Antioxidant properties of South African plants

Abstract

South Africa with its vast biodiversity and use of plants in traditional medicine provides a platform for natural drug research and development through the discovery of new chemical entities. The present study was based on the identification of plants as a natural resource for compounds conveying potential antioxidant properties. Free radicals and related molecules, collectively termed reactive oxygen/nitrogen species (ROS/RNS), in excess, promotes oxidative stress within cells and ultimately neurodegeneration. The role of the antioxidant defenses is to eliminate the excess in ROS/RNS and therefore the interest in prevention and/or treatment of neurodegenerative diseases. These compounds have the ability to scavenge free radicals and prevent oxidative stress and related diseases such as neurodegenerative diseases; Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. For this study, Plumbago auriculata and Tarchonanthus camphoratus were selected from an initial screening (ORAC/FRAP) of a selection of 21 plant species. P. auriculata and T. camphoratus were extracted by Soxhlet extraction, using four solvents, petroleum ether, dichloromethane, ethyl acetate and ethanol, in order of increasing polarity. The extracts were bleached using an ultra-violet irradiation apparatus. Each extract of both plants were then subjected to in vitro assays to determine the antioxidant properties thereof. Both, the thiobarbituric acid (TBA) and nitro-blue tetrazolium (NBT) assays of Ottino & Duncan (1996) were used for antioxidant activity screening of the extracts in rat brain homogenates. The TBA assay relies on the assessment of lipid peroxidation, via hydroxyl anions (OH), on the basis of the complex formation between malondialdehyde (MDA) and TBA, generating a pink colour to be measured spectro-photometrically at 532 nm. The principal of the NBT assay is the reduction of NBT to nitro-blue diformazan (NBD), signified as a purple colour formation, in the presence of the superoxide anions (02 0-). The intensity of the purple colour is then measured spectrophotometrically at 650 nm. The experimental data showed that all of the extracts of P. auricufata and T. camphorates were able to scavenge for OH• and O2.-, which were then correlated to the antioxidant activity of the extracts. The best results were obtained with the EtOAc and EtOH-extracts of T. camphoratus. In comparison to the toxin (H20 2), 0.0270 ± 0.00045 nmole MDAlmg tissue, the extracts showed significant decrease in MDA formation (TBA assay). The EtOH-extract of T. camphoratus attenuated the lipid peroxidation to 0.00386 ± 0.00015 nmole MDAlmg tissue. The extracts were also compared to the toxin (KCN), 30.5006 ± 0.7812 pmole/mg proteins (NBT assay) and showed significant reduction of NBO formation and O2'.scavenging ability. The EtOAc-extract of T. camphoratus obtained the best O2'. Scavenging results 3.5891 ± 0.5029 I-lmole/mg protein reduction in NBO formation. However, based on the correlation of TBA, FRAP and ORAC results the EtOH-extract was chosen for further research. Plant extracts were also subjected to toxicity testing via the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The extracts showing toxicity were; PE (10 mg/ml) of both plants and OCM (10 mg/ml) of T. camphoratus. All other extracts showed non-significant toxicity against HeLa cell growth. The EtOH-extracts of T. camphoratus was submitted to bioassay-guided fractionation to isolate the compound responsible for these properties. Compound MVHS9 was isolated through column chromatography and purified by solid phase extraction. 1H, 13C, COSY NMR and IR spectral data were used for structure elucidation and a phthalate ester was proposed for MVHS9.