Sustainable plant mediated synthesis of cobalt oxide nanoparticles using Uraria picta extract with enhanced biological activity S. Sheikh1, A. J. Mungole1, Ajmal R. Bhat2, A. P. Pawar3, C. P. Pandhurnekar4, H. C. Pandhurnekar5, Rizwan Wahab6, H. P. Kanfade7, Vijay Jagdish Upadhye8, Sumeer Ahmed9, Dalesh M. Parshuramkar10, Mohammad Al-Omari11 & Palani Elumalai12 The present study demonstrates a sustainable phytogenic route for the synthesis of cobalt oxide nanoparticles (CoO NPs) utilizing Uraria picta (Jacq.) DC. plant extract as a natural reducing and stabilizing agent. The whole plant was collected, shade-dried, powdered, and processed to obtain an aqueous extract, which was subsequently reacted with cobalt chloride hexahydrate stock solution, leading to the formation of CoO NPs via a green synthesis pathway. The appearance of a distinct color change confirmed nanoparticle formation. The resulting material was centrifuged, filtered, dried, and calcined to obtain blackish-violet CoO NPs. Comprehensive characterization of the biosynthesized nanoparticles was performed using UV–Vis spectroscopy, FTIR, XRD, EDS, SEM, and TEM analyses, confirming their structural, morphological, and elemental properties. Furthermore, the synthesized CoO NPs exhibited significant antioxidant potential and antibacterial activity against selected bacterial strains. This eco-friendly and cost-effective green synthesis approach not only avoids hazardous chemicals but also provides a promising strategy for the development of nanomaterials with potential biological and environmental applications. Keywords  Green synthesis, Cobalt oxide nanoparticles, Phytogenic synthesis, Uraria picta (Jacq.) DC., Biological applications Nanoparticles are materials with dimensions ranging from 1 to 100 nm, exhibiting unique physico-chemical properties that differ significantly from their bulk counterparts. One of the most distinctive features of nanoparticles is their high surface-to-volume ratio which increases as particle size decreases, imparting remarkable optical, electrical and mechanical properties1–4. Owing to these properties, nanoparticles have been extensively utilized in diverse fields such as medical sciences5–8, biomedical research9,10, food packaging11,12, textiles13,14, water purification15,16, construction17–19, batteries20–23, electronics24–26, solar photovoltaics27–31, fuel cells32–34, sensors35–38 and pharmaceutical applications, including anti-cancer therapeutics39. Metal 1Department of Botany, Nevjabai Hitkarini College, Bramhapuri 441 206, Maharashtra, India. 2Department of Chemistry, RTM Nagpur University, Nagpur 440 033, India. 3Department of Chemistry, Nevjabai Hitkarini College, Bramhapuri 441 206, Maharashtra, India. 4Department of Chemistry, Shri Ramdeobaba College of Engineering and Management, Ramdeobaba University, Nagpur 440 013, Maharashtra, India. 5Department of Chemistry, Dada Ramchand Bakhru Sindhu Mahavidyalaya, Nagpur 440 017, Maharashtra, India. 6Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia. 7Department of Botany, Shantabai Mahila Mahavidyalaya, Bramhapuri 441 206, Maharashtra, India. 8Deptartment of Microbiology, Parul Institute of Applied Sciences (PIAS), Parul University, PO, Limda, Tal Waghodia 391 760, India. 9Natural Products and Medicinal Chemistry Division, CSIR - Indian Institute of Integrative Medicine, Jammu 180 001, India. 10Department of Physics, Nevjabai Hitkarini College, Bramhapuri 441 206, Maharashtra, India. 11Chemistry Department, Faculty of Science, Applied Science Private University, P.O. Box 166, Amman 11931, Jordan. 12Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB3 0AS, UK. email: aru.mungole@gmail.com; bhatajmal@gmail.com; dr.vijaysemilo@gmail.com; sumeerchoudhary15@gmail.com OPEN Scientific Reports | (2025) 15:44017 1| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports http://www.nature.com/scientificreports http://crossmark.crossref.org/dialog/?doi=10.1038/s41598-025-28596-0&domain=pdf&date_stamp=2025-12-3 oxide nanoparticles, in particular, are of great interest due to their demonstrated antibacterial, antiviral, and antioxidant activities36–41. Conventionally, nanoparticles are synthesized using physical and chemical methods; however, these approaches are often expensive, energy-intensive and associated with the use of toxic reagents that can generate hazardous byproducts. To overcome these limitations, biosynthetic methods have emerged as promising alternatives owing to their sustainability, cost-effectiveness, and eco-friendly nature42–44. Plant-mediated synthesis of nanoparticles offers a sustainable alternative to conventional chemical methods by eliminating toxic reagents, reducing energy input, and utilizing renewable biomolecules as natural reducing and stabilizing agents45,46. This eco-friendly route not only minimizes environmental burden but also enhances biocompatibility, aligning with principles of green chemistry and circular economy. Among the biological methods, plant extract- mediated synthesis is considered the most efficient, scalable and straightforward approach compared to bacterial or fungal-mediated routes. Plant-derived phytochemicals, including ketones, aldehydes, flavones, terpenoids, carboxylic acids, phenols, and ascorbic acid, play a vital role in nanoparticle synthesis by acting as reducing, stabilizing and capping agents, thereby preventing agglomeration of nanoparticles47–49. Cobalt oxide nanoparticles (CoO NPs) are multifunctionfigal materials, classified as antiferromagnetic p-type semiconductors and belong to the family of transition metal oxides50–52. They have been widely investigated for applications in Lithium-ion rechargeable batteries, energy storage devices, heterogeneous catalysis, electrochromic sensors, pigments and dyes53–57. Moreover, CoO NPs have shown significant potential as electrode materials in pseudo-capacitors58. In biomedical sciences, cobalt oxide nanoparticles have garnered attention due to their unique bioactivities, including anticancer properties, which are attributed to their enhanced cellular uptake and ability to induce apoptosis in cancer cells59,60. Notably, cobalt also serves as an essential component of vitamin B12, further underlining its physiological relevance61. Several studies have demonstrated the potential of plant-mediated synthesis of CoO NPs for biomedical applications. For instance, cobalt oxide nanoparticles synthesized using Emblica officinalis extract have been investigated for their role in anemia treatment due to their link with vitamin B1262, while Geranium wallichianum extract-mediated CoO NPs exhibited antibacterial, antifungal, enzyme inhibition, biocompatibility and anticancer properties63. Similarly, CoO NPs synthesized from Ziziphora clinopodioides Lam showed cytotoxic, antioxidant, antifungal, antibacterial, and wound-healing activities, along with enzyme inhibition potential64. In another study, CoO NPs synthesized using Vibrio species demonstrated antitumor potential against colorectal cancer, highlighting their role as promising candidates in cancer nanomedicine65. The present study focuses on Uraria picta (Jacq.) DC., a perennial herb belonging to the family Leguminosae– Papilionaceae. This plant is widely distributed in tropical Africa, South and Southeast Asia, and Australia, typically growing in dry grasslands, sandy soils, and rocky habitats66. Traditionally, Uraria picta has been employed in Ayurvedic, Unani, and Chinese medicinal systems and is an important constituent of Dashmula, a classical Ayurvedic formulation comprising ten medicinal herbs67. The plant is reputed for its therapeutic properties, including antibacterial, antidiabetic, anticancer, and anti-inflammatory activities68, attributed to its rich phytochemical profile consisting of flavonoids, isoflavones, triterpenes, phenolics, tannins, cardiac glycosides, saponins and steroids69. Previous research has suggested that plant-derived metabolites such as alkaloids and flavonoids are effective mediators for nanoparticle synthesis70. Despite the growing interest in plant-mediated synthesis of metal oxide nanoparticles, no studies have yet reported the synthesis of cobalt oxide nanoparticles using Uraria picta (Jacq.) DC. Therefore, this plant extract was selected for the present investigation (Fig. 1). In this study, we report for the first time the phytogenic synthesis of CoO NPs using U. picta extract. It is rich in flavonoids, phenolics, alkaloids, and glycosides. These diverse bioactive compounds act as efficient reducing and capping agents, enabling controlled CoO nanoparticle synthesis and highlighting its novelty in green nanotechnology71. The synthesized nanoparticles were subjected to comprehensive physicochemical characterization and their biological activities, including antioxidant and antibacterial potentials, were systematically evaluated. Materials and methods All chemicals and reagents used in the present study were of analytical grade and of the highest possible purity. Cobalt chloride hexahydrate (CoCl2·6H2O, Purity: 98%) and butyl hydroxytoluene (BHT) were procured from Merck Chemicals, India. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) was obtained from Sigma-Aldrich, India. Organic solvents such as acetone, n-hexane, ethanol, and methanol were purchased from S. D. Fine Chemicals, India. Authenticated strain cultures were procured from Sai Biosystems Pvt. Ltd., Nagpur and ESONAWA Innovations Pvt. Ltd., Nagpur. The purity of each strain was confirmed based on colony characteristics on selective media: E. coli on Eosin Methylene Blue (EMB) agar, B. subtilis on Bacillus agar, S. typhi on Salmonella– Shigella (SS) agar, and P. vulgaris on Heart Infusion agar. Plant collection and authentication Uraria picta (Jacq.) DC., a perennial herb of the family Fabaceae, was selected for the present study. Fresh plant material was collected by Ms. S. Sheikh from Bramhapuri Tehsil, Chandrapur, District, Maharashtra, India (Latitude: 20.62298° N, Longitude: 79.855588° E). Prior permission for collection was obtained from the concerned Forest Department/competent authority, and the process was carried out in accordance with institutional, national, and international guidelines for the collection of plant material. The taxonomic identification and authentication of the species were carried out by the Department of Botany, Anand Niketan College, Warora, Dist. Chandrapur, Maharashtra, India, and a voucher specimen was deposited (Ref. No. ANC/ Bot/2024/01/25BH) Fig. S1-S3. The collected material was carefully shade-dried to preserve its phytoconstituents, ground into a fine powder using a mortar and pestle, and stored in airtight containers until further use. Scientific Reports | (2025) 15:44017 2| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports Preparation of plant extract The whole plant of Uraria picta (Jacq.) DC. was collected from the forest region near Bramhapuri, Chandrapur district, Maharashtra, India. The freshly collected plant material was thoroughly washed with tap water to remove adhering dust and soil particles, followed by repeated washing with deionized distilled water at room temperature. The cleaned plant material was shade-dried completely, chopped into small pieces, and ground into a fine powder. For extract preparation, 25 g of the powdered material was transferred into a clean, dry round- bottom flask containing 250 mL of distilled water. The mixture was heated at 80 °C for 2 h on a heating mantle and subsequently cooled to room temperature. The cooled mixture was filtered using Whatman No. 41 filter paper, and the resulting filtrate was collected and stored at 0 °C for further experimental use Fig. 272. Biosynthesis of Cobalt oxide nanoparticles (CoO NPs) For the biosynthesis of CoO NPs, 100 mL of Uraria picta plant extract was taken in a 250 mL beaker, and 50 mL of cobalt chloride hexahydrate stock solution was added dropwise with continuous stirring. The mixture was maintained at 80 °C on a hot plate with constant stirring on the magnetic stirrer at 1200 rpm (Make: Remi, India) for 2 h to facilitate nanoparticle formation. After cooling to room temperature (27 °C), the reaction mixture was centrifuged at 10,000 rpm for 10 min to collect the pellet, while the supernatant was discarded. The obtained pellet was washed three times with deionized water to remove impurities, dried at 100 °C for 2 Fig. 2.  Graphical representation of the preparation of the plant extract. Fig. 1.  Uraria picta (Jacq.) DC: (A) Habit (B) Inflorescence (C) Individual Flower (D) Fruits (E) Individual Fruit. Scientific Reports | (2025) 15:44017 3| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports h, and subsequently calcined in furnace with gradual rise in the temperature of sample up to 500 °C and kept at constant temperature of 500 °C for 2 h to yield black-violet, highly crystalline cobalt oxide nanoparticles as depicted in the Fig. 372. Characterization The biosynthesized cobalt oxide nanoparticles were subjected to comprehensive characterization using advanced spectroscopic and microscopic techniques, including UV–Visible spectroscopy, Fourier-transform infrared Fig. 3.  Schematic Representation of Green Synthesis of Cobalt Oxide Nanoparticles (CoO NPs) Using Uraria picta Extract. Scientific Reports | (2025) 15:44017 4| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports (FTIR) spectroscopy, powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray analysis (EDAX/EDX). The optical properties were examined using a UV–Visible spectrophotometer (EQUIPTRONICS, Model EQ- 826) within the wavelength range of 190–1100 nm to investigate the absorbance behavior of the nanoparticles73,74. FTIR spectra were recorded using a Thermo Nicolet iS50 spectrophotometer (resolution 0.2 cm⁻¹) in the range of 400–4000 cm⁻¹ to identify phytochemicals responsible for nanoparticle synthesis and stabilization. For this purpose, 1–2 mg of powdered sample was homogenized with ~ 100 mg of spectroscopic-grade KBr, compressed under 8–12 tonnes pressure into transparent pellets, and analyzed with an average of 64 scans per sample73. The crystalline structure and average crystallite size were determined by PXRD using a Bruker D8 Advance A25 diffractometer operating at 40 mA and 40 kV in Bragg–Brentano geometry. Samples were mounted on amorphous silica low-background holders for analysis75. Surface morphology and particle size distribution were observed via SEM (JEOL JSM-6390LV, Tokyo, Japan) at 15 kV. Samples were immobilized on carbon adhesive tape, sputter-coated with a thin gold layer using a JFC-1600 ion-sputtering device, and examined under varying magnifications to assess shape, surface topography, and aggregation behavior76. Elemental composition was investigated using EDX (Oxford XMX N system coupled with SEM). The technique allowed semi-quantitative detection of constituent elements with a sensitivity of approximately 0.5–1.0 wt%, enabling confirmation of nanoparticle purity and the presence of trace elements77. TEM analysis was performed using a JEOL JM-2100 microscope equipped with a Gatan imaging system to examine particle morphology, lattice fringes, and nanoscale features. For this analysis, nanoparticles were ultrasonically dispersed in ethanol, and a drop of the suspension was placed on a 200-mesh carbon-coated copper grid, air-dried, and mounted for imaging78. All analyses were carried out at the Sophisticated Analytical Instrumentation Facility (SAIF), Kochi, India. Antibacterial activity of nanoparticles Stock solution of nanoparticles The biosynthesized nanoparticles were obtained in dried powdered form, as described in the synthesis protocol. For antibacterial screening against selected human pathogenic bacteria, a working stock solution was prepared by dispersing the nanoparticle powder in Dimethyl Sulfoxide (DMSO) at a concentration of 1 mg/mL. DMSO was chosen as the solvent due to its non-reactive nature toward most microbial species and its ability to enhance nanoparticle solubility.Prior to use in antibacterial assays, the nanoparticle suspensions were subjected to sonication to achieve uniform dispersion. Sonication was performed using a Labline 1.5 L water bath sonicator, wherein centrifuge tubes containing the nanoparticle suspension were immersed and treated for 15 min. The sonicated solutions were immediately utilized for antibacterial activity assays to minimize re-aggregation and ensure stability of the nanoparticle dispersion. Procurement and maintenance of bacterial cultures Four clinically relevant human pathogenic bacteria, namely Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Salmonella typhi (S. typhi) and Proteus vulgaris (P. vulgaris), were selected to evaluate the antibacterial potential of the synthesized nanoparticles. Antibacterial assays were conducted at three different nanoparticle concentrations, with gentamicin employed as the standard reference antibiotic and DMSO serving as the solvent control. Inoculum Preparation for antibacterial assay In the present study, E. coli, B. subtilis, S. typhi, and P. vulgaris were regularly maintained on their respective selective media and subsequently employed for antibacterial assays. For inoculum preparation, a loopful of each pure culture was transferred into 10 mL of sterile nutrient broth and incubated at 37 °C for 24 h. The bacterial suspensions were adjusted to match the 0.5 McFarland standard (equivalent to 1.0 O.D. at 600 nm), ensuring uniform turbidity prior to use in antibacterial testing. Well diffusion assay for nanoparticles based antibacterial activity Cobalt oxide nanoparticles were prepared at a stock concentration of 1 mg/mL and evaluated for their antibacterial activity against four human pathogenic bacteria, namely E. coli, B. subtilis, S. typhi, and P. vulgaris following standard procedures79. The in vitro antibacterial potential of the nanoparticles was assessed using Mueller–Hinton agar (MHA) and established microbiological techniques. Antibacterial activity assay Mueller–Hinton agar was prepared by dissolving 38 g of agar powder in 1000 mL of purified water. The mixture was boiled until completely dissolved and subsequently sterilized by autoclaving. After cooling to 45–50 °C, the medium was aseptically poured into sterile Petri plates and allowed to solidify. Each plate was inoculated with 10 µL of standardized bacterial broth culture using the pour-plate technique, followed by uniform spreading with a sterile spreader. Sterile steel borers were used to prepare equidistant wells in the solidified agar. The wells were filled with 25, 50, and 100 µL of nanoparticle suspensions, corresponding to 25, 50, and 100 µg/mL, respectively. Gentamicin was used as the positive control (disk diffusion method), while DMSO served as the negative control (well diffusion method). The plates were incubated at 37 °C for 24 h, and the zones of inhibition (mm) were measured to evaluate antibacterial activity. Scientific Reports | (2025) 15:44017 5| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports Antioxidant activity of nanoparticles by DPPH assay Working principle of DPPH assay The DPPH assay was performed following the established protocol for stable free radical scavenging activity80. In this method, the DPPH radical, containing an unpaired electron, exhibits a maximum absorption at 517 nm, producing a characteristic purple color. When antioxidants are present, they donate hydrogen atoms to the DPPH radical, reducing it to its non-radical form (DPPH-H). This reduction leads to a decrease in absorbance at 517 nm81. The color change from purple to yellow, known as decolorization, reflects the extent of radical scavenging. A greater degree of decolorization corresponds to a higher number of electrons or hydrogen atoms donated by the antioxidants, thereby indicating stronger reducing potential82. Methodology The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay was performed using ascorbic acid as the standard reference antioxidant, following a modified protocol83. All test samples were evaluated alongside ascorbic acid. A 0.135 mM DPPH solution was prepared in methanol, and different concentrations of the test samples like 5, 10, 20, 40, 80, 160, and 320 µg/mL were prepared. Each concentration (2.5 mL) was mixed with the DPPH solution, vortexed thoroughly, and incubated at room temperature for 30 min in the dark. The absorbance was then measured at 517 nm using a UV–Vis spectrophotometer. The free radical scavenging activity (%) was calculated using the following equation: % DPPH scavenging = (ODcontrol − ODsample) ODcontrao × 100 Where, OD (control) is the absorbance of the control (DPPH solution without sample) and OD (sample) is the absorbance in the presence of the test sample. Results and discussion The aqueous extract of Uraria picta has been utilized for the phytogenic synthesis of cobalt oxide nanoparticles (CoO NPs). Upon the drop wise addition of cobalt chloride hexahydrate solution to the plant extract under the condition of constant stirring and heating, a distinct color change was observed, signifying the reduction of Co²⁺ ions and the onset of nanoparticle formation. Following centrifugation, washing, drying, and calcination at 500 °C, a black-violet crystalline powder was obtained, confirming the successful synthesis of CoO NPs (Scheme 1). The biomolecules such as flavonoids, phenolics, and alkaloids, present in the Uraria picta extract likely acted as both reducing and stabilizing agents, enabling the formation of nanoparticles without the involvement of toxic chemicals. This green approach not only ensured the eco-friendly and cost-effective synthesis of cobalt oxide but also highlighted the potential role of plant metabolites in nanoparticle stabilization. The crystalline nature of the final product is in agreement with earlier reports on biosynthesized metal oxide nanoparticles. Spectral characterization of Cobalt oxide nanoparticles The UV–Vis spectrum of Uraria picta mediated cobalt oxide nanoparticles exhibited a strong absorption maximum at ~ 300 nm, with a smaller secondary peak near ~ 240 nm (Fig. S4). These features likely arise from electronic transitions within the cobalt–oxygen framework and may reflect the presence of multiple cobalt oxidation states or a distribution of particle sizes. A comparable absorption band at 309 nm was reported for CoO NPs synthesized using Coriandrum sativum seed extract, supporting the assignment of this band to cobalt oxide-related transitions84 and for Allium sativum at ~ 314 nm, Hibiscus rosa-sinensis derived Co3O4 NPs display a strong UV feature near 210 nm respectively85,86. The slight hypsochromic (blue) shift observed here suggests the formation of relatively smaller nanoparticles, consistent with quantum- or size-dependent spectral shifts. The prominent band also corresponds to charge-transfer/metal–oxygen electronic transitions commonly observed for cobalt oxides rather than true localized surface plasmon resonance. UV–Vis scans performed over 200–1100 nm (absorbance ≤ 1.5 OD) confirmed nanoparticle formation and showed no additional broad 1. Reduction Step The phytochemicals present in Uraria picta (such as phenolics, flavonoids, tannins, etc.) act as reducing agents, donating electrons and protons to cobalt ions: 2. Nucleation and Growth The reduced cobalt atoms aggregate to form cobalt nanoparticles, stabilized by plant metabolites: 3. Oxidation and Calcination Upon calcination at 500 °C, cobalt nanoparticles oxidize to cobalt oxide nanoparticles: Scheme 1.  General Reaction Pattern for cobalt oxide nanoparticles. Scientific Reports | (2025) 15:44017 6| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports plasmonic features, reinforcing that the recorded peaks are attributable to cobalt–oxygen electronic processes and size-dependent effects87,88. The FTIR spectrum of Uraria picta-mediated cobalt oxide nanoparticles revealed nine characteristic peaks at 3420.52, 3167.71, 2914.09, 2197.53, 1590.97, 1375.92, 1098.49, 912.17, and 611.76 cm⁻¹ (Fig. 4). Compared to copper nanoparticles, CoO nanoparticles exhibited fewer peaks, suggesting relatively simpler vibrational modes. Interestingly, similar peak patterns were observed across different plant-mediated syntheses, confirming the role of phytochemicals as reducing and stabilizing agents during nanoparticle formation. The broad absorption around 3420–3160 cm⁻¹ corresponds to O–H stretching of hydroxyl groups and N–H stretching of amines, indicating the presence of phenolics and proteins from the extract. Peaks at ~ 2197 cm⁻¹ are attributed to –OH stretching in phosphines, while the sharp band at ~ 1375 cm⁻¹ is associated with C = O stretching of benzophenols and SO₂ vibrations. The band at ~ 1098 cm⁻¹ corresponds to C–O/C–N stretching of aliphatic amines, and the peak at ~ 912 cm⁻¹ is related to –C–O–C linkages. Importantly, the band at ~ 611 cm⁻¹ represents Co–O stretching vibrations, confirming the formation of cobalt oxide nanoparticles. Similar FTIR features were reported for phyto-fabricated CoO NPs synthesized using Psidium guajava and other plant extracts, where strong O–H, N–H, C = O, and Co–O vibrations were observed89–91. These results collectively indicate that the functional groups from plant metabolites not only reduce cobalt ions but also cap and stabilize the nanoparticles, while the Co–O band validates the presence of crystalline cobalt oxide. Scanning Electron Microscopy (SEM) of the green-synthesized CoO nanoparticles revealed irregular morphologies, predominantly ranging from roughly triangular to pyramidal shapes (Fig. 5). Such morphology is consistent with the influence of plant-derived phytochemicals, which act as capping and stabilizing agents during nanoparticle growth. Similar observations have been reported for CoO NPs synthesized using rosemary leaf extract, where semi-triangular to pyramidal particles with a wide size distribution were noted92. In contrast, CoO nanoparticles produced via Fusarium oxysporum exhibited polyshaped crystalline structures, and agglomeration was attributed to their magnetic induction properties93. The current SEM results confirm that the Uraria picta extract not only mediates nanoparticle formation but also influences particle shape and size, leading to irregular yet well-defined nanostructures suitable for various biological and catalytic applications. Energy Dispersive Spectroscopy (EDS) was employed to determine the elemental composition of Uraria picta-mediated CoO nanoparticles, which exhibited a high cobalt content of 79.58 wt% and oxygen 20.42 wt% (Fig. 6). The spectrum showed two major peaks corresponding to cobalt and oxygen, confirming the formation of cobalt oxide, while minor peaks were attributed to residual phytochemicals from the plant extract acting as capping and stabilizing agents. These results are consistent with earlier studies, such as CoO NPs synthesized using Alhagi maurorum, where cobalt and oxygen dominated the composition94, and Euphorbia tirucalli- mediated CoO nanoparticles, where minor peaks represented trace elements from plant metabolites, while cobalt oxide was the primary component95. The EDS analysis thus validates the successful green synthesis of CoO nanoparticles and highlights the role of plant biomolecules in stabilizing the nanostructures. X-ray diffraction (XRD) was employed to investigate the crystalline structure of Uraria picta-mediated CoO nanoparticles, providing insights into their atomic-level organization, lattice parameters, and potential structural defects96–98. The XRD pattern (Fig. 7) displayed distinct diffraction peaks at 2θ = 37.36°, 42.95°, 43.84°, and Fig. 4.  Uraria picta based CoO NPs FTIR pattern. Scientific Reports | (2025) 15:44017 7| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports 61.87°, confirming the formation of highly crystalline cobalt oxide nanoparticles. The observed peaks correspond well with standard reference data from the International Centre for Diffraction Data (JCPDS card 96–152-8839). Comparable XRD patterns have been reported for CoO nanoparticles synthesized using Pedalium murex leaf extract, where well-defined peaks similarly indicated a crystalline structure99. The sharpness and intensity of the diffraction peaks in the present study suggest good crystallinity and uniform particle formation, consistent with the effective reduction and stabilization facilitated by phytochemicals in Uraria picta. Cobalt oxide nanoparticles synthesized using Uraria picta extract exhibited a globular morphology with remarkably small dimensions (Fig. 8). Particle size analysis revealed an average diameter of 5.26 ± 0.74 nm, with a minimum size of 4.01 nm, a median of 5.40 nm, and a maximum of 6.27 nm, as summarized in Table  1. These findings indicate a narrow size distribution, consistent with the influence of plant-derived biomolecules in controlling nanoparticle growth and preventing excessive aggregation. Broadly, CoO NPs synthesized via green methods tend to exhibit sizes in the 4–5 nm range, highlighting the effectiveness of Uraria picta extract in producing uniform, ultra-small nanoparticles. The small size and globular morphology are expected to enhance the surface area and reactivity of the nanoparticles, making them suitable for various catalytic, biomedical, and electronic applications. In a similar studies potential of Transmission Electron Microscope has been put forward to elucidate size of the NPs. CoO NPs with means size of 34 nm synthesized using Hibisus rosa sinensis flower extract able to control bacterial phytopathogens diseases investigated with Rice plants100. Cobalt oxide NPs synthesized from root of Allium cepa (onion bulb) found to be phytotoxic in nature also especially to plants via excessive absorption of cobalt oxide101. Thus, it in importance to screen the potential of such CoO NPs before getting commercialized especially of smaller size nanoparticles ranging between 10 and 15 nm. In a positive size CoO NPs found to be controlling cancer cell growth at 55 µg/mL concentration which was synthesized from plants and able to give sheet shaped cobalt oxide nanoparticles ranging between 15 and 20 nm102. Biological studies Antimicrobial activity The antibacterial activity of biosynthesized cobalt oxide nanoparticles (CoO NPs) was evaluated against four test bacterial strains (E. coli EC23, B. subtilis BS07, S. typhi ST12, and P. vulgaris PV05) using the agar well Fig. 5.  (A) 20 kV X 10,000 resolution, (B) 20 kV X 1,500 resolution, (C) 20 kV X 3,500 resolution and (D) 20 kV X 7,000 resolution display SEM images of Uraria picta-based CoO NPs at low and high resolution. Scientific Reports | (2025) 15:44017 8| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports diffusion method79, and the results were compared with the standard antibiotic gentamicin (120 µg/L). The zone of inhibition (ZOI) values revealed a clear concentration-dependent antibacterial effect of CoO NPs (Table 2 & Fig. S5). At the lowest concentration (25 µL), no inhibition was observed for any of the tested organisms, indicating that a threshold concentration of nanoparticles is necessary to elicit antibacterial action. However, at 50 µL, measurable inhibition zones appeared in all test strains, with maximum sensitivity shown by B. subtilis Fig. 7.  XRD pattern of CoO NPs synthesised by using Uraria picta. Fig. 6.  EDS pattern of Uraria picta based CoO NPs. Scientific Reports | (2025) 15:44017 9| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports Test Organism Zone of Inhibition in mm Zone of Inhibition in mm for Control (Gentamicin 120 µg/L) Mean ± SD Cobalt oxide nanoparticles (CoO NPs) Mean ± SD (mm) 25 µg/mL 50 µg/mL 100 µg/Ml EC23 (E. coli) 0.00 ± 0.00 13.67 ± 0.5774 17.33 ± 0.5774 23.57 ± 1.718 BS07 (B. subtilis) 0.00 ± 0.00 15.33 ± 0.5774 16.67 ± 0.5774 26.57 ± 2.370 ST12 (S. typhi) 0.00 ± 0.00 10.67 ± 0.5774 19.33 ± 1.155 21.86 ± 1.952 PV05 (P. vulgaris) 0.00 ± 0.00 12.00 ± 0.0 11.33 ± 0.5774 19.86 ± 1.952 Table 2.  Anti-bacterial activity of CoO NPs at three concentrations along with standard drug against different bacterial strains. Minimum 4.010 nm 25% Percentile 4.775 nm Median 5.395 nm 75% Percentile 5.798 nm Maximum 6.270 nm Mean ± Std deviation 5.257 nm ± 0.7435 nm Table 1.  Average size of the CoO NPs recorded for the Minimum, Median, Maximum, Average, and standard Deviation. Fig. 8.  Transmission Electron Micrograph of Uraria picta based CoO NPs (A) 10 nm resolution, (B) 20 nm resolution, (C) 20 nm resolution and (D) 10 nm resolution. Scientific Reports | (2025) 15:44017 10| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports (15.33 ± 0.57 mm), followed by E. coli (13.67 ± 0.57 mm), P. vulgaris (12.00 ± 0.00 mm), and S. typhi (10.67 ± 0.57 mm). A further increase in concentration to 100 µL significantly enhanced the antibacterial activity, with B. subtilis (26.57 ± 2.37 mm) and E. coli (23.57 ± 1.71 mm) showing the highest inhibition, exceeding or approaching the activity of gentamicin. S. typhi and P. vulgaris also showed marked inhibition zones of 21.86 ± 1.95 mm and 19.86 ± 1.95 mm, respectively. These results confirm that CoO NPs possess strong antibacterial activity, particularly at higher concentrations, with B. subtilis being the most susceptible strain. The enhanced antibacterial effect may be attributed to the nanoscale size, which facilitates penetration through bacterial cell walls, generation of reactive oxygen species (ROS), and disruption of cellular components as depicted in the Fig. 9. The variation in susceptibility among Gram-positive and Gram-negative bacteria suggests that differences in cell wall structure and permeability play a key role in determining nanoparticle efficacy103–109. Overall, the findings indicate that biosynthesized CoO NPs exhibit promising broad-spectrum antibacterial potential, comparable to conventional antibiotics, and may serve as alternative antimicrobial agents against multidrug-resistant pathogens. Moreover, the antibacterial activity of nanoparticles can be enhanced by carefully controlling their physical and chemical properties. These factors include particle size, shape, surface area, and crystallinity, which influence how the nanoparticles interact with different cells. Furthermore, phase composition, size distribution, and band gap also play crucial roles in determining their efficacy110. Antioxidant activity The antioxidant potential of Uraria picta–mediated cobalt oxide nanoparticles (CoO NPs) was evaluated using the DPPH free radical scavenging assay. The results revealed a dose-dependent response, with the minimum activity recorded as 1.188% inhibition at 5 µg/mL, while the maximum activity reached 8.481% inhibition at 320 µg/mL as depicted in the Fig. 10. These findings indicate that the synthesized CoO NPs exhibit moderate antioxidant activity, as also represented in Table 3 & Table 4. The present findings, highlight that phytogenic synthesis not only facilitates eco-friendly production of CoO NPs but also imparts desirable biological properties. While the antioxidant activity of Uraria picta–derived CoO NPs appears moderate compared to other reports, their biosynthetic potential and biological efficacy provide a promising foundation for further exploration in therapeutic and biomedical applications (Fig. S6). Conclusion The present study demonstrates that Uraria picta extract functions as an efficient bio-reducing and stabilizing agent for the green synthesis of CoO nanoparticles (CoO NPs) via a simple, non-toxic, and environmentally benign protocol. The synthesized CoO NPs, comprehensively characterized by SEM-EDX, FTIR, HR-TEM, UV–Vis spectroscopy, and XRD analyses, exhibited a globular morphology with an average particle size of 5.257 ± 0.743 nm (minimum 4.010 nm, median 5.395 nm, maximum 6.270 nm), consistent with the reported nanoscale range of 4–5 nm. This green synthesis approach offers a rapid, cost-effective, and sustainable strategy for producing CoO NPs with potential applications in biomedical and industrial sectors. The Uraria picta–mediated CoO NPs displayed moderate, dose-dependent antioxidant activity, indicating that phytogenic synthesis confers functional biological properties. Additionally, the nanoparticles exhibited pronounced, concentration-dependent Fig. 9.  Proposed mechanisms of CoO NPs against bacterial cell. Scientific Reports | (2025) 15:44017 11| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://www.nature.com/scientificreports antibacterial activity, with B. subtilis and E. coli demonstrating the highest susceptibility, underscoring their potential as broad-spectrum antimicrobial agents. Collectively, these findings substantiate the utility of U. picta in generating biologically active CoO NPs. Future investigations should aim to elucidate the mechanistic role of specific phytochemicals in nanoparticle formation and stabilization, optimize scale-up strategies for industrial Conc. (µg/mL) % DPPH Inhibition for Ascorbic acid % DPPH Inhibition for CoO NPs 0 0.000 0.000 5 10.136 1.188 10 42.239 2.714 20 67.387 3.859 40 94.275 5.385 80 95.462 6.361 160 96.989 7.760 320 98.219 8.481 Table 4.  Antioxidant activity of nanoparticles via DPPH assay along with standard represents % DPPH inhibition. Experiment Result at 517 nm % DPPH inhibition Uco Conc. µg/mL Singlet Duplicate Triplicate Singlet Duplicate Triplicate Mean SD IC50µg/mL 5 0.775 0.771 0.784 1.399 1.658 0.508 1.188 0.604 > 320 10 0.764 0.763 0.767 2.799 2.679 2.665 2.714 0.074 20 0.758 0.753 0.756 3.562 3.954 4.061 3.859 0.263 40 0.747 0.744 0.74 4.962 5.102 6.091 5.385 0.616 80 0.739 0.733 0.736 5.980 6.505 6.599 6.361 0.334 160 0.725 0.728 0.722 7.761 7.143 8.376 7.760 0.616 320 0.719 0.723 0.716 8.524 7.781 9.137 8.481 0.679 Table 3.  Antioxidant activity of CoO NPs. Fig. 10.  DPPH based assay profile for ascorbic acid showcasing concentration-based antioxidant activity recorded as IC50 value (13.75 µg/mL) and that of Uraria picta cobalt oxide NPs with > 320 µg/mL as IC50 value. 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Also, thanks to SAIF, Cochin, for the characterization of samples. The authors would also like to thank the Ongoing Research Funding Program (ORF-2025-1042), King Saud University, Riyadh, Saudi Arabia. Author contributions S.S.: Writing - Original Draft; A.J.M.: Methodology, Visualization and Experimental design; A.R.B. Project ad- ministration; A.P.P.: Conceptualization; C.P.P.: Methodology; H.C.P.: Data Curation & Biological activities; R.W.: Funding acquisition; H.P.K.: Validation; V.J.U.: Validation & Investigation; S.A.: Writing – Review, Editing & submission; D.M.P.: Result interpretation; M.A‐O.: Formal analysis; P.E.: Review & Editing Manuscript. Funding King Saud University, Riyadh, Saudi Arabia, Ongoing Research Funding Program (ORF-2025-1042) and Parul Institute of Applied Sciences (PIAS), Parul University, PO Limda, Tal Waghodia – 391 760, India. Declarations Consent for publication All of the authors consent to publish this work. Competing interests The authors declare no competing interests. Conflict of interest On behalf of all authors, the corresponding authors states that there is no conflict of interest. Declaration of competing interest There are no pertinent financial or non-financial interests that the authors need to disclose. Consent to participate All of the authors consent to participate in this research work. Ethical approval The collection of plants for this experimental research exploration are in strict agreement with relevant national, international, and institutional regulations and guidelines and complies with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. We also declare herein that our paper is original and unpublished elsewhere and that this manuscript complies to the Ethical Rules applicable for this journal. Additional information Supplementary Information The online version contains supplementary material available at ​h​t​t​p​s​:​/​/​d​o​i​.​o​r​g​/​1​ 0​.​1​0​3​8​/​s​4​1​5​9​8​-​0​2​5​-​2​8​5​9​6​-​0​​​​​.​​ Correspondence and requests for materials should be addressed to A.J.M., A.R.B., V.J.U. or S.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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To view a copy of this licence, visit ​h​t​t​p​:​/​/​c​r​e​a​t​i​v​e​c​o​m​m​o​ n​s​.​o​r​g​/​l​i​c​e​n​s​e​s​/​b​y​-​n​c​-​n​d​/​4​.​0​/​​​​​.​​ © The Author(s) 2025 Scientific Reports | (2025) 15:44017 17| https://doi.org/10.1038/s41598-025-28596-0 www.nature.com/scientificreports/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://www.nature.com/scientificreports Sustainable plant mediated synthesis of cobalt oxide nanoparticles using Uraria picta extract with enhanced biological activity Materials and methods Plant collection and authentication Preparation of plant extract Biosynthesis of Cobalt oxide nanoparticles (CoO NPs) Characterization Antibacterial activity of nanoparticles Stock solution of nanoparticles Procurement and maintenance of bacterial cultures Inoculum Preparation for antibacterial assay Well diffusion assay for nanoparticles based antibacterial activity Antibacterial activity assay Antioxidant activity of nanoparticles by DPPH assay Working principle of DPPH assay Methodology Results and discussion Spectral characterization of Cobalt oxide nanoparticles Biological studies Antimicrobial activity Antioxidant activity Conclusion References