摘要
葛根素(puerarin,4′,7—二羟基—8β—D葡萄糖基异黄酮)是习用中药葛根的主要有效成分,在治疗心血管疾病方面有着广泛的应用,主要用于治疗冠心病、心绞痛、心肌梗死、视网膜动静脉阻塞、突发性耳聋等。
随着临床用药的增多,葛根素注射液不良反应的报道日益增多。不良反应主要包括发热、皮疹、肝肾损伤、头痛头晕、血管内溶血等,其中急性血管内溶血发病较为严重,甚至威胁到患者生命。目前,葛根素注射液致溶血不良反应的具体产生机制不明。针对此研究空白,本课题通过体外细胞试验和动物试验,主要从制剂因素、葛根素药源性溶血反应类型、葛根素对红细胞的作用特点等进行研究,探索葛根素注射液致溶血的发生机制,并研究膜稳定剂牛磺酸对葛根素溶血作用的干预,为探寻解决葛根素注射液不良反应的应对措施提供实验依据。
本课题在《中国药典》2005年版二部溶血检查项方法的基础上建立了偶发性溶血实验方法,通过提高供试品溶液浓度、延长反应时间,研究葛根素注射液酸碱性及其溶媒丙二醇的溶血作用,发现制剂的酸碱性及丙二醇无溶血作用、葛根素自身具有明显的溶血作用。
确定葛根素的溶血作用后,基于临床上药源性溶血反应类型分为:氧化性溶血、免疫性溶血、非免疫性溶血三种,本研究继续考察葛根素注射液药源性溶血反应的类型。按照临床等效剂量,葛根素注射液以50mg/kg的剂量连续静脉给药后,豚鼠体内红细胞过氧化产物丙二醛(MDA)、超氧化物歧化酶(SOD)水平均显著低于空白对照组,表明葛根素注射液能够减少红细胞内自由基的氧化作用,对红细胞无氧化损伤的作用;SOD活性的降低可能与葛根素和SOD中的金属离子形成络合物有关;排除了葛根素注射液致溶血反应属于氧化性溶血的可能性。葛根素注射液连续多次静脉给药,未诱发豚鼠出现过敏反应;致敏前后豚鼠红细胞的溶血率相似,表明无免疫性溶血反应的出现;抗人球蛋白直接试验(Goombs实验)结果亦呈阴性,表明多次静脉给药豚鼠体内未产生葛根素抗体,葛根素注射液诱发的溶血反应主要非免疫因素引起。体外溶血实验显示葛根素均能直接致Beagle犬、豚鼠、人红细胞溶血,溶血率与葛根素浓度呈明显的量效关系;动物实验中葛根素多次静脉注射可直接引起红细胞呈棘状细胞;与空白对照组比较,葛根素注射液给药组动物血清间接胆红素及血红蛋白含量略有升高、红细胞总数下降,但差异无统计学意义;提示葛根素注射液致溶血反应可能与葛根素直接改变红细胞形态、降低红细胞稳定性有关,其溶血反应类型属于药源性非免疫性溶血。
基于体外实验葛根素能直接引起红细胞溶血、静脉给药可引起红细胞形态异常,本研究进一步从生理生化反应角度考察葛根素对红细胞脆性、能量代谢、钙离子浓度等的改变,及从葛根素对膜流动性及膜蛋白的影响分析葛根素与膜的结合特点,并讨论这两方面结果与葛根素致溶血的关系。
浓度范围0.1～1mmol/L葛根素溶液对红细胞渗透脆性无影响;浓度范围在0.1～2mmol/L的葛根素也未影响高温引起的红细胞脆性增大。钙离子通道阻断剂硝苯地平、维拉帕米均未能抑制葛根素的溶血作用;借助荧光探针Fluo 3/AM和流式细胞仪未检测到葛根素引起红细胞钙离子内流。葛根素对红细胞钠-钾-ATP活性无影响;葡萄糖、腺苷亦未能减轻葛根素的溶血作用。以上结果表明葛根素对正常红细胞脆性、细胞内外钙离浓度的平衡、能量代谢均无影响。因此排除葛根素通过引起细胞脆性增加、钙超载或ATP耗损而致红细胞溶血的可能性。
值得注意的是,在生理盐水中(NS,pH 7.0),葛根素的溶血作用随着浓度的增加、反应时间的延长(≥6h)而增大;在等渗磷酸缓冲液中(PBS,pH 7.4),即使葛根素浓度不断增加、反应时间延长,仍未见葛根素有溶血作用。同时,葛根素的溶血作用可被阳离子两亲分子利多卡因所拮抗。
基于任何溶血反应必然涉及红细胞膜的损伤,本课题继续考察葛根素在不同溶媒(NS及PBS)中对膜流动性的改变。在NS(pH 7.0)中,葛根素显著提高膜的流动性;当葛根素浓度＞0.1mmol/L时,膜的流动性未随着葛根素浓度的增加而改变。膜流动性的增加表明葛根素嵌入膜磷脂双层中,可能通过增加膜磷脂分子之间的静电斥力、降低磷脂分子的有序性从而引起膜流动性的增大。在PBS(pH 7.4)中,低浓度的葛根素亦显著提高膜的流动性;但当葛根素浓度＞1mmol/L时,膜的流动性随着葛根素浓度的增加反而出现降低,推测葛根素与膜磷脂分子之间主要通过氢键的形式结合,氢键作用增强膜的规整性从而抑制膜流动性。
由于葛根素属于阴离子两亲分子,与膜磷脂双层的结合部位刚好与阳离子两亲分子利多卡因结合的部位相反,因此对红细胞形态的影响也相反。在本实验中,往含有葛根素的生理盐水溶液中加入利多卡因,可抑制葛根素对红细胞膜流动性的提高,同时亦拮抗葛根素的溶血作用,说明葛根素致溶血的作用与其跟膜的结合特点密切相关。
总结在NS或PBS中、在含有利多卡因的生理盐水溶液中,红细胞膜流动性的改变与葛根素致溶血结果的关系,发现有一个共同的特点:在相同的反应体系中,膜流动性下降,红细胞未出现溶血;相反,膜流动性增大,红细胞破裂。由于在本实验中膜流动性的提高与膜表面电荷、磷脂分子的有序性改变有关,因此推测葛根素的溶血作用同其它阴离子两亲分子一样,与其改变红细胞脂膜表面电荷、结构有关,最终导致红细胞“渗漏”、破裂。并且这种“溶血方式”的特征呈时间依赖性,即反应时间越长,溶血程度越明显。
除了影响红细胞脂膜,葛根素还改变膜蛋白的表达。体外细胞实验中葛根素明显降低膜蛋白尤其是骨架蛋白的表达;动物连续给药后红细胞骨架蛋白中血影蛋白(以β型为主)、锚蛋白、带3、带4.1的表达略有下降。体外实验表明高浓度的葛根素溶液对红细胞膜蛋白的影响较为明显;动物试验中,葛根素静脉注射对红细胞膜蛋白的影响不明显,这可能由于药物快速代谢不易在体内形成持续的高血药浓度有关。但是无论体外体内实验结果均表明一定浓度或剂量的葛根素会破坏红细胞膜的完整性、稳定性。
葛根素与膜稳定剂牛磺酸配伍而成的复方葛根素注射液对红细胞膜流动性的影响与葛根素注射液相似,但未有溶血作用,静脉注射液亦未引起红细胞形态异常。提示牛磺酸能够稳定红细胞膜的完整性,降低葛根素对膜的破坏和抑制葛根素的溶血作用。
总之,本实验从细胞和动物两方面进行了较为系统的葛根素注射液致溶血发生机制的研究,确定了葛根素注射液药源性非免疫性溶血的反应类型,发现高浓度的葛根素改变红细胞膜的结构、规整性是葛根素致溶血的前提。葛根素对膜的作用特点与其它阴离子两亲分子一致,葛根素非特异性作用于红细胞,降低葛根素浓度(剂量)、添加膜稳定剂牛磺酸均可消除葛根素的溶血作用。该实验为临床消除或减少葛根素注射的不良反应的应对措施提供了实验依据。
Puerarin {7-hydroxy,3-(4-hydroxyphenyl)-8β-D-glueopyranosyl},is the most abundant and active ingredient in Radix Puerariae Lobatae which is a very common used herb in China at folk and at Chinese clinic.Puerarin Injection(PI) is widely used for treating coronary heart diseases,angina,high blood pressure,miocardial infarction,retina vas infarction,sudden deafness and so on.Although puerarin is well-known on low toxicity and high therapeutic effect,it has caused a great anxiety because of the serious side effects(ADR) induced by PI. Primary symptoms of ADR induced by PI are fever,anthesma,renal and hepatic injury acute hemolysis,et al.The acute hemolysis usually may be produded quickly and then the risk is dangerous.
Some reports said that the occurrence of ADR is associated with age,manufacturers, dosage,course of and combination of administer.Some researchers considered the hemolysis was the second type of allergic reaction.However,the mechanism for the hemolysis reaction is still unclear.So the aims of this study were to reveal the mechanisms as well as try to bring out measures to decrease the hemolysis incidence induced by PI.
We found that increasing concentration of puerain and extending reaction time,the incidence of hemolysis was obviously higher.The optimal reaction time should be more than 6 hours.Thus hemolysis was tested after 24 hours' incubation in vitro.For the solvent in Puerarin Injection is propylene glycol(PP),hemolysis induced by PP and the acitity of preparation was studied simultaneously.But no hemolysis occurred.Thus it was concluded that hemolysis was mainly caused by peurarin.
Experiments were further carried on to investigate the feature of hemolysis induced by PI at the clinic.There are mainly three mechanisms underlying the drug induced hemolysis, oxidative hemolysis,immune hemolysis as well as non-immune hemolysis.Guinea pigs were administrated intravenously for several days at the dose of 50mg/kg per day which is equal to the dose of about 400 mg/d given at clinic.The malondialdehyde(MDA) and the superoxide dismutase(SOD) in the RBC were tested.The level of MDA obviously decreased compared to the control group so did the SOD too.The lower MDA indicated the fewer free radicals in the RBC.The reduction of SOD could be explained by the formation of complex produced by peurarin and metal ions such as Mg~(2+) or Mn~(2+) from SOD.No allergic reaction and no immune hemolysis occurred.No antibody of globulin was found too.However,the shape of RBC changed after injecting PI.Echinocytes were examed by the microscope.Combining the results in vitro that peurarin could induce RBC of human,dogs or guinea pigs at a dose-dependent manner,the type of PI-caused hemolysis was defined as the non-immune one.
Because of hemolysis induced directly by the PI in vitro and morphological change of RBC in vivo,the influence caused by PI on the RBC fragility,energy metabolism as well as calcium influx was analyzed.Puerarin had no effect on the RBC fragility in the hyposmosis saline solution or hot iso-osmia saline solution at the temperature of 50℃.Neither calcium ionic channel blocker Nifedipine nor Varapamil could inhibit the hemolysis induced by puerarin.Flow cytometer and fluorescent probe Fluo 3/AM were employed to detect the level of calcium ion(Ca~(2+)) in cytoplasma.No elevation of Ca~(2+) in the puerarin group indicated that puerarin did not cause the influx of Ca~(2+),which was a very important contributor for the death of cells.ATP also plays an important role in keeping normal shape and function of RBC. ATP-depleted can cause the erythrocytes become crenated,which are called echinocytes. Puerarin had no effect on the Na~+-K~+-ATP activity and the hemolysis could not be attenuated by the addition of both of glucose and adenosine.Therefore the hypothesis that puerarin inducing hemolysis by accelerating APT-depletion was excluded.
To our surprise,puerarin dissolved in the non-buffered saline solution(pH 7.0) could induce hemolysis and the hemolysis could be inhibited by Lidocaine,while puerarin in phosphate buffered saline(pH 7.4) had no destroy on RBC.
For any hemolytic reaction is inevitably to involve the impairment of erythrocyte membrane.The change of membrane fluidity induced by puerarin was tested too.In non-buffered physiological saline solution(pH 7.0),puerarin dramatically improved the membrane fluidity compared to the control group.When the concentration was more than 1mmol/L,fluidity no longer increased along with the elevation of puerarin concentration. This result indicated that puerarin nonspecifically inserted into the lipidic part of the membrane and changed membrane fluidity.The higher fluidity might be due to the increasing membrane surface electric density and the repulsion of phospholipid molecules and then abrupted the on membrane organization.In PBS iso-osmia solution(pH 7.4),puerarin at the concentration ranging from 0.05～1mmol/L also increased membrane fluidity obviously. However,when the concentration became higher,the fluidity decreased quickly.This could result from the formation of hydrogen bonding between puerarin and phospholipid molecules, which was then packing the membrane bilayer and restraining its movement,and that may be the mechanism why puerarin can not induce hemolysis in PBS.What's more,the addition of Lidocaine could inhibit the increment of membrane fluidity induced by puerarin in the non-buffered saline solution(pH 7.0) and prevented hemolysis.For Lidocine is a cationic compound which interacts with erythrocyte membranes inducing internalization or stomatocytosis by their incorporation in the inner monolayer and performs the opposite effect to the one induced by neutral or ionic drugs such as puerarin,the effect on the membrane fluidity should be taken into consideration as a contributor to hemolysis induced by puerarin.
To further investigate whether membrane protein was damaged by puerain,experiments were carried out In vitro,human erythrocytes were suspended in puerarin injection containing 2mmol/L puerarin and 0.8%PP at 37℃for 2 hours and then membrane protein was extracted by kits and separated by SDS-PAGE.Compared to the normal control groups,the amount of protein expression of band 2(spectrin),band 2.1(ankyrin),band 3,band 4.1,band 5(actin) decreased along with some other unknown bands increased in intensity after staining. Similar result in the PP control group(containing 0.8%PP) but change was much more moderate.Theses results confirmed the effect of pruerin at high concentration on the organization of RBC membrane protein especially the skeleton protein and the PP also had gentle effect too.In vivo,puerarin injection was given at the dose of 50 mg/kg intravenously for ten days.The PP control group was administrated with the same amount of PP.Both in PI or PP group,the change of band 2(spectrin),band 2.1(ankyrin),band 3,band 4.1 was very slight vs.the normal control group which was injected with the same volume of saline.The difference between the results of in vitro to the ones of In vivo might be explained by the quick elimination of puerarin in animals and shorter interaction with erythrocyte membrane. Thus it could be concluded that generally puerarin injected intravenously had slight effect on RBC in vivo.The causes for acute hemolysis induced by PI at the clinic were probably associated with the dysfunction of metabolism of liver and/or kidney in some old patients who were given high dose of puerarin for several days,which resulted into the accumulation of puerarin in the serum and longer disturbance on the RBC and occurrence of hemolysis.
Taurine is a common amine acid in animals or human.It can stabilize the cell membrane. In the study,taurine inhibited the hemolysis induced by purarin and the change in conformation of RBC after the injection of puerarin.This inferred the disturbance on stability of RBC membrane induced by puerarin.
In summary,this study revealed some mechanisms underlying the hemolysis caused by Puerarin Injection through experiments carried out in vitro and in vivo.The feature of the hemolysis seemed to belong to the non-immune one,which showed dose-dependent as well as temporal-dependent manners and had little relationship with oxidation reaction or allergic reaction.A large amount of puerarin attached to membrane and then induced the increment of membrane surface electric density and the repulsion of phospholipid molecules and disturbed membrane organization.Consequently,morphological change of RBC occurred and hemolysis was induced.Reduction on concentration/dose or addition of taurine could prevent against hemolysis.
The present results may contribute to supply some interventions to avoiding hemolysis induced by Puerarin Injection.
引文
[1]李华洲,陈维宁,吕欣然,等.葛根素的毒性研究和安全实验[J].潍坊医学院学报,1985(02).
[2]吴燕红,苏子仁,陈建南,等.从小鼠体内血药浓度时间曲线与组织分布特征评价葛根素的给药途径[J].中药新药与临床药理,2005(02).
[3]杜力军,邢东明,炎彬,等.对葛根素与葛根黄酮体内动力学关系的探讨——兼论中药药代动力学的研究方法[J].世界科学技术-中医药现代化,2004(06).
[4]路玫,李妮,蒙大平,等.葛根素在糖尿病肾病患者中的药动学[J].中国医院药学杂志,2000(12).
[5]《药物不良反应杂志》编辑部.葛根素注射剂致急性血管内溶血[J].药物不良反应杂志,2006(8).
[6]梁勇,程昌盛.葛根素致急性溶血[J].药物不良反应杂志.2001(03).
[7]邓培媛,李群娜,朱玉珍,等.葛根素注射剂不良反应及其影响因素分析[J].药物流行病学杂志.2005(01).
[8]邓培媛,张俊,朱玉珍,等.葛根素注射剂致急性溶血性贫血临床分析[J].中国新药杂志.2004(08).
[9]陈易新,田春华.从药品不良反应信息通报品种看药品风险管理(八)[J].中国药物警戒,2008(06).
[10]Yue P F,Hai-long Y H,Zhu W F,et al.The study to reduce the hemolysis side effect of puerarin by a submicron emulsion delivery system.[J].Biol Pharm Bull.2008,31(1):45-50.
[11]常瑛.药源性血液病(3).药物不良反应杂志[J].2004(5).
[12]Mandal D,Moitra PK,Saha Set al.Caspase 3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes.[J].FEBS Lett,2002,513(2-3):184-88.
[13]Bruce LJ,Beckmann R,Ribeiro ML et al.A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane.[J].Blood,2003,101(10):4180-88.
[14]Oldenborg PA,Zheleznyak A,Fang YF et al.Role of CD47 as a marker of self on red blood cells.[J].Science,2000,288(5473):2051-54.
[15]Lopez-Revuelta A,Sanchez-Gallego JI,Hernandez-Hemandez Aet al.Membrane cholesterol contents influence the protective effects of quercetin and rutin in erythrocytes damaged by oxidative stress.[J].Chem Biol Interact,2006,161(1):79-91.
[16]Pawlikowska-Pawlega B,Gruszecki WI,Misiak Let al.Modification of membranes by quercetin,a naturally occurring flavonoid,via its incorporation in the polar head group.[J].Biochim Biophys Acta,2007,1768(9):2195-4.
[17]Fiorani M,Accorsi A,Cantoni O.Human red blood cells as a natural flavonoid reservoir.[J].Free Radic Res,2003,37(12):1331-38.
[18]温筱煦,程丽静,刘蔚,等.束0五加总黄酮对小鼠红细胞膜流动性的影响[J].中国基层医药,2006(08).
[19]温筱煦,刘蔚,邓锡军,等.刺五加提取物的总黄酮含量测定及其对血液流变学的影响[J].解放军药学学报,2006(03).
[20]Pawlikowska-Pawlega B,Gruszecki WI,Misiak LEet al.The study of the quercetin action on human erythrocyte membranes.[J].Biochem Pharmacol,2003,66(4):605-12.
[21]蒋风萍,周本宏,刘刚,等.葛根素对红细胞膜脂质过氧化损伤的防护作用[J].中药药理与临床,2004(05).
[22]徐邦宁.生物膜与疾病[J].中国乡村医药,1998(01).
[23]王玉环,田清海,孙济川,等.冠心病患者红细胞膜脂微粘度改变与脂质过氧化的关系[J].陕西医学杂志,2002(04).
[24]任爱华,厉朝喜.老年冠心病患者红细胞变形能力观察及其意义[J].浙江医学,1997(06).
[1]闵巍巍,张作法.黄酮类化合物的药理作用[J].蚕桑通报,2007(04).
[2]万明,宋永钢,杨菠.黄酮类化合物的药理作用及其在食品工业中的应用[J].江西食品工业,2007(03).
[3]Oteiza PI,Erlejman AG,Verstraeten SVet al.Flavonoid-membrane interactions:a protective role of flavonoids at the membrane surface?[J].Clin Dev Immunol,2005,12(1):19-25.
[4]Ollila F,Hailing K,Vuorela Pet al.Characterization of flavonoid—biomembrane interactions[J].Arch Biochem Biophys,2002,399(1):103-8.
[5]奚菊群,郭荣.葛根素与卵磷脂囊泡的相互作用[J].科学通报,2007(52).
[6]Arora A,Byrem TM,Nair MGet al.Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids[J].Arch Biochem Biophys,2000,373(1):102-9.
[7]咸德玲,黄可龙,胡卫国,等.脂质体电动色谱法评价阿魏酸与生物膜的相互作用[J].分析化学,2007(10).
[8]Pawlikowska-Pawlega B,Gruszecki WI,Misiak LEet al.The study of the quercetin action on human erythrocyte membranes[J].Biochem Pharmacol,2003,66(4):605-12.
[9]Chaudhuri S,Banerjee A,Basu Ket al.Interaction of flavonoids with red blood cell membrane lipids and proteins:antioxidant and antihemolytic effects[J],Int J Biol Macromol,2007,41(1):42-48.
[10]Maniewska J,Grynkiewicz G,Szeja Wet al.Interaction of genistein benzyl derivatives with lipid bilayers—fluorescence spectroscopic and calorimetric study[J].Bioorg Med Chem,2009,17(6):2592-97.
[11]Lehtonen JY,Adlercreutz H,Kinnunen PK.Binding of daidzein to liposomes[J].Biochim Biophys Acta,1996,1285(1):91-100.
[12]Reinhart WH,Chien S.Red cell rheology in stomatocyte-echinocyte transformation:roles of cell geometry and cell shape[J].Blood,1986,67(4):1110-18.
[13]Fujii T,Sato T,Tamura Aet al.Shape changes of human erythrocytes induced by various amphipathic drugs acting on the membrane of the intact cells[J].Biochem Pharmacol,1979,28(5):613-20
[14]Galati G,O'Brien PJ.Potential toxicity of flavonoids and other dietary phenolics:significance for their chemopreventive and anticancer properties[J].Free Radic Biol Med, 2004,37(3):287-303.
[15]Kerry N,Rice-Evans C.Inhibition of peroxynitrite-mediated oxidation of dopamine by flavonoid and phenolic antioxidants and their structural relationships[J].J Neurochem,1999,73(1):247-53.
[16]Pawlikowska-Pawlega B,Gruszecki WI,Misiak Let al.Modification of membranes by quercetin,a naturally occurring flavonoid,via its incorporation in the polar head group[J].Biochim Biophys Acta,2007,1768(9):2195-204.
[17]Jeong JM,Choi CH,Kang SKet al.Antioxidant and chemosensitizing effects of flavonoids with hydroxy and/or methoxy groups and structure-activity relationship[J].J Pharm Pharm Sci,2007,10(4):537-46.
[18]赵继红,梁宇,颜达予.黄酮类化合物抗氧化活性的结构因素[J].北方工业大学学报,2001(1).
[19]Mukhopadhyay R,Lim HW,Wortis M.Echinocyte shapes:bending,stretching,and shear determine spicule shape and spacing[J].Biophys J,2002,82(4):1756-72.
[20]Suwalsky M,Oyarce K,Avello Met al.Human erythrocytes and molecular models of cell membranes are affected in vitro by Balbisia peduncularis(Amancay) extracts[J].Chem Biol Interact,2009,179(2-3):413-18.
[21]Backman L.Shape control in the human red cell[J].J Cell Sci,1986,80:281-98.
[22]Van Dijk C,Driessen AJ,Recourt K.The uncoupling efficiency and affinity of flavonoids for vesicles[J].Biochem Pharmacol,2000,60(11):1593-600.
[23]Pawlikowska-Pawlega B,Gruszecki WI,Misiak LEet al.The study of the quercetin action on human erythrocyte membranes[J].Biochem Pharmacol,2003,66(4):605-12.
[24]侯少贞,李耿,赖小平,等.葛根素注射液引起急性溶血机制的实验研究[J].药物不良反应杂志,2008(01).
[25]Ahyayauch H,Goni FM,Bennouna M.Interaction of electrically neutral and cationic forms of imipramine with liposome and erythrocyte membranes[J].Int J Pharm,2004,279(1-2):51-58.
[26]Ahyayauch H,Goni FM,Bennouna M.pH-dependent effects of chlorpromazine on liposomes and erythrocyte membranes[J].J Liposome Res,2003,13(2):147-55.
[27]Garcia DA,Quiroga S,Perillo MA.Flunitrazepam partitioning into natural membranes increases surface curvature and alters cellular morphology[J].Chem Biol Interact,2000,129(3):263-77.
[1]刘翎.葛根素注射液不良反应及其相关因素分析[J].中国药物与临床,2004(02).
[2]王慧力,张药.葛根素不良反应[J].中国误诊学杂志,2007(08).
[3]啜丽娟.葛根素不良反应的临床观察[J].护理研究,2004(12).
[4]李梅,杨柳.不同厂家葛根素注射液溶血试验比较[J].广西医科大学学报,2001(18)
[1]陆奇成.葛根素不良反应国内文献的系统性综述[J].药物流行病学杂志,2001(10).
[2]高萧枫,李平.葛根素的不良反应[J].山西医药杂志,2001(30).
[3]高天,王文莉.葛根素注射液不良反应分析[J].中国中医药信息杂志,2002(9).
[4]刘翎,孙亚红,付欣.葛根素注射液不良反应及使用注意事项[J].中国中医基础医学杂志,2001(7).
[5]智丽敏,张志清.葛根素注射液不良反应文献综述[J].河北中医,2002(24).
[6]张志清,王瑞琴.葛根素注射液的不良反应[J].中国医院用药评价与分析,2001(1).
[7]欧阳小青,欧阳亮.121例葛根素注射液不良反应分析[J].中原医刊,2004(31).
[8]全心荣.葛根素不良反应63例文献分析[J].现代中西医结合杂志,2005(14).
[9]张广秋,刘俊东.葛根素注射液的严重不良反应[J].天津药学,2003(15).
[10]张丽娜,雍小兰,金伟华.关注葛根素注射液的不良反应[J].西南军医,2004(6).
[11]国家食品药品监督管理局.《药品不良反应信息通报》(第10期).2005.
[12]张赞玲,尹桃.葛根素注射液引起死亡3例[J].药物不良反应杂志,2004(1).
[13]汪波,朱玉珍,李秀清,等.葛根素致急性血管内溶血5例临床分析[J].北京大学学报(医学版),2004(1).
[14]国家食品药品监督管理局.药品不良反应信息通报(第3期)[R].2003.
[15]国家食品药品监督管理局.药品不良反应信息通报(第8期)[R].2005.
[16]常瑛.药源性血液病(3)[J].药物不良反应杂志,2004(6).
[17]李仪奎.中药药理实验方法学[M].上海:上海科学技术出版社,1991:539-540.
[18]王德才,朱玉云,高允生,等.普乐林注射液的安全性评价[J].泰山医学院院报,2001(22).
[19]张光成,方思鸣.葛根异黄酮的抗氧化的作用[J].中药材,1997(20).
[20]赵雍,杜贵友,崔海峰,等.复方葛根素对脑缺血损伤保护的实验研究[J].中国中药杂志,2005(30).
[21]张远荣,蒋企洲.葛根素的抗氧化活性作用[J].实用临床医药杂志,2005(9).
[22]刘萍,张建益,姜玉亮.葛根素对老年难治性心力衰竭病人血中三种酶活性及脂质过氧化物含量的影响[J].药学服务与研究,2004(4).
[23]朱庆磊,何爱霞,吕欣然.葛根素对氧自由基的清除和抗氧化性损伤作用[J].解放军药学学报,2001(17).
[24]谢雯熙,吴小杨.201例葛根素的不良反应分析[J].海峡药学,2008(03).
[25]叶世泰.变态反应学[M].北京:科学出版社,1998.176.177
[1]Bratosin D,Estaquier J,Petit Fet al.Programmed cell death in mature erythrocytes:a model for investigating death effector pathways operating in the absence of mitochondria[J].Cell Death Differ,2001,8(12):1143-56.
[2]NAKAO M,NAKAO T,YAMAZOE S.Adenosine triphosphate and maintenance of shape of the human red cells[J].Nature,1960,187:945-46.
[3]Sumie Manno,Yuichi Takakuwa NM.Identification of a functional role for lipid asymmetry in biological membranes Phosphatidylserine-skeletal protein interactions modulate membrane stability[J].PNAS,2002,99(4):1943-1948.
[4]王艳伟,林娜,乔利.中药对红细胞膜结构和功能影响的研究进展[J].中国实验方剂学杂志,2006(11)
[5]赵琪,崔乃强,李继坤,吴咸中.寒下药体外质膜保护作用的实验研究[J].中国中西医结合外科杂志,1997(3).
[6]姚伟娟,谢利德,喀蔚波,文宗曜.钙离子对红细胞流变特性的影响及其微观机制分析[J].北京生物医学工程,1999(18).
[7]Backman L.Shape control in the human red cell.[J].J Cell Sci,1986,80:281-98.
[8]刘桂琴,岳慧琴,张勇.荧光偏振法测定生物膜的流动性[J].现代仪器二,2004(6):32-34.
[9]Chaudhuri S,Banerjee A,Basu Ket al.Interaction of flavonoids with red blood cell membrane lipids and proteins:antioxidant and antihemolytic effects[J].Int J Biol Macromol,2007,41(1):42-48.
[10]Cruz SM,Madeira VM,Almeida LMet al.Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure[J].Biochim Biophys Acta,2000,1464(1):49-61.
[11]Fairbanks G,Steck TL,Wallach DF.Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane[J].Biochemistry,1971,10(13):2606-17.
[12]蒋风萍,周本宏,刘刚,胡先明.葛根素对红细胞膜脂质过氧化损伤的防护作用[J].中药药理与临床,2004(5).
[13]Qian Y,Li Z,Huang Let al.Blocking effect of puerarin on calcium channel in isolated guinea pig ventricular myocytes[J].Chin Med J(Engl),1999,112(9):787-89.
[14]Gao Q,Yang B,Ye ZGet al.Opening the calcium-activated potassium channel participates in the cardioprotective effect of puerarin[J].Eur J Pharmacol,2007,574(2-3): 179-84.
[15]Tadahiro Oonishi,Kanako Sakashita ANU.Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways[J].Am J Physiol Cell Physiol,1997,273(6):C1828-C1834.
[16]Mukhopadhyay R,Lim HW,Wortis M.Echinocyte shapes:bending,stretching,and shear determine spicule shape and spacing[J].Biophys J,2002,82(4):1756-72.
[17]Motais R,Baroin A,Motais Aet al.The mechanisms of action of positively and negatively charged drugs[J].Biochim Biophys Acta,1980,599(2):673-88.
[18]Fujii T,Sato T,Tamura Aet al.Shape changes of human erythrocytes induced by various amphipathic drugs acting on the membrane of the intact cells[J].Biochem Pharmacol,1979,28(5):613-20.
[19]Garcia DA,Quiroga S,Perillo MA.Flunitrazepam partitioning into natural membranes increases surface curvature and alters cellular morphology[J].Chem Biol Interact,2000,129(3):263-77.
[20]李雪萍,董果雄.红细胞膜脂流动性研究进展[J].青岛大学医学院学报,2002(38).
[21]奚菊群,郭荣.葛根素与卵磷脂囊泡的相互作用[J].科学通报,2007(52).
[22]Saija A,Bonina F,Trombetta Det al.Flavonoid-biomembrane interactions:A calorimetric study on dipalmitoylphosphatidylcholine vesicles[J].International Journal of Pharmaceutics,1995,124(1):1-8.
[23]Ollila F,Halling K,Vuorela Pet al.Characterization of Flavonoid-Biomembrane Interactions[J].Archives of Biochemistry and Biophysics,2002,399(1):103-8.
[24]卢义钦,刘俊凡.红细胞膜蛋白与膜骨架[J].生命科学研究,2005(9).
[25]王艳伟,林娜,乔利冲药对红细胞膜结构和功能影响的研究进展[J].中国实验方剂学杂志,2006(11).
[26]任绪义,张建鹏.红细胞膜锚蛋白研究进展[J].国外医学.输血及血液学分册,2002(03).
[27]彭激雁,王翔,高玮,等.红细胞膜带4.2蛋白的结构与功能[J].细胞生物学杂志,2007(05).
[28]祁春华,韩凤梅.葛根素与血浆蛋白非共价结合的电喷雾离子阱质谱研究[J].中国药学杂志,2006(23).
[29]Xiao J,Shi J,Cao Het al.Analysis of binding interaction between puerarin and bovine serum albumin by multi-spectroscopic method[J].J Pharm Biomed Anal,2007,45(4):609-15.
[30]许庭郁,杜汴兴.169例葛根素注射液不良反应分析[J].实用药物与临床,2007(04).
[31]徐向辉.对临床使用葛根素注射液出现不良反应的分析[J].上海中医药杂志,2006(08).
[1]Petrosian AM,Haroutounian JE.Taurine as a universal carrier of lipid soluble vitamins:a hypothesis.[J].Amino Acids,2000,19(2):409-21.
[2]周宝宽,林静.牛磺酸膜稳定作用的实验研究[J].中国药理学通报,1997,13(3):277-78.
[3]张秀珍,段建华.牛磺酸对细胞膜脂流动性的影响[J].青岛医学院学报,1996,32(2):106-8.
[4]门秀丽,张连元,等.牛磺酸对肢体缺血再灌注后红细胞的保护作用[J].华北煤炭医学院学报,2002,4(2):137-38.
[5]魏智清,芮键,杨涓.牛磺酸对H_2O_2诱导的红细胞溶血率的影响[J].生物学杂志,2007,24(1):38-40.
[6]O'Leary TJ,Ross PD,Levin IW.Effects of anesthetic tetradecenols on phosphatidylcholine phase transitions.Implications for the mechanism of the bilayer pretransition.[J].Biophys J,1986,50(6):1053-59.
[7]Heimburg T.A model for the lipid pretransition:coupling of ripple formation with the chain-melting transition.[J].Biophys J,2000,78(3):1154-65.
[8]何莹,杜文经,刘雪峰.临床常用的血液流变计算指标的差异分析[J].中国血液流变学杂志,2004(14).
[9]Ahyayauch H,Goni FM,Bennouna M.pH-dependent effects of chlorpromazine on liposomes and erythrocyte membranes.[J].J Liposome Res,2003,13(2):147-55.
[10]Ahyayauch H,Goni FM,Bennouna M.Interaction of electrically neutral and cationic forms of imipramine with liposome and erythrocyte membranes.[J].Int J Pharm,2004,279(1-2):51-58.
[11]Maniewska J,Grynkiewicz G,Szeja Wet al.Interaction of genistein benzyl derivatives with lipid bilayers—fluorescence spectroscopic and calorimetric study.[J].Bioorg Med Chem, 2009,17(6):2592-97.
[12]Lehtonen JY,Adlercreutz H,Kinnunen PK.Binding of daidzein to liposomes.[J].Biochim Biophys Acta,1996,1285(1):91-100.
[13]Timbrell JA,Seabra V,Waterfield CJ.The in vivo and in vitro protective properties of taurine.[J].Gen Pharmacol,1995,26(3):453-62.
[14]Schaffer S,Takahashi K,Azuma J.Role of osmoregulation in the actions of taurine.[J].Amino Acids,2000,19(3-4):527-46.
[15]Hoffmann EK,Lambert IH,Pedersen SF.Physiology of cell volume regulation in vertebrates.[J].Physiol Rev,2009,89(1):193-277.
[16]林琳,巴燕燕,吕品,腾和,等.红细胞变形性及其在血液流变学研究中的意义[J].内蒙古民族大学学报,2006(21)
[17]吴燕红,苏子仁,陈建南,等.从小鼠体内血药浓度时间曲线与组织分布特征评价葛根素的给药途径[J].中药新药与临床药理,2005(02).
[18]祁春华,韩凤梅,曹皴,等.葛根素与血浆蛋白非共价结合的电喷雾离子阱质谱研究[J].中国药学杂志,2006(23).
[19]Van Dijk C,Driessen AJ,Recourt K.The uncoupling efficiency and affinity of flavonoids for vesicles.[J].Biochem Pharmacol,2000,60(11):1593-600.
[20]Bratosin D,Estaquier J,Petit Fet al.Programmed cell death in mature erythrocytes:a model for investigating death effector pathways operating in the absence of mitochondria.[J].Cell Death Differ,2001,8(12):1143-56.
[21]Kon K,Maeda N,Shiga T.The influence of deformation of transformed erythrocytes during flow on the rate of oxygen release.[J].J Physiol,1983,339:573-84.