大青叶水提物对牛传染性鼻气管炎病毒的体外增殖抑制活性评价

doi:10.11843/j.issn.0366-6964.2024.11.044

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (11): 5287-5298.doi: 10.11843/j.issn.0366-6964.2024.11.044

大青叶水提物对牛传染性鼻气管炎病毒的体外增殖抑制活性评价

冯琦¹^,²(), 刘义钢¹^,², 何琴¹^,², 李泽龙¹^,², 马英才¹^,², 易鹏飞¹^,², 李娜¹^,², 孙亚伟¹^,², 陈如龙³, 姚刚¹^,²^,*(), 马雪连¹^,²^,*()

1. 新疆农业大学动物医学学院, 乌鲁木齐 830052
2. 新疆草食动物新药研究与创制重点实验室, 乌鲁木齐 830052
3. 新疆天莱养殖有限责任公司, 博乐 833400

收稿日期:2024-01-11 出版日期:2024-11-23 发布日期:2024-11-30
通讯作者: 姚刚,马雪连 E-mail:fengqi0103@163.com;yg@xjau.edu.cn;13699381790@163.com
作者简介:冯琦(1998-), 女, 新疆克拉玛依人, 硕士生, 主要从事动物保健与畜产品质量安全研究, E-mail: fengqi0103@163.com
基金资助:
新疆维吾尔自治区自然科学基金杰出青年科学基金项目(2024D01E05)

Evaluation of the Inhibitory Activity of Folium Isatidis Aqueous Extract on the in vitro Proliferation of Infectious Bovine Rhinotracheitis Virus

Qi FENG¹^,²(), Yigang LIU¹^,², Qin HE¹^,², Zelong LI¹^,², Yingcai MA¹^,², Pengfei YI¹^,², Na LI¹^,², Yawei SUN¹^,², Rulong CHEN³, Gang YAO¹^,²^,*(), Xuelian MA¹^,²^,*()

1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
2. Xinjiang Key Laboratory of New Drug Research and Creation for Herbivores, Urumqi 830052, China
3. Xinjiang Tianlai Breeding Limited Liability Company, Bole 833400, China

Received:2024-01-11 Online:2024-11-23 Published:2024-11-30
Contact: Gang YAO, Xuelian MA E-mail:fengqi0103@163.com;yg@xjau.edu.cn;13699381790@163.com

摘要/Abstract

摘要：

旨在探究大青叶水提物对牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus, IBRV)体外增殖抑制活性，为临床治疗IBRV感染提供新的参考依据。通过细胞增殖检测试剂盒(CCK-8)和噻唑蓝(MTT)法，结合细胞病变(cytopathic effect, CPE)法确定大青叶水提物对牛肾细胞(Madin-Darby bovine kidney cells, MDBK)的毒性作用，测得药物对细胞的最大安全浓度；将药物最大安全浓度以二倍稀释法稀释3个梯度，通过MTT法和CCK-8法检测大青叶水提物在不同给药方式下对IBRV的抑制活性；计算细胞存活率、药物有效抑制率、药物半数中毒浓度(50%cytotoxic concentration, CC₅₀)和药物半数有效浓度(50% effective drug concentration, EC₅₀)，并以治疗指数(therapeutic index, TI)作为评价指标；通过病毒滴度、实时荧光定量PCR和免疫荧光染色法进一步确定大青叶水提物对IBRV增殖的抑制作用。结果表明，大青叶水提物对MDBK细胞的最大安全浓度为0.8 μg·μL^―1，半数中毒浓度(CC₅₀)为1.937 μg·μL^―1，在最大安全浓度范围内药物浓度越高抑制效果越好；大青叶水提物在不同给药方式对IBRV均有抑制作用，中药和病毒预先作用、先接种病毒后加中药及先加中药后接种病毒三种给药方式的EC₅₀分别为0.292 8、0.350 1、0.416 1 μg·μL^―1，相应的TI分别为6.615 4、5.532 7、4.655 1；通过病毒滴度、实时荧光定量PCR和免疫荧光染色发现，在中药和病毒预先混合作用下，病毒感染36 h药物对病毒抑制作用最显著。大青叶水提物有较好的抗IBRV活性，研究结果可为大青叶水提物在兽医临床应用和深入开发提供科学依据。

关键词: 大青叶水提物, 牛传染性鼻气管炎病毒, 体外抗病毒, 实时荧光定量PCR, 免疫荧光染色

Abstract:

We aimed to investigate the inhibitory activity of Folium Isatidis aqueous extract on the in vitro proliferation of infectious bovine rhinotracheitis virus (IBRV), and to provide new reference data for the clinical treatment of IBRV infection. The cytotoxic effects of Folium Isatidis aqueous extract on Madin-Darby bovine kidney (MDBK) cells were assessed using cell counting kit-8 (CCK-8) and MTT assays, alongside the cytopathic effect (CPE) method, to establish the maximum safe concentration (MSC). The MSC was subsequently diluted in a three-step, two-fold serial dilution to evaluate the extract's inhibitory action on IBRV through both MTT and CCK-8 assays across various administration modes. Metrics such as cell viability, drug efficacy, 50% cytotoxic concentration (CC₅₀), and 50% effective concentration (EC₅₀) were calculated, employing the Therapeutic Index (TI) as a principal evaluative criterion. The inhibitory effect of Folium Isatidis aqueous extract on the proliferation of IBRV was further confirmed by viral titration, real-time quantitative PCR, and immunofluorescence assays. The findings indicate that the MSC of Folium Isatidis aqueous extract on MDBK cells is 0.8 μg·μL^―1, with a CC₅₀ of 1.937 μg·μL^―1. The antiviral efficacy enhances with increasing concentrations within the MSC range. The extract exhibited significant inhibitory effects on IBRV across different administration modes, pre action of medicine and virus, inoculate virus first and then add medicine, add medicine first and then inoculate virus, with EC₅₀ values of 0.292 8, 0.350 1, and 0.416 1 μg·μL^―1, respectively, and corresponding TIs of 6.615 4, 5.532 7, and 4.655 1. Notably, the maximal viral suppression was observed at 36 hours post-infection when the extract was pre-mixed with the virus, as evidenced by real-time fluorescence quantitative PCR and immunofluorescence staining. The aqueous extract of Folium Isatidis demonstrates potent anti-IBRV activity, providing a scientific foundation for its clinical application and further development in veterinary medicine. This study contributes valuable insights into the antiviral properties of Folium Isatidis, underscoring its potential as a therapeutic agent against IBRV infections.

Key words: Folium Isatidis aqueous extract, infectious bovine rhinotracheitis virus (IBRV), in vitro antiviral, real-time fluorescent quantitative PCR, immunofluorescence staining

中图分类号:

冯琦, 刘义钢, 何琴, 李泽龙, 马英才, 易鹏飞, 李娜, 孙亚伟, 陈如龙, 姚刚, 马雪连. 大青叶水提物对牛传染性鼻气管炎病毒的体外增殖抑制活性评价[J]. 畜牧兽医学报, 2024, 55(11): 5287-5298.

Qi FENG, Yigang LIU, Qin HE, Zelong LI, Yingcai MA, Pengfei YI, Na LI, Yawei SUN, Rulong CHEN, Gang YAO, Xuelian MA. Evaluation of the Inhibitory Activity of Folium Isatidis Aqueous Extract on the in vitro Proliferation of Infectious Bovine Rhinotracheitis Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5287-5298.

图/表 7

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检验项目Test items		标准规定 Standard designation	检验结果 Test results
理化项 Physicochemical control	性状Appearance	精细粉末	合格
	颜色Color	棕黄色	合格
	气味Oduor	特有气味	合格
	粒径Granular size	80目通过率大于98%	合格
	干燥失重/% Loss on drying	≤7.0	3.4
	灰分/% Ash	≤5.0	3.10
重金属 Heavy metals	砷/(mg·kg^-1) Arsenic	≤2.0	合格
	铅/(mg·kg^-1) Lead	≤1.0	合格
	汞/(mg·kg^-1) Mercury	≤0.2	合格
微生物 Microbiological control	细菌总数/(CFU·g^-1) Total Bacteria	＜1 000	合格
	霉菌/(CFU·g^-1) Mould	＜50	合格
	大肠杆菌/(CFU·g^-1) Escherichia coli	＜10	合格
	沙门菌Salmonella	不得检出	合格

给药方式 Administration method	浓度/(μg·mL^-1)
给药方式 Administration method	0.1	0.2	0.4	0.8	0(对照组)
中药和病毒预先作用 Pre-interaction between traditional Chinese medicine and virus	10^-5.67	10^-5.41	10^-5.2	10^-4.5	10^-6.19
先接种病毒后加中药 Firstly inoculate the virus and then add traditional Chinese medicine	10^-5.77	10^-5.5	10^-5.41	10^-5.29	10^-6.19
先加中药后接种病毒 Firstly add traditional Chinese medicine and then inoculate the virus	10^-5.9	10^-5.64	10^-5.57	10^-5.4	10^-6.19

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大青叶水提物对牛传染性鼻气管炎病毒的体外增殖抑制活性评价

Evaluation of the Inhibitory Activity of Folium Isatidis Aqueous Extract on the in vitro Proliferation of Infectious Bovine Rhinotracheitis Virus

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