【作者】 江筠；

【导师】 刘方；

【作者基本信息】 南开大学 ， 微生物学， 2010， 博士

【摘要】 天然产物一直是药物和药物先导化合物的重要来源。已有研究表明,抗氧化、抗HIV-1和免疫调节三者之间存在着密切的关系。本研究首先以抗氧化为筛选指标,以人们日常食用的莲藕为研究对象,用大孔树脂NKA从莲藕粗提物L中分离到三个组分L1、L2和L3,活性产物在L2中富集。进一步采用葡聚糖凝胶LH-20从L2中分离到7个组分,分别命名为L2a-L2g,其中L2c和L2f显示出较高的抗氧化活性。经高效液相色谱从L2c中纯化出单一组分L2c-3,从L2f中纯化出单一组分L2f-2和L2f-3。通过质谱、红外光谱、核磁共振等方法确定L2c-3为色氨酸(tryptophan)、L2f-2为没食子儿茶素[(+/-)-gallocatechin]、L2f-3为儿茶素[(-)-catechin]。反应浓度为250μg/ml时,L2c-3、L2f-2和L2f-3对红细胞氧化溶血的抑制率达75.39%、80.26%和79.09%。L2f-2和L2f-3在此浓度下,抑制脂质过氧化率达75.42%和74.48%,显示出极强的抗氧化活性。此外,L2f-2和L2f-3对超氧阴离子具有较强的清除活性(IC50分别为126.2 gM和204.8μM),同时它们还可以有效清除羟基自由基(其ICso分别为102.0μM和234.4μM)。采用50%(v/v)和67%(v/v)的乙醇对莲藕粗品L进行分步沉淀,得到两个组分LA和LB,其中LB的抗氧化活性远高于LA。用葡聚糖凝胶Sephadex G75从LB中分离到两个单峰LBl和LB2,其中LB2为活性成分。用凝胶层析确定LB2的分子量约为18.8 kD。经化学分析,LB2糖含量为40.7%、蛋白含量33.4%,由此推测为多糖蛋白复合物。通过多糖水解和单糖PMP衍生的方法测定LB2中的多糖部分是由甘露糖、鼠李糖、葡萄糖、半乳糖和木糖,按摩尔比2：8：7：8：1组成。红外光谱分析表明LB2是一种同时具有α-和β-吡喃型单糖以及α-呋喃型单糖的硫酸酯多糖。生物活性实验结果显示,LB2的抗氧化活性比粗品提高了3倍,其对红细胞氧化溶血的抑制率为72.2%(125μg/ml),对脂质过氧化的抑制率为83.2%(250μg/ml)。HIV-1整合酶(IN)是逆转录病毒特有的酶,在人体内没有同源类似物,是抗HIV-1药物研究的重要靶点,但目前被FDA批准上市的32种抗HIV-1药物中仅有一种是IN抑制剂。IN抑制剂研究缓慢的重要原因之一就是筛选模型的缺乏。本研究通过创新性设计,构建了以HIV-1 IN 3’切割活性为靶点的新型N抑制剂体外筛选模型和胞内双质粒IN抑制剂筛选模型。在体外筛选模型中,首先通过双重PCR技术在IN中引入两个突变位点F185K和C280S,在大肠杆菌中实现IN的可溶性表达,利用NTA-Ni2+亲和柱从菌体裂解液上清中纯化带His标签的IN,用Western Blot检测确认。重组IN不依赖其他细胞蛋白即可对底物质粒pLTR进行3’切割使其线性化。通过DNA电泳,用凝胶成像系统分析不同构性质粒光密度的变化,计算样品对IN的抑制率。用此模型检测,莲藕中大分子LB2抑制IN的ICso为5.28μM,显示出极强的抗IN作用。同时莲藕中小分子L2f-2(IC50：412.45μM)和L2f-3(IC50：432.40μM)也具有比IN抑制剂标准品Raltegravir (IC50:686.17μM)更强的抗IN活性。在胞内双质粒IN抑制剂筛选模型中,通过利用96孔板-两步生长法测定样品对双质粒菌生长的抑制作用,从而间接显示抗IN活性。用此模型检测,LB2在反应浓度为10μM时对IN的抑制率为47.48%,高于标准品Raltegravir。L2f-3在反应浓度为100μM时,也显示出较高的对IN的抑制作用,其抑制率为48.78%。用MTT法检测样品毒性发现,在相同反应浓度下,Raltergravir处理的正常细胞存活率不到20%;而L2f-3和LB2处理的细胞存活率高达100%,未显示出任何细胞毒性。这两个模型具有操作简单、成本低、检测快速、重复性好等优点,适合大规模筛选IN抑制剂。本研究首次证明了莲藕活性成分的抗HIV-1多效性。HIV-1逆转录酶(RT)和蛋白酶(PR)也是抗艾滋药物研究的有效靶点。L2f-2、L2f-3和LB2均可以有效抑制RT活性,其IC50分别为10.41μM、9.58μM和33.70μM。同时,在反应浓度为70μg/ml时,L2f-2和L2f-3对PR的抑制率达17.61%和15.66%,而市售蛋白酶抑制剂标准品pepstatin A的抑制率仅为7.36%。细胞免疫功能的恢复作为免疫重建的重要组成部分,在对HIV-1感染者的治疗中起着重要的作用,而细胞因子是衡量细胞免疫功能的重要指标。本研究通过Real-time PCR的方法检测莲藕活性成分对重要细胞因子的影响。结果显示,L2f-2和L2f-3可显著上调Bal b/c小鼠脾细胞IL-2和IL-6基因表达水平,同时下调IL-10的表达水平。这种调节可提高细胞免疫应答水平,有利于细胞抵御病毒感染。L2f-3还可以通过下调TNF-a的表达和抑制Tat对病毒LTR的激活直接抑制HIV-1的复制。LB2可以上调Bal b/c小鼠腹腔巨噬细胞IL-2、IL-4和IL-10的表达,从整体上激活免疫系统,有利于改善HIV-1感染后细胞因子普遍降低的状态,维持机体免疫平衡。LB2对TNF-a表达的下调作用使其具有可直接抑制HIV-1复制的能力。本研究首次构建了两种新型HIV-1 IN抑制剂筛选模型,并从抗氧化、抑制HIV-1关键酶、细胞因子调节三方面研究了莲藕中活性成分抗HIV-1作用的机制。与化学合成药物相比,这些天然产物具有更高的抗HIV-1活性和更低的细胞毒性,其抗HIV-1作用的多效性克服了化学合成药物易因病毒株变异而失效的缺点,为开发新型抗HIV-1药物奠定了基础,具有很好的应用前景。更多还原

【Abstract】 Natural product is an important source for drug. Previous researches indicated the close relationship among oxidative damage, immune imbalance and AIDS which suggested that antioxidants might play an important role in the treatment of AIDS. In this study, antioxidation was the breakthrough point to select natural ingredients from Nelumbo nucifera Gaertn. The macroporous resin named NKA was used for chromatography of the aqueous crude extract L. Fraction L2 showed the highest antioxidative activity in the three ingredients named L1, L2 and L3 from L. Chromatographed on Sephadex LH-20, L2 was separated into seven fractions named L2a to L2g. Antioxidative ingredients L2c and L2f were collected from them. High Performance Liquid Chromatography was applied to purify L2c-3 from L2c, while L2f-2 and L2f-3 from L2f. Identified by MS, FTIR and NMR, L2c-3 was tryptophan, L2f-2 was (+/-)-gallocatechin and L2f-3 was (-)-catechin. The inhibitions of L2c-3, L2f-2 and L2f-3 were 7539%,80.26% and 79.09% on erythrocyte hemolysis at 250μg/ml. L2f-2 and L2f-3 exhibited strong antioxidative activities on lipid peroxidation. The inhibitory rates were 75.42% and 74.48% at 250μg/ml. Furthermore, L2f-2 could eliminate superoxide anion and hydroxy radical with the IC50 values of 126.2μM and 102.0μM, while the IC50 values of L2f-3 were 204.8μM and 234.4μM. The crude aqueous extract L was precipitated with 50%(v/v) and then 67%(v/v) ethanol to collect fractions LA and LB. Antioxidative fraction LB2 was extracted from LB on Sephadex-G75 column. LB2 is a polysaccharide-protein complex with a molecular weight of 18.8 kDa. HPLC data showed that LB2 was composed of mannose, rhamnose, glucose, galactose and xylose with a mol ratio 2:8:7:8:1. LB2 was identified as a polysaccharide sulfate containingα/β-pyranoid and a-furanoid monose by FTIR. The antioxidative capability of LB2 on erythrocyte hemolysis assay was 72.2%(125μg/ml) and 83.2%(250μg/ml) on lipid peroxidation.HIV-1 integrase (IN) is a critical enzyme of HIV-1. It is an ideal target for anti-HIV therapy because previous researches have not found any functional integrase analogue in human bodies. But the agents against integrase were developed so slow that there was only one drug against IN named Raltegravir approved by FDA in 32 anti-HIV drugs. Proper screening model is the most important of drug development. In the present studies, two novel drug screening models named 3’-process model and double-plasmid model were established for IN inhibition screening. Two mutant sites F185K and C280S were introduced into cDNA of IN, which could improve the solubility of IN. In the 3’-process model, soluble IN was purified by NTA-Ni2+ from the lysate of pET-IN (BL21) induced by IPTG. Recombinant IN was identified by Western Blot. It showed high 3’-process activity on substrate plasmid pLTRs to make them linearization in vitro. The ratio of linear DNA to total DNA reflected the activity of IN. LB2 exhibited the highest inhibitory activity on IN with an IC50 value of 5.28μM. The inhibitory activities of L2f-2 (IC50: 412.45μM) and L2f-3 (IC50:432.40μM) on IN were higher than that of Raltegravir (IC50:686.17μM). Double-plasmid screening system contained two recombinant plasmids named pET-IN and pLTR in E.coli BL21. IN expressed by pET-IN acted on pLTR in bacteria which could inhibit their growth and samples with potential IN inhibition could recover the growth of bacteria. LB2 showed higher inhibitory rate (47.48%) on IN than Raltegravir (42.92%) at 10μM. The inhibitory rate of L2f-3 on IN was 48.78% at 100μM. The survival rates of splenic cells incubated with 1mM L2f-3 and LB2 were 100% when tested with MTT method, which exhibited that L2f-3 and LB2 did not show any cytotoxicity on normal cells. Raltegravir showed higher cytotoxicity than L2f-3 and LB2. Only 20% cells survived after incubated with 1mM Raltegravir.It was the first time that multiple effects of compounds from Nelumbo nucifera Gaertn were studied. HIV-1 reverse transcriptase (RT) and protease (PR) are other two critical enzymes of HIV-1. L2f-2, L2f-3 and LB2 could strongly inhibit RT with the IC50 values of 10.41μM,9.58μM and 33.70μM. The inhibitory rates of L2f-2 and L2f-3 on PR were 17.61% and 15.66% at 70μg/ml, while the inhibitory rate of Pepstatin A (standard PR inhibition) was only 7.36%. Immune reconstitution of HIV-infected patients was the most important part of immune function recovery. Cytokines were critical regulatory factors and played important roles in immune system. Real-time PCR was used to test the expressive levels of cytokines. The results demonstrated that both L2f-2 and L2f-3 could up-regulate the gene expressive levels of IL-2 and IL-6, while down-regulate IL-10. These regulations would be helpful to increase the organic immunity and balance immune system. Moreover, L2f-3 could inhibit HIV-1 directly by down-regulating TNF-αand inhibiting the activation of Tat on LTR. LB2 exhibited positive regulation on IL-2, IL-4 and IL-10. It could improve the inhibitory state of cytokines in HIV-1 sufferer and balance the immune system.In the present researches, two novel IN inhibitor screening models were established for quick and large scale drug screening. Natural products from Nelumbo nucifera Gaertn were examined in antioxidation, inhibition on the key enzymes of HIV-1 and immune regulation. Most chemically synthetic HIV-1 inhibitor could not deal with mutant virus. Natural products with multiple anti HIV-1 effects would overcome this disadvantage. Therefore, this research is significant for anti HIV-1 drug development and application of natural products to HIV-1 therapy.更多还原