Comparative bioactivity analysis of hydro-methanolic extracts of root, leaf, and inflorescence of Tupistra nutans Wall. ex Lindl: An in vitro study on antioxidant and anti-hepatocellular carcinoma activity

Abstract

Tupistra nutans, native to the Eastern Himalayas, is traditionally used as vegetables and in manaand pressure. Despite being a medicinal agricultural plant, no in-depth antioxidant and anticancer study was done. Therefore, in this study, we investigated antioxidant and anti-hepatocellular carcinoma activities of hydro-methanolic extracts of its root (ETNRt), leaf (ETNLf), and inflorescence (ETNIn). DPPH, ABTS, FRAP, and total antioxidant capacity (TAC) assays revealed that ETNLf had the highest antioxidant potential. Also, extracts showed antioxidant activity in human hepatocellular carcinoma (HepG2) cells by reducing H2O2-induced ROS generation at lower concentrations. However, at higher concentrations, ETNRt, ETNLf, and ETNIn had anticancer effects in HepG2 cells with IC50 (12 h) values of 92.62, 34.60, and 85.55 μg/ml respectively, which was further confirmed by cell migration and clonogenicity reduction. Spectrofluorometric and fluorescence microscopy revealed that ETNRt, ETNLf, and ETNIn increased intracellular ROS, superoxide, and calcium levels and reduced mitochondrial membrane potential and mass. ETNRt, ETNLf, and ETNIn also depleted reduced glutathione levels in HepG2. Acridine orange/propidium iodide and scanning electron microscopy analysis confirmed the membrane damage and apoptotic changes, whereas confocal microscopic analysis with DAPI staining indicated cytoplasmic shrinkage and DNA damage. GC-MS analysis of ETNRt, ETNLf, and ETNIn revealed the differential distribution of anticancer phytochemicals such as beta-citronellol, piperidine, diosgenin, beta-sitosterol, and stigmasterol in Tupistra nutans. Our study suggested that Tupistra nutans had a promising antioxidant and in-vitro anti-hepatocellular carcinoma activity. However, the anticancer potential of inflorescence, used as a vegetable, decreased when it was cooked compared to its hydro-methanolic extract.