| Objective: Resveratrol(RES), an polyphenol compound,is a kind of phytoalexin produced by plant angainst fungi.In 1940,it was named as “resveratrol†after discovered from the root of resveratrol plant by a Japnese.RES has many biological activites including antitumor,regulation immunity,anti-cardiovascular, antioxidant,antimicrobial,antivirus and so on.With the growth of multi resistant Staphylococcus aureus,doctors face more difficulty in how to choose antibiotic reasonablly.RES has a activites of inhibition many microbial,but the study on antibacterial mechanisms was parum reported in recent years. Stapylococcus aureus standard strains(ATCC 25923) is an effective bacterial in this research.By determining the mininmum inhibitory concentration(MIC),drawing the growth curve of staphy,testing the conductivity and content of macromolecules of membrance penetrability cell membrane,observing ultrastructure changes of S.au standard strains,we evaluate the antimicrobial activity of RES in vitro,aim to make further approach to antibacterial activity and mechanism. RES lies widely in many plants,this reseach may make reuse of many waste plants,such peanut root,grape, grape vine,polygonum cuspidatum and so on, Our study will desir to provide foundation for clinical applicationg and new drug research.Method: 1 Minimal inhibitory concentrations and growth curve of RES on Staphycoccus aureus standard strains ATCC 25923(S.au standard strains) We use S.au standard strains as the research subject.Using the microdilution protocol of CLSI M7-A5, Using the broth microdilution method.The minimum inhibitory concentration of RES on S.au standard strains was detected with M-H broth to obtain MIC of RES on S.au standard strains. By drawing the growth curve, observe the growth of S.au standard strains under the environment of four different concentrations of RES,1/4 MIC,1/2 MIC,1MIC and 2 MIC, With containing DMSO and without RES as control groups. 2 Act on cell membrane permeability of S. au standard strains 2.1 Influence of conductivity about RES acting on S.aureus standard strains medium. The same amount of culture medium was added to 3 bottles of LB broth respectively,37℃ concussion incubation.On entering the logarithmic growth stage, 2 bottles were added varying amounts of RES,obtain 1 MIC and 2 MIC of culture solution.1 bottles without RES as control group,37℃ concussion incubation.Get 2 m L culture solution in 0 h,2 h,4 h,6 h,7h,8 h from each groups. Analysis of the changes of conductivity of S.au standard strains culture solution at different concentrations of RES. 2.2 Data analysis at OD260, OD280 of cultures after the action of RES on S.au standard strains. Equal amount of S.au standard strains was inoculated to LB broth,37 ℃ concussion incubation,before entered the logarithmic growth phase,added RES gain of 1 MIC RES of bacteria suspension,37℃ concussion incubation.Get 2 m L culture solution in 0 h, 2 h, 4 h, 6 h, 7h,8 h from each groups,obtain the determination of OD260 and OD280 and analysis. 2.3 Analysis of the changes of the culture solution absorbance value at OD630 S.au standard strains was inoculated to LB broth, concussion incubation, before entered the logarithmic growth phase, 37℃ concussion incubation.Get 2 m L culture solution in 0 h,2 h,4 h,6 h,7h,8 h from each groups,obtain the determination of OD630 No RES culture solution was used as control. 3 The ultrastructural changes of S.au standard strains Electron microscopy specimens were prepared using containing plating method. The final drug concentration of the plate was 0.256 mg/ml. Inoculation of S.au standard strains at 35℃for 18~24 hours. Prepare the scanning and transmission electron microscope sample.According to the conventional method,we observe the changes of ultramicro morphology and cell structure of resveratrol against S.au standard strains by Transmission electron microscope(TEM) and Scanning electron microscope(SEM).Result: 1 The MIC of RES on S.au standard strains were 0.256 mg/ml. The growth of S.au standard strains was suppressed at the concentration of 1/4 MIC and 1/2 MIC. The growth curve was typical, The logarithmic growth phase is slow;S.au standard strains growth was completely inhibited when the concentration of RES was 1 MIC and 2 MIC. Growth curve was flat,the growth of S.au standard strains was suppressed,the group containing DMSO was no difference with control group. DMSO has no effect on the growth of s. au standard strains(Fig.1). 2 The effection of RES on S.au standard strains cell membrane permeability 2.1 The conductivity of culture supernatant with 1 MIC RES in 0 h, 2 h, 4 h, 6 h,7h,8 h time points were 1.13 ms/cm,1.40 ms/cm,1.51 ms/cm,1.73 ms/cm,1.77 ms/cm,1.74 ms/cm, respectively. The conductivity of LB broth with 2 MIC RES in 0h,2h,4h,6h,7h,8h,time points were 1.16 ms/cm,1.45 ms/cm,1.58 ms/cm,1.87 ms/cm,1.93 ms/cm,1.97 ms/cm. The conductivity of control group were 1.13 ms/cm,1.23 ms/cm,1.28 ms/cm,1.29 ms/cm,1.33 ms/cm,1.23 ms/cm. The electrical conductivity of 2 MIC and 1 MIC RES supernatant was significantly higher than that of control group. The change is more obvious after 6 h~8 h. The conductivity of 1MIC is higher than that of control group 1.41%(Table 1 and Fig.2). Using repetitive measure analysis of variance(Spss 13.0),there was significant difference between control and experiment group,P<0.05. 2.2 The OD260 of the culture in experimental group were 0.832,1.106,1.138,1.117,1.989,2.769 respectively. The OD260 of the culture in experimental group were 0.802,0.884,0.850,0.812,1.544,1.893(Table 2 and Fig.3)Repetitive measure analysis of variance was analyzed by SPSS 13.0 software. OD260 in the experimental group was significantly higher than the control group(P<0.05). The OD280 of experimental group is 0.811,1.093,1.122,1.136,1.76,2.629 respectively.The OD280 of control group is 0.601,0.609,1.078,1.235,1.493,2.180 respectively((Table 3and Fig.4).The experimental group was significantly higher than that of control group(P<0.05). So the absorbance is higher than that of control group in both 260 nm and 280 nm. Using repetitive measure analysis of variance(Spss 13.0),there was significant difference between control and experiment group,P<0.05. 2.3 LB broth of 1 MIC RES inoculated S. au standard strains, The OD630 of the culture in contral group were 0.043,0.310,1.149,1.726,1.991,2.270 respectively.The OD630 of the culture in experimental group were 0.137,0.303,0.385,0.437,1.449,1.423.Drawing curve according OD630 data. The control group curve is a typical growth curve of normal bacteria, including a buffer period, logarithmic phase and plateau phase, was a typical growth curve. The experimental group has been in a state of gentle curve, The growth of the standard strains of S.au was completely inhibited by RES(Table 4 and Fig.5). Using repetitive measure analysis of variance(Spss 13.0),there was significant difference between control and experiment group,P<0.05. 3 Transmission electron microscope observation showed that after the act of RES on S.au standard strains,compared with control group(Fig.6), The cell morphology of S.au standard strains changes serious deformation, cells in all shapes and size, cell boundary roughness, cell wall defect and rupture, edge is Osteoporosis, cell membrane thinning, looseness of its structure, cytoplasmic matrix shades, the nuclear membrane rupture, organelle obscures the structures, cytoplasmic inclusions in thin, local showed vacuolar characteristics. Cell ultrastructure was obvious damaged and inhibition. Scanning electron microscope observation found that bacteria was in a uniform size after RES treated, Cell division is blocked, Irregular shape, Folds of cell surface, spines arounding(Fig.7).Conclusion: Resveratrol destruct cell membrane structure of of S.au standard strains,Caused the increase of cell membrane permeability,the contents leakage to the outside of the cell. Normal metabolism and organelles are also affected. RES play its effect of bacteriostatic.Changes of ultrastructure also confirmed the inhibitory effect of resveratrol on S. au standard strains. |
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