| Objective: Resveratrol(Res) is a non-flavonoid polyphenols hydroxyl compounds, widely distributed in grapes, peanuts, gnetum, pine, rhizoma polygoni cuspidati and maackia amurensis and 70 kinds of plants. Resveratrol is colorless needle crystal, in water solubility is very low, but soluble in polar organic solvents(such as methanol, ethanol, ethyl ether, carbon tetrachloride, etc.). Its anti-bacterial, anti-cancer, anti-platelet aggregation, protection of cardiovascular and estrogen like effects and other health functions, has been applied in medicine, functional food. Because resveratrol is difficult to dissolve in water, the half-life is short, the metabolism and elimination rate is quick, the biological utilization degree is low, the stability is bad and so on the characteristic, has greatly restricted its clinical application. In this study, the polymer micelles were prepared according to the hydrophobicity of resveratrol, and the aim was to increase the water solubility, stability and bioavailability of resveratrol significantly.Methods:1 The preparation of resveratrol polymeric micelles:(1) The phospholipid/sodium/PVP three(PC/BS/PVP-MMS) of the insoluble drug was prepared by thin film dispersion method. The prescription of the single factor investigation to determine various preparation conditions, and through the drugs and the phospholipid mass ratio(a), the dosage of PVP(b), phospholipids and DNA sodium cholate molar mass ratio(c) by a factor of 3 different levels of preparation process, the effect of L9(34) orthogonal table of, the solubilization ratio and packet encapsulation rate as a standard screening and to determine the best prescription. In accordance with the proportion of the best prescription said take each component is placed in a round bottom flask, adding a certain amount of organic solvent, ultrasonic dissolving, which is removed by a vacuum rotary evaporation of the organic solvent, add the right amount of saline and uniform shaking water removal of membrane, with0.2?m membrane filter can get resveratrol pbpm polymeric micelles.(2) The block copolymer of praziquantel and dissolved in an appropriate amount of organic solvent, slow dropwise adding distilled water with rapid stirring. At room temperature, stirring volatile organic solvent, the solution of clear and transparent micro strip light blue emulsion was obtained, and the insoluble particles were filtered by 0.45?m filtration membrane, and the filtrate was collected, which was Res/m PEG-PLA micellar solution. 2 The identification method of the polymer micelles of resveratrol: using transmission electron microscope and particle size detection method to verify the formation of resveratrol polymer micelle. 3 The quality of resveratrol polymer micelles: a HPLC method was established for the determination of the drug content of the polymeric micelles of resveratrol. Influencing factor test and accelerated test were carried out in order to investigate the stability of particle size, drug content and encapsulation efficiency. 4 In vitro study on the in vitro hemolysis of resveratrol micelles: preparation of red blood cell suspension from rat blood and preparation of resveratrol micelles according to the formula. The red blood cell suspension was added to the micelle at different concentration, and the A value of each group was observed and measured by spectrophotometry. 5 Pharmacokinetics test: SD rats were used as the subjects, and two administration modes of administration of the stomach and the tail vein were used. HPLC method was used to determine the concentration of the drug in the blood of rats after the injection of resveratrol in the rat's blood. The pharmacokinetics parameters and relative bioavailability were studied. 6 Cell test: the human breast cancer cells MCF-7 after subculture and culture, set the control group, soluble enzyme group, as well as Res and Res-MP micellar group, through inhibition rate to evaluate the inhibitory effect on MCF-7 cell growth. 7 In vivo anti-tumor effects of mices: 30 S180 mice were randomly divided into blank control group, normal saline group(negative control group),cyclophosphamide group(positive control group) and PBPM and m PEG-PLA polymer micelle group. The tumor inhibition rate was observed. The spleen index of each group was used to evaluate the anti-tumor effect of polymeric micelles in mice.Results:1(1) According to the results of single factor investigation, the prescription of soybean lecithin, sodium salt and PVPK30 were selected. According to the L9(34) orthogonal table, the optimal prescription was selected by the criteria of solubilization ratio and encapsulation efficiency:Res:phospholipid:bile salt: PVP =0.025:0.15:0.225:0.05.(2) According to the results of prescription, the copolymer m PEG1900-PLA8000 as carrier, acetone as solvent, the volume ratio of acetone to aqueous phase was 1:4, and the drug loading ratio was 35%. 2 The use of transmission electron microscopy and particle size detection method to verify the resveratrol polymer micelles. Transmission electron microscopy showed that the formation of polymeric micelles, the appearance of a round, uniform size, particle size in the 118 nm or so. Particle size was detected by Malvin particle size analyzer, and the results showed that the particle size was 118-123 nm, which was in accordance with the requirements of polymer micelles. Table understanding of the success of the preparation of the polymeric micelles. 3 HPLC method was established to determine the content of resveratrol. The results showed that the concentration and peak area of resveratrol in the range of 10?g/m L~200?g/m L have good linear relationship, recovery, stability and precision are in line with the requirements of the methodology. The stability test of the polymer micelle of resveratrol showed that the appearance of the polymer was changed into white turbidity at high temperature, and the content of the polymer was also changed obviously. Under the illumination condition, although its appearance has no obvious change, but the content change is obvious, the reason is that the resveratrol in the light condition will take place the CIS trans isomerization and the degradation reaction, therefore needs to avoid the light operation and the preservation.4 In vitro study of the effect of resveratrol on the in vitro hemolysis: the results showed that the PBPM group had a hemolytic effect under the experimental concentration, while the hemolysis rate of Res-MP group was less than 5%. 5 Pharmacokinetics test: Resveratrol extravascular to drugs and intravenous injection are in line with the single compartment model, rat irrigation stomach and tail vein injection Res-MP micelles compared to peak plasma Tmax and MRT has been extended, Cmax and AUC increased significantly. 6 Cell test: Res-MP in the concentration range of 1-1000?mol/L has inhibitory effect on the growth of MCF-7 cells, the inhibition increased with the increase of drug concentration and time. 7 Mice in vivo antitumor effect of research: Res-PM polymeric micelles group have strong anti-tumor effect, polymer Res-PM micelle group tumor rate was 75.86%, anti tumor effect of strong, the function of immune organs no significant effect and safety.Conclusions: The preparation of two kinds of resveratrol micelles were simple and reproducible. Effectively improves the solubility of resveratrol, and improve the resveratrol light easily decomposable properties, significantly improve the biological utilization rate, for the future of resveratrol anti-tumor research, to lay a theoretical and experimental basis. This study showed that the polymer micelles can increase the solubility of insoluble drugs, and further expand the clinical application of soluble drugs. |
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