Studies On Antitumor Mechanism Of Lycium Barbarum Polysaccharide--Apoptosis, Related Factors Gene Expressions

It is widely recognized that Fructus Lycii plays multiple roles in pharmacological and biological functions as a traditional Chinese herbal medicine and also a kind of food. It was proved by our laboratory that Lycium barbarum polysaccharides (LBP) were its important bioactive components. This paper was performed on extracting technology of LBP, physicochemical characteristics of a purified fraction (LBP-X) and antitumor mechanism of LBP-X by apoptosis and related factors gene expressions. Immune function evaluation of Fructus Lycii juice which is a three-generation functional food processed by our laboratory was also conducted. The main results are as follows:1. Extracting technology of LBPFructus Lycii was soaked in 4 volumes of solvent system 1 for 1.5h and mixed. Add solvent system 2 to achieve 50% concentration. The residues were extracted with 3 volumes of solvent system 3 for 3 times at 60℃, each time for 1h. The dried pellets were extracted with 3 volumes of water for 4 times at 100℃, each time for 2h. The extracts were concentrated and free proteins were removed by Sevag method for at least 10 times. LBP were concentrated and add solvent system 4 to achieve 75% concentration. The residues were dehydrated to obtain the total LBP. The yield of the total LBP was 2.35%.The total exchange capacity of DEAE-cellulose (OH') was 0.8482mmol/g and its exchange capacity to LBP was 0.05386g/g. The four fractions of LBP can be separated well under this condition. Each fraction was desalted by Sephadex G25 column or dialysis for at least 2 days with dialysis tubing. The yields of four fractions were 14.85%, 14.06%, 18.43%, 38.41% respectively(compared with the total LBP). Each fraction was further purified on Sephadex G200 column. The yields of two subfractions of LBP-X were 7.8% and 1.0% respectively(compared with the total LBP). The bioactive studies were performed with the first subfraction of LBP-X.The contents of four fractions of LBP in different types were analyzed by spectrometer method. The evaluation of the quality of Fructus Lycii can base on this method to some extent.2. Physicochemical characteristics of LBP-XSDS-PAGE and Sephadex G200 column chromatography illustrated that LBP-X was homogeneous polysaccharide-protein complex. LBP-X was composed of arabine (Ara),rhamnose(Rha), xylose(Xyl), mannose(Man), galactose(Gal), glucose(Glu) and galacturnic acid. Gas chromatography showed that the neutral sugar molar ratio of LBP-X was Ara: Rha: Xyl: Man: Gal: Glu=l.10:1.25:1.76:1.95:1:2.12. Neutral sugar contents of LBP-X were 63.56% while galacturnic acid content was 24.80%. Protein content of LBP-X was 7.63%. Eighteen amino acids were included in LBP-X and the total contents of amino acids were 7.5%. Its weight-average molecular weight (Mw) was 1.569X 105 and the number-average molecular weight (Mn) was 1.422 X 105 by GPC-LLS. The polydispersity index (Mw/Mn) of LBP-X was 1,10. The molecular weight of LBP-X was 38414 by SDS-PAGE. The sugars were linked to peptide chain through Glycan-O-Ser linkage.3. Antitumor mechanism of LBP-X. Function of immune enhancement LBP-X 5, 10, 20mg/ (kg.d) could inhibit the growth of S180 effectively and increase thymus indexes, macrophage phagocytosis, anti-SRBC antibody secreted by spleen cells, lymphocyte proliferation and CTL activity markedly. Effect of LBP-X 10mg/(kg.d) is the best. Its antitumor effect is close to that of positive control Cy and it can nearly restore the immune function of S180-bearing mice to the level of normal mice. Lycium barbarum polysaccharide could inhibit tumor by enhancing immune function.. Apoptosis-inducing in tumor cells 20, 100, 500, 1000mg/L LBP-X inhibited the growth of HL-60 cells in a dose-dependent manner and the membrance fludity of HL-60 cells decreased markedly. The nuclei of HL-60 cells treated with LBP-X for 48h shrinked, condensed and cleaved. Agarose gel electrophoresis of DNA from cells treated with LBP-X revealed "DNA ladder" and TUNEL positive cells were detectable. HL-60 cells exposed to LBP-X showe...