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Title: Targeted screening of novel antiseizure compounds from ethnomedical plants using zebrafish (Danio rerio)
Other Titles: Gericht onderzoek van nieuwe antiepileptische verbingingen uit ethnomedicijnaal planten met gebruik van zebravis (Danio rerio)
Authors: Buenafe, Olivia Erin
Issue Date: 25-Sep-2014
Abstract: The recent decades have witnessed the revival of interest in traditional and complementary alternative medicine to treat various types of disorders. The impetus behind the renewed attention on the subject is the impression that the ethnomedical treatments employed by such practitioners are safe due to long history of human use spanning centuries. Cultures in countries with high biodiversity and with long-standing ethnomedical tradition often maintain oral or written accounts of their practices, including materiae medicae comprising items derived from both plant and animal kingdoms. These serve as a starting point for targeted natural-product drug discovery and development.This has been the most successful strategy so far in producing new medicines for almost a half-century. Despite its apparent success, the tactic only accounts for approximately one-third of the newly-approved drugs in the market, with only a fraction corresponding to antiepileptic drugs (AEDs). The dearth of AEDs developed or derived from natural-products presents an opportunity in expanding natural-products drug discovery and development into the field of epilepsy and seizures.Approaching this gap requires a basic understanding of the disease in question. Epilepsy is a complex neurological disorder that features spontaneous electrical activity called seizures. Its etiology can be traced to various underlying factors—such as existing brain lesions, head trauma, chemical exposure, and genetic predisposition—that affect treatment regimens. Approximately 30% of the affected patients suffer from pharmacoresistant seizures, affecting their quality of life, thus presenting a challenge in developing new AEDs with few or no side effects. In order to discover and develop new AEDs from natural-products in the context of targeted ethnomedical screening, there is a need for an epileptic seizure model appropriate for this general plan. The use of zebrafish (Danio rerio) larvae, which were exposed to proconvulsants like pentylenetetrazol (PTZ), as the first-line in vivo seizure model is compatible to this strategy, as it presents itself as a simple, economical and straightforward approach to AED screening. For this doctoral thesis, plants from Chinese and Philippine ethnopharmacopeia were selected based on their recorded anti-seizure activities—danshen, and pea eggplant.Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is a commonly-prescribed herb in traditional Chinese medicine (TCM) to treat various cardiovascular and neurological disorders, including epileptic seizures. In our study, acetone extracts of danshen demonstrated inhibition of PTZ-induced seizure activity in the larval zebrafish PTZ assay. Four tanshinones with varying degrees of inhibition of PTZ-induced seizures in larval zebrafish were isolated via subsequent zebrafish bioactivity-guided fractionation using HPLC and NMR spectroscopy. One of the active tanshinones, tanshinone IIA—which is a prescribed drug in China to treat cerebral ischemia—demonstrated significant reduction of brain c-fos expression in zebrafish larvae at 80 µM. Tanshinone IIA also showed anticonvulsant activity in the mouse 6-Hz psychomotor seizure model from 0.5-1 mg/kg without any notable side-effects as demonstrated in beam-walking tests. These results indicate the potential of the compound and its possible derivatives to be developed as a novel AED.Solanum torvum, L., or the pea eggplant, is used in Philippine ethnomedical tradition as an anti-seizure agent in patients in a form of a decoction. Both the methanol and aqueous extracts of S. torvum demonstrated reduction of PTZ-induced activity in the larval zebrafish PTZ assay. Microfractionation of the methanolic extract coupled with the larval zebrafish PTZ assay enabled the isolation of various compounds which were identified via UHPLC-TOF-MS as steroidal glycosides. Complete de novo structure elucidation of six isolated compounds was performed, with the generation of their common aglycone, solanolide, by acid-hydrolysis. These steroidal glycosides and the aglycone exhibited various degrees of inhibition of PTZ-induced seizure activity in larval zebrafish. A similarity in structure was noted between the steroidal glycosides (and aglycone) with ganaxolone, a neurosteroid-derived compound which acts as a potent GABAA receptor modulator and is in Phase III clinical trials as potential treatment for epilepsy and Fragile X syndrome. However, these steroidal glycosides and aglycone had less modulatory activity as compared to ganaxolone or other neurosteroids on overexpressed GABA receptors in Xenopus laevis oocytes, indicating that the S. torvum compounds’ mode of action may not wholly involve GABA receptor modulation. As these studies involve ethnomedical plants from different parts of the world, following this general AED discovery strategy in the future may be affected by international legislation such as the Nagoya Protocol, which may add a layer of complexity in further natural-product AED development, especially in terms of source attribution and intellectual property rights.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Biodiscovery (+)

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