Study On The Urinary Excretion Kinetics And Urine Metabolomics Of The Main Components Of Atractylodes Japonica Before And After Processing Based On LC-MS

Purpose:Through the study of urine excretory kinetics of the main components in crude and processed Atractylodis Rhizoma,clarified the differences of the excretion in rat before and after theAtractylodis Rhizoma was processed;Through the metabonomics,the potential biomarkers of spleen-deficiency were found,and then the similarities and differences of the effects of crude and processed Atractylodis Rhizoma on biomarkers were analyzed.The processingmechanism of Atractylodis Rhizoma was clarified by excretory kinetics and metabonomics.Material and method:1 A new UPLC-MS/MS method was developed for simultaneous quantification ofactractyloside A,atractylenolideⅠ,atractylenolideⅡ,and atractylenolideⅢby the multiplereaction monitoring model。Rats were randomly divided into two groups（n=6 per group）and oral administrated crude and processed Atractylodis Rhizoma extracts,respectively.Urinesamples were collected at different time before and after administration and deproteinated with methanol after adding with acetaminophen,then the samples were injected andanalyzed.The excretion parameters of each component were calculated by DAS3.2.8 software,and cumulative excretion curve and excretory rate curve were plotted.Then the differences of excretory regulation of each component between crude and processed Atractylodis Rhizomawere defined.2 The SD rats were randomly divided into the normal group,model group,crude Atractylodis Rhizoma group and processed Atractylodis Rhizoma roup.From the first day to the ninth day,let the model group,crude Atractylodis Rhizoma group and processed Atractylodis Rhizoma roup rats swimming to the endurance limit and eating cabbage in the single days,while lard oil was gavaged to them in the double days.From tenth days to sixteenth days,the rats were gavaged with Folium Sennae.After the spleen-deficiency models were established,the crude Atractylodis Rhizoma group and processed Atractylodis Rhizoma roup were treated with crude Atractylodis Rhizoma and processed Atractylodis Rhizoma,respectively.The behavior of rats in each group was investigated,while the content of GAS,NA⁺-K⁺-ATPase,TRY and AMS in rats serum was determined by ELISA.3 The total ions chromatogram of rat urine was established by UPLC/Q-TOF-MS,then the metabolic spectrum of rat urine was collected.Principal component analysis（PCA）,partial least squares-discriminant analysis（PLS-DA）and orthogonal partial leastsquares-discriminant analysis（OPLS-DA）was used for pattern recognition of normal group and model group.The VIP values（VIP>1）and P values（P<0.05）were used to selecteddifferentially expressed metabolites.And combined with targeting metabonomics,comparing the retention time and mass rationof differentially expressed metabolites and standardproducts that related to the mechanism of spleen deficiency syndrome.4 The urine samples metabolic spectrum of four groups were collected,and the changetrajectory was analyzed by orthogonal partial least squares-discriminant analysis（OPLS-DA）.And the peak area after normalization processing would be used to quantitative analyze the effect of crude and processed Atractylodis Rhizoma on the potential biomarkers。Results:1 Four main componentsn in the rats urine had a good linearity in the certain concentration range（r2≥0.997）,the precision,accuracy,matrix effect,recovery and stability were all in accordance with the requirements of quantitative analysis methods of biological samples,which showed that the method was stable and could be used for the simultaneousdetermination of four components.The excretory kinetic results:The t1/2 of actractyloside A,atractylenolideⅠ,atractylenolideⅡand atractylenolideⅢin crude Atractylodis Rhizoma were12.88 h,32.48 h,32.34 h and 44.58 h,and the total urinary excretion rate were 0.207%,0.051%,0.446%and 0.179%,respectively.And the t_1/2/2 of actractyloside A,atractylenolideⅠ,atractylenolideⅡand atractylenolideⅢin processed Atractylodis Rhizoma were 12.44 h,35.05 h,33.03 h and 46.43 h,while the total urinary excretion rate were0.299%,0.069%,0.523%and 0.212%,respectively.2 The normal group rats had a good condition in the experiment,while the model groupshowed lose weight,loose stools,reduced eating and dry hair.The symptoms were relieved after the treatment of crude and processed Atractylodis Rhizoma extracts,but the condition of the processed Atractylodis Rhizoma group was closer to the normal group.The results ofELISA:compared with the normal group,the content of GAS,NA⁺-K⁺-ATPase,TRY and AMS in the model group decreased significantly（P<0.05）,however these indicators in the crude Atractylodis Rhizom group and processed Atractylodis Rhizom group increasedsignificantly after the intervention,And compared with the crude Atractylodis Rhizom group,the rise of processed Atractylodis Rhizom group is more obvious.3 56 differential metabolites was screened out by nontargeted metabonomics,including amino acids,fatty acids,bile acids,organic acids and so on.Then target metabolomics was used to compare the difference of differential metabolites and standard products,and 13 biomarkers ofspleen deficiency were identified finally.These biomarkers were related to aminoacyl-tRNA biosynthesis,primary bile acid biosynthesis,steroid hormone biosynthesis,arginine and proline metabolism,taurine and hypotaurine metabolism,respectively.The urine metabolism track of the spleen-deficiency syndrome model rats had a tendency to move toward the normal group after the intervention of crude and processed AtractylodisRhizoma,and the processed one is more obvious,which indicates that the effect of tonifying spleen was enhanced after Atractylodis Rhizoma was stir fried with bran;It was found that the effect of processed Atractylodis Rhizoma on cysteine,DL-pipecolic acid,L-Phenylalanine,L-Valine and citrulline was better than the crude one.Conclusion:1 The t_1/2 and Ke of actractyloside A,atractylenolideⅠ,atractylenolideⅡ,and atractylenolideⅢhad no significant difference between crude and processed Atractylodis Rhizoma,but the total excretion rate of them in processed Atractylodis Rhizoma were apparently higher than thecrude ones（p<0.05,p<0.01）,which showed that processing Atractylodis Rhizoma with wheat bran by stir-frying could promote the urinary excretion of them.2 The spleen deficiency syndrome was related to the glycometabolism,lipid metabolism,and protein metabolism disorder,and the effect of processed Atractylodis Rhizoma on proteinmetabolism is better than the crude one,which indicated that the enhancement of tonifying spleen of processed Atractylodis Rhizoma might be related to its better repair of proteinmetabolism disorder.