Prevention of False-Positive Results: Development of an HPTLC Autographic Assay for the Detection of Natural Tyrosinase Inhibitors

Abstract

A simple and rapid high-performance thin-layer chromatography-based autographic assay was established to screen plant extracts for the presence of tyrosinase-inhibiting substances. Three mobile phases were selected for the chromatographic separation of different types of extracts. After development, the plate was sprayed with the substrate solution Levodopa followed by a solution of the enzyme tyrosinase. Several known tyrosinase inhibitors were tested simultaneously as positive controls. They were detected as white spots with white light in remission from the plate as well as with white light transmitted through the plate. Some of the investigated extracts included spots showing a different behaviour; some lipophilic substances appeared as white spots in white light remission but were black in white light transmission. This behaviour, which could lead to false-positive results, was due to poor wettability of the corresponding spots. False-positive results were eliminated by adding Triton X-100 to the Levodopa solution and drying the plate after 10 minutes incubation with a molecular sieve. Tyrosinase inhibitors can be clearly identified as white spots against a dark background in white light remission as well as in white light transmitted through the plate. The established high-performance thin-layer chromatography autographic assay was validated and can be used as a standard method for the detection of tyrosinase inhibitors in plant extracts without causing false-positive results.

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

“…As shown in Figure (A), a gradual increase of peak area was observed with enzyme concentrations from 10 to 75 U/mL, and no significant changes occurred with enzyme concentrations beyond 75 U/mL. The consumption of enzyme was relatively lower than that of the reported method (215–400 U/mL; Wangthong et al ., ; Taibon et al ., ). Similarly, the effect of substrate concentrations on the tyrosinase reaction system was studied using varying concentrations of L‐DOPA (2.0–4.0 mM).…”

“…However, Taibon et al . () reported recently that different silica gel HPTLC plates (including glass‐backed plates, aluminum‐backed sheets with or without a fluorescence indicator) had no effect on their results.…”

“…Thus, 580 nm in the fluorescence mode was selected for spot densitometric scanning. It is worthy of note that, due to the aluminum‐backed plate used, TLC bands in our study were scanned in remission fluorescence mode rather than under white light by transmission or remission mode as in Taibon et al .’s () work.…”

Tyrosinase Inhibitors from the Aerial Parts of Wulfenia carinthiacaJacq.

“…Mushroom tyrosinase, L-DOPA and kojic acid 4.20 AE 6.06 2 0 -O-Acetylplantamajoside (3) 33.07 AE 1.00 Globularin (4) 79.59 AE 1.62 2 0 ,6″-O-Diacetylplantamajoside (5) 29.76 AE 4.24 2 0 -O-Acetylisoplantamajoside (6) 13.50 AE 3.10 Baldaccioside (7) 23.01 AE 3.16 Isoscrophularoside (8) 48.49 AE 2.08 2 0 ,6″-O-Diacetylisoplantamajoside (9) 26.14 AE 3.18 Solvents used for HPLC analysis were obtained from Merck (Darmstadt, Germany).…”

“…[1] Due to the limited local distribution and the attractive intensive blue color of the flowers, especially of Wulfenia carinthiaca, this species was selected as the national flower of the federal state of Carinthia/Austria. The MeOH extract of the aerial parts of W. carinthiaca (Carnic Alps population, cultivated material) showed in an HPTLC-based mushroom tyrosinase inhibition assay [5] a promising inhibitory effect, which was also evaluated in a 96 well based assay. 'Alba' is commercially available from different providers.…”