秦皮水提物和秦皮乙素抗石斑鱼虹彩病毒的研究

Study on the Antiviral Abilities of Cortex Fraxini Water Extracts and Esculetin Against Grouper Iridovirus Infection

为开发绿色、安全、高效的抗石斑鱼虹彩病毒(Singapore Groupe Riridovirus,SGIV)渔用药物,本研究先采用光学显微镜观察、CCK-8细胞活力测定实验确定秦皮水提物(Cortex Fraxini water extracts,CFE)及其主要活性成分秦皮乙素(Esculetin,EC)的细胞安全浓度,并在确定药物安全工作浓度基础上,分别采用光学显微镜观察、实时荧光定量PCR(Realtime Quantitative,RT qPCR)、基于核酸适配体LYGV1的荧光分子探针技术(LYGV1-AFMP)等方法探究CFE和EC抗石斑鱼虹彩病毒的效果。结果表明,当CFE浓度低于1 mg/mL、EC浓度低于10μg/mL时,光学显微镜观察结果显示细胞形态正常,无明显病变效应。光学显微镜观测结果与CCK 8检测细胞存活率结果一致,即秦皮水提物安全工作浓度为1 mg/mL(ρCFE≤1 g/L),秦皮乙素安全工作浓度为≤10μg/mL(ρEC≤0.01 g/L)。光学显微镜观察、RT-qPCR、LYGV1-AFMP这3种检测技术均证实秦皮水提物及秦皮乙素具有明显的抗石斑鱼虹彩病毒效果,且在安全工作浓度下,抗病毒效果与药物剂量呈正相关关系。秦皮水提物及秦皮乙素能够有效抑制石斑鱼虹彩病毒的感染,具有开发成高效抗病毒渔用药物的潜力。

In order to develop green,safe and efficient drugs against grouper iridovirus(SGIV),this article first uses optical microscope observation and CCK-8 cell viability test to determine the safe cell concentration of Cortex Fraxini water extracts(CFE)and its main active ingredient Esculetin(EC).And on the basis of determining the safe working concentration of the drug,the methods of optical microscope observation,real time fluorescent quantitative PCR(Real-time quantitative PCR,RT-qPCR),and fluorescent molecular probe technology(LYGV1-AFMP)based on nucleic acid aptamer LYGV1(LYGV1-AFMP)were used to explore CFE and the effect of EC against grouper iridovirus.The results show that when the CFE concentration was lower than 1 mg/mL and the EC concentration is lower than 10μg/mL,the light microscope observation results show that the cell morphology was normal and there is no obvious pathological effect.The results of optical microscope observation are consistent with the results of CCK-8 detection of cell viability,that was,the safe working concentration of CFE is 1 mg/mL(ρ_(CFE)≤1 g/L),and the safe working concentration of Esculetin is≤10μg/mL(ρ_(EC)≤0.01 g/L).The light microscope observation,RT-qPCR and LYGV1-AFMP all confirm that CFE and Esculetin,have great anti-SGIV effects,and the anti SGIV effects were positively correlated with the drug amount at safe working concentration.Cortex Fraxini water extracts and its main active ingredient Esculetin could effectively inhibit SGIV infection,and have the potential to develop highly effective antiviral drugs for aquaculture.