AMELIORATIVE EFFECT OF MORINGA OLEIFERA LEAVES ON VANADIUM-INDUCED NEUROTOXICITY IN MICE

Abstract

Moringa oleifera (MO) is a shrub belonging to the family Moringaceae and various reports exist on the medicinal usefulness of its crude extract, including effect on some neurodegenerative diseases. Vanadium (V), a transition metal emitted into the atmosphere during fossil burning and gas flaring, is implicated in various neurodegenerative conditions. However, the ameliorative effect of a pure compound from MO has not been documented. This study was therefore designed to assess the neurotherapeutic properties of a pure compound isolated from MO leaves against vanadium-induced neurotoxicity in mice. A bioassay-guided fractionation was employed to separate the fractions of the methanol extract of MO leaves. Ferric reducing antioxidant potential assay was used to assess the fraction with the highest anti-oxidant potential, while preparative HPLC was employed to isolate the compound. Nuclear magnetic resonance was employed to elucidate the structure of the pure compound obtained, which was named MIMO2. Cell culture assays (Dihydroethidium, Micronucleus, and Comet assay tests) using immortalised mouse hippocampal cell lines (HT22) were used to assess the effect of MIMO2 on vanadium neurotoxicity. Eighty-four 2-week old mice were randomly and equally divided into seven groups, and dosed for 14 days, in the following groups: controls were water and DMSO, vanadium 3mg/kg (V), MIMO2 5mg/kg (M5), MIMO2 10mg/kg (M10), M5+V, and M10+V. Route of administration for all groups was intraperitoneal. Hanging wire and open field neurobehavioural tests were carried out on day 14, while all animals were humanely sacrificed and perfused on day 15. Histological examination on the brain included H&E, Cresyl Violet (for hippocampal neuronal count in cornu Ammonis 1 and 3 regions), immunohistochemistry (for microglia and astrocytes, with sterological count for microglia), Black Gold II histochemistry and triple immunofluorescence with confocal imaging. Data were analysed using descriptive statistics and ANOVA at α0.05. The concurrent administration of MIMO2 and V in HT22 cells resulted in a significant reduction of the immuno-expression of reactive oxygen species (33% reduction) and vanadium-induced DNA damage (52% reduction). Administration of M10 resulted in a significant amelioration of iii the neurobehavioural deficits caused by vanadium. In V group, histology showed Purkinje cell degeneration, depletion and focal multiple layering, with cerebral gliosis, neuronal clumping and degeneration. All these neuropathologies were considerably reduced with the administration of M10. Cresyl Violet stain showed significant amelioration of vanadium-induced neuronal loss in the cornu Ammonis 1 region of M10+V (4.9±2.3 x 10-5/sqμm) compared to V (3.9±1.9 x 10-5/sqμm). For H&E and Cresyl Violet, no appreciable differences were observed in M5 and M5+V compared to controls and V group, respectively. The somatosensory cortex showed microglia and astrocytic hyperplasia and hypertrophy evident in the vanadium group (12.5±1.5%, area covered by microglia), which was significantly ameliorated in M10+V (9.3±2.3%). Black Gold II histochemistry showed severe vanadium-induced pantropic demyelination, particularly in the middle band of the corpus callosum, somatosensory and motor cortices, which were significantly alleviated in M10+V. A novel antioxidant compound, MIMO2, was isolated in this study from Moringa oleifera leaves. The MIMO2 ameliorated vanadium-induced neurotoxicity both in vitro and in vivo in mice. Keywords: Moringa oleifera leaves, MIMO2, vanadium neurotoxicity, neurotherapeutic activity Word count: 499