Studies On Effective Constituents And Mechanism Of Lignans In S.chinensis For Anti-alzheimer’s Disease

Lignans in S.chinensis are the main active components that play a role in neuroprotective effects and against cognitive function damage.However,studies on the pharmacodynamic components and therapeutic mechanism of lignans in S.chinensis are far from systematic and complete.The research on the metabolism of Schisandra lignans in vivo generally focused on the pharmacokinetics and identification of metabolites in normal rats.Till now,it has not been explored the metabolism and pharmacokinetics of lignans in Alzheimer’s disease(AD)rats.Hence,modern techniques could be applied to clarify the medicinal components and targets of lignans in S.chinensis for treating AD.It is meaningful to reveal the therapeutic mechanism of lignans in S.chinensis for treating AD.In this paper,the lignans in S.chinensis were isolated,purified and identified.In order to get comprehensively insight on lignans in S.chinensis against AD,a systematic plasma metabolomics study,in-vivo ingredient analysis,target-network pharmacology strategy and pharmacokinetics were carried out,which provided powerful experimental technical support for the development of new drugs.We look forward to roundly and deeply illuminate the pharmacodynamic material basis and mechanism of lignans in S.chinensis for treating AD.This work also provided a new research strategy for investigating the potential multi-compound,multi-target and multi-channel modes of Chinese herbal medicine.The research contents mainly include the following points.Research contents mainly include the following points.1.Separation,purification and identification of lignans from S.chinensisExtraction process of homogenate extraction and traditional solvent extraction was optimized based on orthogonal experiments.Extracting solution was further purified by macroporous resin.Best technology of extraction and purification was decided based on lignans extraction efficiency,yield and content.The results showed that the extraction technology was flash extraction,extraction solvent was 80 % ethyl alcohol,solid-liquid ratio was 1:19,extraction voltage was 100 V and time was 100 s.Then a mass spectral database of lignans in S.chinensis was established according to literatures.Based on LC-MS/MS analysis,15 lignans were identifed.2.Investigation of pathophysiological process and plasma metabolomic analysis of AD rats induced by amyloid beta 25-35(Aβ25-35)Cognitive disorder was one of the main characters in AD whose pathological features included neurofibrillary tangles,amyloid protein deposits and neuron loss.The pathogenesis of AD was not fully understood.Hence,no effective drugs were available for treating AD.Animal model could help for researching occurrence and progress of diseases and discover novel drugs.In this study,the AD rat model was established by injecting Aβ25-35 solution into bilateral hippocampus.Morris water maze(MWM)test,HE staining,immunohistochemistry and plasma metabolomics were applied to research the pathophysiological process and endogenous metabolic profile changes in AD rats.The results suggested that injecting Aβ25-35 solution in rat hippocampus results in irreversible damage to learning and memory abilities of rats.The results showed the injury on the spatial learning ability of AD rats was gradually aggravated within 4 weeks after Aβ25-35 injection,reached the maximum at 4 weeks and then was stable until 8 weeks.During 8 weeks of modeling,the levels of enzymes involved in formed senile plaques and tau phosphorylation were significant increase in the plasma of AD rats.The number of neurons in AD rat hippocampus is gradually reduced.The neurotransmitter dysfunction was mainly involved in glutamic acid(Glu),γ-aminobutyric acid(GABA),5-hydroxytryptamine(5-HT),acetylcholine(Ach),norepinephrine(NE),and dopamine(DA).Meanwhile,based on dynamic metabolomics study throughout the 8 weeks,17 endogenic metabolites were successfully screened out which correlated with AD.These metabolites were mainly involved in lipid metabolism,vitamin metabolism,and amino acid metabolism.3.Plasmatic metabolomics strategy to explore the holistic mechanism of lignans in S.chinensis on treating ADIn this article,MWM test was used to verify the effects of lignans in S.chinensis on learning and memory in AD rats.The results indicated that lignans in S.chinensis showed similar effect as donepezil in improving spatial learning and memory abilities of AD rats.To illuminate the mechanism,we further studied the regulating effects of lignans on deposition of Aβ,hyperphosphorylation of tau,and levels of neurotransmitters and endogenous metabolites.Overall,lignans in S.chinensis might not only deduce the Aβ deposition,tau phosphorylation and neuronal apoptosis,but also could upregulated the levels of GABA,glycine(Gly),5-HT,and Ach,and downregulated acetyl cholinesterase(ACh E)and aspartic acid(Asp).A total of 15 endogenous metabolites associated with ameliorating learning and memory were identified after treatment with lignans in S.chinensis.These metabolites were involved in metabolism of sphingolipids,linoleic acid,alanine,aspartic acid and glutamic acid.All of the above results showed that lignans in S.chinensis could regulate multiple endogenous metabolic pathways in pathological AD,therefore alleviated the neurotoxic effects of neurological inflammation and oxidative stress.4.Correlation analysis of in-vivo ingredients and target-network pharmacology strategy for the anti-Alzheimer’s disease mechanism of lignans in S.chinensisAs achronic disease,the cause of AD was closely associated abnormal expression of a variety of genes and proteins.Thus,it is difficulty for a drug that target at single gene or protein to therapy complicated diseases.In the present study,we analyzed the main efficiency compositions and metabolites that lignans in S.chinensis metabolized in vivo.In total,ten absorbed prototype constituents and 39 metabolites were identified or tentatively characterized in dosed AD rat plasma based on ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS).The formation of these metabolites mainly involved in hydroxylation,demethylation,reduction,phosphorylation and dehydration reaction.Based on effective constituents in vivo,target-network pharmacology(T-NP)approach was adopted,via which pattern 17 targets were identified as potential target shared by lignans in S.chinensis and AD.These targets found to be associated with multiple active pharmaceutical ingredients of lignans in S.chinensis including ACh E,inducible nitric oxide synthase(i NOS),GSK3β,heme oxygenase 1(HMOX1)and so on.Further analysis was continued to find other targets interacted to these 17 targets were also selected as therapeutic targets of lignans in S.chinensis.The new identified targets included APP,neurotransmitter,and inflammatory response and so on.For further recover mechanism,independent verification experiment was applied.The results suggested that lignans could regulate amyloid precursor protein(APP)metabolism,neurofibrillary tangles(NFTs)metabolism,neurotransmitter metabolism,the inflammatory response and antioxidant system.5.Pharmacokinetic study of lignans in AD ratsA combination of UPLC-Q-TOF-MS and ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometer(UPLC-QQQ-MS)was applied together to establish a fast,sensitive and reliable analysis method.Meanwhile,the contents of lignans in plasma samples were measured.The established method was performed to character the pharmacokinetic features of 10 lignans ingredients in AD and healthy control rats.Then drug-time curves and corresponding kinetic parameters were obtained.The results showed that compared with that of healthy control rats,the absorption of lignans in AD rats was obviously improved,which suggested pharmacokinetics of lignans in S.chinensis were changed in AD rats.And it might be one of the reasons for lignans’ therapeutic effect on AD.The above results provided reliable experimental data for the furtherpharmacology and toxicology study about lignans in S.chinensis for treating AD.It might provide useful support for the further study of pharmacodynamic material basis and the mechanisms of lignans in S.chinensis for treating AD.