- Title
- Antidiabetic and toxicological properties of Dianthus thunbergii (Caryophyllaceae) roots and Hypoxis argentea (Hypoxidaceae) corms
- Creator
- Akinrinde, Akinleye Stephen
- Subject
- Medicinal plants--South Africa--Eastern Cape Traditional medicine--South Africa--Eastern Cape Diabetics--Alternative treatment--South Africa
- Date
- 2017
- Type
- Thesis
- Type
- Doctoral
- Type
- Biochemistry
- Identifier
- http://hdl.handle.net/10353/16036
- Identifier
- vital:40606
- Description
- Diabetes mellitus is a chronic metabolic disorder primarily characterized by elevated blood glucose levels. Its rapidly increasing prevalence as a major non-communicable disease is now a growing concern for both developed and developing countries. The development of safe, cost-effective and pharmacologically-effective medications continues to a major challenge of antidiabetic management. Since most conventional antidiabetic drugs are costly and present with numerous adverse effects, alternatives are increasingly being sought from natural sources, most notably medicinal plants, as viable approaches to tackling the scourge of this disease. In the Eastern Cape Province of South Africa, the roots of Dianthus thunbergii and corms of Hypoxis argentea are frequently used traditionally for the treatment of diabetes mellitus, although no scientific evidence exists to validate their effectiveness for diabetes management. The studies conducted in the resent research were, therefore, aimed at investigating the antidiabetic and toxicological properties of these plants, in an attempt to providing data towards validating their use in traditional management of diabetes mellitus. Aqueous and ethanol extracts of the underground parts of D. thunbergii and H. argentea were initially subjected to analysis of their phytochemical composition, relative to standard compounds, and the nature of their in-vitro antioxidant activities using standard spectrophotometric methods. The potentials of these extracts for cytotoxicity and/or cell proliferation were evaluated using MTT assay in HepG2 cells and Crystal violet assay in INS-1 cells. These activities were further examined in INS-1 cells using live cell fluorescence imaging techniques. To evaluate the antidiabetic properties of the extracts, they were screened for their inhibitory effects on the activities of different enzymes including α-amylase, α-glucosidase, porcine pancreatic lipase, Dipeptidyl peptidase IV (DPP-IV), collagenase and the drug metabolizing enzyme, CYP3A4, while also assessing their effects on protein glycation using in- vitro visible and fluorescence spectrophotometric approaches. Cell culture procedures were carried out to evaluate the effects of the extracts on glucose utilization in HepG2 cells and L6 myotubes; nitric oxide production in RAW 264.7 macrophages; glucose metabolism in INS-1 cells, as well as triglyceride accumulation in 3T3-L1 pre-adipocytes. Furthermore, identification of compounds present in the aqueous and ethanol extracts was carried out by Liquid chromatography- Mass spectrometry (LC-MS), while volatile oils extracted from fresh and dried parts of the two plants by hydrodistillation were also analyzed by Gas chromatography-Mass spectrometry (GC-MS). The ethanol extracts of both D. thunbergii and H. argentea contained higher amounts of total phenols, flavonoids, tannins, proanthocyanidins and alkaloids, when compared with the aqueous extracts. This finding was in direct correlation with the antioxidant activities of the extracts, with the ethanol extracts of both plants demonstrating stronger scavenging activities against hydrogen peroxide, nitric oxide, ABTS and DPPH radicals, while also exhibiting higher ferric reducing antioxidant potentials, when compared with the aqueous extracts, and in some cases, the standard antioxidants, Vitamin C, butylated hydroxytoluene and rutin. The aqueous extracts of D. thunbergii exhibited the highest toxicity in HepG2 cells with IC50 < 50 μg/ml, while also producing a concentration-dependent reduction in the viability of INS-1 cells up to 41.81percent at 50 μg/ml. Both extracts of H. argentea, however, did not produce any significant toxicity in these cells. Fluorescence imaging of live INS-1 cells using Hoechst and propidium iodide staining revealed stimulation of cell proliferation by H. argentea, while the cytotoxicity of D. thunbergii was further confirmed. H. argentea caused stimulation of glucose uptake in HepG2 cells up to 119.58 percent at 100 μg/ml and as much as 116.96 percent in L6 myotubes at 50 μg/ml, without showing toxicity to these cells. D. thunbergii produced 18.39 percent increase in L6 glucose uptake above untreated control; although its effect on HepG2 glucose uptake was irrelevant as significant toxicity was produced in these cells. H. argentea produced a concentration-dependent reduction in nitric oxide production in RAW macrophages, although not as effectively as the positive control, aminoguanidine. Again, the toxicity of D. thunbergii to this cell line precludes the relevance of nitric oxide inhibition as an antidiabetic mechanism for this plant. D. thunbergii produced a concentration-dependent increase in 3T3-L1 triglyceride accumulation, as measured by Oil red O staining, compared to untreated cells, while H. argentea exerted no significant alterations in pre- adipocyte differentiation. Generally, the two plants produced weak inhibition of the activities of the various enzymes measured, suggesting that this mechanism may not play a major role in the activities of these plants as possible antidiabetic agents. GC-MS analysis revealed major differences in the volatile oil composition between fresh and dried plant parts for both plants. Most notably, total terpenoid content of D. thunbergii oils reduced significantly from 77.17 percent in the fresh root oil to 47.58 percent in the dried root oil. Total terpenoid content was much lower in H. argentea oils, but similarly reduced from 10.58 percent in the fresh corm oil to 4.00 percent in the dried corm oil. LC-MS analysis enabled the tentative identification of compounds including phenolic glycosides, flavonoids, alkaloids, terpenoids saponins and sapogenins, many of which have been reported in literature to exert bioactivities relevant to the ones elucidated in the present study. Overall, H. argentea exhibited antidiabetic properties that may be mediated by its stimulation of glucose uptake in HepG2 and L6 cells; stimulation of proliferation in INS-1 cells; lack of stimulation of 3T3-L1 triglyceride accumulation and a tendency to reduce nitric oxide production in RAW macrophages. These activities suggest that H. argentea has promise for further investigations as an antidiabetic agent. On the contrary, D. thunbergii exhibited significant toxicity to HepG2 cells, INS-1 cells and RAW macrophages. Its cytotoxicity at the concentrations investigated in the present studies raises significant concerns about any potential antidiabetic applications for this plant.
- Format
- 225 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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