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Wennberg, T; Kreander, K; Lahdevuori, M; Vuorela, H; Vuorela, P
Journal of liquid chromatography & related technologies, 2004, 1/1/2004, 2004-01-00, Letnik: 27, Številka: 16Journal Article
An efficient off-line procedure for primary screening of the bioactivity of a complex mixture was developed. The procedure includes HPLC separation, micro-fractionation, and a bioactivity assay followed by scale up of preparative isolation of active substances. Test compounds were used to develop and validate a chemical assay based on a stable free radical, 2,2′-diphenyl-1-picrylhydrazyl (DPPH). The procedure was equally developed and validated for a cell-based bioactivity assay in which the antimicrobial activity of a test mixture was tested against Streptococcus pyogenes. In the procedure, the analytical-scale sample was separated by HPLC and fractionated into a 96-well microplate. The fractionated microplate was lyophilized and either a chemical reagent or bacterial suspension was added to the plate wells. The UV-absorbance of DPPH and the turbidity of Str. pyogenes were measured using a microplate reader. Three natural extracts (Lythrum salicaria, Linum usitatissimum, and Cladina stellaris) were analyzed by the procedure using both the chemical and cell-based assays. The analytical HPLC separation of the most bioactive components of C. stellaris was further scaled up using a computer assisted simulation program and preparative amounts of the active compounds were isolated and identified by UV, NMR, or MS. In most cases, the on-line screening chromatographic methods are difficult to apply to cell-based bioactivity assays. However, the off-line micro-fractionation procedure presented here was found to be a valuable tool for bioactivity screening, especially for cell-based assays.
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