Study On Extraction,Isolation,Purification And Immunological Activity Of Fucoidan From Pelvetia Siliquosa Tsenget C.F.Chang

Pelvetia siliquosa Tsenget C.F.Chang belongs to Pelvetia, Fucaceae, Fucales, Cyclosporeae, Phaeophyta.Currently, there are not yet on the Extraction,Isolation, purification and biological activity of fucoidan from Pelvetia siliquosa Tsenget C.F.Chang in the reported studies. In this paper, the process of extracting fucoidan from Pelvetia siliquosa Tsenget C.F.Chang, isolation, purification and immunological activity were studied.The article is composed of three parts: Optimization of ultrasonic assisted extraction of fucoidan from Pelvetia Siliquosa Tsenget C.F.Chang, isolation and purification of fucoidan from Pelvetia siliquosa Tsenget C.F.Chang, the immunological activity of fucoidan from Pelvetia Siliquosa Tsenget C.F.Chang.Part Ⅰ: Optimization of ultrasonic assisted extraction of fucoidan from Pelvetia Siliquosa Tsenget C.F.Chang(1) Traditional water extraction: the effects of liquid-solid ratio, water bath temperature and water bath time on the extraction efficiency were studied. The optimum extraction conditions optimized by box benhnken design(BBD) are liquid-solid ratio 32mL/g, water bath temperature 90 °C and water bath time 8.4h. Under this condition, the fucoidin extraction rate reached 5.2005%, compared with an ideal predictive value(5.2153%), the relative error is small;(2) Ultrasonic assisted extraction: the effects of ultrasonic time, liquid-solid ratio, water bath temperature and water bath time on the extraction efficiency were studied. The optimum extraction conditions optimized by box benhnken design(BBD) are ultrasonic time 60 min, liquid-solid ratio 51 m L/g, water bath temperature 91 °C and water bath time 5h. Under this condition, the fucoidin extraction rate reached 5.961%, compared with an ideal predictive value(5.993%), the relative error is less than 5%;Compared with the traditional water extraction, the total time of extraction was shortened by 2.4h, polysaccharide extraction improved 0.79%.Part Ⅱ: Isolation and purification of fucoidan from Pelvetia siliquosa Tsenget C.F.Chang(1) Fucoidan extracted by ultrasound-assisted process was separated by Q-Sepharose Fast flow anion and Superdex-200, Superdex-75 dextran gel column chromatography. The four main fucoidan ingredients have been got: PSF1, PSF2, PSF3 and PSF4.They are high purity and the molecular weight were 407.735 KDa, 7.646 KDa, 18.121 KDa and 20.529 KDa respectively.(2) By infrared spectroscopy found that four fucoidan groups all contain a polysaccharide characteristic peak. The molecular weight of four groups is quite different. Wherein the molecular weight of PSF1 is maximum, also the sulfate content is highest. Four fucoidan components do not contain uronic acid, also PSF4 do not contain sulfate groups. Therefore it is preliminary determined that PSF1, PSF2, PSF3 are the fucoidan of no uronic acid, PSF4 is the fucoidan of no sulfate groups.Part Ⅲ: the immunological activity of fucoidan from Pelvetia Siliquosa Tsenget C.F.ChangThis test mainly determined to measuring the effect of Fucoidan isolated and purified by Q-Sepharose Fast flow, Superdex-200 and Superdex-75 on Raw264.7 cell viability, NO emissions and phagocytic activity. These values compared with each other can reactive immune activity.(1) The MTT method was used to determine the effect of fucoidan on Raw264.7 cell viability.It was found that PSF-W, PSF-0.2N, PSF-0.4N, PSF-0.8N isolated by Q-Sepharose Fast flow act on Raw264.7 cells safety, and is non-cytotoxic within 25-400μg/mL, the PSF1, PSF2, PSF3 and PSF4 purified by Q-Sepharose Fast flow are that.(2) Through the experiment of LPS-induced release of NO of Raw264.7, it is found that the effect of PSF1 and PSF 3 on NO concentration was significantly higher than PSF-W, PSF-0.4N correspondingly. However, the impact of PSF2 and PSF4 on NO concentration has a little difference with PSF-0.2N and PSF-0.8N correspondingly.(3) Through the experiment of Raw264.7 phagocytic activity, it is found that the effect of PSF1, PSF2 and PSF3 on the maximum phagocytic activity was significantly higher than PSF-W, PSF-0.2N and PSF-0.4N correspondingly. However, the impact of PSF4 on the maximum phagocytic activity is similar with PSF-0.8N correspondingly and both of them have weak influence.(4) Through the experiment of griess reagent determination of NO release and neutral red phagocytosis experiment, it is found that PSF1 had the largest molecular weight and maximum sulfate content have the largest NO release and the highest phagocytic activity. The NO release and phagocytic activity of PSF3 and PSF4 had similar molecular weight and low sulfate content are similar. PSF2 had small molecular weight and low sulfate content has the minimum NO emission and the weakest phagocytic activity. Therefore, fucoidan with a high sulfate content and molecular weight have a stronger immune activity.