| Objective:To analyze the nutritional composition of Lycium ruthenicum Murr of different origins and conduct a preliminary study on their biopharmacological characteristics,and to investigate the protective effect of its main component,polysaccharide of Lycium ruthenicum Murr,on cellular damage in retinal pigment epithelium?ARPE-19?caused by oxidative stress.Methods:1.Four different origins were selected,namely,Xinjiang,Ningxia,Gansu and Qinghai,to determine the content of water,ash,total protein,total fat,amino acids and trace elements in accordance with national standards,and to characterize the powder,water test,microscopy and physical and chemical identification.2.The polysaccharides were extracted by water extraction,deproteinized by trichloroacetic acid,decolorized by hydrogen peroxide,separated by DEAE-52 cellulose column,purified and characterized by Sephadex G-100,and the composition and content of polysaccharides were briefly analyzed by gas-phase mass spectrometry.3.CCK-8 method to detect the effect of different concentrations of Lycium ruthenicum Murr polysaccharide on ARPE-19 cell viability;CCK-8 method to detect the viability of ARPE-19 cells induced by different concentrations of H2O2 and construct an oxidative damage model;CCK-8 method to detect the effect of Lycium ruthenicum Murr polysaccharide on the viability of ARPE-19 cells induced by H2O2;Hoechst/PI staining and addition of caspase inhibitor to observe the morphology of ARPE-19 cells after H2O2induction;flow cytometry double staining method to investigate the effect of Lycium ruthenicum Murr polysaccharide on the apoptosis of ARPE-19 cells induced by H2O2;immunoblotting method to detect the differences in NLRP3,caspase-1,IL-1?protein expression levels of normal,H2O2-induced and Lycium ruthenicum Murr polysaccharide intervention cells.Results:1.In this study,there were significant differences in the basic composition of black fruit of different origins?P<0.05?,with the highest content of calcium and zinc in Xinjiang,the highest content of iron in Ningxia and the highest content of magnesium in Qinghai;except for alanine,there were no significant differences in the other amino acids;the differences in microstructure were mainly shown in stonecytes and epidermal cells of the fruit pericarp;the physical and chemical identification of Rf value,color and size of spots were the same as those of controls.2.The crude LMPs was obtained by hydrotitol sinking,the rate of deproteinization by trichloroacetic acid was 75.90%and the rate of depigmentation by hydrogen peroxide was39.90%;four components were separated by DEAE-52 cellulose column,and the purity of Sephadex G-100 chromatography column was identified as a single component with uniform molecular weight.LMP-1-1 monosaccharides were analyzed by gas-mass spectrometry with a molar ratio of 1:1.24;LMP-2-1 monosaccharides were composed of mannose,galactose and arabinose with a molar ratio of 1.73:1.08:1;LMP-3-1monosaccharides were composed of glucose,galactose and arabinose with a molar ratio of2.20:1:1.07;LMP-4-1 monosaccharides were composed of glucose,galactose and arabinose with a molar ratio of 1.77:1:1.07.3.CCK-8 results showed that cell viability was not statistically different from that of the control group?P>0.05?when the concentration of LMPs was 25-450?g·mL-11 and cell viability was inhibited when the concentration of LMPs increased to 500?g·mL-1.H2O2 significantly reduced ARPE-19cell viability,half of ARPE-19 cells survived at a H2O2 concentration of 400?mol·L-1,and cell viability was statistically different from that of the control group?P<0.01?;LMPs was protective at a concentration of 25-400?g·mL-1,and cell viability was statistically different from that of the control group?P<0.01?,and the protective effect was reduced at a concentration of LMPs over 100?g·mL-1.SOD and GSH-Px activity was elevated and MDA decreased in the LMP-2-1 group compared with the model group,and there was a statistical difference?P<0.01?between the detection index and the control group.The total apoptotic rate of ARPE-19 was reduced differently in the model group compared with the model group with low LMP-2-1 and medium dose,and the apoptotic rate was statistically different from the control group?P<0.05?.Compared with the blank group,NLRP3,Caspase-1,and IL-1?expression increased?P<0.05?in the model group,NLRP3,Caspase-1,and IL-1?expression decreased significantly?P<0.01?in the groups of LMP-2-1 low and medium-dose compared to the model group,and NLRP3 protein expression decreased significantly?P<0.01?and IL-1?protein expression decreased?P<0.05?in the high-dose group compared to the model group.Conclusion:1.In this study,the basic composition,metal element content and traits of Lycium ruthenicum Murr of different origins are different.2.Extraction and purification of the Lycium ruthenicum Murr polysaccharide,the composition of which includes glucose,galactose,arabinose,mannose.3.The purified LMPs increased the antioxidant capacity of ARPE-19 cells without any dose-dependence,increased the SOD and GSH-Px activity of H2O2-damaged ARPE-19 cells,decreased the MDA content,and possibly inhibited the pyroptosis of ARPE-19 cells by down-regulating the protein expression of NLRP3,Caspase-1 and IL-1?,thereby preventing age-related macular degeneration. |
PDF Full Text Request