Isolation,Structural Identification And Biological Activity Of Polysaccharides From Angelica Sinensis

Chinese Danggui(Angelica sinensis),also named Gangui or Qinna,is a perennial herb belongs to the Umbelliferon family.Its root can be used as medicine,which is one of the most commonly used traditional Chinese medicines.Angelica polysaccharide is one of the main effective ingredients of Angelica sinensis when it comes to traditional Chinese medicine dosage form-decoction.Modern medicine has proved that Angelica polysaccharide has many pharmacological effects such as anti-anemia,anti-tumor,antiradiation and anti-diabetic.However,due to the characteristics of large molecular weight,complex structure and difficulty in crystallization,the targeted properties of Angelica polysaccharides is rather lagging,which has led to difficulties in the further study of the pharmacological mechanism of Angelica polysaccharides.In addition,due to the variety of polysaccharides in Angelica sinensis,how to separate them from Angelica sinensis adequately and effectively has been attracted more attention.On the current research progress of Angelica polysaccharides of view,the extraction methods are relatively simple,researches on structural analysis are few,and the study of pharmacological activity is insufficient.Moreover,reports on relationship between structure and activity relationships is rarely seen.Therefore,a comprehensive and indepth study of the extraction and separation methods of Angelica polysaccharides and elucidation of their structure and pharmacological effect are of great significance to fully explore the medicinal value of Angelica sinensis and to promote the modernization of traditional Chinese medicine.In this dissertation,to lay a theoretical foundation for further expounding the structure and function mechanism of Angelica polysaccharides and provides a scientific basis for the comprehensive development and utilization of Angelica sinensis,we systematically studied the extraction,purification,structure analysis,hepatic targeting activity and antioxidant activity of polysaccharides extracted from Angelica sinensis(Minxian,Gansu province),and the connection between structure and related activities of Angelica polysaccharide were identified.Part I Preparation of SASP,JASP1-1A,JASP-1B and JASP-2 from Angelica sinensisThe crude polysaccharides were extracted from the root of Angelica sinensis by water extraction and alcohol precipitation method combined with alkali extraction and alcohol precipitation method.After treated with repeated freeze-thaw deproteinization,dialysis,column chromatography separation and purification methods,we finally established a complete,reliable,best-effort extraction,separation and purification route and obtained four kinds of Angelica polysaccharides named SASP,JASP-1A,JASP-1B and JASP-2.The sugar contents of SASP,JASP-1A,JASP-1B and JASP-2 were measured by phenol-sulfuric acid method and it was determined to be 95.6%,91.23%,89.12% and 90.19%,respectively.The content of uronic acid were tested by mhydroxybiphenyl method.No uronic acid was found in JASP-1A and JASP-1B and the uronic acid content in SASP and JASP-2 was 11.5% and 6.5% respectively.Results of UV,Bradford method and elemental analysis showed that the four Angelica polysaccharide are free of nucleic acids and proteins.The results of HPGPC showed that the molecular weights of SASP,JASP-1A,JASP-1B,and JASP-2 were 8.08×104 Da,8.62×105 Da,7.18×104 Da,and 5.64×104 Da,respectively.SASP,JASP-1A,JASP-1B and JASP-2 are all homogenous polysaccharide and of high purity through the identification of the purity,which meets the needs of subsequent polysaccharide structure analysis experiments.Part II Structural characterization of Angelica polysaccharidesThe structure of the four Angelica polysaccharide was analyzed by chemical analysis and spectral analysis.The IR spectrum was used to speculate functional groups from the characteristic absorption peaks.The PMP pre-column derivatization method was used to determine monosaccharide composition.The ingredient of main chains and branches of polysaccharide was confirmed by the partial acid hydrolysis method.The type of sugar residue was determined by methylation analysis combined with NMR analysis.The morphology of polysaccharide in solution was obtained by AFM combined with AFM.The IR spectrums of SASP,JASP-1A,JASP-1B and JASP-2 were typical polysaccharide spectrum.Both of them are contained pyranose ring,and the carboxyl group were found in SASP and JASP-2.The monosaccharide composition of SASP was glucuronic acid: glucose: galactose: arabinose = 1:1.7:5.02:1.85.The monosaccharide composition of JASP-1A is rhamnose: galactose: arabinose = 1:4.55:4.92.JASP-1B and JASP-2 are acidic heteropolysaccharides,which contain mannose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose and arabinose,and the ratios are 2.16: 2.96: 3.31: 2.34: 1: 49.1: 40.5 and 3.32: 7.88: 4.79: 15.9: 0.5: 46.6: 42.1,respectively.The main chain of SASP is mainly composed of glucose and galactose and the branched chain is composed of glucuronic acid and arabinose.The main chain of JASP-1A is mainly composed of rhamnose and galactose,and the branched chain is composed of arabinose.The results of methylation analysis showed that there are 10 and 6 types of sugar residues in SASP and JASP-1A,respectively.The glucuronic acid in SASP exists in the form of T-Glup A.The repeated units of SASP and JASP-1A predicted by NMR spectra were as follows.SASP is a highly branched polysaccharide.Its backbone structure were consists of(1→3)-linked Galp,(1→6)-linked Galp and 2-OMe-(1→6)-linked Galp.Three branches were found in SASP and they were connected to the main chain through the O-3 position of 2-OMe-(1→6)-linked Galp.All arabinose and most of glucose were existed in the branches and all Glcp A were presented as T-Glcp A.The backbone of JASP-1A was composed of 1,4-linked Galp and 1,3-linked Rha.The branches consists entirely of arabinose and linked at the C-6 position of 1,4-linked Galp.The terminal structure of the branches is T-Araf.Part III Study on Liver Targeting Activity of Angelica PolysaccharideIn this part of the experiment,FITC labeling combined with high performance liquid chromatography was used to quantify the concentration of SASP and JASP-1A in biological samples.The results showed that both SASP and JASP-1A were successfully labeled.The substitution degrees of FITC in FS and FJ were 1.18% and 1.76% respectively.The low degree of substitution indicated that the pharmacokinetics of FS and FJ are consistent with SASP and JASP-1A.The liver-targeting activity of SASP and JASP-1A was tested by in vivo distribution experiments and the results showed that the concentration of SASP in liver tissue of mice was much higher than that of other tissues and JASP-1A was mainly distributed in liver and kidney after tail vein injection,indicating the liver targeting activity of SASP is higher than that of JASP-1A,and the emergence of liver-targeting activity may be related to ASGPR.In order to obtain the appropriate antagonists,NGA was synthesized by a five-step method.The analysis of 1H NMR and Moli-Tof-Ms flight mass spectrometry showed that the molar ratio of galactose residue to protein is 51:1 in the successfully synthesized NGA.To investigate the reason of liver targeting activity of FS,the uptake of FS by Hep G2 cells,Bel-7402 cells,and Hela cells under different conditions were studied in in vitro assays.Western blotting results showed that the ASGPR content in the three cells was Bel-7402 cell > Hep G2 cell > Hela cell,and the ASGPR was almost absent in Hela cell,so chose Hep G2 cell and Bel-7402 cell were chose as positive group and Hela cell was chose as a negative group.At the same concentration at different time points or different concentrations at the same time,the situation of endocytosis of FS was Hep G2 cells > Bel-7402 cells > Hela cells.The uptake rate of FS was higher in both Hep G2 cells and Bel-7402 cells,but Hela cells had less than 10% of FS intake.After NGA was added,the uptake rates of FS in Hep G2 cells and Bel-7402 cells were reduced by 56.52% and 76.19%,respectively.Meanwhile,Dextran has no effect on the uptake of cells.To further investigate whether the production of liver-targeting activity is related to ASGPR,the distribution of FS in mouse liver and blood in different conditions were examined in vivo.The results showed that the concentration time curve of FS was a typical two-compartment model and the FS was basically cleared from the blood at 60 min.In the meanwhile,the concentration of FS in liver reaches the maximum.After NGA was added,the clearance rate of FS in blood was slowed down,and the time for maximizing FS in liver was also delayed.After adding dextran,the concentration time curve and liver aggregation of FS remained unchanged.Rats were randomly divided into experimental group,antagonistic group and negative group and injected intravenously with FS(12 mg/kg),FS(12 mg/kg)+ NGA(240 mg/kg)and physiological saline,respectively.The results showed that FS was mainly distributed in hepatocytes and the brightness of FS in hepatocytes was decreased after added NGA.In summary,both SASP and JASP-1A have liver-targeting activity,and livertargeting activity of SASP is stronger than JASP-1A.The reason for the liver-targeting activity is that the polysaccharide can be specifically recognized by the ASGPR on the surface of the liver parenchymal cell membrane and transported to the liver by endocytosis.Part IV Study on antioxidant activity of Angelica polysaccharidesIn this part of the experiment,the activity of SASP and JASP-1A scavenging free radicals in vitro and the protective effect of SASP and JASP-1A on oxidative damage Hep G2 cells were investigated.The content of phenolic compounds in the two polysaccharides was determined by Folin-Ciocalteu method.The results showed that the content of phenols in SASP and JASP-1A was all less than 0.1%.The hydroxyl radical and DPPH free radical scavenging experiments were used to evaluate the in vitro antioxidant activity of SASP and JASP-1A.The results showed that both SASP and JASP-1A had a certain scavenging ability for the two free radicals,and the scavenging ability of SASP was much greater than that of JASP-1A,which could be owe to the different monosaccharides composition and different forms in solution between SASP and JASP-1A.The Hep G2 cell was selected to establish hepatic injury model in vitro by hydrogen peroxide injury,and then the protective effect of SASP and JASP-1A on oxidative damage cells was studied.When the concentration of polysaccharides in the range of 100~3200 μg/m L,there was no effect on the proliferation rate of Hep G2 cells in MTT assay.It was found that both SASP and JASP-1A could significantly increase the survival rate of oxidative damaged cells.The results of ROS assay showed that SASP and JASP-1A can significantly reduce the level of ROS compare to model group.The levels of SOD,MDA,LDH,AST,and ALT were measured.It was found that SASP and JASP-1A have various degrees of improvement on the above indicators.Taken together,both SASP and JASP-1A have good antioxidant activity in vitro and can protect oxidative damaged Hep G2 cells.