| Non-enzymatic glycation of proteins plays a significant role in skin aging.Advanced glycation end products（AGEs）accumulate in aging skin,which can cause changes in skin biomechanical properties and biological changes,causing skin to become dry,thin,inelastic and rigid.Adding safe anti-glycation substances into cosmetics is one of the most effective methods for the development of anti-aging cosmetic products.In this study,the effects and mechanisms of longan seed polyphenols in inhibiting non-enzymatic glycation of BSA were studied through various models.The protective effects of longan seed polyphenols on human skin fibroblasts affected by AGEs were studied by detecting oxidative stress,pro-inflammatory factors and receptor RAGE.Finally,the antiglycation effect of longan seed polyphenols in dermalt was studied by tissue engineering.The main contents are as follows:The inhibitory effect of longan seed polyphenols on non-enzymatic glycation of protein was studied with BSA-glucose as glycation model.It has been proved that longan seed polyphenols did not inhibit the generation of Amadori products in the first stage of glycation through NBT method.HPLC derivatization method showed that longan seed polyphenols could significantly reduce 70%of glyoxal and methylglyoxal in the system and the inhibition of 3-deoxyglucosone was dependent on concentration of longan seed polyphenols.What’s more,fluorescence analysis and enzyme-linked immunosorbent assay showed that the inhibition rates of longan seed polyphenols（5μg/m L）on the production of fluorescent AGEs at excitation/emission wavelengths of 335/385 nm and 370/440 nm were 62.4%and 21.3%,respectively.The inhibition rate on the production of CML was 61.5%.The results showed that longan seed polyphenols have a good anti-glycation effect on protein.Capturing carbonyl group experiment results showed that longan seed polyphenols have good ability to capture glyoxal and methylglyoxal.In the Amadori product reaction model,longan seed polyphenols could produce glyoxal and fluorescent AGEs by inhibiting the oxidation of Amadori products.Comparing the two systems of MGO-BSA and MGO-Arg,the longan seed polyphenols can interact with the protein and better protect the amino group from the attack of dicarbonyl compounds according the to analysis of molecular docking.Fibroblast injury was induced by the synthesis of AGEs and it showed that AGEs have certain cytotoxic effects on fibroblasts and inhibit cell proliferation.In the presence of AGEs（0.5 mg/m L）,the amount of NF-κB p65 increased 13.9%,while after adding 5μg/m L longan seed polyphenols,it decreased to 8.8%,which meant that longan seed polyphenols can inhibit the synthesis of NF-κB p65 in fibroblasts induced by AGEs.In the presence of AGEs（1mg/m L）,the amount of intracellular collagen decreased from 285μg/mgprot to 238μg/mgprot,intracellular ROS increased by 62.5%,intracellular SOD activity decreased from3.26 U/mg to 2.57 U/mg,phosphorylation of NF-κB p65 increased by 49.2%,expression of RAGE increased by 94.4%,suggesting that AGEs can induce oxidative stress in fibroblasts,activate cell signaling pathways and induce cell damage.After 5μg/m L longan seed polyphenols was added,collagen content increased to 285.9μg/mgprot,the increasing rate of ROS decreased to 28%,SOD activity increased to 2.89 U/mg,the increasing rate of phosphorylation of NF-κB p65 decreased to 36.8%,the increasing rate of expression of RAGE decreased to 71.9%.Results showed that longan seed polyphenols can reduce AGE-induced oxidative stress of fibroblasts,increase collagen synthesis,inhibit the continuous activation of NF-κB and decrease the expression of RAGE to protect fibroblasts.The anti-glycation effect of longan seed polyphenols in biomimetic dermal model was evaluated by using collagen gel to form dermal equivalent substance.The results showed that the mixture of collagen formed and pink celled dermal equivalents at presence of 5%CO2（37℃）.In the process of dermal equivalent glycation,longan seed polyphenols could effectively inhibit or eliminate the formation of dicarbonyl compounds.At the excitation/emission wavelengths of 370/440 nm and 335/385 nm,the inhibition rates of 5μg/m L longan seed polyphenols on the formation of fluorescent AGEs were 59.2%and 57.9%,respectively.In the process of collagen preglycation,the inhibition rates of 100μg/m L longan seed polyphenols on the production of fluorescent AGEs at the excitation/emission wavelengths of 370/440 nm and 335/385 nm were 70.0%and 37.4%,respectively.In the cell proliferation experiment of aging dermal equivalent containing cells,the relative activity of dermal equivalent cells treated with longan seed polyphenols was higher than that treated with ribose.Longan seed polyphenols can reduce the effect of collagen glycation on the proliferation of fibroblasts by inhibiting the glycation of collagen in the extracellular matrix,thus protecting the cells. |
