DPPH scavenging assay and reactive oxygen species (ROS)

A DPPH assay was conducted using a slight modification of the method reported by Gyamfi et al. [29]. Reduction of the DPPH free radical was measured by reading the absorbance at 540 nm after adding each reactant. Briefly, various concentrations of samples were incubated with 50 mM Tris-HCl (pH 7.4) and 0.1 mM DPPH ethanol solution for 30 min, in a dark room.

A fluorescent 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay was performed to determine the intracellular ROS concentrations. Murine macrophages were seeded on a 96-well black plate at 1 × 10⁵ cells/ml, and incubated with LPS in the presence or absence of SC-E1. After removing the medium, 10 μM DCFH-DA in PBS was added to each well at 37 °C for 30 min. The fluorescence was measured at excitation and emission wavelength of 480 nm and 530 nm, respectively, using a fluorescence microplate reader (Spectra Gemini, Molecular Devices).