| Objective:With the increasing environmental pollution,the atmospheric ozone layer is getting thinner,and the intensity of ultraviolet radiation increases,skin problems such as chloasma and freckles seriously affect people’s quality of life,so skin care products with whitening effect are sought after by many people.In recent years,it has been reported that flavonoids and isoflavones of pueraria root inhibit tyrosinase activity,but the study on whitening activity of water-soluble total protein of pueraria root has not been reported.Therefore,response surface BBD design experiment was used to obtain the best extraction process of pueraria protein.On the basis of this,its physical and chemical properties and its effect on mouse melanoma B16 cells and its mechanism were studied.Methods:1.After screening and determining whitening active ingredients,single factor test of extraction time,solid-liquid ratio and p H was carried out to determine the response surface level of three factors,and process optimization was carried out according to BBD design.2.Through the kudzu root water-soluble total protein structure,physical and chemical properties and functional characteristics research: using UV,fluorescence spectrum and FTIR and CD spectra analysis of puerarin water-soluble total protein structure,detection of puerarin protein molecular weight protein,amino acid composition,solubility,emulsification,oily and holding water and other physical and chemical properties and functional properties.3.The water-soluble total protein of pueraria root can inhibit the activity of tyrosinase,reduce the production of melanin,and reduce the level of cellular oxidative stress.MITF is the key factor throughout this chapter.The p38 MAPK pathway can activate MITF.The decreased expression of P38 leads to the decreased expression of MITF.The decreased expression of MITF can inhibit the proliferation of mouse melanoma B16 cells and reduce the expression of TYR,TRP-1 and TRP-2,and TRP-1 and TRP-2 are involved in the synthesis of melanin downstream of TYR,so the decreased expression of TRP-1 and TRP-2down-regulates the expression of TYR.Thus reducing the synthesis of melanin.Subsequently,the decreased expression of MITF can also promote cell apoptosis,thus upregulating the ratio of Bax/Bcl-2 and the expression of Caspase-3 and Caspase-9,thus promoting cell apoptosis.Apoptosis not only reduced ATP release and mitochondrial quality,but also inhibited oxidative phosphorylation.Results:1.The water-soluble total protein extracted from pueraria root decoction pieces was screened by inhibition of tyrosinase activity in vitro.The optimal extraction process was determined as extraction time of 4 h,solid-liquid ratio of 1:10,p H8.2.The secondary structure of PLP is mainly composed of β-folding,β-rotation and random crimping.The results of infrared spectrum showed that PLP has characteristic functional groups such as CO,-NH and COO-.Sds-page showed that PLP was mainly composed of proteins with molecular weight less than 26 k Da.The solubility,emulsifying activity and emulsifying stability of PLP decreased first and then increased near the isoelectric point.Its water holding capacity is 0.8 g/g,oil holding value is 2.94 g/g.3.Study on whitening mechanism of pueraria water-soluble total protein(PLP)on B16 cells.Studies have shown that pueraria water-soluble total protein can inhibit tyrosinase activity,reduce cell oxidative stress level,reduce melanin production,and down-regulate the expressions of MITF,TYR,TRP-1,TRP-2 and P38.P38 MAPK pathway can activate MITF,and the down-regulation of MITF expression can inhibit the proliferation of mouse melanoma B16 cells and reduce the expression of TYR,TRP-1 and TRP-2.Moreover,TRP-1 and TRP-2 are involved in the synthesis of melanin downstream of TYR,so the decreased expression of TRP-1 and TRP-2 down-regulates the expression of TYR.Thus reducing the synthesis of melanin.In this study,we also studied the mechanism of PLP promoting apoptosis: apoptosis through mitochondrial membrane potential.Firstly,PLP decreased the content of SDH and the expression of SDHA and SDHB proteins.Subsequently,the decreased expression of MITF can also promote cell apoptosis,up-regulate the ratio of Bax/Bcl-2,and up-regulate the expression of Caspase-3 and Caspase-9 to promote cell apoptosis.Apoptosis not only reduced ATP release and mitochondrial quality,but also inhibited oxidative phosphorylation.Conclusion:The extraction process of water-soluble total protein from pueraria root optimized by response surface methodology showed that it had the physical and chemical properties contained in the protein and could inhibit the proliferation of mouse melanoma B16 cells by promoting cell apoptosis and inhibiting melanin formation. |
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