人参叶多糖的系统分级和抗肿瘤活性研究
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摘要
人参(Panax ginseng C. A. Meyer)是我国传统名贵中药,具有多种功效。多糖是人参的有效成分之一,具有免疫调节、抗肿瘤和降血糖等生物活性。人参根虽有极高的药用价值,但4～6年才收获一次。而人参叶每年都有收获,却没有得到充分的利用。目前,有关人参叶多糖的研究报道已有近20余篇,主要集中在单一级分的分离纯化、结构分析及生物活性等方面,还缺乏对人参叶多糖全面系统的分析。本文用水提方法制备了人参叶水溶性多糖,乙醇沉淀、凝胶过滤和离子交换层析等方法对水溶性人参叶多糖进行了系统的分级,全面分析了各种级分的单糖组成、分子量分布,初步分析了各种级分可能的结构特征,并对各级分的免疫调节和抗肿瘤活性进行了研究,研究结果如下:
干燥的人参叶经过热水煮提、乙醇沉淀、Sevag法脱蛋白得到人参叶水溶性粗多糖GL,HPLC分析GL的单糖组成为Gal、Ara、GalA、Glc、Man和Rha。GL经过DEAE-Cellulose离子交换柱层析分级,得到四个多糖级分GLN、GLA-1、GLA-2、GLA-3。
中性糖含量较高的级分GLN为不均一的多糖混合物,通过DEAE-Sepharose FF柱层析分级得到四个级分GLN-1、GLN-2、GLN-3和GLN-4。GLN-1主要由Gal、Ara、Glc和Man组成,糖含量81.5%,其Gal与Ara的摩尔比接近1:1,推测GLN-1是AG型果胶,分子量大约为4000 Da;GLN-2主要由Gal、Ara、Glc、Man和GalA组成,糖含量86.9%,糖醛酸含量7.7%,分子量大约为9600 Da;GLN-3主要由Gal、Ara、GalA、Glc和Man组成,糖含量为65.4%,糖醛酸含量为22.9%,GLN-3在Sepharose CL-6B上呈现两个峰,分子量分别为80000 Da和6500 Da。GLN-4的单糖组成与GLN-3相似,糖含量为45.1%,糖醛酸含量为17.6%,GLN-4在Sepharose CL-6B上也呈现两个峰,分子量分别是47000 Da和10000 Da。
GLA-1主要含有GalA、Gal和Ara,且GalA含量高达45.1%,推测GLA-1为HG型果胶。GLA-2主要含有Gal、Ara、Rha和GalA,且Rha和GalA含量较高,推测GLA-2是RG-I型果胶。通过DEAE-Sepharose FF柱层析将GLA-1进一步分级,得到四个级分GLA-1aa、GLA-1ab、GLA-1b和GLA-1c,通过HPLC和Sephadex G-75柱层析测定各级分的单糖组成和分子量分布。GLA-1aa级分主要含有Gal,Ara,Glc和Man,其收率很低。GLA-1ab、GLA-1b和GLA-1c主要由Gal、Ara、GalA和Rha构成。根据GLA-1ab和GLA-1b的单糖组成摩尔比分析,推测其可能为RG-I型果胶。GLA-1c的GalA含量为78.1%,推测其可能为HG型果胶。
应用MTT法检测人参叶多糖在体外对人结肠癌HT-29和HCT116细胞增殖的抑制作用。结果表明人参叶多糖GL、GLA-1和GLA-3对HT-29和HCT116细胞,尤其是高转移性的HCT116细胞增殖有显著的抑制作用,且呈浓度依赖关系。通过PI单染流式细胞术检测细胞周期对其机制进行深入的研究,发现人参叶多糖诱导HCT116细胞阻滞在G0/G1期。
采用划痕实验检测人参叶多糖对L929细胞的迁移作用,发现人参叶多糖各级分对细胞迁移均有浓度依赖性的抑制作用。其中人参叶多糖GL的抑制作用最强,可能各级分之间存在着协同作用。
用人参叶多糖GL喂食S-180腋下移植瘤小鼠11天,分别计算抑瘤率、胸腺指数和脾指数,发现三种剂量的人参叶多糖都有不同程度的抑瘤作用,其中200 mg/kg的剂量具有最强的抑制肿瘤生长的功能,抑瘤率为41.76%。另外,GL还能促进荷瘤小鼠的胸腺发育,逆转移植肿瘤对胸腺的抑制作用,同时对脾脏的发育也有明显的促进效果。
Panax ginseng C. A. Meyer (P. ginseng) has been used in China as a traditional medicine for over 2000 years. The polysaccharides from ginseng roots have many bioactivities, such as immunomodulation, anti-tumor and hypoglycemic activities. Up to now, there were about 20 papers reporting the studies of polysaccharides from ginseng leaves on their isolation, structural analysis and bioactivities,but no reports on the total fractionation of polysaccharides from ginseng leaves. In this paper, we reported the results of total fractionation of the polysaccharides from ginseng leaves by a combination of anion-exchange and gel permeation chromatography and the analysis of their sugar composition and molecular weight distribution. Further, their possible structural features were discussed. The immunomodulative and antitumor activities were also studied in mice. The investigation results are as follows:
Water-soluble polysaccharide (GL) was extracted from dried ginseng leaves with hot water, precipitated with ethanol and deproteinized by the Sevag method. The HPLC result showed that GL mainly contained Gal, Ara, GalA, Glc, Man and Rha. After GL was loaded on DEAE-Cellulose column, four polysaccharide fractions GLN, GLA-1, GLA-2 and GLA-3 were obtained.
GLN was a neutral sugar-rich mixture and fractionated into four fractions (GLN-1, GLN-2, GLN-3 and GLN-4) by DEAE-Sepharose FF chromatography. High performance liquid chromatography (HPLC) results showed that GLN-1 mainly contained Gal, Ara, Glc and Man. The total carbohydrate content of GLN-1 was 81.5%. As the molar ratio of Gal and Ara in GLN-1 was nearly 1:1, we speculated it was an arabinogalactan and the molecular weight of it was 4000Da. GLN-2 was mainly contained Gal, Ara, Glc, Man and GalA. The total carbohydrate content of GLN-2 was 86.9%, uronic acid content was 7.7%, and the molecular weight was 9600 Da. GLN-3 was rich in Gal and Ara with some Glc and GalA .The content of total carbohydrate of GLN-3 was 65.4%, uronic acid content was 22.9%. GLN-3 showed two populations on Sepharose CL-6B column, with the molecular weights of 80000 Da and 6500 Da, respectively. GLN-4 was similar with GLN-3 in sugar composition. The content of total carbohydrate of GLN-4 was 45.1%, uronic acid content was 17.6%. GLN-4 also showed two populations on Sepharose CL-6B column, with the molecular weights of 47000 Da and 10000 Da, respectively.
GLA-1 was mainly composed of GalA, Gal and Ara, and GalA content was up to 45.1%, so it might be HG-rich pectin. GLA-2 mainly contained Gal, Ara, Rha and GalA, and the content of Rha and GalA were high, so it might be RG-I-rich pectin. GLA-1 was separated through DEAE-Sepharose FF chromatography eluting by different NaCl concentration to give four fractions (GLA-1aa, GLA-1ab, GLA-1b and GLA-1c). GLA-1aa was mainly contained Gal, Ara, Glc and Man and had a low recovery. GLA-1ab, GLA-1b and GLA-1c were rich in Gal, Ara, GalA and Rha. GLA-1ab and GLA-1b contained 99% and 98.8% total carbohydrate, 34.9% and 49.1% uronic acid, respectively. From the monosaccharide composition features, we speculated GLA-1ab and GLA-1b were RG-I-rich pectins, while GLA-1c was HG-rich pectin.
The direct effects of ginseng leaves polysaccharides on the proliferation of human colon cancer HT-29 and HCT116 cells was measured by MTT assay. The ginseng leaves polysaccharides GL, GLA-1 and GLA-3 had significant dose-dependent anti-proliferative effect on HT-29 cells and HCT116 cells, especially on highly metastatic cell HCT116. To elucidate the mechanism by which ginseng leaves polysaccharides affect cell proliferation, we carried out cell cycle analyses by flow cytometry after staining with PI. The results showed that ginseng leaves polysaccharides induced G0/G1 cell cycle arrest on human colon cancer HCT116 cells.
We invested the migration-inhibitory effect of ginseng leaves polysaccharides on L929 cells by wound healing assay. All of the polysaccharides inhibit migration of L929 cell in a dose-dependent mannar. GL has the most significant inhitory effect. Synergistic inhibition might excist among the polysaccharides.
S-180 tumor cell bearing mice were admistrated with polysaccharide GL for 11 days and then tumor-inhibiting rate, thymus index and spleen index were determined. The results showed that various doses had different effects on tumor-inhibiting rate, thymus index and spleen index. However, the most effective dose was 200 mg.kg-1. Polysaccharide GL inhibited the growth of tumor of S-180 model mice and the tumor-inhibiting rate was 41.76%. GL enhanced the development of thymus as well as spleen of S-180 mice.
干燥的人参叶经过热水煮提、乙醇沉淀、Sevag法脱蛋白得到人参叶水溶性粗多糖GL,HPLC分析GL的单糖组成为Gal、Ara、GalA、Glc、Man和Rha。GL经过DEAE-Cellulose离子交换柱层析分级,得到四个多糖级分GLN、GLA-1、GLA-2、GLA-3。
中性糖含量较高的级分GLN为不均一的多糖混合物,通过DEAE-Sepharose FF柱层析分级得到四个级分GLN-1、GLN-2、GLN-3和GLN-4。GLN-1主要由Gal、Ara、Glc和Man组成,糖含量81.5%,其Gal与Ara的摩尔比接近1:1,推测GLN-1是AG型果胶,分子量大约为4000 Da;GLN-2主要由Gal、Ara、Glc、Man和GalA组成,糖含量86.9%,糖醛酸含量7.7%,分子量大约为9600 Da;GLN-3主要由Gal、Ara、GalA、Glc和Man组成,糖含量为65.4%,糖醛酸含量为22.9%,GLN-3在Sepharose CL-6B上呈现两个峰,分子量分别为80000 Da和6500 Da。GLN-4的单糖组成与GLN-3相似,糖含量为45.1%,糖醛酸含量为17.6%,GLN-4在Sepharose CL-6B上也呈现两个峰,分子量分别是47000 Da和10000 Da。
GLA-1主要含有GalA、Gal和Ara,且GalA含量高达45.1%,推测GLA-1为HG型果胶。GLA-2主要含有Gal、Ara、Rha和GalA,且Rha和GalA含量较高,推测GLA-2是RG-I型果胶。通过DEAE-Sepharose FF柱层析将GLA-1进一步分级,得到四个级分GLA-1aa、GLA-1ab、GLA-1b和GLA-1c,通过HPLC和Sephadex G-75柱层析测定各级分的单糖组成和分子量分布。GLA-1aa级分主要含有Gal,Ara,Glc和Man,其收率很低。GLA-1ab、GLA-1b和GLA-1c主要由Gal、Ara、GalA和Rha构成。根据GLA-1ab和GLA-1b的单糖组成摩尔比分析,推测其可能为RG-I型果胶。GLA-1c的GalA含量为78.1%,推测其可能为HG型果胶。
应用MTT法检测人参叶多糖在体外对人结肠癌HT-29和HCT116细胞增殖的抑制作用。结果表明人参叶多糖GL、GLA-1和GLA-3对HT-29和HCT116细胞,尤其是高转移性的HCT116细胞增殖有显著的抑制作用,且呈浓度依赖关系。通过PI单染流式细胞术检测细胞周期对其机制进行深入的研究,发现人参叶多糖诱导HCT116细胞阻滞在G0/G1期。
采用划痕实验检测人参叶多糖对L929细胞的迁移作用,发现人参叶多糖各级分对细胞迁移均有浓度依赖性的抑制作用。其中人参叶多糖GL的抑制作用最强,可能各级分之间存在着协同作用。
用人参叶多糖GL喂食S-180腋下移植瘤小鼠11天,分别计算抑瘤率、胸腺指数和脾指数,发现三种剂量的人参叶多糖都有不同程度的抑瘤作用,其中200 mg/kg的剂量具有最强的抑制肿瘤生长的功能,抑瘤率为41.76%。另外,GL还能促进荷瘤小鼠的胸腺发育,逆转移植肿瘤对胸腺的抑制作用,同时对脾脏的发育也有明显的促进效果。
Panax ginseng C. A. Meyer (P. ginseng) has been used in China as a traditional medicine for over 2000 years. The polysaccharides from ginseng roots have many bioactivities, such as immunomodulation, anti-tumor and hypoglycemic activities. Up to now, there were about 20 papers reporting the studies of polysaccharides from ginseng leaves on their isolation, structural analysis and bioactivities,but no reports on the total fractionation of polysaccharides from ginseng leaves. In this paper, we reported the results of total fractionation of the polysaccharides from ginseng leaves by a combination of anion-exchange and gel permeation chromatography and the analysis of their sugar composition and molecular weight distribution. Further, their possible structural features were discussed. The immunomodulative and antitumor activities were also studied in mice. The investigation results are as follows:
Water-soluble polysaccharide (GL) was extracted from dried ginseng leaves with hot water, precipitated with ethanol and deproteinized by the Sevag method. The HPLC result showed that GL mainly contained Gal, Ara, GalA, Glc, Man and Rha. After GL was loaded on DEAE-Cellulose column, four polysaccharide fractions GLN, GLA-1, GLA-2 and GLA-3 were obtained.
GLN was a neutral sugar-rich mixture and fractionated into four fractions (GLN-1, GLN-2, GLN-3 and GLN-4) by DEAE-Sepharose FF chromatography. High performance liquid chromatography (HPLC) results showed that GLN-1 mainly contained Gal, Ara, Glc and Man. The total carbohydrate content of GLN-1 was 81.5%. As the molar ratio of Gal and Ara in GLN-1 was nearly 1:1, we speculated it was an arabinogalactan and the molecular weight of it was 4000Da. GLN-2 was mainly contained Gal, Ara, Glc, Man and GalA. The total carbohydrate content of GLN-2 was 86.9%, uronic acid content was 7.7%, and the molecular weight was 9600 Da. GLN-3 was rich in Gal and Ara with some Glc and GalA .The content of total carbohydrate of GLN-3 was 65.4%, uronic acid content was 22.9%. GLN-3 showed two populations on Sepharose CL-6B column, with the molecular weights of 80000 Da and 6500 Da, respectively. GLN-4 was similar with GLN-3 in sugar composition. The content of total carbohydrate of GLN-4 was 45.1%, uronic acid content was 17.6%. GLN-4 also showed two populations on Sepharose CL-6B column, with the molecular weights of 47000 Da and 10000 Da, respectively.
GLA-1 was mainly composed of GalA, Gal and Ara, and GalA content was up to 45.1%, so it might be HG-rich pectin. GLA-2 mainly contained Gal, Ara, Rha and GalA, and the content of Rha and GalA were high, so it might be RG-I-rich pectin. GLA-1 was separated through DEAE-Sepharose FF chromatography eluting by different NaCl concentration to give four fractions (GLA-1aa, GLA-1ab, GLA-1b and GLA-1c). GLA-1aa was mainly contained Gal, Ara, Glc and Man and had a low recovery. GLA-1ab, GLA-1b and GLA-1c were rich in Gal, Ara, GalA and Rha. GLA-1ab and GLA-1b contained 99% and 98.8% total carbohydrate, 34.9% and 49.1% uronic acid, respectively. From the monosaccharide composition features, we speculated GLA-1ab and GLA-1b were RG-I-rich pectins, while GLA-1c was HG-rich pectin.
The direct effects of ginseng leaves polysaccharides on the proliferation of human colon cancer HT-29 and HCT116 cells was measured by MTT assay. The ginseng leaves polysaccharides GL, GLA-1 and GLA-3 had significant dose-dependent anti-proliferative effect on HT-29 cells and HCT116 cells, especially on highly metastatic cell HCT116. To elucidate the mechanism by which ginseng leaves polysaccharides affect cell proliferation, we carried out cell cycle analyses by flow cytometry after staining with PI. The results showed that ginseng leaves polysaccharides induced G0/G1 cell cycle arrest on human colon cancer HCT116 cells.
We invested the migration-inhibitory effect of ginseng leaves polysaccharides on L929 cells by wound healing assay. All of the polysaccharides inhibit migration of L929 cell in a dose-dependent mannar. GL has the most significant inhitory effect. Synergistic inhibition might excist among the polysaccharides.
S-180 tumor cell bearing mice were admistrated with polysaccharide GL for 11 days and then tumor-inhibiting rate, thymus index and spleen index were determined. The results showed that various doses had different effects on tumor-inhibiting rate, thymus index and spleen index. However, the most effective dose was 200 mg.kg-1. Polysaccharide GL inhibited the growth of tumor of S-180 model mice and the tumor-inhibiting rate was 41.76%. GL enhanced the development of thymus as well as spleen of S-180 mice.
引文
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