Syzygium aromaticum-derived triterpenes modulate intestinal glucose handling in streptozotocin-induced diabetic rats.
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Date
2014
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Abstract
Polyphagia in diabetes mellitus ascribed to elevated plasma ghrelin concentrations is associated
with prolonged postprandial hyperglycaemia due to increased activities of intestinal carbohydrate
hydrolyzing enzymes and glucose transporters. Postprandial hyperglycaemia is a major risk
factor in the development of diabetic complications, and as such, should be managed to prevent
chronic vascular complications. Previous studies in our laboratory have shown that Syzygium
aromaticum-derived oleanolic acid (OA) and maslinic acid (MA) use various mechanisms to
lower blood glucose concentrations in experimental diabetes. The effects of these triterpenes,
however, on intestinal glucose handling remain unknown. Accordingly, this study was designed
to investigate the effects of these triterpenes on intestinal glucose handling in STZ-induced
diabetic rats.
Materials and methods
OA and MA were extracted from Syzygium aromaticum cloves using a previously validated
protocol. Briefly, S. aromaticum-derived OA and MA were sequentially extracted with
dichloromethane and ethyl acetate to obtain ethyl acetate solubles which contained mixtures of
OA/ursolic acid (UA) and methyl maslinate/methyl corosolate. These solubles were purified by
silica gel 60 column chromatography with hexane: ethyl acetate solvent systems to produce OA
and MA. The structures of these triterpenes were confirmed by using 1H and 13C Nuclear
Magnetic Resonance (NMR) spectroscopy and were comparable with those previously reported
in literature. The in vitro studies investigated the inhibitory effects of OA and MA against
enzymes such as α-amylase, α-glucosidase and sucrase. Additionally, the effects of various
concentrations of OA and MA (0.82 - 6.56 mmol/L) on intestinal glucose transport were
investigated using the everted intestinal sacs protocol. The in vivo studies investigated the effects
of OA and MA on intestinal carbohydrate handling in separate groups of non-diabetic and STZdiabetic
male Sprague Dawley rats. These studies were subdivided into oral glucose tolerance
(OGT) responses which were carried out over two hours following loading with various
carbohydrates as well as sub-chronic studies that were carried out over 5-weeks where the rats
were kept on standard rat chow. OGT responses were monitored in separate groups of nondiabetic
and STZ-induced diabetic animals the rats treated with OA and MA (80 mg/kg, p.o.).
The rats were loaded with monosaccharides, disaccharides and polysaccharides after an 18-hour
fast. The sub-chronic studies investigated the effects of the triterpenes on blood glucose
concentrations over 5-weeks in groups of non-diabetic and STZ-induced diabetic male Sprague-
Dawley rats. In those animals in which the effects of OA/MA were investigated, the rats were
administered with OA/MA (80 mg/kg, p.o.) twice daily. Blood glucose, body weights as well as
food and water intake were assessed every third day for the duration of the experimental period.
At the end of the experimental period, the rats were killed and blood was collected for plasma
insulin and ghrelin measurements. Furthermore, mid portions of the small intestine were snap
frozen in liquid nitrogen and stored in a BioUltra freezer at -70 °C for Western blot analysis of
glucose transporters, carbohydrate hydrolyzing enzymes and ghrelin expression. Additionally,
the effects of OA and MA on intestinal oxidative stress were evaluated through malondealhyde
(MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) measurements.Results
The in vitro studies revealed that OA and MA possess inhibitory effects on the activity of α-
amylase, α-glucosidase and sucrase comparable with those of the standard drug acarbose. In
addition, OA and MA significantly (p<0.05) inhibited intestinal glucose transport in the everted
intestinal sacs in a dose-independent manner. The OGT studies showed that OA and MA had no
significant effects on blood glucose concentrations in non-diabetic rats loaded with the various
carbohydrates by comparison with the non-diabetic control. However, the triterpene-treated STZdiabetic
rats loaded with the various carbohydrate showed significant (p<0.05) reductions in
blood glucose concentrations by comparison with untreated STZ-diabetic rats. OA and MA
progressively reduced blood glucose concentration as well as food and water intake over the 5-
week study in STZ-induced diabetic rats by comparison with untreated STZ-diabetic rats.
Treatment with OA and MA had no effects on plasma insulin concentration in STZ-induced
diabetic rats. However, these triterpenes significantly (p<0.05) reduced plasma ghrelin
concentrations by comparison with untreated STZ-induced diabetic rats. Furthermore, rats
treated with OA and MA showed significant (p<0.05) decreases in ghrelin, SGLT1, GLUT2, α-
amylase and α-glucosidase expression in the gastrointestinal tract by comparison with untreated
STZ-diabetic rats. This was accompanied by improvements in their intestinal antioxidant status
as there were significant (p<0.05) reductions in MDA concentrations with significant (p<0.05)
increases in SOD and GPx by comparison with the STZ-diabetic control. Additionally, OA and
MA-treated rats showed significant (p<0.05) increases in intestinal glycogen concentrations with
concomitant significant (p<0.05) increases in the intestinal expression of glycogen synthase by
comparison with untreated STZ-diabetic animals.
Discussion
The results of the present study indicate that the blood glucose lowering effects of OA and MA
in STZ -induced diabetic rats are mediated, in part, via modulating postprandial hyperglycaemia.
These findings suggest that this is achieved through the ghrelin-mediated reduction in food
intake leading to decreased expression of intestinal carbohydrate hydrolyzing enzymes as well as
intestinal glucose transporters. This was followed by significant improvements in the antioxidant
status in the rats suggesting that these triterpenes could, by preventing chronic postprandial
hyperglycaemia, prevent the onset of the development of diabetic complications. The results of
this study are of considerable importance as they suggest another mechanism for the anti-diabetic
properties of the triterpenes and further explain the role of the gastrointestinal tract in the
management of diabetes mellitus.
Conclusion
The results of the present study suggest that the S. aromaticum-derived triterpenes possess antidiabetic
properties that arise, in part, through the modulation of intestinal glucose handling.
Description
Ph. D. University of KwaZulu-Natal, Durban 2014.
Keywords
Clove tree., Diabetes--Complications., Intestines--Infections., Medicinal plants., Streptozotocin., Theses--Human physiology.