| The paper mainly include four parts of reseaching in extraction and purification of effective components from the mixed bovine and ovine bile : (1) Established a reliable suitable small-experiment technology route, which is used to be extracted and separated Taurine, Cholic acid, Glycocholic acid and Taurocholic acid, by improving and perfecting the original technology and magnifying the experiment scale at the base of the previous research in our laboratory. (2) Stduied on the recycling and reusing technology of the raw material and reagent at the extracting process. (3) Stduied on the pharmacokinetics of glycocholic acid in rat. Depending on the above research, it can be said that it has been provided experimental basic for the further research in the middling experiment of extracting the active component from the mixed bovine and ovine bile, supplied the reliable theory and possibility for comprehensive delevoping utilizing the bovine and ovine bile, and illustrated the distribution variation law of glycocholic acid in the blood-serum and texture of rat which is regarded to helping developing the biocherical drug from animal organs.The study on the extracting small experiment of TaurineThe extracting small experiment of Taurine was studied by adopting deproteinizatio, depigmentation, alkaline hydrolysis and acidolysis. Bile extraction was qualitatively detected by using the paper chromatography and infrared spectral scanning method, and the results showed that the extraction has the same Rf value and infrared spectra's characteristic absorption peak as the Taurine standard preparation which can prove that this extraction is Taurine. The tlc-scanning experiment showed that the purity coefficient of extraction is up to 98.6% averagely. The recovery rate detected experiment showed that the extraction amount of taurine is 13.1g/L and the recovery rate is 86.7﹪which is consisted with the results of laboratory technology and showed that this technology is reliable suitable.The study on the extracting small experiment of Choleic acid, Glycocholic acid and Taurocholic acid.The extracting small experiments of Choleic acid, Glycocholic acid and taurocholic acid were studied by adopting deproteinizatio, depigmentation, extraction and column chromatographic fractionation.The optimum condition was determined via orthogonal experiments, and the result shows that the technology of bile extraction and purification adopts 10L chromato bar with 3L bile, gel silica as fixed phase, the mixed chloroform and isopropanol as mobile phase, gradient elution with mobile phase (the mobile phase ratio in extraction of cholalic acid and glycocholic acid is 4:1 , and then becomes 3:2 in extraction of glycocholic acid and glycocholic acid), and eluting velocity of 3～5mL/s .These three kinds of Bile extraction were qualitatively detected by using the paper chromatography and infrared spectral scanning method, and the results show that These extractions have the same Rf value and infrared spectra's characteristic absorption peak as choleic acid, glycocholic acid and taurocholic acid standard preparation which can prove that These three kinds of Bile extraction are choleic acid, glycocholic acid and taurocholic acid respectively.The TLC-scanning experiment shows that the purity coefficient of these three kinds of bile extraction are up to 98.0%,98.4%,98.6%averagely, respectively. The recovery rate detected experiment shows that these three kinds of bile extraction amounts are 11.0g/L,9.1g/L,9.3g/L and the recovery rates are 64.4﹪,63.7﹪,67.8﹪respectively which are consisted with the results of laboratory technology and shows that this technology is reliable suitable.The stability of these three kinds of bile extraction is detected with the remaining sample and observing methods at room temperature and 4℃closed lucifuge environment ,and the results show that they have high stability.The study on the recycling and reusing technology of the raw material and reagent The waste ethanol was recovered by rectification method and the result shows that the purity coefficient of recovered ethanol is up to 95% according to the process purity requirement. The waste gel silica was recovered by alkali washing, acid cleaning and deionized water washing, and the results shows that the physical properties and PH of recovered gel silica are not remarkably different from the new gel silica. Recovered ethanol and gel silica were used in the extraction process, and the abtained result is not remarkably different from the previous result of using the new ethanol and gel silica in the process, which indicats that recovered ethanol and gel silica have no significant effect on the extraction of four active components and can be reutilizated.The study on pharmacokinetics of Glycocholic acid in rats Solid extraction -reversed-phase high performance liquid was used to detect the concentration of glycocholic acid in blood serum,liver, kidney and brain tissue in rats.The results manifest that the concentration of glycocholic acid has satisfactory linear correlation whith peak area, regression equation: Y= 39579X + 57823,R2=0.9984;The lowest detected concentration is 0.02μg/mL with S/N=3 ; Average recovery rate is 98.49%; RSD为0.52%～2.00%in day, RSD为0.74%～4.98%between days;The average recovery rate is 99.35%, RSD is 2.24% in the first team, and it is 100.87%, RSD is 4.73% in the second team at the stability test. When the concentration of glycocholic acid was detected in the liver, kidney and brain tissue, corresponding tissue concentration was calculated by preparing the standard curve with the glycocholic acid standard preparation in vitro, but the result can only reflect the relative chang law of the concentration of glycocholic acid in tissue but not in real tissue. The result of tissue stability experiment is consistent with that of the serum experiment. This method has the convenient,stable, accurate and pragmatic characteristics and is suitable to analyze the glycocholic acid content.After the rats was administrated with glycocholic acid on the does of 0.14g/kg·b·w, the concentration of glycocholic acid was detected in the blood serum, liver, kidney and brain tissue and the obtained data was analysed using the pharmacokinetics software of DAS ver1.0.The results show that Serum drug-time curve is consistent with first-order absorptiontwo-compartment model, its kinetic equation is C= 41.17e-1.256t+84.7 e-0.491t-125.87e-0.667t.Liver drug-time curve is consistent with first-order single compartment model, its kinetic equation is C= 18.734(e-0.042t-e-0.733t).Kidney drug-time curve is consistent with first-order single compartment model, its kinetic equation is C= 105.88(e-0.076t-e-0.081t).Brain tissue drug-time curve is consistent with first-order single compartment model, its kinetic equation is C= 5.312(e-0.082t-e-0.098t).The above data demonstrates that glycocholic acid has the high exhaustion, wide distribution and slow elimination characteristcs in rat which is beneficial to exert the clinical pharmacologic action by keeping the drug concentration for a long time.
|
PDF Full Text Request