| According to the characteristics of Spirulina, reaction and determination conditions of gelatin-barium chloride turbidimetric method used for determining sulfate content were investigated systematicly. The conditions of standard curve drawing was determinated as follows:the linear intervals were 0-40μg, using K2SO4 as standard sample, the determination wavelength was 360nm and concentration of HCl was 0.10 mol/L. Furthermore, the sulfate content was little effected by determination conditions when the reaction temperature and time were in the range of 10-40℃and 10-40 min, respectively. Using repeatability and accuracy of determination results of sulfate content in SPS as evaluation index, ampoul was selected as optimum hydrolysis unit from centrifuge tube,15N testing tube and ampoul comparison. Hydrolysis temperature and time of SPS were also studied and the confirmed results were 95℃and 4 h, respectively. Besides, the improved method used for sulfated quantification had been proved stable and reproducible by five replications.We studied the extraction process of Spirulina polysaccharides by spherical optical design using extraction rate as index. The optimal extraction effect with theoretical yield of 3.89% could be obtained when ratio of water to material, extraction temperature and time were 30 ml/g,74℃and 6 h, respectively. Extracting polysaccharides by improved method, the average extraction rate of three replications was 4.02%. After concentrating the crude extraction five times, the pH of alcohol precipitation system and precipitation time were studied using yield and sulfate content as index. The optimum precipitation condition was obtained as follows:pH value at 3.5 and time at 18 h. Although here the recovery of polysaccharides was slightly lower, both the purity and sulfate content of polysaccharides were relatively higher.Considering removal rate of protein, polysaccharides recovery and sulfate content of polysaccharides, protein removal method which is applicable to purification of sulfated polysaccharides from the water extraction of Spirulina was studied and established by comparing Sevag method with TCA method. When the time of Sevag method for removing protein was two, about 50% protein could be removed, and polysaccharides recovery and sulfated content were 76.3% and 1.3%, respectively. And more protein could be removed, but the sulfate content decreased significantly, if the number of removal time was increased. Crude polysccharides (SPS-CE) treated with the method of Sevag were collected. Then four sulfated polysaccharides coded SPS-Ⅰ, SPS-Ⅱ, SPS-ⅢI and SPS-Ⅳwere obtained by using the Q Sepharose Fast Flow column chromatography. The sulfate content of them were 20.7%,11.7%, 8.2% and 13.0%, respectively. The purity of SPS-Ⅰand SPS-Ⅱwere 90.6% and 84.6%, respectively. IR spectrum analysis indicated that the spatial positioning of sulphate groups in SPS-Ⅰis different with that in the others.Antioxidant activity analysis showed that all these polysaccharides, especially SPS-Ⅳexhibited the highest antioxidant activities on OH, O2- and O2 with IC50 values of 48.7μg/ml,22.1μg/ml and 66.7 (μg/ml, which equaled to 3.36,33.1 and 2.76 times the scavenging capacities of SPS-CE respectively. This result indicated that SPS-Ⅳwas major antioxidant component in Spirulina polysaccharides. In view of the relationship between free radicals and tumorigenesis & metastasis, this result perhaps suggested that SPS-Ⅳmay have antitumor effect. Therefor, the anticancer activity of SPS was filtered preliminarily by using Pyriculaia oryzae model. This research indicated that SPS-Ⅳshowed strong deformation activity in a certain concentration range (187.50-375μg/ml), and inhibitory activity with dose increase (>750μg/ml). The result suggested that SPS-Ⅳmay be a potential antimitotic agent and had the ability to inhibit tumor cells by encumbering mitosis. Then the relationship between the molecular properties and antioxidant & anti-Pyricularia oryzae activities was investigated using SAS software auxiliarily. The result indicated that biological activity of SPS was not a function of a single factor but a combination of several factors, such as monosaccharide composition, sulfate group and conformation. |
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