Evaluating antidiabetic properties of selected African medicinal plants in a cell-based model
- Sirkhotte, Saeedah, Reddy, Shanika
- Authors: Sirkhotte, Saeedah , Reddy, Shanika
- Date: 2023-12
- Subjects: Medicinal plants -- South Africa , Insulin resistance , Gluconeogenesis
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/62600 , vital:72827
- Description: The World Health Organisation lists diabetes as one of the major non-communicable diseases affecting the world, and its prevalence is expected to increase rapidly. Type II diabetes mellitus (T2DM) is characterised by insulin resistance and impaired blood glucose control. T2DM is of growing concern within South Africa, with 10.8% of the population currently diagnosed. The popularity, as well as availability, of traditional plant-based medicine in South Africa, might provide a solution. Antidiabetic potential is commonly tested by in vitro assays, however the methods to test this potential via gluconeogenesis are limited. This project aimed to optimise an in vitro hepatic gluconeogenesis model. In addition, this project aimed to determine the antidiabetic properties of Prunus africana, Hypoxis stellipilis, and Eriocephalus africanus by in vitro analysis. These properties were examined in human hepatoma cells (C3A cell line) and rat pancreatic cells (INS1 cell line) and methods included: cytotoxicity analysis, Amplex® red glucose oxidase assay, antibody staining, gene expression analysis by RT-qPCR, oxidative stress analysis, and calcium signaling for insulin release. Insulin resistance was successfully induced by exposing C3A hepatocarcinoma cells to a combination treatment of 50 μM dexamethasone, 1.25 mM fructose and 0.125 mM palmitic acid for a period of three days. Thereafter, gluconeogenesis was assessed using the Amplex® red glucose oxidase assay. The established model was effective in inducing insulin resistance and upregulating gluconeogenesis. Of the tested plant extracts, H. stellipilis showed the most potential as an antidiabetic treatment. It had low toxicity, significantly decreased hepatic glucose production and reduced the amount of phosphoenolpyruvate carboxykinase (PCK) and well as PCK gene expression, and reduced lipid content and reactive oxygen species (ROS) in the C3A cell line. H. stellipilis increased calcium signalling in INS1 rat insulinoma cells, however there was a decrease in expression of genes for insulin and glucose transporter 2 after 6 hr exposure. H. stellipilis appears be beneficial as an antidiabetic treatment. Although antidiabetic studies have been done on other Hypoxis species, this is the first study on the effects of H. stellipilis on gluconeogenesis and diabetes. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
- Authors: Sirkhotte, Saeedah , Reddy, Shanika
- Date: 2023-12
- Subjects: Medicinal plants -- South Africa , Insulin resistance , Gluconeogenesis
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/62600 , vital:72827
- Description: The World Health Organisation lists diabetes as one of the major non-communicable diseases affecting the world, and its prevalence is expected to increase rapidly. Type II diabetes mellitus (T2DM) is characterised by insulin resistance and impaired blood glucose control. T2DM is of growing concern within South Africa, with 10.8% of the population currently diagnosed. The popularity, as well as availability, of traditional plant-based medicine in South Africa, might provide a solution. Antidiabetic potential is commonly tested by in vitro assays, however the methods to test this potential via gluconeogenesis are limited. This project aimed to optimise an in vitro hepatic gluconeogenesis model. In addition, this project aimed to determine the antidiabetic properties of Prunus africana, Hypoxis stellipilis, and Eriocephalus africanus by in vitro analysis. These properties were examined in human hepatoma cells (C3A cell line) and rat pancreatic cells (INS1 cell line) and methods included: cytotoxicity analysis, Amplex® red glucose oxidase assay, antibody staining, gene expression analysis by RT-qPCR, oxidative stress analysis, and calcium signaling for insulin release. Insulin resistance was successfully induced by exposing C3A hepatocarcinoma cells to a combination treatment of 50 μM dexamethasone, 1.25 mM fructose and 0.125 mM palmitic acid for a period of three days. Thereafter, gluconeogenesis was assessed using the Amplex® red glucose oxidase assay. The established model was effective in inducing insulin resistance and upregulating gluconeogenesis. Of the tested plant extracts, H. stellipilis showed the most potential as an antidiabetic treatment. It had low toxicity, significantly decreased hepatic glucose production and reduced the amount of phosphoenolpyruvate carboxykinase (PCK) and well as PCK gene expression, and reduced lipid content and reactive oxygen species (ROS) in the C3A cell line. H. stellipilis increased calcium signalling in INS1 rat insulinoma cells, however there was a decrease in expression of genes for insulin and glucose transporter 2 after 6 hr exposure. H. stellipilis appears be beneficial as an antidiabetic treatment. Although antidiabetic studies have been done on other Hypoxis species, this is the first study on the effects of H. stellipilis on gluconeogenesis and diabetes. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
Evaluation of the antidiabetic potential, safety and efficacy of selected natural products using zebrafish larvae and in vitro bioassays
- Reddy, Shanika, Dambuza, Ntokozo
- Authors: Reddy, Shanika , Dambuza, Ntokozo
- Date: 2023-12
- Subjects: Traditional medicine -- Research -- South Africa , Medicinal plants -- South Africa -- Research , Insulin resistance
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/62575 , vital:72825
- Description: Abstract. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
- Authors: Reddy, Shanika , Dambuza, Ntokozo
- Date: 2023-12
- Subjects: Traditional medicine -- Research -- South Africa , Medicinal plants -- South Africa -- Research , Insulin resistance
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/62575 , vital:72825
- Description: Abstract. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
The hepatoprotective capacity of selected natural products from South Africa
- Authors: Reddy, Shanika
- Date: 2019
- Subjects: Hepatology , Liver -- Physiology Liver -- Pathophysiology Liver -- Diseases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/43622 , vital:36948
- Description: Drug-induced liver injury (DILI) is recognized as a significant clinical problem, which may account for up to 50% of all cases of acute liver failure. DILI is initiated by the bioactivation of parent drug molecules to produce chemically reactive metabolites. These reactive intermediates induce mitochondrial dysfunction and oxidative stress leading to glutathione (GSH) depletion and damage to cellular proteins, lipids and nucleic acids, which eventually culminates in necrotic cell death. The principal objective of this study is to establish an in vitro screening platform to identify potential hepatoprotective natural products (plants and mushrooms) from South Africa (SA). Aqueous plant extracts (Cyclopia intermedia, Opuntia ficus indica and Kigelia africana), and aqueous and ethanolic macrofungal extracts (Ganoderma lucidum, Russula capensis, Pleurotus ostreatus and Lenzites elegans) were prepared and screened against HepG2 and VERO cells to assess their safety using Hoechst 33342-PI dual labelling. A drug-induced hepatotoxic model was established, using the dietary supplement menadione (vitamin K3). Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling was used for necrosis, oxidative stress and mitochondrial membrane potential depolarization (ΔΨm) detection, respectively. The accuracy of the hepatoprotection model was confirmed through HepG2 cellbased assays (Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling) that measured the protective effects of natural products against the menadione-induced toxicity, anti-oxidant assays (DPPH, NO, ORAC, CAPe and FRAP) that measured their anti-oxidant potential and enzyme assays (βglucuronidase, carboxylesterase and CYP450 isoform 3A4) that measured their effects on drug metabolism. Silymarin was used as a positive control for each assay. Menadione displayed significant cell death, increased oxidative stress and decreased ΔΨm at an elevated concentration of 100 μM; confirming the hepatotoxicity model, where necroptosis was suspected to be menadione’s cell death mode. Only ethanolic G. lucidum was cytotoxic. All three aqueous plant extracts demonstrated strong anti-oxidant capacities out of all the tested extracts; where C. intermedia displayed the most promising DPPH, NO, ORAC, CAPe and FRAP activity, followed by aqueous G. lucidum. Aqueous plant and ethanolic macrofungal extracts (C. intermedia, O. ficus indica, K. africana, and ethanolic P. ostreatus, R. capensis) displayed decreased menadione-induced ROS production and protected against menadione-induced ΔΨm depolarization, posing them and aqueous G. lucidum potential therapeutic interventions for DILI. Ethanolic L. elegans demonstrated the highest enzyme inhibition for each assay and presented genotoxicity, ruling it out as a therapeutic strategy against DILI. Together these assays addressed several aspects relating to DILI and hepatoprotection, and served as a good starting point in evaluating the therapeutic value of natural products from South Africa.
- Full Text:
- Date Issued: 2019
- Authors: Reddy, Shanika
- Date: 2019
- Subjects: Hepatology , Liver -- Physiology Liver -- Pathophysiology Liver -- Diseases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/43622 , vital:36948
- Description: Drug-induced liver injury (DILI) is recognized as a significant clinical problem, which may account for up to 50% of all cases of acute liver failure. DILI is initiated by the bioactivation of parent drug molecules to produce chemically reactive metabolites. These reactive intermediates induce mitochondrial dysfunction and oxidative stress leading to glutathione (GSH) depletion and damage to cellular proteins, lipids and nucleic acids, which eventually culminates in necrotic cell death. The principal objective of this study is to establish an in vitro screening platform to identify potential hepatoprotective natural products (plants and mushrooms) from South Africa (SA). Aqueous plant extracts (Cyclopia intermedia, Opuntia ficus indica and Kigelia africana), and aqueous and ethanolic macrofungal extracts (Ganoderma lucidum, Russula capensis, Pleurotus ostreatus and Lenzites elegans) were prepared and screened against HepG2 and VERO cells to assess their safety using Hoechst 33342-PI dual labelling. A drug-induced hepatotoxic model was established, using the dietary supplement menadione (vitamin K3). Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling was used for necrosis, oxidative stress and mitochondrial membrane potential depolarization (ΔΨm) detection, respectively. The accuracy of the hepatoprotection model was confirmed through HepG2 cellbased assays (Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling) that measured the protective effects of natural products against the menadione-induced toxicity, anti-oxidant assays (DPPH, NO, ORAC, CAPe and FRAP) that measured their anti-oxidant potential and enzyme assays (βglucuronidase, carboxylesterase and CYP450 isoform 3A4) that measured their effects on drug metabolism. Silymarin was used as a positive control for each assay. Menadione displayed significant cell death, increased oxidative stress and decreased ΔΨm at an elevated concentration of 100 μM; confirming the hepatotoxicity model, where necroptosis was suspected to be menadione’s cell death mode. Only ethanolic G. lucidum was cytotoxic. All three aqueous plant extracts demonstrated strong anti-oxidant capacities out of all the tested extracts; where C. intermedia displayed the most promising DPPH, NO, ORAC, CAPe and FRAP activity, followed by aqueous G. lucidum. Aqueous plant and ethanolic macrofungal extracts (C. intermedia, O. ficus indica, K. africana, and ethanolic P. ostreatus, R. capensis) displayed decreased menadione-induced ROS production and protected against menadione-induced ΔΨm depolarization, posing them and aqueous G. lucidum potential therapeutic interventions for DILI. Ethanolic L. elegans demonstrated the highest enzyme inhibition for each assay and presented genotoxicity, ruling it out as a therapeutic strategy against DILI. Together these assays addressed several aspects relating to DILI and hepatoprotection, and served as a good starting point in evaluating the therapeutic value of natural products from South Africa.
- Full Text:
- Date Issued: 2019
The hepatoprotective capacity of selected natural products from South Africa
- Authors: Reddy, Shanika
- Date: 2019
- Subjects: Liver -- Physiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/43611 , vital:36929
- Description: Drug-induced liver injury (DILI) is recognized as a significant clinical problem, which may account for up to 50% of all cases of acute liver failure. DILI is initiated by the bioactivation of parent drug molecules to produce chemically reactive metabolites. These reactive intermediates induce mitochondrial dysfunction and oxidative stress leading to glutathione (GSH) depletion and damage to cellular proteins, lipids and nucleic acids, which eventually culminates in necrotic cell death. The principal objective of this study is to establish an in vitro screening platform to identify potential hepatoprotective natural products (plants and mushrooms) from South Africa (SA). Aqueous plant extracts (Cyclopia intermedia, Opuntia ficus indica and Kigelia africana), and aqueous and ethanolic macrofungal extracts (Ganoderma lucidum, Russula capensis, Pleurotus ostreatus and Lenzites elegans) were prepared and screened against HepG2 and VERO cells to assess their safety using Hoechst 33342-PI dual labelling. A drug-induced hepatotoxic model was established, using the dietary supplement menadione (vitamin K3). Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling was used for necrosis, oxidative stress and mitochondrial membrane potential depolarization (ΔΨm) detection, respectively. The accuracy of the hepatoprotection model was confirmed through HepG2 cellbased assays (Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling) that measured the protective effects of natural products against the menadione-induced toxicity, anti-oxidant assays (DPPH, NO, ORAC, CAPe and FRAP) that measured their anti-oxidant potential and enzyme assays (βglucuronidase, carboxylesterase and CYP450 isoform 3A4) that measured their effects on drug metabolism. Silymarin was used as a positive control for each assay. Menadione displayed significant cell death, increased oxidative stress and decreased ΔΨm at an elevated concentration of 100 μM; confirming the hepatotoxicity model, where necroptosis was suspected to be menadione’s cell death mode. Only ethanolic G. lucidum was cytotoxic. All three aqueous plant extracts demonstrated strong anti-oxidant capacities out of all the tested extracts; where C. intermedia displayed the most promising DPPH, NO, ORAC, CAPe and FRAP activity, followed by aqueous G. lucidum. Aqueous plant and ethanolic macrofungal extracts (C. intermedia, O. ficus indica, K. africana, and ethanolic P. ostreatus, R. capensis) displayed decreased menadione-induced ROS production and protected against menadione-induced ΔΨm depolarization, posing them and aqueous G. lucidum potential therapeutic interventions for DILI. Ethanolic L. elegans demonstrated the highest enzyme inhibition for each assay and presented genotoxicity, ruling it out as a therapeutic strategy against DILI. Together these assays addressed several aspects relating to DILI and hepatoprotection, and served as a good starting point in evaluating the therapeutic value of natural products from South Africa.
- Full Text:
- Date Issued: 2019
- Authors: Reddy, Shanika
- Date: 2019
- Subjects: Liver -- Physiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/43611 , vital:36929
- Description: Drug-induced liver injury (DILI) is recognized as a significant clinical problem, which may account for up to 50% of all cases of acute liver failure. DILI is initiated by the bioactivation of parent drug molecules to produce chemically reactive metabolites. These reactive intermediates induce mitochondrial dysfunction and oxidative stress leading to glutathione (GSH) depletion and damage to cellular proteins, lipids and nucleic acids, which eventually culminates in necrotic cell death. The principal objective of this study is to establish an in vitro screening platform to identify potential hepatoprotective natural products (plants and mushrooms) from South Africa (SA). Aqueous plant extracts (Cyclopia intermedia, Opuntia ficus indica and Kigelia africana), and aqueous and ethanolic macrofungal extracts (Ganoderma lucidum, Russula capensis, Pleurotus ostreatus and Lenzites elegans) were prepared and screened against HepG2 and VERO cells to assess their safety using Hoechst 33342-PI dual labelling. A drug-induced hepatotoxic model was established, using the dietary supplement menadione (vitamin K3). Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling was used for necrosis, oxidative stress and mitochondrial membrane potential depolarization (ΔΨm) detection, respectively. The accuracy of the hepatoprotection model was confirmed through HepG2 cellbased assays (Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling) that measured the protective effects of natural products against the menadione-induced toxicity, anti-oxidant assays (DPPH, NO, ORAC, CAPe and FRAP) that measured their anti-oxidant potential and enzyme assays (βglucuronidase, carboxylesterase and CYP450 isoform 3A4) that measured their effects on drug metabolism. Silymarin was used as a positive control for each assay. Menadione displayed significant cell death, increased oxidative stress and decreased ΔΨm at an elevated concentration of 100 μM; confirming the hepatotoxicity model, where necroptosis was suspected to be menadione’s cell death mode. Only ethanolic G. lucidum was cytotoxic. All three aqueous plant extracts demonstrated strong anti-oxidant capacities out of all the tested extracts; where C. intermedia displayed the most promising DPPH, NO, ORAC, CAPe and FRAP activity, followed by aqueous G. lucidum. Aqueous plant and ethanolic macrofungal extracts (C. intermedia, O. ficus indica, K. africana, and ethanolic P. ostreatus, R. capensis) displayed decreased menadione-induced ROS production and protected against menadione-induced ΔΨm depolarization, posing them and aqueous G. lucidum potential therapeutic interventions for DILI. Ethanolic L. elegans demonstrated the highest enzyme inhibition for each assay and presented genotoxicity, ruling it out as a therapeutic strategy against DILI. Together these assays addressed several aspects relating to DILI and hepatoprotection, and served as a good starting point in evaluating the therapeutic value of natural products from South Africa.
- Full Text:
- Date Issued: 2019
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