Tracking of β-lactoglobulin binding compounds with biofingerprinting chromatogram analysis of natural products

The addition of milk to tea has been shown to prevent the vascular protective effects of the tea. Thus we had an interest in evaluating the possible binding of compounds in extracts of black tea, Camellia sinensis (L.) O. Kunze, and nettle, Urtica dioica L, to P-lactoglobulin (beta LG), which is the main proteinaceous component of whey. Since our previously miniaturised 96-well plate method based on fluorescence quenching is suitable only for pure compounds, we have developed a method of ultrafiltration sampling combined with FPLC to study the beta LG-binding property of the extracts. The method development was made with extract of Piper nigrum L. (black pepper), because its major component is piperine, which has been earlier shown to bind to beta LG. The results showed that piperine from P. nigrum was highly and rutin from U. dioica slightly bound to beta LG. No components from C. sinensis were bound to beta LG. We showed for the first time, that the ultrafiltration/HPLC system can be used in beta LG-binding studies with plant extracts. Furthermore, we demonstrated that PLG could bind compounds from extracts and in this way either enhance, or decrease, their health benefits, for example in food preparations.