Rapid purification of antioxidants from Magnolia officinalis by semi-prep-HPLC with a two-step separation strategy guided by on-line HPLC-radical scavenging detection

A rapid and accurate strategy of screening, identification, and purification of antioxidants from natural medicines was put forward in this work, and it was applied to discover the antioxidants from magnolia officinals Rahd. et Wils. On-line HPLC-DPPH combined with electrospray ionization time-of-flight mass spectrometry was employed for the rapid screening and potential identification of the antioxidants. Semi-prep-HPLC with two-step separation procedure was employed for the separation and purification of the representative compounds (the first step: compounds 1-4 were obtained by gradient elution; the second step: compounds 5-7 were obtained by isocratic elution). NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. With this method, 28 compounds with antioxidant activity were discovered in the extraction. After the preparation and purification, seven compounds with the purity over 95% were get, which were identified as syringing, magnoloside B, magnoloside A, magnoloside F, magnolol, obvatol and honokiol. The results of in vitro assay showed that these seven compounds all had higher DPPH scavenging activity. Thus, all the results suggested that this work provide a more rapid, accurate and efficient methodology to achieve the screening, characterization and preparation of antioxidative constituents from complex natural products under active guidance.