Thus, we applied 2. 37 7. 11 mg ml for the following experiments. Mushroom tyrosinase is widely used as the target enzyme in screening potential inhibitors of melanogenesis. It was first observed that the dosage range of the Lycium chinense Miller root SFE could http://www.selleckchem.com/products/AP24534.html not inhibit the activity of mushroom tyrosinase. The Lycium chinense Mill root SFE had in hibitory effect on tyrosinase activity using L DOPA as a substrate. The IC50 of Lycium chinense Mill root SFE was found to be 49. 32 mg mL. Kojic acid had strong inhibitory effect on tyrosinase. Tyrosinase is well known to play an essential role in the first two steps of the melanin synthesis pathway. To elucidate the true inhibitory effect of Lycium chinense Miller root SFE on melanin production, the B16F10 melanin content and intracellular tyrosinase activ ity were determined.

The results shown in Figure 3B indi cate that the Lycium chinense Miller root SFE presents a stronger inhibitory effect on melanin formation than arbu tin does. The data provide evidence that Lycium chinense Miller root SFE truly blocks melanogenesis in melanoma cells. The results shown in Figure 3C are in accord with the results indicated in Figure 3B, which suggests that the Lycium chinense Miller root SFE inhibited intracellular tyrosinase activity and then decreased the melanin con tent. In those experiments, MSH was used as a cAMP inducer to stimulate melanin synthesis. It is reported that MSH can bind melanocortin 1 receptor and activate adenylate cyclase, which in turn catalyzes ATP to cAMP and increases intracellular cAMP levels.

The results reveal that the Lycium chinense Miller root SFE inhibited melanogenesis induced by MSH mediated intracellular cAMP up regulation. It has been reported that binding of the human MC1R by its ligands can activate the cAMP signaling pathway and regulate pigmentation of human melanocytes. Melanin biosynthesis in mammalian cells is directly reg ulated by three major enzymes, tyrosinase, TRP 1 and TRP 2. Furthermore, MITF is well known to be the most important regulator of melanocyte differentiation and pigmentation and is the major transcriptional regu lator of the tyrosinase, TRP 1 and TRP 2 genes. The results shown in Figure 4A and Figure 4B indicate that the Lycium chinense Miller root SFE decreased the protein expression levels of those proteins, then inhibited tyrosin ase activity and finally decreased the melanin content in the B16F10 cells.

The results shown in Figure 4 indicate that Lycium chinense Miller root SFE decreased MC1R ex Drug_discovery pression and further suggests that Lycium chinense Miller root SFE inhibited melanogenesis induced via MSH mediated intracellular cAMP up regulation. Moreover, the results shown in Figure 5 further confirm that Lycium chi nense Miller root SFE inhibited cAMP mediated PKA signaling. It has been reported that MAPKs modulate melanin synthesis.