Comprehensive evaluation of effective polyphenols in apple leaves and their combinatory antioxidant and neuroprotective activities

Apple (Malus pumila Mill.) leaves contain many antioxidant polyphenols that have received considerable attention due to their potential prevention of various chronic human diseases. The present study aimed to provide a comprehensive evaluation of polyphenols in apple leaves. The petroleum ether, ethyl acetate, and 75% ethanol extracts of apple leaves were assessed for their total phenolic and flavonoid contents and antioxidant activities. Among the three extracts, the 75% ethanol extract contained the highest phenolic (56.74 mg/g) and flavonoid (37.56 mg/g) contents and had the highest antioxidant activity (EC50 value 50.96 mg/L) by 2, 2-diphenyl-1-picrylhydrazyl assay. According to high performance liquid chromatography profiling and high-speed countercurrent chromatography separation, the 75% ethanol extract contained five major polyphenols phloridzin (P, 66.1 mg/g), isoquercitrin (IQ, 8.4 mg/g), quercetin 3-O-xyloside (9.5 mg/g), quercetin 3-O-arabinoside (10.7 mg/g), and quercetin 3-O-rhamnoside (28.5 mg/g). Additionally, the five separated polyphenols were investigated for their antioxidant activities and protective effects against hydrogen peroxide-induced oxidative stress in rat hippocampal neurons in vitro. The results showed that all the five polyphenols had antioxidant activities like the synthetic antioxidants butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) and IQ had the highest antioxidant activity (EC50 value 2.92 mg/L) among them. The IQ combination with P or with BHT had significant synergism in antioxidant activity at the ratios from 25:1 to 1:25 (w/w) and the strongest synergism at the ratio of 1:1. The IQ showed significant neuroprotective effect at the concentrations of 0.5-1.0 mg/L, while P and BHT had no neuroprotective effect and TBHQ had neurotoxic effect at high concentrations (0.5-5.0 mg/L). These results indicated that apple leaves could be exploited as a cheap and abundant resource of antioxidants and their utilization could be very attractive for pharmaceutical, food, and cosmetic industries.