UPLC-Q-Exactive/MS-based metabonomics revealed protective effect of Zingiberis rhizome and its processed product on deficiency-cold and hemorrhagic syndrome rats.

Zingiberis rhizome carbonisata (ZRC) is the processed product of Zingiberis rhizome (ZR). ZR is mainly used for warming the spleen and stomach to dispel cold, whereas ZRC is commonly applied as a treatment for deficiency-cold and hemorrhagic syndrome (DCHS). Although they have long been used to serve different clinical purposes, the specific action mechanism of the drugs and molecular changes underlying ZR processing are not clear. In this study, metabolomics study was carried out to analyze the alterations in endogenous metabolites in serum and urine samples of DCHS rat models using ultra-high-performance liquid chromatography coupled with quadrupole-Exactive mass spectrometry technique and constructed principal component analysis score plots that showed that the ZRC group was completely separated from the DCHS and ZR groups but demonstrated a highly close plotting to the normal control group. The results revealed that both ZR and ZRC intervened in the metabolic pathways of DCHS models but to varying degrees and with different influencing factors. In addition, ZRC was found to function as a treatment for the metabolic disorders of DCHS through 15 pharmacodynamic biomarkers involving a series of pathways, such as glycine, serine, and threonine metabolic pathway, as well as arachidonic acid metabolic pathways. This study showed that metabolomics method based on ultra-high-performance liquid chromatography coupled with quadrupole-Exactive mass spectrometry could preliminarily illuminate the therapeutic mechanism of ZR and ZRC on DCHS and the changes in ZR processing from the molecular-level perspective. The results also provided new insight into further research on DCHS treatment.

Identifying of Anti-Thrombin Active Components From Curcumae Rhizoma by Affinity-Ultrafiltration Coupled With UPLC-Q-Exactive Orbitrap/MS.

Recent studies concerning products that originate from natural plants have sought to clarify active ingredients, which both explains the mechanisms of the function and aids in quality control during production. As a traditional functional plant, Curcumae Rhizoma (CR) has been proven to be effective in promoting blood circulation and removing blood stasis. However, the components that play a role in its huge compound library are still unclear. The present study aimed to develop a high-throughput screening method to identify thrombin inhibitors in CR and validate them by in vitro and in vivo experiments. The effect of CR on thrombin in HUVECs cells was determined by ELISA, then an affinity-ultrafiltration-UPLC-Q-Exactive Orbitrap/MS approach was applied. Agatroban and adenosine were used as positive and negative drugs respectively to verify the reliability of the established method. The in vitro activity of the compounds was determined by specific substrate S-2238. The in vivo effect of the active ingredients was determined using zebrafish. Molecular docking was used to understand the internal interactions between compounds and enzymes. ELISA results showed that CR had an inhibitory effect on thrombin. The screening method established in this paper is reliable, by which a total of 15 active compounds were successfully identified. This study is the first to report that C7, 8, and 11 have in vitro thrombin-inhibitory activity and significantly inhibit thrombosis in zebrafish models at a safe dose. Molecular docking studies were employed to analyze the possible active binding sites, with the results suggesting that compound 16 is likely a better thrombin inhibitor compared with the other compounds. Based on the affinity-ultrafiltration-UPLC-Q-Exactive Orbitrap/MS approach, a precisely targeted therapy method using bio-active compounds from CR might be successfully established, which also provides a valuable reference for targeted therapy, mechanism exploration, and the quality control of traditional herbal medicine.