Development Of Cinnamon Twig Injection And Pharmacodynamic Evaluation Of Against Listeria Monocytogenes Infection

Listeria monocytogenes(L.monocytogenes)is a common clinical cause of listeriosis.Listeriosis has a mortality rate of approximately 20-30% and most infected animals present with invasive disease,including encephalitis,meningitis,septicaemia and abortion.L.monocytogenes can survive in a wide range of environments,contaminating farm environments and feeds and causing listeriosis in animals,resulting in serious losses to humans and livestock farming.L.monocytogenes invades host cells by producing a variety of different virulence factors that assist the organism in breaking through the host’s three barriers(gastrointestinal,placental and blood-brain barriers).As the main virulence factor of L.monocytogenes,listeriolysin O(LLO)plays an essential role in the intracellular life cycle,including barrier penetration,colonisation and systemic dissemination of the bacteria.The structural integrity and activity of LLO determines the pathogenicity of L.monocytogenes.Therefore,LLO based on bacterial virulence factor targeting is a potentially important target for anti-L.monocytogenes infection.In recent years,natural compounds and their extracts have attracted much attention in the research of anti-bacterial virulence factors due to their unique advantages,including diverse sources,wide distribution,low toxicity,low residue and no apparent drug resistance.On this basis,the study was conducted to screen a variety of herbal extracts through a laboratory-established LLO inhibitor screening platform and found that Cinnamon Twig extract was able to inhibit the hemolysis activity of LLO protein and L.monocytogenes culture supernatant at a concentration of 32 μg/m L.Therefore,taking Cinnamon Twig as the target medicinal material,the extraction process of Cinnamon Twig extract was optimized,and the optimal extraction process was determined as follows: Extraction was carried out by adding 80% volume fraction of ethanol aqueous solution to 20 times of the material dosage of Cinnamon Twig,extracting twice,for 2 h each time,combining filtrate twice,adding 6 times of pure water,standing at 4℃ for 12 h,filtering filter slag,drying the extract powder of Cinnamon Twig.The content of cinnamaldehyde,the main component of Cinnamon Twig,was taken as the index of Cinnamon Twig extract.We established a HPLC method for the determination of cinnamaldehyde in Cinnamon Twig extract.The applicability and accuracy of the method were investigated.The content of cinnamaldehyde was determined to be 0.76%.The preliminary mechanism analysis of the inhibition of LLO hemolysis activity by Cinnamon Twig extract was carried out by fluorescence quantitative PCR assay and Western blot assay.The results showed that the prepared Cinnamon Twig extract could effect the expression of the hemolysis gene hly in L.monocytogenes and reduce the content of LLO protein in the supernatant of the bacterial culture and significantly weakened the pore-forming activity of LLO.In cell infection experiments,LLOmediated macrophage damage was significantly alleviated and intracellular proliferation of L.monocytogenes was inhibited after treatment with Cinnamon Twig extract.In this study,based on the physicochemical properties and poor absorption of the active ingredients of Cinnamon Twig extract in vivo,the prescription of Cinnamon Twig injection was finally determined as follows: Cinnamon Twig extract as main ingredient,N-N dimethylacetamide as organic solvent,castor oil as injection oil,Tween-80 as organic co-solvent,benzyl alcohol as analgesic and tert-butyl p-hydroxyanisole as antioxidant.Cinnamon Twig injection was successfully prepared.The influencing factor tests revealed that light conditions influenced the content of Cinnamon Twig injection,so the preparation needs to be protected from light during preparation,storage and transportation.The accelerated test investigated the stability of the retention sample of Cinnamon Twig injection,and the results showed that all quality control indicators of Cinnamon Twig Injection meet the requirements and had good stability.The therapeutic study of L.monocytogenes infection in mice was carried out by artificially establishing in L.monocytogenes infection model and using Cinnamon Twig injection.It was found that Cinnamon Twig injection could reduce the mortality rate of bacterialinfected mice and alleviate the bacterial load of liver and kidney of L.monocytogenes infection.In conclusion,this study has successfully prepared the Cinnamon Twig injection through preliminary screening and mechanism confirmation.The prepared Cinnamon Twig injection has quality control,and has good therapeutic effect on mice infected with L.monocytogene,providing a new effective Chinese medicinal preparation for the treatment of L.monocytogene infection in veterinary clinics.