UPLC-Q TOF-MS-Based metabolomics and anti-myocardial ischemia activity of Dioscoreae Nipponicae Rhizoma from different geographical origins.

The dried rhizome of Dioscorea nipponica Makino ("Chuanshanlong" in Chinese) is a medicinal herb with multiple major producing areas. The main objective of this study was the comparative profiling of Dioscoreae Nipponicae Rhizoma (DNR) from various geographical origins. A hypoxia/reoxygenation-induced H9c2 cell injury model was established, and the antimyocardial ischemia activity of DNR samples from different origins was detected using the cell counting kit-8 (CCK-8) method. The result showed that the antimyocardial ischemia potential of DNR samples from the Heilongjiang province was higher than that of the other studied samples. Subsequently, a plant metabolomics technique utilizing ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q TOF-MS) was used to determine the differences in DNR samples from various geographical origins. Forty compounds, including steroidal saponins, free fatty acids, and organic acids, were tentatively identified based on UPLC-Q TOF-MS fragmentation pathways and via comparison with available reference standards. Partial least squares discriminant analysis was performed to estimate the differences in DNR samples from different origins. Five compounds were significantly up-regulated and correlated with antimyocardial ischemia in DNR samples from Heilongjiang province. Molecular docking was used to discern the interactions of key markers with the active sites of the target protein. The findings signified that UPLC-Q TOF-MS metabolomics coupled with molecular docking is a powerful tool to rapidly identify the quality control characteristics of DNR samples and their products. The research provides a direction for the rational utilization of DNR.

The metabolites of Ambinine, a benzo[c]phenanthridine alkaloid, in rats identified by ultra-performance liquid chromatography-quadrupoletime-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS).

Ambinine (AM), one of the major hexahydrobenzo[c]phenanthridine alkaloids from Corydalis ambigua Cham. et Schlecht. var. amurensis Maxim, is considered to be an important compound because of its special content and activity. However, there are few published studies on AM metabolism. In this research, the metabolism of AM in vivo was comprehensively studied for the first time. A total 44 metabolites (including 13 phase I and 31 phase II metabolites) as well as its parent drug in plasma, bile, urine and feces of rats were identified and 41 of them were reported for the first time. The results obtained indicated that demethylation, sulfation and glucuronidation were the major metabolic pathways of AM in vivo. Furthermore, this study provides valuable and new information about the metabolism of AM, which will be very helpful for understanding the safety and efficacy of AM, as well as its analogues.

UPLC/Q-TOFMS-Based Metabolomics Studies on the Protective Effect of Panax notoginseng Saponins on Alcoholic Liver Injury.

Consistent, excessive alcohol consumption leads to liver injury. The aim of the present study is to evaluate the possible efficacy of Panax notoginseng saponins (PNS) against chronic alcohol-induced liver injury using LC-MS-based urinary metabolomics. Mice were fed a Lieber-DeCarli liquid diet containing alcohol or isocaloric maltose dextrin as a control diet with or without PNS (200 mg/kg/BW) for 4 weeks. Treatment with PNS significantly reduced the increases in plasma ALT and AST levels, hepatic levels of reactive oxygen species (ROS) and malondialdehyde (MDA), which induced by chronic alcohol exposure. Conversely, PNS was also found to restore the glutathione (GSH) depletion and increase the superoxide dismutase (SOD) activities. The end-point urine sample of each mouse was collected overnight (24 h) in metabolic cages and their metabolic profiling changes were analyzed using UPLC/Q-TOFMS followed by multivariate statistical analysis. After 4 week of Lieber-DeCarli alcohol diet feeding, the metabolic profile experienced great perturbation in PCA score plot, and the treatment of PNS could assist to regulate the disturbed metabolic profile induced by alcohol exposure. Additionally, sixteen potential biomarkers responsible for derivations of the metabolic profile induced by alcohol exposure were identified, and the alcohol-induced changes in these biomarkers, except hexanoylglycine, could be partially or nearly reversed by PNS treatment. Taken together, PNS protects against chronic alcohol-induced liver injury. Our findings demonstrated that the LC-MS-based metabolomics approach is a useful tool to investigate the efficacy of Chinese medicines.

UPLC-Q-TOF/MS-based screening and identification of two major bioactive components and their metabolites in normal and CKD rat plasma, urine and feces after oral administration of Rehmannia glutinosa Libosch extract.

Rehmannia glutinosa is a widely used traditional Chinese medicine (TCM) in clinical practice to tackle chronic kidney disease for thousands of years. However, the in vivo metabolism of its two major bioactive components (catalpol and acteoside) remains unknown. In this paper, a highly sensitive, rapid and robust ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with MetaboLynx™ software combined with mass defect filtering (MDF) method was established. This validated analysis method was successfully applied to investigate the in vivo metabolic profiles of R. glutinosa extract in normal and chronic kidney disease (CKD) rats. The results showed that a total of 17 metabolites of two parent compounds in normal rats in vivo were tentatively detected and identified according to the characteristics of their protonated ions and relevant literature. While 11 of the metabolites were observed in the CKD rat samples. These metabolites suggested that catalpol was firstly deglycosylated to its aglycone and subsequently to two main metabolites (M1 and M4) by conjugation and hydrogenation respectively and acteoside was mainly metabolized by O-glucuronide conjugation and O-sulphate conjugation. In conclusion, this study showed an insight into the metabolism of R. glutinosa extract in vivo and the proposed metabolic pathways of bioactive components might play a key role in further pharmacokinetic experiments evaluations.

Purification process of epimedin and icariin from Epimedii Folium by macroporous resin / 中草药

Objective: To investigate the best technological conditions for the purification of epimedin and icariin from Epimedii Folium by macroporous resin, and preliminarily characterize the purification fraction of the best technological conditions. Methods: Five kinds of macroporous resins were screened by static adsorption experiment with the content of epimedin A1, epimedin A, epimedin B, epimedin C and icariin as indexes. The best purification conditions were optimized by the concentration of upper column solution, the maximum sample volume, the upper column flow rate, the volume of water washing, the concentration of removing impurity ethanol and elution ethanol, the volume of removing impurity ethanol and elution ethanol, the column diameter-height ratio through dynamic adsorption experiment. Finally, UPLC-Q-TOF/MS, HPLC and ultraviolet spectrophotometry were used to characterize the purification fraction of the best technological conditions. Results: The best macroporous resin was AB-8, column diameter-height ratio was 1:7, 6 BV of upper column solution (crude drug 0.5 g/mL) was used for dynamic adsorption at a flow rate of 6 BV/h, 5 BV of water and 5 BV of 20% ethanol were used for impurity removal, and 6 BV of 50% ethanol was used for elution. The flow rate of impurity removal and elution was 6 BV/h. After purification, the total flavonoids content was 63.29%, the total content of epimedin A1, A, B, C and icariin was 40.48%, the content of epimedin A1, epimedin A, epimedin B, epimedin C and icariin was 1.63%, 2.52%, 16.36%, 5.51% and 14.46%, respectively. Conclusion: The purification process of epimedin and icariin from Epimedii Folium by AB-8 macroporous resin is stable, reasonable and feasible. The chemical characterization indicated that the purification fraction was mainly flavonoids, mainly consisting of epimedin and icariin. The optimized purification process can be used for the purification and enrichment of such ingredients.