Anti-seizure effects of medicinal plants in Malawi on pentylenetetrazole-induced seizures in zebrafish larvae

概要

学 位 論 文 全 文 要 約
Anti-seizure effects of medicinal plants in Malawi on
pentylenetetrazole-induced seizures in zebrafish
larvae
(ペンチレンテトラゾール誘発性ゼブラフィッシュけ
いれん発作モデルによるマラウィ産薬草の抗けいれ
ん作用）
Journal of Ethnopharmacology, 2021, in press.
Mayeso Naomi Victoria Gwedelaa, Haruhi Teraia, Fanuel Lampiaob,
Katsuyoshi Matsunamic, Hidenori Aizawaa

a Department

of Neurobiology, Graduate School of Biomedical and Health Sciences,

Hiroshima University, Hiroshima 734-8553, Japan

b Africa

Centre of Excellence in Public Health and Herbal Medicine, Kamuzu University of

Health Sciences, Private Bag 360, Blantyre, Malawi

c Department

of Pharmacognosy, Graduate School of Biomedical and Health Sciences,

Hiroshima University, Hiroshima 734-8553, Japan

Background and aim
Epilepsy is a neurological disorder characterized by recurrent and unprovoked
seizures. It affects 1% of the global population where up to 70% of patients can be
successfully treated with anti-seizure drugs (ASDs). In Africa however, barriers such as
long distance to health centers, low numbers of neurology medical personnel, high cost and
unavailability of ASDs put the epilepsy treatment gap at 68.5 %. Even when ASDs are
available, many patients use medicinal plants due prevailing cultural beliefs that epilepsy
is caused by supernatural forces. Among the plants used in Malawi are Margaritaria

discoidea, Dalbergia boehmii, Dalbergia nitidula, Catunaregam spinosa, and Lannea
discolor. However, their biological anti-seizure efficacy remains unclear. Using the larval
zebrafish (Danio rerio) pentylenetetrazole (PTZ) chemoconvulsant model, we screened for
the anti-seizure effect of these plants
Methods
Powdered plant material was boiled to produce decoctions and maximum tolerated
tests were conducted in larval zebrafish to determine experimental concentrations. To
validate the seizure model, larvae pre-treated with the ASD diazepam (DZP) as positive
control or vehicle were exposed to PTZ. Total distance travelled was compared between
PTZ-only and vehicle groups to confirm effect of PTZ, and between PTZ-only and DZP pretreated groups to confirm anti-seizure effect of DZP. As a primary screen of plant
decoctions, larvae were pre-treated 18 hours then exposed to PTZ. Decoctions that
significantly suppressed PTZ-induced total distance travelled passed the primary screen
and their effects on development and progression of seizure-like behavior were further
studied using seizure latency and frequency. Next, electrophysiological recordings were
obtained from the tectum of paralyzed larvae to directly measure effects of decoctions on
neuronal activity. To further confirm whether decoctions modified neuronal activation,
expression levels of the immediate early genes c-fos and npas4 were measured 0.5 and 1
hour after PTZ exposure using quantitative PCR (qPCR). Lastly, larval brains were stained
for c-fos protein to explore region-specific effects of decoctions on brain activation in the
telencephalon, midbrain, and hindbrain.
Results and discussion
In the primary assay, decoctions of M. discoidea (male) leaves, D. boehmii roots,
and D. nitidula leaves significantly suppressed PTZ-induced locomotor activity without
sedation. M. discoidea (female) leaves were excluded from further experiments because they
significantly impaired spontaneous larval movement even in the absence of PTZ, suggesting

strong sedative effect. In the locomotor behavior assay, we further used seizure latency and
frequency as measures to determine effects of decoctions on seizure development and
progression. D. boehmii significantly increased the latency to first seizure and reduced
seizure frequency. These results suggested that D. boehmii acted on seizures more
predominantly that the other two decoctions.
Electrophysiological recordings from the optic tectum confirmed that PTZ elicited
frequent seizure-like neural discharges not observed under baseline condition without PTZ.
As anticipated, larvae pre-treated with DZP had significantly lower frequency of such
discharges. Among the three decoctions, larvae pre-treated with D. boehmii and D. nitidula
had a markedly lower number of seizure-like discharges with a large effect size.
As previously reported, application of PTZ to vehicle-treated larvae significantly
increased expression levels of c-fos and npas4 transcripts. This effect was suppressed by
DZP as positive control. Similarly, both D. boehmii and D. nitidula suppressed upregulation
of the two transcripts, although the effect of D. boehmii was longer lasting. Collectively, we
observed a more robust effect of DZP, D. boehmii, and D. nitidula on PTZ-induced npas4
than c-fos. This may be because npas4 induction is more selective to excitatory neurons
than c-fos.
To explore region-specific effects of decoctions on seizure-induced brain activation,
we quantified expression of c-fos protein in the forebrain, midbrain, and hindbrain
respectively. Only the midbrain had significantly higher levels of c-fos protein upon PTZ
application when compared to control larvae. Comparable to DZP, all three decoctions
suppressed this PTZ-induced protein expression in the midbrain. Since a previous study
identified th2-positive dopaminergic neurons responsible for spontaneous larval movement
in the hypothalamus of the midbrain, it would be interesting to examine the midbrain
neuron subtypes responding to PTZ more preferentially.
Although we confirmed anti-seizure activity of three out of seven plants
administered independently, two or more plants are usually prescribed in practice to
increase the potency of treatment. Additionally, M. discoidea and D. boehmii used in
childhood-onset epilepsy whilst D. nitidula is prescribed for epilepsy in adulthood. Thus,
further studies are needed to address whether decoctions examined in the current analysis
as monotherapy and polytherapy have a differential effect on the developmental stages of
zebrafish.
Anti-seizure activities of plants have been attributed to phytocompounds such as
phenols and flavonoids that also have antioxidant and anti-inflammatory effects. Oxidative
stress and inflammation are known to be involved in seizure pathophysiology Previous
studies have profiled phenols and flavonoids in the three decoctions showing anti-seizure

activity. It would therefore be interesting to examine the effects of such isolated
phytocompounds on the larval zebrafish seizure model more specifically.
Conclusion
Our study provides evidence for anti-seizure activity in decoctions of M. discoidea leaves, D.

boehmii roots and D. nitidula leaves from a collection of plants used in Malawian
traditional medicine. Uncovering the mechanism underlying the anti-seizure effect of these
three Malawian herbs would pave the way for understanding the optimal use of a
combination of decoctions dependent upon the subtypes of epilepsy and complications.