Discovery of Anti-Inflammatory Bioactivity Components in Dioscorea Nipponica Makino Based on Hplc-Ms/Ms, Quantitative Analysis of Multiple Components by Single Marker and Chemometric Methods

The rhizome of Dioscorea nipponica Makino (RDN) is a commonly used herbal medicine, which has significant anti-inflammatory activities on various inflammatory diseases. However, the bioactive compositions responsible for the anti-inflammatory effect of RDN are still unknown. The present study aimed to discover the anti-inflammatory bioactivity compounds in RDN using high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q/TOF-MS), quantitative analysis of multiple components by single marker (QAMS) and chemometric methods. Firstly, an HPLC-Q/TOF-MS method was employed for identification of bioactive steroidal saponins in RND, and a total of twelve steroid saponins were identified. Then, QAMS method was established and validated to simultaneously determine seven bioactive steroidal saponins, including protodioscin, protogracillin, methyl protodioscin, pseudoprotodioscin, pseudoprogracillin, dioscin and gracillin in RND samples using dioscin as the reference analyte. The anti-inflammatory activities of RDN samples were also evaluated by inhibition of NO production in LPS-induced RAW264.7 cells. Furthermore, chemometric methods, including Pearson correlation analysis and partial least squares regression (PLSR) were used to investigate the correlations between chemical components and anti-inflammatory activities, and explore the potential anti-inflammatory bioactive compounds of RDN. The results indicated that that there was a close correlation between chemical components and anti-inflammatory activities of RND, and protodioscin, dioscin and gracillin were selected as the major anti-inflammatory compounds in RND. The further verification experiments showed that protodioscin, dioscin and gracillin exhibited strong inhibition on NO production with the IC 50 values of 0.712 μM, 0.469 μM, 0.815 μM, and the three steroid saponins also could significantly reduce the levels of TNF-α, IL-1β, and IL-6 in LPS-stimulated RAW264.7 cells. The present research provided evidences for the anti-inflammatory activity of RND and discovered the anti-inflammatory compounds in RDN, which could be used for exploration of bioactive components and quality control of herbal medicines.