Planta Med 2014; 80 - P2O27
DOI: 10.1055/s-0034-1395019

Development and validation of an HPTLC based assay for the screening and identification of tyrosinase inhibitors

J Taibon 1, A Ankli 2, C Magnenat 3, CA Simões-Pires 3, VI Boka 4, A Argyropoulou 4, N Aligiannis 4, AL Skaltsounis 4, M Cuendet Licea 3, H Stuppner 1, E Reich 2
  • 1Institute of Pharmacy/Pharmacognosy, University of Innsbruck, CCB – Center of Chemistry and Biomedicine Innrain 80/82, 6020 Innsbruck, Austria
  • 2CAMAG Laboratory, Sonnenmattstr. 11, 4132 Muttenz, Switzerland
  • 3School of pharmaceutical sciences, University of Geneva, University of Lausanne, Quai Ernest-Ansermet 30, 1211 Geneva, Switzerland
  • 4Department of Pharmacognosy and Natural Products Chemistry, University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece

An HPTLC-based assay was established to screen plant extracts for the presence of tyrosinase inhibiting substances on the basis of previously reported work [1 – 3]. Three mobile phases, one for lipophilic and two for more polar extracts, were selected for the chromatographic separation of extracts and optimized for compatibility with the subsequent bioassay performed directly on the HPTLC plate. Following chromatography, the plate was sprayed first with the substrate L-Dopa and then with tyrosinase. Tyrosinase inhibitors appear after an incubation time of 10 minutes as white zones against a dark background. Several known tyrosinase inhibitors (e.g. kojic acid) were tested as positive controls, and could be clearly documented as white zones with white light in remission from the plate (WR), as well as with white light transmitted through the plate (WT). When extracts were tested, especially apolar ones, some spots appeared differently; some lipophilic substances of the extracts were seen as white zones in WR but as black spots in WT. This phenomenon could lead to false positive results. It was assumed that the observed behaviour was due to poor wettability of the corresponding zones. The problem was resolved and these false positive results were eliminated by adding Triton X100, a non-ionic surfactant, to the L-Dopa solution. This phenomenon was further reduced by drying the plate after incubation with a molecular sieve. The whole procedure resulted in a very homogeneous background colouration that improved the contrast and thus detectability of inhibition zones. In this optimized assay it is possible to clearly identify tyrosinase inhibitors as white zones in WT as well as in WR. Results were confirmed and the method validated by using several known tyrosinase inhibitors, as well as active and non-active plant extracts.

References:

1. Wangthong, S. et al. (2007) Biomed. Chromatogr. 21: 94 – 100. 2. Momtaz, S. et al. (2008)J. Ethnopharmacol. 119: 507 – 512.

3. Kamagaju, L. et al. (2013)J. Ethnopharmacol. 146: 824 – 834.