Phytochemistry 87 (2013) 8695

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Phytochemistry
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Cytotoxic cardenolide glycosides from the root of Reevesia formosana

Hsun-Shuo Chang a, Michael Y. Chiang b, Hsing-Yu Hsu c, Cheng-Wei Yang c, Chu-Hung Lin d,
Shiow-Ju Lee c,, Ih-Sheng Chen a,d,
a

Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC
Department of Chemistry, National Sun Yat-sen University, Kaohsiung 804, Taiwan, ROC
c
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli 350, Taiwan, ROC
d
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC
b

a r t i c l e

i n f o

Article history:
Received 29 March 2012
Received in revised form 1 October 2012
Available online 11 January 2013
Keywords:
Reevesia formosana
Sterculiaceae
Root
Cardenolide glycosides
Cytotoxicity

a b s t r a c t
Bioassay-guided fractionation of the root tissue of Reevesia formosana led to isolation of 13 cardenolide
glycosides, reevesiosides AI and epi-reevesiosides FI. Their structures were determined by means of
spectroscopic analysis and single-crystal X-ray diffraction was performed using reevesioside A. Reevesioside A, reevesioside F, and epi-reevesioside F displayed especially potent cytotoxicity against the MCF-7
and NCI-H460 cancer cell lines, with IC50 values of 63 2 and 19 1, 72 8 and 20 0, and 34 6 and
10 1 nM, respectively. Identication of the sugar constituents and unusual 18,20-epoxide cardenolide
glycosides are described herein. Cardiac glycosides were previously unknown in the Sterculiaceae family.
2012 Elsevier Ltd. All rights reserved.

1. Introduction

2. Results and discussion

Reevesia formosana Sprague (Sterculiaceae) is an endemic

deciduous tree that grows in southern Taiwan (Li and Lo, 1993).
There are about 25 species of Reevesia worldwide, with two species in Central America, 14 in mainland China, and one in Taiwan
(Tang et al., 2007). The remainder are found mostly in Southeast
Asia. The chemical constituents and biological activities of the
genus Reevesia have not been studied previously, except for a report of ve known compounds, b-sitosterol, daucosterol, betulinic
acid, lupeol, and (+)-catechin, isolated from Reevesia longipetiolata
(Zhu et al., 2003). Over 1300 species of Formosan plants have
been screened in our laboratory for cytotoxicity against MCF-7,
NCI-H460, SF-268 or HepG2 cancer cell lines in vitro, and the R.
formosana extract was found to be one of the most bioactive species. Bioassay-guided fractionation of the active EtOAc-soluble
fraction of the root of this species led to isolation and characterization of 13 new cardiac glycosides, reevesiosides AI (16, 8, 10,
12) and epi-reevesiosides FI (7, 9, 11, 13). The structure elucidation of 113 (Fig. 1) and their cytotoxic evaluation are described
herein.

Compound 1 was obtained as optically active colorless prisms

with a26
28.7 (c 0.80, MeOH). Its molecular formula was
D
determined as C30H42O9 by ESIMS (m/z 569 [M+Na]+) and HRESIMS. The IR spectrum indicated the presence of a hydroxy group
(3518 cm1), an a,b-unsaturated c-lactone ring (1780, 1744, and
1621 cm1), and an aldehyde group (1715 cm1) (Jones and
Gallagher, 1959). This substance was suggested to be a cardenolide
possessing a sugar moiety according to analysis of its MS and IR,
1
H, and 13C NMR spectra. The 1H (Table 1) and 13C (Table 2) NMR
spectra of 1 showed signals characteristic of an a,b-unsaturated
c-lactone ring at d 4.79 (1H, dd, J = 18.0, 1.6 Hz, H-21b), 4.95 (1H,
dd, J = 18.0, 1.6 Hz, H-21a), and 5.86 (1H, s, H-22), an aldehyde at
d 10.0 (1H, s, H-19), a hydroxy group at d 4.25 (1H, s, OH-5), a
methyl group at d 0.85 (3H, s, H-18), and two oxygen-bearing quartenary carbons (d 73.4, C-5; 85.1, C-14), indicating the aglycone of
1 to be similar to those of 3-O-substituted strophanthidins (Pauli
et al., 1993). The identity of aglycone 1 was conrmed by correlations in the HMBC spectrum (Fig. 2) between H-19 and C-10 (d
54.6); OH-5 and C-4 (d 34.5), C-5 (d 73.4), and C-6 (d 36.3); H-18
and C-12 (d 39.7), C-13 (d 49.4), C-14 (d 85.1), and C-17 (50.4);
and H-17 [d 2.75 (1H, dd, J = 9.8, 5.4 Hz)] and C-13, C-14, C-16 (d
26.8), C-20 (d 174.5), C-21 (d 73.3), and C-22 (d 117.7). As
determined by the NOESY spectrum (Fig. 3), the H-19 showed correlations with OH-5 and H-8 (d 1.91, m) while H-18 showed correlations with H-8 and H-22, conrming the relative conguration of

Corresponding authors. Tel.: +886 37 246166x35715; fax: +886 37 586456 (S.-J.

Lee), tel.: +886 7 3121101x2191; fax: +886 7 3210683 (I.-S. Chen).
E-mail addresses: slee@nhri.org.tw (S.-J. Lee), m635013@kmu.edu.tw (I.-S.
Chen).
0031-9422/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.11.024

87

H.-S. Chang et al. / Phytochemistry 87 (2013) 8695

R1
O

23
22
18

19 R1
10
3

R 4O

3'

OH

CHO

OH

COOH

OH

OH

OH

CH3

CH3

1'

6'

6'
4'

2'

3'

b=

OCOCH3

R2

5'

4'

2'

OH

25

CHO

6'

R3

5'

R4

21

OH

6'
4'

a=

17
16

14

24

R3

20

13

CHO

R2

HO
HO
c=

1'

5'
3'

2'

1'

d=

7'

5'
4'

3'

OH

OCH3

OCH3

OH

7'

HO

2'

1'

OCH3

7'

7'

R
O
23

22

O
18

19

6'

10
3

4'

2'

3'

1'

CHO

(20S)

CHO

(20R)

10

COOH

(20S)

11

COOH

(20R)

12

OH

(20S)

13

OH

(20R)

21
20

13

17

14

OH

OH

7'

Fig. 1. Structures of compounds 113.

the aglycone. Thus, the aglycone of 1 was identied as strophanthidin, which was conrmed by COSY, NOESY (Fig. 3), HSQC, and
HMBC (Fig. 2) experiments, and by comparison to literature values
(Pauli et al., 1993). An anomeric proton at d 4.46 (1H, d, J = 6.8 Hz,
H-10 ), a methyl group as a doublet at d 1.22 (3H, d, J = 6.4 Hz, H-60 ),
a methylenedioxy at d 4.88 (1H, s, H-70 b) and 5.21 (1H, s, H-70 a),
three oxymethine protons at d 3.79 (1H, dd, J = 6.8, 5.4 Hz, H-20 ),
3.79 (1H, m, H-50 ), and 4.13 (1H, ddd, J = 5.4, 4.0, 1.8 Hz, H-30 ),
and a methylene group at d 1.76 (1H, m, H-40 b) and 2.12 (1H, m,
H-40 a) in the 1H NMR spectrum suggested that 1 contains a 4,6dideoxypyranosyl sugar moiety. The relative conguration of the
latter sugar moiety was determined from analysis of the NOESY
(Fig. 3) spectrum, where H-10 (d 4.46, d, J = 6.8 Hz) showed a
correlation with H-50 , and H-20 showed a correlation with H-40 a,
conrming that H-10 , H-20 , H-40 a, and H-50 are all axial, suggesting
a b-conguration of the 4,6-dideoxypyranosyl group. Therefore, it
was identied as a rare sugar moiety, namely a 4,6-dideoxy-2,3methylenedioxy-b-allopyranosyl unit. The HMBC spectrum
(Fig. 2) showed correlations between H-10 and C-3 (d 73.0), thus
establishing that it is connected to C-3 of 1. The a-orientation of
the proton at C-3 was supported by the appearance of the H-3 (d
4.18) signal as a broad singlet. The absolute conguration was
established on the basis of the nal renement on the Cu Ka data
resulted in a Flack parameter = 0.12(12) (Flack, 1983). Single

crystal X-ray diffraction of 1 was showed a conguration

consistent with the structure proposed above (Fig. 4). The absolute
conguration is consistent with that of the strophanthidin moiety
in the literature (Crowfoot, 1935). The R-conguration of C-50 of
the 4,6-dideoxypyranosyl group suggested the glycone had a
D-conguration. On the basis of the above results, structure 1
was elucidated as (3S,5S,8R,9S,10S,13R,14S,17R,10 R,20 S,30 S,50 R)strophanthidin-4,6-dideoxy-2,3-methylenedioxy-b-D-allopyranoside and named reevesioside A.
Compound 2 was obtained as an optically active colorless syrup
with a26
D 18.7 (c 0.07, MeOH). The ESIMS and HRESIMS were
used to establish the molecular formula of compound 2 as
C32H44O11. This suggested the presence of an acetoxy group in 2
replacing a proton in 1. The IR, 1H (Table 1), and 13C (Table 2)
NMR spectra of 2 were similar to those of 1, except for the presence
of an acetoxy group [dH 1.98 (3H, s, H-25), dC 21.1 (C-25), 170.5
(C-24)] and an oxymethine [dH 5.45 (1H, ddd, J = 9.6, 8.6, 2.7 Hz,
H-16)] rather than a methylene [dH 1.86 (1H, m, H-16b), 2.14
(1H, m, H-16a) ] in 1. Based on the HMBC spectrum, H-16 showed
a correlation with the carbonyl at C-24, suggesting the presence of
an acetoxy group at C-16. The relative conguration of OAc-16 was
assigned as b due to the correlation between H-25 and H-21, with
there being no correlation between H-25 and H-17 in the NOESY
spectrum. For a b-acetoxy group substituted at C-16 of this

88

H.-S. Chang et al. / Phytochemistry 87 (2013) 8695

Table 1
1
H NMR spectroscopic data for compounds 15.a
Position

1a/b
2a/b
3
4a/b
6a/b
7a/b
8
9
11a/b
12a/b
15a/b

2.22,
1.91,
4.18,
1.97,
2.06,
2.08,
1.91,
1.51,
1.50,
1.51,
2.00,

16a/b
17
18
19
21a/b

2.14, m/1.86, m
2.75, dd (9.8, 5.4)
0.85, s
10.0, s
4.95, dd (18.0, 1.6)/4.79, dd
(18.0, 1.6)
5.86, s

22
25
10
20
30
40 a/b
50
60
70 a/b
OH-5b

4.46,
3.79,
4.13,
2.12,
3.79,
1.22,
5.21,
4.25,

m/1.69,
m/1.51,
br s
m/1.68,
m/1.68,
m/1.23,
m
m
m/1.30,
m/1.32,
m/1.66,

m
m
m
m
m

m
m
m

d (6.8)
dd (6.8, 5.4)
ddd (5.4, 4.0, 1.8)
m/1.76, m
m
d (6.4)
s/4.88, s
s

2.30, m/1.71, m
1.98, m/1.52, m
4.21, br t (2.7)
1.96, m/1.72, m
2.04, m/1.71, m
2.08, m/1.18, m
1.97, m
1.43, m
1.51, m/1.38, m
1.57, m/1.22, m
2.63, dd (15.9, 9.6)/1.76, dd
(15.9,2.7)
5.45, ddd (9.6, 8.6, 2.7)
3.18, d (8.6)
0.94, s
10.0, d (0.8)
4.95, dd (18.3, 1.8)/4.85, dd
(18.3, 1.8)
6.00, s
1.98, s
4.47, d (6.9)
3.82, dd (6.9, 5.4)
4.14, ddd (5.4, 4.2, 1.8)
2.15, m/1.72, m
3.83, m
1.25, d (6.0)
5.23, s/4.90, s
4.30, s

2.23,
1.93,
4.22,
1.94,
2.07,
2.09,
1.92,
1.52,
1.54,
1.52,
2.01,

4
m/1.72,
m/1.54,
br s
m/1.72,
m/1.72,
m/1.25,
m
m
m/1.31,
m/1.33,
m/1.68,

m
m
m
m
m

m
m
m

2.17, m/1.85, m
2.75, dd (9.6, 5.2)
0.86, s
10.05, s
4.95, dd (18.2, 1.6)/4.79, dd
(18.2, 1.6)
5.88, s
4.74,
3.01,
4.27,
1.92,
3.98,
1.19,
3.43,
4.49,

d (7.9)
dd (7.9, 3.0)
m
m/1.49, m
m
d (6.0)
s
s

2.36,
1.94,
4.25,
2.08,
1.77,
2.04,
1.98,
1.52,
2.26,
1.52,
1.93,

5
m/1.46,
m/1.59,
br s
m/1.79,
m/1.69,
m/1.15,
m
m
m/1.86,
m/1.35,
m/1.72,

m
m
m
m
m

m
m
m

1.89,
1.81,
4.17,
1.88,
1.86,
1.87,
1.81,
1.43,
1.58,
1.56,
2.00,

m/1.49, m
m/1.74, m
br t, (2.7)
m/1.45, m
m/1.45, m
m/1.06, m
m
m
m
m/1.43, m
m/1.68, m

2.12, m/1.88, m
2.76, dd (9.2, 4.8)
0.97, s

2.15, m/1.86, m
2.77, dd (9.6, 5.4)
0.92, s

4.98, dd (18.2, 1.4)/4.81, dd

(18.2, 1.4)
5.88, s

4.96, dd (18.0, 1.5)/4.81, dd

(18.0, 1.5)
5.88, s

4.73,
3.03,
4.30,
1.95,
3.99,
1.19,
3.44,

4.74,
3.02,
4.28,
1.93,
3.99,
1.20,
3.47,
4.39,

d (7.9)
dd (7.9, 3.0)
m
m/1.49, m
m
d (6.0)
s

d (8.0)
dd (8.0, 3.3)
m
m/1.47, m
m
d (6.6)
s
s

a 1
b

H NMR data (d) were measured in CDCl3 at 400 MHz for 1, 3, and 4, at 600 MHz for 2 and 5.
D2O exchangeable.

Table 2
13
C NMR spectroscopic data for compounds 17.a
Position

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
10
20
30
40
50
60
70

17.8
25.3
73.0
34.5
73.4
36.3
24.1
41.6
39.3
54.6
21.9
39.7
49.4
85.1
31.9
26.8
50.4
15.5
208.2
174.5b
73.3
117.7
174.3b

18.0
25.3
73.1
34.4
73.2
36.3
23.7
41.5
39.1
54.3
21.4
39.0
49.8
84.0
40.1
73.7
55.6
15.8
208.1
167.2
75.5
121.6
173.9
170.5
21.1
99.3
73.8
74.6
34.3
67.1
20.9
95.4

17.9
25.4
72.0
34.0
73.1
36.5
24.2
41.7
39.3
54.6
21.9
39.7
49.4
85.2
32.0
26.8
50.4
15.6
208.3
174.4b
73.4
117.9
174.1b

21.3
25.7
72.2
33.2
74.6
36.5
23.9
40.7
39.4
53.3
21.8
40.0
49.8
85.3
32.3
26.8
50.4
15.7
176.6
174.7b
73.6
117.6
174.6b

174.44c
73.4
117.7
174.38c

96.5
80.7
64.6
38.1
66.5
20.6
57.6

96.5
80.5
64.3
38.1
66.7
20.6
57.3

96.5
80.7
64.6
38.1
66.5
20.6
57.6

99.1
73.7
74.5
34.3
67.0
20.9
95.4

5
28.6
26.1
72.4
34.3
73.71b
34.5
23.5
40.5
39.8
73.74b
21.0
40.0
49.5
85.2
32.6
26.9
50.6
15.7

30.3
26.6
73.2
29.5
36.4
26.6
21.4
41.8
35.8
35.2
21.1
40.0
49.6
85.6
33.1
26.9
50.9
15.7
23.8
174.54b
73.4
117.7
174.53b

30.2
26.6
73.1
29.5
36.3
26.6
21.3
41.7
35.7
35.1
21.1
39.9
49.6
85.5
33.0
26.8
50.8
15.7
23.8
174.8b
73.5
117.5
174.7b

100.6
83.3
76.3
75.2
71.4
17.6
60.8

97.8
80.2
69.9
72.8
69.5
17.7
59.4

a 13
C NMR data (d) were measured in CDCl3 at 100 MHz for 1, 3, 4, 6, and 7, at
150 MHz for 2 and 5.
b,c
Interchangeable within the same column.

cardenolide, H-17 would be present with a large coupling constant

at about 8 Hz. On the other hand, an a-acetoxy group would result
in a smaller coupling constant of about 4 Hz (Humber et al., 1985).
The H-17 proton of 2 exhibited a large coupling constant at 8.6 Hz,
indicative of a 16b-acetoxy group. The 1H (Table 1), and 13C
(Table 2) NMR spectra of the sugar moiety of 2 were the same as
1. Hence, the glycone, 4,6-dideoxy-2,3-methylenedioxy-b-D-allopyranosyl, was identied, and was connected to C-3 of 2 as
evidenced by the HMBC spectrum showed a correlation between
H-10 (dH 4.47) and C-3 (dC 73.1). According to the above data, the
structure of 2 (reevesioside B) was elucidated as 16b-acetoxystrophanthidin-4,6-dideoxy-2,3-methylenedioxy-b-D-allopyranoside.
Analysis of the ESIMS and HRESIMS of 3 indicated a molecular
formula of C30H44O9, representing nine degrees of unsaturation,
one less than compound 1. The aglycone of 3 was identied as strophanthidin, the same as that of 1, from analysis of the 1H and 13C
NMR spectra, including COSY, NOESY, HSQC, and HMBC experiments. The major differences observed between 1 and 3 were in
the signals of the sugar moiety. The 1H NMR spectrum (Table 1)
of the 4,6-dideoxypyranosyl group of 3 showed resonances characteristic of an anomeric proton at d 4.74 (1H, d, J = 7.9 Hz, H-10 ), a
methyl group as a doublet at d 1.19 (3H, d, J = 6.0 Hz, H-60 ), three
oxymethine protons at d 3.01 (1H, dd, J = 7.9, 3.0 Hz, H-20 ), 3.98
(1H, m, H-50 ), and 4.27 (1H, m, H-30 ), a methoxy group at d 3.43
(3H, s, H-70 ), and a methylene group at d 1.49 (1H, m, H-40 b) and
1.92 (1H, m, H-40 a). The NOESY spectrum (Fig. 3) showed correlations between H-10 (d 4.74, d, J = 7.9 Hz) and H-50 and between
H-20 and H-40 a, with no correlation observed between H-30 and
H-10 , suggesting the 4,6-dideoxypyranosyl sugar of 3 was assigned
to be b as discussed for compound 1. This sugar unit corresponded
to the 4-deoxy derivative of javose (6-deoxy-2-O-methyl-D-allose)
synthesized by Brimacombe and Husain (1967). The D-conguration of the sugar unit could be supported by the isolation of 2-Omethyl-6-deoxy-b-methyl-D-allopyranoside, which had chemically

H.-S. Chang et al. / Phytochemistry 87 (2013) 8695

13

CHO
H
H

H
O

OH

O
OH
H

H
H

HO
HO

H
O

OH

OCH3

6
O

CHO
H
H

H
O

H
O

OH

O
OH

8
Fig. 2. Key HMBC (

) correlations of 1, 6 and 8.

been correlated to javose (Hoffmann et al., 1966), following acid

hydrolysis of compound 7 as discussed later. The a-orientation of
the proton at C-3 was supported by the appearance of the H-3 (d
4.22) signal as a broad singlet. As determined by the above observations, the structure of reevesioside C (3) was assigned as strophanthidin-4,6-dideoxy-2-O-methyl-b-D-allopyranoside.
Compound 4 was obtained as optically active colorless needles
with a26
14.1 (c 0.12, MeOH). Its molecular formula of
D
C30H44O10, with one oxygen more than 3, was established by ESIMS
and HRESIMS. The 1H and 13C NMR spectra were similar to those of
3, except that an aldehyde group [dH 10.05 (1H, s, H-19); dC 208.3
(C-19)] of 3 was replaced by a carboxylic acid group [dC 176.6 (C19)] in 4. This observation was also supported by IR absorptions
at 1730 (C@O) and 25003300 (OH) cm1. Similarly, the 1H and

89

C NMR spectra of 5 showed the presence of a cardenolide similar

to that of 3, except for a hydroxy group in C-10 replacing an aldehyde group, resulting in the C-10 signal at dC 54.6 in 3 being shifted
to dC 73.74 in 5. According to the above evidence, the structures of
4 and 5 were elucidated and named reevesioside D and reevesioside E, respectively.
Compound 6 was obtained as optically active colorless needles
with a26
D 17.6 (c 0.08, MeOH). The ESIMS analysis of 6 showed
the [M+Na]+ ion at m/z 557, in agreement with the molecular formula of C30H46O8, as conrmed by HRESIMS. The 13C NMR (Table 2)
and DEPT spectra indicated that 6 contains four methyls, 10 methylenes, 11 methines, and ve quaternary carbons. The 1H NMR
spectrum (Table 3) of 6 established the presence of a,b-unsaturated c-lactone signals at d 4.81 (1H, dd, J = 18.1, 1.7 Hz, H-21b),
4.99 (1H, dd, J = 18.1, 1.7 Hz, H-21a), and 5.88 (1H, s, H-22). This
observation was also supported by IR absorptions at 1779, 1742,
and 1622 (a,b-unsaturated c-lactone ring) cm1. Furthermore,
two methyl groups at 0.87 (3H, s, H-18), 0.93 (3H, s, H-19) and
an O-bearing quaternary carbon (dc 85.6, C-14) indicated that 6 is
a cardenolide with C-18 and C-19 methyl groups. Thus, the aglycone of 6 was identied as digitoxigenin, which was conrmed
by COSY, NOESY (Fig. 3), HSQC, and HMBC (Fig. 2) experiments
and by comparison to data in literature values (Beale et al.,
1988). A 6-deoxypyranosyl group as indicated by the 1H NMR spectrum of 6 showed signals of an anomeric proton at d 4.34 (1H, d,
J = 7.7 Hz, H-10 ), a methyl group as a doublet at d 1.31 (3H, d,
J = 6.2 Hz, H-60 ), four oxymethine protons at d 2.97 (1H, dd,
J = 9.3, 7.7 Hz, H-20 ), 3.26 (1H, dd, J = 9.3, 8.6 Hz, H-40 ), 3.30 (1H,
dq, J = 9.3, 6.2 Hz, H-50 ), and 3.42 (1H, dd, J = 9.3, 8.6 Hz, H-30 ),
and a methoxy group at d 3.62 (3H, s, H-70 ). The relative conguration of the 6-deoxypyranosyl group of 6 was determined from
analysis of the NOESY (Fig. 3) spectrum, where H-10 (d 4.34, d,
J = 7.7 Hz) showed correlations with H-30 and H-50 ; H-30 showed
a correlation with H-50 , conrming that H-10 , H-30 , and H-50 are
all in an axial position, suggesting a b-conguration of the 6deoxypyranosyl group. Furthermore, H-20 showed a correlation
with H-40 but no correlation with H-10 , suggesting that H-20 and
H-40 are also axial. Evidence for the axial positions of H-10 H-50
was also supported by coupling constants of between 7.7 and
9.3 Hz. Therefore, the sugar moiety in 6 was identied as 6deoxy-2-O-methyl-b-glucopyranosyl, a sugar unit in toxicarioside
B (Carter et al., 1997). The D-conguration of the sugar unit of 6
was deduced by consideration of the literature (Rathore et al.,
1985, 1986). In the literature, these researchers synthesized enantiomeric sugars and connected them to the same aglycone, digitoxigenin.
The
diastereomeric
digitoxigenin-b-glycosides
synthesized displayed opposite [a]D value. In our case, a series of
cardenolide glycosides showed similar negative optical rotatory
powers around 10.3 to 28.7. Hence, the sugar unit of 6 was assigned to be in a b-D-conguration. The HMBC spectrum (Fig. 2)
showed correlations between H-10 and C-3 (d 73.2), thus establishing that the 6-deoxy-2-O-methyl-b-D-glucopyranosyl group is connected at C-3 of the aglycone. Evidence for the a-orientation of the
proton at C-3 of digitoxigenin was supported by the broad singlet
signal of H-3 (d 4.08). Accordingly, the structure of 6 (reevesioside
F) was suggested as digitoxigenin-6-deoxy-2-O-methyl-b-Dglucoside.
Compound 7 was obtained as optically active colorless needles,
with a26
D 16.9 (c 1.20, MeOH). HRESIMS analysis gave a quasimolecular ion peak at m/z 557 [M+Na]+ (calcd. for C30H46O8Na),
consistent with a molecular formula of C30H46O8, the same as 6.
The 1H and 13C NMR spectra of 7 were similar to those of 6, except
for the signals of the 6-deoxypyranosyl group. This functionality
showed 1H NMR resonances for an anomeric proton at d 4.64
(1H, d, J = 7.8 Hz, H-10 ), a methyl group at d 1.25 (3H, d,
J = 6.4 Hz, H-60 ), four oxymethine protons at d 3.02 (1H, dd,

90

H.-S. Chang et al. / Phytochemistry 87 (2013) 8695

Fig. 3. Key NOESY (

) correlations of 1, 3 and 69.

Fig. 4. ORTEP drawing of 1 as determined by X-ray analysis.

91

H.-S. Chang et al. / Phytochemistry 87 (2013) 8695

Table 3
1
H NMR spectroscopic data for compounds 69.a
Position

1a/b
2a/b
3
4a/b
5
6a/b
7a/b
8
9
11a/b
12a/b
15a/b
16a/b
17
18a/b

1.50,
1.69,
4.08,
1.70,
1.72,
1.88,
1.69,
1.56,
1.61,
1.44,
1.52,
2.13,
2.16,
2.78,
0.87,

19
21a/b
22a/b

0.93, s
4.99, dd (18.1, 1.7)/4.81, dd (18.1, 1.7)
5.88, s

0.91, s
4.98, dd (18.0, 1.6)/4.79, dd (18.0, 1.6)
5.85, s

10.07, s
4.33, dd (9.8, 1.5)/3.98, d (9.8)
2.65, d (17.4)/2.59, dd (17.4, 1.2)

10
20
30
40 a/b
50
60
70 a/b
OH-30 b
OH-40 b
OH-5b
OH-14b

4.34,
2.97,
3.42,
3.26,
3.30,
1.31,
3.62,

4.64,
3.02,
4.18,
3.23,
3.61,
1.25,
3.54,
2.90,
2.62,

4.47,
3.79,
4.13,
2.13,
3.81,
1.23,
5.22,

m
m/1.52, m
br s
m/1.23, m
m
m/1.28, m
m/1.26, m
m
m
m/1.26, m
m/1.39, m
m/1.69, m
m/1.88, m
dd (8.8, 5.2)
s

d (7.7)
dd (9.3,
dd (9.3,
dd (9.3,
dq (9.3,
d (6.2)
s

7.7)
8.6)
8.6)
6.2)

1.48,
1.66,
4.03,
1.69,
1.69,
1.84,
1.67,
1.53,
1.59,
1.38,
1.51,
2.11,
2.14,
2.76,
0.85,

8
m
m/1.48, m
br s
m/1.23, m
m
m/1.26, m
m/1.18, m
m
m
m/1.18, m
m/1.40, m
m/1.68, m
m/1.84, m
dd (9.2, 4.8)
s

d (7.8)
dd (7.8, 3.0)
br t (3.0)
ddd (9.6, 9.2, 3.0)
dq (9.6, 6.4)
d (6.4)
s
s
br d (9.2)

2.18,
1.93,
4.18,
1.96,

m/1.70, m
m/1.49, m
br t (2.4)
m/1.71, m

2.20,
1.94,
4.18,
1.96,

2.18,
2.24,
1.90,
1.41,
1.64,
1.72,
1.86,
2.01,
2.12,
4.15,

m/1.72, m
m/1.28, m
m
m
m/0.86, m
m/1.46, m
m/1.71, m
m/1.71, m
m
d (10.2)/3.42, d (10.2)

2.17, m/1.74, m
2.21, m/1.27, m
1.92, m
1.41, m
1.65, m/0.88, m
1.74, m/1.45, m
1.82, m/1.73, m
1.94, m/1.62, m
2.30, dd (9.4, 7.4)
4.16 d (10.2)
3.37 d (10.2)
10.07 s
4.35 s
2.73 d (17.2)
2.43 d (17.2)
4.47 d (6.7)
3.80dd (6.7 5.4)
4.13 m
2.13 m/1.76 m
3.80 m
1.23 d (6.4)
5.22 s/4.89 s

d (7.1)
dd (7.1, 5.7)
m
m/1.78, m
m
d (6.0)
s/4.89, s

4.24, s
2.70, br s

m/1.72, m
m/1.49, m
br s
m/1.73, m

4.24, s
2.71 br s

a 1
b

H NMR data (d) were measured in CDCl3 at 400 MHz for 6, 7, and 9, at 600 MHz for 8.
D2O exchangeable.

J = 7.8, 3.0 Hz, H-20 ), 3.23 (1H, ddd, J = 9.6, 9.2, 3.0 Hz, H-40 ), 3.61
(1H, dq, J = 9.6, 6.4 Hz, H-50 ), and 4.18 (1H, br t, J = 3.0 Hz, H-30 ), a
methoxy group at d 3.54 (3H, s, H-70 ), and two hydroxy groups at
d 2.62 (1H, br d, J = 9.2 Hz, OH-40 ) and 2.90 (1H, s, OH-30 ). The
NOESY spectrum (Fig. 3) showed correlations with H-10 , OH-30 ,
and H-50 ; H-20 and H-40 ; H-30 and H-40 , but no correlation between
H-30 and H-10 , conrming that H-10 , H-20 , H-40 a, and H-50 are all axial and H-30 (br t, 3.0 Hz) is equatorial. The 6-deoxypyranosyl group
of 7 was assigned as b in accordance with the NOESY correlations
and an observed doublet (J = 7.8 Hz) of H-10 (d 4.64) in the 1H
NMR spectrum. Acid hydrolysis of compound 7 in methanol
according to a previous study (Dai et al., 2009) afforded 2-Omethyl-6-deoxy-b-methyl-D-allopyranoside. The methylated sugar
had been correlated to javose by treatment with 1 N H2SO4 (Hoffmann et al., 1966). Hence, the 6-deoxypyranosyl group of 7 was
identied as javose, which had been synthesized by Brimacombe
and Husain (1967). The HMBC spectrum showed correlations between H-10 and C-3 (d 73.1), thus establishing that the 6-deoxy2-O-methyl-b-D-allopyranosyl group is connected to C-3 of the
aglycone. The a-orientation of the proton at C-3 of digitoxigenin
was supported by the H-3 (d 4.03, br s). Therefore, 7 is an epimer
of 6. As determined by the above observations, the structure of
epi-reevesioside F (7) was recommended as digitoxigenin-6deoxy-2-O-methyl-b-D-allopyranoside.
The mixture of 8 and 9 were puried by preparative RP-18 TLC
with the solvent system CH2Cl2acetone (5:1), this being repeated
ve times to yield pure 8 (Rf 0.56) and 9 (Rf 0.58), respectively. The
same molecular formula C30H42O10 of 8 and 9 was established by
the [M+Na]+ ion peak at m/z 585.2673 and 585.2678 in the HRESIMS, respectively. The 1H and 13C NMR spectra of 8 and 9 were
similar to those of 1, but the olenic proton signal [d 5.86 (1H,
s)] at C-22 was absent. Instead, characteristic resonances of three

methylene groups were evident at C-18, C-21, and C-22, suggesting

that 8 and 9 are 18,20-epoxides, as previously reported as being
observed in Apocynum cannabinum (Abe and Yamauchi, 1994).
Upon comparing the IR spectrum of 8 and 9 with that of 1, the substitution of the absorption at 1744 cm1 by that at 1779 cm1 suggested that the a,b-unsaturated c-lactone ring was replaced by a
saturated c-lactone in 8 and 9. The 1H NMR spectra of 8 and 9 (Table 3) were alike, except for three methylene groups at d 3.42 (1H,
d, J = 10.2 Hz, H-18b), 4.15 (1H, d, J = 10.2 Hz, H-18a), 3.98 (1H, d,
J = 9.8 Hz, H-21b), 4.33 (1H, dd, J = 9.8, 1.5 Hz, H-21a), 2.59 (1H,
dd, J = 17.4, 1.2 Hz, H-22b), and 2.65 (1H, d, J = 17.4 Hz, H-22a) in
8, and d 3.37 (1H, d, J = 10.2 Hz, H-18b), 4.16 (1H, d, J = 10.2 Hz,
H-18a), 4.35 (2H, s, H-21), 2.43 (1H, d, J = 17.2 Hz, H-22b), and
2.73 (1H, d, J = 17.2 Hz, H-22a) in 9. The NOESY spectrum (Fig. 3)
of 8 showed correlations between: H-21a and H-18b; H-21b and
H-17; H-22b and H-16b, suggesting that the absolute conguration
of C-20 of 8 is S (Abe et al., 1992). On the other hand, C-20 of 9 was
determined as R from the NOESY spectrum (Fig. 3), showing
correlations between: H-22a and H-18b; H-22b and H-17; H-21
and H-16b, respectively. Signicant differences of the 13C NMR
data between 8 and 9 were found for C-16, C-17, C-20, C-21, and
C-22. The signals at dC 54.9 (C-17) and 37.2 (C-22) in 8 were shifted
downeld to dC 57.1 (C-17) and 40.8 (C-22) in 9, whereas dC 25.4
(C-16), 88.6 (C-20), and 75.6 (C-21) in 8 were shifted upeld to
dC 23.8 (C-16), 87.0 (C-20), and 74.1 (C-21) in 9. This observation
coincides with those of a previous study (Abe et al., 1992). The
assignments of the 1H and 13C NMR spectroscopic data of 8 and
9, as displayed in Tables 3 and 4, were conrmed by COSY, NOESY
(Fig. 3), HSQC, and HMBC (Fig. 2) experiments. Thus, 8 (reevesioside G) and 9 (epi-reevesioside G) were a pair of epimers and
elucidated as (20S)- and (20R)-18,20-epoxystrophanthidin-4,6dideoxy-2,3-methylenedioxy-b-D-allopyranosides, respectively.

92

H.-S. Chang et al. / Phytochemistry 87 (2013) 8695

Table 4
13
C NMR spectroscopic data for compounds 813.a
Position

10

11

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
10
20
30
40
50
60
70

17.9
25.5
72.9
34.5
73.1
36.26
24.7
43.8
39.5
54.6
24.7
36.28
58.6
83.7
34.4
25.4
54.9
71.4
207.9
88.6
75.6
37.2
174.7
99.1
73.8
74.5
34.3
67.0
20.9
95.4

17.9
25.5
73.0
34.5
73.1
36.2
24.59
43.8
39.5
54.6
24.61
36.4
58.8
83.6
34.2
23.8
57.1
71.6
207.9
87.0
74.1
40.8
174.6
99.1
73.8
74.5
34.3
67.0
20.9
95.4

21.9
25.6
73.5
33.7
74.8
36.3
24.4
42.2
39.7
53.2
23.4
36.4
58.8
83.9
35.0
25.3
54.8
71.5
175.7
88.7
75.9
37.2
174.9
99.7
73.6
74.7
34.2
67.4
20.8
95.5

21.9
25.6
73.5
33.8
74.72
36.2
24.4
42.2
39.7
53.2
23.4
36.5
58.9
83.9
34.8
23.7
57.0
71.7
175.7
87.0
74.3
41.0
174.8
99.8
73.6
74.69
34.2
67.3
20.8
95.5

12
28.6
26.1
73.3
34.3
73.6b
34.7
23.8
42.0
39.69
73.9b
23.5
36.6
58.8
83.7
35.0
25.4
55.1
71.6

13

Table 6
Cytotoxicity (IC50 values) of 113 against the MCF-7, NCI-H460, and HepG2 cancer
cell lines.
Compounds

28.6
26.1
73.3
34.3
73.6b
34.7
23.8
42.1
39.66
73.9b
23.5
36.7
58.9
83.7
34.8
23.5
57.2
71.8

Reevesioside A (1)
Reevesioside B (2)
Reevesioside C (3)
Reevesioside D (4)
Reevesioside E (5)
Reevesioside F (6)
epi-Reevesioside F (7)
Reevesioside G (8)
epi-Reevesioside G (9)
Reevesioside H (10)
epi-Reevesioside H (11)
Reevesioside I (12)/epiReevesioside I (13)a
Tylophorineb
Actinimycin Db
a

88.5
75.9
37.2
174.8
99.1
73.8
74.6
34.3
67.1
20.9
95.5

86.9
74.3
40.9
174.8
99.1
73.8
74.6
34.3
67.1
20.9
95.5

a 13
C NMR data (d) were measured in CDCl3 at 100 MHz for 9, 12 and 13, at
150 MHz for 8, 10 and 11.
b
Interchangeable within the same column.

The mixture of 10 and 11 was subjected to MPLC, eluting with

CH2Cl2acetone (2:1), and repeated this step three times to obtain
pure 10 and 11, which were elucidated to be a pair of 18,20-epoxide epimers as 8 and 9 by the following evidence: HRESIMS showed
the same molecular formula C30H42O11 and IR spectra were very
much alike. The 1H (Table 5) and 13C NMR (Table 4) spectra of 10

b
c

IC50 (nM)
MCF-7

NCI-H460

HepG2

63 2
3524 72
2040 170
36400 2600
11800 2200
72 8
34 6
2627 139
2415 125
>500000
45345 881
9350 960

19 1
431 45
208 16
3900 320
1770 35
20 0
10 1
749 33
680 7
8066 30
4638 147
1090 210

368 30
4760 200
3740 260
>500000
40500 6400
836 33.
996 93
8118 536
6215 357
>500000
42840 3332
22700 3500

236 11
101 1

233 24
10 2

215 14
c

Tested as a mixture.
Positive control.
Not determined.

and 11 were similar to those of 8 and 9, except that the aldehyde

group [dH 10.07 (1H, s, H-19); dC 207.9 (C-19)] of 8 and 9 was
replaced by a carboxylic acid group [dC 175.7 (C-19)] in 10 and
11. The same correlations observed in the NOESY spectra as 8
and 9 allowed us to elucidate these to be 20S and 20R, respectively.
Therefore, the structures of 10 and 11 were established as (20S)and (20R)-18,20-epoxycheiranthidin-4,6-dideoxy-2,3-methylenedioxy-b-D-allopyranosides and these compounds were named
reevesioside H and epi-reevesioside H, respectively.
Compounds 12 and 13 were isolated as a mixture in a ratio of
3:2, which could not be separated in our hands. The signals assigned to rings A and B were identical with those of 5. However,
the duplicate pattern of 1H and 13C NMR resonances in addition
to the NOESY and HMBC spectra concerning the 18,20-epoxide
structures of 8 and 9 allowed us to determine the structures of
12 (20S) and 13 (20R) as shown, which were named reevesioside
I and epi-reevesioside I, respectively.

Table 5
1
H NMR spectroscopic data for compounds 1013.a
Position

10

11

12

13

1a/b
2a/b
3
4a/b
6a/b
7a/b
8
9
11a/b
12a/b
15a/b
16a/b
17
18a/b
21a/b
22a/b
10
20
30

2.28, m/1.82, m
1.92, m/1.51, m
4.24, br s
2.08, m/1.74, m
1.89, m/1.62, m
2.05, m/1.16, m
2.01, m
1.43, m
1.89, m/1.59, m
1.74, m/1.43, m
1.82, m/1.79, m
2.01, m/1.67, m
2.08, m
4.28, d (10.2)/3.52, d (10.2)
4.34, d (9.6)/3.98, d (9.6)
2.64, d (18.0)/2.60, d (18.0)
4.45, d (6.9)
3.81, dd (6.9, 5.7)
4.15, ddd
(5.7, 3.6, 1.2)
2.10, m/1.74, m
3.80, m
1.23, d (6.6)
5.24, s/4.92, s

2.29, m/1.87, m
1.94, m/1.53, m
4.24, br s
2.13, m/1.81, m
1.87, m/1.70, m
2.08, m/1.18, m
2.04, m
1.44, m
1.91, m/1.56, m
1.82, m/1.44, m
1.81, m/1.79, m
2.08, m/1.63, m
2.31, m
4.28, d (10.2)/3.48, d (10.2)
4.36, s
2.76, d (17.4)/2.44, d (17.4)
4.45, d (6.8)
3.81, dd (6.8, 5.3)
4.15, ddd
(5.3, 3.6, 1.8)
2.16, m/1.77, m
3.80, m
1.23, d (6.0)
5.23, s/4.92, s

1.93, m/1.47, m
1.87, m/1.76, m
4.15, br s
1.84, m/1.43, m
1.90, m/1.43, m
2.00, m/1.06, m
1.90, m
1.31, m
1.93, m/1.68, m
1.81, m/1.53, m
1.84, m/1.76, m
1.87, m/1.76, m
2.15, m
4.23, d (10.2)/3.48, d (10.2)
4.33, dd (10.2, 0.8)/3.99, d (10.2)
2.66, d (17.8)/2.60, d (17.8)
4.47, d (6.7)
3.81, dd (6.7, 5.3)
4.15, ddd
(5.3, 3.6, 2.0)
2.14, m/1.76, m
3.81, m
1.23, d (6.4)
5.24, s/4.91, s

1.93, m/1.47, m
1.87, m/1.76, m
4.15, br s
1.84, m/1.43, m
1.90, m/1.43, m
2.00, m/1.06, m
1.90, m
1.31, m
1.93, m/1.68, m
1.81, m/1.53, m
1.84, m/1.76, m
1.90, m/1.67, m
2.31, m
4.24, d (10.0)/3.44, d (10.0)
4.37, s
2.74, d (17.2)/2.45, d (17.2)
4.47, d (6.7)
3.81, dd (6.7, 5.3)
4.15, ddd
(5.3, 3.6, 2.0)
2.14, m/1.76, m
3.81, m
1.23, d (6.4)
5.24, s/4.91, s

40 a/b
50
60
70 a/b
a 1

H NMR data (d) were measured in CDCl3 at 400 MHz for 12 and 13, at 600 MHz for 10 and 11.

H.-S. Chang et al. / Phytochemistry 87 (2013) 8695

Compounds 113 were tested for cytotoxicity against the MCF7, NCI-H460, and HepG2 cancer cell lines, with the IC50 data shown
in Table 6. Tylophorine (Yang et al., 2010) and actinimycin D
(Chang et al., 2009) were used as positive reference controls for
the cytotoxicity assays. All 13 new compounds showed strong
cytotoxic potencies against NCI-H460 cell line. Reevesioside F (6)
and epi-reevesioside F (7), with the digitoxigenin as the aglycone
showed more potent cytotoxicities than the strophanthidin glycosides 13, and the 18,20-epoxystrophanthidin derivatives 813.
Reevesiosides CE (35), with the same skeleton but with different
substituents at C-10, exhibited ascending degrees of cytotoxicity in
the order: reevesioside C (3) > reevesioside E (5) > reevesioside D (4).
3. Conclusions
Thirteen new cardenolide glycosides were isolated from the
root of R. formosana. This is the rst report of cardenolide glycosides from a plant in the family Sterculiaceae. Furthermore, all 13
new isolates showed potent cytotoxicity against a small panel of
cancer cell lines in vitro. Reevesioside A (1), reevesioside F (6),
and epi-reevesioside F (7) displayed especially potent cytotoxicity
against the MCF-7 and NCI-H460 cancer cell lines, with IC50
values of 63 2 and 19 1, 72 8 and 20 0, and 34 6 and
10 1 nM, respectively. Two new sugar moieties: 4,6-dideoxy-2,3methylenedioxy-b-D-allopyranosyl and 4,6-dideoxy-2-O-methylb-D-allopyranosyl, together with two rare sugar moieties,
6-deoxy-2-O-methyl-b-D-glucopyranosyl and 6-deoxy-2-O-methylb-D-allopyranosyl are also reported.
4. Experimental
4.1. General experimental procedures
All melting points were determined with a Yanaco micromelting
apparatus and are uncorrected. Optical rotations were measured on
a Jasco P-1020 polarimeter. UV spectra were obtained with a JASCO
V-530 UV/vis spectrophotometer, and IR spectra (KBr) were acquired with a Genesis II FTIR spectrophotometer. 1D (1H, 13C, DEPT)
and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra, using CDCl3 (1H,
d 7.26; 13C, d 77.0) as solvent, were recorded on a Varian Unity Plus
400 spectrometer (400 MHz for 1H NMR, 100 MHz for 13C NMR) and
Varian VNMRS-600 spectrometer (600 MHz for 1H NMR, 150 MHz
for 13C NMR). Chemical shifts are given as d (ppm) using TMS as
the internal standard. Low-resolution mass spectra were obtained
with Micromass Trio-2000 GC/MS, VG Biotech Quattro 5022, and
JEOL-JMS-HX 100 mass spectrometers. HRMS were recorded on
JEOL JMS-SX102A GC/LC/MS and Finnigan MAT-95XL mass
spectrometers. Silica gel (70230 and 230400 mesh; Merck) and
Spherical C18 100 reversed-phase silica gel (RP-18; particle size
2040 lm; Silicycle) were used for column chromatography (CC),
and silica gel 60 F254 (Merck) and RP-18 F254S (Merck) were used
for TLC and preparative TLC. The X-ray diffraction study was performed using a Bruker Smart diffractometer.
4.2. Plant material
The root tissue of R. formosana was collected at Mudan, Pingtung County, Taiwan, in September 2009, and identied by one
of the authors (I.-S.C.). A voucher specimen (Chen 6117) has been
deposited in the Herbarium of the School of Pharmacy, College of
Pharmacy, Kaohsiung Medical University.
4.3. Extraction and isolation
The dried root tissue of R. formosana (6.5 kg) was sliced and extracted with cold MeOH (30 L  3) at room temperature for 9 days,

93

and the solvent was evaporated in vacuo. The MeOH extract

(150 g) was partitioned between EtOAc and H2O to yield EtOAcsoluble (45 g) and H2O-soluble fractions (100 g). Both fractions
showed cytotoxicity against the MCF-7, NCI-H460, and HepG2
cancer cell lines. The survival rates against these three cancer cell
lines of the MeOH extract and the above two fractions were <10%
(at 30 lg/mL). The EtOAc-soluble fraction (45 g) was subjected to
a silica gel CC, eluted with a gradient of hexaneEtOAc, to produce
12 fractions (A-1A-12). Fractions A-9A-12 showed cytotoxicity
against the three cancer cell lines used. Fraction A-10 (3.6 g) was
subjected to passage over a Sephadex LH-20 column, eluting with
MeOH, to yield 13 further fractions (A-10-1A-10-13). Fraction
A-10-3 (0.87 g) was separated over a RP-C18 column, eluting with
MeOHH2O (3:2), to afford 12 fractions (A-10-3-1A-10-3-12), of
which Fraction A-10-3-8 was composed entirely of 1 (460 mg).
Fraction A-10-3-5 (144 mg) was puried by preparative RP-18
TLC with the solvent system CH2Cl2acetone (5:1), this being repeated ve times to yield pure 8 (3.2 mg, Rf 0.56) and 9 (3.0 mg,
Rf 0.58), respectively. The residue was subjected to passage over
a silica gel column, eluting with CH2Cl2acetone (5:1), to obtain seven fractions (A-10-3-5-1A-10-3-5-7). Fraction A-10-3-5-2 was
found to be a mixture of 12 and 13 (1.7 mg), and fraction A-103-5-5 was submitted to MPLC, eluting with CH2Cl2acetone (2:1),
and repeated this step three times to obtain pure 10 (1.5 mg)
and 11 (1.3 mg), respectively. Fraction A-10-3-6 (14.3 mg) was
subjected to silica gel CC, eluting with CH2Cl2acetone (3:1), to
furnish seven fractions (A-10-3-6-1A-10-3-6-7), of which fraction
A-10-3-6-2 was puried further by preparative RP-18 TLC with
acetoneH2O (1:1) to yield 2 (2.0 mg, Rf 0.46). Fraction A-10-3-10
(100 mg) was puried by silica gel CC, eluting with CH2Cl2acetone
(3:1), to yield ve fractions (A-10-3-10-1A-10-3-10-5), of which
fractions A-10-3-10-3 and A-10-3-10-4 contained 7 (63.0 mg)
and 6 (11.0 mg), respectively. Fraction A-11 (13.7 g) was applied
to a Sephadex LH-20 column, eluting with MeOH, to afford nine
fractions (A-11-1A-11-9). Fraction A-11-2 (0.74 g) was submitted
to a RP-C18 column, using MeOH-H2O (1:1) for elution, to obtain 14
fractions (A-11-2-1A-11-2-14). Fraction A-11-2-8 (149 mg) was
subjected to passage over a silica gel column, eluting with CH2Cl2
acetone (3:1), to yield nine fractions (A-11-2-8-1A-11-2-8-9).
Fractions A-11-2-8-7 and A-11-2-8-8 were puried further by preparative TLC with hexaneCH2Cl2acetone (1:1:2) to yield 3
(9.0 mg, Rf 0.68), 4 (14.0 mg, Rf 0.35), and 5 (2.8 mg, Rf 0.33).
4.3.1. Reevesioside A (1)
Colorless prisms (hexaneEtOAc); mp 231232 C; a26
D 28.7
(c 0.80, MeOH); IR (KBr) mmax 3518 (OH), 1780, 1744, 1621 (a,bunsaturated c-lactone ring), 1715 (CHO) cm1; for 1H and 13C
NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z 569
[M+Na]+; HRESIMS m/z 569.2729 (calcd. for C30H42O9Na,
569.2726).
4.3.2. Reevesioside B (2)
Colorless syrup; a26
D 18.7 (c 0.07, MeOH); IR (neat) mmax 3511
(OH), 1775, 1741, 1628 (a,b-unsaturated c-lactone ring) cm1; for
1
H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z
627 [M+Na]+; HRESIMS m/z 627.2777 (calcd. for C32H44O11Na,
627.2781).
4.3.3. Reevesioside C (3)
Colorless needles (hexaneEtOAc); mp 140142 C; a26
D 20.7
(c 0.08, MeOH); IR (KBr) mmax 3498 (OH), 1779, 1744, 1621 (a,bunsaturated c-lactone ring), 1712 (CHO) cm1; for 1H and 13C
NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z 571
[M+Na]+; HRESIMS m/z 571.2881 (calcd. for C30H44O9Na,
571.2883).

94

H.-S. Chang et al. / Phytochemistry 87 (2013) 8695

4.3.4. Reevesioside D (4)

Colorless needles (hexaneEtOAc); mp 160162 C; a26
D 14.1
(c 0.12, MeOH); IR (KBr) mmax 3499 (OH), 1780, 1746, 1621 (a,bunsaturated c-lactone ring), 25003300, 1730 (COOH) cm1; for
1
H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS
m/z 587 [M+Na]+; HRESIMS m/z 587.2836 (calcd. for C30H44O10Na,
587.2832).
4.3.5. Reevesioside E (5)
Colorless needles (hexaneEtOAc); mp 228229 C; a26
D 10.3
(c 0.04, MeOH); IR (KBr) mmax 3479 (OH), 1779, 1745, 1620 (a,bunsaturated c-lactone ring) cm1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z 559 [M+Na]+; HRESIMS
m/z 559.2886 (calcd. for C29H44O9Na, 559.2883).
4.3.6. Reevesioside F (6)
Colorless needles (hexaneEtOAc); mp 241242 C; a26
D 17.6
(c 0.08, MeOH); IR (KBr) mmax 3442 (OH), 1779, 1742, 1622 (a,bunsaturated c-lactone ring) cm1; for 1H and 13C NMR spectroscopic data, see Tables 3 and 2; ESIMS m/z 557 [M+Na]+; HRESIMS
m/z 557.3088 (calcd. for C30H46O8Na, 557.3090).
4.3.7. epi-Reevesioside F (7)
Colorless needles (hexaneEtOAc); mp 216217 C; a26
D 16.9
(c 1.20; MeOH); IR (KBr) mmax 3458 (OH), 1780, 1741, 1621 (a,bunsaturated c-lactone ring) cm1; for 1H and 13C NMR spectroscopic data, see Tables 3 and 2; ESIMS m/z 557 [M+Na]+; HRESIMS
m/z 557.3094 (calcd. for C30H46O8Na, 557.3090).
4.3.8. Acid hydrolysis of 7
Compound 7 (5 mg) was dissolved in MeOH (2 mL) and 5%
H2SO4 solution (2 mL) and hydrolyzed under conditions, reux
at 80 C for 3 h. The mixture was allowed to cool, diluted twofold
with distilled H2O and partitioned between EtOAc and H2O. The
aq. layer was neutralized with aq. NaHCO3 soln. (1.0 M) and evaporated to give 2-O-methyl-6-deoxy-b-methyl-D-allopyranoside: 1H
NMR (400 MHz, CDCl3) d 4.51 (1H, d, J = 7.6 Hz, H-10 ), 4.24 (1H, br
t, J = 3.7 Hz, H-30 ), 3.69 (1H, dq, J = 9.7, 6.4 Hz, H-50 ), 3.53 (3H, s,
OCH3-10 ), 3.52 (3H, s, OCH3-20 ), 3.25 (1H, br td, J = 9.7, 3.7 Hz, H40 ), 3.06 (1H, dd, J = 7.6, 3.7 Hz, H-20 ), 2.55 (1H, br s, OH-30 ), 2.45
(1H, br d, J = 9.7 Hz, OH-40 ), 1.32 (1H, d, J = 6.4 Hz, H-60 ). a26
D
29.3 (c 0.05; MeOH).
4.3.9. Reevesioside G (8)
Colorless syrup; a26
D 17.8 (c 0.03; MeOH); IR (neat) mmax 3518
(OH), 1779 (lactone ring), 1713 (CHO) cm1; for 1H and 13C NMR
spectroscopic data, see Tables 3 and 4; ESIMS m/z 585 [M+Na]+;
HRESIMS m/z 585.2673 (calcd for C30H42O10Na, 585.2676).
4.3.10. epi-Reevesioside G (9)
Colorless syrup; a26
D 121.0 (c 0.03; MeOH); IR (neat) mmax
3497 (OH), 1779 (lactone ring), 1712 (CHO) cm1; for 1H and 13C
NMR spectroscopic data, see Tables 3 and 4; ESIMS m/z 585
[M+Na]+; HRESIMS m/z 585.2678 (calcd. for C30H42O10Na,
585.2676).
4.3.11. Reevesioside H (10)
Colorless syrup; a26
D 4.0 (c 0.03; MeOH); IR (neat) mmax 3520
(OH), 1781 (lactone ring), 25003300, 1729 (COOH) cm1; for 1H
and 13C NMR spectroscopic data, see Tables 5 and 4; ESIMS m/z
601 [M+Na]+; HRESIMS m/z 601.2622 (calcd. for C30H42O11Na,
601.2625).
4.3.12. epi-Reevesioside H (11)
Colorless syrup; a26
D 38.2 (c 0.04; MeOH); IR (neat) mmax 3521
(OH), 1783 (lactone ring), 25003300, 1729 (COOH) cm1; for 1H

and 13C NMR spectroscopic data, see Tables 5 and 4; ESIMS m/z
601 [M+Na]+; HRESIMS m/z 601.2621 (calcd. for C30H42O11Na,
601.2625).
4.3.13. Mixture of reevesioside I (12) and epi-reevesioside I (13)
Colorless syrup; IR (neat) mmax 3520 (OH), 1781 (lactone ring)
cm1; for 1H and 13C NMR spectroscopic data, see Tables 5 and
4; ESIMS m/z 573 [M+Na]+; HRESIMS m/z 573.2679 (calcd. for C29H42O10Na, 573.2676).
4.3.14. X-ray crystallographic study of reevesioside A (1)
Crystal data: C30H42O9, M = 546.64, monoclinic system, space
group
P21
(No.
4),
a = 10.3097(2) ,
b = 13.0707(2) ,
c = 10.7637(2) ,
b = 109.832(1),
V = 1364.44(4) 3,
Z = 2,
Dcalcd. = 1.331 g/cm3. A crystal of dimensions 0.20  0.15 
0.15 mm was used for measurements on a Bruker APEX DUO diffractometer with a montel mirror monochromator (u and x scans,
2hmax = 50.0o), Cu Ka radiation (k = 1.5418 ) at 100 K. The total
number of independent reections measured was 4171, of which
4117 were observed (|F|2 = 2r|F|2). The crystal structure was
solved by the direct method and expanded using difference Fourier
techniques, further rened by the program SHELXTL 97 (Sheldrick,
G.M. University of Gottingen, Gottingen, Germany, 1997) and
full-matrix least-squares calculations with 356 variable
parameters. The non-hydrogen atoms were given anisotropic thermal parameters. Final indices: Rf = 0.0287, Rw = 0.0754 (w = 1/
[r2(F 2o ) + (0.0391P)2 + 0.2963P] where P F 2o 2F 2c =3. The absolute conguration of 1 was determined with Flacks parameter of
0.12(12). The nal difference Fourier map was at, with the highest
and lowest residual peaks of 0.275 and 0.155 e/3, respectively.
Crystallographic data for the structure of reevesioside A (1) have
been deposited with the Cambridge Crystallographic Data Center
as supplementary publication number CCDC-787008. Copies of
the data can be obtained, free of charge, on application to CCDC,
12, Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1223 336033
or e-mail: deposit@ccdc.cam.ac.uk).
4.4. Cytotoxicity assay
Human cancer cells were seeded in 96-well microtiter plates in
100 lL culture medium at cell numbers/well of 6500, 2500 and
7500 for MCF-7 (human breast adenocarcinoma), NCI-H460 (nonsmall-cell lung cancer) and HepG2 (liver hepatocellular cells),
respectively. Maintenance of cell cultures and the cytotoxicity
were performed as described, previously (Chang et al., 2009).
Acknowledgments
This work was supported by the National Science Council of the
Republic of China and by intramural funding of the National Health
Research Institutes of the Republic of China.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
11.024.
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