Research On Extraction, Purification And In Vitro Antitumor Activity Of Polysaccharides From Porphyra Yezoensis

The ultrasonic-assisted or microwave-assisted synchronous extraction parameters of polysaccharide and Phycoerythrin from Porphyra yezoensis were optimized. Trichloroacetic acid (TCA) combined with the denaturation temperature of Phycobiliprotein was used to remove protein and membrane separation technology was used to isolate the polysaccharides. Three different purified fractions PYPSⅡa,PYPS1a and PYPS 2a were furhter obtained based on their different charge characteristic and molecular weight by DEAE C-52 and Sephadex G-100 chromatography, respectively. The basic structures of three main fractions, monosaccharide compositions and antitumor activity of PYPSⅡa and PYPS 2a in vitro were also investigated in the paper. The primary research results were as follows:1. In this paper, the optimum parameters for ultrasonic-assisted synchronous extraction of polysaccharide and Phycoerythrin from Porphyra yezoensis were definitely present in first time. The optimum condition was as follows: ultrasonic power 880W, ultrasonic time 60min, water extracting temperature 30℃for 4 hours, ratio of solid-liquid 1:50, the extraction percent of polysaccharide and Phycobiliprotein was respectively 34.03% and 1.2%, Phycoerythrin purity was 0.356.2. In this paper, the optimum parameters for microwave-assisted synchronous extraction of polysaccharide and Phycoerythrin from Porphyra yezoensis were definitely present in first time. The optimum condition was as follows: microwave power 150W, microwave time 4min, water extracting temperature 30℃for 5 hours, ratio of solid-liquid 1:40, the extraction percent of polysaccharide and Phycobiliprotein was respectively 16.94% and 0.417%, Phycoerythrin purity was 0.255. Compared with control (water extracting temperature 30℃for 5 hours, ratio of solid-liquid 1:40, the extraction percent of polysaccharide and Phycobiliprotein was respectively 10.05% and 0.324%, Phycoerythrin purity was 0.215), the extraction percent of polysaccharide of ultrasonic-assisted extraction and microwave-assissted extraction increased 23.5% and 6.4%, respectively. 3. The optimum condition of removing protein by trichloroacetic acid (TCA) combined with the denaturation temperature of Phycobiliprotein was researched in this paper. The condition was as follows: TCA concentration 2%, temperature50℃, time 20min, the ratio of protein removing was 99.63%, the retention ratio of polysaccharide was 90.49%. The effect was better than traditonal TCA method (TCA concentration 3% placed in One hour, the ratio of protein removing was 91.24%, the retention ratio of polysaccharide was 82.84%), the ratio of protein removing and the retention ratio of polysaccharide increased 8.39% and 7.65%, respectively.4. The membranes with molecular weight cut-off (MWCO) of 50 kDa and 3 kDa were adopted for the ultrafiltration process. Further purification of the polysaccharides was carried on DEAE C-52 and Sephadex G-100 chromatography, and two purified fractions of PYPSⅡa, PYPS 1a and PYPS2a were obtained.5. Ultraviolet spectra of PYPSⅡa, PYPS 1a and PYPS 2a revealed that the three polysaccharides were single composition without nucleic acid and protein. Fourier-transform infrared spectra (FT-IR) of PYPSⅡa, PYPS 1a and PYPS2a revealed typical characteristics of polysaccharides, they were composed of pyranoses, sulfate group and 3,6-inner aether bridge. Gas chromatography indicated PYPS 1a and PYPS 2a were composed of D-galactose, L-rharmnose and three unknown monosaccharides, molar proportions of D-galactose and L-rharmnose were 30: 1 and 12.8:1, respectively. PYPSⅡa was composed of D-galactose, L-rharmnose and four unknown monosaccharides, molar proportions of D-galactose and L-rharmnose were 6.6:1.6. Both PYPSⅡa and PYPS 2a showed a certain extent antitumor activity and the activity was in a dose-dependent manner, at 1 mg/ml, the inhibition rate was 43.1% and 30.0% respectively.