| Clam(Ruditapes philippinarum)is a common marine bivalve,which has both edible value and medicinal value.In this article,the clam was used as the raw material,the three fractions(cytoplasm,nucleus,mitochondrion)were extarcted by papain,and the polysaccharides were purified by the DEAE-Sepharose Fast Flow anion exchange chromatography.The structure and compositions of purified polysaccharide were analyzed by infrared spectral and precolumn derivatization HPLC,respectively.The pre-column derivatization HPLC method was used to determine the monosaccharide composition of three polysaccharides fractions,and the thermal analysis,infrared spectroscopy,and ultraviolet spectroscopy were also performed.Finally,the polysaccharides were studied for their antioxidative,antibacterial and preservative effects.The results were as follows:(1)Centrifugal method was used to separate the different fractions of Ruditapes philippinarum to obtain three components: cytoplasm,nucleus and mitochondria.Three components were separated and purified by enzymatic hydrolysis combined with DEAE-Sepharose Fast Flow anion exchange column.The colorimetric method and pre-column derivatization HPLC results showed that the cytoplasmic and nuclear polysaccharides had little difference in the composition of monosaccharides.The main constituents of the polysaccharides were glucosamine,galacturonic acid and galactose,and the composition of mitochondrial polysaccharides was relatively simple.Only glucose was detected;(2)The structure of three polysaccharides fractionss was identified by infrared spectroscopy.The UV spectra showed that the polysaccharides was a kind of glycoprotein,which contained protein.The results of molecular weight measurement by HPLC indicated that the polysaccharides was a single component.Fourier transform infrared spectroscopy(FT-IR)results showed that the polysaccharide structure contained polyhydroxyl groups,no benzene ring and sulfuric acid group,and the infrared measurement results were consistent with the colorimetric results.(3)Antioxidant activity of the three fractions polysaccharides were studied in vitro.The results showed that the three fractions polysaccharides all have different DPPH radical scavenging abilities,hydroxyl radical scavenging abilities,reducing abilities,and lipid peroxidation inhibitory activities.In the range of 2-12 mg/ml,with the increase of polysaccharide concentration,the antioxidant activity of the threecomponent polysaccharides showed an upward trend.The order of the DPPH free radical scavenging activity was : cytoplasmic polysaccharides> nuclear polysaccharides> mitochondrial polysaccharides.The superoxide anion scavenging ability was : cytoplasmic polysaccharide> nuclear polysaccharide> mitochondrial polysaccharide;reducing power was: cytoplasmic polysaccharide> nuclear polysaccharide> mitochondrial polysaccharide.The differences in the antioxidant activity of the three-component polysaccharides may be due to differences in the monosaccharide composition,molecular weight,degree of polymerization,and main chain characteristics of the polysaccharides.The cytoplasmic polysaccharides 1,2 and 3 of Philippine Ruditapes philippinarum and nucleus polysaccharide 2 all showed different degrees of inhibition on He La cells in a certain concentration range,and the inhibition rate increased with the increase of polysaccharide concentration,showing dose dependence.There was a significant difference in growth inhibitory rate between each concentration(p<0.01),and at the concentration of 1000 ?g/m L,the inhibitory rates of four polysaccharides on Hela cells were 76.57 %,68.78%,24.00% and 64.90%,respectively.Polysaccharide 1 exhibited strong inhibitory activity on Hela cells,the inhibition rate was 10.37% at 15.5 ?g/m L,and the inhibition rate was not significantly increased when the polysaccharide concentration was greater than 500 ?g/m L. |
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