Isolation and characterization of antimicrobial compounds from Funtumia africana (Apocynaceae) leaf extracts

Abstract

Medicinal plants have played an important role in drug discovery, with many pharmaceutical products originating from plants. Isolation and characterization of antibacterial compounds is still relevant today because of continuing development of resistance of bacteria to antibiotics. The aim of the study was to evaluate the antibacterial activity of leaf extracts of nine tree species (Acalypha sonderiana, Androstachys johnsonii, Dracaena mannii, Drypetes natalensis, Funtumia africana, Necepsia casteneifolia, Oncinotus tenuiloba, Turraea floribunda, and Xylia torreana) selected from the Phytomedicine Programme Database based on good antimicrobial activities. The next step was to select the most active plant species and to isolate and characterize the antibacterial compounds. A serial microplate dilution method was used to determine the minimal inhibitory concentration and bioautography was used to determine the number of antibacterial compounds in the extract and their Rf values. Four nosocomial infection pathogens (Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus) were used as test organisms. Extracts of all the plant species were active with average MIC values ranging from 0.13 to 2.0 mg/ml against the four bacteria. MIC values as low as 0.08 mg/ml was obtained with F. africana and O. tenuiloba extracts against S. aureus. In bioautography seven of the nine leaf extracts had activity with clear zones of inhibition on bioautograms against the red background. F. africana was active against all four bacteria while O. tenuiloba had selective activity against P. aeruginosa with clear bands on the bioautogram. F. africana was chosen for further investigation because (a) it had good antibacterial activity against the four tested bacteria with MIC value as low as 0.08 mg/ml, (b) there were several active compounds against all the tested bacteria based on bioautography, (c) it is common in nature, and (d) as far as our literature survey could ascertain there was no published information on the antimicrobial activity of this plant species. The bulk powdered leaves of F. africana were extracted with acetone. The acetone extract was fractionated into five fractions (hexane, chloroform, butanol, H2O and 30% H2O in methanol) using solvent-solvent fractionation, to group the phytochemicals based on their polarity. Hexane and chloroform fractions were the most active with MIC values as low as 0.02 mg/ml for the chloroform fraction. One of the traditional uses of F. africana is to treat burns. As a result, the crude extract and its five fractions were also tested for anti-inflammatory activity using both the COX-1 and COX-2 assays. The crude extract and the hexane and chloroform fraction had moderate activity against both cyclooxygenase 1 and 2. The chloroform fraction was more active than the crude extract (59.7 ± 1.4%)with an inhibition of 68.2 ± 6.6%. Because there was no activity in the aqueous extracts and traditional healers usually use water as extractant, the pain relief experiences traditionally must be due to another anti-inflammatory mechanism. One antibacterial compound was isolated from the hexane fraction using column chromatography with silica gel as the stationary phase and a hexane ethyl acetate gradient as the mobile phase from low to high polarity. The isolated compound was identified as methyl ursolate using nuclear magnetic resonance (NMR) and mass spectrometry. Methyl ursolate has been isolated from a number of plant species. However, this is the first report on the isolation from Funtumia genus and the first report of its antimicrobial activity. Previous phytochemical investigation from the stem bark of F. africana led to the isolation of steroidal alkaloids of the conanine group. Methyl ursolate had a low activity with MIC values of >250 μg/ml against the four tested bacteria, but had better activity against five fungal (Candida albicans, Cryptococcus neomeforms, Fusarium oxysporum, Penicillium janthinellium, and Rhizoctonia solani) species with an MIC value of 63 μg/ml against F. oxysporum. The chloroform fraction had excellent activity with an MIC of 20 μg/ml and may be developed to become a useful complex drug. The more than one hundred fold lower activity of the isolated methyl ursolate compared to the activity of the chloroform fraction from which it was isolated, provides strong evidence of synergism. This may be good model system for studying synergism in antimicrobial preparations.