High-performance liquid chromatography – diode array detection method validation for amentoflavone-type biflavonoids in five Encephalartos species with potential neuroprotective activity

Abstract Ginkgo biloba is a well-known food supplement for enhancing memory and is rich in biflavonoids. Biflavonoids are predominant in the Order Cycadales (cycads). Among cycads, five Encephalartos species, E. ferox , E. kisambo , E. laurentianus , E. natalensis , and E. villosus , were selected to assess the neuroprotective potential by estimation of the antioxidant and acetylcholinesterase (AChE) inhibition activities. E. natalensis and E. ferox strongly inhibited AChE (IC 50 =1.349 ± 0.041 and 1.948 ± 0.06 µg/mL respectively), while E. kisambo and E. ferox were the most potent antioxidant. Accordingly, E. ferox was selected for further investigation. Chromatographic isolation of the ethyl acetate fraction afforded amentoflavone ( 1 ), bilobetin ( 2 ), ginkgetin ( 3 ), naringenin ( 6 ), and apigenin( 7 ). The isolated biflavonoids ( 1–3 ) showed potent AChE inhibitory activity with IC 50 = 2.146 ± 0.086, 0.762 ± 0.039, and 1.474 ± 0.061 µg/mL respectively compared to rivastigmine, the positive control (IC 50 = 3.357 ± 0.103 µg/mL). A validated high-performance liquid chromatography with diode array detection (HPLC-DAD) method was developed for the simultaneous estimation of five biflavonoids: amentoflavone, bilobetin, ginkgetin, isoginkgetin and sciadopitysin in the five Encephalartos species and Ginkgo biloba. In addition, total phenolic and total flavonoid contents were estimated in the selected plants. This study highlights Encephalartos as a potential source of bioactive compounds for further neuroactive drug discovery research.