Research On The Mechanism Of Gentian Wine Production Based On The Difference Of Chemical Composition In Vivo And In Vitro

Purpose:UHPLC-LTQ-Orbitrap MS~n method was used to study the chemical constituents of Gentiana scabra before and after wine-processed,and to determine the difference of constituents between crude and wine-processed Gentiana Radix.HPLC method was used to simultaneously determine the contents of iridoids,and the similarities and differences between the two were clarified.The pharmacokinetics and tissue distribution of gentiopicroside in ratsof Gentiana scabra before and after wine-processed were determined by HPLC.Through the above these studies,the mechanism of the processing Gentiana scabra with wine was clarified from the point of view of chemical constituents,which could provide references for the optimization of processing Gentiana scabra with wine,the establishment of quality standards for processing Gentiana scabra with wine,and clinical medication.Material and method:1.UHPLC-LTQ-Orbitrap MS~n method was used for qualitative analysis of constituents in crude and wine-processed Gentiana Radix.PCA and PLS-DA were used to clarify the effect of processing with wine on the composition of the constituents of Gentiana Radix.2.The contents of VIP and three constituents with larger peak area of total ion flow diagram in the data of liquid chromatography-mass spectrometry(UHPLC-LTQ-Orbitrap MS~n)were determined,they are gentiopicroside,swertiamarin and swertiamarin(three constituents are iridoid terpenoids).HPLC method was used for analysis crude and wine-processed Gentiana Radix in 22 regions,the content differences of iridoid terpenoids in them.3.SD rats of SPF grade were randomly divided into blank control group,crude Gentiana Radix group and wine-processed Gentiana Radix group.these drug groups were given a certain dose of crude and wine-processed Gentiana Radix extract by gavage,while the blank control group was given the same amount of normal saline.At 0.083,0.016,0.25,0.5,1,1,1,5,2,3,4,6,8,10 and 12 hours after administration,rat orbital blood was taken.After sample treatment,the blood concentration of gentiopicroside was determined by HPLC.The data were processed by DAS 3.0 program,the pharmacokinetic parameters of gentiopicroside were compared,and the pharmacokinetic-time curve was drawn to clarify whether processing Gentiana scabra with wine could promote the absorption of the target component(gentiopicroside)and improve its absorption and bioavailability.4.SD rats were randomly divided into blank control group,crude Gentiana Radix group and wine-processed Gentiana Radix group.Crude Gentiana Radix group and wine-processed Gentiana Radix group were given a certain dose of the extract of crude and wine-processed Gentiana Radix by gavage.The blood concentration of gentiopicroside in different tissues(heart,liver,spleen,lung,kidney,stomach,large intestine and small intestine)at different time points was determined by HPLC.The distribution of different time points was calculated and the information of the target organs of raw gentian and wine gentian were obtained.Results:1.The results showed that 44 chemical constituents were identified in crude Gentiana Radix,42 chemical constituents were identified in wine-processed Gentiana Radix,34 of which had total chemical constituents,but 10 only existed in crude Gentiana Radix,and 8 only existed in wine-processed Gentiana Radix,.Because the sampling amount ofcrude and wine-processed Gentiana Radix is the same,the content change of the common chemical constituents can be known by comparing the peak area.When the ratio of peak area of raw gentian to peak area of wine(raw/wine)is less than 0.8,it showed that the content of raw gentian increases after wine-processing,while when the ratio of peak area is more than 1.2,it shows that the content of crude Gentiana Radix decreases after wine-processing.It can be seen that after the wine-processing of Gentiana,7 chemical constituents increased significantly,while 4 chemical constituents decreased significantly.Twenty distinct constituents(VIP>5)were screened from the experimental data.Six of them were found in the positive mode,they are depressoside,swertiamarin,gentianaside,primeverosyl-1-decussatin,6’-O-β-D-glucosyl gentiopicroside and oleanolic acid.Seventheen of them were found in the negative mode,they are gentiopicroside,depressoside,rindoside,8-O-glucosyl bellidifolin,swertiamarin, isoscoparine,6’-O-β-D-glucosyl gentiopicroside,loganic acid,1,3,4-trihydroxy-8-β-D-glucospranosyl-5,6,7,8-tetrahydroxanthone,eustomoside,glucosyl-8-swertianin,isovitexin-2’’-O-β-D-glucoside,isoscoparine-7-O-β-D-glucoside,sweroside,mangiferin,1-O-D-glucopyransylampexine and 1-hydroxy-2,3,4,7-tetramethoxy xanthone.Through the analysis of these different constituents,it was found that wine-processing could change the chemical constituents of Gentiana Radix.2.The contents of gentiopicroside,swertiamarin and sweroside were determined in crude and wine-processed Gentiana Radix in 22 different areas.It was found that the content of gentiopicroside was increased afterwine-processing Gentiana Radix,and the contents of swertiamarin and swertiamarin changed inconsistently.3.The pharmacokinetics of gentiopicroside in Gentiana Radix before and after wine-processing showed that the peak time of gentiopicrin reached earlier and the peak concentration decreased after wine-processing.4.Study the tissue distribution in Gentiana Radix before and after wine-processing showed that the distribution of gentiopicroside in the organs of Shangjiao(heart,liver and lung)was increased by wine-processing.Conclusion:1.It is clear that the difference constituents between crude and wine-processed Gentiana Radix.2.It is clear that gentiopicroside can be elevated after the wine-processing of Gentiana Radix,which lays a foundation for the study of the wine-processing mechanism Gentiana Radix.3.It is clear that gentiopicroside can accelerate the action speed of gentiopicroside after wine-processing Gentiana Radix.4.The distribution of gentiopicroside in the organs of Shangjiao(heart,liver and lung)was increased after wine-processing Gentiana Radix.It was speculated that gentiopicroside was one of the reasons for the promotion uplifting after wine-processing Gentiana Radix.