Cytotoxic triterpenes from the aerial roots of Ficus microcarpa

Article abstract of DOI:10.1016/j.phytochem.2004.12.026

Six triterpenes, 3β-acetoxy-12,19-dioxo-13(18)-oleanene (1), 3β-acetoxy-19(29)-taraxasten-20α-ol (2), 3β-acetoxy-21α, 22α-epoxytaraxastan-20α-ol (3), 3,22-dioxo-20-taraxastene (4), 3β-acetoxy-11α,12α-epoxy-16-oxo-14-taraxerene (5), 3β-acetoxy-25-methoxylanosta-8,23-diene (6) along with nine known triterpenes, 3β-acetoxy-11α,12α-epoxy-14-taraxerene (7), 3β-acetoxy-25-hydroxylanosta-8,23-diene (8), oleanonic acid (9), acetylbetulinic acid (10), betulonic acid (11), acetylursolic acid (12), ursonic acid (13), ursolic acid (14), and 3-oxofriedelan-28-oic acid (15) were isolated from the aerial roots of Ficus microcarpa, and their structures elucidated by spectroscopic methods. The in vitro cytotoxic efficacy of these triterpenes was investigated using three human cancer cell lines, namely, HONE-1 nasopharyngeal carcinoma, KB oral epidermoid carcinoma, and HT29 colorectal carcinoma cells. Compound 8 and pentacyclic triterpenes 9-15 possessing a carboxylic acid functionality at C-28 showed significant cytotoxic activities against the aforementioned cell lines and gave IC₅₀ values in the range 4.0-9.4 μM.

Full text of DOI:10.1016/j.phytochem.2004.12.026

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 495–501
www.elsevier.com/locate/phytochem
Cytotoxic triterpenes from the aerial roots of Ficus microcarpa
a
Yi-Ming Chiang , Jang-Yang Chang , Ching-Chuan Kuo ,
b
b
b
Chi-Yen Chang , Yueh-Hsiung Kuo
c,
*
a
Institute of BioAgricultural Sciences, Academia Sinica, Taipei 115, Taiwan, ROC
b
Division of Cancer Research, National Health Research Institutes, Taipei 115, Taiwan, ROC
c
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan, ROC
Received 27 July 2004; received in revised form 8 November 2004
Available online 29 January 2005
Abstract
Six triterpenes, 3b-acetoxy-12,19-dioxo-13(18)-oleanene (1), 3b-acetoxy-19(29)-taraxasten-20a-ol (2), 3b-acetoxy-21a,22a-epox-
ytaraxastan-20a-ol (3), 3,22-dioxo-20-taraxastene (4), 3b-acetoxy-11a,12a-epoxy-16-oxo-14-taraxerene (5), 3b-acetoxy-25-methoxy-
lanosta-8,23-diene (6) along with nine known triterpenes, 3b-acetoxy-11a,12a-epoxy-14-taraxerene (7), 3b-acetoxy-25-
hydroxylanosta-8,23-diene (8), oleanonic acid (9), acetylbetulinic acid (10), betulonic acid (11), acetylursolic acid (12), ursonic acid
(13), ursolic acid (14), and 3-oxofriedelan-28-oic acid (15) were isolated from the aerial roots of Ficus microcarpa, and their struc-
tures elucidated by spectroscopic methods. The in vitro cytotoxic efficacy of these triterpenes was investigated using three human
cancer cell lines, namely, HONE-1 nasopharyngeal carcinoma, KB oral epidermoid carcinoma, and HT29 colorectal carcinoma
cells. Compound 8 and pentacyclic triterpenes 9–15 possessing a carboxylic acid functionality at C-28 showed significant cytotoxic
activities against the aforementioned cell lines and gave IC50 values in the range 4.0–9.4 lM.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Ficus microcarpa; Moraceae; Triterpenoid; Cytotoxic activity
1. Introduction
and two novel a-tocopheroids from the aerial roots
(Chiang and Kuo, 2003). Triterpenes bearing a carbox-
ylic acid at C-28 have been shown to exhibit cytotoxicity
to tumor cells (Baglin et al., 2003; Lee et al., 2003; Sakai
et al., 2004; Chang et al., 2004a). As part of our research
into anti-tumor effects of plant products, we identified
fifteen triterpenes from the EtOAc-soluble fraction of
the aerial roots of F. microcarpa MeOH extract. Herein,
we describe the structural elucidation of six new triter-
penes (1–6). In addition, the isolated compounds 1, 4,
6, and 8–15 were also evaluated for their in vitro cyto-
toxic efficacy against a small panel of cancer cell lines.
Ficus microcarpa L. f. (Moraceae) is a popular orna-
mental plant in Taiwan. The plantÕs anti-platelet activity
prompted us to investigate its phytochemical constitu-
ents. Phytochemical studies of this species promptly
led to identification of two isoflavones together with
twenty-eight components from the bark (Kuo and Li,
1997; Li and Kuo, 1997); a monoterpenoid, phenoids,
lignans, and a c-lactone from the heartwood (Li and
Kuo, 1998; Li and Kuo, 2000); many types of triterpe-
noids including cycloartanoid, lupanoid, oleanoid, ursa-
noid, taraxastane from the leaves and the aerial roots
(Chiang and Kuo, 2002, and references cited therein);
2. Results and discussion
*
Corresponding author. Tel.: +886 2 23638146; fax: +886 2
Compound 1 was isolated as a colorless solid.
The molecular formula was found to be C32H48O4 by
23636359.
E-mail address: yhkuo@ccms.ntu.edu.tw (Y.-H. Kuo).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.12.026

496
Y.-M. Chiang et al. / Phytochemistry 66 (2005) 495–501
HREIMS. The IR spectrum of 1 showed absorptions
for acetoxyl (1734, 1248 cmꢀ1) and conjugated car-
2.02 (s)], a methylene flanked by a ketone and a ter-
tiary carbon [dH 2.35 (dd, J = 15.6, 5.6 Hz, Ha-11),
2.41 (dd, J = 15.6, 12.4 Hz, Hb-11)], and one methine
proton attached to an acetoxyl group [dH 4.46 (dd,
J = 10.8, 5.2 Hz, H-3)]. The 13C NMR (Table 2) and
DEPT spectra of 1 exhibited one acetoxy (dC 21.2,
bonyl (1697, 1684 cmꢀ1
)
functionalities. The 1H
NMR (Table 1) spectrum in CDCl3 exhibited signals
for eight singlet methyl groups (dH 0.84, 0.85, 0.92,
0.95, 0.97, 1.05, 1.07, 1.23), one acetoxyl group [dH
Table 1
1H-NMR spectroscopic data for compounds 1–6a (400 MHz in CDCl3)
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
1
2
1.57b (Hb),
1.02b (Ha)
1.65b (Hb),
1.00b (Ha)
1.64b (Hb),
1.00b (Ha)
1.92b, 1.40b
1.88 dt
1.68b, 1.26b
(13.6, 3.6 Hb),
1.35b (Ha)
1.74b (Ha),
1.64b (Hb)
1.64b
1.60b
1.60b
2.49 ddd
(16.0, 9.2, 7.2, Hb),
2.42 ddd
1.66b, 1.56b
(16.0, 8.0, 4.8, Ha)
–
3
4.46 dd
(10.8, 5.2)
4.45 dd
(10.8, 5.6)
4.45 dd (10.4, 4.4)
4.50 dd (10.8, 5.2)
4.46 dd (11.2, 4.7)
4
5
–
–
–
0.77 br d (11.2)
–
–
–
0.84b
0.80b
1.35b
0.84b
1.10b
6
1.62b, 1.30b
1.46b
1.51b, 1.36b
1.41b, 1.30b
1.50b, 1.36b
1.46b
1.46b
1.16b
1.64b, 1.46b
2.00b
7
1.37b
–
2.03b (Ha), 1.35b (Hb)
8
–
–
–
–
–
9
1.68b
1.40b
–
1.35b
–
1.37b
1.03b
–
10
11
–
2.41 dd
–
–
3.14 t (4.4)
–
1.44b
1.45b
1.57b, 1.37b
2.01b
(15.6, 12.4, Hb),
2.35 dd
(15.6, 5.6, Ha)
–
12
1.83 br d
(10.8, Hb),
0.79b (Ha)
1.71b
2.03b (Hb),
1.10b (Ha)
1.68b
2.88 d (4.4)
1.60b
13
14
15
–
1.31a
1.93 m
–
1.20b, 1.14b
–
–
–
–
–
–
5.82 s
–
1.14b
1.02b
1.67b (Hb),
1.08b (Ha)
1.78 td
(13.6, 4.0, Ha),
1.26b (Hb)
–
1.13b
16
1.57b
1.47b, 1.23b
1.28b
–
1.90b, 1.30b
17
18
19
20
21
–
–
–
–
1.44b
0.66 s
–
2.42 d (11.6)
–
1.74b
1.45b
1.44b
2.01 m
–
1.66b
1.48b
–
–
0.97 s
1.43b
0.86 d (6.3)
–
–
–
3.09 d (4.0)
1.77b
1.75–1.50b,
1.15–1.00b
1.75–1.50b,
1.15–1.00b
0.82 s
5.70 br s
1.35b
22
23
1.46b
2.89 d (4.0)
0.82 s
–
1.63b, 1.23b
2.15 dd
(13.6, 5.4), 1.74b
5.49 ddd
(15.8, 8.1, 5.4)
5.35 d (15.8)
–
0.85 s
1.07 s
0.86 s
24
25
26
27
28
29
0.84 s
0.92 s
1.07 s
0.97 s
0.95 s
1.23 s
0.82 s
0.86 s
0.81 s
0.83 s
1.02 s
0.94 s
0.88 s
1.10 s
1.14 s
0.78 s
1.14 s
1.00 s
1.03 s
0.99 s
0.64 s
0.99 s
0.94 s
1.08 s
0.92 s
1.21 s
1.21 s
0.93 s
1.01 d (7.2)
0.96 s
1.10 d (6.4)
0.84 s
0.84 s
5.05 s (Ha),
4.48 s (Hb)
1.35 s
30
1.05 s
2.02 s
1.27 s
2.02 s
1.88 br s
1.02 s
2.03 s
0.82 s
2.01 s
3.11 s
CH3CO
OMe
OH
a
2.02 s
2.12 s
Figures in parentheses are coupling constants (J) in Hz.
b
Data obtained from HMQC spectrum.

Y.-M. Chiang et al. / Phytochemistry 66 (2005) 495–501
497
Table 2
13C-NMR spectroscopic data for compounds 1–6 (100 MHz in CDCl3)
1
2
3
4
5
6
1
2
37.9 t
23.4 t
80.3 d
37.7 s
55.3 d
18.0 t
34.6 t
41.3 s
49.8 d
37.3 s
39.3 t
205.2 s
145.3 s
45.1 s
24.8 t
36.6 t
40.1 s
148.0 s
211.4 s
46.3 s
36.3 t
33.7 t
27.9 q
16.5 q
15.9 q
16.9 q
20.6 q
23.1 q
24.7 q
24.5 q
170.9 s
21.2 q
38.4 t
23.7 t
81.0 d
37.8 s
55.5 d
18.2 t
33.6 t
41.1 s
50.9 d
37.1 s
21.0 t
24.7 t
33.0 d
42.1 s
26.5 t
36.6 t
36.2 s
44.5 d
151.5 s
72.8 s
37.8at
37.6at
27.9 q
16.4 q
16.4 q
15.8 q
15.7 q
16.8 q
107.1 t
28.1 q
171.0 s
21.3 q
38.2 t
23.6 t
80.9 d
37.7 s
55.2 d
18.2 t
34.0 t
41.2 s
49.8 d
36.9 s
21.3 t
27.2 t
37.3 d
43.2 s
26.1 t
34.0 t
35.9 s
37.0 d
40.2 d
70.5 s
60.7 d
65.6 d
27.9 q
16.4 q
16.2 q
15.9 q
15.5 q
17.9 q
17.0 q
27.1 q
171.0 s
21.3 q
39.6 t
34.0 t
218.1 s
47.3 s
54.7 d
19.6 t
33.5 t
41.0 s
49.6 d
36.7 s
22.2 t
27.6 t
38.4 d
42.0 s
26.3 t
28.5 t
44.7 s
45.2 d
36.7 d
162.5 s
122.9 d
205.9 s
26.8 q
21.0 q
16.2 q
15.9 q
14.5 q
18.7 q
22.6 q
22.1 q
37.7 t
23.1 t
80.4 d
37.7 s
54.4 d
18.6 t
39.5 t
40.0 s
52.6 d
36.6 s
51.4 d
55.4 d
37.6 s
173.9 s
120.4 d
206.5 s
45.1 s
45.1 d
32.2 t
28.1 s
36.3 t
27.2 t
27.8 q
16.5 q
17.1 q
26.0 q
26.0 q
30.9 q
33.9 q
30.6 q
170.8 s
21.2 q
35.2 t
24.1 t
80.8 d
37.7 s
50.5 d
18.1 t
26.3 t
134.4 s
134.3 s
36.9 s
20.9 t
30.9 t
44.4 s
49.8 s
30.8 t
28.1 t
50.0 d
15.8 q
19.1 q
36.6 d
18.7 q
39.3 t
128.6 d
136.7 d
74.8 s
25.7 q
26.1 q
27.9 q
16.5 q
24.2 q
170.9 s
21.2 q
50.2 q
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
CH3CO
CH3CO
OMe
a
Values may be interchanged.
170.9), one carbon attached to an acetoxyl group [dC
80.3 (C-3)], two quaternary olefinic carbons [dC 145.3
(C-13), 148.0 (C-18)], and two carbonyl carbons [dC
205.2 (C-12), 211.4 (C-19)]. b-Amyrin acetate, an ole-
anane derivative, has similar 13C NMR spectroscopic
data for its A and B rings (Knight, 1974; Mahato
and Kundu, 1994). This suggested that compound 1
and b-amyrin acetate possessed a similar structure on
A and B rings. On the basis of the above evidence,
the structure of compound 1 was proposed as an olean-
ane skeleton containing an acetoxy, a tetra-substituted
olefin, and two ketone functionalities. The double bond
was deduced to be located at C-13 and C-18 because it
was the only possible structure to have a tetra-substi-
tuted olefin. In the HMBC spectrum, the long-range
13C–1H correlations C-12/H2-11 and C-19/H2-21, H3-
29, H3-30 established partial structure of the C, D,
Compound 2, a colorless solid, analyzed for
C32H52O3 on the basis of HREIMS data. Its IR spec-
trum showed hydroxyl (3464 cmꢀ1), a terminal double
bond (3091, 1642, and 900 cmꢀ1), and acetoxyl (1734
1
and 1249 cmꢀ1) functionalities. The H NMR spectrum
(Table 1) of 2 exhibited signals for seven singlet methyl
groups (dH 0.64, 0.82, 0.82, 0.86, 0.99, 1.03, 1.35), one
acetoxyl group [dH 2.02 (s)], an allylic proton [dH 2.42
(d, J = 11.6 Hz, H-18)], one methine proton attached
with the acetoxyl group [dH 4.45 (dd, J = 10.8, 5.6 Hz,
H-3)], and a terminal double bond [dH 4.48 (1H, s,
Hb-29), 5.05 (1H, s, Ha-29)]. The 13C NMR spectrum
exhibited 32 signals (eight CH3, eleven CH2, five CH,
and eight C) including one acetoxyl group (dC 21.3
and 171.0), two carbons to which oxygen was attached
[dC 72.8 (C-20) and 81.0 (C-3)], and a terminal double
bond [dC 107.1 (C-29) and 151.5 (C-19)]. Comparison
of the 13C NMR spectra between 2 and 3b-acetoxy-
20b-H-19(29)-taraxastene (Bhutani et al., 1992) revealed
a difference in the ring E of both the compounds. In the
HMBC spectrum, long-range 13C–1H correlations for C-
19/H-18, H2-29, H3-30 and C-20/H-18, H2-29, H3-30
established a partial structure of the E ring. NOESY
and
E rings. The UV absorption maximum at
252.0 nm was consistent with this partial structure.
The 2D-NMR (HMQC, HMBC, NOESY, and COSY)
analyses also confirmed the assigned structure. There-
fore, compound 1 was assigned as 3b-acetoxy-12,19-di-
oxo-13(18)-oleanene.

498
Y.-M. Chiang et al. / Phytochemistry 66 (2005) 495–501
20-taraxasten-3b-ol indicated a ketone group at C-3 in 4
Ha
instead of a hydroxy group at C-3 in 22-oxo-20-taraxa-
sten-3b-ol. The ketone group caused H2-2 in compound
4 to exhibit a downfield shift in NMR spectrum to dH
2.42 and 2.49. Thus, compound 4 was established as
3,22-dioxo-20-taraxastene.
29
Hb
30
H
H
12
13
18
OH
Compound 5, C32H48O4 (form HREIMS), had IR
absorption bands at 1734, 1247 cmꢀ1 (acetoxyl group)
and 3053, 1683 cmꢀ1 (conjugated carbonyl group),
respectively. The 1H NMR data of compound 5 were sim-
ilar to those of compound 7 (Tanaka and Matsunaga,
1988). The only difference was an addition of a carbonyl
group at C-16 in 5. The UV absorption band at kmax
243.0 nm and an olefinic proton at dH 5.82 (s, H-16) con-
firmed the presence of a b,b-disubstituted cyclohexenone
moiety. Thus, compound 5 was established as 3b-acetoxy-
11a,12a-epoxy-16-oxo-14-taraxerene.
H
(a)
27
30
28
H
19
OH21
H
29
H
H
12
22
O
(b)
1
The H and 13C NMR spectroscopic data of com-
Fig. 1. Key NOESY correlations of compound 2 (a) and 3 (b).
pound 6 were similar to those of compound 8 (Four-
rey et al., 1970) except for the presence of
a
correlations for H-18/H3-27; Ha-29/Hb-29, H3-30; and
Hb-29/Hb-12, H-13, Ha-29 suggested that CH3-30
should be on the b face (Fig. 1(a)). Therefore, com-
pound 2 was assigned as 3b-acetoxy-19(29)-taraxasten-
20a-ol.
methoxyl group [dH 3.11 (s)] in compound 6 instead
of the hydroxyl group found in 8. This evidence
showed that the structure of 6 is 3b-acetoxy-25-meth-
oxylanosta-8,23-diene.
Nine known compounds were identified as 3b-acet-
oxy-11a,12a-epoxy-14-taraxerene (7) (Tanaka and
Matsunaga, 1988), 3b-acetoxy-25-hydroxylanosta-8,23-
diene (8) (Fourrey et al., 1970), oleanonic acid (9)
(Shirane et al., 1996), acetylbetulinic acid (10) (Nom-
HREIMS of compound 3 gave the molecular ion
peak at m/z = 500.3845 corresponding to a molecular
formula of C32H52O4. IR absorption bands at 3513,
1728, and 1247 cmꢀ1 indicated hydroxyl and acetoxyl
1
groups. The H NMR spectrum of 3 exhibited signals
´
ura et al., 1981), betulonic acid (11) (Gonzalez et al.,
for seven singlet methyl groups (dH 0.81, 0.82, 0.83,
0.93, 0.94, 0.99, and 1.27), a doublet methyl group
(dH 1.01), a methine proton associated with an acetoxyl
group (dH 2.02 and 4.45), a D2O exchangeable proton
(dH 2.12), and two oxymethine protons coupled to each
1983), acetylursolic acid (12) (Tkachev and Denisov,
1994), ursolic acid (14) (Fang et al., 1991), and 3-oxo-
friedelan-28-oic acid (15) (Kumar et al., 1985), respec-
tively, by comparing their NMR and MS data as well
as optical rotations with those reported in the litera-
ture. Ursonic acid (13) was identified by direct com-
parison (1H NMR) with an authentic sample, which
was synthesized from ursolic acid (14) by Jones
oxidation.
1
other (dH 2.89 and 3.09). Its H and 13C NMR data
were similar to those of 3b-acetoxy-21a,22a-epoxide-
20(30)-taraxastene (Menichini et al., 1996), except for
the existence of one tertiary hydroxy group at C-20
in compound 3, that was proposed from the hydration
Eleven isolated compounds (1, 4, 6, and 8–15) were
evaluated for their cytotoxicity against HONE-1, KB,
and HT29 cancer cell lines by using methylene blue
dye assay and anti-cancer drugs, etoposide (Chang et
al., 2004b) and cisplatin (Chang et al., 2002), as positive
controls (Table 3). Among them, compounds 8–15
exhibited cytotoxicity against these cell lines and gave
IC50 values in the range 4.0–9.4 lM. On the other hand,
compounds 1, 4, and 6 displayed no cytotoxicity effects
against these cancer cell lines (>10 lM). On the basis of
structure and activity relationships, the pentacyclic tri-
terpenes possessing a carboxylic acid at C-28, including
compounds 9–15, showed more potent cytotoxic efficacy
than that of other tested triterpenes. Triterpenes with a
C-28 carboxylic acid are reported to exhibit cytotoxic ef-
fects (Baglin et al., 2003; Sakai et al., 2004) and this was
also observed in this study.
of
3b-acetoxy-21a,22a-epoxide-20(30)-taraxastene.
NOESY correlations for H3-28/H-19, H-22; H3-29/
Hb-12; and H3-30/H-19, H-21, H-22 suggested that
CH3-30 should be on the same face as H-19, H-21,
H-22, and CH3-28 (Fig. 1(b)). Therefore, compound
3 was assigned as 3b-acetoxy-21a,22a-epoxytaraxa-
stan-20a-ol.
Compound 4 had the molecular formula C30H46O2
on the basis of its HREIMS data. Its IR spectrum indi-
cated the presence of a carbonyl group (1707 cmꢀ1) and
a conjugated carbonyl group (1677 and 1642 cmꢀ1). The
UV absorption at kmax 236.0 nm was consistent with the
presence of a conjugated ketone. The 1H and 13C NMR
spectroscopic data of compound 4 were similar to those
of 22-oxo-20-taraxasten-3b-ol (Kuo and Chiang, 1999).
Comparison of the NMR spectra between 4 and 22-oxo-

Y.-M. Chiang et al. / Phytochemistry 66 (2005) 495–501
499
Table 3
Cytotoxicity of compounds 8–15 against cultured HONE-1, KB, and
HT29 cancer cell lines
30
29
30
OH
29
O
20
20
O
E
a
)
13
13
Compounds
Growth inhibition constant (IC50
[lM]
HT29
26
25
25
17
26
C
D 17
28
28
1
1
HONE-1
KB
3 A 10B
10
Etoposideb
0.5 0.2
3.2 0.5
>10
0.9 0.3
4.4 0.9
>10
2.4 0.5
5.7 1.1
9.3 1.6
>10
27
3
27
AcO
Cisplatinb
AcO
AcO
AcO
6
6
24
24
23
23
8
1
2
9
7.2 1.9
4.7 1.9
4.9 2.1
>10
6.3 1.6
6.7 2.6
8.2 1.8
8.4 2.9
4.0 2.1
8.2 2.7
8.3 2.4
10
11
12
13
14
15
a
>10
>10
OH
>10
O
5.2 0.7
8.8 1.5
9.4 2.8
6.3 1.8
4.7 1.5
>10
O
O
IC50 is defined as the concentration that resulted in a 50%
decrease in cell number and the results are means standard deviation
of three independent replicates. The IC50 greater than 10 lM was
considered to be no cytotoxicity.
3
4
30 29
b
Positive control substance.
22
20
17
21
18
27
20
27
13
O
R
26
19
13
26
25
17
28
1
1
1000 digital polarimeter, whereas IR spectra were re-
corded on a Perkin–Elmer 983G spectrophotometer.
1H and 13C NMR spectra were run on a Varian Unity
Plus 400 spectrometer, whereas EIMS and FABMS
were obtained on a Finnigan TSQ-46C and JEOL
JMS-HX 300 mass spectrometer respectively. Extracts
were purified using Si gel chromatography (Merck 70–
230 mesh, 230–400 mesh, ASTM).
R
10
10
3
30
3
AcO
6
6
29
28
24
23
6
8
R = OMe
R = OH
5
7
R = O
R = H2
OH
OH
3.2. Plant material
O
O
The aerial roots of Ficus microcarpa L. f. were col-
lected on the campus of National Taiwan University,
Taipei, Taiwan, in August 1996. The plant was identified
by Mr. Muh-Tsuen Gun (retired), Department of Bot-
any, National Taiwan University. A voucher specimen
(No. 038671) has been deposited at the Herbarium of
the Department of Botany, National Taiwan University,
Taipei, Taiwan.
O
R
9
10 R = α-H, β-OAc
11 R = O
OH
OH
O
O
3.3. Extraction and isolation
R
O
15
12 R = α-H, β-OAc
13 R = O
The dried aerial roots of F. microcarpa were crushed
to give 18 kg of raw material, which was extracted with
MeOH (150 l) at room temperature (7 days · 2). The
combined extracts were evaporated in vacuo to yield a
residue, which was suspended in H2O (1 l), and this
was then partitioned with ethyl acetate (1 l · 3). The
combined ethyl acetate layer afforded a black syrup
(250 g), which was subsequently applied to a Si gel col-
umn (15 · 60 cm) eluted with hexane-EtOAc mixtures of
increasing polarity (1:0, fr. 1, 23 l; 49:1, fr. 2, 4 l; 97:3, fr.
3, 3 l; 19:1, fr. 4, 13 l; 9:1, fr. 5, 6 l; 4:1, fr. 6, 23 l; 7:3, fr.
7, 19 l; 1:1, fr. 8, 15 l; 3:7, fr. 9 15 l; 0:1, fr. 10, 9 l). Fr. 2
was separated by preparative HPLC [Phenomenex Luna
5 lm Silica (2), 250 mm · 10 mm] eluted with 3% EtOAc
14 R = α-H, β-OH
3. Experimental
3.1. General procedures
Melting points were determined with a Yanagimoto
micromelting point apparatus and are uncorrected.
Optical rotations were measured on a JASCO DIP-

500
Y.-M. Chiang et al. / Phytochemistry 66 (2005) 495–501
in hexane to afford compounds 1 (12 mg), 5 (7 mg), and
7 (11 mg). Fr. 6 was separated by preparative HPLC
[Phenomenex Luna 5 lm Silica (2), 250 mm · 10 mm]
eluted with 20% EtOAc in hexane to afford compounds
2 (11 mg), 4 (5 mg), 6 (33 mg), 8 (18 mg), and 10
(15 mg). Fr. 7 (13.3 g) was subjected to Si gel cc
(5 · 30 cm) eluted with CH2Cl2–MeOH mixtures of
increasing polarity (1:0, fr. 7-1, 500 ml; 49:1, fr. 7-2,
1500 ml; 19:1, fr. 7-3, 1000 ml; 9:1, fr. 7-4, 500 ml). Fr.
7-2 was separated by preparative HPLC [Phenomenex
Luna 5 lm Silica (2), 250 mm · 10 mm] eluted with
35% EtOAc in hexane to afford compounds 3 (9 mg),
9 (6 mg), 11 (11 mg), 12 (20 mg), 13 (10 mg), and 15
(19 mg). Fr. 8 (7.1 g) was subjected to Si gel cc
(3 · 20 cm) eluted with CH2Cl2–MeOH mixtures of
increasing polarity (1:0, fr. 8-1, 250 ml; 49:1, fr. 8-2,
300 ml; 19:1, fr. 8-3, 1000 ml; 9:1, fr. 8-4, 500 ml; 4:1,
fr. 8-5, 500 ml). Fr. 8-3 was separated by preparative
HPLC [Phenomenex Luna 5 lm Silica (2),
250 mm · 10 mm] eluted with 5% acetone in CH2Cl2
to afford compound 14 (14 mg).
3.4.4. 3,22-Dioxo-20-taraxastene (4)
24
D
Colorless solid, mp 245–248 ꢁC; ½aꢁ : +63.9ꢁ
(CHCl3; c 0.2); UV kMeOH nm (log e): 236.0 (3.92); IR
max
1
KBr
max
m
cmꢀ1: 3052, 1707, 1677, 1642, 1384, 738. For H
and 13C NMR spectroscopic data, see Tables 1 and
2. EIMS 70 eV, m/z (rel. int.): 438 [M]+ (100), 423
(13), 395 (6), 368 (11), 353 (6), 243 (7), 236 (10), 219
(13), 205 (33), 189 (10), 163 (14), 152 (13), 107 (14),
97 (24); HREIMS, m/z found: 438.3503; calc. for
C30H46O2: 438.3500.
3.4.5. 3b-Acetoxy-11a,12a-epoxy-16-oxo-14-taraxerene
(5)
24
D
Colorless solid, mp >300 ꢁC; ½aꢁ : ꢀ39.3ꢁ (CHCl3; c
MeOH
max
KBr
max
0.2,); UV k
nm (log e): 243.0 (3.77); IR m
cmꢀ1
:
1
3053, 1734, 1683, 1375, 1247, 1031, 992, 738. For H
and 13C NMR spectroscopic data, see Tables 1 and 2.
EIMS 70 eV, m/z (rel. int.): 496 [M]+ (22), 481 (20),
478 (14), 463 (8), 436 (20), 403 (13), 372 (38), 356 (13),
312 (70), 294 (100), 279 (81), 175 (40), 148 (78), 135
(46); HREIMS, m/z found: 496.3546; calc. for
C32H48O4: 496.3554.
3.4. Compound identification
3.4.6. 3b-Acetoxy-25-methoxylanosta-8,23-diene (6)
26
D
3.4.1. 3b-Acetoxy-12,19-dioxo-13(18)-oleanene (1)
Colorless solid, mp 148–150 ꢁC; ½aꢁ : +30.4ꢁ (CHCl3;
24
D
KBr
max
Colorless solid, mp 244–247 ꢁC; ½aꢁ : ꢀ94.9ꢁ (CHCl3;
c 2.7); IR m
cmꢀ1: 1736, 1371, 1244, 1076, 1028, 979.
MeOH
max
1
c 1.0); UV k
1734, 1697, 1684, 1624, 1394, 1370, 1248, 1030, 1003,
nm (log e): 252.0 (3.83); IR m
cmꢀ1
:
KBr
max
For H and 13C NMR spectroscopic data, see Tables 1
and 2. EIMS 70 eV, m/z (rel. int.): 498 [M]+ (5), 466
(14), 451 (32), 406 (24), 391 (100), 309 (16), 297 (18),
109 (84); HREIMS, m/z found: 498.4073; calc. for
C33H54O3: 498.4075.
971, 902, 737, 705. For H and 13C NMR spectroscopic
1
data, see Tables 1 and 2. EIMS 70 eV, m/z (rel. int.): 496
[M]+ (30), 427 (100), 356 (16), 273 (14), 232 (62), 189
(30), 176 (28), 135 (25); HREIMS, m/z: found:
496.3557; calc. for C32H48O4: 496.3554.
3.4.7. Ursonic acid (13)
25
D
Colorless solid, mp 271–275 ꢁC; ½aꢁ : +56.7ꢁ (CHCl3;
cmꢀ1: 3400–2400, 3065, 1706, 1695, 1379,
KBr
max
3.4.2. 3b-Acetoxy-19(29)-taraxasten-20a-ol (2)
c 0.9); IR m
24
Colorless solid, mp 245–248 ꢁC; ½aꢁ : +55.3ꢁ
1387, 1318, 1278, 1257, 1238, 978, 740; 1H NMR
(400 MHz, CDCl3): 0.80 (s, H3-26), 0.84 (d,
D
(CHCl3; c 0.5); IR mKmaBxr cmꢀ1: 3464, 3091, 1734, 1642,
1380, 1370, 1249, 1037, 982, 900, 739. For 1H and
13C NMR spectroscopic data, see Tables 1 and 2.
EIMS 70 eV, m/z (rel. int.): 484 [M]+ (6), 466 (72),
451 (9), 426 (25), 406 (39), 391 (22), 363 (22), 337
(6), 303 (5), 295 (7), 269 (5), 255 (10), 229 (10), 217
(32), 202 (40), 189 (100), 173 (38), 133 (48), 121 (76),
107 (50); HREIMS, m/z found: 484.3927; calc. for
C32H52O3: 484.3919.
d
J = 6.4 Hz, H3-29), 0.93 (3H, d, J = 6.4 Hz, H3-30),
1.00 (s, H3-24), 1.03 (s, H3-25), 1.06 (s, H3-23 and H3-
27), 2.17 (d, J = 11.2 Hz, H-18), 2.36 (ddd, J = 16.0,
6.8, 3.6 Hz, Ha-2), 2.52 (ddd, J = 16.0, 10.8, 7.2 Hz,
Hb-2), 5.24 (t, J = 3.2 Hz, H-12); FABMS m/z (rel.
int.): 455 [M + H]+ (10), 425 (14), 407 (14), 248 (19),
203 (35), 154 (100). Identified by direct comparison
(1H NMR) with an authentic sample, which was synthe-
sized from ursolic acid (14) by Jones oxidation.
3.4.3. 3b-Acetoxy-21a,22a-epoxytaraxastan-20a-ol (3)
21
D
Colorless solid, mp >300 ꢁC; ½aꢁ : +5.6ꢁ (CHCl3; c
3.5. Antitumoral cytotoxic assay
KBr
max
0.6); IR m
cmꢀ1: 3513, 1728, 1386, 1375, 1247, 1014,
983, 936, 902, 869, 814. For H and 13C NMR spectro-
scopic data, see Tables 1 and 2. EIMS 70 eV, m/z (rel.
int.): 500 [M]+ (4), 482 (6), 466 (10), 442 (8), 440 (12),
422 (16), 411 (12), 379 (10), 353 (6), 299 (8), 257 (10),
234 (16), 203 (100), 189 (84), 175 (38), 135 (48), 121
(52), 95 (57); HREIMS, m/z found: 500.3845; calc. for
C32H52O4: 500.3868.
Human nasopharyngeal carcinoma HONE-1, oral
epidermoid carcinoma KB, and colorectal carcinoma
HT29 cells were maintained in RPMI-1640 medium sup-
plied with 5% fetal bovine serum. Cells in logarithmic
phase were cultured at a density of 5000 cells/ml/well
in a 24-well plate. The cells were exposed to various con-
centrations of the tested drugs for 72 h. The methylene
1

Y.-M. Chiang et al. / Phytochemistry 66 (2005) 495–501
501
cytotoxic compounds on exponentially growing carcinoma cells.
Anal. Biochem. 139, 272–277.
Fourrey, J.L., Rondest, J., Polonsky, J., 1970. Sur quelques oxydations
blue dye assay was used to evaluate the effects of the
tested drugs on cell growth, as described previously
(Finlay et al., 1984). The IC50 value resulting from
50% inhibition of cell growth was calculated graphically
as a comparison with the control.
´ ´
de type biogenetique. Tetrahedron 26, 3839–3847.
´
Gonzalez, A.G., Amaro, J., Fraga, B.M., Luis, J.G., 1983. 3-oxo-6b-
hydroxyolean-18-en-28-oic acid from Orthopterygium huancuy.
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Acknowledgement
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Kuo, Y.H., Chiang, Y.M., 1999. Five new taraxastane-type triterpenes
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498–500.
This research was supported by the National Science
Council of the Republic of China (NSC 92-2323-B-002-
003).
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Cytotoxic triterpenoids from the fruits of Zizyphus jujuba. Planta
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