Isolation, identification, and characterization of α- asarone, from hydromethanolic leaf extract of <i>Acorus calamus</i> L. and its apoptosis-inducing mechanism in A549 cells

Due to the presence of several active secondary metabolites, the traditional Indian and Chinese medicinal herb <i>Acorus calamus</i> L. has been utilized for both medical and culinary purposes since ancient times. A recent report has underscored the promising cytotoxic effect of <i>A. calamus</i> leaves extract against non-small cell lung cancer A549 cells. Thus, we want to separate the bioactive substance from the hydromethanolic extract of <i>A. calamus</i> leaves in the current investigation. Thin-layer chromatography was used to separate the compounds and different spectroscopic methods (UV, FTIR, NMR, and LCMS/MS) were used for the structure prediction. <i>α-</i>asarone was found to be the main bioactive compound present and it was isolated from <i>A. calamus</i> leaves extract. It exerted a good cytotoxic effect with an IC₅₀ value of 21.43 ± 1.27 μM against A549 cells and IC₅₀ value of 324.12 ± 1.32 μM against WI-38 cells. The induction of apoptosis in A549 cells by α<i>-</i>asarone was reaffirmed by the diverse differential staining methods including DAPI, Acridine Orange/Ethidium Bromide, and Giemsa staining. Additionally, α-asarone induced mitochondrial membrane potential (ΔΨ_m) dissipation with a concomitant increase in the production of ROS. Furthermore, it also increased expressions of caspase-3, caspase-9, caspase-8, DR4, and DR5 genes in A549 cells. In conclusion, α-asarone-induced apoptotic cell death in non-small lung cancer cells (A549) as a result of loss of mitochondrial function, increased ROS production, subsequent activation of an internal and extrinsic caspase pathway, and altered expression of genes controlling apoptosis. As a whole, α<i>-</i>asarone is a plausible therapeutic agent for managing lung cancer. HIGHLIGHTSIsolation of bioactive compound from hydromethanolic leaves extract of <i>Acorus calamus</i> L. by thin layer chromatography.Structural elucidation of the bioactive compound was carried out using different methods like UV analysis, FTIR, NMR, and LC-MS/MS analysis.A plausible mode of action revealed that α-asarone can induce apoptosis in lung cancer cells (A549). Isolation of bioactive compound from hydromethanolic leaves extract of <i>Acorus calamus</i> L. by thin layer chromatography. Structural elucidation of the bioactive compound was carried out using different methods like UV analysis, FTIR, NMR, and LC-MS/MS analysis. A plausible mode of action revealed that α-asarone can induce apoptosis in lung cancer cells (A549). Communicated by Ramaswamy H. Sarma