Method for promoting intestinal absorption of dihydroquercetin by using quercetin, composition and application of composition

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a method and a composition for promoting intestinal absorption of dihydroquercetin by using quercetin and application of the method and the composition, and the method comprises the following steps: jointly applying an effective amount of dihydroquercetin and an effective amount of quercetin serving as a synergist to an intestinal environment; wherein the quercetin plays a role through at least one of the following mechanisms: inhibiting the function or expression of P-glycoprotein in intestinal epithelial cells; the tight connection of intestinal epithelial cells is regulated. It is found that quercetin not only can effectively inhibit the excretion function of P-glycoprotein in intestinal epithelial cells, but also can enhance the integrity of intestinal barriers by down-regulating the expression level of the protein and up-regulating the expression of tight junction protein, so that intestinal absorption of dihydroquercetin is synergistically promoted from the multi-mechanism level; in addition, the composition can still maintain a good absorption promoting effect in an inflammatory environment, and shows a synergistic anti-inflammatory effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method, composition, and application of using quercetin to promote the intestinal absorption of dihydroquercetin. Background Technology

[0002] Dihydroquercetin (DHQ) is a natural flavonoid compound widely found in plants such as Douglas fir, larch, onion, grape, and citrus, possessing various biological activities including antioxidant, anti-inflammatory, hepatoprotective, and antitumor effects. However, the oral bioavailability of DHQ is significantly limited by efflux transport proteins in the intestine, such as P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2). P-gp is an ATP-dependent transmembrane efflux pump... ABCB1 The gene encodes a substance that is mainly distributed in the apical membrane of intestinal epithelial cells. This substance can pump drug substrates (such as DHQ) that have been absorbed into the cells back into the intestinal lumen, resulting in reduced absorption efficiency and insufficient blood drug concentration, thereby affecting the exertion of its biological activity.

[0003] Several chemically synthesized P-gp inhibitors (such as verapamil, cyclosporine A, and tamicolam) have been developed to reverse multidrug resistance, but their clinical application is often limited by potential toxic side effects, pharmacokinetic interactions, and adverse reactions. In contrast, natural inhibitors derived from food (such as flavonoids like quercetin) show better application prospects due to their high safety, wide availability, and potential for multi-target regulation. Quercetin (QUE), a typical dietary flavonoid, has been shown to inhibit P-gp function, but the specific mechanism by which it is used in combination with DHQ to promote the latter's intestinal absorption has not yet been systematically elucidated.

[0004] Existing research largely focuses on single inhibitors or single mechanisms, lacking studies that systematically validate the role of quercetin in promoting DHQ absorption and its synergistic anti-inflammatory effects under inflammatory conditions at the cellular, molecular, and isolated organ levels. Therefore, developing a DHQ absorption-promoting strategy based on natural ingredients and employing multiple synergistic mechanisms has significant scientific and clinical application value. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the object of this invention is to provide a method, composition, and application of using quercetin to promote the intestinal absorption of dihydroquercetin.

[0006] To achieve the above objectives, a first aspect of the present invention provides a method for promoting the intestinal absorption of dihydroquercetin, comprising co-administering an effective amount of dihydroquercetin and an effective amount of quercetin as an synergist to the intestinal environment; wherein the quercetin exerts its effect through at least one of the following mechanisms: (a) Inhibits the function or expression of P-glycoprotein in intestinal epithelial cells; (b) Regulate tight junctions of intestinal epithelial cells.

[0007] In some embodiments of the present invention, the quercetin works by downregulating the protein expression level of P-glycoprotein and / or encoding the P-glycoprotein. ABCB1 The expression level of the gene's messenger RNA is used to suppress the expression of the P-glycoprotein.

[0008] In some embodiments of the present invention, the quercetin regulates the tight junction by upregulating the expression of at least one of the following tight junction-related proteins: occlusion protein, tight protein-1, and tight junction protein-1.

[0009] In some embodiments of the present invention, the molar ratio of dihydroquercetin to quercetin is 1:0.4 to 1:1.5.

[0010] To achieve the above objectives, a second aspect of the present invention provides the use of a composition in the preparation of a product for promoting the intestinal absorption of dihydroquercetin, the composition comprising the active ingredients dihydroquercetin and quercetin, wherein the quercetin, as a synergist, promotes the absorption of dihydroquercetin by inhibiting P-glycoprotein and regulating tight junctions.

[0011] In some embodiments of the present invention, the product is a pharmaceutical composition, a health food, or a functional food.

[0012] In some embodiments of the present invention, the composition enables the efflux ratio of dihydroquercetin to be less than 1 in an intestinal epithelial cell model.

[0013] To achieve the above objectives, a third aspect of the present invention provides the use of a composition comprising dihydroquercetin and quercetin in the preparation of a product for preventing or alleviating intestinal inflammation, wherein the quercetin exerts an anti-inflammatory effect by promoting the intestinal absorption of dihydroquercetin.

[0014] In some embodiments of the present invention, the composition exerts its anti-inflammatory effect through at least one of the following mechanisms: (a) Upregulates the expression of heme oxygenase-1; (b) Downregulate the expression of one or more inflammatory factors selected from cyclooxygenase-2, tumor necrosis factor-α, interleukin-1β, interleukin-6 and interleukin-8.

[0015] To achieve the above objectives, a fourth aspect of the present invention provides a pharmaceutical composition comprising dihydroquercetin and quercetin as active ingredients, and pharmaceutically acceptable excipients. The molar ratio of dihydroquercetin to quercetin is 1:0.6 to 1:1.0. The quercetin is used to inhibit the expression of P-glycoprotein and upregulate the expression of lockout protein and tight junction protein-1, thereby improving the intestinal absorption rate of dihydroquercetin.

[0016] The present invention relates to a method, composition, and application of quercetin to promote the intestinal absorption of dihydroquercetin. Through the combined use of quercetin and dihydroquercetin, multiple beneficial effects are observed: quercetin not only effectively inhibits the efflux function of P-glycoprotein in intestinal epithelial cells, but also enhances intestinal barrier integrity by downregulating the expression level of this protein and upregulating the expression of tight junction proteins, thereby synergistically promoting the intestinal absorption of dihydroquercetin from multiple mechanism levels; furthermore, the composition maintains good absorption-promoting efficacy under inflammatory conditions and exhibits synergistic anti-inflammatory effects. Attached Figure Description

[0017] Figure 1 The present invention provides P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MDR) in KB / MDR cells. ABCC2 A schematic diagram of protein and gene expression levels of / MRP2; Figure 2 This is a schematic diagram illustrating the protein expression levels of efflux proteins (such as P-gp) and tight junction proteins (ZO-1, Occludin, Claudin-1) in Caco-2 cells provided by the present invention. Figure 3 It is the efflux transport protein of Caco-2 cells in the Caco-2 / THP-1 macrophage co-culture model provided by this invention. ABCB1 , ABCC1-3 ) and tight junction-related genes ( TJP1 , CLDN1 , OCLN A schematic diagram of mRNA expression levels; Figure 4 The present invention relates to inflammation-related genes in THP-1 cells in the Caco-2 / THP-1 macrophage co-culture model. HO1 , COX2 , TNFA , IL6 , IL8 , IL1B Schematic diagram of mRNA expression and cytokine (IL-8, IL-1β) protein secretion levels; Figure 5This is a schematic diagram of the intestinal absorption of DHQ and QUE (at time points of 45 minutes and 90 minutes) in the everted intestinal sac experiment of SD rats provided by the present invention. Detailed Implementation

[0018] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0019] The following description, with reference to the accompanying drawings, describes an embodiment of the present invention: a method for promoting intestinal absorption of dihydroquercetin; the use of a composition in the preparation of a product for promoting intestinal absorption of dihydroquercetin; the use of a composition comprising dihydroquercetin and quercetin in the preparation of a product for preventing or alleviating intestinal inflammation; and a pharmaceutical composition. Example 1:

[0020] This embodiment aims to evaluate the promoting effect of quercetin QUE on the intestinal absorption of dihydroquercetin DHQ using an in vitro intestinal cell model.

[0021] For example, human colon cancer cells Caco-2 were selected as an in vitro intestinal epithelial cell model in the experiment. They can spontaneously differentiate into a monolayer of cells with brush borders, microvilli, and tight junctions, and are widely used in the study of drug intestinal permeability.

[0022] Optionally, Caco-2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection and routinely cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture conditions were 37°C and 5% CO2 saturated humidity.

[0023] For example, when cells were passaged to the 21st to 35th generation, they were seeded onto Transwell permeable membranes (e.g., polycarbonate membranes with 0.4 μm pore size) at a seeding density of [missing information - likely a density of cells per square centimeter]. 100 cells. During culture, the culture medium was changed every other day, and the transepithelial electrical resistance (TEER) value was measured every three days. When the TEER value stabilized above 600 Ω·cm², it indicated that the Caco-2 cell monolayer was complete and dense, and could be used for subsequent permeability experiments.

[0024] Optionally, prior to the permeability test, remove the culture medium from each well in the Transwell plate and wash the cell monolayer three times with Hank's balanced salt solution (HBSS) preheated to 37°C, soaking for 15 minutes each time, to remove residual culture medium and stabilize the cells.

[0025] For example, the experiment is set up with the following groups: 1. Blank control group: Only HBSS was added as a solvent; 2. DHQ monotherapy group: Dihydroquercetin (DHQ) at a known concentration was added only at the administration side; 3. QUE monotherapy group: Quercetin (QUE) at a known concentration was added only at the administration side, and its concentration was consistent with that of QUE in the combination therapy group; 4. DHQ+QUE combined administration group: Dihydroquercetin DHQ and quercetin QUE were added at the same concentration on the administration side.

[0026] Optionally, all compounds are dissolved in a suitable solvent and formulated into HBSS working solutions. The concentration of DHQ is, for example, 10 μg / mL, and the concentration of QUE is, for example, 10 μg / mL. In the DHQ+QUE co-administration group, the molar ratio of DHQ to QUE is, for example, 1:0.8.

[0027] For example, a two-way permeability experiment was conducted, including a permeation experiment in the absorption direction from the top AP to the base BL and a permeation experiment in the outflow direction from the base BL to the top AP.

[0028] For experiments in the AP→BL direction, the working solution of each group was added to the AP side of the Transwell membrane, and HBSS was added to the BL side of the membrane.

[0029] For the BL→AP direction experiment, the working solution of each group was added to the basal side (BL) of the Transwell membrane, and HBSS was added to the top side (AP).

[0030] Optionally, samples may be collected from the receiving side (the side receiving drug permeation or efflux) at, for example, 0.5 hours, 1 hour, 2 hours, and 4 hours after drug administration, while simultaneously replenishing an equal volume of preheated HBSS. The concentration of DHQ in the collected samples may be determined by high-performance liquid chromatography (HPLC).

[0031] For example, the apparent permeability coefficient and efflux ratio of DHQ are calculated based on the detected drug concentration and time. The formula for calculating the apparent permeability coefficient Papp is as follows: ; Where dQ / dt represents the drug permeation rate on the receiving side, A represents the surface area of ​​the Transwell membrane, and C0 represents the initial drug concentration on the dosing side.

[0032] Alternatively, the formula for calculating the efflux ratio ER is as follows: ; Wherein, Papp(BL→AP) represents the apparent permeability coefficient of DHQ from the basal side to the apical side, and Papp(AP→BL) represents the apparent permeability coefficient of DHQ from the apical side to the basal side. The closer the ER value is to 1, the less obvious the drug efflux phenomenon; the ER value is much greater than 1, indicating that the drug has a significant efflux effect.

[0033] For example, experimental results are expected to show that in the DHQ+QUE combination therapy group, the Papp (AP→BL) value of DHQ from the apical to the basal side is significantly higher than that of the DHQ alone therapy group. Meanwhile, the Papp (BL→AP) value of the DHQ+QUE combination therapy group is significantly lower than that of the DHQ alone therapy group.

[0034] Optionally, further, the DHQ efflux ratio ER value in the DHQ+QUE combination group was significantly lower than that in the DHQ-only group, and the ER value was observed to be less than 1. These results indicate that quercetin can significantly inhibit P-glycoprotein-mediated DHQ efflux, thereby effectively promoting the intestinal absorption of dihydroquercetin. Example 2:

[0035] This embodiment aims to elucidate the mechanism by which quercetin promotes the absorption of dihydroquercetin. Specifically, this embodiment verifies whether quercetin exerts a synergistic effect by inhibiting P-glycoprotein by detecting the expression levels of P-glycoprotein and its encoding gene.

[0036] For example, the cell samples used in this embodiment were directly derived from Caco-2 cells after the permeability experiment in Example 1. After the drug treatment of each group was completed, the Transwell chamber was carefully removed, and the cell monolayer was washed twice with pre-cooled phosphate-buffered saline (PBS) in preparation for subsequent protein and RNA extraction.

[0037] Optionally, the experimental groups are consistent with those in Example 1, including a blank control group, a DHQ-only group, a QUE-only group, and a DHQ+QUE combined group.

[0038] For example, the protein expression level of P-glycoprotein in each group of cells was detected by Western blotting. First, cells were lysed on ice using RIPA lysis buffer containing a protease inhibitor to extract total protein. Subsequently, the protein samples were quantified using the BCA method. Equal volumes of protein were subjected to SDS-PAGE gel electrophoresis, and then the proteins were transferred to a PVDF membrane. After blocking with 5% skim milk powder at room temperature for one hour, primary antibodies against P-glycoprotein and β-actin (internal control protein) were added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed with TBST, and the corresponding horseradish peroxidase-labeled secondary antibody was added, followed by incubation at room temperature for one hour. After washing, the membrane was developed using enhanced chemiluminescence (ECL) reagent, and images were acquired using a gel imaging system. The gray values ​​of the protein bands were analyzed using image analysis software, and the relative expression level of P-glycoprotein was expressed as the ratio of the gray value of the P-glycoprotein band to that of the β-actin band.

[0039] Optionally, the encoding of P-glycoprotein in cells of each group can be detected by real-time quantitative polymerase chain reaction. ABCB1 Messenger RNA (mRNA) expression levels were determined. First, total RNA was extracted from cells in each group using TRIzol reagent. RNA purity and concentration were measured using a micro-spectrophotometer. Then, 1 μg of total RNA was reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit. Using the cDNA as a template, a specific gene expression assay was performed. ABCB1 Gene-specific primers and internal reference genes GAPDH The primers were used for amplification on a real-time quantitative PCR instrument.

[0040] For example, after the PCR reaction is completed, the following steps are taken: Method calculation ABCB1 Relative gene expression levels. This method determines the fold change in gene expression in the experimental group relative to the control group by comparing the difference in cycle threshold (Ct value) between the target gene and the internal reference gene.

[0041] For example, the expected results from Western blotting showed that, compared with the DHQ-only group, the protein band of P-glycoprotein in the DHQ+QUE combination group was significantly lighter and its relative expression level was significantly reduced.

[0042] Optionally, the expected results of qRT-PCR show that, compared with the DHQ alone group, the DHQ+QUE combined treatment group has [a higher concentration of cells in the cells]. ABCB1 The relative expression level of the gene's mRNA was significantly downregulated. These results collectively indicate that quercetin can exert its effect by downregulating the expression of the gene encoding P-glycoprotein. ABCB1The transcriptional level of the gene is reduced, and the protein expression level of P-glycoprotein is further reduced, thereby inhibiting its drug efflux function, which explains one of the molecular mechanisms by which it promotes the intestinal absorption of dihydroquercetin. Example 3:

[0043] This embodiment aims to further elucidate one of the dual mechanisms by which quercetin promotes the absorption of dihydroquercetin, namely, to verify its regulatory effect on tight junction-related proteins in intestinal epithelial cells.

[0044] For example, the cell samples used in this embodiment were also derived from Caco-2 cells after the permeability experiment in Example 1. The experimental groups were consistent with those in Example 1, including a blank control group, a DHQ-only group, a QUE-only group, and a DHQ+QUE combined group.

[0045] Optionally, the expression levels of tight junction-related proteins in each group of cells were detected by Western blotting. The experimental procedures were basically the same as those described in Example 2. The main difference was in the choice of primary antibody. In this example, primary antibodies that specifically recognize occludin, claudin-1, and tight junction protein-1 (ZO-1) were used, and β-actin was used as an internal control.

[0046] For example, after acquiring images using a gel imaging system, image analysis software was used to analyze the gray values ​​of each protein band. The relative expression level of each tight junction-related protein was represented by the ratio of the gray value of the target protein (Occludin, Claudin-1, ZO-1) band to the gray value of the internal reference β-actin band.

[0047] Optionally, the experimental results are expected to show that, compared with the blank control group or the DHQ alone group, the protein expression levels of Occludin and ZO-1 in the cells of the DHQ+QUE combined treatment group are significantly upregulated, while the expression level of Claudin-1 may not change significantly or may also show an upregulated trend.

[0048] For example, the results above indicate that quercetin can enhance the connections between intestinal epithelial cells by upregulating the expression of tight junction proteins such as Occludin and ZO-1, which are key components of the intestinal physical barrier, thereby regulating intestinal permeability. This constitutes another important molecular mechanism by which it promotes the absorption of dihydroquercetin. Example 4:

[0049] This embodiment aims to determine the effective and optimal molar ratio range in which dihydroquercetin (DHQ) and quercetin (QUE) exert a synergistic absorption-enhancing effect.

[0050] For example, this embodiment uses the same Caco-2 cell monolayer model and permeability experiment method as in Example 1. In the experiment, the concentration of DHQ was fixed, for example, 10 μg / mL, while a series of different concentrations of QUE were set to create different DHQ to QUE molar ratios.

[0051] Alternatively, the experimental group design is as follows: 1. DHQ monotherapy group: used as a baseline control; 2. Series ratio combined drug administration groups: The molar ratio of DHQ to QUE was set to 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1.0, 1:1.5 and 1:2.0, respectively.

[0052] For example, following the bidirectional permeability test method described in Example 1, the apparent permeability coefficient Papp (AP→BL) of DHQ from the top side to the base side in each group was measured.

[0053] Optionally, the experimental results are expected to show that, compared with the DHQ monotherapy group, the Papp (AP→BL) value of DHQ is significantly increased when the molar ratio of DHQ to QUE is in the range of 1:0.4 to 1:1.5.

[0054] For example, further, when the molar ratio is in the range of 1:0.6 to 1:1.0, the Papp (AP→BL) value of DHQ reaches its peak or plateau, showing the optimal synergistic absorption-promoting effect. When the molar ratio continues to increase to 1:2.0, the promoting effect may no longer be enhanced or may even decrease slightly. This result provides experimental data support for the ratio range of the two active ingredients in the composition of the present invention. Example 5:

[0055] This embodiment aims to demonstrate that the composition containing dihydroquercetin and quercetin described in this invention has pharmacological activity in preventing or alleviating intestinal inflammation.

[0056] For example, this embodiment uses a lipopolysaccharide (LPS)-induced Caco-2 / THP-1 macrophage inflammation model. Healthy Caco-2 cells were seeded at the top of a Transwell culture plate, and THP-1 macrophages were seeded at the base. After 21 days of culture and cell confluence, the cells were pretreated with drugs.

[0057] Alternatively, the experimental group design is as follows: 1. Normal control group: cultured only with culture medium; 2. Model group: Cells were treated with LPS (e.g., 1 μg / mL) for 3 hours; 3. DHQ treatment group: Pretreated with DHQ for 9 hours, then incubated with LPS for 3 hours; 4. DHQ+QUE combined treatment group: First, pre-treat with a combination of DHQ and QUE (e.g., molar ratio 1:0.8) for 9 hours, then add LPS and incubate together for 3 hours.

[0058] For example, after drug treatment, the cell culture supernatant was collected, and the concentrations of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) were detected by enzyme-linked immunosorbent assay (ELISA).

[0059] Optionally, cells were collected, and total protein and total RNA were extracted. The expression levels of pNF-κB, NF-κB, pIκBα, and IκBα were detected by Western blotting. qRT-PCR was used to detect... HO1 , COX2 , TNFA , IL6 , IL8 , IL1B The mRNA expression levels of cytokines.

[0060] For example, ELISA results are expected to show that, compared with the model group, the DHQ+QUE combination therapy group significantly reduces the secretion of inflammatory factors such as TNF-α, IL-6, and IL-8, and its inhibitory effect is superior to that of the DHQ monotherapy group. Western Blot results are expected to show that the combination therapy group significantly upregulates IκBα expression and downregulates pNF-κb and pIκBα expression. Results are expected to show that the combination therapy group significantly upregulates... HO1 and lowered COX2 , TNFA , IL6 , IL8 and IL1B Gene expression. These results collectively demonstrate that the compositions of the present invention exert a significant anti-intestinal inflammation effect through synergistic action. Example 6:

[0061] This embodiment aims to provide a specific product form of the composition of the present invention, demonstrating that it can be prepared into dosage forms commonly used in the pharmaceutical or food industries, and has the feasibility of industrial application.

[0062] Formulation 1: Tablets of a pharmaceutical composition For example, a tablet comprising the composition of the present invention, wherein the components and their weight parts are as follows: Dihydroquercetin: 100 parts; Quercetin: 105 parts (making the molar ratio approximately 1:0.8); Microcrystalline cellulose: 150 parts; Lactose: 80 parts; Cross-linked polyvinylpyrrolidone: 15 parts; Magnesium stearate: 3 parts.

[0063] Optionally, the preparation method includes: mixing dihydroquercetin, quercetin, microcrystalline cellulose and lactose evenly; adding crospovidone and mixing evenly again; finally adding magnesium stearate and mixing, and then compressing into tablets using a tableting machine.

[0064] Formulation 2: Hard capsules for health food For example, a hard capsule comprising the composition of the present invention, wherein the components and weight of the contents of each capsule are as follows: Dihydroquercetin: 50 mg; Quercetin: 52.5 mg; Pregelatinized starch: 197.5 mg.

[0065] Optionally, the preparation method includes: mixing dihydroquercetin, quercetin and pregelatinized starch in a mixer until uniform, and then filling the mixed powder into No. 0 hard capsule shells using a fully automatic capsule filling machine.

[0066] Formulation 3: Solid Beverages in Functional Foods For example, a solid beverage comprising the composition of the present invention, the components and weight of each sachet are as follows: Dihydroubecortin: 200 mg; Quercetin: 210 mg; Maltodextrin: 2000 mg; Citric acid: 50 mg; Sucralose: 10 mg; Lemon flavoring: as needed.

[0067] Optionally, the preparation method includes: sieving all powdered raw and auxiliary materials, mixing them evenly in a three-dimensional motion mixer, and then packaging them into composite film bags and sealing them. Example 7:

[0068] Corresponding to the above embodiments, this seventh embodiment proposes a series of experimental methods and corresponding experimental results, specifically including the following: Optionally, first confirm the experimental conditions and cell information: 1. Chemicals and reagents Dihydroquercetin (DHQ), quercetin (QUE), and elacridar were purchased from Aladdin Chemicals Ltd. (purity ≥98%, Shanghai, China). Hygromycin was purchased from Roche Group (Hoffmann-Roche Ltd., Switzerland).

[0069] 2. Cell Culture The KB cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The KB / MDR1 cell line, stably expressing human P-glycoprotein (P-gp) cDNA, was constructed based on our previous research. Both KB and KB / MDR cells were cultured in Durbeco Modified Eagle Medium (DMEM, Gibco, Wuhan, China) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% sodium pyruvate, 1% glutamine, and 1% penicillin-streptomycin. KB / MDR cells were maintained at each passage using medium containing 500 μg / mL hygromycin.

[0070] Caco-2 cells were cultured in minimum essential medium (MEM) containing 10% FBS, 1% non-essential amino acids and 1% penicillin-streptomycin.

[0071] The human monocytic leukemia cell line THP-1 was kindly provided by Associate Professor Martin Rousgaard of the Department of Public Health at the University of Copenhagen. The cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Gibco) supplemented with 10% heat-inactivated FBS, 1% 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES, Gibco), 1% sodium pyruvate, and 0.1% gentamicin (Invitrogen).

[0072] All cells were cultured in a humid atmosphere at 37°C, 5% CO2, and 90% relative humidity.

[0073] Specifically, the experimental methods include: 1. Uptake of DHQ and QUE by KB cells and KB / MDR cells KB and KB / MDR cells were seeded in 6-well plates and cultured until confluence. Cells were pre-incubated at 37°C for 30 min with different concentrations of QUE (40 μM and 100 μM) or ectenafil (10 μM). Then, different concentrations of DHQ (40 μM, 60 μM, 80 μM, and 100 μM) and their corresponding inhibitors (QUE and ectenafil) were added to KB or KB / MDR cells, and incubation was continued at 37°C for 2 h. After washing twice with ice-cold phosphate-buffered saline (PBS), cells were collected and lysed. Protein content was determined using the quinoline carboxylic acid (BCA) method. Cell lysates were freeze-dried and stored at -80°C for subsequent high-performance liquid chromatography (HPLC) analysis. Results are expressed in μmol / g.

[0074] 2. DHQ and QUE on P-gp and P-gp in KB and KB / MDR cells ABCC2The effect of (MRP2) expression KB and KB / MDR cells were seeded in 6-well plates and cultured until confluence. Cells were treated with different concentrations of DHQ and their corresponding inhibitors at 37°C for 24 h. After washing twice with ice-cold PBS, total RNA and total protein were extracted.

[0075] 3. Effects of DHQ and QUE on the expression of efflux proteins and tight junction proteins in Caco-2 cells Caco-2 cells ( Cells were seeded in 6-well plates (cells / well), and the culture medium was changed every other day for 21 days. Cells were treated with DHQ and different concentrations of inhibitors at 37°C for 24 h. After washing twice with ice-cold PBS, total RNA and total protein were extracted for the detection of efflux proteins and tight junction proteins.

[0076] 4. Two-way transport and co-culture experiments Caco-2 cells were used Cells were seeded at a density of 12 cells / mL in 12-well Transwell chambers. 0.5 mL of culture medium was added to the top chamber (AP) and 1.5 mL to the basal chamber (BL). The culture medium was changed every two days. Cells were incubated at 37°C with 5% [missing information - likely a specific concentration]. Cultured for 21 days under specific conditions to differentiate into a single layer of epithelium, ensuring that the transepithelial resistance (TEER) value reaches at least 600 Ω·cm².

[0077] In the bidirectional transport experiment, differentiated Caco-2 monolayers were maintained under identical culture conditions. DHQ and QUE were added to the top chamber (AP, 0.5 mL) or the basal side chamber (BL, 1.5 mL), while the corresponding receiving chamber was filled with phenol red-free MEM medium. After 6 h of incubation, 200 μL samples were collected from the receiving chamber at 2 h and 4 h, respectively, with an equal volume (200 μL) of fresh phenol red-free MEM medium added after each collection. At 6 h, all samples from the top and basal side chambers were collected. The concentrations of DHQ and QUE in the samples were determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0078] For the co-culture experiment of Caco-2 and THP-1 macrophages, DHQ and QUE were added to the top chamber of Caco-2 monolayer cells and incubated at 37°C with 5% [temperature missing]. The cells were co-incubated for 12 h under the specified conditions. Simultaneously, lipopolysaccharide (LPS, 100 ng / mL) was added to the basal side chamber. Samples for analysis were collected at different time points: 3 h for quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), and 45 min for Western blot analysis. After treatment, the supernatant from the basal side chamber was collected. One portion was used for ELISA detection, and the other portion was used for LC-MS / MS analysis to determine the concentrations of DHQ and QUE. For THP-1 macrophages co-cultured in the basal side chamber with DHQ and QUE transported through the Caco-2 monolayer, qRT-PCR was used for detection. HO1 , COX2 , TNFA , IL10 , IL6 , IL8 and IL1B The mRNA expression levels of IL-8 and IL-1β were assessed using ELISA.

[0079] 5. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from cells using an RNA extraction kit (Wuhan Adila Biotechnology Co., Ltd., China) according to the manufacturer's instructions. The extracted RNA was reverse transcribed into cDNA. qRT-PCR was then performed using the primers listed in Table 1. The qRT-PCR program was: initial pre-denaturation at 94°C for 30 seconds, followed by 40 cycles of denaturation at 94°C for 10 seconds and annealing / extension at 60°C for 30 seconds. Using 2... -ΔΔCt The method calculates the fold change in target gene expression relative to the control group.

[0080] Table 1 Primer sequences for real-time quantitative polymerase chain reaction (qRT-PCR)

[0081] 6. Western blotting Cells were washed twice with ice-cold PBS and lysed for 30 min in lysis buffer containing phosphorylated protease inhibitors and protease inhibitors. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. After blocking with 5% bovine serum albumin for 1 h, the membranes were incubated overnight at 4°C with primary antibodies [(P-gp, Abcam), (claudin-1, occludin, zonula occludens protein-1 (ZO-1), and GAPDH, Xavier)]. Subsequently, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies for 1 h. Protein bands were visualized using an enhanced chemiluminescence detection kit and a multifunctional imaging system.

[0082] 7. Rat everted intestinal sac experiment Male Sprague-Dawley rats (SPF grade, weighing 280-300 g) were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. All experimental procedures were approved by the Animal Ethics Committee of Hainan University (Approval No.: HNUAUCC-2023-00042).

[0083] Everted intestinal sacs were prepared according to our previous method. Rats were euthanized after being fasted overnight. The small intestine was rapidly removed through a midline abdominal incision and divided into segments of approximately 8 cm. After rinsing with cold Krebs-Ringer buffer, the intestinal segments were completely everted. One end of the intestinal segment was tied with cotton thread, and 1.5 mL of preheated Krebs-Ringer buffer was injected into the sac from the other end, followed by ligation of that end. The prepared everted intestinal sacs were vertically transferred to Krebs-Ringer buffer containing the test compound, continuously purged with an O2 / CO2 gas mixture, and incubated at 37°C for 45 min and 90 min, respectively.

[0084] 8. High Performance Liquid Chromatography (HPLC) The lyophilized samples were reconstituted with methanol, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC. The contents of DHQ and QUE were determined using a Hitachi Chromaster chromatographic system (Tokyo, Japan) equipped with an Agilent HC-C18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, California, USA). The mobile phase consisted of phase A (aqueous solution containing 0.1% formic acid) and phase B (acetonitrile), with a flow rate of 1.0 mL / min. The gradient elution program was as follows: 0–4 min: 85% A; 4–24 min: 85–50% A; 24–32 min: 50–0% A; 32–38 min: 0% A; 38–38.1 min: 0–85% A; 38.1–45 min: 85% A. A diode array detector (DAD) was used to monitor the elution peaks at the maximum absorption wavelength for each compound.

[0085] 9. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) Analysis was performed using a SCIEX Qtrap™ 6500+ LC-MS / MS system (AB Sciex, Prague, Czech Republic). Chromatographic separation was performed using a Kinetex C18 column (1.7 μm, 150 mm × 2.1 mm, Phenomenex, Aschaffenburg, Germany) at a temperature maintained at 40 °C. Detection was performed using a triple quadrupole-linear ion trap mass spectrometer (AB Sciex, Prague, Czech Republic).

[0086] The mobile phase consisted of phase A (an aqueous solution containing 0.1% formic acid) and phase B (acetonitrile). The flow rate was 0.3 mL / min, and the injection volume was 5 μL. The gradient elution program was as follows: 0–1 min, 10% B; 1–3 min, 10% → 33% B; 3–5 min, 33% B; 5–7 min, 33% → 90% B; 7–8.5 min, 90% B; 8.5–10 min, 90% → 10% B; 10–13 min, 10% B.

[0087] Quantitative analysis was performed using multiple reaction monitoring (MRM) mode. In positive ion mode, the ion spray voltage was 4500 V and the ion source temperature was 500 °C; in negative ion mode, the ion spray voltage was -4500 V, the ion source temperature was 500 °C, and the nebulizing gas pressure was 45 psi. The optimized declusivity voltage (DP) and collision energy (CE) values ​​are shown in Table 2.

[0088] Quantitative analysis was performed using the external standard method, with standard curves plotted using standard solutions ranging from 0.05 to 150 μM. Quantification was performed using the precursor ion (MS1) and its corresponding daughter ion (MS2), and the concentration of the target analyte in each sample was calculated by interpolation of the standard curve.

[0089] Table 2. Multiple reaction monitoring (MRM) transition parameters of DHQ and QUE analyzed by LC-MS / MS

[0090] Conversely, the experimental results include: For example, the uptake of DHQ and QUE in KB and KB / MDR cells is shown here.

[0091] Table 3. Intake of DHQ and QUE under different conditions

[0092] Lowercase letters: Comparison of different concentrations of DHQ uptake in KB or KB / MDR cells; Uppercase letters: Comparison of the same DHQ concentration in KB / MDR cells; *: Comparison of DHQ co-incubation with QUE or elacridar and DHQ treatment alone in KB or KB / MDR cells; #: Comparison of DHQ and QUE40 in KB and KB / MDR cells.

[0093] Table 3 above shows the accumulation of DHQ in KB cells and KB / MDR cells.

[0094] In KB cells, when DHQ was incubated alone, the accumulation of DHQ increased significantly with increasing concentration (40-100 μM), reaching a peak of 770.024 nmol / g at 100 μM. When different concentrations of DHQ were co-incubated with QUE40, the accumulation of DHQ also increased significantly with increasing DHQ concentration (except for DHQ100). There was no significant difference in the accumulation of DHQ100 compared to DHQ80, indicating that DHQ accumulation had reached a saturation point at this stage. After co-incubation with QUE40 and DHQ, the accumulation of DHQ was significantly greater than in the DHQ-only treatment group, suggesting that QUE may promote DHQ uptake pathways (e.g., inhibiting other efflux proteins).

[0095] In P-gp-overexpressing KB / MDR cells, the intracellular accumulation of DHQ increased with increasing incubation concentration (40-100 μM), and was significantly lower than that in KB cells at the corresponding concentrations, indicating that P-gp mediates DHQ efflux, leading to decreased uptake. Compared with DHQ alone, co-incubation with the P-gp inhibitor elacridar or QUE40 and QUE100 significantly increased DHQ accumulation. p The value <0.01 indicates that DHQ is a substrate of P-gp, and its efflux process is blocked by these inhibitors. Furthermore, QUE40 is more effective at increasing DHQ uptake in KB / MDR cells than in KB cells; in fact, there was no difference in DHQ uptake when QUE and DHQ were co-incubated in both cell types.

[0096] Under low concentration DHQ (40 μM) conditions, compared with co-incubation with QUE40 (387.196 ± 59.045 nmol / g), QUE100 significantly increased the accumulation of DHQ to 509.601 ± 37.063 nmol / g. pThe concentrations of QUE (<0.01) and DHQ levels were all higher than those of elacridar, indicating that P-gp function may not have been saturated, and the differences in efficacy and affinity among different inhibitors were evident. This suggests that QUE can inhibit P-gp function and promote DHQ accumulation in KB / MDR, and its inhibitory effect is positively correlated with its concentration within a certain range. When the DHQ concentration was increased to 60 μM, there was no statistically significant difference in the accumulation-promoting effect between QUE 40 μM and QUE 100, but both were still significantly higher than the elacridar group. This may mean that at this concentration, the inhibitory effect of QUE has reached a plateau. Interestingly, when the DHQ concentration was further increased to 80 μM, the DHQ accumulation in all inhibitor groups tended to be consistent, with no significant difference. This indicates that P-gp has been saturated by high substrate concentrations, and its efflux capacity has reached its limit. Therefore, regardless of the inhibitor used, the space for further inhibition becomes smaller, and the effects tend to be consistent. At the highest substrate concentration (DHQ100), the DHQ accumulation in the elacridar group was significantly higher than that in the QUE40 and QUE100 groups, and there was no statistically significant difference between QUE40 and QUE100. This indicates that elacridar and QUE have different inhibitory mechanisms. elacridar is a classic third-generation P-gp inhibitor, and its inhibitory effect may be more advantageous at high substrate concentrations (e.g., non-competitive inhibition). QUE, on the other hand, is likely more of a competitive inhibitor; in the presence of high substrate concentrations, the difficulty of competing for binding sites increases, resulting in lower inhibitory efficiency than elacridar.

[0097] The table also reveals that QUE accumulates in KB / MDR cells under co-incubation conditions, with QUE100 accumulation significantly higher than QUE40, indicating a concentration-dependent accumulation of QUE. Furthermore, QUE accumulation is completely unaffected by DHQ concentration. Overall, QUE accumulation is significantly greater than DHQ accumulation.

[0098] For example, the protein and gene expression levels of efflux proteins in KB / MDR cells are shown here.

[0099] Figure 1 AC represents the expression of P-gp protein and mRNA in KB / MDR cells. First, we confirmed that the basal protein expression level and mRNA level of P-gp in KB / MDR cells were significantly higher than those in parental KB cells (…). p <0.05. Compared with KB / MDR cells, the expression levels of P-gp protein and mRNA did not change when incubated with low concentrations (≤60 μM) of DHQ alone. However, when the DHQ concentration was increased to 80 and 100 μM, the expression level of P-gp protein decreased significantly. p<0.05), but there was no statistically significant difference in transcriptional levels. Compared with the DHQ-only treatment group, QUE40 significantly inhibited the expression levels of both P-gp protein and mRNA. p <0.05), and there was no statistically significant difference among the groups when QUE40 was co-incubated with different concentrations of DHQ. When co-incubated with DHQ40, the expression of P-gp protein by QUE100 co-incubated with DHQ40 was consistent with that of QUE40, but ABCB1 The expression level of mRNA was significantly lower than that of QUE40. When QUE100 was combined with DHQ100, the transcriptional level remained consistent with that of the DHQ40+QUE100 group. Figure 1 C). P-gp protein expression did not decrease further; instead, it rebounded to levels comparable to the DHQ80-treated group. When the positive control elacridar was co-incubated with DHQ, it did not regulate either the transcriptional or translational levels of P-gp. These results indicate that P-gp is not affected by the concentration of the substrate DHQ at the transcriptional and translational levels, but is regulated by QUE, especially at the transcriptional level; higher QUE concentrations lead to downregulation. ABCB1 The stronger the ability to detect mRNA levels.

[0100] We also detected DHQ and QUE pairs in KB / MDR cells. ABCC2 The regulatory role of genes ( Figure 1 D). The figure shows that adding DHQ to KB / MDR cells significantly downregulated [the condition / effects]. ABCC2 mRNA levels ( p <0.05), but not affected by DHQ concentration. When DHQ80 and DHQ100 are co-incubated with QUE40, ABCC2 mRNA expression levels were significantly lower in the DHQ-treated group than in the group treated alone. p <0.05). And the DHQ100+QUE100 group... ABCC2 mRNA expression levels were significantly lower in the DHQ100+QUE40 group than in the DHQ100+QUE40 group, indicating that QUE regulates... ABCC2 Gene activity is concentration-dependent. Combined with Table 3, we know that QUE can increase the accumulation of DHQ in KB cells, thus indicating... ABCC2 The transcriptional level may have been inhibited by QUE. When elacridar was co-incubated with DHQ, it was only significantly downregulated at a DHQ concentration of 100 μM. ABCC2 mRNA levels ( p <0.05).

[0101] The results above demonstrate that QUE not only inhibits the function of P-gp, but is also a protein that regulates efflux protein expression at both the transcriptional and translational levels, and its effects are quite different from those of the classic inhibitor elacridar.

[0102] For example, this demonstrates the regulation of efflux proteins and tight junction proteins in Caco-2 cells by DHQ and QUE.

[0103] The expression levels of P-gp protein and mRNA in Caco-2 cells cultured alone are as follows: Figure 2 As shown in AC, compared with the DHQ-only treatment group, QUE (60, 80, and 100 μM) significantly downregulated the transcription and translation levels of P-gp. p <0.05), and furthermore, QUE's inhibition of P-gp protein expression levels was concentration-dependent, with no significant difference between QUE80 and QUE100. Co-incubation of elacridar with DHQ also significantly inhibited P-gp protein expression and... ABCB1 mRNA levels ( p The effect was <0.05%, which was lower than that of QUE, indicating that elacridar not only inhibits P-gp function but also affects its protein expression level.

[0104] Expression of tight junction protein ZO-1 ( Figure 2 D) showed that, compared with the DHQ-only group, there was no significant change when DHQ was co-incubated with QUE40, but QUE60 and QUE80 significantly upregulated ZO-1 expression. p< 0.05), and expression further increased at QUE100 ( p <0.01). elacridar also significantly increased ZO-1 expression ( p <0.05).

[0105] Occludin protein expression ( Figure 2 E) The results showed that, compared with DHQ alone, QUE significantly increased Occludin expression in a concentration-dependent manner, reaching saturation at 60 μM. Co-incubation of elacridar with DHQ significantly decreased Occludin expression. p <0.05 indicates that elacridar may impair tight junction integrity.

[0106] Claudin-1 protein expression ( Figure 2 F) showed that, compared with the DHQ alone, co-incubation with DHQ only significantly upregulated Claudin-1 expression when the QUE concentration reached 80 μM and 100 μM. pThe concentration of QUE (<0.05%) showed saturation at 100 μM, while low concentrations had no effect, indicating that high concentrations of QUE can help improve cell barrier integrity. Co-incubation of elacridar and DHQ had no significant effect on Claudin-1. These results suggest that QUE, while inhibiting P-gp expression, can also enhance the expression of ZO-1, Occludin, and Claudin-1 to maintain intestinal barrier function.

[0107] For example, experimental results of bidirectional transport in a Caco-2 cell monolayer model are shown here.

[0108] Table 4. Inflow and outflow of DHQ under different conditions (×10) -6 μmol)

[0109] Results are expressed as mean ± standard deviation, n=3. Uppercase letters: significant differences at the same time point; lowercase letters: significant differences at different time points.

[0110] Table 4 above shows the bidirectional transport data of the substrate (DHQ) and inhibitor (QUE) in the Caco-2 cell monolayer model. As can be seen from the table, when DHQ was incubated alone, both the influx (inflow rate) and efflux (efflux rate) of DHQ significantly increased with time (2 h to 6 h). p <0.05). Furthermore, when DHQ was incubated alone, its efflux ratio (ER) was as high as 30.573, clearly confirming that DHQ is a substrate of intestinal efflux pumps (such as P-gp).

[0111] At 2 h, QUE40 and QUE60 showed no statistically significant difference in DHQ inflow, while QUE80 and QUE100 were significantly greater than QUE40. p <0.05). However, there was no significant difference in DHQ influx between QUE60 and QUE (80 and 100), indicating that the concentration dependence of QUE on DHQ uptake was not significant in the short term. At 4 h, QUE (40, 60, and 80 μM) showed a significant concentration dependence on DHQ influx. DHQ influx increased significantly with increasing QUE concentration. However, when the QUE concentration increased to 100 μM, DHQ influx reached a saturation point, showing no significant difference from QUE80. At 6 h, DHQ influx increased significantly when the QUE concentration increased from 40 μM to 100 μM. p <0.05), the inflow of DHQ reached its maximum at QUE100 (250.369±23.015 pmol), while there was no significant difference between QUE60 and QUE80.

[0112] At the same QUE concentration, the inflow of DHQ also showed a certain time dependence (except for QUE40 at 2 h and 4 h). The inflow of DHQ gradually increased with time.

[0113] Throughout the efflux process, except for QUE40 which had no inhibitory effect on DHQ efflux, QUE60-100 significantly inhibited DHQ efflux. p <0.05). At 2 h, QUE80 and QUE100 showed significantly greater ability to inhibit DHQ efflux than QUE60 ( p <0.05%, and in the short term, there was no significant difference in the inhibitory effects of QUE80 and QUE100. At 4 h, there was no statistically significant difference in the inhibitory effects of QUE60 and QUE80 on DHQ efflux, but the inhibitory effect on DHQ efflux was significantly enhanced when the QUE concentration reached 100 μM. p <0.05). At 6 h, the inhibitory effect of QUE100 was significantly greater than that of QUE60, while the ability of QUE80 to inhibit DHQ efflux was between that of QUE60 and QUE100, and there was no significant difference between it and either of them.

[0114] Compared with the DHQ-only incubation group, co-incubation with QUE significantly increased DHQ inflow and significantly decreased efflux. Table 2 shows that, except for QUE80 at 2 h and QUE100 at 2 h and 4 h, the ER was greater than 1, indicating that DHQ efflux exceeded inflow. After co-incubation with QUE, the DHQ ER decreased sharply, showing a clear concentration dependence, but the reversal effect weakened with time, manifesting as an increase in ER. At 2 h, QUE80 and QUE100 could reduce the ER to below 1 (e.g., QUE80 group ER = 0.724, QUE100 ER = 0.433). At 4 h, the ER of the QUE100 treatment group was also less than 1 (0.790). ER < 1 means that QUE not only inhibited the efflux pump but also completely reversed the net transport direction of DHQ, changing it from net efflux to net absorption.

[0115] The bidirectional transport results of QUE in the co-culture model are shown in Table 4. QUE uptake was less than that of co-incubated DHQ, as evidenced by extremely low influx and efflux rates. Both influx (except for QUE40 at 4 h and 6 h) and efflux (except for QUE100 at 2 h and 4 h) increased significantly with time, exhibiting a time-dependent relationship.

[0116] At the same time, the efflux of QUE was also minimally affected by its own concentration. Except at 2 h, the efflux of QUE80 was significantly greater than that of QUE60. p<0.05); Under 6 h conditions, the efflux of QUE80 was significantly greater than that of QUE100 μM ( p <0.05).

[0117] For example, the transport results in a Caco-2 / THP-1 macrophage bilayer model are shown here.

[0118] Table 5 Inflow of DHQ and QUE

[0119] Table 5 shows the influx of DHQ into the Caco-2 / THP-1 co-culture model after 12 h of incubation with QUE. Compared with the DHQ-only treatment group, the influx of DHQ into the basal side increased significantly by 2.7-fold after 3 h of LPS stimulation. p <0.01), which may be because LPS disrupts the Caco-2 cell monolayer barrier function and increases the permeability of intestinal epithelial cells.

[0120] Based on this inflammatory model, we investigated the effects of different concentrations of QUE and elacridar on DHQ influx. Compared with the DHQ100 group, co-incubation with low concentrations of QUE (40 and 60 μM) had no significant promoting effect on DHQ influx. When the QUE concentration was increased to 80 and 100 μM, the DHQ influx significantly increased to 76.879±22.481 and 93.991±24.332 μmol / L, respectively. p <0.01), and there was no statistically significant difference between the two, indicating that QUE at high concentrations inhibited efflux pumps (such as P-gp) in Caco-2 cells. Both DHQ and QUE are substrates of P-gp; high concentrations of QUE competitively inhibited P-glycoprotein-mediated DHQ efflux, thereby increasing DHQ influx. Elacridar showed a significant promoting effect, with its DHQ influx significantly higher than that of QUE40 (…). p <0.05), but there was no significant difference compared to the QUE60-100 group.

[0121] In addition, we analyzed the inflow of QUE itself. The inflow of QUE increased significantly with increasing incubation concentration. p< 0.05). At the same QUE concentration (100 μM), the QUE inflow in the co-incubation group was significantly higher than that in the QUE-only incubation group. p< 0.05).

[0122] For example, this demonstrates the regulation of DHQ and QUE on the expression of efflux transporters and the transcription of tight junction-related genes in Caco-2 cells in a co-culture model.

[0123] To elucidate the molecular mechanisms by which DHQ and QUE affect drug transport and barrier function in a Caco-2 / THP-1 co-culture model, we analyzed the effects of DHQ and QUE on drug transport and barrier function in Caco-2 cells using RT-qPCR. ABCB1 MRP2 ABCC2 MRP1 ABCC1 ) and MRP3 ( ABCC3 The mRNA expression level of ). Figure 3 AD) like Figure 3 As shown in Figure A, compared with the DHQ-only treatment group, LPS stimulation significantly upregulated [the condition]. ABCB1 mRNA expression ( p <0.05). However, QUE significantly reversed this upregulation and restored its transcription to the same basal level as the LPS-free DHQ group at 80 μM and 100 μM. Elacridar also produced a similar downregulation effect.

[0124] for ABCC2 The expression of LPS treatment had no significant effect, and co-incubation of QUE and DHQ at various concentrations also had no significant effect. Figure 3 B).

[0125] LPS processing ABCC1 Transcription had no effect. Figure 3 C). QUE at low concentrations (40 μM and 60 μM) has an effect on... ABCC1 Transcription was unaffected, but significantly upregulated at high concentrations (80 μM and 100 μM). ABCC1 mRNA levels. Elacridar also produced the same upregulation effect, but less than QUE80 and QUE100.

[0126] Compared with the DHQ group alone, LPS significantly decreased ABCC3 mRNA levels ( Figure 3 D, p <0.05). All concentrations of QUE, and the elacridar group, showed significant upregulation. ABCC3 The mRNA level was significantly higher than that of the LPS model group. Furthermore, except for Q40, the other three concentrations were more effective than elacridar, but there were no significant differences among them.

[0127] Tight junction protein ZO-1 ( TJP1 Claudin-1 CLDN1 ) and Occludin ( OCLN The mRNA expression level of ) was also measured ( Figure 3EG). LPS stimulation significantly downregulated the mRNA expression of Occludin ( p <0.05, Figure 3 G), but for TJP1 and CLDN1 No significant effect ( Figure 3 (E, F) This indicates that LPS partially disrupts the integrity of tight junctions. This result is consistent with the phenomenon in Table 3 that LPS leads to an increase in DHQ inflow, jointly confirming the impairment of barrier function.

[0128] In the presence of LPS, QUE was significantly upregulated at all concentrations compared to the model group. TJP1 The expression ( Figure 3 E), and higher concentrations (80-100) showed better effects, but there was no significant difference between them. Elacridar, however, had no effect. For CLDN1 LPS did not cause any change in its mRNA levels. Compared with the model group, QUE and elacridar levels were significantly different. CLDN1 There were no significant differences in mRNA levels. Figure 3 F). Lowered by LPS OCLN All concentrations of QUE and the elacridar group significantly reversed the decrease in expression. Figure 3 G), and QUE60, QUE80, and QUE100 are significantly more effective than elacridar.

[0129] These results indicate that, in an inflammatory environment, QUE modulates the drug efflux system and tight junction barrier of Caco-2 cells by inhibiting the dominant efflux pump. ABCB1 The expression of mRNA, the differential regulation of MRP family members, and the upregulation of tight junction protein genes provide key mechanistic evidence at the molecular level for explaining their enhancement of barrier function and promotion of DHQ uptake.

[0130] For example, the expression levels of inflammatory factors in cells are shown here in a co-culture model.

[0131] To evaluate the anti-inflammatory effects of DHQ and QUE in an intestinal barrier-immune cell interaction model, we established a Caco-2 / THP-1 macrophage co-culture system and induced inflammation using LPS. The expression levels of inflammation-related gene mRNAs in THP-1 cells were as follows: Figure 4 As shown.

[0132] Three hours after LPS stimulation, compared with the DHQ treatment alone group, HO1 mRNA expression levels were significantly reduced ( p <0.05, Figure 4 A). However, QUE significantly reversed this trend and was revised upward. HO-1 The mRNA level showed a concentration-dependent effect (40 to 100 μM). Co-incubation of elacridar with DHQ also significantly upregulated [mRNA levels]. HO1 mRNA, with effects comparable to QUE100.

[0133] Compared with the DHQ group alone, LPS successfully induced an inflammatory response and significantly improved [the condition]. COX2 , TNFA , IL6 , IL8 and IL1B mRNA levels ( p <0.05)( Figure 4 BF), the secretion levels of key pro-inflammatory cytokines IL-8 and IL-1β in the cell culture supernatant were also significantly increased ( p <0.05)( Figure 4 HI). Except for QUE40, all concentrations of QUE and elacridar significantly inhibited [the disease / condition]. COX-2 mRNA expression ( p <0.05), and there were no significant differences between groups. For TNFA Compared with the model group, QUE40 promoted the transcription of mRNA, while high concentrations of QUE (60, 80, and 100) significantly inhibited its transcription, with no significant differences among the groups, while elacridar had no effect. Figure 4 C).

[0134] Figure 4 D shows that QUE40 and QUE80 are... IL-6 mRNA was unaffected, but QUE60 was significantly increased. IL-6 Only high concentrations of quercetin (100 μM) and elacridar, when co-incubated with DHQ, could significantly inhibit mRNA. IL-6 mRNA ( p <0.05) LPS stimulation IL10 mRNA expression was significantly increased at all concentrations of QUE and elacridar. IL10 Overexpression of mRNA, with QUE100 showing significantly higher efficacy than the other four intervention groups ( p <0.05, Figure 4 E).

[0135] for IL8 mRNA, QUEQ40 and QUE60 had no effect, while QUEQ80 and QUE100 showed significant inhibitory effects. p <0.05, the effect is comparable to that of the elacridar group ( Figure 4F). Figure 4 H showed that the level of cytokine IL-8 was only significantly suppressed at QUE100.

[0136] Except for QUE60, all other concentrations of QUE were significantly downregulated when co-incubated with DHQ. IL1B mRNA ( p <0.05, the elacridar group showed the strongest inhibitory effect ( Figure 4 (G). Under QUE and elacridar conditions, the secretion level of the cytokine IL-1β showed a significant downregulation. There was no significant difference among the QUE40, 60, and 80 groups, and the expression level in the QUE100 group was significantly lower than that in the QUE40-80 group. Figure 4 I). This demonstrates that the higher the concentration of QUE co-incubated with DHQ, the stronger the inhibitory effect on the expression of pro-inflammatory factors.

[0137] The above results indicate that in the Caco-2 / THP-1 macrophage co-culture model, co-incubation of DHQ and QUE can effectively alleviate LPS-induced inflammatory responses, as evidenced by upregulation. HO1 It inhibits the secretion and expression of various pro-inflammatory factors, and this effect is more pronounced at higher QUE concentrations. This is because DHQ is better absorbed at higher concentrations.

[0138] For example, the intestinal absorption results of DHQ and QUE in SD rats in vitro are shown here.

[0139] Figure 5 The results are from rat intestinal eversion experiments using DHQ (100 μM) as a substrate and different concentrations of QUE (40-100 μM) as inhibitors.

[0140] Figure 5 A represents the absorption of the substrate DHQ at 45 min and 90 min. It is evident that the DHQ absorption at 90 min is significantly higher than that at 45 min (except for DHQ100+QUE100).

[0141] At 45 min, the uptake of DHQ under elacridar and low concentrations of QUE (40 and 60 μM) showed no significant difference compared to the DHQ-only group, and the uptake was less than 2 nmol. This indicates that at short time and low inhibitor concentrations, the efflux of DHQ substrate by efflux proteins may not be inhibited, resulting in low DHQ uptake. However, when the QUE concentration increased to 80 and 100 μM, DHQ uptake significantly increased, and at 100 μM, the uptake reached 6.669 ± 0.201 nmol, significantly higher than that at 80 μM. p<0.05).

[0142] At 90 min, the absorbance of DHQ without inhibitors was only 3.024 ± 0.396 nmol. With increasing QUE concentration (60 to 100 μM), the absorbance of DHQ increased significantly. p <0.05 indicates a higher inhibition efficiency of efflux transporters, and this concentration-dependent effect is observed. However, when the QUE concentration increases to 100 μM, the DHQ absorption is not different from that at 80 μM, indicating that the QUE's effect in inhibiting efflux has reached saturation. Under the action of Elacridar, the DHQ absorption significantly increases to 5.7 ± 0.638 nmol ( p <0.05), comparable to the QUE60 μM group.

[0143] Figure 5 B represents the absorbance of QUE. At 45 min, under co-incubation conditions, there was no significant difference in the absorbance of QUE after co-incubation with low concentrations of QUE (40 and 60 μM) and DHQ. However, when the concentration was increased from 60 μM to 100 μM, the absorbance of QUE showed a concentration-dependent effect and significantly increased to 4.363 ± 0.461 nmol. p <0.05. The lowest absorption was observed when QUE acted alone (2.071 ± 0.068 nmol).

[0144] At 90 min, the absorbance of QUE showed a concentration-dependent decreasing trend with increasing concentration. Furthermore, there was no significant difference in the absorbance of QUE at 80 μM and 100 μM.

[0145] from Figure 5 B also found that when QUE was co-incubated with DHQ at lower concentrations (40 μM and 60 μM), the uptake at 90 min was significantly greater than that at 45 min, while at QUE concentrations of 80 μM and 100 μM, the uptake at 45 min was significantly greater than that at 90 min.

[0146] When QUE was incubated alone, the uptake at 90 min was significantly higher than that at 45 min. These results reveal a more complex mechanism, suggesting that QUE itself may be a substrate of the efflux pump, its uptake at high concentrations may be self-inhibited, and it may interact with DHQ during uptake to compete for the same efflux pump.

[0147] This invention demonstrates that quercetin can enhance cellular uptake of DHQ by inhibiting efflux proteins in KB / MDR cells overexpressing P-glycoprotein by combining quercetin and dihydroquercetin in a specific ratio; and using a Caco-2 cell model, it was found that quercetin can regulate the expression of efflux proteins and tight junction proteins, and bidirectional transport experiments confirmed that it can significantly increase the apparent permeability coefficient of DHQ. Furthermore, in the Caco-2 / THP-1 inflammation co-culture model, it was verified that quercetin could still improve DHQ transport and regulate the expression of related protein genes and inflammatory responses under inflammatory conditions. Finally, through the SD rat intestinal sac inversion experiment, the promoting effect of quercetin on DHQ absorption and its enhanced physiological relevance were confirmed at the isolated organ level.

[0149] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method of promoting intestinal absorption of dihydroquercetin, characterized by, The composition comprises an effective amount of dihydroquercetin and an effective amount of quercetin as a synergist, wherein the quercetin functions through at least one of the following mechanisms: (a) inhibiting the function or expression of P-glycoprotein in intestinal epithelial cells; (b) regulating the tight junction of intestinal epithelial cells.

2. The method of claim 1, wherein, The quercetin inhibits the expression of P-glycoprotein by down-regulating the protein expression level of P-glycoprotein and / or the messenger ribonucleic acid expression level of the gene encoding the P-glycoprotein. ABCB1 The quercetin inhibits the expression of P-glycoprotein by down-regulating the protein expression level of P-glycoprotein and / or the messenger ribonucleic acid expression level of the gene encoding the P-glycoprotein.

3. The method of claim 1, wherein, The quercetin regulates the tight junction by up-regulating the expression of at least one of the following tight junction related proteins: claudin, claudin-1, and occludin.

4. The method of claim 1, wherein, The molar ratio of dihydroquercetin to quercetin is 1:0.4 to 1:1.

5.

5. Use of a composition for the manufacture of a product for promoting the intestinal absorption of dihydroquercetin, characterized in that, The composition comprises dihydroquercetin and quercetin as active ingredients, wherein the quercetin as a synergist promotes the absorption of dihydroquercetin by inhibiting P-glycoprotein and regulating the tight junction.

6. Use according to claim 5, characterized in that, The product is a pharmaceutical composition, a health food, or a functional food.

7. Use according to claim 5, characterized in that, The composition can make the efflux ratio of dihydroquercetin less than 1 in an intestinal epithelial cell model.

8. Use of a composition comprising dihydroquercetin and quercetin for the manufacture of a product for preventing or alleviating inflammation of the intestinal tract, characterized in that, The quercetin synergistically exerts an anti-inflammatory effect by promoting the intestinal absorption of dihydroquercetin.

9. Use according to claim 8, characterized in that, The composition exerts an anti-inflammatory effect through at least one of the following mechanisms: (a) up-regulating the expression of heme oxygenase-1; (b) down-regulating the expression of one or more inflammatory factors selected from the group consisting of cyclooxygenase-2, tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8.

10. A pharmaceutical composition, characterized by, The composition comprises dihydroquercetin and quercetin as active ingredients, and pharmaceutically acceptable excipients; The molar ratio of dihydroquercetin to quercetin is 1:0.6 to 1:1.

0. The quercetin is used to inhibit the expression of P-glycoprotein and up-regulate the expression of claudin and occludin-1, so as to improve the intestinal absorption rate of dihydroquercetin.

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