ABSTRACT
OBJECTIVE：To isolate the water extract of polysaccharide from Cistanche tubulosa ，and to investigate their immunocompetence in vitro . METHODS ：AB-8 macroporous adsorption resin was used to decolorize C. tubulosa polysaccharide. The decolorization process was optimized by orthogonal test with retention rate and decolorization rate of polysaccharide as comprehensive score ，and using adsorption rate ，decolorization time ，sample concentration as factors. The verification tests were conducted. DEAE- 650M ion exchange column was used to separate the water extract of decolorized C. tubulosa polysaccharide. CCK-8 assay was used to detect the effect s of different concentration of polysaccharide （6.25-100 μg/mL）before and after isolation on the proliferation rate of mice macrophage RAW 264.7. Griess method and ELISA assay were adopted to detect the effects of low ， medium and high concentration of polysaccharide （12.5，25，50 μg/mL）on the release of NO ，IL-6 and TNF-α in LPS-induced RAW264.7 cells. RESULTS ：In the optimal decolorization process of AB- 8 macroporous adsorption resin ，the adsorption flow rate was 1.2 BV/h，the decolorization time was 9 h，and sample concentration was 25 mg/mL. The comprehensive scores of 3 times of verification tests were 63.43％，63.29％ and 63.34％，respectively，with an average of 63.35％（RSD＝0.11％，n＝3）. One neutral polysaccharide （CTZ）and 5 acid polysaccharides （CT1，CT2，CT3，CT4，CT5）were isolated from the polysaccharide of C. cistanche ，the contents were 299.2，168.0，123.2，121.6，54.4，11.2 mg/g. Compared with control group ，6.25-100 μg/mL CTZ （except for 6.25 μg/mL），CT2，CT4，CT5 and 6.25 μg/mL CTC（the polysaccharide before seperation ）could significantly increase the proliferation rate of RAW 264.7 cells（P＜0.05），while 6.25-100 μg/mL CT1，CT3 and 50 μg/mL CTC could decrease te proliferation rate of RAW 264.7 cells（P＜0.05）. Compared with LPS group ，the release of NO were decreased significantly in low，medium and high concentration groups of CTC ，CT2，CT3 and CT 5，CTZ low concentration group （P＜0.05），while were increased significantly in high concentration groups of CT 1 and CT 4 （P＜0.05）. The release of IL- 6 （except for CT 1 high concentration group and CT 5 low concentration group ）and TNF-α（except for CT 1 medium concentration group ）were decreased significantly in low ，medium and high concentration groups （P＜0.05）. CONCLUSIONS ：The optimized decolorization technology of macroporous adsorption resin is stable and feasible in the study. One neutral polysaccharide and 5 acidic polysaccharides can be isolated from water extract of C. tubulosa polysaccharides，among which CT 2 polysaccharide has stronger anti-inflammatory ability.