Study On Isolation, Identification And Bioactivity Of Polysaccharides From Duanaliella Salina

Dunaliella salina is a single celled, salt-water micro-algae with no cell wall. The man-cultivation of a massive amount of Duanaliella salina is mainly utilized in production ofβ-carotene. The past studies of D. salina were mainly focused on the biology properties, production and culture technologies, and the extraction and separation ofβ-carotene. However, there were few research reports on the polysaccharides, which consists 10-40% of D. salina. The residue after the extraction ofβ-carotene from D. salina contains massive amounts of polysaccharides, proteins, vitamins and micronutrients. Among them, the polysaccharides possess various biological activities including immunity adjustment, anti-virus, and anti-tumor activity. Therefore, it is very important to study the polysaccharides from D. salina thoroughly in order to utilize the D. salina resources effectively and beneficially. The objective of this dissertation is to study the isolation, physical chemistry property and its analysis methods, structure and bioactivity of polysaccharides from Duanaliella salina. The results are as follows:The primary chemical composition of powder of D. salina after extraction ofβ-carotene were: protein 43.15%, crude fat 0.4%, total sugar 15.40%, crude fibre 0.35%, ash 15.13%, moisture 8.24%.Aiming at the extraction of polysaccharide from Duanaliella salina, experiment factors and levels were firstly selected by single-factor tests. Next, on the basis of this and according to the Box-Benhnken center-united experimental design principles, the method of response surface analysis with 3 factors and 3 levels was adopted. Then, the factors influencing the extraction technology were determined by means of regression analysis, and response surface was graphed with the extraction rate of Duanaliella salina polysaccharide as the response value. The results showed that the optimum conditions were: the extraction temperature of 81℃, pH value of extraction of 8.80, extraction time of 210 min, and that the extraction rate of Duanaliella salina polysaccharide was up to 8.77% while ratio of water to material was 16:1(v/w).The chemical composition of crude polysaccharide products extracted from Duanaliella salina under optimum condition was as follows:polysaccharide content 78.31%, uronic acid content 3.81%, protein content 4.40%, sulfate groups content 3.27%, ash content 7.60%。Under the best condition of basic extraction method, various polysaccharide were all fully extracted,while aqueous extraction method was not only to have a lower rate,but also the amount of crude polysaccharide over 100KDa was less。The crude polysaccharides (PD) was isolated on a ion-exchange column (DEAE Sepharose Fast Flow ) to four fractions including PD1, PD2, PD3, and PD4. Then PD4 was further separated to two fractions PD4a and PD4b by chromatography on a Sepharose CL6B column .The five fractions were shown to be homogeneous by high-performance size-exclusion chromatography (HPSEC),agarose gel electrophoresis and high-performance capillary electrophoresis (HPCE) .A modified procedure of the pre-column derivatization method for simultaneous determination of eight neutral,two acidic and two basic monosaccharides labeled with 1-phenyl-3-methyl-5-pyrazolone(PMP) method has been established by high performance liquid chromatography.The effects of volume proportions of acetonitrile and pH values of mobile phase(0.1mol/L phosphate buffer-acetonitrile) on retention and separation of PMP derivatives of ten monosaccharides were investigated with Eclipse XPB-C18 Column (4.6mm×250mm) screened out suitable to separate the monosaccharide derivatives from six brands of reversed-phase columns.The results shows that optimum volume proportions of acetonitrile and pH values of mobile phase was 17% and 6.7 respectively. Furthermore,the peak area changes of monosaccharides derivatives due to different reaction time , different HCl amounts used for neutralization of the reaction system and different hydrolyzation conditions were compared and therefore better condition of sample preparation was obtained.The developed methods were successfully applied for the analysis of monosaccharide compositions in the polysaccharide fractions PD4a,PD4b from Duanaliella.salina .The results were agreement basically with values obtained by high-performance anion-exchange chromatography with pulsed-amperometric detection.The results obtained by the chemical analysis,HPLC,ultraviolet spectrum and infrared spectrum and hydrolysis with nuclease indicated that PD1 was a sulfated glucan (sulfate groups content 6.93%);PD2 and PD3 were a neutral glucan and a glucan with small amount of peptide chain; PD4a is an acidic heteropolysaccharide containing sulfate groups ,uronic acid , and small amount of peptide chain(sulfate groups content 8.36%).PD4b is an acidic heteropolysaccharide containing nucleic acid (49.26%) and small amount of peptides.Their relative weight mean molecular mass (Mw) were 1548KDa,33kDa,67kDa,424kDa and 10kDa respectively.The monosaccharide compositions of PD4a and PD4b (mol%)were Man:Rib:Rha:GlcUA:GalUA:Glc:Gal:Xgl:Ara:Fuc=5.04:0.71:3.6:2.41:5.26:5.38:42.04:11.56:19.48:4.52 and 3.48:67.80:1.34:2.36:2.70:4.69:8.40:4.25:3.61:1.37. The base compositions of nucleic acid in PD4b (mol%) were G:A:C:U:T=39.18:40.54:10.67:0.75: 8.86. A HPSEC method for the determination of relative mean molecular mass and its distribution of polysaccharides from D. salina was developed by examining their retention behaviors and optimizing the separation conditions of the five polysaccharide fractions on the size-exclusion column (Waters Ultrahydrogel, Linear, 7.8 mm I.D.×300 mm). The secondary effect under different separation conditions, especially the nonspecific interaction of sulfated polysaccharide fraction with the column matrix, and its influence on the retention behaviors of three acidic D. salina polysaccharide factions were investigated. The results illustrated that in order to eliminate to secondary effect of sulfated polysaccharides, it is better to use high concentration NaAc as the mobile phase for high molecular mass polysaccharides, while low NaAc concentration was needed for the analysis of low molecular mass polysaccharides. NaNO3 and NaCl solutions as the mobile phase were not suitable in the HPSEC analysis of polysaccharide from D. salina..There were significant differences between the ion strengths of different salt solutions with same concentration.The structure of PD1 and PD4a were studied by methylation analysis and NMR.The results showed:The main chain of PD1 was composed ofα(-1→4)-D-Glc with some amount of sulfate groups at C6 ,and about 5% of Glc on branch chain. The main chain of PD4a was made up ofα-(1→4)-D-Gal with a lot of branch chain at C2 or C3 and some of nonreducing ends of Ara,Xyl,Rha,Gal,and with some amount of sulfate groups at C2 mainly.There were many of branch chain at 3-O of Gal in main chain of PD4a bound to Ara .In vitro experiment on the proliferation of splenocytes of normal mice, the results indicate that PD2 and PD3 have not evident activity of promoting proliferation. But PD1, PD4, or PD4a and PD4b abstracted from PD4 all have evident activity of promoting proliferation, and show dose-effect relationship. PD1, PD4 and PD4a have evident activity of promoting proliferation of ConA-induced splenocytes of normal mice in vitro, but show no dose-effect relationship. PD4b have not evident activity of promoting proliferation of ConA-induced splenocytes of normal mice in vitro. PD1, PD4a and PD4b have not evident activity of promoting proliferation of LPS-induced splenocytes of normal mice in vitro. In addition, PD4a can significantly promote splenic lymphocytes of normal mice to produce IL-2. Accordingly, PD1, PD4 and PD4a can significantly promote specific immunity function of normal mice, especially specific cell immunity function.In vivo experiment on carbon particle clearance rate in normal mice, the results indicate that PD4b, PD1 and PD4a can significantly enhance englobement function of heterophil granulocyte and macrophage. Thus they can enhance nonspecific immunity function of normal mice. Moreover, PD4a and PD4b can significantly promote serum hemolysin level in normal mice, so they can enhance specific humoral immunity function of normal mice.In vitro experiment on the proliferation of splenocytes of normal mice and in vivo experiment on carbon particle clearance rate in normal mice, all of those results indicate that the sulphuric acid group of PD4a and the nucleinic acid group of PD4b are concerned with their immunologic competence in some degree. Adequate degradation can enhance the immunologic competence of PD4a.In vitro experiment on antiviral activity, PD4a has evident activity of anti- Influenza virus. The relative molecular weight of PD4a is 424KDa, which is accordance with Fabregas's presumption, that is the antiviral active component in dushi algae is polysaccharides via acidation of sulphuric acid and its relative molecular weight is about 500KDa.In vitro experiment on anti-tumor, the results indicate that PD4 has depressant effect on S180, A549 and MDA-MB-231 tumour cell in some degree. The inhibition ratio of PD4 on KB and Lovo tumour cell are much higher, and the highest inhibition ratio is about 60%. Moreover, PD4a and PD4b have depressant effect on KB and Lovo tumour cell in some degree. All of these results indicate that PD4, PD4a and PD4b have cell toxicity on the above-mentioned tumour cells.In vivo experiment on anti-tumor, the results indicate that the high dose group of PD4 have evident depressant effect on S180 tumor carneus, and the inhibition ratio is 30.58%, and can enhance spleen index or thymus index of tumor-bearing mice. The high dose, middle dose and low dose group have depressant effect on Heps tumor in some degree, and the depressant effect in middle dose group is much better, and its inhibition ratio is 31.16%, and can enhance spleen index or thymus index of tumor-bearing mice. The high dose and middle dose group of PD4a have evident depressant effect on S180 tumor carneus, and their inhibition ratio are respectively 32.89% and 37.48%, and can significantly enhance spleen index and thymus index of tumor-bearing mice. The high dose group of PD4b has significant depressant effect on S180 tumor carneus.