Pyrazole alkaloids from watermelon (Citrullus lanatus) seeds

Article abstract of DOI:10.1016/j.phytol.2015.02.017

Abstract Three new compounds, i.e. 1-[2-(5-hydroxymethyl-1H-pyrrole-2-carbaldehyde-1-yl)ethyl]-1H-pyrazole (1), 1-({[5-(α-d-galactopyranosyloxy)methyl]-1H-pyrrole-2-carbaldehyde-1-yl}-ethyl)-1H-pyrazole (2), and (4-hydroxyphenyl)methanol 4-[β-d-apiofuranosyl(1→2)-O-β-d-glucopyranoside] (3), were isolated from an extract of watermelon seeds. Compounds 1 and 2 were pyrazole-alkaloids with a pyrrole ring. This is the first study to show compounds with pyrrole and pyrazole rings in a molecule isolated from natural products. In the evaluation for melanogenesis inhibitory, compound 1 exhibited modest inhibitory activity on melanogenesis without cytotoxicity. Meanwhile compound 2 showed some inhibitory activity accompanied by some cytotoxicity.

Full text of DOI:10.1016/j.phytol.2015.02.017

Phytochemistry Letters 12 (2015) 94–97
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Pyrazole alkaloids from watermelon (Citrullus lanatus) seeds
Takashi Kikuchi, Aoi Ikedaya, Akiko Toda, Kenji Ikushima, Takahiro Yamakawa,
*
Rina Okada, Takeshi Yamada, Reiko Tanaka
Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new compounds, i.e. 1-[2-(5-hydroxymethyl-1H-pyrrole-2-carbaldehyde-1-yl)ethyl]-1H-pyra-
Received 19 January 2015
Received in revised form 16 February 2015
Accepted 20 February 2015
Available online 7 March 2015
zole (1), 1-({[5-(
zole (2), and (4-hydroxyphenyl)methanol 4-[
a
-
D
-galactopyranosyloxy)methyl]-1H-pyrrole-2-carbaldehyde-1-yl}-ethyl)-1H-pyra-
b
-D
-apiofuranosyl(1!2)-O- -glucopyranoside] (3),
b-D
were isolated from an extract of watermelon seeds. Compounds 1 and 2 were pyrazole-alkaloids
with a pyrrole ring. This is the first study to show compounds with pyrrole and pyrazole rings in a
molecule isolated from natural products. In the evaluation for melanogenesis inhibitory, compound 1
exhibited modest inhibitory activity on melanogenesis without cytotoxicity. Meanwhile compound 2
showed some inhibitory activity accompanied by some cytotoxicity.
Keywords:
Citrullus lanatus
Alkaloid
ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Glycoside
Inhibitory activity on melanogenesis
1. Introduction
of compounds 1–3 as well as the inhibitory effects of compounds
from watermelon on melanogenesis in B16 4A5 cells.
Citrullus lanatus (Thunb.) Matsum. and Nakai (Citrullus vulgaris
Schrad, Japanese name: suika) is cultivated throughout the world
for use as a fruit as well as a medicine. Its fruits have been used as a
diuretic, its seed coat has been used to treat hematemesis and
melena, and its kernel has been used as an antihypertensive and in
the treatment of acute cystisis (Shanghai Scientific and Technical
Publishers, Shogakukan, Inc., 1985). Previous studies detected
polyphenols (Lako et al., 2006), flavonols (Lako et al., 2006),
carotenoids (Lako et al., 2006), amino acids (Shanghai Scientific
and Technical Publishers, Shogakukan, Inc., 1985), including
citrulline and arginine, and triterpenes (Ripperger and Seifert,
1975) in the fruits of C. lanatus. The apoptogenic effects of
cucurbitacin L 2-O-glucoside on colon adenocarcinoma, and anti-
inflammatory activities of cucurbitacin E, both isolated from C.
lanatus, have been reported previously (Hassan et al., 2011). We
recently isolated two new cucurbitane-type triterpenes, 24-
2. Results and discussion
Three new compounds (1–3) were isolated from the seeds of C.
lanatus (Fig. 1).
Compound 1 exhibited an [M+H]+ ion in the HR-FAB-MS at m/z
220.1089, which was compatible with the molecular formula
C
11H14N3O2 (calcd 220.1086). Its IR spectrum showed absorption
that indicated a hydroxy group (nmax 3424 cmꢀ1) and carbonyl
group (nmax 1649 cmꢀ1). 1H and 13C NMR spectra indicated the
presence of an oxymethylene [
dH 6.16 (d, J = 4.1 Hz), 7.03 (d, J = 4.1 Hz); dC 111.3 (d), 126.8 (d),
133.4 (s), 145.3 (s)], pyrazole ring [ H 6.19 (t, J = 2.0 Hz), 7.16 (dd,
dH 4.02 (s); dC 55.9 (t)], pyrrole ring
[
d
J = 0.6, 2.0 Hz), 7.49 (dd, J = 0.6, 2.0 Hz); dC 106.9 (d), 131.8 (d),
140.9 (d)], and formyl group [dH 9.45 (s); dC 181.1 (d)]. The
following correlations were observed in the HMBC spectrum; H-10
hydroperoxycucurbita-5,25-dien-3
b-ol and 25-hydroperoxycu-
[
dH 4.52 (t, J = 5.9 Hz)]/C-5 [dC 131.8 (d)]; H-20
J = 5.9 Hz)]/C-200 [ C 133.4 (s)] and C-500 [ C 145.3 (s)]; H-300 [
(d, J = 4.1 Hz)]/C-700 [ C 181.1 (d)]; H-600 [ H 4.02 (s)]/C-400 [
(d)] and 500 [ C 145.3 (s)] (Fig. 2). In the 1H–1H COSY spectrum, H-3
dH 7.49 (dd, J = 0.6, 2.0 Hz)]/H-4 [dH 6.19 (t, J = 2.0 Hz)], H-4/H-5
H 7.16 (dd, J = 0.6, 2.0 Hz)], and H-300 [ H 7.03 (d, J = 4.1 Hz)]/H-400
dH 6.16 (d, J = 4.1 Hz)] were observed (Fig. 2). Therefore, 1
[
dH 4.71 (t,
H 7.03
C 111.3
curbita-5,23-dien-3 -ol, that had hydroperoxy groups on their
b
d
d
d
d
d
side chains from the Et2O fraction of a C. lanatus seed MeOH
extract, and examined their cytotoxic activities against the HL-60,
P388, and L1210 cell lines, and melanogenesis inhibitory activities
d
d
[
[
[
in
a-MSH-stimulated B16 4A5 cells (Kikuchi et al., 2013a). In the
d
d
present study, we describe the isolation and structural elucidation
was established as 1-[2-(5-hydroxymethyl-1H-pyrrole-2-carbal-
dehyde-1-yl)ethyl]-1H-pyrazol (Table 1).
Compound 2 exhibited an [M+H]+ ion in the HR-FAB-MS at m/z
382.1617, which was compatible with the molecular formula
*
Corresponding author. Tel.: +81 72 690 1084; fax: +81 72 690 1084.
E-mail address: tanakar@gly.oups.ac.jp (R. Tanaka).
http://dx.doi.org/10.1016/j.phytol.2015.02.017
1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

T. Kikuchi et al. / Phytochemistry Letters 12 (2015) 94–97
95
Fig. 1. Structures of compounds 1–3 from watermelon seeds.
C17H24N3O7 (calcd for 382.1609). Its IR spectrum showed
absorption that indicated hydroxy groups (3408 cmꢀ1) and a
carbonyl group (1656 cmꢀ1). 1H NMR and 13C NMR spectra
suggested compound 2 was a glycoside of 1 since the signals of 2
were similar to those of 1, except for the sugar moiety. This was
confirmed by HMBC and 1H–1H COSY spectra (Fig. 2). The 1H and
(d)]/C-10; H-30 [
d
H 7.04 (d)]/C-40 [
d
c 158.4 (s)]; H-100 [
d
H 4.95 (d)]/C-
40; H-1000 H 5.46 (d)]/C-200 c 78.6 (d)], C-3000; H-500/C-100, C-400; H-
[d
[d
600/C-400; H-2000/C-3000; H-4000/C-3000; H-5000/C-3000(Fig. 2). On acid
hydrolysis, afforded -glucose and -apiose, which were
identified by derivatization with -cysteine methyl ester hydro-
3
D
D
L
chloride and o-tolylisothiocyanate, and HPLC analysis. Thus,
compound 3 was established as (4-hydroxyphenyl)methanol 4-
13C NMR signals of the sugar moiety [
(d), 71.1 (d), 71.3 (d), 72.9 (d), 99.5 (d)] suggested the presence of a
terminal
-galactopyranose, and its configuration at C-1000 was
established as the -orientation due to the coupling constant of H-
1000 (J = 4.1 Hz). This was confirmed by acid hydrolysis, derivatiza-
tion with -cysteine methyl ester hydrochloride and o-tolyli-
sothiocyanate, and HPLC analysis. Therefore, compound 2 was
established as 1-({[5-( -galactopyranosyloxy)methyl]-1H-pyr-
role-2-carbaldehyde-1-yl}-ethyl)-1H-pyrazol (Table 1). Compounds
1 and 2 are rare pyrazole-alkaloids. Kumar et al. (2013) isolated
amino- -(pyrazolyl-N)-propanoic acid, a naturally occurring amino
dH 4.79 (d); dC 62.8 (t), 70.0
[b
-D
-apiofuranosyl(1!2)-O-
Compounds 1–3 and the positive control, arbutin were
evaluated for there melanogenesis inhibitory effects against a-
b-D-glucopyranoside] (Table 1).
D
a
MSH-induced melanogenesis in B16 4A5 melanomas (Table 2). To
determine safe concentrations, the cytotoxicities of three com-
pounds were examined using the MTT assay. Compound 1 did not
exhibit cytotoxicity against B16 4A5 cells at 3–100
modestly inhibited melanogenesis in B16 4A5 cells (melanin
content: 80.3% at 30 M, 64.1% at 100 M). This compound may
L
a-D
mM. It also
L-a-
m
m
b
be valuable in the development of skin-whitening agents.
acid with a pyrazole ring, from the juice of watermelons. The
pyrazole rings of 1 and 2 may be derived from this amino acid. This is
the firststudyto show compounds withpyrrole and pyrazolerings in
a molecule isolated from natural products; however, pyrrole–
imidazole alkaloids have been isolated from the Mediterranean
sponge (Fattorusso and Taglialatela-Scafati, 2000).
Compound 2 exhibited some melanogenesis inhibitory activity
(melanin content: 86.0% at 30
this was attributed to its cytotoxic effects (cell viability: 92.7% at
30 M, 70.4% at 100 M).
mM, 48.6% at 100 mM); however,
m
m
3. Experimental
Compound 3 exhibited an [M+Na]+ ion in the HR-FAB-MS at m/z
441.1375, which had the molecular formula of C18H26O11Na (calcd
441.1372). Its IR spectrum revealed absorption bands indicating
hydroxy groups (nmax 3392 cmꢀ1). The 13C NMR spectrum of 3
3.1. General
Chemicals and reagents were purchased as follows: fetal bovine
serum (FBS) from Invitrogen Co. (Carlsbad, CA, USA), 3-(4,5-
displayed dC 62.5 (t), 71.4 (d), 78.0 (d), 78.6 (d), 78.7 (d), 101.0 (d)
attributed to inner glucose, and dC 66.1 (t), 75.5 (t), 78.1 (d), 80.8
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide
(s), 110.8 (d) attributed to terminal apiose. In the 1H NMR
(MTT) from Sigma–Aldrich Japan Co. (Tokyo, Japan), Dulbecco’s
modified Eagle’s medium (D-MEM), and antibiotics from Nacalai
Tesque, Inc. (Kyoto, Japan). All other chemicals and reagents were
of analytical grade. Optical rotations were measured with a JASCO
DIP-1000 digital polarimeter. IR spectra were recorded on a Perkin-
Elmer 1720X FTIR spectrophotometer. The 1H (600 MHz) and 13C
spectrum, the A2B2-type aromatic proton signals, [dH 7.04 (d, 8.8)
and 7.27 (d, 8.8)] and hydroxy methylene signal [dH 4.53 (s);
d
c
64.8 (t)] suggested the presence of a hydroxy methyl phenyl
moiety. The following correlations were observed in the HMBC
spectrum: H-1 [dH 4.53 (s)]/C-10 dc 136.5 (s)]; H-20
[ [dH 7.27
Fig. 2. Key HMBC (
) and 1H–1H COSY (
) correlations of compounds 1–3.

96
T. Kikuchi et al. / Phytochemistry Letters 12 (2015) 94–97
Table 1
1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compounds 1–3 (CD3OD).a
No.
1
2
3
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1
3
64.8
t
4.53 s
140.9
106.9
131.8
53.4
d
d
d
t
7.49 dd (0.6, 2.0)
6.19 t (2.0)
140.9
106.9
131.9
53.2
d
d
d
t
7.50 dd (0.9, 2.1)
6.19 t (2.1)
4
5
7.16 dd (0.6, 2.0)
4.52 t (5.9)
7.23 dd (0.9, 2.1)
4.53 t (5.9)
10
20
30
40
50
60
100
200
300
400
500
600
136.5
129.5
117.4
158.4
117.4
129.5
101.0
78.6
s
47.3
t
4.71 t (5.9)
47.4
t
4.75 td (2.0, 5.8)
d
d
s
7.27 d (8.8)
7.04 d (8.8)
d
d
d
d
d
d
d
t
7.04 d (8.8)
7.27 d (8.8)
4.95 d (7.6)
3.63 m
133.4
126.8
111.3
145.3
55.9
s
d
d
s
t
133.8
126.2
113.1
141.0
60.7
s
d
d
s
t
7.03 d (4.1)
6.16 d (4.1)
7.03 d (4.1)
6.26 d (4.1)
78.7
3.58 t (8.5)
71.4
3.39 dd (8.5, 9.7)
3.41 m
78.0b
62.5
4.02 s
9.45 s
A
B
4.03 d (12.9)
4.23 d (12.9)
9.47 s
A
B
3.68 dd (5.3, 12.0)
3.88 dd (2.0, 12.0)
700
1000
2000
3000
4000
181.1
d
181.3
99.5
70.0
71.3
71.1
d
d
d
d
d
4.79 d (4.1)
110.8
78.1b
80.8
d
d
s
5.46 d (1.5)
3.94 d (1.5)
3.77 dd (4.2, 10.3)
3.71 brd (10.3)
3.86 dd (1.2, 3.5)
75.5
t
A
B
A
B
3.78 d (9.5)
4.05 d (9.5)
3.51 d (11.4)
3.54 d (11.4)
5000
6000
72.9
62.8
d
t
3.76 brd (11.7)
66.1
t
A
B
3.68 dd (4.9, 11.1)
3.72 dd (6.7, 11.1)
a
Assignments were based on 1H–1H COSY, HMQC, HMBC, and NOESY spectroscopic data.
Interchangeable.
b
(150 MHz) NMR spectra were recorded on an Agilent vnmrs 600 in
CP3OD with tetramethylsilane as the internal standard. HR-FAB-
MS was recorded on a JEOL JMS-7000 mass spectrometer. Silica gel
60 (230–400 mesh, Nacalai Tesque, Inc., Kyoto, Japan) Diaion HP-
20 (Mitsubishi Chemical Co., Tokyo, Japan), octadecylsilica gel
(ODS;100-200 mesh; Fuji Siliya Chemical, Ltd., Aichi, Japan), and
sephadex LH-20 CGE healthcare Bio-Sciences AB, Uppsala, Sweden).
Were used for column chromatography. HPLC [PU-2086 pump and
RI-2031 refractive index detector (Jasco, Tokyo, Japan)] was carried
out on an ODS column [Cosmosil 5C18-PAQ column (Nacalai Tesque,
Inc., Kyoto, Japan), 25 cm ꢁ 20 mm i.d.] at 35 8C with H2O/MeCN
[20:1 (HPLC system I), 90: 10 (HPLC system II), 85: 15 (HPLC system
III), flow rate 4.0 mL/min].
3.3. Extraction and isolation
The seeds of C. lanatus (10 kg) were pulverized in juice mixer,
and subjected to extraction with MeOH under reflux (10 L, 1 week
4ꢁ). The MeOH extract (250 g) was then partitioned between Et2O
and H2O (1:1, v/v), followed by n-BuOH and H2O (1:1, v/v). The n-
BuOH fraction (17 g) was subjected to Diaion HP-20 CC [Diaion HP-
20 (330 g)] resulting in H2O Fr. (4.0 g), MeOH Fr. (12.1 g), and
acetone Fr. (0.81 g) were eluted with H2O, MeOH, and acetone,
respectively]. A portion of MeOH Fr. (11.7 g) was subjected to
silica-gel CC [silica-gel (200 g)] to yield 5 fractions, A–E [CHCl3/
MeOH (1:1) ! (7:3) ! (9:1) ! (1:0)]. Among them, Fr. C (5.71 g)
was subjected to ODS CC [ODS (50 g)] to yield 6 fractions, C1–C6
[MeOH/H2O (1:1) ! (7:3) ! (9:1) ! (1:0)]. C-1, eluted with
MeOH/H2O (1:1), was carried out ODS CC [ODS (50 g)] to give
sixteen fractions: C1-1–C1-16. C1-5 (312.02 mg) was fractionated
with preparative HPLC (HPLC system III) to nineteen fractions, C1-5-
1–C1-5-19. Purification of C1-5-13 gave 2 (1.47 mg; tR 232.0 min,
HPLC system II). Preparative HPLC of C1-9 (62.6 mg), which was
eluted with MeCN/H2O (1:9), gave 1 (2.1 mg; tR 66.4 min)(HPLC
system III). Fr. D (4.51 g) was subjected to ODS CC [ODS (50 g)] to
3.2. Plant material
The seeds of C. lanatus, obtained in 2011, in the USA, were
purchased from Takada Seeds Co., Ltd. in 2011. A voucher specimen
(CL-S-2011-01) was deposited in the Herbarium of the Laboratory of
Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.
Table 2
yield
6
fractions, D1–D7 [MeOH/H2O (1:1) ! (9:1) ! (1:0)],
Melanogenesis inhibitory activities of compounds 1–3 from watermelon seeds.a,b
followed by D1 (3.12 g) was carried out re-column chromatogra-
phy with ODS to give D1-1–D1-9. Fr. D1-1 (1.67 g) was subjected to
shephadex LH-20 CC [sephadex LH-20 (40 g)] to yield 6 fractions,
D1-1-1–D1-1-7 [MeOH/H2O (0:1) ! (1:0)]. Preparative HPLC of
D1-1-2 (466.6 mg), which was eluted with H2O, 3 (3.3 mg; tR
104.6 min) (HPLC system I).
Melanin content (%)
3
m
M
10
mM
30
m
M
100 mM
1
119.3 ꢂ 5.1*
103.9 ꢂ 0.7
106.6 ꢂ 2.3
98.6 ꢂ 2.5
94.1 ꢂ 9.8
99.7 ꢂ 1.7
100.9 ꢂ 1.6
85.7 ꢂ 6.7*
80.3 ꢂ 9.3*
86.0 ꢂ 2.4**
90.5 ꢂ 1.6*
66.6 ꢂ 6.1**
64.1 ꢂ 5.8**
48.6 ꢂ 0.6**
65.9 ꢂ 2.1**
45.2 ꢂ 2.0**
2
3
Arbutinc
a
3.4. Acid hydrolysis and determination of sugar configuration
Melanin content (%) were determined based on absorbances at 405 nm, and
comparisons with values for vehicle (DMSO) control (100%). Each value represents
the mean ꢂ standard error (S.E.) of three determinations. The concentration of DMSO
in the sample solution was 2 mL/mL. Significant differences from the control group
Determination of the absolute sugar configuration was carried
out according to a method reported previously with a little
modification (Tanaka et al., 2007). A solution of 2 (1 mg) and 3
(1 mg), respectively, in 2 M CF3COOH (1 mL) was heated under
reflux for 3 h. The mixture was extracted with EtOAc (3 times), and
were shown as *p < 0.05, and **p < 0.01.
b
Compound 2, 3, and arbutin exhibited some cytotoxicities at 100
viability 2: 70.4 ꢂ 1.9%; 3; 88.4 ꢂ 0.5%; arbutin: 86.2 ꢂ 1.2%).
mM (cell
c
Positive control.

T. Kikuchi et al. / Phytochemistry Letters 12 (2015) 94–97
97
the H2O layer was concentrated to dryness. The residue was then
dissolved in pyridine (0.1 mL) and stirring with -cystein methyl
[M+H]+; HR-FAB-MS m/z: 220.1089 (calcd for 220.1086:
C11H14N3O2).
L
ester hydrochloride (0.5 mg) at 60 8C for 1 h. The reaction mixture
was treated with o-tolylisothiocyanate, and heated at 60 8C for 1 h.
The reaction mixture was analyzed by reversed-phase HPLC
3.9. 1-({[5-(
a-D-Galactopyranosyloxy)methyl]-1H-pyrrole-
2-carbaldehyde-1-yl}-ethyl)-1H-pyrazole (2)
[column: Cosmosil
250 mm ꢁ 4.6 mm i.d. (5
AcOH (2:8, v/v); detection: refractive index; flow rate: 1.0 mL/min;
column temperature: 35 8C] to identify the derivatives of
galactose in 2, and -glucose and -apiose in 3 by comparison of
their retention times with those of authentic samples (tR: -glucose
-galactose
5
C18-PAQ column (Nacalai Tesque),
EtOH
nm
max
m
m); mobile phase: MeCN–H2O in 1%
Amorphous solid, ½a D27
ꢃ
; 62.4 (c = 0.062, EtOH); UV
l
KBr
(log e): 206.5 (3.66), 256.5 (3.84), 282.0 (3.84), 294.0 (3.70); IR n
max
D
-
cmꢀ1: 3408, 2922, 1743, 1723, 1656, 1640, 1400, 1353, 1400, 1281,
1029; FAB-MS m/z: 382 [M+H]+; HR-FAB-MS m/z: 382.1617 (calcd
for 382.1609: C17H24N3O7).
D
D
D
28.4 min,
22.4 min,
L
-glucose 26.0 min,
D-apiose 45.4 min, D
L
-galactose 23.3 min).
3.10. (4-Hydroxyphenyl)methanol 4-[
b-D-apiofuranosyl
(1 ! 2)-O- -glucopyranoside] (3)
b
-D
3.5. Cell culture
EtOH
nm
max
Amorphous solid; ½a D27
ꢃ
ꢀ85.6 (c = 0.097, EtOH); UV
l
cmꢀ1: 3392, 1612, 1509,
KBr
max
The cell line B16 4A5 cells were grown in Dulbecco’s modified
Eagle medium (D-MEM). The medium was supplemented with 10%
(log
e
): 221.5 (3.95), 270.5 (3.11); IR
n
1225, 1076; FAB-MS m/z: 441 [M+Na]+; HR-FAB-MS m/z: 441.1375
FBS and antibiotics (100 units/mL penicillin and 100
m
g/mL
(calcd for 441.1372: C18H26O11Na).
streptomycin). The cells were incubated at 37 8C in a 5% CO2
humidified incubator.
Acknowledgments
3.6. Viability of melanoma cells
We thank Dr. Katsuhiko Minoura and Dr. Mihoyo Fujitake
(Osaka University of Pharmaceutical Sciences) for the NMR and MS
measurements.
Melanoma cells proliferation was examined according to a
method reported previously (Yamada et al., 2011), with a little
modification. Briefly, B16 4A5 cells [obtained from Riken Cell Bank
Appendix A. Supplementary data
(Tsukuba, Ibaraki, Japan)] (3 ꢁ 103 cells in 100
mL), preincubated
for 24 h, were treated for 72 h with test samples dissolved in
dimethyl sulfoxide (DMSO) at a final concentration of 100, 30, 10,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2015.02.017.
and 3
mM, and MTT solution was added. After 3 h of incubation,
20% NDS containing 0.1 M HCl was added to dissolve the formazan
produced in the cells. The absorbance of each well was read at
570 nm using a microplate reader.
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The assay of melanin content was performed as described
previously (Kikuchi et al., 2013b), with a little modification. B16
4A5 cells (3 ꢁ 104 cells in 1000
mL) were pre-incubated in a-MSH
(100 nM) containing medium. Test samples dissolved in DMSO
were added to the medium and the cells were cultured for 72 h.
The medium was removed and the cells were dissolved in 2 M
NaOH containing 10% DMSO. The amount of melanin was
determined spectrophotometrically by measuring absorbance at
405 nm using a microplate reader. The optical density of control
cells was assumed to be 100%.
Ripperger, H., Seifert, K., 1975. Cucurbitacins from Citrullus lanatus. Tetrahedron 31,
1561–1563.
3.8. 1-[2-(5-Hydroxymethyl-1H-pyrrole-2-carbaldehyde-1-
Shanghai Scientific and Technical Publishers, Shogakukan, Inc. (Eds.), Chuyaku-
daijiten, vol. II. Shogakukan, Inc., Tokyo, pp. 1374–1375.
yl)ethyl]-1H-pyrazole (1)
Tanaka, T., Nakashima, T., Ueda, T., Tomii, K., Kouno, I., 2007. Facile discrimination of
aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 55, 899–901.
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2011. New class azaphilone produced by a marine fish-derived Chaetomium
globosum. The stereochemistry and biological activities. Bioorg. Med. Chem. 19,
4106–4113.
EtOH
max
Amorphous solid, ½a D27
ꢃ
ꢀ2.4 (c = 0.055, EtOH); UV
l
nm
(log e
): 219.0 (3.33), 255.0 (3.26), 292.5 (3.58); IR nKmBarx cmꢀ1: 3480,
3424, 3354, 1649, 1585, 1377, 1157, 1089; FAB-MS m/z: 220