Antioxidant properties of Lippia javanica (Burm.f.) Spreng.
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The evolution of aerobic metabolic processes unavoidably led to the production of reactive oxygen species (ROS). ROS have the ability to cause harmful oxidative damage to biomolecules. Increased ROS generation and subsequent oxidative stress have been associated with aging and neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases as a result of the extreme sensitivity of the central nervous system to damage from ROS. Antioxidant defence systems have co–evolved with aerobic metabolic processes to counteract oxidative damage inflicted by ROS. The impact of neurodegenerative disorders on society is increasing rapidly as the life expectancy of the global population increases. In this day and age, a much younger group of the population is also experiencing neurodegenerative symptoms as a result of the harmful effect of the human immunodeficiency virus (HIV) on the central nervous system. Plants are an invaluable source of medicinal compounds. The use of plants for their healing properties is rooted in ancient times. The aim of this study was to select from twenty one plants, the plant with the most promising antioxidant activity and to determine whether extracts of this plant could act as free radical scavengers, comparing the results to Trolox, a known free radical scavenger. The next step was to isolate and characterize a compound from an extract exhibiting promising antioxidant activity. Bioassay–guided fractionation was followed to achieve this. During screening trials, twenty one plants, namely Berula erecta, Heteromorpha arborescens, Tarchonanthus camphoratus, Vernonia oligocephala, Gymnosporia buxifolia, Acacia karroo, Elephantorrhiza elephantina, Erythrina zeyheri, Leonotis leonurus, Plectranthus ecklonii, P. rehmanii, P. venteri, Salvia auretia, S. runcinata, Solenostemon latifolius, S. rotundifolius, Plumbago auriculata, Clematis brachiata, Vangueria infausta, Physalis peruviana and Lippia javanica were selected from literature, based on reported antioxidant activity within the plant families, for screening of their antioxidant activity. One hundred and ten extracts were prepared from the leaves, using Soxhlet extraction and the solvents petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) and ethanol (EtOH), consecutively. The focus during initial screening trials was on chemistry–based assays. The oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays were employed for the primary screening of the one hundred and ten leaf extracts. The ORAC assay was used to determine whether the plant extracts were able to scavenge peroxyl radicals and the FRAP assay was used to determine the reducing abilities of the extracts. Quantification of the peroxyl radical scavenging activity by the ORAC assay revealed that activity was observed for most of the extracts, with the ethyl acetate and ethanol extracts of L. javanica exhibiting the most promising activity. This pattern of activity was also found with the reducing capacity evaluated by the FRAP assay in which the EtOAc and EtOH extracts of L. javanica also exhibited the most promising activity. L. javanica was selected for further study by screening for biological activity, employing the nitro–blue tetrazolium (NBT) assay and thiobarbituric acid reactive substances (TBARS) assay. Using a cyanide model to induce neurotoxic effects in rat brain homogenate, the neuroprotective properties of the extracts of L. javanica leaves were examined using the NBT assay and compared to that of Trolox. The NBT assay determines the level of superoxide anions. All the extracts of L. javanica significantly reduced superoxide anion generation at all concentrations used. The petroleum ether and ethyl acetate extracts, at all concentrations, reduced superoxide anion generation to values lower than that of the control, suggesting that these extracts may be able to attenuate normal free radical processes in the brain. The petroleum ether extract exhibited the most promising activity at a concentration of 1.25 and 2.5 mg/ml and also exhibited similar results as the ethyl acetate extract at a lower concentration than the ethyl acetate extract (2.5 mg/ml compared to 5 mg/ml). A toxin–solution consisting of hydrogen peroxide (H2O2), iron(III)chloride (FeCl3) and ascorbic acid was used to induce lipid peroxidation and the ability of the extracts of the leaves of L. javanica to attenuate lipid peroxidation was investigated in rat brain homogenate and compared to that of Trolox. All of the extracts of L. javanica significantly attenuated toxininduced lipid peroxidation at all concentrations used. All of the extracts were also able to significantly attenuate toxin–induced lipid peroxidation to values lower than that of the control. These results suggest that all of the extracts of L. javanica possess the ability to attenuate not only toxin–induced lipid peroxidation, but also lipid peroxidation that occurs during normal processes in the brain. The petroleum ether extract was subjected to bioassay–guided fractionation using column and thin–layer chromatography and the NBT and TBARS assays. Fraction DD1 was investigated by means of nuclear magnetic resonance, infrared and mass spectrometry. The exact structure of fraction DD1 was not elucidated. Considering all the results, it is clear that L. javanica shows great potential as a medicinal plant with antioxidant activity and may therefore be beneficial in diminishing the destructive oxidative effects inflicted by free radicals. There are however still many compounds to be isolated from L. javanica. Key words: Verbenaceae, Lippia javanica, antioxidant, neurodegeneration, oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), nitro–blue tetrazolium assay (NBT), thiobarbituric acid reactive substances assay (TBARS).
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