Cytotoxic triterpenoid glycosides from the roots of Gordonia chrysandra

Article abstract of DOI:10.1021/np900089v

Eight new oleanane triterpenoid glycosides, gordonosides A-H (1-8), were isolated from a 50% EtOH extract of the roots of Gordonia chrysandra. Their structures were determined by spectroscopic analysis, including 1D and 2D NMR and ESIMS, and by chemical m

Full text of DOI:10.1021/np900089v

866
J. Nat. Prod. 2009, 72, 866–870
Cytotoxic Triterpenoid Glycosides from the Roots of Gordonia chrysandra
Lei Yu, Jing-Zhi Yang, Xiao-Guang Chen, Jian-Gong Shi, and Dong-Ming Zhang*
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Key Laboratory of BioactiVe
Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Beijing 100050, People’s Republic of China
ReceiVed February 11, 2009
Eight new oleanane triterpenoid glycosides, gordonosides A-H (1-8), were isolated from a 50% EtOH extract of the
roots of Gordonia chrysandra. Their structures were determined by spectroscopic analysis, including 1D and 2D NMR
and ESIMS, and by chemical methods. Among these substances, compounds 1, 3, 5, and 6 exhibited cytotoxic activity
against several human cancer cell lines, with 3 being the most potent.
The genus Gordonia (Theaceae) consists of 40 species, with six
species found in mainland China. Gordonia chrysandra Cowan is
widely distributed in Sichuan, Guizhou, and Yunnan Provinces of
the People’s Republic of China.1 Previous phytochemical studies
on Gordonia species have led to the identification of triterpenoids,2-6
steroids,3 tannins,7 and several other components.8 Some of these
substances have exhibited antifungal8 and apoptosis-inducing
activities.7 In previous work, G. chrysandra roots were extracted
successively with 95% EtOH and 50% EtOH in H2O. The 95%
EtOH extract exhibited hepatoprotective activity, and two active
flavanonol glycosides were isolated.9 The 50% EtOH extract
showed cytotoxic activity against several human cancer cell lines,
and bioassay-guided fractionation of this extract led to the isolation
of eight cytotoxic acylated triterpenoid saponins (1-8). In this
paper, we report the structural elucidation and cytotoxic activities
of these new compounds.
and/or olefinic protons at δ 4.30 (1H, m, H-3), 4.42 (1H, m,
H-16), 6.26 (1H, d, J ) 10.0 Hz, H-22), 6.62 (1H, d, J ) 10.0
Hz, H-21), and 5.37 (1H, brs, H-12). Signals were also observed
assignable to two anomeric protons of two sugar units at δ 5.19
(1H, d, J ) 8.0 Hz, H-1′′′) and 5.26 (1H, d, J ) 7.5 Hz, H-1′′′′),
as well as partially overlapped signals due to the oxymethylenes
and oxymethines of the sugar units between δ 4.01 and 4.51.
Acid hydrolysis of 1 yielded D-glucuronic acid and L-arabinose,
which were confirmed by measuring their specific rotations. The
coupling constants of the anomeric protons (8.0 and 7.5 Hz,
respectively) indicated a ꢀ- and an R-configuration at the
anomeric carbon of the glucuronic acid and arabinose moiety,
respectively.10 In addition, the 1H NMR spectrum exhibited three
pairs of characteristic signals due to two angeloyloxy groups at
δ 1.87 (3H, s, H3-5′′) and 1.96 (3H, s, H3-5′), 2.01 (3H, d, J )
6.5 Hz, H3-4′′) and 2.03 (3H, d, J ) 7.0 Hz, H3-4′), and 5.88
(1H, q, J ) 6.5 Hz, H-3′′) and 5.93 (1H, q, J ) 7.0 Hz, H-3′).11
These data suggested that 1 is a highly oxygenated 23,28-
dihydroxyolean-12-ene diglycoside derivative with ꢀ-D-glucu-
ronyl and R-L-arabinosyl sugar units and two angeloyloxy groups
as additional substituents. This was supported by the 13C NMR
spectroscopic data of 1 (Table 2).
The structure of 1 was finalized by a comprehensive analysis of
its 2D NMR spectroscopic data. The proton and protonated carbon
signals in the NMR spectra of 1 were assigned unequivocally
(Tables 1 and 2) on the basis of TCOSY and HSQC spectroscopic
analysis. In the HMBC spectrum of 1, two- and three-bond
correlations from protons to carbons of the aglycon moiety, together
with the chemical shifts of these protons and carbons, confirmed
that 1 possesses a 23,28-dihydroxyolean-12-ene nucleus with four
oxygenated substituents at C-3, C-16, C-21, and C-22, respectively.
HMBC correlations from H-21 to C-1′ and from H-22 to C-1′′, in
combination with chemical shifts of these protons and carbons,
indicated unambiguously that the two angeloyloxy ester groups are
attached to C-21 and C-22 of the aglycon, respectively. In addition,
HMBC correlations from H-1′′′ to C-3 confirmed that the ꢀ-D-
glucuronopyranosyloxy unit is located at C-3 of the aglycon. A
long-range correlation from H-1′′′′ to C-3′′′ (δ 85.7) suggested that
the R-L-arabinopyranosyl unit is linked to C-3 of the ꢀ-D-
glucuronopyranosyl unit. On taking account of the molecular
composition of 1, its planar structure was elucidated as 3-O-[R-L-
arabinopyranosyl(1f3)-ꢀ-D-glucuronopyranosyl]-21,22-diangeloy-
loxyolean-12-en-16,23,28-triol.
Results and Discussion
Compound 1 was isolated as a white, amorphous powder, and
its IR spectrum displayed absorptions for hydroxy (3391 cm-1
)
and conjugated carbonyl (1676 cm-1) groups. The positive-ion
HRESIMS of 1 showed a quasimolecular ion peak at m/z
1001.5056 [M + Na]+ and indicated a molecular formula of
C51H78O18 (calcd for C51H78O18Na, m/z 1001.5086). The 1H NMR
spectrum of 1 showed signals attributable to six tertiary methyl
groups at δ 0.85 (H3-26), 0.88 (H3-25), 0.92 (H3-24), 1.01 (H3-
29), 1.27 (H3-30), and 1.75 (H3-27), two isolated oxymethylenes
at δ 3.36 and 3.62 (1H each, d, J ) 10.5 Hz, H2-28) and 3.69
and 4.28 (1H each, d, J ) 10.5 Hz, H2-23), and five oxymethine
The relative configuration of 1 was established by a NOE
difference experiment and from the vicinal coupling constants of
related protons. In the NOE difference spectrum of 1, irradiation
of H-23b enhanced the H-3 and H-5 signals, suggesting that the
diglycosyl moiety at C-3 has a ꢀ-orientation. Irradiation of H-28b
gave enhancements of H-16, H-22, and H-26, indicating that both
the hydroxy at C-16 and the angeloyloxy at C-22 possess an
* To whom correspondence should be addressed. Tel: +86 1063165227.
Fax: +86 1063165227. E-mail: zhangdm@imm.ac.cn.
10.1021/np900089v CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/15/2009

Cytotoxic Triterpenoid Glycosides from Gordonia chrysandra
Journal of Natural Products, 2009, Vol. 72, No. 5 867
Table 1. 1H NMR Spectroscopic Data for Compounds 1-8a
position
1
2
3
4
5
6
7
8
3
5
9
12
15
4.30, m
1.62, m
1.73, m
5.37, brs
1.52, m
1.80, m
4.42, m
3.05, m
3.05, m
1.34, m
4.26, m
1.60, m
1.74, m
5.38, brs
1.60, m
1.80, m
5.49, brs
3.03, dd (13.5, 4.5) 3.11, s
2.62, t (14.0)
1.42, m
3.38, dd (12.0, 4.2)
0.79, s
1.72, m
5.42, brs
1.62, m
1.88, m
3.30, dd (11.5, 3.0) 4.18, m
4.47, m
1.68, m
1.80, m
5.39, brs
1.58, m
1.80, m
4.46, m
3.08, m
3.06, m
1.39, m
4.50, m
1.76, m
1.82, m
5.48, brs
4.18, d (3.5)
4.48, m
1.76, m
1.80, m
5.47, brs
4.17, d (4.2)
0.73, m
1.36, m
5.41, brs
1.65, m
1.83, m
5.53, brs
1.38, m
1.78, m
5.40, brs
1.55, m
1.83, m
4.48, brs
16
18
19a
19b
21
4.49, brs
4.41, d (3.5)
3.05, s
3.05, s
1.40, m
6.69, d (10.5) 6.64, d (10.2)
6.31, d (10.5) 6.24, d (10.2)
4.37, d (4.2)
3.06, m
3.06, m
3.06, dd (11.5, 3.0) 3.06, m
3.11, s
1.41, m
6.70, d (10.2)
6.32, d (10.2)
1.31, s
2.67, t (9.0)
1.47, m
3.08, m
1.40, m
1.40, m
6.62, d (10.0) 5.96, d (10.0)
6.26, d (10.0) 6.26, d (10.0)
3.69, d (10.5) 3.71, d (10.5)
4.28, d (10.5) 4.35, d (10.5)
5.99, d (10.5)
6.27, d (10.5)
1.29, s
6.70, d (10.5) 6.67, d (10.5)
6.32, d (10.5) 6.29, d (10.5)
9.74, s
22
23
24
25
26
27
0.92, s
0.88, s
0.85, s
1.75, s
0.92, s
0.86, s
0.76, s
1.38, s
0.99, s
0.82, s
0.86, s
1.87, s
0.97, s
0.78, s
0.76, s
1.51, s
1.30, s
0.81, s
0.81, s
1.83, s
1.43, s
0.83, s
0.83, s
1.81, s
1.43, s
0.84, s
0.98, s
1.82, s
1.44, s
0.85, s
0.99, s
1.82, s
28a
28b
29
3.62, d (10.5) 3.60, d (10.5)
3.36, d (10.5) 3.43, d (10.5)
3.66, d (10.2)
3.41, d (10.2)
1.09, s
3.61, d (10.5)
3.45, d (10.5)
1.08, s
3.62, d (10.5) 3.62, d (10.5)
3.39, d (10.5) 3.39, d (10.5)
3.74, d (10.5) 3.73, d (10.2)
3.49, d (10.5) 3.47, d (10.2)
1.01, s
1.27, s
5.93, q (7.0)
2.03, d (7.0)
1.96, s
1.04, s
1.26, s
5.94, m
2.01, m
1.94, s
1.09, s
1.06, s
1.07, s
1.06, s
30
1.33, s
1.29, s
1.32, s
1.31, s
1.31, s
1.29, s
3′
5.95, q (7.2)
2.07, d (7.2)
2.00, s
5.93, m
2.02, m
1.95, s
5.95, q (7.0)
2.07, d (7.0)
2.00, s
5.95, q (7.5)
2.07, d (7.5)
1.99, s
5.96, q (7.0)
2.08, d (7.0)
2.00, s
6.06, q (7.2)
2.15, d (7.2)
2.03, s
4′
5′
2′′
2.07, q (7.2)
1.21, m 1.58, m
0.66, t (7.2)
1.01, d (7.2)
4.95, d (7.8)
4.02, t (8.4)
4.30, m
3′′
5.88, q (6.5)
2.01, d (6.5)
1.87, s
5.90, m
2.03, m
2.00, s
5.90, q (7.2)
2.04, d (7.2)
1.89, s
5.01, d (7.2)
4.14, t-like (9.0, 7.8) 4.13, t (8.0)
4.40, d (9.6)
4.55, d (9.0)
4.68, d (9.6)
5.36, d (7.2)
4.52, t-like (8.4, 7.2) 4.51, m
4.19, dd (8.4, 3.0)
4.30, brs
5.92, m
2.04, m
2.02, m
4.96, d (7.0)
5.89, q (7.0)
2.03, d (7.0)
1.88, s
5.90, q (7.0)
2.03, d (7.0)
1.88, s
5.78, q (7.0)
1.95, d (7.0)
1.73, s
4′′
5′′
1′′′
2′′′
3′′′
4′′′
5′′′
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
5.19, d (8.0)
4.01, m
4.21, t (9.0)
4.44, m
5.20, d (7.5)
4.12, t (8.0)
4.22, m
4.90, d (8.0)
4.02, t (8.5)
4.31, m
4.95, d (8.0)
4.02, t-like (9.0, 8.0) 4.02, t (8.5)
4.29, m
4.95, d (7.5)
4.38, d (9.0)
4.53, m
4.66, d (9.0)
5.35, d (7.5)
4.29, m
4.48, m
4.50, m
4.47, m
4.47, m
4.51, m
4.51, d (9.5)
5.26, d (7.5)
4.47, d (8.0)
4.15, m
4.54, m
5.31, d (7.0)
4.50, m
4.63, d (9.5)
5.33, d (6.5)
4.52, m
4.62, d (10.0)
5.33, d (7.5)
4.50, m
4.63, d (9.0)
5.34, d (6.5)
4.47, m
4.62, d (9.0)
5.33, d (6.6)
4.50, m
4.21, m
4.19, dd (8.5, 2.5)
4.18, m
4.17, m
4.16, m
4.17, m
4.30, m
4.33, m
4.30, brs
3.80, d (11.5)
4.36, m
4.34, m
4.30, m
4.33, m
4.30, m
3.78, d (11.5) 3.82, d (11.5)
3.81, d (11.4)
4.38, dd (11.4, 3.0)
3.78, d (11.5) 3.78, d (12.0)
3.76, m
3.75, m
4.33, m
4.39, m
2.48, s
4.37, m
4.34, m
4.37, m
4.35, m
Ac
2.52, s
OMe
3.81, s
3.77, s
3.78, s
a
1H NMR data (δ) were measured in C5D5N at 500 MHz for 1, 2, and 4-7, and at 600 MHz for 3 and 8. Coupling constants (J) in Hz are given in
parentheses. The assignments are based on DEPT, H-1H COSY, HSQC, and HMBC experiments.
1
1
R-orientation. In the H NMR spectrum of 1, the vicinal coupling
constant between H-21 and H-22 (10.0 Hz) suggested that these
two protons adopt a trans diaxial orientation. This indicated that
the angeloyloxy group at C-21 is ꢀ-oriented, which was confirmed
by an enhancement of H3-27 caused by irradiation of H-21. Thus,
the structure of 1 was determined as 3ꢀ-O-[R-L-arabinopyrano-
syl(1f3)-ꢀ-D-glucuronopyranosyl]-21ꢀ,22R-diangeloyloxyolean-
12-ene-16R,23,28-triol. This compound has been assigned the trivial
name gordonoside A.
Compound 3 was obtained as a white, amorphous powder. The
positive-ion HRESIMS of 3 showed a quasimolecular ion peak at
m/z 985.5156 [M + Na]+, indicating a molecular formula of
C51H78O17 (calcd for C51H78O17Na, m/z 985.5137), one oxygen atom
less than that of 1. Comparison of the NMR data of 3 and 1 (see
Experimental Section and Tables 1 and 2) indicated that signals of
the oxymethylene group of 1 (CH2-23) were replaced by those of
a methyl group of 3 (δH 1.31, δC 28.1; CH3-23). These data
suggested that 3 is a 23-deoxygenated derivative of 1, which was
confirmed by 2D NMR experiments on 3. Therefore, compound 3
(gordonoside C) was determined as 3ꢀ-O-[R-L-arabinopyrano-
syl(1f3)-ꢀ-D-glucuronopyranosyl]-21ꢀ,22R-diangeloyloxyolean-
12-ene-16R,28-diol.
Compound 4 was obtained as a white, amorphous powder.
The positive-ion HRFABMS of 4 showed a quasimolecular ion
peak at m/z 1027.5258 [M + Na]+, indicating the molecular
formula to be C53H80O18 (calcd for C53H80O18Na, m/z 1027.5242).
The IR and NMR data of 4 resembled those of 3 (see
Experimental Section and Tables 1 and 2). However, the NMR
spectrum of 4 showed additional signals attributed to an acetyl
unit (δH 2.52, and δC 169.9 and 22.0) and a deshielded shift of
H-16 (∆δ 1.04 ppm) as compared to that of 3. These data
suggested that 4 is a 16-acetyl derivative of 3. Therefore,
compound 4 (gordonoside D) was elucidated as 3ꢀ-O-[R-L-
arabinopyranosyl(1f3)-ꢀ-D-glucuronopyranosyl]-16R-acetoxy-
21ꢀ,22R-diangeloyloxyolean-12-en-28-ol.
Compound 2 was obtained as a white, amorphous powder, and
its positive-ion HRESIMS gave a quasimolecular ion peak at m/z
1043.5206 [M + Na]+, which indicated the molecular formula to
be C53H80O19 (calcd for C53H80O19Na, m/z 1043.5192). The IR and
NMR spectroscopic features of 2 were similar to those of 1 (see
Experimental Section and Tables 1 and 2), except for additional
signals [δH 2.48(3H, s), δC 169.9 and 22.0] assignable to an acetyl
group in the NMR spectrum of 2. These data suggested that 2 is
an acetyl derivative of 1, which was confirmed by appropriate 2D
NMR experiments on 2. In the 1H NMR spectrum, H-16 was
deshielded by ∆δ 1.07 ppm as compared to 1, indicating that the
acetyl group is located at C-16 in 2. In the HMBC spectrum of 2,
a long-range correlation from H-16 to the carbonyl carbon of the
acetyl unit confirmed the location of the acetyl unit. In the NOESY
spectrum of 2, correlations between H-16 and H2-28 and H3-26
proved that the acetyloxy at C-16 possesses an R-orientation.
Therefore, compound 2 (gordonoside B) was determined as 3ꢀ-O-
[R-L-arabinopyranosyl(1f3)-ꢀ-D-glucuronopyranosyl]-16R-acetoxy-
21ꢀ,22R-diangeloyloxyolean-12-ene-23,28-diol.
Compound 5 was isolated as a white, amorphous powder. Its
molecular formula, C51H76O18 (calcd for C51H76O18Na, m/z 999.4929),

868 Journal of Natural Products, 2009, Vol. 72, No. 5
Table 2. 13C NMR Spectroscopic Data for Compounds 1-8a
Yu et al.
position
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
38.7
25.9
82.1
43.4
47.4
18.0
32.7
40.0
46.9
36.6
23.7
123.5b
142.6
41.5
34.7
68.6
47.9
40.0
47.0
36.2
78.6
73.5
64.1
13.4
16.1
16.8
27.4
63.5
29.3
20.1
167.5
128.8
136.9
15.6
20.8
168.1
128.8
137.0
15.6
20.6
105.6
74.5
85.7
71.3
77.5
172.1
105.6
72.6
74.2
69.1
67.0
38.6
26.1
81.9
43.5
47.2
17.8
32.7
40.0
46.9
36.5
23.7
125.1
140.9
41.1
30.7
72.0
46.9
39.4
47.0
36.0
78.0
72.4
64.0
13.6
16.1
16.7
27.0
63.3
29.4
19.7
167.7
128.5
138.1
15.9
20.9
167.2
128.3
138.4
15.7
20.8
106.0
74.7
85.6
71.4
77.5
172.3
105.8
72.8
74.4
69.2
67.1
169.9
22.0
38.8
26.7
89.2
39.6
55.7
18.4
33.1
40.1
46.9
36.8
23.9
123.5b
142.8
41.7
34.9
68.7
48.1
40.1
47.2
36.4
78.8
73.7
28.1
17.0
15.7
16.9
27.6
63.7
29.6
20.3
167.7
129.1
137.2
15.94
21.1
168.2
129.1
137.1
15.85
20.9
106.9
74.8
85.9
71.5
77.6
172.3
106.0
72.9
74.5
69.3
67.2
38.6
26.5
89.0
39.5
55.5
18.2
33.0
40.0
46.8
36.7
23.7
125.1
140.9
41.1
30.8
72.1
46.9
39.5
47.1
36.1
78.0
72.4
27.9
16.9
15.6
16.7
27.0
63.4
29.4
19.7
167.7
128.5
138.1
15.9
20.9
167.2
128.4
138.4
15.7
20.8
106.8
74.7
85.8
71.5
77.5
172.3
105.9
72.8
74.4
69.3
67.1
169.9
22.0
38.1
25.2
82.2
55.5
47.5
20.3
32.3
40.3
46.8
35.9
23.7
123.5b
142.8
41.7
34.7
68.5
48.0
40.0
47.1
36.4
78.7
73.5
206.6
10.3
15.7
16.7
27.5
63.5
29.5
20.3
167.7
128.9
137.2
15.9
21.0
168.1
128.9
137.1
15.8
20.8
104.9
74.4
85.4
71.3
77.5
172.1
105.8
72.8
74.4
69.2
67.0
38.5
26.0
85.7
53.6
52.3
21.1
32.7
40.3
47.1
36.5
23.7
123.5b
142.8
41.6
34.8
68.6
48.0
40.0
47.2
36.3
78.7
73.7
178.0
12.2
16.1
16.7
27.5
63.6
29.4
20.2
167.7
129.0
137.1
15.8
20.9
168.2
129.0
137.1
15.7
20.7
105.9
74.2
85.6
71.3
77.4
172.0
105.7
72.8
74.4
69.2
67.0
38.7
26.0
85.4
53.5
52.1
21.4
36.3
41.6
47.2
36.6
23.9
125.0
143.7
47.6
67.4
73.4
48.3
40.9
46.8
36.3
78.5
73.3
178.0
12.3
16.2
17.3
21.2
63.1
29.4
20.2
167.7
128.9
137.4
15.9
21.0
168.0
129.1
136.5
15.7
20.6
105.9
74.2
85.6
71.3
77.5
172.2
105.8
72.9
74.4
69.2
67.0
38.8
26.1
85.6
53.6
52.1
21.5
36.4
41.7
47.3
36.6
24.0
125.1
143.8
47.7
67.5
73.0
48.5
40.8
46.8
36.4
78.6
73.2
178.0
12.3
16.2
17.4
21.2
63.0
29.4
20.2
167.6
128.7
138.6
16.1
21.1
176.6
41.5
26.9
11.9
16.7
105.9
74.3
85.6
71.4
77.6
172.2
105.8
72.9
74.5
69.3
67.1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
2′
3′
4′
5′
1′′
2′′
3′′
4′′
5′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
Ac
OMe
52.1
52.1
52.2
a
13C NMR data (δ) were measured in C5D5N at 125 MHz for 1, 2, and 4-7, and at 150 MHz for 3 and 8. The assignments are based on DEPT,
1H-1H COSY, HSQC, and HMBC experiments. b Signal overlapped by solvent peaks.
was proposed by the positive-ion HRESIMS at m/z 999.4945 [M
+ Na]+. The IR and NMR data of 5 (see Experimental Section
and Tables 1 and 2) were similar to those of 1 except that the
resonances of the oxymethylene of 1 (CH2-23) were replaced by
signals due to an aldehyde unit in 5 (δH 9.74 and δC 206.6). These
spectroscopic data revealed that 5 is a C-23 aldehyde form of 1.
This was proved by 2D NMR experiments on 5, especially by
HMBC correlations from the aldehyde proton to both C-5 and C-24
and from H-3 to both C-1′′′ and the aldehyde carbonyl carbon. This
was further supported by a correlation between H3-24 (δ 1.30) and
H3-25 (δ 0.81) in the NOESY spectrum of 5. Therefore, compound
5 (gordonoside E) was determined as 3ꢀ-O-[R-L-arabinopyrano-
syl(1f3)-ꢀ-D-glucuronopyranosyl]-21ꢀ,22R-diangeloyloxy-23-
oxoolean-12-ene-16R,28-diol.
1029.5035), was assigned by HRFABMS at m/z 1029.5100 [M +
Na]+. The IR and NMR spectroscopic data of 6 were similar to
those of 1 (see Experimental Section, and Tables 1 and 2). However,
the resonances of the oxymethylene (C-23) of 1 were replaced by
resonances of a methoxycarbonyl of 6 (δH 3.81, and δC 178.0 and
52.1). Meanwhile, C-4 of 6 (δC 53.6) was significantly deshielded
as compared to that of 1. These spectroscopic data indicated that 6
is a methyl 23-oic acid ester form of 1.12 This was supported by
correlations from H-3, H-5, H3-24, and the methoxyl protons to
the carbonyl carbon (δC 178.0) in the HMBC spectrum of 6. It
was also confirmed by a NOESY experiment on 6 showing cross-
peaks between H-3 and H-5 and between H3-24 (δ 1.43) and H3-
25 (δ 0.83). Consequently, compound 6 (gordonoside F), was
determined as 3ꢀ-O-[R-L-arabinopyranosyl(1f3)-ꢀ-Dglucuronopy-
ranosyl]-21ꢀ,22R-diangeloyloxy-23-methoxycarbonylolean-12-ene-
16R,28-diol.
Compound 6 was obtained as a white, amorphous powder. Its
molecular formula, C52H78O19 (calcd for C52H78O19Na, m/z

Cytotoxic Triterpenoid Glycosides from Gordonia chrysandra
Journal of Natural Products, 2009, Vol. 72, No. 5 869
Bruker AVANCE DRX 500 spectrometer or a SYSTEM-600 FT
spectrometer, with solvent (pyridine-d5) peaks as references. HRESIMS
and HRFABMS were performed on an Autospec-Ultima ETOF mass
spectrometer. ESIMS data were obtained using an Agilent 1100 series
LC/MSD Trap SL mass spectrometer. Preparative HPLC was carried
out on a Shimadzu LC-6AD instrument with a SPD-10A detector, using
a YMC-Pack ODS-A column (250 × 20 mm, 5 µm). Column
chromatography was performed with macroporous resin D101 (26-60
mesh, Tianjin Haiguang Chemistry Company, Tianjin, People’s Re-
public of China), silica gel (200-300 mesh, Qingdao Marine Chemical
Inc., Qingdao, People’s Republic of China), ODS (50 µm; YMC, Kyoto,
Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala,
Sweden), respectively. TLC was carried out with glass precoated silica
gel GF254 plates. Spots were visualized under UV light or by spraying
with 10% sulfuric acid in EtOH followed by heating.
Plant Material. The roots of Gordonia chrysandra were collected
in Yunnan Province, People’s Republic of China, in May 2005. The
plant material was identified by Prof. Cui Jingyun (Xishuangbanna
Tropical Botanical Garden, Chinese Academy of Sciences). A voucher
specimen (No. 20050512) was deposited at the Institute of Materia
Medica, Chinese Academy of Medical Sciences, Beijing 100050,
People’s Republic of China.
Table 3. Evaluation of the Cytotoxic Potential of Compounds
1-7
cell line IC50 (µM)
compound HCT-8 Bel-7402 BGC-823
A-549
>10
1.8
A2780
1
3
5
6
>10
1.2
2.7
>10
3.6
4.7
0.7
1.1
1.3
6.3
>10
2.5
>10
>10
0.04
5.5
0.4
2.0
2.0
0.9
>10
>10
paclitaxelb
1.0 × 10-3
a Compounds 2, 4, and 7 were inactive against all cell lines tested
(IC50 >10 µM). b Positive control.
Compound 7 was obtained as a white, amorphous powder. Its
molecular formula, C52H78O20 (calcd for C52H78O20Na, m/z
1045.4984), was determined by HRFABMS at m/z 1045.4934 [M
+ Na]+. The IR and NMR spectroscopic features of 7 were almost
identical to those of 6. However, detailed comparison of the NMR
data of 7 and 6 (Experimental Section, and Tables 1 and 2) indicated
that signals of an oxymethine (CH-15) of 7 replaced those of the
methylene (CH2-15) of 6 and that H-16 of 7 was shielded by ∆δH
0.05 ppm. In turn, C-14 and C-16 of 7 were deshielded by ∆δC
6.00 and 4.80 ppm as compared to those of 6, respectively. These
spectroscopic data suggested that 7 is a derivative of 6 with an
additional hydroxy group at C-15. This was proved unambiguously
by 2D NMR experiments on 7. In the HMBC spectrum of 7,
correlations from both H3-27 and H-16 to C-15 (δC 67.4) confirmed
that the additional hydroxy group is located at C-15. In the NOESY
spectrum of 7, correlations between H-15 with H3-26 and H2-28,
together with correlations between H-16 and H2-28, were used to
show that both hydroxy groups at C-15 and C-16 have an
R-orientation.13 Therefore, compound 7 (gordonoside G) was
determined as 3ꢀ-O-[R-L-arabinopyranosyl(1f3)-ꢀ-D-glucuronopy-
ranosyl]-21ꢀ,22R-diangeloyloxy-23-methoxycarbonylolean-12-ene-
15R,16R,28-triol.
Compound 8 was obtained as a white, amorphous powder. Its
molecular formula, C52H80O20 (calcd for C52H80O20Na, m/z
1047.5141), as determined by HRESIMS at m/z 1047.5125 [M +
Na]+, showed two hydrogen atoms more than that of 7. The IR
and NMR spectroscopic data of 8 were similar to those of 7 (see
Experimental Section, and Tables 1 and 2). However, in the NMR
spectra of 8, signals ascribed to a 2-methylbutanoyl unit replaced
those of an angeloyl unit in 7. The NMR data of 8 were assigned
unambiguously by HSQC experiments. In the HMBC spectrum,
long-range correlations of C-1′′ with H-22, H-2′′, H-3′′, and H-5′′
revealed the 2-methylbutanoyl unit to be located at C-22. Thus,
compound 8 (gordonoside H) was determined as 3ꢀ-O-[R-L-
arabinopyranosyl-(1f3)-ꢀ-D-glucuronopyranosyl]-21ꢀ-angeloyloxy-
22R-(2-methylbutanoyloxy)-23-methoxycarbonylolean-12-ene-
15R,16R,28-triol.
Extraction and Isolation. The dried roots (6.4 kg) were extracted
with 95% EtOH in H2O, and then the residues were extracted with
50% EtOH in H2O. After removing the solvent, a brown residue (424
g) was obtained from the 50% EtOH extract. The residue was
partitioned between n-BuOH and H2O. The n-BuOH phase was
concentrated under reduced pressure to yield a n-BuOH-soluble portion
(160 g), which was fractionated by column chromatography over silica
gel, using a solvent system of CH3Cl-MeOH-H2O (7:3:0.5), to yield
12 fractions. The saponin-enriched fraction V (5.70 g) was subjected
to reversed-phase MPLC (YMC-ODS-A 50 µm, 500 mm × 50 mm,
flow rate 20.0 mL/min), eluting with a gradient of increasing methanol
(25%-100%) in H2O, to obtain 10 fractions (Fr. 1-10). Fr. 1 was
subjected to column chromatography over C18 silica gel eluting with
MeOH-H2O (7:3) to afford an amorphous powder, and reversed-phase
HPLC (YMC-ODS-A 5 µm, 250 mm × 20 mm, detection at 210 nm,
flow rate 10.0 mL/min) purification, using MeOH-H2O (4:3) containing
0.05% TFA as mobile phase, yielded compounds 3 (15 mg) and 4 (9
mg). Fr. 2 was separated by reversed-phase HPLC with CH3CN-H2O
(47:53) containing 0.05% TFA as mobile phase to afford compounds
1 (48 mg), 2 (12 mg), 5 (12 mg), and 6 (80 mg). Fr. 3 was subjected
to reversed-phase HPLC separation with CH3CN-H2O (4:6) containing
0.05% TFA as mobile phase, to yield compounds 7 (15 mg) and 8 (6
mg).
Gordonoside A (1): white, amorphous powder; [R]20D -3.5 (c 0.09,
MeOH); UV (MeOH) λmax (log ε) 203 (4.29), 254 (4.05) nm; IR νmax
3391, 2926, 1676, 1437, 1387, 1240, 1202, 1143, 1080, 1044 cm-1
;
1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz),
see Tables 1 and 2, respectively; positive-ion ESIMS m/z 1001 [M +
Na]+; negative-ion ESIMS m/z 977 [M - H]-; positive-ion HRESIMS
m/z 1001.5056 [M + Na]+ (calcd for C51H78O18Na, 1001.5086).
Gordonoside B (2): white, amorphous powder; [R]20D -3.5 (c 0.09,
MeOH); UV (MeOH) λmax (log ε) 210 (4.23), 254 (3.56) nm; IR νmax
3399, 2956, 2929, 1683, 1441, 1381, 1203, 1148, 1081, 1044 cm-1
;
The cytotoxic activities of compounds 1-7 (purity of each
compound >90%) were evaluated against several human cancer cell
lines (HCT-8, Bel-7402, BGC-823, A549, and A2780) with
paclitaxel as a positive control (see Table 3). Compound 3 exhibited
significant cytotoxicity for all the tested human cancer cell lines.
Compounds 1, 5, and 6 showed selective cytotoxicities for the Bel-
7402 and A-2780 cell lines. These results indicate that the free
hydroxy group at C-16 may play an important role in mediating
cytotoxicity. Acetylation of the hydroxy group at C-16 (2 and 4)
and the presence of a hydroxyl group at C-15 (7) decreased the
resultant cytotoxic activity.
1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz),
see Tables 1 and 2, respectively; positive-ion ESIMS m/z 1043 [M +
Na]+; negative-ion ESIMS m/z 1019 [M - H]-; positive-ion HRESIMS
m/z 1043.5206 [M + Na]+ (calcd for C53H80O19Na, 1043.5192).
Gordonoside C (3): amorphous, white powder; [R]20D -5.9 (c 0.12,
MeOH); UV (MeOH) λmax (log ε) 209 (4.34) nm; IR νmax 3397, 2950,
2922, 1700, 1649, 1455, 1384, 1357, 1243, 1164, 1081, 1044, 1021
1
cm-1; H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5,
150 MHz), see Tables 1 and 2, respectively; positive-ion ESIMS m/z
985 [M + Na]+; negative-ion ESIMS m/z 961 [M - H]-; positive-ion
HRESIMS m/z 985.5156 [M + Na]+ (calcd for C51H78O17Na, 985.5137).
Gordonoside D (4): amorphous, white powder; [R]20D -23.0 (c 0.09,
MeOH); UV (MeOH) λmax (log ε) 210 (4.30) nm; IR νmax 3413, 2952,
2924, 1743, 1717, 1646, 1455, 1378, 1236, 1151, 1081, 1043, 1025
Experimental Section
1
General Experimental Procedures. Optical rotations were measured
with a Perkin-Elmer 241 automatic digital polarimeter. UV spectra were
recorded on a Shimadzu UV-300 spectrophotometer. IR spectra were
recorded on a Nicolet 5700 FT-IR spectrometer by a microscope
transmission method. 1D and 2D NMR spectra were obtained at 500
cm-1; H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5,
125 MHz), see Tables 1 and 2, respectively; positive-ion HRFABMS
m/z 1027.5258 [M + Na]+ (calcd for C53H80O18Na, 1027.5242).
Gordonoside E (5): white, amorphous powder; [R]20D +1.2 (c 0.08,
MeOH); UV (MeOH) λmax (log ε) 209 (4.26), 254 (3.46) nm; IR νmax
1
and 125 MHz or 600 and 150 MHz for H and 13C, respectively, on a
3388, 2959, 2923, 1698, 1442, 1387, 1204, 1151, 1082, 1044 cm-1
;

870 Journal of Natural Products, 2009, Vol. 72, No. 5
Yu et al.
1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz),
see Tables 1 and 2, respectively; positive-ion ESIMS m/z 999 [M +
Na]+; negative-ion ESIMS m/z 975 [M - H]-; positive-ion HRESIMS
m/z 999.4945 [M + Na]+ (calcd for C51H76O18Na, 999.4929).
Gordonoside F (6): white, amorphous powder; [R]20D -5.9 (c 0.12,
MeOH); UV (MeOH) λmax (log ε) 210 (4.29), 254 (3.20) nm; IR νmax
3355, 2954, 2923, 1673, 1435, 1388, 1243, 1201, 1143, 1080, 1044
into control wells. The cells were treated with various concentrations
of the test compounds for 96 h, and then cell growth was evaluated by
an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
assay procedure. A 100 µL aliquot of 0.5 mg/mL MTT in RPMI 1640
was added to every well, and the plate was reincubated in 5% CO2 in
air for 4 h at 37 °C. The plate was then centrifuged to precipitate cells
and formazan. An aliquot of 150 µL of the supernatant was removed
from every well, and 150 µL of DMSO was added to dissolve the
formazan crystals. The plate was mixed on a microshaker for 10 min
and then read on a microplate reader at 570 nm. All compounds were
tested at five concentrations, and each concentration of the compounds
was tested in three parallel wells. A dose-response curve was plotted
for each compound, and the IC50 value was calculated as the
concentration of the test compound resulting in 50% reduction of optical
density compared with the control (see Table 3).
1
cm-1; H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5,
125 MHz), see Tables 1 and 2, respectively; positive-ion ESIMS m/z
1029 [M + Na]+; negative-ion ESIMS m/z 1005 [M - H]-; positive-
ion HRFABMS m/z 1029.5100 [M + Na]+ (calcd for C52H78O19Na,
1029.5035).
Gordonoside G (7): white, amorphous powder; [R]20D -1.0 (c 0.10,
MeOH); UV (MeOH) λmax (log ε) 204 (4.19) nm; IR νmax 3263, 2962,
1669, 1435, 1390, 1243, 1185, 1135, 1083, 1045 cm-1 1H NMR
;
(pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz), see
Tables 1 and 2, respectively; positive-ion ESIMS m/z 1045 [M + Na]+;
negative-ion ESIMS m/z 1021 [M - H]-; positive-ion HRFABMS m/z
1045.4934 [M + Na]+ (calcd for C52H78O20Na, 1045.4984).
Acknowledgment. The authors are grateful to the Department of
Instrumental Analysis at the Institute of Materia Medica, Chinese
Academy of Medical Sciences, for all spectroscopic measurements. We
also thank the Department of Pharmacology at the Institute of Materia
Medica, Chinese Academy of Medical Sciences, for the bioactivity tests.
Gordonoside H (8): white, amorphous powder; [R]20D -2.0 (c 0.05,
MeOH); UV (MeOH) λmax (log ε) 203 (4.30) nm; IR νmax 3405, 2961,
2931, 1710, 1680, 1436, 1388, 1243, 1196, 1147, 1078, 1044 cm-1
;
Supporting Information Available: MS and 1D and 2D NMR
spectra of compounds 1-8. This material is available free of charge
via the Internet at http://pubs.acs.org.
1H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz),
see Tables 1 and 2, respectively; positive-ion ESIMS m/z 1047 [M +
Na]+, negative-ion ESIMS m/z 1023 [M - H]-; positive-ion HRESIMS
m/z 1047.5125 [M + Na]+ (calcd for C52H80O20Na, 1047.5141).
Acid Hydrolysis of Compound 1 and Determination of Sugar
Configurations. A solution of compound 1 (25 mg) was hydrolyzed
with 5% aqueous H2SO4-1,4-dioxane (1:1, v/v, 2 mL) under reflux
for 4 h. On cooling, the reaction mixture was extracted with CHCl3 (3
× 1 mL) to yield a CHCl3 extract and a H2O phase.
References and Notes
(1) Institute of Botany, Chinese Academy of Sciences. Iconographia
Cormophytorum Sinicorum Supplementum II; Science Press: Beijing,
1983; p 468.
(2) Herath, H. M. T. B.; Athukoralage, P. S. Nat. Prod. Sci. 1998, 4,
253–256.
(3) Herath, H. M. T. B.; Athukoralage, P. S.; Jamie, J. F. ACGC Chem.
Res. Commun. 1999, 9, 3–8.
(4) Herath, H. M. T. B.; Athukoralage, P. S. Nat. Prod. Sci. 2000, 6,
102–105.
(5) Herath, H. M. T. B.; Athukoralage, P. S.; Jamie, J. F. Phytochemistry
2000, 54, 823–827.
(6) Herath, H. M. T. B.; Athukoralage, P. S.; Jamie, J. F. Nat. Prod. Lett.
2001, 15, 339–344.
The H2O phase of 1 was subjected to passage through an anion-
exchange resin column, eluting with H2O and then with 0.1 N aqueous
HCl. The H2O and aqueous HCl solutions were separately concentrated
to dryness. The residue from the H2O solution was subjected to normal-
phase preparative TLC with CHCl3-MeOH-H2O-AcOH (7:3:0.5:
0.5) as solvent system to yield arabinose (2.14 mg), with [R]23D +99.2
(c 0.21, H2O), and glucuronic acid (1.50 mg), with [R]23 +30.5 (c
D
0.19, H2O), respectively. L-Arabinose (Rf 0.40) and D-glucuronic acid
(Rf 0.12-0.25) were identified by comparison of [R]D and Rf values
with authentic sugar samples.
(7) Wang, C. C.; Chen, L. G.; Yang, L. L. Toxicology 2001, 168, 231–
240.
(8) Athukoralage, P. S.; Herath, H. M. T. B.; Deraniyagala, S. A.;
Wijesundera, R. L. C.; Weerasinghe, P. A. Fitoterapia 2001, 72, 565–
567.
(9) Wang, K.; Yang, J. Z.; Zuo, L.; Zhang, D. M. Chin. Chem. Lett. 2008,
1, 61–64.
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J. F.; Duchamp, O.; Lacaille-Dubois, M. A. J. Nat. Prod. 2007, 70,
1680–1682.
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(12) Morikawa, T.; Li, N.; Nagatomo, A.; Matsuda, H.; Li, X.; Yoshikawa,
M. J. Nat. Prod. 2006, 69, 185–190.
(13) Lu, Y.; Umeda, T.; Yagi, A.; Sakata, K.; Chaudhuri, T.; Ganguly,
D. K.; Sarma, S. Phytochemistry 2000, 53, 941–946.
Cells, Culture Conditions, and Cell Proliferation Assay. HCT-8
(human colon cancer cell line), Bel-7402 (human hepatoma cancer cell
line), BGC-823 (human gastric cancer cell line), A549 (human lung
cancer cell line), and A2780 (human ovarian cancer cell line) were
maintained in the RPMI 1640 medium containing 10% fetal bovine
serum supplemented with L-glutamine, 100 units/mL of penicillin, and
100 µg/mL of streptomycin. Cultures were incubated at 37 °C in 5%
CO2 in air.
HCT-8, Bel-7402, BGC-823, A549, and A2780 cells (1.5 × 103)
were seeded in 96-well tissue culture plates, and 100 µL of cell
suspension was placed in each well. After 24 h, 100 µL of DMSO
solution containing the test compounds was added to give final
concentrations of 0.01-10 µmol/mL; 100 µL of DMSO was added
NP900089V