Studies On Active Constituents Recognition Approaches And The Network Pharmacology Of Traditional Chinese Medicine For Type 2 Diabetes

Diabetes mellitus is one of the major threats to human health, and more than 90% of the diabetic patients are affected with type 2 diabetes mellitus (T2D). The majority of clinical medicines for T2D are single target drugs, which could not suppress the process of this multifactorial complex disease. The drug tolerance and unwanted side effects also could not be ignored in the long-term use. Multi-target drugs and formula, e.g. TCM, show more distinctive superiorities in the treatment of complex diseases, such as T2D, than the single target drugs, which makes them become the hot point in the development of next generation drug. Some CPMs exhibited sound clinical effect in the treatment of T2D, which is called ’Xiaoke’ disease in TCM. However, the anti-diabetic substances and the therapeutic mechanisms of most CPMs for T2D are still unclear.Active constituents of TCM for T2D should be recognized from multiple levels and aspects, as they are multi-target drugs. Network pharmacology offers a network view to understand the relationship between drugs and targets, which provides a new perspective to explain the therapeutic mechanism of TCM.The objective of this thesis is to elucidate the active constituents of the anti-diabetic TCM and the mechanisms of its therapeutic effects. The techniques of TCM chemistry, TCM analysis, TCM pharmacology, bioinformatics, network pharmacology are involved. The efforts were made to establish the methods for discovering active constituents of mulberry leaves and Xiaokean formula in the aspects of glucose absorption reduction, glucose and lipid metabolism regulation, improvement in insulin resistance and antioxidation. It is hoped to elucidate the active constituents of the anti-diabetic TCM from multiple levels and aspects, and illuminate the mechanisms of its integrated regulation in the view of network pharmacology to offer the methodology for the secondary development of CPMs. The main contents and academic contributions of this thesis are summarized as follows:1. The constituents of Xiaokean formula and mulberry leaves were characterized by high performance-liquid chromatography (HPLC) coupled with mass spectrometry. Totally,40 and 12 constituents were identified or tentatively identified, respectively.2. By bioassay-guided screening and isolation, a-glucosidase and tyrosinase inhibitors were identified from mulberry leaves. A series of compounds exerted a-glucosidase and tyrosinase inhibition were discovered from Morus alba. Three new compounds were identified.3. An ultrafiltration HPLC-DAD-MS approach was developed for the rapid screening and identification of ligands for tyrosinase. Twelve compounds with tyrosinase binding activity were found in mulberry leaf extracts. Particularly, two compounds were identified as new tyrosinase inhibitors.4. A DPPH-HPLC-MS approach was developed for the rapid screening and identification of antioxidant compounds from Xiaokean formula.5. By ultrafiltration HPLC-MS approach, a-glucosidase inhibitors from Xiaokean formula were rapidly identified.6. The anti-diabetic components were identified from Xiaokean formula at the cellular level. An insulin-resistant HepG2 cell model was developed, and components alleviating the cell insulin-resistant states were identified from Xiaokean formula. A model of 3T3-L1 cell differentiation was developed, and components reducing the intracellular total cholesterol, intracellular triglyceride and inhibiting cell differentiation were identified.7. The pharmacodynamic studies of Xiaokean formula were performed at multiple levels. Xiaokean formula could exert the activities of antioxidation, a-glucosidase inhibition, improvement of insulin-resistant, reducing the intracellular total cholesterol and triglyceride of 3T3-L1 cell, and inhibiting 3T3-L1 cell differentiation. It also reduced non-fasting blood glucose, fasting blood glucose, serum triglyceride, increased serum high density lipoprotein cholesterol, and improved the glucose tolerance of KKAy mice.8. A comprehensive and up-to-date knowledgebase for T2D, i.e. T2D@ZJU, was developed, which contains three levels of T2D associated data retrieved from pathway databases, PPI databases and literature, respectively. T2D@ZJU will offer a better understanding of T2D for basic and clinical researchers and hereby facilitate the T2D-related studies of network pharmacology and multi-target therapeutics.9. Xiaokean formula was investigated in the view of network pharmacology. Studies of gene expression were performed to reveal the potential anti-diabetic mechanisms and regulation of the T2D network by Xiaokean formula.