| Chapter one, Literature StudyIn Section 1, A synthetical review was given to the domestic and foreign present research of diabetes pathogenesis, diagnostic methods and the progress of treatment research in order to understanding of diabetes, and provide a scientific basis for clinical treatment.In Section 2, To extract prescriptions for the treatment of wasting-thirst in the prescriptions of traditional Chinese medicine database (from the prescriptions of traditional Chinese medicine dictionary) by using computer retrieval, a database, included each single herb in the prescription was built through excel software, drugs pairs and formula composition rules were analyzed with association rules mining method.In Section 3, With deepening the basic research and industrial development of Mori Folium, the bioactive component groups and their action mechanisms of Mori Folium used for the prevention and treatment of diabetes and diabetic complication continue to be revealed. In this paper, based on the previous research, we summarize and analyze the understanding of domestic and foreign scholars about the pathogenesis of diabetes and the active component groups and their action mechanisms of Mori Folium for the prevention and treatment of diabetes and diabetic complication in recent years in order to provide the guidance for the further study and the reference for the development and utilization of Mori Folium resource.Chapter two, The preparation of mulberry leaf extractsIn Section 1, The optimal parameters of the extraction for flavonoids and phenolic acids were: ethanol concentration 70%, ratio of material to liquid was 1:40, extraction time is 45 minutes,2 times of extraction, respectively. Then merged two filtrate, decompression to concentrate without alcohol flavor. Select AB-8 types of macroporous resin for purification. The best technique for purification of the flavonoids with macroporous resin of AB-8 was:the sampling flow rate was controlled at 2 BV·h1, the total flavonoids was eluted by 3 BV and 70% of ethanol in rate of 2 BV·h-1, collect the eluent, enrichment, freeze drying. Flavonoids content was 0.1%, the content of phenolic acids were 0.4%, its content was 5.3% after purification.In Section 2, Mulberry leaf weighing 50 g, and the optimal parameters of the extraction were solid-liquid ratio of 1:12, reflux extraction of 1 h,80% alcohol, supernatant fluid concentration on the cation exchange resin, with 0.5 mmol·L-1 of ammonia in 1.5 mL·min-1 flow rate of elution, collect pH 7-9.4, pH 9.4-10.5, pH 10.5-13 respectively, enrichment, freeze drying purification of alkaloids component. DNJ mainly concentrated in pH 7-9.4 and pH 9.4-10.5, the DNJ content of 19.9% and 39.4%, respectively.In Section 3, Mulberry leaf weighing 50 g, and the optimal parameters of the extraction were solid-liquid ratio of 1:12, reflux extraction of 1 h,80% alcohol sink for 24 h. Using Sevage and AB-8 macroporous resin to purify for polysaccharide, the optimal parameters of the purification of mulberry leaf polysaccharide components were eluting with 0.1 mg·mL-1 concentration of NaCl in 2 mL·min-1 elution rate and collect the eluent. freeze drying.The polysaccharide content increased from 0.69% to 18.9%.Chapter three, Study on the interaction of multi-components in Morus alba leaves extraction in vitro based on a-glucosidase inhibitory activityUsing oxidase colorimetric method and using sucrose as the substrate to measure the inhibitory effects of mulberry leaf extracts and their compatibility on a-glucosidase; Interaction effects of two drugs used in compatibility were measured by an isobole method, Chou-Talalay combination index analysis and Isobolographic analysis to evaluate the inhibitory effects of mulberry leaf extracts and their compatibility on a-glucosidase activity in vitro. The results showed that all the extracts and their combinations in different compatibility had significant or very significant inhibitory effects on a-glucosidase, and the inhibition increases with the increasing concentration. Compatibility of the flavonoids and alkaloid extracts had synergism effect and its activity was better than both single-use of extracts, which was the same as compatibility of the alkaloid and polysaccharide extracts.Chapter four, Study on the action mechanisms of Morus alba leaves multicomponent for the treatment of diabetes based on the network pharmacologyUsing the method of network pharmacology to evaluate eight active ingredients of mulberry leaves for the treatment of diabetes in order to explore the relationship of ingredients-multiple targets-pathways. The results showed that eight kinds of active components in mulberry leaves were concerned with forty-seven targets,63 pathway.These pathways were related to nerve-endocrine-immune, including insulin, MAPK, VEGF, Wnt, Jak-STAT multiple signaling pathways which was associated with diabetes.Chapter five, The metabolism research of multi-component in mulberry leavesA sensitive and rapid UPLC-MS/MS method was developed for simultaneous quantification of the components in rats plasma after oral administration of mulberry leaves alkaloids, flavonoids extract. After validation, this method was successfully applied to a pharmacokinetic study.The results showed that quercetin and kaempferol reached peak after 0.333 h, illustrate the rat after oral mulberry leaf flavonoid constituents absorbed and distributed quickly. About 4 h after taking medicine, both of them reach the peak concentration for the second time, suggests that they stay for a long time, there may be related to a enterohepatic circulation. DNJ and Fagomine in gastrointestinal tract can be quickly absorbed into the blood, the blood drug concentration reach peak after 0.667 h, that then rapid distribution after into the systemic circulation of rats.Chapter six, Study on the action mechanisms of Morus alba leaves extraction for the treatment of diabetes based on plasma and urine metabolomicsIn order to evaluate the effect and mechanism of the mulberry leaf alkaloid, flavones, and polysaccharide intervention on diabetes, the overall metabolite profiling characteristics for the plasma and urine of diabetic mouse was performed by using an ultra-performance liquid chromatography/electrospray-tandem mass spectrometry (UPLC-ESI-MS). The 17 potential biomarkers in diabetic mice plasma and 21 potential biomarkers in urine were found based on the data of MS/MS characteristics obtained from the UPLC-TOF/MS/MS analysis. The biomarkers in plasma mainly involved valine, leucine and isoleucine, tryptophan metabolism and glycerol phospholipid metabolism. The biomarkers in urine were related to phenylalanine, tyrosine and tryptophan biosynthesis and metabolism of phenylalanine, nicotine and nicotinic acid salt metabolism, histidine metabolism and sphingolipid metabolism pathways. The principal component analysis showed that the normal group and model group were obviously distinguished and implied the metabolic disturbance was happened in diabetic mice. The extracts of mulberry leaf flavonoids, polysaccharide, alkaloid had exhibited the effects of callback function for diabetic mice.Chapter seven, Research on the action mechanisms of Morus alba leaves extraction for the treatment of diabetes and its complicationsIn Section 1, Using GLUTag cells as the research object to observe the effect of AGEs in early cell damage, apoptosis, then study the change of the corresponding factor after mulberry leaf alkaloids, flavonoids and polysaccharide extract and monomer DNJ, quercetin intervention in order to explore the action mechanism. According to the results, AGEs, which combine with RAGE, can activate caspase-3,-9, induce intestinal cells apoptosis through p53/Bax pathways and activate the NF-κB factor expression to release of TNF-a, IL-6, IL-1, lead to inflammatory lesions of the intestinal cells. After mulberry leaves multicomponent intervention, various factor return to the normal group, thus improving inflammation damage of intestinal cells and preventing cell apoptosis.In Section 2, Using HepG2 cells as the research object, by comparing the insulin concentration and action time, finally chose the best method to establish HepG2 insulin resistance model, then observed the impact of mulberry leaves on IR-HepG2 cells for absorbing glucose. HepG2 cells induced by different concentration of insulin, action time, insulin resistance model was set up with 3 ug·mL-1 insulin to induce 48 h. Mulberry leaves alkaloids, flavones, polysaccharide extract and monomer DNJ, quercetin can obviously increase glucose consumption of insulin resistance cell, after treating with the extracts and monomers, the absorption of glucose have no significant difference between normal levels, which suggested that the multicomponent of mulberry leaf can play a role in the occurrence of diabetes development process.In Section 3, Using 3T3-L1 cells as the research object, observe cell differentiation, glucose uptake, fat cells factor mRNA expression after inducing with sugar, then study the change of the corresponding factor after mulberry leaf alkaloids, flavonoids and polysaccharide extract and monomer DNJ, quercetin intervention in order to explore the action mechanism. The results showed that mulberry leaves alkaloids, flavones, polysaccharide extract and monomer DNJ, quercetin in low concentration can promote the proliferation of 3T3-L1 fat cells and promote the expression of adipocyte differentiation key factor PPARy, C/EBPa, SREBP-1, thus promote the differentiation of fat cells and glucose uptake, transport, improve adipose tissue insulin sensitivity and improve insulin resistance.In Section 4, Using HK-2 cells as the research object, observe the ROS, NADPH oxidase, the ERK1/2 level after inducing with sugar, then study the change of the corresponding factor after mulberry leaf alkaloids, flavonoids and polysaccharide extract and monomer DNJ, quercetin intervention in order to explore the action mechanism. The results showed that high sugar can increase the expression of NOX1, NOX2, NOX4 in the cells, and induced EMT. After mulberry leaves multicomponent intervention, each factor return to the normal group, indicates mulberry leaves can reduce the production of ROS by regulating NADPH oxidase, and prevent HK-2 cell transdifferentiation. |
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