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PHYTOL 670 1–6
Phytochemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters
jo urnal homepage: www.elsevier.com/locate/phytol
1
2 New antiproliferative and immunosuppressive withanolides from the
3 seeds of Datura metel
a,1 a,1 a b a a
4 Q1 Bing-You Yang , Yong-Gang Xia , Yan Liu , Li Li , Hai Jiang , Liu Yang ,
a a,
5 Qiu-Hong Wang , Hai-Xue Kuang *
a
6 Key Laboratory of Chinese Materia Medica, Heilongjiang University of Chinese Medicine, Ministry of Education, Harbin 150040, People’s Republic of China
b
7 Q2 Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences &
8 Peking Union Medical College, People’s Republic of China
A R T I C L E I N F O A B S T R A C T
Article history: Two new withanolides baimantuoluoside H (1) and baimantuoluoline K (2), and one known withanolide
Received 28 September 2013
glycoside (3) were isolated and identified from the ethyl acetate-soluble fraction of ethanol extract of
Received in revised form 10 February 2014
Datura metel seeds. The structures of the new compounds were determined using 1D and 2D NMR
Accepted 14 February 2014
spectroscopy, and mass spectrometry. All isolated compounds were evaluated for their antiproliferative
Available online xxx
activity against human gastric adenocarcinoma (SGC-7901), human hepatoma (Hepg2), and human
breast cancer (MCF-7) cells, as well as their immunosuppressive properties. It was determined that
Keywords:
compounds 1–3 exhibited medium antiproliferative and potential immunosuppressive effects.
Datura metel L.
ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. Solanaceae Withanolides Antiproliferative Immunosuppressive
9
10 1. Introduction which mainly include withanolides, flavonoids and alkaloids. 28
Many reports have demonstrated that withanolides have anti- 29
11 Q3 Datura metel L. known as baimantuoluo in Chinese, has been proliferative activities in pancreatic cancer cells (Gu et al., 2013) 30
12 used as a popular traditional Chinese medicine for centuries to and HT29 cancer cell lines (Bouzidi et al., 2013), and showed 31
13 treat asthma, rheumatism, convulsions and relieve pain. Previous cytotoxicity against human breast (MCF7), lung (Lu1) and prostate 32
14 pharmacological studies have demonstrated that extracts of D. (LNCaP) cancer cell lines (Veras et al., 2004a,b). Additionally, some 33
15 metel have antifungal, anti-feeding, anti-insect, anti-hyperglycae- withanolides have significant immunosuppressive properties 34
16 mic, narcotic, anodyne and antispasmodic activities (Dabur et al., (Yang et al., 2013; Huang et al., 2009; Mirosława et al., 2001; 35
17 2004; Kaushik and Goyal, 2008; Krishna et al., 2004; Pascual- Solomon, 1997). This is the first report of the chemical constituents 36
18 Villalobos and Robledo, 1998; Zhou et al., 2008). The whole plant is of the seeds of Datura metel. The activities of the isolated 37
19 considered to be as narcotic, anodyne and antispasmodic. compounds were also evaluated. As a result, two new withanolides 38
20 Additionally, the bark, leaves, and seeds of D. metel are used in (1–2) and one known withanolide (3) were isolated and identified 39
21 Ayurveda for treating various diseases. Some studies have from the EtOAc-soluble partition of its ethanol extract. The isolated 40
22 demonstrated that its seeds have effects as an aphrodisiac and compounds were determined to have cytotoxic effects in SGC- 41
23 in treating leucoderma, skin disorders, ulcers, bronchitis, jaundice, 7901 cancer cells and exhibited potential immunosuppressive 42
24 piles and in diabetes (Krishna et al., 2004). Researchers have properties. 43
25 extensively studied the active constituents of the D. metel flower
26 (Yang et al., 2010a,b; Kuang et al., 2008, 2009, 2011; Yang et al.,
2. Results and discussion 44
27 2008; Ma et al., 2006; Manickam et al., 1998; Bellila et al., 2011),
Extracts of D. metel were separated and purified via multiple 45
chromatographic procedures including silica gel and octadecylsilyl 46
(ODS) column chromatography, as well as high-performance liquid 47
* Corresponding author. Tel.: +86 0451 82193001.
chromatography (HPLC), and three withanolides were obtained 48
E-mail addresses: [email protected], [email protected] (H.-X. Kuang).
1
These authors contributed equally to this article. (Compounds 1–3, Fig. 1). 49
http://dx.doi.org/10.1016/j.phytol.2014.02.008
1874-3900/ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Please cite this article in press as: Yang, B.-Y., et al., New antiproliferative and immunosuppressive withanolides from the seeds of
Datura metel. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.02.008
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2 B.-Y. Yang et al. / Phytochemistry Letters xxx (2014) xxx–xxx
1 13
The H and C NMR spectra (Table 1) of 1 displayed 34 carbon 56
signals and several characteristic signals corresponding to the A–D 57
rings of the steroid skeleton. The four signals at dH 0.87 (s, 3H), 1.04 58
(d, J = 6.7 Hz, 3H), 1.25 (s, 3H) and 2.12 (s, 3H) were attributable to 59
1
Me-18, Me-19, Me-21 and Me-28, respectively. The H NMR signals 60
of five olefinic protons at dH 5.89 (d, J = 9.6 Hz, H-2); 7.11 (dd, 61
J = 9.6, 6.2, H-3); 6.04 (d, J = 6.2, H-4); 6.21 (dd, J = 9.9, 2.6 Hz, H-6); 62
5.89 (d, J = 9.6 Hz, H-7) indicated the presence of three olefinic 63
1 13
bonds. The H and C NMR data of 1 indicated the presence of a b- 64
0
configured glucosyl unit [dH 4.32 (d, J = 7.8 Hz, H-1 ), dC 104.0]. 65
Fig. 1. Structures of compounds 1–3. These data indicated that 1 was a withanolide glycoside that was 66
structurally similar to daturametelin H (Ma et al., 2006). The only 67
difference was the configuration of C-14. 68
The configuration of 1 was established based on the results of 69
NOESY experiments and coupling constants. The NOE cross-peaks 70
50 Compound 1 was obtained as an amorphous powder and between Me-18 and H-8, H-14 and H-8, respectively, indicated an 71
51 showed positive results with the Molish reagent and the uncommon withanolide skeleton with H-14 being b-oriented. 72
52 Liebermann-Burchard reaction, which indicated that it might be Assignments of all of the functional groups of 1 were achieved on 73
1 1
53 a triterpenoid or steroid. The molecular formula wsa assigned as the basis of H– HCOSY, HSQC and HMBC data (Fig. 2). Addition- 74
+
54 C34H46O9 based on the [M+Na] signal at m/z 621.3058 in the ally, the NOE correlations (Fig. 3) between H-18 and H-20/H-21/ 75
55 positive mode HRESIMS, indicating 12 degrees of unsaturation. Hb-16, H-20 and Hb-16/H-22 revealed that C-20 of compound 1 76
Table 1
1 13 1 13
H and C NMR data of 1–3 (400 MHz in H NMR and 100 MHz in C NMR, in CD3OD).
No. 1 2 3
dC dH dC dH dC dH
1 207.2 73.5 3.85 (s) 211.5
2 125.8 5.89 d (9.6) 39.1 2.03 (m) 40.6 2.74 dd (4.0,20.4)
1.75 (m) 3.46 dt (3.4,21.2)
3 143.2 7.11 dd (6.2,9.6) 66.7 3.94 (m) 126.4 5.87ddd (3.0,4.6,9.6)
4 118.4 6.04 d (6.2) 42.4 2.35 (m) 130.2 6.18 dd (1.6,10.0)
5 157.5 144.3 146.5
6 128.5 6.21 dd (2.7,9.9) 127.3 5.74 d (5.4) 125.6 6.06 d (5.2)
7 136.2 5.89 d (9.6) 65.9 3.74 t (3.8) 74.0 3.51 t (4.3)
8 39.4 2.38 t (10.3) 35.1 1.92 (m) 38.0 1.64(m)
9 48.7 1.57 (m) 38.9 1.47 (m) 35.7 2.13(m)
10 52.3 43.3 54.2
11 25.0 1.83 (m) 21.0 1.60 (m) 23.4 1.83 (m)
1.36 (m) 1.52 (m) 1.76(m)
12 41.0 2.07 (m) 40.5 2.03 (m) 40.4 1.99 (m)
1.20 (m) 1.24 (m) 1.28 (m)
13 45.0 43.7 43.8
14 55.1 1.27(m) 50.7 1.60 (m) 50.4 1.64 (m)
15 23.7 2.19 (m) 25.2 1.84 (m) 25.1 1.76(m)
1.70 (m) 1.24 (m) 1.28 (m)
16 28.1 1.83(m) 28.3 1.84 (m) 28.2 1.83 (m)
1.46 (m) 1.43 (m) 1.45 (m)
17 53.1 1.26 (m) 53.1 1.24 (m) 53.2 1.28 (m)
18 12.2 0.87 (s, 3H) 12.0 0.80 (s, 3H) 12.0 0.80 (s, 3H)
19 20.5 1.25 (s, 3H) 18.9 1.03 (s, 3H) 20.1 1.39 (s, 3H)
20 40.4 1.98 (m) 40.4 1.97 (m) 40.5 1.99 (m)
21 13.6 1.04 d (6.7,3H) 13.8 1.07 d (6.6, 3H) 13.7 1.07 d (6.6, 3H)
22 80.0 4.49dt (3.4,13.2) 80.2 4.49 dt (3.4,13.3) 80.1 4,51dt (3.4,13.6)
23 30.8 2.55 (m) 30.7 2.56 (m) 30.8 2.60 (m)
2.19 (m) 2.23 (m) 2.25 dd (3.2,18.1)
24 160.3 157.9 160.4
25 123.7 126.4 123.6
26 168.6 168.6 168.6
27 63.6 4.62 d (11.2) 56.4 4.40 d (11.7) 63.6 4.65 d (11.2)
4.46 d (11.2) 4.33 d (11.7) 4.49 d (11.2)
28 20.8 2.12 (s, 3H) 20.3 2.13 (s, 3H) 20.8 2.16 (s, 3H)
0
1 104.0 4.32 d (7.8) 104.0 4.35 d (7.8)
0
2 75.0 3.16 t (8.4) 75.0 3.19 dd (8.0, 8.7)
0
3 78.0 3.34 (m) 78.0 3.36 (m)
0
4 71.6 3.27 (m) 71.5 3.27 (m)
0
5 78.0 3.27 (m) 78.0 3.28 (m)
0
6 62.7 3.85dd (1.8,11.9) 62.7 3.70 dd(1.9,11.9)
3.67dd (5.5,11.6) 3.88 dd (5.2,11.9)
7-OCH3 56.8 3.32(s, 3H)
Please cite this article in press as: Yang, B.-Y., et al., New antiproliferative and immunosuppressive withanolides from the seeds of
Datura metel. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.02.008
G Model
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B.-Y. Yang et al. / Phytochemistry Letters xxx (2014) xxx–xxx 3
1 1
Fig. 2. Key HMBC and H– H COSY correlations of Compound 1.
Fig. 4. Calculated ECD and experimental ECD spectra of compound 1 at the B3LYP
level.
77 was in the S configuration. According to the literatures (Minguzzi except for the replacement of the C-7 methylene group of 106
78 et al., 2002; Ma et al., 2006), an a-oriented H-22 atom gives rise to pubesenolide with an oxygenated methine in 2. The HMBC 107
79 J(22,23) values of 0.5–4 and 9–13.8 Hz, whereas the b-oriented form correlations of H-7/C-5, H-7/C-8 and H-6/C-7 (Fig. 5) confirmed 108
80 exhibits J(22,23) values of 2.5–7.0 and 2.0–5.0 Hz. Thus, the observed that the hydroxyl was attached to C-7. In the NOESY experiments, 109
81 coupling constants (J = 3.4, 13.2 Hz) indicate the presence of an Ha- the NOE cross-peaks of H-18/H-11 and H-7/H-11, indicated that H- 110
82 22 atom, i.e., an R-configuration at C-22. The absolute configura- 7 was b-oriented, and that the C-7 hydroxyl group was a-oriented. 111
83 tions of C-22 in compound 1 was assigned by comparison of the Additionally, the NOE correlations (Fig. 6) between H-20 and Hb- 112
84 experimental with the theoretical electronic circular dichroism 16/H-22, and H-18 and H-20/H-21/Hb-16 revealed that C-20 of 113
85 (ECD) spectra, which was predicted using the time-dependent compound 2 was also in the S configuration. Furthermore, ECD 114
86 density functional theory (TDDFT) method (Crawford, 2006; Ishida experiments and calculations for compound 2 were also con- 115
87 et al., 2006). Compound 1 was split two parts 1A and 1B (Fig. S1). ducted. Similar to the above methods, the absolute configuration of 116
88 All possible conformers (Fig. S2) of 1A and 1B were optimised at the 2 was also determined by comparing its experimental CD data and 117
89 B3LYP/6-31G(d, p) basis set level, and then used to calculate their calculated ECD curve (Fig. 7) of 2A + 2B (Fig.S3). 118
90 ECD spectra (Fig. 4). The results indicated that the calculated ECD From the above data, the structure of 2 was determined to be 119
91 spectrum (Fig. 4) of 1A + 1B was similar to the experimental 1a,3b,7a,20S,22R,27-tetrahydroxy-5,24-dienolide, and named 120
92 spectrum of compound 1. Thus, C-22 of compound 1 was assigned baimantuoluoline K. 121
93 the R configuration. The antiproliferative and immunosuppressive effects of the 122
94 From the above data, the structure of 1 was determined to be three compounds were measured. It was found that compounds 1– 123
95 14b-hydrogen-(20S,22R)-22,26-epoxy-27-[(b-glucopyranosyl)ox- 3 exhibited cytotoxicity against SGC-7901 cancer cells with IC50 124
96 y]ergosta-2,4,6,24-tetraene-1,26-dione, and the molecule was values of 37.7, 29.2 and 39.0 mg/mL, respectively, but very weak 125
97 named baimantuoluoside H. effects on the hepatoma and breast cancer cell lines. The IC50 value 126
98 Compound 2 was obtained as an amorphous powder and the of 5-fluorouracil (5-FU) used as the positive control and was 127
+
99 molecular formula assigned as C28H42O6 from the [M+Na] signal 1.22 mg/mL in SGC-7901. Additionally, 1–3 possessed potential 128
100 at m/z 497.2944 in the positive mode HRESIMS, indicating 8 immunosuppressive properties with IC50 values of 14.0 mg/mL, 129
101 degrees of unsaturation.
1 13
102 The H and C NMR spectra of 2 displayed several characteris-
103 tic signals of the 1a,3b-dihydroxy-5-en-witha in the A and B rings
1 13
104 of withanolides. Structurally, the H and C NMR data (Table 1) of
105 2 was found to be similar to those for pubesenolide (Sahai, 1985),
1 1
Fig. 3. Key NOESY correlations of Compound 1. Fig. 5. Key HMBC and H– H COSY correlations of Compound 2.
Please cite this article in press as: Yang, B.-Y., et al., New antiproliferative and immunosuppressive withanolides from the seeds of
Datura metel. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.02.008
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4 B.-Y. Yang et al. / Phytochemistry Letters xxx (2014) xxx–xxx
China). Silica gel was purchased from Qingdao Marine Chemical 156
Ltd., Qingdao, China. All the solvents used were of analytical grade 157
and were purchased from Tianjinfuyu Company Ltd., Tianjin, 158
China. Cancer cells were obtained from Harbin Medical University, 159
Heilongjiang, China. 160
3.2. Plant material 161
Dry seeds of D. metel L. were collected in October 2010 from 162
Harbin, Heilongjiang Province of China. The plant was identified by 163
Prof. Zhengyue Wang of Heilongjiang University of Chinese 164
Medicine, and its voucher specimen (2010035) was deposited at 165
Fig. 6. Key NOESY correlations of Compound 2.
Heilongjiang University of Chinese Medicine. 166
3.3. Extraction and isolation 167
130 12.3 mg/mL and 13.8 mg/mL, respectively, but were much lower
131 than that of cyclosporine, which served as a positive control.
Dry seeds (5 kg) of D. metel were exhaustively extracted with 168
132 Compounds 1–3 did not show cytotoxicity over the entire range of
95% ethanol and then sequentially with petroleum ether 169
133 tested concentrations in the normal cell lines (HSF and MRC5),
(60–90 8C), EtOAc and n-BuOH. Six fractions (Fr.1-Fr.6) were 170
134 which indicated that the potential immunosuppressive effects
combined and concentrated in vacuo based on their TLCs. Fr.3 (10 g) 171
135 observed in vitro are most likely not due to the toxicity of
was purified by silica gel chromatography, using CHCl3/MeOH 172
136 compounds.
(100:1 to 0:1) as the eluent, to afford sub-fractions B1-B13. Sub- 173
137 Studies on the seeds of D. metel have great potential to discover
fraction B5 (1.1 g) was subjected to ODS column chromatography 174
138 new compounds with medicinal applications and expand the
with MeOH/H2O (2:8 to 1:0) and finally purified by semi-preparative 175
139 existing pool of drug sources. Also, our results indicated that
HPLC on a Hypersil-ODS P column (10 mm, 20 mm 300 mm, flow 176
140 compounds 1–3 could be developed as antiproliferative and
rate, 5 mL/min, UV detection, at 225 nm), to afford 1 (11 mg) and 2 177
141 immunosuppressive drugs. This research also provides guidance
(24 mg). Sub-fraction B6 was purified in a similar manner to that of 178
142 for discovering new compounds that can prevent or modulate
sub-fraction B5, to afford 3 (16 mg). 179
143 immune cell proliferation.
3.3.1. Baimantuoluoside H (1) 180
27
White amorphous powder. 10:7 (c 0.1, MeOH); UV 181
144 3. Experimental ½a D
(MeOH) lmax 226 nm; IR (film) nmax 3385, 2928, 1610, 1400, 1383, 182
1 1 13
1074, 1040 cm ; H and C NMR (MeOH, 400, 100 MHz) data, see 183
145 3.1. General
+
Table 1; HRESIMS m/z 621.3058 [M+Na] (calcd for C18H22N2O4Na, 184
621.3040). 185
146 Optical rotations were recorded on a JASCO P-2000 polarimeter.
147 The UV spectra were recorded on a Shimadzu UV-1601 instrument.
3.3.2. Baimantuoluoline K (2) 186
148 IR spectra were obtained on a Shimadzu FTIR-8400S instrument.
27
White amorphous powder. 13:1 (c 0.32, MeOH); UV 187
149 The NMR spectra were recorded on a Bruker DPX 400 instrument ½a D þ
1 13
(MeOH) 226 nm; IR (film) 3385, 2937, 2876, 1701, 1407, 188
150 (400 MHz for H NMR and 100 MHz for C NMR). Samples were lmax nmax
1 1 13
1159, 1052 cm ; H and C NMR (MeOH, 400, 100 MHz) data, see 189
151 prepared in MeOH with TMS as an internal standard. Coupling
+
Table 1; HRESIMS m/z 497.2944 [M+Na] (calcd for C H O Na, 190
152 constants (J) are given in Hz. The HR-ESI-MS data were acquired on 35 50 10
TM 497.2879). 191
153 an Waters Xevo-TOF-MS instrument. Preparative HPLC (Waters,
154 Delta 600-2487) was performed using a Hypersil-ODS II column
3.3.3. Baimantuoluoside I (3) 192
155 (10 mm, 20 mm 300 mm, Yilite, Da Lian, People’s Republic of
27
White amorphous powder. ½a D 10:4 (c 0.69, MeOH); UV 193
(MeOH) lmax 226 nm; IR (film) nmax 3413, 2937, 2878, 1709, 1398, 194
1 1 13
1078, 1043 cm ; H and C NMR (MeOH, 400, 100 MHz) data, see 195
+
Table 1; HRESIMS m/z 653.3364 [M+Na] (calcd for C35H50O10Na, 196
653.3302). 197
3.4. ECD calculation 198
The theoretical calculations of compounds 1 and 2 were 199
performed using Gaussian 09 (Gaussian 09, revision B.01, 200
Gaussian, Inc., www.gaussian.com.). Conformational analysis 201
was initially carried out using a Maestro7.5 conformational search 202
using the MMFF94 molecular mechanics force field via the MOE 203
software package (MOE2009.10, Chemical Computing Group Inc., 204
www.chemcomp.com.). The MMFF94 conformers were then 205
optimised at the B3LYP/6-31G(d) level. Room-temperature equi- 206
librium populations were calculated according to the Boltzmann 207
distribution law. The theoretical ECD calculation was performed 208
using TDDFT at the B3LYP/6-31G (d, p) level in MeOH with the PCM 209
model. The ECD spectra of compounds 1 and 2 were obtained by 210
Fig. 7. Calculated ECD and experimental ECD spectra of compound 2 at the B3LYP weighing the Boltzmann distribution rate of each geometric 211
level. conformation. 212
Please cite this article in press as: Yang, B.-Y., et al., New antiproliferative and immunosuppressive withanolides from the seeds of
Datura metel. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.02.008
G Model
PHYTOL 670 1–6
B.-Y. Yang et al. / Phytochemistry Letters xxx (2014) xxx–xxx 5
Table 2
added to each well, and the absorbance (A) was evaluated after 245
Immunosuppressive of 1–3 in the experiment of Splenocyte Proliferation of Mouse.
15 min in an ELISA reader at 570 nm with a 630 nm reference 246
a
Growth inhibition constant (IC50 in mg/mL) (Tables 2 and 3). 247
Drugs IC50
b 3
Cyclosporine 4.5 10 Acknowledgments 248
Compound 1 14.0
Compound 2 12.3
This work was financed by the Major State Basic Research 249
Compound 3 13.8
a Development Program of China (973 Program 2006CB504708), the 250
IC50 was defined as the concentration that resulted in a 50% decrease in cell
number National Natural Science Foundation of China (Nos. 30371736, 251
b
Positive control. 30672633), the Special Fund Project of National Excellent Doctoral 252
Dissertation of China (200980) and General Program Projects 253
Funding of Heilongjiang University of Chinese Medicine (200916). 254
Table 3
Appendix A. Supplementary data 255
Antiproliferative of 1–3 against three human cancer cell lines.
a
Drugs Growth inhibition constant (IC50 in mg/mL) Supplementary data associated with this article can be found, in 256
the online version, at http://dx.doi.org/10.1016/j.phytol.2014.02.008. 257
SGC-7901 MCF-7 HepG2
b
5-FU 8.9 32.7 1.4
Compound 1 37.7 >50 >50 References 258
Compound 2 29.2 >50 >50
Compound 3 39.0 >50 >50 Bellila, A., Tremblay, C., Pichette, A., Marzouk, B., Mshvildadze, V., Lavoie, S., Legault, 259
a J., 2011. Cytotoxic activity of withanolides isolated from Tunisian Datura metel 260
IC50 was defined as the concentration that resulted in a 50% decrease in cell
L. Phytochemistry 72, 2031–2036. 261
number
Bouzidi, L., Mahiou-Leddet, V., Bun, S., Larhsini, M., 2013. Cytotoxic withanolides 262
The IC greater than 50 mg/mL was deemed inactive. And/means ineffective.
50 from the leaves of Moroccan Withania frutescens. Pharm. Biol. 51, 1040–1046. 263
b
Positive control. Crawford, T.D., 2006. Ab initio calculation of molecular chiroptical properties. 264
Theor. Chem. Acc. 115, 227–245. 265
Dabur, R., Ali, M., Singh, H., Gupta, J., Sharma, G., 2004. A novel antifungal pyrrole 266
derivative from Datura metel leaves. Die Pharmazie 59, 568–570. 267
Gu, M., Yu, Y., Gumaherath, G., Gunatilaka, A., Li, D., Sun, D., 2013. Structure-activity 268
relationship (SAR) of withanolides to inhibit Hsp90 for its activity in pancreatic 269
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213 3.5. MTT bioassays
Huang, C.-F., Ma, L., Sun, L.-J., Mumtaz, A., Mohammad, A., Liu, J.-W., Hu, L.-H., 2009. 271
Immunosuppressive withanolides from Withania coagulans. Chem. Biodivers. 9, 272
1415–1426. 273
214 Compounds 1–3 (purity >92%, >95%, >95%, by HPLC, respec-
Ishida, K., Maksimenka, K., Fritzsche, K., Scherlach, K., Bringmann, G., Hertweck, C., 274
215 tively) were tested for their antiproliferative effects, against
2006. The boat-shaped polyketide resistoflavin results from re-facial central 275
216 human hepatoma (Hepg2), human breast cancer (MCF-7), and hydroxylation of the discoid metabolite resistomycin. J. Am. Chem. Soc. 128, 276
14619–14624. 277
217 Q4 human gastric adenocarcinoma (SGC-7901) cells using the MTT
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218 method (Wu et al., 2010a,b; Wu et al., 2011) using 5-fluorouracil
potential antibacterial activity. Indian J. Microbiol. 48, 353–357. 279
219 (5-FU) as a positive control. Briefly, 100 mL of test cells in Krishna Murthy, B., Nammi, S., Kota, M., Krishna Rao, R., Koteswara Rao, N., 280
4
Annapurna, A., 2004. Evaluation of hypoglycemic and antihyperglycemic effects 281
220 exponential growth were plated at a final concentration of 5 10
of Datura metel (Linn.) seeds in normal and alloxan-induced diabetic rats. J. 282
221 cells/well in 96-well microtiter plates and cultured in RPMI-1640
Ethnopharmacol. 91, 95–98. 283
222 medium supplemented with 10% foetal bovine serum (FBS), in a Kuang, H., Yang, B., Xia, Y., Feng, W., 2008. Chemical constituents from the flower of 284
Datura metel L. Arch. Pharm. Res. 31, 1094–1097.
223 37 8C incubator under an atmosphere of 5% CO2 for 24 h. Then, the 285
Kuang, H., Yang, B., Xia, Y., Wang, Q., 2011. Two new withanolide lactones from flos 286
224 cells were treated with compounds at varying concentrations,
daturae. Molecules 16, 5833–5839. 287
225 cultivated for 48 h, and 20 mL of MTT (5 mg/mL) was added to each Kuang, H., Yang, B., Tang, L., Xia, Y., Dou, D., 2009. Baimantuoluosides A–C, three new 288
226 well. After 4 h, the culture medium was removed and the formazan withanolide glucosides from the flower of Datura metel L. Helv. Chim. Acta 92, 289
1315–1323. 290
227 crystals were completely dissolved with 150 mL of DMSO in each
Ma, L., Xie, C.M., Li, J., Lou, F.C., Hu, L.H., 2006. Daturametelins H, I, and J: three new 291
228 well by vigorously shaking the plate. The above operations were
withanolide glycosides from Datura metel L. Chem. Biodivers. 3, 180–186. 292
229 carried out under dark conditions. Finally, formazan absorbance Manickam, M., Srivastava, A., Ray, A.B., 1998. Withanolides from the flowers of 293
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Please cite this article in press as: Yang, B.-Y., et al., New antiproliferative and immunosuppressive withanolides from the seeds of
Datura metel. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.02.008