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[Rabindra Nath Acharyya](https://orcid.org/0000-0002-5439-8937), [Md. Abid Muktadir Risha](https://orcid.org/0009-0001-9984-5657), [Novi Dwi Widya Rini](https://orcid.org/0000-0003-1128-7804), [Sarita Manandhar](https://orcid.org/0009-0001-4177-9693), [Sabina Shahi](https://orcid.org/0000-0002-9198-2470), [Biswa Nath Bhadra](https://orcid.org/0000-0001-7268-2563), [Asish Kumar Das](https://orcid.org/0000-0001-7312-1012), [Katsuhiko Ariga](https://orcid.org/0000-0002-2445-2955), Shrabanti Dev, [Lok Kumar Shrestha](https://orcid.org/0000-0003-2680-6291)

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[Anti‐Allergic Potentials of                    <i>Actinodaphne angustifolia</i>                    : Integrated In Vivo Evaluation and In Silico Analysis of Bioactive Compounds](https://mdr.nims.go.jp/datasets/f38f485b-5aff-4dc0-9c0d-6dceccfa6451)

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Anti‐Allergic Potentials of Actinodaphne angustifolia: Integrated In Vivo Evaluation and In Silico Analysis of Bioactive CompoundsChemistry & BiodiversityRESEARCH ARTICLEAnti-Allergic Potentials of Actinodaphne angustifolia:Integrated In Vivo Evaluation and In Silico Analysis ofBioactive CompoundsRabindra Nath Acharyya1,2,3 Md. Abid Muktadir Risha1 Novi Dwi Widya Rini2,3 Sarita Manandhar2,3Sabina Shahi2,3 Biswa Nath Bhadra4 Asish Kumar Das1 Katsuhiko Ariga2,5 Shrabanti Dev1Lok Kumar Shrestha2,61Pharmacy Discipline, Life Science School, Khulna University, Khulna, Bangladesh 2Research Center for Materials Nanoarchitectonics (MANA), NationalInstitute for Materials Science (NIMS), Tsukuba, Ibaraki, Japan 3Graduate School of Sciences and Technology, University of Tsukuba, Tsukuba, Ibaraki,Japan 4Institute Charles Gerhardt Montpellier (ICGM), Centre National De La Recherche Scientifique (CNRS), Montpellier, France 5Department ofAdvanced Materials Science, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwa, Japan 6Department of Materials Science, Institute ofPure and Applied Sciences, University of Tsukuba, Tsukuba, JapanCorrespondence: Rabindra Nath Acharyya (robi12@pharm.ku.ac.bd) Shrabanti Dev (shrabantidev@pharm.ku.ac.bd) Lok Kumar Shrestha(SHRESTHA.Lokkumar@nims.go.jp)Received: 14 April 2026 Revised: 10 August 2026 Accepted: 8 September 2026Keywords: Actinodaphne angustifolia | allergy | in silico molecular docking | phytoconstituentsABSTRACTActinodaphne angustifolia (Lauraceae), traditionally used for treating inflammatory and allergic disorders was investigated for itsanti-allergic potential using integrated in vivo and in silico approaches. Safety evaluation in accordance with OECD guidelinesconfirmed the non-toxic nature of the ethanolic leaf extract. In a toluene 2,4-diisocyanate (TDI)-induced allergic model, theextract reduced allergic symptoms, including sneezing, nasal scratching, and nasal scores, also normalizing elevated white bloodcell counts in blood and bronchoalveolar lavage fluid. A marked reduction in serum IgE levels further indicated suppression ofhypersensitivity responses, with effects comparable to cetirizine. HPLC analysis revealed a rich polyphenolic profile dominated by(−) epicatechin and rutin hydrate, while GC–MS identified 15 additional bioactive constituents. Molecular docking demonstratedstrong binding affinities of these compounds toward key allergic targets, namely histamine H1 receptor, interleukin-4, andphospholipase A2, supporting their mechanistic roles in anti-allergic activity. The findings validate the ethnomedicinal relevanceof A. angustifolia and highlight its potential as a source of plant-derived anti-allergic agents.1MsphomcTm©ChIntroductionedicinal plants have been among the most ancient and reliableources of therapeutic agents, with nearly 80% of the globalopulation still relying on traditional remedies for primaryealthcare [1]. The chemical diversity and structural complexityf plant-derived metabolites make them invaluable resources forodern drug discovery, especially for chronic diseases whereonventional therapies often exhibit limited efficacy and adversehis is an open access article under the terms of the Creative Commons Attribution-NonCedium, provided the original work is properly cited and is not used for commercial purp2026 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AG.hemistry & Biodiversity, 2026; 23:e71727ttps://doi.org/10.1002/cbdv.71727effects [2, 3]. In Bangladesh, a rich heritage of medicinal plantsexists, many of which play a central role in traditional healingsystems. A large proportion of the rural and tribal populationsrelies on these herbal remedies to treat diverse ailments dueto limited access to modern healthcare [4, 5]. Among thesecommunities, the hill tract regions represent an especially valu-able repository of ethnomedicinal knowledge, where plants areroutinely used to address life-threatening diseases, such as dia-betes, asthma, rheumatism, and allergic disorders. This ecologicalommercial License, which permits use, distribution and reproduction in anyoses.1 of 14https://doi.org/10.1002/cbdv.71727https://orcid.org/0000-0002-5439-8937https://orcid.org/0009-0001-9984-5657https://orcid.org/0000-0003-1128-7804https://orcid.org/0009-0001-4177-9693https://orcid.org/0000-0002-9198-2470https://orcid.org/0000-0001-7268-2563https://orcid.org/0000-0001-7312-1012https://orcid.org/0000-0002-2445-2955https://orcid.org/0000-0003-2680-6291mailto:robi12@pharm.ku.ac.bdmailto:shrabantidev@pharm.ku.ac.bdmailto:SHRESTHA.Lokkumar@nims.go.jphttp://creativecommons.org/licenses/by-nc/4.0/https://doi.org/10.1002/cbdv.71727http://crossmark.crossref.org/dialog/?doi=10.1002%2Fcbdv.71727&domain=pdf&date_stamp=2026-09-22dto[eiAhssaiaaThbrlaocaarudctrgaiatpcAmSchlostsataqccmiptCd2 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creativersity, coupled with centuries of empirical use, highlightshe therapeutic potential of hill-tract flora as promising sourcesf novel bioactive compounds for modern drug development6, 7]. Indeed, several pharmaceutical industries have alreadyxplored plant-derived metabolites from such ecosystems forncorporation into commercial therapies.llergic disorders remain a major global health concern, affectingundreds of millions of individuals worldwide. Allergy, or hyper-ensitivity, is defined as a triggered immune response to foreignubstances present in the environment. The consequences ofllergies range from minor symptoms, such as atopic dermatitis,tching, sneezing, and rhinitis, to severe manifestations, suchs asthma and anaphylaxis [8, 9]. Sometimes, the severity ofllergic syndromes is such that it can even result in death.hese allergic reactions are mediated by a biomolecule namedistamine. Histamine, a biogenic amine formed by the decar-oxylation of histidine, plays a central role in mediating allergicesponses through activation of histamine H1 receptors (H1R),eading to symptoms, such as sneezing, nasal irritation, andirway inflammation [10, 11]. In addition to histamine signaling,ther mediators such as interleukins and phospholipase A2 alsoontribute significantly to the allergic cascade. Antihistaminicgents such as cetirizine and loratadine are routinely used tolleviate allergic symptoms, particularly sneezing, itching, andhinitis. [12, 13], despite their clinical effectiveness, prolongedse may be accompanied by undesirable effects, includingrowsiness, central nervous system disturbances, and, in certainircumstances, cardiovascular-related effects. [14]. Experimen-al models using toluene diisocyanate (TDI), a well-knownespiratory sensitizer, further highlight the complexity of aller-ic mechanisms, as exposure can induce allergic rhinitis andsthma-like symptoms [15, 16]. Notably, approximately 15% ofndividuals with chronic occupational exposure to TDI developsthma, emphasizing the urgent need for safer and more effectiveherapeutic alternatives [17]. Hence, exploring and validatinglant-based alternatives holds great promise in addressing thesehallenges.ctinodaphne angustifolia (Lauraceae), locally known as Modonosta, is traditionally used by the indigenous community inoutheast Asia for the management of inflammatory and allergiconditions [18]. Phytochemical studies of Actinodaphne speciesave revealed a structurally diverse range of secondary metabo-ites, including isoquinoline alkaloids, such as aporphines andxoaporphines, lactones, lignans, phenolic amides, flavonoids,terols, and terpenoid constituents [19]. These findings indicatehat A. angustifolia is a rich source of pharmacologically relevantecondary metabolites. Several of these metabolite classes aressociated with biological activities, such as antioxidant, antibac-erial, antifungal, anti-inflammatory, and cytotoxic effects. In A.ngustifolia, previously reported constituents include β-sitosterol,uercetin-3-O-rhamnoside, vitexin, friedelin, and various hydro-arbon derivatives [20]. The presence of these chemically diverseonstituents suggests that the biological effects of the speciesay arise from the combined actions of multiple phytochem-cals rather than from a single constituent. The anti-allergicotential associated with its constituents has not been inves-igated with molecular targets involved in allergic responses.onsequently, the traditional use of this species for allergic disor-ers has remained without experimental validation. Consideringof 14its ethnomedicinal significance and promising phytochemicalcomposition, A. angustifolia was selected for a comprehensiveinvestigation to evaluate its anti-allergic activity and to identifythe bioactive constituents that may contribute to this effect. Here,we investigated the anti-allergic potential of the ethanolic leafextract of A. angustifolia using an integrated in vivo and insilico approach. Anti-allergic activity was evaluated in a toluenediisocyanate (TDI)-induced mouse model by assessing clinicalsymptoms, including sneezing, nasal scratching, and nasal scores,together with hematological parameters, such as white bloodcell (WBC) counts in blood and bronchoalveolar lavage (BAL)fluid and serum IgE levels. HPLC–DAD and GC–MS analyseswere employed to characterize the phytochemical compositionof the extract. The pharmacokinetic and drug-likeness propertiesof selected constituents were further examined through ADMEanalysis. Molecular docking was performed to explore the bindinginteractions of the identified phytoconstituents with key allergy-related targets, including the histamine H1 receptor (PDB ID:3RZE), interleukin-4 (PDB ID: 3BPN), and phospholipase A2(PDB ID: 1DB4), while molecular dynamics simulations wereused to further assess the stability of selected protein–ligandcomplexes. Collectively, the experimental and computationalfindings provide scientific support for the traditional use of A.angustifolia in allergic conditions and offer insights into thephytoconstituents and molecular targets potentially associatedwith its anti-allergic activity.2 Results and Discussion2.1 Phytochemical ScreeningPreliminary phytochemical evaluation of the ethanolic extractof A. angustifolia revealed the presence of diverse classes ofsecondary metabolites, including alkaloids, tannins, flavonoids,glycosides, terpenoids, and reducing sugars, while gums and xan-thoproteins compounds were absent (Table S1). These phytocon-stituents are widely associated with significant pharmacologicalactivities. In particular, flavonoids and phenolic compounds arewell known for their antioxidant and anti-allergic effects, as theyscavenge free radicals and modulate immune responses. Thepresence of bioactive compounds provides a rational basis for A.angustifolia’s observed bioactivities.2.2 Safety Study (Acute and Sub-Acute Toxicity)of the ExtractThe acute toxicity study demonstrated that oral administration ofthe extract up to 3000 mg/kg produced no mortality, behavioralchanges, or visible signs of toxicity during the 14-day observationperiod (Table S2). Body weight remained stable compared tothe control group, indicating the absence of systemic toxicity(Figure S1). Similarly, subacute toxicity assessment (500 mg/kg)showed no significant alterations in physiological behavioror biochemical parameters. Hepatic enzymes (SGPT, SGOT,ALP) and renal function markers (creatinine, urea, bilirubin)remained within normal physiological limits (Figure 1). Thesefindings classify the extract as practically non-toxic under OECDGuideline 425, indicating a wide safety margin for furtherpharmacological use.Chemistry & Biodiversity, 2026ive Commons LicenseFIGURE 1 Safety evaluation of the ethanolic extract of A. angustifolia (EEAA) based on serum biochemical parameters. Effects of EEAA on liverfunction markers: (a) Serum glutamate pyruvate transaminase (SGPT), (b) serum glutamate oxaloacetate transaminase (SGOT), (c) alkaline phosphatase(ALP), and (d) serum bilirubin; and kidney function markers: (e) Serum creatinine, and (f) serum urea. Data are presented as mean ± standard error ofthe mean (SEM) (n = 6). ns: not significant.2Ttnv4apAiaso(5tpSs(eepaTWbiidC 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable .3 Evaluation of Anti-Allergic ActivityDI sensitization successfully induced nasal allergy-like symp-oms in mice, including sneezing, scratching, rhinorrhea, andasal score. The TDI control group exhibited significantly ele-ated sneezing frequency, scratching behavior, and nasal scores of1.2 ± 4.25, 234 ± 19.9, and 2.8 ± 0.20, respectively (Figure 2). Oraldministration of the ethanolic extract of A. angustifolia (EEAA)roduced a marked reduction in all allergy-associated symptoms.t 500 mg/kg, the extract markedly decreased sneezing, scratch-ng, and nasal scores to levels comparable to those of the standardntihistamine cetirizine. Notably, lessened the allergic symptoms,uch as sneezing, scratching, and nasal scores with the valuef (EEAA-500 vs Cetirizine 11.8 ± 2.97 vs 11.2 ± 3.07), scratchesEEAA 500 vs Cetirizine vs 111.2 ± 18.5), and nasal scores (EEAA-00 vs Cetirizine 0.8 ± 0.37 vs 1.3 ± 0.24). These findings indicatehat A. angustifolia possesses substantial anti-allergic potential,ossibly through modulation of histamine-mediated pathways.erum IgE levels, a key biomarker of allergic response, wereignificantly elevated in TDI-treated mice. EEAA administration500 mg/kg) markedly reduced IgE levels, approaching theffect of cetirizine (Figure 2d). This suggests that the extractffectively modulates IgE-mediated hypersensitivity responses,ossibly through regulation of cytokine signaling pathways suchs IL-4.DI exposure significantly increased total and differentialBC counts, including lymphocytes, neutrophils, eosinophils,asophils, and monocytes. EEAA treatment effectively normal-zed these elevated counts (Figure 3), suggesting suppression ofmmune cell activation and infiltration. The extract at 500 mg/kgemonstrated a comparable efficacy to cetirizine, indicatinghemistry & Biodiversity, 2026its capacity to regulate immune cell infiltration and mitigatehypersensitivity responses.Consistent results were observed in bronchoalveolar lavage(BAL) fluid, where TDI-induced elevation of inflammatorycells was significantly reduced following extract treatment(Figure 4). This demonstrates that the extract exerts both systemicand localized anti-inflammatory effects within the respiratorytract.2.4 HPLC Standardization and Quantification ofPolyphenolsHPLC–DAD analysis was performed using a mixed standardcontaining 16 authentic polyphenolic reference compoundsunder identical chromatographic conditions (Figure 5). Thenumber of polyphenolic compounds identified and quantifiedwas based on their retention times (Figures S2, S3). Theidentified compounds included catechol (5.69 mg/100 g), (-)epicatechin (106.55 mg/100 g), rutin hydrate (8.08 mg/100 g),rosmarinic acid (17.92 mg/100 g), myricetin (26.46 mg/100 g), andkaempferol (10.21 mg/100 g). Among these, (-)epicatechin wasthe most abundant, indicating its possible role as a dominantbioactive component (Table S3). These polyphenols are welldocumented for their antioxidant and immunomodulatoryeffects. Polyphenols such as catechol, (-)epicatechin, rutinhydrate, myricetin, and kaempferol are well-documented fortheir immunomodulatory and antihistaminic effects, actingthrough suppression of mast cell degranulation, inhibition ofhistamine release, and modulation of inflammatory mediators[21]. Their combined presence indicates a synergistic mechanism3 of 14Creative Commons LicenseFIGURE 2 Evaluation of the anti-allergic effects of the ethanolic extract of A. angustifolia (EEAA) in mice. The assessed parameters include (A)sneezing frequency, (B) scratching behavior, (C) nasal symptom scores, and (D) serum IgE levels. Data are expressed as mean± standard error of the mean(SEM) (n = 6). Statistical significance was determined using Dunnett’s test: #p < 0.05 versus the control group; *p < 0.05, **p < 0.001, and ***p < 0.0001versus the TDI control group.FIGURE 3 Effect of the ethanolic extract of A. angustifolia (EEAA) on total and differential white blood cell (WBC) counts in mice. Data areexpressed as mean ± standard error of the mean (SEM) (n = 6). Statistical significance was performed using Dunnett’s test: #P < 0.05 versus controlgroup, *p < 0.05, **p < 0.001, and ***p < 0.0001 versus TDI control group.4 of 14 Chemistry & Biodiversity, 2026 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons LicenseFIGURE 4 Effect of ethanolic extract of A. angustifolia (EEAA) total and differential white blood cell (WBC) counts in bronchoalveolar lavage(BAL) fluid of mice. Data are presented as mean ± standard error of the mean (SEM) (n = 6). Statistical significance was performed using Dunnett’s test:#P < 0.05 versus control group, *p < 0.05, **p < 0.001, and ***p < 0.0001 versus TDI control group.FIGURE 5 Chemical structures and quantification of majorpolyphenolic compounds identified in the ethanolic extract of A.angustifolia using HPLC analysis. The detected compounds include (a)catechol, (b) (–)epicatechin, (c) rutin hydrate, (d) rosmarinic acid, (e)myricetin, and (f) kaempferol.uaiC 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creatnderlying the observed anti-allergic activity. GC–MS and FT-IRnalyses were additionally performed to provide complementarynformation on the broader chemical composition of the extract.hemistry & Biodiversity, 20262.5 GC–MS Analysis of BioactivePhytoconstituentsGC–MS analysis tentatively identified fifteen volatile and semi-volatile constituents in the 95% ethanolic leaf extract of A.angustifolia based on their mass spectral fragmentation patternsand comparison with the NIST library. The identified constituentscomprised aliphatic hydrocarbons, terpenoid derivatives, esters,and other volatile or semi-volatile metabolites. (Figure S4 andTable S4). Among them (1S,15S)-bicyclohexadecan-2-one, (2R)-2-[(1R)-4-methylcyclohex-3-en-1-yl] propan-1-ol, 5-methylhept-3-yne, and dibutyl benzene-1,2-dicarboxylate—were found to bedominant (Figure 6). These compounds have been associatedwith diverse pharmacological activities, including antioxidantand anti-inflammatory properties, suggesting a multifactorialmechanism of action for the extract. These compounds areassociated with diverse pharmacological activities, includingantioxidant and anti-inflammatory effects. Notably, (1S,15S)-bicyclohexadecan-2-one, a hydrophobic bioactive molecule, mayplay a complementary role by interacting with non-polar regionsof biological targets, thereby enhancing membrane permeabilityand ligand stability [22]. The coexistence of volatile (GC–MSidentified) and non-volatile (HPLC identified) phytoconstituentsprovides a comprehensive biochemical basis for the biologicalactivities of the extract.2.6 FT-IR AnalysisFT-IR analysis was additionally performed to provide comple-mentary information on the major functional groups present inthe ethanolic leaf extract of A. angustifolia (Figure S5). Sevencharacteristic absorption bands were observed at 847, 1037, 1177,5 of 14ive Commons LicenseFIGURE 6 GC–MS–based identification and structuralcharacterization of bioactive constituents present in the ethanolicextract of A. angustifolia. A total of 15 compounds were identified,among which four major bioactive compounds are highlighted: (a)(1S,15S)-bicyclohexadecan-2-one, (b) (2R)-2-[(1R)-4-methylcyclohex-3-en-1-yl] propan-1-ol, (c) dibutyl benzene-1,2-dicarboxylate, and (d)5-methylhept-3-yne, demonstrating the chemical complexity andpotential pharmacological relevance of the extract.1aCaaTffcii2TaaataAeri(iTsRabSko6 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creat437, 1557, 2328, and 2917 cm−1. These bands were tentativelyssociated with aromatic C–H out-of-plane bending (847 cm−1),–O stretching (1037 and 1177 cm−1), C–H bending (1437 cm−1),romatic C═C stretching (1557 cm−1), atmospheric C═O relatedbsorption (2328 cm−1), and aliphatic C–H stretching (2917 cm−1).he FT-IR spectrum therefore provides complementary evidenceor the presence of diverse oxygenated, aromatic, and aliphaticunctional groups in the extract. However, the FT-IR findings areonsidered supportive evidence of the functional groups presentn the extract and does not by themselves, confirm the identity ofndividual phytochemical compounds..7 In Silico Molecular Docking Studyo elucidate the molecular basis of the observed anti-allergicctivity, molecular docking studies were performed against keyllergic targets: Histamine H1 receptor, Interleukin-4 (IL-4),nd Phospholipase A2 (PLA2). The docking performance ofhe polyphenolic compounds was compared with the standardntihistamine drug cetirizine (Table 1).mong the HPLC-identified compounds, (-) epicatechinxhibited the strongest binding affinity toward the Histamine H1eceptor, with a docking score of −8.8 kcal/mol (Figure 7), whichs significantly better than that of cetirizine (−7.1 kcal/mol)Figure S6). This compound formed multiple stabilizingnteractions with key amino acid residues, including TRP158,YR431, PHE432, TYR458, ASP107, and TYR108, indicatingtrong receptor binding and potential antagonistic activity.utin hydrate (−7.9 kcal/mol) and myricetin (−7.8 kcal/mol)lso demonstrated notable binding affinities, forming hydrogenonds and electrostatic interactions with residues, such asER128, GLU410, ASN63, and ARG409. These residues arenown to be crucial for ligand binding within the active pocketf the receptor. In contrast, catechol showed comparativelyof 14weaker binding (−5.8 kcal/mol), suggesting a contribution toantihistaminic activity. The stronger binding interactions ofphenolic compared to cetirizine suggest that these compoundsmay act as natural H1 receptor antagonists, potentially inhibitinghistamine-mediated allergic responses [23]. The superiorbinding affinity compared to cetirizine highlights its potentialto modulate cytokine-mediated immune responses, which maylead to IL-4-driven IgE production.Docking analysis against Interleukin-4 revealed that rutinhydrate exhibited the highest binding affinity (−9.0 kcal/mol)(Figure 8), among the identified polyphenols and cetirizine(−7.4 kcal/mol). The interaction profile included multiple hydro-gen bonds with residues such as ASP66, GLU45, LYS84, TYR56,and ARG88, indicating strong stabilization within the bindingsite. Similarly, (-) epicatechin (−7.5 kcal/mol), rosmarinic acid(−7.9 kcal/mol), and myricetin (−7.9 kcal/mol) also showedfavorable interactions with key residues involved in cytokinesignaling (Figures S7–S10). These interactions suggest a potentialinhibitory effect on IL-4-mediated responses, which play a centralrole in IgE allergic responses [24].A similar binding pattern was observed in the docking analy-sis with Phospholipase A2. Rutin hydrate again demonstratedthe highest binding affinity (−9.0 kcal/mol) than cetirizine(−7.4 kcal/mol), followed by rosmarinic acid, myricetin, andkaempferol, all of which showed stronger binding. The ligandsinteracted with critical residues, such as ASP66, GLU45, LYS84,TYR56, and ARG88, which are associated with enzymatic activityand inflammatory mediator release. These findings suggest thatthe phytoconstituents may inhibit PLA2 activity, thereby reduc-ing the production of pro-inflammatory lipid mediators involvedin allergic responses [25].Notably, the GC–MS-identified bioactive phytocompounds(1S,15S)-bicyclohexadecan-2-one also demonstrated appreciablebinding interactions with the selected targets (Figure 9).Other potential compounds include (2R)-2-[(1R)-4-methylcyclohex-3-en-1-yl] propan-1-ol), 5-methylhept-3-yne,and dibutyl benzene-1,2-dicarboxylate also show the potentialbinding affinity (Table S5). Although its binding affinity wascomparatively moderate relative to polyphenols, its hydrophobicscaffold enabled favorable interactions within non-polarregions of the receptor binding pockets (Figures S11–S13). Suchhydrophobic interactions are crucial for ligand stabilizationand membrane permeability, suggesting that this compoundmay complement the activity of polar compounds by enhancingoverall binding diversity and pharmacokinetic behavior.2.8 In Silico Drug-Likeness andPharmacokinetic (ADME) AnalysisThe drug-likeness of the selected compounds was evaluatedusing Lipinski’s rule of five (RO5), which is commonly applied toestimate the oral drug-development potential of small molecules(Table 2). According to this guideline, compounds generallyshow favorable drug-like properties when their molecular weightis below 500 Da, logP is ≤5, hydrogen-bond acceptors (HBA) are≤10, and hydrogen-bond donors (HBD) are ≤5, with no morethan one criterion being exceeded. Based on these parameters,Chemistry & Biodiversity, 2026ive Commons LicenseTABLE 1 Interacting amino acid residues of the identified bioactive compounds by HPLC and standard drug (cetirizine) with key antiallergictarget proteins— Histamine H1 receptor (PDB ID: 4RZE), Interleukin-4 (PDB ID: 3BPN), and Phospholipase A2 (PDB ID: 1DB4) —As determined frommolecular docking studies.LigandsHistamine H1 Receptor (3RZE)Docking score Interacted amino acidCatechol −5.8 TRP428, PHE432, SER111, ILE115, ASN198, THR112(-) Epicatechin −8.8 TRP158, ALA195, TYR431, PHE432, TYR 458, ILE454,ASP107, TYR108Rutin hydrate −7.9 SER128, GLU410, ASN63, ARG125, ASN472, THR60, LYS57,ARG409Rosmarinic acid −7.1 ARG409, LYS412, ASN472, GLU410, SER128, THR60,ALA413Myricetin −7.8 GLU447, ASN443, ARG176, LYS442, ILE438, ASP186Kaempferol −7.1 ASN63, ARG125, ALA413, ARG409, ARG139, THR60, LYS 57Cetirizine −7.1 ARG409, LYS412, ARG139, SER128, GLN131, ARG127,ALA413Interleukin 4 (PDB ID: 3BPN)Catechol −5 ILE C176, PHE C183(-) Epicatechin −7.5 GLN A106, GLU A103, LEU A27, ASN C105, THR C104, LYSC109, ARG A64, THR A28, VAL A29Rutin hydrate −9 ASP B66, GLU B45, LEU B39, LYS A84, TYR A56, ARG A88,ASP A87, ARG A81Rosmarinic acid −7.9 ASP B67, ASP B66, PHE B41, ARG A88, TYR A56, LYS A84Myricetin −7.9 TYR B74, ASP B67, ASP B66, LYS A84, SER B44Kaempferol −7.9 HIS B131, LEU C319, GLN C278, ASP C324, LYS C318, GLUA19, LYS A12Cetirizine −7.4 LEU C319, LYS A12, THR A13, ASP B125, ARG A85, LYS C318Phospholipase A2 (PDB ID: 1DB4)Catechol −7.5 ILE C176, PHE C183(-) Epicatechin −7.5 GLN A106, GLU A103, LEU A27, ASN C105, THR C104, LYSC109, ARG A64, THR A28, VAL A29Rutin hydrate −7.9 ASP B66, GLU B45, LEU B39, LYS A84, TYR A56, ARG A88,ASP A87, ARG A81Rosmarinic acid −8.2 ASP B67, ASP B66, PHE B41, ARG A88, TYR A56, LYS A84Myricetin −4.6 TYR B74, ASP B67, ASP B66, LYS A84, SER B44Kaempferol −7.8 HIS B131, LEU C319, GLN C278, ASP C324, LYS C318, GLUA19, LYS A12Cetirizine −7.3 LEU C319, LYS A12, THR A13, ASP B125, ARG A85, LYS C318amdbfgTcpfmoC 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creall examined compounds satisfied the RO5 criteria. Thisinor deviation may still be acceptable because several naturallyerived therapeutic compounds possess relatively high hydrogen-ond donor counts. The predicted pharmacokinetic profilesurther indicated that three of the four compounds exhibited highastrointestinal (GI) absorption and favorable molar refractivity.heir oral drug-likeness was additionally assessed using Veber’sriteria, which consider ≤10 rotatable bonds and a topologicalolar surface area (TPSA) ≤140 Å2 as favorable characteristicsor oral bioavailability. Rotatable bonds provide an indication ofolecular flexibility, whereas TPSA is associated with the abilityf a compound to interact with and cross biological membraneshemistry & Biodiversity, 2026t[26]. Rosmarinic acid showed a slightly higher TPSA than therecommended range, which may account for its comparativelylower predicted GI absorption. The other three compounds metthe Veber criteria, suggesting favorable physicochemical andpharmacokinetic characteristics and supporting their potentialfor further investigation as drug candidates.2.9 Molecular Dynamics (MD) SimulationMolecular dynamics (MD) simulations were performed to furtherassess the stability of the predicted (–) epicatechin–Histamine7 of 14ive Commons LicenseFIGURE 7 In silico molecular docking interactions of (-) epicatechin (PubChem ID: 107957) with key receptor proteins associated with antiallergicactivity. The interactions of (–)-epicatechin with (a) Histamine H1 receptor (PDB ID: 4RZE), (b) Interleukin-4 (PDB ID: 3BPN), and (c) Phospholipase A2(PDB ID: 1DB4) are presented. The upper panel depicts the three-dimensional (3D) docking conformations of the ligand–protein complexes, whereas thelower panel presents the corresponding two-dimensional (2D) interaction diagrams. Interaction types are color-coded as follows: Green, conventionalhydrogen bonds; light green, carbon–hydrogen bonds; red, unfavorable donor–donor or acceptor–acceptor interactions; pink, π–π T-shaped interactions;orange, π–cation interactions, and light pink, π–alkyl interactions.FIGURE 8 In silico molecular docking interactions of Rutin hydrate (PubChem ID: 107957) with key receptor proteins associated with antiallergicactivity. The figure illustrates the binding interactions of Rutin hydrate with (a) Histamine H1 (PDB ID: 4RZE), (b) Interleukin 4 (PDB ID: 3BPN), and(c) Phospholipase A2 (PDB ID: 1DB4). The upper panel presents the three-dimensional (3D) ligand–protein docking complexes, whereas the lowerpanel shows the corresponding two-dimensional (2D) interaction maps. Color codes represent the interaction types: Green, conventional hydrogenbond; light green, carbon-hydrogen bond; red, unfavorable acceptor/donor; pink, π-π T-shaped interaction; orange: π-cation interaction; and light pink,π-alkyl interaction.Hts(8 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable C1 receptor complex, using the cetirizine–Histamine H1 recep-or complex as a reference. The dynamic behavior of bothystems was assessed using RMSD, RMSF, radius of gyrationRg), and solvent-accessible surface area (SASA). As shown inof 14Figure 10a, both complexes exhibited an initial adjustment fol-lowed by fluctuations around relatively stable RMSD values. The(–) epicatechin–Histamine H1 complex maintained an averageRMSD of approximately 3.4 Å, compared with 3.7 Å for theChemistry & Biodiversity, 2026reative Commons LicenseFIGURE 9 In silico molecular docking interactions of (1S,15S)-bicyclohexadecan-2-one (PubChem ID: 107957) with key receptor proteins associatedwith antiallergic activity. The interactions of (1S,15S)-bicyclohexadecan-2-one with (a) Histamine H1 receptor (PDB ID: 4RZE), (b) Interleukin-4 (PDBID: 3BPN), and (c) Phospholipase A2 (PDB ID: 1DB4) are presented. The upper panel depicts the three-dimensional (3D) docking conformations ofthe ligand–protein complexes, whereas the lower panel presents the corresponding two-dimensional (2D) interaction diagrams. Interaction types arecolor-coded as follows: Green, conventional hydrogen bonds; light green, carbon–hydrogen bonds; red, unfavorable donor–donor or acceptor–acceptorinteractions; pink, π–π T-shaped interactions; orange, π–cation interactions, and light pink, π–alkyl interactions.TABLE 2 ADME analysis of selected compounds.Compounds GI absorption MR Lipinski criteria Violation Veber criteriaRange/limitM. Weight HBA HBD Log P RB TPSA40-130 ≤500 ≤10 ≤5 ≤5 ≤1 ≤10 ≤140 Å2Catechol High 79.03 110.12 7 6 0.53 1 1 112.61(-) Epicatechin High 87.30 290.27 7 5 0.99 0Rutin hydrate High 68.03 610.52 7 5 1.22 0 1 131.36Rosmarinic acid Low 91.40 360.31 8 5 1.52 0 7 144.52Myricetin High 72.06 318.23 6 5 0.77 0 1 121.3Kaempferol High 59.33 286.23 7 6 0.91 0 5 127.5ciobd)fmmorit(strwC 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Createtirizine–Histamine H1 complex. The absence of a continuousncrease in RMSD during the simulation suggests that theverall receptor structure remained stable in the presence ofoth ligands. The RMSF profile (Figure 10b) showed differentegrees of flexibility among individual receptor residues. The (–epicatechin–Histamine H1 complex displayed slightly greaterluctuations than the cetirizine complex, with most residue move-ents occurring within approximately 1–5.5 Å. These variationsay reflect local flexibility of the receptor rather than disruptionf the ligand-bound structure [27]. The Rg values (Figure 10c)emained within a relatively narrow range for both complexes,ndicating that the receptor retained its overall compact struc-ure throughout the simulation. Similarly, the SASA profileFigure 10d) showed no substantial or progressive change inolvent exposure, further suggesting that the global structure ofhe receptor was maintained. The RMSD, RMSF, Rg, and SASAesults indicate that (–) epicatechin remained stably associatedith the Histamine H1 receptor during the simulation. Its overallhemistry & Biodiversity, 2026dynamic profile was comparable to that of cetirizine, supportingthe docking prediction and suggesting that the interaction of(–) epicatechin with the H1 receptor is structurally favorable.These computational findings provide additional evidence for thepotential role of (–) epicatechin as a bioactive compound targetingthe Histamine H1 receptor.These findings strongly support the experimental in vivo resultsand provide mechanistic insight into the anti-allergic activity ofA. angustifolia. These findings highlight the therapeutic potentialof its bioactive constituents as promising candidates for thedevelopment of safer, plant-derived anti-allergic agents.3 ConclusionThe present study provides experimental and computationalevidence supporting the anti-allergic potential of the ethanolic9 of 14ive Commons LicenseFIGURE 10 (a) Root-mean-square deviation (RMSD) of the Cα atoms of the Histamine H1 receptor in complexes with (–) epicatechin and cetirizineat 310 K. (b) Root-mean-square fluctuation (RMSF) profiles of the Histamine H1 receptor residues in the (–) epicatechin and cetirizine complexes. (c)Radius of gyration (Rg) profiles of the (–) epicatechin–Histamine H1 and cetirizine–Histamine H1 complexes during the MD simulations at 310 K. (d)Solvent-accessible surface area (SASA) profiles of the (–) epicatechin–Histamine H1 and cetirizine– Histamine H1 complexes.lerrsvpgmsaaedhescuthpp44LT1 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Createaf extract of A. angustifolia. The extract showed beneficialffects in the TDI-induced allergic mouse model, as reflected byeductions in allergic symptoms, WBC counts, BAL inflammatoryesponses, and serum IgE levels. HPLC–DAD and GC–MS analy-es revealed several phytoconstituents, while FT-IR analysis pro-ided complementary evidence for the major functional groupsresent in the extract. The identified compounds also showedenerally favorable drug-likeness and ADME profiles. In silicoolecular docking further supported these findings, revealingtrong binding affinities of major phytoconstituents toward keyllergic targets, including Histamine H1 receptor, Interleukin-4,nd Phospholipase A2. Notably, (-) epicatechin and rutin hydratexhibited superior binding interactions compared to the standardrug cetirizine, supporting their potential role as natural anti-istamine agents. MD simulations further indicated that the (–)picatechin–Histamine H1 receptor complex maintained stabletructural behavior comparable to the reference cetirizine–H1omplex, supporting the persistence of the predicted interactionnder simulated physiological conditions. The findings indicatehat A. angustifolia exerts its anti-allergic effects by suppressingistamine. These results suggest that A. angustifolia extract hasotential for traditional use for allergic disorders and serves as aromising source for isolation of bioactive compounds.Experimental Section.1 Plant Sample and Extraction Processeaves of A. angustifolia were collected from the Chittagong Hillracts region of Bangladesh. The plant material was taxonomi-0 of 14cally identified by experts at the Bangladesh National Herbarium,Mirpur, Dhaka-1216, where a voucher specimen (DACB 65236)was deposited for future reference. The leaves were thoroughlycleaned, shade-dried, and pulverized into coarse powder. Thepowdered leaves were extracted with ethanol using a macerationtechnique at room temperature (sample-to-solvent ratio 1:5, w/v)in a sealed jar. The crude extract was filtered, and the solventwas removed under reduced pressure at 40 ◦C using a rotaryevaporator (Büchi Rotavapor, Switzerland). Ethanol was selectedbecause of its relatively low toxicity, suitability for biologicalstudies, and ability to recover a broad range of moderately polarphytochemicals. Although a single solvent cannot extract allphytochemical constituents present in the plant material, theethanolic extract provides a broad range of bioactive compoundssuitable for evaluating the anti-allergic potential of A. angusti-folia. The dried extract was stored at 2 ◦C in airtight vials untilfurther phytochemical and pharmacological investigations.4.2 Chemicals and ReagentsAll experiments were conducted using analytical-grade reagents.Toluene-2,4-diisocyanate (TDI) was obtained from Wako Chem-icals (Tokyo, Japan). The standard antihistamine drug ceti-rizine was procured from Square Pharmaceuticals Ltd. (Dhaka,Bangladesh). Commercial diagnostic kits for estimating biochem-ical parameters, including alkaline phosphatase (ALP), bilirubin,creatinine, and urea, were obtained from certified suppliers inGermany. All solvents, such as methanol, ethanol, and diethylether, were of HPLC grade and purchased from Loba Chemie(India).Chemistry & Biodiversity, 2026ive Commons License4Sfe1daDAC0n4PApiap4a4T(4wc(31ddap1a4SprgwdTocaatcpC 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creat.3 Experimental Animalswiss albino mice (5–6 weeks old, body weight 22–28 g) were usedor in vivo experiments. Animals were housed under controllednvironmental conditions (24–27 ◦C, relative humidity 55 ± 5%,2 h light/dark cycle) with ad libitum access to standard pelletiet and water. Before the experiments, the animals (mice) werecclimatized for one week in the Pharmacology Laboratory,iscipline of Pharmacy, Khulna University, 9208, Bangladesh.ll experimental protocols were approved by the Animal Ethicsommittee of Khulna University (Approval No.: KUAEC-2025-3-04) and conducted in accordance with institutional andational guidelines for the care and use of laboratory animals..4 Phytochemical Screeningreliminary phytochemical analysis of the ethanolic extract of. angustifolia was performed following standard qualitativerotocols [28]. The presence of major secondary metabolites,ncluding alkaloids, flavonoids, phenolics, saponins, tannins,nd terpenoids, was investigated. A detailed description of testrocedures is provided in the Supporting Information..5 Safety Evaluation of the Actinodaphnengustifolia Extract.5.1 Acute Toxicity Testhe acute oral toxicity of the ethanolic extract of A. angustifoliaEEAA) was evaluated in accordance with OECD Guideline25, with minor modifications [29]. Healthy Swiss albino miceere randomly divided into six groups (n = 6 per group). Theontrol (Group I) received distilled water, whereas the test groupsEEAA-250, EEAA-500, EEAA-1000, EEAA-2000, and EEAA-000) were administered EEAA at graded doses of 250, 500,000, 2000, and 3000 mg/kg body weight via oral gavage. Beforeosing, all animals were individually weighed to ensure accurateose calculations. Following administration by oral gavage, thenimals were carefully monitored for the first 30 min, observederiodically over the subsequent 24 h, and assessed daily for4 consecutive days to detect any behavioral changes, physicalbnormalities, or signs of mortality..5.2 Sub-Acute Toxicity Testub-acute toxicity was assessed following a previously reportedrotocol with minor modifications [30]. 12 Swiss albino mice wereandomly divided into two groups: Group I (control) and a testroup (EEAA-500) (n = 6 per group). The control group receivedater, while the test group was administered the extract orally at aose of 500 mg/kg body weight once daily for 14 consecutive days.hroughout the experimental period, all animals were closelybserved for signs of behavioral abnormalities, physiologicalhanges, and mortality. At the end of the treatment period, thenimals’ body weight was measured. The animals were thennesthetized, and blood samples were collected in heparinizedubes from the cervical vein. The collected blood samples wereentrifuged at 3000 rpm for 10 min to obtain serum. Biochemicalarameters, including hepatic function markers (SGPT, SGOT,hemistry & Biodiversity, 2026and ALP) and renal function markers (creatinine, urea, andbilirubin), were analyzed in triplicate, and the mean values wereused for statistical analysis.4.6 Evaluation of Anti-Allergic Activity4.6.1 TDI Sensitization and Provocation ProtocolAllergic responses were induced using toluene-2,4-diisocyanate(TDI) following a previously established method with minormodifications to optimize experimental conditions [31, 32]. A totalof thirty mice were randomly divided into five groups (n = 6per group): Group I (control), Group II (TDI control), Group III(standard, cetirizine 20 mg/kg), and Groups IV and V (EEAA-treated groups at 250 and 500 mg/kg, respectively). The extractand standard drug were administered orally once daily for threeweeks. Sensitization was carried out by applying 10 μL of 5% TDIsolution (in ethyl acetate) to the nasal vestibule of mice (GroupsII–V) 1 h after treatment, for five consecutive days. Following atwo day interval, the sensitization cycle was repeated for anotherfive days. After a nine days rest period, final provocation wasperformed using the same TDI dose. The negative control groupreceived ethyl acetate alone following the same schedule.4.6.2 Assessment of Allergy-Like SymptomsAllergic symptoms were evaluated immediately after TDI provo-cation and observed for 15 min. Parameters included sneezingfrequency, nasal scratching (rubbing), and the nasal score. Symp-toms were scored using a standardized scale ranging from 0 to 3(0 = no symptoms, 3 = severe symptoms) [33].4.6.3 White Blood Cell (WBC) AnalysisOn day 21 after the final provocation, allergy-like symptoms wererecorded. After 24 h, mice were anesthetized to reduce the pain,and blood samples were collected from the cervical vein. Bloodsamples were diluted (1:10) with 1% acetic acid, and total anddifferential WBC counts were determined using an automatedhematology analyzer (DS-500i, Nihon Kohden, Japan). Eachsample was analyzed in triplicate, and the mean values were usedfor statistical evaluation.4.6.4 Brochoalveolar Lavage Fluid (BAL) AnalysisBAL fluid was collected by intratracheal cannulation with sterilesaline (0.9%), following standard protocol [34]. BAL fluid sampleswere centrifuged at 3000 rpm for 10 min, and the resultingcell pellets were used to prepare Leishman-stained smears fordifferential leukocyte assessment. Each sample was analyzed intriplicate, and the average values were used for statistical analysis.4.7 HPLC Standardization and Quantification ofPolyphenolsPolyphenolic compounds in the ethanolic extract of A. angusti-folia were analyzed by high-performance liquid chromatography11 of 14ive Commons License(eas(Tag26faqsaftc4GpC0C1hoctwh4obI[4Fuclwrwag44PM1 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicableHPLC) using a Shimadzu LC-20A system (Shimadzu, Japan)quipped with dual solvent delivery pumps, an autosampler,nd a photodiode array detector, all controlled by LabSolutionoftware. Separation was performed on a Luna C18 column250 × 4.6 mm, 5 μm; Phenomenex, USA) maintained at 33 ◦C.he mobile phase consisted of solvent A (1% acetic acid incetonitrile) and solvent B (1% acetic acid in water), withradient elution programmed as follows: 0–20 min, 5%–25% A;0–30 min, 25%–40% A; 30–35 min, 40%–60% A; 35–40 min,0%–30% A; 40–45 min, 30%–5% A; and 45–50 min, 5% A. Thelow rate was 0.5 mL/min, the injection volume was 20 μL,nd detection was carried out at 270 nm. Polyphenols wereuantified against calibration curves prepared from authentictandards, and the method was validated for linearity, precision,nd reproducibility. The identity of detected compounds was con-irmed by comparing their retention times and UV spectra withhose of reference standards, and by quantifying major bioactiveonstituents [35]..8 GC–MS Analysisas chromatography–mass spectrometry (GC–MS) analysis waserformed using a Clarus 690 gas chromatograph (PerkinElmer,A, USA) equipped with an Elite-35 column (30 m × 0.25 mm,.25 μm film thickness; PerkinElmer, CA, USA) and coupled to alarus SQ 8C mass spectrophotometer (PerkinElmer, CA, USA).μL sample was injected in splitless mode, with high-purityelium (99.999%) serving as the carrier gas at a constant flow ratef 1 mL/min for a total run time of 40 min. The ionization wasarried out in electron ionization (EI) mode at 70 eV. The inletemperature was maintained at 280 ◦C, while the column ovenas programmed as follows: initial temperature 60 ◦C (0 minold), increased at 5 ◦C/min to 240 ◦C, and maintained formin. The mass spectral data were acquired over an m/z rangef 50–600 with a scan time of 1 s. Compounds were identifiedy comparing their fragmentation patterns with the Nationalnstitute of Standards and Technology (NIST) library database36]..9 FT-IR Analysisourier-transform infrared (FT-IR) spectroscopy was performedsing an ATR-FTIR spectrometer (Nexus 670, Tokyo, Japan) toharacterize the major functional groups present in the ethanoliceaf extract of A. angustifolia. A small amount of the dried extractas directly placed on the ATR crystal, and the spectrum wasecorded over the appropriate mid-infrared range. The spectrumas collected after background correction, and the characteristicbsorption bands were analyzed to assign the major functionalroups present in the extract..10 In Silico Molecular Docking Study.10.1 Ligand Preparationhytochemical constituents identified through HPLC and GC–S, along with the reference drug cetirizine, were retrieved2 of 14 Creafrom the PubChem database [37]. The compounds includedcatechol (CID: 289), (−)-epicatechin (CID: 72276), rutinhydrate (CID: 45479757), rosmarinic acid (CID: 5281792),myricetin (CID: 5281672), kaempferol (CID: 5280863),(1S,15S)-bicyclo[13.1.0]hexadecan-2-one (CID: 13760785),(2R)-2-[(1R)-4-methylcyclohex-3-en-1-yl]propan-1-ol (CID:12571838), 5-methylhept-3-yne (CID: 521962), dibutyl benzene-1,2-dicarboxylate (CID: 3026), and cetirizine (CID: 2678). Thechemical structures were subjected to energy minimizationand geometry optimization using Avogadro (v1.2.0) with theUniversal Force Field (UFF). The optimized ligands weresubsequently saved in Protein Data Bank (.pdb) format forfurther docking analysis.4.10.2 Protein PreparationThe three-dimensional crystal structures of the allergy-relatedtarget proteins- Histamine H1 receptor (PDB ID: 4RZE),Interleukin-4 (PDB ID: 3BPN), and Phospholipase A2 (PDB ID:1DB4) were obtained from the Protein Data Bank [26]. Proteinstructures were prepared by removing co-crystallized ligands,water molecules, and other non-essential components usingPyMOL v2.0 (Schrödinger, LLC). Energy minimization was thenperformed using Swiss-PdbViewer (v4.1) to ensure structuralstability before docking.4.10.3 Molecular Docking and Interaction AnalysisMolecular docking simulations were carried out using AutoDockVina implemented in PyRx 0.8 [27]. Both ligands and receptorswere imported into PyRx and defined appropriately as “ligand”or “macromolecule.” Grid boxes were defined to encompass theactive binding sites of the selected target proteins. The gridbox dimensions were: Histamine H1 receptor (PDB ID: 3RZE),69.29 × 59.35 × 94.71 Å; for Interleukin-4 (PDB ID: 3BPN),97.63 × 64.98 × 121.08 Å; and for Phospholipase A2 (PDB ID:1DB4), 27.23× 29.52× 31.71 Å. The default AutoDock Vina settingswere applied for all docking simulations. The best dockingposes were selected based on binding affinity (lowest bindingenergy scores). The docked ligand–receptor complexes werefurther visualized and analyzed in Discovery Studio Visualizerv4.5 (BIOVIA, Dassault Systèmes) to identify hydrogen bonding,hydrophobic, and electrostatic interactions with amino acidresidues.4.10.4 Drug-Likeness and ADME AnalysisDrug-likeness assessment was performed to estimate the suitabil-ity of the selected compounds as potential orally active drug can-didates. The compounds were evaluated using the SwissADMEweb server based on their physicochemical and pharmacokineticproperties, including absorption, distribution, metabolism, andexcretion (ADME). Their drug-like characteristics were furtherassessed according to Lipinski’s rule of five, which providescommonly used criteria for evaluating oral bioavailability anddrug-development potential.Chemistry & Biodiversity, 2026tive Commons License4TastefsboacocaCatufsbsp4AowfsARgcMaAsfraATBwKFTB2CTC 16121880, 2026, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.71727 by Lok Shrestha - National Institute For , Wiley Online Library on [23/09/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Crea.10.5 Molecular Dynamics (MD) Simulationhe Protein–ligand Complexes showing favorable docking inter-ctions were subsequently subjected to molecular dynamics (MD)imulations to examine their structural stability and conforma-ional behavior under conditions approximating a physiologicalnvironment. Simulations were performed using the AMBER14orce field implemented in YASARA Structure v. 24.4.10. Eachystem was placed in a cubic simulation cell extending 20 Åeyond the complex and solvated with water to achieve a densityf 0.997 G cm−3. The systems were maintained at 310 K, 1 Atm,nd pH 7.4, with 0.9% NaCl to approximate physiological ioniconditions. periodic boundary conditions were applied through-ut the simulations. long-range electrostatic interactions werealculated using the Particle-mesh Ewald (PME) method, whilen 8 Å cutoff was applied for short-range van der Waals andoulombic interactions. A 2.5 Fs integration time step was used,nd trajectory frames were recorded at regular intervals duringhe 00 ns simulation. The resulting trajectories were analyzedsing root-mean-square deviation (RMSD), root-mean-squareluctuation (RMSF), radius of gyration (Rg), solvent-accessibleurface area (SASA), molecular surface area (MolSA), hydrogen-ond interactions, and secondary-structure analysis to assess thetability, flexibility, compactness, and interaction behavior of therotein–ligand complexes..11 Statistical Analysisll experimental results were expressed as mean ± standard errorf mean (SEM). Statistical analyses were performed using one-ay analysis of variance (ANOVA) followed by Dunnets’s t-testor group-wise comparisons. A p-value of < 0.05 was consideredtatistically significant.uthor Contributionsabindra Nath Acharyya: conceptualization, data curation, investi-ation, and writing – original draft. Md. Abid Muktadir Risha: datauration and software. Novi Dwi Widya Rini: formal analysis. Saritaanandhar: investigation and methodology. Sabina Shahi: formalnalysis and methodology. Biswa Nath Bhadra: validation and editing.sish Kumar Das: investigation, validation, review, and editing. Kat-uhiko Ariga: review and editing. Shrabanti Dev: conceptualization,unding acquisition, and review and editing. Lok Kumar Shrestha:eview and editing. The authors declare that the works presented in thisrticle are original.cknowledgmentshe authors are grateful to RIC and the authority of Khulna University,angladesh, for providing funding for this research work. The authorsould like to express our cordial thanks to the Pharmacy Discipline athulna University for providing laboratory facilities.undinghe Research and Innovation Center, Khulna University, Khulna-9208,angladesh, supported the work (Grant No: KU/GACELL04(8)/2000-95).onflicts of Interesthe authors declare no conflicts of interest.hemistry & Biodiversity, 2026tData Availability StatementThe data that support the findings of this study are available from thecorresponding author upon reasonable request.References1. R. 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