Received: 3 July 2020 Revised: 26 August 2020 Accepted: 28 August 2020

DOI: 10.1002/bmc.4979

RESEARCH ARTICLE

UHPLC/GC-TOF-MS metabolomics, MTT assay, and molecular

docking studies reveal physostigmine as a new anticancer
agent from the ethyl acetate and butanol fractions of Kigelia
africana (Lam.) Benth. fruit extracts

Oladapo F. Fagbohun1 | Babatunde Olawoye2 | Adedeji N. Ademakinwa3 |

Olumayowa V. Oriyomi4 | Oladoyin S. Fagbohun5 | Olatomide A. Fadare6 |
Titus A.M. Msagati7

1
Department of Biomedical Engineering, First
Technical University, Ibadan, Nigeria Abstract
2
Department of Food Science and Technology, Kigelia africana plant is widely used as a herbal remedy in preventing the onset and
First Technical University, Ibadan, Nigeria
the treatment of cancer-related infections. With the increase in the research interest
3
Department of Physical and Chemical
Sciences, Elizade University, Ilara-Mokin, of the plant, the specific chemical compound or metabolite that confers its anticancer
Nigeria properties has not been adequately investigated. The ethyl acetate and butanol
4
Institute of Ecology, Obafemi Awolowo
fractions of the fruit extracts were evaluated by 2-(4,5-dimethylthiazol-2-yl)-
University, Ile-Ife, Nigeria
5
Department of Chemical Engineering,
3,5-diphenyl-2H-tetrazolium bromide assay against four different cell lines, with the
Obafemi Awolowo University, Ile-Ife, Nigeria ethyl acetate fraction having inhibition concentration values of 0.53 and 0.42 μM
6
Organic Chemistry Research Laboratory, against Hep G2 and HeLa cells, respectively. More than 235 phytoconstituents were
Department of Chemistry, Obafemi Awolowo
University, Ile-Ife, Nigeria profiled using UHPLC-TOF-MS, while more than 15 chemical compounds were
7
Nanotechnology and Water Sustainability identified using GC–MS from the fractions. Molecular docking studies revealed that
Research Unit, College of Science Engineering
physostigmine, fluazifop, dexamethasone, sulfisomidine, and desmethylmirtazapine
and Technology, University of South Africa
(UNISA), Johannesburg, South Africa could favorably bind at higher binding energies of –8.3, –8.6, –8.2, and –8.1 kcal/mol,
respectively, better than camptothecin with a binding energy of –7.9 kcal/mol. The
Correspondence
Oladapo F. Fagbohun, Department of results of this study showed that physostigmine interacted well with topoisomerase
Biomedical Engineering, First Technical
IIα and had a high score of pharmacokinetic prediction using absorption, distribution,
University, Ibadan, Oyo State, Nigeria.
Email: [email protected] metabolism, excretion, and toxicity profiles, thereby suggesting that drug design
using physostigmine as a base structure could serve as an alternative against the
Funding information
First Technical University, toxic side effects of doxorubicin and camptothecin.

KEYWORDS

camptothecin, doxorubicin, GC–MS, Kigelia africana, physostigmine, UHPLC-TOF-MS

1 | I N T RO D UC TI O N diseases such as diarrhea, rheumatism, psoriasis, and wounds

(Bello, Shehu, Musa, Abdullah, & Mahmud, 2016; Kaur, Shyam, &
Kigelia africana, a quintessential tradomedical plant of African origin, is Amutha, 2012; Neba, 2006). Recent research studies on the plant
widely used in herbal preparations for the treatment of numerous have shown that K. africana (Lam.) Benth. plant has antioxidant

Abbreviations: A375, human melanoma cancer line; HEK 293, human embryonic kidney cell line; HeLa, human cervical cancer cells; Hep G2, human hepatoma cancer line; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Biomedical Chromatography. 2020;e4979. wileyonlinelibrary.com/journal/bmc © 2020 John Wiley & Sons, Ltd. 1 of 18
https://doi.org/10.1002/bmc.4979
2 of 18 FAGBOHUN ET AL.

(Akanni et al., 2017; Farah, Elhussein, Khalid, & Osman, 2018), et al., 2019; Pommier, 2013; Wang, 2002). Clinically successful anti-
antibacterial (Akunyili, Houghton, & Raman, 1991; Binutu, cancer agents, such as doxorubicin and camptothecin, act through the
Adesogan, & Okogun, 1996; Fomogne-Fodjo, Van Vuuren, Ndinteh, poisoning of topoisomerases, leading to the formation of double-
Krause, & Olivier, 2014; Singh, Kumari, Singh, & Singh, 2018), antifun- strand break and ultimately replication fork arrest (Amin, Gali-
gal (Olatunji & Olubunmi, 2009; Owolabi, Omogbai, & Obasuyi, 2005), Muhtasib, Ocker, & Schhneider-stock, 2009; Delgado, Hsieh, Chan, &
antiviral (Chinsembu, 2016; Oladunmoye & Kehinde, 2011), antimalar- Hiasa, 2018; Irfan et al., 2020). These anticancer agents act by interca-
ial (Muthaura, Rukunga, Chhabra, Mungai, & Njagi, 2007; Oladele & lating with DNA and interfere in RNA production as well as DNA
Adewunmi, 2008; Zofou et al., 2011), hepatoprotective (Mülling dos metabolism. The limit in their usefulness is their main adverse effect,
Santos et al., 2014; Sachan et al., 2013), anti-inflammatory (Carey, which is known as cardiotoxicity (Thorn et al., 2011).
Babu, Venkat Rao, & Mohan, 2008; Namita, Mukesh, & Tirath, 2012), Based on the conclusion of Bello et al. (2016) regarding the
analgesic (Owolabi & Omogbai, 2007; William Carey, Venkat Rao, Ravi probing of the existential studies on the phytoconstituents and
Kumar, & Mohan, 2010), antidiarrheal (Agyare et al., 2013; Würger, pharmacological activities with the end goal of unscrambling the
McGaw, & Eloff, 2014), anti-ulcerogenic (Agyare et al., 2013), and pharmacokinetics, pharmacodynamics, contaminants, toxicity, and
antidiabetic (Khan et al., 2016; Muyenga Akapelwa, Muungo, possible clinical significance as well as the side effects of the extracts,
Prashar, & Bwalya, 2015) properties. fractions, and active ingredients in K. africana plant, this study, there-
Pharmaceutical research interests regarding the anticancer and fore, investigated the chemical compounds in the butanol and ethyl
antiproliferative activities of K. africana were fueled by traditional acetate fractions of the fruit through the use of modern omics-based
reports of the medicinal applications of the fruit for the prevention approaches such as gas chromatography time-of-flight coupled mass
and treatment of diseases such as skin neoplasms and melanoma by spectrometry (GC-TOF-MS) and ultra-high-pressure liquid chromatog-
African herbalists (Bello et al., 2016; Houghton & Jäger, 2002; raphy time-of-flight tandem mass spectrometry (UHPLC-TOF-MS),
Ochwang'i et al., 2013; Saini, Kaur, Verma, & Singh, 2008). These which can detect a wide range of chemical compounds in the plant
activities have been extensively studied in various parts of the plant. extract as well as in the soil composition. Subsequently, the study
The seed oil exhibited statistically significant suppression of human tested the fractions on human hepatoma (Hep G2), human melanoma
embryonic kidney (HEK-293) and epithelial colorectal adenocarcinoma (A375), and human cervical (HeLa) cancer cells for their antip-
(Caco2) cell growth (Chivandi, Davidson, & Erlwanger, 2011). Fouche roliferative effects and cell viability studies on human embryonic kid-
et al. (2008) in an investigation on the anticancer activity of the root ney (HEK 293) cells. Molecular docking and in silico pharmacokinetics
and leaves of the plants discovered moderate suppression of these studies were further carried out on the target protein implicated in
extracts against human melanoma (UACC62) and breast (MCF-7) cells cancer and the identified metabolites or chemical compounds of this
with a total growth inhibition of 14.90, 8.82, and 0.01 μg/mL as well study.
as 42.9, 15.0, and 8.02 μg/mL for the root and leaf extracts, respec-
tively. Several antiproliferative activities of the chemical compounds
profiled from the various parts of K. africana were determined by 2 | MATERIALS AND METHODS
2-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-2H-tetrazolium bromide
(MTT) assay against breast cancer and human melanoma cancer cells. 2.1 | Fruit materials
Ferulic acid and hydroxyethyl-naphtho(2,3-b)furan-4,9-dione were
potent with an inhibition concentration (IC50) of less than 1 μg/mL for K. africana fruits were obtained from Ayegunle-Ekiti region of Ekiti
all cells (Micheli, Sanogo, Mobilia, & Occhiuto, 2020). State (70.500 42.000 N, 50.060 36.000 E) and deposited at the Herbarium
Despite a large number of literature studies on the antip- Unit, Department of Botany, Obafemi Awolowo University, for identi-
roliferative activities of the various parts of the plant, data regarding fication and authentication, and a voucher number (IFE-17801) was
the anticancer activities of the fruit are inadequate. In addition, the collected. The fruit materials were rinsed thoroughly under tap water,
relative contribution of the specific chemical compounds to the air-dried at 25 C, and homogenized into powdery form using an
observed anticancer activity remains evasive, partly because either impact grinder at the DPPR Research Unit, Department of Pharmacy,
the active metabolites such as lapachol and norviburtinal discovered Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria.
in previous research studies on the plant are available at a very low
concentration with no modern spectrophotometric or chromato-
graphic techniques available to detect them (Jackson, Houghton, 2.2 | Extraction of K. africana (Lam.) Benth. fruit
Retsas, & Photiou, 2001), or the cytotoxicity of other metabolites is extracts
yet to be determined (Moideen, Houghton, Rock, Croft, & Aboagye-
Nyame, 1999). The development of new anticancer agents with mini- The ground fruits were steeped in analytically graded 70% ethanol for
mal side effects for the management of cancer is an important goal 72 h. Then, the crude ethanol extract was filtered with eight layers of
for scientists. Topoisomerases are enzymes implicated in DNA replica- cheese cloth and separated at 1552g for 20 min using a centrifuging
tion during cell division. They are upregulated in cancer cells and are, machine. The supernatant was collected, evaporated using a rotary
therefore, the targets of anticancer drugs (Azarova et al., 2007; Beebe evaporator system (BUCHI LaborTechnik, Delaware, USA), and
FAGBOHUN ET AL. 3 of 18

freeze-dried using a freeze-dryer (Labconco Corporation, Missouri, 5% diphenyl/95% dimethylpolysiloxane (serial number: 1280721) was
USA) at the Department of Chemistry (Inorganic Laboratory), Univer- used for the separation. The conditions of the TOF were as follows:
sity of South Africa. The resulting ethanolic crude extract was lique- 1 μL of the sample was injected in splitless mode in a GC machine
fied in deionized water and fractionated sequentially with ethyl hyper-aerated to a time-of-flight mass spectrophotometer with a tri-
acetate and butanol in a capped separating funnel. Then, the extract ple axis detector coupled to an autoinjector of a 10-μL syringe. The
and fractions were kept at 4 C in an airtight container for further injector as well as ion source was set at 250 C, while the transfer line
analyses. was set at 350 C. The initial oven temperature of 90 C was
maintained for 1 min and increased by 20 C/min to 200 C followed
by 15 C/ min to 300 C. This temperature was maintained for 8 min,
2.3 | Chemicals making the total run time to be 20 min. The carrier gas was helium
operated at 1.4 mL/ min, and the ion source was operated at 70 eV.
N-Methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA), pyridine, and The period of modulation was 2 s, with 0.6-s hot pulse and 0.4-s lag
analytically graded acetonitrile (HPLC standard) were procured from time between cooling stages. Full-scan detection was started at 1 min
Sigma-Aldrich Chemical Company, MO, USA. Other analytically and mentioned throughout the experiment. Scanning was from 50 to
graded chemicals of purest quality were obtained from BDH Chemical 500 m/z. The mass spectra generated by TOF/MS were compared
Ltd., VWR, UK. with those of standard mass spectra obtained from the National
Institute of Standards and Technology Library (NIST) version 14.

2.4 | Cell culture

2.6 | UHPLC-TOF-MS analyses
Hep G2, A375, HEK 293, and HeLa cells from ATCC (Manassas, VA,
USA) were grown as monolayer culture using Dulbecco's modified 2.6.1 | HPLC sample preparation
eagle's medium (GIBCO, Gaithersburg, MD, USA) in 1640 RPMI
medium comprising 10% fetal bovine serum (Bio West, Nuaillé, Deionized water (5 mL) was mixed with 2.5 g of K. africana butanol
France) and 1% antibiotics (250 μg/L fungizone, 100 μg/mL strepto- and ethyl acetate fractions separately. The resulting mixtures were
mycin, and 100 μg/mL penicillin) (GIBCO, Gaithersburg, MD, USA) for vortexed, acidified with 200 μL of acetic acid, and sonicated to dis-
complete growth medium in 5% CO2 atmosphere and 95% humidity solve the insoluble fraction before extraction into acetonitrile. Two
at 37 C. The cells in growth media were continually subcultured 2–3 grams of NaCl was added to the solution to form layers before being
days after the formation of a confluent monolayer. After washing in passed through sodium sulfate cartridge to remove water without the
phosphate-buffered saline to remove debris, the cells were sonicated loss of the samples. The top layer being the acetonitrile layer was

before transfer to vials and stored at –80 C until further analysis. concentrated to 1 mL using a turbovap at 55 C nitrogen and diluted
to 5 mL with acetonitrile/acetic acid in water.

2.5 | GC-TOF-MS analysis

2.6.2 | HPLC-QTOF-MS conditions
2.5.1 | GC sample preparation
Instrumental analysis was carried out using a 3000 series HPLC+
For GC analyses, the ethyl acetate and butanol fractions of K. africana QTOF Dionex Ultimate equipment (Thermo Scientific Inc., Waltham,
fruit were prepared following the method of Bottinelli, Revelut, MA, USA) with heat-activated electrospray ionization (ESI) coupled to
Hologne, Gaillard, and Bévalot (2019). Briefly, 1 g each of the dried a binary solvent delivery system. The Q-Exactive Plus TOF mass spec-
ethyl acetate and butanol fractions of K. africana fruit extract was trometer system has an X-Bridge C18 chromatography separation col-
steeped in 2 mL each of pyridine and MSTFA separately. The resulting umn (100 × 2.1 mm, 3.5-μm particle size) (Waters, Milford, MA, USA).

mixtures were vortexed, incubated at 40 C for 30 min, and then The injection volume of 5 μL was used. The mobile solvent phase A
cooled at 25 C. Thereafter, the mixtures were filtered with a 0.25 μm for separation was formic acid in water (0.1% v/v), while 0.1% of
polyvinylidene difluoride membrane syringe-like filter and extracted formic acid in acetonitrile served as solvent phase B. The gradient
using a sodium sulfate cartridge to remove water and then concen- flow conditions were 0.5 mL/min which started at 5% B and was

trated to 1 mL with turbovap at 55 C with nitrogen before subjected increased to 50% in 10 min with a further increase to 100% over 4
to GC-TOF-MS analysis. min. After the equilibrium gradient was achieved at 100% B, the col-
umn was re-equilibrated for 3.5 min at 5% B, making a total run time
of 21.5 min. Fragmentor voltage had positive HESI at 120 V, gas flow
2.5.2 | GC condition
at 8 L/min, gas temperature at 320 C, nebulizer pressure at 40 psig,
An Rxi 5-Sil MS capillary column (30-m length × 0.25 mm ID × sheath gas temperature at 380 C, and capillary voltage at 3500 V.
0.25-μm film thickness, from Restek, USA) with a crossband similar to Data were obtained in auto MS/MS data-dependent mode. MS
4 of 18 FAGBOHUN ET AL.

spectra as well as MS/MS spectra were obtained at a rate of 6 Hz in 2.8 | Protein identification and ligand preparation
the mass range of 50–1000 m/z. The detector was operated at 2
GHz and extended at a dynamic range, providing a resolution of To disclose the binding modes of the identified most potent
250,000. Relative abundance was used for precursor selection, and anticancer agent, molecular docking was performed on the target
an intensity threshold of 1000 counts was applied. This specific proteins of cancer cells, DNA topoisomerase-IIα. The structures of
precursor was excluded for 0.03 min after one spectrum from the synthetic anticancer agents (doxorubicin and camptothecin) were
precursor was obtained. The software controlling the instrument also obtained to serve as standards for the identified agent. The
was TraceFinder Acquisition software (version 3.2). Data files identified compounds were constructed using UCSF Chimera
obtained from the experiment consisted of full spectrum MS-only version 1.14, build 42094 (University of California, USA), which was
(MS1) of all ionized compounds and MS2 spectra of the precursors also consistent with the structure obtained using ChemDraw 3DPro
selected for fragmentation (Bian et al., 2020). An additional stage of version 12.0.2.1076 (Bharathi et al., 2014). The obtained structures
MS analysis (MS3) was performed on the primary fragment ion from that are ligands were prepared for docking by adding required
the MS/MS experiment to yield sufficient points for confirmation of charges and hydrogen bonds and equating all the charges using
nonvolatile metabolites (Schmidt et al., 2008; Ulintz et al., 2009). AM1 Hamiltonian in MOPAC software package (Chen et al., 1996).
Samples with large deviations of retention time (tR = >0.2) were not The X-ray structure of DNA topoisomerase-IIα was obtained from
analyzed. A mass accuracy of more than 5 ppm as well as peak Brookhaven Protein Data Bank with corresponding PDB ID 1QZR,
areas from previous equilibrium averages were observed for the with a resolution of 1.90 Å, R-value free of 0.237, R-value work of
compounds in the daily performance sample. The internal scan 0.196, R-value observed of 0.199, total structure weight of 97.53
time was 0.05 s, while the total scan time was 1.0 min per transition kDa, atom count of 6978, and sequence length of 418 (Classen,
with a total run time of 20 min. Nitrogen and argon were used as Olland, & Berger, 2003). PyMOL was used to analyze the binding
cone gas and collision gas, respectively, at a flow rate of 1.5 site and to remove water of crystallization as well as impurities.
mL/min. The analysis was carried out on a modified full single- MGL tools 1.5.4 was used for protein calculations to analyze
ion-monitoring system. docking. Docking calculations were performed using AutoDock Vina
software package (Trott & Olson, 2010).

2.7 | Determination of the antiproliferative activities

of the fractions 2.9 | Binding affinity by molecular docking studies

Antiproliferative activities were measured using MTT assay The crystal structures of the ligands generated by UCSF Chimera
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) analy- were refined by removing unnecessary electrons and water molecules
sis. A stock solution of MTT reagents was prepared in phosphate- as well as repeating coordinates and then saved in a PDBQT format
buffered saline at a concentration of 5 mg/mL. A total of 100 μL of using AutoDock tools 1.5.7rc1. The required charges were assigned to
cells (Hep G2, A375, HEK 293, and HeLa cells) measuring 5 × 103 cells the proteins by Kollman united atoms force field using AutoDock Vina
per well were seeded on 96-well plates and incubated for cell adhe- tools. Gasteiger partial atomic charges were added to the identified

sion at 37 C for 24 h in an atmospheric condition of 5% CO2. The compounds where AUTOTORS in the software tools was used to
fractions were assessed in a concentration range of 10, 50, 100, 500, define the possible flexible torsion angles and subsequently saved for
and 1000 μg/mL alongside reference standard drugs (camptothecin calculations in a PDBQT database format. AutoDock Vina requires a
and doxorubicin). Then, the growth medium of the cell was replaced precalculated grid box, which approximates for one each atom type
with a complete stock solution of MTT at a final concentration of 0.5 present in the docked structure. Intermediary steps were completed
mg/mL and further incubated for 4 h. The mixture was then removed using graphical user interface. AutoGrid was used for grid map prepa-
from the wells and mixed with 100 μL of dimethyl sulfoxide in each ration by a grid box. The grid size was set to 68 × 66 × 76 xyz points
well to dissolve the formazan crystals. The resulting mixture was mea- with an exhaustiveness of 8, and grid center was designated at dimen-
sured at an absorbance of 545 nm using a STAT FAX 2100 Microplate sions of 23.002, 32.703, and 31.241 for x, y, and z, respectively. Dur-
Reader (Awareness Technology, Palm City, FL, USA). The IC50 values ing the docking procedure, both the ligand and protein were
were determined from sigmoidal dose–response curves fitted to the considered as rigid. The results less than 1.0 Å in positional root-
data using OriginPro software (OriginLab Corporation, MA, USA). Per- mean-square deviation was clustered together and represented by
centage cell viability was calculated for HEK 293 cells from OriginPro the most favorable free-binding affinity result. The results with the
software (OriginLab Corporation) using the following formula: lowest energy in comparison with reference standard (camptothecin)
were extracted and aligned with the receptor structure for further
Cell viability ð%Þ = ðAtreated =Auntreated Þ × 100 analyses. The modeled H-bonds and Van der Waals interaction were
obtained by AutoGrid Box. Lamarckian genetic algorithm method in
where Atreated is the absorbance of cells with samples treated and command prompt was used for docking calculations. Analysis of the
Auntreated is the absorbance of untreated samples. docking results was done with consideration to the XP GS with
FAGBOHUN ET AL. 5 of 18

inspection on each visual derivative binding mode carried out by chemical compounds from UPLC/GC–MS were predicted using
PyMOL molecular graphics system (Trott & Olson, 2010). PreADMET server (http://preadmet.bmdrc.org/) and admetSAR
server (http://lmmd.ecust.edu.cn) (Cheng et al., 2012). Various
topologies alongside energy-minimized coordinates in a variety of
2.10 | Pharmacokinetics prediction of the ligands formats, were obtained from PRODRG server (Hurtado-Guerrero
et al., 2009; van Aalten et al., 1996).
The pharmacokinetics of a drug molecule constitute ADMET
(absorption, distribution, metabolism, excretion, and toxicity) analyses
using online and offline computational tools. ADMET profiles of the 2.11 | Statistical analysis
ligands were based on the structures and interaction between the
ligands and the receptor. In this study, significant descriptors of All determinations and extractions were performed in triplicates, and
druglikeness and prediction such as toxicological dosage level the results were expressed as mean ± standard deviation (n = 3).
and mutagenicity at different cellular and tissue levels as well as Hierarchical cluster (HCA) and principal component (PCA) analyses
pharmaceutical and pharmacological properties of the identified were performed on potent anticancer agents using XLSTAT software

F I G U R E 1 UHPLC-TOF-MS chromatograms of ethyl acetate and butanol fractions of Kigelia africana fruit; the positive- and negative-ion
mode chromatogram of nonvolatile chemical compounds where 64 peaks were characterized from the ethyl acetate and butanol fractions,
respectively, with the identification of (a) 235 chemical compounds in ethyl acetate fraction and profiling of (b) 209 secondary metabolites
 6 of 18

TABLE 1a Selected list of chemical compounds in the ethyl acetate fraction of Kigelia africana fruit extract using UHPLC-TOF-MS

Observed mass Calculated mass Mass Exact mass of Reference

tR [M + H]+/ [M + H]+/ tolerance D Elemental metabolites database ID
Number (min) [M – H]− m/z [M – H]− m/z m/z (ppm) MS/MS data composition (Da) (CAS) Detected metabolites
1 4.59 252.9953 252.9941 4.72 103.00, 71.01, 72.99, 101.02, 209.15 C11H8Cl2N2O 254.0013 62865-36-5 Diclomezine
2 10.24 178.1221 178.1226 −3.11 91.05,a 160.11, 132.08, 131.07, 147.08 C11H15NO 177.1153 408332-79-6 Buphedrone
a
3 10.49 199.1798 199.1805 −3.26 72.05, 89.07, 69.07, 156.08, 111.12 C11H22N2O 198.1732 2163-69-1 Cycluron
4 10.24 178.1221 178.1226 −3.11 58.07,a 105.07,a 133.06,a 105.03,a 72.08 C11H15NO 177.1153 35026-77-8 Dimethylcathinone
a
5 13.14 251.1632 251.1642 −4.01 57.07, 55.06, 83.09, 123.08, 129.09 C15H22O3 250.1568 25812-30-0 Gemfibrozil
6 10.24 178.1221 178.1226 −3.11 91.05,a 117.07, 134.01, 133.09, 160.11 C11H15NO 177.1153 134-49-6 Phenmetrazine
7 9.4 252.1486 252.1495 −3.57 195.05, 209.11, 235.12, 58.07, 207.09 C16H17N3 251.1422 61337-68-6 Desmethylmirtazapine
8 1.04 123.0550 123.0553 −2.17 80.05,a 96.04,a 78.03,a 106.03a C6H6N2O 122.0480 98-92-0 Nicotinamide
9 6.58 393.2077 393.2072 1.33 147.08, 237.13, 355.19, 171.08, 337.18 C22H29FO5 392.1999 50-02-2 Dexamethasone
10 11.97 355.2016 355.2016 −0.06 144.08, 212.13, 224.13, 117.07, 134.10 C21H26N2O3 354.1943 146-48-5 Yohimbine
11 14.53 277.2151 277.2162 −3.83 241.20, 235.17, 259.21, 185.13, 201.16 C18H28O2 276.2089 33036-33-8 19-Noretiocholone
12 11.21 301.0740 301.0738 0.68 259.06, 224.09, 105.03, 181.02, 153.02 C16H13ClN2O2 300.0665 22316-47-8 Clobazam
13 11.21 279.0922 279.0910 4.31 124.09, 204.04, 108.04, 156.01, 92.05 C12H14N4O2S 278.0837 515-64-0 Sulfisomidine
14 14.49 276.1710 276.1706 1.12 162.09, 219.11, 176.11, 188.11, 233.13 C15H21N3O2 275.1633 57-47-6 Physostigmine
15 9.61 331.2159 331.2169 −2.83 316.19,a 315.19,a 239.15, 272.14, 209.12 C23H26N2 330.2096 129-73-7 Leucomalachite green
16 14.53 267.1712 267.1703 3.19 91.05,a 123.08, 181.09, 166.06, 136.05 C14H22N2O3 266.1630 5011-34-7 Trimetazidine
17 14.45 328.0801 328.0791 3.11 282.07, 91.05, 238.05, 254.08, 254.04 C15H12F3NO4 327.0718 69335-91-7 Fluazifop
18 14.45 329.0833 329.0818 4.57 86.10, 302.07, 188.99, 141.11, 98.98 C15H18Cl2N2O2 328.0745 115852-48-7 Fenoxanil
19 17.81 379.2366 379.2380 −3.76 97.09, 292.17, 143.09, 167.09, 264.17 C24H30N2O2 378.2307 309-29-5 Doxapram
20 17.31 391.2497 391.2480 4.28 – C23H34O4 374.2457 143-62-4 Digoxigenin
21 8.81 479.2609 479.2604 1.12 57.07, 85.07, 121.06, 355.19, 375.20 C27H36F2O5 478.2530 59198-70-8 Diflucortone pivalate
22 1.21 268.1029 268.1040 −4.08 127.05, 110.02, 142.06, 109.04, 84.04 C10H13N5O4 267.0967 30516-87-1 Zidovudine

Note. CAS, Chemical Abstracts Service database; ppm, part per million; tR, retention time (min).
a
m/z value (mass-to-charge ratio): m/z represents mass divided by charge number and the horizontal axis in a mass spectrum and expressed in units of m/z. Because z is always 1 with MS, the m/z value is often
considered to be mass (experimental).
FAGBOHUN ET AL.
FAGBOHUN ET AL. 7 of 18

version 19 (XLSTAT, New York, USA). Molecular docking and in silico

m/z value (mass-to-charge ratio): m/z represents mass divided by charge number and the horizontal axis in a mass spectrum and expressed in units of m/z. Because z is always 1 with MS, the m/z value is often
Pentedrone
Butylamine
N-Ethylbuphedrone

Phendimetrazine
2,4-Dimethylaniline
pharmacokinetic results were computed and analyzed using Hp work-
station, Intel Core i7, 16 GB DDR4 ram, and 4 GB-dedicated graphics

metabolites
Detected
processing unit.

3 | RESULTS AND DISCUSSION

95-68-1
109-73-9

879722-57-3
634-03-7
1354631-28-9
database ID
3.1 | Characterization of chemical compounds using

Reference
UHPLC-TOF-MS

(CAS)
This study used a more recent UHPLC-TOF-MS2 approach to charac-

metabolites (Da)
terize bioactive phytochemicals present in the ethyl acetate and buta-

Exact mass of
nol fractions of K. africana fruit extracts. The chromatograms obtained

73.0891
121.0891

191.1310
191.1310
191.1310
in the positive- and negative-ion modes (Figure 1) led to the identifi-
cation of 235 and 209 phytochemicals for ethyl acetate and butanol
fractions, respectively, using modern chromatographic equipment
coupled with MS techniques. Selected phytochemicals found in the

composition
C8H11N
C4H11N
C12H17NO
C12H17NO
C12H17NO
Elemental
fractions are summarized in Table 1a, while each compound chro-

Selected list of chemical compounds in the butanol fraction of Kigelia africana fruit extract using UHPLC-TOF-MS
matogram along with the individual peak as well as fragmentation
pattern is provided in the Supporting Information. The bioactive
compounds were characterized based on UHPLC–MS data majorly

91.05, 174.13, 146.10, 145.09, 147.08

91.05,a 174.13, 132.08, 161.10, 131.07
58.07, 117.07, 148.11, 147.10, 146.10
94.07,a 79.05,a 105.07, 107.07, 103.05
parent ions, retention time (tR), molecular formula, MS fragments, and

56.06,a 56.05,a 57.03, 56.94, 55.93

matching data from different databases. The results of the TOF/MS
analysis showed that the fractions contain a complex mixture of
chemical compounds ranging from polyphenols, piperazines, steroids,
amino acids, stimulants or designer drugs, analgesics (nonsteroidal
anti-inflammatory drugs), antibiotics, alkaloids, trace amines, pyre-
MS/MS data

thrins, terpenes, and even contaminants.

Note. CAS, Chemical Abstracts Service database; ppm, part per million; tR, retention time (min).
As shown in Table 1a, compound 1 from ethyl acetate fraction a

a
eluted at tR = 4.59 min and was identified as 6-(3,5-dichlor-4-
methylphenyl)-3(2H)-pyridazinon (known as diclomezine). This com-
pound exhibited [M – H]− ion peak at m/z 253 with molecular formula
Mass tolerance

C11H8Cl2N2O. The mass spectral analysis revealed that diclomezine

D m/z (ppm)

yielded other peaks at 209.15, 103.00, 101.02, 72.99, and 71.01, indi-
−2.52
3.37
−3.49
−3.49
−3.49

cating successive loss of water and methyl groups, and from the
review of literature, this compound is pyridazinone and is used as a
fungicide in the treatment of rice sheath blight caused by Rhizoctonia
Calculated mass

solani (Kim, Kim, Kim, & Park, 2010). Another compound of interest
[M + H]+ m/z

79.0964
122.0964

192.1383
192.1383
192.1383

from the ethyl acetate fraction eluted at tR = 13.14 min (Mänttäri

et al., 1990). Gemfibrozil exhibited other peaks at 129.09, 123.08,
83.09, 57.07, and 55.06 with consequential loss of water and methyl
groups from the [M + H]+ ion peak at m/z 251. This compound is used
Observed mass

considered to be mass (experimental).

in the treatment of abnormal blood lipid levels, thereby decreasing

[M + H]+ m/z

‘bad’ cholesterol levels (Bocos, Orozco, Castro, Quack, &

74.0967
122.0961

192.1376
192.1376
192.1376

Herrera, 1995) and increasing the activity of extrahepatic lipoprotein

lipase leading to the increase in lipoprotein triglyceride lipolysis via
the activation of peroxisome proliferator-activated receptor alpha
5.45

5.28
5.28
5.28
20.25
(min)

(Doummar & Aoun, 2018). Gemfibrozil also acts as an inhibitor of apo-

tR

lipoprotein B (a carrier molecule for very-low-density lipoprotein) by

TABLE 1b

Number

inactivating the synthesis and inhibiting its clearance.

Of importance in this study is the identification of an alkaloid that
1
2
3
4
5

eluted at tR = 14.49 min and deprotonated at [M + H]+ ion m/z

a
8 of 18 FAGBOHUN ET AL.

276 with fragmentation patterns of 233.13, 219.11, 188.11, 176.11, norepinephrine-dopamine reuptake inhibitor without causing their
and 162.09 showing the consequential loss of methyl groups. This release (Maheux & Copeland, 2012), and phendimetrazine, an anti-
compound was identified as physostigmine, which is a parasympatho- obese agent chemically related to amphetamine (Landau, Jackson, &
mimetic alkaloid whose positive medicinal applications were first Gonzalez, 2008), were identified alongside more than 207 chemical
suggested by Thomas Fraser Richard at the University of Edinburgh in compounds (Table 1b).
1862 (Mehta, Adem, & Sabbagh, 2012; Moore, Rasimas, &
Donovan, 2015). Physostigmine functions as an acetylcholinesterase
3.2 | Metabolite fingerprinting of volatile
inhibitor by preventing the hydrolysis of acetylcholine at the transmit-
compounds using GC– TOF-MS
ted sites of acetylcholine and enhancing its effects, making it useful in
the treatment of cholinergic disorders and myasthenia gravis. The Tables 2a and 2b summarize the chemical compounds present in the
results of this study also showed that it can be used as an ethyl acetate and butanol fractions of K. africana fruit extracts. GC–
antiproliferative agent as a result of its binding affinity with MS was used to identify the volatile phytochemicals present in the
topoisomerase-IIα. Other uses of physostigmine suggest its role in the fractions. A total of 15 compounds were characterized from the ethyl
treatment of mydriasis and glaucoma alongside its mitotic function acetate fraction, while 3 secondary metabolites were identified from
causing pupillary constrictions (Proudfoot, 2006; Scheindlin, 2010). In the butanol fraction of K. africana fruit extracts. A peak identification
the same vein, compound 22 from ethyl acetate fraction, in Table 1a, was carried out by comparing mass spectra and retention times (tR)
eluted at tR = 1.21 min and deprotonated at m/z 268 and exhibited with reference standards, NIST database, and literature data. The
other peaks at 142.06, 127.05, 110.02, 109.04, and 84.04. This com- chromatograms in Figure 2 show the peaks of compounds in the frac-
pound was identified as zidovudine, which is used as an antiretroviral tions. The name of the detected and identified compounds, retention
agent (Flint, 1994). time in minutes, peak height, exact mass of the identified compounds,
Other compounds identified from the ethyl acetate and butanol molecular formula, NIST similarity in percentage, and reference
fraction as shown in Table 1a and in the Supporting Information Data database, that is, CAS, were used in the profiling of the chemical
S1 and S2 include desmethylmirtazapine (normirtazapine), an anxio- compounds and are presented in Tables 2a and 2b. Similar results
lytic drug that functions as a tetracyclic antidepressant (Morgan, Tap- were reported from different parts of the plant by other researchers
per, & Spencer, 2003); and trimetazidine, a cytoprotective drug whose (Akunyili et al., 1991; Arkhipov, Shalom, Matthews, & Cock, 2014;
cardiovascular effectiveness has been known in recent years Houghton & Jäger, 2002; Jabeen et al., 2013; Khan et al., 2016). The
(Chrusciel, Rysz, & Banach, 2014). In the butanol fraction (Table 1b), present study agrees with the results from the work of Arkhipov
pentedrone, a stimulant of the cathinone class that acts as a et al. (2014), who discovered several phytochemicals with

TABLE 2a List of compounds identified in the ethyl acetate fraction of Kigelia africana fruit extracts using GC-TOF-MS

tR NIST similarity Exact massa Molecular Reference

Number (min) matching (%) Peak height (m/z value) formula Identified compounds database ID (CAS)
1 4.67 95 248,707 182.2034 C13H26 1-Tridecene 2437-56-1
2 4.86 87 812,140 120.0575 C8H8O Benzofuran-2,3-dihydro 496-16-2
3 5.78 95 1,202,490 150.0680 C9H10O2 2-Methoxy-4-vinylphenol 7786-61-0
4 6.93 87 1,943,830 170.0731 C12H10O Diphenyl ether 101-84-8
5 7.54 86 695,776 146.0367 C9H6O7 Benzofuran-2-carboxaldehyde 4265-16-1
6 8.40 95 256,040 206.3200 C14H22O Phenol-2,4-bis 96-76-4
(1,1-dimethylethyl), −
7 9.34 98 421,839 226.2660 C16H34 Hexadecane 544-76-3
8 12.46 97 2,892,459 284.2715 C18H36O2 Hexadecanoic acid, ethyl ester 628-97-7
9 13.74 99 2,382,546 306.2558 C20H34O2 Linoleic acid ethyl ester 544-35-4
10 13.81 99 4,381,876 306.4828 C20H34O2 9,12,15-Octadecatrienoic acid 1191-41-9
ethyl ester, (Z, Z, Z)-
11 13.97 99 668,094 312.3028 C20H40O2 Octadecanoic acid, ethyl ester 111-61-5
12 15.40 93 533,489 281.2718 C18H35NO 9-Octadecanamide, (Z)- 301-02-0
13 15.57 70 246,010 283.2875 C18H37NO Octadecanamide 124-26-5
14 16.65 98 11,809,660 390.2770 C24H38O4 Bis(2-ethylhexyl)phthalate 117-81-7
15 18.34 96 409,040 337.3344 C22H43NO 13-Docosenamide, (Z)- 112-84-5

Note. CAS, Chemical Abstracts Service database; NIST, National Institute of Standards and Technology; tR, retention time (min).
a
m/z value (mass-to-charge ratio): m/z represents mass divided by charge number and the horizontal axis in a mass spectrum and expressed in units of m/z.
Because z is always 1 with MS, the m/z value is often considered to be mass (experimental).
FAGBOHUN ET AL. 9 of 18

TABLE 2b List of compounds identified in the butanol fraction of Kigelia africana fruit extracts using GC-TOF-MS

tR NIST similarity Exact massa (m/z Molecular Identified Reference database

Number (min) matching (%) Peak height value) formula compounds ID (CAS)
1 5.65 96 9,821,224 230.2053 C12H27BO3 Tributyl borate 688-74-4
2 15.41 98 1,006,014 281.2718 C18H35NO 9-Octadecenamide, 301-02-0
(Z)–
3 15.56 93 275,856 283.2875 C18H37NO Octadecanamide 124-26-5

Note. CAS, Chemical Abstracts Service database; NIST, National Institute of Standards and Technology; tR, retention time (min).
a
m/z value (mass-to-charge ratio): m/z represents mass divided by charge number and the horizontal axis in a mass spectrum and expressed in units of m/z.
Because z is always 1 with MS, the m/z value is often considered to be mass (experimental).

F I G U R E 2 GC–MS chromatograms of ethyl acetate and butanol fractions of Kigelia africana fruit extracts; the GC chromatograms showing
the identification of (a) 15 chemical compounds from ethyl acetate fraction and (b) 3 chemical compounds from butanol fraction from K. africana
fruit extracts
10 of 18 FAGBOHUN ET AL.

antiproliferative activities against different cancer cell lines, which is several chemical compounds could also act in suppressing prolifera-
one of focal points of this study. From the conclusion of Arkhipov tion by cancer cells, highlighting the importance of the secondary
et al. (2014), which suggested further metabolomic profiling studies metabolites present in the fractions of K. africana fruits.
using HPLC–MS/MS coupled with GC–MS, this present study
served to answer the questions raised from their research and have
metabolically profiled (UHPLC-TOF-MS and GC–MS) the fruit 3.3 | Cytotoxicity
extracts of K. africana along with antiproliferative activities using MTT
assay on different cell lines. Cell viability and IC50 values were analyzed using MTT assays. The
Of importance is the identification of 2-methoxyl-4-vinylphenol results are presented in Table 3. IC50 results revealed that ethyl ace-
in the ethyl acetate fraction with a peak height of 1,202,490 eluted at tate fraction had high antiproliferative effects on human hepatoma
tR = 5.78 min and molecular formula C9H10O2. This compound belongs cells (Hep G2) with a value of 0.53 μM at 1000 μg/mL. From the
to guaiacol, a class of phenol, and is used as a pheromone, a flavoring results of MTT assays, doxorubicin (reference standard drug) had the
agent, and a plant metabolite. This polyphenolic compound also plays lowest IC50 values, thereby exhibiting antiproliferative effects against
a role in raising an alarm by signaling a food trail through by triggering three cancer cell lines, but cell viability for HEK-293 cells reduced in a
sexual arousal, thereby delineating territory. It is also used in creating dose-dependent manner, pointing to its toxicity potential on
the bond between mother and offspring. It can induce cell-cycle arrest normal cells. As shown in Table 3, camptothecin and the ethyl acetate
by the blockage of hyper-phosphorylation of retinoblastoma proteins fraction had the same capacity to inhibit proliferation against
in cell lines (Jeong & Jeong, 2010). Kim et al. (2019) discovered the cancel cell lines with IC50 values of 0.55 and 0.53 μM, respectively, at
role of 2-methoxy-4-vinylphenol in the blockage of focal adhesion 1000 μg/mL. Butanol fraction had the least IC50 values, leading to the
kinase (FAK) and protein kinase B (AKT) signaling by attenuating the conclusion that it does not have a strong ability to inhibit proliferation
migration of human pancreatic cancer cells. From the discovery of in cancer-induced cells. The result, therefore, showed that the
several chemical compounds from the fractions of K. africana fruit phytochemicals responsible for antiproliferative activities are found
extracts, it could be concluded that several phytochemicals play abundantly in the ethyl acetate fraction. On the contrary, the fractions
important roles as antiproliferative agents, and thus, a combination of showed higher cell viability potentials against HEK 293 better than

TABLE 3 Determined IC50 values (μg/mL) of ethyl acetate and butanol fractions against different cancer and embryonic cell lines

IC50 (μM)

Fractions Concentrations (μg/mL) Hep G2 HeLa A375 HEK 293 cell viability (%)
Ethyl acetate 10 3.65 ± 0.02 2.49 ± 1.01 5.78 ± 1.03 85.67 ± 2.03
50 3.05 ± 0.14 2.03 ± 0.98 5.23 ± 1.99 83.35 ± 1.98
100 2.01 ± 0.15 1.65 ± 0.35 4.19 ± 1.01 73.34 ± 1.98
500 1.09 ± 0.08 0.97 ± 0.11 4.18 ± 0.99 73.55 ± 2.01
1000 0.53 ± 0.18 0.42 ± 0.15 2.98 ± 0.75 52.35 ± 3.58
Butanol 10 8.83 ± 2.35 10.56 ± 2.45 9.67 ± 3.45 43.76 ± 3.54
50 6.78 ± 1.23 10.36 ± 2.01 8.23 ± 1.34 33.98 ± 1.87
100 6.66 ± 1.26 6.78 ± 1.89 7.77 ± 2.43 32.11 ± 2.30
500 5.98 ± 0.83 5.78 ± 1.90 5.46 ± 2.09 31.23 ± 1.65
1000 4.53 ± 0.98 5.55 ± 2.03 5.03 ± 1.46 29.88 ± 3.67
Doxorubicin 10 1.89 ± 0.04 1.23 ± 0.98 1.89 ± 0.11 13.23 ± 0.67
50 1.23 ± 0.53 1.02 ± 0.55 1.23 ± 0.15 9.58 ± 0.58
100 0.98 ± 0.18 0.98 ± 0.23 1.00 ± 0.09 9.23 ± 0.34
500 0.24 ± 0.08 0.21 ± 0.02 0.98 ± 0.03 5.67 ± 2.30
1000 0.08 ± 0.01 0.09 ± 0.01 0.45 ± 0.01 4.33 ± 1.21
Camptothecin 10 2.01 ± 0.89 1.89 ± 0.09 1.20 ± 0.04 70.34 ± 2.30
50 1.35 ± 0.23 1.09 ± 0.08 0.45 ± 0.02 66.78 ± 1.23
100 1.09 ± 0.15 0.45 ± 0.02 0.23 ± 0.02 63.67 ± 0.13
500 0.55 ± 0.05 0.22 ± 0.02 0.06 ± 0.01 55.34 ± 1.21
1000 0.12 ± 0.02 0.09 ± 0.01 0.02 ± 0.01 48.89 ± 1.01

Note. Data are presented as mean ± standard deviation (n = 3); A375, human melanoma cells; HEK 293, human embryonic kidney cells; HeLa, cervical can-
cer cells; Hep G2, human hepatoma cells; IC50, the half-maximal inhibitory concentration.
 FAGBOHUN ET AL.

TABLE 4 Tabulated druglikeness and ADMET properties from PreADMET server

Properties Druglikeness

Number Chemical compounds BBB Caco2 HIA MDCK PPB Binding affinity (kcal/mol) CMC-like rule Lead-like rule MDDR-like rule Rule of five WDI-like rule
1 19-Noretiocholanolone 3.93 19.49 95.36 184.40 100.00 −7.6 Y N N Y Y
2 Clobazama 2.89 32.71 97.83 46.94 91.21 −7.5 Y Y N Y Y
a
3 Desmethylmirtazapine 0.94 56.25 96.89 288.65 57.79 −8.1 Y Y N Y Y
4 Dexamethasone 0.14 20.35 88.76 11.44 71.75 −8.6 Y N N Y N
5 Diclomezine 0.91 22.48 96.01 223.98 91.34 −7.7 Y N N Y Y
6 Diflucortolone pivalate 0.21 25.77 96.95 0.05 90.39 −8.2 Y N N Y N
7 Digoxigenin 0.56 21.16 88.85 9.56 85.44 −7.7 Y N N Y N
8 Doxapram 0.05 55.54 99.15 217.20 74.76 −7.8 Y N Y Y Y
9 Fenoxanil 0.74 30.01 95.86 69.52 90.43 −7.8 Y N N Y Y
10 Fluazifop 0.15 18.02 97.68 0.06 89.71 −8.6 Y N N Y Y
a
11 Physostigmine 0.55 27.95 95.07 46.65 42.77 −8.3 Y Y N Y Y
12 Sulfisomidine 0.11 0.48 93.05 2.91 45.29 −8.2 Y N N Y Y
13 Yohimbine 2.22 15.68 90.73 26.72 66.43 −7.6 Y N N Y Y
14 Zidovudine 0.13 8.11 32.29 3.05 44.29 −7.9 N N N N N
b
15 Camptothecin 0.23 18.51 96.86 24.47 79.32 −7.9 Y Y N Y Y
16 Doxorubicinb 0.03 17.73 31.95 1.02 32.79 −9.1 N N N N N

Note. BBB, blood–brain barrier; values between 0.1 and 2.0 and above represent better permeability through BBB; Caco2 and MDCK, human intestinal cell line and Madin–Darby canine kidney cell line; values
above 4 and 25, respectively, represent better absorption through cells; HIA, human intestinal absorption; values between 70 and 100 represent better absorption through intestine; PPB, plasma protein binding;
values up to 90 and above represent better bound compounds; druglikeness: Y represents suitability, whereas N represents nonsuitability; binding affinity: values above −7.9 kcal/mol represent better binding
affinity compared with standard anticancer agents.
a
Represents compounds with better affinity compared with standard drugs.
b
Represents standard drugs.
11 of 18
12 of 18 FAGBOHUN ET AL.

reference standard drugs (camptothecin and doxorubicin). This points Although doxorubicin and camptothecin showed better results in
to the fact that the fractions had high cytoprotective activities on nor- inhibiting proliferation in cancer cells, the reduction in cell viability
mal cells, suggesting that camptothecin and doxorubicin exhibited tox- suggests the toxic side effects of these synthetic compounds. Based
icity against normal cells while eliciting their various pharmacological on these outcomes, there is the need to screen herbal preparation
activities. The highest anticancer potential was observed in the ethyl from medicinal plants with minimal side effects to serve as therapeutic
acetate fraction against Hep G2 and HeLa cells (IC50 = 0.53 ± 0.18 and alternatives in the treatment of cancer, which is one of the objectives
0.42 ± 0.15 μM at 1000 μg/mL), which was consistent with the results of this study. Therefore, this study further screened the toxicity
obtained from the studies of Abou Baker (2020). potentials of these synthetic drugs (camptothecin and doxorubicin)
The bioactivity of K. africana fruit could be attributed to the high and the most potent phytochemicals having the highest antip-
percentage of alkaloids, trace amines, phenols, and flavonoids. A roliferative activities with the lowest toxicological potentials using
review of literature shows that phenolic acids have a crucial role in molecular docking and in silico pharmacokinetic studies.
blocking oncogenic pathways (Anantharaju, Gowda, Vimalambike, &
Madhunapantula, 2016). Moreover, a number of clinical studies have
shown that secondary metabolites, mainly coumaric acid, apigenin, 3.4 | Molecular docking study, PCA, and HCA
chlorogenic acid, and polyphenols, exhibited potent activity against
breast, brain, colon, leukemia, and lung cancers (Ali, Hassan, & In the current study, all the phytoconstituents identified from the
Abdrabou, 2016; Xu, Kang, Ren, Li, & Xu, 2013). Consistent with the ethyl acetate and butanol fractions of K. africana fruit extracts were
results of Abou Baker (2020) and Zheng, Chiang, and Lin (2005), tested against topoisomerase-IIα. Four hundred and sixty-four
which reported that secondary metabolites can induce apoptosis in phytoconstituents, including the reference standard drugs (camp-
HeLa cells by inhibiting cell viability (IC50 = 35.89 μM), similar results tothecin and doxorubicin), were molecularly docked against the
were observed from the outcomes of this study, as shown in Table 3. enzyme that has a polar contact of 3.2 Å. For the validation of the

F I G U R E 3 Principal component (PCA) and hierarchical cluster (HCA) analyses of chemical compounds identified from the ethyl acetate and
butanol fractions of Kigelia africana fruit. Four PCA revealed 100% variance, and HCA showed the classification of chemical compounds from
fractions of K. africana fruit extracts having better binding affinity compared with reference standards (camptothecin and doxorubicin). The
classification showed the classes of phytochemicals present in the fruit
FAGBOHUN ET AL. 13 of 18

docking protocol, docking was performed in the enzyme binding sites. characterized by zidovudine, fluazifop, digoxigenin, diflucortolone
The interacting residues and binding orientation were the same as pivalate, desmethylmirtazapine, and 19-noretiocholanolone. Principal
reported in previous literature (Jadhav & Karuppayil, 2016). The bind- component 2, on the contrary, was characterized by yohimbine, doxa-
ing affinities of doxorubicin and camptothecin are −9.1 and −7.9 pram, dexamethasone, and clobazam, while PC3 was characterized by
kcal/mol, respectively. Therefore, the phytoconstituents having a sulfisomidine, diclomezine, and fenoxanil. Surprisingly, physostigmine
binding affinity from −7.5 kcal/mol were selected as being potent for is the only compound that resides at principal component 4
topoisomerase-IIα. The results showed that the binding affinity ranges (Figure 3a,b). Based on the HCA, the compounds were classified or
from −7.5 to −8.6 kcal/mol, as shown in Table 4 with the highest grouped into nine different classes, as shown in Figure 3c. As shown
binding energy observed with fluazifop and the lowest with clobazam. in the plot, compounds that are categorized in class 1 include zidovu-
These results also showed that almost all the compounds exhibited dine, diflucortolone pivalate, and dexamethasone. Class 2 includes
hydrogen bond interactions with amino acids of the active sites along- yohimbine, whereas class 3 includes sulfisomidine. Physostigmine is
side ligand–enzyme complexes stabilized by π–π stacking interactions. the only compound grouped under class 4, while fluazifop and
Furthermore, these compounds were classified using PCA and HCA to fenoxanil are grouped under class 5. Doxapram, digoxigenin,
show the classes of compounds having antiproliferative activities. clobazam, diclomezine, and finally 19-noretiocholanolone were
Among the chemometric techniques used in extracting informa- grouped under classes 6, 7, 8, and 9, respectively. The classification of
tion from the original data, PCA is the most commonly used. In PCA the panelist was based on the Euclidean distance.
(Figure 3a,b), four principal components were extracted, which The PCA and HCA results as well as molecular docking analyses
explained 100% of the total variance for the identified compounds in show that physostigmine is a unique class of compounds having a
which component 1 accounted for 34.77% of the total variation, com- binding affinity of −8.3 kcal/mol and interacting with the following
ponent 2 accounted for 28.52% variation, and component 3 accounted residues: ASN70, ASP73, ASN74, ASN99, ILE104, ILE120, PHE121,
for 21.72% variation. Finally, component 4 accounted for 15.44% of GLY140, ARG141, ASN142, GLY143, LYS143, and polar contacts
the total variance in the experimental data. With respect to the infor- (Figure 4a). Topoisomerase-IIα is a heterotrimer (A2B2) (Figure 4b). In
mation obtained from the factor scores, principal component 1 was cancers, the enzyme is highly expressed in highly proliferating cells

F I G U R E 4 Molecular docking studies on topoisomerase-IIα and the interaction of physostigmine; crystal structure of topoisomerase-IIα
showing polar contact with physostigmine and amino acid residues within 4 Å consisting of ASN70, SER 127, and ASN 129 at binding sites having
interactions with physostigmine at bond distances of 2.5, 3.3, and 3.6 Å, respectively
 14 of 18

TABLE 5 In silico pharmacokinetic prediction of identified compounds

Absorption, toxicity, and distribution profiles obtained from PreADMET server

CYP_2C19 CYP_2C9 CYP_2D6 CYP_2D6 CYP_3A4 CYP_3A4 P-gp Ames hERG

Number Chemical compounds inhibition inhibition inhibition substrate inhibition substrate inhibition test Carcinogenicity inhibition
1 19-Noretiocholanolone Non Inhibitor Non Non Inhibitor Non Inhibitor Mutagen Non Low
2 Clobazam Non Non Non Non Non Substrate Inhibitor Mutagen Non Medium
3 Desmethylmirtazapine Non Non Inhibitor Substrate Non Weak Non Mutagen Non Medium
4 Dexamethasone Non Inhibitor Non Non Inhibitor Substrate Non Non Carcinogen High
5 Diclomezine Inhibitor Inhibitor Non Non Non Non Non Mutagen Non Medium
6 Diflucortolone pivalate Non Inhibitor Non Non Inhibitor Substrate Inhibitor Non Carcinogen Low
7 Digoxigenina Non Inhibitor Non Non Inhibitor Substrate Non Non Non Low
8 Doxapram Non Non Non Weak Inhibitor Substrate Non Mutagen Non Medium
9 Fenoxanil Non Non Non Non Non Substrate Inhibitor Mutagen Non Low
10 Fluazifop Non Inhibitor Non Non Non Non Inhibitor Mutagen Non Medium
11 Physostigminea Non Non Inhibitor Weak Non Substrate Non Non Non Low
12 Sulfisomidine Non Inhibitor Non Non Non Substrate Non Mutagen Non Low
13 Yohimbinea Non Non Inhibitor Substrate Non Substrate Non Non Non Medium
14 Zidovudine Non Non Non Non Inhibitor Substrate Non Mutagen Carcinogen Low
15 Camptothecinb Non Non Non Non Non Weak Non Mutagen Non Medium
b
16 Doxorubicin Inhibitor Inhibitor Non Weak Inhibitor Weak Non Non Non High

Note. Non represents no inhibition against different cytochrome P450 molecules; Ames test is done to detect a probable mutagen; mutagen indicates potential positive toxicity of a molecule; hERG, human
ether-a-go-go-related gene; values with low risk represent nonblockage of hERG gene.
a
Represents compounds with better affinity compared with standard drugs.
b
Represents standard drugs.
FAGBOHUN ET AL.
FAGBOHUN ET AL. 15 of 18

where the high expression of its encoded protein is also associated permeability analysis has a direct correlation with Caco2 test. A value
with poor patient survival. Inhibition of topoisomerase-IIα is essential between 25 and 500 shows medium permeability, while values more
for drug discovery regarding antiproliferation of cancer cells. The than 500 exhibit high permeability. Furthermore, a drug compound
discovery of potent anticancer agents must take into cognizance with high blood–brain barrier (BBB) absorption shows better potency
the suppression of topoisomerase-IIα activities (Hevener, Verstak, with values between 0.1–2.0 and above. Moreover, for better
Lutat, Riggsbee, & Mooney, 2018; Marinello, Delcuratolo, & distribution of a molecule, plasma protein binding and Pgp analyses
Capranico, 2018; McClendon & Osheroff, 2007; Nakopoulou with values more than 90% provide information about the drug
et al., 2000; Nitiss, 2009; Turley et al., 1997). The results of the study activities. Metabolism analysis shows durability of a drug. Drug
showed that physostigmine had the following residues: ASN metabolism is a chemical process in which enzymes are required for
129, SER127, and ASN70 with side chains CONH-CO, CH2OH-CO, the conversion of one species to another, and these metabolic
and H2NCO-NH of esters and amides on ligand and bond distances reactions are directed by the cytochrome P450 family (Dutkiewicz &
2.5, 3.5, and 3.6, respectively (Figure 4c,d). These results show Mikstacka, 2018; Furge & Guengerich, 2006; Zanger &
that physostigmine interacted well with the protein, but the affinity Schwab, 2013). For better metabolism, drugs should be non-inhibitors
as well as the interaction is subjected to in silico pharmacokinetics or non-substrates of CYP_2C6 and 2D6. Finally, the toxicity of drug
prediction using ADMET profiles on the phytoconstituent and the molecules was tested using Ames test (Zeiger, 2019). This test shows
reference standard drugs to further predict the antiproliferative whether a drug is toxic through its mutagenicity. As presented in
activities of physostigmine to determine the compound as a new Table 5, physostigmine, digoxigenin, and yohimbine passed the
anticancer agent. analyses to a considerable extent. Camptothecin and doxorubicin
failed the analyses due to the fact that they failed Ames test.
Therefore, in lieu of the fact that yohimbine and digoxigenin did not
3.5 | In silico pharmacokinetic prediction and pass ADMET tests, physostigmine is suggested as a potent candidate
ADMET profiles acting as an antiproliferative and anticancer agent.

Table 4 presents the ADMET profiles as well as druglikeness analyses

of the identified potent molecules as obtained from the PreADMET 4 | C O N CL U S I O N
server. Druglikeness uses Lipinski's rule of five to determine if a
compound with a certain pharmacological or biological activity has In conclusion, 235 metabolites and 15 chemical compounds were
physical or chemical properties that will make it a likely orally active profiled in the ethyl acetate fraction of K. africana fruit extract,
drug in humans (Lipinski, Lombardo, Dominy, & Feeney, 1997). These with the various phytochemicals showing antiproliferative activities.
rules are based on the observation that most orally administered The results from this study have shown that physostigmine is a potent
drugs are relatively small or moderately lipophilic molecules (Lipinski molecule that could be considered a lead compound against
et al., 1997). The results presented in Table 4 show that three topoisomerase-IIα. Moreover, according to molecular docking studies,
phytoconstituents, namely physostigmine, clobazam, and des- this compound has a higher binding affinity than camptothecin, and
methylmirtazapine, passed Lipinski's rule of five. The findings of this drug design using physostigmine as a base structure could be a great
study showed that doxorubicin failed the rule of five, whereas camp- option to finding a lead molecule against topoisomerase-IIα. Further-
tothecin passed the rule, suggesting that doxorubicin could be toxic to more, K. africana fruit extract could be considered an antiproliferative
normal cells, which was consistent with the results of the MTT assays agent because of the high quantity of phytochemicals and secondary
of this study. Doxorubicin, therefore, does not have the required metabolites profiled in this study using UHPLC-TOF-MS and GC–MS.
chemical or physical properties to make it an orally active drug in Therefore, physostigmine could be considered a new anticancer
humans according to Lipinski's rule of five. For the pharmacokinetic agent. Gene expression studies are underway and will determine the
properties that include absorption, distribution, and metabolism of the drug mechanistic actions.
tested compounds, the limits of the analysis must be passed before
determining the potency of a candidate molecule. In absorption, for a ACKNOWLEDG MENTS
compound to get to the tissue, it must enter the bloodstream before The support of Professor Ayobami Salami, the vice-chancellor of First
being absorbed by the cells. Human intestinal absorption (HIA) Technical University, is gratefully acknowledged. The organizational
analysis is important for a potent compound. A poorly absorbed support from the university is acknowledged as well.
compound has a limit between 0 and 20%, while moderately absorbed
and well-absorbed compounds have values between 20 and 70% and CONFLIC T OF INT ER E ST
70 and 100%, respectively. For Caco2 test, in silico Caco2 absorption The authors declare that there is no conflict of interest to disclose.
is most appropriate. These cells are derived from the human colon
with multiple drug transport pathways. A value ranging between FUNDING INF ORMATI ON
4 and 70 shows medium permeability, while values greater than This study did not receive any specific funding from any agencies in
70 show high permeability. MDCK (Madin–Darby canine kidney) cell the public, commercial, or not-for-profit sectors.
16 of 18 FAGBOHUN ET AL.

ORCID Binutu, O. A., Adesogan, K. E., & Okogun, J. I. (1996). Antibacterial and
Oladapo F. Fagbohun https://orcid.org/0000-0002-1340-6697 antifungal compounds from Kigelia pinnata. Planta Medica, 62,
352–353. https://doi.org/10.1055/s-2006-957900
Babatunde Olawoye https://orcid.org/0000-0002-3089-1196
Bocos, C., Orozco, E., Castro, M., Quack, G., & Herrera, E. (1995). Effect of
Adedeji N. Ademakinwa https://orcid.org/0000-0001-7736-0459 etofibrate on bile production in the normolipidemic rat. General Phar-
macology: The Vascular System, 26, 537–542. https://doi.org/10.1016/
RE FE R ENC E S 0306-3623(94)00225-C
Bottinelli, C., Revelut, K., Hologne, M., Gaillard, Y., & Bévalot, F. (2019).
Abou Baker, D. H. (2020). Achillea millefolium L. ethyl acetate fraction
GC-MS, GC-QTOF and NMR analyses to elucidate composition of
induces apoptosis and cell cycle arrest in human cervical cancer (HeLa)
41 powders from an NPS collector. Toxicologie Analytique et Clinique,
cells. Annals of Agricultural Science, 65, 42–48. https://doi.org/10.
31, 275–282. https://doi.org/10.1016/j.toxac.2019.10.002
1016/j.aoas.2020.03.003
Carey, W. M., Babu, D., Venkat Rao, N., & Mohan, G. (2008).
Agyare, C., Dwobeng, A. S., Agyepong, N., Boakye, Y. D., Mensah, K. B.,
Antiinflammatory activity of the fruit of Kigelia pinnata DC. Pharmacol-
Ayande, P. G., & Adarkwa-Yiadom, M. (2013). Antimicrobial, Antioxi-
ogy, 2, 234–245.
dant, and Wound Healing Properties of Kigelia africana (Lam.) Beneth.
Chen, J., Feng, L., Liao, Y., Han, S., Wang, L., & Hu, H. (1996). Using AM1
and Strophanthus hispidus DC. Advances in Pharmacological Sciences,
hamiltonian in quantitative structure-properties relationship studies of
2013, 692613. https://doi.org/10.1155/2013/692613
alkyl (1-phenylsulfonyl)cycloalkane-carboxylatts. Chemosphere, 33,
Akanni, O., Omotayo, O., Akinbo, D., Monjeed, I., Adebola, O., Bilikis, S., …
537–546. https://doi.org/10.1016/0045-6535(96)00197-X
Krishnamurthy, P. (2017). Kigelia africana Stem Bark, Fruit and Leaf
Cheng, F., Li, W., Zhou, Y., Shen, J., Wu, Z., Liu, G., … Tang, Y. (2012).
Extracts Alleviate Benzene-induced Leukaemia in Rats. Journal of Phar-
admetSAR: a comprehensive source and free tool for assessment of
maceutical Research International, 18, 1–10. https://doi.org/10.9734/
chemical ADMET properties. Journal of Chemical Information and
JPRI/2017/34625
Modeling, 52, 3099–3105. https://doi.org/10.1021/ci300367a
Akunyili, D. N., Houghton, P. J., & Raman, A. (1991). Antimicrobial activities
Chinsembu, K. C. (2016). Ethnobotanical Study of Plants Used in the Man-
of the stembark of Kigelia pinnata. Journal of Ethnopharmacology, 35,
agement of HIV/AIDS-Related Diseases in Livingstone, Southern
173–177. https://doi.org/10.1016/0378-8741(91)90070-t
Province, Zambia. Evidence-based Complementary and Alternative Medi-
Ali, F., Hassan, N., & Abdrabou, R. (2016). Hepatoprotective and antip-
cine, 2016, 4238625. https://doi.org/10.1155/2016/4238625
roliferative activity of moringinine, chlorogenic acid and quercetin.
International Journal of Research in Medical Sciences, 4, 1147–1153. Chivandi, E., Davidson, B., & Erlwanger, K. (2011). Kigelia africana seed:
https://doi.org/10.18203/2320-6012.ijrms20160799 Proximate, mineral, vitamin E, fibre, amino acid and fatty acid composi-
Amin, A., Gali-Muhtasib, H., Ocker, M., & Schhneider-stock, R. (2009). tion. International Journal of Food Science & Technology, 46,
Overview of Major Classes of Plant-Derived Anticancer Drugs. Inter- 2153–2158. https://doi.org/10.1111/j.1365-2621.2011.02730.x
national Journal of Biomedical Science : IJBS, 5, 1–11. Chrusciel, P., Rysz, J., & Banach, M. (2014). Defining the role of
Anantharaju, P. G., Gowda, P. C., Vimalambike, M. G., & trimetazidine in the treatment of cardiovascular disorders: some
Madhunapantula, S. V. (2016). An overview on the role of dietary phe- insights on its role in heart failure and peripheral artery disease. Drugs,
nolics for the treatment of cancers. Nutrition Journal, 15, 99. https:// 74, 971–980. https://doi.org/10.1007/s40265-014-0233-5
doi.org/10.1186/s12937-016-0217-2 Classen, S., Olland, S., & Berger, J. M. (2003). Structure of the topoisomer-
Arkhipov, A., Shalom, J., Matthews, B., & Cock, I. (2014). Metabolomic Pro- ase II ATPase region and its mechanism of inhibition by the chemo-
filing of Kigelia africana Extracts with Anti-Cancer Activity by High therapeutic agent ICRF-187. Proceedings of the National Academy of
Resolution Tandem Mass Spectroscopy. Pharmacognosy Communica- Sciences of the United States of America, 100, 10629–10634. https://
tions, 4, 10–32. https://doi.org/10.5530/pc.2014.4.3 doi.org/10.1073/pnas.1832879100
Azarova, A. M., Lyu, Y. L., Lin, C.-P., Tsai, Y.-C., Lau, J. Y.-N., Wang, J. C., & Delgado, J. L., Hsieh, C. M., Chan, N. L., & Hiasa, H. (2018).
Liu, L. F. (2007). Roles of DNA topoisomerase II isozymes in chemo- Topoisomerases as anticancer targets. The Biochemical Journal, 475,
therapy and secondary malignancies. Proceedings of the National Acad- 373–398. https://doi.org/10.1042/bcj20160583
emy of Sciences, 104, 11014–11019. https://doi.org/10.1073/pnas. Doummar, J., & Aoun, M. (2018). Assessment of the origin and transport
0704002104 of four selected emerging micropollutants sucralose, Acesulfame-K,
Beebe, S., Celestine, M., Bullock, J., Sandhaus, S., Arca, J., Cropek, D., gemfibrozil, and iohexol in a karst spring during a multi-event spring
… Holder, A. (2019). Synthesis, characterization, DNA binding, response. Journal of Contaminant Hydrology, 215, 11–20. https://doi.
topoisomerase inhibition, and apoptosis induction studies of a novel org/10.1016/j.jconhyd.2018.06.003
cobalt (III) complex with a thiosemicarbazone ligand. Journal of Dutkiewicz, Z., & Mikstacka, R. (2018). Structure-Based Drug Design for
Inorganic Biochemistry, 203, 110907. https://doi.org/10.1016/j. Cytochrome P450 Family 1 Inhibitors. Bioinorganic Chemistry and
jinorgbio.2019.110907 Applications, 2018, 3924608. https://doi.org/10.1155/2018/3924608
Bello, I., Shehu, M., Musa, M., Abdullah, M., & Mahmud, R. (2016). Kigelia Farah, H., Elhussein, A., Khalid, H., & Osman, H. (2018). Toxicity of Kigelia
africana (Lam.) Benth. (Sausage tree): Phytochemistry and pharmaco- africana Fruit in Rats. Advances in Research, 12, 1–9. https://doi.org/
logical review of a quintessential African traditional medicinal plant. 10.9734/AIR/2017/38539
Journal of Ethnopharmacology, 189, 253–276. https://doi.org/10. Flint, O. P. (1994). In vitro studies of the toxicity of nucleoside analogues
1016/j.jep.2016.05.049 used in the treatment of HIV infection. Toxicology In Vitro, 8, 677–683.
Bharathi, A., Roopan, S. M., Vasavi, C. S., Munusami, P., Gayathri, G. A., & https://doi.org/10.1016/0887-2333(94)90042-6
Gayathri, M. (2014). <i>In Silico</i> Molecular Docking and <i>In Fomogne-Fodjo, M., Van Vuuren, S., Ndinteh, D., Krause, R., & Olivier, D.
Vitro</i> Antidiabetic Studies of Dihydropyrimido[4,5-<i>a</i>] (2014). Antibacterial activities of plants from Central Africa used
acridin-2-amines. BioMed Research International, 2014, 971569. traditionally by the Bakola pygmies for treating respiratory and
https://doi.org/10.1155/2014/971569 tuberculosis-related symptoms. Journal of Ethnopharmacology, 155,
Bian, Y., Zheng, R., Bayer, F. P., Wong, C., Chang, Y.-C., Meng, C., … 131–155. https://doi.org/10.1016/j.jep.2014.04.032
Kuster, B. (2020). Robust, reproducible and quantitative analysis of Fouche, G., Cragg, G. M., Pillay, P., Kolesnikova, N., Maharaj, V. J., &
thousands of proteomes by micro-flow LC–MS/MS. Nature Communi- Senabe, J. (2008). In vitro anticancer screening of South African plants.
cations, 11, 157. https://doi.org/10.1038/s41467-019-13973-x Journal of Ethnopharmacology, 119, 455–461.
FAGBOHUN ET AL. 17 of 18

Furge, L., & Guengerich, F. (2006). Cytochrome P450 Enzymes in drug special reference to initial triglyceride levels. Atherosclerosis, 81,
metabolism and chemical toxicology. Biochemistry and Molecular Biol- 11–17. https://doi.org/10.1016/0021-9150(90)90054-M
ogy Education: A Bimonthly Publication of the International Union of Bio- Marinello, J., Delcuratolo, M., & Capranico, G. (2018). Anthracyclines as
chemistry and Molecular Biology, 34, 66–74. https://doi.org/10.1002/ Topoisomerase II Poisons: From Early Studies to New Perspectives.
bmb.2006.49403402066 International Journal of Molecular Sciences, 19, 3480. https://doi.org/
Hevener, K., Verstak, T. A., Lutat, K. E., Riggsbee, D. L., & Mooney, J. W. 10.3390/ijms19113480
(2018). Recent developments in topoisomerase-targeted cancer che- McClendon, A. K., & Osheroff, N. (2007). DNA topoisomerase II, gen-
motherapy. Acta Pharmaceutica Sinica B, 8, 844–861. https://doi.org/ otoxicity, and cancer. Mutation Research, 623, 83–97. https://doi.org/
10.1016/j.apsb.2018.07.008 10.1016/j.mrfmmm.2007.06.009
Houghton, P., & Jäger, A. (2002). The sausage tree (Kigelia pinnata): Ethno- Mehta, M., Adem, A., & Sabbagh, M. (2012). New acetylcholinesterase
botany and recent scientific work. South African Journal of Botany, 68, inhibitors for Alzheimer's disease. International Journal of Alzheimer's
14–20. https://doi.org/10.1016/S0254-6299(15)30434-8 Disease, 2012, 728983. https://doi.org/10.1155/2012/728983
Hurtado-Guerrero, R., Schüttelkopf, A. W., Mouyna, I., Ibrahim, A. F. M., Micheli, V., Sanogo, R., Mobilia, M. A., & Occhiuto, F. (2020). Effects of
Shepherd, S., Fontaine, T., … van Aalten, D. M. F. (2009). Molecular Kigelia africana (Lam.) Benth. fruits extract on the development and
mechanisms of yeast cell wall glucan remodeling. The Journal of Biologi- maturation of the reproductive system in immature male rats. Natural
cal Chemistry, 284, 8461–8469. https://doi.org/10.1074/jbc. Product Research, 34, 162–166. https://doi.org/10.1080/14786419.
M807990200 2019.1579809
Irfan, A., Batool, F., Zahra Naqvi, S. A., Islam, A., Osman, S. M., Moideen, S. V., Houghton, P. J., Rock, P., Croft, S. L., & Aboagye-Nyame, F.
Nocentini, A., … Supuran, C. T. (2020). Benzothiazole derivatives as (1999). Activity of extracts and naphthoquinones from Kigelia pinnata
anticancer agents. Journal of Enzyme Inhibition and Medicinal Chemistry, against Trypanosoma brucei brucei and Trypanosoma brucei
35, 265–279. https://doi.org/10.1080/14756366.2019.1698036 rhodesiense. Planta Medica, 65, 536–540. https://doi.org/10.1055/s-
Jabeen, B., Riaza, N., Saleema, M., Naveeda, M., Ahmeda, M., Tahirb, M., … 1999-14011
Jabbara, A. (2013). Isolation and Characterization of Limonoids from Moore, P. W., Rasimas, J. J., & Donovan, J. W. (2015). Physostigmine is the
Kigelia africana. Zeitschrift Fur Naturforschung B, 68, 1041–1048. antidote for anticholinergic syndrome. Journal of Medical Toxicology,
https://doi.org/10.5560/ZNB.2013-3096 11, 159–160. https://doi.org/10.1007/s13181-014-0442-z
Jackson, S., Houghton, P., Retsas, S., & Photiou, A. (2001). In Vitro Cyto- Morgan, P. E., Tapper, J., & Spencer, E. P. (2003). Measurement of total
toxicity of Norviburtinal and Isopinnatal from Kigelia pinnata Against mirtazapine and normirtazapine in plasma/serum by liquid chromatog-
Cancer Cell Lines. Planta Medica, 66, 758–761. https://doi.org/10. raphy with fluorescence detection. Journal of Chromatography B, 798,
1055/s-2000-9778 211–215. https://doi.org/10.1016/j.jchromb.2003.09.046
Jadhav, A. K., & Karuppayil, S. M. (2016). Molecular docking studies on Mülling dos Santos, M., Olaleye, T., Porto Ineu, R., Boligon, A., Linde
thirteen fluoroquinolines with human topoisomerase II a and b. In Silico Athayde, M., Barbosa, N., & Rocha, J. B. (2014). Antioxidant and
Pharmacology, 5, 4–4. https://doi.org/10.1007/s40203-017-0024-2 antiulcer potential of aqueous leaf extract of Kigelia Africana against
Jeong, J. B., & Jeong, H. J. (2010). 2-Methoxy-4-vinylphenol can induce ethanol-induced ulcer in rats. EXCLI Journal, 13, 323–330. https://doi.
cell cycle arrest by blocking the hyper-phosphorylation of retinoblas- org/10.17877/DE290R-15540
toma protein in benzo[a]pyrene-treated NIH3T3 cells. Biochemical and Muthaura, C. N., Rukunga, G. M., Chhabra, S. C., Mungai, G. M., &
Biophysical Research Communications, 400, 752–757. https://doi.org/ Njagi, E. N. M. (2007). Traditional phytotherapy of some remedies
10.1016/j.bbrc.2010.08.142 used in treatment of malaria in Meru district of Kenya. South African
Kaur, B., Shyam, R., & Amutha, R. (2012). Kigelia africana Fruit Carbon as a Journal of Botany, 73, 402–411. https://doi.org/10.1016/j.sajb.2007.
Low Cost Adsorbent for Removal of Copper. Nature, Environment and 03.004
Pollution Technology, 10(3), 419–422. Muyenga Akapelwa, T., Muungo, L., Prashar, L., & Bwalya, A. (2015). The
Khan, T., Fatima, I., Rafay, M., Shabir, S., Akram, M., & Bano, S. (2016). effect of Kigelia Africana fruit extract on blood glucose in diabetes
Evaluation of antibacterial and antioxidant activity of leaves, fruit and induced mice. Journal of Medical Science & Technology, 4, 201–206.
bark of kigelia Africana. Pakistan Journal of Botany, 48, 277–283. Nakopoulou, L., Lazaris, A., Kavantzas, N., Alexandrou, P.,
Kim, H., Kim, T. H., Kim, J., & Park, K.-M. (2010). Diclomezine: Athanassiadou, P., Keramopoulos, A., & Davaris, P. (2000). DNA Topo-
6-(3,5-dichloro-4-methyl-phen-yl)pyridazin-3(2H)-one. Acta isomerase II-Alpha Immunoreactivity as a Marker of Tumor Aggres-
Crystallographica, Section E: Structure Reports Online, 66, siveness in Invasive Breast Cancer. Pathobiology: Journal of
o3257–o3257. https://doi.org/10.1107/S1600536810047409 Immunopathology, Molecular and Cellular Biology, 68, 137–143. https://
Kim, D. H., Han, S. I., Go, B., Oh, U. H., Kim, C. S., Jung, Y. H., … Kim, J. H. doi.org/10.1159/000055914
(2019). 2-Methoxy-4-vinylphenol Attenuates Migration of Human Namita, P., Mukesh, R., & Tirath, K. (2012). Evaluation of anti-inflammatory
Pancreatic Cancer Cells via Blockade of FAK and AKT Signaling. Anti- potential of Kigelia pinnata leaf extract in wistar rats. Asian Journal of
cancer Research, 39, 6685–6691. https://doi.org/10.21873/ Pharmaceutical and Clinical Research, 5, 95–97.
anticanres.13883 Neba, N. (2006). Degradation of useful plants in Oku tropical montane
Landau, D., Jackson, J., & Gonzalez, G. (2008). A case of demand ischemia cloud forest, Cameroon. The International Journal of Biodiversity
from phendimetrazine. Cases Journal, 1, 105–105. https://doi.org/10. Science and Management, 2, 73–86. https://doi.org/10.1080/
1186/1757-1626-1-105 17451590609618100
Lipinski, C., Lombardo, F., Dominy, B. W., & Feeney, P. J. (1997). Experi- Nitiss, J. L. (2009). Targeting DNA topoisomerase II in cancer chemotherapy.
mental and computational approaches to estimate solubility and per- Nature Reviews Cancer, 9, 338–350. https://doi.org/10.1038/nrc2607
meability in drug discovery and development settings. Advanced Drug Ochwang'i, D. D., Kimwele, C., Oduma, J., Gathumbi, P., Mbaria, J., &
Delivery Reviews, 3, 23–25. Kiama, S. (2013). Medicinal plants used in treatment and management
Maheux, C. R., & Copeland, C. R. (2012). Chemical analysis of two new of cancer in Kakamega County, Kenya. Journal of Ethnopharmacology,
designer drugs: buphedrone and pentedrone. Drug Testing and Analysis, 151, 1040–1055. https://doi.org/10.1016/j.jep.2013.11.051
4, 17–23. https://doi.org/10.1002/dta.385 Oladele, A., & Adewunmi, C. O. (2008). Medicinal Plants Used In The Man-
Mänttäri, M., Koskinen, P., Manninen, V., Huttunen, J. K., Heikki agement Of Malaria Among The Traditional Medicine Practitioners
Frick, M., & Nikkilä, E. A. (1990). Effect of gemfibrozil on the concen- (TMP\'s) In South Western Nigeria. African Journal of Infectious Dis-
tration and composition of serum lipoproteins A controlled study with eases, 2, 51. https://doi.org/10.4314/ajid.v2i1.42091
18 of 18 FAGBOHUN ET AL.

Oladunmoye, M. K., & Kehinde, F. (2011). Ethnobotanical Survey of topologies and unique molecular descriptors from coordinates of small
Medicinal Plants Used in Treating Viral Infections among Yoruba Tribe molecules. Journal of Computer-Aided Molecular Design, 10, 255–262.
of South Western Nigeria. African Journal of Microbiology Research, 5, https://doi.org/10.1007/bf00355047
2991–3004. Wang, J. C. (2002). Cellular roles of DNA topoisomerases: a molecular per-
Olatunji, G., & Olubunmi, A. (2009). Comprehensive scientific demystifica- spective. Nature Reviews. Molecular Cell Biology, 3, 430–440. https://
tion of Kigelia africana: A review. African Journal of Pure and Applied doi.org/10.1038/nrm831
Chemistry, 3, 158–164. William Carey, M., Venkat Rao, N., Ravi Kumar, B., & Mohan, G. (2010).
Owolabi, O., & Omogbai, E. K. I. (2007). Analgesic and anti-inflammatory Anti-inflammatory and analgesic activities of methanolic extract of
activities of the ethanolic stem bark extract of Kigelia Africana Kigelia pinnata DC flower. Journal of Ethnopharmacology, 130,
(Bignoniaceae). African Journal of Biotechnology, 6(5), 582–585. 179–182. https://doi.org/10.1016/j.jep.2010.04.023
Owolabi, O., Omogbai, E. K. I., & Obasuyi, O. (2005). Antifungal and Würger, G., McGaw, L. J., & Eloff, J. N. (2014). Tannin content of leaf
antibacterial activities of the ethanolic and aqueous extract of Kigelia extracts of 53 trees used traditionally to treat diarrhoea is an impor-
africana (Bignoniaceae)stem bark. African Journal of Biotechnology, tant criterion in selecting species for further work. South African Jour-
6(14), 582–585. nal of Botany, 90, 114–117. https://doi.org/10.1016/j.sajb.2013.
Pommier, Y. (2013). Drugging Topoisomerases: Lessons and Challenges. 11.003
ACS Chemical Biology, 8, 82–95. https://doi.org/10.1021/cb300648v Xu, R., Kang, Q., Ren, J., Li, Z., & Xu, X. (2013). Antitumor Molecular Mech-
Proudfoot, A. (2006). The early toxicology of physostigmine: a tale of anism of Chlorogenic Acid on Inducting Genes GSK-3 β and APC and
beans, great men and egos. Toxicological Reviews, 25, 99–138. https:// Inhibiting Gene β -Catenin. Journal of Analytical Methods in Chemistry,
doi.org/10.2165/00139709-200625020-00004 2013, 951319–7. https://doi.org/10.1155/2013/951319
Sachan, R., Chauhan, N., Omar, B., Agrawal, S., Ji, O., & Professor, A. Zanger, U. M., & Schwab, M. (2013). Cytochrome P450 enzymes in drug
(2013). Ph ton. International Journal of Medicinal Plants Therapeutic uses metabolism: Regulation of gene expression, enzyme activities, and
of Kigelia pinnata, 105, 163–173. impact of genetic variation. Pharmacology & Therapeutics, 138,
Saini, S., Kaur, H., Verma, B., & Singh, S. (2008). Kigelia africana (Lam.) 103–141. https://doi.org/10.1016/j.pharmthera.2012.12.007
Benth. — An Overview. Natural Product Radiance, 8, 190–197. Zeiger, E. (2019). The test that changed the world: The Ames test and the
Scheindlin, S. (2010). Episodes in the story of physostigmine. Molecular regulation of chemicals. Mutation Research, Genetic Toxicology and
Interventions, 10, 4–10. https://doi.org/10.1124/mi.10.1.1 Environmental Mutagenesis, 841, 43–48. https://doi.org/10.1016/j.
Schmidt, A., Gehlenborg, N., Bodenmiller, B., Mueller, L. N., Campbell, D., mrgentox.2019.05.007
Mueller, M., … Domon, B. (2008). An integrated, directed mass spec- Zheng, P. W., Chiang, L. C., & Lin, C. C. (2005). Apigenin induced apoptosis
trometric approach for in-depth characterization of complex peptide through p53-dependent pathway in human cervical carcinoma cells.
mixtures. Molecular & Cellular Proteomics, 7, 2138–2150. https://doi. Life Sciences, 76, 1367–1379. https://doi.org/10.1016/j.lfs.2004.
org/10.1074/mcp.M700498-MCP200 08.023
Singh, A., Kumari, S., Singh, D. N., & Singh, A. (2018). Ethnopharmacology Zofou, D., Kengne, A., Tene, M., Ngemenya, M., Tane, P., & Titanji, V.
and pharmacology of Kigelia africana (Lam.) Benth. International Jour- (2011). In vitro antiplasmodial activity and cytotoxicity of crude
nal of Green Pharmacy, 11, 23-31. extracts and compounds from the stem bark of Kigelia africana (Lam.)
Thorn, C. F., Oshiro, C., Marsh, S., Hernandez-Boussard, T., McLeod, H., Benth (Bignoniaceae). Parasitology Research, 108, 1383–1390. https://
Klein, T. E., & Altman, R. B. (2011). Doxorubicin pathways: pharmaco- doi.org/10.1007/s00436-011-2363-y
dynamics and adverse effects. Pharmacogenetics and Genomics, 21,
440–446. https://doi.org/10.1097/FPC.0b013e32833ffb56
Trott, O., & Olson, A. J. (2010). AutoDock Vina: improving the speed and
accuracy of docking with a new scoring function, efficient optimiza- SUPPORTING INF ORMATION
tion, and multithreading. Journal of Computational Chemistry, 31, Additional supporting information may be found online in the
455–461. https://doi.org/10.1002/jcc.21334 Supporting Information section at the end of this article.
Turley, H., Comley, M., Houlbrook, S., Nozaki, N., Kikuchi, A.,
Hickson, I. D., … Harris, A. L. (1997). The distribution and expression of
the two isoforms of DNA topoisomerase II in normal and neoplastic How to cite this article: Fagbohun OF, Olawoye B,
human tissues. British Journal of Cancer, 75, 1340–1346. https://doi.
Ademakinwa AN, et al. UHPLC/GC-TOF-MS metabolomics,
org/10.1038/bjc.1997.227
Ulintz, P. J., Yocum, A. K., Bodenmiller, B., Aebersold, R., Andrews, P. C., & MTT assay, and molecular docking studies reveal
Nesvizhskii, A. I. (2009). Comparison of MS(2)-only, MSA, and MS(2)/ physostigmine as a new anticancer agent from the ethyl
MS(3) methodologies for phosphopeptide identification. Journal of Pro- acetate and butanol fractions of Kigelia africana (Lam.) Benth.
teome Research, 8, 887–899. https://doi.org/10.1021/pr800535h
fruit extracts. Biomedical Chromatography. 2020;e4979.
van Aalten, D. M., Bywater, R., Findlay, J. B., Hendlich, M., Hooft, R. W., &
Vriend, G. (1996). PRODRG, a program for generating molecular https://doi.org/10.1002/bmc.4979