Biological activity of five antibacterial flavonoids from Combretum erythrophyllum (Combretaceae)
Introduction
Plants have developed an arsenal of chemicals to survive attacks by microbial invasion (Grayer and Harborne, 1994). These include both physical barriers as well as chemical ones, i.e. the presence or accumulation of antimicrobial metabolites. These metabolites are either preformed in the plant (prohibitins) or induced after infection (phytoalexins). Since phytoalexins can also be induced by abiotic factors such as UV irradiation, they have been defined as ‘antibiotics formed in plants via a metabolic sequence induced either biotically or in response to chemical or environmental factors’. When infection or damage to a plant takes place, a number of processes are activated and some of the compounds produced become activated immediately whereas phytoalexins are produced after two to three days. Sometimes it is difficult to determine whether the compounds are phytoalexins or prohibitins especially as the same compound may be a preformed antimicrobial substance in one species and a phytoalexin in another. The chemical classes in which these substances can be found varies greatly and many are identified as flavonoids (Cowan, 1999).
Combretum erythrophyllum is widely used in traditional medical practice in southern Africa. It has been used for treating abdominal pains and venereal diseases, which indicates the presence of antibacterial compounds in the leaves (Hutchings et al., 1996). The aim of this study was to isolate the antibacterial constituents of this species since leaf extracts showed substantial activity against Staphylococcus aureus, Enterococcus faecalis, Staphylococcus aureus and Pseudomonas aeruginosa (Martini and Eloff, 1998, Eloff, 1999). Bioassay-guided fractionation yielded seven flavonoids, all but one isolated for the first time from Combretaceae (Martini et al., 2004). Flavonoids are well documented for their biological effects, including antimicrobial and cardiovascular activity, which led to the belief that a diet rich in fruit and vegetables contributes to good health (Williamson et al., 2000). Large gaps still exist, however, particularly regarding their pharmacological effects and therefore antimicrobial, antioxidant and anti-inflammatory bioassays were performed on the isolated flavonoids to shed more light on these phenolic metabolites.
There are currently no recommended and acceptable dosing regimens for commercially available “bioflavonoids” and few toxicological reports (Berkoff, 1998). We also attempted to determine the cytotoxicity of the isolated flavonoids using the lactate dehydrogenase (LDH) assay. This is a popular and reliable test for cytotoxicity in immunological as well as biocompatibility studies (Allan and Rushton, 1994). Several studies have been done on kaempferol and apigenin and they were therefore not included in the bioassays.
Section snippets
Plant material, extraction and isolation
The procedure described in Martini et al. (2004) was followed for the isolation work. Briefly leaves were dried, extracted with acetone, separated into fractions by solvent–solvent fractionation, the chloroform fraction was fractionated by open column and closed column Silica gel chromatography and collected by crystallization.
Test organisms used
One fungus and 10 bacterial isolates were used as shown in Table 1. Minimum inhibitory concentrations were determined by a serial dilution microplate assay using
Results
The structures of seven flavonoids isolated from Combretum erythrophyllum (Martini et al., 2004) are shown in Fig. 1, Fig. 2.
Discussion and conclusion
Phenolic substances generally have significant antimicrobial activity and it is assumed that their function in tissues where they accumulate might be to provide chemical barriers to invading microorganisms. It has been postulated that the methylated, lipophilic flavonoids are especially suitable as protection against microorganisms because of the ease with which they penetrate bacterial cells (Williamson et al., 2000). Many possess antifungal properties, especially the nonpolar polymethylated
Acknowledgements
This research was supported by the National Research Foundation, The Technology and Human Resources for Industry Programme and Biomox Pharmaceuticals. Mrs. A. Lombard (Microbiology, U.P.) supplied the microorganisms and Dr. A. Theron (Immunology, U.P.) assisted with the toxicity assays.
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