纳米磁珠辅助红细胞膜垂钓技术及其在当归活性成分筛选中的应用
|
摘要
目的为提高天然产物中活性成分的筛选效率,利用活性成分与细胞膜(CMs)受体间具有特异性亲和的性质,建立一种新型、高效的纳米磁珠(MNPs)辅助CMs垂钓的方法。方法将MNPs与兔红细胞CMs结合形成纳米磁珠-细胞膜复合物(MN-CMs),利用MN-CMs垂钓当归水提取液中的活性成分,通过GC-MS对垂钓结果进行分析。结果自制MNPs平均粒径为(250.6±3.3)nm(n=3),且粒径分散均匀,多分散指数(PDI)为0.010±0.003(n=3),具有较强的磁性和良好的超顺磁性,饱和磁化强度值为83.4 emu/g。MNPs能与CMs稳定结合,10 mg MNPs的最大吸附量为1.02 mg CMs,同时可以有效保持一定的CMs酶活性。GC-MS结果表明,MN-CMs从当归水提取液中垂钓出的潜在活性成分为藁本内酯,出峰时间为20 min,同时该新型垂钓方法能够有效避免非活性成分的干扰。结论建立的MNPs辅助CMs垂钓方法能够特异性垂钓出当归中活性成分藁本内酯,弥补了传统药物筛选方法的缺点,也为其他天然产物的活性成分筛选提供一种新方法。
Objective To improve the screening efficiency of the active ingredients in natural products by building up a kind of novel and efficient magnetic nanoparticle-assisted cell membranes(MN-CMs) fishing assay employing specific affinity interactions between active ingredients and receptors on cell membranes(CMs). Methods The magnetic nanoparticles(MNPs) were combined with erythrocyte membrane of rabbits to fish active ingredients from water extracts of Angelica sinensis, and the fishing results were analyzed by GC-MS. Results The particle size of the self-made magnetic beads was about(250.6 ± 3.3) nm(n = 3) with PDI index at 0.010 ± 0.003(n = 3), and the beads were monodisperse, strongly magnetic and superparamagnetic, the saturation magnetization was 83.4 emu/g. The combination of MNPs and CMs was stable, the maximum combined amount was 1.02 mg CMs/10 mg MNPs, and the combination was able to keep better enzyme activity of CMs in the fishing assay. GC-MS results showed that ligustilide was fished out as the active compound from water extracts of A. sinensis by MN-CMs assay with the retention time at 20 min, and the new established fishing assay could effectively avoid the interference of inactive components. Conclusion Ligustilide, one of the active ingredients in A. sinensis, can be screened out by the established fishing assay of MN-CMs. The developed fishing method in this workmakes up for some deficiencies of traditional screening method and provides a novel and efficient way to screen ingredients from natural products.
Objective To improve the screening efficiency of the active ingredients in natural products by building up a kind of novel and efficient magnetic nanoparticle-assisted cell membranes(MN-CMs) fishing assay employing specific affinity interactions between active ingredients and receptors on cell membranes(CMs). Methods The magnetic nanoparticles(MNPs) were combined with erythrocyte membrane of rabbits to fish active ingredients from water extracts of Angelica sinensis, and the fishing results were analyzed by GC-MS. Results The particle size of the self-made magnetic beads was about(250.6 ± 3.3) nm(n = 3) with PDI index at 0.010 ± 0.003(n = 3), and the beads were monodisperse, strongly magnetic and superparamagnetic, the saturation magnetization was 83.4 emu/g. The combination of MNPs and CMs was stable, the maximum combined amount was 1.02 mg CMs/10 mg MNPs, and the combination was able to keep better enzyme activity of CMs in the fishing assay. GC-MS results showed that ligustilide was fished out as the active compound from water extracts of A. sinensis by MN-CMs assay with the retention time at 20 min, and the new established fishing assay could effectively avoid the interference of inactive components. Conclusion Ligustilide, one of the active ingredients in A. sinensis, can be screened out by the established fishing assay of MN-CMs. The developed fishing method in this workmakes up for some deficiencies of traditional screening method and provides a novel and efficient way to screen ingredients from natural products.
引文
[1]勾明玥,刘梁,张春枝.五倍子醇提物的抗氧化活性[J].大连工业大学学报,2011,30(2):90-93.
[2]郭瑞霞,李力更,王于方,等.天然药物化学史话:天然产物化学研究的魅力[J].中草药,2015,46(14):2019-2033.
[3]吕源.龙脑与樟脑考辨[J].兰台世界,2013,32(16):142-143.
[4]Klayman D L.Qinghaosu(artemisinin):An antimalarial drug from China[J].Science,1985,228(4703):1049-1055.
[5]郭宗儒.经久不衰的阿司匹林[J].药学学报,2015,50(4):506-508.
[6]丁岗,董自波,李智立,等.生物色谱法及其在药物研究中的应用[J].中国药科大学学报,2002,33(4):354-357.
[7]贺浪冲,杨广德,耿信笃.固定在硅胶表面细胞膜的酶活性及其色谱特性[J].科学通报,1999,44(6):632-637.
[8]Liang M J,He L C,Yang G D.Screening,analysis and in vitro vasodilatation of effective components from Ligusticum Chuanxiong[J].Life Sci,2005,78(2):128-133.
[9]Hou X F,Zhou M Z,Jiang Q,et al.A vascular smooth muscle/cell membrane chromatography-offline-gas chromatography/mass spectrometry method for recognition,separation and identification of active components from traditional Chinese medicines[J].JChromatogr A,2009,1216(42):7081-7087.
[10]Dong Z B,Li S P,Zhu Q,et al.Hypothesis of potential active components in Angelica sinensis by using biomembrane extraction and high performance liquid chromatography[J].J Pharm Biomed Anal,2005,38(4):664-669.
[11]Gao J,Gu H,Xu B.Multifunctional magnetic nanoparticles:Design,synthesis,and biomedical applications[J].Accounts Chem Res,2009,42(8):1097-1107.
[12]Kumari A,Yadav S K,Yadav S C.Biodegradable polymeric nanoparticles based drug delivery systems[J].Colloid Surfaces B,2010,75(1):1-18.
[13]Wang Y,Ma X,Ding C,et al.p H-responsive deoxyribonucleic acid capture/release by polydopamine functionalized magnetic nanoparticles[J].Anal Chem,2015,862(67):33-40.
[14]Kang J H,Super M,Yung C W,et al.An extracorporeal blood-cleansing device for sepsis therapy[J].Nat Med,2014,34(4):345-355.
[15]Guardia P,Di Corato R,Lartigue L,et al.Water-soluble iron oxide nanocubes with high values of specific absorption rate for cancer cell hyperthermia treatment[J].ACS Nano,2012,6(4):3080-3091.
[16]张鑫,李鑫钢,姜斌.四氧化三铁纳米粒子的合成及表征[J].化学工业与工程,2006,23(1):45-48.
[17]邱星屏.四氧化三铁磁性纳米粒子的合成及表征[J].厦门大学学报:自然科学版,1999,38(5):711-715.
[18]李冬梅,徐光亮.制备超顺磁性Fe3O4纳米粒子的研究进展[J].中国粉体技术,2008,14(4):55-58.
[19]Deng H,Li Y D,Peng Q,et al.Monodisperse magnetic single-crystal ferrite microspheres[J].Angew Chem Int Ed,2005,44(18):2782-2785.
[20]华永丽,郭延生,邓红娟,等.当归不同药用部位水提液体外清除自由基作用研究[J].时珍国医国药,2010,21(7):1582-1584.
[21]Wang X,Xu L,Zhao X C,et al.An insertion/self-fusion mechanism for cell membrane immobilization on porous silica beads to fabricate biomimic carriers[J].Biomater Sci,2017,5(7):1334-1341.
[22]Marszall M P,Moaddel R,Kole S,et al.Ligand and protein fishing with heat shock protein 90 coated magnetic beads[J].Anal Chem,2008,80(19):7571-7575.
[23]Moaddel R,Marszall M P,Bighi F,et al.Automated ligand fishing using human serum albumin-coated magnetic beads[J].Anal Chem,2007,79(14):5414-5417.
[24]Vanzolini K L,Jiang Z,Zhang X,et al.Acetylcholinesterase immobilized capillary reactors coupled to protein coated magnetic beads:A new tool for plant extract ligand screening[J].Talanta,2013,116(22):647-652.
[25]Cao Y X,Zhang W,He J Y,et al.Ligustilide induces vasodilatation via inhibiting voltage dependent calcium channel and receptor-mediated Ca2+influx and release[J].Vascul Phamacol,2006,45(3):171-176.
[26]王丽莉,张铁军.细胞膜色谱法及其在中药活性成分研究中的应用[J].药物评价研究,2011,34(2):110-114.
[27]Hou X,Wang S,Zhang T,et al.Recent advances in cell membrane chromatography for traditional Chinese medicines analysis[J].J Pharm Biomed Anal,2014,101(3):141-150.
[28]张宇洁,贺浪冲.用细胞膜色谱模型筛选长春七抑制He La细胞增殖的活性成分[J].中国药学杂志,2005,40(6):463-465.
[29]张博,贺浪冲.用细胞膜色谱法分析金刷把中具有细胞毒活性的有效成分[J].现代医药卫生,2006,22(15):2303-2304.
[30]高琨,贺浪冲,杨广德.用细胞膜色谱法筛选研究红毛七中的有效成分[J].中国药学杂志,2003,38(1):16-18.
[31]李洪玲,杨广德,贺浪冲.心康平原料药的鉴别和含量测定[J].药物分析杂志,2001,21(5):348-351.
[32]Bian X,He L G.Synthesis and antihyperglycemic evaluation of various protoberberine derivatives[J].Bioorg Med Chem Lett,2006,16(5):1380-1383.
[33]李菁,葛发欢.超临界CO2萃取当归挥发油的研究[J].中药材,1996,19(4):187-189.
[2]郭瑞霞,李力更,王于方,等.天然药物化学史话:天然产物化学研究的魅力[J].中草药,2015,46(14):2019-2033.
[3]吕源.龙脑与樟脑考辨[J].兰台世界,2013,32(16):142-143.
[4]Klayman D L.Qinghaosu(artemisinin):An antimalarial drug from China[J].Science,1985,228(4703):1049-1055.
[5]郭宗儒.经久不衰的阿司匹林[J].药学学报,2015,50(4):506-508.
[6]丁岗,董自波,李智立,等.生物色谱法及其在药物研究中的应用[J].中国药科大学学报,2002,33(4):354-357.
[7]贺浪冲,杨广德,耿信笃.固定在硅胶表面细胞膜的酶活性及其色谱特性[J].科学通报,1999,44(6):632-637.
[8]Liang M J,He L C,Yang G D.Screening,analysis and in vitro vasodilatation of effective components from Ligusticum Chuanxiong[J].Life Sci,2005,78(2):128-133.
[9]Hou X F,Zhou M Z,Jiang Q,et al.A vascular smooth muscle/cell membrane chromatography-offline-gas chromatography/mass spectrometry method for recognition,separation and identification of active components from traditional Chinese medicines[J].JChromatogr A,2009,1216(42):7081-7087.
[10]Dong Z B,Li S P,Zhu Q,et al.Hypothesis of potential active components in Angelica sinensis by using biomembrane extraction and high performance liquid chromatography[J].J Pharm Biomed Anal,2005,38(4):664-669.
[11]Gao J,Gu H,Xu B.Multifunctional magnetic nanoparticles:Design,synthesis,and biomedical applications[J].Accounts Chem Res,2009,42(8):1097-1107.
[12]Kumari A,Yadav S K,Yadav S C.Biodegradable polymeric nanoparticles based drug delivery systems[J].Colloid Surfaces B,2010,75(1):1-18.
[13]Wang Y,Ma X,Ding C,et al.p H-responsive deoxyribonucleic acid capture/release by polydopamine functionalized magnetic nanoparticles[J].Anal Chem,2015,862(67):33-40.
[14]Kang J H,Super M,Yung C W,et al.An extracorporeal blood-cleansing device for sepsis therapy[J].Nat Med,2014,34(4):345-355.
[15]Guardia P,Di Corato R,Lartigue L,et al.Water-soluble iron oxide nanocubes with high values of specific absorption rate for cancer cell hyperthermia treatment[J].ACS Nano,2012,6(4):3080-3091.
[16]张鑫,李鑫钢,姜斌.四氧化三铁纳米粒子的合成及表征[J].化学工业与工程,2006,23(1):45-48.
[17]邱星屏.四氧化三铁磁性纳米粒子的合成及表征[J].厦门大学学报:自然科学版,1999,38(5):711-715.
[18]李冬梅,徐光亮.制备超顺磁性Fe3O4纳米粒子的研究进展[J].中国粉体技术,2008,14(4):55-58.
[19]Deng H,Li Y D,Peng Q,et al.Monodisperse magnetic single-crystal ferrite microspheres[J].Angew Chem Int Ed,2005,44(18):2782-2785.
[20]华永丽,郭延生,邓红娟,等.当归不同药用部位水提液体外清除自由基作用研究[J].时珍国医国药,2010,21(7):1582-1584.
[21]Wang X,Xu L,Zhao X C,et al.An insertion/self-fusion mechanism for cell membrane immobilization on porous silica beads to fabricate biomimic carriers[J].Biomater Sci,2017,5(7):1334-1341.
[22]Marszall M P,Moaddel R,Kole S,et al.Ligand and protein fishing with heat shock protein 90 coated magnetic beads[J].Anal Chem,2008,80(19):7571-7575.
[23]Moaddel R,Marszall M P,Bighi F,et al.Automated ligand fishing using human serum albumin-coated magnetic beads[J].Anal Chem,2007,79(14):5414-5417.
[24]Vanzolini K L,Jiang Z,Zhang X,et al.Acetylcholinesterase immobilized capillary reactors coupled to protein coated magnetic beads:A new tool for plant extract ligand screening[J].Talanta,2013,116(22):647-652.
[25]Cao Y X,Zhang W,He J Y,et al.Ligustilide induces vasodilatation via inhibiting voltage dependent calcium channel and receptor-mediated Ca2+influx and release[J].Vascul Phamacol,2006,45(3):171-176.
[26]王丽莉,张铁军.细胞膜色谱法及其在中药活性成分研究中的应用[J].药物评价研究,2011,34(2):110-114.
[27]Hou X,Wang S,Zhang T,et al.Recent advances in cell membrane chromatography for traditional Chinese medicines analysis[J].J Pharm Biomed Anal,2014,101(3):141-150.
[28]张宇洁,贺浪冲.用细胞膜色谱模型筛选长春七抑制He La细胞增殖的活性成分[J].中国药学杂志,2005,40(6):463-465.
[29]张博,贺浪冲.用细胞膜色谱法分析金刷把中具有细胞毒活性的有效成分[J].现代医药卫生,2006,22(15):2303-2304.
[30]高琨,贺浪冲,杨广德.用细胞膜色谱法筛选研究红毛七中的有效成分[J].中国药学杂志,2003,38(1):16-18.
[31]李洪玲,杨广德,贺浪冲.心康平原料药的鉴别和含量测定[J].药物分析杂志,2001,21(5):348-351.
[32]Bian X,He L G.Synthesis and antihyperglycemic evaluation of various protoberberine derivatives[J].Bioorg Med Chem Lett,2006,16(5):1380-1383.
[33]李菁,葛发欢.超临界CO2萃取当归挥发油的研究[J].中药材,1996,19(4):187-189.