红景天中苷（苷元）类成分的抗辐射活性及其在Caco-2模型中的转运机制研究
|
摘要
随着我国“神州”载人航天飞行的圆满成功，目前载人航天已成为我国一项重大的系统工程，被列入国家的经济建设计划中。在长期航天飞行中，空间辐射是危害宇航员健康的主要因素之一。提高航天员对空间特殊环境的适应能力和耐受能力，有效保障航天员健康是确保航天任务圆满完成的重要前提。
景天科（Crassulaceae）红景天属（Rhodiola L.）植物为多年生草本或亚灌木植物，已有研究证明其具有明显的抗辐射、抗氧化和调节免疫等作用。但目前对红景天的研究仅限于对其粗提物或红景天苷的研究，对于其它活性成分的作用以及可能的机制则尚无系统的研究报道。苯乙素糖苷-红景天苷(苷元为酪醇)和苯丙素糖苷-洛塞琳、络萨维(苷元为肉桂醇)是红景天属植物的两类主要活性成分，具有独特的药理功能，因而成为近年来红景天活性研究的热点。
本研究针对红景天中苷（苷元）类成分，从辐射防护的抗氧化机制和免疫保护作用两个角度切入，通过建立体外抗氧化模型和辐射所致的淋巴细胞损伤模型，对红景天中苷（苷元）类成分进行辐射防护活性评价，筛选得到抗辐射活性成分。基于筛选评价的结果，通过Caco-2单层细胞体外渗透转运模型对目标抗辐射活性成分的口服吸收情况进行预测，对其转运机制及影响因素进行初步探讨，评估其作为辐射防护剂的可行性，为进一步深入了解其在体内的口服吸收情况以及生物利用度等问题提供实验依据，为下一步开发高效、低毒的辐射防护剂提供基础研究资料，为潜在辐射防护剂的研发和利用奠定实验基础。
本研究主要围绕以下三方面内容展开：
（一）从辐射防护的抗氧化机理出发，利用自由基清除原理建立并优化六种体外抗氧化模型，系统地对红景天中苷（苷元）类成分的抗氧化活性进行评价。
辐射生物学效应产生的最根本原因，是由于射线作用于机体导致的生物分子电离和激发的直接作用，以及由此产生超氧阴离子，羟自由基等活性氧（Reactiveoxygen species，ROS）导致的继发作用，如核酸分子、蛋白质分子以及生物膜的损伤等。因此，清除已经形成的ROS或者抑制其产生，是辐射防护的主要途径之一。本研究建立并优化了DPPH自由基、NBT-NADH-PMS体系、羟自由基、氮氧自由基（NO）、脂质过氧化反应体系和铁离子抗氧化体系六种抗氧化模型，分别测定了芦丁苷、红景天苷、异槲皮苷、酪醇、洛塞琳、熊果苷、络萨维和肉桂醇对DPPH自由基、超氧阴离子、羟自由基和NO自由基四种自由基的清除能力，以及对脂质过氧化的抑制能力和总抗氧化能力。结果表明，红景天中的苷（苷元）类化学成分从不同方面表现出抗氧化活性：红景天苷、酪醇、洛塞琳、络萨维和肉桂醇表现出较强的羟自由基清除能力（IC_(50)分别为18.97μg·mL~(-1)，10.84μg·mL~(-1)，61.84μg·mL~(-1)，81.08μg·mL~(-1)，26.00μg·mL~(-1)）。芦丁苷和异槲皮苷表现出较强的DPPH自由基和超氧阴离子清除能力，抗脂质过氧化和总抗氧化能力；熊果苷的DPPH自由基清除能力和总抗氧化能力较强。
（二）从辐射防护剂的免疫保护作用出发，利用细胞学和分子生物学实验方法建立体外辐射防护模型，在细胞水平上对红景天中苷（苷元）类化合物的体外抗辐射活性进行评价。
免疫系统是辐射损伤的重要靶器官，具有高度的辐射敏感性。淋巴细胞损伤是辐射导致机体免疫功能抑制的重要原因，因此有效地保护淋巴细胞免受辐射损伤对降低机体辐射损伤有重要意义。本研究先通过细胞增殖检测方法比较，细胞辐射敏感性研究，增殖与辐照的量效关系考察等方面，对建立体外辐射防护模型进行了方法学评价；又在确保受试物对细胞无毒的基础上，通过给予人淋巴母细胞(AHH-1)10Gy~(60)Co-γ射线一次性照射建立辐射损伤模型，以MTS法检测红景天苷、芦丁苷、异槲皮苷、酪醇、洛塞琳、熊果苷和络萨维对受到辐射后AHH-1细胞的保护作用。研究结果显示，与模型组比较，异槲皮苷(12.5μg.mL~(-1)，P <0.05)、红景天苷(12.5μg.mL~(-1)，P <0.05)、酪醇(50μg.mL~(-1)，P <0.05)、洛塞琳(25μg.mL~(-1)，P <0.05)、络萨维(12.5μg.mL~(-1)，P <0.01)和熊果苷(12.5μg.mL~(-1)，P <0.01)均能够显著提高辐射损伤AHH-1的增殖活性。基于抗氧化与免疫保护两部分实验结果，确定了红景天苷、酪醇、洛塞琳和络萨维为目标抗辐射活性成分，进行下一步的渗透转运研究。
（三）建立用于药物吸收研究Caco-2渗透转运模型，评价目标抗辐射活性成分的口服吸收情况，初步探讨其转运机制。
通过观察细胞单层形态，监测跨膜电阻（TEER）值的变化以及比较普萘洛尔和阿替洛尔两种标记物在单层模型的转运特性等指标对Caco-2细胞单层模型的可靠性进行评价。结果显示，TEER值在Caco-2细胞单层形成过程中稳定上升，达到实验模型要求标准（>500Ω·cm~2）。胞内转运标记物普萘洛尔（高渗透性药物，表观渗透系数（Papp）=1.01×10~(-5)cm·s~(-1)）和胞间转运标记物阿替洛尔（低渗透性药物，Papp=2.20×10-7cm·s~(-1)）呈现可预见的高、低Papp值，表明建立的Caco-2细胞模型在完整性、通透性等方面均符合小肠吸收机制研究的要求。结合抗氧化与体外AHH-1模型两部分实验结果，选定抗氧化作用和保护~(60)Co-γ射线辐照后AHH-1细胞两方面作用均较强的红景天苷、酪醇、洛塞琳和络萨维为目标抗辐射活性成分，通过体外Caco-2渗透转运模型对这四个成分的肠毒性、吸收情况及转运机制进行研究。本研究通过考察抗辐射活性成分对Caco-2细胞24h的细胞毒性作用，对目标成分的肠毒性进行评估，并推算其用于转运模型的安全剂量；在建立了可靠的转运模型和确保受试物对Caco-2细胞单层无显著毒性的前提下，应用Caco-2单层模型对受试物进行了双向转运研究（肠腔侧向肠壁侧，AP→BL；肠壁侧向肠腔侧，BL→AP），考察了转运时间（60min-180min）和药物浓度（50μM-200μM）对转运的影响。然后，采用反相高效液相色谱法（RP-HPLC）测定并计算受试物在渗透转运模型中的Papp。AP→BL转运数据表明，红景天苷（Papp=0.85±0.12×10~(-6)cm·s~(-1)）、洛塞琳（Papp=0.61±0.10×10~(-6)cm·s~(-1)）和络萨维（Papp=0.74±0.12×10~(-6)cm·s~(-1)）均为难吸收（PappAP→BL<1×10~(-6)cm·s~(-1)）药物。红景天苷的苷元酪醇（Papp=1.52±0.16×10~(-6)cm·s~(-1)）渗透性要好于红景天苷。浓度为200μM时辐射防护活性成分的双向转运数据未表现出明显方向性（0.61×10~(-6)cm·s~(-1) 综上所述，红景天中的苷（苷元）类化学成分从不同方面均表现出一定的抗氧化活性。其中，红景天苷、酪醇、洛塞琳、络萨维和肉桂醇表现出较强的羟自由基清除能力。在淋巴细胞损伤模型中，异槲皮苷、红景天苷、络萨维、洛塞琳、熊果苷和酪醇均能够显著提高辐射损伤的AHH-1细胞的活力。两部分筛选结果提示红景天苷、酪醇、洛塞琳和络萨维可能为红景天中的抗辐射活性成分。在Caco-2单层模型中的转运研究中发现，红景天苷、洛塞琳和络萨维均为难吸收药物；而作为红景天苷的苷元-酪醇，其吸收性优于红景天苷。
As part of national economic initiative, manned spaceflight has become one ofmajor national projects in China. During the space flight, space radiation is verydangerous for human health. Thus, it’s important to keep healthy and improve theadaptability and tolerance of the astronauts when exposure to the outer spaceradiation.
Crassulaceae Rhodiola L. plants are perennial herbage or subshrubs plants withsignificant anti-radiation, antioxidant and immune adjustment effects. Current studiesis only limited to extracts or Salidroside and few investigation has been conducted ontheir other ingredients and mechanism of action. Phenethyl and phenylpropanoidglycosides are two key active ingredients in Rhodiola L.. Due to their uniquebiological activity, Phenethyl and phenylpropanoid glycosides, including Salidroside,Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol have been drawn great attentions bymany researchers.
In this paper, the antioxidant and immune protective effect of glycosides (aglycone)in Rhodiola were investigated for its application in radiation protection. In vitroantioxidant models and radiation-induced cell damage model were established to findits anti-radiation active ingredients. In addition, using Caco-2cell model, thepermeability and mechanism of transportation of the anti-radiation active ingredientswere investigated. Their potential as a radiation protective agent were evaluated.These studies have firstly investigated the oral absorption and bioavailability of itsanti-radiation active ingredients for the further development of these ingredients aspotential radio-protectors. The main studies were conducted as followed:
1. Based on the anti-oxidative radiation protection mechanism using free radicalscavenging, six in vitro antioxidant models were built and optimized foranti-oxidative effect of glycosides (aglycone) in Rhodiola.
Generally, the radiation can cause the ionization and excitation of the targetbiomolecule which lead to the production of reactive oxygen species (ROS). TheseROS, including superoxide anion and hydroxyl radical et al., may contribute to radiation-induced injury of nucleic acid, protein and biofilm. The main mechanism ofradiation protection is clearing the genera ROS or restraining its production. In thisstudy, six in vitro antioxidant models (include DPPH radical scavenging assay,superoxide radical scavenging assay, hydroxyl radical scavenging assay, NO radicalscavenging assay, anti-lipid peroxidation assay, and total antioxidant activity) wereestablished and optimized. The antioxidative activities and the mechanisms action ofRutin, Salidroside, Isoquercitrin, Tyrosol, Rosarin, Arbutin, Rosavin and Cinnamylalcohol (six glycosides and two aglycones) were investigated. These studies haveshowed that eight glycosides (aglycone) showed anti-oxidative activity. Salidroside,Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol exhibited strong hydroxyl radicalscavenging activity (IC_(50)value were18.97μg·mL~(-1),10.84μg·mL~(-1),61.84μg·mL~(-1)，81.08μg·mL~(-1),26μg·mL~(-1)). Rutin and Isoquercitrin showed strong DPPH radical,superoxide radical scavenging and anti-lipid peroxidation and total antioxidativeactivity. Arbutin showed DPPH scavenging activity and total anti oxidative activity.
2. Based on the immune protection of radio-protectors, in vitro anti-radiation modelwas established using cellular and biomedical methods. Glycosides (aglycone) inRhodiola were evaluated for anti-radiation at the cellular level.
Immune system is an important target organ of radiation, which are highly radiationsensitive. Radiation-induced lymphocytes damage will cause the suppression ofimmune function, so protection of lymphocytes from radiation damage is of important.In this study, in vitro anti-radiation model was established by comparing cellproliferation assays, investigating cell radio-sensitivity and investigating therelationship between proliferation and irradiation dose. Followed by toxicity studies,Lymphocytes (AHH-1) were exposed to10Gy60Co γ-ray to induce radiation injurymodel, and then MTS assay was used to detect the proliferation activity of AHH-1after treated by Rutin, Salidroside, Isoquercitrin, Tyrosol, Rosarin, Arbutin andRosavin. Comparing with the model groups, Salidroside (12.5μg.mL~(-1), P <0.05),Isoquercitrin (12.5μg.mL~(-1), P <0.05), Rosavin (12.5μg.mL~(-1), P <0.01), Rosarin (25μg.mL~(-1), P <0.05), Arbutin (12.5μg.mL~(-1), P <0.01) and Tyrosol (50μg.mL~(-1), P <0.05)could significantly improve the viability of radiation-induced AHH-1.
3. Caco-2permeability and transportation assay was established to investigate thepermeability and mechanism of transportation of the anti-radiation active ingredients.
The reliability of Caco-2cell monolayer was evaluated by cell morphology,transepithelial electrical resistance (TEER) and standard markers. Results showed that TEER steady raised in developing process of cell monolayer, and reached the standardvalue(>500Ω·cm~2).The Papp value of Atenolol (low permeability, Papp=2.20×10-7cm·s~(-1)) and Propranolol (high permeability, Papp=1.01×10~(-5)cm·s~(-1)) were all withinthe reported range. So Caco-2cell model was validated for the study of the evaluationof the intestinal absorption. Anti-radiation active ingredients, including Salidroside,Tyrosol, Rosarin and Rosavin were selected for the studies of intestinal toxicity,absorption and transportation using Caco-2permeability and transportation model.The24h cytotoxicity of anti-radiation active ingredients was investigated. Intestinaltoxicity was evaluated and safe dose was calculated. In addition, bidirectionaltransport assay was also conducted using Caco-2monolayer model. Effects oftransport time (60min-180min) and drug concentration (50μM-200μM) wereinvestigated. The Papp values were determined by RP-HPLC methods. Salidroside(Papp=0.85±0.12×10~(-6)cm·s~(-1)), Rosarin (Papp=0.61±0.10×10~(-6)cm·s~(-1)) and Rosavin(Papp=0.74±0.12×10~(-6)cm·s~(-1)) were poor-absorbed ingredients (PappAP→BL<1×10~(-6)cm·s~(-1)). While Tyrosol (Papp=1.52±0.16×10~(-6)cm·s~(-1)) showed higher permeabilitythan its glycosides Salidroside. The bidirectional transport of4glycosides (aglycone)at200μM was comparable, indicating a lack of directional preference; it was alsolinear with time and concentrations, suggesting a passive diffusion mechanism duringtheir transportation across the Caco-2cell monolayer. The poor-absorption ofSalidroside may be related to its efflux.
In summary, glycosides (aglycone) in Rhodiola showed anti-oxidative activity.Salidroside, Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol showed strong hydroxylradical scavenging. Comparing with the model groups, Salidroside, Isoquercitrin,Rosavin, Rosarin, Arbutin and Tyrosol can improve the viability of radiation-inducedAHH-1. The results suggested that salidroside, tyrosol, Rosavin and Rosarin wereanti-radiation active ingredients in Rhodiola. Salidroside, Rosarin and Rosavin werepoor-absorbed ingredients. There was higher permeability for Tyrosol than for itsglycosides Salidroside.
景天科（Crassulaceae）红景天属（Rhodiola L.）植物为多年生草本或亚灌木植物，已有研究证明其具有明显的抗辐射、抗氧化和调节免疫等作用。但目前对红景天的研究仅限于对其粗提物或红景天苷的研究，对于其它活性成分的作用以及可能的机制则尚无系统的研究报道。苯乙素糖苷-红景天苷(苷元为酪醇)和苯丙素糖苷-洛塞琳、络萨维(苷元为肉桂醇)是红景天属植物的两类主要活性成分，具有独特的药理功能，因而成为近年来红景天活性研究的热点。
本研究针对红景天中苷（苷元）类成分，从辐射防护的抗氧化机制和免疫保护作用两个角度切入，通过建立体外抗氧化模型和辐射所致的淋巴细胞损伤模型，对红景天中苷（苷元）类成分进行辐射防护活性评价，筛选得到抗辐射活性成分。基于筛选评价的结果，通过Caco-2单层细胞体外渗透转运模型对目标抗辐射活性成分的口服吸收情况进行预测，对其转运机制及影响因素进行初步探讨，评估其作为辐射防护剂的可行性，为进一步深入了解其在体内的口服吸收情况以及生物利用度等问题提供实验依据，为下一步开发高效、低毒的辐射防护剂提供基础研究资料，为潜在辐射防护剂的研发和利用奠定实验基础。
本研究主要围绕以下三方面内容展开：
（一）从辐射防护的抗氧化机理出发，利用自由基清除原理建立并优化六种体外抗氧化模型，系统地对红景天中苷（苷元）类成分的抗氧化活性进行评价。
辐射生物学效应产生的最根本原因，是由于射线作用于机体导致的生物分子电离和激发的直接作用，以及由此产生超氧阴离子，羟自由基等活性氧（Reactiveoxygen species，ROS）导致的继发作用，如核酸分子、蛋白质分子以及生物膜的损伤等。因此，清除已经形成的ROS或者抑制其产生，是辐射防护的主要途径之一。本研究建立并优化了DPPH自由基、NBT-NADH-PMS体系、羟自由基、氮氧自由基（NO）、脂质过氧化反应体系和铁离子抗氧化体系六种抗氧化模型，分别测定了芦丁苷、红景天苷、异槲皮苷、酪醇、洛塞琳、熊果苷、络萨维和肉桂醇对DPPH自由基、超氧阴离子、羟自由基和NO自由基四种自由基的清除能力，以及对脂质过氧化的抑制能力和总抗氧化能力。结果表明，红景天中的苷（苷元）类化学成分从不同方面表现出抗氧化活性：红景天苷、酪醇、洛塞琳、络萨维和肉桂醇表现出较强的羟自由基清除能力（IC_(50)分别为18.97μg·mL~(-1)，10.84μg·mL~(-1)，61.84μg·mL~(-1)，81.08μg·mL~(-1)，26.00μg·mL~(-1)）。芦丁苷和异槲皮苷表现出较强的DPPH自由基和超氧阴离子清除能力，抗脂质过氧化和总抗氧化能力；熊果苷的DPPH自由基清除能力和总抗氧化能力较强。
（二）从辐射防护剂的免疫保护作用出发，利用细胞学和分子生物学实验方法建立体外辐射防护模型，在细胞水平上对红景天中苷（苷元）类化合物的体外抗辐射活性进行评价。
免疫系统是辐射损伤的重要靶器官，具有高度的辐射敏感性。淋巴细胞损伤是辐射导致机体免疫功能抑制的重要原因，因此有效地保护淋巴细胞免受辐射损伤对降低机体辐射损伤有重要意义。本研究先通过细胞增殖检测方法比较，细胞辐射敏感性研究，增殖与辐照的量效关系考察等方面，对建立体外辐射防护模型进行了方法学评价；又在确保受试物对细胞无毒的基础上，通过给予人淋巴母细胞(AHH-1)10Gy~(60)Co-γ射线一次性照射建立辐射损伤模型，以MTS法检测红景天苷、芦丁苷、异槲皮苷、酪醇、洛塞琳、熊果苷和络萨维对受到辐射后AHH-1细胞的保护作用。研究结果显示，与模型组比较，异槲皮苷(12.5μg.mL~(-1)，P <0.05)、红景天苷(12.5μg.mL~(-1)，P <0.05)、酪醇(50μg.mL~(-1)，P <0.05)、洛塞琳(25μg.mL~(-1)，P <0.05)、络萨维(12.5μg.mL~(-1)，P <0.01)和熊果苷(12.5μg.mL~(-1)，P <0.01)均能够显著提高辐射损伤AHH-1的增殖活性。基于抗氧化与免疫保护两部分实验结果，确定了红景天苷、酪醇、洛塞琳和络萨维为目标抗辐射活性成分，进行下一步的渗透转运研究。
（三）建立用于药物吸收研究Caco-2渗透转运模型，评价目标抗辐射活性成分的口服吸收情况，初步探讨其转运机制。
通过观察细胞单层形态，监测跨膜电阻（TEER）值的变化以及比较普萘洛尔和阿替洛尔两种标记物在单层模型的转运特性等指标对Caco-2细胞单层模型的可靠性进行评价。结果显示，TEER值在Caco-2细胞单层形成过程中稳定上升，达到实验模型要求标准（>500Ω·cm~2）。胞内转运标记物普萘洛尔（高渗透性药物，表观渗透系数（Papp）=1.01×10~(-5)cm·s~(-1)）和胞间转运标记物阿替洛尔（低渗透性药物，Papp=2.20×10-7cm·s~(-1)）呈现可预见的高、低Papp值，表明建立的Caco-2细胞模型在完整性、通透性等方面均符合小肠吸收机制研究的要求。结合抗氧化与体外AHH-1模型两部分实验结果，选定抗氧化作用和保护~(60)Co-γ射线辐照后AHH-1细胞两方面作用均较强的红景天苷、酪醇、洛塞琳和络萨维为目标抗辐射活性成分，通过体外Caco-2渗透转运模型对这四个成分的肠毒性、吸收情况及转运机制进行研究。本研究通过考察抗辐射活性成分对Caco-2细胞24h的细胞毒性作用，对目标成分的肠毒性进行评估，并推算其用于转运模型的安全剂量；在建立了可靠的转运模型和确保受试物对Caco-2细胞单层无显著毒性的前提下，应用Caco-2单层模型对受试物进行了双向转运研究（肠腔侧向肠壁侧，AP→BL；肠壁侧向肠腔侧，BL→AP），考察了转运时间（60min-180min）和药物浓度（50μM-200μM）对转运的影响。然后，采用反相高效液相色谱法（RP-HPLC）测定并计算受试物在渗透转运模型中的Papp。AP→BL转运数据表明，红景天苷（Papp=0.85±0.12×10~(-6)cm·s~(-1)）、洛塞琳（Papp=0.61±0.10×10~(-6)cm·s~(-1)）和络萨维（Papp=0.74±0.12×10~(-6)cm·s~(-1)）均为难吸收（PappAP→BL<1×10~(-6)cm·s~(-1)）药物。红景天苷的苷元酪醇（Papp=1.52±0.16×10~(-6)cm·s~(-1)）渗透性要好于红景天苷。浓度为200μM时辐射防护活性成分的双向转运数据未表现出明显方向性（0.61×10~(-6)cm·s~(-1)
As part of national economic initiative, manned spaceflight has become one ofmajor national projects in China. During the space flight, space radiation is verydangerous for human health. Thus, it’s important to keep healthy and improve theadaptability and tolerance of the astronauts when exposure to the outer spaceradiation.
Crassulaceae Rhodiola L. plants are perennial herbage or subshrubs plants withsignificant anti-radiation, antioxidant and immune adjustment effects. Current studiesis only limited to extracts or Salidroside and few investigation has been conducted ontheir other ingredients and mechanism of action. Phenethyl and phenylpropanoidglycosides are two key active ingredients in Rhodiola L.. Due to their uniquebiological activity, Phenethyl and phenylpropanoid glycosides, including Salidroside,Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol have been drawn great attentions bymany researchers.
In this paper, the antioxidant and immune protective effect of glycosides (aglycone)in Rhodiola were investigated for its application in radiation protection. In vitroantioxidant models and radiation-induced cell damage model were established to findits anti-radiation active ingredients. In addition, using Caco-2cell model, thepermeability and mechanism of transportation of the anti-radiation active ingredientswere investigated. Their potential as a radiation protective agent were evaluated.These studies have firstly investigated the oral absorption and bioavailability of itsanti-radiation active ingredients for the further development of these ingredients aspotential radio-protectors. The main studies were conducted as followed:
1. Based on the anti-oxidative radiation protection mechanism using free radicalscavenging, six in vitro antioxidant models were built and optimized foranti-oxidative effect of glycosides (aglycone) in Rhodiola.
Generally, the radiation can cause the ionization and excitation of the targetbiomolecule which lead to the production of reactive oxygen species (ROS). TheseROS, including superoxide anion and hydroxyl radical et al., may contribute to radiation-induced injury of nucleic acid, protein and biofilm. The main mechanism ofradiation protection is clearing the genera ROS or restraining its production. In thisstudy, six in vitro antioxidant models (include DPPH radical scavenging assay,superoxide radical scavenging assay, hydroxyl radical scavenging assay, NO radicalscavenging assay, anti-lipid peroxidation assay, and total antioxidant activity) wereestablished and optimized. The antioxidative activities and the mechanisms action ofRutin, Salidroside, Isoquercitrin, Tyrosol, Rosarin, Arbutin, Rosavin and Cinnamylalcohol (six glycosides and two aglycones) were investigated. These studies haveshowed that eight glycosides (aglycone) showed anti-oxidative activity. Salidroside,Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol exhibited strong hydroxyl radicalscavenging activity (IC_(50)value were18.97μg·mL~(-1),10.84μg·mL~(-1),61.84μg·mL~(-1)，81.08μg·mL~(-1),26μg·mL~(-1)). Rutin and Isoquercitrin showed strong DPPH radical,superoxide radical scavenging and anti-lipid peroxidation and total antioxidativeactivity. Arbutin showed DPPH scavenging activity and total anti oxidative activity.
2. Based on the immune protection of radio-protectors, in vitro anti-radiation modelwas established using cellular and biomedical methods. Glycosides (aglycone) inRhodiola were evaluated for anti-radiation at the cellular level.
Immune system is an important target organ of radiation, which are highly radiationsensitive. Radiation-induced lymphocytes damage will cause the suppression ofimmune function, so protection of lymphocytes from radiation damage is of important.In this study, in vitro anti-radiation model was established by comparing cellproliferation assays, investigating cell radio-sensitivity and investigating therelationship between proliferation and irradiation dose. Followed by toxicity studies,Lymphocytes (AHH-1) were exposed to10Gy60Co γ-ray to induce radiation injurymodel, and then MTS assay was used to detect the proliferation activity of AHH-1after treated by Rutin, Salidroside, Isoquercitrin, Tyrosol, Rosarin, Arbutin andRosavin. Comparing with the model groups, Salidroside (12.5μg.mL~(-1), P <0.05),Isoquercitrin (12.5μg.mL~(-1), P <0.05), Rosavin (12.5μg.mL~(-1), P <0.01), Rosarin (25μg.mL~(-1), P <0.05), Arbutin (12.5μg.mL~(-1), P <0.01) and Tyrosol (50μg.mL~(-1), P <0.05)could significantly improve the viability of radiation-induced AHH-1.
3. Caco-2permeability and transportation assay was established to investigate thepermeability and mechanism of transportation of the anti-radiation active ingredients.
The reliability of Caco-2cell monolayer was evaluated by cell morphology,transepithelial electrical resistance (TEER) and standard markers. Results showed that TEER steady raised in developing process of cell monolayer, and reached the standardvalue(>500Ω·cm~2).The Papp value of Atenolol (low permeability, Papp=2.20×10-7cm·s~(-1)) and Propranolol (high permeability, Papp=1.01×10~(-5)cm·s~(-1)) were all withinthe reported range. So Caco-2cell model was validated for the study of the evaluationof the intestinal absorption. Anti-radiation active ingredients, including Salidroside,Tyrosol, Rosarin and Rosavin were selected for the studies of intestinal toxicity,absorption and transportation using Caco-2permeability and transportation model.The24h cytotoxicity of anti-radiation active ingredients was investigated. Intestinaltoxicity was evaluated and safe dose was calculated. In addition, bidirectionaltransport assay was also conducted using Caco-2monolayer model. Effects oftransport time (60min-180min) and drug concentration (50μM-200μM) wereinvestigated. The Papp values were determined by RP-HPLC methods. Salidroside(Papp=0.85±0.12×10~(-6)cm·s~(-1)), Rosarin (Papp=0.61±0.10×10~(-6)cm·s~(-1)) and Rosavin(Papp=0.74±0.12×10~(-6)cm·s~(-1)) were poor-absorbed ingredients (PappAP→BL<1×10~(-6)cm·s~(-1)). While Tyrosol (Papp=1.52±0.16×10~(-6)cm·s~(-1)) showed higher permeabilitythan its glycosides Salidroside. The bidirectional transport of4glycosides (aglycone)at200μM was comparable, indicating a lack of directional preference; it was alsolinear with time and concentrations, suggesting a passive diffusion mechanism duringtheir transportation across the Caco-2cell monolayer. The poor-absorption ofSalidroside may be related to its efflux.
In summary, glycosides (aglycone) in Rhodiola showed anti-oxidative activity.Salidroside, Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol showed strong hydroxylradical scavenging. Comparing with the model groups, Salidroside, Isoquercitrin,Rosavin, Rosarin, Arbutin and Tyrosol can improve the viability of radiation-inducedAHH-1. The results suggested that salidroside, tyrosol, Rosavin and Rosarin wereanti-radiation active ingredients in Rhodiola. Salidroside, Rosarin and Rosavin werepoor-absorbed ingredients. There was higher permeability for Tyrosol than for itsglycosides Salidroside.
引文
1.中国植物志.科学出版社,1986,第34卷,第一分册:161-220.
2.国家药典委员会.中国药典一部,化学工业出版社,2010,第一版:144.
3. Ali Z, Fronczek F R, and Khan I A. Phenylalkanoids and monoterpene analoguesfrom the roots of Rhodiola rosea[J]. Planta Med,2008.74(2):178-181.
4. Ma G, Li W, Dou D, et al. Rhodiolosides A-E, monoterpene glycosides fromRhodiola rosea[J]. Chem Pharm Bull (Tokyo),2006.54(8):1229-1233.
5. Rohloff J. Volatiles from rhizomes of Rhodiola rosea L[J]. Phytochemistry,2002.59(6):655-661.
6. Panossian A, Gabrielian E, and Wagner H. On the mechanism of action of plantadaptogens with particular reference to cucurbitacin R diglucoside[J].Phytomedicine,1999.6(3):147-155.
7. Panossian A G, Oganessian A S, Ambartsumian M, et al. Effects of heavyphysical exercise and adaptogens on nitric oxide content in human saliva[J].Phytomedicine,1999.6(1):17-26.
8. Panossian A and Wikman G. Evidence-Based Efficacy of Adaptogens in Fatigue,and Molecular Mechanisms Related to their Stress-Protective Activity[J]. CurrentClinical Pharmacology,2009.4(3):198-219.
9. Perfumi M and Mattioli L. Adaptogenic and central nervous system effects ofsingle doses of3%rosavin and1%salidroside Rhodiola rosea L. extract inmice[J]. Phytotherapy Research,2007.21(1):37-43.
10. Zhang Y and Liu Y. Study on effects of salidroside on lipid peroxidation onoxidative stress in rat hepatic stellate cells[J]. Zhong Yao Cai,2005.28(9):794-796.
11. Parisi A, Tranchita E, Duranti G, et al. Effects of chronic Rhodiola Roseasupplementation on sport performance and antioxidant capacity in trained male:preliminary results[J]. J Sports Med Phys Fitness,2010.50(1):57-63.
12. Olsson E M, von Scheele B, and Panossian A G. A randomised, double-blind,placebo-controlled, parallel-group study of the standardised extract shr-5of theroots of Rhodiola rosea in the treatment of subjects with stress-related fatigue[J].Planta Med,2009.75(2):105-112.
13.李岩,吕晓华.红景天胶囊对小鼠抗疲劳的效应[J].川北医学院学报,2012(06):531-533.
14. Wang H, Ding Y, Zhou J, et al. The in vitro and in vivo antiviral effects ofsalidroside from Rhodiola rosea L. against coxsackievirus B3[J]. Phytomedicine,2009.16(2-3):146-155.
15. Zhang J K, Yang L, Meng G L, et al. Protection by Salidroside against Bone Lossvia Inhibition of Oxidative Stress and Bone-Resorbing Mediators[J]. PLoS One,2013.8(2): e57251.
16.曾满红,黄清松,张德兴.红景天总黄酮对自然衰老大鼠抗氧化和免疫功能的影响[J].解剖学研究,2012(02):135-137.
17. Wojcik R, Siwicki A K, Skopinska-Rozewska E, et al. The effect of Chinesemedicinal herb Rhodiola kirilowii extracts on cellular immunity in mice andrats[J]. Pol J Vet Sci,2009.12(3):399-405.
18. Skopnska-Rozewska E, Wojcik R, Siwicki A K, et al. The effect of Rhodiolaquadrifida extracts on cellular immunity in mice and rats[J]. Pol J Vet Sci,2008.11(2):105-111.
19. Zhong H, Xin H, Wu L X, et al. Salidroside attenuates apoptosis in ischemiccardiomyocytes: a mechanism through a mitochondria-dependent pathway[J]. JPharmacol Sci,2010.114(4):399-408.
20. Zhang J, Liu A, Hou R, et al. Salidroside protects cardiomyocyte againsthypoxia-induced death: A HIF-1α-activated and VEGF-mediated pathway[J].European Journal of Pharmacology,2009.607(1–3):6-14.
21. Arora R, Singh S, Sagar R K, et al. Radiomodulatory and Free-RadicalScavenging Activity of the Fractionated Aquo-Alcoholic Extract of theAdaptogenic Nutraceutical (Rhodiola imbricata)-A Comparative In VitroAssessment with Ascorbate[J]. J Diet Suppl,2008.5(2):147-163.
22.张茗昱,张建,李雪,等.长白山红景天有效成分对X射线辐射防护作用的研究[J].中国医药指南,2012(20)：88-90.
23. Pooja, Bawa A S, and Khanum F. Anti-inflammatory activity of Rhodiola rosea–―a second-generation adaptogen‖[J]. Phytotherapy Research,2009.23(8):1099-1102.
24. Wu X and Prior R L. Identification and Characterization of Anthocyanins byHigh-Performance Liquid Chromatography Electrospray Ionization TandemMass Spectrometry in Common Foods in the United States: Vegetables, Nuts, andGrains[J].J Agric Food Chem,2005.53(8):3101-3113.
25. Wolfe K L and Liu R H. Cellular Antioxidant Activity (CAA) Assay forAssessing Antioxidants, Foods, and Dietary Supplements[J].J Agric Food Chem,2007.55(22):8896-8907.
26. Adom K K and Liu R H. Rapid Peroxyl Radical Scavenging Capacity (PSC)Assay for Assessing both Hydrophilic and Lipophilic Antioxidants[J].J AgricFood Chem,2005.53(17):6572-6580.
27. Moore J, Yin J-J, and Yu L. Novel Fluorometric Assay for Hydroxyl RadicalScavenging Capacity (HOSC) Estimation[J].J Agric Food Chem,2006.54(3):617-626.
28. Huang D, Ou B, and Prior R L. The Chemistry behind Antioxidant CapacityAssays[J].J Agric Food Chem,2005.53(6):1841-1856.
29. MacDonald-Wicks L K, Wood L G, and Garg M L. Methodology for thedetermination of biological antioxidant capacity in vitro: a review[J].J SciFoodAgric,2006.86(13):2046-2056.
30. Ou B, Huang D, Hampsch-Woodill M, et al. Analysis of Antioxidant Activities ofCommon Vegetables Employing Oxygen Radical Absorbance Capacity (ORAC)and Ferric Reducing Antioxidant Power (FRAP) Assays: A ComparativeStudy[J].J Agric Food Chem,2002.50(11):3122-3128.
31. Chaudière J and Ferrari-Iliou R. Intracellular Antioxidants: from chemical tobiochemical mechanisms[J].Food Chem Toxicol,1999.37(9–10):949-962.
32. Benzie IFF. Evolution of dietary antioxidants[J].Comparative Biochemistry andPhysiology Part A: Molecular&Integrative Physiology,2003.136(1):113-126.
33.韦金亮,黄忠.红景天提取物对训练小鼠肝SOD、MDA的影响实验研究[J].内江科技,2012(06):40.
34. Dhar P, Tayade A B, Bajpai P K, et al. Antioxidant capacities and total polyphenolcontents of hydro-ethanolic extract of phytococktail from trans-Himalaya[J]. JFood Sci,2012.77(2):156-161.
35. Bayliak M M and Lushchak V I. The golden root, Rhodiola rosea, prolongslifespan but decreases oxidative stress resistance in yeast Saccharomycescerevisiae[J]. Phytomedicine,2011.18(14):1262-1268.
36.程安玮,王文亮,陈相艳,等.红景天多酚的提取及抗氧化活性研究[J].山东农业科学,2012(09):115-117.
37. Tayade A B, Dhar P, Sharma M, et al. Antioxidant Capacities, Phenolic Contents,and GC/MS Analysis of Rhodiola imbricata Edgew. Root Extracts fromTrans-Himalaya[J]. J Food Sci,2013.78(3): C402-C410.
38.黎明华,汤长发,欧阳江琼.红景天苷对运动后自由基和能量代谢改变的影响[J].中国应用生理学杂志,2012(01):53-56.
39. Li X, Sipple J, Pang Q, et al. Salidroside stimulates DNA repair enzyme Parp-1activity in mouse HSC maintenance[J]. Blood,2012.119(18):4162-4173.
40. Yee S. In vitro permeability across Caco-2cells (colonic) can predict in vivo(small intestinal) absorption in man--fact or myth[J]. Pharm Res,1997.14(6):763-766.
41. Lee W C, Peng C C, Chang C H, et al. Extraction of Antioxidant Componentsfrom Bidens pilosa Flowers and Their Uptake by Human Intestinal Caco-2Cells[J]. Molecules,2013.18(2):1582-1601.
42. Abalos I S, Rodriguez Y I, Lozano V, et al. Transepithelial transport of biperidenhydrochloride in Caco-2cell monolayers[J]. Environ Toxicol Pharmacol,2012.34(2):223-227.
43. Li J, Volpe D A, Wang Y, et al. Use of transporter knockdown Caco-2cells toinvestigate the in vitro efflux of statin drugs[J]. Drug Metab Dispos,2011.39(7):1196-1202.
44. Hessel S, John A, Seidel A, et al. Multidrug resistance-associated proteins areinvolved in the transport of the glutathione conjugates of the ultimate carcinogenof benzo[a]pyrene in human Caco-2cells[J]. Arch Toxicol,2013.87(2):269-280.
45. Elsby R, Gillen M, Butters C, et al. The utility of in vitro data in making accuratepredictions of human P-glycoprotein-mediated drug-drug interactions: a casestudy for AZD5672[J]. Drug Metab Dispos,2011.39(2):275-282.
46. Hughes J and Crowe A. Inhibition of P-glycoprotein-mediated efflux of digoxinand its metabolites by macrolide antibiotics[J]. J Pharmacol Sci,2010.113(4):315-324.
47. Kamiyama E, Nakai D, Mikkaichi T, et al. Interaction of angiotensin II type1receptor blockers with P-gp substrates in Caco-2cells and hMDR1-expressingmembranes[J]. Life Sci,2010.86(1-2):52-58.
48. Hidalgo I J, Raub T J, and Borchardt R T. Characterization of the human coloncarcinoma cell line (Caco-2) as a model system for intestinal epithelialpermeability[J]. Gastroenterology,1989.96(3):736-749.
49.白宇,秦晶,王建新,等.芦丁在Caco-2细胞模型中吸收和外排机制的研究[J].中国医药工业杂志,2011(09):655-659.
50.李素云,李峥,李敬来,等.槲皮素及其糖苷衍生物在Caco-2单层细胞上的吸收特征[J].营养学报,2012(04):358-367.
51. George K, Durante M, Wu H, et al. Chromosome aberrations in the bloodlymphocytes of astronauts after space flight[J]. Radiation Research,2001.156(6):731-738.
52. Ushakov I B and Vasin M V. Radiation protectors within the radiation safetysystem for extended duration exploration missions[J]. Aviakosm Ekolog Med,
2011.45(3):3-12.
1. Joyeux M, Lobstein A, Anton R, et al. Comparative antilipoperoxidant,antinecrotic and scavenging properties of terpenes and biflavones from Ginkgoand some flavonoids[J]. Planta Med,1995.61(2):126-129.
2. Brand-Williams W, Cuvelier M E, and Berset C. Use of a free radical method toevaluate antioxidant activity[J].LWT-Food Science and Technology,1995.28(1):25-30.
3. Sánchez-Moreno C, Larrauri J A, and Saura-Calixto F. A procedure to measurethe antiradical efficiency of polyphenols[J].Journal of the Science of Food andAgriculture,1998.76(2):270-276.
4. De Beer D, Joubert E, Gelderblom W C A, et al. Antioxidant Activity of SouthAfrican Red and White Cultivar Wines: Free Radical Scavenging[J].J Agric FoodChem,2003.51(4):902-909.
5. Singh S and Singh R P. In Vitro Methods of Assay of Antioxidants: AnOverview[J]. Food Reviews International,2008.24(4):392-415.
6. Karadag A, Ozcelik B, and Saner S. Review of Methods to DetermineAntioxidant Capacities[J]. Food Analytical Methods,2009.2(1):41-60.
7. Fukumoto L R and Mazza G. Assessing Antioxidant and Prooxidant Activities ofPhenolic Compounds[J].J Agric Food Chem,2000.48(8):3597-3604.
8. Liu F, Ooi V E C, and Chang S T. Free radical scavenging activities of mushroompolysaccharide extracts[J]. Life Sci,1997.60(10):763-771.
9. Evans P and Halliwell B. Micronutrients: Oxidant/antioxidant status[J]. BritishJournal of Nutrition,2001.85(SUPPL.2): S67-S74.
10. Gilbert D L. Fifty years of radical ideas[J]. Annals of the New York Academy ofSciences,2000.899:1-14.
11.向志军,赵广荣,元英进,等.复方丹参的体外抗氧化活性研究[J].中草药,2006(02):211-213.
12. Paul T. Effect of a prolonged superoxide flux on transferrin and ferritin[J]. ArchBiochem Biophys,2000.382(2):253-261.
13. Imlay J A. Cellular defenses against superoxide and hydrogen peroxide[J]. AnnuRev Biochem,2008.77:755-776.
14. Pacher P, Beckman J S, and Liaudet L. Nitric oxide and peroxynitrite in healthand disease[J]. Physiol Rev,2007.87(1):315-424.
15. Halliwell B, Gutteridge J M, and Aruoma O I. The deoxyribose method: a simple"test-tube" assay for determination of rate constants for reactions of hydroxylradicals[J]. Anal Biochem,1987.165(1):215-219.
16. Halliwell B, Gutteridge J M C, and Aruoma O I. The deoxyribose method: Asimple 'test-tube' assay for determination of rate constants for reactions ofhydroxyl radicals[J]. Anal Biochem,1987.165(1):215-219.
17.陆峰,肖杰,林培英,等.褶合光谱法考察羟自由基清除剂对紫外照射致DNA变异的作用[J].光谱学与光谱分析,2004(01):82-84.
18. Cheeseman K H and Slater T F. An introduction to free radical biochemistry[J].Br Med Bull,1993.49(3):481-493.
19. Slater T F. Free-radical mechanisms in tissue injury[J]. Biochem J,1984.222(1):1-15.
20. Barbosa R M, Lourenco C F, Santos R M, et al., Chapter20In Vivo Real‐TimeMeasurement of Nitric Oxide in Anesthetized Rat Brain, in Methods inEnzymology, C. Enrique and P. Lester, Editors.2008, Academic Press. p.351-367.
21. Wang X, Tanus-Santos J E, Reiter C D, et al. Biological activity of nitric oxide inthe plasmatic compartment[J]. Proc Natl Acad Sci U.S.A.,2004.101(31):11477-11482.
22. Benzie I F and Strain J J. The ferric reducing ability of plasma (FRAP) as ameasure of "antioxidant power": the FRAP assay[J]. Anal Biochem,1996.239(1):70-76.
23. Prior R L, Wu X, and Schaich K. Standardized Methods for the Determination ofAntioxidant Capacity and Phenolics in Foods and Dietary Supplements[J]. JAgric Food Chem,2005.53(10):4290-4302.
24. Huang D, Ou B, and Prior R L. The Chemistry behind Antioxidant CapacityAssays[J].J Agric Food Chem,2005.53(6):1841-1856.
25. Ferreres F, Sousa C, Pereira D M, et al. Screening of Antioxidant PhenolicCompounds Produced by In Vitro Shoots of Brassica oleracea L. var. costataDC[J]. Comb Chem High Throughput Screen,2009.12(3):230-240.
26. Ljubuncic P, Portnaya I, Cogan U, et al. Antioxidant activity of Crataegus aroniaaqueous extract used in traditional Arab medicine in Israel[J]. J Ethnopharmacol,2005.101(1-3):153-161.
27. Fattman C L, Schaefer L M, and Oury T D. Extracellular superoxide dismutase inbiology and medicine[J]. Free Radic Biol Med,2003.35(3):236-256.
1. Ran X Z, Ran X, Zong Z W, et al. Protective effect of atorvastatin onradiation-induced vascular endothelial cell injury in vitro[J]. J Radiat Res,2010.51(5):527-533.
2. Ma Z C, Hong Q, Wang Y G, et al. Ferulic acid attenuates adhesion moleculeexpression in gamma-radiated human umbilical vascular endothelial cells[J]. BiolPharm Bull,2010.33(5):752-758.
3. Chen C, Chen Z, Xu F, et al. Radio-protective effect of catalpol in cultured cellsand mice[J]. J Radiat Res,2013.54(1):76-82.
4.程赢，黄越承，高福，等.富勒烯赖氨酸衍生物对AHH-1细胞防护效应研究[J].辐射研究与辐射工艺学报，2010.28(05):257-261.
5. Zheng H, Chen Z W, Wang L, et al. Radioprotection of4-hydroxy-3,5-dimethoxybenzaldehyde (VND3207) in culture cells is associatedwith minimizing DNA damage and activating Akt[J]. Eur J Pharm Sci,2008.33(1):52-59.
6. O'Toole S A, Sheppard B L, McGuinness E P, et al. The MTS assay as anindicator of chemosensitivity/resistance in malignant gynaecological tumours[J].Cancer Detect Prev,2003.27(1):47-54.
7.陈秀生，方铁兰，蔡瑞波，等.检测白血病细胞增殖力的MTS/pms比色分析法的建立[J].中国实验血液学杂志，2002(05):438-440.
8. Wang Z B, Liu Y Q, Zhang Y, et al. Reactive oxygen species, but notmitochondrial membrane potential, is associated with radiation-induced apoptosisof AHH-1human lymphoblastoid cells[J]. Cell Biol Int,2007.31(11):1353-1358.
9. Arora R, Singh S, Sagar R K, et al. Radiomodulatory and Free-RadicalScavenging Activity of the Fractionated Aquo-Alcoholic Extract of theAdaptogenic Nutraceutical (Rhodiola imbricata)-A Comparative In VitroAssessment with Ascorbate[J]. J Diet Suppl,2008.5(2):147-163.
1. Gao J, Hugger E D, Beck-Westermeyer M S, et al. Estimating intestinal mucosalpermeation of compounds using Caco-2cell monolayers[J]. Curr ProtocPharmacol,2001. Chapter7: Unit7.2.
2. Yang X W, Yang X D, Wang Y, et al. Establishment of Caco-2cell monolayermodel and standard operation procedure for assessing intestinal absorption ofchemical components of traditional Chinese medicine[J]. Zhong Xi Yi Jie HeXue Bao,2007.5(6):634-641.
3. Yee S. In Vitro Permeability Across Caco-2Cells (Colonic) Can Predict In Vivo(Small Intestinal) Absorption in Man—Fact or Myth[J]. Pharmaceutical Research,1997.14(6):763-766.
4. Lennern s H, Palm K, Fagerholm U, et al. Comparison between active andpassive drug transport in human intestinal epithelial (caco-2) cells in vitro andhuman jejunum in vivo[J]. International Journal of Pharmaceutics,1996.127(1):103-107.
5. Artursson P and Karlsson J. Correlation between oral drug absorption in humansand apparent drug permeability coefficients in human intestinal epithelial(Caco-2) cells[J]. Biochemical and Biophysical Research Communications,1991.175(3):880-885.
6. Foger F, Kopf A, Loretz B, et al. Correlation of in vitro and in vivo models forthe oral absorption of peptide drugs[J]. Amino Acids,2008.35(1):233-241.
1. Balimane P V, Han Y H, and Chong S. Current industrial practices of assessingpermeability and P-glycoprotein interaction[J]. AAPS J,2006.8(1): E1-E13.
2. Lennernas H. Intestinal permeability and its relevance for absorption andelimination[J]. Xenobiotica,2007.37(10-11):1015-1051.
3. Giacomini K M, Huang S M, Tweedie D J, et al. Membrane transporters in drugdevelopment[J]. Nature Reviews Drug Discovery,2010.9(3):215-236.
4. Washiyama M, Koizumi N, Fujii M, et al. Percolation analysis in electricalconductivity of madin-darby canine kidney and caco-2cells bypermeation-enhancing agents[J]. Biol Pharm Bull,2013.36(3):384-389.
5. Reis J M, Dezani A B, Pereira T M, et al. Lamivudine permeability study: Acomparison between PAMPA, ex vivo and in situ Single-Pass Intestinal Perfusion(SPIP) in rat jejunum[J]. Eur J Pharm Sci,2013.48(4-5):781-789.
6. Phuong H L, Qiu J, Kuwahara T, et al. Transepithelial transport of6-O-caffeoylsophorose across Caco-2cell monolayers[J]. Food Chem,2013.138(1):101-106.
7. Skolnik S, Lin X, Wang J, et al. Towards prediction of in vivo intestinalabsorption using a96-well Caco-2assay[J]. J Pharm Sci,2010.99(7):3246-3265.
8. Zhang B, Zhu X M, Hu J N, et al. Absorption mechanism of ginsenosidecompound K and its butyl and octyl ester prodrugs in Caco-2cells[J]. J AgricFood Chem,2012.60(41):10278-10284.
9. He Y, Liu Y, and Zeng S. Stereoselective and multiple carrier-mediated transportof cetirizine across Caco-2cell monolayers with potential drug interaction[J].Chirality,2010.22(7):684-692.
10. Paix o P, Gouveia L F, and Morais J A G. Prediction of the in vitro permeabilitydetermined in Caco-2cells by using artificial neural networks[J]. EuropeanJournal of Pharmaceutical Sciences,2010.41(1):107-117.
11. Yan Z, Sun J, Chang Y, et al. Bifunctional peptidomimetic prodrugs of didanosinefor improved intestinal permeability and enhanced acidic stability: synthesis,transepithelial transport, chemical stability and pharmacokinetics[J]. Mol Pharm,2011.8(2):319-329.
12. Marquez Ruiz J F, Kedziora K, Pigott M, et al. A nitrophenyl-based prodrug typefor colorectal targeting of prednisolone, budesonide and celecoxib[J]. BioorgMed Chem Lett,2013.23(6):1693-1698.
13. Tsume Y and Amidon G L. Selection of suitable prodrug candidates for in vivostudies via in vitro studies; the correlation of prodrug stability in between cellculture homogenates and human tissue homogenates[J]. J Pharm Pharm Sci,2012.15(3):433-446.
14. Bimbo L M, M kil E, Laaksonen T, et al. Drug permeation across intestinalepithelial cells using porous silicon nanoparticles[J]. Biomaterials,2011.32(10):2625-2633.
15. Parmentier J, Hartmann F J, and Fricker G. In vitro evaluation of liposomescontaining bio-enhancers for the oral delivery of macromolecules[J]. Eur J PharmBiopharm,2010.76(3):394-403.
16. Omkvist D H, Brodin B, and Nielsen C U. Ibuprofen is a non-competitiveinhibitor of the peptide transporter hPEPT1(SLC15A1): possible interactionsbetween hPEPT1substrates and ibuprofen[J]. Br J Pharmacol,2010.161(8):1793-1805.
17. Kowapradit J, Opanasopit P, Ngawhirunpat T, et al. In vitro permeabilityenhancement in intestinal epithelial cells (Caco-2) monolayer of water solublequaternary ammonium chitosan derivatives[J]. AAPS PharmSciTech,2010.11(2):497-508.
18. Gertz M, Harrison A, Houston J B, et al. Prediction of human intestinal first-passmetabolism of25CYP3A substrates from in vitro clearance and permeabilitydata[J]. Drug Metab Dispos,2010.38(7):1147-1158.
19. Yee S. In vitro permeability across Caco-2cells (colonic) can predict in vivo(small intestinal) absorption in man--fact or myth[J]. Pharm Res,1997.14(6):763-766.
20. Hidalgo I J, Raub T J, and Borchardt R T. Characterization of the human coloncarcinoma cell line (Caco-2) as a model system for intestinal epithelialpermeability[J]. Gastroenterology,1989.96(3):736-749.
21. Cao F, Gao Y, Wang M, et al. Propylene glycol-Linked Amino Acid/DipeptideDiester Prodrugs of Oleanolic Acid for PepT1-Mediated Transport: Synthesis,Intestinal Permeability and Pharmacokinetics[J]. Mol Pharm,2013.10(4):1378-1387.
22. Khomane K S, Nandekar P P, Wahlang B, et al. Mechanistic insights intoPEPT1-mediated transport of a novel antiepileptic, NP-647[J]. Mol Pharm,2012.9(9):2458-2468.
23. Neimark E, Chen F, Li X, et al. Bile acid-induced negative feedback regulation ofthe human ileal bile acid transporter[J]. Hepatology,2004.40(1):149-156.
24. Borthakur A, Priyamvada S, Kumar A, et al. A novel nutrient sensing mechanismunderlies substrate-induced regulation of monocarboxylate transporter-1[J]. Am JPhysiol Gastrointest Liver Physiol,2012.303(10): G1126-G1133.
25. Saksena S, Theegala S, Bansal N, et al. Mechanisms underlying modulation ofmonocarboxylate transporter1(MCT1) by somatostatin in human intestinalepithelial cells[J]. Am J Physiol Gastrointest Liver Physiol,2009.297(5):G878-G885.
26. Tamai I. Oral drug delivery utilizing intestinal OATP transporters[J]. Adv DrugDeliv Rev,2012.64(6):508-514.
27. Lemos C, Faria A, Meireles M, et al. Thiamine is a substrate of organic cationtransporters in Caco-2cells[J]. Eur J Pharmacol,2012.682(1-3):37-42.
28. Zhang D, He K, Herbst J J, et al. Characterization of Efflux TransportersInvolved in Distribution and Disposition of Apixaban[J]. Drug Metab Dispos,2013.41(4):827-835.
29. Wright J A, Haslam I S, Coleman T, et al. Breast cancer resistance protein BCRP(ABCG2)-mediated transepithelial nitrofurantoin secretion and its regulation inhuman intestinal epithelial (Caco-2) layers[J]. Eur J Pharmacol,2011.672(1-3):70-76.
30. Haslam I S, Wright J A, O'Reilly D A, et al. Intestinal ciprofloxacin efflux: therole of breast cancer resistance protein (ABCG2)[J]. Drug Metab Dispos,2011.39(12):2321-2328.
31. Sukhaphirom N, Vardhanabhuti N, Chirdchupunseree H, et al. Phyllanthin andhypophyllanthin inhibit function of P-gp but not MRP2in Caco-2cells[J]. JPharm Pharmacol,2013.65(2):292-299.
32. Hilgendorf C, Ahlin G, Seithel A, et al. Expression of thirty-six drug transportergenes in human intestine, liver, kidney, and organotypic cell lines[J]. Drug MetabDispos,2007.35(8):1333-1340.
33. Volpe D A, Faustino P J, Ciavarella A B, et al. Classification of drug permeabilitywith a Caco-2cell monolayer assay[J]. Clinical Research and Regulatory Affairs,2007.24(1):39-47.
34. Artursson P and Karlsson J. Correlation between oral drug absorption in humansand apparent drug permeability coefficients in human intestinal epithelial(Caco-2) cells[J]. Biochemical and Biophysical Research Communications,1991.175(3):880-885.
35. Volpe D A. Permeability classification of representative fluoroquinolones by acell culture method[J]. AAPS J,2004.6(2):1-6.
36. Lee W C, Peng C C, Chang C H, et al. Extraction of Antioxidant Componentsfrom Bidens pilosa Flowers and Their Uptake by Human Intestinal Caco-2Cells[J]. Molecules,2013.18(2):1582-1601.
37. Abalos I S, Rodriguez Y I, Lozano V, et al. Transepithelial transport of biperidenhydrochloride in Caco-2cell monolayers[J]. Environ Toxicol Pharmacol,2012.34(2):223-227.
38. Guan M, Zhu Q L, Liu Y, et al. Uptake and transport of a novel anticancerdrug-delivery system: lactosyl-norcantharidin-associated N-trimethyl chitosannanoparticles across intestinal Caco-2cell monolayers[J]. Int J Nanomedicine,2012.7:1921-1930.
39. Segawa M, Ogura J, Seki S, et al. Rapid stimulating effect of the antiarrhythmicagent amiodarone on absorption of organic anion compounds[J]. Drug MetabPharmacokinet,2012Sep18.[Epub ahead of print]
40. Togami K, Tosaki Y, Chono S, et al. Enantioselective uptake of fexofenadine byCaco-2cells as model intestinal epithelial cells[J]. J Pharm Pharmacol,2013.65(1):22-29.
41.李晓天,王素军,王广基. HPLC-MS法测定关附甲素及其在Caco-2细胞上的摄取特性[J].中国药理学通报,2013.29(02):286-289.
42. Kis O, Zastre J A, Ramaswamy M, et al. pH dependence of organicanion-transporting polypeptide2B1in Caco-2cells: potential role inantiretroviral drug oral bioavailability and drug-drug interactions[J]. J PharmacolExp Ther,2010.334(3):1009-1022.
43. Annaert P, Ye Z W, Stieger B, et al. Interaction of HIV protease inhibitors withOATP1B1,1B3and2B1[J]. Xenobiotica,2010.40(3):163-176.
44. Elsby R, Surry D D, Smith V N, et al. Validation and application of Caco-2assays for the in vitro evaluation of development candidate drugs as substrates orinhibitors of P-glycoprotein to support regulatory submissions[J]. Xenobiotica,2008.38(7-8):1140-1164.
45. Oga E F, Sekine S, Shitara Y, et al. P-glycoprotein mediated efflux in Caco-2cellmonolayers: the influence of herbals on digoxin transport[J]. J Ethnopharmacol,2012.144(3):612-617.
46. Mease K, Sane R, Podila L, et al. Differential selectivity of efflux transporterinhibitors in Caco-2and MDCK-MDR1monolayers: a strategy to assess theinteraction of a new chemical entity with P-gp, BCRP, and MRP2[J]. J Pharm Sci,2012.101(5):1888-1897.
47. Lin X, Skolnik S, Chen X, et al. Attenuation of intestinal absorption by majorefflux transporters: quantitative tools and strategies using a Caco-2model[J].Drug Metab Dispos,2011.39(2):265-274.
48. Lepist E I, Phan T K, Roy A, et al. Cobicistat boosts the intestinal absorption oftransport substrates, including HIV protease inhibitors and GS-7340, in vitro[J].Antimicrob Agents Chemother,2012.56(10):5409-5413.
49. Ye L, Yang X, Yang Z, et al. The role of efflux transporters on the transport ofhighly toxic aconitine, mesaconitine, hypaconitine, and their hydrolysates, asdetermined in cultured Caco-2and transfected MDCKII cells[J]. Toxicol Lett,2013.216(2-3):86-99.
50. Siissalo S, Hannukainen J, Kolehmainen J, et al. A Caco-2cell based screeningmethod for compounds interacting with MRP2efflux protein[J]. Eur J PharmBiopharm,2009.71(2):332-338.
51. Zhang W, Li J, Allen S M, et al. Silencing the breast cancer resistance proteinexpression and function in caco-2cells using lentiviral vector-based short hairpinRNA[J]. Drug Metab Dispos,2009.37(4):737-744.
52. Pratt J, Venkatraman N, Brinker A, et al. Use of zinc finger nuclease technologyto knock out efflux transporters in C2BBe1cells[J]. Curr Protoc Toxicol,2012.Chapter23: Unit23.2.
53. Graber-Maier A, Gutmann H, and Drewe J. A new intestinal cell culture model todiscriminate the relative contribution of P-gp and BCRP on transport ofsubstrates such as imatinib[J]. Mol Pharm,2010.7(5):1618-1628.
54. Li J, Volpe D A, Wang Y, et al. Use of transporter knockdown Caco-2cells toinvestigate the in vitro efflux of statin drugs[J]. Drug Metab Dispos,2011.39(7):1196-1202.
55. Hessel S, John A, Seidel A, et al. Multidrug resistance-associated proteins areinvolved in the transport of the glutathione conjugates of the ultimate carcinogenof benzo[a]pyrene in human Caco-2cells[J]. Arch Toxicol,2013.87(2):269-280.
56. Yeh T H, Hsu L W, Tseng M T, et al. Mechanism and consequence ofchitosan-mediated reversible epithelial tight junction opening[J]. Biomaterials,2011.32(26):6164-6173.
57. Zhu S, Huang R, Hong M, et al. Effects of polyoxyethylene (40) stearate on theactivity of P-glycoprotein and cytochrome P450[J]. European Journal ofPharmaceutical Sciences,2009.37(5):573-580.
58. Kapitza S B, Michel B R, van Hoogevest P, et al. Absorption of poorly watersoluble drugs subject to apical efflux using phospholipids as solubilizers in theCaco-2cell model[J]. European Journal of Pharmaceutics and Biopharmaceutics,2007.66(1):146-158.
59. Tsume Y, Vig B, Sun J, et al. Enhanced Absorption and Growth Inhibition withAmino Acid Monoester Prodrugs of Floxuridine by Targeting hPEPT1Transporters[J]. Molecules,2008.13(7):1441-1454.
60. Fenner K S, Troutman M D, Kempshall S, et al. Drug-Drug InteractionsMediated Through P-Glycoprotein: Clinical Relevance and In Vitro-In VivoCorrelation Using Digoxin as a Probe Drug[J]. Clin Pharmacol Ther,2008.85(2):173-181.
61. Zheng X and Polli J E. Synthesis and in vitro evaluation of potential sustainedrelease prodrugs via targeting ASBT[J]. International Journal of Pharmaceutics,2010.396(1–2):111-118.
62. Cook J A, Feng B, Fenner K S, et al. Refining the in vitro and in vivo criticalparameters for p-glycoprotein,[I]/IC50and [I2]/IC50, that allow for theexclusion of drug candidates from clinical digoxin interaction studies[J]. MolPharm,2010.7(2):398-411.
63. Collett A, Tanianis-Hughes J, Carlson G L, et al. Comparison ofP-glycoprotein-mediated drug–digoxin interactions in Caco-2with human androdent intestine: Relevance to in vivo prediction[J]. European Journal ofPharmaceutical Sciences,2005.26(5):386-393.
64. Elsby R, Gillen M, Butters C, et al. The utility of in vitro data in making accuratepredictions of human P-glycoprotein-mediated drug-drug interactions: a casestudy for AZD5672[J]. Drug Metab Dispos,2011.39(2):275-282.
65. Hughes J and Crowe A. Inhibition of P-glycoprotein-mediated efflux of digoxinand its metabolites by macrolide antibiotics[J]. J Pharmacol Sci,2010.113(4):315-324.
66. Kamiyama E, Nakai D, Mikkaichi T, et al. Interaction of angiotensin II type1receptor blockers with P-gp substrates in Caco-2cells and hMDR1-expressingmembranes[J]. Life Sci,2010.86(1-2):52-58.
67. Castillo-Garit J A, Marrero-Ponce Y, Torrens F, et al. Estimation of ADMEproperties in drug discovery: Predicting Caco-2cell permeability usingatom-based stochastic and non-stochastic linear indices[J]. J Pharm Sci,2008.97(5):1946-1976.
68. Lai Y, Sampson K E, and Stevens J C. Evaluation of drug transporter interactionsin drug discovery and development[J]. Combinatorial Chemistry and HighThroughput Screening,2010.13(2):112-134.
2.国家药典委员会.中国药典一部,化学工业出版社,2010,第一版:144.
3. Ali Z, Fronczek F R, and Khan I A. Phenylalkanoids and monoterpene analoguesfrom the roots of Rhodiola rosea[J]. Planta Med,2008.74(2):178-181.
4. Ma G, Li W, Dou D, et al. Rhodiolosides A-E, monoterpene glycosides fromRhodiola rosea[J]. Chem Pharm Bull (Tokyo),2006.54(8):1229-1233.
5. Rohloff J. Volatiles from rhizomes of Rhodiola rosea L[J]. Phytochemistry,2002.59(6):655-661.
6. Panossian A, Gabrielian E, and Wagner H. On the mechanism of action of plantadaptogens with particular reference to cucurbitacin R diglucoside[J].Phytomedicine,1999.6(3):147-155.
7. Panossian A G, Oganessian A S, Ambartsumian M, et al. Effects of heavyphysical exercise and adaptogens on nitric oxide content in human saliva[J].Phytomedicine,1999.6(1):17-26.
8. Panossian A and Wikman G. Evidence-Based Efficacy of Adaptogens in Fatigue,and Molecular Mechanisms Related to their Stress-Protective Activity[J]. CurrentClinical Pharmacology,2009.4(3):198-219.
9. Perfumi M and Mattioli L. Adaptogenic and central nervous system effects ofsingle doses of3%rosavin and1%salidroside Rhodiola rosea L. extract inmice[J]. Phytotherapy Research,2007.21(1):37-43.
10. Zhang Y and Liu Y. Study on effects of salidroside on lipid peroxidation onoxidative stress in rat hepatic stellate cells[J]. Zhong Yao Cai,2005.28(9):794-796.
11. Parisi A, Tranchita E, Duranti G, et al. Effects of chronic Rhodiola Roseasupplementation on sport performance and antioxidant capacity in trained male:preliminary results[J]. J Sports Med Phys Fitness,2010.50(1):57-63.
12. Olsson E M, von Scheele B, and Panossian A G. A randomised, double-blind,placebo-controlled, parallel-group study of the standardised extract shr-5of theroots of Rhodiola rosea in the treatment of subjects with stress-related fatigue[J].Planta Med,2009.75(2):105-112.
13.李岩,吕晓华.红景天胶囊对小鼠抗疲劳的效应[J].川北医学院学报,2012(06):531-533.
14. Wang H, Ding Y, Zhou J, et al. The in vitro and in vivo antiviral effects ofsalidroside from Rhodiola rosea L. against coxsackievirus B3[J]. Phytomedicine,2009.16(2-3):146-155.
15. Zhang J K, Yang L, Meng G L, et al. Protection by Salidroside against Bone Lossvia Inhibition of Oxidative Stress and Bone-Resorbing Mediators[J]. PLoS One,2013.8(2): e57251.
16.曾满红,黄清松,张德兴.红景天总黄酮对自然衰老大鼠抗氧化和免疫功能的影响[J].解剖学研究,2012(02):135-137.
17. Wojcik R, Siwicki A K, Skopinska-Rozewska E, et al. The effect of Chinesemedicinal herb Rhodiola kirilowii extracts on cellular immunity in mice andrats[J]. Pol J Vet Sci,2009.12(3):399-405.
18. Skopnska-Rozewska E, Wojcik R, Siwicki A K, et al. The effect of Rhodiolaquadrifida extracts on cellular immunity in mice and rats[J]. Pol J Vet Sci,2008.11(2):105-111.
19. Zhong H, Xin H, Wu L X, et al. Salidroside attenuates apoptosis in ischemiccardiomyocytes: a mechanism through a mitochondria-dependent pathway[J]. JPharmacol Sci,2010.114(4):399-408.
20. Zhang J, Liu A, Hou R, et al. Salidroside protects cardiomyocyte againsthypoxia-induced death: A HIF-1α-activated and VEGF-mediated pathway[J].European Journal of Pharmacology,2009.607(1–3):6-14.
21. Arora R, Singh S, Sagar R K, et al. Radiomodulatory and Free-RadicalScavenging Activity of the Fractionated Aquo-Alcoholic Extract of theAdaptogenic Nutraceutical (Rhodiola imbricata)-A Comparative In VitroAssessment with Ascorbate[J]. J Diet Suppl,2008.5(2):147-163.
22.张茗昱,张建,李雪,等.长白山红景天有效成分对X射线辐射防护作用的研究[J].中国医药指南,2012(20)：88-90.
23. Pooja, Bawa A S, and Khanum F. Anti-inflammatory activity of Rhodiola rosea–―a second-generation adaptogen‖[J]. Phytotherapy Research,2009.23(8):1099-1102.
24. Wu X and Prior R L. Identification and Characterization of Anthocyanins byHigh-Performance Liquid Chromatography Electrospray Ionization TandemMass Spectrometry in Common Foods in the United States: Vegetables, Nuts, andGrains[J].J Agric Food Chem,2005.53(8):3101-3113.
25. Wolfe K L and Liu R H. Cellular Antioxidant Activity (CAA) Assay forAssessing Antioxidants, Foods, and Dietary Supplements[J].J Agric Food Chem,2007.55(22):8896-8907.
26. Adom K K and Liu R H. Rapid Peroxyl Radical Scavenging Capacity (PSC)Assay for Assessing both Hydrophilic and Lipophilic Antioxidants[J].J AgricFood Chem,2005.53(17):6572-6580.
27. Moore J, Yin J-J, and Yu L. Novel Fluorometric Assay for Hydroxyl RadicalScavenging Capacity (HOSC) Estimation[J].J Agric Food Chem,2006.54(3):617-626.
28. Huang D, Ou B, and Prior R L. The Chemistry behind Antioxidant CapacityAssays[J].J Agric Food Chem,2005.53(6):1841-1856.
29. MacDonald-Wicks L K, Wood L G, and Garg M L. Methodology for thedetermination of biological antioxidant capacity in vitro: a review[J].J SciFoodAgric,2006.86(13):2046-2056.
30. Ou B, Huang D, Hampsch-Woodill M, et al. Analysis of Antioxidant Activities ofCommon Vegetables Employing Oxygen Radical Absorbance Capacity (ORAC)and Ferric Reducing Antioxidant Power (FRAP) Assays: A ComparativeStudy[J].J Agric Food Chem,2002.50(11):3122-3128.
31. Chaudière J and Ferrari-Iliou R. Intracellular Antioxidants: from chemical tobiochemical mechanisms[J].Food Chem Toxicol,1999.37(9–10):949-962.
32. Benzie IFF. Evolution of dietary antioxidants[J].Comparative Biochemistry andPhysiology Part A: Molecular&Integrative Physiology,2003.136(1):113-126.
33.韦金亮,黄忠.红景天提取物对训练小鼠肝SOD、MDA的影响实验研究[J].内江科技,2012(06):40.
34. Dhar P, Tayade A B, Bajpai P K, et al. Antioxidant capacities and total polyphenolcontents of hydro-ethanolic extract of phytococktail from trans-Himalaya[J]. JFood Sci,2012.77(2):156-161.
35. Bayliak M M and Lushchak V I. The golden root, Rhodiola rosea, prolongslifespan but decreases oxidative stress resistance in yeast Saccharomycescerevisiae[J]. Phytomedicine,2011.18(14):1262-1268.
36.程安玮,王文亮,陈相艳,等.红景天多酚的提取及抗氧化活性研究[J].山东农业科学,2012(09):115-117.
37. Tayade A B, Dhar P, Sharma M, et al. Antioxidant Capacities, Phenolic Contents,and GC/MS Analysis of Rhodiola imbricata Edgew. Root Extracts fromTrans-Himalaya[J]. J Food Sci,2013.78(3): C402-C410.
38.黎明华,汤长发,欧阳江琼.红景天苷对运动后自由基和能量代谢改变的影响[J].中国应用生理学杂志,2012(01):53-56.
39. Li X, Sipple J, Pang Q, et al. Salidroside stimulates DNA repair enzyme Parp-1activity in mouse HSC maintenance[J]. Blood,2012.119(18):4162-4173.
40. Yee S. In vitro permeability across Caco-2cells (colonic) can predict in vivo(small intestinal) absorption in man--fact or myth[J]. Pharm Res,1997.14(6):763-766.
41. Lee W C, Peng C C, Chang C H, et al. Extraction of Antioxidant Componentsfrom Bidens pilosa Flowers and Their Uptake by Human Intestinal Caco-2Cells[J]. Molecules,2013.18(2):1582-1601.
42. Abalos I S, Rodriguez Y I, Lozano V, et al. Transepithelial transport of biperidenhydrochloride in Caco-2cell monolayers[J]. Environ Toxicol Pharmacol,2012.34(2):223-227.
43. Li J, Volpe D A, Wang Y, et al. Use of transporter knockdown Caco-2cells toinvestigate the in vitro efflux of statin drugs[J]. Drug Metab Dispos,2011.39(7):1196-1202.
44. Hessel S, John A, Seidel A, et al. Multidrug resistance-associated proteins areinvolved in the transport of the glutathione conjugates of the ultimate carcinogenof benzo[a]pyrene in human Caco-2cells[J]. Arch Toxicol,2013.87(2):269-280.
45. Elsby R, Gillen M, Butters C, et al. The utility of in vitro data in making accuratepredictions of human P-glycoprotein-mediated drug-drug interactions: a casestudy for AZD5672[J]. Drug Metab Dispos,2011.39(2):275-282.
46. Hughes J and Crowe A. Inhibition of P-glycoprotein-mediated efflux of digoxinand its metabolites by macrolide antibiotics[J]. J Pharmacol Sci,2010.113(4):315-324.
47. Kamiyama E, Nakai D, Mikkaichi T, et al. Interaction of angiotensin II type1receptor blockers with P-gp substrates in Caco-2cells and hMDR1-expressingmembranes[J]. Life Sci,2010.86(1-2):52-58.
48. Hidalgo I J, Raub T J, and Borchardt R T. Characterization of the human coloncarcinoma cell line (Caco-2) as a model system for intestinal epithelialpermeability[J]. Gastroenterology,1989.96(3):736-749.
49.白宇,秦晶,王建新,等.芦丁在Caco-2细胞模型中吸收和外排机制的研究[J].中国医药工业杂志,2011(09):655-659.
50.李素云,李峥,李敬来,等.槲皮素及其糖苷衍生物在Caco-2单层细胞上的吸收特征[J].营养学报,2012(04):358-367.
51. George K, Durante M, Wu H, et al. Chromosome aberrations in the bloodlymphocytes of astronauts after space flight[J]. Radiation Research,2001.156(6):731-738.
52. Ushakov I B and Vasin M V. Radiation protectors within the radiation safetysystem for extended duration exploration missions[J]. Aviakosm Ekolog Med,
2011.45(3):3-12.
1. Joyeux M, Lobstein A, Anton R, et al. Comparative antilipoperoxidant,antinecrotic and scavenging properties of terpenes and biflavones from Ginkgoand some flavonoids[J]. Planta Med,1995.61(2):126-129.
2. Brand-Williams W, Cuvelier M E, and Berset C. Use of a free radical method toevaluate antioxidant activity[J].LWT-Food Science and Technology,1995.28(1):25-30.
3. Sánchez-Moreno C, Larrauri J A, and Saura-Calixto F. A procedure to measurethe antiradical efficiency of polyphenols[J].Journal of the Science of Food andAgriculture,1998.76(2):270-276.
4. De Beer D, Joubert E, Gelderblom W C A, et al. Antioxidant Activity of SouthAfrican Red and White Cultivar Wines: Free Radical Scavenging[J].J Agric FoodChem,2003.51(4):902-909.
5. Singh S and Singh R P. In Vitro Methods of Assay of Antioxidants: AnOverview[J]. Food Reviews International,2008.24(4):392-415.
6. Karadag A, Ozcelik B, and Saner S. Review of Methods to DetermineAntioxidant Capacities[J]. Food Analytical Methods,2009.2(1):41-60.
7. Fukumoto L R and Mazza G. Assessing Antioxidant and Prooxidant Activities ofPhenolic Compounds[J].J Agric Food Chem,2000.48(8):3597-3604.
8. Liu F, Ooi V E C, and Chang S T. Free radical scavenging activities of mushroompolysaccharide extracts[J]. Life Sci,1997.60(10):763-771.
9. Evans P and Halliwell B. Micronutrients: Oxidant/antioxidant status[J]. BritishJournal of Nutrition,2001.85(SUPPL.2): S67-S74.
10. Gilbert D L. Fifty years of radical ideas[J]. Annals of the New York Academy ofSciences,2000.899:1-14.
11.向志军,赵广荣,元英进,等.复方丹参的体外抗氧化活性研究[J].中草药,2006(02):211-213.
12. Paul T. Effect of a prolonged superoxide flux on transferrin and ferritin[J]. ArchBiochem Biophys,2000.382(2):253-261.
13. Imlay J A. Cellular defenses against superoxide and hydrogen peroxide[J]. AnnuRev Biochem,2008.77:755-776.
14. Pacher P, Beckman J S, and Liaudet L. Nitric oxide and peroxynitrite in healthand disease[J]. Physiol Rev,2007.87(1):315-424.
15. Halliwell B, Gutteridge J M, and Aruoma O I. The deoxyribose method: a simple"test-tube" assay for determination of rate constants for reactions of hydroxylradicals[J]. Anal Biochem,1987.165(1):215-219.
16. Halliwell B, Gutteridge J M C, and Aruoma O I. The deoxyribose method: Asimple 'test-tube' assay for determination of rate constants for reactions ofhydroxyl radicals[J]. Anal Biochem,1987.165(1):215-219.
17.陆峰,肖杰,林培英,等.褶合光谱法考察羟自由基清除剂对紫外照射致DNA变异的作用[J].光谱学与光谱分析,2004(01):82-84.
18. Cheeseman K H and Slater T F. An introduction to free radical biochemistry[J].Br Med Bull,1993.49(3):481-493.
19. Slater T F. Free-radical mechanisms in tissue injury[J]. Biochem J,1984.222(1):1-15.
20. Barbosa R M, Lourenco C F, Santos R M, et al., Chapter20In Vivo Real‐TimeMeasurement of Nitric Oxide in Anesthetized Rat Brain, in Methods inEnzymology, C. Enrique and P. Lester, Editors.2008, Academic Press. p.351-367.
21. Wang X, Tanus-Santos J E, Reiter C D, et al. Biological activity of nitric oxide inthe plasmatic compartment[J]. Proc Natl Acad Sci U.S.A.,2004.101(31):11477-11482.
22. Benzie I F and Strain J J. The ferric reducing ability of plasma (FRAP) as ameasure of "antioxidant power": the FRAP assay[J]. Anal Biochem,1996.239(1):70-76.
23. Prior R L, Wu X, and Schaich K. Standardized Methods for the Determination ofAntioxidant Capacity and Phenolics in Foods and Dietary Supplements[J]. JAgric Food Chem,2005.53(10):4290-4302.
24. Huang D, Ou B, and Prior R L. The Chemistry behind Antioxidant CapacityAssays[J].J Agric Food Chem,2005.53(6):1841-1856.
25. Ferreres F, Sousa C, Pereira D M, et al. Screening of Antioxidant PhenolicCompounds Produced by In Vitro Shoots of Brassica oleracea L. var. costataDC[J]. Comb Chem High Throughput Screen,2009.12(3):230-240.
26. Ljubuncic P, Portnaya I, Cogan U, et al. Antioxidant activity of Crataegus aroniaaqueous extract used in traditional Arab medicine in Israel[J]. J Ethnopharmacol,2005.101(1-3):153-161.
27. Fattman C L, Schaefer L M, and Oury T D. Extracellular superoxide dismutase inbiology and medicine[J]. Free Radic Biol Med,2003.35(3):236-256.
1. Ran X Z, Ran X, Zong Z W, et al. Protective effect of atorvastatin onradiation-induced vascular endothelial cell injury in vitro[J]. J Radiat Res,2010.51(5):527-533.
2. Ma Z C, Hong Q, Wang Y G, et al. Ferulic acid attenuates adhesion moleculeexpression in gamma-radiated human umbilical vascular endothelial cells[J]. BiolPharm Bull,2010.33(5):752-758.
3. Chen C, Chen Z, Xu F, et al. Radio-protective effect of catalpol in cultured cellsand mice[J]. J Radiat Res,2013.54(1):76-82.
4.程赢，黄越承，高福，等.富勒烯赖氨酸衍生物对AHH-1细胞防护效应研究[J].辐射研究与辐射工艺学报，2010.28(05):257-261.
5. Zheng H, Chen Z W, Wang L, et al. Radioprotection of4-hydroxy-3,5-dimethoxybenzaldehyde (VND3207) in culture cells is associatedwith minimizing DNA damage and activating Akt[J]. Eur J Pharm Sci,2008.33(1):52-59.
6. O'Toole S A, Sheppard B L, McGuinness E P, et al. The MTS assay as anindicator of chemosensitivity/resistance in malignant gynaecological tumours[J].Cancer Detect Prev,2003.27(1):47-54.
7.陈秀生，方铁兰，蔡瑞波，等.检测白血病细胞增殖力的MTS/pms比色分析法的建立[J].中国实验血液学杂志，2002(05):438-440.
8. Wang Z B, Liu Y Q, Zhang Y, et al. Reactive oxygen species, but notmitochondrial membrane potential, is associated with radiation-induced apoptosisof AHH-1human lymphoblastoid cells[J]. Cell Biol Int,2007.31(11):1353-1358.
9. Arora R, Singh S, Sagar R K, et al. Radiomodulatory and Free-RadicalScavenging Activity of the Fractionated Aquo-Alcoholic Extract of theAdaptogenic Nutraceutical (Rhodiola imbricata)-A Comparative In VitroAssessment with Ascorbate[J]. J Diet Suppl,2008.5(2):147-163.
1. Gao J, Hugger E D, Beck-Westermeyer M S, et al. Estimating intestinal mucosalpermeation of compounds using Caco-2cell monolayers[J]. Curr ProtocPharmacol,2001. Chapter7: Unit7.2.
2. Yang X W, Yang X D, Wang Y, et al. Establishment of Caco-2cell monolayermodel and standard operation procedure for assessing intestinal absorption ofchemical components of traditional Chinese medicine[J]. Zhong Xi Yi Jie HeXue Bao,2007.5(6):634-641.
3. Yee S. In Vitro Permeability Across Caco-2Cells (Colonic) Can Predict In Vivo(Small Intestinal) Absorption in Man—Fact or Myth[J]. Pharmaceutical Research,1997.14(6):763-766.
4. Lennern s H, Palm K, Fagerholm U, et al. Comparison between active andpassive drug transport in human intestinal epithelial (caco-2) cells in vitro andhuman jejunum in vivo[J]. International Journal of Pharmaceutics,1996.127(1):103-107.
5. Artursson P and Karlsson J. Correlation between oral drug absorption in humansand apparent drug permeability coefficients in human intestinal epithelial(Caco-2) cells[J]. Biochemical and Biophysical Research Communications,1991.175(3):880-885.
6. Foger F, Kopf A, Loretz B, et al. Correlation of in vitro and in vivo models forthe oral absorption of peptide drugs[J]. Amino Acids,2008.35(1):233-241.
1. Balimane P V, Han Y H, and Chong S. Current industrial practices of assessingpermeability and P-glycoprotein interaction[J]. AAPS J,2006.8(1): E1-E13.
2. Lennernas H. Intestinal permeability and its relevance for absorption andelimination[J]. Xenobiotica,2007.37(10-11):1015-1051.
3. Giacomini K M, Huang S M, Tweedie D J, et al. Membrane transporters in drugdevelopment[J]. Nature Reviews Drug Discovery,2010.9(3):215-236.
4. Washiyama M, Koizumi N, Fujii M, et al. Percolation analysis in electricalconductivity of madin-darby canine kidney and caco-2cells bypermeation-enhancing agents[J]. Biol Pharm Bull,2013.36(3):384-389.
5. Reis J M, Dezani A B, Pereira T M, et al. Lamivudine permeability study: Acomparison between PAMPA, ex vivo and in situ Single-Pass Intestinal Perfusion(SPIP) in rat jejunum[J]. Eur J Pharm Sci,2013.48(4-5):781-789.
6. Phuong H L, Qiu J, Kuwahara T, et al. Transepithelial transport of6-O-caffeoylsophorose across Caco-2cell monolayers[J]. Food Chem,2013.138(1):101-106.
7. Skolnik S, Lin X, Wang J, et al. Towards prediction of in vivo intestinalabsorption using a96-well Caco-2assay[J]. J Pharm Sci,2010.99(7):3246-3265.
8. Zhang B, Zhu X M, Hu J N, et al. Absorption mechanism of ginsenosidecompound K and its butyl and octyl ester prodrugs in Caco-2cells[J]. J AgricFood Chem,2012.60(41):10278-10284.
9. He Y, Liu Y, and Zeng S. Stereoselective and multiple carrier-mediated transportof cetirizine across Caco-2cell monolayers with potential drug interaction[J].Chirality,2010.22(7):684-692.
10. Paix o P, Gouveia L F, and Morais J A G. Prediction of the in vitro permeabilitydetermined in Caco-2cells by using artificial neural networks[J]. EuropeanJournal of Pharmaceutical Sciences,2010.41(1):107-117.
11. Yan Z, Sun J, Chang Y, et al. Bifunctional peptidomimetic prodrugs of didanosinefor improved intestinal permeability and enhanced acidic stability: synthesis,transepithelial transport, chemical stability and pharmacokinetics[J]. Mol Pharm,2011.8(2):319-329.
12. Marquez Ruiz J F, Kedziora K, Pigott M, et al. A nitrophenyl-based prodrug typefor colorectal targeting of prednisolone, budesonide and celecoxib[J]. BioorgMed Chem Lett,2013.23(6):1693-1698.
13. Tsume Y and Amidon G L. Selection of suitable prodrug candidates for in vivostudies via in vitro studies; the correlation of prodrug stability in between cellculture homogenates and human tissue homogenates[J]. J Pharm Pharm Sci,2012.15(3):433-446.
14. Bimbo L M, M kil E, Laaksonen T, et al. Drug permeation across intestinalepithelial cells using porous silicon nanoparticles[J]. Biomaterials,2011.32(10):2625-2633.
15. Parmentier J, Hartmann F J, and Fricker G. In vitro evaluation of liposomescontaining bio-enhancers for the oral delivery of macromolecules[J]. Eur J PharmBiopharm,2010.76(3):394-403.
16. Omkvist D H, Brodin B, and Nielsen C U. Ibuprofen is a non-competitiveinhibitor of the peptide transporter hPEPT1(SLC15A1): possible interactionsbetween hPEPT1substrates and ibuprofen[J]. Br J Pharmacol,2010.161(8):1793-1805.
17. Kowapradit J, Opanasopit P, Ngawhirunpat T, et al. In vitro permeabilityenhancement in intestinal epithelial cells (Caco-2) monolayer of water solublequaternary ammonium chitosan derivatives[J]. AAPS PharmSciTech,2010.11(2):497-508.
18. Gertz M, Harrison A, Houston J B, et al. Prediction of human intestinal first-passmetabolism of25CYP3A substrates from in vitro clearance and permeabilitydata[J]. Drug Metab Dispos,2010.38(7):1147-1158.
19. Yee S. In vitro permeability across Caco-2cells (colonic) can predict in vivo(small intestinal) absorption in man--fact or myth[J]. Pharm Res,1997.14(6):763-766.
20. Hidalgo I J, Raub T J, and Borchardt R T. Characterization of the human coloncarcinoma cell line (Caco-2) as a model system for intestinal epithelialpermeability[J]. Gastroenterology,1989.96(3):736-749.
21. Cao F, Gao Y, Wang M, et al. Propylene glycol-Linked Amino Acid/DipeptideDiester Prodrugs of Oleanolic Acid for PepT1-Mediated Transport: Synthesis,Intestinal Permeability and Pharmacokinetics[J]. Mol Pharm,2013.10(4):1378-1387.
22. Khomane K S, Nandekar P P, Wahlang B, et al. Mechanistic insights intoPEPT1-mediated transport of a novel antiepileptic, NP-647[J]. Mol Pharm,2012.9(9):2458-2468.
23. Neimark E, Chen F, Li X, et al. Bile acid-induced negative feedback regulation ofthe human ileal bile acid transporter[J]. Hepatology,2004.40(1):149-156.
24. Borthakur A, Priyamvada S, Kumar A, et al. A novel nutrient sensing mechanismunderlies substrate-induced regulation of monocarboxylate transporter-1[J]. Am JPhysiol Gastrointest Liver Physiol,2012.303(10): G1126-G1133.
25. Saksena S, Theegala S, Bansal N, et al. Mechanisms underlying modulation ofmonocarboxylate transporter1(MCT1) by somatostatin in human intestinalepithelial cells[J]. Am J Physiol Gastrointest Liver Physiol,2009.297(5):G878-G885.
26. Tamai I. Oral drug delivery utilizing intestinal OATP transporters[J]. Adv DrugDeliv Rev,2012.64(6):508-514.
27. Lemos C, Faria A, Meireles M, et al. Thiamine is a substrate of organic cationtransporters in Caco-2cells[J]. Eur J Pharmacol,2012.682(1-3):37-42.
28. Zhang D, He K, Herbst J J, et al. Characterization of Efflux TransportersInvolved in Distribution and Disposition of Apixaban[J]. Drug Metab Dispos,2013.41(4):827-835.
29. Wright J A, Haslam I S, Coleman T, et al. Breast cancer resistance protein BCRP(ABCG2)-mediated transepithelial nitrofurantoin secretion and its regulation inhuman intestinal epithelial (Caco-2) layers[J]. Eur J Pharmacol,2011.672(1-3):70-76.
30. Haslam I S, Wright J A, O'Reilly D A, et al. Intestinal ciprofloxacin efflux: therole of breast cancer resistance protein (ABCG2)[J]. Drug Metab Dispos,2011.39(12):2321-2328.
31. Sukhaphirom N, Vardhanabhuti N, Chirdchupunseree H, et al. Phyllanthin andhypophyllanthin inhibit function of P-gp but not MRP2in Caco-2cells[J]. JPharm Pharmacol,2013.65(2):292-299.
32. Hilgendorf C, Ahlin G, Seithel A, et al. Expression of thirty-six drug transportergenes in human intestine, liver, kidney, and organotypic cell lines[J]. Drug MetabDispos,2007.35(8):1333-1340.
33. Volpe D A, Faustino P J, Ciavarella A B, et al. Classification of drug permeabilitywith a Caco-2cell monolayer assay[J]. Clinical Research and Regulatory Affairs,2007.24(1):39-47.
34. Artursson P and Karlsson J. Correlation between oral drug absorption in humansand apparent drug permeability coefficients in human intestinal epithelial(Caco-2) cells[J]. Biochemical and Biophysical Research Communications,1991.175(3):880-885.
35. Volpe D A. Permeability classification of representative fluoroquinolones by acell culture method[J]. AAPS J,2004.6(2):1-6.
36. Lee W C, Peng C C, Chang C H, et al. Extraction of Antioxidant Componentsfrom Bidens pilosa Flowers and Their Uptake by Human Intestinal Caco-2Cells[J]. Molecules,2013.18(2):1582-1601.
37. Abalos I S, Rodriguez Y I, Lozano V, et al. Transepithelial transport of biperidenhydrochloride in Caco-2cell monolayers[J]. Environ Toxicol Pharmacol,2012.34(2):223-227.
38. Guan M, Zhu Q L, Liu Y, et al. Uptake and transport of a novel anticancerdrug-delivery system: lactosyl-norcantharidin-associated N-trimethyl chitosannanoparticles across intestinal Caco-2cell monolayers[J]. Int J Nanomedicine,2012.7:1921-1930.
39. Segawa M, Ogura J, Seki S, et al. Rapid stimulating effect of the antiarrhythmicagent amiodarone on absorption of organic anion compounds[J]. Drug MetabPharmacokinet,2012Sep18.[Epub ahead of print]
40. Togami K, Tosaki Y, Chono S, et al. Enantioselective uptake of fexofenadine byCaco-2cells as model intestinal epithelial cells[J]. J Pharm Pharmacol,2013.65(1):22-29.
41.李晓天,王素军,王广基. HPLC-MS法测定关附甲素及其在Caco-2细胞上的摄取特性[J].中国药理学通报,2013.29(02):286-289.
42. Kis O, Zastre J A, Ramaswamy M, et al. pH dependence of organicanion-transporting polypeptide2B1in Caco-2cells: potential role inantiretroviral drug oral bioavailability and drug-drug interactions[J]. J PharmacolExp Ther,2010.334(3):1009-1022.
43. Annaert P, Ye Z W, Stieger B, et al. Interaction of HIV protease inhibitors withOATP1B1,1B3and2B1[J]. Xenobiotica,2010.40(3):163-176.
44. Elsby R, Surry D D, Smith V N, et al. Validation and application of Caco-2assays for the in vitro evaluation of development candidate drugs as substrates orinhibitors of P-glycoprotein to support regulatory submissions[J]. Xenobiotica,2008.38(7-8):1140-1164.
45. Oga E F, Sekine S, Shitara Y, et al. P-glycoprotein mediated efflux in Caco-2cellmonolayers: the influence of herbals on digoxin transport[J]. J Ethnopharmacol,2012.144(3):612-617.
46. Mease K, Sane R, Podila L, et al. Differential selectivity of efflux transporterinhibitors in Caco-2and MDCK-MDR1monolayers: a strategy to assess theinteraction of a new chemical entity with P-gp, BCRP, and MRP2[J]. J Pharm Sci,2012.101(5):1888-1897.
47. Lin X, Skolnik S, Chen X, et al. Attenuation of intestinal absorption by majorefflux transporters: quantitative tools and strategies using a Caco-2model[J].Drug Metab Dispos,2011.39(2):265-274.
48. Lepist E I, Phan T K, Roy A, et al. Cobicistat boosts the intestinal absorption oftransport substrates, including HIV protease inhibitors and GS-7340, in vitro[J].Antimicrob Agents Chemother,2012.56(10):5409-5413.
49. Ye L, Yang X, Yang Z, et al. The role of efflux transporters on the transport ofhighly toxic aconitine, mesaconitine, hypaconitine, and their hydrolysates, asdetermined in cultured Caco-2and transfected MDCKII cells[J]. Toxicol Lett,2013.216(2-3):86-99.
50. Siissalo S, Hannukainen J, Kolehmainen J, et al. A Caco-2cell based screeningmethod for compounds interacting with MRP2efflux protein[J]. Eur J PharmBiopharm,2009.71(2):332-338.
51. Zhang W, Li J, Allen S M, et al. Silencing the breast cancer resistance proteinexpression and function in caco-2cells using lentiviral vector-based short hairpinRNA[J]. Drug Metab Dispos,2009.37(4):737-744.
52. Pratt J, Venkatraman N, Brinker A, et al. Use of zinc finger nuclease technologyto knock out efflux transporters in C2BBe1cells[J]. Curr Protoc Toxicol,2012.Chapter23: Unit23.2.
53. Graber-Maier A, Gutmann H, and Drewe J. A new intestinal cell culture model todiscriminate the relative contribution of P-gp and BCRP on transport ofsubstrates such as imatinib[J]. Mol Pharm,2010.7(5):1618-1628.
54. Li J, Volpe D A, Wang Y, et al. Use of transporter knockdown Caco-2cells toinvestigate the in vitro efflux of statin drugs[J]. Drug Metab Dispos,2011.39(7):1196-1202.
55. Hessel S, John A, Seidel A, et al. Multidrug resistance-associated proteins areinvolved in the transport of the glutathione conjugates of the ultimate carcinogenof benzo[a]pyrene in human Caco-2cells[J]. Arch Toxicol,2013.87(2):269-280.
56. Yeh T H, Hsu L W, Tseng M T, et al. Mechanism and consequence ofchitosan-mediated reversible epithelial tight junction opening[J]. Biomaterials,2011.32(26):6164-6173.
57. Zhu S, Huang R, Hong M, et al. Effects of polyoxyethylene (40) stearate on theactivity of P-glycoprotein and cytochrome P450[J]. European Journal ofPharmaceutical Sciences,2009.37(5):573-580.
58. Kapitza S B, Michel B R, van Hoogevest P, et al. Absorption of poorly watersoluble drugs subject to apical efflux using phospholipids as solubilizers in theCaco-2cell model[J]. European Journal of Pharmaceutics and Biopharmaceutics,2007.66(1):146-158.
59. Tsume Y, Vig B, Sun J, et al. Enhanced Absorption and Growth Inhibition withAmino Acid Monoester Prodrugs of Floxuridine by Targeting hPEPT1Transporters[J]. Molecules,2008.13(7):1441-1454.
60. Fenner K S, Troutman M D, Kempshall S, et al. Drug-Drug InteractionsMediated Through P-Glycoprotein: Clinical Relevance and In Vitro-In VivoCorrelation Using Digoxin as a Probe Drug[J]. Clin Pharmacol Ther,2008.85(2):173-181.
61. Zheng X and Polli J E. Synthesis and in vitro evaluation of potential sustainedrelease prodrugs via targeting ASBT[J]. International Journal of Pharmaceutics,2010.396(1–2):111-118.
62. Cook J A, Feng B, Fenner K S, et al. Refining the in vitro and in vivo criticalparameters for p-glycoprotein,[I]/IC50and [I2]/IC50, that allow for theexclusion of drug candidates from clinical digoxin interaction studies[J]. MolPharm,2010.7(2):398-411.
63. Collett A, Tanianis-Hughes J, Carlson G L, et al. Comparison ofP-glycoprotein-mediated drug–digoxin interactions in Caco-2with human androdent intestine: Relevance to in vivo prediction[J]. European Journal ofPharmaceutical Sciences,2005.26(5):386-393.
64. Elsby R, Gillen M, Butters C, et al. The utility of in vitro data in making accuratepredictions of human P-glycoprotein-mediated drug-drug interactions: a casestudy for AZD5672[J]. Drug Metab Dispos,2011.39(2):275-282.
65. Hughes J and Crowe A. Inhibition of P-glycoprotein-mediated efflux of digoxinand its metabolites by macrolide antibiotics[J]. J Pharmacol Sci,2010.113(4):315-324.
66. Kamiyama E, Nakai D, Mikkaichi T, et al. Interaction of angiotensin II type1receptor blockers with P-gp substrates in Caco-2cells and hMDR1-expressingmembranes[J]. Life Sci,2010.86(1-2):52-58.
67. Castillo-Garit J A, Marrero-Ponce Y, Torrens F, et al. Estimation of ADMEproperties in drug discovery: Predicting Caco-2cell permeability usingatom-based stochastic and non-stochastic linear indices[J]. J Pharm Sci,2008.97(5):1946-1976.
68. Lai Y, Sampson K E, and Stevens J C. Evaluation of drug transporter interactionsin drug discovery and development[J]. Combinatorial Chemistry and HighThroughput Screening,2010.13(2):112-134.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |