ANTIDIABETIC ACTIVITY OF THE AQUEOUS EXTRACTS OF SARCOCEPHALUS POBEGUINII (BARKS) AND NAUCLEA DIDERICHII (LEAVES AND BARKS) IN NORMAL AND STREPTOZOTOCIN INDUCED-HYPERGLYCEMIC RATS. Mbot

Mbot Elvis Jolinom 1 , * Agnaniet Huguette 1 , Ngueguim Tsofack Florence 2 , Padzys Guy Stephane 3 and ** DimoTheophile 2 . 1. Laboratory of natural substances and organometallic synthesis, University of Science and Technique of Masuku, Faculty of Science BP. 943, Franceville, Gabon. 2. Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde I, P.o. Box 812, Cameroon. 3. Department of Biology, University of Science and Technique of Masuku, Faculty of Science BP. 943, Franceville, Gabon. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Induction of diabetes Mellitus:-
The animals were acclimated during two weeks in laboratory conditions before experimental work. The healthy animals: albino Wistar rats were acclimated during two weeks in laboratory conditions before experimental work. Then they were fasted overnight for 12 hours. Diabetes mellitus was induced by subcutaneous injection of a single dose of Steptozotocin (55 mg/kg) freshly dissolved in ice citrate buffer (pH 4.5) glucometer and administered subcutaneously to fourty experimental rats. Non diabetic control rats received citrate buffer only, by the same route. Hyperglycemia was confirmed in the streptozotocin-treated rats by measuring (72 h post injection) blood glucose level withdrawn from the vein tail using a glucometer. Only rats in which hyperglycemia had been successfully induced (glucose levels above 250 mg/dL) were kept for 15 days post-injection before the beginning of the experiment to stabilize the hyperglycemic conditions. The rats with fasted blood glucose levels above 250 mg/dL, after this time of stabilisation, were considered to be diabetic and were used in the experiment (Dzeufiet et al., 2006, Kesari et al 2006Ngueguim et al, 2007).
Effects of extract on blood glucose of diabetic rats: The normal and the diabetic rats were each randomly separated into nine groups and each group consisted to five animals: Group 1 and 2 containing normal and diabetic rats respectively received distilled water (10 ml /kg). Group 3 containing diabetic rats, received Glibenclamide (5 mg/kg), representing the positive control. The diabetic others groups 4 to 9 were given 100 and 200 mg / kg of the extracts E 10 , E 14 and E 15 respectively.
Baseline fasting blood glucose levels were initially determined in all the groups. The substances were given for 14 consecutive days. Glucose levels were determined at days 7 and 14. At the end of drugs and plant extracts administration. The rats were fasted for 12 h, sacrificed by decapitation after anesthesia with diethyl ether and blood sample collected in normal tubes. From the clotted blood at room temperature, serum was collected by centrifuging at 3000 g for 10 min to determine the glucose, cholesterol, triglyceride, total protein, creatinin, transaminases, total and direct bilirubin and uric acid levels. Organs such as aorta, heart, liver and kidney were dissected out and washed in ice saline solution to remove the blood., Homogenate (20 %) of each organ was prepared using Tris-HCl buffer 50 mM (pH 7.4). Mc Even solution was used for the aorta and heart homogenates. The mixture obtained was centrifuged at 3500 g for 25 min at 4 0 C. The resulting supernatant were separated for the evaluation of some parameters of oxidative stress (superoxide dismutase, catalase activities; reduced glutathione and nitrite concentrations). Statistical analysis: Results are expressed as the mean ± SEM. Statistical differences between control and treated group were tested by one way analysis of variance (ANOVA) followed by Tukey's test using GraphPad Prism, version 5.00 (Trial). P values less than 0.05 were considered to be significant. Table 1 shows the results of blood glucose monitoring performed for five hours (5 hours). At the doses of 50 and 100 mg/kg, no diminution of blood glucose levels was observed. At dose 200 and 400 mg/kg, a significant decrease of blood sugar is observed respectively from 3and 4hours after administration of extracts until the end of experiment. Thus, 200 and 400 mg/kg are more active than others.

Effects of repeated administration of extracts on blood glucose levels on normal and STZ-induced diabetic rats:-
The variation of blood glucose levels in the experimental rats are given in table 2. After seven days of treatment, there is a significant reduction of blood glucose in some groups of diabetic rats receiving treatment. Among these groups, the extracts E 14 at the doses 100 and 200 mg / kg and E 15 at the dose 200 mg/kg have shown a fall of 78.71%, 73.22% and 65.34% of blood glucose respectively as compared to the initial value (early treatment). Whereas the Glibenclamide induced a non-significant decrease in blood glucose levels (7.67%). However after fourteen days of administration extracts and Glibenclamide, the different groups of rats have shown a significant decrease of glycaemia by 53.95%, for E 10 (100 mg/kg), 81.85%, 78.19% for E 14 (100 and 200 mg/kg), 69.16% for E 15 (200 mg/kg) and 71.31% for Gliben (5 mg/kg). During the 7 and 14 days of treatment with 3 extracts (E 10 at 100 mg/kg, E 14 at 100 and 200 mg/kg and E 15 200 mg / kg), the diabetic rats had improvement in the normalization of the blood glucose levels. Irrespective of de sampling days, E 14 (100 and 200 mg/kg) showed a better antidiabetic action, respectively by 81.85 and 78.19% than the Glibenclamide (71%). E 15 at the dose of 200 mg/kg had the comparable reduction (69.16) with Glibenclamide (71.36). Whereas, E 10 at the dose of 200 mg/kg and E 15 at dose 100 mg/kg appeared weaker (22.90 and 20.05% respectively. After one to two weeks of treatment with the E 10 (100 mg/kg), the E 14 (100 and 200 mg / kg) and the E 15 (200 mg / kg), the blood glucose levels returned to normal indicating that the hypoglycemic effect of the plant extracts, in the case of N. diderrichii (leaves), is achieved through repeated and not single administration.

Effects of the extracts on some biochemical parameters of normal an STZ-induced diabetic rats Lipid profile:-
Studies on lipid profile (Table 3) at the end of 14 th days of the treatment with the aqueous extracts were compared with that of the diabetic control group. There was no significant change in total cholesterol and HDL-cholesterol in the diabetic rats. However significant increase was observed in triglycerides concentration as compared to normal control. When different extracts were administered, a significant decrease of triglycerides was observed with E 10 at the doses of100 mg/kg (p < 0.01), 200 mg/kg (p < 0.05) and withE 15 at the dose of100 mg/kg (p < 0.05). E 14 failed to reduce triglycerides concentration at all doses. The administration of streptozotocin also provoked a significant increase in the LDL-cholesterol concentration. The extract E 15 at the doses of 100 and 200 mg/kg significantly decreased (p < 0.001) LDL-cholesterol while E 10 at the dose of 100 mg/kg and E 14 at the dose of 200 mg/kg). Thus,E 10 and E 15 were more active than E 14. Glibenclamide used in the same conditions as plant extracts significantly reduced LDL-cholesterol but failed to change other parameters. Table 4 shows the effects of different extracts on some parameters of liver and kidney functions. At the end of the experimental period, various plasmatic parameters were measured in all groups.There was a significant increase (p < 0.001) in ALT, AST activities and creatinin concentration as compared to normal control. However, the serum total proteins, total bilirubin, direct bilirubin and uric acid remain (p ˃ 0.05) unchanged when compared to normal control rats. Streptozotocin injection induced about three times the levels of transaminases in diabetic group. After administration of different treatments, significant changes were observed: ALT activity was reduced by E 10 (p < 0.01) at both doses, E14 reduced the ALT activity at doses of 100 (p < 0.05) and 200 mg/kg (p < 0.01) and E 15 at the dose of 100 (p < 0.001) and 200 mg/kg (p < 0.01). Parallel, AST activity was reduced by E 10 at both doses (p < 0.05); by E 14 at the doses of 100 (p < 0.01), 200 mg/kg (p < 0.05) and by E 15 at both doses (p < 0.01). E 10 andE 14 extracts also provoked a significant decrease (p < 0.001), in creatinin at both doses even E 15 at the dose of 100 (p < 0.05) and 200 mg/kg (p < 0.01). Total Proteins and biluribin, direct biluribin and uric acid levels remained unchanged.

Effects of different plant extracts on some parameters of oxidative stress:-
Streptozotocin administration to animals induced variations in some parameters of oxidative stress (Fig1.) depending to the organ. In SOD activity (Fig. 1A) there was a significant increasein aorta (p˂0.05) contrary to the heart, liver and kidney where SOD activity was no significant. The plant extract E 14 at the dose of 200 mg/kg administered for two weeks, provoked a significant increase in the SOD activityin the aorta while the smallest dose significantly reduced this parameter in the heart. The extract E 15 at all doses failed to reduce SOD activity in organs investigated. On other hand, diabetic rats showed a significant increase in the catalase concentration (Fig. 1B) only in the aorta. The extract E 14 and E 15 at all doses significantly reduced (p˂0.001) catalase concentration in the aorta as compared to the diabetic control. These extract also reduced catalase concentration in other organs whenever nonsignificant. There was no variation in GSH concentration in diabetic rat's organs (Fig. 1C). At the dose of 200 mg/kg, only the extract E 15 , induced a significant reduction in GSH concentration in the kidney as compared to the diabetic control. A unique dose of streptozotocin (55 mg/kg) was associated with an increase in nitrites (p˃0.05) in aorta, heart and liver (Fig. 1D). The increase levels of nitrites were attenuated by two weeks administration of the extract E 14 at the dose of 200 mg/kg and E 15 by both doses. Glibenclamide (5 mg/kg) used as reference drug in the same conditions as the extracts, failed to reduced GSH and nitrites concentration in all organs but significantly reduced SOD activity and catalase concentration in the aorta.

Discussion:-
The activity of three plant extracts used in Gabonese traditional medicine to manage diabetes was investigated in the present study. The results indicated that Sarcocephalus pobeguinii aqueous bark (E 10 ) extract, Nauclea diderrichii aqueous leaf (E 14 ) and bark (E 15 ) extracts reduced the glucose levels in normal rats .E 14 showed the lowest activity on blood sugar in normoglycemic rats. At equal dose (200 mg/kg), E 15 extract was more potent than E 10 extract because of the ability of the former to significantly reduce blood glucose levels 3hours post-dosing. However the effect of Glibenclamide used as reference drug was better than that of the extracts.The results also revealed that E 15 (200 mg / kg) was more active than E 15 (400 mg / kg), we can therefore say that the hypoglycemic effect of this extract is optimal at moderate doses. Moreover, these results allowed not only to know the more potent extract with hypoglycemic effect but, also to select the doses to use in the evaluation of anti-diabetic activity on streptozotocininduced diabetic rats. Agnaniet et al., 2016 have shown that Sarcocephalus pobeguinii (barks) Nauclea. Diderrichii (leaves and barks) could be used to manage diabetes state due to their capacity to inhibit α-glucosidase activity. In this study the administration of this different extracts for two weeks to streptozotocin induced-diabetic rats have shown a decrease in blood glucose levels towards normal value at the dose of 100mg/kg with E 14 extract one week post-administration. Parallel, E 15 and E 10 did not bring the glycaemia towards normal value even the treatment was continuing up to two weeks. These results suggest that, E 14 extract is more potent that E 10 and E 15 in diabetic state. Since in acute treatment E 15 was the most potent extract and E 14 was the most potent extract in antidiabetic activity we can therefore say that, there is no correlation between hypoglycemic effect and antidiabetic activity. This allows us to suppose that the mode of action of the extracts is not the same.  Pari and Saravanan, 2002).The administration of plant extracts improved the lipid profile of diabetic rats probably due to their hypoglycemic effect. ALT and AST are key metabolic enzyme makers for liver function.The increase in these two parameters indicates hepatotoxicity which is related to glucose induce-oxidative stress (Poitout and Robertson, 2002). Infact, hyperglycemia induced autoxidation of glucose which in turn generates reative oxygen species, attacks cells and compromised membrane function thus caused leakage of these enzymes into the blood stream (Lery et al., 1999).The improvement of these enzymes by the extracts could be attributed to the presence in these extracts of triterpenes, flavonoids and phenols which are known to have an antioxidant activities (Montilla et al., 2003;Hennebele et al., 2004;Rodrigues et al, 2005). STZ induceddiabetic rats provoked a creatinemia suggesting the increase in muscular activity which was reversed by the plant extracts at all doses. In this study, permanent hyperglycemia provokes inbalance of antioxidant defence system characterized by an increase in SOD, catalase, GSH and NO mainly in the aorta. This increase attests the present of free radicals. However, the administration of the plant extract reduced these different parameters showing that E 14 extract was the best extract to reduce oxidative stress.