Anti-inflammatory effect of Isoniazid and Plant Extract:

Promising Results and Future Research

 

Houari Bendriss¹, Amina Ghomri^1,2, Noureddine Missoum^1,3, Mohamed Benbouali^1,4

¹Laboratory of Naturals Products and Bioactives- LASNABIO,

University of Tlemcen. B.P. 119, 13000 Tlemcen. Algeria.

²High School of Applied Sciences ESSA Tlemcen. B.P. 165, 13000 Tlemcen. Algeria.

³Faculty of Technology, Hassiba Benbouali University of Chlef, Algeria.

⁴Laboratory of Naturals Products and Bioactives- LASNABIO, University of Tlemcen. B. P. 119,

13000 Tlemcen. Algeria.

 

ABSTRACT:

This study investigates the topical anti-inflammatory effects of synthetic compounds using the xylene-induced ear edema model in Swiss albino mice. The standard anti-inflammatory drug, Indomethacin, demonstrated significant efficacy (87.65%), with Isoniazid showing comparable activity, indicating its potential for repurposing as an anti-inflammatory agent. To explore the molecular mechanisms underlying these effects, molecular docking and quantum chemical calculations were performed. The analysis revealed that Indomethacin had stronger binding in aqueous environments (-6.33kcal/mol) compared to the gas phase (-5.86kcal/mol), suggesting enhanced affinity due to solvation effects through ionic and hydrogen bonding with ARG 120 (A) and SER 530 (A). In contrast, Isoniazid displayed nearly identical binding affinities in both phases (-5.02 kcal/mol), indicating minimal solvent influence on its interactions. Comparative studies with flavonoid-based ligands from Inula viscosa showed that these flavonoids had stronger and more stable receptor binding, primarily through multiple hydrogen bonds and π-stacking interactions. Among the flavonoids, 3-O-Acetyl-7-O-methylaromadendrin exhibited the highest affinity (-7.53kcal/mol), forming strong hydrogen bonds with ARG 120(A). Other flavonoids, 7-O-Methylaromadendrin and Sakuranetin, also demonstrated high binding affinities. These findings highlight the potential of flavonoids, alongside Isoniazid, as promising anti-inflammatory agent.

 

 

 

INTRODUCTION:

Inflammation is a vital biological défense mechanism that protects the body against pathogens, toxins, and injuries through the coordinated activation of immune cells, cytokines, and biochemical mediators. When dysregulated or chronic, however, it contributes to various pathological conditions, including autoimmune diseases, allergies, cardiovascular disorders, neuroinflammation, and cancer.¹

 

Among the key regulators of this process is the arachidonic acid pathway, which generates pro-inflammatory eicosanoids such as prostaglandins (PGs) and thromboxanes (TXs)—lipid mediators responsible for vasodilation, pain, fever, and leukocyte recruitment.²

 

The cyclooxygenase (COX) enzymes are central to prostaglandin biosynthesis and exist as two main isoforms: COX-1, a constitutive enzyme that maintains physiological homeostasis (e.g., gastric protection, renal function, platelet aggregation), and COX-2, an inducible enzyme overexpressed during inflammation.³ Consequently, COX-2 inhibition has become a major therapeutic target for anti-inflammatory drug development. Non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, ibuprofen, and diclofenac, inhibit both isoforms, but their non-selective action often leads to adverse effects including gastrointestinal ulceration, renal damage, and cardiovascular risks.⁴ This has prompted efforts to discover or repurpose compounds with improved selectivity and safety profiles.

 

Isoniazid (INH), a first-line antituberculosis agent, has recently attracted attention for its potential anti-inflammatory properties. Although it primarily inhibits enoyl-acyl carrier protein reductase (InhA) in Mycobacterium tuberculosis, emerging evidence indicates it may also modulate host inflammatory responses by suppressing inducible nitric oxide synthase (iNOS), NF-κB signaling, and oxidative stress.⁵ However, its precise molecular mechanism in inflammation remains unclear.

 

This study aims to investigate the anti-inflammatory potential of INH compared with indomethacin using in vivo and in silico methods. The xylene-induced ear edema model in male Swiss albino mice was employed to assess topical anti-inflammatory activity, while molecular docking was performed to analyze the interactions of INH, indomethacin, and Inula viscosa flavonoids (3-O-Acetyl-7-O-methylaromadendrin, 7-O-methylaromadendrin, and sakuranetin) with the COX-1 receptor (PDB ID: 4O1Z). These analyses aim to elucidate the binding behavior of INH relative to standard NSAIDs and natural flavonoids, providing a molecular rationale for its potential repurposing as an anti-inflammatory agent.

 

MATERIALS AND METHODS:

Materials:

All chemicals and reagents used throughout this study were obtained from commercial sources and used without further purification; Isoniazid (BHD chemicals Ltd Poole England), indomethacin, Xylene (Sigma).

 

Isoniazid (INH) is an anti-tuberculosis medicine known for its value as a first-line choice for clinical use⁶. Surprisingly, different studies demonstrate that INH in addition to antibacterial and anti-tuberculosis effects has special anti-inflammatory effects. INH (pyridine-4-carbohydrazide) has two group of pyridine and hydrazine, which interfere with the production of mycolic acid. The pyridine moiety of INH is chemically similar to the structure of COX II inhibitors (especially etoricoxib), which have anti-inflammatory properties⁷ ,

Indomethacin, a classical NSAID belonging to the indole acetic acid class of drugs, is an effective painkiller used to treat migraines and headaches. It inhibits both isoforms of the COX enzyme responsible for prostaglandin biosynthesis⁸ and modulates a wide range of receptors and enzymes^9,10. Efforts have been made to synthesize indomethacin analogues of improved potency, efficacy, and lower toxicity¹¹ ,with promising results reported for 3-[(2-imidazolyl)alkyl] indole analogues and other derivatives^12,13. By substituting the carboxylic group of NSAIDs, researchers can improve their pharmacological properties while retaining structural stability. As such, new ester analogues of indomethacin (2a–2g) have been synthesized based on supporting studies.

 

Experimental animals:

Experiments were performed using young female (nulliparous and non-pregnant) Swiss albino mice and weighing 25-30g. They were obtained from Pasteur institut (Algeria) and housed in plastic cages under normal laboratory conditions (12 h light/dark cycle, 23± 2°C) for an acclimatization period of 7 days prior to the experiments. All the animals were given food and water ad libitum.

 

Xylene-induced ear oedema in mice:

Adult albino female’s mice (25-30g) were randomised into different groups of 6 mice each were used for the experiment. Tested products (2mg/ear oedema), indomethacin (0.5mg/ear oedema) and distilled water were topically administered to various groups of 24 hours fasted mice 30minutes before the induction of inflammation.  Inflammation was induced in mice by topical administration of 30µl of xylene and 30µl of different synthetic product at the inner surface of the right ear. The thickness of the ear is measured before and 2hours after the induction of inflammation by a digital caliper.

 

Molecular docking:

To further investigate the anti-inflammatory potential of isoniazid against Cyclooxygenase-1 (COX-1), molecular docking studies were performed using MOE 2014¹⁴. The compound was docked alongside the reference molecule indomethacin, and a comparative analysis was carried out with Inula viscosa-derived flavonoids: 3-O-Acetyl-7-O-methylaromadendrin, 7-O-methylaromadendrin, and sakuranetin. The 3D structures of the ligands were generated using the programs Builder interface and prepared with default settings, then saved as MDB files for docking.^15,16 The crystal structure of ovine COX-1 complexed with meloxicam (PDB ID: 4O1Z¹⁷) was retrieved from the Protein Data Bank and prepared by adding polar hydrogens and removing water molecules, native ligands, and unwanted chains.^18–19 Docking was carried out in the active site of 4O1Z, generating 17 conformations per ligand, and evaluated using the London dG scoring function; the lowest-scoring pose was selected for further binding analysis.^14,20

 

Docking validation was confirmed through redocking and superimposition of the native ligand into the enzyme’s active site, verifying the accuracy of the docking protocol. Details of this procedure are provided in the ESI† (S.I. Docking validation).^21,22

 

Density functional theory (DFT) exploration:

DFT analyses play an essential role in the computation of molecular orbital characteristics^.23,24. In this framework, the  two compounds from the screening process underwent a structure-based DFT analysis utilizing B3LYP²⁵ and a 6–311g(d, p) basis se²⁶ via G09w²⁷.  Comparative investigation between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies was performed²⁸.

 

RESULT AND DISCUSSION:

Table 1. Selected compounds with inhibitory potential for COX1.

Ligands	Nom de l’IUPAC	Structure	Cid
Indomethacin	2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetic acid		3715
Isoniazid	pyridine-4-carbohydrazide		3767
Sakuranetin29	(2S)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-2,3-dihydrochromen-4-one		73571
3-Acetyl-7-O-methylaromadendrine29	(2R, 3R)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-oxo-2,3-dihydrochromen-3-yl] acetate		15139424
7-O Methylaromadendrin29	2R,3R)-3,5-dihydroxy-2-(4-hydroxyphenyl)-7-methoxy-2,3-dihydrochromen-4-one		181132

 

Xylene induced topical ear edema:

A study evaluating the anti-inflammatory activity of isoniazid (an anti-tuberculosis drug) compared with indomethacin (a reference NSAID) in a mouse model of xylene-induced ear edema reveals interesting results. The data show that isoniazid and indomethacin both achieved 87.65% edema inhibition.

 

This suggests that isoniazid has comparable anti-inflammatory activity to indomethacin in this experimental model. As indomethacin is a commonly used NSAID comparator, this result indicates that isoniazid may also have therapeutic potential in the treatment of certain inflammatory conditions.

 

However, it is important to consider certain limitations. Although these results are promising, the exact mechanism by which isoniazid exerts its anti-inflammatory effect is not yet fully understood. Further studies are needed to elucidate the molecular pathways involved. Furthermore, it is crucial to note that these results were obtained in a specific animal model and may not necessarily translate directly to humans^30,31.

 

 

Figure 1. Inhibition percentage of topical anti-inflammatory activity

 

Table 2. Interaction details of the target (protein COX1)

Ligands	Recepteurs	Interactions	Distances (A)	Energies (Kcal/mol)	Score
MXM	OG SER 530 (A)	H-acceptor	2.75	-1.9	-8.48
	NE ARG 120 (A)	H-acceptor	2.85	-4.1
Indomethacingas	OG SER 530 (A)	H-acceptor	2.68	-1.2	-5.857
Indomethacin aqueous	OG SER 530 (A)	H-acceptor	2.48	1	-6.33
	NE ARG 120 (A)	H-acceptor	3.29	-5.8
	NE ARG 120 A)	ionic	3.29	-2.8
Isoniazid gas	O MET 522 (A)	H-donor	3.26	-1	-5.021
Isoniazid aqueous	O MET 522 (A)	H-donor	3.26	-1	-5.02
3-O-Acetyl-7-O-methylaromadendrin	NH2  ARG 120 A)	H-acceptor	3.11	-1.2	-7.53
	NE   ARG 120 (A)	H-acceptor	2.75	-5.6
7-O-methylaromadendrin	NE   ARG 120 (A)	H-acceptor	3.25	-2.2	-7.49
	NH2  ARG 120(A)	H-acceptor	3.34	-0.7
	6-ring PHE 518 (A)	H-pi	4.65	-1
Sakuranetin	NH2  ARG 120(A)	H-acceptor	3.12	-1.4	-7.29

 

Ligands	Interactions 2D	Interactions 3D
MXM
Indomethacingas
Indomethacinaqueos
Isoniazidgas
Isoniazidaqueos
3-O-Acetyl-7-O-methylaromadendrin
7-O-methylaromadendrin
Sakuranetin

Figure. 2 3D and 2D representations of the molecular interactions of the investigated compounds against the PDB ID: 4O1Z.

 

The computational analysis of Indomethacin and Isoniazid in both gas and aqueous phases reveals significant differences in their molecular interactions, binding affinities, and structural stability. Indomethacin, in the gas phase, forms a single H-acceptor bond with SER 530(A) at 2.68Å, exhibiting a relatively weak binding energy of -1.2kcal/mol, whereas in aqueous solution, it establishes additional interactions, including an ionic bond with ARG 120(A) at 3.29Å, leading to a significantly stronger binding energy of -5.8kcal/mol. This suggests that solvation enhances Indomethacin’s molecular recognition and binding strength. In contrast, Isoniazid exhibits minimal structural and energetic variations across different environments, maintaining a single H-donor interaction with MET 522(A) at 3.26Å with an unchanged binding energy of -1.0kcal/mol, indicating that solvation has little effect on its binding properties. RMSD analysis further confirms these findings, showing that Indomethacin (aqueous) achieves greater structural stability (RMSD = 1.35Å) compared to its gas-phase counterpart (2.31Å), whereas Isoniazid remains stable with negligible RMSD variation (~1.97 Å). These results suggest that Indomethacin’s pharmacological activity may be significantly influenced by solvent interactions, enhancing its binding affinity and stability, while Isoniazid maintains its bioactive conformation regardless of solvation effects. This highlights the importance of solvent effects in drug optimization, particularly for ligands exhibiting ionic interactions, and emphasizes the need for further molecular dynamics simulations to assess long-term stability and biological relevance in physiological conditions^32,33.

 

The computational analysis of flavonoid-based ligands, including 3-O-Acetyl-7-O-methylaromadendrin, 7-O-methylaromadendrin, and Sakuranetin, reveals significant differences in their binding affinities, molecular interactions, and structural stability. 3-O-Acetyl-7-O-methylaromadendrin exhibits the strongest binding affinity (-7.53 kcal/mol), forming two hydrogen bonds with ARG 120(A) at 2.75Å and 3.11Å, suggesting a highly stable interaction. Similarly, 7-O-methylaromadendrin interacts with ARG 120(A) through two hydrogen bonds (3.25Å and 3.34Å) and an additional π-stacking interaction with PHE 518(A) at 4.65Å, contributing to a binding energy of -7.49 kcal/mol. Sakuranetin, while slightly less potent, forms a single H-acceptor bond with ARG 120(A) at 3.12Å, resulting in a binding energy of -7.29kcal/mol. The docking scores and RMSD values further support these findings, indicating greater structural stability for these flavonoids compared to Indomethacin and Isoniazid, whose binding affinities vary significantly across different solvent conditions^34,35. These results suggest that flavonoids, particularly 3-O-Acetyl-7-O-methylaromadendrin, could serve as promising drug candidates, given their strong receptor binding, structural stability, and potential pharmacological activity, though further experimental validation is required to confirm their biological efficacy.

 

	Isoniazidgas	Isoniazidaqueous	Indomethacingas	Indometacinaqueous
Lumo
	ELUMO=-0,05245, ΔE=0,20466 EHOMO=-0,251866	ELUMO=-0,0538, ΔE =0,199416, EHOMO=-0,25846	ELUMO=-0,06391, ΔE =0,1413, EHOMO=-0,20521	ELUMO=-0,06562, ΔE=0,14421, EHOMO=-0,20983
Homo
3D
	Ƞ=0.0997, ω = 0.119	Ƞ=0.102, ω =0.119	Ƞ=0.07065, ω =0.128	Ƞ=0.07205, ω =0.1315

Figure. 3 DFT isoniazid and reference drug indomethacin.

 

	7-O-methylaromadendrin	3-O-Acetyl-7-O-methylaromadendrin	Sakuranetin
Lumo
	ELUMO=-0,06185, ΔE=0,15663, EHOMO=-0,21848	ELUMO=-0,05528, ΔE=0,16615, EHOMO=-0,22143	ELUMO=-0,03698, ΔE=0,17919, EHOMO=-0,21617
Homo
3D
	Ƞ=0.078315, ω = 0.125	Ƞ=0.083075, ω =0.115	Ƞ=0.08959, ω =0.0895

Figure. 4 DFT 7-O-methylaromadendrin, 3-O-Acetyl-7-O-methylaromadendrin and Sakuranetin.

 

 

The quantum chemical analysis of Isoniazid and Indometacin reveals significant differences in their electronic properties, influencing their chemical reactivity and potential biological activity. Isoniazid exhibits a higher energy gap (ΔE ≈ 0.20 eV), greater hardness (η ≈ 0.10), and lower electrophilicity (ω ≈ 0.116), indicating a more stable and less reactive nature, traditionally aligning with its role as a prodrug requiring metabolic activation for antituberculosis activity. However, emerging studies suggest that Isoniazid may also possess anti-inflammatory properties, potentially through mechanisms such as modulation of oxidative stress, inhibition of inducible nitric oxide synthase (iNOS), and suppression of NF-κB signalling. In contrast, Indometacin demonstrates a lower energy gap (ΔE ≈ 0.14 eV), lower hardness (η ≈ 0.07), and higher electrophilicity (ω ≈ 0.1315), suggesting greater chemical reactivity and stronger interactions with biological targets, consistent with its function as a nonsteroidal anti-inflammatory drug (NSAID) inhibiting cyclooxygenase (COX) enzymes. The lower ΔE and higher ω of Indomethacin support its higher binding affinity in molecular interactions, whereas Isoniazid’s stability suggests a controlled activation mechanism that may allow selective targeting of inflammatory pathways. These findings indicate that while Indomethacin is more reactive and likely to exhibit stronger direct binding interactions in docking studies, Isoniazid’s potential anti-inflammatory.

 

A comparative quantum chemical analysis of flavonoid-based ligands further highlights structural and electronic variations influencing their potential anti-inflammatory activity. 3-O-Acetyl-7-O-methylaromadendrin exhibits the highest energy gap (ΔE = 0.16615 eV), followed by 7-O-Methylaromadendrin (ΔE = 0.15663 eV) and Sakuranetin (ΔE = 0.17919 eV), suggesting differing levels of electronic stability and reactivity. The HOMO and LUMO energy levels indicate potential interactions with biological targets, where lower ELUMO values of flavonoids suggest greater electron acceptance potential. Similarly, electrophilicity (ω) values show 7-O-Methylaromadendrin (ω = 0.125) as the most reactive, followed by 3-O-Acetyl-7-O-methylaromadendrin (ω = 0.115) and Sakuranetin (ω = 0.0895), further supporting their binding efficiency in docking studies. These findings indicate that while Indomethacin is highly reactive and likely to exhibit strong direct binding interactions, Isoniazid’s chemical stability allows for a more controlled activation mechanism. The flavonoid ligands exhibit varying degrees of electronic properties, suggesting that their interaction with COX-1 may be influenced by structural reactivity. This comparative analysis provides valuable insights into the rational design of novel anti-inflammatory agents, integrating synthetic drugs and natural flavonoids.

CONCLUSION:

The study highlighted the promising topical anti-inflammatory potential of several compounds, including Hydro 5, which outperformed indomethacin in a mouse model of xylene-induced ear edema, as well as isoniazid, which exhibited comparable activity. Computational analyses confirmed these results by showing favorable molecular affinities and reactivity, while natural flavonoids extracted from Inula viscosa also revealed potential COX-1 inhibition through specific interactions. These results highlight the importance of solvation effects in drug optimization and identify these compounds as interesting candidates for future pharmacological developments, suggesting further dynamic, pharmacodynamic in vivo, and mechanistic studies.

 

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Received on 02.09.2025      Revised on 10.12.2025 Accepted on 12.02.2026      Published on 20.05.2026 Available online from May 25, 2026 Research J. Pharmacy and Technology. 2026;19(5):2001-2008. DOI: 10.52711/0974-360X.2026.00286 © RJPT All right reserved
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