Study On Antimicrobial And Anti-influenza Activities In Vitro Of Extract Of Thesium Chinense Turcz.

Respiratory tract infection（RTI）includes upper respiratory tract infection（URTI）and lower respiratory tract infection（LRTI）.RTIs are short-term,mild and self-limiting.In some cases,however,it can lead to serious complications and place a heavy social and economic burden on individuals and society.Influenza and bacterial respiratory infections are the most common types of acute respiratory infections,affecting all age groups and associated with high mortality during epidemics and sporadic outbreaks.At the same time,viral infection alters the bacterial community in the upper respiratory tract and promotes bacterial colonization in the lower respiratory tract,often resulting in more severe clinical symptoms.Our country has a long history of using traditional Chinese medicine to treat respiratory diseases,which can be considered to have a good theoretical and practical foundation.The bioactive components contained in traditional Chinese medicines are often structurally diverse.When TCM is used for treatment,it can give full play to its multi-component and multi-target effects.According to the report,kaempferol,which can against hand-foot and mouth enterovirus 71（EV71）and Japanese encephalitis virus,is one of the active components of Thesium chinense Turcz.Extracts of T.chinense are also thought to have some antibacterial properties.However,research on the effects of T.chinense against bacteria or against influenza viruses associated with RTIs is lacking.Part 1 In vitro study on the activity against bacteria related to respiratory tract infection of extracts of Thesium chinense Turcz.Objective:T.chinense is known as"natural antibiotics",and it also has the effect of treating RTIs.The aim of this study was to investigate the antibacterial activity of T.chinense extract against Staphylococcus aureus,Streptococcus pneumoniae,Streptococcus pyogenes,and Haemophilus influenzae.Methods:The plants were extracted by ethanol（BRY）,petroleum ether（BS1）,ethyl acetate（BY2）,n-butanol（BZ3,BS4）and dd H2O（BRS）.The extract was made into a powder by rotating evaporation and freeze-drying.The powder was prepared as a solution in sterile double distilled water.The diameter of the inhibitory zone of the T.chinense extract against four species of bacteria was determined using agar punch to verify whether it had an effect on the growth of the bacteria.Then,the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of the effective parts of T.chinense extract against four kinds of bacteria were determined by the broth microdilution method.The OD_630nm value was used to evaluate the effect of the drug on the bacterial growth curve.The effect of active parts of T.chinense extract on bacterial biofilms was determined using crystal violet staining,and bacterial activity on biofilms was detected using MTT.The effect of drugs on bacterial protein synthesis was determined by Coomassie bright blue staining.Scanning electron microscopy was used to observe the effect of drugs on the morphology of bacteria.Transcriptome sequencing was used to screen the possible antibacterial mechanism of T.chinense,and RT-q PCR was used to verify the reliability of the data.Results:1.Agar punching method:the diameters of BRY and BY2 against S.aureus are（9.67±0.24）㎜and（13.27±0.68）㎜.The diameters of the inhibition zone of BY2against S.pneumoniae,S.pyogenes and H.influenzae were（16.4±1.78）㎜,（16.57±0.54）㎜and（17.23±0.34）㎜.No obvious inhibition zone was observed in other parts.2.BMD:The MIC of BRY against S.aureus was 128 mg/m L,and the MBC was 512mg/m L.The MICs of BY2 against S.aureus,S.pneumoniae,S.pyogenes and H.influenzae were 8 mg/m L,4 mg/m L,4 mg/m L,and 4 mg/m L,and the MBCs were 16mg/m L,4 mg/m L,256 mg/m L,64 mg/m L.3.Bacterial growth curve:BRY only showed inhibitory effects on the growth of S.aureus,while BY2 inhibited the growth of all four bacteria.When the concentration was1×MIC,the growth inhibitory effect was obvious.4.The results of crystal violet staining and MTT metabolism detection showed that the formation of bacterial biofilms was inhibited by the drug.After drug treatment,bacterial protein synthesis was inhibited.The result from SEM showed that the drug had a significant effect on the destruction of bacterial morphology.5.Transcriptome sequencing results showed that a total of 1632 genes were changed in S.aureus after BY2 treatment.Among them,804 genes were up-regulated and 828genes showed down-regulated expression.KEGG enrichment analysis showed that the differentially up-regulated genes were significantly enriched in photosynthesis,amino acid biosynthesis and ribosome pathways.The main enrichment pathways of differentially down-regulated genes included citric acid cycle,carbon metabolism,and life span regulation pathways.Conclusion:T.chinense extract significantly inhibited S.aureus,S.pneumoniae,S.pyogenes and H.influenzae in vitro.And the antibacterial mechanism of BY2 against S.aureus may be related to affecting ribosome synthesis,limiting key enzymes of the citric acid cycle,reducing pathogenicity,and affecting biofilm formation.Part 2 In vitro study on the activity against influenza virus of extracts of Thesium chinense Turcz.Objective: The aim of this study is to investigate the anti-influenza virus effect of various extracts of T.chinense in vitro.Methods: Firstly,the cytotoxicity of 6 extracts of T.chinense to human non-small cell lung cancer cells（A549 cells）was determined by CCK-8 method for the half toxic concentration（CC50）.Tissue culture infective dose（TCID50）of the influenza virus on A549 cells was determined by cytopathic effect（CPE）,so as to determine the working concentration of the drug and virus.The haemagglutination of the virus was determined using 1% chicken erythrocytes.Real-time quantitative PCR（RT-q PCR）was used to detect the expression level of M2 gene of influenza virus.Results: 1.Cytotoxicity results: The CC50 values of BRY,BS1,BY2 and BZ3 on A549 cells were 147.6 μg/m L,125.4 μg/m L,12.1 μg/m L,and 45.99 μg/m L,respectively.The CC50 values of BS4 and BRS were both greater than 640 μg/m L.2.Due to the weak virulence of the virus used in this study,no significant CPE phenomenon was observed on A549 cells.Hemagglutination assay results from chicken erythrocytes showed a 1:4 haemagglutination ratio for influenza A/PR/8/34 strain,with influenza B virus showing no haemagglutination.Therefore,follow-up experiments were conducted using only influenza A virus.3.RT-q PCR showed that the M2 gene expression in the BRY group was significantly lower than that in the virus group in the prevention mode.That is,it has an antiviral entry effect.In the treatment mode,BRY had no significant effect on M2 gene expression.When the concentrations of BS1 and BZ3 were 160 μg/m L and 80 μg/m L,the M2 gene expression level could be decreased（P < 0.05）.However,in the treatment mode,BRY had no significant effect on M2 gene expression.Conclusion: BRY,BS1 and BZ3 of T.chinense extract have anti-influenza virus A/PR/8/34 effects in vitro.