Screening Of Natural Acetylcholinesterase Inhibitors In Drynariae Rhizome Based On Uplc-ms/ms

Acetylcholinesterase inhibitors(AChEIs)are currently the most effective class of drugs for the treatment of Alzheimer's Disease(AD)in the world.The development of natural medicines to find AChEIs suitable for AD patients can be taken for a long time,the drug has little toxic and side effects and other advantages has attracted widespread attention from scientific researchers.Drynariae Rhizome is widely distributed in Liao Ning,Shan Dong,Jiang Su and other places in China.As a natural medicinal plant,it is often used clinically to treat cognitive impairment.Ellman's method is the most classic method for screening potential AChEIs.It is mostly used for the determination of acetylcholinesterase(AChE)inhibitory activity in vitro.However,the composition of natural medicinal plants is complex and uncertain,and it is easy to produce false positive or false negative results.Based on Ultra Performance Liquid Chromatography tandem Mass Spectrometry(UPLC-MS/MS),this project established AChE in vivo and in vitro to catalyze the conversion of Iodized acetylcholine(ACh-I)to Iodized choline(Ch-I)high-throughput detection method for evaluating AChE inhibition of bone fragmentation and its main components.1.Construction of screening method for AChE inhibitory activity in vitro.This project uses UPLC-MS/MS,ACQUITY UPLCTM BEH C18(1.7 ?m,50 mm×2.1 mm)column,isocratic elution with 0.5%acetic acid-water and acetonitrile,electrospray ionization source(ESI)combined with multiple reactions Monitor(MRM)positive ion scan mode to detect Ch-I produced after AChE reaction.The conditions such as the amount of AChE,substrate concentration,reaction temperature,and reaction time were optimized,and the optimal conditions for AChE incubation were finally determined.AChE inhibitory activity was evaluated in vitro by calculating the semi-inhibitory concentration(IC50).The positive control galantamine was selected to test the established in vitro screening method,and the IC50 was 1.26±0.15 ?M.2.Guided isolation of natural AChE inhibitor in Drynariae Rhizome.The established in vitro screening method of AChE activity was used to inhibit the activity of Drynariae Rhizome ethanol extract,n-butyl site,petroleum ether site,and water site.By calculating the IC50 value,it was found that the n-butanol site was significantly inhibited.Eight flavonoids were isolated and identified as:Naringenin(28 mg),Eriodictyol(19 mg),Kaempferol(24 mg),Luteolin(9 mg),Astragalin(11 mg),Luteolin-7-O-?-D-glucoside(7 mg),Naringin(51 mg)and Neoeriocitrin(68 mg).Their IC50 values were 3.81 ±0.21 ?M,7.19±0.62 ?M,11.09±1.02 ?M,17.26±0.23 ?M,18.24±2.33 ?M,17.13±1.02 ?M,26.4±1.17 ?M and 22.49±1.25 ?M.Therefore,it was confirmed that the ethanol extract of Drynariae Rhizome has inhibitory AChE activity,and the main component that exerts the inhibitory effect is flavonoids.3.Construction of AChE activity detection method in rats.A method for detecting endogenous AChE activity in rat plasma and brain tissue was established.Betaine was used as the internal standard solution,and promethazine hydrochloride was used as the butyrylcholinesterase inhibitor.The diluted plasma and brain homogenate were mixed with the substrate ACh-I.After the reaction,it was measured by UPLC-MS/MS The amount of product Ch-I in the reaction mixture and the AChE activity was calculated.The AChE activity in the plasma and brain tissue of 6 old rats was tested.The results showed that the AChE activity in the plasma of the old rats was 0.1394-0.1905 U/mL,and the AChE activity in the brain tissue was 0.7015-1.0922 U/mL.4.Effect of total Drynariae Rhizome flavonoids on AChE enzyme activity in blood and brain tissues of aged female rats.The established AChE activity measurement method in rats was used to detect changes in AChE activity in blood and brain tissues of rats after oral administration of Drynariae Rhizome flavone total flavones(total flavonoid content of 85.83%).AChE inhibitory ability of total flavonoids in Drynariae Rhizome.The results showed that the AChE activity values of plasma and brain tissue homogenates of normal age(8 weeks old)rat group were 0.0348 ± 0.002 and 0.3059±0.003 U/mL;AChE activity values were 0.1459±0.023 and 0.9078±0.003 U/mL;after 30 days of oral administration of the total flavonoids of Drynariae Rhizome supplementation in aged rats(25 weeks of age),the AChE activity values of plasma and brain tissue homogenates were 0.0926±0.012 and 0.5310±0.002 U/mL.The results showed that compared with normal rats,the AChE activity in the plasma and brain tissues of the elderly rats was significantly increased(P<0.05).After administering total flavonoids from Drynariae Rhizome supplementation,the AChE activity in plasma and brain tissue of aged rats was significantly reduced(P<0.05).Therefore,it was confirmed that the total flavonoids of Drynariae Rhizome can significantly inhibit the AChE activity of aged rats,and it was confirmed that the total flavone in Drynariae Rhizome is an effective natural AChE inhibitor at the animal level.In this paper,a method for measuring AChE activity in vivo and in vitro based on UPLC-MS/MS was established,and it was determined that the active ingredient that inhibits AChE in Drynariae Rhizome is flavonoids such as naringin.It not only provides a new direction for finding and discovering AChEIs candidate drugs from natural medicinal plants,but also provides more favorable basic conditions for the development of drugs for treating AD.It has a good development prospect and value in the future.