Tyrosinase (90-98)

Tyrosinase is a key enzyme for melanin biosynthesis, and hyperpigmentation disorders are associated with abnormal accumulation of melanin pigments, which can be improved by treatment with depigmenting agents. In the present study, piperlonguminine from Piper longum was discovered to inhibit melanin production in melanoma B16 cells stimulated with alpha-melanocyte stimulating hormone (alpha-MSH), 3-isobutyl-1-methylxanthine or protoporphyrin IX, where the compound exhibited stronger depigmenting efficacy than kojic acid. However, piperlonguminine did not affect 1-oleoyl-2-acetyl-sn-glycerol-induced melanogenesis and did not affect protein kinase C-mediated melanin production. Surprisingly, piperlonguminine did not inhibit the catalytic activity of cell-free tyrosinase from melanoma B16 cells but rather suppressed tyrosinase mRNA expression. This effect was attributed to the inhibitory action of piperlonguminine on alpha-MSH-induced signaling through cAMP to the cAMP responsive element binding protein that in turn regulates the expression of the microphthalmia-associated transcription factor, a key activator of the tyrosinase promoter. This study demonstrates that piperlonguminine is an efficient depigmenting agent with a novel mechanism of action.

In the present study, the immobilization method of physical adsorption combined with covalent crosslinking was developed to avoid the shortcomings of both the noncovalent and covalent coupling methods. For the first time, tyrosinase (TYR) was immobilized on the surface of aminated magnetic nanoparticles (Fe3O4-NH2) by the developed method. TYR was firstly adsorbed on the surface of Fe3O4-NH2 by electrostatic interaction, and then by covalent crosslinking with glutaraldehyde (GA), TYR was firmly immobilized on the supports. The immobilized TYR showed enhanced pH and temperature endurances at the optimum pH of 7.0 and temperature of 35 °C. TYR reusability was significantly improved after immobilization and it retained 61.4 ± 2.3% of its initial activity after 5 repeated cycles. Immobilized TYR also showed improved storage stability (73.2 ± 1.1% after 30 days of storage at 4 °C). In addition, the immobilized TYR showed a higher biological affinity to substrate owing to the stabilization of TYR in its active conformation by electrostatic interaction prior to covalent crosslinking. Finally, the immobilized TYR was used to screen of enzyme inhibitors from 11 traditional Chinese medicines (TCMs) to validate whether this method can be used for enzyme inhibitor screening or not.

Like other members of the Colchicum genus, C. szovitsii subsp. szovitsii is also of medicinal importance in Turkish traditional medicine. However, its biological properties have not been fully investigated. Herein, we focused on the evaluation of the in vitro antioxidant and enzyme inhibitory effects of flower, root and leaf extracts, obtained using different extraction methods. In addition, a comprehensive (poly)-phenolic and alkaloid profiling of the different extracts was undertaken. In this regard, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) allowed us to putatively annotate 195 polyphenols and 87 alkaloids. The most abundant polyphenols were flavonoids (83 compounds), whilst colchicine and 2-demethylcolchicine were some of the most widespread alkaloids in each extract analyzed. However, our findings showed that C. szovitsii leaf extracts were a superior source of both total polyphenols and total alkaloids (being, on average 24.00 and 2.50 mg/g, respectively). Overall, methanolic leaf extracts showed the highest (p < 0.05) ferric reducing antioxidant power (FRAP) reducing power (on average 109.52 mgTE/g) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging (on average 90.98 mgTE/g). Interestingly, each C. szovitsii methanolic extract was more active than the water extracts when considering enzymatic inhibition such as against tyrosinase, glucosidase, and acetylcholinesterase (AChE). Strong correlations (p < 0.01) were also observed between polyphenols/alkaloids and the biological activities determined. Multivariate statistics based on supervised orthogonal projections to latent structures discriminant analysis (OPLS-DA) allowed for the detection of those compounds most affected by the different extraction methods. Therefore, this is the first detailed evidence showing that C. szovitsii subsp. szovitsii might provide beneficial effects against oxidative stress and the associated chronic diseases. Nevertheless, the detailed mechanisms of action need to be further investigated.