Leonotis leonurus: understanding the mechanism of anti-diabetic action and investigating a nano drug delivery system
- Odei-Addo, Frank, Levendal, Ruby-Ann
- Authors: Odei-Addo, Frank , Levendal, Ruby-Ann
- Date: 2016
- Subjects: Diabetes Plant extracts
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/13116 , vital:27153
- Description: Diabetes mellitus is a metabolic disease characterised by hyperglycaemia resulting from defects in insulin secretion, insulin action, or both. The leaf extract of Leonotis leonurus and its active compound marrubiin, have been shown to possess anti-diabetic, antiplatelet, anti-inflammatory and anti-coagulation activity. In the present study, the mechanism by which L. leonurus and marrubiin exert their anti-diabetic properties, the cross-talk between the peripheral tissues and a nano drug delivery system were investigated. Marrubiin in the plant extract was effectively quantified by an optimised reversed phase highperformance liquid chromatography (HPLC) protocol using a pentafluorophenyl (PFP) column with water and acetonitrile (50:50) as mobile phase, and a flow rate of 1ml/min. The chemical structure was determined using liquid chromatography-tandem mass spectroscopy LC-MS/MS. Real-time quantitative polymerase chain reaction (RT-qPCR) gene expression of selected adipokines and proteins implicated in Type-2 diabetes (T2D) were investigated in specific peripheral tissues isolated from an in vivo obese rat model. An in vitro cell culture model was used to determine the crosstalk between the peripheral tissues and pancreatic (INS-1E) β-cells. Various nanoformulations of L. leonurus extract were prepared and their effect on cytotoxicity (in Chang liver and INS-1 cells), insulin-mediated glucose uptake (Change liver cells) and insulin secretion (INS-1) were investigated. The average yield of marrubiin from the plant extract was 10% (n=3), with a molecular mass of 333.20Da and a molecular formula of C20H29O4 +. Results from the in vivo study showed that the L. leonurus extract significantly (p<0.05) enhanced the gene expression of adiponectin, peroxisome proliferator-activated receptor gamma (PPAR-γ), glucokinase (GK), uncoupling protein-2 (UCP-2) and reduced leptin in adipose tissue, but resistin, glucose transporters (GLUT), fatty acid synthase (FAS), insulin receptor substrate -1 (IRS-1) and phosphoenolpyruvate carboxykinase (PEPCK) gene expression were not affected. Marrubiin decreased gene expression of leptin and resistin, and increased IRS-1 and glucokinase in adipose tissue. In liver and muscle tissues, marrubiin and the L. leonurus extract reduced gene expression of PPAR-γ, IRS-1, glucokinase and PEPCK. In the in vitro crosstalk study (under normoglycaemic and hyperglycaemic conditions), conditioned medium from 3T3-L1 cells significantly (p<0.01) enhanced insulin secretion. This was not observed in INS-1E cells exposed to muscle- and liver-conditioned medium, respectively. The in vitro studies using a nanostructured lipid formulation (NLC) of the plant extract was not cytotoxic to either INS-1 and Chang liver cells. The NLC formulation significantly (p<0.05) enhanced glucose uptake in Chang liver cells and improved chronic insulin release in INS-1 cells (p<0.05). Based on the above findings from the in vivo and in vitro studies, both L. leonurus and marrubiin exerted an insulinotropic effect via adipose tissue on pancreatic β-cells. The findings in the in vivo study showed that marrubiin and the L. leonurus extract were employing their major anti-diabetic action via the adipose tissue.
- Full Text:
- Date Issued: 2016
- Authors: Odei-Addo, Frank , Levendal, Ruby-Ann
- Date: 2016
- Subjects: Diabetes Plant extracts
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/13116 , vital:27153
- Description: Diabetes mellitus is a metabolic disease characterised by hyperglycaemia resulting from defects in insulin secretion, insulin action, or both. The leaf extract of Leonotis leonurus and its active compound marrubiin, have been shown to possess anti-diabetic, antiplatelet, anti-inflammatory and anti-coagulation activity. In the present study, the mechanism by which L. leonurus and marrubiin exert their anti-diabetic properties, the cross-talk between the peripheral tissues and a nano drug delivery system were investigated. Marrubiin in the plant extract was effectively quantified by an optimised reversed phase highperformance liquid chromatography (HPLC) protocol using a pentafluorophenyl (PFP) column with water and acetonitrile (50:50) as mobile phase, and a flow rate of 1ml/min. The chemical structure was determined using liquid chromatography-tandem mass spectroscopy LC-MS/MS. Real-time quantitative polymerase chain reaction (RT-qPCR) gene expression of selected adipokines and proteins implicated in Type-2 diabetes (T2D) were investigated in specific peripheral tissues isolated from an in vivo obese rat model. An in vitro cell culture model was used to determine the crosstalk between the peripheral tissues and pancreatic (INS-1E) β-cells. Various nanoformulations of L. leonurus extract were prepared and their effect on cytotoxicity (in Chang liver and INS-1 cells), insulin-mediated glucose uptake (Change liver cells) and insulin secretion (INS-1) were investigated. The average yield of marrubiin from the plant extract was 10% (n=3), with a molecular mass of 333.20Da and a molecular formula of C20H29O4 +. Results from the in vivo study showed that the L. leonurus extract significantly (p<0.05) enhanced the gene expression of adiponectin, peroxisome proliferator-activated receptor gamma (PPAR-γ), glucokinase (GK), uncoupling protein-2 (UCP-2) and reduced leptin in adipose tissue, but resistin, glucose transporters (GLUT), fatty acid synthase (FAS), insulin receptor substrate -1 (IRS-1) and phosphoenolpyruvate carboxykinase (PEPCK) gene expression were not affected. Marrubiin decreased gene expression of leptin and resistin, and increased IRS-1 and glucokinase in adipose tissue. In liver and muscle tissues, marrubiin and the L. leonurus extract reduced gene expression of PPAR-γ, IRS-1, glucokinase and PEPCK. In the in vitro crosstalk study (under normoglycaemic and hyperglycaemic conditions), conditioned medium from 3T3-L1 cells significantly (p<0.01) enhanced insulin secretion. This was not observed in INS-1E cells exposed to muscle- and liver-conditioned medium, respectively. The in vitro studies using a nanostructured lipid formulation (NLC) of the plant extract was not cytotoxic to either INS-1 and Chang liver cells. The NLC formulation significantly (p<0.05) enhanced glucose uptake in Chang liver cells and improved chronic insulin release in INS-1 cells (p<0.05). Based on the above findings from the in vivo and in vitro studies, both L. leonurus and marrubiin exerted an insulinotropic effect via adipose tissue on pancreatic β-cells. The findings in the in vivo study showed that marrubiin and the L. leonurus extract were employing their major anti-diabetic action via the adipose tissue.
- Full Text:
- Date Issued: 2016
Purification and characterization of serine proteinase inhibitors from two South African indigenous plants, Acacia karoo and Acacia schweinfurthii
- Authors: Odei-Addo, Frank
- Date: 2009
- Subjects: Proteinase -- Inhibitors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10319 , http://hdl.handle.net/10948/1291 , Proteinase -- Inhibitors
- Description: Serine proteases are known to perform a wide range of functions essential to life; however there has to be some form of control mechanism in place. One of the many control mechanisms is their specific inhibition by protein proteinase inhibitors. Proteinase inhibitors in plants, present in their seeds, participate in defense mechanisms and their production is induced by herbivory or wounding. Plant proteinase inhibitors have been reported to inhibit a variety of serine proteinases, including enzymes of the blood coagulation cascade. In this study, various indigenous seed extracts were screened for potential serine proteinase inhibition. Acacia schweinfurthii was selected as a potential inhibitor that inhibited trypsin and factor X. The AS inhibitor was successfully purified to homogeneity by precipitating with 80 percent (v/v) acetone and the sequential chromatographic steps including ion-exchange chromatography, size exclusion chromatography, affinity purification on a trypsin-agarose column and RP-HPLC. Reducing SDS-PAGE conditions revealed an inhibitor of two polypeptide chains A and B of approximate molecular weights 16 and 10 kDa, respectively, and under non-reducing conditions, 25 kDa was observed. The inhibitor was shown to inhibit trypsin, chymotrypsin and factor X indicating the dynamic nature of the reactive site. An enzyme: inhibitor ratio of 1:1, and a Ki of 3.45nM was determined for the AS inhibitor on trypsin, and the inhibitor also weakly inhibit chymotrypsin. AS inhibitor and STI inhibited factor X with a Ki values of 13.7nM and 77.5μM respectively. Amino acid analysis revealed Mmin values of the A- and B- chain of 15,000 and 7,800, respectively. The effect of seed extracts on the activated partial thrombin time (APTT) and prothrombin time (PT) was tested. No prolongation of the PT was obtained. For the crude extracts of AK and AS, IC200 values of 4.6 and 189.62 μg/mL, were respectively obtained. For the purified fractions of STI, AS and AK, IC200 values of 51.5, 114.31 and 893.8 μg/ml were observed, respectively. Keywords: proteinase inhibitors, Acacia species, trypsin inhibitor, FX inhibitor.
- Full Text:
- Date Issued: 2009
- Authors: Odei-Addo, Frank
- Date: 2009
- Subjects: Proteinase -- Inhibitors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10319 , http://hdl.handle.net/10948/1291 , Proteinase -- Inhibitors
- Description: Serine proteases are known to perform a wide range of functions essential to life; however there has to be some form of control mechanism in place. One of the many control mechanisms is their specific inhibition by protein proteinase inhibitors. Proteinase inhibitors in plants, present in their seeds, participate in defense mechanisms and their production is induced by herbivory or wounding. Plant proteinase inhibitors have been reported to inhibit a variety of serine proteinases, including enzymes of the blood coagulation cascade. In this study, various indigenous seed extracts were screened for potential serine proteinase inhibition. Acacia schweinfurthii was selected as a potential inhibitor that inhibited trypsin and factor X. The AS inhibitor was successfully purified to homogeneity by precipitating with 80 percent (v/v) acetone and the sequential chromatographic steps including ion-exchange chromatography, size exclusion chromatography, affinity purification on a trypsin-agarose column and RP-HPLC. Reducing SDS-PAGE conditions revealed an inhibitor of two polypeptide chains A and B of approximate molecular weights 16 and 10 kDa, respectively, and under non-reducing conditions, 25 kDa was observed. The inhibitor was shown to inhibit trypsin, chymotrypsin and factor X indicating the dynamic nature of the reactive site. An enzyme: inhibitor ratio of 1:1, and a Ki of 3.45nM was determined for the AS inhibitor on trypsin, and the inhibitor also weakly inhibit chymotrypsin. AS inhibitor and STI inhibited factor X with a Ki values of 13.7nM and 77.5μM respectively. Amino acid analysis revealed Mmin values of the A- and B- chain of 15,000 and 7,800, respectively. The effect of seed extracts on the activated partial thrombin time (APTT) and prothrombin time (PT) was tested. No prolongation of the PT was obtained. For the crude extracts of AK and AS, IC200 values of 4.6 and 189.62 μg/mL, were respectively obtained. For the purified fractions of STI, AS and AK, IC200 values of 51.5, 114.31 and 893.8 μg/ml were observed, respectively. Keywords: proteinase inhibitors, Acacia species, trypsin inhibitor, FX inhibitor.
- Full Text:
- Date Issued: 2009
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