Extraction,Purification And Antioxidant Activity Of Polysaccharides From Synsepalum Dulcificum Leaves

Synsepalum dulcificum,also known as the miracle fruit,belonging to the genus Synsepalum in the family of Sapotaceae Juss.,which is native to the tropical region of West Africa.It is currently grown in Hainan,Yunnan and other regions of China.As a national treasure fruit plant,Synsepalum dulcificum has attracted widespread attention because of its mysterious taste-modifying function.It can not only change sour taste into sweetness,but also has many pharmacological activities,such as reducing blood sugar,lowing uric acid and blood lipid,etc.As a precious medicine and edible plant,Synsepalum dulcificumhas high research and development value.In this paper,Synsepalum dulcificum leaveswere used as the raw material,to optimize the extraction process,purification andanalysis of physicochemical properties of Synsepalum dulcificum leaves polysaccharides(SDLP).The purified polysaccharides were obtained to further complete the preliminary analysis of the structure and antioxidant activity research in the vitro.This study provides scientific theoretical basis and reference materials for the research and application of SDLP in food,biology and other fields.The main results are as follows:1、 Extraction optimization of SDLPThe single factor experiments were carried out on three factors: extraction temperature,extraction time and ratio of liquid to material,and the effects of different conditions on the extraction yield of SDLP were investigated.Based on the results of single factor experiment,the response surface test of 3 factors and 3 levels performed.Through the analysis of regression equation,the reliability of the experimental model was verified.The optimum extraction conditions of SDLP were as follows: extraction temperature 111 ℃,extraction time 2.6 h and liquid-material ratio 39 m L/g.Under these conditions,the yield of Synsepalum dulcificum polysaccharides was 7.28 ± 0.16%.The confirmatory experimental results showed that there was a good agreement between the experimental values and the predicted values.2、 Isolation,purification and physicochemical properties of SDLPThe yield of crude SDLP,SDLP-1,SDLP-2,SDLP-3 and SDLP-4 was 2.4%,8.15%,4.50%,43.55% and 4.25%,after hot water extraction,ethanol precipitation,protein removal,depigmentation and other steps.The crude SDLP was separated by DEAE-52 cellulose anion exchange column to obtain four components,SDLP-1,SDLP-2,SDLP-3 and SDLP-4.SDLP-3 was further separated by Sephacryl-S200 colunmn and yielded two components,SDLP-3a and SDLP-3b.The molecular weights of SDLP-1,SDLP-3a and SDLP-3b are 1481.0 k Da,1286.3 k Da and 44.3 k Da,respectively.The results of physical and chemical analysis showed that the total sugar contents of SDLP-1,SDLP-3a and SDLP-3b were 57.17 ± 2.77%,90.24 ± 0.88%,80.06 ± 2.26%and 65.76 ± 1.55%,respectively.No protein was detected in SDLP-1 and SDLP-3a,indicating that most of the protein was removed.Both crude SDLP and SDLP-3b have low protein content,and some proteins may not be removed or there are binding proteins in polysaccharides.The uronic acid contents of crude SDLP,SDLP-3a and SDLP-3b were 14.26 ± 4.06%,9.31 ± 1.90% and 16.59 ± 2.35%,respectively.No uronic acid was detected by SDLP-1.It can be concluded that SDLP-1 is a neutral polysaccharide,and SDLP-3a and SDLP-3b are acidic polysaccharides.3、 Preliminary structural analysis of SDLPThe SDLP-1 was mainly composed of: mannose: glucose: galactose: arabinose=0.29: 26.95: 2.01: 1.60.The main glycosidic linkage types of SDLP-1 consisted of(1→5)-α-Araf,(1→4)-β-Galp,(1→6)-α-Glcp,(1→4)-α-Glcp,T-β-Glcp,(1→3)-α-Manp and(1→4,6)-α-Galp by FT-IR(fourier transform infrared spectroscopy),methylation analysis and NMR(nuclear magnetic resonance)analysis,the structure of polysaccharide is dominated by(1→4)-α-Glcp.The SDLP-3a was mainly composed of:rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 1.54:1.28: 2.00: 7.16: 4.73: 5.47.The main glycosidic linkage types of SDLP-3a consisted of(1→5)-α-Araf,(1→3)-β-Galp,(1→4)-β-Galp,(1→4,6)-α-Galp,(1→2,4)-α-Galp,(1→3)-α-Rhap,(1→4)-α-Rhap,(1→4)-β-Glcp A,T-Glcp,(1→6)-α-Glcp,(1→4)-α-Glcp,(1→3)-β-Glcp,(1→3,6)-Glcp and(1→3,4)-α-Glcp by FT-IR,methylation analysis and NMR analysis.The SDLP-3b was mainly composed of: rhamnose:glucuronic acid: galacturonic acid: glucose: galactose: arabinose =1.82: 1.84: 5.14: 2.01:7.16: 5.29.The main glycosidic linkage types of SDLP-3b consisted of(1→3)-α-Araf,(1→5)-α-Araf,(1→4)-β-Glcp A,(1→4)-α-Glcp,(1→3)-α-Rhap,(1→4)-α-Rhap,(1→4)-β-Galp,(1→3)-Galp and(1→4)-α-Galp A by FT-IR,methylation analysis and NMR analysis.The Congo red experiment showed that there was three helix spatial exsited in the structures of SDLP-3a different from SDLP-1 and SDLP-3b.4、 Antioxidative activity of SDLPThe antioxidant activities of crude SDLP and purified polysaccharides(SDLP-1,SDLP-3a,SDLP-3b)were studied in vitro.Amongthe four different fractions,crude SDLPshowedagood ability to scavenge ABTS free radicals,superoxide anion free radicals and metal ion chelating ability,and exhibited a certain dose-effect relationship.Compared with SDLP-3a and SDLP-3b,SDLP-1 possessed stronger scavenging ability of DPPH free radical,ABTS free radical,superoxide anion radical and metal ion chelating ability.