Five new steroidal alkaloid glycosides from solanum tuberosum

Article abstract of DOI:10.1002/hlca.201200269

Five new steroidal alkaloid glycosides, 1-5, along with the known analog 6, have been isolated from the aerial parts of Solanum tuberosum. The structures of the new compounds were elucidated by spectroscopic methods, including 1D- and 2D-NMR and HR-ESI-MS

Full text of DOI:10.1002/hlca.201200269

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Five New Steroidal Alkaloid Glycosides from Solanum tuberosum
by Zhi-Qiang Zhang, Jian-Guang Luo, Jun-Song Wang, and Ling-Yi Kong*
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China (phone: þ 86-25-83271405;
fax: þ 86-25-83271405; e-mail: cpu_lykong@126.com)
Five new steroidal alkaloid glycosides, 1 – 5, along with the known analog 6, have been isolated from
the aerial parts of Solanum tuberosum. The structures of the new compounds were elucidated by
spectroscopic methods, including 1D- and 2D-NMR and HR-ESI-MS techniques, as well as by
comparison of the spectral data with those of related compounds. Only compound 6 showed significant
cytotoxicity.
Introduction. – Steroidal alkaloid glycosides are the main chemical components of
Solanum species, and they exhibit various pharmacological activities such as cytotoxic
[1], antiviral [2], anti-inflammatory [3], and antifungal properties [4], making Solanum
tuberosum an attractive target for the search of health-promoting phytochemicals. With
this aim, we investigated the aerial parts of S. tuberosum, and isolated six steroidal
alkaloid glycosides, 1 – 6, including a rare 14a-hydroxy steroidal alkaloid glycoside, 1,
two new ones, 2 and 4, two novel 7-oxo derivatives, 3 and 5, and the known metabolite 6
(Fig. 1). The cytotoxicities of 1 – 4 and 6 against human cancer cell lines SMMC-7721,
NCI-H460, and A-549 were evaluated. In this article, we reported the isolation,
structure elucidation, and the cytotoxicity evaluation of the compounds isolated from
the aerial parts of this plant.
Results and Discussion. – Structure Elucidation. All compounds, 1 – 6, showed
positive LiebermannꢀBurchard, Dragendorff, and Molish reactions, indicating the
steroidal alkaloid glycoside nature of these compounds. The presence of glucose,
rhamnose, and/or galactose in the hydrolysates of each compound was confirmed by co-
TLC with authentic samples. Glucose, rhamnose, and galactose were assigned d-, l-,
and d-forms, respectively, by GC/MS analysis of their silyl derivatives. The b-anomeric
configurations of the d-glucopyranosyl and d-galactosyl moieties were determined by
the coupling constants (3J(1,2) > 7 Hz), respectively. The a-anomeric configuration of
the l-rhamnopyranosyl group was deduced from the small coupling constant of the
anomeric H-atom and the chemical shifts of C(3) and C(5) [5].
Compound 1 was obtained as a white amorphous powder. Its positive-ion-mode
HR-ESI-MS displayed a quasimolecular-ion peak at m/z 722.4481 ([M þ H]þ; calc.
722.4474) indicating the molecular formula C39H63NO11, with nine degrees of
unsaturation. Its IR spectrum revealed the presence of OH groups (3423 cmꢀ1) and
of an olefinic bond (1641 cmꢀ1). Upon acid hydrolysis, compound 1 afforded an
aglycone, along with d-glucose and l-rhamnose, which were identified by GC/MS
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Fig. 1. Structures of 1 – 6
1
comparison with authentic samples. In the H-NMR spectrum of the aglycone of 1
(Table 1), there were signals of two tertiary Me groups at d(H) 0.96 (s, Me(19)) and
1.20 (s, Me(18)), two secondary Me groups at d(H) 0.79 (d, J ¼ 6.4, Me(27)) and 0.96 (d,
J ¼ 4.8, Me(21)), and an olefinic H-atom at d(H) 5.42 (br. s, HꢀC(6)). The 13C-NMR
spectrum of the aglycone (Table 1) displayed 27 C-atom signals, which were classified
into those of four Me, ten CH2, nine CH, and four quaternary C-atoms (one O-bearing
and one olefinic C-atoms) on the basis of DEPT and HSQC spectra. These spectral
features were characteristic for alkaloids of the solanidine group [6]. The HMBCs
(Fig. 2) Me(19)/C(1), C(5), and C(9); HꢀC(4)/C(2), C(3), C(5), and C(6); HꢀC(6)/
C(7), C(8), and C(10); Me(18)/C(12), C(13), C(14), and C(17); HꢀC(17)/C(13) and
C(16); Me(21)/C(17), C(20), and C(22); and Me(27)/C(24), C(25), and C(26)
confirmed this assumption and also located the OH group signal at d(C) 87.0
(C(14)). In the ROESY spectrum (Fig. 2), HꢀC(3) (d(H) 3.84) was assigned a-

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Table 1. 1H- and 13C-NMR (500 and 125 MHz, resp.) Data the Aglycone Moieties of 1 and 2 in
(D5)Pyridine. d in ppm, J in Hz. Assignments are based on HSQC, HMBC, ROESY, and TOCSY
experiments.
Position
1
2
d(H)
d(C)
d(H)
d(C)
1
2
3
4
1.04 (dd, J ¼ 13.4, 3.4), 1.76 – 1.78 (m)
1.69 – 1.71 (m), 2.03 – 2.05 (m)
3.83 – 3.85 (m)
2.47 – 2.49 (m), 2.70 (dd, J ¼ 13.1, 2.3) 39.6 (t) 2.77 (d, J ¼ 11.5),
37.9 (t) 0.95 – 0.97 (m), 1.70 – 1.73 (m) 37.6 (t)
30.5 (t) 1.85 – 1.88 (m), 2.08 – 2.10 (m) 30.6 (t)
78.5 (d) 3.92 – 3.95 (m)
78.3 (d)
38.9 (t)
2.90 (dd, J ¼ 11.5, 2.9)
140.7 (s)
5
6
7
8
9
142.0 (s)
128.9 (d)
73.2 (d)
41.1 (d)
48.9 (d)
37.5 (s)
21.5 (t)
40.4 (t)
41.4 (s)
57.6 (d)
5.42 (br. s)
1.90 – 1.93 (m), 2.51 – 2.54 (m)
2.03 – 2.05 (m)
122.7 (d) 5.74 (br. s)
26.9 (t) 4.03 (d, J ¼ 8.0)
35.9 (d) 1.78 – 1.80 (m)
43.9 (d) 1.08 – 1.10 (m)
37.6 (s)
20.7 (t) 1.41 – 1.43 (m), 1.47 – 1.50 (m)
32.4 (t) 1.13 – 1.15 (m), 1.67 – 1.70 (m)
45.3 (s)
1.80 – 1.82 (m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1.37 – 1.40 (m), 1.55 (br. d, J ¼ 6.4)
1.42 – 1.45 (m), 2.25 – 2.27 (m)
87.0 (s) 1.38 – 1.40 (m)
1.72 – 1.74 (m), 2.08 – 2.11 (m)
3.25 (br. s)
2.47 – 2.50 (m)
1.20 (s)
0.96 (s)
38.9 (t) 1.46 – 1.48 (m), 2.69 – 2.71 (m) 33.4 (t)
69.9 (d) 3.04 (br. s)
59.9 (d) 1.62 – 1.64 (m)
20.5 (q) 0.97 (s)
70.6 (d)
62.4 (d)
18.0 (q)
17.0 (q)
37.2 (d)
19.1 (q)
75.5 (d)
19.6 (q) 1.05 (s)
1.90 – 1.93 (m)
37.2 (d) 1.95 (br. s)
18.6 (q) 0.95 (d, J ¼ 8.0)
75.4 (d) 1.95 (br. s)
0.96 (d, J ¼ 4.8)
1.76 – 1.78 (m)
1.45 (br. d, J ¼ 12.1), 1.75 – 1.77 (m)
0.81 – 0.84 (m), 1.65 – 1.67 (m)
1.82 – 1.84 (m)
1.51 – 1.54 (m), 3.00 (br. s)
0.79 (d, J ¼ 6.4)
29.4 (t) 1.28 – 1.31 (m), 2.23 – 2.26 (m) 30.3 (t)
33.8 (t) 0.83 – 0.85 (m), 1.67 – 1.70 (m) 33.1 (t)
31.3 (d) 1.58 – 1.61 (m)
30.5 (d)
60.1 (t)
19.5 (q)
60.6 (t) 1.69 – 1.71 (m), 3.11 (br. s)
19.8 (q) 0.76 (d, J ¼ 7.0)
configuration due to its correlations with HaꢀC(1) (d(H) 1.04) and HaꢀC(4) (d(H)
2.70). To determine the configuration at C(14), 1D- and 2D-NMR spectra of
compound 1 were recorded with (D6)DMSO as solvent, and the cross-peak between
d(H) 3.21 (HOꢀC(14)), and 3.86 (HꢀC(16)) and 0.96 (HꢀC(21)) in the ROESY
spectrum indicated a-configuration for HOꢀC(14). Two anomeric H-atom signals at
d(H) 4.93 (d, J ¼ 7.7, 1 H) and 5.87 (br. s, 1 H) in the low-field region of the 1H-NMR
spectrum (Table 2) correlated with the corresponding anomeric C-atom signals at d(C)
102.6 (C(1’)) and 102.9 (C(1’’)), respectively (Table 2), in the HSQC spectrum. The
HMBC features were observed (Fig. 2) between d(H) 4.93 (HꢀC(1’)) and d(C) 78.5
(C(3)), confirming that the b-d-Glc unit was attached at OꢀC(3) of the aglycone, as
well as between d(H) 5.87 (HꢀC(1’’)) and d(C) 78.7 (C(4’)), revealing that the
disaccharide chain was of the type a-l-Rha-(1 ! 4)-b-d-Glc. Therefore, the structure of
1 was established as (3b)-14-hydroxysolanid-5-en-3-yl 4-O-(6-deoxy-a-l-mannopyra-
nosyl)-b-d-glucopyranoside.

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Fig. 2. Key HMB (H ! C) and ROESY (H --- ! H) correlations of compound 1
Table 2. 1H- and 13C-NMR (500 and 125 MHz, resp.) Data of the Sugar Moieties of 1 and 2 in
(D5)Pyridine. d in ppm, J in Hz. Assignments are based on HSQC, HMBC, ROESY, and TOCSY
experiments.
Position
1
2
d(H)
d(C)
d(H)
d(C)
Glc
Glc
1’
2’
3’
4’
5’
6’
4.93 (d, J ¼ 7.7)
3.98 (t, J ¼ 7.7)
4.19 (t, J ¼ 7.7)
4.42 (t, J ¼ 7.7)
3.68 – 3.70 (m)
4.10 (dd, J ¼ 11.5, 3.4),
4.25 (d, J ¼ 11.5)
102.6 (d)
75.7 (d)
76.9 (d)
78.7 (d)
77.3 (d)
61.8 (t)
4.93 (d, J ¼ 7.7)
4.20 – 4.23 (m)
4.20 – 4.23 (m)
4.33 – 4.35 (m)
3.63 (dt, J ¼ 8.9, 3.4)
4.10 (dd, J ¼ 12.1, 3.4),
4.19 – 4.21 (m)
100.7 (d)
78.2 (d)
78.3 (d)
79.1 (d)
77.2 (d)
61.7 (t)
Rha
Rha
1’’
2’’
3’’
4’’
5’’
6’’
5.87 (br. s)
102.9 (d)
73.0 (d)
72.8 (d)
74.2 (d)
70.6 (d)
18.7 (q)
5.85 (br. s)
103.2 (d)
73.0 (d)
72.8 (d)
74.2 (d)
70.8 (d)
18.2 (q)
4.58 (dd, J ¼ 9.2, 3.2)
4.68 – 4.70 (m)
4.34 (t, J ¼ 9.2)
4.99 (dq, J ¼ 9.2, 6.2)
1.70 (d, J ¼ 6.2)
4.52 (dd, J ¼ 9.2, 3.3)
4.67 – 4.70 (m)
4.31 – 4.34 (m)
4.89 – 4.91 (m)
1.62 (d, J ¼ 6.2)
Rha
1’’’
2’’’
3’’’
4’’’
5’’’
6’’’
6.38 (br. s)
102.4 (d)
73.2 (d)
72.8 (d)
74.4 (d)
69.8 (d)
18.9 (q)
4.61 (dd, J ¼ 9.2, 3.4)
4.82 – 4.84 (m)
4.38 – 4.40 (m)
4.95 – 4.97 (m)
1.75 (d, J ¼ 6.2)
Compound 2 was isolated as a white amorphous powder. Its positive-ion-mode HR-
ESI-MS displayed a quasimolecular-ion peak at m/z 868.5062 ([M þ H]þ; calc.
868.5053), in accordance with the molecular formula C45H73NO15. The IR spectrum
indicated the presence of OH groups (3426 cmꢀ1) and of an olefinic bond (1642 cmꢀ1).
Upon acid hydrolysis, compound 2 afforded the sugar moieties l-rhamnose and d-
1
glucose in a ratio of 2 :1 based on the GC analysis of their chiral derivatives. The H-

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and 13C-NMR spectra of the aglycone of 2 (Table 1) were compared with those of
solanidine [6], showing considerable structural similarity, except for the absence of a
CH2 C-atom resonance at d(C) 32.1 (C(7)). Instead, the appearance of an O-bearing C-
atom signal at d(C) 73.2 (C(7)), and the observed downfield shifted C-atom resonances
at d(C) 128.9 (C(6) (þ 7.6 ppm)) and d(C) 41.1 (C(8) (þ 8.6 ppm)), suggested a
hydroxylation at C(7), which was confirmed by the observed HMBC features from
d(H) 4.03 (HꢀC(7)) to d(C) 142.0 (C(5)) and 128.9 (C(6)), from d(H) 1.78 (HꢀC(8))
and 1.08 (HꢀC(9)) to d(C) 73.2 (C(7)), respectively. In the ROESY spectrum of 2, the
correlations from HꢀC(7) (d(H) 4.03) to HaꢀC(9) (d(H) 1.08) and HaꢀC(14) (d(H)
1.38) established a-configuration for HꢀC(7) and thus b-configuration HOꢀC(7).
Three anomeric H-atom signals at d(H) 4.93 (d, J ¼ 7.7), 5.85 (br. s), and 6.38 (br.
s), and three anomeric C-atom signals at d(C) 100.7 (C(1’)), 103.2 (C(1’’)), and 102.4
1
(C(1’’’)) were observed in the H- and 13C-NMR spectra, respectively (Table 2). The
HMBC features between d(H) 5.85 (HꢀC(1’’)) and d(C) 79.1 (C(4’)), between d(H)
6.38 (HꢀC(1’’’)) and d(C) 78.2 (C(2’)), and between d(H) 4.93 (HꢀC(1’)) and d(C) 78.3
(C(3)) revealed that the trisaccharide chain was a a-l-Rha-(1 ! 2)-[a-l-Rha-(1 ! 4)]-
b-d-Glc moiety. Therefore, 2 was elucidated as (3b,7b)-7-hydroxysolanid-5-en-3-yl 6-
deoxy-a-l-mannopyranosyl-(1 ! 2)-[6-deoxy-a-l-mannopyranosyl-(1 ! 4)]-b-d-gluco-
pyranoside.
Compound 3 was obtained as a white amorphous powder. Its positive-ion-mode
HR-ESI-MS displayed a quasimolecular-ion peak at m/z 866.4906 ([M þ H]þ; calc.
866.4896), indicating the molecular formula C45H71NO15. The IR spectrum showed the
presence of OH groups (3427 cmꢀ1) and of an a,b-unsaturated ketone unit (1713 and
1
1644 cmꢀ1). The H- and 13C-NMR spectra of the aglycone of 3 (Table 3) resembled
those of 2, except for the absence of the signal of an O-bearing CH group at d(C) 73.2
(C(7)). Instead, the appearance of a C¼O functionality at this position, forming a
conjugated enone group was indicated by the H-atom resonances at d(H) 5.76
(HꢀC(6)) and C-atom resonances at d(C) 201.8 (C(7)), 167.8 (C(5)) and 124.9 (C(6))
(Table 3), and confirmed by the observed HMBCs (Fig. 3) from d(H) 2.53 (HꢀC(8))
to d(C) 124.9 (C(6)) and 201.8 (C(7)), from d(H) 2.75 (HꢀC(4)) to d(C) 167.8 (C(5))
and 124.9 (C(6)), and from d(H) 1.30 (HꢀC(19)) to d(C) 167.8 (C(5)). The NMR
spectroscopic data (Table 4) for the sugar part of 3 resembled a closely those of 2,
revealing that 3 had the same sugar substitution pattern as 2. The HMBC feature
between d(H) 4.56 (HꢀC(1’)) and d(C) 76.5 (C(3)) showed that the linkage of the
sugar chain was at OꢀC(3) of the aglycone. The structure of 3 was thus assigned as
(3b)-7-oxosolanid-5-en-3-yl 6-deoxy-a-l-mannopyranosyl-(1 ! 2)-[6-deoxy-a-l-man-
nopyranosyl-(1 ! 4)]-b-d-glucopyranoside.
Compound 4 was obtained as a white amorphous powder. Its positive-ion-mode
HR-ESI-MS displayed a quasimolecular-ion peak at m/z 886.5166 ([M þ H]þ; calc.
886.5159), in accordance with the molecular formula C45H75NO16. The 1H-NMR
spectra of the aglycone of 4 (Table 3) displayed signals of two tertiary Me groups at
d(H) 0.88 (s, 3 H) and 1.17 (s, 3 H), two secondary Me groups at d(H) 0.91 (d, J ¼ 5.8,
1
3 H) and 0.97 (d, J ¼ 5.6, 3 H). The H- and 13C-NMR spectra of the aglycone of 4
(Table 3) were in good agreement with those of (22R,25S)-solanid-5-enine-3b,5a,6b-
triol [7]. The NMR spectroscopic data for the sugar part of 4 was very similar to those
of 3, revealing that 4 had the same sugar substitution pattern as 3. Based on HSQC and

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Fig. 3. Key HMB (H ! C) and ROESY (H --- ! H) correlations of compound 3
Table 4. 1H- and 13C-NMR (500 and 125 MHz resp.) Data for the Sugar Moieties of 3 – 5 in CD3OD. d in ppm, J
in Hz. Assignments are based on HSQC, HMBC, ROESY, and TOCSY experiments.
Position 3
d(H)
4
5
d(C)
d(H)
d(C)
d(H)
d(C)
Glc
Glc
Gal
1’
2’
3’
4’
5’
6’
4.56 (d, J ¼ 7.8)
3.43 – 3.45 (m)
3.62 – 3.64 (m)
3.51 – 3.53 (m)
3.36 – 3.38 (m)
3.66 – 3.68 (m),
3.81 – 3.83 (m)
99.3 (d) 4.45 (d, J ¼ 7.8)
77.6 (d) 3.37 – 3.39 (m)
76.6 (d) 3.56 (t, J ¼ 8.7)
78.7 (d) 3.51 (t, J ¼ 8.7)
75.2 (d) 3.44 – 3.46 (m)
101.0 (d) 4.52 (d, J ¼ 7.5)
79.5 (d) 3.80 – 3.82 (m)
78.3 (d) 3.75 – 3.77 (m)
80.3 (d) 4.09 – 4.11 (m)
76.7 (d) 3.53 – 3.55 (m)
99.7 (d)
74.0 (d)
84.2 (d)
68.8 (d)
74.7 (d)
61.1 (t)
60.6 (t) 3.64 (dd, J ¼ 12.1, 4.3), 62.1 (t) 3.68 – 3.70 (m),
3.79 (dd, J ¼ 12.1, 1.8)
3.71 – 3.73 (m)
Rha
Rha
Glc
1’’
2’’
3’’
4’’
5’’
6’’
4.85 (d, J ¼ 1.5)
3.84 – 3.86 (m)
3.63 – 3.65 (m)
3.44 – 3.46 (m)
3.93 – 3.95 (m)
1.30 (d, J ¼ 5.8)
101.6 (d) 4.83 (d, J ¼ 1.5)
103.2 (d) 4.47 (d, J ¼ 7.7)
104.4 (d)
73.6 (d)
76.8 (d)
69.8 (d)
76.5 (d)
61.0 (t)
71.0 (d) 3.82 (dd, J ¼ 3.2, 1.5) 72.6 (d) 3.26 – 3.28 (m)
70.8 (d) 3.60 (dd, J ¼ 9.4, 3.2) 72.3 (d) 3.33 – 3.35 (m)
72.3 (d) 3.41 (dd, J ¼ 9.4, 6.2) 73.9 (d) 3.32 – 3.34 (m)
69.3 (d) 3.90 – 3.93 (m)
16.5 (q) 1.26 (d, J ¼ 6.2)
70.9 (d) 3.26 – 3.29 (m)
18.0 (q) 3.65 – 3.68 (m),
3.83 – 3.85 (m)
Rha
Rha
Rha
1’’’
2’’’
3’’’
4’’’
5’’’
6’’’
5.25 (d, J ¼ 1.5)
3.67 – 3.69 (m)
3.93 – 3.95 (m)
3.42 – 3.44 (m)
4.12 (dq, J ¼ 12.3, 6.2) 68.3 (d) 4.12 – 4.15 (m)
1.26 (d, J ¼ 6.2) 16.4 (q) 1.24 (d, J ¼ 6.2)
100.8 (d) 5.20 (d, J ¼ 1.5)
102.4 (d) 5.22 (d, J ¼ 1.5)
100.7 (d)
71.0 (d) 3.91 – 3.93 (m)
72.4 (d) 3.64 (dd, J ¼ 3.3, 1.5) 71.0 (d)
70.7 (d) 3.67 (dd, J ¼ 9.5, 3.3) 72.3 (d) 3.94 (dd, J ¼ 9.4, 3.3) 70.6 (d)
72.5 (d) 3.38 (dd, J ¼ 9.5, 6.2) 74.4 (d) 3.39 – 3.41 (m)
72.6 (d)
69.7 (d) 4.12 (dq, J ¼ 12.3, 6.2) 68.3 (d)
18.1 (q) 1.24 (d, J ¼ 6.2)
16.6 (q)
HMBC evidence, the structure of 4 was determined to be (3b,5a,6b)-5,6-dihydroxy-
solanidan-3-yl 6-deoxy-a-l-mannopyranosyl-(1 ! 2)-[6-deoxy-a-l-mannopyranosyl-
(1 ! 4)]-b-d-glucopyranoside.

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Compound 5 was isolated as a white amorphous powder. Its positive-ion-mode HR-
ESI-MS displayed a quasimolecular-ion peak at m/z 882.4852 ([M þ H]þ; calc.
882.4846), indicating the molecular formula C45H71NO16. The IR spectrum revealed
the presence of OH groups (3441 cmꢀ1) and of an a,b-unsaturated ketone unit (1710
and 1641 cmꢀ1). Upon acid hydrolysis of 5, three sugar monomers were identified as d-
glucose, l-rhamnose, and d-galactose by GC/MS analysis of their silyl derivatives. The
anomeric H-atom signals at d(H) 4.47 (d, J ¼ 7.7), 4.52 (d, J ¼ 7.5), and 5.22 (d, J ¼ 1.5)
correlated with the C-atom resonances at d(C) 104.4 (C(1’’)), 99.7 (C(1’)), and 100.7
(C(1’’’)), respectively, in the HSQC spectrum. The connectivity of the three sugars was
determined by the following HMBC features: from d(H) 4.52 (HꢀC(1’)) to d(C) 76.2
(C(3)) from d(H) 4.47 (HꢀC(1’’)) to d(C) 84.2 (C(3’)), and from d(H) 5.22 (HꢀC(1’’’))
to d(C) 74.0 (C(2’)). The 1H- and 13C-NMR signals (Table 3) for the aglycone of 5 were
in good agreement with those of 3. Therefore, the structure of 5 was elucidated as (3b)-
7-oxosolanid-5-en-3-yl
6-deoxy-a-l-mannopyranosyl-(1 ! 2)-[b-d-glucopyranosyl-
(1 ! 3)]-b-d-galactopyranoside.
The known steroidal alkaloid glycoside a-solanine (6) was identified by comparison
of its NMR and MS data with those reported in the literature [8].
Biological Study. Cytotoxic activities of compounds 1 – 4 and 6 were evaluated in
vitro against SMMC-7721 (human hepatoma), NCI-H460 (non-small cell lung cancer),
and A-549 (human lung adenocarcinoma) cell lines, by the MTT (¼ 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) method. Compound 6 showed
cytotoxicity against to SMMC-7721, NCI-H460, and A-549 cell lines, with IC50 values of
14.4, 39.0, and 35.7 mm, respectively. The other compounds showed no or little cytotoxic
activity (IC50 values > 100 mm) against the tested tumor cells.
This work was financially supported by the Scaling Project for Innovation Scholars, the Natural
Science Foundation of Jiangsu Province, China (BK2008039), the Key Project of National Natural Science
Foundation of P.R.China (30830116), the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD), and the Program for Changjiang Scholars and Innovative Research Team
in University (PCSIRT-IRT1193).
Experimental Part
General. All the reagents and solvents were of the anal. grade (Jiangsu Hanbang Sci. & Tech. Co.,
Ltd., Huaian, China). Column chromatography (CC): silica gel H (SiO2; 100 – 200 and 200 – 300 mesh;
Qingdao Marine Chemical Factory, Qingdao, China), RP-18 (40 – 63 mm; Fuji Silysia Chemical Ltd.),
D101 macroporous resin (The Chemical Plant of Nankai University, Tianjin, China), Sephadex LH-20
(Pharmacia, Amersham Biosciences, S-Uppsala, Sweden). TLC: SiO2 GF254 (Qingdao Marine Chemical
Co., Ltd.). Optical rotations: JASCO P-1020 polarimeter. IR Spectra: Bruker Tensor-27 spectrometer;
KBr pellets; in cmꢀ1. 1D- and 2D-NMR spectra: Bruker AV-500 spectrometer; at 500 (1H) and 125 MHz
(13C); d in ppm rel. to TMS as an internal standard, J in Hz. GC/MS: Agilent 6890 gas chromatograph and
Agilent 5975 mass spectrometer. ESI-MS: Agilent 1100 Series LC/MSD Trap mass spectrometer; in m/z.
HR-ESI-MS: Micro Q-TOF MS instrument; in m/z.
Plant Material. Aerial parts of S. tuberosum were collected from Nanjing City, Jiangsu Province,
China, in May 2009. The identity of the plant was confirmed by Prof. Min-Jian Qin, Department of
Medicinal Plants, China Pharmaceutical University. A voucher specimen (No. 20090525) has been
deposited with the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
Extraction and Isolation. Dried aerial parts of S. tuberosum (10.6 kg) were extracted three times with
90% EtOH (3 ꢁ 40 l) under reflux for 2 h each time. The extract was evaporated under reduced pressure.

Helvetica Chimica Acta – Vol. 96 (2013)
939
Then, the residue (640.8 g) was suspended in H2O and, by standing, partitioned with supernatant and
precipitation successively. The supernatant was passed through a D101 macroporous adsorption resin
column and eluted with EtOH/H2O 0 :100, 30 :70, 70 :30, and 100 :0 to yield four fractions, Frs. 1 – 4, resp.
Fr. 3 (24.2 g) was separated by CC (SiO2; (CHCl3/MeOH/H2O 7:3 :0.2 ! 6 :4 :0.5) to give further ten
subfractions, Subfrs. 3.1 – 3.10. Subfr. 3.9 (3.0 g) was subjected to CC (SiO2; CHCl3/MeOH/NH3 · H2O
7:3 :0.3; and ODS; MeOH/H2O 30 :70, 50 :50, 70 :30) to give 1 (5 mg) and 2 (7 mg), resp. Subfr. 3.10
(2.2 g) was submitted to CC(ODS; MeOH/H2O 45 :55; and SiO2 ; (CHCl3/MeOH/NH3 · H2O
6.5 :3.5 :0.4) to afford 3 (3 mg), 4 (4 mg), and 5 (1.5 mg).
(3b)-14-Hydroxysolanid-5-en-3-yl 4-O-(6-Deoxy-a-l-mannopyranosyl)-b-d-glucopyranoside (1).
White amorphous powder. [a]2D0 ¼ ꢀ16.4 (c ¼ 0.11, MeOH). IR (KBr): 3423, 2925, 1641, 1400, 1066,
1
618. H- and 13C-NMR: Tables 1 and 2. ESI-MS: 722 ([M þ H]þ), 576 ([M ꢀ 146 þ H]þ), 414 ([M ꢀ
146 ꢀ 162 þ H]þ). HR-ESI-MS: 722.4481 ([M þ H]þ, C39H64NO1þ1 ; calc. 722.4474).
(3b,7b)-7-Hydroxysolanid-5-en-3-yl 6-Deoxy-a-l-mannopyranosyl-(1 ! 2)-[6-deoxy-a-l-mannopyr-
anosyl-(1 ! 4)]-b-d-glucopyranoside (2). White amorphous powder. [a]2D0 ¼ ꢀ34.4 (c ¼ 0.10, MeOH).
IR (KBr): 3426, 2938, 1642, 1402, 1044, 620. 1H- and 13C-NMR: Tables 1 and 2. ESI-MS: 868 ([M þ H]þ),
722 ([M ꢀ 146 þ H]þ), 576 ([M ꢀ 146 ꢀ 146 þ H]þ), 414 ([M ꢀ 146 ꢀ 146 ꢀ 162 þ H]þ). HR-ESI-MS:
868.5062 ([M þ H]þ, C45H74NO1þ5 ; calc. 868.5053).
(3b)-7-Oxosolanid-5-en-3-yl 6-Deoxy-a-l-mannopyranosyl-(1 ! 2)-[6-deoxy-a-l-mannopyranosyl-
(1 ! 4)]-b-d-glucopyranoside (3). White amorphous powder. [a]2D0 ¼ ꢀ50.7 (c ¼ 0.09, MeOH). IR
1
(KBr): 3427, 1713, 1644, 1403, 670. H- and 13C-NMR: Tables 3 and 4. ESI-MS: 866 ([M þ H]þ), 720
([M ꢀ 146 þ H]þ), 574 ([M ꢀ 146 ꢀ 146 þ H]þ), 412 ([M ꢀ 146 ꢀ 146 ꢀ 162 þ H]þ). HR-ESI-MS:
866.4906 ([M þ H]þ, C45H72NO1þ5 ; calc. 866.4896).
(3b,5a,6b)-5,6-Dihydroxysolanidan-3-yl 6-Deoxy-a-l-mannopyranosyl-(1 ! 2)-[6-deoxy-a-l-man-
nopyranosyl-(1 ! 4)]-b-d-glucopyranoside (4). White amorphous powder. [a]2D0 ¼ ꢀ34.4 (c ¼ 0.09,
MeOH). IR (KBr): 3425, 2925, 1400, 1046. 1H- and 13C-NMR: Tables 3 and 4. ESI-MS: 886 ([M þ
H]þ), 740 ([M ꢀ 146 þ H]þ), 594 ([M ꢀ 146 ꢀ 146 þ H]þ), 432 ([M ꢀ 146 ꢀ 146 ꢀ 162 þ H]þ). HR-ESI-
MS: 886.5166 ([M þ H]þ, C45H76NO1þ6 ; calc. 886.5159).
(3b)-7-Oxosolanid-5-en-3-yl 6-Deoxy-a-l-mannopyranosyl-(1 ! 2)-[b-d-glucopyranosyl-(1 ! 3)]-
b-d-galactopyranoside (5). White amorphous powder. [a]2D0 ¼ ꢀ37.3 (c ¼ 0.09, MeOH). IR (KBr):
1
3441, 1710, 1641, 1400, 668. H- and 13C-NMR: Tables 3 and 4. ESI-MS: 882 ([M þ H]þ), 736 ([M ꢀ
146 þ H]þ), 574 ([M ꢀ 146 ꢀ 162 þ H]þ), 412 ([M ꢀ 146 ꢀ 162 ꢀ 162 þ H]þ). HR-ESI-MS: 882.4852
([M þ H]þ, C45H72NO1þ6 ; calc. 882.4846).
Absolute Configuration. Each compound (1 – 2 mg) was dissolved in MeOH (4 ml) and treated with
3 ml of 5% H2SO4 at 908 for 2 h. After addition of H2O (3 ml), each mixture was concentrated to 3 ml
under reduced pressure and then neutralized with Amberlite MB-3 resin (D-Darmstadt). Each residue,
evaporated to dryness in vacuo, was mixed with l-cysteine methyl ester hydrochloride (2 mg) and
dissolved in pyridine (2 ml), with the solns. being kept at 608 for 1 h, followed by addition of Me3SiCl
(0.5 ml) and then keeping for 30 min. Each soln. was diluted with H2O and extracted with hexane (1 ml ꢁ
3). Each extract was analyzed by GC/MS [9][10]. The monosaccharides were confirmed as l-rhamnose,
d-glucose, and d-galactose by comparison of the retention times of their derivatives with those of
standard samples (l-rhamnose (14.19 min), d-glucose (15.49 min), and d-galactose (15.77 min), resp).
Cytotoxicity Assay. SMMC-7721 (human hepatoma carcinoma), NCI-H460 (human lung cancer),
and A-549 (human lung adenocarcinoma) cell lines were obtained from the Type Culture Collection of
the Chinese Academy of Sciences (Shanghai, China) and grown in the indicated media supplemented
with 10% FBS and 50 IU penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 378. The
cytotoxicity assay was performed according to the MTT method in 96-well microplates [11]. Briefly,
200 ml of adherent cells were seeded into 96-well cell-culture plates and allowed to adhere for 12 h before
drug addition, while suspended cells were seeded just before drug addition with initial density of 1 ꢁ
105 cells/ml. Each tumor cell line was exposed to the test compound at concentrations of 3.125, 6.25,
12.5, 25, 50, and 100 mm in triplicates for 48 h, with 5-fluorouracil (5-FU, Sigma, USA) as a positive
control. After compound treatment, the optical density was measured at 570 nm using a Spectra Shell
Microplate Reader and a cell growth curve was plotted.

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Helvetica Chimica Acta – Vol. 96 (2013)
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Received May 21, 2012