

DMSOPIP THPrCHP

**

***

**

*

**

0

100

200

300

400

500

600

700

800

900

*

*

**

** *

**

**

(a)

(b)

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

· 200.

(a)

(b)

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.




and melanin synthesis.21,25 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.11,12 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.26

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.27

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.28

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 humans5 and in

mice.15 A similar progression has been observed using therapies

based on UVR.9,29 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 naıve mice. This indicates the possible

presence of poorly melanized melanocytes but in greater num-

bers than in naıve 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.30 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.31–37 It has recently been shown

that some of these molecules are imbalanced in vitiligo skin,38

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.39 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.11

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.

Acknowledgments

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




manuscript. Dr A Soumyanath and Professor AR Young held

joint responsibility for the supervision of this work.

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ISSN 2229-5054 International Journal of Drug Formulation & Research Nov.-Dec. 2010, Vol. 1 (iii) 263-275

Available online on www.ordonearresearchlibrary.org

263

International Journal of Drug Formulation & Research

FORMULATION AND EVALUATION OF 1%PIPERINE PHOSPHATIDYL CHOLINE

COMPLEX EMULSION SYSTEM INTENDED FOR VITILIGO

*Vinod K.R.1, Swetha R1, David banji1

1. Nalanda College of Pharmacy, Nalgonda, Andhrapradesh.

Department of Pharmacy, Nalanda College of Pharmacy, Nalgonda- 508001, AndhraPradesh,

India.

Abstract

Vitiligo is an auto immune disorder where the pigment cells (melanocytes) in various parts of the

skin are destroyed. Use of piperine in vitiligo has been proved by many researches quite recently.

The aim of the present work is to formulate a topical preparation of piperine for vitiligo. This

was achieved by complexing the piperine with an amphiphillic polymer, phosphatidyl choline in

1:1, 1:3, 1:5 ratios in order to improve its localization at dermal layer. The piperine dispersion

complexes are incorporated into topical formulations i.e., emulsion systems and a formulation

containing pure piperine alone, was prepared and are evaluated for drug content, in vitro

diffusion study. By the in vitro study it was noticed that 1:1 type of piperine dispersion complex

emulsion system has shown optimum localization of drug in the dermal region when compared

to piperine cream alone. The selected formulation was then evaluated for TLC, partition

coefficient, FTIR study, X-Ray powder diffraction study, physical stability, accelerated stability

studies. The FTIR study shown no interaction and X-ray diffraction study shown reduction in



264

crystallinity. The globules retained its size more than 4 months and accelerated stability studies

suggested that the formulation retained a shelf life of 17.6 months.

Key words: Vitiligo, Phospholipid complex, X- ray diffraction, SEM, FTIR

Introduction

The corresponding author has reported the herbal initiative of vitiligo therapy1. This is caused by

the loss of pigment, resulting in irregular pale patches of skin. There is some evidence suggesting

it is caused by a combination of auto-immune, genetic, and environmental factors. Vitiligo

develop patches of de-pigmented skin appearing on extremities. The patches may grow or remain

constant in size. It has been reported in recent years about the use of piperine obtained from

Piper nigrum in vitiligo. Conventional treatment of vitiligo involves corticosteroids and UV

radiation. But these have some side effects like Cushing syndrome, skin carcinoma etc. Use of

piperine in vitiligo not only reduces the UV radiation but also avoids side effects.

Phospholipids are an important component of cell membrane and play a major role in drug

delivery technology. It is an important carrier for the drug molecules, which require controlled

release.

Materials and methods

Piperine was extracted and isolated in the laboratory, phosphatidyl choline (LIPOID

phospholipin 80 H) was obtained as a gift sample from LIPOID GmbH Germany. All other

chemicals were of analytical grade. Extraction and isolation of piperine crystals was done based

on the work reported by Carol white, Athens technical college2. Physicochemical tests of

piperine crystals like chemical tests3, Thin layer chromatography4, melting point and UV spectral

analysis5 are done.

FTIR Analysis6

The extracted Piperine was subjected to IR Spectral analysis by using FTIR analysis (Thermo

Nicolet Nexus 670 IR Spectrometer), detector-DTGS KBr Beamsplitter KBr, Source IR.



265

X-Ray Diffraction studies7

Powdered X-Ray diffraction studies of Piperine powder is carried out to study crystalline nature

of the drug. Powdered piperine was dissolved in ethanol by ultrasonicator and then it was

subjected to evaporation, then dried and triturated. D8 Advanced Bruker AXS, instrument was

used for this purpose. Type of radiation used was copper radiation. The diffractograms of

piperine before and after sonication were studied.

Scanning electron microscopy8

Topology studies were carried out for crystals by using scanning electron microscopy. Powders

were coated with platinum in a sputter coater (JFC-1100, Jeol, Japan), and their surface

morphology was viewed and photographed with a Jeol scanning electron microscope (JSM-

5310-LV, Jeol).

Preparation of piperine – phospholipid complexes8

The required amounts of extract and phospholipids were placed in a 100 ml round-bottom flask

and dissolved in anhydrous ethanol. After ethanol was evaporated off under vacuum at 400C, the

dried residues were gathered and placed in desiccators overnight, then sieved through a 100

mesh. The resultant extract–phospholipid complex was transferred into a glass bottle and stored

in the room temperature.

In order to evaluate the effect of polymer on the drug release, three different types of complexes

were prepared, varying the polymer concentration. Complex of drug and polymer in 1:1, 1:3, 1:5

ratios.

Characterization of piperine phospholipid complexes:

Complex prepared were subjected to various characterization parameters like determination of

drug content in the complex by dissolving 100 mg equivalent complex in 100 ml methanol,

stirred diluted and measured UV spectrophotometrically by Lambda25 Perkin Elmer UV/Visible

Spectrophotometer, Interaction studies by FT-IR, topology studies by Scanning Electron

Microscopy (SEM) and X – ray powder diffraction study.



266

Formulation of the complex into topical emulsion system9:

Beeswax (12.5 gm), Lanolin (2 gm) and Stearic acid (2.5 gm) were taken in one beaker.

Glycerine (6.25 gm), water (7.25 gm), triethanolamine (0.437 gm) was taken in another beaker.

Complex was dissolved in ethanol by sonication and maintained at 400c. Both the beakers were

maintained at 60°C and all the ingredients were melted. Then oily phase is added to aqueous

phase and stirred continuously. The complex was added to the mixture when the temperature

dropped to 400c. As the temperature goes down, rose oil was added and mixed well until required

consistency was obtained. Complexes of piperine in phosphatidyl choline were prepared in the

ratio of 1:1, 1:3 and 1:5. Topical preparations were formulated by using Fo (pure piperine), F1

(1:1), F2 (1:3) and F3 (1:5).

Evaluation of the Piperine – phosphatidyl choline complex emulsion system:

Evaluations of the topical preparations were done by performing drug content uniformity10, ex –

vivo permeation study by using pig ear skin as semipermeable membrane11. Drug concentrations

on the dorsal skin surface, within the skin and in buffer solution pH 6.6 were calculated.

Determination of pH, Partition coefficient of cream12 between pH 7.4 phosphate buffer and n –

hexane spectorscopically, Stability Studies by optical microscopy13 (10 X), and accelerated

stability studies by Arrhenius method14, phase separation, were also carried out. Ex-Vivo

diffusion studies were performed to determine the percentage drug release of the drug in 6.6 pH

phosphate buffer at 344 nm using pig skin. Drug concentration with in the skin and on the dorsal

side of the skin were also determined.

Stability studies: As a part of stability studies, physical stability studies like microscopical

determination of globule size, and an accelerated stability study performed. The formulation was

subjected to accelerated stability study by Arrhenius method. Log % retained was calculated and

a graph was plotted between log % retained and time in hours. As it is a straight line, it is

following first order reaction. From the graph the k values are calculated for 55°C and 45°C.

Results and discussion



267

Piperine was extracted from black pepper by using Soxhelet and method and the % practical

yield was found to be 4.4 %. Test for alkaloids was done by DragonDroff’s test, Hager’s test,

Wagner’s test and Mayer’s test and it has given positive reaction for all the reagents confirming

the presence of alkaloids. Thin layer chromatography was carried and from the chromatogram,

the Rf value was calculated and found to be 0.25 which complies with that of the standard

piperine. Melting point of piperine was found to be 129°C which was compared with the

standard value 130°C. From UV spectral analysis, the absorption maximum was found to be 344

nm. Where as absorption maximum of standard piperine is 342 nm. As the value of test is near to

standard value, it is confirmed that the piperine crystals formed are intact. The FTIR spectrum of

the piperine crystals have produced the characteristic peaks at 1634, 2937, 1489, 1442, 1362,

1251, 1027, 3008.2 cm-1 responsible for their respective functional groups. The FTIR spectrum

can be seen in figure 1.From the X – ray diffraction study, it is noticed that the crystallinity was

reduced and solubility was increased after complexation. The diffractograms can be seen in

figure 2. In Scanning electron microscopy (SEM), the surface morphology of the crystals was

found to be needle like in the pictures, which are shown in figure 3.

The prepared complex was dispersed in water, and the pictures were observed under 10x. The

globules showing drug inserted inside were observed, which can be seen in figure 5.

Drug content uniformity of piperine polymer complex shown the drug concentration present in

1:1, 1:3, 1:5 drug, polymer ratio complex is, 249 mg, 247.3 mg, 247.3 mg respectively. Surface

morphology of phospholipid complex as examined by SEM is given in fig.3. Phospholipid

complexes are made up of phospholipid and drug and appeared in column shape. In the FTIR

spectrum of piperine – phosphatidyl choline complex, the peak responsible for the unsaturation

was missing, which indicates that the phospholipid has interacted with piperine through these

hydrogen bonding, in process of complexation. On other hand, physical mixture of PC and

piperine shows all sharp peaks as that of the piperine and a broad peak at 3424.2cm-1 as that of

PC. The spectra can be seen in fig.1,(c), (d).

From the drug content uniformity, the drug concentrations found in F1, F2, F3 formulations are

247.36mg, 239.42 mg and 239.42mg/ml respectively. As the concentrations are near to 250 mg,

it is confirmed that the creams formulated are maintaining good drug content.



268

Table 1: Drug release from different formulations

Formulation %Drug

retained within

the skin

% Drug

remained on the

dorsal side of the

skin

% Drug retained in

the buffer

F1 57.7 4.3 37.63

F2 49.8 20.1 34.88

F3

F0

32.0

51.8

41.9

22.1

23.8

20.8

The intention of the formulation was to retain the drug with in the epidermis of the skin, so that

the drug available for melanocytic proliferation will be more. Phosphatidyl choline as a polymer

was used in the complex formation to see the retention efficacy of the drug, piperine, with in the

skin. Formulation without phosphatidyl choline was incorporated with piperine alone and the

drug retained within the epidermis was found to be 51.8%. But when the complex was prepared,

(piperine – phosphatidyl complex), drug concentration was augmented to57.7%. Almost 6%

increase was there in the retention. It shows that when the drug and polymer were taken in ratio

1:1 i.e., formulation F1 enhances the retention power. Ironically, when the polymer

concentration was increased, as in F2 and F3, the drug retention within epidermis of skin was

proportionally decreasing. Therefore, formulation F1was optimized. F1 was selected as suitable

formulation and allowed for further evaluations.

Curve fitting analysis



269

The values from the diffusion studies were plotted in different kinetic model graphs, the selected

formulation fits the zero order kinetic model best (Table no. 2).

Table 2: Drug release kinetic model fitting

Model F1 F2 F3 F0

Zero order 0.967 0.932 0.833 0.941

First order 0.499 0.717 0.606 0.604

Higuchi

Peppas

0.933

0.735

0.887

0.728

0.805 0.891

0.428 0.495

The pH of the formulation F1 was found to be 5.9 which denotes the cream is slightly acidic n

nature. FTIR Reports of formulation F1, from the graph in fig.1, it is noticed that the peaks

responsible for the piperine are present in the graph and hence it is reported that the piperine has

not undergone any interaction with the excipients of the topical preparation. By performing the

Partition coefficient, the drug concentration found concentration of piperine in organic phase is

0.972mg/ml; concentration of piperine in aqueous phase is 0.02mg/ml. The partition coefficient

value of the cream was found to be 1.68. Phase separation was observed carefully every 24 hrs

for 30 days. The formulated cream was kept intact in a closed container at 25-30°C not exposed

to light. No change in phase separation was observed. A comparative TLC study of piperine and

cream was carried out, which has shown the Rf value of cream as 0.24, which is nearer to the Rf

value of piperine.



270

Table 3: Determination of globule size

Globule

size

1stMonth

(µm)

2ndMonth

(µm)

3rdMonth

(µm)

4thMonth

(µm)

Min size 5.4 5.4 5.42 5.42

Max size 55.81 55.8 55.75 55.75

Average

size

30.6 30.6 30.58 30.55

The globule size of the Formulation F1 has shown no significant change even after 4 months.

Fig 5: Plots of log % drug concentration VS time for different temperatures

1 / T values of 45°C and 55°C in Kelvin are 3.048 × 10-3 and 3.144 × 10 -3.



271

A graph is plotted between the 1 / T values and log k values and the k value obtained by

extrapolating the curve at 27 °C is 0.006 / months.

Fig 6: Plot of log k Vs 1 / T

-2.5

-2

-1.5

-1

-0.5

0

3000 3100 3200 3300 3400

1/T

log

k

From the above k value, the shelf life t can be calculated from the first order equation.

First order reaction: log c = log C0 – k t

2.303

The shelf life of the prepared piperine dispersion complex emulsion system was found to be

17. 65 months if stored at 27 °C.

Fig 1:FTIR reports: (a)piperine (b) phosphatidyl choline (c) physical mixture (d) complex

(a) (b)



272

(c) (d)

Fig 2: X – Ray diffraction reports of Piperine and Piperine polymer complex

(a) Piperine (b) Piperine polymer complex

Fig. 3. SEM pictures of (a) piperine crystals (b) phosphatidyl choline (c) piperine – phosphatidyl Choline complex (d) Microscopical view of piperine- phospholipid complex.

(a)

(b)



273

(a) (b)

(c) (d)

Conclusion

In the present study, piperine phospholipid complex emulsion system was prepared and

evaluated for various physicochemical parameters. It was concluded by good formulation results

and the evaluation studies of the piperine crystals, piperine polymer complex, and finally the

topical preparations F1, F2, F3 gave the satisfactory results. The FTIR, SEM, have shown the

formation of complex and the XRPD studies revealed that there is a decrease in crystallinity after

sonication and after complexation. The main intention of the work i.e., localization of the drug at

the dermal layer was attained by F1 piperine - phosphatidyl choline complex emulsion system.

i.e., the less amount of drug is entered into the buffer solution(37.63 %) and more is localized



274

with in the dermis(57.7%). And the formulation will be safe under preservation at room

temperature for 17.65 months.

Acknowledgment

The authors express indebt gratitude to Nalanda College of Pharmacy for providing facilities of

the Central library, Formulation research laboratory with regard to the successful completion of

the present project.

References

1. Vinod K.R., Sandhya S, Santosha D. A new Herbal Initiative for Vitiligo Therapy,

Indian Drugs, 6, 2009, 5-10.

2. Carol White, Athens Technical College, Athens, GA, Infrared Analysis of Piperine in

Black Pepper, Developed through the National Science Foundation-funded Partnership

for the Advancement of Chemical Technology (PACT).

3. Kokate CK, Practical pharmacognosy, vallabh prakashan publications, Delhi, 2003, 143.

4. Egon Stahl, Thin layer chromatography, a laboratory hand book, 2005, 430.

5. Rajpal V, Standardization of botanicals, Piper nigrum, vol 2, Eastern publications, New

Delhi, 2002, 258 – 267.

6. Robert M. Silverstein, Francis S, Spectrometric identification of organic compounds, 6th

edition, Wiley publishers, 2007, page no 136 – 140.

7. Ajay Semalty, Mona Semalty, Devendra Singh M.S, Rawat. M, “Preparation and

characterization of phospholipid complexes of naringenin for effective drug delivery”, J

Incl Phenom Macrocycl Chem, November 2009, DOI 10.1007/s10847-009-9705-8.

8. Xiao Yanyu, Song Yunmei, Chen Zhipeng, Ping Qineng. The preparation of silybin–

phospholipid complex and the study on its pharmacokinetics in rats, International Journal

of Pharmaceutics, 2006, volume307, 77–82.



275

9. Mehta R.M, Jain M. K, Pharmaceutics II, 4th edition, Vallabh prakashan publications,

Delhi. 2008.

10. Chowdary, KPR, Suresh babu KVV, Evaluation of water soluble cellulose polymers as

carriers for indomethacin solid dispersions. Eastern pharmacist publications, 1992, 181 –

182.

11. Vinod KR, Vikram, SDC – 1T09, B. Pharm project, Osmania University, 2009, 32 – 35.

12. Krishnaiah YSR, Satyanarayana V, Karthikeyan RS, Effect of the solvent system on in

vitro permeability of Nicardipine hydrochloride through excised rat epidermis. Indian

journal of pharmaceutical sciences, 2002.

13. Vijaya raghavan, A Practical book of physical pharnaceutics, 2nd edition, New century

book house (p) ltd, Madras, 2000, 35, 41.

14. Guru Prasad Mohantha, Prabal Kumar manna, Physical pharmacy, pharma book

syndicate, Hyderabad, 2006, 89 – 90.

Research Article

FORMULATION AND EVALUATION OF PIPERINE CREAMA NEW HERBAL DIMENSIONAL APPROACH FOR VITILIGO PATIENTS

*VINOD K.R1, SANTHOSHA D1, ANBAZHAGAN. S2

*1Nalanda College of Pharmacy, Affiliated to Osmania university, Cherlapally, Nalgonda508 001, 2Karuna College of Pharmacy, Iringuttoor, Thirumittacode PO, Pattambi via, Palakad Dist., Kerala.

Email: [email protected]

Received: 17 Dec 2009, Revised and Accepted: 12 Dec 2010

ABSTRACT: Vitiligo also known as leukoderma is a pigmentation disorder in which melanocytes (the cells that make pigment) in the skin are destroyed. As a result, white patches appear on the skin on different parts of the body which affects even the psychology and social status of the patient. In recent years, it has been proved that Piperine, an alkaloid from black pepper has the repigmenting capacity. Use of Piperine in Vitiligo not only reduces UV Radiation but also prevents side effects. The present work is about the extraction of Piperine from Black Pepper and its evaluation followed by formulation and evaluation of cream.

Keywords: Vitiligo, Leukoderma, Pigmentation, Melanocytes, UV Radiation.

INTRODUCTION

Vitiligo is a pigmentation disorder in which melanocytes (the cells that make pigment) in the skin are destroyed.1 As a result, white patches appear on the skin on different parts of the body. Similar patches also appear on both the mucous membranes (tissues that line the inside of the mouth and nose), and the retina (inner layer of the eyeball). The hair that grows on areas affected by Vitiligo sometimes turns white. Vitiligo is not a fatal disease, but it is chronic and progressive. The most important consequence of the disease is probably social and psychological, as people may feel devastated by their changed appearance. 2

There are many treatments available for Vitiligo but has some side effects like Cushing’s syndrome, Skin Cancer , GI disorders etc. Piperine is used as anti Vitiligo agent by reducing the effects of UV radiation and also in avoiding side effects.3‐ 5

Piperine is extracted and isolated from Black pepper by two different methods.6, 7 They are soxhlation and reflux method in which highest %yield is obtained for soxhlation (89.432%).The following evaluation works has been carried out for Piperine .pH, Solubility, thin layer chromatography, X‐Ray diffraction studies, IR spectral analysis, chemical tests, Melting point, Partition coefficient, Particle size and UV Spectral analysis

A 1%Piperine cream was prepared using bees wax as base and it’s evaluation was carried out. Thin layer chromatography, Globule size, Partition coefficient, Scanning electron microscopic studies, pH,

Moisture absorption studies, consistency, Irritancy test, Drug content uniformity, phase separation, Organoleptic characters etc

MATERIALS AND METHODS

A comparative extraction of Piperine and its recrystallization

The piperine was extracted by both Reflux method and Soxhlation method by using 95% ethanol as solvent. The solution was filtered and concentrated under vacuum in a water bath at 60˚C. 50ml alcoholic potassium hydroxide was added to the concentrate and the solution was stirred continuously for 30min. The obtained solution was heated and water was added drop wise until yellow precipitate was formed. Water was added until no more precipitate appeared to form and this was allowed to settle overnight. Needles of Piperine were observed to be separated out. The solid was collected and washed with cold ether 2‐3 times. It was recrystallized by using acetone. For this, dissolve solid in acetone and filter it to remove extraneous matter and keep the filtrate aside for 24hrs so that crystals of Piperine are formed. Yellow coloured rod shaped crystals were recrystallized after 24 hrs.

Analytical works on Piperine

UV spectral analysis

5μg/ml of the drug in ethanol was used for complete scan between 190‐900nm and the maximum absorption was obtained at 344nm as shown in the fig.1 whereas the λ max for standard Piperine is 342nm.8

Fig. 1: Absorption maximum of Piperine

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 3, Suppl 2, 2011

Vinod et al. Int J Pharm Pharm Sci, Vol 3, Suppl 2, 2011, 2933

30

Partition coefficient of Piperine 9

Procedure: The partition coefficient of drug between phosphate buffer solution (PH 6.6) & n‐hexane was determined at 37o ± 0.2o. An excess amount i.e. 50 mg of Piperine was taken in a separating funnel containing 1:1 ratio of buffer 6.6 & n‐hexane & placed in a water bath for 24h. The solution was shaken at regular intervals. Then, both of them were separated & filtered through a 2 μ filter &

the drug concentration in each phase was determined by measuring the absorbance using UV spectrophotometer at 344nm.

Particle size of Piperine using microscope

A small amount of powdered drug is placed on the slide & mounted using glycerin. By using eye piece micrometer, the diameters of 200 particles are determined randomly. Particle size distribution was expressed as histogram (Fig 2) 10‐12.

44

39

11

4 2

0

5

10

15

20

25

30

35

40

45

50

2.5 7.5 12.5 17.5 22.5

mean size range (microns)

No. o

f par

ticle

s in

eac

h ra

nge

Fig. 2: Histogram of Particle size distribution of Piperine

Fig. 3: Xray Diffractograms of Piperine crystals and powder

Solubility of Piperine was determined in water, ethyl alcohol, chloroform and Acetone. Temperature was maintained at 37±0.213. Melting point and TLC studies of piperine were also conducted. The chromatogram was observed under UV lamp (365nm). The alkaloid shows violet colored zone. Rf value was found to be 0.26 (value of standard piperine~0.25)14

XRay diffraction studies

Powdered X‐Ray diffraction studies of Piperine crystals and powdered Piperine by sonication were carried out to study crystalline nature of the drug .D8 Advanced Bruker AXS, instrument was used for this purpose .Type of radiation used was copper

radiation. The diffractograms are shown in fig 3. It shows that the crystallinity reduces for Piperine powdered using ultrasonicator i.e., solubility has increased.

Determination of pH 11, 13

A small quantity of Piperine was dissolved in ethanol and it’s pH was checked by using pH meter and it is found to be 7.9.

Chemical tests

10 mg of Piperine crystals were dissolved in 10ml ethanol and this solution is used as sample for chemical tests15, 16.


31

Test Observation Dragendroff’s test Orange brown ppt is formedMayer’s test Cream coloured ppt was observedHager’s test Yellow ppt was observed Wagner’s test Reddish brown ppt was formed

Fig. 4: IR Spectra of Piperine and its structure 16‐20

Table 1

3008.18cm‐1 Alkenes Present 2932.66cm‐1 CH2‐CH2 ‐CH3 Present1630.82cm‐1 Amines Present1582.55 Ketonic group Present

Fig. 5: SEM picture of cream diluted with glycerin

Formulation of cream 21, 22

Procedure: Beeswax, Lanolin and Stearic were taken in one beaker. In another beaker, Piperine was dissolved in ethanol by sonication and introduced into glycerine, water, triethanolamine. Both the

beakers were maintained at 60°C and all the ingredients were melted. Then oily phase is added to aqueous phase and stirred continuously. As the temperature goes down peppermint oil was added and mixed well until required consistency was obtained.


32

Evaluation of cream

Organoleptic characters were studied by visual appearance, colour and odour23.

By using eyepiece micrometer, the diameters of 200 particles are determined randomly10, 11, 12. Topology studies were carried out for cream by using scanning electron microscopy

Presence of foreign particles/grittiness was observed against diffused light to check for foreign particles13. Drug content uniformity of the cream was also carried out24,25.

Partition coefficient of cream

The partition coefficient of drug between phosphate buffer solution (PH 6.6) & n‐hexane was determined at 37o ± 0.2o. An excess amount i.e. 50 mg of cream was taken in a separating funnel containing 1:1 ratio of buffer 6.6 & hexane & placed in a water bath for 24h. The solution was shaken occasionally. Then, both of them were separated & filtered through a 2 μ filter & the amount solubilized in each phase was determined by measuring the absorbance using UV spectrophotometer at 344nm9.

The formulated cream was kept intact in a closed container at 25‐30°C not exposed to light. Any change in phase separation was checked every 24 hrs for one month.

Irritancy test26

Mark an area (1sq.cm) on the left hand dorsal surface. The cream was applied to the specified area and time was noted. Irritancy, erythma, edema, was checked if any for regular intervals up to 24 hrs and reported.

Rheological studies 27

The formulated cream was found to be non ‐ newtonian. Take a fixed quantity 10gms of cream in a 10ml beaker. Keep it impact for 1 hr. The beaker was inclined to one side see whether the cream is liquefied or not. beaker is shaken to and fro for continuous 5mns and checked whether consistency has changed or not. The beaker was again tilted and checked for pourability of the cream. The formulation showed no thixotropic (shear thinning) characteristics.

Diffusion studies of Piperine incorporated cream using goat’s skin as semipermeable membrane

Fresh goat’s skin was shaved well to remove all the hair and cleaned thoroughly in water and rinsed in isotonic solution and later with 6.6 buffer solution. Fresh skin is used for the study. A student diffusion cell was fabricated and the goat’s skin was used as semi permeable membrane. A 200mg of 1% Piperine cream was placed on the outer layer of the skin which was fixed as to face unit I from inside.

At predetermined intervals, samples 2ml from recipient chamber were withdrawn and transferred to amber coloured ampoules. The samples were suitably replaced. The samples were estimated for drug content, analyzed at maximum absorbance 344nm using UV‐Spectrophotometer ( Elico SL 196).The above work was repeated 3 times and average was calculated .From this, concentration of cream in buffer can be obtained.

After the completion of diffusion studies the skin was taken and dissolved in ethanol and left for 24hrs.Then the absorbance was calculated at 344nm.From this, concentration of cream in skin layer was calculated.

RESULTS AND DISCUSSION

Black pepper which was obtained from the local source was subjected to standardization and the results were found to comply with the standard values. Piperine was extracted from black pepper by using Soxhlet and Reflux method and the % yield was found to be maximum in Soxhlet method i.e., 89.632% whereas with reflux method it is 85%.Yellow coloured rod shaped crystals were found. Melting point determination of Piperine was performed thrice by melting point apparatus and the average was found to be 129˚C which was compared with the standard value 130 ˚C. The alkaloid

shows violet coloured zone under UV Radiation in TLC studies. The Rf value of test (extracted Piperine) is found to be 0.26 which corresponds to the standard value of Piperine (0.25) and only one spot was obtained indicating that it is pure. From UV Spectral analysis absorption maximum was found to be 344nm which almost matched with the standard. Infra red spectra of Piperine were compared with structure and the corresponding bonds were found to present.

Test for alkaloids was done by Dragendroff’s test, Hager’s test, Wagner’s test and Mayer’s test and it has given positive reaction for all the reagents confirming the presence of alkaloids. Assay of Piperine was performed and % purity of Piperine extract was 98.67%. Solubility of piperine was found to be in the order‐ chloroform>ethanol>Acetone but insoluble in water.

X‐Ray diffractograms revealed peak heights was reduced in powder after sonication indicating that crystallinity has reduced by ultrasonication and solubility has increased.

Piperine was formulated into % cream and was subjected to various evaluation works Thin layer chromatographic studies were performed. Clear single spots were obtained and Rf value of Piperine and the formulated cream were found to be correlating with each other. Melting point and Rf values of test(cream) and standard(Piperine) were comparable which indicates there is no change in the physical and chemical nature of the Piperine.This also reflects that the drug is compatable with other excepients like bees wax, lanolin etc. Arithmetic mean particle size of Piperine and globule size was found to 21.6 µ and 28.67µ respectively. Minimum globule size and maximum globule size of cream was found to be 4.72µ and 52.72µ respectively.

Expectation of the topical formulation was that the drug should penetrate the stratum cornium, get into the dermis but should not get into the systemic circulation. The % of Piperine for cream retained in the skin, the site of action was found to be was found to be 69.06% drug released into the buffer was found to 18.62%. it could be postulated that in addition to transcellular permeation, paracellular permeation also is significant through tight junctions. The final preparation was found to be smooth texture and consistency and free from gritty nature with light yellow colour and peppermint odour. The globules retained its size and no coalescence was found. Accelerated stability studies were done which shows no significant change in the concentration of drug which shows stability of the formulation. Moisture absorption studies showed no significant absorption of moisture. pH of the cream was found to be 6.5. Topology studies by SEM revealed almost smooth globules. In addition irritancy test was also performed on rabbits and found no redness, edema, Inflammation and irritation.

CONCLUSION

The formulation of Piperine cream intended for Vitiligo was successfully done and evaluated. Drug targeting at the skin were the melanocytic proliferation is intended was achieved 69.06%. The topical formulation was physically stable throughout the shelf life. Further novel drug delivery formulations are highly recommended to increase the percentage drug targeting.

ACKNOWLEDGMENT

The authors express their gratitude to Nalanda college of Pharmacy, A.P. for the support throughout the project. The authors wish to express Acharya Nagarjuna University, Guntur for providing technical support for this project.

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2. Vitiligo treatment, http://pushpakaran.shoutpost.com‐/archives/2007/May as on 17/7/09.

3. Faas L , Venkatasamy, Hider R C, Young A. R., Soumyanath A . In vivo evaluation of piperine and synthetic analogues as


33

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Archives of Applied Science Research, 2010, 2 (4): 165-169

(http://scholarsresearchlibrary.com/archive.html)

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165 Scholar Research Library

Effect of UV radiation in the antivitiligo therapy by piperine topical formulation

*K. R. Vinod1, S. Anbazhagan2, D. Santhosha3, Otilia Banji1, David Banji1, S. Sandhya1

1Nalanda College of Pharmacy, Nalgonda, Andhra Pradesh, India

2Chilkur Balaji College of Pharmacy, Hyderabad, Andhra Pradesh, India 3RGR Siddhanthi College of Pharmacy, Hyderabad, Andhra Pradesh, India

______________________________________________________________________________ Abstract Vitiligo also known as Leukoderma is caused by the loss of pigment, resulting in irregular pale patches of skin. Vitiligo develops patches of de-pigmented skin appearing on extremities. A team of scientists at King’s College London have discovered that Piperine and its synthetic derivatives can stimulate pigmentation in the skin. The present study evaluates the pharmacological response of repigmentation to a herbal topical formulation incorporated with piperine with and without UV radiation using New Zealand strain rabbits. Although the group deprived of UV radiation took more time for repigmentation, the pattern was more homogenous. Keywords: Vitiligo, melanocytes, UV radiation, herbal cream, evaluation. ______________________________________________________________________________

INTRODUCTION

About 0.5 to 1 percent of the world’s population, or as many as 65 million people, have Vitiligo[1]. Most develop vitiligo before their fortieth birthday! The disorder affects both sexes and all races equally. However, it is more noticeable in people with dark skin[2]. Vitiligo seems to be somewhat more common in people with certain autoimmune diseases. These include hyperthyroidism, adrenocortical insufficiency, alopecia areata and pernicious anemia. In the past two decades, research on the role that melanocytes play in Vitiligo has greatly increased. A variety of technical advances, such as gene mapping and cloning have permitted relatively rapid advances in knowledge of melanocytes at the cellular and molecular levels. People who develop Vitiligo usually first notice white patches (Depigmentation) on their skin. These patches are more commonly found on sun-exposed areas of the body, including the hands, feet, arms, face, and lips. Other common areas where these white patches appear are the armpits and groin, and around the mouth, eyes, nostrils, navel, genitals, and rectal areas. In addition to white patches on the skin, people with Vitiligo may have premature graying of the scalp hair,

K. R. Vinod et al Arch. Apll. Sci. Res., 2010, 2 (4): 165-169 ______________________________________________________________________________

Scholar Research Library 166

eyelashes, eyebrows, and beard. People with dark skin may notice a loss of color inside their mouths. Systemic phototherapy induces cosmetically satisfactory repigmentation in up to 70% of patients with early or localized disease. Narrow-band UV-B phototherapy is widely used and produces good clinical results. Narrow-band fluorescent tubes with an emission spectrum of 310-315 nm and a maximum wavelength of 311 nm are used. Treatment frequency is 2-3 times weekly, but never on consecutive days. This treatment can be safely used in children, pregnant women, and lactating women. Short-term adverse effects include pruritus and xerosis. Several studies have demonstrated the effectiveness of narrow-band UV-B therapy as monotherapy[3]. Groundbreaking new research in the British Journal of Dermatology has revealed that black pepper could provide a new treatment for the skin disease Vitiligo. Current treatments include corticosteroids applied to the skin, and phototherapy using UV radiation (UVR) to re-pigment the skin[4-5]. Both, however, carry possible long-term side effects and are not always effective. In particular, less than a quarter of patients respond successfully to corticosteroids, while UVR causes a re-pigmentation that is spotted and patchy and in the long-term could lead to a higher risk of skin cancer. But now a team of scientists at King’s College London have discovered that Piperine the compound that gives black pepper its spicy, pungent flavour and its synthetic derivatives can stimulate pigmentation in the skin[6].

MATERIALS AND METHODS Piperine was extracted , purified and recrystallised in our lab. Beeswax ), Lanolin and Stearic acid were obtained from S D fine-chem Ltd(Mumbai) were as Triethanolamine Lab company(Hyderabad). 4-Hydroxyanisole was procured from Hychem laboratories. All reagents used in this work is of high quality analytical grade. Process development of topical herbal cream Beeswax (12.5 g), Lanolin (2 g) and Stearic acid (2.5 g) were taken in one beaker. In another beaker, Piperine as API (1 %) was dissolved in ethanol by sonication and introduced into glycerine (6.25 ml), water (7.26 ml), triethanolamine (0. 437 g). Both the beakers were maintained at 60°C and all the ingredients were melted. Then oily phase is added to aqueous phase and stirred continuously. As the temperature goes down peppermint oil was added and mixed well until required consistency was obtained[7-8]. Irritancy Patch test was carried out by applying cream on their ear pinne and dorsal surface to check for the redness or edema of skin. An area (1sq.cm) on the dorsal surface of the shaved rabbit was marked. The cream was applied to the specified area and time was noted. Irritancy, erythma and edema, was checked if any for regular intervals upto 24 hrs and reported[9]. Penetration Penetration of topical formulations was performed on (n=3) after testing for irritancy on rabbits[10]. A specific amount of the drug (1g) was spread evenly on the dorsal surface of rabbits. After 12 hrs the topical preparation was scraped off and made sure that there is no left



over. The quantity scraped off was weighed and subtracted from the initial quantity. The difference is assured to be penetrated and reported. Preparation of animals A protocol hard copy in triplicate was submitted to the institutional animal ethical committee and got approval (code no.2/IAEC).The animals were conditioned to the normal diurnal and nocturnal rhythms. The animals were fed with leafy vegetables and water ad libitum. The average weight of rabbits was 2.5kgs. Three groups of adult rabbits (Average weight 2.5kgs) (New Zealand strain rabbits) were selected containing 6 animals each. All animals were subjected to depigmentation by using 4-hydroxyanisole ointment. Animals were separated into three groups G1, G2 and G3. G1 group of animals under control, G2 for the group of animals were cream was applied and exposed to UV Radiation (Ultraviolet source lamp 15W, Sankyo Denki, Japan ) and G3 for the animals were applied cream alone[11-13].

RESULTS AND DISCUSSION Pharmacological evaluation of the cream was performed to prove the anti Vitiligo efficacy by using adult New Zealand OB Brown rabbits[14-18]. Depigmentation of the animals was successfully carried out. All animals in each group were defoliated and followed by depigmentation by 35 days. Repigmentation was carried out by applying only cream for G2. G3 was subjected to Cream and UV therapy. The group which was subjected to the topical cream therapy was found to commence repigmentation within 59 days.

G1(control) G2 G3

Figure 1. Rabbits before depigmentation

G1(control) G2 G3

Figure 2. Rabbits during repigmentation



G1 (control) G2 (59th day) G3 (50th day)

Figure 3. Rabbits after repigmentation (G2- piperine cream, G3- UV radiation + piperine cream)

CONCLUSION

It is proved that the formulated topical piperine cream was effective in the repigmentation. In UV therapy the repigmentation was faster but was found to be patchy pigmentation. When the repigmentation was performed only with the cream the rate was found to be slow but homogenous. Therefore it can be concluded that the topical treatment can be facilitated even without UV radiation. Acknowledgement The authors express their heartfelt gratitude to Nalanda College of pharmacy for providing information sources, library, internet and electronic facilities.

REFERENCES

[1] EM Shajil; Sreejata chaterjee; Deepali Agarwal; T Bagchi Rasheedunissa Begum, Indian Journal of Experimental Biology, 2006, 44, 526-539. [2] T Caixia ; F Hongwen ; L Xiran., J Dermatol , 1999, 21, 59-61. [3] KR Vinod; S Sandhya; D Santhosha. Indian drugs, 2009, 46, 5-10. [4] M Casacci; P Thomas; A Pacifico; A Bonnevalle; A Paro Vidolin; G Leone. Dermatology clinic, Huriez Hospital. University at life, 2007, 21, 956-963. [5] M L Cucchi ; P Frattini ; G Santargostino; G Orecchia. Pigment cell Res, 2000, 13,28-31. [6] L Faas; R Venkatasamy; RC Hider; AR Young; British J Dermatol, 2008, 158, 5, 941-950. [7] RM Mehta; M K Jain. Pharmaceutics II, 4th ed. Vallabh prakashan, Delhi, 2008, 12-26. [8] DPS Kohli. Drug formulations manual. Ist ed. Eastern publishers, New Delhi, 1991, 26-33. [9] J Jesto Mittal; Indian J. of Nat.Products. 2006, 23, (1), 3. [10] AK Seth. Pharmaceutics II .1st ed. Vikas &co.Publishing house, Jalandar, 2007, 2843- 284. [11] Frank Alena; Walter Dixon; Panakkezhum Thomas and Kowichi Jimbow. The journal of Investigative dermatology, 1995, 104, 5, 793-795. [12] Shosuke Ito; Kowichi Jimbow. The journal of investigative dermatology, 1983, 80, (4), 268-272. [13] Nikolaus Romani; Gerald Schuler; Peter pritsch, Journal of Indian dermatology, 1983, 40(4), 272-277. [14] M H Ahmida; M H Abuzogaya. Archives of applied science Research, 2009, 1, 1, 56-61.



[15] Chitranjib; Pawan dubey; Margret chandira; K.P.Sampath kumar. Archives of applied science Research. 2009, 1, 2, 32-56. [16] H.S.Nagwa Ahmida; Ebtisam EJ-Hasheme; N-E1-Enany; F.Betal. Archives of applied science research. 2009, 1, 2, 1-11. [17] Madhukar Saxena; C.G.Agarwal; Sunaina Gautam, Hemant K.Bid; Monisha Banerjee. Archives of apples science research, 2009, 1, 2, 65-66. [18] Sandhya.S, Chaitanya R.S.N.A.K.K, Vinod K.R et al, Archives of applied science research, 2010, 2, 2, 309-322.

Testing a piperine cream with and without ultraviolet Bphototherapy in 75 patients affected by bilateral vitiligo

G. Menchini, C. ComacchiGISV- Italian Group for the Study and Treatment of Vitiligo

Introduction

The bilateral vitiligo vulgaris (VVB) is an autoimmune disease with clin-ical course somewhat unpredictable. It affects about 1% of the popu-lation without significant differences of race and sex.

To date there is no universally accepted effective treatment. Based onmore than 10 years of study and treatment of these patients and theobservations on piperine in the international literature (1-4), we decid-ed to use a cream with the active principle Piperine to evaluate theireffectiveness. The proposed wording for vititan, the name of the creamhas been developed around the piperine based on observing the teamat King's College London who had tested on animal model the efficacyof the extract of Piper nigrum as a stimulator of melanocytes ( 1). Inthis work, mice were treated with piperine in health with and withoutultraviolet irradiation, which is why we have decided to experiment withand without ultraviolet B irradiation 311nm, now the gold standard oftreatment of vitiligo

Materials & Methods

Patients

75 patients affected by SV underwent topical treatment with piperinecream. Of the 75 patients 39 were males and 36 females aged between18 and 53 years.

The extension of vitiligo ranged between 5 and 35% of the total skinsurface.

32 patients (group A) carry 311nm UVB phototherapy, but the other43 patients not subjected to any form of phototherapy (group B).

Patients enrolled in this open study should satisfy the following rules:

Inclusion criteria:

1. SV with an extension from 5 to 50% of the total skin surface;2. Aged between 18 and 65;3. Compilation of written informed consent.

Exclusion criteria:

1. SV with an area greater than 50% of the total skin surface andother forms of vitiligo (seborrheic, segmental, etc.);

2. Age outside the range between 18 and 65;3. Serious local infection;4. Specific therapies carried out continuously in the two months pre-

ceding the enrollment phase.5. Pregnancy

During the first visit in addition to completing the clinical pictures weremade (with a particular flash to fluorescent lighting and ambient light-ing) for an objective proof of the initial clinical picture and provided allinformation relating to therapy. All patients had given informed consentprior to treatment also includes the possible alternative treatments andpossible side effects and patients recruited for the written declarationthat they were not pregnant. None of the patients had undergone in thetwo months prior to other drug continuous systemic treatment, topicaland / or physical vitiligo. Some patients (3 men and 4 women and 5 menin Group A Group B) were used occasionally in the two months prior totopical corticosteroids. Similarly prohibited was any other medicationand / or physically during and after a minimum of two months from theend of the protocol. The patient history is another important feature, itis essential to discuss the expectations of the patient, medication takenon, assessing the presence of scars, keloids, and local infection, theimmune status of patients, patient's habits than the exposure the sun.

The protocol provided for a single daily application of the cream in themorning to piperine for six consecutive months. Patients were visitedand photographed at the time of first visit and every month until the endof the treatment protocol. Patients were visited and photographed ineach patch; Through a morphometric evaluation of the spots using adedicated software (DDAX-MIPS) were then determined the percent-age of repigmentation of each individual at intervals of photography.

The percentage of repigmentation refers therefore to the extension ofvitiligo as a percentage of taxable value. For ease of viewing and con-formation studies in this field we divided the percentage of repigmen-tation in 4 values: 0 to 25% from 26 to 50% from 51 to 75% and finally76 to 100%.

With regard to phototherapy, all patients in group A performed the stan-dard protocol for UVB phototherapy.

The study lasted 10 months, from July 2008 to May 2009.

Cream

Results

Of the initial 75 patients completed the study 3 did not accurately andwere therefore eliminated from this report, two have performed occa-sionally (one in group A and one from group B) applying the cream forreasons other than itself, a patient but did not show the last two con-trols (group B).

No patient experienced acute or chronic side effects both locally andin general within six months of experimentation. Known only to reporta burning sensation and transient erythema at the application withoutthe skin around the lips and eyelids. The feeling lasted from 5 to 30minutes and was quite bearable, nothing to see on other skin areas.

Consequently, patients in the study of which 72 are: Group A is formedby 30 individuals, 12 males and 18 females while the group B 42 pa-tients including 25 males and 17 females.

The percentage of repigmentation obtained at the end of 6 months oftreatment is shown in Figure 1. As you can see the results are verygood with a repigmentation of between 76 and 100% in 80% (24 pa-tients) in group A and 52.4% (22 patients) of group B. If we look at thetrend over time of two groups, and relatively Figure 2 for group A andFigure 3 for group B, we see a fairly regular pattern of repigmentationin the two groups, with significant prevalence and speed improvementsfor group A. Only one patient showed repigmentation of 72 less than

25% belonged to group B. Over 50% of patients in group A began toshow signs of repigmentation from the first month, while group B thispercentage has been reached between the 2nd and 3rd month. Thepercentage of repigmentation obtained remained stable even after 3and 6 months after the end of the protocol.

Conclusions

The cream in Vititan monoapplicazione daily, has proved highly effec-tive in inducing repigmentation of vitiligo affected skin areas with andwithout stimulation ultraviolet B 311 nm.

The patient compliance was excellent and only transient burning sen-sations were complained by some patients especially during the imple-mentation of the lips and eyelids.

Repigmentation rates were extremely positive, although as always, theface and chest have responded faster and more complete patches ofthe limbs.

We believe that both ultraviolet treatment without the cream vititan isthe real novelty in the treatment of vitiligo.

Fig 2.Before and after of a young woman of group B (Photo made underwood light), a repigmentation rate of 93%

Fig. 3Before and after of a 40 yrs old man 95% of repigmentation (group b)

Fig. 4Repigmentaion of an halo nevus (group A)

Fig 5.Complete repigmenation of eyelids of a young lady, before and aftertreatment (Group B)

References

1. Faas L, Venkatasamy R, Hider RC, Young AR, Soumyanath A. Invivo evaluation of piperine and synthetic analogues as Potentialtreatments for vitiligo using a sparsely pigmented mouse model.Br J Dermatol. 2008 May; 158 (5) :941-50. Epub 2008

2. Lin Z, Liao Y, Venkatasamy R, Hider RC, Soumyanath A. Amidesfrom Piper nigrum L. with dissimilar effects on melanocyte prolifer-ation in vitro. J Pharm Pharmacol. 2007 APR 59 (4) :529-36.

3. Soumyanath A Venkatasamy R, Joshi M, Faas L, Adejuyigbe B,Drake AF, Hider RC, Young AR. UV irradiation affects melanocytestimulatory activity and protein binding of piperine. PhotochemPhotobiol. 2006 Nov-Dec, 82 (6) :1541-8.

4. Lin Z, Hoult JR, Bennett DC, Raman A. Stimulation of mousemelanocyte proliferation by Piper nigrum fruit extract and Its mainalkaloid, piperine. Planta Med 1999 Oct; 65 (7) :600-3.

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Scientific Studies Pigmerise™ · (360 MHz) NMR spectrometer. Reversed-phase HPLC analysis of irra- diated and unexposed piperine was performed with the use of an Alltech Adsorbosphere