Plant-Based Fabrication of Zinc oxide and Copper Nanoparticles Using Shorea robusta Resin: Dual Evaluation of Safety and Antimicrobial Efficacy

Plant-Based Fabrication of Zinc oxide and Copper Nanoparticles Using Shorea robusta Resin: Dual Evaluation of Safety and Antimicrobial Efficacy | 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 Plant-Based Fabrication of Zinc oxide and Copper Nanoparticles Using Shorea robusta Resin: Dual Evaluation of Safety and Antimicrobial Efficacy Shuaib Burgee, Barghavi V., Swati Bhati, S. Shankara Narayanan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7883522/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract In this study, a green, sustainable, and cost-effective approach was employed for the synthesis of zinc oxide (ZnO) and copper (Cu) nanoparticles using Shorea robusta resin extract as a natural agent for reducing, capping, and stabilizing. The synthesis of metal nanoparticles utilizing S. robusta resin is being reported for the first time. The synthesized nanoparticles were characterized using Ultraviolet–Visible spectroscopy (UV-Vis), Fourier Transform Infrared spectroscopy (FT–IR), and Nanoparticle Tracking Analysis (NTA). The phyto-synthesized nanoparticles exhibited significant antibacterial and antifungal activity, particularly against Pseudomonas stutzeri (Gram-negative), Staphylococcus aureus (Gram-positive), and the pathogenic fungi Candida albicans and Aspergillus flavus . Additionally, both ZnO and Cu nanoparticles demonstrated excellent hemocompatibility and cytocompatibility, highlighting their potential for pharmacological and therapeutic applications. Zinc oxide Copper nanoparticles Shorea robusta green synthesis anti-microbial biocompatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Nanoscience and nanotechnology work toward enhancing the optical, electrical, magnetic, and catalytic functionalities of bulk materials to improve human lives[ 1 ]. Due to the damage caused by global warming, scientists worldwide are working hard to create new materials with unique features and minimal environmental hazard[ 2 ]. Nanomaterials are broadly classified into organic and inorganic nanoparticles, with metallic nanoparticles most commonly synthesized through chemical and physical methods[ 3 ]. Growing awareness of environmental degradation has prompted scientists to adopt greener, cost-effective, and non-toxic methods for synthesizing and fabricating nanomaterials[ 1 ]. Nanoparticle synthesis using plants is often favoured over microbial methods due to its simplicity, ease of scaling up, the vast diversity of plant species, and the abundance of phytochemicals they contain, such as alkaloids, steroids, terpenoids, tannins, saponins, polyphenols, and phenolic acids[ 4 , 5 ]. They function as capping or stabilizing agents in addition to reducing agents[ 6 – 8 ]. Typically, metal nanoparticles possess distinct physical, chemical, and biological properties compared to their bulk forms, largely due to their exceptionally high surface-to-volume ratio[ 9 , 10 ]. It has been proposed that the phyto-synthesis of nanoparticles is attributed to phenolic chemicals[ 11 , 12 ]. Due to their antioxidant properties, phenolic compounds can be used as stabilizing and reducing agents while creating nanomaterials[ 13 , 14 ]. Bioactive substances, viz., alkaloids, glycosides, phenols, tannins, steroids, and terpenoids that contribute to strong antibacterial properties, were found in very high concentrations during phytochemical screening of the oleoresin of Shorea robusta [ 15 ]. Zeghoud et al. claim that the plant species used for green synthesis has a minimal influence on the yield and morphology of ZnO nanoparticles, except when the extract lacks sufficient bioactive compounds. [ 16 ]. ZnO is the most widely used of them at the nanoscale due to its remarkable scientific characteristics, including high excitonic binding energy and band gap[ 17 ]. ZnO NPs are well-known among nanometal oxides for their antimicrobial, anti-inflammatory, and anticancer properties[ 18 ]. Moreover, ZnO is also classified as a "GRAS" (Generally Recognized as Safe) chemical by the US Food and Drug Administration (FDA)[ 19 ]. Copper is an abundant metal and serves as an essential trace element in most living organisms[ 20 ]. It has been acknowledged by the U.S. Environmental Protection Agency (EPA) as the first and sole metal officially registered for its antimicrobial activity. [ 21 ]. When reduced to the nanoscale, copper exhibits unique physicochemical properties that make it highly versatile, with applications across various industries such as solar cells, wood preservatives, gas sensors, and high-temperature superconductors. Similarly, the catalytic, high electrical conductivity, optical, antifungal, and antibacterial properties of CuNPs (Copper nanoparticles) make them the most sought-after of the NPs [ 22 ]. This paper describes a green method for synthesising CuNPs and ZnO NPs using resin extract from Shorea robusta . All characterization results provided clear evidence of nanoparticle formation. Its antifungal and antibacterial properties against the pathogenic fungus Aspergillus flavus and Candida albicans , as well as Staphylococcus aureus and Pseudomonas stutzeri were evaluated. The Cu and ZnO nanoparticles were further evaluated for their biocompatibility, specifically focusing on hemocompatibility and cytocompatibility. 2. Materials and Methods 2.1. Materials Shorea robusta (SR) resin was procured from DKC Agrotech Pvt. Ltd., India, and used for nanoparticle synthesis. Ethanol, zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O; QUALIGENS), and copper(II) sulfate pentahydrate (CuSO₄·5H₂O; CAS No. 7758-99-8; Sigma-Aldrich) were employed as precursor materials. Nutrient Agar, Nutrient Broth, and Potato Dextrose Agar (Himedia) were used for microbial culture and antimicrobial assays. The microbial strains utilized in this study included Staphylococcus aureus (MTCC 3160), Pseudomonas stutzeri (MTCC 863), Candida albicans (MTCC 183), and Aspergillus flavus (MTCC 277). All strains were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India. The reagents and consumables used for the assays comprised sterile petriplates (TARSONS), 96-well microtiter plates, phosphate-buffered saline (PBS, pH 7.4), Triton X-100 (Sigma), Amphotericin B (100 units/mL), and Streptomycin (10 µg/mL). For cytotoxicity and bioactivity assessments, MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Himedia), RPMI-1640 culture medium (Himedia), fetal bovine serum (FBS; Sigma), and red cell lysis buffer (RCLB; Sigma) were used. The ELISA readings were recorded using a BioTek 800 TS Absorbance Reader. DMSO (dimethyl sulfoxide; Sigma) was used as a solvent, and Histopaque-1077 (Sigma) was used for cell isolation and density gradient separation. 2.2. Characterization Techniques: The UV–Visible absorption spectra were recorded using a Shimadzu UV-1900i double-beam spectrophotometer. FTIR spectra were obtained using a Nicolet iS50 FTIR spectrometer, from Thermo Scientific. The particle size distribution of the samples was determined through nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument (Malvern Panalytical, Netherlands) equipped with a 488 nm laser and a 500 nm long-pass filter. For NTA measurements, all samples were diluted to a concentration of 10 6 –10 7 particles/mL. Each sample was analyzed in five replicates, and 30–60 s videos were recorded for each run, capturing at least 200 valid particle tracks per analysis. During all measurements, the light scatter mode (LSM) camera level was maintained between 12 and 14. Data acquisition and analysis were performed using Nanosight software (version 3.0) with standard parameters. The mean ± standard deviation values were calculated from three independent video recordings. 2.3. Preparation of ethanolic Shorea robusta resin extract: An ethanolic extract of Shorea robusta resin was made by measuring 10 g and thoroughly crushing it with a mortar and pestle. Pure ethanol was used to dissolve the crushed resin, which was then filtered through Whatman 40 filter paper and utilized as the biogenic source to produce the nanoparticles. 2.4. Green synthesis of ZnO NPs: In brief, 25 mL (0.5 M) zinc acetate dihydrate was mixed with 2.5 mL of Shorea robusta resin extract. The pH of the mixture was 6.13, and to maintain it at 8, 2 M NaOH was added drop by drop. It was then stirred and heated at 70°C for 30 minutes, resulting in complete reduction and the appearance of a white precipitate. The resultant material was then collected by decantation, cleaned of residues using distilled water, and oven-dried for an entire night at 70°C to produce powdered ZnO nanoparticles. For subsequent characterization and application, the material was stored at ambient temperature in an airtight container. [ 23 ]. 2.5. Green Synthesis of CuNPs : 50 mL (5 mM) of copper sulphate solution was combined with 5 mL of Shorea robusta resin extract to create plant extract resin copper nanoparticles. A solution of NAOH (1N) was added to bring the pH down to 7. Furthermore, a green colour mixture solution was produced. After centrifugation, the pellet collected was dried overnight at 60 degrees in a hot air oven to produce powder. Powder was collected and stored at 4 degrees temperature for further use [ 22 ]. 2.6 Agar well diffusion assay: Nutrient agar media was poured into the plates in sterile conditions. In the appropriate conical flasks, nutrient broth was made for the cultures of Pseudomonas stutzeri (MTCC 3160) and Staphylococcus aureus (MTCC 863), which were then maintained in the shaker for a whole day. The cultures were spread throughout the nutrient agar medium using a glass spreader. For the controls and test compounds, Positive control: 10 mg antibiotic streptomycin, Negative control: pure ethanol, Test compounds: ZnO and Cu NPs with various stock concentrations 50, 25, 12.5, 6.25 mg/mL, were used, and the wells were punched using a gel punch. 50 µL of different concentrations of ZnO and CuNPs were loaded into the wells, and the plates were kept in a Biochemical Oxygen Demand (BOD) incubator for 24 hours. Using a vernier calliper, the diameter of the zone (in millimetres) was recorded. The inhibitory zone was calculated using the formula: Zone of inhibition (mm) = Diameter of zone (mm) - Diameter of well (mm). 2.7. Anti-fungal agar well diffusion Assay: We procured Aspergillus flavus (MTCC 277) and Candida albicans (MTCC 183) from the IMTECH Microbial Type Culture Collection in Chandigarh. One colony was inoculated in Potato Dextrose Broth (PDB), and was cultured for overnight at 37°C with continuous shaking at 150 rpm. After transferring 60 µl of the overnight culture into 3 mL of PDB, it was cultivated at 37°C (150 rpm) until the optical density at 600 nm reached the 0.5 McFarland standard. The 100 µL of Candida albicans culture from the previous stage was distributed onto Potato Dextrose Agar ( PDA) using a sterile cotton swab. Following this, the PDA plates were incubated at 37°C for 48 hours. ZnO and Cu NPs doses ranging from 6.25 to 50 mg/mL were added to the wells in 50 µL increments. Amphotericin B (100 units/disc) acted as a positive control, and the solvent as a negative control. The inhibition zone, or clear area around the disc, was measured with a vernier calliper to determine its diameter. 2.8. Hemocompatibility test: Hemolysis assay was used to examine the cytotoxic effects of ZnO and Cu NPs [ 24 ]. The experiment was carried out under the applicable institutional policies and procedures, and informed consent was acquired. The Sharda University School of Medical Sciences and Research (SMS&R) Institutional Ethics Committee examined and accepted the study protocol, assigning approval reference number SU/SMS&R/76-A/2024/125. A healthy individual's freshly obtained blood was collected in an anticoagulant-containing tube, and centrifuged for five minutes at 3000 rpm after adding phosphate-buffered saline (PBS) (v/v; pH 7.2). The pellet was further washed with PBS at 3000 rpm. The RBCs obtained were further diluted with PBS. ZnO and Cu NPs were added to diluted erythrocyte suspensions at concentrations of 50, 75, 100, 250, 500, 750, and 1000 µg/mL, gently mixed, and then incubated for 4 hours at 37°C. Triton X-100 (10%) was used as the positive control, and PBS (normal) as the negative control. The samples were incubated and then centrifuged. The supernatant was then collected, and absorbance at 540 nm was measured using an ELISA reader (BioTek 800 TS). To calculate the percentage hemolysis, the formula was as follows: Hemolysis (%) = [(Mean OD of sample - Mean OD of negative control)/ (Mean OD of positive control - Mean OD of negative control) × 100]. 2.9. Cytocompatibility test In a heparinized vacutainer, human whole blood was drawn. 10 mL of whole blood was diluted with 10 mL of RPMI-1640 before being stacked on top of 12.5 mL Histopaque-1077 gradient in centrifuge tubes. Following centrifugation at 2000 rpm for 20 minutes, the peripheral blood mononuclear cells layer was re-suspended in RPMI-1640 with 10% FBS after being washed with PBS. Different doses of ZnO and Cu NPs were used in the MTT test on peripheral blood mononuclear cells (PBMCs). Cells were seeded at 0.5 x 10 5 cells/mL density in a 96-well plate. After being treated for 24 hours with varying concentrations of (50, 100, 200, 300 µg/mL) of ZnO and Cu NPs, the cells were incubated for 3–4 hours at 37°C in media containing 5 mg/mL of MTT. After dissolving the resultant formazan crystals in DMSO, the absorbances were measured at 570 nm. Cell Viability% = [(Absorbance of treated sample - Absorbance of blank) / (Absorbance of untreated - Absorbance of blank)] * 100. 3. Results and Discussion 3.1 ZnO and Cu NP Nanoparticle Characterizations The UV–Vis absorption spectrum of the prepared ZnO nanoparticles (Fig. 1 a) exhibits a pronounced absorption edge/peak centred at ~ 366 nm. This absorption corresponds to a direct band-to-band transition; using Eg​ (eV) = 1240 / λ (nm) gives an optical band gap of ≈ 3.39 eV. This value is in very good agreement with the expected band gap of wurtzite ZnO (≈ 3.3–3.4 eV) and indicates that the material is phase-pure ZnO with no large amounts of other semiconducting impurities [ 25 ]. The absorption peak at 366 nm is slightly blue-shifted (shorter wavelength) compared with bulk ZnO values reported near ~ 368–380 nm, which can be ascribed to quantum-confinement effects for small nanoparticle sizes. The broad decrease in absorbance toward longer wavelengths and the weak tail extending into the visible region could be attributed to sub-band-gap absorption from defect states (e.g., oxygen vacancies) and to light scattering by nanoparticles in the suspension[ 26 ]. Formore accurate determination of the optical band gap, a Tauc plot analysis (Fig. 1 b) for a direct band transition (αhν) 2 vs hν was performed and the linear extrapolation of the low-energy edge yielded Eg ≈ 3.39 eV, consistent with the value calculated directly from the absorption edge. These observations corroborate the formation of nanoscale ZnO with electronic properties appropriate for UV-active photocatalytic and optoelectronic applications. The UV–Vis absorption spectrum of Cu-NPs is depicted in Fig. 1 c. The spectrum features a band at ≈ 252 nm, which confirms the successful synthesis of Cu NPs. These results corroborate with previously reported observations[ 22 ]. The FTIR spectrum of both the green-synthesized ZnO and Cu nanoparticles were found to be similar and (Fig. 1 d) showed distinct absorption bands at 3295 cm⁻¹, 2117 cm⁻¹, and 1636 cm⁻¹. The broad and intense absorption band around 3295 cm⁻¹ corresponds to the O–H stretching vibrations of hydroxyl groups and adsorbed water molecules present on the nanoparticle surface. This is a common feature in ZnO/Cu nanoparticles synthesized via aqueous or plant-extract-mediated routes, indicating the presence of surface –OH groups and hydrogen-bonded moisture[ 27 ]. The weak absorption observed near 2117 cm⁻¹ can be attributed to C ≡ C stretching or C = O overtone/C–N–C vibrations that arise from organic residues originating from plant phytochemicals [ 28 ]. Such features suggest partial retention of organic molecules that may act as capping or stabilizing agents during the green synthesis process. The peak appearing at 1636 cm⁻¹ is assigned to the H–O–H bending vibration of molecular water and may also overlap with C = O stretching vibrations of amide groups or carboxylate ions from biomolecules in the plant extract, further confirming the interaction between phytochemicals and metallic ions during Cu/ZnO nanoparticle formation [ 29 ]. The particle size distribution of the synthesized ZnO nanoparticles was analyzed using Nanoparticle Tracking Analysis (NTA), as shown in Fig. 2 a. The NTA profile exhibits a prominent peak centred around ~ 120 nm, indicating that majority of the particles fall within this size range. A smaller shoulder observed near 70–80 nm suggests the presence of a minor population of smaller nanoparticles or aggregates. The concentration of nanoparticles at the main peak reaches approximately 3.2 × 10⁸ particles/mL, reflecting a well-dispersed colloidal suspension with good stability. The relatively narrow distribution implies that the synthesis process produced uniformly sized ZnO nanoparticles with minimal aggregation, which is a desirable feature for optoelectronic and photocatalytic applications. Minor variations in particle size can be attributed to the presence of naturally formed agglomerates or residual capping molecules from the green synthesis process. The particle size distribution of the synthesized Cu nanoparticles was also analysed using NTA, as presented in Fig. 2 b. The NTA profile reveals a bimodal distribution, indicating the presence of two predominant nanoparticle populations. The first major peak appears around 95–110 nm, while a secondary peak is observed near 180–200 nm. The total particle concentration reaches approximately 4 × 10⁸ particles/mL, suggesting a high colloidal density of dispersed Cu nanoparticles. The presence of a bimodal distribution may arise from partial aggregation or variation in nucleation and growth rates during synthesis. The smaller size fraction corresponds to well-dispersed primary Cu nanoparticles, while the larger fraction likely represents agglomerated or fused particles formed due to the high surface energy of nanosized Cu. Overall, the observed size range confirms the nanoscale nature of the synthesized Cu nanoparticles, typically below 200 nm. The relatively sharp and well-defined peaks indicate that the colloidal suspension is reasonably monodisperse with moderate stability, suitable for optical and catalytic applications. Representative screenshots from the Nanoparticle Tracking Analysis (NTA) video are shown in Fig. 2 (c) and 2(d), displaying the Brownian motion of ZnO and Cu nanoparticles dispersed in aqueous medium, respectively. Each bright spot corresponds to an individual ZnO/Cu nanoparticle scattering laser light as it moves randomly due to collisions with solvent molecules. The intensity of scattered light varies with particle size —larger particles appear as brighter spots, while smaller ones exhibit weaker scattering. The observed random trajectories confirm that the nanoparticles are well-dispersed and remain colloidally stable without significant sedimentation or aggregation during measurement. This dynamic visualization, recorded by NTA, was used to calculate the hydrodynamic diameter distribution shown in Fig. 2 (a) and 2 (b). The clear and distinct scattering points further validate the nanoscale dimensions and optical activity of the synthesized ZnO and Cu NPs. 3.2. Anti-bacterial activity: The antibacterial activity of synthesized ZnO and Cu nanoparticles was evaluated against selected pathogens, namely Staphylococcus aureus and Pseudomonas stutzeri , using the disc diffusion method. The diameters of the zones of inhibition were measured in millimeters and are presented in Table 1 and Figs. 2 a–d and 3 a–d. In the disc diffusion assay, the synthesized ZnO nanoparticles exhibited significant antibacterial activity against all tested bacterial strains. The results clearly demonstrated that antibacterial activity, expressed as the zone of inhibition, increased with rising concentrations of ZnO nanoparticles (6.25, 12.5, 25, and 50 mg/mL). This effect may be attributed to the higher production of H₂O₂ and the generation of reactive oxygen species (ROS) from the nanoparticle surface at high concentrations. Table 1 Anti-microbial activity of Zinc oxide and Copper nanoparticles synthesized from Shorea robusta resin extracts Micro organisms Zinc Oxide Nanoparticles Copper Nanoparticles Reference Standards Concentration (mg/mL) 50 25 12.5 6.25 50 25 12.5 6.25 Streptomycin 10 mcg Amphotericin-B Gram-positive bacteria Zone (in mm) Staphylococcus aureus 13 11 11 9 13 12 11 11 29 - Gram-negative bacteria Pseudomonas stutzeri 20 15 16 15 17 16 12 11 27 - Yeast Candida albicans 19 18 17 16 13 13 11 11 - 23 Fungi Aspergillus flavus 29 27 20 19 13 12 11 11 - 4 Among the tested pathogens, Pseudomonas stutzeri showed the largest zone of inhibition (20 mm), whereas Staphylococcus aureus exhibited a comparatively smaller zone (12 mm), as shown in Table 1 and Figs. 2 a–d. These findings are consistent with previous studies. For instance, Chinnammal Janaki et al. (2015) reported antibacterial activity of ZnO nanoparticles with inhibition zones of 10 mm against Staphylococcus aureus , 10 mm against Candida albicans , and 12 mm against Penicillium notatum [ 30 ]. Cu nanoparticles also exhibited notable antibacterial activity, with a stronger effect against Pseudomonas stutzeri (17 mm) compared to Staphylococcus aureus (13 mm), as shown in Table 1 and Figs. 3 a–d. The antibacterial action of Cu NPs is primarily attributed to electrostatic interactions with the cell walls of gram-negative bacteria. Their strong affinity for carboxyl and amine groups which are key components of bacterial cell walls further enhances their antimicrobial potential [ 31 ]. At the nanoscale, Cu particles exert antibacterial effects through multiple mechanisms. These include adhesion of NPs to gram-negative bacterial cell walls due to electrostatic attraction, disruption of cell membrane proteins, denaturation of intracellular proteins, and interactions with phosphorus- and sulphur-containing biomolecules such as DNA[ 32 ]. In a comprehensive study, Chatterjee et al. used E. coli as a model system to investigate these mechanisms. They reported that treatment with the minimum bactericidal concentration (MBC) dose of CuNPs induced a 2.5-fold increase in intracellular reactive oxygen species (ROS). This ROS overproduction triggered lipid peroxidation, protein oxidation, and DNA degradation, ultimately leading to bacterial cell death[ 33 ]. 3.3. Anti-fungal activity : The antifungal activity of synthesized ZnO nanoparticles was evaluated against the selected fungal pathogens Aspergillus flavus and Candida albicans using the disc diffusion method. The diameters of the zones of inhibition (in millimeters) are presented in Table 1 and Figs. 3 , 4 (e–h). The synthesized ZnO nanoparticles exhibited significant antifungal activity against both fungal strains. Similar to the antibacterial results, the antifungal activity increased with increasing concentrations of ZnO nanoparticles (6.25, 12.5, 25, and 50 mg/mL). Notably, A. flavus was more sensitive, showing the highest zone of inhibition (29 mm), while C. albicans exhibited a smaller zone (12 mm). By contrast, Cu nanoparticles displayed only moderate antifungal action, with inhibition zones of 13 mm for both fungal species. 3.4. Bio-compatibility: In the present study, ZnO and Cu nanoparticles synthesized using Shorea robusta resin extract were employed for hemolysis testing. Hemolysis results from either direct or indirect damage to the red blood cell (RBC) membrane and is widely accepted as a reliable marker of biological incompatibility. One of the standard methods to evaluate whether a biomaterial is safe for blood-contacting applications is to measure its hemolytic activity. RBCs undergo lysis when their membranes are disrupted, releasing hemoglobin into the medium[ 34 ]. Among the various cell types that can be utilized for assessing nanoparticle-induced toxicity[ 35 ], RBCs remain the most suitable, as nanoparticles, regardless of origin, intended use, or route of administration, eventually reach the blood circulation and react with them, which are the predominant cellular constituent of blood circulation. This interaction often compromises RBC functionality. Since RBCs are structurally well studied, readily available, and simple to handle, they serve as excellent model cells for nanotoxicity studies. Several investigations have examined the impact of nanoparticles on RBCs, particularly their hemolytic activity, highlighting its importance as a key test for nanoparticle safety evaluation. However, comparison across different studies is often challenging due to variations in nanoparticle characterization methods and hemolysis testing protocols [ 36 ]. To address this, the American Society for Testing and Materials (ASTM) issued in 2008 a standardized procedure for evaluating hemolytic properties of nanoparticles [ 37 ], which measures the hemoglobin content released following interaction between nanoparticles and RBCs. In our experiments, hemolysis assays were carried out at nanoparticle concentrations of 50, 75, 100, 250, 500, 750, and 1000 µg/mL. The results demonstrated minimal hemolysis, with the maximum observed at less than 1% for ZnO NPs and 2% for Cu NPs at the highest concentration tested (1000 µg/mL) (Figure. 5). These findings fall well below the 5% threshold defined by ASTM for hemocompatibility, thereby confirming that the synthesized nanoparticles are non-hemolytic. Overall, the results indicate that green-synthesized ZnO and Cu nanoparticles from Shorea robusta resin extract exhibit excellent blood compatibility, supporting their potential for future in vivo applications in drug delivery and other biomedical uses. Primary lymphocytes serve as an effective model for evaluating genotoxic effects, as they exhibit normal cellular responses unlike cancer or transformed cells, and are more likely to encounter nanoparticles in daily life[ 38 ]. The biosynthesized ZnO NPs and Cu NPs were tested for their cytotoxic effects on lymphocytes derived from PBMCs using the MTT assay. The findings in Fig. 6 demonstrate that ZnO NPs and Cu NPs had cytotoxic effects on blood cells that were dose-dependent. Overall, ZnO NPs showed less cytotoxicity compared to CuNPs at the highest concentrations (300 µg/mL). Existing reports on the effects of ZnO NPs on human cells remain inconsistent; while some studies highlight their potential as strong anticancer agents, others describe them as non-toxic, supporting their application in consumer products[ 38 ]. Some findings suggest that ZnO nanoparticles exert low toxicity toward normal immune cells while maintaining significant anticancer activity, supporting their potential as a promising nanotherapeutic agent. ZnO nanoparticles demonstrate selective biological effects, exhibiting minimal cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) even at concentrations as high as 300 µg/mL, thereby underscoring their relative biocompatibility with non-transformed cells[ 39 , 40 ]. Zivari Fard et al. reported concentration-dependent cytotoxicity of CuO nanoparticles in human PBMCs assessed by MTT after exposure to 1–200 µg/mL, with viability decreasing progressively at higher concentrations [ 41 ]. 4. Conclusion The present study demonstrated that ZnO and Cu nanoparticles synthesized using Shorea robusta resin extract possess remarkable antimicrobial properties and excellent biocompatibility. Both types of nanoparticles exhibited concentration-dependent antibacterial and antifungal activity, with ZnO NPs showing larger inhibition zones, particularly against Pseudomonas stutzeri and Aspergillus flavus . Cu NPs also displayed strong activity, mainly through electrostatic interactions and ROS-mediated mechanisms, though their effects were comparatively moderate against fungal pathogens. Importantly, hemolysis assays confirmed that the synthesized nanoparticles were non-hemolytic even at high concentrations, remaining well below the ASTM threshold of 5%. This highlights their blood compatibility and supports their safety for biomedical applications. Furthermore, MTT assays indicated that ZnO NPs were less cytotoxic to blood cells than Cu NPs, suggesting a more favourable safety profile. Overall, these findings indicate that green-synthesized ZnO and Cu nanoparticles not only exhibit potent antimicrobial and antifungal activity but also maintain good hemocompatibility, making them prospective candidates for future applications in the delivery of drugs, infection control, and other biomedical fields. Future research could explore incorporating biogenic ZnO and Cu nanoparticles derived from Shorea robusta resin into advanced biomedical systems such as implant coatings, wound dressings, and controlled drug delivery platforms. Further studies on their long-term stability, in vivo performance, and molecular mechanisms of anti-microbial action would help strengthen their translational potential. Moreover, optimizing synthesis conditions and surface modification strategies could enhance their selectivity and overall performance, paving the way for the development of sustainable and therapeutically relevant nanomaterials. Declarations Conflicts of Interest The authors have declared no conflict of interest. Ethics Declaration This study involving humans was approved by the Sharda University School of Medical Sciences and Research (SMS&R) Institutional Ethics Committee (SU/SMS&R/76-A/2024/125). All participants provided written informed consent to participate in this study. Funding The work was supported by SEED grant from Sharda University (Grant No.SU/SF/2023/11). Author Contribution Shuaib Burgee: Designed and performed the experiments, methodology, data collection, analysis, original draft, and writing.V. Barghavi: Designed the experiments, data collection, calculations, original draft, writing, and formal analysis.Swati Bhati: Designed the experiments and figures.S. Shankara Narayanan: Methodology, formal analysis, writing, review & editing.Soumi Sadhu: Conceptualization, Project Administration, Writing - Review & EditingAll authors have approved the final version of the manuscript. Acknowledgements The authors gratefully acknowledge Sharda University for the sanction of the SEED funding (Grant No. SU/SF/2023/11) awarded to Dr. Soumi Sadhu, which provided crucial support for the studies contributing to this chapter. The authors also extend their gratitude to the DST-FIST facility for providing the necessary infrastructure and support for the experimental work. Data Availability All data supporting the findings of this study have been included in the main text; further inquiries can be directed to the corresponding author. References Firdhouse, M. J., & Lalitha, P. (2015). Biosynthesis of Silver Nanoparticles and Its Applications. Journal of Nanotechnology , 2015 (1), 829526. https://doi.org/10.1155/2015/829526 Sa, K., F, N., S, K., A, I., & G, H. (2018). Green synthesis of ZnO and Cu-doped ZnO nanoparticles from leaf extracts of Abutilon indicum, Clerodendrum infortunatum, Clerodendrum inerme and investigation of their biological and photocatalytic activities. Materials science & engineering. C, Materials for biological applications , 82 . https://doi.org/10.1016/j.msec.2017.08.071 Gurgur, E., Oluyamo, S. S., Adetuyi, A. O., Omotunde, O. I., & Okoronkwo, A. E. (2020). Green synthesis of zinc oxide nanoparticles and zinc oxide–silver, zinc oxide–copper nanocomposites using Bridelia ferruginea as biotemplate. SN Applied Sciences , 2 (5), 911. https://doi.org/10.1007/s42452-020-2269-3 Luzala, M. M., Muanga, C. K., Kyana, J., Safari, J. B., Zola, E. N., Mbusa, G. V.,… Memvanga, P. B. (2022). A Critical Review of the Antimicrobial and Antibiofilm Activities of Green-Synthesized Plant-Based Metallic Nanoparticles. Nanomaterials , 12 (11), 1841. https://doi.org/10.3390/nano12111841. Singh, J., Dutta, T., Kim, K. H., Rawat, M., Samddar, P., & Kumar, P. (2018). Green’ synthesis of metals and their oxide nanoparticles: applications for environmental remediation. Journal of Nanobiotechnology , 16 (1), 84. https://doi.org/10.1186/s12951-018-0408-4 Loganathan, S., Shivakumar, M. S., Karthi, S., Nathan, S. S., & Selvam, K. (2021). Metal oxide nanoparticle synthesis (ZnO-NPs) of Knoxia sumatrensis (Retz.) DC. Aqueous leaf extract and It’s evaluation of their antioxidant, anti-proliferative and larvicidal activities. Toxicology Reports , 8 , 64–72. https://doi.org/10.1016/j.toxrep.2020.12.018 Ismail, N. A., Shameli, K., Che Jusoh, N. W., Ali, R., Mohamad Sukri, R., S. N. A., & Isa, M. (2021). E. D. Preparation of Copper Nanoparticles by Green Biosynthesis Method: A Short Review. IOP Conference Series: Materials Science and Engineering , 1051 (1), 012084. https://doi.org/10.1088/1757-899X/1051/1/012084 Mahmoud, A. E. D., El-Maghrabi, N., Hosny, M., & Fawzy, M. (2022). Biogenic synthesis of reduced graphene oxide from Ziziphus spina-christi (Christ’s thorn jujube) extracts for catalytic, antimicrobial, and antioxidant potentialities. Environmental Science and Pollution Research International , 29 (59), 89772. https://doi.org/10.1007/s11356-022-21871-x Masoudpanah, S. M. (2017). Structural, Magnetic and Photocatalytic Properties of BiFeO3 Nanoparticles. Journal of Nanostructures , 7 (3). https://doi.org/10.18502/jns.v7i3.3 Khan, S. T., Musarrat, J., & Al-Khedhairy, A. A. (2016). Countering drug resistance, infectious diseases, and sepsis using metal and metal oxides nanoparticles: Current status. Colloids and Surfaces B: Biointerfaces , 146 , 70–83. https://doi.org/10.1016/j.colsurfb.2016.05.046 Menazea, A. A., Ismail, A. M., & Samy, A. (2021). Novel Green Synthesis of Zinc Oxide Nanoparticles Using Orange Waste and Its Thermal and Antibacterial Activity. Journal of Inorganic and Organometallic Polymers and Materials , 31 (11), 4250–4259. https://doi.org/10.1007/s10904-021-02074-2 N, M., Ab, G. T. M. C. D. H. S., S., N, D., & O, R. (2019). Green synthesis of silver nanoparticles: effect of synthesis reaction parameters on antimicrobial activity. World journal of microbiology & biotechnology , 35 (6). https://doi.org/10.1007/s11274-019-2664-3 T, L., W, Y., & Q, L. (2010). Comparison of the phenolic content and antioxidant activities of Apocynum venetum L. (Luo-Bu-Ma) and two of its alternative species. International journal of molecular sciences , 11 (11). https://doi.org/10.3390/ijms11114452 Ovais, M., Khalil, A. T., Islam, N. U., Ahmad, I., Ayaz, M., Saravanan, M., … Mukherjee,S. (2018). Role of plant phytochemicals and microbial enzymes in biosynthesis of metallic nanoparticles. Applied Microbiology and Biotechnology , 102 (16), 6799–6814. https://doi.org/10.1007/s00253-018-9146-7. Murthy, K. S. R., Lakshmi, N., & Ramulu, D. R. (2011). Biological activity and phytochemical screening of the oleoresin of Shorea robusta Gaertn. f. Tropical and subtropical agroecosystems , 14 (3), 787–791. Zeghoud, S., Hemmami, H., Ben Seghir, B., Ben Amor, I., Kouadri, I., Rebiai, A.,… Simal-Gandara, J. (2022). A review on biogenic green synthesis of ZnO nanoparticles by plant biomass and their applications. Materials Today Communications , 33 , 104747. https://doi.org/10.1016/j.mtcomm.2022.104747. Chaudhuri, S. K., & Malodia, L. (2017). Biosynthesis of zinc oxide nanoparticles using leaf extract of Calotropis gigantea: characterization and its evaluation on tree seedling growth in nursery stage. Applied Nanoscience , 7 (8), 501–512. https://doi.org/10.1007/s13204-017-0586-7 Nilavukkarasi, M., Vijayakumar, S., & Prathipkumar, S. (2020). Capparis zeylanica mediated bio-synthesized ZnO nanoparticles as antimicrobial, photocatalytic and anti-cancer applications. Materials Science for Energy Technologies , 3 , 335–343. https://doi.org/10.1016/j.mset.2019.12.004 Espitia, P. J. P., Otoni, C. G., & Soares, N. F. F. (2016). Chapter 34 - Zinc Oxide Nanoparticles for Food Packaging Applications. In J. Barros-Velázquez (Ed.), Antimicrobial Food Packaging (pp. 425–431). Academic. https://doi.org/10.1016/B978-0-12-800723-5.00034-6 Salvadori, M. R., Ando, R. A., Nascimento, C. A. O. do, & Corrêa, B. (2014). Intracellular Biosynthesis and Removal of Copper Nanoparticles by Dead Biomass of Yeast Isolated from the Wastewater of a Mine in the Brazilian Amazonia. PLOS ONE , 9 (1), e87968. https://doi.org/10.1371/journal.pone.0087968 Prado, J., Vidal, V. A, R., & Durán, T., C (2012). [Application of copper bactericidal properties in medical practice]. Revista medica de Chile , 140 (10), 1325–1332. https://doi.org/10.4067/s0034-98872012001000014 Mali, S. C., Dhaka, A., Githala, C. K., & Trivedi, R. (2020). Green synthesis of copper nanoparticles using Celastrus paniculatus Willd. leaf extract and their photocatalytic and antifungal properties. Biotechnology Reports , 27 , e00518. https://doi.org/10.1016/j.btre.2020.e00518 Naiel, B., Fawzy, M., Halmy, M. W. A., & Mahmoud, A. E. D. (2022). Green synthesis of zinc oxide nanoparticles using Sea Lavender (Limonium pruinosum L. Chaz.) extract: characterization, evaluation of anti-skin cancer, antimicrobial and antioxidant potentials. Scientific Reports , 12 (1), 20370. https://doi.org/10.1038/s41598-022-24805-2 Liaqat, N., Jahan, N., Khalil-ur-Rahman, Anwar, T., & Qureshi, H. (2022). Green synthesized silver nanoparticles: Optimization, characterization, antimicrobial activity, and cytotoxicity study by hemolysis assay. Frontiers in Chemistry , 10 . https://doi.org/10.3389/fchem.2022.952006 Dejene, F. B. (2022). Characterization of low-temperature-grown ZnO nanoparticles: The effect of temperature on growth. Journal of Physics Communications , 6 (7), 075011. https://doi.org/10.1088/2399-6528/ac8049 Chen, L., Zhai, B., & Huang, Y. M. (2020). Rendering Visible-Light Photocatalytic Activity to Undoped ZnO via Intrinsic Defects Engineering. Catalysts , 10 (10), 1163. https://doi.org/10.3390/catal10101163 Fakhari, S., Jamzad, M., & Kabiri Fard, H. (2019). Green synthesis of zinc oxide nanoparticles: a comparison. Green Chemistry Letters and Reviews , 12 (1), 19–24. https://doi.org/10.1080/17518253.2018.1547925 Wijesinghe, U., Thiripuranathar, G., Menaa, F., Iqbal, H., Razzaq, A., & Almukhlifi, H. (2021). Green Synthesis, Structural Characterization and Photocatalytic Applications of ZnO Nanoconjugates Using Heliotropium indicum. Catalysts , 11 (7), 831. https://doi.org/10.3390/catal11070831 Jaishi, D. R., Ojha, I., Bhattarai, G., Baraili, R., Pathak, I., Ojha, D. R., … Sharma,K. R. (2024). Plant-mediated synthesis of zinc oxide (ZnO) nanoparticles using Alnus nepalensis D. Don for biological applications. Heliyon , 10 (20), e39255. https://doi.org/10.1016/j.heliyon.2024.e39255. Janaki, A. C., Sailatha, E., & Gunasekaran, S. (2015). Synthesis, characteristics and antimicrobial activity of ZnO nanoparticles. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy , 144 , 17–22. https://doi.org/10.1016/j.saa.2015.02.041 Pourmadadi, M., Holghoomi, R., shamsabadipour, A., Maleki-baladi, R., Rahdar, A., & Pandey, S. (2024). Copper nanoparticles from chemical, physical, and green synthesis to medicinal application: A review. Plant Nano Biology , 8 , 100070. https://doi.org/10.1016/j.plana.2024.100070 Mahmoodi, S., Elmi, A., & Hallaj-Nezhadi, S. (2018). Copper Nanoparticles as Antibacterial Agents. Journal of Molecular Pharmaceutics & Organic Process Research , 6 (1), 1–7. https://doi.org/10.4172/2329-9053.1000140 Chatterjee, A. K., Chakraborty, R., & Basu, T. (2014). Mechanism of antibacterial activity of copper nanoparticles. Nanotechnology , 25 (13), 135101. https://doi.org/10.1088/0957-4484/25/13/135101 Neamah, S. A., Albukhaty, S., Falih, I. Q., Dewir, Y. H., & Mahood, H. B. (2023). Biosynthesis of Zinc Oxide Nanoparticles Using Capparis spinosa L. Fruit Extract: Characterization, Biocompatibility, and Antioxidant Activity. Applied Sciences , 13 (11), 6604. https://doi.org/10.3390/app13116604 Cheng, L. C., Jiang, X., Wang, J., Chen, C., & Liu, R. S. (2013). Nano–bio effects: interaction of nanomaterials with cells. Nanoscale , 5 (9), 3547–3569. https://doi.org/10.1039/C3NR34276J Yedgar, S., Barshtein, G., & Gural, A. (2022). Hemolytic Activity of Nanoparticles as a Marker of Their Hemocompatibility. Micromachines , 13 (12), 2091. https://doi.org/10.3390/mi13122091 ASTM E2524-08( (2013). - Standard Test Method for Analysis of Hemolytic Properties of Nanoparticles (Withdrawn 2022). (n.d.). iTeh Standards . Retrieved September 6, 2025, from https://standards.iteh.ai/catalog/standards/astm/86e47339-603a-4ec0-86f9-dacaaddda4db/astm-e2524-08-2013 Bhattacharya, D., Santra, C. R., Ghosh, A. N., & Karmakar, P. (2014). Differential toxicity of rod and spherical zinc oxide nanoparticles on human peripheral blood mononuclear cells. Journal of biomedical nanotechnology , 10 (4), 707–716. Ashoub, M. H., Amiri, M., Fatemi, A., & Farsinejad, A. (2024). Evaluation of ferroptosis-based anti-leukemic activities of ZnO nanoparticles synthesized by a green route against Pre-B acute lymphoblastic leukemia cells (Nalm-6 and REH). Heliyon , 10 (17), e36608. https://doi.org/10.1016/j.heliyon.2024.e36608 Li, Z., Yin, X., Lyu, C., Cui, S., Wang, J., Si, Y., & Xu, R. (2022). PB1961: Zno Nanoparticles Induce Multiple Myeloma Cell Death Based On Reactive Oxygen Species And Caspase Signaling Pathway. HemaSphere , 6 (Suppl), 1834–1835. https://doi.org/10.1097/01.HS9.0000850680.43770.6b Fard, M. Z., Fatholahi, M., Abyadeh, M., Bakhtiarian, A., Mousavi, S. E., & Falahati, M. (2020). The Investigation of the Cytotoxicity of Copper Oxide Nanoparticles on Peripheral Blood Mononuclear Cells. Nanomedicine Research Journal , 5 (4), 364–368. https://doi.org/10.22034/nmrj.2020.04.008 Additional Declarations No competing interests reported. Supplementary Files graphicalabstract2300.tif Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Feb, 2026 Reviews received at journal 04 Feb, 2026 Reviews received at journal 29 Jan, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers agreed at journal 22 Jan, 2026 Reviewers agreed at journal 25 Oct, 2025 Reviewers invited by journal 20 Oct, 2025 Editor assigned by journal 20 Oct, 2025 Submission checks completed at journal 19 Oct, 2025 First submitted to journal 17 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7883522","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":534868421,"identity":"e9d1e8d3-5192-4e50-bc68-4c6ef8c04ece","order_by":0,"name":"Shuaib Burgee","email":"","orcid":"","institution":"Sharda University","correspondingAuthor":false,"prefix":"","firstName":"Shuaib","middleName":"","lastName":"Burgee","suffix":""},{"id":534868422,"identity":"1f5308bf-6715-4016-a7b3-3ffb7ee33495","order_by":1,"name":"Barghavi V.","email":"","orcid":"","institution":"Sharda University","correspondingAuthor":false,"prefix":"","firstName":"Barghavi","middleName":"","lastName":"V.","suffix":""},{"id":534868423,"identity":"3adfaf82-7611-4f6e-a242-7ca6ef423eec","order_by":2,"name":"Swati Bhati","email":"","orcid":"","institution":"Sharda University","correspondingAuthor":false,"prefix":"","firstName":"Swati","middleName":"","lastName":"Bhati","suffix":""},{"id":534868425,"identity":"0c07cf48-4f1b-415c-ad03-04b974ab427e","order_by":3,"name":"S. Shankara Narayanan","email":"","orcid":"","institution":"Sharda University","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"Shankara","lastName":"Narayanan","suffix":""},{"id":534868426,"identity":"37296042-072b-4434-a54c-f9816b5efb67","order_by":4,"name":"Soumi Sadhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYLCCBBBxg4HxAWMDkHEALCZBlBZmA+K1MEC0sEkgacEN5Nt7TDc8YKiT57vdY1b5c4ddPt/xA4wffjBY5OHSYnDmjNmNBIbDhjPvnDG7zXsm2XLmmQRmyR4GiWKcWiRyQFoOMG64kWN2m7GN2cDgQAKDNNAviQ24HDYDrKXOHqSl8GdbvYHB+QfMv/FpYbgB1sKcCNLCwNt22MDgRgIbXlsMzhwru5FgcDh55o20YmnetuMGkjcetln2GOBxWHvztps/Kups+24kb/z4s63agO988uEbQBHcDoPYhcIDxY4BdoWjYBSMglEwCogDAPJBXMozOi4aAAAAAElFTkSuQmCC","orcid":"","institution":"Sharda University","correspondingAuthor":true,"prefix":"","firstName":"Soumi","middleName":"","lastName":"Sadhu","suffix":""}],"badges":[],"createdAt":"2025-10-17 06:53:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7883522/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7883522/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94795816,"identity":"1abe27f8-be48-4343-bedf-592d7d03be64","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173318,"visible":true,"origin":"","legend":"","description":"","filename":"FIg.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/7b9e318399795f5aa21b9d06.tif"},{"id":94826053,"identity":"979c7299-ebd3-47dd-b097-01ade2e8f057","added_by":"auto","created_at":"2025-10-31 06:50:59","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":218354,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/a4ff4369d5d46303f2f6ad21.tif"},{"id":94824752,"identity":"18c9ada8-0d39-4af5-a473-cb471d93efb4","added_by":"auto","created_at":"2025-10-31 06:49:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3336942,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/763bd2eaeecc6d6e5908fec6.docx"},{"id":94795822,"identity":"847804f6-a9a7-4f21-9916-e5d9cb5719bc","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18782,"visible":true,"origin":"","legend":"","description":"","filename":"Table.1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/00e2e9a193825f072f1fd906.docx"},{"id":94826156,"identity":"3436b346-1f5e-41b3-a1ed-41e010af2ae4","added_by":"auto","created_at":"2025-10-31 06:51:11","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1395972,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/e1c418ac3c9463b9c4b1d1ef.tif"},{"id":94826017,"identity":"6cc40462-2cc7-46c9-a0db-b55e056c3568","added_by":"auto","created_at":"2025-10-31 06:50:54","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1250196,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/63a36c38e5037b1ba13e9d33.tif"},{"id":94795830,"identity":"65337e72-22d7-472e-a210-7b400fd405ce","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":368830,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/884a3194a4d3f73bf32fed7e.tif"},{"id":94795828,"identity":"afd05c77-84c3-4536-9d50-ab7364e6f765","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":524684,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/aada8ae5fd2ef3dde00bfb1a.tif"},{"id":94795826,"identity":"ad4f323d-387d-4891-a427-a17771e613a7","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17778,"visible":true,"origin":"","legend":"","description":"","filename":"FigureCaptions.docx","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/54b5594338a7187b4181c497.docx"},{"id":94825985,"identity":"18eba06a-8402-41aa-a62b-9cb37753d7f9","added_by":"auto","created_at":"2025-10-31 06:50:51","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":313930,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract2300.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/f60370d6fe8f3e338b026dc3.tif"},{"id":94795834,"identity":"f51964c9-19a6-4a2f-971a-eb2a36774a4a","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"json","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6388,"visible":true,"origin":"","legend":"","description":"","filename":"7e9a84063c3d48859ccf0f3ffbbb8081.json","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/d0412648662d778a6646aaea.json"},{"id":94825618,"identity":"af3cce11-f6cc-4f38-b329-9b472a485538","added_by":"auto","created_at":"2025-10-31 06:50:31","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133565,"visible":true,"origin":"","legend":"","description":"","filename":"7e9a84063c3d48859ccf0f3ffbbb80811enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/c6eb5dc1655362ff760be737.xml"},{"id":94795836,"identity":"1d13a8ec-c8c6-4c08-9487-fce8cf566458","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173318,"visible":true,"origin":"","legend":"","description":"","filename":"FIg.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/90aea6dd12641452e78fbba8.tif"},{"id":94825686,"identity":"267d9859-9fa8-4f49-8615-3b374800af1b","added_by":"auto","created_at":"2025-10-31 06:50:34","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":218354,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/8d784df394b7d0ea90906369.tif"},{"id":94825261,"identity":"87595d76-5ebe-49d4-a999-fc9e78b4a6cc","added_by":"auto","created_at":"2025-10-31 06:50:01","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1395972,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/b03f13f9aad339c0b8acc2f3.tif"},{"id":94826020,"identity":"eee34061-8732-49b7-902f-1da7d04a2cd5","added_by":"auto","created_at":"2025-10-31 06:50:55","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1250196,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/535eeea9bed012002ad7de35.tif"},{"id":94824840,"identity":"d404b655-0d7e-49c4-b5f2-c8a541059f0b","added_by":"auto","created_at":"2025-10-31 06:49:25","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":368830,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/6bc299d4d803a38a198a4fd3.tif"},{"id":94824673,"identity":"a39aa101-ed59-4cd5-93b6-a946d784eaee","added_by":"auto","created_at":"2025-10-31 06:49:12","extension":"tif","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":524684,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/74a7557eccf35ff21ec3b220.tif"},{"id":94825660,"identity":"f30000c9-5c67-4d59-9217-6ff712d85a42","added_by":"auto","created_at":"2025-10-31 06:50:32","extension":"jpeg","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173318,"visible":true,"origin":"","legend":"","description":"","filename":"FIg.1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/a24dd0b05ee4281a26e14331.jpeg"},{"id":94826044,"identity":"928e48e0-9d6c-451e-a910-aa4be147a538","added_by":"auto","created_at":"2025-10-31 06:50:58","extension":"jpeg","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":218354,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/e919582a6f7bfe5028e062ff.jpeg"},{"id":94795840,"identity":"be31ff5b-f1a6-4ba2-b8bb-96c34a49cba8","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"jpeg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":711262,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/8227a0acbf7ee05b1e184838.jpeg"},{"id":94825750,"identity":"fef1062e-21e0-4e4d-8d3d-fef2f5afe7e6","added_by":"auto","created_at":"2025-10-31 06:50:40","extension":"jpeg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1055152,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/b8442ba5dbd5f1d2534b7635.jpeg"},{"id":94795838,"identity":"ade448b6-eca4-4f19-b1d7-8a86e7c8ab93","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"jpeg","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":294568,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/048990e385bc9f72d0a065c1.jpeg"},{"id":94795845,"identity":"f939a00d-bd9e-479d-9d46-a0651043ec21","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"jpeg","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":202638,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/9bf1c2fa5065c80a9b82c964.jpeg"},{"id":94826251,"identity":"dcda0921-848d-4a6e-9010-0966ca3b9e86","added_by":"auto","created_at":"2025-10-31 06:51:17","extension":"tif","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":313930,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract2300.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/db3ec40ccbee03b3197cc703.tif"},{"id":94825282,"identity":"035ce30a-2a3c-4168-8972-1f5122fcf97c","added_by":"auto","created_at":"2025-10-31 06:50:04","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31116,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/3918569d38798267f721acce.png"},{"id":94795842,"identity":"04cc9f11-c520-4d36-8b03-cde4b0526bcd","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31074,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/035d5b99f0ffe84b2f41f0d4.png"},{"id":94795849,"identity":"d7a6c088-9c4c-4ad3-9bc3-6ad00df808ef","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":401581,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/5f4bd52a66eb6be15d253107.png"},{"id":94795854,"identity":"529a7f21-f78f-41f7-9869-63630e16f2c4","added_by":"auto","created_at":"2025-10-30 19:55:07","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135039,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/a5f8bec2bb59d980dc0436fa.png"},{"id":94825896,"identity":"a4e8eb51-1cee-46a1-a90d-a20e14993574","added_by":"auto","created_at":"2025-10-31 06:50:48","extension":"png","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74085,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/c4abb066b43d14ae3b1992fa.png"},{"id":94795851,"identity":"5ba18a8d-4b7a-435c-b6b3-75cc7f1e9b3d","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29369,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/4fafec5a16ce6b322d84aee8.png"},{"id":94795846,"identity":"299a2a64-cafb-4860-b0dc-73166794b696","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":43409,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegraphicalabstract2300.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/68e378e3e439e97914eba4ee.png"},{"id":94824885,"identity":"cce1187e-ef4f-4460-818e-da0e9cd25aa2","added_by":"auto","created_at":"2025-10-31 06:49:31","extension":"xml","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132846,"visible":true,"origin":"","legend":"","description":"","filename":"7e9a84063c3d48859ccf0f3ffbbb80811structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/b2ee92cc84f0aa7e41f463e1.xml"},{"id":94795852,"identity":"7f432ef5-31ca-4c87-88cd-1869c465c744","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"html","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148261,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/9b2447706119a8c9dfe2b9cb.html"},{"id":94795815,"identity":"943032ec-dee6-4516-8e8a-8182ea8ba1a5","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31116,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV–Vis absorption spectrum of green-synthesized ZnO NPs. (b) The corresponding Tauc Plot for calculating the optical direct band gap of ZnO NPs. (c) UV–Vis absorption spectrum of green-synthesized Cu NPs. (d) FTIR Spectrum of ZnO/Cu synthesized NPs.\u003c/p\u003e","description":"","filename":"OnlineFIg.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/b7fb5edf51b78fed44c80c95.png"},{"id":94795814,"identity":"85815881-a847-4f69-94a3-0363b702c871","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31074,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Nanoparticle Tracking Analysis (NTA) profile of green-synthesized ZnO nanoparticles showing a dominant size distribution centered around ~120 nm with a particle concentration of approximately 3.2 × 10⁸ particles/mL. (b) Nanoparticle Tracking Analysis (NTA) profile of Cu nanoparticles showing a bimodal size distribution with dominant peaks around 100 nm and 190 nm, and a total particle concentration of approximately 4 × 10⁸ particles/mL. Screenshot from the Nanoparticle Tracking Analysis (NTA) video showing the Brownian motion of dispersed (c) ZnO nanoparticles and (d) Cu NPs in aqueous suspension. Each bright spot represents an individual nanoparticle scattering light as it moves randomly, confirming good dispersion and colloidal stability.\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/18499d85d4fe4ee6641a9570.png"},{"id":94795823,"identity":"2990c640-6573-40c1-9ab8-7b93da893dab","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":289221,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of ZnO nanoparticles against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (a,b) and \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e(c,d) at concentrations of 50, 25, 12.5, and 6.25 mg/mL, with streptomycin (10 µg) as control. Antifungal activity of ZnO nanoparticles against \u003cem\u003eAspergillus flavus\u003c/em\u003e (e,f) and \u003cem\u003eCandida albicans\u003c/em\u003e (g,h) at the same concentrations, with amphotericin-B as control.\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/bbe303c8247c580fd5842779.png"},{"id":94795819,"identity":"45d68b6a-d4fe-4f39-a0c6-a7aab9dd9a25","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":170099,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of Cu nanoparticles against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (a,b) and \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (c,d) at concentrations of 50, 25, 12.5, and 6.25 mg/mL, with streptomycin (10 µg) as control. Antifungal activity of Cu nanoparticles against \u003cem\u003eAspergillus flavus\u003c/em\u003e (e,f) and \u003cem\u003eCandida albicans\u003c/em\u003e (g,h) at the same concentrations, with amphotericin-B as control.\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/2d03f6e2b921ac26b24519ca.png"},{"id":94795824,"identity":"e5732b39-75ea-4809-b145-432d7823cd31","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43708,"visible":true,"origin":"","legend":"\u003cp\u003eHemolysis assay of ZnO and Cu nanoparticles showing minimal hemolysis. b. Cu NPs caused \u0026lt;2% hemolysis at 1000 µg/mL, while ZnO NPs showed only 0.5% hemolysis at the same concentration\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/a4c589e307b93596011292f2.png"},{"id":94795817,"identity":"2df83a04-0430-41be-8a96-adf9aa013870","added_by":"auto","created_at":"2025-10-30 19:55:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":62793,"visible":true,"origin":"","legend":"\u003cp\u003eMTT assay showing lower cytotoxicity of ZnO nanoparticles compared to Cu nanoparticles in human white blood cells. At 300 µg/mL, ZnO NPs maintained ~80% cell viability, whereas Cu NPs reduced viability. Even at higher concentrations, ZnO NPs consistently exhibited higher cell viability than Cu NPs.\u003c/p\u003e","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/04e9eb8b5e6dd4b1cc4d8516.png"},{"id":94984695,"identity":"234c9c5b-5a26-47c8-9fca-b7102f49b6b7","added_by":"auto","created_at":"2025-11-03 06:55:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1732627,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/ac0adcbc-4dec-4cc1-8164-8e4c0cebb0bc.pdf"},{"id":94825042,"identity":"bb0c3d74-2dd0-4dba-9dfb-f95f51a51457","added_by":"auto","created_at":"2025-10-31 06:49:46","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":243191,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract2300.tif","url":"https://assets-eu.researchsquare.com/files/rs-7883522/v1/5659cdee1c4c9f3bd206a837.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plant-Based Fabrication of Zinc oxide and Copper Nanoparticles Using Shorea robusta Resin: Dual Evaluation of Safety and Antimicrobial Efficacy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanoscience and nanotechnology work toward enhancing the optical, electrical, magnetic, and catalytic functionalities of bulk materials to improve human lives[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Due to the damage caused by global warming, scientists worldwide are working hard to create new materials with unique features and minimal environmental hazard[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Nanomaterials are broadly classified into organic and inorganic nanoparticles, with metallic nanoparticles most commonly synthesized through chemical and physical methods[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Growing awareness of environmental degradation has prompted scientists to adopt greener, cost-effective, and non-toxic methods for synthesizing and fabricating nanomaterials[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nanoparticle synthesis using plants is often favoured over microbial methods due to its simplicity, ease of scaling up, the vast diversity of plant species, and the abundance of phytochemicals they contain, such as alkaloids, steroids, terpenoids, tannins, saponins, polyphenols, and phenolic acids[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. They function as capping or stabilizing agents in addition to reducing agents[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTypically, metal nanoparticles possess distinct physical, chemical, and biological properties compared to their bulk forms, largely due to their exceptionally high surface-to-volume ratio[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It has been proposed that the phyto-synthesis of nanoparticles is attributed to phenolic chemicals[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Due to their antioxidant properties, phenolic compounds can be used as stabilizing and reducing agents while creating nanomaterials[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Bioactive substances, viz., alkaloids, glycosides, phenols, tannins, steroids, and terpenoids that contribute to strong antibacterial properties, were found in very high concentrations during phytochemical screening of the oleoresin of \u003cem\u003eShorea robusta\u003c/em\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZeghoud et al. claim that the plant species used for green synthesis has a minimal influence on the yield and morphology of ZnO nanoparticles, except when the extract lacks sufficient bioactive compounds. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. ZnO is the most widely used of them at the nanoscale due to its remarkable scientific characteristics, including high excitonic binding energy and band gap[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. ZnO NPs are well-known among nanometal oxides for their antimicrobial, anti-inflammatory, and anticancer properties[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Moreover, ZnO is also classified as a \"GRAS\" (Generally Recognized as Safe) chemical by the US Food and Drug Administration (FDA)[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Copper is an abundant metal and serves as an essential trace element in most living organisms[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It has been acknowledged by the U.S. Environmental Protection Agency (EPA) as the first and sole metal officially registered for its antimicrobial activity. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. When reduced to the nanoscale, copper exhibits unique physicochemical properties that make it highly versatile, with applications across various industries such as solar cells, wood preservatives, gas sensors, and high-temperature superconductors. Similarly, the catalytic, high electrical conductivity, optical, antifungal, and antibacterial properties of CuNPs (Copper nanoparticles) make them the most sought-after of the NPs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis paper describes a green method for synthesising CuNPs and ZnO NPs using resin extract from \u003cem\u003eShorea robusta\u003c/em\u003e. All characterization results provided clear evidence of nanoparticle formation. Its antifungal and antibacterial properties against the pathogenic fungus \u003cem\u003eAspergillus flavus\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e, as well as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e were evaluated. The Cu and ZnO nanoparticles were further evaluated for their biocompatibility, specifically focusing on hemocompatibility and cytocompatibility.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003eShorea robusta\u003c/em\u003e (SR) resin was procured from DKC Agrotech Pvt. Ltd., India, and used for nanoparticle synthesis. Ethanol, zinc acetate dihydrate (Zn(CH₃COO)₂\u0026middot;2H₂O; QUALIGENS), and copper(II) sulfate pentahydrate (CuSO₄\u0026middot;5H₂O; CAS No. 7758-99-8; Sigma-Aldrich) were employed as precursor materials. Nutrient Agar, Nutrient Broth, and Potato Dextrose Agar (Himedia) were used for microbial culture and antimicrobial assays. The microbial strains utilized in this study included \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MTCC 3160), \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (MTCC 863), \u003cem\u003eCandida albicans\u003c/em\u003e (MTCC 183), and \u003cem\u003eAspergillus flavus\u003c/em\u003e (MTCC 277). All strains were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India.\u003c/p\u003e\u003cp\u003eThe reagents and consumables used for the assays comprised sterile petriplates (TARSONS), 96-well microtiter plates, phosphate-buffered saline (PBS, pH 7.4), Triton X-100 (Sigma), Amphotericin B (100 units/mL), and Streptomycin (10 \u0026micro;g/mL). For cytotoxicity and bioactivity assessments, MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Himedia), RPMI-1640 culture medium (Himedia), fetal bovine serum (FBS; Sigma), and red cell lysis buffer (RCLB; Sigma) were used. The ELISA readings were recorded using a BioTek 800 TS Absorbance Reader. DMSO (dimethyl sulfoxide; Sigma) was used as a solvent, and Histopaque-1077 (Sigma) was used for cell isolation and density gradient separation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Characterization Techniques:\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe UV\u0026ndash;Visible absorption spectra were recorded using a Shimadzu UV-1900i double-beam spectrophotometer. FTIR spectra were obtained using a Nicolet iS50 FTIR spectrometer, from Thermo Scientific. The particle size distribution of the samples was determined through nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument (Malvern Panalytical, Netherlands) equipped with a 488 nm laser and a 500 nm long-pass filter. For NTA measurements, all samples were diluted to a concentration of 10\u003csup\u003e6\u003c/sup\u003e\u0026ndash;10\u003csup\u003e7\u003c/sup\u003e particles/mL. Each sample was analyzed in five replicates, and 30\u0026ndash;60 s videos were recorded for each run, capturing at least 200 valid particle tracks per analysis. During all measurements, the light scatter mode (LSM) camera level was maintained between 12 and 14. Data acquisition and analysis were performed using Nanosight software (version 3.0) with standard parameters. The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation values were calculated from three independent video recordings.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Preparation of ethanolic \u003cem\u003eShorea robusta\u003c/em\u003e resin extract:\u003c/h2\u003e\u003cp\u003eAn ethanolic extract of \u003cem\u003eShorea robusta\u003c/em\u003e resin was made by measuring 10 g and thoroughly crushing it with a mortar and pestle. Pure ethanol was used to dissolve the crushed resin, which was then filtered through Whatman 40 filter paper and utilized as the biogenic source to produce the nanoparticles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Green synthesis of ZnO NPs:\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn brief, 25 mL (0.5 M) zinc acetate dihydrate was mixed with 2.5 mL of \u003cem\u003eShorea robusta\u003c/em\u003e resin extract. The pH of the mixture was 6.13, and to maintain it at 8, 2 M NaOH was added drop by drop. It was then stirred and heated at 70\u0026deg;C for 30 minutes, resulting in complete reduction and the appearance of a white precipitate. The resultant material was then collected by decantation, cleaned of residues using distilled water, and oven-dried for an entire night at 70\u0026deg;C to produce powdered ZnO nanoparticles. For subsequent characterization and application, the material was stored at ambient temperature in an airtight container. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.5. Green Synthesis of CuNPs\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e50 mL (5 mM) of copper sulphate solution was combined with 5 mL of \u003cem\u003eShorea robusta\u003c/em\u003e resin extract to create plant extract resin copper nanoparticles. A solution of NAOH (1N) was added to bring the pH down to 7. Furthermore, a green colour mixture solution was produced. After centrifugation, the pellet collected was dried overnight at 60 degrees in a hot air oven to produce powder. Powder was collected and stored at 4 degrees temperature for further use [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Agar well diffusion assay:\u003c/h2\u003e\u003cp\u003eNutrient agar media was poured into the plates in sterile conditions. In the appropriate conical flasks, nutrient broth was made for the cultures of \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (MTCC 3160) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MTCC 863), which were then maintained in the shaker for a whole day. The cultures were spread throughout the nutrient agar medium using a glass spreader. For the controls and test compounds, Positive control: 10 mg antibiotic streptomycin, Negative control: pure ethanol, Test compounds: ZnO and Cu NPs with various stock concentrations 50, 25, 12.5, 6.25 mg/mL, were used, and the wells were punched using a gel punch. 50 \u0026micro;L of different concentrations of ZnO and CuNPs were loaded into the wells, and the plates were kept in a Biochemical Oxygen Demand (BOD) incubator for 24 hours. Using a vernier calliper, the diameter of the zone (in millimetres) was recorded. The inhibitory zone was calculated using the formula:\u003c/p\u003e\u003cp\u003eZone of inhibition (mm)\u0026thinsp;=\u0026thinsp;Diameter of zone (mm) - Diameter of well (mm).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Anti-fungal agar well diffusion Assay:\u003c/h2\u003e\u003cp\u003eWe procured \u003cem\u003eAspergillus flavus\u003c/em\u003e (MTCC 277) and \u003cem\u003eCandida albicans\u003c/em\u003e (MTCC 183) from the IMTECH Microbial Type Culture Collection in Chandigarh. One colony was inoculated in Potato Dextrose Broth (PDB), and was cultured for overnight at 37\u0026deg;C with continuous shaking at 150 rpm. After transferring 60 \u0026micro;l of the overnight culture into 3 mL of PDB, it was cultivated at 37\u0026deg;C (150 rpm) until the optical density at 600 nm reached the 0.5 McFarland standard. The 100 \u0026micro;L of \u003cem\u003eCandida albicans\u003c/em\u003e culture from the previous stage was distributed onto Potato Dextrose Agar \u003cem\u003e(\u003c/em\u003ePDA) using a sterile cotton swab. Following this, the PDA plates were incubated at 37\u0026deg;C for 48 hours. ZnO and Cu NPs doses ranging from 6.25 to 50 mg/mL were added to the wells in 50 \u0026micro;L increments. Amphotericin B (100 units/disc) acted as a positive control, and the solvent as a negative control. The inhibition zone, or clear area around the disc, was measured with a vernier calliper to determine its diameter.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Hemocompatibility test:\u003c/h2\u003e\u003cp\u003eHemolysis assay was used to examine the cytotoxic effects of ZnO and Cu NPs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The experiment was carried out under the applicable institutional policies and procedures, and informed consent was acquired. The Sharda University School of Medical Sciences and Research (SMS\u0026amp;R) Institutional Ethics Committee examined and accepted the study protocol, assigning approval reference number SU/SMS\u0026amp;R/76-A/2024/125. A healthy individual's freshly obtained blood was collected in an anticoagulant-containing tube, and centrifuged for five minutes at 3000 rpm after adding phosphate-buffered saline (PBS) (v/v; pH 7.2). The pellet was further washed with PBS at 3000 rpm. The RBCs obtained were further diluted with PBS. ZnO and Cu NPs were added to diluted erythrocyte suspensions at concentrations of 50, 75, 100, 250, 500, 750, and 1000 \u0026micro;g/mL, gently mixed, and then incubated for 4 hours at 37\u0026deg;C. Triton X-100 (10%) was used as the positive control, and PBS (normal) as the negative control. The samples were incubated and then centrifuged. The supernatant was then collected, and absorbance at 540 nm was measured using an ELISA reader (BioTek 800 TS). To calculate the percentage hemolysis, the formula was as follows:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHemolysis (%) = [(Mean OD of sample - Mean OD of negative control)/ (Mean OD of positive control - Mean OD of negative control) \u0026times; 100].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Cytocompatibility test\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn a heparinized vacutainer, human whole blood was drawn. 10 mL of whole blood was diluted with 10 mL of RPMI-1640 before being stacked on top of 12.5 mL Histopaque-1077 gradient in centrifuge tubes. Following centrifugation at 2000 rpm for 20 minutes, the peripheral blood mononuclear cells layer was re-suspended in RPMI-1640 with 10% FBS after being washed with PBS. Different doses of ZnO and Cu NPs were used in the MTT test on peripheral blood mononuclear cells (PBMCs). Cells were seeded at 0.5 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL density in a 96-well plate. After being treated for 24 hours with varying concentrations of (50, 100, 200, 300 \u0026micro;g/mL) of ZnO and Cu NPs, the cells were incubated for 3\u0026ndash;4 hours at 37\u0026deg;C in media containing 5 mg/mL of MTT. After dissolving the resultant formazan crystals in DMSO, the absorbances were measured at 570 nm.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCell Viability% = [(Absorbance of treated sample - Absorbance of blank) / (Absorbance of untreated - Absorbance of blank)] * 100.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1 ZnO and Cu NP Nanoparticle Characterizations\u003c/h2\u003e\u003cp\u003eThe UV\u0026ndash;Vis absorption spectrum of the prepared ZnO nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) exhibits a pronounced absorption edge/peak centred at ~\u0026thinsp;366 nm. This absorption corresponds to a direct band-to-band transition; using Eg​ (eV)\u0026thinsp;=\u0026thinsp;1240 / λ (nm) gives an optical band gap of \u0026asymp;\u0026thinsp;3.39 eV. This value is in very good agreement with the expected band gap of wurtzite ZnO (\u0026asymp;\u0026thinsp;3.3\u0026ndash;3.4 eV) and indicates that the material is phase-pure ZnO with no large amounts of other semiconducting impurities [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The absorption peak at 366 nm is slightly blue-shifted (shorter wavelength) compared with bulk ZnO values reported near ~\u0026thinsp;368\u0026ndash;380 nm, which can be ascribed to quantum-confinement effects for small nanoparticle sizes. The broad decrease in absorbance toward longer wavelengths and the weak tail extending into the visible region could be attributed to sub-band-gap absorption from defect states (e.g., oxygen vacancies) and to light scattering by nanoparticles in the suspension[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Formore accurate determination of the optical band gap, a Tauc plot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) for a direct band transition (αhν)\u003csup\u003e2\u003c/sup\u003e vs hν was performed and the linear extrapolation of the low-energy edge yielded Eg\u0026thinsp;\u0026asymp;\u0026thinsp;3.39 eV, consistent with the value calculated directly from the absorption edge. These observations corroborate the formation of nanoscale ZnO with electronic properties appropriate for UV-active photocatalytic and optoelectronic applications. The UV\u0026ndash;Vis absorption spectrum of Cu-NPs is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. The spectrum features a band at \u0026asymp;\u0026thinsp;252 nm, which confirms the successful synthesis of Cu NPs. These results corroborate with previously reported observations[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe FTIR spectrum of both the green-synthesized ZnO and Cu nanoparticles were found to be similar and (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) showed distinct absorption bands at 3295 cm⁻\u0026sup1;, 2117 cm⁻\u0026sup1;, and 1636 cm⁻\u0026sup1;. The broad and intense absorption band around 3295 cm⁻\u0026sup1; corresponds to the O\u0026ndash;H stretching vibrations of hydroxyl groups and adsorbed water molecules present on the nanoparticle surface. This is a common feature in ZnO/Cu nanoparticles synthesized via aqueous or plant-extract-mediated routes, indicating the presence of surface \u0026ndash;OH groups and hydrogen-bonded moisture[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The weak absorption observed near 2117 cm⁻\u0026sup1; can be attributed to C\u0026thinsp;\u0026equiv;\u0026thinsp;C stretching or C\u0026thinsp;=\u0026thinsp;O overtone/C\u0026ndash;N\u0026ndash;C vibrations that arise from organic residues originating from plant phytochemicals [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Such features suggest partial retention of organic molecules that may act as capping or stabilizing agents during the green synthesis process. The peak appearing at 1636 cm⁻\u0026sup1; is assigned to the H\u0026ndash;O\u0026ndash;H bending vibration of molecular water and may also overlap with C\u0026thinsp;=\u0026thinsp;O stretching vibrations of amide groups or carboxylate ions from biomolecules in the plant extract, further confirming the interaction between phytochemicals and metallic ions during Cu/ZnO nanoparticle formation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The particle size distribution of the synthesized ZnO nanoparticles was analyzed using Nanoparticle Tracking Analysis (NTA), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. The NTA profile exhibits a prominent peak centred around ~\u0026thinsp;120 nm, indicating that majority of the particles fall within this size range. A smaller shoulder observed near 70\u0026ndash;80 nm suggests the presence of a minor population of smaller nanoparticles or aggregates. The concentration of nanoparticles at the main peak reaches approximately 3.2 \u0026times; 10⁸ particles/mL, reflecting a well-dispersed colloidal suspension with good stability. The relatively narrow distribution implies that the synthesis process produced uniformly sized ZnO nanoparticles with minimal aggregation, which is a desirable feature for optoelectronic and photocatalytic applications. Minor variations in particle size can be attributed to the presence of naturally formed agglomerates or residual capping molecules from the green synthesis process. The particle size distribution of the synthesized Cu nanoparticles was also analysed using NTA, as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. The NTA profile reveals a bimodal distribution, indicating the presence of two predominant nanoparticle populations. The first major peak appears around 95\u0026ndash;110 nm, while a secondary peak is observed near 180\u0026ndash;200 nm. The total particle concentration reaches approximately 4 \u0026times; 10⁸ particles/mL, suggesting a high colloidal density of dispersed Cu nanoparticles. The presence of a bimodal distribution may arise from partial aggregation or variation in nucleation and growth rates during synthesis. The smaller size fraction corresponds to well-dispersed primary Cu nanoparticles, while the larger fraction likely represents agglomerated or fused particles formed due to the high surface energy of nanosized Cu. Overall, the observed size range confirms the nanoscale nature of the synthesized Cu nanoparticles, typically below 200 nm. The relatively sharp and well-defined peaks indicate that the colloidal suspension is reasonably monodisperse with moderate stability, suitable for optical and catalytic applications.\u003c/p\u003e\u003cp\u003eRepresentative screenshots from the Nanoparticle Tracking Analysis (NTA) video are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c) and 2(d), displaying the Brownian motion of ZnO and Cu nanoparticles dispersed in aqueous medium, respectively. Each bright spot corresponds to an individual ZnO/Cu nanoparticle scattering laser light as it moves randomly due to collisions with solvent molecules. The intensity of scattered light varies with particle size \u0026mdash;larger particles appear as brighter spots, while smaller ones exhibit weaker scattering. The observed random trajectories confirm that the nanoparticles are well-dispersed and remain colloidally stable without significant sedimentation or aggregation during measurement. This dynamic visualization, recorded by NTA, was used to calculate the hydrodynamic diameter distribution shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a) and 2 (b). The clear and distinct scattering points further validate the nanoscale dimensions and optical activity of the synthesized ZnO and Cu NPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Anti-bacterial activity:\u003c/h2\u003e\u003cp\u003eThe antibacterial activity of synthesized ZnO and Cu nanoparticles was evaluated against selected pathogens, namely \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e, using the disc diffusion method. The diameters of the zones of inhibition were measured in millimeters and are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;d and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;d. In the disc diffusion assay, the synthesized ZnO nanoparticles exhibited significant antibacterial activity against all tested bacterial strains. The results clearly demonstrated that antibacterial activity, expressed as the zone of inhibition, increased with rising concentrations of ZnO nanoparticles (6.25, 12.5, 25, and 50 mg/mL). This effect may be attributed to the higher production of H₂O₂ and the generation of reactive oxygen species (ROS) from the nanoparticle surface at high concentrations.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnti-microbial activity of Zinc oxide and Copper nanoparticles synthesized from \u003cem\u003eShorea robusta\u003c/em\u003e resin extracts\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMicro organisms\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eZinc Oxide Nanoparticles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c10\" namest=\"c6\"\u003e\u003cp\u003eCopper\u003c/p\u003e\u003cp\u003eNanoparticles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eReference Standards\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration (mg/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e12.5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e6.25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e12.5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e6.25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003eStreptomycin\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e10 mcg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003eAmphotericin-B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGram-positive bacteria\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"11\" nameend=\"c12\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eZone (in mm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGram-negative bacteria\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas stutzeri\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eYeast\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFungi\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAspergillus flavus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAmong the tested pathogens, \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e showed the largest zone of inhibition (20 mm), whereas \u003cem\u003eStaphylococcus aureus\u003c/em\u003e exhibited a comparatively smaller zone (12 mm), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;d. These findings are consistent with previous studies. For instance, Chinnammal Janaki et al. (2015) reported antibacterial activity of ZnO nanoparticles with inhibition zones of 10 mm against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, 10 mm against \u003cem\u003eCandida albicans\u003c/em\u003e, and 12 mm against \u003cem\u003ePenicillium notatum\u003c/em\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCu nanoparticles also exhibited notable antibacterial activity, with a stronger effect against \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (17 mm) compared to \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (13 mm), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;d. The antibacterial action of Cu NPs is primarily attributed to electrostatic interactions with the cell walls of gram-negative bacteria. Their strong affinity for carboxyl and amine groups which are key components of bacterial cell walls further enhances their antimicrobial potential [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt the nanoscale, Cu particles exert antibacterial effects through multiple mechanisms. These include adhesion of NPs to gram-negative bacterial cell walls due to electrostatic attraction, disruption of cell membrane proteins, denaturation of intracellular proteins, and interactions with phosphorus- and sulphur-containing biomolecules such as DNA[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In a comprehensive study, Chatterjee et al. used \u003cem\u003eE. coli\u003c/em\u003e as a model system to investigate these mechanisms. They reported that treatment with the minimum bactericidal concentration (MBC) dose of CuNPs induced a 2.5-fold increase in intracellular reactive oxygen species (ROS). This ROS overproduction triggered lipid peroxidation, protein oxidation, and DNA degradation, ultimately leading to bacterial cell death[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.3. Anti-fungal activity\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003eThe antifungal activity of synthesized ZnO nanoparticles was evaluated against the selected fungal pathogens \u003cem\u003eAspergillus flavus\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e using the disc diffusion method. The diameters of the zones of inhibition (in millimeters) are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (e\u0026ndash;h). The synthesized ZnO nanoparticles exhibited significant antifungal activity against both fungal strains. Similar to the antibacterial results, the antifungal activity increased with increasing concentrations of ZnO nanoparticles (6.25, 12.5, 25, and 50 mg/mL). Notably, \u003cem\u003eA. flavus\u003c/em\u003e was more sensitive, showing the highest zone of inhibition (29 mm), while \u003cem\u003eC. albicans\u003c/em\u003e exhibited a smaller zone (12 mm). By contrast, Cu nanoparticles displayed only moderate antifungal action, with inhibition zones of 13 mm for both fungal species.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Bio-compatibility:\u003c/h2\u003e\u003cp\u003eIn the present study, ZnO and Cu nanoparticles synthesized using \u003cem\u003eShorea robusta\u003c/em\u003e resin extract were employed for hemolysis testing. Hemolysis results from either direct or indirect damage to the red blood cell (RBC) membrane and is widely accepted as a reliable marker of biological incompatibility. One of the standard methods to evaluate whether a biomaterial is safe for blood-contacting applications is to measure its hemolytic activity. RBCs undergo lysis when their membranes are disrupted, releasing hemoglobin into the medium[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Among the various cell types that can be utilized for assessing nanoparticle-induced toxicity[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], RBCs remain the most suitable, as nanoparticles, regardless of origin, intended use, or route of administration, eventually reach the blood circulation and react with them, which are the predominant cellular constituent of blood circulation. This interaction often compromises RBC functionality. Since RBCs are structurally well studied, readily available, and simple to handle, they serve as excellent model cells for nanotoxicity studies. Several investigations have examined the impact of nanoparticles on RBCs, particularly their hemolytic activity, highlighting its importance as a key test for nanoparticle safety evaluation. However, comparison across different studies is often challenging due to variations in nanoparticle characterization methods and hemolysis testing protocols [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To address this, the American Society for Testing and Materials (ASTM) issued in 2008 a standardized procedure for evaluating hemolytic properties of nanoparticles [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which measures the hemoglobin content released following interaction between nanoparticles and RBCs.\u003c/p\u003e\u003cp\u003eIn our experiments, hemolysis assays were carried out at nanoparticle concentrations of 50, 75, 100, 250, 500, 750, and 1000 \u0026micro;g/mL. The results demonstrated minimal hemolysis, with the maximum observed at less than 1% for ZnO NPs and 2% for Cu NPs at the highest concentration tested (1000 \u0026micro;g/mL) (Figure. 5). These findings fall well below the 5% threshold defined by ASTM for hemocompatibility, thereby confirming that the synthesized nanoparticles are non-hemolytic. Overall, the results indicate that green-synthesized ZnO and Cu nanoparticles from \u003cem\u003eShorea robusta\u003c/em\u003e resin extract exhibit excellent blood compatibility, supporting their potential for future in vivo applications in drug delivery and other biomedical uses.\u003c/p\u003e\u003cp\u003ePrimary lymphocytes serve as an effective model for evaluating genotoxic effects, as they exhibit normal cellular responses unlike cancer or transformed cells, and are more likely to encounter nanoparticles in daily life[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The biosynthesized ZnO NPs and Cu NPs were tested for their cytotoxic effects on lymphocytes derived from PBMCs using the MTT assay. The findings in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e demonstrate that ZnO NPs and Cu NPs had cytotoxic effects on blood cells that were dose-dependent. Overall, ZnO NPs showed less cytotoxicity compared to CuNPs at the highest concentrations (300 \u0026micro;g/mL). Existing reports on the effects of ZnO NPs on human cells remain inconsistent; while some studies highlight their potential as strong anticancer agents, others describe them as non-toxic, supporting their application in consumer products[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Some findings suggest that ZnO nanoparticles exert low toxicity toward normal immune cells while maintaining significant anticancer activity, supporting their potential as a promising nanotherapeutic agent. ZnO nanoparticles demonstrate selective biological effects, exhibiting minimal cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) even at concentrations as high as 300 \u0026micro;g/mL, thereby underscoring their relative biocompatibility with non-transformed cells[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Zivari Fard et al. reported concentration-dependent cytotoxicity of CuO nanoparticles in human PBMCs assessed by MTT after exposure to 1\u0026ndash;200 \u0026micro;g/mL, with viability decreasing progressively at higher concentrations [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe present study demonstrated that ZnO and Cu nanoparticles synthesized using \u003cem\u003eShorea robusta\u003c/em\u003e resin extract possess remarkable antimicrobial properties and excellent biocompatibility. Both types of nanoparticles exhibited concentration-dependent antibacterial and antifungal activity, with ZnO NPs showing larger inhibition zones, particularly against \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e and \u003cem\u003eAspergillus flavus\u003c/em\u003e. Cu NPs also displayed strong activity, mainly through electrostatic interactions and ROS-mediated mechanisms, though their effects were comparatively moderate against fungal pathogens. Importantly, hemolysis assays confirmed that the synthesized nanoparticles were non-hemolytic even at high concentrations, remaining well below the ASTM threshold of 5%. This highlights their blood compatibility and supports their safety for biomedical applications. Furthermore, MTT assays indicated that ZnO NPs were less cytotoxic to blood cells than Cu NPs, suggesting a more favourable safety profile. Overall, these findings indicate that green-synthesized ZnO and Cu nanoparticles not only exhibit potent antimicrobial and antifungal activity but also maintain good hemocompatibility, making them prospective candidates for future applications in the delivery of drugs, infection control, and other biomedical fields.\u003c/p\u003e\u003cp\u003eFuture research could explore incorporating biogenic ZnO and Cu nanoparticles derived from \u003cem\u003eShorea robusta\u003c/em\u003e resin into advanced biomedical systems such as implant coatings, wound dressings, and controlled drug delivery platforms. Further studies on their long-term stability, in vivo performance, and molecular mechanisms of anti-microbial action would help strengthen their translational potential. Moreover, optimizing synthesis conditions and surface modification strategies could enhance their selectivity and overall performance, paving the way for the development of sustainable and therapeutically relevant nanomaterials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors have declared no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eEthics Declaration\u003c/h2\u003e\n\u003cp\u003eThis study involving humans was approved by the Sharda University School of Medical Sciences and Research (SMS\u0026amp;R) Institutional Ethics Committee (SU/SMS\u0026amp;R/76-A/2024/125). All participants provided written informed consent to participate in this study.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe work was supported by SEED grant from Sharda University (Grant No.SU/SF/2023/11).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eShuaib Burgee: Designed and performed the experiments, methodology, data collection, analysis, original draft, and writing.V. Barghavi: Designed the experiments, data collection, calculations, original draft, writing, and formal analysis.Swati Bhati: Designed the experiments and figures.S. Shankara Narayanan: Methodology, formal analysis, writing, review \u0026amp; editing.Soumi Sadhu: Conceptualization, Project Administration, Writing - Review \u0026amp; EditingAll authors have approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors gratefully acknowledge Sharda University for the sanction of the SEED funding (Grant No. SU/SF/2023/11) awarded to Dr. Soumi Sadhu, which provided crucial support for the studies contributing to this chapter. The authors also extend their gratitude to the DST-FIST facility for providing the necessary infrastructure and support for the experimental work.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study have been included in the main text; further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFirdhouse, M. J., \u0026amp; Lalitha, P. (2015). Biosynthesis of Silver Nanoparticles and Its Applications. \u003cem\u003eJournal of Nanotechnology\u003c/em\u003e, \u003cem\u003e2015\u003c/em\u003e(1), 829526. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2015/829526\u003c/span\u003e\u003cspan address=\"10.1155/2015/829526\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSa, K., F, N., S, K., A, I., \u0026amp; G, H. (2018). Green synthesis of ZnO and Cu-doped ZnO nanoparticles from leaf extracts of Abutilon indicum, Clerodendrum infortunatum, Clerodendrum inerme and investigation of their biological and photocatalytic activities. \u003cem\u003eMaterials science \u0026amp; engineering. C, Materials for biological applications\u003c/em\u003e, \u003cem\u003e82\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.msec.2017.08.071\u003c/span\u003e\u003cspan address=\"10.1016/j.msec.2017.08.071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGurgur, E., Oluyamo, S. S., Adetuyi, A. O., Omotunde, O. I., \u0026amp; Okoronkwo, A. E. (2020). Green synthesis of zinc oxide nanoparticles and zinc oxide\u0026ndash;silver, zinc oxide\u0026ndash;copper nanocomposites using Bridelia ferruginea as biotemplate. \u003cem\u003eSN Applied Sciences\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e(5), 911. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s42452-020-2269-3\u003c/span\u003e\u003cspan address=\"10.1007/s42452-020-2269-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuzala, M. M., Muanga, C. K., Kyana, J., Safari, J. B., Zola, E. N., Mbusa, G. V.,\u0026hellip; Memvanga, P. B. (2022). A Critical Review of the Antimicrobial and Antibiofilm Activities of Green-Synthesized Plant-Based Metallic Nanoparticles. \u003cem\u003eNanomaterials\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(11), 1841. https://doi.org/10.3390/nano12111841.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh, J., Dutta, T., Kim, K. H., Rawat, M., Samddar, P., \u0026amp; Kumar, P. (2018). Green\u0026rsquo; synthesis of metals and their oxide nanoparticles: applications for environmental remediation. \u003cem\u003eJournal of Nanobiotechnology\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(1), 84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12951-018-0408-4\u003c/span\u003e\u003cspan address=\"10.1186/s12951-018-0408-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoganathan, S., Shivakumar, M. S., Karthi, S., Nathan, S. S., \u0026amp; Selvam, K. (2021). Metal oxide nanoparticle synthesis (ZnO-NPs) of \u003cem\u003eKnoxia sumatrensis\u003c/em\u003e (Retz.) DC. Aqueous leaf extract and It\u0026rsquo;s evaluation of their antioxidant, anti-proliferative and larvicidal activities. \u003cem\u003eToxicology Reports\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, 64\u0026ndash;72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.toxrep.2020.12.018\u003c/span\u003e\u003cspan address=\"10.1016/j.toxrep.2020.12.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIsmail, N. A., Shameli, K., Che Jusoh, N. W., Ali, R., Mohamad Sukri, R., S. N. A., \u0026amp; Isa, M. (2021). E. D. Preparation of Copper Nanoparticles by Green Biosynthesis Method: A Short Review. \u003cem\u003eIOP Conference Series: Materials Science and Engineering\u003c/em\u003e, \u003cem\u003e1051\u003c/em\u003e(1), 012084. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/1757-899X/1051/1/012084\u003c/span\u003e\u003cspan address=\"10.1088/1757-899X/1051/1/012084\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMahmoud, A. E. D., El-Maghrabi, N., Hosny, M., \u0026amp; Fawzy, M. (2022). Biogenic synthesis of reduced graphene oxide from Ziziphus spina-christi (Christ\u0026rsquo;s thorn jujube) extracts for catalytic, antimicrobial, and antioxidant potentialities. \u003cem\u003eEnvironmental Science and Pollution Research International\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e(59), 89772. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-022-21871-x\u003c/span\u003e\u003cspan address=\"10.1007/s11356-022-21871-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMasoudpanah, S. M. (2017). Structural, Magnetic and Photocatalytic Properties of BiFeO3 Nanoparticles. \u003cem\u003eJournal of Nanostructures\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18502/jns.v7i3.3\u003c/span\u003e\u003cspan address=\"10.18502/jns.v7i3.3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan, S. T., Musarrat, J., \u0026amp; Al-Khedhairy, A. A. (2016). Countering drug resistance, infectious diseases, and sepsis using metal and metal oxides nanoparticles: Current status. \u003cem\u003eColloids and Surfaces B: Biointerfaces\u003c/em\u003e, \u003cem\u003e146\u003c/em\u003e, 70\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.colsurfb.2016.05.046\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfb.2016.05.046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMenazea, A. A., Ismail, A. M., \u0026amp; Samy, A. (2021). Novel Green Synthesis of Zinc Oxide Nanoparticles Using Orange Waste and Its Thermal and Antibacterial Activity. \u003cem\u003eJournal of Inorganic and Organometallic Polymers and Materials\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e(11), 4250\u0026ndash;4259. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10904-021-02074-2\u003c/span\u003e\u003cspan address=\"10.1007/s10904-021-02074-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eN, M., Ab, G. T. M. C. D. H. S., S., N, D., \u0026amp; O, R. (2019). Green synthesis of silver nanoparticles: effect of synthesis reaction parameters on antimicrobial activity. \u003cem\u003eWorld journal of microbiology \u0026amp; biotechnology\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11274-019-2664-3\u003c/span\u003e\u003cspan address=\"10.1007/s11274-019-2664-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eT, L., W, Y., \u0026amp; Q, L. (2010). Comparison of the phenolic content and antioxidant activities of Apocynum venetum L. (Luo-Bu-Ma) and two of its alternative species. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(11). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms11114452\u003c/span\u003e\u003cspan address=\"10.3390/ijms11114452\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOvais, M., Khalil, A. T., Islam, N. U., Ahmad, I., Ayaz, M., Saravanan, M., \u0026hellip; Mukherjee,S. (2018). Role of plant phytochemicals and microbial enzymes in biosynthesis of metallic nanoparticles. \u003cem\u003eApplied Microbiology and Biotechnology\u003c/em\u003e, \u003cem\u003e102\u003c/em\u003e(16), 6799\u0026ndash;6814. https://doi.org/10.1007/s00253-018-9146-7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMurthy, K. S. R., Lakshmi, N., \u0026amp; Ramulu, D. R. (2011). Biological activity and phytochemical screening of the oleoresin of Shorea robusta Gaertn. f. \u003cem\u003eTropical and subtropical agroecosystems\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(3), 787\u0026ndash;791.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeghoud, S., Hemmami, H., Ben Seghir, B., Ben Amor, I., Kouadri, I., Rebiai, A.,\u0026hellip; Simal-Gandara, J. (2022). A review on biogenic green synthesis of ZnO nanoparticles by plant biomass and their applications. \u003cem\u003eMaterials Today Communications\u003c/em\u003e, \u003cem\u003e33\u003c/em\u003e, 104747. https://doi.org/10.1016/j.mtcomm.2022.104747.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChaudhuri, S. K., \u0026amp; Malodia, L. (2017). Biosynthesis of zinc oxide nanoparticles using leaf extract of Calotropis gigantea: characterization and its evaluation on tree seedling growth in nursery stage. \u003cem\u003eApplied Nanoscience\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(8), 501\u0026ndash;512. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13204-017-0586-7\u003c/span\u003e\u003cspan address=\"10.1007/s13204-017-0586-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNilavukkarasi, M., Vijayakumar, S., \u0026amp; Prathipkumar, S. (2020). \u003cem\u003eCapparis zeylanica\u003c/em\u003e mediated bio-synthesized ZnO nanoparticles as antimicrobial, photocatalytic and anti-cancer applications. \u003cem\u003eMaterials Science for Energy Technologies\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, 335\u0026ndash;343. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mset.2019.12.004\u003c/span\u003e\u003cspan address=\"10.1016/j.mset.2019.12.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEspitia, P. J. P., Otoni, C. G., \u0026amp; Soares, N. F. F. (2016). Chapter 34 - Zinc Oxide Nanoparticles for Food Packaging Applications. In J. Barros-Vel\u0026aacute;zquez (Ed.), \u003cem\u003eAntimicrobial Food Packaging\u003c/em\u003e (pp. 425\u0026ndash;431). Academic. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B978-0-12-800723-5.00034-6\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-12-800723-5.00034-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSalvadori, M. R., Ando, R. A., Nascimento, C. A. O. do, \u0026amp; Corr\u0026ecirc;a, B. (2014). Intracellular Biosynthesis and Removal of Copper Nanoparticles by Dead Biomass of Yeast Isolated from the Wastewater of a Mine in the Brazilian Amazonia. \u003cem\u003ePLOS ONE\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(1), e87968. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0087968\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0087968\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrado, J., Vidal, V. A, R., \u0026amp; Dur\u0026aacute;n, T., C (2012). [Application of copper bactericidal properties in medical practice]. \u003cem\u003eRevista medica de Chile\u003c/em\u003e, \u003cem\u003e140\u003c/em\u003e(10), 1325\u0026ndash;1332. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4067/s0034-98872012001000014\u003c/span\u003e\u003cspan address=\"10.4067/s0034-98872012001000014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMali, S. C., Dhaka, A., Githala, C. K., \u0026amp; Trivedi, R. (2020). Green synthesis of copper nanoparticles using Celastrus paniculatus Willd. leaf extract and their photocatalytic and antifungal properties. \u003cem\u003eBiotechnology Reports\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e, e00518. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.btre.2020.e00518\u003c/span\u003e\u003cspan address=\"10.1016/j.btre.2020.e00518\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNaiel, B., Fawzy, M., Halmy, M. W. A., \u0026amp; Mahmoud, A. E. D. (2022). Green synthesis of zinc oxide nanoparticles using Sea Lavender (Limonium pruinosum L. Chaz.) extract: characterization, evaluation of anti-skin cancer, antimicrobial and antioxidant potentials. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1), 20370. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-24805-2\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-24805-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiaqat, N., Jahan, N., Khalil-ur-Rahman, Anwar, T., \u0026amp; Qureshi, H. (2022). Green synthesized silver nanoparticles: Optimization, characterization, antimicrobial activity, and cytotoxicity study by hemolysis assay. \u003cem\u003eFrontiers in Chemistry\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fchem.2022.952006\u003c/span\u003e\u003cspan address=\"10.3389/fchem.2022.952006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDejene, F. B. (2022). Characterization of low-temperature-grown ZnO nanoparticles: The effect of temperature on growth. \u003cem\u003eJournal of Physics Communications\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(7), 075011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/2399-6528/ac8049\u003c/span\u003e\u003cspan address=\"10.1088/2399-6528/ac8049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, L., Zhai, B., \u0026amp; Huang, Y. M. (2020). Rendering Visible-Light Photocatalytic Activity to Undoped ZnO via Intrinsic Defects Engineering. \u003cem\u003eCatalysts\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(10), 1163. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/catal10101163\u003c/span\u003e\u003cspan address=\"10.3390/catal10101163\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFakhari, S., Jamzad, M., \u0026amp; Kabiri Fard, H. (2019). Green synthesis of zinc oxide nanoparticles: a comparison. \u003cem\u003eGreen Chemistry Letters and Reviews\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1), 19\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/17518253.2018.1547925\u003c/span\u003e\u003cspan address=\"10.1080/17518253.2018.1547925\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWijesinghe, U., Thiripuranathar, G., Menaa, F., Iqbal, H., Razzaq, A., \u0026amp; Almukhlifi, H. (2021). Green Synthesis, Structural Characterization and Photocatalytic Applications of ZnO Nanoconjugates Using Heliotropium indicum. \u003cem\u003eCatalysts\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(7), 831. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/catal11070831\u003c/span\u003e\u003cspan address=\"10.3390/catal11070831\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJaishi, D. R., Ojha, I., Bhattarai, G., Baraili, R., Pathak, I., Ojha, D. R., \u0026hellip; Sharma,K. R. (2024). Plant-mediated synthesis of zinc oxide (ZnO) nanoparticles using Alnus nepalensis D. Don for biological applications. \u003cem\u003eHeliyon\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(20), e39255. https://doi.org/10.1016/j.heliyon.2024.e39255.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJanaki, A. C., Sailatha, E., \u0026amp; Gunasekaran, S. (2015). Synthesis, characteristics and antimicrobial activity of ZnO nanoparticles. \u003cem\u003eSpectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy\u003c/em\u003e, \u003cem\u003e144\u003c/em\u003e, 17\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.saa.2015.02.041\u003c/span\u003e\u003cspan address=\"10.1016/j.saa.2015.02.041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePourmadadi, M., Holghoomi, R., shamsabadipour, A., Maleki-baladi, R., Rahdar, A., \u0026amp; Pandey, S. (2024). Copper nanoparticles from chemical, physical, and green synthesis to medicinal application: A review. \u003cem\u003ePlant Nano Biology\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, 100070. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.plana.2024.100070\u003c/span\u003e\u003cspan address=\"10.1016/j.plana.2024.100070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMahmoodi, S., Elmi, A., \u0026amp; Hallaj-Nezhadi, S. (2018). Copper Nanoparticles as Antibacterial Agents. \u003cem\u003eJournal of Molecular Pharmaceutics \u0026amp; Organic Process Research\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(1), 1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4172/2329-9053.1000140\u003c/span\u003e\u003cspan address=\"10.4172/2329-9053.1000140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChatterjee, A. K., Chakraborty, R., \u0026amp; Basu, T. (2014). Mechanism of antibacterial activity of copper nanoparticles. \u003cem\u003eNanotechnology\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(13), 135101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/0957-4484/25/13/135101\u003c/span\u003e\u003cspan address=\"10.1088/0957-4484/25/13/135101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNeamah, S. A., Albukhaty, S., Falih, I. Q., Dewir, Y. H., \u0026amp; Mahood, H. B. (2023). Biosynthesis of Zinc Oxide Nanoparticles Using Capparis spinosa L. Fruit Extract: Characterization, Biocompatibility, and Antioxidant Activity. \u003cem\u003eApplied Sciences\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(11), 6604. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/app13116604\u003c/span\u003e\u003cspan address=\"10.3390/app13116604\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng, L. C., Jiang, X., Wang, J., Chen, C., \u0026amp; Liu, R. S. (2013). Nano\u0026ndash;bio effects: interaction of nanomaterials with cells. \u003cem\u003eNanoscale\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(9), 3547\u0026ndash;3569. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C3NR34276J\u003c/span\u003e\u003cspan address=\"10.1039/C3NR34276J\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYedgar, S., Barshtein, G., \u0026amp; Gural, A. (2022). Hemolytic Activity of Nanoparticles as a Marker of Their Hemocompatibility. \u003cem\u003eMicromachines\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(12), 2091. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/mi13122091\u003c/span\u003e\u003cspan address=\"10.3390/mi13122091\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eASTM E2524-08( (2013). - Standard Test Method for Analysis of Hemolytic Properties of Nanoparticles (Withdrawn 2022). (n.d.). \u003cem\u003eiTeh Standards\u003c/em\u003e. Retrieved September 6, 2025, from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://standards.iteh.ai/catalog/standards/astm/86e47339-603a-4ec0-86f9-dacaaddda4db/astm-e2524-08-2013\u003c/span\u003e\u003cspan address=\"https://standards.iteh.ai/catalog/standards/astm/86e47339-603a-4ec0-86f9-dacaaddda4db/astm-e2524-08-2013\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhattacharya, D., Santra, C. R., Ghosh, A. N., \u0026amp; Karmakar, P. (2014). Differential toxicity of rod and spherical zinc oxide nanoparticles on human peripheral blood mononuclear cells. \u003cem\u003eJournal of biomedical nanotechnology\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(4), 707\u0026ndash;716.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAshoub, M. H., Amiri, M., Fatemi, A., \u0026amp; Farsinejad, A. (2024). Evaluation of ferroptosis-based anti-leukemic activities of ZnO nanoparticles synthesized by a green route against Pre-B acute lymphoblastic leukemia cells (Nalm-6 and REH). \u003cem\u003eHeliyon\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(17), e36608. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.heliyon.2024.e36608\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2024.e36608\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, Z., Yin, X., Lyu, C., Cui, S., Wang, J., Si, Y., \u0026amp; Xu, R. (2022). PB1961: Zno Nanoparticles Induce Multiple Myeloma Cell Death Based On Reactive Oxygen Species And Caspase Signaling Pathway. \u003cem\u003eHemaSphere\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(Suppl), 1834\u0026ndash;1835. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/01.HS9.0000850680.43770.6b\u003c/span\u003e\u003cspan address=\"10.1097/01.HS9.0000850680.43770.6b\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFard, M. Z., Fatholahi, M., Abyadeh, M., Bakhtiarian, A., Mousavi, S. E., \u0026amp; Falahati, M. (2020). The Investigation of the Cytotoxicity of Copper Oxide Nanoparticles on Peripheral Blood Mononuclear Cells. \u003cem\u003eNanomedicine Research Journal\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(4), 364\u0026ndash;368. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22034/nmrj.2020.04.008\u003c/span\u003e\u003cspan address=\"10.22034/nmrj.2020.04.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Zinc oxide, Copper nanoparticles, Shorea robusta, green synthesis, anti-microbial, biocompatibility","lastPublishedDoi":"10.21203/rs.3.rs-7883522/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7883522/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, a green, sustainable, and cost-effective approach was employed for the synthesis of zinc oxide (ZnO) and copper (Cu) nanoparticles using \u003cem\u003eShorea robusta\u003c/em\u003e resin extract as a natural agent for reducing, capping, and stabilizing. The synthesis of metal nanoparticles utilizing \u003cem\u003eS. robusta\u003c/em\u003e resin is being reported for the first time. The synthesized nanoparticles were characterized using Ultraviolet\u0026ndash;Visible spectroscopy (UV-Vis), Fourier Transform Infrared spectroscopy (FT\u0026ndash;IR), and Nanoparticle Tracking Analysis (NTA). The phyto-synthesized nanoparticles exhibited significant antibacterial and antifungal activity, particularly against \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e (Gram-negative), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Gram-positive), and the pathogenic fungi \u003cem\u003eCandida albicans\u003c/em\u003e and \u003cem\u003eAspergillus flavus\u003c/em\u003e. Additionally, both ZnO and Cu nanoparticles demonstrated excellent hemocompatibility and cytocompatibility, highlighting their potential for pharmacological and therapeutic applications.\u003c/p\u003e","manuscriptTitle":"Plant-Based Fabrication of Zinc oxide and Copper Nanoparticles Using Shorea robusta Resin: Dual Evaluation of Safety and Antimicrobial Efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 19:55:01","doi":"10.21203/rs.3.rs-7883522/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-11T10:02:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T07:28:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-29T09:42:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60007890948251699839492474490635901028","date":"2026-01-28T09:28:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249698502581173447279437556782692429387","date":"2026-01-26T09:41:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277324042843215399927488340805135744511","date":"2026-01-22T23:32:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72630885309818218982788160143655297964","date":"2025-10-25T13:33:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-20T13:07:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-20T12:54:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-20T01:01:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-10-17T06:47:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"83bdde5b-0d05-4c02-8b70-94604dfee59f","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T06:55:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-30 19:55:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7883522","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7883522","identity":"rs-7883522","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}