Bioassay Guided Fractionation and Determination of Total Phenolic, Flavonoid Contents and in vitro Radical Scavenging Activity of Fractions From A. Macrosperma L, Kiwi FruitAuthor(s): Sujeewa Krishanthi Hettihewa*, Yasine Hemar and HP Vasantha Rupasinghe
This study was designed to successfully use bioassay guided fractionation to purify phenolic antioxidants rich fractions from Actinidia macrosperma fruit. Phenolic compounds were extracted using 70% acetone by steeping technique, and the vacuum manifold solid phase extraction (SPE) using Strata C18 cartridges was used for the preliminary purification to remove more polar compounds such as sugars and proteins from the defatted crude extract resulting in an acidified aqueous fraction (F1), while moderately polar compounds were obtained in an acidified methanol fraction (F2). The total phenolic, total flavonoid contents were determined by using Folin Ciocalteu method and aluminiumchloride method respectively. In vitro radical scavenging activity was evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Phenolics of the extracts were identified using high performance liquid chromatography coupled to diode array detector (HPLC-DAD). Total phenolic content, total flavonoid content and radical scavenging activity determined for the acidified methanol fraction (F2) (476.3 ± 12.7 mg GAE/100 g DW, 106.7 ± 7.5 mg CAE/100 g DW and 2.9 ± 0.1 mmolTrolox equivalents/100 g DW respectively) were significantly (p=0.05) higher than those for the acidified aqueous fraction (F1) (363.2 ± 18.9 mg GAE/100 g DW, 46.2 ± 0.7 mg CAE/100 g DW and 2.0 ± 1.0 mmol Trolox equivalents/100 g DW respectively). The phenolic profile, radical scavenging activity and the HPLC-DAD fingerprints of the SPE–purified extracts showed that the acidified methanol fraction (F2) was rich in flavonoids and was the most active fraction which was subjected to further purification to identify the antioxidants. The presented result along with the qualitative analysis on high-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) revealed that the bioassay guided fractionation was successfully employed to purify phenolics rich fractions from A. macrosperma kiwi fruit.