Isolation and characterization of antibacterial and antioxidant compounds from rinicus communis leaves

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2015

Antioxidants play an important role in living organisms to control level of free radicals and other reactive molecules in the body to reduce oxidative damage. Synthetic antioxidant compounds are used in food industries as food additives to boost our immune systems. These compounds are associated with a number of critical side effects including liver damage and carcinogenesis. Scientists are also concerned about microorganisms that have developed resistant genes against current antibiotics used in hospitals. The aim of the study was to isolate and characterize bioactive compounds from Ricinus communis leaves with activity against Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Consequently, medicinal plants are studied and considered for their efficacy and safety, because they possess bioactive compounds with various biological activities. Leaves of R. communis were collected at the University of Limpopo, Turfloop campus in Limpopo province, South Africa. The leaves were dried and milled to a fine powder. A number of trial extraction methods were employed using various solvents of different polarities on a fine powder leaves to identify the best extraction method. Plant extracts were analyzed by thin layer chromatography (TLC) developed in four mobile phases. To detect separated phytochemical compounds, TLC plates were sprayed with vanillin- sulphuric acid in methanol and heated at 110oC for optimal colour development. Qualitative antioxidant activity was determined by using 2, 2–diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates. Quantitative antioxidant activity was determined by measuring percentages scavenging activity of DPPH and 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) free radical molecules by plant extracts. Antibacterial activity of all extracts was quantified by a serial microbroth dilution method while bioautography was used in qualitative analysis of the active compounds. Cytotoxicity effect of R. communis extracts was evaluated using tetrazolium-based calorimetric assay on human Caucasian skin fibroblast (Bud-8) cell line. Anti-inflammatory activity was assessed using phagoburst kit on Raw 264.7 macrophages cell line. Pure compounds were subjected to nuclear magnetic resonance spectroscopy for 1H, 13C and DEPT experiments to elucidate structures of compounds. 2 During extraction process, methanol was the best extractant, extracting greater amount of extracts than any of the other solvents. Serial exhaustive extraction method was selected as the best extraction method for extracting compounds from ground plant materials. In quantitative antioxidant assays, chloroform and methanol extracts had highest percentage scavenging activity against DPPH free radicals compared to other extracts and vitamin C. Methanol extract had the highest percentage scavenging activity of ABTS free radicals and minimum percentage scavenging activity was in hexane extract. Acetone, ethyl acetate and ethanol extracts showed strong antioxidant activity against DPPH free radicals in qualitative antioxidant assay on TLC plates. In quantitative antibacterial assay, crude extracts showed lowest minimum inhibitory concentration value of 0.13 mg/ml against all tested organisms and the highest was 1.05 mg/ml. Hexane extracts revealed potent antibacterial activity against all tested microorganisms on bioautograms. Hexane and acetone extracts also revealed anti-inflammatory activity and have ability to reduce oxidative stress. In cytotoxicity effect of plant extracts, Methanol extracts had lethal concentration for 50% of the cells (Lc50) of 784 μg/ml on Human Caucasian skin fibroblast (Bud-8) cell line while hexane extracts had Lc50 of 629 μg/ml. Plant extracts with high Lc50 are low toxic to normal cell line and preferable to work with for drug development. Bioassay-guided fractionations results in successful isolation of three antioxidant and two antibacterial compounds from R. communis using column chromatography. Isolated compounds were tested for their biological activities using qualitative DPPH assay on TLC plates for antioxidant activity and bioautography for antibacterial activity. Antioxidant compounds showed strong antioxidant activity after spraying with DPPH in methanol and antibacterial compounds showed less activity compared to the crude extracts. The study suggests the use of crude extracts to fight against pathogenic microorganisms compared to pure compounds. Compound 4 was successful identified as the mixture of stigmasterol and β-sitosterol. The present study recommends the use of R. communis leaves as the potential source of antioxidant, antibacterial and anti-inflammatory compounds. The study serves as a scientific proof for use of this plant in traditional medicine for treatment of various ailments.