Bioactivity-guided Isolation And Purification Of Antibacterial Enhancer From Fructus Crataegi And Their Biological Activities

Objective：Methicillin-resistant Staphylococcus aureus (MRSA) is one of the principal multi-resistant pathogens causing serious infections associated with high morbidity and mortality.Since isolated in1961, MRSA had been one of important gram-positive pathogens in clinic.In fact, MRSA is resistant to almost all kinds of antibiotics especially to β-lactam antibiotics.Vancomycin is the most used antibiotics for MRSA infection, and is considered as “lastdefense line”. However, the strains of Staphylococcus aureus resistant to vancomycin (VRSA)and vancomycin-intermediary staphylococcus aureus (VISA) have been detected in USA andother countries. Therefore, it is very important to search for effective treatments based on thebacterial resistance mechanisms.The main resistance mechanisms of MRSA against antibiotics include:①acquisition ofthe mecA gene that encodes the penicillin-binding protein2a (PBP2a) with low affinity toβ-lactam antibiotics,②large expression of β-lactamase to destroy β-lactams by hydrolysis,and③expressions of efflux pumps in order to extrude antibiotics.To date, there are two strategies to overcome MRSA resistance. One is directlyantimicrobial strategy including new structures’ compounds and modified compoundsderivatived from present antibiotics. However, the discovery of new structures’ compoundsneeds a longer period and derivatives such as teicoplanin doesn’t display much bettereffective than parent compound vancomycin. Another one is indirect antimicrobial strategynamed as antibacterial enhancer. This kind of agents itself has no antibacterial effect but it canrestore the pathogen’s sensitivity to present antibiotics, decreases the dosage of presentantibiotics, and reduces the emergence of resistance bacteria. Therefore, new antibacterialenhancer has become the focus in the field of antibacterial agents. Natural products from plants are the treasure-house of the new antibacterial enhancers.Traditional Chinese herbs have been used for thousands year to treat infection, which is thesource of new antibacterial enhancers.Previously, we have established a directional separation platform for new antibacterialenhancers. PBP2a was coated on the surface of biosensor in order to screen the herbs withaffinity for PBP2a. At the present, MRSA strains were selected as objective. Firstly,Traditional Chinese herbs with high affinity for PBP2a were screened from seventy-eightherbs based on target of PBP2a. Secondary, Fructus Crataegi was screened from the herbswith high affinity for PBP2a under the guide of antibacterial enhancer’s activity. And then thecompounds with good activities was isolated from the extract, their activities wereinvestigated in vitro and in vivo, and the possible mechanisms were also investigated.Methods：1. The directional isolation of compositions and compounds from Fructus Crataegibased on the combination of pharmaceutical chemistry methods and bioactivity assays2. The investigation of compositions and monomers in vitro2.1Determination of combination of compounds with best bioactivity and their ratio, thecombination was named as CE.2.2Determination of FIC of the compounds combined with antibiotics usingmicrodilution method.3. The investigation of compositions in vitroAfter establishment of sepsis mice model challenged with sublethal live MRSA, bloodbacteria number, serum CRP, PCT and TNF-α levels were tested.4. The preliminary investigation of antibacterial enhancement mechanisms4.1Measurement of affinity of CE for PBP2a using affinity biosensor technology.Influences of CE on mecA gene expression were observed using RT-PCR method.4.2Influence of CE on β-lactamase activity was measured by nitrocefin method.4.3Influences of CE on accumulation of daunorubicin within bacteria treated with CEwere observed using both confocal scanning microscopy and fluorospectrophotometry methods.And then effect of CE on efflux system of WHO-2was measured by RT-PCR method.4.4Effect of CE on the autolysis rate of WHO-2treated with Triton-X-100was measured and then effect of CE on the genes expression of autolysins and its regulators weremeasured using RT-PCR method.Results：1. The water extraction of Fructus Crataegi which had stable antibacterial enhancementwas screened out from78Traditional Chinese herbs under the guides of PBPa2-target andbioactivity.2. Three compounds were isolated from water extraction of Fructus Crataegi, they wereidentified as (+)-catechin (C),(-)-epicatechin gallate (ECg) and (-)-epigallatecatechin (EGC)after structure confirmation.3. The investigation of Pharmacological activities of compositions in vitro and in vivo.3.1The combination ratio with best bioactivity was8:1, including128μg/mL of C and16μg/mL of ECg. This combination was named as CE.3.2CE specifically enhanced antibacterial activities of six kinds of β-lactam antibioticsagainst forty-five clinical MRSA strains.3.3CE in combination with oxacillin markedly decreased whole blood bacterial load,CRP, PCT and TNF-α levels of mice challenged with sublethal WHO-2.4. The preliminary investigation of antibacterial enhancement mechanisms4.1C and ECg had no high affinity for PBP2a, and they didn’t inhibit mecA geneexpression.4.2CE had no influence on the activity of β-lactamase.4.3CE increased daunorubicin accumulation within WHO-2, which might be related tothe down-regulation of mRNA expressions of efflux pumps.4.4CE inhibited bacterial division, decrease autolysis rate of WHO-2, which might berelated to the down-regulation of mRNA expressions of autolysins and its regulators.