In vitro antiviral activities of Chinese medicinal herbs against duck hepatitis B virus

Fifty-six different Chinese medicinal herbs from 29 families were evaluated for their antiviral activities against duck hepatitis B virus (DHBV) in vitro. The DHBV DNA level in primary duck hepatocyte cultures was monitored by dot blot hybridization and the cytotoxicity was evaluated by MTT assay. Anti-DHBV activities were found more strongly in the aqueous extracts of Ardisia chinensis and Pithecellobium clypearia with selective indices of 2.6 and >2.7, respectively, which were comparable to that of 2',3'-dideoxycytidine. Further research on the isolation of the active antiviral phytochemicals from these herbs may provide alternative options for the treatment of chronic hepatitis B.     

扶正利湿活血复方对鸭胚肝细胞中鸭乙型肝炎病毒的抑制作用

目的 探讨扶正利湿活血中药复方水提物和醇提物对先天感染乙肝病毒(HBV)的鸭胚肝细胞乙型肝炎病毒(DHBV)的抑制作用.方法 荧光定量PCR技术对鸭胚尿囊液DHBV DNA分析筛选先天感染DHBV的阳性鸭胚,对DHBV阳性的鸭胚肝细胞原代培养;细胞接种后第3天分组加入不同剂量扶正利湿活血复方水提物和醇提物,共6 d,分别于药物干预前、药物干预第3天、第6天、停药第2天收集培养上清;辛酸钠法提取上清DHBV DNA,实时荧光定量PCR(FQ-PCR)检测分析血清DHBV DNA的含量.结果 水提物各剂量组组内不同时间点DHBV DNA载量相比无显著性差异(P﹥0.05),仅水提物低剂量组DHBV DNA载量显著低于相同时间点病毒对照组(P<0.05);醇提物中、低剂量组在给药后和停药后培养上清DHBV均明显下降(组间、组内比较均为P<0.01),扶正利湿活血复方醇提物高剂量组在给药后第3天时DHBV明显下降(组间、组内比较均为P<0.01),第6天时组内比较无明显下降(P>0.05),而在停药后第3天时组内与组间比较又均有显著性差异(均为P<0.01).结论 扶正利湿活血复方醇提物对鸭胚肝细胞中的DHBV有直接抑制作用,水提物在鸭体内可能不是通过直接抑制DHBV而起作用.     

水芹水提物在鸭原代肝细胞培养中对DHBV抑制作用的初步研究

 目的：应用鸭乙型肝炎病毒（DHBV）体外肝细胞培养模型，研究中草药水芹（SQ）抗乙型肝炎病毒（HBV）的作用。方法：制取北京雏鸭原代肝细胞（50万／ml／孔／24孔板）于37℃，CO₂培养24h存活后，除对照组外，给药组每天换药1次，连续加药5d并测定DNA的含量。结果：①在肝细胞毒性实验中，SQ对鸭肝细胞无毒性，半数有毒浓度（CD₅₀）大于10000μg·ml^－1。②在DHBV感染鸭的原代肝细胞培养中能够保护肝细胞，连续给药培养7d，肝细胞的贴壁状态、形态和存活量均优于对照组。③对DHBV DNA有明显的抑制作用，其抑制率（2500μg·ml^－1）为64％，半数有效浓度（ED₅₀）为1120．8μg·ml^－1，选择指数（SI）大于9。结论：实验结果提示，SQ对DHBV有一定的抑制作用。     

葫芦茶苷体内抗鸭乙型肝炎病毒及保肝作用研究＊

目的 考察葫芦茶苷对鸭乙型肝炎病毒(hepatitis B virus，HBV)的体内抑制作用及其保肝作用。方法 以广西麻鸭为实验动物，建立感染鸭乙肝病毒的鸭乙肝动物模型，随机分为模型组、拉米夫定阳性对照组和葫芦茶苷低、中、高剂量组，每组10只，每日灌胃给药1次，连续给药14天，模型组每天灌服等体积的生理盐水。采用ELISA法检测鸭血清乙型肝炎表面抗原(DHBsAg)和鸭血清乙型肝炎E抗原(DHBeAg)的含量。采用实时荧光定量PCR法检测鸭血清鸭乙肝病毒DNA的含量。并检测鸭血清谷丙转氨酶(ALT)和谷草转氨酶(AST)的变化，检测肝组织匀浆超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活性和还原型谷胱甘肽(GSH)、丙二醛(MDA)的含量。苏木精-伊红染色法(HE)观察鸭肝组织病理学变化。结果 与模型组比较，葫芦茶苷各剂量组能显著降低血清DHBsAg和DHBeAg的滴度以及DHBV DNA的含量(P < 0.05或P < 0.01)。而且能降低鸭血清ALT、AST活性和MDA含量，增加肝组织SOD、GSH-PX活性和GSH含量。病理学检查结果提示葫芦茶苷能显著减轻肝细胞损伤程度。结论 葫芦茶苷对鸭乙型肝炎病毒具有体内抑制作用，对DHBV引起肝损伤具有保护作用。     

玉郎伞水提物对慢性鸭乙型肝炎的保护作用

目的:建立鸭乙型肝炎慢性感染模型,研究玉郎伞水提物对鸭慢性乙型肝炎的保护作用。方法:1日龄的广西麻鸭经颈静脉接种0.2 m L的鸭乙型肝炎病毒(DHBV)强阳性血清,7 d后用荧光定量PCR筛选DHBV阳性鸭,随机分为4组,即:模型组,玉郎伞水提物高、低剂量组(按生药量计分别为20,10 g·kg-1)和秋水仙碱组(0.02 g·kg-1)。将未感染鸭作为正常组。除正常组外,其余各组每周均接种0.2 m L的DHBV强阳性血清,直到实验结束,以造成鸭乙型肝炎慢性感染。从造模第25周开始,连续灌胃给药5周。末次给药24 h后,所有动物从颈静脉采血,测定血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)活性,血清鸭乙型肝炎病毒表面抗原(DHBs Ag),DHBV-DNA含量。将所有动物处死,迅速摘取肝组织,测定肝匀浆超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px)的活性,丙二醛(MDA)的含量,肝组织中羟脯氨酸(Hyp)的含量。通过HE染色观察肝细胞损伤程度。结果:与正常组比较,模型组血清ALT,AST活性,HBs Ag,DHBV-DNA含量均显著升高(P0.05,P0.01)。肝组织中SOD,GSP-Px活性均显著降低,MDA和Hyp含量则显著升高(P0.05,P0.01)。与模型组比较,玉郎伞水提物高、低剂量组均能降低血清ALT活性和DHBV-DNA含量(P0.05,P0.01),均能增加肝组织匀浆中GSH-Px活性,降低MDA和Hyp的含量(P0.05,P0.01)。玉郎伞水提物高剂量组能降低血清AST活性和HBs Ag含量,增加肝组织SOD活性(P0.05,P0.01)。HE染色显示玉郎伞水提物能显著减轻肝细胞损伤。结论:玉郎伞水提物对DHBV所致的慢性鸭乙型肝炎有一定的保护作用。     

Antiviral activities of extracts isolated from Terminalis chebula Retz., Sanguisorba officinalis L., Rubus coreanus Miq. and Rheum palmatum L. against hepatitis B virus.

The antiviral effects of aqueous extracts of Terminalis chebula Retz., Sanguisorba officinalis L., Rubus coreanus Miq. and Rheum palmatum L. were examined by a cell culture system using a hepatitis B virus (HBV) producing cell line, HepG2 2.2.15. The extracts were assayed for the inhibition of HBV multiplication by measurement of HBV DNA and surface antigen (HBsAg) levels in the extracellular medium of HepG2 2.2.15 cells after an 8-day treatment. All extracts decreased the levels of extracellular HBV virion DNA at concentrations ranging from 64 to 128 microg/mL and inhibited the secretion of HBsAg dose dependently. Of the four tested plants, Terminalis chebula exhibited the most prominent anti-HBV activities.     

六月青多糖对鸭乙型肝炎病毒诱导肝损伤的保护作用研究

目的:观察六月青多糖(LYQP)对鸭乙型肝炎所致肝损伤的保护作用,为研究和开发新的抗乙型肝炎病毒(HBV)药物提供实验依据.方法:采用鸭乙型肝炎病毒(DHBV)诱导广西麻鸭乙型肝炎病毒肝损伤模型.分别检测给药前(T0)、给药7 d(T7),14 d(T14)及停药后3d(P3)血清中丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)的活性,同时采用酶联免疫吸附( ELISA)法测定上清液鸭乙型肝炎病毒表面抗原(DHBsAg)和e抗原(DHBeAg)的滴度,HE染色观察肝损伤程度.结果:与模型组相比,各给药组鸭血清ALT,AST的活性及血清DHBsAg,DHBeAg的滴度显著降低(P＜0.05或P＜0.01);停药3d后,LYQP高、中剂量组血清ALT,AST活性及血清DHBsAg,DHBeAg的滴度均无反跳现象.HE染色结果显示LYQP可显著减轻鸭肝组织病理损伤程度.结论:LYQP对DHBV所致肝损伤具有保护作用.     

不同产地叶下珠及其联合用药抗鸭乙型肝炎病毒的实验研究

本文应用重庆麻鸭乙型肝为动物模型进行不同产地中草药叶下珠及其联合中药苦参与抗菌药环丙沙星的抗鸭乙型肝炎病毒疗效初探，连续用药１个月后，结果表明；广西叶下珠，云南叶下珠能使鸭血清中鸭乙型肝炎病毒脱氧核糖核酸滴度下降（Ｐ＜０．０５），而重庆叶下珠无此作用（Ｐ＞０．０５）；云南叶下珠停药后有血清ＤＨＢＶ ＤＮＡ回升现象，广西叶下珠与苦参合用，无明显协同作用，但与环丙沙星合用，似有增强抗毒的作用（Ｐ＜０．     

京玉清肝胶囊抗鸭乙肝病毒效应及其机制探讨

目的：研究京玉清肝胶囊抗鸭乙型肝炎病毒（DHBV）的作用。方法：采用重庆麻鸭乙型肝炎动物模型口服灌胃，治疗28d，停药观察7d，检测用药前、后血清中DHBV DNA的改变情况及肝脏病理改变。结果：京玉清肝胶囊用药28d后鸭血清中的DHBV DNA降低有显著意义（P〈0．05），停药7d后未出现反跳。结论：京玉清肝胶囊在鸭体内有抑制鸭乙型肝炎病毒DNA的作用且停药后无反跳。     

复方玉郎伞多糖对鸭乙型肝炎病毒诱导的肝损伤的影响

目的：研究复方玉郎伞多糖（CYLSP）在鸭乙型肝炎病毒（DHBV）持续性感染模型中对DHBV所致的肝损伤的保护作用。方法：采用1 d龄雏鸭接种广西麻鸭乙型肝炎病毒（DHBV）强阳性血清,接种1周后用聚合酶链式反应（PCR）法筛选出DHBV强阳性鸭50只,随机分为5组,每组10只,CYLSP高、中、低剂量组（10,5,2.5 g·kg^-1）、拉米夫定（0.05 g·kg^-1）组和模型组,每日上午ig给药1次,连续14 d。于用药前（T₀）、用药7 d（T₇）和14 d（T₁₄）及停药后3 d（P₃）分别采血,同时检测血清中丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）的活性;在停药3 d后,取肝脏匀浆并除蛋白,检测肝匀浆液中总超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性以及谷胱甘肽（GSH）、丙二醛（MDA）的含量;HE染色处理肝脏组织切片,光镜观察病理学改变。结果：与同组T₀及模型组比较,各给药组鸭血清高、中剂量治疗组给药7 d （T₇）和14 d （T₁₄）即能明显降低血清ALT,AST的活性,停药3（P₃）天后,CYLSP高剂量组仍能显示出持续有效,没有出现反跳现象（P<0.05或P<0.01）;检测肝匀浆液中SOD,GSH-Px活性以及GSH,MDA的含量,CYLSP高、中剂量组和拉米夫定组仍能显示出持续有效,没有出现反跳现象（P<0.05或P<0.01）;HE染色显示CYLSP可显著减轻鸭肝组织病理损伤程度。结论：CYLSP对DHBV所致的肝损伤具有保护作用。     

用嗜肝DNA病毒模型筛选抗病毒中草药

对叶下珠等21种中草药进行抗乙肝病毒药物研究,通过体外及体内逐步筛选发现,叶下珠和虎杖提取物对鸭乙型肝炎病毒(DHBV)及乙型肝炎病毒(HBV)均有较好的抑制效果,旱莲草对HBVDNAp有一定的抑制作用。     

苍耳子提取物抗鸭乙型肝炎病毒作用的实验研究

目的 研究苍耳子提取物对鸭乙型肝炎的抗病毒作用.方法 选1日龄北京雏鸭,人工感染鸭乙型肝炎病毒(DHBV)后随机分为5组:苍耳子提取物3个剂量组(1,0.1和0.01 g·kg-1·d-1),对照组采用生理盐水和拉米夫定(3TC,2 mg·kg-1·d-1),每组6只,灌胃10 d,观察不同剂量苍耳子提取物抗DHBV作用效果.结果 苍耳子提取物(1 g·kg-1·d-1)剂量组对延缓鸭肝病理改变有一定的作用,但对DHBV DNA无作用.结论 苍耳子提取物对控制鸭乙型肝炎病毒引起的病理改变有一定作用.     

苦参碱脂质体抗鸭乙型肝炎病毒的体内实验研究

目的:观察苦参碱脂质体对鸭乙型肝炎病毒的影响。方法:对鸭乙型肝炎病毒(DHBV)实验感染的广州麻鸭雏鸭灌胃口服苦参碱脂质体和苦参碱,前者设20mg·kg~(-1)和10mg·kg~(-2)组,后者设20mg·kg~-组,每天1次,连续给药20天,另设病毒对照组。采用DHBV-DNA Dot Blot法,测定鸭血清DHBV-DNA水平值。结果:苦参碱脂质体20mg·kg~-组在给药后第10、15、20天鸭血清DHBV-DNA水平明显下降(P<0.01),效果优于苦参碱20mg·kg~(-1)组(P<0.05),与病毒对照组比较,差异有非常显著性意义(P<0.01)。苦参碱脂质体10mg·kg~(-1)组在给药后第20天及停药后3天鸭血清DHBV-DNA水平也有下降(P<0.05),与病毒对照组比较,差异有显著性意义(P<0.05),但与苦参碱20mg·kg~(-1)组比较,差异无显著性意义(P>0.05)。苦参碱脂质体2个剂量组停药后3天鸭血清DHBV-DNA水平无回升。结论:苦参碱脂质体对鸭乙型肝炎病毒有较好的抑制作用,优于同等剂量的普通苦参碱,其抗病毒效应相当于普通苦参碱1/2剂量的作用效果。     

复方六月雪对鸭乙型肝炎病毒DNA的抑制作用

目的:观察复方六月雪(CLYX)对鸭乙型肝炎病毒(DHBV)DNA的抑制作用,为研究和开发新的抗HBV药物提供实验依据。方法:采用1日龄雏鸭接种广西麻鸭DHBV强阳性血清,13天后用PCR法筛选出DHBV强阳性鸭。随机分成5组:CLYX高、中、低剂量组、模型组和阳性对照组,每组10只,各组雏鸭均灌胃给药14天。于用药前(T0)、用药7天(T7)、14天(T14)及停药后3天(P3)分别采血,采用荧光定量PCR(FQ-PCR)法检测血清DHBVDNA的含量。结果:CLYX各剂量组用药后血清DHBV DNA含量显著降低(P<0.01),停药3天后,CLYX低剂量组和阳性对照组血清DHBV DNA有反跳现象,而CLYX中、高剂量组血清DHBV DNA没有反跳现象。CLYX抑制DHBV DNA的作用有明显的量效和时效反应关系。结论:CLYX可有效地抑制DHBV DNA的复制。     

Antiviral potential of medicinal plants against HIV,HSV, influenza,hepatitis, and coxsackievirus: A systematic review

Viral infections are being managed therapeutically through available antiviral regimens with unsatisfactory clinical outcomes. The refractory viral infections resistant to available antiviral drugs are alarming threats and a serious health concern. For viral hepatitis, the interferon and vaccine therapies solely are not ultimate solutions due to recurrence of hepatitis C virus. Owing to the growing incidences of viral infections and especially of resistant viral strains, the available therapeutic modalities need to be improved, complemented with the discovery of novel antiviral agents to combat refractory viral infections. It is widely accepted that medicinal plant heritage is nature gifted, precious, and fueled with the valuable resources for treatment of metabolic and infectious disorders. The aims of this review are to assemble the facts and to conclude the therapeutic potential of medicinal plants in the eradication and management of various viral diseases such as influenza, human immunodeficiency virus (HIV), herpes simplex virus (HSV), hepatitis, and coxsackievirus infections, which have been proven in diverse clinical studies. The articles, published in the English language since 1982 to 2017, were included from Web of Science, Cochrane Library, AMED, CISCOM, EMBASE, MEDLINE, Scopus, and PubMed by using relevant keywords including plants possessing antiviral activity, the antiviral effects of plants, and plants used in viral disorders. The scientific literature mainly focusing on plant extracts and herbal products with therapeutic efficacies against experimental models of influenza, HIV, HSV, hepatitis, and coxsackievirus were included in the study. Pure compounds possessing antiviral activity were excluded, and plants possessing activity against viruses other than viruses in inclusion criteria were excluded. Hundreds of plant extracts with antiviral effect were recognized. However, the data from only 36 families investigated through in vitro and in vivo studies met the inclusion criteria of this review. The inferences from scientific literature review, focusing on potential therapeutic consequences of medicinal plants on experimental models of HIV, HSV, influenza, hepatitis, and coxsackievirus have ascertained the curative antiviral potential of plants. Fifty‐four medicinal plants belonging to 36 different families having antiviral potential were documented. Out of 54 plants, 27 individually belong to particular plant families. On the basis of the work of several independent research groups, the therapeutic potential of medicinal plants against listed common viral diseases in the region has been proclaimed. In this context, the herbal formulations as alternative medicine may contribute to the eradication of complicated viral infection significantly. The current review consolidates the data of the various medicinal plants, those are Sambucus nigra, Caesalpinia pulcherrima, and Hypericum connatum, holding promising specific antiviral activities scientifically proven through studies on experimental animal models. Consequently, the original research addressing the development of novel nutraceuticals based on listed medicinal plants is highly recommended for the management of viral disorders.     

藤茶双氢杨梅树皮素抗鸭乙肝病毒的实验研究

目的:研究藤茶双氢杨梅树皮素(APS)体内抗鸭乙肝病毒的作用.方法:选1日龄北京雏鸭,人工感染鸭乙型肝炎病毒(DHBV).感染后第7天APS(70,150,300 mg·kg~(-1))口服给药,每天2次,共10 d.应用斑点杂交法观察用药前与用药后及停药后3 d血清中DHBV-DNA的表达.结果:于药后5,10天,150,300 mg·kg~(-1)APS对感染鸭血清DHBV-DNA水平均有显著的抑制作用,且停药3 d未见明显的反跳现象.结论:APS有抑制乙肝病毒的作用.     

In vitro and in vivo anti-hepatitis B virus activities of the lignan niranthin isolated from Phyllanthus niruri L.

Ethnopharmacological relevance

Niranthin is a lignan isolated from Phyllanthus niruri L. This plant has long been used in folk medicine for liver protection and antihepatitis B in many Asian countries. This study was designed to evaluate the anti-hepatitis B virus activity of niranthin using HepG2.2.15 cells and duck hepatitis B virus (DHBV) infected ducks as in vitro and in vivo models.

Materials and methods

Niranthin was isolated from Phyllanthus niruri L. (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hepatitis B virus activity was evaluated both in vitro and in vivo. The human HBV-transfected liver cell line HepG2.2.15 was used in vitro assay. And the in vivo anti-hepatitis B virus activity was evaluated on the expression of HBV replication, HBsAg, HBeAg, ALT and AST on day 0, 7, 14, 17 after niranthin was dosed intragastricly (i.g.) once a day for 14 days at the dosages of 25, 50 and 100 mg/kg/day in the duck hepatitis B virus (DHBV) infected ducks.

Results

In the human HBV-transfected liver cell line HepG2.2.15, the secretion of HBsAg and HBeAg were significantly decreased after treatment with niranthin for 144 h, with IC₅₀ values for HBsAg of 15.6 µM, IC₅₀ values for HBeAg of 25.1 µM. In DHBV-infected ducklings, niranthin significantly reduced the serum DHBV DNA, HBsAg, HBeAg, ALT and AST. Furthermore, analysis of the liver pathological changes confirmed the hepatoprotective effect of niranthin.

Conclusion

The experimental data demonstrated that niranthin exhibits anti-hepatitis B virus activity both in vitro and in vivo.     

拉米夫定联合原儿茶酸对鸭乙肝病毒的体外抑制

目的:研究原儿茶酸(PA)、拉米夫定(3TC)单用以及联合对鸭HBV(DHBV)感染、复制不同时期的影响。方法:利用感染DHBV的鸭原代肝细胞模型,从病毒感染的不同时期研究PA、3TC单用及合用的抗病毒效果。结果:PA、3TC单用及其合用,分别于DHBV感染前,感染的同时及感染后给药,均可有效抑制DHBV对鸭原代肝细胞的感染。虽然PA与3TC合用对DHBV的预防与阻断作用与单用3TC相比没有显著差异,但对DHBV的吸附与进入,感染活性与复制能力,以及肝细胞中AST,ALT酶活的抑制能力均显著强于单用3TC组。结论:PA与3TC药物组合不仅有比二者单用更强的抗DHBV作用,而且对DHBV所致鸭原代肝细胞损伤也有更强的保护作用。     

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。     

Anti-hepatitis B Virus Activity of 8-epi-Kingiside in Jasminum officinale var. grandiflorum

Objective To evaluate the effect of 8-epi-kingiside (8-Epik) derived from the buds of Jasminum officinale var. grandiflorum (JOG) on hepatitis B virus (HBV) replication in HepG2 2.2.15 cell line in vitro and duck hepatitis B virus (DHBV) replication in ducklings in vivo. Methods The concentration of extracellular hepatitis B e antigen and hepatitis B surface antigen (HBsAg) in cell culture medium was determined by ELISA, respectively. The anti-HBV effects of 8-Epik were also demonstrated in the model of DHBV. 8-Epik was ip given (20, 40, and 80 mg/kg, twice daily) to the DHBV-infected ducklings for 10 d. The isotonic saline liquid diet was ip given as negative control and Lamivudine (50 mg/kg, twice daily) was given as positive control. DHBV DNA was measured at days 0 (T0), 5 (T5), 10 (T10), and day 3 after cessation of treatment (P3) by dot blotting. Results 8-Epik effectively blocked HBsAg secretion in HepG2 2.2.15 cells in a dose-dependent manner [IC 50 = (19.4 ± 1.04) μg/mL]. 8-Epik (40 or 80 mg/kg, ip, twice daily) also reduced viremia in DHBV-infected ducks. Conclusion Therefore, 8-Epik is warranted as a potential therapeutic agent for HBV infection.