Glycosidic inhibitors of melanogenesis from leaves of momordica charantia

Article abstract of DOI:10.1002/cbdv.201100350

Eight glycosidic compounds, 1-8, including two new compounds, (4I)-α-terpineol 8-O-[α-L-arabinopyranosyl-(1→6)-β- D-glucopyranoside] (5) and myrtenol 10-O-[β-D-apiofuranosyl-(1→6)- β-D-glucopyranoside] (7), were isolated from the BuOH-soluble fraction of a MeOH extract of Momordica charantia leaves. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of compounds 1-8 on the melanogenesis in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), these compounds were found to exhibit inhibitory activities with 7.1-27.0% and 23.6-46.4% reduction of melanin content at 30 μM and 100 μM, respectively, with no or almost no toxicity to the cells (80.0-103.5% of cell viability at 100 μM). Western blot analysis showed that compound 7 reduced the protein levels of MITF, tyrosinase, TRP-1, and TRP-2 mostly in a concentration-dependent manner, suggesting that this compound inhibits melanogenesis on the α-MSH-stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP-1, and TRP-2. Copyright

Full text of DOI:10.1002/cbdv.201100350

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Glycosidic Inhibitors of Melanogenesis from Leaves of Momordica charantia
by Takashi Kikuchia)b), Jie Zhanga), Yan Huanga), Kensuke Watanabea), Kenta Ishiia), Ayako
Yamamotoa), Makoto Fukatsua), Reiko Tanakab), and Toshihiro Akihisa*a)
a) College of Science and Technology, Nihon University, 1–8 Kanda Surugadai, Chiyoda-ku,
Tokyo 101-8308, Japan (phone: þ81-3-3259-0806; fax: þ81-3-3293-7572;
e-mail: akihisa.toshihiro@nihon-u.ac.jp)
b) Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Eight glycosidic compounds, 1–8, including two new compounds, (4x)-a-terpineol 8-O-[a-l-
arabinopyranosyl-(1!6)-b-d-glucopyranoside] (5) and myrtenol 10-O-[b-d-apiofuranosyl-(1!6)-b-d-
glucopyranoside] (7), were isolated from the BuOH-soluble fraction of a MeOH extract of Momordica
charantia leaves. The structures of the new compounds were elucidated on the basis of extensive
spectroscopic analyses and comparison with literature. Upon evaluation of compounds 1–8 on the
melanogenesis in B16 melanoma cells induced with a-melanocyte-stimulating hormone (a-MSH), these
compounds were found to exhibit inhibitory activities with 7.1–27.0% and 23.6–46.4% reduction of
melanin content at 30 mm and 100 mm, respectively, with no or almost no toxicity to the cells (80.0–
103.5% of cell viability at 100 mm). Western blot analysis showed that compound 7 reduced the protein
levels of MITF, tyrosinase, TRP-1, and TRP-2 mostly in a concentration-dependent manner, suggesting
that this compound inhibits melanogenesis on the a-MSH-stimulated B16 melanoma cells by, at least in
part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP-1, and
TRP-2.
Introduction. – The plant Momordica charantia L. (bitter gourd; Cucurbitaceae) is
cultivated throughout the world for use as a vegetable as well as medicine. M. charantia
has been used in traditional medicine in developing countries, mostly for healing
diabetes and as a carminative, and in the treatment of colic [1][2]. Previous
investigations have shown that crude extracts of M. charantia fruits possess antidiabetic
activity [3], and many cucurbitane-type triterpenoids have been isolated from various
parts of this plant [4–13]. In search for potential bioactive compounds from natural
sources [14][15], we have isolated 18 cucurbitane-type triterpenoids from the MeOH
extract of the dried fruits of Japanese M. charantia, which is commonly called ꢀnigauriꢁ
or ꢀgoyaꢁ [16][17]. Some of these triterpenoids have been shown to exhibit inhibitory
effects against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced EpsteinꢀBarr
virus early antigen (EBV-EA) activation in Raji cells, and against skin-tumor
promotion in both 7,12-dimethylbenz[a]anthracene (DMBA) and peroxynitrite
(ONOOꢀ )-induced mouse-skin carcinogenesis tests [17]. In addition, our investigation
on a MeOH extract of the leaves of M. charantia has led to the isolation of six new and
eleven known cucurbitane-type triterpenoids. Some of these triterpenoids showed
inhibitory effects against TPA-induced EBV-EA induction and against DMBA-
induced mouse-skin carcinogenesis [18]. In continuing our study on the constituents of
the MeOH extract of M. charantia leaves, we have isolated two new, i.e., 5 and 7, and six
ꢂ 2012 Verlag Helvetica Chimica Acta AG, Zꢃrich

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
known, i.e., 1–4, 6, and 8, glycosidic compounds. Here, we describe the characterization
of these glycosides and the evaluation of their inhibitory effects against melanogenesis
in the a-melanocyte-stimulating hormone (a-MSH)-stimulated B16 melanoma cells.
Results and Discussions. – Melanogenesis Inhibitory Activity of the Extracts of M.
charantia Leaves. The leaves of M. charantia were extracted with MeOH, and the
extract was, then, partitioned into hexane-, MeOH/H2O-, BuOH-, and H2O-soluble
fractions. The MeOH extract and the four fractions were evaluated for their
melanogenesis-inhibitory activities in the a-MSH-stimulated B16 melanoma cells.
The melanin content was reduced to 40.8% by addition of MeOH extract (100 mg/ml)
to the incubation medium of B16 cells (Table 1). Whereas the MeOH/H2O-soluble
fraction, followed by hexane-soluble fraction, reduced melanin content significantly
(25.1 and 35.9%, resp., of melanin content at 100 mg/ml), most of their melanogenesis
inhibitory activities are, however, due to their cytotoxicities (0.3 and 38.4%, resp., of
cell viability at 100 mg/ml). The H2O-soluble fraction showed only slight melanogenesis
inhibition (88.1 of melanin content at 100 mg/ml) with almost no cytotoxicity (101.6% of
cell viability at 100 mg/ml). On the other hand, the BuOH-soluble fraction exhibited
marked melanogenesis inhibitory activity (40.5% of melanin content at 100 mg/ml)
without significant inhibition of cell proliferation (62.1% of cell viability at 100 mg/ml).
The BuOH-soluble fraction was then investigated for its constituents.
Table 1. Melanogenesis-Inhibitory Activities and Cytotoxicities of the Extracts of Momordica charantia
Leavesa) in B16 Mouse Melanoma Cell Line
Extract and fraction
MeanꢁS.E. at 10 mg/ml
MeanꢁS.E. at 100 mg/ml
Melanin content
Cell viability
Melanin content
Cell viability
Control (100% DMSO)
MeOH Extract
Hexane-soluble fraction
MeOH/H2O-Soluble fraction
BuOH-Soluble fraction
H2O-Soluble fraction
100.0ꢁ3.3
108.4ꢁ15.5
58.2ꢁ0.7
95.4ꢁ4.4
85.4ꢁ3.1
96.9ꢁ1.8
100.0ꢁ4.7
78.9ꢁ5.6
94.1ꢁ1.7
115.5ꢁ0.9
118.4ꢁ1.5
116.0ꢁ0.8
100.0ꢁ3.3
40.8ꢁ8.6
35.9ꢁ1.1
25.1ꢁ2.5
40.5ꢁ2.7
88.1ꢁ7.3
100.0ꢁ4.7
10.7ꢁ1.2
38.4ꢁ0.5
0.3ꢁ0.3
62.1ꢁ3.0
101.6ꢁ1.6
a) Melanin content [%] and cell viability [%] were determined based on the absorbances at 405 nm, and
570 (top)–630 (bottom) nm, respectively, by comparison with those for DMSO (100%). Each value
represents the meanꢁstandard error (S.E.) of three determinations. Concentration of DMSO in the
sample solution was 2 ml/ml.
Isolation, Identification, and Structure Elucidation of Compounds from BuOH-
Soluble Fraction. The BuOH-soluble fraction was subjected to successive chromatog-
raphy on Diaion HP-20, SiO2, and octadecyl silica gel (ODS) columns, and to
preparative HPLC, yielding eight glycosides, 1–8, of which 5 and 7 were new
compounds. Identifications of six known compounds, hex-3-en-1-ol 1-O-b-d-glucopyr-
anoside (1) [19], benzyl alcohol 1-O-[a-l-arabinopyranosyl-(1!6)-b-d-glucopyrano-
side] (2) [20], (6S,9R)-roseoside (3) [21], 3-oxo-a-ionol 9-O-b-d-glucopyranoside (4)
[22], sacranoside A (¼ myrtenol 10-O-[a-l-arabinopyranosyl-(1!6)-b-d-glucopyra-

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1223
noside]; 6) [23][24], and myrtenol 10-O-b-d-glucopyranoside (8) [23], were accom-
plished by MS and NMR comparison with corresponding literature data.
Compound 5 exhibited a [MþNa]þ ion peak in the HR-ESI-MS at m/z 471.2206,
compatible with a molecular formula C21H36O10. The 1H-NMR spectrum (Table 2) of 5
displayed two anomeric signals at d(H) 4.29 (d, J¼6.4) and 4.47 (d, J¼7.8 ) . The
13C-NMR (Table 2) of 5 displayed signals at d(C) 66.3 (t), 69.4 (d), 72.2 (d), 74.0 (d),
and 104.9 (d), attributable to a terminal a-arabinopyranose moiety [23][24], and at
d(C) 69.3 (t), 71.5 (d), 75.2 (d), 76.3 (d), 78.1 (d), and 98.6 (d) assigned to an inner b-
glucopyranose. The glycosylation shift of C(6’) signal, compared to the signal of
sacranoside A (6) [23][24], suggested that the terminal arabinose unit was connected to
1
C(6’) of the inner glucose. In addition to signals for sugars, the 13C- and H-NMR
spectra of compound 5 exhibited signals of two tertiary Me groups, a vinyl Me group,
three CH2 groups, a methine group, a trisubstituted C¼C bond, and a tertiary CꢀO
(d(C) 81.1) group, which were in good agreement with those of terpineol moiety of (S)-
a-terpineol [a-l-arabinofuranosyl]-(1!6)-b-d-glucopyranoside [25]. Acid hydrolysis
of 5 with aqueous CF3COOH solution liberated d-glucose and l-arabinose [26], which
were identified by GLC analysis of the trimethylsilyl thiazolidine derivative [27]. The
1
above evidence, coupled with the analysis of DEPT, H,1H-COSY, NOESY, HMQC,
and HMBC data, suggested that 5 has the structure (4x)-a-terpineol 8-O-[a-l-
arabinopyranosyl-(1!6)-b-d-glucopyranoside]. HMBC Experiments showed diag-
nostic cross-correlations of HꢀC(1’) with C(8), and of CH2(6’) with C(1’’) (Fig. 1),
supporting the proposed structure.
The molecular formula of compound 7 was determined as C21H54O10 on the basis of
1
its HR-ESI-MS (m/z 469.4043 ([MþNa]þ )). The H-NMR spectrum of 7 displayed
two anomeric signals at d(H) 4.24 (d, J¼7.8) and 5.00 (d, J¼2.3) suggesting a
disaccharide. The 13C-NMR (Table 2) of 7 showed signals at d(C) 65.6 (t), 75.0 (t), 78.0

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1
Table 2. 13C- and H-NMR Data (at 100 and 400 MHz, resp.; CD3OD) of Compounds 5 and 7 Isolated
from Momordica charantia Leaves
Position
5
7
d(C)
d(H)
d(C) (
d(H)
1
2
3
4
5
134.8 (s)
44.5 (d) 2.24 (dt, J¼1.4, 6.0)
146.3 (s)
121.9 (d) 5.32–5.36 (m)
28.1 (t) 1.73–1.78 (m), 2.02–2.06 (m)
45.1 (d) 1.65–1.72 (m)
25.0 (t) 1.59–1.64 (m), 1.98–2.06 (m)
32.1 (t) 1.86–1.94 (m), 2.01–2.06 (m)
23.6 (q) 1.61 (br. s)
121.0 (d) 5.56–5.59 (m)
32.3 (t) 2.27–2.31 (m, 2 H)
42.2 (d) 2.06–2.12 (m)
32.5 (t) 2.42 (dt, J¼5.5, 8.7)
38.9 (s)
6
7
8
81.1 (s)
26.6 (q) 1.30 (s)
9
23.0 (q) 1.17 (s)
21.6 (q) 0.87 (s)
10
25.1 (q) 1.21 (s)
72.8 (t) 4.00 (dd, J¼1.4, 12.8),
4.19 (dd, J¼1.8, 12.8)
Sugar moiety
1’
2’
3’
4’
5’
6’
98.6 (d) 4.47 (d, J¼7.8)
103.4 (d) 4.24 (d, J¼7.8)
75.1 (d) 3.18 (t, J¼8.9)
78.1 (d) 3.31 (t, J¼8.7)
71.7 (d) 3.26 (t, J¼8.7)
76.9 (d) 3.32–3.38 (m)
68.6 (t) 3.58 (dd, J¼5.9, 11.4),
3.99 (dd, J¼1.9, 11.5)
111.0 (d) 5.00 (d, J¼2.3)
78.0 (d) 3.90 (d, J¼2.3)
80.6 (s)
75.2 (d) 3.13 (t, J¼7.8)
78.1 (d) 3.33 (t, J¼9.0)
71.5 (d) 3.34–3.39 (m)
76.3 (d) 3.30–3.38 (m)
69.3 (t) 3.71 (dd, J ¼ 3.2, 11.3),
4.00 (br. d, J¼11.5)
1’’
2’’
3’’
4’’
104.9 (d) 4.29 (d, J¼6.4)
72.2 (d) 3.58 (t, J¼8.7)
74.0 (d) 3.47–3.56 (m)
69.4 (d) 3.77–3.81 (m)
75.0 (t) 3.76 (d, J¼9.6),
3.96 (d, J¼9.6)
5’’
66.3 (t) 3.53 (dd, J¼3.2, 12.4), 3.86 (dd, J¼3.6, 12.4)
65.6 (t) 3.57 (s, 2 H)
a) Figures in parentheses denote J values in Hz.
(d), 80.6 (d), and 111.0 (d), attributable to a terminal b-apiofuranose moiety1), and
d(C) 68.6 (t), 71.7 (d), 75.1 (d), 76.9 (d), 78.1 (d), and 103.4 (d) assigned to an inner b-
glucopyranose. The glycosylation shift of C(6’) signal, compared to the signals of
everlastosides A–E [28], indicated that the terminal arabinose unit is connected to
C(6’) of the inner glucose. In addition, the 1H- and 13C-NMR of 7 revealed the presence
of two tertiary Me groups, two CH2 groups, a CH2ꢀO group, two CH groups, a
trisubstituted C¼C bond, and a quaternary C-atom (d(C) 38.9; Table 2), which were
almost indistinguishable from those of the myrtenol moiety of myrtenol 10-O-b-d-
glucopyranoside (8) [23]. Acid hydrolysis of 7 liberated d-glucose and d-apiose, which
were identified by GLC analysis of the trimethylsilyl thiazolidine derivatives. The
1
above evidence, in combination with the analysis of DEPT, H,1H-COSY, NOESY,
HMQC, and HMBC data, suggested that 7 possesses the structure myrtenol 10-O-[b-d-
The terminal b-apiofuranosyl moiety of everlatoside B exhibited 13C-NMR signals (CD3OD,
1
)
150 MHz) at d(C) 65.6 (t, C(5’’)), 74.9 (t, C(4’’)), 78.0 (d, C(2’’)), 80.5 (d, C(3’’)), and 110.9 (d, C(1’’))
[28].

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Fig. 1. Key HMBC (H!C) and 1H,1H-COSY (—) correlations for compounds 5 and 7
apiofuranosyl-(1!6)-b-d-glucopyranoside]. HMBC Experiments showed diagnostic
cross-correlations of CH2(10) with C(1’), and of CH2(6’) with C(1’’) (Fig. 1), supporting
the proposed structure.
Melanogenesis-Inhibitory Activity of Compounds. Eight glycosidic compounds, 1–
8, isolated from the BuOH-soluble fraction, were evaluated for their melanogenesis-
inhibitory activities in the a-MSH-stimulated B16 melanoma cells. By addition of these
compounds to an incubation medium of B16 melanoma cells, all of the compounds
showed inhibitory activities (85.1–99.9%, 73.0–92.9%, and 53.6–76.4% of melanin
content at 10, 30, and 100 mm, resp.; Table 3) with no or almost no cytotoxicity (90.0–
115.5, 84.0–115.7, and 80.0–103.5% of cell viability at 10, 30, and 100 mm, resp.). The
inhibitory activity on melanogenesis of these compounds was superior to a known
melanogenesis inhibitor, arbutin (103.5, 100.3, and 86.2% of melanin content at 10, 30,
and 100 mm, resp.), which has been recognized as a useful depigmentation compound
for skin whitening in the cosmetic industry [29]. Among the glycosides tested,
compounds 2 and 7 exhibited inhibition (87.1 and 85.1% of melanin content, resp.) of
melanogenesis even at a lower concentration (10 mm).
Mechanism of Melanogenesis Inhibition by Compound 7. Tyrosinase, and tyrosi-
nase-related protein-1 (TRP-1) and TRP-2 are enzymes responsible for the synthesis of
melanin [30]. Regulations of the transcription and activity of these melanogenic
enzymes are effective for depigmentation [31]. Tyrosinase, a rate limiting enzyme,
catalyzes the hydroxylation of l-tyrosine to l-dihydroxyphenylalanine (l-DOPA), and
the oxidation of l-DOPA to l-DOPA quinone [32]. TRP-2 functions as DOPAchrome
tautomerase, and TRP-1 catalyzes oxidation of 5,6-dihydroxy-1H-indole-2-carboxylic
acid (DHICA) [32]. Transcription for expression of these enzymes is regulated by
microphthalmia-associated transcription factor (MITF) [33]. To clarify the mechanism
involved in the melanogenesis inhibition by compound 7, one of the potent melano-

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table 3. Melanogenesis-Inhibitory Activities and Cytotoxicities of Compounds Isolated from Momordica
charantia Leaves in B16 Mouse Melanoma Cell Linea)
Compound
MeanꢁS.E. at 10 mm
MeanꢁS.E. at 30 mm
MeanꢁS.E. at 100 mm
Melanin
Cell
Melanin
Cell
Melanin
Cell
content
viability
content
viability
content
viability
Control (100% DMSO) 100.0ꢁ5.1 100.0ꢁ2.4 100.0ꢁ5.1 100.0ꢁ2.4 100.0ꢁ5.1 100.0ꢁ2.4
1
2
3
4
5
6
7
8
99.9ꢁ4.4
90.0ꢁ4.6
75.8ꢁ1.6
84.0ꢁ4.4
69.9ꢁ2.3
58.6ꢁ1.6
80.0ꢁ6.7
88.7ꢁ3.5
87.1ꢁ1.7 107.0ꢁ2.5
95.5ꢁ0.9 115.5ꢁ1.7
99.2ꢁ3.0 112.2ꢁ0.7
73.0ꢁ3.5 102.1ꢁ2.6
89.8ꢁ3.1 115.7ꢁ2.2
92.3ꢁ4.8 112.2ꢁ0.8
68.8ꢁ2.2 103.5ꢁ3.5
76.4ꢁ0.9 101.5ꢁ2.4
95.4ꢁ2.3
92.5ꢁ1.6
85.1ꢁ4.7
94.7ꢁ1.0
92.1ꢁ7.2
98.5ꢁ1.2
92.9ꢁ2.3
79.0ꢁ1.5
88.2ꢁ4.7
93.6ꢁ5.9
74.6ꢁ2.2
71.5ꢁ3.3
59.8ꢁ3.3
53.6ꢁ2.6
86.2ꢁ3.0
88.1ꢁ1.2
96.3ꢁ6.5
85.0ꢁ5.7
86.8ꢁ1.5
93.6ꢁ2.4
77.5ꢁ3.2 101.8ꢁ2.9
78.9ꢁ2.8 103.9ꢁ1.7
94.5ꢁ1.3 109.1ꢁ2.3
Arbutinb)
103.5ꢁ2.8 108.7ꢁ3.2 100.3ꢁ1.0 104.7ꢁ1.6
a) Melanin content [%] and cell viability [%] were determined based on the absorbances at 405 nm, and
570 (top) – 630 (bottom) nm, respectively, by comparison with those for DMSO (100%). Each value
represents the meanꢁstandard error (S.E.) of three determinations. Concentration of DMSO in the
sample solution was 2 ml/ml. b) Reference compound.
genesis inhibitors found in this study, the protein levels of tyrosinase, TRP-1, TRP-2,
and MITF were evaluated in B16 melanoma cells treated with compound 7 by Western
blot analysis. Treatment of B16 melanoma cells with compound 7 reduced protein levels
of all of MITF, tyrosinase, TRP-1, and TRP-2 proteins mostly in a concentration-
dependent manner (Fig. 2). These results suggested that compound 7 exhibits
melanogenesis inhibitory activity in the a-MSH-stimulated B16 melanoma cells due
to, at least in part, inhibition of the expression of MITF, followed by a decrease in the
expression of tyrosinase, TRP-1, and TRP-2.
Fig. 2. Effect on expression of MITF, tyrosinase, TRP-1, and TRP-2 in a-MSH-stimulated B16 melanoma
cells treated with compound 7
Conclusions. – The BuOH-soluble fraction separated from a MeOH extract of M.
charantia leaves exhibited melanogenesis-inhibitory activity in the a-MSH-stimulated

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1227
B16 melanoma cells without significant inhibition of cell proliferation. Eight glycosidic
compounds, 1–8, were isolated from the BuOH-soluble fraction, and all of these
compounds exhibited melanogenesis-inhibitory activity with no or almost no cytotox-
icity. Among these compounds, myrtenol 10-O-[b-apiofuranosyl-(1!6)-b-glucopyr-
anoside] (7) has been shown to exert its melanogenesis inhibition, at least in part, by
inhibiting expression of MITF, tyrosinase, TRP-1, and TRP-2. It appears that the
glycosidic compounds isolated from M. charantia leaves in this study may be valuable as
potential skin-whitening agents.
Experimental Part
General. Column chromatography (CC): Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan),
silica gel (SiO2, 230–400 mesh; Merck), and octadecyl silica gel (ODS; Chromatorex-ODS, 100–200
mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). TLC: on Silica gel 60 F254 aluminium sheets (Merck),
eluent: CHCl3/MeOH/AcOH/H2O 15 :8 :3 :2. Reversed-phase (RP) prep. high-performance liquid
chromatography (HPLC): on ODS columns (25 cmꢂ10 mm i.d.) at 258; on a Pegasil ODS-II 5-mm
column (Senshu Scientific Co., Ltd., Tokyo, Japan) with MeOH/H2O/AcOH 50 :50 :0.1 (flow rate 2.0 ml/
min, HPLC system I), or on a Capcell pak AQ 5 mm column (Shiseido Co., Ltd., Tokyo, Japan) with
MeCN/H2O/AcOH 10 :90 :0.1 (flow rate 2.0 ml/min, HPLC system II), or 45 :55 :0.1 (flow rate 2.0 ml/
min, HPLC system III) or 35 :65 :0.1 (flow rate 3.0 ml/min, HPLC system IV). GLC: Shimadzu GC-2014
instrument on a DB-17 fused silica glass cap. column (Agilent Technologies, Inc., Santa Clara, CA, USA;
30 mꢂ0.32 mm i.d.; column temp., 2008; injection and detector temp., 2708; He flow rate, 0.4 ml/min;
split ratio, 1:75). Optical rotations: JASCO P-1020 digital polarimeter. UV Spectra: JASCO V-630Bio
spectrophotometer; lmax (log e) in nm. IR Spectra: JASCO FTIR-300E spectrometer; ˜n in cmꢀ1. 1D- and
2D-NMR Spectra: JEOL ECX-400 (1H, 400 MHz; 13C, 100 MHz) spectrometer at r.t.; CD3OD solns.; d
in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Agilent 1100 LC/MSD TOF (time-of-
flight) system (positive-ionization mode; cap. voltage, 3000 V; fragmentor voltage, 225 V).
Chemicals and Materials. The source of leaves of Momordica charantia was described in [18]. The
chemicals were purchased as follows: fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin
and 100 mg/ml streptomycin) from Invitrogen Co. (Carlsbad, CA, USA), Dulbeccoꢀs modified Eagleꢀs
medium (D-MEM), and d-apiose and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT) from Sigma-Aldrich Japan Co. (Tokyo, Japan). Primary antibodies of anti-MITF, anti-
tyrosinase, anti-TRP-1, and anti-TRP-2 were purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA, USA), anti-b-actin from Cell Signaling Technology (Beverly, MA, USA), l-glucose, l-
arabinose, and TMS-HT kit (hexamethyldisilazaneꢀtrimethylchlorosilane (HMDSꢀTMCS) in anh.
pyridine) from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), d-arabinose from Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan), and d-glucose and l-cysteine methyl ester hydrochloride from Kanto
Chemical Co., Inc. (Tokyo, Japan). All other chemicals and reagents were of anal. grade.
Cell Cultures. Cell line B16 4A5 (mouse melanoma) was obtained from Riken Cell Bank (Tsukuba,
Ibaraki, Japan). The cell line was grown in D-MEM. The medium was supplemented with 10% FBS and
antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were incubated at 378 in a 5% CO2
humidified incubator.
Extraction and Isolation. Chopped and air-dried leaf material of M. charantia (4.8 kg) was extracted
with MeOH (1 kg/4 l) at r.t. (3ꢂ3 d) to give a crude extract (399 g). The extract was suspended in H2O
(40 g/1 l) and then extracted with AcOEt (3 ꢂ 1 l). The AcOEt-soluble fraction was further partitioned
between hexane/H2O/MeOH (19 :19 :1), which yielded hexane- and MeOH/H2O-soluble fractions (48
and 75 g, resp.). On the other hand, the H2O layer was further extracted with BuOH, which yielded
BuOH- and H2O-soluble fractions (40 and 170 g, resp.) [18]. A portion of BuOH-soluble fraction (39 g)
was chromatographed on a Diaion HP-20 (1226 g) column with step gradient MeOH/H2O 1:9, 3 :7, 1:1,
7:3 and 1:0, which yielded three fractions, Frs. A–C. Fr. B (6.9 g), eluted with H2O/MeOH 7:3 and 1:1,
was further chromatographed on a SiO2 (160 g) column (hexane/AcOEt gradient 1:0!0 :1, and AcOEt/

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
MeOH gradient 1:0!0 :1) to yield fifteen fractions, Fr. B1–B15. Prep. HPLC (system I) of Fr. B3
(101 mg), eluted with hexane/AcOEt 3 :7, yielded compound 1 (3.0 mg; retention time (tR) 14.8 min).
Fr. B6 (1608.8 mg), eluted with hexane/AcOEt 7:3, was chromatographed on an ODS (48 g) column
(H2O/MeOH gradient 3 :7!0 :1) to give eight fractions, Frs. B6-1–B6-8. Prep. HPLC (system II) of
Fr. B6-3 (193 mg), eluted with MeOH/H2O 3 :7, afforded compounds 2 (4.4 mg; tR 27.2 min) and 3
(27.5 mg; tR 33.6 min). Fr. C (5.5 g), eluted with H2O/MeOH 3 :7 and 0 :1, was chromatographed on an
ODS (144 g) column to provide nine fractions, Frs. C1–C9. Prep. HPLC (system III) of Fr. C2 (94 mg),
eluted with MeOH/H2O 6 :4, yielded compound 4 (2.1 mg; tR 32.0 min). Fr. C3 (1326 mg) was
chromatographed on an ODS (48 g) column (MeOH/H2O gradient 3 :7!0 :1) to afforded thirteen
fractions, Frs. C3-1–C3-13. Prep. HPLC (system IV) of Fr. C3-3 (97 mg), eluted with MeOH/H2O 1:1,
gave compounds 5 (3.1 mg; tR 25.6 min), 6 (3.2 mg; tR 27.6 min), 7 (6.4 mg; tR 30.4 min), and 8 (7.4 mg; tR
41.6 min). The specific-rotation, IR, and HR-ESI-MS data of two new compounds, 5 and 7, are described
below, while their 13C- and 1H-NMR spectroscopic data are compiled in Table 2.
(4x)-a-Terpineol 8-O-[a-l-Arabinopyranosyl-(1!6)-b-d-glucopyranoside] (¼1-Methyl-1-(4-meth-
ylcyclohex-3-en-1-yl)ethyl 6-O-a-l-Arabinopyranosyl-b-d-glucopyranoside; 5). Amorphous solid.
1
[a]2D2 ¼ ꢀ35.5 (c¼1.07, EtOH). IR (KBr): 3412 (OH), 2925, 1637, 1384, 1227, 1082. H- and 13C-NMR:
see Table 2. HR-ESI-MS: 471.2206 ([MþNa]þ , C21H36NaO1þ0 ; calc. 471.2206).
Myrtenol 10-O-[b-d-Apiofuranosyl-(1!6)-b-d-glucopyranoside] (¼ [(1R,5S)-6,6-Dimethylbicy-
clo[3.1.1]hept-2-en-2-yl]methyl 6-O-[3-C-(Hydroxymethyl)-a-d-erythrofuranosyl]-b-d-glucopyranoside;
7): Amorphous solid. [a]2D2 ¼ ꢀ49.3 (c¼0.30, EtOH). IR (KBr): 3401 (OH), 2927, 2366, 1718, 1650, 1425,
1057. 1H- and 13C-NMR: see Table 2. HR-ESI-MS: 469.4043 ([MþNa]þ , C21H34NaO1þ0 ; calc. 469.4049).
Acid Hydrolysis of 5 and 7, and Sugar Identification. A soln. of 5 (3.2 mg) in H2O (1.1 ml) and 2m aq.
CF3COOH (11.0 ml) was heated under reflux for 2 h [26]. The mixture was diluted with H2O and
extracted with AcOEt (3ꢂ ). The H2O layer was concentrated to dryness by adding repeatedly MeOH to
remove acid, and the residue was subjected to TLC, which afforded two spots with Rf values of 0.32 and
0.42, compatible with those of standard glucose and arabinose, resp. The residue was then dissolved in
pyridine (0.4 ml) and stirred with l-cysteine methyl ester hydrochloride (5 mg) at 608 for 1.0 h [27]. The
mixture was treated with trimethylsilylation reagent HMDS-TMCS (0.3 ml), and the whole was stirred at
608 for 30 min. The mixture was centrifuged, and a portion (1 ml) of the filtrate was subjected to GLC
analysis. The GLC afforded two peaks with tR values of 7.7 and 15.0 min, which were identical with those
of authentic l-arabinose and d-glucose, resp. Authentic d-arabinose and l-glucose eluted with tR values
of 9.5 and 16.3 min, resp., under the same GLC condition. Acid hydrolysis of compound 7 (4.4 mg) in the
similar way as described above yielded the neutralized H2O extract, which exhibited two TLC spots with
Rf values of 0.32 and 0.47, compatible with those of standard glucose and apiose, resp. The GLC of the
trimethylsilyl thiazolidine derivative of the residue afforded two peaks with tR values of 7.1 and 15.0 min,
which were identical with those of authentic d-apiose and d-glucose, resp.
Determination of Cell Proliferation. Cell proliferation was performed according to the method
reported in [34]. Briefly, B16 cells (5ꢂ103 cells/well) were treated with test samples, dissolved in DMSO
at final concentration of 100, 30, and 10 mm, for 96 h, and then, MTT soln. was added. After 3 h of
incubation, i-PrOH containing 0.08m HCl was added to dissolve the formazan produced in the cells. The
absorbance of each well was then read at 570 nm (reference, 630 nm) by using microplate reader.
Assay of Melanin Content. Assay of melanin content was performed according to the method
reported in [34]. B16 Cells were pre-incubated in the same way as described above. Then, the test
samples, dissolved in DMSO and a-MSH (100 nm), were added to the medium, and the cells were
cultured for 96 h. The medium was removed, and the cells were dissolved in 2m NaOH containing 10%
DMSO. The amount of melanin was determined spectrophotometrically by absorbance at 405 nm using
microplate reader. The optical density of control cells was assumed to be 100%.
Western Blot Analysis. Western blot analysis was performed according to the method reported in [35]
with a slight modification. Briefly, B16 melanoma cells (1ꢂ105 cells) were exposed to test compound (10,
30, and 50 mm) for 48 h. Cells were collected and lased. Lysates of total protein were separated by 15%
sodium dodecyl sulfate (SDS)ꢀpolyacrylamide gels and transferred to polyvinylidene difluoride (PVDF)
membranes. After blocking, the membranes were incubated with anti-MITF, anti-tyrosinase, anti-TRP-1,
anti-TRP-2, and anti-b-actin primary antibodies at 48 overnight. The blots were then detected with

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1229
enhanced chemiluminescence (ECL) plus Western blotting detection system (GE Healthcare, Chalfont St
Giles, UK).
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Received October 31, 2011