Studies On The Extraction, Isolation, Purification Of Radix Polygalae Glycoprotein And Its Anti-aging Activity In Vivo And Vitro

Objective:Glycoprotein from Radix Polygalae root,a traditional Chinese medicinal herb,was researched in the present study.Extraction process,purification,structure characterization and anti-aging activity in vivo and in vitro were systematically investigated by using the modern extraction and separation methods,instrument analysis identification technology etc.On this basis,the metabolic pathways of anti-aging activity were explored preliminarily by serum metabolomics technology.And these results will be used to explore a new area of utilizing active materials of Radix Polygalae.Methods:In single factor experiments,protein yield was regarded as an evaluation index.The extraction technology was investigated from five aspects.They were p H volue,temperature,salt concentration,liquid-solid ratio and extraction time.According to the results of single factor experiments,three factors were selected to response the surface experiments.And the best extraction process of Radix polygala glycoprotein(PG)was investigated through the response surface method.The PG was regarded as the research object next.The anti-aging effect of different doses of PG on D-galactose aging mice was studied through pharmacological experiment.After the experiments,the activities of Superoxide Dismutase(SOD),Catalase(CAT)and Methane Dicarboxylic Aldehyde(MDA)content in serum and brain tissue were tested.After the step,DEAE-52 ion exchange chromatography and Sephadex G-25 chromatography were used to purificati PG.The elution curves were drawn with polysaccharide and protein content.The purity and molecular weight of the pure Radix Polygalae glycoprotein(PG-D)were verified by SDS-Page electrophoresis method.First the characterization and composition of PG-D were illustrated by ultra violet(UV),Fourier Transform Infrared Spectroscopy(FTIR)and amino acid automatic analysis methods.And the second PG-D was evaluated its antioxidant capacity in vitro,including the 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical scavenging activity,hydroxyl free radicals and ferric reducing antioxidant power(FRAP)assay.At last,metabolic markers associated with anti-aging activities were determined by gas chromatography-massspectrometry(GC-MS)technology.And the pathways which were relevant of anti-aging effect were explored preliminarily by analysis of principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA),univariate statistical analysis(UVA)and metabolic pathways.Results:The protein yield is 5.96% in the best conditions,with p H=7.85 Tris-HCl buffer(0.1 mol/L Na Cl)to extract 1.5 hours at 33 ?.The results of anti-aging pharmacological experiment with different dose of PG are as following.Comparing with the blank group,the thymus index and spleen index are significantly lower(P<0.05 or P<0.01),the activity of SOD and MDA content in serum and brain tissue are decreased significantly(P<0.05),the activity CAT in serum is significantly lower(P<0.01)in the model group.And comparing with the model group,the activity of SOD in serum is significantly increased(P<0.05)in the high dose group(400 mg /(kg day)),MDA content in brain tissue is significantly lower(P<0.01),the activity of CAT in serum significantly increase(P<0.01)in the middle(200 mg /(kg day))and high dose groups.The results of separation and purification are as following.The symmetric protein and polysaccharide peak in the components are eluted by Buffer D(p H = 7.85 0.1mol/L Tris-HCl Buffer containing 0.3 mol/L Na Cl)with DEAE-52 for ion-exchange filler.The pure Radix Polygalae glycoprotein(PG-D)are pure of SDS-Page electrophoresis,and its molecular weight is 36 k D.The obvious protein and polysaccharide peak are showed in FTIR spectrum at 3230?2930?1640?1550?1458?1030?898cm-1.The UV spectrum shows that the absorption intensity changes in 240 nm before and after ultraviolet beta elimination reaction.So O-sugar peptide bond is existed in the glycoprotein.Amino acid automatic analysis results show that the PG-D contains 18 kinds of amino acids.From the results of antioxidant activity in vitro of PG-D show that the glycoprotein is active in antioxidant activity,and its antioxidant activity is lower than Vc and higher than PG.The results of serum metabolomics show that there are 6 kinds of different metabolites in the control group(CON),the model group(MON)and the middle dose of glycoprotein group(MPGP).Comparing with the CON,Uridine 2,Tagatose 1,N-cyclohexyl formamide 2,Saccharic acid,uracil,and asparagine 3 are increasing in MOD group.Comparing with the MOD,the content is decreasing in MPGP.According to metabolic pathway analysis,PG resist aging in these metabolic pathways.Pyrimidine metabolism,Ascorbate & Aldarate metabolism and Galactose metabolism are involved in nucleotide metabolism,carbohydrate synthesis metabolism and sugar metabolism.Conclusion:Response surface method is adopted to establish the model to optimize the best extraction process of PG.And it has certain anti-aging effect in D-galactose aging mice.The serum metabolomics pathways of anti-aging are relevant to Pyrimidine metabolism,Ascorbate & aldarate metabolism and Galactose metabolism.The result of purification and antioxidant activity experiment show that the antioxidant capacity in PG-D is stronger than PG.These results will provide basic instruction for further clinical application and product development of Radix Polygalae.