Harmal-Plant Extract Yields Stable Copper-Zinc Oxide Nanocomposite With Antimicrobial Activity

Researchers have synthesized a copper oxide-zinc oxide nanocomposite using nothing more elaborate than an aqueous leaf extract of Peganum harmala, the medicinal plant known as harmal or wild rue, and report that the material shows measurable antibacterial and antifungal activity in laboratory assays. The study, published in Discover Nano, combines wet-lab synthesis and characterization with computational modeling, and its authors position the work as a contribution to the search for alternatives to conventional antibiotics at a time when antimicrobial resistance continues to spread.

The synthesis route is deliberately simple. Zinc acetate dihydrate and copper sulfate monohydrate were mixed in a 5:1 zinc-to-copper molar ratio, and 20 milliliters of the plant extract was added dropwise under stirring at room temperature and neutral pH. Over four hours, the solution changed color from pale yellow to a dark greenish-brown, a visual signal the authors interpret as the formation of the nanocomposite. The extract supplies the phytochemistry that makes green synthesis work: preliminary screening detected alkaloids, flavonoids, phenols, tannins, and saponins in the leaves. Polyphenols and flavonoids, rich in hydroxyl groups, can donate electrons to reduce the metal precursors, while tannins coordinate to particle surfaces and form a steric barrier against agglomeration. The authors suggest that protonated alkaloids such as harmine and vasicine electrostatically coat the growing particles, an idea they link to the unusually high positive surface charge they measured later.

Characterization painted a consistent picture of a small, crystalline, and stable material. UV-visible spectroscopy showed a characteristic absorption peak at approximately 372 nanometers. X-ray diffraction revealed peaks matching hexagonal wurtzite ZnO alongside distinct reflections assigned to monoclinic CuO, indicating that copper oxide sits as a secondary phase deposited on the ZnO surface rather than substituted into the zinc oxide lattice; the authors note that no peak shifting or lattice distortion characteristic of copper substitution was observed. Scanning electron microscopy showed near-spherical, well-dispersed particles, and a histogram built from more than 150 measured particles gave an average primary particle size of 29 plus or minus 1.3 nanometers. Dynamic light scattering in suspension yielded a slightly larger Z-average hydrodynamic diameter of 32.3 plus or minus 3.76 nanometers, an expected difference because the hydrodynamic volume includes the hydration layer and the organic capping shell of adsorbed plant phytochemicals.

Two stability-related measurements stand out. Energy-dispersive X-ray analysis quantified copper at 24.25 percent by mass, or 7.09 percent atomic, with elemental mapping showing zinc, copper, and oxygen uniformly distributed across the analyzed region rather than segregated into separate clusters. The zeta potential, a measure of surface charge and therefore of the electrostatic repulsion that keeps particles from clumping, came in at +47.2 plus or minus 4.24 millivolts. Values of that magnitude generally indicate strong colloidal stability, and the authors attribute the positive charge to the alkaloid-rich capping layer, in contrast to chemically synthesized CuO-ZnO systems that often carry low or negative surface charges and aggregate more readily.

The antimicrobial testing was performed against three bacterial strains obtained from culture collections: Staphylococcus epidermidis (ATCC 12228), Salmonella typhi (ATCC 14028), and Klebsiella pneumoniae (ATCC 13883). Using an agar well diffusion method with three biological replicates, the nanocomposite produced concentration-dependent zones of inhibition. At 110 micrograms per milliliter, the zones measured 22.40 plus or minus 0.27 millimeters against S. epidermidis, 21.03 plus or minus 0.20 millimeters against S. typhi, and 22.60 plus or minus 0.23 millimeters against K. pneumoniae. Notably, the zone against S. epidermidis showed no statistically significant difference from the ciprofloxacin positive control at the same concentration, and the nanocomposite matched the standard antibiotic’s performance against K. pneumoniae as well. The plant extract alone was consistently the weakest treatment, which the authors take as evidence that the copper component contributes meaningfully to the activity.

Broth microdilution established minimum inhibitory concentrations of 50 plus or minus 0.50 micrograms per milliliter for S. epidermidis, 70 plus or minus 1.00 for S. typhi, and 80 plus or minus 1.50 for K. pneumoniae. Minimum bactericidal concentrations, the levels at which bacteria are killed rather than merely halted, were higher: 80 plus or minus 1.20 micrograms per milliliter for S. epidermidis, 110 plus or minus 0.80 for S. typhi, and 130 plus or minus 1.40 for K. pneumoniae. The gram-positive S. epidermidis was the most susceptible, a pattern the authors attribute to the lipopolysaccharide-rich outer membrane of gram-negative bacteria restricting nanoparticle penetration. The gap between MIC and MBC values indicates that bactericidal activity required elevated concentrations, consistent with a dose-dependent mechanism the authors propose involves membrane disruption and intracellular reactive oxygen species generation.

Antifungal results were similarly competitive. Using a test tube slant method at a single concentration of 30 micrograms per milliliter, the nanocomposite inhibited linear growth of Aspergillus flavus by 88.67 plus or minus 0.97 millimeters and Fusarium oxysporum by 74.33 plus or minus 0.36 millimeters. Statistical comparison with terbinafine, the reference antifungal, showed no significant difference against A. flavus, while the difference against F. oxysporum was minor but statistically significant. The authors are careful to note a limitation here: the minimum fungicidal concentration was not determined, so the findings demonstrate growth inhibition rather than definitive fungicidal killing, and standardized broth dilution methods will be needed to establish that.

The computational half of the study modeled a simplified 16-atom Cu1Zn7O8 cluster, built from a wurtzite ZnO supercell with a single zinc atom replaced by copper, and optimized it with density functional theory at the PBE level with dispersion correction. The calculated HOMO-LUMO gap of 1.68 electron volts is far below bulk zinc oxide’s roughly 3.3 electron volt bandgap, a narrowing the authors ascribe to quantum confinement, surface dangling bonds, and copper 3d states introducing mid-gap defect levels in the finite cluster; they caution that the physical 29-nanometer particles will retain bandgaps closer to bulk values. Projected density of states analysis suggested overlap between copper 3d and oxygen 2p states near the Fermi level, and the electrostatic potential map showed electrophilic regions around the metal centers that could, in principle, interact with negatively charged microbial cell walls.

Molecular docking of the same simplified cluster against five microbial target proteins produced modeled binding scores ranging from -5.4 to -6.9 kilocalories per mole, with the strongest affinity for squalene synthase of A. flavus. The authors explicitly frame these simulations as speculative, hypothesis-generating indicators of surface affinity rather than proof of enzyme inhibition, since a gas-phase cluster cannot replicate the complex surface topology, organic capping layer, or solvent environment of the real nanoparticles. This hedging matters: docking scores of this kind are qualitative approximations, and the paper’s own language distinguishes them carefully from experimental evidence.

The authors themselves flag the distance between these results and any practical application. No cytocompatibility evaluation, long-term stability assessment, mechanistic investigation in cells, or in vivo validation has yet been performed, and future work should include undoped ZnO and CuO nanoparticle controls to isolate copper’s specific contribution, along with XPS and ICP analyses to pin down copper oxidation states and incorporation mode. What the study does establish is a reproducible green route to a small, crystalline, positively charged CuO-ZnO nanocomposite whose in vitro antimicrobial activity against a small panel of reference strains is competitive with standard drugs at the tested concentrations. Whether that translates into a safe, effective antimicrobial material for biomedical or environmental use remains, as the authors conclude, a question for the substantial additional research that must come first.

Subject of Research: Green synthesis of a CuO-ZnO nanocomposite from Peganum harmala leaf extract and its antibacterial and antifungal activity

Article Title: Computational and experimental study of CuO–ZnO nanocomposite using the plant Peganum harmala L. and their antifungal, antibacterial applications

Article References: Gul, H., Zara, F., Nasreen, Z., Khan, H. A., Assad, N., Khan, M. N., Khalid, S., Adnan, M., Kaplan, A., Zaman, A., Kifle, D., Sindi, A. A., & Tolah, A. M. (2026). Computational and experimental study of CuO–ZnO nanocomposite using the plant Peganum harmala L. and their antifungal, antibacterial applications. Discover Nano, 21(1), 567. https://doi.org/10.1186/s11671-026-04984-x

Image Credits: AI Generated

DOI: 10.1186/s11671-026-04984-x

Keywords: CuO-ZnO nanocomposite, green synthesis, Peganum harmala, antibacterial, antifungal, zeta potential, DFT, molecular docking, nanoparticles, antimicrobial resistance, MIC, XRD