沙漠研究 19-1, 387- 390 (2009 )Journal of Arid Land Studies“Desert Technology IX ”
Refereed Paper

Effect of Mediterranean Medicinal Plant Extracts on Melanogenesis Regulation
Kyoko MATSUYAMA1, Yusaku MIYAMAE2, Yukiko SEKII2, Junkyu HAN1, Manef ABDERRABBA3,Takahiro MORIO1, Hideyuki SHIGEMORI2, Hiroko ISODA1
Abstract: We investigated the melanogenesis regulation effect of Mediterranean medicinal plant
extracts. The melanin synthesis system is known as melanogenesis, and tyrosinase is its key
enzyme. Murine melanoma B16 cells were treated with extracts of medicinal plants’, namely,Capparis spinosa, Erica multiflora and Thymelaea hirsuta, and then cell viability and synthesizedmelanin content were measured. Furthermore, to determine the mechanism of melanogenesis
regulation, we performed western blot analysis and DNA microarray. Results show that C.
spinosa and E. multiflora extracts increased melanin content in B16 cells without cytotoxity. We
also observed that the tyrosinase protein expression level was clearly increased in cells treatedwith the extracts. On the other hand, T. hirsuta extract significantly decreased the synthesizedmelanin content in B16 cells without cytotoxity. We also detected ERK1/2 phosphorylation anddown-regulation of tyrosinase protein expression level. In addition, we identified thatdaphnanes-genkwadaphnin, gnidicin and gnidilatidin from T. hirsuta extract, have theanti-melanogenesis activity. In this research, we report that Mediterranean medicinal plant
extracts have high activities in relation to the stimulation or inhibition of melanogenesis.
Keywords: B16 melanoma, Mediterranean medicinal plant, Melanogenesis

1. Introduction
Pigmentation of the epidermis is caused by melanin synthesis, and melanin plays a major role in theprevention of DNA damage and protection of skin tissues from UV irradiation. The melanin synthesis
system is known as melanogenesis, and it is regulated by melanogenic enzymes, such as tyrosinase,tyrosinase-related protein 1 (TYRP-1) and TYRP-2. Among the three enzymes, tyrosinase is the mostimportant enzyme in melanogenesis. Tyrosinase catalyzes the first two steps in melanin synthesis: thehydroxylation of tyrosine to 3-(3, 4-dihydroxyphenyl)-alanine (DOPA) and the oxidation of DOPA todopaquinone (Kobayashi et al., 1998). Thus, melanin production is mainly dependent on tyrosinase
expression and activation.
Extracellular signal-regulated kinase (ERK) is a key signaling mediator of cell proliferation anddifferentiation. Recently, it has been reported that ERK signaling pathway is related to melanogenesis.
ERK1/2 phosphorylation is related to the inhibition of melanogenesis (Kim et al., 2006).
Medicinal plants are valuable natural products used as raw materials in many fields, including
perfumes, cosmetics, aromatherapy, phototherapy, spices and nutrition. Recently, many papers on thebiological and pharmacological activity of medicinal plants have been published (Cho et al., 2002, Wang etal., 2006). In addition, antimelanogenesis effect of Tunisian herb Thymelaea hirsuta extract was initiallyreported by Kawano et al. (2007).
In this study, we investigated the effects of Medditerranean medicinal plant extracts on melanogenesis
regulation in the murine melanoma B16 cells.
2. Materials and Methods
2.1 Extraction of Mediterranean medicinal plantsAerial leaves of Mediterranean medicinal plants (10 g) were extracted with 70% EtOH (100 ml) for
1-2 weeks at room temperature. After extraction, the extract was filter-sterilized using a 0.45 ㎛ pore size
filter (Millipore, USA) and then stored at -80 ˚C until use.

1Alliance for research on North Africa (ARENA), University of Tsukuba, Ibaraki, JapanGraduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan
3
Unité de Recherche: Physico-chimie et Moléculaire: Institut Préparatoire aux Etudes Scientifiques et Techniques
IPEST, La Marsa, Tunis, TUNISIA
2388

2.2 Measurement of melanin content and cell viability
B16 melanoma cells were seeded onto 100-mm dishes at a density of 5 × 105 cells per dish andcultivated in Dulbecco’s modified Eagle’s medium (Nissui, Japan) supplemented with 10% fetal bovine
serum (Sigma, USA), 4 mM L-gluramine (Sigma, USA), 50 units/ml penicillin and 50 ㎍/ml streptomycin(Cambrex, USA) at 37 ˚C in a humidified atmosphere of 5% CO2. After overnight incubation, the medium
was replaced with sample-containing medium and incubated for 48 hours. The medium was then removed,
and then the cells were washed twice with phosphate-buffered saline and then harvested by trypsinization.
The harvested cells were resuspended in growth medium and stained with the ViaCount reagent (Millipore,USA) according to the manufacturer’s instructions. Cell viability and cell number was determined with
Guava PCA (Millipore, USA) using the ViaCount program for analysis. After determination of cell viability,
the cell membrane was dissolved using 0.1% Triton X-100. The synthesized melanin was then purified andprecipitated in 10% trichloroacetate. The purified melanin was dissolved by incubation in 8 N NaOH for 2 hat 80 ˚C. The absorbance of the solution was measured at 410 nm.
2.3 Western blotting
B16 melanoma cells were seeded by the method described above. After overnight incubation, themedium was replaced with sample-containing medium followed by incubation for 0, 0.25, 0.5, 1, 2, 4, 8,12, 24, 48 and 72 hours. After incubation, the total protein was extracted using RIPA buffer (SIGMA,USA) according to the manufacturer’s instructions. Fifteen micrograms of extracted protein sample wereresolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and blottedwith primary antibodies for tyrosinase (Santa Cruz Biotechnology, USA), ERK1/2 (SIGMA, USA) andphospho-ERK1/2 (SIGMA, USA).
The signal was visualized using Immobilon Western
Chemiluminescent HRP Substrate (Millipore, USA), after reaction with HRP-labeled anti-rabbit IgG
antibody (Santa Cruz Biotechnology, Inc., USA).
2.4 RNA isolation and DNA microarray
B16 melanoma cells were seeded by the method described above. After overnight incubation, themedium was replaced with sample-containing medium followed by incubation for 1hour. Afterincubation, the total RNA was isolated using ISOGEN (Nippon Gene, Japan). The total RNA
concentration and purity were determined by measuring the optical density, OD260 and OD260/280 using a
UV spectrophotometer.
DNA microarray experiments were performed using GeneChip® Mouse Genome 430 2.0 arrays(Affymetrix, Inc., USA). Total RNA samples were processed following a standard one-cycle eukaryotic
target preparation protocol from Affymetrix. Biotinylated cRNA targets were hybridized to GeneChip®
array in a rotating hybridization oven. After hybridization, the arrays were stained with streptavidin-phycoerythrin in the GeneChip® Fluidics station and then scanned using Affymetrix GeneChip® Scanner.
The array image data were acquired, and the fluorescent signal intensities were quantified using
Affymetrix® GCOS v. 1.4 software.

3. Results and Discussion
3.1 Effect of Mediterranean medicinal plant extracts on melanogenesis and cell viability
The effect of C. spinosa (Fig. 1A), E. multiflora (Fig. 1B) and T. hirsuta (Fig. 1C) extract on themelanogenesis of B16 mouse melanoma cells was examined. We observed that B16 mouse melanoma
cells treated with C. spinosa and E. multiflora extract showed an increase in cytoplasmic accumulation ofmelanin in a dose-dependent manner without cytotoxicity. To examine the mechanism underlying thepromotion effect of C. spinosa and E. multiflora extract on melanin synthesis, we determined the tyrosinase
protein expression in B16 cells by western blot analysis. The tyrosinase expression levels of cells treatedwith the extract were highly upregulated in time-dependent manner compared with the control cells.
These results suggest that the melanogenesis promotion effect of C. spinosa and E. multiflora extract on
increased melanin synthesis is due to the up-regulation of tyrosinase, the most important enzyme in
melanogenesis.

389

On the other hand, we observed that B16 mouse melanoma cells treated with T. hirsuta extract
showed a decrease in cytoplasmic accumulation of melanin in a dose-dependent manner without
cytotoxicity. To examine the mechanism underlying the suppressive effect of T. hirsuta extract onmelanin synthesis, we determined the tyrosinase protein expression and ERK1/2 phosphorylation in B16cells by western blot analysis. The tyrosinase expression levels of cells treated with the extract weredown-regulated compared with the control cells. Although the expression levels of ERK1/2 in cultured
B16 cells were not affected by treatment with the extract, the phosphorylation of ERK1/2 was considerablyenhanced after 0.5 hour to 5 hour. These results suggest that the suppressive effect of T. hirsuta extract onmelanin synthesis is due to the down-regulation of tyrosinase and via ERK1/2 phosphorylation.
(A)(B)(C)

Fig. 1. Mediterranean medicinal plants. (A) C. spinosa (B) E. multiflora (C) T. hirsuta.

3.2 Daphnanes having anti-melanogenesis activity in the T. hirsuta extract
To identify the major compound responsible for the activity, leaves of T. hirsuta was extracted in
MeOH and the extract was partitioned with EtOAc, BuOH and water. We examined the effect of eachlayer on the melanogenesis of B16 cells and found that the EtOAc layer showed anti-melanogenesisactivity. The EtOAc layer was fractionated into 17 fractions (Th-EtOAc-1-17) and Th-EtOAc-11
possessed the highest antimelanogenesis activity. Th-EtOAc-11 was further fractionated into six fractions(Th-EtOAc-11-1-6) and Th-EtOAc-11-3 strongly inhibited melanogenesis. We identified that daphnanes,
genkwadaphnin (Fig. 2A), gnidicin (Fig. 2B) and gnidilatidin (Fig. 2C) included in Th-EtOAc-11-3 andthese compounds decreased in cytoplasmic accumulation of melanin without cytotoxicity (Fig. 3).
Moreover, we determined that ginidilatidin, has the strongest effect of anti-melanogenesis activity.
(A)

(B)

(C)

Fig. 2. Structure of major compounds from T. hirsuta extract. (A) genkwadaphnin (B) gnidicin (C) gnidilatidin.

3.3 Molecular mechanism of anti-melanogenesis activity by gnidilatidin
To examine the mechanism underlying the suppressive effect of gnidilatidin on melanin synthesis, wedetermined the tyrosinase protein expression and ERK1/2 phosphorylation in B16 cells by western blot
analysis. The tyrosinase expression levels of cells treated with gnidilatidin were significantly
down-regulated in time-dependent manner compared with the control cells. The phosphorylation of
ERK1/2 was considerably enhanced after 0.25 hours treatment with gnidilatidin and then graduallyattenuated in time dependent manner. These results suggest that the anti-melanogenesis effect ofgnidilatidin in B16 cells is due to the down-regulation of tyrosinase and via ERK1/2 phosphorylation.

390

Fig. 3. Effect of daphnanes from T. hirsuta extract on melanogenesis and cell viability in B16 murine melanoma
cells. Results represent means ± SD of triplicate samples. **Statistically significant (P ＜ 0.001) difference
between daphnanes treated cells and control.

Furthermore, to evaluate more details of the mechanism underlying the effects of gnidilatidin, weperformed DNA microarray analysis and determined comprehensive gene expression level. Of the 34,000
mouse genes examined on the DNA microarray, 606 genes exhibited at least a two-fold change inexpression. There was a two-fold increase in gene expression of 394 genes; on the other hand, there wasa two-fold decrease in 212 gene’s expression. These genes were categorized according to their activity inapoptosis regulation, cell cycle, cell adhesion, cell proliferation and signal transduction.
4. Conclusions
In this research, we found that the extracts of Mediterranean medicinal plants have high activities inrelation to the stimulation or inhibition of melanogenesis. C. spinosa and E. multiflora extract have themelanogenesis stimulation activity. However, we did not identify the major compounds in these plants’extracts. To confirm the melanogenesis stimulation activity by these plant extracts, more analysis shouldbe performed and details of the mechanism should be clarified.
On the other hand, T. hirsuta extract have the melanogenesis inhibition activity and we determinedthat daphnanes contained in T. hirsuta extract has strong effect of antimelanogenesis activity. In addition,
we found that gnidilatidin, one of dapnanes from T. hirsuta extract, has the strongest activity ofanti-melanogenesis. Moreover, we clarified that gnidilatidin induced gene variation in relation to celldifferentiation in B16 cells briefly and then induced phosphorylation of ERK1/2 and downregulation oftyrosinase in protein expression level, followed by melanin synthesis inhibition.
In conclusion, our results suggest that the extracts of Mediterranean medicinal plants have the ability
to maintain skin homeostasis, and can be used as a tanning or whitening agent.
Acknowledgement
This study was supported in part by funds from the Grant-in Aid for Scientific Research (Grant No.
17255011). The photos of flowers were kindly given by Dr. Kawada (ARENA, Japan).
References
Cho S.M., Kwon Y.M., Lee J.H., Yon K.H., Lee M.W. (2002): Melanogenesis inhibitory activities of diarylheptanoids from Alnus
hirsuta Turcz in B16 mouse melanoma cell. Arch. Pharm. Res., 25: 885-888.
Kawano M., Matsuyama K., Miyamae Y., Shinmoto H., Kchouk M.E., Morio H., Shigemori H., Isoda H. (2007): Antimelanogenesis
effect of Tunisian herb Thymelaea hirsuta extract on B16 murine melanoma cells. Exp. Dermatol., 16: 977-984.
Kim D.S., Park S.H., Kwon S.B., Park E.S., Huh C.H., Youn S.W., Park K.C. (2006): Sphingosylphosphorylcholine-induced ERK
activation inhibits melanin synthesis in human melanocytes. Pigment Cell Res., 19: 146-153.
Kobayashi T., Imokawa G., Bennett D.C., Hearing V.J. (1998): Tyrosinase stabilization by Tyrp1 (the brown locus protein). J. Biol.
Chem., 273: 31801-31805
Wang K.H., Lin R.D., Hsu F.L., Huang Y.H., Chang H.C., Huang C.Y., Lee M.H. (2006): Cosmetic applications of selected traditional
Chinese herbal medicines. J. Ethnopharmacol., 106: 353-359.