Callicarpa longissima extract, carnosolrich, potently inhibits melanogenesis in
B16F10 melanoma cells
Minori Yamahara, Koji Sugimura,Ayako Kumagai, Hiroyuki Fuchino,Azusa Kuroi, Mai Kagawa, Yumi Itoh,
Hidehisa Kawahara, et al.
Journal of Natural Medicines
ISSN 1340-3443
Volume 70
Number 1
J Nat Med (2016) 70:28-35
DOI 10.1007/s11418-015-0933-5

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Author's personal copy
J Nat Med (2016) 70:28–35
DOI 10.1007/s11418-015-0933-5

ORIGINAL PAPER

Callicarpa longissima extract, carnosol-rich, potently inhibitsmelanogenesis in B16F10 melanoma cells
Minori Yamahara1,2 • Koji Sugimura3 • Ayako Kumagai4 • Hiroyuki Fuchino5 •Azusa Kuroi2,4 • Mai Kagawa4 • Yumi Itoh4 • Hidehisa Kawahara6 •Yasuo Nagaoka6 • Osamu Iida3,5 • Nobuo Kawahara3,5 • Hiroshi Takemori4 •Hideto Watanabe1

Received: 16 February 2015 / Accepted: 2 August 2015 / Published online: 13 August 2015
Ó The Japanese Society of Pharmacognosy and Springer Japan 2015

Abstract Cosmetic industries focus on developing
materials and resources that regulate skin pigmentation.
Melanin, the major pigment in human skin, protects theskin against damage from ultraviolet light. An ethanolic
extract of the leaves of Callicarpa longissima inhibitsmelanin production in B16F10 mouse melanoma cells by
suppressing microphthalmia-associated transcription factor(MITF) gene expression. Following purification and analysis using liquid chromatography–mass spectrometry (LC–MS), NMR, and biochemical assays, carnosol was determined to be responsible for the major inhibitory effect of
the C. longissima extract on melanin production. Carnosol& Hiroshi Takemori
mukuharu22@yahoo.co.jp
1

Institute for Molecular Science of Medicine, Aichi Medical
University, 1-1, Yazakokarimata, Nagakute, Aichi 480-1195,
Japan

2

Department of Research and Development, Momotanijuntenkan Co. Ltd, 2-4-30, Ichioka, Minato-ku,
Osaka 552-0012, Japan

3

Tanegashima Division, Research Center for Medicinal Plant
Resources, Japan National Institute of Biomedical
Innovation, Health and Nutrition, 17007-2, Matsubarayama,
Noma, Nakatane-cho, Kumage-gun, Kagoshima 891-3604,
Japan

4

Cell Signaling and Metabolic Disease Laboratory,
Department of Fundamental Technological Research, Japan
National Institute of Biomedical Innovation, Health andNutrition, 7-6-8, Asagi-Saito, Ibaraki, Osaka 567-0085, Japan

5

Tsukuba Division, Research Center for Medicinal Plant
Resources, Japan National Institute of Biomedical
Innovation, Health and Nutrition, 1-2, Hachimandai,
Tsukuba, Ibaraki 305-0843, Japan

6

Department of Life Science and Biotechnology, Kansai
University, Suita, Osaka 564-8680, Japan123

is an oxidative product of carnosic acid, whose presence inthe extract was also confirmed by an authentic reference.
The carnosol and carnosic acid content in the extract wasapproximately 16 % (w/w). These results suggest that C.
longissima is a novel, useful, and attractive source of skinwhitening agents.
Keywords Melanogenesis  MITF  Carnosol  Carnosic
acid  B16F10Introduction
Aged human skin is characterized by hyperpigmentation, in
addition to wrinkles and decreased elasticity, leading tofrequent reliance on cosmetics [1]. Hyperpigmentation
results from an increase in the number or activity of melanocytes [2]. Melanin synthesis is regulated by tyrosinase(TYR) (EC 1.14.18.1), which catalyzes hydroxylation of Ltyrosine to 3,4-dihydroxyphenylalanine and its subsequentoxidation to dopaquinone. Dopaquinone is converted
through dopachrome into an indole derivative via autoxidation, and the conjugation of dopaquinone with this indole
derivative produces melanin [3].
TYR activity is regulated by a multi-step process
involving several molecules. Ultraviolet radiation stimulates keratinocytes to synthesize and secrete a-melanocytestimulating hormone [4], a paracrine hormone that binds tothe melanocortin 1 receptor on the melanocyte membrane,increases cAMP levels, and activates protein kinase A(PKA). Active PKA phosphorylates the transcription factor
cAMP response element-binding protein (CREB), up-regulating transcription of microphthalmia-associated transcription factor (MITF) [5, 6]. In turn, MITF binds to the
Tyr promoter region and up-regulates its expression.

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J Nat Med (2016) 70:28–35

Both natural and synthetic sources contain melanin
synthesis inhibitors. Only a few of these have been
approved under strict governmental regulations for use inskin-whitening products. These include linoleic acid [7],hinokitiol [8], kojic acid [9], hydroquinone [10], catechols[11], and salicylate [12]. In a previous study, we identified
the ethanolic extract of Callicarpa longissima [13], which
potently inhibits melanin synthesis in B16F10 mousemelanoma cells by suppressing MITF expression, as a new
resource for skin-whitening compounds. Further analysis
suggested that carnosol and carnosic acid in this extract are
responsible for inhibiting melanin synthesis. The present
study aimed to investigate this relationship.

Materials and methods
Plant extracts and chemicals
Plant materials from fields of the Tanegashima and Tsukuba Divisions of the Research Center for Medicinal Plant
Resources, Japanese National Institute of Biomedical
Innovation, were dried (LC-234 Dryer, ESPEC, Tokyo,
Japan) at 50 °C for 2–3 days and ground into powder. Fivegrams of the powder was soaked in 20 mL of ethanol for
2 days and the extract was then passed through 2 g ofactivated charcoal (Wako Pure Chemicals Co., Ltd., Kyoto,
Japan) to exclude chlorophyll and related compounds. The
ethanol was evaporated and the extracts were resuspended
in 10 mg/mL ethanol. Authentic carnosol and carnosic acid
were obtained from Wako. Antibody reagents of the following were obtained: anti-tyrosinase (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA, USA); anti-MITF(Thermo Fisher Scientific Inc. Waltham, MA, USA); andb-actin, pp38, pERK1/2, pMEK1/2, pp90Rsk, and pCREB(Cell Signaling Technology, Danvers, MA, USA). Antigen–antibody interactions were visualized using ECL
chemiluminescence reagent (GE Healthcare, Uppsala,Sweden) followed by detection with LAS4000 (Fuji Film,
Tokyo, Japan), or use of a chromogenic reaction withimmunostain horseradish peroxidase (HRP) (ATTO Co.,
Ltd., Tokyo, Japan).
Cell culture and reporter assay
Mouse B16F10 melanoma cells from the American Type
Culture Collection (Manassas, VA, USA) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, 5.5 mM Dglucose), supplemented with 10 % fetal bovine serum andantibiotics. The cells were incubated in a humidified
atmosphere with 5 % CO2 at 37 °C and were transferred
every 2 days via trypsinization to new culture dishes. Cells
plated in 6-well dishes were stimulated with forskolin (Fsk,

29

10–15 lM) and cultured for 3 or 48 h for MITF or TYR/
melanin assays, respectively.
To perform reporter analyses, the 2-kb promoter regionof the mouse Tyr gene was amplified by PCR using two
primers: 50 -TCT ATC GAT AGG TAC CGG GTA GAC
AAT AGT CAA ACA G-30 and 50 -CTT AGA TCG CAG
ATC TTC TGC ACC AAT AGG TTA ATG AGT G-30 .
After digestion with Acc65I/BglII, the promoter DNA
fragment was ligated into the corresponding site of thepGL4 firefly luciferase vector and then identified as pGL4mouse tyrosinase.
To prepare a Mitf overexpression vector, the mouse Mitf
cDNA fragment was amplified by PCR using a cDNA poolof B16F10 melanoma cells and two primers: 50 -TTT AGA
TCT ATG CTA GAA TAC AGT CAC TAC CA-30 and
50 -AAA GCG GCC GCT CGC TAA CAC GCA TGC TCC
GT-30 . After digestion with BglII and NotI, the amplified
cDNA fragment was ligated into the corresponding site of
the pTarget vector (Promega, Madison, WI, USA). B16F10
melanoma cells plated onto a 24-well plate were cotransfected with pGL4 mouse tyrosinase (0.2 lg/well) with theinternal reporter pRL-TK (0.03 lg), in the presence orabsence of the Mitf expression vector (pTarget-mouse Mitf,
0.1 lg) using Lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA). After 24 h, the cells were exposed to Fsk(15 lM) and cultured further for 24 h. Reporter activity
was monitored using the Dual Luciferase Reporter Assay
Kit (Promega). The quantitative PCR analyses, including
primer sets, have been described in [6].
Assay for melanin content and tyrosinase activity
Melanin measurement was performed as in [14]. Briefly,
the cells were washed twice with phosphate-buffered saline
and recovered in 2-mL collection tubes, followed by centrifugation at 8000 rpm for 1.5 min. Each cell pellet wassuspended in 300 lL of 1 M NaOH and then lysed byincubation at 45 °C for 2 h. Melanin was extracted with a
2:1 chloroform–methanol mixture and measured with aspectrophotometer (Model 680; Bio-Rad, Hercules, CA,
USA) at 405 nm. The protein concentration of the cellpellets was determined using Bradford reagent (Bio-Rad)and used for normalization of the melanin content.
TYR activity was measured according to the method
described in [15]. Cells in 6-well plates were stimulatedwith 15 lM Fsk for 48 h and collected in 2-mL tubes.
After recovery as cell pellets, the cells were lysed in 500
lL 0.5 % sodium deoxycholate supplemented with
200 lM PMSF at 4 °C for 30 min; thereafter, 80 lL of celllysates were incubated with 400 lL assay buffer (29)(100 mM sodium phosphate buffer, pH 7.4, 4 % N,N0 dimethylformamide), 200 lL of 5 mM L-DOPA, and 200
lL of 20.7 mM 3-methyl-2-benzothiazoline hydrazone at123Author's personal copy
30

37 °C for 30 min. TYR activity was monitored by absorbance (505 nm) and normalized by amount of protein.
Purification of carnosol and its analysis
Forty grams of dried powder made from the leaves of C.
longissima were soaked in 400 mL of ethanol and extracted three times. The resulting extract was concentrated to
100 mL by evaporation and passed through 40 g of activated charcoal. To remove water-soluble compounds,
100 mL of hexane was added to the flow-through fraction
and the viscous solution (approximately 5 mL) was discarded. Ethanol and hexane were evaporated and theextract was dissolved with chloroform and applied onto asilica gel column (100 mL; Nacalai Co. Ltd., Kyoto,
Japan). Melanin-suppressing substances were eluted using
a 9:1 chloroform:methanol solvent. The eluted materials
were first suspended in 20 % acetonitrile–water andapplied onto a C-18 column (4.6 mm 9 50 mm; Cosmosil
MS-II, Nacalai, San Diego, CA, USA), then suspended in
50 % acetonitrile–water of 90 % or greater purity, recovered in ethyl acetate, and analyzed by LC–MS (QSTAR,AB Sciex, Framingham, MA, USA) and 13C-NMR (ECA500, JEOL, Tokyo, Japan). The substance was extracted as
carnosol.
1
H-NMR (CD3OD) d: 0.858, 0.864 (each 3H, s,
H-19,18), 1.18, 1.19 (each 3H, d, J = 6.3 Hz, H-16,17),
1.29 (1H, ddd, J = 16.5, 16.5, 4.1 Hz, H-3ax), 1.49 (1H,
brd, J = 16.5, H-3eq), 1.58 (1H, m, H-2eq), 1.68 (1H, dd,
J = 12.4, 6.9 Hz, H-5), 1.83 (1H, m, H-6ax), 1.90 (1H, m,
H-2ax), 2.18 (1H, m, H-6eq), 2.56 (1H, ddd, J = 14.4,
14.4, 4.6 Hz, H-1eq), 2.79 (1H, d, J = 14.4 Hz, H-1ax),
3.23 (1H, m, H-15), 5.41 (1H, dd, J = 4.9, 2.1 Hz, H-7),
6.68 (1H, s, H-14).
13
C-NMR (CD3OD) d: 30.1 (C-1), 20.1 (C-2), 42.2 (C3), 35.5 (C-4), 47.0 (C-5), 30.8 (C-6), 79.7 (C-7), 133.3 (C8), 122.9 (C-9), 49.5 (C-10), 144.2 (C-11), 144.7 (C-12),
136.0 (C-13), 112.5 (C-14), 27.9 (C-15), 23.21 (C-16),
23.24 (C-17), 32.1 (C-18), 20.1 (C-19), 179.4 (C-20).

Results
Screening of melanogenesis modulators
To identify plant extracts that modulate melanogenesis, wetested the leaf, stem, bark, and fruit of over 200 plants forinhibitory effects on melanin synthesis. The powderedplant materials were soaked in ethanol for 2 days. The
extracts were passed through activated charcoal columns toremove chlorophyll and related compounds and concentrated to 10 mg/mL. B16F10 melanoma cells were treatedwith the extracts (10 lg/mL) in the presence of Fsk

123

J Nat Med (2016) 70:28–35(10 lM), which increases cytoplasmic cAMP levels. After
2 days, our gross visual observations identified 10 candidates with inhibitory effects on melanin synthesis (Fig. 1a).
Further examination of cells treated with the extracts at
1 lg/mL identified three extracts showing significant
inhibitory effects on melanin synthesis (Fig. 1b), of which
the extract from the leaves of C. longissima showed thestrongest effect. Quantification of melanin content (Fig. 1c)and total TYR activity (Fig. 1d) showed significant inhibition of melanin synthesis by C. longissima extract at thelowest concentration of 1 lg/mL and in a dose-dependent
manner. Therefore, subsequent experiments focused on thisextract.
C. longissima extract inhibits the induction of MITF
protein
Next, we investigated the effects of the C. longissima
extract on protein levels of MITF and TYR. When wetreated B16F10 cells with the C. longissima extract in thepresence of Fsk, Western blots revealed that levels of
MITF (Fig. 2a) and TYR (Fig. 2b) decreased in a dosedependent manner. Time-course analysis revealed anincrease in MITF protein level 180 min after Fsk stimulation, which was inhibited by co-treatment with the C.
longissima extract (Fig. 2c). Although we similarly
examined phosphorylation levels of several signal transduction molecules (p38, ERK, MEK, RSK, and CREB)
known to affect MITF expression, we could not identify
any clues to the mechanisms by which the C. longissima
extract inhibited the MITF expression.
Overexpression of MITF rescues C. longissima
extract-dependent suppression of the Tyr promoter
activity
To investigate whether the reduced level of MITF protein
in C. longissima extract-treated cells was the result of
down-regulation of MITF gene expression, we performed
quantitative PCR analysis of Mitf mRNA (Fig. 3a). Treatment with the C. longissima extract reduced Mitf and Tyr
mRNA levels in unstimulated and Fsk-stimulated cells
after 3 and 48 h, respectively (Fig. 3a, b). To investigate
the importance of MITF levels on Tyr gene expression, we
performed a promoter assay using the mouse Tyr promoter
linked with the luciferase gene. Fsk stimulation up-regulated Tyr promoter activity four-fold, and treatment withthe extract down-regulated Tyr promoter activity toapproximately 30 % in both Fsk-stimulated and unstimulated cells (Fig. 3c). Overexpression of MITF in the cells
abolished the inhibitory effect of the extract (Fig. 3d).
Taken together, these results indicate that the C. longissima
extract initially down-regulates MITF gene transcription

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31

Fig. 1 Screening of plant
extracts inhibiting
melanogenesis. a Melanin
production in B16F10
melanoma cells was induced by
10 lM of Fsk in the presence ofa variety of plant extracts(10 lg/mL). After 48 h
incubation, cells were collected
into centrifuge tubes andmelanin contents were judged
by visual evaluation. Asterisk
indicates cells treated with the
C. longissima extract. b Positive
extracts were further evaluatedat 1 lg/mL. ?? indicates morethan 75 % inhibition; ?
indicates 50 % inhibition byvisual evaluation. c Melanincontent was examined after
normalization by protein
concentration (n = 3, means
and SD are indicated),*p \ 0.05, **p \ 0.01,***p \ 0.001 compared with
Fsk only. d Total tyrosinase
enzyme activity in the cells(c) was measured

and that a decrease in MITF leads to downregulation of
TYR gene transcription, diminishing TYR protein levels
and enzymatic activity.
Carnosol in C. longissima extract is the compound
responsible for suppression of melanogenesis
To identify the functional compounds in the C. longissima
extract, we fractionated the extract, evaluated the inhibitory
effects of the fractions on pigmentation, purified a candidate to 90 % homogeneity, and performed LC–MS and
13
C-NMR analyses (see ‘‘Materials and methods’’),resulting in carnosol as a candidate (Fig. 4a). Carnosol isan oxidative product of carnosic acid, abundant in someherbs including rosemary, whose extracts are known tosuppress melanogenesis [16]. The presence of a substantial
amount of carnosic acid in the C. longissima extract wasalso confirmed by an authentic standard. The total content
of carnosol and carnosic acid in the C. longissima extract
was approximately 16 % (w/w).
Compared with carnosol or carnosic acid alone, the C.
longissima extract more strongly inhibited melanogenesis(Fig. 4b, c), suggesting that the extract exerts synergistic
activity of carnosol and carnosic acid and possibly other123Author's personal copy32

J Nat Med (2016) 70:28–35

agents. Such synergism may also be helpful in averting cell
toxicity compared with carnosol or carnosic acid alone(Fig. 4d).

Discussion

Fig. 2 MITF protein level is suppressed by C. longissima extract.
a Western blot analysis of MITF protein in B16F10 melanoma cells
treated with Fsk (15 lM) for 3 h. b Tyrosinase (TYR) protein levels
were evaluated in cells incubated with Fsk for 48 h. c Timedependent induction of MITF protein and phosphorylation status ofsignaling molecules were evaluated. B16F10 melanoma cells wereincubated with the C. longissima extract (3 lg/mL) and Fsk (15 lM)

An ethanol extract of the leaves of C. longissima strongly
inhibited melanin production in mouse B16F10 melanoma
cells. The substances in the extract responsible formelanogenesis inhibition were carnosol and carnosic acid.
Both carnosol and carnosic acid are gaining attention for
their antioxidant properties [17–19]. Their anti-melanogenic potencies have also been reported as suppressors
of TYR expression in B16 melanoma cells [16]. Carnosol
is formed from carnosic acid by autoxidation through its
intermediate carnosic acid quinone [20]. Although carnosic
8-lactone and rosmanol are also produced via the same
oxidative reaction, their contributions to the suppression ofmelanogenesis are as yet unknown, probably owing to their
unstable nature.
Cellular targets of carnosol and carnosic acid havebeen identified. They include CREB-binding protein/E1A

Fig. 3 Mitf expression is critical for C. longissima extract-dependent
suppression of tyrosinase promoter activity. a Total RNA wasextracted from B16F10 melanoma cells that had been treated with Fsk(15 lM for 3 h) together with the C. longissima extract (3 lg/mL),and used as templates for quantitative PCR to measure MITF mRNA(n = 3, means and SD are indicated), *p \ 0.05. b The same
experiment was performed for Tyr mRNA in 48-h treated cells,**p \ 0.01. c pGL4-mouse tyrosinase promoter reporter (fLuc) wastransformed into B16F10 melanoma cells together with an internal
TK reporter (rLuc). After 24 h, Fsk (15 lM) was added to the culture
medium and the cells were incubated further for 24 h (n = 4). d The
expression vector for mouse MITF was co-transformed with the pGL4mouse tyrosinase promoter reporter. After 48 h, reporter activities
were measured

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Fig. 4 C. longissima extract isabundant in carnosol andcarnosic acid. a Structure ofcarnosol. b B16F10 melanoma
cells were treated with Fsk(15 lM) for 48 h together withindicated concentrations ofcarnosol, carnosic acid, and the
C. longissima extract andmelanin content was measured(c). d Mouse B16F10 melanoma
cells (plated on a 96-well plate
at 1 9 104 cells per plate) weretreated with extract,compounds, or DMSO alone for
48 h. Cell viability wasestimated by measurement ofcellular NADH with the WST-8
kit. Values (OD) werenormalized to that of DMSO
alone (0 lg/mL compounds).
n = 4, means and SD are
shown. ***p \ 0.001

binding protein p300 (CBP/p300) [21], kelch-like ECHassociated protein 1 (Keap1)-nuclear factor erythroid
2-related factor 2 (Nrf2) [19], p38 [22], b-catenin [23],nuclear factor jB-matrix metalloprotainase-9 [24, 25],transient receptor potential ankyrin 1 [26], and AMP-activated kinase (AMPK) [27]. CBP/p300 acts as a CREB
coactivator, upregulates MITF gene expression in melanocytes, and promotes MITF-dependent transcription [28].
Carnosol suppressed CBP/p300 on the cyclooxygenase-2
promoter in human 184B5/HER breast epithelial cells [21].
In contrast to CBP/p300, AMPK is proposed to suppress
CREB activity in the liver via the phosphorylation-dependent inhibition of other coactivator CRTCs (CREB-relatedtranscription coactivators) [29], and CRTC1 promotes
MITF gene expression in melanocytes [6]. Although the
AMPK activator metformin was found to suppress CREB
activity in human melanocytes by reducing cAMP levels[30], the metformin-dependent suppression of melanogenesis did not require AMPK, suggesting that AMPK is notessential for the suppressed melanogenesis found for the C.
longissima extract. b-Catenin also acts as a co-activator of
the LEF/TCF complex, which up-regulates MITF gene
expression in Wnt signaling [31]. Carnosol binds b-catenin
and promotes its degradation [23]. These reports suggestthat carnosol and carnosic acid suppress MITF gene
expression through multiple cascades.

In addition to these well-known cascades regulating
Mitf promoter activity, overexpression of Nrf2 was foundto suppress MITF gene expression [32]. In unstressed
cells, Nrf2 binds to Keap1, a recruiter of Nrf2 to theproteasome complex, leading to Nrf2 degradation [33].
Keap1 has free thiol moieties that act as sensors of redox
status. Once the thiol moieties are oxidized, Keap1 isdegraded and releases Nrf2, which induces Nrf2-dependenttranscription of anti-oxidative molecules [34]. Carnosic
acid attacks the thiol moieties of Keap1 and activates theNrf2 pathway [19].
The p38 pathway also has been reported to regulate Nrf2
both positively [35] and negatively [36]. In addition, p38 is
a component of a UV-induced stress signal that promotes
melanogenic programs. Indeed, carnosol up-regulated p38
phosphorylation in B16F10 melanoma cells [37], suggesting the complex roles of p38 in melanogenesis.
Rosemary is rich in carnosol and carnosic acid, and isused for a variety of purposes [17], including in foods [38].
By evaluating anti-melanogenic activity, we found that theethanol extract of C. longissima leaf was also rich in thesesubstances and strongly inhibited MITF gene expression.
Although C. longissima is currently in danger ofextinction in Japan [13], we have successfully cultured thisplant on the experimental farm operated by the Tanegashima Division of the Japan National Institute of123

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34

Biomedical Innovation, Health and Nutrition. This suggeststhat C. longissima may be an attractive source of skinwhitening compounds used in the cosmetic industry. Further study is required to elucidate the molecular mechanism
by which the C. longissima extract inhibits melanogenesis.
Acknowledgments We thank Mrs. Junko Morita, Mr. Sachio Shiga,
Mr. Fumihiro Kamada, Dr. Shigeki Katsuki (National Institute of
Biomedical Innovation), and Mr. Akihiro Hojo (Kansai University)
for providing technical assistance.
Compliance with ethical standards
Conflict of interest MY and AK are employees of Momotani-juntenkan Co. Ltd., which paid 300,000 JP Yen: 2012 towards the costsof this study. Other contributors declare that they have no conflict ofinterest.
Funding This study was funded by Grants-in-Aid for Scientific
Research from the Japan Ministry of Health, Labor, and Welfare/
AMED (2013–2017, 2014–2016 to HT and HW), and Supported
Program for the Strategic Research Foundation at Private Universities(2011–2015: S1101027 to HW and 2013–2017 to HK, YN, and HT)from the Ministry of Education, Culture, Sports Science and Technology Japan.

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