Five new triterpenoid saponins from the Roots of Camellia oleifera C. Abel with cytotoxic activities

Article abstract of DOI:10.1016/j.phytol.2015.08.005

Abstract Five new triterpenoid saponins, oleiferosides P-T (1-5) were isolated from the EtOH extract of the roots of Camellia oleifera C. Abel. The structures of saponins 1-5 were elucidated on the basis of integrated spectroscopic techniques. All the compounds were characterized to be oleanane-type saponins with sugar moieties linked to the C-3 of the aglycone. By using the MTT assay, an in vitro analysis of the cytotoxic activities of these saponins on the human tumor cell lines (lung adenocarcinoma A549 cells, hepatic carcinoma SMMC-7721 cells and breast cancer MCF-7 cells). Among them, compound 4 showed a certain cytotoxic activity against all the tested cell lines.

Full text of DOI:10.1016/j.phytol.2015.08.005

Phytochemistry Letters 13 (2015) 379–385
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Five new triterpenoid saponins from the Roots of Camellia oleifera C.
Abel with cytotoxic activities
a
b
a
a
a,
c,
Jiangping Wu , Jianping Zhao , Yanli Liu , Xiaoran Li , Qiongming Xu *, Yulin Feng *,
a,b
a,c
Ikhlas A. Khan , Shilin Yang
College of Pharmaceutical Science, Soochow University, Suzhou 215123, China
a
b
National Center for Natural Products Research, and Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy,
University of Mississippi, Mississippi 38677, USA
c
Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 4 June 2015
Received in revised form 1 August 2015
Accepted 1 August 2015
Available online xxx
Five new triterpenoid saponins, oleiferosides P–T (1–5) were isolated from the EtOH extract of the roots
of Camellia oleifera C. Abel. The structures of saponins 1–5 were elucidated on the basis of integrated
spectroscopic techniques. All the compounds were characterized to be oleanane-type saponins with
sugar moieties linked to the C-3 of the aglycone. By using the MTT assay, an in vitro analysis of the
cytotoxic activities of these saponins on the human tumor cell lines (lung adenocarcinoma A549 cells,
hepatic carcinoma SMMC-7721 cells and breast cancer MCF-7 cells). Among them, compound 4 showed a
certain cytotoxic activity against all the tested cell lines.
Keywords:
Camellia oleifera
Triterpenoid saponins
Oleiferoside P–T
Cytotoxic activities
ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
1. Introduction
to give five new saponins, named oleiferosides P–T (1–5), the
structures of the newly characterized saponins were elucidated by
Camellia oleifera C. Abel, which is mainly distributed in China,
Japan, India and other South-East Asian countries, is the most
representative and largest genus of the family Theaceae with more
than 120 species recognized currently (Li et al., 2014; Zhao et al.,
means of extensive spectroscopic analyses. Furthermore, the new
compounds were screened for cytotoxity against three tumor cell
lines (A549, SMCC-7721 and MCF-7 cells) by using 3-(4,5-
dimethylthiazol)-2, 5-diphenyl tetrazoliumbromide (MTT) assay.
Herein, we report on the isolation, structural elucidation and
cytotoxity of the five new saponins.
2011). And it has been grown as an oil crop in many countries
including China, Brazil, Philippines, India and South Korea (Lee and
Yen, 2006; Wang et al., 2013). The oil is rich in unsaturated fatty
acids, which is comparable to the olive oil (Lee et al., 2007).
Previous studies on constituents from the Camellia genus led to the
isolation of different compounds such as flavonoids, bibenzyl
glycosides and saponins from the seeds, seed cake or flowers (Chen
et al., 2013; Chen et al., 2011; Sugimoto et al., 2009). Pharmaco-
logical investigations revealed that these aforementioned com-
pounds exhibited antioxidant activity, hepatoprotective activity,
cytotoxic activity and myocardial cell-injury-protective activity
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. The
+
positive-ion HR-ESI–MS spectrum of 1 displayed an [M + Na] peak
at m/z 1101.5229 (calcd. 1101.5246), corresponding to a molecular
formula of C55
hydroxyl group (3456 cm ) and an
(1601, 1603 cm ). The C NMR (Table 1) spectrum showed the
resonances of 55 carbons, ascribable to twelve methyl, nine
methylene, twenty methine and fourteen quaternary carbons as
82
H O21. The IR spectrum showed the presence of a
ꢀ1
a, b-unsaturated ester group
ꢀ1
13
(Chen et al., 2013; Chen et al., 2011; Lee et al., 2007). The EtOH
extract of the roots of C. oleifera C. Abel was successively subjected
to column chromatography over D101 marcroporouse resins, silica
gels and LX-2000 microreticular resin, and semi-preparative HPLC
1
revealed by the HSQC (Fig. 2) experiment. The H NMR (Table 2)
spectrum showed six methyl proton signals at
.92 (Me-25), 1.04 (Me-26), 1.96 (Me-27), 1.09 (Me-29) and 1.32
Me-30), two isolated oxymethylene proton signals at 3.70 (1H,
d, J = 11.5 Hz, H-23) and 4.32 (1H, d, J = 11.5 Hz, H-23), as well as
.73 (1H, d, J = 12.0 Hz, H-28) and 3.89 (1H, d, J = 12.0 Hz, H-28), five
isolated oxygenated methine proton signals at 5.67 (1H, d,
H
d 0.95 (Me-24),
0
(
d
H
d
H
*
Corresponding authors. Fax: +86 512 65882089/791 87119620.
3
E-mail addresses: xuqiongming@suda.edu.cn (Q. Xu), fengyulin2003@126.com
d
H
(
Y. Feng).
http://dx.doi.org/10.1016/j.phytol.2015.08.005
874-3900/ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
1

3
80
J. Wu et al. / Phytochemistry Letters 13 (2015) 379–385
Table 1
H-12). Furthermore, the proton signals of two angeloyl (Ang)
1
3C NMR (125 MHz) spectroscopic data for compounds 1–5 in C
5
D
5
N.
0
groups at
H
d [6.00 (1H, dq, J = 7.0, 1.5 Hz, 21-O-Ang-3 ), 2.05 (3H, d,
0
0
No.
1
2
3
4
5
J = 7.5 Hz, 21-O-Ang-4 ), 2.03 (3H, s, 21-O-Ang-5 )] and
d
H
[5.81 (1H,
0
0
dq, J = 7.0, 1.5 Hz, 22-O-Ang-3 ), 1.97 (3H, d, J = 7.5 Hz, 22-O-Ang-
1
2
3
4
5
6
7
8
9
39.4
26.5
81.8
43.9
47.1
18.8
35.5
42.4
47.3
37.3
24.4
128.1
141.2
47.7
69.9
72.0
48.4
40.7
47.1
36.6
78.2
71.7
64.5
14.2
16.8
18.1
21.4
63.0
30.0
20.1
38.7
26.0
81.2
43.3
47.1
38.5
25.8
80.6
43.2
46.7
17.7
38.8
25.9
80.6
43.2
47.2
18.3
36.6
41.5
45.7
36.7
23.8
38.5
25.8
80.5
43.2
46.9
17.7
0
0
00
4 ), 1.72 (3H, s, 22-O-Ang-5 )] were observed. And the same time
0
the two acetoxy (AC) groups at
d
H
2.02 (3H, s, 15-O-AC-2 ) and 2.53
0
1
(
3H, s, 16-O-AC-2 ) could also be observed. The C NMR data
indicated the presence of two acetoxy (AC) groups at
d
C
171.0 (15-
18.3
35.0
41.5
46.7
36.7
23.8
127.6
140.6
48.0
69.3
71.1
46.5
40.0
45.9
36.0
77.8
71.0
63.8
13.5
16.2
17.5
20.8
62.1
29.2
19.5
0
0
00
32.5
39.8
45.8
36.4
23.6
124.9
140.6
40.9
30.8
71.3
47.0
39.3
46.9
35.8
77.9
72.2
63.8
13.4
16.0
16.6
26.8
63.3
29.2
19.5
32.5
39.8
45.7
36.4
23.6
125.9
140.6
40,9
30.5
71.9
47.5
39.3
47.0
35.9
77.8
72.3
63.8
13.4
16.0
16.6
26.8
63.2
29.2
19.5
O-AC-1 ), 22.27 (15-O-AC-2 ) and d 170.5 (16-O-AC-1 ), 21.98 (16-
C
0
0
O-AC-2 ), the other data were comparable to those reported for
oleiferoside F published in the literature (Li et al., 2014), suggesting
1
0
that compound 1 had the 15a,16a-diacetoxy-21b, 22a-O-dia-
1
1
ngeloyloxy-3 ,23,28-trihydroxyolean-12-ene type of aglycone. In
b
1
2
126.1
1
3
142.2
47.6
66.4
71.7
47.4
40.4
46.9
35.9
77.8
71.3
63.9
13.5
16.2
17.5
20.4
63.1
29.2
19.5
the HMBC spectrum, the correclations were observed from H-21 at
0
1
4
d
5.87 to C-21-Ang-1 at
d
C
168.2 and from H-22 at
d
H
6.32 to C-22-
H
1
5
0
Ang-1 at
C
d 167.7. It was proved that the two Ang groups were
1
6
connected to C-21 and C-22 respectively. The locations of the two
1
7
1
8
AC groups at C-15 and C-16 were also confirmed by the HMBC
0
1
9
(Fig. 2) correlations between H-15 at
71.0 and H-16 at
d
5.62 to C-15-O-AC-1 at
d
H
C
2
0
0
1
d
H
5.89 to C-16-O-AC-1 at
d
C
170.5. A CH
2
OH
2
1
group was located at C-23, which was confirmed by the
correlations between H-23 at 3.70, 4.32 and C-3 at 81.8,
and between H-23 at 3.70, 4.32 and C-24 at 14.16 in the HMBC
spectrum. The relative configuration of 1 was determined from the
NOESY (Fig. 2) experiment. The NOEs from H-29 at 1.09 to H-
2 at 6.32 and from H-30 at 1.32 to H-21 at 5.87 suggested
that H-21 and H-22 are - and -oriented, which was confirmed
that the two Ang groups at C-21 and C-22 were - and -oriented.
The NOEs from H-18 at 1.09 to H-16 at 5.89 and from H-15 at
5.67 to H-16 at 5.89, indicated that H-18, H-15 and H-16 were
co-facial and -oriented, while the two AC groups were on the
opposite side of the molecular plane and thus -oriented. The
anomeric proton signals at 5.18 (1H, d, J = 7.5 Hz, H-1 of
glucuronic acid) and 5.29 (1H, d, J = 7.5 Hz, H-1 of arabinose), which
showed the HSQC (Fig. 2) correlations to 106.2 (C-1 of glucuronic
22
23
24
25
26
27
28
29
30
d
H
d
C
d
H
d
C
d
H
2
d
H
d
H
d
H
a
b
b
a
2
1-O-Ang
d
H
d
H
0
0
0
0
0
1
2
3
4
5
2
1
2
3
4
5
2
1
2
3
4
5
168.2
129.0
139.1
16.5
167.4
128.0
139.5
16.0
167.3
127.9
139.1
15.9
167.2
127.8
140.0
15.9
166.9
128.0
137.8
15.5
d
H
d
H
b
a
21.6
20.9
20.8
20.8
20.6
d
H
2-O-Ang
0
0
0
0
0
0
0
0
0
0
167.7
128.7
138.9
16.2
167.5
138.3
128.2
15.7
d
C
acid) and 106.1 (C-1 of arabinose) respectively, revealling the
presence of two sugar residues. Acid hydrolysis of compound 1 and
GC analysis confirmed one unit of D-glucuronic acid and one unit of
21.4
20.7
2-O-MB
L-arabinose. The HMBC correlations between
glucuronic acid) and 81.8 (C-3 of aglycone) and between
(H-3 of aglycone) and 106.2 (C-1 of glucuronic acid) suggested
d
H
5.18 (H-1 of
0
175.9
41.7
26.6
11.7
175.6
41.5
26.5
11.7
176.0
41.5
26.5
11.7
0
0
0
0
d
C
d
H
4.33
d
C
that the glycosidic chain attached to C-3 of the aglycone. The
linkage of the glycosidic chain was determined by the HMBC
16.8
16.5
16.6
1
1
2
5-O-AC
0
0
correlations between
glucuronic acid), and between
106.1 (C-1 of arabinose). The glucuronopyranosyl unit was in
-anomeric configuration based on the observation of the large
JHꢀ1,Hꢀ2 coupling constant. The -configuration of the arabino-
pyranosyl unit was confirmed by the NOESY correlations between
5.29 (H-1 of arabinose) and 4.20 (H-3 of arabinose). Based on
the above analyses, the structure of 1 was established to be
,16 -diacetoxy-21 , 22 -O-diangeloyloxy-23,28-dihydroxyo-
lean-12-ene -O- -glucurono-
d
H
5.29 (H-1 of arabinose) and
C
d 86.4 (C-3 of
171.0
22.3
169.8
21.7
d
H
4.33 (H-3 of glucuronic acid) and
1
1
2
6-O-AC
d
C
b
0
0
0
0
170.1
22.0
Glc-A
106.2
75.0
86.4
72.0
78.2
nd
169.8
21.7
169.6
21.8
Glc-A
105.1
74.1
85.5
71.2
78.3
nd
170.0
22.0
Glc-A
105.3
73.5
86.0
71.3
78.3
170.5
Ara
169.6
21.8
Glc-A
105.3
74.1
85.5
71.5
77.8
3
a
Sugar
Glc-A
105.6
74.5
85.5
71.4
77.1
172.7
Ara
1
2
3
4
5
6
d
H
d
H
15a
a
b
a
3
b
a
-
L
-arabinopyranosyl-(1 !3)-
b-D
170.2
Ara
pyranoside and named oleiferoside P, as shown in Fig. 1.
Ara
Ara
1
2
3
4
5
106.1
73.2
75.0
69.7
67.6
105.6
72.6
74.3
69.1
67.0
105.1
72.3
74.0
68.9
66.8
105.3
72.3
74.1
68.9
66.8
105.3
72.3
74.1
68.9
66.7
Compound 2 was isolated and purified as white amorphous
powder, and the molecular formula was determined as [M + Na] at
m/z 1103.5385 (calcd. 1119.5403), referring to a molecular formula
+
84
of C55H O21. The IR spectrum showed absorption bands at 3454,
ꢀ
1
1
b
605 and 1602 cm , which were assignable to a hydroxyl and an
a,
Chemical shifts are in ppm, and the assignments were based on HSQC, HMBC, and
NOSEY spectra.
nd = not detected.
1
13
-unsaturated ketone group. Comparison to their H and C NMR
data, 2 was similar to 1, and the main differences in their NMR
0
0
spectra arose from the significant upfield shifts of the C-2
0
0
(
2
ꢀ86.3 ppm) signal at
6.6. With the aid of the H– H COSY, HSQC and HMBC spectra
reveled that an Ang group was replaced to a 2-methylbutanoyl
MB) group, which was attached to C-22 in compound2. It was
d
C
41.7 and C-3 (ꢀ112.9 ppm) signal at
d
C
J = 4.5 Hz, H-15), 5.89 (1H, d, J = 4.5 Hz, H-16), 4.33 (1H, dd, J = 11.0,
.5 Hz, H-3), 6.32 (1H, d, J = 10.5 Hz, H-22) and 5.87 (1H, d,
J = 10.5 Hz, H-21) and one olefinic proton signal at 5.62 (1H, brs,
1
1
4
d
H
(

J. Wu et al. / Phytochemistry Letters 13 (2015) 379–385
381
Table 2
1
5 5
H NMR (125 MHz) spectroscopic data for compounds 1–5 in C D N.
1
2
3
4
5
1
2
3
1.51 m, 0.97 m
2.28 m, 2.00 m
4.33 dd
1.48 m, 0.96 m
2.29 m, 1.98 m
4.34 dd
1.47 m, 0.90 m
2.28 m, 1.96 m
4.29 dd
1.51 m, 0.97 m
2.27 m, 1.99 m
4.28 dd
1.90 m, 1.41 m
2.27 m, 1.94 m
4.25 dd
(
10.0, 5.0)
(10.0, 5.0)
1.52 m
(10.0, 5.0)
1.79 m
(10.0, 5.0)
1.84 m
(10.0, 5.0)
1.75 m
5
6
7
9
1.74 m
1.70 m, 1.3 m
2.01 m, 1.31 m
1.80 m
1.70 m, 1.31 m
2.00 m, 1.29 m
1..80 m
1.71 m, 1.32 m
1.56 m, 1.15 m
2.50 m
1.76 m, 1.38 m
2.18 m, 1.45 m
2.72 m
1.69 m, 1.27 m
1.51 m, 1.10 m
2.86 m
1
12
1
1.95 m, 1.81 m
5.62 brs
1.92 m, 1.78 m
5.60 brs
1.91 m, 1.82 m
5.44 brs
1.93 m, 1.84 m
5.53 brs
1.89 m, 1.75 m
5.42 brs
1
16
5
5.67 brs
5.89 brs
5.62 brs
5.84 brs
1.60 m, 1.90 m
5.61 brs
4.39 brs
5.82 brs
1.59 m, 1.80 m
5.50 brs
18
3.23 m
3.22 m
3.05 m
3.08 m
3.06 m
1
9
2.78 m, 1.54 m
5.87 d (10.0)
6.32 d (10.0)
4.32 d (10.0)
2.73 m, 1.52 m
5.79 d (10.0)
6.27 d (10.0)
4.33 d (10.0)
3.69 d (10.0)
0.94 s
0.90 s
1.02 s
1.70 s
2.65 m, 1.45 m
5.90 d (10.0)
6.23 d (10.0)
4.33 d (10.0)
3.70 d (10.0)
0.94 s
0.91 s
0.83 s
1.45 s
2.78 m, 1.47 m
5.83 d (10.0)
6.17 d (10.0)
4.28 d (10.0)
3.65 d (10.0)
0.94 s
0.95 s
1.06 s
1.76 s
2.63 m, 1.45 m
5.95 d (10.0)
6.25 d (10.0)
4.27 d (10.0)
3.66 d(10.0)
0.90 s
0.87 s
0.79 s
1.40 s
2
2
2
1
2
3
3
.70 d (10.0)
2
2
2
2
2
4
5
6
7
8
0.95
s
0.92 s
1.04 s
1.96 s
3.89 d (10.0)
.73 d (10.0)
3.87 d (10.0)
3.70 d (10.0)
1.05 s
3.68 d (10.0)
3.53 d (10.0)
1.07 s
3.76 d (10.0)
3.52 d (10.0)
1.04 s
3.60 d (10.0)
3.43 d (10.0)
1.05 s
3
2
3
9
0
1.09 s
1.32 s
1.28 s
1.29 s
1.25 s
1.27 s
2
3
1-O-Ang
0
6.00 dq
6.07 dq
(7.0, 1.5)
2.10 d (7.5)
1.70 s
6.11 dq
6.04 dq
(7.0, 1.5)
2.09 d (7.5)
1.96 s
5.96 dq
(
7.0, 1.5)
(7.0, 1.5)
2.14 d (7.5)
2.03 s
(7.0, 1.5)
2.02 d (7.5)
2.00 s
0
0
4
5
2
3
2.05 d (7.5)
2.03 s
2-O-Ang
0
0
5.81 dq
5.92 dq
(7.0, 1.5)
2.04 d (7.5)
1.94 s
(
7.0, 1.5)
0
0
0
0
4
5
2
2
3
4
5
1.97 d (7.5)
1.72 s
2-O-MB
0
2.45 m
2.50 m
2.53 m
0
0
0
1.71 m 1.43 m
0.88 t (7.5)
1.12 d (6.5)
1.62 m 1.49 m
0.91 t (7.5)
1.20 d (6.5)
1.81 m 1.49 m
0.88 t (7.5)
1.16 d (6.5)
1
2
5-O-AC
0
2.02 s
1.99 s
1
2
6-O-AC
0
0
2.53 s
2.51 s
2.54 s
2.49 s
2.48 s
Sugar
Glc-A
Glc-A
Glc-A
Glc-A
Glc-A
1
2
3
4
5
6
5.18 d (10.0)
4.13 m
4.33 m
4.43 m
5.29 d (10.0)
4.13 m
5.19 d (10.0)
4.11 m
4.20 m
4.41 m
4.47 m
nd
5.17 d (10.0)
4.09 m
4.20 m
4.41 m
4.47 m
nd
5.11 d (10.0)
4.07 m
4.18 m
4.29 m
4.50 m
nd
4.22
4.46
m
m
4.48
nd
m
4.47 m
nd
Ara
Ara
Ara
Ara
Ara
1
2
3
4
5
5.29 d (10.0)
4.53 m
5.19 d (10.0)
4.54 m
4.20 m
4.32 m
4.40 dd
(12.5, 3.0)
3.82 dd
(12.5, 2.0)
5.29 d (10.0)
4.55 m
4.20 m
4.33 m
4.42 dd
(12.5, 3.0)
3.81 dd
(12.5, 2.0)
5.27 d (10.0)
4.50 m
4.17 m
4.29 m
4.38 dd
(12.5, 3.0)
3.79 dd
(12.5, 2.0)
5.23 d (10.0)
4.52 m
4.15 m
4.27 m
4.39 dd
(12.5, 3.0))
3.77 dd
(12.5, 2.0)
4.20
m
4.33 m
4.43 dd
(
3
(
12.5, 3.0)
.82 dd
12.5, 2.0)
Chemical shifts are in ppm, and coupling constants (J) in Hz are given in parentheses. The assignments were based on HSQC, HMBC, and NOSEY spectra.
ꢀ
1
readily confirmed 2 had the same relative configuration from the
NOESY experiment. Accordingly, compound 2 was formulated as
1604 and 1602 cm , which were in accordance with a hydroxyl
1
and an
a, b-unsaturated ketone groups. Comparison to their H
and C NMR data, 3 was closely similar to 2, and the main
differences in their NMR spectra were accounted by the absence of
1
3
1
5
a
,16
a
-diacetoxy-21
-O-(2-methylbutanoyloxy) olean 3
-glucuronopyranoside as shown in Fig. 1
b
-O-angeloyloxy-23,28-dihydroxyolean-
12-ene 22
a
b
-O- -arabino-
a
-
L
0
0
pyranosyl-(1 !3)-
b
-
D
the AC group (
in 2 and presences of one methylene group (
30.8, C-15) in 3. The assignment was confirmed by the observation
of HMBC correlations between C-15 at 30.8 and Me-27 at
1.45 s. Thus, the structure of compound 3 was assigned to be 16
acetoxy-21 -O-angeloyloxy-23,28-diahydroxyolean-12-ene 22
d
C
170.3, C-15-O-AC-1 ,
d
H
1.99 s;
d
C
21.3,15-O-AC-2 )
and named oleiferoside Q.
d
H
1.60 m,
d
H
1.90 m;
d
C
Compound 3 was obtained as white amorphous powder. The
+
positive-ion HR-ESI–MS spectrum of 3 showed an [M + Na] peak at
d
C
d
H
m/z 1045.5349(calcd. 1045.5348), referring to a molecular formula
a
a
-
-
of C53
H
82
O19. The IR spectrum showed absorption bands at 3456,
b

3
82
J. Wu et al. / Phytochemistry Letters 13 (2015) 379–385
4
''
O
29
3
0
2
'' 3''
OAng
1
1''
MB=
19
2
5
''
1
3
R1
1
1
25
26
1
7
4'(4'')
'(2'')
Ang= 1'(1'')
5'(5'')
2
8
1
OH
O
O
OAC
2
1
0
15
OH
HO
HO
HO
3'(3'')
3
4
5
7
27
O
O
O
2
R2
O
OH
OH
OH
O
1'''
Ac=
1
2
3
R =OAng
R =OAC
2'''
1
2
R =OMB
R2=OAC
R2=H
1
R =OMB
1
4
R =OMB
R2=OH
R2=H
1
5
R =OAng
1
Fig. 1. The structures of new triterpenoid saponins from the Roots of Camellia oleifera C. Abel.
O-(2-methylbutanoyloxy) olean
3
b
-O-
a
-
L
-arabinopyranosyl-
In conclusion, five isolated new triterpenoid saponins from the
roots of C. oleifera C. Abel (P–U) and their structures were
determined. Preliminary cytotoxicity screening revealed that
oleiferoside S (compound 4) exhibited a certain cytotoxicity
against A549, SMCC-7721 and MCF-7 cells shown in Table 2.
(
1 !3)-
b -D
-glucuronopyranoside as shown in Fig. 1 and named
oleiferoside R.
Compound 4 was obtained as white amorphous powder. Its
82
molecular formula of C53H O20 was determined by the observa-
+
tion of the [M + Na] ion peak at m/z 1061.5247 (calcd.1061.5297) in
the HR–ESI–MS spectrum. The IR spectrum showed absorption
3. Experimental
ꢀ
1
bands at 3455,1603 and 1601 cm , which were in accordance with
a hydroxyl and an -unsaturated ketone group. Comparison to
their H and C NMR data, 4 was closely similar to 2, except for
disappearance of a series of signals of the AC group ( 170.3, C-15-
21.3, 15-O-AC-2 ) and presence of a hydroxyl
group linked to C-15 of 4. The assignment was confirmed by the
observation of HMBC correlations between C-15 at 4.39 and Me-
7 at 20.4 and the only one AC group linked to C-16 by the
a
,
b
3.1. General experimental procedures
1
13
d
C
Optical rotations were measured on a Perkin-Elmer model 241
polarimeter. IR spectra were determined on a PerkinElmer 983 G
spectrometer. NMR spectra were recorded on a Varian Inova
0
0
H C
O-AC-1 , d 1.99 s; d
d
H
5 5
500 spectrometer in C D N using tetramethylsilane (TMS) as the
2
d
C
internal standard. HR–ESI–MS spectra were taken on a Micromass
Q-TOF2 spectrometer. HPLC analysis and purification were carried
out on a SB-C18 semipreparative HPLC column (250 ꢁ 9.4 mm i.d.,
observation of HMBC correlations between H-16 at
H
d 5.82 and
0
C-16-O-AC-1 at
d
C
170.0. Thus, the structure of compound 4
-acetoxy-21 -O-angeloyloxy-15 , 23,
8-trihydroxyolean-12-ene 22 -O-(2-methylbutanoyloxy) olean
-O- -glucuronopyranoside as
was assigned to be 16
a
b
a
5 mm, Agilent Corp. Palo Alto, CA, USA) with a Shimadzu HPLC
2
3
a
system composed of a LC-20AT pump with a SPD-20A detector
(Shimadzu Corp., Kyoto, Japan), and the wavelength for detection
was 203 nm. Medium pressure liquid chromatography (MPLC)
purification was performed on a Büchi Flash Chromatography
system composed of a C-650 pump with a flash column (460 mm
ꢁ 26 mm i.d., Büchi Corp., Flawil, Switzerland). Silica gel (200–
300 mesh) for column chromatography and precoated silica gel
TLC plates were purchased from Qingdao Marine Chemical Factory.
Compounds on TLC were colored by 10% sulfuric acid alcohol
solution. Sephadex LH-20 for column chromatography was
obtained from GE Corp. GC was carried out on a GC-14C (Shimadzu
Corp.) with a flame ionization detector (FID). D101 macroporous
resin and LX-2000 mircroreticular resin (a type of phenol modified
polystyrene-base anion exchange resin) for column chromatogra-
phy were acquired from Xi’an Sunresin New Material Co. Ltd..
b
a-L
-arabinopyranosyl-(1 !3)-
b-D
shown in Fig. 1 and named oleiferoside S.
Compound 5 was obtained as white amorphous powder. The
positive-ion HR-ESI–MS spectrum of 5 showed an [M + Na] peak at
m/z 1043.5184 (calcd. 1043.5192), referring to a molecular formula
+
of C53
80
H O19. The IR spectrum showed absorption bands at 3456,
ꢀ
1
1604 and 1602 cm , which were in accordance with a hydroxyl
1
and an
a
,
b
-unsaturated ester groups. Comparison to their H and
C NMR data, 5 was closely similar to 1, and the main differences in
their NMR spectra were accounted by the absence of the AC group
13
0
0
(
d
C
171.0, C-15-O-AC-1 ,
presence of an methylene group (
C-15) in 5. The assignment was confirmed by the observation of
HMBC correlations between C-15 at 30.5 and Me-27 at 1.40 s,
d
H
2.02 s;
d
C
22.3, 15-O-AC-2 ) in 1 and
d
H
1.59 m, 1.80 m; 30.5,
d
H
d
C
d
C
d
H
and the only one AC group linked to C-16 by the observation of
0
HMBC correlations between H-16 at
d
H
5.50 and C-16-O-AC-1 at
d
C
3.2. Plant material
1
69.6. Therefore, the structure of compound 5 was assigned to be
-acetoxy-21 ,22 -O-diangeloyloxy-23,28-dihydroxyolean-
2-ene 3 -O- -glucuronopyra-
-arabinopyranosyl-(1 !3)-
noside as shown in Fig. 1 and named oleiferoside T.
1
1
6
a
b
a
The roots of C. oleifera C. Abel were collected in Qichun, Hubei
province of China in November 2011, and identified by Professor
Xiao-Ran Li at Soochow University. A voucher sample (No. 11-11-
b
a
-L
b -D

J. Wu et al. / Phytochemistry Letters 13 (2015) 379–385
383
O
O
O
H
H
O
H
H
O
H
O
O
H
O
OH
HO
HO
H
O
H
O
O
GlcA
OH
OH
HO
H
Ara
HO
GlcA
H
O
OH
O
O
Ara
O
H
O
O
O
O
O
HO
O
H
O
O
HO
O
OH
OH
OH
OH
CH2OH
CH2OH
O
O
1
2
O
O
O
O
H
H
H
H
O
O
H
H
O
O
O
O
OH
HO
H
OH
OH
OH
H
GlcA
OH
GlcA
OH
H
Ara
HO
Ara
OH
O
O
O
O
HO
O
O
O
O
O
O
H
O
O
OH
OH
OH
OH
OH
H
CH2OH
CH2OH
3
4
O
O
H
H
O
H
O
O
OH
OH
H
GlcA
OH
Ara
OH
O
O
OH
O
O
O
O
H M B C
OH
OH
H
CH2OH
N O E S Y
5
Fig. 2. Key HMBC and NOE correlations for compounds 1–5.
0
6-01) was deposited in the herbarium of the College of Pharmacy,
2
eluted with a gradient of aqueous MeOH (MeOH–H O 0%, 40%, 70%,
Soochow University.
and 100%, each 40 L). The 70% MeOH fraction (25.2 g) was
separated on a silica gel (200–300 mesh) column (30 ꢁ18 cm),
3.3. Extraction and isolation
3
eluted with a gradient of CHCl –MeOH (100:0, 90:10, 80:20, 70:30,
6
0:40, 50:50, 40:60, 30:70, 0:100, each 6.0 L). The CHCl
(80:20) eluate (4.5 g) was chromatographed by MPLC over ODS,
eluted with MeOH–H O (50:50, 60:40, 70:30, 80:20, 90:10, 100:0,
3
-MeOH
The dried plant material (10 kg) were crushed with a grinder
into fine debris, then extracted twice with 50% EtOH (100 L) at
2
ꢂ
8
0 C under reflux. After removing the solvent under reduced
each 500 mL) at a flow rate of 20.0 mL/min to afford 6 fractions.
pressure to yield the combined extracts (0.45 kg), which was then
dissolved in distilled water and passed through
Fraction 2 (80 mg) was separated by semi-preparative HPLC over
an ODS column, eluted with MeOH–H O (70:30) to yield
2
a
D101 macroporous resin column (i.d. 30 cm ꢁ 200 cm, Xi’an
compound 1 (16 mg, tR 58.7 min), 2 (14 mg, tR 56.4 min) and 4
(12 mg, tR 50.2 min). In a similar way, fraction 4 (62 mg) was
Sunresin New Materials Co. Ltd.), using a sequential mixture of
EtOH-H
80 g) was further applied to a LX-2000 mircroreticular resin
column (26 ꢁ 200 cm; Xi’an Sunresin New Materials Co. Ltd.),
2
O (0%, 30%, 60%, 80%, each 50 L). The 60% EtOH eluate
2
purified by semi-preparative HPLC, eluted with MeOH–H O
(68:32) to yield compound 3 (16 mg, tR 70.7 min) and 5 (18 mg,
tR 75.0 min). All the semi-preparative HPLC separations were
(

3
84
J. Wu et al. / Phytochemistry Letters 13 (2015) 379–385
Table 3
conducted at a flow rate of 2 mL/min and the detection wavelength
was 203 nm. The purities of the isolated saponins were >95% by
HPLC with ELSD detection.
In vitro cytotoxic activities of compounds 1–5 (IC50 values in
mM).
Cell line IC50
A549
(mM)
SMMC-7721
MCF-7
3.4. Oleiferoside P (1)
1
>100
>100
>100
2
3
4
5
>100
>100
>100
(
15
droxyolean-12-ene, 3
curonopyranoside): White amorphous powder; [
a
,16
a
-diacetoxy-21
b
a
,
22
a
-O-diangeloyloxy-23, 28-dihy-
-glu-
+ 6.4 (c
lmax (log e): 206 (3.26), 254 (4.22) nm; IR
93.91 ꢃ 2.63
25.96 ꢃ 0.32
83.73 ꢃ 1.71
4.16 ꢃ 0.28
97.54 ꢃ 1.43
34.85 ꢃ 0.71
88.42 ꢃ 1.57
7.93 ꢃ 0.46
86.78 ꢃ 3.48
25.52 ꢃ 0.82
78.33 ꢃ 2.78
5.17 ꢃ 0.38
b
-O-
-
L-arabinopyranosyl-(1 !3)-
b
]
D
-D
2
0
a
Norcantharidin
0
n
.12, MeOH); UV (MeOH)
ꢀ1
IC50 is means ꢃ standard deviation of five new triterpenoid saponins.
max (KBr): 3456, 2925, 1603, 1601, 1161, 1042 cm ; HR-ESI–MS
M + Na]+ m/z 1101.5229 (calcd. for C55
H
O
21Na, 1101.5246);
1
H
[
82
NMR (C
5
D
5
N, 500 MHz) and 13C NMR (C
5
D
5
N, 125 MHz) data are
given in Tables 1 and 2.
evaporated to give a mixture of monosaccharides. Each residue was
dissolved in dry pyridine (1 mL), then L-cysteine methyl ester
3.5. Oleiferoside Q (2)
hydrochloride in dry pyridine (0.06 M, 2 mL) was added. After
(
15
lean-12-ene 22
nopyranosyl-(1 !3)-
amorphous powder; [
log
602,1180,1046 cm ; HR–ESI–MS [M + Na] m/z 1103.5385 (calcd.
a
,16
a
-diacetoxy-21
b-O-angeloyloxy-23,28-dihydroxyo-
ꢂ
heating at 60 C for 2 h, the solvent was evaporated under N
2
, and
a
-O-(2-methylbutanoyloxy) olean 3
b
-O-a
-L-arabi-
0
.2 mL trimethylsilylimidazole was added. Then the mixture was
b
a
-
D
]2
-glucuronopyranoside):
White
ꢂ
heated at 60 C for another 2 h and partitioned between n-hexane
and water. The organic layer was investigated by GC under the
following conditions: FID detector with a temperature of 280 C;
injector temperature 250 C; initial temperature 160 C, then
raised to 280 C at 5 C/min, final temperature maintained for
0
D
+ 5.3 (c 0.11, MeOH); UV (MeOH)
l
max
(
1
e
): 206 (4.23), 254 (3.84) nm; IR
n
max (KBr): 3454, 2986, 1605,
ꢂ
ꢀ
1
+
ꢂ
ꢂ
21Na, 1103.5403); 1H NMR (C
5 5
D N, 125 MHz) data are given in Tables 1 and 2.
N, 500 MHz) and
13
C
for C55
NMR (C
H
84
O
5
D
5
ꢂ
ꢂ
3
1
0 min; carrier gas N
following sugar units of each compound was identified
by comparison with authentic sample: t (min) 19.770 min
-glucuronic acid), 33.284 min ( -arabinos).
2
(2.0 kg/cm ). Under these conditions, the
3
.6. Oleiferoside R (3)
R
(D
L
(
16
ene 22
nosyl-(1 !3)-
a
-acetoxy-21
-O-(2-methylbutanoyloxy) olean 3
-glucuronopyranoside): white amorphous pow-
+ 2.1 (c 0.10, MeOH); UV (MeOH) max (log ): 208
4.28), 254 (3.85) nm; IR max (KBr): 3456, 2934, 1604, 1602, 1183,
b
-O-angeloyloxy-23,28-diahydroxyolean-12-
a
b
-O- -arabinopyra-
a-L
4
. Antiumoral cytotoxic bioassays
The viability of the cells after treated with the isolated saponins
b-D
2
0
der; [
a
]
D
l
e
(
1
C
n
against A549, SMCC-7721 and MCF-7 human tumor cell lines (Cell
Bank, Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences) was evaluated using the MTT assay performed as
previously reported (Wang et al., 2008). Cells were plated into 96-
ꢀ1
+
048 cm
; HR–ESI–MS [M + Na] m/z 1045.5349 (calcd. for
1
13
53
H
82
O
19Na, 1045.5348); H NMR (C
5
D
5
N, 500 MHz) and
C
NMR (C
5
D
5
N, 125 MHz) data are given in Tables 1 and 2.
5
well flat-bottomed cultured plates at a concentration of 1 ꢁ10
3
.7. Oleiferoside S (4)
cells per well in fresh culture medium. After 24 h, the test solutions
were applied to the cells in different final concentrations at 6.25,
(
16
2-ene, 22
pyranosyl-(1 !3)-
a
-acetoxy-21
-O-(2-methylbutanoyloxy) olean 3
-D-glucuronopyranoside): white amorphous
+ 2.5 (c 0.12, MeOH); UV (MeOH) max (log ): 206
4.29), 254 (2.82) nm; IR max (KBr): 3455, 2732, 1603, 1601, 1182,
b
-O-angeloyloxy-15
a
, 23,28-trihydroxyolean-
1
2.5, 25, 50, 100
m
M. After 24 h, the MTT solution was added to the
1
a
b
-O- -L-arabino-
a
ꢂ
wells and the plates were incubated at 37 C for 4 h. After the
b
medium was removed, 100 mL of DMSO was added to each well.
2
0
powder; [
a
]
D
l
e
The plants were gently against until the color reaction was uniform
and the OD570 was determined using a microplate reader. The 50%
inhibitory concentration (IC50) was defined as the concentration
that reduced the absorbance of the untreated wells by 50% of the
vehicle in the MTT assay(Zhang et al., 2015). The IC50 values (50%
inhibitory concentration) of compounds 1–5 are shown in Table 3.
(
1
n
ꢀ
1
+
046 cm
; HR-ESI–MS [M + Na] m/z 1061.5247 (calcd. for
1
13
C
53
H
82
O
20Na, 1061.5297); H NMR (C
5
D
5
N, 500 MHz) and
C
NMR (C
5
D
5
N, 125 MHz) data are given in Tables 1 and 2.
3
.8. Oleiferoside T (5)
Appendix A. Supplementary data
(
16
lean-12-ene, 3
pyranoside): white amorphous powder; [
MeOH); UV (MeOH) max (log
max(KBr): 3456, 2963, 1604, 1602, 1168, 1040 cm ; HR–ESI–MS
a
-acetoxy-21
b
-
,22
a-O-diangeloyloxy-23, 28-dihydroxyo-
b
-O-a
L-arabinopyranosyl-(1 !3)-
b
-
D
-glucurono-
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2015.08.005.
2
0
a
]
D
+ 5.6 (c 0.12,
l
e): 210 (4.18), 254 (4.34) nm; IR
ꢀ1
n
+
1
[
M + Na] 1043.5184 (calcd. for C53
D
H
D
80
O
19Na, 1043.5192); H NMR
References
13
(
C
5
5
N, 500 MHz) and C NMR (C
5
5
N, 125 MHz) data are given in
Tables 1 and 2.
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