| China has become the first country of the world in the production and consumption of antibiotics, and also become one of the most serious countries using antibiotics indiscriminately. Most antibiotics can not be completely absorbed by the organism and up to85percent of antibiotics discharged into the environment by the patient and animal excreta in prototype or metabolites form, which has aroused attention for the environmental health risks.In order to systematically analyze the environmental behavior of macrolide antibiotics, we investigated the usage of antibiotics in the livestock farming; then selected11kinds of typical macrolide antibiotics to implement their inhibition experiments on the enzyme activity, drawed chemical structure diagrams of macrolide antibiotics by professional software Chemoffice and accomplished the calculations of their molecular structural parameters and builded the relationships between structure and properties of macrolide antibiotics by means of statistical analysis tools. For further study the migration and transformation of macrolide antibiotics in the environment, we established the HPLC method system to detect the content of macrolide antibiotics. The main results are summarized as follows:1. After investigating two large-scale farming companies and few small scattered farmers in the local town of xuancheng city in Anhui province, we found that there are some problems in the use of antibiotics in livestock, mainly in the following six aspects:(1) serious problems of blind and random medication;(2) widely excessive and long-term use of antibiotics;(3) using the forbidden antibiotics;(4) human medicine veterinary;(5) improper drug compatibility;(6) without obeying the medication rules of withdrawal.2. According to the inhibition mechanism of macrolide antibiotics, and analyzing the research status of macrolide antibiotics inhibiting enzyme activity as well, we established the enzyme activity inhibitory of macrolide antibiotics as the breakthrough, selected acetylcholinesterase and glutamic GGT as target enzymes, by testing the inhibitory effect of macrolide antibiotics on acetylcholinesterase (promoting nerve conduction) and glutamyl endopeptidase (promoting the peptide transfering), combined with the structural characteristics of macrolide antibiotics molecules, revealed the mechanism of macrolide antibiotics inhibiting enzyme.(1)Organic solvents would have some impact on the activity of the enzymes, for selecting suitable organic solvents and reducing their disadvantages on the enzyme activity, wen consulted the reference documentation and selected10kinds of organic solvents, such as methanol, ethanol, isopropanol, acetic acid, acetone, acetonitrile, dichloromethane, N, N-dimethylformamid, chloroform and ethyl acetate, and tested their impact on enzyme activity. The sequence of inhibition ability of10kinds of organic solvents on acetylcholinesterase as follows:acetic acid>dichloromethane>chloroform> N, N-dimethylformamide>ethyl acetate>acetone>acetonitrile>isopropanol> methanol> ethanol; the sequence of inhibition ability of10kinds of organic solvents on glutamyl endopeptidase as follows:acetic acid>chloroform>ethanol>N, N-dimethylformamide>acetone>methanol>isopropanol>acetonitrile>ethyl acetate> dichloromethane.The sequences of inhibition of10kinds of organic solvents to both enzymes are mainly consistent, but there is a difference determined by the spatial conformation of the two enzyme molecules. The organic solvents had weak inhibition to both enzymes containing polar functional groups of hydroxyl and cyano group, which can substitute water molecules to maintain the enzyme flexibility for their activity conformational changes by hydrogen bonding forces making enzyme tend to good catalytic state.Based on the above analysis, selected ethanol and acetonitrile as organic solvents in acetylcholinesterase and glutamyl endopeptidase inhibition experiments respectively.(2) We tested series of inhibition levels of the11kinds of typical macrolide antibiotics on acetylcholinesterase and glutamyl endopeptidase and caculated the corresponding IC50by linear interpolation method. The results showed that at low concentrations, macrolide antibiotics had weak inhibition to both enzymes.The sequence of inhibition ability of11macrolide antibiotics on acetylcholinesterase as follows:nystatin<roxithromycin<midecamycin<kitasamycin<acetylspiramycin<azithromycin<erythromycin<tylosin<avermectin<clarithromycin<amphotericin B.The inhibition ability of amphotericin B is455times of nystatins’on acetylspiramycin. The sequence of inhibition ability of11macrolide antibiotics on glutamyl endopeptidase as follows:acetylspiramycin<midecamycin<kitasamycin<nystatin<erythromycin<clarithromycin<roxithromycin <tylosin<azithromycin<avermectin<amphotericin B.the inhibition ability of amphotericin B is950times of acetylspiramycin on glutamyl endopeptidase, and it’s nearly three orders of magnitude. According to literature review and combining molecular conformation, the analysis were as follows:a. Both experiments are in vitro environment, the enzyme activity decreased and their sensitivity to macrolides also decreased; b. Macrolide antibiotics themselves can not change the enzyme activity, they need to be transformated by metabolizing enzymes in vivo or binding to specific binding sites in vivo and then inhibited enzyme activity.c. The organic solvents produced competitive inhibition to enzyme activity. Amphotericin B made the strongest inhibition to both enzymes. It could be seen from its molecular structure that amphotericin B molecule has maximum hydroxys among11macrolide antibiotics, which are easy to bind to enzyme through hydrogen bonding, thus affecting the enzyme molecule and change its conformation, reducing the binding chances of enzyme to substrate or decreasing the binding stability, resulting in reducing enzymatic activity.(3). To further reveal the mechanism of the inhibition of organic solvents and macrolide antibiotics on enzyme activity, the QSAR models of organic solvents and macrolide antibiotics inhibiting the activity of both enzymes have been established. Firstly, dimensional molecular structures and three-dimensional molecular structure diagrams of organic compounds were constructed by ChemDraw Ultra and Chem3D Ultra of ChemOffice2004office software respectively, after energy optimization by the AMI algorithm, generated the input file for DRAGON software, and then caculated the molecular descriptors by DRAGON software, finally received more than1,800molecular descriptors of organic solvents and macrolides. Enzyme inhibition ratios of macrolide antibiotics and organic solvents being dependent variables, their molecular structure parameters as independent variables, using statistical software SPSS, QSAR models were established through multiple linear stepwise regression method and the inhibition mechanisms of organic solvents and macrolide antibiotics on enzyme activity were interpreted by model stability test and the inputed parameters.(1) the QSAR model of organic solvent inhibiting acetylcholinesterase activity as follows:Inhibition Ratio(IR)=-193.939+147.660*(AAC)+112.824*(SEige), n=10, R=0.960, R2=0.921, F=41.055, Sig=0.000, P=0.4.AAC and SEige parameters in the equation are two-dimensional topological index descriptors, AAC indicats atoms composition average information index, SEige indicates the electrical characteristics of the total number of negative values, and the entered two descriptors showed that the atoms composition of organic solvent determines the entire molecular electronegativity of organic solvents, and as the values of the two descriptors increase, inhibitions of organic solvents on enzyme activity enhance, so the inhibition of organic solvents on acetylcholinesterase activity depends on atoms composition and molecules electronegativity. (2) the QSAR model of macrolides inhibiting acetylcholinesterase activity as follows: IC50=9354.291-1216.501*(GGI3)-93.782*(RDF105e), n=11, R=0.934, R2=0.872, F=31.054, Sig=0.000, P=0.2.GGI3as the two-dimensional spatial structure Galvez topological index descriptor indicats topological charge index. RDF105e as the RDF descriptor of three-dimensional structure indicats atomic electronegativity weighted distribution function. The entered two descriptors showed that the inhibitions of macrolides on acetylcholinesterase activity depend on the electronegativity in particular direction of molecular structure, and with the electronegativity increases, the inhibition rate enhances.(3) the QSAR model of organic solvent inhibiting glutamyl endopeptidase activity as follows: IR=68.985-176.102*(E3u), n=10, R=0.920, R2=0.846, F=29.058, Sig=0.000149.E3u parameter as WHIM index indicats unweighted index pointing to the third spindle Z-axis direction, the entered descriptor indicated that the inhibitory effect of organic solvents on glutamyl endopeptidase was mainly affected by its atomic connectivity in particular direction.(4) the QSAR model of macrolides inhibiting glutamyl endopeptidase activity as follows: IC50=41337.302-8477.261*(GGI3)+415.436(RDF060p), n=11, R=0.897, R2=0.805, F=16.531, Sig=0.00, P=0.000.GGI3as topological charge index indicats two-dimensional spatial structure Galvez topological index descriptor. RDF060p as RDF descriptor of three-dimensional structure indicats atomic polarization radial weighted distribution function. The entered two descriptors revealed that the inhibitions of macrolides on glutamyl endopeptidase activity mainly depended on the polarization of molecular structure in the particular direction.The inhibitory effects of organic solvents and macrolides on both enzymes are consistent, mainly depending on the electronegativity and the polarization in particular direction, but the different molecular steric arrangements of the two enzymes resulted in the result that the degree of inhibition of organic solvents on acetylcholinesterase was higher than that of glutamyl endopeptidase; macrolide molecules have different attack points on enzyme molecules, the molecules points are No.105and No.060for acetylcholinesterase and glutamyl endopeptidase, respectively 4. To further systematically analyze the rules of migration and transformation of macrolide antibiotics, and based on the existing laboratory conditions, the detection method of macrolide antibiotics was established. In this study, azithromycin, clarithromycin and roxithromycin detection wavelengths were210nm, erythromycin detection wavelength was205nm, tylosin286nm and avermectin245nm, the above results provided strong technical support for the subsequent detection of macrolide antibiotics residues in the environment. |
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