【作者】 何欣；

【导师】 欧阳五庆；

【作者基本信息】 西北农林科技大学 ， 基础兽医学， 2008， 博士

【摘要】 目的:利用纳米技术,以增强雷公藤多甙药效、降低其毒副作用为主体思路,制备高效低毒的雷公藤多甙纳米乳透皮制剂,并综合运用动物疾病模型、细胞生物学实验以及家兔体内药物代谢动力学实验对该新制剂的稳定性、安全性、有效性以及体内外释药性能进行深入研究。内容与方法:（1）雷公藤多甙纳米乳的制备及质量评价建立用于雷公藤多甙纳米乳含量检测的高效液相检测方法。在处方前研究中,测定雷公藤多甙在几种油和表面活性剂中的溶解度,滴定法绘制伪三元相图。根据相图中纳米乳区的大小、纳米乳的稳定性以及药物在油及表面活性剂中的溶解度,筛选最佳处方,制备雷公藤多甙纳米乳。利用染色法判断纳米乳的类型,利用透射电镜、激光粒度分析仪、黏度计、折光仪和电导仪对其进行质量评价,并对其稳定性进行考察。（2）雷公藤多甙纳米乳的安全性评价利用简化寇氏法对雷公藤多甙纳米乳的经口半数致死量进行测定,并对其皮肤急性毒性、皮肤和眼部刺激性进行考察。（3）雷公藤多甙纳米乳的药效学研究建立二甲苯诱导的小鼠急性耳肿胀和以血管通透性为主要改变的急性炎症模型,考察雷公藤多甙纳米乳的抗炎作用;通过醋酸致小鼠扭体试验和热板试验,研究雷公藤多甙纳米乳的镇痛作用;建立大鼠蛋清性关节炎和佐剂型关节炎模型,对雷公藤多甙纳米乳的抗炎及免疫效果进行深入研究;另外,体外培养家兔滑膜细胞,研究雷公藤多甙纳米乳对滑膜细胞的诱导凋亡作用。（4）雷公藤多甙纳米乳对肝、肾的毒性研究肝、肾是雷公藤多甙的主要毒性作用器官。在确定了该药物的药效基础上,我们对其毒副作用是否降低进行研究。首先观察了动物肝肾的组织病理学变化,并检测血清中AST、ALT、BUN以及尿液中尿蛋白的含量变化。另外,体外培养大鼠肝、肾细胞,从细胞水平考察该纳米乳的肝肾毒性。（5）雷公藤多甙纳米乳的体外释药与药动学研究建立血浆中雷公藤多甙纳米乳的含量检测方法。利用显色法进行雷公藤多甙的含量测定,对雷公藤多甙纳米乳的透皮性能进行研究,比较添加氮酮对透皮性能的影响,另外与雷公藤多甙混悬液的透皮性能以及雷公藤多甙片在模拟胃环境中的体外释药性能进行比较;家兔体外涂抹雷公藤多甙纳米乳和灌胃雷公藤多甙片混悬液后,用高效液相色谱测定血浆中药物含量,利用残数法拟合该纳米乳药物与传统剂型在家兔体内的动力学方程,并对其动力学参数进行比较分析。结果:（1）雷公藤多甙纳米乳的制备及质量评价结果建立了高效液相色谱检测方法,该方法稳定,在125²000 ng/mL范围内线性关系良好,平均回收率为92.14%。综合纳米乳区大小及溶解度测定结果,最终配方选择以聚氧乙烯氢化蓖麻油为表面活性剂,肉豆蔻酸异丙酯为油相,聚氧乙烯氢化蓖麻油:肉豆蔻酸异丙酯:水的质量比为27: 3.3:69.7,其中可添加5%以下的氮酮;制备的雷公藤多甙纳米乳为澄清、透明、流动性良好的棕黄色液体;亚甲基蓝在其中的扩散速度大于苏丹红Ⅲ,为O/W型纳米乳。经考察,此纳米乳粒子呈圆球形,分散性良好,平均粒径为23.6 nm,Zeta电位为（–5.35±0.42）mV,折光率为（1.3617±0.0051）nD²⁰,电导率为（97.6±3.6）μs/cm,黏度为（3.56±0.12）mm²·s^-1;且该纳米乳稳定性参数的平均值为3.13%;室温、40℃环境下放置的样品颜色基本不变;在室温环境下放置9个月,样品含量无明显变化。（2）雷公藤多甙纳米乳的安全性评价结果经简化寇氏法计算雷公藤多甙纳米乳的经口半数致死量为977.24 mg/kg,是雷公藤多甙原料药的3.02倍;所制备的雷公藤多甙纳米乳无皮肤急性毒性;无论是单次还是多次给药,对完整皮肤和破损皮肤均无刺激性,对眼无刺激性。（3）雷公藤多甙纳米乳的药效学研究结果所制备的雷公藤多甙纳米乳可以显著抑制二甲苯所致的耳肿胀并降低毛细血管通透性（P<0.01）,可以减少醋酸所致的小鼠扭体次数,延长热板痛阈时间（P<0.05）。雷公藤多甙纳米乳对蛋清性关节炎的足肿胀具有保护作用,且表现出一定的缓释性;对佐剂型关节炎大鼠原发侧和继发侧足肿胀有明显的抑制作用;可以增强体内炎症因子SOD的活力、提高NO的含量;从脾淋巴细胞增殖试验可以看出,雷公藤多甙片和雷公藤多甙纳米乳对T、B淋巴细胞增殖抑制率分别为15.2%、25.3%和15.8%、22.9%,抑制率相差分别为10.1%和7.1%;雷公藤多甙纳米乳和雷公藤多甙片可以诱导体外培养的滑膜细胞发生凋亡,流式细胞仪结果显示,同等剂量下前者是后者的3.35倍。（4）雷公藤多甙纳米乳对肝、肾的毒性研究结果组织病理学观察发现相同剂量下,雷公藤多甙纳米乳作用组的肝细胞索基本清晰,但肝细胞稍有肿胀,雷公藤多甙片组的大鼠肝细胞索紊乱,肝细胞变形,有的呈空泡状;雷公藤多甙纳米乳组的大鼠肾小管细胞轻微肿胀,雷公藤多甙片组大鼠肾小管上皮增厚,细胞肿胀、脱落,炎性细胞浸润。雷公藤多甙纳米乳组血清中ALT、AST、BUN和尿液中尿蛋白的含量明显低于雷公藤多甙片组（P<0.05）;雷公藤多甙纳米乳对肝细胞的IC50为128.82 mg/mL,是雷公藤多甙片的2.65倍。雷公藤多甙纳米乳对肾细胞的IC50为61.66 mg/mL,是雷公藤多甙片的3.16倍。（5）雷公藤多甙纳米乳体外释药与药动学研究结果建立了血浆中雷公藤内酯甲的含量测定方法,该方法稳定,在62.5¹500 ng/mL线性关系良好,平均回收率为95.41%,可以满足动力学研究需要。通过体外释药性能的研究,雷公藤多甙纳米乳中不含氮酮时稳态渗透速率为75.48μg·cm- 2·h- 1,而当氮酮的含量增加至5%时,稳态渗透速率下降至24.33μg·cm- 2·h- 1,仅是无氮酮配方的32.23%;雷公藤多甙混悬液和雷公藤多甙片透析的稳态渗透速率分别为16.40μg·cm- 2·h- 1和16.15μg·cm- 2·h- 1。通过药动学研究,雷公藤多甙纳米乳透皮给药与雷公藤多甙片口服给药均属于有吸收的二室模型。雷公藤多甙纳米乳的消除半衰期t1/2β为16.956 h,是片剂的1.782倍;雷公藤多甙纳米乳的相对生物利用度是雷公藤多甙片的141.352%,提高了41.35%。结论:制备的雷公藤多甙纳米乳是澄清、透明、流动性良好的棕黄色液体。该纳米乳属于O/W型,粒子形态大多为圆球形,分散性良好,粒径约为23.6 nm,具有良好的稳定性。该纳米乳的半数致死量显著小于雷公藤多甙片,无皮肤急性毒性,无皮肤刺激性和眼部刺激性,具有安全性。该纳米乳具有显著的抗炎和免疫抑制效果,对体外培养的滑膜细胞有较强的诱导凋亡作用。对肝、肾组织以及对肝、肾细胞的毒性显著降低,临床用药安全。在家兔体内的消除半衰期明显长于传统制剂雷公藤多甙片,相对生物利用度提高。雷公藤多甙纳米乳具有高效、低毒的优点,并且具有一定的缓释效应,且雷公藤多甙纳米乳制剂已申请国家发明专利,专利申请号:200610104979.2。更多还原

【Abstract】 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.更多还原