Rapid discovery of antioxidants in Salvia miltiorrhiza by UPLC-DPPH- PDA-ESI-TOF/MS online technique / 中草药

ABSTRACT

Objective: To establish a method for rapid screening and identifying antioxidants in natural products by ultra high performance liquid chromatography-(1,1-dipheny l-2-trinitrophenylhydrazine)-photo diode array-electrospray-time-of-flight mass spectrometry (UPLC-DPPH-PDA-ESI-TOF/MS) and apply it to the rapid discovery of antioxidants from Salviamiltiorrhiza extracts. Methods: The Waters ACQUITY UPLC system and a Waters ACQUITY UPLC HSS T3 column (100 mm×3.0mm,1.8 μm; Waters Technologies, USA) were used to elute the sample at a low flow rate of 0.35 mL/min, which could achieve the rapid separation of S. miltiorrhiza extracts. The elution was joined with DPPH solution (0.3 mL/min) and reacted in the PEEK coil, and then flew into the detector. The anti-oxidation components in S. miltiorrhiza extracts were quickly screened by comparing the chromatograms at 517 nm and 254 nm. Results: The UPLC technology was adopted to realize the efficient separation of samples at low volume flow rate, which avoided the baseline noise caused by high volume flow rate, significantly improved the resolution and sensitivity of the online screening method, and reduced the solvent consumption.The results showed that 31 antioxidant active compounds were screened from S. miltiorrhiza extracts, and 11 of them were identified by PAD-ESI-TOF/MS. Activity verification experiments showed that caffeic acid, salvianolic acid B, salvianolic acid A and danshensu had better antioxidant activity. The IC50 value ofcaffeic acid was 29.00 μmol/L, which was significantly better than the positive control (vitamin C). Conclusion: The method is rapid and efficient, which provides methods and technical support for the rapid discovery of antioxidant components from complex natural products.