Inhibitory Effects And Mechanism Of Traditional Li Herb Dalbergia Pinnata (Lour.) Prain On Methicillin Resistant Staphylococcus Aureus

ObjectiveDifferent extracts from Dalbergia pinnata(Lour.)Prain(D.pinnata),were screened for antibacterial activity against methicillin resistant Staphylococcus aureus.The extraction process was optimized by response surface methodology,and the antibacterial mechanism and the effect of the Optimized Dalbergia pinnata extract(ODPE)on MRS A biofilm were discussed in order to provide new ideas and theoretical basis for new medicinal resources further development of Dalbergia.Methods1.In vitro antibacterial activity of different extracts from D.pinnataDisk diffusion method was used to explore the antibacterial effect of steam distillation volatile oil,supercritical carbon dioxide extraction volatile oil,water extract and alcohol extract from D.pinnata on Staphylococcus aureus,Escherichia coli,Candida albicans and other six strains,and the effect of extract concentration on the antibacterial activity of sensitive strains was also explored.At the same time,microdilution method and plate coating counting method were used to determine the minimum inhibitory concentration and minimum bactericidal concentration.2.Optimization of extraction processTaking the inhibition zone size as the response value,the single factor experiment method was used to investigate the extraction method,extraction temperature,ethanol concentration,extraction time,solid-liquid ratio and ultrasonic power in the extraction process of D.pinnata.On the basis of the results,the response surface method was used to optimize the extraction process,and the extraction conditions with the best inhibition effect were selected.3.Preliminary evaluation of anti-MRSA effect of ODPEThe OD-CFU standard curves and bacterial growth curves were plotted by UV spectrophotometry,and the antimicrobial performance of ODPE extracted by the optimized process was evaluated by microdilution method and plate coating counting method.The induction effect of ODPE on MRSA resistance was also investigated.4.Study on the inhibitory mechanism of ODPE on MRS ABy measuring the content of alkaline phosphatase(AKP),nucleic acid and protein in MRSA culture medium,the utilization of reducing sugar by MRSA,the content of adenosine triphosphate(ATP)in MRSA cells,the content of soluble protein and DNA in MRSA cells,and combining the measurement of surface hydrophobicity and Zeta potential value,the mechanism of the inhibitory effect of ODPE on MRSA was explored.5.Study on the influence of ODPE on MRSA biofilmThe MRSA biofilm model was constructed,and the crystal violet staining method and the micro double dilution method were combined to determine the minimum biofilm inhibition concentration(MBIC),the minimum biofilm removal concentration(MBEC)and the Biofilm Prevention Concentration(BPC).The effect of ODPE on the formation of MRSA biofilm was also observed by microscopy method.Results1.In vitro antibacterial activity of different extracts from D.pinnataDifferent extracts from D.pinnata showed different degrees of antibacterial activity,and the activity was positively correlated with the concentration of the extract.The results showed that the water extract had obvious inhibitory effect on two kinds of Staphylococcus,and slightly weaker inhibitory effect on Enterococcus faecalis.The distilled volatile oil had strong inhibitory effect on Candida albicans,whose diameter of inhibition zone was more than 30 mm.The MIC and MFC of volatile oil of C.albicans were 220 μg·mL-1,while the MIC and MBC of water extract against S.aureus and MRSA were all 625 μg·mL-1.2.Optimization of extraction processThe optimum extraction conditions were as follows:ultrasonic assisted extraction method,ethanol concentration 15%,extraction temperature 30℃,extraction time 1 h,solidliquid ratio 1:10,ultrasonic power 640 W.The antibacterial effect of the extracts obtained was significantly improved,with the antibacterial rate enhanced by about 59.69%,MIC value decreased by 4 times and MBC value decreased by 2 times compared with the extracts before optimization.3.Preliminary evaluation of anti-MRSA effect of ODPEODPE can effectively inhibit the growth of MRSA.The higher the concentration it was,the better the inhibitory effect it could perform.After long-term sub-inhibitory concentration inducting culture,MRSA finally presented drug resistance to ODPE,whose MIC increased by 8 times.4.Study on the inhibitory mechanism of ODPE on MRSAODPE could not cause the leakage of nucleic acid and protein in MRSA bacteria,nor could it cause the outflow of alkaline phosphatase from the cell wall.However,it could significantly reduce the surface hydrophobicity of MRSA and increase its absolute value of Zeta potential.At the same time,ODPE could inhibit the utilization of reducing sugars by MRSA,causing the reduction of ATP in the bacteria,and affect the intracellular protein and nucleic acid of MRSA,resulting in the loss of protein bands and the decrease of DNA concentration.5.Study on the influence of ODPE on MRSA biofilmHigh concentration ODPE could inhibit the formation of MRSA biofilm,and had a certain scavenging effect on the formed biofilm.The BPC,MBIC and MBEC value of ODPE to MRSA biofilm were 1.25 mg·mL-1,1.25 mg·mL-1 and 5 mg·mL-1,respectively.ConclusionThis exploratory study on the in vitro antibacterial activity proved that D.pinnata has excellent antibacterial potential.The extracts obtained by optimizing the process conditions showed good reproducibility of bacterial inhibition and significant increase in inhibition rate.The mechanism study proved that D.pinnata could effectively inhibit the growth of MRSA from several aspects and cause killing effect on it,as well as inhibiting and removing its biofilm.Therefore,D.pinnata is expected to be a new medicinal plant resource against drugresistant bacteria,which is worthy of in-depth development and utilization.