236 Original Papers

Lupenone from Erica multiflora Leaf Extract Stimulates

Melanogenesis in B16 Murine Melanoma Cells through
the Inhibition of ERK1/2 Activation

Authors Myra O. Villareal 1, Junkyu Han 1, 2, Kyoko Matsuyama 2, Yukiko Sekii 2, Abderrazek Smaoui 3, Hideyuki Shigemori 2,
Hiroko Isoda 1, 2

1
Affiliations Alliance for Research on North Africa (ARENA), University of Tsukuba, Tsukuba, Ibaraki, Japan
2
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

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3
Technopole de Borj-Cedria-Centre de Biotechnologie, Hammam-Lif, Tunisia

Key words Abstract nase enzyme activity but can increase tyrosinase
" Erica multiflora L.
l ! expression at both the transcriptional and trans-
l
" Ericaceae
Hypopigmentation diseases are usually managed lational levels. The increase in the tyrosinase
l
" lupenone
using UVB light which increases the patientsʼ risk mRNA expression was most likely due to the in-
l
" B16 murine melanoma cells

for skin cancer. Here, we evaluated the melano- hibited mitogen-activated protein kinase phos-
l
" melanogenesis

l
" tyrosinase genesis stimulatory effects of leaf extracts of Erica phorylated extracellular signal-regulated kinases
l
" pERK1/2 multiflora, a medicinal plant from the Mediterra- 1 and 2 activation. We report for the first time
nean region, and its active component, lup-20 that E. multiflora ethyl acetate extract and its ac-
(29)-en-3-one, as possible therapeutic agents to tive compound lup-20(29)-en-3-one stimulate
address hypopigmentation disorders. B16 murine melanogenesis by increasing the tyrosinase en-
melanoma cells were treated with E. multiflora zyme expression via mitogen-activated protein
extracts or its active component lupenone to eval- kinase phosphorylated extracellular signal-regu-
uate their effects on melanin biosynthesis. The lated kinases 1 and 2 phosphorylation inhibition,
mechanism underlying the observed effects was making it a possible treatment for hypopigmenta-
also determined. Bioactivity-guided fractionation tion diseases.
of fifteen ethyl acetate fractions identified frac-
tion 2 to have melanogenesis stimulatory effects
due to its ability to decrease mitogen-activated Abbreviations
protein kinase phosphorylated extracellular sig- !
nal-regulated kinases 1 and 2 activation. Prepara- α-MSH: [Nle4, D‑Phe7]-α-melanocyte stimu-
tive TLC of ethyl acetate fraction 2 revealed the lating hormone trifluoroacetate salt
presence of lup-20(29)-en-3-one as the major EMEA: Erica multiflora ethyl acetate extract
bioactive component. B16 cells treated with lup- EMME: Erica multiflora methanol extract
received October 14, 2012
20(29)-en-3-one increased melanin content EMBU: Erica multiflora butanol layer
revised Nov. 28, 2012 without cytotoxicity. To determine the mecha- EMWA: Erica multiflora water layer
accepted Dec. 27, 2012 nism for the observed effects of lup-20(29)-en-3- lupenone: lup-20(29)-en-3-one
one, the tyrosinase enzyme activity, the tyrosi- MAPK: mitogen-activated protein kinase
Bibliography
DOI http://dx.doi.org/ nase protein expression, and the activation of p-ERK1/2: phosphorylated extracellular signal-
10.1055/s-0032-1328189 phosphorylated extracellular signal-regulated ki- regulated kinases 1 and 2
Published online February 13, nases 1 and 2 were determined. In addition, the Mitf: microphthalmia-associated transcrip-
2013 expression of the tyrosinase mRNA was quanti- tion factor
Planta Med 2013; 79: 236–243
fied using real-time PCR. Results showed that PMH: progressive macular hypomelanosis
© Georg Thieme Verlag KG
Stuttgart · New York · lup-20(29)-en-3-one has no effect on the tyrosi-
ISSN 0032‑0943

Correspondence
Dr. Hiroko Isoda, Professor Introduction is catalyzed by the enzymes tyrosinase, tyrosi-
University of Tsukuba ! nase-related protein 1, and dopachrome tauto-
Tennodai 1–1–1 Melanin is considered to be the perfect protection merase [3–5] with tyrosinase being regarded as
Tsukuba Ibaraki 305–8572
Japan against UV-induced photodamage and has anti- the key enzyme in melanogenesis. Melanin is the
Phone: + 81 2 98 53 57 75 oxidant and radical scavenging properties [1] as primary determinant of skin color which may be
Fax: + 81 2 98 53 57 76 well as optical and chemical filtering properties altered significantly by diseases or exposure to
isoda.hiroko.ga@
u.tsukuba.ac.jp [2]. The synthesis of melanin in the melanocytes certain substances (drugs, irritants). A minor al-

Villareal MO et al. Lupenone from Erica multiflora … Planta Med 2013; 79: 236–243
 Original Papers 237

teration of cutaneous pigmentation yields unwanted aesthetic ef-

fects that can have major psychological implications [6, 7]. The
changes in skin color are usually caused by hypopigmentation
diseases such as PMH, post-inflammatory pigmentary changes,
or leukoderma. Hypopigmentation in PMH is due to a change in
melanogenesis based on a decrease in melanin formation and a
change in the distribution of melanosomes. The exact patho-
genesis of PMH is yet unknown with some research reporting
the cause to be due to Propionibacterium spp. [8], but incidences
of PMH with no evidence for Propionibacterium spp. have also
been reported [9]. At present, PMH is generally managed using
UVB laser [10] and narrow-band ultraviolet B phototherapy that
managed to reduce the lesions or hypopigmented areas which
actually reappear after a few weeks needing more UVB sessions
[11]. The use of UVB as therapy, however, increases the risk for

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skin cancer and inflammatory responses, particularly neutrophil
infiltration, impairment of the protective effect of TNF-α, and ver-
sican upregulation, are closely involved in UVB/ROS-induced skin
tumorigenesis [12–14] making the search for a treatment from a
natural source a better and safer therapeutic alternative.
Erica multiflora L. is a perennial shrub characterized by a small
leaf size [15] and a deep root system of low diameter [16]. E. mul- Fig. 1 Schematic diagram of extraction of Erica multiflora L. leaves using
tiflora is distributed in the Mediterranean area, mostly in North different solvents.
Africa, where it is used as a folk medicine to treat hyperlipidemia
and as a diuretic and antiseptic agent [17, 18]. The high plant di-
versity of the Mediterranean vegetation is believed to give plants
adaptive strategies to cope with summer drought stress of the glass sample bottle for 2 weeks at room temperature. The extract
Mediterranean climate [19]. The use of Erica species in tradition- was then filtered using a 0.45-µm filter (Millipore) and stored at
al medicines is widespread and includes antibacterial [20] and − 80 °C until use.
anti-inflammatory [21] activities. Although E. multiflora has been
used as a traditional medicine, its ability to stimulate the synthe- Extraction and isolation of lupenone from E. multiflora
sis of melanin in B16 murine melanoma cells and the mechanism ethyl acetate extract
underlying its ability to modulate melanogenesis have not yet Leaves (100 g) of E. multiflora were extracted with MeOH (1.3 L)
been reported. Reports on the chemical constituents of Erica spe- and evaporated to dryness in vacuo at 30 °C. The MeOH extract
cies revealed the presence of flavonoids, anthocyanins [21, 22], (22.7 g) was then partitioned between EtOAc (400 mL × 3) and
coumarins, and triterpenoids [23, 24]. E. multiflora flowers and H2O (400 mL). The H2O-soluble portion was partitioned with n-
leaves were also reported to contain tannins, proanthocyanidols, BuOH (400 mL × 3). The EtOAc-soluble material (3.5 g) was sub-
a number of flavonoids, and cyanogenetic glucosides [18]. Previ- jected to a silica gel column (φ 2.2 × 30 cm, n-hexane/EtOAc,
ously, E. multiflora extract was reported to promote human der- 5 : 1 → 3 : 1 → 1 : 2→CHCl3/MeOH, 8 : 2 → 5 : 5 → 2 : 8 → 0 : 10) to af-
mal papilla cells in vitro and in vivo by induction of the anagen ford 15 fractions (EMEA-1~15). EMEA-2 was separated into 2
phase from the telogen phase [25]. Lupenone has been reported fractions (EMEA2-1~2) by a C8 Sep-Pak cartridge (10 g, n-hex-
to be present in many plants including Sorbus commixta [26], Sa- ane/EtOAc, 10 : 0 → 7 : 1 → 4 : 1 → 2 : 1 → 0 : 10; Waters). EMEA2-1
madera indica [27], yellow birch [28], and Tephrosia villosa [29], was purified by preparative TLC (SiO2 60F254, 20 × 20 cm, n-hex-
but there have been no reports on its presence in E. multiflora. ane/acetone, 98 : 2; Merck). HPLC analysis determined the purity
In this study, the effect of E. multiflora leaf extract on melanogen- of lupenone to be ≥ 90%. l " Fig. 1 presents the schematic diagram

esis in B16 melanoma cells and the mechanism underlying these for the sample preparation from extraction with ethanol until the
effects were investigated. Moreover, the effect of the major active isolation of the active compound.
compound, lupenone, on melanogenesis was also determined.
This is the first report on the presence of lupenone in E. multi- Cell culture
flora and on the mechanism of their melanogenesis stimulatory B16 cells used in the experiment were purchased from the Riken
effects in mammalian cells. Cell Bank in Tsukuba and maintained as a monolayer culture in
Dulbeccoʼs modified Eagleʼs medium (DMEM) (Sigma) supple-
mented with 10 % fetal bovine serum (Sigma) and 4 mM L-gluta-
Materials and Methods mine (Sigma). Cells were incubated at 37 °C in a 95 % air and 5 %
! CO2 incubator.
Plant collection and preparation of ethanol extract Melanin assay: The melanin content synthesized by B16 melano-
E. multiflora leaves were collected from Tunisia in April 2006 and ma cells treated with E. multiflora extract or lupenone was deter-
authenticated by Prof. Abderrazek Smaoui (Technopark Borj Ce- mined as previously described [30]. B16 cells were seeded onto
dria Tunisia). Voucher specimens of the leaf samples (UT-ARE- 100-mm petri dishes at a density of 5 × 105 cells/100-mm petri
NA‑TN‑00 057) were deposited in the Alliance for Research on dish and incubated at 37 °C with 5% CO2 incubator. After over-
North Africa, University of Tsukuba, Japan. Air-dried leaf samples night incubation, the growth medium was replaced with a fresh
(10 g) were macerated in 100 mL of 70 % ethanol and kept in a medium with or without the sample (0.0005, 0.005, 0.05 w/v

Villareal MO et al. Lupenone from Erica multiflora … Planta Med 2013; 79: 236–243
238 Original Papers

ethanol extract; 1 µg/mL EMEA; 0.01, 0.05, or 0.15 % EMEA; 0.1, structions. Synthesized complementary DNA was used for real-
0.5, or 1.0 µmol/L lupenone) and incubated further for 72 h. For time polymerase chain reaction (rt-PCR) analysis performed with
the confirmation of the effect of lupenone on the melanin con- a 7500 Fast Real-Time PCR system using TaqMan Universal PCR
tent of B16 cells compared with EMEA, the dot-blot method was mix and TaqMan probes (Applied Biosystems) to quantify the ex-
performed. B16 cells were seeded onto 96-well plates at a density pression of the tyrosinase gene. Gapdh was used to normalize the
of 8 × 103 cells/well and incubated for 48 h. After cell lysis using tyrosinase gene expression (housekeeping gene). Rt-PCR reac-
10 % SDS (Sigma), the melanin was blotted on a nitrocellulose tions were run on an ABI 7500 Fast (Applied Biosystems) with
membrane (BIO‑RAD) using a vacuum pump. The total protein the following thermal cycling protocol: 95 °C for 10 min followed
was then quantified using the Pierce BCA Protein Assay (Pierce by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, and the data ob-
Biotechnology) and the melanin content was expressed as mela- tained were analyzed using 7500 Fast System SDS Software 1.3.1.
nin content/total protein. α-MSH was used as a positive control (Applied Biosystems).
and was purchased from Sigma with a purity of ≥ 95% (HPLC).
Statistical analysis
Cell viability Results are expressed as mean value ± SD, and differences be-
The cell viability of B16 cells was evaluated using the Guava PCA tween treated samples and the control (untreated cells) were

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(GE Healthcare) using the ViaCount program for analysis and at evaluated for significance using the Studentʼs t-test.
the same time as the melanin assay. B16 cells (5 × 105 cells/dish)
were seeded onto 100-mm dishes and cultivated as described
above. After overnight incubation, the growth medium was re- Results
placed with sample-containing medium and incubated further !
for 3 days. The medium was then removed, and the cells washed Dilutions of the ethanol extract of the E. multiflora leaves were
twice with PBS and harvested by trypsinization. The harvested prepared (5.0 × 10−4 %, 5.0 × 10−3 %, 5.0 × 10−2 % w/v) and used to
cells were then resuspended in growth medium, and a 20-µL ali- treat B16 cells for 48 h. Results showed that the melanin content
quot was stained with the ViaCount reagent (GE Healthcare) fol- of B16 cells was increased in a dose-dependent manner without
lowing the manufacturerʼs instructions. affecting the cell viability (l " Fig. 2 a). B16 cells treated with

−2
5.0 × 10 % w/v of the extract had a 33 % higher melanin content
Tyrosinase activity assay compared with the control.
The tyrosinase enzyme activity was determined following the With the observed increase in melanogenesis in the initial test,
protocol on the tyrosinase activity assay described in Lee et al. EMME was produced and then partitioned into EMEA, EMBU
[31] with some modifications. Briefly, 2 mM L-tyrosine was dis- and EMWA. B16 cells were then treated with the EMME, EMEA,
solved in phosphate sodium buffer, and 0.1, 0.5, or 1.0 µmol/L lu- EMBU, and EMWA layers (1 µg/mL) and incubated further for
penone or 200 µmol/L arbutin (≥ 98 % purity from Sigma) were 72 h to determine the effect of the extracts using different sol-
added to the L-tyrosine solution. Mushroom tyrosinase was then vents on melanin synthesis. Results showed that the EMEA-treat-
added and the mixture incubated for 30 min at 37 °C. The tyrosi- ed cells had the highest melanin content among all the treat-
nase activity was determined based on the differences in the ab- ments or had 38.5 % more melanin compared to the untreated
sorbance (475 nm) between the control and lupenone or arbutin. cells or control (data not shown).
EMEA was then subjected to bioactivity-guided fractionation to
Western blot determine the fraction that contains the compound that was re-
Total protein samples were extracted from B16 cells seeded onto sponsible for the observed stimulated melanogenesis. Results
100-mm dishes at a density of 3 × 106 cells per dish and cultivated showed that cells treated with six out of fifteen fractions (frac-
as described above. After the cells were allowed to attach over- tions 1–5 and 15) had higher melanin content than the control
night, the medium was then replaced with medium containing and fraction 2, and EMEA2, had the highest synthesized melanin
E. multiflora extract or lupenone and incubated for an additional (127 %) compared with the control (100 %) (l " Fig. 2 b). To deter-

24 or 48 h. The medium was then removed, and the cells washed mine if EMEA2 has cytotoxic effect on B16 cells, the Guava Via-
twice with PBS before the total protein was extracted using RIPA Count system for cell viability evaluation was performed, and
buffer (Sigma) according to the manufacturerʼs instructions. Pro- the results showed that EMEA2 does not have any cytotoxic effect
tein sample (15 µg) was resolved in 10% SDS-polyacrylamide gel on B16 cells even after 72 h of treatment (l " Fig. 2 b).

by polyacrylamide gel electrophoresis, transferred to the nitro- Tyrosinase is the major and rate-limiting enzyme of the melano-
cellulose membrane, and blotted with primary antibodies for ty- genesis pathway. The effect of EMEA2 on the expression of the ty-
rosinase, p-ERK1/2, and ERK1/2 (Santa Cruz Biotechnology, Inc.). rosinase enzyme was determined by Western blot analysis, and
The signal was then visualized using Immobilon Western Chem- the results showed that treatment with EMEA2 stimulated the
iluminescent HRP Substrate (Millipore), after reaction with HRP- tyrosinase enzyme expression in B16 cells. This observed in-
labeled anti-rabbit IgG antibody (Santa Cruz Biotechnology, Inc.) crease in the tyrosinase expression, in terms of band density,
and anti-mouse IgG1 (Santa Cruz Biotechnology, Inc.) or with was more than 15 % compared to either the control or α-MSH-
goat anti-mouse IRDye 680LT (LI‑COR), donkey anti-goat IRDye treated cells (l
" Fig. 3 a). To elucidate the mechanism underlying

800CW (LI‑COR), or goat anti-rabbit IRDye 800CW (LI‑COR) melanogenesis stimulation effect of EMEA2, the phosphorylation
of MAPK ERK1/2 proteins was determined. The Western blot re-
Real-time PCR sults revealed that the expressions of phosphorylated ERK 1/2 in
Total RNA (1 µg), extracted using an Isogen kit (Nippon Gene), B16 cells was significantly decreased by EMEA2 treatment com-
was used for reverse transcription polymerase chain reaction pared to α‑MSH treatment and the untreated control, by fivefold
(RT‑PCR) which was carried out with the Superscript III reverse and tenfold, respectively (l " Fig. 3 b). EMEA2 did not have any ef-

transcriptase kit (Invitrogen) following the manufacturerʼs in- fect on the expression of non-phosphorylated ERK1/2 (l " Fig. 3 c).

Villareal MO et al. Lupenone from Erica multiflora … Planta Med 2013; 79: 236–243
 Original Papers 239

Fig. 2 Effect of Erica multiflora extract on the mel-

anin biosynthesis in B16 murine melanoma cells
treated with a ethanol extract (0.0005, 0.005,
0.05 w/v) or b ethyl acetate extract (1 µg/mL). The
melanin content is represented by the bar graph,
while the cell viability is presented as a line graph.
B16 melanoma cells were cultured in a 100-mm
dish at a density of 5 × 105 cells/dish. ** Statistically
significant (p < 0.01) difference between treated
cells and control.

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The active compound present in the EMEA2 sample that caused enzyme activity inhibitor, lupenone cannot increase or has no ef-
the increase in melanin content of B16 cells was determined by fect on the tyrosinase enzyme activity (l " Fig. 5 a). The effect of

subjecting the EMEA2 sample to purification and structural anal- lupenone on the tyrosinase enzyme was also evaluated by ex-
ysis, as reported elsewhere [26, 27]. Lupenone was identified as tracting the total protein, and the expression of the tyrosinase
the compound that was abundant in EMEA2 and about 38 mg/ enzyme determined by Western blotting. Results showed that lu-
100 g of which can be purified from E. multiflora leaves. To verify penone has a dose-dependent stimulatory effect on tyrosinase
if lupenone was the major active compound in EMEA2 that stim- expression (l " Fig. 5 b). α-MSH (200 µmol/L) was used as a posi-

ulated melanogenesis, B16 cells were treated without (Con) or tive control and showed a very significant increase in the tyrosi-
with lupenone (Lup) at 0.1, 0.5, and 1.0 µmol/L and compared to nase expression as expected. To determine what caused the in-
those treated with EMEA2 at 0.01, 0.05, and 0.15 % w/v or 100 nm crease in the expression of the tyrosinase enzyme, the expression
α-MSH and further incubated for 72 h prior to melanin/total pro- of activated or phosphorylated ERK1/2 was also determined
tein assays. Results showed that lupenone can stimulate melano- (l" Fig. 5 c). To verify if the increase in melanin was due to the in-

genesis in B16 cells in a dose-dependent manner, parallel to the crease in the tyrosinase mRNA expression, real-time PCR was
effect of EMEA2 treatment. Lupenone treatment at 0.1 µmol/L performed, and results showed an upregulated tyrosinase mRNA
was comparable to treatment with 100 nmol/L α-MSH, a com- expression (l " Fig. 6).

pound known to increase the melanin content of B16 cells

(l" Fig. 4) without affecting the cell viability (l
" Fig. 4 b).

To find out if lupenone can inhibit the activity of tyrosinase en-

zyme as mushroom tyrosinase, a tyrosinase assay was per-
formed. Results showed that unlike arbutin, a known tyrosinase

Villareal MO et al. Lupenone from Erica multiflora … Planta Med 2013; 79: 236–243
240 Original Papers

Fig. 3 Effect of EMEA2 on the expressions of tyrosinase (a), phosphoryl-

ated extracellular-signal-regulated kinases 1/2 or pERK1/2 (b), ERK1, and
ERK2 (c) proteins. B16 melanoma cells were cultured in a 100-mm dish at a
density of 3 × 106 cells/dish and treated without (Con) or with 100 nmol/L

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α-MSH or 0.1 % v/v of EMEA2. Total proteins were then extracted and re-
solved by SDS-PAGE, and the resolved proteins were then blotted onto a
nitrocellulose membrane. The tyrosinase and phosphorylated ERK1/2 ex-
tracted from the B16 cells were detected by immunoblotting with anti-ty-
rosinase or anti-activated ERK1/2 monoclonal antibody (upper panel) and
anti-ERK1/2 monoclonal antibody (lower panel). The signal was visualized
using the Immobilon Western Chemiluminescent HRP Substrate.

Discussion
!
Erica multiflora is a valuable herbal medicine in North Africa. The Fig. 4 Effect of EMEA2 and lupenone (Lup) on the melanin content (a)
aqueous flower extract of E. multiflora has been observed to ex- and cell viability (b) of B16 murine melanoma cells. B16 melanoma cells
hibit acute diuretic [17] and hypolipidemic [18] activities, but were cultured in a 100-mm dish at a density of 5 × 105 cells/dish and treat-
ed without (Con) or with the sample – 100 nmol/L α-MSH, EMEA2, and lu-
there has been no report on the effect of the leaf extract of E. mul-
penone, and incubated for 72 h after incubation. The dot-blot method for
tiflora on melanogenesis in B16 murine melanoma cells. Current
melanin assay was performed to quantify the melanin content per total
research on the regulation of melanogenesis has focused on the protein, while cell viability was assessed using the ViaCount assay (Guava
use of compounds from natural resources [32–35]. In this study, Technologies). ** Statistically significant (p < 0.01) difference between
treatment with E. multiflora ethanol extract increased the mela- treated cells and control.
nin content of B16 murine melanoma cells in a dose-dependent
manner (l " Fig. 1). Studies on melanogenesis are useful in clarify-

ing the mechanism of action of existing drugs or various com-

pounds on the pigmentation system. When the skin is exposed zyme activity, which shows that it had a melanogenesis stimula-
to UV, ROS are generated in the skin [36] and melanin, a special tory effect by increasing the tyrosinase enzyme expression.
class of compounds that play a crucial role in the absorption of Tyrosinase gene expression is regulated by the Mitf, the master
free radicals in the cytoplasm, counters this effect due to its ra- regulator of melanogenesis [44] and whose phosphorylation is
dioprotective effect and ability to completely remove low-dose linked to the activation of ERK [45, 46]. In mammalian systems
radiation effects [37, 38]. There has been no evidence that in- there are five distinguishable MAPK modules that are important
duced pigmentation can provide protection against UVR-induced in the regulation of cellular processes which include gene expres-
cancer, but results of recent studies show that it provides protec- sion, cell proliferation, cell survival and death, and cell motility
tion against the formation of UVR-induced DNA photoproducts [47]. One of these is the ERK1/2 cascade, which preferentially
[39, 40]. regulates cell growth and differentiation [48] and melanogenesis
Here, we have shown that treatment with E. multiflora ethyl ace- [49, 50]. ERK 1/2 has been reported to be involved in UVA-in-
tate extract caused an increase in the melanin content of B16 cells duced melanogenesis in normal human epidermal melanocytes
(l" Fig. 2 a and b). Succeeding experiments focused on the deter- [51] and in B16 murine melanoma cells [34]. An increase in phos-
mination of the major active compound present in the extract, phorylated ERK1/2 has been reported to inhibit melanogenesis as
lupenone, and its effect on melanogenesis (l " Fig. 5). Melanin bio- activated ERK1/2 induces Mitf phosphorylation, and phosphory-
synthesis or melanogenesis is catalyzed by the enzyme tyrosi- lated Mitf shows an increased transcriptional activity and at the
nase, the key enzyme of the process. Tyrosinase catalyzes the hy- same time an enhanced proteosome-dependent degradation
droxylation of L-tyrosine to L-dihydroxyphenylalanine and the [50–52]. EMEA treatment caused an increase in the expression
subsequent oxidation of this o-diphenol to L-dopaquinone [41, of the tyrosinase enzyme (l " Fig. 3 a) and a decrease in ERK1/2

42], the two rate-limiting steps of melanogenesis [43]. In this phosphorylation (l " Fig. 3 b) as determined by Western blot. In

study, we have shown that lupenone, the active compound in E. the skin, α-MSH stimulates the production and release of mela-
multiflora, has a significant dose-dependent stimulatory effect nin by melanocytes [53]. α-MSH, known to stimulate melanogen-
on tyrosinase expression and has no effect on the tyrosinase en- esis in murine and human melanocytes by increasing the tyrosi-

Villareal MO et al. Lupenone from Erica multiflora … Planta Med 2013; 79: 236–243
 Original Papers 241

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Fig. 6 Effect of lupenone on the expression of TYR gene. B16 cells were
seeded at a density of 3 × 106 cells per 100-mm dish. After overnight incu-
bation, the cells were treated with or without 0.2 µmol/L α-MSH, 0.1, 0.5,
and 1.0 µmol/L lupenone for 4 h after which RNA isolation and then reverse
transcription PCR were carried out to obtain cDNAs that were used for real-
time PCR. Results represent the mean ± SD of two independent experi-
ments. * Statistically significant (p < 0.05) difference between control and
treated cells.

the E. multiflora extract, can decrease ERK1/2 phosphorylation

(l" Fig. 5 c) that led to the upregulation of the expression of the

tyrosinase mRNA (l " Fig. 6) which subsequently caused an in-

crease in the tyrosinase enzyme levels (l " Fig. 5 b). The gene for

tyrosinase is a downstream gene of MITF [59] as evidenced by

Fig. 5 Effect of lupenone on tyrosinase activity (a), tyrosinase protein ex- the fact that promoters of the tyrosinase are transactivated by
pression (b), and pERK1/2 and ERK1 proteins expressions (c). Tyrosinase en- Mitf [60]. The activation or phosphorylation of the Mitf protein
zyme activity was evaluated by a cell-free assay using mushroom tyrosinase causes its degradation leading to the inhibition of the tyrosinase
and arbutin as a standard. For the tyrosinase enzyme and pERK1/2 expres-
gene transcription [61]. In this study, effects of E. multiflora ex-
sions, B16 melanoma cells were cultured in a 100-mm dish at a density of
tract on melanogenesis in B16 cells was determined using differ-
5 × 105 cells/dish and treated without (Con) or with 100 nm α-MSH or 0.1,
0.5, or 1.0 µmol/L lupenone. Total proteins were then extracted and re- ent solvents; the results showed that the ethyl acetate extract
solved by SDS-PAGE, and the resolved proteins were then blotted onto a had a stimulatory effect, and the major active compound was
nitrocellulose membrane. The phosphorylated ERK1/2 and non-phos- identified to be lupenone. Both the ethyl acetate extract and lu-
phorylated ERK1 extracted from the B16 cells were detected by immuno- penone increased the melanin content of the cells without any
blotting with goat anti-mouse IRDye 680LT, donkey anti-goat IRDye cytotoxic effects. Lupenone inhibits the ERK signaling (l " Fig. 5 c)

800CW, or goat anti-rabbit IRDye 800CW.

to effectively stimulate Tyr gene expression (l " Fig. 6) and there-

fore, increase the melanin content of B16 murine melanoma cells.

This report presents a potential molecular target-based thera-
peutic strategy for addressing hypopigmentation. It is important
nase activity [54], was used as a positive control for melanogene- to point out, though, that other compounds that may also be
sis stimulation. present in the E. multiflora ethyl acetate extract could have simi-
This study reports for the first time the use of lupenone lar effect as lupenone on B16 cells and cannot be excluded as con-
(l" Fig. 4 a) from Tunisian E. multiflora as a melanogenesis modu- tributing in its overall melanogenesis stimulatory effect. How-
lator that is not cytotoxic or does not affect the cell viability of ever, the use of lupenone alone can induce the same effect. E.
B16 cells (l " Fig. 4 b). Lupenone is a natural triterpene and classi- multiflora may therefore be explored as a possible repigmenta-
fied as a lupane terpenoid. Recently, natural triterpenoids have tion therapy for hypopigmentation problems such as PMH. The
received an increased interest in the field of pharmacology, and present PMH treatment utilizes ultraviolet therapy either alone
this is because, at present, more than 100 cytostatic agents are or in combination with topical antibacterial agents, but recur-
natural triterpenes [55, 56]. Lupenone can be isolated from many rence is possible after several weeks of treatment discontinua-
plants [26–29], and this report shows that it can also be found in tion, requiring additional UV sessions [62], hence, increasing the
a Mediterranean plant E. multiflora. Reports on the effects of lu- risk for skin cancer. The data obtained in this study provide scien-
penone include protein tyrosine phosphatase 1B inhibition [57], tific evidence on E. multiflora’s effects on melanogenesis stimula-
among several bioactivities. Although lupenone has been re- tion and therefore as a repigmentation agent in hypopigmented
ported to inhibit B162F2 cell proliferation by inducing apoptosis skin and establish E. multiflora extractʼs ethnopharmacological
[58], we did not observe any cytotoxicity in B16 cells treated with importance for the North African region.
1 µM lupenone (l " Fig. 4 b) and even up to 2 µM concentration

(data not shown). Here we have also shown that lupenone, like

Villareal MO et al. Lupenone from Erica multiflora … Planta Med 2013; 79: 236–243
242 Original Papers

Acknowledgments 19 Gratani L, Varone L. Leaf key traits of Erica arborea L., Erica multiflora L.
! and Rosmarinus officinalis L. co-occurring in the Mediterranean ma-
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This research was partially supported by the JST-JICAʼs Science 20 Toro Sainz MV, Garcia Gimenez MD, Pascual Martinez M. Isolation and
and Technology Research Partnership for Sustainable Develop- identification of phenol acids from Erica andevalensis Cabezudo-Ri-
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Conflict of Interest 23 Bennini B, Chulia AJ, Kaouadji M, Delage C. Phytochemistry of the Erica-
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!
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