A new strategy for the preparation of total iridoids from Radix Gentianae Macrophyllae and anti-inflammatory profile digesting by UPLC-Q-TOF-MS characterization coupled with PLS analysis

Abstract

Abstract Radix Gentianae Macrophyllae (RGM) is a well-known traditional Chinese medicine with anti-inflammatory, analgesia and gastric motility strengthening effects. The iridoids are believed to be the RGM bioactive ingredients, however, an effective strategy for its total iridoids extraction and purification is lacking. In addition, a reliable method for the bioactive iridoid constituents digesting needs to be developed. In the present work, followed by process optimization via response surface modeling and Box-Behnken analysis, the maximum yield of total RGM iridoids was predicted to be 7.75 % from rhizomes of Gentiana macrophylla Pall. via three cycles of extraction in 69.16 % ethanol at liquid-solid ratio 12.27 (mL/g) for 110.91\u202fmin. The crude iridoid extract was further purified using HPD-300 macroporous resin to give the enriched product with the iridoid content up to 73.27 %. The anti-inflammatory effects of refined iridoid extracts were quantitatively measured to validate the purification procedure. The refined extract was detected to significantly inhibit the expression of NO through expression inhibition of COX-2, iNOS in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells with the IC50 of 10.49\u202fμg/mL, which was stronger than that of crude extract with the IC50 of 51.11\u202fμg/mL, indicating the iridoid compounds were enriched from crude extract through the purification process. Subsequently, an UPLC-QTOF-MS method was established for qualitative and quantitative analysis of the iridoid constituents in the crude and refined extracts. In addition, based on the UPLC-QTOF-MS data, 6 principle iridoids that give the most important support for the anti-inflammatory activities were digested by means of PCA and PLS analysis. The current study developed a new and effective strategy for optimal extraction, purification and identification of natural bioactive iridoids from Radix Gentianae Macrophyllae, which facilitates its further applications in pharmaceuticals and/or foods industry.