Transepithelial Transport of Caffeoylquinic Acids in Caco-2 Cells: Structure Dependence and Modulation by Dietary Flavonoids

Abstract

Caffeoylquinic acids (CQAs) are polyphenolic compounds widely present in daily diets, but their bioactivities are limited by poor intestinal absorption, the mechanisms of which remain incompletely understood for various isomers. This study investigated the transepithelial transport of three mono-CQAs and three di-CQAs using Caco-2 monolayers. Concurrently, the potential of five dietary flavonoids to enhance intestinal absorption by modulating efflux transporters was evaluated. Results suggest that CQAs were mainly transported via passive paracellular diffusion. The apparent permeability coefficients (P_app) of mono-CQAs were (1.49 ± 0.22) × 10⁻⁶, (1.49 ± 0.25) × 10⁻⁶, and (2.15 ± 0.57) × 10⁻⁶ cm/s, which were significantly higher than those of di-CQAs. And the efflux of 5-CQA, 3,4-diCQA, and 3,5-diCQA was primarily mediated by P-gp. Among the five dietary flavonoids tested for their potential to inhibit this efflux, quercetin and kaempferol exhibited the most potent enhancing CQA uptake. They increased the P_app of 5-CQA from (2.15 ± 0.21) × 10⁻⁶ to (3.05 ± 0.08) × 10⁻⁶ cm/s and (2.57 ± 0.17) × 10⁻⁶ cm/s, respectively. Similar promoting trends were observed for 3,4-diCQA and 3,5-diCQAs. Molecular docking revealed that CQAs and these effective flavonoids share common binding residues within the P-gp pocket, providing a structural basis for the inhibition of efflux. These findings provide insights into the intestinal transport of structurally diverse CQAs and highlight the potential of dietary flavonoids to improve the oral bioavailability of CQAs.

1. Introduction

Caffeoylquinic acids (CQAs), formed by the esterification of caffeic acid and quinic acid, are significant plant secondary metabolites. They are found in a variety of edible and medicinal plants, such as coffee, Flos chrysanthemums Indici, honeysuckle, and artichokes [1,2,3,4,5]. Based on the number of caffeoyl groups attached to the quinic acid backbone, CQAs are classified into mono-, di-, tri-, and multi-CQAs, among which mono- and di-CQAs are the most prevalent in nature (Figure 1) [6]. Research has shown that CQAs exhibit multiple biological activities, particularly exhibiting antioxidant, anti-inflammatory, and metabolic regulatory effects, indicating their potential as bioactive ingredients in functional foods and nutraceuticals [6,7,8,9,10].

The health effects of CQAs are closely related to their bioavailability. However, their poor oral absorption significantly restricts their bioefficacy in vivo. It has been reported that only approximately one-third of ingested CQAs could be absorbed, with the small intestine serving as the primary absorption site [11,12]. Studies utilizing in vitro models (e.g., Ussing chamber and the Caco-2 cell monolayer) have demonstrated that CQA transport involves both passive diffusion (via transcellular and paracellular pathways) and carrier-mediated mechanisms [13,14,15]. During the transcellular transport process, CQAs are regulated by efflux transporters that actively pump absorbed molecules back into the intestinal lumen, thereby significantly reducing their absorption efficiency [16]. Among these transporters, ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP), play a critical role in limiting CQA absorption [17,18]. Therefore, identifying natural compounds that could modulate these transporters has emerged as a promising approach to improve the bioavailability of CQAs.

Flavonoids, a class of dietary polyphenols widely present in fruits, vegetables, and cereals, are generally regarded as safe and possess diverse health benefits [19,20]. Accumulating evidence has indicated that certain flavonoids could regulate the expression or activity of ABC transporters [21,22]. For instance, in vitro studies have demonstrated that epigallocatechin gallate (EGCG), kaempferol, quercetin, and genistein could facilitate the intestinal uptake of 7,8-dihydroxyflavone by inhibiting P-gp [23]. Moreover, quercetin has been reported to improve EGCG absorption by downregulating MRP2 in both Caco-2 cells and human studies [24]. Furthermore, tangeretin has been shown to improve the bioavailability of silybin in rats by inhibiting efflux transporters [25]. Collectively, these findings suggest that flavonoids may enhance CQA absorption by modulating efflux transporter activity.

This study aimed to investigate the transepithelial transport characteristics of the six naturally abundant CQA isomers, including three mono-CQAs (3-CQA, 4-CQA, 5-CQA) and three di-CQAs (3,4-diCQA, 3,5-diCQA, 4,5-diCQA), using the Caco-2 cell monolayer model. It further identified the major efflux transporters involved in CQA absorption and evaluated the capacity of five dietary flavonoids (hesperetin, EGCG, quercetin, kaempferol, and naringenin) to enhance CQA uptake by inhibiting major efflux transporters. Furthermore, molecular docking was performed to clarify the binding interactions of CQAs and flavonoids with major efflux transporters, providing structural insights into the molecular mechanisms of efflux and its inhibition. Overall, this study elucidates the intestinal transport of structurally diverse CQAs and highlights the potential of dietary flavonoids as modulators to improve oral bioavailability, thereby supporting their application in functional foods.

2. Materials and Methods

2.1. Materials and Reagents

Caco-2 cells (human colon adenocarcinoma) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) and Minimum Essential Medium (MEM, high glucose) were purchased from Gibco (Waltham, MA, USA). Hanks’ balanced salt solution (HBSS) and trypsin-EDTA solution (0.25%) were supplied by Soborbio Biotechnology Co., Ltd. (Shanghai, China) and Genom Biotechnology Co., Ltd. (Hangzhou, China), respectively. Transwell inserts (polycarbonate membrane, 0.4 μm pore size, 12 mm diameter) were provided by Corning Inc. (Lowell, MA, USA). The MTT assay reagent was sourced from Gen-View Scientific Inc. (El Monte, CA, USA). Reference standards of 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA, and the flavonoids (hesperetin, EGCG, quercetin, kaempferol, and naringenin) were purchased from Chengdu Puruifa Technology Co., Ltd. (Chengdu, China), with all purity ≥ 98%. MK-571 (≥96%), verapamil (≥98%), Ko143 (≥99%), and EGTA (≥99%) were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). HPLC-grade methanol and acetonitrile were purchased from Fisher Scientific (Waltham, MA, USA), and formic acid (HPLC grade) from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China).

2.2. Cell Culture

Cell Culture Caco-2 cells were cultured following the method previously reported [26]. Cells at passages 40 to 60 were utilized for the experiments [27].

2.3. Cytotoxicity Assay

The cytotoxicity of CQAs and flavonoids was assessed using the MTT assay [28,29], following the procedures described in our previously study [26]. Cells were exposed to individual CQAs or flavonoids at concentrations of 10, 20, 40, 80, and 100 μM for 24 h. Control wells contained vehicle (DMSO, final concentration ≤ 0.1%), while blank wells contained medium without cells. Cell viability (%) was calculated according to the following formula:

$$\mathrm{Cell} \mathrm{viability}\left(\%\right) = ( {\displaystyle \frac{{\mathrm{OD}}_{\mathrm{Sample}}- {\mathrm{OD}}_{\mathrm{Blank}}}{{\mathrm{OD}}_{\mathrm{Control}} - {\mathrm{OD}}_{\mathrm{Blank}}}} ) \times 100\%$$

(1)

2.4. Caco-2 Absorption Model

2.4.1. Model Establishment

Log-phase Caco-2 cells were enzymatically digested using 0.25% trypsin-EDTA, then reconstituted in complete growth medium. After counting, the cells were seeded into the apical (AP) chamber of 12-well Transwell inserts at a density of 2 × 10⁵ cells/mL (0.5 mL per insert), with 1.5 mL of complete medium added to the basolateral (BL) chamber. The cells were maintained at 37 °C in 5% CO₂ for 21 days, with the medium replaced every other day during the first two weeks and daily in the third week [27].

2.4.2. Transepithelial Electrical Resistance (TEER) Measurement

Before conducting transport experiments, the integrity of Caco-2 cell monolayers was assessed by measuring transepithelial electrical resistance (TEER) [30]. Measurements were performed using an epithelial volt/ohmmeter (ERS2, Millicell, Merck Millipore, Berlin, Germany) in three random positions per well, and blank resistance (R₀) was measured using cell-free inserts. TEER values (Ω·cm²) were calculated using the following formula:

$$\mathrm{TEER} = \left({\mathrm{R}}_{\mathrm{t} }- {\mathrm{R}}_0\right) \times \mathrm{S}$$

(2)

where S is the effective membrane area (1.12 cm² in this model).

2.4.3. FD-4 Permeability Test

The paracellular permeability of the monolayers was assessed using fluorescein isothiocyanate–dextran (FD-4) [31]. Standards (0–20 μM) were prepared in MEM medium, and their fluorescence intensity (excitation 495 nm, emission 520 nm) was measured to generate a calibration curve. For permeability assessment, 500 μL of FD-4 solution (100 μM) was applied to the AP side, and 1.5 mL of blank medium was introduced to the BL side. After incubating at 37 °C for 3 h, samples were collected from the BL side, and FD-4 concentrations were quantified using the calibration curve. The apparent permeability coefficient (P_app) was then calculated.

2.5. Transepithelial Transport Experiments of CQAs

Stock solutions of 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA were prepared in DMSO, with the final DMSO concentration not exceeding 0.1%. These solutions were subsequently diluted in HBSS to obtain working concentrations of 10, 20, 40, and 80 μM. Prior to the initiation of transport experiments, the monolayers were rinsed twice with HBSS. Following this, 0.5 mL of HBSS was added to the AP chamber and 1.5 mL to the BL chamber, and the plates were equilibrated at 37 °C for 30 min [32].

2.5.1. Time-Dependent Transport Assay

CQA solution (40 μM, 0.5 mL) was added to the donor chamber, while 1.5 mL of blank HBSS was added to the receiver chamber (AP → BL). For the reverse direction (BL → AP), the donor and receiver chambers were switched accordingly. Samples (200 μL) were collected from the receiver chamber at 30, 60, 90, 120, 150, and 180 min and immediately replaced with an equal volume of fresh HBSS.

2.5.2. Concentration-Dependent Transport Assay

Donor chambers were loaded with CQA solutions at concentrations of 10, 20, 40, and 80 μM. Samples incubated for 120 min were collected from the receiver chamber for quantitative analysis.

2.5.3. Paracellular Transport Evaluation

Caco-2 cell monolayers were incubated with Ca²⁺/Mg²⁺-free HBSS containing 2.5 mmol/L EGTA at 37 °C for 15 min. Subsequently, CQA solution (40 μM) was prepared in Ca²⁺/Mg²⁺-free HBSS and added to the AP chamber, while blank HBSS was added to the BL chamber. After another 120 min, the CQA content in the BL side was measured. Mannitol was utilized as a positive control for paracellular permeability.

2.5.4. Efflux Transporter Involvement Assay

Specific inhibitors [verapamil (100 μM, P-gp inhibitor), MK571 (100 μM, MRP inhibitor), and Ko143 (10 μM, BCRP inhibitor)] were employed to assess the involvement of efflux transporters [32]. Monolayers were pre-incubated with each inhibitor added to both the AP and BL chambers for 30 min. Subsequently, CQA solutions (40 μM) containing the respective inhibitor were applied to the donor chamber (AP for AP → BL transport or BL for BL → AP transport), while blank HBSS was added to the receiver chamber.

P_app was calculated using the formula:

$$P_{\mathrm{app}} = {\displaystyle \frac{∆\mathrm{Q}/∆\mathrm{t}}{\mathrm{A} \times {\mathrm{C}}_0}}$$

(3)

where ΔQ is the amount of compound transported during the time interval Δt, A represents the surface area of the monolayers (1.12 cm²), and C₀ is the initial donor concentration. P_app is expressed in cm/s.

The efflux ratio was calculated as:

$$\mathrm{ER} = {\displaystyle \frac{ P_{\mathrm{app}\mathrm{BA}}}{P_{\mathrm{app}\mathrm{AB}}}}$$

(4)

P_{app BA} refers to the P_app value measured from the BL to the AP direction, while P_{app AB} is the P_app value measured from the AP to the BL direction.

2.6. Transepithelial Transport Experiments to Evaluate Dietary Flavonoids as Potential P-gp Inhibitors

The dietary flavonoids, including hesperetin, EGCG, quercetin, kaempferol, and naringenin, were used to evaluate their potential to enhance the transport of CQAs by inhibiting P-gp. Prior to the transport experiment, cells were pre-incubated with the flavonoids on the AP side for 30 min. The medium was then replaced with HBSS containing equal concentrations of the flavonoid and CQAs (40 μM each) in the donor chamber (AP or BL), while blank HBSS was added to the opposite chamber. After 120 min, samples (200 μL) were collected from the receiver chamber for CQA quantification. Verapamil (100 μM), a well-established P-gp inhibitor, was used as a positive control [23].

2.7. Molecular Docking Analysis

The potential interactions between CQAs, flavonoids, and P-gp were evaluated using molecular docking [33]. The three-dimensional structure of P-gp (PDB ID: 6FN1) was obtained from the RCSB Protein Data Bank (https://www.rcsb.org/, accessed on 10 September 2025), and the 3D structures of the ligands were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/, accessed on 10 September 2025). PyMOL was employed for protein preprocessing, which included the removal of water and native ligands. File format conversion was handled by OpenBabelGUI. The actual molecular docking was performed using AutoDock Vina v.1.5.7, followed by visualization in PyMOL v.2.2.0 and detailed analysis in Discovery Studio 2019.

2.8. UPLC Analysis of CQAs

Quantification of CQAs was carried out on an ACQUITY UPLC H-Class system (Waters, Milford, MA, USA) with an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm; Waters, Wexford, Ireland) [34]. Prior to UPLC analysis, samples collected from the transport experiments were centrifuged at 10,000 rpm for 10 min, and the supernatants were filtered through a 0.22 μm organic membrane filter before injection. The mobile phase consisted of 0.1% (v/v) formic acid in water (solvent A) and acetonitrile (solvent B). The gradient elution program was as follows: 0–1 min, 90% A; 1–4 min, 85% A; 4–5 min, 82% A; 5–18 min, 82% A; 18–19 min, 50% A; 19–21 min, 20% A; 21–23 min, 90% A; 23–25 min, 90% A. The flow rate was 0.2 mL/min, the injection volume was 1 μL, and detection was performed at 325 nm using a PDA detector (Waters, Wexford, Ireland).

2.9. Statistical Analysis

All data obtained from three replicate experiments are presented as the mean ± SD. Statistical analyses were performed using IBM SPSS Statistics 26. Group comparisons involving two sets of data were evaluated using the unpaired two-tailed Student’s t-test. For analyses encompassing more than two groups, results were subjected to one-way ANOVA, followed by Tukey’s honestly significant difference post hoc analysis. Differences were deemed statistically significant at p < 0.05.

3. Results and Discussion

3.1. Validation of Caco-2 Monolayer Integrity

The integrity of the Caco-2 cell monolayers was assessed by measuring TEER, which is a widely recognized marker of tight junction formation [30]. A TEER value exceeding 300 Ω·cm² indicates the formation of a dense and functionally intact epithelial barrier. Over the first 15 days of culture, TEER values gradually increased, reaching a peak of approximately 800 Ω·cm² at 15 days and remaining stable above this threshold until 21 days (Figure 2). This result confirmed the establishment of intact tight junctions.

Monolayer permeability was further evaluated using FD-4, a low-permeability marker [31]. The fluorescence intensity of FD-4 exhibited an excellent linear correlation with concentration (y = 244.38x + 334.69, R² = 0.9993). In our study, the P_app of FD-4 was (0.454 ± 0.076) × 10⁻⁶ cm/s, which was lower than the well-recognized threshold of 0.5 × 10⁻⁶ cm/s, indicating low permeability [31]. These results confirmed the integrity of the monolayer as suitable for transport studies.

3.2. Cytotoxicity of CQAs in Caco-2 Cells

The cytotoxicity of six representative CQAs (3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) was evaluated in Caco-2 cells using the MTT assay to establish suitable concentrations for transport experiments. As shown in Figure 3a,b, cell viability remained above 90% at concentrations up to 80 μM, with no significant differences compared to the control (p > 0.05). A slight reduction in viability was noted at 100 μM for certain CQAs. These results suggested that CQAs exhibited low cytotoxicity within the tested concentration range, supporting the use of ≤80 μM as a safe and reliable concentration for subsequent transepithelial transport studies.

3.3. Effect of Time and Concentration on the Transport of CQAs in Caco-2 Monolayers

The bidirectional transport of six CQAs progressively increased over 180 min, indicating time-dependent transepithelial permeability across Caco-2 monolayers (Figure 4a,b). These findings aligned with previous Ussing chamber studies reporting time-dependent absorption of 5-CQA in intestinal tissue [35]. The transport was also concentration-dependent, showing linear increases from 10 to 80 μM without signs of saturation (Figure 4c,d). This linear, non-saturable profile is consistent with earlier studies that reported non-saturable CQA transport at 10–50 μM and a comparable linear dose–response relationship [18,35]. Collectively, these results suggest that passive diffusion is the predominant transport mechanism of these six CQAs; similar results were found in other phenolic acids [15,36].

The absorption of these six CQAs was further evaluated. According to established Caco-2 criteria, compounds with P_app < 1 × 10⁻⁶ cm/s are considered poorly absorbed, while values between 1 × 10⁻⁶ and 1 × 10⁻⁵ cm/s indicate moderate absorption [14,37]. In our study, all six CQAs at 40 μM exhibited P_app values within the moderate absorption range (

Table 1. The bi-directional P_app and ER values of CQAs at 40 μM after 120 min transport across Caco-2 monolayers.

	Papp AB (×10−6 cm/s)	Papp BA (×10−6 cm/s)	ER
3-CQA	1.49 ± 0.25 b	1.74 ± 0.43	1.16
4-CQA	1.49 ± 0.22 b	1.82 ± 0.09	1.22
5-CQA	2.15 ± 0.57 a	3.83 ± 0.60 *	1.78
3,4-diCQA	0.85 ± 0.23 bc	1.80 ± 0.16 **	2.11
3,5-diCQA	0.96 ± 0.14 c	2.15 ± 0.06 ***	2.24
4,5-diCQA	0.71 ± 0.11 c	1.01 ± 0.11 *	1.41

Data are expressed as mean ± SD (n = 3). Different letters indicate significant differences among CQAs (p < 0.05). * p < 0.05, ** p < 0.01, *** p < 0.005 compared with transport in the AP → BL direction.

). Among them, 5-CQA exhibited the highest AP→BL transport, followed by 4-CQA and 3-CQA, whereas 3,5-diCQA showed the greatest permeability among the di-CQAs. Notably, the ER of 5-CQA (ER = 1.78), 3,4-diCQA (ER = 2.11), and 3,5-diCQA (ER = 2.24) exceeded 1.5, suggesting the involvement of efflux mechanisms in their transport [38]. These results align with previous reports in which active efflux restricted the intestinal absorption of 5-CQA and 3,5-diCQA [18,35,36]. Major efflux transporters involved in intestinal absorption belong to the ABC family and are primarily localized on the apical membrane of intestinal epithelial cells [17,18]. In line with this, the involvement of efflux processes observed in this study suggests that CQAs can access the intracellular compartment during transepithelial passage. Transcellular passive diffusion across the epithelial cell membrane may therefore represent one of the transport routes contributing to CQA transepithelial transport in the Caco-2 monolayer.

In this study, mono-CQAs consistently showed higher transport rates than di-CQAs. This difference could be attributed to the smaller molecular size (MW ≈ 354 g/mol for mono-CQAs and MW ≈ 516 g/mol for di-CQAs) and lower polarity of mono-CQAs, both of which would facilitate passive membrane permeation. Previous studies have shown that intestinal absorption of di-CQAs was markedly lower than that of mono-CQAs, with negligible tissue accumulation [35]. Beyond these general differences, the position of the caffeoyl groups might also influence transport. Specifically, 3,4-diCQA and 3,5-diCQA exhibited elevated ER, suggesting greater recognition by efflux transporters. However, 4,5-diCQA displayed a minimal ER, possibly due to a more compact conformation that reduces transporter accessibility. Collectively, these findings support the fact that both physicochemical properties and transporter interactions are key determinants of intestinal permeability [39].

3.4. Effect of the Paracellular Pathway on the Transport of CQAs in Caco-2 Monolayers

Ca²⁺ plays a crucial role in maintaining the integrity of tight junctions in Caco-2 monolayers. EGTA, a calcium chelator, could disrupt these junctions, thereby increasing paracellular permeability [40]. In this study, the P_app of six CQAs was measured with EGTA treatment to assess the contribution of the paracellular pathway to their transport. Mannitol, a classical paracellular marker, was included as a positive control. The marked increase in mannitol permeability following EGTA exposure confirmed the effective disruption of tight junction (Figure 5). Similarly, all six CQAs showed significant increases in P_app after EGTA treatment, indicating enhanced permeability. These findings suggest that the paracellular pathway may contribute to the passive transport of CQAs. However, given the substantial alteration of tight junction integrity induced by Ca²⁺/Mg²⁺-free HBSS supplemented with EGTA, the increased transport observed in this system primarily reflects the sensitivity of CQA permeability to junctional opening, rather than serving as definitive evidence of physiological paracellular transport. Nevertheless, this behavior is consistent with previous reports on chlorogenic acid and related phenolic acids, in which the paracellular pathway was identified as an important route for intestinal transport in epithelial models [13,15]. To further substantiate this contribution under less disruptive conditions, future studies using graded Ca²⁺ concentrations combined with TEER monitoring would be valuable.

3.5. Effect of Efflux Transporters on the Transcellular Transport of CQAs in Caco-2 Monolayers

Our results suggested that the efflux mechanisms were involved in the transport of 5-CQA, 3,4-diCQA, and 3,5-diCQA (Table 1). Therefore, 5-CQA, 3,4-diCQA, and 3,5-diCQA were chosen for bidirectional transport studies utilizing classical inhibitors [Verapamil (P-gp), MK571 (MRP2), and Ko143 (BCRP)] to further evaluated the contribution of the efflux transporter in their absorption. The results are summarized in

Table 2. Effects of efflux transporter inhibitors on the bi-directional transport of 5-CQA, 3,4-diCQA, and 3,5-diCQA across Caco-2 monolayers.

	Papp (×10−6 cm/s)		ER
	AP → BL	BL → AP
5-CQA
Control	2.14 ± 0.30	3.84 ± 0.14	1.80
Verapamil	3.23 ± 0.41 *	3.26 ± 0.08 ##	1.01 **
MK 571	2.18 ± 0.25	3.80 ± 0.11	1.74
Ko l43	2.23 ± 0.66	3.38 ± 0.22 #	1.52 *
3,4-diCQA
Control	0.84 ± 0.04	1.81 ± 0.17	2.16
Verapamil	0.99 ± 0.04 *	1.05 ± 0.12 ##	1.06 **
MK 571	0.92 ± 0.17	1.61 ± 0.14	1.75
Ko l43	0.80 ± 0.12	1.77 ± 0.20	2.21
3,5-diCQA
Control	0.94 ± 0.03	2.17 ± 0.03	2.32
Verapamil	1.16 ± 0.05 *	1.02 ± 0.07 ###	0.88 **
MK 571	0.96 ± 0.08	2.05 ± 0.09	2.14
Ko l43	1.04 ± 0.15	1.35 ± 0.16 ##	1.30 *

Data are expressed as mean ± SD (n = 3). * p < 0.05 compared with the AP → BL control, ^# p <0.05, ^## p < 0.01, ^### p < 0.005 compared with the BL → AP control, * p < 0.05, ** p < 0.01 compared with the ER control.

.

Verapamil significantly increased the P_{app AB} and decreased the P_{app BA} in 5-CQA, 3,4-diCQA, and 3,5-diCQA compared to those of the Control, with a decrease in ER. MK571 showed no significant effect on these three compounds. Ko143 lowered the P_{app BA} in both 5-CQA and 3,5-diCQA from (3.84 ± 0.14) × 10⁻⁶ to (3.38 ± 0.22) × 10⁻⁶ cm/s and (2.17 ± 0.03) × 10⁻⁶ to (1.35 ± 0.16) × 10⁻⁶ cm/s, respectively. These results indicated that P-gp was the principal efflux transporter that restricted the intestinal uptake of these CQAs, with an additional contribution of BCRP to the efflux of 5-CQA and 3,5-diCQA. A previous study reported that P-gp was the primary efflux transporter of chlorogenic acid in Ussing chamber studies [35], and multiple ABC transporters restricted the intestinal absorption of 3,5-diCQA [36]. The predominance of P-gp might be due to its broad substrate specificity for polyphenols, whereas BCRP preferentially recognizes hydroxyl-rich caffeoyl structures, and MRP2 generally prefers organic anions and conjugated metabolites [33,41].

3.6. Cytotoxicity of Flavonoids in Caco-2 Cells

Having established that P-gp was the primary efflux transporter limiting the uptake of CQA (Table 2), a panel of representative dietary flavonoids were selected based on their structural diversity and documented potential for P-gp inhibition to evaluate their potential modulatory effects on the efflux of CQAs [23,24,25]. These flavonoids included naringenin and hesperetin (both flavanones), epigallocatechin gallate (EGCG, a catechin), and kaempferol and quercetin (both flavonols).

The cytotoxicity of these compounds (10–100 μM) was assessed in Caco-2 cells using the MTT assay. All five flavonoids displayed low cytotoxicity up to 80 μM, with cell viability remaining above 90% (Figure 6). Therefore, 80 μM was selected as the maximum concentration for subsequent transport inhibition studies to minimize cytotoxic interference.

3.7. Effect of Different Flavonoids on Bidirectional Permeation and Efflux of CQAs in Caco 2 Monolayers

The effects of flavonoids on the efflux of CQAs is illustrated in

Table 3. Effects of natural flavonoids on bi-directional P_app and ER of CQAs across Caco-2 monolayers.

	Papp (×10−6 cm/s)		ER
	AP → BL	BL → AP
5-CQA
Control	2.15 ± 0.21	3.86 ± 0.08	1.79
Verapamil	3.25 ± 0.13 **	3.27 ± 0.02 ##	1.01 **
Naringenin	2.37 ± 0.17	3.99 ± 0.11	1.69
EGCG	2.28 ± 0.03	3.64 ± 0.13	1.59
Hesperetin	2.43 ± 0.08	3.53 ± 0.15 #	1.45
Quercetin	3.05 ± 0.08 **	3.37 ± 0.14 ##	1.10 **
Kaempferol	2.57 ± 0.17 *	3.69 ± 0.29	1.44
3,4-diCQA
Control	0.95 ± 0.07	2.11 ± 0.09	2.25
Verapamil	1.17 ± 0.11 **	1.02 ± 0.03 ###	0.87 **
Naringenin	0.95 ± 0.12	2.06 ± 0.08	2.18
EGCG	1.05 ± 0.07	1.73 ± 0.07 ###	1.64
Hesperetin	1.04 ± 0.04	2.00 ± 0.13	1.92
Quercetin	1.14 ± 0.09 *	1.30 ± 0.06 ###	1.15 **
Kaempferol	1.11 ± 0.04	1.33 ± 0.03 ###	1.20 **
3,5-diCQA
Control	0.84 ± 0.03	1.82 ± 0.04	2.16
Verapamil	1.00 ± 0.07 **	1.04 ± 0.07 ##	1.05 **
Naringenin	0.85 ± 0.07	1.73 ± 0.05	2.03
EGCG	0.93 ± 0.09	1.73 ± 0.04	1.86
Hesperetin	0.90 ± 0.12 *	1.69 ± 0.03	1.88
Quercetin	0.95 ± 0.05 **	1.19 ± 0.06 ##	1.26 **
Kaempferol	0.87 ± 0.03 *	1.30 ± 0.06 #	1.50 *

Data are expressed as mean ± SD (n = 3), * p < 0.05, ** p < 0.01 compared with the AP → BL control, ^# p < 0.05, ^## p < 0.01, ^### p < 0.005 compared with the BL → AP control, * p < 0.05, ** p < 0.01 compared with the ER control.

. Co-incubation with flavonoids produced distinct effects on the bidirectional transport of CQAs across Caco-2 monolayers. Quercetin exhibited the most pronounced inhibitory effect on efflux, lowering the ER values of 5-CQA, 3,4-diCQA, and 3,5-diCQA to 1.10, 1.15, and 1.26, respectively. In the case of 5-CQA, P_{app AB} increased from (2.14 ± 0.30) × 10⁻⁶ to (3.05 ± 0.08) × 10⁻⁶ cm/s, while P_{app BA} decreased from (4.09 ± 0.35) × 10⁻⁶ to (3.37 ± 0.14) × 10⁻⁶ cm/s (both p < 0.01), with a reduction in ER from 1.79 to 1.10. Kaempferol also showed significant but more moderate effects, particularly on di-CQAs, decreasing the ER of 3,4-diCQA from 2.25 to 1.20 and that of 3,5-diCQA from 2.16 to 1.50. In contrast, hesperetin and EGCG exhibited only slight improvements in absorptive transport with minimal reductions in ER, and naringenin demonstrated negligible effects.

These results align with previous reports showing that flavonols at equimolar concentrations could enhance the P_{app AB} and reduce the P_{app BA} of 7,8-DHF in Caco-2 cells, leading to a reduction in ER values [23]. The underlying mechanism might involve the inhibition of P-gp activity by flavanols through downregulating MDR1 expression and directly suppressing transporter function. Overall, these findings indicate that flavonols, especially quercetin and kaempferol, would significantly improve CQA transport by reducing efflux, suggesting their potential as dietary strategy adjuvants to enhance bioavailability of CQAs.

3.8. Molecular Docking of CQAs and Flavonoids with P-gp

Molecular docking was performed to further investigate the mechanism by which flavonoids inhibited the efflux of CQAs. Docking analysis revealed that all six CQAs exhibited favorable binding affinities toward P-gp, with binding energies of ≤−5 kcal/mol (

Table 4. Molecular docking information of CQAs and flavonoids with P-gp.

Ligands	Binding Energy (kcal/mol)	Binding Sites
Verapamil	−8.51	LEU-723, ASN-841, GLY-721, GLN-724, ASN-720, GLN-837, PHE-769, VAL-990, PHE-993, MET-298, ASN-295, TRP-231, ALA-986, GLN-989, PHE-302, TYR-306, ILE-305
3-CQA	−5.448	ILE-339, ILE-305, PHE-342, LEU-338, ALA-301, PHE-302, GLN-989, TRP231, MET-875, TYR-309, ALA228, ALA232, GLN346, LEU-235, LEU878, GLU874
4-CQA	−5.238	TYR-309, LEU-338, ILE305, ILE-339, PHE-342, SER-343, GLN-346, ALA-228, MET-875, LEU-235, LEU-878, TRP-231
5-CQA	−6.187	LEU-235, MET-875, TRP-231, PHE-993, GLN-989, MET-298, GLN-837, GLN-772, PHE-769, ASN-720, SER-765, TYR-306, LEU-723, GLN-724, PHE-302, ALA-986, ASN-841, VAL-990
3,4-diCQA	−7.143	GLN-837, ASN-720, GLY-721, LEU-723, ALA-986, MET-298, PHE-993, TRP-231, VAL-990, PHE-302, GLN-989, MET-985, GLN-724, PHE-982, PHE-727, TYR-309, ILE-305, TYR-306, ASN-841
3,5-diCQA	−6.917	MET-298, PHE-993, VAL-990, MET-985, GLN-989, PHE-982, GLN-724, TYR-309, ALA-986, PHE-727, ASN-720, GLY-721, ILE-305, GLN-837, ASN-841, LEU-723, TYR-306, PHE-302
4,5-diCQA	−6.404	TYR-306, GLN-724, GLN-989, PHE-302, PHE-342, ILE-305, TYR-309, ALA-301, ILE-339, LEU-338, GLU-346, ALA-232, LEU-235, LEU-878, TRP-231
Naringenin	−7.252	VAL-990, ALA-990, GLN-837, ASN-295, GLN-772, MET-298, PHE-796, ASN-720, PHE-302, LEU-723, GLU-724, TYR-306, PHE-993, VAL-834
EGCG	−7.001	GLN-989, MET-985, ALA-986, PHE-302, ASN-841, GLN-724, GLY-721, ASN-720, MET-298, PHE-993, VAL-990, PHE-769, GLN-772, GLN-837, ALA-833, TRP-231
Hesperetin	−7.187	VAL-990, ALA-986, VAL-834, ALA-994, ASN-295, MET-298, GLN-772, PHE-769, PHE-302, GLN-724, TYR-306, TEU-723, GLY-721, ASN-720, PHE-993, GLN-837
Quercetin	−8.182	GLN-724, ALA-986, ASN-841, GLY-721, ASN-720, VAL-990, GLN-837, PHE-769, GLN-772, ASN-295, PHE-993, MET-298, PHE-302

). Among these, 5-CQA, 3,4-diCQA, and 3,5-diCQA exhibited the strongest binding, with energies of −6.19, −7.14, and −6.92 kcal/mol, respectively. Structural analysis indicated that 5-CQA formed hydrogen bonding with GLN837 and π-π interactions with GLN989 and PHE993. 3,4-diCQA established a hydrogen bond with GLN989 and π-π stacking with PHE302, along with a hydrophobic bond with VAL990 and ALA986. Similarly, 3,5-diCQA interacted with GLN989 and PHE982 through hydrogen bonds, while engaging VAL990 and ALA986 through π–σ and π–alkyl interactions. Collectively, VAL990, GLN989 and ALA986 emerged as common anchoring residues for CQA binding within P-gp. Conversely, 3-CQA, 4-CQA, and 4,5-diCQA primarily interacted with surface residues, which might account for their lower susceptibility to efflux (Figure 7a–f). These differences could be attributed to structural characteristics: mono-CQAs, containing only one caffeoyl group, offer limited binding opportunities, whereas di-CQAs, which contain two caffeoyl groups, facilitate additional π-π and hydrophobic interactions, thereby enhancing their binding affinity to P-gp [13,42,43,44].

Notably, quercetin and kaempferol exhibited strong affinities (−8.18 and −7.99 kcal/mol, respectively), comparable to that of the inhibitor verapamil (−8.51 kcal/mol) (Table 4). Quercetin formed hydrogen bonds with GLN724 and ASN295, along with hydrophobically interactions with VAL990, ALA986, and MET298. Kaempferol mainly engaged PHE769 via π-π stacking, VAL990 through π-σ interaction, and ALA986 and MET298 through hydrophobic contacts (Figure 7g–l). These interaction patterns suggest that quercetin stabilizes binding mainly through polar–hydrophobic forces, while kaempferol relies more on aromatic–hydrophobic interactions [45,46]. Structurally, both compounds are flavonols featuring a planar aromatic backbone with multiple hydroxyl groups. They share critical amino acid residues with CQAs within the P-gp binding pocket, particularly VAL990 and ALA986. This similarity may explain their pronounced inhibition of CQA efflux in Caco-2 cells. In contrast, the flavanones naringenin and hesperetin, which possess a saturated C-ring and fewer hydroxyl groups, only contacted surface residues and correspondingly showed weak inhibition [47,48]. Although EGCG is highly hydroxylated, its binding stability is compromised by the steric hindrance of the bulky gallate group, resulting in less stable interactions [49].

4. Conclusions

In conclusion, our study demonstrated that the intestinal absorption of CQAs occurs primarily via passive diffusion through the paracellular pathway, with mono-CQAs being more permeable than di-CQAs. However, the absorption of 5-CQA, 3,4-diCQA, and 3,5-diCQA was further restricted by active efflux, predominantly mediated by P-gp with additional involvement of BCRP. Notably, the flavonols quercetin and kaempferol were identified as effective efflux inhibitors that improve the absorption of CQAs by occupying overlapping binding sites on P-gp with CQAs. These findings elucidate the intestinal transport characteristics of structurally distinct CQAs and the limiting effects of efflux transporters. Therefore, this study proposes a feasible strategy to enhance CQA absorption through co-administration with specific dietary flavonoids. Future in vivo and formulation studies are warranted to validate the potential of such dietary combinations in functional foods and nutraceuticals.