Review on the Use of Cell Cultures to Study Metabolism,Transport, and Accumulation of Flavonoids: From Mono‐Cultures to Co‐Culture Systems

The aim of this review is to provide a comprehensive discussion on the various human/animal cell‐based models used to study the absorption, transport, and metabolism of flavonoids. Flavonoids are plant‐based bioactive compounds that have been extensively investigated for their active role in health alleviation and disease prevention. For this purpose, cell lines isolated from various human and animal tissues have been routinely used as an in vitro model to assess the bioavailability and bioactivity of these compounds. This paper reviews for the first time various transporters (SLCT, SGLT, bilitranslocase, and ABC transporters), metabolic routes (deglycosylation, glucuronidation, sulfation, and deconjugation), and accumulation of flavonoids in different cell lines commonly used in flavonoid research. Also, the use of co‐culture systems to study flavonoid bioactivity will be discussed. To date, no definite mono‐culture or co‐culture formulation has been generally accepted to be the most accurate representation of the in vivo situation. Therefore, further investigation and improvement of cell‐based in vitro models for flavonoid research merit further investigation.     

Esterification of Quercetin Increases Its Transport Across Human Caco‐2 Cells

Plant polyphenols showed useful biochemical characteristics in vitro; however, the assessments of their clinical applications in vivo are restricted by their limited bioavailability due to their strong resistance to 1st‐pass effects during absorption. In order to improve the bioavailability of quercetin (QU), the ester derivative of QU (3,3′,4′,5,7‐pentahydroxy flavones, TAQU) was synthesized, followed by examining the oil–water partition coefficient as well as the transport mechanisms of QU and its ester derivative (TAQU) using human Caco‐2 cells. The transport characteristics of QU and TAQU transport under different conditions (different concentrations, time, pH, temperature, tight junctions, and potential transporters) were systematically investigated. Results showed that QU had a lower permeability coefficient (2.82 × 10^?6 cm/s) for apical‐to‐basolateral (AP‐BL) transport over 5 to 50 μM, whereas the transport rate for AP to BL flux of TAQU (5.23 × 10^?6 cm/s) was significantly greater than that of QU. Paracellular pathways were not involved during the transport of both QU and TAQU. QU was poorly absorbed by active transport, whereas TAQU was mostly absorbed by passive diffusion. Efflux transporters, P‐glycoproteins, multidrug resistance proteins were proven to participate in the transport process of QU, but not in that of TAQU. These results suggested that improving the lipophicity of QU by esterification could increase the transport of QU across Caco‐2 cells.     

Polyphenols and phenolic acids from strawberry and apple decrease glucose uptake and transport by human intestinal Caco‐2 cells

The effect of polyphenols, phenolic acids and tannins (PPTs) from strawberry and apple on uptake and apical to basolateral transport of glucose was investigated using Caco‐2 intestinal cell monolayers. Substantial inhibition on both uptake and transport was observed by extracts from both strawberry and apple. Using sodium‐containing (glucose transporters SGLT1 and GLUT2 both active) and sodium‐free (only GLUT2 active) conditions, we show that the inhibition of GLUT2 was greater than that of SGLT1. The extracts were analyzed and some of the constituent PPTs were also tested. Quercetin‐3‐O‐rhamnoside (IC₅₀=31 μM), phloridzin (IC₅₀=146 μM), and 5‐caffeoylquinic acid (IC₅₀=2570 μM) contributed 26, 52 and 12%, respectively, to the inhibitory activity of the apple extract, whereas pelargonidin‐3‐O‐glucoside (IC₅₀=802 μM) contributed 26% to the total inhibition by the strawberry extract. For the strawberry extract, the inhibition of transport was non‐competitive based on kinetic analysis, whereas the inhibition of cellular uptake was a mixed‐type inhibition, with changes in both V_max and apparent K_m. The results in this assay show that some PPTs inhibit glucose transport from the intestinal lumen into cells and also the GLUT2‐facilitated exit on the basolateral side.     

Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro

Scope: Zearalenone (ZEN) and α‐zearalanol (α‐ZAL, zeranol) were studied in differentiated Caco‐2 cells and in the Caco‐2 Millicell^® system in vitro to simulate their in vivo intestinal absorption and metabolism in humans. Methods and results: In addition to metabolic reduction/oxidation, extensive conjugation with glucuronic acid and sulfate of the parent compounds and their phase I metabolites was observed. The positional isomers of the glucuronides and sulfates were unambiguously identified: Sulfonation occurred specifically at the 14‐hydroxyl group, whereas glucuronidation was less specific and, in addition to the preferred 14‐hydroxyl group, involved the 16‐ and 7‐hydroxyl groups. Using the Caco‐2 Millicell^® system, an efficient transfer of the glucuronides and sulfates of ZEN and α‐ZAL and their phase I metabolites into both the basolateral and the apical compartment was observed after apical administration. The apparent permeability coefficients (P_app values) of ZEN, α‐ZAL and the ZEN metabolite α‐zearalenol were determined, using an initial apical concentration of 20 μM and a permeation time of 1 h. Conclusion: According to the P_app values, the three compounds are expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycones and as glucuronide and sulfate conjugates in humans.     

Digestion stability and evaluation of the metabolism and transport of olive oil phenols in the human small-intestinal epithelial Caco-2/TC7 cell line

The aims of this study were to investigate (i) the metabolism of olive oil phenolics by intestinal epithelial cells and (ii) their transport across epithelial cell monolayers. The various conjugates and derivatives produced by the intestinal epithelial cells were identified following separation by ultra-performance liquid chromatography (UPLC), using a combination of UV/visible spectra, mass spectrometry and specific enzyme treatments (β-glucuronidase and aryl-suphatase). Limited metabolism of olive oil phenolics was observed using Caco-2/Tc7 cells as a model of the human intestinal epithelium, and the methyslated conjugates were the major metabolites detected. The results of the transport rate data for phenols and their metabolites to the apical, cellular, and basolateral compartments after apical loading of the phenol at 100 μM showed a time-dependent efflux of various free and conjugated forms of phenols.     

The antioxidant activity and transcellular pathway of Asp‐Leu‐Glu‐Glu in a Caco‑2 cell monolayer

Asp‐Leu‐Glu‐Glu (DLEE) is one of the antioxidant peptides purified from Chinese dry‐cured Xuanwei ham in our previous study. In the current work, the stability in a simulated digestion system, the transportation pathway and the antioxidant ability of DLEE were further investigated in a Caco‐2 cell monolayer. In the simulated gastrointestinal digestion system, no oligopeptides were generated. In the transport trial, the inhibitors cytochalasin D increased the transport of DLEE across the Caco‐2 cell monolayer, with P_app values of 3.22 × 10^?6 cm s^?1. A decreased expression occludin was observed with the DLEE incubation in the cell monolayer, and the antioxidant activity showed to be increased gradually in basolateral side. This study indicates that the absorption of DLEE could mainly occur via paracellular transport and provides information about its antioxidant activity after being absorbed across a cell monolayer.     

The (193–209) 17‐residues peptide of bovine β‐casein is transported through Caco‐2 monolayer

Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. β‐casein (β‐CN, 193–209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the β‐CN (193–209) peptide across Caco‐2 cell monolayer. In addition, we evaluated the possible routes of the β‐CN (193–209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the β‐CN (193–209) peptide resisted the action of brush‐border membrane peptidases, and that it was transported through the Caco‐2 cell monolayer. The main route involved in transepithelial transport of the β‐CN (193–209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight‐junctions could not be excluded. Our results demonstrated the transport of an intact long‐chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.     

Effects of Catechins and Their Related Compounds on Cellular Accumulation and Efflux Transport of Mitoxantrone in Caco‐2 Cell Monolayers

The ability of catechins and their related compounds to inhibit breast cancer resistance protein (BCRP) function in Caco‐2 cell monolayers was investigated with mitoxantrone as a BCRP substrate. The gallate or pyrogallol moiety on the catechin structure seemed to promote increased cellular accumulation and inhibit efflux transport of mitoxantrone. The ability of gallate catechins such as (?)‐epigallocatechin gallate (EGCG) and (?)‐epicatechin gallate (ECG) to increase cellular accumulation and inhibit efflux transport of mitoxantrone was greater than that of nongallate catechins. Gallic acid octyl ester (GAO) also increased intracellular mitoxantrone accumulation. Experiments using GAO derivatives indicated that the gallate moiety required the presence of a long carbon chain for BCRP inhibition. Cellular accumulation and reduced efflux transport of mitoxantrone were greater with epigallocatechin 3‐(3″‐O‐butyl) gallate than with EGCG. EGCG inhibition of BCRP seemed to be restricted by hydrophobicity. The co‐administration of catechins, particularly EGCG and related compounds, with greater hydrophobicity may increase the therapeutic activities of BCRP substrates such as mitoxantrone.     

Cranberries and Wild Blueberries Treated with Gastrointestinal Enzymes Positively Modify Glutathione Mechanisms in Caco‐2 Cells In Vitro

Beneficial health effects of cranberries (CBs) and wild blueberries (BBs), such as reduced levels of oxidative stress, have been demonstrated in feeding studies. These Vaccinium berries contain high levels of flavonoids; however, the bioavailability of flavonoids is generally low. We investigated the in vitro effects of these berries on intestinal cells, focusing on mitigating oxidative stress and associated reactive oxygen species (ROS). First, we simulated the passage of CB and BB through the gastrointestinal (GI) tract by treating berry homogenates to a battery of digestive enzymes. Then, Caco‐2 cells, a model of small intestine epithelial uptake, were exposed to these homogenates for 60 min. Using a cell‐free assay, we found that the antioxidant activity in CB homogenates was not affected by these enzymes, but that BB homogenates treated with gut enzymes had 43% lower free‐radical quenching activity (P < 0.05). However, both of the enzyme‐treated homogenates were still able to counteract the ROS‐generating ability of H₂O₂ added exogenously to Caco‐2 cells. Berry homogenates also increased mitochondrial metabolic rates at 60 min posttreatment, as measured by MTT assays. Enzyme‐treated CB (but not BB) homogenates increased the levels of reduced glutathione (GSH) relative to oxidized glutathione (GSSG), a critical indicator of the cellular redox state (P < 0.05). Our data suggest that CBs do not lose their antioxidant ability when passing through the GI tract, and specifically, digested CB may serve to enhance cytoprotective effects in intestinal cells by reducing potential damage caused by free radicals and ROS derived from other food sources.     

Bioavailability of iron from a heat treated glucose‐lysine model food system: assays in rats and in Caco‐2 cells

The influence of Maillard reaction products (MRP) from glucose–lysine on iron bioavailability was investigated. Equimolar glucose–lysine mixtures (GL, 40% moisture) were heated (150 °C, for 30, 60 and 90 min) to prepare samples GL30, GL60 and GL90, respectively, and were used to study the influence of MRP on iron solubility, iron uptake and transport in Caco‐2 cells and iron balance in rats. After an initial increase with GL30, in vitro iron solubility decreased in the presence of most of the heated samples. The Caco‐2 cell experiments showed that Fe cell content increased in the presence of heated mixtures with respect to GL, whereas transport was less affected. Iron balance was determined in rats fed diets containing the GL30 and GL90 samples (3%) for a 21 day period and compared with that measured in a control group. Consumption of glucose–lysine heated mixtures increased total iron retention, although hemoglobin values decreased. Iron accumulations in organs such as the spleen, kidney and small intestine were observed. Copyright © 2004 Society of Chemical Industry     

Determination of S‐methyl‐L‐methionine (SMM) from Brassicaceae Family Vegetables and Characterization of the Intestinal Transport of SMM by Caco‐2 Cells

The objectives of the current study were to determine S‐methyl‐L‐methionine (SMM) from various Brassicaceae family vegetables by using validated analytical method and to characterize the intestinal transport mechanism of SMM by the Caco‐2 cells. The SMM is well known to provide therapeutic activity in peptic ulcers. The amount of SMM from various Brassicaceae family vegetables ranged from 89.08 ± 1.68 μg/g to 535.98 ± 4.85 μg/g of dry weight by using validated ultra‐performance liquid chromatography‐electrospray ionization‐mass spectrometry method. For elucidating intestinal transport mechanism, the cells were incubated with or without transport inhibitors, energy source, or a metabolic inhibitor. Phloridzin and verapamil as inhibitors of sodium glucose transport protein (SGLT1) and P‐glycoprotein, respectively, were not responsible for cellular uptake of SMM. Glucose and sodium azide were not affected by the cellular accumulation of SMM. The efflux ratio of SMM was 0.26, implying that it is not effluxed through Caco‐2 cells. The apparent coefficient permeability (P _app) of SMM was 4.69 × 10^?5 cm/s, indicating that it will show good oral absorption in in vivo .     

Absorption,conjugation and efflux of the flavonoids,kaempferol and galangin,using the intestinal CaCo-2/TC7 cell model

Flavonoids are biologically active compounds in food with potential health effects. We have used the CaCo-2 cell monolayer model to study the absorption and metabolism of two flavonols, a class of flavonoids, specifically kaempferol and galangin. Metabolism experiments allowed identification of five kaempferol conjugates: 3-, 7- and 4′-glucuronide, a sulphate and a glucurono-sulphate; and four galangin conjugates: 3-, 5- and 7-glucuronides, and a sulphate, using specific enzyme hydrolysis, HPLC–MS, and HPLC with post-column metal complexation/tandem MS. Transport studies showed that the flavonols were conjugated inside the cells then transported across the monolayer or effluxed back to the apical side. Sulphated conjugates were preferentially effluxed back to the apical side, whereas glucuronides were mostly transported to the basolateral side. For kaempferol, a small amount of the unconjugated aglycone permeated in both directions, indicating some passive diffusion. When kaempferol-3-glucuronide and quercetin 7-sulphate were applied to either side of the cells, no permeation in either direction was observed, indicating that conjugates cannot re-cross the cell monolayer. Formation of apical kaempferol-7- and 4′-glucuronides was readily saturated, whereas formation of other conjugates at the apical side and all at the basolateral side increased with increasing concentration of kaempferol, implying different transporters are responsible at the apical and basolateral sides. The results highlight the important but complex metabolic changes occurring in flavonoids during absorption.     

Antioxidant Peptide Fractions Isolated from Wheat Germ Protein with Subcritical Water Extraction and Its Transport Across Caco‐2 Cells

Wheat germ protein (WGP) was extracted with subcritical water and then hydrolyzed with Alcalase 2.4 L to obtain antioxidant hydrolysates. Wheat germ peptides (WG‐P, Mw < 1 kDa) were purified by using Sephadex G‐15 column chromatography. The results showed that WG‐P‐4 possessed the strongest DPPH radical scavenging activity in comparison with other peptides fractions. In addition, free amino acids and LC‐MS/MS analysis showed that Gly‐Pro‐Phe, Gly‐Pro‐Glu, and Phe‐Gly‐Glu were the major peptides of WG‐P‐4. Interestingly, the WG‐P‐4 fractions had good absorption characteristic. Moreover, the ratio of P_app both sides of apical compartment (AP) and basolateral compartment (BL) were between 0.5 and 1.0 on Caco‐2 cell model, which indicated that transmembrane transportation was mainly passive transport. Therefore, WG‐P could exert an effective antioxidant action by across the intestinal epithelium.     

Engineered Nanoparticles as Potential Food Contaminants and Their Toxicity to Caco‐2 Cells

Engineered nanoparticles (ENPs), such as metallic or metallic oxide nanoparticles (NPs), have gained much attention in recent years. Increasing use of ENPs in various areas may lead to the release of ENPs into the environment and cause the contamination of agricultural and food products by ENPs. In this study, we selected two important ENPs (zinc oxide [ZnO] and silver [Ag] NPs) as potential food contaminants and investigated their toxicity via an in vitro model using Caco‐2 cells. The physical properties of ENPs and their effects on Caco‐2 cells were characterized by electron microscopy and energy dispersive X‐ray spectroscopic (EDS) techniques. Results demonstrate that a significant inhibition of cell viability was observed after a 24‐h of exposure of Caco‐2 cells to 3‐, 6‐, and 12‐mM ZnO NPs or 0.5‐, 1.5‐, and 3‐mM Ag NPs. The noticeable changes of cells include the alteration in cell shape, abnormal nuclear structure, membrane blebbing, and cytoplasmic deterioration. The toxicity of ZnO NPs, but not that of Ag NPs after exposure to simulated gastric fluid, significantly decreased. Scanning transmission electron microscopy shows that ZnO and Ag NPs penetrated the membrane of Caco‐2 cells. EDS results also confirm the presence of NPs in the cytoplasm of the cells. This study demonstrates that ZnO and Ag NPs have cytotoxic effects and can inhibit the growth of Caco‐2 cells.     

Bioavailability of zinc from infant foods by in vitro methods (solubility,dialyzability and uptake and transport by Caco‐2 cells)

The aim of this study was to evaluate the bioavailability of zinc from infant foods (adapted, follow‐up and toddler milk‐based formulas and fruit juices containing milk and cereals, FMC) using solubility, dialyzability and a model combining simulated gastrointestinal digestion and zinc uptake and transport by Caco‐2 cells. The greater solubility of zinc from infant formulas compared with fruit juices (FMC) could be due to the greater casein phosphopeptide content resulting from casein hydrolysis. The highest zinc dialysis percentage corresponded to FMC, which on the other hand had the lowest zinc contents of the analyzed samples. The presence of organic acids in samples of this kind favors the formation of soluble low molecular weight complexes with zinc, thereby increasing the solubility of the latter. Bifidobacterium addition exerted no effect upon zinc bioavailability. Transport and uptake efficiency in Caco‐2 cells were significantly greater for toddler formulas, which presented the highest casein contents. The greater efficiency in zinc transport and uptake from the powdered toddler formula compared with the liquid formulation could be explained by the effect of Maillard reaction products. Copyright © 2006 Society of Chemical Industry     

Acetylation of hydroxytyrosol enhances its transport across differentiated Caco-2 cell monolayers

Hydroxytyrosol and hydroxytyrosyl acetate are two well-known phenolic compounds with antioxidant properties that are present in virgin olive oil. Since the in vivo biological activity of polyphenols is dependent on their intestinal absorption and metabolism, the absorption of hydroxytyrosol and hydroxytyrosyl acetate and the extent to which they are conjugated and metabolised during transfer across intestinal Caco-2/TC7 cell monolayers, was investigated. LC-DAD and LC-MS were used for the quantification and identification of metabolites. Further evidence was obtained by observing metabolite susceptibility to β-glucuronidase treatment and by comparison of products of in vitro conjugation reactions of authentic phenolics with those produced by the CaCo-2 cells. Homovanillyl alcohol was the only conjugate detected as a result of hydroxytyrosol metabolism, and accounted for 20% of the total metabolites detected in the basolateral compartment after 2 h of incubation. Hydroxytyrosyl acetate was largely converted into free hydroxytyrosol (38.4%) and subsequently metabolised into homovanillyl alcohol (6.7%). In addition, hydroxytyrosyl acetate glucuronide (17.4%) together with non-metabolised hydroxytyrosyl acetate (37.5%) were also detected. Both hydroxytyrosyl acetate and hydroxytyrosol were transferred across human Caco-2/TC7 cell monolayers, but the acetylated compound exhibited an apparent permeability (Papp_AP→BL/Papp _BL→AP) 2.1-fold higher than free hydroxytyrosol. For both compounds, all conjugates were preferentially transported to the basolateral side. These results show that the acetylation of hydroxytyrosol significantly increases its transport across the small intestinal epithelial cell barrier, and supports further research into hydroxytyrosyl acetate as a hydroxytyrosol prodrug offering enhanced bioavailability.     

Interactions between ABC‐transport proteins and the secondary Fusarium metabolites enniatin and beauvericin

Enniatins (ENN) and beauvericin (BEA) exert cytotoxic properties. Here, we observed that their impact on Ca²⁺‐homeostasis can be reversed by exogenous ATP. Thus, we investigated whether membrane‐located ATP‐binding cassette (ABC) transporters influence ENNs‐ and BEA‐induced cytotoxicity. In short‐term exposure assays breast cancer resistance protein (ABCG2)‐overexpression weakly but significantly reduced the cytotoxic activity of BEA but not ENNs. In contrast, multidrug resistance‐associated protein‐1 (ABCC1)‐ and P‐glycoprotein (ABCB1)‐overexpression was not protective under identical conditions. ABCG2‐mediated resistance against BEA was reversible by ABCG2 modulators. In long‐term exposure assays, ABCG2 and ABCB1 significantly protected against ENNs‐ and to a lesser extent BEA‐induced cytotoxicity. Moreover, both fusariotoxins potently inhibited the ABCG2‐ and ABCB1‐mediated efflux of specific fluorescent substrates, with BEA being more effective. Additionally, ATPase and photoaffinity‐labelling assays proofed interaction of both substances with ABCG2 and ABCB1. Remarkably, 2 years selection of KB‐3‐1 cells against both fusariotoxins resulted only in two‐fold ENNs but negligible BEA resistance. Interestingly, the selected sublines displayed upregulation of multidrug resistance proteins and crossresistance to other chemotherapeutics. Summarizing, ABCG2 and ABCB1 slightly but significantly protect human cells against ENNs‐ and BEA‐induced cytotoxicity. However, both mycotoxins potently interact with ABCB1 and ABCG2 transport functions suggesting influences on bioavailability of xenobiotics and pharmaceuticals.     

Development of a three‐tier in vitro system,using Caco‐2 cells,to assess the effects of lactate on iron uptake and transport from rye bread following in vitro digestion

A three‐tier Caco‐2 cell system was developed to assess simultaneously iron dialysability, uptake and transport across the Caco‐2 monolayer from an in vitro digested food matrix. The effect of lactate (0–200 mmol L⁻¹) on iron absorption from rye bread subjected to simulated peptic (pH 5.5) and pancreatic digestion (pH 6.5) was investigated to model absorption pre and post the sphincter of Oddi. Lactate increased dialysability (11.8%, P < 0.05) in peptic digests whereas it reduced it in pancreatic digests (4.9%, P < 0.001). Iron uptake from the peptic digests was in the region of 39–76 pmol mg⁻¹ protein whereas it decreased from 281 to 51 pmol mg⁻¹ protein in pancreatic digests. Iron transport was calculated for the peptic digests from [¹⁴C]polyethylene glycol movement and only at 200 mmol L⁻¹ lactate was there any detectable transcellular transport (180 pmol mg⁻¹ protein, P < 0.05). Iron absorption was positively correlated to dialysable iron for both digests (R² = 0.48 and 0.41, respectively, P < 0.01) and the effect of lactate was therefore associated mainly with iron bioaccessibility. The three‐tier system showed the potential to obtain detailed insight into each step involved in iron transport across the monolayer from a food mixture. Copyright © 2006 Society of Chemical Industry     

Effect of addition of Agaricus blazei mushroom residue to milk enriched with Omega‐3 on the prevention of lipid oxidation and bioavailability of bioactive compounds after in vitro gastrointestinal digestion

The residue from a hydroalcoholic extract of the mushroom Agaricus blazei (MAR) was evaluated for phenolic compounds, flavonoids and antioxidant activity. The ability of MAR to slow the oxidation of Omega‐3 resulting from light exposure in milk matrix, and its bioavailability after in vitro digestion was investigated. MAR presented phenolic compounds and flavonoids and showed antioxidant activity. At each concentration, addition of MAR to Omega‐3‐supplemented milk inhibited the production of conjugated dienes and malonaldehyde compared with samples without MAR. The bioavailability assay showed that polyphenols were still present after in vitro digestion and had antioxidant activity.     

Antioxidant activity in HepG2 cells,immunomodulatory effects in RAW 264.7 cells and absorption characteristics in Caco‐2 cells of the peptide fraction isolated from Dendrobium aphyllum

DA‐P, fraction of peptides with a molecular weight <1 kDa isolated from Dendrobium aphyllum, was analysed in three types of cell lines to verify its bioactivity and absorptivity. The cellular antioxidant activity of DA‐P in HepG2 cells was used and results revealed an EC50 of 2.88 ± 0.143 mg mL^?1 and a CAA unit of 63.46 ± 2.11 μm QE/100 g peptides. DA‐P treatment enhanced the secretion of cytokines in RAW 264.7 cells. After demonstrating the presence of tight junctions in Caco‐2 monolayers, the absorption was 25.57% ± 0.016% and 19.7% ± 0.012% from different sides. The relatively high absorption indicated that the antioxidant‐relevant immune functions of DA‐P had a greater possibility to be absorbed by Caco‐2 cells. Free amino acids and LC‐MS/MS analysis indicated the degradation and expulsion of components after the absorption of DA‐P, and Ser‐Ser‐Arg was able to come across the monolayers.