Ultrasound-assisted extraction with natural deep eutectic solvents coupled with metabolomic and KEGG pathway analyses for bioactive compounds from thinned young citrus fruits

Thinned young citrus fruits (TYCF), an underutilized agricultural byproduct, contain abundant bioactive phenolic and flavonoid compounds. However, efficient extraction is limited by the environmental and safety issues of conventional solvents. Thus, green and effective methods are increasingly pursued to recover bioactives from citrus byproducts. This study investigated the extraction of bioactive compounds from TYCF using ultrasonic-assisted extraction (UAE) coupled with natural deep eutectic solvents (NADES). The results showed that the NADES system containing choline chloride and glycerol (ChCl-Gly) achieved the highest extraction efficiency under UAE conditions. Metabolomic profiling coupled with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the NADES-based extract exhibited upregulated metabolites, particularly flavonoids, organic acids, phenolic acids, and amino acid derivatives, which were predominantly involved in flavonoid, phenylpropanoid, and flavone and flavonol biosynthesis pathways. FTIR, SEM, and XRD analyses demonstrated that NADES extraction more effectively disrupted cell structures and reduced cellulose crystallinity compared to traditional solvents, resulting in higher yields of bioactive compounds from TYCF. This study highlights the potential of NADES as a green and efficient alternative for valorizing agricultural byproducts.