Evaluation Of Two Natural Chalcones Against Clostridium Difficile And Their Pharmacokinetics In Rats

Clostridium difficile infection（CDI）was prevalent in both human being and animals with diarrhea as main symptom.CDI was closely related to diarrhea of cubs,which was a serious threat to breeding industry.However,high recurrence rate and emerging drug resistance of traditional antibiotics for CDI cannot be ignored.Recently reports showed that natural flavonoids were active against Gram-positive bacteria.This study screened active compounds against C.difficile from Traditional Chinese Medicine（TCM）Psoraleae fructus and natural flavonoids.The in vivo efficacy on CDI mice,antibacterial mechanism and pharmacokinetics of active compounds were investigated.This study provides basis for CDI drug development as well as antibiotics reduction and replacement by TCM.1.Flavanone extract of Psoraleae fructus（FEPF）was prepared and in vitro antibacterial activity against C.difficile was investigated.Isobavachalcone（IBC）was found to be the most active constituent in FEPF.Licochalcone A（LICOA）was the most active compounds screened from 20 natural products.The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of IBC and LICOA were 4μg/m L and 8μg/m L,respectively.IBC and LICOA can quickly sterilize C.difficile,whereas inactive to normal enterobacteria including Bifidobacterium bifidum,Lactobacillus acidophilus,Bacteroides fragilis and Enterococcus faecalis.A high relative,stable and predictable 3D-QSAR model of flavonoids against C.difficile was successfully established.This model was benefit to the rapid discovery of active molecules against C.difficile.2.The damage of bacteria membrane was proved by SEM investigation,propidium iodide（PI）uptake,determination of extracellular ATP,determination of extracellular protein and isothermal titration calorimetry（ITC）.IBC and LICOA may interact with the bacterial membrane constituent phosphatidylglycerol（PG）and lead to the damage of membrane integrity.As a result,the permeability of membrane was increased,which lead to the leak of Intracellular material,change of bacteria morphology and finally the death of C.difficile.This is the possible antibacterial mechanism of IBC and LICOA against C.difficile.Besides,the formation of biofilm of C.difficile was effectively inhibited by IBC and LICOA as well as eradication of generated biofilm.AS a result,living C.difficile in biofilm can be effectively killed.3.CDI model was successfully established on mice.At 50 mg/kg oral dose,IBC and LICOA can significantly alleviate the symptoms of CDI.The dramatic decrease of body weight CDI mice was controlled and the survival rates of IBC and LICOA were 100%.The efficacy of IBC and LICOA were equal or slightly better than vancomycin（50mg/kg）.Meanwhile,IBC and LICOA were found to be less influence on gut microbiota.The diversity of gut microbiota treated by IBC and LICOA were significant high than vancomycin treatment and closer to normal mice.4.The LC-MS/MS method of simultaneous determination of IBC and LICOA in rat serum was successfully established.This method was founded to be sensitive,accurate,stable and easy operation through method validation,which satisfied the requirements of Pharmacokinetic study on rat.At an oral dose of 50mg/kg,the T_1/2,T_max,C_max and AUC of IBC and LICOA were 4.86±3.03 and 3.02±1.90 h,1.83±1.12 and 2.88±2.11h,36.47±21.42 and 217.42±87.96 ng/m L,128.61±59.46 and1769.23±404.19h·ng/m L,respectively.IBC and LICOA were hardly absorbed in the stomach and intestine.The Absolute bioavailability of oral administration of IBC and LICOA on rats were only 1.59±0.73%and 5.31±2.14%,respectively.In conclusion,IBC and LICOA showed significant antibacterial activity against C.difficile with a rapid sterilization characteristic.The possible antibacterial mechanism of IBC and LICOA include the disruption of membrane integrity and inhibition of biofilm.The efficacy of IBC and LICOA on CDI model was remarkable,with low bioavailability and less influence on gut microbiota.Thus,IBC and LICOA may be promising lead compouds or drug candidates for CDI.