Therapeutic Effect And Application Of Licochalcone A Against Clostridium Perfringens Infection

Clostridium perfringens(C.perfringens),also known as Clostridium Welchii,is a gram-positive opportunistic pathogen that is associated with diverse environments(such as soils,sewage,food,faeces,and as a member of the gastrointestinal tract microbial community of healthy humans and animals.In non-diseased animals,C.perfringens has low pathogenicity.When the external environment factors or the feed changed,C.perfringens can grow rapidly,leading to food poisoning and necrotic enteritis(NE),causing serious threats to the poultry.During the pathogenicity,C.perfringens cansecrete an arsenal of toxins.According to the production of typing toxins,C.perfringens strains are typed for five toxinogenic types: A to E.In poultry,NE infection is commonly caused by type A isolates.In recent years,NE incidence has increased since the ban on in-feed “growth promoting” antibiotics.According to estimates,the economical impact of the disease is US$ 2 billion year through.In addition,the large amount of antibiotics used in poultry may pollute the natural environment such as soil and water,resulting in the existence of a large number of antibiotics in the natural environment,which not only contributes to the spread of antibiotic resistance,but also leads to an increasing global threat to human health.Therefore,there is a urgently need to develop new compounds to prevent or control NE,especially developing drugs other than antibiotics to reduce the use of antibiotics and drug resistance.In this study,C.perfringens,the key pathogen of NE was selected as the target pathogens,and the minimum inhibitory concentrations(MIC)assays were used to evaluate the antibacterial effect of tested compounds.Licochalcone A were identified as the potential compound against C.perfringens by broth dilution method.Then,the minimum bactericidal concentration(MBC)assays were employed to evaluate the potential bactericidal activity of licochalcone A against the tested strains.Subsequently,the effect of licochalcone A on the growth of C.perfringens was determined by growth curve assays.The time-killing assays were used to further determine the bactericidal effect of of licochalcone A against C.perfringens.Through cytotoxicity assays,we investigated whether licochalcone A could protect tested cells from C.perfringens-mediated cell injury.In order to obtain licochalcone A on a large scale,this study further studied the extraction method of licochalcone A.Licochalcone A in the Glycyrrhiza uralensis Fisch was extracted and refined by alcohol extraction,and the content of licochalcone A in the extract was detected.In in vivo assays,we established a chicken NE model by challenging a suspension of C.perfringens to broiler chickens and evaluated the protective effect of licochalcone A on C.perfringens infected chickens by administering the liquorices extracts.Intestinal lesions,histopathological changes and other indicators were used to evaluate the therapeutic effect of licochalcone A.In the MIC assays and MBC assays,the MIC value of licochalcone A against tested C.perfringens strains was 4 μg/mL,and the MBC value was 8 μg/ mL.In the growth curve and time-killing assays,licochalcone A significantly inhibited the growth of C.perfringens at a concentration of 4 μg/mL.In cytotoxicity assays,licochalcone A could alleviate the cytotoxicity mediated by C.perfringens.In the extraction method part,we successfully extracted the licochalcone A(with a content of 11.7%)from Glycyrrhiza uralensis Fisch.In the in vivo assays,the liquorices extracts extracted from this study could effectively protect broilers from intestinal damage caused by NE and reduce the expression of TNF-α in small intestine issues.And licochalcone A had no significant effect on organ index of broiler chickens.In conclusion,we found that licochalcone A showed significant antibacterial and bactericidal activity against C.perfringens.Licochalcone A could alleviate cell damage caused by C.perfringens infection and protect broiler chickens from NE caused by C.perfringens infection.In addition,this study established a extraction method for extract licochalcone A from Glycyrrhiza uralensis Fisch,providing a theoretical basis and research foundation for the clinical application of licochalcone A in veterinary medicine.