山楂总黄酮体外抗猪流行性腹泻病毒作用研究

Total Flavonoids from Hawthorn Inhibit Porcine Epidemic Diarrhea Virus Infection in Vitro

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)是一种α-冠状病毒,可引起仔猪急性腹泻、呕吐、脱水,死亡率达到100%。目前,传统PEDV疫苗对当前PEDV流行毒株保护作用有限,因此亟须开发新型抗PEDV药物。黄酮类化合物是多种中草药的重要有效成分,具有抗菌、抗病毒、调节免疫力等作用。山楂富含黄酮类物质,作为“药食同源”植物,价格低廉,广泛存在于我国多个省份。为评价山楂总黄酮的抗PEDV效果,通过乙醇回流、柱色谱法和比色法对山楂总黄酮进行提取、富集和含量测定,通过CCK-8、Western blot、qRT-PCR、TCID50等检测方法检测山楂总黄酮在细胞水平上对PEDV复制影响。结果显示,山楂总黄酮提取率为2.5%,其最佳体外给药浓度为20.0μg·mL^(-1)。与PEDV HM2017感染对照组相比,山楂总黄酮能显著抑制PEDV HM2017的核酸水平(ORF3基因)(P<0.01)、蛋白水平(N蛋白)(P<0.01)以及病毒滴度(P<0.01)。研究结果表明,山楂总黄酮在细胞水平上能显著抑制PEDV HM2017复制,可作为一种针对PEDV的抗病毒候选药物。

Porcine epidemic diarrhea virus(PEDV)is anα-coronavirus which can cause acute diarrhea or vomiting,dehydration in piglets with a mortality rate of 100%.At present,traditional PEDV vaccines have limited protective effect against PEDV endemic strains,and it is urgent to develop new anti-PEDV drugs.Flavonoids are one of the important and effective components of many traditional Chinese medicines,which have antibacterial,antiviral and immunity regulation functions.Hawthorn,as a medicinal and edible plant,contains abundant flavonoids,is inexpensive and widely cultivated in China.The total flavonoids from hawthorn were extracted,enriched and determined by ethanol reflux,column chromatography and colorimetric method.In order to evaluate the anti-PEDV effect of hawthorn flavonoids,CCK-8,Western blot and qRT-PCR were used to determine the effects of total flavonoids from hawthorn on PEDV replication at the cellular level.The results showed that the extraction rate of total flavonoids from hawthorn was 2.5%,and the optimal concentration in vitro was 20.0μg·mL^(-1).Compared with PEDV HM2017-infected control group,the optimal dose of total flavonoids from hawthorn,pretreated Vero E6,could significantly inhibit the protein level(N protein)(P<0.01),nucleic acid level(ORF3 gene)(P<0.01)and virus titer(P<0.01)of PEDV HM2017.It suggested that total flavonoids from hawthorn could significantly inhibit PEDV HM2017 replication at the cellular level,and could be used as an antiviral candidate for PEDV.