Comprehensive profiling of minor tyrosinase inhibitors from Gastrodia elata using an off-line hyphenation of ultrafiltration, high-speed countercurrent chromatography, and high-performance liquid chromatography.

In the present study, a novel hyphenation of ultrafiltration (UF), high-speed countercurrent chromatography (HSCCC), and high-performance liquid chromatography (HPLC) was developed for comprehensive profiling and characterization of the minor tyrosinase inhibitors from Gastrodia elata (GE). A small quantity of GE extract was first fractionated by HSCCC, using elution solvents with a wide range of polarities to enrich minor compounds; then, the fractions were profiled by UF-HPLC to generate a comprehensive 2D chromatogram of the distribution of bioactive components. To determine the binding affinities of these bioactive components, the binding degree (BD%) was calculated by peak area reduction, in which a higher BD% indicates a higher binding affinity to tyrosinase. Among the 212 metabolites, 49 were identified as tyrosinase ligands, 17 of which showed high binding affinity. According to the 2D chromatogram, these 17 candidates were isolated by semiprep-HPLC for characterization of their structure using off-line hyphenated ultraviolet (UV), electron ionized mass spectrometry (EIMS), proton nuclear magnetic resonance (H NMR). Their activities were further validated by functional assays. In conclusion, the approach developed here can comprehensively identify both major and minor bioactive constituents from natural products, and provide meaningful suggestions to direct further research. Compared to conventional approaches, this approach, developed by hyphenating several techniques, is a highly efficient means for comprehensive profiling of potent minor compounds extracted from natural products.