Rapid screening of antioxidant from natural products by AAPH-Incubating HPLC-DAD-HR MS/MS method: A case study of Gardenia jasminoides fruit.

A new AAPH-Incubating HPLC-DAD-HR MS/MS method was developed for the rapid and high-throughput screening of antioxidants directly in natural products and applied to Gardenia jasminoides fruit. This method was assumed that the peak areas of compounds with potential antioxidant activity in HPLC chromatograms would be significantly reduced or disappeared after incubating with the AAPH which can release ROO at physiological conditions (37 °C, pH 7.4). Additionally, the activity of antioxidants can be evaluated by comparing the peak reduction rates and the screened components can be further identified by HRMS/MS. Then, 17 potential natural antioxidants from the crude extract of GJF was screened. Among them, three major components including crocin I, crocin II and crocetin showed excellent ROO scavenging activity, which were further validated by the ORAC assay. In conclusion, our study provided a simple and effective strategy to rapidly screen antioxidants in natural products.

Non-targeted metabolomic analysis of variation of volatile fractions of ginseng from different habitats by HS-SPME-GC-MS coupled with chemometrics.

Cultivated ginseng (CG), transplanted ginseng (TG) and mountain cultivated ginseng (MCG) classified by the habitat type all belong to Panax ginseng and were reported to have similar types of secondary metabolites. Nonetheless, owing to the distinctly diverse habitats in which these ginseng types grow, their pharmacological effects differ. In the present study, an emerging analytical approach involving headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was established to effectively distinguish among CG, TG and MCG. First, the volatile components were analysed and identified by using the NIST library combined with measured retention indices (Kovats', RI), and a total of 78 volatile components were finally characterized, which included terpenes, alcohols, esters, aldehydes and alkynols. Furthermore, multivariate statistical approaches, principal component analysis (PCA) and orthogonal partial least-squares discrimination analysis (OPLS-DA) were subsequently utilized to screen for compounds of significance. Under optimized HS-SPME-GC-MS conditions, 12, 16, and 16 differential markers were screened in the CG-TG, CG-MCG and TG-MCG groups, respectively. Our study suggested that HS-SPME-GC-MS analysis combined with metabolomic analytical methods and chemometric techniques can be applied as potent tools to identify chemical marker candidates to distinguish CG, TG and MCG.

Protective effects of Huang-Lian-Jie-Du Decoction on diabetic nephropathy through regulating AGEs/RAGE/Akt/Nrf2 pathway and metabolic profiling in db/db mice.

BACKGROUND: Diabetic nephropathy (DN) is a severe diabetic complication that is the principal cause of end-stage kidney disease worldwide. Huang-Lian-Jie-Du Decoction (HLJDD) is widely used to treat diabetes clinically. However, the nephroprotective effects and potential mechanism of action of HLJDD against DN have not yet been fully elucidated. PURPOSE: This study aimed to investigate the potential roles of HLJDD in DN and elucidate its mechanisms in db/db mice. METHODS: An integrated strategy of network pharmacology, pharmacodynamics, molecular biology, and metabolomics was used to reveal the mechanisms of HLJDD in the treatment of DN. First, network pharmacology was utilized to predict the possible pathways for DN using the absorbed ingredients of HLJDD in rat plasma in silico. Then, combined with histopathological examination, biochemical evaluation immunohistochemistry/immunofluorescence assay, western blot analysis, and UPLC-Q-Orbitrap HRMS/MS-based metabolomics approach were applied to evaluate the efficacy of HLJDD against DN and its underlying mechanisms in vivo. RESULTS: In silico, network pharmacology indicated that the AGEs/RAGE pathway was the most prominent pathway for HLJDD against DN. In vivo, HLJDD exerted protective effects against DN by ameliorating glycolipid metabolic disorders and kidney injury. Furthermore, we verified that HLJDD protected against DN by regulating the AGEs/RAGE/Akt/Nrf2 pathway for the first time. In addition, 22 potential biomarkers were identified in urine, including phenylalanine metabolism, tryptophan metabolism, glucose metabolism, and sphingolipid metabolism. CONCLUSION: These findings suggest that HLJDD ameliorates DN by regulating the AGEs/RAGE/Akt/Nrf2 pathway and metabolic profiling.

Rapid screening of neuroprotective components from Huang-Lian-Jie-Du Decoction by living cell biospecific extraction coupled with HPLC-Q-Orbitrap-HRMS/MS analysis.

Huang-Lian-Jie-Du Decoction (HLJDD), a well-known traditional Chinese formulation, has been proved to exert neuroprotective effects, however, the bioactive components in HLJDD still remain to be elucidated. In the present study, a rapid and effective method involving live cell biospecific extraction and HPLC-Q-Orbitrap HRMS/MS was utilized to rapidly screen and identify the neuroprotective compounds from the HLJDD crude extract directly. Firstly, sixteen principal components in HLJDD crude extract were identified by HPLC-Q-Orbitrap HRMS/MS analysis. After co-incubation with PC12 cells, which have been validated as the key target cells for neurodegenerative diseases, seven compounds of them were demonstrated to exhibit binding affinity to the target cells. Furthermore, three representative compounds named baicalin, wogonoside, and berberine were subsequently verified to exert cytoprotective effects on PC12 cells injured by hydrogen peroxide via inhibiting oxidative stress and cell apoptosis, indicating that these screened compounds may possess a potential for the treatment of neurodegenerative diseases and were responsible, in part at least, for the neuroprotective beneficial effects of HLJDD. Taken together, our study provides evidence that live cell biospecific extraction coupled with LC-HRMS/MS technique is an efficient method for rapid screening potential bioactive components in traditional Chinese medicines.

Pterostilbene prevents methylglyoxal-induced cytotoxicity in endothelial cells by regulating glyoxalase, oxidative stress and apoptosis.

Methylglyoxal (MGO), a cytotoxic byproduct of glycolysis in biological systems, can induce endothelial cells dysfunction, implicated in diabetic vascular complications. Pterostilbene (PTS), a naturally occurring resveratrol derivative, is involved in various pharmacological activities. This study aimed to explore the effects of PTS on MGO induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms for the first time. In the current study, it has been demonstrated that PTS could enhance the level of glyoxalase 1 (GLO-1) and elevate glutathione (GSH) content to active the glyoxalase system, resulting in elimination of the toxic MGO as well as advanced glycation end products (AGEs) in HUVECs. Meanwhile, PTS could also suppress oxidative stress and thus exert cytoprotective effects by elevating Nrf2 nuclear translocation and the corresponding down-stream antioxidant enzymes in MGO induced HUVECs. In addition, PTS could alleviate MGO induced apoptosis in HUVECs via inhibition of oxidative stress and associated downstream mitochondria-dependent signaling apoptotic cascades, as characterized by preventing caspases family activation. Taken together, these findings suggest that PTS could protect against MGO induced endothelial cell cytotoxicity by regulating glyoxalase, oxidative stress and apoptosis, suggesting that PTS could be beneficial in the treatment of diabetic vascular complications.