Isolation, Purification And Anti - Tumor Mechanism Of Polysaccharide From Abalone

Research shows that edible fungus polysaccharide has the remarkable enhancement immunity, lowering blood pressure, lowering blood lipid, antioxidant and antitumor activity. Pleurotus abalonus Han, K.M.Chen et S.Cheng is a traditional Chinese medicinal and edible fungus distributed in Fujian and Taiwan Province of China, which is a Pleurotus genus fungus of Tricholomataceae belonging to the Basidiomycetes and some biological activities have been reported.At present, the study of P. abalones is still in the initial stage in home and aboard. In this article, we study the extraction, separation and purification, structure and biological activity of polysaccharide from P. abalones.The water-soluble crude polysaccharides were extracted from the fruiting bodies of P. abalonus by hot water extraction and ethanol precipitation. The extracts were further purified through with DEAE-52cellulose column based on the difference of ionic groups present in P. abalonus polysaccharide (PAP) molecules, and three major fractions eluted with0,0.05,0.1M sodium chloride solutions were collected, respectively. The fractionated elution was subjected to a further purification by gel-filtration chromatography on Sephadex G-100column, and then concentrated, dialyzed and lyophilized to afford PAP-1, PAP-2and PAP-3for further bioactivity assay, respectively. To compare the biological activities of the fractions, we evaluated anti-tumor effects. Interestingly, in this study only one of the isolated fractions, namely high-molecular weight of PAP-3, could remarkably suppress the growth of human breast carcinoma MCF-7cells, suggesting that it is highly feasible to improve biological activity of P. abalonus polysaccharides by the separation of more active fractions and even chemical manipulation. The largest PAP-3, an acidic polysaccharide fraction with a molecular mass of3.68×15Da, was the most active in inhibiting MCF-7cancer cells with an IC50of193μg/mL. Furthermore, the HPLC analysis showed that PAP-3was composed of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose, and their corresponding mole percentages were7.8%,1%,2.7%,1.3%,41.7%,29.8%,14.6%and1.1%of all the quantitative monosaccharides, respectively.The changes in cell normal morphology were observed by DAPI staining and the PAP-3-induced apoptosis was confirmed by annexin V/propidium iodide staining. The exposure to200μg/ml PAP-3after48h resulted in about20.48%of the cells going into early apoptotic phase, as well as29.39%of late apoptotic phase. The apoptosis was involved in mitochondria-mediated pathway including the loss of mitochondrial membrane potential (△Ψm), the increase of Bax/Bcl-2ratio, caspase-9/3activation, and poly(ADP-ribose) polymerase (PARP) degradation. PAP-3also induced up-regulation of p53, and cell cycle arrest at the S phase.Compared with the control group, the generation of intracellular H2O2and O2-significantly increased after exposure to PAP-3. To investigate if the mitochondrial dysfunction observed in MCF-7cells treated with PAP-3is promoted by ROS production, we measured ROS levels using the cell-permeable dye DCF-DA and DHE. The results showed that the apoptotic effect of PAP-3on MCF-7cells was associated with an early elevated level of intracellular ROS in a time-or concentration-response manner. The levels of H2O2and O2-in200μg/mL PAP-treated cells were5.4-fold and5-fold higher for24h, respectively, compared to the level of ROS in control cells throughout the experiment. Moreover, to further confirm that the apoptotic effect of PAP-3was mediated by ROS, antioxidants NAC and SOD were also used. Both NAC and SOD inhibited the accumulation of intracellular ROS induced by PAP-3, and both could ensure cell survival as shown by trypan blue exclusion and significantly reduced cell apoptosis as demonstrated by Annexin V-FITC/PI staining. These results indicate that PAP-3-induced apoptosis is associated with ROS generation.We here fractionated the crude polysaccharide preparation from P. abalonus into three fractions, namely PAP-1, PAP-2and PAP-3, and tested these fractions for antiproliferative activity in human breast cancer MCF-7cells. The largest PAP-3, an acidic polysaccharide fraction with a molecular mass of3.68×105Da, was the most active in inhibiting MCF-7cancer cells with an IC50of193μg/mL. The apoptosis caused by PAP-3was involved in mitochondria-mediated pathway, which dependent Intracellular ROS. On the basis of systematic studies of PAP-3antitumor mechanism, for the development of independent intellectual property of the new functional food factors or drugs to provide the theory basis, to play its great potential economic and social benefits.