Abstract:
Previous studies [Martini, 1998] on the leaves of Combretum erythrophyllum (Combretaceae) confirmed the antimicrobial activity but the compounds responsible for the activity could not be identified due to insufficient material. The main aim of this study was therefore to isolate and identify the antimicrobial compounds from Combretum erythrophyllum leaf material. Trees around the Pretoria region were tested for variation in activity and a small difference in bioactivity between plants was noted. Leaf extracts were also tested for free radical scavenging activity exhibiting good antioxidant activity and possible anti-inflammatory activity. Toxicity tests using human lymphocytes showed that compounds isolated were not toxic to these human cells.For preliminary testing four standard organisms were used to compare the activity of antimicrobial components, namely Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa. These are isolates recommended by the National Committee for Clinical Laboratory Standards. The pure compounds isolated were tested using a wider spectrum of bacteria and fungi.Acetone as solvent extracted 14 antibacterial inhibitors in previous studies and was hence used for the crude extraction. The extracts were complex and group separation by solvent/solvent extraction yielded six fractions. The hexane and chloroform fractions were selected for further study. The hexane fraction contained mainly non-polar compounds and the chloroform fraction both non-polar and polar compounds. Both these fractions showed activity against S. aureus.Methods used for compound isolation were mainly column chromatography, preparative TLC and HPLC using solvents with different polarities and selectivity. Column chromatography produced the best results, yielding a number of pure compounds. Although PTLC was not as effective, seven highly active compounds were isolated from the hexane fraction but were too impure and in insufficient quantities for structure elucidation.NMR was the method used for identification of isolated compounds and confirmed by MS. The hexane fraction yielded primarily waxes and fatty acids. Although these compounds are known to exhibit good antimicrobial activity, they have not been used in medicinal research due to their poor pharmacokinetic properties and as a result further research with this fraction was abandoned. Previous work with the chloroform fraction produced triterpenoids but these were in insufficient quantity for identification. This study yielded seven antibacterial flavonoids from the same fraction, possibly due to different extraction techniques. Three of these compounds were flavones, i.e. apigenin, genkwanin and 5-hydroxy-7,4'-dimethoxyflavone and four flavonols were identified i.e. kaempferol, rhamnocitrin, rhamnazin and quercetin-5,3'-dimethylether. Although all these compounds are fairly common flavonoids they are all reported for the first time in Combretum erythrophyllum, and in some cases in the family Combretaceae.Bioassays showed selective antibacterial activity between different microorganisms. In some cases MIC values ranged in the order of 25-100 mg/ml with the overall best activity against Vibrio cholerae. For some of these compounds this is the first report of antibacterial activity.
