Chemical structures and hepatoprotective effects of constituents from the leaves of Salacia chinensis

Article abstract of DOI:10.1248/cpb.59.1020

The methanolic extract from the leaves of Salacia chinensis collected in Thailand was found to show a protective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. From the methanolic extract, eight new glycosides, named foliachinenosides E, F, G, H, and I, and foliasalaciosides J, K and L, were isolated together with 26 known constituents. The structures of new glycosides were determined on the basis of physicochemical and chemical evidence. In addition, the hepatoprotective effects of the isolated compounds on D-galactosamine-induced cytotoxicity were examined. Among them, lignans, eleutheroside E₂ and 7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-β -D-glucopyranoside, were found to show the protective effects [inhibition (%) 41.4±3.6 (p<0.01), 45.5±2.7 (p<0.01) at 100 μM, respectively].

Full text of DOI:10.1248/cpb.59.1020

1020
Regular Article
Chem. Pharm. Bull. 59(8) 1020—1028 (2011)
Vol. 59, No. 8
Chemical Structures and Hepatoprotective Effects of Constituents
from the Leaves of Salacia chinensis
Seikou NAKAMURA,a Yi ZHANG,a Hisashi MATSUDA,a Kiyofumi NINOMIYA,b Osamu MURAOKA,b and
,a
*
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 April 27, 2011; accepted May 26, 2011; published online May 30, 2011
The methanolic extract from the leaves of Salacia chinensis collected in Thailand was found to show a pro-
tective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. From the
methanolic extract, eight new glycosides, named foliachinenosides E, F, G, H, and I, and foliasalaciosides J, K
and L, were isolated together with 26 known constituents. The structures of new glycosides were determined on
the basis of physicochemical and chemical evidence. In addition, the hepatoprotective effects of the isolated com-
pounds on D-galactosamine-induced cytotoxicity were examined. Among them, lignans, eleutheroside E2 and
7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-b-D-glucopyranoside, were found to show the protective effects
[inhibition (%) 41.4ꢀ3.6 (pꢀ0.01), 45.5ꢀ2.7 (pꢀ0.01) at 100 mM, respectively].
Key words Salacia chinensis; Hippocrateaceae; foliachinenoside; foliasalacioside; glycoside; hepatoprotective effect
In the course of our characterization studies on bioactive (4.1%) and an aqueous phase. The aqueous phase was further
constituents from Salacia species (Hippocrateaceae),1—6) we extracted with n-BuOH to give an n-BuOH-soluble fraction
have reported the isolation and absolute stereostructure eluci- (2.4%) and a H2O-soluble fraction (6.6%). The n-BuOH-solu-
dation of 13 megastigmane glycosides, 7 phenolic glycosides, ble fraction was subjected to Diaion HP-20 column chro-
and 11 triterpenes from the leaves of Salacia chinensis col- matography (H2O→MeOH) to give the water- and MeOH-
lected in Thailand together with 40 known constituents.7—11) eluted fractions as previously reported.7) The EtOAc-soluble
As a continuning study on the leaves of Salacia chinensis, fraction and the MeOH-eluted fraction were respectively sub-
the methanolic (MeOH) extract was found to show a protec- jected to normal- and reversed-phase silica gel column chro-
tive effect on D-galactosamine-induced cytotoxicity in pri- matographies, and finally HPLC to give eight new com-
mary cultured mouse hepatocytes. From the MeOH extract, pounds, foliachinenosides E (1, 0.00009%), F (2, 0.00013%),
eight new glycosides, named foliachinenosides E, F, G, H, G (3, 0.00017%), H (4, 0.00048%), and I (5, 0.0018%), and
and I, and foliasalaciosides J, K, and L, were isolated to- foliasalaciosides J (6, 0.00017%), K (7, 0.00016%), and L (8,
gether with 26 known constituents. Furthermore, we exam- 0.00007%), together with 26 known compounds, 3-methyl-2-
ined the hepatoprotective effects of the isolated compounds but-2-en-1-ol 6-O-a-L-arabinopyranosyl-b-D-glucopyranoside
on D-galactosamine-induced cytotoxicity. In this paper, we (9, 0.0017%),12) (3Z)-3-hexen-1-ol 6-O-a-L-arabinopyrano-
describe the isolation and structure elucidation of the new syl-b-D-glucopyranoside (10, 0.0014%),13) 2,4,6-trimethoxy-
constituents (1—8) and the hepatoprotective effects of iso- phenol 1-O-b-D-glucopyranoside (11, 0.00016%),14) benzyl
lated compounds from the leaves of Salacia chinensis. alcohol b-D-glucopyranoside (12, 0.00003%),15) benzyl alco-
The dried leaves of S. chinensis, which were collected at hol 6-O-a-L-arabinopyranosyl-b-D-glucopyranoside (13,
Nakhon Si Thammarat province, Thailand, were finely cut 0.0026%),16) benzyl b-primeveroside (14, 0.00014%),17) vio-
and extracted with MeOH to furnish a MeOH extract lutoside (15, 0.00039%),18) 2-phenethyl alcohol 6-O-a-L-ara-
(13.0%). The MeOH extract was partitioned into an EtOAc– binopyranosyl-b-glucopyranoside (16, 0.00033%),19) 2,6-
H2O (1 : 1, v/v) mixture to furnish an EtOAc-soluble fraction dimethoxy-4-(2-hydroxyethyl)phenol 1-O-b-D-glucopyrano-
Chart 1
∗ To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp
© 2011 Pharmaceutical Society of Japan

August 2011
1021
Chart 2
side (17, 0.00008%),20) eugenyl vicianoside (18,0.00096%),21) H-5), 4.60 (1H, m, H-6)], a quaternary carbon bearing an
2,6-dimethoxy-4-(2-propenyl)phenol 6-O-b-D-glucopyra- oxygen function [dC 69.6 (C-8)] together with a b-D-glu-
nosyl-b-D-glucopyranoside (19, 0.00011%),22) coniferin (20, copyranosyl part [d 5.17 (1H, d, Jꢄ7.9 Hz, H-1ꢁ)]. The pro-
0.00024%),23) syringin (21, 0.0032%),24) cis-syringin (22, ton and carbon signals due to the aglycon part of 1 in the 1H-
0.00024%),25) dihydrosyringin (23, 0.00024%),26) trans-p- and 13C-NMR spectra were similar to those of (1R,4R,
sinapoyl-b-D-glucopyranoside (24, 0.00068%),27) (E)- 5R,8S,9S)-4,11,11-trimethyltricyclo[6.3.1.0.1,9]dodecane-5,8-
coumaroyl-1-O-b-D-glucopyranoside (25, 0.00026%),28) 1- diol 5-O-b-D-glucopyranoside40) except for the 6-position. As
[(2Z)-3-(4-hydroxyphenyl)-2-propenoate]-b-D-glucopyra- shown in Fig. 1, the double quantum filter correlation spec-
noside (26, 0.00005%),28) myzodendrone (27, 0.00024%),29,30) troscopy (DQF COSY) experiment on 1 indicated the pres-
hovetrichoside A (28, 0.00010%),31) 4,7,9-trihydroxy-3,3ꢁ- ence of partial structures written in bold lines (Fig. 1), and in
dimethoxy-8-O-4ꢁ-neolignan-9ꢁ-O-b-D-glucopyranoside[7S, the heteronuclear multiple bond connectivity spectroscopy
8R-erythro form] (29, 0.00019%),32) syringaresinol mono-b- (HMBC) experiment, long-range correlations were observed
D-glucopyranoside (30, 0.00049%),33) eleutheroside E2 (31, between the following protons and carbons: H-1 and C-8, 10;
0.00017%),34) 7R,8S-dihydrodehydrodiconiferyl alcohol 4-O- H-3 and C-15; H-5 and C-3, 15; H-7 and C-6; H-10 and C-1,
b-D-glucopyranoside (32, 0.00028%),35) (2S)-2,3-O-di-(9,12, 8, 13; H-12 and C-1, 10, 11, 13; H-13 and C-1, 10, 11, 12;
15-octadecatrienoyl)-glyceryl-b-D-galactopyranoside (33, H-14 and C-4, 5, 7, 9, 15; H-15 and C-3, 5, 14; H-1ꢁ and C-
0.0066%),36) and 1,2-di-9,12,15-octadecatrienoyl-sn-glycerol 5. In addition, the enzymatic hydrolysis of 1 afforded an
(34, 0.00023%).37)
aglycon, foliachinenol E (1a). Foliachinenol E (1a) was ob-
Structures of Foliachinenosides, E, F, G, H, and I, and tained as colorless oil with positive optical rotation ([a]D23
Foliasalaciosides J, K, and L Foliachinenoside E (1) was ꢂ5.0° in MeOH). The molecular formula C15H26O3 of 1a
obtained as a colorless amorphous powder with positive opti- was determined from a molecular ion peak [m/z 254 (M)ꢂ]
cal rotation ([a]D27 ꢂ9.8° in MeOH). The IR spectrum and by HR-electron ionization (EI)-MS measurement. The
showed absorption bands at 3400 and 1076 cmꢃ1 assignable 1H- (CD3OD) and 13C-NMR (Table 1) spectra39) of 1a
to hydroxyl and ether functions. The positive-ion fast atom showed signals assignable to three methyls [d 1.00, 1.00,
bombardment (FAB) MS of 1 exhibited a quasimolecular ion 0.99 (3H each, all s, H3-12, 13, 15)], two methines bearing an
peak at m/z 439 (MꢂNa)ꢂ. The molecular formula C21H36O8 oxygen function [d 3.01 (1H, d, Jꢄ8.6 Hz, H-5), 3.80 (1H,
of 1 was determined from the quasimolecular ion peak and m, H-6)]. Comparison of the 13C-NMR data of 1 with those
by high-resolution (HR) FAB-MS measurement. Acid hy- of 1a indicated the presence of a glycosidation shift around
drolysis of 1 with aqueous HCl (1.0 m) liberated D-glucose, the 5-position of 1. Thus, the planar structures of 1 and 1a
which was identified in HPLC analysis using an optical rota- were determined as shown. The relative stereostructure of 1
1
tion detector.38) The H-NMR (pyridine-d5) and 13C-NMR was elucidated by using the nuclear Overhauser enhancement
(Table 1) spectra of 1, which were assigned by various NMR spectroscopy (NOESY) experiment, which showed NOE cor-
experiments,39) showed signals assignable to three methyls [d relations between the following proton pairs: H-1 and H-2b,
0.99, 1.02, 1.36 (3H each, all s, H3-13, 12, 15)], two me- H3-12; H-3a and H-6, 9; H-5 and H-7b, H-14b, H3-15; H-6
thines bearing an oxygen function [d 3.61 (1H, d, Jꢄ8.9 Hz, and H-9; H-7a and H-9; H-9 and H-10, H3-13; H-10a and

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Vol. 59, No. 8
H3-13; H-14b and H3-15. On the basis of this evidence, foli- from the positive-ion FAB-MS at m/z 439 (MꢂNa)ꢂ and by
achinenoside E (1) was sesquiterpene glycoside possessing HR-FAB-MS measurement. Acid hydrolysis of 2 with aque-
the rare tricyclo[6.3.1.0.1,9]dodecane skeleton and the struc- ous HCl (1.0 m) liberated D-glucose, which was identified in
1
ture of 1 was determined to be 4,11,11-trimethyltricyclo HPLC analysis using an optical rotation detector.38) The H-
[6.3.1.0.1,9]dodecane-5a,6b,8b-triol 5-O-b-D-glucopyrano- NMR (pyridine-d5) and 13C-NMR (Table 1) spectra of 2,39)
side.
showed signals assignable to two methyls [d 1.16, 1.21 (3H
Foliachinenoside F (2), obtained as a colorless amorphous each, both s, H3-13, 15)], two methines and a methylene
powder with a negative optical rotation ([a]D27 ꢃ22.0° in bearing an oxygen function [d 3.56, 4.10 (1H each, both d,
MeOH), showed the absorption bands at 3400 and 1075 cmꢃ1 Jꢄ9.2 Hz, H2-14), 3.58 (1H, m, H-9), 4.12 (1H, dd like,
ascribable to hydroxyl and ether functions in the IR spec- Jꢄ5.5, 7.9 Hz, H-2)] together with a b-D-glucopyranosyl part
trum. The molecular formula C21H36O8 of 2 was determined [d 4.90 (1H, d, Jꢄ7.9 Hz, H-1ꢁ)]. The proton and carbon sig-
1
nals due to the aglycon part of 2 in the H- and 13C-NMR
spectra were similar to those of clovane-2,9-diol,41) except
for the 14-position. As shown in Fig. 1, the DQF COSY ex-
periment on 1 indicated the presence of partial structures
written in bold lines, and in the HMBC experiment, long-
range correlations were observed between the following pro-
tons and carbons: H-2 and C-1, 4, 11; H2-6 and C-8; H-9 and
C-11, 12; H2-10 and C-1; H2-12 and 5, 9, 11; H3-13 and C-3,
5, 14; H3-15 and C-7, 8, 9, 12; H-1ꢁ and C-14. In addition,
the enzymatic hydrolysis of 2 afforded an aglycon (2a),
which was identified to be (1S,3R,3aR,6S,7S,9aR)-decahy-
dro-1-(hydroxymethyl)-1,7-dimethyl-3a,7-methano-3aH-cy-
clopentacyclooctene41,42) Comparison of the 13C-NMR data
of 2 with those of 2a indicated the presence of a glycosida-
tion shift around the 14-position of 2. Next, the relative ster-
eostructure of 2 was determined by using NOESY experi-
ment, in which NOE correlations were observed between the
following proton pairs: H-2 and H-3b, H3-13; H-3a and H-
6a, H2-14; H-3b and H3-13; H-5 and H-6a, H2-14, H3-15;
H-6a and H-7a; H-6b and H3-13; H-7a and H-9; H-9 and
H3-15. On the basis of this evidence, foliachinenoside F (2)
Fig. 1. Selected HMBC, DQF COSY, and NOE Correlations
was determined as shown.
Table 1. 13C-NMR Data at (125 MHz) for 1—8, 1a, 2a, 6a, and 8a
Position
1a)
1b)
1aa)
2b)
3a)
4a)
5a)
6a)
6ac)
7a)
8a)
8ac)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1ꢁ
2ꢁ
3ꢁ
4ꢁ
5ꢁ
6ꢁ
1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
45.9
22.1
33.3
39.4
96.3
72.3
46.8
70.9
41.3
35.6
35.9
30.8
21.0
48.8
30.2
107.1
76.2
78.5
71.5
78.1
62.7
45.2
21.6
33.3
38.8
97.1
71.6
47.6
69.6
41.1
35.3
35.1
30.7
20.9
49.3
30.0
107.6
76.4
78.8
71.6
78.5
62.8
45.8
22.0
32.5
39.4
84.5
72.0
47.6
71.0
41.1
35.5
36.0
30.8
21.1
48.6
30.1
45.9
80.5
44.2
43.1
46.0
22.0
34.1
35.5
74.5
28.2
27.6
36.6
21.9
80.4
29.5
105.6
75.3
78.8
71.8
78.6
63.0
180.3
29.7
29.0
83.2
36.6
26.5
30.6
30.5
30.5
27.1
30.8
70.9
69.5
38.8
144.0
112.1
23.0
69.5
39.6
35.8
47.9
75.7
43.9
32.2
58.9
133.7
133.9
74.4
67.7
21.7
32.0
21.8
35.0
50.3
66.8
44.7
30.8
57.3
133.9
131.4
73.2
66.8
21.4
31.2
21.2
40.7
43.3
74.2
44.1
78.0
79.1
131.1
136.2
69.6
24.2
26.2
27.6
27.1
44.5
49.4
76.8
48.6
82.2
92.8
126.6
134.3
78.1
21.4
25.9
32.8
31.6
43.5
48.5
75.4
47.7
82.0
90.6
123.5
135.1
68.8
23.7
25.6
32.0
31.5
26.1
23.0d)
23.1d)
104.4
75.2
78.2
71.7
78.0
62.8
104.4
75.1
78.0
71.6
76.9
69.5
105.2
72.4
74.2
69.5
66.7
104.4
75.1
78.0
71.6
76.9
69.5
105.1
72.4
74.2
69.4
66.8
102.8
75.2
78.1
71.8
77.9
62.9
103.2
75.2
78.1
71.9
78.0
63.1
102.8
75.4
78.1
71.5
78.0
62.7
Measured in a) CD3OD, b) pyridine-d5, c) CDCl3, d) interchangeable.

August 2011
1023
Fig. 2. Selected HMBC and DQF COSY Correlations
Foliachinenoside G (3), obtained as an amorphous color-
less powder, showed absorption bands at 3420, 1761, and
1034 cmꢃ1 assignable to hydroxyl, g-lactone, and ether func-
tions in the IR spectrum. The molecular formula C18H32O8 of
3 was determined from the positive-ion FAB-MS at m/z 399
(MꢂNa)ꢂ and by HR-FAB-MS measurement. The acid hy-
drolysis of 3 liberated D-glucose, which was identified by
1
HPLC analysis.38) The H-NMR (CD3OD) and 13C-NMR
(Table 2) spectra39) of 3 showed signals assignable to a meth-
ylene and a methine bearing an oxygen function {[d 3.53,
3.90 (1H each, both td, Jꢄ6.7, 14.0 Hz, H2-12), 4.54 (1H, m,
H-4)]}, a g-lactone carbon [dC 180.3 (C-1)] together with a
b-D-glucopyranosyl part [d 4.24 (d, Jꢄ7.7 Hz, H-1ꢁ)]. The
relative structure of 3 was characterized by DQF COSY and
HMBC experiments as shown in Fig. 2. Finally, the enzy-
matic hydrolysis 3 with b-glucosidase gave an aglycon, foli-
achinenol G (3a), whose molecular formula C12H22O3 was
determined from positive-ion chemical ionization (CI)-MS
{m/z 215 [Mꢂ1]ꢂ} and by HR-CI-MS measurement. The
Fig. 3. Selected HMBC, DQF COSY, and NOESY Correlations
absolute stereostructure of the 3-position of 3 was character- 3.92 (1H each, both td, Jꢄ6.9, 14.7 Hz, H2-1)] together with
ized by comparison of the optical rotation of 3a with those of a b-D-glucopyranosyl and an a-L-arabinopyranosyl parts [4
known g-butylolactones with an alkyl group at the 3-posi- d: 4.27 (1H, d, Jꢄ7.7 Hz, H-1ꢁ), 4.31 (1H, d, Jꢄ6.7 Hz, H-
tion. Namely, the optical rotations of known compounds, (R)- 1ꢅ), 5 d: 4.24 (1H, d, Jꢄ7.6 Hz, H-1ꢁ), 4.31 (1H, d, Jꢄ6.8
g-dodecalactone and (R)-g-undecalactone, with 4R orienta- Hz, H-1ꢅ)]. The positions of the glycoside moieties in 4 and
tion were reported to show positive {[a]D20 ꢂ42.1° (MeOH), 5 were clarified on the basis of a HMBC experiment, which
[a]D20 ꢂ44.8° (MeOH), respectively}, while the optical rota- showed long-range correlations between the following pro-
tions of known compounds, (S)-g-dodecalactone and (S)-g- tons and carbons: H-1ꢁ and C-1; H-1ꢅ and C-6ꢁ. Furthermore,
undecalactone, with 4S orientation were reported to show on the basis of the DQF-COSY and HMBC experiments on 4
negative {[a]D20 ꢃ42.2° (MeOH), [a]D20 ꢃ45.6° (MeOH), re- and 5 (Fig. 2), foliachinenosides I (4) and J (5) were eluci-
spectively}.43) Since 3a showed positive optical rotation dated as shown.
{[a]D27 ꢂ35.7° (MeOH)}, the absolute stereostructure of the
Foliasalacioside J (6) was obtained as a colorless amor-
3-position of 3 was determined to be R orientation. On the phous powder with negative optical rotation ([a]D26 ꢃ26.0° in
basis of this evidence, foliachinenoside G (3) was determined MeOH). The IR spectrum showed absorption bands at 3400
as shown.
and 1076 cmꢃ1 assignable to hydroxyl and ether functions.
Foliachinenosides H (4) and I (5), obtained as a colorless The molecular formula C19H34O8 of 6 was determined from
amorphous powder with negative optical rotation (4: [a]D27 the positive-ion FAB-MS at m/z 413 (MꢂNa)ꢂ and by HR-
ꢃ18.0°; 5: [a]D28 ꢃ19.4° in MeOH), respectively. The IR FAB-MS measurement. The acid hydrolysis of 6 liberated D-
1
spectra of 4 and 5 showed absorption bands due to hydroxyl glucose.38) The H-NMR (CD3OD) and 13C-NMR (Table 1)
and ether functions. The molecular formula (C16H28O10 for 4, spectra39) of 6 showed signals assignable to three methyls [d
C16H30O10 for 5) of 4 and 5 were determined from the posi- 0.86, 0.88 (3H each, both s, H3-11, 12), 0.87 (3H, d,
tive- and negative-ion FAB-MS and by HR-FAB-MS meas- Jꢄ6.7 Hz, H3-13)], a methylene and two methines bearing an
urements. The acid hydrolysis of 4 and 5 liberated D-glucose oxygen function [d 3.43 (1H, dd, Jꢄ7.3, 11.0 Hz, H-10a),
and L-arabinose, which were identified by HPLC analysis.38) 3.48 (1H, dd, Jꢄ4.6, 11.0 Hz, H-10b), 3.87 (1H, m, H-3),
The 1H-NMR (CD3OD) and 13C-NMR (Table 2) spectra39) of 4.10 (1H, m, H-9)] together with a b-D-glucopyranosyl part
4 and 5 showed signals assignable to an aglycon part [4 (3- [d 4.35 (1H, d, Jꢄ8.0 Hz, H-1ꢁ)]. As shown in Fig. 3, the
methyl-3-butenol part) d: 1.76 (3H, s, H3-5), 2.35 (2H, dd, DQF COSY experiment on 6 indicated the presence of par-
Jꢄ7.3, 7.3 Hz, H2-2), 3.65, 3.99 (1H each, both td, Jꢄ7.3, tial structures written in bold lines, and in the HMBC experi-
14.7 Hz, H2-1), 4.74, 4.75 (1H each, both br s, H2-4); 5 (3- ment, long-range correlations were observed between the fol-
methyl-3-butanol part) d: 0.92 (6H, d, Jꢄ6.9 Hz, H3-4, 5), lowing protons and carbons: H-4 and C-2, 6; H-8 and C-6;
1.51 (2H, dd, Jꢄ6.9, 6.9 Hz, H2-2), 1.74 (1H, m, H-3), 3.58, H2-10 and C-8, 9; H3-11 and C-1, 2, 6, 12; H3-12 and C-1, 2,

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Vol. 59, No. 8
6, 11; H3-13 and C-4, 5, 6; H-1ꢁ and C-3. Thus, the planar thermore, on the basis of the DQF-COSY and HMBC exper-
structure of 6 was determined as shown. The relative stereo- iments on 8, the planar structure of 8 was determined as
structure of 6 except for the 9-position was characterized by shown (Fig. 3). Next, the relative stereostructure of 8 except
the NOESY experiment, which showed NOE correlations be- for the 9-position was determined by a NOESY experiment,
tween the following proton pairs: H-2a and H-6, H3-12; H- in which correlations were observed between the following
2b and H-3; H-3 and H-4b, H-5, H3-11; H-4a and H-6; H-4b proton pairs: H-2a and H-3; H-2b and H-3, H3-11; H-3 and
and H-5; H-5 and H-7; H-7 and H3-11. Furthermore, the en- H-4a, H-4b; H-4a and H3-13; H-4b and H3-11; H-7 and H3-
zymatic hydrolysis of 6 with b-glucosidase gave an aglycon, 11, H3-12, H3-13. The configuration at the 9-position of 8
foliasalaciol J (6a). Catalytic reduction of 6a yielded a was characterized by comparison of the carbon signal of the
known megastigmane, sarmentol B.44) Consequently, 6 was 9-position in the 13C-NMR spectrum (CD3OD) with those of
determined to be (3S,5R,6R,7E,9R)-megastigman-7-ene-3,9, known 9-hydroxymegastigman-7-ene 9-O-b-D-glucopyrano-
10-triol 3-O-b-D-glucopyranoside.
sides. Namely, the 13C-NMR signal of 9-hydroxymegastig-
Foliasalacioside K (7) was obtained as a colorless amor- man-7-ene 9-O-b-D-glucopyranosides, ampelopsisionoside
phous powder with negative optical rotation ([a]D27 ꢃ5.6° in (d 77.6) and lauroside B (d 77.0) with the 9R configuration
MeOH). The IR spectra of 7 showed absorption bands due to were reported to be shifted downfield relative to those of lau-
hydroxyl and ether functions. The molecular formula roside A (d 74.8) and lauroside C (d 74.0) with the 9S con-
C19H34O9 was determined from the positive-ion FAB-MS and figuration.50,51) The carbon signal of the 9-position on 8 was
by HR-FAB-MS measurements. The acid hydrolysis of 7 lib- observed at d 78.1, so that the configuration of the 9-position
erated D-glucose, which was identified by HPLC analysis.38) was determined to be R form. Finally, enzymatic hydrolysis
The 1H-NMR (CD3OD) and 13C-NMR (Table 1) spectra39) of of 8 gave an aglycon, 5,6-dihydro-5-hydroxy-3,6-epoxy-b-
7 showed signals assignable to four methyls [d 0.85, 1.14, ionol (8a),48,49) whose relative stereostructure was determined
1.19 (3H each, all s, H3-12, 13, 11), 1.27 (3H, d, Jꢄ6.5 Hz, by X-ray analysis at its nitrobenzoyl derivative. Conse-
H3-10)], two methines bearing an oxygen function [d 4.17 quently, foliasalacioside L (8) was determined to be
(1H, m, H-3), 4.35 (1H, m, H-9)], two olefinic protons [d (3R*,5S*,6S*,7E,9R)-5,6-dihydro-5-hydroxy-3,6-epoxy-b-
5.78 (1H, dd, Jꢄ6.3, 15.9 Hz, H-8), 6.05 (1H, dd, Jꢄ1.1, ionol 9-O-b-D-glucopyranoside.
15.9 Hz, H-7)] together with a b-D-glucopyranosyl part [d
Recently, we have reported the isolation and structure elu-
4.36 (1H, d, Jꢄ7.8 Hz, H-1ꢁ)]. The position of the glycoside cidation of several constituents with hepatoprotective effects
moiety in 7 was clarified on the basis of the HMBC experi- from natural traditional medicine, Hedychium coronarium,52)
ment, which showed long-range correlation between H-1ꢁ Camellia sinensis,53) Sedum sarmentosum,54,55) and Rhodiola
and C-3. Furthermore, on the basis of the DQF-COSY and sachalinensis,56) and Piper chaba.57) Since the MeOH extract
HMBC experiments, the planar structure of 7 was deter- from the leaves of Salacia chinensis showed the protective
mined as shown in Fig. 3 and found to be same as effects on D-GalN-induced cytotoxicity in primary cultured
(3S,5R,6R,7E,9S)-megastigman-7-ene-3,5,6,9-tetraol 3-O-b- mouse hepatocytes [inhibition: 26% (100 mg/ml, pꢀ0.01)],
D-glucopyranoside45) and kiwiionoside [(3S,5R,6R,7E,9R)- the activities of the principal constituents, 3, 5, 9—34, were
megastigman-7-ene-3,5,6,9-tetraol].46) Next, the relative examined. As shown in Table 2, compounds, 12, 23, 29, 31,
stereostructure of 7 except for the 9-position was character- 32, 33, and 34, were found to show the hepatoprotective ef-
ized by the NOESY experiment. Finally, enzymatic hydro- fects. Particularly, lignans, eleutheroside E2 (31) and 7R,8S-
lysis of 7 gave a known megastigmane, (3R,5S,6S,7E,9R)- dihydrodehydrodiconiferyl alcohol 4-O-b-D-glucopyranoside
megastigman-7-ene-3,5,6,9-tetraol (7a).47) Consequently, the (32), significantly showed the protective effects [inhibition
structure of 7 including the absolute configuration was eluci- (%) 41.4ꢆ3.6 (pꢀ0.01), 45.5ꢆ2.7 (pꢀ0.01) at 100 mM, re-
dated and 7 was determined to be (3R,5S,6S,7E,9R)- spectively].
megastigman-7-ene-3,5,6,9-tetraol 3-O-b-D-glucopyranoside.
Experimental
Foliasalacioside L (8) was obtained as an colorless amor-
phous powder with negative optical rotation ([a]D27 ꢃ5.5° in
MeOH). The molecular formula C19H32O8 was determined
from the positive-ion FAB-MS and by HR-FAB-MS meas-
General Experimental Procedures The following instruments were
used to obtain physical data: specific rotations, Horiba SEPA-300 digital po-
larimeter (lꢄ5 cm); IR spectra, Shimadzu FTIR-8100 spectrometer; EI-MS
and high-resolution MS, JEOL JMS-GCMATE mass spectrometer; 1H-NMR
spectra, JEOL EX-270 (270 MHz) and JNM-LA500 (500 MHz) spectrome-
ters; 13C-NMR spectra, JEOL EX-270 (68 MHz) and JNM-LA500 (125
MHz) spectrometers with tetramethylsilane as an internal standard; and
HPLC detector, Shimadzu RID-6A refractive index and SPD-10Avp
UV–VIS detectors. HPLC column, Cosmosil 5C18-MS-II (Nacalai Tesque
Inc., 250ꢇ4.6 mm i.d.) and (250ꢇ20 mm i.d.) columns were used for analyt-
ical and preparative purposes, respectively. The following experimental con-
ditions were used for chromatography: ordinary-phase silica gel column
chromatography, Silica gel BW-200 (Fuji Silysia Chemical, Ltd., Aichi,
Japan, 150—350 mesh); reversed-phase silica gel column chromatography,
Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., Aichi, Japan,
100—200 mesh); TLC plates and 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 HPTLC, with Silica gel
RP-18 WF254S (Merck, 0.25 mm); and detection was achieved by spraying
with 1% Ce(SO4)2–10% aqueous H2SO4 followed by heating.
urements. The acid hydrolysis of 8 liberated D-glucose,
1
which was identified by HPLC analysis.38) The H-NMR
(CD3OD) and 13C-NMR (Table 1) spectra39) of 8 showed sig-
nals assignable to four methyls [d 0.85, 1.18, 1.40 (3H each,
all s, H3-12, 13, 11), 1.30 (3H, d, Jꢄ6.4 Hz, H3-10)], two me-
thines bearing an oxygen function [d 4.34 (1H, m, H-3), 4.38
(1H, m, H-9)], two olefinic protons [d 5.74 (1H, dd, Jꢄ5.8,
15.9 Hz, H-8), 5.80 (1H, d, Jꢄ15.9 Hz, H-7)] together with a
b-D-glucopyranosyl part [d 4.35 (1H, d, Jꢄ8.0 Hz, H-1ꢁ)].
1
The proton and carbon signals of 8 in the H- and 13C-NMR
spectra were similar to those of 5,6-dihydro-5-hydroxy-3,6-
epoxy-b-ionol,48,49) except for the around of the 9-position.
The position of the glycoside moiety in 8 was clarified on the
basis of the HMBC experiment, which showed long-range
correlation between the 1ꢁ-position and the 9-position. Fur-
Plant Material The dried leaves of S. chinensis were collected at
Nakhon Si Thammarat province, Thailand in 2006 and identified by one of

August 2011
1025
Table 2. Effects of Constituents on D-GalN-Induced Cytotoxicity in Primary Cultured Mouse Hepatocytesa)
Inhibition (%)
Treatment
Conc. (mM):
0
3
10
30
100
Foliachinenoside G (3)
12
17
18
0.0ꢆ3.2
0.0ꢆ2.4
0.0ꢆ1.9
0.0ꢆ4.8
0.0ꢆ2.4
0.0ꢆ3.3
0.0ꢆ1.6
0.0ꢆ2.5
0.0ꢆ2.9
0.0ꢆ2.8
0.0ꢆ3.6
0.0ꢆ2.6
0.0ꢆ1.2
0.0ꢆ8.8
0.0ꢆ2.5
0.0ꢆ0.3
1.1ꢆ3.0
ꢃ0.2ꢆ4.0
4.0ꢆ2.0
ꢃ1.8ꢆ5.8
10.1ꢆ5.8
2.5ꢆ2.5
ꢃ1.1ꢆ2.7
9.1ꢆ5.4
ꢃ4.9ꢆ1.7
5.7ꢆ4.5
ꢃ6.6ꢆ2.9
8.0ꢆ6.0
17.5ꢆ6.6
10.7ꢆ2.7
16.8ꢆ3.5*
4.8ꢆ1.1
ꢃ1.4ꢆ3.9
6.6ꢆ9.1
9.8ꢆ3.6
4.2ꢆ2.3
16.8ꢆ6.5
7.9ꢆ1.4
6.0ꢆ3.7
22.5ꢆ6.0*
1.2ꢆ1.8
3.8ꢆ8.4
3.3ꢆ3.4
6.8ꢆ4.6
20.3ꢆ2.7*
10.0ꢆ1.7
6.2ꢆ9.2
14.3ꢆ5.8
6.4ꢆ2.2
7.2ꢆ4.1
24.3ꢆ6.1**
6.6ꢆ2.6
18.5ꢆ2.5**
33.7ꢆ2.8**
13.2ꢆ4.3*
18.6ꢆ3.8
19
30.2ꢆ1.8**
15.4ꢆ4.3**
14.6ꢆ2.5*
30.8ꢆ1.1**
11.8ꢆ2.5*
34.2ꢆ6.6**
16.3ꢆ0.4*
41.4ꢆ3.6**
45.5ꢆ2.7**
34.3ꢆ3.7*
39.1ꢆ4.0**
—
Coniferin (20)
cis-Syringin (22)
Dihydrosyringin (23)
26
29
30
Eleutheroside E2 (31)
32
33
12.1ꢆ5.8
8.8ꢆ1.9
7.2ꢆ2.6
14.1ꢆ5.8
23.4ꢆ6.4
13.6ꢆ8.9
25.0ꢆ2.3**
45.2ꢆ8.8**
20.3ꢆ10.8
14.4ꢆ7.7
17.3ꢆ4.4**
7.7ꢆ0.7
34
Silybin61,b)
Compounds, 5, 9—11, 13—16, 21, 24, 25, 27, and 28, did not show the effects (inhibition: ꢀ12% at 100 mM). a) Each value represents the meanꢆS.E.M. (nꢄ4). Signifi-
cantly different from the control, ∗ pꢀ0.05, ∗∗ pꢀ0.01. (—): Cytotoxicity. b) Commercial silybin (Funakoshi Co., Ltd., Tokyo, Japan) was used as a reference compound.
side (10, 11 mg, 0.00025%), and (E)-coumaroyl-1-O-b-D-glucopyranoside
(25, 9.1 mg, 0.00020%). Fraction 16-3 (403 mg) was subjected to HPLC {[1]
MeOH–H2O (30 : 70, v/v)}, [2] CH3CN–MeOH–H2O (10 : 9 : 81, v/v)] to
furnish (3Z)-3-hexen-1-ol 6-O-a-L-arabinopyranosyl-b-D-glucopyranoside
(10, 11 mg, 0.00025%). Fraction 16-4 (1.3 g) was separated by HPLC {[1]
MeOH–H2O (30 : 70, v/v), [2] [CH3CN–MeOH–H2O (10 : 9 : 81, v/v/v)] to
furnish 7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-b-D-glucopyranoside
(32, 12 mg, 0.00028%).
The n-BuOH-soluble fraction (100.0 g) was subjected to Diaion HP-20
column chromatography (1.5 kg, H2O→MeOH→acetone) to give a H2O-
eluted fraction (49.8 g, 1.19%), a MeOH-eluted fraction (39.2 g, 0.93%) and
an acetone-eluted fraction (11.0 g, 0.26%), respectively. The MeOH-eluted
fraction (39.2 g) was subjected to ordinary-phase silica gel column chro-
matography {480 g, CHCl3–MeOH (10 : 1, v/v)→CHCl3–MeOH–H2O [(10 :
3 : 1, v/v/v, lower layer)→(7 : 3 : 1, v/v/v, lower layer)→(6 : 4 : 1, v/v/v, lower
layer)]→MeOH} to give ten fractions [Fr. 1 (0.5 g), Fr. 2 (0.6 g), Fr. 3
(1.3 g), Fr. 4 (7.3 g), Fr. 5 (3.0 g), Fr. 6 (6.7 g), Fr. 7 (1.6 g), Fr. 8 (2.4 g), Fr. 9
(9.3 g), Fr. 10 (3.5 g)] as reported previously.7) Fraction 3 (1.3 g) was sub-
jected to reversed-phase silica gel column chromatography [480 g,
MeOH–H2O (10 : 90→20 : 80→30 : 70→40 : 60→50 : 50→60 : 40, v/v)→
MeOH] to give five fractions [Fr. 3-1, Fr. 3-2, Fr. 3-3 (389 mg), Fr. 3-4
(131 mg), Fr. 3-5]. Fraction 3-3 (389 mg) was separated by HPLC {[1]
MeOH–H2O (40 : 60, v/v), [2] CH3CN–MeOH–H2O (15 : 8 : 77, v/v/v)} to
furnish eleutheroside E2 (31, 7.0 mg, 0.00017%). Fraction 3-4 (131 mg) was
separated by HPLC {[1] MeOH–H2O (40 : 60, v/v), [2] CH3CN–MeOH–
authors (Rajamangala University of Technology Srivijaya, Pongpiriyadacha
Y.). A voucher of the plant is on file in our laboratory (2006. Thai-06).
Extraction and Isolation The dried leaves of S. chinensis LINN. (5.8 kg)
were finely cut and extracted 3 times with MeOH under reflux for 3 h. Evap-
oration of the solvent under reduced pressure provided a MeOH extract
(756 g, 13.0%). The MeOH extract (712 g) was partitioned into an
EtOAc–H2O (1 : 1, v/v) mixture to furnish an EtOAc-soluble fraction (222 g,
4.1%) and an aqueous phase. The aqueous phase was further extracted with
n-BuOH to give an n-BuOH-soluble fraction (130 g, 2.4%) and a H2O-solu-
ble fraction (361 g, 6.6%). The EtOAc fraction (200 g) was subjected to ordi-
nary-phase silica gel column chromatography [3.8 kg, hexane–EtOAc (40 :
1→10 : 1→5 : 1→1 : 1, v/v)→CHCl3–MeOH–H2O (10 : 3 : 1, v/v/v, lower
layer)→MeOH] to give 16 fractions [Fr. 1 (0.7 g), Fr. 2 (1.3 g), Fr. 3 (28.3 g),
Fr. 4 (0.9 g), Fr. 5 (9.0 g), Fr. 6 (14.9 g), Fr. 7 (3.2 g), Fr. 8 (10.7 g), Fr. 9
(9.1 g), Fr. 10 (6.2 g), Fr. 11 (4.2 g), Fr. 12 (13.8 g), Fr. 13 (4.1 g), Fr. 14
(43.2 g), Fr. 15 (16.9 g), Fr. 16 (26.3 g)]. Fraction 10 (6.2 g) was subjected to
Sephadex LH-20 column chromatography [200 g, MeOH–CHCl3 (1 : 1, v/v)]
to give two fractions [Fr. 10-1, Fr. 10-2 (3.6 g)]. Fraction 10-2 (3.6 g) was
subjected to reversed-phase silica gel column chromatography [120 g,
CH3CN–H2O (75 : 25→85 : 15→90 : 10→100 : 5, v/v)→CH3CN→CHCl3] to
give nine fractions [Fr. 10-2-1, Fr. 10-2-2, Fr. 10-2-3, Fr. 10-2-4, Fr. 10-2-5,
Fr. 10-2-6, Fr. 10-2-7 (49 mg), Fr. 10-2-8, Fr. 10-2-9]. Fraction 10-2-7 (49
mg) was isolated with HPLC [MeOH–H2O (96 : 4, v/v)] to give 1,2-di-9,12,
15-octadecatrienoyl-sn-glycerol (34, 10 mg, 0.00023%). Fraction 14 (43.2 g)
was subjected to reversed-phase silica gel column chromatography [1.2 kg,
MeOH–H2O (60 : 40→70 : 30, v/v)→CH3CN–H2O (65 : 35→75 : 25→85 :
15, v/v)→CH3CN→CHCl3] to give six fractions [Fr. 14-1, Fr. 14-2, Fr. 14-3,
Fr. 14-4, Fr. 14-5 (11.6 g), Fr. 14-6]. Fraction 14-5 (11.6 g) was separated
with silica gel NH column chromatography [400 g, hexane–EtOAc (1 : 10,
v/v)→CHCl3–MeOH (50 : 1→50 : 3→50 : 10, v/v)→MeOH] to give four
fractions [Fr. 14-5-1, Fr. 14-5-2 (5.5 g), Fr. 14-5-3, Fr. 14-5-4]. Fraction 14-
5-2 (5.5 g) was further isolated with reversed-phase silica gel column chro-
matography [160 g, MeOH–H2O (70 : 30→80 : 20→90 : 10, v/v)→MeOH→
CH3CN] to give five fractions [Fr. 14-5-2-1, Fr. 14-5-2-2, Fr. 14-5-2-3, Fr.
14-5-2-4, Fr. 14-5-2-5 (3.9 g)]. A part of fraction 14-5-2-5 (1.9 g) was sub-
jected to HPLC (MeOH) to furnish (2S)-2,3-O-di-(9,12,15-octadeca-
trienoyl)glyceryl]-b-D-galactopyranoside (33, 149 mg, 0.0066%). Fraction
16 (26.3 g) was subjected to Diaion HP-20 column chromatography (1.5 kg,
H2O→MeOH) to give a H2O-eluted fraction (15.2 g) and a MeOH-eluted
fraction (10.8 g), respectively. The MeOH-eluted fraction (10.8 g) was sub-
jected to reversed-phase silica gel column chromatography [480 g, MeOH–
H2O (20 : 80→30 : 70→40 : 60→50 : 50→70 : 30, v/v)→MeOH] to give 11
fractions [Fr. 16-1, Fr. 16-2 (766 mg), Fr. 16-3 (403 mg), Fr. 16-4 (1.3 g), Fr.
16-5, Fr. 16-6, Fr. 16-7, Fr. 16-8, Fr. 16-9, Fr. 16-10, Fr. 16-11]. Fraction 16-
2 (766 mg) was purified by HPLC {[1] MeOH–H2O (30 : 70, v/v), [2]
H2O (15 : 8 : 77, v/v/v)} to furnish foliachinenoside
G (3, 7.0 mg,
0.00017%). Fraction 4 (7.3 g) was subjected to reversed-phase silica gel col-
umn chromatography [220 g, H2O→MeOH–H2O (10 : 90→20 : 80→30 :
70→40 : 60→60 : 40, v/v)→MeOH→CHCl3] to give 10 fractions [Fr. 4-1,
Fr. 4-2, Fr. 4-3 (1.1 g), Fr. 4-4, Fr. 4-5, Fr. 4-6 (652 mg), Fr. 4-7 (1.3 g), Fr. 4-
8 (413 mg), Fr. 4-9, Fr. 4-10]. Fraction 4-3 (1.1 g) was separated by HPLC
{[1] MeOH–H2O (15 : 85 and 20 : 80, v/v), [2] CH3CN–MeOH–H2O
(8 : 8 : 84 and 10 : 8 : 82, v/v/v)} to furnish foliasalacioside J (6, 7.0 mg,
0.00017%), 2,4,6-trimethoxyphenol 1-O-b-D-glucopyranoside (11, 6.9 mg,
0.00016%), benzyl alcohol b-D-glucopyranoside (12, 1.3 mg, 0.00003%), vi-
olutoside (15, 16 mg, 0.00039%), 2,6-dimethoxy-4-(2-hydroxyethyl)phenol
1-O-b-D-glucopyranoside (17, 3.3 mg, 0.00008%), coniferin (20, 10 mg,
0.00024%), syringin (21, 124 mg, 0.0029%), cis-syringin (22, 10.0 mg,
0.00024%), dihydrosyringin (23, 10.1 mg, 0.00024%), (E)-coumaroyl-1-O-
b-D-glucopyranoside (25, 2.5 mg, 0.00006%), 1-[(2Z)-3-(4-hydroxyphenyl)-
2-propenoate]-b-D-glucopyranoside (26, 2.1 mg, 0.00005%), myzodendrone
(27, 10 mg, 0.00024%), and hovetrichoside A (28, 4.4 mg, 0.00010%). Frac-
tion 4-6 (652 mg) was subjected to HPLC {[1] MeOH–H2O (30 : 70 and
26 : 74, v/v), [2] CH3CN–MeOH–H2O (12 : 8 : 80, v/v/v)} to furnish (3Z)-3-
hexen-1-ol 6-O-a-L-arabinopyranosyl-b-D-glucopyranoside (10, 6.8 mg,
0.00016%), syringin (21, 8.7 mg, 0.00021%) and 4,7,9-trihydroxy-3,3ꢁ-
dimethoxy-8-O-4ꢁ-neolignan-9ꢁ-O-b-D-glucopyranoside[7S,8R-erythro
MeOH–H2O (26 : 74, v/v)} to furnish foliachinenoside
I (5, 15 mg,
0.00033%), (3Z)-3-hexen-1-ol 6-O-a-L-arabinopyranosyl-b-D-glucopyrano-

1026
Vol. 59, No. 8
m/z 439.2303 (Calcd for C21H36O8Na [MꢂNa]ꢂ, 439.2308).
Foliachinenoside G (3): An amorphous colorless powder; [a]D29 ꢃ3.0°
(cꢄ0.38, MeOH); IR (KBr) nmax: 3420, 2934, 1761, 1034 cmꢃ1 1H-NMR
form] (29, 8.1 mg, 0.00019%). Fraction 4-7 (1.3 g) was separated by HPLC
{[1] MeOH–H2O (40 : 60, v/v), [2] CH3CN–MeOH–H2O (15 : 8 : 77, v/v/v)}
to furnish foliasalaciosides L (8, 3.1 mg, 0.00007%), (3Z)-3-hexen-1-ol 6-O-
a-L-arabinopyranosyl-b-D-glucopyranoside (10, 5.1 mg, 0.00012%), eugenyl
vicianoside (18, 30 mg, 0.00070%), and syringaresinol mono-b-D-glucopy-
ranoside (30, 17 mg, 0.00040%). Fraction 4-8 (413 mg) was subjected to
HPLC {[1] MeOH–H2O (40 : 60, v/v), [2] CH3CN–MeOH–H2O (15 : 8 : 77,
v/v/v)} to furnish foliachinenoside E (1, 3.9 mg, 0.00009%), eugenyl vi-
cianoside (18, 4.2 mg, 0.00010%) and syringaresinol mono-b-D-glucopyra-
noside (30, 3.5 mg, 0.00008%). Fraction 5 (3.0 g) was subjected to reversed-
phase silica gel column chromatography [90 g, MeOH–H2O (10 : 90→
20 : 80→30 : 70→40 : 60→50 : 50, v/v)→MeOH] to give seven fractions [Fr.
5-1, Fr. 5-2 (196 mg), Fr. 5-3 (212 mg), Fr. 5-4 (224 mg), Fr. 5-5, Fr. 5-6
(400 mg), Fr. 5-7]. Fraction 5-2 (196 mg) was purified by HPLC {[1]
MeOH–H2O (24 : 76, v/v), [2] CH3CN–MeOH–H2O (10 : 8 : 82, v/v/v)} to
furnish foliachinenoside H (4, 11.6 mg, 0.00028%), 3-methylbut-2-en-1-ol
6-O-a-L-arabinopyranosyl-b-D-glucopyranoside (9, 32 mg, 0.00077%), ben-
zyl alcohol 6-O-a-L-arabinopyranosyl-b-D-glucopyranoside (13, 34 mg,
0.00080%). Fraction 5-3 (212 mg) was separated by HPLC {[1] MeOH–H2O
(26 : 74, v/v), [2] CH3CN–MeOH–H2O (10 : 8 : 82, v/v/v)} to furnish foli-
achinenoside I (5, 35 mg, 0.00083%), benzyl alcohol 6-O-a-L-arabinopyra-
nosyl-b-D-glucopyranoside (13, 19 mg, 0.00045%), benzyl b-primeveroside
(14, 6.0 mg, 0.00041%), and trans-p-sinapoyl-b-D-glucopyranoside (24,
29 mg, 0.00068%). Fraction 5-4 (224 mg) was purified by HPLC {[1]
MeOH–H2O (26 : 74, v/v), [2] CH3CN–MeOH–H2O (10 : 8 : 82, v/v/v)} to
furnish (3Z)-3-hexen-1-ol 6-O-a-L-arabinopyranosyl-b-D-glucopyranoside
(10, 34 mg, 0.00080%) and 2-phenethyl alcohol 6-O-a-L-arabinopyranosyl-
b-glucopyranoside (16, 14 mg, 0.00033%). Fraction 5-6 (400 mg) was sub-
jected to HPLC {[1] MeOH–H2O (40 : 60, v/v)}, [2] CH3CN–MeOH–H2O
(15 : 8 : 75, v/v/v)} to furnish foliachinenoside F (2, 5.4 mg, 0.00013%),
eugenyl vicianoside (18, 6.2 mg, 0.00015%), 2,6-dimethoxy-4-(2-pro-
penyl)phenol 6-O-b-D-glucopyranosyl b-D-glucopyranoside (19, 4.6 mg,
0.00011%). Fraction 6 (6.7 g) was subjected to reversed-phase silica gel col-
umn chromatography [220 g, MeOH–H2O (10 : 90→20 : 80→30 : 70→40 :
60→50 : 50→60 : 40→100 : 0, v/v)→CHCl3] to give 11 fractions [Fr. 6-1, Fr.
6-2 (859 mg), Fr. 6-3, Fr. 6-4, Fr. 6-5, Fr. 6-6, Fr. 6-7, Fr. 6-8, Fr. 6-9, Fr. 6-
10, Fr. 6-11]. Fraction 6-2 (859 mg) was separated by HPLC {[1] MeOH–
H2O (25 : 75, v/v), [2] CH3CN–MeOH–H2O (7 : 7 : 86, v/v/v)} to furnish to
foliachinenoside H (4, 9.0 mg, 0.00021%), foliachinenoside I (5, 19 mg,
0.00045%), foliasalacioside K (7, 6.6 mg, 0.00016%), 3-methylbut-2-en-1-ol
6-O-a-L-arabinopyranosyl-b-D-glucopyranoside (9, 38 mg, 0.00089%), and
benzyl alcohol 6-O-a-L-arabinopyranosyl-b-D-glucopyranoside (13, 58 mg,
0.0012%). The known compounds were identified by comparison of their
physical data ([a]D, 1H-NMR, 13C-NMR, and MS) with reported values.
Foliachinenoside E (1): An amorphous colorless powder; [a]D27 ꢂ9.8°
;
(CD3OD, 500 MHz) d: 1.86, 2.33 (1H each, both m, H2-3), 1.35 (6H, m, H2-
7, 8, 9), 1.39 (2H, m, H2-10), 1.39, 1.44 (1H each, both m, H2-6), 1.59, 1.72
(1H each, both m, H2-5), 1.62 (2H, m, H2-11), 2.55 (2H, m, H2-2), 3.53, 3.90
(1H each, both td, Jꢄ6.7, 14.0 Hz, H2-12), 3.66 (dd, Jꢄ5.2, 11.6 Hz, H-6ꢁa),
3.85 (dd, Jꢄ1.5, 11.6 Hz, H-6ꢁb), 4.24 (d, Jꢄ7.7 Hz, H-1ꢁ), 4.54 (1H, m, H-
4); 13C-NMR data (CD3OD, 125 MHz) given in Table 1; Positive-ion FAB-
MS m/z 399 [MꢂNa]ꢂ; HR-FAB-MS m/z 399.2001 (Calcd for C18H32O8Na
[MꢂNa]ꢂ, 399.1995).
Foliachinenoside H (4): An amorphous colorless powder; [a]D27 ꢃ18.0°
(cꢄ0.63, MeOH); IR (KBr) nmax: ;
3440, 2940, 1090 cmꢃ1 1H-NMR
(CD3OD, 500 MHz) d: 1.76 (3H, s, H3-5), 2.35 (2H, dd, Jꢄ7.3, 7.3 Hz, H2-
2), 3.65, 3.99 (1H each, both td, Jꢄ7.3, 14.7 Hz, H2-1), 4.74, 4.75 (1H each,
both br s, H2-4), 4.27 (1H, d, Jꢄ7.7 Hz, H-1ꢁ), 4.31 (1H, d, Jꢄ6.7 Hz, H-1ꢅ);
13C-NMR data (125 MHz, CD3OD) given in Table 1; Positive-ion FAB-MS
m/z 403 [MꢂNa]ꢂ; Negative-ion FAB-MS m/z 379 [MꢃH]ꢃ; HR-FAB-MS
m/z 403.1586 (Calcd for C16H28O10Na [MꢂNa]ꢂ, 403.1580).
Foliachinenoside I (5): An amorphous colorless powder; [a]D28 ꢃ19.4°
(cꢄ1.11, MeOH); IR (KBr) nmax: ;
3450, 2943, 1076 cmꢃ1 1H-NMR
(CD3OD, 500 MHz) d: 0.92 (6H, d, Jꢄ6.9 Hz, H3-4, 5), 1.51 (2H, dd, Jꢄ6.9,
6.9 Hz, H2-2), 1.74 (1H, m, H-3), 3.58, 3.92 (1H each, both td, Jꢄ6.9,
14.7 Hz, H2-1), 4.24 (1H, d, Jꢄ7.6 Hz, H-1ꢁ), 4.31 (1H, d, Jꢄ6.8 Hz, H-1ꢅ);
13C-NMR data (125 MHz, CD3OD) given in Table 1; Positive-ion FAB-MS
m/z 405 [MꢂNa]ꢂ; Negative-ion FAB-MS m/z 381 [MꢃH]ꢃ; HR-FAB-MS
m/z 405.1740 (Calcd for C16H30O10Na [MꢂNa]ꢂ, 405.1737).
Foliasalacioside J (6): An amorphous colorless powder; [a]D26 ꢃ26.0°
(cꢄ0.29, MeOH); IR (KBr) nmax: 3400, 2926, 1634, 1076 cmꢃ1 1H-NMR
;
(CD3OD, 500 MHz) d: 0.86, 0.88 (3H each, both s, H3-11, 12), 0.87 (3H, d,
Jꢄ6.7 Hz, H3-13), 1.02 (1H, m, H-4a), 1.16 (1H, m, H-2a), 1.35 (1H, m,
H-6), 1.57 (1H, m, H-5), 1.85 (1H, m, H-2b), 2.12 (1H, m, H-4b), 3.43 (1H,
dd, Jꢄ7.3, 11.0 Hz, H-10a), 3.48 (1H, dd, Jꢄ4.6, 11.0 Hz, H-10b), 3.87 (1H,
m, H-3), 4.10 (1H, m, H-9), 4.35 (1H, d, Jꢄ8.0 Hz, H-1ꢁ), 5.41 (1H, dd,
Jꢄ5.0, 15.8 Hz, H-8), 5.42 (1H, dd like, Jꢄ10.3, 15.8 Hz, H-7); 13C-NMR
data (CD3OD, 125 MHz) dC: given in Table 1; Positive-ion FAB-MS m/z 413
[MꢂNa]ꢂ; HR-FAB-MS m/z 413.2160 (Calcd for C19H34O8Na [MꢂNa]ꢂ,
413.2151).
Foliasalacioside K (7): An amorphous colorless powder, [a]D26 ꢃ5.6°
(cꢄ0.33, MeOH). IR (KBr): 3430, 2934, 1076 cmꢃ1 1H-NMR (CD3OD,
;
500 MHz) d: 0.85, 1.14, 1.19 (3H each, all s, H3-12, 13, 11), 1.27 (3H, d,
Jꢄ6.5 Hz, H3-10), 1.59 (1H, ddd like, Jꢄ1.9, 4.4, 12.1 Hz, H-2b), 1.68 (1H,
dd, Jꢄ12.1, 12.1 Hz, H-2a), 1.85 (1H, dd, Jꢄ12.8, 12.8 Hz, H-4a), 1.94
(1H, ddd like, Jꢄ1.9, 4.3, 12.8 Hz, H-4b), 4.17 (1H, m, H-3), 4.35 (1H, m,
H-9), 4.36 (1H, d, Jꢄ7.8 Hz, H-1ꢁ), 5.78 (1H, dd, Jꢄ6.3, 15.9 Hz, H-8), 6.05
(1H, dd, Jꢄ1.1, 15.9 Hz, H-7); 13C-NMR data (CD3OD, 125 MHz) dC: given
in Table 1; Positive-ion FAB-MS m/z 429 [MꢂNa]ꢂ; HR-FAB-MS m/z
429.2094 (Calcd for C19H34O9Na [MꢂNa]ꢂ, 429.2101).
(cꢄ0.20, MeOH); IR (KBr) nmax: 3400, 2932, 1076, 758 cmꢃ1 1H-NMR
;
(500 MHz, pyridine-d5) d: 0.99, 1.02, 1.36 (3H each, all s, H3-13, 12, 15),
1.38 (1H each, m, H-2a, 3b), 1.51 (1H, m, H-2b), 1.61 (1H, d, Jꢄ13.4 Hz,
H-14b), 1.68 (1H, dd, Jꢄ8.3, 9.3 Hz, H-10a), 2.01 (1H, dd, Jꢄ9.3, 9.3 Hz,
H-10b), 2.04 (1H, m, H-3a), 2.11 (1H, m, H-1), 2.14 (1H, br d, Jꢄ13.4 Hz,
H-14a), 2.15 (1H, m, H-7b), 2.46 (1H, m, H-9), 2.62 (1H, m, H-7a), 3.61
(1H, d, Jꢄ8.9 Hz, H-5), 4.44 (1H, m, H-6ꢁa), 4.52 (1H, dd, Jꢄ2.4, 11.6 Hz,
H-6ꢁb), 4.60 (1H, m, H-6), 5.17 (1H, d, Jꢄ7.9 Hz, H-1ꢁ); 1H-NMR
(500 MHz, CD3OD) d: 1.00, 1.00, 1.09 (3H each, all s, H3-13, 12, 15), 1.17
(1H, m, H-3b), 1.17 (1H, d, Jꢄ13.7 Hz, H-14b), 1.71 (1H, m, H-3a), 1.79
(1H, br d, Jꢄ13.7 Hz, H-14a), 1.35 (1H, m, H-2a), 1.43 (1H, m, H-7b),
1.49 (1H, m, H-2b), 1.54 (2H, m, H2-10), 1.88 (1H, m, H-1), 2.01 (1H, m,
H-7a), 2.19 (1H, m, H-9), 3.15 (1H, d, Jꢄ9.7 Hz, H-5), 3.69 (1H, dd,
Jꢄ5.5, 11.7 Hz, H-6ꢁa), 3.82 (1H, dd, Jꢄ2.0, 11.7 Hz, H-6ꢁb), 4.05 (1H, m,
H-6), 4.47 (1H, d, Jꢄ7.6 Hz, H-1ꢁ); 13C-NMR data (125 MHz, CD3OD and
pyridine-d5) dC: given in Table 1; Positive-ion FAB-MS m/z 439 [MꢂNa]ꢂ;
HR-FAB-MS m/z 439.2314 (Calcd for C21H36O8Na [MꢂNa]ꢂ, 439.2308).
Foliachinenoside F (2): An amorphous colorless powder; [a]D27 ꢃ22.0°
Foliasalacioside L (8): An amorphous colorless powder; [a]D26 ꢃ5.5°
(cꢄ0.11, MeOH); IR (KBr): 3420, 2934, 1078 cmꢃ1 1H-NMR (CD3OD,
;
500 MHz) d: 0.85, 1.18, 1.40 (3H each, all s, H3-12, 13, 11), 1.30 (3H, d,
Jꢄ6.4 Hz, H3-10), 1.58 (1H, d like, Jꢄ11.6 Hz, H-2b), 1.64 (1H, d like,
Jꢄ12.2 Hz, H-4b), 1.76 (1H, ddd like, Jꢄ2.2, 6.4, 11.6 Hz, H-2a), 1.95
(1H, ddd like, Jꢄ2.2, 5.8, 12.2 Hz, H-4a), 4.34 (1H, m, H-3), 4.35 (1H, d,
Jꢄ8.0 Hz, H-1ꢁ), 4.38 (1H, m, H-9), 5.74 (1H, dd, Jꢄ5.8, 15.9 Hz, H-8),
5.80 (1H, d, Jꢄ15.9 Hz, H-7); 13C-NMR data (CD3OD, 125 MHz) dC: given
in Table 1; Positive-ion FAB-MS m/z 411 [MꢂNa]ꢂ; HR-FAB-MS m/z
411.2000 (Calcd for C19H32O8Na [MꢂNa]ꢂ, 411.1995).
Acid Hydrolyses of 1—8 A solution of 1—8 (1.0 mg each) in 1 M HCl
(1.0 ml) was heated under reflux for 3 h. After cooling, the reaction mixture
was extracted with EtOAc. The aqueous layer was subjected to HPLC analy-
sis under the following conditions, respectively: HPLC column, Kaseisorb
LC NH2-60-5, 4.6 mm i.d.ꢇ250 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 (i) L-arabinose from 4 and 5 and (ii) D-glucose from 1—8
present in the aqueous layer was carried out by comparison of its retention
time and optical rotation with those of authentic sample, tR: (i) 10.2 min (L-
arabinose, positive optical rotation) and (ii) 12.8 min (D-glucose, positive op-
tical rotation).
(cꢄ0.27, MeOH); IR (KBr) nmax: 3400, 2928, 1075, 1036 cmꢃ1 1H-NMR
;
(pyridine-d5, 500 MHz) d: 1.07 (1H, br d, Jꢄ12.5 Hz, H-12a), 1.16, 1.21 (3H
each, both s, H3-13, 15), 1.26 (1H, m, H-7a), 1.40 (1H, m, H-7b), 1.50 (1H,
m, H-6b), 1.59 (1H, m, H-11a), 1.63 (1H, m, H-6a), 1.92 (1H, m, H-10b),
1.95 (1H, dd, Jꢄ4.0, 10.4 Hz, H-5), 2.00 (1H, dd, Jꢄ5.5, 12.5 Hz, H-3b),
2.14 (1H, d, Jꢄ12.5 Hz, H-12b), 2.17 (1H, m, H-10a), 2.23 (1H, dd, Jꢄ7.9,
12.5 Hz, H-3a), 2.40 (1H, m, H-11b), 3.56, 4.10 (1H each, both d,
Jꢄ9.2 Hz, H2-14), 3.58 (1H, m, H-9), 4.12 (1H, dd like, Jꢄ5.5, 7.9 Hz, H-
2), 4.41 (1H, dd, Jꢄ5.5, 11.0 Hz, H-6ꢁa), 4.60 (1H, dd, Jꢄ2.4, 11.0 Hz, H-
6ꢁb), 4.90 (1H, d, Jꢄ7.9 Hz, H-1ꢁ); 13C-NMR data (125 MHz, pyridine-d5)
dC: given in Table 1; Positive-ion FAB-MS m/z 439 [MꢂNa]ꢂ; HR-FAB-MS
Enzymatic Hydrolysis of 1 with b-Glucosidase A solution of 1, 3, 6,
7, and 8 (3.0, 5.1, 5.0, 2.0, 2.0 mg, respectively) in H2O (1.0 ml) was treated
with b-glucosidase (20.6, 10.3, 10.2, 6.5, 6.5 mg, respectively) and the solu-
tion was stirred at 37 °C for 24 h (for 3, 7) or 48 h (for 1, 6, 8). After EtOH

August 2011
1027
was exchanged with 100 ml of the fresh medium, and 10 ml of MTT
(5 mg/ml in phosphate buffered saline) solution was added to the medium.
After 4 h culture, the medium was removed, 100 ml of isopropanol contain-
ing 0.04 M HCl was then added to dissolve the formazan produced in the
cells. The optical density (OD) of the formazan solution was measured by
was added to the reaction mixture, the solution was centrifuged at 4000 rpm
for 10 min. The supernatant solution was concentrated under vacuum to give
a residue, which was purified by HPLC [MeOH–H2O (1, 6, 7: 40 : 60, 3:
50 : 50, 8: 55 : 45, v/v)] to furnish 1a (0.5 mg), 3a (1.8 mg), 6a (1.8 mg), 7a
(0.8 mg), and 8a (1.0 mg), respectively.
Compound 1a: Colorless oil; [a]D23 ꢂ5.0° (cꢄ0.02, MeOH); IR (film) microplate reader at 562 nm (reference: 660 nm). Inhibition (%) was ob-
n
max: 3390, 2932, 1040, 754 cmꢃ1 1H-NMR (500 MHz, CD3OD) d: 1.00,
;
tained by following formula.
1.00, 0.99 (3H each, all s, H3-12, 13, 15), 1.14 (1H, m, H-3b), 1.14 (1H, d,
Jꢄ13.4 Hz, H-14b), 1.65 (1H, m, H-3a), 1.76 (1H, br d, Jꢄ13.4 Hz, Ha-
14), 1.37 (1H, m, H-2a), 1.41 (1H, m, H-7b), 1.50 (1H, m, H-2b), 1.54 (2H,
m, H2-10), 1.90 (1H, m, H-1), 2.01 (1H, m, H-7a), 2.18 (1H, m, H-9), 3.01
(1H, d, Jꢄ8.6 Hz, H-5), 3.80 (1H, m, H-6); 13C-NMR data (125 MHz,
CD3OD) dC: given in Table 1; EI-MS m/z 254 [M]ꢂ (4), 236 (84), 218 (34),
203 (19), 162 (53), 161 (84), 143 (100); HR-FAB-MS m/z 254.1886 (Calcd
for C15H26O3 [M]ꢂ, 254.1882).
inhibition (%)ꢄ[(OD(sample)ꢃOD (control))/(OD (normal)
ꢃOD (control))]ꢇ100
Statistics Values were expressed as meansꢆS.E.M. For statistical analy-
sis, one-way analysis of variance followed by Dunnett’s test was used. Prob-
ability (p) values less than 0.05 were considered significant.
Acknowledgments This research was supported by the 21st COE Pro-
gram, Academic Frontier Project, and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology of
Japan.
Compound 3a: A white powder; [a]D271 ꢂ35.7° (cꢄ0.07, MeOH); IR
(KBr) nmax: 3400, 2936, 1755, 1470 cmꢃ1; H-NMR (CD3OD, 500 MHz) d:
1.86, 2.33 (1H each, both m, H2-3), 1.35 (10H, m, H2-6, 7, 8, 9, 10), 1.52
(2H, m, H2-11), 1.62, 1.72 (1H each, both m, H2-5), 2.55 (2H, m, H2-2), 3.54
(t, Jꢄ6.7 Hz, H2-12), 4.54 (1H, m, H-4); 13C-NMR (CD3OD, 125 MHz) dC:
180.3 (C-1), 29.7 (C-2), 29.0 (C-3), 83.2 (C-4), 36.5 (C-5), 26.5 (C-6), 30.6
(C-7), 30.5 (C-7), 30.5 (C-8), 30.5 (C-9), 27.1 (C-10), 33.7 (C-11), 63.0 (C-
12); Positive-ion CIMS m/z 215 [Mꢂ1]ꢂ (100), 197 (18); HR-CI-MS m/z
215.1639 (Calcd for C12H23O3 [Mꢂ1]ꢂ, 215.1647).
References and Notes
1) Xie W., Tanabe G., Matsuoka K., Amer M. F. A., Minematsu T., Wu
X., Yoshikawa M., Muraoka O., Bioorg. Med. Chem., 19, 2252—2262
(2011).
2) Xie W., Tanabe G., Akaki J., Morikawa T., Ninomiya K., Minematsu
T., Yoshikawa M., Wu X., Muraoka O., Bioorg. Med. Chem., 19,
2015—2022 (2011).
3) Muraoka O., Morikawa T., Miyake S., Akaki J., Ninomiya K., Pong-
piriyadacha Y., Yoshikawa M., J. Nat. Med., 65, 142—148 (2011).
4) Nakamura S., Takahira K., Tanabe G., Morikawa T., Sakano M., Ni-
nomiya K., Yoshikawa M., Muraoka O., Nakanishi I., Bioorg. Med.
Chem. Lett., 20, 4420—4423 (2010).
5) Muraoka O., Xie W., Osaki S., Kagawa A., Tanabe G., Amer M. F. A.,
Minematsu T., Morikawa T., Yoshikawa M., Tetrahedron, 66, 3717—
3722 (2010).
6) Muraoka O., Morikawa T., Miyake S., Akaki J., Ninomiya K.,
Yoshikawa M., J. Pharm. Biomed. Anal., 52, 770—773 (2010), and
references cited therein.
7) Nakamura S., Zhang Y., Pongpiriyadacha Y., Wang T., Matsuda H.,
Yoshikawa M., Heterocycles, 75, 131—143 (2008).
8) Zhang Y., Nakamura S., Pongpiriyadacha Y., Matsuda H., Yoshikawa
M., Chem. Pharm. Bull., 56, 547—553 (2008).
Compound 6a: An amorphous colorless powder; [a]D28 ꢃ9.9° (cꢄ0.09,
MeOH); IR (film) nmax: 3370, 2923, 1640, 754 cmꢃ1 1H-NMR (CDCl3,
;
500 MHz) d: 0.84, 0.85 (3H each, both s, H3-11, 12), 0.85 (3H, d, Jꢄ6.4 Hz,
H3-13), 0.92 (1H, m, H-4a), 1.12 (1H, dd, Jꢄ11.9, 11.9 Hz, H-2a), 1.34
(1H, m, H-6), 1.56 (1H, m, H-5), 1.74 (1H, ddd like, Jꢄ2.4, 4.3, 11.9 Hz, H-
2b), 2.02 (1H, m, H-4b), 3.50 (1H, m, H-10a), 3.65 (1H, m, H-10b), 3.80
(1H, m, H-3), 4.25 (1H, m, H-9), 5.43 (1H, dd, Jꢄ10.3, 15.8 Hz, H-7), 5.44
(1H, dd, Jꢄ5.2, 15.8 Hz, H-8); 1H-NMR (pyridine-d5, 500 MHz) d: 0.88,
0.96 (3H each, both s, H3-11, 12), 0.89 (3H, d, Jꢄ6.6 Hz, H3-13), 1.23 (1H,
m, H-4a), 1.42 (1H, dd, Jꢄ9.2, 10.3 Hz, H-6), 1.46 (1H, dd like, Jꢄ11.7,
11.7 Hz, H-2a), 1.54 (1H, m, H-5), 2.01 (1H, ddd like, Jꢄ2.2, 4.1, 11.7 Hz,
H-2b), 2.24 (1H, m, H-4b), 4.05 (1H, m, H-3), 4.06 (2H, m, H2-10), 4.74
(1H, m, H-9), 5.69 (1H, dd, Jꢄ10.3, 15.8 Hz, H-7), 5.84 (1H, dd, Jꢄ5.2,
15.8 Hz, H-8); 13C NMR data (CDCl3, 125 MHz) dC: given in Table 1; Posi-
tive-ion FAB-MS m/z 251 [MꢂNa]ꢂ; HR-FAB-MS m/z 251.1631 (Calcd for
C13H24O3Na [MꢂNa]ꢂ, 251.1623).
Compound 8a58): An amorphous colorless powder; [a]D27 ꢃ11.4° (cꢄ0.05,
MeOH); IR (film): 3390, 2926, 1638, 1129, 1032, 970 cmꢃ1 1H-NMR
;
9) Nakamura S., Zhang Y., Wang T., Matsuda H., Yoshikawa M., Hetero-
cycles, 75, 1435—1446 (2008).
10) Yoshikawa M. Zhang Y., Wang T., Nakamura S., Matsuda H., Chem.
Pharm. Bull., 56, 915—920 (2008).
11) Zhang Y., Nakamura S., Wang T., Matsuda H., Yoshikawa M., Tetra-
hedron, 64, 7347—7352 (2008).
12) Chassagne D., Crouzet J., Bayonove C. L., Brillouet J. M., Baumes R.
L., Phytochemistry, 41, 1497—1500 (1996).
(CDCl3, 500 MHz) d: 0.88, 1.23, 1.41 (3H each, all s, H3-12, 13, 11), 1.30
(3H, d, Jꢄ6.4 Hz, H3-10), 1.59 (1H, d like, Jꢄ11.6 Hz, H-2b), 1.66 (1H, br d
like, Jꢄ12.2 Hz, H-4b), 1.82 (1H, ddd, Jꢄ2.4, 6.1, 11.6 Hz, H-2a), 2.03
(1H, ddd like, Jꢄ2.4, 6.5, 12.2 Hz, H-4a), 4.37 (1H, m, H-3), 4.38 (1H, m,
H-9), 5.71 (1H, br d, Jꢄ15.9 Hz, H-7), 5.80 (1H, dd, Jꢄ5.8, 15.9 Hz, H-8);
13C-NMR data (CD3OD, 125 MHz) dC: given in Table 1; Positive-ion FAB-
MS m/z 249 [MꢂNa]ꢂ; HR-FAB-MS m/z 249.1471 (Calcd for C13H22O3Na
[MꢂNa]ꢂ, 249.1467).
13) Kishida M., Fujii M., Ida Y., Akita H., Heterocycles, 65, 2127—2137
(2005).
Enzymatic Hydrolysis of 2 with b-Glucosidase and Cellulase (1 : 1)
To a solution of 2 (3.4 mg) in 0.2 M acetate buffer (pH 3.8, 1.0 ml), was
added 8.1 mg of b-glucosidase and 8.1 mg cellulase and stirred at 37 °C for
48 h. After EtOH was added to the reaction mixture, the solution was cen-
trifuged at 4000 rpm for 10 min. The supernatant solution was concentrated
under vacuum to give a residue, which was purified by HPLC [MeOH–H2O
(55 : 45, v/v)] to furnish 2a (1.8 mg).
Hydrogenation of 6 A solution of 6 (1.4 mg) in MeOH (1.0 ml) was
treated with 10% Pd–C (5 mg) and the whole mixture was stirred at room
temperature under an H2 atmosphere for 36 h. The catalyst was filtered off
and the solvent was evaporated under reduced pressure to yield a residue,
which was purified by HPLC [MeOH–H2O (40 : 60, v/v)] to give sarmentol
B (0.5 mg).
Protective Effect on Cytotoxicity Induced by D-GalN in Primary Cul-
tured Mouse Hepatocytes The hepatoprotective effects of the con-
stituents were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) colorimetric assay using primary cultured
mouse hepatocytes. Hepatocytes were isolated from male ddY mice (30—
35 g) by collagenase perfusion method.59,60) The cell suspension at 4ꢇ104
cells in 100 ml William’s E medium containing fetal calf serum (10%), peni-
cillin G (100 units/ml), and streptomycin (100 mg/ml) was inoculated in a
96-well microplate, and precultured for 4 h at 37 °C under a 5% CO2 atmo-
sphere. The fresh medium (100 ml) containing D-GalN (2 mM) and a test
sample were added and the hepatocytes were cultured for 44 h. The medium
14)
Saijo R., Nonaka G., Nishioka I., Phytochemistry, 28, 2443—2446
(1989).
15) Seigler D. S., Pauli G. F., Nahrstedt A., Leen R., Phytochemistry, 60,
873—882 (2002).
16) Cui B., Nakamura M., Kinjo J., Nohara T., Chem. Pharm. Bull., 41,
178—182 (1993).
17) Otsuka H., Takeda Y., Yamasaki K., Phytochemistry, 29, 3681—3683
(1990).
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