Green Synthesis of Copper Oxide Nanoparticles Using Prosopis cineraria Extract: Evaluation of Cytotoxic Antioxidant and Antimicrobial Activity

Green Synthesis of Copper Oxide Nanoparticles Using Prosopis cineraria Extract: Evaluation of Cytotoxic Antioxidant and Antimicrobial Activity | 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 Oxide Nanoparticles Using Prosopis cineraria Extract: Evaluation of Cytotoxic Antioxidant and Antimicrobial Activity Renu Vajjiravelu, Banuppriya Palani, Rajeshkumar Shanmugam, Santhoshkumar Jayakodi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7092059/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 Copper nanoparticles (CuONp) have garnered considerable attention due to their unique thermodynamic properties and broad bioactivities, including antioxidant, antibacterial, and cytotoxic effects. The green synthesis of CuONp, which employs natural plant extracts as reducing and stabilizing agents, is an eco-friendly and sustainable alternative to conventional methods. This study investigates the synthesis of CuONp using an aqueous extract of Prosopis cineraria and evaluates their biological activities. CuONp were synthesized by mixing P. cineraria extract with copper nitrate under magnetic stirring to promote nanoparticle formation. The synthesis was validated using UV-visible spectroscopy, revealing characteristic absorption peaks indicative of CuONp formation. Antioxidant activity was assessed using the DPPH assay, while antibacterial activity was tested against two bacterial strains. Cytotoxicity was evaluated via a brine shrimp lethality assay, measuring nauplii mortality at different CuONp concentrations. UV-visible spectroscopy confirmed the successful synthesis of CuONp. The DPPH assay showed dose-dependent free radical scavenging, with maximum activity at the highest concentration. Antibacterial testing demonstrated significant inhibition zones, indicating strong activity against both bacterial strains. The cytotoxicity assay revealed dose-dependent nauplii mortality, underscoring the cytotoxic potential of CuONp. Green-synthesized CuONp using P. cineraria exhibit strong antioxidant, antibacterial, and cytotoxic properties, highlighting their potential for clinical and therapeutic applications. This sustainable synthesis approach harnesses the bioactive potential of P. cineraria for nanoparticle production. Copper nanoparticles (CuONp) Green synthesis Prosopis cineraria Antioxidant activity Antibacterial activity Cytotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Nanotechnology has revolutionized the field of science and technology by offering innovative solutions across multiple disciplines, including medicine, agriculture, and environmental sciences[ 1 ]. Among the various nanoparticles studied, copper nanoparticles (CuONp) have received considerable interest because of their unique qualities, such as high catalytic activity, conductivity, and antimicrobial efficacy [ 2 ]. Recently, the focus has shifted towards the green synthesis of nanoparticles, as conventional chemical and physical methods often involve toxic chemicals, high energy consumption, and expensive equipment [ 3 ]. Green synthesis, leveraging natural sources such as plant extracts, microorganisms, and biomolecules, provides an eco-friendly and sustainable approach [ 4 , 5 ]. Plants, in particular, are an excellent resource for synthesizing nanoparticles, as they contain a diverse array of phytochemicals such as alkaloids, flavonoids, phenols, and tannins [ 6 ]. These compounds not only reduce metal ions to their nanoparticle form but also stabilize the nanoparticles, imparting additional bioactivity [ 7 ]. Prosopsis cineraria holds immense significance in traditional medicine and is renowned for its pharmacological characteristics, comprising anti-oxidants, antiviral, anti-fungal, and therapeutic activity[ 8 , 9 ]. The phytochemical composition of Prosopis cineraria includes flavonoids, tannins, alkaloids, and phenolic acids, which make it an ideal candidate for the green synthesis of nanoparticles [ 10 ]. Utilizing this plant for synthesizing CuONp combines the inherent bioactivity of the plant with the enhanced properties of nanoparticles, paving the way for innovative applications in health and environmental sciences [ 11 ]. The biological activities of CuONp, such as antioxidant, antimicrobial, and cytotoxic effects, have been widely explored [ 12 ]. Antioxidant activity is an essential characteristic, because the inflammation induced by reactive oxygen species (ROS) is linked to ageing and the development of many illnesses, especially malignancy, neurological diseases, and circulatory maladies [ 13 , 14 ]. CuONp, by their redox potential and the presence of surface-active groups, can effectively scavenge ROS and reduce oxidative damage [ 15 ]. Similarly, the antimicrobial properties of CuONp have demonstrated broad-spectrum efficacy against bacteria, fungi, and viruses [ 16 ]. Copper ions break membranes in microbes, impede cellular respiration, and produce reactive oxygen species, resulting in microbes' cell mortality [ 17 ]. These properties are especially relevant in combating antimicrobial resistance (AMR), which poses a global health challenge, and in food preservation, where microbial contamination is a major concern [ 18 , 19 ]. Furthermore, the cytotoxic potential of CuONp against cancer cells offers promising applications in oncology [ 20 ]. CuONp induce cytotoxicity through mechanisms such as ROS generation, DNA damage, and disruption of mitochondrial function, selectively targeting cancer cells while sparing normal tissues [ 21 ]. This dual action of CuONp, combining therapeutic and diagnostic capabilities, highlights their potential as nanomedicine agents [ 11 , 22 ]. The antioxidant activity was assessed using standard radical scavenging assays to determine their efficacy in neutralizing oxidative stress [ 23 , 24 ]. The ability to inhibit microbes was evaluated towards several harmful microorganisms to investigate how they might help microbial resistance [ 25 ]. The cytotoxic activity was analyzed in vitro against specific cancer cell lines to evaluate their therapeutic applicability [ 14 , 26 ]. The present investigation intends to add to the expanding body of research on green-synthesized nanoparticles in emphasising the beneficial effects of plant-mediated CuONp [ 27 ]. 2. Materials and Methods Extract preparation In the current state investigation, 1gm of Prosopsis cineraria was dissolved in a hundred ml of purified water and boiled for ten to fifteen minutes at 70 degrees Celsius. After heating up, Whatman filtered the plant extract. No.1 filter paper [ 28 ]. In a 250 ml conical flask, 70 ml of 20 millimolar copper nitrate was prepared, and 30 ml of filtered vegetal extract was added and put into an iron magnetic stirrer to produce nanoparticles. After the synthesis of nanoparticles, a colour change was seen [ 29 ]. Antioxidant assay DPPH method The DPPH method was applied to study the protective characteristics of naturally occurring synthesised particles of copper. Copper nitrate nanomaterials have been added with Prosopsis cineraria herb extract (2–10 µg/ml) in 1 ml of 0.1 mm DPPH in methanol and 450 µl of 50 mm Tris HCl buffer (pH 7.4) for ten minutes. Afterwards, the drop of DPPH free radicals was measured using the intensity of absorption at 517 nm. BHT was used as control. [ 30 ]. The following equation determined the level of inhibition, The percentage of inhibition = absorbance of control minus absorbance of test sample divided by 100. H₂O₂ Assay The potential of the biosynthesized tocopherol chitosan biocomposite to scavenge H₂O₂ was evaluated in 40 mM H₂O₂ solution in a pH 7.4 phosphate buffer. 0.6 mL of H₂O₂ solution was mixed with a solution of the test sample (ɑ-tocopherol chitosan biocomposite) and a standard sample of ascorbic acid at different concentrations (10–50µg/mL). Spectrophotometric measurements of the reaction solution absorbance at 532 nm were made following a 10-minute incubation period in a dark environment. The standard used was vitamin C. Using the following formula, the proportion of H₂O₂ scavenging activity was calculated To calculate the percentage of inhibition, divide the absorbance of the control by the absorbance of the sample and multiply by 100. Antimicrobial Assay To manufacture Mueller-Hinton agar, 10 µL of fresh microbial cultures ( C. albicans, E. faeclalis, E. coli, Pseudomonas , and S. aureus ) were injected in a sterile Hi-veg media. A sanitized polystyrene tip was used to make 5 mm wells. Antimicrobial effect was assessed to determine the effectiveness of Prosopsis cineraria -mediated CuONp and biosynthesized copper Particle-mediated nanocomposite material. Wells were filled with varying amounts of three samples (25, 50, and 100 µL) and a conventional test. The specimens of Petri plates and zone inhibition were kept in an incubator at 37 degrees Celsius for 24 hours. The area was evaluated in order to contrast and examine the possible impact of Prosopsis cineraria -mediated copper nanoparticles [ 31 ]. Cytotoxicity assay Brine shrimp eggs were placed in a hatching chamber filled with salty water. After 24 hours, precisely ten hatched larvae (nauplii) were floating in six wells, each holding 10 ml of saltwater. Nanoparticles were scattered in each well at varying quantities (5 µL, 10 µL, 20 µL, 40 µL, and 80 µL), with the very last well serving as a control (no nanoparticles). After 24 hours, the number of remaining nauplii was calculated and noted. 3. Results Visual Observation and UV-Vis Studies for Copper Oxide NPs The development of copper oxide NPs was tracked using a UV-visible spectrophotometer. Prosopsis cineraria leaf extract functions as a stabilizing and diminutive agent. Biological molecules in the leaf extract reduce copper nitrate nanoparticles to copper oxide nanoparticles. The color changes from reddish brown to dark reddish brown after adding leaf extract, and then progressively gets dark brown due to the synthesis of CuONp. (Fig. 1 ) depicts the color variations that occur during nanoparticle production with CuONp. UV-visible data were taken over a period of 0–84 hours. Copper nanoparticles' resonance with the surface exhibited the highest peak at 420 nm, as shown in Fig. 2 ). FTIR Analysis The FTIR spectrum of the sample labelled CuO + PC (Copper oxide nanoparticles synthesised using Prosopsis cineraria extract) offers useful insight into the functional compounds present in the sample, revealing the importance of plant phytochemicals in the green synthesis procedure. A large absorption peak at 3185.8 cm¹ corresponds to O-H stretching vibrations, which are indicative of hydroxyl groups from alcohols or phenolic compounds. This suggests the participation of plant metabolites like flavonoids or polyols in stabilising the nanoparticles. The signal at 2960.3 cm¹ is due to C-H stretching vibrations of aliphatic -CH₂ and -CH₃ groups, suggesting the presence of organic chemicals in the plant extract. A peak at 1557.5 cm¹ may be due to N-H bending oscillations from amide connections or C = C stretching of aromatic rings, which are typically found in proteins or polyphenolic structures. The band at 1385.8 cm¹ may be attributed to C-N stretching or O-H bending, indicating the existence of biomolecules with amine or phenol groups. The absorption at 1034.7 cm-¹ corresponds to C-O stretching vibrations of alcohols, ethers, or esters, suggesting the presence of plant-derived substances such as carbohydrates and flavonoids in the capping material. The peaks at 813.58 cm⁻¹ and 678.32 cm⁻¹ show out-of-plane bending vibrations of aromatic C-H bonds, similar to those found in plant phenols. The high absorption bands at 613.3 cm⁻¹ and 465.03 cm⁻¹ show the production of copper oxide nanoparticles. These data combined show that prosopsis cineraria extract functions as a reducing and capping agent in the green production of CuONp, and the functional groups observed in the FTIR spectrum play an important role in nanoparticle stabilisation and bioactivity. XRD Analysis The X-ray diffraction (XRD) image depicts copper oxide nanoparticles (CuONp) synthesised utilising Prosopis cineraria. The prominent diffraction peaks at specific 2θ values can be indexed to crystalline planes such as (021), (121), (-213), (-204), (-214), (-245), (-107), (018), and (-261), which match the monoclinic phase of CuONP. These peaks are consistent with the JCPDS card No. 00-042-1959, demonstrating the creation of pure, crystalline monoclinic CuONp. The strong and powerful patterns show great crystallinity, and the lack of further patterns shows that the synthesised nanoparticles are pure, without no other copper phases or contaminants[ 32 , 33 ]. SEM Analysis A scanning electron microscope (SEM) image displaying the surface morphology of copper oxide nanoparticles (CuONp). The 30,000× magnification shows a granular structure with irregular particle shapes and distribution. These particles appear to be closely packed, generating clusters of high surface roughness. The shape indicates an enormous surface area, which is useful for catalytic, antibacterial, or cytotoxic applications. The dimensions of the bar represents a length of 300 nm, emphasising the nanoscale size of the particles. This shape is prevalent in biosynthesised nanoparticles and shows the effective generation of CuONp with improved reactivity and surface contact, which are important for biological and environmental purposes. A energy dispersive x-ray spectroscopy (EDX or EDS) spectrum is utilised for identifying the constituent makeup of a material. This spectrum shows particular peaks for copper (Cu) and oxygen (O), demonstrating the existence of both of these substances in the sample. The strong peaks for copper indicate it is the major component, whereas the considerable peak at roughly 0.5 keV for oxygen verifies its relationship, proposing the creation of CuONp. The lack of any major peaks shows that the synthesised substance is highly pure, with negligible pollutants from other components. This EDX study confirms the effective creation of CuONp, which are predicted to contain the desired chemical and functional properties for applications in biomedicine, chemical reaction, and cleaning up the environment. Antioxidant assay DPPH method CuONp synthesized from Prosopsis cineraria showed inhibition rates of 58.65% for 10µL, 69.63% for 20µL, 79.21% for 30µL, 82.5% for 40µL, and 84.1% for 50µL. The standard's inhibition percentages were 76.56% for 10µL, 78.52% for 20µL, 85.63% for 30µL, 88.68% for 40µL, and 90.15% for 50µL. The maximum inhibition was seen at 50µL, indicating a higher concentration. The nanoparticles of copper derived from Prosopis cineraria have significant antioxidant effects and are equivalent to the reference. H₂O₂ Assay Copper nanoparticles synthesized from Prosopsis cineraria showed inhibition rates of 45.6% at 10µL, 50.4% at 20µL, 60.5% at 30µL, 70.8% at 40µL, and 82.54% at 50µL. The standard had a percentage of inhibition ranging from 51.2% for 10µL to 88.6% for 50µL. Maximum inhibition was found at 50µL, indicating a higher concentration. Copper nanoparticles derived from Prosopsis cineraria exhibit high antioxidant activity and are similar to the standard. Antimicrobial Assay Copper nanocomposites antibacterial activity was assessed using an agar well diffusion assay. The antimicrobial effectiveness of Prosopsis cineraria -mediated CuONp nanocomposite was visualized against five clinical pathogens such as C. albicans , E. faeclalis , E. coli , Pseudomonas , and S. aureus , as represented in. A standard control was employed, which was Prosopsis cineraria leaf extract. Staphylococcus aureus , a gram-positive bacterium, and the opportunistic pathogenic yeast C. albicans showed a high inhibition zone. The gram-negative bacteria E. faecalis was shown to have a minimal inhibitory zone. Cytotoxicity assay Demonstrates the cytotoxicity at various conc (5–80 µL). Observations were taken over three days at concentrations of 5 µL, 10 µL, 20 µL, 40 µL, and 80 µL. Control Without CuONp, 10% cell death is seen. This reflects either baseline mortality from the experimental setting or spontaneous cell death. At 5µL, mortality reduces to 7%. Mortality stabilizes at 5% at doses ranging from 10µL to 40µL. At 80µL, the mortality rate drops to 4%. This might imply that CuONp do not cause considerable cytotoxicity at the studied doses, and may even reduce cell death when compared to the control. 4. Discussion Multiple characterisation techniques were used to evaluate the green synthesis of copper oxide nanoparticles (CuONp) from Prosopis cineraria extract of the leaf. The UV-Vis examination showed nanoparticle creation by a prominent plasmon resonance surface peak at 420 nm, followed by visible colour modifications, showing gradual diminution in copper ions[ 34 ]. FTIR examination showed the existence of functional groups as O-H, N-H, and C-O, indicating the participation of plant-based compounds in both diminution and capping of CuONp[ 35 ]. XRD spectra revealed an amorphous character with minimal crystalline peaks, which is regular of biosynthesised nanoparticles[ 36 ]. SEM pictures proved irregular, nanosized particles with significant surface area, while EDX data validated elemental purity, demonstrating the effective synthesis of CuONp[ 37 ]. Antioxidant experiments (DPPH and H₂O₂) showed significant dose-related free radical scavenging activity, equivalent to conventional antioxidants. Antimicrobial investigations revealed substantial suppression of S. aureus and C. albicans , demonstrating excellent antibacterial and antifungal action[ 38 ]. Cytotoxicity experiments demonstrated low toxicity, with fewer fatalities in nauplii even at greater dosages. These results emphasise the variety of uses and biological safety of the synthesised CuONp[ 39 ]. Overall, the work confirms the eco-friendliness and biological potential of P. cineraria-mediated CuONp. 5. Conclusion The work effectively demonstrated the green manufacturing of copper oxide nanoparticles utilising Prosopis cineraria leaf extract, an efficient and environmentally benign strategy. UV-Vis spectroscopy, FTIR, XRD, SEM, and EDX all verify the generation, shape, arrangements, and functionalisation of CuONp. The plant-derived compounds in the extract exert an important role in decreasing and stabilising the nanoparticles, leading to primarily amorphous, nanoscale particles with outstanding surface features. CuONp show significant antioxidant capacity in DPPH and H₂O₂ tests, showing their potential to neutralise free radicals. In addition, they exhibit promise antibacterial effectiveness, notably towards gram-positive bacteria and fungal diseases. The cytotoxicity investigation reveals that they are biocompatible at all investigated levels. These findings demonstrate that the synthesised nanoparticles are secure as well as successful for biological and environmental purposes. This green synthesis approach is a useful complement to established chemical pathways, delivering a low-cost, non-toxic, and sustainable choice for nanoparticle creation. Declarations Funding This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Ethics approval and consent to participate All the data was available from public databases and there is no need for ethics approval and consent. Credit authorship contribution Statement: RV: Writing-Review & editing, Writing Original Draft, Methodology, Data curation, Visualization, Drew the figures and pictures, Conceptualization. BP: Helped in writing the manuscript and revised it. RS: Validation and editing with review of the manuscript. SJ: Designed protocols for all the experiments, supervised, proofread, analyzed the data, and revised the reviewer's comments, investigation, and Conceptualization. All authors reviewed and discussed the results, provided comments on the manuscript, and approved its final published version. Consent for publication Not applicable. 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Ragavendran, C., et al., Synthesis of Lawsonia inermis-encased silver–copper bimetallic nanoparticles with antioxidant, antibacterial, and cytotoxic activity. Green Processing and Synthesis, 2024. 13 (1): p. 20230194. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-7092059","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":484583011,"identity":"e7a1cef5-28a7-4627-b548-df84b5d7ee40","order_by":0,"name":"Renu Vajjiravelu","email":"","orcid":"","institution":"Saveetha Institute of Medical and Technical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Renu","middleName":"","lastName":"Vajjiravelu","suffix":""},{"id":484583012,"identity":"7dd7f06a-1bb1-49f0-889f-9348c5578046","order_by":1,"name":"Banuppriya Palani","email":"","orcid":"","institution":"Saveetha Institute of Medical 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Jayakodi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACxh4wdYCBQYKN8QGQxcNHnJYEsBZmA5AWNoLW8CC0sEmA2AS1MPccf/i58McdOfnZbWmVX3PsZNgYmB8+uoHPYb09xtIzEp4ZG9w5duy27LZkoMPYjI1z8Gnp52GQ5kk4nLhBIr3ttuQ2ZqAWHjZp/FrYH/8GaZk/I72tWHJbPRFaehvMwLY03Eg7xvhx22EitPScMbPmSTtsbHAjLVmacdtxHjZmAn4x7El/fJvH5rCc/Iw0w48/t1Xb87M3P3yMV0sDEocZHEnMeJSDgDyKK38QUD0KRsEoGAUjEwAA55JHTs3LvwMAAAAASUVORK5CYII=","orcid":"","institution":"Saveetha Institute of Medical and Technical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Santhoshkumar","middleName":"","lastName":"Jayakodi","suffix":""}],"badges":[],"createdAt":"2025-07-10 10:38:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7092059/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7092059/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86763802,"identity":"8aa62431-762d-4557-87af-5b515f3c1d41","added_by":"auto","created_at":"2025-07-15 10:44:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206824,"visible":true,"origin":"","legend":"\u003cp\u003eCopper oxide nanoparticles synthesis from copper nitrate with \u003cem\u003eprosopsis cinerar\u003c/em\u003eia plant extractUV-Vis spectrum showing the absorbance of synthesized nanoparticles, with a distinct peak indicating successful synthesis.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/62da058b46e9b5f77d8adfe7.png"},{"id":86762776,"identity":"656e820f-1e88-4238-9ebe-321f6d368814","added_by":"auto","created_at":"2025-07-15 10:36:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32508,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectrum of NPS at different concentrations\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/ae22087cfbb96f0e79072813.png"},{"id":86766187,"identity":"301added-635b-43e1-b52e-a7d2aa722344","added_by":"auto","created_at":"2025-07-15 11:08:17","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":239113,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of copper oxide nanoparticles synthesized using \u003cem\u003eprosopsis cineraria\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/0ba1e5363213bdd382867bb5.jpeg"},{"id":86763804,"identity":"4e7120e5-9db5-4b3b-8c81-439e1aa51d2f","added_by":"auto","created_at":"2025-07-15 10:44:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":325937,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of NPs synthesized using \u003cem\u003eprosopsis cineraria\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/f7a67426d697b4adc7d61ddd.png"},{"id":86762777,"identity":"daef0ece-4b50-47d4-923a-8a511cdc946f","added_by":"auto","created_at":"2025-07-15 10:36:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":44142,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity of copper oxide nanoparticles synthesized using \u003cem\u003eprosopsis cineraria\u003c/em\u003eusing the DPPH assay\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/a70e1d1cc11a1f9202d59493.png"},{"id":86764048,"identity":"58e71836-ea23-4c7c-81a2-3ced5b839747","added_by":"auto","created_at":"2025-07-15 10:52:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":22350,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity of copper oxide NPs synthesized using \u003cem\u003eprosopsis cineraria\u003c/em\u003e using the Hydrogen peroxide assay\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/84bd00d2bb5dedfd411df1d7.png"},{"id":86765588,"identity":"063cfb4f-0e92-436d-a74a-d0c996ad1dbc","added_by":"auto","created_at":"2025-07-15 11:00:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":48747,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-microbial activity of copper nanoparticles synthesized using agar well diffusion assay\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/345dc318db96359145944ab1.png"},{"id":86766188,"identity":"fff4732c-fd36-4ead-ac7b-cddcf0277c6b","added_by":"auto","created_at":"2025-07-15 11:08:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":14386,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity assay demonstrating the mortality of the nauplii at various concentrations.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/a6b3db16092cbff14bfe2169.png"},{"id":86762786,"identity":"5e371939-32cc-4520-86a3-4a685dc9c465","added_by":"auto","created_at":"2025-07-15 10:36:17","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":232122,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Materials and Methods section.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/133214f48c46aac961feba4e.jpeg"},{"id":92358819,"identity":"219fc164-29ff-4460-9e8c-393252475ee4","added_by":"auto","created_at":"2025-09-28 15:46:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1833971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7092059/v1/664d65e5-8f77-4f53-8ead-72dd6ceb5160.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Green Synthesis of Copper Oxide Nanoparticles Using Prosopis cineraria Extract: Evaluation of Cytotoxic Antioxidant and Antimicrobial Activity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanotechnology has revolutionized the field of science and technology by offering innovative solutions across multiple disciplines, including medicine, agriculture, and environmental sciences[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among the various nanoparticles studied, copper nanoparticles (CuONp) have received considerable interest because of their unique qualities, such as high catalytic activity, conductivity, and antimicrobial efficacy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recently, the focus has shifted towards the green synthesis of nanoparticles, as conventional chemical and physical methods often involve toxic chemicals, high energy consumption, and expensive equipment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Green synthesis, leveraging natural sources such as plant extracts, microorganisms, and biomolecules, provides an eco-friendly and sustainable approach [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Plants, in particular, are an excellent resource for synthesizing nanoparticles, as they contain a diverse array of phytochemicals such as alkaloids, flavonoids, phenols, and tannins [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These compounds not only reduce metal ions to their nanoparticle form but also stabilize the nanoparticles, imparting additional bioactivity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eProsopsis cineraria\u003c/em\u003e holds immense significance in traditional medicine and is renowned for its pharmacological characteristics, comprising anti-oxidants, antiviral, anti-fungal, and therapeutic activity[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The phytochemical composition of \u003cem\u003eProsopis cineraria\u003c/em\u003e includes flavonoids, tannins, alkaloids, and phenolic acids, which make it an ideal candidate for the green synthesis of nanoparticles [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Utilizing this plant for synthesizing CuONp combines the inherent bioactivity of the plant with the enhanced properties of nanoparticles, paving the way for innovative applications in health and environmental sciences [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The biological activities of CuONp, such as antioxidant, antimicrobial, and cytotoxic effects, have been widely explored [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Antioxidant activity is an essential characteristic, because the inflammation induced by reactive oxygen species (ROS) is linked to ageing and the development of many illnesses, especially malignancy, neurological diseases, and circulatory maladies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CuONp, by their redox potential and the presence of surface-active groups, can effectively scavenge ROS and reduce oxidative damage [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Similarly, the antimicrobial properties of CuONp have demonstrated broad-spectrum efficacy against bacteria, fungi, and viruses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Copper ions break membranes in microbes, impede cellular respiration, and produce reactive oxygen species, resulting in microbes' cell mortality [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These properties are especially relevant in combating antimicrobial resistance (AMR), which poses a global health challenge, and in food preservation, where microbial contamination is a major concern [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, the cytotoxic potential of CuONp against cancer cells offers promising applications in oncology [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. CuONp induce cytotoxicity through mechanisms such as ROS generation, DNA damage, and disruption of mitochondrial function, selectively targeting cancer cells while sparing normal tissues [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This dual action of CuONp, combining therapeutic and diagnostic capabilities, highlights their potential as nanomedicine agents [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The antioxidant activity was assessed using standard radical scavenging assays to determine their efficacy in neutralizing oxidative stress [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The ability to inhibit microbes was evaluated towards several harmful microorganisms to investigate how they might help microbial resistance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The cytotoxic activity was analyzed in vitro against specific cancer cell lines to evaluate their therapeutic applicability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The present investigation intends to add to the expanding body of research on green-synthesized nanoparticles in emphasising the beneficial effects of plant-mediated CuONp [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cb\u003eExtract preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the current state investigation, 1gm of \u003cem\u003eProsopsis cineraria\u003c/em\u003e was dissolved in a hundred ml of purified water and boiled for ten to fifteen minutes at 70 degrees Celsius. After heating up, Whatman filtered the plant extract. No.1 filter paper [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In a 250 ml conical flask, 70 ml of 20 millimolar copper nitrate was prepared, and 30 ml of filtered vegetal extract was added and put into an iron magnetic stirrer to produce nanoparticles. After the synthesis of nanoparticles, a colour change was seen [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntioxidant assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDPPH method\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe DPPH method was applied to study the protective characteristics of naturally occurring synthesised particles of copper. Copper nitrate nanomaterials have been added with \u003cem\u003eProsopsis cineraria\u003c/em\u003e herb extract (2\u0026ndash;10 \u0026micro;g/ml) in 1 ml of 0.1 mm DPPH in methanol and 450 \u0026micro;l of 50 mm Tris HCl buffer (pH 7.4) for ten minutes. Afterwards, the drop of DPPH free radicals was measured using the intensity of absorption at 517 nm. BHT was used as control. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The following equation determined the level of inhibition,\u003c/p\u003e\u003cp\u003eThe percentage of inhibition\u0026thinsp;=\u0026thinsp;absorbance of control minus absorbance of test sample divided by 100.\u003c/p\u003e\u003cp\u003e\u003cb\u003eH₂O₂ Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe potential of the biosynthesized tocopherol chitosan biocomposite to scavenge H₂O₂ was evaluated in 40 mM H₂O₂ solution in a pH 7.4 phosphate buffer. 0.6 mL of H₂O₂ solution was mixed with a solution of the test sample (ɑ-tocopherol chitosan biocomposite) and a standard sample of ascorbic acid at different concentrations (10\u0026ndash;50\u0026micro;g/mL). Spectrophotometric measurements of the reaction solution absorbance at 532 nm were made following a 10-minute incubation period in a dark environment. The standard used was vitamin C. Using the following formula, the proportion of H₂O₂ scavenging activity was calculated\u003c/p\u003e\u003cp\u003eTo calculate the percentage of inhibition, divide the absorbance of the control by the absorbance of the sample and multiply by 100.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo manufacture Mueller-Hinton agar, 10 \u0026micro;L of fresh microbial cultures (\u003cem\u003eC. albicans, E. faeclalis, E. coli, Pseudomonas\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e) were injected in a sterile Hi-veg media. A sanitized polystyrene tip was used to make 5 mm wells. Antimicrobial effect was assessed to determine the effectiveness of \u003cem\u003eProsopsis cineraria\u003c/em\u003e-mediated CuONp and biosynthesized copper Particle-mediated nanocomposite material. Wells were filled with varying amounts of three samples (25, 50, and 100 \u0026micro;L) and a conventional test. The specimens of Petri plates and zone inhibition were kept in an incubator at 37 degrees Celsius for 24 hours. The area was evaluated in order to contrast and examine the possible impact of \u003cem\u003eProsopsis cineraria\u003c/em\u003e-mediated copper nanoparticles [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxicity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBrine shrimp eggs were placed in a hatching chamber filled with salty water. After 24 hours, precisely ten hatched larvae (nauplii) were floating in six wells, each holding 10 ml of saltwater. Nanoparticles were scattered in each well at varying quantities (5 \u0026micro;L, 10 \u0026micro;L, 20 \u0026micro;L, 40 \u0026micro;L, and 80 \u0026micro;L), with the very last well serving as a control (no nanoparticles). After 24 hours, the number of remaining nauplii was calculated and noted.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003eVisual Observation and UV-Vis Studies for Copper Oxide NPs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe development of copper oxide NPs was tracked using a UV-visible spectrophotometer. \u003cem\u003eProsopsis cineraria\u003c/em\u003e leaf extract functions as a stabilizing and diminutive agent. Biological molecules in the leaf extract reduce copper nitrate nanoparticles to copper oxide nanoparticles. The color changes from reddish brown to dark reddish brown after adding leaf extract, and then progressively gets dark brown due to the synthesis of CuONp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) depicts the color variations that occur during nanoparticle production with CuONp. UV-visible data were taken over a period of 0\u0026ndash;84 hours. Copper nanoparticles' resonance with the surface exhibited the highest peak at 420 nm, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFTIR Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe FTIR spectrum of the sample labelled CuO\u0026thinsp;+\u0026thinsp;PC (Copper oxide nanoparticles synthesised using \u003cem\u003eProsopsis cineraria\u003c/em\u003e extract) offers useful insight into the functional compounds present in the sample, revealing the importance of plant phytochemicals in the green synthesis procedure. A large absorption peak at 3185.8 cm\u0026sup1; corresponds to O-H stretching vibrations, which are indicative of hydroxyl groups from alcohols or phenolic compounds. This suggests the participation of plant metabolites like flavonoids or polyols in stabilising the nanoparticles. The signal at 2960.3 cm\u0026sup1; is due to C-H stretching vibrations of aliphatic -CH₂ and -CH₃ groups, suggesting the presence of organic chemicals in the plant extract.\u003c/p\u003e\u003cp\u003eA peak at 1557.5 cm\u0026sup1; may be due to N-H bending oscillations from amide connections or C\u0026thinsp;=\u0026thinsp;C stretching of aromatic rings, which are typically found in proteins or polyphenolic structures. The band at 1385.8 cm\u0026sup1; may be attributed to C-N stretching or O-H bending, indicating the existence of biomolecules with amine or phenol groups. The absorption at 1034.7 cm-\u0026sup1; corresponds to C-O stretching vibrations of alcohols, ethers, or esters, suggesting the presence of plant-derived substances such as carbohydrates and flavonoids in the capping material.\u003c/p\u003e\u003cp\u003eThe peaks at 813.58 cm⁻\u0026sup1; and 678.32 cm⁻\u0026sup1; show out-of-plane bending vibrations of aromatic C-H bonds, similar to those found in plant phenols. The high absorption bands at 613.3 cm⁻\u0026sup1; and 465.03 cm⁻\u0026sup1; show the production of copper oxide nanoparticles. These data combined show that \u003cem\u003eprosopsis cineraria\u003c/em\u003e extract functions as a reducing and capping agent in the green production of CuONp, and the functional groups observed in the FTIR spectrum play an important role in nanoparticle stabilisation and bioactivity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eXRD Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe X-ray diffraction (XRD) image depicts copper oxide nanoparticles (CuONp) synthesised utilising Prosopis cineraria. The prominent diffraction peaks at specific 2θ values can be indexed to crystalline planes such as (021), (121), (-213), (-204), (-214), (-245), (-107), (018), and (-261), which match the monoclinic phase of CuONP. These peaks are consistent with the JCPDS card No. 00-042-1959, demonstrating the creation of pure, crystalline monoclinic CuONp. The strong and powerful patterns show great crystallinity, and the lack of further patterns shows that the synthesised nanoparticles are pure, without no other copper phases or contaminants[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eSEM Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA scanning electron microscope (SEM) image displaying the surface morphology of copper oxide nanoparticles (CuONp). The 30,000\u0026times; magnification shows a granular structure with irregular particle shapes and distribution. These particles appear to be closely packed, generating clusters of high surface roughness. The shape indicates an enormous surface area, which is useful for catalytic, antibacterial, or cytotoxic applications. The dimensions of the bar represents a length of 300 nm, emphasising the nanoscale size of the particles. This shape is prevalent in biosynthesised nanoparticles and shows the effective generation of CuONp with improved reactivity and surface contact, which are important for biological and environmental purposes.\u003c/p\u003e\u003cp\u003eA energy dispersive x-ray spectroscopy (EDX or EDS) spectrum is utilised for identifying the constituent makeup of a material. This spectrum shows particular peaks for copper (Cu) and oxygen (O), demonstrating the existence of both of these substances in the sample. The strong peaks for copper indicate it is the major component, whereas the considerable peak at roughly 0.5 keV for oxygen verifies its relationship, proposing the creation of CuONp. The lack of any major peaks shows that the synthesised substance is highly pure, with negligible pollutants from other components. This EDX study confirms the effective creation of CuONp, which are predicted to contain the desired chemical and functional properties for applications in biomedicine, chemical reaction, and cleaning up the environment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntioxidant assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDPPH method\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCuONp synthesized from \u003cem\u003eProsopsis cineraria\u003c/em\u003e showed inhibition rates of 58.65% for 10\u0026micro;L, 69.63% for 20\u0026micro;L, 79.21% for 30\u0026micro;L, 82.5% for 40\u0026micro;L, and 84.1% for 50\u0026micro;L. The standard's inhibition percentages were 76.56% for 10\u0026micro;L, 78.52% for 20\u0026micro;L, 85.63% for 30\u0026micro;L, 88.68% for 40\u0026micro;L, and 90.15% for 50\u0026micro;L. The maximum inhibition was seen at 50\u0026micro;L, indicating a higher concentration. The nanoparticles of copper derived from Prosopis cineraria have significant antioxidant effects and are equivalent to the reference.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eH₂O₂ Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCopper nanoparticles synthesized from \u003cem\u003eProsopsis cineraria\u003c/em\u003e showed inhibition rates of 45.6% at 10\u0026micro;L, 50.4% at 20\u0026micro;L, 60.5% at 30\u0026micro;L, 70.8% at 40\u0026micro;L, and 82.54% at 50\u0026micro;L. The standard had a percentage of inhibition ranging from 51.2% for 10\u0026micro;L to 88.6% for 50\u0026micro;L. Maximum inhibition was found at 50\u0026micro;L, indicating a higher concentration. Copper nanoparticles derived from \u003cem\u003eProsopsis cineraria\u003c/em\u003e exhibit high antioxidant activity and are similar to the standard.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCopper nanocomposites antibacterial activity was assessed using an agar well diffusion assay. The antimicrobial effectiveness of \u003cem\u003eProsopsis cineraria\u003c/em\u003e-mediated CuONp nanocomposite was visualized against five clinical pathogens such as \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003eE. faeclalis\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e, as represented in. A standard control was employed, which was \u003cem\u003eProsopsis cineraria\u003c/em\u003e leaf extract. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, a gram-positive bacterium, and the opportunistic pathogenic yeast \u003cem\u003eC. albicans\u003c/em\u003e showed a high inhibition zone. The gram-negative bacteria \u003cem\u003eE. faecalis\u003c/em\u003e was shown to have a minimal inhibitory zone.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxicity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDemonstrates the cytotoxicity at various conc (5\u0026ndash;80 \u0026micro;L). Observations were taken over three days at concentrations of 5 \u0026micro;L, 10 \u0026micro;L, 20 \u0026micro;L, 40 \u0026micro;L, and 80 \u0026micro;L. Control Without CuONp, 10% cell death is seen. This reflects either baseline mortality from the experimental setting or spontaneous cell death. At 5\u0026micro;L, mortality reduces to 7%. Mortality stabilizes at 5% at doses ranging from 10\u0026micro;L to 40\u0026micro;L. At 80\u0026micro;L, the mortality rate drops to 4%. This might imply that CuONp do not cause considerable cytotoxicity at the studied doses, and may even reduce cell death when compared to the control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMultiple characterisation techniques were used to evaluate the green synthesis of copper oxide nanoparticles (CuONp) from Prosopis cineraria extract of the leaf. The UV-Vis examination showed nanoparticle creation by a prominent plasmon resonance surface peak at 420 nm, followed by visible colour modifications, showing gradual diminution in copper ions[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. FTIR examination showed the existence of functional groups as O-H, N-H, and C-O, indicating the participation of plant-based compounds in both diminution and capping of CuONp[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. XRD spectra revealed an amorphous character with minimal crystalline peaks, which is regular of biosynthesised nanoparticles[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. SEM pictures proved irregular, nanosized particles with significant surface area, while EDX data validated elemental purity, demonstrating the effective synthesis of CuONp[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Antioxidant experiments (DPPH and H₂O₂) showed significant dose-related free radical scavenging activity, equivalent to conventional antioxidants. Antimicrobial investigations revealed substantial suppression of \u003cem\u003eS. aureus and C. albicans\u003c/em\u003e, demonstrating excellent antibacterial and antifungal action[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Cytotoxicity experiments demonstrated low toxicity, with fewer fatalities in nauplii even at greater dosages. These results emphasise the variety of uses and biological safety of the synthesised CuONp[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Overall, the work confirms the eco-friendliness and biological potential of P. cineraria-mediated CuONp.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe work effectively demonstrated the green manufacturing of copper oxide nanoparticles utilising Prosopis cineraria leaf extract, an efficient and environmentally benign strategy. UV-Vis spectroscopy, FTIR, XRD, SEM, and EDX all verify the generation, shape, arrangements, and functionalisation of CuONp. The plant-derived compounds in the extract exert an important role in decreasing and stabilising the nanoparticles, leading to primarily amorphous, nanoscale particles with outstanding surface features. CuONp show significant antioxidant capacity in DPPH and H₂O₂ tests, showing their potential to neutralise free radicals. In addition, they exhibit promise antibacterial effectiveness, notably towards gram-positive bacteria and fungal diseases. The cytotoxicity investigation reveals that they are biocompatible at all investigated levels. These findings demonstrate that the synthesised nanoparticles are secure as well as successful for biological and environmental purposes. This green synthesis approach is a useful complement to established chemical pathways, delivering a low-cost, non-toxic, and sustainable choice for nanoparticle creation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All the data was available from public databases and there is no need for ethics approval and consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCredit authorship contribution Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRV: Writing-Review \u0026amp; editing, Writing Original Draft, Methodology, Data curation, Visualization, Drew the figures and pictures, Conceptualization. \u0026nbsp;BP: \u0026nbsp;Helped in writing the manuscript and revised it. \u0026nbsp;RS: \u0026nbsp; Validation and editing with review of the manuscript. SJ: Designed protocols for all the experiments, supervised, proofread, analyzed the data, and revised the reviewer\u0026apos;s comments, investigation, and Conceptualization. \u0026nbsp;All authors reviewed and discussed the results, provided comments on the manuscript, and approved its final published version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSoni, S., et al., \u003cem\u003eHerbal nanogels: Revolutionizing skin cancer therapy through nanotechnology and natural remedies.\u003c/em\u003e European Journal of Medicinal Chemistry Reports, 2023: p. 100126.\u003c/li\u003e\n\u003cli\u003eNoah, N.M. and P.M. 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Auravindam, and T.K. Nailwal, \u003cem\u003eBiomedical Applications of Algae-Synthesized Nanoparticles: Drug Delivery, Anti-Bacterial, Anti-Fungal, Anti-Inflammatory, Anti-Oxidant\u003c/em\u003e, in \u003cem\u003ePhyconanotechnology: Current Research, Challenges, and Prospects\u003c/em\u003e. 2025, Springer. p. 99-115.\u003c/li\u003e\n\u003cli\u003eRagavendran, C., et al., \u003cem\u003eSynthesis of Lawsonia inermis-encased silver\u0026ndash;copper bimetallic nanoparticles with antioxidant, antibacterial, and cytotoxic activity.\u003c/em\u003e Green Processing and Synthesis, 2024. \u003cstrong\u003e13\u003c/strong\u003e(1): p. 20230194.\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":"Copper nanoparticles (CuONp), Green synthesis, Prosopis cineraria, Antioxidant activity, Antibacterial activity, Cytotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-7092059/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7092059/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCopper nanoparticles (CuONp) have garnered considerable attention due to their unique thermodynamic properties and broad bioactivities, including antioxidant, antibacterial, and cytotoxic effects. The green synthesis of CuONp, which employs natural plant extracts as reducing and stabilizing agents, is an eco-friendly and sustainable alternative to conventional methods. This study investigates the synthesis of CuONp using an aqueous extract of \u003cem\u003eProsopis cineraria\u003c/em\u003e and evaluates their biological activities. CuONp were synthesized by mixing \u003cem\u003eP. cineraria\u003c/em\u003e extract with copper nitrate under magnetic stirring to promote nanoparticle formation. The synthesis was validated using UV-visible spectroscopy, revealing characteristic absorption peaks indicative of CuONp formation. Antioxidant activity was assessed using the DPPH assay, while antibacterial activity was tested against two bacterial strains. Cytotoxicity was evaluated via a brine shrimp lethality assay, measuring nauplii mortality at different CuONp concentrations. UV-visible spectroscopy confirmed the successful synthesis of CuONp. The DPPH assay showed dose-dependent free radical scavenging, with maximum activity at the highest concentration. Antibacterial testing demonstrated significant inhibition zones, indicating strong activity against both bacterial strains. The cytotoxicity assay revealed dose-dependent nauplii mortality, underscoring the cytotoxic potential of CuONp. Green-synthesized CuONp using \u003cem\u003eP. cineraria\u003c/em\u003e exhibit strong antioxidant, antibacterial, and cytotoxic properties, highlighting their potential for clinical and therapeutic applications. This sustainable synthesis approach harnesses the bioactive potential of \u003cem\u003eP. cineraria\u003c/em\u003e for nanoparticle production.\u003c/p\u003e","manuscriptTitle":"Green Synthesis of Copper Oxide Nanoparticles Using Prosopis cineraria Extract: Evaluation of Cytotoxic Antioxidant and Antimicrobial Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 10:36:12","doi":"10.21203/rs.3.rs-7092059/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":"cf4b8a38-2669-4276-a0be-03f108ab9d7d","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-28T15:38:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-15 10:36:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7092059","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7092059","identity":"rs-7092059","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}