Exogenous H₂O₂ enhances the quality of Radix Paeoniae Rubra and Radix Paeoniae Alba by activating ROS-mediated secondary metabolism.

Radix Paeoniae Rubra (RPR) and Radix Paeoniae Alba (RPA) are important medicinal herbs widely used in Asian countries. Although both are derived from the dried roots of Paeonia lactiflora Pall., they exhibit distinct pharmacological properties due to differences in germplasm and post-harvest processing methods. However, growing market demand has led to cultivated materials becoming the primary commercial source, resulting in a significant decline in their medicinal quality. Environmental stress induces the substantial production of reactive oxygen species (ROS), which can modulate enzyme activities and enhance secondary metabolism. This study aimed to address this quality decline by treating fresh roots of two P. lactiflora germplasms-one for RPR production (RPR-germplasm) and one for RPA production (RPA-germplasm)-with H₂O₂ solutions at concentrations of 0.0, 0.5, 1.0, or 2.0 mmol/L to induce ROS bursts, thereby mimicking environmental stress and activating cellular oxidative stress defense and secondary metabolic pathways. In the fresh roots of RPR-germplasm treated with 1.0 mmol/L H₂O₂ on day 2, the contents of the secondary metabolites paeoniflorin, albiflorin, paeonol, catechin, gallic acid, benzoylpaeoniflorin, oxypaeoniflorin, and benzoic acid increased by 23.7%, 22.8%, 77.0%, 58.5%, 31.3%, 29.4%, 37.4%, and 43.8%, respectively. In the fresh roots of RPA-germplasm, the contents of the major secondary metabolites albiflorin, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, gallic acid, and catechin increased by 23.0%, 30.1%, 39.4%, 14.1%, 20.3%, and 19.1%, respectively. Furthermore, co-treatment experiments with H₂O₂ and ROS scavengers confirmed that ROS act as the critical signaling mediator connecting exogenous H₂O₂ to the secondary metabolism of P. lactiflora: scavenging ROS significantly attenuated the H₂O₂-induced accumulation of target secondary metabolites. Collectively, these findings demonstrate that exogenous H₂O₂ activates ROS-mediated secondary metabolism to enhance the quality of cultivated RPR and RPA. This study elucidates a "H₂O₂-ROS-secondary metabolism" regulatory axis and provides a practical strategy for improving secondary metabolite accumulation in medicinal plants via controlled oxidative stress.