Evaluation Of Anti-Zika Virus Activity Of Aqueous Extracts Of Cinnamomum Cassia Presl And Prunella Vulgaris L.in Vitro

ObjectiveZika virus(ZIKV)can cause many diseases such as congenital microcephaly and Guillain-Barre syndrome,which are harmful to human beings.In recent years,it has become popular around the world.However,there is no efficient drug to anti-Zika virus,so it is emergency to develop an anti-Zika virus drugs.Using traditional Chinese medicine to some intractable disease has got unique advantages.In the early stage,our research group have established a system for screening traditional Chinese medicine against ZIKV,and found that the aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.may have a good inhibitory effect on Zika virus.In this study,we evaluated the effects of anti-Zika virus on the aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.,trying to explore the possible stage of its anti-Zika virus,laying a foundation for anti-Zika virus of traditional Chinese medicine,and providing reference for clinical application.Methods1.According to the clinical rule of traditional Chinese medicine for prevention and treatment of infectious diseases,we chosen 32-flavored traditional Chinese medicines as research objects in this project.Each samples added 8 folds water,then heated under reflux for 30 min,concentrated in vacuo,and lyophilized to prepare water-dried dry powder.The plaque assay was used to determine the Zika virus titer amplified by C6/36 cells.Using the cytopathic effect(CPE)as an evaluation index,the screening model of Chinese herbal medicine aqueous extract exerts against ZIKV was established,and the effect of the aqueous extract exerts of 32-flavored Chinese herbal against ZIKV was screened.2.Using cell observation method,real-time fluorescent quantitative PCR and Western blotting,comprehensive evaluation of the anti-Zika virus efficacy of the aqueous extract of Cinnamomum cassia Presl and Prunella vulgaris L.in Vero cells.3.Through the inhibition of apoptosis,real-time fluorescent quantitative PCR and Western blotting,the efficacy of anti-Zika virus in the aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.under different modes of administration was evaluated,and the possible phase of anti-Zika virus was discussed.Results1.Thirty-two water-extracted freeze-dried powders were obtained,and the yield were 1.60%～34.24%.The median toxic concentration(TC50)of 32 Chinese herbal extracts in Vero cells was more than 1000μg/ml.The titer of ZIKV was PFU=1.8×108 pfu/mL.The screening results of anti-Zika virus of 32-flavored traditional Chinese medicine aqueous extracts showed that Cinnamomum cassia Presl and Prunella vulgaris L.could significantly alleviate the cell pathological changes in the concentration of 50 to 500μg/mL,which suggesting that this two traditional Chinese medicine aqueous extract might have anti-Zika virus effect.2.The TC50 of the Cinnamomum cassia Presl aqueous extract in Vero cells and HeLa cells were 4500μg/mL and 3396±1.03μg/mL,respectively.The TC50 of the aqueous extract of Prunella vulgaris L.in Vero cells and HeLa cells was 1886±1.11μg/mL and 2112±1.02μg/mL,respectively.Using the whole mode of administration to evaluated the anti-Zika virus effect of of Cinnamomum cassia Presl and Prunella vulgaris L.aqueous extracts.The results show that aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.could significantly reduce the cell pathological changes induced by ZIKV in the concentration of 50-500μg/mL.Moreover they could significantly inhibit the replication of nucleic acid and protein expression of ZIKV at 100μg/mL and 200μg/mL,respectively.It is suggested that the aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L,can effectively inhibit the replication of Zika virus at the cellular level.3.It was found that the aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.had a dose-dependent inhibition effect on the apoptosis induced by Zika virus in different modes of administration.When premixed,the therapeutic index of the aqueous extract of Cinnamomum cassia Presl was in Vero cells beyond 180,and 45.89 in HeLa cells;the aqueous extract of Prunella vulgaris L.was 10.88 in Vero cells,and 46.39 in HeLa cells.When administered simultaneously,the therapeutic index of the aqueous extract of Cinnamomum cassia P.was over 12.33 in Vero cells,and greater than 67.92 in HeLa cells;the aqueous extract of Prunella vulgaris L.was higher than 12.57 in Vero cells,was more than 8.45 in HeLa cells.At the time of therapeutic administration,the therapeutic index of the aqueous extract of Cinnamomum cassia Presl was over 20.93 in Vero cells and 8.89 in HeLa cells;the aqueous extract of Prunella vulgaris L.was 10.33 in Vero cells and 12.14 in HeLa cells.Among the results of different modes of administration,the results showed that the pre-mixed administration of cassia twig aqueous extract was the best for the replication of anti-Zika virus nucleic acid,and the simultaneous administration was the second.For the aqueous extract of Prunella vulgaris L.,the inhibition of ZIKV nucleic acid replication was best,followed by the therapeutic administration.4.Under premixed administration and simultaneous administration,the aqueous extract of Cinnamomum cassia Presl significantly inhibited the expression of Zika virus protein at 25μg/mL,and affected the expression of Zika virus protein and nucleic acid replication at 50μg/mL in the therapeutic administration.During the premixed and therapeutic administration,the aqueous extract of Prunella vulgaris L.significantly inhibited the expression of Zika virus protein at 25μg/mL and 100μg/mL,respectively.The effect of nucleic acid replication and the expression of Zika virus protein is not obvious at the time of simultaneous administration.The results showed that the anti-Zika virus protein expression of the aqueous extract of Cinnamomum cassia Presl was the best in the premixed administration,followed by the simultaneous administration and the anti-Zika virus protein expression of the aqueous extract of Prunella vulgaris L.was the best in premixed administration,followed by therapeutic administration.ConclusionsThis study established a screening system for the anti-Zika virus of aqueous extracts from Chinese herbal medicine,and found that the aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.has good anti-Zika virus effect.The effects of anti-Zika virus on the aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.were evaluated by various methods.The aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.was found that they can reduce the cytopathic effect induced by Zika virus,inhibit the replication of Zika virus nucleic acid and the expression of Zika virus protein.The aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.may exert anti-Zika virus effect by inhibiting the early stage of viral replication.The aqueous extracts of Cinnamomum cassia Presl and Prunella vulgaris L.may exert anti-Zika virus effect by inhibiting the early replication stage of ZIKV.