Hibiscus anthocyanins-rich extract inhibited LDL oxidation and oxLDL-mediated macrophages apoptosis

The oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. Anti-oxidative reagents, which can effectively inhibit LDL oxidation, may prevent atherosclerosis via reducing early atherogenesis, and slowing down the progression to advance stages. As shown in previous studies Hibiscus sabdariffa L. is a natural plant containing a lot of pigments that was found to possess anti-oxidative of activity. Therefore, in this study, we evaluated the anti-oxidative activity of Hibiscus anthocyanins (HAs) by measuring their effects on LDL oxidation (in cell-free system) and anti-apoptotic abilities (in RAW264.7 cells). HAs have been tested in vitro examining their relative electrophoretic mobility (REM), Apo B fragmentation, thiobarbituric acid relative substances (TBARS) and radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay. The anti-oxidative activity of HAs was defined by relative electrophoretic mobility of oxLDL (decrease of 50% at 2 mg/ml), fragmentation of Apo B (inhibition of 61% at 1 mg/ml), and TBARS assay (IC50: 0.46 mg/ml) in the Cu2+-mediated oxidize LDL. Furthermore, the addition of >0.1 mg/ml of HAs could scavenge over 95% of free DPPH radicals, HAs showed strong potential in inhibiting LDL oxidation induced by copper. In addition, to determine whether oxLDL-induced apoptosis in macrophages is inhibited by HAs, we studied the viability, morphology and caspase-3 expression of RAW 264.7 cells. MTT assay, Leukostate staining analysis and Western blotting reveals that HAs could inhibit oxLDL-induced apoptosis. According to these findings, we suggest that HAs may be used to inhibit LDL oxidation and oxLDL-mediated macrophage apoptosis, serving as a chemopreventive agent. However, further investigations into the specificity and mechanism(s) of HAs are needed.