Study On The Antidiabetic Pharmacological Mechanism And Active Components Of Mulberry Leaf

ObjectTo track the hypoglycemic effective parts of mulberry leaf by pharmacodynamics screening,to research the molecular mechanisms of prevention and treatment of type 2 diabetes based onmitochondrial regulation,to study the active components of mulberry leaf,to provide a new me-thod in the treatment of type 2 diabetes and new targets,to provide theoretical basis and practical basis for the development and utilization of mulberry leaves.Method1.The rapid hyperglycemia rat model was established by intraperitoneal injection of high dose glucose,8 Chinese medicinal herbs which contain high levels of melatonin,including lotus leaf,white mulberry root-bark,fructus schisandrae,mulberry leaf,radix scutellariae,turmeric,uncaria,herba violae were screened for their hypoglycemic effects;the water extract and the al-cohol extract of mulberry leaves,which has the best anti-hyperglycemic effect,were screened;70%mulberry leaf ethanol extract with different polarity solvent in turn?petroleum ether,dichlorome-thane,ethyl acetate?extraction,divided into the petroleum ether part,methylene chloride part,ethyl acetate part and water part were compared and screened for the hypoglycemic effects in the fast hyperglycemia rat model,and the glucose tolerance test was used as efficacy evaluation me-thod.2.The rats were divided into Control normal group?Control?,Model group?Model?,high dose of mulberry leaf-ethyl acetate parts group?SY-H?,high dose of mulberry leaf-ethyl acetate parts group?SY-M?and high dose of mulberry leaf-ethyl acetate parts group?SY-L?.Controlgroup and Model group were administrated the same amount of normal saline by gastric gavage,GMR group was administrated the Glimepiride 0.4mg·kg^-1,SY-H,SY-M,SY-L group were admi-nistrated the mulberry leaf-ethyl acetate parts 100mg·kg^-1,50mg·kg^-1 and 25 mg·kg^-1.The volume was 1ml·200g^-1,the experiment lasted for 5 weeks.The water consumption and food intake were recorded daily at regular intervals,and the fasting blood glucose was measured at d7,d14,d21 and d28.At d21,the intraperitoneal glucose tolerance test was performed.At d28,the intraperitoneal insulin tolerance test was performed.After 35 days experiment,all the animals were killed,whole blood was collected and saved at-80?after centrifugation and package.The liver,pancreas,hippocampus,hypothalamus,pituitary,adrenal and adipose tissue were obtained for determina-tion.3.Based on the above mentioned pharmacology experiment,the ATP content,ATPase activi-ty and protein content of hippocampus,liver,muscle tissue in rats were determined.The real-time fluorescent quantitative PCR?Real-time PCR?method was used for detecting the GK mRNA,PEPCK mRNA,G-6-P mRNA,GSK-3?mRNA expression in rat liver,assaying the ERRa,PGC-1?,Drp1,Mfn2,SIRT1 mRNA expression in rat liver,hippocampus,muscle tissues.The Western Blotting method was used for detecting the relative expression of PGC-1?,Drp1,Mfn2proteins in rat liver,hippocampus,muscle tissue.4.The active components of ethyl acetate portion of 70%alcohol extract of mulberry leaves were separated and purified by the medium pressure column chromatography combined with re-verse C18 column chromatography,and tested by HPLC chromatography.The chemical structures were identified by MS,NMR(¹H-NMR,¹³C-NMR),DEPT spectroscopy.Results1.The hypoglycemic effect among lotus leaf,white mulberry root-bark,fructus schisandrae,mulberry leaf,radix scutellariae,turmeric,uncaria and herba violae were compared and the result showed that,compared with normal control group,model group and the other treatment groupsignificantly increased the blood sugar levels at 30 min and the AUC?P<0.01?.In comparison with the model group,the mulberry leaf significantly reduced the blood glucose value by 9.84%?P<0.05?after glucose bolus 30 min.The result demonstrated that anti-hyperglycemic effect of mulberry leaf is one of the best.The alcohol extract of mulberry leaf lowered the glucose contents significantly by 11.05%?P<0.05?at 30min,and decreased the AUC by 8.4%?P<0.05?.The re-sult demonstrated that the glucose-lowering effect of mulberry alcohol extract is better.The an-ti-hyperglycemic effect study of mulberry leaf different parts showed that,the hyperglycemia was decreased markedly by 11.05%?P<0.05?at 30min in ethyl acetate fraction administrated rats.The result demonstrated that the glucose-lowering effect of ethyl acetate fraction is better.2.The ethyl acetate fraction,which could significantly improve glucose tolerance and im-prove insulin resistance,has better glycemic effect than the other fractions.From the first week to the experiment end,compared with normal control group,food intake and water consumption in model groups were significantly increased?P<0.01?,the body weight of T2DM rats tend to de-cline,the levels of TG and LDL-C in model group were significantly higher than normal group?P<0.01?,HDL-C level was significantly reduced by 28.52%?P<0.05?.Insulin levels and HOMA-IR index were significantly increased in type 2 diabetes rats,while insulin sensitivity was decreased.GMR decreased the higher water consumption,higher food intake and hyperglycemia improved the weight loose,the dyslipidemia and insulin resistance,protect the liver and pancreas.After 3 weeks administration,mulberry leaves treated groups decreased the food intake by 1.49%,9.63%?P<0.05?,13.84%?P<0.01?,and decreased water concumption by 1.48%,9.77%?P<0.05?and 13.77%?P<0.01?respectively in low dose,middle dose and high dose group.From 3week to the experiment end,compared with model group,SY-L,SY-M and SY-H group reduced the food intake and water consumption remarkable.Compared with the model group,SY-M and SY-H treatment decreased the FBG by 43.26%?P<0.05?and 48.21%?P<0.05?in the 35th day.The results of IPGTT test showed that the blood glucose levels of rats in the model group were significantly higher than that of normal control group?P<0.01?at different time points of 0,30,60 and 120 min.The AUC of T2DM rats was lowered by 16.33%,24.40%?P<0.01?and 29.21%?P<0.01?respectively after SY-L,SY-M,SY-H treatment.SY-H group has a tendency to reduce TC,TG and LDL-C and increase HDL-C.3.The results of study on hypoglycemic mechanism of mulberry leaf effective parts showed that,the ATP content in type 2 diabetic rats were significantly lower than that of normal rats?P<0.01?,GMR group and different dose of SY groups increased ATP content?P<0.05?significantly.The results of the RT-PCR showed that the PGC-1?,Mfn2,SIRT1,ERR?and SRC mRNA ex-pression were significantly decreased?P<0.01?,while the Drp1 mRNA expression was signifi-cantly increased?P<0.01?compared with normal control rats.After 5 weeks administration of SY and GMR,the PGC-1?,Mfn2,SIRT1,ERR?and SRC mRNA expression were significantly in-creased?P<0.01?,while the Drp1 mRNA expression was significantly decreased?P<0.01?compared with normal control rats.WB protein expression level was the similar with the RT-PCR level,the PGC-1?and Mfn2 protein expression was significantly decreased?P<0.01?,while the Drp1 protein expression was significantly increased?P<0.01?compared with normal control rats.GMR and SY treatment significantly increased the level of the PGC-1?,Mfn2 protein expression and decreased the Drp1 protein expression significantly?P<0.05?.4.The main active compounds in mulberry leaves ethyl acetate parts were isolated and puri-fied rapidly by medium pressure column chromatography technology,after EI-MS,¹H-NMR,¹³C-NMR spectrum analysis,Quercetin-3-O-?-glucopyranoside and Kaempferil-3-?-D-Glucopyra--noside were identified.Though the other compounds were limited by their quantities and impure to identify,these compounds were primarily identified flavonoids.Conclusion1.Mulberry leaf showed anti-hyperglycemic effect among the 8 Chinese traditional Chinese drugs with high melatonin concentration in their fresh medicinal materials.The ethyl acetate of mulberry leaf alcohol extract is the active part have the hypoglycemic activity.2.The ethyl acetate fraction of Mulberry leaf improved the glucose intolerance and insulin resistance in type 2 diabetes rats significantly.3.The ATP content was significantly reduced in T2DM rat liver,demonstrated the impaired mitochondrial function.Meanwhile,molecular study showed that SY improved the mitochondrial dysfunction in T2DM rats with the mitochondrial dysfunction and abnormal mitochondria fusion and fission.4.The method of separation and purification of active components in the ethyl acetate frac-tion from mulberry leaf by medium pressure column chromatography was performed,which is simple and practicable.The ethyl acetate fraction is one of the active hypoglycemic part of Mul-berry leaves and mainly composed by flavonoids.