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

Abstract

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

CLC Number:

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.

Figures/Tables 7

Table 1

Fig. 1

Fig. 2

Fig. 3

Table 2

Fig. 4

Fig. 5

References 28

1	张颖慧, 岳华, 汤承, 等.牛传染性鼻气管炎病毒恒温隔绝式荧光PCR检测方法的建立[J].中国预防兽医学报,2019,41(6):601-604, 640.
	ZHANG Y H , YUE H , TANG C , et al.Development and application of an insulated isothermal PCR (iiPCR) for on-site detecting infectious bovine rhinotracheitis virus[J].Chinese Journal of Preventive Veterinary Medicine,2019,41(6):601-604, 640.
2	MUYLKENS B , THIRY J , KIRTEN P , et al.Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis[J].Vet Res,2007,38(2):181-209. doi: 10.1051/vetres:2006059
3	CHUNG Y C , SHEN H Y , CHENG L T , et al.Effectiveness of a BHV-1/BEFV bivalent vaccine against bovine herpesvirus type 1 infection in cattle[J].Res Vet Sci,2016,109,161-165. doi: 10.1016/j.rvsc.2016.10.004
4	王亚新, 刘春羽, 侯丽娜, 等.牛传染性鼻气管炎病毒SYBR GreenⅠ荧光定量PCR检测方法的建立[J].动物医学进展,2022,43(11):6-11.
	WANG Y X , LIU C Y , HOU L N , et al.Establishment of SYBR green I real-time PCR for detection of infectious bovine rhinotracheitis virus[J].Progress in Veterinary Medicine,2022,43(11):6-11.
5	何小丽, 李凡飞, 张凯, 等.国内外牛传染性鼻气管炎的流行现状及防控措施的研究进展[J].现代畜牧兽医,2018,(6):53-57.
	HE X L , LI F F , ZHANG K , et al.Epidemiology and control of Infectious bovine rhinotracheitis in the domestic and overseas[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2018,(6):53-57.
6	袁铭铭, 李柯城, 代敏, 等.大青叶化学成分的研究[J].中成药,2023,45(9):2923-2929.
	YUAN M M , LI K C , DAI M , et al.Chemical constituents from Folium Isatidis[J].Chinese Traditional Patent Medicine,2023,45(9):2923-2929.
7	LIAO B L , PAN Y J , ZHANG W , et al.Four natural compounds separated from folium isatidis: crystal structures and antibacterial activity[J].Chem Biodivers,2018,15(7):e1800152. doi: 10.1002/cbdv.201800152
8	GAMBARYAN A S , MATROSOVICH T Y , PHILIPP J , et al.Receptor-binding profiles of H7 subtype influenza viruses in different host species[J].J Virol,2012,86(8):4370-4379. doi: 10.1128/JVI.06959-11
9	刘盛, 陈万生, 乔传卓, 等.不同种质板蓝根和大青叶的抗甲型流感病毒作用[J].第二军医大学学报,2000,21(3):204-206.
	LIU S , CHEN W S , QIAO C Z , et al.Antiviral action of Radix Isatidis and Folium Isatidis from different germplasm against influenza A virus[J].Academic Journal of Naval Medical University,2000,21(3):204-206.
10	刘钊, 杨占秋, 肖红.大青叶有效单体抗呼吸道合胞病毒作用的实验研究[J].时珍国医国药,2009,20(8):1977-1979.
	LIU Z , YANG Z Q , XIAO H .Experimental studies on the anti-respiratory syncytial virus effect of effective monomers of Folium Isatidis[J].Lishizhen Medicine and Materia Medica Research,2009,20(8):1977-1979.
11	DENG Y P , LIU Y Y , LIU Z , et al.Antiviral activity of Folium Isatidis derived extracts in vitro and in vivo[J].Am J Chin Med,2013,41(4):957-969. doi: 10.1142/S0192415X1350064X
12	付祥. 抗牛传染性鼻气管炎病毒中草药的筛选[D]. 秦皇岛: 河北科技师范学院, 2021.
	FU X. Screening of traditional Chinese medicine against infectious bovine rhinotracheitis[D]. Qinhuangdao: Hebei Normal University of Science and Technology, 2021. (in Chinese)
13	KAHRS R F .Infectious bovine rhinotracheitis: a review and update[J].J Am Vet Med Assoc,1977,171(10):1055-1064.
14	易鹏飞, 孙磊, 马亚楠, 等.健康安格斯犊牛与IBRV感染犊牛鼻腔菌群变化比较[J].畜牧兽医学报,2024,55(3):1147-1158.
	YI P F , SUN L , MA Y N , et al.Comparative on changes in nasal microbiota between healthy Angus calves and IBRV infected calves[J].Acta Veterinaria et Zootechnica Sinica,2024,55(3):1147-1158.
15	PASTORET P P , THIRY E .Diagnosis and prophylaxis of infectious bovine rhinotracheitis: the role of virus latency[J].Comp Immunol Microbiol Infect Dis,1985,8(1):35-42. doi: 10.1016/0147-9571(85)90052-9
16	杨立国, 王琪, 苏都那布其, 等.菘蓝属植物化学成分及药理作用研究进展[J].中国现代应用药学,2021,38(16):2039-2048.
	YANG L G , WANG Q , SUDUNABUQI , et al.Research progress on chemical constituents and pharmacological effects of Isatis[J].Chinese Journal of Modern Applied Pharmacy,2021,38(16):2039-2048.
17	许涛. 大青叶中4(3H)喹唑酮体外抗甲1型流感病毒和PRRS病毒的研究[D]. 兰州: 甘肃农业大学, 2008.
	XU T. Anti-viral effects of 4(3H)quinazolinone from Folium Isatidis against influenza virus a and PRRS virus in vitro[D]. Lanzhou: Gansu Agricultural University, 2008. (in Chinese)
18	刘相海. 藏药盘花垂头菊多糖提取工艺优化及其对巨噬细胞功能的影响[D]. 成都: 西南民族大学, 2022.
	LIU X H. Optimization of the extraction process of polysaccharide from Tibetan medicine Cremanthodium discoideum and its effect on macrophage function[D]. Chengdu: Southwest Minzu University, 2022. (in Chinese)
19	郭雪萍, 马英才, 李泽龙, 等.牛传染性鼻气管炎病毒诱导MDBK细胞线粒体损伤模型的建立[J].畜牧兽医学报,2022,53(9):3132-3139.
	GUO X P , MA Y C , LI Z L , et al.Establishment of a model of mitochondrial damage induced by bovine infectious rhinotracheitis virus in MDBK cells[J].Acta Veterinaria et Zootechnica Sinica,2022,53(9):3132-3139.
20	王娟娟, 贾山岭, 万新军, 等.MTT法测定棉酚对多浪羊淋巴细胞生长的影响[J].湖北农业科学,2013,52(5):1098-1101.
	WANG J J , JIA S L , WAN X J , et al.The effect of gossypol on lymphocyte growth of Duolang sheep by MTT[J].Hubei Agricultural Sciences,2013,52(5):1098-1101.
21	秦华珍, 时博, 李世阳, 等.南板蓝根和北板蓝根抗甲型流感病毒作用的比较研究[J].中华中医药学刊,2009,27(1):168-169.
	QIN H Z , SHI B , LI S Y , et al.Comparing research in antiviral action of rhizoma et radix baphicacanthis cusiae and Radix Isatidis against influenza A virus[J].Chinese Archives of Traditional Chinese Medicine,2009,27(1):168-169.
22	颜晓维, 何蓉, 刘芳.复方板蓝根颗粒中有效成分研究进展[J].广东化工,2023,50(4):94-95.
	YAN X W , HE R , LIU F .Research orogress of effective components in compound Banlangen granules[J].Guangdong Chemical Industry,2023,50(4):94-95.
23	HSUAN S L , CHANG S C , WANG S Y , et al.The cytotoxicity to leukemia cells and antiviral effects of Isatis indigotica extracts on pseudorabies virus[J].J Ethnopharmacol,2009,123(1):61-67. doi: 10.1016/j.jep.2009.02.028
24	DU Z J , LIU H , ZHANG Z L , et al.Antioxidant and anti-inflammatory activities of Radix Isatidis polysaccharide in murine alveolar macrophages[J].Int J Biol Macromol,2013,58,329-335. doi: 10.1016/j.ijbiomac.2013.04.037
25	刘相文, 田景振, 袁琦, 等.中药金樱子不同溶剂萃取物抗病毒活性分析[J].药物分析杂志,2017,37(12):2196-2201.
	LIU X W , TIAN J Z , YUAN Q , et al.Antiviral activity of different solvent extractions of Rosa laevigata Michx[J].Chinese Journal of Pharmaceutical Analysis,2017,37(12):2196-2201.
26	胡怀月. 杨梅素对伪狂犬病毒的体外抑制活性研究及其对感染小鼠的保护效果评价[D]. 雅安: 四川农业大学, 2022.
	HU H Y. Ininhibitory activity of Myricetin onpseudorabies virus in vitro and its protective effect on infected mice[D]. Yaan: Sichuan Agricultural University, 2022. (in Chinese)
27	张淼丹, 布玮玮, 张春杰.抗猪繁殖与呼吸障碍综合征中药单体成分的体外筛选[J].中国饲料,2022,(8):37-40.
	ZHANG M D , BU W W , ZHANG C J .The research of screening traditional Chinese medicine monomers of anti-porcine reproductive and respiratory syndrome in vitro[J].China Feed,2022,(8):37-40.
28	HAO Q , GUO Q , XU W , et al.Specific inhibitory effect of κ-carrageenan polysaccharide on swine pandemic 2009 H1N1 influenza virus[J].PLoS One,2015,10(5):0126577.

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

[1]	JIANG Jinxiu, ZHANG Jingpeng, LIN Yusheng, LIU Weiwei, HU Qilin, WAN Chunhe. Establishment of a TaqMan Assay for Mycoplasma ovipneumoniae based on the Hsp70 Gene and Analysis of Its Genetic Evolution [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1684-1695.
[2]	WAN Chun-he, LIU Rong-chang, CHENG Long-fei, FU Guang-hua, SHI Shao-hua, CHEN Hong-mei, FU Qiu-ling, HUANG Yu. Establishment of a EvaGreen Real-Time Fluorescent Quantitative PCR Method for Vaccine Strain of Duck Enteritis Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1558-1566.
[3]	DUAN Mei-yan,ZHAO Zhi-dong，LI An-ning，LI Shi-jun,WANG Ming-ming,ZAN Lin-sen. Analysis of Bovine ATP5B Gene Promoter and Tissue Expression [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(3): 382-387.
[4]	HAN Rui, ZHAO Yuan, PAN Li, WANG Yu-qiong, QIN Gui-xin. Effects of Glycinin on Occludin mRNA Expression in Piglet Intestinal Epithelial Cells [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(8): 1258-1262.
[5]	KONG Qun-fang, LUO Fen-hua, WU Sachula, BAO Jia-jing, LIU Dai-yan, WU Ying-ji. Enrichment and Identification of Spermatogonial Stem Cells from Goat Testis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(10): 1554-1560.

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

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 7

References 28

Related Articles 5

Recommended Articles

Metrics

Comments