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INTRODUCTION
Alcoholic Liver Disease (ALD) is a sequence of hepatic diseases, ranging from alcoholic fatty liver to alcoholic hepatitis, ultimately leading to alcoholic cirrhosis, the most advanced and largely irreversible stage of alcohol-induced liver injury (Patel et al., 2024). Alcohol consumption causes about 3.3 million deaths each year worldwide, representing 5.9% of global mortality, and also leads to both acute and chronic liver damage that can result in ALD. (Nagarjuna et al., 2025). The leading cause of cirrhosis (43.2%) in India is alcohol consumption, followed by NAFLD/Cryptogenic liver disease (14.4%), Hepatitis B Virus (11.5%), and Hepatitis C Virus (6.2%), with a significant rise in alcohol-related cirrhosis over recent decades and a concurrent decline in viral etiologies (Elhence et al., 2024).
ALD is considered completely irreversible there are currently no FDA-approved anti-fibrotic therapies for cirrhosis, and therapeutic options remain limited (Nagarjuna et al., 2025; Ohashi et al., 2018). Conventional treatments suggest corticosteroids as the standard treatment for severe alcoholic hepatitis; however, they provide only short-term survival benefit without significant improvement in long-term outcomes, with six-month mortality remaining high (Nagarjuna et al., 2025; Ohashi et al., 2018). There is no treatment for patients with advanced liver disease, liver transplantation is the only option, yet reduced feasibility, rendering the need for alternative and adjunctive therapeutic strategies (Ohashi et al., 2018).
Alcohol Dehydrogenases (ADHs) play an important function in ethanol metabolism. Pharmacological inhibition of ADH using agents such as fomepizole (4-methylpyrazole), a non-specific ADH inhibitor currently used for methanol and ethylene glycol poisoning, has been proposed as a potential strategy to prevent the formation of toxic metabolites in substance intoxication (Di et al., 2021). Traditional Siddha medicine offers various formulations to promote liver health and manage liver diseases, representing the therapeutic strength of this system of medicine (Subramani et al., 2017).
Beyond conventional treatment strategies, there is growing scientific interest in traditional herbal remedies, especially polyherbal formulations, due to their potential to serve as sources of effective therapeutic remedies (Kotmire et al., 2024). This study aimed to evaluate the drug-likeness and inhibitory potential of bioactive compounds from Thaalisaathi Chooranam against human alcohol dehydrogenase using in silico ADME analysis and molecular docking studies.
MATERIALS AND METHODS
In this study, previously published GC-MS data of Thaalisaathi Chooranam (Mudhaliyar, 1992) were analyzed, which identified 43 compounds (Kumarasamy et al., 2016). From these, five small molecules were shortlisted based on peak area percentage after excluding fatty acids, due to their high structural flexibility and nonspecific Hydrophobic Interactions, which may produce unreliable binding scores. The selected compounds were evaluated for pharmacokinetic properties, drug-likeness, and lead-likeness using the SwissADME web tool (http://www.swissadme.ch/) in accordance with Lipinski’s Rule of Five. Compounds that met ADME criteria were subsequently subjected to molecular docking against human alpha-type alcohol dehydrogenase (PDB ID: 1HSO) using iGEMDOCK v2.0 (Yang et al., 2004), and their binding affinities and protein-ligand interactions were analyzed. The method is represented diagrammatically in Figure 1.
Selection of Molecules and Ligand Preparation
The following Five Phyto-compounds (Table 1) were selected as ligands based on peak area percentage for drug-likeness analysis and ADME studies (Figure 2). The 5 selected ligand canonical SMILES were obtained from the PubChem website (https://pubchem.ncbi.nlm.nih.gov/). The canonical SMILES were converted to PDB format using Cactus software (https://cactus.nci.nih.gov/translate/).
| Compounds | 2-Oxo-5,11(13)-eudesmadien-12,8-olide | 4-Cyano-4-(3,4-methylenedioxyphenyl) cyclohexanone | Dihydroqinghaosu | Apiole | Patchoulene |
|---|---|---|---|---|---|
| A | B | C | D | E | |
| IUPAC Name | 5,8a-dimethyl-3-methylidene-3a,5,6,8,9,9a hexahydrobenzo[f][1] benzofuran-2,7-dione | 1-(benzo[d][1,3] dioxol-5-yl)-4-oxocyclohexane-1-carbonitrile | (4S,5R,8S,9R,10S,12R,13R)-1,5,9-trimethyl-11,14,15,16 tetraoxatetracyclo [10.3.1.04,13.08,13] hexadecan-10-ol | 4,7-dimethoxy-5-prop-2-enyl-1,3-benzodioxole | 1,5,11,11-tetramethyltricyclo [6.2.1.02,6] undec-2(6)-ene |
| Canonical SMILES | CC1CC(=O) CC2(C1=CC3C(C2) OC(=O)C3=C)C | N#CC1(C2=C/C3=C(\C=C/2) OCO3) CCC(=O)CC1 | C[C@@H]1CC[C@H]2[C@H] ([C@H] (O[C@H]3[C@@]24[C@H]1 CCC(O3)(OO4)C)O)C | COC1=C2C(=C(C(=C1) CC=C) OC) OCO2 | C[C@@H]1CCC2=C1C[C@@H]3CC[C@@]2(C3(C)C)C |
| Chemical Structure | |||||
| Molecular Formula | C15H18O3 | C14H13NO3 | C15H24O5 | C12H14O4 | C15H24 |
| Molecular Weight | 246.30 | 243.26 | 284.35 | 222.24 | 204.35 |
| H bond donor | 0 | 0 | 1 | 0 | 0 |
| H bond acceptor | 3 | 4 | 5 | 4 | 0 |
| Log p | 2.27 | 2.04 | 2.17 | 2.44 | 3.76 |
| Molar refractivity | 68.15 | 64.03 | 71.34 | 59.59 | 66.88 |
| No.heavy atoms | 18 | 18 | 20 | 16 | 15 |
| No.Aromatic heavy atoms | 0 | 6 | 0 | 6 | 0 |
| Fraction Csp3 | 0.60 | 0.43 | 1.00 | 0.33 | 0.87 |
| No rotatable bonds | 0 | 1 | 0 | 4 | 0 |
Ethical statement
Not applicable as this is an in silico computational study not involving live human or animal subjects.
RESULTS
Selection of Target protein
The human liver alpha-type alcohol dehydrogenase enzyme (PDB ID: 1HSO) protein structure was acquired from the protein data bank (www.rcsb.org). The PDB format extraction was used to study the protein's crystal structure. The structure is represented in Figure 2d. Preliminary virtual screening of the extracted compounds was carried out using the iGEMDOCK v2.0 molecular docking program (Yang et al., 2004). Based on the binding energy scores obtained, the compounds of TSC demonstrating the highest binding affinity were shortlisted and subsequently subjected to detailed molecular docking analysis for further evaluation of their interaction with the target protein.
DISCUSSION
The selection of ADH1A (Hadi et al., 2023) as the molecular target is scientifically coherent, given that its inhibition could reduce the generation of acetaldehyde, a toxic ethanol metabolite central to ALD pathogenesis. The formulation's constituent Triphala Chooranam has documented ADH-inhibitory potential (Banjan et al., 2025), and Coriandrum sativum (Dhanalakshmi et al., 2019) (present in relatively high proportions) has demonstrated beneficial effects in Alcohol Use Disorder clinically. Classical Siddha literature (Siddha Vaithiya Thirattu) (Mudhaliyar et al., 1992) further indicates Thaalisaathi Chooranam in liver cirrhosis (Eeral Ularal). The present in silico investigation evaluated five phytochemical constituents (Compounds A-E) from GC-MS profile of Thaalisaathi Chooranam, a classical Siddha polyherbal formulation, as the inhibitors of human alpha-type alcohol dehydrogenase (ADH1A; PDB ID: 1HSO) (Figure 2).
The physicochemical parameters of Compounds A-E (from Table 1) demonstrated full compliance with Lipinski's Rule of Five (MW ≤ 500 g/mol; Log p≤ 5; HBD ≤ 5; HBA ≤ 10), with molecular weights (204.35-284.35 g/mol) and consensus Log p values (2.04-3.76) falling within the oral bioavailability range of 1-3, associated with successful oral drug candidates in approved pharmaceutical inventories (Lipinski et al., 1997). All compounds additionally satisfied the Ghose, Veber, and Egan filters, reinforcing their pharmacokinetic acceptability. Compounds A-D exhibited moderate lipophilicity (consensus Log P ≈ 2.0-2.4), considered optimal for balancing oral absorption with solubility. These observations agree with the established pharmacokinetic principle that excessive lipophilicity can impair aqueous dissolution and limit bioavailability (Table 3). Predicted aqueous solubility across ESOL, Ali, and Silicos-IT models consistently classified all compounds as "Soluble" (log S>-4), with Compound A demonstrating the highest solubility (ESOL log S =-2.27) and Compound E the lowest (-3.81), still within acceptable drug-like limits (Tables 1 and 2).
| Compounds | 2-Oxo-5,11(13)-eudesmadien-12,8- olide | 4-Cyano-4-(3,4-methylenedioxyphenyl) cyclohexanone | Dihydroqinghaosu | Apiole | Patchoulene | |
|---|---|---|---|---|---|---|
| TPSA | 43.37 Å2 | 59.32 Å2 | 57.15 Å2 | 36.92 Å2 | 0.00 Å2 | |
| iLOGP | 2.18 | 1.95 | 2.49 | 2.85 | 0.00 | |
| XLOGP3 | 1.44 | 1.42 | 2.99 | 2.75 | 4.29 | |
| WLOGP | 2.42 | 2.32 | 2.19 | 2.16 | 4.56 | |
| MLOGP | 2.38 | 1.31 | 1.95 | 1.40 | 5.65 | |
| Silicos-IT Log P | 2.93 | 3.17 | 1.23 | 3.02 | 4.30 | |
| Consensus Log P | 2.27 | 2.04 | 2.17 | 2.44 | 3.76 | |
| ESOL log S | -2.27 | -2.42 | -3.49 | -2.96 | -3.81 | |
| ESOL solubility [mg/mL] | 1.31e+00 mg/mL | 9.17e-01 mg/mL | 9.27e-02 mg/mL | 2.42e-01 mg/mL | 3.17e-02 mg/mL | |
| ESOL solubility [mol/l] | 5.32e-03 mol/l | 3.77e-03 mol/l | 3.26e-04 mol/l | 1.09e-03 mol/l | 1.55e-04 mol/l | |
| ESOL class | Soluble | Soluble | Soluble | Soluble | Soluble | |
| Ali log S | -1.96 | -2.27 | -3.85 | -3.18 | -4.00 | |
| Ali solubility [mg/mL] | 2.72e+00 mg/mL | 1.31+00 mg/mL | 3.98e-02 mg/mL | 1.47e-01 mg/mL | 2.03e-02 mg/mL | |
| Ali solubility [mol/l] | 1.11e-02 mol/l | 5.36e-03 mol/l | 1.40e-04 mol/l | 6.60e-04 mol/l | 9.93e-05 mol/l | |
| Ali class | Very soluble | Soluble | Soluble | Soluble | Moderately Soluble | |
| Silicos-IT log S | -2.94 | -3.79 | -1.34 | -3.26 | -3.97 | |
| Silicos-IT solubility [mg/mL] | 2.83e-01 mg/mL | 3.97e-02 mg/mL | 1.30+01 mg/mL | 1.21e-01 mg/mL | 2.19e-02 mg/mL | |
| Silicos-IT solubility [mol/mL] | 1.15e-03 mol/l | 1.63e-04 mol/l | 4.58e-02 mol/l | 5.45e-04 mol/l | 1.07e-04 mol/l | |
| Silicos-IT class | Soluble | Soluble | Soluble | Soluble | Soluble | |
| Lipinski # violations | Yes; 0 Violation | Yes; 0 violation | Yes; 0 violation | Yes;0 violation | Yes; 1 violation: MLOGP>4.15 | |
| Ghose # violations | Yes | Yes | Yes | Yes | Yes | |
| Veber # violations | Yes | Yes | Yes | Yes | Yes | |
| Egan # violations | Yes | Yes | Yes | Yes | Yes | |
| Muegge # violations | Yes | Yes | Yes | Yes | No;1 violation: Heteroatoms<2 | |
| Bioavailability score | 0.55 | 0.55 | 0.55 | 0.55 | 0.55 | |
| PAINS # alerts | 0 alert | 0 alert | 0 alert | 0 alert | 0 alert | |
| Brenk # alerts | 2 alerts: isolated_alkene, Michael_acceptor_1 | 0 alert | 1 alert: Peroxide | 1 Alert: isolated_ alkene | 1 alert: isolated_ alkene | |
| Leadlikeness # violations | No;1 violation: MW < 250 | No; 1 violation: MW<250 | Yes | No;1 violation: MW<250 | No;2 violations: MW<250, XLOGP3>3.5 | |
| Synthetic accessibility | 4.17 | 2.24 | 6.59 | 2.57 | 5.22 | |
Topological polar surface area values (0.00-59.32 Ų), established intestinal absorption threshold of 140 Ų and the BBB permeability reference limit of 90 Ų, alongside rotatable bond counts (0-4) substantially below Veber's ≤10 threshold, collectively predict high gastrointestinal absorption and favourable membrane permeability for all compounds. Compound E's TPSA of 0.00 Ų reflects complete non-polarity and strong hydrophobic character, consistent with its higher consensus Log P (3.76) and lower predicted aqueous solubility (Table 2).
The absence of PAINS alerts and the uniform Abbott bioavailability score of 0.55 compare favourably against established benchmarks, given that approximately 6-10% of screening library compounds carry PAINS liabilities and a score of ≥0.55 is considered acceptable for early-stage oral candidates (Baell et al., 2010) (Table). Molar refractivity values (59.59-71.34) fell within the Ghose filter range (40-130), and rotatable bond counts (0-4) were well below Veber's threshold of ≤10, indicating optimal molecular flexibility conducive to receptor binding. The Bioavailability Radar profiles (Figure 1) for all compounds predominantly occupied the optimal physicochemical space, and a uniform Abbott bioavailability score of 0.55 was predicted, suggesting a moderate but acceptable probability of oral bioavailability Table 2 (Martin et al., 2005).
All compounds except Compound E show high GI absorption and BBB permeability, indicating favorable oral bioavailability and potential CNS activity; however, Compound B and D exhibit CYP inhibition, suggesting possible drug-drug interactions. Compound E demonstrates poor GI absorption, no BBB penetration, and P-gp efflux, indicating limited systemic and CNS availability despite relatively higher skin permeability (Log Kp) Table 3 (Bosco et al., 2024).
| Compound Name | 2-Oxo-5,11(13)-eudesmadien -12,8-olide | 4-Cyano-4-(3,4-methylenedioxyphenyl) cyclohexanone | Dihydroqinghaosu | Apiole | Patchoulene |
|---|---|---|---|---|---|
| LD50 | 5000 mg/kg | 4000 mg/kg | 567 mg/kg | 1000 mg/kg | 5000 mg/kg |
| Toxicity Class | 5 | 5 | 4 | 4 | 5 |
| Hepatotoxic | No | No | No | No | No |
| Water Solubility (SILICOS-IT) | Very Soluble | Soluble | Soluble | Soluble | Moderately Soluble |
| BBB Permeant | Yes | Yes | Yes | Yes | No |
| GI Absorption | High | High | High | High | Low |
| P-gp Substrate | No | Yes | No | No | Yes |
| Lipinski Rule | Yes; 0 Violation | Yes; 0 Violation | Yes; 0 Violation | Yes; 0 Violation | Yes; 1 Violation: MLOGP > 4.15 |
| CYP1A2 inhibitor | No | Yes | No | Yes | No |
| CYP2C19 inhibitor | No | Yes | No | No | No |
| CYP2C9 inhibitor | No | No | No | No | No |
| CYP2D6 inhibitor | No | No | No | No | No |
| CYP3A4 inhibitor | No | No | No | No | No |
| Log Kp [cm/s] | -6.78 cm/s | -6.78 cm/s | -5.91 cm/s | -5.70 cm/s | -4.50 cm/s |
Molecular docking analysis
Following ADME-guided exclusion of Compounds A and E, three lead compounds -Dihydroqinghaosu, 4-Cyano-4-(3,4-methylenedioxyphenyl) cyclohexanone, and Apiole -were subjected to molecular docking against the ADH1A active site using iGEMDOCK (Kiran, 2022) (population size 200; 70 generations; very slow/accurate mode). Dihydroqinghaosu exhibited the highest binding affinity (-83.54 kcal/mol), with a strong hydrogen bonding contribution of -29.32 kcal/mol involving active site residues CYS46, THR48, HIS51, and the catalytic zinc ion ZN1376 (Figure 3 and Table 4). The prominent involvement of the zinc centre is mechanistically significant, as zinc coordinates the substrate hydroxyl group during catalysis in ADH enzymes; competitive occupancy of this site could directly attenuate enzymatic activity. These interactions suggest that Dihydroqinghaosu may act through a zinc-chelation-mediated inhibitory mechanism, meriting further investigation (Asahi et al., 2023).
| Sl. No. | Compound | Energy | VDW | HBOND | AA-HBONDS |
|---|---|---|---|---|---|
| 1. | 4-Cyano-4-(3,4-methylenedioxyphenyl) cyclohexanone | -82.22 | -71.92 | -10.31 | HIS51, PYZ1378 |
| 2. | Dihydroqinghaosu | -83.54 | -57.22 | -29.32 | CYS46, THR48, HIS51, ZN1376 |
| 3. | Apiole | -80.25 | -61.21 | -19.05 | CYS46, CYS174, LEU319, ZN1376, PYZ1378 |
4-Cyano-4-(3,4-methylenedioxyphenyl) cyclohexanone ranked second (-82.22 kcal/mol), with dominant van der Waals interactions (-71.92 kcal/mol) through hydrophobic contacts with GLY47, THR178, VAL292, and VAL294, though its hydrogen bonding energy (-10.31 kcal/mol) was comparatively lower, implying reduced binding specificity. Apiole demonstrated moderate overall affinity (-80.25 kcal/mol) with balanced van der Waals (-61.21 kcal/mol) and hydrogen bonding (-19.05 kcal/mol) contributions, engaging CYS46, CYS174, LEU319, ZN1376, and PYZ1378 -a broader residue interaction profile suggestive of stable but less specific active-site accommodation.
Toxicity Prediction
Toxicity profiling via the ProTox-II web tool classified all five compounds (Table 3) under GHS Toxicity Classes 4 and 5 (LD50: 567-5000 mg/kg), indicating low to practically non-toxic acute oral toxicity profiles, with no compound demonstrating predicted hepatotoxicity -a critically favourable finding given the hepatic disease context of the study (Venkatachalam et al., 2025; Cheng et al., 2012). Aqueous solubility ranged from Very Soluble to Moderately Soluble, with high GI absorption predicted for Compounds 1-4, while Patchoulene exhibited low GI absorption, moderate solubility, and a single Lipinski violation (MLOGP>4.15), collectively reflecting a lipophilicity-driven bioavailability limitation requiring formulation-level intervention (Lipinski et al., 1997). P-glycoprotein efflux substrate activity predicted for Compounds 2 and 5 suggests potential reduction in intestinal uptake and tissue distribution, Since Compounds 1, 3, and 4 are not P-gp substrates, they are less likely to be actively effluxed, allowing better systemic availability (International Transporter Consortium et al., 2010).
LIMITATIONS
The present work represents a preliminary in silico initiative aimed at identifying potential lead compounds targeting human alcohol dehydrogenase. As the findings are based solely on computational docking predictions, further detailed in vitro and in vivo studies are required to validate the biological efficacy and therapeutic potential of the identified compounds.
CONCLUSION
The present in silico study demonstrated that among the screened compounds, Dihydroqinghaosu exhibited the highest binding affinity toward human alpha-type alcohol dehydrogenase (PDB ID: 1HSO), with the lowest total binding energy (-83.54 kcal/mol). The interaction analysis revealed significant hydrogen bonding with key active site residues, including CYS46, THR48, HIS51, and the catalytic Zinc Ion (ZN1376), indicating strong and specific binding within the enzyme’s active pocket. Compared to 4-Cyano-4-(3,4-methylenedioxyphenyl) cyclohexanone and Apiole, Dihydroqinghaosu showed superior interaction stability due to a higher hydrogen bond contribution along with favorable van der Waals interactions. These findings suggest that Dihydroqinghaosu may act as a potential modulator of alcohol dehydrogenase activity and could be considered a promising lead candidate for further pharmacological investigation in alcohol-induced liver damage.
