Bio-guided isolation of new phenolic compounds from Hippocrepis emerus flowers and investigation of their antioxidant, tyrosinase and elastase inhibitory activities

This study presents the bio-guided chemical investigation of a 80% methanol extract of Hippocrepis emerus flowers, a perennial non-climbing shrub. Liquid-liquid partitioning in solvents of increasing polarity combined to biological screening enabled to determine the EtOAc and n-BuOH soluble fractions as the most active parts of the extract. These fractions were chemically profiled by using a C-13 NMR-based dereplication method, resulting in the identification of twenty-six compounds. The dereplication process was completed by purification of some unknown or minor compounds of the n-BuOH fraction. Three new glycosylated flavonoids, namely kaempferol-3-O-beta-D-glucopyranosyl-7-O-D-glucopyranosyl (1 -> 3)-alpha-L-rhamnopyranoside (1), isorhamnetin 3-O-beta-D-gluco-pyranosyl-7-O-D-glucopyranosyl (1 -> 3)-alpha-L-rhamnopyranoside (2) and quercetin-3-O-D-glucopyranosyl-7,4'-O-alpha-L-dirhamnopyranoside (3), together with twelve known compounds (4 - 15) were isolated. Their structures were elucidated by spectroscopic methods including NMR, HR-ESI-MS and UV. The antioxidant activity of fractions and isolated compounds were evaluated using DPPH and hydroxyl radical scavenging and CUPRAC assays. In parallel, their inhibitory properties against mushroom tyrosinase and human neutrophil elastase enzymes were assessed. Three quercetin glycosides exhibited a significant radical-scavenging activity and two flavonoids showed a moderate elastase inhibitory activity.