翅果藤的细胞毒活性和化学成分研究

对翅果藤(Myriopteron extensum (Wight)K.Schum)的甲醇提取物及其不同的极性部位进行的细胞毒活性筛选发现乙酸乙酯、正丁醇部分具有一定的细胞毒活性。首次对其成分的研究分离得到7个化合物,通过波谱分析鉴定为β—谷甾醇,齐墩果酸,α—香树素乙酸酯,β—香树素乙酸酯,羽扇豆烷乙酸酯,乌沙甙元和胡萝卜甙。进一步的活性测试和化学研究仍在进行。     

苏州西山岛产枇杷叶的药材品质分析

枇杷叶为枇杷〔Eriobotrya japonica(Thunb.)Lindl.〕的干燥叶片,为常用中药材之一[1]。目前已从枇杷叶中分离得到三萜酸类、黄酮类及多酚类等多种有效成分[2-3],其中,三萜酸类是枇杷叶的主要药效物质,具有抗炎、降血糖和抗病毒等活性,主要包括齐墩果酸、熊果酸、蔷薇酸和科罗索酸等成分[4],目前已将齐墩果酸和熊果酸作为枇杷叶质量检测标准[1]。大量的分析结果[5-8]表明:不同产地、不同季节、落叶与生长叶和     

白菊木树皮的化学成分及其抗炎活性研究

为了解白菊木[Gochnatia decora(Kurz)A.L.Cabrera]的化学成分,从其树皮甲醇提取物的石油醚萃取部位分离出11个化合物,分别鉴定为木栓酮(1)、木栓醇(2)、齐墩果酸(3)、齐墩果烯(4)、豆甾醇(5)、β-谷甾醇(6)、棕榈酸(7)、正十五烷酸(8)、十四烷酸-1-甘油酯(9)、对羟基苯甲酸甲酯(10)和没食子酸甲酯(11),所有化合物均为首次从白菊木属植物中发现。采用脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7产生一氧化氮(NO)模型和中性粒细胞弹性蛋白酶抑制试验对单体化合物1~4进行了抗炎活性测试,结果表明,化合物1、3和4具有明显的NO抑制活性(IC5010μmol L–1),化合物2、3和4具有一定的中性粒细胞弹性蛋白酶抑制活性。     

入侵植物猫爪藤体外细胞毒活性成分的筛选分离

为了筛选分离入侵植物猫爪藤的细胞毒活性成分,采用MTT法以75%乙醇提取物的不同组分分别处理人肝癌细胞SMMC7721、Bel7402和正常肝细胞Chang Liver,对他们的体外增殖抑制率进行了研究。结果表明,总醇提物的氯仿组分对肝癌细胞表现出明显的体外增殖抑制作用,其次是石油醚组分。从氯仿萃取组分中分离出具有更强细胞毒活性的成分熊果酸。因此,入侵植物猫爪藤具有体外细胞毒活性,熊果酸是其体外细胞毒活性的主要成分之一。     

反相高效液相色谱法测定鹿衔草中熊果酸和齐墩果酸的含量

建立反相高效液相色谱法测定鹿衔草中熊果酸和齐墩果酸含量,并进行方法学考察.采用Kromasil C18分析柱(4.6 mm×250 mm,5 μm);流动相为甲醇-水(88:12);柱温25 ℃;流速0.8 mL/min;检测波长210 nm.熊果酸进样量在0.922～18.44 μg,齐墩果酸进样量在0.506～10.12 μg范围内呈良好线性关系,其中熊果酸平均回收率为98.5%,RSD为2.13%(n=6);齐墩果酸平均回收率为 101.3%,RSD为1.69%(n=6).本方法使鹿衔草中主要成分熊果酸和齐墩果酸达到基线分离,操作简便、结果可靠,可为鹿衔草质量控制和评价提供有效手段.     

滇南美登木中一个新三萜化合物

以抗肿瘤活性追踪法,利用多种分离手段,对滇南美登木中抗肿瘤活性成分进行了系统研究。结果发现三萜部分是主要活性部分,从中分离得到8个三萜类化合物,经波谱分析分别鉴定为:19α-羟基-3-氧代-12-烯-29-齐墩果酸(1)、22β-羟基-3-氧代-12-烯-29-齐墩果酸(2)、3β-羟基-22-氧代-12-烯-29-齐墩果酸(3)、3β-羟基-12-烯-29-齐墩果酸(4)、3β,22β-二羟基-12-烯-29-齐墩果酸(5)、3α,22α-二羟基-12-烯-29-齐墩果酸(6)、3β,22α-二羟基-12-烯-29-齐墩果酸(美登叶酸)(7)、3α,22β-二羟基-12-烯-29-齐墩果酸(8),其中化合物1为新化合物,化合物1、2、4、6对人宫颈癌He La肿瘤细胞系有显著的细胞毒活性。     

血满草化学成分研究

首次对血满草(Sambucus adnataWall)的乙醇提取物的乙酸乙酯,正丁醇溶解部分进行分离纯化,得到4个化合物,经理化方法和光谱分析,分别鉴定为对羟基苯甲酸(-phydroxybenzoic acid,1)、齐墩果酸(oleanolic acid,2)、熊果酸(ursolic acid,3)、3,5-dimethoxy-4-hydroxy-1-O--βD-glucopyranoside(4)。     

天麻蜜环菌粉正己烷提取物的分离和抗神经性炎活性研究

为了探究天麻蜜环菌粉正己烷提取物抗炎作用的物质基础及抗炎机制,本文首先对天麻蜜环菌粉正己烷提取物进行薄层层析和中低压柱层析分离,获得了具有显著抗炎活性的组分5(Fr.5),进一步通过硅胶柱分离,得到主成分较为集中的组分5-1(Fr.5-1),液质联用(LC-MS)及高效液相色谱分析推测Fr.5-1的主成分为大豆黄素;其次,通过脂多糖(LPS)诱导小胶质细胞(BV2)炎症模型考察Fr.5-1不同剂量(10、30、100μg/m L)对炎症因子一氧化氮(NO)和肿瘤坏死因子(TNF-α)表达的影响,发现中高剂量(30、100μg/m L)的Fr.5-1能显著抑制LPS诱导的BV2细胞炎症因子NO及TNF-α的分泌。研究表明天麻蜜环菌粉提取物Fr.5-1具有抗炎活性,其机制可能是通过抑制炎症因子的表达和分泌。     

219份枣种质资源果实三萜酸含量分析

以219份枣种质脆熟期果实为试材,采用超高效液相色谱(UPLC)法检测不同枣种质果实中的山楂酸、桦木酸、齐墩果酸和熊果酸4种三萜酸组分含量,并进行描述统计分析、相关性分析和聚类分析,进而筛选三萜酸含量较高的优特异种质。结果表明:(1)枣果中总三萜酸(TTA)含量的变化范围95.72~737.82μg·g^-1,平均值306.83μg·g^-1,变异系数为29.02%,含量最高的为‘大荔小圆枣’。4种组分以山楂酸含量最高,其次为桦木酸,齐墩果酸和熊果酸含量较低,各组分在不同枣种质中都存在丰富的遗传多样性,其中熊果酸变异系数最高(60.75%)。(2)正态性检验结果表明,各种质的山楂酸、桦木酸和总三萜酸含量服从正态分布,齐墩果酸和熊果酸含量不服从正态分布。(3)相关性分析结果显示,总三萜酸含量与4个组分均呈极显著正相关关系,组分间也存在相关性,山楂酸含量与桦木酸呈极显著正相关关系,桦木酸含量与齐墩果酸呈极显著正相关关系,齐墩果酸含量与熊果酸呈极显著正相关关系。(4)聚类分析将219份枣种质划分为4大类群,分别为熊果酸含量最高的类群,齐墩果酸和熊果酸含量较高类群,各组分中低含量类群以及山楂酸、桦木酸和总三萜酸含量较高类群。(5)筛选出13份各组分或总三萜酸含量较高的优特异种质,为枣三萜酸相关功能产品的开发提供原料选择。     

北山楂果实中三萜酸资源的研究

山楂是具有消食化积、降血压、降血脂及抗癌等多种疗效的中药,同时又是富含营养成分的水果。山楂中的三萜酸是降压活性成分。经分离和鉴定证明英国山楂(Crataegus oxyacatha)等多种山楂中含有熊果酸、齐墩果酸和山楂酸,但从中国山楂属植物中仅分离鉴定出熊果酸。本文从开发利用北山楂果实中的三萜酸资源出发,应用TLC法     

Diverse effects of cyclosporine on hepatitis C virus strain replication

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.     

Development of mouse hepatocyte lines permissive for hepatitis C virus (HCV)

The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV) infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN) induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko) mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1) or interferon A receptor (IFNAR) were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (h)CD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications.     

Generation of a recombinant reporter hepatitis C virus useful for the analyses of virus entry, intra-cellular replication and virion production

The lack of a culture system that efficiently produces progeny virus has hampered hepatitis C virus (HCV) research. Recently, the discovery of a novel HCV isolate JFH1 and its chimeric derivative J6/JFH1 has led to the development of an efficient virus productive culture system. To construct an easy monitoring system for the viral life cycle of HCV, we generated bicistronic luciferase reporter virus genomes based on the JFH1 and J6/JFH1 isolates, respectively. Transfection of the J6/JFH1-based reporter genome to Huh7.5 cells produced significantly greater levels of progeny virus than transfection of the JFH1 genome. Furthermore, the expression of dominant-negative Vps4, a key molecule of the endosomal sorting complex required for transport machinery, inhibited the virus production of JFH1, but not that of J6/JFH1. These results may account for the different abilities to produce progeny virus between JFH1 and J6/JFH1. Using the J6/JFH1/Luc system, we showed that the two polyanions heparin and polyvinyl sulfate decreased the infectivity of J6/JFH1/Luc virus in a dose-dependent manner. We also analyzed the function of microRNA on HCV replication and found that miR-34b could affect the replication of HCV. The reporter virus generated in this study will be useful for investigating the nature of the HCV life cycle and for identification of HCV inhibitors.     

Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient

Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.     

A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells

The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon’s anti-HCV action and for screening and testing new anti-HCV drugs.     

Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses

The lack of an efficient system to produce hepatitis C virus (HCV) particles has impeded the analysis of the HCV life cycle. Recently, we along with others demonstrated that transfection of Huh7 hepatoma cells with a novel HCV isolate (JFH1) yields infectious viruses. To facilitate studies of HCV replication, we generated JFH1-based bicistronic luciferase reporter virus genomes. We found that RNA replication of the reporter construct was only slightly attenuated and that virus titers produced were only three- to fivefold lower compared to the parental virus, making these reporter viruses an ideal tool for quantitative analyses of HCV infections. To expand the scope of the system, we created two chimeric JFH1 luciferase reporter viruses with structural proteins from the Con1 (genotype 1b) and J6CF (genotype 2a) strains. Using these and the authentic JFH1 reporter viruses, we analyzed the early steps of the HCV life cycle. Our data show that the mode of virus entry is conserved between these isolates and involves CD81 as a key receptor for pH-dependent virus entry. Competition studies and time course experiments suggest that interactions of HCV with cell surface-resident glycosaminoglycans aid in efficient infection of Huh7 cells and that CD81 acts during a postattachment step. The reporter viruses described here should be instrumental for investigating the viral life cycle and for the development of HCV inhibitors.     

Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC(50)s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interaction between NS5A residues 30 and 92. By using a trans-complementation assay with JFH1 replicons encoding inhibitor-sensitive and inhibitor-resistant NS5A proteins, we provide genetic evidence that NS5A performs the following two distinct functions in HCV RNA replication: a cis-acting function that likely occurs as part of the HCV replication complex and a trans-acting function that may occur outside the replication complex. The cis-acting function is likely performed by basally phosphorylated NS5A, while the trans-acting function likely requires hyperphosphorylation. Our data indicate that BMS-790052 blocks the cis-acting function of NS5A. Since BMS-790052 also impairs JFH1 NS5A hyperphosphorylation, it likely also blocks the trans-acting function.     

丙型肝炎病毒F蛋白缺失对病毒复制及感染性的影响

探索了F蛋白缺失及核心蛋白(Core)二级结构改变对丙型肝炎病毒(HCV)复制和感染性的影响．利用定点突变方法,将J6JFH1的核心基因引进5个终止密码子以中断F蛋白的表达,从而获得F蛋白缺失的病毒复制子J6JFH1/ΔF．体外制备RNA转录体,并电穿孔转染Huh7.5.1细胞,采用免疫荧光、实时荧光定量PCR方法以及病毒感染等方法,观察F蛋白缺失对病毒复制、蛋白质表达及转染细胞上清感染性病毒颗粒产生的影响．在此基础上,构建5个单一突变病毒体,对HCV核心蛋白进行二级结构分析,观察核心蛋白二级结构对HCV复制和翻译的影响．结果显示,转染48 h后,J6JFH1/ΔF与野生型J6JFH1相比,J6JFH1/ΔF转染阳性细胞数明显降低,细胞内HCV RNA 水平降低约95%,J6JFH1/ΔF转染后不同时间点细胞上清中HCV RNA拷贝数和病毒颗粒也明显降低．5个单一突变体不影响核心基因二级结构,病毒在细胞内复制和感染性与野生型水平一致．J6JFH1/ΔF所产生的改变可能是由于5处突变导致核心基因二级结构改变而造成的．结果说明,HCV F蛋白缺失不影响病毒的复制翻译及病毒颗粒的包装释放,核心蛋白二级结构的改变对病毒复制和翻译则产生较大影响．     

DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication

DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supernatants were suppressed in DDX3 knockdown cells after inoculation of the cell culture-generated HCVcc. Thus, DDX3 is required for HCV RNA replication.     

Small interfering RNA targeted to hepatitis C virus 5' nontranslated region exerts potent antiviral effect

Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma. Interferon alone or together with ribavirin is the only therapy for HCV infection; however, a significant number of HCV-infected individuals do not respond to this treatment. Therefore, the development of new therapeutic options against HCV is a matter of urgency. In the present study, we have examined vectors carrying short hairpin RNA (shRNA) targeting the 5' nontranslated conserved region of the HCV genome for inhibition of virus replication. Initially, three sequences were selected, and all three shRNAs (psh-53, psh-274, and psh-375) suppressed HCV internal ribosome entry site (IRES)-mediated translation to different degrees in Huh-7 cells. Next, we introduced siRNA into Huh-7.5 cells persistently infected with HCV genotype 2a (JFH1). The most efficient inhibition of JFH1 replication was observed with psh-274, targeted to the portion from subdomain IIId to IIIe of the IRES. Subsequently, Huh-7.5 cells stably expressing psh-274 further displayed a significant reduction in HCV JFH1 replication. The effect of psh-274 on cell-culture-grown HCV genotype 1a (H77) was also evaluated, and inhibition of virus replication and infectivity titers was observed. In the absence of a cell-culture-grown HCV genotype 1b, the effects of psh-274 on subgenomic and full-length replicons were examined, and efficient inhibition of genome replication was observed. Therefore, we have identified a conserved sequence targeted to the HCV genome that can inhibit replication of different genotypes, suggesting the potential of siRNA as an additional therapeutic modality against HCV infection.