The synthesis itself was deliberately simple. Ground carob pods were extracted with 30% aqueous ethanol, a low-toxicity solvent chosen to capture both polar and moderately polar compounds. For the binary composite, silver nitrate was added to a sonicated suspension of anatase TiO2, followed by the plant extract, with stirring at 60 °C for three hours; a color change from pale brown to brown signaled the in-situ reduction of Ag+ ions onto the titania surface. The trimetallic version added a ferric sulfate solution first, with the pH adjusted to 8.0 using sodium hydroxide to promote hydrolysis and surface anchoring of iron species, before the silver salt and a larger dose of extract were introduced. Calcination at 300 °C or 350 °C then burned off residual organics and improved crystallinity.

A battery of characterization techniques confirmed that the chemistry had worked as intended. Fourier-transform infrared spectroscopy detected metal-oxygen lattice vibrations—Ti-O bands near 374 cm−1 in the binary material and Fe-O bands between 598 and 736 cm−1 in the ternary one—alongside residual phytochemical signatures, indicating that plant molecules remained adsorbed on the surfaces. Ultraviolet-visible spectroscopy showed the absorption peak shifting from 395 nm in the crude extract to 471 nm for Ag/TiO2 and 513 nm for Ag/Fe2O3/TiO2, a redshift consistent with the surface plasmon resonance of silver nanoparticles and the visible-light sensitization contributed by iron oxide. Tauc-plot analysis yielded optical band gaps of roughly 0.83 eV for Ag/TiO2 and 1.00 eV for the trimetallic composite, far below the ~3.2 eV typical of pure anatase, reflecting defect states introduced by the silver and iron oxide phases.

High-resolution transmission electron microscopy revealed mostly spherical to quasi-spherical nanoparticles. The Ag/TiO2 material showed a broad size distribution peaking in the 31–35 nm range, with dark-contrast silver particles anchored to the brighter titania matrix and no observable agglomeration. The trimetallic composite was more heterogeneous, spanning 15–40 nm with peaks at 21–25 nm and 31–35 nm, and displayed moderate agglomeration within a networked porous structure. Energy-dispersive X-ray spectroscopy quantified the compositions: the binary material contained 1.71% silver by weight (7.92% normalized) alongside titanium and oxygen, while the ternary sample carried 4.25% silver, 6.61% iron, and notably high carbon content (27.41%) attributed to residual plant-derived organic matter. X-ray diffraction confirmed sharp anatase reflections and face-centered cubic silver peaks in both samples, with additional hematite or maghemite Fe2O3 reflections in the trimetallic pattern.

The phytochemical analysis provided direct evidence for the proposed mechanism. The crude extract contained 171.86 mg of phenolics per gram dry weight (gallic acid equivalents), 123.53 mg of flavonoids (catechin equivalents), 24.87 mg of tannins, and 52.4 mg of carbohydrates. After synthesis, these values dropped substantially—phenolics fell to 113.75 mg/g for Ag/TiO2 and 92.31 mg/g for the trimetallic composite, with similar declines in flavonoids, tannins, and carbohydrates. The authors interpret this depletion as consumption of the biomolecules during metal-ion reduction and capping, with the larger decrease in the ternary system suggesting a stronger interaction with the multi-metallic surface.

Antioxidant testing told a nuanced story. In the DPPH radical-scavenging assay, the crude extract was the strongest performer with an IC50 of 0.146 mg/mL, followed by Ag/Fe2O3/TiO2 (0.254 mg/mL) and Ag/TiO2 (0.301 mg/mL), all trailing the ascorbic acid control at 0.022 mg/mL. The phosphomolybdate total antioxidant capacity assay showed the same hierarchy: 182.6 mg ascorbic acid equivalents per gram for the extract, 97.4 for Ag/TiO2, and 123.5 for the trimetallic composite. The researchers attribute the nanocomposites’ residual activity to two mechanisms working together—surface-bound phytochemicals that donate hydrogen atoms or electrons, and the intrinsic redox cycling of the metal oxides, with Fe2O3 providing Fe3+/Fe2+ cycling sites that explain its edge over the binary material.

Antibacterial results, measured by agar well diffusion against eight pathogenic species, favored the trimetallic composite in nearly every case. Against Escherichia coli, Ag/Fe2O3/TiO2 produced a 16.0 ± 1.23 mm inhibition zone versus 14.0 ± 0.09 mm for Ag/TiO2; Salmonella typhimurium showed 13.0 versus 11.0 mm, and Klebsiella pneumoniae 15.0 versus 13.0 mm, the latter matching the azithromycin control’s 18.0 mm zone more closely. Among Gram-positive bacteria, Staphylococcus aureus was most sensitive to the trimetallic composite (18.0 ± 1.17 mm), though azithromycin outperformed both at 26.0 mm, and B. cereus responded nearly equally to all three. Unexpectedly, neither nanocomposite inhibited Bacillus subtilis at all. The proposed mechanism combines sustained silver-ion release, which binds sulfur- and phosphorus-containing biomolecules and destabilizes membranes, with reactive oxygen species generation from the TiO2 and Fe2O3 phases, potentially amplified by Fenton-like reactions catalyzed by iron oxide.

Eco-safety testing addressed two common concerns about silver nanomaterials: uncontrolled leaching and environmental toxicity. When nanocomposite samples were immersed overnight in distilled water, leached silver rose with loading, reaching 15.6 mg/L for Ag/TiO2 and 16.8 mg/L for the trimetallic material at 0.60 g, but the percentage leached declined with increasing mass, indicating a saturation effect. The ternary composite released a higher fraction at low loading—30% versus 26% for the binary—which the authors link to Fe2O3 enhancing silver mobility. In a germination bioassay with Trigonella foenum-graecum (fenugreek) seeds, the trimetallic nanocomposite produced a hormetic response: at 12.5 mg/L, seedlings grew their longest roots (6.07 ± 0.38 cm) and total lengths (8.92 ± 0.47 cm), a stimulatory effect at sub-toxic concentrations, while above 25 mg/L inhibition became dose-dependent, with germination falling from 92.1% to 63.4% at 100 mg/L and seedling length dropping from 8.30 cm in controls to 5.10 cm.

Molecular docking added a computational layer to the work. Using MOE software, the team docked three carob polyphenols—kaempferol, ferulic acid, and protocatechuic acid—against human dihydrofolate reductase (PDB: 1DRF). Kaempferol bound most strongly at −6.8365 kcal/mol, forming hydrogen bonds with Asp21 and Arg70 plus π-stacking interactions with Ile60 and Phe31; ferulic acid scored −6.3136 kcal/mol and protocatechuic acid −5.7098 kcal/mol, each with specific hydrogen-bond contacts. The authors suggest these polyphenols may contribute to antibacterial activity through enzyme binding, though the docking targeted a human enzyme structure and the findings remain computational predictions requiring in vitro validation. Overall, the study positions carob-derived, plant-synthesized nanocomposites as a greener route to multifunctional antimicrobial and photocatalytic materials, while its own ecotoxicity data underscore that dose—and the delicate balance between stimulation and harm—will govern any real-world deployment.

Subject of Research: Green synthesis of silver-based nanocomposites from carob pod extract with antibacterial, antioxidant, and eco-safety evaluation

Article Title: Phytochemical-guided green synthesis of Ag/TiO2 and Ag/Fe2O3/TiO2 nanocomposites using Ceratonia siliqua pods: comprehensive bioactivity, in silico, and eco-safety assessment

Article References: Aldosari, F. M. (2026). Phytochemical-guided green synthesis of Ag/TiO2 and Ag/Fe2O3/TiO2 nanocomposites using Ceratonia siliqua pods: comprehensive bioactivity, in silico, and eco-safety assessment. Journal of Saudi Chemical Society, 30(6), Article 80. https://doi.org/10.1007/s44442-026-00102-9

Image Credits: AI Generated

DOI: 10.1007/s44442-026-00102-9

Keywords: green synthesis, nanocomposites, silver nanoparticles, carob, Ceratonia siliqua, antibacterial, antioxidant, molecular docking, phytochemicals, eco-toxicity, TiO2, Fe2O3