| Objective:With Oldenlandia as the carrier, flavonoids as the research object, the Oldenlandia diffusa flavonoids extraction and purification, and carries on the preliminary study of in vitro intestinal flora metabolism and pharmacokinetics.Methods:1. Ethanol concentration,dosage of ethanol and extraction time were selected as variables,overall desirability of the content of rutin, isoquercitrin, quercetin as index, to optimize the process of extacting flavonoids from Hedyotis diffusa Wind.by Box-Behnken Design and Response Surface Methodology.2.Adopt fecal incubation in vitro,human,normal rats, pseudo germ free rats intestinal bacteria test solution and Hedyotis diffusa extract, rutosid,isoquercitrin,quercitrin were incubated under anaerobic conditions at 37 ℃. High-performance liquid chromatography with diode-array detection( HPLC-DAD) was used for analysis on components and metabolic product as well as the trend of metabolic transformation of the various components according to the changes in chromatographic peak areas in different incubation times.3.Gastric perfusion of Oldenlandia diffusa flavonoid extract and the corresponding monomer, pseudo germ free rats intragastrically Hedyotis diffusa flavonoid extract, the preliminary study on the pharmacokinetic process.Results:1.Optimal extraction technology was as following:extracted 2 times with 14-fold the amount of 73% ethanol,1.50 h per time,the average extraction of rutin, isoquercitrin, quercetin was 2.529, 0.435, 0.215 mg·g-1,the measured value of overall desirability is 0.9227,the error of prediction values for-1.24%.2.The components of Hedyotis diffusa extract and its monomer were quickly metabolized by human and normal rat intestinal flora. However, they almost were not metabolized by pseudo germ free rats intestinal flora. The metabolism of components intestinal flora is weaker than the corresponding monomers.and the main metabolites is quercetin, With the extension of time of metabolism, metabolites decreased gradually, almost undetectable after 24 h. 3.The established method was applied to research pharmacokinetic of Hedyotis diffusa flavonoid extract, Intragastric administration of Hedyotis diffusa flavonoid extract, the concentration of rutin, isoquercitrin and quercetin is low, the peak time was respectively 1.17, 0.67, 0.85 h and 24 h after three index composition almost undetectable in plasma, showed that flavonoids intestinal absorption faster, then quickly eliminate; rutin absorption slower than the other two, Due to the three kinds of compounds with high similarity in the chemical structure, the blood drug concentration at that time the shape of the curve also shows a certain degree of similarity; come to relative bioavailability of rutin, isoquercetin, quercitrin were about 10.5±9.7%, 24.5±9.7%, 15.5±7.1%. 4.The same dose of Hedyotis diffusa flavonoid extract and the corresponding monomer in plasma of rutin, Isoquercitrin and quercetin main pharmacokinetic parameters T1/2, AUC0-t 和AUC0-∞the difference was statistically significant(P<0.05). 5.After intragastric administration of Hedyotis diffusa flavonoid extrac in normal and pseudo germ free rats, Comparison of pharmacokinetic parameters of rutin, Isoquercitrin, quercitrin in normal and pseudo germ free rats, pharmacokinetic parameters of normal rats, AUC0-∞ and Cmax were significantly higher than that of pseudo infinity germfree rats, with significant difference(P<0.05).Conclusion:To establish a HPLC-DAD method for the determination rutin,isoquercitrin and quercitrin from Oldenlandia diffusa and plasma sample, the method is simple, fast, accurate, and easy to operate. Preliminary research on Hedyotis diffusa flavonoids extraction in vivo and vitro metabolism, to reveal the metabolic rule of in vitro and pharmacokinetics in vivo. To provide a basis for clinical medicine, but also providing a theoretical reference to improve the efficient preparation and bioavailability. |
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