OBJECTIVE:To develop a HPLC method for determination of tinidazole and chlorhexidine acetate in co-tinidazole gargle.METHODS:Tinidazole and chlorhexidine acetate in co-tinidazole gargle were determined by RP-HPLC with metronidazole as the internal standard.ODS was adopted as stationary phase and acetonitrile-water(35∶65)as mobile phase.The detector was operated at UV254nm.RESULTS:The calibration curve of tinidazole was linear within the concen?tration range of(0.01～0.10)mg/ml(r=0.9999);The calibration curve of chlorhexidine acetate was linear within the con?centration range of(0.01～0.10)mg/ml(r=0.9997).The average recoveries of tinidazole and chlorhexidine were(97.06～102.02)%and(97.21～103.40)%,respectively.The within-day coefficients of variance were(0.20～1.26)%and(0.71～1.11)%,respectively.The between-days coefficients of variance were(0.50～2.06)%and(0.80～2.17)%,respective?ly.CONCLUSION:The method is suitable for determination of preparations containing tinidazole and chlorhexidine acetate.

The traditional primary culture methods of human normal epithelial cells have disadvantages of low activity of cultured cells,the low cultivated rate and complicated operation.To solve these problems,researchers made many studies on culture process of human normal primary epithelial cell.In this paper,we mainly introduce some methods used in separation and purification of human normal epithelial cells,such as tissue separation method,enzyme digestion separation method,mechanical brushing method,red blood cell lysis method,percoll layered medium density gradient separation method.We also review some methods used in the culture and subculture,including serum-free medium combined with low mass fraction serum culture method,mouse tail collagen coating method,and glass culture bottle combined with plastic culture dish culture method.The biological characteristics of human normal epithelial cells,the methods of immunocytochemical staining,trypan blue exclusion are described.Moreover,the factors affecting the aseptic operation,the conditions of the extracellular environment,the conditions of the extracellular environment during culture,the number of differential adhesion,and the selection and dosage of additives are summarized.

Glutamate decarboxylase, a unique pyridoxal 5'-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364P was significantly improved, the half-life time (t1/2) at 55 °C and the semi-inactivation temperature (T₅₀ ¹⁵) of the mutant G364P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.
Glutamate Decarboxylase ; Glutamic Acid ; Lactobacillus brevis ; Molecular Dynamics Simulation ; Proline