Anti-Zika Virus Effect Of Cinnamic Acid And Eugenol In Active Components Of Twig

Objective:At present,there is no effective anti-Zika virus specific drug,and it is particularly important to develop safe and effective antiviral drugs at an early date.In the early stage of the laboratory,it was found that the Chinese medicine Cinnamomum cassia Presl has a good anti-Zika virus effect,but the substance basis of the drug effect is not clear.Therefore,In this project,the active ingredients of anti-Zika virus were screened from the traditional Chinese medicine Cinnamomum cassia Presl.Antiviral studies were conducted from in vitro and in vivo experiments,respectively,which provided a certain reference value for the research of anti-Zika virus drugs.Methods:1.Screening of antiviral active ingredients in Cinnamomum cassia PreslThe titer determination of Zika virus(KU963796)expanded in C6/36 cells cultured at 30℃ by plaque experiment;In Vero cells,the antiviral active components of Cinnamomum cassia Presl were preliminarily screened and cinnamic acid and eugenol has an anti-Zika virus effect.2.In vitro study of Eugenol against Zika virusMTT method was used to determine the toxicity of eugenol to Huh-7 and A549 cells;Zika virus infected Huh-7 and A549 cells,and eugenol was administered for 48h,real-time fluorescence quantitative method(qRT-PCR)was used to determine the viral RNA level Western blot was used to determine the effect of eugenol on Zika virus-related proteins.The effect of eugenol on viral proteins was detected by immunofluorescence experiment;Further,the antiviral stage of eugenol was determined through different modes of administration.And administered at different time points(-2,0,2,4,6,8,10h),using qRT-PCR,measurement of apoptosis inhibition rate to confirm the RNA level and apoptosis in cells 48h after virus infection.The effect of eugenol on the early stage of the virus was further verified by adsorption and entry experiments.3.In vitro study of Cinnamic acid against Zika virusMTT method was used to detect the toxicity of cinnamic acid to Huh-7 and A549 cells;Zika virus infection and administration of cinnamic acid for 48h.QRT-PCR method was used to determine the level of viral RNA;western blot method was used to determine the effect of cinnamic acid on Zika virus-related proteins.The effect of cinnamic acid on viral proteins was detected by immunofluorescence experiments;the antiviral stage of cinnamic acid was determined through different modes of administration.And administered at different time points-2-10h,using qRT-PCR,the measurement of apoptosis inhibition rate to confirm the RNA level in the cells and the apoptosis inhibition rate after 48h of virus infection.4.In vivo anti-Zika virus activity studyIn traperitoneal injection of virus(108 PFU·mL-1)was used to infect INFAR1-/-C57BL/6 mice to construct a model,divided into blank group,model group,high-dose group(150 mg·kg-1)and low-dose group(75 mg·kg-1).A high and low dose of cinnamic acid was intragastrically administered for 12 days every day to record the weight change and death protection of the mice.Blood was collected from the fundus venous plexus on the 3rd and 5th day of administration to determine the blood virus content.The brain of the mouse was used for protein extraction to verify the effect of cinnamic acid on the expression of viral NS5 and E proteins in the brain.Results:1.Virus titer determined by plaque method PFU=1×109pfu/mL.Screening the active ingredients in Cinnamomum cassia Presl against Zika virus.Screened out cinnamic acid and eugenol have certain antiviral effects.In Vero cells,Cinnamic acid TC50>4000 μM,EC50=49.55 μM;Eugenol TC50=207.7 μM;EC50=19.88 μM;SI>>2,indicating that cinnamic acid and syringol are highly efficient and low-toxic drugs.2.In vitro study of Eugenol against Zika virus(1)The TC50 of eugenol on Huh-7 and A549 cells was 205.2 μM and 555.6 μM,respectively.For Huh-7 cell,the EC50 of A549 cells were 46.94 μM and EC50=22.72 μM,respectively.(2)Western blot and immunofluorescence experiments show that eugenol can inhibit Zika virus-associated eggs White expression.(3)Preventive administration of eugenol EC50=38.33μM,simultaneous administration of EC50=36.53 μ M,therapeutic administration of EC50=94.29μM.The virus suppression rate was highest when eugenol was administered at Oh.It is preliminarily determined that eugenol acts on the early stage of the virus.In Protein expression level,further verification that eugenol can inhibit the early stage of Zika virus,and can inhibit Zika virus adsorption.3.In vitro study of cinnamic acid against Zika virus(1)The TC50 of cinnamic acid on Huh-7 and A549 cells was 3752 μM and 1665 μ M,respectively.The EC50 of Huh-7 and A549 cells were 83.96 μM and 103.8 μM,respectively.(2)Western blot and immunofluorescence experiments show that cinnamic acid can inhibit the expression of Zika virus-related proteins.(3)Prophylactic administration of cinnamic acid EC50=99.44 μM,simultaneous administration of EC50=76.63 μM,therapeutic administration of EC50=66.79μM.The virus suppression rate was highest when cinnamic acid was administered from 2h-8h.It is preliminarily determined that cinnamic acid acts on the late stage of the virus.Cinnamic acid inhibits the late stage of Zika virus to a certain extent.4.In vivo study of cinnamic acid against Zika virusThe body weight of the mice was counted.Compared with the model group,the cinnamic acid in the high-dose group had a significant protective effect on the body weight of the viral mice(P<0.01),and the low-dose group(P>0.05).Through 12 days of observation,the death protection rate was calculated in the high-dose group(P<0.01)and the low-dose group(P<0.01).The cinnamic acid in the high-dose group can effectively reduce the viral load in the blood of mice infected with virus on the third day(P<0.01),and the low-dose group(P<0.05);Viral load(P<0.05),low dose group(P>0.05);cinnamic acid has a certain inhibitory effect on the expression of viral NS5 and E proteins in the brain.Conclusion:1.Through screening,it was found that both cinnamic acid and eugenol belong to the effective active ingredients of Guizhi against Zika virus.2.In vitro administration of eugenol,detected by WB,qRT-PCR,and immunofluorescence methods,found that eugenol has the effect of inhibiting Zika virus.Eugenol was administered in different modes and at different time points,and it was found that eugenol can inhibit the early stage of Zika virus,and further verified that eugenol acts on the early stage of the virus through adsorption experiments.3.In vitro administration of cinnamic acid,it was found that cinnamic acid has the effect of inhibiting Zika virus.Cinnamic acid administered in different modes has a certain inhibitory effect on viral RNA caused by Zika virus.Cinnamic acid inhibits the late stage of Zika virus to a certain extent.4.In vivo experiments,the cinnamic acid in the high and low dose groups can reduce the mortality of mice caused by Zika virus to a certain extent,and the high dose group can maintain the weight of the mice and reduce the virus content in the blood of the mice.Reduce the content of viral protein in the brain of mice,reduce the damage of virus to mice.In summary,cinnamic acid and eugenol can be used as drug candidates against Zika virus.