- Title
- Studies on the antimicrobial, antioxidant and antiproliferative potential of the ethyl acetate extract and compounds of Peltophorum africanum
- Creator
- Okeleye, Benjamin Ifeoluwa
- Date
- 2014
- Type
- Thesis
- Type
- Masters
- Type
- MSc (Microbiology)
- Identifier
- vital:11282
- Identifier
- http://hdl.handle.net/10353/d1016165
- Description
- Cells are constantly exposed to a variety of oxidizing agents, some of which are necessary for life. Oxidants produced in excess can cause an imbalance, leading to oxidative stress, especially in chronic bacterial, viral, and parasitic infections. This can result to damage of biomolecules such as lipids, proteins, and DNA, hence, an increased risk for cancer. Plants have a long history of use in the treatment of cancer. Plant secondary metabolites have proved to be an excellent reservoir of new medical compounds. Fruits, vegetables, and whole grains contain a wide variety of antioxidant phytochemicals, such as phenolics and carotenoids, and may help protect cellular systems from oxidative damage and also may lower the risk of chronic diseases. Peltophorum africanum, a member of the family Fabaceae (Sond) is also known as the African weeping wattle and is used in traditional medicine in South Africa. This study investigated the antimicrobial, antioxidant and antiproliferative potential of the ethyl acetate extract and compounds of Peltophorum africanum in order to validate its pharmacological use. The study assessed the in vitro antimicrobial activity of ethyl acetate extract (EAE) of Peltophorum africanum stem bark and its fractions by the agar well and macrodilution methods. The toxicity on a normal human liver cell (Chang liver cell) and antiproliferation of human breast (MCF-7), colon (HT-29) and cervical (HeLa) cancer cell lines were determined using the CellTiter-Blue cell viability assay and the mechanism of action delineated using the Nucleic Acid and Protein Purification Nucleospin® Tissue Kit, Scanning Electron Microscopy (SEM), Propidium iodide (PI) and Acridine orange (AO) double-staining techniques, the Cleaved Caspase 3 (Asp 175) Alexa Fluor® 488 Antibody and the Coulter® DNA PrepTM Reagents Kit. Purification and identification of the compounds from EAE and fractions as well as the morphological alteration of bacteria, yeast and cancer cells were determined using thin layer chromatography, infrared spectra fingerprint and GC-MS analysis, micro-dilution and scanning electron microscopy with energy-dispersive X-ray analysis. In vitro antioxidant activity of EAE was determined by means of radical scavenging and ferric reducing power analysis using 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2`-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) kit, hydrogen peroxide (H2O2), iron (iii) chloride (Fe3+) and nitric oxide (NO). To assess the likely effects of secondary metabolites on the activities observed; total proanthocyanidins, phenolics, flavonols, and flavonoids were determined using standard phytochemical methods. Data were analyzed by one way analysis of variance (ANOVA; SPSS Version 17.0, 2011), regression analysis (MINITAB, version 12 for windows), probit analysis test (software NCSS, 2007) and GraphPad Prism4 software package. The p-values < 0.05 were considered significant. Marked activity of the extract was observed against Plesiomonas shigelloides ATCC 51903, with MIC and MLC values of 0.15625 and 0.3125mg/mL, respectively. The extract was both bactericidal (MICindex ≤ 2) and bacteriostatic/fungistatic (MICindex > 2) in activity. Lethal dose at 50 (LD50) showed 82.64 ± 1.40 degree of toxicity at 24 hrs, and 95 percentile of cell death dose activity ranged from log 3.12 ± 0.01 to 4.59 ± 0.03. The activity of the eight fractions tested ranged from 1.0 ± 0.5 to 3.7 ± 1.6 mg/mL (IC50) and from 2.1 ± 0.8 to 6.25 ± 0 mg/mL (IC90) (Chapter 3). Due to the effect of compounds present in the crude extract and fractions, the P. aeruginosa treated with EAE had a reduction of sodium from 5.55 % (untreated) - 1.50 %. For C. albicans, pottasium was reduced from 4.16 % (untreated) - 0.76 % (T1). Remarkable morphological alterations were observed including deformation of the germ tubes and perforation of the cell wall (Chapter 4). Extract scavenging activity of 88.73± 6.69 % (25 μg mL-1), 53.93±1.09 % (25 μg mL-1) were recorded for H2O2 and NO respectively with proanthocyanidins (92.18±4.68 mg/g) occurring more (p < 0.05) in the extract compared to all other phenolics compounds (Chapter 5). Significant reduction in cell viability of the cells was noted as the MCF-7 cells were reduced from 100 - 54.33±1.84 % after 72 hrs of treatment with 5 μg/mL of EAE (P. value < 0.05). TEt10 was cytotoxic against human normal cells (chang liver cell) at EC50 of 37 μg/mL and 74 μg/mL after 24 and 48 h of treatment respectively. Marked antiproliferative activity of 13.2 μg/mL (EC50) was observed when HeLa cells were treated for 48 h. Internucleosomal DNA of MCF-7, HT-29 and HeLa cells randomly fragmented into an uninterrupted spectrum of sizes, complemented by the intercalation of nucleic acid-specific fluorochromes by PI and AO spotting two phases of apoptosis; early (EA) and late (LA) apoptosis. Distinctive ultramorphological changes observed include; cell shrinkage, membrane blebbing, and typical cell induced death. The study also recorded 705.102 ± 28.56 % TEt10 caspase-3 activity compared to curcumin 592.857 ± 165.76 % (positive control) and untreated (negative control; 100 ± 15.81 %) cells. Percentage HeLa cell with Sub-G1 DNA phase increased from 0.13 ± 0.06 % (negative control) to 13.8 ± 3.04 % compared to curcumin (8.17 ± 2.20 %) after treatment with TEt10. The compounds identified in the fractions including Colchicine, N-(trifluoroacetyl)methyl-N-deacetyl-, Lupeol and .gamma.-Sitosterol may be responsible for the induction of apoptosis observed and could be further studied in vivo as a potential template for new anticancer treatment (Chapter 6 & 7).
- Format
- 291 leaves; 30 cm
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science & Agriculture
- Language
- English
- Rights
- University of Fort Hare
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UFH Department of Biochemistry & Microbiology
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