Iridoid and acyclic monoterpene glycosides, kankanosides L, M, N, O, and P from Cistanche tubulosa

Article abstract of DOI:10.1248/cpb.58.1403

Three iridoid glycosides, kankanosides L, M, and N, and two acyclic monoterpene glycosides, kankanosides O and P, were isolated from fresh stems of Cistanche tubulosa (Orobanchaceae) together with eight iridoid glycosides, five acyclic monoterpene glycosides, three phenylpropanoid glycosides, and four lignan glycosides. Their structures were elucidated on the basis of chemical and physicochemical evidence.

Full text of DOI:10.1248/cpb.58.1403

October 2010
Note
Chem. Pharm. Bull. 58(10) 1403—1407 (2010)
1403
Iridoid and Acyclic Monoterpene Glycosides, Kankanosides L, M, N, O,
and P from Cistanche tubulosa
Toshio MORIKAWA,a Yingni PAN,a,b Kiyofumi NINOMIYA,a Katsuya IMURA,a Dan YUAN,b
Masayuki YOSHIKAWA,a,c Takao HAYAKAWA,a and Osamu MURAOKA*,a
a Pharmaceutical Research and Technology Institute, Kinki University; 3–4–1 Kowakae, Higashi-osaka, Osaka 577–8502,
Japan: b School of Traditional Chinese Medicine, Shenyang Pharmaceutical University; 103 Wenhua Rd., Shenyang
c
110016, People’s Republic of China: and Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412,
Japan. Received June 12, 2010; accepted July 14, 2010; published online July 15, 2010
Three iridoid glycosides, kankanosides L, M, and N, and two acyclic monoterpene glycosides, kankanosides
O and P, were isolated from fresh stems of Cistanche tubulosa (Orobanchaceae) together with eight iridoid glyco-
sides, five acyclic monoterpene glycosides, three phenylpropanoid glycosides, and four lignan glycosides. Their
structures were elucidated on the basis of chemical and physicochemical evidence.
Key words Cistanche tubulosa; kankanoside; iridoid; acyclic monoterpene; Orobanchaceae
Cistanche tubulosa (SCHRENK) R. WIGHT (Orobanchaceae) reflux to yield a methanolic extract (8.36% from the fresh
is a perennial parasitic plant growing on roots of Salvadora stems). From the methanolic extract, H2O- and MeOH-eluted
or Calotropis species, distributed in North Africa, Arabia, fractions (5.63% and 2.73%, respectively) were obtained by
and Asian countries.1) Stems of this plant (Kanka-nikujuyou Diaion HP-20 column chromatography (H2O→MeOH) as
in Japanese) have traditionally been used for treatment of im- described previously.5) The MeOH-eluted fraction was sub-
potence, sterility, lumbago, and body weakness as well as a jected to SiO2 and ODS column chromatographies and
promoting agent of blood circulation.1,2) During the course of finally HPLC to furnish kankanosides L (1, 0.0026%), M (2,
our studies on bioactive constituents from stems of C. tubu- 0.0001%), N (3, 0.0007%), O (4, 0.0020%), and P (5,
losa,3—6) we previously reported twenty four phenylethanoid 0.0002%), 6-deoxycatalpol3,7) (6, 0.197%), bartsioside3,7) (7,
oligoglycosides including kankanosides H1, H2, I, J1, J2, K1, 0.0583%), gluroside3,7) (8, 0.0443%), kankanoiside A3) (9,
and K2, and two acylated oligosugars from fresh stems of C. 22.3 mg, 0.0010%), mussaenosidic acid3,7) (10, 0.0056%), 8-
tubulosa.5,6) Furthermore, principal phenylethanoid glyco- epiloganic acid3,8) (11, 0.0023%), 8-epideoxyloganic acid3,7)
sides, echinacoside, acteoside, and isoacteoside, were found (12, 0.0004%), geniposidic acid3,7) (13, 0.0040%), kankanoside
to inhibit increase in serum aspartate aminotransferase E3) (14, 0.0026%), (2E,6Z)-8-b-D-glucopyranosyloxy-2,6-di-
(sAST) and alanine aminotransferase (sALT) levels in the methyl-2,6-octadienoic acid3,9) (15, 31.0 mg, 0.0014%),
mice’s injured liver induced by D-galactosamine (D-GalN)/ (2E,6E)-3,7-dimethyl-8-hydroxyoctadien-1-yl-O-b-D-glu-
lipopolysaccharide at doses of 25—100 mg/kg per os (p.o.). copyranoside10) (16, 0.0082%), 8-hydroxygeraniol 8-O-b-
Structural requirements of the phenylethaonoid glycosides D-glucopyranoside11) (17, 0.0044%), betulalbuside A12)
for the hepatoprotective activity were also elucidated.5) In (18, 0.0004%), coniferin13) (19, 0.0002%), syringin13) (20,
this continuing study on constituents in the fresh stems of C. 0.0015%), sinapic aldehyde 4-O-b-D-glucopyranoside14) (21,
tubulosa, we further isolated eleven iridoid glycosides in- 0.0001%), (ϩ)-pinoresinol O-b-D-glucopyranoside4,8,15) (22,
cluding kankanosides L (1), M (2), and N (3), seven acyclic 0.0010%), eucommin A16) (23, 0.0002%), isoeucommin A17)
monoterpene glycosides including kankanosides O (4) and P (24, 0.0010%), and (ϩ)-syringaresinol O-b-D-glucopyra-
(5), three phenylpropanoids, and four lignans. This paper noside4,8,18) (25, 0.0044%).
deals with isolation and structure elucidation of five new
Structures of Kankanosides L (1), M (2), and N (3)
compounds (1—5).
Kankanoside L (1) was obtained as a white powder with neg-
Fresh stems of C. tubulosa (cultivated in Urumuqi, Xin- ative optical rotation ([a]D26 Ϫ45.7 in MeOH). Its IR spec-
jiang Province, China) were extracted with methanol under trum showed strong absorption band at 3433 and 1080 cmϪ1
suggestive of a glycoside moiety. The fast atom bombard-
ment (FAB)-MS of 1 run in the positive- and negative-ion
modes showed quasimolecular ion peaks at m/z 371 [Mϩ
Na]ϩ and 347 [MϪH]Ϫ, respectively, and the molecular for-
mula was determined as C15H24O9 by high-resolution FAB-
MS measurement. Acid hydrolysis of 1 with 1.0 M hydrochlo-
ric acid (HCl) liberated D-glucose, which was identified by
HPLC analysis using an optical rotation detector.3—6) The 1H-
and 13C-NMR spectra of 1 (CD3OD, Tables 1, 2), which were
assigned by various NMR experiments,19) showed signals as-
signable to four methylenes [d 1.43 (1H, br dd, Jϭca. 5, 13
Hz, 4a-H), 1.73 (1H, br dd, Jϭca. 12, 14 Hz, 6a-H), 1.85
(1H, m, 4b-H), 1.93 (1H, br dd, Jϭca. 8, 14 Hz, 6b-H), 3.50
(1H, ddd, Jϭ2.4, 12.5, 13.0 Hz, 3a-H), 3.83 (1H, br dd, Jϭ
Chart 1
∗ To whom correspondence should be addressed. e-mail: muraoka@phar.kindai.ac.jp
© 2010 Pharmaceutical Society of Japan

1404
Vol. 58, No. 10
Table 2. 13C-NMR Data (150 MHz, CD3OD) for Kankanosides L (1), M
(2), N (3), O (4), and P (5), 4a, and 5a
Position
1
2
3
Position
4
4a
5
5a
1
3
4
5
6
7
8
9
10
11
97.5 174.9
95.2
62.1
38.5
40.0
28.8
32.3
38.0
46.9
17.9
72.1
1
2
3
4
5
6
7
8
9
170.1 172.0 171.0 171.7
128.7 129.5 117.4 118.0
142.3 142.8 160.5 159.4
62.6
26.0
31.7
30.7
68.5
30.7
36.0
39.9
27.2
38.5
28.0
39.2
41.4
26.7
41.5
26.7
60.6 131.9
69.1 138.8
139.9 138.5 128.5 125.2
122.0 125.7 134.0 137.0
43.9
61.7
51.0
68.4
65.5
12.4
16.2
59.4
12.5
16.2
75.6
18.8
14.1
102.5
75.1
78.1
71.7
77.9
62.8
68.8
18.8
13.7
10
Glc-1Ј 99.4
104.3 104.8 Glc-1Ј 102.1
2Ј
3Ј
4Ј
5Ј
6Ј
74.9
77.9
71.8
78.5
63.0
75.3
78.1
71.6
78.0
62.8
75.2
78.1
71.6
77.9
62.7
2Ј
3Ј
4Ј
5Ј
6Ј
74.4
77.4
71.0
77.1
62.3
Chart 2
Table 1. 1H-NMR Data (600 MHz, CD3OD) for Kankanosides L (1), M
(2), and N (3)
1
2
3
Position
d (J Hz)
d (J Hz)
d (J Hz)
1
4.81 (d, 8.9)
3.50
(ddd, 2.4, 12.5, 13.0) (ddd, 3.1, 6.7, 14.3)
3.83 (br dd, ca. 5, 13) 4.29
4.67 (d, 7.4)
3.70 (dd, 3.3, 12.0)
4.35
3a
3.88 (m)
1.69 (m)
3b
(ddd, 2.8, 8.4, 14.3)
4a
4b
5
1.43 (br dd, ca. 5, 13) 1.66 (m)
1.85 (m)
2.23 (m)
1.73
(br dd, ca. 12, 14)
1.93 (br dd, ca. 8, 14) 2.75 (m)
Fig. 1. 1H–1H COSY, HMBC, and NOE Correlations for 1—3
2.11 (m)
2.97 (m)
2.15 (m)
2.16 (m)
1.65 (m)
observed between the following protons and carbons (1-H
and 3-C, 8-C; 3-H and 1-C; 7-H and 8-C, 10-C; 9-H and 8-C;
10-H2 and 7-C, 8-C; 1Ј-H and 1-C) as shown in Fig. 1. Next,
the relative stereostructure of 1 was characterized by phase-
sensitive nuclear Overhauser enhancement spectroscopy
(phase-sensitive NOESY) experiment, which showed NOE
correlations between the following proton pairs (1-H and 3a-
H; 3a-H and 4a-H; 3b-H and 4b-H; 4b-H and 5-H, 9-H; 5-
H and 6b-H, 9-H; 6a-H and 7-H; 7-H and 10-H2), as shown
6a
6b
1.81 (m)
1.37 (m)
1.87 (m)
2.06 (m)
7(a) 3.46 (br s)
7b
8
5.94 (m)
9
2.15 (dd, 7.2, 8.9)
3.78, 4.12
(both d, 13.1)
3.82 (br s)
4.32, 4.51
(both d, 13.1)
1.75 (m)
1.07 (3H, d, 7.2)
10
11
3.65 (dd, 9.1, 9.8)
3.90 (dd, 5.9, 9.8)
4.26 (d, 7.9)
3.17 (dd, 7.9, 9.2)
3.35 (dd, 8.6, 9.2)
3.28 (m)
1
in Fig. 1. The H- and 13C-NMR spectra of 1 were superim-
Glc-1Ј 4.70 (d, 7.9)
4.32 (d, 7.9)
2Ј
3Ј
4Ј
5Ј
3.23 (dd, 7.9, 8.9)
3.37 (dd, 8.8, 8.9)
3.27 (m)
3.19 (dd, 7.9, 9.1)
3.34 (dd, 9.1, 9.1)
3.28 (dd, 9.1, 9.7)
3.24
posable on those of principal iridoid constituent 6-deoxy-
catalpol (6), except for the signals due to the saturated d-lac-
tol moiety. Finally, hydrogenation of 6 gave 1, so that the
stereostructure of kankanoside L was elucidated to be 3,4-
dihydro-6-deoxycatalpol (1).
3.27 (m)
3.25 (m)
(ddd, 2.2, 5.5, 9.7)
6Ј
3.64 (dd, 5.9, 12.0) 3.66 (dd, 5.5, 11.8) 3.67 (dd, 5.5, 11.8)
3.89 (dd, 1.9, 12.0) 3.84 (dd, 2.2, 11.8) 3.86 (dd, 1.9, 11.8)
Kankanoside M (2) was obtained as a white powder with
negative optical rotation ([a]D26 Ϫ18.7 in MeOH). The IR
ca. 5, 13 Hz, 3b-H), 3.78, 4.12 (1H each, both d, Jϭ13.1 Hz, spectrum of 2 showed absorption bands at 3433, 1736, 1655,
10-H2)], two methines [d 2.15 (1H, dd, Jϭ7.2, 8.9 Hz, 9-H) and 1076 cmϪ1 ascribable to hydroxyl, d-lactone, olefin, and
and 2.23 (1H, m, 5-H)], and an acetal group [d 4.81 (1H, d, ether moieties. The positive-ion FAB-MS spectrum of 2
Jϭ8.9 Hz, 1-H)] together with a b-glucopyranosyl moiety [d showed a quasimolecular ion peak at m/z 353 [MϩNa]ϩ, and
1
4.70 (d, Jϭ7.9 Hz, 1Ј-H)]. As shown in Fig. 1, the H–1H the molecular formula was determined as C15H22O8 by high-
correlation spectroscopy (1H–1H COSY) experiment on 1 resolution positive-ion FAB-MS measurement. Acid hydroly-
indicated the presence of partial structures written in bold sis of 2 with 1.0 M HCl liberated D-glucose. The 1H- and 13C-
lines. In the heteronuclear multiple-bond correlation NMR spectra of 2 (CD3OD, Tables 1, 2) showed signals
(HMBC) experiment on 1, long-range correlations were assignable to four methylenes [d 1.66, 2.11 (1H each, both

October 2010
1405
Table 3. 1H-NMR Data (600 MHz, CD3OD) for Kankanosides O (4) and P
(5)
m, 4-H2), 2.15, 2.75 (1H each, both m, 6-H2), 4.29 (1H, ddd,
Jϭ2.8, 8.4, 14.3 Hz, 3b-H), 4.32, 4.51 (1H each, both d, Jϭ
13.1 Hz, 10-H2), 4.35 (1H, ddd, Jϭ3.1, 6.7, 14.3 Hz, 3a-H)],
two methines [d 2.97 (1H, m, 5-H), 3.82 (1H, br s, 9-H)], an
olefin [d 5.94 (1H, m, 7-H)], and a saturated lactone group
(dC 174.9) together with a b-D-glucopyranosyl moiety [d
4.32 (1H, d, Jϭ7.9 Hz, 1Ј-H)]. As shown in Fig. 1, the 1H–1H
COSY experiment on 2 indicated the presence of partial
structures written in bold lines and, in the HMBC experi-
ment, long-range correlations were observed between the fol-
lowing proton and carbon pairs (3-H and 1-C; 7-H and 9-C;
9-H and 1-C, 8-C; 10-H2 and 7-C, 8-C, 9-C; 1Ј-H and 10-C).
The relative stereostructure of 2 was characterized by phase-
sensitive NOESY experiment, which showed NOE correla-
tions between the following proton pairs (3a-H and 4a-H;
3b-H and 4b-H; 4b-H and 5-H; 5-H and 6b-H, 9-H) as
shown in Fig. 1. Thus, the stereostructure of 2 was elucidated
as shown.
4
5
Position
d (J Hz)
d (J Hz)
2
3
4
5
6
7
8
5.67 (br s)
6.75 (tq, 7.2, 1.0)
2.36 (2H, m)
2.19 (2H, br t, ca. 7)
2.24 (2H, m)
2.27 (2H, m)
5.47 (tq, 7.1, 0.9)
5.41 (ddd, 1.2, 6.2, 7.6)
4.24 (dd, 7.6, 12.0)
4.33 (dd, 6.2, 12.0)
1.81 (3H, d, 1.0)
4.05 (br d, ca. 12)
4.20 (br d, ca. 12)
2.14 (3H, br s)
9
10
1.71 (3H, br s)
1.70 (3H, br s)
Glc-1Ј
2Ј
4.34 (d, 7.8)
4.23 (d, 7.7)
3.23 (dd, 7.8, 9.2)
3.45 (dd, 8.8, 9.2)
3.37 (dd, 8.8, 9.6)
3.31 (ddd, 2.6, 5.6, 9.6)
3.69 (dd, 5.6, 12.0)
3.86 (dd, 2.6, 12.0)
3.19 (dd, 7.7, 9.1)
3.34 (dd, 8.9, 9.1)
3.28 (dd, 8.9, 9.5)
3.22 (ddd, 2.2, 5.9, 9.5)
3.66 (dd, 5.9, 11.8)
3.85 (dd, 2.2, 11.8)
3Ј
4Ј
5Ј
6Ј
Kankanoside N (3) was isolated as a white powder with
negative optical rotation ([a]D25 Ϫ24.6 in MeOH). In the posi-
tive-ion FAB-MS of 3, a quasimolecular ion peak was ob-
served at m/z 371 [MϩNa]ϩ. The molecular formula
C16H28O8 was determined by high-resolution FAB-MS meas-
urement. Acid hydrolysis of 3 with 1.0 M HCl liberated D-glu-
1
cose. The H- and 13C-NMR data (CD3OD, Tables 1, 2)
showed signals assignable to a methyl [d 1.07 (3H, d, Jϭ
7.2 Hz, 10-H3)], four methylenes {d 1.37, 1.87 (1H each,
both m, 7-H2), 1.65, 1.81 (1H each, both m, 6-H2), [3.65 (1H,
dd, Jϭ9.1, 9.8 Hz), 3.90 (1H, dd, Jϭ5.9, 9.8 Hz), 11-H2], and
Fig. 2. 1H–1H COSY and HMBC, Correlations for 4 and 5
[3.70 (1H, dd, Jϭ3.3, 12.0 Hz), 3.88 (1H, m), 3-H2]}, four with those of 4a, a glycosylation shift was observed at the 8-
methines [d 1.69 (1H, m, 4-H), 1.75 (1H, m, 9-H), 2.06 (1H, position (dC 4: 65.5; 4a: 59.4). The position of the glucoside
m, 8-H), 2.16 (1H, m, 5-H)], and a hemiacetal group [d 4.67 linkage was also confirmed by HMBC experiments as shown
(1H, d, Jϭ7.4 Hz, 1-H)] together with a b-D-glucopyranosyl in Fig. 2. Consequently, the stereostructure of 4 was clarified
moiety [d 4.26 (1H, d, Jϭ7.9 Hz, 1Ј-H)]. The iridoid struc- to be (2E,6E)-8-b-D-glucopyranosyloxy-2,6-dimethyl-2,6-oc-
1
ture of 3 was clarified by H–1H COSY and HMBC experi- tadienoic acid. On the other hand, the 1H- and 13C-NMR data
ments and the relative stereostructure was characterized by of 5 (CD3OD, Tables 2, 3) indicated the presence of a
phase-sensitive NOESY experiment as shown in Fig. 1. Con- (2E,6E)-8-hydroxy-3,7-dimethyl-2,6-octadienoic acid moiety
sequently, the sterostructure of 3 was elucidated as shown.
[d 1.70 (3H, br s, 10-H3), 2.14 (3H, br s, 9-H3), 2.24 (2H, m,
Structures of Kankanosides O (4) and P (5) Kankano- 4-H2), 2.27 (2H, m, 5-H2), 4.05, 4.20 (1H each, both br d, Jϭ
sides O (4) and P (5), C16H26O8, were also obtained as white ca. 12 Hz, 8-H2), 5.47 (1H, tq, Jϭ7.1, 0.9 Hz, 6-H), 5.67
powders with negative optical rotations (4: [a]D23 Ϫ26.1; 5: (1H, br s, 1-H)] together with a b-D-glucopyranosyl part [d
[a]D21 Ϫ32.7 both in MeOH). The IR spectra of 4 and 5 4.23 (d, Jϭ7.7 Hz, 1Ј-H)]. The connectivity of the b-D-glu-
showed absorption bands at 3433, 1696, 1647, and 1076 copyranosyl moiety in 5 was elucidated on the basis of
cmϪ1 for 4, and at 3434, 1701, 1647, and 1076 cmϪ1 for 5, HMBC experiments as shown in Fig. 2. Furthermore, a typi-
ascribable to glycosidic, carboxyl, and olefin functions. Their cal glycosylation shift was observed for the signals at 8-posi-
UV spectra showed common absorption maximum at 217 nm tion (dC 5: 75.6; 5a: 68.8). On the basis of the above-men-
indicating the presence of an a,b-unsaturated carboxylic acid tioned evidence, the stereostructure of 5 was determined to
moiety in both of them. Acid hydrolysis of 4 and 5 liber- be (2E,6E)-8-b-D-glucopyranosyloxy-3,7-dimethyl-2,6-octa-
ated D-glucose, whereas by the enzymatic hydrolysis with b- dienoic acid.
a
Effects of the Constituents on Tumor Necrosis Factor-
glucosidase, 4 and 5 gave (2E,6E)-8-hydroxy-2,6-dimethyl-
2,6-octadienoic acid20) (4a) and (2E,6E)-8-hydroxy-3,7- (TNF- )-induced Cytotoxicity in L929 Cells TNF-a is
a
dimethyl-2,6-octadienoic acid21) (5a), respectively. H- and known to mediate a variety of organ injury through its induc-
1
13C-NMR data of 4 (CD3OD, Tables 2, 3) showed signals as- tion of cellular apoptosis. In the case of liver, the biological
signable to two methyls [d 1.71 (3H, br s, 10-H3), 1.81 (3H, effects of TNF-a have been implicated in hepatic injury in-
d, Jϭ1.0 Hz, 9-H3)], three methylenes {d 2.19 (2H, br t, Jϭ duced by hepatic toxins, ischemia/reperfusion, viral hepatitis,
ca. 7 Hz, 5-H2), 2.36 (2H, m, 4-H2), [4.24 (1H, dd, Jϭ7.6, and alcohol.22—24) Therefore, TNF-a is considered to be an
12.0 Hz), 4.33 (1H, dd, Jϭ6.2, 12.0 Hz), 8-H2]}, and two important target to discover anti-inflammatory and hepato-
trisubstituted olefins [d 5.41 (1H, ddd, Jϭ1.2, 6.2, 7.6 Hz, 7- protective agents. On the basis of above-mentioned concept,
H), 6.75 (1H, tq, Jϭ7.2, 1.0 Hz, 3-H)] together with a b-D- we investigated protective constituents from naturally occur-
glucopyranosyl part [d 4.34 (d, Jϭ7.8 Hz, 1Ј-H)]. By the ring products on TNF-a-induced cell death in L929 cells, a
comparison of carbon signals in the 13C-NMR spectrum of 4 TNF-a-sensitive cell line.25) Previously, we have reported

1406
Vol. 58, No. 10
Table 4. Inhibitory Effects of Constituents from Stems of C. tubulosa on TNF-a-Induced Cytotoxicity in L929 Cells
Inhibition (%)
10 mM
0 mM
3 mM
30 mM
100 mM
6-Deoxycatalpol (6)
Bartsioside (7)
Kankanoside A (9)
Mussaenosidic acid (10)
8-Epiloganic acid (11)
Geniposidic acid (13)
Kankanoside E (14)
0.0Ϯ1.2
0.0Ϯ0.4
0.0Ϯ0.9
0.0Ϯ3.5
0.0Ϯ1.5
0.0Ϯ2.5
0.0Ϯ3.7
0.0Ϯ1.6
0.0Ϯ1.1
0.0Ϯ3.1
0.0Ϯ1.3
0.0Ϯ0.8
0.0Ϯ4.8
0.0Ϯ1.1
0.0Ϯ1.2
1.3Ϯ2.1
1.4Ϯ2.0
8.8Ϯ2.7
10.8Ϯ6.9
8.0Ϯ2.8
2.3Ϯ1.8
Ϫ5.4Ϯ7.2
5.0Ϯ3.0
Ϫ2.5Ϯ0.5
3.6Ϯ6.0
4.1Ϯ3.3
4.5Ϯ4.5
5.2Ϯ3.5
16.4Ϯ1.3*
Ϫ4.6Ϯ3.5
1.1Ϯ1.9
5.5Ϯ2.2
15.5Ϯ3.1**
26.5Ϯ7.7*
4.3Ϯ1.9
19.8Ϯ3.9**
15.8Ϯ2.9
3.3Ϯ5.0
Ϫ1.5Ϯ1.6
4.4Ϯ7.9
10.1Ϯ5.8
Ϫ1.4Ϯ2.9
22.5Ϯ1.6**
24.1Ϯ4.6**
19.0Ϯ2.6**
Ϫ1.9Ϯ2.4
6.1Ϯ2.0
17.2Ϯ3.7**
72.0Ϯ1.6**
9.7Ϯ2.2*
24.1Ϯ1.5** Ϫ2.5Ϯ3.4
19.3Ϯ2.6
4.2Ϯ2.7
Ϫ2.8Ϯ2.7
9.9Ϯ3.3
13.9Ϯ3.3*
Ϫ0.4Ϯ2.0
45.7Ϯ6.0**
58.4Ϯ2.5**
0.2Ϯ3.5
1.1Ϯ1.6
16.3Ϯ2.0**
44.7Ϯ8.7**
10.7Ϯ0.4**
2.1Ϯ12.1
2.8Ϯ2.7
(2E,6Z)-8-b-D-Glucopyranosyloxy-2,6-dimethyl-2,6-octadienoic acid (15)
(2E,6E)-3,7-Dimethy-8-hydroxyoctadien-1-yl-O-b-D-glucopyranoside (16)
8-Hydroxygeraniol 8-O-b-D-glucopyranoside (17)
(ϩ)-Pinoresinol O-b-D-glucopyranoside (22)
(ϩ)-Syringaresinol O-b-D-glucopyranoside (25)
Echinacoside5)
Ϫ8.1Ϯ2.4
21.3Ϯ2.4**
22.3Ϯ1.6**
Ϫ2.3Ϯ4.3
80.4Ϯ4.5**
91.9Ϯ5.3**
Acteoside5)
Isoacteoside5)
61.9Ϯ5.9** 102.4Ϯ8.7**
Each value represents the meanϮS.E.M. (nϭ4). Significantly different from the control, ∗ pϽ0.05, ∗∗ pϽ0.01.
that several constituents from Piper chaba,26—29) Boesenber-
gia rotunda,30,31) Punica granatum,32) Helichrysum arenar-
ium,33—35) and Sapindus rarak,36—38) were found to show in-
hibitory effects of TNF-a-induced cytotoxicity in L929 cells.
Since the phenylethanoid constituents of C. tubulosa (e.g.
echinacoside, acteoside, and isoacteoside, etc.)5) were also
inhibited this cytotoxicity, we further examined iridoid,
phenylpropanoid, and lignan constituents as shown in Table
4. As the result, kankanoside A (9, inhibition: 16.3Ϯ2.0% at
100 mM), mussaenosidic acid (10, 44.7Ϯ8.7%), 8-epiloganic
acid (11, 10.7Ϯ0.4%), 8-hydroxygeraniol 8-O-b-D-glucopy-
ranoside (17, 21.3Ϯ2.4%), and (ϩ)-pinoresinol O-b-D-glu-
copyranoside (22, 22.3Ϯ1.6%), were found to show signifi-
cant activity. Although their activities were weaker than those
of echinacoside (IC50ϭ31.1 mM), acteoside (17.8 mM), and
isoaceteoside (22.7 mM), the principle phenylethanoid con-
stituents.5)
(167.84 g, 5.63% and 81.21 g, 2.73%, respectively). The MeOH-eluted frac-
tion (61.00 g) was subjected to normal-phase silica gel CC [1.8 kg, CHCl3–
MeOH–H2O (15 : 3 : 0.4→10 : 3 : 0.5→6 : 4 : 1, v/v/v)→MeOH] to give seven
fractions [Fr. 1 (1.12 g), 2 (9.56 g), 3 (0.89 g), 4 (10.69 g), 5 (8.84 g), 6
(12.52 g), and 7 (4.60 g)], as was described previously.5) The fraction 1
(1.12 g) was separated by reversed-phase silica gel CC [55 g, MeOH–H2O
(40 : 60, v/v)→MeOH] to give five fractions [Fr. 1-1 (132.8 mg), 1-2
(267.0 mg), 1-3 (182.8 mg), 1-4 (310.9 mg), and 1-5 (91.4 mg)]. The fraction
1-1 (132.8 mg) was further purified by HPLC [Cosmosil 5C18-MS-II,
CH3CN–1% aqueous AcOH (10 : 90, v/v)] to give kankanoside M (2,
2.8 mg, 0.0001%), bartsioside (7, 5.2 mg, 0.0002%), sinapic aldehyde 4-O-
b-D-glucopyranoside (21, 1.4 mg, 0.0001%). The fraction 1-2 (267.0 mg)
was further purified by HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous
AcOH (7 : 93, v/v)] to give (2E,6E)-3,7-dimethyl-8-hydroxyoctadien-1-yl-O-
b-D-glucopyranoside (16, 15.8 mg, 0.0007%), 8-hydroxygeraniol 8-O-b-D-
glucopyranoside (17, 5.2 mg, 0.0002%), (ϩ)-pinoresinol O-b-D-glucopyra-
noside (22, 22.0 mg, 0.0010%), eucommin A (23, 5.0 mg, 0.0002%), isoeu-
commin A (24, 21.3 mg, 0.0010%), and (ϩ)-syringaresinol O-b-D-glucopy-
ranoside (25, 98.0 mg, 0.0044%). The fraction 2 (9.56 g) was separated by
reversed-phase silica gel CC [400 g, MeOH–H2O (10 : 90, v/v)→MeOH→
acetone] to give five fractions [Fr. 2-1 (55.9 mg), 2-2 (4.48 g), 2-3 (3.42 g),
2-4 (1.16 g), and 2-5 (31.9 mg)], as was described previously.5) The fraction
2-2 (500.0 mg) was subjected to HPLC [Cosmosil 5C18-MS-II, CH3CN–1%
aqueous AcOH (5 : 95, v/v)] to give kankanoside L (1, 6.6 mg, 0.0026%) and
6-deoxycatalpol (6, 455.5 mg, 0.182%). The fraction 2-3 (1.00 g) was sub-
jected to HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (7 : 93,
v/v)] to give seven fractions [Fr. 2-3-1 (66.5 mg), 2-3-2 (20.4 mg), 2-3-3
(26.0 mg), 2-3-4 (136.6 mg), 2-3-5 [ϭ7 (380.6 mg, 0.0581%)], 2-3-6 [ϭ
gluroside (8, 285.1 mg, 0.0435%)], and 2-3-7 (84.9 mg)]. The fraction 2-3-4
(106.6 mg) was further purified by HPLC [Cosmosil pNAP, CH3CN–1%
aqueous AcOH (5 : 95, v/v)] to give 6 (68.4 mg, 0.0134%) together with
salidroside5) (13.7 mg, 0.0027%). The fraction 2-4 (1.16 g) was subjected to
HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (15 : 85, v/v) and
Cosmosil pNAP, CH3CN–1% aqueous AcOH (10 : 90 or 15 : 85, v/v)] to
give kankanosides N (3, 15.6 mg, 0.0007%), O (4, 44.1 mg, 0.0020%), and P
(5, 4.2 mg, 0.0002%), 6 (24.0 mg, 0.0011%), 8 (17.7 mg, 0.0008%),
kankanoiside A (9, 22.3 mg, 0.0010%), 8-epideoxyloganic acid (12, 8.1 mg,
0.0004%), kankanoside E (14, 58.1 mg, 0.0026%), (2E,6Z)-8-b-D-glucopy-
ranosyloxy-2,6-dimethyl-2,6-octadienoic acid (15, 31.0 mg, 0.0014%), 16
(168.6 mg, 0.0075%), 17 (94.3 mg, 0.0042%), betulalbuside A (18, 8.1 mg,
0.0004%), coniferin (19, 3.8 mg, 0.0002%), and syringin (20, 33.9 mg,
0.0015%). The fraction 4 (10.69 g) was separated by reversed-phase silica
gel CC [500 g, MeOH–H2O (30 : 70, v/v)→MeOH→acetone] to give four
fractions [Fr. 4-1 (878.2 mg), 4-2 (7.06 g), 4-3 (1.57 g), and 4-4 (792.8 mg)],
as was described previously.5) The fraction 4-1 (500.0 mg) was purified by
HPLC [Cosmosil 5C18-MS-II, CH3CN–1% aqueous AcOH (10 : 90, v/v)] to
give mussaenosidic acid (10, 39.1 mg, 0.0031%) and geniposidic acid (13,
51.2 mg, 0.0040%). The fraction 5 (8.84 g) was separated by reversed-phase
silica gel CC [400 g, MeOH–H2O (20 : 80→30 : 70, v/v)→MeOH→acetone]
to give seven fractions [Fr. 5-1 (870.2 mg), 5-2 (478.9 mg), 5-3 (3.72 g), 5-4
Experimental
The following instruments were used to obtain spectral and physical data:
specific rotations, Horiba SEPA-300 digital polarimeter (lϭ5 cm); UV spec-
tra, Shimadzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100
spectrometer; 1H- and 13C-NMR spectra, JEOL JNM-ECA600 (600,
150 MHz) and JEOL JNM-ECS400 (400, 100 HMz) spectrometers with
tetramethylsilane as an internal standard; FAB-MS and high-resolution FAB-
MS, JEOL JMS-SX 102A mass spectrometer; HPLC detector, Shimadzu
RID-10A refractive index, Shimadzu SPD-10A UV–VIS, and Shodex OR-2
optical rotation detectors. HPLC column, Cosmosil 5C18-MS-II and pNAP
(Nacalai Tesque Inc., 250ϫ4.6 mm i.d.) and (250ϫ20 mm i.d.) columns
were used for analytical and preparative purposes, respectively.
The following experimental conditions were used for chromatography:
normal-phase silica gel column chromatography (CC), silica gel 60N (Kanto
Chemical Co., Ltd., 63—210 mesh, spherical, neutral); reversed-phase silica
gel CC, Diaion HP-20 (Nippon Rensui) and Chromatorex ODS DM1020T
(Fuji Silysia Chemical, Ltd., 100—200 mesh); normal-phase TLC, pre-
coated TLC plates with silica gel 60F254 (Merck, 0.25 mm); reversed-phase
TLC, pre-coated TLC plates with silica gel RP-18 F254S (Merck, 0.25 mm);
reversed-phase HPTLC, pre-coated TLC plates with silica gel RP-18 WF254S
(Merck, 0.25 mm), detection was achieved by spraying with 1% Ce(SO4)2–
10% aqueous H2SO4, followed by heating.
Plant Material This item was described in a previous report.5)
Extraction and Isolation Fresh stems of C. tubulosa (2.98 kg) were
finely cut and extracted three times with methanol under reflux for 3 h.
Evaporation of the solvent under reduced pressure provided a methanolic
extract (249.1 g, 8.36%). The methanolic extract was subjected to Diaion
HP-20 CC (5.0 kg, H2O→MeOH) to give H2O- and MeOH-eluted fractions

October 2010
1407
(979.9 mg), 5-5 (1.19 g), 5-6 (1.27 g), and 5-7 (130.1 mg)], as was described
previously.5) The fraction 5-1 (870.2 mg) was further purified by HPLC
[Cosmosil pNAP, CH3CN–1% aqueous AcOH (5 : 95, v/v)] to give 10 (55.3
mg, 0.0025%) and 8-epiloganic acid (11, 51.5 mg, 0.0023%).
jiang Staple Medicinal Plants,” ed. by Xinjiang Institute of Traditional
Chinese and Ethnologic Medicines, 2004, pp. 84—88.
3) Xie H., Morikawa T., Matsuda H., Nakamura S., Muraoka O.,
Yoshikawa M., Chem. Pharm. Bull., 54, 669—675 (2006).
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raoka O., Bioorg. Med. Chem., 14, 7468—7475 (2006).
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M., Yuan D., Muraoka O., Bioorg. Med. Chem., 18, 1882—1890 (2010).
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Bull., 33, 3645—3650 (1985).
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Chem. Pharm. Bull., 38, 1927—1930 (1990).
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(2001).
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Koshino H., Biosci. Biotechnol. Biochem., 63, 384—389 (1999).
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O., Yasuhara F., Yamaguchi S., Phytochemistry, 25, 871—876 (1986).
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M., Takeda Y., Chem. Pharm. Bull., 52, 1086—1090 (2004).
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1219 (1993).
Kankanoside L (1): A white powder, [a]D26 Ϫ45.7 (cϭ0.48, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C15H24O9Na [MϩNa]ϩ
371.1318; Found 371.1309. IR (KBr, cmϪ1): 3433, 2928, 1080. 1H-NMR
(600 MHz, CD3OD) d: given in Table 1. 13C-NMR (150 MHz, CD3OD) dC:
given in Table 2. Positive-ion FAB-MS m/z: 371 [MϩNa]ϩ. Negative-ion
FAB-MS m/z: 347 [MϪH]Ϫ.
Kankanoside M (2): A white powder, [a]D26 Ϫ18.7 (cϭ0.11, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C15H22O8Na [MϩNa]ϩ
353.1212; Found 353.1208. IR (KBr, cmϪ1): 3433, 2924, 1736, 1655, 1076.
1H-NMR (600 MHz, CD3OD) d: given in Table 1. 13C-NMR (150 MHz,
CD3OD) dC: given in Table 2. Positive-ion FAB-MS m/z: 353 [MϩNa]ϩ.
Kankanoside N (3): A white powder, [a]D25 Ϫ24.6 (cϭ1.00, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C16H28O8Na [MϩNa]ϩ
1
371.1682; Found 371.1686. IR (KBr, cmϪ1): 3415, 2874, 1076, 1028. H-
NMR (600 MHz, CD3OD) d: given in Table 1. 13C-NMR (150 MHz, CD3OD)
dC: given in Table 2. Positive-ion FAB-MS m/z: 371 [MϩNa]ϩ.
Kankanoside O (4): A white powder, [a]D23 Ϫ26.1 (cϭ2.30, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C16H26O8Na [MϩNa]ϩ
369.1525; Found 369.1528. UV [lmax (log e), MeOH, nm]: 217 (4.04). IR
14) Ida Y., Satoh Y., Ohtsuka M., Nagasao M., Shoji J., Phytochemistry,
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1
(KBr, cmϪ1): 3433, 2926, 1696, 1647, 1076. H-NMR (600 MHz, CD3OD)
d: given in Table 3. 13C-NMR (150 MHz, CD3OD) dC: given in Table 2. Pos-
itive-ion FAB-MS m/z: 369 [MϩNa]ϩ.
Kankanoside P (5): A white powder, [a]D21 Ϫ32.7 (cϭ0.18, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C16H26O8Na [MϩNa]ϩ
369.1525; Found 369.1520. UV [lmax (log e), MeOH, nm]: 217 (4.08). IR
16) Deyama T., Ikawa T., Nishibe S., Chem. Pharm. Bull., 33, 3651—3657
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Bull., 33, 1452—1457 (1985).
1
(KBr, cmϪ1): 3434, 2926, 1701, 1647, 1076. H-NMR (600 MHz, CD3OD)
d: given in Table 3. 13C-NMR (150 MHz, CD3OD) dC: given in Table 2. Pos-
1
19) The H- and 13C-NMR spectra of 1—5 were assigned with the aid of
itive-ion FAB-MS m/z: 369 [MϩNa]ϩ.
distortionless enhancement by polarization transfer (DEPT), double
quantum filter correlation spectroscopy (DQF COSY), heteronuclear
multiple quantum coherence (HMQC), and heteronuclear multiple
bond connectivity (HMBC) experiments.
Acid Hydrolysis of Kankanosides L—P (1—5) Solutions of 1—5
(each 1.0 mg) in 1.0 M HCl (1.0 ml) were heated at 80 °C for 3 h. After being
cooled, the reaction mixture was neutralized with Amberlite IRA-400 (OHϪ
form), and the resins were removed by filtration. After removal of the sol-
vent under reduced pressure, the residue was separated by Sep-Pak C18 car-
tridge column (H2O→MeOH). The H2O-eluted fraction was subjected to
HPLC analysis under following conditions: HPLC column, Kaseisorb LC
NH2-60-5, 4.6 mm i.d.ϫ250 mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); de-
tection, 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 D-glucose present in the H2O-eluted fractions of 1—5 were
carried out by comparison of their retention times and optical rotation with
that of an authentic sample [tR 17.9 min (positive)].
Hydrogenation of 6-Deoxycatalpol (6) A suspension of 6 (12.0 mg)
and 10% palladium carbon (5.0 mg) in MeOH (2.0 ml) was stirred at room
temperature under an H2 atmosphere for 2 h. The catalyst was filtered off,
and the filtrate was condensed under reduced pressure to give 1 (12.0 mg,
quant.).
Enzymatic Hydrolysis of Kankanosides O (4) and P (5) with b-Glu-
cosidase To a solution of 4 (8.0 mg) in H2O (1.5 ml) was added b-glucosi-
dase (4.7 mg, from almond, Oriental Yeast Co., Tokyo, Japan), and the solu-
tion was stirred at 37 °C for 24 h. The reaction was quenched by the addition
of EtOH (5.0 ml), the mixture was condensed under reduced pressure. The
residue was extracted with EtOAc and evaporation of the solvent gave
(2E,6E)-8-hydroxy-2,6-dimethyl-2,6-octadienoic acid20) (4a, 3.9 mg, 92%).
Through a similar procedure, (2E,6E)-8-hydroxy-3,7-dimethyl-2,6-octa-
dienoic acid21) (5a, 0.9 mg, 94%) was obtained from kankanoside P (5, 1.8
mg).
20) Iwagawa T., Asai H., Hase T., Sako S., Su R., Hagiwara N., Kim M.,
Phytochemistry, 29, 1913—1916 (1990).
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Bioassay Method Inhibitory effect on TNF-a-induced cytotoxicity in
L929 cells was assayed by the method described in a previous paper.5)
Statistics Values are expressed as meansϮS.E.M. One-way analysis of
34) Wang L.-B., Morikawa T., Nakamura S., Ninomiya K., Matsuda H.,
Muraoka O., Wu L.-J., Yoshikawa M., Heterocycles, 78, 1235—1242
(2009).
variance (ANOVA) followed by Dunnett’s test was used for statistical analy- 35) Morikawa T., Wang L.-B., Ninomiya K., Nakamura S., Matsuda H.,
Muraoka O., Wu L.-J., Yoshikawa M., Chem. Pharm. Bull., 57, 853—
859 (2009).
sis. Probability (p) values less than 0.05 were considered significant.
Acknowledgements This work was supported by ‘High-Tech Research
Center’ Project for Private Universities: matching fund subsidy from MEXT
(Ministry of Education, Culture, Sports, Science and Technology), 2007—
2011 and a Grant-in Aid for Scientific Research from MEXT.
36) Morikawa T., Xie Y., Asao Y., Okamoto M., Yamashita C., Muraoka
O., Matsuda H., Pongpiriyadacha Y., Yuan D., Yoshikawa M., Phyto-
chemistry, 70, 1166—1172 (2009).
37) Asao Y., Morikawa T., Xie Y., Okamoto M., Hamao M., Matsuda H.,
Muraoka O., Yuan D., Yoshikawa M., Chem. Pharm. Bull., 57, 198—
203 (2009).
References and Notes
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2) Xinjiang Science and Technology Press, “Culture Techniques of Xin-
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(2010).