2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-Oxide (PTIO^•) Radical Scavenging: A New and Simple Antioxidant Assay In Vitro

Abstract

Current in vitro antioxidant assays have several limitations, which frequently cause inconsistent results. The study develops a new antioxidant assay using the 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 h, 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 ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry 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 the tested sample. It can also satisfactorily analyze the antioxidant structure-activity relationship. PTIO scavenging has no stereospecificity and is at least involved in H transfer.

Simultaneous Study of Anti-Ferroptosis and Antioxidant Mechanisms of Butein and (S)-Butin

“…Several reports have suggested that ET is always accompanied by H + -transfer (proton transfer) in biological systems [45][46][47][48]. To evaluate proton transfer with butein and (S)-butin, we used a PTIO • scavenging assay established by our team [49]. In the assay, varying IC 50 values (the concentration with 50% radical inhibition or relative reducing power) were observed for the two isomers at different pH values (4.5, 6.0, and 7.4, shown in Table 1, and Supplementary Materials S1).…”

“…In fact, (S)-butin had a pKa value of 7.11 at 3 -OH [50,51]. On the other hand, the PTIO • scavenging reaction in pH 4.5 aqueous solution is indicated as an ET process [49]. The effectiveness of the two isomers at pH 4.5 implies that they also had the potential for ET.…”

“…The PTIO • -scavenging spectrophotometer assay was conducted in accordance with our method [49,84]. In brief, the test sample solution (x = 0-10 µL, 0.5 mg/mL) was added to (20 − x) µL of methanol, followed by 80 µL of an aqueous PTIO • solution.…”

Inhibitory Effect and Mechanism of Action of Quercetin and Quercetin Diels-Alder anti-Dimer on Erastin-Induced Ferroptosis in Bone Marrow-Derived Mesenchymal Stem Cells

“…The PTIO • -scavenging colorimetric measurement was conducted in accordance with our method [44]. Briefly, the test sample solution (x = 0-10 µL, 1 mg/mL) was added to (20 − x) µL of methanol, followed by 80 µL of an aqueous PTIO • solution.…”

Regulation of ROS–NF‐κB axis by tuna backbone derived peptide ameliorates inflammation in necrotizing enterocolitis

“…The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) assay and 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO•) assay were performed according to a previous study (Li, 2017). In brief, 1 mg DPPH• or 1 mg PTIO• was dissolved into 24 ml ethyl alcohol and the ZHANG ET AL.…”