Hypoglycemic effects in alloxan-induced diabetic rats of the 2 phenolic extract enriching ellagic acid , kaempferol and their 3 derivatives from Mongolian oak cups 4

Our previous in vitro reports showed that crude extract prepared with 50% ethanol 12 (ethanol crude extract, ECE) from Mongolian oak cups possessed excellent antioxidant capacities as 13 well as inhibitory activities against α-glucosidase, α-amylase and protein glycation caused by its 14 enrichment in phenolics, including mainly ellagic acid, kaempferol and their derivatives. 15 Nevertheless, few in vivo studies on antidiabetic activities of these phenolics were conducted. The 16 present study investigated hypoglycemic effects with normal and diabetic rats being administrated 17 orally without or with ECE at 200 and 800 mg/kg for 15 days. In normal rats, no significant 18 differences were exhibited after ECE administration in body weight, fasting blood glucose level, 19 levels of chelesterol, triglyceride, LDL and AST in serum, organ indexes, and levels of GSH and 20 MDA in organs. In diabetic rats, the fasting blood glucose level, indexes of heart and liver, and 21 levels of chelesterol and triglyceride in serum and MDA in heart tissue were significantly 22 decreased. Moreover, HDL levels in serum and SOD activities in the four organs of diabetic rats 23 were significantly improved after ECE administration at 800 mg/kg. Thus, in addition to inhibiting 24 α-glucosidase, α-amylase and protein glycation reported previously, oak cups might contain novel 25 dietary phytonutrients in preventing abnormal changes in blood glucose and lipid profile and 26 attenuating oxidant stress in vivo. The results also implied that it is ellagic acid, kaempferol and 27 their derivatives enriched in ECE that might play vital roles in managing type 1 as well as type 2 28 diabetes. 29


Introduction
. Effects of ECE on body weights and fasting blood glucose levels in normal and alloxan-induced N+E8 rats compared to those in the NC rats, which would cause negative influences upon the treated rats thus need to be investigated further.   both normal and diabetic rats without or with ECE treatments. The AST level extremely significantly 167 increased after the allaxon induction compared to that in NC rats, and showed dose-dependent 168 decreases both in N+E2 and N+E8 rats compared to NC rats and in D+E2 and D+E8 rats compared to 169 DC rats, although no significant differences were observed within the three normal and the four 170 diabetic groups of rats. The results demonstrated that ECE exerted no significant influences on AST 171 level in both normal and diabetic rats, while ECE administration did decrease, although with no 172 significant differences, AST levels of both normal and diabetic rats to some extent during the course 173 of experiment, which likely revealed that ECE might further improve liver conditions in normal rats 174 and alleviate liver damages caused in diabetic rats. Similarly, probiotic Lactobacillus paraplantarum 175 BGCG11 has also showed the ability to attenuate liver damage by reducing AST level in diabetic rats 176 [32]. Therefore, as a byproduct of oak industry, oak cups might hold certain potential to be 177 developed into therapeutic agents for treatment of liver disease associated with diabetes.

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were presented as mean ± SD with five independent experiments (n=5). # and ## indicate significant (P < 0.05) 181 and extremely significant (P < 0.01) between the normal controls (NC) and all other groups, respectively. Not 182 that no significant difference was observed among the three normal and the four diabetic groups of rats. N,

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It had been reported that hyperglycemia could increase oxidative stress and lipid peroxidation 206 and further resulted in tissue damage [6,33]. Therefore, it is possible to deduce from these results 207 that the oxidative stress and lipid peroxidation increased thus caused damage in tissues of diabetic 208 rats after alloxan-induction and ECE could reduce the damage of organs, especially in heart and radical scavenger of cells [7]. It is one of the indicators of antioxidant defence system in vivo, and a Table 2 (DC vs NC), increases of GSH levels were observed in all the four organs after alloxan 225 stimulation, but only with those in heart and spleen being significant. The reason for these increases might be the experiment period being too short [35], that is, 7 days might be not long enough for 227 the decrease of GSH levels to occurr in tissues of diabetic rats, especially in heart and spleen. For 228 more details of the data in Table 2, it is obvious that no significant difference was found in all the 229 four organs among NC, N+E2 and N+E8 rats, indiacting that ECE exerted no significant influence 230 on GSH levels in normal rats. In liver, significant reduction from both of NC and DC was observed 231 only in D+E8 rats. Furthermore, ECE administration to the diabetic rats led to reductions of GSH 232 levels in three organs, with kidney as an exception at the lower dose of 200 mg/kg (i.e., D+E2; 233 1.43±0.36 mol/g prot) which was little higher than that of DC (1.38±0.33 mol/g prot). Moreover, 234 reductions in hearts of both D+E2 and D+E8 rats were extremely significant from that of DC rats, 235 and those in heart and liver of D+E8 rats were even significant from that of NC rats. These results       There are considerable evidences that oxidative stresses would be increased in diabebes [4].  Table 4, with a significant increase of the SOD activity in heart with ECE treatment 286 at the higher dosage (N+E8) as an only exception, no significant changes were observed in all the 287 four organs of all the other normal groups of rats compared to that of NC rats. SOD activities of the 288 DC rats were decreased significantly in heart and spleen and extremely significantly in liver and 289 kidney compared to those of NC rats, and ECE administration to DC rats at both the two dosages 290 (i.e., D+E2 and D+E8) significantly recovered SOD activities to a similar extent in all the four organs, 291 with three organs (heart, kidney and spleen) being restored back to the levels of the NC rats and 292 one organ (liver) even became extremely significantly higher than that of the NC rats. These results

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suggest that ECE could also ameliorate the alloxan-induced type 1 diabetes via improving the 294 reduced SOD activities in DC rats, especially its SOD activity in liver. Furthermore, degradation of 295 antioxidant enzymes such as SOD, catalase and glutathione peroxidase, and their associated 296 co-factors are known to be essential for decrease of pancreatic insulin secretion [41]. Therefore, the 297 recoved or even enhanced (e.g, in liver) SOD activity by ECE might contribute to increase of

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The oak cups were air-dried until equilibrium humidity, ground and screened through a 1-mm 326 mesh, then stored in darkness at room temperature for further use. polyphagia, polydipsia, polyuria, and body weight loss.