Usnic acid contents in acetone extracts of 31 samples of lichen Parmelia flexilis collected from different altitudes were identified using thin layer chromatography (TLC) and determined by high performance liquid chromatography (HPLC). The usnic acid content varied in between highest 5.13% to lowest 1.66% in oven dried (80 °C) lichen samples. The species collected from lower altitudes all show high levels of usnic acid. The negative relationship between usnic acid and altitude was obtained. Statistically, it is revealed that there is a significant difference between average percentages of usnic acid in lichen samples with varying altitudes (p < 0.05). Beside these, the precipitation averages of the regions where the species have been collected were linked with the content of usnic acid. It is clear that lichens from the regions receiving the highest precipitation produced lower amounts of usnic acid. The results suggest that the production of secondary metabolite in lichens is altered due to the climatic variables like temperature and precipitation at different altitude gradients.

Coppers play crucial roles in the maintenance homeostasis in living species. Approximately 20 enzyme families of eukaryotes and prokaryotes are known to utilize copper atoms for catalytic activities. However, small-molecule inhibitors directly targeting catalytic centers are rare, except for those that act against tyrosinase and dopamine-β-hydroxylase (DBH). This study tested whether known tyrosinase inhibitors can inhibit the copper-containing enzymes, ceruloplasmin, DBH, and laccase. While most small molecules minimally reduced the activities of ceruloplasmin and DBH, aside from known inhibitors, 5 of 28 tested molecules significantly inhibited the function of laccase, with the K_i values in the range of 15 to 48 µM. Enzyme inhibitory kinetics classified the molecules as competitive inhibitors, whereas differential scanning fluorimetry and fluorescence quenching supported direct bindings. To the best of our knowledge, this is the first report on organic small-molecule inhibitors for laccase. Comparison of tyrosinase and DBH inhibitors using cheminformatics predicted that the presence of thione moiety would suffice to inhibit tyrosinase. Enzyme assays confirmed this prediction, leading to the discovery of two new dual tyrosinase and DBH inhibitors.