管花肉苁蓉多糖水提物的分离及免疫活性研究

Study on Separation and Immunocompetence of Water Extract of Polysaccharide from Cistanche tubulosa

目的:分离管花肉苁蓉多糖水提物,考察分离物的体外免疫活性。方法:采用AB-8大孔吸附树脂法对管花肉苁蓉多糖进行脱色,以多糖保留率和多糖脱色率为指标进行综合评分,以吸附速率、脱色时间、上样质量浓度为因素,采用正交实验优化脱色工艺并验证。采用DEAE-650M离子交换柱层析柱对脱色后的管花肉苁蓉多糖水提物进行分离。采用CCK-8法检测不用质量浓度(6.25~100μg/mL)分离前、后各种多糖对小鼠巨噬细胞RAW264.7增殖率的影响,采用Griess法和酶联免疫吸附测定法检测低、中、高质量浓度(12.5、25、50μg/mL)分离前、后各种多糖对脂多糖(LPS)诱导RAW264.7细胞一氧化氮(NO)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)释放量的影响。结果:AB-8大孔吸附树脂的最优脱色工艺为吸附流速1.2 BV/h,脱色时间9 h,上样质量浓度25 mg/mL;3次验证实验的综合评分分别为63.43%、63.29%、63.34%,平均值为63.35%(RSD=0.11%,n=3)。从管花肉苁蓉多糖水提物中分离出1种中性多糖(CTZ)和5种酸性多糖(CT1、CT2、CT3、CT4、CT5),含量分别为299.2、168.0、123.2、121.6、54.4、11.2 mg/g。与对照组比较,6.25~100μg/mL的CTZ(6.25μg/m L除外)、CT2、CT4、CT5和6.25μg/mL的CTC(即分离前的多糖)均可显著增加RAW264.7细胞的增殖率(P<0.05),6.25~100μg/mL的CT1、CT3和50μg/mL的CTC均可显著降低RAW264.7细胞的增殖率(P<0.05)。与LPS组比较,低、中、高质量浓度CTC、CT2、CT3、CT5组和低质量浓度CTZ组细胞的NO释放量均显著降低(P<0.05),高质量浓度CT1、CT4组细胞的NO释放量均显著升高(P<0.05);低、中、高质量浓度各组细胞的IL-6(高质量浓度CT1组和低质量浓度CT5组除外)、TNF-α释放量(中质量浓度CT1组除外)均显著降低(P<0.05)。结论:本研究所优化的大孔吸附树脂脱色工艺稳定、可行;管花肉苁蓉多糖水提物中可分离出1种中性多糖、5种酸性多糖,其中酸性多糖CT2的免疫活性较强。

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-650 M ion exchange column was used to separate the water extract of decolorized C.tubulosa polysaccharide.CCK-8 assay was used to detect the effects of different concentration of polysaccharide(6.25-100μg/m L)before and after isolation on the proliferation rate of mice macrophage RAW264.7.Griess method and ELISA assay were adopted to detect the effects of low,medium and high concentration of polysaccharide(12.5,25,50μg/m L)on the release of NO,IL-6 and TNF-αin 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/m L.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,6.25-100μg/m L CTZ(except for 6.25μg/m L),CT2,CT4,CT5 and 6.25μg/m L CTC(the polysaccharide before seperation)could significantly increase the proliferation rate of RAW264.7 cells(P<0.05),while 6.25-100μg/m L CT1,CT3 and 50μg/m L CTC could decrease te proliferation rate of RAW264.7 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 CT5,CTZ low concentration group(P<0.05),while were increased significantly in high concentration groups of CT1 and CT4(P<0.05).The release of IL-6(except for CT1 high concentration group and CT5 low concentration group)and TNF-α(except for CT1 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 CT2 polysaccharide has stronger anti-inflammatory ability.