Structures of acylated sucroses from the flower buds of Prunus mume

Article abstract of DOI:10.1007/s11418-014-0818-z

Seven new acylated sucroses, mumeoses P-V, were isolated from the flower buds of Prunus mume, cultivated in Zhejiang province, China. Their chemical structures were elucidated on the basis of chemical and physicochemical evidence. Moreover, mumeoses C, D,

Full text of DOI:10.1007/s11418-014-0818-z

J Nat Med
DOI 10.1007/s11418-014-0818-z
ORIGINAL PAPER
Structures of acylated sucroses from the flower buds
of Prunus mume
•
Katsuyoshi Fujimoto Seikou Nakamura Takahiro Matsumoto
•
•
•
•
•
•
Tomoe Ohta Masayuki Yoshikawa Keiko Ogawa Eri Kashiwazaki
Hisashi Matsuda
Received: 28 December 2013 / Accepted: 14 January 2014
Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract Seven new acylated sucroses, mumeoses P–V,
were isolated from the flower buds of Prunus mume, cul-
tivated in Zhejiang province, China. Their chemical
structures were elucidated on the basis of chemical and
physicochemical evidence. Moreover, mumeoses C, D, and
R, were shown to substantially inhibit aldose reductase.
seven new acylated sucroses termed mumeoses P (1), Q
(2), R (3), S (4), T (5), U (6) and V (7) from the flower buds
of Chinese Prunus mume. This paper deals with the
structure elucidation of new constituents (1–7) and the
inhibitory effects of the acylated sucroses on aldose
reductase (Fig. 1).
Keywords Prunus mume ꢀ Mumeose ꢀ Acylated sucrose ꢀ
Aldose reductase ꢀ Medicinal flower
Results and discussion
The MeOH extract (30.4 % from the dried flower buds of
P. mume cultivated in Zhejiang province, China) was
partitioned into an EtOAc–H2O (1:1, v/v) mixture to fur-
nish an EtOAc-soluble fraction (6.6 %) and an aqueous
layer. The aqueous layer was further extracted with
1-butanol to give 1-butanol- (7.5 %) and H2O- (13.0 %)
soluble fractions, as reported previously [3–5]. The
1-butanol-soluble and EtOAc-soluble fractions were sub-
jected to normal-phase and reversed-phase silica-gel col-
umn chromatography and repeated high performance liquid
chromatography (HPLC). From those fractions, five new
acylated sucroses, mumeoses P (1, 0.0002 %), Q (2,
0.0003 %), R (3, 0.0010 %), S (4, 0.0020 %), T (5,
0.0002 %), U (6, 0.0012 %), and V (7, 0.0054 %), were
isolated.
Introduction
In the course of our studies on bioactive constituents from
medicinal flower [1–12], we previously reported on the
isolation and structure elucidation of acylated sucroses
termed prunoses I–III, with inhibitory effects on platelet
aggregation and aldose reductase and radical scavenging
effect, from the flower buds of Prunus (P.) mume SIEB. et
ZUCC. (Rosaceae) cultivated in Wakayama prefecture,
Japan [1, 2]. Additionally, 15 new acylated sucroses,
mumeoses A–O, a new acylated flavonol glycoside termed
mumeflavonoside A and three new acylated quinic acid
analogs, mumeic acid-A and its methyl ester and 5-O-(E)-
p-coumaroylquinic acid ethyl ester were isolated from the
flower buds of P. mume cultivated in Zhejiang province,
China [3–5]. The principal constituents, acylated quinic
acid analogs, showed inhibitory effects on melanogenesis
and aldose reductase. As a continuing study, we isolated
Mumeoses P (1) and Q (2), which were obtained as a
white amorphous powder with positive (1: [a]1D5 ?32.0°)
and negative (2: [a]1D5 -30.8°) optical rotations in MeOH,
showed absorption bands due to hydroxy, ester, a,b-
unsaturated ester, aromatic ring, and ether functions in their
IR spectra (1: 3400, 1730, 1716, 1604, 1515 and
1035 cm-1, 2: 3400, 1730, 1717, 1604, 1516 and
1034 cm-1). Their molecular formulas (C25H32O15 of 1,
C29H36O17 of 2) were determined from the quasimolecular
ion peaks (m/z 595 [M ? Na]? for 1, 679 [M ? Na]? for
K. Fujimoto ꢀ S. Nakamura ꢀ T. Matsumoto ꢀ T. Ohta ꢀ
M. Yoshikawa ꢀ K. Ogawa ꢀ E. Kashiwazaki ꢀ H. Matsuda (&)
Department of Pharmacognosy, Kyoto Pharmaceutical
University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
e-mail: matsuda@mb.kyoto-phu.ac.jp
123

J Nat Med
Fig. 1 Structure of acylated
sucroses isolated from flower
buds of P. mume
2) in the positive-ion fast atom bombardment mass spec-
trometry (FABMS) and by high-resolution mass spec-
trometry (HRMS) measurement. Basic hydrolysis of 1 and
2 with a 10 % aqueous KOH-1,4-dioxane (1:1, v/v) mix-
ture yielded sucrose. Acid hydrolysis of 1 and 2 with 5 %
aqueous H2SO4-1,4-dioxane yielded D-glucose and fructose
together with p-coumaric acid [(E)-p-coumaric acid from 1,
and (Z)-p-coumaric acid from 2] [13]. The 1H nuclear
magnetic resonance (NMR) (methanol-d4) and 13C NMR
(Table 1) spectra of 1 showed signals assignable to two
acetyl groups [d 2.07, 2.08 (all s, CH3CO– 9 2)], a (E)-p-
coumaroyl group [d 6.43 (d, J = 15.8 Hz, H-800), 6.80 (d,
J = 8.5 Hz, H-300, 500), 7.52 (d, J = 8.6 Hz, H-200, 600), 7.71
(d, J = 15.8 Hz, H-700)] and a sucrose moiety [d 3.34 (m,
H-40), 3.39 (d, J = 11.9 Hz, H-1a), 3.58 (d, J = 12.2 Hz,
H-1b), 3.78 (m, H-30), 3.80 (m, H2-6), 3.91 (m, H-5), 4.09
(m, H-50), 4.16 (m, H-60a), 4.32 (dd, J = 8.5, 8.5 Hz, H-4),
4.47 (m, H-60b), 4.56 (m, H-20), 5.49 (d, J = 8.6 Hz, H-3),
5.54 (d, J = 3.6 Hz, H-10)]. The positions of two acetyl
groups and a (E)-p-coumaroyl moiety were confirmed by
heteronuclear multiple bond connectivity (HMBC) spec-
troscopy analysis. Namely, long-range correlations were
observed between the following proton and carbon pairs:
H-3 and C-100, H-20, 60 and two acetyl carbonyl carbons.
The 1H NMR (methanol-d4) and 13C NMR (Table 1)
spectra of 2 showed signals assignable to four acetyl
groups [d 1.95, 1.99 9 2, 2.05 (all s, CH3CO– 9 4)], a
(Z)-p-coumaroyl group [d 5.88 (d, J = 12.7 Hz, H-800),
6.76 (d, J = 8.5 Hz, H-300, 500), 7.68 (d, J = 8.8 Hz, H-200,
600), 6.97 (d, J = 12.7 Hz, H-700)] and a sucrose moiety.
The positions of four acetyl groups and a (Z)-p-coumaroyl
moiety were confirmed by HMBC spectroscopy analysis.
Namely, long-range correlations were observed between
the following proton and carbon pairs: H-3 and C-100, H-20,
30, 40, 60 and four acetyl carbonyl carbons. Additionally,
Table 1 13C NMR (125 MHz, CD3OD) spectroscopic data for
compounds 1–7
Position
1
2
3
4
5
6
7
1
64.7
65.0
65.7
65.0
65.1
64.8
64.2
2
105.2 105.5 105.5 106.5 104.4 106.5 106.7
3
78.7
73.9
84.1
63.5
90.5
74.3
72.1
71.7
71.9
65.0
78.6
73.6
84.2
63.3
90.3
71.7
71.4
69.9
69.4
63.3
79.9
74.7
85.0
63.5
92.9
72.9
72.6
72.5
70.1
64.3
77.8
77.3
83.8
63.3
93.4
72.9
72.5
72.3
70.2
64.2
77.4
73.6
82.8
65.0
91.6
74.1
77.4
72.2
71.7
64.0
77.8
77.1
83.6
63.3
93.2
70.8
74.3
70.2
69.9
63.7
77.2
76.6
83.0
63.6
91.0
71.5
71.2
69.9
69.5
63.4
4
5
6
10
20
30
40
50
60
100
200, 600
300, 500
400
700
800
900
127.1 127.5 127.1 127.1 127.0 127.2 127.1
131.5 133.9 131.4 131.5 131.6 131.6 131.5
116.8 115.9 116.9 116.9 116.9 116.8 116.8
161.5 160.3 161.6 161.6 161.7 161.5 161.5
147.6 146.3 147.4 147.8 148.3 148.1 148.0
114.5 115.7 114.7 114.3 113.8 114.2 114.1
168.4 167.4 167.9 167.3 167.8 167.5 167.7
CH3COO– 172.5 171.3 172.0 171.8 171.8 171.5 171.3
172.8 171.4 172.6 172.0 171.9 171.9 172.0
171.8
172.4
172.7 172.7 172.1 172.4
172.9 172.5 172.6
172.8
CH3COO–
20.8
21.0
20.6
20.6
20.7
20.7
20.8
20.8
20.7
20.7
20.7
20.6
20.7
20.7
21.1
20.4
20.7
20.7
20.8
20.4
20.6
20.7
20.7
20.7
123

J Nat Med
500), 7.51 (d, J = 8.6 Hz, H-200, 600), 7.70 (d, J = 15.8 Hz,
H-700)] and a sucrose moiety [d 3.50 (m, H-20), 3.60 (d,
J = 12.2 Hz, H-1a), 3.65 (d, J = 12.2 Hz, H-1b), 3.72 (m,
H-30), 3.80 (m, H2-6), 3.97 (m, H-5), 4.09 (d, J = 10.4 Hz,
H-60a), 4.14 (d, J = 10.4 Hz, H-60b), 4.24 (m, H-50), 4.32
(dd, J = 7.0, 7.0 Hz, H-4), 4.73 (m, H-40),, 5.42 (d,
J = 7.0 Hz, H-3), 5.46 (d, J = 3.7 Hz, H-10)]. The posi-
tions of two acetyl groups and a (E)-p-coumaroyl moiety
were confirmed by HMBC spectroscopy analysis, which
showed long-range correlations between the following
proton and carbon pairs: H-3 and C-100, H-4, 60 and four
1
acetyl carbonyl carbons. The H NMR (methanol-d4) and
13C NMR (Table 1) spectra of 4–7 showed signals
assignable to acetyl groups [d 1.82, 2.06, 2.08 (all s,
CH3CO– 9 3) for 4, 2.06, 2.07, 2.09, 2.13 (all s, CH3CO–
9 4) for 5, 1.71, 2.00, 2.04, 2.08 (all s, CH3CO– 9 4) for
6, 1.76, 1.92, 2.00, 2.01, 2.03 (all s, CH3CO– 9 5) for 7,
respectively], a (E)-p-coumaroyl group and a sucrose
moiety. The positions of acetyl groups and a (E)-p-cou-
maroyl moiety were confirmed by HMBC spectroscopy
analysis. On the basis of all this evidence, the chemical
structures of 3–7 were determined to be 40,60-di-O-acetyl-
3-O-(E)-p-coumaroylsucrose (mumeose R, 3), 4,40,60-tri-O-
acetyl-3-O-(E)-p-coumaroylsucrose (mumeose S, 4), 1, 4,
6, 20-tetra-O-acetyl-3-O-(E)-p-coumaroylsucrose (mume-
ose T, 5), 4, 30, 40,60-tetra-O-acetyl-3-O-(E)-p-coumaro-
ylsucrose (mumeose U, 6) and 4, 20,30,40,60-penta-O-
acetyl-3-O-(E)-p-coumaroylsucrose (mumeose V, 7),
respectively.
Fig. 2 Important two-dimensional (2D) NMR correlations of 1–7
comparisons of the NMR data for 2 with those for 20, 30, 40, 60-
tetra-O-acetyl-3-O-(E)-p-coumaroylsucrose led us to con-
firm the structure of 2 to be the cis–trans isomer of 20, 30, 40,
60-tetra-O-acetyl-3-O-(E)-p-coumaroylsucrose [14]. On the
basis of all this evidence, the chemical structures of mume-
ose P (1) and mumeose Q (2) were determined to be 20,60-di-
O-acetyl-3-O-(E)-p-coumaroylsucrose and 20,30,40,60-tetra-
O-acetyl-3-O-(Z)-p-coumaroylsucrose (Fig. 2).
Aldose reductase, a key enzyme in the polyol pathway,
has been reported to catalyze the reduction of glucose to
sorbitol. Sorbitol does not readily diffuse across cell
membranes, and the intracellular accumulation of sorbitol
has been implicated in the chronic complications of
diabetes such as cataract. Previously, we reported that
various constituents such as flavonoids and terpenoids
from natural medicines and medicinal foods inhibited the
aldose reductase inhibitory effect [15–20]. In addition, we
Mumeoses R (3), S (4), T (5), U (6) and V (7) were
obtained as a white amorphous powder with positive
15
15
15
optical rotations (3: ½aꢁD ?38.5°, 4: ½aꢁD ?30.6°, 5: ½aꢁD
15
15
?28.2°, 6:½aꢁD ?44.2°, 7: ½aꢁD ?28.0° in MeOH).
Absorption bands due to hydroxyl, ester, a,b-unsaturated
ester, aromatic ring, and ether functions are shown in their
IR spectra. Their molecular formulas (3: C25H32O15, 4:
C27H34O16, 5 and 6: C29H36O17, and 7: C31H38O18) were
determined from the quasimolecular ion peaks (m/z
595[M ? Na]? for 3, 637 [M ? Na]? for 4, 679
[M ? Na]? for 5 and 6, and 721 [M ? Na]? for 7) in the
positive-ion FABMS and by HRMS measurement. Basic
hydrolysis of 3–7 with a 10 % aqueous KOH-1,4-dioxane
(1:1, v/v) mixture yielded sucrose. Acid hydrolysis of 3–7
with 5 % aqueous H2SO4-1,4-dioxane yielded D-glucose
and fructose together with (E)-p-coumaric acid [13]. The
1H NMR (methanol-d4) and 13C NMR (Table 1) spectra of
3 showed signals assignable to two acetyl groups [d 1.89,
2.07 (all s, CH3CO– 9 2)], a (E)-p-coumaroyl group [d
6.38 (d, J = 15.8 Hz, H-800), 6.82 (d, J = 8.6 Hz, H-300,
reported that acylated sucroses such as prunose
I
[IC50 = 58 lM], acylated flavonol glycoside such as
mumeflavonoside A [IC50 = 8.2 lM], and acylated qui-
nic acid analogs such as mumeic acid-A [IC50 = 0.4 lM]
showed moderate to strong inhibitory effect [4, 5]. Fur-
thermore, we examined the aldose reductase inhibitory
effects of the isolated acylated sucroses, mumeoses R (3)
and U (6), in this study, and mumeoses C (8) and D (9)
which were previously isolated from the flower buds of
P. mume [3]. As a result, mumeose R (3, IC50 = 22.7
lM), U (6, IC50 = 76.5 lM), C (8, IC50 = 25.5 lM) and
D (9, IC50 = 26.6 lM) showed moderate inhibitory
effects. The aldose reductase inhibitory effects of com-
pounds including acylated sucroses will be the focus of
further studies.
123

J Nat Med
Experimental
(850 mg), Fr.B4 (1.37 g), Fr.B5 (4.70 g), Fr.B6 (7.24 g),
Fr.B7 (41.4 g)], as reported previously [3–5].
General
Fraction B6 (7.24 g) was further separated by silica gel
column chromatography [230 g, CHCl3-EtOAc (1:1,
v/v) ? CHCl3-EtOAc-2-propanol-MeOH (20:20:1:1 ? 15:
15:1:1 ? 10:10:1:1 ? 5:5:1:1 ? 1:1:1:1, v/v) ? MeOH]
to give five fractions [Fr.B6-1, Fr.B6-2, Fr.B6-3,
Fr.B6-4 (920 mg), Fr.B6-5]. Fraction B6-4 (910 mg) was
purified by reversed phase silica gel column chromatog-
raphy [33 g, MeOH–H2O (20:80 ? 30:70 ? 40:60 ?
50:50 ? 60:40, v/v) ? MeOH] to give six fractions
[Fr.B6-4-1, Fr.B6-4-2, Fr.B6-4-3, Fr.B6-4-4 (175 mg),
Fr.B6-4-5, Fr.B6-4-6]. Fraction B6-4-4 (175 mg) was
purified by HPLC {(1) H2O-MeOH-AcOH (700:300:3, v/v/
v), (2) H2O-MeCN-AcOH (830: 170: 3, v/v/v)} to give 1
(2.3 mg) and 3 (10.4 mg). A part of the EtOAc-soluble
fraction (80 g) was subjected to normal phase silica gel
column chromatography [2.4 kg, n-hexane ? n-hexane–
EtOAc (5:1 ? 3:1, v/v) ? CHCl3–MeOH (100:1 ?
50:1 ? 20:1 ? 10:1 ? 4:1 ? 2:1, v/v) ? MeOH] to
give six fractions [Fr.E1, Fr.E2, Fr.E3, Fr.E4 (6.39 g),
Fr.E5 (20.6 g), Fr.E6]. Fraction E4 (6.39 g) was separated
by reversed phase silica gel column chromatography
[200 g, MeOH–H2O (30:70 ? 40:60 ? 50:50 ? 60:40,
v/v) ? MeOH] to give six fractions [Fr.E4-1, Fr.E4-2,
Fr.E4-3 (456 mg), Fr.E4-4 (750 mg), Fr.E4-5, Fr.E4-6].
Fraction E4-3 (456 mg) was further separated by normal
phase silica gel column chromatography [15 g, n-hex-
ane ? n-hexane–EtOAc (10:1 ? 5:1 ? 2:1 ? 1:1, v/v)
? EtOAc ? MeOH] to give five fractions [Fr.E4-3-1,
Fr.E4-3-2, Fr.E4-3-3, Fr.E4-3-4 (122 mg), Fr.E4-3-5].
Fraction E4-3-4 (122 mg) was purified by HPLC {(1) H2O-
MeCN-AcOH (650: 350: 3, v/v/v), (2) H2O-MeCN-MeOH-
AcOH (600: 150: 250: 3, v/v/v/v)} to give 6 (13.7 mg).
Fraction E4-4 (750 mg) was separated by normal phase
silica gel column chromatography [17 g, n-hexane ? n-
hexane–EtOAc (10:1 ? 5:1 ? 2:1 ? 1:1 ? 1:2, v/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 {(1) H2O-MeCN-AcOH (700:300:3,
v/v/v), (2) H2O-MeCN-MeOH-AcOH (650:300: 50:3, v/v/
v)} to give 5 (2.2 mg) and 7 (57.8 mg). Fraction E5
(20.6 g) was separated by normal phase silica gel column
chromatography [720 g, CHCl3 ? CHCl3–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, Fr.E5-7]. Fraction E5-
4 (7.18 g) was further separated by reversed phase sil-
ica gel column chromatography [220 g, MeOH–H2O
(20:80 ? 30:70 ? 40:60 ? 50:50 ? 60:40 ? 80:20 ?
90:10, v/v) ? MeOH] to give five fractions [Fr.E5-4-1,
Fr.E5-4-2, Fr.E5-4-3, Fr.E5-4-4, Fr.E5-4-5 (0.54 g), Fr.E5-
4-6]. Fraction E5-4-5 (0.54 g) was purified by HPLC [H2O-
The following instruments were used to obtain physical
data: specific rotations, a Horiba SEPA-300 digital polar-
imeter (l = 5 cm); IR spectra, a Thermo Electron Nexus
470; FAB-MS and HRFAB-MS, a JEOL JMS-SX 102A
1
mass spectrometer; H NMR spectra, 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
(250 9 4.6 mm i.d., 250 9 10 mm i.d. and 250 9 20 mm
i.d.), 5C18-PAQ (250 9 20 mm i.d.) and 5C18-AR-II
(250 9 4.6 mm i.d.) columns were used for analytical and
preparative purposes. The following materials were used
for chromatography: normal-phase silica gel column
chromatography, Silica gel BW-200 (Fuji Silysia Chemi-
cal, Ltd., 150–350 mesh); reversed-phase silica gel column
chromatography, Chromatorex ODS DM1020T (Fuji Si-
lysia Chemical, Ltd., 100–200 mesh); TLC, precoated TLC
plates with Silica gel 60F254 (Merck, 0.25 mm) (ordinary
phase) and Silica gel RP-18 F254S (Merck, 0.25 mm)
(reversed phase); reversed-phase high-performance thin
layer chromatography ( HPTLC), precoated TLC plates
with Silica gel RP-18 WF254S (Merck, 0.25 mm). Detection
was achieved by spraying with 1 % Ce(SO4)2–10 %
aqueous H2SO4 followed by heating.
Plant material
The dried flower buds of P. mume cultivated in Zhejiang
province, China, were commercial products purchased
from Tochimoto Tenkaido Co. Ltd (Osaka, Japan) in April
2011.
Extraction and isolation
The dried flower buds (2.0 kg) were extracted three times
with methanol under reflux for 3 h. Evaporation of the
solvent under reduced pressure provided a MeOH extract
(608.0 g, 30.4 %). A part of the MeOH extract (486.4 g)
was partitioned into an EtOAc–H2O (1:1, v/v) mixture to
furnish an EtOAc-soluble fraction (106.0 g, 6.6 %) and an
aqueous phase. The aqueous phase was further extracted
with 1-butanol to give a 1-butanol-soluble fraction
(119.3 g, 7.5 %) and an H2O-soluble fraction (256.0 g,
16.0 %). A part of the 1-butanol-soluble fraction (80.0 g)
was subjected to normal phase silica gel column chroma-
tography [2.4 kg, n-hexane ? CHCl3–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
123

J Nat Med
MeCN-AcOH (700:300:3, v/v/v)] to give 7 (65.6 mg).
Fraction E5-5 (3.56 g) was further separated by rever-
sed phase silica gel column chromatography [120 g,
MeOH–H2O (20:80 ? 30:70 ? 40:60 ? 50:50 ? 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, Fr.E5-5-4, Fr.E5-5-5
(313 mg), Fr.E5-5-6, Fr.E5-5-7, Fr.E5-5-8, Fr.E5-5-9].
Fraction E5-5-5 (313 mg) was purified by HPLC {(1) H2O-
MeCN-AcOH (750:250:3, v/v/v), (2) H2O-MeCN-MeOH-
AcOH (700:150:150:3, v/v/v)} to give 4 (24.2 mg) and 2
(3.3 mg).
92), 3.50 (m, H-20), 3.60 (d, J = 12.2 Hz, H-1a), 3.65 (d,
J = 12.2 Hz, H-1b), 3.72 (m, H-30), 3.80 (m, H2-6), 3.97
(m, H-5), 4.09 (m, H-60a), 4.14 (m, H-60b), 4.24 (m, H-50),
4.32 (dd, J = 7.0, 7.0 Hz, H-4), 4.73 (m, H-40), 5.42 (d,
J = 7.0 Hz, H-3), 5.46 (d, J = 3.7 Hz, H-10), 6.38 (d,
J = 15.8 Hz, H-800), 6.82 (d, J = 8.6 Hz, H-300, 500), 7.51
(d, J = 8.6 Hz, H-200, 600), 7.70 (d, J = 15.8 Hz, H-700); 13C
NMR: given in Table 1; positive-ion FAB-MS: m/z 595
[M ? Na]?; HR-FAB-MS: m/z 595.1635 (Calcd for
C25H32O15Na [M ? Na]?: m/z 595.1639).
Mumeose S (4)
Mumeose P (1)
15
White amorphous powder; ½aꢁD ?30.6° (c = 0.11,
15
White amorphous powder; ½aꢁD ?32.0° (c = 0.13,
MeOH); IR(ATR): mmax 3400, 1730, 1716, 1603, 1516,
MeOH); IR(ATR): mmax 3400, 1730, 1716, 1604, 1515,
1457, 1369, 1228, 1152, 1032, 833 cm-1 1H NMR
;
1446, 1368, 1236, 1163, 1035, 835 cm-1
;
1H NMR
(methanol-d4, 500 MHz) d 1.82, 2.06, 2.08 (all s, CH3CO–
9 3), 3.56 (d, J = 12.5 Hz, H-1a), 3.67 (d, J = 12.4 Hz,
H-1b), 3.51 (m, H-20), 3.73 (m, H-30), 3.75–3.86 (m, H2-6),
4.08–4.14 (m, H2-60), 4.16 (m, H-5), 4.18 (m, H-50), 4.75
(m, H-40), 5.44 (m, H-4), 5.45 (d, J = 4.2 Hz, H-10), 5.61
(d, J = 5.4 Hz, H-3), 6.36 (d, J = 16.0 Hz, H-800), 6.82 (d,
J = 8.5 Hz, H-300, 500), 7.51 (d, J = 9.0 Hz, H-200, 600), 7.64
(d, J = 16.0 Hz, H-700); 13C NMR: given in Table 1;
positive-ion FAB-MS: m/z 637 [M ? Na]?; HR-FAB-MS:
m/z 637.1748 (Calcd for C27H34O16Na [M ? Na]?: m/z
637.1745).
(methanol-d4, 500 MHz) d 2.07, 2.08 (all s, CH3CO– 9 2),
3.39 (d, J = 11.9 Hz, H-1a), 3.58 (d, J = 12.2 Hz, H-1b),
3.34 (m, H-40), 3.78 (m, H-30), 3.80 (m, H2-6), 3.91 (m,
H-5), 4.09 (m, H-50), 4.16 (m, H-60a), 4.32 (dd, J = 8.5,
8.5 Hz, H-4), 4.47 (m, H-60b), 4.56 (m, H-20), 5.49 (d,
J = 8.6 Hz, H-3), 5.54 (d, J = 3.4 Hz, H-10), 6.43 (d,
J = 15.8 Hz, H-800), 6.80 (d, J = 8.5 Hz, H-300, 500), 7.52
(d, J = 8.6 Hz, H-200, 600), 7.71 (d, J = 15.8 Hz, H-700); 13C
NMR: given in Table 1; positive-ion FAB-MS: m/z 595
[M ? Na]?; HR-FAB-MS: m/z 595.1644 (Calcd for
C25H32O15Na [M ? Na]?: m/z 595.1639).
Mumeose T (5)
Mumeose Q (2)
15
White amorphous powder; ½aꢁD ?28.2° (c = 0.13,
15
White amorphous powder; ½aꢁD -30.8° (c = 0.15, MeOH)
MeOH); IR(ATR): mmax 3400, 1734, 1717, 1603, 1516,
IR(ATR): mmax 3400, 1730, 1717, 1604, 1516, 1457, 1368,
1457, 1373, 1229, 1150, 1041, 835 cm-1 1H-NMR
;
1220, 1154, 1034, 837 cm-1
;
1H NMR (methanol-d4,
(methanol-d4, 500 MHz) d 2.06, 2.07, 2.09, 2.13 (all s,
CH3CO– 9 4), 3.36 (m, H-50), 3.77 (m, H-60a), 3.81 (m,
H-30), 3.86 (m, H-60b), 4.09 (m, H2-1), 4.10 (m, H-5), 4.11
(m, H-40), 4.46 (m, H2-6), 4.64 (m, H-20), 5.50 (dd,
J = 7.2, 7.2 Hz, H-4), 5.59 (d, J = 3.6 Hz, H-10), 5.61 (d,
J = 7.5 Hz, H-3), 6.39 (d, J = 15.8 Hz, H-800), 6.81 (d,
J = 8.6 Hz, H-300, 500), 7.52 (d, J = 8.5 Hz, H-200, 600), 7.67
(d, J = 15.8 Hz, H-700); 13C NMR: given in Table 1;
positive-ion FAB-MS: m/z 679 [M ? Na]?; HR-FAB-MS:
m/z 679.1855 (Calcd for C29H36O17Na [M ? Na]?: m/z
679.1850).
500 MHz) d 1.95, 1.99 9 2, 2.05 (all s, CH3CO– 9 4),
3.45 (d, J = 11.9 Hz, H-1a), 3.63 (d, J = 11.9 Hz, H-1b),
3.76 (m, H2-6), 3.89 (m, H-5), 4.07–4.15 (m, H2-60), 4.16
(m, H-50), 4.26 (dd, J = 8.5, 8.5 Hz, H-4), 4.81 (m, H-20),
4.98 (dd, J = 8.9, 8.9 Hz, H-40), 5.34 (dd, J = 9.8, 9.8 Hz,
H-300), 5.48 (d, J = 8.3 Hz, H-3), 5.66 (d, J = 3.6 Hz,
H-1 ), 5.88 (d, J = 12.7 Hz, H-800), 6.76 (d, J = 8.5 Hz,
H-300, 500), 6.97 (d, J = 12.7 Hz, H-700), 7.68 (d, J = 8.8 Hz,
H-200, 600); 13C-NMR: given in Table 1; positive-ion FAB-
MS: m/z 679 [M ? Na]?; HR-FAB-MS: m/z 679.1847
(Calcd for C29H36O17Na [M ? Na]?: m/z 679.1850).
Mumeose U (6)
Mumeose R (3)
15
White amorphous powder; ½aꢁD ?44.2° (c = 0.42,
15
White amorphous powder; ½aꢁD ?38.5° (c = 0.33,
MeOH); IR(ATR): mmax 3400, 1732, 1717, 1604, 1516,
MeOH); IR(ATR): mmax 3400, 1730, 1713, 1604, 1516,
1437, 1368, 1226, 1152, 1032, 833 cm-1 1H-NMR
;
1443, 1369, 1238, 1158, 1035, 833 cm-1
;
1H NMR
(methanol-d4, 500 MHz) d 1.71, 2.00, 2.04, 2.08 (all s,
CH3CO– 9 4), 3.60 (d, J = 12.4 Hz, H-1a), 3.68 (d,
(methanol-d4, 500 MHz) d 1.89, 2.07 (all s, CH3CO– 9
123

J Nat Med
J = 12.4 Hz, H-1b), 3.73 (m, H-20), 3.81 (m, H2-6), 4.16
(m, H-5), 4.16 (m, H2-60), 4.35 (m, H-50), 4.88 (m, H-40),
5.23 (m, H-30), 5.47 (dd, J = 6.0, 6.0 Hz, H-4), 5.52 (d,
J = 3.7 Hz, H-10), 5.63 (d, J = 6.0 Hz, H-3), 6.38 (d,
J = 16.1 Hz, H-800), 6.80 (d, J = 8.6 Hz, H-300, 500), 7.54
(d, J = 8.6 Hz, H-200, 600), 7.73 (d, J = 15.8 Hz, H-700); 13C
NMR: given in Table 1; positive-ion FAB-MS: m/z 679
[M ? Na]?; HR-FAB-MS: m/z 679.1855 (Calcd for
C29H36O17Na [M ? Na]?: m/z 679.1850).
1 h. The reaction mixture was analyzed by reversed-phase
HPLC [column: COSMOSIL 5C18-AR-II, 250 9 4.6 mm
i.d.; mobile phase: MeCN– 0.05 M H3PO4 (23 : 77, v/v);
detection: UV (250 nm); flow rate: 0.8 ml/min; column
temperature: 35 °C] to identify the derivative of D-glucose
by comparison of the retention time with those of authentic
samples (tR: L-glucose; 17.8 min, D-glucose; 19.8 min).
Alkaline hydrolysis of 1–7
Mumeose V (7)
Compounds 1–7 (1.0 were treated with 10 % aqueous
KOH-1,4-dioxane (1:1, v/v, 1.0 ml) and stirred at 37 °C for
24 h. Each reaction mixture was neutralized with Dowex
HCR W2 (H? form), and the resin was removed by fil-
tration. Evaporation of the solvent from the filtrate under
reduced pressure yielded a crude product. The crude
product was dissolved in water and applied to normal-
phase HPLC {column: YMC-Pack Polyamine II,
250 9 4.6 mm i.d.; mobile phase: MeCN–H2O (3:1, v/v);
detection: optical rotation [Shodex OR-2 (Showa Denko
Co., Ltd., Tokyo, Japan)]; flow rate: 1.0 ml/min; column
temperature: ambient} to identify D-(?)-sucrose by com-
parison of the retention time and optical rotation with that
of authentic sample (tR = 19.8 min with positive rotation).
15
White amorphous powder; ½aꢁD ?28.0° (c = 0.29,
MeOH); IR(ATR): mmax 3400, 1742, 1716, 1604, 1516,
1435, 1367, 1218, 1150, 1034, 834 cm-1
;
1H-NMR
(methanol-d4, 500 MHz) d 1.76, 1.92, 2.00, 2.01, 2.03 (all
s, CH3CO– 9 5), 3.46 (d, J = 11.9 Hz, H-1a), 3.63 (d,
J = 12.2 Hz, H-1b), 3.74 (m, H2-6), 4.12 (m, H-5), 4.14
(m, H2-60), 4.33 (m, H-50), 4.84 (m, H-20), 4.96 (m, H-40),
5.39 (m, H-30), 5.45 (dd, J = 6.5, 6.5 Hz, H-4), 5.63 (d,
J = 6.5 Hz, H-3), 5.66 (d, J = 3.6 Hz, H-10), 6.30 (d,
J = 15.8 Hz, H-800), 6.77 (d, J = 8.0 Hz, H-300, 500), 7.49
(d, J = 8.0 Hz, H-200, 600), 7.74 (d, J = 15.8 Hz, H-700); 13C
NMR: given in Table 1; positive-ion FAB-MS: m/z 721
[M ? Na]?; HR-FAB-MS: m/z 721.1951 (Calcd for
C31H38O18Na [M ? Na]?: m/z 721.1956).
Effects on rat lens aldose reductase
Acid hydrolysis of 1–7
The experiments were performed as described in our pre-
vious reports [4]. The supernatant fluid of rat lens
homogenate was used as a crude enzyme. The enzyme
suspension was diluted to produce ca. 10 nmol/tube of
NADP in the following reaction. The incubation mixture
contained phosphate buffer 135 mM (pH 7.0), Li2SO4
100 mM, NADPH 0.03 mM, DL-glyceraldehyde 1 mM as
a substrate, and 100 ll of enzyme fraction, with 25 ll of
sample solution, in a total volume of 0.5 ml. The reaction
was initiated by the addition of NADPH at 30 °C. After
30 min, the reaction was stopped by the addition of 150 ll
of HCl 0.5 M. Then, 0.5 ml of NaOH 6 M containing
imidazole 10 mM was added, and the solution was heated
at 60 °C for 20 min to convert NADP into a fluorescent
product. Fluorescence was measured using a fluorescence
microplate reader (FLUOstar OPTIMA, BMG Labtech-
nologies) at an excitation wavelength of 360 nm and an
emission wavelength of 460 nm. Each test sample was
dissolved in DMSO. Measurements were performed in
duplicate, and IC50 values were determined graphically.
Compounds 1–7 (0.8 mg) were dissolved in 5 % aqueous
H2SO4–1,4-dioxane (1:1, v/v, 1.0 ml), and each solution
was heated at 90 °C for 3 h, and neutralized with Amber-
lite IRA-400 (OH- form). After drying in vacuo, a small
part of the residue of 1–7 was dissolved in H2O-MeOH
(1:1, v/v) and analyzed by reversed-phase HPLC [column:
COSMOSIL 5C18-MS-II, 250 9 4.6 mm i.d.; mobile
phase A: H2O-AcOH (1000:3, v/v), B: MeCN-AcOH
(1000:3, v/v), Linear gradient: mobile phase A–B
(90:10 ? 72:28, v/v, in 24 min); detection: UV (300 nm);
flow rate: 1.0 ml/min; column temperature: ambient] to
identify (E)-p-coumaric acid (from 1 and 3–7) and (Z)-p-
coumaric acid (from 2) by comparison of the retention time
with that of authentic sample [tR = 17.1 min for (E)-p-
coumaric acid, 18.5 min for (Z)-p-coumaric acid]. In
addition, glucose and fructose from a small part of the
residue of 1–7 were identified by TLC (ordinary phase)
[CHCl3–MeOH–H2O (30:15:3, v/v/v)]. Furthermore, the
remaining parts of the residue from 1–7 were dissolved in
pyridine (0.1 ml) containing L-cysteine methyl ester
hydrochloride (0.5 mg) and each solution was heated at
60 °C for 1 h. o-Tolyl isothiocyanate (0.5 mg) in pyridine
(0.1 ml) was added to the mixture and heated at 60 °C for
Acknowledgments This research was supported in part by a Min-
istry of Education, Culture, Sports, Science and Technology (MEXT)-
Supported Program for the Strategic Research Foundation at Private
Universities, and by a Grant-in-Aid for Young Scientists (B) from the
Japan Society for the Promotion of Science (JSPS).
123

J Nat Med
Miki H, Matsuda H, Muraoka O, Yoshikawa M (2013) Alkaloid
constituents from flower buds and leaves of sacred lotus (Nel-
umbo nucifera, Nymphaeaceae) with melanogenesis inhibitory
activity in B16 melanoma cells. Bioorg Med Chem 21:779–787
11. Liu J, Nakamura S, Matsuda H, Yoshikawa M (2013) Hyd-
rangeamines A and B, novel polyketide-type pseudoalkaloid-
coupled secoiridoid glycosides from the flowers of Hydrangea
macrophylla var. thunbergii. Tetrahedron Lett 54:32–34
12. Liu J, Nakamura S, Zhuang Y, Yoshikawa M, Hussein GME,
Matsuo K, Matsuda H (2013) Medicinal flowers. XXXX. Struc-
tures of dihydroisocoumarin glycosides and inhibitory effects on
aldose reductase from the flowers of Hydrangea macrophylla var.
thunbergii. Chem Pharm Bull 61:655–661
13. 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
14. Yoshinari K, Sashida Y, Mimaki Y, Shimamura M (1990) New
polyacylated sucrose derivatives from the bark of Prunus padus.
Chem Pharm Bull 38:415–417
15. Matsuda H, Morikawa T, Toguchida I, Harima S, Yoshikawa M
(2002) Medicinal flowers. VI. Absolute stereostructures of two
new flavanone glycosides and a phenylbutanoid glycoside from
the flowers of Chrysanthemum indicum L.: their inhibitory
activities for rat lens aldose reductase. Chem Pharm Bull
50:972–975
16. Matsuda H, Morikawa T, Toguchida I, Yoshikawa M (2002)
Structural requirements of flavonoids and related compounds for
aldose reductase inhibitory activity. Chem Pharm Bull
50:788–795
17. Morikawa T, Kishi A, Pongpiriyadacha Y, Matsuda H, Yoshik-
awa M (2003) Structures of new friedelane-type triterpenes and
eudesmane-type sesquiterpene and aldose reductase inhibitors
from Salacia chinensis. J Nat Prod 66:1191–1196
18. Xie H, Wang T, Matsuda H, Morikawa T, Yoshikawa M, Tani T
(2005) Bioactive constituents from Chinese natural medicines.
XV. Inhibitory effect on aldose reductase and structures of sau-
ssureosides A and B from Saussurea medusa. Chem Pharm Bull
53:1416–1422
References
1. Yoshikawa M, Murakami T, Ishikawa T, Morikawa T, Kagawa
M, Higashi Y, Matsuda H (2002) New flavonol oligoglycosides
and polyacylated sucroses with inhibitory effects on aldose
reductase and platelet aggregation from the flowers of Prunus
mume. J Nat Prod 65:1151–1155
2. Matsuda H, Morikawa T, Ishiwada T, Managi H, Kagawa M,
Higashi Y, Yoshikawa M (2003) Medicinal flowers. VIII. Radical
scavenging constituents from the flowers of Prunus mume:
structure of prunose III. Chem Pharm Bull 51:440–443
3. Nakamura S, Fujimoto K, Matsumoto T, Nakashima S, Ohta T,
Ogawa K, Matsuda H, Yoshikawa M (2013) Acylated sucroses
and acylated quinic acids analogs from the flower buds of Prunus
mume and their inhibitory effects on melanogenesis. Phyto-
chemistry 92:128–136
4. Fujimoto K, Nakamura S, Matsumoto T, Ohta T, Ogawa K,
Tamura H, Matsuda H, Yoshikawa M (2013) Medicinal flowers.
XXXVIII. Structures of acylated sucroses and inhibitory effects
of constituents on aldose reducatase from the flower buds of
Prunus mume. Chem Pharm Bull 61:445–451
5. Nakamura S, Fujimoto K, Matsumoto T, Ohta T, Ogawa K,
Tamura H, Matsuda H, Yoshikawa M (2013) Structures of
acylated sucroses and an acylated flavonol glycoside and inhib-
itory effects of constituents on aldose reductase from the flower
buds of Prunus mume. J Nat Med 67:799–806
6. Matsuda H, Hamao M, Nakamura S, Kon’i H, Murata M, Yos-
hikawa M (2012) Medicinal flowers. XXXIII. Anti-hyperlipi-
demic and anti-hyperglycemic effects of chakasaponins I-III and
structure of chakasaponin IV from flower buds of Chinese Tea
Plant (Camellia sinensis). Chem Pharm Bull 60:674–680
7. Nakamura S, Moriura T, Park S, Fujimoto K, Matsumoto T, Ohta
T, Matsuda H, Yoshikawa M (2012) Melanogenesis inhibitory
and fibroblast proliferation accelerating effects of noroleanane-
and oleanane-type triterpene oligoglycosides from the flower
buds of Camellia japonica. J Nat Prod 75:1425–1430
8. Nakamura S, Fujimoto K, Nakashima S, Matsumoto T, Miura T,
Uno K, Matsuda H, Yoshikawa M (2012) Medicinal flowers.
XXXVI. Acylated oleanane-type triterpene saponins with inhib-
itory effects on melanogenesis from the flower buds of Chinese
Camellia japonica. Chem Pharm Bull 60:752–758
9. Fujimoto K, Nakamura S, Nakashima S, Matsumoto T, Uno K,
Ohta T, Miura T, Matsuda H, Yoshikawa M (2012) Nor-olean-
ane-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
10. Nakamura S, Nakashima S, Tanabe G, Oda Y, Yokota N, Fu-
jimoto K, Matsumoto T, Sakuma R, Ohta T, Ogawa K, Nishida S,
19. Morikawa T, Xie H, Wang T, Matsuda H, Yoshikawa M (2008)
Bioactive constituents from Chinese natural medicines. XXXII.
Aminopeptidase N and aldose reductase inhibitors from Sino-
crassula indica: structures of sinocrassosides B4, B5, C1, and D1–
D3. Chem Pharm Bull 56:1438–1444
20. Matsuda H, Asao Y, Nakamura S, Hamao M, Sugimoto S, Hongo
M, Pongpiriyadacha Y, Yoshikawa M (2009) Antidiabetogenic
constituents from the Thai traditional medicine Cotylelobium
melanoxylon. Chem Pharm Bull 57:487–494
123