Optimization of green ultrasound-assisted extraction of phenolic compounds from Crataegus laciniata leaves and assessing for antioxidant activity, enzyme inhibition, and UPLC-ESI-MS-MS guided identification of metabolites

The wide therapeutic potential attributed to Crataegus laciniata leaves has spurred considerable interest, yet comprehensive studies on the extraction of bioactive compounds, phytochemical composition, and biological properties are scarce. The present study aimed at optimizing ultrasound-assisted extraction of phenolic compounds from C. laciniata leaves (CLL), and assessing their composition, antioxidant potential, and enzyme inhibition. Box-Behnken design was used to estimate the effects of ethanol concentration, time, and temperature on the extraction procedure. Phytochemical composition was analyzed using UPLC-ESI-MS-MS, operating in the multiple reaction monitoring (MRM) mode. Biological properties were tested through antioxidant activity and inhibition potential of three key enzymes implicated in different pathologies. The mathematical model for total phenolic content (TPC) recovery indicated high significance and experimental adequacy with optimal extraction conditions of 51.18%, 54.82 °C, and 35.80 min for ethanol concentration, temperature, and time, respectively. Under these conditions the TPC was 308.25 mg GAE/g. UPLC-ESI-MS-MS analysis led to the identification of 15 phenolic compounds, including 9 flavonoids (quercetin, myricetin, rutin, hesperetin, naringenin, chrysin, vitexin, catechin, and esculin) and 6 phenolic acids (chlorogenic, gallic, ferulic, cinnamic, coumaric and 2-methoxybenzoic acids). The CLL extract exhibited strong antioxidant activity revealed by DPPH, ABTS, OH free radical scavenging activities and the FRAP assay. In addition, the optimal extract showed a fairly good inhibitory potential against urease, α-glycosidase, and acetylcholinesterase, with IC₅₀ values of 133.28, 7.21, and 153.94 μg/mL, respectively. The optimal extract of C. laciniata leaves can be considered a rich and diversified source of phenolic compounds with potent biological activities with great potential for exploration in the pharmacological and food industries.