Bio-panning Of A Tyrosinase Inhibitor And Preparation Of Its Flexible Liposome

Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase, is a copper metalloenzymen which is widely distributed in microorganisms, insects, plants and mammals. The different sources of tyrosinase have similar structures and functional characteristics. Tyrosinase is a key enzyme in melanin synthesis in organisms. Therefore, the study on tyrosinase inhibition has become an important mean of treatment to pigmentation. Tyrosinase inhibitors can also be used in whitening cosmetics, bio-pesticides, fresh preservation of fruits and vegetables and many other fields. Currently, tyrosinase inhibitors have become a hot research area.In this study, we firstly screened a tyrosinase inhibitor from the random phage display peptide library. Secondly, we carried on a series of inhibition kinetics tests in order to explore the inhibition mechanism. At last, we prepared liposome as drug delivery system to study the performance of its penetration.This thesis includes the following contents:Using tyrosinase as a target molecule, we obtained 10 monoclones which had specific affinities on tyrosinase molecule after three rounds of bio-panning. After amplifying and the titer tests of the monoclones, the results of the ELISA showed the highest affinity was the NO.3 clone. After sequencing the extracted plasmid, the encoding gene was inferred, which was a part of genome in the single-stranded DNA of bacteriophage. The sequence of NO.3 monoclone was:Ile-Gln-Ser-Pro-His-Phe-Phe (7AAP), which was displayed on the surface of phage particles.During the enzyme kinetics tests, both L-tyrosine and L-dopa were taken as the substrate. The results verified that 7AAP could inhibited both monophenolase and diphenolase activities of tyrosinase. For monophenolase, the inhibitory effect mainly embodied in extending the lag time significantly. Similarly, the Lineweaver-Burk analysis showed the inhibition of 7AAP belonged to the reversible and competitive-type for diphenolase.In cytological experiments, human A375 melanoma cells were selected as subjected cells. The effects of 7AAP on the A375 melanoma cells (including morphology, proliferation, melanin synthesis and tyrosinase enzyme activity) were discussed by setting different concentrations of 7AAP (0,4.5,9.0,18.0,36.0,72.0, 144.0,288.0,576.0,1152.0μmol/L). Observed under the microscope,7AAP had no effect on the morphology of A375 melanoma cells compared with control group. When concentration of 7AAP below 72μmol/L, it didn't affect A375 human melanoma cells'proliferation significantly. In the range of 4.5-72μmol/L,7AAP existed a concentration-dependent inhibition of melanogenesis. In order to explore whether this inhibition of melanogenesis was related to tyrosinase, further research about 7AAP on melanocyte tyrosinase activity was carried on. We found there was also a concentration-dependent inhibition of tyrosinase activity. The results showed that in the concentration range of 4.5-72μmol/L, the reduction of the relative melanin content was caused by inhibiting tyrosinase activity of A375 melanoma cells, rather than just reducing cell proliferation rate.In the study of 7AAP loaded transdermal drug delivery system, we selected soy lecithin, cholesterol as raw material.7AAP loaded liposomes were successfully prepared by reverse-phase evaporation method, film method and film-ultrasonic method, respectively. The particle size, Zeta potential, recovery ratio and encapsulation efficiency were tested. The results of dynamic light scattering showed the liposomes prepared by reverse evaporation method had the most uniform size distribution. The results of the field emission scanning electron microscopy (FE-SEM) indicated liposomes presented a shape of spherical or ellipsoidal and the majority of liposomes were single-chamber. In order to observe the impact of surfactant for deformation ability of the liposomes, different flexible nano-liposomes were prepared by adjusting the mass ratio of phospholipids:sodium cholate (6:1,8:1,10:1). The results suggested that the solution of pure 7AAP had the fastest rate in permeating the skin, while 7AAP liposomes accumulated in the receiving tank were lower than 7AAP solution. This indicated that liposome could reduce the amount of 7AAP into the systemic circulation. The results showed the flexible liposomes had the lower skin permeation rates than 7AAP solution. So, the flexible liposomes prepared in this thesis can substantially improve the retention amount of 7AAP in the epidermis and dermis layers, and achieved sustained drug release. The biological effects were improved.