The inhibitory effect of Rheum palmatum against coxsackievirus B3 in vitro and in vivo

Coxsackievirus B(3)(CVB(3)) infection is the major cause of viral myocarditis, as well as dilated cardiomypathy. Rhubarb is one of the oldest and best-known traditional Chinese medicines. We initiated this study to determine the antiviral effect of an ethanol extract from the roots and rhizoma of Rheum palmatum (R. palmatum, one of the Chinese Rhubarbs), against CVB(3) in tissue culture cells and in a mouse model. The ethanol extract from R. palmatum showed significant inhibitory activity against CVB(3) on HEp-2 cells when added after infection, with IC(50) of 4 μg/ml, TI of 10. The medicated mouse serum still contained the pharmaceutical compound 24 h after intraperitoneal injection, and exhibited an antiviral effect on CVB(3)-infected cells, especially in the 0.3 and 0.5 g/kg/day treatment groups. Furthermore, the CVB(3)-infected mice were treated with the extract solution with dosages of 0.3 g/kg/day beginning 24 h post-CVB(3) exposures. The ethanol extract treated mice showed alleviated clinical signs, better survival rate, prolonged MTD and decreased viral titers compared to the virus control group. Our results indicate that the ethanol extract from R. palmatum has the anti-CVB(3) activity in vitro and in vivo and thus provides a re-evaluation of this old remedy with a broad therapeutic potential.     

Glycyrrhizic acid as the antiviral component of Glycyrrhiza uralensis Fisch. against coxsackievirus A16 and enterovirus 71 of hand foot and mouth disease

Ethnopharmacological relevance

The radices of Glycyrrhiza uralensis Fisch. and herbal preparations containing Glycyrrhiza spp. have been used for thousands of years as an herbal medicine for the treatment of viral induced cough, viral hepatitis, and viral skin diseases like ulcers in China. Glycyrrhizic acid (GA) is considered the principal component in Glycyrrhiza spp. with a wide spectrum of antiviral activity.

Aim

The present study attempt to validate the medicinal use of Glycyrrhiza uralensis for hand, foot and mouth disease (HFMD) and further to verify whether GA is an active antiviral component in the water extract of Glycyrrhiza uralensis.

Materials and methods

Radices of Glycyrrhiza uralensis Fisch. were extracted with hot water. The chemical contents of the extract were profiled with HPLC analysis. The antiviral activity of the extract and the major components was evaluated against infection of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) on Vero cells. The cytopathic effect caused by the infection was measured with MTT assay. Infectious virion production was determined using secondary infection assays and viral protein expression by immunoblotting analysis.

Results

The extract at 1000 μg/ml suppressed EV71 replication by 1.0 log and CVA16 by 1.5 logs. The antiviral activity was associated with the content of GA in the extract since selective depletion of GA from the extract by acid precipitation resulted in loss of antiviral activity. In contrast, the acid precipitant retained antiviral activity. The precipitant at a concentration of 200 μg/ml inhibited EV71 and CVA16 replication by 1.7 and 2.2 logs, respectively. Furthermore, GA dose-dependently blocked viral replication of EV71 and CVA16. At 3 mM, GA reduced infectious CVA16 and EV71 production by 3.5 and 2.2 logs, respectively. At 5 mM, CVA16 production was reduced by 6.0 logs and EV71 by 4.0 logs. Both EV71 and CVA16 are members of Enterovirus genus, time-of-drug addition studies however showed that GA directly inactivated CVA16, while GA anti-EV71 effect was associated with an event(s) post virus cell entry.

Conclusions

This study validated the medicinal usefulness of radices Glycyrrhiza uralensis against the etiological agents of HFMD. In addition to the identification of GA as the antiviral component of Glycyrrhiza uralensis against EV71 and CVA16 infection, this study also reveals that GA inhibits EV71 and CVA16 with distinct mechanisms.     

羊栖菜提取物的体外抗病毒作用

目的:探讨羊栖菜提取物的体外抗病毒作用。方法:对羊栖菜的提取物进行了常见的第三类病原体病毒呼吸道合胞病毒(RSV)、单纯疱疹病毒1型(HSV-1)、肠道病毒71型(EV71)、柯萨奇病毒A_(16)、柯萨奇病毒B_2、柯萨奇病毒B_3、柯萨奇病毒B_5(CVA_(16)、CVB_2、CVB_3、CVB_5)等7种病毒的抗病毒效应研究,显微镜下观察细胞存活率。结果:羊栖菜提取物对上述7种病毒的抑制能力远远优于广谱抗病毒药物利巴韦林,尤以对EV71的抑制效果最为明显,抑毒指数极高。结论:羊栖菜提取物对这7种病毒均具有良好的抑制作用,为进一步研究羊栖菜提取物对第三类病原体病毒的广谱抗病毒作用奠定基础。     

Antiviral activities of some Ethiopian medicinal plants used for the treatment of dermatological disorders

Acokanthera schimperi (Apocynaceae), Euclea schimperi (Ebenaceae), Inula confertiflora (Asteraceae), Melilotus elegans (Leguminosae), and Plumbago zeylanica (Plumbaginaceae), are some of the medicinal plants used in Ethiopia for treatment of various skin disorders. In this study, the antiviral activities of the 80% methanolic extracts of these plants have been examined against coxsackievirus B3 (CVB3), influenza A virus and herpes simplex virus type1 Kupka (HSV-1) using cytopathic effect (CPE) inhibitory assays in HeLa, MDCK, and GMK cells, respectively. In parallel, the cytotoxicity was quantified using a crystal violet uptake assay. The antiviral activity of the most active compound was confirmed with plaque reduction assays. The results revealed that the extracts of Acokanthera schimperi and Euclea schimperi showed antiviral activity against all three tested viruses albeit with unequal efficacy. Whereas the Acokanthera schimperi extract exhibited the strongest activity against CVB3, the extract of Euclea schimperi inhibited influenzavirus A replication most effectively. A weak anti-influenzavirus A activity was also exhibited by the other plant extracts tested. In addition, CVB3 was inhibited by the extracts of Plumbago zeylanica and HSV-1 by Inula confertiflora. Thus, the extracts of these plants, particularly those of Acokanthera schimperi, Euclea schimperi and Inula confertiflora which showed activity against CVB3 and HSV-1 support their traditional use in the treatment of skin diseases of viral origin.     

甘露消毒丹抗肠道病毒71型作用的体外实验研究

目的：探讨甘露消毒丹体外抗肠道病毒71型（EV71）的作用。方法以病毒唑为对照药,运用细胞培养技术,通过直接抑毒实验、治疗性抑毒实验、预防性抑毒实验、预防＋治疗性抑毒实验4组实验观察甘露消毒丹体外抗EV71的病毒抑制率。结果在直接抑毒实验、治疗性抑毒实验、预防＋治疗性抑毒实验中,甘露消毒丹的EV71病毒抑制率高于病毒唑;在预防性抑毒实验中,甘露消毒丹与病毒唑的EV71病毒抑制率几乎为零。结论甘露消毒丹抗EV71作用强于病毒唑,且其抗EV71效应体现在影响EV71复制的多个环节。     

中药抗EV71病毒活性及其在治疗手足口病中的应用

手足口病是由肠道病毒引起的传染病.柯萨奇病毒A16和肠道病毒71型(EV71)是本病的主要致病原,且后者较凶险,更易导致严重并发症而致死、致残,危害性较大.目前对EV71感染尚缺乏有效的药物,因此研制抗EV71病毒活性的药物意义重大.中国中药资源丰富,具有进一步开发和利用的独特优势,从中筛选出具有抗EV71病毒活性的成分,进而开发出新的抗病毒药物具有很大潜力.同时,我国自1981年在上海始见手足口病以来,中药在临床治疗手足口病方面的报道日益增多且方法多样,大多疗效满意,对提高临床诊治水平、控制疾病的发生发展有着积极作用,展现了其独特的临床优势与特点.     

黄芪对柯萨奇B_3病毒感染培养大鼠心肌细胞Ca~（2＋）内流及该病毒RNA复制

本研究结果发现，黄芪对柯萨奇Ｂ_３病毒（ＣＶＢ_３）感染大鼠心肌细胞及正常对照心肌细胞的Ｃａ~（２＋）内流均呈显著抑制作用（ｐ＜０．０１，Ｐ＜０．０５））；若在病毒感染１ｈ后加黄芪，经４８ｈ培养，对正常对照及感染细胞的Ｃａ~（２＋）内流亦有抑制作用（Ｐ＜０．０５）；同时细胞中ＣＶＢ_３－ＲＮＡ含量显著少于病毒对照组（Ｐ＜０．００１）。提示黄芪具有钙拮抗作用，可减少病毒感染引起的心肌Ｃａ~（２＋）内流量，有可能减轻感染细胞的继发性Ｃａ~（２＋）损伤；且对细胞中病毒ＲＮＡ的复制具有抑制作用，显示了其在病毒性心肌炎临床治疗上的应用价值。     

黄芪对柯萨奇B3病毒感染培养大鼠心肌细胞Ca2＋内流及该病毒RNA复制

本研究结果发现,黄芪对柯萨奇Ｂ３病毒（ＣＶＢ３）感染大鼠心肌细胞及正常对照心肌细胞的Ｃａ＾２＋内流均呈显著抑制作用（Ｐ＜０．０１,Ｐ＜０．０５）;若在病毒感染１ｈ后加黄芪,经４８ｈ培养,对正常对照及感染细胞的Ｃａ＾２＋内流亦有抑制作用（Ｐ＜０．０５）。同时细胞中ＣＶＢ３－ＲＮＡ含量显著少于病毒对照组（Ｐ＜０．００１）。提示黄芪具有钙拮抗作用,可减少病毒感染引起的心肌Ｃａ＾２＋内流量,有可能减轻感染     

益心康对柯萨奇B_(3m)病毒性心肌炎小鼠作用的实验研究

目的 :实验观察中药复方益心康 ,对实验性病毒性心肌炎小鼠心肌细胞保护作用和对心肌细胞内病毒复制的抑制作用。方法 :诱导Balb/c小鼠形成病毒性心肌炎模型 ,分别予益心康、病毒唑稀释液、生理盐水灌胃。测每个小鼠血清磷酸肌酸激酶 (CK)及其同功酶MB(CK -MB) ,测心肌细胞感染CVB3mRNA量。结果 :中药治疗组小鼠的血清CK和CK -MB明显低于其它两个治疗组。中药治疗组小鼠的心肌细胞含有的病毒RNA量明显少于其它两个治疗组。结论 :益心康对柯萨奇B3m病毒性心肌炎小鼠有保护作用 ,并抑制柯萨奇病毒B3m在心肌细胞中的复制。     

热毒宁注射液组分抗CoxA16和EV71病毒活性研究

目的:明确热毒宁注射液组分抗柯萨奇病毒A16(coxsackievirus A16,Cox A16)和肠道病毒71(enterovirus 71,EV71)病毒的活性。方法:考察热毒宁注射液1~19号组分(RND1~19)对非洲绿猴肾细胞(Vero)的毒性,明确半数中毒浓度(TC50);进一步利用病毒感染的Vero细胞模型研究各组分的抗病毒活性及量效关系。采用50倍半数组织培养感染量(TCID50)Cox A16和EV71病毒分别感染Vero细胞(1万个/孔),热毒宁注射液组分(15.625~500 mg·L-1)给药3 d后,考察抗Cox A16和EV71病毒活性(n=3)。活性组分(最佳浓度)给药8 h后,检测病毒载量。结果:热毒宁注射液各组分的Vero细胞毒性,TC50从1 000 mg·L-1到221 mg·L-1不等;抗病毒活性及量效关系研究表明,RDN7和RDN18具有较好的抗Cox A16活性,半数有效浓度(EC50)分别为460,92 mg·L-1,治疗指数(TI)分别为1.44,2.40;RDN6和RDN17具有较好的抗EV71活性,EC50分别为395,378 mg·L-1,TI分别为2.22,1.10;病毒载量研究表明,RDN7极显著降低Vero细胞中Cox A16病毒载量(P0.01),RDN6,RDN17极显著地降低Vero细胞中EV71病毒载量(P0.01),而RDN18则没有上述活性。结论:明确了热毒宁注射液组分的抗Cox A16和EV71病毒活性。     

广枣总黄酮对CVB3病毒引起细胞凋亡的影响

目的:建立病毒性心肌炎的体外模型,用新的化学方法提取到含量较高的广枣总黄酮,研究广枣总黄酮抗病毒的效果.方法:化学法提取广枣总黄酮,高效液相法测定其含量.培养HeLa细胞和乳鼠心肌细胞,在HeLa细胞和心肌细胞水平上对柯萨奇B3病毒进行毒力滴定,并测定广枣总黄酮的药物毒性,建立体外病毒性心肌炎模型,通过细胞病变抑制实验...     

In vitro anticancer activity of aqueous extracts and ethanol extracts of fifteen traditional Chinese medicines on human digestive tumor cell lines

The purpose of this study was to investigate the anticancer activity of 15 traditional Chinese medicines which are usually used for tumor patients in China. The MTT (methylthiazolyldiphenyl-tetrazolium bromide) method was applied to compare the antitumoral activity of the aqueous crude extracts and the ethanol crude extracts of these drugs on six human digestive tumor cell lines: human liver carcinoma cell lines (HepG-2 and SMMC-7721), human gastric cancer cell line (BGC-823), human colon adenocarcinoma cell lines (LoVo and SW-116) and esophagus adenocarcinoma cell line (CaEs-17). Most ethanol extracts demonstrated a more powerful inhibitory effect than aqueous extracts. Their IC50 values were between 10 microg/mL and 500 microg/mL. Among these drugs, Paris polyphylla Smith showed a predominant inhibitory effect on all the cell lines with IC50 values ranging from 10 microg/mL to 30 microg/mL. The findings in this study suggested that traditional Chinese medicines, especially Paris polyphylla Smith, might have potential anticancer activity on digestive cancer and its mechanism needs further study.     

重楼总皂苷提取工艺研究

目的:优选重楼总皂苷提取工艺。方法:以总皂苷得率为评价指标,采用单因素试验和正交试验优选最佳提取工艺。结果:优化后重楼总皂苷的提取工艺为:70%乙醇提取两次,第一次用10倍量提取2 h,第二次用8倍量提取1.5 h;按优选出的工艺进行提取,总皂苷得率为4.24%,重楼总苷Ⅰ、Ⅱ总提取率为93.28%。结论:验证试验结果表明,优选出的工艺稳定、合理、可行。     

薯蓣皂苷的分离提取研究

目的:以太白灯台七为原料,研究薯蓣皂苷的提取工艺.方法:加热回流溶剂萃取,沉淀分离.结果:对提取效果影响较大的因素有提取溶剂、提取次数、回流时间等.以甲醇回流提取2次,每次8h,提取效果较佳.并从粗皂苷中分离出一种白色片状结晶,经TLC和熔点鉴定为薯蓣皂苷.结论:该工艺简单易行,粗皂苷中薯蓣皂苷的含量可达64.9%,为开发利用太白灯台七植物资源提供了依据.     

重楼的基源、生长条件与质量相关性研究

目的 比较不同基源、生长条件及采收季节的重楼皂苷含量,为重楼的引种栽培提供科学依据.方法 采集不同基源、不同生长年限、不同海拔及不同采收季节的重楼药材,按2015年版《中华人民共和国药典》重楼总皂苷含量测定方法进行测定.色谱柱为ACQUITY UPLC?HSS T3(3.0 mm×100 mm,1.8μm),流动相为乙腈-水,梯度洗脱,检测波长203 nm,进样量5μl,流速0.5 ml/min,柱温35℃.结果 在所测样品中,总皂苷含量范围在1.35%~3.89%之间,含量顺序为:多叶重楼>滇重楼>华重楼;3~10年生的华重楼,生长年限越长,总皂苷含量越高;在海拔400~800 m生长的华重楼,总皂苷含量随着海拔上升而增加;春季采收的云南重楼其皂苷含量高于其他季节.结论 不同基源、生长条件、采收季节的重楼皂苷含量不同,引种栽培重楼应综合考虑.     

益心康对感染柯萨奇病毒B3的大鼠体外心肌细胞CVB3 mRNA复制及相关细胞因子的影响

[目的]通过检测益心康药液作用于感染柯萨奇病毒B3(CVB3)后的乳鼠心肌细胞中柯萨奇病毒mRNA的复制情况及其所释放的肿瘤坏死因子-α(TNF-α)及转化生长因子-β(TGF-β)表达情况,进一步探讨益心康对病毒性心肌炎的病毒复制及相关细胞因子的作用机制。[方法]使益心康的药液作用于乳鼠心肌细胞,用逆转录-聚合酶链反应(RT-PCR)法检测心肌细胞中柯萨奇病毒mRNA的复制情况及其所释放的TNF-α及TGF-β的表达水平。[结果]益心康在CVB3感染的病毒性心肌炎的发病及发展过程中有抑制CVB3复制的作用,同时可以减少相关细胞因子TNF-α、TGF-β的分泌。益心康组(D组)、益心康加地塞米松组(E组)的细胞中的CVB3 mRNA、TNF-α、TGF-β表达水平明显低于病毒感染组(P<0.01),同时低于利巴韦林组(P<0.05),均有统计学意义;D组、E组之间P>0.05,无统计意义。[结论]益心康对柯萨奇病毒感染心肌细胞具有显著保护作用。     

蜂胶提取物对荷瘤小鼠肿瘤免疫系统的影响研究

目的观察蜂胶提取物对荷瘤小鼠免疫系统的影响。方法通过将荷瘤小鼠移植S180肉瘤,观察蜂胶提取物对荷瘤小鼠NK细胞活性,淋巴细胞增殖以及淋巴细胞IL-2诱生的影响。结果蜂胶提取物对NK细胞杀伤活性有明显增强作用,能够显著提高淋巴细胞增殖能力和IL-2的含量(P<0.01)。结论蜂胶提取物能够显著提高荷瘤小鼠免疫系统的活性。     

抗柯萨奇B病毒性心肌炎胶囊复方提取物对病毒性心肌炎小鼠血清AST、LDH的影响

目的:观察抗柯萨奇B病毒性心肌炎胶囊复方(K-CoxB-JN)提取物对病毒性心肌炎小鼠心肌细胞的保护作用,并探讨其作用机制。方法:采用肢体Ⅱ导联心电图正常的雄性Balb/c小鼠腹腔注射CVB3,建立实验性小鼠病毒性心肌炎模型。将120只造模成功的雄鼠随机分模型组、黄酮低剂量组、多糖中黄酮中剂量组、原方水煎组,每组30只,各组小鼠在腹腔接种CVB3稀释液半小时后即给予相应药物(模型组灌胃生理盐水);另选取10只健康雄鼠作为正常对照组,腹腔接种同体积的生理盐水后给予生理盐水。将接种病毒当天定为第实验的1天,每日给药1次,连续给药14天,于第15天处死。观察小鼠生存状态及体重的变化,统计小鼠死亡率,计算心重指数,用分离的血清检测心肌酶CK、LDH的活性。结果:K-CoxB-JN各提取物组均能显著降低小鼠血清中AST、LDH的活性(P<0.05),改善小鼠心功能及病变小鼠体重情况、减少死亡率,其中多糖中黄酮中剂量组、原方水煎组效果最佳。结论:K-CoxB-JN提取物对病毒性心肌炎小鼠心肌细胞有很好的保护作用。