Differential distribution of main active components in Dalitong Granules in gastrointestinal tissues of normal and functional dyspepsia model rats
Objective The aim of this study was to explore the gastrointestinal tissue distribution differences of the main active compounds from Dalitong granules in normal and functional dyspepsia model rats.Method The rat model of FD was established by tail clipping stimulation combined with irregular diet(alternate day fasting).12 rats(half of them as controls and half as models)were used to validate the FD model,and gastric and small intestinal emptying experiments were conducted.After HE staining,pathological observations were made on the gastrointestinal tissues.36 rats(half as controls and half as models)were used for the study of differential distribution of gastrointestinal tissues,and they were fasted but not water-restricted for 18 hours.They were given Daliting Granules at a dose of 22.4 g·kg-1(8 times the clinical equivalent dose)once,and the gastric,duodenum,jejunum,ileum,and colon were collected from each group of rats at 1,3,and 6 hours after the administration of the drug.The contents of vitexin-4"-O-glucoside,protopine,coptisine,berberine,sinensetin,nobiletin,tangeretin,5-O-demethylnobiletin,costunolide,dehydrocostuslactone,protocatechuic acid,umbelliferone,cynaroside,hesperidin,luteolin,naringin,hesperetin,and chrysin were detected by UPLC-QTRAP-MS/MS method in the gastric,duodenum,jejunum,ileum,and colon of each group of rats.UPLC-QTRAP-MS/MS was used.Column was an ACQUITY HPLCTM HSS T3(50 mm × 2.1 mm,1.8 μm)and set at 40 ℃.Gradient elution was performed using 0.1％ formic acid water and acetonitrile as the mobile phase,the flow rate was 0.3 mL·min-1 and the injection volume was 2 μL.Results Compared with the control group,the body mass of rats in the model group was significantly decreased(P＜0.001),the emptying rate of the stomach and small intestine was significantly decreased(P＜0.001),the close connection of the duodenal mucosa was destroyed,but no organic lesions of the gastrointestinal tissue were found,which was consistent with the pathological characteristics of FD.For the established UPLC-QTRAP-MS/MS method,the correlation between the response and concentration of the compounds in gastrointestinal tissues met R2 ≥ 0.990 0.The results including the specificity,precision and accuracy,recovery rate,stability and matrix effects of the established method all met the requirements.The results showed that the concentration of the components varied greatly in the control group and the functional dyspepsia model group,and there were significant differences in the time points at which the highest concentration was reached in different tissues.In the control gastric tissue of rats,the tested compounds quickly reached the highest concentration within 1 h.However,the concentration of compounds decreased significantly,which is partially due to slow gastrointestinal motility in the model group.Hesperidin,hesperetin,naringin,protocatechuic acid,dehydrocostuslactone,nobiletin,5-O-demethylnobiletin,berberine,and tangeretin were found to be exposed at higher levels in the stomach,duodenum,jejunum,ileum and colon,and the concentration differences between the control group and the model group at each time point were significant.Conclusion The established method has high sensitivity,specificity and high accuracy,which was suitable for the tissue distribution study of the main active compounds of Dalitong granules in the rat gastrointestinal tissues.Significant differences were showed in the tissue of the functional dyspepsia model rats compared to control.
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