Indiosides G-K: Steroidal glycosides with cytotoxic and anti-inflammatory activities from Solanum violaceum

Article abstract of DOI:10.1021/np200877u

Five new steroidal glycosides (1-5) and nine known compounds were isolated from Solanum violaceum. Indiosides G (1) and H (2) are spirostene saponins with an iso-type F ring, indioside I (3) is a spirostane saponin, and indiosides J (4) and K (5) are unusual furostanol saponins with a deformed F ring. These structures represent rare naturally occurring steroidal skeletons. The structures of the new compounds were elucidated using 1D and 2D spectroscopic techniques and acid hydrolysis. Compounds 2, 3, and 7-9 exhibited cytotoxic activity against six human cancer cell lines (HepG2, Hep3B, A549, Ca9-22, MDA-MB-231, and MCF-7) with IC₅₀ values of 1.83-8.04 μg/mL. Steroidal saponins 3, 8, and 9 showed inhibitory effects on superoxide anion generation with IC₅₀ values of 2.84 ± 0.18, 0.62 ± 0.03, and 1.62 ± 0.59 μg/mL, respectively. Saponins 8 and 9 also inhibited elastase release with IC₅₀ values of 111.05 ± 7.37 and 4.04 ± 0.51 μg/mL, respectively. Structure-activity relationship correlations of these compounds with respect to cytotoxic and anti-inflammatory effects are discussed.

Full text of DOI:10.1021/np200877u

Article
pubs.acs.org/jnp
Indiosides G−K: Steroidal Glycosides with Cytotoxic and Anti-
inflammatory Activities from Solanum violaceum
Chiao-Ting Yen,† Chia-Lin Lee,‡,§ Fang-Rong Chang,† Tsong-Long Hwang,⊥ Hsin-Fu Yen,∥
Chao-Jung Chen,‡,∇ Shu-Li Chen,† and Yang-Chang Wu*,†,‡,§,O
†Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80708, Taiwan, Republic of China
‡School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan, Republic of China
§Natural Medicinal Products Research Center, China Medical University Hospital, Taichung 40402, Taiwan, Republic of China
⊥Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan, Republic of China
∥Botanical Garden, National Museum of Natural Science, Taichung, 40453, Taiwan, Republic of China
∇Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taichung, 40402, Taiwan,
Republic of China
OCenter for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan, Republic of China
S
* Supporting Information
ABSTRACT: Five new steroidal glycosides (1−5) and nine known
compounds were isolated from Solanum violaceum. Indiosides G (1) and H
(2) are spirostene saponins with an iso-type F ring, indioside I (3) is a
spirostane saponin, and indiosides J (4) and K (5) are unusual furostanol
saponins with a deformed F ring. These structures represent rare naturally
occurring steroidal skeletons. The structures of the new compounds were
elucidated using 1D and 2D spectroscopic techniques and acid hydrolysis.
Compounds 2, 3, and 7−9 exhibited cytotoxic activity against six human cancer
cell lines (HepG2, Hep3B, A549, Ca9-22, MDA-MB-231, and MCF-7) with
IC50 values of 1.83−8.04 μg/mL. Steroidal saponins 3, 8, and 9 showed
inhibitory effects on superoxide anion generation with IC50 values of 2.84
0.18, 0.62 0.03, and 1.62 0.59 μg/mL, respectively. Saponins 8 and 9 also
inhibited elastase release with IC50 values of 111.05 7.37 and 4.04 0.51 μg/
mL, respectively. Structure−activity relationship correlations of these compounds with respect to cytotoxic and anti-inflammatory
effects are discussed.
he genus Solanum from the family Solanaceae contains
about 1500 species, which are distributed mostly in
biological effects. The EtOAc fraction of S. violaceum was active
against various human cancer cell lines (HepG2, Hep3B, A549,
Ca9-22, MDA-MB-231, and MCF-7) with IC50 < 20 μg/mL
and also showed significant anti-inflammatory effects on
superoxide anion generation and elastase release in fMLP/
CB-induced human neutrophils.
T
tropical and subtropical regions. An estimated 13 species of
genus Solanum are present in Taiwan, including Solanum
violaceum Ortega (S. indicum auct. non L.), called Indian
Nightshade, an erect medium size shrub growing from low to
medium latitudes in southern Taiwan.1 It has been used in
Chinese folk medicine to eliminate toxins from the body and as
an analgesic and anti-inflammatory agent for rhinitis, toothache,
and breast cancer.2 In Thailand, the fruit of S. violaceum has
been used as both a food and an anticarcinogen.3 However,
only a few phytochemical studies have been performed on S.
violaceum. Thus far, the isolated active components from this
plant include 10 steroidal glycosides,2,3 one solafuranone, and
three phenolic derivatives.2 Among them, indioside D was the
induced feeding preference of larvae of the moth Mamduca
sexta (tobacco hornworm),4 and solavetivone was cytoxic to
OVCAR-3 cells.2 The interesting biological activities and the
limited number of phytochemical reports on S. violaceum
encouraged us to pursue an intensive investigation to isolate
other active components from S. violaceum and determine their
RESULTS AND DISCUSSION
■
The EtOAc fraction of a MeOH extract of S. violaceum was
subjected to CC on silica gel, C18, Sephadex LH-20, and HPLC
on ODS-C18 to give five new compounds, indiosides G−K (1−
5), as well as the known borassoside D (6),5 yamogenin 3-O-α-
L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (7),5 borasso-
side E (8),5 3-O-chacotriosyl-25(S)-spirost-5-en-3β-ol (9),6
sitosterol 3-O-β-D-glucopyranoside (10),5 7-hydroxysitosterol-
3-O-β-D-glucopyranoside (11),7 N-p-coumaroyltyramine (12),8
trans-N-feruloyloctopamine (13),9 and tricalysioside U (14).10
Received: October 31, 2011
Published: March 13, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
636
dx.doi.org/10.1021/np200877u | J. Nat. Prod. 2012, 75, 636−643

Journal of Natural Products
Article
Chart 1
The HRESIMS spectrum of 1 showed a molecular formula of
C38H60O12 with a pseudomolecular ion at m/z 731.3966 [M +
Na]+. The IR spectrum displayed absorption bands for hydroxy
(3404 cm−1) and C−O (1030 cm−1) functionalities. The 1D
NMR (Tables 1 and 2) and HMQC data revealed the presence
of four methyl groups [δH 0.84/δC 16.4 and δH 0.90/δC 19.4
(3H each, both s, CH3-18 and CH3-19); δH 0.70/δC 17.3 and
δH 1.15/δC 15.0 (3H each, both d, J = 6.9 Hz, CH3-27 and
CH3-21)] and an oxymethylene [δH 3.51 (1H, t, J = 10.8 Hz),
3.59 (1H, br d, J ≈ 10.8 Hz)/δC 66.8, CH2-26]. The NMR data
also showed two oxymethines [δH 3.95 (1H, m, H-3) and 4.52
(1H, m, H-16)] along with their corresponding carbons at δC
78.2 and 81.1, respectively, and an olefinic proton [δH 5.33
(1H, d, J ≈ 5.4 Hz)/δC 121.8, CH-6], together with two
anomeric signals at δH 5.05 (1H, d, J = 7.8 Hz, δC 102.7) and
5.37 (1H, d, J = 7.8 Hz, δC 106.5). Twenty-seven carbon
signals, including four methyl, 10 methylene, nine methine, and
four quaternary carbons, were observed in the 13C NMR and
DEPT spectra (Table 1). Among the four quaternary carbon
signals, the signal at δC 109.2 was identified as a hemiketal (C-
22) and the signal at δC 140.8 was assigned as an olefinic (C-5)
carbon. The aforementioned data suggested that 1 is possibly a
spirostanol glycoside with the aglycone having a 27-carbon
skeleton along with two sugar moieties.5 The acidic hydrolysis
of 1 with HCl in 1,4-dioxane gave D-glucose and D-xylose,
which were identified by GC analysis of the acetylated sugar
residues.11 In the HMBC spectrum of 1, the xylosyl H-1″ and
glucosyl H-1′ protons exhibited 3J interactions with the glucosyl
C-3′ (δ 87.8) and C-3 (δ 78.2) carbons of the aglycone moiety,
respectively. These correlations led to the assignment of the
glycosidic moiety as xylopyranosyl-(1→3)-glucopyranosyl,
which was connected to C-3 of the aglycone moiety through
an O-linkage, as shown in Figure 1. The aglycone of 1 could
not be isolated due to its decomposition during acid hydrolysis.
The NMR data of 1 were similar to those of known
compounds 75 and 9,6 indicating that the three compounds
shared a basic steroidal nucleus. However, differences were
present in the 1H and 13C chemical shifts corresponding to the
E and F rings (Table 1). In 1, the C-26 protons appeared in
close proximity at δH 3.51 (1H, t, J = 10.8 Hz) and 3.59 (1H, br
d, J ≈ 10.8 Hz), indicating that 1 has an iso-type F ring.12 The
CH3-27 protons were shifted upfield from δH 1.07 in 7 and 9 to
δH 0.70 in 1, and the corresponding carbon signal was shifted
downfield from δC 15.8 to 17.3, indicative of an equatorial α-
orientation for CH3-27 in 1,13 rather than the axial β-
orientation of the same group in 7 and 9. Additionally, H-20
was observed at higher field in 1 (δH 1.96) compared with 7
and 9 (δH 2.47), suggesting that H-20 and the F ring oxygen in
1 have a trans-relationship.12 The stereostructure of 1 was
confirmed by ROESY analysis, as shown in Figure 2.
Consequently, compound 1 was characterized as indioside G,
3-O-[β-D-xylopyranosyl-(1→3)-β-D-glucopyranosyl]-(22S,25S)-
spirost-5-en-3β-ol. The steroidal saponin of 1 has been studied
and described as having antifungal, antiviral, and hemolytic
activities.14,15 However, the glycoside indioside G (1) has not
been isolated previously as a natural product.
Compounds 2, 6, and 7 have the same molecular formula,
C39H62O12, HRESIMS m/z 745.4241 [M + Na]+. The IR
spectrum of 2 displayed absorptions for hydroxy (3402 cm−1)
and C−O (1048 cm−1) functional groups. The 1D NMR and
HMQC spectra showed two anomeric proton signals at δH 5.05
(d, J = 7.3 Hz)/δC 100.4 and δH 6.39 (br s)/δC 102.1,
637
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Journal of Natural Products
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1
Table 1. H and 13C NMR (Pyridine-d5) Data of Indioside G−K (1−5) (Aglycone Part)
1
2
3
4
5
position
1
δH (J Hz)
0.94 (m)
δC
δH (J Hz)
0.98 (m)
δC
δH (J Hz)
0.82 (m)
δC
δH (J Hz)
δC
δH (J Hz)
δC
37.0
37.5
37.2
0.99 (m)
1.75 (m)
1.86 (m)
2.09 (m)
3.88 (m)
2.73 (m)
2.80 (m)
37.0
0.99(m)
1.73(m)
1.85(m)
2.08(m)
3.91 m)
2.72 (m)
2.75 (m)
37.5
1.70 (m)
1.67 (m)
2.10 (m)
3.95 (m)
2.44 (m)
2.72 (m)
1.75 (m)
1.87 (m)
2.05 (m)
3.95 (m)
2.76 (m)
2.83 (m)
1.59 (m)
1.79 (m)
2.02 (m)
3.92 (m)
1.65 (m)
1.95 (m)
0.87 (m)
1.12 (2H, m)
1.53 (m)
2.00 (m)
1.39 (m)
2
30.2
31.8
29.9
29.7
30.2
3
4
78.2
39.3
77.9
39.0
77.2
34.5
77.5
38.5
78.1
39.0
5
6
7
140.8
121.8
32.2
140.9
121.7
32.3
44.6
28.9
32.4
140.3
121.3
32.0
140.8
121.8
32.5
5.33 (d, 5.4)
1.44 (m)
1.85 (m)
1.49 (m)
0.85 (m)
5.32 (d, 5.1)
1.44 (m)
1.87 (m)
1.46 (m)
0.90 (m)
5.31 (m)
1.53 (m)
2.00 (m)
1.53 (m)
0.92 (m)
5.30 (m)
1.55 (m)
2.02 (m)
1.52 (m)
0.90 (m)
a
a
a
a
a
8
31.6
50.2
37.4
21.1
31.7
50.3
37.2
21.1
35.3
54.4
35.9
21.3
31.2
49.9
36.6
20.6
31.7
50.3
37.1
21.1
9
0.52 (td, 11.2, 3.9)
10
11
1.39 (m)
1.44 (m)
1.26 (m)
1.67 (m)
1.37 (m)
1.40 (m)
1.11 (m)
1.73 (m)
1.21 (m)
1.43 (m)
1.03 (m)
1.77 (m)
1.47 (2H, m)
1.52 (2H, m)
12
39.8
39.9
40.1
1.03 (m)
1.77 (m)
38.4
1.13 (m)
1.76 (m)
39.8
13
14
15
40.4
56.6
32.2
40.5
56.7
32.2
40.8
56.5
32.1
40.5
56.1
31.8
41.0
56.6
32.1
1.03 (m)
1.53 (m)
2.07 (m)
4.52 (m)
1.81 (m)
0.84 (s)
1.09 (m)
1.58 (m)
2.15 (m)
4.56 (m)
1.83 (m)
0.84 (s)
1.00 (m)
0.80 (m)
1.39 (m)
4.51 (m)
1.90 (m)
0.82 (s)
1.11 (m)
0.80 (m)
1.39 (m)
5.00 (m)
1.92 (m)
0.98 (s)
1.08 (m)
1.59 (m)
1.90 (m)
4.90 (m)
1.94 (m)
0.96 (s)
a
a
a
a
16
17
18
19
20
21
22
23
81.1
62.8
16.4
19.4
42.0
15.0
109.2
31.8
81.1
62.9
16.3
19.4
42.0
15.0
109.3
29.3
81.2
62.9
16.6
12.4
42.5
14.8
109.7
26.4
80.9
63.5
15.8
18.9
37.6
15.9
108.9
82.7
81.4
64.0
16.3
19.4
38.2
15.8
109.1
84.3
0.90 (s)
1.07 (s)
0.86 (s)
1.06 (s)
1.06 (s)
1.96 (m)
1.15 (d, 6.9)
1.96 (m)
1.15 (d, 6.9)
1.90 (m)
1.14 (d, 6.9)
2.50 (t, 6.7)
1.32 (d, 6.7)
2.40 (t, 6.5)
1.31 (d, 6.9)
1.51 (2H, m)
1.57 (2H, m)
1.54 (2H, m)
1.46 (2H, m)
1.87 (m)
4.56 (m)
4.39(m)
2.14 (m)
a
24
29.2
30.2
1.34 (m)
26.2
1.34 (m)
1.45 (m)
2.40 (m)
4.86 (m)
31.6
1.54 (m)
2.02 (m)
2.18 (m)
4.98 (m)
32.8
1.45 (m)
25
26
1.58 (m)
30.6
66.8
1.59 (m)
30.6
66.9
1.59 (m)
27.5
65.1
39.4
39.5
3.51 (t, 10.8)
3.59 (br d, 10.8)
0.70 (d, 6.9)
3.51 (t, 11.2)
3.59 (br d, 11.2)
0.71 (d, 5.6)
3.36 (d, 11.0)
4.05 (dd, 11.0, 7.6)
1.08 (d, 7.1)
109.9
105.4
27
17.3
17.3
16.3
0.96 (d, 7.1)
3.44 (m)
16.4
67.4
1.10 (d, 6.7)
3.37 (m)
12.9
68.0
26-O-1
3.92 (m)
3.86 (m)
2
1.36 (m)
19.2
13.5
1.34 (m)
19.6
14.0
1.38 (m)
1.38 (m)
3
0.83 (t, 7.4)
0.81 (t, 7.4)
a
Signals may be interchanged.
corresponding to the glycone part. Two tertiary methyl proton
signals at δH 0.84/δC 16.3 and δH 1.07/δC 19.4, two secondary
methyl proton signals at δH 0.71 (d, J = 5.6 Hz)/δC 17.3 and
δH 1.15 (d, J = 6.9 Hz)/δC 15.0, and an olefinic proton signal at
δH 5.32 (d, J = 5.1 Hz)/δC 121.7 were observed, corresponding
to the aglycone part. The NMR spectra of 2 and 7 were quite
similar, with the only difference evident in the signals for H2-26.
As also found in 1, the presence of H2-26 proton signals at δH
3.51 (1H, t, J = 11.2 Hz) and 3.59 (1H, br d, J ≈ 11.2 Hz)
indicated an iso-type F ring in 2, while the same proton signals
were further apart at δH 3.37 (1H, d, J = 10.2 Hz) and 4.06
(1H, d, J = 10.2 Hz) in 7, indicative of a normal-type F ring.
Acidic hydrolysis of 2 afforded D-glucose and L-rhamnose. The
HMBC spectrum showed a correlation between the rhamnose
H-1″ (δH 6.38) and the glucose C-2′ (δC 78.0), as well as
between the aglycone H-3 (δH 3.95) and the anomeric carbon
(δC 100.4) of glucose (Figure 1). Consequently, 2 was
identified as 3-O-[α-L-rhamnopyranoside-(1→2)-β-D-glucopyr-
anosyl]-(22S,25S)-spirost-5-en-3β-ol and named indioside H.
Compound 3 has the molecular formula C45H74O16, as
established by HRESIMS (m/z 893.4862 [M + Na]+). IR
absorptions at 3445 and 1054 cm−1 indicated the presence of
1
hydroxy and ether functionalities. The H NMR spectrum
displayed signals corresponding to four steroidal methyl groups
[δH 0.82, 0.86 (3H each, both s, H3-18, H3-19), 1.08, 1.14 (3H
each, both d, J = 7.1, 6.9 Hz, H3-27, H3-21)], a methylene [δH
3.36 (1H, d, J = 11.0 Hz), 4.05 (1H, dd, J = 11.0, 7.6 Hz), H2-
26], and two oxymethines [δH 3.92 (1H, m, H-3) and 4.51 (1H,
m, H-16)]. D-Glucose and L-rhamnose in a 1:2 ratio were
obtained upon acidic hydrolysis of 3, as indicated by GC.
638
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Journal of Natural Products
Article
1
Table 2. H and 13C NMR (Pyridine-d5) Data of Indioside G−K (1−5) (Sugar Part)
1
2
3
4
5
position
δH (J Hz)
δC
δH (J Hz)
δC
δH (J Hz)
δC
δH (J Hz)
δC
δH (J Hz)
δC
3-O-Glc-1′
5.05 (d, 7.8)
4.10 (m)
4.31 (m)
4.23 (m)
4.17 (m)
4.36 (m)
4.56 (m)
5.37 (d, 7.8)
4.09 (m)
4.17 (m)
4.30 (m)
3.74 (m)
4.34 (m)
102.7
74.4b)
87.8
5.05 (d, 7.3)
4.28 (m)
100.4
78.0
79.7
71.9
78.3
62.7
4.96 (d, 7.8)
4.21 (m)
3.72 (m)
4.38 (m)
4.28 (m)
4.11 (m)
4.26 (m)
99.9
78.1
77.0
78.8
78.0
61.4
5.00 (overlap)
4.24 (overlap)
3.66 (overlap)
4.41 (m)
99.8
77.3
76.5
78.2
77.6
60.8
4.92 (m)
4.15 (m)
3.71 (m)
4.28 (m)
4.16 (m)
4.08 (m)
4.22 (m)
100.3
77.9
77.0
78.7
77.8
61.3
2′
3′
4′
5′
6′
4.30 (m)
70.9
4.18 (t, 8.9)
3.90 (m)
78.2
4.24 (m)
62.4
4.37 (m)
4.11 (m)
4.53 (m)
4.22 (m)
3′-O-Xyl-1″
106.5
75.5 b)
78.2
2″
3″
4″
5″
69.5
67.4
2′-O-Rha-1″
6.39 (s)
102.1
72.6 b)
72.9 b)
74.2
6.27 (s)
102.1
72.5
72.7
73.9
69.5
18.5
102.9
72.5
72.8
74.1
70.4
18.6
6.42 (s)
101.6
72.0
72.3
73.4
69.9
18.0
102.4
72.0
72.3
73.7
69.0
18.1
6.42 (s)
102.0
72.5
74.1
72.7
69.5
18.6
102.4
72.5
73.9
72.8
70.4
18.5
2″
4.81 (m)
4.83 (m)
4.61 (br s)
4.34 (m)
4.91 (m)
1.75 (d, 6.2)
5.85 (s)
4. 85 (m)
4. 53 (br s)
4.35 (m)
4. 69 (m)
4. 33 (br s)
4.35 (m)
3″
4.64 (dd, 3.3, 9.2)
4.35 (m)
4″
5″
4.91 (m)
69.5
4.94 (m)
5.00 (overlap)
1.78 (d, 6.1)
5.88 (s)
6″
1.79 (d, 6.2)
18.7
1.65 (d, 5.9)
5.88 (s)
4′-O-Rha-1′″
2′″
3′″
4′″
5′″
6′″
4.69 (m)
4.55 (br s)
4.35 (m)
4.92 (m)
1.63 (d, 6.2)
4. 70 (m)
4. 64 (br s)
4.37 (m)
4.69 (m)
4.64 (br s)
4.56 (m)
4.94 (m)
5.00 (overlap)
1.64 (d, 6.2)
1.78 (d, 5.9)
Analysis of NMR data indicated the presence of three sugar
units in a β-chacotriosyl moiety, with three anomeric protons
[δH 4.96 (1H, d, J = 7.8 Hz, H-1′ of β-D-glucopyranosyl), 5.85
(1H, br s, H-1′″ of α-L-rhamnopyranosyl) and 6.37 (1H, br s,
H-1″ of α-L-rhamnopyranosyl)] with correlations to carbon
signals at δC 99.9, 102.9, and 102.1, respectively.5 In the HMBC
spectrum of 3, a correlation between H-1′ of the β-D-
glucopyranosyl moiety at δH 4.96 and C-3 at δC 77.2 indicated
that the aglycone is glycosylated at C-3. The 1H and 13C NMR
spectra of 3 were similar to those of immunoside,12,16 with the
exception of the A/B ring carbon signals, leading to a molecular
structure as shown. The α-orientation of H-5 was determined
by ROESY analysis (Figure 2). Finally, the full structure of 3
(indioside I) was elucidated as 3-O-{α-L-rhamnopyranoside-
(1→2)-O-[α-L-rhamnopyranoside-(1→4)]-β-D-glucopyrano-
syl}-(22R,25S)-spirost-5α-an-3β-ol.
corresponding to five methyl groups [δC 13.5
(OCH2CH2CH3), 15.8 (C-18), 15.9 (C-21), 16.4 (C-27),
18.9 (C-19)], one trisubstituted double bond (δC 140.3 and
121.3), one oxymethylene (δC 67.4), three oxymethines (δC
77.6, 80.9, and 82.7), one hemiketal (δC 108.9), one hemiacetal
(δC 109.9), and three anomeric carbons (δC 99.8, 101.6, and
102.4). The HMBC data (Figure 1) revealed that the
triglycoside unit [δH 5.00 (1H, overlapped with proton signals
of water, inner glucosyl H-1′)] was connected to the aglycone
at C-3 (δC 77.5), a double bond was located between C-5 and
C-6 [H2-4/C-5, C-6; H-6/C-4, C-10; H3-19/C-1, C-5, C-9],
and hemiketal and hemiacetal carbons were present at C-22
[H-20/C-13, C-17, C-22; H3-21/C-17, C-20, C-22] and C-26
[H-26/C-24, C-25; H3-27/C-24, C-25, C-26], respectively.
Moreover, ROESY correlations (Figure 2) were observed
between H-23 (δH 4.56) and H3-27 (δH 0.96) and between H-
26 (δH 4.86) and H3-27 (δH 0.96). The 1D NMR assignments
were analogous to those of anguivioside XI17 and solasodoside
F;18 however, the O-glucosyl group attached to C-26 in
solasodoside F was absent, while an n-propyl group was present
in 4. Accordingly, the structure of compound 4 (indioside J)
was elucidated as 3-O-{α-L-rhamnopyranoside-(1→2)-O-[α-L-
rhamnopyranoside-(1→4)]-β-D -glucopyranosyl}-
(22S,23S,25R,26S)-23,26-epoxy-26β-propoxy-3β,22α,26-trihy-
droxyfurost-5-ene.
Compounds 4 and 5 were separated readily by HPLC with
distinctly different retention times (4, tR = 32.90 min; 5, tR =
50.75 min, 80% MeOH(aq)), although they showed similar
spectroscopic data. Compounds 4 and 5 have the same
molecular formula of C48H78O18 (4: m/z 965.5055 [M + Na]+;
5: m/z 965.5102 [M + Na]+) based on HRESIMS analysis.
Similar specific rotations (4: [α]D26 −68.9 (c 0.47, MeOH); 5:
[α]D26 −65.2 (c 0.37 MeOH) were also observed. The IR spectra
of the two compounds also had similar absorption bands [4:
3438 (OH), 1038 (C−O) cm−1; 5: 3438 (OH), 1038 (C−O)
cm−1].
The 1H and 13C NMR spectra of 5 were similar to those of 4
(Tables 1 and 2), except the CH3-27 proton signal was shifted
downfield from δH 0.96 to 1.10 and the corresponding carbon
signal was shifted upfield from δC 16.4 to 12.9. The molecular
structure of 5 was determined on the basis of analysis of
1H−1H COSY and HMBC experiments (Figure 1). Cross-
peaks between H-23 (δH 4.39) and H-26 (δH 4.98) and
between H-26 and H3-27 (δH 1.10, d, J = 6.7 Hz) were
1
The H NMR spectrum of 4 showed two tertiary methyl
groups (δH 0.99 and 1.06), two secondary methyl groups [δH
0.96 (d, J = 7.1 Hz) and 1.32 (d, J = 6.7 Hz)], one olefinic
proton (δH 5.31), and three monosaccharide moieties; the
sugar signals were essentially identical to those of 3. Analysis of
the 13C NMR spectrum of 4 indicated 48 carbon signals
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Journal of Natural Products
Article
Figure 1. Key HMBC and COSY correlations of compounds 1−5.
a few iso-type substances have been identified.12,16 Our
investigation of S. violaceum has led to the isolation of two
new spirostene saponins, 1 and 2, with an iso-type F ring, and a
sprirostane saponin, 3, with a normal-type F ring. Interestingly,
new compounds 4 and 5 possess an unusual furostanol saponin
skeleton with a deformed F ring. Only two additional
furostanols, anguivioside XI17 and solasodoside F,18 with a
deformed F ring have been reported previously. The nine
known constituents were isolated previously from different
genera of Solanaceae (Lycium chinense),9 Arecaceae (Borassus
flabellifer5 and B. decumbens6), Rubiaceae (Tricalysia dabia),10
and Urticaceae (Urtica dioica)7 families, but are first reported
herein from S. violaceum.
Several prior studies have highlighted a close relationship
between cytotoxic and anti-inflammatory effects.19 Kawabata
and his co-workers20 proposed that steroidal saponins can
prevent production of inflammatory cytokines, such as TNF-α,
in a monocytic cell line and melanin synthesis in melanoma
cells. In the present investigation, the bioassay-guided
fractionation of the EtOAc fraction of S. violaceum led to the
isolation of compounds 1−14, which were evaluated for
cytotoxic activity against six human cancer cell lines [HepG2
and Hep3B (hepatoma), A549 (lung), Ca9−-22 (oral), and
MDA-MB-231 and MCF-7 (breast)] and anti-inflammatory
effects on superoxide anion generation and elastase release
induced by fMLP/CB in human neutrophils.
Figure 2. Key ROESY correlations of compounds 1−5 (aglycone
part).
identified in the ROESY experiments. On the basis of the above
data, indioside K (5) is a C-25 epimer of 4. It was characterized
as 3-O-{α-L-rhamnopyranoside-(1→2)-O-[α-L-rhamnopyrano-
side-(1→4)]-β-D-glucopyranosyl}-(22S,23S,25S,26S)-23,26-
epoxy-26β-propoxy-3β,22α,26-trihydroxyfurost-5-ene.
Two different structures, normal- and iso-type F rings, occur
in steroidal saponins.13 The normal-type F ring is found most
commonly in naturally occurring spirostanols, and thus far, only
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Journal of Natural Products
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Table 3. MTT Cytotoxicity of Compounds 1−14
a
cancer cell line (IC50 value, μg/mL)
MCF7 A549
NT NT
compound
HepG2
NT
Hep3B
NT
Ca9-22
NT
MDA-MB-231
1
NT
2
2.22 0.01
5.33 0.16
2.95 0.02
3.32 0.42
NT
4.78 0.02
11.57 0.70
NT
3.09 0.02
7.27 0.07
NT
2.95 0.07
6.76 0.15
NT
6.12 0.15
8.04 0.12
NT
3
c
4
NT
5
NT
NT
NT
NT
NT
NT
6
>20
>20
>20
>20
>20
>20
7
6.48 0.01
1.83 0.12
6.44 0.45
>20
6.98 0.05
2.03 0.03
2.87 0.04
>20
5.84 0.04
2.61 0.10
8.84 0.12
>20
4.26 0.02
2.34 0.02
4.09 0.08
>20
4.51 0.24
2.33 0.02
3.77 0.02
>20
7.25 0.15
2.75 0.10
5.84 0.06
>20
8
9
10
11
>20
>20
>20
>20
>20
>20
12
9.57 0.26
8.18 0.01
>20
11.23 0.02
15.31 0.21
>20
>20
15.21 0.12
17.53 0.01
>20
12.28 0.07
16.84 0.12
>20
>20
13
>20
>20
14
>20
>20
b
doxorubicin
0.18 0.00
1.31 0.12
0.80 0.03
1.40 0.02
0.31 0.01
1.39 0.00
a
Human cancer cell lines used were HepG2 and Hep3B (liver), A549 (lung), Ca9-22 (oral), MDA-MB-231 and MCF-7 (breast). Results are
b
c
presented as mean SEM (n = 3). Doxorubicin was used as positive control. “NT” means not tested.
The cytotoxicity data are shown in Table 3. Compound 2
showed moderate activity against all six cancer cell lines
(HepG2, Hep3B, A549, Ca9-22, MCF-7, and MDA-MB-231
with IC50 values of 2.22 0.01, 2.95 0.02, 3.09 0.07, 2.95
Table 4. Effects of Compounds 1−14 on Superoxide Anion
Generation and Elastase Release in fMLP/CB-Induced
Human Neutrophils
superoxide anion
elastase release
0.07, 4.78
0.02, and 6.12
0.15 μg/mL, respectively).
a
compound
IC50 (μg/mL) or (Inh %)
IC50 (μg/mL) or (Inh %)
Compound 8 showed moderate cytotoxicity against six cancer
cell lines with IC50 values of 1.83−2.75 μg/mL. Compounds 3
and 9 showed selective cytotoxicity against the Hep3B cancer
cell line (3: 3.32 0.42 μg/mL; 9: 2.87 0.04 μg/mL), which
is a p53 mutant cell line.21 Compounds 2 and 7, as well as 8
and 9, have the same sugar moieties, but differ in the
spirostanol F ring. Comparison of the data for the paired
compounds (2/7 and 8/9) showed that the normal
spirostanols (7 and 9) were less active than the iso-compounds
(2 and 8). Among the nonsteroidal saponins, compounds 12
and 13 exhibited weak cytotoxic activity with IC50 values of
8.18−17.53 μg/mL against four cancer cell lines (HepG2,
Hep3B, A549, Ca9-22), but not MCF7 and MDA-MB-231
breast cancer cell lines.
Regarding the anti-inflammatory data (Table 4), steroidal
saponins 3, 8, and 9 showed selective inhibition of superoxide
anion generation with IC50 values of 2.84 0.18, 0.62 0.03,
and 1.62 0.59 μg/mL, respectively. In the human neutrophil
elastase release assay, the normal spirostanol 9 was considerably
more active (IC50: 4.04 0.51 μg/mL) than the iso-spirostanol
8 (IC50: 111.05 7.37 μg/mL) and also was 5-fold more active
than phenylmethylsulfonyl fluoride, the positive control.
However, compounds 3, 8, and 9 also reduced the cell viability,
as measured by LDH release (see Supporting Information). In
contrast, treatment with compounds 12 and 13 resulted in only
slight inhibition of superoxide anion generation and elastase
release, but did not increase LDH release compared to control
values. Thus, phenolic components 12 and 13 exhibited mild
anti-inflammatory effects without toxicity toward human
neutrophils.
1
NT
NT
NT
2
NT
d
3
2.84 0.18
NT
(38.90 3.34)
4
NT
NT
NT
NT
5
NT
6
NT
7
NT
e
8
0.62 0.03
1.62 0.59
111.05 7.37
9
4.04 0.51
(1.58 1.72)
NT
b
10
11
12
13
14
(24.86 6.08)
NT
c
(23.24 3.66)
(−0.17 3.13)
(24.05 5.59)
c
b
(22.58 4.22)
NT
NT
f
DPI
0.22 0.13
f
PMSF
22.80 5.07
a
Percentage of inhibition (Inh %) at 10 μg/mL concentration. Results
are presented as mean SEM (n = 3). p < 0.05. p < 0.01. p < 0.001
compared with the control value. NT means not tested. Compound 8
alone induced elastase release by human neutrophils. The results are
expressed as a percentage of the fMLP/CB-activated data.
Diphenyleneiodonium (DPI) and phenylmethylsulfonyl fluoride
(PMSF) were used as positive controls for superoxide anion
generation and elastase release, respectively.
b
c
d
e
f
furostanol saponins were characterized, and the stereoisomeric
compounds showed distinct differences in bioactivity.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a JASCO P-1020 digital polarimeter (JASCO Inc.,
Tokyo, Japan). The IR spectra were measured on a Perkin-Elmer 2000
FT-IR spectrophotometer (Perkin-Elmer, Norwalk, CT, USA). 1D
(1H, 13C, DEPT) and 2D (COSY, HSQC, HMBC, ROESY) NMR
spectra using pyridine-d5 as solvent were obtained on a Bruker Magnet
System 500 MHz Ultrashield Plus 600 NMR spectrometer (Bruker
To date, the major constituents of S. violaceum had not been
evaluated previously for cytotoxic and anti-inflammatory
activities. Thus, our study provides the first scientific evidence
to support the traditional use of S. violaceum in Chinese folk
medicine. Most importantly, unique spirostanol and unusual
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Journal of Natural Products
Article
Instruments, Billerica, MA, USA). Chemical shifts are reported in parts
per million (δ), and coupling constants (J) are expressed in Hz and
were internally referenced to the solvent signals in pyridine-d5 (1H, δH
7.21, 7.58, 8.74; 13C, δC 123.5, 136.0, 150.3). Low-resolution ESIMS
were measured on a Bruker Daltonics EsquireHCT ultra high capacity
trap mass spectrometer (Bruker Instruments, Billerica, MA, USA).
HRESIMS were obtained on a Bruker Daltonics APEX II 30e
spectrometer (Bruker Instruments, Billerica, MA, USA). A Shimadzu
LC-20AT pump, a Shimadzu RID-10A refractive index detector
(Shimadzu Inc., Kyoto, Japan), and a Supelco Ascentis ODS 5 μm
(250 × 10 mm i.d.) column were used for HPLC (Supelco, Bellefonte,
PA, USA).
Plant Material. Whole plants of S. violaceum Ortega were collected
from Yunlin, Taiwan (Sept 2010), and identified by one of the authors
(H.-F.Y.). The samples (SIEM 201009) were authenticated and
deposited at the Natural Medicinal Products Research Center,
Taichung.
HRESIMS m/z 722.4241 [M + Na]+ (calcd for C38H62O12Na,
722.9024).
Indioside I (3): white, amorphous powder; [α]D27 −68.9 (c 0.47,
1
MeOH); IR (neat) νmax 3445 (OH), 2931, 1054 cm−1; 13C and H
NMR data, see Tables 1 and 2; ESIMS m/z 893 [M + Na]+;
HRESIMS m/z 893.4862 [M + Na]+ (calcd for C45H74O16Na,
893.0413).
Indioside J (4): white, amorphous powder; [α]D26 −68.9 (c 0.47,
MeOH); IR (neat) νmax 3438 (OH), 2929, 1258, 1271, 1039 cm−1;
1
13C and H NMR data, see Tables 1 and 2; ESIMS m/z 965 [M +
Na]+; HRESIMS m/z 965.5055 [M + Na]+ (calcd for C48H78O18Na,
965.1040).
Indioside K (5): white, amorphous powder; [α]D26 −65.2 (c 0.37,
MeOH); IR (neat) νmax 3438 (OH), 2929, 1258, 1271, 1039 cm−1;
1
13C and H NMR data, see Tables 1 and 2; ESIMS m/z 965 [M +
Na]+; HRESIMS m/z 965.5102 [M + Na]+ (calcd for C48H78O18Na,
965.1040).
Acid Hydrolysis. A solution of steroidal glycoside (8, 2 mg; 1−5, 1
mg) was dissolved in 2 M HCl/1,4-dioxane (1:1, v/v, 1.0 mL) and
stirred overnight at 90 °C. After cooling, the mixture was partitioned
with CH2Cl2/H2O (1:1, v/v), and the aqueous layer was neutralized
with Na2CO3 and filtered. The filtrate was dissolved in pyridine (0.2
mL), and Ac2O was added. The mixture was stirred overnight at 60
°C. After cooling, the reaction mixture was dried under vacuum, and
the residue was extracted with CH2Cl2 and H2O. The CH2Cl2 layer
was subjected to GC analysis (DSQ II Single Quadrupole GC/MS,
Thermo Fisher Scientific Inc., Rockford, IL, USA) under the following
conditions: H2 flame ionization detector; DB-5MS capillary column
(Agilent J&W Scientific, Palo Alto, CA, USA) (30 m × 0.25 mm ×
0.25 μm); column temperature, 100−200 °C increased at a rate of 4
°C/min; carrier gas, He (1 mL/min); injector temperature, 200 °C;
ion source temperature, 200 °C; EI, 70 eV; injection volume, 5 μL;
mass range, m/z 50−1000. The acetate derivatives of D-glucose, L-
rhamnose, and D-xylose had the following retention times, tR (min): D-
glucose (27.32), L-rhamnose (20.76), and D-xylose (20.70). Identi-
fication of the sugar derivative was based on a comparison of mass
spectra with those of authentic samples, data from Wiley/NBS
Registry of Mass Spectral Data (V. 5.0)/National Institute of
Standards and Technology (NIST) MS Search V. 2.0.
MTT Cytotoxicity Assay. Fractions and isolates were tested
against six human cancer cell lines [HepG2 and Hep3B (hepatoma),
A549 (lung), Ca9-22 (oral), MDA-MB-231 and MCF-7 (breast)]. The
inhibitory effect of test drug on the cell viability was measured by the
MTT colorimetric method as described previously.22 Cells were
seeded at densities of 5000−10 000 cells/well in 96-well tissue culture
plates. On day 2, cells were treated with test drug for various time
periods. After drug treatment, attached cells were incubated with MTT
(0.5 mg/mL, 1 h) and subsequently solubilized in DMSO. The
absorbency at 550 nm was then measured using a microplate reader.
The IC50 is the concentration of agent that reduced the cell viability by
50% under the experimental conditions. Experiments were performed
in triplicate, and the values are the averages of three (n = 3)
independent experiments. Doxorubicin was used as the positive
control.
Human Neutrophil Superoxide Anion Generation. Human
neutrophils were obtained by means of dextran sedimentation and
Ficoll centrifugation. Superoxide anion production was assayed by
monitoring the superoxide dismutase-inhibitable reduction of
ferricytochrome c.23,24 In brief, after supplementation with 0.5 mg/
mL ferricytochrome c and 1 mM Ca2+, neutrophils were equilibrated at
37 °C for 2 min and incubated with drug for 5 min. Cells were
incubated with cytochalasin B (CB) (1 μg/mL) for 3 min, before
activation by the tripeptide N-formyl-L-methionyl-L-leucyl-L-phenyl-
alanine (fMLP) (100 nM) for 10 min. Changes in absorbance with the
reduction of ferricytochrome c at 550 nm were continuously
monitored in a double-beam, six-cell positioner spectrophotometer
(Hitachi U-3010, Tokyo, Japan) with constant stirring. Calculations
were based on differences in the reactions with and without superoxide
dismutase (100 U/mL) divided by the extinction coefficient for the
reduction of ferricytochrome c (ε) 21.1 mM/10 mm). Diphenyle-
Extraction and Isolation. The air-dried whole plants (5 kg) were
extracted with MeOH (3 × 20 L) overnight at room temperature. The
combined MeOH extracts were evaporated under vacuum to give
243.9 g of dried crude extract. The MeOH extract was partitioned
between EtOAc and H2O (1:1, v/v) to give an EtOAc-soluble fraction
(103.9 g) and an aqueous phase (140 g). The EtOAc-soluble fraction
was further extracted with n-hexane and 90% MeOH (1:1, v/v) to
yield n-hexane (15.7 g) and 90% MeOH (88.2 g) fractions. The 90%
MeOH fraction was separated on silica gel column chromatography
(840 g, 10 × 25 cm, 70−230 mesh) and eluted with a gradient mixture
of CHCl3/MeOH (50:1 → 0:1, v/v) to give 10 fractions (A1−A10).
Fraction A3 (8.9 g) was recrystallized from MeOH and filtered to
afford sitosterol 3-O-β-D-glucopyranoside (10, 2.5 g). The mother
liquid was concentrated under reduced pressure and chromatographed
on silica gel (500 g, 8 × 17 cm, 230−400 mesh) with a gradient elution
of CHCl3/MeOH (50:1 → 0:1, v/v) to provide seven subfractions
(A3.1−A3.7, 2 L/each). Subfraction A3.4 (1.35 g) was subjected to
column chromatography on Sephadex LH-20 (820 g, 5 × 57 cm) with
CHCl3/MeOH (1:1, v/v) and further purified by silica gel column
chromatography (120 g, 3.5 × 27 cm, 230−400 mesh, EtOAC) to give
N-p-coumaroyltyramine (12, 324.7 mg). Subfraction A3.5 (1.1 g) was
chromatographed on Sephadex LH-20 (820 g, 5 × 57 cm) with
CHCl3/MeOH (1:1, v/v) to furnish trans-N-feruloyloctopamine (13,
14.1 mg). Fraction A4 (3.8 g) was chromatographed on silica gel with
a gradient of CHCl3/MeOH (25:1 → 15:1 → 0:1, v/v) to give four
subfractions (A4.1−A4.4, 1.2 L/each). Subfractions A4.2 (2.3 g) and
A4.3 (1.2 g) were combined and chromatographed repeatedly on
Sephadex LH-20 [820 g, 5 × 45 cm, CHCl3/MeOH (1:1)], silica gel
[300 g, 2 × 24 cm, 230−400 mesh, CHCl3/MeOH (20:1, v/v)], and
RP-RI-HPLC (MeOH/H2O, 9:1, v/v) to give indioside G (1, 4.0 mg),
borassoside D (6, 8.5 mg), and 7-hydroxysitosterol-3-O-β-D-glucopyr-
anoside (10, 2.2 mg). Fraction A5 (4.0 g) was chromatographed on
Sephadex LH-20 (820 g, 5 × 50 cm) with MeOH to give four
subfractions (A5.1−A5.4, 1.5 L/each). Subfraction A5.2 (1.8 g) was
purified using ODS HPLC to furnish tricalysioside U (14, 2.1 mg),
borassoside E (8, 43.4 mg), 3-O-chacotriosyl-25(S)-spirost-5-en-3β-ol
(9, 25.4 mg), indioside H (2, 4.4 mg), indioside I (3, 8.1 mg), and
yamogenin 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (7,
2.2 mg). Fraction A6 (8.13 g) was chromatographed on Sephadex LH-
20 (820 g, 5 × 57 cm) with CHCl3/MeOH (1:1, v/v) to give four
subfractions (A6.1−A6.4, 2.0 L/each). Subfraction A6.3 (591.4 mg)
was chromatographed on RP-18 (50 g, 40−63 μm, 2 × 26 cm, 90%
MeOHaq, v/v) and further purified using ODS HPLC (MeOH/H2O,
4:1, v/v) to give indioside J (4, 3.5 mg) and indioside K (5, 2.2 mg).
Indioside G (1): white, amorphous powder; [α]D26 −92.4 (c 0.3,
MeOH); IR (neat) νmax 3402 (OH), 2922, 2841, 1274, 1259, 1030
cm−1; 13C and 1H NMR data, see Tables 1 and 2; ESIMS m/z 731 [M
+ Na]+; HRESIMS m/z 731.3966 [M + Na]+ (calcd for C38H60O12Na,
731.4085).
Indioside H (2): white, amorphous powder; [α]D26 −89.2 (c 0.52,
1
MeOH); IR (neat) νmax 3402 (OH), 2915, 1048 cm−1; 13C and H
NMR data, see Tables 1 and 2; ESIMS m/z 722 [M + Na]+;
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neiodonium (a NADPH oxidase inhibitor) was used as the positive
control.
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T.; Aramoto, M.; Takeda, Y.; Kanchanapoom, T. Chem. Pharm. Bull.
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Measurement of Elastase Release. Degranulation of azurophilic
granules in human neutrophils was determined by elastase release as
described previously.25 Experiments were performed using MeO-Suc-
Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate. After supple-
mentation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM),
neutrophils (6 × 105/mL) were equilibrated at 37 °C for 2 min and
incubated with each test compound for 5 min. Cells were activated by
fMLP (100 nM)/CB (0.5 μg/mL), and changes in absorbance at 405
nm were monitored continuously for elastase release. The results were
expressed as the percentage of the initial rate of elastase release in the
fMLP/CB-activated, test compound-free (DMSO) control system.
Phenylmethylsulfonyl fluoride (a serine protease inhibitor) was used as
the positive control.
Lactate Dehydrogenase (LDH) Release. LDH release was
determined by a commercially available method (Sigma). Cytotoxicity
was represented by LDH release in the cell-free medium as a
percentage of the total LDH release. The total LDH release was
determined by lysing cells with 0.1% Triton X-100 for 30 min at 37
°C.26
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Planta Med. 2011, 77, 705−710.
ASSOCIATED CONTENT
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S
* Supporting Information
1
The H, 13C, and 2D NMR data of all new compounds and
LDH experiment are available free of charge via the Internet at
http://pubs.acs.org.
(21) Shin, D. Y.; Kim, G. Y.; Kim, N. D.; Jung, J. H.; Kim, S. K.;
Kang, H. S.; Choi, Y. H. Oncol. Rep. 2008, 19, 517−526.
(22) Yen, C.-T.; Wu, C.-C.; Lee, J.-C.; Chen, S.-L.; Morris-Natschke,
S. L.; Hsieh, P.-W.; Wu, Y.-C. Eur. J. Med. Chem. 2010, 45, 2494−
2502.
AUTHOR INFORMATION
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Corresponding Author
*Tel: +886-4-22057153. Fax: +886-4-22060248. E-mail:
yachwu@mail.cmu.edu.tw.
(23) Hwang, T.-L.; Leu, Y.-L.; Kao, S.-H.; Tang, M.-C.; Chang, H.-L.
Free Radical Biol. Med. 2006, 41, 1433−1441.
(24) Babior, B. M.; Kipnes, R. S.; Curnutte, J. T. J. Clin. Invest. 1973,
52, 741−744.
Funding
The authors declare no competing financial interest.
(25) Sklar, L. A.; McNeil, V. M.; Jesaitis, A. J.; Painter, R. G.;
Cochrane, C. G. J. Biol. Chem. 1982, 257, 5471−5475.
(26) Hwang, T.-L.; Fang, C.-L.; Al-Suwayeh, S. A.; Yang, L.-J.; Fang,
J.-Y. Toxicol. Lett. 2011, 203, 172−180.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We gratefully acknowledge financial support for this project
from the Department of Health, Executive Yuan, Taiwan
(DOH101-TD-C-111-004), and sincerely appreciate Dr. S. L.
Morris-Natschke for English revision (Natural Products
Research Laboratories, UNC).
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