Current in vitro antioxidant assays have several limitations, which frequently cause inconsistent results. The study develops a new antioxidant assay using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical (PTIO?). After the investigation of various factors, the experimental protocol was briefly recommended as follows: PTIO? and the sample solution were added to phosphate buffer (pH 7.4, 50 mM), incubated at 37°C for 2 hours, and then spectrophotometrically measured at 557 nm. The validation test based on 20 pure compounds and 30 lyophilized-aqueous-extracts suggested that PTIO?-scavenging had a good linear relationship, stability, and reproducibility. In the UPLC-ESI-Q-TOF-MS/MS analysis, PTIO? was observed to give m/z 234 when encountering L-ascorbic acid. As an antioxidant assay, PTIO?- scavenging possesses four advantages, i.e., oxygen-centered radical, physiological aqueous solution, simple and direct measurement, and less interference from tested-sample. It can also satisfactorily analyze the antioxidant structure-activity relationship. PTIO?-scavenging has no stereo-specificity and is at least involved in H+-transfer.
... 85 (Chengdu, China). 3,5-Dicaffeoylquinic acid, baicalein, scutellarein, and baicalin were purchased 86 from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).
