Study On Iridoids And Anti-inflammatory Activity In The Rhizomes Of Gentiana Macrophylla Pall.

Gentiana macrophylla Pall.is a well-known traditional Chinese medicine with the anti-inflammatory,analgesia and gastric motility strengthening effects.Although the iridoids are believed to be the bioactive composition of Gentiana macrophylla,the optimum extraction of these constituents and related product quality control or bioactivity evaluation is rarely reported.(1)Following process optimization by response surface modeling and Box-Behnken analysis,the iridoids were extracted with the yield of 7.75± 0.032%from rhizomes of Gentiana macrophylla Pall.in three cycles of extraction for 110.91 min in 69.16%ethanol at liquid-solid ratio 12.27(mL/g).The crude iridoids extract was further refined using HPD-300 resin to give the enriched product with the iridoids content up to 73.27%.(2)In the subsequent quality control research,7 iridoid constituents in the crude extract and refined product were identified and quantified by UPLC-Q-TOF-MS,respectively.At the same time,19 iridoid compounds with similar structure were identified by deducing retention time and mass spectrometry pyrolysis law.(3)The anti-inflammatory effects of the iridoids extracts were evaluated.Strikingly,the crude extract and refined samples significantly inhibited the expression of NO in lipopolysaccharide(LPS)-treated RAW264.7 macrophage cells,and there were significant differences.By means of western blotting analysis,the refined iridoid product was demonstrated to exert the anti-inflammatory bioactivity through significantly inhibited the expression of COX-2,iNOS.(4)On the basis of determining the difference of anti-inflammatory activity,principal component analysis(PCA)and partial least squares(PLS)were used to explore the difference between crude and refined samples,and the difference of metabolites between the two groups was analyzed.The results showed that there were 6 different metabolites in crude and refined samples,which meant that these different metabolites were the cause of the difference in anti-inflammatory activity between the two samples.