Study On The Structure Identification And In Vivo Distribution Of Ginseng Glycopeptide And The Preparation Of Its Dropping Pills

Objective:Alzheimer’s disease is a kind of senile neurolethal disease.At present,more and more people suffer from AD in China,which brings serious influence and burden to the society.Previous studies have found that PGG can improve the learning and memory function of mice,but its structure-activity relationship and tissue distribution have not been clearly reported.In this paper,we studied the structure identification,purification,structure-activity relationship,fluorescence labeling and tissue distribution of PGG.In order to improve the bioavailability,we prepared dropping pills to lay the foundation for the development of drugs for the treatment of AD.Methods:（1）PGG was extracted by water decoction and purified by ultrafiltration and dialysis.（2）Physicochemical properties of PGG were determined by phenol sulfuric acid method,m-hydroxybiphenyl method and Lowry method.（3）PGG molecular weight distribution was determined by high performance liquid chromatography（HPLC）.（4）PGG monosaccharide and amino acid compositions were determined by HPLC.（5）The glycoside bonding mode of each component of PGG was determined by methylation and GC-MS method.（6）PGG was purified by semi-preparative liquid phase method.（7）Cell viability and apoptosis were detected by MTT method and Annexin V-FITC/PI double staining method.（8）The structure of each component of PGG was analyzed by LC-MS/MS method.（9）PGG was labeled by FITC.Fluorescence spectrophotometry and in vivo animal imaging system were used to detect the tissue distribution of PGG.（10）The optimum preparation process of PGG dropping pills was determined by single factor investigation and orthogonal experiment.The structure of PGG dropping pills was characterized by differential scanning calorimetry and infrared spectrophotometry.Results:（1）PGG obtained three parts of PGG-1,PGG-2,PGG-3 samples by purification（2）The neutral sugar content of PGG,PGG-1,PGG-2,PGG-3 was 45.4%,15.2%,11.2%,14.5%;the acid sugar content was 4.3%,7.6%,7.9%,8.5%;the protein content was 51.1%,28.2%,9.4%,95.3%（3）PGG,PGG-1,PGG-2,PGG-3 weight average molecular weight was7892 Da,4443 Da,3409 Da,16650 Da（4）PGG was composed of Gal,Glc,Man,Rha,Fuc,Gal A,Glc NAc,Gal NAc;PGG-1 was composed of Gal A,Glc A,Glc,Glc NAc,Gal NAc,Fuc;PGG-2 was composed of Gal A,Glc,Glc NAc,Gal NAc,Fuc;PGG-3 was composed of Gal A,Glc A,Glc and Glc NAc（5）PGG was composed of Thr,Asp,Ser,Ala,Gly,Val,Leu,Phe,Ile,Glu,His,Lys,Met,Arg,NH₄（6）The PGG main chain was composed of→4)-Rha-（1→,→4）-Fuc-（1→,→6）-Gal-（1→,→4）-Gal A-（1→,→4）-Glc NAc-（1→,→4）-Gal NAc-（1→,the branched chain was mainly composed of→3,6）-Man-（1→,the terminal chain was mainly composed of 1→）-Fuc,1→)-Glc,1→)-Glc NAc or 1→)-Gal NAc（7）All components of PGG have cytoprotective effects,but the effects of PGG and PGG-3 are more obvious（8）PGG has shorter peptide chain and longer sugar chain,while PGG-3 has longer peptide chain and shorter sugar chain.Compared with PGG,PGG-3 has higher stability,thermal stability and stronger hydrophobicity（9）The content of PGG-3-FITC in mouse brain was significantly higher than that of FITC（P<0.05）,with the trend of brain targeting（10）The best preparation technology of PGG-3 dropping pills was as follows:the matrix is PEG4000,the ratio of drug to matrix is 1:4,and the dripping speed is 60 d/min.Conclusion:Through the analysis of the structure-activity relationship of each component of PGG,the relationship between its structure and its activity was clarified;the tracer problem of PGG in vivo was solved by fluorescence labeling,and its tissue distribution was clarified;PGG was prepared into dropping pills by solid dispersion technology,which laid the foundation for the development of drugs for the treatment of AD.