Background: Evidence from our laboratories indicates that triterpene constituents of Syzygium cordatum (Hochst.) [Myrtaceae] crude leaf extracts can be used to treat diabetes mellitus. For the plant derived triterpenes to have further potential in diabetes management, they should, however, additionally alleviate or prevent some of the complications of diabetes mellitus such as impaired kidney function and cardiovascular disorders. Accordingly, this study was designed to isolate the triterpene, oleanolic acid (OA) from S. cordatum leaves and evaluate its effects on blood glucose, renal function and blood pressure in streptozotocin (STZ)-induced diabetic rats. OA was studied because it is the major constituent of many African plant species used in traditional medicine. Materials and Methods S. cordatum crude leaf ethyl acetate solubles (EAS) were obtained after defatting the leaves with hexane followed by dichloromethane before maceration with ethyl acetate. Preliminary experiments indicated that EAS contained triterpenes with hypoglycaemic properties. Solvent extraction and fractionation of EAS yielded mixtures of oleanolic acid/ursolic acid (OA/UA) and methyl maslinate/methyl corosolate. Recrystallisation of the OA/UA mixture using ethanol yielded OA, the structure of which was confirmed by NMR spectroscopy ('H & 13C). Oral glucose tolerance test (OGTT) responses to various doses of OA (40, 80 and 120 mg/kg) were monitored in separate groups of non-diabetic and STZ-induced diabetic rats given a glucose load (0.86 g/kg, p.o.) after an 18-h fast. Rats treated with deionized water (3 ml/kg p.o.), or standard drugs, (insulin, 200 Hg/kg, s.c; metformin, 500 mg/kg, p.o. and glibenclamide, 500 f^g/kg, p.o.) acted as untreated and treated positive controls, respectively. To investigate the possible interaction between OA and standard drugs in lowering blood glucose, OGTT responses were studied in separate groups of animals simultaneously treated with OA at either 40 or 80 mg/kg and insulin (100 or 200 ug/kg, s.c), metformin, (250 or 500 mg/kg, p.o.) or glibenclamide (250 or 500 mg/kg, p.o.). Blood glucose was monitored at 15-min intervals for the first hour, and hourly thereafter for 3 h. Plasma insulin concentrations were measured in separate parallel groups of rats prepared as for OGTT studies to examine whether there was an association between OA treatment and pancreatic insulin secretion. Acute effects of OA on kidney function and mean arterial blood pressure (MAP) were investigated in anaesthetized rats challenged with hypotonic saline after a 3'/2-h equilibration for 4 h consisting of 1 h control, 1XA h treatment and 1 Vi h recovery periods. OA was added to the infusate during the treatment period. Short-term effects of OA were studied in individually-caged rats treated twice daily with OA (80 mg/kg, p.o.) for 5 weeks. Results OA decreased blood glucose concentrations of both non-diabetic and diabetic rats, as did some standard drugs except glibenclamide which did not exhibit any effects in STZ-induced diabetic animals. The blood glucose lowering effects were most potent in STZ-induced rats treated with combined OA and insulin by comparison with all other treatments. Short-term treatment of non-diabetic and STZ-induced diabetic rats with OA alone for 5 weeks decreased blood glucose concentrations, but the reduction in non-diabetic rats was to values that did not achieve statistical significance. Except for non-diabetic rats treated with insulin alone or in combination with OA, plasma insulin concentrations were not altered by treatment in non-diabetic and STZ-induced diabetic animals. Hepatic glycogen concentrations of non-diabetic and STZ-induced diabetic rats were significantly increased by all treatments at the end of 5 weeks. Acute intravenous infusion of OA in anaesthetized rats significantly increased Na+ excretion outputs of non-diabetic and STZ-induced diabetic rats without affecting urine flow, K+ or CI" excretion rates. Similarly, daily OA treatment (80 mg/kg, p.o.) significantly increased Na+ excretion rates of non-diabetic and STZ-induced diabetic rats throughout the 5 week experimental period without affecting urine flow, K+ or CI" excretion rates. By comparison with respective control animals, Short-term administrations of OA significantly (p<0.05) increased GFR of non-diabetic (2.88±0.14 vs 3.71±0.30 ml/min) and STZ-diabetic rats (1.81± 0.32 vs 3.07±0.16 ml/min, n=6 in all groups) with concomitant reduction of plasma creatinine concentrations. Acute and Short-term administrations of OA non-diabetic and STZinduced diabetic rats reduced mean arterial blood pressure by comparison with respective control animals. Discussion The results suggest that S. cordatum leaf derived OA not only has the potential to lower blood glucose in diabetes, but also has beneficial effects on kidney function and blood pressure. We suggest that the hypoglycaemic effects of OA mimic those of metformin as evidenced by the fact that neither of these treatments altered plasma insulin concentration of non-diabetic rats. OA-evoked increases in urinary Na+ outputs of STZ-diabetic rats and elevation of GFR suggest up-regulation of renal function by the triterpene. The findings are of considerable importance because they suggest the hypoglycaemic, renal and hypotensive effects of OA in the management of diabetes mellitus. Conclusion The results demonstrated that the oleanolic acid extracted from S. cordatum leaf has blood glucose-lowering effects comparable to standard anti-diabetic drugs in STZ-induced diabetic rats. Furthermore, OA augmented the hypoglycaemic effects of insulin in STZ-induced diabetic rats. These findings suggest that OA may have beneficial effects on some of the processes that are associated with renal derangement in STZ-induced diabetic rats. The results introduce the first in vivo evidence that OA ameliorates kidney function in STZinduced diabetic rats. Keywords: Renal function; diabetes mellitus; triterpenoids; oleanolic acid, hypoglycaemia / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, 2008.

Glucose is a principle precursor for milk lactose and de novo synthesis of milk fat; therefore exercising during lactation could create competition for glucose between exercising muscle and lactating mammary gland. This study investigated the combined effects of maternal dietary glucose (20%, 40%, 60%), exercise (chronically exercised, sedentary) and litter size (8, 12 pups) on rat mammary gland composition, milk composition, milk yield and pup growth. Chronic exercise increased milk fat concentrations and an interaction between chronic exercise and 20% dietary glucose decreased milk lactose concentrations compared to 40% or 60% glucose diets. Restricting maternal dietary glucose also decreased milk fat concentrations and exercise decreased mammary fat. In addition, pups of dams fed the 40% glucose diet were heavier on lactation day 15 than pups of dams fed the 60% diet. These results suggest that competition for glucose occurs and that a 40% glucose maternal diet may be more appropriate for pup growth.

Thèse (M.Ps.)--Université Laval, 2003. / Bibliogr.: f. 82-114. Publié aussi en version électronique.

Thesis (M.S.)--State University of New York at Binghamton, Department of Psychology, 2008. / Includes bibliographical references.

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Lau San Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Title Page --- p.1 / List of Thesis Committee --- p.2 / Declaration Page --- p.3 / Acknowledgements --- p.4 / Table of Contents --- p.5 / Abstract --- p.10 / 論文撰要 --- p.12 / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Drug Metabolism --- p.14 / Chapter 1.2 --- Glucuronidation --- p.16 / Chapter 1.3 --- UDP-glucuronosyltransferase (UGTs) / Chapter 1.3.1 --- Nomenclature --- p.18 / Chapter 1.3.2 --- Tissue Distributions of UGTs --- p.20 / Chapter 1.3.3 --- Genetics --- p.26 / Chapter 1.3.4 --- Evolution of UGTs --- p.28 / Chapter 1.4 --- UDP-glucuronosyltransferase related Human Diseases --- p.33 / Chapter 1.4.1 --- Hyperbilirubinemia --- p.33 / Chapter 1.4.2 --- Cancer --- p.37 / Chapter 1.5 --- Rattus norvrgicus UDP-glucuronosyltransferase 1A8 --- p.38 / Chapter 1.6 --- Aims of the Project --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 1. --- Rat liver mRNA Extraction --- p.43 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.43 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.43 / Chapter 4. --- Construction of bacterial expression vector --- p.43 / Chapter 5. --- Expression of recombinant protein in E.coli --- p.44 / Chapter 6. --- Purification of protein with Ni column --- p.44 / Chapter 7. --- Purification of protein with gel filtration column --- p.44 / Chapter 8. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 9. --- Concentration and Desalting of protein --- p.45 / Chapter 10. --- Enzyme activity of glucuronidation --- p.45 / Chapter 11. --- Near UV and far UV circular dichroism (CD) spectroscopy --- p.45 / Chapter 12. --- Fluorescent properties studies --- p.45 / Chapter 13. --- Western Blotting --- p.46 / Chapter 14. --- 3D modeling of UGT1A8 and interactions with ligands --- p.46 / Chapter 2.2 --- Methods / Chapter 1. --- Rat liver mRNA extraction --- p.46 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.47 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.48 / Chapter 4. --- Cloning of UGT1A8 PCR product into expression vector pRSet B --- p.49 / Chapter 5. --- Confirmation of the presence of insert in the plasmid --- p.51 / Chapter 6. --- Sequence checking for UGT1A8 gene in the pRSet B vector --- p.52 / Chapter 7. --- Expression of recombinant protein in E.coli JM109(DE3) cell strain --- p.52 / Chapter 8. --- Purification of recombinant protein by Ni-column --- p.53 / Chapter 9. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.53 / Chapter 10. --- Recombinant protein purification by gel filtration column --- p.54 / Chapter 11. --- Concentration or Desalting of Purified Protein --- p.54 / Chapter 12. --- Determination of Protein Concentration --- p.55 / Chapter 13. --- Far- UV Circular dichroism spectroscopy --- p.55 / Chapter 14. --- Intrinsic Fluorescence Studies of Proteins --- p.57 / Chapter 15. --- Chemical denaturation stability studies --- p.58 / Chapter 16. --- Glucuronidation protein activity assay --- p.59 / Chapter 17. --- Mutagenesis --- p.60 / Chapter 18. --- Western Blotting for the presence of protein --- p.61 / Chapter 19. --- Protein Modeling with Insight II / Chapter 19.1 --- Construction of substrate 1-napthol structure --- p.62 / Chapter 19.2 --- Obtaining UDP-glucuronic acid in PDB file --- p.63 / Chapter 19.3 --- Obtaining rat UGT1A8 model structure in PDB file --- p.63 / Chapter 19.4 --- Optimization of rat UGT1A8 structure --- p.63 / Chapter 19.5 --- Docking studies of interaction between ligands and protein / Chapter 19.5.1 --- Setting up a Grid --- p.66 / Chapter 19.5.2 --- Docking of 1-napthol to UGT1A8 --- p.67 / Chapter 19.5.3 --- Docking of UDP-glucuronic acid in the complex of UGT1A8 and1- napthol --- p.68 / Chapter 19.5.4 --- Definition of Subsets --- p.68 / Chapter Chapter 3 --- Results --- p.70 / Figure 3.1 The extracted RNA from rat liver tissue --- p.76 / Figure 3.2 DNA gel of PCR amplified gene product --- p.77 / Figure 3.3 Colony PCR of UGT1 A8-pRSetB transformed DH5 a bacteria --- p.78 / Figure 3.4 The alignment of amplified gene sequence with the rat UGT1A8 sequence on NCBI database --- p.79 / Figure 3.5 SDS-PAGE of cell lysates with different expression temperature and time duration --- p.82 / Figure 3.6 SDS-PAGE of bacterial cell lysates --- p.83 / Figure 3.7 SDS-PAGE of Ni-column eluted protein --- p.84 / Figure 3.8 Elution Profile of Gel Filtration Chromatography --- p.85 / Figure 3.9 SDS-PAGE analysis of UGT1A8 fractions from Ni-column and gel filtration column --- p.86 / Figure 3.10 Sequence Alignment of UGTs in the rat UGT1A family and 2D structure prediction of UGT1A8 --- p.88 / Figure 3.11 Circular Dichroism (CD) measurements on rat UGT1A8 --- p.89 / Figure 3.12 Western Blotting of UGT1A8 wild-type and mutant proteins --- p.91 / Table 3.1 The specific activity of wild-type and mutated proteins --- p.92 / Figure 3.13 Fluorescence spectrum of wild type and two charged-residue mutants ofUGTlA --- p.93 / Figure 3.14 Fluorescence spectrum of wild type and Trp mutants of UGT1A8 --- p.94 / Figure 3.15 Chemical denaturation of wild type and Trp-mutated UGT1A8 proteins --- p.95 / Figure 3.16 Resolved Stern-Volmer plot of UGT1A8 on acrylamide quenching --- p.96 / Figure 3.17 The 3D modeling structure of rat UGT1A8 --- p.97 / Figure 3.18 Modeling simulated the interaction between UDP-glucuronic acid and UGT1A8 --- p.98 / "Figure 3.19 Modeling simulated the interaction between UDP-glucuronic acid, 1-napthol and UGT1A8" --- p.99 / Chapter Chapter 4 --- Discussion / Chapter 1. --- Successful Expression of Rat UGT1A8 --- p.100 / Chapter 2. --- The recombinant rat UGT1A8 protein was properly folded and enzymatic functioning --- p.102 / Chapter 3. --- Purified recombinant rat UGT1A8 protein contained well-ordered structure --- p.103 / Chapter 4. --- "Relative positions of Trp38, Trp64, Trp98 and Trp208 in the protein" --- p.105 / Chapter 5. --- Contribution of Trp residues in the folding and stability of the protein --- p.106 / Chapter 6. --- Probing of substrate coupling region by mutagenesis --- p.108 / Chapter 7. --- Interaction studies of substrates and UDP-glucuronic acid with UGT1A8 by computer modeling and docking simulation --- p.109 / Chapter Chapter 5 --- Conclusion --- p.111 / Chapter Chapter 6 --- References --- p.113

Issued also as thesis - Univ. of Chicago. / Also available on the Internet.

Thesis (M. Sc.)--York University, 1999. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 82-92). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ56172.

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