Medicinal flowers. XXXVIII.1) structures of acylated sucroses and inhibitory effects of constituents on aldose reducatase from the flower buds of Prunus mume

Article abstract of DOI:10.1248/cpb.c12-01068

The methanolic extract from the flower buds of Prunus mume, cultivated in Zhejiang province, China, showed an inhibitory effect on aldose reductase. From the methanolic extract, five new acylated sucroses, mumeoses F-J, were isolated together with 29 known compounds. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of the isolated compounds on aldose reductase were also investigated. Acylated quinic acid analogs, which are one of the major compounds of the flower buds of P. mume, were shown to substantially inhibit aldose reductase. In particular, mumeic acid-A was found to exhibit a potent inhibitory effect [IC₅₀=0.4 μm].

Full text of DOI:10.1248/cpb.c12-01068

April 2013
Regular Article
Chem. Pharm. Bull. 61(4) 445–451 (2013)
445
1)
Medicinal Flowers. XXXVIII. Structures of Acylated Sucroses and
Inhibitory Effects of Constituents on Aldose Reducatase from the Flower
Buds of Prunus mume
Katsuyoshi Fujimoto, Seikou Nakamura, Takahiro Matsumoto, Tomoe Ohta, Keiko Ogawa,
Haruka Tamura, Hisashi Matsuda, and Masayuki Yoshikawa*
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received December 13, 2012; accepted January 25, 2013
The methanolic extract from the flower buds of Prunus mume, cultivated in Zhejiang province, China,
showed an inhibitory effect on aldose reductase. From the methanolic extract, five new acylated sucroses,
mumeoses F–J, were isolated together with 29 known compounds. The chemical structures of the new com-
pounds were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of the
isolated compounds on aldose reductase were also investigated. Acylated quinic acid analogs, which are one
of the major compounds of the flower buds of P. mume, were shown to substantially inhibit aldose reductase.
In particular, mumeic acid-A was found to exhibit a potent inhibitory effect [IC =0.4µm].
5
0
Key words Prunus mume; mumeose; acylated sucrose; medicinal flower; aldose reductase; acylated quinic
acid analog
In the course of our studies on bioactive constituents from silica-gel column chromatography and repeated HPLC. From
2
–6)
medicinal flower,
structure elucidation of acylated sucroses named prunoses I F (1, 0.00117%), mumeose G (2, 0.0017%), H (3, 0.0005%),
11), II (12), and III (13) with inhibitory effects on platelet ag- I (4, 0.0003%), J (5, 0.0033%) was isolated together with 29
gregation and aldose reductase and radical scavenging effect known compounds, 2′,3′,4′,6′-tetra-O-acetyl-3-O-(E)-p-cou-
we previously reported the isolation and those fractions, five new acylated sucroses called mumeose
(
9
)
1)
from the flower buds of Prunus (P.) mume Sieb. et Zucc. maroylsucrose (14, 0.0127%), mumeic acid-A (15, 0.0039%),
Rosaceae) cultivated in Wakayama prefecture, Japan.
addition, the methanolic (MeOH) extract from the flower buds coumaroylquinic acid (17, 0.0132%), chlorogenic acid (18,
of P. mume cultivated in Zhejiang province, China, showed 0.0120%), 5-O-(E)-p-coumaroylquinic acid methyl ester (19,
inhibitory effects on melanogenesis. From the MeOH extract, 0.0129%), chlorogenic acid methyl ester (20, 0.0346%),
five new acylated sucroses, mumeoses A (6), B (7), C (8), D 5-O-(E)-feruloyl quinic acid methyl ester (21, 0.0129%),
9), and E (10), and three new acylated quinic acid analogs, 5-O-(E)-p-coumaroylquinic acid ethyl ester (22, 0.0031%),
mumeic acid-A (15) and its methyl ester (16) and 5-O-(E)-p- chlorogenic acid ethylester (23, 0.0378%), (E)-p-coumaric
7,8)
1)
(
In mumeic acid-A methyl ester (16, 0.0034%), 5-O-(E)-p-
10)
11)
12)
13)
14)
1)
(
15)
16)
17)
coumaroylquinic acid ethyl ester (22), were isolated and their acid (24, 0.0007%), (E)-caffeic acid (25, 0.0016%), (E)-
structures as well as the inhibitory effects on melanogenesis ferulic acid (26, 0.0015%), (S,R)-1-O-caffeoylglycerol (27,
were characterized.
16)
1)
18)
19)
0.0011%), (S,R)-1-O-feruloylglycerol (28, 0.0043%), eu-
2
0)
As a continuing study, five new acylated sucroses termed genyl β-d-glucopyranoside (29, 0.0006%), 1-O-(3-hydroxy-
21)
mumeoses F (1), G (2), H (3), I (4), and J (5) were isolated 4-methoxybenzoyl)-β-d-glucopyranose
(30,
quercetin 3′-O-(2″-O-acetyl)-
quercetin 3-O-(6″-O-acetyl)-β-d-
This paper deals with the structure elucidation of new acyl- glucopyranoside, isorhamnetin 3-O-β-d-glucopyranoside,
0.0005%),
1)
from the flower buds of Chinese Prunus mume together with mumeoses A–E (6−10),
2
2)
2
9 known compounds including acylated quinic acid analogs. β-d-glucopyranoside,
2
3)
24)
2
5)
ated sucroses (1–5) and the inhibitory effects of principle quercetin 3-O-(6″-O-benzoyl)-β-d-galactopyranoside,
constituents on aldose reductase.
iso-
2
6)
27)
rhamnetin 3-O-β-d-galactopyranoside,
d-mandelic acid,
2
7)
The MeOH extract (30.4% from the dried flower buds of P. and quercetin. In this isolation procedure, acylated sucroses,
mume cultivated in Zhejiang province, China) was found to prunoses I (11), II (12), and III (13), which were isolated from
show an inhibitory effect on aldose reductase. [IC ꢀ3.0 µg/ the flower buds of Japanese P. mume, could not be isolated
5
0
mL]. The MeOH extract was partitioned into an EtOAc–H O from the flower buds of Chinese P. mume.
2
(1:1, v/v) mixture to furnish an EtOAc-soluble fraction (6.6%)
Structures of Mumeoses F–J (1–5) Mumeose F (1) was
and an aqueous layer. The aqueous layer was further extracted isolated as a white amorphous powder with positive optical ro-
1
5
with 1-butanol to give 1-butanol- (7.5%) and H O- (13.0%) sol- tation (1: [α]D +40.8°, in MeOH). Its IR spectrum showed ab-
2
−1
uble fractions. The 1-butanol- and the EtOAc-soluble fractions sorption bands at 3400, 1730, 1716, 1604, 1515, and 1031cm
were found to have significant inhibitory effects on aldose re- due to hydroxyl, ester, α,β-unsaturated ester, aromatic ring,
ductase [IC ꢀ1.7, 1.5 µg/mL, respectively], whereas the H O- and ether functions. The positive-ion mode FAB-MS of 1 re-
5
0
2
+
soluble fraction [IC ꢀ3.5 µg/mL] showed weaker effects than vealed a quasimolecular ion peak ([M+Na] ) at m/z 637 from
those of the 1-butanol- and the EtOAc-soluble fractions.
5
0
which the molecular formula C H O was determined by
27 34 16
The 1-butanol- and EtOAc-soluble fractions with the in- high-resolution (HR) MS measurement. Treatment of 1 with a
hibitory activity were subjected to normal- and reversed-phase 10% aqueous KOH–1,4-dioxane (1:1, v/v) mixture yielded su-
crose, which was identified by HPLC analysis. Acid hydrolysis
The authors declare no conflict of interest.
of 1 with 5% aqueous H SO –1,4-dioxane yielded d-glucose
2 4
*
To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp
© 2013 The Pharmaceutical Society of Japan

4
46
Vol. 61, No. 4
Fig. 1. Structure of Constituents Isolated from the Flower Buds of P. mume
Fig. 2. Important 2D-NMR Correlations of 1–5
and fructose together with (E)-p-coumaric acid. d-Glucose of three acetyl groups and a (E)-p-coumaroyl moiety were
was identified by HPLC of the tolylthiocarbamoyl thiazoli- confirmed based on heteronuclear multiple bond connectivity
2
8)
dine derivative. (E)-p-Coumaric acid was also identified by (HMBC) spectroscopy analysis. Namely, long-range correla-
1
13
HPLC. The H-NMR (methanol-d ) and C-NMR (Table 1) tions were observed between the following proton and carbon
4
spectra of 1, which were assigned by various NMR experi- pairs: H-3 and C-9″, H-3′, 4′, 6′ and three acetyl carbonyl car-
2
9)
ments, showed signals assignable to three acetyl groups [δ bon, H-2″, 6″ and C-1″, 7″, H-3″, 5″ and C-4″, H-7″ and C-9″,
1
.78, 1.98, and 2.06 (all s, CH CO– ×3)] and a (E)-p-couma- H-8″ and C-9″, three acetyl proton and three acetyl carbonyl
3
royl group [δ 6.42 (d, Jꢀ15.9Hz, H-8″), 6.80 (d, Jꢀ8.2Hz, carbon. On the basis of all this evidence, the chemical struc-
H-3″, 5″), 7.74 (d, Jꢀ15.9Hz, H-7″), 7.54 (d, Jꢀ8.2Hz, H-2″, ture of mumeose F (1) was determined to be 3′,4′,6′-tri-O-
6
(
″)] and a sucrose moiety [δ 3.59 (d, Jꢀ12.2Hz, H-1a), 3.68 acetyl-3-O-(E)-p-coumaroylsucrose.
d, Jꢀ12.2Hz, H-1b), 3.71 (dd, Jꢀ3.7, 9.0Hz, H-2′), 3.80 (m,
H -6), 3.98 (m, H-5), 4.15 (m, H -6′), 4.32 (m, H-4), 4.32 white amorphous powder with positive optical rotations (2:
Mumeoses G (2) and H (3), which were obtained as a
2
2
15
15
(
m, H-5′), 4.88 (m, H-4′), 5.22 (dd, Jꢀ9.0, 9.0Hz, H-3′), 5.45 [α] +47.3°; 3: [α]D +29.0°, in MeOH), showed absorption
D
(d, Jꢀ7.1Hz, H-3), 5.62 (d, Jꢀ3.7Hz, H-1′)]. The positions bands due to hydroxyl, ester, α,β-unsaturated ester, aromatic

April 2013
447
13
Table 1.
C-NMR (125MHz) Spectroscopic Data for Compounds 1–5 in CD OD
3
Position
1
2
3
4
5
1
2
3
4
5
6
65.4
105.6
79.3
74.6
84.2
64.6
105.6
77.3
76.4
82.5
63.5
106.5
76.8
76.8
82.5
65.3
105.8
79.3
74.8
81.9
63.4
105.0
78.1
76.7
83.6
63.5
64.9
63.8
65.3
63.4
1
2
3
4
5
6
1
″, 6″
″, 5″
4
7
8
9
′
′
′
′
′
′
″
92.8
70.9
74.4
70.3
69.7
63.8
127.3
131.5
116.8
161.4
147.7
114.6
168.1
171.5
93.3
71.2
76.7
69.8
72.3
64.6
127.2
131.6
116.7
161.4
147.9
114.3
168.1
172.0
172.7
172.8
91.4
72.3
73.8
69.2
74.1
61.7
127.1
131.6
116.8
161.5
148.0
114.1
168.1
172.0
172.2
172.3
92.7
70.9
74.4
70.4
69.7
64.0
127.2
131.5
116.8
161.5
147.7
114.5
167.9
171.5
172.2
172.5
91.3
71.5
71.1
69.9
69.8
65.4
127.1
131.7
116.8
161.6
148.3
113.9
167.5
171.3
171.5
171.8
171.8
2
3
″
″
″
″
CH COO–
3
1
1
72.1
72.5
1
72.6
1
1
71.9
72.5
20.3
20.5
20.6
20.7
CH COO–
20.5
20.7
20.7
21.1
20.7
20.9
20.9
20.4
20.7
20.7
3
2
2
0.7
0.8
2
0.8
2
2
0.7
0.8
ring, and ether functions in their IR spectra. In the FAB-MS around the glucose part of 3. The positions of acetyl groups
spectra of 2 and 3, the common quasimolecular ion peak was of 3 were confirmed based on HMBC spectroscopy. Namely,
+
observed at m/z 637 ([M+Na] ) and the molecular formula long-range correlations were observed between the following
C H O was determined by HR-MS measurement. The al- proton and carbon pairs: H-4, 2′, 3′ and three acetyl carbonyl
2
7
34 16
kaline hydrolysis of 2 and 3 yielded sucrose, whereas the acid carbons. On the basis of all this evidence, the chemical struc-
hydrolysis of 2 and 3 yielded d-glucose and fructose together tures of mumeose G (2) and mumeose H (3) were determined
2
8)
1
with (E)-p-coumaric acid. The H-NMR (methanol-d ) and to be 4,3′,6′-tri-O-acetyl-3-O-(E)-p-coumaroylsucralose and
4
13
29)
C-NMR (Table 1) spectra of 2 showed signals assignable 4,2′,3′-tri-O-acetyl-3-O-(E)-p-coumaroylsucrose, respectively.
to three acetyl groups [δ 2.06, 2.06, and 2.10 (all s, CH CO–
Mumeoses I (4) and J (5), obtained as a white amorphous
3
15
15
×
6
7
3)], a (E)-p-coumaroyl group [δ 6.38 (d, Jꢀ16.1Hz, H-8″), powder with positive optical rotations (4: [α]D +38.9°, 5: [α]D
.79 (d, Jꢀ8.3Hz, H-3″, 5″), 7.53 (d, Jꢀ8.3Hz, H-2″, 6″), +28.9°, in MeOH), showed absorption bands due to hydroxyl,
.65 (d, Jꢀ16.1Hz, H-7″)] and a sucrose moiety [δ 3.48 (dd, ester, α,β-unsaturated ester, aromatic ring and ether functions
Jꢀ9.4, 9.4Hz, H-4′), 3.60 (m, H -1), 3.64 (m, H-2′), 3.77 (m, in their IR spectra. Their molecular formulas (C H O of
2
29 36 17
H-6a), 3.89 (m, H-6b), 4.13 (m, H-5), 4.20 (m, H-5′), 4.20 (m, 4, C H O of 5) were determined from the quasimolecular
33
40 19
+
+
H-6′a), 4.48 (m, H-6′b), 5.23 (dd, Jꢀ9.4, 9.4Hz, H-3′), 5.51 (d, ion peaks (m/z 679 [M+Na] for 4, 763 [M+Na] for 5) in
Jꢀ3.4Hz, H-1′), 5.53 (m, H-4), 5.70 (d, Jꢀ7.5Hz, H-3)]. The the positive-ion FAB-MS and by HR-MS measurement. The
positions of three acetyl groups and a (E)-p-coumaroyl moiety alkaline hydrolysis of 4 and 5 yielded sucrose and the acid
were confirmed by HMBC spectroscopy analysis. Namely, hydrolysis of 4 and 5 yielded d-glucose, fructose, and (E)-p-
long-range correlations were observed between the following coumaric acid, respectively.
proton and carbon pairs: H-3 and C-9″, H-4, 3′, 6′ and three and C-NMR (Table 1) spectra of 4 and 5 showed signals
acetyl carbonyl carbons. The H-NMR (methanol-d ) and assignable to four acetyl groups [δ 1.76, 1.99, 2.06, and 2.08
2
8)
1
The H-NMR (methanol-d4)
13
29)
1
4
13
C-NMR (Table 1) spectra of 3 showed signals assignable (all s, CH CO– ×4) for 4] and six acetyl groups [δ 1.78, 1.94,
3
to three acetyl groups [δ 2.03, 2.05, and 2.05 (all s, CH CO- 2.04, 2.07, 2.09, and 2.09 (all s, CH CO– ×6) for 5], respec-
3
3
×
3)], a (E)-p-coumaroyl group [δ 6.39 (d, Jꢀ16.0Hz, H-8″), tively, together with a (E)-p-coumaroyl group and a sucrose
.79 (d, Jꢀ8.6Hz, H-3″, 5″), 7.52 (d, Jꢀ8.6Hz, H-2″, 6″), 7.65 moiety. The positions of acetyl groups of 4 and 5 were con-
d, Jꢀ16.0Hz, H-7″)] and a sucrose moiety. The proton and firmed based on HMBC spectroscopy. Namely, long-range
6
(
1
13
carbon signals in the H- and C-NMR (Table 1) spectra of correlations were observed between the following proton and
were superimposable on those of 2, except for the signals carbon pairs [H-6, 3′, 4′, 6′ and four acetyl carbonyl carbons
3

4
48
Vol. 61, No. 4
for 4, H-1, 4, 2′, 3′, 4′, 6′ and six acetyl carbonyl carbons for detector; and HPLC column, Inertsil ODS-3 (150×3.0mm
]. Consequently, the chemical structures of mumeoses I (4) i.d.), COSMOSIL 5C18-MS-II (250×4.6mm i.d., 250×10mm
and J (5) were characterized to be 6,3′,4′,6′-tetra-O-acetyl- i.d. and 250×20mm i.d.), 5C18-PAQ (250×20mm i.d.) and
5
3
3
-O-(E)-p-coumaroylsucrose and 1,4,2′,3′,4′,6′-hexa-O-acetyl- 5C18-AR-II (250×4.6mm i.d.) columns were used for ana-
-O-(E)-p-coumaroylsucrose, respectively. lytical and preparative purposes. The following experimental
Inhibitory Effect on Rat Lens Aldose Reductase Al- materials were used for chromatography: normal-phase silica
dose reductase, a key enzyme in the polyol pathway, has gel column chromatography, Silica gel BW-200 (Fuji Silysia
been reported to catalyze the reduction of glucose to sorbitol. Chemical, Ltd., 150–350 mesh); reversed-phase silica gel
Sorbitol does not readily diffuse across cell membranes, and column chromatography, Chromatorex ODS DM1020T (Fuji
the intracellular accumulation of sorbitol has been implicated Silysia Chemical, Ltd., 100–200 mesh); TLC, precoated TLC
in the chronic complications of diabetes such as cataract. plates with Silica gel 60F254 (Merck, 0.25mm) (ordinary
Previously, we have reported that various constituents such phase) and Silica gel RP-18 F254S (Merck, 0.25mm) (reversed
as flavonoids and terpenoids from natural medicines and me- phase); reversed-phase HPTLC, precoated TLC plates with
3
0–35)
dicinal foods inhibited aldose reductase inhibitory effect.
In addition, we reported that acylated sucrose, prunose I (11), achieved by spraying with 1% Ce(SO ) –10% aqueous H SO
4
Silica gel RP-18 WF254S (Merck, 0.25mm); and detection was
4
2
2
7
)
exhibited moderate inhibitory effect [IC ꢀ58µm] and chlo- followed by heating.
rogenic acid methyl ester (20) showed strong inhibitory effect
IC ꢀ1.3 µm]. In the present study, the inhibitory effects on vated in Zhejiang province, China, were the commercial prod-
aldose reductase of the principal isolated constituents from the ucts purchased from Tochimoto Tenkaido Co., Ltd. (Osaka,
5
0
Plant Material The dried flower buds of P. mume culti-
32)
[
50
flower buds of P. mume cultivated in China were examined Japan) in April 2011.
for structure–activity relationship study (Table 2). Among
Extraction and Isolation The dried flower buds (2.0kg)
these principal constituents, acylated quinic acid analogs were were extracted three times with methanol under reflux for 3h.
shown to substantially inhibit aldose reductase. In paricular, Evaporation of the solvent under reduced pressure provided a
mumeic acid-A (15) and mumeic acid-A methyl ester (16), MeOH extract (608.0g, 30.4%). A part of the MeOH extract
which are quinic acids with benzoyl and cafferoyl groups, (486.4g) was partitioned into an EtOAc–H O (1:1, v/v) mix-
2
were found to exhibit a potent inhibitory effects [IC ꢀ0.4µm ture to furnish an EtOAc-soluble fraction (106.0g, 6.6%) and
5
0
for 15, 0.6µm for 16]. The biological effects were equal to an aqueous phase. The aqueous phase was further extracted
or stronger than that of a reference compound, chlorogenic with 1-butanol to give a 1-butanol-soluble fraction (119.3g,
acid [18, IC ꢀ0.7 µm], which is reported to have inhibi- 7.5%) and an H O-soluble fraction (256.0g, 16.0%). A part of
5
0
2
3
6)
tory effect. On the other hand, 20 showed the similar value the 1-butanol-soluble fraction (80.0g) was subjected to normal
compared with the previous reported data. With regard to phase silica gel column chromatography [2.4kg, n-hexane→
32)
structural requirements of acylated quinic acid analogs 15–23 CHCl –MeOH (50:1→20:1→10:1→5:1→10:3, v/v)→MeOH]
3
for the effect, quinic acid analogs (15, 16, 18, 20, 23) with a to give seven fractions [Fr. B1 (450mg), Fr. B2 (960mg), Fr.
cafferoyl group showed stronger inhibitory effect than quinic B3 (850mg), Fr. B4 (1.37g), Fr. B5 (4.70g), Fr. B6 (7.24g), Fr.
1)
acid analogs (17, 19, 21, 22) with a coumaroyl or a feruloyl B7 (41.4g)] as reported previously.
group, (E)-p-coumaric acid (24), (E)-caffeic acid (25), and Fraction B5 (4.70g) was further separated by reversed
(
E)-p-coumaric acid (26). These findings suggest that the caf- phase silica gel column chromatography [132g, MeOH–H O
2
feroyl group is essential for the effect, and the quinic acid part
is important for the enhancement of the effect. On the other Table 2. Inhibitory Effects on Aldose Reductase of Compounds from the
Flower Buds of P. mume
hand, acylated sucrose 1, 2, 3, and 5 exhibited moderate in-
hibitory effect [IC ꢀ23–51 µm].
5
0
Sample
IC50 (µm)
In conclusion, five new acylated sucroses, mumeose F (1),
G (2), H (3), I (4), and J (5), were isolated from the flower
buds of P. mume cultivated in Zhejiang province, China. In
addition, acylated quinic acid analogs, which are one of the
major compounds of the flower buds of P. mume, substantially
exhibited the inhibitory effects on aldose reductase. Further
study for the development of acylated quinic acid analogs as
potent anti-cataract agents are expected.
Mumeose F (1)
Mumeose G (2)
Mumeose H (3)
Mumeose J (5)
Mumeic acid-A (15)
44.7
51.0
41.7
22.9
0.4
0.6
7.5
8.9
Mumeic acid-A methyl ester (16)
5
5
-O-(E)-p-Coumaroylquinic acid (17)
-O-(E)-p-Coumaroylquinic acid methyl ester (19)
Chlorogenic acid methyl ester (20)
5-O-(E)-Feruloylquinic acid methyl ester (21)
5-O-(E)-p-Coumaroylquinic acid ethyl ester (22)
Chlorogenic acid ethyl ester (23)
2.4
72.2
7.0
Experimental
General Experimental Procedures The following in-
struments were used to obtain physical data: specific rota-
tions, Horiba SEPA-300 digital polarimeter (lꢀ5cm); IR
spectra, a Thermo Electron Nexus 470; electron ionization
1.5
(E)-p-Coumaric acid (24)
>100
30.9
>100
36.9
>100
9.7
(
(
(
E)-Caffeic acid (25)
E)-Ferulic acid (26)
S,R)-1-O-Feruloylglycerol (28)
(
EI)-MS and HR-EI-MS, JEOL JMS-GCMATE mass spec-
trometer; FAB-MS and HR-FAB-MS, a JEOL JMS-SX 102A
mass spectrometer; H-NMR spectra, JEOL JNM-LA 500
1
Eugenyl β-d-glucopyranoside (29)
30
13
(
(
500MHz) spectrometer; C-NMR spectra, JEOL JNM-LA
125MHz) spectrometer with tetramethylsilane as an internal
standard; HPLC detector, Shimadzu SPD-10AVP UV/VIS
Chlorogenic acid (18) (positive control)
0.7
Each value represents the mean of 2–4 experiments.

April 2013
449
(20:80→30:70→40:60→50:50→60:40, v/v)→MeOH] to Fraction E5-4 (7.18g) was further separated by reversed
give ten fractions [Fr. B5-1 (0.29g), Fr. B5-2 (0.73g), Fr. B5-3 phase silica gel column chromatography [200g, MeOH–H O
2
(0.15g), Fr. B5-4 (0.26g), Fr. B5-5 (0.32g), Fr. B5-6 (0.28g), (20 : 80→30 : 70→40 : 60→50 : 50→60 : 40→80 : 20→90 : 10,
Fr. B5-7 (0.22g), Fr. B5-8 (0.10g), Fr. B5-9 (0.17g), Fr. B5-10 v/v)→MeOH] to give nine fractions [Fr. E5-4-1, Fr. E5-4-2
(
0.46g)]. Fraction B5-4 (260mg) was purified by HPLC [H O– (959mg), Fr. E5-4-3, Fr. E5-4-4 (220mg), Fr. E5-4-5 (162mg),
2
MeOH–AcOH (700:300:3, v/v/v), COSMOSIL 5C18-MS-II] Fr. E5-4-6 (560mg), Fr. E5-4-7, Fr. E5-4-8 (538mg), Fr.
to give 25 (17mg) and 27 (12mg). Fraction B5-9 (170mg) was E5-4-9 (170mg)]. Fraction E5-4-2 (959mg) was purified by
purified by HPLC [H O–MeOH–AcOH (650:350:3, v/v/v), HPLC [H O–MeCN–AcOH (830:170:3, v/v/v), COSMOSIL
2
2
COSMOSIL 5C18-MS-II] to give two fractions [Fr. B5-9-1 5C18-MS-II] to give 25 (168mg). Fraction E5-4-4 (220mg)
(
61mg), Fr. B5-9-2 (39mg)]. Fraction B5-9-2 (39mg) was was purified by HPLC [H O–MeCN–AcOH (800:200:3,
2
purified by HPLC [H O–MeCN–AcOH (800:200:3, v/v/v), v/v/v), COSMOSIL 5C18-MS-II] to give 28 (8.4mg). A part
2
COSMOSIL 5C18-MS-II] to give 1 (13mg) and 29 (6.1mg). of fraction E5-4-6 (200mg) was purified by HPLC [H O–
2
Fraction B6 (7.24g) was further separated by silica gel column MeCN–MeOH–AcOH (650:200:150:3, v/v/v/v), COSMOSIL
chromatography [230g, CHCl –EtOAc (1:1, v/v)→CHCl – 5C18-PAQ] to give 14 (37mg). Fraction E5-5 (3.56g) was
3
3
EtOAc–2-propanol–MeOH (20:20:1:1→15:15:1:1→10:10: further separated by reversed phase silica gel column chro-
:1→5:5:1:1→1:1:1:1, v/v)→MeOH] to give five frac- matography [120g, MeOH–H O (20:80→30:70→40:60→
1
2
tions [Fr. B6-1 (70mg), Fr. B6-2 (1.56g), Fr. B6-3 (3.83g), Fr. 50:50→60:40→80:20→90:10, v/v)→MeOH] to give nine
B6-4 (0.92g), Fr. B6-5 (0.49g)]. Fraction B6-3 (3.83g) was fractions [Fr. E5-5-1 (147mg), Fr. E5-5-2 (207mg), Fr. E5-5-3
purified by reversed phase silica gel column chromatogra- (326mg), Fr. E5-5-4 (594mg), Fr. E5-5-5 (313mg), Fr. E5-5-6
phy [132g, MeOH–H O (20:80→30:70→40:60→50:50→ (152mg), Fr. E5-5-7 (261mg), Fr. E5-5-8 (114mg), Fr. E5-5-9
2
6
0:40, v/v)→MeOH] to give nine fractions [Fr. B6-3-1 (614mg)]. Fraction E5-5-5 (313mg) was further separated by
(
186mg), Fr. B6-3-2 (2.47g), Fr. B6-3-3, Fr. B6-3-4 (520mg), HPLC [H O–MeCN–AcOH (750:250:3, v/v/v), COSMOSIL
2
Fr. B6-3-5 (158mg), Fr. B6-3-6, Fr. B6-3-7, Fr. B6-3-8 (51mg), 5C18-MS-II] to give three fractions [Fr. E5-5-5-1 (65mg),
Fr. B6-3-9]. Fraction B6-3-1 (186mg) was purified by HPLC Fr. E5-5-5-2 (16mg), Fr. E5-5-5-3 (13mg)]. Fraction E5-5-5-1
[
H O–MeOH–AcOH (800:200:3, v/v/v)] to give 30 (5.4mg). (65mg) was purified by HPLC [H O–MeCN–MeOH–AcOH
2
2
A part of the EtOAc-soluble fraction (80.0g) was subjected (700:150:150:3, v/v/v/v), COSMOSIL 5C18-MS-II] to give
to normal phase silica gel column chromatography [2.4kg, 2 (20mg) and 3 (5.7mg). Fraction E5-5-6 (152mg) was puri-
n-hexane→n-hexane–EtOAc (5:1→3:1, v/v)→CHCl –MeOH fied by HPLC [H O–MeCN–AcOH (700:300:3, v/v/v/v),
3
2
(100:1→50:1→20:1→10:1→4:1→2:1, v/v)→MeOH] to give COSMOSIL 5C18-MS-II] to give 14 (47mg). The isolation
six fractions [Fr. E1 (15.89g), Fr. E2 (4.13g), Fr. E3 (9.73g), methods of several known compounds were not shown in “Ex-
Fr. E4 (6.39g), Fr. E5 (20.57g), Fr. E6 (13.33g)]. Fraction E4 traction and Isolation.” Those isolation methods were showed
(
6.39g) was further separated by reversed phase silica gel col- in ref. 1.
umn chromatography [200g, MeOH–H O (30:70→40:60→ Mumeose F (1): A white amorphous powder; [α]D +40.8°
0:50→60:40, v/v)→MeOH] to give six fractions [Fr. E4-1 (cꢀ0.73, MeOH); IR (attenuated total reflection (ATR)): νmax
0.40g), Fr. E4-2 (0.20g), Fr. E4-3 (0.46g), Fr. E4-4 (0.60g), 3400, 1730, 1716, 1604, 1515, 1441, 1367, 1228, 1164, 1031,
15
2
5
(
−1
1
Fr. E4-5 (0.25g), Fr. E4-6 (1.50g)]. Fraction E4-3 (0.46g) was 833 cm ; H-NMR (methanol-d , 500MHz) δ: 1.78, 1.98,
4
further separated by normal phase silica gel column chro- 2.06 (all s, CH CO– ×3), 3.59 (d, Jꢀ12.2Hz, H-1a), 3.68
3
matography [15g, n-hexane→n-hexane–EtOAc (10:1→5:1→ (d, Jꢀ12.2Hz, H-1b), 3.71 (dd, Jꢀ3.7, 9.0Hz, H-2′), 3.80 (m,
2
:1→1:1, v/v)→EtOAc→MeOH] to give five fractions [Fr. H -6), 3.98 (m, H-5), 4.15 (m, H -6′), 4.32 (m, H-4), 4.32 (m,
2
2
E4-3-1 (69mg), Fr. E4-3-2 (70mg), Fr. E4-3-3 (27mg), Fr. H-5′), 4.88 (m, H-4′), 5.22 (dd, Jꢀ9.0, 9.0Hz, H-3′), 5.45 (d,
E4-3-4 (122mg), Fr. E4-3-5 (63mg)]. Fraction E4-3-2 (70mg) Jꢀ7.1Hz, H-3), 5.62 (d, Jꢀ3.7Hz, H-1′), 6.42 (d, Jꢀ15.9Hz,
was purified by HPLC [H O–MeCN–AcOH (770:230:3, H-8″), 6.80 (d, Jꢀ8.2Hz, H-3″, 5″), 7.54 (d, Jꢀ8.2Hz, H-2″,
2
13
v/v/v), COSMOSIL 5C18-MS-II] to give 24 (7.7mg) and 6″), 7.74 (d, Jꢀ15.9Hz, H-7″); C-NMR: given in Table 1;
6 (17mg). Fraction E4-3-4 (122mg) was purified by positive-ion FAB-MS: m/z 637 [M+Na] ; high-resolution pos-
+
2
HPLC [H O–MeCN–AcOH (650:350:3, v/v/v), COSMOSIL itive-ion FAB-MS: m/z 637.1747 (Calcd for C H O Na [M+
2
27 34 16
+
5
C18-MS-II] to two fractions [Fr. E4-3-4-1 (37mg), Fr. Na] : m/z 637.1745).
E4-3-4-2 (27mg)]. Fraction E4-3-4-1 (37mg) was purified by
1
D
5
Mumeose G (2): A white amorphous powder; [α] +47.3°
HPLC [H O–MeCN–MeOH–AcOH (600:150:250:3, v/v/v/v), (cꢀ0.65, MeOH) IR (ATR): νmax 3400, 1733, 1716, 1604, 1516,
2
−1 1
COSMOSIL 5C18-MS-II] to give 4 (3.6mg). Fraction E4-5 1457, 1364, 1227, 1155, 1032, 832cm ; H-NMR (methanol-d ,
4
(
0.25g) was further separated by normal phase silica gel 500MHz) δ: 2.06, 2.06, 2.10 (all s, CH CO– ×3), 3.48 (dd,
3
column chromatography [7.5g, n-hexane→n-hexane–EtOAc Jꢀ9.4, 9.4Hz, H-4′), 3.60 (m, H -1), 3.64 (m, H-2′), 3.77 (m,
2
(10:1→5:1→2:1→1:1→1:2, v/v)→EtOAc→MeOH] to give H-6a), 3.89 (m, H-6b), 4.13 (m, H-5), 4.20 (m, H-5′), 4.20 (m,
three fractions [Fr. E4-5-1 (33mg), Fr. E4-5-2 (113mg), Fr. H-6′a), 4.48 (m, H-6′b), 5.23 (dd, Jꢀ9.4, 9.4Hz, H-3′), 5.51
E4-5-3 (35mg)]. Fraction E4-5-2 (113mg) was purified by (d, Jꢀ3.4Hz, H-1′), 5.53 (m, H-4), 5.70 (d, Jꢀ7.5Hz, H-3),
HPLC [H O–MeCN–AcOH (620:380:3, v/v/v), COSMOSIL 6.38 (d, Jꢀ16.1Hz, H-8″), 6.79 (d, Jꢀ8.3Hz, H-3″, 5″), 7.53
2
1
3
5
C18-MS-II] to give 5 (42mg). Fraction E5 (20.57g) was (d, Jꢀ8.3Hz, H-2″, 6″), 7.65 (d, Jꢀ16.1Hz, H-7″); C-NMR:
separated by normal phase silica gel column chromatogra- given in Table 1; positive-ion FAB-MS: m/z 637 [M+Na] ;
phy [720g, CHCl →CHCl –MeOH (50:1→20:1→ 15:1→ high-resolution positive-ion FAB-MS: m/z 637.1738 (Calcd for
0:1→5:1, v/v)→MeOH] to give seven fractions [Fr. E5-1 C H O Na [M+Na] : m/z 637.1745).
40mg), Fr. E5-2 (580mg), Fr. E5-3 (950mg), Fr. E5-4
+
3
3
+
1
(
(
27 34 16
1
5
Mumeose H (3): A white amorphous powder; [α] +29.0°
D
7.18g), Fr. E5-5 (3.56g), Fr. E5-6 (3.80g), Fr. E5-7 (1.29g)]. (cꢀ0.24, MeOH); IR (ATR): νmax 3400, 1733, 1717, 1603, 1516,

4
50
Vol. 61, No. 4
−1
1
1
436, 1373, 1228, 1150, 1014, 833cm ; H-NMR (methanol- the retention time with that of authentic sample (t : 19.8min).
R
d , 500MHz) δ: 2.03, 2.05, 2.05 (all s, CH CO– ×3), 3.56 (d,
Alkaline Hydrolysis of 1–5 Compounds 1–5 (1.5mg)
4
3
Jꢀ12.2Hz, H-1a), 3.63 (d, Jꢀ12.2Hz, H-1b), 3.66 (dd, Jꢀ9.0, were treated with 10% aqueous KOH–1,4-dioxane (1:1, v/v,
9
.0Hz, H-4′), 3.83 (m, H -6′), 3.79 (m, H -6), 4.00 (m, H-5′), 1.0mL) and stirred at 37°C for 24h. Each reaction mixture
2
2
+
4
.10 (m, H-5), 4.76 (dd, Jꢀ3.9, 9.0Hz, H-2′), 5.40 (dd, Jꢀ9.0, was neutralized with Dowex HCR W2 (H form) and the resin
9
.0Hz, H-3′), 5.56 (dd, Jꢀ7.8, 7.8Hz, H-4), 5.73 (d, Jꢀ7.8Hz, was removed by filtration. Evaporation of the solvent from the
H-3), 5.74 (d, Jꢀ3.9Hz, H-1′), 6.39 (d, Jꢀ16.0Hz, H-8″), 6.79 filtrate under reduced pressure yielded a crude product. The
(d, Jꢀ8.6Hz, H-3″, 5″), 7.52 (d, Jꢀ8.6Hz, H-2″, 6″), 7.65 (d, crude product was dissolved in water and applied to normal-
13
Jꢀ16.0Hz, H-7″); C-NMR: given in Table 1; positive-ion phase HPLC {column: YMC-Pack Polyamine II, 250×4.6mm
+
FAB-MS: m/z 637 [M+Na] ; high-resolution positive-ion i.d.; mobile phase: MeCN–H O (3:1, v/v); detection: opti-
2
+
FAB-MS: m/z 637.1744 (Calcd for C H O Na [M+Na] : m/z cal rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo,
2
7
34 16
6
37.1745).
Mumeose I (4): A white amorphous powder; [α]D +38.9° to identify d-(+)-sucrose by comparison of the retention time
cꢀ0.16, MeOH); IR (ATR): νmax 3400, 1730, 1716, 1604, 1515, and optical rotation with that of authentic sample (t ꢀ19.8min
Japan)]; flow rate: 1.0mL/min; column temperature: ambient}
15
(
R
−1
1
1446, 1368, 1228, 1157, 1034, 833cm ; H-NMR (methanol- with positive rotation).
d , 500MHz) δ: 1.76, 1.99, 2.06, 2.08 (all s, CH CO– ×4), 3.60
Effects on Rat Lens Aldose Reductase The experi-
4
3
3
0)
(
m, H-1a), 3.69 (m, H-1b), 3.70 (m, H-2′), 4.15 (m, H -6′), 4.13 ments were performed as described in our previous reports.
2
(
m, H-5), 4.36 (m, H -6), 4.36 (m, H-4), 4.37 (m, H-5′), 4.82 The supernatant fluid of rat lens homogenate was used as a
2
(m, H-4′), 5.20 (dd, Jꢀ9.6, 9.6Hz, H-3′), 5.46 (d, Jꢀ7.0Hz, crude enzyme. The enzyme suspension was diluted to pro-
H-3), 5.49 (d, Jꢀ3.6Hz, H-1′), 6.42 (d, Jꢀ16.0Hz, H-8″), 6.80 duce ca. 10nmol/tube of nicotinamide adenine dinucleotide
(
d, Jꢀ8.3Hz, H-3″, 5″), 7.54 (d, Jꢀ8.3Hz, H-2″, 6″), 7.74 (d, phosphate (NADP) in the following reaction. The incubation
13
Jꢀ16.0Hz, H-7″); C-NMR: given in Table 1; positive-ion mixture contained phosphate buffer 135mm (pH 7.0), Li SO
FAB-MS: m/z 679 [M+Na] ; high-resolution positive-ion 100 mm, reduced nicotinamide adenine dinucleotide phosphate
FAB-MS: m/z 679.1855 (Calcd for C H O Na [M+Na] : m/z (NADPH) 0.03mm, dl-glyceraldehyde 1mm as a substrate,
6
2
4
+
+
2
9
36 17
79.1850).
and 100µL of enzyme fraction, with 25µL of sample solution,
1
5
Mumeose J (5): A white amorphous powder; [α] +28.9° in a total volume of 0.5mL. The reaction was initiated by the
D
(
1
cꢀ0.21, MeOH); IR (ATR): νmax 3400, 1741, 1715, 1604, 1515, addition of NADPH at 30°C. After 30min, the reaction was
−1 1
437, 1367, 1220, 1149, 1033, 834cm ; H-NMR (methanol-d , stopped by the addition of 150µL of HCl 0.5m. Then, 0.5mL
4
5
00MHz) δ: 1.78, 1.94–2.04, 2.07, 2.09, 2.09 (all s, CH CO– of NaOH 6m containing imidazole 10mm was added, and the
3
×
6), 3.78 (m, H -6), 4.11 (m, H-5), 4.16 (m, H -1), 4.21 (m, solution was heated at 60°C for 20min to convert NADP into
2
2
H -6′), 4.33 (m, H-5′), 4.89 (m, H-2′), 4.99 (dd, Jꢀ9.2, 9.2Hz, a fluorescent product. Fluorescence was measured using a
2
H-4′), 5.41 (m, H-3′), 5.46 (m, H-4), 5.56 (m, H-3), 5.70 (d, fluorescence microplate reader (FLUOstar OPTIMA, BMG
Jꢀ3.6Hz, H-1′), 6.40 (d, Jꢀ15.9Hz, H-8″), 6.81 (d, Jꢀ7.7Hz, Labtechnologies) at an excitation wavelength of 360nm and
H-3″, 5″), 7.53 (d, Jꢀ7.7Hz, H-2″, 6″), 7.71 (d, Jꢀ15.9Hz, an emission wavelength of 460nm. Each test sample was dis-
13
H-7″); C-NMR: given in Table 1; positive-ion FAB-MS: solved in DMSO. Measurements were performed in duplicate,
+
m/z 763 [M+Na] ; high-resolution positive-ion FAB-MS: m/z and IC50 values were determined graphically.
+
7
63.2055 (Calcd for C H O Na [M+Na] : m/z 763.2061).
33
40 19
Acid Hydrolysis of 1–5 Compounds 1–5 (1.5mg) were
Acknowledgments This research was supported in part
dissolved in 5% aqueous H SO –1,4-dioxane (1:1, v/v, by a Ministry of Education, Culture, Sports, Science and
2
4
1
.0mL), and each solution was heated at 90°C for 3h, and Technology (MEXT)-Supported Program for the Strategic
−
neutralized with Amberlite IRA-400 (OH form). After drying Research Foundation at Private Universities, by a Grant-in-Aid
in vacuo, a small part of the residue was dissolved in H O– for Young Scientists (B) from the Japan Society for the Pro-
2
MeOH (1:1, v/v) and analyzed by reversed-phase HPLC [col- motion of Science (JSPS).
umn: COSMOSIL 5C18-MS-II, 250×4.6mm i.d.; mobile phase
A: H O–AcOH (1000:3, v/v), B: MeCN–AcOH (1000:3, v/v), References and Notes
2
Linear gradient: mobile phase A–B (90:10→72:28, v/v, in
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2
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[
tRꢀ17.1min]. In addition, the remaining parts of the residue
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3
2
3) Nakamura S., Fujimoto K., Nakashima S., Matsumoto T., Miura
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dine (0.1mL) was added to the mixture and heated at 60°C
for 1h. The reaction mixture was analyzed by reversed-phase
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6
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5
6
)
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3
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