Preparation method and application of flavonoid compound in dihydroquercetin oral absorption enhancer by inhibiting P-glycoprotein and pharmaceutical composition

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a preparation method and application of a flavonoid compound in a dihydroquercetin oral absorption enhancer by inhibiting P-glycoprotein and a pharmaceutical composition, and the preparation method comprises the following steps: the flavonoid compound is selected from at least one of quercetin, kaempferol, luteolin and flavone; the flavonoid compound is quercetin; the flavonoid compound promotes oral absorption of dihydroquercetin by inhibiting mRNA (messenger Ribonucleic Acid) and / or protein expression of P-glycoprotein and / or by competitively binding a substrate binding site of the P-glycoprotein. According to the invention, the excretion function of P-glycoprotein in intestinal tracts can be safely and effectively inhibited, and the expression level of the P-glycoprotein is reduced, so that the oral absorption and bioavailability of dihydroquercetin are remarkably improved; wherein quercetin is proved to be a natural accelerant with the best effect, and a brand new solution is provided for developing dihydroquercetin medicines or functional foods with high bioavailability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing, application, and pharmaceutical composition of a flavonoid compound by inhibiting P-glycoprotein in an oral absorption enhancer of dihydroquercetin. Background Technology

[0002] Dihydroquercetin (also known as piperidine) is a natural flavonoid compound with various physiological activities, including antioxidant, anti-inflammatory, antibacterial, and cardiovascular protective effects. However, its extremely low oral bioavailability severely limits its application and commercial development. Studies have shown that the absolute bioavailability of 99% pure dihydroquercetin is only 0.49% after oral administration; while the absolute bioavailability of dihydroquercetin dissolved in solution is even lower, at only 0.17%.

[0003] One of the key reasons for the poor oral absorption of dihydroquercetin is the efflux mediated by P-glycoprotein (P-gp) highly expressed on the intestinal epithelial cell membrane. P-glycoprotein is an important drug efflux pump that actively pumps absorbed substrate drugs out of the cell, thereby reducing their blood concentration and tissue exposure. Our previous studies have confirmed that the efflux ratio of dihydroquercetin in the Caco-2 cell monolayer model is as high as 4.13; in KB / MDR cells overexpressing P-glycoprotein, the uptake of dihydroquercetin is significantly lower than that in parental KB cells, which fully demonstrates that dihydroquercetin is a substrate of P-glycoprotein.

[0004] To address this issue, using P-glycoprotein inhibitors is an effective strategy. Currently, some chemical inhibitors (such as ecrizotinib) have been reported, but they may have toxic side effects and safety concerns, making them unsuitable for functional foods or long-term supplements. Therefore, developing safe, natural P-glycoprotein inhibitors is a current research hotspot.

[0005] Flavonoids are widely found in fruits, vegetables, and traditional Chinese medicine, and are generally considered safe for consumption. Multiple studies have reported that some flavonoids (such as tangerine flavonoids and genistein flavonoids) inhibit P-glycoprotein-mediated efflux. However, due to structural differences, different flavonoids may exhibit significantly different inhibitory efficacy and mechanisms of action (e.g., competitive inhibition, non-competitive inhibition, or downregulation of P-glycoprotein expression). Currently, there is a lack of in-depth research on the systematic comparison of the absorption-promoting effects, structure-activity relationship, and molecular mechanisms of four common dietary flavonoids—quercetin, kaempferol, luteolin, and flavonoids—on dihydroquercetin as a specific substrate, their inhibitory effects on P-glycoprotein, and their specific absorption-promoting effects.

[0006] Therefore, there is an urgent need in the field for a solution that can effectively and safely improve the oral bioavailability of dihydroquercetin, especially for screening highly effective P-glycoprotein inhibitors with well-defined mechanisms of action from natural and safe dietary components. Summary of the Invention

[0007] 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 for preparing a flavonoid compound that inhibits P-glycoprotein in a dihydroquercetin oral absorption enhancer, its application, and a pharmaceutical composition to improve the oral absorption and bioavailability of dihydroquercetin.

[0008] To achieve the above objectives, a first aspect of the present invention provides a method for preparing an oral absorption enhancer of dihydroquercetin, comprising the following steps: Provides flavonoids as active ingredients; The flavonoids are mixed with pharmaceutical excipients to prepare an oral formulation; The flavonoids are selected from at least one of quercetin, kaempferol, luteolin, and flavonoids. The oral absorption enhancer is used to improve the oral bioavailability of dihydroquercetin by inhibiting the efflux function of P-glycoprotein.

[0009] In some embodiments of the present invention, the flavonoid compound is quercetin.

[0010] In some embodiments of the present invention, the mass ratio of the flavonoid compound to dihydroquercetin in the oral absorption enhancer is from 0.5:1 to 3:1.

[0011] In some embodiments of the present invention, the mass ratio of the flavonoid compound to dihydroquercetin is 1:1 to 2.5:1.

[0012] In some embodiments of the present invention, the oral preparation is a tablet, capsule, granule, powder, or oral liquid.

[0013] To achieve the above objectives, a second aspect of the present invention provides the application of a flavonoid compound in the preparation of a pharmaceutical or functional food composition for improving the oral bioavailability of dihydroquercetin, wherein the flavonoid compound is selected from at least one of quercetin, kaempferol, luteolin, and flavonoids.

[0014] In some embodiments of the present invention, the flavonoid compound is quercetin.

[0015] In some embodiments of the present invention, the flavonoids promote the oral absorption of dihydroquercetin by inhibiting the mRNA and / or protein expression of P-glycoprotein and / or by competitively binding to the substrate binding site of P-glycoprotein.

[0016] To achieve the above objectives, a third aspect of the present invention provides a pharmaceutical composition for improving the oral bioavailability of dihydroquercetin, comprising dihydroquercetin, an oral absorption enhancer prepared by the above method, and pharmaceutical excipients.

[0017] In some embodiments of the present invention, the concentration of dihydroquercetin in the composition is from 40 μM to 100 μM, and the concentration of the flavonoid compound is from 40 μM to 120 μM.

[0018] This invention utilizes specific flavonoid compounds as P-glycoprotein inhibitors in the dihydroquercetin administration system, which can safely and effectively inhibit the efflux of P-glycoprotein in the intestine and downregulate its expression level, thereby significantly improving the oral absorption and bioavailability of dihydroquercetin. Quercetin has been proven to be the most effective natural promoter, providing a novel solution for developing highly bioavailable dihydroquercetin drugs or functional foods. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the KB cell viability detection results (MTT method) provided by the present invention; Figure 2 This is a schematic diagram of the KB / MDR cell viability detection results (MTT method) provided by the present invention; Figure 3 This is a schematic diagram illustrating the effect of flavonoid inhibitors provided by the present invention on dihydroquercetin uptake in KB / MDR cells; Figure 4 This is a schematic diagram illustrating the uptake of co-incubated flavonoid inhibitors by KB / MDR cells provided by the present invention; Figure 5 This is a schematic diagram illustrating the effect of quercetin on P-glycoprotein mRNA and protein expression provided by the present invention; Figure 6 This is a schematic diagram illustrating the effect of kaempferol on P-glycoprotein mRNA and protein expression provided by the present invention; Figure 7 This is a schematic diagram illustrating the effect of luteolin on P-glycoprotein mRNA and protein expression provided by the present invention; Figure 8 This is a schematic diagram illustrating the effect of flavonoids on P-glycoprotein mRNA and protein expression provided by the present invention; Figure 9This is a schematic diagram illustrating the effect of co-incubation of flavonoid inhibitors and dihydroquercetin provided by the present invention on the expression of P-glycoprotein; Figure 10 This is a schematic diagram illustrating the effect of flavonoids provided by this invention on the viability of Caco-2 cells (MTT assay); Figure 11 This is a schematic diagram of the molecular docking model of the binding site between flavonoids and P-glycoprotein substrates provided by the present invention; Figure 12 This is a schematic diagram of the molecular docking model of the binding site between flavonoids and P-glycoprotein inhibitors provided by the present invention. Detailed Implementation

[0020] 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.

[0021] The following description, with reference to the accompanying drawings, describes the preparation method of flavonoids in the preparation of oral absorption enhancers for dihydroquercetin, the application of flavonoids in the preparation of pharmaceutical or functional food compositions for improving the oral bioavailability of dihydroquercetin, and compositions for improving the oral bioavailability of dihydroquercetin. Example 1:

[0022] This embodiment presents a method for preparing an oral absorption enhancer of dihydroquercetin, fully demonstrating each step of the preparation process, including the screening and provision of flavonoids, the determination of their mass ratio with dihydroquercetin, the selection and mixing of pharmaceutical excipients, and the final molding process of the oral formulation.

[0023] Optionally, this embodiment first relates to the step of providing flavonoids as active ingredients. The selected flavonoids are chosen from at least one of quercetin, kaempferol, luteolin, and flavonoids. These compounds are all naturally occurring dietary components with high safety for consumption. Specifically, quercetin, kaempferol, luteolin, and flavonoids are all commercially available, for example, from Aladdin Chemicals Ltd., with a purity of not less than 98%. When selecting, single or multiple components can be used in combination based on the expected inhibitory effect and cost. In particular, quercetin has been shown to be the most effective natural promoter in inhibiting the efflux function of P-glycoprotein.

[0024] Therefore, in a preferred embodiment, the selected flavonoid compound is quercetin. Its mechanism of action lies in its ability to reduce the pumping of dihydroquercetin out of cells by inhibiting the efflux function of P-glycoprotein, which is highly expressed on the intestinal epithelial cell membrane, thereby increasing its exposure and bioavailability in vivo.

[0025] For example, after identifying the active ingredient, this embodiment further clarifies the mass ratio between flavonoids and dihydroquercetin. This mass ratio ranges from 0.5:1 to 3:1.

[0026] More preferably, the mass ratio ranges from 1:1 to 2.5:1. This range was determined based on extensive dose-response studies in in vitro experiments. For example, in the KB / MDR cell uptake assay, when flavonoids were co-incubated with dihydroquercetin at different mass ratios, it was observed that within the specified range, the cellular uptake of dihydroquercetin was significantly increased without significant cytotoxicity.

[0027] In practical application, for example, 100 mg of quercetin can be weighed and mixed with 100 mg of dihydroquercetin to achieve a 1:1 mass ratio; or 125 mg of quercetin can be weighed and mixed with 50 mg of dihydroquercetin to achieve a 2.5:1 mass ratio. This ratio range ensures that the flavonoids can effectively exert their P-glycoprotein inhibitory effect, while forming a synergistic effect with dihydroquercetin, optimizing the absorption-promoting effect without significantly increasing the total dose.

[0028] Optionally, the core step of this embodiment involves mixing flavonoids with pharmaceutical excipients and preparing a formulation suitable for oral administration. The oral formulation may be tablets, capsules, granules, powders, or oral liquids. The selection of pharmaceutical excipients should meet pharmaceutical standards and ensure the stability, manufacturability, and patient compliance of the formulation. Commonly used pharmaceutical excipients include, but are not limited to, fillers such as microcrystalline cellulose, lactose, or starch; binders such as polyvinylpyrrolidone or hydroxypropyl methylcellulose; disintegrants such as croscarmellose sodium or carboxymethyl starch sodium; lubricants such as magnesium stearate or silica; and, if necessary, flavoring agents, coloring agents, and stabilizers. These excipients should have good compatibility with the active ingredient and should not affect its biological activity.

[0029] For example, the following describes the preparation process of tablets in detail. First, according to the aforementioned determined mass ratio, a specified amount of flavonoid active ingredients, such as quercetin and dihydroquercetin, is accurately weighed. Next, the weighed active ingredients, along with predetermined amounts of filler microcrystalline cellulose and disintegrant croscarmellose sodium, are placed in a high-efficiency mixer and thoroughly dry-mixed to ensure uniform distribution of each component. Then, an appropriate amount of pre-prepared binder solution, such as a 10% concentration of polyvinylpyrrolidone aqueous solution, is slowly added, and wet granulation is performed under stirring. The resulting wet granules are then transferred to a fluidized bed dryer and dried at 50 to 60 degrees Celsius until the moisture content of the granules drops to below 5%. The dried granules are then sized by passing them through a vibrating sieve to obtain an intermediate with a uniform particle size distribution. Finally, the sized granules are mixed evenly with the lubricant magnesium stearate in a double-cone mixing drum to obtain the final tableting mixture. The mixture is compressed into tablets using a rotary tablet press, controlling tablet weight variation within legally permissible limits and ensuring the tablets have appropriate hardness and friability, with disintegration time meeting pharmacopoeia requirements. Tablets prepared in this way contain a specific ratio of flavonoids and dihydroquercetin per unit dose.

[0030] Optionally, this embodiment is also applicable to the preparation of other oral dosage forms besides tablets. For capsules, the prepared tableting mixture or similar granule mixture can be directly filled into gelatin capsules or hydroxypropyl methylcellulose capsule shells, and the filling and sealing can be completed using a fully automated capsule filling machine. For granules, after wet granulation and drying, tableting can be avoided, and the granules can be directly dispensed into aluminum-plastic bags or composite film bags according to dosage. For powders, the active ingredient and excipients can be mixed, pulverized and sieved to the specified fineness, and then dispensed. For oral liquids, the active ingredient needs to be mixed with suitable solvents such as water, glycerin, propylene glycol, etc., as well as necessary solubilizers such as polysorbate, stabilizers such as ascorbic acid, and flavoring agents such as steviol glycosides. The mixture needs to be stirred, homogenized, or even micronized to ensure the formation of a uniform solution or suspension, and then filled into oral liquid bottles or ampoules. Regardless of the dosage form, the core principle is to ensure that flavonoids and dihydroquercetin coexist in a specific mass ratio in the final dosing unit.

[0031] For example, the oral absorption enhancer prepared in this embodiment functions primarily by inhibiting the efflux function of P-glycoprotein through its contained flavonoids, thereby improving the oral bioavailability of dihydroquercetin. For instance, in the classic in vitro intestinal absorption model of the Caco-2 cell monolayer, dihydroquercetin exhibits extremely low influx and high efflux ratio when present alone, confirming that its poor absorption is mainly attributed to the active efflux of P-glycoprotein. However, when co-incubated with flavonoids, especially quercetin, the influx of dihydroquercetin significantly increases, the efflux significantly decreases, and the efflux ratio drops dramatically, directly demonstrating the improved absorption. Molecular docking studies further revealed its mechanism of action at the molecular level, indicating that flavonoids such as quercetin can competitively bind to the substrate binding site of P-glycoprotein, or interfere with the efflux process of dihydroquercetin by downregulating the mRNA and protein expression levels of P-glycoprotein.

[0032] Optionally, process parameters can be optimized and adjusted when implementing this preparation method. For example, during mixing, the mixing time and speed can be determined based on the properties of the materials and batch size to ensure uniform mixing. During granulation, the amount and rate of binder addition can be controlled to obtain particles of the ideal size. During drying, the drying temperature and time settings need to balance efficiency with the protection of heat-sensitive active ingredients. During tableting or filling, machine parameters such as pressure and speed need to be adjusted to ensure the appearance and internal quality of the product. All these process steps and parameter selections aim to ensure that the stability, content uniformity, and in vitro dissolution behavior of the active ingredients in the final formulation meet design requirements.

[0033] For example, to verify the efficacy of the oral absorption enhancer prepared in this embodiment, in vitro evaluation methods can be referenced. For instance, the transport behavior of the formulation in a simulated intestinal environment can be assessed using a Caco-2 cell model; or the content and dissolution rate of the active ingredient in the formulation can be determined by high-performance liquid chromatography or liquid chromatography-tandem mass spectrometry. These quality control measures help ensure that each batch of product consistently performs its intended absorption-enhancing function. Example 2:

[0034] This embodiment presents the application of flavonoids in the preparation of pharmaceutical or functional food compositions that improve the oral bioavailability of dihydroquercetin. The aim is to demonstrate how flavonoids can effectively promote the intestinal absorption of dihydroquercetin through specific molecular mechanisms, thereby significantly improving its oral bioavailability, and to provide practical guidance and theoretical support for the development of related therapeutic drugs or daily health care products.

[0035] Optionally, the flavonoids used in this embodiment are selected from at least one of quercetin, kaempferol, luteolin, and flavonoids. These compounds are widely found in natural diets, such as fruits, vegetables, and tea, and have a recognized safety profile and good biocompatibility, making them ideal for long-term use in pharmaceuticals or functional foods. Based on systematic experimental comparisons, including cellular uptake kinetics and dose-response studies, quercetin has been shown to be the component that best promotes the absorption of dihydroquercetin. For example, in the KB / MDR cell model, quercetin most significantly increased the cumulative uptake of 40 μmol of dihydroquercetin at all tested concentrations, with the order of potency being quercetin > luteolin > kaempferol > flavonoids. Therefore, in a preferred embodiment, the flavonoid is quercetin to maximize the absorption-promoting effect.

[0036] For example, flavonoids promote the oral absorption of dihydroquercetin by inhibiting the mRNA and / or protein expression of P-glycoprotein and / or by competitively binding to the substrate binding site of P-glycoprotein. At the gene and protein levels, real-time quantitative polymerase chain reaction and Western blotting results showed that quercetin, kaempferol, luteolin, and flavonoids, whether alone or co-incubated with dihydroquercetin, significantly reduced the mRNA and protein expression levels of P-glycoprotein in KB / MDR cells.

[0037] For example, quercetin exhibits inhibitory effects on P-glycoprotein expression in the concentration range of 40 to 100 μmol, showing a dose-dependent effect at certain concentrations. Simultaneously, molecular docking experiments revealed a competitive binding mechanism: quercetin forms multiple hydrogen bonds and aromatic hydrocarbon-hydrogen interactions with the substrate binding site of P-glycoprotein, its conformation being similar to that of dihydroquercetin, thus competitively occupying the binding site and preventing the efflux of dihydroquercetin. This dual mechanism of action—downregulating P-glycoprotein expression and directly competitively inhibiting its function—synergistically reduces the efflux of dihydroquercetin from intestinal cells, increasing its influx and intracellular accumulation.

[0038] Optionally, in pharmaceutical compositions, flavonoids and dihydroquercetin can be integrated into an oral delivery system to form a stable formulation. For example, in the preparation of tablets or capsules, quercetin can be used as the active ingredient, mixed with dihydroquercetin, pharmaceutical excipients such as diluents, binders, and lubricants, and formulated into a unit dose form using standard pharmaceutical processes. The concentration of dihydroquercetin in the composition can be set to between 40 and 100 micromoles, and the concentration of the flavonoids can be set to between 40 and 120 micromoles.

[0039] In specific applications, this pharmaceutical composition can be used to treat or prevent conditions related to oxidative stress, inflammation, or cardiovascular disease. Dihydroquercetin acts as the main active ingredient, while flavonoids act as absorption enhancers. Through the above mechanism, the efficient absorption of dihydroquercetin in the intestine is ensured.

[0040] For example, in functional food compositions, flavonoids and dihydroquercetin can be added to food or health supplement matrices, such as fortified beverages, dietary supplement granules, or healthy snacks. Because flavonoids themselves possess antioxidant and anti-inflammatory properties, their combination with dihydroquercetin not only enhances bioavailability by inhibiting P-glycoproteins but may also produce additional health benefits. Conventional food processing techniques, such as mixing, granulation, or emulsification, can be used in preparation to ensure uniform distribution and stability of the active ingredients, thereby enabling them to effectively promote absorption in the intestinal environment after ingestion.

[0041] Optionally, those skilled in the art can adjust the types and combinations of flavonoids according to the actual application scenario. For example, considering cost or raw material availability, one or more of kaempferol, luteolin, or a mixture of flavonoids can be used, although their effects may be inferior to quercetin, they can still achieve significant absorption improvement. During application, it is necessary to ensure that the flavonoids and dihydroquercetin coexist adequately in the formulation and maintain their activity so as to effectively interact in the intestinal tract during digestion and jointly address the challenge of P-glycoprotein-mediated efflux. Example 3:

[0042] This embodiment presents a pharmaceutical composition for improving the oral bioavailability of dihydroquercetin, the pharmaceutical composition comprising a therapeutically effective amount of dihydroquercetin, an oral absorption enhancer prepared by the method in Example 1, and pharmaceutical excipients.

[0043] Optionally, the core active ingredients in the composition include dihydroquercetin and flavonoids as oral absorption enhancers. The concentration of dihydroquercetin in the composition is from 40 μmol to 100 μmol, and the concentration of flavonoids is from 40 μmol to 120 μmol. For example, in dynamic bidirectional transport experiments in Caco-2 monolayer cells, 40 μmol and 100 μmol of dihydroquercetin were used as model substrate concentrations, while 100 μmol of flavonoid inhibitors (quercetin, kaempferol, luteolin, flavonoids) were shown to significantly improve the absorption of dihydroquercetin, manifested as increased influx and decreased efflux. In practice, when preparing a certain volume or mass of the final composition, it is necessary to ensure that the final concentrations of dihydroquercetin and flavonoids fall within the above-mentioned ranges.

[0044] For example, the active ingredient of an oral absorption enhancer is at least one flavonoid compound selected from quercetin, kaempferol, luteolin, and flavonoids.

[0045] In a preferred embodiment, quercetin is selected as the sole active ingredient for the absorption enhancer. Prior to mixing with dihydroquercetin, the enhancer may have been premixed with some pharmaceutical excipients to form an intermediate, such as the particulate mixture prepared according to the method of Example 1. In the final composition, the oral absorption enhancer, along with a therapeutically effective amount of dihydroquercetin and additional pharmaceutical excipients, constitutes a unified drug delivery system.

[0046] Optionally, pharmaceutical excipients include one or more of the following pharmaceutically acceptable carriers, diluents, binders, disintegrants, lubricants, flavoring agents, colorants, and stabilizers. The selection and dosage of these excipients must ensure the stability, manufacturability, palatability, and bioavailability of the active ingredient in the composition. For example, in preparing a composition in tablet form, dihydroquercetin raw material, pre-prepared quercetin promoter granules, fillers such as microcrystalline cellulose, disintegrants such as croscarmellose sodium, and lubricants such as magnesium stearate can be mixed and then directly compressed into tablets. The specific form of the composition is not limited to tablets; it can also be any suitable oral dosage form such as capsules, granules, powders, or oral liquids.

[0047] As an example, the following provides a specific example of tablet composition preparation. First, a precise amount of dihydroquercetin raw material is weighed to ensure a concentration of 40 micromoles in the final tablet. Simultaneously, intermediate granules of an oral absorption enhancer containing quercetin as the active ingredient, prepared by wet granulation, are weighed, ensuring a quercetin concentration of 100 micromoles in the final tablet. The dihydroquercetin raw material, the enhancer granules, an appropriate amount of microcrystalline cellulose as a filler, and croscarmellose sodium as a disintegrant are placed together in a mixer and thoroughly mixed. Then, magnesium stearate is added as a lubricant for final mixing to form a homogeneous tableting mixture. Finally, the mixture is compressed into tablets using a tableting machine. In this tablet composition, both dihydroquercetin and the flavonoid quercetin are present at precisely controlled concentrations, working together to exert therapeutic and absorption-enhancing effects.

[0048] Optionally, in the Caco-2 monolayer cell model, the test groups containing 40 μmol and 100 μmol of dihydroquercetin showed a significantly reduced efflux ratio (from 27.24 to 1.86) and a substantial increase in influx transport compared to the control group using dihydroquercetin alone. This fully demonstrates that the composition can effectively overcome P-glycoprotein-mediated efflux and significantly improve the intestinal absorption of dihydroquercetin. Flavonoids create a more favorable absorption environment for dihydroquercetin by inhibiting the expression and / or competitively inhibiting the function of P-glycoprotein. Example 4:

[0049] Corresponding to the above embodiments regarding the preparation method, application, and composition, this embodiment presents 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 Flavonoids (dihydroquercetin, flavonoids, luteolin, quercetin, kaempferol) were purchased from Aladdin Chemical Co., Ltd. (purity ≥98%, Shanghai, China). Elacridar (GF120918A) was purchased from MedChem Express Ltd. (Juncushion, New Jersey, USA). Hygromycin was purchased from Roche Group (Hoffmann-LaRoche Ltd., Switzerland).

[0050] 2. Cell Culture KB cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). KB / MDR1 cells were obtained through stable transfection with human P-glycoprotein (P-gp) cDNA, based on previous research by our group. Both KB and KB / MDR cells were cultured in Duchenne basal medium (DMEM, Gibco, Wuhan, China) containing 10% fetal bovine serum (Gibco), 1% penicillin (100 U / mL), and streptomycin (100 μg / mL) at 37°C and 5% CO2. KB / MDR cells were cultured in medium containing 500 μg / mL hygromycin after each passage.

[0051] Caco-2 cells were cultured in MEM medium containing 10% fetal bovine serum, 1% non-essential amino acids, 1% penicillin, and streptomycin, under conditions of 5% CO2 and 37°C. The cells used in this study were passaged between 45 and 55 times.

[0052] Specifically, the experimental methods include: 1. Cell viability detection Cells were seeded in 96-well plates and cultured for 24 hours, followed by treatment with a flavonoid compound for 3 hours. Then, 0.5 mg / mL of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) was added to the wells, and incubation continued for 4 hours. After dissolving the formazan crystals formed within the cells with dimethyl sulfoxide, the absorbance was measured at 490 nm using a microplate reader. A significant difference in cell viability compared to the control group indicated that the compound was cytotoxic.

[0053] 2. Flavonoid uptake experiment in KB cells and KB / MDR cells KB / MDR cells with Cells were seeded at a density of cells / well in 12-well plates and cultured until confluence. Cells were pre-incubated for 30 min with different concentrations (40 μM, 60 μM, 80 μM, 100 μM, 120 μM) of inhibitors (quercetin, kaempferol, luteolin, flavonoids). Then, 40 μM of dihydroquercetin was added, and the cells were incubated with the corresponding inhibitors at 37°C for different times (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h).

[0054] Cells were washed twice with phosphate-buffered saline (PBS) in an ice bath, collected, and lysed by sonication. The lysates were freeze-dried and stored at -80°C for analysis by high-performance liquid chromatography (HPLC). The cellular uptake of each flavonoid compound was normalized to the protein content determined by the dicaprylic acid (BCA) method, and the results are expressed as the compound concentration per gram of protein.

[0055] 3. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from cells using an RNA extraction kit (Adley Biotechnology Co., Ltd., Wuhan, China) according to the kit instructions. The RNA was reverse transcribed into complementary DNA (cDNA) and then subjected to qRT-PCR. The primers used in the experiment were as follows: human... ABCB1 The primers for the gene (encoding P-glycoprotein) are 5'-CCTTCACCCAGGCAATGATG-3' (upstream) and 5'-TGGGCTGCTGATATTTTTGGC-3' (downstream); the internal reference gene is β-actin. ACTB The primers were 5'-AGCGAGCATCCCCCAAAGTT-3' (upstream) and 5'-GGGCACGAAGGCTCATCATT-3' (downstream).

[0056] The qRT-PCR reaction conditions were: 94℃ pre-denaturation for 30 seconds; followed by 40 cycles, each cycle consisting of 94℃ denaturation for 10 seconds, annealing at 60℃, and extension for 30 seconds. The ΔΔCt method was used [ΔΔCt=2]. （ΔCtACTB-ΔCtABCB1） ] Calculate the target gene ( ABCB1 The fold change in expression relative to the control group.

[0057] 4. Western blotting Cells were washed twice with ice-bathed phosphate-buffered saline (PBS) and then lysed in radioimmunoprecipitation (RIPA) lysis buffer containing protease inhibitors and phosphatase inhibitors (Servicebio Technology Co., Ltd., Wuhan, China).

[0058] After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 5% bovine serum albumin (BSA) for 1 h. The membrane was then immersed in a primary antibody solution (P-glycoprotein / P-gp or β-actin / β-actin) and incubated overnight at 4°C. After incubation with secondary antibody for 1 h, the protein bands were visualized using enhanced chemiluminescence detection reagents (Thermo Fisher Scientific, USA) and a GE Amersham Imager 600 imaging system (GE Healthcare Corporate, Wisconsin, USA).

[0059] 5. Experiment on the transport of flavonoids in Caco-2 monolayer cells Caco-2 cells were seeded in 12-well Transwell chambers and cultured for 21 days to form a Caco-2 monolayer cell model. Monolayer cell models with transepithelial resistance (TEER) values ​​above 600 Ω·cm² before and after the transport experiment were retained for data acquisition.

[0060] After washing cells with phenol red-free MEM medium, dihydroquercetin and its inhibitor were added to the top side (AP side, 0.5 mL) or the basal side (BL side, 1.5 mL). Phenol red-free MEM medium was added to the receiving chamber, and the cells were incubated for 6 h. At 1 h, 2 h, and 4 h of incubation, 200 μL of sample was collected from the receiving chamber, and 200 μL of phenol red-free MEM medium was added. At 6 h of incubation, all samples from the top and basal sides were collected. All samples were immediately frozen, lyophilized, and stored at -80°C for analysis by liquid chromatography-mass spectrometry (LC-MS / MS).

[0061] 6. High-performance liquid chromatography (HPLC) analysis of flavonoid uptake in cells. The lyophilized samples were reconstituted with methanol, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC. The content of flavonoids was 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.

[0062] 7. Determination of flavonoid transport in Caco-2 monolayer cells by 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).

[0063] 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.

[0064] 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 1.

[0065] Quantitative analysis was performed using the external standard method, with standard curves constructed using standard solutions ranging from 0.05 to 150 μM. Quantification was performed using the parent 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.

[0066] Table 1. Multiple reaction monitoring (MRM) transition parameters of flavonoids in liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) analysis.

[0067] 8. Molecular docking The 3D structures of ligands (dihydroquercetin and four flavonoid inhibitors) were plotted and optimized in MOE 2024 software, and a ligand database was established. Two human P-glycoprotein 3D structures (Protein Data Bank PDB numbers 9CTF and 9CTC, respectively) were selected for docking: 9CTF is the co-crystal structure of P-glycoprotein and the substrate taxol, used for substrate docking; 9CTC is the co-crystal structure of P-glycoprotein and the inhibitor zosuquidar, used for inhibitor docking.

[0068] After preprocessing the 9CTF and 9CTC structures, docking calculations were performed: the docking "site" was changed from "ligand atoms" to "selected atoms of co-crystal ligand", the "ligand" was set to the established ligand database, and all other parameters were set to default.

[0069] Conversely, the experimental results include: For example, the effects of flavonoids on the viability of KB cells and KB / MDR cells are shown here.

[0070] Effects of tested flavonoids on the viability of KB cells and KB / MDR cells, such as Figure 1 and Figure 2 As shown, the maximum non-toxic concentrations of dihydroquercetin in KB cells and KB / MDR cells were 100 μM and 200 μM, respectively. This result may be due to the efflux of dihydroquercetin by P-glycoprotein (P-gp) in KB / MDR cells, leading to a decrease in the effective intracellular concentration of dihydroquercetin.

[0071] The maximum non-toxic concentration of quercetin in KB / MDR cells (>400 μM) was higher than its concentration in KB cells (100 μM), indicating that quercetin may also be pumped out of the cells by P-glycoproteins. Meanwhile, the maximum non-toxic concentrations of kaempferol, luteolin, and flavonoids in KB / MDR cells (all 120 μM) were consistent with their concentrations in KB cells (all 120 μM), suggesting that P-glycoprotein-mediated efflux did not occur in these three compounds.

[0072] Based on the viability test results of KB / MDR cells, all flavonoids were used at the same non-toxic concentration (maximum 120 μM) in subsequent KB / MDR cell uptake experiments.

[0073] For example, the effect of flavonoid inhibitors on dihydroquercetin uptake in KB / MDR cells is shown here.

[0074] Dynamic uptake of 40 μM dihydroquercetin (dihydroquercetin 40) by KB / MDR cells is as follows: Figure 3 As shown. Throughout the dynamic process, the uptake of dihydroquercetin 40 remained low, with significant increases only observed at 2 h (6.78 ± 0.52 nmol / g) and 2.5 h (11.38 ± 2.15 nmol / g). p <0.05).

[0075] The P-glycoprotein inhibitor ecrizotin significantly increased the uptake of dihydroquercetin 40 at all tested time points. p <0.05), with a maximum of 171.29 nmol / g, especially with more significant effects after incubation for 2 h and 2.5 h.

[0076] Four flavonoid inhibitors also significantly increased the uptake of dihydroquercetin 40 throughout the entire dynamic process. p <0.05), with a maximum of 448.59 nmol / g. It is noteworthy that, except for flavonoids, the other three flavonoid inhibitors were more effective than ecrizotinib. Among them, quercetin significantly increased the uptake of dihydroquercetin 40 than kaempferol and luteolin.

[0077] In terms of the rate of increase in uptake, flavonoids and quercetin showed the highest rate of increase in dihydroquercetin 40 uptake during the incubation period of 1.5 h to 2 h; while kaempferol and luteolin showed the highest rate of increase during the incubation period of 0 h to 0.5 h. During the incubation period of 1 h to 1.5 h, the five inhibitors (four flavonoid inhibitors + ectelide) had little effect on dihydroquercetin 40 uptake, and the cumulative uptake of dihydroquercetin 40 at these two time points was similar.

[0078] In the presence of quercetin or flavonoids, the uptake of dihydroquercetin 40 approached a plateau; however, in the presence of luteolin or kaempferol, the uptake of dihydroquercetin 40 continued to increase during the 2-2.5 h incubation period. These results indicate that all four flavonoid inhibitors can effectively and dynamically reverse P-glycoprotein-mediated dihydroquercetin 40 efflux, with quercetin exhibiting the best inhibitory effect on P-glycoprotein.

[0079] The dose-effects of four flavonoid inhibitors on dihydroquercetin 40 uptake are as follows: Figure 3 As shown in B. When used alone, dihydroquercetin 40 has extremely low uptake at low concentrations (≤100 μM) (≤57.35 nmol / g), with moderate uptake only occurring at 120 μM (≤183.21 nmol / g).

[0080] At all tested concentrations (except for 120 μM flavonoids and luteolin), the uptake of dihydroquercetin 40 was significantly increased in a non-dose-dependent manner in the presence of each flavonoid inhibitor. p <0.05), with a maximum of 428 nmol / g. Among them, flavonoids showed the best effect on increasing the uptake of dihydroquercetin 40 at 60 μM; luteolin showed the best effect at 80 μM; quercetin showed the best effect at 100 μM; and kaempferol showed the best effect at 120 μM.

[0081] Clearly, at all tested concentrations (except 120 μM), quercetin significantly outperformed the other three flavonoid inhibitors in enhancing dihydroquercetin 40 uptake in a dose-dependent manner. Only at low concentrations (40 μM and 60 μM) did the reversal effect of flavonoids on dihydroquercetin 40 uptake show a dose-dependent effect, with the lowest overall effect. Luteolin significantly increased dihydroquercetin 40 uptake at low concentrations (≤100 μM). p <0.05), but had no significant effect at 120 μM. Kaempferol increased the uptake of dihydroquercetin 40 at all tested concentrations, but was unaffected by concentration changes.

[0082] The above results fully demonstrate that, over a wide concentration range, quercetin exhibits the most significant dynamic inhibitory effect on P-glycoprotein.

[0083] For example, this section demonstrates the uptake of flavonoid inhibitors by KB / MDR cells after co-incubation.

[0084] Understanding how dihydroquercetin affects the uptake of co-incubated flavonoid inhibitors (FIs) is equally important for elucidating their potential inhibitory mechanism against P-glycoproteins. Therefore, this study measured the dynamic uptake of flavonoid inhibitors, and the results are as follows: Figure 4 As shown.

[0085] The dynamic uptake of all inhibitors (0.65-61.38 μmol / g) was higher than that of 40 μM dihydroquercetin (dihydroquercetin 40), indicating that dihydroquercetin has a weaker cellular uptake capacity, while flavonoid inhibitors have a stronger cellular uptake capacity. During the incubation period of 0.5 h to 1.5 h, the autologous uptake of the five inhibitors (four flavonoid inhibitors + ectelida) did not change significantly, and the cumulative uptake at these three time points was similar. The uptake of flavonoids and luteolin approached a plateau, while the uptake of quercetin and kaempferol continued to increase during the incubation period of 2 h to 2.5 h. Figure 4 A).

[0086] The uptake of 10 μM ectelida was low (≤2.2 nmol / g) before 1.5 h of incubation, and increased significantly only at 2 h (3.76±0.34 nmol / g) and 2.5 h (5.93±0.38 nmol / g). p <0.05). Throughout the entire dynamic process, quercetin had the lowest uptake (≤6.34 nmol / g), while kaempferol had the highest uptake (up to 61.38 nmol / g).

[0087] The above results suggest that quercetin dynamically reverses P-glycoprotein-mediated dihydroquercetin efflux, possibly through a competitive inhibition mechanism. Specifically, quercetin replaces its substrate (dihydroquercetin) and is pumped out of the cell by P-glycoprotein, thereby increasing the intracellular accumulation of dihydroquercetin 40. The uptake of luteolin and flavonoids showed moderate stability with minimal time influence, indicating that the intracellular accumulation of these two flavonoid inhibitors remained stable at a concentration of 100 μM.

[0088] Similarly, this study also examined the dose-effect of four co-incubated flavonoid inhibitors on uptake, and the results were as follows: Figure 4 As shown in B. At all tested concentrations, the uptake of all inhibitors (0.43–63.74 μmol / g) was higher than that of dihydroquercetin 40. Overall, the uptake of the four flavonoid inhibitors increased in a dose-dependent manner within the concentration range of 40–120 μM, with luteolin and quercetin (except at 120 μM) showing the most significant dose dependence.

[0089] At low concentrations (≤80 μM), the uptake of flavonoids and kaempferol was not affected by concentration; however, uptake increased significantly at 100 μM and remained stable at 120 μM. Quercetin showed the lowest uptake at all tested concentrations (≤3.84 nmol / g). These results further confirm that quercetin exerts the strongest inhibitory effect on P-glycoprotein-mediated dihydroquercetin efflux through a competitive inhibition mechanism. Luteolin showed the highest uptake at low concentrations (≤80 μM), while kaempferol showed the highest uptake at high concentrations (≥80 μM).

[0090] For example, the effects of flavonoid inhibitors on P-glycoprotein mRNA and protein expression are shown here.

[0091] This study evaluated the effects of flavonoid inhibitors alone on the mRNA and protein expression of P-glycoprotein in KB / MDR cells. The results are as follows: Figure 5-7 As shown, compared with parental KB cells, the mRNA and protein expression levels of P-glycoprotein in KB / MDR cells were significantly increased, which confirms the overexpression characteristic of P-glycoprotein in KB / MDR cells.

[0092] 40 μM dihydroquercetin or ecrizotin had no effect on the mRNA and protein expression of P-glycoprotein in KB / MDR cells. However, at all tested concentrations, all flavonoid inhibitors, when used alone, significantly reduced the mRNA and protein expression levels of P-glycoprotein in KB / MDR cells. Specifically, the inhibitory effect of quercetin on P-glycoprotein mRNA and protein expression gradually weakened as the concentration increased from 40 μM to 80 μM, and remained stable at 100 μM. The inhibitory effects of kaempferol and luteolin on P-glycoprotein mRNA and protein expression were not affected by concentration. High concentrations (80-100 μM) of flavonoids showed significantly better inhibitory effects on P-glycoprotein mRNA and protein expression than low concentrations (40-60 μM) of flavonoids.

[0093] To evaluate the expression of P-glycoprotein in a substrate-inhibitor co-incubation system, this study also measured the effects of co-incubation of 40 μM or 100 μM flavonoid inhibitors with 40 μM dihydroquercetin on the mRNA and protein expression of P-glycoprotein in KB / MDR cells. The results are as follows: Figure 9 As shown in AF. Dihydroquercetin alone had no effect on the mRNA and protein expression of P-glycoprotein in KB / MDR cells; however, compared to dihydroquercetin alone, all flavonoid inhibitors, when co-incubated with dihydroquercetin at concentrations of 40 μM and 100 μM, significantly reduced the mRNA and protein expression levels of P-glycoprotein. p <0.05).

[0094] At a concentration of 40 μM, there was no significant difference in the inhibitory effects of different types of flavonoid inhibitors on P-glycoprotein expression. Figure 9 AC). At a concentration of 100 μM, flavonoids showed the strongest inhibitory effect on P-glycoprotein expression, while kaempferol and luteolin showed moderate inhibition, and quercetin showed the weakest inhibitory effect. Figure 9 DF). At a concentration of 40 μM, quercetin showed the strongest inhibitory effect on P-glycoprotein mRNA transcription, while kaempferol and flavonoids showed moderate inhibition, and luteolin showed the weakest inhibitory effect. Figure 9 C). At a concentration of 100 μM, except for kaempferol, different types of flavonoid inhibitors showed no significant difference in their inhibitory effects on P-glycoprotein transcription. Figure 9 F).

[0095] For example, the effect of flavonoid inhibitors (dihydroquercetin) on the viability of Caco-2 cells is shown here.

[0096] The effects of tested flavonoids on Caco-2 cell viability, such as Figure 10 As shown in the figure. At all tested concentrations ranging from 40 to 200 μM, none of the five flavonoids were toxic to differentiated Caco-2 cells. Based on the concentrations used in the above uptake experiments, in subsequent Caco-2 cell-related experiments, the concentrations of dihydroquercetin were set at 40 μM and 100 μM, and the concentrations of the four inhibitors were all set at 100 μM.

[0097] For example, the effect of flavonoid inhibitors on bidirectional transport of dihydroquercetin in Caco-2 monolayer cells is shown here.

[0098] To verify whether flavonoid inhibitors effectively inhibit P-glycoprotein-mediated dihydroquercetin efflux in an environment more closely resembling the real gut, this study used Caco-2 monolayer cells—a classic in vitro model of intestinal absorption—as the research subject. The dynamic bidirectional transport of dihydroquercetin after co-incubation with different concentrations of dihydroquercetin (40 μM, dihydroquercetin 40; 100 μM, dihydroquercetin 100) and a flavonoid inhibitor (100 μM) was measured (Table 2). Influx of dihydroquercetin was assessed by measuring the amount transported from the apical to the basal side, while efflux was assessed by measuring the amount transported from the basal to the apical side.

[0099] When used alone, the influx of dihydroquercetin 40 was extremely low (maximum only 25.05 pmol), and the transport rate was slow throughout the experiment; however, its efflux transport increased significantly and rapidly from 2 h to 6 h, reaching 688.22 pmol at 6 h. The efflux ratio of dihydroquercetin 40 at 6 h was 27.24, indicating a significant efflux phenomenon. Overall, all five inhibitors (four flavonoid inhibitors + ectoderma) significantly increased the influx of dihydroquercetin 40 at all time points (except for flavonoids at 1 h and 2 h), and significantly reversed the efflux transport of dihydroquercetin 40 at each time point (except for ectoderma at 1 h and 2 h).

[0100] Eclareta significantly increased the influx of dihydroquercetin 40 at every time point, reaching a maximum of 127.04 pmol at 6 h; and significantly reversed P-glycoprotein-mediated dihydroquercetin 40 efflux at 4 h (decreasing from 122.59 pmol to 94.98 pmol) and 6 h (decreasing from 688.22 pmol to 412.36 pmol). The efflux ratio of dihydroquercetin 40 at 6 h decreased from 27.24 to 3.25. These results indicate that this inhibitor can improve the intestinal absorption of dihydroquercetin 40 by increasing influx and reversing P-glycoprotein-mediated efflux.

[0101] Quercetin significantly increased the influx of dihydroquercetin 40 at all time points (except at 1 h, where its effect was superior to ectoda), with the highest influx reaching 167.62 pmol at 6 h. Furthermore, it significantly reversed the efflux of dihydroquercetin 40 at all time points, particularly at 4 h (52.83 pmol) and 6 h (311.77 pmol). The efflux ratio of dihydroquercetin 40 decreased from 27.24 to 1.86 at 6 h. These results indicate that quercetin effectively improves the intestinal absorption of dihydroquercetin 40 by enhancing influx (superior to the other four inhibitors) and reducing efflux (superior to ectoda).

[0102] Kaempferol and luteolin significantly increased the influx of dihydroquercetin 40 (approximately 80 pmol) at each time point and significantly reversed its efflux at each time point with a similar mechanism of action, particularly at 4 h (75.86 pmol with kaempferol and 47.89 pmol with luteolin) and 6 h (approximately 300 pmol with both flavonoid inhibitors). The efflux ratio of dihydroquercetin 40 decreased to 3.26 (with kaempferol) and 3.94 (with luteolin) at 6 h, respectively. These results indicate that both kaempferol and luteolin can improve the intestinal absorption of dihydroquercetin 40 by increasing influx and inhibiting efflux (with better efflux inhibition than ectoderma), but their overall effect is weaker than that of ectoderma.

[0103] Flavonoids significantly increased the influx of dihydroquercetin 40 only at 4 h and 6 h, showing the weakest effect among the five inhibitors; however, they significantly reversed the efflux of dihydroquercetin 40 at all time points, especially at 4 h (30.89 pmol) and 6 h (126.55 pmol), where their efflux inhibition was superior to the other four inhibitors. The efflux ratio of dihydroquercetin 40 decreased to the lowest level of 1.54 at 6 h. These results indicate that flavonoids can improve the intestinal absorption of dihydroquercetin 40 by increasing influx (weaker than ectoda) and weakening efflux (better than the other four inhibitors).

[0104] The translocation capacity of dihydroquercetin 40 at 1 h was extremely low; therefore, this study only measured the influx and efflux translocation of dihydroquercetin 100 within 2–6 h (Table 2). Compared with dihydroquercetin 40, the influx translocation capacity of dihydroquercetin 100 increased only slightly at 4 h (12.01 pmol) and 6 h (36.23 pmol). p <0.05), but its external discharge and transfer volume increased significantly from 2 h to 6 h ( p <0.05), reaching as high as 867.28 pmol at 6 h. Although the efflux ratio of dihydroquercetin 100 at 6 h (23.94) was slightly lower than that of dihydroquercetin 40, the former's efflux transport was significantly higher than that of the latter. p <0.05), and the influx transport of the two is similar, indicating that the efflux of dihydroquercetin is aggravated at high concentrations.

[0105] Overall, all five inhibitors significantly increased the influx of dihydroquercetin 100 at all time points. p <0.05), and the increase was significantly higher than that of dihydroquercetin 40 influx; in addition, except for ecrizotin at 2 h, all five inhibitors significantly inhibited the efflux transport of dihydroquercetin 100 at all time points.p <0.05), and the inhibitory effect was also superior to the inhibitory effect on the efflux of dihydroquercetin 40. Eclatide significantly increased the influx of dihydroquercetin 100 and reversed its efflux at each time point, indicating that at high concentrations, clatide has a stronger inhibitory effect on P-glycoprotein-mediated dihydroquercetin efflux. Except for flavonoids, the other four inhibitors all reduced the efflux ratio of dihydroquercetin 100 at 6 h, indicating that at high concentrations, these inhibitors have a more significant effect on improving the intestinal absorption of dihydroquercetin 100.

[0106] Quercetin showed the best effect in improving the influx of dihydroquercetin 100 at all time points, and the best inhibitory effect on the efflux of dihydroquercetin 100 was observed at 4 h. The increase in influx and reversal of efflux of dihydroquercetin 100 by quercetin were both greater than those by dihydroquercetin 40. Kaempferol, luteolin, and flavonoids significantly increased the influx of dihydroquercetin 100 and significantly reversed its efflux at all time points, and their effects on dihydroquercetin 100 were superior to those on dihydroquercetin 40.

[0107] The above results indicate that the four flavonoid inhibitors have a better effect on improving the intestinal absorption of dihydroquercetin 100 than on dihydroquercetin 40.

[0108] Table 2 Dynamic bidirectional transport of dihydroquercetin in Caco-2 monolayer cells

[0109] Dihydroquercetin 40: 40 μM dihydroquercetin. Dihydroquercetin 100: 100 μM dihydroquercetin. Dihydroquercetin was co-incubated with an efflux transporter inhibitor (100 μM) in Caco-2 monolayer cells. Uppercase letters indicate significant differences at different time points under the same compound treatment conditions. Lowercase letters indicate significant differences between different inhibitors at the same time point. "*" and "**" indicate that the difference between dihydroquercetin 40 and dihydroquercetin 100 was statistically significant under the same treatment conditions ("*" corresponds to...). p <0.05, "**" corresponds to p <0.01).

[0110] For example, this demonstrates the bidirectional transport of flavonoid inhibitors in Caco-2 monolayers co-incubated with dihydroquercetin.

[0111] To investigate the inhibitory mechanism of flavonoid inhibitors on P-glycoprotein, this study also measured the bidirectional transport of four flavonoid inhibitors in Caco-2 monolayer cells when co-incubated with dihydroquercetin 40 or dihydroquercetin 100 (Table 3).

[0112] In the system co-incubated with dihydroquercetin 40: flavonoids exhibited the highest influx transport (up to 125.06 nmol), luteolin showed a moderate influx transport, and kaempferol and quercetin had the lowest influx transport (<0.65 nmol). The order of efflux transport for the four inhibitors was consistent with the order of influx transport. Compared to adjacent time points, the influx transport of the four flavonoid inhibitors dynamically increased at each time point. p <0.05 (except for flavonoids at 1 h), the trend of efflux transport was consistent with that of influx transport. Except for kaempferol (efflux ratio of 5.63), the efflux ratios of the other three inhibitors were all less than 1, indicating that the influx transport of these three inhibitors was higher than that of efflux transport.

[0113] In the system co-incubated with dihydroquercetin 100, the order of endo- and efflux transport of the four flavonoid inhibitors was consistent with that when co-incubated with dihydroquercetin 40. Compared with adjacent time points, the endo- and efflux transport of the four inhibitors changed significantly at each time point. Compared with the system co-incubated with dihydroquercetin 40, the efflux ratios of quercetin, luteolin (all efflux ratios > 1), and flavonoids increased when co-incubated with dihydroquercetin 100, while the efflux ratio of kaempferol decreased. Notably, except for kaempferol at 4 h and 6 h, the endo-transport of the four flavonoid inhibitors co-incubated with dihydroquercetin 100 was significantly lower at each time point than when co-incubated with dihydroquercetin 40, indicating that these inhibitors have a competitive transport relationship with dihydroquercetin. In addition, the efflux transport of the four flavonoid inhibitors co-incubated with dihydroquercetin 100 was significantly lower at each time point than when co-incubated with dihydroquercetin 40.

[0114] Table 3. Dynamic bidirectional transport of flavonoid inhibitors during co-incubation in Caco-2 monolayer cells.

[0115] Dihydroquercetin 40: 40 μM dihydroquercetin. Dihydroquercetin 100: 100 μM dihydroquercetin. Dihydroquercetin was co-incubated with an efflux transporter inhibitor (100 μM) in Caco-2 monolayer cells. Uppercase letters indicate significant differences at different time points under the same compound treatment conditions. Lowercase letters indicate significant differences between different inhibitors at the same time point. "*" and "**" indicate that the difference between dihydroquercetin 40 and dihydroquercetin 100 was statistically significant under the same treatment conditions ("*" corresponds to...). p <0.05, "**" corresponds to p <0.01).

[0116] For example, here are the results of molecular docking research.

[0117] To investigate the interaction between substrates or inhibitors and P-glycoprotein, this study docked five flavonoids (dihydroquercetin and flavonoid inhibitors) to the substrate binding sites of the transmembrane domains (TMDs) of P-glycoprotein (using the 9CTF structure containing paclitaxel cocrystal ligands as the substrate binding site model). Figure 11 ) or inhibitor binding site (using the 9CTC structure containing the zolsudil co-crystal ligand as the inhibitor binding site model, Figure 12 ).

[0118] like Figure 11 As shown, dihydroquercetin exhibits a moderate interaction with P-glycoprotein. Its carbonyl group at position 4 (4-C=O) forms hydrogen bonds with glutamine 838 (Gln838), and its hydroxyl group at position 3 (3-OH) forms hydrogen bonds with asparagine 721 (Asn721), respectively. Simultaneously, its A ring forms an aromatic hydrocarbon-hydrogen interaction with valine 991 (Val991). This indicates that dihydroquercetin readily occupies the substrate binding site of P-glycoprotein, thus enabling its transport by P-glycoprotein.

[0119] Quercetin and dihydroquercetin have similar conformations, both forming hydrogen bonds with glutamine 838 (Gln838) via the carbonyl group at position 4 (4-C=O) and forming an aromatic hydrocarbon-hydrogen interaction with valine 991 (Val991) via ring A. Furthermore, quercetin's ring B also forms an additional aromatic hydrocarbon-hydrogen interaction with phenylalanine 770 (Phe770). This indicates that quercetin can competitively occupy the binding site of dihydroquercetin, thus forming a moderate affinity for the substrate binding site of P-glycoproteins.

[0120] For the other three flavonoid inhibitors, their interactions with the P-glycoprotein substrate binding sites were weaker and fewer in number. Kaempferol and luteolin formed only one hydrogen bond with glycine 300 (Gly300) via the carbonyl group at position 4 (4-C=O); flavonoids formed only weak aryl-aryl interactions. This suggests that these three flavonoid inhibitors have low affinity for the P-glycoprotein substrate binding sites, indicating that they may occupy the dihydroquercetin substrate binding site in a non-competitive manner.

[0121] like Figure 12 As shown, dihydroquercetin has a very low affinity for the binding site of the P-glycoprotein inhibitor, and only forms a weak aromatic-hydrogen interaction with the hydrogen of leucine 65 (Leu65) through the A ring.

[0122] The interaction between quercetin and P-glycoprotein is characterized by hydrogen bonds formed between its 3-hydroxyl group (3-OH) and the key residue glutamic acid 875 (Glu875), and between its 7-hydroxyl group (7-OH) and histidine 1 (His61), respectively. Simultaneously, the B ring forms an aromatic hydrocarbon-hydrogen interaction with isoleucine 340 (Ile340). This indicates that quercetin can bind to the inhibitory binding site of P-glycoprotein.

[0123] Kaempferol and luteolin form a moderate affinity for P-glycoprotein. Kaempferol forms a hydrogen bond with threonine 199 (Thr199) through a 4'-hydroxyl group (4'-OH), and its A ring forms an aromatic-hydrogen interaction with leucine 65 (Leu65). Luteolin forms a hydrogen bond with threonine 199 (Thr199) through a 7-hydroxyl group (7-OH), and its B ring forms an aromatic-hydrogen interaction with leucine 65 (Leu65).

[0124] Because flavonoids lack specific functional groups, they have the lowest affinity for P-glycoproteins. They only form an aromatic hydrocarbon-aromatic hydrocarbon interaction with phenylalanine 983 (Phe983) through the A ring, and an aromatic hydrocarbon-hydrogen interaction with tyrosine 950 (Tyr950) through the B ring.

[0125] The above results explain why quercetin has the strongest inhibitory effect on P-glycoprotein-mediated dihydroquercetin efflux, while flavonoids have the weakest inhibitory effect.

[0126] This invention investigates the dose- and time effects of flavonoids on the inhibition of P-glycoprotein (P-gp) efflux and the structure-activity relationship of P-glycoprotein expression inhibition, aiming to identify the flavonoid inhibitor with the best efflux effect. The activity mechanics of P-gp-ATPase was analyzed, and the cellular accumulation of dihydroquercetin was monitored to determine the inhibitor's mechanism of action. Molecular simulations were used to reveal the molecular mechanism by which the inhibitor intervenes in the interaction between dihydroquercetin and P-glycoprotein. Furthermore, the dynamic bidirectional transport of dihydroquercetin after co-incubation with four flavonoid inhibitors was measured in Caco-2 monolayer cells.

[0127] All of the above research was funded by the National Natural Science Foundation of China (Project No.: 32302002).

[0128] 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 for preparing an oral absorption enhancer of dihydroquercetin, characterized in that, Includes the following steps: Provides flavonoids as active ingredients; The flavonoids are mixed with pharmaceutical excipients to prepare an oral formulation; The flavonoids are selected from at least one of quercetin, kaempferol, luteolin, and flavonoids. The oral absorption enhancer is used to improve the oral bioavailability of dihydroquercetin by inhibiting the efflux function of P-glycoprotein.

2. The method according to claim 1, characterized in that, The flavonoid compound is quercetin.

3. The method according to claim 1 or 2, characterized in that, In the oral absorption enhancer, the mass ratio of the flavonoid compound to dihydroquercetin is 0.5:1 to 3:

1.

4. The method according to claim 3, characterized in that, The mass ratio of the flavonoids to dihydroquercetin is from 1:1 to 2.5:

1.

5. The method according to claim 1, characterized in that, The oral preparations are tablets, capsules, granules, powders, or oral liquids.

6. The use of a flavonoid compound in the preparation of a pharmaceutical or functional food composition for improving the oral bioavailability of dihydroquercetin, characterized in that, The flavonoids are selected from at least one of quercetin, kaempferol, luteolin, and flavonoids.

7. The application according to claim 6, characterized in that, The flavonoid compound is quercetin.

8. The application according to claim 6, characterized in that, The flavonoids promote the oral absorption of dihydroquercetin by inhibiting the mRNA and / or protein expression of P-glycoprotein and / or by competitively binding to the substrate binding site of P-glycoprotein.

9. A pharmaceutical composition for improving the oral bioavailability of dihydroquercetin, characterized in that, It comprises dihydroquercetin, an oral absorption enhancer prepared by the method of any one of claims 1-5, and pharmaceutical excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, The concentration of dihydroquercetin in the composition is from 40 μM to 100 μM, and the concentration of the flavonoid compound is from 40 μM to 120 μM.

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