Reverse phase compatible TLC-bioautography for detection of tyrosinase inhibitors

Abstract

Introduction Reverse phase chromatography and bioautographic assays are key tools for natural product bioguided isolation; however, their direct coupling has not been fully achieved. Objectives To develop a bioautographic assay to detect tyrosinase inhibitors present in complex matrices sorbed on reverse phase (RP) TLC-plates that can be used for bioguided isolation of bioactive compounds. Methods Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The gel turns into a brown skin like colour due to tyrosinase catalysed oxidation of L-tyrosine. The inhibitors are visualised as clear spots against a brown coloured background. Results The assay was able to localise cinnamaldehyde in Cinnamomum cassia essential oil, as its main constituent with known tyrosinase inhibition properties. The assay allowed the detection of 0.03% (w/w) of kojic acid co-spotted with a methanolic extract of Sphaeralcea bonariensis and chromatographed on RP-TLC.Conclusion The developed assay is able to detect, with high sensitivity, tyrosinase inhibitors present in complex matrices that were chromatographed in RP-TLC. Results can be easily read by colour change, inhibitors appear as clear spots in a darker background.