Systematical Fractionation Of Polysaccharides From Panax Ginseng Leaves And Their Antitumor Activities

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.