Abstract
Leaves of custard apple are widely used in many places as a popular dietary supplement for the treatment of diabetes. Flavonoids are known to have anti-diabetic activity. In this study, the main flavonoid epimers were separated. The crude extract was first screened by HPLC-DAD before and after incubation with DPPH method to evaluate the antioxidants. An efficient extraction method was employed to remove non-flavonoid components. Subsequently, five main flavonoids with two pairs of epimers including quercetin-3-O-robinobioside, rutin, quercetin-3-O-β-D-glucoside, kaempferol-3-O-robinobioside, and kaempferol-3-O-rutinoside were successfully separated by high-speed counter-current chromatography with ethyl acetate/n-butanol/water (4:1:5, v/v) coupled with online-storage inner-recycling mode. The structures of the separated compounds were identified by spectral techniques. The purity of the separated flavonoid glycosides was over 98%, as determined by HPLC. The separated pure constituents were found to possess the antioxidant capacities following DPPH radical scavenging protocol. The compounds (1-3) exhibited better antioxidant activity. Furthermore, the glucose uptake of crude flavonoid extract had better results than the crude ethanol extract. The present study demonstrates that the efficacy of custard apple leaves in lowering glucose level, and antioxidant capacities of separated pure compounds probably appear to be predominantly responsible for hypoglycaemic properties on HepG2 cells.
Highlights
Leaves of custard apple are widely used in many places as a popular dietary supplement for the treatment of diabetes
Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), phosphate-buffered saline (PBS), streptomycin, and penicillin were purchased from Jinan Kang-Mai Science & Technology Co., Ltd. (Jinan, China)
The high performance liquid chromatography (HPLC)-diode array detector (DAD) before and after incubation with DPPH is a rapid screening method to understand the antioxidant capacity which is evaluated by the decrease of peak areas in the HPLC chromatogram after reaction with DPPH
Summary
Leaves of custard apple are widely used in many places as a popular dietary supplement for the treatment of diabetes. Oxidative stress has a significant contribution to the pathogenesis of diabetic nephropathy26,27 In this case, the prominent antidiabetic www.nature.com/scientificreports activity may be caused by the antioxidants present in custard apple leaves. Traditional separation methods using silica gel, preparative HPLC or TLC for separation of complex mixtures, such as that in a crude extract of custard apple leaves, usually require multiple chromatographic steps This leads to long separation times, consumption of large amount of solvent, a high risk of irreversible sample adsorption, and low sample recovery. The structures of the isolated compounds were elucidated by ESI-MS, 1H- and 13C NMR spectra Their antioxidant and antidiabetic activities were evaluated in vitro by DPPH radical scavenging and HepG2 cells respectively. This is, to the best of our understanding, the first report of the evaluation of antioxidant and antidiabetic activities with monomer compounds from custard apple leaves
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