Modulatory and antidiabetic effects of vindoline and Catharanthus roseus in type 2 diabetes mellitus induced male Wistar rats and in RIN-5F cell line

Abstract

Diabetes mellitus (DM) is a group of metabolic disorders characterised by persistent high blood glucose levels together with abnormal metabolism of macromolecules. If the hyperglycemia is not controlled, adverse metabolic changes could occur leading to the progressive development of severe complications. Formation of reactive oxygen/nitrogen species and inflammatory responses are principal mechanisms that have been implicated in the development of hyperglycemia-induced tissue damage. The commercially available drugs utilised in the treatment of diabetes have been linked to detrimental side effects hence the need to discover alternative medicines especially from medicinal plants. Catharanthus roseus is both a medicinal and ornamental plant that is traditionally used to treat various diseases. It has been reported to possess antidiabetic, anticancer, antimicrobial and antioxidant properties. The plant has been shown to possess more than 100 monotepernoid indole alkaloids which were linked to the plants’ antihyperglycemic and antioxidant effects. Therefore, this study was carried out to investigate the effect of vindoline; a bioactive compound derived from C. roseus against type 2 diabetes–induced complications. The study also investigated the effects of Catharanthus roseus extracts in RIN-5F cell line. The study was carried out in two parts: viz in vitro and the in vivo assessments. The in vitro study initially investigated the polyphenolic content and antioxidant activities of vindoline and the 3 extracts (methanolic, aqueous and the dichloromethane) of C.roseus. The assays used to evaluate the antioxidant capacity of the extracts include oxygen radical absorbance capacity (ORAC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibitory assay. Among the evaluated extracts, the methanolic extract demonstrated both high total polyphenolic content and antioxidant capacity. The HLPC analysis of the extracts was performed and showed highest concentrations of vindoline in the dichloromethane extract and the aqueous extract exhibited the least. The antioxidant activities of vindoline were determined and compared to a known antioxidant, ascorbic acid. Vindoline revealed stronger ORAC activity than ascorbic acid however the ferric reducing antioxidant power did not show any significant differences (p < 0.05). Insulin secretion studies were performed in a β-cell insulinoma cell line- RIN-5F exposed to different concentrations of glucose (high, low and in the absence of glucose). The studies were carried out to compare the β-cell stimulatory effect of vindoline to the extracts. After performing cytotoxic experiments, concentrations that resulted in about 80% cell viability were used to determine the insulin secretory effects. In cells that exposed to glucotoxicity (50 mM glucose), vindoline showed the highest β-cell stimulatory effect (p < 0.05) when compared to the untreated controls and to the cells that were treated with the methanolic extract. In cells that were exposed to a low glucose concentration, vindoline additionally showed significant β-cell stimulatory effect at p < 0.05 when compared to the aqueous and the methanolic extracts. Thereafter, the intracellular reactive oxygen species assay (ROSA) was performed in glucotoxicity-induced cells after treatment with vindoline and the respective extracts. The results were compared to the untreated control: vindoline, methanolic and the dichloromethane extracts indicated significant reduction in ROS generation (p < 0.05). Further measurement of the release of TNF-α, a pro-inflammatory cytokine in the cells following treatment, the results were not significant among the groups at p < 0.05. The carbohydrate enzymes inhibitory activity of vindoline and extracts of C.roseus (50, 25, 12.5 and 6.125 mg/ml) were measured. The alpha glucosidase inhibitory activities of the extracts at 50 mg/ml resulted in < 30% enzyme inhibition with no significant differences among the groups at p < 0.05. At lower concentrations, the dichloromethane extract exhibited significantly lower inhibitory activities when compared to the methanolic and the aqueous extract (p < 0.05). The alpha amylase inhibitory activity of the methanolic extract was significantly increased at all concentrations; recording the highest enzyme inhibition of approximately 40% (p < 0.5). However, the dichloromethane extract did not show any enzyme inhibitory activity. The enzyme inhibitory activity of vindoline was compared to acarbose-a known standard drug, for both enzymes; vindoline did not show appreciable enzyme inhibition when compared to acarbose (p < 0.05). In vivo studies were performed in a type 2 diabetes (T2DM) rat model in which T2DM was induced in 6 weeks old male Wistar rats by having them drink 10% fructose solution ad libitum for 14 days followed by a single intraperitoneal injection of streptozotocin (STZ 40 mg/kg) in freshly prepared 0.1 M citrate buffer (pH 4.5). Animals were randomly divided into six groups (n=8) and received daily treatments for 6 weeks with the vehicle, vindoline (20 mg/kg) or glibenclamide (5 mg/kg) via oral gavage. The effects of the treatments on blood glucose, insulin, body weight, organ weight, serum biochemical parameters, oxidative status, inflammatory markers and tissue histology were assessed in diabetic and non-diabetic rats. Administration of vindoline significantly (p < 0.05) reduced the fasting blood glucose in diabetic rats by 15% and significantly increased serum insulin levels when compared to the diabetic controls. Vindoline and glibenclamide significantly (p < 0.05) reduced the levels of circulating hepatic enzymes in T2DM; the results were significant when compared to the diabetic controls. Treatment with vindoline significantly improved the hepatic antioxidant status as indicated by increased ORAC, superoxide dismutase and catalase activities, indicative of the protective effect of vindoline in diabetes-induced hepatic injury. Assessment of the levels of pro-inflammatory cytokines in the hepatic tissue indicated remarkable reduction of TNF-ɑ by (-41%) and IL-6 (-28%) in diabetic rats treated with vindoline when compared to the diabetic controls (p < 0.05). The serum lipid profile showed marked increases in the levels of serum lipids (triglycerides, low density lipoproteins, total cholesterol and very low density lipoproteins) in diabetic controls when compared to all treatment groups (p < 0.05). Therefore, vindoline and glibenclamide showed possible protective effects against diabetes-induced cardiovascular disease. Kidney function assessment revealed increased levels of urea and creatinine in the diabetic control group. Vindoline and glibenclamide significantly reduced the urea and creatinine levels in diabetic rats. Vindoline additionally improved the FRAP in diabetic hearts. The SOD activity and ORAC were increased while lipid peroxidation was reduced in the kidneys of diabetic rats treated with vindoline when compared to the diabetic control (p < 0.05). Histopathological assessment in diabetic rats showed severe damage of the liver, kidney and pancreas. Treatment of diabetic rats with vindoline restored the structure of these organs which was indicated by minimum structural changes. The expression of pro-apoptotic marker caspase 9 in response to glucose stress was significantly higher in the diabetic control group when compared to all the treatment groups. Treatment with vindoline showed remarkable reduction of caspase 9 expression in the diabetic rats. In conclusion, persistent high blood glucose levels resulted in free radical induced tissue damage in the type 2 diabetes rat model. Vindoline demonstrated protective effects against diabetes induced hepatic, cardiac, pancreatic and nephritic injuries. In addition, vindoline improved insulin secretion in both in vitro and in vivo setups hence the findings suggest that vindoline could be an important agent that can be considered in the treatment and management of diabetes and diabetic complications.