Study On Antibacterial Activity Of Euphorbia Humifusa Willd In Vitro

Objective(s):1.To determine the in vitro antimicrobial activities of ethanol extracts from 19 Chinese herbal medicines such as Euphorbia humifusa Willd.,Berberis sargentiana Schneid.,Thalictrum petaloideum L.against common clinical pathogenic microorganisms of Staphylococcus aureus(SA),Escherichia coli(EC),Pseudomonas aeruginosa(PA),Candida albicans(CA),and screen out the active extracts.2.To track and separate monomer compounds from the active extracts from Euphorbia humifusa Willd.and indentify the structure of active monomer compounds.3.To determine the in vitro antimicrobial activities of monomer compounds against the pathogen,and evaluate effects of active compounds combined with conventional antibacterial agents.Methods:1.The dried powder of the collected 19 Chinese herbal medicine samples were extracted with 80%ethanol at room temperature and the solvent was evaporated under reduced pressure to get the ethanol extracts.In this study,each extract was screened for antimicrobial activity using the agar diffusion method,and minimum inhibitory concentration(MIC)and minimum bactericidal/fungicidal concentration(MBC/MFC)were determined by serial microdilution method.The Chinese herbal medicines with better antimicrobial activity were screened as the further separation target.2.Bioactivity-guided fractionation was used to isolate and purify monomer compounds from active fractions of the extracts by combining microbial susceptibility tests with chromatographic separation using silica gel,polyamide and Sephadex LH-20.The structures of the compounds were identified by physicochemical methods and spectroscopic analysis techniques.3.Using disk agar diffusion method to screen antimicrobial agents which showed combined effects.Then the combined activity was determined by Checkerboard microdilution method and evaluated by fractionary inhibitory concentration index(FICI).Time-kill curves was used to observe dynamic bactericidal efficacy.Results:1.Based on the results of antimicrobial activity of Chinese herbal medicine,Euphorbia humifusa Willd.was identified as the target Chinese medicine for further separation.2.Four compounds were isolated from the ethyl acetate layer of Euphorbia humifusa Willd.Their structures were identified as gallic acid,methyl gallate,ethyl stearate and palmitic acid.3.The results of monomer compounds combined with clinical commonly antibacterial agents showed the effects against pathogens:(1)The disk diffusion tests screened out?-lactams,macrolides and aminoglycosides.(2)The results of microdilution checkerboard method exhibited that methyl gallate combined with ceftriaxone sodium against MRSA,5 strains were synergistic,and 2 strains were additive;methyl gallate combined with ceftazidime pentahydrate against MRSA,2 strains were synergistic,and 3 strains were additive;methyl gallate combined with imipenem cilastatin against MRSA,5 strains were synergistic,and 3 strains were additive;methyl gallate combinated with ampicillin against MRSA,4 strains were synergistic,and 5 strains were additive;methyl gallate combined with penicillin against MRSA,7 strains were synergistic,and 1 strain were additive;methyl gallate combined with amikacin against MRSA,6 strains were synergistic,and 1 strain were additive.When the compounds were combined with various antibacterial agents against standard Escherichia coli,only brevifolin carboxylic acid combinated with penicillin showed synergistic effect,brevifolin carboxylic acid combined with cefperazone-sulbacta showed additive effect.Compounds were combined with various antibacterial agents against kanamycin-resistant Escherichia coli,brevifolin carboxylic acid combined with ceftazidime pentahydrate,amikacin and cefperazone-sulbacta showed synergistic effect,brevifolin carboxylic acid combined with ceftriaxone and penicillin showed additive effect;methyl gallate in combination with ceftazidime pentahydrate,amikacin and ceftriaxone sodium showed synergy or additivity.(3)The results of time-killing curves showed that:methyl gallate in combination with imipenem cilastatin against MRSA166 showed additivity,methyl gallate in combination with ampicillin and penicillin against MRS A166 showed synergy,methyl gallate in combination with ceftriaxone sodium against MRSA70 showed synergy;methyl gallate and brevifolin carboxylic acid in combination with ceftazidime pentahydrate,amikaxin and cefperazone-sulbacta against kanamycin-resistant Escherichia coli showed synergy.Conclusion(s):1.Through studying on the antimicrobial activity of 19 extract of Chinese herbal medicines in vitro,the ethanol extracts from Thalictrum petaloideum L,Cuminum cyminum L,Euphorbia humifusa Willd,Curcuma kwangsiensis S.G.Leeet C.F.Liang,Andrographis paniculata(Burm.f.)Nees,Leonurus artemisia(Laur.),Evodia rutaecarpa(Juss.)Benth,Rumex madaio Mak,Helwingia japonica(Thunb)DIETR,Hymenodictyon flaccidum Wall,Pteris multifida Poir and Berberis sargentiana Schneid were shown to have significant antimicrobial activity against pathogens.Euphorbia humifusa Willd possessed the best antimicrobial activity against SA,EC and PA.2.In the study,we made activity tracking against bacteria or fungi for extracts from Euphorbia humifusa Willd,the compounds obtained from its active ectract of ethyl acetate layer were gallic acid,methyl gallate,ethyl stearate and palmitic acid.3.Methyl gallate,phlorizin and Phloretin had different degrees of antimicrobial activities.Methyl gallate and brevifolin carboxylic acid in combination with ceftazidime pentahydrate,amikaxin and cefperazone-sulbacta against kanamycin-resistant Escherichia coli showed synergy.