Study On The Neuroprotective Mechanism Of Active Ingredients In Ginkgo Biloba Leaves

With the continuous progress of scientific technology and the constant change of market demand,the development of global chemical industry is changing,and Chemical Engineering is also gradually expanding from Process Engineering to Product Engineering.The Drug,as one of the research objects of Chemical Product Engineering(CPE),studying the mechanism of its action is beneficial to the development of CPE.Meanwhile,Traditional Chinese Medicine(TCM),as an active natural product,has been favored by people because of its good efficacy and fewer toxic side effects,and has been increasingly accepted by the international community.However,due to the “multi-components”,“multi-targets” and “multi-pathways” features of TCM,studying its molecular mechanism will face more difficulties,problems and challenges than studying the molecular mechanism of chemical drugs.Therefore,there is still a need to apply new theories,new methods and new technologies to the field.This dissertation takes the Ginkgo biloba leaves(GBLs)with clinical curative effects on many diseases as an example,to systematically study the neuroprotective mechanism of active constituents in GBLs.Firstly,using various chemoinformatics methods,a systemic pharmacology technology which integrated drug ADME screening,target prediction,network construction,pathway enrichment and drug-disease correlation analysis,was developed.Based on this technology,the potential active ingredients of GBLs were screened and their corresponding targets were identified.Subsequently,analysis of the biological function of the obtained targets revealed that the active components of GBLs are most closely related to ischemic stroke(IS)and depression in nervous system diseases(NSDs),and also found that the insomnia target,i.e.,Histamine Receptor H1(HRH1),has the highest number of molecules.Finally,based on these findings,we take the anti-insomnia,anti-IS and anti-depression effects of active ingredients in GBLs as examples,to study the neuroprotective mechanism of GBLs from both theoretical and experimental aspects.The main contents and results are as follows:(1)Study on the anti-insomnia mechanism of active ingredients in GBLs based on molecular simulation.Employing a series of novel antagonists of the insomnia target HRH1,an optimal 3D-QSAR model with good predictive ability was obtained.Combining the optimal model and molecular docking,11 potential anti-insomnia active ingredients in GBLs were screened.Through molecular dynamics simulation,the specific binding modes of these hit compounds to their targets were systematically studied.Finally,the binding free energy calculation of the ligands to receptors shows that in all simulations systems,the electrostatic and van der Waals interactions play important roles in the combination of the system.All these results reveal the neuroprotective mechanism of GBLs for treating insomnia at the molecular level,and also provide the research basis for further development of anti-insomnia molecules.(2)Further study on the anti-IS mechanism of active ingredients in GBLs based on the animal model of cerebral ischemia.Through the rat transient middle cerebral artery occlusion(tMCAO)model,the effects of ingredients of GBLs,i.e.,ginkgolide A,B,C(GA,GB,GC)and Ginkgo diterpene lactone meglumine injection(GDJ)mainly prepared from GA,GB and GC,on cerebral infarction volume,inflammatory factors and apoptotic proteins in IS rats were observed.TTC staining results show that GDJ,GA,GB and GC decrease the volume of cerebral infarction caused by tMCAO in a dose-dependent manner.Western blot demonstrates that the neuroprotective effect of GDJ on IS may be through activating the PI3K/AKT,inhibiting NF-κB signaling pathways,validating the previous theoretical hypothesis.Finally,the ELISA results depict that the main mechanism of GDJ on preventing ischemic stroke is anti-inflammatory and anti-apoptotic response.The above results reveal the neuroprotective mechanism of GBLs on ischemic stroke from the levels of whole animals,tissues and molecular proteins.(3)Based on the above research basis and animal model of depression,the antidepressant mechanism of active ingredients in GBLs was further explored.Using the chronic unpredictable mild stress(CUMS)model of rats,the neuroprotective effect of active molecule GB on depression in GBLs was observed.The results of body weight,sucrose preference test,elevated plus maze,open field test and forced swim test show that GB can significantly reverse the depression-like behavior induced by CUMS in rats.In addition,LC-MS/MS results indicate that GB can change the concentrations of monoamine neurotransmitters in hippocampus of depression model rats returned to steady state,indicating that the antidepressant mechanism of GB may be related to restoring neurotransmitter system function in rats.Moreover,western blot results show that the antidepressant effect of GB may be through activating NGF/ERK/CREB/BDNF in the hippocampus of CUMS rats,revealing the neuroprotective mechanism of GBLs from the molecular protein levels.Overall,this dissertation developed a systemic pharmacology technology using various chemoinformatics methods and applied it to the traditional botanical GBLs.Taking insomnia,IS and MDD as examples,the neuroprotective mechanism of the active ingredients in GBLs was systematically studied from different levels of whole animal,tissue and molecular proteins.All the results provide a new framework for the study of the neuroprotective mechanism of natural products,and also offer a new approach for humans to explore drug interventions for complex diseases.Hopefully,the work of this dissertation may promote the extensive research and application of active natural products,as well as promote the vigorous development of the pharmaceutical industry in the motherland for the benefit of mankind.