Comparative Effects of Ethanol Concentration on Phytochemical Profiles and In Vitro Bioactivities of Lime (Citrus aurantifolia) and Lemon (Citrus limon) Peel Extracts

This study comparatively evaluated the effects of ethanol concentration on the phytochemical characteristics and in vitro biological responses of lime (Citrus aurantifolia) and lemon (Citrus limon) peel extracts. Extractions were performed using 50, 70, and 95% (v/v) ethanol. Extraction yield, total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity using DPPH and ABTS assays, nitric oxide (NO) production and cell viability in LPS-stimulated RAW 264.7 macrophages, and cellular metabolic activity in HeLa and A549 cancer cells were evaluated. Targeted phenolic and flavonoid profiles were also characterized using UHPLC–ESI–QTOF–MS. Lemon peel extracts consistently showed higher extraction yields than lime peel extracts across the tested ethanol concentrations, with the highest yield obtained using 50% ethanol (16.94% for lemon and 6.66% for lime). In contrast, lime peel extracts showed higher TPC values across all ethanol concentrations, with the highest TPC observed in the 95% ethanol lime peel extract (18.08 mg GAE/g extract), whereas the 95% ethanol lemon peel extract showed the highest TFC among all samples (27.82 mg QE/g extract). Among the tested extracts, the 95% ethanol lime peel extract exhibited the strongest antioxidant activity, with DPPH and ABTS IC50 values of 1.04 and 0.53 mg/mL, respectively. The 95% ethanol lime peel extract also showed a lower NO level (55.36 µM) than the LPS control while maintaining moderate cell viability (65.39%). The 95% ethanol lemon peel extract showed a lower NO level (61.41 µM), but this was accompanied by lower cell viability (43.65%), indicating that reduced cellular metabolic activity may have contributed to the lower measured NO level. Targeted UHPLC–ESI–QTOF–MS analysis revealed species- and ethanol concentration-dependent differences in the measured phenolic and flavonoid profiles. Hesperidin and rutin were detected at higher concentrations in lime peel extracts, particularly in the 95% ethanol extract, whereas gallic acid was detected only in lemon peel extracts. Lime peel extracts also generally produced greater reductions in cellular metabolic activity than lemon peel extracts, particularly the 50 and 70% ethanol extracts under selected treatment conditions. Overall, both citrus species and ethanol concentration influenced the chemical composition, antioxidant activity, NO responses, and cellular metabolic activity of the peel extracts. These findings support further investigation of citrus peel extracts as sources of bioactive compounds, while additional analytical and biological studies are required to clarify the contributions of individual constituents and their potential applications.