【作者】 黄皓；

【导师】 干信；

【作者基本信息】 华中农业大学 ， 食品科学， 2008， 博士

【摘要】 随着人们对健康的日益关注,安全、有效、特异的抗病毒生化药物研究成为热点。多糖作为生物活性物质能够提高机体免疫力,并具有低毒和低耐药性等特点,其中,硫酸化多糖又不同于一般多糖的免疫调节机制,而是通过其硫酸聚阴离子对病毒感染细胞进行抑制作用发挥更强的抗病毒活性,显示了广阔的抗病毒药物开发前景。为此,本论文以我国西部丰富的特产资源魔芋葡甘聚糖（KonjacGlucomannan,KGM）为原料,在已有的丙酯和硫酸酯化基础上,采用离子交换树脂层析对制备得到的魔芋葡甘低聚糖醛酸丙酯硫酸酯（Hydroxylpropyl Oligo-konjacGlucomannannuroate Sulfate,HOGS）分离纯化,并运用葡聚糖凝胶层析、醋酸纤维素薄膜电泳及高效液相色谱鉴定其纯度。采用气相色谱（GC）、凝胶渗透色谱（GPC）、傅立叶红外光谱（FT-IR）、激光拉曼光谱（RAMAN）、光电子能谱（XPS）和高分辨¹H核磁共振波谱（¹H-NMR）等现代分析手段和技术,对其进行了结构表征。通过MTT法测定HOGS的各种病毒研究对象的宿主细胞Hela、Vero、HepG₂-2.2.15、MDCK的细胞毒性,确定其最大无毒浓度。按直接抑制病毒包膜蛋白、抑制病毒在胞内复制和表达、抑制病毒吸附细胞和阻断病毒受体（保护细胞不受病毒侵染）四种不同作用方式分组进行抗病毒特性研究。通过细胞病变（CPE）观察、MTT法测定细胞存活率等方法确定HOGS抗柯萨奇3型病毒、脊髓灰质炎I型病毒的作用特性;通过ELASA法测定乙肝表面抗原（HBsAg）、e抗原（HBeAg）的含量、荧光定量PCR法检测病毒DNA含量等方法研究HOGS抑制乙肝病毒抗原表达和DNA复制的过程;通过血凝活性、病毒表面神经氨酸酶活性测定及荧光RT-PCR法检测病毒RNA含量等方法研究HOGS抗甲型流感病毒血凝效价,直接抑制病毒进入细胞后释放、扩散和在胞内复制过程的作用机理。研究的主要结果如下:（1）二次硫酸化制备的HOGS经过乙醇二次沉淀后采用离子交换树脂将硫酸化与未硫酸化的样品分离纯化,结果显示,离子交换洗脱曲线出现的糖峰为单一峰,表明硫酸化HOGS组分均一,达到了分离纯化的目的。葡聚糖凝胶层析纯度鉴定结果显示,洗脱曲线出现的糖峰为单一对称峰,可以判断HOGS为分子大小和形状均一的组分;醋酸纤维膜电泳结果为一条单一谱带,样品已达到电泳纯,表明样品是单一组分;高效液相色谱图结果显示,经过离子交换纯化的HOGS得到单一样品峰,表明其杂质含量较低,面积归一化法计算纯度为96.39±0.52%。分离纯化及纯度鉴定表明HOGS已达到较高纯度,为后续研究奠定基础。（2）硫酸化多糖的结构显著影响其抗病毒活性。气相色谱法分析HOGS的魔芋葡甘低聚糖部分由甘露糖、葡萄糖聚合而成,其摩尔比约为:Man:Glu=1.8:1。凝胶渗透色谱分子量分布测定结果显示,色谱图呈正态分布,多分散系数MW/Mn=1.01,HOGS的相对分子质量峰值Mp为1608D,重均相对分子质量MW为1595D,数均相对分子质量Mn为1572D,相对分子质量分布宽度指数σn为3.62×10⁴,制得的HOGS纯品为一种相对分子质量分布很窄的硫酸化多糖。FT-IR和RAMAN结果显示,由于硫酸基团特征吸收峰的出现,分子已接上硫酸基团。XPS分析显示,HOGS已引入硫元素,纯化HOGS的S元素的相对含量质量分数为58.79%,其S元素含量与未纯化HOGS相比,提高约40%,同时,对S₂P进行曲线拟合,根据结合能数据分析,对于S原子仅可能以-OSO₃^-形式存在。¹H-NMR分析显示,糖环的C₂和C₃位在硫酸化修饰后,化学位移发生漂移变化,提示硫酸基可能连接在C₂和C₃位,而且甲基氢的化学位移发生改变,表明在C₆位连接的羟丙基上的羟基可能接有硫酸基团。（3）HOGS阻断CVB₃病毒侵染作用研究结果表明,HOGS分别与细胞作用4h、24h,再加入CVB₃,能够显著减轻细胞病变程度,减少病变细胞数量,对CVB₃侵染细胞起到一定的阻断作用,保护了Hela细胞不受病毒的感染,且随着HOGS浓度的增加,其保护细胞作用增强。根据细胞存活率计算半数抑制浓度IC₅₀及抑制指数TI,阻断病毒侵染组（4h）:IC₅₀为0.1483mg/mL,TI值为4.50;阻断病毒侵染组（24h）:IC₅₀为0.1517mg/mL,TI值为4.40。直接抑制病毒组（1h）:IC₅₀为0.2338 mg/mL,TI值为2.86;直接抑制病毒组（3h）:IC₅₀为0.2242mg/mL,TI值为2.97;直接抑制病毒组（5h）:IC₅₀为0.2445mg/mL,TI值为2.73。结果显示HOGS在阻断CVB₃侵染细胞的作用下,HOGS抗CVB₃的抑制指数TI较高,抑制效果较好。初步推断可能是HOGS竞争结合了细胞表面的柯萨奇病毒受体所致。（4）HOGS抑制PV₁病毒胞内复制作用研究结果表明,PV₁病毒先感染细胞,作用2h后,再加入HOGS,对细胞病变的程度和数量均有明显的抑制作用,即使在较低的浓度下,细胞病变也能受到有效抑制,说明HOGS抑制PV₁的主要作用特性是在PV₁进入细胞后,对其RNA在胞内的释放和生物合成过程的抑制作用。选择分组试验中效果较好的两组,即抑制PV₁胞内复制和保护细胞两组,进一步比较其抑制细胞病变效果,确定最小有效浓度:CPE结果显示,PV₁作用Vero细胞2h后,再加入HOGS一组的细胞病变明显少于另一组,进一步说明HOGS抗PV₁的主要作用特性是通过抑制病毒RNA在胞内的释放和复制过程,影响PV₁在细胞内的增殖,最小有效浓度为0.0095mg/mL。（5）HOGS对HBV抗原（HBsAg、HBeAg）表达的抑制作用显示:作用120h,最大无毒浓度HOGS对HBsAg的抑制率为65.27±5.04%,抑制HBsAg表达效果优于对照药物IFNα-2b,半数有效浓度IC₅₀为0.2675mg/mL,抑制指数TI为5.52。各浓度HOGS对HBeAg的抑制作用不显著。作用120h,最大无毒浓度HOGS抑制率为14.76±3.01%,抑制效果小于IFNα-2b。HOGS对HBV-DNA复制的抑制作用显示:作用144h,最大无毒浓度的HOGS对胞外HBV-DNA的抑制率为37.04±3.11%;作用144h,最大无毒浓度的HOGS对胞内的HBV-DNA的抑制率为59.25±6.32%。说明HOGS对HBV-DNA的复制和释放均具有一定抑制作用,且对胞内DNA复制的抑制效果更显著。提示HOGS抗乙肝病毒的作用特性是抑制了HBV-DNA的复制水平（转录前水平）,或干扰了包括逆转录酶和抗原在内的蛋白质合成及后加工。（6）HOGS分组抑制IAV病毒血凝活性（血凝效价）结果显示:病毒与细胞先作用8h,后加HOGS试验组血凝效价与病毒组比较,显著降低,最大无毒浓度HOGS组的病毒血凝效价由128下降到4,说明HOGS能明显减轻病毒感染细胞后,在胞内释放、扩散和复制的过程。HOGS先与病毒作用2h,再加入细胞试验组在一定程度上降低病毒的血凝效价,与病毒组比较,血凝效价从320降低到24,说明HOGS可能通过直接抑制病毒包膜蛋白来减轻病毒进入细胞后,颗粒的释放、扩散,使新的病毒颗粒产生自我凝集,从而减少病毒的数量。选择上述两组,进一步比较抗病毒活性,根据CPE观察,细胞先感染病毒,再加HOGS一组,最小有效浓度为0.0625mn/mL;HOGS与病毒作用2h后,再感染细胞一组,最小有效浓度为0.03125mg/mL。HOGS抑制流感病毒表面神经氨酸酶活性结果显示,随着浓度的增加,其抑制NA活性增强,最高抑制率可达53±2.47%,说明HOGS体外抑制甲型流感病毒的机理之一是通过对流感病毒进入宿主细胞后在细胞内释放、扩散过程的阻断实现病毒颗粒的自我凝集作用。HOGS抑制病毒RNA复制结果显示:最大无毒浓度HOGS作用下,144h后,对甲型流感病毒在细胞内总RNA合成的抑制率达到59.25±6.32%以上,效果明显,说明HOGS体外抑制甲型流感病毒机理的另一方面是在病毒吸附和侵入宿主细胞后,对病毒在细胞内RNA合成过程的抑制。通过HOGS在体外对不同类型病毒的抗病毒特性研究,证实HOGS能够通过多种方式抑制病毒,其作用特性大致有3种,即与细胞表面的病毒受体相互作用,保护细胞并阻断病毒侵染细胞;直接与病毒的包膜蛋白作用,抑制病毒进入细胞后的释放、扩散;抑制病毒新颗粒在胞内的复制、翻译过程。因而HOGS作为广谱抗病毒药物的开发具有良好的前景。更多还原

【Abstract】 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.更多还原