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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
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
87
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'
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
88
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
4.73,
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.
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.
89
90
91
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
92
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.
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.
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.
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
94
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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