In this article, we determine the pH effect and chemical mechanism of antioxidant higenamine by using four spectrophotometric assays: (1) 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical (PTIO•)-scavenging assay (at pH 4.5, 6.0, and 7.4); (2) Fe3+-reducing power assay; (3) Cu2+-reducing power assay; and (4) 1,1-diphenyl-2-picryl-hydrazyl (DPPH•)-scavenging assay. The DPPH•-scavenging reaction product is further analyzed by ultra-performance liquid chromatography, coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) technology. In the four spectrophotometric assays, higenamine showed good dose-response curves; however, its IC50 values were always lower than those of Trolox. In UPLC-ESI-Q-TOF-MS/MS analysis, the higenamine reaction product with DPPH• displayed three chromatographic peaks (retention time = 0.969, 1.078, and 1.319 min). The first gave m/z 541.2324 and 542.2372 MS peaks; while the last two generated two similar MS peaks (m/z 663.1580 and 664.1885), and two MS/MS peaks (m/z 195.9997 and 225.9971). In the PTIO•-scavenging assays, higenamine greatly decreased its IC50 values with increasing pH. In conclusion, higenamine is a powerful antioxidant-it yields at least two types of final products (i.e., higenamine-radical adduct and higenamine-higenamine dimer). In aqueous media, higenamine may exert its antioxidant action via electron-transfer and proton-transfer pathways. However, its antioxidant action is markedly affected by pH. This is possibly because lower pH value weakens its proton-transfer pathway via ionization suppression by solution H⁺, and its electron-transfer pathway by withdrawing the inductive effect (-I) from protonated N-atom. These findings will aid the correct use of alkaloid antioxidants.
Keywords: antioxidant; higenamine; mechanistic chemistry; pH effect.