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Theses and dissertations (Applied Sciences)

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    Biosynthesis and antibacterial activity of silver and gold nanoparticles from the leaf and callus extracts of Amaranthus dubius, Gunnera perpensa, Ceratotheca triloba and Catharanthus roseus
    (2013-09-17) Patel, Naazlene; Odhav, Bharti
    The biosynthesis of NPs has many advantages over the tedious, expensive and toxic physical and chemical methods of synthesis. Plants are stocked with valuable metabolites that are capable of reducing metal salts to form NPs. In this study, aqueous leaf extracts of A. dubius, G. perpensa, C. roseus and C. triloba were reacted with AgNO3 and HAuCl4 to determine the plants reducing abilities and hence synthesis of Ag and Au NPs capabilities. The synthesis reactions were carried out at different temperatures and extract concentrations for optimization. The goal was to form NPs within the specific wavelength range. Polar solvents: methanol and ethyl acetate extractions were carried out at the optimized conditions to evaluate the best solvent for the extraction of phytochemicals from the plants. The plant leaf extracts that were successful (A. dubius, G. perpensa and C. triloba) in synthesizing NPs were then micropropagated to form callus cultures. The reducing abilities of these callus cultures extracts were determined by varying temperature and concentration parameters. Characterization of the NPs formed by the different extracts was performed using UV-vis, TEM and FTIR. UV-vis spectrophotometry was used as a confirmatory as well as characterizing tool. TEM analysis was able to provide a description on the size and shape of the NPs whereas FTIR provided information on the biomolecules responsible for synthesis and capping of NPs. The stability of the NPs was determined by UV-vis scans over a period of 30 days which allowed observation of the alteration in peak shape and absorbance and hence condition of particles. Phytochemical tests were performed on the leaf extracts of the four plants to elucidate possible phytochemicals responsible for the reduction of metal salts. Antibacterial activity of the NPs was evaluated by using the disk diffusion assay and MICs were determined by the broth dilution method against pathogenic bacteria. A. dubius, G. perpensa and C. triloba were capable of synthesizing Ag NPs and Au NPs which were indicated by yellowish orange and reddish purple colour changes respectively. G. perpensa was able to spontaneously form Ag and Au NPs without any addition of heat whereas A. dubius and C. triloba required heat to form Au NPs. As the temperature of the reactions increased, the absorbance and possibly the number of NPs produced, increased. When the concentration of the extract was doubled, the absorbance was seen to decrease. C. roseus did not produce any Ag or Au NPs with any of the leaf extracts. Only A. dubius and C. triloba callus extracts were investigated for NP synthesis and it was found that A. dubius callus extracts were unsuccessful in synthesizing NPs and C. triloba callus extracts were able to form unstable Ag and Au NPs. The spherical Ag NPs that were formed from aqueous extracts of A. dubius were slightly larger than the methanolic Ag NPs. The Ag NPs produced by G. perpensa were in the same size range for aqueous and methanolic extracts. C. triloba Ag NPs formed from the methanolic extract were closer in size to A. dubius aqueous Ag NPs but the C. triloba aqueous extract produced much larger Ag NPs than the other extracts. The Ag NPs produced from A. dubius aqueous and methanolic extracts as well as C. triloba methanolic extracts exhibited the longest stability of 30 days. Ag NPs from G. perpensa aqueous extracts had the least stability. G. perpensa did not form any hexagonal Au NPs and the spherical and triangular Au NPs were smaller unlike in A. dubius and C. triloba Au NPs. The Au NPs formed by the aqueous extracts of A. dubius and C. triloba were larger in comparison to their methanolic counterparts. The Au NPs produced from G. perpensa aqueous and methanolic extracts as well as A. dubius and C. triloba methanolic extracts exhibited the longest stability of 30 days. Au NPs were stable for longer in comparison to Ag NPs. FTIR provided evidence that Ag and Au NPs have a chemical bond with the amide group in amino acids. However the intensities of biomolecules for Au NPs are more pronounced compared to the Ag NPs. It was also found that the Ag NPs synthesized by methanolic leaf extracts have slightly higher intensities than Ag NPs synthesized from aqueous leaf extracts. Phytochemical screening showed the absence of tannins in the C. roseus leaf, A. dubius and C. triloba callus extracts and presence in the other three plants. C. triloba methanolic extract Ag NPs showed the highest activity against Gram-positive S. aureus. Aqueous and methanolic Ag NPs from G. perpensa and C. triloba as well as A. dubius methanolic Ag NPs had activity against all fourteen bacteria. A. dubius aqueous Ag NPs had no activity against Enterobacter spp. and a strain of Klebsiella pneumoniae. G. perpensa Ag NPs had better antibacterial activity and lower MICs against Gram-positive and Gram-negative pathogenic bacteria compared to A. dubius and C. triloba. There was no antibacterial activity seen with Au NPs. The size and shape of NPs are the keys to their biomedical properties. Green synthesis of NPs is a feasible way for the future. This study showed that NPs can be synthesized very easily and economically. A key finding of this study is that different plants produce varying sizes and aggregation of NPs.
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    Screening, in-vitro propagation and bioaugmentation of Ceratotheca triloba for the production of secondary metabolites
    (2010) Mohanlall, Viresh; Odhav, Bharti; Okole, Blessed
    Ceratatheca triloba (Bernh.) E. Mey. Ex Hook. f. is one of four species that is common to the summer rainfall areas in South Africa, especially the grasslands. It is used in traditional medicine to treat stomach cramps, nausea, fever and diarrhea. Like many other plants used in the traditional medicine system, these uses are not justified through scientific investigations. This study was undertaken to characterize the functionality of the main bioactive compounds from Ceratatheca triloba. This was achieved by isolating and identifying predominant chemicals from the non polar extracts using conventional chromatography techniques. Once identified the crude extracts and identified compounds were tested for their antimicrobial, anti-oxidant activity, anti-inflammatory activity and anticancer activity. This was followed by investigating the safety of the crude extracts and the purified compounds by the Brine shrimp lethality assay, and its toxicity to HepG2 cells and the Salmonella mutagenecity test. For large scale production, we set up a protocol to produce 9, 10 anthracenedione in a cell suspension culture system. Following the complete chemical profile of the roots, stems, flowers and leaves the predominant compounds were isolated, characterized and identified by UV-Vis, IR, EI-LCMS and NMR (COSY, HMQC, HMBC and DEPT). Three anthraquinone derivatives and one steroid, 9, 10 anthracenedione, 1-hydroxy-4-methylanthraquinone, 5, 8-dimethoxy-2, 3, 10, 10a-tetrahydro-1H-phenanthrene-4, 9-dione and androst-5-ene-3, 17, 19-triol were determine by analysis of spectral data (UV, 1H NMR, 13C NMR and EI-LC-MS) 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone showed antibacterial activity against S.aureus, M. luteus, B cureus and E. coli. Due to the synergistic effect of the individual compounds, the crude extract exhibited good potency (>500) against S.aureus and M. luteus, medium potency against E. coli. and S. typhimurium (<100) and very low potency against B cureus (<10). Although a similar trend was observed for 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone unlike the crude extract. A very low potency against S.aureus for 9, 10 anthracenedione and a high potency for 1 hydroxy-4-methylanthraquinone. Thus 9, 10 anthracenedione is an effective drug against E. coli and S. typhimurium and 1 hydroxy-4-methylanthraquinone is effective against S.aureus and M. luteus. The crude root extracts and 9, 10 anthracenedione, 1 hydroxy-4-methylanthraquinone, 8-dimethoxy-2, 3, 10, 10a-tetrahydro-1H-phenanthrene-4 showed a ± 50% reduction of the free radicals. No anti-inflammatory activity was observed. The purified extracts showed moderate toxicity against HepG2 cells at high concentrations and no toxicity was observed against brine shimp larvae. No mutagenecity was observed with the crude extracts using the Ames test. All purified and crude extracts showed potent inhibition of the human topoisomerase II enzyme. In conclusion, although this study does not indicate any relationship to its traditional usage it provides valuable information that paves a way for commercial exploitation of C. triloba. 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone can be used as antibacterial agents. Their antioxidative potential can be exploited for anti-cancer as in many cancers reactive oxygen species are implicated in the aetiology of these cancers. Furthermore, in this study 9, 10 anthracenedione was produced from both callus cultures and cell suspension cultures. This compound demonstrates potent anti-topoisomerase II activity which is vital to cancer treatment. Thus, the synergistic effect of 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone as antibacterial, anti-oxidative and anti-cancer compounds demonstrate the importance of C. triloba.