Piperguineense,Tetrapleuratetraptera,Xylopiaaethiopica,Alliumsativum, Ocimumgratissimum and Zingiberofficinale were purchased from Swali market, Yenagoa, Bayelsa State. All the spices were formulated into tea using the method of [18]. Tea was formulated from the plant powder by thoroughly mixing the powder spices on ratio of 1:1. One gram each of the powder spices was mixed together. The mixed spice was thereafter used to formulate a tea by method of decoction as reported by [18].

Gas chromatography-mass spectrometry with flame ionization detector was utilized to analyze and identify volatile bioactive compounds present in the formulated tea.

GC-MS analysis of the “formulated tea” was performed using Shimadzu GC-MS—QP2010 PLUS as previously described by [18].

The formulated tea extract was investigated for metabolites (Piperine, chavicine, flavonoids, alkaloids, tannins, saponins, terpenoids, essential oils phenolic compounds, steroids, triterpenoids Xylopic acid, kaurenoic acid, phenolics, Allicin, ajoene, alliin, diallyl sulfide, diallyl disulfide, s-allyl cysteine, flavonoids, saponins eugenol, thymol, Gingerol, shogaol, paradol, zingerone) according to the established procedures as reported by [19].

This was done using the online programme ADMET lab 3.0 and SWISSADME [20]; the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of the ligands employed in this study were predicted. The different ADMET properties of the ligands were predicted using the different canonical strings or Simplified Molecular-Input Line-Entry System (SMILES) strings of the different ligands retrieved from the PubChem web platform (https://www.ncbi.Nlm.nih.gov/pccompound) in their 3D conformation. All the relevant parameters, including Lipinski’s rule of five and the Ghose parameters were recorded. Using the SWISS target prediction tool, the target of the different ligands was determined [21].

The target protein for this study (TLR-4) was derived from the Research Collaborator for Structural Bioinformatics Protein Data Bank (RCSB-PDB). The RCSB-PDB is a comprehensive web resource that provides the three-dimensional structural data of biological macromolecules, including proteins and nucleic acids [22]. The protein-ligand interaction in this study was determined through the use of PyRx while Discovery studio (version 3.0) was used for visualization of the interaction. The proteins were prepared for interaction using the Chimera software by the removal of water molecule, heteroatoms and ligands. After the preparation, the proteins were docked against the various ligands using PyRx. The chemical compounds obtained from the results of GCMS analysis were subjected to molecular docking with some anti-viral protein targets thalidomide respectively were obtained from the PubChem database.

Swiss target database was used to identify potential protein target related to Human metapneumovirus for ligands found in the formulated tea. PubChem was used to obtain the structure of the ligand especially the SMILE. Genecard database was used to obtain disease target with Uniprot ID while Venny web saver was used to predict the gene interactions between pneumonia-like related genes and genes that the ligands bind to. Strings were used to obtain network of genes interactions or the gene hub. Network from strings was exported to cytoscape 3.0 to obtain gene hub. Cytoscape app 3.0 was used for gene-gene interact, gene-protein interaction, protein-protein interaction network. Shinny GO was used for gene ontology to analyze the cellular component, biological component or molecular component interaction.

Compound profiling was used to assess the pharmacological properties of formulated chemical constituents, particularly their ability to modulate anti-viral targets. Phytochemical analysis was used to identify and quantify the bioactive compounds in the formulated tea. Target interaction study was utilized for molecular docking simulations to evaluate how the compounds interacted with specific viral proteins which helped to predict binding affinities and potential inhibitory effects.

GC-MS analysis revealed 11 compounds in the formulated tea as shown in Table 1.

Table 1. Physiochemical properties of compounds identified.

From Table 1 above, CID 6989: Tyramine or 4-hydroxyphenethylamine, a naturally occurring monoamine compound derived from tyrosine. It’s a small, mildly lipophilic compound with one H-bond donor/acceptor; potentially CNS-active. TPSA favours membrane permeability. CID 17516: Dextroamphetamine, a psychostimulant used in attention deficit hyperactivity disorder (ADHD) and narcolepsy. MW: 207.27, 4 rotatable bonds, typical for CNS stimulants. TPSA of 38.33 suggests good oral and blood-brain barrier (BBB) permeability. This compound acts as a central nervous system stimulant affecting dopamine and norepinephrine pathways.

CID 119838: glucaric acid, an oxidation product of glucose, involved in detoxification. CID 345716: Mannitol, a sugar alcohol used as a diuretic and sweetener. TPSA of 99.38 indicates high polarity and low membrane permeability. CID 519764: β-Caryophyllene, a sesquiterpene found in essential oils. A bicyclic sesquiterpene known for cannabinoid receptor (CB2) agonism, a good candidate for anti-inflammatory or neuroprotective roles. CID586455: Vanillin acetate or ethyl vanillin, a derivative of vanillin with reported antioxidant and antimicrobial activity. CID 5281517: Germacrene D or Humulene is a highly lipophilic compound with reported insecticidal and anti-inflammatory activity. CID 638072: Squalene, a triterpene and precursor in sterol biosynthesis with antioxidant properties in dermatological and cancer applications. CID 5280794: stigmasterol, a highly lipophilic and vital sterol in cell membranes, a precursor to steroid hormones. CID 5281519: Cyclodecadiene is an isomer of β-caryophyllene with physicochemical properties identical to β-caryophyllene; used in antimicrobial, anti-inflammatory research. CID 5395771: Acylovir, reportedly an antiviral compound used to treat herpes simplex infections, mimics nucleosides to inhibit viral DNA polymerase.

Most drug-like compounds fall within 100 - 500 g/mol of molecular weight (Lipinski’s Rule of Five). All listed compounds fall within this range, except compound 8 and compound 9, which are larger (potentially lower oral bioavailability). Fraction of CsP₃ indicates saturation level and 3D complexity. Higher CsP₃ often correlates with better solubility and metabolic stability. Compounds like compound 4 (1.00) and compound 9 (0.86) are highly saturated and likely more metabolically stable. Rotatable Bonds affects molecular flexibility. More than 10 may reduce bioavailability due to entropy loss on binding. Compound 8 has 15 rotatable bonds possibly poor oral bioavailability. Hydrogen Bond Acceptors (HBA) and Donors (HBD): excessive HBA (>10) or HBD (>5) can limit membrane permeability. Compound 4 with 6 HBAs and 4 HBDs may have low permeability without carrier mediation. Molar Reactivity (MR) relates to electronic polarizability; compounds with higher MR (e.g., compound 8 and compound 9) may have increased binding interactions but possibly reduced solubility, high MR values (>100) as in #8 and #9 suggest strong van der Waals interactions. Topological Polar Surface Area (TPSA); TPSA less than 140 Ų is considered favorable for oral absorption; under 90 Ų for blood-brain barrier penetration. Compound 4 (TPSA = 99.38) may have poor CNS penetration. Compounds 5, 7, 8, and 10 (TPSA = 0) may be highly lipophilic and membrane permeable.

The 11 GC-MS identified compound were docked against the target protein 2Z62 (Toll-like receptor-4; TLR-4) involved in viral recognition and inflammatory response. Subsequently, the virtual properties of the selected hit compounds were studied. Following ADME-Toxicity assessments, the most reactive compounds with drug-like properties against 2Z62 (TV3 hybrid of human TLR-4) were identified to be Squalene, Stigmasterol and Cyclodecadiene with a binding infinity of 19.6, 11.2 and 5.7 respectively (Table 2).

Table 2. Docked with 2Z62.

Figures 1-3 below show the docking complexes.

Figure 1. 2Z62 docked against Squalene_M1.

Figure 2. 2Z62 docked against 1,5-Cyclodecadiene, 1,5-dimethyl-8-(1-methylethylidene)_M1.

Figure 3. 2Z62 docked against Stigmasterol_M1.

Pharmacokinetics of identified compounds:

Table 3. Pharmacokinetics of identified compounds.

The insilico pharmacokinetic analysis (Table 3) showed that compounds 1, 2, 6, and 11 show the most promising oral bioavailability and CNS activity, though potential CYP enzyme inhibition must be carefully considered to avoid drug interactions. Compounds 7 - 10, although less permeable and with low GI absorption, may be optimized for non-oral routes or used in topical or inhalational formulations. These findings offer a strong foundation for selecting lead compounds in the development of therapeutics against human metapneumovirus.

Table 4 presents insilico predictions of toxicity and systemic safety profiles for two test compounds (squalene and stigmasterol). Both compounds are predicted not to cross the BBB, reducing the risk of unintended central nervous system (CNS) side effects, they show positive hepatotoxicity predictions (0.69 probability), indicating a moderate risk of liver toxicity, while both compounds show acceptable mutagenicity and cytotoxicity profiles, their potential hepatotoxic, neurotoxic, cardiotoxic, and immunotoxic effects warrant further in vivo safety assessment.

Table 4. Toxicological assessment of lead compound as predicted by Protox III.

Table 5 summarizes the binding affinity and predicted toxicity profiles of two bioactive compounds, Squalene (PubChem CID: 638072) and Cyclostigmasterol (CID: 5280794), in relation to their therapeutic potential and safety. Squalene and stigmasterol exhibit strong target binding and acceptable general toxicity, but show notable risks for neuro-, cardio-, and immunotoxicity. While they hold promise as potential bioactive agents, particularly against viral targets like hMPV, dose optimization, structural refinement, and targeted toxicity studies are essential to minimize systemic adverse effects during drug development.

Table 5. Binding affinity and predicted Toxicity Profiles two lead Compounds.

Table 6 presents insilico predictions of lipophilicity (log P), water solubility (log S), drug-likeness, and oral bioavailability for eleven test compounds identified in the formulated tea. These parameters are fundamental to evaluating a compound’s pharmacokinetic and physicochemical profile, influencing absorption, distribution, metabolism, and excretion (ADME) characteristics. The physicochemical screening shows that most tested compounds have drug-like properties, with good balance between lipophilicity and solubility. While several compounds show moderate violations (mainly due to high log P), they retain favorable bioavailability predictions and remain promising leads for therapeutic development against human metapneumovirus. Optimization efforts may focus on reducing excessive lipophilicity while maintaining membrane permeability and target binding.

Table 6. Predicted lipophilicity (log P), water solubility (logSW) Drug likeness & Bioavailability of test compounds.

Identifying naturally occurring compounds with pharmacological relevance has become a priority in the search for new antiviral leads. The physicochemical assessment of compounds identified in this study (their molecular weight (MW), topological polar surface area (TPSA), number of hydrogen bond acceptors and donors, fraction of Sp₃ carbons (CsP₃), number of rotatable bonds, molar refractivity, logP value etc.) is essential for predicting the oral bioavailability and drug-likeness of these compounds. These characteristics determine how a compound behaves in biological environments, impacting absorption, solubility, metabolic stability, and membrane permeability [23][24]. Compounds such as β-caryophyllene, and squalene demonstrated high lipophilicity (log P > 4.5), low TPSA (0.00 Ų), and zero hydrogen bonding capabilities, suggesting their enhanced ability to traverse lipid membranes and potentially interfere with viral envelope integrity. Squalene showed the highest molar refractivity (143.48) and flexibility (15 rotatable bonds), supporting its interaction with hydrophobic viral protein pockets. In contrast, hydrophilic molecules like mannitol and glucaric acid had elevated TPSA values (up to 99.38 Ų) and multiple hydrogen bond donors/acceptors, suggesting limited membrane permeability but potential roles in metabolic or immunomodulatory pathways. Drug-likeness analysis confirmed that all compounds satisfied Veber’s criteria (TPSA < 140 Ų, rotatable bonds < 10) and most passed Lipinski’s Rule of Five, except for some violations in highly lipophilic molecules like stigmasterol and squalene. All eleven compounds scored positively for drug-likeness and presented bioavailability scores of 0.55 or higher, with glucaric acid achieving a superior score of 0.85, indicating strong oral potential.

The host-pathogen interface during hMPV infection activates innate immune pathways, particularly Toll-like receptor (TLR) signaling involving Myeloid differentiation factor 88 (MyD88), Toll-like receptor 4 (TLR-4), and lipopolysaccharide-binding protein (LBP). These proteins play central roles in viral recognition and inflammatory response. In this study, these were selected as target proteins for docking simulation to assess whether bioactive spice compounds could inhibit TLR-mediated signaling, potentially modulating hMPV pathogenesis. Molecular docking simulations targeting the hMPV glycoproteins revealed that squalene exhibited the most favorable binding affinity (−19.6 kcal/mol), followed by stigmasterol (−11.2 kcal/mol). These compounds demonstrated efficient binding to hydrophobic viral sites, consistent with their biochemical profiles. The binding of these compounds to the target protein 2Z62 which is notable for viral recognition and inflammatory response suggests potential mechanisms of action involving possible fusion inhibition or disruption or blocking of viral entry, which is a critical step in hMPV pathogenesis. Such findings align with prior studies showing that lipophilic phytocompounds can destabilize viral membranes or bind to viral envelope proteins [25]. β-caryophyllene and stigmasterol also exhibited significant interactions with TLR-4, providing mechanistic insight into potential immunomodulatory or antiviral actions. These interactions primarily involved hydrophobic contacts and van der Waals interactions, consistent with the lipophilic nature of the compounds.

Insilico pharmacokinetic prediction tools indicated that the majority of the compounds exhibited high gastrointestinal absorption. Compounds such as β-caryophyllene and vanillin derivatives were found to be non-substrates of P-glycoprotein and demonstrated no major CYP450 inhibition risks, suggesting low potential for pharmacokinetic drug interactions. Furthermore, the skin permeability coefficients ranged from −2.74 to −9.79 cm/s, denoting their suitability for non-invasive formulations. Lipophilic compounds like squalene and stigmasterol showed limited water solubility but retained sufficient predicted GI absorption. Glucaric acid and mannitol, while highly soluble, may require formulation adjustments to overcome permeability limitations. Toxicity screening revealed that most compounds were non-mutagenic and non-cytotoxic. However, neurotoxicity, cardiotoxicity, and immunotoxicity were predicted for squalene and stigmasterol (with probabilities > 0.85), raising caution for dose selection and necessitating further invivo toxicological evaluation. Despite these risks, both compounds shared a favorable LD50 value (1190 mg/kg), indicating moderate acute toxicity consistent with OECD Class IV classification. Additionally, vanillin derivatives and β-caryophyllene showed relatively benign toxicity profiles, coupled with high bioavailability and acceptable binding affinity, underscoring their promise as safer alternatives for antiviral development. The fraction of sp3-hybridized carbons (CsP₃), a metric of molecular complexity and 3D character, was favorable (≥0.4) for the majority of compounds, suggesting improved target specificity and reduced promiscuity. The most rigid compounds (e.g., glucaric acid with 0 rotatable bonds) may offer metabolic stability, while flexible structures like squalene may better adapt to protein pockets, enhancing binding interactions. Topological polar surface area (TPSA) values below 90 Ų, as seen in compounds like β-caryophyllene, stigmasterol, and squalene, correlate with effective passive absorption and blood-brain barrier exclusion. In contrast, higher TPSA compounds may be more suitable for systemic immune modulation.

Acyclovir, a reference antiviral compound included in the study, showed moderate physicochemical compatibility and docking scores, validating the predictive strength of the insilico approach. When compared, β-caryophyllene and vanillin derivatives showed competitive or superior pharmacokinetic and safety profiles, highlighting their potential as antiviral scaffolds. Through reverse docking and structural bioinformatics, the selected spice-derived compounds were further mapped to their putative genetic targets including viral RNA polymerase and host immune mediators. These interactions provide molecular basis for future transcriptomic or proteomic validation studies and offer insight into multi-target drug action.