Journal of Ethnopharmacology 206 (2017) 78–91
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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep
Anticonvulsant activity of Pseudospondias microcarpa (A. Rich) Engl. MARK
hydroethanolic leaf extract in mice: The role of excitatory/inhibitory
neurotransmission and nitric oxide pathway
Donatus W. Adongoa,⁎, Priscilla K. Manteb, Kennedy K.E. Kukuiac, Robert P. Bineyd,
Eric Boakye-Gyasib, Charles K. Bennehb, Elvis O. Ameyawe, Eric Woodeb
a Department of Pharmacology, School of Medicine, University of Health and Allied Sciences, Ho, Ghana
b Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and
Technology, Kumasi, Ghana
c Department of Pharmacology and Toxicology, University of Ghana School of Pharmacy, University of Ghana, Accra, Ghana
d Department of Pharmacology, School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana
e Department of Biomedical and Forensic Sciences, School of Biological Science, University of Cape Coast, Cape Coast, Ghana
A R T I C L E I N F O A B S T R A C T
Chemical compounds: Ethnopharmacological relevance: Pseudospondias microcarpa (A. Rich) Engl. is a plant used for managing
Diazepam (PubChem CID: 3016) various diseases including central nervous system disorders.
Pentylenetetrazole (PubChem CID: 73422) Aim of the study: This study explored the anticonvulsant activity of P. microcarpa hydroethanolic leaf extract
Picrotoxin (PubChem CID: 5311359) (PME) as well as possible mechanism(s) of action in animal models.
Isoniazid (PubChem CID: 3767) Methods: Effects of PME was assessed in electroconvulsive (the maximal electroshock and 6-Hz seizures) and
Strychnine (PubChem CID: 441079)
chemoconvulsive (pentylenetetrazole-, picrotoxin-, isoniazid-, 4-aminopyridine-, and strychnine-induced
4-AP (PubChem CID: 1727)
L-NAME (PubChem CID: 39836) seizures) models of epilepsy. In addition, effect of the extract on the nitric oxide pathway and GABAA receptor
L-Arginine (PubChem CID: 6322) complex was evaluated.
−1
Methylene blue (PubChem CID: 6099) Results: The extract (30, 100 and 300 mg kg , p.o.) significantly delayed the onset as well as decreased the
Sildenafil (PubChem CID: 5212) duration and frequency of pentylenetetrazole-, picrotoxin- and strychnine-induced seizures. In addition, PME
Carbamazepine (PubChem CID: 2554) pre-treatment significantly improved survival in the 4-aminopyridine- and isoniazid-induced seizure tests.
Valproic acid (PubChem CID: 16760703) Furthermore, the extract protected against 6-Hz psychomotor seizures but had no effect in the maximal
Keywords: electroshock test. The anticonvulsant effect of PME (100 mg kg−1, p.o.) was also reversed by pre-treatment with
Pseudospondias microcarpa flumazenil, L-arginine or sildenafil. However, L-NAME or methylene blue (MB) augmented its effect.
Anticonvulsant Conclusion: Results show that PME has anticonvulsant activity and may probably be affecting GABAergic,
Nitric oxide (NO) glycinergic, NMDA, K+ channels and nitric oxide-cGMP pathways to exert its effect.
Pentylenetetrazole (PTZ)
Psychomotor seizures
1. Introduction cognitive, psychological, and social disturbances (Raol and Brooks-
Kayal, 2012). Epilepsy is the second most common neurological
Epilepsy, a brain disorder, is characterized by occurrence of more disorder, with 0.5% prevalence, and a 2–3% life time risk of being
than one seizure with a persistent predisposition to generate subse- diagnosed of it (Browne and Holmes, 2001; Löscher, 2002a). More
quent epileptic seizures. This is associated with neurobiological, than 80% of people with epilepsy live in developing countries, where
Abbreviations: AED, Antiepileptic drug; CNS, Central Nervous System; PME, Pseudospondias microcarpa extract; CBZ, Carbamazepine; FMZ, Flumazenil; VPA, Valproic acid;
KNUST, Kwame Nkrumah University of Science and Technology; PTZ, Pentylenetetrazole; PTX, Picrotoxin; Hz, Hertz; GABA, Gamma amino butyric acid; NO, Nitric oxide; 4-AP, 4-
aminopyridine; STN, Strychnine; INH, Isoniazid; cGMP, cyclic GMP; cAMP, cyclic AMP; sGC, soluble guanylyl cyclase; PDE5, Phosphodiesterase V; L-NAME, L-Nitro Arginine Methyl
Ester; MB, Methylene Blue; DZP, Diazepam; NOS, Nitric oxide synthase; ANOVA, Analysis of variance; GAD, Glutamic acid decarboxylase; L-ARG, L-Arginine; BZD, Benzodiazepine;
NMDA, N-Methyl D-Aspartate; MES, Maximal electroshock seizure
⁎ Corresponding author.
E-mail addresses: donatusadongo@yahoo.com, dadongo@uhas.edu.gh (D.W. Adongo), pkmante.chs@knust.edu.gh (P.K. Mante), edemkennedy@yahoo.com (K.K.E. Kukuia),
rpbiney@hotmail.com (R.P. Biney), ebgyasi.pharm@knust.edu.gh (E. Boakye-Gyasi), benneh.ck@outlook.com (C.K. Benneh), elvisameyaw@gmail.com (E.O. Ameyaw),
ewoode.pharm@knust.edu.gh (E. Woode).
http://dx.doi.org/10.1016/j.jep.2017.05.017
Received 16 January 2017; Received in revised form 9 May 2017; Accepted 10 May 2017
Available online 17 May 2017
0378-8741/ © 2017 Elsevier B.V. All rights reserved.
D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
the condition remains largely undiagnosed, poorly untreated and hence the vivarium of the Department of Pharmacology, KNUST. The animals
results in a poor prognosis (de Boer et al., 2008). were housed in groups of five (5) in stainless steel cages
Despite the availability of many antiepileptic drugs (AEDs), nearly (34 cm×47 cm×18 cm) with soft wood shavings as bedding. Housing
one in three patients with epilepsy who have access to current AEDs conditions of mice were as follows: controlled–temperature maintained
show less than satisfactory prognosis and a similar proportion experi- at 24–25 °C, relative humidity 60–70%, and 12 h light-dark cycle. All
ence unacceptable AED-related adverse effects (Brodie, 2005; Löscher, mice had free access to food and water ad libitum. A period of at least
2002b). Thus, the need to source for clinically efficacious and safer one week for acclimatization to the laboratory environment was
AEDs with improved clinical profiles is still valid. Plant extracts are allowed. All laboratory procedures were conducted in accordance with
attractive sources of new drugs, and have shown to produce promising accepted principles for laboratory animal use and care (NRC, 2010).
results for the treatment of epilepsy. Examples of plants that have Approval for this study was obtained from the Faculty Ethics
shown promise as a source of good pharmacological properties include: Committee.
Passiflora incarnate (LINN.), Berberis vulgaris (LINN.), Butea mono-
sperma (Lam.) Taub. and Cymbopogon winterianus (Jowitt.) 2.4. Drugs and chemicals
(Bhutada et al., 2010; Kasture et al., 2002; Nassiri-Asl et al., 2007;
Quintans-Junior et al., 2008). Pentylenetetrazole (PTZ), picrotoxin (PTX), 4-aminopyridine (4-
Pseudospondias microcarpa has been extensively used in Ghana AP), strychnine (STN), isoniazid (INH), N-nitro-L-arginine methyl
and other parts of Africa as medication for different diseases. The plant ester (L-NAME), L-arginine (L-arg) and methylene blue (MB) (Sigma-
is suspected of having a sedative effect on those who sit or sleep under Aldrich Inc., St. Louis, MO, USA); diazepam, DZP (INTAS, Gujarat,
it, hence the Ghanaian name katawani, literally meaning “close your India); sildenafil, SIL (Pfizer, U.S.A); carbamazepine, CBZ (Tegretol®,
eyes”. It is therefore used traditionally as a sedative and for treating Novartis, Basel, Switzerland); flumazenil, FMZ (Anexate®, Roche
general central nervous system (CNS) disorders (Burkill, 1985). products Ltd., Herts, England); sodium valproate, VPA (Epilim®,
Preliminary studies from our laboratory showed that the hydroetha- Sonofi-Synthelabo Ltd-UK).
nolic leaf extract of P. microcarpa (PME) possesses sedative effects
(Adongo et al., 2014), confirming the traditional use of the plant. 2.5. Pentylenetetrazole-induced seizures
Moreover, in this study, we indicated the presence of some phyto-
chemical constituents which were reported earlier (Yondo et al., 2009). Clonic convulsions was induced using Pentylenetetrazole
The antioxidant (Yondo et al., 2009), antimicrobial (Kisangau et al., (60 mg kg−1, s.c.) according to methods described by Oliveira et al.
2008), cytotoxic and antiplasmodial (Malebo et al., 2009) effects of the (2001). Mice were divided into 7 groups (n=8) and received PME (30,
plant have also been reported. We also showed in our preliminary 100 or 300 mg kg−1, p.o.), diazepam (0.1, 0.3 or 1 mg kg−1, i.p.) or
studies that PME protected against convulsions induced by pentylene- vehicle (normal saline; 10 mL kg−1 i.p.) 30 min (i.p.) or 1 h (p.o.)
tetrazole (PTZ) (Adongo et al., 2014). Therefore, this study further before subcutaneous injection of pentylenetetrazole (PTZ), respec-
explored the anticonvulsant activity of PME and possible mechanism(s) tively. Immediately after subcutaneous injection of PTZ, animals were
in mice models. placed in Perspex-walled testing chambers (15 cm×15 cm ×15 cm)
with a mirror angled at 45° below the floor of the chamber to allow a
2. Materials and methods complete view of convulsive events, if present. The convulsive beha-
viour was captured with a camcorder placed at a favourable distance
2.1. Collection of plant material and extraction directly opposite to the mirror. Video outputs of each 30 min session
was later scored using JWatcher™ Version 1.0 (University of
Fresh leaves of P. microcarpa were collected from the campus of California, Los Angeles, USA and Macquarie University, Sidney,
Kwame Nkrumah University of Science and Technology (KNUST), Australia available at http://www.jwatcher.ucla.edu/.) for behavioural
Kumasi (6° 40.626′N, 1° 34.041′W). The plant was authenticated at parameters including: latency, frequency and duration of clonic
the Department of Herbal Medicine, KNUST, Kumasi, Ghana. A convulsions. The observed clonic seizures were characterized for the
voucher specimen (KNUST/HM1/2013/L005) was subsequently kept appearance of facial myoclonus, forepaw myoclonus and forelimb
at the herbarium of the Faculty. Leaves of the plant were room-dried clonus. The ability of a drug/extract to reduce or prevent the seizures
for seven days and pulverised into fine powder. The powder was or delay/prolong the latency or onset of the clonic convulsions was
extracted by cold percolation with 70% (v/v) ethanol in water over a considered as an indication of anticonvulsant activity.
period of 72 h and the resulting extract concentrated into a syrupy
mass under reduced pressure at 60 °C in a rotary evaporator. It was 2.6. Picrotoxin-induced seizures
further dried in a hot air oven at 50 °C for 7 days and kept in a
refrigerator for use with a yield of 20.5% (w/w). The crude extract is Anticonvulsant testing method by Leewanich et al. (1996) was
subsequently referred to as (Pseudospondias microcarpa extract) PME modified and adopted for this test. Briefly, mice were divided into 7
or simply, extract. groups (n=8) and received PME (30, 100 or 300 mg kg−1, p.o.),
diazepam (0.1, 0.3 or 1 mg kg−1, i.p.) or vehicle (normal saline;
2.2. FT-IR analysis of crude extract 10 mL kg−1 i.p.) 30 min (i.p.) or 1 h (p.o.) before the injection of
picrotoxin (3 mg kg−1, i.p.) respectively. The latency to, frequency and
To identify the possible functional groups that may be present in duration of clonic convulsions were recorded for 30 min.
the sample, a triplicate FT-IR (PerkinElmer UATR Two) spectra was
generated and baseline corrected. The spectra between 400 and 2.7. Isoniazid-induced seizures
1400 cm−1 is usually considered as the unique region for every
compound/compound mixtures and hence can be used for identifica- Mice were divided into seven groups (n=10) and received PME (30,
tion and quality control. 100 or 300 mg kg−1, p.o.), vehicle or the standard drug diazepam (0.1,
0.3 or 1.0 mg kg−1, i.p.). One hour (p.o.) or 30 min (i.p.) after
2.3. Animals administration of test compounds, animals were injected with isoniazid
(300 mg kg−1, s.c.). Thereafter, mice were observed for 120 min for
Male ICR mice (20–25 g) were purchased from the Noguchi characteristic behavioural signs, such as intermittent forelimb exten-
Memorial Institute for Medical Research, Accra, Ghana and kept in sion, clonic seizures, tonic seizures and death. The latencies to the
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D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
onset of the convulsive episode (clonic or tonic) and death were 2.12. Effect of PME on GABAA
recorded as indicators of pro- or anticonvulsive effect of compounds.
To investigate the possible involvement of γ-aminobutyric acid
2.8. Strychnine-induced seizures (GABA)A receptors in the anticonvulsant activity of PME, mice were
pre-treated with flumazenil (2 mg kg−1, i.p.), a selective benzodiazepine
The method described by Porter et al. (1984) was employed. Mice receptor antagonist or vehicle 15 min before PME (100 mg kg−1, p.o.)
received PME (30, 100 or 300 mg kg−1, p.o.), diazepam (0.1, 0.3 or or diazepam (0.3 mg kg−1, i.p.) administration. After 45 min, mice were
1 mg kg−1, i.p.) or vehicle (normal saline; 10 mL kg−1 i.p.) 30 min (i.p.) challenged subcutaneously with PTZ (60 mg kg−1, s.c.) and assessed
or 1 h (p.o.) before the injection of STN (0.5 mg kg−1, i.p.), respectively. 30 min for latency, frequency and duration of clonic convulsions.
Latency, frequency and duration of clonic convulsions were assessed
for 30 min. 2.13. Effect of PME on L-arginine-nitric oxide-cGMP pathway
2.9. 4-Aminopyridine-induced seizures Doses of the modulators were chosen based on pilot experiments
and previous reports (Akula et al., 2008; Bahremand et al., 2010). To
The method as described by Rogawski and Porter (1990) was investigate the possible involvement of the L-arginine-NO-cGMP path-
adopted for this test.. Mice received PME (30, 100 or 300 mg kg−1, way in the anticonvulsant action of PME, mice were pre-treated with
p.o.), vehicle or the standard drug carbamazepine (30, 100 or sub-effective doses of −1L-arginine [150 mg kg , i.p., a precursor of nitric
300 mg kg−1, p.o.). One hour after administration of test compounds, oxide (NO)], L-NAME [30 mg kg−1, i.p., a non-selective nitric oxide
animals were treated with a single injection of 4-AP (12 mg kg−1, i.p.). synthase (NOS) inhibitor], methylene blue [1 mg kg−1, i.p., an inhibitor
Thereafter, mice were observed 60 min for both clonic and tonic of NO synthase and an inhibitor of soluble guanylyl cyclase (sGC)],
seizures. Clonic seizures were characterized as described in Section sildenafil [5 mg kg−1, i.p., a phosphodiesterase 5 (PDE 5) inhibitor] or
2.5 and tonic seizures were characterized as explosive clonic seizures vehicle 15 min before PME (100 mg kg−1, p.o) administration. After
with wild running and tonic forelimb and hind limb extension. 45 min, mice were challenged subcutaneously with PTZ (60 mg kg−1)
Latencies for the onset of convulsive episodes (clonic or tonic) and and assessed 30 min for latency, frequency and duration of clonic
death were recorded as indicators of pro- or anticonvulsive effect of convulsions.
compounds.
2.14. Grip-strength test
2.10. Maximal electroshock seizure test
The effects of PME and diazepam on skeletal muscular strength in
Electroconvulsions were produced by application of electric current mice were quantified by the grip-strength test of Meyer et al. (1979).
(60 Hz, 50 mA, 0.2 s) delivered via ear-clip electrodes with an ECT Unit The grip-strength apparatus (BioSeb, Chaville, France) comprised a
7801 (Ugo Basile, Comerio, Italy). This current intensity elicited wire grid (8 cm×8 cm) connected to an isometric force transducer.
complete tonic extension of the hind limbs in saline treated mice. Mice were randomly divided into eight groups (n=6): saline-treated
Mice received PME (30, 100 or 300 mg kg−1, p.o.), carbamazepine (3, control group; diazepam group (0.1, 0.3, 1 and 3 mg kg−1, i.p.) and
10 or 30 mg kg−1, p.o.) or vehicle (normal saline; 10 mL kg−1, p.o.) PME group (30, 100 and 300 mg kg−1, p.o.). Thirty minutes after i.p.
60 min before tonic hind limb convulsions were induced. Protection and 1 h after oral administration of the test compounds, mice were
against tonic hind limb seizures was determined. An animal was lifted by the tails so that their forepaws could grasp the grid. Mice were
considered to be protected if the characteristic electroshock convulsive then gently pulled backward by the tail until the grid was released. The
seizure pattern was absent. maximal force exerted by the mouse before losing grip was recorded.
The mean of 4 measurements for each animal was calculated and
2.11. 6 Hz seizure test subsequently, the mean maximal force was determined. Skeletal
muscular strength in mice was expressed in Newton (N).
The 6-Hz seizure model was performed according to methods
previously described (Brown et al., 1953; Luszczki et al., 2012). 2.15. Statistical analysis
Psychomotor (limbic) seizures were induced via trans auricular stimu-
lation (6 Hz, 0.2 ms rectangular pulse width, 32 mA, 3 s duration) In all experiments, a sample size of six to ten (n=6–10) was used.
delivered by an ECT Unit 5780 (Ugo Basile, Comerio, Italy). Normal Data are presented as mean ± SEM. To compare differences between
saline (0.9%) was used to wet the electrodes immediately before testing groups, one-way analysis of variance (ANOVA) was performed with
to ensure good electrical contact. Animals were manually restrained Newman-Keuls’ test as post hoc. In analysing the possible role of
and released immediately after the stimulation and recorded the GABAergic and nitric oxide mechanisms in the anticonvulsant effect of
presence or absence of psychomotor seizure activity. Immediately the extract, two-way ANOVA with the Bonferroni's post hoc test
following stimulation, mice were placed separately in Plexiglas cages (treatment × dose) was performed. In the 4-aminopyridine and
(25 cm×15 cm ×10 cm) for behavioural observation. After stimulation, isoniazid seizure tests, the Kaplan-Meier method was used in estimat-
the animals exhibited behavioural signs of psychomotor seizures— ing survival relative to time and survival differences were analysed with
behavioural arrest, forelimb clonus, twitching of the vibrissae, and the log-rank test. GraphPad® Prism Version 5.0 (GraphPad Software,
Straub tail—that lasted for 60–120 s in untreated animals. Animals San Diego, CA, USA) was used for all statistical analysis. P < 0.05 was
resumed normal exploratory behaviour after seizure induction. The considered statistically significant for all test.
experimental endpoint was protection against the seizure: an animal
was considered to be protected if it resumed its normal exploratory 3. Results
behaviour within 10 s after stimulation. Protection in the 6 Hz model
was defined as the absence of a seizure. Mice not experiencing seizures 3.1. IR analysis
exhibited normal exploratory behaviour when placed in the cages. Mice
were divided into 8 groups (n=10) and received PME (30, 100, 300 or FT-IR spectroscopy was used for the distinct functional group
1000 mg kg−1, p.o.), sodium valproate (100, 200 or 400 mg kg−1, p.o.) identification and run under IR region between the ranges of 400
or vehicle (normal saline; 10 mL kg−1, p.o.) 60 min before psychomotor and 4000 cm−1. The characteristic spectra in the region from 400 to
seizures were induced. 1400 cm−1 was as a fingerprint region for subsequent comparison of
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future extracts. Baseline corrected Infrared spectra and peak values, (F3,36=6.117, P=0.0025; Fig. 2b) of clonic convulsions induced by
with labels, is provided in the Appendix A. picrotoxin. In addition, PME significantly delayed the onset of clonic
convulsions (F3,28=6.117, P=0.0028) with statistical significance ob-
3.2. Pentylenetetrazole-induced seizures served at 300 mg kg
−1 (P < 0.01). Diazepam produced effects similar to
that of the extract. It significantly delayed the onset of convulsions
The extract significantly and dose-dependently delayed the onset of (F3,36=9.7118, P < 0.0001; Fig. 2c) and reduced the frequency
clonic convulsions (F =3.009, P=0.0469; Fig. 1a) with statistical (F3,36=9.131, P < 0.0001; Fig. 2c) and duration (F3,36=15.63, P <3,28
signi cance at 300 mg kg−1 (P < 0.05). One-way ANOVA revealed that 0.0001; Fig. 2d) of convulsions.fi
PME also significantly reduced the frequency of clonic convulsions
(F3,28=6.947, P=0.0012; Fig. 1a) at all tested doses (P < 0.05 at
30 mg kg−1; P < 0.01 at 100 and 300 mg kg−1). Newman-Keuls’ post
hoc test indicated a statistical significant reduction in the duration of 3.4. Isoniazid-induced seizures
clonic convulsions by the extract at all the doses used (P < 0.05 at
30 mg kg−1; P < 0.001 at 100 and 300 mg kg−1). The reference antic- Isoniazid (300 mg kg−1, s.c.) elicited clonic convulsions followed by
onvulsant, diazepam, delayed the onset of clonic convulsions tonic hind limb extensions and mortality in mice. Fig. 3 indicates that
(F3,36=24.76, P < 0.001; Fig. 1c) with statistical significance at 0.3 treatment of mice with PME (30–300 mg kg−1, p.o.) significantly
and 1.0 mg kg−1 (both P < 0.001). Also, diazepam caused significant delayed the onset to both clonic (F3,36=12.90, P < 0.0001) and tonic
and dose-dependent reduction in the frequency (F3,36=38.01, P < (F3,36=15.63, P < 0.0001) convulsions as compared to vehicle-treated
0.001; Fig. 1c) and duration of clonic convulsions (F3,36=49.60, P < mice. Furthermore, in Fig. 4, PME significantly (P=0.0005, χ2 (df =3)
0.0001; Fig. 1d). =17.65) improved survival of the animals after induction of convul-
sions. As compared to vehicle-control group, mice treated with
3.3. Picrotoxin-induced seizures diazepam (0.1–1.0 mg kg−1, i.p.) showed significant protection against
INH-induced mortality (P=0.003, χ2 (df=3) =13.90). In addition, it
As shown in Fig. 2, the extract significantly and dose-dependently significantly delayed onset of convulsions [clonic (F3,36=19.69, P <
reduced the frequency (F3,36=5.071, P=0.0063; Fig. 2a) and duration 0.0001) and tonic (F3,36=25.46, P < 0.0001)].
Fig. 1. Effect of PME (30–300 mg kg−1) and diazepam (0.1–1.0 mg kg−1) on frequency (a, c), latency (a, c) and duration (b, d) of PTZ-induced clonic seizures in mice. Data are
expressed as mean ± SEM. Each group consisted of 8 mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared to vehicle-treated group (One-way ANOVA followed by Newman-Keuls’ post
hoc test).
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D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
Fig. 2. E ect of PME (30–300 mg kg−1) and diazepam (0.1–1.0 mg kg−1ff ) on frequency (a, c), latency (a, c) and duration (b, d) of picrotoxin-induced clonic seizures in mice. Data are
expressed as mean ± SEM (n=8). *P < 0.05; **P< 0.01; ***P < 0.001 compared to vehicle-treated group (One-way ANOVA followed by Newman-Keuls’ post hoc test).
3.5. Strychnine-induced seizures revealed that the extract exhibited a dose-dependent effect against
strychnine-induced clonic seizures by significantly increasing latency to
Fig. 5 shows the e −1ffects of PME (30–300 mg kg , p.o.) and convulsions (F3,24=4.208, P=0.0159) and reducing the frequency
diazepam (0.1–1 mg kg−1, i.p.) on latency, frequency and duration of (F3,24=7.569, P=0.0010). In addition, PME provided 29% (at
clonic convulsions induced by strychnine in mice. One-way ANOVA 30 mg kg−1), 43% (at 100 mg kg−1) and 57% (at 300 mg kg−1) protec-
Fig. 3. Effect of PME (30–300 mg kg−1) and diazepam (0.1–1.0 mg kg−1) on latency to isoniazid-induced seizures in mice. Data are expressed as mean ± SEM (n=10). *P < 0.05; **P <
0.01; ***P < 0.001 compared to vehicle-treated group (One-way ANOVA followed by Newman-Keuls’ post hoc test).
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D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
Fig. 4. Kaplan–Meier estimates of overall survival of animals treated with PME (30, 100 and 300 mg kg−1) and diazepam, DZP (0.1, 0.3 and 1 mg kg−1) in the isoniazid-induced seizure
test over a 2 h observation period (n=10).
tion against strychnine-induced seizures. Duration of clonic seizures Diazepam significantly delayed the onset of convulsions (F3,24=13.32,
was also reduced by the extract even though this was not statistically P < 0.0001) and reduced the frequency (F3,24=7.768, P=0.0009) and
significant as compared to the control (F3,24=2.593, P=0.0761). duration (F3,24=6.721, P=0.0019) of convulsions. Acute treatment with
Fig. 5. Effect of PME (30–300 mg kg−1) and diazepam (0.1–1.0 mg kg−1) on frequency (a and b), latency (a and b) and duration (c and d) of strychnine-induced clonic seizures in mice.
Data are expressed as mean ± SEM (n=8). *P < 0.05; **P< 0.01; ***P < 0.001 compared to vehicle-treated group (One-way ANOVA followed by Newman-Keuls’ post hoc test).
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Fig. 6. E ect of PME (30–300 mg kg−1ff ) and carbamazepine (30–300 mg kg−1) on latency of 4-AP-induced seizures in mice. PME and carbamazepine were administered p.o. 60 min
before behavioural assessments for 1 h. Data are expressed as mean ± SEM (n=10). **P < 0.01; ***P < 0.001 compared to vehicle-treated group (One-way ANOVA followed by Newman-
Keuls’ post hoc test).
diazepam provided 29% (at 0.1 mg kg−1), 71% (at 0.3 mg kg−1) and protection against 4-AP-induced clonic seizures. Furthermore, it
100% (at 1 mg kg−1) protection against strychnine-induced clonic provided 10% (at 30 mg kg−1), 60% (at 100 mg kg−1) and 80% (at
seizures. 300 mg kg−1) protection against 4-AP-induced tonic seizures in mice.
The extract significantly (P < 0.0001, χ2 (df=3) =30.27) improved
3.6. 4-Aminopyridine-induced seizures survival of the animals after induction of convulsions. Carbamazepine
also produced similar effects on survival (P < 0.0001, χ2 (df=3)
The effects of PME and carbamazepine on 4-AP-induced seizures =36.61).
are shown in Figs. 6 and 7. A single administration of 4-AP
(12 mg kg−1, i.p.) caused clonic and tonic seizures as well as death in 3.7. Effect on maximal electroshock seizures
all saline-treated mice. In contrast, pre-treatment of animals with PME
(30–300 mg kg−1, p.o.) caused a significant delay in the latency to both Electrical stimulation produced tonic hind limb extensions (HLEs)
clonic (F3,36=10.81, P < 0.0001) and tonic (F3,36=9.513, P < 0.0001) in all saline-control mice (Table 1). The extract did not protect against
seizures. The effects of the extract on 4-AP-induced seizures decreased tonic hind limb extensions. In contrast to PME, carbamazepine at the
−1
with increasing dose. PME provided 40% (at 30 mg kg−1), 30% (at dose of 30 mg kg , completely protected mice against tonic hind limb
100 mg kg−1) and 0% (at 300 mg kg−1) protection against 4-AP-in- extensions. In addition, no deaths were recorded at 10 and 30 mg kg
−1.
duced clonic seizures. Furthermore, the extract provided 50% (at Data shows the percentage of mice (n=10) that produced tonic
30 mg kg−1), 50% (at 100 mg kg−1) and 20% (at 300 mg kg−1) protec- convulsions and deaths.
tion against 4-AP-induced tonic seizures in mice. Carbamazepine
(CBZ) produced effects analogous to the extract in the 4AP-induced 3.8. Effect on psychomotor seizures
seizure test but the effects increased with increasing dose. It caused a
significant delay in the latency of clonic (F3,36=9.040, P < 0.0001) and In Table 2, it was observed that all control mice exhibited
tonic seizures (F3,36=12.06, P < 0.0001). Carbamazepine provided 10% psychomotor seizures (0% protection) after electrical stimulation that
(at 30 mg kg−1), 50% (at 100 mg kg−1) and 60% (at 300 mg kg−1) lasted 60–120 s. However, acute treatment with PME protected against
Fig. 7. Kaplan–Meier estimates of overall survival of animals treated with PME (30, 100 and 300 mg kg−1) and carbamazepine (30, 100 and 300 mg kg−1) in the 4-aminopyridine
seizure test over a one hour observation period (n=10).
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D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
Table 1 latency, duration and frequency of convulsions as compared with saline
Effects of PME and carbamazepine (CBZ) on maximal electroshock (MES)-induced (vehicle)-treated animals. However, pre-treatment with flumazenil
seizures in mice. significantly reversed the effect of PME (100 mg kg−1, p.o) by decreas-
Group Dose (mg kg−1) Incidence of tonic extensions (%) Death (%) ing latency (F1,36=36.48, P < 0.0001) as well as increasing duration
(F1,36=24.04, P < 0.0001) and frequency (F1,36=23.10, P < 0.0001) of
Control 100 33.3 clonic seizures induced by PTZ. Similar results were obtained for
PME 30 100 33.3 diazepam.
100 100 50
300 100 33.3
1000 100 33.3
3.10. Effect of PME on L-arginine-NO-cGMP pathway
CBZ 3 100 16.7
10 83.3 0
30 0 0 Figs. 9 and 10 show the effects of L-NAME, methylene blue, L-
arginine or sildenafil on the actions of PME in the PTZ-induced seizure
test. Acute PME (100 mg kg−1, p.o) treatment significantly increased
Table 2 latency and decreased both frequency and duration of clonic convul-
Effects of PME and sodium valproate (VPA) in the mouse 6 Hz-induced limbic seizure sions. Administration of L-arginine (150 mg kg−1, i.p.) had no effect on
model. latency, frequency and duration of convulsions compared with saline
Group Dose (mg kg−1) % protection (vehicle)-treated animals. However, pre-treatment with L-arginine
significantly inhibited PME action by decreasing latency (P < 0.01)
control 0 and increasing duration (P < 0.05) of clonic seizures as revealed by post
PME 30 30 hoc analysis. Pre-treatment with either L-NAME (30 mg kg−1, i.p.) or
100 40
methylene blue (1 mg kg−1, i.p.) significantly potentiated PME action
300 60
1000 90 as evident from the delayed expression of clonic convulsions [L-NAME
(F1,24=4.474, P < 0.05); MB (F1,24=6.005, P < 0.05)] induced by PTZ.
VPA 100 20 Fig. 10 shows the effect of pre-treatment of sildenafil, a phospho-
200 60 diesterase 5 inhibitor on PTZ-induced clonic seizures. Concomitant
400 90
administration with sildenafil (5 mg kg−1, i.p.) significantly reversed
Data indicates the percentage of mice (n=10) that were protected. the effect of PME. This is observed as a decrease in the onset (P < 0.05)
and increase in the duration of clonic (P < 0.05) convulsions induced by
6 Hz-induced seizures. It provided 20% (at 30 mg kg−1), 40% (at PTZ.
100 mg kg−1), 60% (at 300 mg kg−1) and 90% (at 1000 mg kg−1)
protection against 6 Hz-induced seizures. Sodium valproate (VPA),
the reference anticonvulsant, produced effects similar to the extract as 3.11. Grip-strength test
it provided 10% (at 100 mg kg−1), 60% (at 200 mg kg−1) and 90% (at
400 mg kg−1) protection against 6 Hz-induced seizures. Fig. 11 shows the results of the effect of PME and diazepam on
skeletal muscle strength in the grip-strength test. One-way ANOVA
revealed that pretreatment of mice with PME (30–300 mg kg−1, p.o.)
3.9. Effect on GABAA did not significantly affect the grip-strength in mice (P > 0.05). In
contrast to PME, diazepam (0.1–3 mg kg−1, i.p.) significantly and dose-
As shown in Fig. 8, PME (100 mg kg−1, p.o) significantly increased dependently decreased (F3,16=4.308, P=0.0113) the grip-strength in
latency and decreased both frequency and duration of clonic convul- mice with Newman-Keuls’ post hoc analysis revealing a significant
sions. Administration of flumazenil (2 mg kg−1, i.p.) had no effects on effect at the dose of 3 mg kg−1 (P < 0.05).
Fig. 8. E ect of umazenil on the latency (a), frequency (b), and duration of seizures (c) of PME (100 mg kg−1, p.o.) and diazepam (0.3 mg kg−1ff fl , i.p.) in PTZ-induced seizures. Data are
presented as mean ± SEM (n=7). *?? < 0.05 and ***?? < 0.001 compared to vehicle-treated group (One-way analysis of variance followed by Newman-Keuls’ post hoc test). ††?? < 0.01
and †††?? < 0.001 (two-way ANOVA followed by Bonferroni's test).
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D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
Fig. 9. Effects of pre-treatment of L-NAME [30 mg kg−1, i.p., a non-selective nitric oxide synthase (NOS) inhibitor] and methylene blue [1 mg kg−1, i.p., an inhibitor of NO synthase and
an inhibitor of soluble guanylyl cyclase (sGC)] on the anticonvulsant e ect of PME (100 mg kg−1ff , p.o.) in the PTZ-induced seizure test. L-NAME, MB or saline were administered 15 min
before administration of PME and 45 min before determination of PTZ-induced seizures. Data are presented as group mean ± SEM (n=7). *P < 0.05, **P < 0.01 versus vehicle-treated
animals (One-way ANOVA followed by Newman Keuls’ test). Significant difference between treatments: †P < 0.05 (Two-way ANOVA followed by Bonferroni's test).
4. Discussion said that the extract exhibits anticonvulsant effect against PTZ-induced
clonic seizures probably due to interference with GABAergic mechan-
Results of the present study provide evidence that a hydroethanolic ism(s). The significant effect of diazepam as evident in the PTZ-induced
leaf extract of the Pseudospondias microcarpa possess anticonvulsant convulsions agrees with its enhancing effects in GABAergic neuro-
activity in pharmacologically validated experimental animal models. transmission (Patil et al., 2011). It has also been suggested that PTZ-
The GABAergic system is implicated in epilepsy since enhancement induced clonic seizures model myoclonic seizures (Kubova, 2009).
and inhibition of the neurotransmission of GABA will attenuate and Thus, the extract could possibly protect against myoclonic seizures.
enhance convulsions respectively (Quintans-Junior et al., 2008). Similar to PTZ, picrotoxin exerts its convulsant action via blockade
Pentylenetetrazole (PTZ), a GABAA blocker, is the most frequently of the GABAA receptor-linked chloride ion channel, which normally
used epileptogenic agent employed in the search for new antiepileptic opens to allow increased chloride ion conductance following the
drugs (AEDs) (Löscher, 2011). It blocks GABA-mediated chloride ion activation of GABAA receptors by γ-aminobutyric acid (GABA)
influx through an allosteric interaction in the Cl– channel, thus leading (Nicoll, 2001; Velíšek, 2006). Data from this study shows that PME
to induction of convulsions in animals (Kubova, 2009; Velíšek, 2006). and diazepam exhibited anticonvulsant activity against picrotoxin-
Accordingly, drugs such as benzodiazepines and phenobarbitone which induced seizures. Attenuation of picrotoxin-induced convulsions by
enhance GABAA receptor-mediated inhibitory neurotransmission can PME further indicates possible GABAergic neurotransmission in its
block PTZ-induced clonic seizures (Macdonald and Kelly, 1995). In this anticonvulsant action. In addition, similar to diazepam, PME signifi-
study, acute administration of PME and the benzodiazepine diazepam, cantly delayed convulsions (clonic and tonic) and reduced mortality
exhibited anticonvulsant activity against PTZ-induced seizures. Ability against INH-induced convulsions. Isoniazid induces convulsions by
of an agent to prevent or delay the onset of convulsions induced by PTZ inhibiting GABA synthesis (Costa et al., 1975). It inhibits glutamic acid
in animals is an indication of anticonvulsant activity. Thus, it could be decarboxylase (GAD) activity (enzyme involved in GABA synthesis),
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D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
Fig. 10. E ects of pre-treatment of -arginine [150 mg kg−1, i.p., a precursor of nitric oxide (NO)] and sildena l [5 mg kg−1ff L fi , i.p., a phosphodiesterase 5 (PDE 5) inhibitor] on the
anticonvulsant effect of PME (100 mg kg−1, p.o.) in the PTZ-induced seizure test. L-arginine, sildenafil or saline were administered 15 min before administration of PME and 45 min
before determination of PTZ-induced seizures. Data are presented as group mean ± SEM. *P < 0.05 versus vehicle-treated animals (One-way ANOVA followed by Newman Keuls’ test).
Significant difference between treatments: †P < 0.05, ††P< 0.01 (Two-way ANOVA followed by Bonferroni's test).
Fig. 11. Behavioural effects of PME and DZP on muscle relaxant activity in the grip-strength test in mice. Data are expressed as group mean ± SEM (n=6). *P < 0.05 compared to control
group (One-way ANOVA followed by Newman-Keuls’ post hoc test).
87
D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
resulting in decreased levels of GABA (Raygude et al., 2012; Vergnes In contrast to the MES test, the extract protected against seizures in
et al., 2000). Anticonvulsant effect against INH-induced convulsions the 6-Hz seizure test. This test uses a low-frequency, long-duration
further confirm the GABA-enhancing activity of the plant extract. stimulation paradigm to induce psychomotor seizures that involve
Additionally, the anticonvulsant effect of the extract was blocked by forelimb clonic convulsions and stereotyped behaviours similar to
flumazenil, a specific antagonist of the benzodiazepine site in the those seen in complex partial epilepsy (Giardina and Gasior, 2001).
GABAA-Benzodiazepine receptor complex (Brogden and Goa, 1991; Protection of PME against 6 Hz-induced psychomotor seizures there-
File and Pellow, 1986). The extract, like diazepam, may therefore be fore suggests anticonvulsant activity against complex partial seizures.
acting via direct activation of benzodiazepine site of the GABAA In addition, it has been suggested that the 6-Hz seizure test might
receptor complex. This further gives confirmation to the hypothesis identify anticonvulsant compounds with novel mechanisms of action
that PME may be affecting GABAergic mechanism(s) to exert its and serve as a test of human drug-resistant epilepsy (Barton et al.,
anticonvulsant activity. 2001; Duncan and Kohn, 2005). Thus, like levetiracetam (Surges et al.,
Activation of glycine receptors results in an influx of chloride ions 2008), PME could as well possess a distinct profile of activity from the
into the neuron, which is then hyperpolarized and inhibited. commonly used antiepileptic drugs, implying possible efficacy in
Strychnine acts as a selective competitive antagonist that blocks pharmacoresistant epilepsies.
inhibitory effect of the chloride channel associated with glycine at all Various preclinical studies suggest nitric oxide (NO) as a modulator
glycine receptors (Curtis et al., 1971; Patil et al., 2011), resulting in of seizure activity with both anticonvulsant (Noh et al., 2006) and
seizures. The extract exhibited anticonvulsant activity against seizures proconvulsant (Royes et al., 2007) effects. These effects have been
induced by strychnine by decreasing frequency and duration of shown to be based on the type of seizure, route of administration,
convulsions. In addition, latency to convulsions was significantly source of NO and other neurotransmitter systems involved (Riazi et al.,
delayed. Thus, the observed protection of the extract against strych- 2006). Osonoe et al. (1994), demonstrated that decreased NO levels
nine-induced seizures is mediated through the glycinergic pathway. results in suppression of convulsions, and inhibition of nitric oxide
Pre-treatment of PME and carbamazepine before administration of synthase (NOS) activity shows anticonvulsant property against penty-
4-AP significantly increased the latency to seizures and reduced the lenetetrazole-induced seizures in rats. Studies have therefore shown
incidence of mortality indicating anticonvulsant activity against 4-AP- that L-NAME, inhibitor of both endothelial and neuronal NOS activity
induced seizures. Action of 4-AP occurs through a K+-channel blockade (Rees et al., 1990; Talarek and Fidecka, 2003), inhibits pentylenete-
at the presynaptic neuronal level (Brito et al., 2009). As a result, efflux trazole and strychnine-induced seizures in mice (Kaputlu and Uzbay,
of intracellular K+ is suppressed and calcium influx is enhanced, 1997). In the present study, a sub-effective dose of L-NAME given
leading to an increase in neurotransmitter release (Molgo et al., alone did not influence pentylenetetrazole-induced seizures. However,
1985). At the neuronal level, K+ channels are involved in neuronal it potentiated the anticonvulsant effect of PME by delaying latency as
excitability (Pongs, 1999). Thus, direct activation of voltage dependent well as decreasing frequency and duration of PTZ-induced clonic
K+ channels hyperpolarizes the neuronal membrane and limits the seizures. This potentiating effect has been observed for anticonvulsants
firing of an action potential (Porter and Rogawski, 1992). Accordingly, that act via the GABAergic pathway such as the benzodiazepines
K+ channel activators possess anticonvulsant effects in some seizure (Talarek and Fidecka, 2003). This shows that PME also elicits its
models (Rostock et al., 1996). Therefore, the extract being able to anticonvulsant effect in the pentylenetetrazole seizure model probably
protect against 4-AP-induced seizures may probably be due to direct by interacting with the nitric oxide pathway.
activation of K+ channels and could therefore contribute to membrane L-arginine administered at high doses enhances seizure suscept-
hyperpolarization (Herrero et al., 2002). Moreover, since GABAergic ibility through excessive release of nitric oxide in chemical seizure
receptor activation can result in enhancement of potassium conduc- models induced by GABA antagonists, likely due to hyperexcitability
tance, it is therefore possible that PME may be acting indirectly to (Riazi et al., 2006). Results showed that L-arginine [nitric oxide
enhance potassium ion conductance. Currently, retigabine is the only synthase (NOS) substrate] at the dose used attenuated the antic-
approved antiepileptic drug which activates potassium currents (Hecht, onvulsant activity of PME by significantly decreasing latency and
2012; Rundfeldt, 1997). The present study therefore gives PME a increasing duration of convulsions. This is consistent with various
distinct profile than the widely used antiepileptic agents, since it may reports where L-arginine decreased the antiepileptic effect of com-
have the potential to activate potassium channels and also be a source pounds in the pentylenetetrazole model of epilepsy (Bahremand et al.,
of anticonvulsant compounds that will enhance potassium conduc- 2010; Gholipour et al., 2008). This further confirms the possible
tance. involvement of the nitric oxide pathway in the anticonvulsant effect
Furthermore, the convulsant effect of 4-AP is due to the release of of PME.
excitatory neurotransmitters such as glutamate and this results in over Nitric oxide stimulates cGMP generation via soluble guanylyl
activation of mainly the N-methyl-D-aspartate (NMDA)‐type receptors cyclase, which plays a major role in seizure (Snyder and Bredt,
(Morales-Villagrán et al., 1996). Indeed, an enhancement in the 1991). Methylene blue inhibits soluble guanylyl cyclase (Meller and
glutamatergic neurotransmission has been linked to the 4‐AP con- Gebhart, 1993), and it has widely been applied in experiments to
vulsant action (Tapia et al., 1999), since the administration of NMDA determine the contribution of the cGMP pathway in the effects of
receptor antagonists protects against 4‐AP induced seizures (Fragoso- nitricoxidergic system (Talarek and Fidecka, 2003). Although methy-
Veloz and Tapia, 1992). Anticonvulsant activity of PME against lene blue was not effective in antagonizing clonic seizures induced by
seizures induced by 4-AP may also possibly be due to its inhibition of PTZ when administered alone, it potentiated the anticonvulsant effect
the glutamate signal pathway: NMDA receptors and could therefore of PME. This confirms the role of the nitric oxide pathway in the
protect against neuronal excitotoxicity. anticonvulsant effect of PME. The potentiating effect of methylene blue
As a well-validated preclinical model, the maximal electroshock has also been demonstrated in the anticonvulsant effect of diazepam
seizure (MES) test predicts anticonvulsant drug efficacy against gen- and clonazepam in PTZ-induced seizures in mice (Talarek and Fidecka,
eralized tonic-clonic (grand mal) seizures (Holmes, 2007; Löscher, 2003).
1998). In addition, it permits the evaluation of the ability of a Intra-cellular concentrations of cGMP are also regulated not only by
substance to prevent seizure spread through neural tissue (Castel- soluble guanylate cyclase (sGC), but also by Phosphodiesterase V (PDE
Branco et al., 2009). Since the extract produced no anticonvulsant 5) enzyme, which catalyses the hydrolysis of the second messengers
effect against MES-induced tonic seizures in mice, it therefore indicates cAMP and cGMP (Akula et al., 2008). Sildenafil is a PDE 5 inhibitor
its inability to protect against generalized tonic-clonic seizures as well and enhance the NO-mediated effects by inhibiting cGMP degradation
as prevent seizure spread. in target tissues (Gholipour et al., 2009; Jackson et al., 1999). Sildenafil
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D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
inhibited the anticonvulsant effect of PME by significantly delaying based on the natural tendency of the mouse to grasp a grid when it is
latency and increasing duration of clonic convulsions. This is in suspended by the tail. PME had no significant impairment effect on
agreement with reports establishing the proconvulsant effect of silde- skeletal muscular strength. In contrast, diazepam at the highest dose
nafil (Akula et al., 2008; Riazi et al., 2006). Results of this study (3 mg kg−1) impaired neuromuscular strength by decreasing the force
therefore suggest possible contribution of the cGMP pathway in the (N). This effect is in agreement with various reports in which some
anticonvulsant effect of the extract. classical and second generation antiepileptic drugs—diazepam, carba-
Several studies have demonstrated potential levels of interaction mazepine, valproate, clonazepam, phenytoin, phenobarbital, lamotri-
between GABA and the NO system in the regulation of seizure gine, oxcarbazepine and topiramate—produced impairment of skeletal
susceptibility (Paul, 2003; Paul and Subramanian, 2002). For instance, muscular strength in mice in a dose-dependent manner (Łuszczki et al.,
increased synthesis of NO can decrease GABA-stimulated chloride ion 2008; Zadrozniak et al., 2009).
influx by inhibiting GABAA receptor function (Gholipour et al., 2008;
Zarri et al., 1994). In addition, release of endogenous NO participates 5. Conclusion
in the excitatory transmission through NMDA receptors (Riazi et al.,
2006), where activation of NMDA-type glutamate receptors causes a Findings of the present study suggests that administration of
reduction in the effect of GABA (Talarek and Fidecka, 2003). Moreover, Pseudospondias microcarpa hydroethanolic leaf extract has antic-
NMDA receptor blockade or suppression has been shown to decrease onvulsant activity and may probably be affecting GABAergic, glyciner-
susceptibility to seizure development (Ahmed et al., 2005; Ghasemi gic, NMDA, K+ channels and nitric oxide-cyclic GMP pathways to exert
et al., 2010). Just like the benzodiazepines, PME has shown to possess its effect.
anticonvulsant activity against convulsions induced by GABA antago-
nists probably by interacting with GABAA receptors. The extract has Conflict of interest
also shown to elicit its anticonvulsant effect probably via an interaction
with the NMDA receptor complex. Therefore, the influence of NO The authors declare no conflicts of interest.
modulators in the anticonvulsant activity of PME could possibly be
through its interaction with GABA and/or NMDA receptors. Acknowledgements
In this study, the neuromuscular tone of animals was evaluated by
use of the grip-strength test. This test is a widely-used non-invasive We are grateful to Messrs Thomas Ansah, Gordon Darku, Prosper
method designed to evaluate mouse limb strength and has been used to Akortia, Edmond Dery and Prince Okyere of the Department of
investigate the effects of neuromuscular disorders and drugs. It is Pharmacology for their technical assistance.
Appendix A
.
Infrared spectra of the hydroethanolic leaf extract of P. microcarpa (PME).
Peak table for IR spectra of the hydroethanolic leaf extract of P. microcarpa (PME).
Peak X (cm−1) Y (%T) Peak X (cm−1) Y (%T) Peak X (cm−1) Y (%T) Peak X (cm−1) Y (%T)
1 3223.67 94.07 2 2923.60 94.62 3 2852.65 95.47 4 2323.74 98.30
5 2162.45 98.77 6 2106.77 98.86 7 1980.62 98.99 8 1780.04 96.23
9 1707.78 94.24 10 1604.01 88.97 11 1518.16 94.40 12 1438.55 90.43
13 1340.89 89.66 14 1196.32 86.68 15 1030.95 82.51
89
D.W. Adongo et al. Journal of Ethnopharmacology 206 (2017) 78–91
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