Journal of Ethnopharmacology 243 (2019) 112117

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

Ameliorative influence of Cnestis ferruginea vahl ex DC (Connaraceae) root T

extract on kainic acid-induced temporal lobe epilepsy in mice: Role of
oxidative stress and neuroinflammation
Emmanuel S. Ojoa, Ismail O. Isholaa, Benneth Ben-Azub,e, Olasunmbo O. Afolayanc,
Ayorinde B. Jamesd, Abayomi M. Ajayib, Solomon Umukorob, Olufunmilayo O. Adeyemia,*
a
Department of Pharmacology, Therapeutics and Toxicology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos State, Nigeria
b
Neuropharmacology Unit, Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria
c
Department of Anatomy, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos State, Nigeria
d
Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos State, Nigeria
e
Department of Pharmacology, Faculty of Basic Medical Sciences, PAMO University of Medical Sciences, Port Harcourt, Rivers State, Nigeria

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

Keywords: Ethnopharmacology relevance: the root decoction of Cnestis ferruginea Vahl ex DC (Connaraceae) is widely used
COX-2 in traditional African medicine for the treatment of various ailments including pain, inflammation and epilepsy.
NF-κB We have earlier reported anticonvulsant effect of Cnestis ferruginea root extract in mice.
Hippocampus Aim of the study: to evaluate the effect of ethanolic root extract of Cnestis ferruginea (CF) on kainic acid (KA)-
Oxidative stress
induced temporal lobe epilepsy (TLE) in mice as well as the involvement of inflammatory mediators and oxi-
Neuroinflammation
dative stress.
Materials and methods: mice were randomly divided into preventive treatment (vehicle (normal saline) or CF
(400 mg/kg, p.o.) for 3 consecutive days before KA (5 mg/kg, i.p.) on days 4 and 5. In the reversal model, KA
(5 mg/kg, i.p.) was administered on days 1 and 2 before vehicle or CF (400 mg/kg) administration on days 3–5.
The effect of treatments on seizure severity was recorded using Racine scale. Animals were euthanized on day 5,
6 h after last KA exposure in preventive model and 1 h after CF administration in reversal model to estimate
markers of oxidative stress and neuroinflammation.
Results: exposure of mice to KA induced TLE evidenced in increased severity of seizures which was significantly
reduced by the pre- and post-treatment of mice with CF. Moreso, KA-induced malondialdehyde/nitrite gen-
eration and GSH deficit in the brain were attenuated by CF treatments. KA-induced up-regulation of in-
flammatory transcription factors; cyclooxygenase-2 (COX-2) and nuclear facor-kappaB (NF-κB) in the CA1, CA2,
CA3 and dentate gyrus (DG) regions of the hippocampus regions were attenuated by CF treatments.
Conclusion: findings from this study showed that Cnestis ferruginea root extract ameliorated KA-induced TLE
through enhancement of antioxidant defense mechanism and attenuation of neuro-inflammatory transcription
factors. Thus, could possibly be a potential phytotherapeutic agent in the management of temporal lobe epilepsy.

1. Introduction
Epilepsy is a chronic neurological disorders characterized by re-
current unprovoked seizures with variation in occurrence and severity
(Szilágyi et al., 2014). Temporal Lobe Epilepsy (TLE) being the most
common form of partial epilepsy (Téllez-Zenteno and Hernández-
Ronquillo, 2012), is characterized by epileptic seizures originating
mainly in temporal lobe areas. One of the major pathological findings in
TLE is hippocampal sclerosis, characterized by massive neuronal loss
and severe gliosis in the hippocampal formation and in the other re-
gions of temporal lobe (Fernandes et al., 2015; Huang et al., 2015). The
epileptogenesis process of TLE usually starts with initial precipitating
insults, followed by neurodegeneration, abnormal hippocampus cir-
cuitry reorganization, and the formation of hypersynchronicity (Huang
et al., 2015. The mechanism by which inciting injury induces epi-
leptogenesis, involves multiple cellular, molecular, and physiological
changes resulting in altered hyperexcitable circuitry. Imaging and
tissue analysis of TLE patients indicate mitochondrial impairment

*
Corresponding author.
E-mail address: ooadeyemi@cmul.edu.ng (O.O. Adeyemi).

https://doi.org/10.1016/j.jep.2019.112117
Received 1 May 2019; Received in revised form 18 July 2019; Accepted 23 July 2019
Available online 24 July 2019
0378-8741/ © 2019 Elsevier B.V. All rights reserved.
E.S. Ojo, et al.
(Rowley et al., 2013). Moreso, animal studies showed oxidative damage
to cellular macromolecules during different phases of epileptogenesis
(Rowley et al., 2013, 2015).
Several studies have shown the potential role of neuroinflammation
in epileptogenesis. Moreso, excessive activation of inflammatory sig-
nalling pathways seems to be the hallmark of epileptogenesis
(Marcheselli and Bazan, 1996; Takemiya et al., 2006; Rojas et al., 2014;
Russmann et al., 2017). It is well known that COX-1 is constitutively
expressed in neurons, astrocytes, and microglial cells (Marcheselli and
Bazan, 1996). Several reports have shown that seizures induced COX-2
in hippocampal neurons within hours (Marcheselli and Bazan, 1996;
Takemiya et al., 2006; Rojas et al., 2014). Moreover, anti-epileptic ef-
fect of COX-2 inhibitors are well reported, in addition, COX-2 is upre-
gulated in cells under pathophysiological conditions (Citraro et al.,
2015). In hippocampal long-term potentiation, COX-2, PGE synthase,
and PGE2 are induced in post-synaptic neurons (Citraro et al., 2015;
Yagami et al., 2016). Pronounced increases in the expression of key
inflammatory mediators (COX-2, NF-κB, TNF-α) after seizures may
cause secondary damage in the brain and increase the likelihood of
repetitive seizures (Rojas et al., 2014). The increased level of COX-2 in
specific areas of the epileptic brain could assist in the identification of
regions of seizure-induced neuroinflammation. The kainic acid (KA)
model presents with neuropathological and electroencephalographic
features that are seen in patients with TLE (characterized by a latent
period that follows the initial precipitating injury (i.e., status epi-
lepticus) until the appearance of recurrent seizures, as observed in the
human condition) (Lévesque and Avoli, 2013). Moreover, systemic
administration of KA causes neuronal cell death, mossy fibre sprouting
and spontaneous seizure in rodents (Ben-Ari et al., 1978).). In 50–70%
of people with TLE the condition cannot be adequately treated by the
present antiepileptic drugs (van Vliet et al., 2014). Thus, the need for
better therapeutic options, natural product could be a good source of
drug development for epilepsy owing to their polypharmacologic or
multi-targets activities. Cnestis ferruginea DC (Connaraceae) is widely
used in Traditional Africa medicine for the treatment of conjunctivitis,
syphilis, gum pain, wounds, dysentery, epilepsy and gonorrhoea
(Burkill et al., 2000). We have earlier reported antioxidant (Ishola et al.,
2013a), anti-inflammatory, analgesic (Ishola et al., 2011, 2013b), an-
ticonvulsant (Ishola et al., 2014), antidepressant/anxiolytic (Ishola
et al., 2012, Ishola et al., 2016) effects of CF and its active constituent,
amentoflavone using in vitro and in vivo models. We have earlier re-
ported that Cnestis ferruginea root extract; reduced the duration of
tonic hind limb extensions evoked by electrical stimuli, delayed la-
tencies to strychnine, picrotoxin, isoniazid, and yohimbine-induced
seizures. In addition, the extract completely inhibited bicucculine-in-
duced seizures in mice indicative of its ability to enhance glycinergic
and GABAergic neurotransmission (Ishola et al., 2014). Hence, this
study sought to evaluate the ameliorative influence of ethanol root
extract of Cnestis ferruginea on kainic acid-induced temporal lobe
epilepsy in mice as well as the role of oxidative stress and neuroin-
flammation.
2. Materials and methods
2.1. Drugs and chemicals
Kainic acid, ethanol, hydrogen peroxide, sodium azide, xylene,
thiobarbituric acid, trichloroacetic acid, Griess reagent (Sigma Aldrich,
St. Loiuis MO, USA.), normal goat serum (Thermo-Fischer Scientific
USA.), phosphate buffer (Gibco Life Technologies, USA.), liquid ni-
trogen (BOC gas Nigeria limited), Rabbit COX-2 (E-AB-17010,
Elabscience, USA), NF-κB p65 (ab 16502, Abcam, USA), 3,3-diamino-
benzidine, secondary antibody, Vectastain ABC Elite kit (Vector
Laboratories, USA), normal saline (Unique Pharmaceuticals Ltd,
Nigeria).
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Journal of Ethnopharmacology 243 (2019) 112117
2.2. Laboratory animals
Eight weeks old male Swiss male albino mice (20–25g) used in this
study were obtained from the Laboratory Animal Centre of the College
of Medicine, University of Lagos, Nigeria. The animals were kept in
polyacrylic cages containing 6 animals per cage and maintained under
standard housing conditions (room temperature 24–27 °C and humidity
60–65%) with a 12 h light and dark cycle. Food, in the form of dry
pellets, and water were available ad libitum. The experimental proce-
dures adopted were in compliance with the ethical approval obtained
from Health Research Ethics Committee of the College of Medicine,
University of Lagos, Nigeria (CMUL/HREC/01/19/482) and in ac-
cordance with the United States National Institutes of Health Guidelines
for Care and Use of Laboratory Animals in Biomedical Research (NIH,
2011).
2.3. Extraction procedure
The dried roots of Cnestis ferruginea (484g) were pulverized. The
powdered root was macerated in 2.5 L of ethanol for 72 h with inter-
mittent agitation, after which the filtrate decanted and filtered. The
filtrate obtained was evaporated to dryness in an oven at 40 °C. The
weight of the dried extract was 12g (percentage yield = 2.48%w/w).
moreover, Gas chromatography-mass spectroscopic analysis revealed
the presence of 9,12-octadecanoic acid, 9-octadecanoic acid, 9,12,15-
octadecanoic acid, linoleic acid, sitosterol, and stigmasterol
(Supplementary Fig. 1).
2.4. Treatment regimen
Mice were divided into 5 groups (n = 12) as follows; Group I: ve-
hicle (10 ml/kg, p.o., 5 consecutive days); Group II: CF (400 mg/kg,
p.o.) + vehicle (10ml/kg, 5 consecutive days), Group III: vehicle 10ml/
kg + KA (5mg/kg, i.p., days 4 and 5), Group IV: CF (400 mg/kg, p.o., 3
consecutive days) + KA (5mg/kg, i.p., days 4 and 5), Group V: KA
(5mg/kg, i.p., days 1 and 2) + CF (400 mg/kg, p.o., days 3–5) (see
Fig. 1). The choice of CF (400mg/kg) was based on our previous study
(Ishola et al., 2014). Moreso, CF (400 mg/kg, p.o.) does not affect
spontaneous locomotor activity nor induce mortality (Ishola et al.,
2011), while KA (5 mg/kg, i.p.) was given in divided doses to reduce
mortality based on our preliminary study and in accordance with the
study of Sumanont et al. (2007). Several studies have shown that status
epilepticus occurs approximately 1 h after KA injection and loss of
pyramidal cells of the hippocampal CA1, CA2, CA3 and DG regions
peaked 6 h post KA exposure (Sumanont et al., 2007, Lévesque and
Avoli, 2013, Park et al., 2016).
2.5. Behavioural study
After kainic acid administration, the animals were place in-
dividually in a polyacrylic cage and monitored for 4 h. The duration and
severity of seizures were scored according to the rating scale of Sperk
(1994): 0- normal behaviour; 1 = wet dog shakes, 2 = strongly in-
creased frequency of wet dog shakes, mild myoclonic twitching of head,
face/forelimbs; 3 = clonic seizures and mouth foaming; 4- prolonged,
generalized severe seizures with frequent rearing and falling over to
one side; 5- generalized seizures (status epilepticus) with early death.
2.6. Dissection and tissue perfusion/homogenization
Six hours after KA administration, animals were anaesthetized with
chloral hydrate (300mg/kg, i.p.), then perfused with normal saline
(biochemical assay) and 4% paraformaldehyde (immunohistochemical
assay). The skull was cut open and the brain was exposed from its dorsal
side. The whole brain was quickly removed on an ice-cold plate. The
isolated brains were weighed and homogenized in 0.03 M sodium
E.S. Ojo, et al. Journal of Ethnopharmacology 243 (2019) 112117

phosphate buffer, pH-7.4 with an Ultra-Turrax T25 (USA) homogenizer

at a speed of 9500 rpm and kept at −20 °C for biochemical assay. The
brains for immunohistochemistry were kept at −80 °C until processed.

2.7. Estimation of biochemical markers of oxidative and nitrosative stress

Malondialdehyde (MDA) was spectrophotometrically measured

using the thiobarbituric acid assay procedure (Ohkawa et al., 1979),
GSH (endogenous antioxidant) was determined by its reaction with 5,
5′-dithiobis (2-nitrobenzoic acid) (Ellman’s reagent) to yield a yellow
chromophore while nitrite level (indicator of nitric oxide production)
was estimated using the method of (Green et al., 1982).

2.8. Immunohistochemical assessment

Coronal sections of the frozen brain (10 μm thick) were prepared

with cryostat and every sixth sections were fixed with 4% paraf-
ormaldehyde for 10 min, followed by washing in PBS (pH 7.4). H2O2
(0.3% in methanol) solution was used to quench endogenous perox-
idase for 30 min, followed by washing in PBS (3x, 5 min each). The
sections were incubated for 1 h in 5% normal goat serum in 0.6% Triton
X-100 in PBS. The sections were incubated overnight at 4 °C with the
selective antibody directed against COX-2 (E-AB-17010, Elabscience,
USA) or NF-κB p65 (ab 16502, Abcam, USA). Following 24 h of in-
cubation, the sections were washed 3 times with PBS (5 min each), then
incubated with biotinylated secondary antibody (1: 500, anti-IgG an-
tibody, Vector Laboratories, USA) for 1 h at room temperature, washed
in PBS for 5 min each three times. The sections were processed with
avidin-biotinylated horseradish peroxidase complex (1: 250 in PBS,
Vectastain Elite ABC kit, Vector Laboratories) for 1 h, after washing 3
times in PBS (5 min each). Antibody visualization was carried out with
3,3-diaminobenzidine (DAB kit (chromogen), Vector Laboratories,
USA). The sections were then rinsed in water and counterstained in
haematoxylin. The sections were rinsed in water and dehydrated in
Fig. 2. A–B: Effect of CF on KA-induced (a) seizures severity and (b) duration of
graded ethanol solutions, cleared in xylene before cover slips were
seizures in mice. Values are expressed as mean ± SEM (n = 12). Values are
mounted.
expressed as mean ± SEM (n = 5). **p < 0.01,***p < 0.001 versus
vehicle + KA using two way ANOVA followed by Sidak’s post hoc multiple
2.9. Statistical analysis comparison test.

Results are expressed as Mean ± SEM. The data were analyzed

lobe epilepsy evidenced in increased severity of seizures which was
using one-way or two-way ANOVA followed by Tukey’s post hoc mul-
significantly reduced by the pretreatment of mice with CF [F
tiple comparison test using Graphpad Prism (Graphpad software, ver-
(1,16) = 39.20,P < 0.001] (Fig. 2A). Moreover, KA-induced prolonga-
sion 6.0, CA, USA) .
tion of seizures was ameliorated by CF pretreatment (p < 0.001)
(Fig. 2B) on days 1 and 2.
3. Results

3.1. Effect of CF on kainic acid-induced behavioural seizure 3.2. Effect of CF on in-vivo antioxidant activities in the brain of mice

As shown in Fig. 2A, two way ANOVA revealed no significant in- Fig. 3Ashowed that the intraperitoneal injection of KA-induced
teraction between CF pretreatment and KA administration [F significant increase in MDA generation in comparison to vehicle control
(1,16) = 0.0,P = 0.99]. However, KA administration induced temporal treated group [F (4, 20) = 39.35, P < 0.0001]. However, Sidak’s post

Fig. 1. A–B: Illustration of treatment regimen (A) pretreatment of mice with CF before KA exposure and (B) treatment of mice with CF after KA exposure.

3
E.S. Ojo, et al.
Fig. 3. A–C: Effect of CF treatment on KA-induced (a) lipid peroxidation, (b)
nitrite generation and (c) GSH level in mice brain. Values are expressed as
mean ± SEM (n = 5). #p < 0.001 versus vehicle, **p < 0.01,***p < 0.001
versus vehicle + KA using one way ANOVA followed by Turkey post hoc
multiple comparison test.
hoc analysis showed that pretreatment or post-KA treatment of mice
with CF significantly attenuated KA-induced increased MDA level. In
another experiment, KA–induced nitrite generation was attenuated by
preventative and reversal treatment of mice with CF in mice [F
(4,20) = 16.20 p = 0.0002] (Fig. 3B). KA-induced deficits in GSH level
(2 folds) was reversed by pre- or post-KA treatment of mice with CF [F
(4,20) = 30.00,P < 0.001] (Fig. 3C).
3.3. Effect of CF on KA-induced NF-κB protein expression
Exposure of mice to KA significantly increased NF-κB protein ex-
pression in the CA1 hippocampus regions by 2.06 folds compared with
vehicle treated control (Fig. 4A–F). However, the post-treatment of
mice with CF after KA exposure reduced NF-κB protein expression in
the CA1 hippocampus region (Fig. 4A–F). Similarly, intraperitoneal
4
Journal of Ethnopharmacology 243 (2019) 112117
Fig. 4. A–F: Representative photomicrographs of the effect of CF on kainic acid-
induced expressions of NF-κB protein in the CA1 hippocampus regions.
A = Vehicle, B = KA (5 mg/kg), C = CF 400 mg/kg, D = CF (400 mg/
kg) + KA (5 mg/kg), and E = KA (5 mg/kg) + CF (400 mg/kg). Vertical arrow
indicates: high immunoreactivity of NF-κB protein; horizontal arrow indicates:
Low immunoreactivity of NF-κB protein. × 400. F = Effect of CF on NF-κB
immunoreactivity intensity scores in the CA1 hippocampus regions. . Values are
expressed as mean ± SEM (n = 5). #p < 0.001 versus vehicle-treated control,
b
p < 0.01, cp < 0.001 versus vehicle + kainic acid. Statistical level of sig-
nificance analysis by one way ANOVA followed by Tukey post hoc multiple
comparison test.
Fig. 5. A–F: Representative photomicrographs of the effect of CF on kainic acid-
induced expressions of NF-κB protein in the CA2 hippocampus regions.
A = Vehicle, B = KA (5 mg/kg), C = CF 400 mg/kg, D = CF (400 mg/
kg) + KA (5 mg/kg), and E = KA (5 mg/kg) + CF (400 mg/kg). Vertical arrow
indicates: high immunoreactivity of NF-κB protein; horizontal arrow indicates:
Low immunoreactivity of NF-κB protein. × 400. F = Effect of CF on NF-κB
immunoreactivity intensity scores in the CA2 hippocampus regions. . Values are
expressed as mean ± SEM (n = 5). #p < 0.001 versus vehicle-treated control,
b
p < 0.01, cp < 0.001 versus vehicle + kainic acid. Statistical level of sig-
nificance analysis by one way ANOVA followed by Tukey post hoc multiple
comparison test.
injection of mice with KA significantly increased NF-κB protein ex-
pression in the CA2 (Fig. 5A–F), CA3 (Fig. 6A–F) and DG (Fig. 7A–F)
hippocampus regions by 1.30, 2.20, and 2.25 folds, respectively, com-
pared with vehicle treated control. However, preventative treatment of
mice with CF reversed the KA-induced elevation of NF-κB protein ex-
pression in the hippocampal CA3 and DG regions by 1.50 and 1.90
E.S. Ojo, et al.
Fig. 6. A–F: Representative photomicrographs of the effect of CF on kainic acid-
induced expressions of NF-κB protein in the CA3 hippocampus regions.
A = Vehicle, B = KA (5 mg/kg), C = CF 400 mg/kg, D = CF (400 mg/
kg) + KA (5 mg/kg), and E = KA (5 mg/kg) + CF (400 mg/kg). Vertical arrow
indicates: high immunoreactivity of NF-κB protein; horizontal arrow indicates:
Low immunoreactivity of NF-κB protein. × 400. F = Effect of CF on NF-κB
immunoreactivity intensity scores in the CA3 hippocampus regions. . Values are
expressed as mean ± SEM (n = 5). #p < 0.001 versus vehicle-treated control,
a
p < 0.05, bp < 0.01, cp < 0.001 versus vehicle + kainic acid. Statistical level
of significance analysis by one way ANOVA followed by Tukey post hoc mul-
tiple comparison test.
Fig. 7. A–F: Representative photomicrographs of the effect of CF on kainic acid-
induced expressions of NF-κB protein in the DG hippocampus regions.
A = Vehicle, B = KA (5 mg/kg), C = CF 400 mg/kg, D = CF (400 mg/
kg) + KA (5 mg/kg), and E = KA (5 mg/kg) + CF (400 mg/kg). Vertical arrow
indicates: high immunoreactivity of NF-κB protein; horizontal arrow indicates:
Low immunoreactivity of NF-κB protein. × 400. F = Effect of CF on NF-κB
immunoreactivity intensity scores in the DG hippocampus regions. . Values are
expressed as mean ± SEM (n = 5). #p < 0.001 versus vehicle-treated control,
b
p < 0.01, cp < 0.001 versus vehicle + kainic acid. Statistical level of sig-
nificance analysis by one way ANOVA followed by Tukey post hoc multiple
comparison test.
folds, respectively, but not in the CA1 and CA2, compared with ve-
hicle + KA treated (Figs. 4–7). In the reversal study, post-treatment of
mice with CF after KA administration attenuated NF-κB protein ex-
pression in the hippocampal CA1, CA2, CA3 and DG regions by 3.60,
4.30, 5.10 and 6.00 folds, respectively, compared with KA-vehicle
treated (Figs. 5–7). Moreso, two way ANOVA revealed significant effect
5
Journal of Ethnopharmacology 243 (2019) 112117
Fig. 8. A–F: Representative photomicrographs of the effect of CF on kainic acid-
induced expressions of COX-2 protein in the CA1 hippocampus regions.
A = Vehicle, B = KA (5 mg/kg), C = CF 400 mg/kg, D = CF (400 mg/
kg) + KA (5 mg/kg), and E = KA (5 mg/kg) + CF (400 mg/kg). Vertical arrow
indicates: high immunoreactivity of COX-2 protein; horizontal arrow indicates:
Low immunoreactivity of COX-2 protein. × 400. F = Effect of CF on COX-2
immunoreactivity intensity scores in the CA1 hippocampus regions. . Values are
expressed as mean ± SEM (n = 5). #p < 0.001 versus vehicle-treated control,
a
p < 0.05, bp < 0.01, cp < 0.001 versus vehicle + kainic acid. Statistical level
of significance analysis by one way ANOVA followed by Tukey post hoc mul-
tiple comparison test.
of treatment [F(4,56) = 199.80,p < 0.001] and significant interaction
between CF × KA treatments [F(12,56) = 7.47,p < 0.001] ((Figs. 5–7).
3.4. Effect of CF on kainic acid-induced COX-2 protein expression
Pre- and post-treatment of mice with KA significantly (p < 0.001)
increased COX-2 protein expression by 1.30, 2.10, 1.20, and 1.20 folds,
respectively, in the hippocampal CA1, CA2, CA3 and DG regions com-
pared to vehicle control group (Figs. 8–11). However, preventative
treatment of mice with CF reversed the KA-induced elevation of COX-2
protein expression in the hippocampal CA1 (Fig. 8A–F), CA2
(Fig. 9A–F), CA3 (Fig. 10A–F) and DG (Fig. 11A–F) by 1.30, 1.30, 1.20
and 1.20 folds, respectively, compared with KA-vehicle treated group.
In the reversal study, post-treatment of mice with CF after KA admin-
istration attenuated COX-2 protein expression in the hippocampal CA1,
CA2, CA3 and DG regions by 1.40, 1.50, 1.40 and 1.50 folds, respec-
tively, compared with KA-vehicle treated. Moreover, two way ANOVA
revealed significant effect of treatment [F(4,56) = 171.80,p < 0.001]
and significant interaction between CF × KA treatments [F
(12,56) = 6.34,p < 0.001] (Figs. 8–11).
4. Discussion
This present study showed that systemic administration of kainic
acid produce various behavioral manifestations such as wet dog shakes,
twitching of head, face and forelimbs resembling status epilepticus
which was ameliorated by pre- and post-treatment of mice with CF.
Moreso, kainic acid induced neuroinflammation, oxidative and ni-
trosative stress were also attenuated by administration of CF.
Kainic acid (KA) as a tool for studying temporal lobe epilepsy has
contributed massively to the understanding of cellular and molecular
mechanisms underlying the transition and progression of temporal lobe
epilepsy. The neuropathological, behavioral and electroencephalo-
graphic features present in patients with temporal lobe epilepsy cor-
relates with kainic acid model which makes it a reliable tool in the
discovery of potential therapeutic agents for the management of the
E.S. Ojo, et al.
Fig. 9. A–F: Representative photomicrographs of the effect of CF on kainic acid-
induced expressions of COX-2 protein in the CA2 hippocampus regions.
A = Vehicle, B = KA (5 mg/kg), C = CF 400 mg/kg, D = CF (400 mg/
kg) + KA (5 mg/kg), and E = KA (5 mg/kg) + CF (400 mg/kg). Vertical arrow
indicates: high immunoreactivity of COX-2 protein; horizontal arrow indicates:
Low immunoreactivity of COX-2 protein. × 400. F = Effect of CF on COX-2
immunoreactivity intensity scores in the CA2 hippocampus regions. . Values are
expressed as mean ± SEM (n = 5). #p < 0.001 versus vehicle-treated control,
b expressed as mean ± SEM (n = 5). #p < 0.001 versus vehicle-treated con-
p < 0.01, cp < 0.001 versus vehicle + kainic acid. Statistical level of sig-
nificance analysis by one way ANOVA followed by Tukey post hoc multiple
comparison test.
Fig. 10. A–F: Representative photomicrographs of the effect of CF on kainic
acid-induced expressions of COX-2 protein in the CA3 hippocampus regions.
A = Vehicle, B = KA (5 mg/kg), C = CF 400 mg/kg, D = CF (400 mg/
kg) + KA (5 mg/kg), and E = KA (5 mg/kg) + CF (400 mg/kg). Vertical arrow
indicates: high immunoreactivity of COX-2 protein; horizontal arrow indicates:
Low immunoreactivity of COX-2 protein. × 400. F = Effect of CF on COX-2
immunoreactivity intensity scores in the CA3 hippocampus regions. . Values are
expressed as mean ± SEM (n = 5). #p < 0.001 versus vehicle-treated control,
a
p < 0.05, bp < 0.01, cp < 0.001 versus vehicle + kainic acid. Statistical level
of significance analysis by one way ANOVA followed by Tukey post hoc mul-
tiple comparison test.
neurological disorder (Lévesque and Avoli, 2013). KA is well known to
cause sustained or repetitive depolarization and firing of neurons which
eventually leads to neuronal loss (Liu, 1994). Previous reports showed
that intraperitoneal administration of KA (5 mg/kg) for two consecutive
days produced a well characterized complex seizure in mice (Miller
et al., 2014). In this study, intraperitoneal administration of KA in-
creased seizures episode and duration when compared with the vehicle
6
Journal of Ethnopharmacology 243 (2019) 112117
Fig. 11. A–F: Representative photomicrographs of the effect of CF on kainic
acid-induced expressions of COX-2 protein in the CA3 hippocampus regions.
A = Vehicle, B = KA (5 mg/kg), C = CF 400 mg/kg, D = CF (400 mg/
kg) + KA (5 mg/kg), and E = KA (5 mg/kg) + CF (400 mg/kg). Vertical arrow
indicates: high immunoreactivity of COX-2 protein; horizontal arrow indicates:
Low immunoreactivity of COX-2 protein. × 400. F = Effect of CF on COX-2
immunoreactivity intensity scores in the CA3 hippocampus regions. . Values are
trol,bp < 0.01, cp < 0.001 versus vehicle + kainic acid. Statistical level of
significance analysis by one way ANOVA followed by Tukey post hoc multiple
comparison test.
group. Forelimb clonus and rearing were the most prominent features
observed which was ameliorated by CF administration.
Emerging evidence confirms oxidative stress as a key mechanism in
several neurological disorders. Oxidative stress occurs after the first
seizure insult which then becomes the cause of epileptogenesis
(Pearson-Smith et al., 2017). Kainic acid induces oxidative stress
through disruption of calcium homeostasis and mitochondrial functions
in the hippocampus (Liang et al., 2007). Moreover, metabolic para-
meters such as gluthatione concentration, lipid peroxidation have been
proposed as one of the biomarkers for prediction of epileptogenesis
(Pitkänen et al., 2016). Findings from this study showed that the in-
traperitoneal injection of KA increased malondialdehyde and nitrite
formation as well as decrease in GSH levels indicative of nitrosative and
oxidative stresses. However, the pretreatment or post-treatment of mice
with CF before or after KA exposure attenuated membrane lipid per-
oxidation and nitration. This findings corroborated our earlier findings
of the potential of CF in the enhancement of antioxidant defense me-
chanism (Ishola et al., 2016). Neuroinflammation plays a central role in
epileptogenesis. The evidence of inflammatory processes in the clinical
manifestations and neuropathological sequelae of epilepsy have accu-
mulated in the last decade (Oprica et al., 2003). Interestingly, several
studies have shown that pro-inflammatory cytokines are the most up-
regulated biological process during epileptogenesis (Aronica et al.,
2017). Neuroinflammation molecules enhances synaptic transmission
strength through modulation of glutamate receptors expression leading
to increased neuronal excitability which contributes to decrease seizure
threshold (Vezzani and Granata, 2005). Hence, one of the aim of this
study was to examine the role of NF-κB and COX-2 in KA-induced TLE.
NF-κB a transcription factor whose upregulation have been found to be
responsible for increasing the expression of pro-inflammatory cytokines
in neuronal excitability and hippocampal gliosis, thus, making it a
feasible therapeutic targets in the treatment of TLE (Wang et al., 2017).
In this study, we examined the expression of NF-κB in hippocampi CA1,
CA2, CA3 and DG regions in KA-induced TLE. Our results showed that
NF-κB was significantly over-expressed in KA-induced TLE hippocampi
when compared with vehicle control normal. However, the
E.S. Ojo, et al.
pretreatment of mice with CF or reversal treatment attenuated KA-in-
duced up-regulation of NF-κB in the hippocampi regions.
Cyclooxygenase-2 is the most ubiquitous isoform expressed in the
brain following synaptic activity such as seizures (Kawaguchi et al.,
2005; Manabe et al., 2004; Das et al., 2012). Interestingly, arachidonic
acid as well as reactive oxygen/nitrogen species are released after sei-
zures episode or brain insult (Kawaguchi et al., 2005; Drion et al.,
2018). Moreover, COX-2, PGD2, PGE2, PGF2α, and TXA2 are elevated
in epilepsy (Marcheselli and Bazan, 1996). Several reports have shown
that seizures induced COX-2 in hippocampal neurons within hours
(Marcheselli and Bazan, 1996; Takemiya et al., 2006; Rojas et al.,
2014).interestingly, anti-epileptic effect of COX-2 inhibitors are well
reported (Citraro et al., 2015). Furthermore, COX-2 inhibitors exhibit
neuroprotective effects in vivo and in vitro model of stroke, amyo-
trophic lateral sclerosis, and epilepsy, suggesting neurotoxicities of COX
products (Yagami et al., 2016). Results from this present study revealed
that the exposure of mice to KA-induced COX-2 protein expression in
the hippocampal CA1, CA2, CA3 and DG regions suggestive of neu-
roinflammation. However, pre- and post-KA treatment with CF atte-
nuated COX-2 generation in the hippocampal regions corroborating our
earlier reported anti-neuroinflammatory effect of CF (Ishola et al.,
2013c). These findings showed that the anti-seizure effect of CF could
results from its anti-neuroinflammatory activity through inhibition of
NF-κB and COX-2 activities in the hippocampus. Thus, showed the
potential ameliorative influence of CF in KA-induced TLE, neuroin-
flammation and oxidative stress. To corroborate these findings we have
earlier reported antioxidant and anti-inflammatory effects of Cnestis
ferruginea and its bioactive flavonoid (amentoflavone) as well as co-
morbid psychiatric conditions (depression, anxiety and cognitive im-
pairments). Moreover, GC-MS analysis revealed the presence of 9,12-
octadecanoic acid, 9-octadecanoic acid, 9,12,15-octadecanoic acid, li-
noleic acid, sitosterol, and stigmasterol. Interestingly, neuroprotective,
anti-apoptosis and anti-neuroinflammatory effects of linoleic acid, β-
sitosterol and stigmasterol have been reported through regulation of
oxidative stress, apoptotic responses and pro-inflammatory mediators
(Brimson et al., 2012; Lee et al., 2018). Hence, further study is ongoing
to evaluate the protective effect of the extract in KA-induced neuronal
degeneration, micro gliosis and astrogliosis.
5. Conclusion
Findings from this showed that Cnestis ferruginea ameliorates KA-
induced temporal lobe epilepsy through enhancement of antioxidant
defence mechanism and attenuation of COX-2 and NF-κB signaling.
Hence, Cnestis ferruginea root extract could be a novel phytother-
apeutic agent in the management of epilepsy.
Author’s contribution
Ojo ES, Ishola IO, Ben-AZu B, Ajayi AM, James AB, Afolayan O are
involved in laboratory bench work while Ojo ES, Ishola IO, Umukoro S,
and Adeyemi OO are involved in study conceptualization and manu-
script writing.
Conflicts of interest
We do not have any conflict of interest to declare.
Acknowledgement
The authors are grateful to University of Lagos Central Research
grant committee, Lagos, Nigeria (CRC/2017/06) for the financial as-
sistance.
7
Journal of Ethnopharmacology 243 (2019) 112117
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jep.2019.112117.
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