TCM Serum Pharmacochemistry And Network Pharmacology-based Analysis Of Quality Markers Of Pericarpium Citri Reticulatae

Pericarpium Citri Reticulatae(PCR),a common Chinese herbal medicine,was the dried and mature peel of Citrus reticulata Blanco and its cultivated varieties.It has the function of regulating qi,eliminating phlegm,strengthening spleen and stopping diarrhea.At present,the quality control indexes of PCR were only hesperidin and synephrine,which were lack of specificity and were not strong correlation with specific diseases in the compound compatibility environment,so it was difficult to accurately evaluate the effectiveness of PCR.The identification of pharmacodynamic material basis was the key to improve the quality standard of traditional Chinese medicine(TCM).Based on the TCM theory,Q-Marker focused on the overall quality characterization and diversified control from the perspective of effectiveness,with a view to establishing the quality control of the whole process of TCM with specificity,transferability,and traceability.Therefore,the research method of Q-markers of PCR was established by the integration strategy of TCM serum pharmacochemistry and network pharmacology based on Ultra performance liquid chromatography tandem quadrupole time of flight mass spectrometry technology(UPLC-Q-TOF-MS),providing new ideas for the selection of quality control indicators of TCM,and providing a experimental basis for further improving the quality control level of PCR.Objective:In accordance with the research method of “spectrum-effect-net” and“Q-Marker” theory,the whole component analysis of PCR medicinal materials,PCR decoction pieces and Chenpi Huoxiang Decoction(CHD)was analyzed.On the premise of effectiveness,serum pharmacochemistry and network pharmacology techniques were integrated to screen the pharmacodynamic material basis of PCR in the compatible environment of CHD.The Q-markers of PCR were predicted based on the five principles of “Q-Marker”,which provided theoretical reference for improving the quality standard of PCR.Methods:1.The diarrhea rat model was reproduced by intragastric administration of senna leaf decoction(0.5 g/ml).The general morphological characteristics,body weight,diarrhea rate,diarrhea index,intestinal ink propelling rate and histopathological morphology of small intestine and colon of rats were used as evaluation indexes to evaluate the success of the model and the effectiveness of CHD.2.A UPLC-Q-TOF-MS technology equipped with Waters ACQUITY UPLC(?)HSS T3 1.8 μm(2.1×100 mm)column was used for samples analysis.The column temperature at 30℃,injection volume at 4μl and flow rate at 0.3 ml/min were set.0.1% formic acid acetonitrile(A)and 0.1% formic acid water(B)were used for gradient elution.The compounds of PCR medicinal materials,PCR decoction pieces and CHD were characterized based on reference substance,literatures and Natural products HR-MS/MS spectra database search.3.TCM serum pharmacochemistry combined with UPLC-Q-TOF-MS technology and multivariate pattern recognition method was used to conduct a comprehensive analysis of the blood components of CHD.Significant variables were discovered by comparing the serum of the administration group and model group.The prototype blood components were screened based on the previous identification results of the whole components of PCR in vitro.4.Taking the blood component as the research object,the blood component and diarrhea related targets were retrieved,and the GO and KEGG signal pathway enrichment analysis was performed on the intersection targets of the two.According to network topology parameters,the top active ingredients and targets were determined,and the molecular virtual docking was carried out.The binding energy of less than-5Kcal/mol was used to determine the binding ability of blood components and proteins.Finally,the predicted key targets were verified by enzyme linked immunosorbent assay(ELISA),and then the pharmacodynamic material basis of CHD in the treatment of diarrhea was clarified.5.According to the five principles of “Q-Marker”,the effectiveness of CHD was clarified by combining compound compatibility and pharmacodynamic evaluation of animal experiments.The whole chain of PCR medicinal materials,PCR decoction pieces and CHD and blood components were used to investigate the transmission and traceability of pharmacodynamic material basis.The specificity and measurability of pharmacodynamic material basis were determined by combining with literature studies,and finally the Q-markers of PCR were determined.Results:1.Compared with the blank group,the model group showed loose and haggard hair,poor glossiness,redness and swelling around the anus,lethargy,loss of appetite,emaciation and other symptoms.The body weight gain was slow or even stagnant,the diarrhea rate and diarrhea index were increased,the small intestine ink propelling rate decreased.The small intestine showed mucosal congestion and edema,villi exfoliation,mucosal structural defects and a large number of inflammatory cell infiltration.The colon showed mucosal epidermal cell necrosis and shedding,intestinal gland atrophy,the number of cup cells decreased,and inflammatory cell infiltration in lamina propria.Compared with model group,the spirit,hair gloss,diet and body weight of rats in PCR group and CHD group were significantly improved.The diarrhea rate and diarrhea index were decreased,and the intestinal ink propelling rate was increased.Although both groups could improve the intestinal peristalsis function of rats,the efficacy of the CHD group was better than that of PCR group in the diarrhea index and intestinal ink propulsion function on the first day of administration.In addition,the histopathologic changes of the small intestine and colon were significantly alleviated,the structure was clear and complete,the congestion and edema of the intestinal tissue were greatly improved,the villi were basically not shed,the villi and goblet cells were evenly arranged,and a small amount of lymphocytes were infiltrated,indicating the effectiveness of CHD.2.85 chemical constituents of PCR medicinal materials,85 constituents of PCR decoction pieces,and 115 constituents of CHD were identified by UPLC-Q-TOF-MS technique.All three mainly included flavonoids,sugars,amino acids,alkaloids,organic acids,such as hesperidin,neohesperidin,naringin,caffeoylglucose,nomilin glucoside,synephrine,etc.,among which flavonoids were the main components of PCR.3.According to the theory of serum pharmacochemistry,15 blood components from PCR single decoction and 18 blood components from CHD were characterized.The prototype blood components in PCR single decoction were hesperidin,neohesperidin,naringin,nobiletin,3,5,6,7,8,3’,4’-hepmethoxyflavone,naringenin 7-O-glucoside etc.And 18 prototype blood components in CHD included hesperidin,naringin,neohesperidin,nobiletin,3,5,6,7,8,3’,4’-hepmethoxyflavone,isorhamnetin-3-O-rutinoside,naringenin 7-O-glucoside,phloretin-3’5’-di-C-glucoside,lonicerin,sinapinic acid,etc.4.Through database screening,6243 diarrhea targets,468 targets of18 blood-entering components,and 177 intersection targets of blood components and diarrhea were screened out.GO and KEGG bioenrichment analysis were performed.It was concluded that hesperidin,nobiletin,3,5,6,7,8,3’,4’-heptemthoxyflavone,naringin,neohesperidin and other active ingredients in CHD,which might act on PI3K-Ak,JAK-STAT,HIF-1,MAPK,AGE-RAGE,TNF and other signaling pathways by regulating ALB,TNF,IL-6,AKT1,EGFR and other target proteins,thus can inhibit intestinal inflammatory response and protect intestinal mucosa.5.Referring to the five principles of “Q-Marker”,hesperidin,nobiletin,3,5,6,7,8,3’,4’-heptemthoxyflavone,naringin,and neohesperidin were preliminatively identified as Q-Markers for PCR.Conclusion:In this study,the diarrhea model was successfully replicated by senna leaf,and the therapeutic effect of CHD on diarrhea was confirmed by the diarrhea rate,diarrhea index,intestinal ink propelling rate,and histopathological morphological indexes.The pharmacochemical material basis of PCR medicinal materials,PCR decoction pieces and CHD was characterized by UPLC-Q-TOF-MS-based serum pharmacochemistry and network pharmacology.Finally,according to the five principles of Q-marker,hesperidin,nobiletin,neohesperidin,naringin,3,5,6,7,8,3’,4’-heptemthoxyflavone were taken as Q-markers of PCR,providing theoretical methods and experimental basis for quality control of PCR.