| Objective:In this paper,matrix tablets and osmotic pump tablets of Ginkgo biloba extract were used as model drug.Two biological activity indexes those are closely related with clinical effect:antioxidant ability and inhibition of platelet aggregation and two chemical compositions:bilobalides and flavones were selected. Investigating the influence factors on experimental result,optimizing the method and determination conditions,setting up biological activity indexes evaluation method.The dose-effect relationship were studied Using those methods to determinate biological effects of dissolved liquid and plasma of Beagle dog that took tablets orally.Discuss the technical feasibility of using biological activity indexes to evaluate sustained release tablets. Drawing release curves and biological effects curves, discussing their correlation. A good correlation is observed, illustrating that biological activity indexes can evaluate sustained release tablets.Methods:(1)Determining the content of bilobalide and flavones in ginkgo biloba extracts. Through the screening of biological activity indexes, determine two biological activity indexes:antioxidant ability and antiplatelet aggregation. Confirming the best experiment schemes, and the standard method by investigating the influence of different influence factors on experimental results. Systematic methodology was studied to ascertain the feasibility of the method.(2) Through preliminary experiment, screening conditions for releasing in vitro:with pure water (containing0.5%SDS) as the dissolution medium,100r·min-1,37℃for ordinary tablets, skeleton and osmotic pump tablets of ginkgo biloba extract, the content of bilobalide and flavones in dissolution fluid were determined, drawing the cumulative release degree-time curve. Simultaneously determined two biological activity indexes of dissolution fluid, rendering time-biological effect curve. Take ginkgo skeleton and osmotic pump tablets compared with ordinary tablets, investigated slow-release effect and the correlation between compositions and biological activity indexes.(3) Established UPLC-MS/MS method for determination of bilobalidein Beagle plasma:Chromatographic column for the Acquity; C]s(2.1mm*100mm,and1.7microns);mobile phase:6mmol·L-1ammonium acetate-acetonitrile solution in gradient elution;flow rate:0.1mL·min-1;the column temperature was40℃when the sample room temperature was4℃;ion source:ESI;internal standard was domperidone. Ginkgo bilobalide A:407.1→351.13, ginkgo bilobalide B:423.2→367.1, ginkgo bilobalide C:439.1-383.1, bilobalide:325.1→163.0, domperidone:424.1-166.9.(4) Beagle were chosed as experimental animals.6Beagle were given gingko ordinary tablets, skeleton and osmotic pump tablets, took samples at different time point respectively. Determined drug concentration in plasma and the two biological activity indexes, using Kinetica software for processing for the pharmacokinetic parameters, and considering the correlation between drug concentration and biological activity indexes.Result:(1)The detecting wavelength, reaction time, reaction temperature, different solvents was investigated in antioxidant experiment, and determine the detection wavelength of522nm, the reaction time for40min, the reaction temperature at25℃, anhydrous ethanol as the solvent. In the range of122～816mg·mL-1a good linear relation(r=0.999)was observed with good precision, and good repeatability. The dosage of PAF, centrifugal time, centrifugal rotational speed, the longest time for saving sample of antiplatelet aggregation experiment were studied. The dosage of platelet activating factor (PAF) was15μL; centrifugal time was6min,;the centrifugal rotational speed was800r·min-1The inducer dosage were investigated15μL,20μL,25μL, there was no significant difference. To save the dosage used15μL. In the range0.12～2.7mg·mL-1a good linear relation(r=0.999)was observed with good precision and good repeatability.(2) The cumulative release of flavones of ordinary tablets was70%in40minutes, and more than90%when60minutes. The cumulative release of skeleton tablets was70%in8h, and more than90%when12h. The cumulative release of osmotic pump tablets was50%in8h, and more than80%when14h. The skeleton, osmotic pump tablets were played a slow-release effect, and the effect of osmotic pump tablet is better. Antioxidant biological indexes of Ordinary tablets have reached the maximum when60min while the skeleton was8h, the osmotic pump tablets was I Oh.The biological indexes also suggested the skeleton and osmotic pump tablets had slow-release effect. The correlation between biological indexes and component index of skeleton is0.992. The correlation between biological indexes and component index of osmotic pump tablets is0.991.The cumulative release of bilobalide of ordinary tablets was70%in40minutes, and more than90%when60minutes. The cumulative release of skeleton tablets was70%in8h, and more than90%when12h. The cumulative release of osmotic pump tablets was50%in8hours, and more than80%when14h. The skeleton, osmotic pump tablets were played a slow-release effect, and the effect of osmotic pump tablet is better. Antiplatelet aggregation indexes of Ordinary tablets have reached the maximum when60min while the skeleton was8h, the osmotic pump tablets was1Oh.The biological indexes also suggested the skeleton and osmotic pump tablets had slow-release effect. The correlation between biological indexes and component indexes of skeleton is0.969. The correlation between biological indexes and component index of osmotic pump tablets is0.986.(3) Compared with ordinary tablets.ginkgo skeleton and osmotic pump tablets’ Tmax extended and Cmax was lower. But the change tendency of the biological index was not observed. Conclusion:The experiment suggested that there is a good correlation between biological indexes and component indexes.The method was stable and feasible,but in the evaluation of Beagle plasma after the treatment, the biological indexes did not show the trend that changes with drug concentration, remained to be further experiment to optimize the methods. |
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