Australasian Journal of Dermatology (2011) 52, 172–178
ORIGINAL RESEARCH
Insights into the mechanism of Piper betle leaf-induced
contact leukomelanosis using C57BL/6 mice as the
animal model and tyrosinase assays
Han-Nan Liu,1,2 Tsung-Yun Liu,3 Chih-Chiang Chen,1 Ding-Dar Lee1 and Yun-Ting Chang1
1
Department of Dermatology, Taipei Veterans General Hospital and National Yang-Ming University,
2
Department of Dermatology, National Defence Medical Centre, and 3Department of Medical Research and
Education, Taipei Veterans General Hospital, Taipei, Taiwan
ABSTRACT
Background/Objectives: Steamed piper betle
leaves (PBL) were once used by many Taiwanese
women to treat pigment disorders on the face. Most
women claimed a quick, favourable response at first,
only to be overcome with facial leukomelanosis later.
Methods: C57BL/6 mice were randomly assigned to
different groups to study if PBL could cause the fol-
lowing effects: contact dermatitis, leukomelanosis, or
hair bleaching. Intracellular melanin content was
measured by tyrosinase assays.
Results: Most steamed PBL-treated mice developed
contact dermatitis and postinflammatory hyperpig-
mentation (PIH) on their shaved backs. About half
developed bleached hair to varying extents. The
steamed PBL did not only bleach the hairs, but also,
unexpectedly, stimulated melanocyte replication,
indicated by the fact that the number of functional
melanocytes in the tail epidermis increased signifi-
cantly after treatment (P = 0.007). Using tyrosinase
assays PBL extract at the undiluted concentration
showed limited inhibition of melanogenesis, probably
via melanocytotoxicity.
Conclusions: The leukomelanosis observed in
patients might be the consequence of PIH combined
with a mixed reaction (hyper- and hypopigmenta
tion), probably due to the different volatile chemicals
Correspondence: Dr Han-Nan Liu, Department of Dermatology,
Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road,
Taipei, Taiwan. Email: hnliu@vghtpe.gov.tw
Han-Nan Liu, MD. Tsung-Yun Liu, PhD. Chih-Chiang Chen, MD.
Ding-Dar Lee, MD. Yun-Ting Chang, MD, PhD.
Submitted 2 August 2010; accepted 21 October 2010.
© 2011 The Authors
Australasian Journal of Dermatology © 2011 The Australasian College of Dermatologists
doi: 10.1111/j.1440-0960.2010.00724.
ajd_724 172..178
that surface after steaming the PBL. This conflicting
mixed reaction suggests that counteractive ingredi-
ents might exist in PBL. PBL, if purified, might be a
promising source of a novel bleaching agent.
Key words: hydroquinone, melanogenesis, piper
betle leaves, postinflammatory hyperpigmenta-
tion.
INTRODUCTION
In early 1997 a new facial bleaching remedy involving
topical dressing with steamed piper betle leaves (PBL)
became popular in Taiwan among women suffering from a
variety of pigment disorders such as melasma, freckles,
solar lentigo and periorbital darkening on the face.1–3 Ini-
tially most patients claimed a quick favourable response to
the treatment. Several months later, however, these patients
began seeking advice from dermatologists island-wide for
severe facial leukomelanosis – hyperpigmentation inter-
spersed with hypopigmentation (Fig. 1)
PBL is one of the ingredients in betel quid. Chewing betel
quid as a recreational activity is popular in many Asian
countries including Taiwan. PBL comes from the betel
pepper plant Piper betle. PBL is generally thought to be
non-carcinogenic, although the leaves may contain a
significant amount (15 mg/g) of Safrole, a carcinogen.4,5
Abbreviations:
PBL Piper Betle Leaves
PIH Postinflammatory Hyperpigmentation
HQ Hydroquinone
MC Melanocytes
DMEM Dulbecco’s Modified Eagle’s Medium
EDTA Ethylenediaminetetraacetic Acid
 Piper betle leaf-induced leukomelanosis
Figure 1 A female patient with Piper betle leaf-induced contact
leukomelanosis (Courtesy of Dr Lih-Jen Yang, Department of Der-
matology, Chang Gung Memorial Hospital, Taipei, Taiwan).1
Safrole is quickly metabolised by the human body into
hydroxychavicol and eugenol, which are excreted along
with urine.6
The purpose of this study was to explore the mechanism
of PBL-induced contact leukomelanosis.
MATERIALS AND METHODS
Topical treatment on C57BL/6 mice
Female C57BL/6 mice (6–8 weeks old) were obtained from
the National Laboratory Animal Centre. All animal proto-
cols were approved by the Institutional Animal Care and
Use Committee of Taipei Veterans General Hospital.
The experiment was partitioned into three repetitive ses-
sions, with the same experiment being carried out in each
of the three sessions. In each session 10 mice were ran-
domly distributed into two groups of five animals per group.
For group 1, PBL were steamed in a cooker for 10 min. The
mice’s shaved backs and tails (the most cephalic portion of
the tail, about 3 cm in length) were then wrapped with
strips of steamed PBL for four consecutive days in a week
for six weeks. In group 2, mice’s shaved backs and tails
were treated with 4% hydroquinone cream (HQ) (Panion &
BF Biotech Inc., Taipei, Taiwan) for four consecutive days in
a week for six weeks. The numbers of mice that actually
completed the experiment at the end in groups 1 and 2 were
15 and 14, respectively. A separate group (group 3) of seven
Australasian Journal of Dermatology © 2011 The Australasian College of Dermatologists
173
mice was designated as a control, receiving no treatment at
all. Signs of dermatitis such as redness, vesicles, scaling or
flaking were recorded daily. The treatment days were
counted as effective only when the surgical paper adhesive
tape that held the PBL on the site did not fall off. One week
after completion of the experiments full-thickness skin
biopsies were harvested from the centre of each test site
and control site for histological evaluation and dopa reac-
tion. To test if the surgical tape would cause dermatitis, five
separate mice were wrapped with the tape for six weeks.
Throughout the experimental period, when mice were
wrapped with PBL or photographed, they were tranquillised
with Zoletil (60 mg/kg, IM, Virbac Laboratories, Carros,
France).
The present study used shaved backs to provide a broader
area for visually evaluating the treatment effects.7 Given the
fact that there are no epidermal melanocytes (MC) in the
back skin when C57BL/6 mice are older than six weeks old,
the bleaching effect was determined by the appearance of
white hairs on the treated sites.
Histological evaluation and dopa reaction
Skin samples were fixed with 10% buffered formalin and
embedded in paraffin. They were sliced into 4-mm vertical
sections and then stained with H&E as well as Fontana-
Masson stain. Skin samples collected from tails were
snap-frozen in liquid nitrogen and processed for dopa his-
tochemistry as previously described.8
We used an eyepiece counting grid at x100 magnification
to quantitate functional MC in the epidermal sheet. The grid
provided a viewing field covering 1 mm2. Every specimen
had a minimum of nine randomly selected areas for viewing
and counting. We counted the numeric change of MC on the
mouse tail, treated site (upper segment) versus non-treated
site (lower segment). There was at least a 2 cm of buffer
zone between the two sites. We examined tails of the same
mice for such a comparison due to the fact that the number
of functional MC on mice tails was steady throughout the
length of tail (Table 1). However, this number could vary
significantly among different batches of mice (from 120 to
360/mm2).
PBL extract preparation
One hundred grams of fragmented PBL was steamed in a
cooker for 10 min, left to cool, and then blended with 30 mL
sterile water until creamy. This was then filtered through
gauze.
Cells, culture techniques and treatments
Pigmented murine melanoma cells B16-F0 were grown in a
humidified atmosphere with 10% CO2 at 37°C. Cells were
routinely passaged, harvested, and resuspended in Dulbec-
co’s modified Eagle’s medium (DMEM) and seeded at
~2 ¥ 104 cells/well in 6-well plates as described previously.9
For treatment of the cells, PBL extract and HQ (Sigma-
Aldrich Corporation, St. Louis, MO, USA) were added 24 h
© 2011 The Authors
174 H-N Liu et al.

Table 1 The profile of C57BL/6 mice after different treatments

The number of mice (%) that developed treatment The number of dopa (+) cells on mouse tail
effects on their shaved back epidermis (melanocytes/mm2)

Group Dermatitis PIH Hair bleaching Upper† Lower‡ P value*

1 (n = 15) 13 (86.7%) 12 (80%) 8 (53.3%) 346 ⫾ 72 254 ⫾ 33 0.007

2 (n = 14) 0 (0%) 0 (0%) 14 (100%) 116 ⫾ 15 175 ⫾ 23 0.000
3 (n = 7) 0 (0%) 0 (0%) 0 (0%) 137 ⫾ 22 136 ⫾ 15 0.909

Group 1: steamed Piper betle leaf treatment. Group 2: 4% hydroquinone cream treatment. Group 3: no treatment. *Paired t-test (two-sided)
of the difference between upper and lower segments of tails. The results for dopa (+) cells were expressed as means ⫾ SD. †Upper treated
segment of tail. ‡Lower untreated segment of tail. PIH, postinflammatory hyperpigmentation.

after seeding in duplicate wells at different concentrations.
Equivalent volumes and concentrations of diluents were
added to duplicate control wells. Cells were treated with
compounds for four days. After allowing one day for cell
attachment, the medium was changed and fresh PBL
extract and HQ were added in fresh DMEM. Four days later,
cells were photographed and then harvested after 3 min
treatment with 0.5 mL trypsin/ethylenediaminetetraacetic
acid (EDTA). After dislodging the cells with occasional agi-
tation, 2 mL of fresh DMEM was added to inactivate the
trypsin and 100 ml aliquots were seeded into flat-bottom
96-well plates for the 3-[4,5 dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide (MTT) assay.
Cell viability and proliferation
And MTT assay kit (Boehringer Mannheim Biochemica,
Mannheim, Germany) was used to determine cellular
viability and proliferation. Because a spontaneous HQ oxi-
dation at a higher concentration may boost the discoloration
and render a higher than expected reading, a trypan blue
exclusion test for viability was added.
Intracellular melanin content assays
Spectrophotometric tyrosinase assay
Melanin content was measured using a modified Virador’s
method.9 Cells collected after trypsin/EDTA treatment were
counted using a trypan blue exclusion method. Pellets of
5 ¥ 105 cells were solubilized in boiling 1N NaOH for 30 min.
The measurement of melanin content was performed by
spectrophotometric analysis at 405 nm absorbance.10
Dot-blot radiometric tyrosinase assay
Modified radiometric tyrosinase assay was used for measur-
ing PBL effects on melanocytes.11,12 The assay was per-
formed in quadruplicate in 96-well microtiter plates by
adding 10 ml PBL and 20 ml purified tyrosinase (Sigma, St.
Louis, MO, USA). After 30 min pre-incubation at 23°C, 10 ml
of L-[3,5-3H] tyrosine (Amersham Biosciences, Amersham,
UK) was added along with 10 mL of L-dopa cofactor in
1 M sodium phosphate buffer, pH 7.2, containing 0.01%
albumin. It was then incubated for 1 h at 37°C, followed by
the addition of 100 mL 0.1 M HCl with excess unlabeled
L-tyrosine. The contents of each well were removed with a
multichannel pipette to a dot-blot apparatus (GenePure,
Bio-East Inc., Taipei, Taiwan). The acid-insoluble radioac-
tive melanin and melanin precursors were bound to Zeta-
Probe blotting membranes (Perkin-Elmer, Boston, MA,
USA) for 15 min at 23°C. The membranes were then dried
under a vacuum and washed three times with 250 mL
0.1 M HCl with excess unlabeled tyrosine. The membranes,
removed from the apparatus and washed three more times,
20 min each with 100 mL 0.1 M HCl, were air-dried and
exposed to a Kodak Biomax MS film in TranScreen low
energy intensifying screen (Carestream Health, Cedex,
France). The membranes were converted to greyscale
digital images with an Epson Stylus CX3900 scanner
(600 ¥ 1200 dpi resolution; Epson Taiwan Technology
&Trading LTD, Taipei, Taiwan.). The images were imported
to an ImageJ (NIH, Bethesda, MD, USA) computer imaging
software program. The greyscale (range 1–256 intensities of
grey) was measured.
Statistical calculations
SPSS-17 (SPSS, Inc., Chicago, IL, USA) was used to analyse
the data. Densities of MC on mouse tails were evaluated
with a two-sided paired t-test. Analysis of variance followed
by post hoc test was used to analyse the significance of
difference between means of control and various treatment
groups.
RESULTS
Contact dermatitis and PIH
Shaved backs
Group 1: Thirteen of the 15 C57BL/6 mice (86.7%) devel-
oped contact dermatitis on their shaved backs (Fig. 2a), 9 to
20 days (median, 11 days) after PBL treatment. The majority
of dermatitis mice (12/13) developed PIH 5 to 7 days later.
The band-like shape of dermatitis and PIH corresponded to
that of the PBL strip (Fig. 2b).
The dermatitis mice presented grossly with scaling,
flaking and slight swelling without accompanying blisters.
Histopathological examination revealed subacute inflam-
mation (Fig. 3) with clumps of melanin and heavily pig-
mented spindle cells in the dermis (Fig. 4).

© 2011 The Authors

Australasian Journal of Dermatology © 2011 The Australasian College of Dermatologists
 Piper betle leaf-induced leukomelanosis
Figure 2 (a) A C57BL/6 mouse developed band-like dermatitis
(between arrows) with scaling on its shaved back after 5 weeks’
treatment with steamed Piper betle leaf (12 accumulative days). (b)
Band-like hyperpigmentation developed 1 week later, representing
postinflammatory hyperpigmentation and growing pigmented
anagen hair underneath.
Figure 3 (a) Normal mouse skin with hair follicles in the telogen
phase. (b) After 6 weeks’ treatment (20 accumulative days), the skin
biopsy showed a pattern of subacute dermatitis with compact par-
akeratosis, epidermal hyperplasia, dilatation of blood vessels and
mononuclear infiltration. (H & E stain, original magnification ¥400).
Group 2: None of 14 mice that finished 4% HQ cream
treatment developed dermatitis or PIH.
Group 3: None of seven control mice developed dermatitis
or PIH.
Mice tails
The tail was an area in which it was hard to determine
dermatitis or PIH. We did not grossly detect any visible PIH
on the tails. Since the shaved back was a more reliable site
than the tail to study dermatitis/PIH, details about
dermatitis/PIH on the tail are not provided.
Hair bleaching
Group 1: Eight out of 15 (53.3%) mice developed hair
bleaching of varying extent after 9 to 24 accumulative days
(median, 14 days) of steamed PBL treatment (Fig. 5). In
Australasian Journal of Dermatology © 2011 The Australasian College of Dermatologists
175
Figure 4 Steamed Piper betle leaf-treated areas (6 weeks’ treat-
ment, 13 accumulative days). Postinflammatory hyperpigmentation
was evident: clumps of melanin and heavily pigmented spindle cells
in the papillary and upper dermis. The overlying epidermis was
moderately hyperplastic, a sign of chronic dermatitis. The hair fol-
licles were heavily pigmented. (Fontana-Masson stain). (H&E, origi-
nal magnification ¥400).
contrast to black hairs in areas not treated (Fig. 6a) or with
PIH (Fig. 6b), bleached hairs were evident microscopically
by diminished melanin pigments in the hair bulbs of the
anagen (Fig. 6c). However, the bleaching effect varied
greatly among the different batches of mice; almost all the
mice in one batch developed hair bleaching, while only one
in another batch did.
Group 2: All 14 mice treated with 4% HQ cream showed
prominent hair bleaching after 7 to 24 accumulative days
of treatment (median, 12 days).
Group 3: No hair bleaching was observed in controls.
None of the five surgical tape-treated mice developed
dermatitis.
Dopa reaction of epidermal melanocytes (MC) in
tail skin
The dopa-positive cells grouped to form rows of somewhat
ovoid islets (Fig. 7a) which were fenced and permeated
by hairs. HQ significantly diminished the density of func-
tional MC (Table 1) (Fig. 7b). However, the steamed PBL
increased notably the average number of functional MC in
the tail epidermis (Table 1) (Fig. 7c). A diverse density of
MC was noted from area to area. The ovoid islets housing
MC did not hold their normal pattern any longer, MC in
some islets had become denser, with new MC appearing in
the surrounding areas, while MC in other islets had become
dispersed and formed blank spots.
Cell viability, proliferation, and melanin content
HQ at the concentration of 0.01 mg/mL significantly inhi
bited melanogenesis and diminished dramatically the
© 2011 The Authors
176 H-N Liu et al.
Figure 5 Patches of bleached hairs among steamed Piper betle
leaf-induced synchronized growth of hair follicles in the anagen
phase (5 weeks’ treatment, 12 accumulative days). *Denotes the
area with homogeneous pink skin colour reflecting that underneath
hair follicles were in the telogen phase.
viability (by trypan blue exclusion test)(Fig. 8a) and the
proliferation (by MTT assay) of MC (Fig. 8b). The rough
PBL extract showed significant inhibition of melanogenesis
only with the undiluted solution (Fig. 8c), while the viability
and proliferation of MC dropped significantly only with the
concentration of 1:10 (Fig. 8a,b)
Dot-blot radiometric assay
HQ inhibited radioactive melanin formation significantly at
the concentration of 0.1 mg/mL, while PBL extract showed
a slight reduction of melanin production only at the undi-
luted concentration (Fig. 9). On the other hand, the undi-
luted PBL was better than nothing (the control) in inhibiting
melanogenesis (P = 0.053).
DISCUSSION
Contact dermatitis from PBL has been rarely reported in the
literature. In 1997, Yang et al. reported 137 patients who
© 2011 The Authors
Australasian Journal of Dermatology © 2011 The Australasian College of Dermatologists
Figure 6 (a) Normal mouse back skin. Almost all the hair follicles
were in the telogen phase, which can be identified by its dermal
location and non-pigmented hair shaft. (b) Steamed Piper betle leaf
(PBL)-treated back skin with subacute dermatitis and postinflam-
matory hyperpigmentation (arrows) in the dermis. The hair follicles
were in the anagen phase, which can be identified by their subcu-
taneous location and heavily pigmented hair shaft (6 weeks’ treat-
ment, 13 accumulative days). (c) Steamed PBL-treated back skin
with bleached hairs. Most hair follicles were in the anagen phase,
but the melanin pigment in the hair bulbs was markedly diminished
(6 weeks’ treatment, 11 accumulative days) (Fontana-Masson
stain). Scale bar = 1 mm.
developed leukomelanosis after use of steamed PBL for
treating pigmented spots on the face.1 Eighty-five percent of
them reported a history of prior contact dermatitis present-
ing as erythema, burning, stinging or vesicle formation. The
authors proposed that the steamed PBL might cause phyto-
phototoxic dermatitis and subsequent severe hyperpigmen-
tation with a confetti-like appearance (leukomelanosis). In
1999, Liao et al. reported another 15 patients and suggested
that the evolution of this pigmented disorder can be divided
into three stages: immediate bleaching stage; prominent
hyperpigmentation stage; and confetti-like depigmentation
stage.3 In the present study, most steamed PBL-treated mice
developed contact dermatitis, supporting the hypothesis
 Piper betle leaf-induced leukomelanosis 177

(a)
120 control
1x
100 ** 10x
** 100x
80 1000x

% viability
10000x
60
1
0.1
40
* 0.01
0.001
20 * *

0
Control PBL HQ (mg/ml)
(b)
2.5

Figure 7 Dopa (+) cells in the mouse tail epidermis. Hair had been ** control
plucked for better visualization of cells. (a) Normal control. Most 2.0 1x

Abs (562-690 nm)

cells grouped together, forming rows of somewhat ovoid islets with 10x
a few cells scattered in the areas between the islets. Inset: dendritic 100x
1.5 1000x
cells in the islet. Some cells in the periphery are out of focus
10000x
because of the thickness of the epidermal sheet. (b) the hydro-
1.0 1
quinone cream-treated group. The cell density dropped signifi- 0.1
cantly, leaving many blank spots in the islets (arrows). Inset: note * * 0.01
the acelluar area in the islet. (c) Steamed Piper betle leaf-treated
0.5 0.001
group. The density of cells varied greatly from area to area. Inset: In
the darker area mainly on the right, many islets were crowded with * *
cells. Scale bar = 0.5 mm and 0.1 mm (inset). 0.0
Control PBL HQ (mg/ml)
(c) 0.5
that steamed PBL is an irritant. However, the dermatitis/PIH
is probably not phytophotodermatitis with subsequent PIH 0.4
control
as suggested previously.1–3 All caged mice were only 1x
Abs (405 nm)

exposed to regular fluorescent lamps in the animal house, 0.3 10x

but most mice (86.7%) in group 1 still developed contact
dermatitis and PIH.
We were unable to reproduce leukomelanosis on the
shaved backs of mice. One possible explanation may be
linked to the fact that there is no epidermal MC in C57BL/6
mice’s back skin when they reach 6 weeks of age. Nor did
we observe leukomelanosis on the tail skin, where func-
tional MC are abundant. Nevertheless, the steamed PBL
treatment unexpectedly increased functional MC in the tail
epidermis significantly (Table 1, Fig. 7). The grouping MC
varied in density notably from area to area, which seemed
to bear superficial resemblance to PBL-induced leukomel-
anosis. It was interesting to note that the steamed PBL could
not only bleach the hairs, but also stimulate melanocyte
replication simultaneously. These conflicting findings
suggest that counteractive ingredients might exist in PBL.
The bleaching effect of steamed PBL varied greatly
among different batches of mice. All mice, however,
responded readily to the treatment with HQ cream, a puri-
fied compound. This discrepancy suggested that hot steam-
ing was not a constantly repeatable way to draw out the
potential whitener of PBL.
PBL may be a promising source of a novel bleaching
agent since steamed PBL could bleach human skin, as dis-
cussed in prior reports, and bleach mouse hairs, as demon-
strated in the present study. However, further fractionation
of PBL extract has to be done to find the candidate com-
pound(s), which ideally should be non-irritative, non-
100x
1000x
0.2 10000x
1
0.1
** * * 0.01
0.1 * 0.001
0.0
Control PBL HQ (mg/ml)
Figure 8 (a) Cell viability on trypan blue exclusion test (n = 3).
After treatments with Piper betle leaf (PBL) or hydroquinone cream
(HQ), the viability of melanoma cells B16-F0 dropped significantly
at the 1:10 dilution and at the concentration of 0.01 mg/mL, respec-
tively. (b) Cell proliferation measured by the MTT assay displayed a
similar pattern to (a), but when the concentration of HQ reached
1 mg/mL, the discoloration due to spontaneous HQ oxidation
became obvious and rendered the reading higher than expected
(PBL: n = 12; HQ: n = 7). (c) Total intracellular melanin content,
measured by absorbance at 405 nm (n = 6). The inhibition of mel-
anogenesis seemed to correspond relatively well to melanotoxicity,
as demonstrated in (a) and (b). *P < 0.001, **P < 0.05.
melanocytotoxic, non-carcinogenic and non-mutagenic.
PBL has been credited with exerting diverse biological
effects including antifungal, antiseptic, antihelmintic,
antidiabetic and radioprotective functions.13
PBL contain volatile oils, nitrate, and small quantities of
sugar, starch and tannin. The most important constituents
of betel leaves may be the various chemicals in the essential
oil,14,15 which are volatile and can be extracted from within

© 2011 The Authors

Australasian Journal of Dermatology © 2011 The Australasian College of Dermatologists
178 H-N Liu et al.
Figure 9 In vitro dot-blot radiometric assay. Purified tyrosinase
was pre-incubated with either Piper betle leaf (PBL) extract (non-
diluted to 1000X dilution) or hydroquinone cream (HQ) (0.001 to
1 mg/mL). This was then assayed in quadruplicate for tyrosinase
activity using the [3H] tyrosine dot-blot assay on a Kodak Biomax MS
film (Carestream Health, Cedex, France). Radiolabelled melanin
dramatically decreased when the concentration of HQ was
ⱖ0.1 mg/mL. PBL rough extract led to mild inhibition of melanin
production only in the undiluted concentration.
PBL to the surface after steaming. The content of essential
oil varies from 0.7 to 2.6 per cent depending upon the vari-
eties of leaves. Some of these constituents are actually
derivatives of phenol (e.g. eugenol, carvacrol, and chavi-
col), catechol (e.g. allylpyrocatechol), or benzene (e.g.
chavibetol, p-cymene, and anethole).2,16 The largest class of
chemicals known to trigger contact/occupational vitiligo is
the phenolic/catecholic derivatives.17 Many have been dem-
onstrated to be preferentially cytotoxic to melanocytes, with
high-dose exposure resulting in the initiation of apoptosis.
The in vitro spectrophotometric tyrosinase assay and
radiometric tyrosinase analysis in our study showed that
PBL extract could only inhibit melanogenesis at the undi-
luted concentration, probably via its melanocytoxicity. This
finding seems to suggest that PBL extract is not such a
promising bleacher as previously suggested.1–3 But we have
to take into consideration the findings that PBL could bleach
the hairs and increase the number of functioning melano-
cytes simultaneously, probably via different constituents of
PBL. The counteractive constituents in PBL extract, when
acting together, might result in only mild inhibition of mel-
anogenesis. Future studies to fractionate PBL extract to
obtain the purified compounds are necessary to solve this
problem.
In conclusion, PBL could cause irritant contact dermatitis,
but not phytophotodermatitis. Steamed PBL could stimulate/
inhibit the activities of MC simultaneously, probably via
different compounds. Although there are limits to extrapo-
lating findings from mouse models to humans, the leukomel-
anosis observed in people after applying steamed PBL may
be a PIH plus a mixed reaction (hyper- and hypopigmenta-
tion). PBL could still be a promising bleaching agent if the
effective bleaching fractionation of PBL extract could be
isolated and be non-irritative, non-melanocytotoxic, non-
carcinogenic and non-mutagenic.
© 2011 The Authors
Australasian Journal of Dermatology © 2011 The Australasian College of Dermatologists
ACKNOWLEGEMENTS
This work was supported by Taipei Veterans General Hos-
pital (grants V96C1-154, V97C1-154 and V98C1-164). We
thank Mr Matt Nicodemus for proofreading the manuscript.
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