艾叶挥发油纳米结构脂质载体的制备及其抗乙肝病毒活性的研究

发布时间：2018-07-26 17:22

【摘要】：艾叶为菊科多年生草本植物艾(Artemisia argyi Lévl.et Vant.)的干燥叶,是临床常用传统中药。艾叶挥发油是其主要有效成分群,具抗菌抗病毒、平喘镇咳祛痰、抗过敏、免疫、护肝利胆等作用[1-9]。本课题组前期研究,首次发现艾叶挥发油具有抗乙肝病毒活性[10]。为研究艾叶挥发油抗乙肝病毒(HBV)新药,又鉴于艾叶挥发油水难溶、易挥发、不稳定,影响药效,本文对艾叶挥发油纳米结构脂质载体(Nanostructured lipid carrier system,NLC)进行了研究,包括:艾叶挥发油NLC的制备、质量评价、药代动力学、组织分布和药效学。此研究未见报道。1.艾叶挥发油NLC的制备及质量评价采用加热熔融-超声分散法制备艾叶挥发油NLC,通过单因素实验和正交试验优化制备工艺及处方。最佳制备工艺为:初乳搅拌10min,超声时间15min,超声振幅100%。最佳处方为:波洛沙姆188 0.45g,蛋黄卵磷脂0.15g,单硬脂酸甘油酯0.1g,辛酸/癸酸甘油三酯0.1g,艾叶挥发油0.1g,蒸馏水加至20m L。通过气相色谱法建立了艾叶挥发油NLC的多成分定量测定方法。方法精密度高,重现性好,桉油精、樟脑、龙脑加样回收率均在95%?105%之间。测得的桉油精、樟脑、龙脑的含量分别为16.34%、3.32%、5.92%。采用纳米粒度分析仪测定粒径与Zeta电位,懫用指纹图谱进行质量整体描述及工艺评价,并从包封率、载药量、体外释放度及稳定性来评价制剂的特性。艾叶挥发油NLC平均粒径为(72.33±1.93)nm,PDI为(0.273±0.0045),Zeta电位为(-30.59±1.42)mv。桉油精、樟脑、龙脑包封率分别为87.49%、86.45%、92.12%,载药量分别为8.25%、2.00%、3.38%。指纹图谱得出十批制剂的相似度均大于0.990,说明十批制剂一致性好,工艺质量稳定。从体外释放度结果可知,在120h时,艾叶挥发油NLC的释放度为61.12%,而原料药的释放度为72.46%,相同时间制剂的释放度比原料药的低,说明艾叶挥发油NLC具有缓释性。药物稳定性实验表明,艾叶挥发油NLC在高温、强光照条件下易发生变化,应放置在4℃条件下避光保存。2.艾叶挥发油与其NLC的药代动力学及组织分布研究本文首次建立了艾叶挥发油及NLC在血浆中桉油精含量的GC测定方法,并对其在大鼠体内的药代动力学进行了研究。艾叶挥发油以1000mg/kg的剂量灌胃、140mg/kg的剂量尾静脉注射给药,艾叶挥发油NLC以同等于艾叶挥发油剂量给药,给药后于不同时间点取血,GC法检测桉油精的血药浓度,采用DAS3.0药代动力学软件拟合,得药代动力学参数。灌胃4h后,艾叶挥发油NLC达到最大血药浓度(Cmax)11.4835μg/L,半衰期(t?)为8.727h,而艾叶挥发油Cmax为9.1064μg/L,t?为7.779h;艾叶挥发油NLC灌胃后的药时曲线面积AUC_(0-∞)为160.283μg/L·h,艾叶挥发油AUC_(0-∞)为103.681μg/L·h。结果表明,艾叶挥发油NLC延长了艾叶挥发油在体内的作用时间,提高了药物生物利用度,改变了药物的药代动力学特征。本文通过小动物活体成像跟踪纳米粒在不同时间不同组织中的分布情况。设立A、B、C三组,A组为生理盐水组,B组为Di R染料组,C组为Di R-NLC组,于不同时间点麻醉,放置于小动物活体成像仪中进行观察拍照,并于24h后取出裸鼠的心、肝、脾、肺、肾各组织,放置到小动物活体成像仪中进行观察拍照,研究纳米粒在裸鼠体内不同组织中的分布情况。结果表明,随着时间的延长,纳米粒逐渐在肝部位蓄积,且从体外不同器官的荧光强度图可知,Di R-NLC组肝脏部位荧光强度比染料组强,因此表明艾叶挥发油NLC具有肝脏靶向性。3.艾叶挥发油及其NLC抗HBV药效学研究采用鸭乙肝病毒模型来观察艾叶挥发油及其NLC的抗HBV作用。1日龄麻鸭正常饲养3天后静脉注射含DHBV的强阳性血清进行攻毒,7天后使用PCR法筛选出感染乙肝病毒的阳性鸭,然后取阳性鸭112只随机分成8组,给予不同剂量的艾叶挥发油及NLC,连续给药15天,于给药后5d、10d、15d及停药后3d检测血清和肝脏组织中DHBV DNA水平,评价艾叶挥发油及其NLC的抗HBV活性。结果表明,艾叶挥发油及NLC对鸭血清及肝脏组织中HBV均有抑制作用,高剂量抑制作用更明显,且艾叶挥发油NLC抑制作用比原料药更显著。本文首次研究了艾叶挥发油NLC,为艾叶挥发油抗HBV新药的研究提供了科学依据。
[Abstract]:Artemisia Artemisia is the dry leaf of Artemisia argyi L e vl.et Vant. of perennial perennial herb of the Compositae. It is a traditional traditional Chinese medicine. The essential oil of Artemisia Artemisia is its main effective component group, which has antivirus and antivirus, antiasthmatic, antitussive and expectorant, anti allergy, immunity, liver protection and gallbladder, and so on. It was first found that the volatile oil of Artemisia Artemisia has resistance to the first group. Hepatitis B virus active [10]. is a new drug to study the anti hepatitis B virus (HBV) of essential oil of Artemisia Artemisia, and in view of the insoluble, volatile, unstable and influence effects of the volatile oil of AI leaf, the Nanostructured lipid carrier system (NLC) is studied in this paper, including the preparation, quality evaluation and medicine of the volatile oil NLC of the leaf leaf. The preparation and quality evaluation of.1. leaf volatile oil NLC was not reported in this study. The preparation process and prescription of the volatile oil NLC were prepared by the heating melting ultrasonic dispersion method. The optimum preparation process was: primary milk stirring 10min, ultrasonic time 15min, ultrasonic amplitude 10 The best prescription of 0%. is: Polo Shameem 188 0.45g, egg yolk lecithin 0.15g, glycerol monostearate 0.1g, octanoic acid / decyl triglyceride 0.1g, volatile oil 0.1g, distilled water and 20m L. to establish a multi component quantitative determination method of volatile oil NLC in the leaf of argyrum, high density, good reproducibility, eucalyptus, camphor, and borneol The recovery rate was between 95% and 105%. The content of the measured eucalyptus, camphor and borneol were 16.34%, 3.32%. 5.92%. was used to determine the particle size and Zeta potential by the nanoscale analyzer. The quality of the preparation was evaluated by the fingerprint and the properties of the preparation were evaluated from the encapsulation rate, the load, the release degree and stability. The average particle size of oil NLC was (72.33 + 1.93) nm, PDI was (0.273 + 0.0045), Zeta potential was (-30.59 + 1.42) mv. eucalyptus, and camphor and borneol encapsulation rates were 87.49%, 86.45%, 92.12% respectively, and the drug loading was 8.25%, 2%, respectively, and the similarity of the ten batches of preparations were all greater than 0.990, indicating that ten batches of preparations were in good agreement and the process quality was stable. From the results of the release in vitro, the release degree of the volatile oil NLC was 61.12% at 120h, and the release degree of the API was 72.46%. The release degree of the preparation was lower than that of the raw material, indicating that the NLC of the volatile oil of Artemisia Artemisia had sustained release. The experiment of drug stability showed that the Ai Yehui oil NLC was easy to change under the condition of high temperature and strong illumination. Study on the pharmacokinetics and tissue distribution of the volatile oil in.2. leaves and its NLC under the condition of 4 degrees C, the GC method for determining the content of the essential oil and NLC in the plasma was established for the first time, and the pharmacokinetics of the volatile oil in the rat was studied in the rat. Dose tail vein injection, NLC of essential oil of AI leaf is equal to the dose of essential oil of Artemisia leaf. After administration, blood is taken at different time points. GC method is used to detect the blood concentration of Eucalyptus, and the pharmacokinetic parameters are fitted with DAS3.0 pharmacokinetics software. After 4h, the Ai Yehui oil NLC reaches the maximum blood concentration (Cmax) 11.4835 mu g/L, half T (t?) was 8.727h, and the essential oil of AI leaf was 9.1064 mu g/L and t? 7.779h; the curve area AUC_ (0-) of the volatile oil after NLC was AUC_ (0-) was 160.283 mu g/L. H, and the volatile oil of AI leaf was 103.681 micron. A, B, C three groups, A group as physiological saline group, B group Di R dye group, C group Di R-NLC group, C group Di R-NLC group, were observed and photographed in small animal living imaging apparatus at different time points. After 24h, the heart, liver, spleen, lung, and kidney tissues were taken out to observe and photograph the small animal living body imaging apparatus. The distribution of nanoparticles in different tissues of nude mice was studied. The results showed that the nanoparticles gradually accumulated in the liver of the nude mice, and the fluorescence intensity map of different organs in vitro was known, Di R-NLC The fluorescence intensity of liver site was stronger than that of the dyestuff group. Therefore, it was indicated that the volatile oil NLC of the livers had the liver targeting.3. leaf volatile oil and its NLC anti HBV Pharmacodynamics Study using the duck hepatitis B virus model to observe the anti HBV effect of the volatile oil and NLC of NLC,.1 day age ducks of.1 were fed with the strong positive sera containing DHBV in the static vein for 3 days. 7 days later, PCR method was used to screen positive ducks infected with HBV. Then 112 positive ducks were randomly divided into 8 groups, giving different doses of volatile oil and NLC for 15 days. 5D, 10d, 15d and 3D were used to detect DHBV DNA in serum and liver tissues after administration, and the anti HBV activity of the volatile oil and NLC was evaluated. The volatile oil and NLC of Artemisia Artemisia had inhibitory effect on HBV in the serum and liver tissues of ducks. The inhibitory effect of high dose was more obvious, and the inhibitory effect of NLC on the essential oil of Artemisia Artemisia was more significant than that of the drug. This paper first studied the NLC of the volatile oil of Artemisia Artemisia, and provided a scientific basis for the study of the anti HBV drug of the volatile oil of Artemisia Artemisia.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R283.6;R285

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