薏苡仁油脂质体的研究
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摘要
作为一种新型的纳米胶囊制备技术,脂质体技术在改善脂溶性营养物的水溶性方面表现出独特的优越性；作为一种定向药物载体,脂质体能够把药物输送到靶部位。薏苡仁油具有增强免疫力和抗肿瘤等多方面生理活性功能,薏苡仁油中不饱和脂肪酸含量超过80%,易被氧化,从而降低其营养和生物效价。本课题旨在研究乙醇注入-超声法制备薏苡仁油脂质体,并考察均质前和均质后的脂质体悬浮液的质量评价及稳定性；采用冷冻干燥技术制备薏苡仁油前体脂质体,考察其特性；同时进行薏苡仁油脂质体悬浮液的体外释放研究。
以包封率、粒径、制备初期和室温静置一天后的外观为指标,对以蛋黄卵磷脂和大豆卵磷脂为主要壁材制备的脂质体进行比较分析,结果表明,蛋黄卵磷脂对薏苡仁油的包埋效果相对较好。选取蛋黄卵磷脂作为薏苡仁油脂质体的主要壁材,采用正交试验优化薏苡仁油脂质体的制备工艺和配方。结果表明,最佳工艺为：水化温度45℃,乙醇用量2.0ml,水化时间20 min,超声强度90%；最佳配方为：蛋黄卵磷脂浓度20 mg/ml,蛋黄卵磷脂、薏苡仁油与胆固醇的质量比3：2：1,磷酸缓冲液pH值6.7,磷酸缓冲液用量50 ml。在此条件下制备的薏苡仁油脂质体包封率为76.34%,载量为25.68%,平均粒径为207.4nm。
选取35 Mpa均质压力对薏苡仁油脂质体进行均质处理,并对均质前和均质后的薏苡仁油脂质体悬浮液进行质量评价,以外观、T500nm、pH值、丙二醛含量和保留率为指标进行稳态化研究,结果表明,均质前后包封率均在70～80%之间；外观为乳白色、均一,对照组空白脂质体均质前后外观为透明、均一；电镜照片显示脂质体呈类圆形；平均粒径分别为207.4 nm和198.9 nm,Zeta电位分别为-36.43 mV和-27.25 mV;薏苡仁油脂质体双分子层的稳定性与贮存温度密切相关,4℃避光保存稳定性较好,但均质后稳定性有所下降。
选择聚乙二醇(分子量4000)为最佳冻干保护剂,聚乙二醇与蛋黄卵磷脂的质量比为4：1,成功制备薏苡仁油前体脂质体,外观饱满、致密平滑、颜色均匀,25℃蒸馏水复水水化后包封率为48.57%,薏苡仁油包封率的保留率达到80.25%；冻干前和复水后脂质体的平均粒径为260.6 nm和286.2 nm。
考察薏苡仁油前体脂质体的特性,扫描电镜观察到前体脂质体的颗粒分布较均匀,结构完整,成圆球形,表面比较光滑；差示扫描量热法分析得到未添加和添加聚乙二醇的薏苡仁油前体脂质体的主相转变温度分别为82.76℃和77.02℃,添加聚乙二醇后相转变温度有所降低；X-射线分析结果表明空白前体脂质体、未添加和添加聚乙二醇的薏苡仁油前体脂质体的衍射图谱特征峰相同,只是峰强度有些差异；傅里叶变换红外光谱分析显示蛋黄卵磷脂的极性头基和聚乙二醇之间发生了作用,形成氢键,这与“水置换”假说一致。
对薏苡仁油溶解液、薏苡仁油脂质体悬浮液和聚乙二醇(分子量为4000)包覆薏苡仁油脂质体悬浮液进行体外释放实验。累积释放曲线表明：脂质体具有缓释效果,聚乙二醇包覆薏苡仁油脂质体的长效缓释效果更加明显；释放方程拟合结果表明：薏苡仁油脂质体和聚乙二醇包覆薏苡仁油脂质体的体外释放规律符合Higuchi方程,而薏苡仁油溶解液的体外释放规律更符合一级动力学方程。
As a new technology for preparating nano-capsule, liposome technology had a unique advantage in improving the solubility of fat-soluble nutrients; as a targeted drug delivery system, liposomes could be able to delivery drugs to the target site. Coix seed oil(CSO) had the physiological activity functions of enhancing body's immunity, anti-tumor and many others. The content of unsaturated fatty acid was more than 80 percent in CSO, which was extremely susceptible to oxidation, thus reducing its nutritional and biological functions. This paper aimed at studying the preparation of CSO liposomes with ethanol injection-sonication method, evaluating the quality and stability of liposomes before and after homogenization, and preparing CSO proliposomes with the freeze-drying technology; while liposomes'property and release in vitro were investigated.
Egg phosphatidylcholine(EPC) and soybean phosphatidylcholine (SPC) were chosen as leading wall material to prepare liposomes, comparative analysis were done with encapsulation efficiency, particle size and appearance before and after one day at room temperature as indexes. The results indicated that encapsulation efficiency, particle size and appearance of CSO liposomes with EPC as leading wall material were better than those of CSO liposomes with SPC. The preparation techniques and formulation were optimized with encapsulation efficiency as criteria by orthogonal array design. The optimum conditions of CSO liposomes were as follows:hydration temperature 45℃, ethanol volume 2.0 ml, hydration time 20 min, ultrasonic intensity 90%; the concentration of EPC was 20 mg/ml, EPC/CSO /cholesterol (3:2:1) with phosphate buffer solution (pH6.7,50 ml) as hydration media. Under optimum conditions, the encapsulation efficiency and mean particle size of CSO liposomes was 76.34% and 207.4 nm, respectively.
CSO liposomes was homogenized with 35 Mpa, the liposomes before and after homogenization were assessed according to quality evaluation and stability, and stability was investigated with appearance, T500nm, pH value, malondialdehyde(MDA) content and retention rate as the indexes. The results indicated that encapsulation efficiency of CSO liposomes before and after homogenization were both 70～80%; the suspension was a uniform milky, while the suspension of blank liposomes as control was transparent and uniform; CSO liposomes before and after homogenization both were uniform and round, mean particle size was 207.4 nm and 198.9 nm, and Zeta potential was -36.43 mV and -27.25 mV, respectively. The stability of bilayer structure was closely related to storage temperature, the optimal storage condition was light-avoiding at 4℃, but the stability decreased after homogenization.
Polyethlene glycol with the molecular weight of 4000(PEG4000) was selected as the optimal cryoprotectant to prepared CSO proliposomes.CSO proliposomes prepared with the mass ratio of PEG4000 to EPC of 4:1 looked full, compact, smooth and color uniform, the encapsulation efficiency reached 48.57% after rehydration with distilled water at 25℃, its retention rate was higher than 80.25%; mean particle size of liposomes before freeze-drying and after rehydration was 260.6 nm and 286.2 nm, respectively.
The characteristics of CSO proliposomes were investigated, the proliposomes observed by scanning electron microscope (SEM) were uniform, round and relatively smooth with structural integrity; DSC analysis indicated that the main phase transition temperatures of CSO proliposomes with and without PEG4000 were 82.76℃and 77.02℃, the phase transition temperature of proliposomes with PEG4000 was decreased; X-ray analysis showed that the blank proliposomes, CSO proliposomes with and without PEG4000 had the same characteristic diffraction peaks, just differed a little in peak intensity; Fourier Transform Infrared Spectroscopy (FTIR) indicated that PEG and the polar head group of EPC interacted and hydrogen bonds created, which was consistent with water-replacement hypothesis.
Release of CSO from samples, which included dissolved CSO, CSO liposomes PEG4000-coated CSO liposomes was investigated in vitro. Cumulative release curves of liposomes showed significant slow releasing, long-acting effects of PEG4000-coated CSO liposomes was more evident. The release rule of liposomes in vitro was subject to Higuchi equation, while the release rule of dissolved CSO in vitro was subject to the first order equation.
以包封率、粒径、制备初期和室温静置一天后的外观为指标,对以蛋黄卵磷脂和大豆卵磷脂为主要壁材制备的脂质体进行比较分析,结果表明,蛋黄卵磷脂对薏苡仁油的包埋效果相对较好。选取蛋黄卵磷脂作为薏苡仁油脂质体的主要壁材,采用正交试验优化薏苡仁油脂质体的制备工艺和配方。结果表明,最佳工艺为：水化温度45℃,乙醇用量2.0ml,水化时间20 min,超声强度90%；最佳配方为：蛋黄卵磷脂浓度20 mg/ml,蛋黄卵磷脂、薏苡仁油与胆固醇的质量比3：2：1,磷酸缓冲液pH值6.7,磷酸缓冲液用量50 ml。在此条件下制备的薏苡仁油脂质体包封率为76.34%,载量为25.68%,平均粒径为207.4nm。
选取35 Mpa均质压力对薏苡仁油脂质体进行均质处理,并对均质前和均质后的薏苡仁油脂质体悬浮液进行质量评价,以外观、T500nm、pH值、丙二醛含量和保留率为指标进行稳态化研究,结果表明,均质前后包封率均在70～80%之间；外观为乳白色、均一,对照组空白脂质体均质前后外观为透明、均一；电镜照片显示脂质体呈类圆形；平均粒径分别为207.4 nm和198.9 nm,Zeta电位分别为-36.43 mV和-27.25 mV;薏苡仁油脂质体双分子层的稳定性与贮存温度密切相关,4℃避光保存稳定性较好,但均质后稳定性有所下降。
选择聚乙二醇(分子量4000)为最佳冻干保护剂,聚乙二醇与蛋黄卵磷脂的质量比为4：1,成功制备薏苡仁油前体脂质体,外观饱满、致密平滑、颜色均匀,25℃蒸馏水复水水化后包封率为48.57%,薏苡仁油包封率的保留率达到80.25%；冻干前和复水后脂质体的平均粒径为260.6 nm和286.2 nm。
考察薏苡仁油前体脂质体的特性,扫描电镜观察到前体脂质体的颗粒分布较均匀,结构完整,成圆球形,表面比较光滑；差示扫描量热法分析得到未添加和添加聚乙二醇的薏苡仁油前体脂质体的主相转变温度分别为82.76℃和77.02℃,添加聚乙二醇后相转变温度有所降低；X-射线分析结果表明空白前体脂质体、未添加和添加聚乙二醇的薏苡仁油前体脂质体的衍射图谱特征峰相同,只是峰强度有些差异；傅里叶变换红外光谱分析显示蛋黄卵磷脂的极性头基和聚乙二醇之间发生了作用,形成氢键,这与“水置换”假说一致。
对薏苡仁油溶解液、薏苡仁油脂质体悬浮液和聚乙二醇(分子量为4000)包覆薏苡仁油脂质体悬浮液进行体外释放实验。累积释放曲线表明：脂质体具有缓释效果,聚乙二醇包覆薏苡仁油脂质体的长效缓释效果更加明显；释放方程拟合结果表明：薏苡仁油脂质体和聚乙二醇包覆薏苡仁油脂质体的体外释放规律符合Higuchi方程,而薏苡仁油溶解液的体外释放规律更符合一级动力学方程。
As a new technology for preparating nano-capsule, liposome technology had a unique advantage in improving the solubility of fat-soluble nutrients; as a targeted drug delivery system, liposomes could be able to delivery drugs to the target site. Coix seed oil(CSO) had the physiological activity functions of enhancing body's immunity, anti-tumor and many others. The content of unsaturated fatty acid was more than 80 percent in CSO, which was extremely susceptible to oxidation, thus reducing its nutritional and biological functions. This paper aimed at studying the preparation of CSO liposomes with ethanol injection-sonication method, evaluating the quality and stability of liposomes before and after homogenization, and preparing CSO proliposomes with the freeze-drying technology; while liposomes'property and release in vitro were investigated.
Egg phosphatidylcholine(EPC) and soybean phosphatidylcholine (SPC) were chosen as leading wall material to prepare liposomes, comparative analysis were done with encapsulation efficiency, particle size and appearance before and after one day at room temperature as indexes. The results indicated that encapsulation efficiency, particle size and appearance of CSO liposomes with EPC as leading wall material were better than those of CSO liposomes with SPC. The preparation techniques and formulation were optimized with encapsulation efficiency as criteria by orthogonal array design. The optimum conditions of CSO liposomes were as follows:hydration temperature 45℃, ethanol volume 2.0 ml, hydration time 20 min, ultrasonic intensity 90%; the concentration of EPC was 20 mg/ml, EPC/CSO /cholesterol (3:2:1) with phosphate buffer solution (pH6.7,50 ml) as hydration media. Under optimum conditions, the encapsulation efficiency and mean particle size of CSO liposomes was 76.34% and 207.4 nm, respectively.
CSO liposomes was homogenized with 35 Mpa, the liposomes before and after homogenization were assessed according to quality evaluation and stability, and stability was investigated with appearance, T500nm, pH value, malondialdehyde(MDA) content and retention rate as the indexes. The results indicated that encapsulation efficiency of CSO liposomes before and after homogenization were both 70～80%; the suspension was a uniform milky, while the suspension of blank liposomes as control was transparent and uniform; CSO liposomes before and after homogenization both were uniform and round, mean particle size was 207.4 nm and 198.9 nm, and Zeta potential was -36.43 mV and -27.25 mV, respectively. The stability of bilayer structure was closely related to storage temperature, the optimal storage condition was light-avoiding at 4℃, but the stability decreased after homogenization.
Polyethlene glycol with the molecular weight of 4000(PEG4000) was selected as the optimal cryoprotectant to prepared CSO proliposomes.CSO proliposomes prepared with the mass ratio of PEG4000 to EPC of 4:1 looked full, compact, smooth and color uniform, the encapsulation efficiency reached 48.57% after rehydration with distilled water at 25℃, its retention rate was higher than 80.25%; mean particle size of liposomes before freeze-drying and after rehydration was 260.6 nm and 286.2 nm, respectively.
The characteristics of CSO proliposomes were investigated, the proliposomes observed by scanning electron microscope (SEM) were uniform, round and relatively smooth with structural integrity; DSC analysis indicated that the main phase transition temperatures of CSO proliposomes with and without PEG4000 were 82.76℃and 77.02℃, the phase transition temperature of proliposomes with PEG4000 was decreased; X-ray analysis showed that the blank proliposomes, CSO proliposomes with and without PEG4000 had the same characteristic diffraction peaks, just differed a little in peak intensity; Fourier Transform Infrared Spectroscopy (FTIR) indicated that PEG and the polar head group of EPC interacted and hydrogen bonds created, which was consistent with water-replacement hypothesis.
Release of CSO from samples, which included dissolved CSO, CSO liposomes PEG4000-coated CSO liposomes was investigated in vitro. Cumulative release curves of liposomes showed significant slow releasing, long-acting effects of PEG4000-coated CSO liposomes was more evident. The release rule of liposomes in vitro was subject to Higuchi equation, while the release rule of dissolved CSO in vitro was subject to the first order equation.
引文
[1]国家药典委员会编.中华人民共和国药典(一部)[S].2005年版.北京：化学工业出版社,2005：附262.
[2]吕峰,杨彩霞,姜艳梅,黄一帆.不同产地薏苡仁的保健与加工品质[J].福建农林大学学报,2007,37(4)：431～434.
[3]范世忠.药食兼用薏苡仁[J].药膳食疗,2002,21(5)：334.
[4]国家中医药管理局《中华本草》编委会.中华本草[M].上海：海科学技术出版社,1993：329.
[5]胡军,金国梁.薏苡仁的营养与药用价值[J].中国食物与营养,2007,13(6)：57.
[6]回瑞华,侯冬岩,郭华,等.薏米中营养成分的分析[J].食品科学,2005,26(8)：375.
[7]张明发,沈雅琴.薏苡仁药理研究进展[J].上海医药,2007,28(8)：360.
[8]赵素霞,程再兴,李连珍,等.薏苡仁药理研究新进展[J].河南中医,2004,24(2)：83.
[9]八木晟.薏苡仁的抗癌消炎活性[J].国外医药·植物药分册,1989,4(2)：75.
[10]Mitsuhiro N, Akihnko Y, Atsuko M, et al. Antitumor components isolated from the Chinese herbal medicine[J]. Coi x lachryma-jobi [J]. Plant Med,1994,60:356.
[11]陈宁,李毓,邱健行,等.薏苡仁酯对52氟脲嘧啶抑制人鼻咽癌细胞增殖的增效作用[J].中医药研究,1999,15(6)：45.
[12]曹国春,梁军,侯亚义.薏苡仁油诱导乳腺癌细胞系MCF-7细胞的凋亡及机理研究[J].实用临床医药杂志,2007,11(2)：1.
[13]谭煌英,李园,于莉莉,等.康莱特对大鼠的镇痛作用及其对促炎细胞因子的影响[J].中国中西医结合外科杂志,2007,13(2)：152.
[14]关云.薏苡仁的药理作用[J].中成药,1990,12(12)：38.
[15]Tokuda H, Matsumoto T, Konoshima T, et al. Inhibitory effects on epstein-barr virus activation and anti-tumor promoting activities of Coix seed[J]. Plant Med,1990,56:653.
[16]Bangham ad. diffusion of univalent ions aeross the lamellae of swollen phosphlipids[J]. Mol. Biol,1965,13:238～252.
[17]翟光喜,张天民.脂质体的研究与应用概况[J].山东食品科技,2004,4：15～18.
[18]陆彬.药物新剂型与新技术[M].第二版.北京：人民卫生出版社,2005.5.
[19]吴小宁.氟苯尼考脂质体的制备及药效研究[D].西北农林科技大学,2005.
[20]邓英杰.脂质体技术[M].北京：人民卫生出版社,2005.5.
[21]张灵芝.脂质体制备及其在生物医学中的应用.第一版.北京医科大学、北京协和医科大学联合出版社,1998：1～53.
[22]Simons K, Vaz WCL. Model systems,lipid rafts,and cell membranes[J]. Ann Rev Biophys Biomol Struc,2004.33:269.
[23]李新中,刘韶,雷鹏,等.薄膜-超声法制备黄芩苷固体脂质纳米粒的工艺研究[J].中 国医学工程,2004,12(5)：21～23.
[24]Hodoshima N, Udagawa C, Ando H, et al. Lipid nanoparticles for delivering antitumor drugs[J]. Int J Pharm,1997,146(1):81.
[25]Allen T M chota. A Large unilia mallar liposomes with the uptake into the reticuliendothelial system[J]. FEBS. Lett:1997,223:42～46.
[26]陈小军,孙志良,唐小武,等.硫酸阿米卡星脂质体的制备及包封率测定[J].中国兽医科技,2004,34(12)：38～41.
[27]Cortesi R,Esposito E, Marietti A, et al. Formulation study for the antitumor drug camptothecin:liposomes, micellar solutions and a microemulsion[J]. Int J Pharm,1997,159 (1):95～103.
[28]韩光星.脂质体制备方法的研究进展[J].黑龙江中医药,2007,1：53～54.
[29]夏书芹,许时婴.微胶囊化硫酸亚铁脂质体的制备及其在液态奶中的应用[J].食品科技,2003,(8)：24～27.
[30]Pons M, Foradada M, Estelrieh J. Liposomes obtained by the ethanol injeetion method. Int J Pharm199,95:51～56.
[31]Xia S Q, Xu S Y, Zhang X M. OptimiZation in the preparation of coenzyme Q10 nanoliposomes. J Agric Food Chem2006,54(17):6358～6366.
[32]Kalko N, Brandl M, Becirevie-LacanM, et al. Liposomes with nifedipine and nifedipine-cyelodextrin complex:calorimetrical and plasma stability comparison. Eur J Pharm Sci1996,4:359～366.
[33]Batzri S, Korn E D. Single bilayer liposomes prepared without sonieation. Bioehim Biophys Acta1973,298:1015～1019.
[34]Kemer J M H, Van der Esker M W, Pathmamanoharan C, et al. Biochemistry1977,16(17): 3932～3933.
[35]夏书芹,许时婴.不同的壁材对辅酶Q10纳米脂质体包埋效果的影响[J].食品科学,2006,27(7)：149～154.
[36]Mayer L D, Baly M B, HoPe K J, et al. Techniques for encapsulation bioactive agents into liposomes. Chem.Phys. Lipids 1986,40:333～345.
[37]Miyajima K. Role of saccharides for the freeze- thawing and freeze drying of liposome. Advanced Drug Delivery Reviews 1997,24:151～159.
[38]Ohtake S, Schebor C, de Pablo J J. Effects of trehalose on the phase behavior of DPPC-cholesterol unilamellar vesicles.Biochmica et Biophyslca Acta2006,1758:65～73.
[39]丁丽燕,杨春,李学明,等.乙醇注入法制备司帕沙星脂质体[J].南京工业大学学报,2007,29(1)：32～35.
[40]Crowe L M, Crowe J H, Rudoiph A, et al. Preservation of freeze-dried liposomes by Trehalose. Arch Biochem Biophys 1985,242 (1):240～247.
[41]van Winden EC A, Zhang W, Crommelin DJA. Effect of freezing rate on the stability of liposomes during freeze-drying and rehydration[J]. Pharm Res,1997,14 (9):1151～1160.
[42]Isele U, van Hoogevest P, Hilfiker R, et al. Large-scale production of liposomes containing monomeric zinc phthalocyanine by controlled dilution of organic solvents[J]. J PharmSci, 1994,83(11):1608～1616.
[43]禹玉洪.注射用柴胡挥发油脂质体制备工艺研究[J].中国中药杂志,2004,29(6)：521～525.
[44]Hailong Peng, Hua Xiong, Jinhua Li, et al. Vanillin cross-linked chitosan microspheres for controlled release of resveratrol[J]. Food Chemistry,2010,121:23～28.
[45]邓英杰等.沈阳药学院学报,1985,2(3)：190.
[46]祝青哲,夏书琴,许时婴.辅酶Q10纳米脂质体强化运动型饮料对小鼠的抗疲劳作用[J].食品与机械,2007,23(4)：44～48.
[47]Ola Loa Energy Drink Orange[EB/OL]. http://www.vitacost.com/ Ola-Loa-Energy-Drink, 2006.
[48]Bangs W E and Reineccius G A. Characterization of Selected Material for Lemon oil Encapsulation by Spray Drying [J]. Food Sci,1990,55(5):1356.
[49]KELLER B C. Liposomes in Nutrition [J]. Trends in Food Science & Technology,2001, (12):25～31.
[50]Lasic D D. Injection methods. In: Lasic D D (ed) Liposomes:from physics to applications.Elsevier Science Publishers,1993,88～90.
[51]李亮,熊华,黄军,等.薏苡仁油脂质体制备工艺研究[J].粮食与油脂,2008,33(12)：162～165.
[52]夏书芹,许时婴.辅酶Q10纳米脂质体的制备[J].食品工业科技,2006,2：164～168.
[53]Imura T, Otake K, Hashimoto S, et al. Preparation and Physicoehemical properties of various soybean lecithin liposomes using supercritical reverse phase evapo ration method. Coll and Surf B 2002,27:133～140.
[54]CODERCH L, FONOLLOSA J, DE PERA M, et al. Influence of cholesterol on liposome fluidity by EPR relationship with percutaneous absorption[J]. J of Controlled Release,2000, 68:85～95.
[55]SUKOWSKIA W W, PENTAK D, NOWAK K, et al. The influence of temperature, cholesterol content and pH on liposome stability[J]. Journal of Molecular Structure,2005, 744～747.
[56]Patel V B, Misra A N. Encapsulation and stabllity of clofazimine liposomes. J microencapsul 1999,16:357～367.
[57]Grit M, Underberg WJM, Crommelin DJA. Hydrolysis of saturated soybean phosphatidylcholine in liposome dispersions. J Pharm Sci,1993,82(4):362.
[58]Grit M, de Smidt J H, Struijke A. et al. Hydrolysis of phosphatidylcholine in queous liposome dispersions. Int J Pharm,1989,50:1.
[59]Grit M, Crommelin DJA. The effect of surface charge on the hydrolysis kinetics of partially hydrogenated egg phosphatidylcholine and egg phosphatidylglycerol in aqueous liposome dispersions. Biochem Biophys Acta,1993,1167:49.
[60]MALGORZATA J R, KRUK J, SKOWRONEK M, et al. Location of Ubiquinone Homologues in Liposome Membranes Studied by Fluorescence Anisotropy of Diphenyl-Hexatriene and Trimethylammonium-Diphenyl-Hexatriene [J]. Chemistry and Physics of Lipids,1996,79 (1):55～63.
[61]Nagayasu A, Uchiyama K, Kiwada H. The size of lipasomes:a factor which affects their targeting efficieney to tumors and therapeutic activity of liposomal antitumor dnrgs [J]. Adavanced Drug Delivery Reviews,1999,40:75.
[62]Wolfgeng M, Karsten M. Solid lipid nanoparticles production, characterization and applications [J]. Adv Drug Deliv Rev,2001,47:165.
[63]Yukiko N, Hiroyuki Y. Lymphatic targeting with nanooparticulate system [J]. Adv Drug Deliv Rev,2001,47:55.
[64]XIA S Q, XU S Y. Ferrous Sulfate Liposomes:Preparation, Stability and Application in Fluid Milk [J]. Food Research International,2005,38:289～296.
[65]HEURTAULT B, SAULNIER P, PECH B, et al. Physico-chemical Stability of Colloidal Lipid Particles [J]. Biomaterials 2003,24:4283～4300.
[66]DAAN J A. Influence of Lipid Composition and Ionic Strength on the Physical Stability of Liposomes[J]. Pharmaceutical Sci,1984,73(11):1559～1563.
[67]GRIT M, UNDERBERG W J M, CROMMELIN D J A. Hydrolysis of Saturated Soybean Phosphatidyl Choline in Aqueous Liposome Dispersions[J]. Pharmaceutical Sci,1993,82 (4):362～366.
[68]张小宁,张郁,姬海红,等.微射流法制备莪术油纳米脂质体的研究[J].中国药学杂志,2004,39(5)：356～358.
[69]GRAZIA S, GERALD W. Phospholipid Spherulers(Liposomes) as a Model for Biological Membranes[J].Lipid Research,1968,9 (3):310～318.
[70]Zhang W, van Winden ECA, Bouwstra JA, et al. Enhanced permeability of freeze-dried liposomal bilayers upon. Chemistry,2000,69:237～244.
[71]Cavalli R, Gasco MR, Chetoni P, et al. Solid lipid nanoparticles (SLN) as ocular delivery system for tobramycin[J]. Int J Pharm,2002,238 (1-2):241～245.
[72]Nagayasu A, Uchiyama K, Kiwada H. The size of lipasomes:a factor which affects their targeting efficieney to tumors and therapeutic activity of liposomal antitumor dnrgs [J]. Adavanced Drug Delivery Reviews,1999,40:75.
[73]杨明华,刘建平,黄文兰.丹参酮前体脂质体的研制[J].药学进展,2004,28(6)：267～271.
[74]王建,李明轩.冷冻干燥法对提高脂质体稳定性的研究概况[J].中国医药工业杂志,2005,36(9)：576～580.
[75]Zingel C, SachseA, Roessling GL, et al. Lyophilization and rehydration of iopromide-carrying liposomes[J]. Int J Pharm,1996,140(1):13～24.
[76]Zhang W, van Winden ECA, Bouwstra JA, et al. Enhanced permeability of freeze-dried liposomal bilayers upon rehydration[J]. Cryobiology,1997,35 (3):277～289.
[77]Crowe JH, Oliver AE, Hoekstra FA, et al. Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose:the role of vitrification[J]. Cryobiology,1997,35(1): 20～30.
[78]丁劲松,杨敏,陈琼.聚乙二醇衍生化磷脂与脂质体立体稳定性[J].中国医药工业杂志,2004,35(1)：55～58.
[79]Mobley W C, Schreier H. Phase transition temperature reduction and glass formation in dehydroprotected lyophilized liposomes[J]. Journal of Controlled Release,1994,31(1): 73～87.
[80]王建,李明轩.冷冻干燥法对提高脂质体稳定性的研究概况[J].中国医药工业杂志,2005,36(9)：576～580.
[81]国家药典委员会编.中华人民共和国药典(二部)[S].2005年版.北京：化学工业出版社,2005：附73.
[82]Winden ECA, Talsma H, Crommelin DJA, Thermal analysis of freeze-dried liposome-carbohydrate mixtures with modulated temperature differential scanning Calorimetry, J Pharm Sci,1998,87 (2),231～237.
[83]Crowe J H, Whittam M A, Crowe I M. Interactions of Phospholipid Monolayers With Carbohydrates. Biocbom Biophys Acta,1984,769:151.
[84]王玮,李来明,席时权.红外光谱法在双亲性化合物水溶液体系研究中的应用[J].分析化学,1994,22(12)：1273～1281.
[85]Casal H L, Mantsch H H. Polymorphic phase behaviour of phospholipid membranes studied by infrared spectroscopy[J]. Biochimica et Biophysica Acta (BBA)-Reviews on Biomembranes,1984,779 (4):381～401.
[86]Umemura J, Cameron D G, Mantsch H H. A Fourier transform infrared spectroscopic study of the molecular interaction of cholesterol with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes,1980,602(1): 32～44.
[2]吕峰,杨彩霞,姜艳梅,黄一帆.不同产地薏苡仁的保健与加工品质[J].福建农林大学学报,2007,37(4)：431～434.
[3]范世忠.药食兼用薏苡仁[J].药膳食疗,2002,21(5)：334.
[4]国家中医药管理局《中华本草》编委会.中华本草[M].上海：海科学技术出版社,1993：329.
[5]胡军,金国梁.薏苡仁的营养与药用价值[J].中国食物与营养,2007,13(6)：57.
[6]回瑞华,侯冬岩,郭华,等.薏米中营养成分的分析[J].食品科学,2005,26(8)：375.
[7]张明发,沈雅琴.薏苡仁药理研究进展[J].上海医药,2007,28(8)：360.
[8]赵素霞,程再兴,李连珍,等.薏苡仁药理研究新进展[J].河南中医,2004,24(2)：83.
[9]八木晟.薏苡仁的抗癌消炎活性[J].国外医药·植物药分册,1989,4(2)：75.
[10]Mitsuhiro N, Akihnko Y, Atsuko M, et al. Antitumor components isolated from the Chinese herbal medicine[J]. Coi x lachryma-jobi [J]. Plant Med,1994,60:356.
[11]陈宁,李毓,邱健行,等.薏苡仁酯对52氟脲嘧啶抑制人鼻咽癌细胞增殖的增效作用[J].中医药研究,1999,15(6)：45.
[12]曹国春,梁军,侯亚义.薏苡仁油诱导乳腺癌细胞系MCF-7细胞的凋亡及机理研究[J].实用临床医药杂志,2007,11(2)：1.
[13]谭煌英,李园,于莉莉,等.康莱特对大鼠的镇痛作用及其对促炎细胞因子的影响[J].中国中西医结合外科杂志,2007,13(2)：152.
[14]关云.薏苡仁的药理作用[J].中成药,1990,12(12)：38.
[15]Tokuda H, Matsumoto T, Konoshima T, et al. Inhibitory effects on epstein-barr virus activation and anti-tumor promoting activities of Coix seed[J]. Plant Med,1990,56:653.
[16]Bangham ad. diffusion of univalent ions aeross the lamellae of swollen phosphlipids[J]. Mol. Biol,1965,13:238～252.
[17]翟光喜,张天民.脂质体的研究与应用概况[J].山东食品科技,2004,4：15～18.
[18]陆彬.药物新剂型与新技术[M].第二版.北京：人民卫生出版社,2005.5.
[19]吴小宁.氟苯尼考脂质体的制备及药效研究[D].西北农林科技大学,2005.
[20]邓英杰.脂质体技术[M].北京：人民卫生出版社,2005.5.
[21]张灵芝.脂质体制备及其在生物医学中的应用.第一版.北京医科大学、北京协和医科大学联合出版社,1998：1～53.
[22]Simons K, Vaz WCL. Model systems,lipid rafts,and cell membranes[J]. Ann Rev Biophys Biomol Struc,2004.33:269.
[23]李新中,刘韶,雷鹏,等.薄膜-超声法制备黄芩苷固体脂质纳米粒的工艺研究[J].中 国医学工程,2004,12(5)：21～23.
[24]Hodoshima N, Udagawa C, Ando H, et al. Lipid nanoparticles for delivering antitumor drugs[J]. Int J Pharm,1997,146(1):81.
[25]Allen T M chota. A Large unilia mallar liposomes with the uptake into the reticuliendothelial system[J]. FEBS. Lett:1997,223:42～46.
[26]陈小军,孙志良,唐小武,等.硫酸阿米卡星脂质体的制备及包封率测定[J].中国兽医科技,2004,34(12)：38～41.
[27]Cortesi R,Esposito E, Marietti A, et al. Formulation study for the antitumor drug camptothecin:liposomes, micellar solutions and a microemulsion[J]. Int J Pharm,1997,159 (1):95～103.
[28]韩光星.脂质体制备方法的研究进展[J].黑龙江中医药,2007,1：53～54.
[29]夏书芹,许时婴.微胶囊化硫酸亚铁脂质体的制备及其在液态奶中的应用[J].食品科技,2003,(8)：24～27.
[30]Pons M, Foradada M, Estelrieh J. Liposomes obtained by the ethanol injeetion method. Int J Pharm199,95:51～56.
[31]Xia S Q, Xu S Y, Zhang X M. OptimiZation in the preparation of coenzyme Q10 nanoliposomes. J Agric Food Chem2006,54(17):6358～6366.
[32]Kalko N, Brandl M, Becirevie-LacanM, et al. Liposomes with nifedipine and nifedipine-cyelodextrin complex:calorimetrical and plasma stability comparison. Eur J Pharm Sci1996,4:359～366.
[33]Batzri S, Korn E D. Single bilayer liposomes prepared without sonieation. Bioehim Biophys Acta1973,298:1015～1019.
[34]Kemer J M H, Van der Esker M W, Pathmamanoharan C, et al. Biochemistry1977,16(17): 3932～3933.
[35]夏书芹,许时婴.不同的壁材对辅酶Q10纳米脂质体包埋效果的影响[J].食品科学,2006,27(7)：149～154.
[36]Mayer L D, Baly M B, HoPe K J, et al. Techniques for encapsulation bioactive agents into liposomes. Chem.Phys. Lipids 1986,40:333～345.
[37]Miyajima K. Role of saccharides for the freeze- thawing and freeze drying of liposome. Advanced Drug Delivery Reviews 1997,24:151～159.
[38]Ohtake S, Schebor C, de Pablo J J. Effects of trehalose on the phase behavior of DPPC-cholesterol unilamellar vesicles.Biochmica et Biophyslca Acta2006,1758:65～73.
[39]丁丽燕,杨春,李学明,等.乙醇注入法制备司帕沙星脂质体[J].南京工业大学学报,2007,29(1)：32～35.
[40]Crowe L M, Crowe J H, Rudoiph A, et al. Preservation of freeze-dried liposomes by Trehalose. Arch Biochem Biophys 1985,242 (1):240～247.
[41]van Winden EC A, Zhang W, Crommelin DJA. Effect of freezing rate on the stability of liposomes during freeze-drying and rehydration[J]. Pharm Res,1997,14 (9):1151～1160.
[42]Isele U, van Hoogevest P, Hilfiker R, et al. Large-scale production of liposomes containing monomeric zinc phthalocyanine by controlled dilution of organic solvents[J]. J PharmSci, 1994,83(11):1608～1616.
[43]禹玉洪.注射用柴胡挥发油脂质体制备工艺研究[J].中国中药杂志,2004,29(6)：521～525.
[44]Hailong Peng, Hua Xiong, Jinhua Li, et al. Vanillin cross-linked chitosan microspheres for controlled release of resveratrol[J]. Food Chemistry,2010,121:23～28.
[45]邓英杰等.沈阳药学院学报,1985,2(3)：190.
[46]祝青哲,夏书琴,许时婴.辅酶Q10纳米脂质体强化运动型饮料对小鼠的抗疲劳作用[J].食品与机械,2007,23(4)：44～48.
[47]Ola Loa Energy Drink Orange[EB/OL]. http://www.vitacost.com/ Ola-Loa-Energy-Drink, 2006.
[48]Bangs W E and Reineccius G A. Characterization of Selected Material for Lemon oil Encapsulation by Spray Drying [J]. Food Sci,1990,55(5):1356.
[49]KELLER B C. Liposomes in Nutrition [J]. Trends in Food Science & Technology,2001, (12):25～31.
[50]Lasic D D. Injection methods. In: Lasic D D (ed) Liposomes:from physics to applications.Elsevier Science Publishers,1993,88～90.
[51]李亮,熊华,黄军,等.薏苡仁油脂质体制备工艺研究[J].粮食与油脂,2008,33(12)：162～165.
[52]夏书芹,许时婴.辅酶Q10纳米脂质体的制备[J].食品工业科技,2006,2：164～168.
[53]Imura T, Otake K, Hashimoto S, et al. Preparation and Physicoehemical properties of various soybean lecithin liposomes using supercritical reverse phase evapo ration method. Coll and Surf B 2002,27:133～140.
[54]CODERCH L, FONOLLOSA J, DE PERA M, et al. Influence of cholesterol on liposome fluidity by EPR relationship with percutaneous absorption[J]. J of Controlled Release,2000, 68:85～95.
[55]SUKOWSKIA W W, PENTAK D, NOWAK K, et al. The influence of temperature, cholesterol content and pH on liposome stability[J]. Journal of Molecular Structure,2005, 744～747.
[56]Patel V B, Misra A N. Encapsulation and stabllity of clofazimine liposomes. J microencapsul 1999,16:357～367.
[57]Grit M, Underberg WJM, Crommelin DJA. Hydrolysis of saturated soybean phosphatidylcholine in liposome dispersions. J Pharm Sci,1993,82(4):362.
[58]Grit M, de Smidt J H, Struijke A. et al. Hydrolysis of phosphatidylcholine in queous liposome dispersions. Int J Pharm,1989,50:1.
[59]Grit M, Crommelin DJA. The effect of surface charge on the hydrolysis kinetics of partially hydrogenated egg phosphatidylcholine and egg phosphatidylglycerol in aqueous liposome dispersions. Biochem Biophys Acta,1993,1167:49.
[60]MALGORZATA J R, KRUK J, SKOWRONEK M, et al. Location of Ubiquinone Homologues in Liposome Membranes Studied by Fluorescence Anisotropy of Diphenyl-Hexatriene and Trimethylammonium-Diphenyl-Hexatriene [J]. Chemistry and Physics of Lipids,1996,79 (1):55～63.
[61]Nagayasu A, Uchiyama K, Kiwada H. The size of lipasomes:a factor which affects their targeting efficieney to tumors and therapeutic activity of liposomal antitumor dnrgs [J]. Adavanced Drug Delivery Reviews,1999,40:75.
[62]Wolfgeng M, Karsten M. Solid lipid nanoparticles production, characterization and applications [J]. Adv Drug Deliv Rev,2001,47:165.
[63]Yukiko N, Hiroyuki Y. Lymphatic targeting with nanooparticulate system [J]. Adv Drug Deliv Rev,2001,47:55.
[64]XIA S Q, XU S Y. Ferrous Sulfate Liposomes:Preparation, Stability and Application in Fluid Milk [J]. Food Research International,2005,38:289～296.
[65]HEURTAULT B, SAULNIER P, PECH B, et al. Physico-chemical Stability of Colloidal Lipid Particles [J]. Biomaterials 2003,24:4283～4300.
[66]DAAN J A. Influence of Lipid Composition and Ionic Strength on the Physical Stability of Liposomes[J]. Pharmaceutical Sci,1984,73(11):1559～1563.
[67]GRIT M, UNDERBERG W J M, CROMMELIN D J A. Hydrolysis of Saturated Soybean Phosphatidyl Choline in Aqueous Liposome Dispersions[J]. Pharmaceutical Sci,1993,82 (4):362～366.
[68]张小宁,张郁,姬海红,等.微射流法制备莪术油纳米脂质体的研究[J].中国药学杂志,2004,39(5)：356～358.
[69]GRAZIA S, GERALD W. Phospholipid Spherulers(Liposomes) as a Model for Biological Membranes[J].Lipid Research,1968,9 (3):310～318.
[70]Zhang W, van Winden ECA, Bouwstra JA, et al. Enhanced permeability of freeze-dried liposomal bilayers upon. Chemistry,2000,69:237～244.
[71]Cavalli R, Gasco MR, Chetoni P, et al. Solid lipid nanoparticles (SLN) as ocular delivery system for tobramycin[J]. Int J Pharm,2002,238 (1-2):241～245.
[72]Nagayasu A, Uchiyama K, Kiwada H. The size of lipasomes:a factor which affects their targeting efficieney to tumors and therapeutic activity of liposomal antitumor dnrgs [J]. Adavanced Drug Delivery Reviews,1999,40:75.
[73]杨明华,刘建平,黄文兰.丹参酮前体脂质体的研制[J].药学进展,2004,28(6)：267～271.
[74]王建,李明轩.冷冻干燥法对提高脂质体稳定性的研究概况[J].中国医药工业杂志,2005,36(9)：576～580.
[75]Zingel C, SachseA, Roessling GL, et al. Lyophilization and rehydration of iopromide-carrying liposomes[J]. Int J Pharm,1996,140(1):13～24.
[76]Zhang W, van Winden ECA, Bouwstra JA, et al. Enhanced permeability of freeze-dried liposomal bilayers upon rehydration[J]. Cryobiology,1997,35 (3):277～289.
[77]Crowe JH, Oliver AE, Hoekstra FA, et al. Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose:the role of vitrification[J]. Cryobiology,1997,35(1): 20～30.
[78]丁劲松,杨敏,陈琼.聚乙二醇衍生化磷脂与脂质体立体稳定性[J].中国医药工业杂志,2004,35(1)：55～58.
[79]Mobley W C, Schreier H. Phase transition temperature reduction and glass formation in dehydroprotected lyophilized liposomes[J]. Journal of Controlled Release,1994,31(1): 73～87.
[80]王建,李明轩.冷冻干燥法对提高脂质体稳定性的研究概况[J].中国医药工业杂志,2005,36(9)：576～580.
[81]国家药典委员会编.中华人民共和国药典(二部)[S].2005年版.北京：化学工业出版社,2005：附73.
[82]Winden ECA, Talsma H, Crommelin DJA, Thermal analysis of freeze-dried liposome-carbohydrate mixtures with modulated temperature differential scanning Calorimetry, J Pharm Sci,1998,87 (2),231～237.
[83]Crowe J H, Whittam M A, Crowe I M. Interactions of Phospholipid Monolayers With Carbohydrates. Biocbom Biophys Acta,1984,769:151.
[84]王玮,李来明,席时权.红外光谱法在双亲性化合物水溶液体系研究中的应用[J].分析化学,1994,22(12)：1273～1281.
[85]Casal H L, Mantsch H H. Polymorphic phase behaviour of phospholipid membranes studied by infrared spectroscopy[J]. Biochimica et Biophysica Acta (BBA)-Reviews on Biomembranes,1984,779 (4):381～401.
[86]Umemura J, Cameron D G, Mantsch H H. A Fourier transform infrared spectroscopic study of the molecular interaction of cholesterol with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes,1980,602(1): 32～44.