Isolation and characterization of antioxidant compounds from Combretum apiculatum (Sond.) subsp apiculatum leaf extracts

Abstract

Combretum species are used in many cultures in folk medicine for treatment of microbial infections and several inflammatory conditions (abdominal pains, headache and toothache). There are two possible mechanisms to explain the use of plants extracts to treat microbial infections. A direct effect involves the action of active agents in the extracts on the microorganism tested and the indirect effect involves the stimulation of the host immune system to overcome the effects of microorganisms via the host immune system. Traditional healers use mainly aqueous extracts and in all Combretaceae we have studied, these extracts had hardly any in vitro antibacterial activity. A search of the literature confirmed our observations, aqueous plants extracts of many plant species usually have very little direct in vitro antimicrobial activity. One would expect that aqueous extracts would have higher anti-oxidant activities and may stimulate the immune system of patients thereby combating the infection indirectly. This possibility prompted the investigation of the antioxidant potential of more polar extracts of Combretum species. Methanol extracts of leaves of ten different Combretum species were evaluated for qualitative antioxidant activity by spraying TLC chromatograms of leaf extracts with 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Compounds with anti-oxidant activity bleached the purple colour. Leaf extracts of Combretum apiculatum subspp apiculatumhad the most antioxidant compounds. This species was consequently selected for further examination. Anti-oxidant directed fractionation of the leaf extracts of C. apiculatum led to the isolation of four antioxidant compounds from ethyl acetate and butanol soluble fractions. The structures of the compounds were determined by spectral analyses (1H-NMR, 13C-NMR and MS) and identified as cardamomin (1), pinocembrin (2), quercetrin (3) and kaempferol (4). These compounds occur commonly in plant extracts, but the anti-oxidant activities of all these compounds were not known previously. In a quantitative antioxidant assay using DPPH with L-ascorbic acid as positive control, the more polar fractions (ethyl acetate and butanol) obtained by solvent-solvent fractionation had the highest activity among the extracts with EC50 values of 3.91 ± 0.02 and 2.44 ± 0.02 µg/mL respectively. Of the isolated compounds, quercetrin (3) and kaempferol (4) had strong antioxidant activity with EC50 values of 11.81 ± 85 and 47.36 ± 0.03 µM respectively. Cardamomin (1) and pinocembrin (2) did not have strong activity as these compounds could not scavenge 50% of the DPPH radical at the highest concentration (200 µM) tested. L-ascorbic acid was used standard antioxidant agent (EC50 = 13.37 ± 0.20 µM or 2.35 µg/mL). The antioxidant activity of the isolated compounds supported structure-activity relationships developed by other authors. The cytotoxicity of cardimonin and pinocembrim was evaluated using MTT assay with, berberine as positive control and DMSO as negative control. At higher concentrations than 50 µg/ml of cardimomin or pinocembrin the cells were not viable. Cardimomin was more toxic to the cells (LC50 of 1.97 µg/ml) than pinocembrin (LC50 of 29.47 µg/ml) and even the positive control, berberine (LC50 of 12.35 µg/ml). The presence of these antioxidants could provide a rationale for the ethnomedicinal use of this species for the treatment of inflammatory conditions in traditional medicine. It appears that the antimicrobial activity of aqueous plant extracts may be related to the antioxidant activity leading to a stimulated immune system rather than antimicrobial activity per se Because the crude polar extract had an antioxidant activity half that of L ascorbic acid, the rationale for using these plants by traditional healers becomes clear. The toxicity of cardomomin is a warning that the safety of these extracts should be evaluated in in vivo assays.