Two new bufadienolides from the rhizomes of helleborus thibetanus with inhibitory activities against prostate cancer cells

Article abstract of DOI:10.1080/14786419.2014.891200

Two new bufadienolide glycosides (1 and 2) with an A/B trans ring fusion together with nine known compounds (3-11)were isolated fromthe rhizomes of Helleborus thibetanus. The structures of new compounds were elucidated by extensive spectroscopic analyses in combinationwith single-crystalX-ray diffraction. The bufadienolides 1 and 3-6 exhibited potent cytotoxic activities against the prostate cancer cells.

Full text of DOI:10.1080/14786419.2014.891200

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Two new bufadienolides from the
rhizomes of Helleborus thibetanus with
inhibitory activities against prostate
cancer cells
Wei Chengab, Ya-Fang Tanb, Hai-Yan Tianb, Xiang-Wen Gongb, Ke-Li
Chena & Ren-Wang Jiangb
a Key Laboratory of Traditional Chinese Medicine Resource and
Compound Prescription, Ministry of Education, Hubei University of
Traditional Chinese Medicine, Wuhan 430065, P.R. China
b Institute of Traditional Chinese Medicine and Natural Products,
Jinan University, Guangzhou 510632, P.R. China
Published online: 04 Apr 2014.
To cite this article: Wei Cheng, Ya-Fang Tan, Hai-Yan Tian, Xiang-Wen Gong, Ke-Li Chen & Ren-
Wang Jiang (2014) Two new bufadienolides from the rhizomes of Helleborus thibetanus with
inhibitory activities against prostate cancer cells, Natural Product Research: Formerly Natural
Product Letters, 28:12, 901-908, DOI: 10.1080/14786419.2014.891200
To link to this article: http://dx.doi.org/10.1080/14786419.2014.891200
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Natural Product Research, 2014
Vol. 28, No. 12, 901–908, http://dx.doi.org/10.1080/14786419.2014.891200
Two new bufadienolides from the rhizomes of Helleborus thibetanus with
inhibitory activities against prostate cancer cells
Wei Chengab1, Ya-Fang Tanb1, Hai-Yan Tianb, Xiang-Wen Gongb, Ke-Li Chena* and
Ren-Wang Jiangb
*
aKey Laboratory of Traditional Chinese Medicine Resource and Compound Prescription, Ministry
of Education, Hubei University of Traditional Chinese Medicine, Wuhan 430065, P.R. China;
bInstitute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou
510632, P.R. China
(Received 19 January 2014; final version received 2 February 2014)
Two new bufadienolide glycosides (1 and 2) with an A/B trans ring fusion together with
nine known compounds (3–11) wereisolated fromthe rhizomesof Helleborus thibetanus.
The structures of new compounds were elucidated by extensive spectroscopic analyses in
combinationwithsingle-crystalX-raydiffraction. Thebufadienolides1 and 3–6 exhibited
potent cytotoxic activities against the prostate cancer cells.
Keywords: Helleborus thibetanus; bufadienolide glycosides; X-ray crystallographic
analyses; cytotoxic activity
1. Introduction
Helleborus thibetanus (Ranunculaceae family) is a Chinese special local plant (Tie-Kuai-Zi in
Chinese), distributed along the Qinling Mountain, northwest of China. The rhizome part of this
plant has been locally used for the treatment of asthma, cystitis, urethritis, sores and traumatic
injury (Nanjing University of Traditional Chinese Medicine 2006). In recent years, the chemical
constituents and biological activities of the Helleborus genus have attracted wide attention
because of the existence of diverse array of natural products. Previous phytochemical
investigations on the Helleborus distributed in Southeast Europe and West Asia have led to the
isolation of bufadienolides (Watanabe et al. 2003; Bassarello et al. 2008), spirostanol saponins
(Muzashvili et al. 2006; Bassarello et al. 2008), ecdysteriods (Akin & Anil 2007), furostanol
saponins (Braca et al. 2004), phytosterol (Bingol & Sener 1986) and flavonoids (Males & Medic-
Saric 2001). Pharmacological studies suggested that this plant possesses antitumour (Kerek
2004), antibacterial (Rosselli et al. 2007), immune-regulation (Littmann et al. 2008) and
cytotoxic properties (Rosselli et al. 2009). Especially, the bufadienolides were found to exhibit
potent positive iontropic, surface anaesthetic, blood pressure elevation properties and
antitumour activities in vitro and in vivo (Gao et al. 2011).
During our continuing search for structurally unique and biologically interesting
bufadienolides from the plant kingdom, two new bufadienolide glycosides 1 and 2, together
with nine known compounds 3–11 (Figure 1) were isolated from H. thibetanus, we reported
the isolation, structural elucidation and inhibitory activities against the prostate cancer cells
herein.
*Corresponding authors. Email: kelichen@126.com; trwjiang@jnu.edu.cn
q 2014 Taylor & Francis

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W. Cheng et al.
O
24
O
O
O
21
20
O
O
23
18
12
13
22
11
9
17
19
HO
H
1
R2
OHC
14
16
2
15
8
10
OH
O
OH1´
5
R1
7
3
OH
O
OH
O
HO
HO
4
6
HO
R
H
OH
O
1 R1 = OH, R2= H
2 R1 = H, R2= H
6
HO
4 R =
5 R = OH
HO
OH
3 R1 = OH, R2= OH
OH
OH
O
OH
OH
OH
OH
OH
HO
HO
HO
HO
HO
HO
OH
OH
R
O
H
O
O
9
10
7 R = H
8 R = OH
O
O
HO
11
Figure 1. Structures of compounds 1–11.
2. Results and discussion
Compound 1 was obtained as needle-shaped crystals from MeOH solution. The molecular
formula was determined to be C30H44O9 based on its HR-ESI-MS data (m/z 549.3063
[M þ H]þ, calculated for 549.3058). Its IR spectrum revealed absorption bands for hydroxyl
1
(3437 cm21) and carbonyl groups (1708 cm21). The H NMR spectrum of 1 indicated the
presence of a 2H-pyran-2-one unit [dH 6.38 (1H, d, J ¼ 9.9 Hz, H-23), 7.49 (1H, br s, H-21) and
8.25 (1H, dd, J ¼ 2.4, 9.9 Hz, H-22)], two angular methyl resonances at dH 0.65 (3H, s, H-19)
and 0.89 (3H, s, H-18) and a number of saturated methine and methylene signals at dH 0.60–
2.80. The 13C NMR revealed a total of 30 carbon signals including 2 methyls, 10 methylenes, 13
methines and 5 quaternary atoms. The four olefinic carbon signals at dc 123.3 (C-20), 150.6 (C-
21), 147.9 (C-22), 115.3 (C-23) and a carbonyl signal at dc 162.4 (C-24) were typical for a 2H-
pyran-2-one moiety. These evidences indicated that compound 1 possessed a bufadienolide
skeleton (Tian et al. 2010).
The five oxygenated signals dc 75.6, 78.9, 72.0, 78.7 and 62.9 together with the anomeric
methine signal at dc 101.9 (C-10) suggested the presence of one hexose moiety in the molecule of
1, which was confirmed by the fragment ion at m/z 387 [M þ H-162]þ corresponding to the loss
of glucosyl unit in the ESI-MS spectrum. The sugar moiety was further corroborated as
D-glucose by co-TLC of the hydrolysed product with an authentic sample. Large coupling
constant of the anomeric proton at dH 5.05 (1H, d, J ¼ 7.8 Hz, H-10) suggested the

Natural Product Research
903
b-configuration of the glycosidic bond. The HMBC (Supplementary Figure S13) correlation
between H-10 (dH 5.05, d, J ¼ 7.8 Hz, H-10) and C-3 (dc 77.3) confirmed that the glucose moiety
was linked to the glycone at C-3. Similarly, HMBC correlations H-18 (dH 0.89), H-8 (dH 1.67)
and H-15b (dH 1.86) to the oxygenated quaternary carbon C-14 (dc 84.7) confirmed that the
hydroxyl group was attached at C-14.
The NMR spectra for the aglycone of compound 1 were very similar to that of bufalin;
however, in the ROESY spectrum, the NOE correlations between H-3 (dH 4.04) and H-5 (dH
0.87) suggested that H-5 was a-oriented and the A/B ring fusion was trans (Supplementary
Figure S13) because the a-oriented H-3 was commonly observed for all natural bufadienolides
(Steyn & van Heerden 1998; Gao et al. 2011). Accordingly, compound 1 was identified as 14b-
hydroxy-3b-(b-D-glucopyranosyloxy)-5a-bufa-20,22-dienolide.
The molecular structure and stereochemistry were further confirmed by X-ray crystal-
lographic analysis (Figure 2), which clearly revealed a skeleton of bufadienolide glucoside. The
asymmetric unit contained one molecule of 1 and two water molecules linked by hydrogen
bonds. The glycone unit consisted of three cyclohexane rings (A, B and C), a five-membered ring
(D) and a six-membered lactone ring (E). Rings A, B and C had normal chair conformations, ring
D adopted an envelope conformation, while ring E was planar. The ring junctions were A/B
trans, B/C trans and C/D cis. In addition, X-ray analysis confirmed the b-configuration of the
glycosidic bond, a-oriented H-3, b-oriented 14-OH, and revealed that the glucose unit adopted a
normal chair conformation. The small Flack parameter 0.0 (2) together with the known
configurations at C-10 and C-17 indicated the absolute configuration of 1 as shown in Figure 2.
Compound 2, an amorphous powder, exhibited HR-ESI-MS data (m/z 533.3109 [M þ H]þ,
calcd for 533.3109), corresponding to a molecular formula C30H44O8 with one oxygen less than
1
that of 1. The H, 13C and DEPT NMR spectra of 2 were similar to those of 1, except for the
replacement of the oxygenated quaternary carbon atom (C-14) with a methine group (dc 51.6),
indicating that 2 was the 14-dehydroxy analog of 1. The full assignments of NMR data were
achieved by extensive analyses of 2D spectra (COSY, HSQC, HMBC and ROESY) as shown in
Supplementary Figure S14. Accordingly, the structure of 2 was established as 3b-(b-D-
glucopyranosyloxy)-5a,14b-bufa-20,22-dienolide.
Figure 2. X-ray structure of 1 with atom labelling scheme. The dashed lines represent hydrogen bonds in the
asymmetric unit.

904
W. Cheng et al.
The known compounds were identified by comparing their spectral data with reported values
in the literatures, and identified to be 14b,16b-dihydroxy-3b-(b-D-glucopyranosyloxy)-5a-
bufa-20,22-dienolide (3) (Yang et al. 2010), deglucohellebrin (4) (Watanabe et al. 2003),
3b,5b,14b-trihydroxy-19-oxo-bufa-20, 22-dienolide (5) (Watanabe et al. 2003), gamabufotalin
(6) (Tashmukhamedov et al. 1995), 20-hydroxyecdysone (7) (Lafont et al. 2002), polypodine B
(8) (Bassarello et al. 2008), stachysterone B (9) (Simon et al. 2007), shidasterone (10) (Sena
Filho et al. 2008) and schidigeragenin A (11) (Petricic 1974), (Figure 1). Compounds 6, 9 and 10
were isolated from Helleborus for the first time.
Bufadienolides have been found to exist in both animals and plants. In animals, all the
bufadienolides demonstrate an A/B cis ring fusion. In the plant kingdom, bufadienolides are
found in a number of families, for example Crassulaceae, Hyacinthaceae, Iridaceae,
Melianthaceae, Ranunculaceae and Santalaceae. Unlike the animals, plant-derived bufadieno-
lides have both A/B cis and A/B trans ring fusion modes (Yang et al. 2010). Helleborus is the
only genus in Ranunculaceae family that was found to contain bufadienolides with both A/B cis
and A/B trans ring fusion modes (Kren & Kopp 1998), which might serve as a chemotaxonomic
marker for the genus Helleborus.
MTT colorimetric assay was performed to test the antitumour activities of 1, 3–11 in
prostate cancer cells (DU145 and PC3) as described earlier. As shown in Table 1, bufadienolides
1 and 3–6 exhibited potent cytotoxic effect against DU145 (IC50 values in the range 0.02–
0.29 mM) and PC3 (IC50 values in the range 0.17–2.3 mM), which were comparable to the
cytotoxic effects of similar bufadienolides from Venom of Bufo bufo gargarizans (Tian et al.
2013). It can be observed that the bufadienolide glycones 4–6 exhibited five fold more potent
activities than those of bufadienolide glycosides 1 and 3. The sterol 9 with a long conjugated
system exhibited moderate cytotoxic effect with IC50 values of 15.0, 49.2 mM against the DU145
and PC3 cancer cells. In contrast, the sterols 7, 8, 10 and 11 were not active with IC50 values over
100 mM.
3. Experimental
3.1. General experimental procedures
Optical rotations were carried out using a JASCO P-1030 automatic digital polarimeter
(JASCO Analytical Instruments, Easton, Maryland, USA). IR spectra were measured on a
JASCO FT/IR-480 plus infrared spectrometer (JASCO Analytical Instruments, Easton,
Maryland, USA) with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AV-
400 spectrometer (Bruker Corporation, Billerica, Massachusetts, USA) with TMS as the
internal standard, and chemical shifts were expressed in d values (ppm). HR-ESI-MS data
were detected on an Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies,
Santa Clara, California, USA). Silica gel (200–300 mesh, Qingdao Marine Chemical, Inc.,
Qingdao city, China), ODS silica gel (50 mm, YMC, Kyoto, Japan) and Sephadex LH-20
Table 1. Inhibitory activities of compounds 1 and 3–11 against the prostate cancer cells (IC50 in mM).
No.
DU145 (mmol/L)
PC3 (mmol/L)
No.
DU145 (mmol/L)
PC3 (mmol/L)
1
3
4
5
0.15 ^ 0.02
0.29 ^ 0.03
0.02 ^ 0.003
0.06 ^ 0.01
0.03 ^ 0.005
0.10 ^ 0.01
2.35 ^ 0.17
1.20 ^ 0.14
0.33 ^ 0.02
0.48 ^ 0.03
0.14 ^ 0.05
0.12 ^ 0.02
7
8
9
10
11
.100
.100
14.85 ^ 1.20
.100
.100
.100
.100
49.6 ^ 2.6
.100
.100
6
Taxol

Natural Product Research
905
(Pharmacia, Uppsala, Sweden) were used for column chromatography. Preparative HPLC was
carried out on a WUFENG instrument equipped with a UV detector (WUFENG, Shanghai
city, China) and a reverse phase C-18 column (5 mm, 20 £ 250 mm; Cosmosil, Kyoto, Japan)
TLC was performed using precoated silica gel plates (GF254, Yantai Institute of Chemical
Industry, Yantai city, Shandong province, China). X-ray crystallographic data collection was
performed on an Agilent Gemini S Ultra Sappharie CCD (Agilent Technologies, Yarnton,
Oxfordshire, United Kingdom) using CuKa radiation.
3.2. Plant material
The herbal materials of H. thibetanus were collected in Taibai County, Shanxi Province, China
in 2012. The roots were identified by Prof. Guang-Xiong Zhou (Jinan University, China). A
voucher specimen (Tkz-001) has been deposited with the College of Pharmacy, Jinan
University.
3.3. Extraction and isolation
The air-dried rhizomes of H. thibetanus (20.0 kg) were extracted twice using 95% EtOH and 60%
EtOH, consecutively. The ethanol extract was evaporated to obtain a residue that was suspended in
water and then extracted successively with petroleum ether, EtOAc and n-BuOH. The EtOAc
extract (82.0 g) was subjected to silica gel column (200–300 mesh, 1.5 kg) eluting with gradient
CHCl3–MeOH (100:1 to 70:30) and 14 fractions (fractions 1–14) were obtained. Faction 4 (8.0g)
was chromatographed on a silica gel column (200–300 mesh, 165 g) eluted with cyclohexane–
acetone (10:1 to 6:4) to afford seven subfractions (41–47). Subfraction 42 was subjected to an
ODS column using gradient mixtures of MeOH–H2O (70:30 to 95:5) as eluents to yield compounds
3(7.2mg) and5(10.0 mg). Fraction6(7.5g)waschromatographedonasilicagelcolumn(200–300
mesh, 160.0 g) eluted with CHCl3–MeOH (15:1 to 7:3) to afford six subfractions (61–66).
Subfraction 62 was purified by recrystallisation in acetone to yield compound 4 (30.0 mg).
Subfraction 63 was subjected to Sephadex LH-20 (CHCl3–MeOH, 1:1) chromatography followed
by preparative HPLC (MeOH–H2O, 60:40) to yield compounds 2 (5.3mg) and 9 (15.0 mg).
Compound 6 (18.0 mg) was obtained from subfraction 64 by recrystallisation. Similarly, fraction 8
(5.0g) was fractionated by a silica gel column (200–300 mesh, 100.0 g) chromatography eluted
with CHCl3–MeOH (10:1 to 7:3) to afford five subfractions (81–85). Subfraction 83 was purified by
RP-18 column (MeOH–H2O, 45: 55 to 100% MeOH) followed by preparative HPLC (MeOH–
H2O, 50:50) to yield compounds 1 (13.0 mg), 11 (8.0mg), 10 (12.0 mg) and 8 (8.5mg). Fraction 10
(4.1g) was subjected to an ODS column using gradient MeOH–H2O (30:70 to 95:5) as eluents to
afford six subfractions (101–106). Compound 7 (40.0 mg) was obtained from subfraction 104 by
recrystallisation in methanol.
3.3.1. 14b-hydroxy-3b-(b-D-glucopyranosyloxy)-5a-bufa-20,22-dienolide (1)
C30H44O9, white needle crystal. HR-ESI-MS: (m/z 549.3063 [M þ H]þ, calcd for 549.3058). 1H
NMR (C5D5N) d: 7.49 (1H, br s, H-21), 8.25 (1H, dd, J ¼ 2.4, 9.9 Hz, H-22), 6.38 (1H, d,
J ¼ 9.9 Hz, H-23), 0.89 (3H, s, H-18), 0.65 (3H, s, H-19), 1.85 (1H, m, H-17), 0.87 (1H, m, H-9),
1.67 (1H, m, H-8), 0.87 (1H, overlapped, H-5), 4.04 (1H, m, H-3), 0.86 (1H, overlapped, H-1a),
1.61 (1H, overlapped, H-1b), 1.64 (1H, m, H-2a), 2.16 (1H, overlapped, H-2b), 1.40 (1H,
overlapped, H-4a), 1.86 (1H, m, H-4b), 0.97 (1H, m, H-6a), 1.23 (1H, m, H-6b), 1.10 (1H,
overlapped, H-7a), 2.35 (1H, overlapped, H-7b), 1.15 (1H, m, H-11a), 1.39 (1H, m, H-11b), 1.26
(1H, overlapped, H-12a), 1.38 (1H, overlapped, H-12b), 1.99 (1H, m, H-15a), 1.86 (1H,
overlapped, H-15b), 2.07 (1H, overlapped, H -16a), 1.86 (1H, m, H-16b), 5.05 (1H, d,

906
W. Cheng et al.
J ¼ 7.8 Hz, H-10), 3.98 (1H, m, H-20), 4.29 (1H, dd, J ¼ 3.0, 9.0 Hz, H-30), 4.25 (1H, dd, J ¼ 3.0,
9.0 Hz, H-40), 4.01 (1H, m, H-50), 4.61 (1H, brd, J ¼ 11.0 Hz, H-60a), 4.41 (1H, dd, J ¼ 11.5,
5.5 Hz, H-60b); 13C NMR (C5D5N) d: 37.6 (C-1), 30.1 (C-2), 77.3 (C-3), 34.8 (C-4), 44.4 (C-5),
29.4 (C-6), 28.3 (C-7), 42.3 (C-8), 50.2 (C-9), 36.2 (C-10), 22.0 (C-11), 40.8 (C-12), 49.0 (C-13),
84.7 (C-14), 32.9 (C-15), 29.8 (C-16), 51.6 (C-17), 17.5 (C-18), 12.4 (C-19), 123.3 (C-20), 150.6
(C-21), 147.9 (C-22), 115.3 (C-23), 162.4 (C-24), 101.9 (C-10), 75.6 (C-20), 78.9 (C-30), 72.0 (C-
40), 78.7 (C-50), 62.9 (C-60).
3.3.2. 3b-(b-D-glucopyranosyloxy)-5a,14b-bufa-20,22-dienolide (2)
C30H44O8, amorphous white powder. HR-ESI-MS: (m/z 533.3109 [M þ H]þ, calcd for
533.3109). 1H NMR (C5D5N) d: 7.49 (1H, br s, H-21), 7.31 (1H, dd, J ¼ 2.4, 9.6 Hz, H-22), 6.44
(1H, d, J ¼ 9.6 Hz, H-23), 0.65 (3H, s, H-18), 0.44 (3H, s, H-19), 1.16 (1H, m, H-17), 1.18 (1H,
m, H-9), 1.25 (1H, m, H-8), 0.92 (1H, m, H-5), 4.05 (1H, m, H-3), 2.15 (1H, m, H-14), 0.92 (1H,
overlapped, H-1a), 1.58 (1H, overlapped, H-1b), 1.68 (1H, m, H-2a), 2.12 (1H, overlapped, H-
2b), 1.39 (1H, m, H-4a), 1.88 (1H, overlapped, H-4b), 1.15 (1H, m, H-6a), 1.76 (1H, m, H-6b),
1.16 (1H, overlapped, H-7a), 1.61 (1H, overlapped, H-7b), 1.20 (1H, overlapped, H-11a), 1.45
(1H, overlapped, H-11b), 0.99 (1H, m, H-12a), 1.45 (1H, m, H-12b), 2.10 (1H, overlapped, H-
15a), 1.85 (1H, m, H-15b), 2.06 (1H, overlapped, H-16a), 1.82 (1H, m, H-16b), 5.07 (1H, d,
J ¼ 7.8 Hz, H-10), 3.99 (1H, t, J ¼ 7.8 Hz, H-20), 4.27 (1H, dd, J ¼ 3.0, 9.0 Hz, H-30), 4.19 (1H,
dd, J ¼ 3.0, 9.0 Hz, H-40), 3.97 (1H, m, H-50), 4.56 (1H, dd, J ¼ 11.5, 2.0 Hz, H-60a), 4.41 (1H,
dd, J ¼ 11.5, 5.5 Hz, H-60b); 13C NMR (C5D5N) d: 37.6 (C-1), 30.3 (C-2), 77.3 (C-3), 35.1 (C-
4), 45.1 (C-5), 26.1 (C-6), 25.2 (C-7), 36.3 (C-8), 54.7 (C-9), 36.3 (C-10), 21.5 (C-11), 37.6 (C-
12), 44.5 (C-13), 51.6 (C-14), 32.6 (C-15), 29.5 (C-16), 56.0 (C-17), 12.6 (C-18), 13.2 (C-19),
119.0 (C-20), 149.6 (C-21), 146.1 (C-22), 115.5 (C-23), 162.3 (C-24), 102.3 (C-10), 75.7 (C-20),
79.1 (C-30), 72.2 (C-40), 78.8 (C-50), 63.4 (C-60).
3.4. Acid hydrolysis of 1 and 2
Methanol solutions of 1 (5 mg) and 2 (4 mg) were added to 2 M HCl and refluxed for 2 h. After
cooling, the reaction mixture was diluted with H2O and extracted with ethyl acetate. The
aqueous layer was neutralised with Na2CO3 and then concentrated to 2 mL. The D-glucose in the
aqueous layer was identified by co-TLC with authentic sugar samples using CHCl3–MeOH–
H2O (16:9:1) as the mobile phase.
3.5. X-ray crystallographic analysis of 1
˚
Data were collected using an Agilent Sapphire CCD with a CuKa radiation, l ¼ 1.54184 A at
173.0(2) K. Crystal data were C30H44O9·2H2O, monoclinic, and space group P212121; unit cell
˚
˚
˚
dimensions were determined to be a ¼ 6.34339(13) A, b ¼ 21.4039(5) A, c ¼ 21.9012(6) A,
V ¼ 2973.61(12) A , Z ¼ 4, Dx ¼ 1.306 g/cm , F (000) ¼ 1264, m (CuKa) ¼ 0.817 mm21. A
total of 6375 reflections were collected until umax ¼ 62.768, in which 3434 independent,
reflections were observed [F 2 . 4s(F 2)]. The structure was solved by direct methods using the
SHELXS-97 program. The final refinement gave R ¼ 0.0510, RW ¼ 0.0640 and S ¼ 1.074.
Crystal data of compound 1 have been deposited with the Cambridge Crystallographic Data
Centre (CCDC 955228).
3
3
˚
3.6. MTT assay
The MTT assay was carried out as described previously (Mosmann 1983), with taxol serving as
the positive control. Briefly, the prostate cancer cells were seeded into the 96-well plates at a

Natural Product Research
907
density of 3 £ 103 cells per well DU145 and PC3 cancer cell lines overnight. Then, the cells were
treated with a series concentration of compounds 1 and 3–11 (compound 2 was not tested due to
the limited sample). Following incubation for 48 h, 20 mL of the MTT solution (5 mg/mL in
phosphate buffered saline) was added to each well, and the cells were further incubated for 4 h.
Then, the medium was removed and replaced by 150 mL of DMSO in each well to dissolve the
formazan crystals. The relative cell viability was determined by measuring the optical densities
at 570 nm on microplate reader (SPECTRAmax 250, Molecular Devices, Sunnyvale, California,
USA). The experiments were performed three times, each in triplicate.
Supplementary material
1
1
Supplementary material relating to this article is available online, alongside H, 13C, H–1H–
COSY, HSQC, HMBC and ROESY spectra of compounds 1 and 2.
Acknowledgements
This work was supported by the Guangdong High Level Talent Scheme to R.W.J., the Fundamental
Research Funds for the Central Universities (11612603), the National Natural Science Foundation of China
(81102518) and Postdoctoral Granted Financial Support (20110490915).
Note
1. These authors contributed equally to this work.
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