| In order to explore a novel antibacterial which could be used to treat effectively clinical mastitis in dairy cows, the advanced nanotechnology was utilized to prepare compound Amoxicillin and Levofloxacin hydrochloride nanoemulsion(AMX-LH-NE). Amoxicillin (AMX) and Levofloxacin hydrochloride(LH) were principal ingredient, and the prescription of AMX-LH-NE was screened and optimized by pseudo-ternary phase diagrams. Then the stability, safety, antibacterial effect, therapeutic effect against bovine clinical mastitis and residue elimination of AMX-LH-NE were systematically studied.Part 1 Preparation and evaluation of AMX-LH-NE.1. Analytical method establishment of AMX-LH-NEIn order to prepare and control quality of AMX-LH-NE, two different analytical methods were respectively established for the content determination of AMX and LH by high performance liquid chromatograph. The results showed that two good linearity were obtained by AMX in the range of 0.5～50μg·mL-1 and by LH in the range of 5～60μg·mL-1, and the average recovery, relative standard deviation (RSD), average retention time, RSD of the with-in-day precision, RSD of the day-to-day precision of AMX and LH were (99.27±1.26)% and (99.65±1.51)%, 1.27% and 1.52%, (10.22±0.13) min and (7.15±0.13) min, 1.56% and 1.75%, 2.46% and 2.62%, respectively. The two analytical methods of high performance liquid chromatography (HPLC) which established in this research possessed good specificity, high recovery rate, repetitiveness and precision. It could offer a method of content determination for the preparation and stability investigation of AMX–LH-NE.2. Preparation and quality evaluation of AMX-LH-NEThe effect of some main factors such as surfactant, oil phage and solution adjuvant on formation of the nanoemulsion was investigated by pseudo-ternary phase diagrams. The composition of blank nanoemulsion was determined initially. The physicochemical property change of AMX and LH was studied by HPLC after compatibility. The optimal match of AMX and LH was determined by Kirby-Baueer test. The prescription of AMX-LH-NE was optimized finally and AMX-LH-NE was prepared. Then the structure type of AMX-LH-NE was judged by centrifugation, staining method and dilution. Its appearance and particle diameter distribution was investigated by transmission electron microscope and laser particle size analysator respectively. The dissolubility of AMX and stability of preparation were studied by established high performance liquid chromatography (HPLC). The results showed that the optimal prescription of AMX-LH-NE was glacial acetic acid as solution adjuvant, Cremophor EL-40 as surfactant phase, isopropyl myristate as oil phase and distilled water as aqueous phase. The proportion of glacial acetic acid, Cremophor EL-40, isopropyl myristate and distilled water (w/w) was 10.5:31.5:3.5:54.5. Chemical incompatibility didn't appear between AMX and LH. The determined match between two principal ingredient was 1:1 against mastitis-causing pathogens. The prepared AMX-LH-NE was the type of oil-in-water and the liquid was pallideflavens, clear and uniform, well-distributed with good flowability. The nanoemulsion drop presented spherical shape, and the drop size averaged 14.30 nm with a polydispersity index of 0.03. Particle diameter of 96% was less than 18 nm. The dissolubility of AMX was 15.80 mg·mL-1 in the nanoemulsion. The stability was good, and the expiration date was 20 months. The results indicated that the quality of AMX-LH-NE was stable.3. Safety evaluation of AMX-LH-NEThe safety of AMX-LH-NE was evaluated in vivo and in vitro by acute toxicity test, skin irritation test and cytotoxic test of mammary epithelial cells. The results showed that the toxicity of AMX-LH-NE was low and its half lethal dose was impossibly measured. The maximum tolerable dose to mice was 426 times. Obvious irritation didn't appear regardless of single or multiple dosing when AMX-LH-NE was used on complete and damaged skin of rabbits respectively. There was no obvious influence of AMX-LH-NE on growth and appearance of mammary epithelial cells in the range of 10～150μg·mL-1, and its toxicity grade was 0 or 1. In other words, AMX-LH-NE had no cytotoxicity against mammary epithelial cells. So AMX-LH-NE was safe, nontoxic and low-irritative, and it was consistent with clinical medication.Part 2 Pharmacodynamics study of AMX-LH-NE on bovine clinical mastitis.1. Combination antibacterial effect of AMX-LH-NE against mastitis-causing pathogens in vitro Combination antibacterial effect of AMX-LH-NE was investigated against three main mastitis-causing pathogens including Staphylococcus.aureus, Streptococcus.agalactia, Escherichia.coli in vitro by single drug bridging test, compound drug susceptibility test, combination susceptibility test and Kirby-Baueer test. The results indicated that the synergistic effect was generated when AMX and LH were combined. MIC of the mixed AMX and LH against Staphylococcus aureus was respectively 1/4 and 1/8 times compared with single drug, MIC against Streptococcus agalactiae was both 1/4 times, and MIC against Escherichia coli was both 1/8 times. Each of fractional inhibitory concentration index was 0.375, 0.5 and 0.25 respectively. The disc diffusion zone diameters of AMX-LH-NE against bacteria were all highly significant difference(P<0.01) compared with other each group. These results suggested that antibacterial activity of AMX-LH-NE was strong against main mastitis-causing pathogens.2. Therapeutic efficacy of AMX-LH-NE against bovine clinical mastitisThe high, middle and low dose of AMX-LH-NE and mycillin was respectively infused into udder quarters to treat bovine clinical mastitis in lactation period. It was studied on their therapeutic efficacy after treating. The results indicated that cure rate of AMX-LH-NE in high, middle and low dose group was 95.16%, 89.66% and 42.31% respectively, and total effective rate was 100%, 93.10% and 61.54% respectively. The cure rate and total effective rate of mycillin was 68.52% and 81.48% respectively. The results of the statistical analysis showed the therapeutic efficacy of high and middle dose group were both obviously better than low dose group and mycillin group(P<0.05). Clinical recommended dose was middle on these grounds when AMX-LH-NE was used to treat bovine clinical mastitis. Furthermore drug consumption could increase fairly when clinical mastitis symptom was more severe.Part 3 Residue elimination of AMX-LH-NE in milk.In order to research residue elimination of AMX-LH-NE in milk from cows with mastitis disease, two different HPLC analytical methods for residual concentration monitoring of AMX and LH were established respectively. Then withdrawal time was decided by the residue elimination trial. The results showed that two good linearity were obtained by AMX in the range of 7.5～400 ng·mL-1 and by LH in the range of 40～600 ng·mL-1 respectively, and the average recovery, RSD, average retention time, RSD of the with-in-day precision, RSD of the day-to-day precision of AMX and LH were (99.72±1.46)% and (99.65±1.53)%, 1.46% and 1.54%, (11.59±0.21)min and (6.87±0.02)min, 1.59% and 1.39%, 3.17% and 3.29%, respectively. The two HPLC analytical methods possessed good specificity, high recovery rate, repetitiveness and precision respectively, it could offer the detection approach for residue monitoring of AMX–LH-NE in milk. The results of the residue elimination experiment indicated that residual AMX and LH quickly cut down with prolongation of withdrawal time in milk respectively. Withdrawal time of AMX was 35.96 h, while that of LH was 32.94 h after withdraw. So withdrawal time of AMX-LH-NE was recommended according to that of AMX in milk.AMX-LH-NE is a novel nanometer compound antibacterial with broad-spectrum, high-performance, safety and stabilization. According to clinical curative effect and residue elimination of AMX-LH-NE in milk, its recommended dose regimen is 20 mL in each udder quarters (AMX was 200 mg) every time,twice every day, intramammary infusion after appropriate dilution, and 36 h of clinical withdrawal time when it is used to treat bovine clinical mastitis in lactation period.
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