Studies On The Chemical Constituents And Mechanism Of Anti-Inflammatory And Analgesic Effects Of Zhuang Medicine Stahlianthus Involucratus(King Ex Bak.) Craib.

Objective:（1）The IR fingerprint of Stahlianthus involucratus（King ex Bak.）Craib.was established.（2）Chemical constituents from the ethanol part,petroleum ether parts and ethyl acetate parts of Stahlianthus involucratus（King ex Bak.）Craib.were isolated and identified by UPLC-Q-Orbitrap HRMS.（3）Through in vitro experiments and in vivo experiments,to study on anti-inflammation and analgesic effect of Stahlianthus involucratus（King ex Bak.）Craib.,to study anti-inflammatory effect and analgesic effect and pharmacodynamic material basis for it.（4）Through in vitro cell experiments,to study anti-inflammatory effect of Stahlianthus involucratus（King ex Bak.）Craib.alcohol extract on blood-heat combined hemorrhage model rats,and to study anti-inflammatory effect,analgesic effect and pharmacodynamic material basis for it.Methods:（1）The fingerprint of 7 batches of Stahlianthus involucratus（King ex Bak.）Craib was established by IR.Used the OMNIC softwarewas to obtain the contrast IR fingerprint and the common characteristic peak of Stahlianthus involucratus（King ex Bak.）Craib.The 7 batches of samples were analyzed by similarity analysis（SA）,hierarchical cluster analysis（HCA）and principal component analysis（PCA）to evaluate the quality.（2）Chemical constituents from the ethanol parts,petroleum ether parts and ethyl acetate parts of Stahlianthus involucratus（King ex Bak.）Craib.were isolated and identified by UPLC-Q-Orbitrap HRMS.Stahlianthus involucratus（King ex Bak.）Craib was isolated on a Thermo Hypersil Gold C18 column（2.1mm×100mm,1.9μm）.The mobile phase was acetonitrile containing 0.1%formic acid and water containing 0.1%formic acid（Containing 10 mmol ammonium acetate）by gradient elution.Chemical constituents of the ethanol parts,petroleum ether parts and ethyl acetate parts of Stahlianthus involucratus（King ex Bak.）Craib were rapidly isolated and identified by HRMS in the positive and negative ion mode using both full scan and two stage threshold-triggered mass modes.（3）（1）The cultured cells were treated with different concentrations of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib（0,12.5,25,50,100,200μg/m L）for 24h,48h,72h respectively,and cell viability was determined by CCK-8 reagent,to detect the optimal administration concentration and time of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib.CCK-8 reagent was used to detect the cell viability of RAW264.7 cells damaged by lipopolysaccharide（LPS）.（2）Cotton pellet granuloma of the mice and contents of IL-6,TNF-α,PGE₂and IL-1βwere measured by ELISA method was used to detect the anti-inflammatory activity.Acetic acid writhing method and contents of IL-6,TNF-α,5-HT and c AMP were measured by ELISA method was used to study the analgesic activity.（4）To preparation of Stahlianthus involucratus（King ex Bak.）Craib.alcohol extract on blood-heat combined hemorrhage model rats.The cultured cells were treated with different concentrations of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib alcohol extract on blood-heat combined hemorrhage model rats（0%,2%,4%,6%,8%,10%,15%,20%）for 24 h,48h,72h respectively,and cell viability was determined by CCK-8 reagent,to detect the optimal administration concentration and time of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib alcohol extract on blood-heat combined hemorrhage model rats.CCK-8 reagent was used to detect the cell viability of RAW264.7 cells damaged by lipopolysaccharide（LPS）.The contents of NO,TNF-α,IL-1β,IL-6 in the supernatant were measured by enzyme-linked immunosorbent assay（ELISA）.Results:（1）There were 10 common peaks in the IR fingerprint of 7batches Stahlianthus involucratus（King ex Bak.）Craib,the similarity of 7batches samples were over 0.9.The 7 batches of Stahlianthus involucratus（King ex Bak.）Craib could be divided into 3 grades in the result of HCA.PCA got 4 principal components through dimension reduction.The PCA spatial score plot was consistent with the results of hierarchical cluster analysis.（2）Based on UPLC-Q-Orbitrap HRMS technical analysis and identification,a total of 14 Stahlianthus involucratus（King ex Bak.）Craib compounds were obtained.（1）Terpenoid:Curcumol,Curdione,Camphor,α-Cyperone;（2）Organic acids:Azelaic acid,Citric acid;（3）Phenolic compound:Protocatechualdehyde;（4）Flavanoid:Isoschaftoside;（5）Nitrogenous compound:Uridine,L-Tyrosine,Nicotinamide,L-Tryptophan;（6）Others:Mannitol,3-Butylidenephthalide.（3）（1）Study on the vitro cell anti-inflammatory effect of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib:The optimal concentration of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib was 12.5、25、50μg/m L and72h.Compared with the LPS group,the high,medium and low doses of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib can enhance the cell activity.（2）Study on the vivo animal anti-inflammatory effect of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib:Compared with the normal group,the model group contents of IL-6,TNF-α,PGE₂,IL-1β,5-HT in serum of the mice were increased,the contents of c AMP in serum of the mice were decreased,with significant statistical difference（P<0.01）.Compared with the model group,Stahlianthus involucratus high,middle and low dose groups（52.25、34.83、26.13 g crude drug/kg）showed obvious inhibitory effects in cotton pellet granuloma and significant decrease in writhing frequency,with significant statistical difference（P<0.05,P<0.01）.The contents of IL-6,TNF-α,PGE₂,IL-1β,5-HT in serum of the mice were decreased,the contents of c AMP in serum of the mice were increased,with significant statistical difference（P<0.05,P<0.01）.（4）Study on the in vitro action of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib extract rats:The optimal concentration of headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib extract rats was 4%、15%and 24h.Compared with the LPS group,the drug-containng serum can ehhance the cell activity and reduction of NO、TNF-α、IL-1β、IL-6 content in each dose group（P<0.01）.Conclusion:（1）The fingerprints of Stahlianthus involucratus（King ex Bak.）Craib from 7 places have good similarities with the fingerprints in the control group,and there are certain differences in the types and contents of chemical components among Stahlianthus involucratus（King ex Bak.）Craib from different places.（2）The chemical constituents of Stahlianthus involucratus（King ex Bak.）Craib are mainly include terpenes and organic acids,most of them have anti-inflammatory and analgesic effects.（3）The headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib shows stronger anti-inflammatory and analgesic activities,the anti-inflammatory mechanism may be related to decreasing the cell resistance to damage and the contents of IL-6,TNF-α,IL-1β,PGE₂in serum;the analgesia mechanism may be related to decreasing the contents of IL-6,TNF-α,5-HT and increasing the content of c AMP in serum.（3）The headquarters parts of Stahlianthus involucratus（King ex Bak.）Craib extract rats has protective effect on RAW264.7 cells of LPS injury,the mechanism may be related to decreasing the cell resistance and reducing the NO、IL-6、IL-1βand TNF-αof inflammatory cytokines in vivo to damage.