艾葉揮發(fā)油納米結(jié)構(gòu)脂質(zhì)載體的制備及其抗乙肝病毒活性的研究
發(fā)布時(shí)間：2018-07-26 17:22
【摘要】：艾葉為菊科多年生草本植物艾(Artemisia argyi Lévl.et Vant.)的干燥葉,是臨床常用傳統(tǒng)中藥。艾葉揮發(fā)油是其主要有效成分群,具抗菌抗病毒、平喘鎮(zhèn)咳祛痰、抗過敏、免疫、護(hù)肝利膽等作用[1-9]。本課題組前期研究,首次發(fā)現(xiàn)艾葉揮發(fā)油具有抗乙肝病毒活性[10]。為研究艾葉揮發(fā)油抗乙肝病毒(HBV)新藥,又鑒于艾葉揮發(fā)油水難溶、易揮發(fā)、不穩(wěn)定,影響藥效,本文對艾葉揮發(fā)油納米結(jié)構(gòu)脂質(zhì)載體(Nanostructured lipid carrier system,NLC)進(jìn)行了研究,包括:艾葉揮發(fā)油NLC的制備、質(zhì)量評價(jià)、藥代動(dòng)力學(xué)、組織分布和藥效學(xué)。此研究未見報(bào)道。1.艾葉揮發(fā)油NLC的制備及質(zhì)量評價(jià)采用加熱熔融-超聲分散法制備艾葉揮發(fā)油NLC,通過單因素實(shí)驗(yàn)和正交試驗(yàn)優(yōu)化制備工藝及處方。最佳制備工藝為:初乳攪拌10min,超聲時(shí)間15min,超聲振幅100%。最佳處方為:波洛沙姆188 0.45g,蛋黃卵磷脂0.15g,單硬脂酸甘油酯0.1g,辛酸/癸酸甘油三酯0.1g,艾葉揮發(fā)油0.1g,蒸餾水加至20m L。通過氣相色譜法建立了艾葉揮發(fā)油NLC的多成分定量測定方法。方法精密度高,重現(xiàn)性好,桉油精、樟腦、龍腦加樣回收率均在95%?105%之間。測得的桉油精、樟腦、龍腦的含量分別為16.34%、3.32%、5.92%。采用納米粒度分析儀測定粒徑與Zeta電位,懫用指紋圖譜進(jìn)行質(zhì)量整體描述及工藝評價(jià),并從包封率、載藥量、體外釋放度及穩(wěn)定性來評價(jià)制劑的特性。艾葉揮發(fā)油NLC平均粒徑為(72.33±1.93)nm,PDI為(0.273±0.0045),Zeta電位為(-30.59±1.42)mv。桉油精、樟腦、龍腦包封率分別為87.49%、86.45%、92.12%,載藥量分別為8.25%、2.00%、3.38%。指紋圖譜得出十批制劑的相似度均大于0.990,說明十批制劑一致性好,工藝質(zhì)量穩(wěn)定。從體外釋放度結(jié)果可知,在120h時(shí),艾葉揮發(fā)油NLC的釋放度為61.12%,而原料藥的釋放度為72.46%,相同時(shí)間制劑的釋放度比原料藥的低,說明艾葉揮發(fā)油NLC具有緩釋性。藥物穩(wěn)定性實(shí)驗(yàn)表明,艾葉揮發(fā)油NLC在高溫、強(qiáng)光照條件下易發(fā)生變化,應(yīng)放置在4℃條件下避光保存。2.艾葉揮發(fā)油與其NLC的藥代動(dòng)力學(xué)及組織分布研究本文首次建立了艾葉揮發(fā)油及NLC在血漿中桉油精含量的GC測定方法,并對其在大鼠體內(nèi)的藥代動(dòng)力學(xué)進(jìn)行了研究。艾葉揮發(fā)油以1000mg/kg的劑量灌胃、140mg/kg的劑量尾靜脈注射給藥,艾葉揮發(fā)油NLC以同等于艾葉揮發(fā)油劑量給藥,給藥后于不同時(shí)間點(diǎn)取血,GC法檢測桉油精的血藥濃度,采用DAS3.0藥代動(dòng)力學(xué)軟件擬合,得藥代動(dòng)力學(xué)參數(shù)。灌胃4h后,艾葉揮發(fā)油NLC達(dá)到最大血藥濃度(Cmax)11.4835μg/L,半衰期(t?)為8.727h,而艾葉揮發(fā)油Cmax為9.1064μg/L,t?為7.779h;艾葉揮發(fā)油NLC灌胃后的藥時(shí)曲線面積AUC_(0-∞)為160.283μg/L·h,艾葉揮發(fā)油AUC_(0-∞)為103.681μg/L·h。結(jié)果表明,艾葉揮發(fā)油NLC延長了艾葉揮發(fā)油在體內(nèi)的作用時(shí)間,提高了藥物生物利用度,改變了藥物的藥代動(dòng)力學(xué)特征。本文通過小動(dòng)物活體成像跟蹤納米粒在不同時(shí)間不同組織中的分布情況。設(shè)立A、B、C三組,A組為生理鹽水組,B組為Di R染料組,C組為Di R-NLC組,于不同時(shí)間點(diǎn)麻醉,放置于小動(dòng)物活體成像儀中進(jìn)行觀察拍照,并于24h后取出裸鼠的心、肝、脾、肺、腎各組織,放置到小動(dòng)物活體成像儀中進(jìn)行觀察拍照,研究納米粒在裸鼠體內(nèi)不同組織中的分布情況。結(jié)果表明,隨著時(shí)間的延長,納米粒逐漸在肝部位蓄積,且從體外不同器官的熒光強(qiáng)度圖可知,Di R-NLC組肝臟部位熒光強(qiáng)度比染料組強(qiáng),因此表明艾葉揮發(fā)油NLC具有肝臟靶向性。3.艾葉揮發(fā)油及其NLC抗HBV藥效學(xué)研究采用鴨乙肝病毒模型來觀察艾葉揮發(fā)油及其NLC的抗HBV作用。1日齡麻鴨正常飼養(yǎng)3天后靜脈注射含DHBV的強(qiáng)陽性血清進(jìn)行攻毒,7天后使用PCR法篩選出感染乙肝病毒的陽性鴨,然后取陽性鴨112只隨機(jī)分成8組,給予不同劑量的艾葉揮發(fā)油及NLC,連續(xù)給藥15天,于給藥后5d、10d、15d及停藥后3d檢測血清和肝臟組織中DHBV DNA水平,評價(jià)艾葉揮發(fā)油及其NLC的抗HBV活性。結(jié)果表明,艾葉揮發(fā)油及NLC對鴨血清及肝臟組織中HBV均有抑制作用,高劑量抑制作用更明顯,且艾葉揮發(fā)油NLC抑制作用比原料藥更顯著。本文首次研究了艾葉揮發(fā)油NLC,為艾葉揮發(fā)油抗HBV新藥的研究提供了科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R283.6;R285
本文編號：2146732
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R283.6;R285
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