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Improved TLC Bioautographic Assay for Qualitative and Quantitative Estimation of Tyrosinase Inhibitors in Natural Products.

INTRODUCTION
TLC bioautography for tyrosinase inhibitors has made recent progress; however, an assay with a relative low consumption of enzyme and quantitative capability would greatly advance the efficacy of related TLC bioautographic assays.


OBJECTIVE
An improved TLC bioautographic assay for detecting tyrosinase inhibitors was developed and validated in this study.


METHODS
L-DOPA (better water-solubility than L-tyrosine) was used as the substrate instead of reported L-tyrosine. The effects of enzyme and substrate concentrations, reaction temperatures and times, and pH values of the reaction system as well as different plate types on the TLC bioautographic assay were optimised. The quantitative analysis was conducted by densitometric scanning of spot areas, and expressed as the relative tyrosinase inhibitory capacity (RTIC) using a positive control (kojic acid) equivalent.


RESULTS
The limit of detection (LOD) of this assay was 1.0 ng for kojic acid. This assay has acceptable accuracy (101.73-102.90%), intra- and inter-day, and intra- and inter-plate precisions [relative standard deviation (RSD), less than 7.0%], and ruggedness (RSD, less than 3.5%). The consumption of enzyme (75 U/mL) is relatively low. Two tyrosinase inhibitory compounds including naringenin and 1-O-β-D-glucopyranosyl-4-allylbenzene have been isolated from Rhodiola sacra guided by this TLC bioautographic assay.


CONCLUSION
Our improved assay is a relatively low-cost, sensitive, and quantitative method compared to the reported TLC bioautographic assays. Copyright © 2016 John Wiley & Sons, Ltd.