大蒜素在Caco-2细胞模型转运特征的研究

目的研究大蒜素在Caco-2细胞的转运特征。方法应用Caco-2细胞模型考察转运时间、药物浓度时大蒜素吸收的影响,采用高效液相色谱法测定大蒜素浓度,计算其表观渗透系（Papp）。结果随着浓度增加和时间延长,大蒜素累积通透量逐渐增加;大蒜素的Papp在2个方向比值[Papp（BL→AP）,Papp（AP→BL）]为1．80,存在方向性差异。结论大蒜素的转运属于双向转运,且吸收良好。     

HPLC考察小檗碱和黄连提取物中小檗碱在Caco-2细胞模型的转运

目的同时考察小檗碱和黄连提取物中小檗碱在Ca-co-2细胞模型的转运。方法以Caco-2细胞单层模型研究药物的双向转运,首先系统考察时间、药物浓度、抑制剂、pH、温度对小檗碱单体吸收转运的影响,然后比较相同浓度的小檗碱单体与黄连提取物中的小檗碱在Caco-2细胞模型的转运差异。运用高效液相色谱法检测药物浓度,计算其表观渗透系数。结果相同浓度的小檗碱从底端(BL)到顶端(AP)的转运量大于顶端(AP)到底端(BL)的转运量,在AP→BP和BP→AP方向,其转运量随时间呈增长趋势。维拉帕米可以明显降低其在BL→AP方向的转运量;在AP→BL方向,其在pH=5的HBSS中的转运量最大;在BL→AP方向的转运对温度有较高的依赖性。在AP→BL和BL→AP方向,提取物中的小檗碱比单一的小檗碱的转运量要低。结论小檗碱在AP→BL方向以被动转运为主,在BL→AP方向以主动转运为主;同时还受到P-gp糖蛋白的调节。提取物中的小檗碱在Caco-2模型中转运的影响因素呈多样化。     

布洛芬在Caco-2细胞模型中的转运行为考察

采用Caco-2细胞单层模型考察了布洛芬的吸收机制.用HPLC法测定了磷酸盐缓冲液中布洛芬的转运量.结果显示,布洛芬的转运量受培养时间、浓度和P-糖蛋白抑制剂维拉帕米的影响,其表观渗透系数为1×10-6～7×10-6cm/s,且双向转运的表观渗透性存在方向性差异.这提示布洛芬在Caco-2细胞模型中以主动转运和被动扩散两种机制吸收,且存在P-糖蛋白介导的外排机制.     

RP-HPLC法测定白头翁汤中白头翁皂苷B4的含量

目的 建立白头翁汤中白头翁皂苷B4含量测定的RP-HPLC法.方法 采用Hypersil C18色谱柱(250 mm×4.6 mm,5 μm),以乙腈-水(体积比28:72)为流动相,流速为1.0 mL·min-1,检测波长205 nm,柱温30℃,进样量10μL.结果 白头翁皂苷B4质量浓度在0.1～1.5 g·L-1内线性关系良好(r=0.999 8);平均加样回收率为103.1%,RSD为1.2%(n=9);重复性RSD为1.6%(n=5).结论 该方法简便、快速、准确、重现性好,为白头翁汤及其新剂型改进提供了一种质量控制方法.     

金丝桃苷在Caco-2细胞模型中的跨膜转运方式

目的研究金丝桃苷在Caco-2细胞模型中的吸收机制。方法用Caco-2细胞单层模型研究金丝桃苷的双向转运,考察pn、药物质量浓度、方向、温度、抑制剂对金丝桃苷细胞转运的影响。采用HPLC法检测金丝桃苷的含量,计算其表观渗透系数(P_app)。结果金丝桃苷的细胞转运P_app。具有pH依赖性。金丝桃苷肠腔（A）侧→基底（B）侧P_app>B→A,并且随着金丝桃苷质量浓度的增大而减小,具有浓度依赖性。P-gp抑制剂维拉帕米增加金丝桃苷的细胞正向转运P_app,降低了其逆向转运P_app。金丝桃苷较高浓度时,MRPl抑制剂吲哚美辛和ATP抑制剂叠氮化钠显著降低了金丝桃苷的转运量。结论金丝桃苷在Caco-2细胞单层模型中的转运具有pH依赖性和浓度依赖性,是以主动转运为主,被动扩散为辅,同时涉及外排蛋白作用的转运方式。     

Caco-2细胞对酸枣仁皂苷A的跨膜转运

目的研究酸枣仁皂苷A（Jujuboside A,JuA）在Caco-2细胞的跨膜转运特性。方法采用体外培养的Caco-2细胞单层模型,考察时间、介质pH值、药物浓度、抑制剂对JuA在Caco-2细胞上转运的影响。结果 Caco-2细胞转运JuA呈时间及浓度依赖性;在pH为5.0～8.0范围内,Caco-2细胞对JuA的转运不受pH值的影响;P-糖蛋白（P-glycoprotein,P-gp）抑制剂维拉帕米（Verapamil,Ver）对Caco-2细胞转运JuA无影响;线粒体呼吸链复合体Ⅳ抑制剂叠氮化钠（Sodium azide）对Caco-2细胞转运JuA有抑制作用;JuA的AP-BL侧的Papp与BL-AP侧的Papp的两组均数比较无统计学意义。结论 JuA不是P-gp的底物,其跨膜转运是被动转运与主动转运共同参与的过程。     

熊果酸在Caco-2细胞单层模型中的跨膜转运

熊果酸(Ursolic acid, UA),又名乌索酸,乌苏酸,属a-香树脂醇(a-amyri)型五环三萜类化合物.在自然界分布非常广泛,据目前所知,至少存在于26个科70多种天然植物中[1].大量的研究表明,UA具有广泛的药理作用和重要的生物活性,尤其在抗炎、护肝、抗肿瘤以及机体免疫调节等方面已经显现出令人关注的药理特性[2,3].体内药代动力学研究表明, UA的生物利用度低[4],口服吸收情况较差,而且其在体内吸收转运的方式和机制也并不清楚.Caco-2细胞系来源于人结肠类腺癌细胞,其结构和生化作用类似于人小肠上皮细胞,含有与小肠刷状缘上皮相关的转运系统以及一     

胡桃醌经大鼠肠道及Caco-2细胞模型转运特征的研究

目的研究胡桃醌经大鼠肠道及Caco-2细胞的转运特征。方法应用Caco-2细胞模型和翻转肠囊法考察转运时间、药物浓度对胡桃醌吸收的影响,采用高效液相色谱法测定胡桃醌浓度,计算其表观渗透系数（Papp）。结果随着浓度增加和时间延长,胡桃醌累积通透量逐渐增加;胡桃醌浓度为100μmol L-1时,在Caco-2细胞模型表观渗透系数为1.4×10-7cm s-1;在翻转肠囊模型表观渗透系数2.0×10-7cm s-1。结论胡桃醌的转运符合被动扩散的性质,经肠道吸收率较低。     

鲑鱼降钙素在Caco-2细胞模型中的吸收机制

目的 研究鲑鱼降钙素(SCT)在Caco-2细胞模型的吸收机制.方法 采用人源结肠癌细胞系Caco-2细胞模型研究不同浓度SCT由绒毛面(AP侧)到基底面(BL侧)的转运过程,用HPLC法测定SCT在转运液中的浓度.结果 SCT 0.5～ 1.5 mg·mL-1对其表观渗透系数(Papp)无显著影响.结论 SCT在小肠上皮细胞主要以被动转运的方式吸收,有望将其开发成生物利用度较高的口服给药系统.     

麦冬多糖MDG-1在Caco-2细胞模型中转运机制研究

目的研究麦冬多糖抗心肌缺血活性成分MDG-1在Caco-2细胞模型中转运机制。方法以Caco-2细胞作为转运研究模型,分别测定改变转运方向,使用P糖蛋白(P-gp)外排泵专属抑制剂维拉帕米(verapam il),以及改变给药浓度各种条件下,MDG-1的跨细胞转运情况。结果麦冬多糖MDG-1的分泌转运(BL-AP)的表观渗透系数Papp并未数倍于吸收转运(AP-BL),两者相近,同时P-gp抑制剂维拉帕米加入与否对麦冬多糖MDG-1转运没有影响;在考察的系列药物浓度范围内,MDG-1的转运随着药物浓度的增加而呈线性增加。结论麦冬多糖MDG-1在Caco-2细胞模型中的转运机制很可能是以被动扩散为主,并且以未降解的药物形式转运,无P-gp外排泵参与。     

仙茅苷在Caco-2细胞模型中跨膜转运特征研究

目的：采用Caco-2细胞Transwell模型研究仙茅苷的跨膜转运机制。方法：建立Caco-2细胞Transwell吸收模型,用聚酯碳酸酯膜培养Caco-2细胞21天,形成致密的单层细胞模型。研究浓度、时间、温度、细胞旁路转运及跨膜转运蛋白（P-糖蛋白,多药耐药蛋白,乳腺癌耐药蛋白）在仙茅苷跨膜转运中的作用。结果：仙茅苷在Caco-2细胞模型中的跨膜转运存在一定的浓度及时间依赖性,细胞旁路转运不参与其转运,仙茅苷的外排转运存在一定的能量依赖性,且由P-gp参与。结论：仙茅苷在Caco-2细胞模型上主要以主动转运方式作跨膜转运,且P-gp参与其跨膜转运。     

Transport Characteristics of Fexofenadine in the Caco-2 Cell Model

PURPOSE: To investigate the membrane transport mechanisms of fexofenadine in the Caco-2 model. METHODS: Transport studies were performed in Caco-2 cell monolayers 21-25 days after seeding. The apparent permeability (Papp) of fexofenadine was determined in the concentration range 10-1000 microM in the basolateral-to-apical (b-a) and 50-1000 microM in the apical-to-basolateral (a-b) direction. The concentration-dependent effects of various inhibitors of P-glycoprotein (P-gp) (GF120918, ketoconazole, verapamil, erythromycin), multidrug resistant associated protein (MRP) (indomethacin, probenecid), and organic anion transporting polypeptide (OATP) (rifamycin SV) on the bidirectional transport of 150 microM fexofenadine were also examined. RESULTS: Fexofenadine displayed polarized transport, with the Pappb-a being 28- to 85-fold higher than the Papp(a-b). The Papp(a-b) was independent of the concentration applied, whereas Pappb-a decreased with increasing concentration (Vmax = 5.21 nmol cm(-2)s(-1) and K(M) = 150 microM), suggesting saturation of an apical efflux transporter. All four P-gp inhibitors had a strong, concentration-dependent effect on the Papp of fexofenadine in both directions, with GF 120918 being the most specific among them. The IC50 of verapamil was 8.44 microM on the P-gp-mediated secretion of fexofenadine. The inhibitors of OATP or MRP appeared not to affect the Papp(a-b) of fexofenadine in the Caco-2 model. CONCLUSIONS: This study clearly indicates that P-gp was the main transport protein of fexofenadine in the Caco-2 model. Even though P-gp was completely inhibited, fexofenadine was predicted to have a low fraction dose absorbed in humans due to poor intestinal permeability, and low passive diffusion seems to be the major absorption mechanism.     

人参皂苷Rb_3在Caco-2单细胞层模型上的吸收特征研究

目的:研究人参皂苷Rb3在Caco-2单细胞层模型上的吸收特征。方法:人参皂苷Rb3细胞样品经高速离心后取上清液,以乙腈-1mmo·lL-1甲酸铵水溶液(34∶66)为流动相,以人参皂苷Rg2为内标,采用电喷雾离子源(ESI),在负离子条件下以多重离子反应监测模式(MRM)检测。检测离子:m/z1077.7→m/z783.4(人参皂苷Rb3),m/z783.6→m/z475.1(人参皂苷Rg2)。测定透过Caco-2单细胞层的人参皂苷Rb3浓度并计算其表观渗透系数(Papp)。结果:人参皂苷Rb3的线性范围为50～2000ng·mL-1,日间、日内精密度均小于15%。Papp从基顶侧(AP)到基底侧(BL)为3.22×10-6cm·s-1,从BL侧到AP侧为6.0×10-6cm·s-1,其P(BL-AP)/P(AP-BL)比值为1.86。结论:本方法简单、灵敏度高,可用于研究人参皂苷Rb3在Caco-2单细胞层模型上的吸收特征。     

LC-MS测定雷公藤甲素及其在Caco-2细胞上的转运特征

目的：建立体外模拟体内肠道细胞的Caco-2细胞Transwell模型,以此研究雷公藤甲素在Caco-2细胞模型上的跨膜转运特征。方法：采用聚酯碳酸酯膜连续培养Caco-2细胞21天,形成致密的单层细胞模型。然后对影响雷公藤甲素在Caco-2细胞模型上转运特征的因素包括浓度、时间及跨膜转运蛋白（P-糖蛋白,多药耐药蛋白,乳腺癌耐药蛋白）进行考察;同时采用LC-MS对溶液中的雷公藤甲素的含量进行测定。结果：雷公藤甲素主要以主动转运的方式进行吸收,且随着时间和药物浓度的增加,转运量明显增加。结论：雷公藤甲素在Caco-2细胞上转运存在一定的浓度及时间依赖性,且P-gp介导雷公藤甲素在Caco-2细胞上转运。     

Evaluation of the Caco-2 Monolayer as a Model Epithelium for Iontophoretic Transport

Purpose. To assess the Caco-2 monolayer as a model for iontophoresis of drugs across a model epithelium. Methods. The apparent permeability co-efficient (Papp) of mannitol, thyrotrophin releasing hormone (TRH), dexamethasone and a range of sizes of fluorescein isothiocyanate (FITC) dextrans across Caco-2 monolayers was measured under passive and electrically stimulated conditions. Trans-epithelial electrical resistance (TEER) was determined throughout. Transmission electron micrographs (TEM) of the monolayers were taken. Confocal laser scanning microscopy (CLSM) was used to visualize the iontophoretic transport route of FITC-Dextran (MW = 20 kDa) across a Caco-2 monolayer. Results. Application of 14.3 -Eq.cm^–2 across the monolayer evoked a transient drop in TEER. The drop in TEER was accompanied by statistically significant increases in fluxes of all the agents in the mucosal to serosal direction except for FD-70. TEM of test samples exhibited tight junction dilatation, in addition to intracellular vacuolisation. The iontophoresis of FD-20 was visualised with confocal laser scanning microscopy and was localised in paracellular spaces of the monolayer. Conclusions. The fluxes of mannitol, TRH, dexamethasone, FD-4, FD-10 and FD-20 across the Caco-2 monolayer were significantly enhanced when electric field was applied. The iontophoretic effect appeared to be directly upon tight junctions     

蒙药塔布森-2中9种苯丙素类成分在Caco-2细胞中的吸收特性研究

目的 研究蒙药塔布森-2(Tabson-2 decoction,TBD)中苯丙素类成分在Caco-2细胞中的吸收特性,初步阐明TBD的口服吸收机制。方法 采用Caco-2细胞单层细胞模型,通过UPLC-MS/MS分析TBD在Caco-2细胞中的摄取成分,并建立UPLC-MS/MS分析方法测定TBD中吸收最好的9种苯丙素类成分原儿茶酸、新绿原酸、绿原酸、隐绿原酸、1,5-二咖啡酰奎宁酸、异绿原酸C、咖啡酸、二氢咖啡酸、3-羟基肉桂酸在Caco-2细胞中的含量,分别考察时间、浓度和P糖蛋白抑制剂对各成分的吸收影响。结果 研究发现咖啡酸、二氢咖啡酸的总摄取量在0~180 min为上升趋势,未呈现饱和;3-羟基肉桂酸在90 min左右吸收恒定趋于饱和;隐绿原酸、1,5-二咖啡酰奎宁酸、异绿原酸C、新绿原酸、绿原酸、原儿茶酸的摄取量约从90 min开始,随时间的增加先下降后上升。与苯丙素类成分相比,加入P-糖蛋白抑制剂维拉帕米及环孢菌素A后对二氢咖啡酸的吸收产生了影响,说明二氢咖啡酸是P-糖蛋白底物。结论 TBD主要苯丙素类成分进入Caco-2细胞以被动扩散为主,主动转运为辅,且除二氢咖啡酸外其他8个成分的吸收过程不受P-糖蛋白的外排作用影响。     

坤复康片中芍药内酯苷在Caco-2细胞模型中的吸收特征研究

目的 研究坤复康片中的芍药内酯苷在Caco-2细胞模型中的跨膜吸收特征,探讨人小肠对芍药内酯苷的吸收和转运。方法 研究了不同药物浓度、pH值、温度和抑制剂对芍药内酯苷在Transwell细胞培养板中从顶膜(apical,AP)到基底(basolateral,BL)的双向渗透吸收的影响。结果 芍药内酯苷以3种测试浓度给药时(5,20和100 μmol·L^-1),从AP到BL方向的表观渗透系数(P_app)值范围为(3.27~5.37)×10^-7 cm·s^-1。相反,从BL到AP,P_app值范围为(6.64~9.63)×10^-7 cm·s^-1。数据表明,芍药内酯苷转运具有pH和温度依赖性。芍药内酯苷的细胞通透性受多种抑制剂影响,包含Na⁺/K⁺离子通道抑制剂钒酸钠,多耐药蛋白抑制剂MK571和吲哚美辛及乳腺癌耐药蛋白抑制剂芹菜素。当坤复康溶液加入后,芍药内酯苷从AP到BL方向的P_app值显著增加。结论 芍药内酯苷在Caco-2细胞模型中的肠道吸收较差,其吸收机制可能涉及多种蛋白介导的主动转运。     

Caco-2 Cell Culture as a Model for Famotidine Absorption

The aim of the study was to determine penetration properties of Famotidine fro the formulations through colon adenocarcinoma (Caco)-2 cell monolayers and to compare in vitro with in vivo test results. It also aimed to determine the effect of particle size on the penetration properties of Famotidine when microsphere formulations were used. Famotidine was chosen as a model drug and Caco-2 cell culture model was used. Biodegradable Famotidine microspheres of poly(lactide-co-glycolide)(PLGA) polymer (50:50) were prepared by using multiple emulsion technique. Microspheres were coded according to their particle size and polymer[LHIV:60 μm Famotidine-PLGA(high viscosity), SHIV:6 μm Famotidine PLGA(high viscosity), LLIV:60 μm Famotidine-PLGA (low viscosity), SLIV:6 μm Famotidine-PLGA (low viscosity)]. Famotidine solution(5 mg/ml) and microsphere formulations were administered orally to mice and blood drug levels were determined and compared with the Caco-2 cell experiments. Permeability values of Famotidine through Caco-2 cells from various formulations were determined (log k_solution = 7, 274 ± 0, 010, log k_SHIV = -3, 884 ± 0, 033, log k_LHIV = -2, 300 ± 0, 009, log k_SLIV = -4, 076 ± 0, 208, log k_LLIV = 3, 525 ± 0, 045). Our results showed that H₂ receptor antagonists alter the barrier properties of the Caco-2 cell monolayer by causing an increment in the tightness of the tight junctions. Therefore, amount of the H₂ receptor antagonist-like drug at the site of action was found to be important as well as polymer type and particle size of microspheres for drug permeation. Permeation of the drug was lower when higher amounts of Famotidine were present at the diffusion site. A controlled release dosage form of H₂ receptor antagonist-like drugs may be beneficial for long-term treatments.     

蛇床子素及其制剂在Caco-2细胞模型中转运机制的研究

I摘要】目的：研究蛇床子素及其制剂在Caco-2细胞模型中的摄取、转运机制。方法：建立Caco-2细胞单层模型,研究蛇床子素、蛇床予素β-环糊精包合物及其包合物分散片的摄取和跨膜转运,考察其时间、温度、浓度、吸收促进剂和抑制剂对药物摄取及跨膜吸收的影响,并比较原料药与制剂吸收过程的差异。结果：Caco-2细胞对蛇床子素及其制剂溶液的摄取与时间、温度、浓度均呈正相关,p-糖蛋白抑制剂（CyA）与能量抑制剂（NaN3）对摄取无显著性影响,其摄取量的大小依次为分散片〉包合物〉原料药;转运实验中,随浓度和温度的增加,蛇床子素及其制刑的溶液在Caco-2细胞中的转运量均增加,而只r与Rt比值无明显变化,P-糖蛋白抑制剂（CyA）、能量抑制剂（NaN,）、细胞内吞抑制剂．氧化苯砷（oxophenylarsine）7L胞旁路转运促进剂一去氧胆酸钠（sodiumdeoxycholate,SDCh）对蛇床子素的转运无影响,而去氧胆酸钠对包合物和分散片的转运有明显的影响,3种制刑AP—BL方向上的Papp为分散片〉包合物〉原料药。结论：蛇床子素主要以被动扩散的方式被吸收,包合物和分散片中的药物以被动转运为主,少部分以细胞旁路转运途径被细胞吸收;难溶性药物经包合物可促进其吸收,制剂辅料对药物的吸收有一定的促进作用。     

A Validated HPLC Method for Zanamivir and its Application to In vitro Permeability Study in Caco-2 Culture Model

A simple HPLC method was developed and validated for the quantification of zanamivir in permeability studies using Caco-2 cell culture model. Chromatographic resolution was achieved using 98% (v/v) ultrapure water and 2% (v/v) acetonitrile as mobile phase with flow rate of 0.5 ml/min on a BDS Hypersil Cyano column (length 250 mm; internal diameter 4.6 mm; particle size 5 μm) and UV detection at 230 nm. The method was linear for the quantification of zanamivir at concentration ranging from 0.1-10 μg/ml with coefficient of determination greater than 0.999. The recovery of zanamivir was in the range of 99.76-105.08%. The relative standard deviations of the within-day precision and between-day precision were lower than 10.32 and 14.33%, respectively. The permeability of zanamivir was independent of the transport direction and zanamivir concentrations, indicating a passive transport of zanamivir across Caco-2 cells. With the absence of Ca(2+) in transport medium, the permeability values of zanamivir increased 56.21 and 57.20 fold in the directions of apical to basolateral and basolateral to apical, respectively. On the basis of these results, zanamivir was found to be predominantly transported across Caco-2 monolayers via the passive paracellular pathway.