Use of quinic acid in the preparation of an anti-glycation inhibitor and a medicament for treating neurocellular inflammation

Abstract

The application discloses application of quinic acid in preparation of anti-glycation inhibitors and drugs for treating nerve cell inflammation and belongs to the technical field of organic acids. Quinic acid can precisely capture dicarbonyl compounds (GO / MGO) by virtue of low steric hindrance and strong nucleophilic alcohol hydroxyl groups, so that efficient chemical blocking is realized. In the cell physiological end (intrinsic physiological environment), aiming at part of the end products (CML) which escape and induce cytotoxicity, quinic acid can compete with RAGE receptors (binding energy-5.13 kcal / mol) to effectively block the AGEs-RAGE pathogenic signal axis, so that BV2 cells are protected from oxidative stress damage. The finding constructs a whole-chain intervention strategy from chemical blocking at the food processing end to biological protection at the cell end.

Description

Technical Field

[0001] This invention relates to the field of organic acid technology, and more specifically to the use of quinic acid in the preparation of anti-glycation inhibitors and drugs for treating neuronal inflammation. Background Technology

[0002] Currently, heat-processed foods (especially aquatic products rich in protein and unsaturated fatty acids) are highly susceptible to the formation of advanced glycation end products (AGEs) during high-temperature processing. Nε-carboxymethyl lysine (CML) is one of the representative AGEs. Numerous studies have shown that dietary AGEs can accumulate in the body after intestinal absorption. By binding to receptors for advanced glycation end products (RAGEs) on cell membranes, they induce excessive production of intracellular reactive oxygen species (ROS) and lipid peroxidation (elevated malondialdehyde (MDA)), thereby triggering oxidative stress and neuronal inflammatory damage. This is closely related to the development and progression of diabetic complications, Alzheimer's disease, and other neurodegenerative diseases.

[0003] To reduce the formation of AGEs in food and their health risks, existing technologies mainly employ two strategies: one is to add exogenous inhibitors (such as chemically synthesized inhibitors like aminoguanidine and natural plant polyphenols) during food processing to reduce AGE formation at the source; the other is to block the binding of AGEs to RAGEs at the physiological level, thereby mitigating their damage to cells. However, existing technologies still have the following shortcomings:

[0004] (1) Traditional anti-glycation inhibitors focus on the phenolic hydroxyl structure, neglecting organic acid components. Currently reported anti-glycation active substances are mostly concentrated in polyphenolic compounds such as flavonoids and anthocyanins containing phenolic hydroxyl groups, which are considered to be the core structure for scavenging free radicals and inhibiting AGEs. However, these substances are easily consumed by a large number of free radicals in lipid-rich thermal processing systems, resulting in a significant decrease in their ability to capture dicarbonyl precursors. In contrast, small molecule organic acids rich in alcoholic hydroxyl groups (such as quinic acid) may exhibit better targeted capture efficiency in lipid-rich thermal processing systems due to their low steric hindrance and strong nucleophilicity, but existing studies have paid very little attention to this.

[0005] (2) Existing anti-glycation studies are mostly limited to aqueous models, which are disconnected from real fat-rich food systems. Most in vitro screenings rely on simple aqueous systems such as bovine serum albumin-fructose, while the thermal processing of real meat products (especially fish) involves a multiphase complex matrix of proteins, reducing sugars, and polyunsaturated fatty acids. Existing technologies have failed to fully reveal the core mechanism of lipid oxidation driving AGEs formation, resulting in a significant reduction in the efficacy of many highly active polyphenols screened in aqueous models in real meat products.

[0006] (3) A complete chain of evidence from food processing to physiology has not yet been established. Existing studies mostly focus on the chemical blocking of AGEs in food, and few studies have explored whether active components can alleviate AGEs-induced oxidative stress damage at the cellular level based on the clear inhibitory effect of AGEs in food systems. Furthermore, there is a lack of targeted intervention programs for specific AGEs (such as CML)-mediated neuronal inflammation.

[0007] Therefore, how to develop a safe and natural active ingredient that can both effectively inhibit the formation of AGEs in heat-processed high-fat foods and effectively alleviate the inflammatory damage to nerve cells caused by AGEs is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide the use of quinic acid in the preparation of anti-glycation inhibitors and medicaments for treating neuronal inflammation, in order to overcome the shortcomings of the prior art.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention seeks protection for the use of quinic acid in the preparation of anti-glycation inhibitors and medicaments for treating neuronal inflammation.

[0010] Furthermore, the aforementioned anti-glycation inhibitors are food ingredients or functional food components used to inhibit the formation of advanced glycation end products in thermally processed food systems.

[0011] Furthermore, the dosage forms of the aforementioned anti-glycation inhibitors are powders, solutions, or microcapsules.

[0012] Furthermore, the aforementioned drugs for treating neurocellular inflammation are preparations used to alleviate neurocellular toxicity and oxidative stress damage induced by CML exposure.

[0013] Furthermore, the dosage forms of the aforementioned drugs for treating neuronal inflammation are injection solutions, lyophilized powder injections, or oral preparations.

[0014] Furthermore, among the aforementioned drugs for treating neuronal inflammation, the effective concentration of quinic acid is 1 μM to 3 μM.

[0015] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention reveals the structure-activity relationship of mulberry polyphenols (quinic acid) in lipid-rich complex systems for anti-glycation and establishes for the first time its dual targeting mechanism of "external inhibitory precursor + internal inhibitory receptor". At the food processing end (external matrix environment), facing the massive amounts of dicarbonyl compounds generated by lipid oxidation, traditional polyphenols lose their anti-glycation efficacy due to excessive consumption of phenolic hydroxyl groups by free radicals; while quinic acid, with its low steric hindrance and strong nucleophilic alcohol hydroxyl groups, precisely captures dicarbonyl compounds (GO / MGO), achieving highly efficient chemical blocking. At the cellular physiological end (internal physiological environment), targeting some escaped end products (CML) that induce cytotoxicity, quinic acid can competitively bind to RAGE receptors (binding energy -5.13 kcal / mol), strongly blocking the AGEs-RAGE pathogenic signaling axis and protecting BV2 cells from oxidative stress damage. This discovery constructs a full-chain intervention strategy from chemical blocking at the food processing end to biological protection at the cellular end. Attached Figure Description

[0016] Figure 1 The content of GO and MGO in the simulation system; Figure 2 The values ​​represent the content of FI-AGEs and CML in the simulated system; Note: Significant differences were observed between different treatments (p<0.05); Figure 3 The values ​​of GO and MGO in the simulated system after the addition of mulberry polyphenols; Note: Significant differences were found between different treatments (p<0.05); Figure 4 The values ​​of FI-AGEs and CML in the simulated system after the addition of mulberry polyphenols; Note: Significant differences were found between different treatments (p<0.05); Figure 5 To screen the safe dosage of quinic acid in BV2 cells and its protective effect against CML-induced damage; (a) shows the effect of different concentrations (1–20 μM) of quinic acid on the relative viability of BV2 cells; (b) shows the protective effect of 3 μM quinic acid against BV2 cell damage induced by CML equivalent to different processed fish paste (raw, grilled, fried, deep-fried); Note: Data are expressed as mean ± standard deviation (n = 3), and different lowercase letters above the bars indicate significant differences between groups (p < 0.05). Figure 6This study investigated the effect of quinic acid on the levels of intracellular oxidative stress markers induced by CML in BV2 cells. (a) represents the degree of cellular lipid peroxidation (MDA content); (b) represents the relative fluorescence intensity of intracellular ROS. Note: BV2 cells were exposed to CML standards equivalent to the CML content in raw, baked, fried, and deep-fried fish paste, with intervention groups including those with and without 3 μM quinic acid. Legend: The left-hand column represents the CML damage model group, and the right-hand column represents the 3 μM quinic acid + CML damage model group. Data are expressed as mean ± standard deviation (n=3). Different lowercase letters above the bars indicate significant differences between treatment groups (p<0.05). Figure 7 The diagram shows a molecular docking simulation of quinic acid with the RAGE receptor (PDBID: 2MOV). (a) is a 3D conformation diagram of quinic acid binding to the active pocket of the RAGE receptor, where the yellow stick-like structures represent quinic acid ligands, the green / cyan bands and stick-like structures represent receptor amino acid residues, and the yellow dashed lines represent intermolecular interactions. (b) is a 2D interaction diagram of quinic acid with key amino acid residues of the receptor, where the green dashed lines represent conventional hydrogen bonds formed between the ligand and receptor residues. RAGE: AGEs receptor, GLU: glutamate, GLY: glycine.

[0017] Figure 8 The effect of different amounts of quinic acid added on the CML content in real fried fish paste was investigated; different lowercase letters above the bars represent significant differences between treatment groups (p<0.05). Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention focuses on quinic acid, a highly effective anti-glycation monomer, and systematically verifies its actual intervention efficacy and cell protection mechanism in a fat-rich thermal processing system. First, a "myofibrillar protein-fructose-fish oil" composite simulation system was constructed to mimic the multiphase environment of real fish processing. The inhibitory effects of different mulberry polyphenol monomers on the formation of GO, MGO, FI-AGEs, and CML were compared, and the structure-activity relationship of alcoholic and phenolic hydroxyl groups in the complex matrix was analyzed in depth. Second, a CML-induced BV2 microglia injury model was established to assess the safe dosage of quinic acid and its regulatory effects on cell viability, ROS, and MDA levels. Finally, molecular docking technology was used to predict the binding mode of quinic acid to RAGE receptors, and its anti-glycation application effect was verified in a real fried fish paste system. This invention aims to fully elucidate the dual-targeting protective mechanism of quinic acid—"external inhibition of precursors and internal inhibition of receptors"—from two dimensions: "chemical nucleophilic capture" and "receptor competitive blockade."

[0020] 1. Materials and Instruments 1.1 Main Reagents Table 1 Main Reagents

[0021] 1.2 Main Instruments Table 2 Main Instruments

[0022] 2. Test methods 2.1 Construction of the myofibrillar protein-fructose-fish oil model simulation system Preparation of myofibrillar protein: Tilapia meat was mixed with 8% (w / v) NaCl solution and cold deionized water in a ratio of 1:2:1 (weight / volume / volume). The mixture was homogenized at 10,000 r / min for 1 min under ice bath conditions. It was then shaken for 10 min and centrifuged at 10,000 r / min for 15 min at 2 °C. The supernatant was collected, diluted 10-fold with cold deionized water, and stirred at 4 °C for 30 min to precipitate the protein. The precipitate was freeze-dried and stored at -80 °C for later use.

[0023] The simulation system was constructed and substrates were selected based on the fact that tilapia muscle tissue is rich in highly unsaturated fatty acids. To extreme and amplify the driving effect of lipid peroxidation on the protein glycosylation cascade in an in vitro model, and to more sensitively and clearly observe the formation and evolution of the core dicarbonyl intermediate (GO / MGO) and AGEs, fish oil (EPA 36%, DHA 24%), which is highly oxidizable, was chosen as a typical representative of the lipid substrates in the simulation system.

[0024] Specific construction process: Using 5 mL of phosphate buffer as the reaction medium, 0.24 g of fish myofibrillar protein, 0.24 g of fructose, and 0.11 g of fish oil were added to prepare the following seven different reaction systems: protein alone, fructose alone, fish oil alone, protein-fructose, protein-fish oil, fructose-fish oil, and protein-fructose-fish oil complex. Each sample was placed in a beaker, sealed with aluminum foil, stirred thoroughly with a glass rod, and then heated in a 230℃ oven for 6 min. After cooling to room temperature, it was used for subsequent experiments.

[0025] Based on the aforementioned myofibrillar protein-fructose-fish oil complex system, six different types of polyphenol solutions (6-methylcoumarin, quinic acid, isochlorogenic acid B, isochlorogenic acid C, isorhamnetin, and hyperoside) were added to the reaction system. Each polyphenol solution was added to the reaction system at a volume of 1 mL and a concentration of 1 μg / mL. After mixing, the mixture was subjected to heat treatment under the same conditions and cooled to room temperature before being used in subsequent experiments.

[0026] 2.2 Determination of GO and MGO content in the simulation system The contents of GO and MGO in tilapia samples were determined by high performance liquid chromatography (HPLC). 2 g of sample was accurately weighed and 10 mL of distilled water was added as the extraction solvent. After vortexing and ultrasonic extraction for 20 min, 10 mL of methanol was added to precipitate the protein. After standing at 4℃ for 30 min, the sample was centrifuged at 8000 r / min for 10 min, and the supernatant was collected. 1 mL of o-phenylenediamine derivatization reagent (0.2%, w / v) was added to 1 mL of the supernatant, and the mixture was reacted in a 37℃ water bath for 2 h in the dark. The reaction was immediately terminated in an ice bath after derivatization. The sample was diluted with 2 mL of 50% methanol aqueous solution (v / v), mixed, filtered through a 0.22 μm organic phase filter membrane, and transferred to an HPLC vial for analysis.

[0027] Chromatographic conditions: Agilent 1260 high-performance liquid chromatography system equipped with a Waters Xbridge C18 column (4.6 mm × 250 mm, 5 μm); column temperature 40℃; detection wavelength 315 nm; injection volume 10 μL. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol; gradient elution program: 0–10 min, 30%–60% B; 10–12 min, 60%–30% B; 12–15 min, hold at 30% B; flow rate 1.0 mL / min. Quantitative analysis was performed using the external standard method.

[0028] 2.3 Determination of FI-AGEs in the Simulation System The content of FI-AGEs in the samples was determined by fluorescence spectrophotometry. 4 g of tilapia sample was accurately weighed, and 20 mL of phosphate buffer (0.2 mol / L, pH 8.2) was added. After homogenization at 10,000 rpm for 1 min, 40 μL of alkaline protease solution (≥2.4 U / g) was added, and the mixture was incubated at 55℃ for 1 h. After hydrolysis, the hydrolysate was centrifuged at 8000 rpm for 15 min, and the supernatant was collected.

[0029] Take an appropriate amount of supernatant and place it in a quartz cuvette. Use a fluorescence spectrophotometer to measure the fluorescence intensity at an excitation wavelength of 347 nm and an emission wavelength of 415 nm (both with a slit width of 5 nm). The results are expressed as a percentage of fluorescence intensity relative to the quinine sulfate standard solution (the fluorescence intensity of 1 μg / mL quinine sulfate solution is defined as 100%).

[0030] 2.4 CML determination in the simulation system The CML content in the samples was determined using an enzyme-linked immunosorbent assay (ELISA) kit. Sample pretreatment: 200 mg of sample was accurately weighed, and 2 mL of borate buffer (0.2 mol / L, pH 9.2) and 1 mL of sodium borohydride solution (1 mol / L) were added. After mixing, the mixture was incubated at 4 °C for 12 h for reduction. After reduction, 4 mL of hydrochloric acid solution (12 mol / L) was added to the system, and the mixture was hydrolyzed at 110 °C for 24 h. The hydrolysate was filtered through filter paper into a 25 mL volumetric flask, and the volume was adjusted to the mark with ultrapure water and mixed thoroughly.

[0031] Solid-phase extraction: Accurately transfer 500 μL of the above solution and evaporate to dryness in a 60℃ oven. Redissolve the residue in 1 mL of ultrapure water and vortex thoroughly. Activate and equilibrate the Sep-Pak MCX solid-phase extraction column sequentially with 3 mL of methanol and 3 mL of ultrapure water. Load the redissolved sample solution onto the column at a flow rate of 1 mL / min. After loading, elute the target analyte with 3 mL of methanol solution containing 5% ammonia and collect the eluent. Perform assays according to the instructions of the CML enzyme-linked immunosorbent assay kit.

[0032] 2.5 Establishment of the BV2 cell model The BV2 mouse microglia cell line is a commonly used in vitro model for studying AGEs-induced neuroinflammatory responses. This invention uses CML (cell-derived AGEs) instead of mixed AGEs as an inducer to more accurately explore the cytotoxicity of specific AGE monomers (CML) and their intervention mechanisms.

[0033] BV2 cells were cultured in DMEM complete medium containing 10% FBS and incubated at 37°C in a 5% CO2 incubator. The experimental procedure is as follows: Cell resuscitation: Preheat the culture medium to 37°C, and sterilize centrifuge tubes, pipette tips, and other consumables with UV for 30 min. Remove cells from liquid nitrogen, thaw rapidly in a 37°C water bath, centrifuge at 800 rpm for 3 min, discard the supernatant, resuspend in 1 mL of culture medium, inoculate into culture dishes, and change the medium after 24 h.

[0034] Cell passage: When the cell density reaches 80%-90%, wash twice with PBS, digest with 0.25% trypsin for 1 min, stop with complete culture medium, centrifuge at 800 rpm for 3 min, discard the supernatant, resuspend and passage at 1:3~1:4, and maintain the density after passage at 30%-40%.

[0035] Cell cryopreservation: After digestion and centrifugation, cells were resuspended in cryopreservation buffer (90% FBS + 10% DMSO), aliquoted into cryovials, and subjected to gradient cooling: 4℃, 5 min; -20℃, 30 min; -80℃, 24 h; and finally transferred to liquid nitrogen for long-term storage.

[0036] Cell plating: 96-well plate: After digestion and counting, dilute with complete culture medium to 4 × 10⁻⁶. 4 Add 100 μL (4 × 10⁻⁶ cells / mL) to each well. 3 (cells / well), cultured for 24 h.

[0037] 6-well plate: After digestion and counting, dilute to 1×10⁻⁶. 5 Add 2 mL per well (i.e., 2 × 10⁻⁶ cells / mL) 5 (cells / well), cultured for 24 h.

[0038] Cell drug administration: CML and polyphenol stock solution were diluted with culture medium according to the experimental concentration. When investigating the protective effect of polyphenol, cells were pretreated with different concentrations of polyphenol for 4 h, and then CML was added and incubated for 24 h. When investigating the optimal concentration of CML to induce an inflammation model, CML was directly added at gradient concentrations for 24 h.

[0039] 2.6 BV2 cell viability assay Cells were seeded in 96-well plates and treated with the drug for 24 h. Cell proliferation activity was then assessed using the CCK8 assay. 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37°C for 1.5 h. After incubation, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula: Cell viability rate = (Experimental group absorbance - Blank group absorbance) / (Normal group absorbance - Blank group absorbance) × 100%; In the formula: blank group: cell-free and drug-free group; normal group: blank control group without drug treatment; experimental group: drug-treated group.

[0040] 2.7 Detection of ROS activity in BV2 cells This invention employs the DCFH-DA fluorescent probe method to detect intracellular ROS levels. The principle is based on the fact that after DCFH-DA enters the cell, it is hydrolyzed by esterases to DCFH, which is then oxidized to the fluorescent substance DCF in the presence of ROS. The fluorescence intensity is directly proportional to the ROS content. The procedure was followed according to the Beyotime ROS detection kit instructions: DCFH-DA was diluted 1:4000 with serum-free DMEM to a final concentration of 3 μmol / L; the culture medium in the 6-well plate was aspirated, and 1.5 mL of diluted DCFH-DA was added to each well to cover the cells; the plate was incubated at 37°C for 20 min; the cells were washed three times with serum-free culture medium to remove any probes that had not entered the cells; immediately after washing, the fluorescence intensity was detected using a microplate reader, and the results were analyzed.

[0041] 2.8 Detection of MDA activity in BV2 cells Determine MDA content according to the kit instructions. Add pre-cooled extraction buffer to cell or tissue samples at a ratio of 100 mg: 1 mL, sonicate on ice, and centrifuge at 13000 g for 10 min at 4°C to collect the supernatant. Transfer an appropriate amount of supernatant to a centrifuge tube, add working solution according to the kit instructions, mix well, and incubate at 95°C for 30 min. After the reaction, quickly cool on ice and centrifuge at 10000 g for 10 min at 25°C. Transfer 200 μL of the centrifuged supernatant to a 96-well plate, and measure the absorbance of the reaction solution at 532 nm and 600 nm using a microplate reader. Subtract background interference from the absorbance at 600 nm. The calculation formula is as follows: ΔA=ΔA 532 -Δ A 600 ; ΔΔ A=Δ A 测定 -Δ A 空白 ; C MDA =51.6×ΔΔ A / C pr ; In the formula: C MDA The sample contains MDA content (nmol / mg prot); A 532 and A 600 51.6 represents the absorbance value at the corresponding wavelength; C is a calculation constant. pr The protein concentration (mg / mL) of the sample.

[0042] 2.9 Molecular docking To explore the possible binding modes of quinic acid and the RAGE receptor, molecular docking simulations were performed. The three-dimensional crystal structure of the RAGE receptor V domain was downloaded from the RCSB PDB database (PDB ID: 2MOV). The three-dimensional structure of quinic acid was obtained from the PubChem database (CID: 6508). Molecular docking calculations were performed using AutoDock Vina software. Before docking, the receptor protein underwent routine pretreatment such as dehydration and hydrogenation; the ligand molecule was optimized for energy minimization using the software's built-in force field. A docking pocket was set to cover the active site region of the RAGE receptor, and the exhaustiveness of the search was set to 32. The docking results were evaluated using binding energy (kcal / mol) to assess the affinity between the ligand and the receptor; a lower binding energy indicates a more stable binding. PyMOL and Discovery Studio Visualizer software were used for three-dimensional and two-dimensional visualization analysis of the optimal docking conformation, demonstrating the interaction between the ligand and key amino acid residues of the receptor.

[0043] 2.10 Construction of an anti-glycation validation model for quinic acid in a real tilapia surimi system To verify the anti-glycation efficacy of polyphenol monomers in real complex food systems, quinic acid, which exhibits the best anti-glycation activity, was selected for a real-world fish paste frying experiment. Tilapia were skinned, deboned, chopped, and then ground into a uniform paste using a high-speed disperser. 5% (w / w) salt was added to the fish paste, and quinic acid powder was added at 0.1%, 0.15%, and 0.3% (w / w) of the fish paste mass, respectively. A control group was used without added quinic acid. The mixture was thoroughly stirred, placed in a sealed bag, and left to stand at 4°C for 4 hours to allow the polyphenols to fully penetrate and bind to the protein matrix. After standing, the fish paste was processed as follows: 20 mL of cooking oil was preheated in an electric frying pan (800W), and the fish paste cakes were fried for 6 minutes. After frying, residual oil was removed from the surface using a stainless steel filter. The mixture was cooled to room temperature, individually packaged, and stored at -80°C for subsequent CML content determination. 200 mg of the fried fish paste sample was accurately weighed, and 2 mL of borate buffer (0.2 mol / L, pH 9.2) and 1 mL of sodium borohydride solution (1 mol / L) were added. After mixing, the mixture was subjected to a reduction reaction at 4°C for 12 h. Then, 4 mL of hydrochloric acid solution (12 mol / L) was added, and the mixture was hydrolyzed at 110°C for 24 h. The hydrolysate was filtered and brought to a final volume of 25 mL. 500 μL of the solution was evaporated to dryness at 60°C. The residue was reconstituted with 1 mL of ultrapure water, purified using a Sep-Pak MCX solid-phase extraction column (activated sequentially with 3 mL of methanol and 3 mL of ultrapure water), and eluted with 3 mL of methanol solution containing 5% ammonia. The eluent was collected. Finally, the CML content was determined according to the instructions of the Nε-carboxymethyl lysine (CML) enzyme-linked immunosorbent assay kit.

[0044] 2.11 Data Analysis Unless otherwise specified, all indicators were measured in triplicate. The experimental data were analyzed using Excel and SPSS 26.0 statistical analysis software, expressed as mean ± standard deviation (sad), and plotted using GraphPad Prism.

[0045] 3. Results and Discussion 3.1 Generation Analysis of AGEs Precursors (GO / MGO) in the Simulation System To further elucidate the contributions and interactions of different components to the formation of AGEs precursors during thermal processing, this invention determined the contents of key active dicarbonyl compounds GO and MGO in different simulation systems. For example... Figure 1 As shown, the production of GO and MGO in the single-component system was at an extremely low baseline level. Specifically, the GO and MGO of the protein group alone were only 0.26 μg / mL and 0.48 μg / mL, respectively, and the sugar group alone was only 0.46 μg / mL and 1.10 μg / mL, respectively. This indicates that the self-degradation or oxidation capacity of a single substance under thermal conditions is very limited (Note: the differences between different treatments are significant (p<0.05)).

[0046] When different components interact, the formation of dicarbonyl compounds increases significantly. Notably, in the two-component system, the 'protein + oil' group exhibits higher reactivity, with GO and MGO accumulations significantly increasing to 2.51 μg / mL and 2.22 μg / mL, respectively, higher than the 'glycan + protein' group based on the classical Maillard reaction (GO 0.86 μg / mL, MGO 1.61 μg / mL). This data confirms that in the composite system constructed in this invention, the lipid oxidation pathway contributes significantly more to the formation of reactive dicarbonyl compounds than the simple sugar-induced Maillard reaction pathway, and lipid degradation products are an important source of AGEs precursors in the system. When all three components are present simultaneously, multiple degradation pathways interact, and the accumulation of GO and MGO in the system reaches its highest level, exhibiting a significant synergistic effect.

[0047] Further observation revealed that the GO and MGO contents in the "sugar + oil" group reached 2.89 μg / mL and 4.66 μg / mL, respectively, significantly higher than those in the "sugar + protein" group and even exceeding those in the "protein + oil" group. This strongly suggests a synergistic promoting effect between lipid oxidation and glucose auto-oxidation pathways, with free radicals generated by lipid peroxidation greatly accelerating the auto-oxidative cleavage of carbohydrates. When all three components were present simultaneously (in the "sugar + protein + oil" composite group), multiple degradation pathways intersected, resulting in peak accumulations of GO and MGO in the system, reaching 3.47 μg / mL and 6.18 μg / mL, respectively, exhibiting a significant additive effect. Comparison of various values ​​revealed that in each interaction group containing sugar or oil, the final absolute yield of MGO was significantly higher than that of GO in the same group. This is mainly attributed to the difference in molecular formation pathways: compared to GO, the reaction pathway for MGO generation through glucose auto-oxidation and lipid peroxidation is shorter and more direct, requiring a lower energy barrier to overcome. MGO is mainly generated by the spontaneous degradation of triose phosphate in physiological systems, while GO has more diverse sources (such as lipid peroxidation, carbohydrate degradation, etc.). Therefore, under the same heat treatment conditions, MGO exhibits higher generation and accumulation efficiency.

[0048] In summary, the specific data from the simulation system accurately reproduced and verified the evolutionary patterns in the real fish meat system, clearly indicating the core driving role of lipid oxidation and its synergistic reaction with carbohydrates in promoting the generation of AGEs active precursors.

[0049] 3.2 Generation and Analysis of AGEs (FI-AGEs / CML) in the Simulation System To further investigate the evolution of the reaction precursors into AGEs, this invention quantitatively analyzed FI-AGEs and typical non-fluorescent AGEs (CML) in the simulated system. Figure 2As shown, the fluorescence intensity of the single-component system was extremely low (1231.33 for the sugar group alone, 937.67 for the protein group alone), while the oil group alone produced a high background fluorescence (6852.67) due to the high-temperature oxidative polymerization of lipids themselves. In the two-component interaction system, the FI-AGEs production of the "protein + oil" group surged to 13500.67, reaching the highest peak among all treatment groups, and its fluorescence intensity was nearly 5 times that of the conventional Maillard reaction "sugar + protein" group (2758.00). This again confirms that the primary and secondary products of lipid oxidation (such as various reactive aldehydes and ketones) have extremely high reactivity in cross-linking with proteins to form fluorescent polymers. However, it is worth noting that when sugar, protein, and oil are present in the system simultaneously (complex group), the FI-AGEs content (9991.00) is significantly lower than that of the "protein + oil" group. This phenomenon suggests that the involvement of reducing sugars may involve a "competitive binding" of lipid oxidation intermediates to free amino groups of proteins. Since the fluorescence intensity of sugar-derived Maillard crosslinks is usually weaker than that of lipid-protein crosslinks, the competitive reaction of sugars alters the overall reaction pathway to some extent, thereby reducing the total fluorescence response of the system.

[0050] In stark contrast to the competitive inhibition phenomenon of FI-AGEs, the formation of the specific end product CML exhibits an extremely strong positive synergistic effect in the three-component system. For example... Figure 2 As shown in the right figure, the CML content in the "protein + oil" group (0.21 μg / mL) of the two-component system is still higher than that in the "sugar + protein" group (0.11 μg / mL), proving that the lipid-derived GO pathway is an important source of CML generation. When sugar, protein, and oil coexist, the amount of CML generated increases significantly to 0.60 μg / mL, which is higher than the simple summation of single pathways. Combined with the analysis results of dicarbonyl compounds mentioned above, the potential mechanism of this phenomenon is that the large number of free radicals generated by the high-temperature oxidation of lipids promotes the oxidative degradation of sugars, leading to an increase in the content of key active dicarbonyl precursors such as GO; at the same time, the addition of sugars consumes the amino sites of proteins, inhibiting the formation of complex macromolecular fluorescent cross-linked compounds (i.e., the decrease of FI-AGEs), and instead directs the reaction funnel to the pathway of generating small molecule, non-fluorescent monomer adducts such as CML. This result is also consistent with reports that lipids contribute more to CML than sugars in food systems.

[0051] In summary, the data from the simulation system profoundly reveal that in the real fatty fish system, lipids and carbohydrates compete for substrates and form a strong oxidative synergistic network in driving protein glycosylation, ultimately leading to the rapid accumulation of specific AGEs, represented by CML.

[0052] 3.3 Inhibitory effect of mulberry polyphenols on GO and MGO formation To verify the anti-glycation potential of mulberry polyphenols in complex, fat-rich food matrices, this invention introduced six representative polyphenol monomers (6-methylcoumarin, quinic acid, isochlorogenic acid B, isochlorogenic acid C, isorhamnetin, and hyperoside) into a "sugar + protein + oil" composite simulation system, and quantitatively analyzed the content evolution of the core active dicarbonyl compounds (GO and MGO).

[0053] like Figure 3 As shown, when lipid oxidation was introduced into the reaction system, the inhibitory effects of these six polyphenols on GO and MGO showed significant differences (p<0.05). Although all polyphenol groups significantly reduced the levels of GO (3.47 μg / mL) and MGO (6.20 μg / mL) in the control group, quinic acid and isochlorogenic acid C exhibited the best inhibitory activity in terms of specific efficacy. Quinic acid reduced the production of GO and MGO to 0.10 μg / mL and 0.55 μg / mL, respectively; isochlorogenic acid C was the second best (GO 0.13 μg / mL, MGO 1.16 μg / mL); 6-methylcoumarin and isochlorogenic acid B showed moderate inhibitory levels; while hyperoside (GO 0.73 μg / mL, MGO 2.32 μg / mL) and isorhamnetin showed relatively weak inhibitory effects in this system.

[0054] This experiment profoundly reveals the structure-activity relationship between the molecular structure of polyphenols and their ability to cope with the dual stresses of oxidation and glycation in complex systems. In a complex system dominated by lipid oxidation, not only are there glycogenic dicarbonyl compounds, but also a massive amount of lipid-derived reactive carbonyl groups and free radicals. Quinic acid stands out, confirming that its molecular structure does not contain traditional phenolic hydroxyl groups. However, in a lipid-dominated system, its unique multiple densely distributed alcoholic hydroxyl groups endow it with extremely strong, low-steric-hindrance nucleophilic capture ability of carbonyl groups, targeting and quenching them at the first moment of GO / MGO generation. The efficient inhibition of isochlorogenic acid C further confirms the success of the "dual-effect synergistic mechanism": its structure not only retains the alcoholic hydroxyl groups of the quinic acid core (responsible for nucleophilic capture of carbonyl groups), but also contains phenolic hydroxyl groups of caffeoyl groups, which can directly block the free radical chain reaction of lipid peroxidation. This rational spatial arrangement of the caffeoyl group away from the carboxyl group avoids the electron-withdrawing effect, achieving a two-pronged approach of "antioxidant source interruption + nucleophilic capture interception".

[0055] Conversely, flavonoids that performed well in single models (such as hyperoside and isorhamnetin) showed significantly reduced effectiveness in this model. This is mainly because, in the intense lipid peroxidation environment, the phenolic hydroxyl groups on their molecular structure are largely consumed to scavenge massive amounts of free radicals, resulting in a sharp reduction in their effective active sites for subsequent carbonyl capture. At the same time, the large steric hindrance of flavonoid glycosides (such as hyperoside) may severely limit their mass transfer efficiency at the oil-water-protein multiphase interface, hindering their effective contact with lipid-protein crosslinks.

[0056] In summary, the data from the simulation system not only confirmed the potent blocking effect of polyphenols on AGEs precursors at the molecular level, but also proved that in real high-fat heat-processed foods, polyphenols with "high nucleophilicity or excellent synergistic spatial structures" (such as quinic acid and its derivatives) must be prioritized to achieve precise intervention in lipid oxidation-driven quality degradation.

[0057] 3.4 Inhibition and structure-activity mechanism of mulberry polyphenols on AGEs (FI-AGEs and CML) in simulated systems To comprehensively evaluate the actual effectiveness of mulberry polyphenols in blocking the end of the glycosylation cascade reaction, this invention quantitatively analyzed the final amounts of FI-AGEs and CML generated in the "sugar + protein + oil" complex system after the addition of different polyphenols. For example... Figure 4 As shown, all six polyphenols significantly reduced the accumulation level of terminal AGEs in the system (p<0.05), but their inhibitory efficacy showed a significant differentiation pattern that was highly consistent with the previous inhibition of GO / MGO.

[0058] It is noteworthy that the molecular weight, galacturonic acid content, and degree of esterification of the polysaccharide significantly affect its inhibitory effect on AGEs. Specifically, a lower molecular weight helps improve the inhibition rate of early and mid-stage glycosylation products (Amadori products and dicarbonyl compounds), but reduces the inhibition rate of late-stage AGEs; while an increased galacturonic acid content comprehensively improves antioxidant and anti-glycation activities. This structure-activity relationship is highly consistent with the findings in this invention: quinic acid, as a small-molecule polyphenol (molecular weight only 192 Da), exhibits a highly efficient ability to capture GO / MGO (…). Figure 3 While polyphenols exhibit relatively limited inhibition of already formed AGEs (Advanced Glycation End Products), isochlorogenic acid C and proanthocyanidins, with their relatively larger molecular weights, show stronger inhibitory effects on late-stage AGEs. This suggests that in complex food thermal processing systems, the anti-glycation activity of polyphenols / polysaccharides depends not only on their functional group type but also on their molecular weight. For example, small-molecule phenolic compounds (such as pyrogallol and catechins) have been shown to efficiently capture active precursors such as methylglyoxal due to their small molecular weight and low steric hindrance.

[0059] In summary, the results of different polyphenols inhibiting AGEs in simulated systems indicate that in complex lipid-rich reaction systems, the anti-glycation activity of polyphenols depends not only on the phenolic hydroxyl groups that scavenge precursor free radicals, but also on whether they possess alcoholic hydroxyl groups with low steric hindrance and strong nucleophilic activity. Furthermore, molecular weight is also a key factor affecting their function at different glycosylation stages.

[0060] In the control group without polyphenol intervention, strong lipid-carbohydrate synergistic degradation resulted in FI-AGEs as high as 9991.00 and CML accumulation of 1.19 μg / mL.

[0061] After the addition of polyphenols, quinic acid and isochlorogenic acid C again showed significant inhibitory advantages: quinic acid controlled FI-AGEs and CML at 3636.33 and 0.32 μg / mL, respectively; isochlorogenic acid C also showed high activity, controlling both at 4566.67 and 0.38 μg / mL, respectively. In contrast, the inhibitory effects of 6-methylcoumarin, isochlorogenic acid B, isorhamnetin, and hyperoside were significantly weakened, with the hyperoside treatment group showing the highest relative levels of FI-AGEs (9525.67) and CML (0.60 μg / mL) among all polyphenol treatment groups.

[0062] This difference in activity observed in complex lipid systems reveals the crucial role of the specific three-dimensional structure and functional group properties of polyphenol molecules in their anti-glycation process. Lipid-rich reaction matrices generate a large number of peroxidation radicals and derived carbonyl compounds under heat treatment, placing higher demands on the structural characteristics of polyphenols. Quinic acid exhibits excellent inhibitory effects in this model, mainly attributed to its specific molecular structure: it lacks easily oxidized phenolic hydroxyl groups but contains multiple alcoholic hydroxyl groups. This structural feature reduces the consumption of quinic acid during free radical scavenging. Simultaneously, due to its low steric hindrance and the strong nucleophilicity of its alcoholic hydroxyl groups, quinic acid can effectively capture reactive dicarbonyl compounds (GO / MGO) generated from lipid and carbohydrate degradation, thereby blocking or delaying their further cross-linking with protein amino groups.

[0063] Furthermore, isochlorogenic acid C and isochlorogenic acid B, as isomers (both formed by the condensation of caffeic acid and quinic acid), exhibited significant differences in inhibitory effects, indicating that the spatial configuration of the molecule significantly affects its activity. Isochlorogenic acid C (4,5-O-dicaffeoylquinic acid) has phenolic hydroxyl groups at both ends of its caffeoyl group, which is beneficial for scavenging free radicals and blocking lipid oxidation chain reactions; simultaneously, its quinic acid core retains alcoholic hydroxyl groups that can undergo nucleophilic addition. This spatial conformation relatively reduces the binding of intramolecular hydrogen bonds, exhibiting better synergistic antioxidant and anti-glycation effects in complex systems. In contrast, the different esterification sites of isochlorogenic acid B may lead to steric hindrance of some nucleophilic hydroxyl groups, thereby reducing its capture efficiency for active dicarbonyl compounds.

[0064] For flavonoids such as hyperoside and isorhamnetin, they exhibit high inhibition rates in the single-phase aqueous BSA-fructose model, but their activity is relatively weak in this lipid-rich model. This is mainly related to their oxidative consumption and steric hindrance in complex systems. On the one hand, flavonoid molecules are relatively large, which may lead to certain mass transfer resistance in oil-water-protein multiphase systems. On the other hand, in environments with high lipid peroxidation, the phenolic hydroxyl groups on flavonoid molecules are preferentially oxidized (e.g., to generate quinones) to scavenge free radicals in the system. This self-consumption of the structure reduces their ability to participate in subsequent nucleophilic addition reactions and inhibit late cross-linking.

[0065] In summary, the inhibitory effects of different polyphenols on AGEs in simulated systems indicate that in complex, lipid-rich reaction systems, the anti-glycation activity of polyphenols depends not only on the phenolic hydroxyl groups that scavenge precursor free radicals, but also on the presence of alcoholic hydroxyl groups with low steric hindrance and strong nucleophilic activity. The results of this invention can provide a theoretical reference for mitigating the quality degradation of heat-processed meat products using plant polyphenols with specific configurations.

[0066] The aforementioned research results based on the "myofibrillar protein-fructose-fish oil" composite simulation system indicate that quinic acid and other mulberry polyphenols can effectively block the formation of AGEs by nucleophilically capturing active dicarbonyl compounds (GO / MGO), demonstrating excellent chemical blocking ability. However, in real dietary exposure scenarios, some AGEs may still escape the chemical interception during food processing, be absorbed into the body through the intestines, and then bind to RAGE receptors on the cell membrane surface, inducing oxidative stress damage. Therefore, it is necessary to further verify at the cellular level whether quinic acid can alleviate the cytotoxicity induced by existing AGEs (represented by CML) and explore its potential targeted protective mechanism.

[0067] 3.5 Safety dose screening of quinic acid in BV2 cells and its protective effect against CML-induced damage. To assess the anti-glycation and cell-protective potential of quinic acid at the cellular level, this invention first used the CCK-8 assay to detect the effects of different concentrations of quinic acid on the viability of BV2 mouse microglia, in order to determine its safe effective dose. Figure 5As shown in Figure (a), when the quinic acid concentration was in the range of 1–3 μM, the relative viability of BV2 cells remained at approximately 109%, which was not significantly different from the control group (100%), and showed a slight trend of cell proliferation promotion (p>0.05). However, when the treatment concentration was increased to 4–5 μM, cell viability began to decline (approximately 80%). When the concentration was further increased to 10 μM and 20 μM, cell viability significantly decreased to 65.0% and 62.5% (p<0.05), indicating that high concentrations of quinic acid have certain cytotoxic effects on BV2 cells. Therefore, based on safety and efficacy considerations, this invention selected 3 μM as the maximum non-toxic dose of quinic acid for subsequent cell protection experiments.

[0068] Based on a clearly defined safe dosage, this invention further explores the protective effect of quinic acid against AGEs-induced cell damage. To make the cell model more closely resemble the actual risks of food processing and intake, this experiment used the CML content of surimi samples processed by different methods (raw, baked, pan-fried, deep-fried) as a benchmark. Corresponding concentrations of CML standards were added to the cell culture medium to create a damage model, and a control group was set up with and without the addition of 3 μM quinic acid. Figure 5 As shown in (b), in the groups without added quinic acid, the introduction of CML standard significantly reduced BV2 cell viability. The group corresponding to the CML concentration from "fried fish paste" showed the most severe cell damage, with viability dropping to 80.6%, significantly lower than the equivalent group from "raw fish paste" (87.2%). This downward trend in cell viability is highly consistent with the confirmed conclusion that "frying produces the highest level of CML," demonstrating that exposure to high concentrations of CML leads to impaired cell viability.

[0069] Notably, after simultaneous intervention with 3 μM quinic acid, cell viability in all damaged groups was significantly restored. Specifically, cell viability in the "raw, grilled, fried, and deep-fried" groups recovered to 97.1%, 92.5%, 92.0%, and 91.7%, respectively, showing a significant improvement compared to the group without quinic acid (p<0.05). Particularly in the most severely damaged group (equivalent to fried fish paste), the addition of quinic acid increased cell viability by approximately 11.4 percentage points, essentially restoring it to levels close to the normal control group. These results indicate that high concentrations of CML exposure in the environment cause significant cytotoxic damage to microglia, and quinic acid, at a safe dose, can effectively alleviate this cytotoxicity mediated by AGEs (Advanced Glycation End Products), demonstrating a good cell-targeted protective function.

[0070] 3.6 Effects of quinic acid on CML-induced oxidative stress levels in BV2 cells To further elucidate the potential mechanism by which quinic acid alleviates CML-induced microglial cell damage, this invention assessed the levels of intracellular oxidative stress markers. AGEs typically trigger a cascade of oxidative stress responses and lead to cell damage within cells by binding to their receptor (RAGE). This experiment quantitatively measured the content of MDA, the end product of intracellular lipid peroxidation, and the relative levels of total intracellular ROS.

[0071] MDA is a secondary product generated after cell membrane polyunsaturated fatty acids are attacked by free radicals, and it directly reflects the severity of lipid oxidative damage in cells. For example... Figure 6 As shown in (a), compared with the normal control group (0.20 nmol / mg prot), the intracellular MDA content in all CML-damaged groups without quinic acid intervention was increased to varying degrees. Specifically, the MDA content in the CML-treated groups corresponding to "fried fish paste" and "deep-fried fish paste" significantly increased to 0.48 nmol / mg prot and 0.51 nmol / mg prot, respectively (p<0.05), confirming that high concentrations of CML indeed induced severe lipid peroxidation damage in BV2 cells. After simultaneous intervention with 3 μM quinic acid, the MDA accumulation in all groups was effectively inhibited. Particularly in the more severely damaged "fried" equivalent group, the addition of quinic acid significantly reduced the MDA content to 0.34 nmol / mg prot, showing a significant decreasing trend compared to the untreated group. This result indicates that quinic acid can effectively interrupt the CML-induced lipid peroxidation chain reaction and reduce damage to cell membrane structure.

[0072] In the detection of relative fluorescence intensity of intracellular ROS ( Figure 6In (b), high concentrations of CML treatment (such as the decoction group) led to an increase in intracellular ROS levels compared to the control group, but the overall difference between the treatment groups was not significant. Meanwhile, the content of downstream lipid peroxidation product MDA decreased significantly after quinic acid intervention. This phenomenon suggests that quinic acid may primarily act on the propagation stage of the lipid peroxidation chain reaction, rather than directly clearing initially generated ROS, which is consistent with the downstream signal blocking mechanism expected by the "competitive binding to RAGE receptor" in molecular docking. This phenomenon has been occasionally reported in cell experiments involving polyphenols, and its mechanism may be: on the one hand, the specificity of the DCFH-DA fluorescent probe is relatively weak, possibly affected by changes in the polyphenol molecule's own structure or early transient redox signals within the cell; on the other hand, this further suggests that the core protective mechanism of quinic acid in BV2 cells may not be simply direct quenching of free ROS, but rather, by leveraging its structural advantages, precisely blocking the key step in the attack of free radicals on cell membrane lipids (i.e., inhibiting the downstream lipid peroxidation pathway). The combined results of significantly improved CCK-8 cell viability and MDA confirm that quinic acid can effectively block CML-mediated cytotoxicity and lipid degradation, thus exerting a definite cell-protective function.

[0073] 3.7 Analysis of molecular docking and interaction mechanism between quinic acid and RAGE receptor AGEs primarily activate downstream oxidative stress and inflammation pathways by binding to specific receptors (RAGE) on the cell membrane, leading to cell damage. To explore the potential targeting mechanism of quinic acid in alleviating CML-induced oxidative stress damage in BV2 cells at the molecular level, this invention used AutoDock Vina software to perform molecular docking simulations of quinic acid with the RAGE receptor V domain (PDBID: 2MOV).

[0074] Binding energy is a core indicator for assessing the affinity between ligands and receptors. Generally, a binding energy less than 0 kcal / mol is considered to indicate that the ligand and receptor can bind spontaneously, and the lower the value, the stronger the affinity. Docking results showed that the lowest binding energy between quinic acid and the RAGE receptor was -5.13 kcal / mol, indicating that quinic acid molecules can spontaneously and stably enter and bind into the active pocket of the RAGE receptor.

[0075] Further through three dimensions ( Figure 7 a) and two-dimensional ( Figure 7 (b) Visualization analysis revealed the specific interaction patterns between quinic acid and amino acid residues within the RAGE receptor pocket. For example... Figure 7As shown, quinic acid is tightly anchored to the RAGE receptor primarily through a strong network of conventional hydrogen bonds. Specifically, the abundant free hydroxyl and carboxyl groups in the quinic acid molecule form multiple hydrogen bonds with key residues in the receptor's active pocket, such as glutamic acid (GLU 74), glutamic acid (GLU 105), and glycine (GLY 36). This dense network of multiple hydrogen bonds mediated by hydroxyl groups greatly enhances the structural stability of the complex.

[0076] Quinic acid's targeted protective mechanism against BV2 cells may be dual: on the one hand, it can directly capture AGE precursors (such as GO / MGO) in vivo through nucleophilic reactions; on the other hand, it can stably bind to the active site of RAGE receptors on the cell surface due to its good spatial affinity. This binding may create steric hindrance and competitive inhibition of AGEs in the environment, blocking the activation of the AGEs-RAGE signaling axis, thereby interrupting the cascade damage pathway of downstream lipid peroxidation (increased MDA) and excessive ROS production at its source. Similarly, natural products such as sesamin have also been reported to alleviate AGEs-induced inflammatory responses in BV2 cells by regulating RAGE expression.

[0077] 3.8 Verification of the anti-glycation effect of quinic acid in a real-world pan-fried tilapia system The complex physicochemical changes during the heat processing of real fatty meat products, such as moisture migration, structural damage, and mass transfer resistance, can significantly affect the actual activity of polyphenols. Therefore, this invention uses "fried fish paste," which has the highest risk of AGEs formation, as a real food intervention model to evaluate the inhibitory efficacy and dose-response effect of quinic acid in actual processing.

[0078] like Figure 8 As shown, the amount of CML generated in the fried fish paste system showed a significant dose-dependent decreasing trend with increasing quinic acid addition (0.1%, 0.15%, 0.3%). In the control group without added polyphenols, the CML content was 3.88 μg / mL, further verifying the intensity of lipid-protein cross-polymerization induced by the frying process. After adding 0.1% quinic acid, the CML content significantly decreased to 1.14 μg / mL, with an inhibition rate of 70.6% (p<0.05). When the addition amount increased to 0.15%, the CML content further decreased to 0.076 μg / mL (inhibition rate>98%). When the quinic acid concentration continued to increase to 0.3% (CML content 0.017 μg / mL), its inhibitory effect was not significantly different from that of the 0.15% group (p>0.05), indicating that the addition amount of 0.15% had reached the anti-glycation saturation dose in this reaction system.

[0079] Structure-activity relationship and intervention mechanism analysis: The above results indicate that quinic acid still possesses highly efficient targeted anti-glycation activity in real complex food systems. Its intervention mechanism is mainly attributed to the synergistic effect of its physicochemical properties: at the physical level, 4℃ marinating pretreatment promotes the uniform penetration of low molecular weight quinic acid into porous surimi matrix and oil-water multiphase interfaces, effectively overcoming mass transfer resistance; at the chemical level, facing the intense lipid peroxidation microenvironment induced by high-temperature frying, quinic acid, with its low steric hindrance and polyol hydroxyl structure, efficiently nucleophilically captures the surge of dicarbonyl compounds (GO / MGO) in the system, thereby in situ interrupting the formation pathway of terminal CML.

[0080] Furthermore, the dose saturation effect observed between the 0.15% and 0.3% groups indicates that the total amount of active carbonyl precursors generated within the system is limited at specific thermal processing intensities (800 W, 6 min) and substrate concentrations. Therefore, the nucleophilic sites provided by 0.15% quinic acid are sufficient to adequately capture these precursors, thereby completely inhibiting the formation of AGEs.

[0081] In conclusion, quinic acid significantly inhibited the formation of CML in the pan-fried tilapia system, and 0.15% can serve as an effective intervention dose for the development of low-AGEs healthy aquatic products. This result successfully validates the feasibility of translating the in vitro anti-glycation mechanism into real-world food industry applications, providing scientific data support for the development of natural anti-glycation ingredients.

[0082] 4. Summary (1) In lipid-rich complex systems, lipid oxidation is the core driving force for the formation of reactive dicarbonyl compounds and AGEs. The results of the simulation system show that the formation of GO, MGO, and the specific end product CML in the "protein + oil" two-component and "sugar + protein + oil" three-component systems are significantly higher than those in the traditional "sugar + protein" system based on Maillard reaction. This further verifies that in lipid-rich environments such as meat heat processing, reactive carbonyl intermediates derived from lipid peroxidation replace carbohydrate degradation products and become the dominant precursors driving protein glycosylation crosslinking.

[0083] (2) The anti-glycation efficacy of polyphenols in complex lipid systems exhibits a high degree of structure dependence (structure-activity relationship). In the intense lipid peroxidation microenvironment (i.e., the food processing end), traditional flavonoid polyphenols (such as hyperoside) suffer from severe "target-competitive consumption" due to the large consumption of their phenolic hydroxyl groups in scavenging lipid free radicals, leading to a significant decrease in their subsequent efficacy in blocking AGEs. In contrast, quinic acid, with its lack of phenolic hydroxyl groups, effectively avoids this ineffective consumption and utilizes the advantages of low steric hindrance and polyol hydroxyl groups to precisely achieve targeted "snap-up" and efficient nucleophilic capture (i.e., externally hindered precursor) of active dicarbonyl compounds (GO / MGO).

[0084] (3) Quinic acid can effectively alleviate cytotoxicity and oxidative stress damage induced by CML exposure. Cell experiments showed that modeling with CML concentrations equivalent to those from heat-processed (especially pan-fried) surimi significantly reduced the relative viability of BV2 cells and induced severe lipid peroxidation (significantly increased MDA content). However, at a safe intervention dose of 3 μM, quinic acid significantly restored the viability of damaged cells (from 80.6% to 92.0% in the pan-fried group) and significantly reduced intracellular MDA levels, demonstrating that it can effectively block the downstream lipid peroxidation chain damage induced by CML.

[0085] (4) Molecular docking confirmed the dual mechanism of quinic acid's cytoprotective effect: nucleophilic capture + receptor competition. Computational simulations showed that quinic acid can spontaneously enter the active pocket of the RAGE receptor with a low binding energy (-5.13 kcal / mol) and form a stable multi-hydrogen bond network. This indicates that quinic acid can not only directly chemically quench AGE precursors in the extracellular environment, but also competitively bind to the RAGE receptor on the cell membrane surface for the small number of AGE end products that escape and enter the body, forming steric hindrance, thereby blocking the cellular oxidative stress pathway (i.e., internal resistance receptor) mediated by the "AGEs-RAGE" signaling axis from the source.

[0086] In summary, this invention successfully connects the complete action chain from the "food processing end" to the "cellular physiology end," clearly proposing a dual targeting mechanism of quinic acid: "external inhibition precursor (chemical nucleophilic capture) and internal inhibition receptor (biological competitive blockade)." This discovery deeply integrates the mechanistic network of lipid oxidation-induced AGEs generation and polyphenol targeted intervention, providing strong theoretical support for the application of natural polyphenols in complex food matrices.

[0087] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of quinic acid in the preparation of anti-glycation inhibitors and drugs for treating neuronal inflammation.

2. The application according to claim 1, characterized in that, The anti-glycation inhibitor is a food ingredient or functional food component used to inhibit the formation of advanced glycation end products in thermally processed food systems.

3. The application according to claim 1 or 2, characterized in that, The anti-glycation inhibitor is available in the form of powder, solution, or microcapsule.

4. The application according to claim 1, characterized in that, The drug used to treat neurocellular inflammation is a preparation for alleviating neurocellular toxicity and oxidative stress damage induced by CML exposure.

5. The application according to claim 4, characterized in that, The dosage form of the drug for treating neuronal inflammation is an injection, a lyophilized powder for injection, or an oral preparation.

6. The application according to claim 1, 4, or 5, characterized in that, In the aforementioned drug for treating neuronal inflammation, the effective concentration of quinic acid is 1 μM to 3 μM.

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