Persistent free radicals in leaves as a stable standard for quantifying free radicals

This study explored plant-derived biogenic persistent free radicals (BPFRs) in crape myrtle leaves as an alternative standard to 2,2-diphenyl-1-picrylhydrazyl (DPPH) for quantifying organic radicals. Conventional methods rely on DPPH as a standard but are prone to degradation due to light, temperature, and humidity fluctuations. We performed electron spin resonance (ESR) measurements on both DPPH and leaf samples at various masses, temperatures (22 °C and 35 °C), and relative humidity (∼100 % RH) to evaluate radical stability. We observed consistent linear responses with increasing sample mass for crape myrtle leaves, similar to the behavior of DPPH. However, the BPFRs remained more stable under high temperature and humidity over seven days, retaining most of their radical signals compared to DPPH. The g-factor of crape myrtle leaves remained nearly constant, indicating no significant alteration in the paramagnetic center. The peak-to-peak linewidth varied slightly, reflecting minor environmental and sample preparation differences. These findings suggest that BPFRs in plant tissue are more robust standards. Implementing leaf-derived radicals as calibration references may enhance reproducibility in free radical quantification, reduce artifacts from DPPH degradation, and support broader environmental or biological applications.•BPFRs in crape myrtle leaves exhibited excellent stability under elevated temperatures and humidity compared to DPPH, maintaining their radical signals over seven days.•BPFRs demonstrated a consistent linear response with increasing sample mass, similar to DPPH, making them a viable alternative for free radical quantification.•Using leaf-derived radicals as calibration standards may enhance reproducibility in free radical quantification and mitigate artifacts from the degradation of DPPH.