Extraction Process,Structure Identification And Biological Activity Verification Of Ginseng Glycoprotein

Objective:With the rapid development of China's aging population,the number of patients with alzheimer's disease is on the rise year by year.This disease not only seriously affects people's work and life,but also brings great pressure to the family of each patient and the whole society.Ginseng as a valuable traditional Chinese medicine in jilin province,has a unique medicinal value and health effects,has been deeply people's attention.Previous studies have shown that Ginseng glycoprotein GGP has a biological activity that significantly improves learning and memory ability.This experiment will further optimize the extraction process of GGP,analyze its physical and chemical properties and structure,and verify its biological activity,laying a foundation for the development of anti-senile dementia drugs.Methods:GGP components were obtained from ginseng residue after water extraction,ultrafiltration and dialysis.The extraction process was screened and optimized to determine the best extraction process of GGP.The content of neutral sugar was determined by phenol sulfuric acid method with glucose as the standard,acid sugar was determined by interhydroxy biphenyl method with glucuronic acid as the standard,and protein was determined by Lowry method with bovine serum albumin as the standard.The HPLC system was used to make the standard curves with bovine serum albumin?66430Da?,VB12?1355.37Da?,l-tryptophan?204.23Da?,protease inhibitor?6500Da?and cytochrome C?13,000Da?as the reference materials.The GPC software was used to calculate the molecular weight distribution of GGP components.The monosaccharide derivatives were used as reference materials,and the GGP component PMP-precolumn derivative products were determined by high performance liquid chromatography?HPLC?.The form of glycosidic linkage was determined by-elimination experiments.Part of the methylated and part of the acetylated products of the glycosylated fragments were prepared by methylation method.MTT assay was used to determine the effect of GGP on cell viability,Annexin v-Fitc/PI double staining method was used to detect the effect of GGP on apoptosis of SH-SY5Y cells induced by A?_25-35,and flow cytometry was used to detect cell cycle.Results:The optimal extraction process of GGP components was as follows:100 g ginseng medicinal materials were taken and 8 times the amount of 75%ethanol was heated for reflux extraction for 3 times,respectively 2 h,1.5 h,1.5 h,and filtrate was collected.Centrifugation at 4000 RPM for 15 min,the supernatant was removed and freeze-dried.The freeze-dried powder obtained above was dissolved by adding 400 mL of water,and the ultrafiltration fluid and the ultrafiltration fluid were obtained by retaining the hollow fiber ultrafiltration column with a molecular weight of 2.8 kDa for 3 h.The ultrafiltration fluid is concentrated and lyophilized.The solution was dissolved with 150mL of water,and the dialysis bag with a molecular weight of 0.1 kDa was retained for 12h to obtain the dialysis fluid and the dialysis fluid,which were named T-1 and T-2respectively,and T-1 was the GGP component.The results of physical and chemical properties showed that the content of neutral sugar,acid sugar and protein in T-1 was37.92%,4.58%and 40.11%,respectively.The molecular weight distribution results showed that the molecular weight distribution range was 778 Da-44325 Da,and the average weight was 16605 Da.Mainly composed of sugar,according to the results of T-1 contains 8 kinds of sugar,Mannose,respectively?Mannose,Man?,glucuronic acid?glucuronic acid,GlcA?,rhamnose?rhamnose,Rha?,galacturonic acid?galacturonic acid,GalA?,glucose?glucose,Glc?,N-acetyl glucose?azeglucose,GlcNAc?,N-acetyl galactose?GalNAc?and fucose?fucose,Fuc?,The contents were 4.96%,3.31%,10.58%,2.98%,49.85%,14.62%,2.51%and 0.55%,respectively-elimination results showed that the GGP polysaccharides and proteins were connected by o-glycosidic bond,which was identified as GGP component.The results of cell activity experiment showed that GGP can effectively inhibit apoptosis of SH-SY5Y cells treated with A?_25-35,and has A neuroprotective effect.Conclusion:In this study,the optimal preparation process of GGP with the ability to improve learning and memory was defined,and its physical and chemical properties and structure were analyzed to verify its biological activity,laying a foundation for the subsequent development of anti-senile dementia drugs.