Application of robiniatin in preparation of medicine for selectively inhibiting COX-2

Abstract

The invention discloses an application of robiniaflavin in preparation of a COX-2 selective inhibitor. The robiniaflavin can specifically inhibit the expression of COX-2 and reduce the expression of iNOS, COX-2 and TNF-alpha in an inflammation model. The robiniatin has remarkable anti-inflammatory and analgesic effects in cells and animals, and the effect is equivalent to or better than that of ibuprofen. The robiniatin has good safety in cells and animals.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to the application of robinia xanthophyllin in the preparation of anti-inflammatory and analgesic drugs. Background Art

[0002] Inflammation and pain are common pathophysiological processes in humans and are closely related to a variety of diseases. Nonsteroidal anti-inflammatory drugs (NSAIDs) are currently the most commonly used anti-inflammatory and analgesic drugs in clinical practice. They primarily work by inhibiting cyclooxygenase (COX). COX-1 is divided into two subtypes: COX-1 and COX-2. COX-1 is primarily involved in the synthesis of physiological prostaglandins, maintaining gastrointestinal mucosal integrity and renal function. COX-2 is primarily induced in inflammatory states and participates in inflammatory responses and pain signaling.

[0003] Traditional NSAIDs, such as aspirin and ibuprofen, inhibit both COX-1 and COX-2. While they offer excellent anti-inflammatory and analgesic effects, COX-1 inhibition weakens the protective effect of the gastrointestinal mucosa, leading to long-term use and serious adverse reactions such as gastric ulcers and gastric bleeding. To address this issue, researchers have developed selective COX-2 inhibitors, such as celecoxib and rofecoxib. These drugs significantly reduce the incidence of gastrointestinal adverse reactions while maintaining their anti-inflammatory and analgesic effects.

[0004] However, existing selective COX-2 inhibitors still have some limitations: long-term use may increase the risk of adverse cardiovascular events, such as myocardial infarction and stroke, leading to the withdrawal of some drugs such as rofecoxib from the market; they have a certain impact on kidney function, which may lead to water and sodium retention and increased blood pressure; some patients may experience abnormal liver function; there are individual differences, and some patients have poor efficacy or develop drug resistance.

[0005] In recent years, natural products and their derivatives have attracted widespread attention as important sources for drug development.

[0006] How to develop a flavonoid COX-2 inhibitor with clear structure, high selectivity and good safety is of great significance for enriching the types of anti-inflammatory and analgesic drugs, improving therapeutic effects and reducing adverse reactions. Summary of the Invention

[0007] In view of this, the main purpose of the present invention is to provide a use of acaciatin in the preparation of drugs for selectively inhibiting COX-2.

[0008] The structural formula of quercetin is shown in Formula 1 below:

[0009]

[0010] Its basic features include:

[0011] 1. Basic skeleton:

[0012] Quercetin belongs to the flavonoids, and its core structure is 3-hydroxyflavone (flavonol), which is a benzo-γ-pyrone (C6-C3-C6) skeleton.

[0013] 2. Functional group distribution:

[0014] Phenolic hydroxyl group (-OH): distributed in multiple positions of the molecule, with a total of 5 hydroxyl groups.

[0015] Ring A (left-side benzene ring): has two hydroxyl groups (3' and 4' positions).

[0016] Ring B (right-side benzene ring): has three hydroxyl groups (5-position, 7-position, and 3-position).

[0017] Carbonyl (C=O): located at the 2-position of the pyrone ring.

[0018] Ether bond (COC): An oxygen atom on the pyran ring connecting two benzene rings.

[0019] 3. Conjugated system:

[0020] The molecule has a highly conjugated double bond structure, which gives it antioxidant activity and the ability to absorb ultraviolet light.

[0021] The present invention systematically optimizes the molecular structure of robinia xanthophylls and reveals the key structural elements of its COX-2 inhibition activity:

[0022] 1. The number and position of hydroxyl groups at specific sites on the flavonoid core skeleton are crucial for COX-2 selectivity;

[0023] 2. The 3' and 4' hydroxyl groups on the A ring are essential for hydrogen bonding with the COX-2 active pocket;

[0024] 3. Structural modifications at specific positions on the C ring can further improve selectivity and bioavailability.

[0025] Robinetin belongs to the flavonoids, and its core structure is a 2-phenyl-4H-1-benzopyran-4-one skeleton with multiple hydroxyl substituents.

[0026] Through molecular docking studies, it was found that the present invention (acacia flavonoids molecule) can precisely bind to the active pocket of the COX-2 enzyme, forming stable hydrogen bonds and hydrophobic interactions, while significantly reducing the binding affinity with COX-1, thereby achieving highly selective inhibition of COX-2.

[0027] The second object of the present invention is to provide the use of acaciatin in the preparation of drugs that selectively promote COX-2 degradation.

[0028] The third object of the present invention is to provide the use of acaciatin in the preparation of drugs for inhibiting iNOS expression.

[0029] The present invention also aims to provide the use of acaciatin in the preparation of drugs for inhibiting TNF-α expression.

[0030] The present invention also aims to provide a use of acaciatin as a selective inhibitor of COX-2 in the screening of anti-inflammatory and analgesic drugs.

[0031] Furthermore, the drug is used for treating anti-inflammatory and analgesic effects.

[0032] Furthermore, the medicine is a preparation prepared using acaciaxanthin as a raw material.

[0033] Furthermore, the preparation is an oral preparation.

[0034] Furthermore, the preparation is an injection preparation.

[0035] Furthermore, the preparation is an inhalation preparation.

[0036] The present invention discovers and confirms for the first time that robinia xanthophyllin can be used as a highly selective COX-2 inhibitor for anti-inflammatory and analgesic treatment.

[0037] The selective inhibition ratio of robinsonin on COX-2 (COX-1IC50 / COX-2IC50) is greater than 50, which is significantly higher than most existing selective COX-2 inhibitors.

[0038] Robinia xanthophylls exert their effects through a dual mechanism of directly binding to the COX-2 active site and promoting COX-2 protein degradation.

[0039] The present invention discloses the specific binding mechanism of robinsonin to COX-2, including:

[0040] 1. The high affinity binding of robinsonin to COX-2 was confirmed by SPR technology;

[0041] 2. The key molecular interactions between robinsonin and COX-2 include hydrogen bonds and hydrophobic interactions at specific amino acid residues (such as Arg120, Tyr355, Val523, etc.);

[0042] 3. The conformational changes caused by the binding of robinsonin to COX-2 lead to structural changes in the enzyme active site and decreased catalytic activity;

[0043] 4. The molecular mechanism by which robinsonin promotes ubiquitination and proteasomal degradation of COX-2 protein.

[0044] The beneficial effects of the present invention include at least:

[0045] The present invention is based on an in-depth study of the structure-activity relationship of flavonoid compounds. While maintaining good anti-inflammatory and analgesic effects, it significantly reduces the adverse reactions of traditional NSAIDs and existing COX-2 inhibitors, providing a new technical solution and theoretical basis for the development of safer and more effective anti-inflammatory and analgesic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the structural formula of acaciatin.

[0047] Figure 2 Molecular docking and binding kinetics analysis of robinsonin. A) Binding site analysis; B) COX-2 protein structure; C) Simulation of robinsonin binding to COX-2 protein; D) Ibuprofen binding kinetics curve; E) robinsonin binding kinetics curve.

[0048] Figure 3 Figures show the results of in vitro evaluation of robinsonin. A) Cytotoxicity test results; B) Western blot test results; C) Immunofluorescence results.

[0049] Figure 4 The left picture shows the results of the writhing test; the right picture shows the histopathological results of major organs. DETAILED DESCRIPTION

[0050] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0051] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0052] The experimental materials and experimental steps used in the present invention are as follows:

[0053] 1. Surface plasmon resonance (SPR) experiment

[0054] (1) Experimental materials: purified COX-1 and COX-2 proteins, acacia yellow sample, celecoxib (as a positive control), buffer and reagents.

[0055] (2) Experimental equipment: SPR instrument (such as Biacore system), sensor chip (CM5)

[0056] (3) Experimental steps

[0057] Sensor chip surface activation: Use EDC / NHS mixture to activate the CM5 chip surface;

[0058] Protein immobilization: Purified COX-1 and COX-2 proteins were immobilized on the chip surface of different flow cells;

[0059] Block unreacted active groups: Use ethanolamine to block unreacted active groups;

[0060] Sample preparation: Prepare acaciatin solutions of different concentrations (0.1-100 nM);

[0061] Binding kinetics: The sample was passed through the chip surface at a flow rate of 30 μL / min, and the changes in the resonance signal during the binding process were recorded to determine the binding affinity (KD) and kinetic parameters (kon and koff) of acaciatin to COX-1 and COX-2.

[0062] Competitive binding experiment: Celecoxib and acacia yellow were mixed in advance, and the changes in the binding signal between the mixture and COX-2 were measured to analyze the competitive binding between acacia yellow and celecoxib.

[0063] 2. Cell experiments

[0064] (1) Experimental materials: RAW264.7 macrophages, lipopolysaccharide (LPS), acacia yellow sample, cell culture medium and reagents, antibodies (anti-COX-1, anti-COX-2, anti-inflammatory markers, etc.)

[0065] (2) Experimental steps

[0066] Cell culture: RAW264.7 cells were cultured in DMEM medium containing 10% FBS. Experiments were performed when the cell density reached 70-80%.

[0067] COX-2 expression and activity inhibition experiment: The cells were divided into control group, LPS stimulation group, and different concentrations of robinsonin treatment group (0.1-50 μM).

[0068] Pretreatment cells: first treated with different concentrations of acaciatin for 2 h;

[0069] LPS stimulation: add 1 μg / mL LPS for 24 hours;

[0070] Cells and culture supernatants were collected, and COX-2 protein expression levels were detected by Western blot, and prostaglandin E2 (PGE2) production was detected by ELISA to evaluate COX-2 activity;

[0071] Protein degradation experiment: LPS-stimulated cells were treated with acacia yellow, and cells were collected at different time points (0, 2, 4, 8, and 12 hours). The changes in COX-2 protein levels were analyzed by Western blot, and the half-life of COX-2 protein was calculated.

[0072] Analysis of inflammatory marker expression: LPS-stimulated cells were treated with acacia yellow, total cell protein was extracted, and the expression levels of inflammatory markers (IL-1β, IL-6, TNF-α, iNOS, etc.) were detected by Western blot;

[0073] 3. Animal experiments:

[0074] (1) Experimental materials: healthy adult male mice (20-25 g), acacia yellow sample, positive control drug (ibuprofen), acetic acid solution (0.6%), hot plate apparatus, drug administration tools and reagents were selected for the experiment;

[0075] (2) Experimental steps:

[0076] Animal groups: control group (solvent), low-, medium- and high-dose groups of robinsonin (such as 10, 30, and 100 mg / kg), and positive control group (such as ibuprofen 10 mg / kg).

[0077] Acetic Acid Writhing Test: The acetic acid writhing test was used to evaluate the anti-inflammatory pain effects of the drug. The corresponding drug was administered (intraperitoneally or orally) according to the group. After 30 minutes of drug absorption, 0.6% acetic acid solution (10 mL / kg, intraperitoneally) was injected. The number of writhing movements in the mice was recorded within 15 minutes after injection. The inhibition rate was calculated as (number of writhings in the control group - number of writhings in the drug group) / number of writhings in the control group × 100%.

[0078] The present invention proposes a novel flavonoid compound, acacia flavonoid, for use as a selective COX-2 inhibitor, and its anti-inflammatory and analgesic effects and mechanism of action are verified through systematic in vitro and in vivo experiments.

[0079] The solution proposed by the present invention is described in detail below through specific embodiments:

[0080] Example 1 Molecular docking and binding dynamics

[0081] For robinia xanthophyllin (structural formula Figure 1 ) were subjected to molecular docking analysis with COX-2 protein, and the results were as follows Figure 2As shown in A, the results show that the compound forms multiple interactions with key amino acid residues in the active pocket of the COX-2 enzyme, including interactions with amino acid residues such as TYR, PHE, VAL, MET, LEU121, and SER322. The hydroxyl group of the compound forms hydrogen bond interactions with key amino acid residues, enhancing binding stability. COX-2 protein ( Figure 2 B) presents a typical tertiary structure dominated by α-helices, with a clear active pocket region. The structure shows good stability and ligand binding potential. Molecular docking simulation ( Figure 2 C) shows that the compound has an ideal spatial orientation at the active site. The ligand-receptor complex exhibits good complementarity, supporting its potential as a COX-2 inhibitor.

[0082] The binding kinetics of robinsonin and ibuprofen were evaluated by surface plasmon resonance (SPR). Figure 2 D) It showed concentration-dependent binding in the concentration range of 0.078125-10 μM, with a maximum response value (RU) of approximately 30 units. The binding kinetics curve showed rapid binding characteristics; Figure 2 E) It exhibited significant concentration-dependent binding within the concentration range of 0.21875-14 μM, with a maximum response of approximately 40 RU, higher than the positive control ibuprofen. The binding curve showed a rapid binding phase, a stable plateau phase, a high response value, and a slow dissociation rate, indicating good binding affinity. Experimental and computational chemistry results demonstrated that robinsonin binds to the COX-2 protein with high affinity, with a dissociation constant (KD) of 5.8 nM, significantly lower than its binding affinity for COX-1 (KD = 320 nM). Binding kinetic analysis revealed that robinsonin binds to COX-2 with rapid binding (kon) and slow dissociation (koff), indicating that it forms a stable complex with the target. Competitive binding experiments confirmed that robinsonin competes with the known COX-2 inhibitor celecoxib for binding sites, further confirming its target.

[0083] Example 2 Cell experiment

[0084] (1) Security Verification

[0085] The cell viability of RAW264.7 cells was detected using CCK8 assay, with a concentration range of 0-100 μM and a treatment time of 24 hours. Figure 3 A) shows that the cell viability was maintained above 90% within the tested concentration range, and good cell compatibility was still observed at the highest concentration of 100 μM. Statistical analysis showed no significant difference between the treated group and the control group (p>0.05).

[0086] (2) COX-2 expression and activity inhibition experiments

[0087] In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages ( Figure 3 A) Robinia yellow inhibits COX-2 expression and activity in a dose-dependent manner with an IC50 value of 0.75 μM, significantly lower than its inhibitory effect on COX-1 (IC50>50 μM). Robinia yellow exhibits a selective inhibition ratio for COX-2 (COX-1 IC50 / COX-2 IC50) greater than 50, significantly higher than most existing selective COX-2 inhibitors.

[0088] Western blot results are as follows Figure 3 As shown in Figure B, iNOS (131kDa) showed significant dose-dependent downregulation, COX-2 (69kDa) expression decreased significantly with increasing concentration, TNF-α (15kDa) expression levels were significantly reduced in the high-concentration group, and β-Actin (45kDa), an internal control protein, maintained stable expression. These results indicate that robinin not only inhibits COX-2 activity but also promotes COX-2 protein degradation, demonstrating a dual inhibitory mechanism.

[0089] (3) Immunofluorescence experiment

[0090] Immunofluorescence was used to investigate the effect of robinsonin on the expression levels of inflammatory markers. Figure 3 As shown in C:

[0091] CTRL group (control group): normal cell morphology and weak fluorescence signal;

[0092] LPS group (inflammation model group): obvious green fluorescence signal enhancement was observed on the cell membrane, indicating that the expression of inflammation-related proteins was significantly upregulated;

[0093] LPS+ibuprofen group (positive control group): The fluorescence signal intensity was significantly weakened compared with the LPS group, showing a good anti-inflammatory effect;

[0094] LPS+Robinetin group (experimental group): The fluorescence signal intensity was significantly reduced, and the inhibitory effect was better than or equivalent to that of the positive control ibuprofen, confirming its significant anti-inflammatory activity.

[0095] In summary, these experimental results confirm that robinsonin has: good cellular safety; significant anti-inflammatory activity; exerts its effects by inhibiting the expression of iNOS, COX-2, and TNF-α; its effect is better than that of the commonly used clinical drug ibuprofen, significantly reducing the expression of inflammatory markers.

[0096] Example 3 Animal Experiment

[0097] (1) Animal efficacy experiments

[0098] The present invention evaluated the anti-inflammatory and analgesic effects of robinsonin in a classic animal model of acute pain (acetic acid writhing test). The acetic acid writhing test is a classic model for evaluating the analgesic effect of drugs, and is mainly used to screen drugs with peripheral analgesic effects. The principle of this experiment is to stimulate the abdominal cavity of mice by intraperitoneal injection of diluted acetic acid solution, causing a chemical pain reaction, which manifests as characteristic abdominal twisting and hind limb extension movements (i.e., "writhing" reaction). The analgesic effect of the drug is evaluated by comparing the degree of reduction in the number of writhing times of mice in the drug-treated group and the control group. The results are as follows: Figure 4 As shown in A, the control group exhibited virtually no writhing reactions, the model group experienced approximately 32 writhing reactions, the ibuprofen group experienced approximately 12 writhing reactions, and the robinetin group experienced approximately 6 writhing reactions. There was a significant difference between the model group and the control group (**p < 0.01), as well as a significant difference between the drug-treated group and the model group (**p < 0.01). There was no significant difference between the robinetin group and the ibuprofen group (ns). These results demonstrate that robinetin exhibits significant analgesic effects in animal studies, comparable to or superior to the positive control drug ibuprofen.

[0099] (2) Safety in animals

[0100] A long-term high-dose drug administration experiment was conducted in mice. The drug administration scheme was: high-dose gavage. After the drug administration was completed, the mouse viscera were taken for HE staining to evaluate the safety. The results are as follows Figure 4 As shown in B.

[0101] Heart: The myocardial fibers of the three groups of samples were regularly arranged, with no obvious pathological changes or inflammatory cell infiltration.

[0102] Liver: The hepatocytes are morphologically normal, the liver lobule structure is intact, and there is no obvious necrosis or inflammatory reaction;

[0103] Spleen: The white pulp and red pulp structures are clear, the lymphoid follicles are normal in morphology, and there is no abnormal proliferation or atrophy;

[0104] Lung: The alveolar structure is intact, the bronchial wall is normal, and there is no obvious inflammatory infiltration;

[0105] Kidney: The glomeruli and renal tubules are structurally normal, with no pathological changes or signs of tissue damage.

[0106] Gastric: The gastric mucosa is intact, the glands are arranged regularly, and there is no ulcer or inflammation;

[0107] Intestine: The intestinal villi are intact, the submucosal tissue is normal, and there are no signs of inflammation or damage.

[0108] The results showed that robinia xanthin showed significant analgesic effect, which was better than the positive control drug ibuprofen; in long-term high-dose administration experiments, robinia xanthin did not cause significant gastrointestinal mucosal damage, and the incidence of gastric ulcers was significantly lower than that of traditional NSAIDs and some selective COX-2 inhibitors. No obvious toxic reactions or pathological changes were observed in major organs, indicating that robinia xanthin has good safety; acute toxicity experiments determined that the LD50 of robinia xanthin was >2000 mg / kg, indicating that it has a high safety margin.

[0109] In summary, this study validates the anti-inflammatory and analgesic effects of robinsonin as a novel selective COX-2 inhibitor and its mechanism of action through SPR technology, cell-based assays, and animal model systems. Compared to existing COX-2 inhibitors, robinsonin exhibits improved safety and efficacy, particularly in reducing cardiovascular and gastrointestinal adverse reactions, providing a new treatment option for inflammatory and pain-related diseases.

[0110] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0111] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. Application of robinia xanthophyllin in the preparation of drugs that selectively inhibit COX-2 expression.

2. Application of robinia xanthophylls in the preparation of drugs that selectively promote COX-2 degradation.

3. Application of robinia xanthophyllin in the preparation of drugs for inhibiting iNOS expression.

4. Application of robinia xanthophyllin in the preparation of drugs for inhibiting TNF-α expression.

5. Application of robinia xanthophyllin as a selective inhibitor of COX-2 in the screening of anti-inflammatory and analgesic drugs.

6. The use according to any one of claims 1 to 4, characterized in that The medicine is used for treating anti-inflammatory and analgesic effects.

7. The use according to any one of claims 1 to 4, characterized in that The medicine is a preparation prepared by taking acaciaxanthin as a raw material.

8. The use according to claim 6, characterized in that The preparation is an oral preparation.

9. The use according to claim 6, characterized in that The preparation is an injection preparation.

10. The use according to claim 6, characterized in that The preparation is an inhalation preparation.

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