Bioactive constituents from chinese natural medicines. XXXII.1 aminopeptidase N and aldose reductase inhibitors from sinocrassula indica: Structures of sinocrassosides B4, B5, C1, and D1-D3

Article abstract of DOI:10.1248/cpb.56.1438

From the methanolic extract of the whole plant of Sinocrassula indica (Crassulaceae), six new flavonol glycosides, sinocrassosides B₄ (1), B₅ (2), C₁ (3), D₁ (4), D₂ (5), and D₃ (6), were isolated together with 30 compounds. The structures of 1-6 were elucidated on the basis of chemical and physicochemical evidence. In addition, several constituents were found to show inhibitory effects on aminopeptidase N and aldose reductase.

Full text of DOI:10.1248/cpb.56.1438

1438
Chem. Pharm. Bull. 56(10) 1438—1444 (2008)
Vol. 56, No. 10
Bioactive Constituents from Chinese Natural Medicines. XXXII.1)
Aminopeptidase N and Aldose Reductase Inhibitors from Sinocrassula
indica: Structures of Sinocrassosides B4, B5, C1, and D1—D3
a
a
a
,a
Toshio MORIKAWA,a,b Haihui XIE, Tao WANG, Hisashi MATSUDA, and Masayuki YOSHIKAWA
*
a Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan: and b Pharmaceutical Research and
Technology Institute, Kinki University; 3–4–1 Kowakae, Higashi-osaka, Osaka 577–8502, Japan.
Received June 7, 2008; accepted July 22, 2008; published online August 7, 2008
From the methanolic extract of the whole plant of Sinocrassula indica (Crassulaceae), six new flavonol glyco-
sides, sinocrassosides B4 (1), B5 (2), C1 (3), D1 (4), D2 (5), and D3 (6), were isolated together with 30 compounds.
The structures of 1—6 were elucidated on the basis of chemical and physicochemical evidence. In addition, sev-
eral constituents were found to show inhibitory effects on aminopeptidase N and aldose reductase.
Key words Sinocrassula indica; sinocrassoside; aminopeptidase N inhibitor; aldose reductase inhibitor; Crassulaceae
During the course of our studies on bioactive constituents were purified using normal- and reversed-phase silica gel
from Chinese natural medicines,1—17) we found that the chromatographies and finally HPLC.
methanolic extract of the whole plant of Sinocrassula indica
Structures of Sinocrassosides B4 (1), B5 (2), C1 (3), D1
(Crassulaceae) inhibited the increase of serum glucose levels (4), D2 (5), and D3 (6) Sinocrassoside B4 (1) was isolated
in both sucrose- and glucose-loaded rats.2) From the as a yellow powder with negative optical rotation ([a]D25
methanolic extract, 15 acylated flavonol glycosides, sinocras- ꢀ47.8° in MeOH). In the UV spectrum of 1, absorption
sosides A1—A12 and B1—B3 (7—21), were isolated together maxima were observed at 257 (log e 4.49) and 358 (4.37)
with 11 flavonoids (22—32), 2 megastigmanes (33, 34), L- nm. The IR spectrum of 1 showed absorption bands at 1725,
phenylalanine, and guanosine1—3) and several fravonoid con- 1655, and 1599 cmꢀ1 assignable to ester carbonyl and g-py-
stituents were found to show hepatoprotective effects on D- rone functions and aromatic ring and strong absorption bands
galactosamine-induced cytotoxicity in primary cultured at 3432 and 1076 cmꢀ1 suggestive of a glycoside moiety. The
mouse hepatocytes.1) As a continuing study on the con- positive- and negative-ion FAB-MS of 1 showed quasimolec-
stituents from S. indica, we additionally isolated six new ular ion peaks at m/z 879 (MꢁNa)ꢁ and m/z 855 (MꢀH)ꢀ,
flavonol glycosides, sinocrassosides B4 (1), B5 (2), C1 (3), D1 respectively. The molecular formula, C38H48O22, of 1 was de-
(4), D2 (5), and D3 (6). This paper deals with the isolation termined by high resolution FAB-MS measurement. Alkaline
and structure elucidation of 1—6 as well as aminopeptidase hydrolysis of 1 with 10% aqueous potassium hydroxide
N and aldose reductase inhibitory activities of the isolated (KOH)–50% aqueous 1,4-dioxane (1 : 1, v/v) furnished de-
compounds.
sacyl-sinocrassoside B4 (1a) together with S-(ꢁ)-2-methyl-
The methanolic extract from the dried whole plant of S. in- butyric acid, which was identified by HPLC analysis using an
dica (7.7% from the dried plant) was partitioned into an optical rotation detector.1—3) Acid hydrolysis of 1a with 1.0 M
EtOAc–H2O (1 : 1, v/v) mixture to furnish an EtOAc-soluble HCl liberated quercetin (28), together with L-rhamnose and
fraction (2.5%) and an aqueous phase. The aqueous phase D-glucose, which were identified by HPLC analysis.1—7,9—13)
was further extracted with n-BuOH to give an n-BuOH-solu- The 1H- (DMSO-d6, Table 1) and 13C-NMR (Table 2) spectra
ble fraction (1.7%) and an H2O-soluble fraction (3.4%), 1a, which were assigned by various NMR experiments,
which was described previously.2) From the EtOAc- and n- showed signals assignable to meta-coupled aromatic protons
BuOH-soluble fractions, 1 (0.0068%), 2 (0.0007%), 3 [d 6.47, 6.83 (1H each, both d, Jꢂ1.8 Hz, 6, 8-H)] and ortho-
(0.0027%), 4 (0.0006%), 5 (0.0074%), and 6 (0.0010%), and meta-coupled ABC-type protons [d 6.85 (1H, d,
Chart 1
∗ To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp
© 2008 Pharmaceutical Society of Japan

October 2008
1439
Chart 2
Jꢂ8.2 Hz, 5ꢃ-H), 7.62 (1H, dd, Jꢂ2.1, 8.2 Hz, 6ꢃ-H), 7.63 6.52, 6.89 (1H each, both d, Jꢂ1.8 Hz, 6, 8-H), 6.86 (1H, d,
(1H, d, Jꢂ2.1 Hz, 2ꢃ-H)] together with two b-D-glucopyra- Jꢂ8.2 Hz, 5ꢃ-H), 7.62 (1H, dd, Jꢂ2.1, 8.2 Hz, 6ꢃ-H), 7.63
nosyl and a a-L-rhamnopyranosyl moieties [d 1.14 (3H, d, (1H, d, Jꢂ2.1 Hz, 2ꢃ-H)] and two b-D-glucopyranosyl and a
Jꢂ5.6 Hz, Rha-6-H3), 4.48 (1H, d, Jꢂ7.6 Hz, 7-O-terminal- a-L-rhamnopyranosyl moieties [d 1.18 (3H, d, Jꢂ5.8 Hz,
Glc-1-H), 5.49 (1H, d, Jꢂ7.3 Hz, 3-O-Glc-1-H), 5.59 (1H, Rha-6-H3), 4.47 (1H, d, Jꢂ7.6 Hz, 7-O-terminal-Glc-1-H),
br s, Rha-1-H)]. The connectivities of glycopyranosyl parts 5.49 (1H, d, Jꢂ7.0 Hz, 3-O-Glc-1-H), 5.70 (1H, br s, Rha-1-
were determined by a heteronuclear multiple-bond correla- H)]} together with (2S)-2-methylbutyryl moiety [d 0.93 (3H,
tions (HMBC) experiment on 1a. Namely, long-range corre- dd, Jꢂ7.4, 7.4 Hz, 2MB-4-H3), 1.12 (3H, d, Jꢂ7.0 Hz, 2MB-
lations were observed between the following protons and car- 5-H3), 1.50, 1.64 (1H each, both ddq, Jꢂ7.0, 14.0, 7.4 Hz,
bons [3-O-Glc-1-H and 3-C (dC 133.5), Rha-1-H and 7-C 2MB-3-H2), 2.45 (1H, ddq, Jꢂ7.0, 7.0, 7.0 Hz, 2MB-2-H)].
(dC 161.2), and 7-O-terminal-Glc-1-H and Rha-3-C (dC In the HMBC experiment of 1, a long-range correlation was
80.6)]. Consequently, the structure of 1a was determined as observed between the Rha-2-proton [d 5.36 (1H, br s)] and
quercetin 3-O-b-D-glucopyranosyl-7-O-b-D-glucopyranosyl- the (2S)-2-methylbutyryl ester carbonyl carbon (dC 175.3),
1
(1→3)-a-L-rhamnopyranoside. The H- (DMSO-d6, Table 1) so that the structure of sinocrassoside B4 was elucidated as
and 13C-NMR (Table 2) spectra of 1 showed signals assigna- quercetin 3-O-b-D-glucopyranosyl-7-O-b-D-glucopyranosyl-
ble to a desacyl-sinocrassoside B4 part {a quercetin part [d (1→3)-a-L-[2-O-(2S)-2-methylbutyryl]-rhamnopyranoside

1440
Vol. 56, No. 10
(1).
quercetin 3-O-b-D-glucopyranosyl-7-O-b-D-(6-O-3-hydroxy-
Sinocrassoside B5 (2), C42H54O24, was also observed as a isobutyryl)-glucopyranosyl-(1→3)-a-L-[2-O-(2S)-2-methyl-
yellow powder with negative optical rotation ([a]D25 ꢀ64.5° butyryl]-rhamnopyranoside (2).
in MeOH). Alkaline hydrolysis of 2 with 10% KOH–50%
Sinocrassoside C1 (3) was isolated as a yellow powder
aqueous 1,4-dioxane (1 : 1, v/v) furnished 1a together with with negative optical rotation ([a]D25 ꢀ16.0° in MeOH). In
(S)-(ꢁ)-2-methylbutyric acid and 3-hydroxyisobutyric acid, the positive- and negative-ion FAB-MS of 3, the quasimolec-
which were identified by HPLC analysis of their p-nitroben- ular ion peaks were observed at m/z 633 (MꢁNa)ꢁ and m/z
zyl derivatives1—4,6,7,12) and/or using an optical rotation detec- 609 (MꢀH)ꢀ, and the molecular formula C27H30O16 was de-
1
tor. The proton and carbon signals in the H- (DMSO-d6, termined by high resolution FAB-MS measurement. Acid hy-
Table 1) and 13C-NMR (Table 2) spectra of 2 were superim- drolysis of 3 with 1.0 M HCl liberated herbacetin18) (35) to-
posable on those of 1, except for the signals due to an addi- gether with L-rhamnose and D-glucose, which were identified
tional 3-hydroxyisobutyryl ester moiety [d 1.03 (3H, d, by HPLC analysis. The H- (DMSO-d6, Table 1) and 13C-
1
Jꢂ7.0 Hz, 3HIB-4-H3), 2.50 (1H, ddq, Jꢂ6.1, 6.1, 7.0 Hz, NMR (Table 2) spectra of 3 showed signals assignable to
3HIB-2-H), 3.42, 3.50 (1H each, both dd, Jꢂ6.1, 13.4 Hz, herbacetin (35) part [d 6.61 (1H, s, H-6), 6.91, 8.13 (2H
3HIB-3-H2)]. In the HMBC experiment of 2, long-range cor- each, both d, Jꢂ8.9 Hz, 3ꢃ,5ꢃ,2ꢃ,6ꢃ-H)] together with a b-D-
relations were observed between the 7-O-Rha-2-proton [d glucopyranosyl and a a-L-rhamnopyranosyl moieties [d 1.12
5.36 (1H, br s)] and the (2S)-2-methybutyryl ester carbonyl (3H, d, Jꢂ6.1 Hz, Rha-6-H3), 5.47 (1H, d, Jꢂ7.3 Hz, Glc-1-
carbon (dC 174.8) and between the 7-O-terminal-Glc-6-pro- H), 5.50 (1H, br s, Rha-1-H)]. Finally, the connectivities of
tons [d 3.87 (1H, dd, Jꢂ5.8, 11.0 Hz), 4.47 (1H, dd, Jꢂ1.8, the b-D-glucopyranosyl and the a-L-rhamnopyranosyl moi-
11.0 Hz)] and the 3-hydroxyisobutyryl ester carbonyl carbon eties in 3 were elucidated on the basis of HMBC experiment,
(dC 174.1). Thus, sinocrassoside B5 was elucidated as which showed long-range correlations were observed be-
Table 1. 1H-NMR (500 MHz) Data of 1—6 and Related Compound (1a)
1
1a
H (J Hz)
2
3
4
5
6
Position
d
H (J Hz)
d
d
H (J Hz)
d
H (J Hz)
d
H (J Hz)
d
H (J Hz)
d
H (J Hz)
6
6.52 (d, 1.8)
6.89 (d, 1.8)
7.63 (d, 2.1)
6.47 (d, 1.8)
6.83 (d, 1.8)
7.63 (d, 2.1)
6.48 (d, 1.9)
6.87 (d, 1.9)
7.64 (d, 1.9)
6.61 (s)
6.60 (s)
6.62 (s)
6.59 (s)
8
2ꢃ
3ꢃ
5ꢃ
6ꢃ
8.13 (d, 8.9)
7.63 (d, 2.1)
7.68 (d, 2.1)
7.64 (d, 2.1)
6.91 (d, 8.9)
6.86 (d, 8.2)
6.85 (d, 8.2)
6.85 (d, 8.3)
6.91 (d, 8.9)
6.86 (d, 8.2)
6.88 (d, 9.2)
6.85 (d, 9.2)
7.62 (dd, 2.1, 8.2)
7.62 (dd, 2.1, 8.2)
12.63 (br s)
7.62 (dd, 1.9, 8.3)
12.61 (br s)
8.13 (d, 8.9)
7.62 (dd, 2.1, 8.2)
11.90 (br s)
7.67 (dd, 2.1, 9.2)
12.06 (br s)
7.63 (dd, 2.1, 9.2)
12.06 (br s)
5-OH 12.61 (br s)
12.03 (br s)
(3-O-Glc)
(3-O-Glc)
(3-O-Glc)
(3-O-Glc)
(3-O-Glc)
(3-O-Glc)
1
2
3
4
5
6
5.49 (d, 7.0)
5.49 (d, 7.3)
5.49 (d, 7.0)
5.47 (d, 7.3)
5.48 (d, 7.6)
5.54 (d, 7.9)
3.26 (dd, 7.0, 8.5)
3.22 (dd, 8.5, 9.2)
3.09 (m)
3.26 (dd, 7.3, 8.5)
3.22 (dd, 8.5, 9.2)
3.10 (m)
3.26 (dd, 7.0, 8.5)
3.19 (dd, 8.5, 9.2)
3.09 (m)
3.19 (dd, 7.3, 9.2)
3.22 (dd, 9.2, 9.5)
3.09 (m)
3.28 (dd, 7.6, 9.2)
3.25 (dd, 9.2, 9.5)
3.10 (m)
4.79 (dd, 7.9, 9.2)
3.43 (dd, 9.2, 9.2)
3.17 (m)
3.10 (m)
3.11 (m)
3.10 (m)
3.09 (m)
3.12 (m)
3.21 (m)
3.33 (dd, 5.5, 11.2)
3.58 (dd, 1.8, 11.2)
(7-O-Rha)
3.34 (dd, 5.5, 11.6)
3.58 (dd, 1.8, 11.6)
(7-O-Rha)
3.33 (dd, 5.8, 11.0)
3.60 (dd, 1.8, 11.0)
(7-O-Rha)
3.34 (dd, 5.8, 11.9)
3.56 (dd, 2.5, 11.9)
(7-O-Rha)
3.34 (dd, 5.5, 11.0)
3.58 (dd, 2.1, 11.0)
(7-O-Rha)
3.38 (dd, 5.8, 11.5)
3.60 (dd, 2.2, 11.5)
(7-O-Rha)
(7-O-Rha)
1
2
3
4
5
6
5.70 (br s)
5.59 (br s)
5.70 (br s)
5.50 (br s)
5.51 (br s)
5.50 (d, 1.8)
5.47 (br s)
5.36 (br s)
4.11 (br s)
5.36 (br s)
3.95 (br s)
4.15 (br d, ca. 3)
5.11 (dd, 3.4, 9.8)
3.58 (dd, 9.5, 9.8)
3.76 (dq, 9.5, 6.1)
1.16 (d, 6.1)
3.97 (dd, 1.8, 3.0)
3.85 (dd, 3.0, 9.0)
3.34 (dd, 9.0, 9.5)
3.55 (dq, 9.5, 6.1)
1.14 (d, 6.1)
3.96 (br s)
4.00 (dd, 3.4, 9.5)
3.50 (dd, 9.5, 9.5)
3.60 (dq, 9.5, 5.8)
1.18 (d, 5.8)
(7-O-Rha3–1Glc)
4.47 (d, 7.6)
3.78 (dd, 3.4, 9.5)
3.52 (dd, 9.5, 9.5)
3.64 (dq, 9.5, 5.6)
1.14 (d, 5.6)
(7-O-Rha3–1Glc)
4.48 (d, 7.6)
3.96 (dd, 3.4, 9.5)
3.48 (dd, 9.5, 9.5)
3.59 (dq, 9.5, 5.8)
1.17 (d, 5.8)
(7-O-Rha3–1Glc)
4.47 (d, 7.7)
3.83 (dd, 2.8, 9.5)
3.32 (dd, 8.9, 9.5)
3.52 (dq, 8.9, 6.1)
1.12 (d, 6.1)
3.83 (dd, 3.4, 9.2)
3.32 (dd, 9.2, 9.2)
3.54 (dq, 9.2, 6.1)
1.13 (d, 9.1)
1
2
3
4
5
6
3.00 (dd, 7.6, 9.5)
3.18 (dd, 8.9, 9.5)
3.01 (dd, 8.9, 9.5)
3.19 (m)
3.11 (dd, 7.6, 9.5)
3.17 (dd, 8.9, 9.5)
3.10 (dd, 8.9, 9.5)
3.19 (m)
3.02 (dd, 7.7, 9.5)
3.21 (dd, 8.9, 9.5)
3.04 (dd, 8.9, 9.5)
3.42 (m)
3.41 (dd, 5.8, 11.0)
3.67 (dd, 2.0, 11.0)
3.47 (dd, 5.8, 11.5)
3.69 (dd, 1.9, 11.5)
3.87 (dd, 5.8, 11.0)
4.47 (dd, 1.8, 11.0)
acyl
2
2.45 (ddq, 7.0, 7.0, 7.0)
1.50 (ddq, 7.0, 14.0, 7.4)
1.64 (ddq, 7.0, 14.0, 7.4)
0.93 (dd, 7.4, 7.4)
2.41 (ddq, 7.0, 7.0, 6.7)
1.50 (ddq, 7.0, 13.7, 7.4)
1.61 (ddq, 7.0, 13.7, 7.4)
0.91 (dd, 7.4, 7.4)
2.44 (ddq, 7.0, 7.0, 7.0)
1.48 (ddq, 7.3, 15.0, 7.0)
1.64 (ddq, 7.3, 15.0, 7.0)
0.90 (dd, 7.3, 7.3)
2.03 (s)
3
4
5
2
3
1.12 (d, 7.0)
1.09 (d, 6.7)
1.12 (d, 7.0)
2.50 (ddq, 6.1, 6.1, 7.0)
3.42 (dd, 6.1, 13.4)
3.50 (dd, 6.1, 13.4)
1.03 (d, 7.0)
4
Measured in DMSO-d6.

October 2008
1441
Table 2. 13C-NMR (125 MHz) Data of 1—6 and Related Compound (1a)
1
dC
1a
dC
2
dC
3
dC
4
dC
5
dC
6
dC
Position
2
3
4
5
6
156.7
133.6
177.5
160.8
99.3
156.6
133.5
177.5
160.8
99.4
156.8
133.7
177.6
160.8
99.5
156.6
133.1
177.8
152.0
98.7
147.8
135.8
176.2
151.6
98.8
156.7
133.2
177.8
152.0
98.6
156.9
132.8
177.5
152.0
98.7
7
8
9
10
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
161.2
94.4
161.2
94.4
160.8
94.5
150.4
127.0
144.4
105.4
120.9
131.0
115.0
160.0
115.0
131.0
(3-O-Glc)
100.7
74.1
150.0
127.3
144.8
104.8
122.0
115.4
144.9
147.5
115.4
120.3
150.4
127.0
144.4
105.4
121.2
116.5
144.7
148.5
115.0
121.8
(3-O-Glc)
100.7
74.0
150.6
127.1
144.5
105.4
120.8
116.4
144.9
148.9
115.1
122.0
(3-O-Glc)
98.5
155.8
105.7
120.9
116.3
144.8
148.6
115.1
121.6
(3-O-Glc)
100.6
74.0
155.8
105.6
120.9
116.2
144.8
148.6
115.1
121.6
(3-O-Glc)
100.6
74.0
155.8
105.9
120.9
116.3
144.8
148.7
115.2
121.7
(3-O-Glc)
100.8
74.0
1
20
3
74.2
74.0
76.4
76.4
76.5
76.3
76.4
4
69.9
69.8
69.9
69.8
69.8
70.0
5
77.5
77.5
77.4
77.4
77.4
77.5
6
60.9
60.9
60.9
60.7
60.9
60.7
(7-O-Rha)
94.7
(7-O-Rha)
98.2
(7-O-Rha)
94.9
(7-O-Rha)
99.3
(7-O-Rha)
99.4
(7-O-Rha)
99.3
(7-O-Rha)
99.5
1
2
3
4
5
6
70.5
77.8
70.6
69.3
68.9
80.6
70.4
69.5
70.8
78.2
70.5
69.3
69.8
69.9
71.6
69.8
67.4
73.1
68.8
69.9
69.8
69.9
71.7
69.8
69.8
69.9
71.7
69.8
17.8
17.8
17.8
17.8
17.7
17.8
17.8
(7-O-Rha3–1Glc) (7-O-Rha3–1Glc) (7-O-Rha3–1Glc)
1
2
3
4
5
104.3
73.9
76.0
70.1
76.5
61.4
104.5
73.9
76.1
69.8
76.7
60.9
104.4
73.8
75.9
70.1
74.0
63.6
6
acyl
1
2
3
4
5
1
2
3
175.3
40.1
26.2
11.1
16.1
174.8
40.1
26.2
11.0
16.0
174.1
42.1
63.1
13.2
175.6
40.2
26.2
11.2
16.3
169.3
20.9
4
Measured in DMSO-d6.
tween the Glc-1-proton and the 3-carbon (dC 133.1) and be- part [d 6.60 (1H, s, 6-H), 6.86 (1H, d, Jꢂ8.2 Hz, 5ꢃ-H), 7.62
tween the Rha-1-proton and the 7-carbon (dC 150.4). Conse- (1H, dd, Jꢂ2.1, 8.2 Hz, 6ꢃ-H), 7.63 (1H, d, Jꢂ2.1 Hz, 2ꢃ-H)]
quently, the structure of 3 was constructed as herbacetin 3-O- and an a-L-rhamnopyranosyl moiety [d 1.16 (3H, d, Jꢂ6.1
b-D-glucopyranosyl-7-O-a-L-rhamnopyranoside (3).
Hz, Rha-6-H3), 5.51 (1H, br s, Rha-1-H)] together with (2S)-
Sinocrassoside D1 (4) was also isolated as a yellow powder 2-methylbutyryl moiety [d 0.90 (3H, dd, Jꢂ7.3, 7.3 Hz,
with positive optical rotation ([a]D23 ꢁ60.2° in MeOH). The 2MB-4-H3), 1.12 (3H, d, Jꢂ7.0 Hz, 2MB-5-H3), 1.48, 1.64
UV and IR spectra of 4 indicated the flavonoid glycoside (1H each, both ddq, Jꢂ7.3, 15.0, 7.0 Hz, 2MB-3-H2), 2.44
structure and the molecular formula, C26H28O13, was deter- (1H, ddq, Jꢂ7.0, 7.0, 7.0 Hz, 2MB-2-H)]. Finally, the posi-
mined by the quasimolecular ion peaks in positive- and nega- tion of (2S)-2-methylbutyryl group in 4 was confirmed by
tive-ion FAB-MS and by high resolution FAB-MS. Alkaline the HMBC experiment, which showed a long-range correla-
hydrolysis of 4 with 10% KOH–50% aqueous 1,4-dioxane tion between the Rha-3-proton [d 5.11 (1H, dd, Jꢂ3.4,
(1 : 1, v/v) furnished gossypetin 7-O-a-L-rhamnopyranoside 9.8 Hz)] and the (2S)-2-methylbutyryl ester carbonyl carbon
(36)19,20) together with S-(ꢁ)-2-methylbutyric acid, which (dC 175.6). Consequently, the structure of sinocrassoside D1
was identified by HPLC analysis using an optical rotation de- was determined as gossypetin 7-O-a-L-[3-O-(2S)-2-methyl-
1
tector. The H- (DMSO-d6, Table 1) and 13C-NMR (Table 2) butyryl]-rhamnopyranoside (4).
spectra of 4 showed signals assignable to gossypetin (37)
Sinocrassosides D2 (5) and D3 (6) were also isolated as a

1442
Vol. 56, No. 10
white powder with positive optical rotation (5: [a]D25 ꢁ71.3°, the Glc-2-proton [d 4.79 (1H, dd, Jꢂ7.9, 9.2 Hz)] and the
25
6: [a]D ꢁ150.6° both in MeOH), respectively. In the posi- acetyl carbonyl carbon (dC 169.3). On the basis of this evi-
tive- and negative-ion FAB-MS of 5, the quasimolecular ion dence, the structure of sinocrassoside D3 was determined
peaks were observed at m/z 649 (MꢁNa)ꢁ and m/z 625 as gossypetin 3-O-b-D-(2-O-acetyl)-glucopyranosyl-7-O-a-
(MꢀH)ꢀ, and the molecular formula C27H30O17 was deter- L-rhamnopyranoside (6).
mined by high-resolution FAB-MS measurement. The acid
Inhibitory Effects on Aminopeptidase N Activity
hydrolysis of 5 liberated gossypetin18) (37) together with L- Aminopeptidase N (APN) is a Znꢁ-dependent metallopro-
rhamnose and D-glucose. The proton and carbon signals in tease and plays an important role in tumor-cell invasion, ex-
1
the H- (DMSO-d6, Table 1) and 13C-NMR (Table 2) spectra tracellular matrix degradation by tumor cells and tumor
of 5 showed signals due to a gossypetin moiety [d 6.62 (1H, metastasis.21,22) Recently, APN was identified on the endothe-
s, 6-H), 6.88 (1H, d, Jꢂ9.2 Hz, 5ꢃ-H), 7.67 (1H, dd, Jꢂ2.1, lial cell surface as a receptor for a tumor-homing peptide
9.2 Hz, 6ꢃ-H), 7.68 (1H, d, Jꢂ2.1 Hz, 2ꢃ-H)] together with a motif, NGR (asparagines-glycine-arginine, Asn-Gly-Arg),
b-D-glucopyranosyl and a a-L-rhamnopyranosyl moieties [d which is capable of homing selectively to the tumor vascula-
1.14 (3H, d, Jꢂ6.1 Hz, Rha-6-H3), 5.48 (1H, d, Jꢂ7.6 Hz, ture.23) This evidence indicated that APN plays a critical role
Glc-1-H), 5.50 (1H, d, Jꢂ1.8 Hz, Rha-1-H)]. The connectivi- in angiogenesis. Accordingly, APN is considered as an im-
ties of the b-D-glucopyranosyl and a-L-rhamnopyranosyl portant therapeutic target for tumor angiogenesis and metas-
moieties in 5 were clarified by HMBC experiment, in which tasis. Previously, we have reported the isolation and structure
long-range correlations were observed between the Glc-1- elucidation of several flavonoids with APN inhibitory activ-
proton and the 3-carbon (dC 133.2) and between the Rha-1- ity from the rhizomes of Boesenbergia rotunda.24) As shown
proton and the 7-carbon (dC 150.4). Thus, the structure of in Table 3, most of them except for megastigmane con-
sinocrassoside D2 was determined as gossypetin 3-O-b-D- stituents (33, 34) were found to inhibit APN activity.3) These
glucopyranosyl-7-O-a-L-rhamnopyranoside (5). On the other activities were equivalent for that of curcumin (31.9ꢄ1.6%
hand, the molecular formula, C29H32O18, of 6 was determined at 30 mM), which is known to show APN inhibitory activity.25)
by high resolution FAB-MS. Alkaline hydrolysis of 6 with
Inhibitory Effects on Rat Lens Aldose Reductase As a
10% KOH–50% aqueous 1,4-dioxane (1 : 1, v/v) furnished 5 key enzyme in the polyol pathway, aldose reductase has been
together with acetic acid, which was identified by HPLC reported to catalyze the reduction of glucose to sorbitol. Sor-
analysis of its p-nitrobenzyl derivative. In the HMBC experi- bitol does not readily diffuse across cell membranes, and the
ment of 6, a long-range correlation was observed between intracellular accumulation of sorbitol has been implicated in
Table 3. Inhibitory Effects of Constituents from S. indica on Aminopeptidase N Activity
Inhibition (%)
0 mM
10 mM
30 mM
Sinocrassoside B4 (1)
Sinocrassoside B5 (2)
Sinocrassoside C1 (3)
Sinocrassoside D1 (4)
Sinocrassoside D3 (6)
Sinocrassoside A1 (7)
Sinocrassoside A2 (8)
0.0ꢄ0.9
0.0ꢄ1.2
0.0ꢄ1.2
0.0ꢄ1.2
0.0ꢄ2.0
0.0ꢄ2.0
0.0ꢄ2.0
0.0ꢄ2.0
0.0ꢄ0.9
0.0ꢄ0.9
0.0ꢄ0.9
0.0ꢄ0.9
0.0ꢄ1.2
0.0ꢄ0.9
0.0ꢄ0.9
0.0ꢄ0.9
0.0ꢄ0.4
0.0ꢄ0.4
0.0ꢄ0.4
0.0ꢄ0.4
0.0ꢄ1.7
0.0ꢄ1.2
0.0ꢄ1.7
0.0ꢄ1.7
0.0ꢄ1.7
0.0ꢄ1.2
0.0ꢄ0.3
0.0ꢄ1.2
0.0ꢄ1.2
0.0ꢄ1.7
12.0ꢄ1.0**
20.9ꢄ1.2**
5.6ꢄ1.1
32.1ꢄ1.2**
28.3ꢄ1.3**
12.4ꢄ1.3**
35.1ꢄ0.9**
23.2ꢄ0.8**
31.4ꢄ0.2**
35.7ꢄ0.4**
26.9ꢄ1.0**
21.7ꢄ0.5**
16.4ꢄ1.0**
23.7ꢄ0.8**
19.9ꢄ0.6**
28.7ꢄ1.1**
33.9ꢄ1.0**
39.4ꢄ0.4**
36.2ꢄ0.4**
13.7ꢄ0.5**
31.7ꢄ0.7**
12.5ꢄ1.4**
19.1ꢄ1.4**
32.8ꢄ1.4**
13.8ꢄ0.6**
18.1ꢄ3.0**
41.8ꢄ0.9**
12.6ꢄ1.1
25.6ꢄ3.8**
11.9ꢄ1.1**
37.2ꢄ0.8**
32.6ꢄ0.9**
26.8ꢄ0.6**
15.7ꢄ1.0**
12.4ꢄ0.7**
11.3ꢄ0.3**
7.7ꢄ0.5*
33.3ꢄ1.1**
27.5ꢄ3.7**
37.4ꢄ1.7**
22.7ꢄ1.0**
6.8ꢄ1.3*
7.1ꢄ1.8**
6.2ꢄ0.8*
6.9ꢄ2.7*
16.3ꢄ2.7*
10.1ꢄ2.9**
6.2ꢄ1.6
Sinocrassoside A4 (10)3)
Sinocrassoside A5 (11)3)
Sinocrassoside A6 (12)3)
Sinocrassoside A7 (13)3)
Sinocrassoside A9 (15)
Sinocrassoside A10 (16)
Sinocrassoside B1 (19)
Sinocrassoside B2 (20)
Sinocrassoside B3 (21)3)
Kaempferol (22)
Kaempferol 3-O-Glc (23)
Kaempferol 7-O-Rha (24)
Kaempferol 3-O-Glc 7-O-Rha (25)
Multiflorin B (26)
Kaempferol 3-O-Rha-(1→4)-Glc 7-O-Rha (27)
Quercetin (28)
Quercetin 3-O-Glc (29)
Quercetin 3-O-Glc 7-O-Rha (30)
Multinoside A (31)
12.6ꢄ8.2
4.4ꢄ2.3
ꢀ0.8ꢄ1.9
19.2ꢄ1.2**
ꢀ2.9ꢄ1.9
ꢀ1.0ꢄ0.8
13.3ꢄ0.9**
15.3ꢄ1.2**
60.2ꢄ1.7**
ꢀ0.8ꢄ3.6
ꢀ1.1ꢄ2.1
31.9ꢄ1.6**
Luteolin (32)
trans-(3S,5R,6R,9S)-Megastigman-7-ene-3,5,6,9-tetraol 3-O-Glc (33)
trans-(3S,5R,6R,9S)-Megastigman-7-ene-3,5,6,9-tetraol 9-O-Glc (34)
Curcumin3)
Each value represents the meanꢄS.E.M. (nꢂ4). Significantly different from the control, ∗ pꢅ0.05, ∗∗ pꢅ0.01.

October 2008
1443
0.0008%), and multinoside A (31, 27.6 mg, 0.0023%).
the chronic complications of diabetes such as cataract. Previ-
ously, we reported that various flavonoid and terpenoid con-
stituents from several natural medicines and medicinal foods
inhibited aldose reductase inhibitory activity.4,26—31) Since
Sinocrassoside B4 (1): A yellow powder, [a]D25 ꢀ47.8° (cꢂ2.00, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C38H48O22Na (MꢁNa)ꢁ
879.2535; Found 879.2540. UV [lmax (log e), MeOH]: 257 (4.49), 358
(4.37) nm. IR (KBr, cmꢀ1): 3432, 1725, 1655, 1599, 1076. 1H-NMR
several flavonoid constituents were isolated from the whole (500 MHz, DMSO-d6) d: given in Table 1. 13C-NMR (125 MHz, DMSO-d6)
dC: given in Table 2. Positive-ion FAB-MS m/z: 879 (MꢁNa)ꢁ. Negative-ion
plants of S. indica, the inhibitory effects of the principal
flavonoid constituents on rat lens aldose reductase were ex-
FAB-MS m/z: 855 (MꢀH)ꢀ.
Sinocrassoside B5 (2): A yellow powder, [a]D25 ꢀ64.5° (cꢂ2.00, MeOH).
amined. Among the flavonoid constituents from S. indica,
kaempferol (22, IC50ꢂ10 mM), quercetin (28, 2.2 mM),
quercetin 3-O-b-D-glucopyranoside (29, 4.5 mM), and luteolin
(32, 0.45 mM), showed strong activity in agreement with the
previous study.4,28) Additionally, sinocrassosides D2 (5,
IC50ꢂ47 mM) and A2 (8, 31 mMM), multiflorin B (26, 2.7 mM),
and quercetin 3-O-b-D-glucopyranosyl-7-O-a-L-rhamnopyra-
noside (30, 56 mM) were found to inhibit rat lens aldose re-
ductase.
High-resolution positive-ion FAB-MS: Calcd for C42H54O24Na (MꢁNa)ꢁ
965.2903; Found 965.2909. UV [lmax (log e), MeOH]: 257 (4.34), 357
(4.22) nm. IR (KBr, cmꢀ1): 3432, 1736, 1728, 1655, 1597, 1076. H-NMR
1
(500 MHz, DMSO-d6) d: given in Table 1. 13C-NMR (125 MHz, DMSO-d6)
dC: given in Table 2. Positive-ion FAB-MS m/z: 965 (MꢁNa)ꢁ. Negative-ion
FAB-MS m/z: 941 (MꢀH)ꢀ.
Sinocrassoside C1 (3): A yellow powder, [a]D25 ꢀ16.0° (cꢂ1.50, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C27H30O16Na (MꢁNa)ꢁ
633.1432; Found 633.1425. UV [lmax (log e), MeOH]: 277 (4.28), 330
(4.11) nm. IR (KBr, cmꢀ1): 3432, 1655, 1603, 1057. 1H-NMR (500 MHz,
DMSO-d6) d: given in Table 1. 13C-NMR (125 MHz, DMSO-d6) dC: given
in Table 2. Positive-ion FAB-MS m/z: 633 (MꢁNa)ꢁ. Negative-ion FAB-MS
Experimental
The following instruments were used to obtain physical data: specific ro- m/z: 609 (MꢀH)ꢀ.
tations, Horiba SEPA-300 digital polarimeter (lꢂ5 cm); UV spectra, Shi-
Sinocrassoside D1 (4): A yellow powder, [a]D23 ꢁ60.2° (cꢂ0.80, MeOH).
madzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100 spectrome- High-resolution positive-ion FAB-MS: Calcd for C26H28O13Na (MꢁNa)ꢁ
ter; EI-MS and high-resolution MS, JEOL JMS-GCMATE mass spectrome- 571.1428; Found 571.1422. UV [lmax (log e), MeOH]: 261 (4.08), 344
ter; FAB-MS and high-resolution MS, JEOL JMS-SX 102A mass spectrom-
eter; 1H-NMR spectra, JEOL EX-270 (270 MHz) and JNM-LA500
(500 MHz) spectrometers; 13C-NMR spectra, JEOL EX-270 (68 MHz) and
(3.83) nm. IR (KBr, cmꢀ1): 3410, 1719, 1655, 1611, 1075. 1H-NMR
(500 MHz, DMSO-d6) d: given in Table 1. 13C-NMR (125 MHz, DMSO-d6)
dC: given in Table 2. Positive-ion FAB-MS m/z: 571 (MꢁNa)ꢁ. Negative-ion
JNM-LA500 (125 MHz) spectrometers with tetramethylsilane as an internal FAB-MS m/z: 547 (MꢀH)ꢀ.
standard; and HPLC detector, Shimadzu RID-6A refractive index and SPD-
10Avp UV–VIS detectors. HPLC column, Cosmosil 5C18-MS-II (Nacalai
Sinocrassoside D2 (5): A yellow powder, [a]D25 ꢁ71.3° (cꢂ2.00, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C27H30O17Na (MꢁNa)ꢁ
Tesque Inc., 250ꢆ4.6 mm i.d.) and (250ꢆ20 mm i.d.) columns were used for 649.1381; Found 649.1385. UV [lmax (log e), MeOH]: 276 (4.26), 350
analytical and preparative purposes, respectively.
(4.08) nm. IR (KBr, cmꢀ1): 3410, 1655, 1605, 1059. 1H-NMR (500 MHz,
The following experimental conditions were used for chromatography: or- DMSO-d6) d: given in Table 1. 13C-NMR (125 MHz, DMSO-d6) dC: given
dinary-phase silica gel column chromatography, Silica gel BW-200 (Fuji in Table 2. Positive-ion FAB-MS m/z: 649 (MꢁNa)ꢁ. Negative-ion FAB-MS
Silysia Chemical, Ltd., Aichi, Japan, 150—350 mesh); reversed-phase silica m/z: 625 (MꢀH)ꢀ.
gel column chromatography, Chromatorex ODS DM1020T (Fuji Silysia
Chemical, Ltd., Aichi, Japan, 100—200 mesh); TLC, precoated TLC plates
with Silica gel 60F254 (Merck, 0.25 mm) (ordinary phase) and Silica gel RP-
Sinocrassoside D3 (6): A yellow powder, [a]D25 ꢁ150.6° (cꢂ0.70,
MeOH). High-resolution positive-ion FAB-MS: Calcd for C29H32O18Na
(MꢁNa)ꢁ 691.1486; Found 691.1477. UV [lmax (log e), MeOH]: 259
18 F254S (Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC, pre- (4.19), 349 (4.03) nm. IR (KBr, cmꢀ1): 3432, 1736, 1655, 1605, 1059. H-
1
coated TLC plates with Silica gel RP-18 WF254S (Merck, 0.25 mm); and de-
NMR (500 MHz, DMSO-d6) d: given in Table 1. 13C-NMR (125 MHz,
tection was achieved by spraying with 1% Ce(SO4)2–10% aqueous H2SO4 DMSO-d6) dC: given in Table 2. Positive-ion FAB-MS m/z: 691 (MꢁNa)ꢁ.
followed by heating.
Negative-ion FAB-MS m/z: 667 (MꢀH)ꢀ.
Plant Material The whole plants of S. indicum cultivated at Guangxi
province in China were purchased via Tochimoto Tenkaido Co., Ltd., Osaka,
Alkaline Hydrolysis of 1, 2, 4, and 6 A solution of 1, 2, 4, and 6 (each
5.0 mg) in 10% aqueous potassium hydroxide (KOH)–50% aqueous 1,4-
Japan. The plant material was identified by one of authors (T. W.). A dioxane (1 : 1, v/v, 1.0 ml) was stirred at 37 °C for 1 h. Removal of the sol-
voucher specimen (2004.02. Guangxi-02) of this plant is on file in our labo-
vent under reduced pressure gave a reaction mixture. A part of the reaction
mixture was dissolved in dichloroethane (2.0 ml) and the solution was
treated with p-nitrobenzyl-N-Nꢃ-diisopropylisourea (10 mg), then the whole
ratory.2)
Extraction and Isolation Fraction 5-2 (770 mg) obtained from the
EtOAc-soluble fraction and fractions 4-8 (970 mg), 5-5 (1980 mg), 5-6 was stirred at 80 °C for 1 h. The reaction solution was subjected to HPLC
(1030 mg), and 5-8 (1310 mg) from the n-BuOH-soluble fraction of the analysis [column: YMC-Pack ODS-A, 250ꢆ4.6 mm i.d.; mobile phase:
methanolic extract from the whole plant of S. indica (9.9 kg, cultivated in MeOH–H2O (70 : 30, v/v); detection: UV (254 nm); flow rate: 0.9 ml/min] to
Guangxi province, China) as reported previously.2)
identify the p-nitrobenzyl esters of acetic acid (a, tR 6.2 min) from 6, 3-hy-
Fraction 5-2 (770 mg) from the EtOAc-soluble fraction was purified by droxyisobutyric acid (b, tR 10.5 min) from 2, and 2-methylbutyric acid (c, tR
HPLC [MeOH–H2O (55 : 45, v/v)] to give sinocrassoside D1 (4, 12.1 mg, 16.9 min) from 1, 2, and 4, which were carried out by comparison of their
0.0006%) together with sinocrassosides A7 (13, 23.0 mg, 0.0011%) and B1 retention time with those of commercially obtained standard samples. The
(19, 17.6 mg, 0.0009%) and quercetin (28, 96.6 mg, 0.0046%). Fraction 4-8
rest of the reaction mixture was neutralized with Dowex HCR W2 (Hꢁ
(970 mg) from the n-BuOH-soluble fraction was further purified by HPLC form) and the resin was removed by filtration. Evaporation of the solvent
[MeOH–H2O (55 : 45, v/v)] to give sinocrassosides B4 (1, 348.6 mg, from the filtrate under reduced pressure yielded a product. A part of the
0.0068%) and B5 (2, 35.4 mg, 0.0007%) together with sinocrassosides A8
(14, 72.0 mg, 0.0014%) and A9 (15, 150.4 mg, 0.0029%). Fraction 5-5
product was subjected to HPLC analysis [column: YMC-Pack ODS-AQ,
250ꢆ4.6 mm i.d.; mobile phase: CH3CN–0.1% aqueous H3PO4 (20 : 80,
(265 mg) from the n-BuOH-soluble fraction was subjected to HPLC v/v); detection: optical rotation [Shodex OR-2 (Showa Denko Co., Ltd.,
[MeOH–H2O (35 : 65, v/v)] to give sinocrassoside D2 (5, 50.6 mg, 0.0074%). Tokyo, Japan)]; flow rate: 1.0 ml/min] to identify S-(ꢁ)-2-methylbutyric
Fraction 5-6 (160 mg) from the n-BuOH-soluble fraction was subjected to acid [cꢃ, tR 15.4 min (positive)] from 1, 2, and 4, which was carried out by
HPLC [MeOH–H2O (40 : 60, v/v)] to give sinocrassoside C1 (3, 23.0 mg, comparison of its retention time and optical rotation with that of commer-
0.0027%) together with quercetin 3-O-b-D-glucopyranosyl-7-O-a-L-
rhamnopyranoside (30, 14.7 mg, 0.0017%). Fraction 5-8 (300 mg) from the
n-BuOH-soluble fraction was further purified by HPLC [MeOH–H2O
(40 : 60, v/v)] to give sinocrassoside D3 (6, 11.5 mg, 0.0010%) together with
cially obtained standard sample. The rest of the product was subjected to
HPLC [MeOH–H2O (50 : 50 v/v)] to give quercetin 3-O-b-D-glucopyra-
nosyl-7-O-b-D-glucopyranosyl(1→3)-a-L-rhamnopyranoside (1a, 1.0 mg
from 1, 1.0 mg from 2), gossypetin 7-O-a-L-rhamnopyranoside19,20) (36,
sinocrassosides A11 (17, 15.8 mg, 0.0013%) and A12 (18, 13.1 mg, 0.0011%), 1.8 mg from 4), and 5 (1.8 mg, from 6).
kaempferol
3-O-b-D-glucopyranosyl-7-O-a-L-rhamnopyranoside
(25,
Quercetin 3-O-b-D-Glucopyranosyl-7-O-b-D-glucopyranosyl-(1→3)-a-
L-rhamnopyranoside (1a) A yellow powder, [a]D23 ꢀ108.2° (cꢂ0.50,
MeOH). High-resolution positive-ion FAB-MS: Calcd for C33H40O21Na
(MꢁNa)ꢁ 795.1960; Found 795.1967. UV [lmax (log e), MeOH]: 257
17.9 mg, 0.0015%), quercetin 3-O-b-D-glucopyranosyl-7-O-a-L-rhamnopy-
ranoside (30, 35.3 mg, 0.0030%), kaempferol 3-O-b-D-glucopyranosyl-
(1→4)-a-L-rhamnopyranosyl-7-O-a-L-rhamnopyranoside (27, 9.6 mg,

1444
Vol. 56, No. 10
1
(4.40), 358 (4.37) nm. IR (KBr, cmꢀ1): 3432, 1655, 1599, 1076. H-NMR
(500 MHz, DMSO-d6) d: given in Table 1. 13C-NMR (125 MHz, DMSO-d6)
dC: given in Table 2. Positive-ion FAB-MS m/z: 795 (MꢁNa)ꢁ. Negative-ion
FAB-MS m/z: 771 (MꢀH)ꢀ.
2) Yoshikawa M., Wang T., Morikawa T., Xie H., Matsuda H., Chem.
Pharm. Bull., 55, 1308—1315 (2007).
3) Morikawa T., Xie H., Wang T., Matsuda H., Yoshikawa M., Chem.
Biodiver., (2008), in press.
4) Xie H., Wang T., Matsuda H., Morikawa T., Yoshikawa M., Tani T.,
Chem. Pharm. Bull., 53, 1416—1422 (2005).
5) Morikawa T., Xie H., Matsuda H., Yoshikawa M., J. Nat. Prod., 69,
881—886 (2006).
6) Morikawa T., Xie H., Matsuda H., Wang T., Yoshikawa M., Chem.
Pharm. Bull., 54, 506—513 (2006).
7) Xie H., Morikawa T., Matsuda H., Nakamura S., Muraoka O.,
Yoshikawa M., Chem. Pharm. Bull., 54, 669—675 (2006).
8) Yoshikawa M., Matsuda H., Morikawa T., Xie H., Nakamura S., Mu-
raoka O., Bioorg. Med. Chem., 14, 7468—7475 (2006).
9) Matsuda H., Sugimoto S., Morikawa T., Matsuhira K., Mizuguchi E.,
Nakamura S., Yoshikawa M., Chem. Pharm. Bull., 55, 106—110
(2007).
Acid Hydrolysis of 3, 5, and 1a A solution of 3, 5, and 1a (each
1.0 mg) in 1 M HCl (2.0 ml) was heated under reflux for 3 h. After cooling,
the reaction mixture was extracted with EtOAc. The EtOAc-soluble fraction
was subjected to HPLC analysis under the following conditions, respec-
tively: HPLC column, YMC-Pack ODS-A, 4.6 mm i.d.ꢆ250 mm (YMC Co.,
Ltd., Ktoyo, Japan); detection, UV (254 nm); mobile phase, MeOH–1%
aqueous AcOH (60 : 40, v/v); flow rate 1.0 ml/min]. Identification of
gossypetin (37, from 5), herbacetin (35, from 3), and quercetin (28, from 1a)
present in the EtOAc-soluble fraction was carried out by comparison of their
retention time with those of authentic samples (tR: 37, 5.0 min; 35, 7.5 min;
and 28, 9.0 min). On the other hand, the aqueous layer was subjected to
HPLC analysis under the following conditions, respectively: HPLC column,
Kaseisorb LC NH2-60-5, 4.6 mm i.d.ꢆ250 mm (Tokyo Kasei Co., Ltd.,
Tokyo, Japan); detection, optical rotation [Shodex OR-2 (Showa Denko Co.,
Ltd., Tokyo, Japan); mobile phase, CH3CN–H2O (85 : 15, v/v); flow rate
0.8 ml/min]. Identification of L-rhamnose (i) and D-glucose (ii) from 3, 5,
and 1a present in the aqueous layer was carried out by comparison of their
retention time and optical rotation with those of an authentic samples [tR: (i)
7.8 min (negative optical rotation) and (ii) 13.9 min (positive optical rota-
tion)].
Bioassay Method. Effects on Aminopeptidase N Inhibitory Activity
Aminopeptidase N activity was assayed by the method described in a previ-
ous report.24) Briefly, 0.2 mM of the enzyme substrate, L-alanine-4-methyl-
coumaryl-7-amide (Ala-MCA, Peptide Institute) in 50 mM Tris–HCl buffer
containing 200 mM NaCl (pH 8.0, 100 ml/well) and sample solution (20 ml)
in 96-well black microplates. The reaction mixture was initiated by adding
an enzyme solution 100 ml/well (rat, 1 mU, Calbiochem) and incubated at
37 °C for 1 h to convert Ala-MCA to a fluorescent product, 7-amino-4-
methylcoumarin (AMC). The reaction mixture was mixed with 0.1 M EDTA
(50 ml/well) to stop the reaction. Fluorescence was measured using a fluores-
cence microplate reader (FLUOstar OPTIMA, BMG Labtechnologies) at an
excitation wavelength of 380 nm and an emission wavelength of 460 nm.
Each sample was dissolved in dimethyl sulfoxide (DMSO) and diluted with
PBS (final DMSO concentration in the incubation mixture was 0.1%). Cur-
cumin was isolated from Thai Zedoary (the rhizomes of Curcuma zedoaria
originating in Thailand)32) and used as a reference compound.
10) Yoshikawa M., Morikawa T., Zhang Y., Nakamura S., Muraoka O.,
Matsuda H., J. Nat. Prod., 70, 575—583 (2007).
11) Morikawa T., Zhang Y., Nakamura S., Matsuda H., Muraoka O.,
Yoshikawa M., Chem. Pharm. Bull., 55, 435—441 (2007).
12) Ninomiya K., Morikawa T., Zhang Y., Nakamura S., Matsuda H., Mu-
raoka O., Yoshikawa M., Chem. Pharm. Bull., 55, 1185—1191 (2007).
13) Zhang Y., Morikawa T., Nakamura S., Ninomiya K., Matsuda H., Mu-
raoka O., Yoshikawa M., Heterocycles, 71, 1565—1576 (2007).
14) Nakamura S., Li X., Matsuda H., Ninomiya K., Morikawa T., Yam-
aguti K., Yoshikawa M., Chem. Pharm. Bull., 55, 1505—1511 (2007).
15) Nakamura S., Li X., Matsuda H., Yoshikawa M., Chem. Pharm. Bull.,
56, 536—540 (2008).
16) Li X., Nakamura S., Matsuda H., Yoshikawa M., Chem. Pharm. Bull.,
56, 612—615 (2008).
17) Yoshikawa M., Nakamura S., Li X., Matsuda H., Chem. Pharm. Bull.,
56, 695—700 (2008).
18) These standard samples were purchased from Apin Chemicals Ltd.
(U.K.).
19) Zapesochnaya G. G., Kurkin V. A., Schavlinskii A. N., Khim. Prir.
Soedin., 1985, 496—507 (1985).
20) Yoshikawa M., Shimada H., Shimoda H., Murakami N., Yamahara J.,
Matsauda H., Chem. Pharm. Bull., 44, 2086—2091 (1996).
21) Look A. T., Ashmum R. A., Shapiro L. H., Peiper S. C., J. Clin.
Invest., 83, 1299—1307 (1989).
22) Saiki I., Fujii H., Yoneda J., Abe F., Nakajima M., Tsuruo T., Azuma
I., Int. J. Cancer, 54, 137—143 (1993).
23) Pasqualini R., Koivunen E., Kain R., Lahdenranta J., Sakamoto M. M.,
Stryhn A., Ashmum R. A., Shapiro L. H., Arap W., Ruoslahti E., Can-
cer Res., 60, 722—727 (2000).
24) Morikawa T., Funakoshi K., Ninomiya K., Yasuda D., Miyagawa K.,
Matsuda H., Yoshikawa M., Chem. Pharm. Bull., 56, 956—962
(2008).
25) Shim J. S., Kim J. H., Cho H. Y., Yum Y. N., Kim S. H., Park H.-J.,
Shim B. S., Choi S. H., Kwon H. J., Chem. Biol., 10, 695—704 (2003).
26) Yoshikawa M., Morikawa T., Murakami T., Toguchida I., Harima S.,
Matsuda H., Chem. Pharm. Bull., 47, 340—345 (1999).
27) Matsuda H., Morikawa T., Ueda H., Yoshikawa M., Heterocycles, 55,
1499—1504 (2001).
Effects on Aldose Reductase Inhibitory Activity Aldose reductase ac-
tivity was assayed by the method described previously.4,26—31) The super-
natant fluid of rat lens homogenate was used as a crude enzyme. The incuba-
tion mixture contained 135 mM Na, K-phosphate buffer (pH 7.0), 100 mM
Li2SO4, 0.03 mM NADPH, 1 mM DL-glyceraldehyde as a substrate, and
100 ml of enzyme fraction, with or without 25 ml 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 ml of
0.5 M HCl. Then, 0.5 ml 6 M NaOH containing 10 mM imidazole was added,
and the solution was heated at 60 °C for 20 min to convert NADP to a fluo-
rescent product. Fluorescence was measured using a fluorophotometer (Lu-
minescence Spectrometer LS50B, Perkin Elmer, U.K.) at an excitation wave-
length of 360 nm and an emission wavelength of 460 nm.
Statistics Values are expressed as meansꢄS.E.M. One-way analysis of
variance followed by Dunnett’s test was used for statistical analysis.
28) Matsuda H., Morikawa T., Toguchida I., Yoshikawa M., Chem. Pharm.
Bull., 50, 788—795 (2002).
29) Matsuda H., Morikawa T., Toguchida I., Harima S., Yoshikawa M.,
Chem. Pharm. Bull., 50, 972—975 (2002).
30) Yoshikawa M., Murakami T., Ishiwada T., Morikawa T., Kagawa M.,
Higashi Y., Matsuda H., J. Nat. Prod., 65, 1151—1155 (2002).
31) Morikawa T., Kishi A., Pongpiriyadacha Y., Matsuda H., Yoshikawa
M., J. Nat. Prod., 66, 1191—1196 (2003).
Acknowledgments M. Yoshikawa and H. Matsuda were supported by
the 21st COE Program, Academic Frontier Project, and a Grant-in Aid for
Scientific Research from the Ministry of Education, Culture, Sports, Science
and Technology of Japan (MEXT). T. Morikawa was supported by High-tech
Research Center Project (2007—2011) and a Grant-in Aid for Young Scien-
tists from MEXT.
32) Matsuda H., Tewtrakul S., Morikawa T., Nakamura A., Yoshikawa M.,
Bioorg. Med. Chem., 12, 5891—5898 (2004).
References and Notes
1) Part XXXI: Ninomiya K., Morikawa T., Xie H., Matsuda H.,
Yoshikawa M., Heterocycles, 75, 1983—1995 (2008).