| Slug is a molluscs with lung,belong to Limax in the family of Gastropoda and has been used as a traditional Chinese medicine for years in oriental countries.In folk medicine,slug has been used in the treatment of malignant tumors.Recently,several studies have reported that crude extracts from slug presented significant antitumor activities,and the main active ingredient may be polysaccharides.However,few data has been reported on the chemical composition,structure and biological activity of polysaccharide from slugs.In order to study the structures and structure-function relationship of active polysaccharides from slugs,the extraction,isolation,purification,structure elucidation,chemical modification,antitumor activity,antioxidant activity and immunoregulatory activity in vitro were performed on the polysaccharides from Philomycus bilineatus.We isolated and purified a novel highly active glycoprotein(PBPa11)from Philomycus bilineatus,it provided the theoretical basis and technical support for the further development of slug drugs.There are five parts in this dissertation.Studies on isolation,purification and physic-chemical character,structure elucidation,chemical modification and activities in vitro of polysaccharides from Philomycus bilineatus were discussed in this dissertation.1.Optimization of extraction parameters of polysaccharides from Philomycus bilineatusIn order to optimize trypsin hydrolysis and flash extraction conditions for the polysaccharide from Philomycusbilineatus,we used single-factor experiments and response surface analysis(Box-Behnken Design).The optimized trypsin hydrolysis condition was as follows:solution pH 8.0,solution temperature 45 ? enzyme addition 1.8%,material ratio 1:1,hydrolysis time 4 h.The optimized flash extraction condition was as follows:extracting temeratre 63?,rotation speed 22000 rpm,material ratio 1:1.6,extracting time 33 min.Under the above mentioned conditions,the actual yield of target polysaccharide was 5.19%.Antitumor results showed that the effective polysaccharide was present in its body tissue.2.Studies on isolation,purification and physic-chemical character of polysaccharides from Philomycus bilineatusGuided by inhibition of A549 cell proliferation in vitro,PBPn11,PBPa11,PBPa21 and PBPal2 were isolated and purified from Philomycus bilineatus through DEAE Sepharose Fast Flow and Sepharose Fast Flow column.As to PBPn11,PBPa11,PBPa21 and PBPa12,the yield was 4.52%,1.73%,2.15%and 0.31%,respectively.Polysaccharides content was 58.22%,48.45%,51.65%and 50.13%,respectively.Protein content was not detected,22.34%,27.99%and not detected,respectively.Uronic acid content was 7.48%,4.42%,not detected and 30.12%,respectively.Hexosamine content was 38.56%,20.32%,18.56%and 40.40%,respectively.The HPLC profile showed the relative molecular weight of PBPn11,PBPa11,PBPa21 and PBPal2 was 3.330×106 Da,2.338×106 Da,2.338×106 Da and 4.1 × 104 Da,respectively.3.Studied on structures of the homogeneous polysaccharide from Philomycus bilineatusTheir structures were detailedly identified on the basis of chemical methods(sugar composition analysis,PMP derivatization analysis,glycosyl residue linkage analysis,etc.),and spectral technologies(FT-IR,GC-MS,LC-MS,1D NMR:1H NMR,13C NMR,DEPT spectra,2D NMR:1H-1H COSY,TOCSY,HMQC,NOESY and HMBC spectra,etc.)Sugar analysis showed that PBPn11 was composed of unknown T3 sugar,N-acetyl-glucosamine and galactose in a molar ratio of 1.00:0.27:0.16.Methylation analysis showed it contained terminal galactose,1,4 N-acetyl-glucosamine linkage.NMR analysis further showed that it contained a ?4)-?-GlcpNAc-(1? residue.HPLC-TOF/MS and NMR analysis showed that T3 was sialic acid derivatives,its molecular weight was 355.3Da and possible structure was(2S,4R,5R,6R)-5-(2,3-dihydroxypropanamido)-2,4-dihydroxy-6-(1,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylic acid.PBPa11 was composed of mannose,glucose,galactose,sorbitol,N-acetylglucosamine,T3 sugar and a little 3(4)-O-methyl-hexose.Methylation analysis showed it contained terminal galactose,1,4 N-acetyl-glucosamine linkage,2,6 sorbitol linkage.?-elimination reaction and hydrazine experiments proved that PBPa11 contained O-glycopeptides and N-glycopeptides,and its antitumor activity is closed related with its O-glycopeptides.PBPa21 was major composed of glucose,galactose,N-acetylglucosamine,a little mannose and 3(4)-O-methyl-hexose.4.Studied on sulfated polysaccharide and seleno-polysaccharide from Philomycus bilineatus(1)With S03-Pyrcom complex,a water-soluble sulfated derivative PBPn11SO4 was obtained from PBPn11.FT-IR and UV spectrum showed that PBPn11SO4 contained characteristicl absorption of sulfate ester bond.Its sulfate content was 15.8%and the DS of the derivative was 1.61.(2)Partical seleno-polysaccharide PBPn11Seland total seleno-polysaccharide PBPn11Se2 were prepared from PBPn11 with isoselenocyanates.Their selenium content were 8.22%and 19.53%,respectively.5.In vitro bioactivity of polysaccharide from Philomycus bilineatus(1)The antitumor activity in vitro of polysaccharide from Philomycus bilineatus was tested by MTT method,and the results presented that PBPn11,PBPal 1,PBPa21,PBPn11SO4,PBPn11Se1 and PBPn11Se2 could inhibit the proliferation of MCF-7,Hela,A549,S-180 and HL-60 cells in vitro in a dose-dependent manner.The inhibition rates of these fractions tested decreased in the order of PBPa11>PBPn11 Se2>PBPn11Se1>PBPn11SO4>PBPa21>PBPn11.PBPa11 presented significant higher antitumor activities against A549,MCF-7,Hela,S-180 and HL-60 cells in vitro,and the IC50 values were 87 ?g/mL,440 ?g/mL,324 ?g/mL,268 ?g/mL and 528 ?g/mL,respectively.The cell toxicity selection index(SI)of PBPa11 were 8.655,1.710,2.324,2.810 and 1.426,respectively.(2)The antioxidant activities in vitro were studied by different methods,including ABTS assay,DPPH assay,hydroxyl radical assay,superoxide radical assay and reducing power assay.In vitro antioxidant tests indicated that all the fractions exhibited significant antioxidant activities in a dose-dependent manner.PBPn11Se1 and PBPn11Se2 presented relative stronger antioxidant activity among the six polysaccharides,It suggests that selenylation can significantly increase the antioxidant activity of polysaccharide.(3)Immunomodulating activities in vitro of PBPn11,PBPa11,PBPa21,PBPn11SO4 and PBPn11Se2 were evaluated by using the cell model experiments.To varying degree,all polysaccharides could stimulate lymphocytes proliferation and improve the content of IFN-a in THP-1 cells.With the stimulating indexes 2.53 and 2.48,PBPa11 and PBPa21 exhibited much higher promoting function than the others.Results showed that polysaccharide immunomodulating activities could be improved by sulfated and selenylation. |
PDF Full Text Request