The anti-proliferative, antioxidative and anti-inflammatory properties of the D2 fraction and HPLC semi-purified sub-fractions of dicerocaryum senecioides

Abstract

Dicerocaryum senecioides is a crawling herb that is found growing mostly in sandy areas of southern and south-eastern Africa and its small, hairy leaves have been used over the years as food, shampoo, and for treatment of various ailments. In this study, the dichloromethane (D2) fraction was prepared from a crude methanol extract of D. senecoides leaves, and its effect on the proliferation of RAW 264.7 murine macrophages was investigated. Treatment of the macrophages with the extract resulted in a dose- and time-dependent decrease in cell viability as determined by the MTT assay and real time cell analysis. Cytotoxicity of the D2 fraction on the macrophages was demonstrated to be due to apoptosis by staining the cells with DAPI nucleic acid stain. Anti-inflammatory activity of D2 fraction on RAW cells was determined by evaluating intracellular ROS production by the DCFH-DA fluorescent assay. Cells treated with the D2 fraction and stimulated with PMA were found to have a lower fluorescence intensity compared to untreated, stimulated cells; thus mimicking the response observed in the resting cells. The percentage fluorescence in untreated, stimulated cells doubled, while no significant change was observed in the D2-treated cells. The effect of the D2 fraction on iNOS activity was also assessed. The fraction reduced the NO synthesised by iNOS in cells treated with the D2 fraction and stimulated with LPS dose-dependently. The D2 fraction was further fractionated by semi-preparative HPLC; and thin layer chromatography was used to analyse phytocompounds of the 96 HPLC sub-fractions as well as to screen these sub-fractions for anti-oxidative activity. Sub-fractions 1-7 and 33-39 showed an intensely pronounced DPPH-scavenging compound and this scavenging ability was confirmed by a quantitative DPPH assay that provided parallel results. The reducing potential of the sub-fractions was assessed by evaluating their Fe3+-reducing ability through the FRAP assay. Sub-fractions 1-7 and 33-39 displayed remarkable reducing potential. Taken together with the DPPH-scavenging activity, these findings suggest that HPLC sub-fractions 1-7 and 33-39 possess a compound(s) with impressive antioxidant activity. These findings merit the D2 fraction as an extract that can be used to control chronic inflammation as it does not only inhibit free radical production, but also scavenges excessive ROS and has the ability to induce apoptosis in the macrophages responsible for dysregulated production of the free radicals. The extract also has commendable chemoprotective and chemotherapeutic potential as it demonstrated pro-apoptotic activity along with prevention of excess free-radical production.