Quality Evaluation And Pharmacological Study Of Three "Dida" Original Plants

Objective The efficacy and material basis of Swertia plant,one of the original plants of"Dida",was investigated based on in vitro liver injury model.The result will provide reference for promoting the development of medicinal resources and clinical application.Methods（1）Delphi method was adopted.We invited experts who engaged in Tibetan medicine.The problems existing in the utilization of"Dida"medicinal resources and clinical application were evaluated by the experts until a consensus was reached.（2）The related targets of 26 Swertia species for treating icteric hepatitis were selected by network pharmacological method.The protein-protein interaction network was constructed by String,the network topology map was analyzed by Cytoscape 3.7.1software.The gene ontology（GO）and kyoto encyclopedia of genes and genomes（KEGG）enrichment analysis were analyzed by DAVID 6.8 platform.The cell viability and related gene level of LO2 hepatocyte injury model induced by H₂O₂ were measured by MTT and quantitative real-time PCR（q RT-PCR）.（3）The main iridoid glycosides in three Swertia species,Swertia bimaculata（Sieb.et Zucc.）Hook.f.et Thoms.ex C.B.Clark water extracts（SBH）,Swertia mussotii Franch water extracts（SMF）and Swertia pseudochinensis Hara water extracts（SPH）,were determined by high pressure liquid chromatography.The cell viability of LO2 hepatocyte injury model induced by CCl₄was measured by CCK-8.The EC₅₀values were calculated.The level changes of relevant m RNA in the cells were detected by q RT-PCR.The material basis and spectrum-activity relationship were further assessed by grey relational analysis combined with molecular docking.（4）SD rats were randomly divided into 5 groups.A prophylactic 7-day gavage dosing regimen was used in the experiment.The bile flow rate of rats was calculated by weight analysis,histopathological changes of liver were observed by H&E staining,and serum biochemical indexes were detected by blood sampling from the femoral artery of rats.The changes of relevant m RNA levels in liver tissues were detected by q RT-PCR.Results（1）A total of 33 experts participated in the two rounds of consultation.A consensus was finally reached on 16 items,and 2 items related to the characteristics and compatibilities of"Dida"had not reach common views.The consensuses of items mainly cover the original name of"Dida"and the evolution of geographic information,the rational selection of medicinal materials,the effectiveness and safety of clinical use.（2）A total of 52 active components were screened.The number of intersection targets with icteric hepatitis were 189.A total of119 signaling pathways were screened.The core targets such as MAPK1,PIK3CA,PIK3R1,HSP90AA1,NFκB1,which were mainly enriched in PI3K-Akt signaling pathway,hepatitis B,Ras signaling pathway.Luteolin（LUT）,oleanolic acid（OA）,sweroside（SD）,swertiamarin（SM）,gentiopicroside（GP）,and amarogentin（AG）improved liver injury by acting on the PI3K-Akt pathway.（3）SMF contained the highest amount of active components,especially SM.The EC₅₀ values of SBH,SMF and SPH were 0.3099,0.1029 and 0.4531,respectively.The three water extracts attenuated LO2 hepatocyte injury by regulating the m RNA levels of TGF-β1,Smad3,Smad4,Smad7,TRAF6 and TAK1.The docking score of SM was the largest and it had the strongest correlation with the pharmacodynamic index.（4）SBH significantly increased the bile flow rate of the cholestatic liver injury in rats,significantly reduced the ALT,AST,γ-GT,ALP,DBIL,TBIL and TBA levels in the serum,significantly up-regulated the m RNA levels of FXR,MRP2,BSEP,NTCP,and SHP,and significantly down-regulated the m RNA level of CYP7A1.Conclusions Swertia plants from"Dida"showed good efficacy in different hepatic injury models.Swertia bimaculata（Sieb.et Zucc.）Hook.f.et Thoms.ex C.B.Clark from Hubei has potential values to replace other species.This study provides a reference for the quality evaluation,resource sustainability and clinical application of Swertia plants from"Dida".