Thin-Layer Chromatography - Bioassay as Powerful Tool for Rapid Identification of Bioactive Components in Botanical Extracts

Copyright: © 2015 Agatonovic-Kustrin S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The plant kingdom, both terrestrial and marine, is an underestimated pool of secondary metabolites some of which have enormous therapeutic potential to be an important source of new and unique agents. The identification of active compounds from complex botanical extracts is the main challenge in natural products discovery. The strategy used to discover active substances has been considerably changed in the last decade from the expensive and laborious methods in which bioactivity was studied only after isolation and identification of the components. The new strategy involves only the effective components being isolated and identified. Since chemical separation of the whole sample does not provide information about biological effects, neither do bioassays of the whole sample extracts provide information on observed therapeutic effects for compounds found in these extracts. However, if chemical separation techniques are combined with bioassays the therapeutic effects of individual compounds in a plant extract are able to be assessed. The use of planar chromatography to first separate plant extracts into individual components followed by a bioassay offers a number of advantages. Many samples can be analyzed in parallel on the same chromatographic plate keeping analysis times short and costs low. The high flexibility in detection is accomplished with the use of various derivatization reagents. Hence, direct bioassay on the chromatographic plate makes Thin Layer Chromatography (TLC) screening a powerful tool for rapid identification of bioactive components in crude plant extracts. To search for bioactive compounds in plant extracts with a targeted activity, TLC is hyphenated with an appropriate bioassay enabling direct in vitro biological study of the components that have been previously separated on the plate. Hyphenation of TLC with bioassays, micro chemical detection and further Mass Spectroscopy (MS) identification enables targeted identification of substances from plant extracts. Post chromatographic derivatization by dipping or spraying can be done with either a universal micro chemical derivatization using for example anisaldehyde/sulfuric acid for detection of phenols, sugars, steroids, and terpenes [1] or with selective micro chemical derivatization. The diphenylpicrylhydrazyl (DPPH*) free radical can be used to detect antioxidants or free radical scavengers [2] while fast blue salt B is a stain that can used to detect various enzymes. Other reagents can be used to detect specific functional groups of phytochemical interest like natural products reagent (2-aminoethyl diphenylborinate) for flavonoids [2] or Brady’s reagent (2,4‐dinitrophenylhydrazine) for aldehydes and ketones [3].