SUPPLEMENTARY MATERIAL
Sensitization of ovarian cancer cells to cisplatin by flavonoids
from Scutellaria barbata
Jie Lia,1, Yun Wanga,1, Jia-Chuan Leib, Yang Haoa, Yuan Yanga, Cheng-Xiong Yangc,
Jian-Qing Yua,*
a
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan
University), Ministry of Education, School of Pharmaceutical Sciences, Wuhan
University, Wuhan 430071, China
b
Renmin Hospital, Wuhan University, Wuhan 430060, China; cJingchu University of
Technology, Jingmen 448000，China
*Corresponding author. E-mail address: jqyu@whu.edu.cn
Abstract
Combination of natural components with anticancer drugs is a new strategy for cancer
chemotherapy to increase antitumor responses. In this study, we investigated the
sensitization effects of nine flavonoids from Scutellaria barbata to cisplatin (CDDP)
on human ovarian cancer cells. The combinations of three flavonoids with CDDP
showed the synergistic effects. The intracellular reactive oxygen species (ROS) and
the antioxidant activity were measured. The data suggest that the synergistic effects of
flavonoids with CDDP on ovarian cancer cells did not directly correlate with their
redox properties, but could be associated with the positions of hydroxyl group and
methoxy group of flavonoids.
Keywords: Scutellaria barbata; flavonoids; cisplatin; ovarian cancer; antioxidant
Experimental
Extraction and isolation
S. barbata (10 kg) was extracted with 95%, 75% and 50% ethanol, respectively. The
extract was concentrated under reduced pressure to yield an ethanol extract, which
was absorbed by macroporous resin (D101) and eluted successively with water, 75%
and 95% ethanol, respectively. Each fraction was concentrated and dried to obtain the
water (Fr.1), 75% ethanol (Fr.2) and 95% ethanol (Fr.3) fractions.
Fr.2 (100g) was then subjected to silica gel column chromatography (CC) eluted
successively with petroleum ether–acetone-methanol[(1:4:1, Fr.2.1); (1:1:1, Fr.2.2 );
(1:1:4, Fr.2.3)] and methanol (Fr.2.4) to give four fractions. Fr.2.2 (22g) was separated
by repeated silica gel column and further purified by sephadex LH-20 column to give
compounds 1, 3, 5, 6, 8 and 9. Fr.2.4 (35g) was chromatographed over a silica gel
column followed by sephadex LH-20 CC to afford compounds 2, 4 and 7.
Structural identification
The identification of compounds was based on the spectral analysis, as well as, by
comparison of their spectral data with literatures.
Compound1: 1H-NMR (400 Hz, DMSO-d6) δ 12.98 (1H, s, 5-OH), 10.72 (1H, s,
7-OH), 9.68 (2H, brs, 3’-OH, 4’-OH), 6,67 (1H, s, H-3), 7.40 (1H, d, J=2.0 Hz, H-2’),
7.43 (1H, dd, J=2.0, 8.4 Hz, H-6’), 6.88 (1H, d, J=8.4 Hz, H-5’), 6.20 (1H, d, J=2.0,
H-6), 6.45 (1H, d, J=2.0 Hz, H-8); 13C-NMR (DMSO-d6) δ 181.7 (4-C), 164.1 (C-2),
163.8 (C-7), 161.4 (C-5), 157.2 (C-9), 149.7 (C-4’), 145.8 (C-3’), 103.4 (C-3), 98.8
(C-6), 94.8 (C-8), 103.6 (C-10), 121.9 (C-1’), 113.9 (C-2’), 114.9 (C-5’), 118.9 (C-6’)
The data were also compared with those in the literature (Miyazawa & Hisama, 2003).
Compound 2: 1H-NMR (400 Hz, DMSO-d6) δ12.50 (1H, s, 5-OH), 10.80 (1H, s,
7-OH), 9.61 (1H, s, 3-OH), 9.36 (1H, s, 3’-OH), 9.33 (1H, s, 4’-OH), 7.68 (1H, d,
J=2.0 Hz, H-2’), 7.54 (1H, dd, J=8.4, 2.0 Hz, H-6’), 6.88 (1H, d, J=8.4 Hz, H-5’), 6.41
(1H, d, J=2.0 Hz, H-8), 6.19 (1H, d, J=2.0 Hz, H-6). 13C-NMR (DMSO-d6) δ 175.5
(C-4), 163.8 (C-7), 60.7 (C-5), 156.1 (C-9), 147.7 (C-3’), 146.7 (C-2), 144.9 (C-4’),
135.7 (C-3), 121.9 (C-1’), 119.9 (C-6’), 115.7 (C-5’), 115.0 (C-2’), 102.9 (C-10), 98.1
(C-6), 93.3 (C-8). The data were also compared with those in the literature (Chen et
al., 2008).
Compound 3: 1H-NMR (400Hz, DMSO-d6) δ 12.98(1H, s, 5-OH), 10.85 (1H, s,
7-OH), 10.38 (1H, s, 4’-OH), 7.94 (2H, d, J=8.0Hz, H-2’, H-6’), 6.93 (2H, d, J=8.0Hz,
H-3’, H-5’) , 6.80 (1H, s, H-3), 6.49 (1H, d, J=2.0 Hz, H-8), 6.20 (1H, d, J=2.0 Hz,
H-6);
13
C-NMR (DMSO-d6) δ 181.0 (C-4), 164.1 (C-7), 163.7 (C-2), 161.4 (C-5),
161.1 (C-4’), 157.3 (C-9), 128.4 (C-2’, C-6’), 115.9 (C-3’, C-5’), 121.1 (C-1’), 103.7
(C-3), 102.8 (C-10), 98.8 (C-6), 93.9 (C-8). The data were also compared with those
in the literature (Miyazawa et al., 2003).
Compound 4: 1H-NMR (400z, DMSO-d6) δ 12.75 (1H, s, 5-OH), 10.37 (1H, s,
4’-OH), 8.61 (1H, s, 7-OH), 7.94 (2H, d, J=8.8Hz, H-2’, H- 6’), 6.94 (2H, d, J=8.8Hz,
H-3’, H-5’), 7.00 (1H, s, H-8), 6.83 (1H, s, H-3), 5.22 (1H, d, J=6.8Hz, anomeric H);
C-NMR(DMSO-d6) δ 182.5 (C-4), 161.4 (C-2), 102.7 (C-3), 147.7 (C-5), 130.6
13
(C-6), 164.2 (C-7), 93.6 (C-8), 151.2 (C-9), 106.1 (C-10), 121.3 (C-1’), 128.6 (C-2’,
C-6’), 116.4 (C-3’, C-5’), 149.3 (C-4’), 100.3 (C-1’’), 72.8 (C-2’’), 75.4 (C-3’’), 71.2
(C-4’’), 75.7 (C-5’’), 170.2 (C-6’’). The data were also compared with those in the
literature (Chen, Cui, Duan, Ma, & Zhong, 2006).
Compound 5: 1H-NMR (400 Hz, DMSO-d6) δ 12.92 (1H, s, 5-OH), 10.58 (1H, s,
7-OH), 10.36 (1H, s, 4’-OH), 8.71 (1H, s, 6-OH), 6.69 (1H, s, 3-H), 6.52 (1H, s, 8-H),
7,88 (2H, d, J=8.4 Hz, H-2’, H-6’), 6.79 (2H, d, J= 8.4 Hz, H-3’, H-5’);
13
C-NMR
(DMSO-d6) δ 181.8 (C-4), 163.8 (C-2), 101.7 (C-3), 152.6 (C-5), 129.1 (C-6), 146.6
(C-7), 93.3 (C-8), 148.8 (C-9), 103.4 (C-10), 121.1 (C-1’), 128.7 (C-2’, C-6’), 115.2
(C-3’, C-5’), 160.4(C-4’). The data were also compared with those in the literature
(Wang, Liu, Dong, Lv, & Xu, 2012).
Compound 6: 1H-NMR (CDCl3) δ 12.51 (1H, s, 5- OH), 10.66 (1H, s, 7- OH)，
7.93 (2H, m, H-2', H-6'), 7.57 (3H, m, H-3', H-4'，H-5'), 6.70 (1H, s, H-3), 6.45 (1H,
s, H-6), 4.05 (3H, -OCH3); EI-MS [M]+ m/ z 284. The data were also compared with
those in the literature (Harrison, Sia, & Sim, 1994).
Compound 7: 1H-NMR (400 Hz, DMSO-d6) δ 12.43 (1H, s, 5-OH), 10.84(1H, s,
7-OH), 9.54(1H, s, H-3’), 9.01(1H, s, H-4’), 7.72 (1H, d, J=2.4 Hz, H-2’), 7.60 (1H,
dd, J=8.4, 2.0 Hz, H-6’), 7.25 (1H, d, J=8.4 Hz, H-5’)，6.47 (1H, d, J=2.0 Hz, H-8),
6.21 (1H, d, J=2.0 Hz, 6-H), 4.88 (1H, d, J=8.0 Hz, anomeric H);
13
C-NMR
(DMSO-d6) δ 146.7(C-2), 136.4 (C-3), 176.0 (C-4), 160.7(C-5), 98.2(C-6), 164.
0(C-7), 93.5(C-8), 156.2(C-9), 103.1(C-10), 125.1 (C-1’), 115.8(C-2’), 145.9 (C-3’),
146.3 (C-4’), 115. 1(C-5’), 119.5(C-6’), 101.3 (C-1’’) , 77.2 (C-2’’), 75.9 (C-3’’),
73.2 (C-4’’), 69.75 (C-5’’), 60.6 (C-6’’). The data were also compared with those in
the literature (van der Woude, Boersma, Vervoort, & Rietjens, 2004; Yang et al.,
2012).
Compound 8: 1H-NMR (400 Hz, (CD3 ) 2 CO) δ 11.80 (1H, s, 5-OH), 8.67 (1H,
s, 4’ - H), 7.30 (1H, s, 7-OH), 7.41 (2H, d, J=8.0 Hz, H-2’, H-6’), 6.89 (2H, d,=8.0 Hz,
H-3’, H-5’), 6.17 (1H, s, H-6), 5.46 (1H, dd, J=12.0, 2.0 Hz, H-2), 3.90 (3H, s,
-OCH3), 3.20 (1H, dd, J=17.2, 12.0 Hz, trans 3-H), 2.77 (1H, dd, J=17.2, 2.0 Hz, cis
3-H);
13
C-NMR ((CD3)2CO): δ 197.0 (C-4), 102.5 (C-10), 92.4 (C-6), 55.7 (OCH3),
43.0 (C-3), 79.23 (C-2), 156.5 (C-5), 158.0 (C-4’), 156.7 (C-9), 129.9 (C-1’), 128.1
(C-2’, C-6’), 115.2 (C-3’, C-5’), 127.2 (C-8), 147.7 (C-7) [8]. The data were also
compared with those in the literature (Tomimori, Miyaichi, Imoto, & Kizu, 1986).
Compound 9: 1H-NMR (400 Hz, DMSO-d6) δ 11.82 (1H, s, 5-OH), 9.58
(1H,s,7-OH), 8.11 (1H, s, 4’-OH), 7.34 (2H, d, J=8.4 Hz, H-2’, H-6’ ), 6.79 (2H, d,
J=8.4 Hz, H-3’, H-5’), 6.20 (1H, s, H-8), 5.43 (1H, dd, J=12.4, 2.8 Hz, H-2), 3.26 (1H,
dd, J=17.2, 12.4 Hz, trans 3-H), 2.74 (1H, dd, J=17.2, 2.8 Hz, cis 3-H), 3.83 (3H, s,
OCH3);
C-NMR (DMSO-d6) δ 197.5 (C-4), 42.2 (C-3), 78.4 (C-2), 56.6 (OCH3),
13
158.3 (C-5), 129.0 (C-6), 157.1 (C-7), 92.8(C-8),
148.7 (C-9), 103. 6 (C-10), 128.9
(C-1’), 127.0 (C-2’), 115.7 (C-3’), 156.9 (C-4’), 115.7 (C-5’), 127.0 (C-6’). The data
were also compared with those in the literature (Lee, Duan, Lee, Lee, Hong, & Lee,
2010).
Assay of DPPH radical-scavenging activity
The DPPH radical-scavenging activities of flavonoids and CDDP were measured
according to the method of Hu, Shen, and Wang, (2009), with some modifications.
Briefly, 150 μL of 0.2mM DPPH radical solution (in methanol) was mixed with 150
μL of sample solution containing flavonoids (60 μM) and/or CDDP (60 μM). The
mixture was left to stand at room temperature for 30 min. Then, the absorbance was
measured at 517nm.
Assay of superoxide anion-scavenging activity
Superoxide anion-scavenging activity was determined by measuring the inhibition of
the auto-oxidation of pyrogallol using a slightly modified method (Siegel et al., 2004).
10 μL sample solution containing flavonoids (600 μM) and/or CDDP(600 μM) and
280 μL of 50 mM Tris–HCl buffer (pH 8.2) were added to a freshly prepared 10 μL of
30 mM pyrogallol solution (dissolved in 10 mM HCl). The inhibition rate of
pyrogallol auto-oxidation was measured at 420 nm. Absorbance of each sample was
recorded at every 0.5 min interval for 5 min.
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Cell viability (%)
100
C1
C2
C3
C4
C5
C6
C7
C8
C9
80
60
40
0
30
60
90
120
150
180
210
Compound concentrationM
Figure S1. Effects of nine flavonoids on viability of OVCAR-3 cells. The cells were
treated with vehicle (0.5% DMSO) and various concentrations of flavonoids for 24 h,
and cell viability was determined by MTT assay. Results are representative of three
independent experiments.
Figure S2. The ROS changes in OVCAR-3 cells with treatments of CDDP (60 μM)
alone, flavonoids (160 μM) alone, and their combinations for 3 h. The images were
acquired by fluorescence microscopy (Olympus IX51S8F). Control: cells treated with
0.5% DMSO.
DPPH radical-scavenging
activity (%)
80
60
40
20
C8
C8+P
C9
C9+P
C6
C6+P
C7
C7+P
C3
C3+P
C4
C4+P
C5
C5+P
C2
C2+P
P
C1
C1+P
0
Figure S3. DPPH radical-scavenging activities of CDDP (30 μM) alone, flavonoids
(30 μM) alone, and their combinations. Results are representative of three
independent experiments. (P), CDDP.
80
0
0
1
2
3
Time (min)
4
5
Superoxide anion-scavenging
activity (%)
Superoxide anion-scavenging
activity (%)
C4 (20 
CDDP (20 
C4 (20 ) + CDDP (20 
40
20
0
0
1
2
3
4
Time (min)
5
6
Superoxide anion-scavenging
activity (%)
Superoxide anion-scavenging
activity (%)
C7 (20 
CDDP (20 
C7 (20  + CDDP (20 
40
20
0
1
2
3
Time (min)
1
2
4
5
6
3
Time (min)
4
5
40
20
20
1
2
3
Time (min)
4
5
20
1
2
3
Time (min)
3
4
Time (min)
5
6
C6 (20 
CDDP (20 
C6 (20  + CDDP (20 
20
0
1
2
3
4
Time (min)
5
6
80
40
0
2
40
6
C8 (20 
CDDP (20 
C8 (20  + CDDP (20 
60
1
60
0
0
0
80
40
0
C3 (20 
CDDP (20 
C3 (20 ) + CDDP (20 
60
0
6
C5 (20 
CDDP (20 
C5 (20  + CDDP (20 
60
0
0
0
80
80
60
20
80
80
60
40
0
6
Superoxide anion-scavenging
activity (%)
20
C2 (20 
CDDP (20 
C2 (20  + CDDP (20 
60
Superoxide anion-scavenging
activity (%)
40
80
Superoxide anion-scavenging
activity (%)
C1 (20 
CDDP (20 
C1 (20  + CDDP (20 
60
Superoxide anion-scavenging
activity (%)
Superoxide anion-scavenging
activity (%)
80
4
5
6
C9 (20 
CDDP (20 
C9 (20  + CDDP (20 
60
40
20
0
0
1
2
3
4
Time (min)
5
6
Figure S4. Superoxide anion-scavenging activities of CDDP alone, flavonoids alone,
and their combinations. Results are representative of three independent experiments.