Eco-Friendly Zinc Oxide Nanoparticles from Peppermint: Synthesis, Characterization, and Antimicrobial Evaluation

Eco-Friendly Zinc Oxide Nanoparticles from Peppermint: Synthesis, Characterization, and Antimicrobial Evaluation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Eco-Friendly Zinc Oxide Nanoparticles from Peppermint: Synthesis, Characterization, and Antimicrobial Evaluation Shahira Al-Reisis, Arshad Ali, Maryam Al-Zadjali, Ali- Al-Subhi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6539690/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2026 Read the published version in BioNanoScience → Version 1 posted 12 You are reading this latest preprint version Abstract This research presents an eco-friendly technique for synthesizing zinc oxide nanoparticles (ZnONPs) using peppermint (Mentha Piperita) extract as both a reducing and stabilizing agent. The synthesis involved a controlled reaction between zinc nitrate hexahydrate [Zn(NO 3 ) 2 .6H 2 O] and the Peppermint extract. The resulting ZnONPs were characterized using SEM, EDXS, TEM, XRD, FTIR, and UV-Vis spectroscopy. SEM revealed well-defined triangular crystals, while TEM showed spherical nanoparticles sized 20–50 nm. EDXS confirmed the elemental composition as Zn = 80.4% and O = 19.6%. XRD analysis validated the crystalline structure, and FTIR identified biomolecules involved in the synthesis. UV-Vis spectra displayed an absorption peak at 220 nm. Antimicrobial testing using the well diffusion method showed inhibitory zones of 0.2, 0.1, and 0.05 mm at zinc nanoparticle (ZnONPs) concentrations of 35, 25, and 15 mg/ml, respectively. The results demonstrate effective bacterial inhibition, with the highest concentration (35 mg/ml) producing the largest zone of inhibition. This eco-friendly synthesis method for ZnONPs is sustainable and holds promise for applications in antibacterial technologies and various other industries. The ZnONPs demonstrated significant antibacterial properties, suggesting their potential for further research in biological and materials science. Peppermint (Mentha Piperita) Zinc oxide nanoparticles SEM EDXS TEM XRD FTIR UV-VIS spectrum Antibacterial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Nanotechnology is a prominent area of research in contemporary materials science. The field of nanotechnology is an evolving technology that has the potential to bring about a revolution in every scientific discipline. This technology could offer diverse and new applications in areas such as advanced fabric compositions, food processing, agricultural production, optics, electronics, biological sciences, and advanced medical treatments (Malik, 2023). Nanotechnology encompasses the processes of synthesizing, characterizing, and investigating materials within the nanometer range, which spans from 1 to 100 nm (Mohammad et al., 2022 ; Sirelkhatim et al., 2015 ). The 21st century has seen no more inventive discipline than nanotechnology. Globally, extensive research is being carried out with the objective of commercializing nanoproducts. Nanoparticles have gained significant importance due to their unique features, especially when compared to their larger counterparts (Sabir, Arshad, and Chaudhari, 2014 ). With the world's population growing, agriculture is becoming more and more important to all cultures. The first and most important need of all people is to eat, and agriculture is directly and indirectly linked to food production for people. In developing countries, it is important for the agricultural industry to grow in order to reach their development goals. Therefore, bio- and nanotechnologies are examples of new technologies that can help farms make more food and make it better. A common belief is that emerging technology will effectively address the increasing global food demands while also offering numerous advantages in terms of good health, the natural environment, and economic growth. Accordingly, nanotechnology is considered as an emerging and highly influential technology with the capacity to bring about significant transformations in the methods and practices of food production and agriculture. It has shown that it belongs in agricultural studies and related fields. It is an interdisciplinary technology that has helped solve problems and fill gaps. Nanotechnology can be used in many ways during all steps of growing, processing, storage, packaging, and transporting food. Nanotechnology is likely to yield positive benefits for the environment, particularly in agriculture and forestry applications. There is increasing attention on employing nanotechnology in farming due to its promising potential. Researchers are developing nanomaterials aimed at enhancing the efficiency and safety of pesticides, herbicides, and fertilizers. These advancements enable precise control over the timing and location of their release, offering greater management capabilities. Nanotechnology has been proposed for various applications across agriculture, food science, and animal science. Its potential impact on the agriculture and food industry is significant, offering novel approaches for disease detection and precise treatment. Furthermore, it can enhance plant nutrient absorption, disease resistance, and resilience to environmental stresses, while also revolutionizing food processing, storage, and packaging methods. Additionally, nanotechnology enables the rational selection of raw materials and the enhancement of their processing, thereby improving the quality of plant-based products. It has effectively addressed challenges in both plant science and food science, particularly concerning post-harvest products (Mousavi and Rezaei, 2011 ). Zinc oxide (ZnO) is categorized as a II-VI semiconductor in materials science due to its composition of zinc and oxygen. Zinc belongs to Group 12, while oxygen belongs to Group 16 of the periodic table. ZnO semiconductors provide unique features including remarkable electron mobility, a large gap in the band, robust luminescence at usual temperatures, and excellent transparency. The special characteristics of liquid crystal displays, such as their translucent electrodes, contribute to their energy-saving and heat-protecting capabilities. These features also enable their application in many technical devices and windows. ZnO is practically water insoluble and has a white powdery appearance. Though naturally occurring as the zincite mineral in Earth's crust, the vast majority of commercially utilized ZnO is synthesized (Wang et al., 2018 ; Wang et al., 2004 ). Methods for environmentally friendly synthesis involve employing plants to produce nanoparticles. Green synthesis techniques utilize eco-friendly chemicals to produce nanostructures with reduced pollution. This approach involves the use of environmentally benign and non-toxic solvents like water and natural extracts. Utilizing microbial and botanical technologies, along with plant extracts, has been proposed as safe alternatives to chemical methods for manufacturing metal nanoparticles. Various biological systems, such as bacteria, fungi, and yeast, have been successfully utilized in the biogenic production of nanoparticles (Fig. 1). However, producing nanoparticles using microorganisms poses challenges due to the complex processes required to maintain cell cultures, achieve intracellular synthesis, and employ various purification methods (Alagumuthu and Kirubha, 2012 ). Using botanical extracts is an environmentally sustainable and cost-effective approach that eliminates the need for intermediary compounds. This method is rapid, does not rely on expensive equipment or substances, and results in a highly pure and concentrated product, free from impurities. Plants emerge as the best option for nanoparticle synthesis due to their ability to generate a significant quantity of nanoparticles efficiently (Alsaiari et al., 2023 ; Rani et al., 2023 ). Moreover, they produce nanoparticles that exhibit stability and are available in a variety of shapes and sizes. Bio-reduction is a chemical process wherein metal ions or metal oxides are converted into metal nanoparticles with a zero valence. This conversion is facilitated by phytochemicals, such as amino acids, polyphenolic compounds, vitamins, polysaccharides, terpenoids, and alkaloids, naturally synthesized by plants. Medicinal herbs are widely used worldwide due to their lack of adverse side effects and cost-effectiveness compared to antibiotics. Mentha piperata , a perennial plant cultivated extensively for its applications in the culinary, cosmetics, medicinal, and pharmaceutical industries, is a prime example (Shah and Mello, 2004 ). Mentha piperata is frequently employed for its therapeutic properties, including antibacterial, antispasmodic, sedative, antioxidant, urinary tract infection-fighting, anti-inflammatory, and antiallergenic effects. Due to their diverse range of benefits spanning pharmaceuticals, colorants, fragrances, pesticides, insecticides, and flavorings, they hold significant commercial value for human use (Bupesh et al., 2007 ). By manipulating production factors of ZnONPs such as size, shape, and surface changes, it is possible to precisely adjust the optical characteristics of ZnO nanoparticles. ZnONPs possess a range of characteristics that render them highly versatile materials suitable for a variety of applications, such as sensors, imaging, photodetectors, and light-emitting devices (Balen et al., 2016 ). ZnONPs possess a broad bandgap, often ranging from 3.3 to 3.4 electron volts (eV) for a large quantity of ZnO. Quantum confinement, a phenomenon, enables the nanoparticle size to affect the bandgap of ZnONPs. Decreasing the particle size generally leads to an increase in the bandgap (Javed et al., 2016 ). Moreover, it demonstrates a significant capacity for absorbing ultraviolet (UV) radiation. This characteristic finds application in various fields, including the incorporation of UV-blocking elements in sunscreens and the development of UV-shielding coatings (Wang et al., 2018 ). Photoluminescence refers to the emission of visible light by ZnONPs when they are subjected to UV radiation. Controlling the size, shape, and flaws in the nanoparticles allows for the manipulation of the emission properties (Wang et al., 2018 ). ZnONPs exhibit nonlinear optical characteristics, including the phenomenon of second-harmonic production. This characteristic is utilized in applications such as frequency-doubling for laser sources (Patil, Lakshminarasimhan, and Santhosh, 2021 ). Although ZnO exhibits considerable absorption in the UV area, it demonstrates transparency in the visible region. Transparent conductive films and optoelectronic devices make use of this transparency (Balen et al., 2016 ). ZnONPs can display localized surface plasmon resonance (LSPR) either in the ultraviolet (UV) or visible spectrum, depending on their dimensions and morphology. This feature is significant for applications in sensors and imaging (Wang et al., 2018 ). Additionally, ZnONPs have the ability to emit light when excited, displaying fluorescence. Several factors, including the process of synthesis and the features of nanoparticles, can influence both the color and intensity of the produced light (Kukreja, Barik, and Misra, 2004 ). Nanoparticles possessing antibacterial properties and spanning a wide range of sizes can readily infiltrate the peptidoglycan layer of cell membranes, leading to significant damage to bacterial cells. Gram-positive bacteria demonstrate greater resistance to nanoparticles due to structural disparities in cell walls between gram-positive and gram-negative bacteria. Moreover, the presence of an additional lipopolysaccharide layer intensifies the direct contact between the outer layer of a bacterial and the nanoparticles themselves (Feng et al., 2000 ). The negative charge of lipopolysaccharide causes attraction forces with positively charged nanoparticles, resulting in an augmentation in the absorption of metal ions. The absorption of this substance is harmful to the bacterial cell. Various techniques have been employed to assess the antibacterial efficacy of ZnONPs against both pathogenic and non-pathogenic microorganisms. Several methodologies, including the disk diffusion method, microtiter plate methods, viability counts using agar dilution, and broth dilution, are employed (Wahab et al., 2010 ). This work examines the synthesis of ZnONPs utilizing peppermint extract as a biogenic precursor. Peppermint extract, known for its rich assortment of phytochemicals, provides an intriguing alternative to traditional chemical ingredients. The phytoconstituents included in peppermint act as both reducing agents and assist in stabilizing the nanoparticles that are formed. In this scenario, employing peppermint extract serves a dual purpose. It not only enables the environmentally friendly synthesis of ZnONPs but also imparts beneficial properties to the nanoparticles. Moreover, the antibacterial effectiveness of ZnONPs was assessed against Gram-positive bacteria. Materials and Methods Materials The precursor zinc nitrate hexahydrate [Zn(NO 3 ) 2 •6H 2 O], and sodium hydroxide (NaOH) were all obtained from Sigma-Aldrich (St. Louis, Missouri, United States). Peppermint leaves used in the experiment were sourced from Lulu Supermarket in Muscat, Oman. Deionized water, produced using the PURELAB Option Ultra-pure Water System from [Plant Pathology Research Lab, SQU], was utilized to prepare all solutions throughout the experiment. Preparation of M. Piperita leaf extract Peppermint leaves are a rich source of functional organic molecules, such as ascorbic acid, which can reduce zinc ions in zinc nitrate solutions (Fig. 2). Peppermint extract was prepared by washing peppermint leaves well a minimum of three times with regular water to eliminate dust and any other unwanted residue. The peppermint leaves were immersed in a small bowl filled with purified water for ten minutes to ensure that the leaves were free of any impurities. Subsequently, they were decanted to remove excess water and allowed to air dry at room temperature for fifteen minutes. Thirty grams of peppermint leaves were blended with 450 ml of distilled water in an electric blender. The resulting mixture was then transferred to a 500 ml beaker and heated on a hot plate while stirring at 80°C for 30 minutes. After cooling to room temperature, the mixture was filtered through No. 1 filter paper and subjected to three additional filtrations to obtain a clear extract. The extract was then centrifuged at 14,000 rpm for 10 minutes (Abdullah, Bakar, and Bakar, 2020 ). Synthesis of Zinc Oxide Nanoparticles (ZnONPs) Zinc oxide nanoparticles were synthesized using zinc nitrate hexahydrate as a precursor. To prepare a stock solution, 17.86 grams of Zn(NO₃) ₂ .6H₂O were dissolved in 300 ml of deionized water to achieve a final concentration of 0.2 M. Next, 90 ml of the peppermint extract was placed in a conical flask, and 150 ml of the stock solution was gradually added while stirring. After adding the extract to the Zn(NO₃)₂ solution, the mixture began to change from colourless to light brown within half an hour, indicating the formation of nanoparticles. The mixture was stirred for 10 minutes, and then the pH was adjusted to 12.0 by adding NaOH. The solution was then stirred for 2.5 hours at room temperature. Afterward, the precipitate was filtered using No. 1 filter paper and rinsed several times with deionized water. The collected precipitates were dried in an oven at 60°C overnight (Fig. 2). The ZnONPs were then ground into a light-yellow powder and stored at -80°C for further analysis (Abdullah, Bakar, and Bakar, 2020 ). Characterization of M. Piperita ZnONPs The synthesized green ZnONPs underwent several analyses to determine their properties and validate their efficacy. The Perkin Elmer UV-Vis spectrophotometer was employed to characterize the nanoparticles by measuring their absorption and transmission of ultraviolet (UV) and visible (Vis) light, thereby providing insights into their optical properties. FTIR spectroscopy was utilized to identify the chemical bonds and surface functional groups present on the nanoparticles. To confirm the pore structure and size distribution of the ZnONPs synthesized biologically, transmission electron microscopy (TEM) was performed using an FEI Tecnai 20 transmission electron microscope operating at 200 kV (Lab6 emitter) with a JEOL JSM 7800F. The optical characteristics of the ZnONPs were examined using a UV/VIS spectrometer (Thermo Scientific™) within the 200 to 700 nm wavelength range. The functional groups present in the ZnONPs were identified using Fourier-transform infrared spectroscopy (FT-IR) with a Bruker Alpha spectrometer, covering the wavelength range from 400 to 4000 cm⁻¹. Energy-dispersive X-ray spectroscopy (EDXS) with an Oxford Instrument X-MaxN was conducted to determine the elemental composition of the ZnO nanoparticles. The crystallographic structure of the ZnONPs was characterized using X-ray diffraction (XRD) at the Earth Sciences Research Centre. Antimicrobial activity test The antimicrobial activity of ZnONPs was examined through agar well diffusion assay. A single colony from a fresh bacterial culture plate was transferred to 5 ml of nutrient broth and incubated overnight at 37°C, 160 rpm. 1.5 ml of the overnight culture was centrifuged at 10,000 rpm for 2 minutes. After discarding the supernatant, another 1.5 ml was added to the pellet and centrifuged at 10,000 rpm for 2 minutes. The pellet was collected and 200 µl of culture was added. It was mixed until the pellet completely dissolved in the added amount of culture. The mixture was poured in a nutrient agar plate and spread using a spreader. Three concentrations of ZnONPs: 15 mg/ml, 25 mg/ml, 35 mg/ml were prepared. Five wells of 6 mm were bored in the plate containing the concentrated bacterial culture using a sterile cork-borer. The wells were filled with 100 µl of different liquids: AP the positive control (Amoxicillin antibiotic), negative control (distilled water), A (15 mg/ml concentration of ZnONPs), B (25 mg/ml concentration of ZnONPs), C (35 mg/ml concentration of ZnONPs), respectively. The plate was incubated at 37°C for 18–24 hours. After incubation, the formation of zones around the wells that correspond to the antimicrobial activity of tested nanoparticles was observed. The observed zone of inhibition (ZOI) was measured in mm. The petri dishes were placed in an incubator and kept at a temperature of 37°C for the duration of one night. The dimensions of the zones adjacent to the wells were determined using a ruler. The antibacterial trials were carried out three times. Results and Discussion Scanning electron microscopy analysis The SEM images illustrate stable, hollow spherical structures composed of interconnected nanoparticles (Fig. 3). The ZnONPs exhibited a sieve-like arrangement, adhering to surfaces and creating interior cavities and loose structures. Agglomeration was also noted, attributed to the reduced particle size, increased surface area, and the presence of biomolecules. Image J software (version 1.53v) was utilized to determine the particle size distribution (Fig. 3), revealing an average diameter of approximately 42.7 nm. Peppermint extracts resulted in the production of well-defined ZnONPs with a triangular shape, consistent with the findings of (Doğaroğlu et al., 2023 ). SEM images reveal that the synthesized zinc oxide nanoparticles from peppermint are organized in aggregated and agglomerated clusters, as similarly reported by (Kulkarni and Shirsat, 2015 ). The SEM analysis demonstrated that the choice of precursors affects the dimensions and morphology of the nanoparticles (Fakhari, Jamzad, and Kabiri Fard, 2019 ). For instance, when zinc acetate is used as a precursor, zinc oxide molecules develop slowly, forming compact spherical structures that cluster into bullet-shaped formations (Visinescu et al., 2018 ). Conversely, using zinc nitrate as a precursor produces rod-shaped ZnONPs (Tsai et al., 2012 ). The clustering is attributed to polarity and electrostatic interactions among ZnONPs (Abegunde, Olasehinde, and Adebayo, 2024 ). However, the ZnONPs in this study displayed a unique nanorod-like structure (Chowdhury et al., 2021 ). Some nanorods exhibited clustering, while others remained isolated. The formation and development of zinc oxide nanorods are mainly driven by the facet with the lowest surface energy. Furthermore, the ZnONPs possess pyramidal symmetry (Ahmad et al., 2023 ). Regarding size, the average dimensions of ZnONPs from peppermint extract were 73.76 nm (Doğaroğlu et al., 2023 ), whereas the particle size observed in SEM images in this study was approximately 20–50 nm, about 30% larger. These findings suggest that the shape and composition of the synthesized nanoparticles vary depending on the specific type of salt, extract, and plant used in the synthesis process. Consequently, the size of the produced particles also differs based on the materials employed and their eventual form. High-magnification TEM images (Fig. 4) show that the pores in the freshly synthesized ZnONPs derived from peppermint extract predominantly consist of spherical particles with sizes ranging from 20 to 50 nm, indicating variability in particle dimensions within the sample (Figs. 4). In the large-area TEM image, some nanoparticles are observed to aggregate into chains. According to previously reported TEM research by (Patel et al., 2022 ), the ZnONPs exhibit a spherical form with sizes ranging from 60 to 160 nm. Furthermore, the TEM technique was utilized to determine the shape and size of the zinc nanoparticles (ZnNPs) produced using peppermint (Mentha piperita) extract. TEM analysis revealed the presence of nanoparticles with globular and oblong shapes, measuring between 15 and 27 nm, with an average particle size of 18 nm, as determined from TEM micrographs (Bandyopadhyay et al., 2012 ; Mohan and Renjanadevi, 2016 ). This analysis confirmed the effectiveness of peppermint extract in synthesizing ZnNPs. Therefore, both studies are consistent with the ZnONPs results obtained from this experiment, verifying the reliability of the outcomes (Ahmad et al., 2023 ). Chemical composition of ZnONPs analysis Energy-dispersive X-ray spectroscopy (EDS) was performed in conjunction with scanning electron microscopy (SEM) to analyze the elemental composition of the synthesized ZnO nanoparticles (ZnONPs). The EDS analysis covered an energy range from 0 to 8 keV, revealing distinct peaks that confirm the presence of zinc and oxygen in the nanoparticles. As illustrated in Fig. 5, the data show zinc constitutes 73.1% of the weight fraction, while oxygen accounts for 26.9%. Further elemental composition analysis of the ZnONPs indicates a weight percentage of 80.4% zinc and 19.6% oxygen, aligning closely with the expected composition from the Zn(NO₃)₂ precursor. The sharp peaks observed within the 0 to 2 keV range and between 6 and 8 keV are indicative of the crystalline structure of the ZnONPs. This structural characterization underscores the porous nature of the nanopowder, which enhances its surface area. The EDS data also revealed spherical-triangular clusters of zinc nanoparticles (Fig. 5a). Notably, the analysis identified only two peaks, corresponding to zinc and oxygen, with no additional contaminants detected. The observed nearly stoichiometric composition of the ZnONPs is consistent with the expected values. A recent study by Chowdhury et al. ( 2021 ) reported an EDS-derived mass percentage composition of 80.12% zinc and 19.88% oxygen for a zinc oxide sample, confirming its purity. Similarly, Ahmed et al. (2023) found that the 3–4 keV energy range indicated a zinc content of approximately 85.71%, highlighting the high purity of the zinc element in their samples (Fig. 5b). XRD Analysis X-ray diffraction pattern (XRD) spectrum of ZnONPs synthesized utilizing extracts derived from peppermint leaves. The diffraction peaks were discerned at specific 2-theta values: 31.69°, 34.37°, 36.21°, 47.47°, 56.49°, 62.73°, 66.29°, 67.85°, and 68.99°, which coincided with peaks corresponding to (100), (002), (101), (102), (110), (103), (200), (112), and (201) respectively (Fig. 6). Recent studies have reported the XRD spectra of ZnONPs synthesized using leaf extracts from Mentha viridis (Chowdhury et al., 2021 ). Distinct diffraction peaks were observed at specific 2θ values, including 31.73°, 34.42°, 36.25°, 47.53°, 56.56°, 62.78°, 66.33°, 67.82°, and 69.1°. These peaks correspond to the lattice planes with Miller indices of (100), (002), (101), (102), (110), (103), (200), (112), and (201), respectively. The presence of these well-defined diffraction peaks confirms the crystalline nature of the biosynthesized ZnO. Additionally, no diffraction peaks were detected apart from ZnO, indicating the formation of pure ZnO (Fig. 6). These findings align closely with previous results, corroborating the crystalline structure of the ZnONPs (Fig. 6). According to Fakhari et al. ( 2019 ), the XRD pattern of the synthesized ZnONPs clearly demonstrates the presence of a distinct crystalline structure within the nanoparticles. These distinct diffraction peaks correspond to the crystalline planes of the synthesized ZnONPs, providing valuable insights into their structural characteristics and crystallographic orientation. These sharp peaks confirm that the synthesized ZnONPs are crystalline, as shown in Fig. 6. The diffraction 2θ values correspond to the face-centered cubic crystalline phase of zinc oxide. The 2θ values were recorded, and distinct diffraction peaks were observed at angles of 31.46°, 34.29°, 36.33°, 47.51°, 56.50°, 62.84°, 67.79°, and 76.83°. These peaks correspond to the diffraction lattice planes (100), (002), (101), (102), (110), (103), (112), and (202), indicating the hexagonal wurtzite structure of the synthesized nanoparticles. The average size of the ZnONPs was determined by analyzing the most intense peak (101) using the Debye-Scherrer equation. This equation incorporates several variables: λ (lambda) represents the X-ray wavelength from Cu-Kα (1.540560 Å), β (beta) is the full width at half maximum (FWHM) of the diffraction peak in radians, θ (theta) is Bragg's angle in degrees, and K is the shape factor, which has a value of 0.9 (Fakhari et al., 2019 ). XRD analyses indicated that nanoparticles synthesized from zinc acetate and zinc nitrate precursors exhibited average sizes of 21.49 nm and 25.26 nm, respectively. Both studies supported the findings of this research. The average size was determined by applying the Eq. 1yielding an average size of 41.55 nm (Fakhari 2019). D = (K λ/ β cos θ) (1) FTIR analysis The role of biomolecules in the synthesis of ZnONPs was investigated and identified using FTIR spectroscopy across the spectrum range of 4000 to 400 cm⁻¹ (Fig. 7). The FTIR analysis of the peppermint-extract-derived ZnONPs revealed distinctive absorption peaks at specific wavelengths, including 3450 cm − 1 , 1650 cm − 1 , 1380 cm − 1 , 890 cm − 1 , and 500 cm − 1 . These absorption peaks signify the presence of functional groups and biomolecules associated with the synthesis process, providing valuable insights into the chemical composition and bonding characteristics of the synthesized ZnONPs. The FTIR spectra of the synthesized ZnONPs have been detailed by (Javed et al., 2016 ). Additionally, a hydroxyl group shows an O-H bending vibration at 574 cm⁻¹. Peaks in the 2850 to 2930 cm⁻¹ range are associated with the C-H stretching vibration. The peaks at 1576 and 1420 cm⁻¹ correspond to the asymmetrical and symmetrical stretching of zinc nitrate hexahydrate (Zn(NO₃)₂•6H₂O), respectively. The bond at 1025 cm⁻¹ can be attributed to the C-O stretching vibration. An absorption peak at 857 cm⁻¹ indicates the formation of tetrahedral coordination of Zn. A significant peak at 1047 cm⁻¹ was observed in the peppermint sample (Koli, 2022). A recent study used FTIR analysis within the wavelength range of 4000 to 400 cm⁻¹ to identify the functional groups and biomolecules involved in the production of ZnONPs (Doğaroğlu et al., 2023 ). The synthesis involves Zn²⁺ ions from zinc acetate binding with polyphenols in the plant extract, converting Zn²⁺ to Zn⁺ through reduction—a process described as the synthesis of Zn⁺ polyphenols in the reaction solution. Both the original ZnONPs and those derived from plant extracts exhibited similar FTIR peaks, suggesting a common synthesis mechanism. The M-ZnONPs show absorption bands at 3837, 3383, 2979, 2903, 1563, 1405, 1252, 1055, 889, 688, 543, and 484 cm⁻¹. In contrast, the B-ZnONPs have absorption peaks at 3385, 1563, 1414, 877, 688, 582, 552, and 481 cm⁻¹. The distinct peaks at 3383 and 3385 cm⁻¹ are likely due to hydroxyl groups present in alcohols and phenolic compounds in plant extracts. Peaks between 2979 and 2903 cm⁻¹ are attributed to the elongation of carbon-hydrogen bonds in alkanes (Abdullah, Bakar, and Bakar, 2020 ). Peaks at 1563 cm⁻¹ indicate C = C stretching vibrations in aromatic rings within polyphenolic compounds (Matinise et al., 2017 ). The peaks at 1405 and 1414 cm⁻¹ relate to the symmetrical stretching mode of the COO⁻ group in acids. The peak at 1252 cm⁻¹ is linked to the C-O stretching (Bharathi and Bhuvaneshwari, 2019 ). The dominant peak at 1055 cm⁻¹ in the peppermint FTIR spectrum results from the elongation of C–O, C-C, and C-O-C bonds in saturated esters, alcohols, phenols, cycloalkanes, and acid anhydrides. The primary absorption band for ZnO appears in the 400–600 cm⁻¹ region, with the wide and strong band at 500 cm⁻¹ representing Zn-O vibration (Tantiwatcharothai and Prachayawarakorn, 2019 ). Bending vibrations of alkanes and alkenes, particularly C = C bending, are noted at 889, 877, and 688 cm⁻¹. UV-Vis Spectrum The color gradually darkened over time, becoming dark brown after four hours of incubation. The ZnONPs exhibited a prominent absorption peak at approximately 220 nm, highlighting the wavelength where the nanopowder most strongly absorbs light, thereby reflecting its optical properties. This spectral absorption characteristic is crucial for understanding the material's behavior and potential applications in fields such as optoelectronics, photovoltaics, and photocatalysis. The color change of the reacting mixture likely corresponds to the surface plasmon resonance of zinc nanoparticles. The formation of nanoparticles in the mixture was monitored using a Perkin Elmer UV-Vis spectrophotometer at regular intervals (every hour). A peak absorbance observed near 450 nm further confirmed the production of ZnONPs. The UV-Vis spectra obtained at different time intervals are illustrated in Fig. 8, depicting changes in peak absorbance over the course of the reaction. Furthermore, the peak absorbance increased almost linearly with reaction time until two hours of incubation, likely due to the continued production of nanoparticles. After four hours, the rate of formation saturated, indicating the reaction had reached completion. The absorption spectra of ZnONPs synthesized from peppermint exhibit a characteristic peak at a wavelength of 281.5 nm, signifying the presence of ZnO. Previous studies have identified a more pronounced absorption peak at 385 nm in ZnO nanoparticles (Ahmad et al., 2023 ). The band gap energy (E) was measured by (Loh et al., 2023 ) through an analysis of the maximum wavelength at which the ZnONPs absorbed light. The ZnONPs were determined to have a band gap energy of 3.37 eV, calculated using Eq. (2). This value aligns with literature values, which typically indicate a broad bandgap ranging from 3.3 to 3.4 eV (Javed et al., 2016 ). The equation for E is given by E = hc/λ, where h is Planck's constant (6.626×10 − 34 J), c is the speed of light (3.0×10 8 m/s), and λ is the maximum absorption wavelength (in nm). At a wavelength of 220 nm, the energy of the photons is approximately 5.64 eV. using h = 6.626×10 − 34 J, c = 3.0×10 8 m/s, and λ = 220 nm in E = hc/λ​ equation. This calculation is based on the relationship between energy and wavelength in the electromagnetic spectrum, as previously discussed. The bandgap in this study varies from prior findings due to differences in absorbance. The stable synthesis of ZnONPs is typically confirmed by examining its distinctive absorption spectrum, using a scanning range of 600–200 nm, as reported by (Chowdhury et al., 2021 ). E = hc/λ​ (2) Antimicrobial Activity The agar well diffusion method revealed clear zones of inhibition around the wells containing ZnONPs for bacteria, demonstrating the nanoparticles' effectiveness against the bacterium pathogen. Significant inhibition zones were observed around wells containing ZnONPs for bacteria. The agar well diffusion method confirmed the antimicrobial activity of ZnONPs against bacterial pathogens (Padmavathy and Vijayaraghavan, 2008 ). Figure 9 illustrates the zone of inhibition obtained through a well diffusion assay. The results indicate that bacterial growth was effectively inhibited by varying concentrations of zinc nanoparticles (ZnONPs). The highest concentration used, 35 mg/ml, produced the largest zone of inhibition. As shown in the figure (Fig. 9), there is a clear trend of increasing inhibition with higher concentrations of ZnONPs. Specifically, the concentrations of 35, 25, and 15 mg/ml resulted in zones of inhibition measuring 0.2 mm, 0.1 mm, and 0.05 mm, respectively. Pillai et al. (2020) also demonstrated the biological efficacy of ZnONPs against E. coli , S. aureus , Candida albicans , and Aspergillus niger . (Doğaroğlu et al., 2023 ) found that S. aureus was more susceptible to ZnONPs compared to E. coli , with the activity level of E. coli being greater for ZnONPs than for B-ZnO (Basil) and M-ZnO (Peppermint) nanoparticles. Conversely, the activity level of S. aureus was greater when exposed to ZnONPs compared to M-ZnO and B-ZnO nanoparticles. According to (Venkataraju et al., 2014 ), Gram-negative bacteria require ZnONPs to penetrate both the outer membrane and the thin peptidoglycan layer, whereas Gram-positive bacteria possess a thicker peptidoglycan layer (approximately 30 nm) in addition to the outer membrane. This explains the observed findings in this study. Conclusion The successful synthesis of ZnONPs using peppermint extract through eco-friendly methods has been validated by comprehensive characterization techniques, including SEM, EDXS, TEM, XRD, FTIR, and UV-Vis spectroscopy. These analyses confirm the production of crystalline ZnONPs with distinct morphological features. The environmentally friendly synthesis process, coupled with the detailed characterization, highlights the potential of these nanoparticles for diverse applications, such as catalysis, sensors, and biomedical devices. Additionally, the ZnONPs exhibit significant antibacterial activity against a broad range of bacterial species, as evidenced by clear inhibition zones observed in the well diffusion assays. Overall, ZnONPs represent a promising integration of green synthesis and effective antimicrobial properties, making them an attractive option for use in antimicrobial technologies and biomedicine. Declarations Acknowledgements Sultan Qaboos University financially supported this research through a research grant number. Author contributions: CRediT SAR: Investigation, Conceptualization, Software, Validation, Visualization, Writing – original draft. AA: Data curation, Investigation, Software, Visualization, Writing – original draft. MAB: Investigation, Software, Visualization. AAS: Conceptualization, Software, Validation. AMAS: Conceptualization, Validation, Visualization, Writing – review & editing. TA: Conceptualization, Software, Validation, Visualization, Writing – review & editing. BL: Resources, Conceptualization, Investigation, Supervision, Visualization, Writing – review & editing. MSS: Resources, Conceptualization, Validation, Visualization, Writing – review & editing. All authors reviewed and approved the final manuscript. Funding sources: Sultan Qaboos University and Zhejiang University provided the funding through the co-funding project (CL/SQU-ZJU/AGR/23/01). Supplementary information accompanies this paper Declaration of competing interests: The authors declare no competing financial interests or personal relationships that could have influenced the work presented. Ethics declaration : not applicable Data availability : the datasets generated and/or analyzed during the current study are included in this article. References Abdullah, F., Bakar, N. A., Bakar, M. A. 2020. Low temperature biosynthesis of crystalline zinc oxide nanoparticles from Musa acuminata peel extract for visible-light degradation of methylene blue. Optik 206, 164279. Abegunde, S. 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Sirelkhatim, A., Mahmud, S., Seeni, A., Kaus, N. H. M., Ann, L. C., Bakhori, S. K. M., Hasan, H., Mohamad, D. 2015. Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism. Nano-micro letters 7, 219-242. Tantiwatcharothai, S., Prachayawarakorn, J. 2019. Characterization of an antibacterial wound dressing from basil seed (Ocimum basilicum L.) mucilage-ZnO nanocomposite. International Journal of Biological Macromolecules 135, 133-140. Tsai, M., Huang, C., Lee, Y., Yang, C., Yu, H., Lee, J., Hu, S., Chen, C. 2012. A study on morphology control and optical properties of ZnO nanorods synthesized by microwave heating. Journal of luminescence 132(1), 226-230. Venkataraju, J. L., Sharath, R., Chandraprabha, M., Neelufar, E., Hazra, A., Patra, M. 2014. Synthesis, characterization and evaluation of antimicrobial activity of zinc oxide nanoparticles. Journal of Biochemical Technology 3(5), 151-154. Visinescu, D., Hussien, M. D., Moreno, J. C., Negrea, R., Birjega, R., Somacescu, S., Ene, C. D., Chifiriuc, M. C., Popa, M., Stan, M. S. 2018. Zinc oxide spherical-shaped nanostructures: investigation of surface reactivity and interactions with microbial and mammalian cells. Langmuir 34(45), 13638-13651. Wahab, R., Kim, Y.-S., Mishra, A., Yun, S.-I., Shin, H.-S. 2010. Formation of ZnO micro-flowers prepared via solution process and their antibacterial activity. Nanoscale research letters 5, 1675-1681. Wang, J., Chen, R., Xiang, L., Komarneni, S. 2018. Synthesis, properties and applications of ZnO nanomaterials with oxygen vacancies: A review. Ceramics International 44(7), 7357-7377. Wang, X., Ding, Y., Summers, C. J., Wang, Z. L. 2004. Large-scale synthesis of six-nanometer-wide ZnO nanobelts. The Journal of Physical Chemistry B 108(26), 8773-8777. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2026 Read the published version in BioNanoScience → Version 1 posted Editorial decision: Revision requested 14 Jun, 2025 Reviews received at journal 22 May, 2025 Reviews received at journal 22 May, 2025 Reviews received at journal 17 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers invited by journal 13 May, 2025 Editor assigned by journal 07 May, 2025 Submission checks completed at journal 07 May, 2025 First submitted to journal 27 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6539690","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456153819,"identity":"448187e7-a307-4023-ba93-2aff4506654c","order_by":0,"name":"Shahira Al-Reisis","email":"","orcid":"","institution":"Sultan Qaboos University","correspondingAuthor":false,"prefix":"","firstName":"Shahira","middleName":"","lastName":"Al-Reisis","suffix":""},{"id":456153820,"identity":"c6f7c420-2a42-4f0c-aa5f-842ed32c0333","order_by":1,"name":"Arshad Ali","email":"","orcid":"","institution":"Sultan Qaboos University","correspondingAuthor":false,"prefix":"","firstName":"Arshad","middleName":"","lastName":"Ali","suffix":""},{"id":456153821,"identity":"9b441201-2046-47ba-af40-f25daee6ea8d","order_by":2,"name":"Maryam Al-Zadjali","email":"","orcid":"","institution":"Sultan Qaboos University","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Al-Zadjali","suffix":""},{"id":456153822,"identity":"c1a5a297-b118-45b2-ac0f-e2771d5eeec8","order_by":3,"name":"Ali- Al-Subhi","email":"","orcid":"","institution":"Sultan Qaboos University","correspondingAuthor":false,"prefix":"","firstName":"Ali-","middleName":"","lastName":"Al-Subhi","suffix":""},{"id":456153823,"identity":"3ae9aa16-a92a-4170-9f2f-b3e2ef2b3c69","order_by":4,"name":"Abdullah M. Al-Sadi","email":"","orcid":"","institution":"Sultan Qaboos University","correspondingAuthor":false,"prefix":"","firstName":"Abdullah","middleName":"M.","lastName":"Al-Sadi","suffix":""},{"id":456153824,"identity":"b9b9e4c0-f1d8-4f13-a9ed-56df5e2ab8cc","order_by":5,"name":"Muhammad Shafiq Shahid","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYFACHgZmBgNmOQiHjQQtxqRqYWBObCBai27/2YOfCwqs0+e35xgwfCg7zMAvdgC/FrMbecnSMwzSczeceWPAOOPcYQbJ2QmEtPAYSPMYHM7dIJFjwMzbdpjB4DYhLefPGP8GakmXnwHU8pcoLQdyzEC2JDDcAGphJErLjRwzax6DdMMNZ54VHOw5l85D2C9Ah93m+WMtL9+evPHBjzJrOX5pAlqQQALDAQZQNJEAiDd8FIyCUTAKRhgAAH05QGQsxYFuAAAAAElFTkSuQmCC","orcid":"","institution":"Sultan Qaboos University","correspondingAuthor":true,"prefix":"","firstName":"Muhammad","middleName":"Shafiq","lastName":"Shahid","suffix":""}],"badges":[],"createdAt":"2025-04-27 10:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6539690/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6539690/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12668-025-02316-4","type":"published","date":"2026-01-16T16:28:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82828363,"identity":"00f04e29-2ce9-45ef-bd2b-6a043bd14ae8","added_by":"auto","created_at":"2025-05-15 16:37:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117408,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of various eco-friendly sources used for the production of ZnONPs.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/b6e39149465b67b372a430d8.jpg"},{"id":82828125,"identity":"3a480f5c-40d1-4627-9e8b-5b0529a62b64","added_by":"auto","created_at":"2025-05-15 16:29:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199033,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of ZnONPs synthesized via a green method using peppermint extract.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/d4f1d90eea85dd0a2252e06e.jpg"},{"id":82828118,"identity":"0caa6b42-4b1a-4853-8f87-bd3b3c6fa441","added_by":"auto","created_at":"2025-05-15 16:29:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":190131,"visible":true,"origin":"","legend":"\u003cp\u003eScanning Electron Microscope (SEM) images illustrating ZnONPs at different magnifications: (a) 10x (1μm), (b) 30x (100nm), (c) 60x (100nm), (d) 80x (100nm), (e) 120x (100nm), (f) 150x (100nm).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/69ae6789246fe71e40469cac.jpg"},{"id":82828366,"identity":"663fc943-f0b1-4e52-85ef-8ca181261b1b","added_by":"auto","created_at":"2025-05-15 16:37:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":260972,"visible":true,"origin":"","legend":"\u003cp\u003eTEM\u003cstrong\u003e \u003c/strong\u003eimagesof ZnONPs at a resolution: \u003cstrong\u003e(a)\u003c/strong\u003e20nm, and \u003cstrong\u003e(b)\u003c/strong\u003e 50nm.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/ec1b07c962d6f57c501177e4.jpg"},{"id":82828122,"identity":"1e69cf0c-1d7e-408b-b44d-ca6dbd71ff7d","added_by":"auto","created_at":"2025-05-15 16:29:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":82072,"visible":true,"origin":"","legend":"\u003cp\u003eEnergy-dispersive X-ray spectroscopy \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003emapping illustrating the distribution of Zn (a), and EDS spectra confirming the presence of Zn and O (b).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/5dc6589dcb8d13bfc6d8cf61.jpg"},{"id":82829009,"identity":"c74b1705-e742-4585-85b9-377240f5c2e5","added_by":"auto","created_at":"2025-05-15 16:45:38","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":56016,"visible":true,"origin":"","legend":"\u003cp\u003eXRD analysis of ZnONPs synthesized using peppermint extract.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/78187116edb7917016b5398f.jpg"},{"id":82829010,"identity":"657d411c-8cb6-45e1-ba98-48c93cede7e8","added_by":"auto","created_at":"2025-05-15 16:45:38","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":29169,"visible":true,"origin":"","legend":"\u003cp\u003eFourier Transform Infrared (FTIR) spectra of ZnONPs.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/4f55b5b2ef51e65ed93acd76.jpg"},{"id":82828129,"identity":"ed6455e5-01c8-4850-bf42-985334b500ab","added_by":"auto","created_at":"2025-05-15 16:29:38","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":69730,"visible":true,"origin":"","legend":"\u003cp\u003eEvolution of UV-Vis absorption spectrum of ZnONPs under visible light irradiation.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/11c11b826b777704b90ab139.jpg"},{"id":82828136,"identity":"2fb61167-1c9e-413f-8b3d-f47ae78b22f0","added_by":"auto","created_at":"2025-05-15 16:29:38","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":88611,"visible":true,"origin":"","legend":"\u003cp\u003eThe antimicrobial activity of the synthesized ZnONPs was evaluated. The ZnONPs were applied at a concentration of 15 mg/ml (A), 25 mg/ml (B), 35 mg/ml (C). For comparison, a positive control (+ve) containing 50 μg of amoxicillin and a negative control with 10% DMSO were also used. The effectiveness of the ZnONPs was measured by assessing the inhibition zones around the wells.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/d7abdd58692154f930cb89c7.jpg"},{"id":100614370,"identity":"6dd7c792-096a-44f3-b73b-a4870211e450","added_by":"auto","created_at":"2026-01-19 17:19:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1750517,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6539690/v1/06320e13-4854-405d-837e-6bc6b84bc67f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Eco-Friendly Zinc Oxide Nanoparticles from Peppermint: Synthesis, Characterization, and Antimicrobial Evaluation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanotechnology is a prominent area of research in contemporary materials science. The field of nanotechnology is an evolving technology that has the potential to bring about a revolution in every scientific discipline. This technology could offer diverse and new applications in areas such as advanced fabric compositions, food processing, agricultural production, optics, electronics, biological sciences, and advanced medical treatments (Malik, 2023). Nanotechnology encompasses the processes of synthesizing, characterizing, and investigating materials within the nanometer range, which spans from 1 to 100 nm (Mohammad et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sirelkhatim et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The 21st century has seen no more inventive discipline than nanotechnology. Globally, extensive research is being carried out with the objective of commercializing nanoproducts. Nanoparticles have gained significant importance due to their unique features, especially when compared to their larger counterparts (Sabir, Arshad, and Chaudhari, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). With the world's population growing, agriculture is becoming more and more important to all cultures. The first and most important need of all people is to eat, and agriculture is directly and indirectly linked to food production for people. In developing countries, it is important for the agricultural industry to grow in order to reach their development goals. Therefore, bio- and nanotechnologies are examples of new technologies that can help farms make more food and make it better. A common belief is that emerging technology will effectively address the increasing global food demands while also offering numerous advantages in terms of good health, the natural environment, and economic growth. Accordingly, nanotechnology is considered as an emerging and highly influential technology with the capacity to bring about significant transformations in the methods and practices of food production and agriculture. It has shown that it belongs in agricultural studies and related fields. It is an interdisciplinary technology that has helped solve problems and fill gaps. Nanotechnology can be used in many ways during all steps of growing, processing, storage, packaging, and transporting food. Nanotechnology is likely to yield positive benefits for the environment, particularly in agriculture and forestry applications. There is increasing attention on employing nanotechnology in farming due to its promising potential. Researchers are developing nanomaterials aimed at enhancing the efficiency and safety of pesticides, herbicides, and fertilizers. These advancements enable precise control over the timing and location of their release, offering greater management capabilities. Nanotechnology has been proposed for various applications across agriculture, food science, and animal science. Its potential impact on the agriculture and food industry is significant, offering novel approaches for disease detection and precise treatment. Furthermore, it can enhance plant nutrient absorption, disease resistance, and resilience to environmental stresses, while also revolutionizing food processing, storage, and packaging methods. Additionally, nanotechnology enables the rational selection of raw materials and the enhancement of their processing, thereby improving the quality of plant-based products. It has effectively addressed challenges in both plant science and food science, particularly concerning post-harvest products (Mousavi and Rezaei, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eZinc oxide (ZnO) is categorized as a II-VI semiconductor in materials science due to its composition of zinc and oxygen. Zinc belongs to Group 12, while oxygen belongs to Group 16 of the periodic table. ZnO semiconductors provide unique features including remarkable electron mobility, a large gap in the band, robust luminescence at usual temperatures, and excellent transparency. The special characteristics of liquid crystal displays, such as their translucent electrodes, contribute to their energy-saving and heat-protecting capabilities. These features also enable their application in many technical devices and windows. ZnO is practically water insoluble and has a white powdery appearance. Though naturally occurring as the zincite mineral in Earth's crust, the vast majority of commercially utilized ZnO is synthesized (Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Methods for environmentally friendly synthesis involve employing plants to produce nanoparticles. Green synthesis techniques utilize eco-friendly chemicals to produce nanostructures with reduced pollution. This approach involves the use of environmentally benign and non-toxic solvents like water and natural extracts. Utilizing microbial and botanical technologies, along with plant extracts, has been proposed as safe alternatives to chemical methods for manufacturing metal nanoparticles. Various biological systems, such as bacteria, fungi, and yeast, have been successfully utilized in the biogenic production of nanoparticles (Fig.\u0026nbsp;1). However, producing nanoparticles using microorganisms poses challenges due to the complex processes required to maintain cell cultures, achieve intracellular synthesis, and employ various purification methods (Alagumuthu and Kirubha, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUsing botanical extracts is an environmentally sustainable and cost-effective approach that eliminates the need for intermediary compounds. This method is rapid, does not rely on expensive equipment or substances, and results in a highly pure and concentrated product, free from impurities. Plants emerge as the best option for nanoparticle synthesis due to their ability to generate a significant quantity of nanoparticles efficiently (Alsaiari et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rani et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, they produce nanoparticles that exhibit stability and are available in a variety of shapes and sizes. Bio-reduction is a chemical process wherein metal ions or metal oxides are converted into metal nanoparticles with a zero valence. This conversion is facilitated by phytochemicals, such as amino acids, polyphenolic compounds, vitamins, polysaccharides, terpenoids, and alkaloids, naturally synthesized by plants. Medicinal herbs are widely used worldwide due to their lack of adverse side effects and cost-effectiveness compared to antibiotics. \u003cem\u003eMentha piperata\u003c/em\u003e, a perennial plant cultivated extensively for its applications in the culinary, cosmetics, medicinal, and pharmaceutical industries, is a prime example (Shah and Mello, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cem\u003eMentha piperata\u003c/em\u003e is frequently employed for its therapeutic properties, including antibacterial, antispasmodic, sedative, antioxidant, urinary tract infection-fighting, anti-inflammatory, and antiallergenic effects. Due to their diverse range of benefits spanning pharmaceuticals, colorants, fragrances, pesticides, insecticides, and flavorings, they hold significant commercial value for human use (Bupesh et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). By manipulating production factors of ZnONPs such as size, shape, and surface changes, it is possible to precisely adjust the optical characteristics of ZnO nanoparticles. ZnONPs possess a range of characteristics that render them highly versatile materials suitable for a variety of applications, such as sensors, imaging, photodetectors, and light-emitting devices (Balen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). ZnONPs possess a broad bandgap, often ranging from 3.3 to 3.4 electron volts (eV) for a large quantity of ZnO. Quantum confinement, a phenomenon, enables the nanoparticle size to affect the bandgap of ZnONPs. Decreasing the particle size generally leads to an increase in the bandgap (Javed et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, it demonstrates a significant capacity for absorbing ultraviolet (UV) radiation. This characteristic finds application in various fields, including the incorporation of UV-blocking elements in sunscreens and the development of UV-shielding coatings (Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Photoluminescence refers to the emission of visible light by ZnONPs when they are subjected to UV radiation. Controlling the size, shape, and flaws in the nanoparticles allows for the manipulation of the emission properties (Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). ZnONPs exhibit nonlinear optical characteristics, including the phenomenon of second-harmonic production. This characteristic is utilized in applications such as frequency-doubling for laser sources (Patil, Lakshminarasimhan, and Santhosh, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although ZnO exhibits considerable absorption in the UV area, it demonstrates transparency in the visible region. Transparent conductive films and optoelectronic devices make use of this transparency (Balen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). ZnONPs can display localized surface plasmon resonance (LSPR) either in the ultraviolet (UV) or visible spectrum, depending on their dimensions and morphology. This feature is significant for applications in sensors and imaging (Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, ZnONPs have the ability to emit light when excited, displaying fluorescence. Several factors, including the process of synthesis and the features of nanoparticles, can influence both the color and intensity of the produced light (Kukreja, Barik, and Misra, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Nanoparticles possessing antibacterial properties and spanning a wide range of sizes can readily infiltrate the peptidoglycan layer of cell membranes, leading to significant damage to bacterial cells. Gram-positive bacteria demonstrate greater resistance to nanoparticles due to structural disparities in cell walls between gram-positive and gram-negative bacteria. Moreover, the presence of an additional lipopolysaccharide layer intensifies the direct contact between the outer layer of a bacterial and the nanoparticles themselves (Feng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The negative charge of lipopolysaccharide causes attraction forces with positively charged nanoparticles, resulting in an augmentation in the absorption of metal ions. The absorption of this substance is harmful to the bacterial cell. Various techniques have been employed to assess the antibacterial efficacy of ZnONPs against both pathogenic and non-pathogenic microorganisms. Several methodologies, including the disk diffusion method, microtiter plate methods, viability counts using agar dilution, and broth dilution, are employed (Wahab et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This work examines the synthesis of ZnONPs utilizing peppermint extract as a biogenic precursor. Peppermint extract, known for its rich assortment of phytochemicals, provides an intriguing alternative to traditional chemical ingredients. The phytoconstituents included in peppermint act as both reducing agents and assist in stabilizing the nanoparticles that are formed. In this scenario, employing peppermint extract serves a dual purpose. It not only enables the environmentally friendly synthesis of ZnONPs but also imparts beneficial properties to the nanoparticles. Moreover, the antibacterial effectiveness of ZnONPs was assessed against Gram-positive bacteria.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe precursor zinc nitrate hexahydrate [Zn(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;6H\u003csub\u003e2\u003c/sub\u003eO], and sodium hydroxide (NaOH) were all obtained from Sigma-Aldrich (St. Louis, Missouri, United States). Peppermint leaves used in the experiment were sourced from Lulu Supermarket in Muscat, Oman. Deionized water, produced using the PURELAB Option Ultra-pure Water System from [Plant Pathology Research Lab, SQU], was utilized to prepare all solutions throughout the experiment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of M. Piperita leaf extract\u003c/h3\u003e\n\u003cp\u003ePeppermint leaves are a rich source of functional organic molecules, such as ascorbic acid, which can reduce zinc ions in zinc nitrate solutions (Fig.\u0026nbsp;2). Peppermint extract was prepared by washing peppermint leaves well a minimum of three times with regular water to eliminate dust and any other unwanted residue. The peppermint leaves were immersed in a small bowl filled with purified water for ten minutes to ensure that the leaves were free of any impurities. Subsequently, they were decanted to remove excess water and allowed to air dry at room temperature for fifteen minutes. Thirty grams of peppermint leaves were blended with 450 ml of distilled water in an electric blender. The resulting mixture was then transferred to a 500 ml beaker and heated on a hot plate while stirring at 80\u0026deg;C for 30 minutes. After cooling to room temperature, the mixture was filtered through No. 1 filter paper and subjected to three additional filtrations to obtain a clear extract. The extract was then centrifuged at 14,000 rpm for 10 minutes (Abdullah, Bakar, and Bakar, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSynthesis of Zinc Oxide Nanoparticles (ZnONPs)\u003c/h3\u003e\n\u003cp\u003eZinc oxide nanoparticles were synthesized using zinc nitrate hexahydrate as a precursor. To prepare a stock solution, 17.86 grams of Zn(NO₃)\u003csub\u003e₂\u003c/sub\u003e.6H₂O were dissolved in 300 ml of deionized water to achieve a final concentration of 0.2 M. Next, 90 ml of the peppermint extract was placed in a conical flask, and 150 ml of the stock solution was gradually added while stirring. After adding the extract to the Zn(NO₃)₂ solution, the mixture began to change from colourless to light brown within half an hour, indicating the formation of nanoparticles. The mixture was stirred for 10 minutes, and then the pH was adjusted to 12.0 by adding NaOH. The solution was then stirred for 2.5 hours at room temperature. Afterward, the precipitate was filtered using No. 1 filter paper and rinsed several times with deionized water. The collected precipitates were dried in an oven at 60\u0026deg;C overnight (Fig.\u0026nbsp;2). The ZnONPs were then ground into a light-yellow powder and stored at -80\u0026deg;C for further analysis (Abdullah, Bakar, and Bakar, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCharacterization of M. Piperita ZnONPs\u003c/h3\u003e\n\u003cp\u003eThe synthesized green ZnONPs underwent several analyses to determine their properties and validate their efficacy. The Perkin Elmer UV-Vis spectrophotometer was employed to characterize the nanoparticles by measuring their absorption and transmission of ultraviolet (UV) and visible (Vis) light, thereby providing insights into their optical properties. FTIR spectroscopy was utilized to identify the chemical bonds and surface functional groups present on the nanoparticles. To confirm the pore structure and size distribution of the ZnONPs synthesized biologically, transmission electron microscopy (TEM) was performed using an FEI Tecnai 20 transmission electron microscope operating at 200 kV (Lab6 emitter) with a JEOL JSM 7800F. The optical characteristics of the ZnONPs were examined using a UV/VIS spectrometer (Thermo Scientific\u0026trade;) within the 200 to 700 nm wavelength range. The functional groups present in the ZnONPs were identified using Fourier-transform infrared spectroscopy (FT-IR) with a Bruker Alpha spectrometer, covering the wavelength range from 400 to 4000 cm⁻\u0026sup1;. Energy-dispersive X-ray spectroscopy (EDXS) with an Oxford Instrument X-MaxN was conducted to determine the elemental composition of the ZnO nanoparticles. The crystallographic structure of the ZnONPs was characterized using X-ray diffraction (XRD) at the Earth Sciences Research Centre.\u003c/p\u003e\n\u003ch3\u003eAntimicrobial activity test\u003c/h3\u003e\n\u003cp\u003eThe antimicrobial activity of ZnONPs was examined through agar well diffusion assay. A single colony from a fresh bacterial culture plate was transferred to 5 ml of nutrient broth and incubated overnight at 37\u0026deg;C, 160 rpm. 1.5 ml of the overnight culture was centrifuged at 10,000 rpm for 2 minutes. After discarding the supernatant, another 1.5 ml was added to the pellet and centrifuged at 10,000 rpm for 2 minutes. The pellet was collected and 200 \u0026micro;l of culture was added. It was mixed until the pellet completely dissolved in the added amount of culture. The mixture was poured in a nutrient agar plate and spread using a spreader. Three concentrations of ZnONPs: 15 mg/ml, 25 mg/ml, 35 mg/ml were prepared. Five wells of 6 mm were bored in the plate containing the concentrated bacterial culture using a sterile cork-borer. The wells were filled with 100 \u0026micro;l of different liquids: AP the positive control (Amoxicillin antibiotic), negative control (distilled water), A (15 mg/ml concentration of ZnONPs), B (25 mg/ml concentration of ZnONPs), C (35 mg/ml concentration of ZnONPs), respectively. The plate was incubated at 37\u0026deg;C for 18\u0026ndash;24 hours. After incubation, the formation of zones around the wells that correspond to the antimicrobial activity of tested nanoparticles was observed. The observed zone of inhibition (ZOI) was measured in mm. The petri dishes were placed in an incubator and kept at a temperature of 37\u0026deg;C for the duration of one night. The dimensions of the zones adjacent to the wells were determined using a ruler. The antibacterial trials were carried out three times.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy analysis\u003c/h2\u003e \u003cp\u003eThe SEM images illustrate stable, hollow spherical structures composed of interconnected nanoparticles (Fig.\u0026nbsp;3). The ZnONPs exhibited a sieve-like arrangement, adhering to surfaces and creating interior cavities and loose structures. Agglomeration was also noted, attributed to the reduced particle size, increased surface area, and the presence of biomolecules. Image J software (version 1.53v) was utilized to determine the particle size distribution (Fig.\u0026nbsp;3), revealing an average diameter of approximately 42.7 nm. Peppermint extracts resulted in the production of well-defined ZnONPs with a triangular shape, consistent with the findings of (Doğaroğlu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). SEM images reveal that the synthesized zinc oxide nanoparticles from peppermint are organized in aggregated and agglomerated clusters, as similarly reported by (Kulkarni and Shirsat, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The SEM analysis demonstrated that the choice of precursors affects the dimensions and morphology of the nanoparticles (Fakhari, Jamzad, and Kabiri Fard, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For instance, when zinc acetate is used as a precursor, zinc oxide molecules develop slowly, forming compact spherical structures that cluster into bullet-shaped formations (Visinescu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Conversely, using zinc nitrate as a precursor produces rod-shaped ZnONPs (Tsai et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The clustering is attributed to polarity and electrostatic interactions among ZnONPs (Abegunde, Olasehinde, and Adebayo, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, the ZnONPs in this study displayed a unique nanorod-like structure (Chowdhury et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Some nanorods exhibited clustering, while others remained isolated. The formation and development of zinc oxide nanorods are mainly driven by the facet with the lowest surface energy. Furthermore, the ZnONPs possess pyramidal symmetry (Ahmad et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Regarding size, the average dimensions of ZnONPs from peppermint extract were 73.76 nm (Doğaroğlu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), whereas the particle size observed in SEM images in this study was approximately 20\u0026ndash;50 nm, about 30% larger. These findings suggest that the shape and composition of the synthesized nanoparticles vary depending on the specific type of salt, extract, and plant used in the synthesis process. Consequently, the size of the produced particles also differs based on the materials employed and their eventual form.\u003c/p\u003e \u003cp\u003eHigh-magnification TEM images (Fig.\u0026nbsp;4) show that the pores in the freshly synthesized ZnONPs derived from peppermint extract predominantly consist of spherical particles with sizes ranging from 20 to 50 nm, indicating variability in particle dimensions within the sample (Figs.\u0026nbsp;4). In the large-area TEM image, some nanoparticles are observed to aggregate into chains. According to previously reported TEM research by (Patel et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the ZnONPs exhibit a spherical form with sizes ranging from 60 to 160 nm. Furthermore, the TEM technique was utilized to determine the shape and size of the zinc nanoparticles (ZnNPs) produced using peppermint (Mentha piperita) extract. TEM analysis revealed the presence of nanoparticles with globular and oblong shapes, measuring between 15 and 27 nm, with an average particle size of 18 nm, as determined from TEM micrographs (Bandyopadhyay et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mohan and Renjanadevi, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This analysis confirmed the effectiveness of peppermint extract in synthesizing ZnNPs. Therefore, both studies are consistent with the ZnONPs results obtained from this experiment, verifying the reliability of the outcomes (Ahmad et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemical composition of ZnONPs analysis\u003c/h3\u003e\n\u003cp\u003eEnergy-dispersive X-ray spectroscopy (EDS) was performed in conjunction with scanning electron microscopy (SEM) to analyze the elemental composition of the synthesized ZnO nanoparticles (ZnONPs). The EDS analysis covered an energy range from 0 to 8 keV, revealing distinct peaks that confirm the presence of zinc and oxygen in the nanoparticles. As illustrated in Fig.\u0026nbsp;5, the data show zinc constitutes 73.1% of the weight fraction, while oxygen accounts for 26.9%.\u003c/p\u003e \u003cp\u003eFurther elemental composition analysis of the ZnONPs indicates a weight percentage of 80.4% zinc and 19.6% oxygen, aligning closely with the expected composition from the Zn(NO₃)₂ precursor. The sharp peaks observed within the 0 to 2 keV range and between 6 and 8 keV are indicative of the crystalline structure of the ZnONPs. This structural characterization underscores the porous nature of the nanopowder, which enhances its surface area.\u003c/p\u003e \u003cp\u003eThe EDS data also revealed spherical-triangular clusters of zinc nanoparticles (Fig.\u0026nbsp;5a). Notably, the analysis identified only two peaks, corresponding to zinc and oxygen, with no additional contaminants detected. The observed nearly stoichiometric composition of the ZnONPs is consistent with the expected values. A recent study by Chowdhury et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported an EDS-derived mass percentage composition of 80.12% zinc and 19.88% oxygen for a zinc oxide sample, confirming its purity. Similarly, Ahmed et al. (2023) found that the 3\u0026ndash;4 keV energy range indicated a zinc content of approximately 85.71%, highlighting the high purity of the zinc element in their samples (Fig.\u0026nbsp;5b).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eXRD Analysis\u003c/h2\u003e \u003cp\u003eX-ray diffraction pattern (XRD) spectrum of ZnONPs synthesized utilizing extracts derived from peppermint leaves. The diffraction peaks were discerned at specific 2-theta values: 31.69\u0026deg;, 34.37\u0026deg;, 36.21\u0026deg;, 47.47\u0026deg;, 56.49\u0026deg;, 62.73\u0026deg;, 66.29\u0026deg;, 67.85\u0026deg;, and 68.99\u0026deg;, which coincided with peaks corresponding to (100), (002), (101), (102), (110), (103), (200), (112), and (201) respectively (Fig.\u0026nbsp;6). Recent studies have reported the XRD spectra of ZnONPs synthesized using leaf extracts from \u003cem\u003eMentha viridis\u003c/em\u003e (Chowdhury et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Distinct diffraction peaks were observed at specific 2θ values, including 31.73\u0026deg;, 34.42\u0026deg;, 36.25\u0026deg;, 47.53\u0026deg;, 56.56\u0026deg;, 62.78\u0026deg;, 66.33\u0026deg;, 67.82\u0026deg;, and 69.1\u0026deg;. These peaks correspond to the lattice planes with Miller indices of (100), (002), (101), (102), (110), (103), (200), (112), and (201), respectively. The presence of these well-defined diffraction peaks confirms the crystalline nature of the biosynthesized ZnO. Additionally, no diffraction peaks were detected apart from ZnO, indicating the formation of pure ZnO (Fig.\u0026nbsp;6). These findings align closely with previous results, corroborating the crystalline structure of the ZnONPs (Fig.\u0026nbsp;6). According to Fakhari et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the XRD pattern of the synthesized ZnONPs clearly demonstrates the presence of a distinct crystalline structure within the nanoparticles. These distinct diffraction peaks correspond to the crystalline planes of the synthesized ZnONPs, providing valuable insights into their structural characteristics and crystallographic orientation. These sharp peaks confirm that the synthesized ZnONPs are crystalline, as shown in Fig.\u0026nbsp;6. The diffraction 2θ values correspond to the face-centered cubic crystalline phase of zinc oxide. The 2θ values were recorded, and distinct diffraction peaks were observed at angles of 31.46\u0026deg;, 34.29\u0026deg;, 36.33\u0026deg;, 47.51\u0026deg;, 56.50\u0026deg;, 62.84\u0026deg;, 67.79\u0026deg;, and 76.83\u0026deg;. These peaks correspond to the diffraction lattice planes (100), (002), (101), (102), (110), (103), (112), and (202), indicating the hexagonal wurtzite structure of the synthesized nanoparticles. The average size of the ZnONPs was determined by analyzing the most intense peak (101) using the Debye-Scherrer equation. This equation incorporates several variables: λ (lambda) represents the X-ray wavelength from Cu-Kα (1.540560 \u0026Aring;), β (beta) is the full width at half maximum (FWHM) of the diffraction peak in radians, θ (theta) is Bragg's angle in degrees, and K is the shape factor, which has a value of 0.9 (Fakhari et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). XRD analyses indicated that nanoparticles synthesized from zinc acetate and zinc nitrate precursors exhibited average sizes of 21.49 nm and 25.26 nm, respectively. Both studies supported the findings of this research. The average size was determined by applying the Eq.\u0026nbsp;1yielding an average size of 41.55 nm (Fakhari 2019).\u003c/p\u003e \u003cp\u003eD = (K λ/ β cos θ) (1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFTIR analysis\u003c/h2\u003e \u003cp\u003eThe role of biomolecules in the synthesis of ZnONPs was investigated and identified using FTIR spectroscopy across the spectrum range of 4000 to 400 cm⁻\u0026sup1; (Fig.\u0026nbsp;7). The FTIR analysis of the peppermint-extract-derived ZnONPs revealed distinctive absorption peaks at specific wavelengths, including 3450 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1380 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 890 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These absorption peaks signify the presence of functional groups and biomolecules associated with the synthesis process, providing valuable insights into the chemical composition and bonding characteristics of the synthesized ZnONPs. The FTIR spectra of the synthesized ZnONPs have been detailed by (Javed et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, a hydroxyl group shows an O-H bending vibration at 574 cm⁻\u0026sup1;. Peaks in the 2850 to 2930 cm⁻\u0026sup1; range are associated with the C-H stretching vibration. The peaks at 1576 and 1420 cm⁻\u0026sup1; correspond to the asymmetrical and symmetrical stretching of zinc nitrate hexahydrate (Zn(NO₃)₂\u0026bull;6H₂O), respectively. The bond at 1025 cm⁻\u0026sup1; can be attributed to the C-O stretching vibration. An absorption peak at 857 cm⁻\u0026sup1; indicates the formation of tetrahedral coordination of Zn. A significant peak at 1047 cm⁻\u0026sup1; was observed in the peppermint sample (Koli, 2022). A recent study used FTIR analysis within the wavelength range of 4000 to 400 cm⁻\u0026sup1; to identify the functional groups and biomolecules involved in the production of ZnONPs (Doğaroğlu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The synthesis involves Zn\u0026sup2;⁺ ions from zinc acetate binding with polyphenols in the plant extract, converting Zn\u0026sup2;⁺ to Zn⁺ through reduction\u0026mdash;a process described as the synthesis of Zn⁺ polyphenols in the reaction solution. Both the original ZnONPs and those derived from plant extracts exhibited similar FTIR peaks, suggesting a common synthesis mechanism. The M-ZnONPs show absorption bands at 3837, 3383, 2979, 2903, 1563, 1405, 1252, 1055, 889, 688, 543, and 484 cm⁻\u0026sup1;. In contrast, the B-ZnONPs have absorption peaks at 3385, 1563, 1414, 877, 688, 582, 552, and 481 cm⁻\u0026sup1;. The distinct peaks at 3383 and 3385 cm⁻\u0026sup1; are likely due to hydroxyl groups present in alcohols and phenolic compounds in plant extracts. Peaks between 2979 and 2903 cm⁻\u0026sup1; are attributed to the elongation of carbon-hydrogen bonds in alkanes (Abdullah, Bakar, and Bakar, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Peaks at 1563 cm⁻\u0026sup1; indicate C\u0026thinsp;=\u0026thinsp;C stretching vibrations in aromatic rings within polyphenolic compounds (Matinise et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The peaks at 1405 and 1414 cm⁻\u0026sup1; relate to the symmetrical stretching mode of the COO⁻ group in acids. The peak at 1252 cm⁻\u0026sup1; is linked to the C-O stretching (Bharathi and Bhuvaneshwari, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The dominant peak at 1055 cm⁻\u0026sup1; in the peppermint FTIR spectrum results from the elongation of C\u0026ndash;O, C-C, and C-O-C bonds in saturated esters, alcohols, phenols, cycloalkanes, and acid anhydrides. The primary absorption band for ZnO appears in the 400\u0026ndash;600 cm⁻\u0026sup1; region, with the wide and strong band at 500 cm⁻\u0026sup1; representing Zn-O vibration (Tantiwatcharothai and Prachayawarakorn, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Bending vibrations of alkanes and alkenes, particularly C\u0026thinsp;=\u0026thinsp;C bending, are noted at 889, 877, and 688 cm⁻\u0026sup1;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eUV-Vis Spectrum\u003c/h2\u003e \u003cp\u003eThe color gradually darkened over time, becoming dark brown after four hours of incubation. The ZnONPs exhibited a prominent absorption peak at approximately 220 nm, highlighting the wavelength where the nanopowder most strongly absorbs light, thereby reflecting its optical properties. This spectral absorption characteristic is crucial for understanding the material's behavior and potential applications in fields such as optoelectronics, photovoltaics, and photocatalysis. The color change of the reacting mixture likely corresponds to the surface plasmon resonance of zinc nanoparticles. The formation of nanoparticles in the mixture was monitored using a Perkin Elmer UV-Vis spectrophotometer at regular intervals (every hour). A peak absorbance observed near 450 nm further confirmed the production of ZnONPs. The UV-Vis spectra obtained at different time intervals are illustrated in Fig.\u0026nbsp;8, depicting changes in peak absorbance over the course of the reaction. Furthermore, the peak absorbance increased almost linearly with reaction time until two hours of incubation, likely due to the continued production of nanoparticles. After four hours, the rate of formation saturated, indicating the reaction had reached completion. The absorption spectra of ZnONPs synthesized from peppermint exhibit a characteristic peak at a wavelength of 281.5 nm, signifying the presence of ZnO. Previous studies have identified a more pronounced absorption peak at 385 nm in ZnO nanoparticles (Ahmad et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The band gap energy (E) was measured by (Loh et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) through an analysis of the maximum wavelength at which the ZnONPs absorbed light. The ZnONPs were determined to have a band gap energy of 3.37 eV, calculated using Eq.\u0026nbsp;(2). This value aligns with literature values, which typically indicate a broad bandgap ranging from 3.3 to 3.4 eV (Javed et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The equation for E is given by E\u0026thinsp;=\u0026thinsp;hc/λ, where h is Planck's constant (6.626\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;34\u003c/sup\u003e J), c is the speed of light (3.0\u0026times;10\u003csup\u003e8\u003c/sup\u003e m/s), and λ is the maximum absorption wavelength (in nm). At a wavelength of 220 nm, the energy of the photons is approximately 5.64 eV. using h\u0026thinsp;=\u0026thinsp;6.626\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;34\u003c/sup\u003e J, c\u0026thinsp;=\u0026thinsp;3.0\u0026times;10\u003csup\u003e8\u003c/sup\u003e m/s, and λ\u0026thinsp;=\u0026thinsp;220 nm in E\u0026thinsp;=\u0026thinsp;hc/λ​ equation. This calculation is based on the relationship between energy and wavelength in the electromagnetic spectrum, as previously discussed. The bandgap in this study varies from prior findings due to differences in absorbance. The stable synthesis of ZnONPs is typically confirmed by examining its distinctive absorption spectrum, using a scanning range of 600\u0026ndash;200 nm, as reported by (Chowdhury et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eE\u0026thinsp;=\u0026thinsp;hc/λ​ (2)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial Activity\u003c/h2\u003e \u003cp\u003eThe agar well diffusion method revealed clear zones of inhibition around the wells containing ZnONPs for bacteria, demonstrating the nanoparticles' effectiveness against the bacterium pathogen. Significant inhibition zones were observed around wells containing ZnONPs for bacteria. The agar well diffusion method confirmed the antimicrobial activity of ZnONPs against bacterial pathogens (Padmavathy and Vijayaraghavan, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Figure\u0026nbsp;9 illustrates the zone of inhibition obtained through a well diffusion assay. The results indicate that bacterial growth was effectively inhibited by varying concentrations of zinc nanoparticles (ZnONPs). The highest concentration used, 35 mg/ml, produced the largest zone of inhibition. As shown in the figure (Fig.\u0026nbsp;9), there is a clear trend of increasing inhibition with higher concentrations of ZnONPs. Specifically, the concentrations of 35, 25, and 15 mg/ml resulted in zones of inhibition measuring 0.2 mm, 0.1 mm, and 0.05 mm, respectively.\u003c/p\u003e \u003cp\u003ePillai et al. (2020) also demonstrated the biological efficacy of ZnONPs against \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eCandida albicans\u003c/em\u003e, and \u003cem\u003eAspergillus niger\u003c/em\u003e. (Doğaroğlu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that \u003cem\u003eS. aureus\u003c/em\u003e was more susceptible to ZnONPs compared to \u003cem\u003eE. coli\u003c/em\u003e, with the activity level of \u003cem\u003eE. coli\u003c/em\u003e being greater for ZnONPs than for B-ZnO (Basil) and M-ZnO (Peppermint) nanoparticles. Conversely, the activity level of \u003cem\u003eS. aureus\u003c/em\u003e was greater when exposed to ZnONPs compared to M-ZnO and B-ZnO nanoparticles. According to (Venkataraju et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), Gram-negative bacteria require ZnONPs to penetrate both the outer membrane and the thin peptidoglycan layer, whereas Gram-positive bacteria possess a thicker peptidoglycan layer (approximately 30 nm) in addition to the outer membrane. This explains the observed findings in this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe successful synthesis of ZnONPs using peppermint extract through eco-friendly methods has been validated by comprehensive characterization techniques, including SEM, EDXS, TEM, XRD, FTIR, and UV-Vis spectroscopy. These analyses confirm the production of crystalline ZnONPs with distinct morphological features. The environmentally friendly synthesis process, coupled with the detailed characterization, highlights the potential of these nanoparticles for diverse applications, such as catalysis, sensors, and biomedical devices. Additionally, the ZnONPs exhibit significant antibacterial activity against a broad range of bacterial species, as evidenced by clear inhibition zones observed in the well diffusion assays. Overall, ZnONPs represent a promising integration of green synthesis and effective antimicrobial properties, making them an attractive option for use in antimicrobial technologies and biomedicine.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSultan Qaboos University financially supported this research through a research grant number.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions: CRediT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSAR: Investigation, Conceptualization, Software, Validation, Visualization, Writing \u0026ndash; original draft. AA: Data curation, Investigation, Software, Visualization, Writing \u0026ndash; original draft. MAB: Investigation, Software, Visualization. AAS: Conceptualization, Software, Validation. AMAS: Conceptualization, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing. TA: Conceptualization, Software, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing. BL: Resources, Conceptualization, Investigation, Supervision, Visualization, Writing \u0026ndash; review \u0026amp; editing. MSS: Resources, Conceptualization, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources:\u0026nbsp;\u003c/strong\u003eSultan Qaboos University and Zhejiang University provided the funding through the co-funding project (CL/SQU-ZJU/AGR/23/01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e accompanies this paper\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing financial interests or personal relationships that could have influenced the work presented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e \u003cstrong\u003edeclaration\u003c/strong\u003e: not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: the datasets generated and/or analyzed during the current study are included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdullah, F., Bakar, N. 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Large-scale synthesis of six-nanometer-wide ZnO nanobelts. \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e 108(26), 8773-8777.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Peppermint (Mentha Piperita), Zinc oxide nanoparticles, SEM, EDXS, TEM, XRD, FTIR, UV-VIS spectrum, Antibacterial activity","lastPublishedDoi":"10.21203/rs.3.rs-6539690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6539690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis research presents an eco-friendly technique for synthesizing zinc oxide nanoparticles (ZnONPs) using peppermint (Mentha Piperita) extract as both a reducing and stabilizing agent. The synthesis involved a controlled reaction between zinc nitrate hexahydrate [Zn(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO] and the Peppermint extract. The resulting ZnONPs were characterized using SEM, EDXS, TEM, XRD, FTIR, and UV-Vis spectroscopy. SEM revealed well-defined triangular crystals, while TEM showed spherical nanoparticles sized 20\u0026ndash;50 nm. EDXS confirmed the elemental composition as Zn\u0026thinsp;=\u0026thinsp;80.4% and O\u0026thinsp;=\u0026thinsp;19.6%. XRD analysis validated the crystalline structure, and FTIR identified biomolecules involved in the synthesis. UV-Vis spectra displayed an absorption peak at 220 nm. Antimicrobial testing using the well diffusion method showed inhibitory zones of 0.2, 0.1, and 0.05 mm at zinc nanoparticle (ZnONPs) concentrations of 35, 25, and 15 mg/ml, respectively. The results demonstrate effective bacterial inhibition, with the highest concentration (35 mg/ml) producing the largest zone of inhibition. This eco-friendly synthesis method for ZnONPs is sustainable and holds promise for applications in antibacterial technologies and various other industries. The ZnONPs demonstrated significant antibacterial properties, suggesting their potential for further research in biological and materials science.\u003c/p\u003e","manuscriptTitle":"Eco-Friendly Zinc Oxide Nanoparticles from Peppermint: Synthesis, Characterization, and Antimicrobial Evaluation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 16:29:33","doi":"10.21203/rs.3.rs-6539690/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-14T05:08:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-22T08:35:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-22T06:42:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-18T01:56:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56647333053668349498741897681143008473","date":"2025-05-13T19:42:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325274444191599104599732425308891296156","date":"2025-05-13T16:57:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113846503623984703552759437404265963266","date":"2025-05-13T15:49:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122077986874405387677250891531209414019","date":"2025-05-13T15:48:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T15:41:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-07T13:20:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-07T11:34:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-04-27T10:01:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"806db5d6-988a-41aa-9eff-1d48af9af0fe","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:44:56+00:00","versionOfRecord":{"articleIdentity":"rs-6539690","link":"https://doi.org/10.1007/s12668-025-02316-4","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2026-01-16 16:28:59","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-05-15 16:29:33","video":"","vorDoi":"10.1007/s12668-025-02316-4","vorDoiUrl":"https://doi.org/10.1007/s12668-025-02316-4","workflowStages":[]},"version":"v1","identity":"rs-6539690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6539690","identity":"rs-6539690","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}