| Objective To screen and determine the prescription of ornidazole sustained-release film, establish its quality control standards, and study its antibacterial effect. Methods The main drug and accessories were screened with L9(34) orthogonal experiment method to determine the best prescription. Appearance of prepared films and the release rate of ornidazole were used as the determinant factors for the prescription selection. According to Chinese Pharmacopoeia (2010edition), Appendix IV A, the quantity of ornidazole was assessed by UV spectrophotometry; the uniformity of the film was determined following the method "content uniformity test" in the Chinese Pharmacopoeia (2010edition), Appendix XI E; The in vitro release rate of ornidazole was determined with the "basket method" as mentioned in Chinese Pharmacopoeia (2010edition), Appendix XC;"Microbial limit test" was used to detect the number of bacteria, mold count, yeast and controlling the number of bacteria as described in Chinese Pharmacopoeia (2010edition), Appendix XI J. Prepared films were cut into6x6mm2and incubated in0.9%saline at37℃, with or without0.5%lysozyme. Samples were taken periodically, collected samples were washed and dried until the weight no longer changes. The weight were then taken to calculate the in vitro degradation rate of the film.16SPF Kunming male mice were randomly divided into four groups,4x4mm2films were sterilized with UV irradiation and implanted into thigh muscles of mice and then sutured. At14,2842or56day post implantation, one group of mice were randomly sacrificed, films were removed to calculate the in vivo degradation rate. The in vitro antibacterial activity against Prevotella intermedia and Fusobacterium nucleatum were assessed with solid medium culture. Statistical analysis were performed with SPSS11.0. Results The best prescription determined with L9(3) orthogonal experiment method includes:ornidazole0.5g, chitosan1.6g, carboxymethyl chitosan1.6g, glycerol2.5ml and20x volume of water. The maximum absorption of omidazole solution was observed at wavelength319nm in UV spectrophotometry, with the linear regression equation of A=0.023C-0.0007, r=0.9999,indicating a good linear relationship of ornidazole reference solution within the concentration range of6-20ug/ml; The average content of ornidazole in film was100.04%(ratio of the actual amount to the labeled amount), RSD=0.98%."Content uniformity test" got a result of A+1.80S=7.59,indicating that the content uniformity meets the requirement of Chinese Pharmacopoeia; The cumulative release rate of ornidazole in distilled water for10minutes,30minutes,one hour,two hours,four hours,six hours,eight hours and ten hours were13.9%,39.0%,59.3%,66.5%,69.5%,73.3%,77.1%and80.2%,respectively, as determined with the "basket method’VThe degradation rate of ornidazole sustained-release film in saline was70.42%after seven day incubation in saline. Addition of0.5%lysozyme to the saline resulted in a degradation rate of83.21%. The in vivo degradation rates as determined with mouse experiment were30.21%,62.45%,82.68%and98.80%after14,28,42or56days, respectively. The inhibition zone diameter of the film against Prevotella intermedia was23±0.84mm,while the inhibition zone diameter of the film against Fusobacterium nucleatum was26±0.71mm. Both were significantly higher than the results of blank films (14±0.71mm fox Prevotella intermedia and17±0.45mm for Fusobacterium nucleatum). Conclusions Both appearance and cumulative release rate of the ornidazole sustained-release film prepared with L9(34) orthogonal-optimized prescription meet the requirement of Chinese Pharmacopoeia. The preparation is simple,quality control methods are accurate and reliable.The film has good antibacterial activity against Prevotella intermedia and Fusobacterium nucleatum. |
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