Green extraction is aimed at reducing energy consumption by using renewable plant sources and environmentally friendly bio-solvents. Lime (Citrus aurantifolia) is a rich source of flavonoids (e.g., hesperidin) and limonoids (e.g., limonin). Manufacturing of lime products (e.g., lime juice) yields a considerable amount of lime peel as food waste that should be comprehensively exploited. The aim of this study was to develop a green and simple extraction method to acquire the highest yield of both limonin and hesperidin from the lime peel. The study method included ethanolic-aqueous extraction and variable factors, i.e., ethanol concentrations, pH values of solvent, and extraction temperature. The response surface methodology was used to optimize extraction conditions. The concentrations of limonin and hesperidin were determined by using UHPLC-MS/MS. Results showed that the yields of limonin and hesperidin significantly depended on ethanol concentrations and extraction temperature, while pH value had the least effect. The optimal extraction condition with the highest amounts of limonin and hesperidin was 80% ethanol at pH 7, 50 °C, which yields 2.072 and 3.353 mg/g of limonin and hesperidin, respectively. This study illustrates a green extraction process using food waste, e.g., lime peel, as an energy-saving source and ethanol as a bio-solvent to achieve the highest amount of double bioactive compounds.

Lime peels are food waste from lime product manufacturing. We previously developed and optimized a green extraction method for hesperidin-limonin-rich lime peel extract. This study aimed to identify the metabolomics profile of phytochemicals and the anti-cancer effects of ethanolic extract of lime (Citrus aurantifolia) peel against liver cancer cells PLC/PRF/5. The extract’s metabolomics profile was analyzed by using LC-qTOF/MS and GC-HRMS. The anti-cancer effects were studied by using MTT assay, Annexin-PI assay, and Transwell-invasion assay. Results show that the average IC50(s) of hesperidin, limonin, and the extract on cancer cells’ viability were 165.615, 188.073, and 503.004 µg/mL, respectively. At the IC50 levels, the extract induced more apoptosis than those of pure compounds when incubating for 24 and 48 h (p < 0.0001). A combination of limonin and hesperidin showed a synergistic effect on apoptosis induction (p < 0.001), but the effect of the combination was still less than that of the extract at 48 h. Furthermore, the extract significantly inhibited cancer cell invasion better than limonin but equal to hesperidin. At the IC50 level, the extract contains many folds lower amounts of hesperidin and limonin than the IC50 doses of the pure compounds. Besides limonin and hesperidin, there were another 60 and 22 compounds detected from the LCMS and GCMS analyses, respectively. Taken altogether, the superior effect of the ethanolic extract against liver cancer cells compared to pure compound likely results from the combinatorial effects of limonin, hesperidin, and other phytochemical components in the extract.