Phytochemical Enrichment of Carrot Seed Extracts by Ethanol-Modified Supercritical Fluid Extraction: Antimicrobial, Enzyme-Inhibitory, Butyrylcholinesterase Inhibition and Molecular Docking Investigations

This study explored the impact of ethanol as a co-solvent in supercritical fluid extraction on the recovery of bioactive compounds from carrot seeds and assessed the resulting extracts for antimicrobial, α-amylase and α-glucosidase, and butyrylcholinesterase inhibitory potential. Ethanol supplementation significantly improved extraction performance, with the yield increasing from 110 mg in the absence of ethanol to 134 mg at 5% ethanol, followed by a slight decrease to 132 mg at 10%. High-performance liquid chromatography (HPLC) revealed pronounced phytochemical enrichment at 5% ethanol, particularly for chlorogenic acid (1541.24 µg/g), gallic acid (1279.27 µg/g), and hesperetin (1513.68 µg/g), indicating enhanced recovery of phenolic and flavonoid constituents. The 5% ethanol extract demonstrated superior antimicrobial activity, producing inhibition zones of 19 mm against Enterococcus faecalis, 26 mm against Klebsiella pneumoniae, 25 mm against Staphylococcus aureus, and 29 mm against Candida albicans. Values of both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were markedly reduced, while antibiofilm activity reached 93.11% for E. faecalis and 91.00% for K. pneumoniae. The extract also exhibited potent inhibitory effects with IC50 values of 7.74 and 13.37 µg/mL, against α-amylase and α-glucosidase, correspondingly, as well as strong butyrylcholinesterase inhibition (IC50 = 2.51 µg/mL), highlighting promising α-amylase/α-glucosidase and butyrylcholinesterase inhibitory potential. Molecular docking further supported these findings, showing that chlorogenic acid bound more strongly than vanillin to OmpK36, lysosomal acid-α-glucosidase, and butyrylcholinesterase, with docking scores ranging from −6.1 to −6.9 kcal/mol. These findings identify ethanol-modified supercritical fluid extraction as a sustainable and effective green strategy for improving the recovery of carrot seed bioactives and enhancing their multifunctional in vitro biological properties. Notably, this study provides the first comprehensive evidence that 5% ethanol modification selectively enriches key phenolic constituents, including chlorogenic acid, gallic acid, and hesperetin, in carrot seed extracts, with corresponding enhancement of α-amylase, α-glucosidase, and butyrylcholinesterase inhibitory activities.