Evaluation Of The Neuroprotective Effect And Biosafety Assessment Of Fermented Virgin Coconut Oil: Importance Of Anti-Inflammatory And Anti-Angiogenic Mediated Properties
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Date
2015-06
Authors
H. IBRAHIM, AHMAD
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Abstract
Neurodegenerative diseases can be characterized by progressive neuronal loss and dysfunction of the nervous system. In Alzheimer’s disease as well as optic neuropathies hypoxia-ischaemia, angiogenesis and inflammation induce neurodegenerative changes initiated by acute or chronic intermittent insults including oxidative damage. Current neuroprotection strategies aim to minimize cellular damage as a result of these processes. Virgin Coconut Oil (VCO) is widely utilized in the South Asian region for food and medicinal applications. Variations in its extraction process influence the levels and types of active phytochemicals present. This study aims to evaluate the neuroprotective efficacy of a commercially developed Fermented Virgin Coconut Oil (FVCO), derived from Cocos nucifera.
FVCO was screened for its neurotoxic properties on Retinal Ganglion Cells (RGC-5). Microscopic and in vitro biochemical analysis revealed no toxicity up to 100 μg/ml. In addition in vivo acute and subchronic toxicology studies in rats showed that FVCO is non-toxic up to 5g/kg. In vitro ischaemia studies revealed 3.1 μg/ml to 50 μg/ml was most effective in rescuing RGC-5 cells from the detrimental effect of 1) 48hr serum deprivation and 2) 24hr chemical hypoxia exposure of 300 μM cobalt (II) chloride (CoCl2). Furthermore FVCO significantly maintained cell viability, decreased apoptotic markers caspases 3/7, 8 and 9 and significantly reduced
microscopic apoptotic RGC-5 cell counts. FVCO’s role in attenuating intracellular oxidative stress conditions was evaluated by the 2’,7’-dichlorodihydrofluorescin (DCFH) diacetate method and its ability to stimulate endogenous glutathione (GSH) and glutathione-S-transferase (GST). FVCO significantly scavenged 1mM H2O2, KO2 and H2O2 plus 100μM ferrous perchlorate (II)-induced reactive oxygen species (ROS). FVCO also significantly stimulated the activity of GSH and GST. Screening with GC/MS revealed high amounts of the antioxidants lauric acid (45%) and myristic acid (21.8%). FVCO’s angiogenic activity was assessed by cell migration analysis of human umbilical vein endothelial cells (HUVEC), microvessel formation using rat aorta ring assay (RARA) and VEGF activity in HUVECs by ELISA method. The proliferation and viability of HUVEC’s were assessed by MTS assay after treatment with FVCO (6-200μg/ml). FVCO significantly stimulated vessel growth, stimulated HUVEC cell migration (12-25 μg/ml) and stimulated VEGF activity (6-50 μg/ml). In-vivo wound excision model studies in Sprague Dawley rats confirmed its pro-angiogenic property. FVCO treated wounds showed faster contraction and epithelialisation (16 days) compared to control (20 days). In albino Balb/c mice ischemic optic neuropathy, ProSense750 fluorescence probe was used to detect the activation of the inflammatory mediator pan-cathepsin, monitored using the Fluorescence Molecular Tomography (FMT) in vivo imaging system. FVCO treated mice (100 & 200 mg/kg) showed almost a 50% and 90% reduction respectively of the fluorescent signals. Optic nerve axonal protein NF-H was also drastically reduced by ischemia but FVCO treatment attenuated this reduction. FVCO thus exhibits its neuroprotective activities by 1) inhibiting caspase dependent apoptosis in ischaemic
and hypoxic RGCs, 2) modulating VEGF dependent angiogenic response on endothelial cells, 3) neutralizing free radicals and 4) inhibiting ischaemia-reperfusion induced neuroinflammation in the optic nerve.
Keywords: Neuroprotection; Ischaemia, Hypoxia, Retinal Ganglion Cell; Mice Ischaemic Optic Neuropathy Model; Fluorescence Molecular Tomography; Fermented Virgin Coconut Oil.; Traditional medicine
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Evaluation Of The Neuroprotective Effect And Biosafety Assessment Of Fermented Virgin Coconut Oil , Importance Of Anti-Inflammatory And Anti-Angiogenic Mediated Properties