Polyherbal-Mediated Synthesis of Copper Nanoparticles Using Hygrophila auriculata and Leucas aspera: Cytotoxicity, Antioxidant Effect, and Antibacterial Potential Against Healthcare-Associated Pathogens

An eco-friendly green synthesis approach was employed to produce copper nanoparticles (CuNPs) using a polyherbal extract derived from two medicinally important plant species, Hygrophila auriculata (Schumach.) Heine and Leucas aspera (Willd.) Link. The plant extracts were initially subjected to phytochemical screening to identify bioactive constituents potentially involved in nanoparticle synthesis. The synthesized CuNPs were characterized using UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), gas chromatography–mass spectrometry (GC-MS), field-emission scanning electron microscopy coupled with energy-dispersive X-ray analysis (FESEM-EDAX), X-ray diffraction (XRD), and thin-layer chromatography (TLC). UV-visible spectroscopy revealed a characteristic absorption peak at 233.6 nm. FTIR analysis indicated the presence of functional groups associated with nanoparticle reduction and stabilization, whereas FESEM imaging showed predominantly spherical particles with sizes ranging 63–68 nm. Elemental composition was confirmed using EDAX analysis. XRD analysis demonstrated polycrystalline nature of the CuNPs, with an average crystallite size of 11.5 nm. GC-MS analysis and phytochemical screening further confirmed the presence of bioactive compounds, whereas TLC analysis revealed differences in mobility between the plant extract and synthesized CuNPs. Antibacterial activity of the synthesized CuNPs was evaluated using the agar well diffusion method against clinically relevant bacterial strains, including those of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Streptococcus pyogenes. The polyherbal-derived CuNPs produced larger inhibition zones than the individual plant extracts, particularly against multidrug-resistant pathogens such as P. aeruginosa and S. aureus. Additionally, the nanoparticles exhibited concentration-dependent antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl assay at concentrations ranging 10–50 mg/mL, with radical scavenging activity increasing from 29.9% to 76.5% and a corresponding decrease in absorbance from 0.698 to 0.234 (p < 0.05). Cytotoxic evaluation in HepG2 cells after 48 h of exposure demonstrated dose-dependent morphological changes and reduced cell viability. These findings suggest that polyherbal-derived CuNPs possess antibacterial, antioxidant, and cytotoxic properties with potential relevance for biomedical applications.