Synthesis,Metabolism And Anti-Alzheimer’s Disease Investigations Of Crocetin Glucuronide

Objective:This article synthesized CM,then verified its existence in plasma and investigated its pharmacokinetics after oral administration.To investigate biological effect of this component,the pharmacological activities of CM by amyloid-beta（Aβ）toxicity in PC-12 cells,drosophila models,anti-AChE activity and virtual screening（Docking）were assessed.Methods:1.Preparation and analysis of crocetinThe dried gardenia fruits were ground to a coarse powder and extracted with 60%ethanol（v/v）by cold percolation,concentrated,and then eluted through HPD-100macroporous resin and 60%ethanol to obtain total crocins.Total crocins were hydrolyzed by 10%KOH,acidified by phosphoric acid,and crocetin portion was obtained.The crocetin portion was dissolved in methanol and centrifuged to obtain pure crocetin.The structure was identified by ¹H-NMR,¹³C-NMR and MS,repectively.The purity was determined by PLC.2.Synthesis of crocetin-monoglucuronideThe crocetin and D-glucuronic acid were used as starting materials,and the fluorochemical a leaving group.The crocetin-monoglucuronide was designed and synthesized.The structure was identified by ¹H-NMR,¹³C-NMR and MS,and whether CM can be hydrolyzed byβ-glucuronidase was also confirmed.Furthermore,purity was determined by PLC.3.The pharmacokinetic investigation of crocin-1 and CMThe crocin-1 was intragastrically administered to SD rats,and blood samples and organs heart,liver,spleen,lung,kidney and brain were quantitatively analyzed at different time.The target compound in blood was identified with reference to the prepared CM by MS and MS².The standard curves of CM and crocin-1 were used to calculate the contents of these two components in the blood sample,as well as heart,liver,spleen,lung,kidney and brain;The pharmacokinetic parameters of these two compounds in the blood sample were calculated by DAS2.0.4.Study on Anti-AD activity of CM（1）Aβ_1-42-42 inducted PC12 cells,cell survival rate was determined by MTT;（2）Inhibiting AChE activity was measured by modified Ellman method;Docking studies were carried out to investigate the binding pocket of CM in hrAChE to verify its activity;（3）In vivo experiment on AD Drosophila model with overexpression of human Aβ₄₂2 was carried out.Behavioral index（PI）was measured to evaluate the salvage effect of crocetin-monoglucuronide on learning and memory deficits in AD model.Results:1.Preparation and analysis of crocetinThe results of MS showed that the main molecular ion peak was consistent with the molecular weight of crocetin;the structure was confirmed by ¹H-NMR and¹³C-NMR.2.Synthesis of CMThe results of HR-MS showed that the main molecular ion peaks was consistent with the theoretical molecular weight of CM;the structure was identified by ¹H-NMR and ¹³C-NMR;the chromatogram was obtained by PLC analysis,and the peak area normalization method was used to calculate the purity to be 95.66%.In addition,data from those incubated byβ-glucuronidase and analyzed by LC-MS revealed that the molecular peaks disappeared after incubation.3.The pharmacokinetic investigation of crocin-1 and CMThe data of MS and MS² showed that the fragment ion peaks of prepared CM is consistent with that of metabolites in rats after intragastric administration.The pharmacokinetic results showed that the linear relationships between peak areas and concentrations of these two components are good,and both R²were greater than0.999.Both RSD of intra-day precision and day-to-day precision were≤14.9%,accuracy RE were≤13.1%;recovery rate were 70.18¹05.51%;stability RE were≤20%.In addition,after administration,quantitative and qualitative analysis of rat heart,liver,spleen,lung,kidney and brain by LC-MS were carried out.The results showed that CM is present in all organs,and the highest concentrations were found in liver.The pharmacokinetic parameters showed that the concentration of CM was significantly higher than crocin-1（7.48 times）,and the elimination half-life of CM(t_1/2=11.26 h)was longer than crocin-1(t_1/2=0.34 h).4.Study on the Anti-AD activity of CM（1）The results of pharmacological activity of PC12 cells showed that 10?M CM could effectively mitigate the damage of PC12 cells induced by Aβ_1-42.In addition,cytotoxicity was not detected with 100?M CM.（2）The Ellman method showed that CM can effectively inhibit AChE(IC₅₀=22.46?M).In addition,docking data showed that CM could bind to anion sites around AChE and inhibit its activity.（3）Further animal experiments showed that crocetin-monoglucuronide possesses significant memory-deficient effects on AD drosophila,with rescue rates of 50%and 73%at 10and100?M,respectively.Conclusion:1.First synthesized CM and the structure was confirmed by HR-MS,¹H-NMR and¹³C-NMR;CM was present in plasma and heart,liver,spleen,lung and kidney brain after intragastric administration of crocin-1 and the concentration of CM was higher than crocin-1（7.48 times）,and the elimination half-life of CM(t_1/2=11.26 h)was longer than crocin-1(t_1/2=0.34 h).The stability,precision,accuracy and recovery of CM analysis method are good.2.CM is not toxic to PC12 cells,and possesses protective effect on PC12 cell injury induced by Aβ_1-42;By docking and analysis of anti-AChE activity,CM binds to anion sites around AChE and inhibits its activity;CM possesses an mitigating effect on learning and memory behavior of AD drosophila model.This study demonstrated that CM may be one of the effective molecules of crocin-1 in the treatment of AD.