Evaluation And Preliminary Study On The Of Anti-photoaging Activity Of Ultrafiltration Components Of Dendrobium Officinale In Vitro

Skin aging is mainly caused by internal aging and external aging,and ultraviolet light aging is the main cause of external aging.A large number of UVB irradiation leads to the accumulation of reactive oxygen species（ROS）in the skin,consumes intracellular antioxidants,causes oxidative stress,and causes oxidative damage to the skin.Dendrobium Officinale Kimura Et Migo,as a natural and precious traditional Chinese medicine,polysaccharides and alkaloids are the main active substances,in which polysaccharides play an excellent role in moisturizing,antioxidant and anti-aging.However,there are few studies on the anti-photoaging activity of Dendrobium officinale polysaccharides,and the mechanism is not clear.In this study,the ultrafiltration components with different molecular weights of D.officinale polysaccharides as the main components were used as the research objects,and UVB-induced photoaging human keratinocytes（HaCaT）were used as the model to screen the active components with anti-photoaging ability,evaluate their activities,and explore their possible mechanisms,so as to provide a theoretical basis for the further development of D.officinale as a raw material for skin care.The main research results are as follows:The dry stems of Dendrobium Officinale were used as raw materials to obtain crude polysaccharides by solvent extraction.Through membrane filtration,WEA（>10 KDa）,WEB（1–10 KDa）,WEC（300 Da-1 KDa）and WED（<300 Da）were obtained respectively.Component analysis,content determination,and molecular weight detection were performed on the soluble part of each component.The soluble fraction of WEA mainly contained 92.41%polysaccharides,the average molecular weight of 78%macromolecular polysaccharides was299.67 KDa,and the average molecular weight of 14.4%oligosaccharide was 1.1 KDa.The soluble fraction of WED mainly contains 64.81%oligosaccharides.The average molecular weight of oligosaccharides was 990 Da.The monosaccharide composition of each component was determined by ion chromatography,which was composed of glucose,mannose and galactose,and contained a small amount of xylose and arabinose.Firstly,the photoaging model of HaCaT cells induced by UVB was established.The irradiation dose of UVB was 60 m J·cm^-2,and the intervention time of the tested components was controlled at 12 h.Based on this model,cell viability was measured by CCK-8 method,and cell morphology was observed under inverted microscope.Compared with the model group,50μg·m L^-1 WEA and 100μg·m L^-1 WED could restore cell viability by 40.54%and 54.09%（P<0.05,P<0.01）,and 200μg·m L^-1 WEA and WED could significantly improve cell morphology.Secondly,reactive oxygen species（ROS）was determined by flow cytometry,and malondialdehyde（MDA）,superoxide dismutase（SOD）and total antioxidant capacity（T-AOC）were detected by biochemical chromogenic reaction.Compared with the model group,WEA and WED decreased ROS content by 38.65%and 26.74%at 100μg·m L^-1 and 50μg·m L^-1,respectively（P<0.01,P<0.05）.200μg·m L^-1 WEA and 100μg·m L^-1 WED decreased MDA（P<0.05）and increased SOD and T-AOC activities（P<0.05）.It indicated that WEA and WED components could inhibit the production of ROS in photoaging HaCaT cells and increase the content of intracellular antioxidants.RT-qPCR was used to detect the transcription levels of keratinocyte barrier-related proteins,moisturizing-related proteins,inflammatory factors and matrix metalloproteinase（MMPs）.The results showed that the minimum effective concentration of WEA was 100μg·m L^-1.Under this concentration,WEA significantly up-regulated the m RNA expression of FLG,LOR and INV,and restored the transcription levels of AQP-3,Caspase-14 and HAS2（P<0.05）.WED component could significantly down-regulate the m RNA levels of IL-1β,IL-6,TNF-α,IL-8,MMP-1 and MMP-3 in HaCaT cells,and the minimum effective concentration was 50μg·m L^-1（P<0.05）.200μg·m L^-1 WEA could also reduce the transcription of IL-1βand MMP-1.WEA showed excellent barrier repair effect,and WED showed remarkable anti-inflammatory and anti-matrix metal enzyme effects.High concentration also has certain anti-inflammatory and anti-matrix metal enzyme activity.Because the water-soluble fraction of WEA contains 14.4%oligosaccharides and the monosaccharide composition is close to WED,it was speculated that the anti-inflammatory and anti-matrix metal enzyme activities of WEA at high concentrations are related to the oligosaccharides.Finally,the key signal proteins in MAPK signaling pathway and NF-κB signaling pathway were detected by Western blotting.200μg·m L^-1WEA and 100μg·m L^-1 WED could reduce the phosphorylation of p38,JNK,c-jun and c-fos（P<0.01,P<0.01,P<0.05,P<0.01）.WEA and WED significantly inhibited the phosphorylation of IκBαand p65/NF-κB（P<0.05,P<0.01）,and decreased the phosphorylation and non-phosphorylation ratio of IκBα.These results suggest that WEA and WED components alleviate photoaging by reversing UV-induced activation of MAPK and NF-κB signaling pathways,reducing inflammation and matrix metalloproteinase expression.