Naturally derived anti-hepatitis B virus agents and their mechanism of action

Despite that some approved drugs and genetically engineered vaccines against hepatitis B virus(HBV)are available for HBV patients,HBV infection is still a severe public health problem in the world.All the approved therapeutic drugs(including interferonalpha and nucleoside analogues)have their limitations.No drugs or therapeutic methods can cure hepatitis B so far.Therefore,it is urgently needed to discover and develop new anti-HBV drugs,especially nonnucleoside agents.Naturally originated compounds with enormous molecular complexity and diversity offer a great opportunity to find novel anti-HBV lead compounds with specific antiviral mechanisms.In this review,the natural products against HBV are discussed according to their chemical classes such as terpenes,lignans,phenolic acids,polyphenols,lactones,alkaloids and flavonoids.Furthermore,novel mode of action or new targets of some representative anti-HBV natural products are also discussed.The aim of this review is to report new discoveries and updates pertaining to anti-HBV natural products in the last 20years,especially novel skeletons and mode of action.Although many natural products with various skeletons have been reported to exhibit potent anti-HBV effects to date,scarcely any of them are found in the list of conventional anti-HBV drugs worldwide.Additionly,in anti-HBV mechanism of action,only a few references reported new targets or novel mode of action of antiHBV natural products.

Analysis of Oseltamivir Resistance Substitutions in Influenza Virus Glycoprotein Neuraminidase using a Lentivirus-Based Surrogate Assay System

Influenza A virus poses a great threat to global health, and oseltamivir (trade marked as Tamiflu), which targets influenza surface glycoprotein neuraminidase (NA), is used clinically as a major anti-influenza treatment. However, certain substitutions in NA can render an influenza virus resistant to this drug. In this study, using a lentiviral pseudotyping system, which alleviates the safety concerns of studying highly pathogenic influenza viruses such as avian influenza H5N1, that utilizes influenza surface glycoproteins (hemagglutinin or HA, and NA) and an HIV-core combined with a luciferase reporter gene as a surrogate assay, we first assessed the functionality of NA by measuring pseudovirion release in the absence or presence of oseltamivir. We demonstrated that oseltamivir displays a dose-dependent inhibition on NA activity. In contrast, a mutant NA (H274Y) is more resistant to oseltamivir treatment. In addition, the effects of several previously reported substitution NA mutants were examined as well. Our results demonstrate that this lentivirus-based pseudotyping system provides a quick, safe, and effective way to assess resistance to neuraminidase inhibitors. And we believe that as new mutations appear in influenza isolates, their impact on the effectiveness of current and future anti-NA can be quickly and reliably evaluated by this assay.

木犀草素通过激活GSK3β/Nrf2通路抑制感染性炎症反应的研究

目的观察木犀草素对流感病毒感染引起的细胞炎症反应的影响,探讨木犀草素调控流感病毒引发的免疫反应的作用机制。方法①用细胞计数试剂盒(CCK-8)法检测不同浓度木犀草素(5、10、25、30μmol/L)对小鼠巨噬细胞RAW 264.7存活率的影响。②咪喹莫特(R837)刺激RAW 264.7或原代腹腔巨噬细胞的同时,用不同浓度木犀草素(5、10、20μmol/L)干预细胞3、6、18 h后,用荧光定量逆转录聚合酶链式反应(RT-qPCR)法和酶联免疫吸附测定(ELISA)法检测白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)、IL-1β、β干扰素(IFN-β)、干扰素诱导蛋白-10(IP-10)以及血红素加氧酶-1(HO-1)基因及TNF-α、IL-6的表达水平。③R837刺激RAW 264.7细胞的同时,用不同浓度木犀草素(5、10、20μmol/L)干预细胞1、3 h,用Western blot法检测细胞核因子-κB(NF-κB)p65蛋白、磷酸化p65(p-p65)蛋白、磷酸化糖原合成酶激酶3β(p-GSK3β)蛋白、Kelch样ECH关联蛋白1(Keap1)和核内核因子红细胞2相关因子(Nrf2)蛋白的表达水平。④用Nrf2抑制剂(ML385)处理RAW 264.7细胞12 h后,加入R837和木犀草素(5μmol/L)继续培养6 h,用RT-qPCR法检测细胞IL-6、TNF-α、IL-1β、IFN-β、IP-10 mRNA的表达水平。⑤流感病毒A/PR/8(PR8)感染A549细胞2 h,同时用不同浓度木犀草素(5、10、20μmol/L)处理细胞10 h,用RT-qPCR法检测细胞IL-6、TNF-α、IL-1β、IFN-β、IP-10和单核细胞趋化蛋白-1(MCP-1)mRNA的表达水平。结果①30μmol/L以内各浓度木犀草素对RAW 264.7细胞存活率没有明显影响(P>0.05)。②木犀草素能够显著降低R837诱导的RAW 264.7细胞IL-6、TNF-α的表达水平和IL-1β、IFN-β、IP-10 mRNA表达水平,且木犀草素也能显著降低原代腹腔巨噬细胞IL-6、TNF-α的表达水平。③木犀草素可以促进Nrf2入核,上调HO-1 mRNA表达并抑制p-p65表达,促进糖原合成酶激酶3β(GSK3β)的磷酸化。④在R837诱导的巨噬细胞炎症反应中,ML385可恢复木犀草素抑制的RAW 264.7细胞炎症因子IL-6、IL-1β、IFN-β和IP-10 mRNA的表达水平。⑤木犀草素抑制PR8感染引起的A549细胞炎症因子的产生。结论木犀草素可通过抑制GSK3β活性促进Nrf2/HO-1通路,从而抑制流感病毒感染引起的炎症反应。

Research progress in human infection with avian influenza H7N9 virus

Since the identification of the novel reassortant avian influenza A(H7N9) virus in China in 2013, until Jun 30, 2017, the virus has caused five epidemic waves leading to a total of 1,552 human infections, with a fatality rate of about 40%. In the spring of2017, highly pathogenic avian influenza(HPAI) H7N9 virus emerged and has caused 25 human infections. The HPAI H7N9 virus has some biological differences from the LPAI one, such as its multiple basic amino acid residues on HA leading to its independence on trypsin for replication. The pathogenicity of the HPAI H7N9 virus to experimental animals or humans is still unclear. A(H7N9) vaccine development for pandemic preparedness is ongoing, including the reassortment(H7N9/PR8)reverse genetic based vaccine, the virus like particle(VLP) vaccine, the intranasal live attenuated influenza vaccine(LAIV),the non-adjuvant Vero cell culture-derived inactivated whole-virus vaccine, the MDCK culture-derived vaccine, the H7 DNA vaccine and the recombinant replicative H7N9 virus \(H7N9-53 TM\) vaccine. Five neuramidinase resistant sites of A(H7N9)virus isolated from patients have been reported. Some alternative drugs have been studied, such as DAS181(Fludase), ribavirin,troglitazone and minocycline. Persistent surveillance and enhanced global control are essential to fight against human infections with A(H7N9) virus.

奥司他韦与帕拉米韦治疗儿童流感病毒性肺炎的临床效果比较

目的 探讨奥司他韦和帕拉米韦治疗儿童流感病毒性肺炎的临床效果及不良反应发生情况。方法 选取2022年12月至2023年12月张家港市第五人民医院收治的120例流感病毒性肺炎患儿,抽签随机分为观察组(n=60)和对照组(n=60)。两组患儿均进行常规治疗,同时,观察组加用帕拉米韦,对照组加用奥司他韦,治疗5 d后,比较两组患儿的临床疗效、临床相关指标(体温恢复时间、咳嗽消失时间、肺部啰音消失时间、住院时间及用药成本)、免疫功能指标(CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+))、炎症因子[白细胞介素-6(IL-6)、C反应蛋白(CRP)、肿瘤坏死因子-α(TNF-α)]水平及不良反应发生情况。结果 观察组总有效率91.67%(55/60)高于对照组85.00%(51/60),差异无统计学意义(χ^(2)=1.294,P>0.05);观察组治疗后用药成本、CD4^(+)、CD4^(+)/CD8^(+)分别为(685.55±125.76)元、(38.01±2.76)%、(1.54±0.52),高于对照组[(157.85±24.86)元、(34.88±2.62)%、(1.32±0.36)],差异具有统计学意义(t=31.886,P<0.01;t=6.371,P<0.01;t=2.694,P<0.01);观察组体温恢复时间、咳嗽消失时间、肺部啰音消失时间、住院时间、CD8^(+)、TNF-α、CRP、IL-6分别为(2.68±0.87) d、(9.01±1.25) d、(6.57±0.75) d、(7.75±1.55) d、(24.76±1.54)%、(6.34±1.57) ng·L^(-1)、(5.18±1.24) mg·L^(-1)、(11.58±2.04) ng·L^(-1),均短于或少于对照组[分别为:(4.78±1.06) d、(11.46±1.57) d、(10.75±1.74) d、(12.76±1.88) d、(26.88±1.65)%、(9.48±2.35) ng·L^(-1)、(6.62±1.64) mg·L^(-1)、(14.03±3.16) ng·L^(-1)],差异具有统计学意义(t=11.862,P<0.01;t=9.456,P<0.01;t=17.088,P<0.01;t=15.927,P<0.01;t=7.276,P<0.01;t=8.606,P<0.01;t=5.425,P<0.01;t=5.046,P<0.01);观察组和对照组的不良反应发生率分别为15.00%(9/60)和13.33%(8/60),差异无统计学意义(χ^(2)=0.069,P>0.05)。结论 奥司他韦和帕拉米韦治疗儿童流感病毒性肺炎均可获得良好效果,安全性好,但帕拉米韦可快速缓解患儿症状,有效改善免疫炎症反应,用药成本偏高。

Roles of the hemagglutinin of influenza A virus in viral entry and development of antiviral therapeutics and vaccines

Seasonal influenza epidemics and influenza pandemics caused by influenza A virus(IAV)has resulted in millions of deaths in the world.The development of anti-IAV vaccines and therapeutics is urgently needed for prevention and treatment of IAV infection and for controlling future influenza pandemics.Hemagglutinin(HA)of IAV plays a critical role in viral binding,fusion and entry,and contains the major neutralizing epitopes.Therefore,HA is an attractive target for developing anti-IAV drugs and vaccines.Here we have reviewed the recent progress in study of conformational changes of HA during viral fusion process and development of HA-based antiviral therapeutics and vaccines.

PA-E18G substitution in influenza A virus confers resistance to ZX-7101,a cap-dependent endonuclease inhibitor

Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(BXM),was approved in Japan and the US in 2018 and several other countries subsequently.Along with the clinical use of BXM,the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern.Herein,we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A,an analogue of BXM.The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes,including pH1N1,H3N2,H7N9 and H9N2,in MDCK cells,and the 50%effective concentration(EC_(50))was calculated to nanomole level and comparable to that of baloxavir acid(BXA),the active form of BXM.Furthermore,in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice,with reduced viral RNA loads and alleviated pulmonary damage.Importantly,serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage.Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA.Taken together,our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance,which provides critical clues for future drug development and drug resistance surveillance.

Discovery of the anti-influenza A virus activity of SB216763 and cyclosporine A by mining infected cells and compound cellular signatures

In this study,SB216763 and cyclosporine A were identified as anti-influenza A virus(IAV)agents by tran-scriptome signature reversion(TSR)analysis through deep mining of the cellular transcriptome of hu-man airway and lung cell lines infected with 3 strains of IAV and the chemical perturbations library.A synergistic effect of SB216763 and cyclosporine A against influenza A was disclosed by quantification of the network-based relationship,which was validated in vitro.Along with burgeoning omics approaches,transcriptome-based drug development is flourishing,which provides a novel insight into antivirals dis-covery with comprehensive cellular transcriptional information of disease and chemical perturbations in multicomponent intervention.This strategy can be applied as a new approach in discovering multitar-get antiviral agents from approved drugs,clinical compounds,natural products or other known bioactive compounds.

奥司他韦、扎那米韦和玛巴洛沙韦的不良事件信号挖掘与分析

目的挖掘3种抗甲型流感病毒药物(奥司他韦、扎那米韦、玛巴洛沙韦)的不良事件(ADE)信号,为临床安全用药提供参考。方法收集美国FDA不良事件报告系统(FAERS)2004年第1季度至2022年第3季度上报的奥司他韦、扎那米韦、玛巴洛沙韦ADE数据,采用报告比值比(ROR)法进行数据挖掘,评估指定医疗事件(DME),利用《国际医学用语词典》(25.0版)药物ADE术语集中的系统器官分类(SOC)进行分类统计。结果分别检索到奥司他韦、扎那米韦、玛巴洛沙韦ADE报告12636、1749、1283例,分别累及26、16、17个SOC。奥司他韦与睡惊症、异常行为、幻觉、谵妄的关联性较强;扎那米韦涉及的异常行为、谵妄、语无伦次、意识状态改变信号强度突出;玛巴洛沙韦与缺血性结肠炎、出血性膀胱炎、多形性红斑、黑便的关联性较强。3种药物的DME均检出多形性红斑,且信号较强。结论临床应用奥司他韦等3种药物时,除关注常见ADE外,还应加强关注药品说明书中未提及的ADE。对于奥司他韦应警惕急性肾损伤、暴发性肝炎,定期监测患者的肝肾功能;对于扎那米韦应警惕与呼吸系统相关的ADE,包括急性呼吸窘迫综合征、呼吸衰竭,并密切关注患者的呼吸状况;对于玛巴洛沙韦应警惕多形性红斑、横纹肌溶解等ADE。

玛巴洛沙韦:首个靶向于Cap-依赖性核酸内切酶的抗流感药物

玛巴洛沙韦是首个Cap-依赖性内切酶抑制剂,通过抑制流感病毒RNA聚合酶酸性蛋白亚基内切酶活性,从而抑制流感病毒的复制。玛巴洛沙韦于2022年8月通过美国食品药品监督管理局(FDA)审批,在临床上用于治疗5岁及以上、流感症状不超过48 h的急性无并发症流感患者,并于2023年3月获得中国国家药品监督管理局正式批准。临床试验表明,玛巴洛沙韦具有单剂量给药、安全性高等优势,将成为儿童流感患者的替代药物。

Advanced researches on the inhibition of influenza virus by Favipiravir and Baloxavir

Anti-influenza drugs are one of themost critical pathways for control of influenza virus infection.Drugs that have been developed or are developing may function via different mechanisms,and so far,inhibitors of influenza virus polymerase are among the most promising types of drugs.Favipiravir and Baloxavir,also named T-705 and Xofluza respectively,have been approved for influenza treatment in Japan and the United States.Favipiravir effectively and selectively inhibits the RNA-dependent RNA polymerase(RdRp)of RNA viruses while Baloxavir specifically targets the cap-dependent endonuclease PA of influenza viruses.These two drugs have been suggested as the first candidate drugs for influenza infection treatment,especially for strains resistant to other anti-influenza drugs.This review will focus on the pharmaceutical mechanisms and anti-influenza activity of these two drugs.

黄芩苷与利巴韦林联用体内外抗流感病毒作用

目的评价黄芩苷与利巴韦林联用体内外抗流感病毒作用,为其临床上联用提供实验依据。方法 CPE法和MTT法测定药物单用及联用对感染3种不同亚型流感病毒MD-CK细胞的保护效果,分别应用合并指数法(combination in-dex,CI)和MacSynergyⅡ分析法,分析黄芩苷与利巴韦林体外联用的相互作用。神经氨酸酶(NA)抑制试验测定黄芩苷对流感病毒NA活性的抑制作用。应用H1N1亚型流感病毒(FM1株)人工感染BALB/c小鼠模型,评价黄芩苷与利巴韦林联用对感染小鼠的保护作用。结果体外黄芩苷与利巴韦林联用对H1N1和H5N1病毒的抑制表现为相加作用,对H9N2病毒的抑制表现为协同作用。黄芩苷对3种亚型流感病毒NA的半数抑制浓度(IC50)在0.29～0.49 mmol.L-1范围。利巴韦林与黄芩苷联合给药与单独给药相比,提高感染小鼠的存活率,延长其存活时间。结论黄芩苷对流感病毒NA活性具有抑制作用。黄芩苷与利巴韦林在体外联用,对不同亚型流感病毒的抑制表现为相加或协同作用;二者联用对感染流感小鼠的保护效果明显优于单用。

重楼皂苷的制备及其抗A型流感病毒活性

目的研究重楼皂苷的制备及体内外对A型流感病毒(IAV)活性的影响。方法 采用柱层析和反相液相色谱分离技术,从重楼中提取重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ,应用高效液相色谱法测定其纯度。采用MTT比色法测定重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ的细胞毒性,并检测其体外对IAV的直接灭活作用、对IAV吸附和侵入细胞的阻断作用及对IAV在靶细胞内增殖的抑制作用。用IAV滴鼻法制备IAV感染小鼠模型,模型制备4 h后分别每天ig给予重楼皂苷Ⅰ5和10 mg.kg-1及阳性对照药奥司他韦3 mg.kg-1,每天分2次给药,连续5 d,自给药后每天观察并记录小鼠发病情况和死亡数,共观察14 d,计算小鼠死亡率和生命延长率,评价其体内抗IAV作用。结果 提取分离得到的重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ的纯度分别为95.3%,90.2%,92.4%和86.8%。重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ浓度分别低于50,12.5,6.25和12.5 mg.L-1时,未见对靶细胞MDCK的细胞毒性。重楼皂苷Ⅰ6.25,12.5,25和50 mg.L-1,重楼皂苷Ⅱ6.25和12.5 mg.L-1,重楼皂苷Ⅵ3.13和6.25 mg.L-1,重楼皂苷Ⅶ6.25和12.5 mg.L-1及奥司他韦40 mg.L-1对IAV均有显著的直接灭活作用(P<0.01),对IAV吸附和侵入靶细胞亦具有一定的阻断作用(P<0.01),对IAV在靶细胞内的增殖具有抑制作用(P<0.01)。重楼皂苷Ⅰ5和10 mg.L-1可明显降低IAV感染小鼠的死亡率(P<0.01),由流感病毒感染组的8/10均下降到2/10;并可延长IAV感染小鼠的存活时间(P<0.01),存活时间由(8.5±0.3)d分别延长到12.7±0.4和(13.2±0.5)d(P<0.01),生命延长率分别为49.4%和55.3%,效果与奥司他韦近似。结论 从植物重楼中分离到高纯度的重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ,重楼皂苷Ⅱ,Ⅵ和Ⅶ在体外具有较好的抗IAV活性,重楼皂苷Ⅰ在体内外均具有较好的抗IAV活性。

2017年江苏省H3N2流感病毒血凝素与神经氨酸酶分子特征及耐药性分析

目的流行性感冒(简称流感)耐药性监测目前已成为流感防控中的一项重要组成部分。文中旨在分析江苏省2017年H3N2亚型流感病毒基因变异及耐药性情况。方法从江苏省13个流感监测点流感毒株中选取17株H3N2亚型毒株,其中12株为2017年夏季高峰期分离株,4株为非高峰期分离株,1株为2016年度分离株(作为1株参考株)。采用逆转录聚合酶链反应(RT-PCR)对H3N2亚型流感毒株进行序列测定,从全球共享流感序列网站GISAID下载构建甲型H3N2亚型流感毒株HA(血凝素)系统进化树的骨架参考序列,使用MAGE7.0软件进行序列比对和系统进化分析。通过神经氨酸酶(NA)抑制试验,分析病毒耐药情况。结果16株病毒HA和NA基因核苷酸相似性分别为98.9%~99.6%、99.2%~99.3%,同源性较高。HA进化树分析显示,16株分离株中15株位于3C.2a分支,13株病毒3个C3.2a1分支关键位点(N171K、I406V和G484E)未发生变化,另外3株毒株关键位点发生变异,1株毒株发生该标志性氨基酸变异,1株毒株发生(N121K、T135K)的免疫相关突变。与2017-2018年度疫苗株推荐株A/HongKong/4801/2014相比,16株分离株抗原位点突变包括:A138S 1株,R142K 2株,R142K、R261Q 9株,R142KS144R、R261Q 1株,R142K、S144K、R261Q 1株。16株病毒的NA片段分析未发现NA耐药位点。基因型和表型分析表明,16株H3N2亚型流感毒株对抗流感药物奥司他韦及扎那米韦均敏感,均发现与M2基因耐药有关A30T、S31N分子位点的改变。结论2017年江苏H3N2亚型流行株HA和NA基因表现出异质性特点及发生基因重配现象,随着H3N2流感病毒变异持续发生,应密切监测病毒遗传进化和耐药基因的变化情况。

抗流行性感冒病毒药物研究进展

流行性感冒(流感)是由流感病毒引起的传染性呼吸道疾病。虽然接种疫苗是减轻流感流行的有效手段,但面对新的大流行病毒的传播和复杂住院流感患者,抗流感病毒药物的应用必不可少。目前,正处于非临床和临床研究阶段的抗流感病毒药物主要包括血凝素抑制剂、M2离子通道蛋白抑制剂、RNA依赖性RNA聚合酶(RdRp)抑制剂和神经氨酸酶抑制剂等。与其他3种抑制剂相比,RdRp抑制剂可快速降低病毒载量,具有广谱抗病毒作用,且不易产生耐药性,但其临床疗效需进一步评估。神经氨酸酶抑制剂是防治流感的常用药物。本文综述抗流感病毒药物的抗病毒活性、作用机制和临床应用的研究进展,以期为研发具有广谱活性的新型抗流感病毒药物提供参考。

热毒宁对流感病毒FM_1的抑制作用

目的:研究中草药热毒宁注射液对流感病毒FM_1的抑制作用。方法:以利巴韦林为阳性对照药,采用细胞培养技术及四氮唑蓝(MTT)比色法,观察在热毒宁干预下,受FM_1感染的犬肾传代细胞(MDCK细胞)的病变效应(CPE)及光密度值(D),计算各组细胞的病毒抑制率。比较不同药物剂量及3种不同给药方式(预防给药、直接灭活及治疗给药)下热毒宁对流感病毒FM_1的抑制作用。结果:热毒宁注射液半数中毒浓度(TC_(50))为10.11g·L^(-1)。预防给药、直接灭活及治疗给药方式下,CPE法测得其抗流感病毒FM_1的半数有效浓度(EC_(50))分别为0.625、0.725、0.629 g·L^(-1),治疗指数(TI)分别为16.17、13.94、16.07;MTT比色法测得其抗流感病毒FM_1的EC_(50)分别为1.135、0.770、0.661 g·L^(-1),TI分别为8.91、13.12和15.29。3种方式给药,MTT法测定时,热毒宁对流感病毒FM_1的抑制作用无差异;CPE法测定时,热毒宁浓度在0.812 51 g·L^(-1)及以上对流感病毒FM_1的抑制作用无差异。结论:热毒宁对流感病毒FM_1有直接灭活及抑制增殖作用,对其感染靶细胞有阻断作用,且呈明显量效关系。

黄芩总黄酮和栀子总环烯醚萜对含药小鼠血清体外抗病毒作用

目的探讨黄芩总黄酮、栀子总环烯醚萜苷及其组合物含药小鼠血清体外抗甲型流感病毒(H1N1)的作用。方法采用细胞病变法测定黄芩总黄酮、栀子总环烯醚萜苷及其组合物含药血清对人肺腺癌细胞(A549)的最大无毒浓度和H1N1对A549细胞的半数组织感染量(TCID50);采用四甲基偶氮唑蓝比色法检测黄芩总黄酮、栀子总环烯醚萜苷及其组合物含药血清对H1N1的体外抑制作用和直接杀灭作用。结果黄芩总黄酮、栀子总环烯醚萜苷及其组合物含药血清对A549细胞的TC0均为12.5%;H1N1对A549细胞的TCID50为10-2.9/0.1 mL;黄芩总黄酮含药血清12.5%、黄芩总黄酮和栀子总环烯醚萜苷组合物含药血清6.25%和12.5%,分别对20倍量TCID50H1N1的细胞内增殖具有明显的抑制作用,且对H1N1具有直接的杀灭作用;黄芩总黄酮、栀子总环烯醚萜苷单用的药效作用弱于组合使用。结论黄芩总黄酮和栀子总环烯醚萜苷组合物具有明显的抗病毒效应,其作用机制可能是抑制病毒在细胞内的增殖和对病毒的直接杀灭作用。

飞龙掌血抗A型流感病毒活性的鉴定

目的:鉴定中药飞龙掌血体外抗流感病毒(A/PR/8/34H1N1)活性。方法:用四唑氮化合物(MTS)测定流感病毒诱导细胞病变(CPE)方法,初筛200余种中草药提取物抗A型流感病毒活性。复筛中样品的抗病毒活性被进一步用荧光定量聚合酶链反应(PCR)测试方法验证。结果:飞龙掌血在MTS方法和定量PCR中均显示很强的抗H1N1型流感病毒活性,半数有效剂量(EC50)在MTS方法和定量PCR中分别为4.7,0.9mg·L-1。毒性实验显示半数细胞毒剂量(CC50)为187.2mg·L-1,选择指数(SI)在定量PCR测试中大于206。药物作用时间实验显示飞龙掌血与病毒同时加入细胞可获得最佳抗病毒效果,病毒感染前、后24h加入飞龙掌血仍然具有一定抗病毒作用。结论:飞龙掌血可作为抗流感病毒候选治疗药物。

青天葵抗甲、乙型流感病毒作用研究

目的研究青天葵在体外抗甲、乙型流感病毒株作用。方法选用狗肾细胞培养法考察青天葵不同极性部位对甲型(FM1)、乙型(昆40B)流感病毒的作用。结果青天葵水溶性部位对甲型流感病毒FM1有抑制作用。结论青天葵具有体外抗甲型流感病毒作用。

荆芥、桂枝挥发油含药血清体外抗病毒的实验研究

目的探讨荆芥、桂枝挥发油含药血清体外抗甲型流感病毒(H1N1)的作用。方法采用细胞病变法(CPE)检测荆芥、桂枝挥发油含药血清对狗肾传代细胞(MDCK)的最大无毒浓度(TC0);采用血凝试验测定甲型流感病毒对MDCK细胞的感染性;采用血凝试验和四甲基偶氮唑蓝(MTT)比色法测定荆芥、桂枝挥发油含药血清对甲型流感病毒的体外抑制作用。结果①荆芥、桂枝挥发油含药血清对MDCK细胞的TC0均为5%,即在相应培养基中血清含量为5%。②甲型流感病毒鼠肺适应株A/PR/8/34(H1N1)对MDCK细胞的TCID50(病毒半数组织感染量)为10-3.5/0.1 ml。③荆芥、桂枝挥发油含药血清5%、3.5%、2.5%浓度对10 TCID50的甲型流感病毒的增殖均有显著抑制作用,且5%、3.5%浓度的荆芥、桂枝挥发油含药血清对甲型流感病毒具有直接杀灭作用。结论荆芥、桂枝挥发油含药血清对甲型流感病毒在MDCK细胞中的增殖具有显著抑制作用,并具有一定直接杀灭作用,结果与挥发油的体外、体内实验一致,进一步明确证实了两者的抗流感病毒效应。