| Vernonia amygdalina (VA) is a tropical African plant of the Asteraceae family used as a dietary supplement and medicinal herb. In this study, liquid-liquid extraction, chromatographic and spectroscopic techniques were used to extract, fractionate and characterize the major active fractions in VA. In addition, the antiproliferative activities, cellular and molecular mechanisms of action of fractions on cancer cell lines were investigated. Immunocytochemical and fluorescence techniques were used to examine tubulin assembly, cell proliferation, apoptosis, and cancer cell death after exposure to active VA fractions. The antiproliferative and antitubulin actions of VA fractions were compared to actions of Taxol and Nocodazol.;Our results indicate that sample fractionation by solvent partitioning followed by repeated chromatographic and spectroscopic analyses revealed presence of fatty acid esters, fatty acid amide, triterpene, diterpene alcohols, and phytostanols as the major chemical groups in the aqueous and organic fractions of VA leaf extracts. Their structures were elucidated, on the basis of GC-MS data, as hexanedioic acid, bis (2-ethylhexyl) ester (2a), erucamide (2b) and squalene (2c) in the water fraction; hexadecanoic acid methyl ester (3a), 9(Z)12(Z)15(Z)-octadecatrienoic acid, methyl ester (3b) and phytol (3c) in methanol fraction; phytol (4a), squalene (4b) and 7,22-ergostadienol (4c) in the petroleum ether fraction. Acid-base extraction of alkaloids and FPLC-purification of protein fractions revealed Topotecan, and fractions A, B, C, and D, respectively.;In vitro evaluation of anti-proliferative activities of sample extracts on human estrogen-receptor negative breast cancer (MDA-MB-231), estrogen receptor positive breast cancer (MCF-7), and androgen-independent prostate cancer (DU-145) cell lines revealed significant growth inhibition by organic fractions (P<0.001) compared to aqueous fraction (P<0.05). Alkaloidal compound, Topotecan, also induced significant growth inhibition on DU-145 (IC50 2.512e-007g/mL), and MCF-7 (P<0.0001) cells. Immunocytochemical analysis of tubulin design indicates that organic and aqueous fractions, as well as Topotecan abrogated the steady state-microtubule pattern to a defused disassembled form normally characterized with Nocodazol treatment. In contrast, Taxol induced aggregation and crystallization of tubulin. Further examination of cellular mechanism of action reveals dose-related induction of apoptotic and necrotic deaths in MCF-7 and DU-145 cells treated with alkaloidal, aqueous and organic fractions of VA extracts, with greater quantities of apoptotic phenotypes observed in MCF-7 than in DU-145 cells. FPLC-purified VA protein fractions also induced apoptotic death, with significant effects observed for fractions A and B (47%) compared to fractions C and D (41% and 4%), in MCF-7 cells. Manifestations of apoptosis were marked by membrane blebs, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and formation of apoptotic bodies. Taken together, these observations demonstrate that VA contains biologically active components capable of inhibiting growth and proliferation of cancer cells; and that these active constituents exert their antineoplastic properties via mechanisms that target and trigger disruption of microtubule organization, effectively causing apoptotic and necrotic cell death. Commercially available Topotecan, a prominent alkaloid in the VA fraction, proved to be a potent antiproliferative and anticancer agent with antitubulin and apoptotic-inductive actions similar to Nocodazol and Taxol. |
