Acylated sucroses and acylated quinic acids analogs from the flower buds of Prunus mume and their inhibitory effect on melanogenesis

Article abstract of DOI:10.1016/j.phytochem.2013.04.012

The methanolic extract from the flower buds of Prunus mume, cultivated in Zhejiang Province, China, showed an inhibitory effect on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From the methanolic extract, five acylated sucroses, mumeoses A-E, and three acylated quinic acid analogs, 5- O-(E)-p-coumaroylquinic acid ethyl ester, and mumeic acid-A and its methyl ester, were isolated together with 13 known compounds. The chemical structures of the compounds were elucidated on the basis of chemical and physicochemical evidence. Inhibitory effects of the isolated compounds on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were also investigated. Acylated quinic acid analogs substantially inhibited melanogenesis. In particular, 5-O-(E)-feruloylquinic acid methyl ester exhibited a potent inhibitory effect [inhibition (%): 21.5 ± 1.0 (P < 0.01) at 0.1 μM]. Moreover, its biological effect was much stronger than that of the reference compound, arbutin [inhibition (%): 10.6 ± 0.6 (P < 0.01) at 10 μM]. Interestingly, the obtained acylated quinic acid analogs displaying melanogenesis inhibitory activity showed no cytotoxicity [cell viability >97% at 10 μM]. It is concluded that acylated quinic acid analogs are promising therapeutic agents for the treatment of skin disorders.

Full text of DOI:10.1016/j.phytochem.2013.04.012

Phytochemistry 92 (2013) 128–136
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Acylated sucroses and acylated quinic acids analogs from the flower buds
q
of Prunus mume and their inhibitory effect on melanogenesis
Seikou Nakamura, Katsuyoshi Fujimoto, Takahiro Matsumoto, Souichi Nakashima, Tomoe Ohta,
⇑
Keiko Ogawa, Hisashi Matsuda, Masayuki Yoshikawa
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The methanolic extract from the flower buds of Prunus mume, cultivated in Zhejiang Province, China,
showed an inhibitory effect on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From
the methanolic extract, five acylated sucroses, mumeoses A–E, and three acylated quinic acid analogs, 5-
O-(E)-p-coumaroylquinic acid ethyl ester, and mumeic acid-A and its methyl ester, were isolated together
with 13 known compounds. The chemical structures of the compounds were elucidated on the basis of
chemical and physicochemical evidence. Inhibitory effects of the isolated compounds on melanogenesis
in theophylline-stimulated B16 melanoma 4A5 cells were also investigated. Acylated quinic acid analogs
substantially inhibited melanogenesis. In particular, 5-O-(E)-feruloylquinic acid methyl ester exhibited a
Received 28 December 2012
Received in revised form 1 March 2013
Accepted 22 April 2013
Available online 18 May 2013
Keywords:
Prunus mume
Rosaceae
potent inhibitory effect [inhibition (%): 21.5 ± 1.0 (P < 0.01) at 0.1
much stronger than that of the reference compound, arbutin [inhibition (%): 10.6 ± 0.6 (P < 0.01) at
10 M]. Interestingly, the obtained acylated quinic acid analogs displaying melanogenesis inhibitory
activity showed no cytotoxicity [cell viability >97% at 10 M]. It is concluded that acylated quinic acid
analogs are promising therapeutic agents for the treatment of skin disorders.
Ó 2013 Elsevier Ltd. All rights reserved.
lM]. Moreover, its biological effect was
Mumeose
Mumeic acid
Medicinal flower
Melanogenesis inhibitor
l
l
1
. Introduction
As a continuation of our studies on inhibitors of melanogenesis de-
rived from medicinal flowers, it was found that a methanolic
(MeOH) extract from flower buds of Chinese P. mume showed
inhibitory effects on melanogenesis. From the MeOH extract, five
new acylated sucroses, mumeoses A (1), B (2), C (3), D (4), and E
(5), and three new acylated quinic acid analogs, 5-O-(E)-p-couma-
roylquinic acid ethyl ester (6), mumeic acid-A (7), and mumeic
acid-A methyl ester (8) were isolated, together with 13 known
compounds (Fig. 1). In this paper, the isolation and structural
elucidation of 1–8 are described, as well as inhibitory effects of
acylated quinic acid analogs on melanogenesis in theophylline-
stimulated B16 melanoma 4A5 cells.
Prunus (P.) mume SIEB. et ZUCC. (Rosaceae) has been widely cul-
tivated as an ornamental plant in Japan, Taiwan, China, and Korea,
and its fruit has been used as a food garnish (pickled ume) and
drink (ume brandy). In addition, the flowers, immature fruit,
leaves, branches, seeds, and roots of P. mume have been exploited
as traditional Chinese medicines. In particular, the flowers have
been prescribed for treatment of skin disorders and eye pain and
for detoxification, stomachic, expectorant, and sedative purposes.
Previous studies have focused on the constituents from P. mume
and their associated bioactivities [e.g., anticancer activity (Jeong
et al., 2006), effect on adrenocorticotropic hormone and catechol-
amine levels in plasma (Ina et al., 2004), radical scavenging activity
2
. Results and discussion
(
(
Matsuda et al., 2003), inhibitory activity on squalene synthase
Choi et al., 2007) and inhibitory activity on aldose reductase and
2.1. Isolation of compounds from the flower buds of P. mume
platelet aggregation (Yoshikawa et al., 2002)].
Identifying inhibitors of melanin production derived from natu-
ral medicines is of interest (Fujimoto et al., 2012; Matsuda et al.,
A MeOH extract of the dried flower buds (30.4%) of P. mume
(
cultivated in Zhejiang Province, China) showed melanogenesis
inhibitory activity [inhibition (%): 31.1 ± 1.1 (P < 0.01) at 100 g/
mL]. The MeOH extract was partitioned into an EtOAc–H O (1:1,
2
009; Nakamura et al., 2010, 2012a,b; Nakashima et al., 2010).
l
2
q
v/v) mixture to furnish an EtOAc-soluble fraction (6.6%) and an
aqueous layer. The latter was further extracted with 1-butanol to
give 1-butanol- (7.5%) and H O- (13.0%) soluble fractions. The
2
This paper is No. 41 in our series ‘‘Medicinal Flowers’’.
Corresponding author. Tel.: +81 75 959 4633; fax: +81 75 595 4768.
E-mail address: myoshika@mb.kyoto-phu.ac.jp (M. Yoshikawa).
⇑
0
031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.04.012

S. Nakamura et al. / Phytochemistry 92 (2013) 128–136
129
Fig. 1. Structures of compounds isolated from the flower buds of P. mume.
0
00
1
-butanol- and the EtOAc-soluble fractions were found to have sig-
nificant inhibitory effects on melanogenesis [inhibition (%):
7.5 ± 6.8 (P < 0.01), 41.5 ± 1.9 (P < 0.01), respectively at 100 g/
mL], but the H O-soluble fraction showed no detectable effect even
at 100 g/mL. The 1-butanol- and the EtOAc-soluble fraction were
3 -O-(2 -O-acetyl)-b-
D
-glucopyranoside (15, 0.0016%), quercetin
0
0
3-O-(6 -O-acetyl)-b-
2008), isorhamnetin 3-O-b-
and Häberlein, 1999), quercetin 3-O-(6 -O-benzoyl)-b-
pyranoside (18, 0.00059%) (Singh et al., 2009), isorhamnetin
3-O-b- -galactopyranoside (19, 0.0006%) (Hsich et al., 2004), and
quercetin (0.0068%). In this case, acylated sucroses, prunoses I
(21), II (22), III (23), which were isolated from the flower buds of
Japanese P. mume (Yoshikawa et al., 2002; Matsuda et al., 2003),
were not detected in the flower buds of Chinese P. mume.
D
-glucopyranoside (16, 0.0010%) (Wang et al.,
2
l
D
-glucopyranoside (17, 0.017%) (Beck
0
0
2
D-galacto-
l
then subjected to normal- and reversed-phase silica-gel column
chromatography and repeated HPLC. From the 1-butanol-soluble
fraction, three new acylated sucroses, mumeose A (1, 0.00032%),
B (2, 0.0010%), and C (3, 0.00050%), and a new acylated quinic acid
analog, 5-O-(E)-p-coumaroylquinic acid ethyl ester (6, 0.0031%),
were isolated together with eight known compounds, 5-O-(E)-p-
coumaroyl quinic acid (9, 0.015%) (Parejo et al., 2004), chlorogenic
acid (10, 0.11%) (Yoshikawa et al., 1999), 5-O-(E)-p-coumaroyl qui-
nic acid methyl ester (11, 0.0013%) (Jaiswal and Kuhnert, 2011),
chlorogenic acid methyl ester (12, 0.11%) (Zhu et al., 2005), 5-O-
D
2.2. Structures of mumeoses A–E (1–5), 5-O-(E)-p-coumaroylquinic
acid ethyl ester (6), mumeic acid-A (7), and mumeic acid-A methyl
ester (8)
(
E)-feruloylquinic acid methyl ester (13, 0.0013%) (Smarrito et al.,
Mumeose A (1) was isolated as a white amorphous powder with
1
5
2
1
0
0
008), chlorogenic acid ethyl ester (14, 0.038%) (Abe and Marumo,
positive optical rotation (1: [
a
]
D
+114.8, in MeOH). Its IR spec-
0
00
972), quercetin 3 -O-(2 -O-acetyl)-b-
.0011%) (Machida et al., 2009), and
.047%). From the EtOAc-soluble fraction, two new acylated suc-
D
-glucopyranoside (15,
trum showed absorption bands at 3400, 1730, 1697, 1603, 1515,
ꢀ1
D
-mandelic acid (20,
and 1033 cm due to hydroxy, ester, a,b-unsaturated ester, aro-
matic ring, and ether functions. FABMS in the positive-ion mode
+
roses, mumeoses D (4, 0.00047%) and E (5, 0.00023%) were iso-
lated, as well as two new acylated quinic acid analogs, mumeic
acid-A (7, 0.0039%) and mumeic acid-A methyl ester (8, 0.0034%),
together with seven known compounds, 5-O-(E)-p-coumaroylqui-
nic acid methyl ester (11, 0.0014%), chlorogenic acid methyl ester
gave a quasimolecular ion peak ([M+Na] ) at m/z 553, from which
the molecular formula C23
tion (HR) MS. Treatment of 1 with a 10% aqueous KOH-1,4-dioxane
(1:1, v/v) mixture yielded -sucrose, which was identified by com-
parison of the retention time and optical rotation (t : 19.8 min
with positive rotation) with that of an authentic sample on
30
H O14 was determined by high-resolu-
D
R
(12, 0.016%), chlorogenic acid ethyl ester (14, 0.0027%), quercetin

1
30
S. Nakamura et al. / Phytochemistry 92 (2013) 128–136
reversed-phase HPLC analysis using an optical rotation detector.
Acid hydrolysis of 1 with a 5% aqueous H SO -1,4-dioxane yielded
-glucose and fructose together with (E)-p-coumaric acid. -Glu-
cose was changed to the tolylthiocarbamoyl thiazolidine derivative
by reaction with -cysteine methyl ester and o-torylisothiocyanate
and identified by comparison of its retention time (t : 19.7 min)
with that of authentic sample on reversed-phase HPLC analysis
Tanaka et al., 2007). (E)-p-Coumaric acid was also identified by
comparison of its retention time (t : 17.1 min) with that of an
authentic sample on reversed-phase HPLC analysis. The H NMR
Mumeoses B (2), C (3), and D (4), which were obtained as white
15
2
4
amorphous powders with positive optical rotations (2: [a]
D
1
5
15
D
D
+46.9, 3: [
bands due to hydroxy, ester,
32 15
tionalities in their IR spectra. Their molecular formulas (C25H O
a
]
D
+54.0, 4: [
a
]
D
+6.0, in MeOH), showed absorption
a
,b-unsaturated ester, and ether func-
L
R
34 38
of 2, C27H O16 of 3, C31H O18 of 4) were determined from the
quasimolecular ion peaks (m/z 595 [M+Na] for 2, 637 [M+Na]+
+
+
(
for 3, 721 [M+Na] for 4) in the positive-ion FABMS and by HRMS
R
measurement. Basic hydrolysis of 2–4 with a 10% aqueous KOH-
1
1,4-dioxane (1:1, v/v) mixture yielded
of 2–4 with a 5% aqueous H SO -1,4-dioxane yielded
and fructose together with (E)-p-coumaric acid, as well as 1. The
D-sucrose. Acid hydrolysis
1
3
(
methanol-d
4
) and C NMR (Table 1) spectra of 1, which were as-
2
4
D-glucose
signed by various NMR experiments (DEPT, DQF COSY, HMQC and
HMBC spectra), showed signals assignable to an acetyl group [d
1
13
H NMR (methanol-d
4
) and C NMR (Table 1) spectra of 2, which
2
.07 (s, CH
3
CO–)] and a (E)-p-coumaroyl group [d 6.43 (d,
were assigned by various NMR experiments, showed signals
00
00
00
J = 16.2 Hz, H-8 ), 6.81 (d, J = 8.6 Hz, H-3 , 5 ), 7.51 (d, J = 8.6 Hz,
H-2 , 6 ), 7.70 (d, J = 16.2 Hz, H-7 )] and a sucrose moiety [d 3.43
assignable to two acetyl groups [d 1.98, 2.08 (s, CH
(E)-p-coumaroyl group [d 6.43 (d, J = 15.8 Hz, H-8 ), 7.71 (d,
3
CO– ꢁ 2)], a
0
0
00
00
00
0
0
00
00
00
(
m, H-4 ), 3.45, 3.61 (both d-like, H
2
-1), 3.74 (m, H-6 a), 3.78 (m,
J = 15.8 Hz, H-7 ), 7.53 (d, J = 8.6 Hz, H-2 , 6 ), 6.61 (d, J = 8.6 Hz,
0
0
0
00
00
0
H
2
-6, 3 ), 3.89 (m, H-5, 5 , 6 b), 4.36 (dd, J = 8.3, 8.3 Hz, H-4), 4.56
H-3 , 5 )] and a sucrose moiety [d 3.46 (dd, J = 9.4, 9.4, H-4 ),
-1, H-2 ), 3.83 (m, H
H-5), 4.18 (m, H-5 , 6 a), 4.34 (dd, J = 7.9, 7.9 Hz, H-4), 4.46 (m H-
6 b), 5.18 (dd, J = 9.4, 9.4, H-3 ), 5.49 (d, J = 7.9 Hz, H-3), 5.50 (d,
J = 3.1 Hz, H-1 )]. The proton and carbon signals in the H and
0
0
(
dd, J = 3.7, 10.1 Hz, H-2 ), 5.47 (d, J = 8.3 Hz, H-3), 5.55 (d,
3.59 (m, H
2
2
-6), 3.94 (ddd, J = 3.1, 3.1, 7.9 Hz,
0
0
0
J = 3.7 Hz, H-1 )]. The linkage between two monosaccharides, as
well as the positions of the acetyl group and (E)-p-coumaroyl
moiety, were confirmed based on heteronuclear multiple bond
connectivity (HMBC) spectroscopy. Namely, long-range correla-
tions were observed between the following proton and carbon
pairs: H-3 and C-9 , H-1 and C-2, H-2 and an acetyl carbonyl
carbon. On the basis of this evidence, the chemical structure of
mumeose A (1) was determined to be 2 -O-acetyl-3-O-(E)-p-
0
0
0
1
13
C
NMR (Table 1) spectra of 2 were superimposable on those of 1, ex-
cept for the resonances around the glucose part of 2. The linkage
between two monosaccharides, as well as the positions of two
acetyl groups and a (E)-p-coumaroyl moiety, were confirmed based
on HMBC spectroscopy. Namely, long-range correlations were
observed between the following proton and carbon pairs: H-3
00
0
0
0
coumaroylsucrose.
Table 1
1
H and 13C NMR (125 MHz) spectroscopic data for 1–5 in CD
3
OD (d in ppm, J in Hz).
Position
1
2
3
4
5
d
C
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
d
H
Sucrose part
1
63.1
3.45 d like
65.3
3.59 m
64.5
3.45 d (11.6)
3.60 d (11.6)
–
5.53 d (8.6)
4.32 dd (8.6, 8.6)
3.92 m
3.81 m
5.62 d (3.7)
4.73 dd (3.7, 10.1)
5.32 dd (10.1, 10.1) 71.3
3.56 dd (10.1, 10.1) 70.0
4.17 m
4.20 m
4.47 m
66.3
4.09 d (11.7)
4.21 d (11.7)
–
5.38 d (7.8)
4.31 dd (7.8, 7.8)
3.93 m
3.78 m
5.68 d (3.6)
4.88 m
5.39 dd (10.1, 10.1) 71.3
4.98 dd (10.1, 10.1) 69.8
4.34 m
4.18 m
66.0
4.20 m
3
–
.61 d like
2
3
4
5
6
105.2
78.9
73.6
83.9
64.7
90.9
74.6
72.2
71.3
74.2
62.3
104.9
79.5
74.1
84.2
63.7
92.9
–
105.4
78.6
73.9
103.9
79.8
73.9
84.5
63.4
90.6
71.6
103.8
79.2
73.4
84.4
63.2
90.3
71.6
–
5.47 d (8.3)
4.36 dd (8.3, 8.3)
3.89 m
3.78 m
5.55 d (3.7)
5.49 d (7.9)
4.34 dd (7.9, 7.9)
3.94 ddd (3.1, 3.1, 7.9) 84.2
3.83 m
5.50 d (3.1)
3.59 m
5.18 dd (9.4, 9.4)
3.46 dd (9.4, 9.4)
4.18 m
5.37 d (8.0)
4.25 dd (8.0, 8.0)
3.89 m
3.76 m
5.65 d (3.9)
4.84 m
5.34 dd (9.9, 9.9)
4.99 m
4.14 m
63.7
90.5
72.2
73.7
69.7
71.8
64.6
0
1
0
2
4.56 dd (3.7, 10.1) 71.3
0
3
4
3.78 m
3.43 m
3.89 m
3.74 m
76.9
69.8
72.1
65.0
0
0
5
69.7
63.6
69.6
63.3
0
6
4.18 m
4.46 m
4.09 m
3.89 m
p-Coumaroyl part
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
7
8
9
127.2
–
127.3
–
127.2
–
127.1
–
127.5
–
00
00
,6
,5
131.5 7.51 d (8.6)
116.8 6.81 d (8.6)
161.5
147.6 7.70 d (16.2)
114.5 6.43 d (16.2)
131.5 7.53 d (8.6)
116.7 6.61 d (8.6)
161.3
147.6 7.71 d (15.8)
114.7 6.43 d (15.8)
131.6 7.53 d (8.6)
116.8 6.79 d (8.6)
161.4
147.7 7.71 d (15.8)
114.5 6.44 d (15.8)
131.6 7.53 d (8.6)
116.8 6.81 d (8.6)
161.6
148.0 7.74 d (16.1)
114.2 6.43 d (16.1)
134.0 7.67 d (8.6)
115.9 6.76 d (8.6)
160.4
146.7 6.99 d (13.0)
115.4 5.89 d (13.0)
168.5
–
168.6
–
168.6
–
168.2
–
167.3
–
Acyl part
CH
3
COO– 172.7
–
172.8
–
–
172.0
172.1
–
–
–
171.3
171.5
171.8
–
–
–
–
171.3
171.6
171.8
172.1
172.4
–
–
–
–
–
1
72.8
172.7
1
1
20.4
20.5
20.6
72.0
72.4
–
a
2.00b
2.02b
2.07b
c
1.83d
e
f
CH
3
COO– 21.0
2.07 s
20.8
1.1
1.98 s
2.08 s
20.8
20.8
2
s
s
s
s
s
s
s
s
20.6
20.6
20.6
20.7
1.96 s
2.00 s
2.02 s
2.05 s
a
a
c
c
c
c
d
e
f
2
1.93
2.04d
2.06d
2.09d
e
f
0.9
e
f
2
2
0.7
0.7
e
f
20.7
2.08 s
a–f
Interchangeable.

S. Nakamura et al. / Phytochemistry 92 (2013) 128–136
131
0
0
0
0
0
0
0
0
0
and C-9 , H-1 and C-2, H-3 , 6 and two acetyl carbonyl carbons.
7.44 (d, J = 8.6 Hz, H-2 , 6 ), 6.54 (d, J = 8.6 Hz, H-3 , 5 )] and an ethyl
ester part [d 1.23 (t, J = 7.0 Hz, OCH CH ), 4.13 (q, J = 7.0 Hz, OCH2-
CH )]. The proton and carbon resonances in the H and C NMR
1
13
The H NMR (methanol-d
and 4 showed signals assignable to three acetyl groups [d 2.00,
4
) and C NMR (Table 1) spectra of 3
2
3
1
13
3
2
.02, 2.07 (s, CH
3
CO– ꢁ 3) for 3] and five acetyl groups [d 1.83,
spectra of 6 were superimposable on those of 11, except for the sig-
nals around the ethyl ester part of 6. The positions of the (E)-p-cou-
maroyl group and ethyl ester part were confirmed based on DQF
COSY and HMBC spectroscopy. Namely, long-range correlations
1
.93, 2.04, 2.06, 2.09 (s, CH
3
CO– ꢁ 5) for 4], respectively, together
with a (E)-p-coumaroyl group and a sucrose moiety. The proton
and carbon resonances in the 1H and C NMR (Table 1) spectra
of 3 were superimposable on those of 1, except for the signals
around the glucose part of 3. The H and C NMR chemical shift
values of the 3 and 6 positions of the glucose part on 3 were ob-
13
were observed between the following proton and carbon pairs:
1
13
0
H-5 and C-9 , OCH
2
CH
3
and C-7. Consequently, the chemical struc-
0
0
ture of 6 was determined to be 5-O-(E)-p-coumaroylquinic acid
ethyl ester.
Mumeic acid-A (7) and mumeic acid-A methyl ester (8), which
were obtained as white amorphous powders with negative optical
served at lower fields compared with those of 1. By acetylation of
one or two hydroxy groups on the sugar moiety, the 1H and
13
C
chemical shift values of the neighboring sugar-skeleton protons
and carbon of the connected acetyl groups were generally shifted
downfield. Therefore, 3 was suggested to possess two acetyl groups
at the 3 and 6 positions of 1. In addition, since the H and C NMR
chemical shift values of the 1 position of 4 were observed at the
lower fields compared with those of 3, the existence of an acetyl
group at the 1 position of the fructose part of 4 was suggested.
Next, positions of acetyl groups of 3 and 4 were confirmed based
on HMBC spectroscopy. Namely, long-range correlations were ob-
rotations, showed absorption bands due to hydroxy, benzoyl,
unsaturated ester, and aromatic ring moieties in their IR spectra.
The molecular formulas, C23 10, were deter-
mined by HRMS. Acid hydrolysis of 7 and 8 with a 5% aqueous
SO -1,4-dioxane yielded
-(ꢀ)-quinic acid, benzoic acid, and
(E)-p-caffeic acid.
a,b-
0
0
1
13
22 24
H O10 and C24H O
H
2
4
D
D
-(ꢀ)-Quinic acid was identified by comparison
of the analytical data with that of the authentic sample as well
as 6. Benzoic acid and (E)-p-caffeic acid were identified by compar-
0
0
0
served between the following proton and carbon pairs [H-2 , 3 , 6
R R
ison of their retention times (t : 23.0 min for benzoic acid, t :
0
0
0
0
and three acetyl carbonyl carbons for 3, H-1, 2 , 3 , 4 , 6 and five
acetyl carbonyl carbons for 4]. Consequently, the chemical struc-
tures of mumeoses B (2), C (3), and D (4) were characterized to
be 3 ,6 -di-O-acetyl-3-O-(E)-p-coumaroylsucrose, 2 ,3 ,6 -tri-O-
acetyl-3-O-(E)-p-coumaroylsucrose, and 1,2 ,3 ,4 ,6 -penta-O-acet-
yl-3-O-(E)-p-coumaroylsucrose, respectively.
12.1 min for (E)-p-caffeic acid) with those of authentic samples
1
on reversed-phase HPLC analysis. The H NMR (methanol-d
4
) and
C NMR (Table 1) spectra of 7 showed signals assignable to a
(ꢀ)-quinoyl moiety [d 2.11 (m, H-2a), 2.26 (m, H-2b, H -6), 4.43
(m, H-3), 5.20 (br d, J = 8.3, H-4), 5.71 (m, H-5)], a benzoyl group
1
3
D-
0
0
0
0
0
2
0
0
0
0
0
0
0
[d 7.37 (dd, J = 7.5, 7.5 Hz, H-3 , 5 ), 7.50 (m, H-4 ), 7.99 (d,
(E)-p-caffeoyl group [d 6.09 (d,
J = 15.8 Hz, H-8 ), 7.43 (d, J = 15.8 Hz, H-7 ), 6.91 (br s, H-2 ),
0
0
Mumeose E (5), obtained as a white amorphous powder with a
J = 7.5 Hz, H-2 , 6 )] and
a
15
00
00
00
negative optical rotation ([
tion bands due to hydroxy, ester,
functionalities in the IR spectrum. Its positive FABMS showed a
quasimolecular ion peak at m/z 721 [M+Na] and a molecular for-
a]
D
ꢀ30.7 in MeOH), showed absorp-
0
0
00
a
,b-unsaturated ester, and ether
6.68 (d, J = 8.1 Hz, H-5 ), 6.81 (br d, J = 8.1 Hz, H-6 )]. The proton
and carbon resonances of the quinic acid part in the 1H and
NMR spectra of 7 were superimposable on those of 4,5-O-trans-
p-dicaffeoyl- -quinic acid (Shi et al., 2007). The positions of a (E)-
13
C
+
mula C31
hydrolysis of 5 yielded
-glucose, fructose, and (Z)-p-coumaric acid, respectively. (Z)-p-
Coumaric acid was identified by comparison of its retention time
: 18.5 min) with that of an authentic sample on reversed-phase
H
38
O18 was determined by HRMS measurement. Basic
D
D
-sucrose and acid hydrolysis of 5 yielded
p-caffeoyl group and a benzoyl group in 7 were also confirmed
D
by HMBC experiments, which showed long-range correlations be-
0
tween the following proton and carbon pairs; H-4 and C-7 , H-5
0
0
1
13
(t
R
and C-9 . The H NMR (methanol-d
showed signals assignable to a methyl ester part [d 3.72 (s,
C(@O)OCH )] together with a
4
) and C NMR spectra of 8
1
13
HPLC analysis. The H NMR (methanol-d
spectra of 5 showed signals assignable to five acetyl groups [d
.96, 2.00, 2.02, 2.05, 2.08 (s, CH
CO– ꢁ 5)] and a (Z)-p-coumaroyl
4
) and C NMR (Table 1)
3
D
-(ꢀ)-quinoyl moiety, a benzoyl
1
3
group, and a (E)-p-caffeoyl group. This result and the detailed
DQF COSY and HMBC experiments led us to confirm the structure
of 8 to be the methyl ester derivative of 7. Consequently, the
chemical structures of mumeic acid-A (7) and mumeic acid-A
methyl ester (8) were 4-O-benzoylchlorogenic acid and 4-O-ben-
zoylchrologenic acid methyl ester.
00
00
00
group [d 5.89 (d, J = 13.0 Hz, H-8 ), 6.76 (d, J = 8.6 Hz, H-3 , 5 ),
00
00
00
6
.99 (d, J = 13.0 Hz, H-7 ), 7.67 (d, J = 8.6 Hz, H-2 , 6 )] together
with a sucrose moiety. Comparison of the NMR spectroscopic data
for 5 with those for 4 led us to confirm the structure of 5 to be the
cis–trans isomer of 4. Consequently, the chemical structure of
mumeose E (5) was characterized to be 1,2 ,3 ,4 ,6 -penta-O-acet-
yl-3-O-(Z)-p-coumaroylsucrose.
0
0
0
0
2.3. Inhibitory effects of the compounds on melanogenesis in B16
melanoma 4A5 cells
5
-O-(E)-p-Coumaroylquinic acid ethyl ester (6) was isolated as a
1
5
white amorphous powder with negative optical rotation ([a]
D
ꢀ
8.9 in MeOH). Its IR spectrum showed absorption bands at
Melanin production, which is principally responsible for skin
color, is a major defense mechanism against harmful ultraviolet
rays in sunlight. However, excess production of melanin after long
periods of exposure to the sun can cause dermatological disorders
such as melasma, freckles, postinflammatory melanoderma, and
solar lentigines. To develop inhibitors of melanogenesis, the inhib-
itory effects of several diarylheptanoids, flavonoids, sterol glyco-
sides, and acylated triterpene glycosides were examined in
theophylline-stimulated B16 melanoma 4A5 cells (Fujimoto et al.,
2012; Matsuda et al., 2009; Nakashima et al., 2010; Nakamura
et al., 2010, 2012a,b). As a continuation of these studies, the inhib-
itory effects of constituents from the flowers buds of P. mume on
melanogenesis were examined. Among the isolates, acylated quinic
acid analogs 6–14 significantly inhibited melanogenesis (Table 3).
Indeed, 6–14 each displayed greater potency for inhibiting melano-
genesis than that of the reference compound, arbutin (Fujimoto
ꢀ
1
3
400, 1720, 1690, 1603 and 1515 cm
due to hydroxy, ester,
a,b-unsaturated ester, and aromatic ring moieties. The EIMS of 6
+
showed a molecular ion peak at m/z 366 [M] and the molecular
formula C18 was determined by HRMS measurement. Treat-
ment of 6 with a 5% aqueous H
nic acid and (E)-p-coumaric acid.
by comparison of the analytical data (NMR and MS spectra and
optical rotation) with that of an authentic sample. (E)-p-Coumaric
22 8
H O
2
SO
D
4
-1,4-dioxane yielded
-(ꢀ)-Quinic acid was identified
D
-(ꢀ)-qui-
acid was identified by comparison of its retention time (t
R
:
1
7.1 min) with that of an authentic sample on reversed-phase
1
13
HPLC analysis. The H NMR (methanol-d
spectra of 6 showed signals assignable to a
d 2.00 (m, H -2), 2.18 (m, H -6), 3.72 (dd, J = 3.1, 7.6 Hz, H-4),
.10 (m, H-3), 5.28 (ddd, J = 4.6, 7.6, 7.6 Hz, H-5)], a (E)-p-couma-
4
) and C NMR (Table 1)
D
-(ꢀ)-quinoyl moiety
[
4
2
2
0
0
royl group [d 6.28 (d, J = 15.8 Hz, H-8 ), 7.52 (d, J = 15.8 Hz, H-7 ),

1
32
S. Nakamura et al. / Phytochemistry 92 (2013) 128–136
et al., 2012). Particularly, 5-O-(E)-feruloylquinic acid methyl ester
13) exhibited a potent inhibitory effect on melanogenesis [inhibi-
tion (%): 21.5 ± 1.0 (P < 0.01) at 0.1 M]. Next, the cytotoxic effects
of the constituents on B16 melanoma 4A5 cells were investigated.
Many of the compounds displaying melanogenesis inhibitory ef-
fects, such as arbutin, diarylheptanoids, flavonoids and saponins,
5
l
m) and 20 mm I.D. – 250 mm (particle size: 5
lm)] columns
(
were used for analytical and preparative purposes.
l
The following experimental materials were used for chromatog-
raphy: normal-phase silica gel column chromatography (cc), Silica
gel BW-200 (Fuji Silysia Chemical Ltd., 150–350 mesh); reversed-
phase silica gel cc, Chromatorex ODS DM1020T (Fuji Silysia Chem-
ical Ltd., 100–200 mesh); TLC, precoated TLC plates with Silica gel
60F254 (Merck, 0.25 mm) (ordinary phase) and Silica gel RP-18
have cytotoxic action at concentrations greater than 10 lM (Fujim-
oto et al., 2012; Matsuda et al., 2009; Nakashima et al., 2010;
Nakamura et al., 2012a,b). Interestingly, acylated quinic acid ana-
logs 6–14 showed potent inhibitory effects on melanogenesis,
F
254S (Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC,
precoated TLC plates with Silica gel RP-18 WF254S (Merck,
0.25 mm). Detection was achieved by spraying with 1% Ce(SO
10% aqueous H SO followed by heating.
but displayed no cytotoxicity [cell viability >97% at 10
lM]. There-
4 2
) –
fore, acylated quinic acid analogs are promising therapeutic agents
for the treatment of skin disorders. By contrast, acylated sucroses
2
4
1–5, flavonol glycosides 15–19, and
D-mandelic acid (20) had no
4.2. Plant material
inhibitory effects on melanogenesis at 1–100
lM.
Dried flower buds of P. mume cultivated in Zhejiang Province,
China, were commercial products purchased from Tochimoto Ten-
kaido Co. Ltd. (Osaka, Japan) in April 2011. The botanical identifica-
tion was undertaken by one of the authors (M.Y.). A voucher of the
plant is on file in our laboratory (KPU Medicinal Flower-PM 2011).
2
1
.4. HPLC profile comparison of acylated quinic acid analogs 6, 8, 11–
4
In the present study, nine acylated quinic acid analogs including
four methyl esters (8, 11–13) and two ethyl esters (6 and 14) were
isolated from the MeOH extract of flower buds of P. mume. In order
to establish whether these methyl- or ethyl esters were artifacts
generated during separation procedures, the HPLC profiles of the
4
.3. Extraction and isolation
Dried flower buds (2.0 kg) were extracted with MeOH (10 L ꢁ 3)
under conditions of reflux for 3 h. Evaporation of the solvents un-
der reduced pressure provided a MeOH extract (608.0 g, 30.4%). An
aliquot of the MeOH extract (486.4 g) was suspended in distilled
1
-butanol- and EtOAc-soluble fractions from the MeOH-extract
were compared with those fractions from the MeCN/water (1:1,
v/v)-extract of the flower buds of P. mume. The 1-butanol- and
EtOAc-soluble fractions of MeCN/water (1:1, v/v)-extract were pre-
pared without using methanol nor ethanol. A portion of the 1-
butanol-soluble and EtOAc-soluble fractions of the MeOH- and
MeCN/water (1:1, v/v)-extracts were dissolved in MeCN and sub-
jected to reversed-phase HPLC. Acylated quinic acid analogs (6,
H
2
O (20 L), and partitioned with EtOAc (20 L ꢁ 3) to furnish an
EtOAc-soluble fraction (106.0 g, 6.6%) and an aqueous phase. The
latter was further partitioned with 1-butanol (10 L ꢁ 3) to give a
1
2
-butanol-soluble fraction (119.3 g, 7.5%) and an H O-soluble frac-
tion (256.0 g, 16.0%). An aliquot of the 1-butanol-soluble fraction
80.0 g) was subjected to normal phase silica gel cc [Silica gel
BW-200 (2.4 kg, 100 mm I.D. – 180 mm), Hexane ? CHCl –MeOH
50:1 ? 20:1 ? 10:1 ? 5:1 ? 10:3, v/v) ? MeOH] to give seven
fractions [Fr.B1 (450 mg), Fr.B2 (960 mg), Fr.B3 (850 mg), Fr.B4
(
8–14) isolated from the MeOH extract were detected in the
MeCN/water (1:1, v/v) extract by comparing their retention times
with those of authentic samples. In addition, the HPLC analysis of
3
(
1
-butanol- and EtOAc-soluble fractions of MeOH- and the MeCN/
(
(
[
1.37 g), Fr.B5 (4.70 g), Fr.B6 (7.24 g), Fr.B7 (41.4 g)]. Fraction B5
4.70 g) was further separated by reversed phase silica gel cc
Chromatorex ODS DM1020T (132 g, 30 mm I.D.
water (1:1, v/v) extracts showed comparable HPLC profiles. Based
on these results, the methyl- and ethyl ester derivatives of quinic
acid (6, 8, 11–14), which were isolated from a MeOH extract of
flower buds of P. mume, seem to be genuine natural products.
–
80 mm),
v/
2
MeOH–H O
(20:80 ? 30:70 ? 40:60 ? 50:50 ? 60:40,
v) ? MeOH] to give 10 fractions [Fr.B5-1, Fr.B5-2 (730 mg),
Fr.B5-3, Fr.B5-4 (260 mg), Fr.B5-5, Fr.B5-6, Fr.B5-7, Fr.B5-8, Fr.B5-
3
. Conclusion
9
[
(
(170 mg), Fr.B5-10]. Fraction B5-2 (108 mg) was purified by HPLC
COSMOSIL 5C18-MS-II (preparative column), H O–MeOH–AcOH
750:250:3, v/v/v)] to give 20 (68 mg). Fraction B5-4 (260 mg)
2
Five new acylated sucroses, mumeoses A (1), B (2), C (3), D (4)
and E (5), and three new acylated quinic acid analogs, 5-O-(E)-p-
coumaroylquinic acid ethyl ester (6), and mumeic acid-A (7) and
its methyl ester (8), were isolated from the flower buds of P. mume
cultivated in Zhejiang Province, China. Acylated quinic acid analogs
was purified by HPLC [COSMOSIL 5C18-MS-II (preparative col-
umn), H O–MeOH–AcOH (700:300:3, v/v/v)] to give 10 (129 mg).
Fr.B5-9 (170 mg) was purified by HPLC [COSMOSIL 5C18-AR-II
preparative column), H O–MeOH–AcOH (650:350:3, v/v/v)] to
2
(
2
6–14 significantly inhibited melanogenesis without inducing cyto-
give two fractions [Fr.B5-9-1 (61 mg), Fr.B5-9-2 (39 mg)]. Fraction
B5-9-1 (61 mg) was purified by HPLC [COSMOSIL 5C18-AR-II (pre-
toxicity. Further structure activity relationship studies and elucida-
tion of the inhibitory mechanism are warranted.
parative column), H
14 mg) and 13 (14 mg). Fraction B5-9-2 (39 mg) was purified by
HPLC [COSMOSIL 5C18-MS-II (preparative column), H O–MeCN–
2
O–MeOH–AcOH (650:350:3, v/v/v)] to give 11
(
4
. Experimental
2
AcOH (800:200:3, v/v/v)] to give 3 (5.3 mg). Fraction B6 (7.24 g)
4.1. General experimental procedures
was further separated by normal phase silica gel cc [Silica gel
BW-200 (230 g, 40 mm I.D.
3
– 110 mm), CHCl –EtOAc (1:1,
The following instruments were used to obtain physical data:
v/v) ? CHCl –EtOAc–2-propanol–MeOH (20:20:1:1 ? 15:15:1:1
3
specific rotations,
a
Horiba SEPA-300 digital polarimeter
? 10:10:1:1 ? 5:5:1:1 ? 1:1:1:1, v/v) ? MeOH] to give five frac-
tions [Fr.B6-1, Fr.B6-2 (1.56 g), Fr.B6-3 (3.83 g), Fr.B6-4 (920 mg),
Fr.B6-5]. Fraction B6-2 (1.56 g) was purified by reversed phase sil-
ica gel cc [Chromatorex ODS DM1020T (66 g, 20 mm I.D. – 90 mm),
(
l = 5 cm); IR spectra, a Thermo Electron Nexus 470; FABMS and
1
HRFABMS, a JEOL JMS-SX 102A mass spectrometer; H NMR spec-
tra, JEOL JNM-LA 500 (500 MHz) spectrometer; 13C NMR spectra,
JEOL JNM-LA 500 (125 MHz) spectrometer; HPLC, a Shimadzu
SPD-10AVP UV–VIS detector. COSMOSIL 5C18-MS-II [4.6 mm I.D.
MeOH–H
2
O
(20:80 ? 30:70 ? 40:60 ? 50:50 ? 60:40, v/v) ?
MeOH] to give seven fractions [Fr.B6-2-1, Fr.B6-2-2 (368 mg),
Fr.B6-2-3 (63 mg), Fr.B6-2-4 (840 mg), Fr.B6-2-5 (62 mg), Fr.B6-
2-6, Fr.B6-2-7]. Fraction B6-2-2 (37 mg) was purified by HPLC
–
250 mm (particle size: 5
lm) and 20 mm I.D. – 250 mm (particle
size: 5 m)] and 5C18-AR-II [4.6 mm I.D. – 250 mm (particle size:
l

S. Nakamura et al. / Phytochemistry 92 (2013) 128–136
133
[
(
COSMOSIL 5C18-MS-II (preparative column), H
700:300:3, v/v/v)] to give 9 (15 mg) and 10 (14 mg). Fraction
2
O–MeOH–AcOH
v) ? MeOH] to give nine fractions [Fr.E5-4-1, Fr.E5-4-2, Fr.E5-4-
3, Fr.E5-4-4 (220 mg), Fr.E5-4-5 (162 mg), Fr. E5-4-6, Fr.E5-4-7,
Fr.E5-4-8 (538 mg), Fr.E5-4-9 (170 mg)]. Fraction E5-4-4
(220 mg) was purified by HPLC [COSMOSIL 5C18-MS-II (prepara-
B6-2-3 (63 mg) was purified by HPLC [COSMOSIL 5C18-MS-II (pre-
parative column), H O–MeOH–AcOH (700:300:3, v/v/v)] to give 9
18 mg) and 10 (5.6 mg), and 12 (16 mg). Fraction B6-2-4
20 mg) was purified by HPLC [COSMOSIL 5C18-MS-II (preparative
O–MeOH–AcOH (650:350:3, v/v/v)] to give 12 (8.7 mg)
and 14 (9.5 mg). Fraction B6-2-5 (62 mg) was purified by HPLC
2
(
(
tive column), H
(25 mg). Fraction E5-4-5 (162 mg) was purified by HPLC [COSMO-
SIL 5C18-AR-II (preparative column), H O–MeCN–MeOH–AcOH
2
O–MeCN–AcOH (800:200:3, v/v/v)] to give 12
column), H
2
2
(700:150:150:3, v/v/v/v)] to give 11 (17 mg) and 14 (32 mg). Frac-
[
COSMOSIL 5C18-MS-II (preparative column), H
2
O–MeOH–AcOH
tion E5-4-8 (538 mg) was purified by HPLC [COSMOSIL 5C18-MS-II
(
550:450:3, v/v/v)] to give 6 (33 mg) and 14 (8.4 mg). Fraction
(preparative column), H
give quercetin (68 mg). Fraction E5-4-9 (170 mg) was purified by
HPLC [COSMOSIL 5C18-MS-II (preparative column), H O–MeCN–
AcOH (700:300:3, v/v/v)] to give (12 mg) and quercetin
2
O–MeCN–AcOH (700:300:3, v/v/v)] to
B6-3 (3.83 g) was purified by reversed phase silica gel cc [Chromat-
orex ODS DM1020T (132 g, 25 mm I.D. – 110 mm), MeOH–H
20:80 ? 30:70 ? 40:60 ? 50:50 ? 60:40, v/v) ? MeOH] to give
2
O
2
(
8
nine fractions [Fr.B6-3-1 (186 mg), Fr.B6-3-2 (2.47 g), Fr.B6-3-3,
Fr.B6-3-4 (520 mg), Fr.B6-3-5 (158 mg), Fr.B6-3-6, Fr.B6-3-7,
Fr.B6-3-8 (51 mg), Fr.B6-3-9]. Fraction B6-3-1 (186 mg) was puri-
(14 mg). Fraction E5-5 (3.6 g) was further separated by reversed
phase silica gel cc [Chromatorex ODS DM1020T (120 g, 25 mm
I.D.
2
– 100 mm), MeOH–H O (20:80 ? 30:70 ? 40:60 ? 50:50
fied by HPLC [COSMOSIL 5C18-MS-II (preparative column), H
MeOH–AcOH (800:200:3, v/v/v)] to give 10 (17 mg). Fraction B6-
-2 (100 mg) was purified by HPLC [COSMOSIL 5C18-MS-II (pre-
parative column), H O–MeOH–AcOH (700:300:3, v/v/v)] to give
0 (28 mg) and 12 (24 mg). Fraction B6-3-4 (26 mg) was purified
by HPLC [COSMOSIL 5C18-MS-II (preparative column), H O–
MeOH–AcOH (700:300:3, v/v/v)] to give 12 (8.6 mg). Fraction B6-
-5 (158 mg) was purified by HPLC [COSMOSIL 5C18-MS-II (pre-
2
O–
? 60:40 ? 80:20 ? 90:10, v/v) ? MeOH] to give nine fractions
[Fr.E5-5-1, Fr.E5-5-2, Fr.E5-5-3 (326 mg), Fr.E5-5-4, Fr.E5-5-5,
Fr.E5-5-6 (152 mg), Fr.E5-5-7, Fr.E5-5-8 (114 mg), Fr.E5-5-9]. Frac-
tion E5-5-3 (326 mg) was purified by HPLC [COSMOSIL 5C18-MS-II
3
2
1
(preparative column), H
give 12 (193 mg). Fraction E5-5-6 (152 mg) was purified by HPLC
[COSMOSIL 5C18-MS-II (preparative column), H O–MeCN–AcOH
2
O–MeCN–AcOH (800:200:3, v/v/v)] to
2
2
3
(700:300:3, v/v/v)] to give 7 (47 mg). Fraction E5-5-8 (114 mg)
parative),
H
2
O–MeOH–AcOH (650:350:3, v/v/v)] to give
9
was purified by HPLC [COSMOSIL 5C18-MS-II (preparative col-
(
4.0 mg) and 12 (10 mg). Fraction B6-3-8 (51 mg) was purified by
2
umn), H O–MeCN–AcOH (700:300:3, v/v/v)] to give 8 (30 mg)
HPLC [COSMOSIL 5C18-MS-II (preparative column), H
2
O–MeOH–
and 18 (7.1 mg). Fraction E5-6 (3.80 g) was separated by reversed
AcOH (650:350:3, v/v/v)] to give 15 (12 mg) and 17 (30 mg). Frac-
phase silica gel cc [Chromatorex ODS DM1020T (123 g, 25 mm
tion B6-4 (910 mg) was purified by reversed phase silica gel cc
2
I.D. – 100 mm), MeOH–H O (20:80 ? 30:70 ? 40:60 ? 50:50
[
33 g, MeOH–H
v) ? MeOH] to give six fractions [Fr.B6-4-1, Fr.B6-4-2, Fr.B6-4-3
366 mg), Fr.B6-4-4 (175 mg), Fr.B6-4-5 (48 mg), Fr.B6-4-6]. Frac-
tion B6-4-3 (37 mg) was purified by HPLC [COSMOSIL 5C18-MS-II
preparative column), H O–MeOH–AcOH (700:300:3, v/v/v)] to
give 9 (6.2 mg) and 10 (15 mg). Fraction B6-4-4 (175 mg) was puri-
fied by HPLC [COSMOSIL 5C18-MS-II (preparative column), H O–
MeOH–AcOH (700:300:3, v/v/v)] to give 1 (3.4 mg), 2 (11 mg), 9
13 mg), 10 (18 mg), and 12 (8.1 mg). Fraction B6-4-5 (48 mg)
was purified by HPLC [COSMOSIL 5C18-MS-II (preparative col-
umn), H O–MeOH–AcOH (650:350:3, v/v/v)] to give 14 (11 mg).
2
O (20:80 ? 30:70 ? 40:60 ? 50:50 ? 60:40, v/
? 60:40 ? 80:20, v/v) ? MeOH] to give seven fractions [Fr.E5-6-
1, Fr.E5-6-2, Fr.E5-6-3 (214 mg), Fr.E5-6-4 (187 mg), Fr.E5-6-5,
Fr.E5-6-6, Fr.E5-6-7]. Fraction E5-6-3 (214 mg) was purified by
HPLC [COSMOSIL 5C18-MS-II (preparative column), H O–MeCN–
2
AcOH (800:200:3, v/v/v)] to give 16 (12 mg), 17 (108 mg), and 19
(
(
2
(6.6 mg). Fraction E5-6-4 (187 mg) was purified by HPLC [COSMO-
2
SIL 5C18-MS-II (preparative column),
2
H O–MeCN–AcOH
(750:250:3, v/v/v)] to give 15 (69 mg) and 17 (19 mg).
(
4.4. Mumeose A (1)
2
A part of the EtOAc-soluble fraction (80 g) was subjected to normal
phase silica gel cc [Silica gel BW-200 (2.4 kg, 100 mm I.D. –
1
5
White amorphous powder (3.4 mg, 0.00032%); [
.16, MeOH); UV [MeOH, nm (log
a
]
D
+114.8 (c
0
(
1
e)]: 229 (4.22), 315 (4.40); IR
max 3400, 1730, 1697, 1603, 1515, 1448, 1369, 1233,
162, and 1033 cm ; for H and C NMR spectroscopic data,
see Table 1; positive-ion FABMS: m/z 533 [M+Na] ; HRFABMS:
1
3
80 mm), n-hexane ? n-hexane–EtOAc (5:1 ? 3:1, v/v) ? CHCl –
ATR):
m
MeOH (100:1 ? 50:1 ? 20:1 ? 10:1 ? 4:1 ? 2:1, v/v) ? MeOH]
to give six fractions [Fr.E1 (15.9 g), Fr.E2 (4.13 g), Fr.E3 (9.73 g),
Fr.E4 (6.39 g), Fr.E5 (20.6 g), Fr.E6 (13.3 g)]. Fraction E4 (6.39 g)
was further separated by reversed phase silica gel cc [Chromatorex
ꢀ
1
1
13
+
+
m/z 553.1573 (Calcd for C23
30
H O
14Na [M+Na] : m/z 553.1533).
ODS DM1020T (200 g, 30 mm I.D.
2
– 120 mm), MeOH–H O
(
30:70 ? 40:60 ? 50:50 ? 60:40, v/v) ? MeOH] to give five frac-
4
.5. Mumeose B (2)
tions [Fr.E4-1, Fr.E4-2, Fr.E4-3, Fr.E4-4 (750 mg), Fr.E4-5 (1.82 g)].
Fraction E4-4 (750 mg) was further separated by normal phase sil-
ica gel cc [Silica gel BW-200 (17 g, 15 mm I.D. – 60 mm), n-hex-
15
White amorphous powder (11 mg, 0.0010%); [
.33, MeOH) UV [MeOH, nm (log
max 3400, 1730, 1704, 1604, 1515, 1450, 1371, 1238,
a
]
D
+46.9 (c
0
e)]: 228 (4.34), 316 (4.56); IR
ane ? n-hexane–EtOAc
v) ? EtOAc ? MeOH] to give three fractions [Fr.E4-4-1, Fr.E4-4-2
596 mg), Fr.E4-4-3]. Fraction E4-4-2 (596 mg) was purified by
HPLC [COSMOSIL 5C18-MS-II (preparative column), H O–MeOH–
AcOH (700:300:3, v/v/v) and O–MeCN–MeOH–AcOH
650:300:50:3, v/v/v/v)] to give 4 (5.6 mg) and 5 (2.8 mg). Fraction
E5 (20.6 g) was separated by normal phase silica gel cc [Silica gel
BW-200 (720 g, 50 mm I.D. – 210 mm), CHCl ? CHCl –MeOH
50:1 ? 20:1 ? 15:1 ? 10:1 ? 5:1, v/v) ? MeOH] to give seven
fractions [Fr.E5-1, Fr.E5-2, Fr.E5-3, Fr.E5-4 (7.18 g), Fr.E5-5
3.56 g), Fr.E5-6 (3.80 g), Fr.E5-7 (1.29 g)]. Fraction E5-4 (7.18 g)
was further separated by reversed phase silica gel cc [Chromatorex
ODS DM1020T (200 g, 30 mm I.D. 120 mm), MeOH–H
20:80 ? 30:70 ? 40:60 ? 50:50 ? 60:40 ? 80:20 ? 90:10,
(10:1 ? 5:1 ? 2:1 ? 1:1 ? 1:2,
v/
(
ATR):
m
ꢀ1
1
13
1
167, and 1034 cm ; for H and C NMR spectroscopic data,
(
+
see Table 1; positive-ion FABMS: m/z 595 [M+Na] ; HRFABMS:
m/z 595.1636 (Calcd for C25
2
+
32
H O15Na [M+Na] : m/z 595.1639).
H
2
(
4.6. Mumeose C (3)
3
3
15
(
White amorphous powder (5.3 mg, 0.00050%); [
0.39, MeOH); UV [MeOH, nm (log )]: 228 (4.30), 316 (4.55); IR
(ATR): max 3400, 1730, 1713, 1604, 1515, 1443, 1369, 1226,
1162, and 1038 cm ; for H and C NMR spectroscopic data,
see Table 1; positive-ion FABMS: m/z 637 [M+Na] ; HRFABMS:
a
]
D
+54.0 (c
e
(
m
ꢀ
1
1
13
+
–
2
O
+
(
v/
m/z 637.1747 (Calcd for C27
H
34
O
16Na [M+Na] : m/z 637.1745).

1
34
S. Nakamura et al. / Phytochemistry 92 (2013) 128–136
4.7. Mumeose D (4)
4.11. Mumeic acid-A methyl ester (8)
1
5
15
White amorphous powder (5.6 mg, 0.00047%); [
a]
D
+6.0 (c
White amorphous powder (42 mg, 0.0034%); [
0.81, MeOH); UV [MeOH, nm (log
a
]
D
ꢀ11.9 (c
0
.21, MeOH); UV [MeOH, nm (log
e
)]: 229 (4.24), 316 (4.39); IR
e)]: 232 (4.20), 328 (4.48). IR
(
1
ATR):
m
max 3400, 1742, 1717, 1604, 1516, 1437, 1368, 1220,
(ATR): max 3400, 1730, 1716, 1698, 1601, 1508, 1456, 1160, and
m
158, and 1033 cm ; for 1H and C NMR spectroscopic data,
ꢀ
1
13
1110 cm ; for H and C NMR spectroscopic data, see Table 2; po-
ꢀ1
1
13
+
+
see Table 1; positive-ion FABMS: m/z 721 [M+Na] ; HRFABMS:
m/z 721.1948 (Calcd for C31
sitive-ion FABMS: m/z 495 [M+Na] ; HRFABMS: m/z 495.1264
+
+
H
38
O
18Na [M+Na] : m/z 721.1956).
(Calcd for C24
H
24
O
10Na [M+Na] : m/z 495.1262).
4.8. Mumeose E (5)
4.12. Acid hydrolysis of 1–8
1
5
White amorphous powder (2.8 mg, 0.00023%); [
a
]
D
ꢀ30.7° (c
Compounds 1–8 (1–5:1.5 mg, 6–8:10 mg) were dissolved in 5%
aqueous H SO –1,4-dioxane (1:1, v/v, 1–5: 2.0 mL, 6–8:10.0 mL),
2 4
0
(
1
.13, MeOH); UV [MeOH, nm (log )]: 229 (4.20), 314 (4.42); IR
e
ATR):
m
max 3400, 1740, 1717, 1604, 1515, 1456, 1368, 1220,
and each solution was heated at 90 °C for 3 h, and neutralized with
152, and 1035 cm ; for 1H and C NMR spectroscopic data,
ꢀ1
13
ꢀ
Amberlite IRA-400 (OH form). After drying in vacuo, a small ali-
+
see Table 1; positive-ion FABMS: m/z 721 [M+Na] ; HRFABMS:
m/z 721.1959 (Calcd for C31H O18Na [M+Na] : m/z 721.1956).
38
quot of the residue was dissolved in H
lyzed by reversed-phase HPLC to identify (E)- or (Z)-p-coumaric,
E)-p-caffeic and benzoic acids, respectively, [column: COSMOSIL
C18-MS-II, 4.6 mm I.D. – 250 mm; mobile phase A: H O–AcOH
2
O–MeOH (1:1, v/v) and ana-
+
(
5
2
4
.9. 5-O-(E)-p-Coumaroyl quinic acid ethyl ester (6)
(1000:3, v/v), B: MeCN–AcOH (1000:3, v/v), Linear gradient: mo-
bile phase A–B (90:10 ? 72:28, v/v, in 24.0 min); detection: UV
(300 nm); flow rate: 1.0 mL/min; column temperature: ambient].
(E)-p-Coumaric acid (from 1–4, 6), (Z)-p-coumaric acid (from 5),
(E)-p-caffeic acid (from 7 and 8) and benzoic acid (from 7 and 8)
were identified by comparison of their retention times with those
1
5
White amorphous powder (33 mg, 0.0031%); [
c = 0.29, MeOH); UV [MeOH, nm (log )]: 228 (4.34), 314 (4.56);
max 3400, 1720, 1690, 1603, 1515, 1444, 1166, and
a
]
D
ꢀ8.9°
(
e
IR (ATR):
m
ꢀ1
1
13
1
079 cm ; for H and C NMR spectroscopic data, see Table 2;
+
EIMS: m/z 366 [M] ; HREIMS: m/z 366.1308 (Calcd for C18
H
22
O
8
of authentic samples [(E)-p-caffeic acid: t
maric acid: t = 17.1 min, (Z)-p-coumaric acid: t
zoic acid: t = 23.0 min]. In addition, the remaining parts of
residue from 1–5, from which glucose and fructose were identified
by TLC (ordinary phase) [CHCl –MeOH–H O (30:15:3, v/v/v)]],
were dissolved in pyridine (0.1 mL) containing -cysteine methyl
R
= 12.1 min, (E)-p-cou-
+
[
M] : m/z 366.1314).
R
R
= 18.5 min, ben-
R
4.10. Mumeic acid-A (7)
3
2
L
1
5
White amorphous powder (47 mg, 0.0039%); [
a]
D
ꢀ82.9 (c
ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h. o-torylis-
othiocyanate (0.5 mg) in pyridine (0.1 mL) was added to the mix-
ture and heated at 60 °C for 1 h. The reaction mixture was
analyzed by reversed-phase HPLC [column: COSMOSIL 5C18-AR-
0
.22, MeOH); UV [MeOH, nm (log
e)]: 232 (4.28), 329 (4.41); IR
(
ATR):
m
max 3400, 1720, 1703, 1695, 1599, 1510, 1455, 1150, and
ꢀ1
1
13
1
109 cm ; for H and C NMR spectroscopic data, see Table 1; po-
+
sitive-ion EIMS: m/z 458 [M] ; HREIMS: m/z 458.1204 (Calcd for
C H O10 [M] : m/z 458.1212).
23 22
II, 4.6 mm I.D. – 250 mm; mobile phase: MeCN–0.05 M H
3 4
PO
+
(23: 77, v/v); detection: UV (250 nm); flow rate: 0.8 mL/min; col-
Table 2
1
H and 13C NMR (125 MHz) spectroscopic data for 6–8 in CD
3
OD (d in ppm, J in Hz).
Position
6
7
8
d
C
d
H
d
C
d
H
d
C
d
H
Quinic acid part
Quinic acid part
1
2
3
4
5
6
7
75.8
38.0
70.5
72.8
72.2
37.9
175.0
–
1
2
3
4
5
6
7
76.2
38.4
69.3
76.7
68.8
39.5
177.0
–
75.8
38.4
68.5
75.8
68.9
38.7
175.2
–
2.00 m
4.10 m
3.72 dd (3.1, 7.6)
5.28 ddd (4.6, 7.6, 7.6)
2.18 m
–
2.11, 2.26 m
4.43 m
5.20 br d (8.3)
5.71 m
2.26 m
–
2.30 m
4.43 m
5.24 br d (7.8)
5.64 m
2.26 m
–
p-Coumaroyl part
Benzoyl part
0
0
1
127.1
116.9
131.2
161.4
146.8
115.2
168.3
62.5
–
1
131.1
130.8
129.5
134.4
167.5
–
131.1
130.8
129.5
134.4
167.4
–
0
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
7
,6
,5
7.44 d (8.6)
6.54 d (8.6)
–
7.52 d (15.8)
6.28 d (15.8)
–
2 ,6
7.99 d (7.5)
7.37 dd (7.5, 7.5)
7.50 m
8.05 d (7.3)
7.44 dd (7.3, 7.3)
7.57 m
0
3 ,5
0
4
0
7
–
–
p-Caffeoyl part
00
1
127.6
115.2
146.8
149.6
116.4
123.0
147.5
114.6
168.2
–
127.5
115.1
146.8
149.8
116.5
123.1
147.6
114.5
167.8
53.1
–
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
OCH
OCH
2
CH
CH
3
3
4.13 q (7.0)
1.23 t (7.0)
2
3
4
5
6
7
8
9
6.91 br s
–
–
6.68 d (8.1)
6.81 br d (8.1)
7.43 (15.8)
6.09 d (15.8)
–
6.97 br s
–
–
6.74 d (7.5)
6.89 br d (7.5)
7.47 d (15.8)
6.13 d (15.8)
–
2
14.3
C(O)OCH
3
3.72 s

S. Nakamura et al. / Phytochemistry 92 (2013) 128–136
135
Table 3
Inhibitory effects of compounds 6–14 isolated from flower buds of P. mume on melanogenesis in B16 melanoma 4A5 cells.
Sample (
lM)
Inhibition (%)
Control
0.1
0.3
1
3
10
⁄
⁄
⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
6
7
8
9
0.0 ± 6.7
0.0 ± 1.4
0.0 ± 5.9
0.0 ± 5.2
0.0 ± 6.5
0.0 ± 6.5
0.0 ± 8.3
0.0 ± 2.1
0.0 ± 5.9
14.3 ± 1.0
19.1 ± 0.4
3.1 ± 2.2
9.1 ± 1.4
15.4 ± 3.1
5.5 ± 1.6
11.5 ± 3.1
21.5 ± 1.0
6.0 ± 1.8
21.1 ± 2.8
23.3 ± 4.7
13.2 ± 6.6
18.5 ± 2.4
38.1 ± 2.6
14.3 ± 6.2
25.0 ± 3.1
22.4 ± 0.3
21.3 ± 1.1
43.3 ± 1.0
36.4 ± 8.4
23.9 ± 2.6
39.1 ± 4.8
56.4 ± 2.9
26.5 ± 1.7
43.6 ± 3.9
38.9 ± 1.0
44.0 ± 3.8
51.1 ± 2.8
43.3 ± 4.7
34.2 ± 1.6
43.9 ± 2.2
58.7 ± 3.5
34.1 ± 2.4
53.0 ± 2.3
52.4 ± 3.6
42.3 ± 6.1
61.2 ± 2.6
47.4 ± 3.9
27.2 ± 0.6
47.1 ± 2.4
58.1 ± 2.9
47.6 ± 4.1
57.9 ± 2.9
63.8 ± 1.8
51.9 ± 3.0
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄⁄
⁄
⁄
⁄
⁄
⁄
⁄
⁄⁄
⁄⁄
1
1
1
1
1
0
1
2
3
4
⁄
⁄
⁄
⁄
⁄⁄
⁄⁄
⁄⁄
⁄
⁄
⁄⁄
⁄
⁄
Sample (
Arbutina
lM)
Inhibition (%)
Control
0.0 ± 1.4
10
30
100
⁄⁄
⁄⁄
⁄⁄
10.6 ± 0.6
20.4 ± 0.5
38.1 ± 0.9
Each value represents the mean ± S.E.M. (n = 4).
Significantly different from the control, P < 0.05, P < 0.01.
⁄
⁄⁄
Compounds, 1–5, 15–20, had no inhibitory effects on melanogenesis at 1–100
The cell viabilities at 10 M of compounds 6–14 are more than 97.4%.
The cell viabilities at 3 M are more than 95.6%.
Reference compound (Fujimoto et al., 2012).
lM.
l
l
a
umn temperature: 35 °C] to identify the derivatives of constituent
monosaccharides in 1–5 by comparison of their retention times
A–B (0.0–10.0 min: linear gradient with 90–10 ? 82–18, 10.0–
30.0 min: isocratic with 82–18, 30.0–70.0 min: linear gradient
with 82–18 ? 0–100); detection: UV (265 nm); flow rate:
0.45 mL/min; column temperature: 25 °C]. Acylated quinic acid
analogs (6, 8–14) isolated from the 1-butanol-soluble fraction of
the MeOH extract were also detected in the 1-butanol-soluble frac-
with those of authentic samples (t
et al., 2007). On the other hand, the remaining parts of residue
from 6–8 were dissolved in water, and purified by HPLC [H O–
MeOH–AcOH (980: 20:3, v/v/v), COSMOSIL 5C18-PAQ] to give
R
: D-glucose; 19.7 min) (Tanaka
2
D
-
(
ꢀ)-quinic acid (6: 2.5 mg, 7: 2.0 mg, 8: 2.1 mg), which was identi-
tion of the MeCN/H
with those of authentic samples (t
12.0 min, 11: 27.0 min, 12: 17.7 min, 13: 26.4 min, 14: 27.8 min).
Furthermore, a portion of the EtOAc-soluble fraction of the MeOH-
and MeCN/water-extracts was dissolved in MeCN (1.0 mg/mL) and
subjected to reversed-phase HPLC [column: Inertsil ODS-3, 3.0 mm
2
O extract by comparing their retention times
6: 42.6 min, 9: 17.0 min, 10:
fied by comparison of their analytical data (NMR and MS spectra
and optical rotation) with that of the authentic sample.
R
4
.13. Alkaline hydrolysis of 1–5
I.D. – 150 mm; mobile phase A: H
MeCN–AcOH (1000:3, v/v), gradient: mobile phase A–B (0.0–
5.0 min: linear gradient with 80–20 ? 50–50); detection: UV
(241 nm); flow rate: 0.45 mL/min; column temperature: 25 °C].
Compound 8 isolated from the EtOAc-soluble fraction of the MeOH
2
O–AcOH (1000:3, v/v), B:
Compounds 1–5 (1.5 mg) were individually treated with a 10%
aqueous KOH–1,4-dioxane (1:1, v/v, 1.0 mL) and stirred at 37 °C
4
for 24 h. Each reaction mixture was neutralized with Dowex HCR
+
W2 (H form) and the resin was removed by filtration. Evaporation
of the solvent from the filtrate under reduced pressure yielded a
extract was identified in that of the MeCN/water extract (t
R
8:
crude product. Each crude product was dissolved in H
plied to normal-phase HPLC [column: YMC-Pack Polyamine II,
.6 mm I.D. – 250 mm; mobile phase: MeCN–H O (3:1, v/v); detec-
2
O and ap-
2
2.6 min). In addition, the HPLC analysis of the 1-butanol- and
EtOAc-soluble fractions of the MeOH- and MeCN/H
2
O-extracts
4
2
showed comparable HPLC profiles.
tion: optical rotation [Shodex OR-2 (Showa Denko Co. Ltd., Tokyo,
Japan); flow rate: 1.0 mL/min; column temperature: ambient] to
identify
retention times and optical rotation with that of authentic sample
= 19.8 min with positive rotation).
D
-sucrose as a constituent of 1–5 by comparison of their
4.15. Reagents for bioassays
(t
R
D
-Glucopyranose, sucrose,
D
-(ꢀ)-quinic acid, benzoic acid, (E)-p-
caffeic acid and (E)-p-coumaric acid were purchased from Tokyo
Chemical Industry. (Z)-p-Coumaric acid was prepared from (E)-p-
coumaric acid by UV irradiation according to the reported proce-
dure (Kort et al., 1996). Dulbecco’s modified Eagle’s medium
4.14. HPLC profile comparison
2
Dried flower buds (40.0 g) were extracted with MeCN/H O
(
(
DMEM, 4500 mg/L glucose) was purchased from Sigma–Aldrich
St. Louis, MO, USA); fetal bovine serum (FBS), penicillin, and strep-
(
800 mL ꢁ 3, 1:1, v/v) under reflux for 3 h. Evaporation of the sol-
vent under reduced pressure provided MeCN/H extract
16.2 g, 40.5%). The MeCN/H O extract (16.2 g) was suspended in
distilled H
a
2
O
tomycin were purchased from Gibco (Invitrogen, Carlsbad, CA,
USA); the Cell Counting Kit-8™ was from Dojindo Lab. (Kumamoto,
Japan); and the other chemicals were purchased from Wako Pure
Chemical Co. Ltd. (Osaka, Japan).
(
2
2
O (500 mL), and partitioned with EtOAc (500 mL ꢁ 3)
to furnish an EtOAc-soluble fraction (1.8 g, 4.5%). The aqueous
phase was further extracted with 1-butanol (500 mL ꢁ 3) to give
2
a 1-butanol-soluble fraction (10.3 g, 25.8%) and an H O-soluble
fraction (4.2 g, 10.5%). An aliquot of the 1-butanol-soluble fraction
of the MeOH- and MeCN/H O-extracts was dissolved in MeCN
2.0 mg/mL) and subjected to reversed-phase HPLC [column: Inert-
sil ODS-3, 3.0 mm I.D. – 150 mm; mobile phase A: H O–AcOH
1000:3, v/v), B: MeCN–AcOH (1000:3, v/v), gradient: mobile phase
4.16. Cell culture
2
(
Murine B16 melanoma 4A5 cells (RCB0557) were obtained from
Riken Cell Bank (Tsukuba, Japan), and grown in DMEM supple-
mented with 10% FBS, penicillin (100 U/mL), and streptomycin
2
(

1
36
S. Nakamura et al. / Phytochemistry 92 (2013) 128–136
Beck, M.A., Häberlein, H., 1999. Flavonol glycosides from Eschscholtzia californica.
(
100 lg/mL) at 37 °C in 5% CO2/air. The cells were harvested by
Phytochemistry 50, 329–332.
incubation in phosphate-buffered saline (PBS) containing 1 mM
EDTA and 0.25% trypsin for ca. 5 min at 37 °C and used for the sub-
sequent bioassays.
Choi, S.W., Hur, N.Y., Ahn, S.C., Kim, D.S., Lee, J.K., Kim, D.O., Park, S.K., Kim, B.Y., Baik,
M.Y., 2007. Isolation and structural determination of squalene synthase
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Matsuda, H., Yoshikawa, M., 2012. Medicinal flowers: XXXV. Nor-oleanane-type
and acylated oleanane-type triterpene saponins from the flower buds of
Chinese Camellia japonica and their inhibitory effects on melanogenesis. Chem.
Pharm. Bull. 60, 1188–1194.
Hsich, T.J., Wu, Y.C., Lo, W.L., Chen, S.C., Kuo, S.H., Chen, C.Y., 2004. Chemical
constituents from the leaves of Mahonia japonica. Chin. Pharm. J. 56, 17–24.
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and chlorogenic acid from Prunus mume on adrenocorticotropic hormone
4
.17. Melanogenesis
4
Melanoma cells (2.0 ꢁ 10 cells/400
lL/well) were seeded into
2
4-well multiplates. After 24 h of culture, a test compound and
theophylline 1 mM were added and incubated for 72 h. Cells were
harvested by incubating with PBS containing 1 mM EDTA and
(
ACTH) and catecholamine levels in plasma of experimental menopausal model
0
.25% trypsin, and cells were washed with PBS. Cells were treated
with NaOH 1 M (120 L/tube, 80 °C, 30 min) to yield a lysate. An
aliquot (100 L) of this was transferred to a 96-well microplate,
rats. Biol. Pharm. Bull. 27, 136–137.
l
Jaiswal, R., Kuhnert, N., 2011. How to identify and discriminate between the methyl
quinates of chlorogenic acids by liquid chromatography–tandem mass
spectrometry. J. Mass Spectrom. 46, 269–281.
Jeong, J.T., Moon, J.H., Park, K.H., Shin, C.S., 2006. Isolation and characterization of a
new compound from Prunus mume fruit that inhibits cancer cells. J. Agric. Food
Chem. 54, 2123–2128.
Kort, R., Vonk, H., Xu, X., Hoff, W.D., Crielaard, W., Hellingwerf, K.J., 1996. Evidence
for trans–cis isomerization of the p-coumaric acid chromophore as the
photochemical basis of the photocycle of photoactive yellow protein. FEBS
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Yoshikawa, M., 2003. Medicinal flowers: VIII. Radical scavenging constituents
from the flowers of Prunus mume: structure of prunose III. Chem. Pharm. Bull.
l
and the optical density of each well measured with a microplate
reader (Model 550, Bio-Rad Laboratories) at 405 nm (reference:
6
concentration of DMSO in the medium was 0.1%. The production of
melanin was corrected based on cell viability. Inhibition (%) was
calculated using the following formula, and IC50 values were deter-
mined graphically.
55 nm). The test compound was dissolved in DMSO, and the final
Inhibition ð%Þ ¼ ½ðA ꢀ BÞ=Aꢂ=ðC=100Þ ꢁ 100
where A and B indicate the optical density of vehicle- and test com-
pound-treated groups, respectively, and C indicates cell viability (%).
51, 440–443.
Machida, K., Matsuoka, E., Kikuchi, M., 2009. Five new glycosides from Hypericum
erectum Thunb.. J. Nat. Med. 63, 223–226.
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Melanogenesis inhibitors from the rhizomes of Alpinia officinarum in B16
melanoma cells. Bioorg. Med. Chem. 17, 6048–6053.
4
.18. Cell viability
Nakamura, S., Chen, G., Nakashima, S., Matsuda, H., Pei, Y., Yoshikawa, M., 2010.
Brazilian natural medicines: Part 4. New noroleanane-type triterpene and
ecdysterone-type sterol glycosides and melanogenesis inhibitors from the roots
of Pfaffia glomerata. Chem. Pharm. Bull. 58, 690–695.
3
The melanoma cells (5.0 ꢁ 10 cells/100
into 96-well microplates and incubated for 24 h. After 70 h incuba-
tion with theophylline 1 mM and a test compound, 10 L of WST-8
solution (Cell Counting Kit-8™) was added to each well. After a fur-
ther 2 h in culture, the optical density of the water-soluble forma-
zan produced by the cells was measured with a microplate reader
lL/well) were seeded
Nakamura, S., Moriura, T., Park, S., Fujimoto, K., Matsumoto, T., Ohta, T., Matsuda, H.,
Yoshikawa, M., 2012a. Melanogenesis inhibitory and fibroblast proliferation
accelerating effects of noroleanane- and oleanane-type triterpene
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Acknowledgments
This research was supported in part by a Ministry of Education,
Culture, Sports, Science and Technology (MEXT)-Supported Pro-
gram for the Strategic Research Foundation at Private Universities,
by a Grant-in-Aid for Young Scientists (B) from the Japan Society
for the Promotion of Science (JSPS).
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1999. Medicinal flowers: I. Aldose reductase inhibitors and three new
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