| Recently, it witnesses an increase in prevalence of diabetes mellitus year on year all over the world. Diabetes is defined as a group of metabolic disease that is characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic high-level blood glucose could induce the glucose toxity which shows the positive relationship with the diabetic complications. Therefor, strict glycemic control is the primary requirement to treat biabetes mellitus. It is very significant to find convenient, economic and safe means to prevent and treat the diabetes mellitus.Psidium guajava Linn. leaves (PGL) showed the hypoglycemic activity, but its blood glucose-lowering components and mechanisms had not been defined completely. There are abundant resources of Psidium guajava Linn. in our country, planted artificially or deserted to be wild in such province or autonomous region as Hainan, Guangdong, Guangxi, Taiwan, Fujian, Yunnan, Guizhou, Sichuan and so on. The hypoglycemic activity of wild Psidium guajava Linn. in Panzhihua in Sichuan province has not been studied as yet.In the present study, the hypoglycemic activity and mechanisms of wild Psidium guajava Linn. in Panzhihua were studied to provide the data and evidence for exploiting the resource of wild PGL and clarifying the hypoglycemic mechanisms of PGL.The present sudy is composed of the following three parts: 1 Preparation of the PGL extracts and comparison of their hypoglycemic activitiesObjectives To prepare the water-soluble, 65% alcohol and 95% alcohol-soluble extract from wild Psidium guajava Linn. leaves (PGL), compare their hypoglycemic activities and screen out the blood glucose-lowering extract with less toxity. Methods PGL, after being picked up in Panzhihua, were cleaned, dried and shredded at once. Shredded leaves were submersed in distill water, 65% and 95% edible alcohol respectively. The water-soluble extract (WE), 65% alcohol-soluble extract (AE65) and 95% alcohol-soluble extract (AE95), with the ester-soluble substances removed from the alcohol-soluble extracts, were gained when the filtrate were dried and grained after being condensed. Diabetes was induced in 18 hours fasted male Kunming mice given twice intraperitoneal injection of streptozotocin (STZ) at a dose of 80 mg/kg body weight. Blood glucose concentration was measured after oral administration (gastrogavage) of WE, AE65 and AE95, with Glipizide as the positive control for blood glocose-lowering activity. The effects of the three extracts on blood glucose and growth of diabetic mice were compared. Results The gain rate of the water-soluble extract (WE), 65% alcohol-soluble extact (AE65) and 95% alcohol-soluble extract (AE95) is 14.5%, 22.0% and 11.0% respectively. WE is brown and prone to be agglomerated resulting from the deliquescence, while AE65 and AE95 are light brown and incompact. At the dose of 1.25g/kg, WE, AE65 and AE95 significantly decreased the blood glucose level in STZ-induced diabetic mice fasted for 3 hours, by 36.3%, 33.5% and 31.3% respectively. The blood glucose-lowering rate was significantly lower induced by AE95 than by Glipizide (43.2%). Although the blood glucose-lowering rates was lower induced by WE and AE65 than by Glipizide (P <0.05), there were no significant difference among their blood glucose-lowering rates. Furthermore, among three extracts, WE showed little influence on the body weight of diabetic mice. For WE, it showed no significant difference of blood glucose-lowering rate between low dose(1.25g/kg) and medial dose(2.5g/kg) and similar effect on the mice body weight of diabetic model control group (DM), Glipizide group, low-dose WE group and medial-dose WE group (P>0.05) . The high-dose WE showed similar blood glucose-lowering rate (45.4%) to Glipizide(P>0.05), higher than the medial-dose WE, but it effected the growth of diabetic mice significantly than the low and medial-dose WE. Conclusion WE, AE65 and AE95 possessed hypoglycemic activities with different degree. WE showed hypoglycemic activity and higher safety than AE65 and AE95.2 Study on the blood glucose-lowering activities of the PGL water-soluble extract2.1 Effects of the PGL water-soluble extract on alpha-glucosidase activity in the small intestineObjective To investigate the inhibitory effect of the PGL water-soluble extract (WE) on alpha-glucosidase activity in the small intestine. Methods The diabetes of male Kunming mice was induced by intraperitoneal injection of STZ (see part 1). The small intestinal mucosa of mice was scraped and made into the homogenates for the preparation of alpha-glucosidase. In vitro, the homogenates were incubated with sucrose and maltose respectively. The formed glucose represented the activities of alpha-glucosidase. The alpha-glucosidase activities of normal mice and diabetic mice were determined respetively, and further, the Lineweaver-Burk plot was applied to determine alpha-glucosidase activity-inhibiting type. Results It was showed WE significantly inhibited, in the dose-dependent manner, the activities of alpha-glucosidase from the small intestinal mucosa of diabetic mice. The 50% inhibiting concentration (IC50) to sucrase and maltase was 0.1 g/L and 3.0 g/L respectively. The mixed inhibition type was showed to be the competitive and non-competitive inhibition. Conclusion The PGL water-soluble extract possesses the potential inhibitory effect on the alpha-glucosidase activity from the small intestinal mucosa of diabetic mice. It may be one of the hypoglycemic mechanisms to inhibit the intestinal assimilation of carbohydrates.2.2 Effects of the PGL water-soluble extract on hepatic glucose metablism Objectives To study the effects of the PGL water-soluble extract (WE) on the hepatic glucose metablism of diabetic mice and its mechanism at the molecular biology level. Methods Male Kunming mice were used in this study. The mice with diabetes were induced by the intraperitoneal injection of STZ (see part 1). The diabetic mice were divided into diabetic mice model (DM) group and WE-administrated diabetic mice group with the gastrogavage at the dose of 2.50 g/kg, while the normal mice were set as the control. After the gastrogavage for 4 weeks, the oral glucose tolerance test (OGTT) and the glycogenesis was tested by the gastrogavage of glucose and by the intraperitoneal injection of alanine after 12 hours fast. After the mice were killed by the cervical spine dislocation, the hepatic glycogen was determined, and the hepatic mRNAs contents of glucokinase (GK), glucose-6-phophatase (G6pc) and glycogen synthase-2 (Gys-2) were determined by the real-time fluorescence-quantified PCR (Real-time PCR). Results Compared with the normal mice in the Ccontrol group, the 3 hours and 12 hours fasted blood glucose level (21.29 and 12.76 mmol/L respectively), the AUC of OGTT (48.11 h·mmol·L-1), the glycogenesis rate 1h and 2h after the injection of alanine (36.5% and 59.1% respectively) were showed significant increase in the diabetic mice of in DM group, while the hepatic glycogen content (3.01 mg/kg) significantly decreased. In the WE-treated diabetic mice, the 3 hours and 12 hours fasted blood glucose level (14.09 and 8.04 mmol/L respectively), the AUC of OGTT (34.62 h·mmol·L-1), the glycogenesis rate lh and 2h after the injection of alanine (23.7% and 38.5% respectively) were showed significant decrease while the hepatic glycogen content (3.01 mg/kg) significantly increased, than those of the diabetic mice model. In the diabetic mice in DM group, the hepatic glucokinase (GK) mRNA content was 1.3% of the normal mice, the hepatic glucose-6-phophatase (G6pc) mRNA content 3.0 fold that of the normal mice, while hepatic glycogen synthase-2 (Gys-2) mRNA showed no significant change. In the WE-treated diabetic mice, their hepatic GK mRNA content increased to 6 fold that of the diabetic mice in DM group, and the G6pc mRNA content showed a decreasing trend, Gys-2 mRNA a increasing trend.Conclusion The PGL water-soluble extract may increase the absorption and mebablism of glucose in various tissues by increasing glycogen synthesis, decreasing glycogenesis and improving insulin secretion by adjusting the expression of the key enzyme gene of hepatic glucose metablism.2.3 Anti-oxidative activities and protecting effects of the PGL water-soluble extract on the pancreas isletObjectives To find the evidence of |3 cell-protecting effect of the PGL water-soluble extract (WE) by investigating the anti-oxdative activities of WE and observing the histological change of pancrea islet of diabetic mice and WE-treated diabetic mice. Methods In vitro, the scavenging rate to hydroxyl radicals and inhibiting rate to lipid peroxidation were analyzed. The contents of SOD, CAT and GSH in liver were determined after the mice were killed (see part 2.2). Their pancreas were fixed in the 4% neutral formalin solution (pH7.2) at once, and then dehydrated, embedded in paraffin and cut into tissue slices. The slices were stained with Hematoxlin and Eosin (HE) and immunostained with anti-insulin respectively. The histological morphology was observed through the light microscope and the insulin expression were quantitatively analyzed with the image grey. Results WE showed the effects on scavenging hydroxyl radicals and inhibiting lipid peroxidation in the dose-dependent manner. Compared with the normal mice in the Ccontrol group, the contents of hepatic SOD and GSH (163.46 U/mg protein and 27.96 mg/g protein respectively) decreased significantly and CAT content (389.4 U/g protein) showed a decreasing trend(P >0.05)in the diabetic mice of DM group. In the WE-treated diabetic mice, the contents of hepatic GSH (32.79 mg/g protein) increased significantly, the hepatic SOD and CAT content (199.23 and 411.7 U/g protein respectively) showed a increasing trend (P>0.05) .There were significant histological lesions in the diabetic mice: the number, density and volume of the pancreas islets were significantly decreased in the diabetic mice. And in the pancreas islets of the diabetic mice, the number of endocrine cells and insulin-expressing beta-cells were decreased, the endocrine cells suffered metamorphosis, and the volume and intensity of insulin expression significantly decreased. The WE-treated diabetic mice showed less histological lesions than the diabetic mice in DM group: the number, density and volume of the pancreas islets were significantly increased, the number of insulin-expressing beta-cells inceased in the pancreas islets, and intensity of insulin expression significantly increased. Conclusion The PGL water-soluble extract possesses the potential antioxidative activities. It may be one of the various blood glucose-lowering mechanisms, by protecting pancreas beta-cells and alleviating their lesions, to improve their function of insulin synthesis and secretion. The PGL water-soluble extrac may play the important role through its anti-oxidative effect in this process.3 Primary analysis of the potential hypoglycemic components in PGLObjective To study the potential blood glucose-lowering components in the extracts from PGL. Methods The contents of total flavonoids in the extracts were determined by ultraviolet spectrophotometry, and the components of the total flavonoids were primarily identified by high performance liquid chromatography (HPLC) and ultraviolet-visible absorption Spectrometry (UV). The contents of Mg, Cr and V were determined by ICP-AES method. Results The total flavonoids in PGL may consist of the flavonoid with quecertin as the aglycone. The content of total flavonoids in WE, AE65 and AE95 was 3.28, 30.71 and 55.98 g/kg respevtively. There were abundant quantities of Mg, Cr and V. In the PGL water-soluble extract, the content of Mg, Cr and V in WE was 9259.08, 11.41 and 49.54 mg/kg respectively, all at high level in the three extracts. Conclusion The PGL extracts contain abundant quantities of the flananoids and minerals such as Mg, Cr, V, and so on, each of which may play the corresponding role in the blood glucose-lowering mechanisms.In general, the wild Psidium guajava Linn. leaves in Panzhihua possesses blood glucose-lowering activity. There are abundant flavonoids and some trace elements such as Mg, Cr, V and so on. The hypoglycemic mechanisms may include inhibiting intestinal assimilation of carbohydrates, adjusting hepatic glucose metabolism and relative gene expression of key enzymes, protecting the functions of pancreas islet, and so on. The flavonoids and some trace elements such as Mg, Cr, V may be the functional components. It shoud be further studied, for giving the data and evidences to the development of Psidium guajava Linn. leaves, to identify the hypoglycemic ingredients and clarify other mechanisms of the blood glucose-lowering activities. |
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