Green synthesis of multifunctional Ag/CeO2 and NiO/CeO2 nanocomposites using Matricaria chamomilla extract as promising agents for combating microbial infections and oxidative stress

Fatmah O. Sefrji; Albandary Almahri; Mansoor Alsahag; Ali Alisaac; Abdulmajeed Fahad Alrefaei; Hawra A. Alghasham; Wael M. Alamoudi; Majid A. Bamaga

doi:10.25259/AJC_315_2024

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Original Article

2025

:18;

3152024

doi:

10.25259/AJC_315_2024

Green synthesis of multifunctional Ag/CeO₂ and NiO/CeO₂ nanocomposites using Matricaria chamomilla extract as promising agents for combating microbial infections and oxidative stress

Fatmah O. Sefrji^a, Albandary Almahri^b, Mansoor Alsahag^c, Ali Alisaac^c, Abdulmajeed Fahad Alrefaei^d, Hawra A. Alghasham^e, Wael M. Alamoudi^f, Majid A. Bamaga^g,*

aDepartment of Biology, College of Science, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia

bDepartment of Chemistry, College of Science and Humanities in Al-Kharj, Prince Sattam Bin Abdulaziz, Al-Kharj, Saudi Arabia

cDepartment of laboratory Medicine, Faculty of Applied Medical Sciences, Al-Baha University, Saudi Arabia

dDepartment of Biology/Genetic and Molecular Biology Central Laboratory (GMCL), Jamoum University College, Umm Al-Qura University, Makkah 2203, Saudi Arabia

eDepartment of Physics, Faculty of Sciences, Umm Al-Qura University, Makkah, Saudi Arabia

fDepartment of Biology, College of Science, Umm Al-Qura University, Makkah 24230, Saudi Arabia

gDepartment of Environment and Health Research, Umm Al-Qura University, Makkah, Saudi Arabia

* Corresponding author: E-mail address: mabamaga@uqu.edu.sa (M. Bamaga)

Received: 2024-12-24, Accepted: 2025-05-08, Epub ahead of print: 2025-08-20, Published: 2025-09-20

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Abstract

The research evaluated the effect of using silver instead of nickel during nanocomposite (NC) formation (Ag-CeO₂ and NiO-CeO₂) on their resulting properties. Matricaria chamomilla extract with rich contents of phenolics and other phytochemical components functioned as the main category of components in the green synthesis of NCs. Ag-CeO₂ NC demonstrated double the concentration of phenolics, flavonoids, and tannins compared to NiO-CeO₂ NC. During biosynthesis, the functional groups of nanoparticles (NPs) indicated forming chemical bonds with particular phytochemical components present in the solution. The IC₅₀ value for Ag-CeO₂ NC was 0.086 mg/mL, indicating higher antioxidant activity compared to NiO-CeO₂ NC, which exhibited a higher IC₅₀ value of 0.142 mg/mL, reflecting lower scavenging efficiency. Ag-CeO₂ NPs exhibited better bacterial inhibition of both Gram-negative and Gram-positive bacterial strains through their superior antioxidant properties. The MIC of Ag-CeO₂ NC against K. pneumoniae and S. aureus strains was 110 μg/mL, which is four times lower than the minimum inhibitory concentration (MICs) observed against S. typhimurium and B. cereus (440 μg/mL). The significant antifungal activity of Ag/CeO₂ NC alongside NiO/CeO₂ NCs against Rhizoctonia solani fungal species was detected using Transmittance electron microscope (TEM) analysis. The work was intended to study the protein-PDB: 2I80 residues binding interactions between plant bioactive molecules through molecular docking analysis. Notably, the results proposed that M. chamomilla-derived NCs retain various biological properties with impending applications in several areas, such as medicine and agriculture.

Keywords

Antibacterial

Antioxidant activities

Characterization of nanocomposites

Chlorogenic acid

Green synthesis

Matricaria chamomilla

Phytochemical analysis

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1. Introduction

The field of nanotechnology receives medical transformations from science to develop advanced treatments for critical health issues that include medication delivery systems and antimicrobial resistance (AMR), and oxidative stress management [1]. Nanoparticles (NPs) deliver medications effectively to targeted areas because of their diminutive size, which allows them to reach a broad contact surface area for minimized treatment side effects and enhanced medication efficiency [2]. Two other AMR solutions rely on NPs that contain antimicrobial properties, like silver and copper oxide NPs, to substitute traditional antibiotics [3]. Scientists study cerium oxide NPs among many nanomaterials because this material eliminates oxidative stress, which protects against disease development caused by cellular injury [4]. The medical application of nanotechnology presents significant potential because it delivers solutions for complex problems, which generates advances in medicine as well as enhances treatment results [5].

Long-standing research has established that microbial infections, together with oxidative stress, negatively affect human health. Bacterial, fungal, and parasite infections, along with viral infections, maintain their status as primary causes of death worldwide. These once-powerful antibiotics struggle to fight against microbial adaptations, thus creating worry about antibiotic effectiveness [6]. The cellular damage occurs from oxidative stress, which emerges when harmful free radicals surpass the body’s antioxidant defense systems [7]. The pathogenesis of various ongoing medical conditions stems from exposure to oxidative stress [8]. Such widespread threats require new and effective methods that can efficiently fight them [9].

Most prevailing methods to fight microbial infections and oxidative stress suffer from known restrictions [10,11]. Researchers use antibiotics as a foundation for treating bacterial infections [12]. The improper application and excessive use of antibiotics have triggered the creation of antibiotic-resistant microorganisms, which makes these treatments ineffective under certain conditions [13]. Toxicological assessments have revealed that several antibiotic medicines lead to gastrointestinal distress and allergic symptoms in patients [14]. People commonly use antioxidants to capture dangerous free radicals, which occur during oxidative stress [15]. Some synthetic antioxidant compounds demonstrate restricted efficiency or present potential medical issues according to scientific research [16]. People with persistent oxidative stress cannot reach appropriate levels of antioxidants through food consumption alone [17].

The application of nanotechnology provides promising solutions for fighting microbial infections and controlling oxidative stress based on the reported research [18]. Nanotechnology provides useful properties to medical applications because of NPs [19]. The precision targeting abilities of designed NPs significantly outperform traditional antibiotics when it comes to specific antimicrobial functions [20]. NPs insert deeply into tissues and biofilm layers because of their small dimensions [21]. Loading NPs with antimicrobial agents makes them more potent, which leads to decreased medicinal requirements [22,23]. With its promise to protect cells from damage, nanotechnology uses specific NPs as free radical scavengers to directly neutralize harmful molecules during oxidative stress [24]. NPs work as carriers to transport antioxidant enzymes together with protective molecules straight to oxidative stress locations, thus surpassing the effectiveness of conventional antioxidant supplement delivery [25].

Authors conduct research into developing innovative NCs through nanotechnology that fight against both microbial infections and oxidative stress. The NPs include metal/metal oxide materials, either silver (Ag-) or nickel oxide (NiO), which receive a cerium oxide (CeO₂) coating (Ag/CeO₂ and NiO/CeO₂). The NC structure brings multiple advantageous features to the entire system. Silver exhibits natural antimicrobial attributes, while the cerium oxide competently removes free radicals that cause oxidative stress. The projected mechanisms from both structure components enable fast concomitant targeting of the threats by the NPs. The CeO₂ serves two beneficial roles by guiding the metal core release while reducing drug-related side effects. The research adopts an environmentally sustainable method for synthesizing NPs using M. chamomilla extract, which goes by the name of chamomile extract. The extract includes natural substances that function as reducing and capping agents in this synthesis process without demanding any detrimental chemicals, thus minimizing both chemical and ecological impacts.

M. chamomilla extract contains phenolic acids and flavonoids (Figure 1) [26,27] that perform dual functions during this research. Phytochemicals act as natural reducing and capping agents during the green synthesis of the nanocomposites (NCs), excluding harsh chemicals and controlling particle size [28]. The biomolecules potentially enhance the NPs’ ability to combat infections and oxidative stress [29]. These compounds might synergistically improve the antimicrobial features of the metal/metal oxide and contribute their antioxidant activity, working alongside the cerium oxide to scavenge free radicals and protect cells from damage [30,31]. The role of phytochemical contents highlights the multifaceted benefits of this plant in developing these multifunctional NCs.

The reactive biomolecules of the M. chamomilla extract.

Modern medicine uses NPs significantly because these method-specific NPs enable both targeted cell and pathogen distribution, and metallic, polymeric, and carbon NP systems. Because of their antibacterial properties, metallic NPs demonstrate efficient resistance against multiple drug-resistant bacteria [32,33]. When NPs were used in their polymeric form, drug delivery systems reduced the rapidity of drug distribution, thereby achieving superior therapeutic results [34,35]. The most optimal use of carbon nanomaterials occurs during medical imaging procedures while transporting therapeutic agents for the treatment of cancer. NPs show multiple diagnostic capabilities together with therapeutic abilities by fulfilling diverse operational tasks [36]. In light of these promising features, the purpose of this research is to evaluate the potential of these green-synthesized Ag/CeO₂ and NiO/CeO₂ NCs as antimicrobial and antioxidant agents. By assessing their efficacy against various pathogens and their ability to scavenge free radicals, this study aims to contribute to the development of novel therapeutic strategies for combating microbial infections and oxidative stress.

2. Materials and Methods

2.1. Reagents

All reagents used in this study were of analytical grade. Detailed information regarding their purity and sources is provided in the supplementary file (Section S1).

Section S1

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2.2. Instruments

NCs were characterized using various analytical techniques such as UV-Vis spectroscopy, scanning electron microscopy/energy dispersive X-ray (SEM/EDX), high-resolution transmission electron microscopy (HR-TEM), TEM, X-ray diffraction (XRD), and Fourier transform infrared (FTIR). Comprehensive information on the instrumentation and analysis parameters is available in the Supplementary file (Section S1).

2.3. Preparation of plant extract

The dried Matricaria chamomilla flowers were supplied by the local market. 10 g of the dried plant material were added to a 250 mL conical flask (250 mL) containing 100 mL of bidistilled water (w/v 1:10). The mixture was shaken on a horizontal water bath shaker (Memmert WB14, Schwabach, Germany) for 20 mins at 70^°C. After cooling to room temperature, the mixture was filtered using Whatman No. 1 filter paper [37].

2.4. Green synthesis of NCs

Silver nitrate solution (10 mL, 1 mM) was prepared in bidistilled water and stirred at room temperature. 50 mL of the freshly prepared M. chamomilla extract (20 mg/mL) was added dropwise to the silver nitrate solution. The temperature was increased gradually up to 60^°C until the solution color turned dark brown. To prepare Ag/CeO₂, a sonicated solution of cerium dioxide (30 mg) was prepared in ethanol (70%, 10 mL) and subsequently added portion-wise to the silver NP solution. The mixture was stirred for 3 h until a remarkable change in the color was noticed (light brown color). The mixture was then sonicated (Elma Schmidbauer GmbH, Gottlieb-Daimler-Straße 17, Singen, Germany) at 60^°C for an additional 4 h. To prepare NiO/CeO₂, similar steps were accomplished using nickel nitrate instead of silver nitrate. The NC solutions were centrifuged (Beckman Coulter Allegra X-15R Centrifuge, Beckman Coulter, Inc., California, USA) at 10,000 ppm for 10 min to separate the solid particles, which were washed with ethanol thrice to remove impurities, residual carbons, and oil contaminants [38,39].

2.5. Phytochemical analyses

The phytochemical analyses, such as phenolic, flavonoid, and tannin contents, were investigated by colorimetric methods to estimate the antioxidant capacity of the biomolecules extracted from M. chamomilla.

2.5.1. Total tannin contents

The tannin content was measured using the vanillin-hydrochloride assay [40], in which the absorbance of the samples was measured after treatment with freshly prepared vanillin-hydrochloride. The attained values of tannin contents for the NCs are expressed as mg of tannic acid equivalents/gram of the dried sample. The capacity of tannins of the investigated samples was calculated from the tannic acid standard curve (y = 0.0009x; R²= 0.955).

2.5.2. Total phenolic contents

The test was run for the NCs to quantify the phenolic content. Folin-Ciocalteu (F-C) assay was used [41,42], in which the standard curve of Gallic acid was used to calculate the characteristic values as milligrams Gallic acid equivalents/gram of the dried sample. The process involved the use of a Gallic acid standard curve (y = 0.0062x, R² = 0.987).

2.5.3. Total flavonoid contents

The flavonoid contents are expressed as grams of catechin acid equivalents per gram of the dry sample. The test was run for the NCs using the aluminum chloride colorimetric assay [43], using the standard curve of Catechin. The total flavonoids were estimated from the following standard curve (y = 0.0028 x, R²= 0.988).

2.6. Antioxidant 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

The antioxidant capacity of the NCs was investigated following the DPPH^• assay using ascorbic acid as a standard [44]. A serial dilution of each sample was prepared by mixing 1 mL of each sample with 1 mL of methanol. 0.135 mM DPPH^• solution (1 mL) was prepared and mixed with each tube in the serial dilution. The tubes were kept in the dark for 30 min at room temperature. The absorbance was measured at 517 nm, and the % remaining DPPH^• was calculated by the following equation (Eq. 1):

(1)

% {DPPH}^{•} remaining =   {[{DPPH}^{•}]}_{T} /   {[{DPPH}^{•}]}_{T =   0} x 1 00

The values of % DPPH^• remaining were plotted versus mg extract/mL using an exponential curve to identify the effective concentration “IC₅₀”. IC₅₀ indicated the amount of antioxidants needed to decrease the initial concentration of DPPH^• solution by 50%. The values of IC₅₀ point out the inverse relationship with the antioxidant capacity of the tested sample [45].

2.7. Antimicrobial assessment

2.7.1. Antibacterial activity

The negative control was applied as distilled water, and gentamicin was chosen as a positive control. Agar well-diffusion assay was employed following the reported procedure [46]. The bacterial strains required subculture for Mueller-Hinton broth incubation at 37°C for 18 to 24 h to reach the exponential growth stage before testing. A sterile saline solution was utilized to adjust bacterial suspensions until they reached a 0.5 McFarland standard (approximating 1.5 × 10⁸ CFU/mL). Each standardized bacterial culture was applied with a uniform distribution through swabbing across Mueller-Hinton agar plates.

The minimum inhibitory concentration (MIC) values were estimated by preparing serial dilutions of each NC against a specific type of bacterial species. For NiO/CeO₂ NC, the serial dilution was prepared in a concentration range of 17620-137.656 μg/mL. For Ag/CeO₂ NC, the samples were prepared in serial dilutions with a concentration range of 7040-13.75 μg/mL. The experiment included additional test tubes that contained only bacterial broth without NC additions. The incubation took place at 37°C for 24 h under conditions where MIC became apparent through the absence of turbidity, together with 600 nm optical density validation [47].

2.7.2. Antifungal activity

Rhizoctonia solani (ARC-NW23), as a plant fungal pathogen, was obtained from the Seed Pathology Research Department, Plant Pathology Research Institute, Agricultural Research Centre, Egypt. The MIC was estimated using a broth microdilution assay [48]. The fungal strain was sub-cultured on potato dextrose agar (PDA) and incubated at 25°C for 5-7 days. A fungal inoculum was prepared by cutting 1 mm agar discs from actively growing colonies using a sterile cork borer. Various concentrations of the NCs were prepared, and each was transferred into the molten agar before pouring into Petri dishes. Subsequently, a 1 mm diameter disc of each previously grown fungus was individually inoculated onto the center of each plate. The MIC was estimated as the lowest concentration of NCs that entirely inhibited visible fungal growth in comparison to a control plate containing only a PDA medium.

2.8. Statistical analysis

Experiments were performed in triplicate for each sample. Data were analyzed utilizing IBM SPSS Statistics version 26 (Armonk, NY, USA). All the results are accessible as mean ± standard deviation (SD) from at least three independent experiments [49].

2.9. In silico molecular docking study

The molecular docking studies were performed using the M.O.E. 2019.0102 program to identify the binding interactions between ligands and the PDB. For instance, we used the 2I80 protein to inhibit d-alanine ligase (DDl), an essential enzyme in the biosynthesis of Staphylococcus aureus bacterial cell walls [50].

3. Results and Discussion

3.1. The plausible mechanism of formation of NCs

The formation of Ag/CeO₂ and NiO/CeO₂ NCs using aqueous M. chamomilla extract, silver nitrate, nickel nitrate solutions, and cerium oxide likely comprises a multi-step process.

3.1.1. Reduction of metal precursors:

The M. chamomilla extract acts as a reducing agent, containing biomolecules like phenolics or flavonoids with reductant functional groups (represented as R-OH). These groups donate electrons to silver ions (Ag⁺) from silver nitrate, leading to the formation of silver NPs (Ag NPs) [51].

(2)

{2AgNO}_{3} +  R - OH  \to  2Ag  (NPs)   + {NO}^{3 -} +  R - C = O  + H^{+}

Similar to silver, biomolecules in the extract can reduce nickel ions (Ni²⁺) from nickel nitrate. However, the reduction process might involve multiple steps depending on the specific biomolecules present. Here’s a simplified representation:

(3)

Ni {({NO}_{3})}_{2} +  R - OH  \to  Ni  (NPs)   + {NO}^{3 -} +  R - C = O + H^{+}

3.1.2. Interaction with CeO₂:

The cerium oxide (CeO₂) NPs act as nucleation sites for the reduced metal ions (Ag or Ni). The surface hydroxyl groups (-OH) on CeO₂ can interact with the metal ions through electrostatic forces or coordination bonds [52].

(4)

M^{n +} ({Ag}^{+} {or Ni}^{2 +}) + {CeO}_{2} - OH  \to M^{n +} - {CeO}_{2} - OH  (M  =  Metal)

3.1.3. NP growth and stabilization:

As more metal ions get reduced and interact with CeO₂, they nucleate and grow on the CeO₂ surface, forming the metal-ceria (Ag/CeO₂ or NiO/CeO₂) NC. Biomolecules from the extract might get adsorbed onto the surface of the growing NPs, acting as capping agents. These capping agents help prevent excessive aggregation and stabilize the size distribution of the NPs in the final composite [53,54]. The specific equations and reaction mechanisms might vary depending on the detailed composition of the M. chamomilla extract and the reaction conditions (e.g., temperature, pH).

3.2. UV-visible spectroscopy

The UV-visible absorption spectra shown in Figure 2 reveal electronic transitions that occur in M. chamomilla extract and Ag/CeO₂ and NiO/CeO₂ NCs. The extract shows two prominent absorption peaks at 480 nm and 332 nm because n→π* and/or π→π* transitions occur in the chromophoric structures present in the extract. The UV-visible absorbing chromophores show properties of π-electron-rich organic molecules that perform electron transitions between occupied and unoccupied orbitals [55]. Some water interaction with ionic components inside NCs results in solution-based dissolution, which changes the number of light-absorbing species and modifies the recorded absorbance values [56]. The extent of these interactions depends on the unique molecular structures of the extract, with the surface chemical properties found in the NPs contained in the NCs. The surface plasmon resonance (SPR) of silver NPs generated a weak absorption maximum at 520 nm, which appeared in Ag/CeO₂ NC spectra. The surface of metal NPs displays collective electron oscillation behavior through surface plasmon resonance after light interacts with them (Figure 2). An indistinct SPR peak in the measurement indicates that silver NPs show irregular morphology or substantial size. NiO/CeO₂ NC exhibits more intense and broader absorbance spanning the UV-visible spectrum than Ag/CeO₂ NC in the light absorption capacity of the material. The strong light absorption of NiO is attributed to electronic transition processes that occur within the nickel oxide (NiO). Additional data analysis is required to establish the specific impact of CeO₂ on the combined absorption behavior because the obtained data does not clarify its unique role. The measured spectra indicate both extract chromophores and electronic transitions occurring inside metal and metal oxide NPs of the NCs.

UV-visible spectroscopy of M. chamomilla extract, Ag/CeO2, and NiO/CeO2 NCs.

3.3. FTIR spectral analyses

The study utilizes FTIR data to analyze functional groups in M. chamomilla extract and Ag/CeO₂ NC and NiO/CeO₂ NC (Figure 3, with supporting data in Table S1). The FTIR analysis presents wavenumber values (cm^-1) for functional group identifications of each tested sample. The FTIR spectroscopy evaluation of M. chamomilla extract detected various important functional group structures. The wide peak at 3295 cm^-1 reveals O-H stretching vibrations that might exist in alcohol or phenol compounds as well as carboxylic acids. The various molecules present in the extract can be attributed to their hydroxyl functional group content [57]. The stretch from aliphatic groups, which construct numerous organic molecules, appears at 2936 and 2860 cm^-1. The carbonyl C=O functional group appears through a 1670 cm^-1 absorption band, which could indicate ketone chemicals in the extract. The aromatic ring stretching vibrations in the range of 1601 to 1405 cm^-1 indicate that aromatic compounds exist within the chamomile extract. The spectral bands located at 1344 and 1267 cm^-1 indicate both C-H bending modes and C-O stretching modes, which align with alcohol or phenol functional groups discovered previously. The complex chemical framework of chamomile extract contains carbohydrates or esters based on the C-O stretching vibrations found at 1104, 1073, and 1018 cm^-1. The absorption peaks at 820 cm^-1, together with peaks at 756, 669, 615 cm^-1, can be explained by the bending modes of aromatic compounds as well as alkenes’ C-H bending.

Table S1-12

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FTIR spectroscopy charts of M. chamomilla extract, Ag/CeO2 NC, and NiO/CeO2 NC.

FTIR analysis of Ag/CeO₂ NC revealed a wide band at 3290 cm^-1, which demonstrates O-H stretching vibrations that are probably from hydroxyl groups. Two absorption bands at 2938 and 2881 cm^-1 represent C-H stretching vibrations, although they appear as a result of organic substances that stick to the NC surface. The 1462 cm^-1 absorption peak in the spectrum shows characteristics of C-H bending while indicating its presence. The formation of NC’s structure was established through the appearance of four peaks at 1111, 1034, 991, and 922 cm^-1 that show the vibrations of M-O metal-oxygen bonds found in the CeO₂ lattice structure. Four absorption bands appear with less clarity at 857, 756, and 676 cm^-1, along with 560 cm^-1.

The FTIR analysis of NiO/CeO₂ NC showed a wide peak at 3324 cm^-1, indicating O-H stretching vibrations mainly from hydroxyl groups or surface-adsorbed water molecules. The NC surface displays C-H stretching vibrations, which are seen through peaks at 2939 and 2881 cm^-1, while potential organic groups function as the origin of these peaks. The affinities between NiO/CeO₂ NC and a carbonyl group appear through a faint peak at 1662 cm^-1, although this feature remains less than those in the chamomile extract. The C-H bending vibrations are revealed through bands located at 1465 and 1411 cm^-1. The most prominent features of the NiO/CeO₂ NC spectrum lie between 1331 and 922 cm^-1 [58]. Like in Ag/CeO₂ NC, this spectrum area shows metal-oxygen bond vibrations which occur throughout the CeO₂ crystal structure. The spectrum contains five less distinctive peaks located at 858, 753, 675, 564, and 484 cm^-1. The NC’s spectrum shows bands that could indicate metal-oxygen bond vibrations, which involve both the CeO₂ lattice and NiO component of the NC. Multiple possible out-of-plane bending vibrations and NiO metal-oxygen vibrations may exist within the absorption bands located at 858, 753, 675, 564, and 484 cm^-1. Characteristic bands related to the metal-oxygen bonds in the CeO₂ lattice structure were identified in both NCs.

3.4. Zeta potential analyses

Zeta potential analysis examined the surface charge and stability of Ag-CeO₂ and NiO-CeO₂ NCs. Both Ag-CeO₂ and NiO-CeO₂ NCs exhibited a negative zeta potential value (-20.8 mV and -20.4 mV, respectively) (Figure 4). A negative zeta potential indicates a predominance of negatively charged groups on the NP surfaces. The negative zeta potential values suggest that the surfaces of both NCs are dominated by negatively charged groups. The negative zeta charge can arise from the adsorption of hydroxyl groups (OH^-) at the NP surface in water, or from other charged species present during synthesis [59].

Zeta potential analyses of NCs. (a) Ag-CeO2 NC, (b) NiO-CeO2 NC. NCs: nanocomposites.

A repulsive force between the negatively charged particles aids in maintaining their suspended state because two similar charges naturally repel one another. The similarity between zeta potential values indicates equal levels of electrostatic stabilization between Ag-CeO₂ and NiO-CeO₂ NCs. The minimal changes in zeta potential values impact how NCs interact with each other, particularly during altered environmental and application conditions. Ag-CeO₂ possesses a slightly more negative surface charge, which provides improved stability for dispersed colloids in various media primarily used for biomedical or catalytic applications. Electronic parameters indicate that surface negativity exists to an almost equal extent for Ag-CeO₂ (-20.8 mV) and NiO-CeO₂ (-20.4 mV). Based on the electrophoretic mobility measurements, Ag-CeO₂ yielded -0.000161 cm²/Vs, and NiO-CeO₂ showed -0.000157 cm²/Vs, which confirms the surface charge presence simultaneously with negative zeta potential. Although the difference is small, it implies subtle variations in surface charge distribution and hydration layers, potentially affecting interactions with target molecules or substrates. The Ag-CeO₂ NCs demonstrate a higher electrophoretic mobility compared to NiO-CeO₂ NCs, although this parameter measures the velocity of particles under electric field conditions. The electrophoretic mobility values suggest that surface charge distribution, together with particle hydration, shows a subtle difference between these two NC types. The dispersion medium exhibited enhanced conductivity value at 0.953 mS/cm for NiO-CeO₂ NCs when measured against 0.493 mS/cm for Ag-CeO₂ NCs. The elevated dispersion medium conductivity of NiO-CeO₂ NCs compared to Ag-CeO₂ NCs may result from added ions in the synthesis process, and NiO will influence the medium properties. Such differences will affect the performance of ionic strength-dependent solutions as well as influence electrostatic screening mechanisms in compound formulations. The suspending stability of the Ag-CeO₂ and NiO-CeO₂ NCs exists at moderate levels since negative electrostatic repulsions occur between charged particles, per their measured negative zeta potential values. The moderate negative values of zeta potential indicate a proper electrostatic stabilization for both NCs, though their minimal differences in surface charge could lead to functional changes in aggregation patterns and dispersion performance, and functional integration levels in practical applications.

3.5. Dynamic light scattering (DLS) analysis

Analysis by DLS showed heterogeneous distribution of particle sizes, which resulted in high polydispersity indexes of 0.401 for Ag-CeO₂ and 0.622 for NiO-CeO₂, indicating moderately broad and broad size distributions (Figure 5). High PDI values suggest a wide range of particle sizes, which can lead to uneven sedimentation rates and increased aggregation over time, ultimately reducing colloidal stability. The synthesis procedure could result in different growth rates and the formation of small particle clusters. The final size of NiO-CeO₂ NCs reached 627.6 nm, while Ag-CeO₂ NCs had a size of 582.3 nm because of inherent metal oxide characteristics, which affect their final dimensions [39]. The end size of NPs can be shaped by the density of materials and the way they grow. The size distributions remained similar due to the equivalent viscosities of 0.897 mPa.s for Ag-CeO₂ and 0.899 mPa.s for NiO-CeO₂ in the dispersion medium. A broad size distribution also implies that smaller particles with larger surface‐to‐volume ratios will dominate surface‐dependent processes (e.g., catalysis or scavenging of ROS), and sedimentation of large aggregates will reduce the available active surface area. For that reason, control of polydispersity is critical to achieve uniform performance, stable reliability, and reproducible functional behavior in applications such as catalysis, drug delivery, and antimicrobial treatment.

Size distribution analyses of NCs. (a) Ag-CeO2 NC, (b) NiO-CeO2 NC. NCs: nanocomposites.

3.6. HR-TEM

HR-TEM micrographs revealed distinct morphologies between Ag-CeO₂ and NiO-CeO₂ nanocrystals, according to Figure 6. Observational investigations confirmed that the NPs maintained spherical structures but displayed key shape variations between them. The Ag-CeO₂ NCs displayed various sizes that extended from 7.32 nm to 25.1 nm while showing a main surface smoothness. The presence of non-aggregated Ag-CeO₂ NPs suggests a high accessible surface area, which can enhance catalytic turnover and adsorption processes. The NiO-CeO₂ NCs showed a single spherical shape, ranging from 33.0 nm to 70.4 nm, while existing in mostly aggregated forms. Such extensive aggregation in NiO-CeO₂ likely reduces effective surface exposure and may impair mass transfer in catalytic or sensing applications.

HR-TEM micrographs of NCs. (a) Ag-CeO2 NC, (b) NiO-CeO2 NC.

Results from Ag-CeO₂ non-aggregated NP studies through HR-TEM showed an extraordinary outcome that went beyond the detection of both primary and secondary nanoclusters. Some aggregated NPs showed either close interfacial bonds or a complete mixture at the interface between Ag and CeO₂ components during analysis. The observed characteristics indicate strong physical bonds form between the Ag NPs and CeO₂ support, which goes beyond basic Ag NP coating on the CeO₂ support. The distinct synthesis methods, as well as natural interaction phenomena between Ag and CeO₂ phases, may influence this potential phase intermixing process [37,38].

Selected area electron diffraction (SAED) serves as a strong analytical instrument to determine the crystal structures of materials present in NCs. Analysis of Debye-Scherrer rings enables the determination of d-spacing, which helps identify crystal structures by pattern matching to reference information through the interaction between electrons and material crystal planes. The SAED pattern from Ag-CeO₂ NC should display rings that represent both cerium oxide (CeO₂) fluorite cubic structure and silver (Ag) face-centered cubic (fcc) pattern. The crystal pattern analysis using Debye-Scherrer rings would display weak rings of silver fcc structure when Ag metal NPs exist along with CeO₂ within the NC. The SAED pattern of an NiO-CeO₂ NC would demonstrate rings that represent the d-spacing values of both nickel oxide (NiO) and CeO₂. The Debye-Scherrer rings show a spotty appearance in small NC NPs because of limited grain dimensions [37-39]. This crystallographic confirmation supports the potential for phase-dependent catalytic selectivity and stability under reaction conditions. HR-TEM and SAED examinations show that Ag-CeO₂ and NiO-CeO₂ have different structural attributes that can directly influence functional properties such as surface reactivity and electron mobility, along with long-term stability.

3.7. SEM

SEM analysis was applied to scrutinize the shape/morphology, homogeneity, and particle size distribution of the solid surface of NCs [60]. The SEM images of Ag-CeO₂ as well as NiO-CeO₂ NCs appear in Figure 7. The Ag-CeO₂ NC contains NPs of irregular form that link together into porous networks, while the majority of particles exist in nanometer dimensions but demonstrate visible clustering. The dispersion level and surface area availability of Ag-CeO₂ NCs exceed those of NiO-CeO₂ NCs because Ag-CeO₂ displays both decreased aggregate quantity and size. The clumped appearance of Ag-CeO₂ NPs seems to stem from plant biomolecule-based capping agents, while their decreased agglomeration indicates stronger colloidal stability. The NiO-CeO₂ material featured both highly porous and regular structures, together with embedded NiO particles throughout the CeO₂ framework, with reduced pore sizes compared to NiO-CeO₂. The metal oxide NPs of both composites are distributed evenly throughout the ceria framework structure. Improved surface-dependent properties emerge due to better exposure of active sites, which result from increased dispersal in Ag-CeO₂. The greater surface area of Ag-CeO₂ generates better performance for photocatalysis and enzyme immobilization than NiO-CeO₂ NC. The porous CeO₂ brought about rough surface textures that researchers observed in both forms of materials [61].

SEM images of NCs. SEM: Scanning electron microscope. (a) Ag-CeO2 NC, (b) NiO-CeO2 NC.

3.8. EDX

EDX is a valuable performance applied in combination with SEM to investigate the elemental composition of the nanomaterials [37,38]. The EDX data of Ag-CeO₂ and NiO-CeO₂ NCs have been shown in Figure 8 & Table S2. The analysis of Ag-CeO₂ NC revealed the detection of Oxygen (O), Chlorine (Cl), Silver (Ag), and Cerium (Ce). The most abundant element was Ce with elemental composition by weight percentage (63.78%), followed by Oxygen (O) at 15.06%. The high percentage of cerium in the cerium dioxide form supports the anticipation for this nanomaterial. The analysis also revealed the presence of silver with 18.31% of weight percentage in addition to the presence of chlorine (2.86 wt%) as a contaminant. In line with the results of Ag-CeO₂ NC, the EDX analysis of NiO-CeO₂ NC demonstrated the presence of cerium (68.85 wt%) as the most abundant element, followed by oxygen (22.91 wt%), specifying the high content of cerium dioxide. Nickel oxide appeared at 5.46 wt% with a lower content compared to the case of silver. Phosphorus and calcium appeared with rare percentages as contaminants (1.61 and 1.17 wt%, respectively). In both samples, the absence of carbon elements might be attributed to the possible encapsulation of the carbon with the metal oxide structure since the thermal decompositions are excluded through the preparation process [39]. The carbon is also not present on the surface of NPs in enough quantities for detection by EDX.

EDX analyses of (a) Ag-CeO2 and (b) NiO-CeO2 NCs.

3.9. XRD

The XRD pattern showed peaks at specific 2θ values, corresponding to the crystal planes in the material’s structure. The XRD data recommended the presence of both cerium oxide (CeO₂) and silver (Ag) in the analysis of Ag/CeO₂ NC (Figure 9a & Table 1). The results of the XRD pattern of Ag/CeO₂ NC verified a good crystallinity with a strong and sharp peak attributed to the (111) plane of CeO₂. Another prominent peak for the (200) plane of CeO₂ was noticed at 2θ 32.9790. Two weak and broad peaks at 5.45° and 38.27° might be attributed to silver based on their positions. The results of 2θ values (28.4358, 32.9790, 38.2698, and 56.2111) are closely matched with the standard reference pattern for silver in its fcc form [38].

XRD analyses of (a) Ag-CeO2 and (b) NiO-CeO2 NCs.

Table 1. The results of XRD analysis of Ag-CeO₂ NCs.

Pos. [°2Th.]	Height [cts]	FWHM [°2Th.]	d-spacing [Å]	Rel. Int. [%]	Tip width [°2Th.]	Particle Size (nm)	Matched by
5.4495	6.43	0.9446	16.21745	2.44	1.1336	8.42	-
28.4358	263.32	0.1574	3.13886	100.00	0.1889	52.07	01-089-2046
32.9790	76.33	0.1968	2.71610	28.99	0.2362	42.10	01-089-2046
38.2698	3.18	0.9446	2.35190	1.21	1.1336	8.90	01-089-2046
47.3733	114.18	0.2362	1.91902	43.36	0.2834	36.73	01-075-0121
56.2111	71.97	0.1574	1.63647	27.33	0.1889	57.22	01-089-2046
59.0515	10.17	0.3149	1.56435	3.86	0.3779	29.00	01-075-0121
69.3803	11.47	0.3936	1.35457	4.36	0.4723	24.55	01-075-0121
76.5573	20.65	0.3149	1.24448	7.84	0.3779	32.14	01-075-0121
79.0163	13.56	0.3149	1.21181	5.15	0.3779	32.70	01-075-0121
88.3529	20.17	0.4800	1.10537	7.66	0.5760	23.08	01-075-0121

The results of XRD analysis of NiO-CeO₂ NCs (Table 2 & Figure 9b) revealed a peak at 2θ 28.5230° attributed to CeO₂ (111), NiO (111), which is the most prominent due to the high intensity and common (111) reflection in cubic structures. The peak at 2θ 33.0527° is due to CeO₂ (200), and NiO (200) is matched by 01-081-0792. A combination of phases in cubic fluorite CeO₂ (Cerium(IV) Oxide) and cubic NiO (Nickel(II) Oxide) specified the presence of both NiO and CeO₂ in the NC. A possible reflection for both phases was verified for the peak at 2θ 47.4560° for CeO₂ (220), NiO (220), while the peak at 2θ 56.3078° for CeO₂ (311), NiO (311) presented a less intense reflection. The sharp peaks verified the crystalline nature and purity of both NCs. The d-spacing values calculated for each peak deliver data around the crystal lattice spacing in the NiO and CeO₂ phases. Otherwise, the peaks at 2θ 28.5230, 47.4560, 59.1460, and 69.4443 indicated good intensity and were well-matched with the reported planes of CeO₂ (JCPDS #01-081-0792). The peak at 2θ 33.0527 is attributed to the reflection of NiO (111) (Tables S3 and S4).

Table 2. The results of XRD analysis of NiO-CeO₂ NCs.

Pos. [°2Th.]	Height [cts]	FWHM [°2Th.]	d-spacing [Å]	Rel. Int. [%]	Tip width [°2Th.]	Particle Size (nm)	Matched by
28.5230	240.39	0.1181	3.12946	100.00	0.1417	74.32	01-081-0792
33.0527	86.17	0.0984	2.71021	35.85	0.1181	89.89	01-081-0792
47.4560	112.97	0.1968	1.91587	46.99	0.2362	42.13	01-081-0792
56.3078	85.50	0.1968	1.63389	35.57	0.2362	42.13	01-081-0792
59.1460	13.55	0.3149	1.56208	5.63	0.3779	-	01-081-0792
69.4443	15.24	0.3149	1.35348	6.34	0.3779	29.03	01-081-0792
76.6951	25.62	0.3936	1.24258	10.66	0.4723	24.59	01-081-0792
79.0338	14.60	0.3936	1.21158	6.07	0.4723	24.59	01-081-0792
88.3564	18.14	0.4800	1.10533	7.55	0.5760	23.09	01-081-0792

3.9.1. Phytochemical analyses

Figure 10 presents the phytochemical data obtained from Ag-CeO₂ and NiO-CeO₂ NCs synthesized through M. chamomilla extract. The phytochemical content of Ag-CeO₂ NC surpasses NiO-CeO₂ NC concerning all detected substances. Ag-CeO₂ NC contains higher amounts of phytochemical components, with phenolics appearing at 87.87 mg/g, while flavonoids reach 59.14 mg/g, and tannins are found at 35.38 mg/g. These concentrations surpass those of NiO-CeO2 NC, where phenolic levels are 23.66 mg/g and flavonoid contents measure 5.799 mg/g, together with tannin levels at 7.004 mg/g. More favorable interactions between surface functional groups and specific phytochemicals occur. The synthesis process could have selected for the retention of particular compounds, such as phenolics, in Ag-CeO₂ NC.

A comparison between the phytochemical contents of M. chamomilla extract and the synthesized NCs.

3.9.2. Potential antioxidant activity

A DPPH assay measured antioxidant activity for NC Ag-CeO₂ NC and NiO-CeO₂ NC samples, and the data appear in Table 3. The studies confirmed that the NC solution concentration directly influenced its capability to eliminate DPPH free radicals. The Ag-CeO₂ NC NCs demonstrated better antioxidant capability than NiO-CeO₂ NC NCs did. The analysis through DPPH assay revealed that Ag-CeO₂ NC demonstrated superior antioxidant properties at all tested concentrations since it maintained lower DPPH percent remaining alongside higher scavenging activity percentages. Ag-CeO₂ NC showed superior free radical scavenging capabilities because its IC₅₀ value (0.086 mg/mL) was lower than that of NiO-CeO₂ NC (0.516 mg/mL).

Table 3. The antioxidant results of the greenly synthesized NCs using DPPH assay.

Sample	Concentrations (mg/mL)	% Remaining DPPH	% Scavenging activity	IC₅₀ (mg/mL)
Ag-CeO₂ NC	0.21	13.98	86.02	0.086
	0.105	36.59	63.41
	0.053	77.24	22.76
	0.026	94.47	5.528
NiO-CeO₂ NC	0.843	31.38	68.62	0.516
	0.421	60.0	40.0
	0.211	76.75	23.25
	0.105	83.09	16.91
Ascorbic acid	0.062	15.267	84.733	0.0222
	0.031	39.084	60.916
	0.016	61.069	38.931
	0.008	74.809	25.191

Several studies have demonstrated that NPs can improve the bioavailability and stability of antioxidants, ensuring more effective modulation of oxidative stress, which is a major contributor to various diseases [62]. Additionally, by incorporating therapeutic enzymes into NP systems, enhanced enzyme activity and prolonged therapeutic effects are achieved, making them a promising platform for advanced medical treatments [63]. These mechanisms highlight the crucial role of NPs in modern therapeutic strategies, particularly in the context of enzyme delivery and antioxidant therapy. On the other hand, the transfer of a hydrogen atom from the NC functional group through the hydrogen atom transfer (HAT) mechanism donates the hydrogen atom to DPPH^• radicals [64]. The hydrogen atom donation separates the double bond in DPPH^• through the production of a stable molecule. The surface of NCs contains phenols or hydroxyl functional groups, which function as donors of hydrogen atoms. The NCs containing CeO₂ and similar substances enable metal ion chelation through their presence of Ce or similar elements [65]. Transition metal ions present in the NCs show the ability to catalyze Fenton reactions, which yield redox and oxidative stress (ROS) that cause oxidative stress. The nano-compositing technology works by binding metal ions, which indirectly block the development of ROS. The antioxidant activities of NC products are potentially achieved through their combined electron transfer (ET) and hydrogen atom transfer (HAT) functions or through these mechanisms working in conjunction with each other.

The IC₅₀ values for CeO₂ NPs produced by green methods span from 0.103 to 0.188 mg/mL, depending on the biological reducing agents employed, according to Naidi et al. [66]. The studies by Parab et al. [67] showed that Ag-modified CeO₂ NPs possessed superior antioxidant capabilities with slightly higher IC₅₀ values between 0.091 and 0.130 mg/mL than Ag-CeO₂ NCs obtained in this research work. The research by Khan et al. [68] demonstrated that green-synthesized CeO₂ NPs show IC₅₀ activity between 0.13-0.19 mg/mL, comparable to the Ag-CeO₂ NCs synthesized in our study. Experimental data show that incorporating Ag or NiO into CeO₂ leads to higher antioxidant properties, where Ag-CeO₂ NCs demonstrate the best scavenging activity through our green synthesis process.

3.9.3. Antimicrobial activity

3.9.3.1. Antibacterial activity

The antibacterial evaluation of M. chamomilla extract and Ag/CeO₂ NC, together with NiO/CeO₂ NC alongside gentamicin, took place against pathogenic bacteria through the agar well diffusion method. No antibacterial activity occurred when testing M. chamomilla extract against different pathogenic bacteria, since none of the bacteria showed any response to this extract. The antibacterial activity spectrum of Ag/CeO₂ NC demonstrated the largest observed inhibition zones between 16-18 mm against every Gram-negative bacterial strain. Information retrieved from Table 4 and Figure 11 demonstrated that NiO/CeO₂ NC effectively inhibited Gram-negative bacteria and produced inhibition zone diameters between 15 and 17 mm. In addition, the positive control gentamicin created inhibition zones from 21 to 25 mm across all tested Gram-negative bacteria. Ag/CeO₂ NC presented antibacterial properties for all Gram-positive bacteria, causing inhibition zone diameters between 13 and 20 mm. NiO/CeO₂ NC showed antibacterial effects against all Gram-positive bacteria except Bacillus cereus, but inhibited them with ring diameters between 14 and 16 mm.

Table 4. The antibacterial results of the investigated NCs against various pathogenic bacteria.

Microorganisms	Inhibition zones in mm
Microorganisms	M. chamomilla extract	Ag/CeO₂ NC	NiO/CeO₂ NC	Control (dist. H₂O)	Gentamycin
Gram-negative bacteria
Escherichia coli (ATCC 10536)	-ve	16±1.06	17±0.88	-ve	25±1.54
Salmonella typhimurium (ATCC 25566)	-ve	15±1.28	15±1.19	-ve	21±1.07
Klebsiella pneumonia (ATCC 10031)	-ve	18±0.59	17±1.72	-ve	22±1.83
Gram-positive bacteria
Bacillus subtilis (DMS 1088)	-ve	13±1.77	16±1.25	-ve	29±2.07
Bacillus cereus (EMCC number 1080)	-ve	16±1.38	14±1.71	-ve	18±1.86
Staphylococcus aureus (ATCC 6538)	-ve	20±1.90	16±1.55	-ve	25±2.26

The results of inhibition zones in mm are expressed as the mean value ± standard deviation (SD).

The experiments were run in triplicate.

The petri dishes indicated the antibacterial activity of the NCs against various bacterial species. (1) referred to M. chamomilla extract. (2) referred to Ag/CeO2 NC, and (3) referred to NiO/CeO2 NC. (w) referred to the negative control DW (distilled water), and (Ab) to the antibiotic.

Gentamicin had the largest inhibition zone diameters (18-29 mm) against all Gram-positive bacteria except B. cereus. The antibacterial effectiveness tests showed improved NC activity against bacterial pathogens while reaching antibacterial outcomes similar to those of Gentamicin in some cases. The antibacterial effects of both NCs proved their potential use against an array of bacterial infections, including Gram-negative (E. coli, S. typhimurium, and K. pneumoniae) and Gram-positive species (B. subtilis, B. cereus, and S. aureus). Synthesized NCs (Ag/CeO₂ and NiO/CeO₂ NCs) exhibited antibacterial action similar to previous research concerning different strain bacteria groups [69-72]. The lack of antibacterial activity detected for M. chamomilla extract aligns with some studies that report limited or inconsistent antibacterial effects against specific bacterial strains [73]. The results support the use of Ag/CeO₂ NC as a hopeful candidate for combating diverse bacterial infections. Studies recommend that Ag/CeO₂ NCs disrupt bacterial membranes owing to their small size, while released silver ions can inactivate essential bacterial enzymes [74,75]. Otherwise, definite metal oxides such as CeO₂ can generate ROS under specific conditions, resulting in the damage of the cellular components within bacteria, leading to cell death.

In contrast to earlier findings, Negi et al. [69] found Ag-CeO₂ nanostructures to possess 13-18 mm inhibition zones for E. coli and S. aureus, which is in very good correlation with the current findings. Similarly, Khan et al. [71] established 15-19 mm inhibition zones for Ag@CeO₂ NCs against various bacterial strains, corroborating the efficacy established in the current findings. Ahmad et al. [76] demonstrated excellent antibacterial activity of green-synthesized Ag-CeO₂ NPs with inhibition zones of up to 21 mm against S. aureus and E. coli, which are closest to our highest values observed. Tao et al. [77] also reported that Ag/CeO₂-loaded graphene aerogels exhibited >20 mm inhibition zones against multidrug-resistant bacteria, which was due to the synergistic effect from the release of Ag⁺ ions and ROS generation. These comparisons highlight that the Ag-CeO₂ and NiO-CeO₂ NCs synthesized in our research not only have similar or improved antibacterial activity to those already reported but also validate the green synthesis route and potential for biomedical and environmental applications.

3.9.3.2. Minimum inhibitory concentration (MIC)

Table 5 demonstrates the MIC results (μg/mL) of the biosynthesized NCs (Ag/CeO₂ and NiO/CeO₂ NCs) against various pathogenic bacteria. Remarkably, NiO/CeO₂ NC exhibited a predominantly strong inhibitory effect against B. subtilis and E. coli with MIC values of 1101.25 and 8810 μg/mL, respectively. The most effective potency of Ag/CeO₂ NC was noticed with much lower MIC values (110 μg/mL) against K. pneumoniae and S. aureus, as well as against Salmonella typhimurium, and B. cereus with MIC values of 440 μg/mL (Tables S5-S10 & Figure S1).

Figure S1

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Table 5. The MIC (μg/mL) results of the most potent NCs against pathogenic bacteria.

Bacterial species	Concentration (μg/mL)
Bacterial species	Ag/CeO₂ NC	NiO/CeO₂ NC
*Escherichia coli*	NT	8810
*Salmonella typhimurium*	440	NT
*Klebsiella pneumonia*	110	NT
*Bacillus subtilis*	NT	1101.25
*Bacillus cereus*	440	NT
*Staphylococcus aureus*	110	NT

NT: refer to not tested samples.

3.9.3.3. Antifungal activity

3.9.3.3.1. Radial fungal growth

The antifungal activity was evaluated against targeted pathogenic plant fungi: Rhizoctonia solani. The MIC of each nanomaterial was assessed (Table 6). Against R. solani, the MIC values were 221 and 1016 μg/mL for Ag/CeO₂ and NiO/CeO₂ NCs, respectively. This indicates that the silver NC was roughly five equivalents as effective as nickel oxide NC in inhibiting fungal growth. Furthermore, Ag/CeO₂ NC displayed concentration-dependent antifungal activity. Remarkably, even at its MIC threshold of 221 μg/mL, Ag/CeO₂ NC inhibited the growth of R. solani by 75.3%. Generally, this study recommends significant antifungal activity of these nanomaterials depending on the metal type and particle size. Previous studies have shown that NCs can inhibit fungal growth through various mechanisms, including the generation of reactive oxygen species (ROS), damage to the fungal cell membrane, disruption of cell wall architecture, interaction with fungal structures, inhibition of spore germination, and regulation of protein and gene expression [78]. Science shows that the antifungal effects of Silver NPs include their ability to fight Saccharomyces cerevisiae (MIC₅₀ = 2 µg/mL) together with Rhizoctonia solani and Aspergillus flavus [79,80]. A particular morphological characteristic combination of size and shape within NPs directly affects their antifungal abilities [81]. Scientific evidence indicates that both large NPs tend to have bigger surface areas, which enables better contact with fungal cell structures. The superior cell interaction created by this mechanism might result in cellular content leaking, which means fungal development would slow down. Indeed, research has shown that variations in NP preparation methods can result in a range of particle sizes, directly impacting their antifungal abilities [82]. NPs with a larger surface area due to a smaller particle size have been observed to exhibit significantly stronger antifungal properties [83].

Table 6. MIC values (μg/mL) of Ag/CeO₂ and NiO/CeO₂ NCs against pathogenic fungi.

Fungi	Ag/CeO₂ NC	NiO/CeO₂ NC
*Rhizoctonia solani*	221	1016

3.9.3.3.2. TEM of the NCs-affected fungi

The TEM micrograph of untreated R. solani cells (Figure 12a) shows healthy fungal cells that have clear cell walls and dense cytoplasmic contents. The cytoplasm, a jelly-like internal compartment, houses various cellular components essential for the fungus’s function. The micrograph shows all necessary cellular features, which include mitochondria along with ribosomes, and a nucleus, along with a vacuole. The TEM micrograph exhibits multiple small vesicles that appear inside the fungal cell cytoplasm. TEM analysis of R. solani cells treated with Ag/CeO₂ NC showed major structural damage via Figure 12(b) when compared to untreated cells in Figure 12(a). The cellular cytoplasm of this organism contains many dark and tiny dots, which appear to be processed NPs. Different hole formations and deteriorations in the cell wall structure become evident under inspection. Research findings indicate that Ag/CeO₂ NC may disrupt cell wall structures, which renders the fungus defenseless to additional harm [84].

TEM micrographs illustrate the antifungal activity of Ag/CeO2 and NiO/CeO2 NCs against R. solani. Panel (a) displays an intact cell wall structure of untreated R. solani hyphae. In contrast, panels (b) and (c) reveal significant damage to the cell wall and membranes of R. solani treated with Ag/CeO2 and NiO/CeO2 NCs, respectively.

A TEM investigation of NiO/CeO₂ NC-treated R. solani organisms showed cellular damage that exceeded the damage levels observed in untreated fungi (Figure 12c). The cytoplasm contains numerous small dark inclusions, which might function as internalized NPs. The cells displayed considerable damage through numerous holes that appeared in the cell walls. The NC shows potential in damaging R. solani cell walls, thus making the fungus more vulnerable to incremental harm [85]. The observed harm progresses from the cell wall into multiple internal organelles, which reside within the cytoplasm. The structure of some mitochondria appears swollen, while their shapes become distorted, which suggests potential damage to their energy production functions. Researchers are currently working to determine how NCs fight fungal infection through their antifungal mechanism, although various theories exist about potential pathways. ROS forms inside fungal cells because of the NPs and disrupts their essential cellular components. The NPs can engage directly with R. solani membranes, thus leading to their structural obstruction and impaired functioning [37]. The NPs display the potential to enter cells, after which they can interact with essential cellular components, including DNA, thus potentially inflicting more harm [86,87]. Several different mechanisms jointly operate to cause the antifungal impact of NCs during R. solani infections.

3.10. Molecular docking study

Here, we used a molecular docking study to uncover the interaction between the targeted ligands and the targeted PDB: 2I80 protein (Table S11). Some of the interactions that apigenin had were hydrogen bonds and π interactions. These included O 18 of the phenol ring with GLU213 (H-donor, 3.00 Å), O 4 of the carbonyl group with ALA218 (H-acceptor, 3.30 Å), Phenol ring with GLY215 (π-H, 4.52 Å), and Phenol ring with PHE175 (π-π, 3.94 É), and RMSD = 1.9009 (Figure 13a). However, caffeic acid displayed a modest binding score (S = -5.2195 kcal/mol) resulting from binding interactions among C 2 of the styryl group with Met128 (H-donor, 4.02 Å), C 5 of the catechol ring with MET128 (H-donor, 3.84 Å), and O 13 of the carboxylic acid with GLU16 (H-donor, 2.81 Å) through RMSD = 0.8112 (Figure 13b). Meanwhile, Chlorogenic acid exhibited the best binding score (S = -7.3531 kcal/mol), with an interaction including O 12 of the hydroxyl group with LEU94 (H-donor, 2.98 Å) and O 24 of the catechol ring with SER127 (H-donor, 3.19 Å). The RMSD was 1.7235 (Figure 13c). Also, ferulic acid showed a weak binding score (S = -5.0256 kcal/mol) with an interaction resulted from bindings between O 13 of the carboxylic acid with ALA309 (H-acceptor, 2.93 Å), O 13 of the carboxylic acid with MET310 (H-acceptor, 3.13 Å), and C 8 of the methoxy group with HIS96 (H-π, 3.86 Å) over RMSD = 1.1747 (Figure 13d). Furthermore, luteolin revealed a moderate binding score (S = -5.6926 kcal/mol) by one interaction between O 4 of the carbonyl group and LYS241 (H-acceptor, 3.63 Å) through RMSD = 1.1237 (Figure 13e).

(a-e) The binding images between the extracted compounds and the PDB: 2I80 protein is of “Allosteric inhibition of Staphylococcus aureus”.

4. Conclusions

The research demonstrated successful findings about metal selection effects on the properties of bio-synthesized NCs. The metal selection process for Ag-CeO₂ with silver inclusion led to enhanced levels of essential phytochemicals (phenolics, flavonoids, tannins) and superior free radical protection than NiO-CeO₂ (nickel-based). Research data indicate Ag-CeO₂ holds potential uses as an antioxidant therapeutic substance. The Ag-CeO₂ NC showed great potential as an antimicrobial solution because it successfully fought against different bacterial strains and the fungus Rhizoctonia solani with greater effectiveness. The research findings demonstrate the potential of bio-synthesized NCs to serve multiple healthcare applications and different applications beyond healthcare. Subsequent investigations need to build upon the existing data regarding mechanisms by refining the development of specialized NCs for particular applications. Experimental research should focus on examining the interactions between plant extract, metal NPs, and CeO₂ while investigating various synthesis conditions. Eventually, this point of research holds significant aptitude for the development of novel, multifunctional NC materials for various applications. The molecular docking analysis showed that chlorogenic acid had the strongest binding affinity of all the compounds tested. Lutein, apigenin, caffeic acid, and ferulic acid came in that order. Chlorogenic acid’s superior binding score and effective hydrogen donor interactions with critical residues indicate its potential as a prospective antibacterial agent.

CRediT authorship contribution statement

Fatmah O. Sefrji, Albandary Almahri: Data curation, formal analysis, methodology, and software; Mansoor Alsahag, Ali Alisaac: Investigation and writing – review & editing; Abdulmajeed F. Alrefaei, Hawra A. Alghasham: formal analysis, investigation, writing-original draft. Wael M. Alamoudi, Majid A. Bamaga: Supervision and administration of research group.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All relevant data are within the manuscript and available from the corresponding author upon request.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Supplementary data

Supplementary material to this article can be found online at https://dx.doi.org/10.25259/AJC_315_2024.

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Green synthesis of multifunctional Ag/CeO2 and NiO/CeO2 nanocomposites using Matricaria chamomilla extract as promising agents for combating microbial infections and oxidative stress

Abstract

Keywords

Antibacterial

Antioxidant activities

Characterization of nanocomposites

Chlorogenic acid

Green synthesis

Matricaria chamomilla

Phytochemical analysis

1. Introduction

2. Materials and Methods

2.1. Reagents

2.2. Instruments

2.3. Preparation of plant extract

2.4. Green synthesis of NCs

2.5. Phytochemical analyses

2.5.1. Total tannin contents

2.5.2. Total phenolic contents

2.5.3. Total flavonoid contents

2.6. Antioxidant 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

2.7. Antimicrobial assessment

2.7.1. Antibacterial activity

2.7.2. Antifungal activity

2.8. Statistical analysis

2.9. In silico molecular docking study

3. Results and Discussion

3.1. The plausible mechanism of formation of NCs

3.1.1. Reduction of metal precursors:

3.1.2. Interaction with CeO2:

3.1.3. NP growth and stabilization:

3.2. UV-visible spectroscopy

3.3. FTIR spectral analyses

3.4. Zeta potential analyses

3.5. Dynamic light scattering (DLS) analysis

3.6. HR-TEM

3.7. SEM

3.8. EDX

3.9. XRD

3.9.1. Phytochemical analyses

3.9.2. Potential antioxidant activity

3.9.3. Antimicrobial activity

3.9.3.1. Antibacterial activity

3.9.3.2. Minimum inhibitory concentration (MIC)

3.9.3.3. Antifungal activity

3.9.3.3.1. Radial fungal growth

3.9.3.3.2. TEM of the NCs-affected fungi

3.10. Molecular docking study

4. Conclusions

CRediT authorship contribution statement

Declaration of competing interest

Data availability

Declaration of Generative AI and AI-assisted technologies in the writing process

Supplementary data

References

Suggested read for related articles:

Green synthesis of multifunctional Ag/CeO₂ and NiO/CeO₂ nanocomposites using Matricaria chamomilla extract as promising agents for combating microbial infections and oxidative stress

3.1.2. Interaction with CeO₂: