Three new triterpenoid saponins from Ardisia crenata

Article abstract of DOI:10.1002/hlca.201000285

Three new triterpenoid saponins, ardisicrenoside I (1), ardisicrenoside J (2), and ardisicrenoside M (3), along with eight known compounds, were isolated from the roots of Ardisia crenata Sims. Their structures were elucidated as 16α-hydroxy-30,30-dimethoxy-3β-O-{β-D-xylopyranosyl-(1→2) -β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)] -α-L-arabinopyranosyl}-13β,28-epoxyoleanane (1), 16α-hydroxy- 30,30-dimethoxy-3β-O-{α-L-rhamnopyranosyl-(1→2) -β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)] -α-L-arabinopyranosyl}-13β,28-epoxyoleanane (2), 30,30-dimethoxy-16- oxo-3β-O-{β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl- (1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl} -13β,28-epoxyoleanane (3), ardisiacrispin A (4), ardisiacrispin B (5), ardisicrenoside B (6), ardisicrenoside A (7), ardisicrenoside H (8), ardisicrenoside G (9), cyclamiretin A-3β-O-β-D-xylopyranosyl- (1→2)-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranoside (10), and cyclamiretin A-3β-O-α-L-rhamnopyranosyl-(1→2)-β-D- glucopyranosyl-(1→4)-α-L-arabinopyranoside (11) by means of chemical and spectral analysis, and their cytotoxicities were evaluated in vitro. Copyright

Full text of DOI:10.1002/hlca.201000285

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Three New Triterpenoid Saponins from Ardisia crenata
by Dai-Lin Liu*a)b), Nai-Li Wangb), Xue Zhangb), and Xin-Sheng Yao*b)
a) Department of Pharmacognosy, Tianjin Key Laboratory for Biomarkers of Occupational and
Environmental Hazard, Medical College of Chinese Peopleꢀs Armed Police Forces, Tianjin, 300162,
P. R. China (phone: þ 86-22-60578194; fax: þ 86-22-60578193; e-mail: dailinlch@163.com)
b) Department of Natural Products Chemistry, Shenyang Pharmaceutical University,
No. 103 Wen-Hua Road, Shenhe District, Shenyang 110016, P. R. China
(phone: þ 86-24-23993994; fax: þ 86-22-23993994; e-mail: liudailinlch@hotmail.com)
Three new triterpenoid saponins, ardisicrenoside I (1), ardisicrenoside J (2), and ardisicrenoside M
(3), along with eight known compounds, were isolated from the roots of Ardisia crenata Sims. Their
structures were elucidated as 16a-hydroxy-30,30-dimethoxy-3b-O-{b-d-xylopyranosyl-(1 ! 2)-b-d-glu-
copyranosyl-(1 ! 4)-[b-d-glucopyranosyl-(1 ! 2)]-a-l-arabinopyranosyl}-13b,28-epoxyoleanane
(1),
16a-hydroxy-30,30-dimethoxy-3b-O-{a-l-rhamnopyranosyl-(1! 2)-b-d-glucopyranosyl-(1 ! 4)-[b-d-glu-
copyranosyl-(1 ! 2)]-a-l-arabinopyranosyl}-13b,28-epoxyoleanane (2), 30,30-dimethoxy-16-oxo-3b-O-
{b-d-xylopyranosyl-(1 ! 2)-b-d-glucopyranosyl-(1 ! 4)-[b-d-glucopyranosyl-(1 ! 2)]-a-l-arabinopyr-
anosyl}-13b,28-epoxyoleanane (3), ardisiacrispin A (4), ardisiacrispin B (5), ardisicrenoside B (6),
ardisicrenoside A (7), ardisicrenoside H (8), ardisicrenoside G (9), cyclamiretin A-3b-O-b-d-
xylopyranosyl-(1 ! 2)-b-d-glucopyranosyl-(1 ! 4)-a-l-arabinopyranoside (10), and cyclamiretin A-3b-
O-a-l-rhamnopyranosyl-(1 ! 2)-b-d-glucopyranosyl-(1 ! 4)-a-l-arabinopyranoside (11) by means of
chemical and spectral analysis, and their cytotoxicities were evaluated in vitro.
Introduction. – Ardisia is a large genus of the family Myrsinaceae, comprising more
than 300 species distributed around the world. Previous chemical studies showed that
triterpenoid saponins were the main components from the plants of this genus [1][2],
which have showed a wide range of bioactivities: utero-contracting activity, inhibitory
activity on cAMP phosphodiesterase, cytotoxicity, anti-HIV and anticancer activities,
etc. [3 – 7].
The roots of Ardisia crenata Sims are used as the traditional Chinese medicine ꢁZhu
Sha Genꢀ for the treatment of respiratory tract infections and menstrual disorders in
China [8]. We have reported several antifungal triterpenoid saponins from this plant in
our previous study [7]. The EtOH extract of Ardisia crenata Sims also showed good
cytotoxic activity on the cancer cell lines in our further study. In search for further
bioactive chemical constituents of this plant, three new compounds, ardisicrenoside I
(1), ardisicrenoside J (2), and ardisicrenoside M (3), along with eight known
compounds, were isolated. In this article, we report the isolation and structure
elucidation of these compounds, as well as their cytotoxic activities against the MCF-7,
NCI-H460, SF-268, HepG2, HepG2-r, and 293 cell lines.
Results and Discussion. – The roots of A. crenata Sims were extracted with 60%
EtOH. The EtOH extract was dissolved in H2O, and then partitioned with AcOEt and
ꢂ 2011 Verlag Helvetica Chimica Acta AG, Zꢃrich

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BuOH. The BuOH-soluble portion was subjected to column chromatography on
Diaion HP-20 and MPLC RP-18. The saponin-containing fractions were further
purified by repeated HPLC on ODS column, to afford three new triterpenoid saponins,
ardisicrenosides I – M (1 – 3, resp.), together with eight known compounds, were
isolated.
Compound 1, a white powder, showed positive to LibermannꢀBurchardꢀs reaction
and Molish test. The HR-ESI mass spectrum (positive-ion mode) revealed the quasi-
molecular-ion peak at m/z 1129.5822 ([M þ Na]þ), corresponding to the molecular
1
formula C54H90O23. The H-NMR spectrum of compound 1 in (D5)pyridine exhibited
signals of the six tertiary Me groups at d(H) 1.56, 1.36, 1.26, 1.21, 1.09, and 0.87, and two

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MeO groups at d(H) 3.54 (s) and 3.52 (s), and four anomeric H-atom signals of sugar
moieties at d(H) 5.48 (d, J ¼ 7.6), 4.98 (d, J ¼ 7.7), 4.92 (d, J ¼ 7.3), and 4.78 (br. s). The
above data suggested that ardisicrenoside I contains four sugars moieties and a
triterpenoid aglycone. Complete analysis of the 13C-NMR and DEPT spectra of
compound 1 revealed 54 C-atom signals, 32 of them were assigned to the aglycone part
and 22 to the sugar moiety. 13C-NMR Data of compound 1 were similar to those of the
known saponin ardisiacrispin A [9]. As listed in Table 1, there was a signal at d(C) 108.7
(CH, by DEPT) of compound 1 instead of a signal at d(C) 207.2 due to the 30-CHO
group of ardisiacrispin A. Furthermore, in the HMBC spectrum of compound 1, the C-
atom signal at d(C) 108.7 not only correlated with Me(29) but also with the two MeO
signals, respectively, confirming that d(C) 108.7 was the signal of C(30), and the two
MeO groups were located at C(30). The O-bearing groups at C(3) and C(16) in the
aglycone were deduced from the signals at d(C) 88.9 and 76.9, respectively. The
configuration at C(16) was determined using a NOESYexperiment. NOE Correlations
of the signals at d(H) 3.17 (HaxꢀC(3)) with those at 1.21 (Me(23)) and 0.70 (HꢀC(5)),
and of the signal at d(H) 4.24 (HꢀC(16)) with that at 3.41 (CH2(28)), indicate an a-
orientation for the H-atom at C(3) and a b-orientation for the H-atom at C(16). The a-
orientation of OH at C(16) was determined by comparing the chemical shift of C(16)
(d(C) 77.3) with that in the literature (d(C) 77.0; HObꢀC(16), d(C) 64.0) [2]. From the
above evidences, the structure of the new sapogenin was established as 3b,16a-
dihydroxy-30,30-dimethoxy-13b,28-epoxyoleanane.
On acid hydrolysis of compound 1, arabinose, glucose, and xylose were identified by
co-TLC with authentic samples. The ESI mass spectrum (positive-ion mode) exhibited
a single predominant peak at m/z 1129 ([M þ Na]þ), giving rise to the peaks at m/z 997
([M þ Na ꢀ 132]þ) and 835 ([M þ Na ꢀ 132 ꢀ 162]þ) in its ESI-MS2. The peak at m/z
835 gave a prominent ion peak at m/z 673 ([M þ Na ꢀ 132 ꢀ 162 ꢁ 2]þ) in the ESI-MS3.
In combination with the ESI-MS results, it could be concluded that the ratio of
arabinose, glucose, and xylose in the molecule of compound 1 was 1:2 :1. 1H,1H-COSY,
TOCSY, HMBC, and NOESY analyses were used to determine the sequence of the
oligosaccharide chain in 1. From the relatively large anomeric HꢀC(1) coupling
constants (7.6, 7.7, and 7.3 Hz), both glucose moieties and the xylose moiety should be b-
configured [10]. The small HꢀC(1) coupling constant of arabinose indicated that it was
possess a-oriented. Based on these results and comparison of the signals of the sugars
moieties of compound 1 in the 13C-NMR spectrum with those in the literature [7][9],
the four sugars and their anomeric configurations in 1 were determined as an a-l-
arabinopyranose, two b-d-glucopyranoses, and a b-d-xylopyranose. The sequence of
the oligosaccharide chain was deduced by comparing the chemical shifts of their
individual sugar residues with those of published compounds [10], and confirmed by
HMBC and NOESY experiments. The C(1) of arabinose was attached to C(3)ꢀO of
the aglycone, as indicated by the correlation between the anomeric H-atom signal at
d(H) 4.78 (Ara HꢀC(1)) and the signal at d(C) 88.9 (C(3)) in HMBC spectrum, and as
well as with the signal at d(H) 3.17 (HꢀC(3)) in NOESY spectrum. From the HMBC of
1 (Fig.), the following correlations were also observed: d(H) 5.48 (terminal Glc
HꢀC(1))/d(C) 79.7 (Ara C(2)), d(H) 4.98 (inner Glc HꢀC(1))/d(C) 78.6 (Ara C(4)),
and d(H) 4.92 (Xyl HꢀC(1))/d(C) 85.4 (inner Glc C(2)). On the basis of the analyses
above, compound 1 was identified to be 16-hydroxy-30,30-dimethoxy-3b-O-{b-d-

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Figure. Key HMBCs (H ! C) for compounds 1, 2, and 3
xylopyranosyl-(1 ! 2)-b-d-glucopyranosyl-(1 ! 4)-[b-d-glucopyranosyl-(1 ! 2)]-a-l-ara-
binopyranosyl}-13b,28-epoxyoleanane, named as ardisicrenoside I.
Compound 2 obtained as a white powder, showed positive to both the Lieber-
mannꢀBurchard reaction and the Molish test. The molecular formula C55H92O23 was
established by HR-ESI-MS (positive-ion mode), which exhibited a quasi-molecular-

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1
ion peak at m/z 1143.5934 ([M þ Na]þ). The H-NMR spectrum of compound 2 in
(D5)pyridine displayed signals for six tertiary Me groups at d(H) 1.55, 1.35, 1.25, 1.16,
1.03, and 0.87; a Me group of rhamnose at d(H) 1.80 (d, J ¼ 6.0); two MeO groups at
d(H) 3.49 and 3.48, and four anomeric H-atom signals from sugars at d(H) 6.39 (br. s),
1
5.36 (d, J ¼ 8.0), 5.23 (d, J ¼ 7.6), and 4.98 (br. s). Its H- and 13C-NMR spectra
indicated that compound 2 had the same aglycone as that of compound 1, but differed
in the oligosaccharide part (see Table 1). So, the structure of sapogenin of 2 is
determined as 3b,16a-dihydroxy-30,30-dimethoxy-13b,28-epoxyoleanane.
The ESI mass spectrum of 2 exhibited a single predominant peak at m/z 1143,
assigned to [M þ Na]þ, which gave ion peaks at m/z 997 ([M þ Na ꢀ 146]þ) and 835
([M þ Na ꢀ 146 ꢀ 162]þ) in the ESI-MS2 (positive-ion mode). The ion peak at m/z 835
gave rise to the ion peak at m/z 673 ([M þ Na ꢀ 146 ꢀ 2 ꢁ 162]þ) in the MS3. The ion
peak at m/z 1119 ([M ꢀ H]ꢀ) gave ion peaks at m/z 973 ([M ꢀ H ꢀ 146]ꢀ) and 811
([M ꢀ H ꢀ 146 ꢀ 162]ꢀ) in the ESI-MS2 (negative-ion mode), and the MS3 of m/z 811
gave a prominent ion peak at m/z 649 ([M ꢀ H ꢀ 146 ꢀ 2 ꢁ 162]ꢀ). On acid hydrolysis,
2 afforded arabinose, glucose, and rhamnose in a ratio of 1:2 :1 (analyzed by the same
method as that for 1). By comparing the vicinal coupling constant of anomeric H-atoms
with those of model compounds reported in [10], the three sugars were determined as
a-l-arabinopyranose, b-d-glucopyranose, and a-l-rhamnopyranose. According to the
same methods as those for 1, the HMBC cross-peaks d(H) 4.98 (Ara HꢀC(1))/d(C)
89.1 (C(3)), d(H) 5.36 (terminal Glc HꢀC(1))/d(C) 80.7 (Ara C(2)), d(H) 5.23 (inner
Glc HꢀC(1))/d(C) 74.7 (Ara C(4)), and d(H) 6.39 (Rha HꢀC(1))/d(C) 77.3 (inner Glc
C(2)) were observed, confirming the sugar sequence and the glycosylation position.
Based on the above evidences, the structure of 2 was elucidated as 16a-hydroxy-30,30-
dimethoxy-3b-O-{a-l-rhamnopyranosyl-(1 ! 2)-b-d-glucopyranosyl-(1 ! 4)-[b-d-glu-
copyranosyl-(1 ! 2)]-a-l-arabinopyranosyl}-13b,28-epoxyoleanane, named ardisicre-
noside J.
Compound 3 was obtained as a white powder. The positive LibermannꢀBurchard
reaction and Molish test suggested it to be also a triterpenoid glycoside. Its HR-ESI-MS
(positive-ion mode) displayed a quasi-molecular-ion peak at m/z 1127.5682 ([M þ
1
Na]þ), which indicated the molecular formula C54H88O23. The H-NMR spectrum
exhibited signals due to six tertiary Me groups at d(H) 0.83, 1.04, 1.07, 1.09, 1.22, and
1.31, two MeO groups at d(H) 3.48 and 3.49, and four anomeric H-atom signals from
sugars at d(H) 5.49 (d, J ¼ 7.6), 5.00 (d, J ¼ 7.8), 4.91 (d, J ¼ 7.2), and 4.78 (d, J ¼ 5.9).
The 13C-NMR spectrum showed the signals of a CO group at d(C) 212.6, of an acetal C-
atom at d(C) 108.0, of two MeO groups at d(C) 58.4 and 58.3, and of four anomeric C-
atoms at d(H) 107.7, 104.9, 104.6, and 104.1. All these data suggest that compound 3
contains four sugars moieties and a triterpenoid aglycone. The 1H- and 13C-NMR data
indicated that the aglycone of 3 was the same as that of ardisicrenoside K, which we
published before [7], but the oligosaccharide part was different. So the aglycone of 3
was elucidated as 3b-hydroxy-30,30-dimethoxy-16-oxo-13b,28-epoxyoleanane.
After acid hydrolysis, the sugar moieties were determined as arabinose, glucose, and
xylose by TLC comparison with authentic samples. According to ESI-MS fragments
analyzed by the same methods as those for 1, it can be concluded that the ratio of
arabinose, glucose, and xylose in the molecule of 3 was 1:2 :1. b-Configuration at the
anomeric position was inferred from the values of the coupling constants for both

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glucopyranosyl units (7.8 and 7.6 Hz) and xylopyranosyl unit (7.2 Hz). Analyzed by the
same methods as those for 1, it was concluded that the anomeric position of
arabinopyranosyl unit should possess a-configuration. The 13C-NMR spectral data for
four sugars in 3 were also in good agreement with those of 1. By HMBC analysis, the
correlations d(H) 4.78 (Ara HꢀC(1))/d(C) 88.8 (C(3)), d(H) 5.49 (terminal Glc
HꢀC(1))/d(C) 79.7 (Ara C(2)), d(H) 5.00 (inner Glc HꢀC(1))/d(C) 78.5 (Ara C(4)),
and d(H) 4.91 (Xyl HꢀC(1))/d(C) 85.4 (inner Glc C(2)) were observed, which
confirmed the sugar sequence and the glycosylation position. Based on these data, 3
was determined as 30,30-dimethoxy-16-oxo-3b-O-{b-d-xylopyranosyl-(1 ! 2)-b-d-glu-
copyranosyl-(1 ! 4)-[b-d-glucopyranosyl-(1 ! 2)]-a-l-arabinopyranosyl}-13b,28-ep-
oxyoleanane and named ardisicrenoside M. Complete assignment of 3 was achieved
1
with the aid of the H,1H-COSY, HMQC, and HMBC spectra.
The MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)
method was used to test the cytotoxicity of these compounds on MCF-7, NCI-H460, SF-
268, HepG2, and HepG2R cancer cell lines, and HEK 293 cell line. The bioassay results
of all isolated compounds are collected in Table 2. From these results, it was concluded
that compound 1, 2, 4, 5, and 6 exhibited very strong cytotoxicities against cancer cell
lines, compound 10 and 11 had relatively weak activities against MCF-7, NCI-H460, and
SF-268 cancer cell lines, compound 3 and 7 had selective inhibitory activity against
NCI-H460 cell line, and compound 8 and 9 showed no activity. It is remarkable that
compound 2 and 4 had very strong activity against cancer cell lines but no cytotoxicity
against HEK 293 cell line.
Table 2. The IC50 Values [mm] for the Inhibition of the Growth of Cancer Cell Lines
MCF-7
NCI-H460
SF-268
HepG2
HepG2R
HEK 293
1
2
3
4
5
6
7
8
14.04
> 22.32
–
4.72
5.77
11.21
–
14.01
> 22.32
12.36
> 23.58
12.10
10.87
14.51
–
9.04
11.34
–
2.36
3.72
5.40
–
16.27
15.18
–
6.60
4.19
11.21
–
9.04
–
15.37
–
–
> 23.58
3.72
6.29
–
–
> 23.58
8.38
7.39
–
–
–
–
–
–
9
10
11
–
–
–
–
–
–
24.12
> 27.84
13.16
4.51
> 27.41
10.92
–
–
–
On the basis of cytotoxic activity, the triterpenoid saponins from A. crenata Sims
display following structureꢀactivity relationships: the activity of 3, with a C(16)¼O
group, was lower than those of 1, 2, 4, and 5. The activities of 10 and 11, with three sugar
moieties at C(3), were decreased. A C(12)¼C(13) bond and a COOH group at C(30)
diminished the activities of 8 and 9. These results indicated that the sugar chain at C(3)
of aglycone, the different substituents at C(30), at 16a-OH and 13,28-epoxy groups
played crucial roles in the cytotoxicity.
The authors gratefully acknowledge Prof. Qishi Sun at the Department of Pharmacognosy, Shenyang
Pharmaceutical University for identification of the plant, and Prof. Mengsu Yang and his staff at the
Department of Biology and Chemistry, City University of Hong Kong, for the bioactivity assay of

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compounds. Thanks are also extended to the Shanghai Institute of Materia Medica of CAS for recording
the HR-ESI-MS.
Experimental Part
General. TLC: Silica gel 60F254 (SiO2; Qingdao Haiyang Chemical Co., Ltd., P. R. China),
visualization by spraying with 10% H2SO4 and heating. Column chromatography (CC): Diaion HP-20
(Mitsubishi Kasei, Japan) and ODS (40 – 63 mm, Merck, USA). Prep. HPLC: ODS column (C-18, 250 ꢁ
20 mm, Shimadzu Pak; Detector: RID); flow rate, 10 ml/min. M.p.: Yanaco MP-S3 micromelting point
1
apparatus; uncorrected. Optical rotations: P-1020 digital polarimeter (Jasco Corporation, Japan). H-
and 13C-NMR, along with 2D-NMR spectra: Bruker AV-400 (at 400 (1H) and 100 MHz (13C))
spectrometer; TMS as an internal standard; chemical shifts (d) in ppm and coupling constants (J) in Hz.
ESI-MS: Bruker esquire 2000 mass spectrometer.
Plant Material. The roots of Ardisia crenata Sims were collected from Guangxi Province of China in
2000, and identified by Prof. Qishi Sun. A voucher specimen (YL-2001-113) has been deposited with the
Shenyang Pharmaceutical University, Liaoning Province of China.
Extraction and Isolation. The air-dried roots of Ardisia crenata Sims (7 kg) were extracted with 60%
EtOH (2 ꢁ 10 l) for 2 h under reflux to give 500 g of crude extract. A portion of the crude extract (200 g)
was then suspended in H2O, and subsequently partitioned with AcOEt and BuOH. The BuOH extract
was subjected to Diaion HP-20 chromatography eluted stepwise with H2O, 30% EtOH, 50% EtOH, 70%
EtOH, and 95% EtOH to afford seven fractions, Frs. ACB-1 – ACB-7). Fr. ACB-5 (20 g) was separated
by an ODS open column with H2O/MeOH gradient to yield six subfractions, Frs. ACB-51 – ACB-56.
Fr. ACB-54 (2.6 g), eluted with 50% MeOH, was further separated by prep. HPLC with MeOH/H2O 3 :2
to give eight subfractions, Frs. ACB-541 – ACB-548. The purification of Fr. ACB-543 (40 mg) on prep.
HPLC with MeOH/H2O 2 :3 yielded 8 mg of compound 8. Prep. HPLC of Fr. ACB-544 (56 mg) with
MeOH/H2O 2 :3 afforded 10 mg of compound 9. By the same method, Frs. ACB-545 (310 mg) and ACB-
546 (440 mg) were purified by prep. HPLC with MeOH/H2O 1:1 to furnish 6 (135 mg) and 7 (160 mg).
ODS Chromatography of a portion of Fr. ACB-55 (1 g) with MeOH/H2O 3 :2, followed by crystallization
from MeOH, afforded compounds 4 (520 mg) and 5 (165 mg). Fr. ACB-6 (7 g) was submitted to CC
(ODS; 60% MeOH) and further purified by prep. HPLC (55% MeOH) to afford compounds 1 (90 mg),
2 (65 mg), and 3 (15 mg). Fr. ACB-7 (11 g) was subjected to CC (SiO2; CHCl3/MeOH of increasing
polarity) to give twelve fractions, Frs. ACB-71 – ACB-712. Fr. ACB-711 was further separated by prep.
HPLC (MeOH/H2O 7:3) to afford compounds 10 (22 mg) and 11 (34 mg).
Ardisicrenoside I (¼(3b,16a)-16-Hydroxy-30,30-dimethoxy-13,28-epoxyoleanan-3-yl b-d-Glucopyr-
anosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-b-d-glucopyranosyl-(1 ! 4)]-a-l-arabinopyranoside; 1).
1
White amorphous powder (MeOH). M.p. 2898. [a]2D5 ¼ þ12.2 (c ¼ 0.117, MeOH). H- and 13C-NMR:
see Table 1. ESI-MS (pos.): 1129. ESI-MS (neg.): 1105. HR-ESI-MS (pos.): 1129.5822 (C54H90NaO2þ3
;
calc. 1129.5771).
Ardisicrenoside J (¼(3b,16a)-16-Hydroxy-30,30-dimethoxy-13,28-epoxyoleanan-3-yl b-d-Glucopy-
ranosyl-(1 ! 2)-[a-l-rhamnopyranosyl-(1 ! 2)-b-d-glucopyranosyl-(1 ! 4)]-a-l-arabinopyranoside; 2).
White amorphous powder (MeOH). M.p. 294 – 2958. [a]2D5 ¼ ꢀ15.7 (c ¼ 0.124, MeOH). 1H- and
13C-NMR: see Table 1. ESI-MS (pos.): 1143. ESI-MS (neg.): 1119. HR-ESI-MS (pos.): 1143.5934
(C55H92NaOþ23 ; calc. 1143.5927).
Ardisicrenoside M (¼(3b)-30,30-Dimethoxy-16-oxo-13,28-epoxyoleanan-3-yl b-d-Glucopyranosyl-
(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-b-d-glucopyranosyl-(1 ! 4)]-a-l-arabinopyranoside; 3). White
amorphous powder (MeOH). M.p. 271 – 2728. [a]2D5 ¼ ꢀ34.6 (c ¼ 0.102, MeOH). 1H- and 13C-NMR:
see Table 1. ESI-MS (pos.): 1127. ESI-MS (neg.): 1105. HR-ESI-MS (pos.): 1127.5682 (C54H88NaO2þ3
;
calc. 1127.5614).
Acid Hydrolysis. Compound 2 (ca. 2 mg) was heated in 1m HCl at 808 for 10 h. The mixture was then
neutralized with 1m NaOH and filtered. The filtrate was partitioned with CHCl3. The H2O layer was
concentrated, and rhamnose, glucose, and arabinose were identified by TLC comparison with authentic
samples. Compounds 1 (2 mg) and 3 (ca. 2 mg) were treated in the same way, and xylose, glucose, and
arabinose were identified by TLC analysis.

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Cytotoxicity Assay. Cancer cells were cultured in PRMI-1640 medium supplemented with 5% fetal
bovine serum. The cultures were incubated at 378 in a 5% CO2 humidified incubator and subcultured
every 2 d to maintain them in a state of logarithmic growth. Then, the cells were seeded into 96-well
microtiter plates (1 ꢁ 104 cells per well). Compounds were dissolved in DMSO and added to the 96-well
microtiter plates 24 h after seeding. The cells were incubated for 2 d in the presence of sample. For the
evaluation of in vitro cytotoxicity, MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide) assay was used. The anticancer drug cis-dichlorodiamine platinum (cis-DDP) was used as the
pos. control.
REFERENCES
[1] M. Vermeersch, K. Foubert, R. I. da Luz, L. Van Puyvelde, L. Pieters, P. Cos, L. Maes, Phytother Res.
2009, 23, 1404.
[2] D.-L. Liu, X.-M. Zhang, N.-L. Wang, X.-S. Yao, J. Shenyang Pharm. Univ. 2004, 21, 394.
[3] Y. Tian, H.-F. Tang, F. Qiu, X.-J. Wang, X.-L. Chen, A.-D. Wen, Planta Med. 2009, 75, 70.
[4] H.-F. Tang, J. Yun, H.-W. Lin, X.-L. Chen, X.-J. Wang, G. Cheng, Chem. Biodiversity 2009, 6, 1443.
[5] P.-C. Zhang, C.-F. Zheng, C.-S. Wu, Y.-X. Sheng, J.-L. Zhang, J. Asian Nat. Prod. Res. 2010, 12, 64.
[6] P. Wen, X.-M. Zhang, Z. Yang, N.-L. Wang X.-S. Yao, J. Asian Nat. Prod. Res. 2008, 10, 873.
[7] D.-L. Liu, N.-L. Wang, X. Zhang, H. Gao, X.-S. Yao, J. Asian Nat. Prod. Res. 2007, 9, 119.
[8] Jiangsu New Medical College, ꢁDictionary of Chinese Traditional Medicineꢀ, Shanghai Science and
Technology Press, 1986, pp. 2358 – 2360.
[9] C. Jansakul, H. Baumann, L. Kenne, G. Samuelsson, Planta Med. 1987, 53, 405.
[10] P.-K. Agrawal, Phytochemistry 1992, 31, 3307.
Received July 29, 2010