Preparation And Whitening Activity Evaluation Of Cyclo-γ-polyglutamic Acid

Cyclo-γ-Polyglutamic acid(cyclo-γ-polyglutamic acid,cyclo-γ-PGA)is a cyclic small molecule peptide extracted from the jellyfish gill sac,which has eight structures from four to eleven rings.It has the advantages of increasing the hydrophilicity and stability of protein,non-toxicity,biodegradability and good biocompatibility,and has the potential as a high-quality biomaterial.In this paper,three different molecular weights(high,medium,and low)of cyclo-γ-PGA were synthesized in vitro using chemical synthesis methods,and through microbial fermentation,a group of control substances were obtained by degradation:γ-PGA with a moderate average molecular weight(γ-polyglutamic acid,γ-PGA).characterization of the content and molecular weight of the four products obtained.Finally,according to the whitening activity evaluation standard method,the methods include the determination of the inhibition rate of mushroom tyrosinase activity in vitro,the determination of the hydroxyl radical scavenging rate in vitro,the MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)detection method,the determination of the inhibition rate of B16 cell tyrosinase activity and the inhibition rate of melanogenesis,the whitening activities of the above three types of cyclo-γ-PGA were discussed.The main conclusions are as follows:1.In this thesis,three different molecular weights of cyclo-γ-PGA: tetrapeptide cyclo-γ-PGA,octapeptide cyclo-γ-PGA,and decapeptide cyclo-γ-PGA were prepared from glutamic acid(Fmoc Glu OTBU)by separation and purification.The purity of the three products obtained were 98.31%,95.29%,and 95.67%,respectively,and their molecular weights were 516.39,1032.83,and 1291.28,respectively.2.A group of control substances were prepared through microbial fermentation by Bacillus subtilis,isolation,purification,and high-temperature acid precipitation: γ-PGA with moderate average molecular weight.The purity was 97.18% and the average molecular weight was 271 k Da by HPLC and GPC.3.Using γ-PGA as a reference substance,the whitening activity of three types of cyclo-γ-PGA with different molecular weights previously prepared was evaluated.Through in vitro and intracellular experiments,the effects and inhibitory effects of cyclo-γ-PGA on mushroom tyrosinase,tyrosine monophenolase,tyrosine diphenolase,hydroxyl radicals,and intracellular tyrosinase activity and melanin production were investigated.The results show that,at the same concentration,three different molecular weights of cyclo-γ-PGA have different degrees of inhibition on mushroom tyrosinase,tyrosine monophenolase,and tyrosine diphenolase in vitro.The inhibition degree of cyclo-γ-PGA on mushroom tyrosinase,tyrosine monophenolase,and tyrosine diphenolase in vitro increases with the increase of molecular weight.The inhibition rate of low molecular weight cyclo-γ-PGA on mushroom tyrosinase,tyrosine monophenolase,and tyrosine diphenolase in vitro is much lower than that of high molecular weight cyclo-γ-PGA.In addition,the inhibition rate of tyrosine diphenolase with the same molecular weight cyclo-γ-PGA is greater than that of tyrosine monophenolase,and its whitening activity is mainly achieved by inhibiting the activity of tyrosine diphenolase.In the experiment of determining hydroxyl radical scavenging rate in vitro,the scavenging rate of cyclo-γ-PGA on hydroxyl radicals in vitro increases with the increase of molecular weight.However,it is similar to control substanceγ-PGA,and there is basically no in vitro hydroxyl radical scavenging rate at low concentrations.Even at high concentrations,the in vitro hydroxyl radical scavenging rate is far less than Vc and arbutin.In the MTT experiment,tetrapeptide cyclo-γ-PGA and octapeptide cyclo-γ-PGA had almost no toxicity to mouse melanoma cells,and when the concentration reached 10 mg/m L,the cell survival rate was also above 90%.Decapeptide cyclo-γ-PGA and control substance γ-PGA are non-toxic to cells at concentrations below 1mg/m L,with a cell survival rate of over 90%.At higher concentrations,the cell survival rate is lower.Therefore,cell experiments were conducted at concentrations below 1 mg/m L.Finally,the inhibition of intracellular tyrosinase activity and melanogenesis was measured.Within the safe concentration range,the inhibition rate of intracellular tyrosinase activity and melanogenesis of cyclo-γ-PGA increased with the increase of molecular weight and concentration,with decapeptide cyclo-γ-PGA having the highest inhibition rate at 1 mg/m L.When the concentration was the same,the inhibition rates of intracellular tyrosinase activity and melanogenesis of three groups cyclo-γ-PGA were greater than that of control substanceγ-PGA.In this study,three different molecular weight cyclo-γ-PGA and control substances were prepared: a group of γ-PGA with an average molecular weight of 271 k Da.It has been proved that cyclo-γ-PGA has whitening activity,which increases with the increase of molecular weight,and performs better in intracellular characteristics.Based on the results of both in vitro and intracellular experiments,it is concluded that the whitening activity of cyclo-γ-PGA is higher than that of cyclo-γ-PGA,providing a reference and theoretical basis for the subsequent application of cyclo-γ-PGA in the field of skin whitening and cosmetics.