Study On The Detection Of Influenza Viral Neuraminidases And The Inhibition Of Ginseng Polysaccharides On Them

Influenza is one of most serious infectious diseases which lead to high disease rate, acutecontagiousness, and extensive pathogenic field. It poses serious threats to public health andlife, inducing great number of economy lost and social problem throughout the world. It isworth noting that the high pathology avian influenza H5N1and H1N1influenza pandemicrecently has added weight to concerns regarding the influenza. Thus the detection of influenzaand anti-influenza drugs have attached intensive interests. A rapid detection method has beenestablished for influenza viral neuraminidase thus for influenza virus, and it can be used forrapid screening neuraminidase inhibitor by employment of this method. In this thesis, thestudy on the inbihition of panax ginseng polysaccharides towards neuraminidases wasconducted. The results of investigations, which provided a sensitive method for rapiddetection of influenza virus and a candidate for potent antiviral drugs, were outlined as below:1、Preparation of substrates for the detection of neuraminidaseThe sialic acid (Neu5Ac) as starting materials was protected by esterification,methylation, acetalation, acetylation, halogenation to give chlor-sialic acid derivatives.D-luciferin was prepared based on commercial available D-cysteine hydrochloridemonohydrate and2-cyano-6-hydroxybenzothiazole. And then chlor-sialic acid derivativeswere linked to D-luciferin by Williamson ether synthesis, followed by deprotection to providetwo novel compounds–luciferyl N-acetylneuraminic acid (1) and luciferyl4,7-di-O-methyl-N-acetylneuraminic acid (2) with the yield of43%and28%, respectively.All compounds involved in the reactions have been characterized by element analysis, IR,NMR and MS. In addition, the crystal structures of the intermediate8,9-O-isopropylidine-Neu5Ac-methylester-methyl-ketoside and4,7–di-O-methyl8,9-O-isopropylidine Neu5Ac methyl ester methyl ketoside were achieved, which providedconfiguration and theory directions for anti-influenza drug with sialic acid derivatives asprecursor.2、Establishment of a rapid detection method-bioluminescent assay for influenza viralneuraminidase with compound1and2as substratesNeuraminidase is a key enzyme in the life cycle of influenza virus. Conjugate1or2iscleaved by neuraminidase to liberate luciferin, which was subsequently oxidized to givedetectable luminescence signal. The intensity of signal is dependent on neuraminidase activity,therefore substrates1and2enable the detection of the neuraminidase. The detectioncondtions and capital components of the detection mixture were optimized by univariateanalysis and orthogonal experiments. The optimal incubation temperature was37oC. Theoptimal pH value was6.5for the reaction of neuraminidases from H1N1and H5N1, and5.0 for that of neuraminidases from A. ureafaciens and C. perfringens. Moreover，The sensitivityand specificity of the assays with compound1or2as the substrates for detection ofneuraminidases from influenza virus (H1N1and H5N1) and bacteria (A. ureafaciens and C.perfringens) were evaluated. Compound1was sensitive to neuraminidases from bothinfluenza virus and bacteria. Bioluminescent assay of this compound with H1N1and H5N1neuraminidases were approximately20-and16-fold more sensitive, respectively, than thefluorescent method with the commercial substrate4-MUNANA. Therefore, substrate1can beused for detection of bacterial and influenza viral neuraminidase. In contrast, compound2was only sensitive to the neuraminidases from influenza virus, showing approximately10-and8-fold greater sensitivity than4-MUNANA for the detection of H1N1and H5N1neuraminidases, respectively. The data showed that compound2revealed excellent sensitivityand specificity towards influenza viral neuraminidases. Thus, the substrate2could be used inassays for detection of influenza virus.3、Study of the inhibitory effect of ginseng polysaccharides towards neuraminidase in vitro bybioluminescent assayWhen there are inhibitors in the system, the luminescence signal became weak. Theinhibition activity of inhibitors can be detected by the change in the intensity of signal, thusbioluminescent assay enable the screening of neuraminidase inhibitors.The water-soluble polysaccharides from the roots of ginseng were extracted with hotwater, precipitated by ethanol according to our previous methods. The polysaccharides werethen fractionated on DEAE-Cellulose column to obtain neutral fraction and acidic fractions.Finally, the acidic fractions were further separated on DEAE-Cellulose column and SepharoseCL-6B into fractions–the type-I rhamnogalacturonan (RG-I) domain-rich pectins,arabinogalactans (AG), and the homogalacturonan (HG) domain-rich pectins.The inhibition of nine fractions of ginseng polysaccharides towards H1N1, H5N1neuraminidases was studied by our bioluminescent assay and typical fluorescent method. Theassay indicated that the inhibition activity tendency with acidic fraction of ginseng> ginsengpolysaccharides> neutral fraction of ginseng. WGPA-1-HG and WGPA-4-HG had betterinhibition activity. Their IC50were below4mg/mL. Conclutions were consistent with thesetwo methods, which revealed that it was rational for screening neuraminidase inhibitors withbioluminescent assay. Moreover, the bioluminescent assay took20min, was more rapid thantypical fluorescent method.In summary, a rapid and sensitive detection method for neuraminidases was established,which could be applied for diagnosis of influenza and high-through drug screening.