Pharmaco-chemical investigation of Erythrina caffra: extracts, isolated compounds and their biological activities
- Authors: Nogqala, Simnikiwe
- Date: 2023-03-29
- Subjects: Coast coral tree , Traditional medicine South Africa , Antibacterial agents , Antineoplastic agents , Organic compounds
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422459 , vital:71944
- Description: In this study, secondary metabolites isolated from Erythrina caffra, a medicinal plant indigenous to South Africa, were investigated. E. caffra is well-known for its healing properties and it is traditionally used for treating bacterial infections like tuberculosis (TB), abscesses, tooth aches and ear infections. Its extracts have also been used to treat cancer. Though many studies have been done on this plant, most of them tended to focus solely on the isolated compounds. In the present study however, extracts, fractions and isolated compounds from E. caffra were evaluated for their anticancer, anti-oxidant, anti-enzymatic, antibacterial and cytotoxicity. The methanol crude extract (B1) from the stem bark of E. caffra was used to extract alkaloidic fractions (B2 and B3) using ethyl acetate and n-butanol respectively, a third fraction (B4) was also extracted using ethyl acetate this fraction was called a neutral fraction. The neutral fraction (B4) was fractionated and through a sequence of column chromatography three active secondary metabolites were isolated. The isolated compounds included Lupeol (1), stigmasterol (2) and 5,7-Dihydroxy-4'-methoxy-3',5'-diprenylflavanone (3). These isolated compounds were characterized and identified using spectroscopic techniques including IR, NMR and high-resolution Mass Spectrometry. Using the cell line HCC-70, isolated from a primary ductal carcinoma, in vitro anticancer assays were carried out on the crude extract from the bark, fractions, isolated compounds and an unseparated mixture of two compounds. These samples were also evaluated for their anti-oxidant, anti-enzymatic, antibacterial and cytotoxicity activities. The crude extract inhibited the cell viability by over 30% and had no effect on the HeLa cells at concentrations of 20μM. Abyssinone V’ 4-methyl-ether (3) and the mixture of stigmasterol (2) and an unidentified compound exhibited potent anticancer activity against the HCC-70 cell line with IC50 of 18.05μM and 9.04μM respectively. Antibacterial assays were also carried out on the crude extracts, fractions and concoctions made from the fractions with the best activity combined with the ones that performed poorly. The concoctions were prepared as two separate series (S and N series). The crude extract inhibited more than 80% of the Staphylococcus aureus cells at a concentration of 20μM with only minimal damage to the HeLa cells. In the concoctions however, the N series managed to inhibit over 96% of the S. aureus while exhibiting no cytotoxicity towards HeLa cells. The extract and its fractions also showed good anti-oxidant activities. Molecular docking of these compounds was done on the Human estrogen receptor (PDB ID:3ERT) and Abyssinone V’ 4-methyl-ether (3) showed the best docking score of -6.6 Kcal/mol, for the simulation against Epidermal growth factor receptor (PDB ID: 1M17) Stigmasterol (2) showed the best docking score of -3.8 Kcal/mol. In silico docking on 3ERT and 1M17 were done to test the binding affinity of the isolated compounds to the proteins which are well known to be overexpressed in some types of cancer. Flavonoids isolated from Erythrina species have been reported to possess good antiplasmodial activity. However, due to the minute amounts isolated in the present study in-vitro assays could not be carried out. Nevertheless, in-silico assays were conducted on the most prominent protozoal parasite which causes malaria in the majority of African countries. In-silico simulations were done against Plasmodium falciparum protein (PDB ID: 7KJH), of the tested compounds Abyssinone V’ 4-methyl-ether (3) was found possess the best docking score of -4.4 Kcal/mol. The molecular docking of 7KJH was done to assess the inhibitory potential of the isolated compounds on protozoal parasites. Pharmacokinetic properties of the isolated compounds were also assessed in silico to assist in evaluating the drug likeness of these compounds. The compounds showed a percent human oral absorption of 100% except for Abyssinone V’ 4-methyl-ether (3), which showed 93.83%, this indicates a remarkable oral bioavailability. Stigamsterol (2) exhibited a Caco-2 cell permeability (QPPCaco) greater than 500 which indicates outstanding results for good intestinal absorption. The compounds also displayed a blood-brain partition co-efficient (QPlogBB) ranging from -1.433 to 0.128 suggesting they will have less potential to cross the blood-brain barrier, thus reducing any CNS related toxicity. Molecular networking of the crude extracts and the fractions was done through GNPS which allowed the identification of known compounds including one isolated in the present study, Abyssinone V’ 4-methyl-ether (3). Possible derivatives that have not been isolated from this plant before were also putatively identified. , Thesis (MSc) -- Faculty of Science, Chemistry, 2023
- Full Text:
- Date Issued: 2023-03-29
- Authors: Nogqala, Simnikiwe
- Date: 2023-03-29
- Subjects: Coast coral tree , Traditional medicine South Africa , Antibacterial agents , Antineoplastic agents , Organic compounds
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422459 , vital:71944
- Description: In this study, secondary metabolites isolated from Erythrina caffra, a medicinal plant indigenous to South Africa, were investigated. E. caffra is well-known for its healing properties and it is traditionally used for treating bacterial infections like tuberculosis (TB), abscesses, tooth aches and ear infections. Its extracts have also been used to treat cancer. Though many studies have been done on this plant, most of them tended to focus solely on the isolated compounds. In the present study however, extracts, fractions and isolated compounds from E. caffra were evaluated for their anticancer, anti-oxidant, anti-enzymatic, antibacterial and cytotoxicity. The methanol crude extract (B1) from the stem bark of E. caffra was used to extract alkaloidic fractions (B2 and B3) using ethyl acetate and n-butanol respectively, a third fraction (B4) was also extracted using ethyl acetate this fraction was called a neutral fraction. The neutral fraction (B4) was fractionated and through a sequence of column chromatography three active secondary metabolites were isolated. The isolated compounds included Lupeol (1), stigmasterol (2) and 5,7-Dihydroxy-4'-methoxy-3',5'-diprenylflavanone (3). These isolated compounds were characterized and identified using spectroscopic techniques including IR, NMR and high-resolution Mass Spectrometry. Using the cell line HCC-70, isolated from a primary ductal carcinoma, in vitro anticancer assays were carried out on the crude extract from the bark, fractions, isolated compounds and an unseparated mixture of two compounds. These samples were also evaluated for their anti-oxidant, anti-enzymatic, antibacterial and cytotoxicity activities. The crude extract inhibited the cell viability by over 30% and had no effect on the HeLa cells at concentrations of 20μM. Abyssinone V’ 4-methyl-ether (3) and the mixture of stigmasterol (2) and an unidentified compound exhibited potent anticancer activity against the HCC-70 cell line with IC50 of 18.05μM and 9.04μM respectively. Antibacterial assays were also carried out on the crude extracts, fractions and concoctions made from the fractions with the best activity combined with the ones that performed poorly. The concoctions were prepared as two separate series (S and N series). The crude extract inhibited more than 80% of the Staphylococcus aureus cells at a concentration of 20μM with only minimal damage to the HeLa cells. In the concoctions however, the N series managed to inhibit over 96% of the S. aureus while exhibiting no cytotoxicity towards HeLa cells. The extract and its fractions also showed good anti-oxidant activities. Molecular docking of these compounds was done on the Human estrogen receptor (PDB ID:3ERT) and Abyssinone V’ 4-methyl-ether (3) showed the best docking score of -6.6 Kcal/mol, for the simulation against Epidermal growth factor receptor (PDB ID: 1M17) Stigmasterol (2) showed the best docking score of -3.8 Kcal/mol. In silico docking on 3ERT and 1M17 were done to test the binding affinity of the isolated compounds to the proteins which are well known to be overexpressed in some types of cancer. Flavonoids isolated from Erythrina species have been reported to possess good antiplasmodial activity. However, due to the minute amounts isolated in the present study in-vitro assays could not be carried out. Nevertheless, in-silico assays were conducted on the most prominent protozoal parasite which causes malaria in the majority of African countries. In-silico simulations were done against Plasmodium falciparum protein (PDB ID: 7KJH), of the tested compounds Abyssinone V’ 4-methyl-ether (3) was found possess the best docking score of -4.4 Kcal/mol. The molecular docking of 7KJH was done to assess the inhibitory potential of the isolated compounds on protozoal parasites. Pharmacokinetic properties of the isolated compounds were also assessed in silico to assist in evaluating the drug likeness of these compounds. The compounds showed a percent human oral absorption of 100% except for Abyssinone V’ 4-methyl-ether (3), which showed 93.83%, this indicates a remarkable oral bioavailability. Stigamsterol (2) exhibited a Caco-2 cell permeability (QPPCaco) greater than 500 which indicates outstanding results for good intestinal absorption. The compounds also displayed a blood-brain partition co-efficient (QPlogBB) ranging from -1.433 to 0.128 suggesting they will have less potential to cross the blood-brain barrier, thus reducing any CNS related toxicity. Molecular networking of the crude extracts and the fractions was done through GNPS which allowed the identification of known compounds including one isolated in the present study, Abyssinone V’ 4-methyl-ether (3). Possible derivatives that have not been isolated from this plant before were also putatively identified. , Thesis (MSc) -- Faculty of Science, Chemistry, 2023
- Full Text:
- Date Issued: 2023-03-29
The de novo biosynthesis of biotin is required for the optimal growth of Salmonella enterica serovar Typhimurium in the intracellular environment
- Authors: McLaughlin, Claire
- Date: 2021-10-29
- Subjects: Salmonella , Biotin , Biosynthesis , Salmonella typhimurium , Antibacterial agents , Anti-infective agents , Pathogenic bacteria , Salmonella food poisoning
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192097 , vital:45195
- Description: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a foodborne pathogen infecting humans and animals, contributing to significant morbidity and mortality worldwide each year. The increase in antibiotic-resistant S. Typhimurium infections in recent years has highlighted the need for new antibacterial drugs and drug targets. S. Typhimurium can acquire biotin through de novo biosynthesis or via transport from its extracellular environment. The importance of the vitamin for bacterial survival, coupled with the absence of the biotin biosynthetic pathway in humans, makes the biotin biosynthetic enzymes attractive targets for drug discovery. The study's primary aim was to determine the relative importance of the biotin biosynthesis and transport pathways for the in vitro and ex vivo growth and survival of S. Typhimurium, with the goal of validating the pathways as valid targets for antimicrobial drug development. In order to achieve this aim, we generated S. Typhimurium mutant strains harbouring deletions in either the biotin biosynthetic gene, bioB, or putative high-affinity biotin transporter, yigM (ΔbioB and ΔyigM, respectively), as well as a double mutant in which the two mutations were combined (ΔbioB ΔyigM). Since the simultaneous disruption of biotin biosynthesis and transport in the double mutant may form a synthetic lethal combination, preventing further analysis of the strain, we also constructed a conditional mutant in which the promoter of the yigM gene was replaced by the arabinose-regulatable, PBAD promoter in the ΔbioB background (ΔbioB PBAD::yigM). Since the expression of the YigM in this strain is arabinose-regulatable, its role as a biotin transporter can be evaluated by altering the arabinose concentration in the growth media. Once the mutant strains were isolated and verified genetically, their growth and that of their genetically complemented counterparts were analysed in liquid and/or solid M9 minimal medium in the absence of biotin. Consistent with previous observations, the ΔbioB auxotrophic mutant's growth was severely compromised in minimal media in the absence of biotin. The growth of the strain could, however, be restored by supplementation with exogenous biotin or expression of the wild type bioB gene from an episomal plasmid. The ability of biotin to reverse the growth defect of the ΔbioB mutant strain was, however, dependent on the presence of a functional YigM, since biotin supplementation did not affect the growth of the ΔbioB ΔyigM double mutant strain. The introduction of a second copy of the yigM gene in the ΔbioB ΔyigM background, however, restored the growth of the strain in the presence, but not absence, of biotin. The dependence of the double mutant on both YigM and biotin for growth supports the idea that the protein functions as the sole or primary biotin transporter in S. Typhimurium, as it has recently been shown for E. coli (Ringsletter, 2010; Finkenwirth et al, 2013). The essentiality of YigM for biotin transport was subsequently verified by two independent means. Firstly, the growth of the ΔbioB PBAD::yigM promoter-replacement mutant was strictly dependent on the inclusion of arabinose in biotin-supplemented M9 minimal media supplemented, indicating that the expression of YigM from the PBAD promoter is essential for biotin transport. Secondly, following treatment with a known small-molecule inhibitor of the biotin biosynthesis, MAC-13772, exogenous biotin was capable of restoring the growth defect of the YigM+ wild type S. Typhimurium strain, but not the YigM− ΔyigM mutant. Taken together, these findings confirm that YigM serves as the biotin transporter for S. Typhimurium and that the corresponding ΔyigM mutant is, as a result, defective for biotin transport. Having confirmed the genotypes and phenotypes of the ΔbioB, ΔyigM, and ΔbioB ΔyigM mutants, we next analysed the importance of the biotin biosynthesis and transport pathways for the growth and survival of S. Typhimurium within the intracellular environment. To this end, we determined the proliferation of each of the mutant strains following infection of HeLa epithelial and RAW264.7 macrophage-like cell lines. Our results revealed that the de novo biosynthesis of biotin is required for the optimal growth of S. Typhimurium following infection of both epithelial and macrophage-like cell lines. Disruption of biotin transport, by contrast, had no significant effect on the intracellular proliferation of S. Typhimurium when a functional pathway for the biosynthesis of biotin was present. The simultaneous disruption of biotin biosynthesis and transport, however, resulted in significant attenuation of S. Typhimurium in epithelial cells, while bacterial survival in macrophages decreased to below the limit of detection. Overall, our results suggest the S. Typhimurium relies primarily on biotin produced by the de novo biosynthesis pathway to support its growth in the intracellular environment. While YigM-mediated biotin transport is essential for sustaining the viability of intracellular S. Typhimurium in the absence of de novo biosynthesis, it appears to play a relatively minor role in the acquisition of biotin during growth in the nutrient-limited Salmonella containing vacuole. Our findings suggest that inhibiting biotin biosynthesis may be a viable strategy for combating systemic infections caused by Salmonella, as has been recently proposed for other medically important bacterial pathogens (Carfrae et al., 2020). , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: McLaughlin, Claire
- Date: 2021-10-29
- Subjects: Salmonella , Biotin , Biosynthesis , Salmonella typhimurium , Antibacterial agents , Anti-infective agents , Pathogenic bacteria , Salmonella food poisoning
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192097 , vital:45195
- Description: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a foodborne pathogen infecting humans and animals, contributing to significant morbidity and mortality worldwide each year. The increase in antibiotic-resistant S. Typhimurium infections in recent years has highlighted the need for new antibacterial drugs and drug targets. S. Typhimurium can acquire biotin through de novo biosynthesis or via transport from its extracellular environment. The importance of the vitamin for bacterial survival, coupled with the absence of the biotin biosynthetic pathway in humans, makes the biotin biosynthetic enzymes attractive targets for drug discovery. The study's primary aim was to determine the relative importance of the biotin biosynthesis and transport pathways for the in vitro and ex vivo growth and survival of S. Typhimurium, with the goal of validating the pathways as valid targets for antimicrobial drug development. In order to achieve this aim, we generated S. Typhimurium mutant strains harbouring deletions in either the biotin biosynthetic gene, bioB, or putative high-affinity biotin transporter, yigM (ΔbioB and ΔyigM, respectively), as well as a double mutant in which the two mutations were combined (ΔbioB ΔyigM). Since the simultaneous disruption of biotin biosynthesis and transport in the double mutant may form a synthetic lethal combination, preventing further analysis of the strain, we also constructed a conditional mutant in which the promoter of the yigM gene was replaced by the arabinose-regulatable, PBAD promoter in the ΔbioB background (ΔbioB PBAD::yigM). Since the expression of the YigM in this strain is arabinose-regulatable, its role as a biotin transporter can be evaluated by altering the arabinose concentration in the growth media. Once the mutant strains were isolated and verified genetically, their growth and that of their genetically complemented counterparts were analysed in liquid and/or solid M9 minimal medium in the absence of biotin. Consistent with previous observations, the ΔbioB auxotrophic mutant's growth was severely compromised in minimal media in the absence of biotin. The growth of the strain could, however, be restored by supplementation with exogenous biotin or expression of the wild type bioB gene from an episomal plasmid. The ability of biotin to reverse the growth defect of the ΔbioB mutant strain was, however, dependent on the presence of a functional YigM, since biotin supplementation did not affect the growth of the ΔbioB ΔyigM double mutant strain. The introduction of a second copy of the yigM gene in the ΔbioB ΔyigM background, however, restored the growth of the strain in the presence, but not absence, of biotin. The dependence of the double mutant on both YigM and biotin for growth supports the idea that the protein functions as the sole or primary biotin transporter in S. Typhimurium, as it has recently been shown for E. coli (Ringsletter, 2010; Finkenwirth et al, 2013). The essentiality of YigM for biotin transport was subsequently verified by two independent means. Firstly, the growth of the ΔbioB PBAD::yigM promoter-replacement mutant was strictly dependent on the inclusion of arabinose in biotin-supplemented M9 minimal media supplemented, indicating that the expression of YigM from the PBAD promoter is essential for biotin transport. Secondly, following treatment with a known small-molecule inhibitor of the biotin biosynthesis, MAC-13772, exogenous biotin was capable of restoring the growth defect of the YigM+ wild type S. Typhimurium strain, but not the YigM− ΔyigM mutant. Taken together, these findings confirm that YigM serves as the biotin transporter for S. Typhimurium and that the corresponding ΔyigM mutant is, as a result, defective for biotin transport. Having confirmed the genotypes and phenotypes of the ΔbioB, ΔyigM, and ΔbioB ΔyigM mutants, we next analysed the importance of the biotin biosynthesis and transport pathways for the growth and survival of S. Typhimurium within the intracellular environment. To this end, we determined the proliferation of each of the mutant strains following infection of HeLa epithelial and RAW264.7 macrophage-like cell lines. Our results revealed that the de novo biosynthesis of biotin is required for the optimal growth of S. Typhimurium following infection of both epithelial and macrophage-like cell lines. Disruption of biotin transport, by contrast, had no significant effect on the intracellular proliferation of S. Typhimurium when a functional pathway for the biosynthesis of biotin was present. The simultaneous disruption of biotin biosynthesis and transport, however, resulted in significant attenuation of S. Typhimurium in epithelial cells, while bacterial survival in macrophages decreased to below the limit of detection. Overall, our results suggest the S. Typhimurium relies primarily on biotin produced by the de novo biosynthesis pathway to support its growth in the intracellular environment. While YigM-mediated biotin transport is essential for sustaining the viability of intracellular S. Typhimurium in the absence of de novo biosynthesis, it appears to play a relatively minor role in the acquisition of biotin during growth in the nutrient-limited Salmonella containing vacuole. Our findings suggest that inhibiting biotin biosynthesis may be a viable strategy for combating systemic infections caused by Salmonella, as has been recently proposed for other medically important bacterial pathogens (Carfrae et al., 2020). , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
Analysis of bacterial Mur amide ligase enzymes for the identification of inhibitory compounds by in silico methods
- Chamboko, Chiratidzo Respina
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
Extraction, isolation and characterization of oleanolic acid and its analogues from Syzygium aromaticum (cloves) and evaluation of their biological activities
- Authors: khwaza, Vuyolwethu
- Date: 2019
- Subjects: Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/19076 , vital:39882
- Description: Pathogenic microorganisms have serious impact on people's lives. Every year, millions of people around the world die of bacterial infections. Resistance to common antibacterial drugs has proven to be a challenging problem in control of bacterial infections. In an attempt to develop an effective and affordable treatment for bacterial infections, oleanolic acid isolated from syzygium aromaticum conjugates incorporating other pharmaceutical scaffolds such as chloroquine derivatives, curcumin, and ergocalciferol etc have been developed. Based on the previous successes of testing combination of antimicrobial drugs and pharmaceutical drugs which appeared to be the promising strategy to overcome treatment failure; a series of hybrid compounds containing oleanolic acid and other pharmaceutical scaffolds were synthesized. 4- Aminoquinoline derivatives were first hybridized with selected organic compounds to form a class of hybrid compounds containing either amide bond or ester bond as a linker between the precursor molecules. Analogues/hybrid compounds can overcome the disadvantages of combination therapy such as drug-drug interaction. The structural effects of this type of conjugation of oleanolic acid and other pharmaceutical scaffolds were characterised by FTIR, Mass Spec and NMR spectroscopy. These compounds were studied along with the monosubstituted oleanolic acid analogues and the organic components in order to compare the effects of the substitution on their biological response.‖ All the synthesized analogues were tested against 11 bacterial strains on both Gram-positive and Gram-negative bacteria. The synthesized compounds showed selectivity and higher activity against Enterococcus faecalis (EF), Klebsiella oxytoca (KO), Escherischia coli (EC), Staphylococcus aureous (SA), Proteus vulgaris (PV) and Bacillus subtilis (BS) with MIC values; ranging between of 1.25 mg/mL to 0.072 mg/mL
- Full Text:
- Date Issued: 2019
- Authors: khwaza, Vuyolwethu
- Date: 2019
- Subjects: Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/19076 , vital:39882
- Description: Pathogenic microorganisms have serious impact on people's lives. Every year, millions of people around the world die of bacterial infections. Resistance to common antibacterial drugs has proven to be a challenging problem in control of bacterial infections. In an attempt to develop an effective and affordable treatment for bacterial infections, oleanolic acid isolated from syzygium aromaticum conjugates incorporating other pharmaceutical scaffolds such as chloroquine derivatives, curcumin, and ergocalciferol etc have been developed. Based on the previous successes of testing combination of antimicrobial drugs and pharmaceutical drugs which appeared to be the promising strategy to overcome treatment failure; a series of hybrid compounds containing oleanolic acid and other pharmaceutical scaffolds were synthesized. 4- Aminoquinoline derivatives were first hybridized with selected organic compounds to form a class of hybrid compounds containing either amide bond or ester bond as a linker between the precursor molecules. Analogues/hybrid compounds can overcome the disadvantages of combination therapy such as drug-drug interaction. The structural effects of this type of conjugation of oleanolic acid and other pharmaceutical scaffolds were characterised by FTIR, Mass Spec and NMR spectroscopy. These compounds were studied along with the monosubstituted oleanolic acid analogues and the organic components in order to compare the effects of the substitution on their biological response.‖ All the synthesized analogues were tested against 11 bacterial strains on both Gram-positive and Gram-negative bacteria. The synthesized compounds showed selectivity and higher activity against Enterococcus faecalis (EF), Klebsiella oxytoca (KO), Escherischia coli (EC), Staphylococcus aureous (SA), Proteus vulgaris (PV) and Bacillus subtilis (BS) with MIC values; ranging between of 1.25 mg/mL to 0.072 mg/mL
- Full Text:
- Date Issued: 2019
Antibacterial activities of both combined and individual medicinal plants extracts traditionally used to treat pneumonia
- Authors: Mhango, Immaculate
- Date: 2017
- Subjects: Antibacterial agents , Pneumonia -- Treatment
- Language: English
- Type: Thesis , Masters , MA
- Identifier: http://hdl.handle.net/10948/19762 , vital:28959
- Description: Pneumonia is one of the five major leading causes of death in children under-fives years and the elderly worldwide. Antibiotics used for its treatment are less potent due to bacteria development of bacteria resistant to antibiotics. This has led to a surge in search of novel drugs. There are already some drugs in clinical use that have natural products and derivatives such as quinine, morphine, vincristine, and taxol among others. The healing value of medicinal plants has been well accepted since Stone Age across the globe. This plant therapy has been prescribed and prepared independently or in combination. The following plants: Terminalia sericea, Warburgia salutaris, Dodonea angustofolia, Eucalyptus camaldulensis, Ballota africana, Kigelia africana and Acorus gramineus. These plants are most commonly used for treatment of pneumonia and other ailment, were studied to validate their antimicrobial activity based on scientific determination. The primary aim of this study was to evaluate the efficacy of these plants against bacteria pneumonia pathogens. Seven medicinal plants, independently and in combinations were relatively analysed for their antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, and Klebsiella pneumoniae. Ground plant material of roots, bark and leafs were prepared with acetone, ethanol and distilled water. Dimethyl sulfoxide (10 &100%) was used as a reconstitution solvent and ciprofloxacin (10 %) as a positive control. The antimicrobial efficacy was determined using agar well diffusion and microtiter plate methods. Interaction between plants was evaluated by calculating fraction inhibitory concentration index (ΣFIC). Noteworthy activity for individual studies with all test organisms was observed with T. sericea. However, highest ZOI (30 mm) was observed for B. africana ethanol extract for S. pyogenes. Weak microbial activity was noted in W. salutaris and D. angustofolia extracts with all test organisms. Good antimicrobial activity was observed in combination studies with all organisms. The potency of different plant combinations varied with highest ZOI observed with B. africana and W. salutaris ranging from 33-35 mm, conversely ZOI of 35 mm was also noted for S. aureus in B. africana and E. camaldulensis ethanol extract. Noteworthy antimicrobial activity was observed in T. sericea and D. angustofolia against all test pathogens. weak antimicrobial activity with highest MICs was observed in combinations where W. salutaris was involved. After calculating ΣFICs, strongest synergistic effect was displayed for W. salutaris and D. angustofolia against all test organisms (lowest ΣFICs 0.0491). Most plant extract combinations, displayed either synergistic, additive or indifferent effect, with few demonstrating antagonistic interactions. Significant antagonism effect was noted for S. pyogenes with T. sericea ethanol extract ΣFIC value of 15.51. Based on results of this study use of plants in combination increase antimicrobial efficacy. The antimicrobial activities; synergistic and additive effects observed adds credibility in the use of plant combination for therapeutic value in treatment of pneumonia. Future studies are recommended to identify and isolate specific active compounds involved in plant combination interactions. The importance of combination studies for possible development of new antimicrobials that can succumb bacterial resistance need to be highlighted.
- Full Text:
- Date Issued: 2017
- Authors: Mhango, Immaculate
- Date: 2017
- Subjects: Antibacterial agents , Pneumonia -- Treatment
- Language: English
- Type: Thesis , Masters , MA
- Identifier: http://hdl.handle.net/10948/19762 , vital:28959
- Description: Pneumonia is one of the five major leading causes of death in children under-fives years and the elderly worldwide. Antibiotics used for its treatment are less potent due to bacteria development of bacteria resistant to antibiotics. This has led to a surge in search of novel drugs. There are already some drugs in clinical use that have natural products and derivatives such as quinine, morphine, vincristine, and taxol among others. The healing value of medicinal plants has been well accepted since Stone Age across the globe. This plant therapy has been prescribed and prepared independently or in combination. The following plants: Terminalia sericea, Warburgia salutaris, Dodonea angustofolia, Eucalyptus camaldulensis, Ballota africana, Kigelia africana and Acorus gramineus. These plants are most commonly used for treatment of pneumonia and other ailment, were studied to validate their antimicrobial activity based on scientific determination. The primary aim of this study was to evaluate the efficacy of these plants against bacteria pneumonia pathogens. Seven medicinal plants, independently and in combinations were relatively analysed for their antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, and Klebsiella pneumoniae. Ground plant material of roots, bark and leafs were prepared with acetone, ethanol and distilled water. Dimethyl sulfoxide (10 &100%) was used as a reconstitution solvent and ciprofloxacin (10 %) as a positive control. The antimicrobial efficacy was determined using agar well diffusion and microtiter plate methods. Interaction between plants was evaluated by calculating fraction inhibitory concentration index (ΣFIC). Noteworthy activity for individual studies with all test organisms was observed with T. sericea. However, highest ZOI (30 mm) was observed for B. africana ethanol extract for S. pyogenes. Weak microbial activity was noted in W. salutaris and D. angustofolia extracts with all test organisms. Good antimicrobial activity was observed in combination studies with all organisms. The potency of different plant combinations varied with highest ZOI observed with B. africana and W. salutaris ranging from 33-35 mm, conversely ZOI of 35 mm was also noted for S. aureus in B. africana and E. camaldulensis ethanol extract. Noteworthy antimicrobial activity was observed in T. sericea and D. angustofolia against all test pathogens. weak antimicrobial activity with highest MICs was observed in combinations where W. salutaris was involved. After calculating ΣFICs, strongest synergistic effect was displayed for W. salutaris and D. angustofolia against all test organisms (lowest ΣFICs 0.0491). Most plant extract combinations, displayed either synergistic, additive or indifferent effect, with few demonstrating antagonistic interactions. Significant antagonism effect was noted for S. pyogenes with T. sericea ethanol extract ΣFIC value of 15.51. Based on results of this study use of plants in combination increase antimicrobial efficacy. The antimicrobial activities; synergistic and additive effects observed adds credibility in the use of plant combination for therapeutic value in treatment of pneumonia. Future studies are recommended to identify and isolate specific active compounds involved in plant combination interactions. The importance of combination studies for possible development of new antimicrobials that can succumb bacterial resistance need to be highlighted.
- Full Text:
- Date Issued: 2017
Synthetic analogues of marine bisindole alkaloids as potent selective inhibitors of MRSA pyruvate kinase
- Veale, Clinton Gareth Lancaster
- Authors: Veale, Clinton Gareth Lancaster
- Date: 2014 , 2014-04-02
- Subjects: Alkaloids , Pyruvate kinase , Staphylococcus aureus , Antibiotics , Sponges -- South Africa , Imidazoles , Biological assay , Antibacterial agents
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4563 , http://hdl.handle.net/10962/d1020893
- Description: Globally, methicillin resistant Staphylococcus aureus (MRSA) has become increasingly difficult to manage in the clinic and new antibiotics are required. The structure activity relationship (SAR) study presented in this thesis forms part of an international collaborative effort to identify potent and selective inhibitors of an MRSA pyruvate kinase (PK) enzyme target. In earlier work the known marine natural product bromodeoxytopsentin (1.6), isolated from a South African marine sponge Topsentia pachastrelloides, exhibited selective and significant inhibition of MRSA PK (IC₅₀ 60 nM). Accordingly bromodeoxytopsentin provided the initial chemical scaffold around which our SAR study was developed. Following a comprehensive introduction, providing the necessary background to the research described in subsequent Chapters, this thesis has been divided into three major parts. Part one (Chapter 2) documents the synthesis of two natural imidazole containing topsentin analogues 1.40, 1.46, five new synthetic analogues 1.58—1.61, 2.104. In the process we developed a new method for the synthesis of topsentin derivatives via selenium dioxide mediated oxidation of N-Boc protected 3-acetylindoles to yield glyoxal intermediates which were subsequently cyclized and deprotected to yield the desired products. Interestingly we were able to demonstrate a delicate relationship between the relative equivalents of selenium dioxide and water used during the oxidation step, careful manipulation of which was required to prevent the uncontrolled formation of side products. Synthetic compounds 1.40, 1.46, 1.58—1.61 were found to be potent inhibitors of MRSA PK (IC₅₀ 238, 2.1, 23, 1.4, 6.3 and 3.2 nM respectively) with 1000-10000 fold selectivity for MRSA PK over four human orthologs. In the second part of this thesis (Chapter 3) we report the successful synthesis of a cohort of previously unknown thiazole containing bisindole topsentin analogues 1.62—1.68 via a Hantzsch thiazole synthesis. Bioassay results revealed that these compounds were only moderate inhibitors of MRSA PK (IC₅₀ 5.1—20 μM) which suggested that inhibitory activity was significantly reduced upon substitution of the central imidazole ring of topsentin type analogues with a thiazole type ring. In addition in Chapter 3 we describe unsuccessful attempts to regiospecifically synthesize oxazole and imidazole topsentin analogues through a similar Hantzsch method. As a consequence of our efforts in this regard we investigated three key reactions in depth, namely the synthesis of 2.2, 3.38, 3.40, 3.41 via α-bromination of 3-acetylindole and the synthesis of indolyl-3-carbonylnitriles 2.13, 3.45—3.47 and α-oxo-1H-indole-3-thioacetamides 3.48—3.51. The investigation of the latter led to the isolation and elucidation of two anomalous N,N-dimethyl-1H-indole-3-carboxamides 3.52 and 3.53. Finally the third part of this thesis (Chapter 4) deals with in silico assessment of the binding of both the imidazole and thiazole containing bisindole alkaloids to the MRSA PK protein which initially guided our SAR studies. In this chapter we reveal that there appears to be no correlation between in silico binding predictions and in vitro MRSA PK inhibitory bioassay data. Superficially it seems that binding energy as determined by the docking program used for these studies correlated with the size of the indole substituents and did not reflect IC₅₀ MRSA PK inhibitory data. Although this led us to computationally explore possible alternative binding sites no clear alternative has been identified.
- Full Text:
- Date Issued: 2014
- Authors: Veale, Clinton Gareth Lancaster
- Date: 2014 , 2014-04-02
- Subjects: Alkaloids , Pyruvate kinase , Staphylococcus aureus , Antibiotics , Sponges -- South Africa , Imidazoles , Biological assay , Antibacterial agents
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4563 , http://hdl.handle.net/10962/d1020893
- Description: Globally, methicillin resistant Staphylococcus aureus (MRSA) has become increasingly difficult to manage in the clinic and new antibiotics are required. The structure activity relationship (SAR) study presented in this thesis forms part of an international collaborative effort to identify potent and selective inhibitors of an MRSA pyruvate kinase (PK) enzyme target. In earlier work the known marine natural product bromodeoxytopsentin (1.6), isolated from a South African marine sponge Topsentia pachastrelloides, exhibited selective and significant inhibition of MRSA PK (IC₅₀ 60 nM). Accordingly bromodeoxytopsentin provided the initial chemical scaffold around which our SAR study was developed. Following a comprehensive introduction, providing the necessary background to the research described in subsequent Chapters, this thesis has been divided into three major parts. Part one (Chapter 2) documents the synthesis of two natural imidazole containing topsentin analogues 1.40, 1.46, five new synthetic analogues 1.58—1.61, 2.104. In the process we developed a new method for the synthesis of topsentin derivatives via selenium dioxide mediated oxidation of N-Boc protected 3-acetylindoles to yield glyoxal intermediates which were subsequently cyclized and deprotected to yield the desired products. Interestingly we were able to demonstrate a delicate relationship between the relative equivalents of selenium dioxide and water used during the oxidation step, careful manipulation of which was required to prevent the uncontrolled formation of side products. Synthetic compounds 1.40, 1.46, 1.58—1.61 were found to be potent inhibitors of MRSA PK (IC₅₀ 238, 2.1, 23, 1.4, 6.3 and 3.2 nM respectively) with 1000-10000 fold selectivity for MRSA PK over four human orthologs. In the second part of this thesis (Chapter 3) we report the successful synthesis of a cohort of previously unknown thiazole containing bisindole topsentin analogues 1.62—1.68 via a Hantzsch thiazole synthesis. Bioassay results revealed that these compounds were only moderate inhibitors of MRSA PK (IC₅₀ 5.1—20 μM) which suggested that inhibitory activity was significantly reduced upon substitution of the central imidazole ring of topsentin type analogues with a thiazole type ring. In addition in Chapter 3 we describe unsuccessful attempts to regiospecifically synthesize oxazole and imidazole topsentin analogues through a similar Hantzsch method. As a consequence of our efforts in this regard we investigated three key reactions in depth, namely the synthesis of 2.2, 3.38, 3.40, 3.41 via α-bromination of 3-acetylindole and the synthesis of indolyl-3-carbonylnitriles 2.13, 3.45—3.47 and α-oxo-1H-indole-3-thioacetamides 3.48—3.51. The investigation of the latter led to the isolation and elucidation of two anomalous N,N-dimethyl-1H-indole-3-carboxamides 3.52 and 3.53. Finally the third part of this thesis (Chapter 4) deals with in silico assessment of the binding of both the imidazole and thiazole containing bisindole alkaloids to the MRSA PK protein which initially guided our SAR studies. In this chapter we reveal that there appears to be no correlation between in silico binding predictions and in vitro MRSA PK inhibitory bioassay data. Superficially it seems that binding energy as determined by the docking program used for these studies correlated with the size of the indole substituents and did not reflect IC₅₀ MRSA PK inhibitory data. Although this led us to computationally explore possible alternative binding sites no clear alternative has been identified.
- Full Text:
- Date Issued: 2014
- «
- ‹
- 1
- ›
- »