Journal of Ethnopharmacology 206 (2017) 184–192

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Anti-tumor effect of Scutellaria barbata D. Don extracts on ovarian cancer MARK

and its phytochemicals characterisation
Lin Zhanga,1, Baoyin Renb,1, Jing Zhangb, Likun Liub, Jia Liub, Guoqiang Jiangb, Man Lic,
⁎
Yuejia Dinga, Weiling Lib,
a
Academy of Integrative Medicine, Dalian Medical University, Dalian 116044, China
b
Department of Biotechnology, Dalian Medical University, Dalian 116044, China
c
Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA

A R T I C L E I N F O A BS T RAC T

Keywords: Ethnopharmacological relevance: Scutellaria barbata D. Don is a widely used medicinal herb in China. It
Scutellaria barbata D. Don possess various medicinal properties including antioxidative, anti-inflammatory and anti-cancer effects. The
Ovarian cancer aim of this study was to explore whether Scutellaria barbata D. Don could inhibit the growth of ovarian cancer
Apoptosis cells in vitro and further investigate the underlying mechanisms.
Migration
Materials and methods: Effects of Scutellaria barbata D. Don on the viability of ovarian cancer A2780 cells
were measured by MTT assay. Apoptosis was measured by cell morphologic observation through DAPI staining
and Annexin V-FITC staining assay for apoptosis analysis. The migration of ovarian cancer cells which exposed
to Scutellaria barbata D. Don were measured by wound healing and transwell chamber assays. The protein
levels of caspase 3/9, Bcl-2 and MMP-2/9 in human ovarian cancer cells treated with Scutellaria barbata D.
Don were assessed by western blotting analysis. The potential bioactive compounds were characterized by
HPLC-Q-TOF-MS.
Results: The present study was to investigate the anticancer effects of crude extracts from Scutellaria barbata
D. Don on ovarian cancer A2780 cells by MTT, DAPI staining, wound healing assay, transwell migration assay
and western blotting analysis. Our study showed that Scutellaria barbata D. Don reduced the viability of A2780
cells and induced apoptosis by down-regulated Bcl-2 protein and increased Caspase 3/9 proteins. Furthermore,
migration of A2780 cells were significantly inhibited by Scutellaria barbata D. Don and the underlying
mechanism may be related to the decrease of MMP-2/9. The main constituents from Scutellaria barbata D.
Don were identified to be thirteen flavonoids. A HPLC-Q-TOF-MS analysis of Scutellaria barbata D. Don
indicated the presence of 14 flavonoids compounds, which may contribute to the anticancer activity of the
Scutellaria barbata D. Don.
Conclusions: Scutellaria barbata D. Don could inhibit proliferation and induce apoptosis in A2780 cells
through mitochondrial pathway. Moreover, the inhibitory effect of Scutellaria barbata D. Don on the migration
of ovarian cancer cells was associated with the down-regulation of MMP-2/9 expression. These findings could
shed a light on the therapy of ovarian cancer.

1. Introduction
Ovarian cancer is one of the most lethal malignancies, and the fifth
leading cause of cancer death from gynecological tumors (Onnis et al.,
1992). As lack of early typical symptoms and effective screening
strategies, approximately 70% of the patients have been diagnosed at
advanced stage (Keller et al., 2016). Although traditional surgery and
chemotherapy based on cisplatin and paclitaxel are applied in ovarian
cancer treatment, it is found that the 5-year survival rate was only
about 30% (Wang et al., 2016). Therefore, development of novel
therapy for ovarian cancer patients is urgent.
Traditional herbal medicines are increasingly being utilized to treat
a variety of cancers (Yamaguchi, 2015). Scutellaria barbata D. Don
(SB), a traditional Chinese medicine, widely distributed in southern
China and Korea (Lee et al., 2006). The chemical constituents of SB
mainly contain flavonoids, alkaloids and saccharides (Liu et al., 2015).

⁎
Corresponding author.
E-mail address: liweiling2004@163.com (W. Li).
1
Contributed equally.

http://dx.doi.org/10.1016/j.jep.2017.05.032
Received 21 April 2017; Received in revised form 26 May 2017; Accepted 27 May 2017
Available online 29 May 2017
0378-8741/ © 2017 Elsevier B.V. All rights reserved.
L. Zhang et al.
Fig. 1. Analytical HPLC-Q-TOF-MS chromatogram of SB performed in positive and negative modes.
Previous researches reported that the main physiological active sub-
stances of SB are flavonoids (Yang et al., 2015). SB has been used to
treat lung cancer, breast cancer and colorectal cancer or combined with
other traditional Chinese medicine such as Hedyotis diffusa Willd (Gao
et al., 2014; Goh et al., 2005; Perez et al., 2010; Yin et al., 2004).
However, the antitumor effect of SB on ovarian carcinoma is still not
elucidated.
Therefore, our goal of this study was to explore whether SB could
inhibit the growth of ovarian cancer cells in vitro and further
investigate the underlying mechanism. In order to identify the phyto-
chemicals responsible for the activity, we analyzed the contents of SB
extracts by HPLC-Q-TOF-MS. Our research findings might contribute
to the improvement of care for ovarian cancer patients.
2. Materials and methods
2.1. Regents and materials
Dulbecco's modified Eagle's medium (DMEM), double-antibody
and pancreatic enzyme were obtained from Gibco, American. Fetal
bovine serum (FBS) was obtained from TransGencompany. Matrigel
and transwells were purchased from BD Biosciences, American. MTT
(3-(4,5-dimethyl-2-yl)-2,5-diphenyl-tetrazolium bromide) and DAPI
(2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) were ob-
tained from Sigma Chemical Co. (St. Louis,MO). DMSO, crystal violet
and methanol were from Romeo reagent company, Tianjin. Protein
maker was obtained from Thermocompany. Antibodies against MMP-
2/9 and β-actin were obtained from Ptoteintech, China. Actonitrile and
methanol (HPLC-MS grade) were purchased from sigma-Aldrich (St.
Louis, MO, USA). Deionized water for HPLC analysis was purified by a
Milli-Q system (Millipore, Milford, MA, USA). The dry grass of
Scutellaria barbata D. Don was obtained from Dalian Metro pharma-
ceutical (Dalian, Liaoning province, China), and authenticated by prof.
Lin Zhang (Dalian Medical University). Voucher specimens were
deposited at the laboratory of authors. Standards of were purchased
from Chengdu Pufeide Biological Technology Co., Ltd. (Sichuan,
China). The purity of standard compounds was higher than 97%
confirmed by UHPLC-DAD analysis.
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Journal of Ethnopharmacology 206 (2017) 184–192
2.2. Preparation of samples for analysis
The drying herb of Scutellaria barbata D. Don (20 g) was extracted
2 times in a reflux extraction device with 300 ml ethanol each time.
After the extracting solutions were mixed and filtered, 100 ml water
was added to the filtrate, and then concentrated in the 60 °C water bath
on the rotary evaporation apparatus until there was no alcohol left. The
concentrated mixture was frozen inside a −80 °C refrigerator (Thermo
company) and freeze dried in a freeze drier (Beijing, China) to give a
fine powder (1.48 g). Accurately weighed extraction powder (0.1 g) was
transferred into a 100 ml Teflon-lined extraction vessel and methanol
was added to the scale. The solution was filtered through 0.22 µm filter
before being injected to HPLC-Q-TOF-MS for analysis.
2.3. Cell line and cell culture
Human ovarian cancer cell line A2780 was purchased from
American Type Culture Collection (ATCC) and cultured in Dulbecco
modified Eagle medium supplemented with 10% FBS (both obtained
from Gibco BRL.,Grand Island, NY, USA) at 37 °C in 5% CO2. Normal
ovarian epithelial cell line IOSE80 was obtained by Canadian Ovarian
Tissue Bank. SK-OV-3 cell was donated by prof. Pixu Liu (Dalian
Medical University). Cells were digested with 0.25% trypsin and treated
with different concentrations of SB with appropriate controls.
2.4. Chromatographic and mass spectrometric conditions
HPLC-DAD-Q-TOF-MS/MS analysis was carried out using a
Shimadzu HPLC 20ADXR LC (Shimadzu, Kyoto, Japan) system in-line
with an AB-Sciex 5600 Triple TOF mass spectrometer (AB SCIEX,
Florida, American) equipped with an electrospray (ESI) interface. The
mobile phase consisted of solvent A (water contained 0.1% formic acid)
and solvent B (acetonitrile contained 0.1% formic acid), and the
gradient elution conditions were from 35% B to 70% in 0–15 min,
95% B for 18–20 min, return to initial conditions in 21 min, and
equilibrate for 4 min before the next sample injection. The flow rate
was 0.45 ml/min. Chromatographic separation was carried out at 40 °C
using a Kromasil 100-5-C18 (5 µm, 4.6 mm × 150 mm, Bohus,
Sweden). The MS detection parameters were optimized as follows:
drying gas temperature, 550 °C; the declustering potential was set to 65
and −65 for positive and negative respectively, and the collision energy
L. Zhang et al. Journal of Ethnopharmacology 206 (2017) 184–192

was set to 5 and −5 for positive and negative respectively. Mass spectra

269.0432[M-H]-, MS/MS: 195.0430, 171.0434, 167.0483, 136.9864, 111.0084, 65.0045; 271.0608 [M+H]+, MS/MS: 253.0497, 169.0136,
271.0586 [M-H]-, MS/MS: 177.0177, 151.0025, 119.0496, 107.0135, 93.0347; 273.0759 [M+H]+, MS/MS: 153.0185, 147.0441, 119.0495
were recorded in the range of m/z 200–1000 with accurate mass
measurement of all mass peaks. Each sample was analyzed in both

609.1447 [M-H]-, MS/MS: 300.0267, 271.0234, 255.0286, 178.9987, 151.0027; 611.1620 [M+H]+, MS/MS: 465.1046, 303.0517

301.0332 [M-H]-, MS/MS: 273.0381, 178.9968, 151.0024, 121.0286, 107.0134; 3.3.0506 [M+H]+, MS/MS: 229.0494, 153.0179

253.0477 [M-H]-, MS/MS: 143.0487, 119.0493, 101.0392, 63.0261; 255.0659 [M+H]+, MS/MS: 153.0183, 129.0340, 103.0552
positive (Fig. 1A) and negative (Fig. 1B) modes to give abundant
information for structural identification.
463.0858 [M-H]-, MS/MS: 300.0258, 271.0226, 255.0271, 178.9975, 151.0028; 465.1034 [M+H]+, MS/MS: 303.0504

285.0384 [M-H]-, MS/MS: 163.8851, 137.0229, 117.0337;287.0545 [M+H]+, MS/MS: 269.0449, 169.0129, 123.0079

269.0432 [M-H]-, MS/MS: 225.0534, 151.0024, 117.0338, 107.0131;271.0607 [M+H]+, MS/MS: 153.0183, 119.0496
2.5. MTT assay

285.0368 [M-H]-, MS/MS: 151.0019, 133.0282, 107.0129; 287.0561 [M+H]+, MS/MS: 153.0183, 135.0449 Cell proliferation was measured by MTT assay. Briefly, we collected

283.0589 [M-H]-, MS/MS: 268.0357, 163.0020, 110.0002;285.0769 [M+H]+, MS/MS: 270.0536, 179.0493
and seeded the logarithmic phase of A2780 cells and IOSE80 cells at a
concentration of 1 × 104/well in a 96-well plate. These cells were then
grown in 200 μl DMEM medium containing different concentrations
447.0917 [M-H]-, MS/MS: 285.0394, 174.9553, 130.9659; 449.1104 [M+H]+, MS/MS: 287.0565

445.0756 [M-H]-, MS/MS: 269.0438, 175.0234, 113.0230; 447.0906 [M+H]+, MS/MS: 271.0593

(0, 50, 100, 200, 400, 600 μg/ml) of SB. 24 h and 48 h later, the MTT
solution (5 mg/ml) was added to each well and incubated for 4 h.
150 μl DMSO was added to each well and the solution was mixed for
10 min. Absorbance of the solution was determined by a Multiskan
Ascent plate reader at a wavelength of 492 nm.
431.0955 [M-H]-, MS/MS: 268.0362; 433.1128 [M+H]+, MS/MS: 271.0597

2.6. DAPI staining assay

475.0885 [M-H]-, MS/MS: 299.0563, 284.0330, 175.0246, 113.0250

In brief, the A2780 cells were collected and seeded into 24-well
plates. After treatment with different concentrations (0, 100, 200,
400 μg/ml) of SB for 24 h, the cells were fixed with 3.7% formaldehyde
and stained with DAPI. These samples were then washed and photo-
graphed by fluorescence microscopy.

2.7. Annexin V-FITC staining assay for apoptosis analysis

Apoptosis of A2780 cells was evaluated using Annexin V-FITC and

propidium iodide (PI) staining. A2780 cells were first treated with
various concentrations (0, 100, 200, 400 μg/ml) of SB for 24 h. The
cultured cells were then collected, washed with PBS three times and
stained with Annexin V-FITC and PI for 5 min in the dark, and finally
123.0081, 103.0548

analyzed by a FACS/Calibur flow cytometer (Becton Dickin-son,

Franklin Lakes, NJ, USA).
Productions

2.8. Wound healing assay

A2780 cells were seeded into 24-well plates with the density of 5 ×
Molecular Weight

104/ml. The wound was scratched using a 200 μl sterile pipette tip.
Cells were washed twice with PBS and then replaced with fresh DMEM
medium. The cells were treated with different concentrations (0, 50,
464.10
448.10
610.15
432.11
476.10
446.08
286.05
286.05
302.04
270.05
272.07
272.07

284.07
254.06

100 μg/ml) of SB for 24 h, the photographs before and after treatment

Chromatographic MS and MS/MS data of compounds identified in the extract of SB.

were taken and the distance of cells migration was measured and
quantified with computer-assisted microscope at 100 × magnification.
Molecular formulas

2.9. Transwell chamber assay

C21H20O12
C21H20O11
C27H30O16
C21H20O10
C22H20O12
C21H18O11
C15H10O6
C15H10O6
C15H10O7
C15H10O5
C15H12O5
C15H12O5

C16H12O5
C15H10O4

We used transwell chamber assay to detect the migration ability of

A2780 cells. A2780 cells were treated with various concentrations (0,
50, 100 μg/ml) of SB for 24 h. The cells were then seeded to the upper
quercretin-3-O-β-D-glucopyranside

apigenin-7-O-β-D-glucopyranoside

chamber with serum-free DMEM medium and serum-containing

luteilin-7-O-β-D-glucopyranside

hispidulin-7-O-β-D-glucuronide

DMEM medium was added to the lower chamber as a chemoattractant

for 24 h. After incubation, the cells on the upper surface of membrane
were removed with cotton swabs and the cells under the membrane
were stained with 0.1% crystal violet and photographed. The migrated
cells of random six fields of each Transwell membrane were counted
under a microscope at 100 × magnification field.
scutellarein
Compound

naringenin
quercetin

baicalein
apigenin

wogonin
baicalin

luteolin

chrysin
rutin

2.10. Western blot analysis

Rt (min)

After incubation with indicated concentrations of SB for 48 h, the

10.22

13.71
13.83

total proteins were obtained. The total cell lysates were separated by
3.78
3.79
3.80
4.04
4.11
5.01
5.75
6.79
7.00
8.52
8.91

10% SDS-PAGE and then transferred to nitrocellulose membrane by

Table 1

semi-dry apparatus for 35 min. The membrane was blocked with 5%

No.

10
11
12

13
14
1
2
3
4
5
6
7
8
9

non-fat milk for 1.5 h and then the blotted membrane was incubated

186
L. Zhang et al.
Fig. 2. The influence of SB treatment on the proliferation of A2780 and SK-OV-3 cells. (A) Determined by MTT experiments to detect A2780 cell and SK-OV-3 cell. activity after 48 h
incubation with concentrations of SB (0, 100, 200, 300, 400, 500, 600 μg/ml) for 24 h and 48 h. The assays were performed in triplicate. *p < 0.05, **p < 0.01,***p < 0.001 compared
with the controls. (B) 24 h the action of the A2780 and SK-OV-3 cells with SB (0, 100, 200, 400 μg/ml) and cells were photographed with inverted contrast miroscopy (magnification, 40
×).
with the specific primary antibody solution overnight at 4 °C. After
incubation with secondary antibody for 1 h the next day, the immuno-
blotting signals were visualized by ECL chemiluminescence.
2.11. Statisticalanalysis
Data were presented as mean ± SD and analyzed by SPSS 15.0
software, and all the raw data was carried on the normal distribution
test. Student’ t-test was performed to assess the statistical significance.
p values < 0.05 was considered as statistically significant.
3. Results
3.1. Phytochemical screening
Compounds 1–14 could easily be identified as Quercretin-3-O-β-D-
glucopyranside(1), luteilin-7-O-β-D-glucopyranside(2), rutin(3), Apigenin-
7-O-β-D-glucopyranoside (4), hispidulin-7-O-β-D-glucuronide (5), baicalin
(6), scutellarein (7), luteolin (8), quercetin (9), apigenin (10), naringenin
(11), baicalein (12), wogonin (13) and chrysin (14) by comparing retention
time and the fragmentation behaviors with the standard sample solution,
respectively as shown in Fig. 1. Their retention time and product ions were
summarized in Table 1.
3.2. Growth inhibitory effect of SB on human ovarian cancer cells
The anti-proliferative effect of SB on A2780 and SK-OV-3 cells was
detected by MTT assay. A2780 cells and SK-OV-3 cells were treated
with different concentrations of SB for 24 h or 48 h. The experiment
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Journal of Ethnopharmacology 206 (2017) 184–192
showed that SB significantly decreased the survival rate of A2780 cells
and SK-OV-3 cells in a dose and time dependent manner (p < 0.05). But
have no significantly effect on IOSE80 cells (p > 0.05)(Fig. 2A).
Moreover, A2780 cells and SK-OV-3 cells treated with SB start to
shrink, became round and dose-dependently detached from the flasks
compared to untreated cells (Fig. 2B). These data suggested that SB
inhibited the viability of A2780 cells and SK-OV-3 cells.
3.3. SB induces ovarian cancer cells apoptosis
The apoptosis of ovarian cancer cells was observed after treatment
with various concentrations of SB for 48 h. The Annexin V-FITC and
presidium iodide (PI) staining were performed to separate the apopto-
tic cells from viable cells. And the Annexin V-positive/PI-negative or
Annexin V/ PI double positive population indicates early or late
apoptosis respectively (Fig. 3B). As shown in figure, the rate of early
and late apoptosis cells was gradually increased with the increasing
concentrations of SB (p < 0.05) (Fig. 3C). In addition, nuclear staining
with DAPI of A2780 cells following SB treatment was more intense
than untreated cells (Fig. 3A). These observations indicated that SB
could induce the ovarian cancer cells apoptosis.
3.4. SB increased apoptosis-related proteins and inhibited Bcl-2
expression
According to the results of the flow cytometry assay, we further
investigated the effects of SB on protein expression in ovarian cancer
cells. Our data showed that SB dramatically derived caspase-3 and
caspase-9 activation in a dose-dependent pattern (p < 0.05) (Fig. 4A
L. Zhang et al. Journal of Ethnopharmacology 206 (2017) 184–192

Fig. 3. SB induce apoptosis of A2780 cells. SB (0, 100, 200, 400 μg/ml) is used to treat ovarian cancer cells for 24 h. (A) Using fluorescent microscope DAPI staining to detect the
morphology of the nucleus. Nuclear condensation and apoptotic bodies were indicated by the arrows (magnification, 100 ×). (B) Cell apoptosis was determined with flow cytometry and
analyzed by Annexin V-FITC/PI double staining. (C)Treatment of A2780 cells with SB resulted in dramatical increase in the percentages of apoptotic cells (including early stage and late
stage). The experiments were repeated three times. *p ;< 0.05, **p < 0.01, ***p < 0.001 compared with the controls.

and C). Besides Caspase members, the expression of anti-apoptotic 50 μg/ml and 100 μg/ml SB respectively with the control group. The
protein Bcl-2 in A2780 cells treated with SB was also detected by results suggested that SB inhibited the migration and invasion of
western-blotting assay. We found that SB significantly down-regulated A2780 cells (Fig. 5B).
the Bcl-2 protein level (p < 0.05) (Fig. 4B).

3.5. SB suppressed migration of ovarian cancer cells

Cell migration is one of the hallmarks of tumor cells. To investigate
the effect of SB on the migration of A2780 cells, healing and transwell
migration assay were conducted. It was found that SB decreased the
movement of A2780 cells in a dose-dependent manner. The wound
closure rates of SB (50 μg/ml and 100 μg/ml) treated cells were 40%
and 20% respectively compared to non-treated cells (p < 0.05)
(Fig. 5A). In addition, the migration of A2780 cells into transwell
chamber was significantly suppressed by SB. The numbers of migrated
cells was reduced by 75% and 87.5% comparing the cells treated with
3.6. SB inhibited the expression of MMP-2/9
Next, we looked into the underlying molecular mechanisms that SB
suppressed the migration on A2780 cells. MMP-2/9 level was exam-
ined by Western blotting. Our experiment showed that the level of
MMP-2/9 dose-dependently decreased after SB treatment (Fig. 6A).
These results suggested that the inhibitory effects of SB on migration of
ovarian cancer cells maybe related to the inhibition of MMP-2/9
protein expression (Fig. 6B).

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L. Zhang et al. Journal of Ethnopharmacology 206 (2017) 184–192

Fig. 4. The influence of SB treatment on apoptosis-related proteins. Cells were treated with indicated doses of SB for 48 h. (A) The expression of Caspase-3 and Caspase-9 were also
measured in the study. The results showed that the cleaved-Caspase-3 and cleaved-Caspase-9 expression levels were upregulated in the osthole treated ovarian cancer cells. The level of
β-actin served as internal control. The experiments were repeated at least three times. (B) Activation of Bcl-2 protein expression by SB in A2780 cells. (C) Histograms show mean ( ± SD)
level of caspase-3, caspase-9 and Bcl-2 from three independent experiments. The caspase-3, caspase-9 and Bcl-2 expression levels were expressed relative to β-actin and standardized to
non-treated control group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the controls.

4. Discussion natural components so that they possess comprehensive functions (Shi

The herbal medicines have drawn more and more attention as
holistically effective patterns of drugs for cancer treatment, due to severe
side effects and drug resistance of the traditional chemotherapeutics
(Agbarya et al., 2014). The traditional herbal medicines consist of multiple
et al., 2016). Scutellaria barbata D. Don is used to treat numerous types
of cancer in China for long time (Jiang et al., 2015). In this study, our data
showed that SB exhibited anti-tumor effects on ovarian cancer cells.
Moreover, we analyzed principal phytochemicals which may contribute to
the anti-tumor activity of the SB.

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Fig. 5. Influence of SB treatment on the migration of A2780 cells. Monolayers of A2780 cells were scratched with a pipette tip and treated with the indicated doses of SB for 24 h. (A)
The migration of A2780 cells before and after injury are showed under the microscope at 100 × magnification field of the scratch. The migration of A2780 cells was quantified by
measuring wound closure areas before and after injury. (B) These photos represented cell migration or invasion onto the underside of transwell membrane under microscope at 100 ×
magnifcation field.The number of cells that crossed the transwell invasion chamber of each field was counted and averaged. Data was representative of at least three independent
experiments. ***p < 0.001compared with the controls.

Previous study showed that SB could effectively inhibit the pro-
liferation and induce the apoptosis of colon carcinoma in vitro (Tao and
Balunas, 2016). In addition, SB significantly inhibited the apoptosis
and invasion of hepatocarcinoma in a dose-dependent manner (Pan
et al., 2016). Our results also showed that SB could dose-dependently
inhibit the growth of ovarian cancer cells. However, SB did not
significantly affect the growth of normal ovarian cells. It indicated that
SB could selectively suppressed the proliferation of ovarian cancer.
Apoptosis is necessary to maintain the cellular homeostasis and
death (Chen et al., 2013). Many natural compounds preventing growth
of tumor cells through inducing apoptosis (Xu et al., 2017). In present
study, we demonstrated that SB treatment could dramatically increase
the percentage of early and late apoptotic cells. To better under-
standing the underlying mechanisms, we further investigate the
molecular changes during this process. As we known, the apoptosis
pathway triggered apoptotic activity through the regulation of apopto-
sis related proteins, such like Caspase and Bcl-2 family members (Lee,
2016). Caspase-3 and caspase-9 proteins are involved in regulating the
mitochondrial pathway of apoptosis (Dong et al., 2016). Bcl-2 family
members have been reported to possess the ability of maintaining cell
viability by preventing mitochondrial membrane potential (Buranrat
et al., 2016). Our results showed that SB induced apoptotic activation
through up-regulation of caspase-3/9 and down regulation of Bcl-2
(Thao do et al., 2015). Based on these data, SB may induce A2780 cells
apoptosis in a mitochondria-associated pathway.
Most phytochemicals detected in SB by HPLC-Q-TOF-MS chroma-
togram are flavonoids which may contribute to the anti-tumor effects
(Srivastava et al., 2016). For instance, scutellarein could inhibit the
growth of tongue cancer cells in vitro and regulate the cell adhesion. In
addition, apigenin could suppress the proliferation of the human breast
cancer cells (Zhang et al., 2016). Moreover, baicalin and baicalein also
showed inhibitory effects on ovarian cancer cells (Li et al., 2013). In our
study, the presence of these pytochemicals in SB may be related to its
cytotoxic activity on ovarian cancer cells. These flavonoids from SB
may be promising drugs for ovarian cancer patients.
The migration of cancer cells is the first step of metastasis (Du et al.,
2016). Our results demonstrated that SB significantly inhibited A2780
cells migration. It may be associated with the downgrade expression of
MMP-2/9. Matrix metalloproteins (MMPs) play an important role in
tumor cells migration and invasion by promoting the interaction
between tumor cells and their local microenvironment (Chen et al.,
2016). MMP-2/9 is expressed at a higher level in ovarian cancer cells
than in normal cells and overexpression of MMP-2/9 could contribute
to metastasis of ovarian cancer (Wen et al., 2014). SB may inhibit
migration of ovarian cancer cells by down-regulation of MMP-2/9.
In conclusion，our study demonstrated that SB could inhibit
proliferation and induce apoptosis in A2780 cells through mitochon-
drial pathway. In addition, SB suppressed the migration of ovarian
cancer cells through down-regulation of MMP-2/9 expression
(Shortrede et al., 2016). The flavonoids detected from SB might be
the active components. This finding suggested that SB may be used as
an anti-ovarian cancer agent for future treatment of ovarian cancer.
Conflict of interest
The authors have declared that there is no conflict of interest.

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L. Zhang et al.
Fig. 6. Inhibition of MMP-2/9 activity and expression in A2780 cells by SB. Cells were treated with indicated doses of SB for 48 h. MMP-2/9 protein in A2780 cells was detected by
Western blot. The ratio of MMP-2/9 and β-actin was presented. Data are presented as means ± SD of three experiments. *p < 0.05, **p < 0.01 compared with the controls.
Acknowledgments
Funding: This work was supported by the National Natural Science
Foundation of China [grant numbers No. 81302282, No. 81403306];
and by the Natural Science Foundation of Liaoning Province [grant
numbers 2014023056].
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