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Efficacy of mycosynthesized zinc oxide nanoparticles against bacterial infections and oxidative stress
* Corresponding author E-mail address: ff7902533@gmail.com (F. Fatima); danish.khan@bpc.edu.sa (D. Iqbal)
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Received: ,
Accepted: ,
Abstract
Biologically mediated zinc oxide nanoparticles (ZnO NPs) were synthesized from Aspergillus flavus extract and evaluated to check their antibacterial and antioxidant properties, particularly against Mycobacterium fortuitum and Serratia marcescens, two significant healthcare-associated pathogens. For this purpose, ZnO NPs were synthesized using the extracellular supernatant of A. flavus, where a pale white color was observed after 24 hrs, indicating successful nanoparticle formation. Various characterization techniques were employed to analyze the synthesized nanoparticles, where UV-Vis spectroscopy revealed a strong absorption peak at 375 nm, confirming ZnONP formation. Differential light scattering (DLS) analysis showed two peaks, one between 30–100 nm and another from 200–1000 nm, indicating size variation and possible aggregation. Fourier transform infrared (FTIR) spectroscopy displayed peaks at 3326 cm-1, 2990 cm-1, 2361 cm-1, 1640.07 cm-1, 1053.87 cm-1, and 547 cm⁻1, while scanning electron microscopy (SEM) confirmed spherical nanoparticles sized 30–100 nm. Our results demonstrated a significant antibacterial effect that increased with higher nanoparticle concentrations, where ZnO-NPs at 150 μg/disc showed inhibition zones of 22 mm against M. fortuitum and 13 mm against S. marcescens. In the reactive nitrogen assay, the IC50 values for ZnO NPs and gallic acid were found to be 130 μg/mL and 100 μg/mL, respectively, while in the reactive oxygen assay, ZnO NPs showed an IC50 value of approximately 115 μg/mL, comparable to that of gallic acid thus prooving that these NPs can effectively scavenge free radicals such as superoxide and nitric oxide. These findings suggest that ZnO NPs synthesized using A. flavus are not only environmentally friendly and non-toxic but also hold great promise as effective antimicrobial and antioxidant agents for potential biomedical applications.
Keywords
Antioxidant activity
Aspergillus flavus
Pathogenic microbes
Zinc oxide nanoparticles
1. Introduction
Various diseases, such as bacterial and fungal infections, contribute to adverse impacts on human health. Despite the formulation of numerous therapeutic drugs to combat these infections, their practical application faces significant limitations (Denning 2024; Ikuta et al. 2022). Due to resilience towards chemicals drugs and ability to build biofilms, most bacterial strains such as Mycobacterium fortuitum (Gram positive, non-tuberculous mycobacterium) that can cause diseases of the skin, lungs, and surgical sites, particularly among immunocompromised individuals whereas and Serratia, marcescens, a member of the Enterobacteriaceae family of gram-negative bacteria, is harmful in healthcare settings due to its ability to cause a variety of diseases, such as respiratory and urinary tract infections (Kakoullis et al. 2024). Common antibacterial drugs like penicillin, ciprofloxacin, and amoxicillin are effective but face limitations due to rising antibiotic resistance and side effects such as allergies or toxicity. These issues reduce their overall effectiveness and safety in long-term use (Naghavi et al. 2024).
Reactive oxygen species (ROS) and reactive nitrogen species (ROS and RNS) scavenging assays are vital in medicinal and anticancer research as they assess a compound’s antioxidant potential, helping to develop therapeutic strategies. These assays provide crucial insights into a substance’s ability to neutralize oxidative stress, which contributes to microbial cell damage. Understanding ROS and RNS assays is key for creating antimicrobial drugs that target oxidative stress mechanisms in pathogens, with potential applications in both infection control and cancer treatment (Donkor 2023). As a result, understanding the RNS test and ROS is essential for locating and enhancing substances that have the potential to treat cancer as well as microbial infections.
Recent advancements in nanoparticle development offer enhanced antibacterial action by penetrating biofilms and disrupting bacterial cell walls, reducing resistance development might be due to their small size, which allows targeted delivery, and minimizing side effects (Pathakoti et al. 2019). However, chemical and physical methods for nanoparticle synthesis have been identified as expensive, hazardous, and chemically damaging in nature. Biological methods for nanoparticle synthesis are eco-friendly and cost-effective, utilizing natural resources. They offer improved biocompatibility and reduce the need for harmful chemicals. Moreover, natural products and their derived products are valuable in treating various infectious and non-communicable diseases (Bhattacharjee et al. 2023). They help to combat antibiotic resistance through diverse mechanisms of action by disrupting bacterial cell walls, inhibiting protein synthesis, or interfering with DNA replication, effectively hindering bacterial growth and survival (Bano et al. 2023).
Fungi are ideal for nanoparticle synthesis due to their ability to tolerate metals, produce large amounts of reducing enzymes, and act as eco-friendly bioreactors. They offer a low-cost, non-toxic, and energy-efficient alternative to chemical methods, supporting green chemistry (Chauhan et al. 2023; Elazab et al. 2023). The rationale for forming nanoparticles using fungi lies in the principles of green chemistry, which emphasize sustainability and reduced environmental impact (Zafar and Iqbal 2024). They produce nanoparticles under mild conditions without harmful solvents or byproducts. This biological approach also enables better control over size, shape, and functionality, making it highly suitable for medical and environmental applications (Zafar et al. 2023). Bioactive metabolites and nanoparticles from natural sources exhibit antibacterial, antifungal, antioxidant, and anticancer effects. Zinc oxide nanoparticles (ZnO NPs), with their high surface area and unique properties, show strong antimicrobial activity, are human-compatible, and have low toxicity, making them excellent antibacterial agents (Divya et al. 2024). Thus, we hypothesize that these biologically synthesized ZnO NPs using Aspergillus flavus exhibit significant antibacterial and antioxidant activities due to their nanoscale properties and bioactive surface chemistry, making them effective agents against healthcare-associated pathogens and oxidative stress.
In this study, A. flavus was utilized for the eco-friendly formation of ZnO NPs, as highly promising nanomaterials. Our green synthesis method, utilizing extracellular materials, focuses on exploring the antibacterial, antifungal, and antioxidant potentials of these mycologically synthesized ZnO NPs.
2. Materials and Methods
2.1 Chemicals
In the current research, all the reagents and chemicals used were of analytical grade. Malt extract broth [HiMedia], zinc nitrate hexahydrate [Sisco Research Laboratories Pvt. Ltd., Maharashtra, India.], NB media [Hi-media], ciprofloxacin disc [HiMedia, Maharashtra, India], potato dextrose agar [HiMedia Laboratories, Maharashtra, India,], amphotericin B antibiotic [HiMedia, Maharashtra, India], Dulbecco’s modified Eagle’s medium (DMEM) [Thermo- Fischer, Hyderabad, Chennai], sodium nitroprusside (SNP) [Otto Chemie Pvt. Ltd. Tardeo Road, Mumbai], Griess Illosvoy [Thermo- Fischer, Hyderabad, Chennai], phosphate-buffered saline [Sigma-Aldrich, St. Louis, MO, USA], sulfanilamide [Thermo- Fischer, Hyderabad, Chennai,], glacial acetic acid [Thermo- Fischer, Hyderabad, Chennai], napthylethylenediamine dihydrochloride NED [Thermo- Fischer, Hyderabad, Chennai], Nitro-Blue-Tetrazolium (NBT) [HiMedia, Maharashtra, India], phenazine-methosulfate (PMS) [Hi-Media, Maharashtra, India], nicotinamide adenine dinucleotide (NADH) [HiMedia, Maharashtra, India] were used in present study.
2.2 Biosynthesis of ZnO NPs by A. flavus
A morphologically and molecularly characterized A. flavus isolate was previously isolated was used for this study (Fatima et al. 2016). The isolated Aspergillus flavus was grown in malt extract broth for 5 days at 28°C (±2°C). After that, the mycelial biomass was collected, filtered, and washed with sterile water. About 20 g of this biomass was added to 150 mL of double-distilled water. This mixture was placed in an Erlenmeyer flask and shaken at 120 rpm for 72 hrs at 28°C (±2°C) and pH 7 to allow the fungus to release proteins into the liquid. The resulting liquid (supernatant) was then mixed with 1 mM zinc nitrate hexahydrate in a total volume of 150 mL. This mixture was stirred continuously and kept at pH 9 and 28°C for another 72 hrs to allow the formation of ZnO NPs. (Alqurashi et al. 2023). A light white precipitate was obtained, which was dried further at 150°C, rinsed, and then dried again at 80°C. The dried material was ground into a powder and calcined at 400°C for 2 hrs to remove impurities, confirming the formation of ZnO NPs.
2.3 ZnO NPs Characterization
2.3.1 Ultraviolet–visible spectroscopy
The formation of ZnO nanoparticles was confirmed using a Beckman DU-20 spectrophotometer, which measured absorbance in the range of 300 to 500 nm. The software “UVWinlab” (Version 1.05) was used to study and record the results in more detail. Distilled water was used as the standard during the test, and the salt-free fungal supernatant was used as the control. The UV–Visible spectrometer used for this analysis was from Spectra-Lab Scientific Inc, Markham, Canada, ZnO NPs (Abdel-Hadi et al. 2023).
2.3.2 Differential light scattering (DLS)
To measure the size of the ZnO nanoparticles, water viscosity at 25°C was used for calculations. A Malvern Zeta-Sizer Nano (ZSTM) with Dispersion Technology Software v.5.1 was used to check the size distribution, average particle size, and polydispersity index (PDI). Before testing, each sample was kept at 25°C for 2 mins, diluted to 0.5% with deionized water, and then sonicated for 1 min to mix well. The refractive index (RI) of the ZnO nanoparticles was found to be 1.78.
2.3.3 SEM with energy dispersive X-ray spectroscopy (SEM-EDX) analysis
The produced nanoparticles were mixed with DMSO and subsequently dried on a glass slide to form a thin layer that could be further studied. To ascertain the chemical characteristics of a nanomaterial and its composition, energy dispersive spectrometry was used with an X-ray diffractometer D-8 ADVANCE ECO BRUKER, Billerica, Massachusetts. Schottky Field Emission Scanning-Electron Microscope [JSM-IT800] from JEOL Ltd., Japan was used to take ZnO NP images (Mohan and Renjanadevi 2016).
2.3.4 Fourier-Transform-Infrared (FTIR) spectroscopy
FTIR spectroscopy was used to identify the metabolites present in the fungal extract. The extract was first cooled to preserve its chemical properties and then mixed with potassium bromide (KBr) in a 1:100 ratio to prepare it for analysis. The mixture was either pressed into a pellet or directly applied to the attenuated total reflectance (ATR) crystal, depending on the method used. The FTIR analysis was performed using a Perkin-Elmer Spectrum instrument with Universal ATR accessories. The spectra were recorded in the range of 400 to 4000 cm⁻1 to detect various functional groups. The results were saved and analyzed using Spectrum 10 software to determine the presence of specific metabolites in the fungal extract.
2.4 Antibacterial assay
The current research used M. fortuitum [ATCC 6841] and S. marcescens [NCIM 2078], purchased from the National Centre for Cell Science (NCCS), Pune, India. Bacterial strains were initially maintained at 4°C on nutritional agar slants and were subsequently transferred to nutrient broth [HiMedia] for ZnO NPs antibacterial evaluation. The bacterial cultures were prepared by adjusting their turbidity to 0.8 at OD600, which corresponds to approximately 1.5 × 10⁸ CFU/mL. For the antibacterial test, paper discs were loaded with 10 µL of ZnO NPs at different concentrations ranging from 0 to 150 µg/mL. To prepare the stock solution, 1 mg of ZnO NPs was accurately weighed using an analytical balance and dissolved in 1 mL of a suitable solvent that ensured complete dissolution. This stock was then diluted to obtain the required concentrations for testing. For the assay, each disc was placed on an agar plate inoculated with the bacterial culture. As a positive control, discs containing 200 µg/mL of ciprofloxacin were used. The salt-free fungal supernatant was used as a negative control. All plates were incubated at 37°C for 24 hrs (± 4°C). After incubation, the antibacterial activity was determined by measuring the clear zones around each disc, called zones of inhibition, in millimeters. Each experiment was performed in triplicate, and the average zone of inhibition was recorded for analysis.
2.5 Antioxidant assay
2.5.1 Cell lines
The HCT116 human colorectal cancer cell line was obtained from the NCCS, Pune, India, and maintained at the Animal Tissue Culture facility of Integral University. The cell lines were sub-cultured in DMEM containing 10% fetal calf serum (FCS), 1% antibiotic–antimycotic, and 2 mM glutamine. Further cell cultures were kept in 75 cm culture flasks at 37°C with 5% CO2. After 10 days, the cells were allowed to move to 96-well microtiter plates having fresh growth medium (Mukherjee et al. 2023). The cells were reseeded at a density of 10 × 103 cells per well and incubated at 37°C for 24 hrs in a CO₂ incubator with less than 5% CO₂. During this period, the cells were treated with varying concentrations of ZnO NPs ranging from 0 to 250 µg/mL.
2.5.2 Reactive nitrogen oxide scavenging assay
Nitric oxide was generated by mixing SNP in phosphate buffer (pH 7.4) with different concentrations of ZnO NPs (0–250 μg/mL) in a total volume of 3 mL. After 150 mins of incubation at 25°C ± 4, 1 mL of 0.33% sulfanilamide (in 20% glacial acetic acid) was added to 0.5 mL of the reaction mixture, followed by 1 mL of 0.1% NED. The mixture was incubated again for 30 mins at 25°C. Nitrite formation was measured at 540 nm using the Griess reaction, with gallic acid as the standard. All tests were performed in triplicate ZnO NPs (Rojas-García et al. 2022). The cells were given treatment with ZnO NPs at varying doses, ranging from 0 to 250 µg/mL.
2.5.3 Super oxide anion radical scavenging assay
The activity was evaluated by measuring the reduction of Nitro-Blue Tetrazolium (NBT) into purple formazan, indicating the presence of superoxide radicals. These radicals were generated through a reaction between NADH and phenazine methosulfate (PMS). The reaction mixture contained phosphate buffer (PBS), PMS, NBT, NADH, and ZnO-NP samples at concentrations ranging from 0 to 250 μg/mL. After 5 mins, the absorbance was measured at 560 nm using an ELISA plate reader. Gallic acid was used as the positive control, and all experiments were conducted in triplicate (Salman et al. 2025; Zheng et al. 2016).
2.6 Statistical analysis
Every experiment was conducted in triplicate, and the mean ± standard deviation was used to report the findings. For p < 0.001, significance levels were shown as ***, indicating a substantial difference from the control group. Values with a normal distribution were analyzed using ANOVA, and homogenous variance was examined using a t-test.
3. Results
3.1 Fungal mediated ZnO NPs synthesis
The synthesis of ZnO NPs occurred by mixing the fungal extract with a solution of Zn(NO3)2.6H2O]. The color of the solution changed from light yellow to pale white within 24 hrs. A control solution containing a solution of 1 mM of Zn(NO3)2.6H2O that showed no color change under similar conditions (Vijayakumar et al. 2022). The observation of pure ZnO NPs as a white powder suggests the initial formation of nanoparticles (Fig. 1a). These nanoparticles, synthesized using an environmentally friendly method, displayed a white crystalline structure and were used to assess absorption spectra. The quantity of zinc nanoparticles formed from the zinc nitrate hexahydrate salt in this solution was 9.8 mg.
![(a) Conical flask showing zinc nitrate hexahydrate [Zn(NO3)2.6H2O], fungal filtrate, and 1 mM solution ZnO-NPs [150 mL of reaction volume] (b) UV–Vis absorption spectra of ZnO-NPs, zinc salt, and fungal filtrate.](/content/185/2025/37/2/img/JKSUS-37-4252025-g1.png)
- (a) Conical flask showing zinc nitrate hexahydrate [Zn(NO3)2.6H2O], fungal filtrate, and 1 mM solution ZnO-NPs [150 mL of reaction volume] (b) UV–Vis absorption spectra of ZnO-NPs, zinc salt, and fungal filtrate.
3.2 ZnO NPs characterization
3.2.1 Ultraviolet-visible spectroscopy
Within the spectrum of 200–450 nm, there is no detectable absorbance observed for the fungal extract alone. Nevertheless, when fungal extracts are subjected to treatment with Zinc precursor salt, a distinct absorption peak emerges prominently at 375 nm. This observation strongly indicates the successful synthesis of ZnO NPs. Consistent findings have been investigated in prior studies, demonstrating that the spectral absorbance range of of ZnO NPs falls between 200–450 nm (Fig. 1b). Previous studies have shown similar results, indicating the absorbance spectra of ZnO NPs between the range of 200–450 nm (Jamdagni et al. 2018).
3.2.2 DLS
The results depict the scattering intensity plotted against the logarithm of the diameters of particles (Fig. 2). There are two peaks in the distribution, with the larger diameter having higher intensity. On average, the particle size is 300 nm, and they have a spherical shape with clusters of nanocrystals. Two peaks spanning from 30-100 nm and 200-1000 nm were observed in the DLS parameter, suggesting that the larger size might be attributed to nanoparticle aggregation. The synthesized ZnO NPs demonstrated polydispersity, evident from their PDI value of 0.3.
- DLS data of ZnO NPs.
3.2.3 SEM- EDX analysis
Pictures were captured from the surface coated with ZnO NPs, uniformly distributed across the grid, further corroborating the circular morphology observed in SEM micrographs. The sizes ranged from 30 nm to 100 nm, with the larger sizes likely resulting from nanoparticle aggregation (Fig. 3a), a phenomenon evident in the DLS data as well. EDX analysis was further utilized to examine the elemental constituents of ZnO NPs, where the EDX spectra confirmed the presence of oxygen and zinc elements. Element characterization of ZnO NPs disclosed a weight percentage of 70.57% zinc and 29.43% oxygen, while the atomic percentage revealed 36.98% zinc and 63.02% oxygen (Fig. 3b).
- (a) SEM images of ZnO-NPs ranging from 30 nm to 100 nm (b) EDX spectrum of synthesized ZnO NPs
3.2.4 FTIR spectroscopy
The characterization of fungal extract and ZnO NPs was examined by using FTIR spectroscopy. Absorption bands occurring within the region <1000 cm−1 are indicative of interatomic vibrations. The FTIR analysis of the fungal extract revealed absorption spectral peaks at 3195.81, 2781.5, 1658.84, 1448.78, 1239.80, and 527 cm−1, while the FTIR analysis of the ZnO NPs revealed absorption peaks at 3326, 2990, 2361, 1640.07, 1053.87, and 547 cm−1. Notably, the peaks were present approximately at 1053 cm−1 with distinctive features, signifying absorption related to Zn-O bonding. The absorption peak at 1643 cm−1 depicted the stretching vibration of C=C. Concurrently, the peaks at 1053.87 and 547.88 cm−1. The absorption peaks around 1640.07 cm−1 denoted C=C stretching, while the peak present at 3326 cm−1 indicated stretching vibrations of the -OH group. Additionally, the presence of C-O was indicated by showing a peak at 2361 cm−1. The peaks at 3326 cm−1 were initially observed and gradually diminished. The FTIR spectra clearly depicted an interference pattern, highlighting the peaks of Zn-O at 1053 cm−1, thereby affirming the ZnO NPs (Fig. 4).
- FTIR analysis of (a) Fungal extract of A. flavus and (b) ZnO-NPs (Wavelength cm-1)
3.3 Antibacterial activity
The inhibition zones of ZnO NPs at a concentration of 150 μg/disc against M. fortuitum and S. marcescens were determined to be 22 mm and 13 mm (Fig. 5(a) and (b)). Moreover, the fungal extract did not exhibit a zone of inhibition against the test strains. This implies that the effective antibacterial action is due to the ZnO NPs. Significant increases in antibacterial activity were noted with increasing ZnO NPs concentration. According to a comparative investigation, ZnO NPs had better antibacterial activity against M. fortuitum, followed by S. marcescens. A statistically significant variance from the untreated group is indicated by values highlighted with three asterisks (***), with a p-value of less than 0.001. This level of significance suggests that the observed differences are highly unlikely to occur by chance alone, indicating a strong effect or relationship between the variables being studied.
- (a) Antibacterial activity of ZnO-NPs against pathogenic bacteria, where “C” acts as 10 µL of fungal extract only and antibiotic as “+ive Control”. The data represents the mean ± standard deviation of three different experiments, where *** represents significantly different from the control group (** p < 0.001). Disc diffusion assay on (b) M. fortuitum (c) S. mercesens.
3.4 Antioxidant assay
3.4.1 Reactive nitrogen oxide scavenging assay
Mammalian cells generate nitric oxide, a free radical that is involved in controlling a number of physiological functions such as neurotransmission, vascular homeostasis, and antibacterial and anticancer properties. But excessive NO generation is linked to a number of disorders. The percentage of nitric oxide radical inhibition by ZnO NPs was found to be 70%, which is comparable to the standard gallic acid (100%) at 250 μg/mL. The IC50 value of ZnO NPs and gallic acid was 130 μg/mL and 100 μg/mL. The results suggested that ZnO NPs have a low nitric oxide radical scavenging effect when compared with gallic acid (Fig. 6). A statistically significant difference from the control group is indicated by values with three asterisks (***), and the p-value is less than 0.001. It indicates a substantial influence or association between the variables under study and signifies that the observed differences are very unlikely to be the result of random fluctuation.
- Reactive nitrogen oxide scavenging activity (%) of ZnO NPs. Statistically significant difference from the control group is indicated by values with three asterisks (***), and the p-value is less than 0.001.
3.4.2 Superoxide anion radical scavenging assay
Superoxide anions can cause direct or indirect damage to biomolecules by generating hydrogen peroxide. These anions have been demonstrated to initiate lipid peroxidation. A dose-dependent relationship was found between the scavenging abilities of gallic acid and ZnO NPs on superoxide radicals, with a maximum scavenging activity of 90% and 85% at 250 μg/mL, respectively. Our results showed that the ZnO NPs IC50 value is nearly 115 μg/mL, which is comparable to gallic acid. Consequently, ZnO NPs synthesized from A. flavus displayed notable superoxide scavenging activity (Fig. 7). With a p-value of less than 0.001, values indicated with three asterisks (***) indicate a statistically significant difference from the control. This suggests a strong influence or relationship between the variables under study because it is extremely unlikely that the observed differences are the result of random variation.
- Superoxide anion radical scavenging activity (%) of ZnO NPs. Statistically significant difference from the control group is indicated by values with three asterisks (***), and the p-value is less than 0.001.
4. Discussion
Fungal sources, due to their ability to withstand the effects of metallic ions, have become attractive options for the NPs synthesis (Wahab et al. 2023). In the biological synthesis method, the culture filtrate of fungi serves as a crucial component. The fungal extract, rich in bioactive compounds and proteins, is collected and then combined with a zinc precursor, commonly zinc nitrate hexahydrate (Huang et al. 2021). The enzyme NADH/NADPH acts as reducing agents, and the other bioactive compounds facilitate in the reduction of Zn2⁺ in the precursor solution to elemental zinc (Zn) nanoparticles (Ahmad and Kalra 2020; Ovais et al. 2018). A. flavus produces various biomolecules that play an important key roles in nanoparticle synthesis and biological activity. Proteins like laccases and peroxidases reduce metal ions and stabilize nanoparticles, while polysaccharides and phenolic compounds (flavonoids, tannins) aid stabilization and act as reducing agents. Other secondary metabolites, including alkaloids and terpenoids, enhance nanoparticle bioactivity, making them valuable for therapeutic and antimicrobial applications.
In our study, ZnO NPs were synthesized using the fungal extract of A. flavus as both a capping and reducing agent, which contained bioactive metabolites. The absorbance spectrum displayed a surface plasmon resonance (SPR) peak at 375 nm. Likewise, bioinspired ZnO NPs derived from Fraxinus rhynchophylla and Aspergillus terreus exhibited a maximum SPR peak at 370 nm. In DLS analysis, a size distribution ranging from 30 nm to 100 nm and 200-1000 nm was noted, potentially arising from nanoparticle aggregation. The variation in size is attributed to the formation of nanostructures via the metallic salt’s oxidation in the presence of proteins (Gahlawat and Choudhury 2019; Pandit et al. 2022). SEM analysis also revealed that the ZnO-NPs were spherical in shape with sizes ranging from 30 to 100 nm. The size variability is associated with the reduction of metals into their nanoparticles, influenced by the enzyme. Additionally, the presence of various functional groups that are responsible for the capping on nanoparticles is further confirmed by FTIR spectrum analysis. In our study, the FTIR spectrum of ZnO nanoparticles synthesized by A. flavus, the peaks at around 547.8 cm⁻1 represent the Zn-O bond stretching, while the peaks at ∼1640.07 cm⁻1 and ∼3326 cm⁻1 correspond to amide I (proteins), C=O bond, C=H bond, and hydroxyl groups (OH), respectively. These functional groups play an important role in the reduction and stabilization of nanoparticle synthesis. Earlier reports have discussed the FTIR analysis of fungal extracellular protein in the context of A. flavus, demonstrating comparable peaks to those identified in our findings (Chatterjee et al. 2020). These findings suggest that the nanoparticles produced in our investigation are coated with a fungal extracellular matrix that includes metabolites and proteins. The EDX analysis exhibits a strong peak for zinc, confirming the presence and synthesis of ZnO NPs, while other peaks correspond to oxygen.
The antibacterial efficacy of synthesized NPs was evaluated against M. fortuitum and Serratia marcescens, bacterial strains often responsible for opportunistic infections in healthcare settings, renowned for their resistance to many antibiotics and ability to form biofilms. We observed the optimal effectiveness of ZnO NPs at a concentration of 150 μg/ml against pathogenic bacterial strains. The selection of 150 μg/ml for testing the antimicrobial effect was likely based on prior research and optimization studies indicating this dosage as effective in inhibiting microbial growth. This specific amount provides a balance between achieving a measurable antimicrobial response while minimizing potential toxicity. The effectiveness of ZnO NPs against various bacterial species like Pseudomonas aeruginosa, Staphylococcus aureus, Serratia liquefaciens, and Lysinibacillus fusiformis is influenced by their concentration, particle size, and surface modifications. Different studies have tested varying concentrations, often finding that antibacterial activity improves with higher concentrations due to the release of Zn2⁺ ions, which disrupt bacterial cell membranes, induce oxidative stress, and interfere with cellular processes. Additionally, ZnO NPs can disrupt bacterial cell membranes, resulting in increased permeability and eventual cell lysis. Furthermore, ZnO may interfere with essential metabolic processes by releasing zinc ions, which can disrupt enzymatic functions and inhibit bacterial growth (El-Fallal et al. 2023; Geetha et al. 2020; Maruthupandy et al. 2018).
The assays of superoxide anion radical scavenging and reactive nitrogen oxide scavenging are crucial in the study of oxidative stress because they provide important information about the possible health advantages (Chaudhary et al. 2023). Finding substances that can eliminate these extremely ROS and display strong antioxidant qualities is made possible by the superoxide anion radical scavenging assay. This is crucial for cellular protection, as oxidative stress is implicated in various diseases and aging processes (Hajam et al. 2022; Tan et al. 2018) (Al-Shehri 2021; Ilari et al. 2020). Thus, according to our findings, A. flavus extract helped create ZnO NPs, which have a specific antioxidant effect on HCT116 cancer cells. Despite their promising potential, ZnO NPs derived from fungal sources have received limited attention for their antibacterial and antioxidant effects against HCT116 cell lines. The lack of thorough investigations indicates that further research is needed to fully uncover their medicinal potential.
5. Conclusions
We effectively utilized the fungus A. flavus to create a simple, dependable, and eco-friendly method for producing ZnO NPs. Using UV-visible spectroscopy, the intensity of the pale white color was examined across wavelengths ranging from 200 to 450 nm, identifying a peak at 375 nm. DLS and scanning electron microscopy (SEM) analyses confirmed that the synthesized ZnO NPs were spherical and ranged from 30 nm to 100 nm, with some aggregation observed between 200 nm and 1000 nm. The prepared ZnO NPs demonstrated effective antibacterial efficacy against pathogenic bacterial strains and exhibited scavenging activity against reactive nitrogen oxide and superoxide anion radicals in human colorectal cell lines. Future studies should focus on optimizing synthesis conditions to minimize aggregation and enhance the uniformity of ZnO NPs. Additionally, further exploration of the mechanisms underlying the antibacterial and antioxidant properties of these nanoparticles is needed, along with in vivo studies to assess their therapeutic potential. Overall, the fungal strain utilized presents several advantages, including efficient ZnO-NP production with microbicidal and antioxidant capabilities, offering a cost-effective, safe, and biocompatible approach for ZnO NP synthesis. This method contributes to developing disease therapies with reduced environmental impact.
Acknowledgments
The authors extend their appreciation to the deputyship for Research & Innovation, Ministry of education in Saudi Arabia for funding this research work through the project number (IFP-2022-07).
CRediT authorship contribution statement
Ahmed Abdel-Hadi: Designed the concept of this study; Raed Alharbi and Awatif B. Albaker: Data analysis and interpretation; Sadaf Jahan and Mohammed Alsaweed: Conceptualization; Omar Darwish and Danish Iqbal: Investigate the research; Yahya Madkhali: Data curation; Manikanadan Palanisamy: Data collection; Faria Fatima: Original draft; Bader Mohammed Alshehri and Sahar Aldosari: Validation of manuscript.
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.
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.
Funding
This research was funded by deputyship for Research & Innovation, Ministry of education in Saudi Arabia, grant number “IFP-2022-07.
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