Natural Product Research

Formerly Natural Product Letters

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A new steroid glycoside from Begonia sp.: cytotoxic

activity and docking studies

Muhammad Sulaiman Zubair, Walied M. Alarif, Mohamed A. Ghandourah &

Syariful Anam

To cite this article: Muhammad Sulaiman Zubair, Walied M. Alarif, Mohamed A. Ghandourah &
Syariful Anam (2019): A new steroid glycoside from Begonia�sp.: cytotoxic activity and docking
studies, Natural Product Research, DOI: 10.1080/14786419.2019.1669026

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1669026

A new steroid glycoside from Begonia sp.: cytotoxic

activity and docking studies
Muhammad Sulaiman Zubaira, Walied M. Alarifb, Mohamed A. Ghandourahb and
Syariful Anama
a
Faculty of Science, Department of Pharmacy, Tadulako University, Palu, Indonesia; bFaculty of
Marine Science, Department of Marine Chemistry, King Abdul Aziz University, Jeddah, Saudi Arabia

ABSTRACT ARTICLE HISTORY

Chemical investigation on the ethyl acetate extract of the aerial Received 8 July 2019
parts of Begonia sp. afforded a new steroid glycoside, Accepted 4 September 2019
9(11)a,16(17)a-dioxirane-20,25-dihydroxy-b-sitosterol-3-O-b-gluco-
pyranoside (1) along with a known steroidal glycoside, b-sitos- KEYWORDS
terol-3-O-b-D-glucopyranoside (2). The Chemical structures were Begonia sp.; steroid
glycoside; spectroscopy;
elucidated by 1D and 2D NMR and mass spectroscopic analysis. molecular docking;
Cytotoxicity against four different cancer cell lines (HeLa, T47D, breast cancer
WiDr and Vero) was assessed. Compound 1 was more potent and
selective against breast cancer cell line (T47D) than other cell lines
with an IC50 value of 0.16 mg/mL. Further docking study of 1
exhibited the preference of molecule to bind in the epidermal
growth factor receptor tyrosine kinase (EGFR-TK) binding pockets
with docking scores of
97.8800 (PLANTS) and
3.56 kcal/mol
(AutoDock 4.2.6).

CONTACT Muhammad Sulaiman Zubair [email protected]; [email protected]

Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1669026.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 M. S. ZUBAIR ET AL.

29
21 OH 28

18 26
20 22 25
12 24
11 O 23
OH
19 13 17
O 27
16
1 9 14 15
10 8
3
O 5 O
Glc 6 Glc
1 2

Figure 1. Structure of steroid glycosides 1 and 2.

1. Introduction
The Begonia (Begoniaceae) is one of the largest genera of the flowering plants that
has leaves and flowers of beautiful shapes and various colours (Hartutiningsih et al.
2011). Previous phytochemical studies on 10 Begonia species revealed the presence of
flavonoids, triterpenoids, steroids, glycosides and alkaloids (Zubair et al. 2016).
Reported biological activities of these plants include antiviral and cytotoxic activities
(Doskotch and Hufford 1970; Frei et al. 1998; Wu et al. 2004), antihyperglycaemic
(Pandikumar et al. 2009), antibacterial (Ramesh et al. 2002; Solomon and Johnson
2012), and antioxidant properties (Velusamy and Veerabahu 2012).
Benalu Batu (Begonia sp.), has been used by Wana tribe in Morowali, Central
Sulawesi, Indonesia, to treat several diseases, including cancer. Our previous study
established the anticancer activity of the methanol extract of this plant against breast
and cervical cancer cell lines (T47D and HeLa cells) (Anam et al. 2014). Therefore, the
aim of the present study was to further investigate the phytochemicals responsible for
its anticancer property.
A preliminary docking study to early identify the potential anticancer compounds
by using database of secondary metabolites reported from the genus of Begonia was
performed. The docking result prompted us for the isolation of glycosides from the
Begonia plant extract (Zubair et al. 2016). Previously, 2-O-b-glucopyranosyl-cucurbitacin
D was reported and it showed potent inhibitory activity against colon cancer cells,
HCT-116 (Zubair et al. Forthcoming). Further investigation on the ethyl acetate fraction
led to the isolation of a new steroid glycoside, 9(11)a-16(17)a-dioxirane, 20,25-dihy-
droxy-b-sitosterol-3-O-b-glucopyranoside (1) (Figure 1) and the known glycoside-
b-sitosterol-3-O-b-D-glucopyranoside (2). Cytotoxic activity of the isolated new com-
pound was assessed against four different cell lines. Molecular docking study of the
new compound is also discussed.

2. Results and discussion

Compound 1 was isolated as an amorphous white powder. A reddish spot appeared
on TLC plate after spraying with p-anisaldehyde-sulphuric acid suggesting the com-
pound being a terpenoid/steroid. The Liebermann-Burchard test gave a green colour
supporting the steroidal nature of the compound. Moreover, the analysis of HRESI-MS
 NATURAL PRODUCT RESEARCH 3

at m/z 637.3840 [M þ H]þ supporting the molecular structure of glycosidal steroid with
the molecular formula of C35H56O10.
The 13C NMR spectrum (Supplementary Table S1) of compound 1 exhibited 35 sig-
nals of carbon atoms which were categorised by DEPT into 6 methyls, 10 methylenes,
12 methines and 7 quaternary carbons. Three of the eight degrees of unsaturation as
calculated from its molecular formula were attributed to one carbon–carbon double
bond (dC 139.9 and 120.1 ppm) assigned for D5 sterol and two oxirane rings repre-
sented by signals resonating at dC 51.3, 57.9, 69.2 and 76.4 ppm. The presence of glu-
copyranosyl moiety was supported by the resonance of the anomeric carbon at dC
103.3, together with four oxymethines at dC 79.4, 77.2, 76.2, 73.7 ppm and one methy-
lene carbon at dC 62.1 ppm. Thus, the molecule was established to be pentacyclic in
which one was for a glucose unit. Additional two oxygenated quaternary carbons
could be deduced from two signals at dC 69.9 and 76.4 ppm.
The 1H NMR (Supplementary Table S1) revealed the skeleton of a stigmasterol par-
tial structure, proven by the presence of six methyl signals characteristic for steroids
resonating at dH 0.67 and 1.06 ppm assigned for two methyls at C-18 and C-19, one
triplet for a methyl at dH 0.89 ppm for C-29 and three downfield signals corresponding
to methyl protons resonating at dH 1.15, 1.28 and 1.29 ppm, as assignable to methyl
groups attached to oxygenated carbons. A broad singlet at dH 5.76 was assigned for
H-6. Two broad singlets at dH 3.08 and 3.59 ppm were characteristic for protons of
two oxirane rings. The shape and position of the signal observed at dH 4.67was char-
acteristic for 3-hydroxylated steroids (John Goad and Akihisa 1997) suggesting the glu-
cose unit was linked to the C-3. The monosaccharide moiety (glucose) was confirmed
from the anomeric proton signal at dH 4.37 (dd, 1.2, 3.0), together with five signals of
oxygenated carbons in the range 3.42–3.76 ppm. From the 1H-1H COSY correlation, a
spin system between H-C3 and H2-C2, H2-C4 and proton anomeric H-C10 was
observed, along with the presence of cross-peaks between H2-C2 and H2-C1. Long-
range C-H correlation (HMBC) observed between Me-19 (dH 1.15, br s) and C-2 (29.7),
C-4 (34.3), C-10 (33.7) and C-8 (34.0) established the closing of ring A, connected by
glycosidic linkage at C-3 (73.7) position. The position of first oxirane ring at C-9 and C-
11 was proven by the 1H-1H COSY correlation between H-C11 and H2-C12, also sup-
ported by the long-range C-H correlation (HMBC) between Me-18 (dH 0.67 ppm, s) and
C-13 (48.5), C-12 (47.3), C-11 (57.9) and C-16 (51.3), suggesting the position of the
second oxirane ring at C-16 and C-17 as well. The connection between steroid skel-
eton and side chain was observed by HMBC long-range correlation between Me-21
(dH 1.06 ppm, s) and C-18 (dC 18.8).The consecutive protons were observed from H2-C-
22, H2-C-23, H-C-24, H2-C-28 and H3-C-29. Long-range C-H correlation (HMBC) was
observed between Me-21 and C-23 (18.8), between Me-29 and C-28 (22.7), C-23 (18.8),
and C-22 (34.7), between Me-26/Me-27 and C-24 (51.2), C-23 (18.8), C-22 (34.7), C-28
(22.7), C-29 (14.1) (Supplementary Figure S2). The position of six hydroxyl groups (four
for the sugar and the remaining for steroid side chain attached to C-20 and C-25), was
determined by examining the chemical shift from 1H and 13C spectral data and sup-
ported by 1H-1H COSY and 1H-13C HMBC spectral data. The relatively large coupling
constant value of H-11 (J ¼ 6.0 Hz) implied axial–equatorial orientation (a-position) of
H-11, hence the C-9-C-11 oxirane ring occupies b-position (Sanap et al. 2010).
4 M. S. ZUBAIR ET AL.

Meanwhile, the second oxirane ring occupies b-position owing to the biogenetic rule
of these steroids (John Goad and Akihisa 1997). Based on above data and database
searching from science finder indicated compound 1 was a new steroid and it was
assigned as 9(11)a,16(17)b-dioxirane-20,25-dihydroxy-b-sitosterol-3-O-b-glucopyrano-
side (Figure 1). Compound 2 was identified by comparison with the published data for
b-sitosterol-3-O-b-D-glucopyranoside (Mizushina et al. 2006; Khatun et al. 2012).
Compounds 1 and 2 were tested for cytotoxic activity towards four different cancer
cell lines (T47D, HeLa, WiDr and Vero) (Supplementary Table S2). Compound 1 showed
higher cytotoxicity only against breast cancer cells (T47D) than compound 2 with the
IC50 value of 0.16 mg/mL. It also possessed high selectivity as this compound did not
show toxicity to Vero cell line. Based on our previous study where methanol extract of
Begonia sp. had high inhibition on breast cancer cells proliferation (T47D cell lines), we
could conclude that compound 1 might be the bioactive compound that was respon-
sible for this specific and selective activity against breast cancer cells.
Further molecular docking study was performed for compound 1 on EGFR-TK pro-
tein targets. Compound 1 was found to have better interaction on EGFR-TK receptors
than native ligand erlotinib. The binding mode of compound 1 on EGFR-TK revealed
the formation of hydrogen bonds with the residues Lys 721 and Asp 831 with the
docking energy score of
97.880, which was higher than native ligand erlotinib
(Supplementary Table S3). The skeleton of steroid was found to insert on the hydro-
phobic pocket on EGFR-TK affording a hydrophobic interaction and the presence of
sugar moiety increase the docking energy score in which the hydroxyl group of sugar
can form hydrogen form with Lys 721, which was located in the phosphate-binding
region along the sugar pocket. Hydrogen bonding was also observed between
hydroxyl group of sugar with Asp 831 which is located in helix aC (Supplementary
Figure S3). It was clearly shown that the docking energy score of compound 1 had
lower than native ligand erlotinib affording the high affinity and selectivity only on
EGFR-TK receptor that responsible for breast cancer cell lines. This might be because
of the presence of two extra oxirane rings and two extra hydroxyl groups attached to
side chain of steroid skeleton prompting for further investigation for the in vitro mech-
anism of compound 1 as a selective and specific agent for breast cancer.

3. Experimental
3.1. General
TLC aluminium sheets 20  20 cm silica gel 60 F254 was used. Silica gel 60 (Merck) for
vacuum liquid column chromatography (230–400 mesh) was used. Pre-coated TLC
glass plates SIL G-25 UV254, 0.25 mm silica gel and Sephadex LH-20 (Sigma, St. Louis,
MO, USA) were used for isolation and purification of the compounds. Spots on TLC
were visualised by using spraying reagent of methanol-sulphuric acid and p-anisalde-
hyde-sulphuric acid for terpenoid/steroid detection. Nuclear magnetic resonance
(NMR) was recorded for 1D and 2D on AvanceIII Bruker WM 600 MHz for 1H and
150 MHz for 13C. Chemical shifts are given d (ppm) relative to TMS as internal standard
and deuterated chloroform was used as a solvent.
 NATURAL PRODUCT RESEARCH 5

The aerial parts of Begonia sp., growing in the mountain, were collected from
Morowali, Central Sulawesi, on April 2014. The plant species were identified at
Biodiversity Unit, Tadulako University, Central Sulawesi, Indonesia and deposited as a
dried specimen (BSP 00020414) at Phytochemistry Laboratory, Department of
Pharmacy, Tadulako University.

3.2. Extraction and isolation

The aerial parts were shade-dried. The dried sample (250 g) was extracted with ethanol
96% (3  0.8 L, 24 h for each batch) at room temperature. The solvent was removed in
vacuo until reached a residue (15 g) referred to as crude ethanol extract. The ethanol
extract (10 g) was suspended in water and successively partitioned with n-hexane and
ethyl acetate to obtain n-hexane and ethyl acetate soluble fraction. The ethyl acetate
fraction (3 g) was chromatographed on silica gel (60–120 mesh) and the packed col-
umn was eluted by employing gradient system of solvent of n-hexane 100%, n-hex-
ane/ethyl acetate mixture, followed by ethyl acetate/methanol mixture and methanol
100%. A total of 40 fractions of 50 mL each were collected. Similar fractions were col-
lected together according to TLC pattern in eight fractions (F1–F8). The fraction 3
(eluted by ethyl acetate:methanol (4:1, 60 mg)), which was found to contain steroidal
skeleton, was continued to further isolation using successive preparative TLC with
chloroform:methanol (1:9) as a mobile phase. The first band with Rf ¼ 0.92 (purple col-
our with sulphuric acid-methanol) was taken to give compound 2 as amorphous white
powder (23 mg). The fraction 4 (eluted by ethyl acetate:methanol (4:1, 55 mg) was also
continued to further isolation using successive preparative TLC preparative with chlor-
oform:methanol (2:8). The first band with Rf ¼ 0.36(purple colour with sulphuric acid-
methanol) was taken to give compound 1 as amorphous white powder (10 mg).
9(11)a,16(17)a-dioxirane-20,25-dihydroxy-b-sitosterol-3-O-b-glucopyranoside (1).
Amorphous white powder (10 mg, 0.04% of dry weight); HRESI-MS m/z 637.3840
[M þ H]þ (calculated 637.3873 for C35H56O10); H NMR (CDCl3, 600 MHz): d ¼ 1.34 (2 H,
m, H-1), 1.26 (2 H, m, H-2), 4.67–4.70 (1 H, m, H-3), 2.83–2.84 (1 H, dd, J ¼ 1.2, 7.8 Hz, H-
4), 5.76 (1 H, m, H-6), 1.26 (2 H, m, H-7), 2.32 (1 H, m, H-8), 3.08–3.10 (1 H, t, J ¼ 6 Hz, H-
11), 2.56–2.57 (1 H, d, J ¼ 10.2 Hz, H-12a), 3.29–3.30 (1 H, d, J ¼ 10.2 Hz, H-12b),
2.00–2.03 (1 H, dd, J ¼ 4.8, 13.2, H-14), 1.22 (2 H, m, H-15), 3.60 (1 H, m, H-16), 0.67 (3 H,
s, H-18), 1.15 (3 H, s, H-19), 1.06 (3 H, s, H-21), 1.34 (2 H, m, H-22), 1.26 (2 H, m, H-23),
1.57 (1 H, m, H-24), 1.28 (3 H, s, H-26), 1.29 (3 H, s, H-27), 1.22 (2 H, m, H-28), 0.89 (3 H,
t, J ¼ 5.4 Hz, H-29), 4.37 (1 H, dd, J ¼ 1.2, 3.0 Hz, H-10 ), 3.58 (1 H, m, H-20 ), 3.45 (1 H, m,
H-30 ), 3.42 (1 H, m, H-40 ), 3.60 (1 H, m, H-50 ), 3.76 (2 H, m, H-60 ); 13C NMR (CDCl3,
150 MHz): d ¼ 23.9 (CH2, C-1), 29.7 (CH2, C-2), 73.7 (CH, C-3), 34.3 (CH2, C-4), 139.9 (C,
C-5), 120.1 (CH, C-6), 31.9 (CH2, C-7), 34.0 (CH, C-8), 69.2 (C, C-9), 33.7 (C, C-10), 57.9
(CH, C-11), 47.3 (CH2, C-12), 48.5 (C, C-13), 42.8 (CH, C-14), 29.4 (CH2, C-15), 51.3 (CH,
C-16), 76.4 (C, C-17), 18.8 (CH3, C-18), 19.9 (CH3, C-19), 69.9 (C, C-20), 18.1 (CH3, C-21),
34.7 (CH2, C-22), 18.8 (CH2, C-23), 51.2 (CH, C-24), 76.4 (C, C-25), 21.3 (CH3, C-26), 21.2
(CH3, C-27), 22.7 (CH2, C-28), 14.1 (CH3, C-29), 103.3 (CH, C-10 ), 76.2 (CH, C-20 ), 79.4 (CH,
C-30 ), 76.4 (CH, C-40 ), 77.2 (CH, C-50 ), 62.1 (CH2, C-60 ).
6 M. S. ZUBAIR ET AL.

3.3. Cell culture

Human ductal breast epithelial tumour cell line (T47D), human cervical cancer cell line
(HeLa), colon adenocarcinoma cell line (WiDr) and green African monkey renal epithe-
lial cell (Vero) were obtained from Laboratory of Parasitology, Faculty of Medicine,
Gadjah Mada University. Cells were maintained in RPMI-1640 medium supplemented
with 100 mg/mL streptomycin, 100 units/mL penicillin and 10% foetal bovine serum
(FBS) in 5% CO2 atmosphere at 37  C.

3.4. Cytotoxicity test

Cytotoxicity test was performed on HeLa, T47D, WiDr and Vero cancer cell lines by
MTT method. The stock samples of all compounds were diluted with RPMI-1640
medium to desired concentrations (1000 mg/mL). The final concentration of dimethyl
sulphoxide (DMSO) in each sample did not exceed 1% v/v. The cancer cells were batch
cultured for 10 days, then seeded in 96 well plates of 10  103 cells/well in fresh com-
plete growth medium in 96-well microtitre plastic plates at 37  C for 24 h under 5%
CO2 using a water-jacketed carbon dioxide incubator (Shedon.TC2323.Cornelius, OR,
USA). The medium (without serum) was added and cells were incubated either alone
(negative control) or with different concentrations of sample to give a final concentra-
tions of (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.7812, 0.3906, 0.1953 mg/mL). Cells
were suspended in RPMI-1640 medium, complemented with 5% FBS and kanamycin
(100 mg/mL) in 96-well flat-bottom microplates at 37  C under 5% CO2. After 24 h of
incubation, cells were added with 10 mL/well of MTT (5 mg/mL) and incubated 4 h in
incubator at 37  C in 5% CO2-humidified atmosphere. The reaction was stopped by
100 mL SDS (10% in HCl 0.01 N). The plate was then shaken in shaker for 10 min and
incubated overnight in the shaded room. The absorbance of each well was read at
550 nm wavelength in ELISA Reader (Biorad), using wells without cells as blanks. All
experiments were performed in triplicate. The data are represented as mean ± SD. The
effect of compounds on the growth inhibition of cancer cells was expressed as the %
cytoviability, using the following formula: % cytoviability ¼ absorbance of treated cells/
absorbance of control cells  100%.

3.5. Molecular docking

Molecular docking protocols were performed by using software combination of
SPORES, Open babel and PLANTS1.2 as described by Yuniarti et al. (2011). The pdb file
was split to the protein file and co-crystallised ligand file using the splitpdb module in
SPORES. The protein was then recognised, protonated and stored as protein.mol2,
while the co-crystallised ligand was also recognised, protonated and stored as
ligand_AQ4999_0.mol2. The co-crystallised ligand subsequently underwent conform-
ational search to find the most stable conformer from 10 seeds, followed by 1000
steps energy minimisation using obconformer module in Open Babel (O’Boyle et al.
2011). The optimised structure of co-crystallised ligand was subjected to SPORES
before submitted to the docking simulations using PLANTS1.2 (Korb et al. 2009; ten
Brink and Exner 2009). The binding site in the docking configuration file was defined
 NATURAL PRODUCT RESEARCH 7

as 5 Å from the coordinates of the location where the co-crystallised ligand was
located in the co-crystallised crystal structure. The bind module of PLANTS1.2 was
used to automatically identify the binding site. The RMSD (root mean square devi-
ation) value between the docked pose and the crystal structure pose was calculated
using rms_cur module in PyMol (Seeliger and de Groot 2010). This procedure was per-
formed iteratively 10 times. The lowest docking energy score with the RMSD < 2 Å
was chosen as selected molecular docking protocol. To confirm the docking results, an
additional docking simulation by using AutoDock 4.2.6 was performed by targeting
the whole protein (Morris et al. 2009). Preparation for receptor and ligand were per-
formed by using AutoDockTools (ADT) software packages. Gasteiger charges were
added, grid boxes was set to 54, 60 and 44 points spaced 1.0 Å, Lamarckian Genetic
Algorithm (LGA) parameters were used as follow: population size of 50, elitism of 1,
mutation rate of 0.02, crossover rate of 0.80, local search rate of 0.06, 250,000 energy
evaluations, and 100 search runs. The final docked conformations were clustered using
a cluster tolerance of 2.0 Å RMSD. The ligand pose with the lowest predicted free
binding energy was used for subsequent analysis.
The chemical structure of compound 1 was built by MarvinSketch (Chemaxon,
Dordrecht, Netherlands) (Bennett et al. 2009). The structure was protonated at pH 7.4
and saved as mrv format file. After that, the file was subjected to conformational
search to gain 10 different conformers and then saved as mol.2 format file.
Obconformer module in open babel was then used to continue the ligand preparation
by re-conformational search to find the most stable conformer from 10 seeds and suc-
cessively minimise the energy of each structure by 1000 steps. The minimised struc-
ture was then subjected to the docking molecular simulation by using PLANTS1.2 and
AutoDock 4.2.6 software. The docking score obtained was then compared to the dock-
ing score of the co-crystallised ligand erlotinib.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The authors would like to greatly acknowledge the Ministry of Research, Technology and Higher
Education, Republic of Indonesia for supporting this study through INSINAS 2015 [grant No. RD-
2015-0106].

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