| Subject: Using the nanotechnology, prepare the nanoemulsion of tripterygium wilfordii polyglycoside in order to strengthen the drug action and degrade the adverse reaction. Study the stability, safety, efficacy and the pharmacokinetics and in vitro release of this praeparatum through animal disease model, cytobiology experiment and pharmacokinetic experiment in rabbit.Method: (1) The preparation and quality on the tripterygium wilfordii polyglycoside nanoemulsion. To establish the HPLC analytical method for content determine of tripterygium wilfordii polyglycoside nanoemulsion. Preformulation study, determine the dissolubility of tripterygium wilfordii polyglycoside in different oil and surfactant. Draw the three initiatives phase diagram titration. The prescription was screened according to the size of nanoemulsion district and the dissolubility and then the tripterygium wilfordii polyglycoside nanoemulsion was prepared. Judge the type of nanoemulsion using staining method. The quality was evaluated using transmission electron microscope, laser particle size analysis, particle size analysis, refractometer and conductimeter. The stability was investigated. (2) The safety evaluation on tripterygium wilfordii polyglycoside nanoemulsion. Evaluate the safety by acute toxicity (simplify Karber's method), skin acute toxicity, skin irritation and eye irritation (single and several). (3) The pharmacodynamics evaluation on tripterygium wilfordii polyglycoside nanoemulsion. Investigate the anti-inflammatory action through acute inflammation model including acute swelling of ear induced by dimethyl benzene and the changes of vasopermeability. Investigate the analgesic effect through mouse writhing test and hot plate test. Study the anti-inflammatory action and immunization action through the model of albumen arthritis and adjuvant arthritis. Otherwise, in vitro culture the rabbit synoviocyte, study the enhancement Apoptosis action of tripterygium wilfordii polyglycoside nanoemulsion. (4) Toxicity on liver and kidney of tripterygium wilfordii polyglycoside nanoemulsion. First, observe the histopathological change of liver and kidney and detect the content of AST,ALT,BUN in serum and the content of Urine protein in urine. Second, study the cytotoxicity through in vitro culture the hepatocyte and renal tubular epithelial cell. (5) The pharmacokinetics and in vitro release of tripterygium wilfordii polyglycoside nanoemulsion. To establish the HPLC analytical method was used in dynamic. Study the penetrate efficiency, researched the influence of adding azone in prescription and the release behavior difference between the prescription, suspl of tripterygium wilfordii polyglycoside and tripterygium wilfordii polyglycoside tablets. After painting the tripterygium wilfordii polyglycoside nanoemulsion and intragastric administration tripterygium wilfordii polyglycoside tablets in rabbits, determine the content of wilforlide A in blood plasma. Using method of residual fitting the kinetic equation and then calculate the parameter of dynamics. So study the differences between nanoemulsion and tablets.Results: (1) The results of preparation and quality on tripterygium wilfordii polyglycoside nanoemulsion. The HPLC analytical method for content determine of wilforlide A was stabilize. The good line range was 1251500 ng/mL. The mean recovery was 92.14%, it could be used in quality control research. According to the size of nanoemulsion district and the dissolubility, RH-40 and IPM were choosed the surfactant and the oil phase of the optimization prescription. And the proportion by weight of RH-40, IPM and water was 27: 3.3:69.7. Content of azone in prescription should lower 5%. tripterygium wilfordii polyglycoside nanoemulsion was a kind of clear, transparent, satis flowability buffy liquid. Spreading rate of methane blue was higher than the Sudan redⅢ. So the nanoemulsion was assessed O/W form. The nanoemulsion particle was globular, satis dispersibility, the mean diameter was 23.6 nm, Zeta electric potential was (–5.35±0.42) mV, refractive index was (1.3617±0.0051) nD20, conductivity was (97.6±3.6)μs/cm, viscosity was (3.56±0.12) mm2·s-1, stability parameter was 3.13%. The color of nanoemulsion was invariably in room temperature and 40℃, the content of nanoemulsion was invariably in room temperature in nine months. (2) The results of safety evaluation on tripterygium wilfordii polyglycoside nanoemulsion. The median lethal dose of tripterygium wilfordii polyglycoside nanoemulsion was 977.24 mg/kg, this was 3.02 times of tripterygium wilfordii polyglycoside crude drug. tripterygium wilfordii polyglycoside nanoemulsion was no skin acute toxicity; there were no stimulation to the party of rabbit's skin and eye no matter single or multiple dosing. (3) The results of pharmacodynamics evaluation on tripterygium wilfordii polyglycoside nanoemulsion. It could striking inhibit acute swelling of ear induced by dimethyl benzene and degrade the vasopermeability (P<0.01). It could decrease the writhing times and lengthen the time of pain threshold (P<0.05). It could protect the feet swell of albumen arthritis mouse and show the delayed release character. Conspicuous depressant effect to feet swell of adjuvant arthritis were found both primarily side and opposite side. It could reinforce the energy of SOD, increase the content of NO. Inhibition ratio to T,B lymphocyte of tripterygium wilfordii polyglycoside tablets and tripterygium wilfordii polyglycoside nanoemulsion were 15.2%,25.3% and 15.8%,22.9%, the differences were 10.1%and 7.1% respective. It could enhance the apoptosis action of in vitro culture the rabbit synoviocyte. According to the result of flow cytometry, the former was 3.35 times to the latter. (4) The results of toxicity on liver and kidney. In the same dosage, the liver cell cord was clear, the hepatocyte and renal tubular cell were slightly swell in tripterygium wilfordii polyglycoside nanoemulsion group; moreover the liver cell cord was chaotic and the hepatocyte cytomorphosis, sometimes the hepatocyte showed vacuole, the nephric tubule endepiderm is become thickening, cellular swelling, even shedding and inflammatory cell infiltrate in tripterygium wilfordii polyglycoside tablets. The content of ALT,AST,BUN in serum and urine protein in tripterygium wilfordii polyglycoside nanoemulsion group were obviously lower than the group of tripterygium wilfordii polyglycoside tablets (P<0.05). tripterygium wilfordii polyglycoside nanoemulsion's IC50 was 128.82 mg/mL to hepatocyte, it was 2.65 times to tripterygium wilfordii polyglycoside tablets. tripterygium wilfordii polyglycoside nanoemulsion's IC50 was 61.66 mg/mL to renal cell, it was 3.16 times to tripterygium wilfordii polyglycoside tablets. (5) The results of pharmacokinetics and in vitro release of tripterygium wilfordii polyglycoside nanoemulsion. The HPLC analytical method for content determine in plasma of wilforlide A was stabilize. The good line range was 62.51500 ng/mL. The mean recovery was 95.41%, it could be used in dynamics research. Homeostasis permeate velocity was 75.48μg·cm- 2·h- 1when there were no azone in prescription. Homeostasis permeate velocity was 24.33μg·cm- 2·h- 1when there were 5% azone in prescription, this was 32.23% of former. Homeostasis permeate velocity of the crude drug and the tripterygium wilfordii polyglycoside tablets were 16.40μg·cm- 2·h- 1 and 16.15μg·cm- 2·h- 1. tripterygium wilfordii polyglycoside nanoemulsion and tripterygium wilfordii polyglycoside tablets were all fit for the two room open model. Elimination half life of tripterygium wilfordii polyglycoside nanoemulsion was 16.956 h, it was 1.782 times to tripterygium wilfordii polyglycoside tablets. tripterygium wilfordii polyglycoside nanoemulsion raised relative bioavailability to 141.35%. It was an increase of 41.35% over tripterygium wilfordii polyglycoside tablets.Conclude: tripterygium wilfordii polyglycoside nanoemulsion is a kind of clear, transparent, satis flowability buffy liquid. It is assessed O/W form. The particle is globular, satis dispersibility, the mean diameter was 23.6 nm. The nanoeulsion have satisfactory stability. The median lethal dose of tripterygium wilfordii polyglycoside nanoemulsion is significant lower than tablets. There is no skin acute toxicity; no stimulation to the party of rabbit's skin and eye no matter single or multiple dosing. Tripterygium wilfordii polyglycoside nanoemulsion has noticeable anti-inflammatory and immune suppression effect and it can enhance the apoptosis action of in vitro culture the rabbit synoviocyte. Toxicity to liver, kidney, hepatocyte and renal tubular epithelial cell are lower than tablets. So the tripterygium wilfordii polyglycoside nanoemulsion is safety. Elimination half life of tripterygium wilfordii polyglycoside nanoemulsion is longer than the tablets, in addition, relative bioavailability is raised than tablets. High performance, low toxical and delayed release are the character of tripterygium wilfordii polyglycoside nanoemulsion.The research outcome has been applied for Nation invention patent, apply order is 200610104979.2.
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