Chemical and biological characterization of antibacterial compounds present in Ochna pretoriensis (Ochnaceae) leaf extracts

Abstract

In preliminary work done in a tree leaf screening project in the Phytomedicine Programme (www.up.ac.za/phyto) Ochna pretoriensis acetone leaf extracts had good antibacterial activity against several important bacterial pathogens. The main aim of this study was to isolate and characterize antibacterial compounds present in the acetone leaf extract of Ochna species growing in South Africa. In a preliminary screening, the minimum inhibitory concentration of acetone leaf extracts of Ochna natalitia, Ochna pretoriensis, Ochna pulchra, Ochna gamostigmata, and Ochna. Serullata, against Staphylococcus aureus, Escherichia coli, Enterococcus faecalisand Pseudomonas aeruginosa were determined by using a serial microplate dilution assay. The number of antibacterial compounds in the extracts was also determined by bioautography against the same bacteria. The MIC values of the five species ranged from 0.039 mg/ml to 1.25 mg/ml. The lowest average MIC values observed were for O. Pretoriensis especially against E. Faecalis and E. Coli. The most sensitive organism to all the plants was E. coli. O. Pretoriensis had the lowest average MIC value and the highest total activity value of 1538 ml/g. Based on bioautography some of the Ochna species had antibacterial compounds with similar Rf values. The thin layer chromatography chemical profiles of the five plant extracts may be useful in the taxonomy of the genus. O. Pretoriensis was chosen for fractionation and isolation of antibacterial compound because it has the lowest average MIC values and highest total activity especially against E. Faecalis and E. coli. The acetone extract of O. Pretoriensis was fractionated into seven fractions (hexane, carbon tetrachloride, chloroform, ethyl acetate, 35% water in methanol, 70% water in methanol and butanol) by solvent-solvent fractionation. Only three of the seven fractions (carbon tetrachloride, chloroform, ethyl acetate fractions) had clearly defined antibacterial spots/lines on bioautograms. The three fractions were further fractionated using column chromatography from which three compounds were successfully isolated. The chemical structures of the isolated compounds were determined using NMR spectroscopy as β-Sitosterol (SS), ochnaflavone (OF) and ochnaflavone 7-O- methyl ether (OFME). Compounds that are related to sitosterol have activity against neurodegenerative disorders as well as estrogenic, analgesic, anti-inflammatory, anthelminthic and antimutagenic activity. OF and OFME are biflavonoids which belong to the group ochnaflavones previously characterized from Ochna obtusata. These compounds have anti-atherosclerotic, anti-inflammatory, and anti-tumor activity. They also inhibit lymphocytes proliferation, archidonic acid release and phospholipase activity. Moreover, OFME was reported to inhibit HIV-1 activity as well as HIV-1 reverse transcriptase activity. The antibacterial activity, and potential cytotoxic, genotoxic and antigenotoxic effects of the isolated compounds were determined. The MIC values ranged from 31.3 to 250 μg/ml. SS was more active against P. Aeruginosa with an MIC of 62.5 μg/ml, OF against P. Aeruginosa and E. Faecalis with MICs of 0.03 mg/ml and OFME against P. Aeruginosa with an MIC of 31.3 μg/ml. The isolated compounds were much less active than the positive control gentamycin. The compounds had low cytotoxic activity, with LC50 values of 193.8 μg/ml for β-Sitosterol, 125.9 μg/ml for OF and 125.9 μg/ml for OFME against Vero cells. The therapeutic indexes of the crude extract and the isolated compounds varied between 0.77 and 3.27, which is an indication of non-specific antibacterial activity i.e. general toxicity, thus the crude plant extract and compounds isolated from O. Pretoriensis can only be recommended for external applications. e.g. topical treatments. None of the compounds tested had potential genotoxic and/or antigenotoxic effects. The number of revertants in the mutagenicity experiments was less than twice the number of revertants in the negative control. The percentage inhibition of 4NQO in the antimutagenicity experiments were less than 45%. The results obtained in this case may be principally associated with the general toxicity of the test samples to the bacteria used in this study. Comparison of the total activity of the crude extract and the fractions gave a clear indication of synergic interaction of compounds in the crude extract to successfully inhibit the growth of the test pathogens. Approximately 76% of activity was lost in the 34% of dry mass lost during fractionation. Twelve percent of activity was present in the chloroform fraction and 6% in the carbon tetrachloride fraction. Despite the evidence for synergistic activity, the crude extract was also relatively toxic to the Vero cells with a therapeutic index of 0.8. As far as could be established, the antibacterial activity of members of the Ochna genus and the cytotoxicity of ochnaflavones were determined for the first time in the current study. The two most active antibacterial compounds (ochnaflavone and ochnaflavone 7-O- methyl ether) are being reported from this species for the first time. The relative safety of the crude extract and the compounds isolated from this plant was relatively low. Preparations of O. Pretoriensis may be safe in a topical application but internal use cannot be recommended for treating antibacterial infections before animal toxicity studies have been carried out. Caution is also required in using the isolated compounds or crude extracts for other applications. Copyright