- Title
- Extraction and characterization of antimicrobial compounds from selected marine invertebrates collected from Phillip's Reef, Port Elizabeth in the Eastern Cape province of South Africa
- Creator
- Kibungu, Cuthbert Wasswa
- Subject
- Invertebrates Bioactive compounds Anti-infective agents
- Date
- 2018
- Type
- Thesis
- Type
- Masters
- Type
- Microbiology
- Identifier
- http://hdl.handle.net/10353/11751
- Identifier
- vital:39103
- Description
- Introduction: The marine environment is rich in the biodiversity of medically significant invertebrate species such as sponges, ascidians, molluscs and bryozoans. Such a diverse environment is a potential source of a great variety of secondary metabolite production. Research in marine natural product discovery is gaining ground with the development of new therapeutic drugs of antimicrobial potential. Aim: This study seeks to evaluate, and identify bioactive compounds obtained from marine invertebrates collected from Phillip’s Reef in Algoa Bay. Methodology: Marine invertebrates were collected from the Phillip’s Reef in Algoa Bay, Port Elizabeth, at a depth of 12 m and the physichochemical parameters at the site of sample collection were recorded onsite. Sampling was done once over the four seasons in South Africa. Sections of each specimen were sent for identification, and extraction of compounds was done using organic solvents [methanol (95percent), hexane (95percent), petroleum ether (95percent), chloroform (95percent) and ethyl acetate (95percent)]. A ratio of 95percent the respective solvents was achieved by the following (1:19), organic solvent combinations [dichloromethane: methanol (1:1), hexane: chloroform: methanol (1:2:1)] and aqueous solution of water. Solvent optimization was done by measuring the absorbance of S. aureus (ATCC 49476) in Mueller Hinton Broth containing crude extracts at 600 nm. The appropriate solvent was then selected for further extraction of bioactive compounds from the specimens. Antimicrobial activity of the crude extracts was evaluated using the standard agar well diffusion method on test pathogens, namely, methicillin-resistant Staphylococcus aureus (ATCC 49476), clostridium difficile (ATCC 9689), Pseudomonas aeruginosa (ATCC 10145), Candida albicans (ATCC 10231) and Aspergillus fumigatus (ATCC 204305). Different concentrations of the crude extracts were used [100percent, 80percent, 60percent, 40percent and 30percent (v:v)] and the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined. Selected bioactive crude extracts were further evaluated for phytochemicals before being purified by subjecting them to Sephadex-G75 column chromatography, using 75percent ethyl acetate as the mobile phase. Bioactive crude extracts fractions obtained following column chromatography were subjected to Electron Spray Ionization Time of Flight (ESI-TOF) analysis using The Bruker Daltonics maXis compact ESI Quadrupole TOF instrument to detect and identify compounds present. Peak identification was done using the blast software contained in Open Chrom Community Edition (Alder), open source software for chromatography and mass spectrometry spectrum analysis. Results: Sixty-seven different marine invertebrate species were collected over the 4 seasons. A mean temperature of 17.35oC ± 2.06 was recorded over all four seasons with the Autumn exhibiting the highest temperature (20oC). Salinity readings obtained ranged between 35.12 psu and 35.27 psu. A stable mean pH range of 8.08–8.78 was also recorded across all four seasons. Salinity and pH readings conformed to the standards set by the South African Water Quality Guidelines. The Spring season exhibited the highest turbidity of 2.97 NTU compared to other seasons. Ethyl acetate and dichloromethane: methanol (1:1) extracts yielded the best inhibitory results. Approximately 60percent (4/67) of the marine invertebrate specimens showed bioactivity against some of the test pathogens. Both ethyl acetate and dichloromethane methanol crude extracts of S14 and S34 showed a broad spectrum of antimicrobial activity against both bacteria and fungi. The C6 ethyl acetate crude extracts were highly active against MRSA, as inhibition zones of 45 mm were observed. MIC at 30percent concentrations was observed for very few crude extracts against the five pathogens. Sixty-three ethyl acetate crude extracts contained phytochemicals while only 35 dichloromethane: methanol crude extracts contained phytochemicals. Neither ethyl acetate and dichloromethane: methanol crude extracts of S34 (Psammopsinia sp. 1) that were biologically active had any phytochemicals. Canadine, Ginkolide A, Apigenin, Amygdalin, 10-hydroxy-Camptothecin, Catechin, Beta-Peltatin and 6 Ethoxy-3(4'-hydroxyphenyl)-4-methylcoumarin were identified in the crude extracts. Conclusion: Marine invertebrate species located at Phillip’s Reef produce compounds with antimicrobial potential. These compounds are biologically active against human pathogenic microorganisms. The best solvent to use for the extraction of these biologically significant metabolites is ethyl acetate, as this study achieved outstanding results from ethyl acetate crude extracts in the form of high zones of inhibition, for example the sponge Ectyonopsis flabellate, which exhibited mean inhibition zones of 45 mm against Aspergillus fumigatus. Similar bioactivity was also observed with Tedania (Tedania) Stylonychaeta against Methicillin Resistant Staphylococcus aureus. This is the first study done on some of the marine sponges and ascidians in the Phillip’s Reef, and therefore further evaluation of the purified compounds is necessary in order to obtain the identity of the pure compounds and possibly determine the mechanism of action, and synthesize structural analogues for further studies.
- Format
- 95 leaves
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science and Agriculture
- Language
- English
- Rights
- University of Fort Hare
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UFH Department of Biochemistry & Microbiology
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| View Details | SOURCE1 | Kibungu, C (Microbiology) - Oct Grad 2018.pdf | 3 MB | Adobe Acrobat PDF | View Details |