Study On Phenolic Substance Basis Of Gentianae Radix Et Rhizoma And Its Anti-inflammatory Activity

Gentianae Radix et Rhizoma,as a traditional Chinese medicine,has the effect of protecting liver and gallbladder.The 2020 edition of the Pharmacopoeia is only based on Gentiopicroside,an iridoid,to evaluate the quality of Gentianae Radix et Rhizoma medicinal materials,which was not enough to reflect the efficacy and resources.In this paper,the Gentianae radix et rhizoma phenolic components(GPC)and their biological activities were systematically studied,hoping to further analyze the material basis of its clinical efficacy and provide a reference method for the evaluation of Gentianae Radix et Rhizoma.Firstly,by UPLC-Q-TOF-MS/MS analysis,four phenolic compounds,Mangiferin,Esculetin,Ferulic acid and Kaempferol,were screened from GPC.Based on the four index components and total phenolic content of extract,a HPLC method was established for quantitative determination of the four index components.Optimization of extraction process of phenolic components by Box-Behnken response surface methodology: concentration of hydrochloric acid was 0.06 mol/L,ratio of liquid-solid was 132:1,and extraction time was6.6 hours.The total amount of four indicator components was 37.484 μg/g,and the error was only 1.27%.The maximum content of total phenol was10.03 mg/g.At the same time,16 batches of 4 species of Gentianae Radix et Rhizoma from different origins were evaluated for resources,and the analysis methods of ring heat map analysis,principal component analysis and Q-type hierarchical clustering were used to prove that the four indexes could effectively control the quality of Gentianae Radix et Rhizoma.Secondly,the anti-inflammatory effects and mechanisms of Gentianae radix et rhizoma phenolic extract(GPE)were studied at the cellular and whole animal levels.At the cellular level,GPE could effectively inhibit LPS-induced nitric oxide production in RAW264.7 macrophages,reduce inflammatory cytokines IL-6,IL-1β,TNF-α and increase IL-10 content.At the whole animal level,GPE effectively inhibited carrageenan-induced toe swelling in Mus musculus,and the inhibition rate was close to Aspirin and exceeded that of the same dose of Gentiopicroside.Further analysis of the toe tissue showed that the m RNA transcription levels of inflammatory cytokines TNF-α,IL-1β,IL-6,and IL-10 had the same trend as the results of the cellular inflammation model;Through Western Blotting analysis,it was found that GPE effectively reduced the expression of i NOS,COX-2,p65,p-p65 and p-IκB proteins.Therefore,it is speculated that the anti-inflammatory activity of GPE may play a role by inhibiting the i NOS/COX-2/NF-κB signaling pathway.Finally,by UPLC-Q-TOF-MS/MS analysis,a systematic chemical constituent study of representative GPE.59 compounds were qualitatively identified through Mass Bank,Sci Finder and Self-built database.The phenolic extract was administered to Mus musculus by gavage and its blood components were studied,and the phenolic components were analyzed in the blood components,and fourteen prototype components were identified,such as Swertianolin,Acacetin,Umbelliferone,caffeic acid.These compounds are predicted to play a key role in anti-inflammatory activity.