Protocol for Evaluation of Benzofuran from European Butterbur (Petasites hybridus) Plant Bioactivity as Anticancer and Immunomodulatory Phytochemical Against Cells’ ROS Species

Biochemical researchers have recently been quite interested in benzofuran compounds. Benzofuran molecules have been shown in various studies to possess strong biological effects, such as antitumor, antibacterial, antioxidative, and antiviral activities. The antioxidant activity of benzofuran compounds has been shown in numerous studies. Despite benzofuran biological activities having been the subject of numerous investigations, its activity on metabolism and mitochondria activity have received less attention, considering that most of the free radical and reactive oxygen species production occurs in mitochondria. In this chapter, we will go over the DPPH assay to determine benzofurane antioxidant activity against ROS as well as the techniques for determining how benzofuran affects mitochondria bioenergetics, using extracellular flux analyzer assay including MTT the cytotoxicity assay as the first screening assay, which relies on the activity of NADPH dehydrogenase, a mitochondrial oxidative phosphorylation enzyme.