HPTLC bioautography as a potent tool for the discovery of new tyrosinase inhibitors from Greek flora

Tyrosinase is an enzyme which is responsible for melanogenesis in animals and browning in plants and fungi. Since nature offers a unique set of active compounds that could be a source of new leads for skin-care cosmetics, there is a great interest in tyrosinase inhibitors originating from natural products [1,2]. The aim of the present study was to discover and isolate natural compounds with anti-hyperpigmenting activity from plant extracts. A total of about 120 plant species from Greek flora were collected and extracted producing around 240 extracts. Extracts were initially studied for their phytochemical profile with three different methods developed on HPTLC based on their chemical characteristics. Thereafter the extracts were in vitro evaluated for their activity against tyrosinase. Based on the above results (chemical profile & biological activity) seven most promising extracts were selected for further study. At this point there was a need for a technique which integrates chromatographic separation and biological detection of active compounds. This was achieved by developing an HPTLC bioautographic method for the detection of tyrosinase inhibitors. Directly after the chromatographic separation the plate was sprayed homogenously with L-Dopa and tyrosinase. After incubation the plate was captured in white light and the active compounds appeared as white spots against a dark background [3]. The main advantage of this method in comparison with in vitro assays, is the ability to detect minor active metabolites from plant extracts. After the detection of the active compounds several chromatographic techniques and especially FCPC were used for the isolation of the most promising ones. Several compounds belonging to various chemical categories were purified and their tyrosinase activity was evaluated. In conclusion, HPTLC combined with enzymatic assays represents an efficient technique in the bio-guided isolation of natural compounds.

Keywords: HPTLC, Tyrosinase, FCPC

References:

[1] Favre-Godal Q. et al. Latest Developments in Assessing Antifungal Activity Using TLC-Bioautography: A Review. J. AOAC Int.. 2013; 96:1175 – 1188

[2] Seo S.Y. et al. Mushroom Tyrosinase: Recent Prospects. J. Agric. Food Chem. 2003, 51, 2837 – 2853

[3] Wangthong S. et al. Post TLC developing technique for tyrosinase inhibitor detection. Biomed. Chromatogr. 2007; 21:94 – 100