Assessment of phytochemicals and developmental toxicity of Litsea cubeba (Lours.) Pers. fruit extract using zebrafish embryo as a model

Litsea cubeba (Lours.) Pers. is an aromatic plant whose fruits are extensively used in traditional medicine and as flavouring ingredients. Although widely used, the information regarding its phytochemical constituents and toxicity remains scarce. This study focused on identifying the phytochemicals present in the methanolic fruit extract of L. cubeba, as well as evaluating the embryotoxicity and developmental toxicity using zebrafish embryos as a model. Six hr post fertilisation (hpf) embryos were treated with 5, 10, 20, 30, 40 and 50 μg/mL of L. cubeba fruit extract (LCFE). Hatching rate was determined at 72 hpf while mortality rate and developmental abnormalities were determined at 96 hpf. Teratogenic index (TI) was calculated as the ratio of LC50/EC50. A teratogenic index value of ≥ 2 signifies teratogenic potential. To investigate the mechanism of toxicity, reactive oxygen species (ROS) production and cell death were evaluated by staining techniques. Phytochemical analysis by gas chromatography-mass spectrometry (GC-MS) identified 35 compounds, including linoleic acid, retinal, n-hexadecanoic acid, undec-10-ynoic acid, tridec-2-yn-1-yl ester, neral and seneciphylline N-oxide, which were previously reported for their toxic properties. Exposure of LCFE to zebrafish embryos caused embryotoxicity and developmental toxicity in a dose-dependent manner. The TI value of 2.3 suggested significant teratogenic potential of LCFE. Furthermore, LCFE-treated zebrafish embryos showed significant ROS production and elevated apoptosis, suggesting oxidative stress-mediated apoptosis might be the reason for the observed embryotoxicity and developmental malformations. However, further studies are required to elucidate the mechanisms that lead to the disruption of key developmental pathways and to identify the safe exposure levels of LCFE.