Hplc isolate from the leaves of kaffir lime (Citrus hystrix dc) exhibits selective cytotoxicity against human breast adenocarcinoma cell line, mcf-7

Abstract

The increasing mortality and morbidity rate caused by breast cancer warrants the need to screen for compounds ideal for chemotherapeutic application. This study evaluated the cytotoxicity of Citrus hystrix leaves utilizing a bioactivity-guided fractionation scheme. Using various chromatographic techniques, the fractions were subjected to MTT [3-(4, 5-dimethylthiazol-2-yl)-2, diphenyltetrazo-lium bromide] assay to determine cytotoxicity and bioassays to confirm apoptosis as the mode of cell death. The results revealed that the crude extract was moderately cytotoxic against human breast adenocarcinoma (MCF-7) but not to other cancer cell lines tested. Solvent partitioning of the crude extract revealed that hexane partition exhibited the highest cytotoxicity (IC50: 23 µg/mL) to MCF-7. Sequentially, vacuum liquid chromatography (VLC) of the hexane partition separated VLC 7 (IC50: 17.2 µg/mL) with the highest percent yield. Hence, VLC 7 was subjected to gravity column chromatography (GCC), where GCC 7.4 exhibited promising cytotoxicity to MCF-7 (IC50: 14.6 µg/mL). Selectivity index (SI) indicated that GCC 7.4 was highly selective against MCF-7 (SI > 2), relative to non-cancer cell lines NIH3T3, HDFn, and AA8. Interestingly, GCC 7.4 induced apoptosis as confirmed by several markers including phosphatidylserine (PS) membrane translocation, mitochondrial membrane depolarization, nuclear condensation, and DNA fragmentation. To further purify the components present in GCC 7.4, high-performance liquid chromatography (HPLC) was performed. HPLC 7.4.5 was active against MCF-7 (IC50: 5.5 µg/mL) without being cytotoxic against the non-cancer cell line NIH3T3. Overall, the results demonstrated that C. hystrix leaves possess promising compounds that are highly selective and cytotoxic against MCF-7 by inducing apoptosis.