Quantitative Comparison of Bile Acid Distribution and Intestinal Transport from Native Cow-bezoar and Artificial and in vitro Cultured Substitutes using Caco-2 Cell Monolayer Model

Objective This study was conducted to examine the absorption and translocation of conjugated bile acids(BAs)in Calculus bovis and its substitutes to detect differences in these materials.Methods A Caco-2 monolayer cell model was used to compare the apparent permeability coefficient(Papp)value and efflux ratio(ER)of BAs in natural cow-bezoar(NCB),artificial cow-bezoar(ACB),and in vitro cultured cow-bezoar(Ivt-CCB).Papp and ER values were determined by liquid chromatography-mass spectrometry.Samples were separated on an analytical column.Results The distribution of BAs in NCB was significantly different from that in ACB and Ivt-CCB.The percentages of conjugated BAs were significantly higher in NCB than in the two substitutes.The distribution differences of conjugated and unconjugated BAs can be used to distinguish costly NCB from relatively inexpensive substitutes.Conclusion The transport characteristics of BAs in Ivt-CCB were more consistent with NCB than with ACB,even when the proportions of BAs in Ivt-CCB were closer to those of ACB.

Intestinal permeability of liquiritin and isoliquiritin in the Caco-2 cell monolayer model

The intestinal permeability of two flavonoid compounds liquiritin （LQ） and isoliquiritin （ILQ） was investigated using the Caco-2 cell monolayer model. In order to evaluate the permeability and predict the absorption mechanism of the two compounds, the study on bidirectional permeability from the apical （AP） side to the basolateral （BL） side as well as from the BL side to the AP side was carried out. The determination was performed by HPLC-UV method. And the permeability parameters, especially the apparent permeability coefficients （Papp）, were then calculated. The Papp values of LQ and ILQ are （5.40±0.16）× 10^-7 and （8.69±0.15）× 10^-7 cm/s, respectively. The results of time- and concentration-dependent transport experiments indicate that both LQ and ILQ are poor absorbed mainly through passive diffusion.

Intestinal permeability of atractylenolides across the human Caco-2 cell monolayer model

The intestinal permeability of three sesquiterpene lactones, atractylenolide Ⅰ, Ⅱ, and Ⅲ, was investigated using the human Caco-2 cell monolayer model. The bidirectional permeability of the three compounds from the apical （AP） to the basolateral （BL） side and in the reserved direction was studied. The three compounds were assayed using HPLC. The Papp values of atractylenolide Ⅰ, Ⅱ, and Ⅲ were all at the level of 10^-5 cm/s, suggesting high intestinal permeability and good absorption. The bidirectional transport of the three compounds was time- and concentration-dependent, and indicated the main mechanism of the passive diffusion of the three compounds across the intestinal epithelium membrane. Moreover, atractylenolide Ⅰ might be partly actively transported.

HPLC-MS analysis of Schisandra lignans and their metabolites in Caco-2 cell monolayer and rat everted gut sac models and in rat plasma

The absorption profiles of Schisandra chinensis were evaluated using the human Caco-2 cell monolayer and rat everted gut sac models,as well as in rat plasma.By analyzing the chromatographic and MSn characteristics of individual peak acquired by HPLC-DAD-APCI-MS^(n) determination,thirteen lignans were identified as the major in vitro absorbable components of the Schisandra extract.Most of these compounds were also detected and identified in rat plasma after an oral administration of the Schisandra extract,except for angeloyl(tigloyl)gomisin H and angeloyl(tigloyl)gomisin Q,whose structures possess an ester group at the cyclooctadiene ring.In addition,four metabolites,corresponding to the hydroxylation and demethylation products of schisandrin and the hydrolysis derivative of angeloyl(tigloyl)gomisin Q,were tentatively identified.The results demonstrate that Schisandra lignans are the major absorbable components of this crude drug,and hydroxylation,demethylation and hydrolysis are important metabolic transformations of the absorbable lignans.

Study on intestinal transport of Veratrum alkaloids compatible with Panax ginseng across the Caco-2 cell monolayer model by UPLC-ESI-MS method

The Caco-2 cells have been recognized as effective tools to be applied to imitate the drug absorption in human intestine for the transport of drug. In this study, Caco-2 cell monolayer model was used to study compatibility of the transport of the Veratrum alkaloids in different proportions with Panax ginseng. A specific ultra-high performance liquid chromatographic-electrospray ionization-mass spectrometric（UPLC-ESI-MS） method is developed for the semi-quantitative determination of Veratrum alkaloids on intestinal transport with berberine as internal standard（IS）. In the Caco-2 model constructed, three influencing factors are investigated, including time, concentration and recovery rates of the Veratrum alkaloids during the uptake from AP（apical side） to BL（basolateral side）. The results suggest that the flux of Veratrum alkaloids is time dependent and concentration dependent. And the absorption of all eight Veratrum alkaloids increase after compatibility with Panax ginseng compared to the single Veratrum nigrum extraction. This research was studied from the perspective of intestinal absorption by the UPLCESI-MS method. This method was successfully applied to transport studies of the Veratrum alkaloids and the interaction mechanism between Veratrum nigrum and Panax ginseng.

Studies on Intestinal Transport of Ginsenoside Compatibility with Veratrum nigrum via Caco-2 Cell Monolayer Model Coupled with UPLC-ESI-MS Method

The Caco-2 cells have been recognized as effective tools to be applied to imitating the drug absorption in human intestine for the transport of drug. Herein, Caco-2 cell monolayer model was used to study the transport of the ginsenoside compatibility with Veratrum nigrum in different proportions. A specific high performance liquid chro- matography-electrospray ionization-mass spectrometry（HPLC-ESI-MS） method was developed for the semiquantita- tive determination of ginsenoside in intestinal transport with Dioscin as an internal standard. For the Caco-2 model constructed, two influencing factors were investigated, including time and concentration. The results suggest that the absorption of ginsenoside Re, Rgl, Rbl, Rc, Rb2 and Rd are time- and concentration-dependent and the excretions of Rbl, Rc, Rb2 and Rd have a relatronship with some transport proteins. The bioavailability of the ginsenosides has reduced compared to the single Panax ginseng extract when compatibility with a certain amount of Veratrum nigrum.

Studies on Absorption and Tansport of Limoninoids from Fructus Evodiae in Caco-2 Cell Monolayer Model

Objective To study the intestinal absorption and transepithelial transport of three limoninoids: evodol (EVO), limonin (LIM), and shihulimonin A (SHIA), isolated from Fructus Evodiae [the unripe fruit of Evodia rutaecarpa and Evodia rutaecarpa var. bodinieri] in the human intestine. Methods The in vitro cultured human colon carcinoma cell line, Caco-2 cell monolayer model, was applied to studying the absorption and transepithelial transport of the three limoninoids from apical (AP) to basolateral (BL) side and from BL to AP side. The three limoninoids were measured by reversed-phase high performance liquid chromatography coupled with ultraviolet absorption detector. Transport parameters and apparent permeability coefficients (Papp) were then calculated and compared with those of Propranolol as a control substance of high permeability and Atenolol as a control substance of poor permeability. Results The Papp value of EVO and LIM from AP to BL side for absorption and transport were 1.78 × 10-5 cm/s and 1.16 × 10-5 cm/s, respectively, which was comparable to that of Propranolol with Papp 2.18 × 10-5 cm/s. Conclusion The absorption and transport of both EVO and LIM are main passive diffusion as the dominating process in Caco-2 cell monolayer model, and they were estimated to be high absorbed compounds. SHIA in Caco-2 cell monolayer model may be involved in metabolism in the transport processes.

Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs transport across Caco-2 cell monolayers

This study aimed to determine the transepithelial transport characteristics of chiral drug enantiomers across Caco-2 cell monolayers,a model of human intestinal epithelial membrane.Six chiral aryloxy enantiomers(atenolol,sotalol,celiprolol,carvedilol,metoprolol and propafenone)were tested in bi-directional transport studies.The separation and quantitation of these enantiomers were performed by reversed-phase high-performance liquid chromatography(RP-HPLC)using 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate(GITC)as a pre-column derivatizing agent.Bi-directional transport studies demonstrated that celiprolol and carvedilol exhibited significant enantioselectivity in polarized transport at the concentration range tested.The efflux ratio(ER)for(R)-(t)-celiprolol was 8.96,but it was much lower for(S)-(-)-celiprolol which is 3.42 at the concentration of 96.0 mM;carvedilol had the same transport behavior as celiprolol while the difference between the ER values of two enantiomers was not as significant as celiprolol at the concentration of 5.0 mM.They are 2.41 for(R)-(t)-carvedilol and 1.98 for(S)-(-)-carvedilol.But in the transport studies of racemic atenolol,sotalol,metoprolol and propafenone,no enantioselective transport were observed over the concentration range tested.Because P-glycoprotein(P-gp)is highly expressed in Caco-2 cells,we inferred that P-gp might participate in the transport processes of celiprolol and carvedilol in chirally discriminative ways.

Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations (】 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION :Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Uptake and transport of furanodiene in Caco-2 cell monolayers： a comparison study between furanodiene and furanodiene loaded PLGA nanoparticles

目的 Furanodiene (FDE ) 与高 lipophilicity 和差的稳定性拥有多样的药理学活动。这研究准备了 FDE 由自发的乳剂溶剂散开方法装载了 PLGA nanoparticles (FDE-PLGA-NPs ) 和 PEGylated PLGA nanoparticles (FDE-PEG-PLGA-NPs ) 改进 FDE 的稳定性和 bioavailability。方法 FDE-PLGA-NPs 和 FDE-PEG-PLGA-NPs 为尺寸被描绘并且缩放分发，表面形态学，希腊语的第六个字母潜力和陷阱效率。在生理的液体(PBS 和人工的胃肠的液体) 的 FDE， FDE-PLGA-NPs 和 FDE-PEG-PLGA-NPs 的稳定性被评估。在 vitro，细胞的举起和运输研究用 Caco-2 房间单层被执行。结果 FDE-PLGA-NPs 和 FDE-PEG-PLGA-NPs 的尺寸从 110-140 nm ，分别地，陷阱效率是 87.3% 和 89.2% ，稳定性与 FDE 相比显著地被提高。FDE-PLGA-NPs 和 FDE-PEG-PLGA-NPs 能被 Caco-2 房间自由地收起并且搬运单层到对面。当 FDE 几乎没到达到受体方面时，它能被收起进 Caco-2 房间单层。这些结果显示了那 FDE-PLGA-NPs 的结论，特别 FDE-PEG-PLGA-NPs，能提高 FDE 的稳定性和 hydrophilicity 并且越过 Caco-2 房间单层增加 FDE 的浸透。

Effect of bioadhesive excipients on absorption of total flavonids from Puerariae Lobatae Radix transporting across Caco-2 cell monolayer

Objective: Pueraria total flavonids(PTF) can treat cardiovascular and cerebrovascular diseases, but it has poor membrane permeability and oral bioavailability. Some excipients, such as carbomer, chitosan, and hydroxypropyl methylcellulose, can improve the oral bioavailability. Traditional in vitro evaluation techniques, including the rat intestinal perfusion and cell line models, cannot evaluate PTF absorption and holistic transporters.Methods: This study evaluated excipients' adhesiveness and effect on PTF transport across Caco-2 cell monolayer. cDNA microarrays identified gene expression changes in Caco-2 cells exposed to PTF and PTF with excipients, and revealed the mechanism underlying the effect of excipients on PTF absorption.Results: In vitro adhesion and transport experiments across Caco-2 showed that excipients had higher adhesiveness to gastric mucosa and transport efficiency across Caco-2 cells than PTF alone. The interaction of PTF with excipients significantly changed the expression of some genes, which might influence the absorption rate of PTF.Conclusion: Different bioadhesive polymers can improve intestinal absorption of PTF, which was related to some genes affiliated to the ATP-binding cassette(ABC) and solute carrier transporter(SLC) to some extent.

Monolayer MoS_(2)/n-Si Heterostructure Schottky Solar Cell

Monolayer MoS_(2)has a promising optoelectronics property,with a bandgap in the visible range;the material is a potential candidate for solar cell applications.In this work,we grew MoS_(2)monolayers using a low-pressure chemical vapor deposition approach.To produce uniform wafer-scale MoS_(2)monolayer films,precursors molybdenum dioxide(MoO_(2))and sulfur(S)are utilized.Atomic force microscopy was used to quantify the thickness of the monolayers,and the result was validated by Raman spectroscopy.Transmission electron microscopy(TEM)was used to confirm the crystalline quality of the monolayers,and photoluminescence spectroscopy was used to evaluate their optical properties.We were able to create a Schottky solar cell with a MoS_(2)monolayer up to 1 cm2 area by transferring monolayer film to n-type silicon.The MoS_(2)/n-Si Schottky solar cell demonstrated photovoltaic characteristics with a short circuit current density of 14.8 mA cm^(-2)and an open-circuit voltage of 0.32 V under 100 mW cm^(-2)illumination.The fill factor and energy conversion efficiency were 53%and 2.46%,respectively,with the highest external quantum efficiency at 530 nm being 44%.

Effect of dephytinization on bioavailability of iron,calcium and zinc from infant cereals assessed in the Caco-2 cell model

AIM： To test the effect of the dephytinization of three different commercial infant cereals on iron, calcium, and zinc bioavailability by estimating the uptake, retention, and transport by Caco-2 cells. METHODS： Both dephytinized （by adding an exogenous phytase） and non-dephytinized infant cereals were digested using an in vitro digestion protocol adapted to the gastrointestinal conditions of infants younger than 6 too. Mineral cell retention, transport, and uptake from infant cereals were measured using the soluble fraction of the simulated digestion and the Caco-2 cells. RESULTS： Dephytinization of infant cereals significantly increased （P 〈 0.05） the cell uptake efficiency （from 0.66%-6.05% to 3.93%-13%）, retention （from 6.04%-16.68% to 14.75%-20.14%） and transport efficiency （from 0.14%-2.21% to 1.47%-6.02%）, of iron, and the uptake efficiency （from 5.0%-35.4% to 7.3%-41.6%） and retention （from 4.05%-20.53% to 14.45%-61.3%） of zinc, whereas calcium only cell uptake showed a significant increase （P 〈 0.05） after removing phytate from most of the samples analyzed. A positive relationship （P 〈 0.05） between mineral solubility and the cell uptake and transport efficiencies was observed. CONCLUSION： Removing phytate from infant cereals had a beneficial effect on iron and zinc bioavailability when infant cereals were reconstituted with water. Since in developing countries cereal-based complementary foods for infants are usually consumed mixed with water, exogenous phytase additions could improve the nutritional value of this weaning food.

Caco-2细胞单层缺氧再复氧损伤的模型构建

目的构建Caco-2细胞单层缺糖、缺血清、缺氧,再复糖、复血清、复氧的损伤模型,探究一氧化碳释放分子3(CORM-3)对Caco-2细胞单层缺氧再复氧损伤模型的影响。方法首先,培养Caco-2细胞,于37℃、含5%CO_(2)、1%O_(2)与94%N_(2)的培养箱中缺氧,建立Caco-2细胞单层缺糖、缺血清、缺氧/复糖、复血清、复氧(H/R)模型。根据缺氧时间分4组,空白对照组(Control组)、H/R 1组(缺氧4 h、复氧4 h)、H/R 2组(缺氧8h、复氧4h)和H/R 3组(缺氧12 h、复氧4 h)。其次,溶解CORM-3药物粉末并制备无活性的CORM-3(iCORM-3),按药物浓度分6组,空白对照组(Control组)、缺氧、复氧损伤模型组(H/R组)、H/R+300μmol/L CORM-3(H/R+C1组)、H/R+400μmol/L CORM-3(H/R+C2组)、H/R+500μmol/L CORM-3(H/R+C3组)和H/R+500μmol/L iCORM-3(H/R+C4组)。主要采用Cell counting kit-8(CCK8)试剂盒检测细胞活力,倒置显微镜观察细胞形态变化,测定Caco-2细胞单层渗透性。结果随着缺氧时间的延长,可观察到Caco-2细胞间连接消失,细胞皱缩从培养瓶底脱落漂浮,细胞活力下降,细胞单层通透性增大。给予CORM-3药物干预处理后,相比于未干预损伤组Caco-2细胞均有不同程度的改善,细胞活力随着药物浓度的增加而上升。结论本研究成功构建Caco-2细胞单层缺氧再复氧的损伤模型,并发现CORM-3对Caco-2细胞单层H/R损伤模型可能有保护作用。

Effects of extracellular iron concentration on calcium absorption and relationship between Ca^(2+) and cell apoptosis in Caco-2 cells

AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase Ⅺ and dispase Ⅰ. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control, n = 150) to 35.71±13.99 (n = 150, P<0.01), 72.19±35.40 (n = 150, P<0.01) and 211.34±29.03 (n = 150,P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 μmol/L). The FI value of Caco-2 cells treated by FAC was 185.85±33.77 (n = 150, P<0.01), 122.73±58.47 (n = 150, P<0.01), and 53.29±19.82 (n= 150,P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187(0.1,1.0 and 10 μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n = 150) to 45.19±21.95 (n = 150, P<0.01), 89.87±43.29 (n = 150, P<0.01) and 104.64±51.07 (n = 150,P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. CONCLUSION: Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

Assessment of Iron Bioavailability in Ten Kinds of Chinese Wheat Flours Using an in vitro Digestion/Caco-2 cell Model

Abstract Objective To compare iron bioavailability （Fe BV） from ten selected kinds of Chinese wheat flours in order to provide scientific basis for further human trials and enable plant breeding programs to screen biofortified wheat cultivars. Methods An in vitro digestion/Caco-2 cell model was used to assess Fe BV of ten flour samples from six leading Chinese wheat cultivars and the stability of Fe BV in one cultivar was studied across three growing environments. Results Significant differences were observed in both Fe BV and Fe bioavailability per gram of food （Fe BVPG） among cultivars （P〈0.01） grown at the same location with the same flour extraction rate. Zhongyou 9507 and Jingdong 8 had Fe BV 37%-54% and Fe BVP（3 103%-154% higher than the reference control. In the Anyang environment, Zhongyou 9507 had a higher wheat flour-Fe level and Fe BVPG. Differences in Fe BV were detected in cultivars with different flour extraction rates. Conclusion Zhongyou 9507 and Jingdong 8 were identified as the most promising cultivars for further evaluation of efficacy by using human subjects. The growing environments had no effect on Fe BV, but did have a significant effect on Fe BVPG. Fe bioavailabilities in low-extraction （40%） flours were higher than those in high-extraction （78%） flours.

Gastric digestion of pea ferritin and modulation of its iron bioavailability by ascorbic and phytic acids in caco-2 cells

AIM： To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model.METHODS： Pea seed ferritin was purified using salt fractionation followed by gel filtration chromatography.The bioavailability of ferritin iron was assessed using coupled in vitro digestion/Caco-2 cell model in the presence or absence of ascorbic acid and phytic acid.Caco-2 cell ferritin formation was used as a surrogate marker of iron uptake. Structural changes of pea ferritin under simulated gastric pH were characterized using electrophoresis, gel filtration and circular dichroism spectroscopy.RESULTS： The caco-2 cell ferritin formation was significantly increased （P 〈 0.001） with FeSO4 （19.3±9.8 ng/mg protein） and pea ferritin （13.9 ± 6.19 ng/mg protein） compared to the blank digest （3.7 ± 1.8 ng/mg protein）. Ascorbic acid enhanced while phytic acid decreased the pea ferritin iron bioavailability. However,either in the presence or absence of ascorbic acid, the ferritin content of caco-2 cells was significantly less with pea ferritin than with FeSO4. At gastric pH, no band corresponding to ferritin was observed in the presence of pepsin either on native PAGE or SDS-PAGE. Gel filtration chromatography and circular dichroism spectroscopy revealed a pH dependent loss of quaternary and secondary structure.CONCLUSION： Under gastric conditions, the iron core of pea ferritin is released into the digestive medium due to acid induced structural alterations and dissociation of protein. The released iron interacts with dietary factors leading to modulation of pea ferritin iron bioavailability,resembling the typical characteristics of non-heme iron.

Tartary buckwheat(Fagopyrum tataricum(L.)Gaertn)protein-derived antioxidant peptides:mechanisms of action and structure-activity relationship in Caco-2 cell models

Excessive reactive oxygen species(ROS)can cause oxidative damage and lead to various metabolic disease.Tartary buckwheat(Fagopyrum tataricum(L.)Gaertn)is a new kind of protein-rich functional food,the protein in which has been proved to have good antioxidant capacity.In this study,in order to further explore the antioxidant mechanism of Tartary buckwheat protein,4 peptides(CR-8,LR-8,GK-10 and SR-12)were isolated and identified from it.H2 O2 was used to induce oxidative damage to Caco-2 cells to evaluate antioxidant capacity of these peptides.The results of superoxide dismutase(SOD),total antioxidant capacity(T-AOC)and mitochondrial membrane potential etc.showed that these peptides have superior antioxidant capacity.CR-8 has the best antioxidant capacity.In order to further clarify the antioxidant mechanism of CR-8,metabolomics was used to analyze related metabolites and metabolic pathways.The results showed that after CR-8 intervention,the content of metabolites such as L-acetyl carnitine has increased.This indicated that CR-8 can improve the antioxidant capacity of damaged cells by intervening in multiple metabolic pathways.This also revealed the anti-oxidant mechanism of tartary buckwheat protein.In conclusion,it provided a theoretical basis for further studying the activity of tartary buckwheat portein and utilizing buckwheat resources.

Molecular analysis and anticancer properties of two identified isolates,Fusarium solani and Emericella nidulans isolated from Wady El-Natron soil in Egypt against Caco-2(ATCC) cell line

Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sub>50 value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.

Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells： an insight into the efflux-metabolism alliance

Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients （Papp） of （ - ） and （ ＋ ）CLA were higher in the ab- sorptive direction than those in the secretory direction with efflux ratios（ER） of 0. 709 ± 0.411 and 0. 867± 0. 250 （ Х10^-6 -1 cm · s ）, respectively. Their bidirectional transports were significantly reduced by （75.6 ± 87.5）% af- ter treatment with verapamil （ a P-glycoprotein inhibitor） that increased the rate of metabolism by CYP3 A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of （ - ） and （ ＋ ） CLA with ERs being 2. 934 ± 1. 432 and 1. 877 ± 0. 148 （ Х 10^-6 cm/s） respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enanti- omers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following （ - ） or （ ＋ ）-isomer treatment alone. Furthermore, the uptake of （ - ）CLA was more than that of （ ＋ ）CLA in the transfected cells. Incubation with ketoeonazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexametha- sone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharma- cology of （？） CLA as a candidate drug for treatment of Alzheimer＇ s disease.