The Quantitative Analysis Of The Main Active Component Of A New Antidepressant Drug Of Anyupeibo Capsule In Vivo And Study Of Targeted Metabolomics

Objective: To study the exposure concentration of K6,the main active ingredient of Anyupeibo capsule,a new antidepressant traditional Chinese medicine,in subjects with depression,and to explore the biomarker of Anyupeibo capsule in the treatment of depression and elucidate its possible metabolic pathways through targeted metabolomics technology.Methods: A method for quantifying the concentration of K6 concentration in plasma samples was developed based on LC-MS/MS in positive ion mode.The mobile phase of the method was 40% ammonium acetate aqueous solution(A)containing 0.1 mmol/L and60% acetonitrile(B).Tinidazole was used as the internal standard,and Agilent agilent ZORBAX SB-C18 column(2.1 × 150 mm,3.5 μm)was used to separate K6 from the internal intereferents.The sample pretreatment method was based on solid-phase extraction,and the extracted filtrate was concentrated and volatilized.The residue was redissolved by the initial mobile phase,followed by sufficient vortices,and the supernatant was centrifuged for sample analysis.The methodology verification was completed according to the relevant guiding principles of quantitative analysis method verification of biological samples,and the exposure concentration of the collected subject samples(V0/V4/V6)was measured.In the meantime,bile acid and amino acid targeted metabolomics were further studied on blood samples(V0/V6)of subjects before and after taking placebo and Anyupeibo capsule respectively in the clinical trial on the strength of the results and platforms of our research group.Through Graph Pad Prism 8.0.2,SPSS 25.0,SIMCA 14.1and other software to process the data,analyze the changes of metabolic spectrum in subjects after taking Anyupeibo capsule and the individual difference compared with placebo,find out the relevant small molecule differential metabolites reflecting the efficacy of Anyupeibo,enrich the metabolic pathways that may be involved,and study the reasons why the drug treatment of depression may be different from placebo,It may be related to the clinical trial results that the efficacy of Anyupeibo group is different from that of placebo.Results: In this analytical method,the substance to be measured could be quickly separated from other intereferents within 3 minutes,with good peak shape.It was validated that the recovery rate of K6 extraction was high(more than 85%),the matrix effect ranged from 87.15% to 100.28%,and the stability is good,the intra-day and inter-day precision and accuracy met the relevant guiding principles.In the measured samples,the K6 in three blood sampling points(V0/V4/V6)in the placebo group and V0 in the administration group were lower than the lower limit of quantification.The average value of K6 in V4 samples in the test group was about 1.72 ng/m L,and the maximum concentration was 28.48 ng/m L.The maximum concentration of V6 samples was 92.34ng/m L,and the mean was 20.26 ng/m L.There were significant differences in drug exposure concentrations among individuals,which may be used for the correlation analysis of phase III efficacy and adverse reactions.A total of 254 tubes of blood in the placebo tranch and the experimental tranch were studied by bile acid and amino acid targeted metabolomics.The differences 23-NCA,3β-OH-D5,GCDCA,TCDCA,Apo-CA,GCA,DCA,GHCA,TCA,12 KCDCA,TLCA,GDCA,GLCA,NDCA,Arg,Gln,His,Val,Tyr,Met,Ala,Asp,Leu,Phe,Ile,Pro,Opr,Orn,Thr,which had VIP-value greater than 1 and P-value less than 0.05 were screened.Based on the enrichment analysis of KEGG database,it was found that the study drug may involve Aminoacyl-t RNA biosynthesis,Valine,leucine and isoleucine biosynthesis,Phenylalanine,tyrosine and tryptophan biosynthesis,D-Glutamine and D-glutamate metabolism,Primary bile acid biosynthesis,Alanine,aspartate and glutamate metabolism,Arginine and proline metabolism,Histidine metabolism,Pantothenate and Co A biosynthesis,beta-Alanine metabolism and other pathways.At the same time,the metabolic spectrum changes of Anyupeibo capsule group were compared with those of placebo group,and 15 differential metabolites were analyzed,which may be related to the difference of curative effect between the two groups.Conclusion: The method builded in this experiment can be used to determine the content of K6 in the plasma samples from depression patients after taking Anyupeibo capsule,which may provide a basis for phase III clinical trial.At the same time,29 possible biomarkers of Anyupeibo capsule in the treatment of depression were found through targeted metabolomics technology,and 15 different compound metabolic spectrum changes different from placebo were found,which further revealed the possible metabolic pathways involved in the new drug.