知母宁体外抗甲型流感病毒作用研究

目的:考察知母宁体外抗甲型流感病毒作用.方法:以利巴韦林作阳性对照药物,用中性红染色法测定知母宁不同加药方式对甲型人流感病毒H1N1的体外抑制作用及其时效关系.结果:知母宁对甲型人流感病毒具明显的综合抑制作用及抑制病毒吸附后的复制增殖作用,这两种作用的半数有效浓度(EC50)分别为0.70及0.76 mg·ml-1.知母宁对病毒吸附的预防作用弱,无直接杀伤病毒的作用.随药物作用时间延长,知母宁在低浓度抗病毒有效率呈减小趋势,在高浓度时(≥1.4 mg·ml-1)其抗病毒能力基本不变.同时知母宁可明显降低病毒的感染性.结论:知母宁具有明显的体外抗甲型人流感病毒作用.     

金莲花醇提物体外抗甲型流感病毒作用研究

目的研究金莲花醇提物的抗呼吸道甲型流感病毒作用。方法采用鸡胚培养流感病毒,并用鸡红血球凝集试验检测药物对病毒在鸡胚尿囊液中增殖的抑制作用。结果在药物和病毒先在体外直接作用后的共同接种试验中,金莲花醇提物对甲型流感病毒具有体外直接灭活作用;在甲型流感病毒先感染鸡胚后再接种药物试验中,金莲花醇提物可抑制病毒在鸡胚内的增殖。结论金莲花醇提物在体外对甲型流感病毒具有直接杀灭作用,对甲型流感病毒在鸡胚中的增殖具有抑制作用。     

中西医结合体外抗甲型流感病毒的实验研究

目的研究中西医结合药物体外对甲型流感病毒的抑制作用。方法体外接种甲型流感病毒鼠肺适应株H1N1于狗肾细胞（MDCK）,观察各组药物体外抗病毒作用。结果根据实验,测得各组药物对甲型流感病毒的选择指数,利巴韦林注射液组为4.68;痰热清注射液组为5.50;达菲组为5.53;中西医结合组为14.48。结论中西医结合组具有明显的体外抗甲型流感病毒的作用。     

抗病毒口服液在鸡胚内的抗病毒作用研究

目的观察抗病毒口服液在鸡胚内的抗病毒作用.方法采用鸡胚法(体内及体外)研究了抗病毒口服液对三种病毒(甲3型流行性感冒病毒IFV株、亚甲型流行性感冒病毒鼠适应的FM1株及新城疫鸡瘟病毒NDVⅡ系病毒株)的作用.结果抗病毒口服液对三种病毒株在鸡胚体外有明显的抑制作用或杀灭作用,在鸡胚内对鸡胚尿囊液HA效价有明显抑制趋势,但统计学处理无明显差异.结论抗病毒口服液在体外对甲3型流感病毒株、亚甲型FM1株和NDVⅡ系病毒株有明显的抑制作用,抗甲3型流感病毒和亚甲型FM1株的作用比抗NDVⅡ系病毒株的作用略强,但该药在鸡胚内抗病毒作用仅有抑制趋势.     

复方抗病毒制剂体外抗流感病毒和呼吸道合胞病毒作用及毒性研究

目的 测定复方抗病毒制剂体外对流感病毒和呼吸道合胞病毒的抗病毒作用. 方法 用鸡红细胞血凝素滴定法测定不同浓度复方抗病毒制剂在狗肾传代细胞（MDCK）中对流感病毒的抑制作用;用中和实验测定复方抗病毒制剂在Hep 2细胞中对呼吸道合胞病毒的活性. 结果 复方抗病毒制剂浓度＜1.5 mg•mL^-1时对MDCK细胞及Hep 2细胞无明显毒性. 浓度＞0.15 mg•mL^-1能抑制流感病毒的血凝活性和呼吸道合胞病毒对靶细胞的攻击作用. 结论 复方抗病毒制剂体外对流感病毒和呼吸道合胞病毒具有较强的抑制作用,且有较大的安全性.     

腔肠素抗流感病毒研究

目的 对海洋来源的化合物腔肠素进行体外抗流感病毒活性研究。方法 细胞病变效应(CPE)抑制实验评价腔肠素对不同流感病毒株的抗病毒作用。CPE实验检测腔肠素细胞毒性和抗病毒作用方式并结合血凝抑制实验、神经氨酸酶(NA)活性实验及微基因组实验初步探究腔肠素的作用靶点。结果 腔肠素体外对不同流感病毒株均有良好抗病毒作用,其中对PR8的抑制作用最强,IC₅₀为5.0μmol·L^-1且细胞毒较小,CC₅₀为144.1μmol·L^-1。通过不同作用方式以及不同作用时间的实验显示,腔肠素预先处理流感病毒以及在病毒感染后加入均有良好抗病毒作用,并且在病毒吸附后0～3 h的抑制效果较好。用腔肠素预处理病毒后可以呈剂量依赖性地抑制血凝素(HA)的活力。故腔肠素可能是通过与病毒HA相互作用来阻断流感病毒的进入,其作用位点可能为HA蛋白HA2链的72、74位谷氨酸。结论 腔肠素具有良好的体外抗甲型流感病毒效果,为海洋来源的小分子化合物的开发与改造提供了借鉴,为寻找新型抗流感病毒药物提供新思路。     

柴黄双解颗粒抗菌、抗病毒作用的研究

目的：观察柴黄双解颗粒抗菌、抗病毒的作用。方法体外观察柴黄双解颗粒对常见致病菌抑制作用、对流感病毒甲1型、甲3型和腺病毒致细胞病变作用的影响。结果柴黄双解颗粒体外对所试病毒所致的细胞病变及所试常见致病菌均显示抑制作用。结论柴黄双解颗粒体外有抗甲型流感病毒、腺病毒及抗菌的作用。     

银翘散主要药物提取物体外抑制流感病毒作用的比较研究

目的:观察并比较银翘散主要药物金银花、连翘、牛蒡子及其配伍提取物体外抗甲型流感病毒FM1株作用.方法:采用血凝试验对中药不同浓度、不同时间点进行体外抗甲型流感病毒的效价测定.结果:金银花、连翘、牛蒡子及其配伍提取物可直接抑制甲型流感病毒FM1株的活性,作用时间从1 h可持续到24 h;各时间点各中药提取物在最大不凝血浓度即金银花25 mg/mL、连翘50 mg/mL、牛蒡子100 mg/mL、配伍组50 mg/mL时均能完全抑制病毒的增殖,其对病毒的抑制作用随着药物浓度的降低而逐渐减弱.结论:金银花、连翘、牛蒡子及其配伍提取物可有效抑制甲型流感病毒FM1株体外增殖.     

桑白皮抗病毒有效成分的提取分离及体外抗病毒活性研究

目的研究桑白皮中化学成分的抗病毒作用。方法利用细胞病变方法进行体外抗病毒筛选试验。结果桑白皮中分离得到的化合物1和化合物3在体外有较好的抑制副流感病毒、流感病毒的致病作用;化合物1具有抗呼吸道合胞病毒作用,延缓腺病毒Ⅲ、HSV Ⅰ致病作用;化合物2和化合物5也有部分抗病毒作用;化合物4能抑制腺病毒Ⅲ、柯萨奇病毒B3 、HSV Ⅰ、副流感病毒的致细胞病变作用。结论桑白皮提取物对呼吸道常见病毒有抑制作用。     

天然多糖在体外抗甲型流感病毒作用的普遍性

目的研究各种天然多糖在体外抗甲型流感病毒的作用,探索多糖未来的抗病毒药学用途.方法 以云芝、虫草、灵芝、猴头菇4种天然多糖和葡聚糖为研究对象,观察天然多糖对狗肾传代细胞(MDCK)上甲型流感病毒的直接灭活作用、治疗作用、预防作用和综合阻断作用:观察天然多糖对鸡胚中甲型流感病毒的直接灭活作用.结果 云芝、虫草、灵芝、猴头菇4种天然多糖对甲型流感病毒有体外灭活作用,这种作用呈剂量和时间依赖关系;毒性低,对病毒感染有一定的治疗作用,安全指数均大于14;没有明显的预防作用;有一定程度的综合阻断作用.结论 在随机获取的5种多糖中,云芝等4种天然多糖具有抗甲型流感病毒的活性,说明天然多糖分子可能普遍具有抗病毒活性,如何在实际中运用多糖这一特征防治病毒感染值得进一步研究.     

通窍止咳液增强顺铂对肿瘤细胞内p38活性的研究

目的:研究通窍止咳液增强顺铂对肿瘤细胞内p38的活性。方法:通过Western-Blot检测通窍止咳液对人鼻咽癌细胞CNE2、人肺癌细胞A549内p38活性的变化;通过MTT比色法检测通窍止咳液单用以及联合顺铂对CNE2、A549细胞生长的影响。结果:Western-Blot检测结果显示,通窍止咳液能够激活CNE2和A549细胞内p38,增高细胞内p38的磷酸化水平;MTT检测结果显示高浓度的通窍止咳液可以抑制CNE2、A549细胞的生长,通窍止咳液能增强顺铂对CNE2细胞的细胞毒作用,而对A549细胞则无此作用。结论:通窍止咳液增强顺铂的细胞毒作用可能与其激活p38有关,这一作用为逆转顺铂耐药提供了新的治疗方案。     

In vitro inhibition of human influenza A virus infection by fruit-juice concentrate of Japanese plum (Prunus mume SIEB. et ZUCC)

Using a plaque reduction assay, treatment of human influenza A viruses with the fruit-juice concentrate of Japanese plum (Prunus mume SIEB. et ZUCC) showed strong in vitro anti-influenza activity against human influenza A viruses before viral adsorption, but not after viral adsorption, with 50% inhibitory concentration (IC50) values against A/PR/8/34 (H1N1) virus, A/Aichi/2/68 (H3N2) virus and A/Memphis/1/71 (H3N2) virus of 6.35+/-0.17, 2.84+/-1.98 and 0.53+/-0.10 microg/ml, respectively. The plum-juice concentrate exhibited hemagglutination activity toward guinea pig erythrocytes. Its hemagglutination activity was inhibited by the monosaccharide N-acetylneuraminic acid and a sialoglycoprotein (fetuin), but not by the other tested monosaccharides (mannose, galactose, glucose and N-acetylglucosamine), suggesting the presence of a lectin-like molecule(s) in the Japanese plum-juice concentrate. Our findings suggest that the fruit-juice concentrate of Japanese plum may prevent and reduce infection with human influenza A virus, possibly via inhibition of viral hemagglutinin attachment to host cell surfaces by its lectin-like activity.     

Clarithromycin inhibits progeny virus production from human influenza virus-infected host cells

In vitro effects of macrolide clarithromycin (CAM) on influenza A virus-infected cells were examined using plaque reduction assay by treating cells either before or after viral adsorption. The significant inhibitory effect on influenza virus infection was detected only when the cells were treated with CAM after viral adsorption. The predominant inhibitory effect was observed during 4-7th hour after viral adsorption using viral production assay. CAM did not exhibit inhibitory effects on influenza virus hemagglutination, membrane fusion and viral sialidase activities. These findings indicate that CAM acts on a middle to late stage of the viral replication cycle resulting in inhibition of progeny virus production from the infected cells.     

In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03

The sulfated polysaccharide, p-KG03, purified from the marine microalga, Gyrodinium impudium, is a unique compound comprising homogenous galactose units conjugated to uronic acid and sulfated groups. Although previous studies showed that p-KG03 suppresses tumor cell growth and infection by encephalomyocarditis virus, its effect against enveloped virus infection and the biological mechanism of action have not been elucidated. In this report, the inhibitory activity of p-KG03 against influenza virus was examined and compared with that of other sulfated polysaccharides (fucoidan and pentosan polysulfate) and antiviral agents (oseltamivir phosphate, oseltamivir carboxylate, amantadine, and ribavirin). The results of a cytopathic effect reduction assay using MDCK cells demonstrated that p-KG03 exhibited the 50% effective concentration (EC(50)) values of 0.19-0.48 μg/ml against influenza type A virus infection (selectivity index >200) but not all influenza type B viruses. Mechanism studies showed that inhibition of influenza virus replication was maximized when p-KG03 was added during or within 6 h after viral infection, suggesting that mainly the viral adsorption and internalization steps are targeted by this compound. The results of influenza virus binding assay to p-KG03 and fluorescence microscopy indicate that the antiviral activity of p-KG03 is directly associated with its interaction with viral particles. The sulfated polysaccharide p-KG03 is a potent and specific influenza A viral entry inhibitor and may be a candidate for antiviral drug development.     

海洋来源的吲哚生物碱衍生物抗流感病毒作用研究

目的 对海洋来源的吲哚生物碱类化合物HDYL-GQQ-1932进行体外抗流感病毒活性研究。方法 CPE抑制实验测定HDYL-GQQ-1932对多种病毒株的增殖抑制作用。CPE实验结合血凝抑制和神经氨酸酶活性实验探究HDYL-GQQ-1932 作用病毒入侵细胞的方式以及细胞毒性,并且初步探究HDYL-GQQ-1932的作用靶点。 结果 HDYL-GQQ-1932体外对多种流感病毒株均有增殖抑制作用,其中对H1N1的抑制作用最强,IC₅₀为26.1μM ,且无明显的细胞毒性,CC₅₀为3313.2μM。通过不同作用方式以及不同作用时间的实验显示,感染后加入 HDYL-GQQ-1932能够很好的抑制病毒增值,且用HDYL-GQQ-1932预处理细胞后可明显降低病毒增殖。此外结果显示HDYL-GQQ-1932在病毒吸附后6-9 h有很好的抑制作用,且剂量依赖性地抑制神经氨酸酶(NA)的活力。故 HDYL-GQQ-1932可能是通过结合病毒神经氨酸酶并抑制其活性来阻断IAV的释放过程。结论 HDYL-GQQ-1932体外对甲型流感具有较好的抑制效果。本研究为海洋来源的该类小分子化合物的药物开发与理性改造提供了思路与方向。     

Activity of Melaleuca alternifolia (tea tree) oil on Influenza virus A/PR/8: study on the mechanism of action

Our previous study demonstrated that Melaleuca alternifolia (tea tree) oil (TTO) had an interesting antiviral activity against Influenza A in MDCK cells. In fact, when we tested TTO and some of its components, we found that TTO had an inhibitory effect on influenza virus replication at doses below the cytotoxic dose; terpinen-4-ol, terpinolene, and alfa-terpineol were the main active components. The aim of this study was to investigate the mechanism of action of TTO and its active components against Influenza A/PR/8 virus subtype H1N1 in MDCK cells. None of the test compounds showed virucidal activity nor any protective action for the MDCK cells. Thus, the effect of TTO and its active components on different steps of the replicative cycle of influenza virus was studied by adding the test compounds at various times after infection. These experiments revealed that viral replication was significantly inhibited if TTO was added within 2h of infection, indicating an interference with an early step of the viral replicative cycle of influenza virus. The influence of the compound on the virus adsorption step, studied by the infective center assay, indicated that TTO did not interfere with cellular attachment of the virus. TTO did not inhibit influenza virus neuraminidase activity, as shown by the experiment measuring the amount of 4-methylumbelliferone, cleaved by the influenza virus neuraminidase from the fluorogenic substrate 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid. The effect of TTO on acidification of cellular lysosomes was studied by vital staining with acridine orange using bafilomycin A1 as positive control. The treatment of cells with 0.01% (v/v) of TTO at 37°C for 4h before staining inhibited the acridine orange accumulation in acid cytoplasmic vesicles, indicating that TTO could inhibit viral uncoating by an interference with acidification of intralysosomal compartment.     

Biological evaluation of anti-influenza viral activity of semi-synthetic catechin derivatives

Catechin derivatives with different alkyl chain length and aromatic ring substitutions at the 3-hydroxyl group were synthesized from epigallocatechin (EGC) and (+)-catechin (C) and their anti-influenza viral activity were evaluated in vitro and in ovo. Pronounced antiviral activity was observed for derivatives carrying moderate chain length (7-9 carbons) as compared to those with aromatic rings, whereas the 5'-hydroxyl group of the trihydroxy benzyl moiety did not significantly contribute to antiviral activity. The derivatives exerted inhibitory effects for all six influenza subtypes tested including three major types of currently circulating human influenza viruses (A/H1N1, A/H3N2 and B type), H2N2 and H9N2 avian influenza virus. The compounds strongly inhibited adsorption of the viruses on red blood cell (RBC). They also restricted the growth of avian influenza virus in ovo with minimum inhibition concentration (MIC) of 5-10 microM far exceeding the neuraminidase (NA) inhibitor oseltamivir or M2 proton channel inhibitor amantadine. The antiviral activity appears to be mediated by interaction with hemagglutinin (HA)/viral membrane rendering HA less fusogenic at the initial stage of infection. The broad spectrum activity against various subtypes of influenza viruses may complement the limitations of current antivirals and contribute for managing potentially emerging influenza pandemic. The structure-activity data of catechin derivatives may usefully guideline future research endeavors for applying green tea catechins as alternative anti-viral agents.     

In vitro anti-influenza virus and anti-inflammatory activities of theaflavin derivatives

The theaflavins fraction (TF80%, with a purity of 80%) and three theaflavin (TF) derivatives from black tea have been found to exhibit potent inhibitory effects against influenza virus in vitro. They were evaluated with a neuraminidase (NA) activity assay, a hemagglutination (HA) inhibition assay, a real-time quantitative PCR (qPCR) assay for gene expression of hemagglutinin (HA) and a cytopathic effect (CPE) reduction assay. The experimental results showed that they all exerted significant inhibitory effects on the NA of three different subtypes of influenza virus strains [A/PR/8/34(H1N1), A/Sydney/5/97(H3N2) and B/Jiangsu/10/2003] with 50% inhibitory concentration (IC(50)) values ranging from 9.27 to 36.55 μg/mL, and they also displayed an inhibitory effect on HA; these inhibitory effects might constitute two major mechanisms of their antiviral activity. Time-of-addition studies demonstrated that TF derivatives might have a direct effect on viral particle infectivity, which was consistent with the inhibitory effect on HA. Subsequently, the inhibitory effect of TF derivatives on the replication of the viral HA gene as assayed by qPCR and on the nuclear localization of the influenza virus vRNP further demonstrated that they may primarily act during the early stage of infection. Interestingly, besides the activity against functional viral proteins, TF derivatives also decreased the expression level of the inflammatory cytokine IL-6 during viral infection, expression of which may result in serious tissue injury and apoptosis. Our results indicated that TF derivatives are potential compounds with anti-influenza viral replication and anti-inflammatory properties. These findings will provide important information for new drug design and development for the treatment of influenza virus infection.     

Inhibition of influenza A virus replication by a kanamycin derivative

We studied the antiviral activity and the mechanism of action of a new antiviral agent and kanamycin derivative, 1-N-eicosanoyl-3"-N-trifluoroacetyl kanamycin A (ETKA), against influenza A virus. From yield reduction assays with VERO cells, ETKA showed a significant antiviral activity with negligible cytotoxic effect. In the presence of 20 micrograms/ml of ETKA at which VERO cell growth was not inhibited, virus titer was suppressed to 11.2% of control, and at 100 micrograms/ml virus production was suppressed to more than 99%. ETKA markedly inhibited viral protein synthesis when cells were pretreated with the drug before infection, but there was no inhibition when the drug was added 15 min post-infection. ETKA did not inhibit virus adsorption and penetration. Nor did it affect the activity of viral RNA polymerase in vitro. We found that the drug had a direct inactivating effect on influenza A virus under acidic conditions. These results suggest that ETKA exerts its antiviral action mainly in the early stage, prior to uncoating by direct inactivation of the virus due to the acidic environment of the endocytic vesicle. Aerosol treatment with the drug protected mice against a lethal influenza A virus infection.     

临床常用中成药的体外抗流感病毒活性评价

本文旨在研究我国临床常用清热解毒中成药的体外抗病毒活性。通过应用已建立的流感病毒神经氨酸酶 (neuraminidase, NA) 抑制剂筛选模型, 评价了33种临床常用中成药的NA抑制活性, 应用已建立的流感病毒诱导的细胞病变效应模型, 对具有NA抑制作用的部分中成药进行了体外抗病毒活性评价。结果表明, 临床常用中成药液体制剂的NA抑制活性普遍较高, 包括双黄连口服液、清开灵口服液、清热解毒口服液和热毒宁注射液, 其中双黄连口服液在细胞水平对于病毒感染所致细胞病变效应 (CPE) 也具有较高抑制活性; 中成药固体制剂中, 双黄连注射粉针的NA抑制活性最高, 而复方鱼腥草片则显示较高的体外抗病毒活性。综合分析表明, 大多数中成药具有一定的NA抑制活性, 但只有部分药物具有明显的体外抗病毒活性, 其活性结果对于中成药的活性物质基础研究和临床用药将具有指导意义。