| Objective: To study the acute toxicity of total flavonoids from Melastoma dodecandrum Lour.(MDF)administration to Kunming mice.Establish type 1 diabetic mouse model and type 2 diabetic rat model to investigate anti-diabetic activity of the MDF.Use UHPLC/QTOF-MS-based metabolomics reveals the therapeutic mechanism of MDE in T2 DM rats.Methods:(1)Acute toxicity study of MDF on mice: 40 Kunming mice(half male and half female mice)was randomly divided into the blank male group,the blank female group,the male administration group,and the female administration group(n=10).The mice in administration group were orally administered twice within 24 hours with the maximal gavage volume(0.4 m L/10 g)and the maximum concentration(140 mg/m L)of the MDF.The blank group were given an equal volume of distilled water.Observed the general conditions of the mice in each groups(including body weight,feed intake,water intake).After 14 days,all mice was sacrificed after blood extraction.The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood creatinine(Scr),and urea nitrogen(BUN)inthe serum were measured.(2)The effect and mechanism of MDF on type 1 diabetic mice(T1DM):70 male Kunming mice were randomly divided into the blank group(n=10)and the model group(n=60).The mice in the model group were fasted for10 hours and intraperitoneally injected with 1% streptozotocin solution(150mg/kg).After 72 hours,the fasting blood glucose(FBG)of mice were measured.If the FBG >11.1 mmol/L,the establishment of models was successful.T1 DM mice were randomly divided into the diabetes group,the MDF high-dose group(1.2 g/kg),the MDF medium-dose group(0.8 g/kg),the MDF low-dose group(0.53 g/kg)and the metformin group(0.6 g/kg),10 rats in each group.The general condition and FBG change of the mice were recorded during the administration.After 21 days,the mice in each group were sacrificed after blood extraction.The serum insulin,high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),total cholesterol(TC),triglycerides(TG),malonaldehyde(MDA),superoxide dismutase(SOD),and catalase(CAT),nitric oxide(NO),BUN,Scr levels were measured.Calculated each organ index and made HE stained sections of pancreas in each group of mice.(3)UHPLC/QTOF-MS-based metabolomics reveal the effect of MDF in type 2 diabetic rats(T2DM): 60 Sprague-Dawley rats were randomly divided into the blank group(n=10)and the model group(n=50).The modelrats were fed a high-fat diet for 6 consecutive weeks,followed by intraperitoneal injection of streptozotocin(STZ)(30 mg/kg)to induce T2 DM.Diabetic rats were randomly divided into diabetes group,the MDF high-dose group(0.6 g/kg),the MDF medium-dose group(0.45 g/kg),the MDF low-dose group(0.34 g/kg)and the metformin group(0.25 g/kg),6rats in each group.The general condition and FBG change of the rats were recorded during the administration.After 35 days,the rats in each group were sacrificed after blood extraction.The insulin,HDL-C,LDL-C,very low density lipoprotein cholesterol(VLDL-C),TC,TG,MDA,SOD,CAT,NO,glutathione peroxidase(GSH-Px),ALT,AST,BUN,and Scr levels were measured.Calculated each organ index and made HE stained sections of pancreas in each group of rats.Serum samples were evaluated via ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry(UHPLC/QTOF-MS),followed by principal components analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).A metabolic pathway network was constructed through a database to clarify the regulatory mechanism of MDF on T2 DM metabolic disorders.Results:(1)Acute toxicity study of MDF on mice: The mice in the administration group did not die after administration of the MDF.There was no significant difference in body weight,water intake,feed intake,organindex,ALT,AST,BUN,and Scr levels of the mice in the administration group compared with the blank group(P >0.05).The maximum dosage of the MDF to kunming mice was 11.2 g/kg(equivalent to 33.6 g/kg of crude drug),which was 107 times the dosage of clinical adults.(2)The effect and mechanism of MDF on T1DM: At 7 days after administration,there was no significant difference in body weight between mice in each administration group and model group(P >0.05).Water consumption and feed intake of the high-dose group were significantly reduced(P <0.05).At 14 days after administration,the body weight of each administration group was lower than that in the model group(P <0.01).The water consumption of mice in each administration group was significantly reduced(P <0.05),and the feed intake of mice in the high-dose group and metformin group was significantly lower than that in the model group(P<0.05).At 21 days after administration,There was a significant difference in body weight between the middle-dose group and the low-dose group compared with the model group(P <0.05).The water consumption and food intake of the mice in each administration group were lower than those in the model group(P <0.05 or P <0.01).At 1 day of administration,there was no significant difference in the FBG of the mice in each administration group compared with the model group(P >0.05).At 10 days after administration,the FBG of the high-dose group,the middle-dose group,and metformingroup were significantly reduced(P <0.05 or P <0.01).At 20 days after administration,the FBG of the mice in each administration group was significantly lower than that of the model group(P <0.05 or P <0.01).In the oral glucose tolerance experiment,the blood glucose of each administration group was significantly lower than that of the model group within 0-2 h(P<0.05 or P <0.01);the area under the oral glucose tolerance curve(AUC)of the mice in each administration group were significantly reduced(P <0.05 or P <0.01).In the serum biochemical test,the insulin level of the model group was significantly lower than that of the blank group(P <0.01).There was no significant difference in serum insulin level in the administration group compared with the model group(P >0.05).The TG level of the high-dose in mice was significantly reduced(P <0.05).The HDL-C level of the high-dose groups and the middle-dose groups were significantly increased(P <0.05).The LDL-C level of the high-dose groups and metformin groups were significantly reduced(P <0.05).The SOD level of the high-dose groups and the middle-dose groups were significantly increased(P <0.05).The MDA content in each administration group was significantly reduced(P <0.05 or P <0.01).The CAT level of high-dose group,the medium-dose group and the metformin group were significantly increased(P <0.05 or P <0.01).The level of NO in each administration group had no significant change(P >0.05).The Scr content in the high-dosegroup,the middle-dose group and the low-dose group was significantly increased(P <0.05 or P <0.01).The level of BUN in the high-dose group,the middle-dose group and the metformin group was significantly increased(P <0.05 or P <0.01).After 20 days of administration,there was no significant difference in urine protein content between the each administration groups and the model group(P> 0.05).showed that the islets of mice in each administration groups showed vacuoles due to streptozotocin damage,but the lesions were milder than the model group.(3)UHPLC/QTOF-MS-based metabolomics reveal the effect of MDF in T2DM: After 7 days of administration,there was no significant difference in body weight between the administration groups and the model group(P >0.05).After 14 days of administration,the body weight of the metformin group significantly increased(P <0.05).After 21 days of administration,the body weight of the high-dose group,the medium-dose group and the metformin group were significantly increased(P <0.05 or P<0.01).After 28 days of administration,the body weight of the each administration group was significantly higher than that in the model group(P <0.05 or P <0.01).Before administration,the FBG of the rats in each administration group was not significantly different from that of the model group(P >0.05).After 14 days of administration,the FBG in the middle-dose group and metformin group were significantly reduced(P<0.05);After 21 and 35 days of administration,the FBG of rats in each administration group was significantly lower than that in the model group(P<0.05 or P <0.01).In the oral glucose tolerance test,the blood glucose of each administration group was significantly lower than that of the model group at 0-2h(P <0.05 or P <0.01).The AUC of each administration group was significantly reduced(P <0.01).In the serum biochemical test,the insulin level of the model group was significantly higher than that of the blank group(P <0.01).The insulin level was significantly reduced(P <0.05 or P <0.01).Compared with the model group,the levels of TG,TC,LDL-C and VLDL-C in the each administration group were significantly reduced,and the levels of HDL-C were significantly increased(P <0.05 or P <0.01);The SOD,GSH-Px,and CAT levels in the serum of each administration group were significantly increased(P <0.01),and the MDA level were significantly reduced(P <0.01).The level of NO in the high-dose group and the middle-dose group were significantly reduced(P <0.05).The BUN,ALT and AST levels of each administration group were significantly reduced(P<0.05 or P <0.01).Pathological observations showed that the degree of pancreatic islet lesions in each administration group was less than that in the model group.The 17 identified potential biomarkers were including taurine,nicotinic acid,cholic acid,phosphopyruvate,hippuric acid,arachidonic acid,tyrosine,phenylalanine,and glucoaldehyde Acid,PGE2,carnitine,phosphatidylcholine(PC),phosphatidylethanolamine(PE),and phosphatidylinositol(PI).The pathways were including phenylalanine,tyrosine,and tryptophan biosynthetic pathways,glycerophospholipid metabolism pathways,arachidonic acid metabolism pathways,taurine and taurine metabolism pathways,bile acid biosynthesis pathways,and niacin metabolisms.Conclusions: The maximum dose of MDF is 11.2 g/kg,and no target toxicity has been found.The MDF can reduce blood glucose,improve dyslipidemia and oxidative stress disorders,alleviate liver and kidney function damage and pancreatic pathological damage caused by the development of diabetes in T1 DM mice and T2 DM rats.The MDF possesses substantial antidiabetic activity.Metabolomics studies show that phenylalanine,tyrosine,and tryptophan biosynthetic pathways,glycerophospholipid metabolic pathways,arachidonic acid metabolic pathways,taurine and taurine metabolic pathways,and bile acid biosynthesis pathway and the niacin and nicotinamide pathway may be the main pathways for the MDF to exert anti-diabetic pharmacological activities. |
PDF Full Text Request