Protective effect of aqueous methanolic bark extracts of Alstonia scholaris (L.) R. Br. against RSL3-induced ferroptosis

Ferroptosis is an iron-dependent form of non-apoptotic cell death that has been implicated in various human pathophysiological conditions and its regulation may have therapeutic potential. Plants have been a rich source of bioactive compounds targeting various diseases, and extracts from Alstonia scholaris (L.) R. Br., locally known as “Dita”, are known for their biological activities such as antioxidant, anti-inflammatory, and hepatoprotective properties. However, its ability to regulate cell death, specifically ferroptosis, has not yet been explored. In this study, the antioxidant potential of A. scholaris bark extract and its effects in the execution of ferroptosis in HT-29 cell line were investigated. 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay demonstrated that A. scholaris extract possesses significant radical scavenging activity (IC₅₀ = 0.24 mg/mL) which may be accounted for by its modest amount of total phenolic (26.83 ± 3.51 mg gallic acid equivalent/g crude extract) and flavonoid (7.59 ± 1.59 mg quercetin equivalent/g crude extract) compounds. At a concentration of 0.1875 mg/mL, the extract rescued HT-29 cells against RAS-selective lethal 3 (RSL3)-induced ferroptosis to levels comparable with the standard inhibitor ferrostatin-1. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analyses suggest the possible presence of quinic acid and magnolol, respectively, both of which could contribute to the observed antioxidant property of the A. scholaris extract. Finally, to explore the possible role of quinic acid and magnolol in inhibiting ferroptosis, molecular docking simulations with heme oxygenase 1 (HO-1) were carried out which demonstrated preferable interactions that were comparable with a co-crystalized inhibitor (binding free energy, BFE = −7.64 kcal/mol), particularly magnolol (BFE = −7.22 kcal/mol). These results demonstrate the potential of finding novel ferroptosis regulators in A. scholaris extracts, nevertheless, further studies are needed to conclusively confirm both the presence and activity of these compounds in the extract.