Recovery of Bioactive Fractions From <i>Citrus sinensis</i> L. Epicarp Wastes by Bioguided Assays

ABSTRACT The epicarp of Citrus sinensis L. is an agro‐industrial waste, containing compounds that may be beneficial to health. The study focused on the extraction and fractionation of epicarp waste, as well as its biological effects using bioguided assays. The epicarp was extracted in sequence using hexane, acetone, and methanol. The hexane extract demonstrated the highest activity against Artemia salina and was fractionated through vacuum liquid chromatography (VLC), inverted column chromatography (ICC), and thin‐layer chromatography (TLC). Fractionation was bioguided by the effect on A. salina . The most active fractions were then evaluated on B16F10, L5178Y cancer cells lines and also in peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers. The isolated VLC fractions were analyzed through qualitative phytochemical analysis. The TLC fractions underwent assays for total phenols, flavonoids, DPPH, and FRAP content. Qualitative phytochemical analysis revealed the presence of sugars, phenolic compounds, coumarins, and saponins. TLC 3 2 showed the highest total polyphenol content with 128.01 ± 0.90 mg GAE/g, as well as an antioxidant effect with 236.95 ± 0.78 mg Trolox/g and 641.54 ± 1.40 mM FE(II)/g. EHX, TLC 2 3, TLC 3 2, and TLC 3 6 induced an LC 50 of 11.63 ± 0.01, 50.54 ± 1.34, 50.54 ± 1.34, and 50.55 ± 1.33 µg/respectively on B16F10 cell line, compared to with doxorubicin showing an LC 50 of 30.72 ± 0.27 µg/mL. On the other hand, EHX, TLC 2 3, TLC 3 2 , and TLC 3 6 showed an LC 50 of 418.2 ± 40.84, 482.5 ± 68.02, y 266 ± 23.04 µg/mL on L5178Y cell line, while doxorubicin presented an LC 50 in 24.25 ± 0.00 µg/mL. The LC 50 of EHX, TLC 2 3, TLC 3 2 and TLC 3 6 in PBMC were 11.64 ± 0.02, 611.95 ± 0.01, 247.55 ± 0.00 and 171.42 ± 0.02, µg/mL, while doxorubicin showed LC 50 of 30.72 ± 0.48 µg/mL. Additionally, EHX induces greater effect on cancer cells without affecting PBMC viability, compared to the fractions tested. This could be related to a synergistic effect between the EHX compounds. Furthermore, the TLC fractions demonstrate selectivity for cancer cells with minimal impact on healthy cells. Therefore, the study demonstrated the potential of C. sinensis agro‐industrial waste for the recovery of bioactive fractions and the design of anticancer products.