DNA polymerase beta lyase inhibitors from Maytenus putterlickoides

During a survey of plant secondary metabolites for DNA polymerase beta lyase inhibitors, we found that a crude methyl ethyl ketone extract prepared from Maytenus putterlickoides showed strong inhibition of the lyase activity of DNA polymerase beta in an in vitro assay. Bioassay-guided fractionation of the extract, using an in vitro assay, resulted in the discovery of a new active principle, 30-(4'-hydroxybenzoyloxy)-11alpha-hydroxylupane-20(29)-en-3-one (1), as well as a known compound, (-)-epicatechin (2). Compounds 1 and 2 exhibited DNA polymerase beta lyase inhibitory activity with IC50 values of 62.8 and 18.5 microM, respectively. Compound 2 was capable of potentiating the action of the monofunctional methylating agent methyl methanesulfonate in cultured human cancer cells, consistent with the possible utility of inhibitors of this type in vivo.     

氧化苦参碱体外抗乙型肝炎病毒作用

目的:体外观察氧化苦参碱(OM)抗乙型肝炎病毒(HBV)作用,并初步探讨其作用机制.方法:用125,250,500,1000,2000 mg·L-1OM连续作用于90％汇合度的HepG2.2.15细胞9d,以MTT比色法观察药物细胞毒性;用酶联免疫法测定细胞上清液中乙型肝炎病毒e抗原(HBeAg),乙型肝炎病毒s抗原(HBsAg);采用荧光定量PCR法(FQ-PCR)测定细胞上清液中HBV DNA,细胞中HBV DNA和共价闭合环状DNA(cccDNA).结果:OM对细胞内HBV DNA和cccDNA,以及细胞外HBV DNA均有抑制作用,随着浓度升高抑制作用加强,2000 mg·L-1OM对细胞内HBV DNA,cccDNA和细胞外HBV DNA的抑制率分别为64.56％,52.12％,54.25％;对HBsAg和HBeAg的分泌也有抑制作用,且浓度越高、处理时间越长,抑制作用越明显;在相同条件下对HBsAg的抑制作用强于HBeAg,OM浓度为2000 mg·L-1时对HBsAg和HBeAg的抑制率分别为51.59％,17.88％.结论:OM能有效抑制HepG2.2.15细胞中HBV复制,该作用是抑制病毒核酸复制和基因表达的结果.     

Investigation of korean plant extracts for potential phytotherapeutic agents against B-virus hepatitis

The present study was undertaken to identify potential phytotherapeutic agents among the aqueous extracts of 151 herbal medicinal plants of Korea. Extracts were assayed for (1) binding potency to hepatitis B-virus surface antigen (HBsAg), and (2) inhibition of serum hepatitis B-virus DNA polymerase activity. Of the 151 aqueous plant extracts tested, 33 demonstrated a positive precipitation reaction with HBsAg⁺ serum, and 15 of these specifically bound HBsAg in serum as well as recombinant HBsAg (Hepavax®). Nine extracts competitively inhibited >80% of anti-HBsAg antibody binding to Hepavax® HBsAg. Furthermore, nine of these 15 plant extracts also exhibited greater than 25%—50% inhibition of hepatitis B-virus (HBV) DNA polymerase activity, and also significantly stimulated production of the potent cytokine, tumour necrosis factor (TNF). None of the extract concentrations used altered liver function parameters of normal rats.     

Bioactive isomalabaricane triterpenoids from Rhabdastrella globostellata that stabilize the binding of DNA polymerase beta to DNA

Four isomalabaricane triterpenoids were isolated from an extract of the sponge Rhabdastrella globostellata that was active in an assay measuring stabilization of the binding of DNA with DNA polymerase beta. The known compounds stelliferin riboside (1) and 3-epi-29-acetoxystelliferin E (2) were shown to induce 29% and 23% binding, respectively, at 28 microg/mL, while the new compound stellettin J (3) induced 5% binding at 28 microg/mL. The new compound stellettin K (4) had no activity in the binding assay. The compounds were characterized by spectroscopic methods. These compounds displayed varying levels of activity toward the A2780 ovarian cancer cell line, revealing structure-based effects on both the level of cytotoxicity and DNA-polymerase beta binding. This is the first report of natural products with the ability to promote stabilization of the DNA-polymerase beta covalent binary complex.     

Antiviral and anti-proliferative glycoproteins from the rhizome of Smilax glabra Roxb (Liliaceae)

The glycoproteins possessing antiviral and anti-proliferative activities were isolated from the Chinese medicinal herb Smilax glabra (known as tufuling), by extraction with 0.2 M NaCl, ammonium sulfate precipitation, fetuin-agarose affinity chromatography and gel filtration. The molecular mass of the fetuin-binding glycoprotein (designated SGPF2) was estimated to be about 58 kDa, with a major protein subunit of 26 kDa. The non-fetuin binding glycoproteins (in the unadsorbed fraction) were further separated into 5 different subfractions (SGPF1a-SGPF1e) with anion-exchange chromatography, all of which also contained the major band at 26 kDa. All the isolated proteins of 26 kDa had similar N-terminal amino acid sequences, implying that they were probably the isoforms originated putatively from a multigene family with different binding affinity and ionic strength. The glycoprotein SGPF2 exhibited antiviral activity against respiratory syncytial virus (RSV) with a median inhibitory concentration (IC(50)) of 62.5 microg/ml and Herpes simplex virus type 1 (HSV-1) had an IC(50) of 31.3 microg/ml. The glycoprotein potencies for antiviral activity appeared to depend on the molecules' binding affinity for fetuin, that is, the fetuin-binding protein was more potent than the non-fetuin binding proteins. Further examination revealed that these glycoproteins also had the ability to suppress the proliferation of MCF-7 cells. The possible mechanism of anti-proliferative action as analyzed by DNA flow cytometry indicated that they could induce apoptosis mediated via sub-G(1) phase of the MCF-7 cell cycle. For example, there was an increase by 75.8% of the control level of apoptosis after incubation with SGPF1a.     

赋肝能抗病毒作用实验研究

 目的研究中药制剂赋肝能(由植物鹅绒蒌陵菜Potentilla asserina L.分离提取而得的主要活性成分)对乙型肝炎病毒(HBV)复制的抑制作用。方法建立以HBV转染的人肝癌细胞系(Hep G2)2.2.15细胞系为体外模型,HBV静脉感染、血清DHBV DNA(duck hepatitis B virus DNA)呈强阳性的北京鸭为体内模型,分别观察2.2.15细胞含药培养基中HBsAg和HBeAg及鸭血清中DHBV-DNA水平。结果 体外实验中,8 d时,各剂量组赋肝能对HBsAg和HBeAg均有明显抑制作用。大剂量赋肝能对HBsAg和HBsAg有显著抑制作用(P＜0.01)。体内实验观察,大剂量和中剂量赋肝能对DHBV-DNA有明显抑制作用(P＜0.05,P＜0.01),其抑制作用呈剂量依赖性和时间依赖性。结论赋肝能对乙型肝炎病毒具有明显抑制作用,可能成为较好的治疗乙型肝炎的临床用药。     

Hepatoprotective and antiviral properties of isochlorogenic acid A from Laggera alata against hepatitis B virus infection

Ethnopharmacological relevance

The aim of this study was to determine the anti-hepatitis B effect of isochlorogenic acid A isolated from Laggera alata (Asteraceae), a traditional Chinese herbal medicine.

Materials and methods

The anti-hepatitis B activity of isochlorogenic acid A was evaluated by the d-galactosamine (D-GalN)-induced HL-7702 hepatocyte damage model and the HBV-transfected HepG2.2.15 cells.

Results

Isochlorogenic acid A significantly improved HL-7702 hepatocyte viability and markedly inhibited the productions of HBsAg and HBeAg. The inhibitory rates of isochlorogenic acid A on the HBsAg and HBeAg expressions were 86.9% and 72.9%, respectively. In addition, isochlorogenic acid A declined markedly the content of hepatitis B virus covalently closed circular DNA (HBV cccDNA) and induced significantly the heme oxygenase-1 (HO-1) expression in HepG2.2.15 cells.

Conclusions

Isochlorogenic acid A was verified to possess the potent anti-hepatitis B activity. The anti-HBV target of isochlorogenic acid A is probably associated with blocking the translation step of the HBV replication. Overexpression of HO-1 may contribute to the anti-HBV activity of isochlorogenic acid A by reducing the stability of the HBV core protein and thus blocking the refill of nuclear HBV cccDNA. Additionally, the hepatoprotective effect of isochlorogenic acid A could be achieved by its antioxidative property and induction of HO-1.     

山芝麻含药血清对HepG2.2.15细胞HBV复制的抑制作用

目的观察山芝麻体外抗乙型肝炎病毒(HBV)的作用。方法采用血清药理学方法,在HBV的体外细胞培养系统中(2.2.15细胞)进行山芝麻抗HBV作用观察。结果山芝麻含药血清在HepG2.2.15细胞培养中可有效地抑制细胞HBV DNA的复制,其作用呈明显的量效和时效反应关系。结论山芝麻在体外能明显抑制HBV。     

地五养肝方对HBeAg阴性慢性乙型肝炎患者外周血T淋巴细胞亚群和细胞因子的影响

目的 观察地五养肝方对HBeAg阴性慢性乙型肝炎（CHB）患者外周血T淋巴细胞亚群和细胞因子的影响.方法 将103例患者随机分为对照组和治疗组,对照组予以恩替卡韦治疗,治疗组在对照组治疗基础上加用地五养肝方.两组共治疗48周.观察两组患者HBV DNA转阴率,检测外周血细胞因子IL-17、IL-10的水平,并用流式细胞技术检测外周血CD3＋、CD4圾CD8＋T淋巴细胞的表达情况.结果 治疗48周后,治疗组患者HBV DNA转阴率显著高于对照组（P＜0.05）,CD8＋水平较对照组明显下降（P＜0.01）,CD4＋及CD4＋/CD8＋水平较对照组升高更显著（P＜0.05）,IL-17、IL-10水平较对照组降低更明显（P＜0.05）.结论 T淋巴细胞和细胞因子在HBeAg阴性CHB的发病及转归中起到非常重要的作用,地五养肝方与恩替卡韦联用能显著改善外周血T淋巴细胞亚群状态,调节细胞因子IL-17、IL-10的水平,提高抗病毒疗效.     

叶下珠复方肝丹体外抗HBV作用特点的实验研究

目的探讨叶下珠复方肝丹对ＨＢＶ的体外作用特点。方法通过２．２．１５细胞系模型的改进实验方法,连续每天测定相关数据,动态观察药物对ＨＢＶ的作用情况。结果有如下特征：①有１个药物作用曲线,即从开始用药到见到效果有个过程,需４ｄ～５ｄ,较强的抑制ＨＢＶ作用可维持３ｄ～４ｄ,然后逐渐减弱、消失;②在ＨＢＶ分泌、复制旺盛的时期,药物对ＨＢＶ的抑制作用表现得越强,越能显示出药物的作用;③药物的作用具有呈正比的量效关系。结论体外实验证实肝丹具有抗ＨＢＶ的作用。     

乙型肝炎抗病毒治疗停药后复发与血清 HBV DNA 水平的相关性分析

目的探讨乙型肝炎（HB）抗病毒治疗停药后复发与血清HBVDNA水平的相关性。方法选取180例接受阿德福韦酯治疗的HB患者,检测患者治疗前及治疗4周后血清HBVDNA水平。结果HBeAg（＋）时,治疗前HBVDNA水平〉10±7copyies／mL复发率是56％,10±6～10±7copies／mL复发率是36％,〈10。copies／mL复发率是10％,HBVDNA水平〉10^7copies／mL停药复发率最高,〈10^6copies／mL最低。治疗4周后,HBVDNA水平〈10±3copies／mL复发率是18％,较基线下降幅度≥3lgcopies／mL者复发率是20％,较基线下降幅度2lgcopies／mL者复发率是21％,较基线下降幅度1lgcopies／mL者复发率是37％,HBVDNA水平〈10±3copies／mL复发率最低,较基线下降幅度1lgcopies／mL者复发率最高。HBeAg（-）时,治疗前HBVDNA水平〉10^7copies／mL复发率是27％,10^6～10^7copies／mL复发率是11％,〈10^6copies／mL复发率是7％,HBVDNA水平〉10。copies／mL停药复发率最高。治疗4周后,HBVDNA水平〈10^3copies／mL复发率是6％,较基线下降幅度≥3lgcopies／mL者复发率是10％,较基线下降幅度2lgcopies／mL者复发率是11％,较基线下降幅度llgcopies／mL者复发率是63％,HBVDNA水平〈10^3copies／mL复发率最低。较基线下降幅度1lgcopies／mL者复发率最高。结论HB患者抗病毒治疗停药后复发和患者治疗前的血清HBVDNA水平、治疗早期HBVDNA水平及血清HBVDNA水平降幅存在相关性。     

Mulberrofuran G Protects Ischemic Injury‐induced Cell Death via Inhibition of NOX4‐mediated ROS Generation and ER Stress

The aim of this study was to investigate the neuroprotective effect of mulberrofuran G (MG) in in vitro and in vivo models of cerebral ischemia. MG was isolated from the root bark of Morus bombycis. MG inhibited nicotinamide adenine dinucleotide phosphate oxidase (NOX) enzyme activity and oxygen–glucose deprivation/reoxygenation (OGD/R)‐induced NOX4 protein expression in SH‐SY5Y cells. MG inhibited the expression of activated caspase‐3 and caspase‐9 and cleaved poly adenine dinucleotide phosphate‐ribose polymerase in OGD/R‐induced SH‐SY5Y cells. In addition, MG protected OGD/R‐induced neuronal cell death and inhibited OGD/R‐induced reactive oxygen species generation in SH‐SY5Y cells. In in vivo model, MG‐treated groups (0.2, 1, and 5 mg/kg) reduced the infarct volume in middle cerebral artery occlusion/reperfusion‐induced ischemic rats. The MG‐treated groups also reduced NOX4 protein expression in middle cerebral artery occlusion/reperfusion‐induced ischemic rats. Furthermore, protein expression of 78‐kDa glucose‐regulated protein/binding immunoglobulin protein, phosphorylated IRE1α, X‐box‐binding protein 1, and cytosine enhancer binding protein homologous protein, mediators of endoplasmic reticulum stress, were inhibited in MG‐treated groups. Taken together, MG showed protective effect in in vitro and in vivo models of cerebral ischemia through inhibition of NOX4‐mediated reactive oxygen species generation and endoplasmic reticulum stress. This finding will give an insight that inhibition of NOX enzyme activity and NOX4 protein expression could be a new potential therapeutic strategy for cerebral ischemia. Copyright © 2016 John Wiley & Sons, Ltd.     

六月青总皂苷对HepG2.2.15细胞HBV复制的抑制作用

目的 观察六月青总皂z苷(the terpenoids of Liuyueqing,TLYQ)体外抗乙型肝炎病毒(HBV)的作用.方法 采用血清药理学方法 ,在HBV的体外细胞培养系统中(2.2.15细胞)进行TLYQ抗HBV作用观察.结果 TLYQ含药血清在HepG2.2.15细胞培养中可有效地抑制细胞HBV DNA的复制,其作用呈明显的量效和时效反应关系.结论 TLYQ在体外能明显抑制HBV,是六月青主要活性成分之一.     

DNA polymerase beta inhibitors from Baeckea gunniana

Crude plant extracts were surveyed for their ability to inhibit DNA polymerase beta. A methyl ethyl ketone extract prepared from Baeckea gunniana was identified as a potent inhibitor of the enzyme. Bioassay-guided fractionation of the extract, using an assay to monitor the inhibitory potential of individual fractions toward DNA polymerase beta, led to the isolation of four active ursane and oleanane triterpenoids (1-4). Inhibitory principle 1 is a new natural product, and 2 is a novel compound. Their structures were established as 3 beta-hydroxyrus-12,19(29)-dien-28-oic acid (1) and 3 beta-hydroxyrus-18,20(30)-dien-28-oic acid (2) by spectroscopic analysis and by comparison with the data for the structurally related compound ursolic acid (4). Also isolated as a DNA polymerase beta inhibitor was oleanolic acid (3). Compounds 1-4 had IC50 values of 5.3-8.5 microM as inhibitors of polymerase beta in the presence of bovine serum albumin (BSA) and 2.5-4.8 microM in the absence of BSA.     

白藜芦醇及其衍生物抗乙型肝炎病毒体外实验研究

 目的 探讨白藜芦醇(resveratrol,反式3,4′,5-三羟基-二苯乙烯)及其衍生物体外抗乙型肝炎病毒(HBV)的作用,及相关 的量效关系与构效关系。方法 将白藜芦醇及其衍生物作用于HepG2.2.15细胞系,MTT法检测样品对HepG2.2.15细胞的毒 性,ELISA法检测细胞上清中HBsAg,HBeAg的变化,实时荧光定量PCR法检测细胞与其上清中总HBV DNA含量,流式细胞仪 检测白藜芦醇时HepG2.2.15的凋亡作用,进行综合评价。结果 白藜芦醇对抑制HepG2.2.15细胞分泌HBsAg,HBeAg治疗指 数(TI)分别为(3.26±0.39),(2.91±0.12);在低毒或无毒浓度(0.11 mol·L^-1)下降低DNA拷贝数;呈浓度依赖性诱导 HepG2.2.15细胞凋亡。结论 白藜芦醇及其衍生物在体外有一定的抗乙型肝炎病毒作用,可能是通过抑制HBV DNA复制, 诱导感染HBV的HepG2.2.15细胞凋亡而发挥抗病毒活性。综合评价白藜芦醇及其衍生物的活性,即白藜芦醇苷(Rn1)、甲氧 基取代羟基的苷[4′-甲氧基-3,3′,5-二苯乙烯-3-葡萄糖苷(Rn3)]效果较好。     

新的核苷类化合物β-L-D4A及其异构体体外抗HBV作用

 目的探索比较β-L-D4A(L-D4A)及其异构体体外抗HBV作用,以期获得高效、高治疗指数(TI)的新一类抗HBV化合物。方法L-D4A及其异构体干预2.2.15细胞(Hep G2细胞进行HBV基因组转染后所得)培养,ELISA法检测上清HBs Ag, HBe Ag;³²p标记HBV DNA为探针,Southern印迹法检测上清液及细胞内HBV DNA,再以计算机图像处理进行定量分析,求出50%抑制的药物浓度(ED₅₀)。洗去HBV探针后,以³²P标记细胞色素氧化酶ⅢDNA为探针再次进行杂交,以探索线粒体的毒性。以MTT法检测不同浓度药物的细胞毒性,求出50%细胞抑制的药物浓度(ID₅₀)。结果上清液病毒DNA的Southern印迹结果显示,L型异构体较之D型异构体具有更强的HBV DNA抑制作用,L-D4A最为突出,并呈显明显的浓度依赖性,计算出ED₅₀为0.2μmol·L^-1;D-D4A具有明显的线粒体毒性,L-D4A则不明显;L-D4A细胞毒性实验显示ID₅₀为200μmol·L^-1;低浓度L-D4A对上清HBs Ag,HBe Ag无明显的影响,高浓度时有显著的抑制作用。结论体外实验显示L-D4A具有明显的抑制病毒DNA复制作用,且无明显的细胞毒性和线粒体毒性,TI(ID₅₀/ED₅₀)值为1 000,高于拉米夫定组(TI值为750),将有望进一步得到开发。     

Effects of Rubiadin isolated from Prismatomeris connata on anti‐hepatitis B virus activity in vitro

Prismatomeris connata was a kind of Rubiaceae plant for treatment of hepatitis, hepatic fibrosis and silicosis. Whereas, the effective components of Prismatomeris connata remains unexplored. The aim of this study was to investigate the inhibitory effects and mechanisms of Rubiadin isolated from Prismatomeris connata against HBV using HepG2.2.15 cells. The levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme‐linked immunosorbent assay. HBV DNA was qualified q‐PCR. Rubiadin was isolated by silica gel column. The structure of the compound was elucidated by HPLC, FT‐IR, ¹H‐NMR, ¹³C‐NMR and identified as 1,3‐Dihydroxy‐2‐methyl‐9, 10‐anthraquinone. Rubiadin significantly decreased HBeAg,HBcAg secretion level and inhibit HBV DNA replication. Rubiadin inhibits the proliferation of the cells and HBx protein expression in a dose‐dependent manner. The intracellular calcium concentration was significantly reduced. These results demonstrated that Rubiadin could inhibit HepG2.2.15 cells proliferation, reduce the level of HBx expression, and intracellular free calcium, which might become a novel anti‐HBV drug candidate.     

pQE-hbFGF表达系统的构建及其蛋白质的表达和纯化

 目的　构建pQE-hishbFGF表达载体,通过一步纯化得到6×HishbFGF蛋白质。方法　用PCR扩增目的基因hbFGF ,连接到pQE40载体上,转化到Top10菌株筛选重组子,经测序后将质粒转化到表达菌M15上,经IPTG诱导表达,超声波破菌后通过镍离子螯合层析一步纯化得6×HishbFGF蛋白质,ELISA鉴定产物 ,用MTT法检测产物促细胞增殖的生物活性。结果　hbFGF基因插入pQE载体中,在M15中6×HishbFGF的表达量达到20%,且为可溶性表达。经一步纯化后得到N端带 6个组氨酸的hbFGF蛋白,纯度为96%,6×HishbFGF具有免疫原性及促进NIH3T3细胞增殖的活性。结论　 6×HishbFGF蛋白质在M15中可溶性地高效表达,并经一步亲和层析得到纯度高活性高的产物,降低了生产成本,为大规模生产hbFGF及hbFGF的应用研究奠定了基础。