High-performance liquid chromatography - diode array detection method validation for amentoflavone-type biflavonoids...

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 (IC₅₀=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 IC₅₀= 2.146 ± 0.086, 0.762 ± 0.039, and 1.474 ± 0.061 µg/mL respectively compared to rivastigmine, the positive control (IC₅₀= 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.