Preparation Of Ginkgo Biloba Extract Gel Matrix Tablets And Evaluation Of Its Biological Activity In Vitro

Objective:This paper selected Ginkgo biloba extract as a model drug, preparing Ginkgo biloba extract hydrophilic matrix tablets by a single factor study and orthogonal experiment, utilizing accumulative release degree of Ginkgo biloba extract total flavonol glycosides at different time points for the evaluation index, and using a dissolution tester for determination of release degree in vitro; selecting antioxidant and dilation of blood vessels which are pharmacological targets closely related to the clinical application of the model drug, using pharmacological experimental method to establish a good linear relationship between model change of the pharmacological targets and the concentration of the model drug, carrying out the determination of release degree in vitro on the homemade hydrophilic matrix tablets by the selected biological activity index, establishing the relevance between the release degree in vitro measured by the component index and the release degree in vitro measured by the biological activity index in order to establish a simple and rapid means and method for evaluation of the release degree in vitro characteristics of the preparation.Methods:(1) Take accumulative release degree of total flavonol glycosides at different time points as an index, have a separate study on species and dosage of the matrix material, the type of filler, tablet hardness, and tabletting method by the ultraviolet spectrophotometry, make similarity judgments in combination with the f2 similarity factor method, so as to identify the factors having a significant effect on the drug release behavior; take the accumulative release percentage at different time points as the study index by utilizing the orthogonal experiment, determine the final prescription of the hydrophilic matrix tablets by the formulation optimization of three factors having a significant effect on the drug release behavior, respectively measure the accumulative drug release degree at different time points by the high-performance liquid chromatography and ultraviolet spectrophotometry, so as to validate the feasibility of the accumulative release degree measured by the ultraviolet spectrophotometry in the formulation screening of the early stage; (2) the establishment of dose-effect relationships between the antioxidant biological activity index and the extract:use DPPH method, ABTS and FRAP assay method, establish linear correlation between the biological activity index and drug concentration; the establishment of dose-effect relationships between the blood vessel dilation bioactive index and the extract:establish culture in vitro of human umbilical vein endothelial cells (HUVEC-T1), seed in 96-well plates by 2.5* 104 mL-1, 37℃, after 24 h in 5% CO2 incubator, discard the old culture medium, divide the cells into:a normal cell group,1 mM CoCl2 damage group,1 mM CoCl2 damage plus Ginkgo biloba extract 100 μg. ml-1 group,1 mM CoCl2 damage plus Ginkgo biloba extract 50 μg. ml-1 group, 1 mM CoCl2 damage plus Ginkgo biloba extract 25 μg. ml-1 group,1 mM CoCl2 damage plus Ginkgo biloba extract 12.5 μg. ml-1 group,1 mM CoCl2 damage plus Ginkgo biloba extract 6.25μg. ml-1 group,1 mM CoCl2 damage plus Ginkgo biloba extract 3.125 μg. ml-1 group, collect the culture supernatant after 24 h, measure NO content in the supernatant by the classical Griess method, measure ET-1 content in the supernatant by the ELISA kit method, and analyze the relevance between the drug concentrations and NO, ET-1 content; regard the linear correlation coefficient r of equation established according to the biological activity index and drug concentration, the lowest concentration when in linear correlation, complicated or simple operation, repeatability as the study index, and select the relatively appropriate measuring method; (3) determine the release degree in vitro of homemade hydrophilic matrix tablets by the use of high performance liquid chromatography (HPLC), take ABTS test on the dissolution liquid at different time points, measure the free radical scavenging rate of ABTS, establish correlation between the component index and the biological activity index of release degree in vitro for Ginkgo biloba extract hydrophilic matrix tablets.Results:(1) For screening the prescription of Ginkgo biloba extract hydrophilic matrix tablets, based on the results of the single factor study, two matrix materials mixing (A), the mixing ratio (B), the filler amount (C) were further chose as three factors, three levels of the factors were:three levels for factor A are K4M-K15M mixed use, K4M-K100M mixed use and K15M-K100M mixed use, three levels for factor B were 4:1,3:1,2:1, the three levels for factor C are 5%,7%,10%, formulation optimization made by choosing a L9 (34) orthogonal table, the influence order of various factors on the drug release degree:HPMC mixing> filler amount> mixing ratio, the order of merits for each level of each factor was:A:2> 3> 1, B:2> 3> 1, C:3> 1> 2, the best combination was optimized as A2B2C3. The accumulative release degree of three batches of matrix tablets at different time points are measured by using the ultraviolet spectrophotometry and HPLC respectively, and calculated f2> 50 indicates the accumulative release behaviors of drugs measured by the two methods are similar, therefore the prescription screening is feasible by using the ultraviolet spectrophotometry in the early stage; (2) results for measuring method of the biological activity indexes of three antioxidants: ① taking DPPH free radical scavenging rate as a vertical axis, the drug concentrations as a horizontal axis, the correlation coefficient r= 0.9993 was obtained in the linear equation, its concentration range was 200-1200 μg.ml-1, the lowest concentration for reaching linear correlation was 200 μg.ml-1; ② taking ABTS free radical scavenging rate as a vertical axis, the correlation coefficient r= 0.9998 was obtained in the linear equation, its concentration range is 6.25-100 μg.ml-1, the lowest concentration for reaching the linear correlation was 200 μg.ml-1; ③ taking reducing capacity as a vertical axis, the extract concentrations as a horizontal axis, the correlation coefficient r= 0.9437 was obtained in the linear equation, its concentration range is 1.5-50μg.ml-1, the lowest concentration for reaching the linear correlation was 1.5μg.ml-1; (3) as observed through an inverted microscope:normal human umbilical vein endothelial cells were of uniform size, plump cells, strong stereoscopic impressions, clearer nucleus and the edge thereof, transparent and bright cells which are cobblestone-like tightly arranged, CoCl2 hypoxia can make cell activity reduced and cell morphology changed, after CoCl2 0.1mM induction for 24h, the vast majority of cells can maintain normal growth; 0.2-0.5 mM group was observed with the phenomenon that a few cells appeared with a shrunken cell wall, and synaptic growth slowed down, after 1 mM pretreatment for 24 h, it was observed that the number of cells with changes above appeared more significantly increased, and the synaptic retract appeared, empty bubbles appeared in the cells, the stereoscopic impression of the cell body become weak, cell deformation and cell debris appeared; after 2.5-5 mM treatment for 24 h, it was observed with rounded cell bodies, synapses disappeared, and cell shrinkage occurred basically. In addition to 0.1mM, compared with the control group, the cobalt chloride concentration groups were observed with decreased cell activity (P<0.05), there were significant differences among the concentration groups (P<0.05), the concentrationl mM was selected in the experiment, NO content in the cell supernatant of pure hypoxia group was significantly lowered (P<0.05), ET-1 was significantly increased (P<0.05), prompting the establishment of in vitro models; compared with normal cell groups, Ginkgo biloba extract concentration groups had NO secretion with significant difference (P<0.05), except when GBE concentration was 3.125μg. ml-1, the extract concentration groups had significant differences compared with the damage group (P<0.05); ET-1 concentrations of each drug group in Ginkgo biloba extract had significant differences compared with that of the normal group (P<0.05); compared with the normal group, each drug group and damage group had significant differences (P< 0.05); Ginkgo biloba extract can significantly interfere with the release of the cell damage model NO, ET-1, and the interference effect and Ginkgo biloba extract concentration had a certain linear relationship, the correlation analysis was made with the Ginkgo biloba extract concentration and corresponding after-administration cell supernatant NO, ET-1 content to show the correlation coefficients between the logarithm of the drug concentrations and NO, ET-1 content in the after-administration cell supernatant were r= 0.9759 and r= 0.9379, the final selection was the antioxidant biological activity index combined with the correlation coefficient of the equation obtained by the method of antioxidant biological activity measurement, repeatability, operation complexity or simpleness of the linear minimum concentration meter, and economical efficiency, and ABTS method was selected for the determination method; (4) ABTS clearance rate of in vitro dissolution of Ginkgo biloba extract sustained-release tablets was a dependent variable Y, the accumulative drug release degree was an independent variable X, the regression equation was established with the correlation coefficient r= 0.998, the results showed a good correlation between the two, the clearance rate of the biological effects was characterized as the drug accumulative release percentage, the f2 similarity factor method was used for comparing whether the biological effects resulting clearance rate and chemical effects resulting clearance rate were similar, and the calculated f2= 66.40> 50 indicated that there was similarity between the drug accumulative release percentage obtained by chemical analysis method and the drug accumulative release percentage obtained by the biological effect method, suggesting that ABTS radical scavenging test method can be used as in line with the characteristics of traditional Chinese medicine which has a multi-component a simple means and method for a simple and rapid evaluation of release characteristics of Ginkgo biloba extract release tablets.Conclusion:The experiment showed that the in vitro drug release behavior was basically consistent with characteristics of the sustained release preparation after the component measurement on the homemade Ginkgo biloba extract hydrophilic matrix tablets by HPLC, thus ABTS free radical scavenging test method can be used as a simple means and method for a simple and rapid evaluation of in vitro sustained release characteristics of Ginkgo biloba extract release tablets.