Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 879, Issue 2, Pages 191-196Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2010.12.004
Keywords
Selaginella sinensis; Antioxidant; DPPH-HPLC; HSCCC; Flavonoid; Biflavonoid
Funding
- National Scientific Foundation of China [20775092]
- Natural Science Foundation of Hunan Province of China [10114006]
- Science and Technology Program of Hunan Province of China [2009RS3030]
- Central South University Science Development Foundation [10SDF21]
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Selaginella sinensis (Selaginellaceae) is used extensively in traditional Chinese medicine (TCM) for the treatment of many kinds of chronic diseases. In this study, fractionation of the methanol extract of S. sinensis by different polarity solvents indicated the ethyl acetate fraction exhibited an potent 1,1-diphenyl-2-picryhydrazyl (DPPH) radical scavenging activity with the IC50 value of 44.9 mu M. In order to evaluate the scientific basis, antioxidant peaks were firstly screened using DPPH spiking test through high performance liquid chromatography (DPPH-HPLC). Under the target-guidance of DPPH-HPLC experiment, two flavonoids and six biflavonoids, quercetin (1), apigenin (2), amentoflavone (3). robustaflavone (4), 2,3-dihydroamentaflavone (5), hinokiflavone (6), 4'-O-methyl-robustaflavone (7) and ginkgetin (8) were separated by high-speed counter-current chromatography (HSCCC) method using n-hexane-ethyl acetate-methanol-water (8:8:9:7) as the solvent system with purities 98.2%, 97.6%, 99.4%, 92.3%, 98.5%, 98.9% and 99.6%, respectively. The structures were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Antioxidant activity of eight isolated compounds was assessed by the radical scavenging effect on DPPH radical, compound 1 showed strongest antioxidant activities with IC50 values of 3.2 mu M, while compounds 2-8 showed weak antioxidant activities. This is the first report on simultaneous separation of eight antioxidant compounds from S.sinensis by HSCCC, moreover, apigenin and 4'-O-methyl-robustaflavone were first identified from this plant. Results of the present study indicated that the combinative method using DPPH-HPLC and HSCCC could be widely applied for rapid screening and isolating of antioxidants from complex TCM extract. (C) 2010 Published by Elsevier B.V.
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