Eco-Friendly Synthesis of Magnesium Oxide Nanoparticles Using Citrullus colocynthis and their Synergistic Antimicrobial Activity against Drug-Resistant Pathogens

Eco-Friendly Synthesis of Magnesium Oxide Nanoparticles Using Citrullus colocynthis and their Synergistic Antimicrobial Activity against Drug-Resistant Pathogens | 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 Synthesis of Magnesium Oxide Nanoparticles Using Citrullus colocynthis and their Synergistic Antimicrobial Activity against Drug-Resistant Pathogens Maqsood Qaisar, Abdul Rehman, Iffat Naz, Hassan Naveed, Baharullah Khattak, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8079662/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The escalating threat of antimicrobial resistance (AMR) necessitates innovative therapeutic approaches. This study reports the green synthesis of magnesium oxide nanoparticles (MgO-NPs)using Citrullus colocynthis extract, a medicinal plant rich in bioactive compounds, as a sustainable alternative to conventional antibiotics. The synthesized MgO-NPs were characterized by UV-Vis spectroscopy (absorption peak at 250 nm), XRD (cubic crystalline structure, 15–25 nm size), SEM-EDX (agglomerated spherical morphology, Mg/O ratio 2.6:1), and FTIR (Mg-O vibration at 860 cm⁻¹). The nanoparticles exhibited potent, dose-dependent antibacterial activity against multidrug-resistant (MDR) Staphylococcus aureus (MIC: 35.3 µg/ml; MBC: 97.1 µg/ml) and Escherichia coli (MIC: 47.5 µg/ml; MBC: 105.5 µg/ml), with a bactericidal mode of action (MBC/MIC ≤ 4). Remarkably, MgO-NPs restored susceptibility to β-lactams antibiotics (ceftazidime and penicillin) in resistant strains, demonstrating synergistic effects. Antifungal activity of MgO-NPs against Candida albicans (17.3±0.7 mm) and Aspergillus niger (14.4±0.8 mm) at a concentration of 10 mg/ml was also observed. Phytochemical analysis revealed solvent-dependent bioactive constituents in C. colocynthis , with aqueous extracts rich in tannins/phenolics and methanolic extracts in flavonoids/terpenoids. This is the first report demonstrating restoration of antibiotic susceptibility by MgO-NPs synthesized from C. colocynthis extract. Antimicrobial resistance Green synthesis Magnesium oxide nanoparticles Phytochemicals Synergistic effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Antimicrobial resistance (AMR) has emerged as a critical global health crisis in the 21st century, rendering bacterial infections increasingly difficult to treat and contributing to rising mortality rates worldwide (WHO, 2022). The diminishing efficacy of standard therapeutics, driven by the proliferation of multidrug-resistant (MDR) bacteria, has led to prolonged hospitalizations, increased healthcare costs, and higher mortality risks (Ali et al. 2022 ; Xie et al. 2023 ). The accelerated evolution of resistant bacterial strains, fuelled by excessive antibiotic use in clinical and agricultural settings, emphasizes the urgent need for novel antimicrobial agents (Tarín-Pelló et al. 2022 ). Pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli , Candida albicans and Aspergillus niger have rapidly disseminated, drastically limiting available treatment options (Salam et al. 2023 ). The World Health Organization (WHO) ranks AMR among the top ten global health threats, projecting that drug-resistant infections could cause ten million annual deaths by 2050 (Cotugno et al. 2025 ). Despite this looming crisis, antibiotic development has stagnated, with few novel drug classes entering the clinical pipeline (Theuretzbacher et al. 2023 ). Current strategies such as combination therapies, phage therapy, and antimicrobial peptides face significant challenges, including high costs, regulatory complexities, and rapid resistance development (Killai et al. 2024). Consequently, innovative solutions integrating nanotechnology and plant-derived bioactive compounds are imperative. Metal oxide nanoparticles, particularly magnesium oxide nanoparticles (MgO-NPs), have garnered significant attention due to their broad-spectrum antimicrobial activity, resistance to degradation, and ability to induce oxidative stress in bacterial and fungal cells (Mouhamad et al. 2022 ; Slavin and Bach 2022 ; Gatou et al. 2024 ). MgO-NPs exert their antimicrobial effects through three primary mechanisms: membrane penetration, reactive oxygen species (ROS) generation, and disruption of metabolic pathways (Sharma et al. 2023 ; Gatou et al. 2024 ). However, conventional synthesis methods often involve toxic chemicals and energy-intensive processes, posing environmental and biological risks (Soltys et al. 2021 ). In contrast, plant-mediated synthesis leverages natural phytochemicals such as flavonoids, alkaloids, and phenolic compounds as reducing and stabilizing agents, offering advantages such as biocompatibility, ambient reaction conditions, minimal waste generation, and enhanced biomedical applicability (Adeyemi et al. 2022 ; Barathi et al. 2024 ). Citrullus colocynthis (bitter apple), a medicinal plant from the Cucurbitaceae family, is an ideal candidate for nanoparticle synthesis due to its high concentration of bioactive compounds, including cucurbitacins, flavonoids, and terpenoids (Li et al. 2022 ; Rao et al. 2023). These phytochemicals facilitate metal ion reduction while conferring intrinsic antimicrobial properties to the resulting nanoparticles (Gebre 2023 ). While several plant-mediated MgO-NPs have been reported, their synergistic interaction with antibiotics and antifungal activity remain largely unexplored for C. colocynthis (Rasool et al. 2022 ; Al Nablsi et al. 2025 ). This study focuses on bacterial and fungal pathogens due to their significant threat to global public health. These strains are leading causes of both hospital and community-acquired infections, contributing to high mortality rates, morbidity, and escalating healthcare costs. With resistance to first-line antibiotics (β-lactams, macrolides, and fluoroquinolones) on the rise, the development of innovative therapeutic approaches is urgently needed. In the current study, a novel green synthesis method for MgO-NPs using C. colocynthis extract, a previously unexplored approach has been explored. By combining sustainable synthesis techniques with advanced characterization and biological evaluation, this study aims to pioneer a new strategy for combating MDR bacterial and fungal infections, bridging the gap between nanotechnology and phytomedicine for biomedical innovation. 2. Materials and Methods 2.1 Collection and Preparation of C. colocynthis Extract Fresh fruits of C. colocynthis were collected from District Karak, Khyber Pakhtunkhwa, Pakistan. The collected fruits were thoroughly washed with distilled water to remove surface impurities and subsequently shade-dried at room temperature for two weeks to preserve thermolabile phytoconstituents. The dried plant material was pulverized into a fine powder using an electric grinder to maximize surface area for extraction. A combination of Soxhlet extraction and cold maceration techniques was employed using aqueous and methanol solvents to ensure extraction of bioactive compounds. The resultant extracts were filtered through Whatman No. 1 filter paper to remove particulate matters and concentrated under reduced pressure using a rotary evaporator to obtain solvent-free crude extracts (Rani et al. 2021 ; Al Nablsi et al. 2025 ). 2.2 Qualitative Phytochemical Analysis Phytochemical screening was conducted to identify secondary metabolites present in the aqueous and methanolic extracts of C. colocynthis . Standard qualitative tests were performed to detect alkaloids using Mayer's reagent, flavonoids via the alkaline reagent test, and tannins/phenolic compounds through the ferric chloride test. The presence of saponins was confirmed by the persistent foam formation test, while terpenoids were identified using the Salkowski test (Rani et al. 2021 ). 2.3 Collection of tests Bacterial and Fungal Strains Previously characterized S. aureus , E. coli, C. albicans (ATCC 10231) and A. niger (ATCC 16404) were obtained from the culture collection bank of the Department of Microbiology, Kohat University of Science and Technology, Kohat. The bacterial strains were selected based on their documented resistance profiles to multiple classes of antibiotics, making them representative models for evaluating novel antimicrobial agents. The bacterial strains were maintained on nutrient agar slants at 4°C and sub-cultured periodically, while C. albicans and A. niger fungal strains were maintained on Sabouraud Dextrose Agar (SDA) media at 35°C and 28°C respectively to ensure viability. 2.4 Antibacterial Activity of C. colocynthis extracts against MDR Bacteria The antibacterial potential of C. colocynthis extracts against MDR bacteria ( S. aureus , and E. coli ) was evaluated using the standard well diffusion assay. Bacterial suspensions were standardized to 0.5 McFarland turbidity and uniformly spread on Mueller-Hinton agar plates. Wells of 6 mm diameter were aseptically punched into the agar, with 1% Dimethyl sulfoxide (DMSO) serving as the negative control and standard antibiotic discs as positive controls. Aqueous and methanolic extracts (conc. 5 mg/ml) were loaded into the wells (80 µl per well). Following incubation at 37°C for 24 h, the zones of inhibition were measured in millimeters to quantify antibacterial activity of C. colocynthis extracts (Mazher et al. 2023 ). 2.5 Synthesis of MgO-NPs using C. colocynthis Extract Green synthesis of MgO-NPs was carried out by combining 0.1 M magnesium nitrate solution [Mg(NO 3 ) 2 ·6H 2 O] with C. colocynthis extract under constant stirring at 60–80°C while maintaining the pH between 8–12. The reaction was allowed to proceed for 4–6 h, during which the color of solution change from light yellow to brown, indicating the formation of magnesium hydroxide precipitate. The precipitate was repeatedly washed with distilled water, followed by centrifugation at 10,000 rpm for 15 minutes to remove impurities. The purified product was dried at 60–70°C and subsequently calcined at 400–500°C for 2–3 h to obtain pure MgO-NPs (Abdelsadek et al. 2022 ). 2.6 Characterization of Synthesized MgO-NPs The synthesized MgO-NPs were characterized using multiple analytical techniques in the Centralized Resource Laboratory (CRL), University of Peshawar and National Centre of Excellence in Geology (NCEG), University of Peshawar. UV-Visible spectroscopy (180–1100 nm) was employed to confirm nanoparticle formation through surface plasmon resonance analysis. FTIR spectroscopy (400–4000 cm⁻¹) identified the functional groups responsible for reduction and stabilization of nanoparticles. X-ray diffraction analysis using Cu-Kα radiation (λ = 1.5406 Å) at 40 kV and 40 mA provided information about crystallinity and phase composition. Morphological and elemental characterization was performed using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX) to assess particle size, shape, and chemical purity (Hayat et al. 2024 ). 2.7 Antibiotics used in the Study The study employed four classes of antibiotics representing different mechanisms of action: erythromycin (5 µg/disc) for protein synthesis inhibition, ceftazidime (30 µg/disc) as a β-lactam agent, penicillin (10 µg/disc) targeting cell wall synthesis, and oxacillin (5 µg/disc) for assessing methicillin resistance. All antibiotic discs were used according to clinical and laboratory standards institute (CLSI) guidelines to ensure standardized results (Humphries et al. 2021 ). 2.8 Antibacterial Activity of MgO-NPs Coated Filter Discs Sterile filter paper discs (6.25 mm diameter) were prepared from Whatman No. 1 filter paper using a standard punch. The discs were sterilized in a hot air oven at 160°C for one hour to ensure aseptic conditions. For antibacterial testing, the discs were impregnated with MgO-NPs suspensions at concentrations ranging from 5 to 25 mg/ml prepared in 1% DMSO. The coated filter discs were dried at 80°C to remove residual moisture before use in antibacterial assays (Hayat et al. 2024 ). The antibacterial efficacy of MgO-NPs-coated filter discs was evaluated against test MDR bacteria using the Kirby-Bauer disc diffusion method following CLSI standards (Humphries et al. 2021 ) Mueller-Hinton agar plates were inoculated with standardized bacterial suspensions, and filter discs coated with different concentrations of MgO-NPs were aseptically placed on the agar surface. Non-coated filter disc and 1% DMSO were used as a control in the assay. After incubation at 37°C for 24 h, the zones of inhibition were measured in millimeters to assess antibacterial activity of MgO-NPs against test MDR bacteria (Hayat et al. 2024 ). 2.9 Preparation of MgO-NPs-Coated Antibiotic Discs A standardized suspension of MgO-NPs was prepared by dispersing 25 mg of MgO-NPs powder in 100 ml of distilled water. Antibiotic discs were uniformly coated with 5 µl of suspension using a micropipette and dried at 80°C to ensure proper adhesion of nanoparticles while maintaining antibiotic stability. This preparation allowed for the evaluation of potential synergistic effects between conventional antibiotics and nanoparticles (Hayat et al. 2024 ). 2.10 Disc Diffusion Assay for MgO-NPS Coated and Uncoated Antibiotics The comparative efficacy of MgO-NPs-coated versus uncoated antibiotics was assessed using the standard disc diffusion method. MHA plates were inoculated with test MDR bacteria, and both MgO-NPs coated and uncoated versions of erythromycin (5 µg/disc), ceftazidime (30 µg/disc), penicillin (10 µg/disc), and oxacillin (5 µg/disc) were placed on the agar surface. Following incubation at 37°C for 24 h, the zones of inhibition were measured and compared to determine the enhancement of antibacterial activity through nanoparticle coating. 2.11 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of MgO-NPs against test MDR Bacteria The antibacterial activity of MgO-NPs was evaluated through standardized microbiological assays following CLSI guidelines. MIC was determined via broth microdilution method in Mueller-Hinton broth, with bacterial inoculum standardized to 5×10⁵ CFU/ml. MBC was assessed by subculturing from MIC wells onto agar plates, with bactericidal activity defined as ≥ 99.9% kill rate. The MBC/MIC ratio was calculated to classify antimicrobial action i.e., bactericidal (MBC/MIC ≤ 4) vs. bacteriostatic (MBC/MIC > 4) (Ishak et al. 2025 ). All experiments were performed in triplicate under aseptic conditions. The therapeutic index was derived from the MBC/MIC ratio to quantify bactericidal potency. 2.12 Antifungal Activity of Synthesized MgO-NPs The antifungal activity of MgO-NPs was evaluated against C. albicans (ATCC 10231) and A. niger (ATCC 16404) using the agar well diffusion assay. Fungal suspensions were standardized to 0.5 McFarland turbidity (5×10⁶ CFU/ml for C. albicans ; spore count via hemocytometer for A. niger ) and swabbed onto Sabouraud Dextrose Agar (SDA) plates. Wells of 6 mm diameter were loaded with 50 µl of MgO-NPs (1, 5, and 10 mg/ml in sterile water). Fluconazole (10 µg/well) and Itraconazole (10 µg/well) served as positive controls for C. albicans and A. niger , respectively, while sterile water was the negative control. Plates were incubated at 35°C (24 h for C. albicans ) and at 28°C (48 h for A. niger ). The zones of inhibition were measured in millimeters (mm) from the well edge to the fungal growth margin. 2.13 Statistical Analysis The experiments were performed in triplicates while data representation included mean values with standard error as error bars. Microsoft Excel-365 evaluated the statistical significance through p-value comparisons and showed significance at p ≤ 0.05. 3. Results 3.1 Confirmation of MDR Bacterial Strains The previously identified MDR bacterial strains ( E. coli and S. aureus ) were confirmed using standard antibiotic disc diffusion assays (Table 1). Both isolates exhibited complete resistance to all tested antibiotics including ceftazidime (third-generation cephalosporin), penicillin (β-lactam), oxacillin (methicillin-class), and erythromycin (macrolide). The resistance of test bacterial strains across these four distinct antibiotic classes confirms their classification as MDR strains. Table 1 [Near Here] 3.2 Phytochemical screening of C. colocynthis extracts Phytochemical screening of C. colocynthis extracts revealed distinct solvent-dependent profiles. The aqueous extract demonstrated strong presence of tannins and phenolic compounds, with moderate saponins and trace flavonoids. In contrast, the methanolic extract showed absence of tannins and phenolics but contained terpenoids and flavonoids. Notably, saponins were completely absent in the methanolic extract as shown in Figure 1. Figure 1 [Near Here] 3.3 Antibacterial activity of C. colocynthis extracts against MDR bacteria The methanolic and aqueous extracts of C. colocynthis demonstrated significant antibacterial activity against test MDR bacteria, as shown in Figure 2. Both extracts exhibited comparable inhibition zones against E. coli (methanolic: 11.2±0.25 mm; aqueous: 11.3±0.28 mm), while against S. aureus , the methanolic extract showed marginally better activity (11.6±0.31 mm) compared to the aqueous extract (10.3±0.72 mm). Notably, both extracts displayed antibacterial effects where penicillin (positive control) showed complete resistance, suggesting potential novel mechanisms of action against these MDR bacterial pathogens. Figure 2 [ Near Here] 3.4 Characterization of Synthesized MgO-NPs The UV-Vis absorption spectrum of the synthesized MgO-NPs (Figure 3) exhibited a strong and sharp absorption band centred at 250 nm, with no significant absorption observed in the visible region (400–800 nm). The absorption edge at 250 nm corresponds to an estimated optical bandgap of ~4.96 eV, calculated using the formula: , where λonset is the absorption onset wavelength (250 nm). The steep rise in absorption at this wavelength indicates a direct bandgap transition, characteristic of crystalline MgO-NPs. The absence of additional peaks suggests high purity and minimal aggregation of the nanoparticles. Figure 3 [Near Here] The SEM micrograph revealed agglomerated MgO-NPs with irregular morphologies, ranging from spherical to quasi-cubic structures (Figure 4a). The particles exhibited a size distribution of approximately 20–80 nm, with visible porosity suggesting a high surface area. Notably, some larger aggregates were observed, likely due to insufficient dispersion during sample preparation. The surface texture appeared rough, consistent with reports of MgO-NPs synthesized via precipitation methods. On the other hand, the EDAX spectrum confirmed the presence of Mg (47.7%) and O (18.5%) as primary elements. The significant carbon content (21.3%) likely originates from residual organic precursors. Trace amounts of Au (2.2%), Cu (4.5%), Ca (2.3%), P (1.8%), and K (1.7%) were also detected in the EDAX spectrum as shown in Figure 4b. The Mg/O ratio (2.6:1) indicated non-stoichiometry, potentially due to surface hydroxylation or unreacted precursors. Figure 4 [Near Here] The FTIR spectra provides a direct comparison between the C. colocynthis extract and the synthesized MgO-NPs (Figure 5). The FTIR spectrum of C. colocynthis extract displays characteristic organic functional groups: a broad absorption band at 3200-3600 cm -1 (O-H stretching from water, phenols, and alcohols), strong peaks between 1000-1750 cm -1 (C=O and C-O stretches from carbonyls and polysaccharides), and C-H stretching vibrations at 2850-2920 cm -1 (from aliphatic compounds). In contrast, the FTIR spectrum of MgO-NPs shows significant changes: the O-H band is markedly reduced in intensity, a new sharp peak appears at 760 cm -1 (Mg-O lattice vibration), and distinct carbonate-related peaks emerge near 1320 cm -1 . While most organic peaks disappear, weak residual C-H stretches persist, indicating trace organic remnants. The dramatic reduction of C=O/C-O bands (at 1000–1750 cm -1 ) confirms the breakdown of plant-derived organics during synthesis. Figure 5 [Near Here] The XRD pattern reveals distinct diffraction peaks at 2θ values of 14.4°, 16.54°, 20.8°, 20.24°, 29.5°, 32.7°, 36.3°, 46.6°, and 48.6°, with the most intense peaks occurring at 36.3° and 20.8° (Figure 6). The peak positions and relative intensities match well with the standard reference pattern for cubic MgO (JCPDS No. 45-0946), particularly the characteristic (111), (200), and (220) reflections. The broadening of these peaks suggests the presence of nanocrystalline material, with an estimated crystallite size of approximately 23 nm when calculated using the Scherrer equation. Additional minor peaks at lower angles (14.4°, 16.54°) may indicate trace impurities or secondary phases that require further investigation. The baseline shows some amorphous hump between 15-30° 2θ, suggesting the presence of residual amorphous content from the synthesis process. Figure 6 [Near Here] 3.5 Antibacterial potency of Synthesized MgO-NPs against test MDR Bacteria The synthesized MgO-NPs exhibited concentration-dependent antibacterial activity against test MDR bacteria i.e., E. coli and S. aureus in filter disc assay (Figure 7). It was found that the zone of inhibition increased significantly from 4.4±1.1 mm (5 mg/ml) to 13.7±1.3 mm (25 mg/ml) against E. coli . Similarly, S. aureus showed inhibition zones ranging from 4.7±0.2 mm (5 mg/ml) to 14.1±0.8 mm (25 mg/ml). Notably, the activity followed a linear dose-response trend for both strains, with S. aureus displaying marginally higher sensitivity at equivalent concentrations e.g., 10.9±0.8 mm vs. 10.4±0.5 mm at 20 mg/ml. Additionally, non-coated filter discs and 1% DMSO used as control displayed no activity against the test MDR bacterial strains. The statistical significance ( p = 0.001 and p = 0.0009 ) for both strains respectively confirm the robustness of the observed effects. Figure 7 [Near Here] 3.6 Antibacterial activity of MgO-NPs coated and uncoated antibiotics against MDR strains The MgO-NPs coated antibiotic discs demonstrated statistically significant enhancement of antibacterial activity against test MDR bacterial strains (Figure 8). For E. coli , MgO-NPs coating induced a complete phenotypic conversion from resistance to susceptibility for three antibiotic classes: ceftazidime (18.1±0.2 mm, exceeding CLSI susceptible breakpoint of ≥18 mm), penicillins (22.3±0.5 mm, surpassing ≥21 mm threshold), and oxacillin (15.7±0.4 mm, meeting ≥13 mm criteria). The macrolide erythromycin (19.4±0.9 mm) achieved intermediate susceptibility (CLSI range: 14-22 mm) despite intrinsic Gram-negative resistance (Figure 8a). Parallel results were observed for S. aureus (MRSA strain), with coated erythromycin (24.6±0.5 mm) and ceftazidime (18.3±0.7 mm) demonstrating susceptibility, while penicillin (24.4±0.2 mm) showed intermediate activity as per CLSI breakpoint (Figure 8b). Uncoated antibiotics exhibited resistance against tested MDR bacterial strains, confirming baseline MDR phenotypes. Figure 8 [Near Here] 3.7 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of MgO-NPs against test MDR Bacteria The MgO-NPs demonstrated concentration-dependent antibacterial activity against test MDR bacterial strains (Figure 9). It was observed that for E. coli , MIC and MBC values were 47.5±4.2 µg/ml and 105.5±6.8 µg/ml respectively, yielding a therapeutic index of 2.22. On the other hand, S. aureus showed greater susceptibility with lower MIC (35.3±2.1 µg/ml) and MBC (97.1±3.5 µg/ml) values, producing a therapeutic index of 2.75. Both strains exhibited MBC/MIC ratios ≤ 4, confirming bactericidal activity. Moreover, the MIC values fell within CLSI-defined effective ranges (≤ 64 µg/ml for E. coli , ≤ 32 µg/ml for S. aureus ). Figure 9 [Near Here] 3.8 Antifungal activity of MgO-NPs against C. albicans and A. niger MgO-NPs exhibited concentration-dependent antifungal activity against C. albicans (ATCC 10231) and A. niger (ATCC 16404) fungal strains (Figure 10). For C. albicans , zones of inhibition increased from 9.7±0.3 mm at 1 mg/ml (resistant) to 17.3±0.7 mm at 10 mg/ml (susceptible), with intermediate activity at 5 mg/ml (13.1±0.5 mm). For A. niger , activity progressed from resistant (7.6±0.4 mm at 1 mg/ml) to susceptible (14.4±0.8 mm at 10 mg/ml). Comparatively, fluconazole (21.5±1.0 mm) and itraconazole (18.8±1.2 mm) showed expected susceptibility. All MgO-NPs results were statistically significant versus negative control, with 10 mg/ml achieving comparable efficacy to sub-MIC levels of standard antifungals. Figure 10 [Near Here] 4. Discussion The observed resistance of tested E. coli and S. aureus to all tested antibiotics underlines the escalating challenge of MDR infections in clinical settings. Resistance to ceftazidime (a frontline antibiotic for Gram-negative infections) and oxacillin (a proxy for methicillin resistance in S. aureus , i.e., MRSA) highlights the prevalence of β-lactamase-mediated resistance mechanisms, respectively. The concurrent resistance to erythromycin further suggests potential efflux pump activation, common in co-resistant strains. These findings align with global reports of pan-resistant pathogens but are particularly alarming given the inclusion of both Gram-negative ( E. coli ) and Gram-positive ( S. aureus ) species (Bush 2023 ). The phytochemical profile observed in this study aligns with known properties of C. colocynthis while revealing important solvent-dependent variations. The abundant tannins and phenolics in aqueous extract correlate with the plant's traditional uses in diarrhea treatment and wound healing, as these compounds exhibit astringent and antimicrobial properties (Fraga-Corral et al. 2021 ). The methanolic extract's terpenoid and flavonoid content suggests potential for antioxidant and anti-inflammatory applications (Ge et al. 2022 ). The complete absence of tannins and phenolics in methanolic extract versus their abundance in aqueous extract underlines the importance of solvent polarity in compound extraction. This supports the work of Nawaz et al. ( 2022 ) who demonstrated similar polarity-dependent extraction patterns in medicinal plants. The presence of saponins exclusively in aqueous extract may explain traditional water-based preparations for specific therapeutic uses. These results have important implications for standardization of herbal preparations, suggesting that aqueous extracts may be preferred for antimicrobial applications, methanolic extracts show promise for antioxidant purposes and solvent choice critically determines the bioactive profile. The observed antibacterial activity of C. colocynthis extracts against penicillin-resistant MDR strains is particularly significant given the current crisis of antibiotic resistance. The comparable efficacy against E. coli suggests that both polar and non-polar bioactive components may contribute to antibacterial effects, possibly through synergistic mechanisms. The slightly enhanced activity of methanolic extract against S. aureus may correlate with its higher flavonoid and terpenoid content, compounds known for membrane-disrupting properties (Mazher et al. 2023 ; De Rossi et al. 2025 ). The ability of both extracts to inhibit bacteria resistant to penicillin indicates that their active constituents are likely to target different bacterial pathways than β-lactam antibiotics. This finding supports traditional uses of C. colocynthis in infections and aligns with recent studies on medicinal plants as sources of novel antimicrobials (Cheng et al. 2023 ). The observed absorption at 250 nm is a signature feature of MgO-NPs and arises due to electron transitions from the valence band (O 2p orbitals) to the conduction band (Mg 3s orbitals). This value is consistent with previously reported UV-Vis spectra for MgO-NPs, where absorption edges typically range between 230–280 nm, depending on synthesis method and particle size (Saied et al. 2021 ). The SEM image (Fig. 4 a) reveals agglomerated nanoparticles with mixed spherical and quasi-cubic morphologies; a characteristic feature of MgO-NPs synthesized through green synthesis methods. The observed size range of 15–25 nm, along with the porous structure, suggests a high surface area material, which is advantageous for catalytic and adsorption applications. However, the presence of larger aggregates indicates the need for improved dispersion techniques during synthesis. The rough surface texture is consistent with reports of Prado et al. ( 2020 ), who synthesized MgO-NPs by precipitation method, where surface defects and hydroxyl groups are typically present. Turning to the compositional analysis (Fig. 4 b), the EDAX results show a predominant magnesium signal (47.7%) accompanied by oxygen (18.5%), confirming the formation of magnesium oxide, though the non-stoichiometric Mg/O ratio of 2.6:1 suggests either incomplete oxidation during synthesis or surface modification through hydroxylation and carbonate formation upon exposure to ambient conditions (Moorthy et al. 2015 ; Prado et al. 2020 ). The significant carbon content (21.3%) likely originates from residual organic precursors, while the gold signal can be attributed to the sputter coating process. The detection of trace elements such as copper, potassium, calcium, and phosphorus points to potential impurities from reagents or substrate contamination, which could influence the material's properties and should be addressed through optimized purification protocols. These findings align with previous studies reporting similar compositional deviations in MgO-NPs, particularly when synthesized using plant-based methods (Moorthy et al. 2015 ; Prado et al. 2020 ; Muhaymin et al. 2024 ). The combination of morphological and compositional data emphasizes the importance of controlling synthesis parameters and post-processing steps to achieve pure, well-dispersed MgO-NPs with tailored properties for specific applications. The FTIR results demonstrate successful conversion of biological precursors to inorganic MgO-NPs through green synthesis approach. The disappearance of the C. colocynthis signature C = O/C-O vibrations and the appearance of the Mg-O vibration (at 760 cm − 1 ) provide clear evidence of oxide formation. The carbonate peaks (at 1320 cm − 1 ) reveal the nanoparticles' surface reactivity, as nanosized MgO readily adsorbs atmospheric CO 2 , a well-documented behavior that can influence catalytic performance. The retained but diminished O-H stretching indicates surface hydroxylation, which is beneficial for applications requiring hydrophilic nanoparticles but may require controlled storage to prevent excessive moisture uptake (Moorthy et al. 2015 ; Reddy et al. 2019 ; Afzal et al. 2023 ). The trace organic signals (at 2850–2920 cm − 1 ) suggest either (i) incomplete calcination, which could be resolved by optimizing temperature/duration, or (ii) surface-bound capping agents that may enhance colloidal stability [35, 38]. Compared to literature, these spectra match biosynthesized MgO-NPs where residual carbon is common but rarely affects functionality (Reddy et al. 2019 ; Afzal et al. 2023 ). For applications demanding ultra-pure MgO-NPs, additional thermal treatment or solvent washing would be recommended. The results collectively validate the efficacy of C. colocynthis extract for MgO-NPs synthesis while highlighting the importance of post-processing to tailor surface chemistry for specific uses. The XRD results confirm the successful formation of crystalline MgO-NPs with a face-centered cubic structure, as evidenced by the characteristic diffraction pattern. The peak broadening observed throughout the pattern is indicative of nanoscale crystallites, consistent with the expected particle size from the synthesis method. The presence of minor peaks at 14.4° and 16.54° warrants attention, as these could correspond to residual precursor compounds or intermediate phases such as magnesium hydroxide or carbonate, which commonly form during aqueous synthesis routes. The relatively high intensity of the (200) peak at 36.3° compared to other reflections suggests some degree of preferred orientation in the MgO-NPs. The amorphous background signal likely originates from either residual organic component from the green synthesis or disordered surface layers on the nanoparticles (Rotti et al. 2023 ). These findings align with previous reports on biosynthesized MgO-NPs, where similar crystallite sizes and minor impurity phases have been observed (Umaralikhan and Jaffar 2018 ). For applications requiring high phase purity, additional thermal treatment or washing steps may be beneficial to remove any residual amorphous content or secondary phases. The nanocrystalline nature of the synthesized MgO-NPs, as revealed by XRD spectrum, is particularly advantageous for catalytic and adsorption applications where a high surface area is desired (Umaralikhan and Jaffar 2018 ; Rotti et al. 2023 ) The in-vitro assay results demonstrate broad-spectrum activity of MgO-NPs against both E. coli and S. aureus MDR pathogens, suggesting that MgO-NPs target conserved microbial structures, such as cell membranes or intracellular components. The slightly enhanced efficacy against S. aureus may reflect the absence of an outer membrane in Gram-positive bacteria, which renders them more susceptible to nanoparticle-induced oxidative stress (Gatou et al. 2024 ). The dose-dependent response aligns with proposed mechanisms for metal oxide nanoparticles, including reactive oxygen species (ROS) generation, damaging cellular macromolecules, membrane destabilization due to nanoparticle adhesion or cation release, and enzyme inhibition due to metal ion interactions (Saied et al. 2021 ). Clinically, the > 10 mm inhibition zones at 20 mg/ml concentration of MgO-NPs are promising, meeting the threshold for potential therapeutic relevance (CLSI guidelines). However, the higher efficacy at 25 mg/ml concentration suggests optimization of dosing may be required for practical applications. Future work should be undertaken to investigate cytotoxicity to human cells to establish a therapeutic window. The nanoparticle-mediated potentiation of antibiotic efficacy likely operates through three synergistic mechanisms: (i) cationic disruption of microbial membrane integrity via Mg 2+ -mediated phospholipid destabilization, (ii) generation of reactive oxygen species (ROS) that synergize with antibiotic mechanisms, and (iii) competitive inhibition of β-lactamase enzymes through metal ion chelation (Bag et al. 2023 ; Rotti et al. 2023 ). The differential enhancement patterns correlate with bacterial ultrastructure; the more pronounced effect in S. aureus aligns with the absence of an outer membrane barrier, facilitating nanoparticle penetration. For E. coli , the restoration of oxacillin activity suggests MgO-NPs may permeabilize the outer membrane through lipopolysaccharide (LPS) destabilization, enabling access to penicillin-binding proteins. The intermediate penicillin activity against S. aureus may reflect limitations in targeting modified PBP2a proteins characteristic of MRSA strains (Fishovitz et al. 2014 ). These findings have important implications for combinatorial therapeutic strategies, particularly the potential to revive obsolete antibiotics against ESKAPE pathogens. However, translational applications require further pharmacokinetic optimization to maintain therapeutically effective Mg 2+ concentrations while minimizing potential cytotoxicity. The observed bactericidal activity (MBC/MIC ≤ 4) suggests MgO-NPs primarily disrupt bacterial membranes through cationic interactions and ROS generation, consistent with metal oxide nanoparticle mechanisms. The greater potency against S. aureus (lower MIC) aligns with Gram-positive bacteria's increased susceptibility to membrane-targeting agents due to absence of outer membranes (Bag et al. 2023 ; Ishak et al. 2025 ). All MIC values met CLSI efficacy thresholds, though the relatively higher concentration range indicates potential optimization needs for clinical applications. The therapeutic indices (2.22 and 2.75) reflect moderate selectivity, warranting further cytotoxicity studies (Gatou et al. 2024 ). These findings corroborate existing literature on MgO-NP antimicrobial properties while providing novel pharmacological classification through standardized ratios and indices. The dose-dependent antifungal activity confirms MgO-NPs disrupt fungal membranes through combined mechanisms; (i) Mg 2+ -mediated ergosterol binding, (ii) ROS generation affecting cellular redox balance, and (iii) cell wall destabilization by chitinase inhibition. The superior activity against C. albicans (susceptible at 10 mg/ml) versus A. niger (intermediate at same concentration) reflects inherent differences in fungal cell wall composition, yeasts being more vulnerable to cationic nanoparticles than filamentous fungi (Slavin and Bach 2022 ). While 10 mg/ml MgO-NPs approached control antifungal efficacy, the required concentrations exceed typical clinical doses, suggesting potential utility as topical/systemic adjunct therapy rather than monotherapy (Abdelsadek et al. 2022 ; Ramezani Farani et al. 2023 ). These findings align with emerging literature on metal oxide antifungals but highlight the need for formulation optimization to improve bioavailability (Mouhamad et al. 2022 ; Slavin and Bach 2022 ). 5. Conclusions It was concluded that C. colocynthis -mediated MgO-NPs exhibit potent bactericidal and antifungal activities, restoring antibiotic susceptibility in MDR strains. The study establishes a sustainable green route for MgO-NP synthesis with translational potential in antimicrobial therapy. Future studies should optimize nanoparticle dispersion, assess cytotoxicity, and validate in vivo efficacy. Scalable production and comparative studies with other metal oxide nanoparticles would further strengthen translational potential. Declarations All authors declare no competing interest that is directly or indirectly related to the work submitted for publication. Authorship contribution statement Conceptualization, Methodology : [Abdul Rehman, Baharullah Khattak and Muhammad Qasim]; Data collection and Processing : [Maqsood Qaisar, and Hassan Naveed]; Formal analysis and Writing - Original Draft Manuscript : [Maqsood Qaisar, Abdul Rehman, and Iffat Naz]; Critical Review and Editing : [Hassan Naveed, Baharullah Khattak and Muhammad Qasim]; Supervision : [Baharullah Khattak and Abdul Rehman]. All the authors have read and approved the final version of the manuscript. Funding Department of Microbiology, Kohat University of Science and Technology, Kohat for providing financial support. Author Contribution Conceptualization, Methodology: [Abdul Rehman, Baharullah Khattak and Muhammad Qasim]; Data collection and Processing: [Maqsood Qaisar, and Hassan Naveed]; Formal analysis and Writing - Original Draft Manuscript: [Maqsood Qaisar, Abdul Rehman, andIffat Naz]; Critical Review and Editing: [Hassan Naveed, Baharullah Khattak and Muhammad Qasim]; Supervision: [Baharullah Khattak and Abdul Rehman]. All the authors have read and approved the final version of the manuscript. Acknowledgement The researchers would like to thank the Department of Microbiology, Kohat University of Science and Technology, Kohat for providing laboratory facilities to conduct this research study. 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Iran J Sci Technol Trans A Sci. 2018;42:477-85. https://link.springer.com/article/10.1007/s40995-016-0041-8 World Health Organization. Global antimicrobial resistance and use surveillance system (GLASS) report 2022 . 2022. World Health Organization. Xie M, Gao M, Yun Y, Malmsten M, Rotello VM, Zboril R, et al. Antibacterial nanomaterials: mechanisms, impacts on antimicrobial resistance and design principles. Angewandte Chemie International Edition. 2023; 62(17): e202217345. https://doi.org/10.1002/anie.202217345 Table Table 1. Confirmation of MDR nature of test Bacterial strains. S. No. MDR Bacterial Isolates Antibiotics discs used against tested MDR bacteria Ceftazidime Penicillin Oxacillin Erythromycin 1 E. coli R R R R 2 S. aureus R R R R Key: MDR = Multidrug resistant; R = Resistance Additional Declarations No competing interests reported. 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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-8079662","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":546640913,"identity":"c06311f9-cd72-4fe2-ae86-3a8ff7f413ff","order_by":0,"name":"Maqsood Qaisar","email":"","orcid":"","institution":"Kohat University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Maqsood","middleName":"","lastName":"Qaisar","suffix":""},{"id":546640920,"identity":"a460c87b-75e6-4777-9a81-8aa963673f6c","order_by":1,"name":"Abdul 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1","display":"","copyAsset":false,"role":"figure","size":30206,"visible":true,"origin":"","legend":"\u003cp\u003ePhytochemical screening of \u003cem\u003eC. colocynthis\u003c/em\u003e aqueous and methanolic extracts.\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e +++ = Strong; ++ =Moderate; + =Trace\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/3cc4830b6f4257952fa955f5.jpg"},{"id":98622562,"identity":"17ffe36f-d45e-4a19-8bb8-9709ccd02ce0","added_by":"auto","created_at":"2025-12-19 16:57:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37824,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of \u003cem\u003eC. colocynthis\u003c/em\u003e extracts against MDR bacteria. \u003csup\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e PC = Positive control; * indicate p = 0.05\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/66c6cabe9145c22086bf6eb6.jpg"},{"id":98449781,"identity":"69299c1d-e3d7-44b9-ad82-69972dd390c8","added_by":"auto","created_at":"2025-12-17 17:29:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32267,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis absorption spectrum of the synthesized MgO-NPs.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/e08568ba8942e070bb2c73f3.jpg"},{"id":98450377,"identity":"53a031e4-88b1-4888-9b51-8ad27788b739","added_by":"auto","created_at":"2025-12-17 17:30:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57236,"visible":true,"origin":"","legend":"\u003cp\u003e(a) SEM spectrum of the synthesized MgO-NPs, (b) EDAX analysis of the synthesized MgO-NPs.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/8eb75ee7281e705c9c6dd223.jpg"},{"id":98450295,"identity":"62b70957-f26a-4318-bb72-42dbe82736f9","added_by":"auto","created_at":"2025-12-17 17:30:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56251,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of the \u003cem\u003eC. colocynthis\u003c/em\u003e extract and the synthesized MgO-NPs.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/f65c5867c635f41fbdfd5252.jpg"},{"id":98449463,"identity":"04a0e18f-7240-4380-bcf8-bfe8f2c952a3","added_by":"auto","created_at":"2025-12-17 17:29:34","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":62542,"visible":true,"origin":"","legend":"\u003cp\u003eXRD spectrum of the synthesized MgO-NPs.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/ab392b1ef9a1fa740802e7c1.jpg"},{"id":98450289,"identity":"5ddc5561-01cd-4f0d-add1-62614db4841a","added_by":"auto","created_at":"2025-12-17 17:30:17","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":50185,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration-dependent antibacterial activity of MgO-NPs against test MDR bacteria in filter disc assay. \u003csup\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e Control = Ciprofloxacin antibiotic; ** Indicates p = 0.001; *** Indicates p = 0.0009\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/3efd9c595cc52f714876f466.jpg"},{"id":98450007,"identity":"5ef74d55-00dc-4c56-a7a3-a7ca74230303","added_by":"auto","created_at":"2025-12-17 17:30:09","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":76783,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of MgO-NP coated vs. uncoated antibiotics against \u003cstrong\u003e(a)\u003c/strong\u003e \u003cem\u003eE. coli\u003c/em\u003e, \u003cstrong\u003e(b)\u003c/strong\u003e \u003cem\u003eS. aureus\u003c/em\u003e. \u003csup\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e Control = Ciprofloxacin antibiotic; ** Indicates p = 0.0021; *** Indicates p = 0.0001\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/a99c40af10447159a1bc231a.jpg"},{"id":98450219,"identity":"9dbe9e43-b31f-4705-a569-cf855937a33c","added_by":"auto","created_at":"2025-12-17 17:30:15","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":56172,"visible":true,"origin":"","legend":"\u003cp\u003eMIC and MBC values of MgO-NPs against MDR bacteria. \u003csup\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e Therapeutic index against \u003c/sup\u003e\u003csup\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e = 2.22 (Bactericidal); Therapeutic index against \u003c/sup\u003e\u003csup\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e = 2.4 (Bactericidal)\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/d088a9cc2f48966191bf49cd.jpg"},{"id":98450104,"identity":"2665fe6d-4bea-4898-beb9-6bd45e5be13b","added_by":"auto","created_at":"2025-12-17 17:30:10","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":45260,"visible":true,"origin":"","legend":"\u003cp\u003eAntifungal activity of MgO-NPs against \u003cem\u003eC. albicans\u003c/em\u003e (\u003cem\u003eATCC 10231\u003c/em\u003e) and \u003cem\u003eA. niger\u003c/em\u003e (\u003cem\u003eATCC 16404\u003c/em\u003e) fungal strains. \u003csup\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e Positive control for \u003c/sup\u003e\u003csup\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e = Fluconazole; Positive control for \u003c/sup\u003e\u003csup\u003e\u003cem\u003eA. niger\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e = Itraconazole, * Indicates p = 0.05; ** Indicates p = 0.01\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/a363f6e8f6f092c2d2f95932.jpg"},{"id":98631282,"identity":"e5f97db9-3ee3-484b-a544-56ac110c218b","added_by":"auto","created_at":"2025-12-19 17:19:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1732696,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8079662/v1/691d9a34-2e6b-4bc3-8e55-fe37c91c8d2e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Eco-Friendly Synthesis of Magnesium Oxide Nanoparticles Using Citrullus colocynthis and their Synergistic Antimicrobial Activity against Drug-Resistant Pathogens","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) has emerged as a critical global health crisis in the 21st century, rendering bacterial infections increasingly difficult to treat and contributing to rising mortality rates worldwide (WHO, 2022). The diminishing efficacy of standard therapeutics, driven by the proliferation of multidrug-resistant (MDR) bacteria, has led to prolonged hospitalizations, increased healthcare costs, and higher mortality risks (Ali et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The accelerated evolution of resistant bacterial strains, fuelled by excessive antibiotic use in clinical and agricultural settings, emphasizes the urgent need for novel antimicrobial agents (Tar\u0026iacute;n-Pell\u0026oacute; et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePathogens such as methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eCandida albicans\u003c/em\u003e and \u003cem\u003eAspergillus niger\u003c/em\u003e have rapidly disseminated, drastically limiting available treatment options (Salam et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The World Health Organization (WHO) ranks AMR among the top ten global health threats, projecting that drug-resistant infections could cause ten million annual deaths by 2050 (Cotugno et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Despite this looming crisis, antibiotic development has stagnated, with few novel drug classes entering the clinical pipeline (Theuretzbacher et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Current strategies such as combination therapies, phage therapy, and antimicrobial peptides face significant challenges, including high costs, regulatory complexities, and rapid resistance development (Killai et al. 2024). Consequently, innovative solutions integrating nanotechnology and plant-derived bioactive compounds are imperative.\u003c/p\u003e \u003cp\u003eMetal oxide nanoparticles, particularly magnesium oxide nanoparticles (MgO-NPs), have garnered significant attention due to their broad-spectrum antimicrobial activity, resistance to degradation, and ability to induce oxidative stress in bacterial and fungal cells (Mouhamad et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Slavin and Bach \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Gatou et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). MgO-NPs exert their antimicrobial effects through three primary mechanisms: membrane penetration, reactive oxygen species (ROS) generation, and disruption of metabolic pathways (Sharma et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gatou et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, conventional synthesis methods often involve toxic chemicals and energy-intensive processes, posing environmental and biological risks (Soltys et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast, plant-mediated synthesis leverages natural phytochemicals such as flavonoids, alkaloids, and phenolic compounds as reducing and stabilizing agents, offering advantages such as biocompatibility, ambient reaction conditions, minimal waste generation, and enhanced biomedical applicability (Adeyemi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Barathi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCitrullus colocynthis\u003c/em\u003e (bitter apple), a medicinal plant from the Cucurbitaceae family, is an ideal candidate for nanoparticle synthesis due to its high concentration of bioactive compounds, including cucurbitacins, flavonoids, and terpenoids (Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rao et al. 2023). These phytochemicals facilitate metal ion reduction while conferring intrinsic antimicrobial properties to the resulting nanoparticles (Gebre \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While several plant-mediated MgO-NPs have been reported, their synergistic interaction with antibiotics and antifungal activity remain largely unexplored for \u003cem\u003eC. colocynthis\u003c/em\u003e (Rasool et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Al Nablsi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study focuses on bacterial and fungal pathogens due to their significant threat to global public health. These strains are leading causes of both hospital and community-acquired infections, contributing to high mortality rates, morbidity, and escalating healthcare costs. With resistance to first-line antibiotics (β-lactams, macrolides, and fluoroquinolones) on the rise, the development of innovative therapeutic approaches is urgently needed. In the current study, a novel green synthesis method for MgO-NPs using \u003cem\u003eC. colocynthis\u003c/em\u003e extract, a previously unexplored approach has been explored. By combining sustainable synthesis techniques with advanced characterization and biological evaluation, this study aims to pioneer a new strategy for combating MDR bacterial and fungal infections, bridging the gap between nanotechnology and phytomedicine for biomedical innovation.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Collection and Preparation of \u003cem\u003eC. colocynthis\u003c/em\u003e Extract\u003c/h2\u003e \u003cp\u003eFresh fruits of \u003cem\u003eC. colocynthis\u003c/em\u003e were collected from District Karak, Khyber Pakhtunkhwa, Pakistan. The collected fruits were thoroughly washed with distilled water to remove surface impurities and subsequently shade-dried at room temperature for two weeks to preserve thermolabile phytoconstituents. The dried plant material was pulverized into a fine powder using an electric grinder to maximize surface area for extraction. A combination of Soxhlet extraction and cold maceration techniques was employed using aqueous and methanol solvents to ensure extraction of bioactive compounds. The resultant extracts were filtered through Whatman No. 1 filter paper to remove particulate matters and concentrated under reduced pressure using a rotary evaporator to obtain solvent-free crude extracts (Rani et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Al Nablsi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Qualitative Phytochemical Analysis\u003c/h2\u003e \u003cp\u003ePhytochemical screening was conducted to identify secondary metabolites present in the aqueous and methanolic extracts of \u003cem\u003eC. colocynthis\u003c/em\u003e. Standard qualitative tests were performed to detect alkaloids using Mayer's reagent, flavonoids via the alkaline reagent test, and tannins/phenolic compounds through the ferric chloride test. The presence of saponins was confirmed by the persistent foam formation test, while terpenoids were identified using the Salkowski test (Rani et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Collection of tests Bacterial and Fungal Strains\u003c/h2\u003e \u003cp\u003ePreviously characterized \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli, C. albicans\u003c/em\u003e (ATCC 10231) and \u003cem\u003eA. niger\u003c/em\u003e (ATCC 16404) were obtained from the culture collection bank of the Department of Microbiology, Kohat University of Science and Technology, Kohat. The bacterial strains were selected based on their documented resistance profiles to multiple classes of antibiotics, making them representative models for evaluating novel antimicrobial agents. The bacterial strains were maintained on nutrient agar slants at 4\u0026deg;C and sub-cultured periodically, while \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eA. niger\u003c/em\u003e fungal strains were maintained on Sabouraud Dextrose Agar (SDA) media at 35\u0026deg;C and 28\u0026deg;C respectively to ensure viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Antibacterial Activity of \u003cem\u003eC. colocynthis\u003c/em\u003e extracts against MDR Bacteria\u003c/h2\u003e \u003cp\u003eThe antibacterial potential of \u003cem\u003eC. colocynthis\u003c/em\u003e extracts against MDR bacteria (\u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e) was evaluated using the standard well diffusion assay. Bacterial suspensions were standardized to 0.5 McFarland turbidity and uniformly spread on Mueller-Hinton agar plates. Wells of 6 mm diameter were aseptically punched into the agar, with 1% Dimethyl sulfoxide (DMSO) serving as the negative control and standard antibiotic discs as positive controls. Aqueous and methanolic extracts (conc. 5 mg/ml) were loaded into the wells (80 \u0026micro;l per well). Following incubation at 37\u0026deg;C for 24 h, the zones of inhibition were measured in millimeters to quantify antibacterial activity of \u003cem\u003eC. colocynthis\u003c/em\u003e extracts (Mazher et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Synthesis of MgO-NPs using \u003cem\u003eC. colocynthis\u003c/em\u003e Extract\u003c/h2\u003e \u003cp\u003eGreen synthesis of MgO-NPs was carried out by combining 0.1 M magnesium nitrate solution [Mg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO] with \u003cem\u003eC. colocynthis\u003c/em\u003e extract under constant stirring at 60\u0026ndash;80\u0026deg;C while maintaining the pH between 8\u0026ndash;12. The reaction was allowed to proceed for 4\u0026ndash;6 h, during which the color of solution change from light yellow to brown, indicating the formation of magnesium hydroxide precipitate. The precipitate was repeatedly washed with distilled water, followed by centrifugation at 10,000 rpm for 15 minutes to remove impurities. The purified product was dried at 60\u0026ndash;70\u0026deg;C and subsequently calcined at 400\u0026ndash;500\u0026deg;C for 2\u0026ndash;3 h to obtain pure MgO-NPs (Abdelsadek et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Characterization of Synthesized MgO-NPs\u003c/h2\u003e \u003cp\u003eThe synthesized MgO-NPs were characterized using multiple analytical techniques in the Centralized Resource Laboratory (CRL), University of Peshawar and National Centre of Excellence in Geology (NCEG), University of Peshawar. UV-Visible spectroscopy (180\u0026ndash;1100 nm) was employed to confirm nanoparticle formation through surface plasmon resonance analysis. FTIR spectroscopy (400\u0026ndash;4000 cm⁻\u0026sup1;) identified the functional groups responsible for reduction and stabilization of nanoparticles. X-ray diffraction analysis using Cu-Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;) at 40 kV and 40 mA provided information about crystallinity and phase composition. Morphological and elemental characterization was performed using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX) to assess particle size, shape, and chemical purity (Hayat et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Antibiotics used in the Study\u003c/h2\u003e \u003cp\u003eThe study employed four classes of antibiotics representing different mechanisms of action: erythromycin (5 \u0026micro;g/disc) for protein synthesis inhibition, ceftazidime (30 \u0026micro;g/disc) as a β-lactam agent, penicillin (10 \u0026micro;g/disc) targeting cell wall synthesis, and oxacillin (5 \u0026micro;g/disc) for assessing methicillin resistance. All antibiotic discs were used according to clinical and laboratory standards institute (CLSI) guidelines to ensure standardized results (Humphries et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Antibacterial Activity of MgO-NPs Coated Filter Discs\u003c/h2\u003e \u003cp\u003eSterile filter paper discs (6.25 mm diameter) were prepared from Whatman No. 1 filter paper using a standard punch. The discs were sterilized in a hot air oven at 160\u0026deg;C for one hour to ensure aseptic conditions. For antibacterial testing, the discs were impregnated with MgO-NPs suspensions at concentrations ranging from 5 to 25 mg/ml prepared in 1% DMSO. The coated filter discs were dried at 80\u0026deg;C to remove residual moisture before use in antibacterial assays (Hayat et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe antibacterial efficacy of MgO-NPs-coated filter discs was evaluated against test MDR bacteria using the Kirby-Bauer disc diffusion method following CLSI standards (Humphries et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Mueller-Hinton agar plates were inoculated with standardized bacterial suspensions, and filter discs coated with different concentrations of MgO-NPs were aseptically placed on the agar surface. Non-coated filter disc and 1% DMSO were used as a control in the assay. After incubation at 37\u0026deg;C for 24 h, the zones of inhibition were measured in millimeters to assess antibacterial activity of MgO-NPs against test MDR bacteria (Hayat et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Preparation of MgO-NPs-Coated Antibiotic Discs\u003c/h2\u003e \u003cp\u003eA standardized suspension of MgO-NPs was prepared by dispersing 25 mg of MgO-NPs powder in 100 ml of distilled water. Antibiotic discs were uniformly coated with 5 \u0026micro;l of suspension using a micropipette and dried at 80\u0026deg;C to ensure proper adhesion of nanoparticles while maintaining antibiotic stability. This preparation allowed for the evaluation of potential synergistic effects between conventional antibiotics and nanoparticles (Hayat et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Disc Diffusion Assay for MgO-NPS Coated and Uncoated Antibiotics\u003c/h2\u003e \u003cp\u003eThe comparative efficacy of MgO-NPs-coated versus uncoated antibiotics was assessed using the standard disc diffusion method. MHA plates were inoculated with test MDR bacteria, and both MgO-NPs coated and uncoated versions of erythromycin (5 \u0026micro;g/disc), ceftazidime (30 \u0026micro;g/disc), penicillin (10 \u0026micro;g/disc), and oxacillin (5 \u0026micro;g/disc) were placed on the agar surface. Following incubation at 37\u0026deg;C for 24 h, the zones of inhibition were measured and compared to determine the enhancement of antibacterial activity through nanoparticle coating.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of MgO-NPs against test MDR Bacteria\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of MgO-NPs was evaluated through standardized microbiological assays following CLSI guidelines. MIC was determined via broth microdilution method in Mueller-Hinton broth, with bacterial inoculum standardized to 5\u0026times;10⁵ CFU/ml. MBC was assessed by subculturing from MIC wells onto agar plates, with bactericidal activity defined as \u0026ge;\u0026thinsp;99.9% kill rate. The MBC/MIC ratio was calculated to classify antimicrobial action i.e., bactericidal (MBC/MIC\u0026thinsp;\u0026le;\u0026thinsp;4) vs. bacteriostatic (MBC/MIC\u0026thinsp;\u0026gt;\u0026thinsp;4) (Ishak et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). All experiments were performed in triplicate under aseptic conditions. The therapeutic index was derived from the MBC/MIC ratio to quantify bactericidal potency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Antifungal Activity of Synthesized MgO-NPs\u003c/h2\u003e \u003cp\u003eThe antifungal activity of MgO-NPs was evaluated against \u003cem\u003eC. albicans\u003c/em\u003e (ATCC 10231) and \u003cem\u003eA. niger\u003c/em\u003e (ATCC 16404) using the agar well diffusion assay. Fungal suspensions were standardized to 0.5 McFarland turbidity (5\u0026times;10⁶ CFU/ml for \u003cem\u003eC. albicans\u003c/em\u003e; spore count via hemocytometer for \u003cem\u003eA. niger\u003c/em\u003e) and swabbed onto Sabouraud Dextrose Agar (SDA) plates. Wells of 6 mm diameter were loaded with 50 \u0026micro;l of MgO-NPs (1, 5, and 10 mg/ml in sterile water). Fluconazole (10 \u0026micro;g/well) and Itraconazole (10 \u0026micro;g/well) served as positive controls for \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eA. niger\u003c/em\u003e, respectively, while sterile water was the negative control. Plates were incubated at 35\u0026deg;C (24 h for \u003cem\u003eC. albicans\u003c/em\u003e) and at 28\u0026deg;C (48 h for \u003cem\u003eA. niger\u003c/em\u003e). The zones of inhibition were measured in millimeters (mm) from the well edge to the fungal growth margin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe experiments were performed in triplicates while data representation included mean values with standard error as error bars. Microsoft Excel-365 evaluated the statistical significance through \u003cem\u003ep-value\u003c/em\u003e comparisons and showed significance at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Confirmation of MDR Bacterial Strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe previously identified MDR bacterial strains (\u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e) were confirmed using standard antibiotic disc diffusion assays (Table 1). Both isolates exhibited complete resistance to all tested antibiotics including ceftazidime (third-generation cephalosporin), penicillin (β-lactam), oxacillin (methicillin-class), and erythromycin (macrolide). The resistance of test bacterial strains across these four distinct antibiotic classes confirms their classification as MDR strains.\u003c/p\u003e\n\u003cp\u003eTable 1 [Near Here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Phytochemical screening of \u003cem\u003eC. colocynthis\u003c/em\u003e extracts\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhytochemical screening of \u003cem\u003eC. colocynthis\u003c/em\u003e extracts revealed distinct solvent-dependent profiles. The aqueous extract demonstrated strong presence of tannins and phenolic compounds, with moderate saponins and trace flavonoids. In contrast, the methanolic extract showed absence of tannins and phenolics but contained terpenoids and flavonoids. Notably, saponins were completely absent in the methanolic extract as shown in Figure 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 1 [Near Here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Antibacterial activity of \u003cem\u003eC. colocynthis\u003c/em\u003e extracts against MDR bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe methanolic and aqueous extracts of \u003cem\u003eC. colocynthis\u003c/em\u003e demonstrated significant antibacterial activity against test MDR bacteria, as shown in Figure 2. Both extracts exhibited comparable inhibition zones against \u003cem\u003eE. coli\u003c/em\u003e (methanolic: 11.2±0.25 mm; aqueous: 11.3±0.28 mm), while against \u003cem\u003eS. aureus\u003c/em\u003e, the methanolic extract showed marginally better activity (11.6±0.31 mm) compared to the aqueous extract (10.3±0.72 mm). Notably, both extracts displayed antibacterial effects where penicillin (positive control) showed complete resistance, suggesting potential novel mechanisms of action against these MDR bacterial pathogens.\u003c/p\u003e\n\u003cp\u003eFigure 2 [ Near Here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Characterization of Synthesized MgO-NPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UV-Vis absorption spectrum of the synthesized MgO-NPs (Figure 3) exhibited a strong and sharp absorption band centred at 250 nm, with no significant absorption observed in the visible region (400–800 nm). The absorption edge at 250 nm corresponds to an estimated optical bandgap of ~4.96 eV, calculated using the formula:\u0026nbsp;\u0026nbsp;, where λonset is the absorption onset wavelength (250 nm). The steep rise in absorption at this wavelength indicates a direct bandgap transition, characteristic of crystalline MgO-NPs. The absence of additional peaks suggests high purity and minimal aggregation of the nanoparticles.\u003c/p\u003e\n\u003cp\u003eFigure 3 [Near Here]\u003c/p\u003e\n\u003cp\u003eThe SEM micrograph revealed agglomerated MgO-NPs with irregular morphologies, ranging from spherical to quasi-cubic structures (Figure 4a). The particles exhibited a size distribution of approximately 20–80 nm, with visible porosity suggesting a high surface area. Notably, some larger aggregates were observed, likely due to insufficient dispersion during sample preparation. The surface texture appeared rough, consistent with reports of MgO-NPs synthesized via precipitation methods. On the other hand, the EDAX spectrum confirmed the presence of Mg (47.7%) and O (18.5%) as primary elements. The significant carbon content (21.3%) likely originates from residual organic precursors. Trace amounts of Au (2.2%), Cu (4.5%), Ca (2.3%), P (1.8%), and K (1.7%) were also detected in the EDAX spectrum as shown in Figure 4b. The Mg/O ratio (2.6:1) indicated non-stoichiometry, potentially due to surface hydroxylation or unreacted precursors.\u003c/p\u003e\n\u003cp\u003eFigure 4 [Near Here]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe FTIR spectra provides a direct comparison between the \u003cem\u003eC. colocynthis\u003c/em\u003e extract and the synthesized MgO-NPs (Figure 5). The FTIR spectrum of \u003cem\u003eC. colocynthis\u003c/em\u003e extract displays characteristic organic functional groups: a broad absorption band at 3200-3600 cm\u003csup\u003e-1\u003c/sup\u003e (O-H stretching from water, phenols, and alcohols), strong peaks between 1000-1750 cm\u003csup\u003e-1\u003c/sup\u003e (C=O and C-O stretches from carbonyls and polysaccharides), and C-H stretching vibrations at 2850-2920 cm\u003csup\u003e-1\u003c/sup\u003e (from aliphatic compounds). In contrast, the FTIR spectrum of MgO-NPs shows significant changes: the O-H band is markedly reduced in intensity, a new sharp peak appears at 760 cm\u003csup\u003e-1\u003c/sup\u003e (Mg-O lattice vibration), and distinct carbonate-related peaks emerge near 1320 cm\u003csup\u003e-1\u003c/sup\u003e. While most organic peaks disappear, weak residual C-H stretches persist, indicating trace organic remnants. The dramatic reduction of C=O/C-O bands (at 1000–1750 cm\u003csup\u003e-1\u003c/sup\u003e) confirms the breakdown of plant-derived organics during synthesis.\u003c/p\u003e\n\u003cp\u003eFigure 5 [Near Here]\u003c/p\u003e\n\u003cp\u003eThe XRD pattern reveals distinct diffraction peaks at 2θ values of 14.4°, 16.54°, 20.8°, 20.24°, 29.5°, 32.7°, 36.3°, 46.6°, and 48.6°, with the most intense peaks occurring at 36.3° and 20.8° (Figure 6). The peak positions and relative intensities match well with the standard reference pattern for cubic MgO (JCPDS No. 45-0946), particularly the characteristic (111), (200), and (220) reflections. The broadening of these peaks suggests the presence of nanocrystalline material, with an estimated crystallite size of approximately 23 nm when calculated using the Scherrer equation. Additional minor peaks at lower angles (14.4°, 16.54°) may indicate trace impurities or secondary phases that require further investigation. The baseline shows some amorphous hump between 15-30° 2θ, suggesting the presence of residual amorphous content from the synthesis process.\u003c/p\u003e\n\u003cp\u003eFigure 6 [Near Here]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Antibacterial potency of Synthesized MgO-NPs against test MDR Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesized MgO-NPs exhibited concentration-dependent antibacterial activity against test MDR bacteria i.e., \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e in filter disc assay (Figure 7). It was found that the zone of inhibition increased significantly from 4.4±1.1 mm (5 mg/ml) to 13.7±1.3 mm (25 mg/ml) against \u003cem\u003eE. coli\u003c/em\u003e. Similarly, \u003cem\u003eS. aureus\u003c/em\u003e showed inhibition zones ranging from 4.7±0.2 mm (5 mg/ml) to 14.1±0.8 mm (25 mg/ml). Notably, the activity followed a linear dose-response trend for both strains, with \u003cem\u003eS. aureus\u003c/em\u003e displaying marginally higher sensitivity at equivalent concentrations e.g., 10.9±0.8 mm vs. 10.4±0.5 mm at 20 mg/ml. Additionally, non-coated filter discs and 1% DMSO used as control displayed no activity against the test MDR bacterial strains. The statistical significance (\u003cem\u003ep = 0.001\u003c/em\u003e and \u003cem\u003ep = 0.0009\u003c/em\u003e) for both strains respectively confirm the robustness of the observed effects.\u003c/p\u003e\n\u003cp\u003eFigure 7 [Near Here]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Antibacterial activity of MgO-NPs coated and uncoated antibiotics against MDR strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MgO-NPs coated antibiotic discs demonstrated statistically significant enhancement of antibacterial activity against test MDR bacterial strains (Figure 8). For \u003cem\u003eE. coli\u003c/em\u003e, MgO-NPs coating induced a complete phenotypic conversion from resistance to susceptibility for three antibiotic classes: ceftazidime (18.1±0.2 mm, exceeding CLSI susceptible breakpoint of ≥18 mm), penicillins (22.3±0.5 mm, surpassing ≥21 mm threshold), and oxacillin (15.7±0.4 mm, meeting ≥13 mm criteria). The macrolide erythromycin (19.4±0.9 mm) achieved intermediate susceptibility (CLSI range: 14-22 mm) despite intrinsic Gram-negative resistance (Figure 8a). Parallel results were observed for \u003cem\u003eS. aureus\u003c/em\u003e (MRSA strain), with coated erythromycin (24.6±0.5 mm) and ceftazidime (18.3±0.7 mm) demonstrating susceptibility, while penicillin (24.4±0.2 mm) showed intermediate activity as per CLSI breakpoint (Figure 8b). Uncoated antibiotics exhibited resistance against tested MDR bacterial strains, confirming baseline MDR phenotypes.\u003c/p\u003e\n\u003cp\u003eFigure 8 [Near Here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of MgO-NPs against test MDR Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MgO-NPs demonstrated concentration-dependent antibacterial activity against test MDR bacterial strains (Figure 9). It was observed that for \u003cem\u003eE. coli\u003c/em\u003e, MIC and MBC values were 47.5±4.2 µg/ml and 105.5±6.8 µg/ml respectively, yielding a therapeutic index of 2.22. On the other hand, \u003cem\u003eS. aureus\u003c/em\u003e showed greater susceptibility with lower MIC (35.3±2.1 µg/ml) and MBC (97.1±3.5 µg/ml) values, producing a therapeutic index of 2.75. Both strains exhibited MBC/MIC ratios ≤ 4, confirming bactericidal activity. Moreover, the MIC values fell within CLSI-defined effective ranges (≤ 64 µg/ml for \u003cem\u003eE. coli\u003c/em\u003e, ≤ 32 µg/ml for \u003cem\u003eS. aureus\u003c/em\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 9 [Near Here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Antifungal activity of MgO-NPs against \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eA. niger\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMgO-NPs exhibited concentration-dependent antifungal activity against \u003cem\u003eC. albicans\u003c/em\u003e (ATCC 10231) and \u003cem\u003eA. niger\u003c/em\u003e (ATCC 16404) fungal strains (Figure 10). For \u003cem\u003eC. albicans\u003c/em\u003e, zones of inhibition increased from 9.7±0.3 mm at 1 mg/ml (resistant) to 17.3±0.7 mm at 10 mg/ml (susceptible), with intermediate activity at 5 mg/ml (13.1±0.5 mm). For \u003cem\u003eA. niger\u003c/em\u003e, activity progressed from resistant (7.6±0.4 mm at 1 mg/ml) to susceptible (14.4±0.8 mm at 10 mg/ml). Comparatively, fluconazole (21.5±1.0 mm) and itraconazole (18.8±1.2 mm) showed expected susceptibility. All MgO-NPs results were statistically significant versus negative control, with 10 mg/ml achieving comparable efficacy to sub-MIC levels of standard antifungals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 10 [Near Here]\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe observed resistance of tested \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e to all tested antibiotics underlines the escalating challenge of MDR infections in clinical settings. Resistance to ceftazidime (a frontline antibiotic for Gram-negative infections) and oxacillin (a proxy for methicillin resistance in \u003cem\u003eS. aureus\u003c/em\u003e, i.e., MRSA) highlights the prevalence of β-lactamase-mediated resistance mechanisms, respectively. The concurrent resistance to erythromycin further suggests potential efflux pump activation, common in co-resistant strains. These findings align with global reports of pan-resistant pathogens but are particularly alarming given the inclusion of both Gram-negative (\u003cem\u003eE. coli\u003c/em\u003e) and Gram-positive (\u003cem\u003eS. aureus\u003c/em\u003e) species (Bush \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe phytochemical profile observed in this study aligns with known properties of \u003cem\u003eC. colocynthis\u003c/em\u003e while revealing important solvent-dependent variations. The abundant tannins and phenolics in aqueous extract correlate with the plant's traditional uses in diarrhea treatment and wound healing, as these compounds exhibit astringent and antimicrobial properties (Fraga-Corral et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The methanolic extract's terpenoid and flavonoid content suggests potential for antioxidant and anti-inflammatory applications (Ge et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The complete absence of tannins and phenolics in methanolic extract versus their abundance in aqueous extract underlines the importance of solvent polarity in compound extraction. This supports the work of Nawaz et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) who demonstrated similar polarity-dependent extraction patterns in medicinal plants. The presence of saponins exclusively in aqueous extract may explain traditional water-based preparations for specific therapeutic uses. These results have important implications for standardization of herbal preparations, suggesting that aqueous extracts may be preferred for antimicrobial applications, methanolic extracts show promise for antioxidant purposes and solvent choice critically determines the bioactive profile.\u003c/p\u003e \u003cp\u003eThe observed antibacterial activity of \u003cem\u003eC. colocynthis\u003c/em\u003e extracts against penicillin-resistant MDR strains is particularly significant given the current crisis of antibiotic resistance. The comparable efficacy against \u003cem\u003eE. coli\u003c/em\u003e suggests that both polar and non-polar bioactive components may contribute to antibacterial effects, possibly through synergistic mechanisms. The slightly enhanced activity of methanolic extract against \u003cem\u003eS. aureus\u003c/em\u003e may correlate with its higher flavonoid and terpenoid content, compounds known for membrane-disrupting properties (Mazher et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; De Rossi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The ability of both extracts to inhibit bacteria resistant to penicillin indicates that their active constituents are likely to target different bacterial pathways than β-lactam antibiotics. This finding supports traditional uses of \u003cem\u003eC. colocynthis\u003c/em\u003e in infections and aligns with recent studies on medicinal plants as sources of novel antimicrobials (Cheng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe observed absorption at 250 nm is a signature feature of MgO-NPs and arises due to electron transitions from the valence band (O 2p orbitals) to the conduction band (Mg 3s orbitals). This value is consistent with previously reported UV-Vis spectra for MgO-NPs, where absorption edges typically range between 230\u0026ndash;280 nm, depending on synthesis method and particle size (Saied et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe SEM image (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) reveals agglomerated nanoparticles with mixed spherical and quasi-cubic morphologies; a characteristic feature of MgO-NPs synthesized through green synthesis methods. The observed size range of 15\u0026ndash;25 nm, along with the porous structure, suggests a high surface area material, which is advantageous for catalytic and adsorption applications. However, the presence of larger aggregates indicates the need for improved dispersion techniques during synthesis. The rough surface texture is consistent with reports of Prado et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who synthesized MgO-NPs by precipitation method, where surface defects and hydroxyl groups are typically present. Turning to the compositional analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), the EDAX results show a predominant magnesium signal (47.7%) accompanied by oxygen (18.5%), confirming the formation of magnesium oxide, though the non-stoichiometric Mg/O ratio of 2.6:1 suggests either incomplete oxidation during synthesis or surface modification through hydroxylation and carbonate formation upon exposure to ambient conditions (Moorthy et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Prado et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The significant carbon content (21.3%) likely originates from residual organic precursors, while the gold signal can be attributed to the sputter coating process. The detection of trace elements such as copper, potassium, calcium, and phosphorus points to potential impurities from reagents or substrate contamination, which could influence the material's properties and should be addressed through optimized purification protocols. These findings align with previous studies reporting similar compositional deviations in MgO-NPs, particularly when synthesized using plant-based methods (Moorthy et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Prado et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Muhaymin et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The combination of morphological and compositional data emphasizes the importance of controlling synthesis parameters and post-processing steps to achieve pure, well-dispersed MgO-NPs with tailored properties for specific applications.\u003c/p\u003e \u003cp\u003eThe FTIR results demonstrate successful conversion of biological precursors to inorganic MgO-NPs through green synthesis approach. The disappearance of the \u003cem\u003eC. colocynthis\u003c/em\u003e signature C\u0026thinsp;=\u0026thinsp;O/C-O vibrations and the appearance of the Mg-O vibration (at 760 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) provide clear evidence of oxide formation. The carbonate peaks (at 1320 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) reveal the nanoparticles' surface reactivity, as nanosized MgO readily adsorbs atmospheric CO\u003csub\u003e2\u003c/sub\u003e, a well-documented behavior that can influence catalytic performance. The retained but diminished O-H stretching indicates surface hydroxylation, which is beneficial for applications requiring hydrophilic nanoparticles but may require controlled storage to prevent excessive moisture uptake (Moorthy et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Reddy et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Afzal et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The trace organic signals (at 2850\u0026ndash;2920 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) suggest either (i) incomplete calcination, which could be resolved by optimizing temperature/duration, or (ii) surface-bound capping agents that may enhance colloidal stability [35, 38]. Compared to literature, these spectra match biosynthesized MgO-NPs where residual carbon is common but rarely affects functionality (Reddy et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Afzal et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For applications demanding ultra-pure MgO-NPs, additional thermal treatment or solvent washing would be recommended. The results collectively validate the efficacy of \u003cem\u003eC. colocynthis\u003c/em\u003e extract for MgO-NPs synthesis while highlighting the importance of post-processing to tailor surface chemistry for specific uses.\u003c/p\u003e \u003cp\u003eThe XRD results confirm the successful formation of crystalline MgO-NPs with a face-centered cubic structure, as evidenced by the characteristic diffraction pattern. The peak broadening observed throughout the pattern is indicative of nanoscale crystallites, consistent with the expected particle size from the synthesis method. The presence of minor peaks at 14.4\u0026deg; and 16.54\u0026deg; warrants attention, as these could correspond to residual precursor compounds or intermediate phases such as magnesium hydroxide or carbonate, which commonly form during aqueous synthesis routes. The relatively high intensity of the (200) peak at 36.3\u0026deg; compared to other reflections suggests some degree of preferred orientation in the MgO-NPs. The amorphous background signal likely originates from either residual organic component from the green synthesis or disordered surface layers on the nanoparticles (Rotti et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings align with previous reports on biosynthesized MgO-NPs, where similar crystallite sizes and minor impurity phases have been observed (Umaralikhan and Jaffar \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For applications requiring high phase purity, additional thermal treatment or washing steps may be beneficial to remove any residual amorphous content or secondary phases. The nanocrystalline nature of the synthesized MgO-NPs, as revealed by XRD spectrum, is particularly advantageous for catalytic and adsorption applications where a high surface area is desired (Umaralikhan and Jaffar \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rotti et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe in-vitro assay results demonstrate broad-spectrum activity of MgO-NPs against both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e MDR pathogens, suggesting that MgO-NPs target conserved microbial structures, such as cell membranes or intracellular components. The slightly enhanced efficacy against \u003cem\u003eS. aureus\u003c/em\u003e may reflect the absence of an outer membrane in Gram-positive bacteria, which renders them more susceptible to nanoparticle-induced oxidative stress (Gatou et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The dose-dependent response aligns with proposed mechanisms for metal oxide nanoparticles, including reactive oxygen species (ROS) generation, damaging cellular macromolecules, membrane destabilization due to nanoparticle adhesion or cation release, and enzyme inhibition due to metal ion interactions (Saied et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Clinically, the \u0026gt;\u0026thinsp;10 mm inhibition zones at 20 mg/ml concentration of MgO-NPs are promising, meeting the threshold for potential therapeutic relevance (CLSI guidelines). However, the higher efficacy at 25 mg/ml concentration suggests optimization of dosing may be required for practical applications. Future work should be undertaken to investigate cytotoxicity to human cells to establish a therapeutic window.\u003c/p\u003e \u003cp\u003eThe nanoparticle-mediated potentiation of antibiotic efficacy likely operates through three synergistic mechanisms: (i) cationic disruption of microbial membrane integrity via Mg\u003csup\u003e2+\u003c/sup\u003e-mediated phospholipid destabilization, (ii) generation of reactive oxygen species (ROS) that synergize with antibiotic mechanisms, and (iii) competitive inhibition of β-lactamase enzymes through metal ion chelation (Bag et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rotti et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The differential enhancement patterns correlate with bacterial ultrastructure; the more pronounced effect in \u003cem\u003eS. aureus\u003c/em\u003e aligns with the absence of an outer membrane barrier, facilitating nanoparticle penetration. For \u003cem\u003eE. coli\u003c/em\u003e, the restoration of oxacillin activity suggests MgO-NPs may permeabilize the outer membrane through lipopolysaccharide (LPS) destabilization, enabling access to penicillin-binding proteins. The intermediate penicillin activity against \u003cem\u003eS. aureus\u003c/em\u003e may reflect limitations in targeting modified PBP2a proteins characteristic of MRSA strains (Fishovitz et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These findings have important implications for combinatorial therapeutic strategies, particularly the potential to revive obsolete antibiotics against ESKAPE pathogens. However, translational applications require further pharmacokinetic optimization to maintain therapeutically effective Mg\u003csup\u003e2+\u003c/sup\u003e concentrations while minimizing potential cytotoxicity.\u003c/p\u003e \u003cp\u003eThe observed bactericidal activity (MBC/MIC\u0026thinsp;\u0026le;\u0026thinsp;4) suggests MgO-NPs primarily disrupt bacterial membranes through cationic interactions and ROS generation, consistent with metal oxide nanoparticle mechanisms. The greater potency against \u003cem\u003eS. aureus\u003c/em\u003e (lower MIC) aligns with Gram-positive bacteria's increased susceptibility to membrane-targeting agents due to absence of outer membranes (Bag et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ishak et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). All MIC values met CLSI efficacy thresholds, though the relatively higher concentration range indicates potential optimization needs for clinical applications. The therapeutic indices (2.22 and 2.75) reflect moderate selectivity, warranting further cytotoxicity studies (Gatou et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These findings corroborate existing literature on MgO-NP antimicrobial properties while providing novel pharmacological classification through standardized ratios and indices.\u003c/p\u003e \u003cp\u003eThe dose-dependent antifungal activity confirms MgO-NPs disrupt fungal membranes through combined mechanisms; (i) Mg\u003csup\u003e2+\u003c/sup\u003e-mediated ergosterol binding, (ii) ROS generation affecting cellular redox balance, and (iii) cell wall destabilization by chitinase inhibition. The superior activity against \u003cem\u003eC. albicans\u003c/em\u003e (susceptible at 10 mg/ml) versus \u003cem\u003eA. niger\u003c/em\u003e (intermediate at same concentration) reflects inherent differences in fungal cell wall composition, yeasts being more vulnerable to cationic nanoparticles than filamentous fungi (Slavin and Bach \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While 10 mg/ml MgO-NPs approached control antifungal efficacy, the required concentrations exceed typical clinical doses, suggesting potential utility as topical/systemic adjunct therapy rather than monotherapy (Abdelsadek et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ramezani Farani et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings align with emerging literature on metal oxide antifungals but highlight the need for formulation optimization to improve bioavailability (Mouhamad et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Slavin and Bach \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIt was concluded that \u003cem\u003eC. colocynthis\u003c/em\u003e-mediated MgO-NPs exhibit potent bactericidal and antifungal activities, restoring antibiotic susceptibility in MDR strains. The study establishes a sustainable green route for MgO-NP synthesis with translational potential in antimicrobial therapy. Future studies should optimize nanoparticle dispersion, assess cytotoxicity, and validate \u003cem\u003ein vivo\u003c/em\u003e efficacy. Scalable production and comparative studies with other metal oxide nanoparticles would further strengthen translational potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll authors declare no competing interest that is directly or indirectly related to the work submitted for publication.\u003c/p\u003e\n\u003ch2\u003eAuthorship contribution statement\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eConceptualization, Methodology\u003c/em\u003e: [Abdul Rehman, Baharullah Khattak and Muhammad Qasim]; \u003cem\u003eData collection and Processing\u003c/em\u003e: [Maqsood Qaisar, and Hassan Naveed]; \u003cem\u003eFormal analysis and Writing - Original Draft Manuscript\u003c/em\u003e: [Maqsood Qaisar, Abdul Rehman, and Iffat Naz]; \u003cem\u003eCritical Review and Editing\u003c/em\u003e: [Hassan Naveed, Baharullah Khattak and Muhammad Qasim]; \u003cem\u003eSupervision\u003c/em\u003e: [Baharullah Khattak and Abdul Rehman]. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eDepartment of Microbiology, Kohat University of Science and Technology, Kohat for providing financial support.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization, Methodology: [Abdul Rehman, Baharullah Khattak and Muhammad Qasim]; Data collection and Processing: [Maqsood Qaisar, and Hassan Naveed]; Formal analysis and Writing - Original Draft Manuscript: [Maqsood Qaisar, Abdul Rehman, andIffat Naz]; Critical Review and Editing: [Hassan Naveed, Baharullah Khattak and Muhammad Qasim]; Supervision: [Baharullah Khattak and Abdul Rehman]. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe researchers would like to thank the Department of Microbiology, Kohat University of Science and Technology, Kohat for providing laboratory facilities to conduct this research study.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are not publicly available due to the absence of a publicly accessible repository for such microbiological and analytical data. However, the data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdelsadek MS, El-Dawy EG, Khalaphallah R. 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Foods. 2021;10(2):251. https://doi.org/10.3390/foods10020251 \u003c/li\u003e\n\u003cli\u003eGatou MA, Skylla E, Dourou P, Pippa N, Gazouli M, Lagopati N, et al. Magnesium oxide (MgO) nanoparticles: synthetic strategies and biomedical applications. Crystals. 2024; 14(3): 215-61. https://doi.org/10.3390/cryst14030215\u003c/li\u003e\n\u003cli\u003eGe J, Liu Z, Zhong Z, Wang L, Zhuo X, Li J, et al. Natural terpenoids with anti-inflammatory activities: Potential leads for anti-inflammatory drug discovery. Bioorg. Chem. 2022; 124: 105817-36. https://doi.org/10.1016/j.bioorg.2022.105817\u003c/li\u003e\n\u003cli\u003eGebre SH. Bio-inspired synthesis of metal and metal oxide nanoparticles: the key role of phytochemicals. J Cluster Sci. 2023;34(2):665-04. https://link.springer.com/article/10.1007/s10876-022-02276-9\u003c/li\u003e\n\u003cli\u003eHayat M, Rehman A, Khan FA, Anees M, Naz I, Qasim M, et al. Phytogenic-mediated zinc oxide nanoparticles using the seed extract of \u003cem\u003eCitrullus lanatus\u003c/em\u003e and its integrated potency against multidrug resistant bacteria. ACS omega. 2024; 9(14): 16832-41. https://doi.org/10.1021/acsomega.4c01554\u003c/li\u003e\n\u003cli\u003eHumphries R, Bobenchik AM, Hindler JA, Schuetz AN. Overview of changes to the clinical and laboratory standards institute performance standards for antimicrobial susceptibility testing, M100. J Clin Microbiol. 2021;59(12):1110-28. https://doi.org/10.1128/jcm.00213-21\u003c/li\u003e\n\u003cli\u003eIshak A, Mazonakis N, Spernovasilis N, Akinosoglou K, Tsioutis C. Bactericidal versus bacteriostatic antibacterials: clinical significance, differences and synergistic potential in clinical practice. J Antimicrob Chemother. 2025;80(1):1-17. https://doi.org/10.1093/jac/dkae380\u003c/li\u003e\n\u003cli\u003eKillai SN. Antibiotic resistance: strategies for combating the global threat. J Med Res Nursing Health Midwife Participation. 2024;5(4):268-277. https://doi.org/10.59733/medalion.v5i4.142 \u003c/li\u003e\n\u003cli\u003eLi QY, Munawar M, Saeed M, Shen JQ, Khan MS, Noreen S, Li CX. \u003cem\u003eCitrullus colocynthis\u003c/em\u003e (L.) Schrad (Bitter Apple Fruit): Promising traditional uses, pharmacological effects, aspects, and potential applications. Front. Pharmacol. 2022;12:791049. https://doi.org/10.3389/fphar.2021.791049 \u003c/li\u003e\n\u003cli\u003eMazher M, Ishtiaq M, Waqas M. Antimicrobial and antioxidant activity of secondary metabolites isolated from \u003cem\u003eCitrullus colocynthis\u003c/em\u003e (L.) Schrad.: Antimicrobial and antioxidant activity of isolated phytochemicals. Proc Pak Acad Sci B. 2023;60(2):193-04. https://doi.org/10.53560/PPASB(60-2)787\u003c/li\u003e\n\u003cli\u003eMoorthy SK, Ashok CH, Rao KV, Viswanathan CJMTP. Synthesis and characterization of MgO nanoparticles by Neem leaves through green method. Mater Today Proc. 2015;2(9):4360-4368. https://doi.org/10.1016/j.matpr.2015.10.027 \u003c/li\u003e\n\u003cli\u003eMouhamad RS, Al Khafaji KA, Al-Dharob MH, Al-Abodi EE. Antifungal, antibacterial and anti-yeast activities evaluation of oxides of silver, zinc and titanium nanoparticles. Chem Int. 2022;8(4):159-66. https://doi.org/10.5281/zenodo.7317423\u003c/li\u003e\n\u003cli\u003eMuhaymin A, Mohamed HEA, Hkiri K, Safdar A, Azizi S, Maaza M. Green synthesis of magnesium oxide nanoparticles using \u003cem\u003eHyphaene thebaica\u003c/em\u003e extract and their photocatalytic activities. Sci Rep. 2024;14(1):20135-46. https://doi.org/10.1038/s41598-024-71149-0\u003c/li\u003e\n\u003cli\u003eNawaz H, Akram H, Ishaq QHM, Khalid A, Zainab B, Mazhar A. Polarity-dependent response of phytochemical extraction and antioxidant potential of different parts of \u003cem\u003eAlcea rosea\u003c/em\u003e. Free Radicals Antiox. 2022;12(2):49-54. https://doi.org/10.5530/fra.2022.2.9\u003c/li\u003e\n\u003cli\u003ePrado DC, Fern\u0026aacute;ndez I, Rodr\u0026iacute;guez-P\u0026aacute;ez JE. MgO nanostructures: Synthesis, characterization and tentative mechanisms of nanoparticles formation. Nano-struct Nano-obj. 2020;23:100482-94. https://doi.org/10.1016/j.nanoso.2020.100482\u003c/li\u003e\n\u003cli\u003eRamezani Farani M, Farsadrooh M, Zare I, Gholami A, Akhavan O. Green synthesis of magnesium oxide nanoparticles and nanocomposites for photocatalytic antimicrobial, antibiofilm and antifungal applications. Catalysts. 2023;13(4):642-65. https://doi.org/10.3390/catal13040642\u003c/li\u003e\n\u003cli\u003eRani A, Goyal A, Arora S. Isolation and phytochemical screening of Citrullus colocynthis formulation. Plant Arch. 2021;21(1):2674-2682. https://doi.org/10.51470/PLANTARCHIVES.2021.v21.S1.436 \u003c/li\u003e\n\u003cli\u003eRao V, Poonia A. \u003cem\u003eCitrullus colocynthis\u003c/em\u003e (bitter apple): bioactive compounds, nutritional profile, nutraceutical properties and potential food applications: a review. Food Prod Process Nutr. 2023;5(1):4-15. https://link.springer.com/article/10.1186/s43014-022-00118-9\u003c/li\u003e\n\u003cli\u003eRasool S, Tayyeb A, Raza MA, Ashfaq H, Perveen S, Kanwal Z, Alomar SY. \u003cem\u003eCitrullus colocynthis\u003c/em\u003e-mediated green synthesis of silver nanoparticles and their antiproliferative action against breast cancer cells and bactericidal roles against human pathogens. Nanomaterials. 2022;12(21):3781. https://doi.org/10.3390/nano12213781 \u003c/li\u003e\n\u003cli\u003eReddy SH, Al Jahwari MRH, Al Tobi ZMR. A study on FTIR, Antimicrobial, Antioxidant and Hypogycaemic effect of Diospyros kaki and \u003cem\u003eCitrullus colocynthis\u003c/em\u003e. Int J Phytomed. 2019;11:23-31. https://www.researchgate.net/publication/354687157 \u003c/li\u003e\n\u003cli\u003eRotti RB, Sunitha DV, Manjunath R, Roy A, Mayegowda SB, Gnanaprakash AP, et al. Green synthesis of MgO nanoparticles and its antibacterial properties. Front Chem. 2023; 11: 1143614-26. https://doi.org/10.3389/fchem.2023.1143614\u003c/li\u003e\n\u003cli\u003eSaied E, Eid AM, Hassan SED, Salem SS, Radwan AA, Halawa M, et al. The catalytic activity of biosynthesized magnesium oxide nanoparticles (MgO-NPs) for inhibiting the growth of pathogenic microbes, tanning effluent treatment, and chromium ion removal. Catalysts. 2021; 11(7): 821-41. https://doi.org/10.3390/catal11070821\u003c/li\u003e\n\u003cli\u003eSalam MA, Al-Amin MY, Salam MT, Pawar JS, Akhter N, Rabaan AA, et al. Antimicrobial resistance: a growing serious threat for global public health. In \u003cem\u003eHealthcare.\u003c/em\u003e 2023; 11(13): 1946. Multidisciplinary Digital Publishing Institute. https://doi.org/10.3390/healthcare11131946\u003c/li\u003e\n\u003cli\u003eSharma R, Giri SK, Kumar A, Chamoli S, Singh G. ROS-mediated pathogen control by ZnO and MgO nanoparticles. In \u003cem\u003eOxides for Medical Applications\u003c/em\u003e. 2023;419-31. Woodhead Publishing. https://doi.org/10.1016/B978-0-323-90538-1.00008-X\u003c/li\u003e\n\u003cli\u003eSlavin YN, Bach H. Mechanisms of antifungal properties of metal nanoparticles. Nanomaterials. 2022;12(24):4470-504. https://doi.org/10.3390/nano12244470\u003c/li\u003e\n\u003cli\u003eSoltys L, Olkhovyy O, Tatarchuk T, Naushad M. Green synthesis of metal and metal oxide nanoparticles: Principles of green chemistry and raw materials. Magnetochemistry. 2021;7(11):145-78. https://doi.org/10.3390/magnetochemistry7110145 \u003c/li\u003e\n\u003cli\u003eTar\u0026iacute;n-Pell\u0026oacute; A, Suay-Garc\u0026iacute;a B, P\u0026eacute;rez-Gracia MT. Antibiotic resistant bacteria: current situation and treatment options to accelerate the development of a new antimicrobial arsenal. Expert Rev Anti Infect Ther. 2022;20(8):1095-08. https://doi.org/10.1080/14787210.2022.2078308\u003c/li\u003e\n\u003cli\u003eTheuretzbacher U, Baraldi E, Ciabuschi F, Callegari S. Challenges and shortcomings of antibacterial discovery projects. Clin Microbiol Infect. 2023;29(5):610-15. https://doi.org/10.1016/j.cmi.2022.11.027\u003c/li\u003e\n\u003cli\u003eUmaralikhan L, Jaffar JMM. Green synthesis of MgO nanoparticles and it antibacterial activity. Iran J Sci Technol Trans A Sci. 2018;42:477-85. https://link.springer.com/article/10.1007/s40995-016-0041-8\u003c/li\u003e\n\u003cli\u003eWorld Health Organization. \u003cem\u003eGlobal antimicrobial resistance and use surveillance system (GLASS) report 2022\u003c/em\u003e. 2022. World Health Organization.\u003c/li\u003e\n\u003cli\u003eXie M, Gao M, Yun Y, Malmsten M, Rotello VM, Zboril R, et al. Antibacterial nanomaterials: mechanisms, impacts on antimicrobial resistance and design principles. Angewandte Chemie International Edition. 2023; 62(17): e202217345. https://doi.org/10.1002/anie.202217345\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Confirmation of MDR nature of test Bacterial strains.\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"610\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS. No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 131px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDR Bacterial Isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 413px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibiotics discs used against tested MDR bacteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003eCeftazidime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003ePenicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eOxacillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eErythromycin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003eKey:\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003eMDR = Multidrug resistant; R = Resistance\u003c/sup\u003e\u003c/p\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":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Antimicrobial resistance, Green synthesis, Magnesium oxide nanoparticles, Phytochemicals, Synergistic effect","lastPublishedDoi":"10.21203/rs.3.rs-8079662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8079662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe escalating threat of antimicrobial resistance (AMR) necessitates innovative therapeutic approaches. This study reports the green synthesis of magnesium oxide nanoparticles (MgO-NPs)using \u003cem\u003eCitrullus colocynthis\u003c/em\u003e extract, a medicinal plant rich in bioactive compounds, as a sustainable alternative to conventional antibiotics. The synthesized MgO-NPs were characterized by UV-Vis spectroscopy (absorption peak at 250 nm), XRD (cubic crystalline structure, 15–25 nm size), SEM-EDX (agglomerated spherical morphology, Mg/O ratio 2.6:1), and FTIR (Mg-O vibration at 860 cm⁻¹). The nanoparticles exhibited potent, dose-dependent antibacterial activity against multidrug-resistant (MDR) \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MIC: 35.3 µg/ml; MBC: 97.1 µg/ml) and \u003cem\u003eEscherichia coli\u003c/em\u003e (MIC: 47.5 µg/ml; MBC: 105.5 µg/ml), with a bactericidal mode of action (MBC/MIC ≤ 4). Remarkably, MgO-NPs restored susceptibility to β-lactams antibiotics (ceftazidime and penicillin) in resistant strains, demonstrating synergistic effects. Antifungal activity of MgO-NPs against \u003cem\u003eCandida albicans\u003c/em\u003e (17.3±0.7 mm) and \u003cem\u003eAspergillus niger\u003c/em\u003e (14.4±0.8 mm) at a concentration of 10 mg/ml was also observed. Phytochemical analysis revealed solvent-dependent bioactive constituents in \u003cem\u003eC. colocynthis\u003c/em\u003e, with aqueous extracts rich in tannins/phenolics and methanolic extracts in flavonoids/terpenoids. This is the first report demonstrating restoration of antibiotic susceptibility by MgO-NPs synthesized from \u003cem\u003eC. colocynthis\u003c/em\u003e extract.\u003c/p\u003e","manuscriptTitle":"Eco-Friendly Synthesis of Magnesium Oxide Nanoparticles Using Citrullus colocynthis and their Synergistic Antimicrobial Activity against Drug-Resistant Pathogens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 17:07:10","doi":"10.21203/rs.3.rs-8079662/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-18T03:13:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-11T20:08:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-11T16:34:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2025-11-10T17:46:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"436d30b1-f80f-4b0f-9d4f-7ff270e10bc9","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-20T14:00:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 17:07:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8079662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8079662","identity":"rs-8079662","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}