Effect of tea phenolics on iron uptake from different fortificants by Caco-2 cells

The in vitro effects of tea phenolics on Fe uptake from different fortificants (FeSO₄, FeCl₃, FeEDTA) by Caco-2 cells were compared. Cell cultures were exposed to catechin, tannic acid, green or black tea solutions, added within Fe-containing solution, or used to pre-treat cell cultures before Fe-exposure. Cell ferritin formation was used as a measure of Fe uptake. Reverse phase chromatography was used to identify specific phenolics in tea solutions, and the Fe-binding catechol and galloyl groups were determined spectrophotometrically. The results showed a positive effect of catechin on Fe uptake only from dissociable Fe sources, and a marked inhibitory effect of tannic acid regardless of the Fe source. Tea phenolics exhibit similar inhibitory patterns on Fe uptake from FeCl₃ and FeEDTA solutions; however, the Fe uptake from FeSO₄ solutions was significantly less affected. These data improve the understanding of interactions by which tea phenolics affect Fe uptake at the intestinal level.     

Availability of iron from milk-based formulas and fruit juices containing milk and cereals estimated by in vitro methods (solubility,dialysability) and uptake and transport by Caco-2 cells

Iron solubility, dialysability and transport and uptake (retention + transport) by Caco-2 cells as indicators of iron availability have been estimated in the in vitro gastrointestinal digests of infant foods (adapted, follow-up and toddler milk-based formulas and fruit juices containing milk and cereals (FMC)). Low correlation coefficients (in all cases R-squared ? 37.1%) were obtained between iron solubility or dialysability versus transport or uptake efficiency – a fact emphasizing the importance of incorporating Caco-2 cell cultures to in vitro systems in order to adapt the conditions to those found in in vivo assays. The highest uptake efficiency corresponded to FMC (25.6–26.1%) and toddler formulas (32.1–41.9%), the samples with the highest ascorbic acid contents and ascorbic acid/iron molar ratios. In addition, the toddler formulas contained caseinphosphopeptides with the cluster sequence SpSpSpEE, representing the binding site for minerals. In adapted formulas, greater iron uptake efficiency was obtained for the formulation containing ferrous lactate (22.7%) versus ferrous sulfate (4.7%).     

Antioxidant effect derived from bioaccessible fractions of fruit beverages against H2O2-induced oxidative stress in Caco-2 cells

This work evaluates the effect of bioaccessible fractions from fruit beverages against oxidative stress (OS) in Caco-2 cells. A fruit beverage (grape + orange + apricot) (with/without milk and/or iron/zinc) was subjected to in vitro gastrointestinal digestion, and bioaccessible fractions were incubated with Caco-2 cell cultures. Following preincubation, OS was induced with 5 mM H₂O₂. Intracellular reactive oxygen species (ROS), mitochondrial potential (Δψ_m), mitochondrial metabolism (MTT test), intracellular reduced glutathione (GSH) and superoxide dismutase activity (SOD) were measured. The data evidenced viable cultures with increased mitochondrial metabolism and GSH-Rd activities, without alteration in SOD activity. Accordingly, more preserved mitochondrial integrity was also evidenced, allowing the action of antioxidant systems in preincubated cultures. Based on these data, we can conclude that a cytoprotective effect is derived from bioaccessible fractions of fruit beverages, though this effect failed to prevent intracellular ROS accumulation in Caco-2 cell cultures exposed to 5 mM H₂O₂.     

Ferritin synthesis by Caco-2 cells as an indicator of iron bioavailability: Application to milk-based infant formulas

The bioavailability of iron from milk-based infant formulas was estimated by an in vitro system including enzymatic digestion, iron uptake by Caco-2 cells and ferritin determination via an enzymoimmunoassay (ELISA). Positive correlations (p < 0.01) were found between the Fe(II) added to Caco-2 cells and ferritin synthesis and between the amount of dialyzed iron added to the cell culture and ferritin synthesis. The comparison of the bioavailability of iron from different milk-based formulas showed that adapted formulas having the same composition but differing in the iron salts added yielded similar ferritin levels. The same happened with follow-up formulas differing only in the presence or absence of bifidobacterium added. However, significant differences in the amount of ferritin synthesized were recorded between the two analyzed toddler formulas. Such differences could be attributed firstly to the ascorbic acid content and perhaps also to the manufacturing process involved, because one formula was in liquid form while the other was powdered.     

Iron and calcium availability from digestion of infant cereals by Caco-2 cells

An adequate mineral intake during infancy is needed for normal growth and development. This study investigated the effect of dephytinization of four infant cereals and the use of water and follow-on formula as the liquid of reconstitution on the intestinal cell uptake of iron and calcium from infant cereals using a model that combines a simulated gastrointestinal digestion adapted to the gastrointestinal conditions of infants younger than 6 months and the Caco-2 cell line. Iron and calcium uptake by Caco-2 cells from most infant cereals was significantly (p < 0.05) improved when a phytase was added. When infant cereals were reconstituted with water, dephytinization increased iron (3.2–19.5 vs. 3–10%) and calcium (0.66–2.3 vs. 0.35–0.59%) availability compared to the same infant cereals reconstituted with a follow-on formula. We can conclude that dephytinization of infant cereals and water addition improved iron and calcium availability, depending on the infant cereal used.     

Process optimisation of haemoglobin hydrolysis by complex proteases to produce haem-enriched peptides and its iron uptake property evaluation by Caco-2 cell model

In this study, haemoglobin was hydrolysed by compound protease to produce haem-enriched peptides in order to improve the bio-absorptivity of haem iron. The hydrolysis process was optimised through an orthogonal experiment. The molecular weight, solubility and absorptivity of haem-enriched peptides were also investigated. The optimal hydrolysis process was papain/alkaline protease ratio 6:4, enzyme content 9000 U g⁻¹, hydrolysis at 50 °C and pH 8 conditions for 4 h. After hydrolysis at this condition, the haem released amount and haem/peptide ratio were 89.6% and 28.2%, respectively. The molecular weight of the haem-enriched peptides was mainly distributed in 3–14.4 kD, and the solubility increased significantly with pH increasing. Caco-2 cell absorption experiment demonstrated that the iron absorption of haem-enriched peptides was significantly higher (50.25 ng ferritin per mg protein) than haemin chloride, haemoglobin and collagen plus haemin chloride complex. This study provides a very promising way for the development of iron supplement products.     

Effect of soluble soybean protein hydrolysate-calcium complexes on calcium uptake by Caco-2 cells

Soybean protein hydrolysates (SPHs) bind with calcium, forming soluble SPH-calcium complexes via the carboxyl groups of glutamic and aspartic acid residues. However, their effect on calcium uptake is still unclear. In this study, Caco-2 cells were used to estimate the effect of SPH-calcium complexes with different molecular weights on calcium uptake in vitro. The changes in intracellular calcium ion concentration were measured by Fura-2 loading and expressed in fluorescence intensity. SPH-calcium complexes could promote calcium uptake. Improved fluorescence intensity was significantly different in SPH-calcium complexes (10 to 30 kDa), SPH-calcium complexes (3 to 10 kDa), and SPH-calcium complexes (1 to 3 kDa). The maximum levels of relative fluorescence intensity (18.3) occurred with SPH-calcium complexes (10 to 30 kDa). The effect of SPH-calcium complexes (10 to 30 kDa) on Ca(2+) increase was determined to be concentration dependent in the range of 0.5 to 4 mg/mL. Our results indicate that soybean protein itself might be responsible for promoting calcium absorption.     

Investigation of the cytotoxicity of food-grade nanoemulsions in Caco-2 cell monolayers and HepG2 cells

Nanoemulsions represent one of the emerging formulations for nutraceutical delivery. However, the possible toxicity associated with the small droplet size (diameter <200 nm) is still unknown. In this study, three nanoemulsions emulsified by modified starch, Tween 20 and whey protein isolate, respectively, were prepared and their cytotoxicity was examined by comparing with the corresponding micron-sized emulsions. Caco-2 cell monolayers were used to mimic the small intestine epithelium. Integrity of the cell membrane and tight junctions was tested by measuring the lactate dehydrogenase leakage and transepithelial electrical resistance, respectively. All three nanoemulsions did not reveal significant difference from their micron-sized counterparts, suggesting no apparent toxicity of the nanoemulsions on the small intestine. Meanwhile, the possible hepatic toxicity was investigated using MTT assay on HepG2 cells. It was found that nanoemulsions made with modified starch and whey protein isolate, but not Tween 20, affected the cell viability/proliferation more than did the micron-sized emulsions. Further in vivo investigation is required to examine the possible hepatic toxicity of nanoemulsions.     

Effect of phytate reduction of sorghum,through genetic modification,on iron and zinc availability as assessed by an in vitro dialysability bioaccessibility assay,Caco-2 cell uptake assay,and suckling rat pup absorption model

Improved iron and zinc availability from sorghum, a commonly consumed staple, will benefit many malnourished communities in rural Africa burdened with high prevalence of iron and zinc deficiency. This research compared the effect of genetic phytate reduction in sorghum on iron and zinc bioaccessibility and uptake measured by in vitro dialysability and Caco-2 cell uptake assays to that of iron and zinc absorption measured by a suckling rat pup model. The phytate reduction (80–86%) in these sorghums significantly increased zinc availability. The Caco-2 cell method, but not the dialysability assay, proved useful in estimating zinc absorption. The measured increase in iron availability differed between the methods, possibly due to the effect of varying mineral (Ca, Fe, Zn, P) contents of the sorghums. This effect was most prominent in the iron uptake results. More research is needed to determine the effect of naturally occurring variations in mineral contents of sorghum on the iron uptake by Caco-2 cells.     

Cellular antioxidant activity and Caco-2 cell uptake characteristics of flavone extracts from Labisia pumila

The structures of two extracts of flavone , obtained from the stem (L40) and leaf (S40) of Labisia pumila (LP), were identified through UHPLC-MS/MS analysis. An 2,2′-azobis[2-methylpropionamidine] dihydrochloride (AAPH)-induced hemolysis model was used and the results indicate the protective effects of flavone extracts on erythrocytes against AAPH-induced oxidative stress, in which L40 treatment performed the better. Quantitative analysis of variations in antioxidative-associated enzyme and non-enzyme systems within the erythrocytes were conducted and led to the identification of protective pathways in which flavone extracts could pass through the cell membrane and act as AAPH-free radical scavengers. Absorptivity of flavone extracts was characterised using a Caco-2 monolayer model, in which the apparent permeability coefficient indicated that both types of flavone extracts were readily absorbed compounds, while L40 showed better intestinal absorptivity. Therefore, we conclude that flavonoid extracts from the leaf of LP show remarkable potential functional effects on human health.     

应用Caco-2细胞模型评价真菌毒素毒性研究进展

真菌毒素是一些病原真菌在侵染作物过程中产生的次级代谢产物, 许多食品和饲料在生产、加工、贮存和流通过程中都有可能受到真菌毒素的污染。人和动物摄入真菌毒素后, 体内能够引发多种生理毒性反应, 如肝肾毒性、致癌性、肠道损伤及炎症、中枢神经系统异常、生殖紊乱等。肠上皮细胞是分隔机体内部环境与外界的屏障, 在真菌毒素的毒性评价中, 对真菌毒素引起肠上皮细胞损伤的评价是一个重要方面。人结肠癌Caco-2细胞系常用于建立体外肠道屏障模型, 该模型可应用于体外评价药物或毒素在小肠粘膜或上皮的吸收转运效率, 以及对肠道屏障功能的影响。本文综述了Caco-2细胞单层模型的建立和评价指标, 应用该模型评价几种常见真菌毒素对肠上皮细胞的运输、屏障功能的影响, 以及肠上皮细胞毒性等研究进展, 为进一步研究多种真菌毒素对肠上皮损伤的机制提供支持。     

Stability of sunflower protein hydrolysates in simulated gastric and intestinal fluids and Caco-2 cell extracts

The in vitro stability of bioactive properties in sunflower protein hydrolysates and purified peptides was studied by using simulated gastric fluid, simulated intestinal fluid, and Caco-2 cells extracts. Protein hydrolysates that inhibit the angiotensin converting enzyme and copper chelating peptides produced by hydrolysis with the microbial protease alcalase were partially resistant to incubation with simulated gastric and intestinal fluids. These hydrolysates and others produced by hydrolysis using pepsin plus pancreatin were also partially resistant to incubation with Caco-2 cells extracts. In addition, the ACE inhibitory peptide FVNPQAGS that is generated by extensive hydrolysis using pepsin and pancreatin, was found to be resistant to hydrolysis by Caco-2 cells extracts. These results suggest that stability in the digestive tract should not be a problem for the bioavailability of these bioactive peptides.     

Tea components influencing bioavailability of fluoride and potential transport mechanism in the Caco-2 cell line model

Previous work showed that the bioavailability of fluoride in dark tea was lower than NaF solution. However, limited information is available indicating the effects of tea components on the fluoride bioavailability. In this study, the effects of the components in tea on the bioavailability of fluoride were evaluated in the Caco-2 cell line model. Additionally, the mechanism of effect of aluminium on fluoride transport was investigated. The result showed that 10–100 μm of epigallocatechin gallate (EGCG) could not influence fluoride transport. Al³⁺ significantly decreased fluoride transport in both apical-basolateral and basolateral-apical directions. Moreover, aluminium could form different forms of aluminium fluoride complexes, which were transported through Caco-2 cells by different pathways. F⁻ transport was mainly dependent on the paracellular pathway and active transport involving Cl⁻ channels. The paracellular pathway played a predominant role in transport of AlF₃. The paracellular pathway and active transport both participated in AlF₂⁺ transport.     

肝脏含铁提取物的制备及对Caco-2细胞吸收铁的促进作用

以新鲜猪肝脏为原料,采用中性蛋白酶和风味酶两步水解工艺制备肝脏含铁提取物,经喷雾干燥的提取物粉末的铁含量约为475.3μg/g,且溶解性良好.采用Caeo-2细胞作为铁吸收的评价模型,与无机铁Fecl3 溶液比较,该含铁提取物粉末具有显著促进铁吸收的作用.这为研究生物利用度高、易于吸收的有机铁提供了一种新来源.     

儿童配方奶粉对Caco-2细胞钙转运及对成骨细胞增殖、矿化的影响

为评价某市售儿童成长配方奶粉的补钙和促进骨骼生长能力,以常见市售纯牛奶A作为对照,运用人结肠腺癌细胞（Caco-2）单层模型,研究其对钙离子转运的作用;运用成骨细胞（MC3T3-E1）矿化模型,研究其对成骨细胞增殖与矿化的影响。Caco-2细胞模型结果表明,各剂量组儿童配方奶粉组的钙转运量均高于市售纯牛奶A,但相较空白组无显著性差异（p<0.05）。成骨细胞增殖实验结果表明,5~50 μg/mL的儿童配方奶粉对成骨细胞均有促进增殖作用,增殖速度均在30%以上;并测定细胞碱性磷酸酶活性,当儿童配方奶粉质量浓度为50 μg/mL时,酶活达7.79 U/g蛋白,相较空白组极显著提高（p<0.01）,较市售纯牛奶A的效果更稳定。成骨细胞矿化结果表明,5~50 μg/mL儿童配方奶粉能显著促进成骨细胞产生矿化结晶,50 μg/mL时茜素红吸光值均为0.180,对比空白组提高50%以上,较市售纯牛奶A矿化程度更稳定。综上实验结果,儿童配方奶粉虽对钙在小肠上皮的转运吸收无明显促进作用,但很可能通过促进骨生长而对儿童生长发育起作用。     

熊猫豆提取物抑制肿瘤活性及诱导结肠癌细胞Caco-2凋亡机制研究

为了探究熊猫豆的抑制肿瘤活性及其诱导Caco-2凋亡的机制,采用80%丙酮浸提法制备熊猫豆粗提物,通过MTT法测定其对人结肠癌细胞Caco-2增殖的抑制效果,琼脂糖凝胶电泳、流式细胞术、酶联免疫分析等技术与方法检测细胞周期、DNA及凋亡相关蛋白相对表达量的变化。结果发现,与空白对照组相比,0.1~1.0 mg/m L熊猫豆丙酮提取物对Caco-2细胞增殖有明显的抑制作用,且呈现剂量效应关系,IC50为0.72 mg/m L。0.4、0.6、0.8 mg/m L熊猫豆丙酮提取物处理72 h后的Caco-2细胞,可以观察到典型的凋亡细胞的梯状DNA条带,细胞周期G1期延长,Cyt C、caspase-9、caspase-3蛋白的相对表达量均有显著上升。熊猫豆丙酮能够抑制结肠癌细胞Caco-2增殖,这种抑制作用是通过阻滞细胞周期和诱导线粒体通路介导的细胞凋亡实现的。     

Supplemental inulin does not enhance iron bioavailability to Caco-2 cells from milk- or soy-based,probiotic-containing,yogurts but incubation at 37 °C does

The in vitro effects of supplemental inulin (4%) on iron (Fe) availability in two different probiotic-containing yogurts were examined. Milk or soy-based yogurts, with and without inulin, were incubated (37 °C) for 48 h or without any incubation before comparison by an in vitro gastrointestinal digestion/Caco-2 cell culture model was used to assess iron bioavailability. The dialysable Fe fraction, cell ferritin formation, and cell associated Fe were monitored. Supplemental inulin decreased dialysable Fe only in non-incubated milk-based yogurt. In both yogurts incubation by itself increased dialysable Fe, and inulin increased the latter only in soy-based yogurt. Cellular ferritin concentration were higher after exposure to non-incubated milk-based than soy-based yogurt, although, after incubation the latter induced the highest ferritin formation. These data suggest that inulin does not have a direct effect on Fe bioavailability in the small intestine, and that probiotic bacteria play an enhancing role on Fe bioavailability.     

Molecular mechanisms of the naringin low uptake by intestinal Caco-2 cells

Naringin, the main flavanone of grapefruit, was reported to display numerous biological effects: antioxidant, hypocholesteremic, anti-atherogenic and favoring drug absorption. Naringin absorption mechanisms were studied in Caco-2 cells (TC7 clone). We investigated the possible involvement of several membrane transporters implicated in polyphenolic compounds intestinal transport (sodium-dependent glucose transporter 1, monocarboxylate transporter, multidrug-associated resistance proteins 1 and 2, and P-glycoprotein). Naringin was poorly absorbed by Caco-2 cells, according to its low value of apparent permeability coefficient (P(app) = 8.1 +/- 0.9 x 10(-8) cm/s). In the presence of verapamil, a specific inhibitor of P-glycoprotein, cellular uptake was increased by almost threefold after 5 min, and P(app) was doubled after 30 min. Our results indicated the involvement of P-glycoprotein, an ATP-driven efflux pump, capable of transporting naringin from the Caco-2 cell to the apical side. This phenomenon could explain, at least in part, the low absorption of this flavanone at the upper intestinal level.