ABTS+ scavenging potency of selected flavonols from Hypericum perforatum L. by HPLC-ESI/MS QQQ: Reaction observation, adduct characterization and scavenging activity determination

Hypericum perforatum L. (HPL) has been used as a beneficial herb on menopause related syndromes for many years due to its antioxidant activity by inhibiting lipid oxidation. The major bioactive gradients of HPL are flavonoids. To date, extensive researches have been focused on its antioxidant effects, however, their exact antioxidant properties remain unclear. For this purpose, this study monitored the antioxidant ability of four common and representative HPL flavonols (rutin, hyperoside, quercitrin and quercetin) to interfere with the reaction of 2, 2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) free radical (ABTS+) based on the kinetic spectrophotometric free radical processing measurement method. In addition, to characterize the antioxidant reaction paths, the above-mentioned products were identified by high performance liquid chromatography-electrospray ionization/mass spectrometer triple quad (HPLC-ESI/MS QQQ) analysis, which was superior to gas chromatography-mass spectrometer (GC-MS) in that no derivation resulted. After the adduct products were identified in the MS2 scan mode, a multiple reaction monitoring (MRM) based LC-MS method for the ABTS+ scavenging determination was developed and validated. The results of free radical scavenging kinetic evaluation showed that the ABTS+ scavenging process of the four flavonols was relatively slow and steady. The degradation products of reactions between flavonols and ABTS+ were mainly a combination of flavonols with the ABTS molecule by reducing 3-ethyl-6-benzothiazolinone imine. Flavonols substituted by glycosides tended to follow a RDA cleavage fragmentation path. With a good linearity regression coefficient (r2 = 0.995) and a good accuracy of 96.5-106.2%, the free radical scavenging ability of rutin, hyperoside, quercitrin and quercetin was 19.0%, 36.1%, 40.2% and 39.5%, respectively. The kinetic scavenging features and specific fragment features deduced here of the four flavonols in vitro could shed light on HPL's antioxidant properties.