title: Target-guided isolation and purification of antioxidants from Selaginella sinensis by offline coupling of DPPH-HPLC and HSCCC experiments.
abstract: 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 IC(50) value of 44.9 μM. 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 IC(50) values of 3.2 μM, 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.
authors.affiliation: School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China.
authors.forename: Yuping
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shuyun
authors.initials: S
authors.lastname: Shi
authors.forename: Yuanxi
authors.initials: Y
authors.lastname: Wang
authors.forename: Kelong
authors.initials: K
authors.lastname: Huang
mesh_terms: D000085:Acetates; D000975:Antioxidants / Q000737:chemistry / Q000302:isolation & purification*; D001713:Biphenyl Compounds / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003377:Countercurrent Distribution / Q000379:methods*; D005419:Flavonoids / Q000737:chemistry / Q000302:isolation & purification*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010851:Picrates / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry; D032503:Selaginellaceae / Q000737:chemistry*; D012997:Solvents
chemical_list: D000085:Acetates; D000975:Antioxidants; D001713:Biphenyl Compounds; D005419:Flavonoids; D010851:Picrates; D010936:Plant Extracts; D012997:Solvents; C007650:ethyl acetate; C004931:1,1-diphenyl-2-picrylhydrazyl