Bioactive constituents from Chinese natural medicines. XXXVI.1) four new acylated phenylethanoid oligoglycosides, kankanosides J1, J2, K1, and K2, from stems of cistanche tubulosa

Article abstract of DOI:10.1248/cpb.58.575

Four new acylated phenylethanoid oligoglycosides, kankanosides J ₁ (1), J₂ (2), K₁ (3), and K₂ (4), were isolated from stems of Cistanche tubulosa (Orobanchaceae) together with isocampneoside I (5). Their struct

Full text of DOI:10.1248/cpb.58.575

April 2010
Notes
Chem. Pharm. Bull. 58(4) 575—578 (2010)
575
1)
Bioactive Constituents from Chinese Natural Medicines. XXXVI.
New Acylated Phenylethanoid Oligoglycosides, Kankanosides J , J ,
Four
1
2
K , and K , from Stems of Cistanche tubulosa
1
2
a,b
a
a
a
b
Yingni PAN, Toshio MORIKAWA, Kiyofumi NINOMIYA, Katsuya IMURA, Dan YUAN,
,
a,c
,a
Masayuki YOSHIKAWA,* and Osamu MURAOKA*
a
Pharmaceutical Research and Technology Institute, Kinki University; 3–4–1 Kowakae, Higashi-osaka, Osaka 577–8502,
b
Japan: School of Traditional Chinese Medicine, Shenyang Pharmaceutical University; 103 Wenhua Rd., Shenyang
1
c
10016, People’s Republic of China: and Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412,
Japan. Received November 28, 2009; accepted February 2, 2010; published online February 4, 2010
Four new acylated phenylethanoid oligoglycosides, kankanosides J (1), J (2), K (3), and K (4), were iso-
1
2
1
2
lated from stems of Cistanche tubulosa (Orobanchaceae) together with isocampneoside I (5). Their structures
were elucidated on the basis of chemical and physicochemical evidence. Among them, 3—5 were found to inhibit
D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes.
Key words Cistanche tubulosa; kankanoside; phenylethanoid glycoside; Orobanchaceae; hepatoprotective activity
During the course of our studies on bioactive constituents rings. The positive- and negative-ion FAB-MS spectra of 1
1
—3)
ꢁ
from Chinese natural medicines,
we found that methano- showed quasimolecular ion peaks at m/z 719 (MꢁNa) and
ꢀ
lic extract of dried stems of Cistanche tubulosa (SCHRENK) R. m/z 695 (MꢀH) , and the molecular formula was deter-
WIGHT (Orobanchaceae) showed vasorelaxant and hepato- mined as C H O by high-resolution positive-ion FAB-MS
protective activities. From the dried stems of C. tubulosa, measurement. The H- and C-NMR spectra of 1 (CD OD,
4)
3
2
40 17
1)
1
13
3
five iridoids, kankanosides A—D and kankanol, a monoter- Tables 1, 2), which were assigned by various NMR experi-
7)
pene glycoside, kankanoside E, two phenylethanoid oligogly- ments, showed signals assignable to a methoxy group [d
cosides, kankanosides F and G, and an acylated oligosugar, 3.21 (3H, s, 7-OCH )], a methylene and a methine bearing an
3
kankanose, were isolated together with 30 known con- oxygen function [d 3.58, 4.00 (1H each, both m, 8-H ), 4.18
2
4,5)
stituents.
Recently, we additionally isolated 19 phenyl- (1H, dd-like, Jꢂca. 4, 8 Hz, 7-H)], ortho- and meta-coupled
ethanoid oligoglycosides including kankanosides H , H , and ABC-type aromatic protons [d 6.63 (1H, dd, Jꢂ1.8, 8.2 Hz,
1
2
1)
I and two acylated oligosugars from fresh stems of C. tubu- 6-H), 6.74 (1H, d, Jꢂ1.8 Hz, 2-H), 6.74 (1H, d, Jꢂ8.2 Hz, 5-
1)
losa. Furthermore, principal phenylethanoid glycosides, H)], a b-D-glucopyranosyl moiety [d 4.54 (1H, d, Jꢂ7.8 Hz,
echinacoside, acteoside, and isoacteoside, were found to in- Glc-1-H)], and an a-L-rhamnopyranosyl moiety [d 1.07 (3H,
hibit increase in serum aspartate aminotransferase (sAST) d, Jꢂ6.4 Hz, Rha-6-H ), 4.80 (1H, br s, Rha-1-H)] together
3
and alanine aminotransferase (sALT) levels in liver injured with an acetyl group [d 2.00 (3H, s)] and a trans-caffeoyl
mice induced by D-galactosamine (D-GalN)/lipopolysaccha- group {an trans-olefin [d 6.26, 7.59 (1H each, both d,
ride at doses of 25—100 mg/kg per os (p.o.), and structural Jꢂ16.0 Hz, 8-, 7-H)] and ortho- and meta-coupled ABC-type
requirements of phenylethanoid glycosides for the hepato- aromatic protons [d 6.77 (1H, d, Jꢂ8.2 Hz, 5-H), 6.95 (1H,
1)
protective activity were elucidated. As a continuing study dd, Jꢂ1.8, 8.2 Hz, 6-H), 7.04 (1H, d, Jꢂ1.8 Hz, 2-H)]}. The
1
13
on constituents from the fresh stems of C. tubulosa, we fur-
ther isolated four new acylated phenylethanoid oligoglyco- of campneoside I
sides, kankanosides J (1), J (2), K (3), and K (4). This acetyl group. Connectivities of the oligoglycoside and acyl
H- and C-NMR spectra of 1 were superimposable on those
1
,8—10)
(6), except for the signals due to the
1
2
1
2
paper deals with isolation and structure elucidation of 1—4.
moieties in 1 were confirmed by the heteronuclear multiple
Fresh stems of C. tubulosa (cultivated in Urumuqi, Xin- bond correlation (HMBC) experiment, which showed long-
jiang Province, China) were extracted with methanol under range correlations between the following proton and carbon
reflux to yield a methanolic extract (8.36% from the fresh pairs: 7-OCH and 7-C (d 83.3); Glc-1-H and 8-C (dC
3
C
stems). From the methanolic extract, H O- and MeOH-eluted
2
fractions (5.63% and 2.73%, respectively) were obtained by
Diaion HP-20 column chromatography (H O→MeOH) as
2
1)
was described previously. By the intensive chromatogra-
phies on the MeOH-eluted fraction, four new phenylethanoid
oligoglycosides, kankanosides J1 (1, 0.0002%), J2 (2,
0
.0002%), K (3, 0.0002%), and K (4, 0.0005%) together
1
2
6)
with isocampneoside I (5, 0.0006%) were isolated.
Structures of Kankanosides J1 (1) and J2 (2)
Kankanoside J (1) was obtained as a white powder with neg-
1
2
5
ative optical rotation ([a] ꢀ6.5° in MeOH). The IR spec-
trum of 1 showed absorption bands at 3414, 1734, 1719,
701, 1638, 1508, 1159, 1067, and 1046 cm ascribable to
D
ꢀ1
1
hydroxyls, ester carbonyls, ether functions, and aromatic
Chart 1
∗
To whom correspondence should be addressed. e-mail: muraoka@phar.kindai.ac.jp; myoshika@mb.kyoto-phu.ac.jp © 2010 Pharmaceutical Society of Japan

5
76
Vol. 58, No. 4
1
Table 1. H-NMR Data (600 MHz, CD OD) for Kankanosides J (1), J (2), K (3), and K (4)
3
1
2
1
2
1
2
3
4
Position
dH (J Hz)
dH (J Hz)
dH (J Hz)
dH (J Hz)
2
5
6
7
8
6.74 (d, 1.8)
6.74 (d, 8.2)
6.63 (dd, 1.8, 8.2)
4.18 (dd-like, ca. 4, 8)
3.58 (m)
6.73 (br s)
6.73 (d, 8.2)
6.80 (d, 2.0)
6.76 (d, 8.1)
6.78 (d, 1.9)
6.76 (d, 8.2)
6.62 (dd, 1.8, 8.2)
4.22 (dd, 3.2, 8.2)
3.63 (m)
3.83 (dd, 3.2, 11.0)
3.24 (3H, s)
6.68 (dd, 2.0, 8.1)
4.34 (dd, 3.4, 8.1)
3.62 (dd, 3.4, 11.0)
4.02 (dd, 8.1, 11.0)
3.23 (3H, s)
6.67 (dd, 1.9, 8.2)
4.37 (dd, 3.1, 9.1)
3.72 (dd, 9.1, 11.0)
3.84 (dd, 3.1, 11.0)
3.25 (3H, s)
4
.00 (m)
7
-OCH3
3.21 (3H, s)
8-O-Glc
1
2
3
4
5
6
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
4.54 (d, 7.8)
4.64 (d, 8.2)
4.91 (dd, 8.2, 9.2)
4.04 (dd, 9.2, 9.6)
5.00 (dd, 9.6, 9.6)
3.61 (m)
4.40 (d, 7.9)
4.44 (d, 7.9)
4.91 (dd, 7.8, 9.2)
4.01 (dd, 9.2, 9.6)
4.99 (dd, 9.2, 9.6)
3.56 (m)
3.43 (dd, 7.9, 9.2)
3.81 (dd, 9.2, 9.7)
5.00 (dd, 9.7, 9.7)
3.77 (m)
3.64 (dd, 5.5, 11.6)
3.92 (dd, 2.0, 11.6)
3.43 (dd, 7.9, 9.1)
3.82 (dd, 9.1, 9.7)
5.02 (dd, 9.7, 9.8)
3.77 (ddd, 2.2, 5.5, 9.8)
3.62 (dd, 5.5, 11.9)
3.94 (dd, 2.2, 11.9)
3.51 (m)
3.52 (m)
3.61 (m)
3
.61 (m)
3
ꢃ-O-Rha
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
6ꢄ
4.80 (br s)
3.25 (m)
3.61 (m)
3.25 (m)
3.55 (m)
4.82 (br s)
3.65 (m)
3.52 (m)
3.26 (m)
3.52 (m)
5.19 (d, 1.7)
5.20 (d, 1.6)
3.91 (dd, 1.7, 3.3)
3.57 (dd, 3.3, 9.5)
3.28 (m)
3.55 (dq, 9.5, 6.4)
1.08 (3H, d, 6.4)
3.92 (dd, 1.6, 3.2)
3.57 (dd, 3.2, 9.4)
3.28 (m)
3.55 (dq, 9.1, 6.2)
1.08 (3H, d, 6.2)
1.07 (3H, d, 6.4)
1.07 (3H, d, 6.4)
6
ꢃ-O-Glc
1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
6ꢅ
4.31 (d, 7.9)
4.26 (d, 7.7)
3.19 (dd, 7.7, 9.3)
3.32 (m)
3.25 (m)
3.22 (m)
3.19 (dd, 7.9, 9.1)
3.34 (dd, 8.8, 9.1)
3.26 (dd, 8.8, 9.6)
3.22 (m)
3.63 (dd, 5.7, 12.1)
3.62 (dd, 5.5, 12.0)
3.82 (dd, 2.1, 12.0)
3
.83 (dd, 2.2, 12.1)
2
ꢃ-O-Ac
2.00 (3H, s)
2.11 (3H, s)
4
ꢃ-O-trans-Caf
2
5
6
7
8
7.04 (d, 1.8)
6.77 (d, 8.2)
6.95 (dd, 1.8, 8.2)
7.59 (d, 16.0)
6.26 (d, 16.0)
7.04 (d, 1.8)
6.77 (d, 8.2)
6.95 (dd, 1.8, 8.2)
7.54 (d, 16.0)
6.27 (d, 16.0)
7.05 (d, 1.9)
6.78 (d, 8.3)
6.96 (dd, 1.9, 8.3)
7.60 (d, 15.8)
6.27 (d, 15.8)
7.05 (d, 1.9)
6.78 (d, 8.4)
6.96 (dd, 1.9, 8.4)
7.60 (d, 15.8)
6.28 (d, 15.8)
1
1
Fig. 1. H– H COSY and HMBC Correlations for 1—4
7
4.1); Glc-2-H [d 4.91 (1H, dd, Jꢂ7.8, 9.2 Hz)] and the dated to be 2-methoxy-2-(3,4-dihydroxyphenyl)ethyl O-a-L-
acetyl carbonyl carbon (d 171.4); Glc-4-H [d 4.99 (1H, dd, rhamnopyranosyl-(1→3)-2-O-acetyl-4-O-trans-caffeoyl-b-D-
Jꢂ9.2, 9.6 Hz)] and the trans-caffeoyl carbonyl carbon (d
C
glucopyranoside (1).
C
1
68.1); and Rha-1-H and Glc-3-C (d 80.5) (Fig. 1). Finally,
Kankanoside J (2) was isolated as a white powder with
C
2
2
5
alkaline hydrolysis of 1 with 5% potassium hydroxide (KOH) negative optical rotation ([a] ꢀ18.1° in MeOH). By high-
liberated trans-caffeic acid, which was identified by HPLC resolution positive-ion FAB-MS measurement, the molecular
D
1
analysis, together with a deacylated product. The deacylated formula of 2 was found to be the same as that of 1. The H-
product was successively treated with 1.0 M hydrochloric acid and C-NMR data of 2 (CD OD, Tables 1, 2) were very sim-
1
3
3
(HCl) to liberate L-rhamnose and D-glucose, which were ilar to those of 1, except for the signals due to the ethyl
identified by HPLC analysis using an optical rotation de- bridge of the aglycone moiety {a methoxy group [d 3.24
1—5)
tector.
Thus, the structure of kankanoside J was eluci- (3H, s, 7-OCH )], a methylene [d 3.63 (1H, m), 3.83 (1H, dd,
1
3

April 2010
Table 2.
577
13
C-NMR Data (150 MHz, CD OD) for Kankanosides J (1), J
11.0 Hz), 4.02 (1H, dd, Jꢂ8.1, 11.0 Hz), 8-H ], 4.34 (1H, dd,
Jꢂ3.4, 8.1 Hz, 7-H); 4: d [3.72 (1H, dd, Jꢂ9.1, 11.0 Hz),
.84 (1H, dd, Jꢂ3.1, 11.0 Hz), 8-H ], 4.37 (1H, dd, Jꢂ3.1,
9.1 Hz, 7-H)}, ortho- and meta-coupled ABC-type aromatic
protons [3: d 6.68 (1H, dd, Jꢂ2.0, 8.1 Hz, 6-H), 6.76 (1H, d,
Jꢂ8.1 Hz, 5-H), 6.80 (1H, d, Jꢂ2.0 Hz, 2-H); 4: d 6.67 (1H,
dd, Jꢂ1.9, 8.2 Hz, 6-H), 6.76 (1H, d, Jꢂ8.2 Hz, 5-H), 6.78
1H, d, Jꢂ1.9 Hz, 2-H)], two b-D-glucopyranosyl moieties
3: d 4.31 (1H, d, Jꢂ7.9 Hz, terminal-Glc-1-H), 4.40 (1H, d,
Jꢂ7.9 Hz, inner-Glc-1-H); 4: d 4.26 (1H, d, Jꢂ7.7 Hz, ter-
minal-Glc-1-H), 4.44 (1H, d, Jꢂ7.9 Hz, inner-Glc-1-H)], and
an a-L-rhamnopyranosyl moiety [3: d 1.08 (3H, d, Jꢂ6.4 Hz,
Rha-6-H ), 5.19 (1H, d, Jꢂ1.7 Hz, Rha-1-H); 4: d 1.08 (3H,
d, Jꢂ6.2 Hz, Rha-6-H ), 5.20 (1H, d, Jꢂ1.6 Hz, Rha-1-H)]
3
1
2
2
(
2), K (3), and K (4)
1 2
3
2
1
2
3
4
Position
dC
dC
dC
dC
1
2
3
4
5
6
7
8
131.7
115.3
146.5
146.4
116.3
120.2
83.3
131.2
115.0
146.5
146.3
116.2
119.7
84.3
131.2
115.2
146.5
146.4
116.3
120.2
83.5
130.7
115.1
146.5
146.5
116.4
119.9
84.4
(
[
74.1
74.9
74.7
75.2
7-OCH3
56.8
57.1
56.7
56.7
3
8-O-Glc
1
2
3
4
5
6
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
101.7
75.1
80.5
70.7
76.3
62.3
102.2
75.3
80.5
70.7
76.2
62.2
104.1
76.0
81.5
70.6
74.9
69.4
104.5
76.3
81.4
70.5
74.7
69.5
3
together with a trans-caffeoyl group {an trans-olefin [3: d
6
7
.27, 7.60 (1H each, both d, Jꢂ15.8 Hz, 8-, 7-H); 4: d 6.28,
.60 (1H each, both d, Jꢂ15.8 Hz, 8-, 7-H)] and ortho- and
meta-coupled ABC-type aromatic protons [3: d 6.78 (1H, d,
Jꢂ8.3 Hz, 5-H), 6.96 (1H, dd, Jꢂ1.9, 8.3 Hz, 6-H), 7.05 (1H,
d, Jꢂ1.9 Hz, 2-H)]; 4: d 6.78 (1H, d, Jꢂ8.4 Hz, 5-H), 6.96
1H, dd, Jꢂ1.9, 8.4 Hz, 6-H), 7.05 (1H, d, Jꢂ1.9 Hz, 2-H)}.
The proton and carbon signals in the H- and C-NMR
3
ꢃ-O-Rha
1
2
3
4
5
6
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
103.4
72.7
71.9
73.7
70.8
18.5
103.3
72.6
71.9
73.6
70.8
18.5
103.0
72.4
72.1
73.8
70.4
18.4
103.0
72.4
72.0
73.8
70.4
18.4
(
1
13
spectra of 3 and 4 were superimposable on those of echina-
1
,4,12)
coside,
except for the signals due to the 7-methoxy
6
ꢃ-O-Glc
group. The connectivities of the trans-caffeoyl group and the
glycosyl moieties in 3 and 4 were elucidated on the basis of
HMBC experiments as shown in Fig. 1. Finally, alkaline hy-
drolysis of 3 and 4 with 5% KOH gave the deacylated prod-
ucts together with trans-caffeic acid. Those deacylated prod-
ucts were successively treated with 1.0 M HCl to liberate L-
rhamnose and D-glucose, respectively. Consequently, the
structure of kankanosides K and K were determined to be
1
2
3
4
5
6
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
104.6
75.1
77.8
71.5
77.9
62.6
104.7
75.1
77.8
71.5
77.9
62.6
2
ꢃ-O-Ac
1
2
20.9
171.4
21.0
171.5
1
2
2
-methoxy-2-(3,4-dihydroxyphenyl)ethyl O-a-L-rhamnopy-
4
ꢃ-O-Caf
1
2
3
4
5
6
7
8
9
127.7
115.3
146.9
149.9
116.6
123.3
148.2
114.6
168.1
127.6
115.2
146.5
149.9
116.5
123.2
148.2
114.6
168.1
127.6
115.3
146.9
149.9
116.5
123.3
148.2
114.7
168.5
127.6
115.3
146.8
149.9
116.5
123.3
148.3
114.7
168.5
ranosyl-(1→3)-[b-D-glucopyranosyl-(1→6)]-4-O-trans-caf-
feoyl-b-D-glucopyranoside (3 and 4).
11,13)
Previously, methanolic extract from stems of C. tubulosa
and several phenylethanoid constituents such as echinaco-
side, acteoside, and isoacteoside were found to show hepato-
protective effects on D-galactosamine (D-GalN)/lipopoly-
saccharide-induced liver injury in mice and inhibitory effect
on D-GalN-induced cytotoxicity in primary cultured mouse
1)
hepatocytes. We further examined inhibitory effects of
kankanosides K (3) and K (4), and isocampneoside I (5) on
1
2
Jꢂ3.2, 11.0 Hz), 8-H ] and a methine bearing an oxygen D-GalN-induced cytotoxicity in primary cultured hepato-
2
function [d 4.22 (1H, dd, Jꢂ3.2, 8.2 Hz, 7-H)]}. Alkaline cytes. Although their activities were weaker than those
hydrolysis of 2 with 5% KOH liberated trans-caffeic acid of echinacoside (IC ꢂ10.2 mM), acteoside (4.6 mM), and
5
0
together with a deacylated product, and the deacylated prod- isoacteoside (5.3 mM), the principle phenylethanoid con-
1)
uct was successively treated with 1.0 M HCl to liberate L- stituents from stems of C. tubulosa, 3—5 showed moderate
14)
rhamnose and D-glucose. As shown in Fig. 1, the same long- activity.
range correlations as in the case of 1 were observed in the
Experimental
HMBC experiment. Consequently, the planar structure of
The following instruments were used to obtain spectral and physical data:
kankanoside J (2) was revealed to be the same as that of 1, specific rotations, Horiba SEPA-300 digital polarimeter (lꢂ5 cm); UV spec-
2
1
1)
tra, Shimadzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100
and was elucidated to be 7-isomer of 1.
Structures of Kankanosides K1 (3) and K2 (4)
Kankanosides K (3) and K (4), C H O , were also ob-
1
13
spectrometer; H- and C-NMR spectra, JEOL JNM-ECA600 (600,
50 MHz) and JEOL JNM-ECS400 (400, 100 MHz) spectrometers with
tetramethylsilane as an internal standard; FAB-MS and high-resolution FAB-
tained as white powders with negative optical rotations (3: MS, JEOL JMS-SX 102A mass spectrometer; HPLC detector, Shimadzu
1
1
2
36 48 21
2
5
25
1
[
a] ꢀ75.3°; 4: [a]D ꢀ7.4° both in MeOH). The H- and RID-10A refractive index, Shimadzu SPD-10A UV–VIS, and Shodex OR-2
18
D
1
3
optical rotation detectors. HPLC column, Cosmosil 5C -MS-II and pNAP
C-NMR spectra of 3 and 4 (CD OD, Tables 1, 2) showed
3
(Nacalai Tesque Inc., 250ꢆ4.6 mm i.d.) and (250ꢆ20 mm i.d.) columns
signals assignable to a methoxy group [3: d 3.23 (3H, s, 7-
were used for analytical and preparative purposes, respectively.
The following experimental conditions were used for chromatography:
OCH ); 4: d 3.25 (3H, s, 7-OCH )], a methylene and a me-
3
3
thine bearing an oxygen function {3: d [3.62 (1H, dd, Jꢂ3.4, normal-phase silica gel column chromatography (CC), silica gel 60N (Kanto

5
78
Vol. 58, No. 4
Chemical Co., Ltd., 63—210 mesh, spherical, neutral); reversed-phase silica
gel CC, Diaion HP-20 (Nippon Rensui) and Chromatorex ODS DM1020T
Alkaline and Acid Hydrolysis of Kankanosides J (1), J (2), K (3),
1
2
1
and K (4) Solutions of 1—4 (each 1.0 mg) in 5% aqueous potassium hy-
2
(Fuji Silysia Chemical, Ltd., 100—200 mesh); normal-phase TLC, pre-
droxide (KOH, 0.5 ml) were stirred at 40 °C for 1 h. Each solution was neu-
ꢁ
coated TLC plates with silica gel 60F254 (Merck, 0.25 mm); reversed-phase tralized with Dowex HCR W2 (H form), and the resin was removed by fil-
TLC, pre-coated TLC plates with silica gel RP-18 F254S (Merck, 0.25 mm); tration. Evaporation of the solvent from the filtrates under reduced pressure
reversed-phase HPTLC, pre-coated TLC plates with silica gel RP-18 yielded the corresponding deacylated products, which were subjected to
WF254S (Merck, 0.25 mm), detection was achieved by spraying with 1% HPLC analysis [column: Cosmosil pNAP, 250ꢆ4.6 mm i.d.; mobile phase:
Ce(SO ) –10% aqueous H SO , followed by heating.
Plant Material This item was described in a previous report.
Extraction and Isolation Fresh stems of C. tubulosa (2.98 kg) were
CH CN–1% aqueous AcOH (15 : 85, v/v); detection: UV (254 nm); flow
rate: 1.0 ml/min] to give trans-caffeic acid (tR 9.9 min from 1—4). Then
each was dissolved in 1.0 M HCl (1.0 ml) and heated at 80 °C for 3 h. After
4
2
2
4
3
1)
finely cut and extracted three times with methanol under reflux for 3 h. being cooled, the reaction mixture was neutralized with Amberlite IRA-400
ꢀ
Evaporation of the solvent under reduced pressure provided a methanolic ex- (OH form), and the resins were removed by filtration. After removal of the
tract (249.1 g, 8.36%). The methanolic extract was subjected to Diaion HP- solvent under reduced pressure, the residue was separated by Sep-Pak C18
2
0 CC (5.0 kg, H O→MeOH) to give H O- and MeOH-eluted fractions cartridge column (H O→MeOH). The H O-eluted fraction was subjected to
2
2
2
2
(167.84 g, 5.63% and 81.21 g, 2.73%, respectively). The MeOH-eluted frac- HPLC analysis under following conditions: HPLC column, Kaseisorb LC
tion (61.00 g) was subjected to normal-phase silica gel CC [1.8 kg, CHCl3– NH -60-5, 4.6 mm i.d.ꢆ250 mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); de-
2
MeOH–H O (15 : 3 : 0.4→10 : 3 : 0.5→6 : 4 : 1, v/v/v)→MeOH] to give seven tection, optical rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo,
2
fractions [Fr. 1 (1.12 g), 2 (9.56 g), 3 (0.89 g), 4 (10.69 g), 5 (8.84 g), 6 Japan); mobile phase, CH CN–H O (85 : 15, v/v); flow rate 0.8 ml/min].
3
2
1)
(
(
(
12.52 g), and 7 (4.60 g)], as was described previously. The fraction 4
Identification of L-rhamnose (i) and D-glucose (ii) from 1—4 present in the
10.69 g) was separated by reversed-phase silica gel CC [500 g, MeOH–H O
30 : 70, v/v)→MeOH→acetone] to give four fractions [Fr. 4-1 (878.2 mg), and optical rotation with those of authentic samples [i, t 9.9 min (negative)]
-2 (7.06 g), 4-3 (1.57 g), and 4-4 (792.8 mg)]. The fraction 4-3 (1.57 g) was
H O-eluted fractions were carried out by comparison of their retention times
2
2
R
4
and [ii, t 17.9 min (positive)].
R
purified by HPLC [Cosmosil 5C -MS-II, CH CN–1% aqueous AcOH
1
8
3
(20 : 80, v/v)] to give 11 fractions {Fr. 4-3-1 (30.4 mg), 4-3-2 (55.2 mg), 4-3-
Acknowledgements T. M., K. N., and O. M. were supported by ‘High-
3
0
[ꢂcampneoside I (6, 22.1 mg, 0.0010%)], 4-3-4 [ꢂacteoside (224.6 mg,
.010%)], 4-3-5 (27.4 mg), 4-3-6 (43.6 mg), 4-3-7 [ꢂisoacteoside sidy from Ministry of Education, Culture, Sports, Science and Technology
tech Research Center’ Project for Private Universities: matching fund sub-
(
(
(
825.0 mg, 0.037%)], 4-3-8 [ꢂsyringalide A 3ꢃ-O-a-L-rhamnopyranoside
(MEXT) of Japan, 2007—2011 and also supported by a Grant-in Aid for
37.6 mg, 0.0017%)], 4-3-9 (39.8 mg), 4-3-10 [ꢂ2ꢃ-acetylacteoside Scientific Research from MEXT. M. Y. and H. M. were supported by the
1)
85.4 mg, 0.0038%)], and 4-3-11 (64.6 mg)}, as was described previously.
21st COE program, Academic Frontier Project, and a Grant-in Aid for Sci-
entific Research from MEXT.
The fraction 4-3-5 (27.4 mg) was further purified by HPLC [Cosmosil
pNAP, CH CN–1% aqueous AcOH (18 : 82, v/v)] to give isocampneoside I
3
(
5, 8.5 mg, 0.0004%). The fraction 4-3-6 (43.6 mg) was further purified by
References and Notes
HPLC [Cosmosil pNAP, CH CN–1% aqueous AcOH (18 : 82, v/v)] to give
1) Part XXXV: Morikawa T., Pan Y., Ninomiya K., Imura K., Matsuda
H., Yoshikawa M., Yuan D., Muraoka O., Bioorg. Med. Chem., 18,
1882—1890 (2010).
3
1)
5
(3.7 mg, 0.0002%) together with kankanosides H (17.0 mg, 0.0008%)
1
1)
and H2 (3.3 mg, 0.0001%). The fraction 4-3-9 (39.8 mg) was further puri-
2)
Morikawa T., Xie H., Wang T., Matsuda H., Yoshikawa M., Chem.
Biodiv., 6, 411—420 (2009).
fied by HPLC [Cosmosil pNAP, CH CN–1% aqueous AcOH (18 : 82, v/v)]
3
to give kankanosides J (1, 3.7 mg, 0.0002%) and J (2, 3.5 mg, 0.0002%) to-
1
2
1)
1)
3) Muraoka O., Morikawa T., Zhang Y., Ninomiya K., Nakamura S., Ma-
gether with kankanoside I (15.4 mg, 0.0007%) and isoacteoside (3.1 mg,
.0001%). The fraction 5 (8.84 g) was separated by reversed-phase silica gel
tsuda H., Yoshikawa M., Tetrahedron, 65, 4142—4148 (2009).
Yoshikawa M., Matsuda H., Morikawa T., Xie H., Nakamura S., Mu-
raoka O., Bioorg. Med. Chem., 14, 7468—7475 (2006).
Xie H., Morikawa T., Matsuda H., Nakamura S., Muraoka O.,
Yoshikawa M., Chem. Pharm. Bull., 54, 669—675 (2006).
0
4
)
)
CC [400 g, MeOH–H O (20 : 80→30 : 70, v/v)→MeOH→acetone] to give
2
seven fractions [Fr. 5-1 (870.2 mg), 5-2 (478.9 mg), 5-3 (3.72 g), 5-4
5
(979.9 mg), 5-5 (1.19 g), 5-6 (1.27 g), and 5-7 (130.1 mg)]. The fraction 5-3-
4
(72.3 mg) was further purified by HPLC [Cosmosil pNAP, CH CN–1%
6) Si C.-L., Liu Z., Kim J.-K., Bae Y.-S., Holzforschung, 62, 197—200
3
aqueous AcOH (10 : 90, v/v)] to give kankanosides K (3, 5.1 mg, 0.0002%)
and K (4, 10.6 mg, 0.0005%) together with campneoside II (7, 10.6 mg,
(2008).
1
1)
1
13
7) The H- and C-NMR spectra of 1—4 were assigned with the aid of
distortionless enhancement by polarization transfer (DEPT), double
quantum filter correlation spectroscopy (DQF COSY), heteronuclear
multiple quantum coherence (HMQC), and heteronuclear multiple
bond correlation (HMBC) experiments.
2
0
.0005%).
2
5
Kankanoside J1 (1): A white powder, [a]D ꢀ6.5° (cꢂ0.25, MeOH).
ꢁ
High-resolution positive-ion FAB-MS: Calcd for C H O Na (MꢁNa)
3
2
40 17
7
19.2163; Found 719.2170. UV [l
(log e), MeOH]: 291 (sh, 3.68), 333
max
ꢀ1
8) Imakura Y., Kobayashi S., Mima A., Phytochemistry, 24, 139—146
(3.83) nm. IR (KBr, cm ): 3413, 1734, 1719, 1701, 1638, 1508, 1159,
1
13
(1985).
1
067, 1046. H-NMR (600 MHz, CD OD) d: given in Table 1. C-NMR
3
9)
Wu J., Huang J., Xiao Q., Zhang S., Xiao Z., Li Q., Long L., Huang L.,
(150 MHz, CD OD) d : given in Table 2. Positive-ion FAB-MS m/z: 719
3 C
ꢁ ꢀ
Magn. Res. Chem., 42, 659—662 (2004).
0) Kitagawa S., Tsukamoto H., Hisada S., Nishibe S., Chem. Pharm.
(MꢁNa) . Negative-ion FAB-MS m/z: 695 (MꢀH) .
1
2
5
Kankanoside J2 (2): A white powder, [a]D ꢀ18.1° (cꢂ0.23, MeOH).
Bull., 32, 1209—1213 (1984).
ꢁ
High-resolution positive-ion FAB-MS: Calcd for C H O Na (MꢁNa)
11) Stereochemistries of 7-position in 1—4 have not been determined.
12) Kobayashi H., Oguchi H., Takizawa N., Miyase T., Ueno A., Usmang-
hani K., Ahmad M., Chem. Pharm. Bull., 35, 3309—3314 (1987).
3
2
40 17
7
19.2163; Found 719.2167. UV [l
(log e), MeOH]: 291 (sh, 3.99), 333
max
ꢀ1
(4.15) nm. IR (KBr, cm ): 3418, 1734, 1717, 1638, 1509, 1159, 1070,
1
13
1
046. H-NMR (600 MHz, CD OD) d: given in Table 1. C-NMR 13) Campneoside I (6), a methylated version of campneoside II (7), was
3
(
150 MHz, CD OD) d : given in Table 2. Positive-ion FAB-MS m/z: 719
reported to be isolated from water extracts of leaves of Campsis
8) 10)
3
C
ꢁ
ꢀ
chinensis and fruit of Forsythia viridissima. In the present study,
6 could not be produced by treatment of campneoside II (7) with
methanol under reflux for more than 24 h. These findings suggest that
(MꢁNa) . Negative-ion FAB-MS m/z: 695 (MꢀH) .
2
5
Kankanoside K1 (3): A white powder, [a]D ꢀ75.3° (cꢂ0.16, MeOH).
ꢁ
High-resolution positive-ion FAB-MS: Calcd for C H O Na (MꢁNa)
3
6
48 21
1
—4 are not artificially produced during the extraction procedure.
8
39.2586; Found 839.2589. UV [l
(log e), MeOH]: 289 (sh, 4.05), 335
max
1
4) Inhibitory effects of presently isolated kankanosides K (3) and K (4),
ꢀ1
1
2
(4.24) nm. IR (KBr, cm ): 3415, 1734, 1717, 1686, 1636, 1614, 1509,
and isocampneoside I (5, IC ꢂ81.6 mM) on D-GalN-induced cytotox-
1
13
50
1
159, 1074. H-NMR (600 MHz, CD OD) d: given in Table 1. C-NMR
3
icity in primary cultured mouse hepatocytes were examined [inhibition
(
150 MHz, CD OD) d : given in Table 2. Positive-ion FAB-MS m/z: 839
3 C
ꢁ
(%) 3: 0.0ꢇ1.1, 5.0ꢇ1.2, 10.2ꢇ2.3*, 16.8ꢇ2.9**, and 31.0ꢇ3.7**;
(MꢁNa) .
Kankanoside K2 (4): A white powder, [a]D ꢀ7.4° (cꢂ0.35, MeOH).
4
: 0.0ꢇ0.6, 5.2ꢇ0.1, 10.3ꢇ0.7**, 18.0ꢇ1.1**, and 24.9ꢇ3.4**; 5:
2
5
0.0ꢇ1.0, 6.0ꢇ0.4*, 16.6ꢇ0.4**, 31.7ꢇ0.9**, and 53.7ꢇ2.5** at 0, 3,
10, 30, and 100 mM, respectively]. Values are expressed as meansꢇ
S.E.M. (nꢂ4). For statistical analysis, one-way analysis of variance
(ANOVA) followed by Dunnett’s test was used. Probability (p) values
less than 0.05 were considered significant (∗ pꢈ0.05, ∗∗ pꢈ0.01). The
ꢁ
High-resolution positive-ion FAB-MS: Calcd for C H O Na (MꢁNa)
3
6
48 21
8
39.2586; Found 8392594. UV [l
(log e), MeOH]: 291 (sh 4.06), 334
max
ꢀ1
(4.23) nm. IR (KBr, cm ): 3415, 1734, 1717, 1686, 1636, 1614, 1509,
1
13
1
159, 1079. H-NMR (600 MHz, CD OD) d: given in Table 1. C-NMR
3
1)
bioassay method was described previously.
(150 MHz, CD OD) d : given in Table 2. Positive-ion FAB-MS m/z: 839
3 C
ꢁ
(MꢁNa) .