Antioxidant Activity and Simultaneous Estimation of Phenolic Compounds in Citrus medica Leaf Extracts Using Isocratic HPLC-PAD

Citrus medica, a member of the Rutaceae family, is renowned for its rich phytochemical profile and therapeutic potential. Its leaves, a less explored part of the plant, are abundant in phenolic compounds known for their antioxidant, antidiabetic, and cardiovascular benefits. This study aimed to extract, isolate, and characterize six phenolic compounds from C. medica leaves, including diosmin, naringenin, hesperidin, quercetin, naringin, and p-coumaric acid. Employing Soxhlet extraction with 80% ethanol, we optimized the isolation process to ensure maximal yield and purity.

The antioxidant activity of the ethanolic extract was assessed via DPPH (2,2-diphenyl-1-picrylhydrazyl) and hydrogen peroxide radical-scavenging assays. In the DPPH assay, the extract exhibited a maximum inhibition of 74.8%, while ascorbic acid, used as the standard, achieved 89.2% inhibition. Similarly, the hydrogen peroxide scavenging assay demonstrated 69.1% inhibition for the extract compared to 85.1% for ascorbic acid, highlighting its significant free radical-scavenging potential.

A novel isocratic high-performance liquid chromatography–photodiode array detection (HPLC-PAD) method was developed for the simultaneous quantification of the six phenolic compounds. This method demonstrated exceptional specificity, linearity, sensitivity (LOD and LOQ), precision, and accuracy. Unlike gradient-based approaches, this isocratic method offers simplicity, reproducibility, and compatibility with systems featuring isocratic pumps. It enables efficient analysis in a single run, addressing a significant gap in the literature, as previous methods primarily targeted fruits and peels while neglecting the phytochemical richness of leaves.

Validation confirmed the robustness and suitability of the method for industrial applications, ensuring high-quality phenolic profiling for the food, pharmaceutical, and nutraceutical sectors. The findings underscore the potential of Citrus leaves as a valuable source of bioactive compounds and the utility of the developed method for advancing plant-based analytical research.