Identification of Antioxidant Components and Tyrosinase Specific Inhibitors from Osmanthus fragrans Flower by Using Online UPLC-ABTS⁺·-assay and UF- LC-MS Technology

Objective: To investigate and analyze antioxidant activities and tyrosinase inhibition of the aqueous extract of Osmanthus fragrans flower (O. fragrans), as well as its active ingredients. Methods: In this study, antioxidant activities of extract of O. fragrans were carried out by using DPPH·, ABTS⁺· radical scavenging assays and total antioxidant activity (FRAP). Qualitative analysis of major active components was performed by UPLC-PDA-QDa-ABTS⁺· online method and UF-LC-MS method were used to qualitatively identify the antioxidant components and tyrosinase inhibitors from O. fragrans extract respectively. Oxidative damage model of skin fibroblasts induced by H₂O₂ was used to evaluate the protective effect of oxidative stress of O. fragrans aqueous extract and its chemical components. Results: Aqueous extract of O. fragrans had good scavenging effect on DPPH· and ABTS⁺· free radical in vitro, and its EC₅₀ values of free radical scavenging effect on DPPH· and ABTS⁺· were 37.66, 38.32 μg/mL, respectively. When the concentration of O. fragrans extract reached 2 mg/mL, FRAP value could be as high as 3.17. The EC₅₀ of tyrosinase diphenoloxidase inhibition of O. fragrans extract was 595.9 μg/mL. Through analysis of UPLC-Triple-TOF/MS, a total of 28 chemical compositions were preliminarily identified. Five antioxidant active compounds which also greatly combined with tyrosinase were screened out from O. fragrans extract by simultaneously using UPLC-PDA-QDa-ABTS⁺· online method and UF-LC-MS method. Among them, the main active ingredient was verbascoside. Antioxidant and tyrosinase assay showed that verbascoside had significant antioxidant activity and tyrosinase inhibitory activity. EC₅₀ values of verbascoside of scavenging effect on DPPH· and ABTS⁺· were 10.27, 14.96 μg/mL, respectively. When the concentration of verbascoside was 1 mg/mL, FRAP value could be up to 4.22. The EC₅₀ of tyrosinase monophenoloxidase inhibition of verbascoside was 477.5 μg/mL, but with little diphenoloxidase inhibitiory effect. The oxidative damage model induced by H₂O₂ was used to further verify the significant protective effects of O. fragrans extract and verbascoside on oxidative damage of skin fibroblasts. Conclusion: The aqueous extract of O. fragrans and its main active component, verbascoside, had good antioxidant activity, tyrosinase inhibitory activity and oxidative stress protection ability.