| Background & objectivePhospholipid-complex which was composed of a certain proportion of drug and phospholipid was synthesized in non-proton transfer solvents. Since phospholipid molecules were amphiphilic, phospholipid was not only a very effective emulsifier, but also an important component of cell membranes. Phospholipid-complex can penetrate the skin barrier and had strong and lasting efficacy. It can improve the absorption and bioavailability of certain medicine, furthermore, delay the release of the drug and the speed of elimination.GBE50 was extracted from Ginkgo biloba leaf and refined, formed the production of second-class Chinese traditional medicine approved by the State Food and Drug Administration. GBE50 was composed of the two main ingredients: flavonoids and ginkgolide. Flavonoids include quercitrin,nimbecetin and bisflavones. It can remove the free radical and superoxide anion radical in order to prevent from the damage of oxygen free radical and injury of lipid peroxidation. It can not only significantly lower the blood lipids,cholesterol and myocardial oxygen consumption, but also increase the blood flow of coronary and improve the microcirculation. It mainly used to prevent and treat the cardiovascular and cerebrovascular diseases. But its poor absorption and less bioavailability limited its clinical application. This study was to synthesize phospholipid-complex, hoping to improve the dissolubility,absorption and bioavailability. Main ContentThe complexing rate was usually used to evaluate the preparation technology of phospholipid-complex. There are many factors such as the composition and quality of the phospholipids, the selection of solvent, the ratio of drug and phospholipids, the reacting time and temperature, the concentration of drug will affect the complexing-rate. The preparation conditions for Ginkgo biloba L-phospholipid complex were optimized by means of single factor study and orthogonal design, and took the complexing rate of total flavonoides as assessment criteria.The results of the single factor study were as follows:solvent was Tetrahydrofu-ran, the reacting time was 6h, the reacting temperature was 40℃, the concentration ofGinkgo biloba L was 20mg/ml, the ratio of Ginkgo biloba L to phospholipids was 1 to 2.According to the results of single factor study, the factors of the ratio of Ginkgo biloba L to phospholipids, the concentration of GBE50 and the reacting temperature were choosing to optimized preparation conditions by orthogonal design. The optimization results were as follow:the ratio of Ginkgo biloba L to phospholipids was 1 to l,the concentration of GBE50 was 20mg/ml, the reacting temperature was 30℃.The preparation process of GBE50-phospholipid complex was as follow:A certain amount of phospholipids was weighed, solved in the tetrahydrofuran, theGBE50 was added, the solution was put n into the magnetic agitator and was agitated for 6h at 30℃,After the filtering and drying in vacuum, using methylene chloride the inert solvent to refinethe product, then vacuum drying and got the GBE50-phospholipids complex.DSC and Infrared spectrophotometry were chosen to identify the phospholipid complex.:The peak of drug and pho-spholipid in the DSC curve of phospholipid complex were disappeared, and the phase-transition temperature of complex was lower than that of the phospholipids, this result may be due to the interaction happened between of GBE50 and the polar part of phos-pholipids. The chains of hydrocarbon in phospholipids were in the free rotation state enveloped the polar parts the GBE50, so that the ordering of the chain was decreased and the second adsorhed peak of phosph-olipid was disappeared. But the change also appeared in the mixture of GBE50 and phospholipids, the reason was may be that certain interaction between them was occurred when the mixture was detected, so that further identification should be done by Infrared spectrophotometry. In the infrared spectrum, the wide peak of the phospholipids changed to shap one between 3500-2800 cm-1, and this situation doesn't happened the mixture wasn't changed. The profile and width of the peak also changed at 1782,1514 cm-1 by the influence of GBE50. The phospholipid complex also retained the main peaks of GBE50, but some changes have taken place such as the weaken of the peak at 1782,1514 cm-1, the disappearance of the peak at 631 cm-1, it can be illustrated that some interaction between GBE50 and phospholipids was happened. The GBE50-phospholipid complex was formed.The apparent oil water distribution coefficients of GBE50-phospholipid complex was improved compared with GBE50. Generally speaking, the higher distribution coefficients, the more easily the drug penetrate the cell membrane. So it will increase the possibility of higher bioavailability.Re-HPLC was used to determine the content of flavonol glycosides in the GBE50-phospholipid complex with choosing querceti,kaempferide,isorhamnetin as the reference. The mobile phase was methanol and 0.5% phosphoric acid (54:46) at the speed of lml/min, the detective wavelength was 368nm. The standard curve of quercetin was that:y=9E+07x+19403 (R2=0.9996),the linear range was from 0.00350 mg/ml to 0.11200 mg/ml, the average recovery was 99.39% and RSD was0.95%. The standard curve of kaempferide was that:y=1E+08x+12672 (R2=0.9996),the linear range was from 0.00160 mg/ml to 0.05120 mg/ml, the average recovery was 99.71% and the RSD was 0.76%. And the standard curve of isorhamnetin was that:y=7E+07X-1046.3 (R2=0.9997),the line range was between 0.00025 mg/ml and 0.00800 mg/ml, the average recovery was 99.20% and the RSD was 0.69%.The content of flavonol glycosides in the GBE50-phospholipid complex was 11.54%.The permeability of GBE50-phospholipid complex via intestine membranes was evaluated in vitro diffusion chamber system. flavonol glycosides were taken in the content of in the receptor which determined by Re-HPLC as assessment criteria, the best intestine was selected from duodenum, jejunum, ileum and colon by comparing with the cumulative permeation amount and the Papp. It was also used to compare the GBE50-phospholipid complex with the GBE50 and positive control group. The results show that the best intestine was jejunum and the permeability of GBE50-phospholipid complex in jejum was better than that of the other two group(P0.01).The protective effect of GBE50-phospholipid complex to cardiomyocytes was evaluated by determining the leakage of lactic acid dehydrogenase (LDH), metabolism activity (MTT)of the cardiomyocytes treated by H2O2. There were three groups including GBE50-phospholipid complex, GBE50 and the positive control group. the cell viability of model group was significantly lower than that of compared with normal control group (P< 0.001).furthermore, the higher concentration of GBE50-phospholipid complex, the better cell viability of the cardiomyocytes. The middle concentration of GBE50-phospholipid complex (3.63μg/ml) was not different from the positive control group (4.2μg/ml), but the concentration of flavonol glycosides was lower, perhaps it was due to the phospholipids. Compared with normal control group(56.7±1.53 U/L), the leakage of LDH of model group(84±2.64 U/L) was obviously increased(P< 0.001). The low concentration of GBE50-phospholipid complex(2.1μg/ml) was not different from that of the GBE50 group, The middle concentration of GBE50-phospholipid complex (3.63μg/ml) was not different from the positive control group (4.2μg/ml), but the concentration of flavonol glycosides was lower, so it can conclude that the protective effect of GBE50-phospholipid complex to cardiomyocytes treated by H2O2 was stronger than the other groups. |
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