| Aims:The aims of this study were to (1) establish a rat model of type 2 diabetes mellitus (T2DM) with hyperglycemia, oxidative damage, insulin resistance and insulin secretion deficiency as main characteristics, (2) screen total saponins extracted from eleven traditional Chinese medicines for their activities in antioxidation, free radical scavenging, antiglycation,α-amylase inhibition and stimulation of insulin release fromβTC3 cell, (3) observe the curative effect of the optimal total saponin on oxidative damage and T2DM, and (4) isolate the chemical constituents from the optimal total saponin and examine their antidiabetic activity.Methods:1. Activity screen of total saponinsTotal saponins were prepared by the routine method and their contents were determined approximately using the vanillin-sulfuric acid colorimetry. The antioxidant activity of total saponin was evaluated by studying the inhibition of lipid peroxidation in rat liver microsomes induced by ascorbate/Fe2+, cumine hydroperoxide(CHP) or CCl4/reduced form of nicotinamide adenine dinucleotide phosphate(NADPH). The free radical scavenging activity was evaluated by studying the scavenging of 2, 2′-diphenyl-1- picrylhydrazyl(DPPH) free radical. The antiglycation activity of total saponin was evaluated byδ-gluconolactone(δ-Glu) assay, bovine serum albumin(BSA)-glucose assay and N-acetyl-glycyl-lysine methyl ester(GK)- ribose assay. Theα-amylase inhibitory activity was evaluated by detecting the maltose content. The activity of total saponin in stimulating insulin release was evaluated by detecting the amount of insulin released fromβTC3 cell.2. Reproduction of the rat model of T2DMT2DM was induced by feeding rats with antioxidant-deprived diet of high fat and high sugar and injecting them intraperitoneally with streptozocin(STZ) in low doses for four times. Insulin resistance was judged through the calculation of insulin sensitive index(ISI) and insulin secretion was observed using the perfused pancreas technique.3. Pharmacodynamical study of the optimal total saponin in T2DM ratsT2DM rats were treated for 30 days with the optimal total saponin by intragastric administration at doses of 62.5mg/kg, 125.0mg/kg and 250.0mg/kg, respectively, once per day. Plasma, liver, skeletal muscle and adipose tissue were taken and the relevant indexes of oxidative stress were examined. Pancreas was taken for pathologic evaluation.4. Isolation and activity screen of the chemical constituents from the optimal total saponinThe chemical constituents from the optimal total saponins were isolated by the methods of silica gel column chromatography and preparative high performance liquid chromatography(HPLC). Their structures were elucidated on the basis of spectrum technology and these compounds were examined for their activities in antioxidation, free radical scavenging, antiglycation and stimulation of insulin release fromβTC3 cell by using different experiment models in vitro and ex vivo.Reulsts:1. Activity screen of the total saponinsThe content determination of the total saponins showed that the extract of Aralia taibaiensis possessed higher content of saponin (45.93%, w/w) than those of the other traditional Chinese medicines. All these total saponins extracted from the eleven traditional Chinese medicines showed different pharmacological activities. Based on a comprehensive evaluation, the total saponin extracted from Aralia taibaiensis was determined as the medicine valuable for further chemical constituents isolation.2. Reproduction of the rat model of T2DMOxidative stress in rats was induced by feeding them with antioxidant-deprived diet of high fat and sugar. Several typical symptoms of oxidative stress were observed, including increased oxygen free radical, decreased antioxidation, and enhanced lipid peroxidation. As a result, insulin resistance was formed in the rats. Further, with the effect of STZ, insulin secretion deficiency and hyperglycemia were successfully induced in the rats.3. Pharmacodynamical study of the optimal total saponin in T2DM ratsThe total saponin extracted from Aralia taibaiensis showed positive effects on treating T2DM rats in terms of reducing oxidative damage to different extents at all the three dose levels. Although the total saponin extracted from Aralia taibaiensis did not reduce the blood glucose of T2DM rats to the normal level, the 250.0mg/kg dose group of T2DM rats showed significantly lowered level of blood glucose when compared with T2DM model group(P<0.05). Morphology experiment showed that the pancreas of the treated T2DM rats recovered gradually but the function of the pancreas had no significant improvement.4. Isolation and activity screen of the chemical constituents from the optimal total saponinTwelve chemical constituents were isolated from the total saponin extracted from Aralia taibaiensis. The results of bioassay indicated that TA35 had strong activities in antiglycation and stimulation of insulin release fromβTC3 cell line. Conclusions:This study systematically investigated the antidiabetic activities of the total saponins extracted from eleven traditional Chinese medicines commonly used in medical practice. Through a comprehensive evaluation, the total saponin extracted from Aralia taibaiensis proves to be a most promising compound that could be developed into new drugs for antidiabetes. The T2DM model that this study established is not able to reproduce the complexity of human T2DM, nevertheless this T2DM model can be helpful for understanding at least the role of oxidative stress in the development of T2DM. The total saponin from Aralia taibaiensis can reduce the oxidative damage and blood glucose level to some extent, but its therapeutic effect on the T2DM still needs to be investigated in the future study. TA35 isolated from Aralia taibaiensis shows strong activities in antiglycation and stimulation of insulin release fromβTC3 cell line. This study advances the knowledge of the role of oxidative stress in the development of T2DM and makes basic contributions to the screening of safe and effective drugs for T2DM. |
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