Ultrasonic-assisted Extraction,Identification And Action Mechanism Of Glycosylation Inhibitors From Lotus Leaves

Lotus is a well-known plant with the homology of medicine and food in China.It has the functions of clearing heat and dampness,clearing heart and removing heat,stopping bleeding and improving water.Studies have shown that its leaves have the functions of reducing fat and weight,anti-inflammation,anti-oxidation,anti-cancer,lowering blood pressure,inhibiting atherosclerosis and anti-diabetes,and flavonoids are the main active components.In China,the planting area of lotus is more than 10 million mu,but the utilization rate of lotus leaf resources is very low,less than 1% of its total output,serious waste of resources.Previous studies showed that lotus leaf extract can effectively inhibit the formation of Advanced glycation end-products(AGEs)in the BSA-fructose glycosylation system,and the polyphenols enriched in lotus leaf can also effectively inhibit the formation of AGEs in the ovalbumin-fructose system.Therefore,based on the lotus leaf as raw material,USES the ultrasonic assisted extraction and optimization of the extraction methods of glycosylation inhibitor in the lotus leaf,using macroporous adsorption resin separation and enrichment of glycosylation inhibitor in the lotus leaf,then the use of chromatographic separation technology and half the main compounds in the optimum component type liquid purification,and by using nuclear magnetic resonance(NMR),mass spectrometry and other technology on the structure characterization,In this study,OVA-fructose and β-LG-fructose systems were used as glycosylation models to study the effects of representative inhibitors on the glycosylation reaction of proteins.Spectroscopic techniques,mass spectrometry and molecular docking techniques were used to elucidate the process of inhibition of protein glycosylation,and finally the mechanism of action was identified.The completion of this study can provide theoretical support for the high-value utilization of lotus leaf resources and the directional regulation of AGEs formation in the food processing process,and also has a certain significance for improving the theoretical system of inhibiting AGEs formation with extracts from natural plant sources.The main research results are as follows:(1)The optimal conditions for ultrasonic-assisted extraction of glycation inhibitors from lotus leaves were as follows: solid-liquid ratio of 1:30(g:mL),ethanol concentration of 70%,ultrasonic time of 40 min,ultrasonic temperature of 50℃,and ultrasonic power of 400 W.The AGEs inhibitory activity of the extracts increased from 21.91% to 75.47%.Macroporous adsorption resin was used to separate and concentrate AGEs inhibitors in lotus leaves.The optimal elution component was80HY(6.6g),and the AGEs inhibitory activity(concentration was 0.2 mg/mL),total phenols and total flavonoids contents were obtained.From 2.43%,175.80 mg GAE/g and 13.06 mg QuE /g to 62.84%,393.64 mg GAE/g and 167.98 mg QUE /g,respectively.(2)Four compounds were isolated and purified at 80 HY,and they were hyperoside(1),isoquercitrin(2),tredaidin(3),and shividin(4).The antiglycosylation activity of the four compounds was stronger than that of the positive control aminoguanidine,among which isoquercitrin had the best activity.(3)The inhibitory effect of Isoquecitrin(ISQ)on the glycosylation of Ovalbumin(OVA)was investigated,and the interaction between OVA and ISQ was studied by spectroscopy and molecular docking.The effects of ISQ on the structure,number of glycosylation sites and degree of glycosylation of OVA were analyzed by spectroscopic method and mass spectrometry.The results showed that ISQ significantly inhibited the formation of AGEs in the OVA glycosylation system,effectively reduced the glycosylation reaction of OVA,and weakened the conformational changes caused by glycosylation of OVA.Nano LC-Orbitrap MSN analysis showed that ISQ did not decrease or increase the number of OVA glycosylation sites,but affected the glycosylation locations and reduced the degree of glycosylation at most sites.ISQ quenched the fluorescence of OVA by a static mechanism,which exposed the fluorescent amino acid tryptophan residues in OVA to a more hydrophobic environment,thus changing its conformational structure.The formation of OVA-ISQ complex is an endothermic process,and the main forces are hydrophobic interaction,van der Waals force and hydrogen bonding.Molecular docking analysis showed that ISQ was inserted into the hydrophobic pocket of the OVA with six hydrogen bonds,leading to changes in the microenvironment of some amino acids,thus affecting the glycosylation activity of the glycosylation sites.(4)The inhibitory effect of ISQ on the glycosylation of β-lactoglobulin(β-LG)was evaluated,and its mechanism was elucidated by fluorescence spectroscopy,Nano LC-Orbitrap MSN and molecular docking.Fluorescence spectrum analysis showed that ISQ could effectively slow down the formation of AGEs,stabilize the conformational structure of glycosylated β-LG(G-β-LG),and change the microenvironment near the chromophore.SDS-PAGE and mass spectrometry showed that ISQ inhibited G-β-LG crosslinking.After the addition of ISQ,the glycosylation sites on G-β-LG were reduced from 10 to 8,and the relative glycosylation degree of most of the glycosylation sites was also significantly reduced.Fluorescence spectra and molecular docking showed that ISQ quenched β-LG fluorescence by a static mechanism,and the binding was an endothermic process.Isq interacts with β-Lg to form an isomolar complex.Isq interacts with Lys69(4.96A)on β-Lg to form A hydrogen bond in which hydrophobicity,hydrogen bond and van der Waals forces are the main forces.