| Sisal(Agave sisalana)is one of the first fiber plants specifically cultivated for fiber,but the fibres account for only about 5%of the plant by weight,this means that a large amount of sisal waste was produced in the production of fibres.If the sisal waste is arbitrarily discarded,it will not only pollute the environment but also waste valuable resources.In recent years,polysaccharides from plants,animals and microorganisms have aroused the interest of many researchers,due to their many biological activities,such as antioxidant,antitumor,antiviral,anticoagulant,and immune-stimulating.However,no report on polysaccharides from sisal was published so far.In the paper,three new water-soluble polysaccharides from the sisal waste were isolated and purified,and their structural characterization and some biological activities were also first investigated.The research contents and conclusions of the paper were as follows:The crude polysaccharide was obtained from the residue after extracting fiber by hot water extraction,ethanol precipitation and deproteinization,and its yield,sugar content,and protein levels were 2.49%,59.36%,and 1.62%.respectively.After destaining and separating by DEAE-cellulose anion exchange chromatography,the three crude polysaccharides(CSP1,CSP2,and CSP3)were obtained from the water eluate,0.3 M NaCl eluate,and 0.4 M NaCl eluate.And then,with successive separation by Sephacryl S-300 HR gel filtration chromatography,three new pure water-soluble polysaccharides were obtained,and named SP1,SP2,and SP3.The extraction processes of sisal polysaccharides were researched and the optimal process conditions were established by means of single-factor experiments and orthogonal experiments.They were that the ratio of water to raw material was 20:1,the reaction temperature was 85?,the reaction time was 3h,and the extraction times was two times.Under the optimized process conditions,the extraction rate of sisal polysaccharides was 2.49%.The effects of deproteinization times on removal rate of protein and recovery rate of polysaccharides were investigated.The results showed that the removal rate of protein increased with the increasing of deproteinization times and the recovery rate of polysaccharides decreased.Therefore,the deproteinization times should be controlled at 7.The structures of three polysaccharides were investigated by FT-IR,UV,NMR,GC-MS,HPLC,acid hydrolysis,methylation analysis,periodate oxidation and Smith degradation.SP1 with an average molecular mass of about 11800 Da consisted of galactose,glucose,mannose,rhamnose,galacturonic acid and arabinose in a molar ratio of 3.0:2.4:1.4:1.0:0.2:0.2.Structure analysis indicated that SP2 possessed a backbone composed of 1,4-linked-?-D-Galp,1,4-linked-?-D-Glcp,1,2-linked-?-D-Manp,and 1,2-linked-?-L-Rhap in the ratio of 3:1:1:1.Part of Glcp,Manp,and Rhap residues in the backbone chain were branched.A single 1-linked ?-D-Arap residue or 1-linked a-D-GalpA residue was attached to the O-4 position of 1,2-linked ?-L-Rhap residue.The side chain composed of a 1-linked(3-D-Glcp and a 1,3-linked ?-D-Glcp was attached to the O-4 position of 1,3-linked(3-D-Glcp.The Manp residue was substituted at O-4 by 1,3-linked?-D-Manp,and the latter was substituted at O-3 by single-unit ?-D-Galp A or?-D-Arap as non-reducing ends.SP2 with an average molecular weight of about 9200 Da was composed of Rha,GalA,Gal,Ara,and Glc in a molar ratio of 1.8:1.7:1.0:0.23:0.15.The structure analysis indicated that the SP2 was comprised of a backbone of alternating 1,2-linked-?-L-Rhap and 1,4-linked-?-D-GalpA units,and the neutral polysaccharide side chains composed of Galp,Ara,and Glcp residues were attached to the O-4 position of 1,2-linked-?-L-Rhap residues.SP3 with an average molecular weight of about 16700 Da was composed of Rha,GalA,Gal,Man,Glc,and Xyl in a molar ratio of 1.73:1.00:1.08:0.10:0.20:0.09.The structure analysis indicated that the SP3 was comprised of a backbone of alternating 1,2-linked-a-L-Rhap,1,4-linked-a-D-GalpA,and 1,4-linked-a-D-Galp residue in a molar ratio of 7.0:3.0:4.0.Around 25%of rhamnose of the backbone and 46%galactose were branched,a single Manp or Xyl residue was attached to the O-4 position of 1,2-linked-a-L-Rhap residues,and a single 1-?-D-Galp residue was attached to the O-2 position of 1,4-linked-?-D-Galp residues.In addition,some hydroxyl group on the O-2 position of 1,4-linked-a-D-Galp was substituted by methylamino.The synthetic process of phosphorylated sisal polysaccharides was researched and established.It is that the sisal polysaccharides was used as raw material,POCl3 was used as phosphorylated reagent,and TMP and CH3CN were used as solvent,pyridine was added to adjust and keep the pH=6.0.After the reaction was terminated,the reaction mixture was filtered,the precipitate was dissolved and dialyzed against water for 48h,and then the dialyzate was concentrated and lyophilized to obtain phosphorylated sisal polysaccharides.The optimal synthetic technological conditions of phosphorylated polysaccharides were established through single-factor experiments and orthogonal experiments.They were that the reaction temperature was 50?,the liquid-solid ratio of POCl3 and SP was 3:1 and the reaction time was 3h.Under the process conditions,the phosphorus content of the phosphorylated sisal polysaccharide was 12.75%.The structure of phosphorylated sisal polysaccharides synthesized was verified by infrared spectroscopy.Through comparing the infrared spectroscopy of the pre and post polysaccharides modification,a conclusion could be drawn that the structure of sisal polysaccharides itself was not destroyed,and the phosphorylation modification has been successful due to the presence of two characteristic absorbing peaks-stretching vibrations of P=O at 1253.88cm-1 and P-O-C at 1090.25cm-1.SP2 could stimulate ConA-induced T lymphocyte proliferation in test concentrations with a concentration dependent manner.MPSP had immunosuppressive activity,but the relationship between activity and concentration was not very regular.The rest of tested samples(SP1,SP3,LPSP,and HPSP)exhibited different immune activity with different concentrations,even no immune regulating effect at some concentration.So we could conclude that SP1,SP3,LPSP,and HPSP had no immune activity.Sisal polysaccharides and its phosphorylated derivatives had scavenging effects on DPPH radical and hydroxyl radical,and their scavenging abilities rose with the increasing of the sample concentrations,but were less than that of ascorbic acid.With regard to the scavenging activity,that of SP2 was the strongest,following by SP3 and SP1,and the scavenging activity was positively correlated to the content of uronic acid,which suggested that uronic acid was contributed to the expression of antioxidant activity.After the sisal polysaccharides were modified with phosphorylation,their scavenging abilities on DPPH radical and hydroxyl radical were significantly less than that of acidic polysaccharides-SP2 and SP3.Moreover,their scavenging abilities reduced with the increase of phosphorus content,implying that phosphorylation modification was not conducive to the expression of antioxidant activity,which may be related to the decrease of hydroxyl group caused by phosphorylation modification.Sisal polysaccharides and its phosphorylated derivatives had reducing power,and their reducing power increased with the increase of sample concentrations,but they were less than that of ascorbic acid.Their reducing capacities from big to small were SP2,SP3,SP1,LPSP,MPSP,and HPSP,this trend was consistent with that of their abilities on free radical scavenging,suggesting that reducing power could contribute to scavenging ability. |
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