抗病毒抗生素16704A的研究Ⅰ.产生菌的分类、发酵及活性成分的提取、分离与鉴别

在筛选抗病毒抗生素的过程中,从云南土壤分离出的一株链霉菌16704,其发酵液抗HSV-1病毒活性较强,我们对链霉菌16704产生菌的初步分类,发酵,活性成分的提取,分离和鉴别等进行了研究,通过大孔树脂吸附层析,有机溶剂提取,RP-8反相柱层析等方法分离得到16704A纯品,经过对化合物16704A理化性质的测定和对其UV、FAB-Mass、EI-Mass和1H-NMR谱的分析,将其鉴别为已知化合物Bafilomycin A1.     

菌毒清颗粒体外抗病毒的药效学研究

目的:研究菌毒清颗粒体外抗病毒的药效学。方法:将柯萨奇病毒B族3型(Cox B3)Nancy株、呼吸道合胞病毒(RSV)分别接种于人喉癌上皮Hep2细胞以复制细胞病毒模型。菌毒清颗粒原液(2 mg/ml)与氯原酸原液(2 mg/ml)以1∶2~1∶256倍比稀释后作用于病毒模型细胞,以Reed-Muench法计算50%细胞毒性浓度(TC50)和最大无毒浓度(TC0)。以1.25、0.625、0.312 5、0.15625 mg/ml菌毒清颗粒溶液与0.5、0.25、0.125、0.062 5 mg/ml绿原酸溶液培养病毒模型细胞,显微镜下评价细胞病变程度。以1.25、0.625、0.312 5、0.156 25、0.078 125 mg/ml菌毒清颗粒溶液与0.5、0.25、0.125、0.062 5、0.031 25 mg/ml绿原酸溶液作用于病毒模型细胞,Reed-Muench法计算对RSV、Cox B3细胞的50%抑制浓度(IC50)和治疗指数(TI)。结果:菌毒清颗粒TC50、TC0分别为1.79、1.25mg/ml,绿原酸TC50、TC0分别为0.71、0.5 mg/ml。当菌毒清颗粒溶液质量浓度为1.25 mg/ml时病毒感染细胞生长正常。菌毒清颗粒IC50均为0.22 mg/ml,TI均为8.14;绿原酸IC50分别为0.18、0.36 mg/ml,TI分别为3.94、1.97。结论:菌毒清颗粒具有体外抗Cox B3和RSV病毒的作用。     

抗生素17997抗病毒作用机制的研究

报道由我国云南省土壤中分离的一株放线菌所产生的广谱抗病毒抗生素１７９９７［１］的细胞毒性及作用机制研究。用氚标记物参入法测定抗生素１７９９７对ＶＥＲＯ细胞ＤＮＡ、ＲＮＡ及蛋白质合成的ＴＤ５０（５０％毒性剂量）分别为７６．８±２．２、１３０．９±１９．５及１４４．２±１７．１μｍｏｌ／Ｌ。抗生素１７９９７无毒浓度不影响单疱病毒Ⅰ型的吸附及释放；对病毒无直接灭活作用；感染后６ｈ给药无抑制病毒作用；对病毒逆转录酶及ＤＮＡ聚合酶均无抑制作用。抗生素１７９９７可能作用于病毒复制早期阶段。     

黄蜀葵花总黄酮体外抗单纯疱疹病毒作用

目的研究黄蜀葵花总黄酮(Total flavone ofabelm oschus manihot L m edic,TFA)的体外抗病毒作用。方法在Hep-2细胞上传代单纯疱疹病毒Ⅰ型(Herpes simp lex virus-Ⅰ,HSV1)和单纯疱疹病毒Ⅱ型(Herpes simp lex virus-Ⅱ,HSV2)后滴定其50%组织细胞感染剂量(50%tissue cell infection dose,TC ID50),测定TFA的半数有效量(50%inh ib ition concentration,IC50)值及治疗指数(TI)值。结果TFA对HSV1和HSV2的IC50分别为1.01±0.39 mg.L-1和1.21±0.42 mg.L-1;TFA对HSV1和HSV2的TI值分别为12.09±2.70和10.83±1.44。结论TFA对HSV1和HSV2均有一定的抑制病毒作用,而对HSV1和HSV2病毒之间作用没有差别。     

广谱抗病毒抗生素17997体内外抗病毒活性研究

从我国云南省土壤中分离到１株放线菌，基们生的抗生素１７９９７具有广谱抗病毒作用在细菌培养内对单纯疱疹病毒１型，单纯疱疹病毒２型，人免疫缺陷病毒１型，水疱性口炎病毒及柯萨奇病毒Ｂ３均有显著抑制活性，ＩＣ５０在μｍｏｌ／Ｌ水平。其在小鼠体内对单纯疱疹病毒１型及２型所致疱疹性脑炎有确切治疗效果，对死亡保护率平均生存日与对照组相比有统计意义的显著差别。     

环氧酶抑制剂对大鼠血小板聚集的影响

目的：研究环氧酶抑制剂艾瑞昔布、氯吲昔布、美洛昔康、塞来昔布和吲哚美辛对环氧酶-1（COX-1）和环氧酶-2（COX-2）的选择性及对大鼠血小板聚集的影响，探讨环氧酶选择性抑制与血小板聚集之间的关系。方法：利用A23187刺激的小鼠腹腔巨噬细胞模型检测COX-1表达的变化；利用LPS诱导的小鼠腹腔巨噬细胞模型检测COX-2表达的变化；利用花生四烯酸和胶原作为刺激剂诱导血小板聚集模型检测化合物对大鼠血小板聚集的影响。结果：上述化合物对COX-1和COX-2的表达均有明显的抑制作用，其对COX-1和COX-2抑制率IC50的比值分别为：艾瑞昔布IC50 coxl／IC50 cox2为6．7；氯吲昔布IC50 cox-1／IC50cox-2为3．5；美洛昔康抑制IC50cox-1／IC50COX-2为3．0；塞来昔布C50cox-1／IC50cox-2为61．5；吲哚美辛抑制IC50cox-1／IC50COX-2为0．66。艾瑞昔布、氯吲昔布、美洛昔康和吲哚美辛抑制花生四烯酸诱导的大鼠血小板聚集的IC50分别为2．98×10-6 mol／L、1．13×10-5 mol／L、6．80×10-6mol／L和1．82×10-6mol／L；其抑制胶原诱导的大鼠血小板聚集的Ic50分别为2．89×10-6mol／L、3．24×10-6mol／L、5.4×10-8mol／L和1．87×10-7mol／L。塞来昔布在10-5mol／L，浓度时对花生四烯酸和胶原诱导的血小板聚集无明显抑制作用。结论：上述化合物对环氧酶选者性抑制南强到弱的顺序依次为塞来昔布、艾瑞昔布、氯吲昔布、美洛昔康和吲哚美辛。随环氧酶抑制的选择性降低，化合物对血小板聚集制作用逐渐增强。     

香雪抗病毒口服液体外抗甲型流感病毒及炎症因子的作用机制研究

目的 探讨香雪抗病毒口服液对甲型流感病毒的活性及其在炎症免疫应答中的潜在作用机制。方法 采用噻唑蓝（MTT）染色法检测香雪抗病毒口服液对MDCK细胞和A549细胞的细胞毒性作用。采用细胞病变抑制法（CPE）及空斑减少实验对香雪抗病毒口服液进行抗病毒活性检测。采用免疫荧光法检测NP蛋白。实时荧光定量PCR测定炎症细胞因子的表达情况。采用蛋白印迹实验检测香雪抗病毒口服液对炎症相关信号通路的蛋白表达的影响。结果 香雪抗病毒口服液对甲型流感病毒A/PR/8/34(H1N1)和A/Aichi/2/68(H3N2)均有一定的抗病毒抑制作用,半数细胞毒性浓度（TC50）分别为42.12、79.36 mg/mL,半数抑制浓度（IC50）分别为8.665、5.260 mg/mL,并可减少H1N1病毒空斑形成。香雪抗病毒口服液能呈剂量相关性地降低H1N1诱导的A549细胞炎症因子肿瘤坏死因子-α(TNF-α)、视黄酸（维甲酸）诱导基因蛋白I(RIGI)、趋化因子（CC基序）配体5(CCL5)、巨噬细胞炎症蛋白-1β(MIP-1β)、干扰素诱导蛋白-10(IP-10)、白细胞介素-10(IL-10)、λ干扰...     

评价HIV-1抗病毒药物的重组假病毒法的建立及其初步应用

目的：探讨重组假病毒方法应用于评价抗病毒药物的可行性。方法：采用pSG3△env／TZM—bl系统，制备假病毒毒种，在不同细胞、不同重组假病毒接种量的条件下，应用重组假病毒法测定AZT的抗病毒效果，以此选择最适的细胞接种量、病毒接种量，评价检测方法的重复性，用建立的假病毒检测法测定AZT、3TC、d4T、ddI的抗病毒效果。结果：细胞接种量对检测结果有一定的影响，最适细胞接种量为每孔10^4个细胞；重组假病毒接种量对检测结果没有明显的差异；重复性检测结果显示，该方法的RSD为11．52％；用该法检测AZT、3TC、d4T、ddI的50％抑制浓度（IC50值）分别为0．0074μmol·L^-1、0．5312μmol·L^-1、0．0868μmol·L^-1、3．4364μmol·L^-1。结论：重组假病毒法可用于评价抗HIV-1药物，具有快速、简便的优点。     

新抗病毒抗生素变活霉素A

变活霉素A为一新的蒽环类广谱抗病毒抗生素,在组织培养内对4株单纯疱疹Ⅰ型病毒(HSV-1),4株单纯疱疹Ⅱ型病毒,流感甲_3病毒及柯萨奇B_6病毒的致细胞病变作用均有抑制活性,IC_(50)为6.25～12.5μg/ml。相同剂量可降低HSV-1繁殖量 1.5～3.5个对数值(log10)。细胞感染HSV-1后 4～10小时再用变活霉素A处理,仍可抑制,推迟细胞病变的发展.250～500μg/ml变活霉素A在无细胞系统内对HSV-2 DNA聚合酶,人免疫缺陷病毒(HIV-1)逆转录酶,鸭乙型肝炎病毒DNA聚合酶及牛胸腺DNA聚合酶α均无抑制活性。     

接受抗病毒治疗的乙型肝炎患者妊娠期耐药20例分析

目的探讨乙型肝炎患者妊娠期耐药的原因及管理策略。方法收集2007年1月1日至2012年12月31日在北京地坛医院足月分娩、妊娠期服用拉米夫定或替比夫定出现耐药的20例乙型肝炎患者的病历资料进行回顾性分析,主要分析指标为妊娠前后抗病毒药物应用情况、妊娠期出现耐药的时间及应对措施、HBVDNA载量、肝功能以及母婴结局等。结果拉米夫定组患者12例,年龄26～38岁,平均（31±3）岁,妊娠前应用拉米夫定100mg／d,治疗时间1．5—8．0年;其中3例在应用恩替卡韦2年后改用拉米夫定。替比夫定组患者8例,年龄27—34岁,平均（31±2）岁,妊娠前应用替比夫定600mg／d,治疗时间2．0～4．5年;其中1例在应用恩替卡韦2年后改用替比夫定。耐药发生在妊娠0～12、13—27、28—40周者拉米夫定组分别为1、2、9例,替比夫定组分别为1、1、6例,发生在妊娠28～40周者共15例（75％）。拉米夫定组12例中4例改用替诺福韦300ml/d单药治疗,3例在妊娠28周后加用阿德福韦酯10mg／d联合治疗;4例继续用拉米夫定,产后加用阿德福韦酯治疗;1例改用替比夫定无效,产后加用阿德福韦酯治疗。替比夫定组8例中2例改用替诺福韦300mg,／d单药治疗,其余6例继续服用替比夫定,产后加用阿德福韦酯10mg／d联合治疗。20例孕妇均单胎足月顺产。拉米夫定组1例新生儿出生时静脉血HBVDNA为1．56×10^6拷贝／ml,诊断为宫内感染,考虑与母亲耐药有关。替比夫定组2例新生儿中1例出生时右耳附耳,另1例出现颅内出血及贫血,均与药物及耐药无关。对加名新生儿随访2—57个月,均生长发育正常。结论接受抗病毒治疗的乙型肝炎孕妇,尤其是妊娠前抗病毒治疗时间较长者,在妊娠期应加强病毒耐药监测。耐药可能导致孕妇肝病加重,并增加乙型肝炎病毒母婴传播的风险。出现耐药后应密切监测患者肝功能,且在充分告知风险、权衡利弊、患者知情同意的情况下改变治疗方案。替诺福韦是妊娠期拉米夫定或替比夫定耐药患者的最佳选择。     

Inhibition of herpes simplex virus type 1 and adenovirus type 5 by heterocyclic Schiff bases of aminohydroxyguanidine tosylate

Eleven heterocyclic Schiff bases of aminohydroxyguanidine tosylate (SB-AHGs), compounds I-XI, were tested for antiviral activity against herpes simplex virus type 1 (HSV-1) and adenovirus type 5 (Ad 5) via plaque reduction and virus yield reduction assays. This work was undertaken to test the hypothesis that low molecular weight SB-AHGs (MW < 235 for the free SB) make better antiviral agents than high MW SB-AHGs (MW > 300). The plaque reduction assay method demonstrated that three compounds, I, VII and IX, had moderate activity against HSV-1, with 50% inhibitory concentration (IC50) values of 38.0, 23.5 and 52.1 microM, respectively. Against Ad 5, compounds I, VIII and XI exhibited moderate activity, with IC50 values of 52.7, 19.3 and 5.1 microM, respectively. Among the compounds screened, compound I (1-[(3'-hydroxy-6'-methyl-2'-pyridyl)methylene]amino-3-hydroxyguanidi ne tosylate) was the most promising antiviral candidate, with selectivity indices (SI) of 10.2 (HSV-1) and 7.6 (Ad 5), respectively. Virus yield reduction assays indicated that compound I had less antiviral potency against HSV-1 than against Ad 5. The antiviral effects of compound I at a high input virus multiplicity of infection (MOI > 5) indicated that compound I had effective anti-adenoviral activity at 24 h post infection. This work demonstrated that some of SB-AHGs only have moderate antiviral activities against Ad 5 and HSV-1 viruses. In general, low MW SB-AHGs have low cytotoxicities to the host cells.     

Antiviral activity of serum from the American alligator (Alligator mississippiensis)

Serum from wild alligators was collected and tested for antibiotic activity against three enveloped viruses using cell-based assays. Alligator serum demonstrated antiviral activities against human immunodeficiency virus type 1 (HIV-1; IC50=0.9%), West Nile virus (WNV; IC50=4.3%), and Herpes simplex virus type 1 (HSV-1; IC50=3.4%). The inhibitory concentration (IC50) is defined as the concentration of serum that inhibits 50% of viral activity. The antiviral effects of the alligator serum were difficult to evaluate at high concentrations due to the inherent toxicity to the mammalian cells used to assay viral activities. The TC50 (serum concentration that reduces cell viability to 50%) values for the serum in the HIV-1, WNV, and HSV-1 assays were 32.8, 36.3 and 39.1%, respectively. Heat-treated serum (56 degrees C, 30 min) displayed IC50 values of >50, 9.8 and 14.9% for HIV-1, WNV and HSV-1 viruses, respectively. In addition, the TC50 values using heat-treated serum were substantially elevated for all three assays, relative to untreated serum (47.3 to >50%). Alligator serum complement activity has been shown to be heat labile under these conditions. HIV-1 antiviral action was heat-sensitive, and thus possibly due to the action of serum complement, while the anti-WNV and anti-HSV-1 activities were not heat labile and thus probably not complement mediated.     

Antiviral sulfated polysaccharide from Navicula directa, a diatom collected from deep-sea water in Toyama Bay

A sulfated polysaccharide named naviculan was isolated from a diatom, Navicula directa (W. SMITH) RALFS, collected in deep sea water from Toyama Bay. The polysaccharide consisted of fucose, xylose, galactose, mannose, rhamnose and sulfate with an apparent molecular weight of 220000. It showed antiviral activities against herpes simplex viruses type 1 and 2, and influenza A virus with selectivity indices (CC50/IC50) of 270, 510 and 32, respectively. Naviculan also showed an inhibitory effect on cell-cell fusion between CD4-expressing and human immunodeficiency virus (HIV) gp160-expressing cells that was used as a model system of infection with HIV.     

Antiviral activity of the marine alga Symphyocladia latiuscula against herpes simplex virus (HSV-1) in vitro and its therapeutic efficacy against HSV-1 infection in mice

The antiviral activities of extracts from 5 species of marine algae collected at Haeundae (Pusan, Korea), were examined using plaque reduction assays. Although the activity of a methanol (MeOH) extract of Sargassum ringoldianum (Sargassaceae) was the most potent against several types of viruses, it was also cytotoxic. A MeOH extract of Symphyocladia latiuscula (Rhodomelaceae) and its fractions exhibited antiviral activities against acyclovir (ACV) and phosphonoacetic acid (PAA)-resistant (AP(r)) herpes simplex type 1 (HSV-1), thymidine kinase (TK(-)) deficient HSV-1 and wild type HSV-1 in vitro without cytotoxicity. The major component, 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (TDB) of a CH(2)Cl(2)-soluble fraction was active against wild type HSV-1, as well as AP(r) HSV-1 and TK(-) HSV-1 (IC(50) values of 5.48, 4.81 and 23.3 microg/ml, respectively). The therapeutic effectiveness of the MeOH extract and TDB from S. latiuscula was further examined in BALB/c mice that were cutaneously infected with HSV-1 strain 7401H. Three daily oral administrations of the MeOH extract and TDB significantly delayed the appearance of score 2 skin lesions (local vesicles) and limited the development of further score 6 (mild zosteriform) lesions in infected mice without toxicity compared with controls. In addition, TDB suppressed virus yields in the brain and skin. Therefore TDB should be a promising anti HSV agent.     

Studies on new dehydropeptidase inhibitors. I. Taxonomy, fermentation, isolation and physico-chemical properties

WS1358A1 (FR104007) and B1 (FR104008), new potent inhibitors of renal dehydropeptidase, were isolated from the culture broth of strain No. 1358 which was identified as Streptomyces parvulus subsp. In vitro inhibitory activities (IC50 value) of WS1358A1 and B1 against porcine renal DHP were 3 and 600 nM, respectively.     

Anthranoid compounds with antiprotozoal activity from Vismia orientalis

A phytochemical investigation of the 80% ethanolic extract of stem bark of Vismia orientalis Engl. (Guttiferae or Clusiaceae), a plant used in traditional medicine in Tanzania, resulted in the isolation and spectroscopic characterisation of 3-geranyloxy-6-methyl-1,8-dihydroxyanthraquinone, emodin, vismione D and bianthrone A1. Vismione D exhibited a broad range of antiprotozoal activities against Trypanosoma brucei rhodesiense and T. cruzi (IC50 < 10 micrograms/mL), Leishmania donovani (IC50 0.37 micrograms/mL) and Plasmodium falciparum strain K1 (IC50 1.0 microgram/mL). However, it was also slightly cytotoxic against human L6 cells (IC50 4.1 micrograms/mL). Emodin showed antileishmanial activity (IC50 2.0 micrograms/mL), while its IC50 against L6 cells was 20.3 micrograms/mL. Other antiprotozoal activities observed for emodin against both Trypanosoma species and P. falciparum, for bianthrone A1 against T. b. rhodesiense and P. falciparum, and for 3-geranyloxy-6-methyl-1,8-dihydroxyanthraquinone against T. b. rhodesiense, L. donovani and P. falciparum were in the range of 10 to 50 micrograms/mL. None of the compounds showed antibacterial or antiviral (including also HIV) activity.     

Synthesis,antimicrobial and antiviral testing of some new 1-adamantyl analogues

A new series of 1-adamantyl derivatives was designed, synthesized and evaluated for their antimicrobial and antiviral activities. Representative derivatives of the newly synthesized compounds were tested. Ampicillin, clotimazole and the antiviral antibiotic aphidicolin were used as positive controls. Compound 18 proved to be the most active member of this series as antimicrobial against Staphylococcus aureus and Candida albicans and as antiviral with IC₅₀ value of 0.21 mg/ml and CD₅₀ value of 0.02 mg/ml while compound 19 proved to be the most active member of this series as antiviral with IC₅₀ value of 0.21 mg/ml and CD₅₀ value of 0.01 mg/ml.     

Two new cytotoxic quinone type compounds from the halotolerant fungus Aspergillus variecolor

Two new quinone type compounds, variecolorquinones A (1) and B (2) together with eleven known related compounds 3 approximately 13 have been isolated from the metabolites produced by the halotolerant fungal strain Aspergillus variecolor B-17. The structures of 1 and 2 were determined by spectroscopic methods. 1 exhibited selective cytotoxicity against A-549 cells with the IC50 values of 3.0 microM. 2 showed cytotoxicity against HL60 and P388 cells with the IC50 values of 1.3 and 3.7 microM, respectively.     

高寒草甸链霉菌Qhu-M197活性次级代谢产物的研究

摘要：目的 从高寒草甸来源的链霉菌Qhu-M197中发现具有抗菌和抗肿瘤活性的次级代谢产物。方法 首先扩增菌株Qhu-M197的16S rRNA基因并基于测序结果,构建系统发育树,初步开展菌株分类鉴定;其次基于色谱分离技术,结合高分辨质谱和核磁共振波谱学数据,对活性次级代谢产物进行分离和结构鉴定。最后采用微量稀释二倍法及CCK8法对分离化合物进行抗菌和抗肿瘤活性体外评价。结果 菌株Qhu-M197为链霉菌,其16S rRNA基因序列与Streptomyces phaeoluteigriseus DSM 41896T相似度为100.00%。菌株Qhu-M197的MS固体培养基发酵产物对金黄色葡萄球菌和枯草芽胞杆菌具有显著的抑菌效果。从其发酵产物中分离得到4个单体化合物,分别是mithramycin、aerugine、aeruginol以及2, 3, 4, 5-tetrahydro-3, 5-dihydroxy-6H-1, 5-benzoxazocin-6-one,其中mithramycin的抗菌和抗肿瘤活性最强,对金黄色葡萄球菌和枯草芽胞杆菌的最小抑制浓度(MIC)分别为0.012和0.095 μg/mL,对HepG2人肝癌细胞株和MCF-7人乳腺癌细胞株的半数抑制浓度IC50分别为0.45和0.46 μmol/L。结论 以链霉菌Qhu-M197为代表的青藏高原高寒草甸放线菌是抗菌和抗肿瘤活性次级代谢产物的重要来源。     

The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell

Two homogeneous sulfated polysaccharides obtained from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata, the kappa/iota/nu carrageenan G3d and the dl-galactan hybrid C2S-3, were assayed for their antiviral properties against the four serotypes of dengue virus (DENV) in different host cell types. Both seaweed derivatives were selective inhibitors of DENV-2 multiplication in Vero cells with inhibitory concentration 50% (IC50) values around 1 microg/ml and selectivity indices > 1000. The compounds had a lower antiviral effect against DENV-3 (IC50 values in the range 13.9-14.2 microg/ml), an even lower effect against DENV-4 (IC50 values in the range 29.3 to > 50 microg/ml) and were totally inactive against DENV-1. With respect to the host cell, the polysulfates were inhibitors of DENV-2 and DENV-3 in the human hepatoma HepG2 and foreskin PH cells, with similar antiviral effectiveness as in Vero cells, but were totally inactive in mosquito C6/36 HT cells. Mechanistic studies demonstrated that G3d and C2S-3 were active DENV-2 inhibitors only when added together with the virus or early after infection, and both initial processes of virus adsorption and internalization are the main targets of these compounds. Therefore, the variations in antiviral activity of the polysaccharides depending on the viral serotype and the host cell may be ascribed to differences in the virus-cell interaction leading to virus entry.