Optimization and pharmacological validation of a leukocyte migration assay in zebrafish larvae for the rapid in vivo bioactivity analysis of anti-inflammatory secondary metabolites.

Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and anti-inflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented anti-inflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassay-guided isolation of anti-inflammatory secondary metabolites from complex NP extracts.

Anticonvulsant activity of bisabolene sesquiterpenoids of Curcuma longa in zebrafish and mouse seizure models.

Turmeric, obtained from the rhizomes of Curcuma longa, is used in South Asia as a traditional medicine for the treatment of epilepsy. To date, in vivo studies on the anticonvulsant activity of turmeric have focused on its principal curcuminoid, curcumin. However, poor absorption and rapid metabolism have limited the therapeutic application of curcumin in humans. To explore the therapeutic potential of turmeric for epilepsy further, we analyzed its anticonvulsant activity in a larval zebrafish seizure assay. Initial experiments revealed that the anticonvulsant activity of turmeric in zebrafish larvae cannot be explained solely by the effects of curcumin. Zebrafish bioassay-guided fractionation of turmeric identified bisabolene sesquiterpenoids as additional anticonvulsants that inhibit PTZ-induced seizures in both zebrafish and mice. Here, we present the first report of the anticonvulsant properties of bisabolene sesquiterpenoids and provide evidence which warrants further investigation toward the mechanistic understanding of their neuromodulatory activity.