Identification Of Metabolites And Pharmacokinetics Of Total Alkaloids Of Lotus Plumule In Rats

The main components of total alkaloids of Lotus Plumule are neferine,liensinine and isoliensinine.Its chemical structure is the structure of bis-benzylisoquinoline alkaloids.It is the main active ingredient of alkaloids in lotus seeds and has antihypertensive and hypoglycemic properties.Treatment of neurodegenerative diseases and other extensive pharmacological activities.In this research,macroporous adsorption resin method was used to isolate and prepare the total alkaloids from lotus seeds.The HPLC/Q-TOF MS technique was used for the first time to identify the rat plasma,feces and urine after intragastric administration of total alkaloids.The prototype of the drug and its 13 metabolites speculated the possible metabolic pathways of total alkaloids in rat kidney,and studied the pharmacokinetics of the prototype total alkaloid and its 2 metabolites.It provided a theoretical and technical foundation for exploring and discovering and separating new active metabolites,and provides the correct data basis and scientific basis for the clinical research and application of monomers and total alkaloids.The main contents and results are as follows:1.The silca gel column combined with D101 macroporous adsorption resin was used for the preparation and purification technology.The total alkaloids from lotus seed were isolated and purified from lotus seeds.The content and purity were determined by high performance liquid chromatography.The final results showed that the purity of the total alkaloids obtained from the lotus root was greater than 95%.2.By using HPLC/Q-TOF MS technology chromatographic analysis method,by comparing the retention time with the known standard three alkaloids and analyzing the MS fragmentation rule,the rat's series of metabolites were collected after intragastric administration of total alkaloids in lotus roots.Including: plasma,urine,feces and cerebrospinal fluid,from these metabolites identified and speculated that the total alkaloids in rat plasma,feces,urine prototype drug and 13 major metabolites,but not found in cerebrospinal fluid Three alkaloids and metabolites.Among them,neferine,liensinine and isoliensinine were identified by comparison with the retention time of the standard and the related mass spectrometry information.Other metabolites were initially identified by Massfragment and Masshunter software,and most were the first reported total alkaloids in vivo metabolites.Based on the above 13 metabolites,the possible metabolic pathways of total alkaloids in rats were inferred.3.Using tetrandrine as the internal standard,the prototype drug three alkaloids and their two metabolites of liensinine glucuronidation metabolites and isoliensinine glucuronidation metabolites were determined by HPLC/Q-TOF MS.The content was confirmed by methodology.The glucuronidation metabolites of liensinine and isoliensinine were identified for the first time and their plasma content was determined.There was no relevant standard and only semi-quantitative was performed.The mobile phase was a gradient elution of acetonitrile-water(0.1% FA)system,neferine was 0.00025-5?g/ml,liensinine was 0.00025-5?g/ml,and isoliensinine was 0.00025-5?g/ml.Linear relationship,other metabolites are semi-quantitative.In the process of validation of the detection methodology,the intra-day and inter-day precisions were less than 15%,with the accuracy of neomycin base at-2.4% to 5% and the accuracy of neferine at-1.6% to 2%.The accuracy of islanine was between 0.5% and 4.8%.The recoveries of the three analysis and the internal standard tetrandrine in rat plasma were all higher than 86.0%.The matrix effect is in the range of 95.4-113.2%.The stability test conditions of the three kinds of test substances were stored at –20°C for 20 days.After 3 freeze-thaw cycles and room temperature for 24 hours,the stability of the three alkaloid quality control samples under three conditions was tested well.The analytical quantitative method established in this experiment measured the concentrations of the three alkaloid prototype drugs in rats and the concentration of liensinine glucuronidation metabolites and isoliensinine glucuronidation metabolites in rats,and produced a series of related pharmacokinetic parameters of them in intragastric administrated rats were measured.The relevant data were fitted by DAS.2.0 software.And the data of all of the five compounds met the two-compartment model.Two absorption peaks appeared in the three alkaloids' drug-time curves,indicating that there may be hepatoenteral circulation.The pharmacokinetic parameters of three alkaloids and metabolites were as follows:liensinine,Tmax was 0.75 h,Cmax was 0.05 ?g/ml,AUC(0-?)was 0.114 ?g/ml*h,t1/2 was 2.571(h)The Tmax of isoliensinine was 0.75 h,Cmax was 0.08 ?g/ml,AUC(0-?)was 0.167 ?g/ml*h,t1/2 was 2.382(h);neferine was Tmax was 0.75 h,and Cmax was 0.13 ?g/ml,AUC(0-?)was 0.333 ?g/ml*h,t1/2 was 2.221(h);For glycoacetaldehyde acidizing metabolite of liensinine Tmax was 2 h,Cmax was 0.013?g/ml,AUC(0-?)was 0.053 ?g/ml*h,t1/2 was 3.466(h);For glycoacetaldehyde acidizing metabolite of isoliensinine Tmax was 2.01 h,Cmax was 0.003 ?g/ml,andAUC(0-?)was 0.019 ?g/ml *h,t1/2 is 4.632(h).From the data analysis results as above,it could be concluded that the main metabolic pattern of liensinine and isoliensinine is two-phase glycoacetaldehyde acidizing metabolism.