返魂草多糖中免疫增强活性有效组分的筛选及表征

Screening and Characterization of Effective Components of Immunopotentiating Activity in Senecioniscannabifolii Herba

目的:筛选并表征返魂草多糖组分中具有免疫增强活性的有效组分。方法:通过水提醇沉法提取返魂草浸膏(SCHE)多糖并制得50%醇沉样品(SCHE-1)和80%醇沉样品(SCHE-2)。将小鼠单核巨噬细胞系RAW264.7细胞分为空白组(不含血清培养基)、阴性对照组(含血清培养基)、脂多糖组(LPS,阳性对照,1μg/m L)以及SCHE-1、SCHE-2低、高剂量(0.5、1 mg/mL)组。采用四甲基偶氮唑盐(MTT)法检测RAW264.7细胞的细胞活性,采用酶联免疫吸附测定法(ELISA)检测RAW264.7细胞中白介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)水平,以考察SCHE-1和SCHE-2对RAW264.7细胞的免疫增强活性。采用分子排阻色谱法测定SCHE-1的分子量及其分布;采用高效液相色谱(HPLC)柱前衍生化法测定SCHE-1的单糖组成。采用NaOH法对SCHE-1进行甲基化分析。结果:与阴性对照组比较,LPS组及SCHE-1各剂量组均可显著增强RAW264.7细胞的活性并显著提高细胞中IL-1β、IL-6、TNF-α的水平(P<0.01),SCHE-1是具有免疫增强活性的有效组分。SCHE-1含中性糖40.05%、糖醛酸35.62%、蛋白质8.89%;SCHE-1为混合物,分子量范围为62～6 119 Da,单糖组成主要为半乳糖醛酸、阿拉伯糖和半乳糖;SCHE-1的中性糖连接方式以半乳糖的1→3、1→4和1→6连接为主,在1→3连接的O-6位上有分支,非还原末端主要为阿拉伯糖。结论:SCHE-1可能是返魂草免疫增强活性的有效组分,其主要由多糖类成分组成;SCHE-1可能通过激活巨噬细胞释放IL-1β、IL-6、TNF-α而发挥调节机体免疫功能的作用。

OBJECTIVE:To screen and characterize effective components of immunopotentiating activity in Senecionis cannabifolii Herba.METHODS:The polysaccharide components were obtained by water extraction and alcohol precipitation method to yield 50%alcohol precipitation sample(SCHE-1)and 80%alcohol precipitation sample(SCHE-2).The cells from mice mononuclear macrophage line RAW264.7 were divided into blank group(medium without serum),negative control group(medium with serum),lipopolysaccharide group(LPS,positive control drug,1μg/mL),SCHE-1 and SCHE-2 low-dose and high-dose groups(0.5,1 mg/mL).The cell viability of RAW264.7 cells was detected by MTT assay.The levels of IL-1β,IL-6 and TNF-αin RAW264.7 were detected by ELISA.These were used to investigate the effects of SCHE-1 and SCHE-2 on the immunological enhancing activity of RAW264.7 cells.The molecular weight and distribution of SCHE-1 were determined by size exclusion chromatography;the monosaccharide composition of SCHE-1 was determined by HPLC pre-column derivatization.Methylation analysis of SCHE-1 was conducted by NaOH method.RESULTS:Compared with negative control group,the activity of RAW264.7 cells was enhanced significantly in SCHE-1 groups and LPS group,which also significantly increased the levels of IL-1β,IL-6 and TNF-αin cell culture fluids(P<0.01).SCHE-1 was an effective component with immunopotentiating activity.The neutral sugar content of SCHE-1 was 40.05%,the uronic acid was 35.62%,and the protein was 8.89%.SCHE-1 was a mixture,molecular weight of which was 62-6 119 Da;monosaccharide was mainly composed of galacturonic acid,arabinose(Ara)and galactose(Gal).The results of methylation analysis showed that the backbone was composed of 1→3,1→4 and 1→6 linked Gal,and branches were on the O-6 position of the 1→3 linked Gal,and the non-reducing terminals were Ara.CONCLUSIONS:SCHE-1 may be the effective component of immuno potentiating activity,and main component of SCHE-1 is polysaccharide.SCHE-1 may regulate the immune function by activating macrophages to release IL-1β,IL-6 and TNF-α.