Study On The Inhibition Of Flavonoid Compounds On Acetylcholinesterase,Structure-Activity Relationship And Protection Of PC12 Cells From Oxidative Damage Induced By Aβ_25-35

Alzheimer’s disease（AD）is a neurodegenerative disease and the most common form of dementia worldwide.Patients with AD suffer from cognitive loss,memory loss,and deterioration of everyday behaviors,which severely deteriorate their quality of life and eventually lead to disability and death.AD has become the fourth leading cause of death in the elderly after heart disease,cancer,and stroke.The causes of AD include Tau protein aggregation,impaired cholinergic neurotransmission,β-amyloid（Aβ）deposition,oxidative stress,and neuroinflammation.Acetylcholinesterase（AChE）is a serine protease that catalyzes the hydrolysis of acetylcholine（ACh）,an important neurotransmitter in the body,into acetate and choline,thereby aborting the transmission of nerve impulses.Based on the"cholinergic hypothesis",AChE inhibitors developed by targeting AChE are the main drugs used in the clinical treatment of AD,such as donepezil and galantamine.However,these drugs in clinical use have disadvantages such as low bioavailability and significant side effects,which limit their application.Compared to synthetic drugs,active ingredients in natural products have fewer side effects and lower toxicity,making them suitable for long-term use.Therefore,the search for AChE inhibitors with better inhibitory activity,less toxic and side effects of natural source is of great importance for the prevention and treatment of AD.Flavonoids,as important active ingredients in"food and medicine"plant resources,have various biological activities such as neuroprotection,antitumor,antioxidant,anti-inflammatory,and cardioprotection,and have no obvious cytotoxicity,which has become a treasure trove of anti-AD drugs and functional food research both domestically and internationally.Based on this,three representative dietary flavonoids（baicalin,quercetin,and myricetin）were selected for researching.Multispectroscopic methods and computer simulation techniques were used to investigate the inhibitory activities,binding properties,binding sites,and conformational changes of AChE induced by the three flavonoids.In all these ways the mechanism of the inhibitory effect of the three flavonoids on AChE was deeply explored.The inhibitory effects of flavonoids on AChE activity were evaluated in pairs and in combination with the positive drug galantamine hydrobromide.Three models based on comparative molecular field analysis（CoMFA）,comparative molecular similarity index analysis（CoMSIA）and Topomer comparative molecular field analysis（CoMFA）were established to analyze the three-dimensional quantitative structure-activity relationship（3D-QSAR）of flavonoids on AChE inhibition.The effects of different pH,heating,and microwave pretreatments on the inhibitory activity of quercetin were investigated,and an simulated digestion model was established to determine the bioaccessibility of quercetin in vitro and its inhibitory activity of AChE before and after treatment.An AD（cell）model was established with Aβ_25-35induced damage to PC12 cells,and the neuroprotective effects of quercetin and baicalin on oxidative damage in PC12 cells were investigated.The main studies and results are as follows:（1）The effects of AChE concentration,substrate concentration,pH,temperature,reaction time,organic solvent,and metal ion concentration on AChE activity were investigated.The reaction conditions were determined as follows:enzyme concentration in the final reaction system was 0.57 nM,the concentration of thiothiocarbophosphate iodide（ATCI）was 0.25 mM,and the reaction was carried out in PBS buffer at pH 7.6 and the reaction temperature was 25℃.Organic solvents have a strong influence on the viability of AChE,and the amount of ethanol in the system should be controlled within 3%when ethanol is the solvent.Different metal ions(Ca²⁺,Mg²⁺,Co²⁺,Mn²⁺,Zn²⁺,Cu²⁺,Ba²⁺,Ni²⁺)have certain effects on AChE activity.（2）Baicalein,quercetin,and myricetin showed strong inhibitory effects on AChE activity with semi-inhibitory concentrations(IC₅₀)of 6.42±0.07μM,4.59±0.27μM,and 2.57±0.13μM,respectively.The types of inhibition of AChE by the three were competitive,mixed,and non-competitive inhibition with inhibition constants of 4.32±0.26μM,6.17±0.18μM,9.21±0.22μM,respectively.Inactivation of AChE induced by the three flavonoids was a first-order monophasic process.The combination of baicalein with myricetin and quercetin with myricetin showed additive effects,while the combination of the three flavonoids with galanthamine hydrobromide showed synergistic inhibition of AChE.（3）Baicalin,quercetin,and myricetin were able to significantly quench the endogenous fluorescence of AChE statically.The binding constants between them were on the order of 10⁴L mol^-1,indicating that the three flavonoids had a moderate binding affinity with AChE.Each of them bound to AChE at one binding site,suggesting that there was only one or one class of sites for the binding of flavonoids in AChE.The binding of baicalein and myricetin to AChE was driven by hydrogen bonding and hydrophobic interactions,while quercetin interacted with AChE via van der Waals forces and hydrogen bonding.Simultaneous fluorescence and circular dichroism showed that all three flavonoids caused changes in the microenvironment and secondary structure of AChE.Molecular simulations showed that all three flavonoids bound to the peripheral anion site（PAS）of AChE and formed stable complexes with the surrounding amino acid residues.The binding caused a conformational shift in the enzyme and tightened the structure of the gorge entrance,preventing the substrate from entering and binding with the enzymes active site,then eventually inhibiting AChE activity.Meanwhile,quercetin and myricetin might extend to the AChE’s active center.It generates hydrogen bonds with Glu202 and Tyr133,further reducing the catalytic activity of AChE by destroying the stability of the hydrogen bond network in the active center of the enzyme.Molecular dynamics simulations showed that the complexes formed by the three flavonoids and AChE had a more stable structure than free AChE,and complex formation reduced the flexibility of the molecules,making the structure more compact.（4）3D-QSAR models were constructed for 32 flavonoid AChE inhibitors based on CoMFA,CoMSIA,and Topomer CoMFA.The cross-validation coefficients（q²）of the 3D-QSAR models constructed by CoMFA,CoMSIA,and Topomer CoMFA were0.645,0.560,and 0.689,respectively.The non-cross-validation coefficients（r²）were0.917,0.881,and 0.920,respectively.The internal and external validation parameters indicated that the generated 3D-QSAR models all had good predictive power and significant statistical reliability.The 3D-QSAR equipotential plots obtained from the three models provided sufficient information for understanding the conformational relationships,which was helpful for further design and development of functional components and derivatives of food-derived flavonoids with AChE inhibitory activity.（5）The effects of pH,heating,and microwave treatment on the AChE inhibitory activity of quercetin were investigated.The results showed that quercetin was relatively stable in an acid environment and had a high inhibition rate for AChE.Microwave treatment and heating were able to enhance the inhibitory activity of quercetin against AChE,but heating at 100℃ for more than 40 min resulted in a substantial decrease in the inhibition rate of AChE.Quercetin still quenched the endogenous fluorescence of AChE by a static-type approach after treatment with both processing conditions.The binding affinity between quercetin and AChE showed an enhanced trend after both heating and microwave treatment,and the binding driving forces were still hydrogen bonding and van der Waals forces.The UPLC-Q-TOF-MS results showed that after heating and microwave treatment,quercetin underwent oxidation,cleavage,and reaction with nucleophilic reagents to produce 7 new products,including 3,4-dihydroxybenzoic acid,flavonoids,and hesperidin.Structural analysis and identification of these compounds were also conducted respectively.The order of bioaccessibility of quercetin after simulated in vitro digestion was:untreated>microwave treated>heat treated quercetin.The digested quercetin still had good AChE inhibitory activity,but the inhibitory activities were all reduced compared with the undigested samples,while the microwave-treated quercetin had relatively stronger AChE inhibitory ability than heated and untreated quercetin after simulated digestion.（6）A model of Aβ_25-35-induced oxidative damage was established in neural cell PC12.MTT test showed that baicalein and quercetin did not produce toxic effect in PC12 cells.The cell survival rate was significantly reduced after treated with different concentrations of Aβ_25-35for 24 h and 48 h.When PC12 cells were treated with 20μM Aβ_25-35for 24 h,the cell survival rate was（54.85±1.92）%,which was used as the best condition for modeling cell oxidative damage.Both baicalein and quercetin could significantly increase the survival rate of PC12 cells reduced by Aβ_25-35and inhibit lactate dehydrogenase（LDH）release in the supernatant.Baicalein and quercetin could protect PC12 neuronal cells by increasing intracellular levels of the antioxidant enzyme reduced glutathione（GSH）,decreasing lipid peroxidation end product malondialdehyde（MDA）content,and reducing intracellular ROS levels,while avoiding apoptosis caused by ROS accumulation.