Synthesis of flavonoid derivatives as anti-rheumatoid arthritis agent: A systematic literature review and meta-analyses

Study selection process

The selection process was conducted systematically in two stages. In the first stage, an initial screening was performed to identify and exclude duplicates and irrelevant studies. Subsequently, in the second stage, a full-text evaluation was conducted for studies that met the inclusion criteria. This process ensured that only studies relevant to and supportive of the research objectives were included in the analysis. The complete search syntaxes used for all databases are provided in Supplementary File S3.

Data extraction

Data from each selected study were systematically extracted by two independent reviewers to maintain objectivity and accuracy. Extracted information included study identifiers (authors, year of publication, title, and journal), research design (type of study, intervention, and control groups), participant characteristics (sample size, age, gender, disease duration, and concomitant treatments), and details of the intervention (type of flavonoid, dosage, and duration of administration). Additionally, primary outcomes, such as effects on inflammatory parameters like TNF-α and IL-6 levels, and secondary outcomes, such as pain, physical function, and adverse effects, were also recorded. The data extraction form and final dataset of included studies are available as Supplementary File S4.

Risk of bias assessment

The risk of bias was assessed using the Newcastle–Ottawa Scale, a validated and reliable tool for evaluating the methodological quality of studies. Publication bias was assessed using both funnel plot analysis and Egger’s regression test. These methods were applied to examine asymmetry and detect potential small-study effects. This assessment included an evaluation of participant selection, outcome measurement, and data analysis to ensure the reliability of the findings. Additionally, to address potential publication bias and enhance the robustness of the meta-analysis, funnel plot analysis was conducted to visually assess asymmetry in effect sizes, which could indicate systematic bias in published studies. Sensitivity analyses were also performed by systematically excluding individual studies to determine their influence on overall results, ensuring that the conclusions drawn remained consistent and were not disproportionately affected by any single study. These strategies help reinforce the credibility of the synthesized evidence on flavonoid derivatives as anti-RA agents.

Quantitative synthesis

Where feasible, a meta-analysis was conducted to combine the results of multiple homogeneous studies. Of the 33 studies included in the systematic review, all 33 studies provided sufficient quantitative data and were included in the meta-analysis. Before this process, a heterogeneity test was performed to ensure compatibility between the results of the studies to be combined. The meta-analysis aimed to provide more accurate and precise effect estimates compared to single-study analyses. A detailed Statistical Analysis Plan is available as Supplementary File S5.

Through this systematic approach, this research aims to provide an in-depth understanding of the potential of flavonoids as therapeutic agents in the management of RA and to evaluate their effectiveness based on existing scientific evidence.

Distribution of research papers by year

Table 4 below presents the distribution of research studies conducted over several years. This distribution provides insights into research trends over time, showing the volume of academic activity during different periods. By examining the frequency and percentage distribution, we can observe patterns that may indicate an increased focus or interest in research during specific years.

Table 4. Frequency distribution of research papers by year. [Click here to view]

Based on Table 4, it can be observed that the year 2021 had the highest number of publications, accounting for 23.53% of the total studies in the past decade, followed by 2022 with 17.65%. This indicates a peak in research activity during those years, which may reflect an increased interest or advancements in the related field at that time. Table 4 also shows a significant rise in publications between 2017 and 2022. This is likely driven by the growing interest of researchers in the potential of flavonoids as anti-inflammatory agents, particularly in the context of autoimmune diseases such as RA. Advances in organic synthesis technology and natural compound analysis may have facilitated more in-depth research on flavonoids. Furthermore, the increasing global prevalence of RA has likely prompted researchers to seek more effective treatment alternatives, contributing to the observed significant upward trend.

The year 2020 marked the onset of the COVID-19 pandemic. Despite this, research on flavonoids actually increased in 2021. This may be attributed to several factors, such as heightened public awareness of the importance of health and immunity, which in turn stimulated pharmaceutical research. Additionally, governments and research institutions may have allocated more funding to health-related research, including studies on natural compounds such as flavonoids. Furthermore, flavonoids are known to possess immunomodulatory properties, and in the context of the pandemic, research on these compounds may have been seen as relevant due to their potential impact on the immune system. However, after the peak of the pandemic, research funding allocations may have returned to normal levels, leading to a decrease in research activities. A possible contributing factor to this decline is that researchers may have shifted their focus to other topics deemed more relevant or compelling. The pandemic may have also disrupted the supply chains of chemicals and reagents required for research, thus hindering research progress.

Distribution of research papers based on research sample

The following table presents the distribution of research methods used across various studies. This distribution categorizes the methodologies, highlighting the prevalence of each method and associated References for quick reference.

Table 5 classifies the research samples based on the synthesis methods used to obtain flavonoid compounds. Three main categories of samples are employed for each method: (1) samples from conventional chemical synthesis methods, which involve a series of chemical reactions to produce flavonoid compounds; (2) enzymatic samples from synthesis methods that utilize enzymes as catalysts to facilitate the chemical reactions leading to flavonoid formation; and (3) nanoparticle powder samples, which involve the use of physical techniques or nanoparticles to generate flavonoid compounds.

Table 5. Distribution of research samples based on sample type. [Click here to view]

Table 5 provides data on the distribution of samples used in the studies. Based on Table 5, it can be seen that the conventional chemical synthesis method has the highest distribution percentage, accounting for 78.79% of the total samples analysed. This indicates that the majority of the samples in these studies were derived from traditional or conventional chemical synthesis processes. Next, flavonoid compound synthesis using enzymatic reactions from microorganisms ranks second, with a distribution percentage of 15.15%. This suggests that a significant portion of the remaining samples was obtained through the isolation of flavonoid compounds, a common method used to extract bioactive compounds from natural sources. In contrast, the category of samples derived from nanoparticle synthesis of flavonoid derivatives shows a much smaller distribution percentage of only 6.06%. This indicates that although all three categories are present in the research, their contribution to the overall sample distribution is limited compared to the previous two categories (conventional chemical synthesis and enzymatic synthesis).

Distribution of research papers based on flavonoid compound synthesis methods

Table 6, which illustrates various flavonoid derivative compounds and the synthesis methods used, provides an overview of the most commonly employed synthesis techniques for these flavonoid compounds. From Table 6, it is evident that the synthesis method with the highest distribution for flavonoid derivatives is the Claisen–Schmidt synthesis method for chalcone compounds, with a percentage of 23.81%. Following this, the second most common methods are microwave radiation synthesis for chalcone compounds and cyclization oxidation synthesis for flavone compounds, both with a distribution percentage of 14.29%.

Table 6. Distribution of flavonoid compound synthesis methods. [Click here to view]

Distribution of research papers based on stages of flavonoid derivative synthesis research

Table 7 shows the distribution of synthesis stages in research related to the synthesis of flavonoid derivatives and RA. Several papers indicate that the most frequent synthesis stage in these studies was a one-step synthesis, accounting for 45.24%. This means that nearly half of the studies reviewed used a one-step synthesis method. This figure suggests that many researchers prefer relatively simple or direct synthesis methods, which may be more efficient or easier to implement in the development of flavonoid compounds for the treatment of RA. Following one-step synthesis, more complex synthesis stages, such as two-step (2-step), three-step (3-step), and four-step (4-step) processes, are ranked according to their frequency. According to Table 7, the specific percentages for each stage, aside from one-step synthesis, indicate that as the number of steps involved in the synthesis increases, the number of studies using these methods decreases. This likely reflects the increasing complexity, higher costs, or longer time required for synthesizing flavonoid compounds with more than one step.

Table 7. Distribution of synthesis stages in research. [Click here to view]

Distribution of research papers based on inflammatory mediators in RA cases

Table 8 presents data on the distribution of inflammatory mediators involved in RA cases. Table 8 also provides information on various inflammatory mediators identified in RA-related studies and their respective roles. It shows that several inflammatory mediators are interconnected in the inflammatory process occurring in RA. These interconnections highlight how immune cells interact or influence each other in mediating the inflammatory response, which is crucial in the progression of RA.

Table 8. Distribution of inflammatory mediators in RA cases. [Click here to view]

TNF-α is one of the most frequently mentioned inflammatory mediators in the studies presented in Table 8. It is noted that TNF-α plays a role in 30% of RA cases, making it the dominant mediator in these studies. TNF-α is a proinflammatory cytokine that plays a significant role in the inflammatory process in RA by inducing the activation of other immune cells and causing joint tissue damage. Following TNF-α, the mediator cyclooxygenase-2 (COX-2) is cited with a percentage of 26.67%, involved in the production of prostaglandins, which contribute to inflammation and pain in RA. In third place are xanthine oxidase (XO) and COX-1, each with an equal percentage of 6.67%. XO is another proinflammatory cytokine that contributes to joint damage in RA. Their nearly equal distribution suggests that both also play a significant role in the inflammatory response in RA, although not as much as TNF-α and COX-2. Other inflammatory mediators involved in RA have lower percentages, ranging from 10% to 3.33%. These figures indicate that while other mediators are involved, they have a smaller role or are less frequently mentioned in the studies referenced.

Distribution of research papers based on bioactivity testing in RA cases

The analysis of Table 9 demonstrates the diversity of approaches used to evaluate the effectiveness of compounds against RA. The majority of studies (51.61%) relied on in vitro testing at the molecular and cellular levels to investigate the underlying mechanisms of action. In vivo testing on animal models was used to observe the direct effects of compounds on RA symptoms, representing 48.39% of the approaches. This indicates the importance of animal models in understanding disease mechanisms and screening potential compounds. On the other hand, in silico testing, which employs computer simulations, has also started to be used, although its percentage remains relatively small. The use of computational models allows for early predictions of a compound’s biological activity, thereby optimizing the drug discovery process. Overall, the combination of in silico, in vitro, and in vivo testing provides a more comprehensive understanding of the therapeutic potential of a compound in treating RA.

Table 9. Frequency distribution of bioactivity testing in RA cases. [Click here to view]

The analysis of Table 10 provides a detailed overview of the various methods used to evaluate the effectiveness of compounds against RA. The dominance of in vivo methods using Carrageenan (CARR)-induced inflammation/Cyclophosphamide (CPA) as an inflammatory inducer indicates that animal models remain a cornerstone in RA research. The Complete Freund’s Adjuvant (CFA) method is also quite popular, reflecting the variation in approaches used to induce disease models in animals. This is evident from the percentage data in Table 10, where the largest percentage is occupied by in vivo testing with the CPA model, reaching 20%, followed by the CFA model at 12%.

Table 10. Distribution of methods in various bioactivity tests. [Click here to view]

In addition to in vivo testing, in vitro testing plays the most significant role among the methods, surpassing both in vivo and in silico approaches. The ELISA method and tests using RAW 264.7 cells are the most frequently employed, with a percentage of 36%. ELISA allows researchers to quantitatively measure various inflammatory biomarkers, while RAW 264.7 cells, as a macrophage cell model, provide in-depth insights into inflammatory mechanisms at the cellular level.

The importance of computational approaches is increasingly evident with the rising use of in silico methods such as docking, quantitative structure–activity relationship (QSAR) and molecular dynamics. These methods enable early predictions of a compound’s biological activity virtually, thus optimizing the drug discovery process. However, it is important to note that there is still considerable variation in the methods used. This reflects the lack of standardization in RA research, which may hinder the comparison of results across studies. Standardizing methods would be highly beneficial in enhancing the reproducibility of research findings and facilitating the development of consensus within the scientific community.

Comparative efficacy with standard RA treatments

Compared to standard treatments such as DMARDs and biological agents, flavonoid derivatives offer a promising alternative with notable advantages. While conventional drugs like methotrexate or TNF inhibitors are effective in suppressing inflammation, they often come with significant side effects, including immunosuppression, liver toxicity, and increased infection risk. In contrast, flavonoids demonstrate a broader safety margin with fewer adverse effects in preclinical models, making them attractive candidates for long-term management of RA (Table 11).

Table 11. Comparison of flavonoid effectiveness versus DMARDS/biologics. [Click here to view]

Emerging studies suggest that certain flavonoid subclasses—such as chalcones and flavonols—not only reduce key pro-inflammatory markers (e.g., TNF-α and IL-6), but also modulate oxidative stress and immune responses more holistically than many synthetic drugs. Although their potency may vary depending on structural modifications and bioavailability, the multifunctional properties of flavonoids position them as more than just anti-inflammatory agents—they could serve as disease modulators with fewer side effects.

Moreover, synthetic derivatization and formulation strategies are rapidly evolving, improving the pharmacokinetic profiles of flavonoids and enhancing their therapeutic efficacy. With optimized delivery systems and structural enhancements, flavonoid derivatives have the potential to match or even surpass traditional RA treatments in both efficacy and safety. Future studies should consider flavonoids not just as supplementary to standard RA therapies, but as agents with the potential to transform treatment paradigms—especially for patients requiring safer, long-term therapeutic options.

Synthesis of flavonoid derivatives

The synthesis of flavonoid compounds as potential anti-RA agents involves various methods, each with its advantages. Organic synthesis remains a common approach, allowing the creation of flavonoid derivatives through specific modifications that enhance their biological activity [47]. Semi-synthesis, which involves modifying natural flavonoids, has been effective in improving bioactivity and solubility, as exemplified by the transformation of hesperidin into hesperidin methyl chalcone [15]. Furthermore, biosynthesis through microbial systems, including yeast and bacteria, leverages natural metabolic pathways to produce flavonoids with high purity and yields [9]. Enzymatic synthesis offers an environmentally friendly alternative, using enzymes to catalyze the formation of flavonoids, thus reducing environmental impact [43]. A combination of these methods, such as enzymatic conversion with green synthesis, offers a sustainable approach to producing flavonoids while maintaining therapeutic efficacy [36]. Overall, these various synthesis strategies provide a comprehensive toolkit for developing flavonoid-based therapies targeting RA.

Additionally, some of the most commonly used methods for chalcone synthesis include the Claisen-Schmidt method, which involves cross-aldol condensation between substituted acetophenones and substituted benzaldehydes under basic conditions [17,53]. Base catalysts such as NaOH, KOH, or Ba(OH)₂ are used to facilitate this reaction, typically carried out in polar solvents such as ethanol or methanol. This reaction produces chalcone, a basic compound that can then be further modified into flavonoids or other derivatives. To enhance efficiency and yields, microwave-assisted synthesis has also been applied [43]. The use of microwaves accelerates reactions and improves synthesis yields in a shorter time, reducing traditional reaction times from hours to minutes. In addition, various catalyst and solvent variations, such as using K₂CO₃ as a base catalyst or THF as a solvent, have been explored to further enhance chalcone synthesis efficiency. A general reaction pathway for flavonoid synthesis is shown in Figure 5.

Figure 5. General reaction for the synthesis of flavonoid derivative compounds [58] . [Click here to view]

Once chalcone has been successfully synthesized, the next step is to convert it into flavone. One common method for flavone synthesis is oxidative cyclization of chalcone, which is done using reagents such as I₂/DMSO, H₂O₂/NaOH, or SeO₂ [46]. This reaction transforms the chalcone structure into flavone by forming an additional ring bond. As with chalcone synthesis, microwave-assisted methods have also been employed in flavone synthesis, improving reaction speed and yields in a more efficient manner [15]. Furthermore, the use of acid catalysts such as CH₃SO₃H or acetic acid can assist in the cyclization process, providing a simpler and faster alternative. Other approaches also allow direct synthesis of flavones from appropriate precursors without first forming chalcone [19,32]. This method often involves multicomponent reactions utilizing specific catalysts to yield flavones in a more efficient and targeted manner [22].

In addition to flavones, flavanon compounds are also flavonoid derivatives that can be synthesized from chalcone. The flavanon synthesis process generally involves the cyclization of chalcone with acid catalysts, such as H₂SO₄, in alcohol solvents. The use of acid catalysts facilitates the formation of the flavanon structure through cyclization reactions. As with chalcone and flavone synthesis, microwave-assisted synthesis is also applied to flavanon synthesis to enhance reaction speed and yields [25,26]. Additionally, base catalysts such as NaOAc can be used to direct the cyclization reaction of chalcone into flavanon, offering flexibility in selecting the most appropriate reaction conditions [47].

Besides synthesis, structural modification of flavonoids is also an important step in enhancing their biological activity. One of the most common modifications is the addition of functional groups to the A and B rings, which may include hydroxyl, methoxy, halogen, or nitro groups [12]. These additions are achieved through various reactions such as alkylation, acylation, halogenation, and nitration. These modifications aim to improve the solubility, stability, or pharmacological potential of flavonoids, which in turn can enhance their therapeutic activity, including in the treatment of inflammatory diseases and cancer [21]. Other modifications include replacing aromatic rings with heterocyclic rings, such as pyridine, which can alter the chemical properties and biological activity of flavonoids, opening new potential in drug design [55].

Overall, flavonoid synthesis and structural modifications offer many opportunities to enhance the efficiency of their production and improve their biological activity. Through various synthetic approaches and structural modifications, flavonoid derivatives can be tailored for broader applications, especially in treating diseases related to inflammation, infection, and cancer.

Synthesis of flavonoid compounds as anti-RA agents

Flavonoids are a class of natural plant compounds known for their various pharmacological properties, especially anti-inflammatory and antioxidant effects. These compounds have drawn significant research interest due to their potential applications in treating chronic inflammatory diseases such as RA. Table 12 summarizes the various flavonoid subclasses, their synthesis methods, and key findings related to their anti-RA activity. By understanding these aspects, researchers can further explore and optimize flavonoids as natural alternatives or adjunct therapies for RA management.

Table 12. distribution of research on flavonoid compounds with various mechanistic pathways of anti-inflammatory activity in RA cases. [Click here to view]

Flavonoids are a diverse group of phytonutrients found in various plants, exhibiting numerous bioactive properties that are beneficial for human health. These compounds are particularly recognized for their antioxidant and anti-inflammatory effects, which play a crucial role in their application against diseases such as RA. Flavonoid subclasses, including chalcones, flavones, flavanols, flavanonols, and flavonols, possess unique structural characteristics that contribute to their bioactive activities. For instance, chalcones synthesized through Claisen–Schmidt condensation and microbial systems demonstrate significant ability to inhibit pro-inflammatory cytokines such as TNF-α and IL-6, which are essential in the development of RA, by reducing oxidative stress and modulating the immune response [12].

Flavonoid synthesis varies according to its specific subclass. Chalcones and flavonols are synthesized via organic methods and biosynthesis, often employing yeast-mediated or plant-based techniques [19]. Flavones, on the other hand, can be synthesized through chemical and enzymatic processes, with methods like methylation and glycosylation enhancing their stability and bioavailability [37]. Flavanols utilize an environmentally friendly synthesis approach using plants, enabling better antioxidant capacity [43]. Semi-synthetic approaches for flavanonols show flexibility in synthetic methods aimed at optimizing the effectiveness of these compounds in therapeutic applications against RA.

Regarding anti-RA activity, flavonoid compounds exhibit various mechanisms. Flavones and isoflavonoids, for example, inhibit NF-kB, STAT signaling pathways, and key inflammatory drivers in RA, effectively reducing joint inflammation and cartilage degradation [45]. Flavanols increase antioxidant enzyme levels, helping reduce free radical-induced damage and improving joint function [26]. Flavonols reduce joint swelling and pain by inhibiting inflammatory mediators, particularly IL-6 and TNF-α, demonstrating their potential as adjunct therapies in RA treatment [14]. Furthermore, flavanonols and chalcones exhibit immunomodulatory and anti-inflammatory effects that may modify the progression of RA, highlighting flavonoids as promising natural agents for managing RA symptoms and improving patient quality of life.

Modification of flavonoid derivatives and functional groups with effective anti-inflammatory activity in RA cases

Research conducted by several scientists, as presented in Table 1, shows that structural modifications of flavonoid derivatives, particularly on functional groups attached to the aromatic rings, such as the addition of hydroxyl, methoxy, and halogen groups, can enhance anti-inflammatory activity in RA [9,12,39]. This research focuses on how structural changes in flavonoids affect their ability to reduce inflammation in RA. The findings suggest that some flavonoid derivatives with hydroxyl groups at positions 5 and 7 on ring A, and at positions 3’ and 4’ on ring B, exhibit superior anti-inflammatory activity in combating RA inflammation. For instance, a study by Miao et al. [37] demonstrated that the anti-inflammatory activity test of the compound 3-(4-aminophenyl)-coumarin showed an average IC₅₀ value of 10 μM. In a rat arthritis model, this compound significantly reduced joint swelling and tissue damage [36]. The addition of hydroxyl groups at these positions enhances the solubility of the compound in water, facilitating penetration into inflammatory cells and increasing interaction with inflammatory molecular targets such as COX-2 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) [5].

Several studies on the SAR have been conducted by Emam et al. [5]. In Figure 4 presented in this study, the relationship between the chemical structure of chalcone compounds and their biological activity is clearly illustrated, with a focus on the inhibition of nitric oxide (NO) production and COX-2 enzyme activity [5]. Several key points can be systematically analyzed based on Figure 6.

Figure 6. Structure-activity relationship of chalcone derivatives synthesis [5] . [Click here to view]

First, the influence of functional groups on the chalcone structure appears to play a critical role in determining the biological activity of this compound. In Figure 6, it can be observed that functional groups such as hydroxyl (-OH) and methoxy (-OCH₃) located on the aromatic ring of chalcone, as well as variations in the R group on the side chain, significantly affect the compound’s ability to inhibit NO production and COX-2 activity. Specifically, the hydroxyl group at certain positions on the chalcone aromatic ring seems to contribute significantly to the inhibition of NO production, indicating that this group plays an important role in the anti-inflammatory mechanism of chalcone compounds.

Furthermore, the anti-NO activity influenced by the hydroxyl group can be clearly observed in Figure 6. The addition of a hydroxyl group at specific positions, indicated by the green arrow, tends to increase the effectiveness of the chalcone compound in inhibiting NO production. NO is an essential mediator in the inflammatory process, and the inhibition of NO production by chalcone compounds demonstrates their potential as a potent anti-inflammatory agent. This suggests that chalcone compounds with hydroxyl groups have a better capacity to reduce oxidative stress and inflammatory responses induced by NO.

Next, the anti-COX-2 activity, also shown in Figure 6, indicates that chalcone compounds with specific structures, again highlighted by the green arrows, exhibit an increased ability to inhibit the COX-2 enzyme. COX-2 is an enzyme involved in the synthesis of prostaglandins, which are key players in inflammation. Chalcone structures with high affinity for the COX-2 active site, as seen in Figure 6, can effectively inhibit the enzyme’s activity, subsequently reducing inflammation caused by prostaglandin production [5]. This highlights chalcone’s potential as a therapeutic agent in treating inflammatory disorders such as arthritis.

Finally, Figure 6 also demonstrates a clear SAR between the chemical structure of chalcone compounds and their biological activity. Small changes in the chalcone structure, such as the addition or removal of specific functional groups, can significantly affect its ability to inhibit NO production and COX-2 activity. This reinforces the importance of understanding and utilizing this relationship in designing compounds with enhanced anti-inflammatory activity.

Overall, the analysis derived from Figure 6 provides a comprehensive understanding of how structural changes in chalcone affect two major inflammatory pathways, namely NO production and COX-2 inhibition, and demonstrates the great potential of chalcone in the development of natural compound-based anti-inflammatory drugs.

The study conducted by Sinyeue et al. [24] also conducted an in-depth investigation into the evaluation of various flavonoid derivatives modified at rings A and B, assessing their ability to inhibit NO production as an inflammatory marker [24]. The main findings from this study indicate that the presence and position of certain functional groups, such as the carboxyl group at the ortho position on ring B, halogen groups on ring B, and the methoxy group at position 5 on ring A, significantly influence the enhancement of the flavonoid anti-inflammatory activity (Fig. 7). The mechanism of action of the tested flavonoid compounds is believed to be related to their ability to inhibit NO production, a molecule that plays an important role in inflammation. Inhibition of NO production by these compounds may potentially reduce inflammation.

Figure 7. Structure-activity relationship in the synthesis of flavanone derivatives [24] . [Click here to view]

Similarly, the structural and activity analysis of flavonoid compounds in a study conducted by Bano et al. [19] provides valuable insights into how chemical structure affects the compound’s ability to inhibit the COX-2 enzyme activity, which is a primary target in the inflammatory process [19,24]. The flavonoid structures analysed in this study contain several key elements contributing to their biological activity.

As shown in Figure 8, the influence of structure and the presence of substituents affect the resulting anti-inflammatory activity. Chalcone structure exhibits the highest anti-inflammatory activity among other flavonoid derivatives, such as flavone and flavanone. The presence of an aromatic ring in the flavonoid structure provides rigidity to the molecule and allows strong interactions with target proteins through hydrogen bonding and van der Waals forces. Furthermore, the chalcone structure with functional group substituents, such as hydroxyl (-OH) and methoxy (-OCH3) on the aromatic ring, is crucial in determining the biological properties of the compound [19]. This is demonstrated by the significant anti-inflammatory activity values observed among other structures, indicating that the addition of these substituents enhances pharmacological activity. The hydroxyl group can act as a hydrogen bond donor, improving the compound’s affinity for the target enzyme, while the methoxy group helps increase the lipophilicity of the compound, affecting its ability to penetrate cell membranes. Additionally, the conjugated double-bond system in the flavonoid structure provides molecular stability and allows electron delocalization, enhancing interactions with target proteins. As a primary target, the COX-2 enzyme plays a role in the production of prostaglandins, which are inflammatory mediators. By inhibiting COX-2, flavonoid compounds can reduce prostaglandin production, thereby decreasing inflammation [19].

Figure 8. Structure-activity relationship in anti-inflammatory activity in the case of RA [19]. [Click here to view]

Based on the explanation, Bano et al. [19] outline that in terms of the SAR, the hydroxyl group on ring B most likely interacts with amino acid residues at the COX-2 active site through hydrogen bonding, increasing the compound’s binding affinity and inhibiting enzyme activity. The aromatic ring in the flavonoid structure can interact with aromatic amino acid residues at the enzyme’s active site using π-π interactions, which enhances the stability of the compound-enzyme complex. The methoxy group, which affects the compound’s lipophilicity, plays a role in enhancing cell membrane permeability and compound distribution within the body, as well as modulating interactions with target proteins. Moreover, variations in the length and type of side chains in the flavonoid structure can influence interactions with the COX-2 enzyme and the physicochemical properties of the compound, collectively improving the anti-inflammatory therapeutic potential of the flavonoid compounds.

Based on several research summaries, the addition of polar groups in the structure of flavonoid derivatives plays a critical role in enhancing anti-inflammatory activity, particularly in relation to bioavailability and molecular interactions with inflammatory targets in the body. Flavonoids, as phenolic compounds, have structures that are generally non-polar, which may hinder their solubility in water and reduce their effectiveness in the body. Therefore, the addition of polar groups, such as hydroxyl (-OH), methoxy (-OCH3), or carboxyl (-COOH) groups in existing studies, can increase the solubility of the compounds, improve interactions with target proteins or enzymes, and enhance their biological activity [12,59]. The presence of these polar groups enhances the flavonoid’s ability to interact with molecular targets involved in the inflammatory process, such as the COX-2 enzyme, NF-kB transcription factors, cell membranes and surface receptors, as well as immune cells involved in inflammation [12]. These interactions can modulate inflammatory signaling pathways involved in the inflammatory process, such as inhibiting NF-kB activation or reducing COX-2 expression, which are two key targets in the treatment of RA [7,42].

The position and number of methoxy groups attached to the flavonoid structure also influence anti-inflammatory activity. Studies show that the addition of methoxy groups at specific positions, such as on ring B (e.g., at positions 3’ and 4’), can enhance the antioxidant potential of flavonoids, which plays a role in reducing oxidative stress associated with inflammation in RA [15]. Oxidative stress contributes to the activation of inflammatory pathways and tissue damage in joints, so reducing inflammation can improve the condition of RA patients [9].

Other modifications, such as the addition of halogen groups (e.g., fluorine or chlorine) at strategic positions in the flavonoid structure, have also been found to enhance biological activity. Halogens in the flavonoid structure can increase compound stability and alter interactions with inflammation-related receptors or enzymes [53]. Such modifications often aim to improve the compound’s affinity for specific inflammatory molecular targets and prolong its half-life in the body [57].

In addition to molecular structure, understanding the bioavailability of flavonoids is also important in enhancing their therapeutic potential. Structural modifications to flavonoids, such as the addition of certain groups, can help address bioavailability challenges by improving compound solubility or stability in the body, allowing the compound to reach sufficient therapeutic concentrations in target tissues, such as joints affected by inflammation in RA [36].

Overall, research into flavonoid structural modifications provides valuable insights for developing new therapeutic compounds for RA treatment. By modifying specific groups within the flavonoid structure, it is hoped that compounds with stronger, more selective, and more effective anti-inflammatory effects can be discovered, ultimately reducing RA inflammation symptoms while minimizing unwanted side effects.

Mechanism of inhibition of inflammatory activity in RA cases by flavonoid derivative compounds

In the research conducted, several possibilities can be predicted regarding the mechanisms of action of flavonoid compounds in anti-inflammatory activity in RA through various interconnected molecular pathways and processes [60]. One of the main mechanisms of flavonoid compounds in anti-inflammatory activity is the inhibition of COX-2 enzymes [19,20,33]. COX-2 is an enzyme involved in the synthesis of prostaglandins, which are key mediators in the inflammatory process [5]. In RA, COX-2 levels are often elevated, contributing to pain and inflammation in the joints. Several flavonoid compounds with hydroxyl and methoxy groups, such as kaempferol, apigenin, luteolin, quercetin, and chrysin, bind to the active site on COX-2, inhibiting the conversion of arachidonic acid to prostaglandin E2, which in turn reduces inflammation [19]. The addition of polar groups such as hydroxyl enhances the affinity of flavonoids for COX-2, making them more effective in inhibiting this enzyme’s activity.

The second pathway involves inhibition through the NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway. In this pathway, the transcription factor involved in regulating inflammatory genes is activated. NF-kB activation leads to increased production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, all of which play a role in the pathogenesis of RA [14]. Flavonoid compounds have been shown to inhibit the NF-kB pathway, reducing the expression of inflammatory genes. Derivatives of flavonoids, such as naringenin, genistein, luteolin, and quercetin, can inhibit the degradation of IκBα (an inhibitor of NF-kB), which prevents the activation of NF-kB and reduces the transcription of pro-inflammatory genes [19]. Structural modifications of flavonoids, such as the addition of hydroxyl or methoxy groups, can enhance their ability to inhibit NF-kB activation and reduce the expression of inflammatory cytokines [42,61].

The third pathway involves the reduction of oxidative stress. This pathway plays an important role in the inflammatory process, particularly in RA, where the production of reactive oxygen species (ROS) contributes to tissue damage and inflammation [45]. Flavonoids act as antioxidants by scavenging free radicals and lowering oxidative stress levels. Several flavonoid derivatives with hydroxyl substituents on both rings interact with free radicals and ROS, neutralizing them before they can damage cells or worsen inflammation [12]. Flavonoid compounds containing hydroxyl groups at specific positions, such as on ring B, have been shown to be effective in counteracting excessive ROS in RA.

The fourth pathway involves the modulation of immune cell activity [33]. Immune cells such as macrophages and T lymphocytes play a key role in the inflammatory process in RA [33]. Flavonoids can modulate the activity of these immune cells by reducing the production of inflammatory cytokines or by inhibiting the migration of immune cells to the site of inflammation. Some flavonoids can suppress macrophage activation, reducing the production of cytokines such as TNF-α and IL-6, which are heavily involved in joint inflammation in RA [14]. These compounds can also inhibit the transmigration of immune cells to infected or inflamed areas.

Flavonoids exhibit significant therapeutic potential as anti-RA agents by targeting multiple inflammatory mechanisms. Flavonoids have been shown to inhibit pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6, which are critical in the inflammatory process of RA [62]. Furthermore, flavonoids increase antioxidant enzyme levels, thereby reducing oxidative stress and free radical damage associated with joint damage in RA patients [56]. Flavonoids also possess strong anti-inflammatory effects by inhibiting key signaling pathways, including NF-κB and MAPK, which are central to the inflammatory cascade in RA [6,56]. This inhibition leads to reduced joint inflammation and cartilage degradation, ultimately improving joint function and alleviating pain [62]. Moreover, flavonoids have immunomodulatory properties that can suppress inflammatory pathways, potentially offering disease-modifying effects on the progression of RA [62]. Overall, the multifaceted actions of flavonoids highlight their potential as adjunct therapies for RA, capable of alleviating symptoms and slowing disease progression.

Limitations and challenges in the clinical translation of flavonoid derivatives

Despite these promising findings, several critical challenges remain in translating flavonoid derivatives into effective clinical treatments for RA. One notable limitation is the inconsistency in their efficacy across different in vitro and in vivo models. Variability in chemical structure, dosage, and model organisms contributes to inconsistent outcomes, making it difficult to generalize findings or establish standard treatment guidelines.

Another major obstacle lies in the poor bioavailability of many flavonoid compounds due to rapid metabolism and limited absorption. These pharmacokinetic limitations hinder their therapeutic effectiveness in vivo and must be addressed through advanced delivery systems or chemical modification. Additionally, clinical data on flavonoid-based therapies for RA remain scarce, highlighting the gap between experimental success and real-world applicability. Therefore, future research should prioritize comparative studies with standard RA drugs, optimize synthesis for bioactivity and bioavailability, and investigate translational models that closely mimic human RA pathology.

The increasing interest in diverse synthesis strategies, including structural modifications and eco-friendly catalytic systems, has significantly expanded the availability and biological potential of flavonoid derivatives. With these innovations, flavonoids are not only easier to produce in high yield and purity, but also more tunable in terms of potency and pharmacokinetics. This positions them as competitive—if not superior—alternatives to current standard therapies in the management of chronic diseases such as RA. Building on this foundation, the following section discusses how various subclasses of flavonoids act against RA, the results of meta-analysis on their efficacy, and how they compare to existing therapies in terms of safety and therapeutic value.