【作者】 刘恩绪；

【导师】 周义发；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 山茶(Camellia japonica)属于山茶科山茶属植物,根和花可入药。山茶蜂花粉是一种营养丰富的物质,具有很好的保健作用。目前,山茶蜂花粉多糖的研究主要集中在活性上,对山茶蜂花粉多糖分离纯化上的研究尚未见报道。本论文主要研究了山茶蜂花粉多糖的分离纯化及免疫活性。山茶蜂花粉经过热水煮提,过滤,浓缩,乙醇沉淀,获得山茶蜂花粉总多糖(WCPP),经分析得到山茶蜂花粉多糖的单糖组成为:2.5% Man、7.8% Rha、3.0% GlcA、37.0% GalA、12.5% Glc、15.1% Gal和22.1% Ara,并通过凝胶柱对其进行检测,发现分子量分布不均一。本论文采用两种方法对WCPP进行分级:第一,采用乙醇沉淀的方法对WCPP进行分级,分别用30%、50%、70%和80%乙醇对WCPP进行沉淀,获得四个级份WCPP-30、WCPP-50、WCPP-70和WCPP-80。单糖组成分析表明:WCPP-30含有4.3% Man、7.4% Rha、45.4% GalA、10.6% Glc、10.3% Gal、2.1% Xyl和20.2% Ara;WCPP-50含有2.0% Man、9.1% Rha、42.7% GalA、8.8% Glc、11.5% Gal、2.0% Xyl和23.9% Ara;WCPP-70含有2.1% Man、8.7% Rha、1.9% GlcA、15.5% GalA、16.3% Glc、20.6% Gal、1.5% Xyl和33.5% Ara;WCPP-80含有6.3% Man,4.7% Rha、4.8% GalA、42.6% Glc、12.2% Gal和29.4% Ara。采用凝胶柱层析方法对四个级份进行了均一性鉴定,发现其分子量分布范围较宽,不易根据分子量进一步分级。第二,采用离子交换柱层析方法对WCPP进行分级,分别用水、0.1 mol/l NaCl、0.3 mol/l NaCl和0.5 mol/l NaCl进行洗脱,获得四个级份:WCPP-N、WCPP-1、WCPP-2和WCPP-3。对四个级份进行单糖组成分析得到:WCPP-N含有4.1% Man、1.0% Rha、69.0% Glc、13.4% Gal和12.7% Ara;WCPP-1含有5.4% Man、4.3% Rha、2.5% GlcA、45.0% GalA、22.7% Gal、18.0% Ara和2.0% Glc;WCPP-2含有1.0% Man、9.5% Rha、1.9% GlcA、30.2% GalA、1.9% Glc、19.3% Gal和36.5% Ara;WCPP-3含有2.1% Man、18.8% Rha、1.1% GlcA、33.2% GalA、17.9% Glc、19.1% Gal和23.9% Ara。凝胶柱层析检测发现四个级份分子量分布比较广泛。WCPP-1和WCPP-3含量比较低,未对其进行分离纯化,主要对WCPP-N和WCPP-2进行了进一步纯化。采用Sephadex G-100凝胶柱层析方法对WCPP-N进行分离纯化,获得WCPP-N-Ⅰ和WCPP-N-Ⅱ两个级份。经检测发现,WCPP-N-Ⅱ的分子量均一性比WCPP-N-Ⅰ好,因此对WCPP-N-Ⅱ进行了结构分析,核磁结果显示,这个级分的主链由α-1,4-Glucan,侧链由α-1,6-Glucan构成,具有支链淀粉样结构。采用Sepharose Cl-6B凝胶柱层析方法对WCPP-2进行纯化,得到WCPP-2-Ⅰ、WCPP-2-Ⅱ、WCPP-2-ⅡⅠ、WCPP-2-ⅠV和WCPP-2-V。经检测发现,WCPP-2-Ⅰ比较均一,其他四个级份不均一。因此对WCPP-2-Ⅰ进行了结构分析,核磁结果显示,WCPP-2-Ⅰ可能含有α-1,2-Rha或α-1,2,4-Rha与α-1,4-GalA交替形成的主链,Rha的4位带有侧链分支,侧链主要为α-1,5-Ara形成的阿拉伯聚糖,还有少量β-1,4-Gal形成的半乳聚糖。通过体内和体外巨噬细胞吞噬实验可知,山茶蜂花粉WCPP-3级份可以促进机体的巨噬细胞吞噬,但是这种促进作用并不是直接作用在细胞上,可能通过作用于其他细胞,产生相应的细胞因子,最终促进了巨噬细胞的吞噬功能。更多还原

【Abstract】 Camellia japonica belongs to the Camellia of Theaceae. Camellia japonica bee pollen is well known as a natural nutrition and health food. The research about polysaccharides of camellia japonica bee pollen is rare and mainly focuses on their biological activity, while the study about isolation and purification of polysaccharides from camellia japonica is very rare. In this paper, we reported the isolation and purification of camellia japonica pollen polysaccharides and immunological activity.Camellia japonica pollen polysaccharides (WCPP) was extracted with hot water,filtered, consentrated and precipitated with 80% ethanol. The monosaccharide composition of WCPP is 2.5% Man, 7.8% Rha, 3.0% GlcA, 37.0% GalA, 12.5% Glc, 15.1% Gal和22.1% Ara. The molecular weight distribution of WCPP was very wide and heterogeneous.Two methods were adopted in this paper to fractionate WCPP.First, stepwise ethanol precipitation (30%, 50%, 70% and 80%) was performed to fractionate WCPP and four fractions WCPP-30, WCPP-50, WCPP-70 and WCPP-80 were obtained. Monosaccharide composition analysis indicated that WCPP-30 was composed of 4.3% Man, 7.4% Rha, 45.4% GalA, 0.6% Glc, 10.3% Gal, 2.1% Xyl and 20.2% Ara; WCPP-50 was composed of 2.0% Man, 9.1% Rha, 42.7% GalA, 8.8% Glc, 11.5% Gal, 2.0% Xyl and 23.9% Ara; WCPP-70 was composed of 2.1% Man, 8.7% Rha, 1.9% GlcA, 15.5% GalA, 16.3% Glc, 20.6% Gal, 1.5% Xyl and 33.5% Ara; WCPP-80 was composed of 6.3% Man, 4.7% Rha, 4.8% GalA, 42.6% Glc, 12.2% Gal and 29.4% Ara. Gel permeation chromatography results implied that the molecular weight distributions of the four fractions were very wide and heterogeneous, so this method was not suitable for fractionation of WCPP.Next, anion-exchange chromatography was performed and eluted with H2O, 0.1 mol/l NaCl, 0.3 mol/l NaCl and 0.5 mol/l NaCl. Four fractions WCPP-N, WCPP-1, WCPP-2 and WCPP-3 were obtained. Monosaccharide composition analysis indicated that WCPP-N was composed of 4.1% Man, 1.0% Rha, 69.0% Glc, 13.4% Gal and 12.7% Ara; WCPP-1 was composed of 5.4% Man, 4.3% Rha, 2.5% GlcA, 45.0% GalA, 22.7% Gal, 18.0% Ara and 2.0% Glc; WCPP-2 was composed of 1.0% Man, 9.5% Rha, 1.9% GlcA, 30.2% GalA, 1.9% Glc, 19.3% Gal and 36.5% Ara; WCPP-3 was composed of 2.1% Man, 18.8% Rha, 1.1% GlcA, 33.2% GalA, 17.9% Glc, 19.1% Gal and 23.9% Ara. Gel permeation chromatography results implied that the molecular weight distributions of the four fractions were heterogeneous. In this paper, WCPP- 1 and WCPP-3 have a little content, WCPP-N and WCPP-2 were further purified mainly.WCPP-N was separated by Sephadex G-100 and two fractions WCPP-N-I and WCPP-N-II were obtained. WCPP-N-II was homogeneous and NMR results indicated that it containedα-1,4-Glucan as the main chain, andα-1,6-Glucan as side chains. So WCPP-N-II had an amylopectin structure. WCPP-2 was separated by Sepharose CL-6B and five fractions WCPP-2-I, WCPP-2-II, WCPP-2-III, WCPP-2-IV and WCPP-2-V were obtained. Among these fractions, WCPP-2-I was homogeneous and NMR results indicated that the main chain containedα-1,2-Rha andα-1,4-GalA repeating units, and the side chains ofα-1,5-Arabinan and minorβ-1,4-Galactan were linked to the C-4 of Rha. So WCPP-2-I was a RG-I type pectin.WCPP-3 could promote macrophage phagocytosis in vivo, but not in vitro. This result implied that WCPP-3 did not act directely on macrophages. It might work via stimulating other cells to produce cell factors which affected macrophage phagocytosis.更多还原