Study On Antibacterial Activity Of Combination Of Flemingia Philippinensis Roxb. Ex Ait. Compounds And Antibiotics In Vitro

Objective:Antibiotic resistance causes the increases of effective dose of antibiotics,leading to escalation of toxicity and side effects of the antibiotics.In order to find the compounds which can reduce the dosage of antibiotics and achieve synerg-istic and toxic reducing effects on antibiotics,from Chinese herbal medicines,we pre-pared the extracts from 23 traditional Chinese herbs and determined the antimicrobial activities against Staphylococcus aureus,Pseudomonas aeruginosa,Escherichia coli and Candida albicans in vitro.Then the active ingredients were used in combination with selected antibiotics to investigate the antimicrobial properties on S.aureus and methicillin-resistant S.aureus（MRSA）.Methods:Firstly,we used 80%ethanol cold soaking and 95%ethanol reflux extraction to prepared the extracts.The extracts were subjected to determine the bacteriostatic zone using agar diffusion method,and the minimum inhibitory concentration（Minimum Inhibitory Concentration,MIC）and the minimum bactericidal/fungicidal concentration（Minimum Bactericidal Concentrati-on/Minimum fungicide Concentration,MBC/MFC）using microdilution method.The Chinese herbal medicines with better antimicrobial activity were screened as the further separation target.Secondly,Bioactivity-guided fractionation was used to isolate and purify compounds from active fractions of the extracts by combining bacterial susceptibility tests with chromatographic separation using silica gel,polyamide and Sephadex LH-20.The structures of the compounds were identified by physicochemiccal methods and spectroscopic analysis techniques.The MIC/MBC（MFC）of the compounds were also determined.Finally,the active compounds were used in combination with antibiotics commonly used in clinic.The combined activity was determined by Checkboard microdilution method and evaluated by fractionary inhibitory concentration index（FICI）.Time-kill curves was used to observe dynamic bactericidal efficacy.Results:Flemingia philippinensis Rox-b.ex Ait.（Leguminosae）,Illiciumverum Hook.f.（Magnoliaceae）,Entada phaseoloides（L.）Merr（Leguminosae）,Myrsine africana L.（Myrsinaceae）,Dioscorea bulbifera L.（Dioscoreaceae）showed good antimicrobial activity.Combining with the antimicrobial activity of Chinese herbal medicine and the structure type of compounds,F.philippinensis was selected for further investigation.Seven compounds were isolated,i.e.,3-（3,4-dihydroxyphen--yl）-2-hydroxy-1-（2-methoxy-4-hydroxy）-acetone（1）,epicatechin（2）,2,4-dihydroxy-b-enzoic acid（3）,protocatechuic acid（4）,epifisetinidol（4→8）epicatechin（5）,p-metho-xy-phenylacrylic acid（6）and daucosterol（7）.Compounds 1,2,3,5 and 6showed anitimicrobial activity against S.aureus,especially compound 5 had strong activity against S.aureus and MRSA.The MIC values were between 64 and 500μg/mL.Compund 5 was found to show synergistic effects in combination with six antibiotics penicillin,ampicillin,piperacillin/tazobactam,imipenem cilastatin,amikacin and ceftazidime against most of the MRSA strains,respectively.When combined with penicillin,4 strains showed synergy FICI values ranging from 0.3125to 0.5,and 5 strains showed additive effects.When combined with ampicillin,3strains showed synergy FICI values ranging from 0.282 to 0.375,and 8 strains showed additive effects.When combined with piperacillin/tazobactam,7 strains showed synergy FICI values ranging from 0.249 to 0.378.When combined with imipenem cilastatin,7 strains showed synergy FICI values ranging from 0.266 to0.375,and 3 strains showed additive;when combined with amikacin,6 strains showed synergy FICI values ranging from 0.187 to 0.378,and 3 strains showed additive;when combined with ceftazidime,8 strains showed synergy FICI values ranging from0.133 to 0.5,and 2 strains showed additive.The Time-kill curves of compound 5 in combination with the six antibiotics against MRSA15 and MRSA202 were also tested.The results showed that the combination of compound 5 with amikacin decreased2.48 and 2.09 Log10CFU/ML,respectively,compared with the group with the best alone activity.Compound 5 combined with five other antibiotics decreased<2Log₁₀CFU/ML,which was indifference.Conclusion:In the thesis,we screened out a kind of Chinese herbal medicine F.philippinensis root with good in vitro activity from 23 of Chinese herbal medicines.Then we systematically studied the antimicrobi-ial activity of its 80%ethanol extract and isolated a polyphenol compound 5 with good activity.We also studied the combination of this compound with six commonly used antibiotics against S.aureus.Compound 5 was found to show synergy with Piperacillin/Tazobactam,imipenem cilastatin,amikacin and ceftazidime against mo-st of MRSA strains（n=10）,and combined with penicillin and ampicillin mainly showed additive effect.The results provide some data for further development and utilization of antimicrobial Chinese herbal medicines and their synergistic effects on antibiotics.