Identification of extracted antioxidants from Citrus aurantium 'Changshanhuyou' residue against digestive enzyme activities and airway smooth muscle cells proliferation using UPLC-MS/MS

Rich in secondary metabolites with high nutritional value, the residue of Huyou (Citrus aurantium 'Changshanhuyou') is limitedly utilized, representing economic loss and damage to soil and environment. The present study valorized the residue by optimizing extraction conditions, identifying extracted antioxidants, and evaluating diverse bioactivities. The extraction conditions were optimized by single-factor and orthogonal array experimental design. Meanwhile, secondary metabolites were identified by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Also, the bioactivities of the extract against digestive enzyme activities and airway smooth muscle cells (ASMCs) proliferation were investigated. In the single factor experiments, a water-solid ratio of 7:1, a 90-min time, and a temperature below 80 degrees C were found to maximize antioxidant activities, respectively. The orthogonal array analysis results indicated that antioxidant activities reduced significantly as temperature increased, whereas other parameters had little effect. The optimal extraction conditions were determined to be 40 degrees C for 30 min with a water-solid ratio of 7:1, balancing antioxidant activity with energy efficiency. 29 bioactive secondary metabolites were identified as phenolic acids, flavonoids, terpenoids, alkaloids, coumarins, and tannins. Among them, flavonoids and limonoids were further quantified by high-performance liquid chromatography (HPLC) due to their variety and typicalness. Their roles as antioxidants were compared along three dimensions, including ridge regression, Pearson correlation, and the percentage of each flavonoid content. Moreover, the extracts inhibited alpha-glucosidase and pancreatic lipase activities by more than 80% and restored ASMCs proliferation to a control level. This study demonstrated that Huyou residue holds significant potential for applications in functional food.