6种中药成分对猪传染性胃肠炎病毒的体外抑制作用

doi:10.3864/j.issn.0578-1752.2016.11.019

摘要/Abstract

摘要： 【目的】为探讨连翘苷等6种中药成分抗猪传染性胃肠炎病毒（TGEV）的作用机制，明确其体外抗病毒作用效果，为抗TGEV的药物筛选提供依据。【方法】利用动物细胞培养技术，采用MTT比色法和细胞病变(CPE)观察法相结合，测定连翘苷、连翘苷元、绿原酸、咖啡酸、丁香酚、丹皮酚6种中药成分不同浓度下对猪睾丸细胞（ST）的毒性作用，通过观察细胞CPE情况，确定药物对细胞作用的最大安全浓度；同时测定病毒的TCID₅₀，用细胞维持液配成100·TCID₅₀病毒悬液备用；将6种药物在最大安全浓度范围内连续2倍倍比稀释后，分别采用先感染病毒后加药、先加药后感染病毒、药与病毒混合后感染细胞3种不同作用方式进行体外增殖抑制试验，各试验组均设置正常细胞对照组和病毒对照组，每组设置3个重复，通过酶标仪测得630 nm处OD值，计算出不同作用方式下中药成分分别对TGEV的抑制率，筛选出抗TGEV活性较好的中药成分，并分别记录抗病毒效果最佳时药物的浓度；6n>种中药成分与病毒作用后，测定TGEVRT-PCR鉴定各个药物单体对病毒RNA合成的抑制情况，进一步明确各中药成分对TGEV的抑制作用。【结果】连翘苷、连翘苷元、绿原酸、咖啡酸、丁香酚、丹皮酚的最大安全浓度分别为320、200、80、125、100、200 μmol·L^-1；抗病毒效果最佳时药物的浓度分别为：160、100、20、62.5、25、100 μmol·L^-1。根据Karber法计算出初始TGEV的TCID₅₀为10^-6.25/0.1 mL；6种中药成分对TGEV在ST细胞上均有一定的增殖抑制作用，其中咖啡酸浓度为62.5 μmol·L^-1时与100·TCID₅₀病毒混合作用后的病毒增殖抑制效果最好，相互作用72 h时，细胞形态依然能够保持圆滑、无固缩、完整，且细胞间轮廓清晰，仅有少量细胞脱落、死亡；此时测上清病毒的TCID₅₀为10^-3.75/0.1 mL，结果显示咖啡酸组病毒含量比病毒对照组10^-6.45/0.1 mL差异极显著（P＜0.01）；通过酶标仪测得630 nm处OD值计算出抑制率能够达到84.4%，咖啡酸直接灭活病毒作用极其显著。其次是连翘苷、连翘苷元、绿原酸、丹皮酚及丁香酚，其病毒滴度分别为：10^-4.75、10^-5.55、10^-5.55、10^-5.65、10^-5.75/0.1 mL，但是这几种中药成分对病毒的抑制作用不够显著，抑制率大多在50%以下。另外，各中药成分对TGEV在ST细胞上的增殖抑制作用均为对TGEV的直接灭活作用最好，其次为对TGEV吸附阻断作用，最后为对TGEV的复制阻断作用。RT-PCR鉴定中药成分对病毒抑制作用，结果显示咖啡酸组条带与病毒对照组相比较暗，病毒效价较低，对病毒抑制作用效果显著；其次是连翘苷、连翘苷元、绿原酸、丹皮酚及丁香酚。【结论】6种中药成分在ST细胞上对TGEV均有一定的增殖抑制作用，其中咖啡酸直接灭活病毒作用最显著，有望被开发成抗病毒药物。

关键词: 中药成分, 猪传染性胃肠炎病毒, 抗病毒作用, 咖啡酸

Abstract: 【Objective】To evaluate the antiviral activity mechanism action of phillyrin and other Chinese traditional medicine components to swine transmissible gastroenteritis virus (TGEV), to confirm the antiviral effect of the drug components in vitro, and therefore to provide scientific basis for screening drugs of antiviral activity to TGEV.【Method】Animal cell culture techniques, MTT detection and cytopathic effect (CPE) methods were used to determine the toxic effect of six Chinese traditional medicine components, including phillyrin, forsythiaside, chlorogenic acid, caffeic acid, eugenol and paeonol, on Swine testis (ST) cells. The maximum safe concentrations of the drugs on ST cells were determined by observing CPE of cells, virus TCID₅₀ was determined at the same time, and 100·TCID₅₀ with cell culture maintenance medium was prepared. The original solutions of the 6 drug components were diluted within the maximum safe concentration scope, and their antiviral effects for cell growth inhibition in vitro were measured with three arrangement routes: TGEV before drugs adding, TGEV after drugs adding and TGEV-and-drugs at the same time. Normal cell control and virus control were arranged for each testing group, with three replications for all treatments. The OD₆₃₀ was determined by using Eliasa approach, the rate of inhibition of six Chinese traditional medicine components to TGEV in different ways was determined, and the inhibition of the best Chinese traditional medicine component on the replication of TGEV was screened, the best antiviral effect concentration of each drug component was recorded. After the six Chinese traditional medicine components reacted with the TGEV, the TCID₅₀, RT-PCR was used to evaluate the viral titer in the supernatant. Furthermore, the proliferation inhibition of six Chinese traditional medicine components on TGEV in ST cell was determined.【Result】The results showed that the highest safe concentrations of Phillyrin, Forsythiaside, Chlorogenic acid, Caffeic acid, Eugenol, Paeonol were 320, 200, 80, 125, 100, 200 μmol·L^-1, respectively; the best antiviral effect concentration were 160, 100, 20, 62.5, 25, 100 μmol·L^-1 , respectively. By using Karber method, the TCID₅₀ of initial TGEV was estimated to be 10^-6.25/0.1 mL. All the six Chinese traditional medicine components had inhibition effects on TGEV in ST cells in vitro. In particular, caffeic acid at 62.5 μmol·L^-1 mixed with TGEV at 100·TCID₅₀ showed the best inhibition effect. With this caffeic acid treatment, 72 h after the test started, the tested cells could still keep smooth, without pycnosis, complete, and profile clear between cells, with only a few cells fell off, or death. At that moment, the measured TGEV TCID₅₀ was 10^-3.75/0.1 mL in the supernatants. The results showed that the virus in treatment with caffeic acid was significantly different, compared to the virus control group with 10^-6.45/0.1 mL (P＜0.01). The inhibition rate reached 84.4% according to the OD₆₃₀ value in the direct inactivation experiment. For other tested components, the TGEV TCID₅₀ of phillyrin, forsythiaside, chlorogenic acid, paeonol and eugenol were 10^-4.75, 10^-5.55, 10^-5.55, 10^-5.65, 10^-5.75/0.1 mL, respectively, with no significant difference, compared to the virus control group, with most of the rate of inhibition below 50%. In addition, all of the six Chinese traditional medicine components had proliferation inhibition on TGEV in ST cells, directly killing effect was the most affective one, followed by adsorpt blocking effect, with replicate blocking effect behind. RT-PCR was used to evaluate the viral titer in the supernatant, the result showed that caffeic acid group was dark compared with the virus control group, the virus titer was low, inhibition effect was remarkable to virus. The next were Phillyrin, Forsythiaside, Chlorogenic acid, Paeonol and Eugenol.【Conclusion】All the six Chinese traditional medicine components had proliferation inhibition on TGEV in ST cells, and caffeic acid showed the best inhibition effect. It has the potential to develop into the antiviral drugs.

Key words: Chinese traditional medicine component, swine transmissible gastroenteritis, antiviral effects, caffeic acid

金修哲，何雷，程相朝，张春杰，余祖华，韩海锋，杨丹芳，梅京京，李银聚. 6种中药成分对猪传染性胃肠炎病毒的体外抑制作用[J]. 中国农业科学, 2016, 49(11): 2234-2244.

JIN Xiu-zhe, HE Lei, CHENG Xiang-chao, ZHANG Chun-jie, YU Zu-hua, HAN Hai-feng, YANG Dan-fang, MEI Jing-jing, LI Yin-ju. Effect of Six Chinese Traditional Medicine Components on Inhibiting Swine Transmissible Gastroenteritis Virus in vitro[J]. Scientia Agricultura Sinica, 2016, 49(11): 2234-2244.

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