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Thesis (MSc (Chemistry and Biochemistry))--University of Limpopo, 2009. / Rhus leptodictya is a member of the Anacardiaceae (mango family) and is used for treating bacteria related disease by indigenous cultures in South Africa. Domestic animals feed on the tree during times of drought and this apparently does not cause as much tainting of milk, as when stock feed on the related Rhus lancea. Beer is brewed from the fruits of R. leptodictya and various parts are used in traditional medicine. The Manyika people use powdered roots of R. leptodictya for acute pain in the chest and abdominal areas. The Xhosa people use roots for gall sickness in cattle. A lotion of branches and smoke from burning is used for eye complaints by Swati people. The main aim of the study was to evaluate the antibacterial activity of extracts of the leaves and twigs of R. leptodictya in order to confirm the traditional use and then to isolate and characterize antibacterial compounds. To determine the best extractants for the extraction of antibacterial compounds from the dried leaves and twigs of R. leptodictya, the efficacy of several extractants was determined. Seven different solvents of varying polarity hexane (Hex), chloroform (Chl), dichloromethane (DCM), ethyl acetate (Eth), acetone (Act), methanol (Met) and water (Wat) were used in serial extraction to extract compounds from the leaves and twigs. The mass extracted from 4g by different solvents ranged from 43-965 mg of dry weight. The plant extracts with the highest yield was the DCM extract followed by the methanol and acetone extracts. Thin layer chromatography (TLC) was used to analyze the chemical composition of the extracts using three eluent solvent systems of varying polarities i.e. CEF, BEA and EMW and sprayed with vanillin-sulphuric acid. The chemical composition of the different extracts was vii similar with the exception of methanol and water extracts, which had only one or two visible compounds after treating with vanillin spray reagent. Chromatograms developed in three different solvent systems (CEF, BEA and EMW) were sprayed with 2, 2-diphenylpicrylhydrazyl (DPPH) to evaluate antioxidant activity. There were few antioxidant compounds present. To evaluate the number of antibacterial compounds present in the fractions, bioautography was used against four most important nosocomial microorganisms. S. aureus, E. faecalis, P. aeruginosa and E. coli. Nearly all the crude serial extraction fractions contained compounds that inhibited the growth of S. aureus. The acetone fraction had the most lines of inhibition (6) followed by ethyl acetate (5). For quantitative evaluation of antibacterial activity, minimum inhibitory concentration (MIC) values were determined using a serial microplate dilution method. The MIC values for all the fractions against all the bacterial pathogens varied from 0.04-0.63 mg/ml. The acetone extract was the most active against four bacteria with the average MIC value of 0.36 mg/ml. Leaves and twigs were extracted in bulk with acetone and solvent-solvent extraction was employed which yielded seven fractions. Bioassay guided fractionation against S. aureus was used to isolate antibacterial compounds. The largest number of antibacterial compounds occurred in the carbon tetrachloride fraction. This fraction was subjected to silica gel column chromatography eluting with eluents of increasing polarity. Two pure compounds were isolated. These compounds were identified using NMR spectroscopy and mass spectroscopy, as 2, 3 dihydro-amentoflavone and lutein. The compounds had good activity against different bacteria with MIC values ranging from 20 to 60 μg/ml. Isolated compounds were investigated for cytotoxicity against Vero cells with an LC50 of 9.4 μg/ml for lutein and 9.8 μg/ml for 2.3- viii dihydro amentoflavone which indicated toxicity. The much higher toxicity against mammalian cells than again bacterial cells indicate that these compounds do not have a good therapeutic potential but support the external use for the treatment of wounds. This is the first report of these compounds from Rhus leptodictya. The results provide scientific support for the use of R. leptodictya in treating bacterial infection in humans and animals.

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 / Dissertation (MSc)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2015 / Five plant species: Ziziphus mucronata, Senna italica, Lantana camara, Ricinus communis and Lippia javanica, were selected for this study based on their use in traditional medicine. In preliminary screening, crude extracts were prepared using hexane, dichloromethane (dcm), acetone and methanol. Phytochemical profiles on Thin Layer Chromatography plates of the extracts were obtained by developing the plates in mobile phases of varying polarity. Tests for compounds such as tannins, flavonoids, alkaloids, phlobatannins, terpenes, steroids, cardiac glycosides and saponins were carried out. Antibacterial activity of the extracts was carried out using microdilution assay for Minimum Inhibitory Concentration and bioautography against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis. Antioxidant activity of the extracts was performed using the 2, 2, diphenyl-1-picrylhydrazyl (DPPH) assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay and Phagoburst test were used to investigate the toxic effects and anti-inflammatory activity of the extracts on mouse Raw 264.7 macrophage cells, respectively. The presence of phytochemicals was observed on the chromatograms after the plates were sprayed with vanillin sulphuric acid reagent. The dcm extracts of the plants showed antibacterial activity against the selected bacterial species on the bioautograms. Senna italica and Z. mucronata showed the most activity bands on the bioautograms. Lippia javanica had the lowest MIC average of 0.56 mg/ml. Antioxidant activity was observed in the extracts of L. javanica and R. communis. The extracts promoted proliferation of the mouse macrophage cells Raw 264.7 at concentrations ranging from 0.31 mg/ml and 0.08 mg/ml. Senna italica leaves were selected for isolation of antibacterial compounds. The isolated compound was analysed on 1H and 13C nuclear magnetic resonance (NMR) and Mass Spectrometry (MS) for structural analysis. The structure could not be elucidated due to impurities in the compound but the tentative structure is a branched chain alkane with at least one ether linkage per repeating unit. Therefore the study shows that there are plant components with biological activities against wound infecting bacteria and a single lead compound was identified. / the National Research Foundation