In Silico, Chemical Profiling and In Vitro Analysis of Ethanolic Preparation of Hydrocotyle Asiatica Q and 6C Against Staphylococcus aureus

Abstract Antimicrobial resistance is a critical global health threat, with Staphylococcus aureus serving as a primary pathogen in diverse human infections. This study aimed to characterize the bioactive constituents of Hydrocotyle asiatica (Mother Tincture Q and 6C) and evaluate their antimicrobial efficacy against S. aureus. Metabolic profiling was performed using ultra-performance liquid chromatography (LC)-quadrupole time-of-flight mass spectrometry (MS). Pharmacokinetic viability and drug-likeness were assessed via pkCSM and Lipinski's “Rule of Five.” Molecular docking was conducted using the PyRx software against eight S. aureus target proteins. In vitro antimicrobial activity was determined through disk diffusion and broth microdilution assays (minimum inhibitory concentration) against S. aureus (ATCC [American Type Culture Collection] 6538), with statistical significance evaluated via analysis of variance and Dunnett's post hoc test (p < 0.05). LC-MS analysis identified 15 compounds in Hydrocotyle asiatica Q and 6 in 6C, with Alpha-chamigrene, Apigenin, and Bayogenin meeting all drug-likeness criteria. Bayogenin exhibited the highest binding affinity (–9.7 kcal/mol) against the toxic shock syndrome toxin-1 (Protein Data Bank: 2QIL). In vitro assays demonstrated that Hydrocotyle asiatica Q produced a significant zone of inhibition (15.3 ± 0.573 mm), representing 42.27% inhibition relative to the positive control (gentamicin). Broth microdilution confirmed that 100 µL of Mother Tincture (Q) and 50 and 100 µL of 6C potency exhibited statistically significant bacterial growth inhibition (p < 0.016) compared with negative ethanol controls. The findings suggest that homoeopathic preparations of Hydrocotyle asiatica possess measurable antimicrobial properties and favorable pharmacokinetic drug properties. The study provides a scientific rationale for its potential as a source of bioactive antimicrobial agents.