In this research, we compared the phenotypical characters, total anthocyanins content, total phenols content, and antioxidant activity of red-fleshed apple cultivars 'XJ4', 'QN-5', 'DH' and 'HX1' at three fruit developmental stages. A further flavonoids metabolites study was conducted in 'XJ4' and 'DH'. We found broader variation of total anthocyanins content in the peel of the four cultivars, which might result in larger differences of free radicals scavenging rate. The most significant difference in fruit phenotype, anthocyanins content, and DPPH scavenging rate was observed between 'XJ4' and 'DH' at mature stage. Therefore, the flavonoids metabolites of 'XJ4' and 'DH' at mature stage were compared to unveil the details of anthocyanins compounds. The unique compounds pelargonidin 3-O-β-d-glucoside and cyanidin-3-O-malonylhexoside were detected only in peel and flesh of 'XJ4' but not in 'DH', which might contribute to the purple peel and dark-red flesh color of 'XJ4'. Significantly decreased upstream metabolites in the early biosynthetic genes regulated domain were found only in 'XJ4' peel but not in the flesh. This might explain why the anthocyanins content in 'XJ4' peel was decreased largely at the mature stage. Taken together, our findings will give some insight into the metabolites study in flavonoid biosynthetic pathway of red-fleshed apple. Molecules 2020, 25, 0 2 of 22The number of hydroxyl groups on two aromatic benzene rings and the double bond positions on oxygenated heterocycle have a great impact on the sensitivity of anthocyanins to oxidation [9].As strong antioxidants, anthocyanins have a strong DPPH, ·OH, and O 2 ·radicals scavenging ability and the related studies have been intensively reported in many horticultural crops [10]. Previous study found the extract of anthocyanins from purple cabbage had strong capacity to scavenge free radicals [11]. Lyophilized pomegranate anthocyanins extract had strong ability to scavenge hydroxyl and superoxide anions [12]. Purple cauliflower had high total anthocyanins contents and scavenging rate of DPPH radical [13]. Red-fleshed apples (Malussieversii f. Neidzwetzkyana (Dieck) Langenf) are valuable resources attracting much more attention because they contain extremely high levels of anthocyanins compared to white and off-white fleshed apples [14]. The enriched anthocyanins properties of red-fleshed apple made it show stronger antioxidant activity. Our previous study indicated red-fleshed apple cultivar 'QN-5' exhibited stronger capacity for DPPH, OH, and O 2 ·radicals scavenging when compared to vitamin C [15]. A recent study found red-fleshed apples can inhibit the proliferation of human breast cancer MCF-7 and MDA-MB-231 cells through studying the antioxidant and anti-proliferation properties, compared with a traditional cultivar 'Fuji' [16]. In addition, it's reported anthocyanins extract from red-fleshed apple could eliminate ROS induced oxidative damage in porcine cells [17]. Interestly, it has been found that the antioxidant activity and phenolic conten...

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