Study On Isolation, Purification Of Polysaccharides From Adlay Seeds And Their Induction Of Apoptosis In A549 Cells

Adlay seeds are commonly used in traditional Chinese medicine or functional food. The polysaccharides from adlay seeds are used to reduce blood sugar and antioxidant etc. In this study the structure and the bioactivity of the polysaccharides from adlay seeds was investigated and its effectsof apoptotic induction of lung cancer A549 cells was discussed.The polysaccharides were extracted from the adlay seeds with decoction and alcohol sedimentation techniques, followed by protein removal, pigment removal, dialysis,freeze-drying, and finally achieved by sequential column chromatography with DEAE-52 and Sephadex G-50.Purity of polysaccharides was determined by spectroscopy with ultravioletvisible full wavelength scanningand HPLC, which was free of nucleic acids and proteins, the optical rotation was+180, determined by an automatic optical rotation photometer. The analysis of monosaccharide composition of the polysaccharides was carried out by GC based pre-column derivatization with acetic acid. The neutral sugar components were rhamnose, arabinose, xylose, glucose and galactos.The relative molecular weight was determinated by the gel filtration and it wasaboutl.2×104 in molecular weight. The structure was analyzed by infrared spectroscopy. The IR spectrum indicated the polysaccharide carry a β-D-glucopyranosewith carboxylic acid.Cell viability was determined by the MTT assay, andcoix-polysaccharide exerted remarkable inhibitory effects on A549 cell proliferation. Apoptotic bodies of cell morphology were observed by scanning electronic microscopy. The cell cycle and apoptotic rate was analyzed by flow cytometry. Apoptotic induction was also verified by DNA accumulation using propidium iodide nucleus staining in the S phase by flow cytometry, and the apoptotic rate was correlated with the concentration of the polysaccharide at range of 0-300μg/mL.. The mitochondrial membrane potential of cells was decreased in the same range of the polysaccharidesby laser scanning confocal microscope.The gene expression of caspase-3 and caspase-9 was increased after coix-polysaccharide treatment by RT-PCR. The expression of protein of caspase-3 and caspase-9 was increased by Western blot.The apoptotic characterization was also determined by single cell gel electrophoresis (comet assay), which the polysaccharide extended tail distance, indicating that the DNA was fragmented.The polysaccharide isolated from the adlay seed show petent induction of apoptosis in lung cancer cell A549, which provide a basis for pharmacological activity of the coix-polysacchaaride.