| Squid ink is a kind of potential natural medicine resources, but usually discarded as waste in the processing of squid. In recent years, it has been demonstrated that squid ink polysaccharide has a variety of physiological activities, such as promoting immunity, antitumor, anti-oxidation, protecting intestional mucosae, improving the mucosal immunity and so on. At present, most researches about squid ink polysaccharide focused on its biological activity, few research is about digestion and absorption. This study aims to explore the mechanism of squid ink polysaccharide underlying its digestion and absorption both in vivo and in vitro. The main works of this project are as following:1) Purification of polysaccharide from squid ink and preparation its antibody: Crude melanin-free polysaccharide of Ommastrephes bartrami was obtained after freeze drying yield is about 1.5%(m/v). Crude polysaccharide was then fractionated by gel permeation chromatography and ion-exchange chromatography classification, purity up to more than 95%. The concentration of four rabbit antibody are rabbit A 2.506mg/mL, rabbit B 2.942mg/mL, rabbit C 2.602mg/mL, rabbit D 2.605mg/mL, respectively. Using ELISA assay, the optimal titer of rabbit A 1:3200 was determined.2) Research on the tranctytosis of polysaccharide in MDCK cells with the specific polysaccharide antibody:squid ink polysaccharide (SIP) incubate with MDCK cells then fixed by paraformaldehyde, tracking localization of SIP in MDCK cells combined with immunohistochemical method through SIP antibody. Results showed that after SIP (200?g/mL) incubation for 1h, weak SIP signal in MDCK cells was identified with laser confocal microscope. Next step is to do research on digestion and absorption mechanism of SIP with fluorescence labeling technique and chromatography.3) Fluorescence labeling of SIP and detecting its chemical property:TLC shows fluorescence labeling is successfully. Compared with SIP, SIP-FTSC produced characteristic absorption peak in 410?530 nm range, fluorescent properties of SIP-FTSC is similar to FTSC, Ex= 495 nm Em= 518 nm, then Confirmed by RID and FLD detector in HPLC. Finally, the optimization of mark condition is FTSC:SIP= 0.06:1,12 h, EDC:NHS:SIP= 4:4:1.4) Research on polysaccharide in transport across the plasma membrane of Caco-2 cells and absorption in mice by SIP-FTSC:In 200?g/mL concentrations, SIP-FTSC fluorescent signal could be tracked in rare Caco-2 cells in 15 min, and actin, ZO-1, E-cadherin are all at intact state; SIP-FTSC content is extremely low in BL side, transfer capacity, transport rate and Papp increases over time,120 min are 1.921?g/cm2,1.06% and 1.4394×10-6cm/s respectively. After gavage the mice with SIP-FTSC, fluorescent signal could be detected in mice blood, peak appeared after 1 h. Fluorescent signal is much stronger in feces compared with blood. FL-HPLC results showed part of SIP-FTSC was degradation, and the signal in the feces of cyclophosphamide injured mice is higher than in normal mice (P< 0.05). |
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