Evaluation Of Neuroprotective Properties Of Echium Amoenum L. Ethanolic Extract
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Date
2015-04
Authors
Behnammanesh, Ghazaleh
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Abstract
In neurodegenerative diseases, ischemia and hypoxia, oxidative stress, excitotoxicity, and inflammation can all lead to neuronal loss. Ganglion cell death as a result of optic nerve degeneration can be caused by intermittent ischemic insults over defined periods. Neuroprotection aims to prevent, retard or reverse neuronal damage as a result of the underlying pathophysiological state. The goal of the present work is to evaluate the neuroprotective properties of Echium amoenum L. an Iranian native plant. Studies were carried out to deduce whether E. amoenum ethanol extract can blunt the negative influences of serum deprivation to retinal ganglion cells (RGC-5) in culture and a defined ischemic insult to the optic nerve in mice. Its anti-apoptotic, antioxidant, angiogenic response and anti-inflammatory activities were all investigated. Further studies were then undertaken to screen the active medicinal entities present by the use of Gas Chromatography-Mass Spectrometry (GC/MS). Petals of E.amoenum chloroform, ethanol, n-hexane, and aqueous extract were prepared via maceration technique and screened on RGC-5 for its neurotoxic properties. Ethanol and water extracts were non-toxic. However in vitro ischaemia studies revealed only the ethanol extract of E. amoenum at 5 μg/mL was effective in rescuing RGC-5 cells from the detrimental effect of serum deprivation by significantly maintaining viability (MTT assay), decreasing apoptotic cells (Hoescht staining) and their markers caspases 3/7, 8 and 9 (Caspase assay). E. amoenum ethanol extract neuroprotective properties in attenuating oxidative stress conditions was simulated by testing its antioxidant capacities using DPPH, ABTS, and β-carotene bleaching tests and its ability to increase the levels of glutathione (GSH) and glutathione-S-transferase (GST). The IC50 of radical scavenging activities were 10.89 ± 0.082 μg/mL, 15.91 ± 0.10 μg/mL and 9.49 ± 0.12 μg/mL respectively. E. amoenum ethanol extract at 5 μg/mL significantly increased the levels of GSH and GST. Screening with GC/MS revealed high amounts of phenolics and flavonoids supporting its potent antioxidant property. Its effect on angiogenesis was evaluated in SD-Rat aortic ring assay (RARA), E. amoenum ethanol extract at 100 μg/mL inhibited of 93.22 ± 0.127% blood vessel formation and significantly decreased the activity of the hypoxia induced factor, VEGF concentration in HUVECs lysates from 240.89 ± 0.00 to 68.15 ± 9.56 pg/mL. Furthermore, E. amoenum ethanol extract was cytotoxic to HUVECs in proliferation assay. In albino Balb/c mice ischaemic optic neuropathy model, ProSense 750 was used to probe the activation of the inflammatory mediator cathepsin and its activity monitored using the Fluorescence Molecular Tomography (FMT) in vivo imaging system. E. amoenum ethanol extract treated mice (200 and 400 mg/kg) had 61.43% and 75.63% reduction of ischaemia induced inflammatory response respectively. E. amoenum ethanol extract showed potent neuroprotective effect in in vitro and in vivo models of retinal neurodegeneration. This is likely due to its anti-apoptotic, antioxidant activity, ability to enhance cellular endogenous antioxidant species, modulation of angiogenesis, and anti-inflammatory properties.
Keywords: Neuroprotection; RGC; Mice Ischaemic Optic Neuropathy Model; FMT; Echium amoenum L.; Traditional medicine
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Pharmacy