苦参素脂质体的制备以及质量研究
摘要
目的:本课题研究以大豆卵磷脂等为载体材料制备苦参素(Marine)脂质体,探索出一种较稳定的、包封率较高的苦参素脂质体的制备方法,并对苦参素脂质体进行质量评价。
方法:采用氧化指数法对购得的载体材料进行检测;通过测定苦参素的表观正辛醇.水分配系数,得到苦参素的表观油/水分配系数;考察薄膜分散法、逆相蒸发法、熔融法、复乳法、pH梯度法、硫酸铵梯度法制备苦参素脂质体的可行性,选择其中包封率高的制备方法,进一步优选制备工艺;建立脂质体中苦参素含量测定的高效液相色谱法(HPLC);用分段收集流分进行HPLC测定的方法考察了葡聚糖凝胶过滤法分离含药脂质体与游离药物的可行性。
选择复乳法作为苦参素脂质体的制备方法,以外观形态和包封率为考察指标优选最佳处方及最佳工艺,首先采用以单因素方法进行初步筛选,再用正交试验设计方法确定最佳制备工艺和处方。研制了苦参素多囊脂质体。用倒置相差显微镜观察脂质体的形态;用纳米粒度分析仪测定平均粒度和粒度分布;通过测定脂质体的渗漏率初步考察脂质体的稳定性;用动态透析法考察苦参素多囊脂质体的体外释药特性。
结果:用氧化指数测定合格的大豆卵磷脂作为载体材料制备苦参素脂质体,以复乳法制备的苦参素多囊脂质体包封率最高,为50%以上;制得的苦参素多囊脂质体呈乳白色、均匀、混悬状态;在显微镜下观察为圆形、椭圆形粒子,平均粒度为745nm,80～89%的粒子粒度在500～800nm之间;初步稳定性试验研究表明,本制剂在5℃条件下放置一个月较稳定;苦参素多囊脂质体体外释放度试验研究表明,0.5小时内药物的累积释放量约40%,累积药物释放百分含量与Higuchi方程吻合良好。
结论:复乳法制备苦参素多囊脂质体的工艺可行,大大提高了苦参素脂质体的包封率,体外释放度试验表明本制剂有一定的缓释性,基本达到试验设计的预期目的。
Objective:To study the preparation technology packaged marine with soya bean phosphatidylcholine et al.To explore the more stable and higher entrapment efficiency(EE,%)preparation method of the marine liposomes and evaluate their quality.
Methods:Firstly,we studied the membrane material purchased in the market by oxidizing index method,the result showed that the soybean phosphatide fulfiled the requirement.Determined by the apparent n-octyl alcohol/water distribution coefficient,we attained the apparent octanol/water distribution coefficient of marine.The liposomes were prepared by the following methods:film dispersion technology,reverse-phase evaporation technology,melting technology, reduplicative emulsification technology,pH gradient technology,ammonium sulfate gradient technology.EE was determined with HPLC of continuous flowing liquid from the column of Sephadex G50.
To determine the encapsulation of liposomes we used the reduplicative emulsification technology,choosing the single factors firstly,optimized method by orthogonal design and came out the best technology.Weighed by appearance and EE of in liposome,we prepared the marine-multivesicular liposomes (marine-MVL).In this paper,we observed the appearance by inverted phase contrast microscope,measured the particle diameter by nanometer particle size apparatus,examined the dissolution profiles in vivo using dynamoic dialyzetion.
Results:Research indicated,the EE measuration was reliable,and measured the average EE of marine-MVL was more than 50%.The results showed that using the defined method,we prepared good marine-MVL,it had cream colored、well-distributed、suspensoid appearance;round and oval granule appearance through inverted phase contrast microscope,and the equally diameter was 745nm,well-distributed particle size distribution,as 80%～89%was during 500～800nm.The initial stability test displayed that the product was stable in 5℃for one month.In vivo experiment showed that the relation of culminateve drug and time accord to Higuchi equation.
Conclusions:The experiments make clear that the reduplicative emulsification technology was feasible and repeatable.The vivo experiment proved they had a good slow-release capability.For basic,we had attained what we want as we expected.
方法:采用氧化指数法对购得的载体材料进行检测;通过测定苦参素的表观正辛醇.水分配系数,得到苦参素的表观油/水分配系数;考察薄膜分散法、逆相蒸发法、熔融法、复乳法、pH梯度法、硫酸铵梯度法制备苦参素脂质体的可行性,选择其中包封率高的制备方法,进一步优选制备工艺;建立脂质体中苦参素含量测定的高效液相色谱法(HPLC);用分段收集流分进行HPLC测定的方法考察了葡聚糖凝胶过滤法分离含药脂质体与游离药物的可行性。
选择复乳法作为苦参素脂质体的制备方法,以外观形态和包封率为考察指标优选最佳处方及最佳工艺,首先采用以单因素方法进行初步筛选,再用正交试验设计方法确定最佳制备工艺和处方。研制了苦参素多囊脂质体。用倒置相差显微镜观察脂质体的形态;用纳米粒度分析仪测定平均粒度和粒度分布;通过测定脂质体的渗漏率初步考察脂质体的稳定性;用动态透析法考察苦参素多囊脂质体的体外释药特性。
结果:用氧化指数测定合格的大豆卵磷脂作为载体材料制备苦参素脂质体,以复乳法制备的苦参素多囊脂质体包封率最高,为50%以上;制得的苦参素多囊脂质体呈乳白色、均匀、混悬状态;在显微镜下观察为圆形、椭圆形粒子,平均粒度为745nm,80～89%的粒子粒度在500～800nm之间;初步稳定性试验研究表明,本制剂在5℃条件下放置一个月较稳定;苦参素多囊脂质体体外释放度试验研究表明,0.5小时内药物的累积释放量约40%,累积药物释放百分含量与Higuchi方程吻合良好。
结论:复乳法制备苦参素多囊脂质体的工艺可行,大大提高了苦参素脂质体的包封率,体外释放度试验表明本制剂有一定的缓释性,基本达到试验设计的预期目的。
Objective:To study the preparation technology packaged marine with soya bean phosphatidylcholine et al.To explore the more stable and higher entrapment efficiency(EE,%)preparation method of the marine liposomes and evaluate their quality.
Methods:Firstly,we studied the membrane material purchased in the market by oxidizing index method,the result showed that the soybean phosphatide fulfiled the requirement.Determined by the apparent n-octyl alcohol/water distribution coefficient,we attained the apparent octanol/water distribution coefficient of marine.The liposomes were prepared by the following methods:film dispersion technology,reverse-phase evaporation technology,melting technology, reduplicative emulsification technology,pH gradient technology,ammonium sulfate gradient technology.EE was determined with HPLC of continuous flowing liquid from the column of Sephadex G50.
To determine the encapsulation of liposomes we used the reduplicative emulsification technology,choosing the single factors firstly,optimized method by orthogonal design and came out the best technology.Weighed by appearance and EE of in liposome,we prepared the marine-multivesicular liposomes (marine-MVL).In this paper,we observed the appearance by inverted phase contrast microscope,measured the particle diameter by nanometer particle size apparatus,examined the dissolution profiles in vivo using dynamoic dialyzetion.
Results:Research indicated,the EE measuration was reliable,and measured the average EE of marine-MVL was more than 50%.The results showed that using the defined method,we prepared good marine-MVL,it had cream colored、well-distributed、suspensoid appearance;round and oval granule appearance through inverted phase contrast microscope,and the equally diameter was 745nm,well-distributed particle size distribution,as 80%～89%was during 500～800nm.The initial stability test displayed that the product was stable in 5℃for one month.In vivo experiment showed that the relation of culminateve drug and time accord to Higuchi equation.
Conclusions:The experiments make clear that the reduplicative emulsification technology was feasible and repeatable.The vivo experiment proved they had a good slow-release capability.For basic,we had attained what we want as we expected.
引文
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[14]张秀琴,马华芳.苦参素胶囊对慢性乙型肝炎的疗效观察[J].时珍国医国药,2003,14(7):407-409.
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[17]王明雷,周秋丽,王本祥.氧化苦参碱肠内菌代谢及吸收入血活性成分的研究[J].中国中药杂志,2001,26(4):272-274.
[18]Xinan WU,Fumiyoshi Yamashita.Determination of matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies[J].Talanta,2003,59:965-971.
[19]Sujun Wang,Guangji Wang.Simultaneous determination of oxymatrine and its active metabolite matrine in dog plasma by liquid chromatography-mass spectrometry and its application to pharmacokinetic studies[J].Journal of Chromatography B,2005,817:319-325.
[20]中华人民共和国药典:2005年版二部.附录XC第182页.
[21]王毅,孟根达莱,郑文艳,等.氧化苦参碱注射液的人体药动学[J].中国临床药理学杂志,2003,19(4):301-305.
[22]费艳秋,安富荣,孙黎,等.苦参素注射液的人体药动学[J].中国医院药学杂志,2003,23(1):21-22.
[23]王秀敏,邓英杰,赵春杰,等.β-榄香烯油水分配系数的测定[J].沈阳药 科大学学报,2005,22(4):289-290.
[24]Defrise Q,Chatelein FP,Delmelle M,et al.Model studies for drug entrapment and liposome stability[A].Gregoriadis G.Lliposome technology:incorporation of drugs,proteins,and genetic material[M].Boca Raton:CRC Press,1984:1-17.
[25]陆彬.药物新剂型与新技术.第1版,人民卫生出版社,1998.
[26]邓英杰,史淑芬,顾学裘.油酸脂质体(139)注射液包封率测定方法的研究[J].药学学报,1988,23(7):539-544.
[27]旷英姿,马全红,郝小祯,等.茶多酚脂质体的制备研究[J].中国生化药物杂志,2006,27(1):28-31.
[28]沈洁,栾立标.甘草酸二铵脂质体的制备及其性质的研究[J].中国新药杂志,2006,15(5):361-364.
[29]张青,朱家壁,邱丽梅.盐酸左氧氟沙星脂质体的包封率和体外释放研究[J].中国药科大学学报,2004,35(6):508-512.
[30]Ye Q,Asherman J,Stevenson M,et al.Depofoam technology:a vehicle for controlled delivery of protein and peptide drugs[J].J Controlled Release,2000,64(1-3):155-166.
[31]严文伟,肖超菊,齐宪荣,等.水溶性药物8-氯腺苷多囊脂质体的制备[J].北京大学学报(医学版),2002,34(4):399-400.
[32]肖超菊,齐宪荣,艾尼瓦尔,等.顺铂缓释多囊脂质体的制备和体外释放性能研究[J].药学学报,2003,38(2):133-137.
[33]杨莉,齐宪荣,石靖,张强.8-氯腺苷多囊脂质体的制备[J].中国新药杂志,2004,13(10):898-902.
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[2]薛敏.慢性乙型肝炎治疗进展[J].井冈山医专学报,2001,8(2):29-30.
[3]Brook MG.Randomised controlled trial of interferon alfa 2A for the treatment of chronic hepatitis B virus(HBV)infection:factors that inflence response.Gut,1989,30:1116-1169.
[4]Lok ASF,et al.Long-term follow-up in randomized controlled trial of recombinant alpha-interferon in Chinese patients with chronic hepatitis B virus infection.Lancet,1988,2:298-300.
[5]代秀芬,张维.苦参素治疗慢性病毒性肝炎近况.综述,2004.
[6]殷忠东,孙学刚,金君梅.苦参药理研究进展[J].中国中医药信息杂志,1996,6(9):17-19.
[7]李丹,王平全,张楠森.苦参碱类生物碱的研究进展及临床应用[J].中草药,1996,27(5):308-310.
[8]张凤玲,唐永,张景梅.苦参碱、氧化苦参碱的药理作用及其制剂的研究进展[J].河南中医学院学报,2004,19(112):84-86.
[9]马方励,程怡.苦参碱多种制剂的药效学研究进展[J].中成药,2004,26(5):420-422.
[10]Fiume L,Bonino F,Mattioli A,et al.Inhibition of HBV relication by vidaratine monophosphate conjugated with lactosa minated serum albumin[J].Lancet,1998,332:13-16.
[11]Plourde R,Wu GY.Targeted therapy for viral hepatitis[J].Adv Drug Deliv, 1995,17(3):311-314.
[12]任进余,陈义,吴斌,等.病毒性肝炎的药物靶向治疗研究[J].中西医结合肝病杂志,1997,7(1):1-4.
[13]郑志想,付红刚.苦参素胶囊治疗慢性乙肝疗效总结[J].中国医学丛刊,2003,3(10):30-32.
[14]张秀琴,马华芳.苦参素胶囊对慢性乙型肝炎的疗效观察[J].时珍国医国药,2003,14(7):407-409.
[15]常萍,袁永荣,王本杰.苦参素片人体生物等效性研究[J].山东大学学报(医学版),2004,42(4):484-486.
[16]李俊生,鲍玉琳,温绪东,郭瑞臣.苦参素片,苦参素胶囊人体生物等效性研究[J].中国药师,2004,7(8):582-584.
[17]王明雷,周秋丽,王本祥.氧化苦参碱肠内菌代谢及吸收入血活性成分的研究[J].中国中药杂志,2001,26(4):272-274.
[18]Xinan WU,Fumiyoshi Yamashita.Determination of matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies[J].Talanta,2003,59:965-971.
[19]Sujun Wang,Guangji Wang.Simultaneous determination of oxymatrine and its active metabolite matrine in dog plasma by liquid chromatography-mass spectrometry and its application to pharmacokinetic studies[J].Journal of Chromatography B,2005,817:319-325.
[20]中华人民共和国药典:2005年版二部.附录XC第182页.
[21]王毅,孟根达莱,郑文艳,等.氧化苦参碱注射液的人体药动学[J].中国临床药理学杂志,2003,19(4):301-305.
[22]费艳秋,安富荣,孙黎,等.苦参素注射液的人体药动学[J].中国医院药学杂志,2003,23(1):21-22.
[23]王秀敏,邓英杰,赵春杰,等.β-榄香烯油水分配系数的测定[J].沈阳药 科大学学报,2005,22(4):289-290.
[24]Defrise Q,Chatelein FP,Delmelle M,et al.Model studies for drug entrapment and liposome stability[A].Gregoriadis G.Lliposome technology:incorporation of drugs,proteins,and genetic material[M].Boca Raton:CRC Press,1984:1-17.
[25]陆彬.药物新剂型与新技术.第1版,人民卫生出版社,1998.
[26]邓英杰,史淑芬,顾学裘.油酸脂质体(139)注射液包封率测定方法的研究[J].药学学报,1988,23(7):539-544.
[27]旷英姿,马全红,郝小祯,等.茶多酚脂质体的制备研究[J].中国生化药物杂志,2006,27(1):28-31.
[28]沈洁,栾立标.甘草酸二铵脂质体的制备及其性质的研究[J].中国新药杂志,2006,15(5):361-364.
[29]张青,朱家壁,邱丽梅.盐酸左氧氟沙星脂质体的包封率和体外释放研究[J].中国药科大学学报,2004,35(6):508-512.
[30]Ye Q,Asherman J,Stevenson M,et al.Depofoam technology:a vehicle for controlled delivery of protein and peptide drugs[J].J Controlled Release,2000,64(1-3):155-166.
[31]严文伟,肖超菊,齐宪荣,等.水溶性药物8-氯腺苷多囊脂质体的制备[J].北京大学学报(医学版),2002,34(4):399-400.
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