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The flavonoids were extracted from alfalfa using ethanol assisted with ultrasonic extraction and purified by D101 macroporous resin column chromatography. The chemical composition and content of ethanol elution fractions (EEFs) were assessed by ultrahigh-performance liquid chromatography and hybrid quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS) and aluminum nitrate-sodium nitrite-sodium hydroxide colorimetric method. The in vitro antioxidant activity of two EEFs was conducted by scavenging DPPH free radical, and the main antioxidants of 75% EEFs were screened using DPPH-UHPLC. Moreover, the in vivo antioxidant activity of 75% EEFs and the growth performance of broilers were studied. The results showed that the content of 30% and 75% EEFs was 26.20% and 62.57%. Fifteen compounds were identified from 75% EEFs, and five of them were reported in alfalfa for the first time. The scavenging activity of 75% and 30% EEFs (200 μg/mL) against DPPH was 95.51% and 78.85%. The peak area of 5,3′,4′-trihydroxyflavone and hyperoside was decreased by 82.69% and 76.04%, which exhibited strong scavenging capacities. The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) level of three treated groups against the normal control group (NC) fed with basal diet significantly increased by 3.89-24.49%, 0.53-7.39%, and 0.79-11.79%, respectively. While the malondialdehyde (MDA) decreased by 0.47-18.27%. Compared with the NC, the feed to gain ratio (F : G) of three treated groups was lowered by 2.98-16.53% and survival rate of broilers significantly increased. Consequently, 75% EEFs extracted from alfalfa exhibited powerful antioxidant activities and might be a potential feed additive to poultry and livestock.

Investigation of Chemical Composition, Antioxidant Activity, and the Effects of Alfalfa Flavonoids on Growth Performance