Analysis Of Components Of Lotus Leaf Extract And Its Effects On Glucose And Lipid Metabolism In Type 2 Diabetic Animal Models

Background:Diabetes mellitus(DM)is a metabolic disease characterized by chronic hyperglycemia.The worldwide epidemic of DM and its complications is a longlasting catastrophe involving all levels of society in every country and becoming an important public health issue.In our country,over 90% of DM cases are type 2 diabetes mellitus(T2DM),which poses a major global health threat.Clinically,it is found that there are certain limitations in the regulation of blood glucose with western medicine alone,while the treatment with traditional Chinese medicine is based on the whole-body situation and has the advantages of protecting islet function,improving insulin sensitivity and alleviating symptoms.In the past decades,the combination of Chinese and Western drugs in the treatment of T2 DM has drawn a lot of attention.Lotus leaf with hypolipidemic effect is commonly used as herbal tea or herbal compound preparations to reduce lipid levels and control body weight.Metformin,as the first-line drug in the treatment of T2 DM,has good efficacy and safety as monotherapy or combination therapy.Whether lotus leaf combined with metformin can better improve lipid and glucose metabolism of T2 DM needs further study.Objective:(1)Identification of main chemical components from Lotus leaf extract(LLE);(2)LLE monotherapy and its combination with metformin on lipid and glucose metabolism in T2 DM animal models.Methods:(1)The HPLC-TripleTOF method was used to analyze and screen the main chemical components of LLE;(2)Effects of LLE on glucose metabolism in spontaneous T2 DM model db/db mice.Six wild type mice were used as control group(CON).Eighteen db/db mice were randomly divided into 3 groups(n = 6 for each group):db/db mice treated with vehicle(Model group,MOD,saline),db/db mice treated with metformin(Metformin group,MET,200 mg/kg)and db/db mice treated with LLE(LLE group,LLE,1 g/kg).The intervention time was eight weeks.Daily diet and water intake,weekly body weight and fasting blood glucose(FBG)were observed and recorded.Intraperitoneal insulin tolerance test(IPITT)was performed during the 8th week of intervention,and the area under the glycemic curve(AUC)of IPITT was evaluated.After that,mice in each group were sacrificed.Blood samples were exsanguinated via the abdominal aorta.The whole liver,pancreas and kidney tissues were collected.Part of the tissues were frozen in the refrigerator under-80 ℃,and part of those tissues were fixed with paraformaldehyde;(3)LLE monotherapy and LLE in combination with metformin on lipid and glucose metabolism in T2 DM rats.T2 DM rats were induced by high fat diet and streptozotocin(STZ)multiple injection(25 mg/kg)in male SD rats.Seven healthy male SD rats was used as control group(CON).28 T2 DM rats were randomly divided into 4 groups(n = 7 for each group): T2 DM rats treated with vehicle(Model group,MOD,saline),T2 DM rats treated with metformin(Metformin group,MET,300 mg/kg),T2 DM rats treated with LLE(LLE group,LLE,2 g/kg).T2 DM rats treated with LLE and metformin(Combination group,LLE+MET,LLE,2 g/kg + MET,200 mg/kg).The intervention time was four weeks.Daily diet and water intake,weekly body weight and FBG were observed and recorded.Four weeks later,rats were administrated with intraperitoneal glucose tolerance test(IPGTT)and IPITT assay,and the area under the glycemic curve was assessed respectively.After that,rats in each group were sacrificed.Blood samples were collected via the abdominal aorta and serum levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL)and high-density lipoprotein(HDL)were measured.The whole liver,pancreas and kidney tissues were collected.Part of the tissues were frozen in the refrigerator under-80 ℃,and part of those tissues were fixed with paraformaldehyde.Hematoxylin-ensin(HE)staining was used for pancreas histological analysis.The levels of insulin and phosphatidylinositol 3-kinases(PI3K)were detected by immunohistochemistry.Results:(1)A total of 23 chemical components in lotus leaves were identified by high resolution mass spectrometry,among which 13 alkaloids were identified including hexanylline,N-norheverine,O-norheverine,etc.,and 10 flavonoids were identified including quercetin-3-O-glucuronide,isoquercetin,kaoneferol-3-O-glucoside,etc.(2)Both LLE and MET could reduce food and water intake of db/db mice,but did not change their body weight.There was no significant effect of LLE and MET on FBG as well as the blood glucose at each time point of IPITT tests and AUC values in db/db mice.(3)Both LLE and LLE in combination with MET could reduce food and water intake of T2 DM rats,but did not change their body weight.Within 2 weeks of intervention,FBG of T2 DM rats with drug intervention was decreased,especially in MET and MET+LLE.What’s more,the FBG in LLE+MET group was lower than that in MET group(p<0.05).With the extension of intervention,FBG of T2 DM rats in MET group and LLW+MET group increased rapidly at the 3rd and 4th week,and there was no significant difference with MOD.LLE,MET and their combination had no significant effects on blood glucose at different time points of IPGTT test and IPITT test as well as AUC values.Compared with MOD,the TG levels in T2 DM rat plasma in MET,LLE and LLE+MET groups were significantly decreased(p<0.05),but there was no significant effect on TC,LDL and HDL levels.Compared with the model group,the volume of islets in LLE,MET and LLE+MET group was increased,and the boundary was more regular,but the size of islet cells was still heterogeneous,and there was no improvement in vacuolar degeneration.Compared with the model group,the expression of insulin in pancreatic islets of rats in LLE,MET and LLE+MET group was higher than that in the model group,but there was no significant change in the expression of PI3 K.Conclusion:(1)In this study,23 chemical constituents including 13 alkaloids and10 flavonoids were identified in the extracts of lotus leaves.(2)LLE in combination with MET can control the blood glucose level of diabetic rats in the early stage,and the effect of LLE+MET is better than MET alone.(3)Both LLE and LLE+MET could improve lipid metabolism,especially hypertriglyceridemia in T2 DM rats.(4)Both LLE and LLE+MET could improve the pathological morphology of pancreas and promote insulin secretion in T2DM rats.