Green Synthesis of Copper Nanoparticles with Adhatoda Vasica: Antibacterial and Antioxidant Study

Green Synthesis of Copper Nanoparticles with Adhatoda Vasica: Antibacterial and Antioxidant Study | 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 Green Synthesis of Copper Nanoparticles with Adhatoda Vasica: Antibacterial and Antioxidant Study Faruk Arodiya, Nahid Malik, Chirag Makvana, Kokila Parmar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6911253/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The burgeoning field of green nanotechnology has spurred the interest of researchers towards environmentally responsible nanoparticle production. In this study, Aradusi leaf extract was utilized for the synthesis of stable copper nanoparticles, subsequently functionalized with Polyvinyl Pyrrolidone (PVP) polymer. A comprehensive characterization of these biosynthesized nanoparticles was conducted using UV–Visible spectrophotometry, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). The UV–visible absorption spectra of bio-reduced copper nanoparticles were analyzed to assess their stability, while their antibacterial activity was evaluated against both gram-negative and gram-positive microbes. Additionally, their antioxidant potential was determined through DPPH free radical scavenging assays. Aradusi leaf extract demonstrated proficient reduction of copper ions into copper nanoparticles. Consequently, this methodology offers a rapid and environmentally benign route for the synthesis of stable copper nanoparticles exhibiting antibacterial and antioxidant activities within the size range of 10-100 nm, showcasing their potential applications in medical science. Antibacterial Antioxidant Nanoparticles Aradusi Polymer functionalized Green Synthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Nanostructures, characterized by their diverse physical, chemical, and electrical properties, have found widespread applications across various fields including antimicrobial treatments, optics, electronics, catalysis, energy conversion, storage devices, and biotechnology [ 1 – 4 ]. Among the plethora of materials available, copper and its alloys have garnered significant attention due to their versatile properties, finding applications in electrical engineering, catalysis, optics, and as potent antibacterial and antifungal agents [ 5 ]. The unique properties of copper nanoparticles position them as promising alternatives to noble metals such as gold, palladium, silver, and platinum, with applications spanning biosciences, biomedicine, catalysis, dielectrics, imaging, magnetism, and beyond [ 6 ]. In the quest for sustainable and environmentally benign nanoparticle synthesis methods, the utilization of medicinal plants has emerged as a cost-effective, abundantly available, and non-toxic approach suitable for industrial-scale production. In recent years, a variety of biological entities including algae [ 7 ], bacteria [ 8 ], fungi [ 9 ], mushrooms [ 10 ], enzymes [ 11 ], and plant leaf extracts [ 12 ] have been harnessed for the fabrication of metallic nanoparticles, offering advantages such as non-toxicity, energy efficiency, cost-effectiveness, and eco-friendliness. Plants, in particular, offer a favorable platform for nanoparticle synthesis as they inherently lack hazardous chemicals and possess natural capping agents, thereby eliminating the need for synthetic stabilizers. Furthermore, the use of plant extracts reduces the costs associated with microbial isolation and culture media, enhancing the cost-competitive viability of microorganism-based nanoparticle synthesis. In this study, we focus on the rapid synthesis of copper nanoparticles utilizing extract from Ocimum sanctum leaves. Ocimum sanctum, commonly known as Tulsi, is a traditional Indian medicinal plant renowned for its potent bio-reduction and stabilization properties. Tulsi leaves contain a rich array of bioactive compounds including alkaloids, glycosides, tannins, saponins, aromatic compounds, and essential minerals such as calcium, manganese, copper, zinc, phosphorus, potassium, sodium, and magnesium, with copper content notably higher compared to other leaf sources, standing at 12.31 mg/kg [ 13 ]. Among its constituents, urosolic acid emerges as a primary active ingredient, contributing to Tulsi's therapeutic properties and its efficacy as a reducer. The aqueous chemistry of Tulsi extract, powered by compounds like gallic acid, has been instrumental in reducing silver ions to silver nanoparticles, highlighting its potential as a versatile reducing agent [ 14 ]. Recent studies have also demonstrated the efficacy of Ocimum sanctum leaf extracts in the synthesis of silver and gold nanoparticles, leveraging its inherent bio-reducing and stabilizing capabilities [ 15 ]. Given copper's well-established antimicrobial properties, various plant extracts including Citrus Lemon fruit, Green coffee bean, Neem flower, Citrus paradisi fruit peel, Hibiscus rosa sinensis, Ocimum sanctum, Syzygium aromaticum (Cloves), Vitis vinifera, Eucalyptus, Cassia alata, Centella asiatica, Malva sylvestris, and others, have been employed for the synthesis of copper nanoparticles [ 20 ]. Notably, capsicum frutescens leaf extract has also been explored for this purpose [ 21 ]. Material and Methods Preparation of Leaf Extract: Fresh Aradusi leaves (5 g) were thoroughly washed with distilled water twice and dried on filter paper to remove residual moisture. Subsequently, the leaves were placed in a clean beaker, and 100 mL of distilled water was added using a measuring cylinder. The mixture was heated to obtain the leaf extract, which was then stored in an amber-colored bottle in a refrigerator. Synthesis of Copper Nanoparticles: A 25 mL portion of the Aradusi leaf extract was mixed with 100 mL of a 1 mM aqueous solution of copper sulphate pentahydrate (CuSO4•5H2O) under continuous stirring. After complete mixing of the leaf extract with the precursor, the mixture was incubated at 31°C for 24 hours. The formation of copper nanoparticles was indicated by a color change from pale green to light yellowish. Subsequently, the solution was centrifuged at 6000 rpm for 30 minutes, and the pellet obtained was re-dispersed in deionized water to remove any unwanted biological contaminants. Synthesis of Polymer Functionalized Copper Nanoparticles: In 100 mL of ultra-pure water, 0.2 g of Polyvinyl Pyrrolidone (PVP) was dissolved and stirred for 1 hour at 80°C. The resulting solution was gradually added to the homogeneous solution of copper nanoparticles obtained from the leaf extract. After 1 hour, the light yellowish color of the mixture turned into a dark yellow hue. The reaction mixture was allowed to cool for 10 minutes before being centrifuged at 10000 rpm for 15 minutes. The precipitates formed were washed with deionized water and then dried in a 70°C oven for 24 hours. Characterization of green CuNPs and PVP functionalized CuNPs Characterization of the green CuNPs and PVP-functionalized CuNPs involved employing various contemporary techniques. The production of CuNPs and polymer-functionalized CuNPs was verified using a UV-visible spectrophotometer (Perkin Elmer USA). Additionally, FTIR analysis spanning the 500–4000 cm^-1 range was conducted to confirm the presence of functional biomolecules associated with both types of nanoparticles. To ensure purity, XRD technique was utilized with a Rigaku D/max 40 kV X-ray diffraction spectrometer. Furthermore, the structural morphology of the synthesized nanoparticles was analyzed using high-resolution transmission electron microscopy (HR-TEM). Anti-microbial activity The antibacterial activity of the synthesized CuNPs was assessed using a modified version of the well diffusion method outlined by Hulikere et al. [ 22 ]. Overnight cultures of all test bacterial strains were grown in nutrient broth at 37°C and adjusted to a McFarland standard of 0.5. Under sterile conditions, 100 µL of each Gram-positive strain (Bacillus subtilis and Staphylococcus aureus) and each Gram-negative strain (Pseudomonas aeruginosa and Escherichia coli) were spread onto individual nutrient agar plates. Using a cork borer, wells with a diameter of 10 mm were punched into the agar plates, and the synthesized CuNPs and PVP-functionalized CuNPs were inoculated into each well. Additionally, 100 µL of streptomycin (1 mg/mL) served as a positive control. The plates were then incubated at 37°C for 24 hours, after which the antibacterial activity was assessed by measuring the diameter of the inhibition zone using a zone scale (HiMedia). Antioxidant activity The antioxidant properties of the synthesized CuNPs and PVP-functionalized CuNPs were evaluated using the DPPH method [ 23 ], with ascorbic acid chosen as the standard due to its high antioxidant activity. Standard solutions of ascorbic acid, as well as various concentrations (10, 20, 30, 40, 50, 75, 100 µg/mL), were prepared. DPPH was prepared by dissolving 20 mg of the compound in 100 mL of methanol. Subsequently, 1 mL of the various concentrations of CuNPs, PVP-functionalized CuNPs, and the standard ascorbic acid solution were separately mixed with 1 mL of the DPPH solution and incubated for 30 minutes. The absorbance was then measured using a UV-Visible Spectrophotometer at 517 nm. The free radical scavenging activity was expressed as the percentage of inhibition, calculated using the following formula. Result and Discussion UV–Visible Spectroscopic Analysis UV–visible spectroscopy confirmed the formation of copper nanoparticles (CuNPs) via aqueous-phase reduction. A distinct color change from light yellow to dark yellow was observed, attributed to the excitation of surface plasmon resonance (SPR), confirming nanoparticle synthesis. Absorbance peaks for CuNPs and PVP-functionalized CuNPs were observed at 322 nm and 247 nm, respectively. These variations reflect the influence of nanoparticle size and morphology on SPR behavior, consistent with previous studies [ 24 – 28 ]. FTIR Analysis FTIR spectroscopy was used to identify functional groups involved in the bioreduction and stabilization processes. Characteristic absorption bands appeared at 1653 cm⁻¹ (C = C stretching), 1100 and 1700 cm⁻¹ (C–O and C = O stretching), 610 cm⁻¹ (indicative of CuNP formation), 1480–1320 cm⁻¹ (C–H bending), and 1024 cm⁻¹ (C–X stretching). These results suggest the presence of bioactive molecules, such as amino acids and phenolic compounds, that act as natural capping agents, enhancing nanoparticle stability and preventing aggregation [ 29 – 31 ]. X-Ray Diffraction (XRD) Analysis XRD analysis of the PVP-CuNPs revealed sharp peaks at 2θ = 19.64°, 41.46°, 45.47°, and 72.28°, corresponding to crystal planes (100), (111), (200), and (311), confirming the crystalline nature of the particles. Using the Debye–Scherrer equation, the average particle size was calculated to be approximately 70.20 nm. These results were in close agreement with the HR-TEM findings, supporting the successful formation of polymer-stabilized nanoparticles [ 32 – 34 ]. HR-TEM Analysis HR-TEM imaging revealed uniformly distributed, spherical PVP-CuNPs with an average particle size of 73.50 nm. The selected area electron diffraction (SAED) pattern exhibited well-defined circular spots, further confirming the crystalline nature of the synthesized nanoparticles. Antibacterial Activity The synthesized CuNPs and PVP-functionalized CuNPs exhibited significant antibacterial activity against both Gram-positive and Gram-negative bacteria. PVP-CuNPs showed superior inhibition zones compared to CuNPs and even surpassed streptomycin in some cases. The enhanced efficacy of PVP-CuNPs may be attributed to the improved dispersion, bioavailability, and surface reactivity conferred by polymer functionalization. The data clearly indicate that PVP-functionalized CuNPs exhibited the highest antibacterial activity against all tested organisms, with inhibition zones ranging from 18 to 22 mm. This suggests that the polymeric capping significantly enhances the antimicrobial efficacy of CuNPs. Among the bacterial strains, Pseudomonas aeruginosa showed the greatest susceptibility to PVP-CuNPs (22 mm), comparable to the standard antibiotic streptomycin (23 mm), followed by Escherichia coli (22 mm) and Bacillus subtilis (19 mm). In contrast, the CuSO₄ solution and plant extract exhibited relatively lower antibacterial effects, with inhibition zones not exceeding 18 mm. The enhanced antibacterial activity of PVP-CuNPs can be attributed to several synergistic factors: Improved dispersion and stability of the nanoparticles in aqueous media due to PVP coating, which increases surface availability and interaction with bacterial cells. Sustained release of copper ions (Cu²⁺) from the nanoparticle core, prolonging their bactericidal action. Electrostatic and hydrogen-bonding interactions between PVP functional groups and bacterial membranes, which may facilitate enhanced uptake or membrane disruption. Table:1 Antibacterial activity of CuNPs and PVP CuNPs Sr.No. Organism Zone of Inhibition (In mm) CuSO 4 Solution (1 mM) Plant extract CuNPs PVP CuNPs Streptomycin (1 mg/ml) 1 Escherichia coli 16 16 21 22 18 2 Staphylococcus aureus 14 15 15 18 19 3 Bacillus subtilis 14 15 18 19 16 4 Pseudomonas Aeruginosa 15 18 17 22 23 Antioxidant activity of CuNPs and Polymer functionalized CuNPs • 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method The antioxidant potential of synthesized CuNPs and PVP-functionalized CuNPs was assessed using a DPPH free radical scavenging assay and compared against standard ascorbic acid across concentrations ranging from 10 to 100 µg/mL (Figure). The results reveal that both nanoparticle formulations exhibit dose-dependent scavenging activity, with a notable enhancement upon PVP functionalization. At lower concentrations (10–30 µg/mL), PVP-CuNPs exhibited higher scavenging activity than both CuNPs and ascorbic acid, indicating superior free radical neutralization efficiency at minimal doses. This suggests that PVP not only improves nanoparticle stability but may also contribute synergistically to antioxidant activity, possibly by facilitating better electron donation or enhancing radical interaction at the nanoparticle surface. At higher concentrations (40–50 µg/mL), both PVP-CuNPs and CuNPs achieved peak scavenging activity, with values reaching approximately 60%. Remarkably, this activity was comparable to or even slightly higher than that of ascorbic acid at the same concentrations, emphasizing the strong antioxidant capability of the nanoparticle systems. Beyond 50 µg/mL, a slight decline in activity was observed for all samples, which may be attributed to saturation effects or potential agglomeration at higher concentrations, limiting effective interaction with DPPH radicals. The overall trend demonstrates that PVP-CuNPs consistently outperform CuNPs and ascorbic acid, particularly at lower and moderate concentrations, highlighting the efficacy of PVP as a functionalizing agent in enhancing antioxidant behavior. These findings suggest that PVP-CuNPs hold significant promise as potent antioxidant agents, potentially useful in therapeutic applications targeting oxidative stress-related pathologies.[ 35 ] Conclusion In conclusion, this study successfully demonstrated the synthesis of copper nanoparticles (CuNPs) using an extract of Aradusi leaves and CuSO4•5H2O salt solution. Subsequently, the CuNPs were further functionalized with polyvinylpyrrolidone (PVP) to enhance biocompatibility, without the use of any harmful or toxic materials. The confirmation of CuNPs formation was validated by UV-visible spectroscopy, which exhibited a characteristic color change to dark brown and a peak at 247 nm after 24 hours. FTIR spectra analysis elucidated the various functional groups present in the Aradusi extract responsible for the biogenic synthesis of CuNPs and polymer-functionalized CuNPs. X-ray diffraction (XRD) examination confirmed the crystalline nature of the nanoparticles and revealed an average particle size of 70.20 nm for the polymer-capped CuNPs. High-resolution transmission electron microscopy (HR-TEM) imaging depicted spherical nanoparticles with sizes ranging from 10 to 100 nm. Moreover, both CuNPs and polymer-capped CuNPs exhibited significant antibacterial and antioxidant activities. Overall, this study highlights an environmentally friendly and cost-effective biological approach for synthesizing polymer-capped nanoparticles with potent antibacterial and antioxidant properties. Declarations Conflict of Interest Statement: The authors declare that there is no conflict of interest regarding the publication of this paper. Funding Statement: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution Author ContributionsFaruk Arodiya and Kokila Parmar contributed equally to this work. They were responsible for conceptualization, methodology, experimental investigation, data collection, and original draft preparation.Chirag Makvana contributed to formal analysis, data interpretation, and writing – review & editing, and also provided supervision throughout the project.Nahid Amlik was involved in resources provision, literature review, and assisted in data curation and visualization.All authors have read and agreed to the published version of the manuscript. References Ashar A., Iqbal, M., Bhatti, I. A., Ahmad, M. Z., Qureshi, K., Nisar, J., &Bukhari, I. H. (2016) . 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2","display":"","copyAsset":false,"role":"figure","size":8741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV Visible spectrum\u003c/strong\u003e of [A] CuNPs [B] PVP CuNPs\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6911253/v1/066e07767e26b09b7d92405f.png"},{"id":85265976,"identity":"db1a6576-2099-4c05-bfd8-c958ffdd7f2b","added_by":"auto","created_at":"2025-06-24 05:30:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":376380,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR Spectrum of [A] Aradusi leaf extract [B] CuNPs [C] PVP CuNPs\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6911253/v1/c9ab2755f71e2b8c5081fd07.png"},{"id":85266001,"identity":"a061f871-fd15-4237-9440-a39642483d84","added_by":"auto","created_at":"2025-06-24 05:30:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD\u003c/strong\u003e pattern of PVP functionalized CuNPs\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6911253/v1/aa5c6b6173fe3628fe981c07.png"},{"id":85265986,"identity":"cf9117fa-527b-47c9-813a-5c64782b658a","added_by":"auto","created_at":"2025-06-24 05:30:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":493127,"visible":true,"origin":"","legend":"\u003cp\u003eHR-TEM image [A], [B], [C] and SAED image [D] of PVP CuNPs\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6911253/v1/052d859a18077f7ba515b88a.png"},{"id":85265982,"identity":"5124c5f7-b009-4f42-9775-e2ee6d9ac99d","added_by":"auto","created_at":"2025-06-24 05:30:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":100284,"visible":true,"origin":"","legend":"\u003cp\u003eThe size distribution curve from the TEM analysis of PVP functionalized CuNPs\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6911253/v1/af78bdc321b09f0d941de265.png"},{"id":85265980,"identity":"4d5c21a2-9767-4d88-8128-bf9f96e21e19","added_by":"auto","created_at":"2025-06-24 05:30:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":334116,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial study of biosynthesized CuNPs and PVP CuNPs against pathogenic bacteria [A] \u003cem\u003eEscherichia coli\u003c/em\u003e [B] \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e [C] \u003cem\u003eBacillus subtilis\u003c/em\u003e[D] \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6911253/v1/36bbf21169c6f06764996224.png"},{"id":85266470,"identity":"937840be-fdda-46a5-b9ed-c32209affc3c","added_by":"auto","created_at":"2025-06-24 05:38:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":16534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibacterial\u003c/strong\u003e zone of inhibition of CuNPs and PVP CuNPs in Comparison with standard streptomycin\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6911253/v1/f009639eb81b1add40bf625c.png"},{"id":86496947,"identity":"c24e4839-35f0-4308-ac1a-b86e194be6b4","added_by":"auto","created_at":"2025-07-11 10:17:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2389729,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6911253/v1/271138a3-a482-4c0a-bae1-321e274c1c82.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGreen Synthesis of Copper Nanoparticles with Adhatoda Vasica: Antibacterial and Antioxidant Study\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanostructures, characterized by their diverse physical, chemical, and electrical properties, have found widespread applications across various fields including antimicrobial treatments, optics, electronics, catalysis, energy conversion, storage devices, and biotechnology [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among the plethora of materials available, copper and its alloys have garnered significant attention due to their versatile properties, finding applications in electrical engineering, catalysis, optics, and as potent antibacterial and antifungal agents [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The unique properties of copper nanoparticles position them as promising alternatives to noble metals such as gold, palladium, silver, and platinum, with applications spanning biosciences, biomedicine, catalysis, dielectrics, imaging, magnetism, and beyond [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the quest for sustainable and environmentally benign nanoparticle synthesis methods, the utilization of medicinal plants has emerged as a cost-effective, abundantly available, and non-toxic approach suitable for industrial-scale production. In recent years, a variety of biological entities including algae [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], bacteria [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], fungi [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], mushrooms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], enzymes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and plant leaf extracts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] have been harnessed for the fabrication of metallic nanoparticles, offering advantages such as non-toxicity, energy efficiency, cost-effectiveness, and eco-friendliness. Plants, in particular, offer a favorable platform for nanoparticle synthesis as they inherently lack hazardous chemicals and possess natural capping agents, thereby eliminating the need for synthetic stabilizers. Furthermore, the use of plant extracts reduces the costs associated with microbial isolation and culture media, enhancing the cost-competitive viability of microorganism-based nanoparticle synthesis. In this study, we focus on the rapid synthesis of copper nanoparticles utilizing extract from Ocimum sanctum leaves. Ocimum sanctum, commonly known as Tulsi, is a traditional Indian medicinal plant renowned for its potent bio-reduction and stabilization properties. Tulsi leaves contain a rich array of bioactive compounds including alkaloids, glycosides, tannins, saponins, aromatic compounds, and essential minerals such as calcium, manganese, copper, zinc, phosphorus, potassium, sodium, and magnesium, with copper content notably higher compared to other leaf sources, standing at 12.31 mg/kg [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Among its constituents, urosolic acid emerges as a primary active ingredient, contributing to Tulsi's therapeutic properties and its efficacy as a reducer.\u003c/p\u003e \u003cp\u003eThe aqueous chemistry of Tulsi extract, powered by compounds like gallic acid, has been instrumental in reducing silver ions to silver nanoparticles, highlighting its potential as a versatile reducing agent [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Recent studies have also demonstrated the efficacy of Ocimum sanctum leaf extracts in the synthesis of silver and gold nanoparticles, leveraging its inherent bio-reducing and stabilizing capabilities [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Given copper's well-established antimicrobial properties, various plant extracts including Citrus Lemon fruit, Green coffee bean, Neem flower, Citrus paradisi fruit peel, Hibiscus rosa sinensis, Ocimum sanctum, Syzygium aromaticum (Cloves), Vitis vinifera, Eucalyptus, Cassia alata, Centella asiatica, Malva sylvestris, and others, have been employed for the synthesis of copper nanoparticles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Notably, capsicum frutescens leaf extract has also been explored for this purpose [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of Leaf Extract:\u003c/h2\u003e\n \u003cp\u003eFresh Aradusi leaves (5 g) were thoroughly washed with distilled water twice and dried on filter paper to remove residual moisture. Subsequently, the leaves were placed in a clean beaker, and 100 mL of distilled water was added using a measuring cylinder. The mixture was heated to obtain the leaf extract, which was then stored in an amber-colored bottle in a refrigerator.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSynthesis of Copper Nanoparticles:\u003c/h3\u003e\n\u003cp\u003eA 25 mL portion of the Aradusi leaf extract was mixed with 100 mL of a 1 mM aqueous solution of copper sulphate pentahydrate (CuSO4\u0026bull;5H2O) under continuous stirring. After complete mixing of the leaf extract with the precursor, the mixture was incubated at 31\u0026deg;C for 24 hours. The formation of copper nanoparticles was indicated by a color change from pale green to light yellowish. Subsequently, the solution was centrifuged at 6000 rpm for 30 minutes, and the pellet obtained was re-dispersed in deionized water to remove any unwanted biological contaminants.\u003c/p\u003e\n\u003ch3\u003eSynthesis of Polymer Functionalized Copper Nanoparticles:\u003c/h3\u003e\n\u003cp\u003eIn 100 mL of ultra-pure water, 0.2 g of Polyvinyl Pyrrolidone (PVP) was dissolved and stirred for 1 hour at 80\u0026deg;C. The resulting solution was gradually added to the homogeneous solution of copper nanoparticles obtained from the leaf extract. After 1 hour, the light yellowish color of the mixture turned into a dark yellow hue. The reaction mixture was allowed to cool for 10 minutes before being centrifuged at 10000 rpm for 15 minutes. The precipitates formed were washed with deionized water and then dried in a 70\u0026deg;C oven for 24 hours.\u003c/p\u003e\n\u003ch3\u003eCharacterization of green CuNPs and PVP functionalized CuNPs\u003c/h3\u003e\n\u003cp\u003eCharacterization of the green CuNPs and PVP-functionalized CuNPs involved employing various contemporary techniques. The production of CuNPs and polymer-functionalized CuNPs was verified using a UV-visible spectrophotometer (Perkin Elmer USA). Additionally, FTIR analysis spanning the 500\u0026ndash;4000 cm^-1 range was conducted to confirm the presence of functional biomolecules associated with both types of nanoparticles. To ensure purity, XRD technique was utilized with a Rigaku D/max 40 kV X-ray diffraction spectrometer. Furthermore, the structural morphology of the synthesized nanoparticles was analyzed using high-resolution transmission electron microscopy (HR-TEM).\u003c/p\u003e\n\u003ch3\u003eAnti-microbial activity\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of the synthesized CuNPs was assessed using a modified version of the well diffusion method outlined by Hulikere et al. [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Overnight cultures of all test bacterial strains were grown in nutrient broth at 37\u0026deg;C and adjusted to a McFarland standard of 0.5. Under sterile conditions, 100 \u0026micro;L of each Gram-positive strain (Bacillus subtilis and Staphylococcus aureus) and each Gram-negative strain (Pseudomonas aeruginosa and Escherichia coli) were spread onto individual nutrient agar plates. Using a cork borer, wells with a diameter of 10 mm were punched into the agar plates, and the synthesized CuNPs and PVP-functionalized CuNPs were inoculated into each well. Additionally, 100 \u0026micro;L of streptomycin (1 mg/mL) served as a positive control. The plates were then incubated at 37\u0026deg;C for 24 hours, after which the antibacterial activity was assessed by measuring the diameter of the inhibition zone using a zone scale (HiMedia).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eAntioxidant activity\u003c/h2\u003e\n \u003cp\u003eThe antioxidant properties of the synthesized CuNPs and PVP-functionalized CuNPs were evaluated using the DPPH method [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e], with ascorbic acid chosen as the standard due to its high antioxidant activity. Standard solutions of ascorbic acid, as well as various concentrations (10, 20, 30, 40, 50, 75, 100 \u0026micro;g/mL), were prepared. DPPH was prepared by dissolving 20 mg of the compound in 100 mL of methanol. Subsequently, 1 mL of the various concentrations of CuNPs, PVP-functionalized CuNPs, and the standard ascorbic acid solution were separately mixed with 1 mL of the DPPH solution and incubated for 30 minutes. The absorbance was then measured using a UV-Visible Spectrophotometer at 517 nm. The free radical scavenging activity was expressed as the percentage of inhibition, calculated using the following formula.\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg 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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Result and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eUV\u0026ndash;Visible Spectroscopic Analysis\u003c/h2\u003e\n \u003cp\u003eUV\u0026ndash;visible spectroscopy confirmed the formation of copper nanoparticles (CuNPs) via aqueous-phase reduction. A distinct color change from light yellow to dark yellow was observed, attributed to the excitation of surface plasmon resonance (SPR), confirming nanoparticle synthesis. Absorbance peaks for CuNPs and PVP-functionalized CuNPs were observed at 322 nm and 247 nm, respectively. These variations reflect the influence of nanoparticle size and morphology on SPR behavior, consistent with previous studies [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eFTIR Analysis\u003c/h2\u003e\n \u003cp\u003eFTIR spectroscopy was used to identify functional groups involved in the bioreduction and stabilization processes. Characteristic absorption bands appeared at 1653 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;C stretching), 1100 and 1700 cm⁻\u0026sup1; (C\u0026ndash;O and C\u0026thinsp;=\u0026thinsp;O stretching), 610 cm⁻\u0026sup1; (indicative of CuNP formation), 1480\u0026ndash;1320 cm⁻\u0026sup1; (C\u0026ndash;H bending), and 1024 cm⁻\u0026sup1; (C\u0026ndash;X stretching). These results suggest the presence of bioactive molecules, such as amino acids and phenolic compounds, that act as natural capping agents, enhancing nanoparticle stability and preventing aggregation [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eX-Ray Diffraction (XRD) Analysis\u003c/h2\u003e\n \u003cp\u003eXRD analysis of the PVP-CuNPs revealed sharp peaks at 2\u0026theta;\u0026thinsp;=\u0026thinsp;19.64\u0026deg;, 41.46\u0026deg;, 45.47\u0026deg;, and 72.28\u0026deg;, corresponding to crystal planes (100), (111), (200), and (311), confirming the crystalline nature of the particles. Using the Debye\u0026ndash;Scherrer equation, the average particle size was calculated to be approximately 70.20 nm. These results were in close agreement with the HR-TEM findings, supporting the successful formation of polymer-stabilized nanoparticles [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eHR-TEM Analysis\u003c/h2\u003e\n \u003cp\u003eHR-TEM imaging revealed uniformly distributed, spherical PVP-CuNPs with an average particle size of 73.50 nm. The selected area electron diffraction (SAED) pattern exhibited well-defined circular spots, further confirming the crystalline nature of the synthesized nanoparticles.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eAntibacterial Activity\u003c/h2\u003e\n \u003cp\u003eThe synthesized CuNPs and PVP-functionalized CuNPs exhibited significant antibacterial activity against both Gram-positive and Gram-negative bacteria. PVP-CuNPs showed superior inhibition zones compared to CuNPs and even surpassed streptomycin in some cases. The enhanced efficacy of PVP-CuNPs may be attributed to the improved dispersion, bioavailability, and surface reactivity conferred by polymer functionalization.\u003c/p\u003e\n \u003cp\u003eThe data clearly indicate that \u003cstrong\u003ePVP-functionalized CuNPs exhibited the highest antibacterial activity\u003c/strong\u003e against all tested organisms, with inhibition zones ranging from 18 to 22 mm. This suggests that the polymeric capping significantly enhances the antimicrobial efficacy of CuNPs.\u003c/p\u003e\n \u003cp\u003eAmong the bacterial strains, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e showed the greatest susceptibility to PVP-CuNPs (22 mm), comparable to the standard antibiotic streptomycin (23 mm), followed by \u003cem\u003eEscherichia coli\u003c/em\u003e (22 mm) and \u003cem\u003eBacillus subtilis\u003c/em\u003e (19 mm). In contrast, the CuSO₄ solution and plant extract exhibited relatively lower antibacterial effects, with inhibition zones not exceeding 18 mm.\u003c/p\u003e\n \u003cp\u003eThe \u003cstrong\u003eenhanced antibacterial activity of PVP-CuNPs\u003c/strong\u003e can be attributed to several synergistic factors:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cstrong\u003eImproved dispersion and stability\u003c/strong\u003e of the nanoparticles in aqueous media due to PVP coating, which increases surface availability and interaction with bacterial cells.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cstrong\u003eSustained release of copper ions (Cu\u0026sup2;⁺)\u003c/strong\u003e from the nanoparticle core, prolonging their bactericidal action.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cstrong\u003eElectrostatic and hydrogen-bonding interactions\u003c/strong\u003e between PVP functional groups and bacterial membranes, which may facilitate enhanced uptake or membrane disruption.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003e\u003cstrong\u003eTable:1\u0026nbsp;\u003c/strong\u003eAntibacterial activity of CuNPs and PVP CuNPs\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"618\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSr.No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrganism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 400px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZone of Inhibition (In mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCuSO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eSolution\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(1 mM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlant extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCuNPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePVP CuNPs\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStreptomycin\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(1 mg/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas Aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eAntioxidant activity of CuNPs and Polymer functionalized CuNPs\u003c/h2\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e\u0026bull; 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method\u003c/h2\u003e\n \u003cp\u003eThe antioxidant potential of synthesized CuNPs and PVP-functionalized CuNPs was assessed using a DPPH free radical scavenging assay and compared against standard ascorbic acid across concentrations ranging from 10 to 100 \u0026micro;g/mL (Figure). The results reveal that both nanoparticle formulations exhibit dose-dependent scavenging activity, with a notable enhancement upon PVP functionalization.\u003c/p\u003e\n \u003cp\u003eAt lower concentrations (10\u0026ndash;30 \u0026micro;g/mL), PVP-CuNPs exhibited higher scavenging activity than both CuNPs and ascorbic acid, indicating superior free radical neutralization efficiency at minimal doses. This suggests that PVP not only improves nanoparticle stability but may also contribute synergistically to antioxidant activity, possibly by facilitating better electron donation or enhancing radical interaction at the nanoparticle surface.\u003c/p\u003e\n \u003cp\u003eAt higher concentrations (40\u0026ndash;50 \u0026micro;g/mL), both PVP-CuNPs and CuNPs achieved peak scavenging activity, with values reaching approximately 60%. Remarkably, this activity was comparable to or even slightly higher than that of ascorbic acid at the same concentrations, emphasizing the strong antioxidant capability of the nanoparticle systems.\u003c/p\u003e\n \u003cp\u003eBeyond 50 \u0026micro;g/mL, a slight decline in activity was observed for all samples, which may be attributed to saturation effects or potential agglomeration at higher concentrations, limiting effective interaction with DPPH radicals.\u003c/p\u003e\n \u003cp\u003eThe overall trend demonstrates that PVP-CuNPs consistently outperform CuNPs and ascorbic acid, particularly at lower and moderate concentrations, highlighting the efficacy of PVP as a functionalizing agent in enhancing antioxidant behavior. These findings suggest that PVP-CuNPs hold significant promise as potent antioxidant agents, potentially useful in therapeutic applications targeting oxidative stress-related pathologies.[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study successfully demonstrated the synthesis of copper nanoparticles (CuNPs) using an extract of Aradusi leaves and CuSO4\u0026bull;5H2O salt solution. Subsequently, the CuNPs were further functionalized with polyvinylpyrrolidone (PVP) to enhance biocompatibility, without the use of any harmful or toxic materials. The confirmation of CuNPs formation was validated by UV-visible spectroscopy, which exhibited a characteristic color change to dark brown and a peak at 247 nm after 24 hours. FTIR spectra analysis elucidated the various functional groups present in the Aradusi extract responsible for the biogenic synthesis of CuNPs and polymer-functionalized CuNPs. X-ray diffraction (XRD) examination confirmed the crystalline nature of the nanoparticles and revealed an average particle size of 70.20 nm for the polymer-capped CuNPs. High-resolution transmission electron microscopy (HR-TEM) imaging depicted spherical nanoparticles with sizes ranging from 10 to 100 nm. Moreover, both CuNPs and polymer-capped CuNPs exhibited significant antibacterial and antioxidant activities. Overall, this study highlights an environmentally friendly and cost-effective biological approach for synthesizing polymer-capped nanoparticles with potent antibacterial and antioxidant properties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor ContributionsFaruk Arodiya and Kokila Parmar contributed equally to this work. They were responsible for conceptualization, methodology, experimental investigation, data collection, and original draft preparation.Chirag Makvana contributed to formal analysis, data interpretation, and writing \u0026ndash; review \u0026amp; editing, and also provided supervision throughout the project.Nahid Amlik was involved in resources provision, literature review, and assisted in data curation and visualization.All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAshar A., Iqbal, M., Bhatti, I. A., Ahmad, M. Z., Qureshi, K., Nisar, J., \u0026amp;Bukhari, I. H. \u003cstrong\u003e(2016)\u003c/strong\u003e. 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Green synthesis of silver nanoparticles using Catharanthusroseus: Its antioxidant and antibacterial properties. \u003cem\u003eNanomedicine Research Journal\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(1), 17-27.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antibacterial, Antioxidant, Nanoparticles, Aradusi, Polymer functionalized, Green Synthesis","lastPublishedDoi":"10.21203/rs.3.rs-6911253/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6911253/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe burgeoning field of green nanotechnology has spurred the interest of researchers towards environmentally responsible nanoparticle production. In this study, Aradusi leaf extract was utilized for the synthesis of stable copper nanoparticles, subsequently functionalized with Polyvinyl Pyrrolidone (PVP) polymer. A comprehensive characterization of these biosynthesized nanoparticles was conducted using UV–Visible spectrophotometry, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). The UV–visible absorption spectra of bio-reduced copper nanoparticles were analyzed to assess their stability, while their antibacterial activity was evaluated against both gram-negative and gram-positive microbes. Additionally, their antioxidant potential was determined through DPPH free radical scavenging assays. Aradusi leaf extract demonstrated proficient reduction of copper ions into copper nanoparticles. Consequently, this methodology offers a rapid and environmentally benign route for the synthesis of stable copper nanoparticles exhibiting antibacterial and antioxidant activities within the size range of 10-100 nm, showcasing their potential applications in medical science.\u003c/p\u003e","manuscriptTitle":"Green Synthesis of Copper Nanoparticles with Adhatoda Vasica: Antibacterial and Antioxidant Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-24 05:29:49","doi":"10.21203/rs.3.rs-6911253/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0d7548a-3cae-4178-8992-f9e16872e061","owner":[],"postedDate":"June 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-11T10:09:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-24 05:29:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6911253","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6911253","identity":"rs-6911253","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}