Anti-inflammatory and antioxidant pharmaceutical composition using formononetin as active ingredient

Abstract

The invention relates to an anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient, the pharmaceutical composition takes FMN as the active ingredient, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The FMN shows wide beneficial effects in regulating macrophage inflammatory response induced by lipopolysaccharide (LPS), and by inhibiting a TLR4 signal and activating Nrf2 and AMPK signal channels, ROS generation is reduced, antioxidant defense is enhanced, and inflammatory response and oxidative stress are effectively relieved. The FMN also regulates the function of the macrophage, promotes the macrophage to be converted to an anti-inflammatory phenotype, and influences the metabolic state and function of the cells by forming an immune-metabolic circuit. In addition, FMN, as an activator of AMPK, plays a positive role in maintaining cell energy balance and metabolic health. Due to the effects, the FMN is expected to become a novel medicine for treating inflammatory diseases such as arthritis and cardiovascular diseases.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, in particular to an anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient. Background Art

[0002] Inflammation and oxidative stress are part of the body's response to external and internal insults, involving a variety of cell types and molecular mediators. Inflammation is a protective response of the body to injury or infection, aimed at eliminating harmful factors and promoting the repair process. However, improper inflammatory response may lead to a variety of diseases, such as atherosclerosis, arthritis, enteritis, etc. Oxidative stress is a state of cell damage caused by excessive production of reactive oxygen species (ROS) or insufficient antioxidant defense mechanisms, and is associated with a variety of chronic diseases and aging.

[0003] At present, the treatments for inflammation and oxidative stress mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, antioxidant supplements, etc. Although these treatments have certain effects, long-term use may bring side effects, such as gastrointestinal damage, immunosuppression, metabolic disorders, etc. Therefore, the development of new, safe and effective therapeutic drugs is an important direction of current medical research.

[0004] Formononetin (FMN) is a naturally occurring isoflavone compound found in a variety of plants and has shown a variety of biological effects, including anti-cancer, anti-cardiovascular disease, anti-inflammatory and antioxidant effects. Studies have shown that FMN affects inflammation and oxidative stress responses by regulating multiple signaling pathways and molecular targets, such as TLR4, Nrf2, and AMPK. However, the specific mechanism of action, clinical application potential, and appropriate pharmaceutical preparations of FMN need further exploration and verification.

[0005] There are many challenges in developing FMN-based pharmaceutical compositions, including ensuring the stability of FMN, optimizing drug release and absorption, and evaluating long-term safety and efficacy. At the same time, the use of modern pharmaceutical preparation technology and molecular biology methods is expected to overcome these challenges and develop new therapeutic drugs to meet the clinical demand for high-efficiency, low-side effect treatments.

[0006] The purpose of the present invention is to provide an anti-inflammatory and antioxidant pharmaceutical composition based on formononetin, aiming to address the limitations of existing treatment methods and utilize the biological activity of formononetin to provide a new strategy for the treatment of inflammation and oxidative stress-related diseases. Summary of the invention

[0007] In order to solve the problems of the prior art, the present invention provides an anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient.

[0008] In order to solve the above technical problems, the present invention is achieved through the following technical scheme: an anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient, the pharmaceutical composition using formononetin as an active ingredient, and the pharmaceutical composition also comprising a pharmaceutically acceptable carrier; formononetin is present in an effective amount to inhibit inflammatory response by regulating the expression of TLR4 and ER and activating the cAMP-PKA-LKB1-AMPK signaling axis.

[0009] The concentration of formononetin is sufficient to inhibit the expression of pro-inflammatory cytokines COX-2, IL-6 and NLRP3.

[0010] The composition has the ability to regulate ROS-dependent Nrf2 signaling.

[0011] The preparation method comprises the step of mixing formononetin with a pharmaceutically acceptable carrier.

[0012] It also includes formulating the composition into a dosage form suitable for oral administration; it also includes formulating the composition into a dosage form suitable for parenteral administration.

[0013] Used for treating or preventing diseases caused by inflammation and oxidative stress; use in preparing drugs for treating inflammatory diseases and oxidative stress-related diseases.

[0014] The inflammatory diseases include autoimmune diseases, cardiovascular diseases or neurodegenerative diseases; the oxidative stress-related diseases include disorders associated with ROS-mediated tissue damage.

[0015] The beneficial effects of the present invention are:

[0016] 1. FMN (flavin mononucleotide) in the present invention exhibits a wide range of beneficial effects in regulating the inflammatory response of macrophages induced by LPS (lipopolysaccharide). It reduces ROS production and enhances antioxidant defense by inhibiting TLR4 signaling and activating Nrf2 and AMPK signaling pathways, effectively alleviating inflammatory responses and oxidative stress. FMN also regulates the function of macrophages, promotes their transformation to an anti-inflammatory phenotype, and affects the metabolic state and function of cells by forming an immune-to-metabolic loop. In addition, as an activator of AMPK, FMN has a positive effect on maintaining cellular energy balance and metabolic health. These effects make FMN a promising new drug for the treatment of inflammatory diseases such as arthritis, cardiovascular disease, etc.

[0017] 2. FMN effectively alleviated the inflammatory response by inhibiting TLR4 signaling and activating Nrf2 and AMPK signaling pathways, reducing the production of ROS and increasing the expression of anti-inflammatory cytokines; FMN significantly reduced the production of pro-inflammatory cytokines such as COX-2, IL-6 and NLRP3 in LPS-stimulated macrophages, which are key factors leading to the aggravation of the inflammatory response;

[0018] 3. As an effective ROS scavenger, FMN can reduce oxidative stress and protect cells from free radical damage; by activating the Nrf2 signaling pathway, FMN enhances the antioxidant defense system in cells and further improves the ability of cells to resist oxidative stress;

[0019] 4. FMN regulates the polarization state of macrophages by affecting ER / TLR4 signal transduction, promoting their transformation to an anti-inflammatory phenotype, and helping to maintain the balance of the immune system; it forms an immune-to-metabolism loop, and affects the function of immune cells and the generation of inflammatory responses by regulating their metabolic state;

[0020] 5. FMN, as an activator of AMPK, regulates cellular energy metabolism and plays a positive role in maintaining intracellular energy balance and metabolic health; by regulating AMPK and cAMP-dependent protein kinase, FMN may affect multiple metabolic pathways, including fatty acid oxidation and glucose metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the effect of formononetin (FMN) of the present invention on the expression of Toll-like receptor 4 (TLR4) and estrogen receptor (ER) in ANA-1 macrophages stimulated by lipopolysaccharide (LPS).

[0022] Figure 2 The present invention is a schematic diagram of the signal transduction of formononetin (FMN) regulating cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-liver kinase B1 (LKB1)-5'-adenosine monophosphate activated protein kinase (AMPK) axis.

[0023] Figure 3 It is a schematic diagram of immunofluorescence images of the regulation of formononetin (FMN) of the present invention on phosphorylated (p)-5'-adenylic acid-activated protein kinase (AMPK) in LPS-induced ANA-1 macrophages.

[0024] Figure 4 It is a schematic diagram of the regulation of ROS and Nrf2 signal transduction by formononetin (FMN) of the present invention in LPS-stimulated ANA-1 macrophages.

[0025] Figure 5It is a schematic diagram of the bidirectional signal interaction between AMPK and ROS of formononetin (FMN) of the present invention in LPS-stimulated ANA-1 macrophages.

[0026] Figure 6 It is a schematic diagram of the inhibitory effect of FMN on proinflammatory cytokines COX-2, IL-6 and NLRP3 in LPS-stimulated ANA-1 macrophages of the present invention and its combined regulation with NAC, compound C and AMPK siRNA.

[0027] Figure 7 It is a schematic diagram of immunofluorescence imaging of formononetin (FMN) of the present invention on COX-2 in LPS-induced ANA-1 cells.

[0028] Figure 8 It is a schematic diagram of the signal transduction pathway by which formononetin (FMN) of the present invention exerts anti-inflammatory effects. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] like Figure 1-8 The anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient is shown.

[0031] Experimental methods of cell culture and reagents used,

[0032] Main reagents:

[0033] Dimethyl sulfoxide (DMSO): Used as a solvent to dissolve other chemicals, such as FMN and LPS.

[0034] Lipopolysaccharide (LPS): An immunostimulant that activates macrophages.

[0035] FMN (Formononetin): a natural product extracted from the root of Astragalus membranaceus, is the main research object of this experiment.

[0036] Cell culture:

[0037] Cell source: Mouse ANA-1 macrophages were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0038] Culture medium: 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic cocktail (penicillin / streptomycin) was used.

[0039] Culture conditions: Cells were cultured at 37°C in a humidified atmosphere of 5% CO2.

[0040] Passaging: Cells were passaged once every 24 hours for a total of three times.

[0041] Experimental design:

[0042] Seeding density: Cells were seeded at different densities in 96-well and 6-well plates.

[0043] Experimental Grouping:

[0044] Blank Group

[0045] DMSO group (50 μmol·L^-1)

[0046] LPS group (1 μg·mL^-1)

[0047] FMN group (0.08-10 μmol·L^-1)

[0048] LPS+FMN group

[0049] Cell proliferation ratio measurement: Cell Counting Kit-8 (CCK-8) was used to measure the cell proliferation ratio.

[0050] This experiment was designed to study the effect of FMN on LPS-activated ANA-1 macrophages. The changes in cell proliferation were observed by treating with different concentrations of FMN and stimulating with LPS to evaluate the immunomodulatory effect of FMN.

[0051] Regulation of TLR4 and ER expression by FMN

[0052] Method Overview:

[0053] Western Blotting (WB): used to detect the expression levels of TLR4 and ER.

[0054] Comparative analysis: Compare the differences between the LPS stimulation group and the LPS stimulation plus FMN treatment group.

[0055] Specific steps:

[0056] Cell collection and lysis: Treat cell samples with a specific lysis buffer and phosphatase inhibitor cocktail.

[0057] Protein quantification: performed using the BCA protein assay kit.

[0058] Electrophoresis and membrane transfer: The proteins were separated by SDS-PAGE and then transferred to nitrocellulose and PVDF membranes.

[0059] Immunodetection: Incubation with specific antibodies against TLR4 and ER, followed by treatment with secondary antibodies and chemiluminescent detection.

[0060] Data analysis: ImageJ software was used for quantitative analysis and the results were normalized with the internal reference β-actin.

[0061] FMN activates the cAMP-PKA-LKB1-AMPK signaling axis

[0062] Method Overview:

[0063] ELISA: Used to quantify cAMP levels.

[0064] Western blotting: detection of background and phosphorylated forms of PKA, LKB1, and AMPK.

[0065] Specific steps:

[0066] Sample preparation: Obtain supernatant by centrifugation.

[0067] cAMP quantification: Use ELISA kit according to the manufacturer's protocol.

[0068] Protein detection: Specific antibodies were used to detect the levels of PKA, p-PKA, LKB1, p-LKB1, AMPK, and p-AMPK.

[0069] Immunofluorescence detection: Measure the immunofluorescence intensity of p-AMPK.

[0070] Regulation of ROS-Nrf2 signaling pathway by FMN

[0071] Method Overview:

[0072] Fluorescent probe method: used to detect ROS levels.

[0073] Real-time quantitative PCR and Western blotting were used to detect the expression and activation levels of Nrf2.

[0074] Specific steps:

[0075] ROS detection: using a fluorescent probe method and quantified by a fluorescence microplate reader.

[0076] Nrf2 mRNA detection: performed by qRT-PCR using specific primer sequences.

[0077] Nrf2 protein detection: Detection was performed by WB using specific antibodies.

[0078] Together, these approaches were used to study how FMN affects inflammatory responses and other biological processes in macrophages at the molecular level.

[0079] Bidirectional regulation of p-AMPK and ROS

[0080] Method Overview:

[0081] Western Blotting (WB): used to detect the level of p-AMPK.

[0082] Drug treatment: AICAR, CC, NAC, and FMN were used alone or in combination, as well as AMPK-targeting and non-targeting siRNAs.

[0083] Specific steps:

[0084] The regulation of p-AMPK by ROS was evaluated by LPS stimulation and NAC treatment, as well as the combination treatment with FMN.

[0085] The regulation of ROS by p-AMPK was evaluated by LPS stimulation, AICAR and AICAR+FMN treatment, CC and CC+FMN treatment, and AMPK-targeted / non-targeted siRNA+FMN treatment.

[0086] WB detection: Use specific antibodies to detect the level of p-AMPK.

[0087] Drug interaction studies: Comparative experiments were performed using the FMN analog 4',7-dimethoxyisoflavone (DMIF) and the NAC derivative N,N'-dimethylthiourea (DMTU).

[0088] Regulation of pro-inflammatory cytokines by FMN

[0089] Method Overview:

[0090] Real-time quantitative PCR (qRT-PCR) and Western blotting (WB) were used to detect the mRNA and protein levels of COX-2, IL-6, and NLRP3.

[0091] Specific steps:

[0092] Sample preparation: LPS-stimulated cells were collected and treated with AICAR, FMN+CC, FMN+NAC and FMN+AMPK siRNA respectively.

[0093] qRT-PCR detection: Specific primer sequences were used to detect the mRNA levels of COX-2, IL-6, and NLRP3.

[0094] WB detection: Specific antibodies were used to detect the protein levels of COX-2, IL-6, and NLRP3.

[0095] Fluorescence staining: FITC staining was used to observe the effect of FMN on COX-2 inhibition and imaging was performed using a fluorescence microscope.

[0096] Data analysis: FITC immunofluorescence intensity was calculated and expressed as mean units per cell.

[0097] Statistical analysis

[0098] Method Overview:

[0099] Repeat experiments: All experiments were repeated at least three times.

[0100] Data processing: GraphPad Prism 7.04 software was used to process the data.

[0101] Statistical tests: One-way analysis of variance (ANOVA) with Tukey's post hoc test was used to compare differences among groups.

[0102] Specific steps:

[0103] Data presentation: The results are expressed as mean ± standard deviation (SD).

[0104] Significance judgment: Statistical significance was judged based on the p value (p<0.05, p<0.01, p<0.001).

[0105] result

[0106] FMN decreases TLR4 but increases ER expression in LPS-stimulated ANA-1 cells

[0107] Cell proliferation and survival: When ANA-1 macrophages were stimulated with 1 μg / mL LPS for 24 h, cell proliferation decreased. However, FMN treatment did not affect cell survival compared with the blank control group. CCK-8 kit detection showed that the toxicity of FMN treatment was negligible, with a level below 50 μmol / L (Figure S1).

[0108] Expression of TLR4 and ER: Figure 1As shown, LPS stimulation increased the expression of TLR4 (relative to blank), but had no effect on ER. This result indicates that inflammatory signals are initiated in cells through TLR4 rather than ER (Cadwell, 2016; Choudhary et al., 2020). As for FMN treatment, it can dose-dependently antagonize the stimulatory effect of LPS on TLR4 expression, but activate ER expression. Analysis of three repeated data showed that when treated with 10 μmol / L FMN, there were statistical differences (p = 0.027 relative to LPS stimulation) and extremely significant statistical differences (p = 8.5E-7 relative to LPS stimulation) for TLR4 and ER, respectively. Obviously, FMN reversely regulated the expression of TLR4 and ER in LPS-stimulated macrophages.

[0109] FMN activates the cAMP-PKA-LKB1-AMPK signaling axis

[0110] cAMP production: The production of cAMP is catalyzed by adenylate cyclase in the ER signaling complex. Figure 2 A shows that LPS did not stimulate cAMP production compared to blank. This indicates that there is no signaling between LPS and ER. However, dose-dependent treatment with 0.4-10 μmol / L FMN promoted cAMP content, which is consistent with the effect of FMN on ER regulation. The results showed that FMN treatment activated cAMP dependent on ER signaling. Analysis of triplicate data of FMN treatment (relative to LPS stimulation) showed statistical differences (p<0.05) and extremely significant statistical differences (p<0.001) at 2 and 10 μmol / L FMN, respectively.

[0111] Activation of protein kinases: The activation of p-PKA, p-LKB1, and p-AMPK depends on the production of cAMP (Chen et al., 2019; Fu et al., 2011). We found that FMN treatment did not affect the background form of protein kinases (Figures S2 and S3). LPS stimulation did not change the phosphorylated form of protein kinases relative to the blank (Figure S2 and S3). Figure 2 B, C and D). This result is consistent with the result that LPS stimulation does not affect cAMP regulation ( Figure 2 A). Notably, FMN treatment upregulated p-AMPK in blank cells (Figure S3). In LPS-stimulated cells, FMN treatment promoted p-PKA ( Figure 2 B) p-LKB1( Figure 2 C) and p-AMPK ( Figure 2 D) Expression of p-AMPK. The regulatory effect of FMN on p-AMPK (in both empty and LPS-stimulated conditions) was similar to that of the commercial AMPK activator AICAR (Figure S4). Figure 2E and F show that when FMN is combined with the AMPK inhibitor CC or siRNA, it antagonizes the regulation of p-AMPK. Both CC and siRNA antagonize the effect of FMN treatment on p-AMPK regulation. Figure 2 E, The explanation for the antagonistic effect is that in LPS-stimulated cells, the order of p-AMPK regulation is: FMN treatment > FMN + CC combination > CC treatment. The CC combination weakened the activity of FMN on p-AMPK regulation. Figure 2 In F, the effect of AMPK-targeting siRNA was more significant than that of non-targeting siRNA (negative control). p<0.05, 0.01, or 0.001 between FMN treatment and LPS stimulation was obtained. Immunofluorescence assay showed that FMN treatment increased p-AMPK ( Figure 3 ). All the results indicate that FMN is an activator of cAMP-PKA-LKB1-AMPK signaling.

[0112] Regulation of ROS-dependent Nrf2 signaling:

[0113] LPS stimulation led to upregulation of ROS (reactive oxygen species), which was inhibited by FMN treatment.

[0114] As a ROS scavenger, FMN has an effect similar to that of NAC (N-acetylcysteine).

[0115] FMN dose-dependently promoted the upregulation of Nrf2 expression, indicating that FMN is an activator of Nrf2 signaling in LPS-stimulated macrophages.

[0116] Two-way cross-talk between p-AMPK and ROS:

[0117] The use of the AMPK activator AICAR or inhibitor CC dose-dependently reduced or increased ROS production in LPS-stimulated cells.

[0118] Targeted silencing of the AMPK gene antagonized the effect of FMN on ROS scavenging.

[0119] NAC alone increased p-AMPK levels, but combined treatment with NAC+FMN at least partially antagonized the effects of NAC or FMN alone on p-AMPK regulation.

[0120] FMN inhibits pro-inflammatory cytokines:

[0121] FMN reduced proinflammatory cytokines such as COX-2, IL-6, and NLRP3 in a dose-dependent manner.

[0122] High-content imaging analysis showed that COX-2 immunofluorescence intensity decreased with FMN treatment.

[0123] Protein WB detection showed that FMN had an anti-inflammatory effect similar to that of AICAR.

[0124] Further WB detection revealed that inflammation was suppressed relative to NAC treatment alone; but in LPS-stimulated cells, the FMN+CC combination relative to CC treatment alone, and FMN+AMPK siRNA relative to siRNA treatment alone, all showed an antagonistic effect on inflammation suppression.

[0125] When exploring the role of FMN (flavin mononucleotide) in regulating the inflammatory response of macrophages induced by LPS (lipopolysaccharide), we found that FMN has multiple pharmacological activities and can regulate the inflammatory response by affecting multiple signaling pathways and molecular mechanisms. The following is a comprehensive description of these complex effects:

[0126] Cross-talk between TLR4 / ER and ROS / cAMP-dependent pathways:

[0127] FMN treatment not only inhibited TLR4 activity and reduced ROS generation, but also activated Nrf2 and cAMP-dependent protein kinase, indicating that FMN is involved in multiple signaling pathways in regulating inflammatory responses.

[0128] AMPK activation and ROS clearance:

[0129] FMN acts as an activator of AMPK and a scavenger of ROS, showing its important role in regulating cellular energy metabolism and oxidative stress. This dual role enables FMN to effectively alleviate the inflammatory response induced by LPS.

[0130] Inhibition of pro-inflammatory cytokines:

[0131] By affecting the above signaling pathways, FMN can significantly reduce the production of proinflammatory cytokines such as COX-2, IL-6 and NLRP3 in macrophages stimulated by LPS, further confirming its potential in anti-inflammatory treatment.

[0132] Cross-talk between signaling pathways:

[0133] FMN treatment enhanced the bidirectional cross-talk between p-AMPK and ROS, which not only increases our understanding of the mechanism of action of FMN but also provides a new perspective for studying inflammation regulation.

[0134] Transformation of immune to metabolic signals:

[0135] The anti-inflammatory effects of FMN involve the formation of a long signaling axis connecting ER / TLR4 signaling with cAMP / ROS-dependent pathways, forming an immune-to-metabolic signaling loop that is critical for understanding the broad biological effects of FMN.

[0136] In summary: Multiple pharmacological activities: FMN exhibited regulatory effects on TLR4 and ER expression, cAMP and ROS production, and AMPK and Nrf2 signaling, demonstrating its multifaceted potential in anti-inflammatory therapy.

[0137] Cross-talk of signaling pathways: FMN forms a long signaling axis by affecting the bidirectional signaling cross-talk between p-AMPK and ROS, which is a key part of the immunity-to-metabolism circuit achieved through ER / TLR4 signaling.

[0138] Inhibition of proinflammatory cytokines: FMN treatment significantly inhibited the production of proinflammatory cytokines such as COX-2, IL-6, and NLRP3 in LPS-stimulated macrophages, indicating its ability to alleviate inflammatory responses.

[0139] Convergence of signaling pathways: The anti-inflammatory effect of FMN involves the interaction of multiple signaling pathways, which converge to lead to the inhibition of proinflammatory cytokines, thereby alleviating the LPS-induced inflammatory response.

[0140] Future research directions: Future studies should further explore the effects of FMN in different inflammatory models, as well as the combination effects with other potential therapeutic agents, in order to develop new therapeutic strategies for the treatment of inflammation-related diseases.

[0141] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient, Features: The pharmaceutical composition uses formononetin as an active ingredient and further comprises a pharmaceutically acceptable carrier; Formononetin is present in an effective amount for inhibiting inflammatory response by regulating the expression of TLR4 and ER receptors and activating the cAMP-PKA-LKB1-AMPK signaling axis.

2. The anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient according to claim 1, Features: The concentration of formononetin is sufficient to inhibit the expression of pro-inflammatory cytokines COX-2, IL-6 and NLRP3.

3. The anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient according to claim 1, Features: The composition has the ability to modulate ROS-dependent Nrf2 signaling.

4. The anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient according to claim 1, Features: The preparation method comprises the step of mixing formononetin with a pharmaceutically acceptable carrier.

5. The anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient according to claim 4, Features: It also includes formulating the composition into a dosage form suitable for oral administration; It is also contemplated that the compositions are formulated in a dosage form suitable for parenteral administration.

6. The anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient according to claim 1, Features: For the treatment or prevention of diseases caused by inflammation and oxidative stress; Use in preparing medicines for treating inflammatory diseases and oxidative stress-related diseases.

7. The anti-inflammatory and antioxidant pharmaceutical composition using formononetin as an active ingredient according to claim 6, Features: The inflammatory disease includes an autoimmune disease, a cardiovascular disease, or a neurodegenerative disease; The oxidative stress-related diseases include disorders associated with ROS-mediated tissue damage.

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