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.

This study was prompted by the inconsistent reports and apparent controversies that exist in the biomedical literature on the responses of diabetic bladder strips to cholinergic nerve stimulation or exogenous administration of muscarinic agonists, especially acetylcholine (ACh), in vitro. In the present study, acetylcholine-induced contractions of urinary bladders isolated from normoglycaemic (normal) and streptozotocin-treated, diabetic Wistar rats were examined under physiological conditions. Mechanical contractile changes of the isolated urinary bladders of STZ-treated, diabetic rats in response to bath-applied acetylcholine were compared with those obtained from isolated urinary bladders of normal, age-matched, control rats. Results obtained show that urinary bladders from diabetic rats consistently weighed more, and were always more spontaneously active after mounting, than those of the age-matched normal, control rats. ft A Acetylcholine (ACh, 10" -10" M) provoked concentration-related, atropine-sensitive contractions of the isolated urinary bladders of both diabetic and age-matched normal, control rats. However, acetylcholine always induced more powerful and greater contractions of the diabetic bladders compared with bladders from the age-matched normal, control rats. The enhanced contractile responses of the diabetic bladder strips to bath-applied ACh were detected soon after induction of diabetes, and the magnitude and/or intensity of the enhanced contractile responses to ACh continued to increase as the diabetic state of the animals progressed. Although this preliminary study could not establish the mechanism of the increased contractile responsiveness of the diabetic bladders to the muscarinic agonist (ACh) used, the results tend to suggest that alterations in diabetic urinary bladder synaptosomal, vesicle-bound neurotransmitter (ACh) concentrations and the compensatory increase in the density of muscarinic M3-receptor population in diabetic bladders are two of the most attractive plausible mechanisms of the increased diabetic bladder responsiveness to bath-applied acetylcholine. / Thesis (M.Sc.Pharm.)-University of KwaZulu-Natal, Westville, 2006.