The imbalance between the cellular biosynthesis and accumulation of reactive oxygen species (ROS) and the organism's antioxidant defense mechanism leads to oxidative stress, which in turn causes cellular and tissue damage (H Sies, 2020; G Pizzino, 2017). Despite the role of several enzymes that can produce ROS, four major systems significantly catalyze the ROS production in humans: NADPH oxidases, xanthine oxidase (XO), uncoupled NO synthase, and the mitochondrial respiratory chain (M El Assar et al., 2017). XO (EC 1.17.3.2) is a molybdoflavoprotein enzyme that catalyzes purine catabolism by oxidizing hypoxanthine to xanthine and xanthine to uric acid, producing ROS during this process (MG Battelli et al., 2016; N Liu et al., 2021). Increased activity or hyperactivity of XO is associated with elevated uric acid levels (hyperuricemia), gout, cardiovascular diseases, and other oxidative stress–related pathologies (N Liu et al., 2021). Hyperuricemia has become a global threat due to its association with other metabolic syndromes (Z Ullah et al., 2024). Given the versatile role of XO in disease conditions, targeting XO with inhibitors to reduce both uric acid accumulation and ROS represents a promising therapeutic strategy (Z Ullah et al., 2024). Currently, several synthetic XO inhibitors, such as allopurinol, topiroxostat, and febuxostat, are clinically effective for ROS suppression but are greatly linked with adverse side effects like polyarthritis (Z Ullah et al., 2024). This has raised concern for using natural products as alternative XO inhibitors with little or no side effects. Senna auriculata (L.) Roxb., commonly known as Avarampoo, belongs to the family Fabaceae and is a shrub that can grow to a height of 500 m (Guruprasad C. Nill et al., 2021). This plant has been well documented for its diverse pharmacological properties, including anti-inflammatory, antioxidant, antimicrobial, antiparasitic, antidiuretic, and antidiabetic activities (Mihir Kumar Purkait et al., 2023). Different parts of S. auriculata, including the flowers, seeds, leaves, bark, stem, wood, and roots, are reported to contain diverse pharmacological properties. The leaves are traditionally used in the treatment of several skin disorders, such as leprosy and acne, as well as in managing eye diseases (e.g., conjunctivitis), and they also exhibit antioxidant, anticytotoxic, and antiulcer activities (Mihir Kumar Purkait et al., 2023; HH Nandhini et al., 2024). The roots are used in the treatment of asthma, diabetes mellitus, and hemorrhagic conditions (Guruprasad C. Nill et al., 2021; HH Nandhini et al., 2024). The flowers are reported to have antidiabetic, antimicrobial, antioxidant, and antihyperlipidemic activities (AFK Rahman et al., 2024; HH Nandhini et al., 2024; N. Jeyashanthi et al., 2010). The stem has demonstrated antioxidant properties, while both the bark and seeds are associated with antidiabetic and antioxidant activities (Guruprasad C. Nill et al., 2021; HH Nandhini et al., 2024).

Several metabolomic studies have been conducted on S. auriculata to identify its bioactive constituents, revealing the presence of flavonoids, terpenoids, alkaloids, terpenoids, phenols, and tannins, which are associated with its therapeutic potential against various human diseases (Murugan Prasathkumar et al., 2021; S Revathi et al., 2025). In particular, phytochemicals such as luteolin, quercetin, and kaempferol have been identified in this plant, and these compounds are well known for their strong antioxidant potential (OS Oladeji et al., 2021). Recent metabolomic investigations of S. auriculata (Syn. Cassia auriculata) have revealed diverse primary and secondary metabolites, including flavonoids with known antidiabetic and antioxidant activities, providing a molecular basis for its traditional use in diabetes management (Z Tietel et al., 2021). Previous studies aimed to underscore the antioxidant potential of compounds identified from the S. auriculata; however, the inhibitory potential of S. auriculata phytochemicals against XO has not been systematically explored. This study aims to identify potential XO inhibitors from S. auriculata phytochemicals through ADMET analysis and molecular interaction studies, thereby linking traditional knowledge with modern computational approaches for drug discovery.

2. MATERIALS AND METHODS

2.1 Collection of Phytochemicals from S. auriculata and ADMET analysis

To screen the XO inhibitory potential of phytochemicals present in S. auriculata, a comprehensive library of S. auriculata phytochemicals was developed. The list of curated compounds, spanning various plant parts including the flowers, fruit, seeds, leaves, wood, and bark, was retrieved from the IMPAAT: Indian Medicinal Plants Phytochemicals Database (https://cb.imsc.res.in/imppat/; K Mohanraj et al., 2018). Further, collected compounds were subjected to ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis using the ADMETLabs 3.0 platform (https://admetlab3.scbdd.com/). Only those phytochemicals that qualified ADMET parameters and satisfied the criteria for drug-likeness (e.g., Lipinski's Rule of Five) were retained and taken for the subsequent molecular docking analysis.

2.2. Ligand Preparation

 The 2D structures of the phytochemicals were retrieved from PubChem in SDF format and subsequently converted into 3D structures using the OpenBabel module in PyRx (https://pyrx.sourceforge.io/). Energy minimization was performed using the Universal Force Field (UFF). The Steepest Descent algorithm was employed with a maximum of 200 steps. The energy-minimized ligands were converted into PDBQT format.

2.3. Protein Preparation

The crystal 3D structure of Xanthine Oxidase (PDB ID: 2E1Q) was retrieved from the Protein Data Bank (https://www.rcsb.org/). Further, protein was prepared for docking by the removal of water molecules and heteroatoms, addition of polar hydrogens, and optimization for docking using Biovia Discovery Studio (https://discover.3ds.com/discovery-studio-visualizer-download) and PyRx. Cofactors of the protein, such as Molybdenum, FAD, and Fe-S clusters essential for activity, were retained in the macromolecule. The crystal structure of XO was comprised of four chains (A, B, C, D). However, as the biologically functional unit of mammalian XO is known to be a homodimer, only Chain A was selected and retained for the molecular docking studies.

2.4. Active site prediction

The binding pockets containing the active sites of the Xo protein were predicted using the Computed Atlas of surface topography of proteins - CASTp 3.0 server (http://sts.bioe.uic.edu/) with default parameters (W Tian et al., 2018).

2.5. Molecular Docking and Visualization

Molecular docking was carried out using AutoDock Vina integrated in PyRx. A grid box encompassing the active site residues of XO was defined using Autogrid, and docking was performed using Autodock. Based on the binding affinity (kcal/mol) best confirmation was selected and visualized using Discovery Studio Visualizer to examine hydrogen bond and other hydrophobic interactions. In addition to the phytochemical library of S. auriculata, allopurinol was also included in the docking study as a positive control.

3. RESULTS

3.1. Drug-likeness of S. auriculata phytochemical library

The phytochemical library for in silico screening comprised 20 compounds identified across different parts of S. auriculata. The distribution of these compounds across the plant parts was as follows: 5 from the seed, 6 from the fruit, 7 from the leaf, 4 from the flower, 2 from the bark, and 1 from the wood (Table 1). All 20 compounds were subjected to ADMET analysis to predict their pharmacokinetic properties. Out of collected 20 compounds tested for their pharmacokinetic properties (ADMET analysis), 16 (80%) successfully met the criteria for drug-likeness, specifically adhering to Lipinski's Rule of Five. In detail, Kaempferol, Cyanidin Chloride, (-)-β-Sitosterol, Cianidanol, Chrysophanol, Rubiadin, Nonacosane, Emodin, Nonacosan-6-one, 1-Tetradecanol, Anthraquinone, Stearic Acid, Palmitic Acid, Oleic Acid, Linolenic Acid, and Anthocyanins, were found to comply with the Lipinski Rule of Five. This indicates that each compound has a molecular weight ≤500 Da, a LogP value ≤5, no more than five hydrogen bond donors, and no more than ten hydrogen bond acceptors, suggesting favorable physicochemical properties and potential oral bioavailability. These 16 compounds were thus deemed suitable ligands for the subsequent molecular docking study against XO. 

3.2. Active Site of Xo

The prediction of the XO active site binding pocket, performed using the CASTp 3.0 server, identified a primary pocket encompassing six amino acid residues. This predicted binding pocket exhibited an Area (SA) of 23.975 Ų and a Volume (SA) of 6.989 Ų. The specific amino acid residues constituting the active sites were identified as LYS552, ASP553, ARG997, LYS1173, LEU1175, and ILE1238. Subsequently, a computational grid box for molecular docking was successfully generated to fully encompass these identified active site residues, ensuring accurate sampling during the docking process.

3.3 Molecular Interaction of Phytochemicals with XO

Among the compounds tested, rutin exhibited the highest binding affinity to XO  (-8.5 kcal/mol), followed by kaempferol (-7 kcal/mol), flavylium (-6.9 kcal/mol), and cianidanol (-6.9 kcal/mol). Several other compounds, including emodin, rubiadin, anthraquinone, and beta-sitosterol, also showed moderate binding affinities ranging from -6.7 to -6.3 kcal/mol. Notably, kaempferol, flavylium, cianidanol, and most of the top hits comply with Lipinski’s rule of five, suggesting better drug-likeness and potential oral bioavailability, whereas rutin, beta-sitosterol-beta-D-glucoside, and quercetin-3-glucoside do not, likely limiting their absorption. The standard inhibitor allopurinol displayed a lower binding affinity (-5.6 kcal/mol) than several natural compounds, highlighting the promising inhibitory potential of these flavonoids and other S. auriculata-derived molecules against XO (Table 2).

The docking analysis further revealed key hydrogen bond interactions between the ligands and amino acid residues of XO (Figure 1). Interaction analysis was carried out for compounds that showed binding affinities better than allopurinol, which was used as the positive control. Rutin formed multiple hydrogen bonds with ASP553, ARG997, GLU1164, LYS1173, GLN551, and GLU991, consistent with its strong binding affinity. Kaempferol interacted with GLN551 and ASN992, while cianidanol formed bonds with ASN992 and ASP1171. Emodin established hydrogen bonds with GLU984, ARG997, and LEU1175, and rubiadin with ASP553 and ARG997. Quercetin-3-glucoside interacted with GLN551, ASP553, ARG997, and LYS1173, whereas chrysophanol showed interactions with ASP553, ARG997, and LEU1175. Beta-sitosterol-beta-D-glucoside and beta-sitosterol formed hydrogen bonds with PRO554 and LEU1175, and GLU1239, respectively. The standard inhibitor allopurinol was stabilized through interactions with ASP553, ARG997, and GLU1239 (Table 3). In addition to hydrogen bonding, the docking analysis showed several other stabilizing interactions between the ligands and XO. These included π–anion, π–alkyl, π–sigma, π–cation, π–π T-shaped interactions, carbon–hydrogen bonds, and van der Waals contacts, which collectively contributed to the stabilization of the ligand–protein complexes within the active site. Such diverse non-covalent interactions further support the strong binding affinities observed for the top compounds compared to the standard inhibitor allopurinol. Notably, all of the top-binding compounds formed hydrogen bonds with the identified active site residues of XO (LYS552, ASP553, ARG997, LYS1173, LEU1175, and ILE1238). Rutin interacted with ASP553, ARG997, and LYS1173, while emodin and rubiadin established contacts with ARG997, ASP553, and LEU1175. Quercetin-3-glucoside and chrysophanol formed hydrogen bonds with ASP553, ARG997, and LYS1173, further confirming their stable binding within the catalytic active sites. Beta-sitosterol-beta-D-glucoside interacted with LEU1175, and beta-sitosterol showed proximity to ILE1238. Importantly, allopurinol, the positive control, also interacted with ASP553 and ARG997, validating the relevance of these residues in ligand stabilization.

4. DISCUSSION 

S. auriculata is a medicinally valuable plant with tremendous pharmacological properties. The present study highlights the inhibitory potential of S. auriculata phytochemicals against xanthine oxidase (XO), an essential enzyme in uric acid biosynthesis and ROS production. Molecular docking and ADMET analyses revealed that several compounds, particularly rutin, kaempferol, flavylium, and cianidanol, exhibited high binding affinities (-8.5 to -6.9 kcal/mol) and favorable drug-likeness profiles. Notably, 16 out of 20 screened phytochemicals satisfied Lipinski’s Rule of Five, indicating good oral bioavailability and physicochemical compatibility for drug development. The predominance of flavonoids and anthraquinones among the active compounds aligns with earlier reports emphasizing these structural classes as major contributors to natural XO inhibition (Yu et al., 2024; Mehmood et al., 2019). Kaempferol, one of the top hits in this study, has been repeatedly identified as a potent competitive XO inhibitor. Similar observations were made for kaempferol-3′-sulfonate and quercetin derivatives, which were shown to reduce uric acid levels through stable binding to the catalytic residues of XO (Wang et al., 2023; Cao et al., 2014).  Active-site mapping using CASTp identified six key residues: LYS552, ASP553, ARG997, LYS1173, LEU1175, and ILE1238, forming the primary binding pocket of XO. The consistent involvement of ASP553 and ARG997 across top-binding ligands and the reference inhibitor allopurinol confirms the catalytic relevance of these residues. Similar binding patterns have been reported for natural flavonoids such as chrysoeriol, quercetin, and 5,7-dihydroxycoumarin, which interact with analogous residues (ARG880, ASN768, GLU802, and PHE914) in bovine XO (Yu et al., 2024). Such overlap supports the hypothesis that S. auriculata compounds occupy the catalytic channel responsible for xanthine oxidation, thereby preventing uric acid formation through competitive inhibition.

Beyond hydrogen bonding, the docking analysis revealed multiple stabilizing non-covalent interactions: π–anion, π–alkyl, π–cation, van der Waals, and hydrophobic contacts that collectively enhanced ligand stability within the active site. These interaction modes are consistent with the multi-residue engagement described for natural XO inhibitors, where hydrophobic packing around the flavonoid core contributes significantly to binding free energy and inhibitory potency (Feng et al., 2022). The stronger binding affinities observed for rutin and kaempferol compared with allopurinol (–5.6 kcal/mol) emphasize the superior stabilization achieved by polyhydroxylated aromatic systems over synthetic heterocycles.

Nevertheless, pharmacokinetic limitations such as low permeability and high molecular weight, particularly for glycosylated flavonoids like rutin, may restrict their oral absorption. Yu et al. (2024) similarly noted that many natural XO inhibitors exhibit excellent binding activity but suboptimal ADME properties, highlighting the need for semi-synthetic optimization to improve lipophilicity and metabolic stability. Natural XO inhibitors from plants provide a safer alternative to synthetic drugs, with added benefits of antioxidant properties that can alleviate oxidative stress. However, in silico findings need to be validated through enzymatic inhibition assays and in vivo studies. Despite these challenges, compounds that both satisfy drug-likeness criteria and exhibit high docking affinity, such as kaempferol and cianidanol, hold promise as lead scaffolds for designing safer and more potent XO inhibitors.

5. CONCLUSION

This study demonstrates that phytochemicals from Senna auriculata (Avarampoo) exhibit promising xanthine oxidase inhibitory potential as revealed by molecular docking analysis. The results underscore the ethnopharmacological relevance of S. auriculata and suggest its potential as a source of natural therapeutics for oxidative stress-related diseases. Future studies should focus on in vitro validation, pharmacological evaluations, and bioavailability assessments of the identified lead compounds, including rutin, kaempferol, flavylium, cianidanol, and emodin, to further explore their therapeutic potential.

Acknowledgments
The authors acknowledge the use of freely available databases and software tools. The authors are thankful to the Director of the National Institute of Siddha, Chennai, India, for providing facilities to conduct this research. Selvalakshmi Selvamani gratefully acknowledges the AYUSH and the National Institute for Siddha for providing a stipend to support her postgraduate studies. She would also like to thank the Department of Gunapadam, National Institute of Siddha, Chennai, India, for their constant support.

Conflict of Interest

 The authors declare no conflict of interest.

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Figure 1. Molecular docking interactions of top xanthine oxidase (XO) inhibitors. A–L show the binding modes of the tested compounds: (A) Rutin (–8.5 kcal/mol); (B) Kaempferol (–7 kcal/mol); (C) Flavylium (–6.9 kcal/mol); (D) Cianidanol (–6.9 kcal/mol); (E) Emodin (–6.7 kcal/mol); (F) β-Sitosterol-β-D-glucoside (–6.6 kcal/mol); (G) Rubiadin (–6.5 kcal/mol); (H) Quercetin-3-glucoside (–6.5 kcal/mol); (I) Anthraquinone (–6.5 kcal/mol); (J) Chrysophanol (–6.3 kcal/mol); (K) β-Sitosterol (–6.3 kcal/mol); (L) Allopurinol (–5.6 kcal/mol, positive control). Hydrogen bond interactions are highlighted in green, showing contacts with active site residues, while other non-covalent interactions (π–π, π–alkyl, π–cation, van der Waals, etc.) are depicted in their respective representations. These panels illustrate the diverse binding patterns and stabilizing interactions of the compounds within the XO active site.

Table 1. Phytochemicals of S. auriculata collected from the IMPPAT database

Table 2. In silico ADMET properties and xanthine oxidase binding affinities of S. auriculata phytochemicals

Table 3. Details of amino acid (AA) residues interacting with ligands through hydrogen bonds