Biomedicine & Pharmacotherapy 116 (2019) 108961

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Biomedicine & Pharmacotherapy

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

Nephroprotective activity of Combretum micranthum G. Don in cisplatin T

induced nephrotoxicity in rats: In-vitro, in-vivo and in-silico experiments
⁎⁎ ⁎
Mabozou Kpemissia,b,c, , Kwashie Eklu-Gadegbekua, Veeresh P. Veerapurc, , Mihai Negrub,
Marian Taulescub, Vivek Chandramohand, Jagadheshan Hiriyane, Siddalingesh M. Banakare,
Thimmaiah NVe, Doddamavattur Shivalingaiah Suhasc, Tumbadi Adinarayanashetty Puneethc,
Sachidananda Vijayakumarc, Kossi Metowogoa, Kodjo Aklikokoua
a
Faculty of Sciences, University of Lomé, Togo
b
University of Agricultural Science and Veterinary Medicine, Manastur Street. 3-5, 400372, Cluj-Napoca, Romania
c
Sree Siddaganga College of Pharmacy, B.H. Road, Tumkur, 572 102, Karnataka, India
d
Department of Biotechnology, Siddaganga Institute of Technology, Tumkur, 572103, Karnataka, India
e
Anthem Biosciences Pvt. Ltd., Industrial Area Phase I, Bommasandra, Hosur Road, Bangalore, 560099, India

A R T I C LE I N FO A B S T R A C T

Keywords: Nephrotoxicity is known to be a major complication during cisplatin chemotherapy in cancer patients. In the
Kidney present study, the protective effect of a hydroalcoholic extract of Combretum micranthum (CM) against cisplatin
Nephrotoxicity (CP)-induced renal damage was evaluated using in-vitro human embryonic kidney (HEK)-293 cells and in-vivo
HEK-293 experiments. Further, in-silico molecular docking and dynamic experiments were carried out with bioactive
Oxidative stress
compounds of the title plant against nuclear factor kappa B (NF-κB) and soluble epoxide hydrolase (sEH).
Molecular docking
Incubation of HEK-293 cells with cisplatin resulted in a significant increase in cell death with changes in normal
Molecular dynamic studies
cellular morphology. Co-treatment of HEK-293 cells with CP and CM extract at varying concentrations resulted
in significant enhancement of cell growth compared to CP treatment indicating the cytoprotective activity of CM
with an EC50 8.136 μg/mL. In vivo nephroprotective activity was evaluated by administering CM (200 and
400 mg/kg, p.o) to rats for 10 days followed by single intraperitonial injection of CP (7.5 mg/kg) on the 5th day
of the experiment. Nephrotoxicity induced by CP was apparent by elevated levels of serum and urine kidney
function markers, transaminases, oxidative stress markers and histopathological alterations in kidney. Pre-
treatment with CM normalized the renal function at both the doses by ameliorating the CP-induced renal damage
markers, oxidative stress and histopathological variations. In-silico studies showed that, out of the thirty
bioactive compounds, isovitexin and gallic acid exhibited a higher docking score of −22.467, −21.167 kcal/mol
against NF-κB. Cianidanol and epicatechin exhibited a higher docking score of −14.234, −14.209 kcal/mol
against sEH. The protective effect of CM extract in CP-induced nephrotoxicity might be attributed to its anti-
oxidant, anti-inflammatory activity by inhibiting NF-κB and sEH upregulation.

1. Introduction effectively, it exhibits a narrow and unfavourable therapeutic index and

Cisplatin (CP) (cis-diamminedichloroplatinum(II)) is effective
against a variety of cancer conditions such as solid tumors, haemato-
logical malignancies, lymphoma, osteosarcoma, bladder, oesophageal,
gastric, pulmonary, testicular, ovarian, head and neck cancers [1]. CP is
a potent chemotherapeutic agent, which causes acute kidney injury
(AKI) by inducing the oxidative stress, inflammation, and cell apop-
tosis. Although cisplatin therapy is able to reduce tumor burden
can lead to severe tissue damage [2]. One such major damage is renal
injury which limits the use of cisplatin in the clinic. Furthermore, 70%
of patients undergoing CP chemotherapy showed signs of ne-
phrotoxicity [3]. Due to the higher benefit to risk ratio, CP is still an
integral part of a combination therapeutic treatment regimen used
against various cancers [3]. There is a need of preventive treatment to
ameliorate the toxicity of CP without compromising the potency [2].
Presently, there is a paucity of an effective treatment to prevent renal

⁎
Corresponding author.
⁎⁎
Corresponding authors at: Laboratory of Physiology/Pharmacology, Department of Animal Physiology, Faculty of Sciences, University of Lomé, Togo.
E-mail addresses: mabozou@gmail.com (M. Kpemissi), veeresh36@gmail.com (V.P. Veerapur).

https://doi.org/10.1016/j.biopha.2019.108961
Received 4 April 2019; Received in revised form 2 May 2019; Accepted 8 May 2019
0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M. Kpemissi, et al.
damage caused by cisplatin [4]. Therefore, there is an unmet need for a
novel therapeutic treatment regimen to combat the development of
nephrotoxicity during cisplatin chemotherapy [4].
Plants derived from an ethnomedical/folklore background are an
important source of preventive medicines to treat a variety of human
ailments. Many reports support the usage of natural products like
polyphenolic compounds against oxidative stress induced organ
pathologies. Natural antioxidants are more preferred over synthetic
compounds due to their safety and the fact that they are derived from
natural sources [5]. Natural products derived from plant sources are
known to provide a source of inspiration for novel druggable com-
pounds and this is sequel to the fact that medicines derived from plants
have made large contributions to human health and well-being [6].
Many herbal medicines are known to have various types of poly-
phenolic compounds which may be safe and effective in reducing the
nephrotoxic effects of cisplatin [7,8]. Several ethnobotanical and eth-
nopharmalogical surveys on Combretum micranthum, suggesting that it
contains polyphenols [9,10] and possesses various biological activities
such as antioxidant [11,12], anti-inflammatory [13] and anti-diabetic
properties [14]. In the recent past, researchers have been utilizing in-
silico tools to identify potential drug candidates derived from plant
sources by screening against several molecular targets during the drug
discovery process [15,16]. In-silico experiments like molecular docking
and dynamic studies of bioactive markers against diverse molecular
targets have been reported [17,18]. The preliminary work on protective
effect of Combretum micranthum in scavenging several biologically re-
levant free radicals (in-vitro experiment), oxidative stress-induced lipid
peroxidation in kidney tissue (ex-vivo experiment) and high glucose-
induced cell death in HEK-293 cell line [19] prompted us to undertake
the present study. Further, there are no literature reports on beneficial
effect of Combretum micranthum in experimentally induced kidney da-
mage. Taking all these facts into consideration, we thought it worth-
while to undertake a detailed study employing drug discovery strategies
such as in-silico, in-vitro and in-vivo experiments to demonstrate the
effectiveness of Combretum micranthum against cisplatin-induced renal
toxicity. The objective of the present work was to investigate the
therapeutic potential of hydroalcoholic extract of Combretum mi-
cranthum (CM) against cisplatin-induced renal damage in HEK-293 cells
and in a rat model of cisplatin induced nephrotoxicity. In addition, to
investigate the mechanism of action of CM, in-silico molecular docking
and dynamic experiments with the bioactive compounds were carried
out against nuclear factor kappa B (NF-κB; protein data bank ID: 1NFK)
and soluble epoxide hydrolase (sEH; protein data bank ID: 3ANS).
2. Material and methods
2.1. Chemicals and reagents
Cisplatin (CP), Dimethyl sulfoxide-bio reagent, DMEM growth
medium, Fetal bovine serum (FBS), L-Glutamine, MTT [3-(4,5-
Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide], MEM-
non-essential amino acid solution, reduced glutathione (GSH), Greiss
reagent, were procured from Sigma-Aldrich (St. Louis, MO, USA).
Commercial reagent kits for determination of creatinine (CRE), urea
(UR), uric acid (UA), total protein (TP), albumin (Alb), calcium (Ca2+),
Magnesium (Mg2+), phosphorus (P), alanine aminotransferase (ALT),
aspartate aminotransferase (AST), alkaline phosphatase (ALP) and
gamma glutamyl transferase (γGT) activities were purchased from
Biolabo S.A. (Paris, France). All other chemicals and reagents used were
of the analytical grade.
2.2. Plant material
Fresh leaves of Combretum micranthum (CM) were collected in
December 2016 from Alibi I, a locality at North West of Tchamba
(Togo). Botanical authentication was confirmed at the Laboratory of
2
Biomedicine & Pharmacotherapy 116 (2019) 108961
Botany and Vegetable Ecology, Faculty of Science, University of Lomé,
Togo and the voucher specimen was deposited at the herbarium (N°
TOGO151085). The leaves were washed, dried under shade and were
coarsely powdered.
2.3. Extraction
The powder (830 g) was macerated at room temperature with 5 L of
ethanol-water (8:2 v/v) for 72 h. The filtrate was evaporated under
vacuum at 45 °C by a rotary evaporator (Rotavapor Buchi R100). The
yield of hydroalcoholic extract of Combretum micranthum (CM) was
12.15%.
2.4. In-vitro cell line studies
2.4.1. Cell culture
Variety of supplements such as 10% Fetal Bovine Serum (FBS), 1X
Penicillin-Streptomycin solution, non-essential amino acids, 2 mM L-
glutamine, 1 mM sodium pyruvate and 1500 mg/L sodium bicarbonate
were added to Dulbecco's Modified Eagle's medium (DMEM) growth
medium and filtered through 0.2 μm filter using a filtration unit fitted
to a vacuum pump. Filtered media was stored at 4 °C. Human em-
bryonic kidney cell line (HEK-293; ATCC®, CRL-1573™) obtained from
ATCC used for the study. Cells stored in liquid nitrogen were thawed
and revived as per recommended methods. Cells were cultured and
expanded in complete DMEM growth medium. Sub-confluent mono-
layers of cells were harvested, pelleted and re-suspended in growth
medium prior to counting on a haemocytometer by Trypan blue ex-
clusion method.
2.4.2. Effect of CM extract in cisplatin-induced toxicity in HEK-293 cells
HEK-293 cells were cultivated in DMEM supplemented with 10%
heat-inactivated fetal bovine serum in a CO2 incubator (5% CO2 in air)
at 37 °C. The cells with 70–80% confluency were trypsinized and suf-
ficient media added to inactivate the trypsin activity. The cells were
centrifuged at 1200 rpm for 5 min, supernatant was discarded and re-
suspended the pellet in media prior to counting on a haemocytometer
by Trypan blue exclusion method. The cells were diluted in media to get
desired number of cells. For cell growth studies, the final seeding
density was kept 10,000 cells/ well in a 96-well flat bottomed micro-
titer plate. Post 24 h of cells seeding, cells were untreated, treated or co-
treated with CP (20 μM) and CM extract (5, 10, 25, 50, 100 and 200 μg/
mL) for 24 h. After 24 h of treatment, cell viability assay and cell
morphological analysis were performed [20].
2.4.3. Cell viability test
Cell viability was measured using 3-(4, 5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) bioassay, which gives a sensitive
measurement of the normal metabolic status of cells. Post treatment at
24 h, MTT solution (5 mg/ml) added to the wells of respective treated
cells and incubated for 3 h. The dark-blue formazan formed in the well
was dissolved in DMSO and the absorbance was measured at 570 nm
using a microtiter plate reader.
2.4.4. Cell morphological evaluation
Morphological changes in HEK-293 cells were examined using
compound microscope post-exposure to [Cisplatin (20 μM) alone, or
combination of Cisplatin (20 μM) and CM extract (5–200 μg/ml) for
24 h] at 37 °C.
2.5. Animals
Male Albino Wistar rats of 6–8 weeks weighing 200–250 g were
procured from Nigerian Institute of Medical Research, Lagos, Nigeria.
Animals were housed in standard cages and maintained under standard
laboratory conditions. Experimental protocols adopted were based on
M. Kpemissi, et al.
Fig. 1. Experimental design of cisplatin-induced nephropathy model.
World Health Organization Guidelines for care and use of laboratory
animals. The experimental usage of the animals was approved by the
Ethics Committee of the University of Lomé, a branch of the National
Ethics Committee for control and supervision of experiments on ani-
mals (N° SBM/UL/14/NS0004). They were acclimatized for two weeks
before the experiments and fed with normal pellet diet and water ad
libitum.
2.6. Effect of CM extract in cisplatin-induced toxicity in Wistar rats
The dose and time schedule of cisplatin administration was adopted
in the present study were based on the pilot study done by Kumar et al.,
[21]. The schematic diagram of experimental design has been shown in
(Fig. 1). A total of 32 animals were randomized based to their body
weight into four groups, comprising of eight rats in each group. Group I
(Normal control): Normal saline was administered orally for 10 days
and a single intraperitoneal injection (i.p) of 0.5 ml normal saline on
the 5th day; Group II (Cisplatin control): Normal saline was adminis-
tered orally for 10 days and a single dose of cisplatin (7.5 mg/kg, i.p)
dissolved in normal saline was administered on the 5th day; Group III
(CM 200 mg/kg): Rats were treated with CM (200 mg/kg/day) orally
for 10 days and a single dose of cisplatin (7.5 mg/kg, i.p) was ad-
ministered on the 5th day, 1 h after the CM dose; Group IV (CM
400 mg/kg): Rats were treated with CM (400 mg/kg/day) orally for 10
days and a single dose of cisplatin (7.5 mg/kg, ip) on the 5th day, 1 h
after dosing the extract.
The body weight of the rats was measured daily during the course of
treatment. On Day 11 (5 days post cisplatin administration), urine
samples were collected by keeping the animal on a plastic plate and
awaiting spontaneous micturition [22]. The urine samples were col-
lected and centrifuged for 5 min at 1500 rpm and the clear supernatant
was stored at −20 °C and thawed just before use for the biochemical
analysis. After urine collection, animals were anesthetized with ether,
and blood samples were collected from the orbital sinus in centrifuge
tubes and allowed to clot for 20 min at room temperature. The samples
were centrifuged at 4000 rpm for 10 min at 4 °C, and the resultant
serum was separated and stored at −20 °C and thawed just before use.
Rats were euthanized by an over dose of thiopentone sodium and
3
Biomedicine & Pharmacotherapy 116 (2019) 108961
kidney tissues were dissected and weighed. The left kidney was fixed in
10% neutral buffered formalin solution for histopathology and the right
kidney was used for biochemical estimations.
2.6.1. Renal function tests
Serum and urine biochemical markers of renal injury and serum
hepatic injury markers such as creatinine (CRE), urea (UR), uric acid
(UA), total protein (TP), albumin (Alb), calcium (Ca2+), Magnesium
(Mg2+), phosphorus (P), alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase (ALP) and gamma glu-
tamyl transferase (γGT) were estimated using Biolabo commercial kits
(Biolabo S.A., Paris, France) according to the manufacturer’s manual
and URIT 8021Aautomated analyser (URIT Medical Electronic Group
Co., Ltd).
2.6.2. Endogenous antioxidant content in kidney
Kidney tissue homogenates (10% w/v) were prepared in tris phos-
phate buffer (50 mM, pH 7.4) and then centrifuged at 3000 × g for
10 min at 4 °C. The supernatant was used for the spectrophotometric
assays for determination of glutathione (GSH), malondialdehyde
(MDA), Tissue nitric oxide (NO) levels and ferric reducing activity of
plasma (FRAP).
2.6.2.1. Determination of total protein content in renal tissue. The protein
content was measured by the method of Bradford using crystalline BSA
as a standard. The homogenate or BSA (15 μL) and 750 μL of the
bradford reagent were added and optical density was read five minutes
after at 595 nm [23].
2.6.2.2. MDA measurement. Malondialdehyde (MDA) content, as a
marker of lipid peroxidation, was estimated as previously explained
[24]. Briefly, 0.65 ml of 10.3 mM 1-methyl-2-phenyl-indole in
acetonitrile was diluted with methanol containing 32 μM FeCl3 (3:1)
was added to 0.3 ml of kidney homogenate samples or standards with
vortexing. After adding 0.15 ml of 37% (v/v) HCl, samples were mixed
well, incubated at 45 °C for 60 min and cool. The samples were
centrifuged at 4000 g for 10 min and the absorbance was
spectrophotometrically measured at 586 nm. A standard curve
M. Kpemissi, et al. Biomedicine & Pharmacotherapy 116 (2019) 108961

Table 1 ranging from 10 to 2000 μM/L. Working reagent was prepared by

Reported bioactive compounds of Combretum micranthum. mixing 25 mL of acetate buffer; 2.5 mL of Fe3+-TPTZ (10 mM/L in
Compounds (Ligands) Classes References 40 mM/L of HCl); and 2.5 ml of FeCl3-6H2O (20 mM/L).

Gallic acid Flavonoids [13,29] 2.6.3. Histopathological analysis

Combretine (Turicine) [30]
Tissue samples from kidneys were collected and set in 10% phos-
Quercitrin [13]
myricetin-3-O-rutinoside [13,29] phate-buffered formalin (pH 7.0) for 24 h, routinely processed, em-
Rutin trihydrate [29,31] bedded in paraffin wax, cut into 2–3 micrometers (μm) sections and
(+)-Catechin (cianidanol) [29,31] stained with hematoxylin and eosin (H&E). The photomicrographs were
Vitexin [29,31] taken using an Olympus SP 350 digital camera and Stream Basic ima-
Isovitexin [29,31]
ging software (Olympus Corporation, Tokyo, Japan). Two serial sec-
Orientin [29]
Homoorientin (isoorientin) [29] tions for each individual sample were evaluated using a standardized
Myricetin-3-O-glucoside [29] protocol in order to identify the major morphological features asso-
(-)-Epigallocatechin [29] ciated with experimentally-induced kidney disease. Using a microscope
(-)-Epicatechin [29]
ocular with a FN of 22 mm, the area of a “high power field
(-)-3',4',5',5,7-pentahydroxyf lavan (Luteoforol) [29]
(-)-3',4',5,7-tetrahydroxyf lavan [29] (40x)” = 0.237 mm2, therefore by examining 10 HPF a total of
2"-O-galloylvitexin [29] 2.37 mm2/slide have been evaluated. The histological changes of en-
2"-O-galloylisovitexin [29] dothelial, glomerular, tubular, and interstitial components were eval-
2"-O-galloylorientin [30,38] uated and scored according to Usman et al. [28].
Lupeol [30,38]
α-Tocopherol [30,38]
α -amyrin [30] 2.7. In silico experiments
Palmitic acid [38]
Oleic acid Acid Gras [38] Molecular modelling studies were performed for the identified
Linolenic acid [30]
bioactive compounds from the CM extract in order to assess the ability
β-Sitosterol [38]
Choline [38,49] of bioactive molecules for inhibitory activity against transcription
Betulin Alkaloids [38] factor NF-κB and soluble epoxide hydrolase (sEH).
Betonicine [13,30]
Stachydrine [30] 2.7.1. Profile of bioactive compounds
4-Hydroxyproline betaine [13]
Betaine [13,30]
The several reports confirmed the presence of phenolic compounds
in the extract by IR and high performance liquid chromatography
techniques [12,29–31]. In addition, in our previous preliminary work
comprising of different concentrations of 1,1,3,3-tetra-methoxypropane also confirms the presence of phenolic, tannin and flavonoidal content
was used for the quantification of MDA. in extract [19]. Further, based on HPLC experiments, the polyphenolic
compounds have been identified such as hyperozide, quercitrine, caf-
taric acid, gentisic acid, caffeic acid and chlorogenic acid by UV and MS
2.6.2.3. GSH measurement. Kidney reduced glutathione (GSH) was
detectors (Under communication). Based on literature reports men-
measured as previously reported [24].The reaction mixture
tioned in Table 1, Thiry bioactive compounds were selected for in silico
containing 0.25 ml of supernatant or standards, 1 ml of 0.2 M
experiments.
tris−HCl (pH 8.9) and 0.05 ml of 10 mM 5,5′-dithiobis-(2-
nitrobenzoic acid) [DTNB] in absolute methanol was kept at room
2.7.2. Protein preparation
temperature for 5 min, and the yellow colour developed was measured
The X-ray crystallographic structure of NF-κB-DNA (PDB ID:1NFK)
spectrophotometrically at 412 nm. Results were calculated from a
p50 homodimer with the resolution of 2.30 Å and human soluble ep-
standard GSH calibration curve and expressed as nM/mg protein.
oxide hydrolase (PDB ID:3ANS) in complex with synthetic inhibitor
with the resolution of 1.98 Å was downloaded from the Research
2.6.2.4. Nitric oxide (NO) measurement. Kidney or serum NO levels Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank
were indirectly evaluated by measuring the nitrite levels using a (PDB) [32,33]. The co-crystallized DNA macromolecule from 1NFK and
colourimetric method based on the griess reaction [25]. Briefly, synthetic inhibitor from 3ANS was removed from the structures. Crys-
100 μL of the analyzed sample was added to 600 μL of the griess tallographic water molecules from the target proteins were deleted to
reagent (equi-volumes of 2% sulfanilamide in 2.5% phosphoric acid obtain clean protein and then the hydrogen atoms were added to both
and 0.1% w/v N’1-(1-naphthyl)-N-2-diethylethylenediamine in distilled the proteins using CHARMm force field in order to stabilize the target
water were mixed just before use), the mixture was mixed and after proteins. Energy minimization of the targets was performed with the
10 min, the absorbance at 540 nm was measured. NO contents were help of standard dynamics cascade protocol of Discovery Studio 3.5.
expressed as μM/g tissue protein. Binding site residues were selected for molecular docking studies [33].

2.6.2.5. Determination of FRAP activity. The reducing ability of
biological sample was determined as previously described [26]. FRAP
activity was analyzed by measuring the total antioxidant potential of
kidney homogenate and serum [27]. A working reagent (prepared by
mixing 25 mL of acetate buffer; 2.5 mL of 10 mM/L Fe3+-TPTZ in
40 mM of HCl; and 2.5 mL of FeCl3-6H2O) (300 μL) was mixed with
10 μL of sample (kidney homogenate or serum) and 30 μL of distilled
water. In this assay the electron-donating capability of the antioxidant
was measured by the change in absorbance at 593 nm, when a blue-
coloured Fe2+ -tripyridyltriazine (Fe2+ TPTZ) compound is formed
from a colourless oxidized Fe3+ form [27]. Calibration curves were
generated from aqueous solution of FeSO4 at different concentrations
2.7.3. Ligand preparation
Thirty bioactive compounds of CM extract were screened for the in-
silico work (Table1). The structures of 30 bioactive compounds were
obtained from the NCBI PubChem Compound database. Energy mini-
mization for the selected bioactive compounds was done to remove
clashes among atoms of the ligand and develops a reasonable stable
confirmation. Ligands optimization was carried out using Chemistry at
Harvard Molecular Mechanics (CHARMm) force field followed by en-
ergy minimization protocol [33]. Several ligand conformations were
generated based on bond energy, CHARMm energy, dihedral energy,
electrostatic energy, initial potential energy, and initial RMS gradient
values. Based on this the lowest conformer from all the ligands were

4
M. Kpemissi, et al.
Fig. 2. Effect of Combretum micranthum (CM) in cisplatin (CP)-induced toxicity in HEK-293 cells. [A] HEK-293 cells were treated with CM (5–200 μg/mL) for 24 h,
and subjected to MTT assay. [B] HEK-293 cells were treated with CP (20 μM) and CM extract (5–200 μg/mL) for 24 h and evaluated for cytotoxicity by MTT assay. [C]
Microscopic images of HEK-293 cells morphology after treatment with CP and CPeCM for 24 h. The results are expressed as Mean ± SEM. One-way ANOVA
followed by Dunnett’s Test. Compared to normal control group: ### P < 0.001; Compared to Cisplatin control group: * P < 0.05, ** P < 0.01, *** P < 0.001.
selected to perform molecular docking with the target protein.
2.7.4. Molecular docking
Molecular docking studies were performed in order to exhibit in-
hibitory binding mode of least energy ligands from the generated
conformations with the target proteins. Molecular docking studies were
carried out using the LeadIT 2.2.0 software, which uses flexible docking
approach [33]. Molecular docking provides preliminary information
about the binding modes of ligand molecules in and around receptor
cavities as well as predicts possible intermolecular non-covalent
bonding. Binding modes may then be used to predict the strength of
association between the ligands and receptors using certain scoring
functions that include number and types of bonds, hysteric clashes and
electronic repulsions as parameters. The Flex docking score correlates
with the binding affinity of the molecules with the target. The docking
scores were recorded, and docking poses were saved for reference. The
docking simulation results were prepared using Discovery Studio 3.5.
Compounds were docked and the highest scoring pose (Top1 and Top2)
were selected for each protein for Molecular dynamics. The best
docking poses are predicted to be the most stable conformation of each
compound for binding to the protein-active site.
2.7.5. Molecular dynamics simulation
After validation, top two poses based on binding energies were
generated and ligand protein interactions were assessed using Desmond
software and the tools associated to Schrodinger suite 2017 [32,33].
Protein ligand complexes were pre-processed using protein preparation
wizard prior to solvation. The system for simulation was prepared using
the ‘system builder’ utility and the system was solvated by TIP3P to
stimulate water molecules. Orthorhombic water box was used by
keeping 10 Å distance away from each edge of the box. Na+ and Cl−
ions were added automatically to ensure the overall neutrality of the
systems. The model system was relaxed using the default protocol im-
plemented in Desmond, utilized to prepare systems for production si-
mulation. The relaxation protocol implemented in Desmond involved a
series of minimizations followed by a series of short MD runs to relax
5
Biomedicine & Pharmacotherapy 116 (2019) 108961
the model systems. The temperature and pressure conditions were
stabilized under NpT (isothermal and isobaric)-ensemble using default
protocol of Desmond. The solvated systems after a minimization over a
maximum of 2000 steps, was ready for trajectory production. Finally,
systems were simulated under biological conditions in the NpT-en-
semble using OPLS-3 (optimized potentials for liquid simulations) force
field parameters [34]. The run time for each complex was 20 ns, re-
corded every 0.2 ps for a total of 100 frames [35].
2.8. Statistical analysis
All the values are expressed as mean ± SEM (n = 8). Statistical
analysis was performed in Graph Pad Prism 7 software (San Diego, CA,
USA) using One-way analysis of variance (ANOVA) followed by Tukey’s
test as a post hoc analysis. The value of P < 0.05 was considered to be
statistically significant.
3. Results
3.1. Effect of CM extract in cisplatin-induced toxicity in HEK-293 cells
The efficacy of the plant extract (CM) was evaluated in cisplatin
induced cytotoxicity in human embryonic kidney (HEK-293) cells. Cell
viability was evaluated to assess the cytoprotective effect of CM extract
in cisplatin treated HEK-293 cells. HEK-293 cells were treated with
various concentrations of CM extract (5, 10, 25, 50, 100 and 200 μg/
mL) alone or in combination with Cisplatin (20 μM) for 24 h. Treatment
with CM extract alone did not induce any overt detrimental effect on
cell viability. Cisplatin treatment significantly (P < 0.001) reduced the
cell viability and was associated with morphological changes such as
cell shrinkage, rounded cell shape and cytoplasmic vacuolation com-
pared to normal control. To measure the effects of CM extract on the
growth of CP-treated renal cells, HEK-293 cells were treated with CP
(20 μM) and/or different concentration of CM extract. Cell viability was
significantly improved when cells were co-treated with CM extract and
CP. The cell viability was improved by 7–25% with CM treatment
M. Kpemissi, et al.
compared to cisplatin control. The EC50 was determined to be 8.136 μg/
mL (Fig. 2).
3.2. Effect of CM extract in Cisplatin-induced toxicity in Wistar rats
3.2.1. Body weight changes
The administration of CP resulted in significant (P < 0.001) loss in
body weight as compared to normal control group (Fig. 3). However,
CM administration attenuated CP induced loss of body weight.
3.2.2. Serum biochemical markers
After a single dose injection of cisplatin, serum levels of creatinine,
urea and uric acid (Fig. 4A–C) in the cisplatin-control group were
significantly increased (P < 0.001) compared to the normal control
group, while the levels of total protein (TP), albumin (Alb) (Fig. 5A and
B) and electrolytes (calcium, Magnesium and phosphorus) (Fig. 6
A,B,C), were significantly (P < 0.01) decreased. In addition, the serum
levels of AST, ALT, ALP and γGT enzymes activity values were also
significantly (P < 0.01) increased in the CP-treated group compared to
Fig. 4. Effect of Combretum micranthum (CM) on Serum Creatinine [A], Urea [B], Uric Acid [C] and Urine Creatinine [D], Urea [E], Uric Acid [F] in CP-induced
nephrotoxicity. Data are expressed as mean ± SEM (n = 8). One-way ANOVA followed by Tukey's multiple comparisons test. Compared to normal control group:
## P < 0.01, ### P < 0.001; Compared to Cisplatin control group: * P < 0.05, ** P < 0.01, *** P < 0.001.
6
Biomedicine & Pharmacotherapy 116 (2019) 108961
Fig. 3. Effect of Combretum micranthum (CM)
extract on body weight [A] and relative weight
of kidney [B] in cisplatin (CP)-induced ne-
phrotoxicity in rats. Data are expressed as
Mean ± SEM (n = 8). One-way ANOVA fol-
lowed by Tukey's multiple comparisons test.
Compared to normal control group: ##
P < 0.01, ### P < 0.001; Compared to
Cisplatin control group: * P < 0.05, ***
P < 0.001.
the normal control (Fig. 7). These elevated levels of renal function
parameters as a result of cisplatin treatment confirmed the induction of
nephrotoxicity. However, pre-treatment with different doses of CM
extract (200 and 400 mg/kg) resulted in significant (P < 0.001) re-
duction in kidney function biomarkers such as serum creatinine, urea,
uric acid and liver function biomarkers such as ALT, AST, ALP, γGT. In
addition, the protective effect of CM extract was also confirmed by
significantly (P < 0.01; P < 0.001) restored levels of electrolytes,
total protein and albumin as compared to cisplatin-control group.
3.2.3. Urine biochemical markers
Single-dose administration of cisplatin resulted in significant
(P < 0.001; P < 0.001) alteration in kidney function urine bio-
markers such as creatinine, urea, uric acid, total protein, albumin
(Fig. 4D–F; Fig. 5C and D) and electrolytes compared to the normal
control group (Fig. 6D–F). Pre-treatment of different doses of CM ex-
tract exhibited significant (P < 0.001; P < 0.01; P < 0.05) normal-
ization of all the kidney function parameters as compared to cisplatin-
control group. Moreover, both kidney and liver function tests using
M. Kpemissi, et al. Biomedicine & Pharmacotherapy 116 (2019) 108961

Fig. 5. Effect of Combretum micranthum (CM)

on Serum Total Protein [A], Albumin [B] and
Urine Total Protein [C], Albumin [D] in CP-
induced nephrotoxicity. Data are expressed as
Mean ± SEM (n = 8). One-way ANOVA fol-
lowed by Tukey's multiple comparisons test.
Compared to normal control group: ##
P < 0.01, ### P < 0.001; Compared to
Cisplatin control group: * P < 0.05, ***
P < 0.001.

serum and urine samples revealed that pre-treatment of CM extract
accelerated recovery from CP-induced nephrotoxicity and hepatotoxi-
city.
3.2.4. Oxidative-stress parameters
Cisplatin-challenged rats exhibited significantly (P < 0.001;
P < 0.01) higher levels of tissue MDA (a lipid peroxidation marker)
and NO, while significant (P < 0.001) reduction in both tissue and
serum antioxidants such as GSH, NO and FRAP respectively as com-
pared to normal rats (Fig. 8). Pre-treatment with both the doses of CM
extract showed significantly (P < 0.001; P < 0.01; P < 0.05) altered
levels of enzymatic and non-enzymatic endogenous antioxidant levels
compared to cisplatin-control group.
3.2.5. Histopathological outcomes
The histological sections of renal tissue in the control group (dis-
tilled water) exhibited normal architecture of renal tubules, en-
dothelium and no inflammatory cell infiltration within the interstitium.
The glomeruli showed thin walled Bowman’s capsule and no tuft re-
traction (medium score = 0) (Fig. 9A). The examination of the kidney
sections from cisplatin-control group showed marked tubular dilation,
extensive epithelial necrosis and denudation of the epithelium,

Fig. 6. Effect of Combretum micranthum (CM) on Serum Calcium [A], Magnesium [B], Phosphorus [C] and Urine Calcium [D], Magnesium [E] and Phosphorus [F] in
CP-induced nephrotoxicity. Data are expressed as Mean ± SEM (n = 8). One-way ANOVA followed by Tukey's multiple comparisons test. Compared to normal
control group: ## P < 0.01, ### P < 0.001; Compared to Cisplatin control group: * P < 0.05, ** P < 0.01, *** P < 0.001.

7
M. Kpemissi, et al. Biomedicine & Pharmacotherapy 116 (2019) 108961

Fig. 7. Effect of Combretum micranthum (CM)

on Serum ALAT [A], ASAT [B], GGT [C] and
PAL [D] in CP-induced nephrotoxicity. Data are
expressed as Mean ± SEM (n = 8). One-way
ANOVA followed by Tukey's multiple compar-
isons test. Compared to normal control group:
###
P < 0.001; Compared to Cisplatin control
group: ** P < 0.01, *** P < 0.001.

affecting more than 60% of the epithelial cells; tubular cast formation,
dystrophic mineralization, epithelial regeneration and mild neutrophil
infiltration were also noted at the level of the renal convoluted tubules.
The small blood vessels were multifocally affected by endothelial
swelling, necrosis and occasional thrombosis. No important changes of
glomeruli were noted. The tubulointerstitial space was mildly distended
by congestion, edema and mixed inflammatory infiltrates, affecting less
than 25% of renal tissue (medium score = 4) (Fig. 9B and C). The
histological changes of renal tissues from pre-treated rats with CM ex-
tract (200 mg/kg) consisted of multifocal epithelial coagulative ne-
crosis, affecting less than 60% of renal tubules, cast formation, thick-
ening of the renal tubular basement membrane, vascular endothelial
cell swelling, mild interstitial congestion and edema, and multifocal
mixed inflammatory infiltrates. The glomeruli showed no significant
changes (medium score = 2) (Fig. 9D). The renal tissue of pre-treated
rats with CM extract (400 mg/kg) showed loss of brush border, mild

Fig. 8. Effect of Combretum micranthum (CM) on kidney MDA [A], FRAP [B], NO [C), GSH [D] and Serum FRAP [E], NO [F] in CP-induced nephrotoxicity. Data are
expressed as Mean ± SEM (n = 8). One-way ANOVA followed by Tukey's multiple comparisons test. Compared to normal control group: ## P < 0.01, ###
P < 0.001; Compared to Cisplatin control group: * P < 0.05, ** P < 0.01, *** P < 0.001.

8
M. Kpemissi, et al.
Fig. 9. Histopathological study of renal tissues of experimental animals (n = 5). No changes were noticed in the normal control group (A); the kidney samples of the
cisplatin control group showed extensive epithelial cell necrosis (arrows), vacuolar degeneration, and severe interstitial congestion and edema (B&C); the kidney
samples from the CM 200 mg/kg group showing focal, mild to moderate hydropic degeneration and necrosis of renal tubule epithelium (arrows) (D); no histological
changes have been identified in the CM 400 mg/kg group (E&F).
Fig. 10. Effect of Combretum micranthum (CM) on kidney Tubular injury,
Endothelial injury, Glomerular injury and Tubulointerstitial changes in CP-in-
duced nephrotoxicity. Data are expressed as Mean ± SEM (n = 5). One-way
ANOVA followed by Tukey's multiple comparisons test. Compared to normal
control group: ## P < 0.01, ### P < 0.001; Compared to Cisplatin control
group: *** P < 0.001.
vacuolar (hydropic) degeneration and occasional individual cell ne-
crosis of the proximal renal tubular epithelium, affecting less than 25%
of tubular cells. No significant histological changes of the endothelial,
glomerular and interstitial components were identified (medium
score = 1) (Fig. 9E and F). Overall, CP administration significantly
(P < 0.001) increased the injuries of tubular, endothelial and tubu-
lointerstitial changes compared to normal control group (Fig. 10).
Treatment of CM extract exhibited dose dependent protection against
9
Biomedicine & Pharmacotherapy 116 (2019) 108961
CP-induced renal damages (Fig. 10).
3.3. In silico studies
3.3.1. Molecular docking
All thirty bioactive compounds of CM were docked into the binding
site of transcription factor NF-κB (1NFK) and soluble epoxide hydrolase
(sEH; 3ANS) (Table 2). Based on docking score and binding site inter-
actions with both the proteins, the two HIT (top ranked) compounds
such isovitexin (−22.467 kcal/mol), gallic acid (−21.167 kcal/mol)
for 1NFK and cianidanol (−14.234 kcal/mol), epicatechin
(−14.209 kcal/mol) for 3ANS were subjected to molecular dynamic
experiments. The hydrogen bonding interactions of all the four HIT
bioactive compounds were depicted in Fig. 11A and B and Fig. 12 A&B.
Also, the docking poses of Protein-ligand interaction are depicted in
Fig. 11C for NF-κB and Fig. 12C for sEH.
3.3.2. Molecular dynamics
During dynamic simulation period, the percentage interactions be-
tween ligands and amino acids of active site of NF-κB (Fig. 11D and E)
and sEH (Fig. 12D and E) were recorded and represented. Furthermore,
to assess the dynamic behaviour and conformational stability of the
docked ligands into the binding site of proteins, molecular dynamic
simulation of NF-kB bound with isovitexin and gallic acid and sEH
bound with cianidanol and epicatechin were examined by monitoring
the RMSD of protein alone and its respective docked complexes. Fur-
ther, in order to identify the deviation of the ligand with respect to its
initial pose as well as extent of movement of various residues of NF-kB
and sEH, RMSF values of tested protein as well as respective protein-
ligand complexes were also computed by averaging overall conforma-
tions during 20 ns simulation (Fig. 13). The root mean square deviation
M. Kpemissi, et al. Biomedicine & Pharmacotherapy 116 (2019) 108961

Table 2 simulation period, there was a slight increase in RMSD values (4.5 Å).
Docking score of bioactive compounds from Combretum micranthum against This small contrary observation can be revalidated by undertaking
nuclear factor kappa B (NF-κB) and soluble epoxide hydrolase (sEH). higher simulation period. Furthermore, complex of epicatechin with
NF-κ B [1NFK] Docking sEH [3ANS] Docking sEH was found to be unstable up to 10 ns and attained better stability in
score score rest of the simulation period with an RMSD of 4.5 Å (Fig. 13B). From
RMSD analysis, two ligands (gallic acid and cianidanol) exhibited good
Isovitexin −22.467 Cianidanol −14.234
interactions with the key residues of respective proteins and remained
Gallic acid −21.167 Epicatechin −14.209
Epigallocatechin −19.972 Epigallocatechin −12.932
bounded in the binding site throughout simulation period (Fig. 13 A
Luteoforol −19.895 Luteoforol −12.705 and B).
Quercitrin −19.766 Gallic acid −10.903 As can be seen from Fig. 13C, RMSF value of native protein NF-κB
4-Hydroxyproline −19.729 Lupeol −9.302 was around 5.0 Å with fluctuation at some regions. However, the
betaine
binding of isovitexin and gallic acid individually with NF-κB showed an
Turicine −19.534 α-Tocopherol −8.986
Stachydrine −19.515 Vitexin −8.955
average RMSF of 2.0 Å and a marked reduction in fluctuation as com-
Epicatechin −18.141 Turicine −7.754 pared to the native protein. For sEH protein, the native protein did not
Cianidanol −17.722 Quercitrin −6.821 show any fluctuations and stayed in the range of 1 Å, but in complex
Betaine −16.793 Stachydrine −6.528 with epicatechin showed higher fluctuation with an RMSF value of
Vitexin −16.549 4-Hydroxyproline_betaine −6.526 4.4 Å. Whereas, cianidanol complex exhibited an RMSF value of 2.5 Å
Linolenic acid −10.761 Betulin −6.503
Lupeol −9.986 Isovitexin −6.35
and less fluctuation compared to epicatechin complex (Fig. 13D).
Betulin −8.893 Betaine −5.888 Hydrogen-bonds play an important role in ligand and protein
Oleic acid −8.421 Choline −3.426 binding. These interactions including hydrophobic and ionic interac-
Palmitic acid −8.395 β-Sitosterol −2.883 tions resulted in selectivity, specificity and important physicochemical
Choline −7.327 Linolenic_acid −1.786
properties triggering pharmacological actions of ligand under in-
β-Sitosterol −6.859 Palmitic acid 1.174
α-Tocopherol −5.477 Oleic acid 1.644 vestigation. isovitexin posses hydrogen bond and hydrophobic inter-
actions with twelve and five amino acids respectively, while gallic acid
interacted with four and two amino acids in the pocket of DNA binding
(RMSD) of protein backbone atoms of native protein NF-κB exhibited site of NF-κB respectively (Fig. 14 A and B). Further, epicatechin
average RMSD of 7.5 Å. The native protein and its complex with iso- showed hydrogen and hydrophobic bonds each with nine amino acids,
vitexin were not much stable throughout the simulation, however, this while cianidanol interacted each with ten amino acids associated with
complex was found to be similar as that of native protein (Fig. 13A). In the active site of sEH (Fig. 14 C and D).
case of gallic acid-NF-κB complex, average RMSD value was found to be
4 Å and this complex attained stability from 5 ns to end of the simula- 4. Discussion
tion period. The gallic acid-NF-κB complex shows stable confirmation
as compare to native protein (Fig. 13A). Cisplatin is the first-line drug in platinum-based chemotherapy for
The protein backbone atoms of sEH exhibited RMSD value of 3.5 Å the treatment of various cancers [20]. Cisplatin exposure is usually
and maintain stability throughout simulation period. The most stable associated with nephrotoxicity, caused by activation of stress, in-
confirmation was observed for sEH-cianidanol than sEH-epicatechin flammation and apoptosis via generation of reactive oxygen species
complex. Further, sEH-cianidanol complex attained stability after (ROS). Therefore, strategies to target multiple pathophysiological pro-
equilibration of 5 ns and this stability were maintained throughout the cesses, including the suppression of oxidative stress, inflammation and
simulation period with an RMSD value of 4 Å but at the end of the renal cytoprotection to prevent cisplatin-induced nephrotoxicity [36].

Fig. 11. Stereo-view of [A] isovitexin; [B] gallic acid docked in to the DNA binding region of NF-κB (1NFK) and their hydrogen bond interactions; [C] Best docked
poses of isovitexin & gallic acid in the active site of 1NFK; [D & E] The percentage hydrogen bond interactions of isovitexin and gallic acid with amino acid residues
present in the active site of 1NFK throughout the dynamic simulation period of 20 ns.

10
M. Kpemissi, et al. Biomedicine & Pharmacotherapy 116 (2019) 108961

Fig. 12. Stereo-view of [A] epicatechin; [B] cianidanol docked in to the active site of sEH (3ANS) and their hydrogen bond interactions; [C] Best docked poses of
epicatechin & cianidanol in the active site of 3ANS; [D & E] The percentage hydrogen bond interactions of epicatechin and cianidanol with amino acid residues
present in the active site of 3ANS throughout the dynamic simulation period of 20 ns.

Based on traditional medicine uses [37], chemical compositions
[9,10,29,38], antioxidant [10,12,39] and anti-inflammatory [13]
properties of Combretum micranthum (CM), form the basis for the pre-
sent study conducted to examine the protective effects of CM against
cisplatin (CP)-induced toxicity in HEK-293 cells and kidney injury in
Rats. Favourably, the study reveals that CM extract per se has no cy-
totoxic effect. However, cisplatin led to a significant increase in cell
death with changes in normal cellular morphology in HEK-293 cells.
HEK-293 cells treated with CP and CM extract resulted in significant
enhancement of cell growth compared to CP control indicating the
cytoprotective activity of CM-extract against cisplatin induced
cytotoxicity. In addition, CP-treated rats depicted typical clinical and
pathological symptoms such as increased relative kidney weight, al-
tered kidney function parameters such as creatinine, urea, uric acid,
total protein, albumin and electrolytes [21]. Further, increased levels of
creatinine, urea, and uric acid in animals treated with cisplatin in-
dicated a reduction in glomerular filtration rate. Similarly, the increase
in the relative weight of the kidneys is attributed to the retention of
urine due to tubular obstruction caused by plasters [36]. Our results are
similar to previous findings on cisplatin induced nephrotoxicity.
Treatment with CM extract resulted in marked amelioration of these
altered parameters in HEK-293 cells and kidney injury in Rats. Thus,

Fig. 13. Molecular dynamic simulation: [A] & [C] Root means standard deviation (RMSD) and Root means standard fluctuations (RMSF) of wild type NF-κB (1NFK)
alone and in complex with isovitexin & gallic acid; [B] & [D] Root means standard deviation (RMSD) and Root means standard fluctuations (RMSF) of wild type
soluble epoxide hydrolase (3ANS) alone and in combination with epicatechin & cianidanol. [E] Structural representation of four hit compounds.

11
M. Kpemissi, et al.
Fig. 14. Interaction fractions of amino acid residues of protein with ligands. [A] isovitexin & [B] gallic acid with NF-κB (1NFK) and [C] epicatechin & [D] cianidanol
with sEH (3ANS).
the models of CP-induced cell death in HEK-293 cells and kidney injury
in Rat were successfully established and the results confirm the ne-
phroprotective activity of CM extract.
Oxidative stress plays a central role in cisplatin-induced renal
physiopathology and is likely to cause cellular lesions and necrosis of
the kidneys [3]. Several studies previously carried out have shown the
key role of free radicals in lipid peroxidation and that of antioxidant
enzymes such as superoxide dismutase and catalase in counteracting
their actions [21]. Antioxidant enzymes such as CAT, SOD and GPx
neutralize free radicals and are considered the first cellular defense
barrier against oxidative stress. The second non-enzymatic defense
barrier, such as the GSH, reinforces the first [21]. The results of our
study showed that the administration of cisplatin significantly increased
lipid peroxidation by production of MDA and NO and caused depletion
of GSH and FRAP. CM extract administration significantly reduced
MDA and enzyme levels of renal toxicity and increased FRAP activity,
comparable to normal animals. In addition, free radical scavengers are
known to prevent acute renal failure through attenuation of tubular
damage, enhanced regenerative response of tubular cells and pre-
servation of renal blood flow [36]. Thus the antioxidant activity of CM
was confirmed in our studies and the results suggest the implication of
this antioxidant property contributing to its nephroprotective activity
[10,12,39].
In the present study, cisplatin administration resulted in severe
nephropathy, with impaired histologic features of the kidneys and loss
of body weight. Histological examination by H&E staining showed
cisplatin-induced lesions and damage as evident by moderate to severe
hydropic degeneration and necrosis of both proximal and distal renal
tubules. Severe interstitial congestion and edema in the cortical and
medullar areas have also been noted. Evaluations of animals in the CM
extract (400 mg/kg) treated group indicate that the extract combats
deleterious effects of cisplatin and kidney histology revealed near to
normal histological architecture. Histological findings are in agreement
with those of biochemical data and are consistent with the previous
studies [21]. Cisplatin administration is known to produce hepato-
toxicity [40] as is evident by the significant increase in liver function
parameters such as hepatic transaminases seen in the present study.
12
Biomedicine & Pharmacotherapy 116 (2019) 108961
Treatment with CM extract resulted in normalisation of liver function
marker levels and suggested it’s hepatoprotective activity in addition to
nephroprotective activity.
Numerous physiological studies have shown that urinary excretion
of water and major electrolytes (Na+, K+, Cl−, H+, Ca2+, Mg2+ and
phosphate) are due to kidney physiology [41]. Administration of cis-
platin to the rats resulted in higher levels of electrolytes in urine,
whereas lower levels in serum. Both doses of the CM significantly re-
duced acute cisplatin-induced nephrotoxicity by restoring the altered
levels of electrolytes such as magnesium, calcium and phosphorus in
urine and serum samples.
NF-κB is a ubiquitously expressed transcription factor that mediates
signal-induced expression of many genes involved in different biolo-
gical processes, including immune and inflammatory responses. In ad-
dition, NF-κB is an essential mediator of signal transduction stimulated
by several pro-inflammatory cytokines, thus participating in the ef-
fector phase of inflammation [42,43]. Further, NF-κB is activated by
different pathophysiological processes in renal cells and responsible for
inflammation in kidney diseases [42,43]. Chronic inflammation and
oxidative stress, processes closely associated with NF-κB activation,
play a key role in the development and progression of chronic renal
disease. NF-κB has also been implicated in cisplatin nephrotoxicity
[2,42–44]. Beneficial effects in experimental kidney injury have been
reported for agents that inhibit or antagonize NF-κB activating stimuli
[42]. Several studies showed that many herbal medicines protected CP-
induced kidney injury by inhibiting NF-κB activation [44]. It is there-
fore clear that the targeting of inflammation (NF-κB) represents an at-
tractive therapeutic approach in the treatment of kidney disease [43].
It was initially believed that soluble epoxide hydrolase (sEH) was
involved only in the metabolism of xenobiotics. Now it is well estab-
lished that fatty acid epoxides are excellent substrates for this enzyme
[45]. As a component of the arachidonic acid cascade, sEH plays an
important role in the metabolism of eicosanoid epoxides [45,46]. sEH
catalyzes the hydrolysis of epoxyeicosatrienoic acids (EETs) into the
corresponding dihydroxyeicosatrienoic acids and is expressed in var-
ious cells and tissues, including liver, kidney, vascular endothelium,
leukocytes, and adipocytes [45]. EETs have many biological functions,
M. Kpemissi, et al.
such as anti-inflammatory, antihypertensive, cardioprotective, re-
noprotective, vasodilator effects [45,47]. In addition, several studies
have reported other potential anti-inflammatory effects of EETs, in-
cluding decreased nitric oxide production and pro-inflammatory med-
iators [45,47]. The sEH inhibitors have been found to have good anti-
inflammatory and nephroprotective activities in several studies
[45–47]. Several experiments have shown that EETs provide ne-
phroprotective potential against cisplatin via their antioxidative, anti-
inflammatory and antiapoptotic activities [45,48]. sEH inhibitors re-
present a novel approach in cisplatin nephrotoxicity [47]. The above
mentioned facts prompted us to investigate the molecular docking and
dynamic behaviour of thirty bioactive compounds reported from the
title plant against NF-κB and sEH. The in silico experiments reveal that
gallic acid and cianidanol exhibited potent inhibition of NF-κB and sEH
protein respectively with better binding affinity and structure stability
inside the binding pockets during dynamic simulation period.
CM extract has a good nephroprotective effect which could be at-
tributed to bioactive compounds such as isovitexin, gallic acid, ciani-
danol and epicatechin present in the extract. Our results are in con-
cordance with literature reports, which indicate that flavonoids are
good nephroprotective compounds [8]. Indeed, these compounds ap-
pears to be involved in several pharmacological pathways such us anti-
inflammatory, antioxidant, anti-apoptotic [2,8]. Our results suggest
that NF-κB and sEH inhibition might attenuated cisplatin-induced
kidney injury.
5. Conclusion
Our results demonstrated that CM extract exerts its renoprotective
effect against CP-induced toxicity in both in vitro and in vivo models
established in HEK-293 cells and rat kidney. We propose that CM ex-
tract can be considered as a safe nephroprotective supplement during
cisplatin chemotherapy. This observed beneficial actions can be cred-
ited to the suppression of inflammation by inhibiting NF-κB and sEH
upregulation, oxidative stress and autophagy activations.
Competing interest
The authors declare no competing financial interest.
Acknowledgements
This work was partly supported by Department of Science &
Technology, Government of India through CV Raman International
Fellowship for African Researchers (DST/INT/CVRF/2016 dated 01/
08/2017). The authors also appreciate Sree Siddaganga College of
Pharmacy, Tumakuru, Department of Biotechnology, Siddaganga
Institute of Technology, Tumakuru and Biotechnology Skill
Enhancement Programme (BiSEP) funded by KBITS, Government of
Karnataka, India for their support in providing the required computa-
tional resources for carrying out part of this project.
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