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Inhibitory Effect of Artocarpanone from Artocarpus heterophyllus on Melanin

Biosynthesis

Article  in  Biological & Pharmaceutical Bulletin · October 2006

DOI: 10.1248/bpb.29.1966 · Source: PubMed

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1966 Notes Biol. Pharm. Bull. 29(9) 1966—1969 (2006) Vol. 29, No. 9

Inhibitory Effect of Artocarpanone from Artocarpus heterophyllus on

Melanin Biosynthesis
Enos Tangke ARUNG, Kuniyoshi SHIMIZU, and Ryuichiro KONDO*
Department of Forest and Forest Products Sciences, Faculty of Agriculture, Kyushu University; 6–10–1 Hakozaki, Higashi-
ku, Fukuoka 812–8581, Japan. Received February 13, 2006; accepted May 29, 2006

In our previous efforts to find new tyrosinase inhibitory materials, we investigated 44 Indonesian medicinal
plants belonging to 24 families. Among those plants, the extract of Artocarpus heterophyllus was one of the
strongest inhibitors of tyrosinase activity. By activity-guided fractionation of A. heterophyllus wood extract, we
isolated artocarpanone, which inhibited both mushroom tyrosinase activity and melanin production in B16
melanoma cells. This compound is a strong candidate as a remedy for hyperpigmentation in human skin.
Key words tyrosinase; melanin; antioxidant; artocarpanone; hyperpigmentation; Artocarpus heterophyllus

Tyrosinase (EC 1.14.18.1; PPO) is known to be a key en-
zyme involved in melanin biosynthesis in plants, microor-
ganisms and mammalian cells. This enzyme catalyses two
different reactions: the hydroxylation of monophenols to o-
diphenols (monophenolase activity), and the oxidation of o-
diphenols to o-quinones (diphenolase activity), which, in
turn, are polymerized to brown, red or black pigments.1)
Many tyrosinase inhibitors have been tested in cosmetics and
pharmaceuticals as a way of preventing overproduction of
melanin in epidermal layers.2) Alterations in melanogenesis
may be responsible for some of the clinical and histopatho-
logical features unique to malignant melanoma,3) a cancer
with a rapidly increasing incidence.4)
These observations led us to focus on the exploration of
tyrosinase inhibitors from natural products. Several chemi-
cals of plant origin have been reported as tyrosinase in-
hibitors. Arbutin,5) ellagic acid,6) oxyresveratrol,7) chloro-
phorin and norartocarpanone8) have all been studied for
their tyrosinase inhibition properties.
Tropical forests constitute 40—50% of the entire forested
area of the world. Indonesia has an extraordinarily rich flora
and great diversity of vegetation types that parallel the di-
verse physiography of the land, which is largely covered by
evergreen rain forest. The great diversity of tropical plants is
reflected in the qualitative and quantitative diversity of plant
extracts, or, from a chemical point of view, of their compo-
nents, which have been used increasingly as medicines in re-
cent years. In Indonesia, several plants are used in a tradi-
tional medicine called “Jamu”. The uses of Jamu can be dis-
tinguished into four categories of medicine: health care,
beauty aids (cosmetics), tonics, and protection (a prophylac-
tic against various kinds of disease).9) Such traditional me-
dicinal knowledge as that in Indonesia offers researchers an
excellent opportunity to find new bioactive components for
medicines.
In this paper, we evaluate the effects of artocarpanone iso-
lated from extracts of A. heterophyllus on melanin biosynthe-
sis (mushroom tyrosinase activity and melanin production in
B16 melanoma cells) and its antioxidant activity.
MATERIALS AND METHODS
Plant Material The wood (sapwood and heartwood) of
Artocarpus heterophyllus was collected at Samarinda city,
∗ To whom correspondence should be addressed. e-mail: kondo@agr.kyushu-u.ac.jp
Indonesia in August 2003. The plant was indentified by
the Laboratory of Dendrology and a voucher specimen
(FHT.LA.13.1H) was deposited at the Laboratory of Wood
Anatomy, Forestry Faculty, Mulawarman University, Indo-
nesia.
Extraction and Bioactivity-Guided Fractionation
Based on our screening, the sapwood extract was more po-
tent than the heartwood extract at inhibiting tyrosinase activ-
ity (IC50
7 and 125 m g/ml respectively). Thus, we focused
on sapwood for further investigation. The wood meal of sap-
wood of A. heterophyllus (2.3 kg) was extracted repeatedly
with methanol at room temperature. The methanol extract
was concentrated in vacuo to yield a residue of 60.6 g (2.6%
of w/w). A portion of the extract (43.1 g) was suspended in
methanol–water (1 : 2) and partitioned with n-hexane, diethyl
ether and ethyl acetate. The tyrosinase inhibitory activity of
the n-hexane, diethyl ether, ethyl acetate, and aqueous solu-
ble fractions were 52, 94, 72, and 49% at 100 m g/ml concen-
tration, respectively. The diethyl ether soluble fraction
(16.3 g, 37.8% of w/w) was applied to a silica gel column
(Wakogel C-200, 1190 cm) and eluted with n-hexane/ethyl
acetate (8 : 2, 7 : 3, 6 : 4, 5 : 5, 4 : 6, 3 : 7, 2 : 8, 1 : 9, 0 : 10 and
methanol, each 800 ml) to give sixteen fractions (Fr 1 to Fr
16). Fraction 7 (2.10 g, 12.9% of w/w) which inhibited ty-
rosinase activity by 97% was repeatedly chromatographed
over silica gel (Wakogel C-200, 650 cm) and eluted with n-
hexane/ethyl acetate (8 : 2, 7 : 3, 6 : 4, 5 : 5, 4 : 6, 3 : 7, 2 : 8, 1 :
9, 0 : 10, and 5 : 5 of ethyl acetate/methanol, each 600 ml and
methanol 1200 ml) to give nine fractions (Fr 7-1 to Fr 7-9).
Thus, Fr 7-3 (400 mg, 19.0% of w/w), which inhibited tyrosi-
nase activity by 99%, was applied to preparative HPLC, elut-
ing with a flow rate of 10 ml/min of aqueous/methanol (0.1%
trifluoroacetic acid) to give compound 1 which was identified
as artocarpanone (13.5 mg, 3.4% of w/w) by comparing with
published NMR and MS data.10,11) The [a ]D24 of this com-
pound was 18° (MeOH, c
0.09) measured using a DIP
370 Digital Polarimeter (JASCO, Japan).
Tyrosinase Enzyme Assay The sample was first dis-
solved in DMSO and used for the actual experiment at 30
times dilution. The assay was performed as previous de-
scribed by Shimizu et al.8) with minor modification.12) First,
333 m l of 2.5 mM L-DOPA solution was mixed with 600 m l of
0.1 M phosphate buffer (Na2HPO4 · 12H2O–NaH2PO4 · 2H2O)
(pH 6.5), and incubated at 25 °C. Then, 33 m l of the sample
© 2006 Pharmaceutical Society of Japan
September 2006
solution and 33 m l of the aqueous solution of mushroom ty-
rosinase (1380 units/ml) were added to the mixture and the
initial rate of linear increase in optical density at 475 nm was
immediately measured on the basis of the formation of
dopachrome. The data was calculated with the following
equation:
tyrosinase activity (%)
(Atest sample /Acontrol)100
[Where, A
absorbance (average value)]
The extent of inhibition by the addition of samples is ex-
pressed as the concentration necessary for 50% inhibition
(IC50)
Melanin Biosynthesis Assay This assay was performed
as previously described by Dooley et al.13) and Virador et
al.14) with slight modifications.15)
Cell Culture A mouse melanoma cell line, B16, was ob-
tained from RIKEN Cell Bank. The cells were maintained in
EMEM supplemented with 10% (v/v) fetal bovine serum
(FBS) and 0.09 mg/ml theophylline. Cells were incubated at
37 °C in a humidified atmosphere of 5% CO2.
Inhibitory Effect of Melanin Biosynthesis Using Cul-
tured B16 Melanoma Cells Confluent cultures of B16
melanoma cells were rinsed in phosphate-buffered saline
(PBS) and lysed with 0.25% trypsin/EDTA. Cells were
placed into 24-well plastic culture plates at a density of
1105 cells/well and incubated for 24 h in media prior to
treatment with sample. After 24 h, the media was replaced
with 998 m l of fresh media and 2 m l of DMSO with or with-
out (control) the test sample at various concentrations was
added. The cells were incubated for an additional 48 h and
then the media was replaced with fresh media. After 24 h, the
remaining adherent cells were assayed (see below). Thus, the
cells were continuously exposed to the test samples for 3 d
duration.
Determination of Melanin Content in B16 Melanoma
Cells The melanin content of cells after treatment was de-
termined as follows. After removing the media and washing
the cells with PBS, the cell pellet was dissolved in 1.0 ml of
1N NaOH. The crude cell extracts were assayed using a
micro plate reader (Bio-Tek, U.S.A.) at 405 nm to determine
melanin content. Results from samples were analyzed as per-
cent of control culture. Arbutin (100 ppm, Tokyo Kasei
Kogyo Co.) was used as a positive standard. The data were
analyzed with the two-tailed Student’s t-test against control.
Cell Viability Cell viability was determined using a
micro culture tetrazolium technique (MTT). The MTT assay
provides a quantitative measure of the number of viable cells
by determining the amount of formazan crystals produced by
metabolic activity in treated versus control cells. Culture was
initiated in 24-well plates at 1105 cells per well. After incu-
bation, 50 m l of MTT reagent [3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide in PBS (5 mg/ml)] was
added to each well. The plates were incubated in a humidi-
fied atmosphere of 5% of CO2 at 37 °C for 4 h. After remov-
ing the media, formazan crystals were dissolved in 1.0 ml of
0.04 N HCl, and the absorbance was measured at 570 nm rel-
ative to 630 nm. The data were analyzed with the two-tailed
Student’s t-test against control.
Antioxidant Assay The sample was first dissolved in
DMSO and used for the actual experiment at 30 times dilu-
tion. The assay was performed as previously described by
1967
Shimizu et al.16) with minor modification. The reaction mix-
ture contained 967 m l of 60 m M DPPH (1,1-diphenyl-2-picryl-
hydrazyl) in ethanol and 33 m l of sample solution in DMSO.
After the reaction was carried out at room temperature for 20
min, the free radical scavenging activity of the sample was
quantified by the decolorization of DPPH at 514 nm.
RESULTS
Compound 1 (Fig. 1) was isolated and identified as arto-
carpanone by comparing its data with published NMR and
MS data. The [a ]D24 value of 18° was different from pub-
lished data.11) The CD spectrum was measured using a J-
720W Spectropolarimeter (JASCO, Japan) and the result in-
dicated that this compound was optically inactive. It was con-
cluded that this compound is a racemic mixture.
The melanin biosynthesis activity (mushroom tyrosinase
enzyme activity and melanin production in B16 melanoma
cells) of artocarpanone is shown in Table 1. Artocarpanone
inhibited both mushroom tyrosinase enzyme activity and
melanin production in B16 melanoma cells with IC50 values
of 80.8 and 89.1 m M, respectively. Arbutin as a positive con-
trol showed a lack of mushroom tyrosinase enzyme in-
hibitory activity (IC50
104000 m M) but strongly suppressed
melanin production (IC50
111 m M). On the other hand, kojic
acid inhibited mushroom tyrosinase activity potently
(IC50
15.5 m M) but weakly suppressed melanin production
(IC50
3521 m M).
The antioxidant activity of artocarpanone was studied
using DPPH (1,1-diphenyl-2-picrylhydrazyl). Artocarpanone
showed antioxidant activity at 135.8 m M (IC50), which was
weaker than that of the positive control, quercetin (10.3 m M
of IC50).
DISCUSSION
In Indonesia, some plants are used in a traditional medi-
cine called “Jamu”. The uses of Jamu can be distinguished
into four categories of medicine: health care, beauty aids
Fig. 1. The Structure of Artocarpanone
Table 1. Effects of Artocarpanone on Mushroom Tyrosinase, and Melanin
Biosynthesis and Cell Proliferation of B16 Melanoma Cells
Melanin Cell
Tyrosinasea)
inhibition viability
Compounds tested
IC50 (m M) IC50 (m M) (% vs. control)b)
Artocarpanone 80.8 89.1 90
Arbutin (positive control) 104000 111.0 90
Kojic acid (positive control) 15.5 >3521c) 90 at 3521 m M
a) substrate: L-DOPA. b) cell viability (%) at the concentration of IC50 for melanin
production in B16 melanoma cells. c) 40% inhibition of melanin production at 3521
m M.
1968
(cosmetics), tonics, and protection (disease prevention).
Based on our screening data on 44 medicinal plants from In-
donesia that are commonly used in “Jamu”, we found that
Artocarpus plants showed potent tyrosinase inhibitory activ-
ity which is useful in anti-browning and whitening agents
(cosmetic materials).11) Among the Artocarpus plants, A. het-
erophyllus was one of the most potent and this result led us
to focus on this plant. A. heterophyllus has been reported as
having some parts that are functional for medicines: the pulp
and seed for cooling, tonics, and pectorial; roots for treating
diarrhea and fever; wood for a sedative during convulsions;
leaves for activating milk production in women and animals,
antisyphilic treatment and vermifuge; and leaf ash for treat-
ing ulcers and wound.17) Some scientists have reported the
isolation of compounds with biological activities such as an-
tibacterial,18) antiplatelet,19) antioxidant,20) antidiabetic21) and
recently, antiinflammatory22) properties.
To date, research on regulation of melanogenesis has fo-
cused on factors that affect tyrosinase, the rate-limiting en-
zyme in the melanogenic pathway, including research on
chemicals that inhibit tyrosinase function. Considering the
importance of counteracting oxidative stress caused by ultra
violet (UV) radiation as a means of preventing skin damage,
it is also important to find a multifunctional skin-whitening
agent with antioxidant properties able to inhibit melanin
biosynthesis. Artocarpanone, which we isolated from A. het-
erophyllus, has several biologically activities, including an-
tiplatelet19) and antiinflammatory22) properties. It should be
noted that other compounds such as artocarpin, albanin A,
cudraflavone C and B, kuwanon C, norartocarpin, 6-preny-
lapigenin, and brosimone I have been isolated, but did not
show tyrosinase inhibitory activity. Therefore, in the present
study, we focused on artocarpanone to evaluate its melanin
biosynthesis inhibitory activity and its antioxidant properties.
To our knowledge, no such study has been previously re-
ported. In this work, artocarpanone showed more potent ty-
rosinase inhibitory activity than arbutin, but it was weaker
than kojic acid. In in vitro experiments using B16 melanoma
cells, artocarpanone was more potent at down-regulating
melanin production in the cells than the two positive con-
trols, arbutin and kojic acid. In this study, we used arbutin
and kojic acid as positive controls because they are used in
cosmetic products.2,14,23) At the IC50 of melanin inhibition
(Table 1), artocarpanone had a lower cytotoxic effect on cell
proliferation (about 10%). Based on these results, it appears
artocarpanone may be a candidate for practical use in the fu-
ture as long as its safety is guaranteed. However, the ability
of artocarpanone to reduce melanin biosynthesis in mush-
room tyrosinase assay may be related to the presence of a
4-substituted resorcinol moiety in its chemical structure.
As suggested by Shimizu et al.,24) Jimenez and Garcia-Car-
mona,25) and Chen et al.,26) a 4-substituted resorcinol moiety
inhibits tyrosinase activity. The mechanism of inhibition of
melanin production in B16 melanoma cells by artocarpanone
is still unclear, but although we believe that a 4-substituted
resorcinol moiety may be related to the inhibitory activity.
Therefore, further experiments are needed in order to deter-
mine the exact mechanism of this compound. Since arto-
carpanone has showed promising results as a whitening
agent, we conducted an antioxidant assay study in order to
determine its ability to counteract oxidative stress from UV
Vol. 29, No. 9
radiation. Artocarpanone showed potent antioxidant activity,
but it was weaker than the positive control, quercetin (135.8
and 10.3 m M of IC50, respectively). The potency of arto-
carpanone as an antioxidant might be related to the presence
of hydroxyl groups in the form of a resorcinol moiety. It was
reported by Ko et al.,20) and Cao et al.,27) that the number of
hydroxyl groups in flavonoids increases the antioxidant abil-
ity, however, the most important is the presence of a catechol
moiety. The presence of a resorcinol moiety also seems to
play a role in the antioxidant activity.
In conclusion, artocarpanone is a promising compound
that could be useful for treating hyperpigmentation, as a
skin-whitening agent, and as well as an antioxidant. How-
ever, it should be noted that safety is a primary consideration
for its practical use in humans. Still, our findings are in line
with the traditional uses of medicinal plants in Indonesia for
their beauty aid (cosmetic) functions.
Acknowledgements We are very grateful for the techni-
cal assistance of Dr. Koki Fujita (FAB-MS measurement); to
Mrs. Nani Husein and Dr. Rudianto Amirta for identifying
and registering the voucher specimen of A. heterophyllus;
and to Dr. Hiroshi Furuno, Dr. Masanori Inagaki, and Dr. To-
mofumi Miyamoto for their technical assistance with the
[a ]D24 and CD spectra measurements.
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