返魂草多糖中免疫增强活性有效组分的筛选及表征
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摘要
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,1 μg/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-α in 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-α in 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.
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,1 μg/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-α in 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-α in 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.
引文
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[13]艾雪,鞠成国,贾坤静,等.仙茅不同炮制品对巨噬细胞免疫活性的影响[J].中成药,2017,39(3):616-620.
[14]武俊华,罗成.Viili多糖对巨噬细胞RAW264.7激活及分泌细胞因子的影响[J].现代免疫学,2013,33(2):113-118.
[15]谢燕霞,安利国,杨桂文.植物多糖对巨噬细胞的免疫调节作用[J].中国生物化学与分子生物学报,2008,24(4):307-314.
[16]尚庆辉,解玉怀,张桂国,等.植物多糖的免疫调节作用及其机制研究进展[J].动物营养学报,2015,27(1):49-58.
[2]赵春芳,李庆杰,王莲萍,等.返魂草的高效液相色谱法指纹图谱研究及成分分析[J].分析化学研究简报,2013,41(1):133-136.
[3]张宏梅,崔佰吉,李景华,等.大孔吸附树脂分离、纯化返魂草有效成分的工艺研究[J].中国药房,2015,26(31):4402-4405.
[4]吴一飞,巩江,令恒莉,等.返魂草的药学研究进展[J].山东中医杂志,2009,28(6):436-437.
[5]赵赫,王晓波,王媛媛,等.HPLC法同时测定返魂草药材及其颗粒中四种酚酸类成分含量[J].亚太传统医药,2017,13(6):29-32.
[6]马鸿雁,杨莉,王长虹,等.HPLC法同时测定返魂草颗粒中6种活性成分[J].中成药,2012,34(8):1496-1500.
[7]李明娟,谭安雄.天然多糖抗炎活性及其应用的研究进展[J].邵阳学院学报(自然科学版),2017,14(4):102-106.
[8]张伟杰,王鹏,林茜,等.3种中药多糖的提取工艺[J].中国实验方剂学杂志,2011,17(16):19-23.
[9]姜瑞芝,王颖,陈英红,等.猴头菌寡糖的化学研究[J].中国药学杂志,2008,43(5):341-344.
[10]吕京南,罗浩铭,陈英红,等.人参非皂苷成分中具有治疗老年痴呆作用的活性成分筛选及其结构表征[J].时珍国医国药,2015,26(11):2561-2563.
[11]王颖,高其品,李桂荣,等.人参糖肽PG-g的分离纯化与结构分析[J].天然产物研究与开发,2010,22(6):1053-1056.
[12]姜瑞芝,姜翔之,曹传英,等.东北红豆杉多糖的化学研究[J].天然产物研究与开发,2012,24(4):440-443.
[13]艾雪,鞠成国,贾坤静,等.仙茅不同炮制品对巨噬细胞免疫活性的影响[J].中成药,2017,39(3):616-620.
[14]武俊华,罗成.Viili多糖对巨噬细胞RAW264.7激活及分泌细胞因子的影响[J].现代免疫学,2013,33(2):113-118.
[15]谢燕霞,安利国,杨桂文.植物多糖对巨噬细胞的免疫调节作用[J].中国生物化学与分子生物学报,2008,24(4):307-314.
[16]尚庆辉,解玉怀,张桂国,等.植物多糖的免疫调节作用及其机制研究进展[J].动物营养学报,2015,27(1):49-58.