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.

Multiresponse Optimisation Applied to the Development of a TLC Autography for the Detection of Tyrosinase Inhibitors.

INTRODUCTION: Autographic methods are useful tools to detect bioactive compounds in complex matrixes. Experimental design and optimisation techniques were implemented for the development of an autographic assay suitable for the detection of tyrosinase inhibitors. OBJECTIVES: To develop an autographic assay to detect tyrosinase inhibitors using gel entrapped enzyme, experimental design and response surface methodology (RSM) to optimise conditions with a minimum number of experiments. METHODS: Gel entrapment was used for the assay and the effects of four factors on the sensitivity and the detection limit for known inhibitors of the enzyme were evaluated. The factors were: tyrosinase amount (TA), L-tyrosine amount (LTA), incubation time and incubation temperature. RESULTS: The assay allowed the detection of kojic acid in an extract of Calamagrostis viridiflavescens (Poir.) Steud spiked with 0.1% w/w. CONCLUSION: The developed assay is able to detect tyrosinase inhibitors present in complex matrixes in a reproducible way.