Microvascular Research 107 (2016) 17–33

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Microvascular Research

journal homepage: www.elsevier.com/locate/ymvre

Scopoletin, an active principle of tree tobacco (Nicotiana glauca) inhibits

human tumor vascularization in xenograft models and modulates ERK1,
VEGF-A, and FGF-2 in computer model
Yasser M. Tabana a,⁎, Loiy Elsir A. Hassan a, Mohamed B. Khadeer Ahamed b, Saad S. Dahham a,
Muhammad Adnan Iqbal b, Mohammed A.A. Saeed c, Md Shamsuddin S. Khan a, Doblin Sandai d,
Aman S. Abdul Majid e, Chern Ein Oon f, Amin Malik S.A. Majid a,⁎
a
EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia
b
EMAN Biodiscoveries Sdn. Bhd. Suite 126, Level 1, EUREKA Complex, Universiti Sains Malaysia (USM) Campus, Minden 11800, Penang, Malaysia
c
Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia
d
Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200 Bertam, Penang, Malaysia
e
Department of Pharmacology, Quest International University, Perak, Malaysia
f
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-
Received 10 August 2015 angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-
Revised 19 April 2016 dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable
Accepted 24 April 2016
human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical inves-
Available online 29 April 2016
tigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-
Keywords:
angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Final-
Scopoletin ly, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using
Nicotiana glauca athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized
Xenograft model using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic ex-
Antineovascularization plants with IC50 (median inhibitory concentration) 0.06 μM. Scopoletin (100 and 200 mg/kg) strongly inhibited
Angiogenic mitogens (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft
model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200 mg/kg, respec-
tively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining
of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor
vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the fol-
lowing angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast
growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong
anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2.
© 2016 Elsevier Inc. All rights reserved.

Introduction
Targeting angiogenesis via suppressing angiogenic factors is one of
the most recent successful strategies for cancer treatment. Cancer cells
produce various angiogenic factors to initiate angiogenesis. Vascular
endothelial growth factor A (VEGFA), fibroblast growth factor 2
(FGF2) and extracellular signal-regulated kinases-1 (ERK-1) are com-
monly expressed in most of the cancers and their expression levels
have been correlated with tumorigenesis and metastasis (Cao et al.,
⁎ Corresponding authors.
E-mail addresses: Yasser.tabana@hotmail.com (Y.M. Tabana),
aminmalikshah@gmail.com (A.M.S.A. Majid).
2012; Wang and Zheng, 2012). For centuries, natural products have
played a strategic role in maintaining human health, and they have
been utilized as a major source of remedies in all human civilizations.
Thus, researchers have been looking for angiogenesis inhibitors and
promoters from natural products for the past 15 years (Wang et al.,
2004).
Tree tobacco, botanically known as Nicotiana glauca (Solanaceae), is
a plant widely used in traditional medicine to treat various infectious
and inflammatory diseases. N. glauca is native to central northwest
Argentina and Bolivia, however later on the plant has invaded Central
and North America (Mexico, southern US states including California)
and Africa, specifically Morocco, South Africa and Namibia (Florentine
and Westbrooke, 2005; Ibrahim, 2012). The plant can also be found in

http://dx.doi.org/10.1016/j.mvr.2016.04.009
0026-2862/© 2016 Elsevier Inc. All rights reserved.
18 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33
Fig. 1. Isolation of scopoletin. Schematic diagram showing the bioassay guided sequential extraction and fractionation of N. glauca leading to isolation of scopoletin.
Australia and is widely distributed from mesic to arid environments,
including the Negev desert of Israel. Extracts from this plant have
been reported to have dynamic biological activities and are commonly
used as a tonic, paste, or drink to treat general pain, boils, headaches,
piles, sores, and wounds. The extracts also were reported to have strong
antimicrobial and cytotoxic activities (Mdee et al., 2009). In a previous
study, we reported the strong anti-angiogenic effect of the n-hexane ex-
tract of N. glauca leaves on sprouting of blood vessels in the rat aorta
(Hassan et al., 2014). However, the active compound responsible for
the bioactivity of the herb was not known.
Chemically, N. glauca is rich in coumarin-derived compounds.
Scopoletin is a 6-methoxy-7-hydroxycoumarin isolated from N. glauca.
Our previous study showed that scopoletin has promising anti-
angiogenic activity (Beh et al., 2012). Several other studies showed
that scopoletin possesses potent hepatoprotective (Kang et al., 1998),
anti-oxidant (Shaw et al., 2003), and spasmolytic (Oliveira et al.,
2001) activities. Liu et al. (2001) reported that scopoletin inhibited
cell proliferation by inducing cell cycle arrest and increasing apoptosis
in human prostate tumor cells. Recently, scopoletin was reported to
inhibit in vitro proliferation, migration and tube formation properties
of human endothelial cells (Pan et al., 2009). As scopoletin could
prove to be useful as a systemic chemotherapeutic agent against
angiogenesis-dependent malignancies, hence the anti-angiogenic prop-
erty of scopoletin was needed to be confirmed using advanced and suit-
able animal models of angiogenesis and tumorigenesis. Based on this
background, the present study was conducted to identify the active
principle (scopoletin) of N. glauca extract which is responsible for its
strong anti-angiogenic property. Furthermore, an attempt was made
to characterize the anti-angiogenic activity of scopoletin using ex vivo,
in vivo and in computer modeling experimental systems. Finally, the
anti-angiogenic property of scopoletin was exploited to study the po-
tential in vivo anti-tumor activity using human tumor xenograft model.
Materials and methods
Chemicals, cell culture, and reagents
The chemicals used in the cell culture study (penicillin/streptomycin
(PS), phosphate buffered saline (PBS), diaminobenzidine, hematoxylin,
trypsin, avidin–biotin complex and MTS (3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) were
procured from Sigma Aldrich (Darmstadt, Germany). Xylene, 3% H2O2,
antigen retrieval solution pH 6 and anti-rabbit biotinylated antibody
 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33 19

Fig. 2. Anti-angiogenic effect of scopoletin. Photomicrographic images show inhibition of sprouting of microvessels in rat aortic explants. The aortic rings were photographed at 20×
magnification after 5 days of treatment with scopoletin and the standard reference, suramin. A: control. B: scopoletin (0.06 μM). C: scopoletin (0.12 μM). D: scopoletin (0.25 μM).
E: scopoletin (0.5 μM). F: suramin (0.5 μM). G: graphical representation of the anti-angiogenic activity of scopoletin in comparison with that of the standard reference, suramin. All
values are expressed as mean ± SEM (n = 8). *p b 0.05, **p b 0.01.
20 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33

were purchased from (Dako, USA). CD34 primary antibody was procured
from (ABCAM, UK).
The reagents for the ex vivo culture study (aprotinin, fibrinogen, bo-
vine serum albumin (BSA), thrombin, matrigel, suramin, and vincristine)
also were purchased from Sigma. HCT 116 (human colon cancer) and
human umbilical vein endothelial cells (HUVECs), colon cancer (HCT
116) and normal (CCD-18Co) cells were purchased from American
Type Culture Collection (ATCC, Manassas, VA, USA) and grown in suitable
cell medium supplemented with 5% heat inactivated fetal bovine serum
(HIFBS), both purchased from ScienCell (St. Louis, MO, USA), and 1% PS.
Other chemicals used were either high performance liquid chromatogra-
phy (HPLC) grade or analytical grade. Methanol and orthophosphoric
acid were procured from QRec (New Zealand) and acetonitrile HPLC
grade was purchased from Merck (Darmstadt, Germany). Thin layer
chromatography (TLC) plates coated with 0.25 mm Silica Gel 60 F254
were obtained from Merck. An ultraviolet (UV) fluorescence cabinet
CM-10 (New York, USA) was used to visualize chromatograms on the
TLC plates. A Sony Cyber-shot DSC-T33 digital camera was used to obtain
the TLC photographs.
Plant material
The plant material was collected from the Botanical Garden at Kassala,
Eastern Sudan during the period of March to July 2013 with permission
from the authorities of the garden. The taxonomic authentication of
N. glauca was performed by Dr. Wail Alsadig at the Medicinal and
Aromatic Plants Research Institute, National Center for Research,
Sudan. A voucher specimen (voucher reference number: MAPRI/NB-
53b) was deposited at the herbarium of the institute (Tabana et al.,
2015). All experiments were carried out in the EMAN (Experimental
Medicine & Advanced Natureceutical) Lab at Universiti Sains Malaysia
(USM) with permission from the director of the laboratory. The present
work did not involve any special locations or activities that required
permission. Furthermore, the present study did not involve endangered
or protected species.
model was approved with the approval reference number: USM/Animal
Ethics Approval/2014/(94) (589).
Acute toxicity of scopoletin
The acute toxic effect of scopoletin was assessed using a single dose
administration test, which was employed using OECD guideline-425
(Khadeer Ahamed et al., 2010).
Ex vivo rat aortic ring assay
The rat aortic ring assay was performed following (Al-Salahi et al.,
2013) with slight modifications. Different concentrations (0.03125,
0.0625, 0.125, 0.25, and 0.5 μM) of scopoletin were used to determine
the IC50 (median inhibitory concentration) values using regression
equation obtained from the concentration-activity curve. Suramin
(0.03 to 0.5 μM) and DMSO (0.1%) were used as positive and negative
controls, respectively (Dahham et al., 2015a, 2015b; Nassar et al., 2011)
MTS assay
MTS assay was performed to assess the anti-proliferative effect of
scopoletin on human endothelial (HUVECs), normal colon (CCD-18Co)
and colon cancer (HCT 116) cells. The assay plate was read using Tecan
Infinite® 200 PRO microplate reader (HIDEX, Finland) at 570 nm absor-
bance. DMSO (0.1%) was used as a negative control. Suramin and 5-
fluorouracil were used as positive controls for HUVEC and HCT 116
cells, respectively. Dose–response curves were plotted and IC50 values
were calculated using the regression equation (Dahham et al., 2015a,
2015b; Tabana et al., 2015). The selectivity index (SI), which indicates
the cytotoxic selectivity of the compound against HUVECs and its ineffec-
tive toward the other cell types, was determined from the ratio of
the IC50 of scopoletin on colon fibroblasts (CCD-18Co) versus IC50 on
HUVECs.
In vivo matrigel plug assay

Isolation and structural confirmation of scopoletin
Isolation of scopoletin was carried out using bioassay guided frac-
tionation of n-hexane extract of N. glauca (Fig. 1). The details of extrac-
tion, fractionation and column chromatographic procedures are given in
the Supplementary information. The structure of scopoletin was
confirmed using FT-IR and NMR spectroscopic techniques (refer to the
Supplementary information).
Experimental animals
NU/Nu immunocompromised nude mice were injected subcutane-
ously with 0.3 ml of matrigel supplemented with 100 μl of HCT-116
cells (106 × 1 cells/ml) near the abdominal midline (Passaniti et al.,
1992). Mice were then treated orally with 100 and 200 mg/kg of
scopoletin (n = 6) daily for 7 days. Vehicle (0.1% Tween 80) and imatin-
ib (100 mg/kg) were used as the negative and positive controls, respec-
tively. At the end of the experiment, all the animals were euthanized
and matrigel plugs were carefully excised and sectioned at 5 μm thick-
ness, then stained with hematoxylin and eosin (H&E). The number of
blood vessels in all sections was counted.

Healthy adult female Sprague Dawley (SD) rats (225–250 g) were
used in the acute toxicity study. For the rat aortic ring assay,
8–10 week old healthy male SD rats (200–230 g) were used. The rats
were obtained from the Animal House Facility, USM. For the xenograft
model, either sex of athymic NCR-nu/nu (nude) mice (8–12 weeks
old) was used. All experiments were conducted according to the guide-
lines of the Institutional Animal Ethics Committee. The study protocol
was approved by the Animal Ethics Committee, USM [approval refer-
ence number: PPSG/07(A)/044/(2010) (61)]. The tumor xenograft
In vivo assessment of tumor angiogenesis in nude mouse xenograft model
Human colorectal tumors were grown in athymic (Ncr-Nu/Nu)
nude mice by injecting HCT-116 cells (107 cells/150 μl DMEM) sub-
cutaneously on the dorsal side of the animals (Ahamed et al.,
2012). Scopoletin was administered to mice orally once daily in
three different doses: 50, 100, and 200 mg/kg (n = 10). Tween 80
(0.1%) and imatinib (100 mg/kg) were used as negative and positive
controls, respectively. The animals from the negative control group

Fig. 3. H&E stained cross-sections taken from matrigel plugs implanted subcutaneously in representative groups of animals. A: Section of the matrigel from the negative control group
shows high vascularization (arrows) (original magnification of 20×) and more proliferation of HCT-116 cells compared to the other treated groups. B: Magnified section (40×) of a rep-
resentative matrigel plug from the negative control group shows abundance of blood vessels (indicated by the arrows). C: Section (20×) of a matrigel plug harvested from a scopoletin
(100 mg/kg)-treated animal demonstrates significant reduction in vascularization. D: Higher magnification (40 ×) of a section of the matrigel plug harvested from a scopoletin
(100 mg/kg)-treated animal reveals drastic reduction of blood vessels in the matrigel implant. E: A representative matrigel implant from a scopoletin (200 mg/kg)-treated mouse shows
complete inhibition of blood vessels (original magnification of 20×). F: Magnified image (40×) of the matrigel section showing the strong anti-angiogenic effect of scopoletin (200 mg/kg)
treatment. Most of the matrigel implant from this group showed complete inhibition of neovascularization. G: Matrigel section (20×) showing the anti-angiogenic effect of the standard
reference drug, imatinib (100 mg/kg). H: Magnified (40×) photomicrographic image of the matrigel section shows the potent anti-angiogenic effect of imatinib. I: Graphical representa-
tion of the effect of scopoletin on the mean blood vessel count in matrigel sections (* = p b 0.05, ** = p b 0.01, n = 6, values are mean ± SEM of 10 low power microscopic fields).
Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33 21
22 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33

Fig. 4. In vivo antitumor and anti-angiogenic activities of scopoletin determined using a human tumor xenograft model in athymic nude mice bearing HCT-116 tumors at day 28 post-
inoculation. A: Animals from the vehicle-treated group (negative control). B: Animal treated with 50 mg/kg scopoletin. C: Animals treated with 100 mg/kg scopoletin. D: Animals
treated with 200 mg/kg scopoletin. E: Animals treated with 100 mg/kg imatinib. F: Tumor morphology of the representative groups of animals: (a) negative control, (b) 50 mg/kg of
scopoletin, (c) 100 mg/kg scopoletin, (d) 200 mg/kg scopoletin, and (e) 100 mg/kg imatinib (arrows indicate prompt and well-developed blood vessels in the control, and arrowheads
indicate the reduction of tumor vasculature).

were euthanized when the tumor volume reached the maximum
level of 1000 mm3 in size. The tumor-bearing animals were treated
until either the tumor volume reached 1000 mm3 or for 21 days.
Tumor tissue was collected from the euthanized animals for histo-
logical studies. The number of blood vessels in all tissue sections
was counted.
Visualization of tumor vasculature in immunohistochemistry
The paraffin sections were deparaffinized at 60 °C for 10 min
followed by rehydration in xylene and a series of graded alcohol. In-
hibition for endogenous peroxidase was carried out using 3% H2O2
(Dako). Antigen retrieval was performed using antigen retrieval so-
lution pH 6 (Dako) for 15 min using the microwave. Sections were
then incubated with 15% bovine serum albumin (SIGMA) for 1 h at
room temperature followed by CD34 primary antibody (ABCAM)
overnight at 4 °C. The slides were rinsed with PBS and incubated
with anti-rabbit biotinylated antibody (Dako) for 30 min. Sections
were then rinsed and incubated with standard avidin–biotin com-
plex (ABC; SIGMA) for 30 min. This is followed by incubation with di-
aminobenzidine (SIGMA) for 5 min and hematoxylin (SIGMA) for
1 min. Sections were finally mounted in mounting media (Dako)
and imaged using a brightfield microscope.
In addition, pericytes were identified by labeling with affinity-
purified rabbit polyclonal antibodies against the NG2 proteoglycan
according to the manufacturer's instruction (Merck, Germany).
Tools for the molecular docking study
Ligand crystal structures of scopoletin and suramin were
downloaded from the ChemSpider database. Protein 3D structures of
mitogen-activated protein kinase (ERK1/2) (Kinoshita et al., 2008), vas-
cular endothelial growth factor A (VEGF-A) (Mandal and Kent, 2011),
and fibroblast growth factor 2 (FGF-2) were downloaded from the
RCSB protein data bank. Suramin was used as the positive control in
this docking study.
Molecular docking study
Specific active site-target and automated docking systems were used
to determine the binding affinity and ligand efficiency of scopoletin
with the major angiogenic mitogens ERK1/2, VEGF-A, and FGF-2. The
activity of scopoletin was compared with that of the standard reference,
suramin. The two different methods were employed using different al-
gorithms to analyze the comparative activity of scopoletin and suramin.
LeadIT — FlexX
BioSolveIT's LeadIT is a specific active site-target program that is
used to dock the compounds as a flexible function of the FlexX algo-
rithm (Böhm, 1998). This program detects the binding site according
to a reference ligand by superimposition of the experimented ligand.
 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33 23

Fig. 5. Illustration of the tumor growth profile in the respective test groups. A: Graphical illustration of the dose-dependent suppression effect of scopoletin on HCT-116 cell tumorigenesis
in nude mice in comparison to the negative and positive controls (Scop = scopoletin; PC = imatinib; NC = vehicle-treated negative control; the numbers in brackets represent the dose in
mg/kg). All values are expressed as mean ± SEM (n = 10). B: Effect of scopoletin on the average body weight of treated animals in comparison to the negative control (HD = high dose
(200 mg/kg) of scopoletin; MD = mid-dose (100 mg/kg) of scopoletin; LD = low dose (50 mg/kg) of scopoletin; PC = positive control (imatinib at 100 mg/kg); NC = vehicle-treated
negative control). All values are mean ± SEM (n = 10).

The active site was defined by selecting the residue of the protein
with 10 Å at the center of the ligand. The top 10 poses were selected
to analyze the free binding energy (ΔG) of the protein–ligand com-
plex and the ligand efficiency using Hyde assessment (Rarey et al.,
1996).
SYBYL Surflex Dock
Statistical analysis
All results are expressed as the mean ± standard error (± SEM).
Statistical differences between the test compounds and the negative
control were analyzed by one-way analysis of variance (ANOVA)
followed by Tukey's multiple comparison test using IBM SPSS software
(Version 20). Differences with p b 0.05 and b0.01 were considered to
be statistically significant.

Surflex Dock is an automated docking program that is used to simu-
late the interaction mode between compounds and protein bio-markers
in SYBYL-X 2.1. The active sites of the protein were selected as the
amino acids containing atoms within 6 Å. Five scoring functions of
Dock (DScore) (Meng et al., 1992), Gold (GScore) (Jones et al., 1997),
PMF (Pscore) (Muegge and Martin, 1999), ChemScore (Eldridge et al.,
1997), and Consensus (CSCORE) were selected to calculate the correla-
tion. CSCORE was used to find the correlation with other scores. Only
the topmost pose was selected to compute the correlation with the
reported binding affinity.
Results and discussion
LD50 value of scopoletin is N2000 mg/kg in SD rats
The acute toxicity test showed that scopoletin did not produce
treatment-related mortality at the limit test dose (2000 mg/kg). Oral
administration of scopoletin in rats did not produce any considerable
toxic behavioral changes such as apathy, hyperactivity, or morbidity.
Body weight, respiration rate, and heart rate in all treated rats were
normal. These results suggest that scopoletin is safe at dose level of
24 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33
 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33 25

Fig. 7. Photomicrographic images display immunofluorescence staining of differentiation molecule CD34 and the chondroitin sulfate proteoglycan NG2 receptors in the tumor histological
sections. A) Photomicrographic image showing NG2 immunofluorescence staining in negative control group (magnification ×400 μm). B) Photomicrographic image of tissue section from
imatinib treated animal showing NG2 immunofluorescence staining (magnification ×400 μm). C) Photomicrographic image showing NG2 immunofluorescence staining in scopoletin
(100 mg/kg) treated group (magnification × 200 μm). D) Photomicrographic image showing NG2 immunofluorescence staining in scopoletin (200 mg/kg) treated group
(magnification ×200 μm). E) Photomicrographic image showing NG2 immunofluorescence staining in negative control group (magnification ×20 μm). F) Photomicrographic image of
tissue section from imatinib treated animal showing NG2 immunofluorescence staining (magnification ×20 μm). G) Photomicrographic image showing NG2 immunofluorescence
staining in scopoletin (100 mg/kg) treated group (magnification ×20 μm). H) Photomicrographic image showing NG2 immunofluorescence staining in scopoletin (200 mg/kg) treated
group (magnification ×20 μm).

Table 1
Summary of the docking scores and reported binding affinities of scopoletin and suramin with ERK1, VEGF, and FGF2 in the docking analysis using the LeadIT FlexX scoring functions (A =
total scores; B = match score; C = lipophilic area score; D = ambiguous area score, E = receptor-protein overlap score; F = ROT score; RMSD b 0).

Receptor–ligand Binding affinity energy, Ligand efficiency Total Match Lipophilic Ambiguous Receptor–ligand Rotational RMSD
complex (LeadIT) ΔG (Kcal/Mol) (Kcal/mol per heavy atom) score score area score area score interaction score bond score

ERK1-scopoletin −19 0.32 −13.14 −16.73 −5.88 −2.52 3.80 2.80 0.00
ERK1-suramin −10 0.03 −22.05 −21.21 −12.98 −12.69 11.04 8.40 0.00
FGF2-scopoletin −14 0.23 −8.9782 −11.5746 −−4.2712 −5.8630 4.5307 2.8000 0.00
FGF2- suramin No binding – – – – – – – –
VEGF-scopoletin −11 0.20 −3.29 −5.60 −4.0682 −4.90 3.08 2.80 0.00
VEGF-suramin −16 0.04 −28.12 −19.43 −16.55 −10.94 5.0047 8.4000 0.00

2000 mg/kg, thus the LD50 value of scopoletin for oral toxicity is consid-
ered to be N 2000 mg/kg.
Scopoletin inhibits sprouting of microvessels in rat aortic explants
Results of the rat aortic ring assay demonstrated that scopoletin
has significant anti-angiogenic activity. Enormous outgrowth of
microvessels was recorded in the vehicle-treated (negative control)
aortic explants (Fig. 2A). In contrast, scopoletin inhibited sprouting of
microvessels (Fig. 2B–E) at the IC50 of 0.06 μM. The standard reference,
suramin, also demonstrated a significant inhibitory effect on
microvessel formation from the rat aorta (Fig. 2F) at the IC50 of
0.22 μM. All tested doses of scopoletin affected the length and density
of microvessels in aortic explants. Fig. 2G illustrates the dose-
dependent inhibitory effect of scopoletin on sprouting of microvessels
in rat aortic explants.
Scopoletin selectively inhibits proliferation of HUVECs
To characterize the mechanism of scopoletin's inhibitory action
on sprouting of aortic microvessels, a series of in vitro angiogenesis
assays were conducted. Formation of blood vessels primarily de-
pends on proliferation of endothelial cells, thus scopoletin was test-
ed for its effect on proliferation of HUVECs. The results showed that
scopoletin possesses significant (p b 0.01) antiproliferative ability
toward endothelial cell proliferation with an IC50 of 0.73 μM. Howev-
er, the antiproliferative effect of scopoletin was poor on other cell
types (Thani et al., 2010). A study of Pan et al. (2009) reported that
scopoletin shows cytotoxicity against human endothelial cells only
at higher concentration and therefore the study could not estimate
the IC50 value of scopoletin on the cells. However, contrary to the
findings of Pan et al. (2009) the results of the present study revealed
that scopoletin possesses a significant cytotoxicity with low IC50
value.

Fig. 6. Necrotic changes in tumor histology and evidence of the anti-angiogenic effect of scopoletin. At the termination of the study, H&E stained tumor sections were examined. A: Tumor
section of a representative animal in the negative control group stained with H&E (original magnification 20×). The photomicrographic image of the tumor section is fully composed of
viable tumor cells (indicated by “t”), a large number of blood vessels (arrows), and virtually no necrosis. B: Magnified (40×) tumor section of the negative control shows well-organized,
densely packed, hexagonal shaped viable tumor cells (t) and blood vessels (arrows). C: Significant changes in tumor histology were noted in immunocompromised animals treated with
scopoletin (100 mg/kg), such as loss of the compact arrangement of tumor cells, and moderate to severe necrotic regions (N) were visible all over the tumor section. Reduced tumor
vasculature was observed as a significantly number of reduced blood vessels (arrows) (original magnification 20×). D: Higher magnification (40×) of a section of the tumor harvested
from a scopoletin (100 mg/kg)-treated animal showing clear evidence of necrosis (N) caused by the treatment. E: A representative tumor from the scopoletin (200 mg/kg)-treated
group (original magnification 20×). The photomicrographic image of the section shows peripheral necrosis, which is evident from the patches of viable tumor cells (t) surrounded by
pink colored dead tissue (necrotic region, N). F: Magnified image (40 ×) of the tumor section show the antitumorigenic and anti-angiogenic effects of scopoletin (200 mg/kg)
treatment. In this image, the peripheral necrotic region can be seen clearly as a small area of live tumor tissue surrounded by dead tissue. A single blood vessel (arrow) can also be
seen at the center of the live tumor tissue area. G: Tumor section (20×) showing the effect of imatinib (100 mg/kg) treatment. Significant necrotic damage can be observed due to the
treatment. H: Magnified (40×) photomicrographic image of the tumor section shows the antitumorigenic effect of imatinib (100 mg/kg). I: Graphical representation of the inhibitory
effect of scopoletin on the mean blood vessel count in HCT-116 tumor sections (*p b 0.05, n = 10, values are mean ± SEM of 10 low power microscopic fields). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)
26 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33

Table 2
Summary of the docking scores and reported binding affinities of scopoletin and suramin with ERK1, VEGF, and FGF2 in the docking analysis using the SYBYL-X Surflex scoring functions (A
= total scores; B = DScore; C = PMF score; D = GScore, E = ChemScore; F = CSCORE; RMSD b 0.5); (C) GOLD Genetic Algorithm Scoring functions (A = gold scores; B = chem score;
RMSD b 0.5).

Receptor–ligand complex Total Crash Polar DScore PMF GScore ChemScore CSCORE COMFA COMSIA COMSIA
(SYBYL) score score STERIC ELECTROSTATIC

ERK1-suramin −0.64 −3.54 6.03 −700.36 −120.47 268.78 −34.74 5 206 7.62 1.01
ERK1-scopoletin 2.02 −0.15 1.69 −417.18 −49.18 5.18 −13.37 4 70 9.59 1.45
FGF2-scopoletin −8.00 −8.10 0 −664.27 108.76 −9.12 −27.48 5 37 8.89 1.35
VEGF-scopoletin 4.17 −0.53 3.30 −53.37 −50.82 −26.75 −14.68 5 35 8.79 1.51
VEGF-suramin 1.90 −3.62 5.63 −158.30 −120.13 226.38 −33.81 5 208 8.33 1.084
FGF2-suramin – – – – – – – No binding – – –

Scopoletin inhibits matrigel induced vasculature in nude mice pellets was clearly evident in untreated mice (Fig. 3A and B). In contrast,
scopoletin (100 mg/kg) treatment significantly (p b 0.05) reduced the
Seven days after implantation of matrigel plugs supplemented with neovascularization process, which was evident from the H&E stained
HCT-116 cells, the formation of hemorrhagic lesions in the matrigel cross-sections of the excised matrigel implants (Fig. 3C and D).

Fig. 8. Visualization of ligands and protein interaction profile. Surface visualization of proteins and active site residue interactions of protein and hydrophobic interactions are shown in the
active pockets of the ligands: A: scopoletin 2D view. B: suramin 2D view. C: FGF2 (cartoon pattern)-scopoletin (ball-stick), SYBYL. D: ERK1 (cartoon pattern)-scopoletin (ball-stick), SYBYL.
E: VEGF (wareframe)-suramin (ball-stick), SYBYL. F: cartoon VEGF-suramin, SYBYL.
 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33 27

Microscopic examination of the blood vessels from the matrigel
plug sections showed that treatment with the high dose (200 mg/kg)
of scopoletin caused strong inhibition of vascularization in matrigel
plugs compared to the untreated control (Fig. 3E and F). The
antineovascularization effect of scopoletin can be compared with that
of the standard reference, imatinib (100 mg/kg). Fig. 3G and H shows
the images of matrigel sections depicting the potent anti-angiogenic ef-
fect of imatinib.
The mean blood vessel count of 10 low power fields in matrigel
sections from representative groups is shown in a graphical representa-
tion in Fig. 3I. The graph shows that the mean blood vessel count in the
negative control group was 106 ± 8 and that treatment with scopoletin
significantly (p b 0.01) inhibited formation of new blood vessels in the
matrigel implants, as the total mean count of blood vessels was 38 ±
4 and 10 ± 2 at 100 and 200 mg/kg, respectively. The value from
matrigel implants from mice treated with imatinib was 14 ± 3.
Scopoletin inhibits in vivo tumor vasculature in the xenograft model
To evaluate in vivo inhibitory effect of scopoletin on tumor vascula-
ture, xenografts of HCT-116 cells were implanted into immunocompro-
mised mice. Fig. 4A shows the representative animals from the negative
control bearing enlarged tumors. Fig. 4B–D depicts the dose-dependent
suppression of growth of the tumor xenograft in scopoletin-treated
mice. All tested doses of scopoletin displayed a remarkable suppression
of HCT-116 tumor growth compared to that of the vehicle-treated con-
trol. Scopoletin displayed 33.01, 34.4, and 94.7% inhibition at 50, 100,
and 200 mg/kg doses, respectively. These results are comparable to
that of imatinib (Fig. 4E), which had a potent antitumor effect
with 84.3% inhibition at 100 mg/kg. Morphological investigation of the
harvested tumors demonstrated a remarkable inhibitory effect of
scopoletin on tumor vasculature (Fig. 4F). The density of blood vessels
in the tumors collected from the treated animals was drastically

Fig. 9. Visualization of ligands and protein interaction profile. Surface visualization of proteins and active site residue interactions of protein and hydrophobic interactions are shown in the
active pockets of the ligands: A: ERK1-scopoletin surface, LeadIT. B: ERK1-scopoletin active site residue interaction, LeadIT. C: VEGF-suramin surface, LeadIT. D: VEGF-suramin active site
residue interaction, LeadIT.
28 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33

reduced compared to that of the tumors collected from the negative
control group. Fig. 5A shows the tumor growth profile in the respective
test groups. The average body weight for the respective groups is shown
in Fig. 5B.
Histological findings
Photomicrographs of all tumor sections excised from the mice were
stained with H&E. The sections were examined to quantify the blood
vessels and viable and necrotic tissue. Histological sections of the tu-
mors from negative control mice (Fig. 6A) were full of growing tissue
with plenty of viable cells arranged compactly and resembling the
vigorous proliferation of cancerous cells. Higher magnification micro-
scopic evaluation revealed that the viable tissue was composed of highly
compact sheets of polygonal-shaped cells with prominent nuclei and
numerous blood vessels (arrows in Fig. 6B). Either little or no necrotic
areas were observed in the tissue sections of untreated mice (Fig. 6B).
The tumors in the scopoletin treatments showed strong evidence of
an anti-angiogenic effect (Fig. 6D–F). Due to reduced blood and oxygen
supply, the tumors in scopoletin-treated animals exhibited loss of
compactness of the viable cells with moderate to severe necrosis. The
necrotic areas with reduced density of viable cells are visible as pink
colored regions in the tumor sections. Small regions of viable tissue
with cells can be seen in the treated tumor sections (Fig. 6D). Treat-
ment with imatinib (Fig. 6G–H) resulted in significant (p b 0.01)
antitumorigenic activity and reduction in blood vessel density
compared to the control. The mean blood vessel counts of 10 different
low power microscopic fields (Fig. 6I) in the tumor tissues from
scopoletin-treated mice were 24 ± 6 and 11 ± 2 for the 100 and
200 mg/kg doses, respectively. Negative control mice had a considerably
higher number of blood vessels (84 ± 11).
Immunohistochemical findings
Immunohistochemistry of blood-vessels is widely used in different
areas of ongoing biomedical research, particularly within the scope of
tumor angiogenesis research. The techniques are based on immunohis-
tochemical labeling of the endothelium by antibodies directed against
pan-endothelial markers such as von Willebrand factor, CD31, and
CD34, followed by light microscopic evaluation. The qualitative assess-
ment of the microvascular structures of the tissue leads to study the ex-
tent and nature of vasculature. CD34 is an antigen located in

Fig. 10. Visualization of ligands and protein interaction profile. Surface visualization of proteins and active site residue interactions of protein and hydrophobic interactions are shown in
the active pockets of the ligands: A: ERK1-suramin surface, LeadIT. B: ERK1-suramin active site residue interaction, LeadIT.
 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33 29

hematopoietic progenitor cells and endothelial cells. Anti-CD34 anti-
body is a highly sensitive marker for endothelial cell differentiation
and has also been used as a marker for vascular tumors (Vieira et al.,
2005). In the present study, the tumor sections were subjected to im-
munohistochemical staining using CD34 antibody to locate the tumor
vasculature. The findings revealed that the tumor sections of negative
control showed extensive vasculature with well-established networks
of blood vessels (arrows in Fig. 7A). Thus, the cellular architecture in
negative control tumors found to be compact with live cells (Fig. 7A).
Whereas, the positive control group treated with imatinib, showed sig-
nificant inhibition of vasculature (Fig. 7B) and displayed necrosis (N).
Similarly, scopoletin (100 mg/kg) exhibited enormous necrotic tissue
(N) and reduced vascularization (Fig. 7C), while at higher dose
(200 mg/kg), scopoletin demonstrated drastic inhibitory effect on
tumor vascularization and necrosis (Fig. 7D).
Additionally, the antivascular property of scopoletin was further
confirmed by immunofluorescence staining with NG2-positive
areas in the histological sections. NG2 is a chondroitin sulfate proteo-
glycan usually used to identify pericytes in tissue sections. It is
expressed on the surface of nascent pericytes during the early stages
of vasculogenic and angiogenic processes. Studies have reported the
role of tumor pericytes in promoting endothelial cells proliferation,
differentiation and promoting the tumor vasculature through a vari-
ety of signaling networks (Raza et al., 2010). Findings of the present
study showed that, in the negative control group (Fig. 7E), the area of
NG2-positive pericytes was abundant indicating the enriched vascu-
lature in the tumor tissue. Whereas, treatment with the standard ref-
erence drug, imatinib demonstrated reduction in the fluorescent
signal (Fig. 7F). Similarly, the treatment with scopoletin revealed
dose dependent antivascular effect in the immunohistological findings,

Fig. 11. Visualization of ligands and protein interaction profile. Surface visualization of proteins and active site residue interactions of protein and hydrophobic interactions are shown in
the active pockets of the ligands: A: FGF2-scopoletin surface, LeadIT. B: FGF2-scopoletin active site residue interaction, LeadIT.
30 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33

as the area of NG2-positive pericytes was reduced with increasing the
dose of scopoletin (Fig. 7G and H).
Scopoletin has potential binding efficiency toward ERK1, FGF2, and VEGF
The docking study results revealed that scopoletin displayed
more efficient activity than suramin. When the LeadIT FlexX meth-
od was used, scopoletin showed significant binding affinity toward
ERK1, FGF2, and VEGF with docked energies (ΔG) of − 19, − 14, and
− 11 Kcal/mol, respectively (Table 1). The ligand efficiencies
calculated upon docking of scopoletin with ERK1, FGF2, and VEGF
were 0.32, 0.23, and 0.20 Kcal/mol per heavy atom (Table 1).
These results can be compared with those of the standard reference,
suramin. Table 1 also lists parameters such as match score, lipophil-
ic area score, ambiguous area score, and receptor–ligand interac-
tion score used in the comparative evaluation of scopoletin and
suramin.
Similar results were observed when the SYBYL Surflex dock method
was used. Table 2 illustrates the potent docking efficiency of scopoletin
with all tested pro-angiogenic ligands.

Fig. 12. Visualization of ligands and protein interaction profile. Surface visualization of proteins and active site residue interactions of protein and hydrophobic interactions are shown in
the active pockets of the ligands: A: VEGF-scopoletin surface, LeadIT. B: VEGF-scopoletin active site residue interaction, LeadIT.
 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33
Figs. 8–12 illustrate the amino acids (Leu124, Met125, Ala69, Val56,
Leu173, and ll48) in the active pockets of the ligands (FGF2, ERK1 and
VEGF) displayed strong interactions with the phenyl group of
scopoletin, which indicates that the binding cavity of ERK1 might be a
good fit for scopoletin. The analysis revealed that scopoletin was more
significantly enfolded in the entire active pocket of the ligand compared
to suramin (Figs. 8–10). The size of suramin might be the reason that it
does not properly fit within any of the cavities of ERK1.
The overall results reveal that the docked energy and ligand binding
efficiency of scopoletin were more pronounced than those of the stan-
dard reference, suramin. The high molecular weight and bulky size of
suramin may hamper effective binding and affinity with the ligands,
particularly FGF2. This would explain the lack of interaction between
suramin and FGF2 in both docking analyses (Fig. 13).
In the present study scopoletin demonstrated potent inhibitory ef-
fect on sprouting of microvessels in rat aortic explants. Rat aortic ring
assay is a 3-D quantitative ex vivo angiogenesis model, where develop-
ment of microvessels takes place via the main features of neovasculari-
zation similar to that observed in vivo. The microvessels that grow out
from aortic explants recruit chiefly the endothelial cells through prolif-
eration, migration and differentiation of the cells. General cytotoxic
compounds affect majority of the cells in aortic tissue and thereby kill
the entire aortic tissue, whereas the antiangiogenic compounds selec-
tively target the endothelial functions. In the present study, in vitro pro-
liferation test showed that scopoletin exhibited selective toxicity on
endothelial cells which was evident through the highest selectivity
index on other cell types (Kurdekar et al., 2014; Thani et al., 2010).
The selective cytotoxicity of scopoletin on endothelial cells could be
the principle reason for the anti-angiogenic effect on the rat aorta. The
Fig. 13. Graphical representation of the estimated binding activity (A) and ligand
efficiency (B) of scopoletin and suramin with ERK1, FGF2, and VEGF in LeadIT.
31
endothelial functions are highly controlled and regulated by the angio-
genic mitogens. Therefore, anti-angiogenic compounds either directly
interfere with endothelial cell functions or block the mitogen functions.
It has been reported that scopoletin possesses strong ability to inhibit
the vital functions of endothelial cells, such as tube formation, differen-
tiation and migration (Pan et al., 2009). In the present study, an in vivo
physiologic model of angiogenesis was employed using matrigel, an ex-
tract composed of angiogenesis-inducing compounds. Assessment of
angiogenesis in the matrigel implants was determined by scoring the
histological sections for vascular density. The results revealed that
scopoletin showed clear evidence of inhibition of vascularization in
matrigel plugs implanted in the treated mice.
To verify that the anti-angiogenic property of scopoletin can be use-
ful in suppression of tumor growth, a human tumor xenograft model
was employed. The in vivo tumor model demonstrated a clear inhibito-
ry effect of scopoletin on the tumorigenesis process in tumor-bearing
mice. In addition, the findings provided clear evidence of the anti-
angiogenic property of scopoletin, as it significantly suppressed the vas-
cular supply to the growing tumor. It is well known that during tumor-
igenesis the endothelial cells divide more rapidly and spread much
more quickly than they would during the normal course of blood vessel
formation (Maeshima et al., 2002). Several recent reports recommend-
ed further studies of scopoletin to investigate its anticancer properties
(Liu et al., 2012; Liu et al., 2001; Zhao et al., 2014). These preliminary
studies reported the in vitro anticancer potential of scopoletin and dem-
onstrated that it could induce cell cycle arrest at the S and G2 phases and
activate apoptosis in tumor cells via the caspase pathway (Liu et al.,
2012; Liu et al., 2001; Zhao et al., 2014).
Malignant tumors require blood vessels for growth, and the main
aim of antiangiogenic therapy is to abolish the pathological vasculature
of tumor. However, recent data indicate that a suppression in tumor
vessels induced by antiangiogenic treatment using antiangiogenic
agents results in an inhibition of tumor growth but on the other hand
it permits tumor invasiveness (Pàez-Ribes et al., 2009). This increase
in tumor invasiveness is most probably due to increased hypoxia within
the tumor during antiangiogenic therapy (Rapisarda and Melillo, 2009).
Such tumor vessels are characterized by a leaky, disorganized and ab-
normal morphology (Mazzone et al., 2009). Subsequently, these patho-
logical tumor vessels impair blood flow into the tumor leading to a
hypoxic microenvironment rendering the tumor unresponsive to tradi-
tional chemotherapeutic agents. In addition, this abnormal phenotype
of tumor vasculature supports tumor progression and resistance to
treatment. However, Several studies have shown that antiangiogenic
agents prune the pathological vasculature and thus organize the con-
trolled and orderly arranged vasculature which helps to maintain a ho-
mogenous blood flow to the tumor tissue, and eventually leads to a
reduction in tumor hypoxia (Campbell et al., 2011; Russell et al.,
2015). Angiogenesis is an intricate process of signal transduction that
involves several mitogens (VEGF and FGF) and receptor protein kinases
(ERK1/2). Therefore, VEGF, FGF, and ERK1/2 are considered to be the
chief angiogenic factors that are essentially required for initiation and
prolongation of vascularization (Meadows et al., 2001). In the present
study, findings of the molecular docking analyses revealed that
scopoletin has the potential to interact with ERK1, VEGF-A, and FGF-2
and modulate their functions. These results support the findings of pre-
vious studies that reported the antagonistic effect of scopoletin toward
FGF-2 and ERK1/2 (Pan et al., 2009; Pan et al., 2011). However, Pan
et al. (2009) reported that scopoletin inhibits the phosphorylation of
ERK1/2, while the present study reported the binding and blocking effi-
ciencies of scopoletin toward ERK1/2.
In conclusion, the present work provides strong supporting evidence
that scopoletin possesses strong anti-angiogenesis activity in animal
models. Further, the inhibitory effect of scopoletin was exploited to
demonstrate its remarkable antitumorigenic activity in a human
tumor xenograft model. Histological and immunohistochemical analy-
sis excised tumors revealed that scopoletin displayed drastic
32 Y.M. Tabana et al. / Microvascular Research 107 (2016) 17–33
suppression of tumor vasculature. In addition, the computational simu-
lation models showed that scopoletin has strong binding efficiency with
the angiogenic factors ERK1, VEGF-A, and FGF-2 and that it is configura-
tionally compatible with the active sites of the tested angiogenic li-
gands. Thus, it can be concluded that the anti-angiogenic effect of
scopoletin functions by regulating ERK1, VEGF-A, and FGF-2 signaling
pathways.
Conflict of interest
The authors declare no conflict of interest.
Submission declaration
The authors declare that the work described has not been published
previously.
Author contributions
Tabana, Amin, Hassan and Ahamed designed the experiments.
Tabana and Hassan carried out the isolation of the compound. Tabana
and Dahham conducted in vitro biological experiments. Iqbal per-
formed the structural elucidation of the compound. Tabana and Saeed
carried out HPLC analysis. Khan conducted the molecular docking
study. Tabana and Hassan carried out the in vivo study. Chern Ein Oon
conducted immunohistochemistry of tumor vessel analysis. Doblin
Sandai help in the revision, Tabana, Ahamed and Dahham interpreted
the results. Tabana, Ahamed, Amin and Aman drafted the manuscript.
All the authors read and approved the final manuscript.
Acknowledgments
The first author would like to sincerely acknowledge University
Sains Malaysia (USM) for postgraduate fellowship. All authors acknowl-
edge the Ministry of Agriculture, Malaysia under NRGS grant no: 304/
PFARMASI/650583/K123. The authors also wish to acknowledge
Universiti Sains Malaysia (USM) for research university team grant no.
1001/PFARMASI/851001. The authors would like to acknowledge
Cheng Wei Kang for help in optimization of antibody staining for CD34.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.mvr.2016.04.009.
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