Reverse Phase Compatible TLC-Bioautography for Detection of Tyrosinase Inhibitors.

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. Copyright © 2016 John Wiley & Sons, Ltd.