The complexity of ingredients in traditional Chinese medicine (TCM) makes it challenging to clarify its efficacy in an acceptable and scientific approach. The present study was aimed to use quantification results from targeted cellular metabolomics to evaluate anti-aging efficacy of a famous Chinese medicine formula, Erzhi Wan (EZW), and screen possible effective extracts, depending on the developed strategy integrating multivariate receiver operating characteristic (ROC) curve and analytic hierarchy process (AHP). In this study, senescent NRK cells induced by D-galactose were treated with drug-containing serum of EZW and four kinds of extracts (petroleum ether, ethyl acetate, butanol and water). Intermediates of two major metabolic pathways for energy synthesis, tricarboxylic acid (TCA) cycle and glycolysis, were accurately quantified by GC–MS/MS to identify discriminate metabolites for clarifying therapeutic mechanism of EZW based on multivariate statistical analysis. Senescent and non-senescent cells were successfully distinguished using these metabolites by ROC curve analysis. Next, these metabolites were used as evaluation indexes to quantitatively reflect different effect of EZW and its extracts, according to the role of them in distinguishing groups and in conjunction with AHP. In vitro detection of senescence-associated β-galactosidase (SA-β-gal) activity was used to verify the reliability of evaluation results. The reversal after treatment of drug-containing serum of EZW and extracts was observed, and the petroleum ether extract might be the potential active extract responsible for the major anti-aging effect of EZW, which was in agreement with in vitro experiments. Altogether, metabolomics was a powerful approach for evaluation efficacy and elucidation action mechanisms of TCM. The integrated evaluation strategy in this paper with properties of high practicality, feasibility and effectivity was expected to provide a new insight into comprehensive and quantitative efficacy evaluation.
Ethnopharmacological relevance Erzhi Wan (EZW), as a prescription of traditional Chinese medicine , has been used for tonifying the liver and kidney. Although past studies have shown that EZW has potential anti-aging effect, the mechanisms associated with cellular metabolomics and lipidomics are not fully understood. Aim of the study This study aimed to evaluate the anti-aging effect of EZW and investigate the mechanisms associated with cellular metabolomics and lipidomics. Materials and methods EZW solution at dosage of 3.6 g/kg in Sprague-Dawley rats was orally administered twice a day for 7 days and serum containing EZW was then collected. NRK cell senescence model induced by D-galactose was established in vitro , and non-contact co-culture cell assay was performed between senescent NRK cells and BRL cells intervened by serum containing EZW. The anti-aging effect of EZW on NRK cells was evaluated by metabolites identification, differential metabolites screening and metabolic pathways analysis through cellular metabolomics with GC-MS and lipidomics with UHPLC-Q-Exactive Orbitrap/MS. Results Serum containing EZW indicated a protective effect through intervening BRL cells in non-contact co-culture system with D-gal-induced senescent NRK cells. For metabolic profiles, 71 endogenous metabolites were identified, among which 24 significantly differential metabolites were screened as metabolomics potential biomarkers. For lipidic profiles, 64 lipid components were identified in NRK cell samples under positive ion mode, among which 24 potential biomarkers of lipids were screened, mainly including PC and PE. 127 lipid components were identified in NRK cell samples under negative ion mode, among which 59 potential biomarkers of lipids were screened, including FA, PC, PE, PI and PS . Metabolic pathway analysis demonstrated that the identified differential metabolites found mainly involved in amino acids metabolism, energy metabolism and phospholipid biosynthesis pathways. Conclusion Serum containing EZW exhibited protective effect on D-gal-induced senescent NRK cells through intervening BRL cells by mainly regulating amino acids metabolism, energy metabolism and phospholipid biosynthesis pathways to possess its anti-aging function, providing a theoretical basis for clinical treatment of EZW.
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