Antioxidant mechanism of polyphenol-rich Nymphaea nouchali leaf extract protecting DNA damage and attenuating oxidative stress-induced cell death via Nrf2-mediated heme-oxygenase-1 induction coupled with ERK/p38 signaling pathway

This study investigates the polyphenolic composition and antioxidant mechanism of an ethyl acetate fraction of Nymphaea nouchali leaves (NNLE). Various in vitro assays were performed using RAW 264.7 cells to assess the antioxidant effects of NNLE and to understand the underlying molecular mechanism. High-performance liquid chromatography analysis revealed the presence of gallic acid, catechin, epigallocatechin, epicatechin gallate, caffeic acid, luteolin, and kaempferol as the key polyphenolic composition of NNLE. NNLE had a potent ability to scavenge numerous free radicals through hydrogen atom transfer and/or electron donation. In addition, NNLE prevented the damage of DNA and quenched t-BHP induced generation of ROS without showing toxicity. NNLE was found to combat oxidative stress by enhancing the transcription and translation of both primary antioxidant enzymes and phase-II detoxifying enzymes, especially heme-oxygenase-1 (HO-1). NNLE treatment enhanced Nrf2 accumulation in the nucleus and post-translational phosphorylation level of p38 kinase and extracellular signal-regulated kinase (ERK) in RAW 264.7 cells. Treatment with p38 and ERK inhibitors completely suppressed NNLE-induced Nrf2 and HO-1 expression. We also found that p38 and ERK inhibitors significantly antagonized the increase in cell viability and cellular ROS scavenging activity induced by NNLE. The findings of this study provide scientific evidence on the potential of NNLE as a cost-effective and readily available source of natural phytochemicals, along with the strategy to prevent diseases associated with oxidative stress through attenuating disease progression.