Neurotrophic Effects of Ethanol Extracts on Hippocampal Neurons: Role of Anethole in Neurite Outgrowth and Synaptic Development

Mill, commonly known as fennel, is an aromatic herb traditionally used for culinary and medicinal purposes, with potential therapeutic effects on neurological disorders. However, limited research has focused on its neurotrophic impact, particularly on neuronal maturation and synaptic development. This study investigates the neurotrophic effects of ethanol extracts (FVSE) on the maturation of rat primary hippocampal neurons. Results show that FVSE and its prominent component, anethole, significantly promote neurite outgrowth in a dose-dependent manner. Optimal axonal and dendritic growth occurred at concentrations of 40 µg/mL FVSE and 20 µM anethole, respectively, without causing cytotoxicity, underscoring the safety of FVSE for neuronal health. Additionally, FVSE enhances the formation of synapses, essential for neuronal communication. Network pharmacology analysis revealed that FVSE components influence critical neurotrophic pathways, including PI3K-AKT and Alzheimer's disease pathways. Specifically, FVSE modulates key proteins, including tropomyosin receptor kinase (Trk), glycogen synthase kinase 3 (GSK3β), phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated protein kinase (Erk1/2). Anethole was found to play a key role in regulating these pathways, which was confirmed by immunocytochemistry experiments demonstrating its effect on promoting neuronal growth and synaptic development. In conclusion, this study highlights the neurotrophic properties of FVSE, with anethole emerging as a critical bioactive compound. These findings provide valuable insights into the therapeutic potential of fennel in treating neurological disorders, offering a basis for future research into interventions promoting neuronal growth and survival.