Developmental toxicity of Peganum harmala seed extractsin zebrafish embryos

Abstract

Introduction: Peganum harmala L. is a widely utilized plant in traditional medicine in many countries, including Qatar. Nevertheless, there have been several reports of toxic effects associated with the use of P. harmala in humans. Investigation of P. harmala toxicity in animal models is limited, and there are no reports on its toxicity in zebrafish. This has prompted the investigation of the toxicity of P. harmala in the zebrafish embryo model as well as in cell culture using human cell lines. Aim: This study aimed to examine the cytotoxic effects of P. harmala seed extracts on human cell lines (breast MDA-MB 231 and lung A549 cancer cell lines) and the zebrafish embryo. Methods: P. harmala ethanolic and methanolic seed extracts and their alkaloids-enriched fractions were prepared and tested for their cytotoxicity against breast (MDA-MB-231) and lung (A549) cancer cell lines using the Alamar blue (resazurin) assay. HPLC was performed to separate and identify P. harmala compounds. Survival and hatching rates and teratogenic defects of P. harmalaethanolic extract and the alkaloids fraction only were recorded at 24, 48, 72, and 96 hpf in zebrafish embryos to calculate lethal concentration 50 (LC50) and No-effect concentration (NOEC). At 72 hpf, cardiac function was assessed through 10-second videos of blood flow. The effects of P. harmala on locomotion were assessed at 96 hpf using a stereomicroscope and analyzed via ViewPoint ZebraLab software. Results: P. harmala ethanol and methanol extracts and alkaloids fraction reduced the viability of MDA-MB-231 and A549 cells. P. harmala ethanol extract and alkaloids fraction reduced the viability of zebrafish in a concentration and time-dependent manner. The ethanol extracts and the alkaloids caused mortality of zebrafish embryos, and the LC50 for P. harmala alkaloid fraction was lower than that of the ethanolic extract at 72 and 96 hpf. P. harmala ethanol extract and the alkaloids caused a developmental delay, as shown by the reduced hatching rate. P. harmala ethanol extract and alkaloid fraction were found to induce neurotoxicity. However, only the P. harmala alkaloids fraction notably affected all cardiac parameters. Conclusion: This is the first study of the in vivo toxicity of P. harmala in the zebrafish embryo model. P. harmala ethanol and methanol extracts and the alkaloids fraction were cytotoxic to MDA-MB-231 and A549 cell lines. P. harmala ethanol extract and alkaloids fraction reduced the survival and hatching rate and caused teratogenicity and neurotoxicity in zebrafish embryos. In addition, only the alkaloids fraction affected all the tested cardiac parameters. HPLC analysis revealed the presence of P. harmala secondary metabolites that could explain the cytotoxicity of P. harmala. Additional investigations including molecular assays and individual organ toxicity assessments are required.