四种根茎类食物活性多糖的研究

【作者】 赵国华；

【导师】 陈宗道； 李志孝；

【作者基本信息】 西南农业大学 ， 农产品贮藏加工， 2001， 博士

【摘要】 本文以山药、百合、甘薯和芋头为原料，利用多糖分离纯化技术、现代医学分析技术、法定的保健食品功能评价方法、仪器分析技术和生物大分子化学改性方法对根茎类食物中的活性多糖进行了系统研究，主要获得如下结果： （1）通过对四种根茎类食物多糖分离纯化的系统研究发现，四种根茎类食物经水浸提、乙醇沉淀、Sevag法脱蛋白、DEAE-52纤维素杜层析和SephadexG-100柱层析，得到的多糖组分经Sepharose CL-6B柱层析鉴定为纯品，以粗多糖为起始原料计算，四种纯品多糖的得率分别为：甘薯多糖63.85％，百合多糖80.1％，芋头多糖76.4％，山药多糖78.9％。经凝胶层析测定，四种活性多糖的分子量分别为：甘薯多糖53 200Dal，百合多糖30 200Dal，芋头多糖65 300Dal，山药多糖42 200Dal。经分析发现四种根茎类食物活性多糖中都不含蛋白质、游离单糖、淀粉和酚类物质，四种多糖均为中性纯粹多糖。 （2）利用法定的保健食品评价方法，对四种根茎类食物多糖纯品的增强免疫调节功能、抗肿瘤功能、降血脂功能和抗突变功能的研究发现： （a）在增强免疫调节功能实验中250 mg/kg^-1的SppS-Ⅰ-Fr-Ⅱ、150mg/kg^-1和250mg/kg^-1的LBPS-Ⅰ以及50 mg/kg^-1、150 mg/kg^-1和250 mg/kg^-1的RDPS-Ⅰ对小鼠体内巨噬细胞吞噬功能有明显地增强作用，而TPS-Ⅰ对巨噬细胞吞噬功能无影响。50 mg/kg^-1、150 mg/kg^-1和250 mg/kg^-1剂量的SPPS-Ⅰ-Fr-Ⅱ、LBPS-Ⅰ和RDPS-Ⅰ能明显地提高小鼠体内T淋巴细胞的活性，其中150 mg/kg^-1的作用最强。150 mg/kg^-1的TPS-Ⅰ也对小鼠T淋巴细胞增殖能力有明显地促进作用。150mg/kg^-1和250mg/kg^-1的SPPS-Ⅰ-Fr-Ⅱ和TPS-Ⅰ以及50mg/kg^-1、150mg/kg^-1和250 mg/kg^-1的LBPS-Ⅰ和RDPS-Ⅰ对小鼠体内NK细胞活性有明显的增强作用，四者在剂量为150 mg/kg^-1时对NK细胞的作用最强。150 mg/kg^-1和250 mg/kg^-1的四种实验根茎类食物多糖都能明显地提高小鼠血清溶血素的活性。150mg/kg^-1和250 mg/kg^-1的SPPS-Ⅰ-Fr-Ⅱ、LBPS-Ⅰ和TPS-Ⅰ以及50 mg/kg^-1、150 mg/kg^-1和250 mg/kg^-1的RDPS-Ⅰ能显著地提高小鼠血清中IgG的含量。可见，四种根茎类食物多糖能全面增强机体的细胞免疫和体液免疫功能。就对小鼠体内免疫调节功能的增强作用来说，在相同剂量下，四种多糖的强弱顺序为RDPS-Ⅰ＞LBPS-Ⅰ＞SPPS-Ⅰ-Fr-Ⅱ≈TPS-Ⅰ。而且，中等剂量的作用高于低剂量和高剂量。 （b）对四种根茎类食物活性多糖抗肿瘤功能的实验研究发现，RDPS-Ⅰ、LBPS-Ⅰ、SPPS-Ⅰ-Fr-Ⅱ在体内对B16黑色素瘤和Lewis肺癌均有显著的抑制作用对B16黑色素瘤抑制的强弱顺序为：RDPS-Ⅰ＞LBPS-Ⅰ＞SPPS-Ⅰ-Fr-Ⅱ，对Lewis肺癌而言，此顺序为LBPS-Ⅰ＞RDPS-Ⅰ＞SPPS-Ⅰ-Fr-Ⅱ，SPPS-Ⅰ-Fr-Ⅱ的体内抗肿瘤功能较弱，而TPS-Ⅰ实际无体内抗肿瘤功能。相关分析发现，RDPS-Ⅰ和LBPS-Ⅰ对荷瘤小鼠体内T淋巴细胞增殖能力、NK细胞活性、TNF-α活性及IL-2活性的影响与其抗肿瘤功能之间有基本相同的规律，因此，可以认为四种根茎类食物多糖的体内抗肿瘤功能是通过增强机体与抗肿瘤有关的免疫调节功能而实现的。但是四种根茎类食物活性多糖在体外对B16黑色素瘤和Lewis肺癌均无抑制作用。 ＊)在四种根茎类食物降血脂实验中发现，本实验所用的高脂饲料能成功地诱发大鼠高血脂的形成，可建立良好的大鼠高血脂模型。四种根茎类食物中，RDPS－I和LBPS工对实验性大鼠高脂血症有较强的降血脂功能或防止高血脂形成的作用，相比之下，TPS＊和SPPS．1．Fr－11的这方面的作用则较弱。 u)四种根茎类食物抗突变研究发现，实验所用的鼠伤寒沙门氏菌 TAI 00和TA98符合抗突变实验要求菌株的特性；类食物活性多糖对鼠伤寒沙门氏菌自发突变菌株无抑制作用，也无致突变作用；25 mgghate”’剂量的 SPPSJFr二、15mgglate”‘剂量和 25mgglate“’剂量的 LBPS一I气 15mgglate”‘剂量的 pPS一I和TPSJ的抗突变活性呈弱阳性；而25mgglate”’剂量的RDPSJ和TPSJ肯定具有抗突变作用；smgglate”‘剂量的根茎类食物多糖不具有抗突变作用。 （3）首次利用多糖化学结构分析的化学方法（甲基化分析、高碘酸氧化、Smith降解、完全酸水解等）和现代仪器分析方法（气相色谱、红外光谱、气相色谱．质谱连用、它和’七核磁共振，旋光分析等人明确提出了三种具有较强保健作用的根茎类食物多糖的化学结构。 SPPS－LFr－11是aJ，6－连接的毗哺葡聚糖，其结构如下： 一［一6）一Q—D—GICP一（1一］。一 LBPS｛的结构大致如下： 、｛QD’GI。p‘、［OD‘GI。p’、QD’GICp’、OD’GICp’刁。、QD’GICp‘）。。 t’t‘ a－D－‘Glcp Q－D－’Glcp t；’t．’ O－D—GICp O－D－GICp RDPS工的结构可能如下：、｛［ODaGICp’、」xQDaGICp‘、?更多还原

【Abstract】 On the bases of research papers investigation, the advances in isolation, purification, bioactive function, structure determination, structure-activity relationship of polysaccharides, and the polysaccharide research situation and prospect in our country were reviewed. Then, four polysaccharides were isolated and purified from four root foodstuff of Rhizoma Diosoreae (RDPS-I), lily bulb(LBPS-I), sweet potato(SPPS-I-Fr-Il) and taro(TPS-I), and their immune enhancing activity, antitumor activity and its mechanism, reducing blood lipids activity, antimutagenicity, structure and structure-antitumor activity relationship were investigated with polysaccharides isolation and purification technique, medical analysis technique, function evaluation methods of healthy food, instrument analysis technique and chemical modification technique of biomaromoleule in this paper. The results indicated: (1 )Four kinds of polysaccharides were isolated from lily bulb, Rhizoma Diosoreae, sweet potato, and taro with water extraction, alcohol sedient, Sevag deprotein, DEAE-52 column and Sephadex G- 100 column, their purity were identified by Sepharose CL-6B. They were named as LBPS-l, RDPS-I, SPPS-I-Fr-II, and TPS-I, and the extraction ratio of these four polysaccharides were 80.1%, 78.9%, 63.8%, and 76.4%. The molecular weight of LBPS-l, RDPS-I, SPPS-I-Fr-I1, and TPS-l were determined with column chromography as 30 200Dal, 42 200Dal, 53 200Dal, and 65 300Dal respectively. Physico-chemiacl characteristics analysis indicated that four polysaccharides do not contain protein, free monosaccharose, starch, polyphenols and uronic acid. Therefore. it can be concluded that they are neutral pure polysaccharides. (2)Amor1g the four polysaccharides isolated from root foodstuff, SPPS-I-Fr-II with a dose of 250 mgkg LBPS-I with a dose of 150 mgkgand 250 mgkgand RDPS-I with a dose of 50 mgkg 150 mgkgand 250 mgkgcould significantly enhance the phagocytolysis of macrophage in mice in vivo. However, TPS did not has any effect on phagocytolysis of macrophage in mice in vivo. When the dose is 50 mgkg 150 mgkg抋nd 250 mgkg, SPPSrI LBPS and RDPS could significantly increase the T splenic lymphocyte proliferation activity in mice in vivo, and the dose of 150 mgkghas the highest effect. With a dose of 150 mgg TPS-l could also significantly increase the T splenic lymphocyte proliferation activity in mice in vivo. As to the effects of polysaccharides on NK activity, with a dose of 150 mgkgand 250 mgkg SSPS--FrI and TPS could significantly enhance the NK activity in mice in vivo. The experimented dose ( 50 mg4cg 150 mgkgand 250 mgkg of LBPS and RDPS could significantly enhanced the NK activity in mice in vivo. It also indicated that among the dose used in experiment 150 mgkgas the strongest effect on NK activity. With a dose of 50 mgkg 150 mgkgand 250 mgkg four polysaccharides isolated from root foodstuff could significantly increase the serum hemolysin in mice in vivo. When four polysaccharides used with a dose of 150 mgkgand 250 mgkg,and RDPS used with a dose of 50 mgkgcould significantly increase thd serum lgG content in mice in vivo. Therefore, It can be concluded with a certain dose used that polysaccharides could significantly increase both the cell immune function and humor immune function in mice in vivo. As to immune enhancing effect among the four polysaccharides, under the same dose, this sequence is RDPS> LBPS> SPPSr-J ITPS-I. (3)With a cell cytocity experiment, four polysaccharides of root foodstuff were confirmed as non-in更多还原