| Objective: In our previous research, using aqueous extraction, silica gel chromatography, ion exchange chromatography, HPLC extraction and other technics, we got the water soluble anti-LPS fraction A (ASLA) that could specifically bind to LPS and its active region Lipid A. In in vitro experiments, ASLA could bind to LPS with high affinity. It also could neutalize LPS and inhibit the LPS-induced TNF-αand IL-6 release. In in vivo experiments, ASLA could protect animals from an LPS challenge. However, there still existed some problems in the preparation of ASLA. The first problem is, the yield of ASLA was not controled very well. In duration of dehydrated alcohol to deposit polysaccharides, the purposed product was easily glued up by polysaccharides deposition. Therefore, the amount of the purposed product markedly decreased. If delete this step, polysaccharides would block the silica gel column, then interrupting the process. The second problem is, the previous work cycle was too long, and the yield rate was not high. The product couldn't satisfy the later work. Therefore, the previous method to get ASLA should be improved now.Methods: using polyamide gel chromatography, silica gel chromatography, ion exchange HPLC, Bio-sensor, RP-HPLC extraction and other technologies, the fraction binding to LPS was directional separation. The abilities of main fractions to antagonize LPS were evaluated in vitro and in vivo.Results:①The fraction 2 (DS2) with high binding activity was prepared by water extraction, polyamide gel chromatography, silica gel chromatography, ion exchange HPLC, affinity biosensor technology.②DS2 had high lipid A- binding activity, and could effectively protect mice from lethal LPS challenge.③Fraction DS2 could be further seperated into four main extrations after HPLC preparation. Among them, the main fraction B could obviously neutralize LPS in vitro, and inhibited LPS induced TNF-αrelease from RAW264.7 cells.④After futher separation of fraction B, we found fraction B1 could... |
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