Optimization of the extraction process and metabonomics analysis of uric acid-reducing active substances from Gymnadenia R.Br. and its protective effect on hyperuricemia zebrafish

BACKGROUND: As Gymnadenia R.Br. (Gym) has an obvious uric acid-lowering effect, but its specific bioactive substances and mechanism are still unclear. The key metabolites and pathways used by Gym to reduce uric acid (UA) were identify. METHODS: An optimized extraction process for urate-lowering active substances from Gym was firstly been carried out based on the xanthine oxidase (XOD) inhibition model in vitro; then, the Ultra-high-performance liquid chromatography and Q-Exactive mass spectrometry (UHPLC-QE-MS) based on non-targeted metabolomics analysis of Traditional Chinese Medicine were performed for comparison of Gym with ethanol concentration of 95% (low extraction rate but high XOD inhibition rate) and 75% (high extraction rate but low XOD inhibition rate), respectively; finally, the protective effect of ethanolic extract of Gym on zebrafish with Hyperuricemia (referred to as HUA zebrafish) was explored. RESULTS: We found that the inhibition rate of Gym extract with 95% ethanol concentration on XOD was 84.02%, and the extraction rate was 4.32%. Interestingly, when the other conditions were the same, the XOD inhibition rate of the Gym extract with 75% ethanol concentration was 76.84%, and the extraction rate was 14.68%. A total of 539 metabolites were identified, among them, 162 different metabolites were screened, of which 123 were up-regulated and 39 were down-regulated. Besides significantly reducing the contents of UA, BUN, CRE, ROS, MDA, and XOD activity in HUA zebrafish by Gym and acutely reduce the activity of SOD. CONCLUSION: Along with the flavonoids, polyphenols, alkaloids, terpenoids, and phenylpropanoids, the ethanolic extract of Gym may be related to reduce the UA level of Gym.