Abstract
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.
Acknowledgment
This research was supported by the National Technology Agency in Finland (grants no. 40045/02 and 40030/03).