【作者】 夏仁飞；

【导师】 周迎春；

【作者基本信息】 南方医科大学 ， 中西医结合临床内科学， 2009， 硕士

【摘要】 研究背景艾滋病（acquired immunodeficiency syndrome,AIDS）又称获得性免疫缺陷综合征,是由人类免疫缺陷病毒（human immunodeficiency virus,HIV）引起的传染性疾病。自从20世纪80年代首次报告艾滋病临床病例以来,艾滋病在世界迅速蔓延,目前在全球已呈爆发性流行趋势,对人口平均寿命和世界经济发展产生了巨大的影响,成为21世纪人类面临的最重大公共健康问题。艾滋病已经成为人类前所未有的最具毁灭性的疾病。至今,发达国家已投入了大量的资金及人力资源,对HIV本身以及抗HIV的药物进行研究。目前被批准用于临床的治疗AIDS的药物主要分为核苷类逆转录酶抑制剂（nucleocide reversetranscriptase inhibitors,NRTI）、非核苷类逆转录酶抑制剂（Non-Nucleoside ReverseTranscriptase Inhibitors,NNRTI）、蛋白酶抑制剂（Protease Inhibitor,PI）、整合酶抑制剂和进入抑制剂5大类,这些药物主要是通过和底物竞争结合酶或结合到酶上使之变性,抑制酶的活性,从而破坏HIV的生活周期,达到抑制病毒复制的目的。这些药物可以在一定程度上延缓病人病情的恶化,但并不能彻底清除患者体内的病毒。大部分病人一直生活在病毒低水平复制的状态中,长期用药患者将产生抗药性,并且一旦停止用药,病人体内病毒水平很快又恢复到甚至超过用药前的水平。在HIV感染过程中,首先是HIV包被膜与靶细胞的细胞膜发生融合。HIV与靶细胞融合主要由包膜糖蛋白gp160（由包膜表面糖蛋白gp120和跨膜糖蛋白gp41非共价结合而成）介导。gp120与靶细胞上的CD4分子和辅助受体（趋化因子受体CCR5或CXCR4等）依次结合,导致gp41的构型发生改变,形成6股α-螺旋束核心结构,将病毒包膜与靶细胞膜拉近并发生融合,完成病毒进入宿主细胞的感染过程。因此,抑制融合过程中的任何一个环节,就能抑制HIV进入靶细胞,从而预防和治疗HIV的感染。gp120、gp41、CD4、趋化因子受体（CXCR4或CCR5等）均可作为HIV进入抑制剂的药物作用靶点。由于化学药物在临床治疗中疗效不甚理想,而且副作用较大,于是我国医药工作者从祖国中医药宝库中寻求中医中药对艾滋病的治疗,从而推动世界各国从植物中筛查预防与治疗艾滋病的药物研究。在对中草药有效成分的分析研究中发现,一些中草药的多糖、皂苷、生物碱、黄酮以及蛋白质类物质可以抑制HIV复制,具有很好的抗HIV病毒活性。黄酮类（flavonoids）是一大类天然存在的具有抗病毒活性的化合物,研究发现其显示出对HIV良好的抑制活性。皂苷（saponin）是天然产物中一类重要的化学成分。皂苷具有多种多样且相当重要的药理活性和生物活性,如抗肿瘤、抗真菌、防治心血管疾病、降血糖和免疫调节等,从而显示出广阔的应用前景。挥发油也称精油（volatile oil）,是存在于植物体中的一类具有挥发性、可随水蒸气蒸馏出来的油状液体的总称。挥发油在植物界分布极广,作为中药使用的植物就有数百种含有挥发油,尤其在菊科、芸香科、伞形科、姜科等科属中较为常见。挥发油具有抗肿瘤、抗菌、抗过敏和抗病毒作用,并具有一定的酶抑制活性。本实验主要研究从番石榴叶中提取的三种成分皂苷、黄酮及挥发油抑制HIV包膜蛋白亚基gp41六股螺旋束形成的作用。目的从番石榴叶中提取出皂苷、黄酮及挥发油,研究三种提取物对HIV包膜蛋白亚基gp41六股螺旋束的抑制作用,为寻求新的抗HIV病毒进入抑制剂提供实验依据。方法1.实验用HeLa细胞传代按1×10⁵个/ml的密度用10%完全培养基接种于96孔培养板中,每孔种植100μl。继续培养24h后,细胞随机（随机方法参照文献[21]）分组:对照组（control）及其他不同剂量组（皂苷、黄酮分为1 mg/ml、0.5 mg/ml、0.25 mg/ml、0.125 mg/ml、0.0625 mg/ml、0.03 125 mg/ml;挥发油分为50μg/ml、25μg/ml、10μg/ml、1μg/ml、0.1μg/ml）,每组设4孔。用RPMI-1640基础培养基配制不同浓度药物,每孔加入100μl,干预24h后,MTT法测OD值,求出其半数细胞毒性浓度(median toxic concentration,TC₅₀)。2.予TC₅₀以下的不同浓度的皂苷、黄酮及挥发油,用夹心酶联免疫测定法（ELISA）检测番石榴叶提取物对HIV包膜蛋白亚基gp41六股螺旋束结构形成的抑制作用。N36和C34是从HIV gp41的NHR和CRH区衍生出来的多肽,二者在体外能形成类似gp41 NHR和CRH结合的螺旋结构。因此,抗gp41六螺旋束核心结构的活性可以用抑制N36和C34结合的作用来测定。3.统计方法:运用SPSS13.0统计软件进行统计,所有数据均以均数±标准差（（?）±S）表示。各组MTT比色法结果比较采用单因素方差分析（One-wayANOVA）。组间均数两两比较,若方差齐性,用LSD（Least Significant Difference）法;若方差不齐性,用Dunnett’s T3方法。P＜0.05表示有显著差异。结果1.MTT法测得各个不同样品浓度作用后的细胞存活率,药物的半数细胞毒性浓度(TC₅₀)用PHARM/PCS-VERSION4软件计算得出:番石榴叶的三种提取物皂苷、黄酮、挥发油的半数细胞毒性浓度(TC₅₀)分别为137.6μg/ml,71.62μg/ml,23.63μg/ml。皂苷、黄酮在最大浓度（1mg/ml）时细胞存活率分别为17.28%、14.53%,在最低浓度（0.03125mg/ml）时细胞存活率分别为93.32%、85.64%。挥发油在最大浓度（50μg/ml）和最低浓度（0.1μg/ml）时细胞存活率分别为25.39%、82.21%。2.应用基于抗原抗体反应的夹心ELISA法对皂苷、黄酮及挥发油三种化合物进行了筛选,在终浓度为25μg/ml时皂苷与黄酮对HIV gp41六螺旋束结构形成有明显抑制作用,二者的抑制率都大于50%,而挥发油在终浓度为25μg/ml时的抑制率较低,低于50%,而且皂苷和黄酮组的抑制率显著高于挥发油组。挥发油组、皂苷组与黄酮组三组均数间都有显著差异（P＜0.0001）。组间两两比较显示,挥发油组与皂苷组、黄酮组比较均有显著差异（P＜0.001）,皂苷组与黄酮组相比无显著性差异（P=0.507）。故在初步筛选后弃掉挥发油,皂苷和黄酮进一步做梯度稀释计算半数抑制浓度(IC₅₀)。皂苷、黄酮稀释为25μg/ml、15μg/ml、2.5μg/ml、1μg/ml、0.25μg/ml五个浓度梯度,二者均在25μg/ml的时候抑制大部分gp41六螺旋束结构的形成,皂苷在2.5μg/ml时以及黄酮在1μg/ml时能够抑制部分六螺旋束结构的形成。二者相比较,黄酮的抑制作用比皂苷稍强。通过计算得皂苷和黄酮的半数抑制浓度(IC₅₀)分别为5.65μg/ml、0.87μg/ml。这说明了皂苷和黄酮类化合物能够作用于HIV gp41抑制其形成六螺旋束核心结构,可能也在抑制HIV与靶细胞融合中发挥作用。结论通过对提取的皂苷、黄酮类化合物及挥发油的筛选,发现皂苷及黄酮具有良好的抑制HIV gp41六螺旋束结构形成的作用。本研究为从中草药等天然产物来源寻找HIV进入抑制剂提供了借鉴和思路。更多还原

【Abstract】 BackgroundAIDS(acquired immunodeficiency syndrome) are communicable diseases caused by human immunodeficiency virus(HIV).Since the initial report of clinical cases of AIDS at 80’s the 20th century,the diseases have spread rapidly allover the world, which turned out to be a global outbreak trend recently.It has been put a dramatic impact upon the average life expectancy of the population and the world’s economic development,and become the most major public health problem in the 21st century. AIDS has become the most destructive and unprecedented disease.So far,developed countries have invested substantial funds and human resources in the research of HIV and anti-HIV drugs.At present,drugs for the clinical treatment of AIDS are reverse transriptase inhibitor and proteinase inhibitor.By binding on the enzyme or competitive binding on the enzyme with the substrate,these drugs make enzyme denatured and inhibit activity of enzyme,and thus destroy HIV’s life cycle and inhibit viral replication.To some extent,these drugs can delay the deterioration of patients,but it can not completely wipe out the virus in patients.A majority of patients have been living at a low level virus replication status.Patients with long-term use these drugs will result in resistance,and if the medication stopped, virus levels in patients restore soon,even higher than the level before treatment. In the course of HIV infection,the first procedure is the fusion of HIV envelope membrane and target cells.This fusion is mainly mediated by gp160.Gp 120 combine CD4 on target cells with supporting elements receptor(chemokine receptor CCR5 or CXCR4,etc.) by turns.Then it resulted in a change in the structure of gp41,and the formation of 6 shares the coreα-helix bundle structure.The 6 shares the coreα-helix bundle structure narrowed the virus envelope with the target cell membrane,and induces fusion.Therefore,inhibiting the process of integration of any one link,you can suppress HIV to enter target cells and thus the prevention and treatment of HIV infection.Gp120,gp41,CD4,chemokine receptor(CXCR4 or CCR5,etc.) can serve as HIV entry inhibitors target the drug.Chemical drugs for AIDS are not efficient enough and have lots of side effects. So many medicine practitioners seek treatments from Chinese traditional medicine for AIDS,so as to promote the research of plants,which used to screen,prevent and treat AIDS.In the analysis of the active ingredients of Chinese herbal medicine,it finds that some polysaccharides,saponins,alkaloids,flavonoids and protein in some Chinese herbs can inhibit HIV replication material,and show good activity against HIV virus.Flavonoids are a large class of compounds which show anti-virus activity.Some studies find that flavonoids show good inhibitory activity against HIV.Saponins are a essential class of natural products in the chemical composition.Saponins have diverse and important pharmacological activity and biological activity,such as anti-tumor, anti-fungal,prevention and treatment of cardiovascular disease,lowering blood sugar and immune regulation and thus show a broad application prospects.Volatile oil,also known as essential oil,are a class of general term of volatile oil-like liquid,which can be distilled from the plants.Distribution of volatile oil in a wide range of plants, hundreds of plants used as traditional Chinese medicine contain volatile oil, particularly in the Compositae,Rutaceae,Umbelliferae,Zingiberaceae.Volatile oil show anti-tumor,anti-bacterial,anti-allergy and anti-viral effect,and may have enzyme inhibitory activity.In the experiments,we extracted flavonoids,saponins and volatile oil from Psidium Guajava Leaves,and studied their inhibitory activity on the HIV gp41 six-helix bundle formation.ObjectivesFlavonoids,saponins and volatile oil isolated from the Psidium Guajava Leaves, were used as a compound library to screen and identify compounds which can effectively inhibit the formation of HIV gp41 core structure,the six-helix bundle structure.So as to provide experimental basis for searching a new efficacious drug for treatment of AIDS.Method1.HeLa Cells were transferred of culture into the 96-well culture plate by the density of 1×10~5/ml,100μl per well.After cells are cultivated 24 hours,the different groups treated with different drugs which were disposed by RPMI-1640 medium: control group and other group of different concentration(flavonoids and saponins are divided into 1mg/ml、0.5mg/ml、250μg/ml、125μg/ml、62.5μg/ml、31.25μg/ml, volatile oil are divided into 50μ/ml、25μg/ml、10μg/ml、1μg/ml、0.1μg/ml).Each group included 4 wells,and 100μl different drugs were added into each well.Being incubated for 24 hours,the cells were detected the optical density which reacts the quantity of living cells by technology of MTT,and get the median toxic concentration (TC50).2.The inhibitory activity of those compounds(saponins,flavonoids and volatile oil) on HIV gp41 six-helix bundle formation was screened by enzyme linked immunosorbent assay(ELISA).Peptide C34 derived from HIV gp41 CHR region and N36 from NHR region,can form a similar helix bundle in vitro like the six-helix bundle formed by CHR and NHR of gp41.Therefore,the inhibitory activity of HIV gp41 six-helix bundle can be determined by inhibiting the interaction of C34 with N36.3.Statistical Analysis:All data used SPSS 13.0 statistical analysis software. Measurement data are expressed as mean±standard deviation((?)±S).Differences among groups were analyzed by One-way ANOVA.LSD(Least Significant Difference)method should be used in the case of homogeneity of variance;otherwise Dunnett’s T3 methods should be used in the case of variance nonhomogeneity. P＜0.05 was considered statistically significant.Result1.MTT method measured the cell survival rate,and the relationship between the concentration of the samples is calculated by the PHARM/PCS-VERSION4 software. By calculated we find the TC50 of saponin,flavonoids and volatile oil on HeLa cells is137.6μg/ml,71.62μg/ml,23.63μg/ml respectively.When saponins and flavonoids are at the greatest concentration(1mg/ml) the cell survival rate was 17.28%and 14.53%,at the lowest concentration(0.03125mg/ml) the cell survival rate was 93.32%and 85.64%.When the concentration of volatile oil is 50μg/ml and 0.1μg/ml the cell survival rate was 25.39%and 82.21%,respectively.2.Based on the antigen-antibody reaction of the sandwich ELISA method, saponins,flavonoids and volatile oil were screened at final concentration of 25μg/ml. It showed that saponin and flavone had obvious inhibitory activity on the formation of HIV gp41 six helix bundle structure,and their inhibition rate were more than 50%.While the volatile oil in the final concentration of 25μg/ml showed lower inhibition rate than saponins and flavonoids,which was lower than 50%.Saponins and flavonoids inhibit group was significantly higher than that of volatile oil group. The differences among volatile oil group and the group of saponins,flavonoids group were significant(P＜0.001).However,saponin and flavonoid group compared with no significant difference(P = 0.507).So,after the initial screening volatile oil was discarded,left saponins and flavonoids to do further calculation of the IC50.Saponins, flavonoids diluted into five concentration,25μg/ml,15μg/ml,2.5μg/ml,1μg/ml, 0.25μg/ml gradient.At the concentration of 25μg/ml both of them inhibited most of the formation of gp41 six helix bundle structure.Saponins in the concentration of 2.5μg/ml and flavonoids in the concentration of 1μg/ml at some time be able to suppress six helical bundle structure.Compared to the inhibitory activity on the formation of HIV gp41 six helix bundle structure,flavonoids slightly stronger than the saponin.Through calculated the IC50 of saponins and flavonoids inhibitory concentration were 5.65μg/ml,0.87μg/ml.It showed that saponins and flavonoids could target HIV gp41 and inhibit the formation of the core six-helix bundle structure, might also play a role in blocking HIV fusion with target cells.ConclusionWe had screened saponins,flavonoids and volatile oil isolated from the Psidium Guajava Leaves on the effects of inhibiting the formation of HIV gp41 six-helix bundle.Saponins and flavonoids showed favourable inhibitory activities.Thy study shows a light on how to find HIV fusion inhibitors from Chinese herbs and other natural resourses.更多还原