Screening Technique To Research Natural Skin-lightening Agent And The Lightening Mechanism Of Dendrobium Officinale Polysaccharide

The stricter safety and efficiency of cosmetic products are required,while the study of skin-whitening products is far from enough.The purpose of this study is to establish a technique of researching and developing new material for skin-whitening products,including preliminary screening,functional evaluation,and mechanism research based on Chemistry,cell biology and animal experiments.Firstly,evaluating the efficacy of the chemicals for whitening-cosmetics through detecting their inhibition activities for tyrosinase and antioxidation activities of scavenging the free radicals from hydroxyl,Super-oxide anion,and DPPH.Second,the assessment model using cell was established.The cell research is composed of two parts: 1.B16 murine melanoma showed good cellular morphology,high cell viability,rapid proliferation and subcultured continuously on the both two media,Gibco-(RPMI-1640)-31800 and Gibco-DMEM-12800.However,melanin was abundant in the B16 cells cultured in DMEM medium,while only small amount was produced in RPMI-1640 medium.Notably,there is no difference in the transcriptional levels of the mRNA of tyrosinase,Tyrosinase-related protein 1,and Tyrosinase-related protein 2 in these two medium detected by RT-PCR.But the levels of these three proteases were obvious higher in DMEM medium than in RPMI-1640 medium detected by Western blot.Furthermore,the cells cultured in DMEM medium had more proteomic diversity detected through two-dimensional electrophoresis.2.Streptomyces bikiniensis was used as supplementary for screening lightening materials.At last,the assessment model using zebrafish was also established.The melanin synthesis in zebrafish starts at over 10 hours of embryos fertilization.It is easily observed due to the embryos are transparent.In addition,we prefered zebrafish as animal experiment model for whitening products research because it have high homology with human genes,easily to be cultured,short experimental period,and so on.Based on the screening and evaluation method mentioned aboved,10 extracts from plants were studied for the skin-whitening activity.The results showed that: 1.The aqueous extracts from Anoectochilus roxburghi and Dendrobium candidum stalk had obvious antioxidation activity as well as tyrosinase inhibitory activity in a dose-dependant manner.2.The aqueous extracts of Lysimachia capillipes,D.candidum stalk and protocorm could restrained the synthesis of melanin in both B16 cells and Streptomyces bikiniensis.While the extrct of A.roxburghi could obviously inhibit melanin synthesis in B16 cells,but it showed no inhibitory activity to S.bikiniensis.The B16 cells were treated with the aqueous extracts from A.roxburghi and D.candidum,and the cytotoxicity,cell cycle,and melanin synthesis were evaluated.The results showed that both extracts at 200 ?g/ml did not affect the normal growth of B16 cells,additionally they reduced the melanin synthesis obviously.3.A.roxburghi,D.candidum stalk and protocorm were proved to be efficient in inhibiting melanin synthesis in zebrafish,while L.capillipes displayed almost no inhibition.Furthemore,the mechanism of melanin synthesis inhibitory activity of polysaccharide from D.officinale to B16 cells were investigated by Western blot,RT-PCR,and two-dimensional electrophoresis.It revealed that the polysaccharide could reduce the transcriptional level of TRP 1,and TRP 2,while had no effect on tyrosinase.It could obviously reduce the translational levels of tyrosinase and TRP 2,while increase the level of TRP 1.Additionally,2-D electrophoresis was used to analyze the influence of D.officinale polysaccharide to the B16 proteome,six protein spots were found,and this result will be investigated in further study.