Research On The Processing Mechanism Of Atractylodes Japonicus Based On Metabolomics And Main Component In Vivo Dynamics

Purpose:On the basis of identifying the biomarkers of spleen deficiency,to study the different regulaory effect of crude Atractylodes Lancea（AL）and bran-processed AL on the endogenous metabolites composition and level in the serum and urine of rats with spleen deficiency,to clarify mechanisms of bran-processed AL with better curative effect and the difference of potential biomarkers in rats after administration of crude and bran-processed AL;to study the similarities and differences on the parameters of pharmacokinetic and excretory kinetics in rats after administration of crude and bran-processed AL,to clarify the synergism of bran-processed AL from the point of main component metabolism and elimination.In summary,this paper is to clarify the processing mechanisms of AL by means of metabolomics,pharmacokinetics and excretory kinetics.Material and method:1 MaterialThe crude AL was purchased from Beijing Tong Ren Tang Pharmacy of Shenyang Branch,and authenticated by Prof.Feng Li,from the School of Pharmacy,Liaoning University of Traditional Chinese Medicine.According to the procedures described in Chinese Pharmacopoeia（2015）,the bran-processed AL was stir-fried with wheat bran.Sennae folium was purchased from Cheng Da Fang Yuan of chain pharmacies in Shenyang shop.Butyramine was purchased from Xi’an Janssen Pharmaceutical Co.,Ltd.Shen Ling Bai Zhu powder was purchased from Shanxi Hua Kang Pharmaceutical Co.,Ltd.ELISA kits were purchased from Shanghai Lian Shuo biological technology Co.,Ltd.TRIzol was purchased from Texas mamba trading company.Reverse transcription kit was purchased from Takara.Methanol and acetonitrile were purchased from merck,germany.Formic acid was purchased from Shanghai CNW company.AtractylosideⅠ,atractylosideⅡ,atractylosideⅢ,atractyloside A,acetaminophen were purchased from Jiangsu Yong medical technology Co.,Ltd.,the purities of these compounds were more than 98%.2 Method2.1 The pharmacodynamics study of the crude AL and bran-processed AL on rats with spleen deficiency120 SPF SD rats were randomly divided into 10 groups（n=12）:control group（CG）,model group（MD）,Shen Ling Bai Zhu Powder group（SL）,domperidone group（DO）,bran-processed AL at a high dose group（HP）,bran-processed AL at a medium dose group（MP）,bran-processed AL at a low-dose group（LP）,crude AL at a high-dose group（HC）,crude AL at a medium dose group（MC）,and crude AL at a low-dose group（LC）.For the spleen deficiency modeling,the rats in model group were treated by excessive exertion combined with irregularly dieting,and extract of sennae folium were used for intragastric administration.The crude and bran-processed AL were prepared into high,medium and low concentrations and gavaged for 10 days.Enzyme-linked immunosorbent assay（ELISA）was used to determine the contents of trypsin and amylase in the serum of rats related to changes in digestive enzyme activity,the content of vasoactive intestinal peptide,somatostatin,gastrin and substance P associated with gastrointestinal hormone levels,the content of,NA⁺-K⁺-ATPase related to membrane protein function,and the content of succinate dehydrogenase related to mitochondrial enzyme activity.The relative expression of vasoactive intestinal peptide,somatostatin,gastrin,and substance P in the small intestine of rats was measured by reverse transcription-polymerase chain reaction（RT-PCR）.2.2 Metabolomic study on spleen deficiency rats after administration of crude AL and bran-processed ALThe serum and urine of these rats from CG（n=10）,MD（n=10）,HP（n=10）and HC（n=10）were collected,and analyzed by means of UPLC-Q/TOF-MS.Chromatographic conditions were optimized,the type of ion source were determined,MassLynx 4.1software was applied to the peak identification and peak matching.After the normalization of integral data,the multidimensional statistical analysis was made by SIMCA_P.The unsupervised pattern recognition（principal component analysis PCA）,supervised pattern recognition（partial least square discriminate analysis PLS-DA）and orthogonal partial least squares-discriminate analysis OPLS-DA)were used to analyze the datas.The score map,OPLS-DA load map and VIP value（VIP>1）were used to find the difference variable,then t-test was used to verify the selected difference variables.Only the difference variables with p<0.05 were considered as differential metabolite.Based on these differences,combined with references and METLIN,HMDB and KEGG databases,differential metabolites were identified.Firstly the differential metabolites between the blank control group and the model group were identified,and then the metabolic pathways were analyzed according to these differential metabolites.Furthermore,the potential biomarkers which were adjusted by crude and bran-processed AL were identified respectively,and applied the metabolic pathway the metabolic pathways were analyzed according to these differential potential biomarkers.2.3 Pharmacokinetics of main components in rats after administration of crude AL and bran-processed ALThe rats were randomly divided into crude AL group（n=6）,bran-processed AL group（n=6）.Rats were gavaged with the crude AL and bran-processed AL（2.5 g/mL）extract.Bloods were collected from the retro-orbital plexus after administration of crude AL and bran-processed AL at 0.083,0.016,0.25,0.5,1,1.5,2,3,4,6,8,10,12 h.The methol was used to precipitaing protein;paracetamol was as an internal standard.The mobile phase was eluted with 0.1%formic acid in water and acetonitrile by gradient elution with a flow rate of 0.3 mL/min.The column was Waters ACQUITY UPLC BEH C18（1.7μm,2.1×100 mm）with temperature at 40℃.Electro spray ionization（ESI⁺）and multiple reactions monitoring（MRM）were used for detection.After the rats were gavaged with the crude AL and bran-processed AL extract,by means of above methods,The concentrations of atractyloside I,atractyloside II,atractyloside III and atractyloside A in rat plasma were determined.All the results were processed by DAS3.2.8 software to calculate the pharmacokinetic parameters（peak time,peak concentration,elimination time and so on）of above components,the drug concentration-time curves were plotted.2.4 Excretary Kinetics of Main Components in rats after administration of crude AL and bran-processed ALThe rats were randomly divided into crude AL group（n=6）,bran-processed AL group（n=6）.Rats were gavaged with the crude AL and bran-processed AL（2.5 g/mL）extract.Urine were collected at 0,3,6,9,12,24,36,48,60,72 h.The methol was used to precipitaing protein,paracetamol was as an internal standard.The mobile phase was eluted with 0.1%formic acid in water and acetonitrile by gradient elution with a flow rate of 0.3 mL/min.The column was Waters ACQUITY UPLC BEH C18（1.7μm,2.1×100 mm）with temperature at 40℃.Electro spray ionization（ESI⁺）and multiple reactions monitoring（MRM）were used for detection.After the rats were gavaged with the crude AL and bran-processed AL extract,by means of above methods,the concentrations of atractyloside I,atractyloside II,atractyloside III and atractyloside A in rat plasma were determined.All the results were processed by DAS3.2.8 software to calculate the excretion dynamics parameters based on the excretion-time data,and plotted the cumulative excretion-time curve.Results:1.Pharmacodynamics study of crude AL and bran-processed AL on rats with spleen deficiency.Compared with the GC,the levels of trypsin,amylase,vasoactive intestinal peptide,somatostatin,gastrin,substance P,NA⁺-K⁺-ATPase,and succinate dehydrogenase in the MD were significantly decreased（p<0.05）,each drug delivery group could significantly increase the content of the above indicators（p<0.05）.After further comparison of the same dose of crude AL and bran-processed AL,it was found that the contents of the above indicators increased more significantly after the AL were processed with bran.Compared with the blank GC,the relative expression levels of vasoactive intestinal peptide,somatostatin,gastrin and substance P in the MD were significantly decreased（p<0.05）,each drug delivery group could significantly increase the relative expression levels of the above indicators（p<0.05）.After further comparison of the same dose of crude AL and bran-processed AL,it was found that the relative expression level of the above indicators increased more significantly after the AL were processed with bran.2.Metabolomic study on spleen deficiency rats after administration of crude AL and bran-processed ALIn the PLS-DA model construction,the GC and the MD were well separated.Each drug delivery group was separated from the MD and were close to the GC,which indicated that the characteristics of the metabolites in the drug delivery groups were significantly different from the MD.Compared with the GC,the levels of D-urobilinogen and 7 substances in the serum increased significantly（p<0.05）,while the levels of L-tyrosine and 7 substances significantly decreased（p<0.05）.These substances were speculated to be differential metabolites associated with spleen deficiency.After analysis of differential metabolites,these 14 compounds were found to be related to the10 metabolic pathways.On the basis of clarifying the differential metabolites and metabolic pathways associated with the spleen deficiency,the serums of rats after administration of crude AL and bran-processed AL were studied.Both crude and bran-processed AL can regulate some potential biomarkers,but only in the regulation of inosine triphosphate and leukotriene C₄,crude AL is superior to bran-processed AL.In the regulation of bilirubin,S-adenosylhomocysteine,tyramine,phosphocholine and S-adenosylmethionine,bran-processed AL is better than crude AL.From the perspective of serum metabolomics,the mechanisms of processing AL with bran were explained.In the PLS-DA model construction,the GC and the MD were well separated.Each drug delivery group was separated from the MD and were close to the GC,which indicated that the characteristics of the metabolites in the drug delivry group were significantly different from the MD.Compared with the GC,the levels of L-proline and10 substances significantly increased（p<0.05）;while the levels of cysteine and 16substances significantly cecreaesd（p<0.05）.These substances were speculated to be differential metabolites associated with spleen deficiency.Analysis of different metabolites revealed that the 26 compounds were associated with the 16 metabolic pathways.On the basis of clarifying the different metabolites and metabolic pathways associated with spleen deficiency,the urines of rats after administration of crude AL and bran-processed AL were studied.Both crude AL and bran-processed AL can regulate some potential biomarkers,but only in the regulation of citicoline and cysteine,crude AL is better than bran-processed AL.In the regulation of L-fucose-1-phosphate,argininosuccinate and L-cystine,bran-processed AL is better than crude AL.From the perspective of urine metabolomics,the mechanisms of processing AL with bran were explained.3.Pharmacokinetics of main components in rats after administration of crude AL and bran-processed ALAtractyloside I,atractyloside II,atractyloside III and atractyloside A had a good linearity（R²≥0.9919）in the range of determination concentration,the precision and accuracy were all within±15%,which accorded with the requirements of the Biological Samples Quantitative analysis method validation guidelines in Chinese Pharmacopoeia 2015 edition.This method is of high specificity,high sensitivity and good precision.It can be used in the pharmacokinetic study of atractyloside I,atractyloside II,atractyloside III and atractyloside A in the serums of rats after administration of crude AL and bran-porcessed AL.Compared with the crude AL group,the Cmax of atractyloside I,atractyloside II,atractyloside III,and atractylenoside A in the bran-porcessed AL were all significantly increased（p<0.05）;the T_1/2 of atractyloside I,atractyloside II,atractyloside III and atractylenoside A in the bran-porcessed AL were significantly shortened（p<0.05）;the AUC_（0-t）of atractyloside I,atractyloside II,and atractyloside III in bran-processed AL were significantly increased（p<0.05）.The pharmacokinetic parameters showed that the bioavailability of 4 components in the plasma of bran-processed was higher than that of crude AL.4.Excretary kinetics of main components in rats after administration of crude AL and bran-processed ALAtractylosideⅠ,atractylosideⅡ,atractylosideⅢand atractyloside A had good linearity（R²≥0.9949）within the range of determination concentration,the precision and accuracy were all within±15%,which accorded with the requirements of the biological samples quantitative analysis method validation guidelines in Chinese Pharmacopoeia2015 edition.This method is of high specificity,high sensitivity and good precision.It can be used in the study on excretary dynamics of atractyloside I,atractyloside II,atractyloside III and atractyloside A in the urines of rats after administration of crude AL and bran-processed AL.Compared with the crude AL group,the total urine output of atractyloside I,atractylosideⅡand atractyloside A in bran-processed AL significantly increased（p<0.05）;the total urine output rate of atractyloside I,atractyloside II,atractyloside III and atractyloside A in bran-processed AL also significantly increased（p<0.05）.The excretary kinetics showed that the excretion rates of above 4components in the urine of excretion rate was higher than that of crude AL.Conclusion:1.Both crude and bran-processed AL alleviated symptoms of spleen deficiency.Comparing with crude AL,bran-processed AL was more effective in treating spleen deficiency.The efficacy of AL could be partly attributed to increase the digestive enzyme activity,gastrointestinal hormone levels,membrane protein activity and mitochondrial activity.2.L-tyrosine,D-urobilinogen,Bilirubin and other substances may be the characteristic metabolites of spleen deficiency.Aminoacyl-tRNA biosynthesis,alanine,aspartate and glutamate metabolism,tyrosine metabolism,et al pathway may be characteristic metabolic pathways in rats with spleen deficiency.Both crude and bran-processed AL show regulating effects on potential biomarkers,but bran-processed AL have better regulating effects than crude AL.3.Processing AL with bran can promote the absorption of atractylosideⅠ,atractylosideⅡ,atractylosideⅢand atractyloside A in the serums of rats.4.Processing AL with bran can promote the excretion of atractylosideⅠ,atractylosideⅡ,atractylosideⅢand atractyloside A in the urine of rats.The above conclusions elucidate the mechanism of processing AL with bran from the aspects of metabolomics,pharmacokinetics and excretary kenitics.