New triterpenoid glycosides from the roots of camellia oleifera abel

Article abstract of DOI:10.1002/hlca.201400208

Five new triterpenoid saponins, oleiferosides I-M (1-5, resp.) were isolated from the roots of Camellia oleifera Abel. Their structures were elucidated by a combination of 1D- and 2D-NMR spectroscopy, mass spectrometry, and chemical methods. All the compounds were identified as oleanane-type saponins with sugar moieties linked to C(3) of the aglycone. In addition, cytotoxic activities of these saponins were evaluated against four human tumor cell lines (A549, B16, BEL-7402, and MCF-7) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro assay. All of the compounds showed significant cytotoxic activities against the tested cell lines.

Full text of DOI:10.1002/hlca.201400208

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Helvetica Chimica Acta – Vol. 98 (2015)
New Triterpenoid Glycosides from the Roots of Camellia oleifera Abel
a
b
a
a
a
b
a b
by Xia Li ), Jianping Zhao ), Xiaoran Li ), Yanli Liu ), Qiongming Xu* ) ), Ikhlas A. Khan ) ), and
a
Shilin Yang )
a) College of Pharmaceutical Science, Soochow University, Suzhou 215123, P. R. China
(
phone: þ 86-512-69561421; fax: þ 86-512-65882089; e-mail: xuqiongming@suda.edu.cn)
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
Five new triterpenoid saponins, oleiferosides I – M (1 – 5, resp.) were isolated from the roots of
Camellia oleifera Abel. Their structures were elucidated by a combination of 1D- and 2D-NMR
spectroscopy, mass spectrometry, and chemical methods. All the compounds were identified as oleanane-
type saponins with sugar moieties linked to C(3) of the aglycone. In addition, cytotoxic activities of these
saponins were evaluated against four human tumor cell lines (A549, B16, BEL-7402, and MCF-7) by
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro assay. All of the
compounds showed significant cytotoxic activities against the tested cell lines.
Introduction. – The genus Camellia (Theaceae family) consists of 119 species,
which are widely distributed in China, Japan, India, and many other South-East Asian
countries [1]. As a woody crop, C. oleifera Abel is widely cultivated in P. R. China,
Japan, and Southeast Asia for producing cooking oil from the seeds [2]. The oil is rich in
unsaturated fatty acids and comparable to olive oil [3]. Not only a crop, the plant is also
used for treatment of diseases in folk medicine. Phytochemical studies led to the
isolation of flavonoids, bibenzyl glycosides, and saponins from the seeds, seed cake, or
flowers [4 – 7]. Pharmacological investigations revealed antioxidant, hepatoprotective,
cytotoxic, and cell-injury-protective activities for these compounds [3 – 7]. The roots of
C. oleifera have long been used as a traditional Chinese medicine for the treatment of
common cold, ardent fever, urinary tract infection, nephritis, edema, and threatened
abortion [8]. According to the Traditional Chinese Medicine, the roots, which are bitter
and cool-natured, can invigorate lung and kidney, and can expel ꢀwindꢁ and ꢀheatꢁ.
Significant cytotoxic activities against A549 human lung adenocarcinom, B16 mouse
melanoma, BEL-7402 human hepatocarcinoma, and MCF-7 human breast carcinoma
cell lines were found for the extract of the roots of C. oleifera in our previous
investigation [9]. To date, 17 triterpenoids have been identified from the root extract,
and many of them showed potent cytotoxic activities [8][9]. Here, we describe the
isolation and structure elucidation of five new triterpenoid saponins, 1 – 5 (Fig. 1).
Their cytotoxic activities against A549, B16, BEL-7402, and MCF-7 human tumor cell
lines were evaluated by using MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide) assay.
ꢂ 2015 Verlag Helvetica Chimica Acta AG, Zürich

Helvetica Chimica Acta – Vol. 98 (2015)
497
Fig. 1. Structures of compounds 1 – 5
Results and Discussion. – Compound 1 was obtained as white amorphous powder.
The negative-ion HR-ESI-mass spectrum of 1 exhibited a quasi-molecular-ion peak
À
[
M À H] at m/z 993.5087, indicating the molecular formula of C H O . The IR
5
1
78 19
À1
spectrum showed the presence of OH (3456 cm ) and a,b-unsaturated ester groups
1601, 1603 cm ). The C-NMR (Table 1) spectrum exhibited the resonances of 51 C-
À1
13
(
atoms, ascribable to ten Me, ten CH , twenty CH groups, and eleven quaternary C-
2
1
atoms as revealed by the HSQC experiment. The H-NMR (Table 2) spectrum showed
six Me signals at d(H) 0.70 (Me(24)), 0.87 (Me(29)), 0.99 (Me(25)), 1.00 (Me(26)),
1
1
.09 (Me(30)), and 1.42 (Me(27)), two isolated CH ÀO signals at d(H) 3.34 (d, J ¼11.5,
2
H, CH (23)) and 3.64 (d, J ¼11.5, 1 H, CH (23)), as well as d(H) 3.05 (d, J ¼12.0, 1 H,
2
2
CH (28)) and 3.30 (d, J ¼12.0, 1 H, CH (28)), five CHÀO signals at d(H) 3.76 (d, J ¼
2
2
4
1
5
.5, HÀC(15)), 3.55 (d, J ¼ 4.5, HÀC(16)), 3.65 (dd, J ¼ 11.0, 4.5, HÀC(3)), 5.58 (d, J ¼
0.5, HÀC(22)) and 5.93 (d, J ¼ 10.5, HÀC(21)), and one olefinic H-atom signal at d(H)
.49 (t, J ¼ 4.0, HÀC(12)). Furthermore, the H-atom signals of two angeloyl (Ang)
groups (d(H) 6.05 (dq, J ¼ 7.0, 1.5, HÀC(3’), 21-O-Ang), 1.92 (d, J ¼ 7.5, Me(4’), 21-O-
Ang) and 1.82 (s, Me(5’), 21-O-Ang), and d(H) 5.94 (dq, HÀC(3’’), J ¼ 7.0, 1.5, 22-O-
Ang), 1.90 (d, Me(4’’), J ¼ 7.5, 22-O-Ang) and 1.82 (s, Me(5’’), 22-O-Ang)) were
observed. The NMR data were similar to those reported for oleiferoside E [9], and the
3
main difference was that two sp -C-atoms were replaced by olefinic C-atoms, which was
indicated by the significant down-field shifts of the C(2’’) signal of 22-O-Ang (Dd(C)
þ 88.0 ppm) at d(C) 129.6 and the C(3’’) signal of 22-O-Ang (Dd(C) þ 112.5 ppm) at
1
1
d(C) 139.5. The analyses of the H, H-COSY, HSQC, and HMBC spectra revealed that
an Ang group was attached at C(22) in compound 1, instead of a 2-methylbutanoyl
(
3
MB) group in oleiferoside E. Therefore, compound 1 had a 21b,22a-bis(angeloyloxy)-
b,15a,16a,23a,28-pentahydroxyolean-12-ene type of aglycone. The locations of the
two Ang groups at C(21) and C(22) were confirmed by the HMBC experiment (Fig. 2).
The presence of a OH group at C(23) was confirmed by the correlations between
CH (23) (d(H) 3.34 and 3.64) and C(4) (d(C) 44.1), and between CH (23) and C(24)
2
2
(d(C) 13.7) in the HMBC spectrum, as well as the correlation between HÀC(3) (d(H)

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Helvetica Chimica Acta – Vol. 98 (2015)
Table 1. 13C-NMR Data (125 MHz) ) of Compounds 1 – 5. d in ppm.
a
Position
d(C)
1b
)
2c)
39.1
3c)
38.9
4c)
38.3
5c)
39.0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
40.1
26.7
83.6
44.1
48.4
19.4
36.9
42.6
48.2
38.0
25.1
127.3
143.9
48.8
68.9
74.5
48.7
41.9
47.8
37.1
79.9
74.2
65.2
13.7
17.0
18.2
21.4
64.1
29.9
20.5
26.7
89.7
39.7
55.7
19.0
36.8
41.6
47.2
37.1
24.1
125.7
143.8
47.9
67.7
73.8
48.5
41.1
47.0
36.5
78.8
73.5
28.2
17.0
16.0
17.7
21.4
63.3
29.7
20.4
26.4
89.4
39.5
55.5
18.7
36.6
41.4
46.7
36.8
23.9
125.3
143.5
47.6
67.4
73.0
48.4
40.7
47.0
36.2
78.5
73.0
27.9
16.8
15.7
17.4
21.1
62.8
29.3
20.1
25.4
84.7
55.3
48.5
20.4
32.5
41.8
46.9
36.2
23.9
123.7
143.0
40.4
34.9
68.7
48.2
40.2
47.3
36.5
78.9
73.8
210.5
11.2
16.0
17.0
27.7
63.8
29.7
20.4
26.7
89.8
39.8
55.9
18.6
33.3
41.8
47.0
36.9
24.0
123.7
143.0
40.2
35.0
68.5
48.3
40.2
47.3
36.6
78.9
73.7
28.3
17.0
15.8
17.1
27.7
63.8
29.7
20.5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
2
1
2
3
4
5
1-O-Ang
’
’
’
’
’
169.5
129.6
139.3
16.3
167.9
129.1
137.5
16.1
167.5
128.6
138.4
15.9
167.8
129.1
137.3
16.0
167.7
128.9
138.6
16.2
21.2
21.1
20.9
21.1
21.2
2
1
2
3
4
5
2-O-Ang
’’
’’
’’
’’
’’
169.8
129.6
139.5
16.2
168.3
129.3
136.7
15.8
168.3
129.1
137.3
15.9
21.1
20.8
21.0
2
1
2
3
4
5
2-O-MB
’’
’’
’’
’’
’’
176.5
41.4
26.8
11.7
16.5
176.9
41.8
27.1
12.1
16.9

Helvetica Chimica Acta – Vol. 98 (2015)
499
Table 1 (cont.)
Position
d(C)
1b
)
2c)
3c)
4c)
5c)
Sugar units
GlcA
105.8
74.9
86.7
72.0
GlcA
105.5
79.0
84.5
70.4
GlcA
105.2
78.5
84.2
70.1
GlcA
104.0
78.3
85.1
70.7
GlcA
105.6
79.0
84.8
71.4
1
2
3
4
5
6
76.7
172.9
76.8
172.5
76.7
77.2
77.1
173.2
d
d
nd )
nd )
Ara
105.8
74.8
73.1
69.9
67.7
Gal 1
101.9
83.6
75.1
71.5
Gal 1
101.6
83.4
74.8
71.2
Gal 1
101.8
84.1
75.1
71.0
Gal 1
101.9
83.8
75.1
70.1
77.0
62.0
1
2
3
4
5
6
76.8
62.1
76.2
61.8
77.1
62.0
Xyl
Xyl
Xyl
Xyl
1
2
3
4
5
107.5
76.2
78.3
70.9
67.7
107.2
76.0
78.1
70.7
67.4
107.8
76.4
78.4
71.0
67.7
107.6
76.2
78.4
70.9
67.7
Gal 1’
103.2
74.0
75.2
70.4
Gal 1’
102.9
73.7
75.0
70.1
Gal 1’
103.0
73.9
75.5
70.0
Gal 1’
103.2
74.0
75.2
70.4
1
2
3
4
5
6
76.4
62.3
76.2
62.0
76.5
62.3
76.5
62.3
a) The assignments were based on HSQC, HMBC, and NOSEY spectra. b) The NMR spectra were
c
d
recorded in CD OD. ) The NMR spectra were recorded in (D )pyridine. ) nd, Not detected.
3
5
3
.65) and CH (23) (d(H) 3.34 and 3.64) in the NOESY spectrum (Fig. 2). The
2
anomeric H-atom signals at d(H) 4.50 (d, J ¼ 7.5, 1 H) and 4.50 (d, J ¼ 7.5, 1 H), which
showed the HSQC correlations to the signals at d(C) 105.8 and 105.8, respectively,
indicated the presence of two sugar residues. Acid hydrolysis of compound 1 and GC
analysis revealed the presence of one d-glucuronic acid and one l-arabinose moieties.
The HMBC correlations between d(H) 4.50 (HÀC(1) of glucuronic acid) and d(C) 83.6
(
(
C(3) of aglycone), and between d(H) 3.65 (HÀC(3) of aglycone) and d(C) 105.8
C(1) of glucuronic acid) (Fig. 2) suggested that the glycosidic chain was located at
C(3) of the aglycone. The linkage of the glycosidic chain was determined by the HMBC
correlations between d(H) 4.50 (HÀC(1) of arabinose) and d(C) 86.7 (C(3) of
glucuronic acid), and between d(H) 3.56 (HÀC(3) of glucuronic acid) and d(H) 105.8
(
C(1) of arabinose). The b-anomeric configuration of the glucuronopyranosyl unit was
3
determined from the observation of the large J(1,2) coupling constant, and the a-

5
00
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Helvetica Chimica Acta – Vol. 98 (2015)
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5
02
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Fig. 2. Key HMBCs (H ! C) and NOESY (H
H) correlations of compounds 1 – 5

5
04
Helvetica Chimica Acta – Vol. 98 (2015)
anomeric configuration of arabinopyranosyl unit was assigned according to the
NOESY correlations between d(H) 4.50 (HÀC(1) of arabinose) and d(H) 3.65
(
HÀC(3) of arabinose) (Fig. 2). On the basis of the above analysis, the structure of 1
was elucidated as (3b,15a,16a,21b,22a)-15,16,23,28-tetrahydroxy-21,22-bis{[(2Z)-2-
methylbut-2-enoyl]oxy}olean-12-en-3-yl 3-O-a-l-arabinopyranosyl-b-d-glucopyrano-
siduronic acid, and named as oleiferoside I.
Compound 2 was obtained as white amorphous powder. Its molecular formula of
À
C H O was deduced from the negative-ion-mode peak [M À H] at m/z 1301.6198 in
63
98 28
À1
its HR-ESI mass spectrum. The IR spectrum indicated the presence of OH (3453 cm )
and a,b-unsaturated ester groups (1602, 1604 cm ). The aglycone structure of 2 was
À1
similar to that of 1, and the main difference arose from the significant chemical-shift
change of C(23), which was not hydroxylated in 2. The assignment was confirmed by
the observation of HMBC correlations between C(23) at (d(C) 28.2) and Me(24)
(
3
d(H) 1.23). Thus, the aglycone of 2 was elucidated as 21b,22a-bis(angeloyloxy)-
b,15a,16a,28-tetrahydroxyolean-12-ene. The H-NMR spectrum of 2 indicated the
1
presence of four sugar residues, which were identified as one d-glucuronic acid, two d-
1
galactoses, and one d-xylose by the acid hydrolysis and GC analysis. The H- and
13
C-NMR data for the sugar units of 2 were the same as those of oleiferoside A [9],
suggesting that the glycosidic chain was b-d-galactopyranosyl-(1 ! 2)-[b-d-xylopyra-
nosyl-(1 ! 2)-b-d-galactopyranosyl-(1 ! 3)]-b-d-glucuronopyranosyl. The b-anomeric
configurations of the four sugar units were deduced from the observation of their large
3
J(1,2) coupling constants. Therefore, the structure of 2 was elucidated as
(
3b,15a,16a,21b,22a)-15,16,28-trihydroxy-21,22-bis{[(2Z)-2-methylbut-2-enoyl]oxy}o-
lean-12-en-3-yl b-d-galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-b-d-galacto-
pyranosyl-(1 ! 3)]-b-d-glucopyranosiduronic acid, and named as oleiferoside J.
Compound 3 was obtained as white amorphous powder. Its molecular formula,
À
C H O , was determined by the quasi-molecular-ion peak [M À H] at m/z
63
100 28
1
303.6311 in its HR-ESI mass spectrum. The IR spectrum indicated the presence of
À1
À1
À1
OH (3452 cm ), C¼O (1720 cm ), and a,b-unsaturated ester groups (1600 cm ). The
NMR spectra data of 3 were closely similar to those of 2, except that two olefinic C-
3
atoms were replaced by sp -C-atoms, which suggested that a MB instead of a Ang group
was attached at C(22). The assignment was supported by the HMBC correlations
between d(H) 6.32 (HÀC(22)) and d(C) 176.5 (C(1’’) of MB), between d(H) 0.73
(
HÀC(4’’) of MB) and d(C) 41.4 (C(2’’) of MB) and 26.8 (C(3’’) of MB), and between
d(H) 1.07 (HÀC(5’’) of MB) and d(C) 176.5 (C(1’’) of MB), 41.4 (C(2’’) of MB), and
2
1
6.8 (C(3’’) of MB). Thus, the structure of 3 was determined as (3b,15a,16a,21b,22a)-
5,16,28-trihydroxy-22-[(2-methylbutanoyl)oxy]-21-{[(2Z)-2-methylbut-2-enoyl]oxy}-
olean-12-en-3-yl b-d-galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-b-d-galac-
topyranosyl-(1 ! 3)]-b-d-glucopyranosiduronic acid, and named as oleiferoside K.
Compound 4 was obtained as white amorphous powder. Its molecular formula,
À
C H O , was deduced from the negative-ion-mode peak [M À H] at m/z 1299.5965
6
3
96 28
in its HR-ESI mass spectrum. The IR spectrum indicated the presence of OH
3452 cm ) and a,b-unsaturated ester groups (1602, 1604 cm ). The NMR spectra
À1
À1
(
data of 4 were similar to those of 2, and one major difference was the significant
chemical-shift change of the signals of C(15) (Dd(C) À 32.8 ppm) at d(C) 34.9, of
C(14) (Dd(C) À 7.5 ppm) at d(C) 40.4, and of C(16) (Dd(C) À 5.1 ppm) at d(C) 68.7,

Helvetica Chimica Acta – Vol. 98 (2015)
505
due to the presence of a CH (15) group in compound 4 rather than a CH(15) group in
2
compound 2. Another difference was the significant chemical-shift change of C(23),
which was part of an aldehyde group in compound 4. The assignment was confirmed by
the observation of HMBC correlations between C(23) (d(C) 210.5) and Me(24) (d(H)
1.55). Therefore, the structure of 4 was elucidated as (3b,16a,21b,22a)-16,28-
dihydroxy-21,22-bis{[(2Z)-2-methylbut-2-enoyl]oxy}-23-oxoolean-12-en-3-yl b-d-gal-
actopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-b-d-galactopyranosyl-(1 ! 3)]-b-d-
glucopyranosiduronic acid, and named as oleiferoside L.
Compound 5 was obtained as white amorphous powder. The negative-ion-mode
À
HR-ESI-MS of 5 exhibited a quasi-molecular-ion peak [M À H] at m/z 1287.6391,
indicating the molecular formula C H O . The IR spectrum showed the signals of
6
3
100 27
À1
À1
À1
OH (3450 cm ), C¼O (1720 cm ), and a,b-unsaturated ester group (1600 cm ). The
NMR data of 5 were similar to those of 3, and the main differences arose from the
significant upfield shifts of the signals of C(15) (Dd(C) À 32.4 ppm) at d(C) 35.0, of
C(14) (Dd(C) À 7.4 ppm) at d(C) 40.2, and of C(16) (Dd(C) À 4.5 ppm) at d(C) 68.5,
suggesting that the HÀC(15) in 3 was replaced by a CH (15) group in 5. Moreover, the
2
aglycone structure of 5 was also confirmed by comparing its spectroscopic data with
those of 21b-(angeloyloxy)-22a-(2-methylbutanoyloxy)-3b,16a,28-trihydroxyolean-
12-ene [8] and aesculioside IVc [10]. Therefore, the structure of 5 was elucidated
as (3b,16a,21b,22a)-16,28-dihydroxy-22-[(2-methylbutanoyl)oxy]-21-{[(2Z)-2-methyl-
but-2-enoyl]oxy}olean-12-en-3-yl b-d-galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-
(
1 ! 2)-b-d-galactopyranosyl-(1 ! 3)]-b-d-glucopyranosiduronic acid, and named as
oleiferoside M.
Bioactivities. Cytotoxic activities of these saponins against four human tumor cell
lines (A549, B16, BEL-7402, and MCF-7) were evaluated by using the MTT in vitro
assay. All compounds had significant cytotoxic activity against A549 (human lung
adenocarcinoma, B16 (mouse melanoma), BEL-7402 (human hepatocarcinoma), and
MCF-7 (human breast carcinoma) (Table 3).
A variety of triterpenoid saponins have been isolated from Camellia plants, and C.
sinensis and C. sinensis var. assamica are the two species mostly investigated [1]. The
present report on five new triterpenoid saponins from C. oleifera and their cytotoxic
activities may enrich our knowledge about the constituents of the genus Camellia from
a chemotaxonomic perspective.
Table 3. In vitro Cytotoxic Activities of Compounds 1 – 5
Compound
Cell line IC50 [mm]
A549
B16
7402
MCF-7
1
2
3
4
23.45 Æ 0.28
11.37 Æ 0.14
29.73 Æ 1.48
10.63 Æ 0.07
12.03 Æ 0.24
4.16 Æ 0.28
20.46 Æ 0.65
13.15 Æ 0.95
19.47 Æ 1.51
13.14 Æ 0.24
10.81 Æ 0.53
3.93 Æ 0.22
15.71 Æ 1.72
12.02 Æ 0.16
28.12 Æ 1.85
12.73 Æ 0.84
12.59 Æ 0.16
3.45 Æ 0.26
17.22 Æ 2.42
11.95 Æ 0.45
14.00 Æ 1.92
11.95 Æ 1.20
12.76 Æ 0.42
5.17 Æ 0.38
5
Norcantharidin

5
06
Helvetica Chimica Acta – Vol. 98 (2015)
This work was supported by the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD). We are grateful to Dr. Jon Parcher at the University of Mississippi for
helpful discussions.
Experimental Part
General. TLC: Pre-coated silica-gel plates (SiO ; Qingdao Marine Chemical Factory); visualization
2
with 10% H SO alcohol soln. Column chromatography (CC): silica gel (SiO , 200 – 300 mesh; Qingdao
2
4
2
Marine Chemical Factory) and Sephadex LH-20 (GE Healthcare Corp., Beijing, P. R. China). GC/MS:
GC-14C (Shimadzu Corp.) with a flame ionization detector (FID). HPLC: Agilent Corp. SB-C18 semi-
prep. HPLC column (250 Â 9.4 mm i.d., 5 mm; Agilent Corp., Palo Alto, CA, USA) on a Shimadzu HPLC
system composed of a LC-20AT pump with a SPD-20A detector (Shimadzu Corp., Kyoto, Japan); the
wavelength for detection, 203 nm. Medium pressure liquid chromatography (MPLC): Büchi flash
chromatography system composed of a C-650 pump with a flash column (460 mm  26 mm i.d., Büchi
Corp., CH-Flawil) and ODS (Merck KGaA, DE-Darmstadt). Specific rotations: PerkinÀElmer model
2
41 polarimeter (PerkinElmer Inc., Waltham, MA, USA). IR Spectra: PerkinÀElmer 983 G spectrom-
1 13
eter (PerkinElmer Inc., Waltham, MA, USA). H-, C-, and 2D-NMR spectra: Varian Inova 500
spectrometer (Varian Inc., Palo Alto, CA, USA) in (D )pyridine or CD OD; d in ppm rel. to Me Si as
5
3
4
internal standard, J in Hz. HR-ESI-MS: Micromass Q-TOF 2 spectrometer (Waters Corp., London, UK);
in m/z.
Plant Material. The roots of C. oleifera Abel were collected in Qichun, Hubei province of P. R.
China in November 2011, and identified by Prof. Xiao-Ran Li at Soochow University. A voucher sample
(
No. 11-11-06-01) was deposited with the herbarium of the College of Pharmacy, Soochow University.
Extraction and Isolation. The air-dried roots (9.3 kg) of C. oleifera were crushed with a grinder into
fine debris, and extracted twice with 70% EtOH (2 Â 50 l) at 808 under reflux. The solvent was
subsequently removed under reduced pressure to yield a residue (0.45 kg), which was then dissolved in
dist. H O and passed through a D101 macroporous resin column (i.d. 30 cm  200 cm; Xiꢀan Sunresin
2
New Materials Co. Ltd.). The column was eluted with gradient aq. EtOH (0, 30, 60, and 80%; each 40 l).
The 60% EtOH eluate (42 g) was further vacuum chromatographed (SiO (60 – 100 mesh; 30 Â 18 cm);
2
CHCl /MeOH 100 :0, 90 :10, 80 :20, 70 :30, 60 :40, 50 :50, 40 :60, 30 :70, 0 :100; each 6.0 l). The CHCl /
3
3
MeOH 60 :40 eluate (4.5 g) was subjected to MPLC (ODS; MeOH/H O 50 :50, 60 :40, 70 :30, 80 :20,
2
9
0 :10, 100 :0; each 500 ml; flow rate, 20.0 ml/min) to afford six fractions. Fr. 3 (500 – 1000 ml, 68 mg) was
separated by semi-prep. HPLC (ODS; MeOH/H O 70 :30) to yield compounds 5 (t 50.7 min; 12 mg), 2
2
R
(tR 56.4 min; 16 mg), and 3 (tR 58.2 min; 12 mg). In a similar way, Fr. 5 (125 mg) was separated by semi-
prep. HPLC (MeOH/H O 60 :40) to give compound 1 (t 66.5 min; 12 mg). Fr. 4 was purified by semi-
2
R
prep. HPLC (MeOH/H O 65 :35) to yield compound 4 (t 70.7 min; 16 mg). The purities of the isolated
2
R
1
saponins were > 93% (determined by H-NMR).
Oleiferoside I ( ¼ (3b,15a,16a,21b,22a)-15,16,23,28-Tetrahydroxy-21,22-bis{[(2Z)-2-methylbut-2-
enoyl]oxy}olean-12-en-3-yl 3-O-a-l-Arabinopyranosyl-b-d-glucopyranosiduronic Acid; 1). White amor-
2
0
phous powder. [a] ¼ þ6.6 (c ¼ 0.11, MeOH ). UV (MeOH): 206 (4.26), 254 (4.22). IR (KBr): 3456
D
1
(
OH), 2963, 2925, 2850, 1603 (C¼CÀC¼O), 1601 (C¼CÀC¼O), 1460, 1372, 1161, 1042. H- (500 MHz,
1
3
À
CD OD) and C-NMR (125 MHz, CD OD): Tables 2 and 1, resp. HR-ESI-MS: 993.5087 ([M À H] ,
3
3
À
77 19
C H O ; calc. 993.5059).
51
Oleiferoside J (¼(3b,15a,16a,21b,22a)-15,16,28-Trihydroxy-21,22-bis{[(2Z)-2-methylbut-2-enoyl]-
oxy}olean-12-en-3-yl b-d-Galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-b-d-galactopyranosyl-
2
0
(
1 ! 3)]-b-d-glucopyranosiduronic Acid; 2). White amorphous powder. [a] ¼ þ1.2 (c ¼ 0.10, MeOH).
D
UV (MeOH): 209 (4.25), 254 (3.84). IR (KBr): 3453 (OH), 2960, 2918, 2850, 1604 (C¼CÀC¼O), 1602
1
13
(
(
C¼CÀC¼O), 1460, 1374,1180, 1046. H- (500 MHz, (D )pyridine) and C-NMR (125 MHz,
5
À
À
D )pyridine): Tables 2 and 1, resp. HR-ESI-MS: 1301.6198 ([M À H] , C H O ; calc. 1301.6166).
5
63 97 28
Oleiferoside
K
(¼(3b,15a,16a,21b,22a)-15,16,28-Trihydroxy-22-[(2-methylbutanoyl)oxy]-21-
{
(
[(2Z)-2-methylbut-2-enoyl]oxy}olean-12-en-3-yl b-d-Galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-
1 ! 2)-b-d-galactopyranosyl-(1 ! 3)]-b-d-glucopyranosiduronic Acid; 3). White amorphous powder.

Helvetica Chimica Acta – Vol. 98 (2015)
507
2
0
[
a] ¼ þ1.0 (c ¼ 0.10, MeOH ). UV (MeOH): 210 (4.26), 254 (3.85). IR (KBr): 3452 (OH), 2960, 2916,
D
1
2
852, 1720 (C¼O), 1600 (C¼CÀC¼O), 1460, 1376,1183, 1048. H- (500 MHz, (D )pyridine) and
5
13
À
À
;
63 99 28
C-NMR (125 MHz, (D )pyridine): Tables 2 and 1, resp. HR-ESI-MS: 1303.6311 ([M À H] , C H O
5
calc. 1303.6323).
Oleiferoside L (¼(3b,16a,21b,22a)-16,28-Dihydroxy-21,22-bis{[(2Z)-2-methylbut-2-enoyl]oxy}-23-
oxoolean-12-en-3-yl b-d-Galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-b-d-galactopyranosyl-
2
0
(
1 ! 3)]-b-d-glucopyranosiduronic Acid; 4). White amorphous powder. [a] ¼ À2.6 (c ¼ 0.10, MeOH).
D
UV (MeOH): 208 (4.29), 254 (3.82). IR (KBr): 3452 (OH), 2960, 2912, 2856, 1604 (C¼CÀC¼O), 1602
1
13
(
(
C¼CÀC¼O), 1462, 1379, 1182, 1046. H- (500 MHz, (D )pyridine) and C-NMR (125 MHz,
5
À
À
D )pyridine): Tables 2 and 1, resp. HR-ESI-MS: 1299.5965 ([M À H] , C H O28 ; calc. 1299.6010).
5
63 95
Oleiferoside M (¼(3b,16a,21b,22a)-16,28-Dihydroxy-22-[(2-methylbutanoyl)oxy]-21-{[(2Z)-2-
methylbut-2-enoyl]oxy}olean-12-en-3-yl b-d-Galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-b-d-
2
0
galactopyranosyl-(1 ! 3)]-b-d-glucopyranosiduronic Acid; 5). White amorphous powder. [a] ¼ þ4.6
D
(
(
(
1
c ¼ 0.12, MeOH ). UV (MeOH): 211 (4.18), 254 (4.34). IR (KBr): 3450 (OH), 2963, 2920, 2850, 1720
1
13
C¼O), 1600 (C¼CÀC¼O), 1441, 1382, 1168, 1040. H- (500 MHz, (D )pyridine) and C-NMR
5
À
À
63 99 27
125 MHz, (D )pyridine): Tables 2 and 1, resp. HR-ESI-MS: 1287.6391 ([M À H] , C H O ; calc.
5
287.6374).
Acid Hydrolysis and Sugar Analysis of Compounds 1 – 5. Each saponin (2 mg) was dissolved in 2n
HCl (2 ml) and stirred at 808 for 4 h. The mixture was extracted with CHCl , and the aq. layer was
3
evaporated to give a mixture of monosaccharides. The residue was dissolved in anh. pyridine (1 ml),
followed by the addition of 2 mg of l-cysteine methyl ester hydrochloride (Tokyo Chemical Industry,
9
9%). After heating at 608 for 2 h, the solvent was eliminated under N , and 0.2 ml 1-trimethylsilyl-1H-
2
imidazole (Tokyo Chemical Industry, 99%) was added. Then, the mixture was heated at 608 for another
h, and partitioned between hexane and H O. The org. layer was investigated by GC under the following
2
2
conditions: cap. column, HP-5 (30 m  0.25 mm  0.25 mm; Dikma); FID detector with a temp. of 2808;
injection temp., 2508; initial temp., 1608, then raised to 2808 at 58/min, final temp., maintained for 10 min;
carrier gas, N . The standard sugars experienced the same reaction and GC conditions. The retention
2
times (t ) of d-glucuronic acid, d-galactose, d-xylose, and l-arabinose were 19.770, 25.643, 32.257, and
R
3
3.284 min, resp.
Cytotoxicity Assay. The MTT colorimetric assay was performed to evaluate the cytotoxic activities of
the isolated saponins against A549, B16, BEL-7402, and MCF-7 human tumor cell lines (Cell Bank,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). Cells were placed into 96-
5
well flat-bottomed culture plates at a concentration of 2 Â 10 cells per well in complete RPMI 1640
culture medium. After 24 h, the medium containing fetal calf serum was removed, and the test solns. were
applied to the cells in different final concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 50, 100 mm. After 24 h,
MTT soln. was added to the wells, and the plates were incubated at 378 for 4 h. The positive control group
was treated with norcantharidin (purity higher than 99.0% as determined by HPLC; Nanjing Zelang
Medical Technology Co., Ltd., Nanjing, China). The amount of formazan was determined by photometry
at 570 nm. The results were expressed as the percentage of absorbance in the control cells in comparison
to that in the drug-treated cells. The IC50 values (50% inhibitory concentration) of compounds 1 – 5 are
compiled in Table 3. The experiment was performed in triplicate for each sample.
REFERENCES
[
1] P. Zhao, D. F. Gao, M. Xu, Z. G. Shi, D. Wang, C. R. Yang, Y. J. Zhang, Chem. Biodiversity 2011, 8,
931.
1
[
[
[
2] C. P. Lee, G. C. Yen, J. Agric. Food Chem. 2006, 54, 779.
3] C. P. Lee, P. H. Shih, C. L. Hsu, G. C. Yen, Food Chem. Toxicol. 2007, 45, 888.
4] D. F. Gao, M. Xu, P. Zhao, X. Y. Zhang, Y. F. Wang, C. R. Yang, Y. J. Zhang, Food Chem. 2011, 124,
432.
[
[
5] Y. Chen, L. Tang, B. Feng, L. Shi, H. Wang , Y. Wang, Fitoterapia 2011, 82, 481.
6] L. Chen, J. Chen, H. Xu, Fitoterapia 2013, 84, 123.

5
08
Helvetica Chimica Acta – Vol. 98 (2015)
[
7] S. Sugimoto, G. Chi, Y. Kato, S. Nakamura, H. Matsuda, M. Yoshikawa, Chem. Pharm. Bull. 2009, 57,
69.
8] S. L. Wang, Z. Chen, X. J. Tong, Y. L. Liu, X. Li, Q. M. Xu, X. R. Li, S. L. Yang, Helv. Chim. Acta
013, 96, 1126.
9] X. Li, J. Z. Zhao, C. P. Peng, Z. Chen, Y. L. Liu, Q. M. Xu, I. A. Khan, S. L. Yang, Planta Med. 2014,
0, 590.
10] Z. Z. Zhang, S. Y. Li, Phytochemistry 2007, 68, 2075.
2
[
[
2
8
[
Received June 18, 2014