Analysis of selected medicinal plants as antioxidants with therapeutic potential for treating diseases related to free radical damage

Oxidative damage is implicated in the pathogenesis of a number of diseases. Scientific research shows positive links between accumulated free radical damage and age-related diseases such as atherosclerosis and osteoarthritis. There is great interest in the possibility that the antioxidant potential of plant-derived compounds such as flavonoids may reduce the risk of developing these conditions. The aim of this study was to evaluate the antioxidant activity of selected non-food plants, traditionally used by herbalists in their treatment of osteoarthritis, using crude plant extracts and herbal tinctures, the most commonly used form of plant extract. As herbalists traditionally argue that herbs used in combinations or formulae will increase in efficacy when used together, an exploratory study was further carried out to investigate whether the antioxidant activity of two herbs tested in combination was greater than the sum of both herbs tested singly.
Eight plants were selected for phytochemical analysis and investigation for antioxidant activity, based on discussions with clinic supervisors from four herbal medicine training clinics and a review of patient's case notes. The prescriptions from a pilot study investigating outcomes for the herbal treatment of osteoarthritis were used as selection criteria.
Chromatographic analysis of each plant by TLC, HPLC and GCMS confirmed the presence of a number of flavonoids reported in the literature and of other compounds which were not possible to identify. Previous studies have established that certain flavonoids in vitro can exert pro-oxidant or antioxidant effects according to the concentration and presence of transition metal ions such as copper and iron. In view of the pro-oxidant effects observed for some extracts during biochemical analysis, metal analysis by ICP was carried out on the selected plant material to test for the presence of selected metal ions known to catalyse free radical reactions. ICP analysis showed the presence of most of the selected metals in all the plant samples.
Several pathways, by which flavonoids and other plant phenolics may exert their effects on chemical oxidation have been identified, one of which is their free radical scavenging capacity to halt the propagation stage of lipid peroxidation. Since lipid peroxidation is implicated in the pathogenesis of osteoarthritis, assays to measure this in vitro were investigated and the following two assays selected: - lipid peroxide assay using the ferric thiocyanate method for the detection of peroxides and an assay using the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) an established method for investigating the potential free radical scavenging activity of plant extracts. The lipid peroxide assay and method of analysis was re-evaluated and a standardised procedure established.
All eight crude plant extracts showed marked antioxidant activity in both assays. Results for the crude plant extract in the lipid peroxide assay varied according to concentration, with 0.1% w/v giving the best results. The crude plant extracts in almost all cases seemed to be more active as antioxidants than tinctures (fluid extracts). When combinations of crude plant extracts were tested in pairs for antioxidant activity, results demonstrated synergy from five of the pairs and antagonism from three, approximately one third of the possible 28 two-herb combinations tested. The synergistic interactions observed could form the foundation for the future development of an antioxidant formula to offset the effects of free radical damage.

A thesis submitted to Middlesex University in partial fulfilment of the requirements for the degree of Doctor of Philosophy.