Eco‑Friendly Copper Oxide Nanoparticles Doped with Ginger and Garlic Extracts Exhibit Potent Antibacterial Activity Against Escherichia coli

Abstract

The rise of antibiotic‑resistant bacteria and the persistence of drug residues in food chains pose a global public‑health crisis. Conventional antibiotics are increasingly ineffective, demanding rapid development of novel antimicrobials. Copper oxide (CuO) nanoparticles (NPs) offer a versatile inorganic platform with proven antimicrobial properties. In this study, CuO NPs were synthesized via a green route and doped with varying concentrations of ginger (Zingiber officinale, ZO) and garlic (Allium sativum, AS) extracts. The resulting biogenic NPs were evaluated against a pathogenic strain of Escherichia coli isolated from bovine mastitis. The data confirm that ginger‑doped CuO NPs, especially at a 6 mL : 1 extract ratio, provide superior bactericidal activity, highlighting a cost‑effective, eco‑friendly alternative to conventional antibiotics.

Introduction

Herbal plants such as garlic and ginger are rich in phenolic compounds that confer potent antioxidant and antibacterial effects. Ginger contains gingerol, shogaol, and curcumin, while garlic is a source of allicin and other sulfur‑containing molecules that exhibit broad‑spectrum activity, including against multidrug‑resistant (MDR) strains (refs [1]–[6]).

Nanotechnology enables the design of materials with unique physicochemical properties. Copper oxide nanoparticles have been investigated for diverse applications—antimicrobial agents, drug delivery, photocatalysts, and sensors—thanks to their semiconductor nature and ability to generate reactive oxygen species (ROS) that disrupt bacterial membranes (refs [10]–[15]).

Escherichia coli is a key pathogen in veterinary and human medicine. In dairy cattle, MDR E. coli strains producing extended‑spectrum β‑lactamases (ESBLs) or AmpC β‑lactamases cause mastitis and significant economic loss (refs [18]–[25]). These challenges underscore the urgency of new antimicrobials that circumvent traditional resistance mechanisms.

Here, we explore green‑synthesised CuO NPs, doped with ginger and garlic extracts, as potential alternatives to antibiotics for controlling E. coli infections.

Methods

The objective was to assess the bactericidal efficacy of phytochemically reduced CuO NPs against ginger and garlic root extracts and a bovine mastitis‑derived E. coli isolate.

Materials

Chemically synthesized CuO NPs (Sigma‑Aldrich) and dried ginger and garlic roots (Lahore market) were used. Standard growth media and analytical reagents were employed without modification.

Aqueous Extraction of ZO and AS Roots

Roots were powdered, mixed with distilled water (1:10 ratio), stirred for 30 min at 70 °C, filtered (Whatman No. 1), cooled, and stored at 4 °C (Fig. 1a).

Overview of (a) aqueous extraction of ZO and AS roots, (b) green synthesis of CuO NPs

Green Synthesis of CuO

A 0.1 M cupric nitrate solution was mixed with 3, 6, or 12 mL of ZO or AS extract under continuous stirring. The pH was adjusted to 12 with 2 M NaOH, heated at 90 °C for 2 h to form precipitates, which were centrifuged (10,000 rpm, 20 min), washed, and dried overnight at 90 °C (Fig. 1b).

Characterization

UV–vis absorption (200–500 nm) was recorded using a GENESYS‑10S spectrophotometer. X‑ray diffraction (XRD) was performed on a Bruker D2 Phaser (Cu Kα, λ = 1.540 Å). Fourier‑transform infrared (FTIR) spectra were obtained via ATR‑FTIR. Scanning electron microscopy (SEM) and energy‑dispersive X‑ray spectroscopy (EDS) were carried out on a JSM‑6610LV. High‑resolution TEM (HRTEM) and selected‑area electron diffraction (SAED) were performed on a JEOL JEM‑2100F. X‑ray photoelectron spectroscopy (XPS) was used to confirm elemental states.

E. coli Isolation and Identification

Collection of Samples

Milk samples were collected from cows and buffaloes with clinical mastitis across several farms.

E. coli Isolation

MacConkey agar was used to culture samples; colonies were sub‑cultured and subjected to antibiotic susceptibility testing following NCCLS guidelines.

Identification of E. coli

Gram staining, morphological assessment, and biochemical tests (methyl‑red, catalase) confirmed E. coli isolates, corroborated by growth on EMB agar.

In Vitro Antibacterial Evaluation

Disk‑diffusion assays were conducted against 10 pathogenic E. coli isolates. Petri dishes were seeded with 1.5 × 10⁸ CFU/mL and 6‑mm wells were filled with 100 µL of each test solution (extracts, doped NPs, or chemically synthesised CuO). Plates were incubated at 37 °C for 24 h, and zones of inhibition (mm) were measured with a Vernier caliper. Data were analysed by one‑way ANOVA (p < 0.05).

Results and Discussion

UV–vis spectra revealed a colour shift from wine to coal‑black during CuO NP formation. Extracts showed maxima at 275 nm (ZO) and 280 nm (AS). Doped CuO NPs exhibited λmax at 250 nm, with optimal absorption at a 6 mL : 1 extract ratio (Fig. 2a,b).

Absorption spectra of CuO NPs doped with (a) ZO and (b) AS extracts; PL spectra of CuO NPs with (c) ZO and (d) AS extracts.

XRD patterns confirmed the monoclinic CuO phase (JCPDS 00‑002‑1040). Crystallite sizes were 24.7 nm (ZO‑doped), 47.6 nm (AS‑doped), and 27.4 nm (pristine) (Fig. 3a,b). FTIR spectra showed characteristic O‑H, C=O, and Cu–O vibrations, confirming phytochemical capping (Fig. 4a,b).

XRD patterns of CuO NPs: (a) AS‑doped, (b) ZO‑doped.

SEM images displayed spherical nanoparticles with agglomerates <1 µm in size; EDS confirmed high purity of Cu and O, with minor Zn and S signals from the extracts (Fig. 5,6).

FE‑SEM images: (a) pristine CuO, (b–d) ZO‑doped, (b′–d′) AS‑doped.

EDS spectra of CuO NPs: (a) pure, (b–d) ZO‑doped, (b′–d′) AS‑doped.

HRTEM images and lattice fringes for (a–f) ZO‑doped and (g–l) AS‑doped CuO NPs.

XPS analysis: (a) C 1s, (b) Cu 2p.

Disk‑diffusion assays demonstrated that ZO‑doped CuO NPs at a 6 mL : 1 ratio produced the largest inhibition zones (up to 2.30 mm) and a bactericidal efficacy of 54.1 % (Fig. 9a–c). AS‑doped NPs exhibited modest activity, with a maximum 1.00 mm zone and 23.5 % efficacy at the highest extract concentration (Fig. 9b–d). The positive control (ciprofloxacin) produced a 4.25 mm zone, while the negative control (DIW) showed no inhibition.

In vitro antibacterial activity: (a) ZO‑doped (low/high dose), (b) AS‑doped (low/high dose), (c) efficacy % for ZO‑doped, (d) efficacy % for AS‑doped.

The antibacterial mechanism is attributed to ROS generation and Cu²⁺ ion release, leading to membrane disruption, protein denaturation, and DNA damage (Fig. 10). Smaller NPs produce higher ROS levels, enhancing bactericidal efficiency.

Illustration of the bactericidal action of CuO NPs.

Conclusions

Green‑synthesised CuO NPs doped with ginger and garlic extracts exhibit significant antibacterial activity against pathogenic E. coli from bovine mastitis. Ginger‑doped NPs, particularly at a 6 mL : 1 ratio, delivered the highest efficacy, likely due to synergistic interactions between flavonoids and CuO. Structural analyses confirmed a monoclinic phase, spherical morphology, and nanometric size (24.7 nm for ZO‑doped, 47.6 nm for AS‑doped). These findings support the use of plant‑based CuO NPs as economical, environmentally friendly alternatives to conventional antibiotics, addressing the growing issue of antibiotic resistance and residue contamination.

Availability of data and materials

All data are fully available without restriction.

Abbreviations

EDS:

Energy‑dispersive X‑ray spectroscopy

fcc:

Face‑centered cubic

FTIR:

Fourier transform infrared spectroscopy

G + ve:

Gram positive

G − ve:

Gram negative

JCPDS:

Joint committee on powder diffraction standards

CuO:

Copper oxide

nm:

Nanometer