Related Study Of Binary Release System Of Alliin/Alliinase

Objective:A methodology regarding certification of certified reference material (CRM) of alliin was presented according to the requirements of Standardization Administration of the People’s Republic of China. Allicin and other typical sulfide are main efficacious ingredients of garlic, which is acceptable internationally. In this work, we studied the catalytic kinetics of alliin/alliinase system by using of fiber-optic in-situ monitoring system to improve the reaction efficiency of alliinase. Alliin/allinase enteric-coated tablets were developed according to the principle of catalytic kinetics of alliin/alliinase system. Effective quality control methods of alliin/allinase enteric-coated tablets were established to controlled quality effectively. The in vitro distribution of alliin in rat blood was studied to illuminate the working process and mechanism of this tablet. Methods:1.The purity of the CRM was determined by area normalization method. Homogeneity and long term stability of sample were investigated. A cooperative certification was conducted by9laboratories.2. According to Lambert-Beer’s law and with assistance of computational relationship of chemical equation, we established two mathematical models, including obtaining absorptivity of each substance in the reaction and looking on products as a whole. According to the UV spectrums, the optical probe specification and detecting wavelength were determined. Then we determined the value of every parameter in mathematical model at detecting wavelength and recorded information into the workstation. Certain concentration of alliinase was well-mixed with series alliin solution in volume ratio1:1and the concentration variation of alliin was monitored in real time. The maximum reaction rate under different concentrations of the substrate was obtained using Origin7.5software, respectively. To this end, double-reciprocal plot was used to calculate the parameters of catalytic kinetics of alliin/alliinase system, including Michaelis constant (Km) and maximum velocity (vmax), which was compared to HPLC.3. Alliin and potential allicin were identified by TLC. The basket method was used to determine the in vitro release of the preparation by Chinese Pharmacopoeia (2010). The content of alliin and potential allicin were determined by HPLC.4. A reversed-phase high performance liquid chromatography combined with precolumn derivatization with O-phthalaldehyde and fluorescence detection was set up for the qualitatively and quantitatively analysis of alliin in blood. The plasma protein binding rate of alliin was determined by ultrafiltration and microdialysis to evaluate the feasibility of in vivo microdialysis. The in vitro distribution of alliin in rat blood was studied by determining the content of alliin in whole blood, plasma and blood cells after incubation. Results:1.The results of homogeneity were validated by F-test statistical method and showed that the homogeneity of samples was good. The results of long term stability for alliin were validated by T-test statistical method and indicated that the period for alliin of storage was43months at4℃. The property value is (99.5±0.95)%.2. The optical probe of5mm gap was chosen for measurement and the230nm was chosen for detecting wavelength during the catalysis reaction. We can effectively obtain the progress curve of catalytic reaction of Alliin/Alliinase system through the mathematic model. There were no significant differences in Km values, not in Vmax values among the3methods.3. The spots of alliin and allicin in TLC were clear. Allicin was not detected after2hours in hydrochloric acid solution (pH=1.0), but in phosphate buffer (pH=6.8), the accumulation release could reach over70%in2hours. The contents of the alliin per tablet in three batches of samples were55.18mg and55.24mg, and the allicin were13.73mg and21.08mg, respectively. No interference from excipients was found throughout the study.4. With validation, this method was proved to be accurate, stable and reproducible for the analysis of alliin in blood. There were no significant differences in plasma protein binding rate by ultrafiltration and microdialysis. And the plasma protein binding rate was connected with concentration. Alliin was dispersed in both plasma and blood cells. Compared with addition of alliin, the content of alliin in whole blood was less. Conclusion:1. The CRM has passed the evaluation of Standardization Administration of the People’s Republic of China and has been applied in the actual detection.2. Compared with traditional sampling analysis, fiber-optic in-situ analysis was a new method, without the need for time-consuming manual sample preparation, for kinetics study and simpler than sampling analysis with less error.3. The quality standard (draft) of alliin/allinase enteric-coated tablets were established according to pharmaceutics regulates. It can be used for the quality control and the development of the preparation.4. In vivo microdialysis was practicable. Alliin metabolism may happen. The metabolites of alliin and the correlation between metabolism and blood cells need further study.