In vitro pharmacological screening of thiazolidinedione-derivatives on diabetes and Alzheimer’s potential therapeutic targets
- Authors: Arineitwe, Charles
- Date: 2022-10-14
- Subjects: Diabetes Treatment , Alzheimer's disease Treatment , Antioxidants Therapeutic use , Rosiglitazone , Hypoglycemic agents , In vitro screening , Thiazolidinedione Derivatives
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/232171 , vital:49968
- Description: There is an increased prevalence of diabetes and other non-communicable diseases in Sub-Saharan Africa and globally. In South Africa, the prevalence of type 2 Diabetes mellitus is currently estimated at 9.0% in people aged 30 and older and is expected to increase. Diabetes-related complications result in acute alterations in the mental state due to poor metabolic control as well as greater rates of decline in cognitive functioning with age, higher prevalence of depression and increased risk of Alzheimer’s disease. Alzheimer’s disease is the most common form of dementia in older adults and possibly contributes to 60 - 70% of cases. Alzheimer’s disease remains incurable, its progression inevitable with the currently available symptomatic therapies being palliative while the treatment of diabetes relies on insulin preparations and other glucose-lowering agents. Current treatment options have numerous side effects such as hypoglycaemia, diarrhoea, weight gain and abnormal liver function. This has geared the investigation of new generations of small molecules which exhibit improved efficacy and safety profiles. On this basis, several studies have shown that thiazolidinediones and their corresponding derivatives exhibit a broad spectrum of biological activities including anti-inflammatory, and antioxidant activities. Furthermore, recent evidence from experimental, epidemiological, and clinical studies highlight the utility of antioxidants for treating diabetes and its complications. Interestingly, there is increasing evidence that links diabetes and Alzheimer’s disease due to their pathophysiology and suppressing glycaemia has been shown to be beneficial in Alzheimer’s disease treatment. Accordingly, the aim of this study, was to evaluate the anti-diabetic and anti-Alzheimer’s properties of four novel synthesized thiazolidinedione-derivatives owing to their antioxidant properties. Methods The aim of this study was achieved through performing ferric reducing antioxidant power activity, 2,2’-Diphenyl-1-Picry Hydrazyl radical scavenging activity, α-amylase inhibition, α-glucosidase inhibition, aldose reductase inhibition, protein tyrosine phosphatase-1B inhibition, dipeptidyl peptidase-4 inhibition, acetylcholinesterase inhibition, matrix metalloproteinase-1 inhibition, and β-amyloid aggregation inhibition assays. In addition, peroxisome proliferator-activated receptor-γ activation was performed through docking studies. To establish physicochemical properties of TZD derivatives investigated, further in-silico studies were done using SwissADME tools. Results To this end, in-vitro and in-silico studies were successfully performed. In-silico ADME profiling predicted these derivatives to be drug-like with moderate to good solubility in the GI and not blood-brain barrier permeable. Furthermore, docking of these molecules against PPARγ predicted a similar mode of action to that of thiazolidinediones using Rosiglitazone as the standard drug with TZDD2 and TZDD4 forming equivalent conformations to that of Rosiglitazone in the same binding site and TZDD3 having an equivalent LBE to that of Rosiglitazone (-8.84 and -8.63kcal/mol respectively). In-vitro evaluation predicted a moderate antioxidant activity with TZDD2 and 3 exhibiting the highest FRAP activity and DPPH radical scavenging activity. Furthermore, enzymatic inhibition assays showed a relative inhibition activity with TZDD3 exhibited > 100% inhibition in concentrations ≥ 30 μg/mL and TZDD1, 2 and 4 exhibited ≥ 50% inhibition activity in all the concentrations (10, 20, 30, 40 and 50 μg/mL) in the α-amylase inhibition assay. Similarly, in the α-glucosidase inhibition assay, all the four derivatives exhibited a concentration dependent activity with TZDD3 showing the most activity. All the four derivatives exhibited ≥ 30% inhibition in the aldose reductase inhibition assay except TZDD1 at 10 μg/mL. TZDD4 exhibited a concentration dependent inhibition activity in the protein tyrosine phosphatase-1B inhibition assay. Interestingly, TZDD3 showed a decreasing inhibition activity as its concentration increased from 10 μg/mL through to 50 μg/mL. In the dipeptidyl peptidase–4 inhibition assay, TZDD2 and TZDD4 exhibited ≥ 20% inhibition activity across all the concentrations and in the acetylcholinesterase assay, TZDD1, 3 and 4 exhibited ≥ 25% across all the concentrations. Interestingly, in the matrix metalloproteinase-1 inhibition assay, some of these derivatives exhibited partial activation activity and partial inhibition with TZDD1 showing activation in concentrations 10 and 20 μg/mL and inhibition in concentrations 30, 40 and 50 μg/mL. TZDD4 showed activation in all the concentrations. In the β-amyloid aggregation assay, all the four derivatives showed inhibition activity ≥ 10% except TZDD1 at 50 μg/mL. Conclusions Diabetes mellitus and Alzheimer’s disease are a type of pathology of global concern, and several researchers worldwide have strived to search for novel therapeutic treatments and prevention for diabetes as well as Alzheimer’s disease. Recent studies provide a direct link v between diabetes mellitus and Alzheimer’s disease, and the need to find novel drugs that can mitigate these two is of increasing interest. In our search for antidiabetic and anti-Alzheimer’s activity, TZD derivatives (TZDD1, TZDD2, TZDD3 and TZDD4) exhibited good antioxidant activity, anti-hyperglycaemic activity and a relatively promising anti-Alzheimer’s activity. This was observed from the in vitro evaluation performed which included α – amylase, α – glucosidase, aldose reductase, PTP1B, DPP4, amyloid β aggregation, and AChE inhibition assays. Furthermore, docking of the derivatives against PPARγ predicted a similar molecular interaction to that of thiazolidinediones using Rosiglitazone as the standard drug. Furthermore, in silico ADME profiling predicted the derivatives to have moderate to good solubility in the GI (good GI bioavailability), and also exhibited excellent drug likeness. However, they are predicted not permeate the BBB. Further in silico studies and in vivo should be conducted to establish toxicities, as well as drug delivery to the brain for effective therapeutic effect against Alzheimer’s disease. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacology, 2022
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- Date Issued: 2022-10-14
Comprehensive characterization of the antidiabetic potential of selected plants and macrofungi from Africa using an in vitro target-directed screening platform and cellomics
- Authors: Pringle, Nadine Alex
- Date: 2020
- Subjects: Hypoglycemic agents
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/46750 , vital:39653
- Description: Several synthetic antidiabetic drugs have been developed to date, however, most are accompanied by adverse side-effects while remaining expensive and largely inaccessible to the vast majority of those who need it. To provide enough scientific evidence to support the inclusion of more affordable African antidiabetic medicinal plants and macrofungi into healthcare programs, this study sought out to develop a comprehensive in vitro antidiabetic target-directed screening platform incorporating high content screening and analysis/ cellomics. To test the success of this model, the potential antidiabetic mechanisms of five plants (Aspalathus linearis, Brachylaena discolor, Carpobrotus deliciosus, Sutherlandia frutescens and Tarchonanthus camphoratus) and two macrofungal species (Ganoderma lucidum and Hericium erinaceus) were explored. The screening model consisted of approximately 22 assays exploring the antidiabetic effects of selected aqueous and ethanolic extracts in five well-characterised antidiabetic targets: the intestine, liver, skeletal muscle, adipose tissue/ obesity and pancreatic β-cells. These targets were further categorised and scored under three mechanistic classes/ therapeutic targets (postprandial hyperglycaemia; insulin resistance and inflammation; pancreatic β-cell function) to elucidate their potential mechanisms of action and select appropriate animal models for future studies. Almost any normal or diabetic rodent model would be suitable to explore the antidiabetic potential of extracts such as A. linearis, B. discolor ethanol, C. deliciosus ethanol or T. camphoratus which obtained high cumulative scores under postprandial hyperglycaemia while high fat diet and genetic models of obesity appear more suited towards extracts such as H. erinaceus aqueous that obtained their highest cumulative score under insulin resistance. In general, a combination of rodent models ranging from non-obese models to models of obesity and β-cell destruction presenting symptoms from all three mechanistic classes should be considered due to the pleiotropic nature of the tested extracts, however, establishing appropriate experimental designs is crucial. To demonstrate the versatility of the screening platform and emphasise the importance of in vitro screening pertaining to diabetic complications, a more detailed biochemical investigation into the potential therapeutic benefits of A. linearis in the treatment of diabetic wounds was conducted. Several properties supporting the therapeutic potential of rooibos were highlighted with the green and fermented extracts presenting distinctly different characteristics. The pro-inflammatory nature of fermented rooibos may have therapeutic value for wounds characterised with a delayed initial inflammatory phase, such as early diabetic wounds while the green extract appears more suited to wounds burdened with excessive inflammation as it attenuated COX-2 levels and effectively protected fibroblasts against oxidative stress. To date, this appears to be the most comprehensive antidiabetic screening platform in existence and consequently provides the only feasible solution that will enable natural product antidiabetic research to progress to the point where natural products can be commercialised and incorporated into meaningful healthcare programs. Future research should be focussed towards further expanding this model by incorporating additional targets, more sophisticated cell culturing techniques, multiplexed high content screening assays and carrying out combination treatments that explore the antidiabetic effects of two or more crude extracts to establish whether they are capable of acting in a synergistic manner.
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- Date Issued: 2020
In silico and in vitro screening of marrubiin and marrubiin derivatives for antidiabetic activity on PTP1ß, C2C12 myocytes, chang liver hepatocytes and 3T3-L1 adipocytes
- Authors: Nicholas, Rudi Berto
- Date: 2013
- Subjects: Hypoglycemic agents , Diabetes -- Treatment -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10347 , http://hdl.handle.net/10948/d1020638
- Description: Diabetes mellitus (DM) is a life changing disease which affects a large portion of the population and the economy through high medical costs and loss of productivity. Marrubiin (MAR), a diterpenoid isolated from Leonotis leonurus, a plant indigenous to Southern Africa, is used by traditional healers to alleviate DM symptoms. This study aims to screen the inhibitory potential of MAR and MAR derivatives on PTP1β and glucose uptake properties of Chang liver, C2C12 and 3T3-L1 cells. Marrubiin and 19 of its derivatives were tested to determine the inhibition constants for PTP1β. A Ki of 21 μM and 0.047 μM was detected for oleanolic acid in silico and in vitro, respectively. All other diterpene derivatives did not display substantial levels of inhibition of PTP1β. Treatment of Chang liver cells with the various MAR derivatives (10 μM) did not significantly increase glucose uptake beyond metformin, a known antidiabetic drug. The various treatments showed a protective/proliferative effect on the C2C12 muscle cells with two MAR treatments (DC16 and DC18) significantly increasing glucose uptake as compared to metformin in C2C12 muscle cells. It was noted that DC17, DC18 and MAR significantly increased glucose uptake in 3T3-L1 adipocytes, relative to the control. Contrary to cytotoxicity studies with Chang liver and C2C12 muscle cells, adipocytes displayed no cytotoxicity to treatments while a significant increase in cell viability was seen for DC9 and DC15. To unravel the mechanism of action, Western blotting analysis was completed and an increased expression of PTP1β was observed for treatments with DC17 and DC6 was seen in adipocytes, while DC18 and metformin decreased expression significantly. This correlated with a significant decrease of Ser 612 phosphorylation of insulin receptor substrate (IRS1) for DC17. Real time qPCR of IRS1 and GLUT4 highlighted that DC17 and MAR were able to significantly increase expression of IRS1 and GLUT4, respectively. The results show that MAR and the selected derivatives (DC6, DC17, DC18) have been found to increase glucose uptake in peripheral tissue types with IRS1, GLUT4 and PTP1β being associated with the mechanism of action. However, a complete understanding of the mechanisms is yet to be established.
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- Date Issued: 2013
Optimisation of an in vitro model for anti-diabetic screening
- Authors: Wilson, Gayle Pamela
- Date: 2006
- Subjects: Hypoglycemic agents , Diabetes -- Treatment -- South Africa , Materia medica, Vegetable -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10308 , http://hdl.handle.net/10948/428 , Hypoglycemic agents , Diabetes -- Treatment -- South Africa , Materia medica, Vegetable -- South Africa
- Description: The need for alternative strategies for the prevention and treatment of diabetes is growing rapidly as type II diabetes is reaching epidemic status in our society. This need was the basis for the creation of this study, as it was necessary to start looking towards medicinal plants as potential antidiabetic treatment and no comprehensive in vitro model existed. In creating a model for determining the effects of alternative traditional medicines as antidiabetic potentiates, it was necessary that two metabolic pathways, namely glucose uptake and insulin secretion, which play a significant role in glucose homeostasis, be at the centre of our investigations. The objective of this project was to optimize the methodology required to screen and ultimately determine the effectiveness of the plant extracts Kankerbos and MRC2003, as antidiabetic potentiates, through observing their effects on glucose utilisation and insulin secretion. If these medicinal plants are going to make a positive contribution to the health of type II diabetic South Africans, then the determination of their efficacy is essential. The cell lines used in this study included 3T3-L1 preadipocytes, Chang liver, C2C12 muscle and INS-1 rat pancreatic cells. Each cell line represents a different in vivo organ that is known to have an influence on glucose homeostasis in our bodies, each with its own unique metabolic pathways and mechanisms of activity, thereby making each one a vital component in the study. The positive controls for the two models were insulin and metformin (glucose utilisation) and glibenclamide (insulin secretion). Insulin was shown to provide a significant increase in the amount of glucose taken up in C2C12 muscle and Chang liver cells for acute conditions. Chronic treatments with metformin provided a significant increase in glucose utilised by Chang liver cells. Glibenclamide was an effective positive control for stimulating insulin secretion by INS-1 cells under acute conditions as there was a significant increase in the amount of insulin secreted. MRC2003 did not show any significant antidiabetic activity. Sutherlandia frutescens (Kankerbos) showed biological activities comparable to some of the more recognized antidiabetic compounds throughout the study. With regards to the glucose utilisation model, Kankerbos was seen to have both acute and chronic effects in different cell lines. In the C2C12 muscle cell line, Kankerbos significantly increased glucose uptake when they were exposed to acute conditions. Kankerbos also had a significant effect on the Chang liver cells as it was observed that under both acute and chronic conditions, this plant extract induced the uptake of glucose into these cells. With respect to the insulin secretion model involving INS-1 cells, no significant effect was seen during acute exposure with Kankerbos treatment. However during chronic exposure, an increase in insulin secretion was initiated by this plant extract. Overall, the results of this study suggest that Kankerbos has a twofold mechanism of action for its glucose-lowering effects. Given that Kankerbos is widely available in South Africa, this study was valuable as it provided an indication that Kankerbos has antidiabetic activities and could possibly be used as an alternative antidiabetic medication.
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- Date Issued: 2006