Studies On Antiviral Characteristic And Mechanism Of Action Of HOGS

Along with the attention to the health daily,the security,effective and special anti-viral biochemistry medicine research becomes the hot spot.The polysaccharide can enhance organism immunity as the biological activity material,and has low poisonous and low drug resistance characteristics,among which,the sulphating polysaccharide display the stronger anti-viral activeness through the direct inhibitory action to the cell viral infection,which is different with the common immunity adjustment mechanism to demonstrate the broad antivirotic medicine development prospect.Therefore,the paper takes konjac glucomannan as raw material which is rich in the west of our country as special resources.On the basis of sulfated,we purify HOGS by the ion exchange resin chromatographic analysis and appraise its purity respectively by glucosan gelatin chromatographic analysis,acetyl cellulose thin film electrophoresis and highly effective liquid chromatography.The structure attribute was studied by modem analysis methods and technology including gas chromatography(GC),gelatin seepage chromatograph (GPC),Fourier infrared spectrum(FT-IR),laser Laman spectrum(RAMAN), photoelectron power spectrum(XPS) and high resolution ¹H-nuclear magnetic resonance spectrum(1H-NMR).The toxicity of host cell including Hela,HepG2-2.2.15 and MDCK cell caused by HOGS was determined through the MTT method to revelate the most greatly non-toxic density of HOGS.We carry on the anti-viral characteristic research by grouping experiment according to four kind of different function ways,including direct inhibition on virus package of membrane protein,inhibition on viruse duplication and expression,inhibition on virus adsorpting cell and blocking the viral acceptor(protect cell not to be invaded by virus).The best function way of CVB₃ virus and polio virus was determined by cell pathological change(CPE) observation and cell survival percentage determination.The inhibition on hepatitis B virus antigen expression and DNA duplication of HOGS was studied respectively through determing the content of hepatitis B surface antigen(HBsAg),e antigen(HBeAg) by ELISA method and examinating the content of DNA by fluorescence quota PCR methods.The action mechanism about inhibition on the effect price of blood congeal and virus release,proliferation,duplication was studied respectively through determing activity of viral surface neuraminic acid enzyme and examinating the content of by fluorescence RT-PCR.The main result of research is as follows:(1) Prepared HOGS was purified by the ion exchange resin to separate sulphated sample with non-sulphated sample after ethyl alcohol precipitation twice.The result demonstrated that the peak of ionic exchange elutes which the curve appears was a sole peak,indicating that we had purified HOGS as one component.The purity appraisal result from glucosan gelatin chromatographic analysis showed that the peak which the curve appears was a sole symmetrical peak,indicating that HOGS had the same molecular size and shape as one component;The purity appraisal result from acetyl cellulose thin film electrophoresis showed a sole band,indicating that the sample was one component;The high performance liquid chromatography result showed that the peak was a sole one after ionic exchange purification,indicating that the foreign inclusion of HOGS was lower.The purity was 96.39±0.52%by area normalization method.The separation,purification and the purity appraisal indicated that HOGS had achieved high-purity,which layed the foundation for the following research.(2) The structure of sulphated polysaccharide remarkably affected its anti-viral activity.From the gas phase chromatography analysis,we know that HOGS was composed of mannose and glucose which was polymerized.The ratio of mannose and glucose was approximately 1.8:1.The molecular weight determination result by gelatin seepage chromatograph showed normal distribution,Mw/Mn=1.01,Mp 1,608D,Mw 1,595D,Mn 1,572D,on 3.62×104,and the relative molecular mass distribution of HOGS was narrow.The characteristic absorption peak of the sulfuric acid groups and bases analyzed by FT-IR and RAMAN showed that HOGS was successfully sulphated.The XPS analysis demonstrated that HOGS had introduced the sulfur element,the comparative quality content of which of purified HOGS was 58.79%.Its S element content enhanced approximately 40%compared with non-purified HOGS According to binding energy data analysis,we regarded S atom exist possibly by -OSO₃-form through carrying on the curve fitting to S₂P.The ¹H-NMR analysis demonstrated that chemistry displacement drifted indicating the molecular had already linked sulphate group on C₂,C₃ position.Moreover the methyl hydrogen chemistry displacement had changed,indicating the C₆ position had connected sulfuric acid groups and bases.(3) Results from the experiment of blocking CVB3 virus to invade cells indicated that HOGS can remarkably reduce the quantity and degree of pathological cell change when infected with CVB3 after function to cells for 4h/24h demonstrating HOGS had protected the HeLa cell not to be infected by virus.Also along with HOGS density increasing,its protective function enhanced.The cell survival percentage result showed that the group of blocking the virus to invade cells(4h):IC₅₀ was 0.1483mg/mL,TI was 4.50;(24h):IC₅₀ was 0.1517mg/mL,TI was 4.40.The group of direct inhibition on virus(1h):IC₅₀ was 0.2338 mg/mL,TI was 2.86;(3h):IC₅₀ was 0.2242 mg/mL,TI was 2.97;(5h):IC₅₀ was 0.2445mg/mL,TI was 2.73.Results showed that TI of HOGS blocking CVB₃ to invade cells was higher,and the inhibition effect was better.We preliminary inference HOGS possibly competted operating on the virus acceptor on the cell surface.(4) Results from the experiment of inhibition on duplication of PV virus indicated that HOGS had obvious inhibitory action to the degree and quantity of cell pathological change when add HOGS after virus affecting for 2 hours,even if under lower density,the cell pathological change also can be inhibited effectively,which demonstrated the main function way of HOGS inhibiting virus possibly was inhibitory action on its RNA release and biosynthesis.Chose two groups of better effect,namely inhibition on PV duplication and protection to cell,comparing its inhibitory effect on cell pathological change,determing the smallest effective concentration:CPE result showed that cells pathological change of group of inhibition on PV duplication obviously was less than another group,further explaining that the main function way of HOGS anti- PV virus was inhibitory action on its RNA release and biosynthesis and affecting virus's multiplication in cells.The smallest effective concentration was 0.0095mg/mL.(5) The inhibitory action of HOGS to HBV antigen(HBsAg,HBeAg) expressing demonstrated that after affecting for 120h,inhibitory rate on HBsAg and HBsAg of HOGS of the most greatly non-toxic density was respectively 65.27±5.04%,14.76±3.01%. The inbibitory effect on HBsAg expression was better than that of IFN a-2b,50% inhibiting concentration EC50 was 0.2675mg/mL,TI was 5.52.The inbibitory effect on HBeAg expression was less than that of IFN a -2b.The inhibitory action to HBV-DNA duplication of HOGS demonstrated that inhibitory rate of the most greatly non-toxic density HOGS on the extracellular and inxtracellular HBV-DNA was 37.04±3.11%and 59.25±6.32%after affecting for 144h, which explaining that HOGS had the certain inhibitory action to the duplication and release of HBV-DNA,especially of inxtracellular HBV-DNA.We conclude that HOGS possible direct act(copy level) on HBV-DNA,or disturbed protein synthesis process including enzyme and antigen.(6) Result of HOGS grouping inhibition on blood congeal activity of IAV virus (blood congeal titer) demonstrated that when added on HOGS after virus affecting cell for 8h,the blood congeal titer remarkably reduced,which declined from 128 to 4 of most greatly non-toxic density HOGS,explaining HOGS could obviously reduce release, proliferation and duplication process of virus infection.When virus affecting cell after HOGS mixed with virus for 2h,the blood congeal titer declined from 320 to 24 of most greatly non-toxic density HOGS,explaining that HOGS possibly reduced virus through direct inhibition on virus package of membrane protein to enter the cell,release, proliferate,causeing new viral pellet to have self-agglutination,thus reduced virus's quantity.Chose two groups above,comparing anti-virus activity according to CPE observation.The smallest effective concentration when added HOGS after virus infecting was 0.0625mn/mL;The smallest effective concentration when infecting cells after HOGS mixed with virus was 0.03125mg/mL.Result of inhibition on neuraminic acid enzyme activity on virus surface of HOGS showed that along with the density increasing,the highest inhibitory rate might reach 53±2.47%,explaining one of action mechanisms of in vitro inhibition was through blocking release and diffusion process of virus after entering the host cell to cause virus pellet to have self-agglutination.Result of inhibition on virus RNA duplication showed that under function of the most greatly non-toxic density HOGS,inhibitory rate to the total RNA synthesis achieved above 59.25±6.32%,indicating that another action mechanism was inhibition on RNA process after the viral adsorption and invasion to host cell.From the research about in vitro anti-viral characteristic of HOGS to different type of virus,HOGS could inhibit virus activity by 3 kinds of ways,namely operating on the virus acceptor of cell surface to block virus invading cells;Directly action to protein on virus surface to inhibit virus release,proliferation after entering the cell;Inhibition on duplication,translation process of new virus pellet.Thus HOGS had good development prospect to be taken as broad spectrum anti-virus medicine.