Bioactivity of Piper crocatum Extract: Apoptosis Induction and Anticancer Activity in A549 Cell Line Supported by Multi Target Molecular Docking

Abstract

Piper crocatum Ruiz & Pav. (red betel) is a medicinal plant known for its antioxidant and anticancer properties. However, its apoptosis-inducing activity associated with mitochondrial dysfunction and its antiproliferative effects against A549 human lung cancer cells remain insufficiently explored. This study aimed to evaluate the phytochemical composition, antioxidant activity, apoptosis-inducing potential, antiproliferative activity, and molecular interactions of P. crocatum extract and fractions. Leaves of P. crocatum were extracted with 70% ethanol and fractionated into n-hexane, ethyl acetate, and aqueous fractions. Phytochemical constituents, including phenolics, flavonoids, and alkaloids, were determined, and antioxidant activity was evaluated using the DPPH assay. Apoptosis-like activity was assessed in Saccharomyces cerevisiae through petite-frequency and rhodamine B fluorescence assays, while antiproliferative activity against A549 lung cancer cells was determined using the MTT assay. In addition, LC-MS-identified phytochemicals were subjected to multi-target molecular docking against EGFR, BCL-2, and KRAS G12C to investigate their potential molecular interactions.The ethyl acetate fraction exhibited the highest phytochemical contents and strong antioxidant activity. In yeast, the crude extract at 10 ppm induced the highest petite frequency and the greatest reduction in rhodamine B fluorescence, indicating pronounced mitochondrial dysfunction and apoptosis-like induction. The ethyl acetate fraction also showed the strongest antiproliferative activity against A549 cells, with an IC50 of approximately 111 ppm. Molecular docking demonstrated that several compounds, particularly Vitexin 2''-O-ß-L-rhamnoside, Kaempferitrin, Vitexin, Flemiphilippinin, and Fusarin C, exhibited favorable binding affinities toward EGFR, BCL-2, and KRAS G12C, suggesting their potential to regulate multiple pathways involved in cancer progression and apoptosis.Overall, the findings demonstrate that P. crocatum possesses significant antioxidant, apoptosis inducing, and antiproliferative activities. The integration of in vitro experiments with in silico multi-target molecular docking provides mechanistic insight into its anticancer potential and supports the development of P. crocatum as a promising natural source of multitarget anticancer agents.