Preparation of the Soybean Lecithin-Gallic Acid Complex and the Hepatoprotective Effect on Iron Overload in Mice with Alcoholic Liver Disease

Author:Wu Xiang Qun

Supervisor:liu zuo

Database:Doctor

Degree Year:2019

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Pages:160

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Objective:Gallic acid(GA)is a commonly used antioxidant in food.The development and application of its biological activity have not been carried out in depth.This study intends to improve the bioavailability of gallic acid by synthesizing soybean lecithin-GA complex(SL-GAC),and to analyze the possible bioactivities of the complex by computer simulation,so as to provide new ideas and research basis for the practical application of this natural plant active ingredient.In addition,alcohol-related cancer mortality accounts for 5.8%of all cancer mortality worldwide,and alcoholic liver disease(ALD)has a very high morbidity and mortality in modern society.In this study,the protective effect of gallic acid-lecithin complex on iron overload in ALD was studied,and its possible mechanism was explored to provide experimental data and theoretical basis for the clinical treatment of ALD.Methods:(1)Through one-factor experiments,the factors affecting the preparation of SL-GAC which soybean lecithin was used as carriers were studied.Optimized preparation designs of Soybean lecithin-GA complex was established by response surface methodology(RSM).Ultraviolet spectrophotometry(UV),infrared spectrophotometry(IR),differential scanning calorimetry(DSC),X-ray diffraction phase analysis(X-ray)and field emission scanning electron microscopy(FESEM)were used to characterize and analyze the synthesized substances.Finally,the liposolubility of the complex was observed by measuring its apparent oil-water partition coefficient.(2)Establishing a computer simulation model of predictable intermolecular interaction by theoretical chemistry method to investigate the molecular structure of the complex,clarify the binding mode between the gallic acid molecule and lecithin molecule in the complex.To investigate the affinity between the complex and its target protein(hPTP),we conducted MD simulations for the apo-hPTP and complex-hPTP systems in both an aqueous and an ethanol environment.(3)The antioxidant activities of SL-GAC in vitro were investigated from five aspects:DPPH radical scavenging capacity,ABTS radical scavenging capacity,linoleic acid autoxidation inhibition capacity,Fenton hydroxyl radical scavenging capacity and Fe3+reduction capacity.(4)The acute toxicity of SL-GAC was investigated by oral acute toxicity test,and its safety was analyzed.A mouse model of ALD with iron overload was established by gavage administration of alcohol,and the complex was administrated on different doses.Biochemical indexes such as serum aminotransferase,hepatic lipid,antioxidant activity and iron metabolism were detected,and the protective effects of SL-GAC on ALD iron overload were observed.The possible mechanism of its hepatoprotective effect on ALD iron overload was preliminarily explored by analyzing the expression of iron-related proteins in liver.Results:(1)The results of one-factor experiments and response surface optimization showed that the factors affecting the preparation of SL-GAC were the mass ratio of GA to lecithin,reaction temperature,reaction time and the mass concentration of GA.In this study,the mathematical model of response surface optimization and laboratory conditions were combined to establish the preparation conditions of SL-GAC:15 mL ethanol was used as the reaction solvent(GA mass concentration was 12.5 mg/mL),1:1 as the mass ratio of GA to phospholipid,50°C as the reaction temperature,and 3 h as the reaction time.The results of characterization experiments confirmed that SL-GAC can be prepared effectively under the optimized conditions determined by the study.The physical and chemical properties of SL-GAC are different from those of GA or phospholipid monomers,and the combination between gallic acid and lecithin monomers may be throught the interaction between polar ends.The results of the determination of apparent oil-water partition coefficient showed that SL-GAC had good liposolubility in the observed pH range(-1<lg P<2).(2)The binding mechanism of GA with lecithin in SL-GAC was proved by GaMD and molecular dynamics simulation,that is,they were combined by strong hydrogen bond,and the phospholipid molecules wrapped the gallic acid molecules through the curl of their fat chains.GA molecules were completely dispersed in the phase of phospholipids.By modeling the complex-target protein system,molecular dynamics simulations show that ethanol environment has a significant impact on the structure of hPTP protein,and the complex plays an active role in maintaining the configuration stability of its target protein,hPTP,in both ethanol and aqueous environments.The calculation of free energy was carried out using molecular mechanics/generalized Born surface area(MM/GBSA)methods,it is found that Van der Waals interaction energy(?Gvdw),Coulomb force free energy(?Gele),non-polar solvent free energy(?Gsurf)and entropy value(T?S)all contribute to the binding of complex-hPTP system,while polar solvent free energy(?GGB)is not conducive to the stability of the system.Van der Waals action can provide the main energy for the formation of the whole system.The results of free energy decomposition showed that the amino acid residues Ile34,Ile181,Val49,Val96,Val189,Arg71,Trp94,Tyr107,Tyr148,Leu152,Met166,Pro172 and Phe192 all had higher binding free energy,which indicated that they might be involved in the close binding of the complex to the target protein.(3)The antioxidant activities of SL-GAC were lower than that of the gallic acid monomer except for the autoxidation inhibition test of linoleic acid.The results of inhibiting ability of linoleic acid autoxidation system showed that SL-GAC inhibiting curve fluctuated most gently with the prolongation of induction time,and its antioxidant stability was higher than that of gallic acid monomer.The scavenging ability of Fenton hydroxyl radical and the reduction ability of Fe3+showed that the complex still had good iron chelating properties.(4)In vivo studies,the acute oral toxicity test of SL-GAC was carried out.The results showed that no adverse reactions and deaths occurred during the experimental observation period in mice with dosage of 5000 mg/kg BW,indicating that the complex was a non-toxic substance.Then,according to the literature and the pre-experimental results,100 mg/kg BW,200 mg/kg BW and 400 mg/kg BW were selected as the dosage of SL-GAC.After 12 weeks of alcohol administration,compared with the blank control group,the ALD model mice had elevated serum aminotransferase(P<0.05),increased liver lipid peroxidation(P<0.05),increased lipid level(P<0.05),increased hepatic ROS level and iron content(P<0.05).Pathological examination with hematoxylin–eosin staining revealed numerous fatty vacuoles and inflammatory cells infiltrating the hepatic tissue in the model group,and even had fibrotic changes.It indicated that the method of this study could successfully establish the mouse model of iron overload with ALD.Compared with the model group,the complex could effectively alleviate liver injury and iron overload induced by alcohol in mice(P<0.05).The results of the medium dose group(200 mg/kg BW)were the closest to that of the blank control group.Compared with the blank group,ethanol could increase the expression of Ferritin-light and TfR1 and decrease the expression of hepcidin(P<0.05).Compared with the model group,SL–GAC ameliorated alcohol-induced liver iron overload,possibly via the above-mentioned proteins.Conclusions:(1)Through one-factor experiments and response surface optimization,the optimum conditions for the preparation of SL-GAC were determined.SL-GAC was characterized by analytical chemistry methods,and its physical and chemical properties were found to be different from those of the raw materials.Through the computer simulation study of the molecular structure of SL-GAC,it is confirmed that GA molecule and lecithin molecule are formed by strong hydrogen bonds,and the molecular configuration of the complex is obtained.Molecular dynamics simulation shows the complex has good affinity with hPTP.Furthermore,we found that the complex stabilized the structure of hPTP in an aqueous and an ethanol environment because of its phospholipid complex molecular structure.the result indicated that SL-GAC may have active pharmacological activities in vivo.(2)The antioxidant activity of SL-GAC was studied in vitro.It was found that SL-GAC can effectively scavenge DPPH radical,ABTS radical and hydroxyl radical produced by Fenton reaction,and can effectively inhibit the autoxidation of linoleic acid,and has a good ability to reduce Fe3+.(3)The mouse model of iron overload in ALD mice was successfully established.SL-GAC inhibited oxidative stress and lipid peroxidation and alleviated hepatic injury and iron accumulation induced by ALD.(4)SL-GAC ameliorated alcohol-induced liver iron overload,possibly via the downregulation of Ferritin-light and TfR1 and the upregulation of hepcidin.Furthermore,200 mg/kg BW dose group of SL-GAC showed a better protective effect on the liver iron overload of ALD mice in this study.