Coreopsis tinctoria Nutt. is an important medicinal plant in traditional Uyghur medicine. The skin-lightening potential of the flower has been recognized recently; however, the active compounds responsible for that are not clear. In this work, tyrosinase, a target protein for regulating melanin synthesis, was immobilized on the Whatman paper for the first time to screen skin-lightening compounds present in the flower. Quercetagetin-7-O-glucoside (1), marein (2), and okanin (3) were found to be the enzyme inhibitors. The IC₅₀ values of quercetagetin-7-O-glucoside (1) and okanin (3) were 79.06 ± 1.08 μM and 30.25 ± 1.11 μM, respectively, which is smaller than 100.21 ± 0.11 μM of the positive control kojic acid. Enzyme kinetic analysis and molecular docking were carried out to investigate their inhibition mechanism. Although marein (2) showed a weak inhibition effect in vitro, it inhibited the intracellular tyrosinase activity and diminished melanin production in melanoma B16 cells as did the other two inhibitors. The paper-based ligand fishing method developed in this work makes it effective to quickly screen tyrosinase inhibitors from natural products. This is the first report on the tyrosinase inhibitory effect of those three compounds, showing the promising potential of Coreopsis tinctoria for the development of herbal skin-lightening products.

Ligand fishing is a promising strategy for the screening of active ingredients from complex natural products. In this work, human tyrosinase (hTYR) was displayed on the surface of Chinese hamster ovary (CHO) cells for the first time; it was then used as bait to develop a new method for ligand fishing. The localization of hTYR on the CHO cell surface was verified by an enzyme activity test and fluorescence microscopy. The displayed tyrosinase (CHO@hTYR) maintained relatively stable enzymatic activity (82.59 ± 2.70%) within 7 days. Furthermore, it can be reused for fishing five times. Guided by the proposed ligand fishing method, four tyrosinase inhibitors, including 4-methoxy-5-methyl coumarin (1), cupressuflavone (2), amentoflavone (3), and 3,4-dimethoxy-5-methyl coumarin (4), were isolated from Alhagi sparsifolia, and the active fraction with low polarity was isolated from Coffea arabica; these two medicinal plants possess skin-lightening potential. All the isolated tyrosinase inhibitors significantly reduced the intracellular tyrosinase activity and melanin level in B16 cells enhanced by α-MSH. Meanwhile, the active fraction (100 μg/mL) from C. arabica exhibited stronger inhibitory effects than the positive controls (α-arbutin and kojic acid) by recovering them to the normal levels. This work demonstrated the promising application of the cell surface display in the field of ligand fishing and is helpful in unveiling the chemical basis of the skin-lightening effect of A. sparsifolia and C. arabica.