| Influenza is a serious infectious disease which leads to high disease rate, it posesserious threats to the health of the public, inducing a great number of economy lostand social problem throughout the world. It is worth noting that the high pathologyavian influenza H5N1and H1N1influenza pandemic recently has added weight to theconcerns regarding the influenza. Thus anti-influenza drugs have attached intensiveinterests. Radix isatidis plays an important role as antiviral antibiotic in clinic, has adecisive position in the proprietary Chinese medicine. Therefore radix isatidis is thepreferred raw material to exploit anti-influenza drugs. As the main active componentof radix isatidis, the polysaccharides of radix isatidis are the focus of anti-influenzavirus. In this paper, water-soluble radix isatidis polysaccharides were extracted fromradix isatidis by hot water and precipitated by ethanol. Then, the polysaccharides werefractionated by ion-exchange to obtain two homogeneous polysaccharides. Further,we studied the structural features of these fractions and their inhibition towardsneuraminidases of influenza virus. Make sure to identify the active ingredient ofanti-influenza virus in the radix isatidis polysaccharides, figure out the relationshipbetween the structure of polysaccharides and the inhibition towards neuraminidases ofinfluenza virus.Firstly, the water-soluble polysaccharides were extracted from the radix isatidiswith hot water, precipitated by80%ethanol. The polysaccharide mixture (IRP) wasobtained in a yield of10.0%(w/w). Phenol-sulfuric acid method determined the totalcarbohydrate content of IRP was75.9%, iodine chromogenic method determined thestarch content of IRP was62.5%, m-hydroxydiphenyl method determined the uronicacid content of IRP was8.3%, coomassie brilliant blue method determined theprotein content was1.5%. Sugar composition analysis by high performance liquidchromatography (HPLC) indicated that IRP was consisted of glucose (65.9%),galacturonic acid (11.7%), rhamnose (2.2%), arabinose (10.9%), galactose (6.2%)and xylose (1.9%).Secondly, IRP was separated on DEAE-Cellulose column into two fractions: anunbound fraction (IRPN) by water elution and a bound fraction (IRPA) by0.5MNaCl elution. The homogeneity of IRPN and IRPA was analyzed by chromatography and HPLC, their sugar composition analysis was also analysed by HPLC, the majorstructural were elucidated using Fourier transform infrared spectroscopy and13C-nuclear magnetic resonance spectrometer. The results showed that IRPN werestarch-like glucans, showed a wide peak on Sepharose CL-6B chromatographycolumn, suggesting that IRPN is inhomogenous in molecular weight. IRPA showed asingle narrow peak in Sepharose CL-6B and DEAE-Sepharose fast flow, and thedistribution of total sugars was consistent with that of uronic acid, implied that IRPAwas homogeneous fractions related to molecular weight and charge. The result ofsugar composition analysis showed that IRPA contained homogalacturonan (HG) andrhamnogalacturonan I (RG-I) with sidechains of arabinan, galactan, type-Iarabinogalactan and type-II rhamnogalacturonan, with a low degree of esterification.Thirdly, the inhibition of three fractions of radix isatidis towards H1N1, H5N1neuraminidases was studied by bioluminescent assay and typical fluorescent method.Conclutions were consistent with the two methods. The assay indicated that theinhibition activity tendency with IRPA>IRP>IRPN. IC50of IRPA towards H1N1,H5N1neuraminidases was below6mg/mL, and the inhibition activity toward H5N1neuraminidase was better than H1N1. It was determined that the acidic fraction wasthe principal active component of polysaccharides of radix isatidis in inhibitiontoward neuraminidases of influenza virus, and its higher inhibition depended on thecarboxyl group in structure. |
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