Five different flavonoids were isolated from licorice after multistep chromatographic fractionation. The aim was to identify and characterize active components in licorice responsible for antibrowning activities and to seek new tyrosinase inhibitors for applications as antibrowning and depigmenting agents in the food and cosmetic industries. The isolated flavonoids were identified as liquiritin, licuraside, isoliquiritin, liquiritigenin (from Glycyrrhiza uralensis Fisch.), and licochalcone A (from Glycyrrhiza inflate Bat.) by UV, MS, (1)H NMR, and (13)C NMR analyses. The inhibitory potencies and capacities of these flavonoids toward monophenolase activity of mushroom tyrosinase were investigated. The IC(50) values of licuraside, isoliquiritin, and licochalcone A for monophenolase activity were 0.072, 0.038, and 0.0258 mM, respectively. A study of the mechanisms of monophenolase inhibition by these flavonoids indicated that they are all competitive inhibitors. Different from the above flavonoids, no inhibitory activity was observed for liquiritin, whereas liquiritigenin activated the monophenolase activity as a cofactor. The inhibitory effect of licuraside, isoliquiritin, and licochalcone A on diphenolase activity with l-DOPA as the substrate was much lower than those with l-tyrosine. Results suggest that licuraside, isoliquiritin, and licochalcone A have the high potential to be further developed into effective antibrowning and depigmenting agents.

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Solid-phase microextraction (SPME) is a promising technique for determining organic contaminants within biotic systems. Existing in vivo SPME-kinetic calibration (SPME-KC) approaches are unwieldy due to the necessity of predetermining a distribution coefficient for the analyte of interest in the tissue and the preloading of a calibrating compound to the fiber. In this study, a rapid and convenient SPME alternative calibration method for in vivo analysis, termed SPME-sampling rate (SPME-SR) calibration, was developed and validated under both laboratory and field conditions to eliminate such presampling requirements. Briefly, the SPME probe is inserted into tissue, in this study fish dorsal-epaxial muscle, for 20 min allowing the concentrations of target analytes in the fish muscle to be determined by the extracted amount of analyte and the predetermined sampling rates. Atrazine, carbamazepine, and fluoxetine were detected nonlethally in the low ppb levels within fish muscle, with both laboratory and field-derived results obtained by in vivo SPME-KC comparable (within a factor of 1.27) to those obtained by lethal sampling followed by tissue liquid extraction. The technique described in this study represents an important advance which broadens the application of SPME in vivo sampling technology.

The space-resolved solid-phase microextraction (SR-SPME) technique was employed to study the tissue-specific bioconcentration of pharmaceuticals in live fish. The segmented design of the SPME fibers allowed for the simultaneous determination of pharmaceutical residues in fish dorsal-epaxial muscle and adipose tissue with a single SPME fiber. The miniaturized fiber endowed the technique with high spatial resolution allowing for quantification of analytes within adjacent, relatively small tissues of immature rainbow trout. The pre-equilibrium sampling and kinetic calibration approach yielded efficient and accurate quantitation of pharmaceuticals in fish tissue. The ability of the SPME method to repeatedly sample the same fish circumvents problems arising from interanimal variation, thus improving the precision of generated bioconcentration kinetic profiles. In vivo monitoring with SR-SPME was validated with in vitro liquid extraction of tissue samples using methanol. Of the nine compounds evaluated, five (atrazine, gemfibrozil, carbamazepine, ibuprofen, and fluoxetine) bioconcentrated in adipose and muscle tissue over the eight exposure days. Although the accumulation of analytes in both tissues was positively correlated, each compound partitioned with differing affinities as modified by their hydrophobicity and unique molecular structure. Water samples analyzed using the SPME technique yielded results similar to those determined by solid-phase extraction (SPE); however, SPME was more rapid and operationally much simpler. This study illustrates the application conditions for in situ SR-SPME while demonstrating the potential of these miniaturized SPME fibers for simultaneous in vivo repeated sampling of multiple tissues.