Antimycobacterial Activity of Bioactive Compounds from Eucalyptus globulus against Mycobacterium tuberculosis Aspartate-semialdehyde Dehydrogenase: In silico Analysis

Antimycobacterial Activity of Bioactive Compounds from Eucalyptus globulus against Mycobacterium tuberculosis Aspartate-semialdehyde Dehydrogenase: In silico Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antimycobacterial Activity of Bioactive Compounds from Eucalyptus globulus against Mycobacterium tuberculosis Aspartate-semialdehyde Dehydrogenase: In silico Analysis Jimoh Salamah Mopelola, Memunat Alake Bankole, Nurudeen Owolabi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7337220/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Tuberculosis is the leading cause of death from a single infectious agent worldwide. Tuberculosis accounted for around 1.25 million deaths globally in 2023. The rise of Multidrug-resistant tuberculosis and the reported side effects of these available anti-TB drugs has complicated the control of the disease. This highlights the urgent need to discover and develop safer and more effective therapies for tuberculosis. Eucalyptus globulus is a tree widely distributed across tropical and subtropical regions with numerous medicinal benefits. Results The antimycobacterial potential of Eucalyptus globulus against Mycobacterium tuberculosis Aspartate-semialdehyde dehydrogenase (ASADH); an enzyme responsible for the bacterium’s growth and virulence was accessed using in silico studies. Fifteen bioactive compounds from Eucalyptus globulus were selected based on literature. Isoniazid; a first line anti-TB drug was used as our control. SwissADME online server was used to evaluate pharmacokinetic properties of the compounds. The compounds were further prepared to obtain the best docking poses using LigPrep module in the Maestro’s Schrodinger software. The compounds were docked into the protein's active site using Schrödinger's Maestro software’s Glide module and the resulting protein-ligand complexes was studied. Drug-likeness screening showed that only M-cymene violated Lipinski’s rule. Molecular docking revealed that thymol (-5.445) and sabinyl acetate (-5.097) has a higher docking score than isoniazid (-5.040), indicating a better binding affinity and biological activity against the target compared to the control (-5.040). Hydrogen bonds interaction of suggesting high strength of binding and molecular interactions that occurred between the protein-ligand complexes suggesting a strong antimycobacterial inhibitory effect. Conclusions In silico analysis from this study identifies thymol and sabinyl acetate from Eucalyptus globulus as promising candidates for further development as anti-TB treatments and show better docking with the target protein compared to isoniazid. Tuberculosis Eucalyptus globulus Antimycobacterial activity In silico drug discovery ADMET Figures Figure 1 Figure 2 BACKGROUND Tuberculosis (TB), caused by Mycobacterium tuberculosis , is the deadliest disease resulting from a single infectious agent ( 1 ) and one of the oldest diseases known to affect humans ( 2 ). Poverty significantly contributes to the persistence of tuberculosis worldwide ( 3 ). Nearly a quarter of the world's population has been infected with TB, with a 5–10% lifetime risk of developing TB disease. In 2023, approximately 1.25 million deaths from TB were reported. India, Indonesia, China, the Philippines, Pakistan, Nigeria, Bangladesh, and the Democratic Republic of the Congo accounted for more than two-thirds of global TB cases ( 4 ). Isoniazid and rifampicin (RFP) are first-line medications used to treat tuberculosis ( 5 ). Isoniazid, also known as isonicotinic acid hydrazide (INH), is a prodrug that suppresses the growth of mycobacteria ( 6 ). Although its precise mechanism of action is not fully understood, it targets fatty acid synthetase in mycobacteria, which plays a role in mycolic acid biosynthesis—essential for cell wall formation (Bereda, 2022). Rifampicin inhibits bacterial DNA-dependent RNA synthesis by targeting bacterial RNA polymerase ( 6 ). However, TB treatment becomes significantly more complex due to the emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR) strains of Mycobacterium tuberculosis ( 7 ). Resistance to at least both INH and RIF is classified as multidrug-resistant TB (MDR-TB), while additional resistance to fluoroquinolones and aminoglycosides is referred to as extensively drug-resistant TB (XDR-TB). Global data from 2023 indicates that 400, 000 people developed multidrug- or rifampicin-resistant tuberculosis (MDR/RR-TB) (WHO,2023). The improper use of antimycobacterial medications has contributed to the widespread emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. Patients with MDR-TB are essentially untreatable with standard first-line TB medications. Resistance to INH arises mainly due to mutations of katG gene which is responsible for encoding catalase-peroxidase, an enzyme responsible for activating isoniazid inside bacterial cell. Among these, the Ser315Thr (S315T) mutation is the most frequently observed, occurring in around 40% of resistant strains. This mutation results in a catalase-peroxidase enzyme that is unable to activate isoniazid, rendering the drug ineffective. However, despite losing its ability to activate the drug, the mutated enzyme retains approximately half of its normal catalase-peroxidase activity. This residual activity allows the bacterium to maintain oxidative stress defense mechanisms, enabling it to neutralize host-derived antibacterial radicals ( 8 , 9 ). Rifampicin resistance is most commonly observed in strains that are also resistant to isoniazid, making it a reliable indicator for identifying multidrug-resistant (MDR) Mycobacterium tuberculosis strains ( 9 ). Rifampicin inhibits RNA synthesis by targeting DNA-dependent RNA polymerase. Resistance to rifampicin is due to mutations of the rpoB gene which encodes the β-subunit of RNA polymerase ( 10 ). As a result, the rise of MDR-TB presents a serious public health challenge, especially in high-burden regions, highlighting the urgent need for the discovery and development of safer and more effective tuberculosis treatments. Phytochemicals are naturally occurring plant compounds with various therapeutic benefits. They have long been utilized for their medicinal properties and remain a crucial healthcare solution for people in rural areas with limited access to modern medicine and technology ( 11 ). Numerous plant-derived drugs have been developed to treat a range of diseases. Advancements in analytical techniques and methodologies have accelerated the discovery of bioactive compounds from plants ( 12 ). Given their broad applications, accessibility, and safety as well as the growing prevalence of antibiotic-resistant bacteria ( 13 ), there is increasing interest in researching, enhancing, and applying these plant-derived compounds to combat antibiotic-resistant bacterial strains ( 14 , 15 ). Eucalyptus globulus is a woody shrub or flowering tree belonging to the Myrtaceae family. This aromatic plant is native to Australia and is widely distributed across tropical and subtropical regions ( 16 ). Its therapeutic properties are primarily found in its essential oil, which is extracted from the leaves ( 17 ). This oil is rich in phytochemicals, including tannins, saponins, terpenoids, glycosides, alkaloids, phenolic compounds, steroids, cardiac glycosides, terpenes, reducing sugars, carbohydrates, resins, acidic compounds, and flavonoids ( 18 ). Key constituents of Eucalyptus globulus essential oil include 1,8-cineole, α-pinene, γ-terpinene, α-phellandrene, β-pinene, limonene, camphene, and sabinene ( 19 , 20 ). Eucalyptus essential oil has a long history of use in traditional medicine for treating respiratory conditions such as colds, coughs, runny nose, sore throat, asthma, nasal congestion, bronchitis, and sinusitis. It is also known for its diverse biological activities, including antibacterial, antioxidant, anticancer, diaphoretic, disinfectant, antimalarial, antiseptic, antidiabetic, analgesic, anti-inflammatory, and expectorant properties ( 17 , 18 , 20 , 21 ). Additionally, eucalyptus essential oil and its primary component, 1,8-cineole, have demonstrated strong antibacterial effects, particularly against the biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) strains ( 21 ). Aspartate-semialdehyde dehydrogenase (ASADH) is a crucial enzyme involved in the biosynthesis of amino acids and the cell wall of microorganisms. This enzyme catalyzes the reduction and dephosphorylation of aspartyl-phosphate to aspartate-semialdehyde within the aspartate biosynthetic pathway ( 22 ). Aspartate-semialdehyde can subsequently be reduced to homoserine, which serves as a precursor for the synthesis of methionine, threonine, and isoleucine—all of which are essential amino acids. Alternatively, when aspartate-semialdehyde undergoes condensation with pyruvate and cyclization within the aspartate pathway, it forms dihydrodipicolinate, which is further converted into diaminopimelate ( 23 ). Diaminopimelate (DAP) is a key metabolite required for the cross-linking of peptidoglycan polymers during bacterial cell wall synthesis ( 24 ). Additionally, S-adenosylmethionine, another important product of this pathway, acts as a precursor for quorum-sensing molecules in Gram-negative bacteria, playing a vital role in virulence and infection. Given its essential role in the survival and virulence of Mycobacterium tuberculosis , the ASADH enzyme represents a promising drug target for the development of novel antimicrobial therapies. An increasingly important strategy in drug discovery involves the use of in silico studies ( 25 , 26 ). This computational approach predicts how molecular ligands interact with protein targets to form stable complexes. In silico methods are widely employed in drug discovery due to their cost-effectiveness, time efficiency, and high predictive accuracy ( 27 ). Therefore, this study aimed to evaluate the Mycobacterium tuberculosis inhibitory potential of bioactive compounds from Eucalyptus globulus by targeting Aspartate-semialdehyde dehydrogenase (ASADH) using an in silico approach. METHODS Ligand Selection Bioactive compounds obtained from Eucalyptus globulus essential oil was used in this study. Fifteen ( 15 ) bioactive compounds including cineole (2758), alpha-phellandrene (CID: 7460), D-Limonene (CID: 440917), m-cymene (CID: 10812), terpinen-4-ol (CID: 11230), globulol (CID: 12304985), spathulenol (CID: 92231), carvacrol (CID: 10364), carvotanacetone (CID: 6432475), thymol (CID: 6989 ), linalool (CID: 6549), 6-Camphenol (CID: 180537), 6-Camphenone (CID: 14088350 ), piperitone (CID: 6987) and Sabinyl acetate (CID: 94266) from eucalyptus essential oil were selected based on literature survey( 19 , 20 ). Isoniazid (CID; 3767) was selected as the control ligand. The selected bioactive compounds and control ligands are shown in Table 1 . The 3D structures of all the ligands molecules in structure data format (SDF) and canonical SMILES were obtained from the PubChem database ( https://pubchem.ncbi.nlm.nih.gov ) ( 28 ). Table 1 Selected bioactive compounds and the control drug S/N Name CID FORMULA CANONICAL SMILES 1 Cineole 2758 C 10 H 18 O CC1(C2CCC(O1)(CC2)C)C 2 Alpha-Phellandrene 7460 C 10 H 16 CC1 = CCC(C = C1)C(C)C 3 D-Limonene 440917 C 10 H 16 CC1 = CCC(CC1)C(= C)C 4 M-cymene 10812 C 10 H 14 CC1 = CC(= CC = C1)C(C)C 5 Terpinen-4-ol 11230 C 10 H 18 O CC1 = CCC(CC1)(C(C)C)O 6 Globulol 12304985 C 15 H 24 O CC1CCC2C1C3C(C3(C)C)CCC2(C)O 7 Spathunelol 92231 C 15 H 24 O CC1(C2C1C3C(CCC3(C)O)C(= C)CC2)C 8 Carvacrol 10364 C 10 H 14 O CC1 = C(C = C(C = C1)C(C)C)O 9 Carvotanacetone 6432475 C 10 H 16 O CC1 = CCC(CC1 = O)C(C)C 10 Thymol 6989 C 10 H 14 O CC1 = CC(= C(C = C1)C(C)C)O 11 Linalool 6549 C 10 H 18 O CC(= CCCC(C)(C = C)O)C 12 6-Camphenol 180537 C 10 H 16 O CC1(C2CC(C1 = C)C(C2)O)C 13 6-Camphenone 14088350 C 10 H 14 O CC1(C2CC(C1 = C)CC2 = O)C 14 Piperitone 6987 C 10 H 16 O CC(C)C12CC1C(= C)C(C2)OC(= O)C 15 Sabinyl acetate 94266 C 12 H 18 O 2 CC(C)C12CC1C(= C)C(C2)OC(= O)C 16 Isoniazid 3767 C 6 H 7 N 3 O C1 = CN = CC = C1C(= O)NN Protein Target Selection The target protein, Mycobacterium tuberculosis Aspartate-semialdehyde dehydrogenase (ASADH) was selected using literature ( 29 ). The 3D crystal structures of the protein (PDB: 3TZ6) in the form of atomic coordinates was obtained from Protein Data Bank ( https://www.rcsb.org ) ( 30 ). Figure 1 shows the structure of Mycobacterium tuberculosis Aspartate-semialdehyde dehydrogenase (ASADH). Protein targets preparation The 3D crystallized structure of the target protein; Mycobacterium tuberculosis Aspartate-semialdehyde dehydrogenase (ASADH), was accomplished using the protein preparation module of Schrödinger’s molecular modeling suit. Protein preparation was carried out by removal of unwanted water molecules, heteroatoms, and co-crystallized ligands coordinates to obtain a more suitable and stable conformation. The protein was minimized for molecular docking using OPLS 4 force field and saved in PDB format. Active Site Generation The grid orientation of the active site was simulated from the binding conformation of the target protein co-crystallized ligand using the grid module of Schrödinger’s molecular modeling suit. X, Y, Z coordinates of the gird box are 238.99, 50.38, and 75.95 respectively. Drug-likeness Screening The fifteen ( 15 ) bioactive compounds and the two control drugs were screened for their drug likeness properties using the SwissADME ( http://swissadme.ch/ ) online server. These compounds were subjected to drug-likeness screening using the parameters hypothesized by Lipinski, Verber, and Egan. Fourteen ( 14 ) out of the fifteen ( 15 ) selected bioactive compounds did not violate Lipinski rule and were subjected to molecular docking. Ligand Preparation The fourteen screened compounds were imported into the LigPrep workflow. OPLS4 forcefield was used due its accuracy in obtaining improved docking poses and conformational analyses ( 31 ). Tautomers were generated for each ligand and the PH specified for generation of ionization state was 7.0 +_ 2.0 Molecular Docking Grid module of Schrödinger’s molecular modeling suit was used to for molecular docking of the protein target and ligands. The prepared ligands and the generated grid file were imported into the program. Standard Precision (SP) docking mode was used and docking was performed flexibly. ADMET Evaluation The absorption, distribution, metabolism, excretion and toxicity (ADMET) of the top ligands obtained from the molecular docking and the control was evaluated using ADMETLab online server ( https://admetmesh.scbdd.com/service/evaluation/cal ) ( 32 ). The pharmacokinetic properties were then predicted from the results. RESULTS Drug-likeness Screening Drug likeness screening results as shown in Table 2 indicated that only M-cymene out of the fifteen bioactive compounds selected from Eucalyptus essential oil violated Lipinski’s drug likeness rule and was therefore eliminated for further analysis. The control ligand; Isoniazid passed the drug likeness screening. The remaining fourteen compounds that passed the drug-likeness screening and the control ligand were subjected for docking analysis with Mtb -ASADH. Molecular Docking Results from the molecular docking study of the fourteen bioactive compounds that passed the drug-likeness screening and the control ligand revealed their docking score, ligand interaction, as well as their hydrogen binding affinities. The results of the bioactive compounds from eucalyptus plant presented in Table 3 predicts their binding affinity and suggest potential biological activity against the target protein Mtb -ASADH. Docking score indicates that thymol (-5.445) had the highest docking score, followed by Sabinyl acetate (-5.097). These two ligands had a higher docking score than Isoniazid (-5.040) indicating a better binding affinity and potential biological activity against the target. Amino acids residues observed in the hydrogen bond interaction of sabinyl acetate with the target protein were Asn 94 and Arg 99 while amino acids residues observed with the hydrogen bond interaction of thymol with the target protein were Arg 99, Asn 94 and Lys 227. Figure 2 shows the interaction of thymol, sabinyl acetate and isoniazid with the target. Table 2 Screening results of drug-likeness bioactive compounds and control drug using SwissADME web tool Molecule MW XLOGP TPSA Lipinski #Violation Ghose #Violation Veber #Violation Egan Violation Meugge #Violation Bioavalability Score Cineole 154.25 2.74 9.23 0 1 0 0 2 0.55 Alpha-Phellandrene 136.23 3.21 0 0 1 0 0 2 0.55 D-Limonene 136.23 4.57 0 0 1 0 0 2 0.55 M-cymene 134.22 4.5 0 1 1 0 0 2 0.55 Terpinen-4-ol 154.25 3.26 20.23 0 1 0 0 2 0.55 Globulol 222.37 3.74 20.23 0 0 0 0 1 0.55 Spathunelol 220.35 3.11 20.23 0 0 0 0 1 0.55 Carvacrol 150.22 3.49 20.23 0 1 0 0 2 0.55 Carvotanacetone 152.23 2.5 17.07 0 1 0 0 2 0.55 Thymol 150.22 3.3 20.23 0 1 0 0 2 0.55 Linalool 154.25 2.97 20.23 0 1 0 0 2 0.55 6-Camphenol 152.23 1.78 20.23 0 1 0 0 2 0.55 6-Camphenone 150.22 1.5 17.07 0 1 0 0 2 0.55 Piperitone 152.23 2.85 17.07 0 1 0 0 2 0.55 Sabinyl acetate 194.27 2.36 26.3 0 0 0 0 1 0.55 Isoniazid 137.14 0.7 68.01 0 3 0 0 1 0.55 Table 3 Molecular docking score of the bioactive compounds and the control drug S/N Name CID Docking Score 1 Cineole 2758 -4.294 2 Alpha-Phellandrene 7460 4.986 3 D-Limonene 440917 -3.849 4 M-cymene 10812 -4.715 5 Terpinen-4-ol 11230 -4.799 6 Globulol 12304985 -4.470 7 Spathunelol 92231 -4.519 8 Carvacrol 10364 -4.901 9 Carvotanacetone 6432475 -4.956 10 Thymol 6989 -5.445 11 Linalool 6549 12 6-Camphenol 180537 -4.552 13 6-Camphenone 14088350 -4.689 14 Piperitone 6987 -4.982 15 Sabinyl acetate 94266 -5.097 16 Isoniazid 3767 -5.040 ADMET STUDY The predicted absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles of the two top-scoring bioactive compounds, in comparison with the control drug, are presented in Table 4 . For classification model endpoints such as blood-brain barrier permeability (BBB), human intestinal absorption (H-HT), and hERG channel inhibition, prediction probabilities were categorized into six symbolic levels: 0–0.1 (−−−), 0.1–0.3 (−−), 0.3–0.5 (−), 0.5–0.7 (+), 0.7–0.9 (++), and 0.9–1.0 (+++). Generally, ‘+++’ or ‘++’ indicates a higher likelihood of toxicity or undesired effects, whereas ‘−−−’ or ‘−−’ suggests that the molecule is likely to be nontoxic or favorable (Xiong et al., 2021). Regarding absorption and distribution, all top-hit compounds, including the control, were predicted to cross the blood-brain barrier (BBB). The predicted plasma protein binding (PPB) values were expressed as percentages. Sabinyl acetate and thymol exhibited PPB values of 47.56% and 93.90%, respectively, whereas isoniazid had a PPB value of 9%. In terms of metabolism, sabinyl acetate posed a lower risk of cytochrome P450 (CYP)-related drug interactions, as it showed no inhibition for any of the tested CYP enzymes. Thymol exhibited no inhibition for three out of the five tested CYP enzymes. Isoniazid, on the other hand, showed inhibition specifically for the CYP1A2 enzyme. The predicted elimination half-life (t₁/₂) values provided insights into drug excretion rates. Thymol has a half-life of 0.098, sabinyl acetate; 0.0682 while isoniazid had a half-life of 0.715. In terms of toxicity, none of the compounds demonstrated hERG inhibition, indicating a lower risk of cardiac side effects. Additionally, all compounds tested negative in the Ames mutagenicity assay, suggesting a lack of mutagenic potential. Furthermore, none of the top-hit compounds exhibited rat oral acute toxicity. However, sabinyl acetate was predicted to have carcinogenic potential. Table 4 ADMET profiling of Drug Candidates and Control Drugs ADMET MODELS Isoniazid Thymol Sabinyl acetate Absorption & Distribution HIA --- --- --- P-gp substrate --- --- --- PPB 9.00% 93.90% 47.56% Metabolism CYP1A2 inhibitor ++ +++ -- CYP2C19 inhibitor -- ++ --- CYP2C9 inhibitor --- - --- CYP2D6 inhibitor -- ++ --- CYP3A4 inhibitor - -- -- Excretion T1/2 0.715 0.682 0.098 Toxicity Ames toxicity --- --- --- Carcinogenicity -- -- ++ Rat oral acute toxicity ++ -- - H-HT - --- -- DILI +++ -- ++ hERG blockers --- --- --- Respiratory toxicity + + +++ SR-p53 --- --- + DISCUSSION Drug-likeness screening The drug-likeness rules are important in discriminating ideal drug-like compounds from false positives. This assertion is supported by a study conducted by ( 33 ). The drug likeness properties of the bioactive compounds and the control were determined using Lipinski’s, Ghose’s, Veber’s, Egan’s and Muegge’s drug likeness rule. The top rank hit bioactive compounds for this study was selected from compounds that passed at least three out of the five rules. These drug-likeness rules are major determinants of oral bioavailability potential of any drug molecule i.e., the probability of drug molecule being readily available to target tissues after being absorbed into the body system. This concept is correlated with previous studies( 34 – 36 ). The control in this study; isoniazid, violated two of the rules. Sabinyl acetate, thymol and the rest of the bioactive compounds excluding M-cymene passed at least three of the drug-likeness rules including the Lipinski’s rule of five indicating that these compounds can potentially be suitable for oral use. Lipinski’s rule is a set of conditions that defines oral bioavailability of a compound based on its chemical and physical properties of the compound ( 37 – 39 ). According to the rule, a drug is considered to be suitable for oral activity if it has no more than five hydrogen bond donors, no more than ten hydrogen bond acceptors, molecular mass ≤ 500 Daltons and an octanol water partition co-efficient (XlogP) not greater than five. Saganuwan ( 40 ), reported that the molecular mass of a compound exerts an influence on a compound’s efficacy as a drug, the molecular weight of a drug determines its oral use and cellular permeability. Bioactive compound with molecular weight within the Lipinski’s range has an optimal permeability that makes it easy to cross cellular membranes and reach its target within a short time. XlogP is a measure of a compound’s hydrophobicity and it represents the ratio of the concentration of a compound in octanol to its concentration in water. XlogP is a relevant parameter that influences the drug-likeness property of a compound by determining its permeability through cell membranes and thereby aiding their absorption( 41 ). The result presented in Table 1 indicates that the top hit selected bioactive compounds in this study has a good druggability and can be used as oral drugs. Molecular docking Molecular docking result indicated that all 14 compounds selected for the study docked to the active site of the target protein. However, Sabinyl acetate and thymol were the only ligands with docking score higher than the control. The docking score of Sabinyl acetate and thymol was − 5.097 and − 5.445 and respectively while the docking score of the Isoniazid was − 5.040 as shown in Table 1 . This observation indicates a strong binding affinity between Mycobacterium Tuberculosis ASADH and the two bioactive compounds and suggests that these compounds can effectively interact with ASADH, inhibit its action, modulate its function and elicit a potentially strong therapeutic action. Previous studies have reported that binding affinity reflects the strength of interaction and the ability of ligand molecules to modulate biological functions ( 42 , 43 ). Furthermore, Sabinyl acetate interacted with Mycobacterium Tuberculosis ASADH active site with 2 hydrogen bonds at amino acid residues; Arg99 and Asn94, Thymol interacted with the protein’s active site with 1 hydrogen bond at amino acid residue; Arg99, while isoniazid interacted with the protein’s active site with 3 hydrogen bonds at amino acid residue; Arg99, Lys227 and Asn94. The H-bond interactions observed in Sabinyl acetate and thymol indicates a high degree of binding and molecular interactions between the protein-ligand complex. This suggests a high degree of binding activity and molecular interaction. This aligns with previous studies ( 44 – 46 ) that reported hydrogen bond is fundamental, enhances stability and essential of effective binding. Moreover, the amino acid residues in the key active site of the protein interacting with the ligands through hydrogen bonding correlates with the studies done by ( 47 , 48 ) where they showed that Arg99, Lys227 and others are the interacting amino acid residues of Mtb -ASADH. Therefore, binding the compounds to Mtb -ASADH could offer some antimycobacterial effects in tuberculosis treatment by the inhibition of Mtb -ASADH. Figures 1 and 2 shows interaction of compound in sabinyl acetate, thymol and isoniazid with the target protein . ADMET The absorption, distribution, metabolism, excretion and toxicity profiles of abinyl acetate, thymol and the control are presented in Table 3 . ADMET profiling is very essential because it determines the pharmacokinetics of our hit compounds, analyzes its possible action in the body and helps assess its efficacy ( 49 ). Human intestinal absorption (HIA) is a crucial parameter in evaluating the oral bioavailability of a compound, as it determines how effectively a drug enters the systemic circulation following administration. High HIA is generally associated with improved absorption, enhanced therapeutic efficacy, and a more favorable dosing regimen ( 50 ). In this study, both thymol and sabinyl acetate were predicted to have favorable levels of HIA, indicating absorption through the gastrointestinal tract. This may increase their suitability for oral delivery as antimycobacterial agents. Another important pharmacokinetic property is blood–brain barrier (BBB) permeability, which restricts the entry of compounds into the central nervous system (CNS) ( 51 ) and presents a major challenge in treating infections such as CNS tuberculosis (CNS-TB), a severe and often fatal manifestation of TB caused by the infiltration of Mycobacterium species into the CNS ( 52 ). Both thymol and sabinyl acetate, along with the control drug isoniazid, were predicted to be BBB permeant, suggesting that they have the ability to cross the BBB and may therefore be effective in the treatment of CNS-TB. Plasma protein binding (PPB) is another important factor influencing the pharmacokinetics of drug candidates, as it determines the fraction of drug available in its free, active form to interact with the biological target. Proteins such as serum albumin and α1-acid glycoprotein are known to bind a wide range of drugs, often affecting their efficacy. In general, a PPB value exceeding 90% can reduce the amount of free drug available and potentially attenuate its therapeutic effect. In this study, sabinyl acetate exhibited a moderate PPB score of 47.56%, indicating that a significant proportion of the compound remains unbound and available for therapeutic action. Isoniazid showed a low PPB score of 9.00%, further confirming its availability in free form. Thymol, however, had a high PPB score of 93.90%, which may limit its immediate bioavailability. Nonetheless, studies suggest that high PPB does not always correlate directly with reduced potency, as several clinically effective drugs exhibit high plasma protein binding yet retain strong pharmacological activity( 53 ). The cytochrome P450 (CYP) family of enzymes, primarily expressed in the liver, plays a central role in drug metabolism. Inhibition of these enzymes can lead to drug–drug interactions, reduced drug clearance, accumulation of toxic metabolites, or decreased therapeutic efficacy ( 54 ). In this study, sabinyl acetate was predicted not to inhibit any of the major CYP isoforms, suggesting a low potential for enzyme-related interactions. Thymol was also predicted not to inhibit CYP3A4, one of the most clinically significant isoforms. These findings indicate that both compounds are unlikely to cause CYP-mediated drug–drug interactions, thereby supporting their metabolic stability and safety in potential combination therapies. Half-life is another critical pharmacokinetic parameter that reflects the duration a drug remains active in the body and influences dosing frequency. Based on empirical classification, compounds with a half-life between 0 and 0.3 hours are considered to have excellent metabolic clearance, those between 0.3 and 0.7 hours are moderate, while values between 0.7 and 1.0 hours are considered poor ( 41 ).Thymol and sabinyl acetate recorded half-lives of 0.098 and 0.0682 respectively, which fall within the excellent category, suggesting rapid clearance from the system. This indicates favorable metabolic processing. In terms of toxicity, both thymol and sabinyl acetate demonstrated favorable safety profiles. The two compounds showed low predicted binding affinity to the human ether-à-go-go-related gene (hERG) potassium channel, which plays a vital role in the repolarization of cardiac cells. Inhibition of this channel can result in delayed cardiac repolarization, leading to conditions such as long QT syndrome (LQTS), arrhythmias, and Torsade de Pointes (TdP) ( 55 ). The low hERG inhibition scores suggest a reduced risk of cardiotoxicity, making both compounds safer for potential therapeutic use. Furthermore, all three compounds, including the control, were Ames-negative, indicating a low likelihood of causing DNA mutations, and therefore a reduced risk of carcinogenicity or heritable genetic damage. In addition, thymol and sabinyl acetate were predicted to be non-hepatotoxic, signifying minimal potential for liver damage or dysfunction, which further supports their safety for therapeutic development. However, it is important to note that sabinyl acetate was predicted to be carcinogenic, unlike thymol and isoniazid, which were both classified as non-carcinogenic. Thus, the findings of this study suggest that the active compounds thymol and sabinyl acetate may exhibit strong in silico inhibitory activity against Mycobacterium tuberculosis aspartate-semialdehyde dehydrogenase, as evidenced by molecular docking analysis. However, these computational results require further validation through molecular dynamics simulations and experimental (in vitro and in vivo) studies to confirm their therapeutic potential. CONCLUSIONS Tuberculosis (TB) remains the leading cause of death from a single infectious agent globally. The emergence of multidrug-resistant TB (MDR-TB) has intensified the urgency to develop more effective and safer therapeutic alternatives. This study evaluated the antimycobacterial potential of bioactive compounds derived from Eucalyptus globulus against Mycobacterium tuberculosis aspartate-semialdehyde dehydrogenase, using isoniazid, a frontline anti-TB drug, as the control. Drug-likeness screening and molecular docking analyses identified thymol and sabinyl acetate as the top-performing compounds, both demonstrating strong binding affinity and stable hydrogen interactions with the Mtb -ASADH. ADMET profiling further supported their potential as promising drug candidates. The compounds were predicted to cross the blood–brain barrier, showed no significant inhibition of cytochrome P450 enzymes, and exhibited favorable half-lives. Although thymol displayed high plasma protein binding and sabinyl acetate showed carcinogenic potential, their overall pharmacokinetic and safety profiles suggest a strong therapeutic promise. The findings from this in silico study provide a foundation for further validation through in vitro and in vivo experiments to confirm their efficacy and safety as novel antimycobacterial agents. Declarations Ethics approval and consent to participate – Not Applicable Consent for publication – Not Applicable Availability of data and materials – All data generated and analysed during this study are included in this published article. Competing interests – The author(s) declare(s) that they have no competing interests. Funding – Not Applicable Authors’ contributions – SMJ designed and executed experiments, performed the docking analysis and wrote the manuscript. MAB and NA designed and executed experiments, obtained protein and ligand structures and edited the manuscript. IA input on data analysis and revised the manuscript. All authors read and approved the final manuscript. Acknowledgments – Not Applicable References World Health Organization. Tuberculosis (TB) [Internet]. 2024 [cited 2025 Jun 19]. Available from: https://www.who.int/news-room/fact-sheets/detail/tuberculosis Natarajan A, Beena PM, Devnikar AV, Mali S. A systemic review on tuberculosis. Indian J Tuberc. 2020;67(3):295–311. Hargreaves JR, Boccia D, Evans CA, Adato M, Petticrew M, Porter JDH. The Social Determinants of Tuberculosis: From Evidence to Action. Am J Public Health. 2011;101(4):654–62. World Health Organization. 1.1 TB incidence [Internet]. 2023 [cited 2025 Jun 19]. Available from: https://www.who.int/teams/global-programme-on-tuberculosis-and-lung-health/tb-reports/global-tuberculosis-report-2023/tb-disease-burden/1-1-tb-incidence Bereda G. First Line Anti-Tuberculosis Medication: Current and Ongoing Clinical Management. 2022. Kumar M, Singh SK, Singh PP, Singh VK, Rai AC, Srivastava AK, et al. Potential Anti-Mycobacterium tuberculosis Activity of Plant Secondary Metabolites: Insight with Molecular Docking Interactions. Antioxidants. 2021;10(12):1990. Nahid P, Mase SR, Migliori GB, Sotgiu G, Bothamley GH, Brozek JL, et al. Treatment of Drug-Resistant Tuberculosis. An Official ATS/CDC/ERS/IDSA Clinical Practice Guideline. Am J Respir Crit Care Med. 2019;200(10):e93–142. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis : 1998 update. Tuber Lung Dis. 1998;79(1):3–29. Somoskovi A, Parsons LM, Salfinger M. The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respir Res. 2001;2(3):164. Telenti A, Imboden P, Marchesi F, Matter L, Schopfer K, Bodmer T, et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet. 1993;341(8846):647–51. Garcia-Campoy A, Garcia E, Muñiz-Ramirez A. Phytochemical and Pharmacological Study of the Eysenhardtia Genus. Plants. 2020;9(9):1124. Chihomvu P, Ganesan A, Gibbons S, Woollard K, Hayes MA. Phytochemicals in Drug Discovery—A Confluence of Tradition and Innovation. Int J Mol Sci. 2024;25(16):8792. Abdallah EM, Alhatlani BY, De Paula Menezes R, Martins CHG. Back to Nature: Medicinal Plants as Promising Sources for Antibacterial Drugs in the Post-Antibiotic Era. Plants. 2023;12(17):3077. Thangaraj PMR. Anti-Bacterial Attributes of Phytochemicals from Bougainvillaea spectabilis: Computational Approach. Pharmacogn Res. 2024;16(2):384–90. Tiwana G, Cock IE, Cheesman MJ. Combinations of Terminalia bellirica (Gaertn.) Roxb. and Terminalia chebula Retz. Extracts with Selected Antibiotics Against Antibiotic-Resistant Bacteria: Bioactivity and Phytochemistry. Antibiotics. 2024;13(10):994. Potts B, Vaillancourt R, Jordan GJ, Dutkowski GW, da Costa e Silva J, McKinnon GE et al. Exploration of the Eucalyptus globulus gene pool. 2004 Jan 1 [cited 2025 Jun 19]; Available from: https://figshare.utas.edu.au/articles/conference_contribution/Exploration_of_the_Eucalyptus_globulus_gene_pool/23213207/1 Silva J, Abebe W, Sousa SM, Duarte VG, Machado MIL, Matos FJA. Analgesic and anti-inflammatory effects of essential oils of Eucalyptus. J Ethnopharmacol. 2003;89(2):277–83. Mishra AK, Sahu N, Mishra A, Ghosh AK, Jha S, Chattopadhyay P. Phytochemical Screening and Antioxidant Activity of essential oil of Eucalyptus leaf. Pharmacogn J. 2010;2(16):25–8. Shala AY, Gururani MA. Phytochemical Properties and Diverse Beneficial Roles of Eucalyptus globulus Labill. Rev Horticulturae. 2021;7(11):450. Usman LA, Oguntoye OS, Ismaeel RO, Usman LA, Oguntoye OS, Ismaeel RO, PHYTOCHEMICAL PROFILE, ANTIOXIDANT AND ANTIDIABETIC POTENTIAL OF ESSENTIAL OIL FROM FRESH AND DRIED LEAVES OF Eucalyptus globulus. J Chil Chem Soc. 2022;67(1):5453–61. Sharma AD, Farmaha M, Kaur I, Singh N. Phytochemical analysis using GC-FID, FPLC fingerprinting, antioxidant, antimicrobial, anti- inflammatory activities analysis of traditionally used Eucalyptus globulus essential oil. Drug Anal Res. 2021;5(1):26–38. Viola RE. The Central Enzymes of the Aspartate Family of Amino Acid Biosynthesis. Acc Chem Res. 2001;34(5):339–49. Hadfield A, Shammas C, Kryger G, Ringe D, Petsko GA, Ouyang J, et al. Active Site Analysis of the Potential Antimicrobial Target Aspartate Semialdehyde Dehydrogenase. Biochemistry. 2001;40(48):14475–83. van Heijenoort J. Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat Prod Rep. 2001;18(5):503–19. Roney M, Mohd Aluwi MFF. The importance of in-silico studies in drug discovery. Intell Pharm. 2024;2(4):578–9. Sabe VT, Ntombela T, Jhamba LA, Maguire GEM, Govender T, Naicker T, et al. Current trends in computer aided drug design and a highlight of drugs discovered via computational techniques: A review. Eur J Med Chem. 2021;224:113705. Meng XY, Zhang HX, Mezei M, Cui M. Molecular Docking: A Powerful Approach for Structure-Based Drug Discovery. Curr Comput Aided Drug Des. 2011;7(2):146–57. Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, et al. PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Res. 2021;49(D1):D1388–95. Meng J, Yang Y, Xiao C, Guan Y, Hao X, Deng Q, et al. Identification and Validation of Aspartic Acid Semialdehyde Dehydrogenase as a New Anti-Mycobacterium Tuberculosis Target. Int J Mol Sci. 2015;16(10):23572–86. Berman HM. The Protein Data Bank. Nucleic Acids Res. 2000;28(1):235–42. D’Amore L, Hahn DF, Dotson DL, Horton JT, Anwar J, Craig I, et al. Collaborative Assessment of Molecular Geometries and Energies from the Open Force Field. J Chem Inf Model. 2022;62(23):6094–104. Dong J, Wang NN, Yao ZJ, Zhang L, Cheng Y, Ouyang D, et al. ADMETlab: a platform for systematic ADMET evaluation based on a comprehensively collected ADMET database. J Cheminformatics. 2018;10(1):29. Mahgoub RE, Mohamed FE, Alzyoud L, Ali BR, Ferreira J, Rabeh WM, et al. The Discovery of Small Allosteric and Active Site Inhibitors of the SARS-CoV-2 Main Protease via Structure-Based Virtual Screening and Biological Evaluation. Molecules. 2022;27(19):6710. Mahgoub RE, Mohamed FE, Ali BR, Ferreira J, Rabeh WM, Atatreh N, et al. Discovery of pyrimidoindol and benzylpyrrolyl inhibitors targeting SARS-CoV-2 main protease (Mpro) through pharmacophore modelling, covalent docking, and biological evaluation. J Mol Graph Model. 2024;127:108672. Srivastava V, Yadav A, Sarkar P. Molecular docking and ADMET study of bioactive compounds of Glycyrrhiza glabra against main protease of SARS-CoV2. Mater Today Proc. 2022;49:2999–3007. Halder SK, Elma F. In silico identification of novel chemical compounds with anti-TB potential for the inhibition of InhA and EthR from Mycobacterium tuberculosis [Internet]. Bioinformatics; 2020 [cited 2025 Jun 17]. Available from: http://biorxiv.org/lookup/doi/ 10.1101/2020.12.04.411967 Lipinski CA. Drug-like properties and the causes of poor solubility and poor permeability. J Pharmacol Toxicol Methods. 2000;44(1):235–49. Lipinski CA. Rule of five in 2015 and beyond: Target and ligand structural limitations, ligand chemistry structure and drug discovery project decisions. Adv Drug Deliv Rev. 2016;101:34–41. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2012;64:4–17. Saganuwan S. Structure-activity relationship of pharmacophores and toxicophores: the need for clinical strategy. Daru J Fac Pharm Tehran Univ Med Sci [Internet]. 2024 [cited 2025 Jun 17]; Available from: https://consensus.app/papers/structureactivity-relationship-of-pharmacophores-and-saganuwan/497397cd29f3587f82fcccf6219706ec /. Xiong G, Wu Z, Fu L, Yang Z, Hsieh C, Yin M, et al. ADMETlab 2.0: an integrated online platform for accurate and comprehensive predictions of ADMET properties. Nucleic Acids Res. 2021;49(W1):W5–14. Friedman R. Computational studies of protein–drug binding affinity changes upon mutations in the drug target. WIREs Comput Mol Sci. 2022;12(1):e1563. Fuji H, Qi F, Qu L, Takaesu Y, Hoshino T. Prediction of Ligand Binding Affinity to Target Proteins by Molecular Mechanics Theoretical Calculation. Chem Pharm Bull (Tokyo). 2017;65(5):461–8. Dhakal A, McKay C, Tanner JJ, Cheng J. Artificial intelligence in the prediction of protein–ligand interactions: recent advances and future directions. Brief Bioinform. 2022;23(1):bbab476. Madushanka A, Moura RT, Verma N, Kraka E. Quantum Mechanical Assessment of Protein–Ligand Hydrogen Bond Strength Patterns: Insights from Semiempirical Tight-Binding and Local Vibrational Mode Theory. Int J Mol Sci. 2023;24(7):6311. Patil R, Das S, Stanley A, Yadav L, Sudhakar A, Varma AK. Optimized Hydrophobic Interactions and Hydrogen Bonding at the Target-Ligand Interface Leads the Pathways of Drug-Designing. Hannenhalli S, editor. PLoS ONE. 2010;5(8):e12029. Kumar R, Garg P, Bharatam PV. Shape-based virtual screening, docking, and molecular dynamics simulations to identify Mtb -ASADH inhibitors. J Biomol Struct Dyn. 2015;33(5):1082–93. Kumar R, Garg P, Bharatam PV. Pharmacoinformatics analysis to identify inhibitors of Mtb -ASADH. J Biomol Struct Dyn. 2016;34(1):1–14. Daoud NEH, Borah P, Deb PK, Venugopala KN, Hourani W, Alzweiri M, et al. ADMET Profiling in Drug Discovery and Development: Perspectives of In Silico, In Vitro and Integrated Approaches. Curr Drug Metab. 2021;22(7):503–22. Fagerholm U. Prediction of human pharmacokinetics —gastrointestinal absorption. J Pharm Pharmacol. 2007;59(7):905–16. Alavijeh MS, Chishty M, Qaiser MZ, Palmer AM. Drug metabolism and pharmacokinetics, the blood-brain barrier, and central nervous system drug discovery. NeuroRX. 2005;2(4):554–71. Be N, Kim K, Bishai W, Jain S. Pathogenesis of Central Nervous System Tuberculosis. Curr Mol Med. 2009;9(2):94–9. Trainor GL. The importance of plasma protein binding in drug discovery. Expert Opin Drug Discov. 2007;2(1):51–64. Esteves F, Rueff J, Kranendonk M. The Central Role of Cytochrome P450 in Xenobiotic Metabolism—A Brief Review on a Fascinating Enzyme Family. J Xenobiotics. 2021;11(3):94–114. Finlayson K, Witchel HJ, McCulloch J, Sharkey J. Acquired QT interval prolongation and HERG: implications for drug discovery and development. Eur J Pharmacol. 2004;500(1–3):129–42. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7337220","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501543728,"identity":"e729fda9-6094-4f76-91cb-e17e6f9acfb9","order_by":0,"name":"Jimoh Salamah Mopelola","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYFAC5gYgcYCZjYGBjeEDkMnGTlALI0IL4wyQFmYitTCAFfOArSWgQb79YOOnGzV32PnEDh97bPNrmzwfMwPjh485uLUYnElsls459oyZTTot3Ti377ZhGzMDs+TMbXi0MCQ2SOewHQZqyTGTzu25zQjUwsbMi0eLfP/D5t85/6BaLHtu2xPUwnAjsU06tw2qheHH7USCWgxuPGyzzu0DaUlLk+xtuJ3cxszYjNcv8v3Jh2/nfDucLD87+ZjEjz+3bee3Nx/88BGfw6AgGUwytoHJBsLqgcAOQv0hSvEoGAWjYBSMMAAAcf5Oont+spcAAAAASUVORK5CYII=","orcid":"","institution":"University of Ilorin","correspondingAuthor":true,"prefix":"","firstName":"Jimoh","middleName":"Salamah","lastName":"Mopelola","suffix":""},{"id":501543729,"identity":"9c12e160-2c08-4dd7-a8c9-56b6b2f22ac7","order_by":1,"name":"Memunat Alake Bankole","email":"","orcid":"","institution":"Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Memunat","middleName":"Alake","lastName":"Bankole","suffix":""},{"id":501543730,"identity":"1dc13a25-7412-4060-8a92-f7e8eb083e35","order_by":2,"name":"Nurudeen Owolabi","email":"","orcid":"","institution":"Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Nurudeen","middleName":"","lastName":"Owolabi","suffix":""},{"id":501543731,"identity":"c0647cda-5d38-41d8-be85-d9d45703719e","order_by":3,"name":"Ibrahim Ajadi","email":"","orcid":"","institution":"Kwara State University","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Ajadi","suffix":""}],"badges":[],"createdAt":"2025-08-10 06:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7337220/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7337220/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89668708,"identity":"5c343e6f-c705-4eb2-af3c-59b759e239de","added_by":"auto","created_at":"2025-08-22 12:37:54","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75196,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of the protein \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eAspartate-semialdehyde dehydrogenase\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7337220/v1/113a91d8129ab1731af16d1a.jpeg"},{"id":89668711,"identity":"4cbe1e87-3739-418c-99f5-06964fa80122","added_by":"auto","created_at":"2025-08-22 12:37:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":654250,"visible":true,"origin":"","legend":"\u003cp\u003eBinding Configuration of Sabinyl acetate (2a), Thymol (2b) and Isoniazid (2c) in the active site of \u003cem\u003eMtb\u003c/em\u003e-ASADH as obtained from molecular docking using Grid module of Schrödinger’s molecular modelling suit.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7337220/v1/6c53d6160769ec0bb352ffa0.png"},{"id":90286775,"identity":"f37a87fe-d055-4a50-b165-21d1b9d05a12","added_by":"auto","created_at":"2025-09-01 06:24:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1413748,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7337220/v1/fabd574c-8206-47f7-8189-2c3399855768.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antimycobacterial Activity of Bioactive Compounds from Eucalyptus globulus against Mycobacterium tuberculosis Aspartate-semialdehyde Dehydrogenase: In silico Analysis","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eTuberculosis (TB), caused by \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e, is the deadliest disease resulting from a single infectious agent (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) and one of the oldest diseases known to affect humans (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Poverty significantly contributes to the persistence of tuberculosis worldwide (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Nearly a quarter of the world's population has been infected with TB, with a 5\u0026ndash;10% lifetime risk of developing TB disease. In 2023, approximately 1.25\u0026nbsp;million deaths from TB were reported. India, Indonesia, China, the Philippines, Pakistan, Nigeria, Bangladesh, and the Democratic Republic of the Congo accounted for more than two-thirds of global TB cases (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIsoniazid and rifampicin (RFP) are first-line medications used to treat tuberculosis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Isoniazid, also known as isonicotinic acid hydrazide (INH), is a prodrug that suppresses the growth of mycobacteria (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Although its precise mechanism of action is not fully understood, it targets fatty acid synthetase in mycobacteria, which plays a role in mycolic acid biosynthesis\u0026mdash;essential for cell wall formation (Bereda, 2022). Rifampicin inhibits bacterial DNA-dependent RNA synthesis by targeting bacterial RNA polymerase (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, TB treatment becomes significantly more complex due to the emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR) strains of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Resistance to at least both INH and RIF is classified as multidrug-resistant TB (MDR-TB), while additional resistance to fluoroquinolones and aminoglycosides is referred to as extensively drug-resistant TB (XDR-TB). Global data from 2023 indicates that 400, 000 people developed multidrug- or rifampicin-resistant tuberculosis (MDR/RR-TB) (WHO,2023). The improper use of antimycobacterial medications has contributed to the widespread emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. Patients with MDR-TB are essentially untreatable with standard first-line TB medications.\u003c/p\u003e\u003cp\u003eResistance to INH arises mainly due to mutations of katG gene which is responsible for encoding catalase-peroxidase, an enzyme responsible for activating isoniazid inside bacterial cell. Among these, the Ser315Thr (S315T) mutation is the most frequently observed, occurring in around 40% of resistant strains. This mutation results in a catalase-peroxidase enzyme that is unable to activate isoniazid, rendering the drug ineffective. However, despite losing its ability to activate the drug, the mutated enzyme retains approximately half of its normal catalase-peroxidase activity. This residual activity allows the bacterium to maintain oxidative stress defense mechanisms, enabling it to neutralize host-derived antibacterial radicals (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Rifampicin resistance is most commonly observed in strains that are also resistant to isoniazid, making it a reliable indicator for identifying multidrug-resistant (MDR) \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e strains (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Rifampicin inhibits RNA synthesis by targeting DNA-dependent RNA polymerase. Resistance to rifampicin is due to mutations of the rpoB gene which encodes the β-subunit of RNA polymerase (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAs a result, the rise of MDR-TB presents a serious public health challenge, especially in high-burden regions, highlighting the urgent need for the discovery and development of safer and more effective tuberculosis treatments.\u003c/p\u003e\u003cp\u003ePhytochemicals are naturally occurring plant compounds with various therapeutic benefits. They have long been utilized for their medicinal properties and remain a crucial healthcare solution for people in rural areas with limited access to modern medicine and technology (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Numerous plant-derived drugs have been developed to treat a range of diseases. Advancements in analytical techniques and methodologies have accelerated the discovery of bioactive compounds from plants (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Given their broad applications, accessibility, and safety as well as the growing prevalence of antibiotic-resistant bacteria (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), there is increasing interest in researching, enhancing, and applying these plant-derived compounds to combat antibiotic-resistant bacterial strains (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eEucalyptus globulus\u003c/em\u003e is a woody shrub or flowering tree belonging to the Myrtaceae family. This aromatic plant is native to Australia and is widely distributed across tropical and subtropical regions (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Its therapeutic properties are primarily found in its essential oil, which is extracted from the leaves (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). This oil is rich in phytochemicals, including tannins, saponins, terpenoids, glycosides, alkaloids, phenolic compounds, steroids, cardiac glycosides, terpenes, reducing sugars, carbohydrates, resins, acidic compounds, and flavonoids (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Key constituents of \u003cem\u003eEucalyptus globulus\u003c/em\u003e essential oil include 1,8-cineole, α-pinene, γ-terpinene, α-phellandrene, β-pinene, limonene, camphene, and sabinene (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEucalyptus essential oil has a long history of use in traditional medicine for treating respiratory conditions such as colds, coughs, runny nose, sore throat, asthma, nasal congestion, bronchitis, and sinusitis. It is also known for its diverse biological activities, including antibacterial, antioxidant, anticancer, diaphoretic, disinfectant, antimalarial, antiseptic, antidiabetic, analgesic, anti-inflammatory, and expectorant properties (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Additionally, eucalyptus essential oil and its primary component, 1,8-cineole, have demonstrated strong antibacterial effects, particularly against the biofilm formation of methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) strains (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAspartate-semialdehyde dehydrogenase (ASADH) is a crucial enzyme involved in the biosynthesis of amino acids and the cell wall of microorganisms. This enzyme catalyzes the reduction and dephosphorylation of aspartyl-phosphate to aspartate-semialdehyde within the aspartate biosynthetic pathway (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Aspartate-semialdehyde can subsequently be reduced to homoserine, which serves as a precursor for the synthesis of methionine, threonine, and isoleucine\u0026mdash;all of which are essential amino acids. Alternatively, when aspartate-semialdehyde undergoes condensation with pyruvate and cyclization within the aspartate pathway, it forms dihydrodipicolinate, which is further converted into diaminopimelate (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Diaminopimelate (DAP) is a key metabolite required for the cross-linking of peptidoglycan polymers during bacterial cell wall synthesis (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Additionally, S-adenosylmethionine, another important product of this pathway, acts as a precursor for quorum-sensing molecules in Gram-negative bacteria, playing a vital role in virulence and infection. Given its essential role in the survival and virulence of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e, the ASADH enzyme represents a promising drug target for the development of novel antimicrobial therapies.\u003c/p\u003e\u003cp\u003eAn increasingly important strategy in drug discovery involves the use of in silico studies (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). This computational approach predicts how molecular ligands interact with protein targets to form stable complexes. In silico methods are widely employed in drug discovery due to their cost-effectiveness, time efficiency, and high predictive accuracy (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Therefore, this study aimed to evaluate the \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e inhibitory potential of bioactive compounds from \u003cem\u003eEucalyptus globulus\u003c/em\u003e by targeting Aspartate-semialdehyde dehydrogenase (ASADH) using an in silico approach.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eLigand Selection\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBioactive compounds obtained from \u003cem\u003eEucalyptus globulus\u003c/em\u003e essential oil was used in this study. Fifteen (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) bioactive compounds including cineole (2758), alpha-phellandrene (CID: 7460), D-Limonene (CID: 440917), m-cymene (CID: 10812), terpinen-4-ol (CID: 11230), globulol (CID: 12304985), spathulenol (CID: 92231), carvacrol (CID: 10364), carvotanacetone (CID: 6432475), thymol (CID: 6989 ), linalool (CID: 6549), 6-Camphenol (CID: 180537), 6-Camphenone (CID: 14088350 ), piperitone (CID: 6987) and Sabinyl acetate (CID: 94266) from eucalyptus essential oil were selected based on literature survey(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Isoniazid (CID; 3767) was selected as the control ligand. The selected bioactive compounds and control ligands are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The 3D structures of all the ligands molecules in structure data format (SDF) and canonical SMILES were obtained from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSelected bioactive compounds and the control drug\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS/N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFORMULA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCANONICAL SMILES\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCineole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1(C2CCC(O1)(CC2)C)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlpha-Phellandrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7460\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1\u0026thinsp;=\u0026thinsp;CCC(C\u0026thinsp;=\u0026thinsp;C1)C(C)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD-Limonene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e440917\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1\u0026thinsp;=\u0026thinsp;CCC(CC1)C(=\u0026thinsp;C)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM-cymene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10812\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1\u0026thinsp;=\u0026thinsp;CC(=\u0026thinsp;CC\u0026thinsp;=\u0026thinsp;C1)C(C)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTerpinen-4-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11230\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1\u0026thinsp;=\u0026thinsp;CCC(CC1)(C(C)C)O\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGlobulol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12304985\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1CCC2C1C3C(C3(C)C)CCC2(C)O\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpathunelol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e92231\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1(C2C1C3C(CCC3(C)O)C(=\u0026thinsp;C)CC2)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCarvacrol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10364\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1\u0026thinsp;=\u0026thinsp;C(C\u0026thinsp;=\u0026thinsp;C(C\u0026thinsp;=\u0026thinsp;C1)C(C)C)O\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCarvotanacetone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6432475\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1\u0026thinsp;=\u0026thinsp;CCC(CC1\u0026thinsp;=\u0026thinsp;O)C(C)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThymol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6989\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1\u0026thinsp;=\u0026thinsp;CC(=\u0026thinsp;C(C\u0026thinsp;=\u0026thinsp;C1)C(C)C)O\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLinalool\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6549\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC(=\u0026thinsp;CCCC(C)(C\u0026thinsp;=\u0026thinsp;C)O)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6-Camphenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e180537\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1(C2CC(C1\u0026thinsp;=\u0026thinsp;C)C(C2)O)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6-Camphenone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14088350\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC1(C2CC(C1\u0026thinsp;=\u0026thinsp;C)CC2\u0026thinsp;=\u0026thinsp;O)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePiperitone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6987\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC(C)C12CC1C(=\u0026thinsp;C)C(C2)OC(=\u0026thinsp;O)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSabinyl acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e94266\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC(C)C12CC1C(=\u0026thinsp;C)C(C2)OC(=\u0026thinsp;O)C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIsoniazid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3767\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC1\u0026thinsp;=\u0026thinsp;CN\u0026thinsp;=\u0026thinsp;CC\u0026thinsp;=\u0026thinsp;C1C(=\u0026thinsp;O)NN\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProtein Target Selection\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe target protein, \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e Aspartate-semialdehyde dehydrogenase (ASADH) was selected using literature (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The 3D crystal structures of the protein (PDB: 3TZ6) in the form of atomic coordinates was obtained from Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the structure of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e Aspartate-semialdehyde dehydrogenase (ASADH).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eProtein targets preparation\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe 3D crystallized structure of the target protein; \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e Aspartate-semialdehyde dehydrogenase (ASADH), was accomplished using the protein preparation module of Schr\u0026ouml;dinger\u0026rsquo;s molecular modeling suit. Protein preparation was carried out by removal of unwanted water molecules, heteroatoms, and co-crystallized ligands coordinates to obtain a more suitable and stable conformation. The protein was minimized for molecular docking using OPLS 4 force field and saved in PDB format.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eActive Site Generation\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe grid orientation of the active site was simulated from the binding conformation of the target protein co-crystallized ligand using the grid module of Schr\u0026ouml;dinger\u0026rsquo;s molecular modeling suit. X, Y, Z coordinates of the gird box are 238.99, 50.38, and 75.95 respectively.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eDrug-likeness Screening\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe fifteen (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) bioactive compounds and the two control drugs were screened for their drug likeness properties using the SwissADME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swissadme.ch/\u003c/span\u003e\u003cspan address=\"http://swissadme.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) online server. These compounds were subjected to drug-likeness screening using the parameters hypothesized by Lipinski, Verber, and Egan. Fourteen (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) out of the fifteen (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) selected bioactive compounds did not violate Lipinski rule and were subjected to molecular docking.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eLigand Preparation\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe fourteen screened compounds were imported into the LigPrep workflow. OPLS4 forcefield was used due its accuracy in obtaining improved docking poses and conformational analyses (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Tautomers were generated for each ligand and the PH specified for generation of ionization state was 7.0 +_ 2.0\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMolecular Docking\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eGrid module of Schr\u0026ouml;dinger\u0026rsquo;s molecular modeling suit was used to for molecular docking of the protein target and ligands. The prepared ligands and the generated grid file were imported into the program. Standard Precision (SP) docking mode was used and docking was performed flexibly.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eADMET Evaluation\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe absorption, distribution, metabolism, excretion and toxicity (ADMET) of the top ligands obtained from the molecular docking and the control was evaluated using ADMETLab online server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://admetmesh.scbdd.com/service/evaluation/cal\u003c/span\u003e\u003cspan address=\"https://admetmesh.scbdd.com/service/evaluation/cal\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The pharmacokinetic properties were then predicted from the results.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDrug-likeness Screening\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eDrug likeness screening results as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicated that only M-cymene out of the fifteen bioactive compounds selected from Eucalyptus essential oil violated Lipinski\u0026rsquo;s drug likeness rule and was therefore eliminated for further analysis. The control ligand; Isoniazid passed the drug likeness screening. The remaining fourteen compounds that passed the drug-likeness screening and the control ligand were subjected for docking analysis with \u003cem\u003eMtb\u003c/em\u003e-ASADH.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMolecular Docking\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eResults from the molecular docking study of the fourteen bioactive compounds that passed the drug-likeness screening and the control ligand revealed their docking score, ligand interaction, as well as their hydrogen binding affinities.\u003c/p\u003e\u003cp\u003eThe results of the bioactive compounds from eucalyptus plant presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e predicts their binding affinity and suggest potential biological activity against the target protein \u003cem\u003eMtb\u003c/em\u003e-ASADH. Docking score indicates that thymol (-5.445) had the highest docking score, followed by Sabinyl acetate (-5.097). These two ligands had a higher docking score than Isoniazid (-5.040) indicating a better binding affinity and potential biological activity against the target. Amino acids residues observed in the hydrogen bond interaction of sabinyl acetate with the target protein were Asn 94 and Arg 99 while amino acids residues observed with the hydrogen bond interaction of thymol with the target protein were Arg 99, Asn 94 and Lys 227.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the interaction of thymol, sabinyl acetate and isoniazid with the target.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eScreening results of drug-likeness bioactive compounds and control drug using SwissADME web tool\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMolecule\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eXLOGP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTPSA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLipinski\u003c/p\u003e\u003cp\u003e#Violation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGhose\u003c/p\u003e\u003cp\u003e#Violation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eVeber\u003c/p\u003e\u003cp\u003e#Violation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eEgan\u003c/p\u003e\u003cp\u003eViolation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eMeugge\u003c/p\u003e\u003cp\u003e#Violation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eBioavalability\u003c/p\u003e\u003cp\u003eScore\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCineole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlpha-Phellandrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e136.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD-Limonene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e136.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM-cymene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e134.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTerpinen-4-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlobulol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e222.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpathunelol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e220.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarvacrol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e150.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarvotanacetone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e152.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThymol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e150.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLinalool\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6-Camphenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e152.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6-Camphenone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e150.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePiperitone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e152.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSabinyl acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e194.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsoniazid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e137.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMolecular docking score of the bioactive compounds and the control drug\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS/N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDocking Score\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCineole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.294\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlpha-Phellandrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7460\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.986\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD-Limonene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e440917\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-3.849\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM-cymene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10812\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.715\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTerpinen-4-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11230\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.799\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGlobulol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12304985\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.470\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpathunelol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e92231\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.519\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCarvacrol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10364\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.901\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCarvotanacetone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6432475\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.956\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThymol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6989\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-5.445\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLinalool\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6549\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6-Camphenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e180537\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.552\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6-Camphenone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14088350\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.689\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePiperitone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6987\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.982\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSabinyl acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e94266\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-5.097\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIsoniazid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3767\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-5.040\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eADMET STUDY\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe predicted absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles of the two top-scoring bioactive compounds, in comparison with the control drug, are presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. For classification model endpoints such as blood-brain barrier permeability (BBB), human intestinal absorption (H-HT), and hERG channel inhibition, prediction probabilities were categorized into six symbolic levels: 0\u0026ndash;0.1 (\u0026minus;\u0026minus;\u0026minus;), 0.1\u0026ndash;0.3 (\u0026minus;\u0026minus;), 0.3\u0026ndash;0.5 (\u0026minus;), 0.5\u0026ndash;0.7 (+), 0.7\u0026ndash;0.9 (++), and 0.9\u0026ndash;1.0 (+++). Generally, \u0026lsquo;+++\u0026rsquo; or \u0026lsquo;++\u0026rsquo; indicates a higher likelihood of toxicity or undesired effects, whereas \u0026lsquo;\u0026minus;\u0026minus;\u0026minus;\u0026rsquo; or \u0026lsquo;\u0026minus;\u0026minus;\u0026rsquo; suggests that the molecule is likely to be nontoxic or favorable (Xiong et al., 2021).\u003c/p\u003e\u003cp\u003eRegarding absorption and distribution, all top-hit compounds, including the control, were predicted to cross the blood-brain barrier (BBB). The predicted plasma protein binding (PPB) values were expressed as percentages. Sabinyl acetate and thymol exhibited PPB values of 47.56% and 93.90%, respectively, whereas isoniazid had a PPB value of 9%.\u003c/p\u003e\u003cp\u003eIn terms of metabolism, sabinyl acetate posed a lower risk of cytochrome P450 (CYP)-related drug interactions, as it showed no inhibition for any of the tested CYP enzymes. Thymol exhibited no inhibition for three out of the five tested CYP enzymes. Isoniazid, on the other hand, showed inhibition specifically for the CYP1A2 enzyme.\u003c/p\u003e\u003cp\u003eThe predicted elimination half-life (t₁/₂) values provided insights into drug excretion rates. Thymol has a half-life of 0.098, sabinyl acetate; 0.0682 while isoniazid had a half-life of 0.715.\u003c/p\u003e\u003cp\u003eIn terms of toxicity, none of the compounds demonstrated hERG inhibition, indicating a lower risk of cardiac side effects. Additionally, all compounds tested negative in the Ames mutagenicity assay, suggesting a lack of mutagenic potential. Furthermore, none of the top-hit compounds exhibited rat oral acute toxicity. However, sabinyl acetate was predicted to have carcinogenic potential.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eADMET profiling of Drug Candidates and Control Drugs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eADMET MODELS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIsoniazid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eThymol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSabinyl acetate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAbsorption \u0026amp; Distribution\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHIA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP-gp substrate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePPB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e93.90%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47.56%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMetabolism\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCYP1A2 inhibitor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCYP2C19 inhibitor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCYP2C9 inhibitor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCYP2D6 inhibitor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCYP3A4 inhibitor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eExcretion\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT1/2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.715\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.682\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.098\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eToxicity\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmes toxicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarcinogenicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRat oral acute toxicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH-HT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDILI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehERG blockers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRespiratory toxicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSR-p53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eDrug-likeness screening\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe drug-likeness rules are important in discriminating ideal drug-like compounds from false positives. This assertion is supported by a study conducted by (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The drug likeness properties of the bioactive compounds and the control were determined using Lipinski\u0026rsquo;s, Ghose\u0026rsquo;s, Veber\u0026rsquo;s, Egan\u0026rsquo;s and Muegge\u0026rsquo;s drug likeness rule. The top rank hit bioactive compounds for this study was selected from compounds that passed at least three out of the five rules. These drug-likeness rules are major determinants of oral bioavailability potential of any drug molecule i.e., the probability of drug molecule being readily available to target tissues after being absorbed into the body system. This concept is correlated with previous studies(\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The control in this study; isoniazid, violated two of the rules. Sabinyl acetate, thymol and the rest of the bioactive compounds excluding M-cymene passed at least three of the drug-likeness rules including the Lipinski\u0026rsquo;s rule of five indicating that these compounds can potentially be suitable for oral use. Lipinski\u0026rsquo;s rule is a set of conditions that defines oral bioavailability of a compound based on its chemical and physical properties of the compound (\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). According to the rule, a drug is considered to be suitable for oral activity if it has no more than five hydrogen bond donors, no more than ten hydrogen bond acceptors, molecular mass\u0026thinsp;\u0026le;\u0026thinsp;500 Daltons and an octanol water partition co-efficient (XlogP) not greater than five. Saganuwan (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), reported that the molecular mass of a compound exerts an influence on a compound\u0026rsquo;s efficacy as a drug, the molecular weight of a drug determines its oral use and cellular permeability. Bioactive compound with molecular weight within the Lipinski\u0026rsquo;s range has an optimal permeability that makes it easy to cross cellular membranes and reach its target within a short time. XlogP is a measure of a compound\u0026rsquo;s hydrophobicity and it represents the ratio of the concentration of a compound in octanol to its concentration in water. XlogP is a relevant parameter that influences the drug-likeness property of a compound by determining its permeability through cell membranes and thereby aiding their absorption(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The result presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicates that the top hit selected bioactive compounds in this study has a good druggability and can be used as oral drugs.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eMolecular docking\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMolecular docking result indicated that all 14 compounds selected for the study docked to the active site of the target protein. However, Sabinyl acetate and thymol were the only ligands with docking score higher than the control. The docking score of Sabinyl acetate and thymol was \u0026minus;\u0026thinsp;5.097 and \u0026minus;\u0026thinsp;5.445 and respectively while the docking score of the Isoniazid was \u0026minus;\u0026thinsp;5.040 as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This observation indicates a strong binding affinity between \u003cem\u003eMycobacterium Tuberculosis\u003c/em\u003e ASADH and the two bioactive compounds and suggests that these compounds can effectively interact with ASADH, inhibit its action, modulate its function and elicit a potentially strong therapeutic action. Previous studies have reported that binding affinity reflects the strength of interaction and the ability of ligand molecules to modulate biological functions (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Furthermore, Sabinyl acetate interacted with \u003cem\u003eMycobacterium Tuberculosis\u003c/em\u003e ASADH active site with 2 hydrogen bonds at amino acid residues; Arg99 and Asn94, Thymol interacted with the protein\u0026rsquo;s active site with 1 hydrogen bond at amino acid residue; Arg99, while isoniazid interacted with the protein\u0026rsquo;s active site with 3 hydrogen bonds at amino acid residue; Arg99, Lys227 and Asn94. The H-bond interactions observed in Sabinyl acetate and thymol indicates a high degree of binding and molecular interactions between the protein-ligand complex. This suggests a high degree of binding activity and molecular interaction. This aligns with previous studies (\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) that reported hydrogen bond is fundamental, enhances stability and essential of effective binding. Moreover, the amino acid residues in the key active site of the protein interacting with the ligands through hydrogen bonding correlates with the studies done by (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) where they showed that Arg99, Lys227 and others are the interacting amino acid residues of \u003cem\u003eMtb\u003c/em\u003e-ASADH. Therefore, binding the compounds to \u003cem\u003eMtb\u003c/em\u003e-ASADH could offer some antimycobacterial effects in tuberculosis treatment by the inhibition of \u003cem\u003eMtb\u003c/em\u003e-ASADH. Figures\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows interaction of compound in sabinyl acetate, thymol and isoniazid with \u003cem\u003ethe target protein\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eADMET\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe absorption, distribution, metabolism, excretion and toxicity profiles of abinyl acetate, thymol and the control are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. ADMET profiling is very essential because it determines the pharmacokinetics of our hit compounds, analyzes its possible action in the body and helps assess its efficacy (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHuman intestinal absorption (HIA) is a crucial parameter in evaluating the oral bioavailability of a compound, as it determines how effectively a drug enters the systemic circulation following administration. High HIA is generally associated with improved absorption, enhanced therapeutic efficacy, and a more favorable dosing regimen (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). In this study, both thymol and sabinyl acetate were predicted to have favorable levels of HIA, indicating absorption through the gastrointestinal tract. This may increase their suitability for oral delivery as antimycobacterial agents.\u003c/p\u003e\u003cp\u003eAnother important pharmacokinetic property is blood\u0026ndash;brain barrier (BBB) permeability, which restricts the entry of compounds into the central nervous system (CNS) (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) and presents a major challenge in treating infections such as CNS tuberculosis (CNS-TB), a severe and often fatal manifestation of TB caused by the infiltration of Mycobacterium species into the CNS (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Both thymol and sabinyl acetate, along with the control drug isoniazid, were predicted to be BBB permeant, suggesting that they have the ability to cross the BBB and may therefore be effective in the treatment of CNS-TB.\u003c/p\u003e\u003cp\u003ePlasma protein binding (PPB) is another important factor influencing the pharmacokinetics of drug candidates, as it determines the fraction of drug available in its free, active form to interact with the biological target. Proteins such as serum albumin and α1-acid glycoprotein are known to bind a wide range of drugs, often affecting their efficacy. In general, a PPB value exceeding 90% can reduce the amount of free drug available and potentially attenuate its therapeutic effect. In this study, sabinyl acetate exhibited a moderate PPB score of 47.56%, indicating that a significant proportion of the compound remains unbound and available for therapeutic action. Isoniazid showed a low PPB score of 9.00%, further confirming its availability in free form. Thymol, however, had a high PPB score of 93.90%, which may limit its immediate bioavailability. Nonetheless, studies suggest that high PPB does not always correlate directly with reduced potency, as several clinically effective drugs exhibit high plasma protein binding yet retain strong pharmacological activity(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe cytochrome P450 (CYP) family of enzymes, primarily expressed in the liver, plays a central role in drug metabolism. Inhibition of these enzymes can lead to drug\u0026ndash;drug interactions, reduced drug clearance, accumulation of toxic metabolites, or decreased therapeutic efficacy (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). In this study, sabinyl acetate was predicted not to inhibit any of the major CYP isoforms, suggesting a low potential for enzyme-related interactions. Thymol was also predicted not to inhibit CYP3A4, one of the most clinically significant isoforms. These findings indicate that both compounds are unlikely to cause CYP-mediated drug\u0026ndash;drug interactions, thereby supporting their metabolic stability and safety in potential combination therapies.\u003c/p\u003e\u003cp\u003eHalf-life is another critical pharmacokinetic parameter that reflects the duration a drug remains active in the body and influences dosing frequency. Based on empirical classification, compounds with a half-life between 0 and 0.3 hours are considered to have excellent metabolic clearance, those between 0.3 and 0.7 hours are moderate, while values between 0.7 and 1.0 hours are considered poor (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).Thymol and sabinyl acetate recorded half-lives of 0.098 and 0.0682 respectively, which fall within the excellent category, suggesting rapid clearance from the system. This indicates favorable metabolic processing.\u003c/p\u003e\u003cp\u003eIn terms of toxicity, both thymol and sabinyl acetate demonstrated favorable safety profiles. The two compounds showed low predicted binding affinity to the human ether-\u0026agrave;-go-go-related gene (hERG) potassium channel, which plays a vital role in the repolarization of cardiac cells. Inhibition of this channel can result in delayed cardiac repolarization, leading to conditions such as long QT syndrome (LQTS), arrhythmias, and Torsade de Pointes (TdP) (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). The low hERG inhibition scores suggest a reduced risk of cardiotoxicity, making both compounds safer for potential therapeutic use. Furthermore, all three compounds, including the control, were Ames-negative, indicating a low likelihood of causing DNA mutations, and therefore a reduced risk of carcinogenicity or heritable genetic damage. In addition, thymol and sabinyl acetate were predicted to be non-hepatotoxic, signifying minimal potential for liver damage or dysfunction, which further supports their safety for therapeutic development. However, it is important to note that sabinyl acetate was predicted to be carcinogenic, unlike thymol and isoniazid, which were both classified as non-carcinogenic. Thus, the findings of this study suggest that the active compounds thymol and sabinyl acetate may exhibit strong in silico inhibitory activity against \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e aspartate-semialdehyde dehydrogenase, as evidenced by molecular docking analysis. However, these computational results require further validation through molecular dynamics simulations and experimental (in vitro and in vivo) studies to confirm their therapeutic potential.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTuberculosis (TB) remains the leading cause of death from a single infectious agent globally. The emergence of multidrug-resistant TB (MDR-TB) has intensified the urgency to develop more effective and safer therapeutic alternatives. This study evaluated the antimycobacterial potential of bioactive compounds derived from \u003cem\u003eEucalyptus globulus\u003c/em\u003e against \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e aspartate-semialdehyde dehydrogenase, using isoniazid, a frontline anti-TB drug, as the control. Drug-likeness screening and molecular docking analyses identified thymol and sabinyl acetate as the top-performing compounds, both demonstrating strong binding affinity and stable hydrogen interactions with the \u003cem\u003eMtb\u003c/em\u003e-ASADH. ADMET profiling further supported their potential as promising drug candidates. The compounds were predicted to cross the blood\u0026ndash;brain barrier, showed no significant inhibition of cytochrome P450 enzymes, and exhibited favorable half-lives. Although thymol displayed high plasma protein binding and sabinyl acetate showed carcinogenic potential, their overall pharmacokinetic and safety profiles suggest a strong therapeutic promise. The findings from this in silico study provide a foundation for further validation through in vitro and in vivo experiments to confirm their efficacy and safety as novel antimycobacterial agents.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e– Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e – Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e– All data generated and analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e– The author(s) declare(s) that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e – Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u0026nbsp;\u003c/strong\u003e– SMJ designed and executed experiments, performed the docking analysis and wrote the manuscript. MAB and NA designed and executed experiments, obtained protein and ligand structures and edited the manuscript. IA input on data analysis and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e – Not Applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization. Tuberculosis (TB) [Internet]. 2024 [cited 2025 Jun 19]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/news-room/fact-sheets/detail/tuberculosis\u003c/span\u003e\u003cspan address=\"https://www.who.int/news-room/fact-sheets/detail/tuberculosis\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNatarajan A, Beena PM, Devnikar AV, Mali S. A systemic review on tuberculosis. 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Eur J Pharmacol. 2004;500(1\u0026ndash;3):129\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tuberculosis, Eucalyptus globulus, Antimycobacterial activity, In silico drug discovery, ADMET","lastPublishedDoi":"10.21203/rs.3.rs-7337220/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7337220/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eTuberculosis is the leading cause of death from a single infectious agent worldwide. Tuberculosis accounted for around 1.25\u0026nbsp;million deaths globally in 2023. The rise of Multidrug-resistant tuberculosis and the reported side effects of these available anti-TB drugs has complicated the control of the disease. This highlights the urgent need to discover and develop safer and more effective therapies for tuberculosis. \u003cem\u003eEucalyptus globulus\u003c/em\u003e is a tree widely distributed across tropical and subtropical regions with numerous medicinal benefits.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe antimycobacterial potential of \u003cem\u003eEucalyptus globulus\u003c/em\u003e against \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e Aspartate-semialdehyde dehydrogenase (ASADH); an enzyme responsible for the bacterium\u0026rsquo;s growth and virulence was accessed using in silico studies. Fifteen bioactive compounds from \u003cem\u003eEucalyptus globulus\u003c/em\u003e were selected based on literature. Isoniazid; a first line anti-TB drug was used as our control. SwissADME online server was used to evaluate pharmacokinetic properties of the compounds. The compounds were further prepared to obtain the best docking poses using LigPrep module in the Maestro\u0026rsquo;s Schrodinger software. The compounds were docked into the protein's active site using Schr\u0026ouml;dinger's Maestro software\u0026rsquo;s Glide module and the resulting protein-ligand complexes was studied. Drug-likeness screening showed that only M-cymene violated Lipinski\u0026rsquo;s rule. Molecular docking revealed that thymol (-5.445) and sabinyl acetate (-5.097) has a higher docking score than isoniazid (-5.040), indicating a better binding affinity and biological activity against the target compared to the control (-5.040). Hydrogen bonds interaction of suggesting high strength of binding and molecular interactions that occurred between the protein-ligand complexes suggesting a strong antimycobacterial inhibitory effect.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eIn silico analysis from this study identifies thymol and sabinyl acetate from \u003cem\u003eEucalyptus globulus\u003c/em\u003e as promising candidates for further development as anti-TB treatments and show better docking with the target protein compared to isoniazid.\u003c/p\u003e","manuscriptTitle":"Antimycobacterial Activity of Bioactive Compounds from Eucalyptus globulus against Mycobacterium tuberculosis Aspartate-semialdehyde Dehydrogenase: In silico Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 12:37:49","doi":"10.21203/rs.3.rs-7337220/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41791d00-8f1b-4769-ace0-9ac77dc0948d","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T06:24:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 12:37:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7337220","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7337220","identity":"rs-7337220","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}