The Neuroprotective Effects of Litsea garciae Bark Extract in Zebrafish Alzheimer's Model

Abstract

Neurodegenerative disease, particularly Alzheimer's disease (AD), leads to a gradual decline in memory and other cognitive functions among the elderly. Developing strategies to prevent oxidative damage is crucial for the therapeutic efficacy of addressing AD, with antioxidants playing a pivotal role. Scientific reports recognize the Litsea genus as a valuable source of natural antioxidants, highlighting its significance in therapies against age-associated diseases, particularly neurodegenerative conditions linked to oxidative stress. Hence, this research aimed to evaluate the neuroprotective effect of Litsea garciae (LG) bark extracts in zebrafish model. This study assessed the antioxidant properties and phytochemical composition of LG bark, including hexane, chloroform, methanol, and aqueous extracts. The antioxidant capability and capacity of various LG bark extracts were evaluated through DPPH, FRAP, Folin-Ciocalteu and aluminium chloride colorimetric assays, respectively and their secondary metabolite composition through UHPLC-MS analysis. The methanolic extract exhibited the highest total phenolic content, consistent with positive outcomes in phytochemical screening. It demonstrated the lowest IC50 value in antioxidant assay, signifying notable antioxidant capacity. These findings imply that the significant free radical scavenging activity may be linked to the high phenolic content. Major phenolic compounds identified include N-trans-feruloyl-4-O-methyldopamine, N-cisferuloyltyramine, epicatechin-(4ß->6)-epicatechin-(2ß->7,4ß->8)-epicatechin, 7-hydroxy-3-(4-methoxyphenyl)-4-propyl-2H-1-benzopyran-2-one, and 9-O-methylneodunol. Consequently, the methanolic extract was chosen for acute toxicity testing on adult zebrafish following OECD Test Guideline No. 203 to determine the optimal dosage for further in vivo investigation. Zebrafish were observed for 96 hours to identify any instances of mortality or abnormalities. The findings revealed an absence of deaths or abnormalities with a maximum dose of 100 mg/l methanolic LG bark. The zebrafish model of AD-like behaviour was validated and optimized before proceeding to investigate the potential of the methanolic LG bark extract. Two potential neurotoxins, aluminium chloride (AlCl3) and okadaic acid (OKA), were used to induce AD-like behaviour in zebrafish. The experimental design spanned 21 days and involved testing with two behavioural tasks: a 6-minute novel tank test followed by a 5-minute T-maze test. Based on the results obtained from the endpoints measured, it was confirmed that the administration of 100 nM of OKA for a 21-day duration successfully enhanced AD-like behaviour in zebrafish. The potential of the methanolic LG bark crude extract was investigated through cognitive studies, oxidative stress assessment, and examination of the GSK3? pathway. Behavioural tasks confirmed the significant (p < 0.05) capacity of the LG bark’s methanolic extract to reduce anxiety, enhance locomotor activity, memory, and learning in a zebrafish model of AD-like behaviour compared to control. The investigation continued with further exploration, involving the measurement of oxidative stress and GSK3?. Results from both evaluations significantly (p < 0.05) supported the potential of LG bark crude methanolic extract to exhibit neuroprotective properties compared to control. In conclusion, this study underscores the novelty of presenting the LG bark crude methanolic extract as a potential antioxidant, capable of controlling oxidative stress and inhibiting the activation of GSK3?.