Biosynthesis of nickel oxide nanoparticles from piper nigrum leaf Extract and their enhanced antioxidant, antibacterial, and enzyme Inhibition Activities

Biosynthesis of nickel oxide nanoparticles from piper nigrum leaf Extract and their enhanced antioxidant, antibacterial, and enzyme Inhibition Activities | 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 Biosynthesis of nickel oxide nanoparticles from piper nigrum leaf Extract and their enhanced antioxidant, antibacterial, and enzyme Inhibition Activities Hajra Akram, Ammar Tariq, Ayesha Khalid, Iqra Saddique, Sirajul Haq, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5337394/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 In recent years, the use of eco-friendly and sustainable methods for synthesizing nanoparticles has gained significant attention due to environmental concerns associated with conventional chemical approaches. This study explores the preparation of nickel oxide (NiO) NPs using Piper nigrum leaf extract sourced from Muzaffarabad Azad Kashmir, Pakistan a novel and environmentally benign approach. These NPs were studied using various techniques to determine their morphology, size, and structural properties. Furthermore, the biological activity of the freshly prepared NPs was evaluated, focusing on their inhibitory effect on alpha-amylase, a key enzyme related to diabetes management. The NPs exhibited excellent antioxidant properties, with a peak scavenging activity of 78% at 80 µg/mL, and an IC50 value of 28 µg/mL. In alpha-amylase inhibition assays, NiO NPs demonstrated significant enzyme inhibition with an IC50 of 1.18 µg/mL. Antibacterial tests revealed strong activity against Gram-positive bacteria, with peak inhibition zones of 18 mm for Streptococcus pyogenes and 13 mm for Staphylococcus aureus. These results highlight the NPs potential for biological applications. Nickel oxide nanoparticles Antibacterial activity Green synthesis Piper nigrum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction In the realm of biomedical applications, there is an escalating need for innovative materials that address persistent challenges such as bacterial resistance, toxicity, and inefficiency of current treatments [ 1 – 2 ]. Despite advancements in conventional therapies, issues such as drug resistance and limited efficacy of antibacterial agents continue to challenge the field, underscoring the need for novel solutions [ 3 ]. Researchers have increasingly turned to nanomaterials for their potential to overcome these limitations. Among the diverse array of nanomaterials, metal oxide nanoparticles (NPs) have gained prominent position due to their distinctive characteristics and multifunctional applicability [ 4 ]. Numerous metal oxides, for example zinc oxide (ZnO), nickel oxide (NiO), and titanium dioxide (TiO₂) have been subject to extensive research aimed at elucidating their photocatalytic, antimicrobial, antibacterial, and various other therapeutic functionalities. [ 5 – 8 ]. Traditional methods for synthesizing these metal oxide nanoparticles include techniques like sol-gel, hydrothermal, and microwave-assisted synthesis. These methods often involve hazardous chemicals, high energy consumption, and environmental concerns [ 9 ]. In contrast, green synthesis, utilizing biological systems for NPs production, offers a sustainable alternative. This approach leverages natural extracts as reducing and capping agents, reducing the environmental impact and enhancing the safety profile of the nanoparticles [ 10 ]. Recent studies have demonstrated the efficacy of various plant extracts in NPs synthesis, highlighting their role in creating materials with enhanced biological and catalytic properties. For instance, NiO NPs synthesized by using E. heterophylla leaf extract exhibited a particle size of 12–15 nm and an FCC structure. These NPs showed significant antibacterial activity, particularly against E. coli , with inhibition zones increasing at higher concentrations (200, 400, and 600 µg/µL). The NPs also demonstrated notable cytotoxicity against A549 and HepG2 cancer cell lines. Their non-toxic nature on human erythrocytes and strong bactericidal properties makes them promising candidates for biomedical applications like antimicrobial coatings and cancer treatments [ 11 ]. Similarly, Vijayakumar et al ., synthesized Ln-ZnO NPs by following green synthesis with leaf extract of the local plant Laurus nobilis , yielding flower-like structures with average grain size of 45 nm. The antibacterial efficacy of these NPs was notably higher against Staphylococcus aureus (Gram-positive), exhibiting excellent inhibition zones diameter of 11.4, 12.6, and 14 mm at varying concentrations. In contrast, for Pseudomonas aeruginosa (Gram-negative), the observed inhibition zones were 9.8, 10.2, and 11.3 mm at corresponding concentrations. At a concentration of 75 mg/mL, this material effectively curtailed biofilm development, and at 80 mg/mL, they demonstrated cytotoxic effects on A549 lung cancer cells, while exhibiting no toxicity towards normal murine macrophage cells. These results underscore their promising potential for applications in the pharmaceutical and biomedical fields [ 12 ]. Ezhilarasi et al ., reported the environmentally friendly synthesis of NiO NPs using Moringa oleifera extract. These NPs were then characterized by XRD, FTIR, HRTEM, EDX, and PL spectroscopy. NiO NPs exhibited significant photoluminescence at 305.46 nm and 410 nm along with strong cytotoxic effects on HT-29 colon cancer cells and effective antibacterial properties [ 13 ]. Moreover, Subhapriya et al ., reported the biosynthesis of TiO₂ NPs using Trigonella foenum-graecum leaf extract, producing spherical NPs ranging from 20–90 nm. These TF-TiO₂ NPs exhibited significant antimicrobial activity, with inhibition zones of 10.6 mm for Yersinia enterocolitica , 10.8 mm for Escherichia coli , 11.2 mm for Staphylococcus aureus , and 11.6 mm for Streptococcus faecalis . The antimicrobial effect was stronger against Gram-positive bacteria because of their thicker cell walls. The bactericidal activity of TiO₂ NPs is due to reactive oxygen species that disrupt bacterial membranes and cause cell death, indicating its potential for antimicrobial treatments like infections and wound care [ 14 ]. By considering the above survey, our work focuses on the preparation of NiO NPs using Piper nigrum leaf extract, evaluating their biological applications in terms of antioxidant activity and antibacterial activity. Piper nigrum, commonly known as black pepper, is one such plant with substantial potential for green synthesis. The leaf extract of Piper nigrum contains bioactive compounds that can facilitate the preparation of NiO NPs. The use of Piper nigrum leaf extract, particularly from Muzaffarabad Azad Kashmir, Pakistan is novel and represents a notable advancement for green synthesis. By leveraging the natural properties of this extract, we aim to develop a more sustainable method for producing NiO NPs and to explore their potential in addressing critical biological challenges. 2. Experimental 2.1. Preparation of Leaf Extract Initially, Piper nigrum leaves were collected from Muzaffarabad, Azad Kashmir. The leaves were air-dried at room temperature for 8 h. Further in a clean beaker, 20 g of the dried leaves were washed twice with distilled water to remove surface contaminants. After washing, these leaves were dried further on filter paper to remove excess moisture. To prepare the leaf extract, 500 mL of deionized water was incorporated with the leaves, which were subsequently subjected to boiling to yield a concentrated extract. 2.2. Synthesis of NiO NPs A 1 mM nickel sulfate solution was created by dissolving 1.3 g of NiSO₄6H₂O in 500 mL of deionized water to create a 10 mM solution, which was then diluted to 1 mM. The 1 mM nickel sulfate solution was added to the freshly prepared Piper nigrum leaf extract. The mixture was continuously stirred and heated to facilitate the reduction of nickel ions and the formation of NiO NPs. After the synthesis process, the resultant product underwent an extensive washing procedure utilizing distilled water in order to eliminate any residual solution, and the synthesized NPs were subsequently collected for further characterization. 3. Characterization The synthesized NiO NPs underwent characterization through an array of analytical methodologies to elucidate their structural, morphological, and chemical attributes. The crystal architecture of prepared material was scrutinized utilizing X-ray diffraction (XRD) spectroscopy, employing a Bruker D8 X-ray diffractometer, which yielded significant insights into the phase composition and crystallinity of the material. The morphological characteristics and elemental constituents were investigated through Field Emission Scanning Electron Microscopy (FESEM) in conjunction with Energy-Dispersive X-ray Spectroscopy (EDX), facilitating comprehensive examination of particle shape, size, and elemental distribution. Fourier Transform Infrared Spectroscopy (FTIR) was employed to ascertain the functional groups present and to validate the existence of nickel-oxygen bonding interactions. Thermogravimetric Analysis (TGA) was performed to examine the thermal stability profile of the NiO NPs. 4. Biological Activities 4.1. Alpha Amylase Inhibition Activity The alpha-amylase inhibition activity of NiO NPs was assessed by pre-incubating various concentrations (100–500 µg/mL) of the nanoparticles with amylase enzyme and starch solution. After incubation, the reaction mixture was subjected to treatment with NaOH and DNS, followed by analysis of absorbance at λ = 540 nm. The inhibition % was determined using \(\:({A}_{c}-{A}_{s})/{A}_{c}\times\:100\) . In this formula, ​ \(\:{A}_{c}\) and \(\:{A}_{s}\:\) represent the absorbance of the control sample and the absorbance in the presence of nanoparticles, respectively. 4.2. Antibacterial Activity The antibacterial potential of Piper nigrum-derived NiO NPs was investigated by well-diffusion method against bacterial strains including Staphylococcus aureus , Serratia marcescens , Streptococcus pyogenes , Pseudomonas aeruginosa , and Klebsiella pneumoniae . 4.3. Antioxidant Activity The antioxidant activity of as prepared NPs was analyzed using the ABTS free radical neutralization method, with the percentage neutralization evaluated by comparing the absorbance of the control and NPs samples. Similarly, the metal chelating activity was determined by incubating the NPs with ferrous sulfate and ferrozine, and calculating the chelating percentage based on the absorbance at 517 nm. Both activities were quantified using: \(\:({A}_{o}-{A}_{i})/{A}_{o}\times\:100\) where A 0 is the control absorbance and A i is the absorbance with NPs. 5. Results and Discussion 5.1. Structural Analysis Figure 1 displays the XRD results of NPs within 2θ range of 20° to 70°. The spectrum reveals distinct diffraction peaks at 2θ values of 37.48°, 43.49°, and 62.94°. These prominent peaks correspond to the (111), (200), and (220) Brags planes, respectively. These diffraction peaks, corresponding to their respective hkl planes, match the reference data from JCPDS card no. 01-073-1519, confirming the synthesized NiO NPs exhibit a cubic crystal structure, as shown in the inset of Fig. 1 . The observed sharpness of the peaks indicates high crystallinity and well-defined crystal planes [ 15 ]. The absence of any additional impurity peaks further supports the purity of the synthesized material, suggesting that the synthesis process was successful, and the NPs are free from contamination. The crystallite size of the NiO NPs yielded 22.37 nm calculated by Debye-Scherrer equation. This relatively small crystallite size indicates that the nanoparticles have a high degree of crystallinity and a well-defined structure. The small size of the crystallites is likely to enhance the biological activity of the NiO NPs due to the increased surface area and reactivity [ 16 ]. All lattice parameters are tabulated in Table 1 . Overall, these results indicate the successful preparation of high-quality NiO NPs with a cubic structure, which is expected to positively impact their biological activity. Table 1 Lattice constant, crystallite size, and X-ray density of NiO Sample Lattice Constant (Å) a = b = c Crystallite Size (nm) X-ray Density (g/ cm 3) NiO 4.18 22.37 6.82 5.2. Morphological Study The morphology of the prepared sample was extensively studied by FESEM at 20,000x as illustrated in Fig. 2 (a). These visuals revealed that the NiO NPs exhibit significant agglomeration, with small particles forming spherical shapes with pronounced porosity within the sample, and numerous visible voids, indicating a high degree of porosity. To quantify the particle size, ImageJ software was employed, yielding an average grain size of 65 nm. This size estimation supports the observed morphology and confirms the presence of small, spherical particles with notable agglomeration and porosity. Further confirmation of the particle morphology was obtained through Scanning Transmission Electron Microscopy (STEM). The STEM analysis provided a clearer view of the spherical grains, confirming an average grain size of 40 nm as illustrated in Fig. 2 (b). The STEM images corroborated the FESEM findings, showing a high level of porosity and spherical particle shapes, enhancing the overall understanding of the material’s morphology. The EDX spectra of NiO NPs is depicted Fig. 2 (c). The analysis reveals that the primary elements are nickel and oxygen, confirming the nickel oxide composition. Prominent peaks for nickel and oxygen validate the material's identity. Small peaks at 0.25 and 0.5 keV are due to C and O, while the main peaks at 0.90, 7.5, and 8.3 keV correspond to nickel. Minor peaks at 1.1, 1.8, and 2.6 keV indicate the presence of sodium, silicon, and chlorine. The elemental composition is summarized in Table 2 . Carbon, chlorine, silicon, and sodium are likely residuals from the synthesis using plant materials. Table 2 Elemental composition of prepared NPs Sample Wt.% Ni Wt.% O Wt.% C Wt.% Cl Wt.% Si Wt.% Na NiO 72.5 21.2 3.4 2.2 0.6 0.6 5.3. FTIR Analysis FTIR works by using IR photons to vibrate atoms within chemical bonds, causing vibrational transitions at specific energy levels. Molecules absorb infrared light at particular wavelengths, and the resulting absorption spectrum identifies functional groups and compounds. Contaminants also emit unique IR bands, allowing for impurity detection. Fourier transformation is used to decode individual frequencies, and a computer processes the data to provide spectral information for analysis [ 17 ]. The FTIR spectrum of NiO NPs as illustrated in Fig. 3 exhibits multiple distinctive peaks. The prominent peak observed at 3701 cm − 1 is ascribed to the presence of water molecules. A broad absorbance band spanning the range of 3565 − 3398 cm − 1 indicating the stretching vibrations of (O-H) groups [ 18 ]. The peak around 1655.81 cm − 1 is attributed to the bending vibration of O-H, indicating the existence of H 2 O molecules in the sample. The peak at 1383.31 cm − 1 is due to the nitro group (NO₃), which originate from the precursor used in the synthesis process. Two prominent peaks at 1104.24 cm − 1 and 1068.89 cm − 1 are designated to the robust stretching vibrations of the Ni–O bond, signifying the formation of nickel oxide. Additionally, the peak at 898.85 cm − 1 represents the stretching vibrations of Ni–O [ 19 ]. Further, the bands at 545.2 cm − 1 and 447.64 cm − 1 correspond to the bending and wagging vibrations of Ni–O, confirming the presence of NiO in the sample [ 20 ]. 5.4. Thermogravimetric Analysis Thermogravimetric analysis (TGA) monitors a sample's mass change with temperature and time, providing accurate compositional data for quality and process control. However, it cannot identify volatile compounds without additional analytical tools. TGA can also determine oxidation induction time (OIT) by heating a small sample (typically < 10 mg) in oxygen at an isothermal temperature, generally around 200°C, and observing the mass gain when oxidation occurs [ 21 ]. The TGA curve of NiO NPs presented in Fig. 4 shows two significant stages of weight loss. The first sharp weight loss occurs between 160°C and 275°C, which corresponds to the evaporation of physically absorbed water molecules. This is typical for samples synthesized using plant-based methods, where moisture remains after synthesis. The second stage, occurring between 276°C and 540°C, reflects a gradual weight loss due to the removal of chemically adsorbed water and the decomposition of organic compounds. These organic compounds are likely residuals from the plant extract used in the green synthesis process. After 540°C, the weight stabilizes, indicating that all volatile and organic components have been fully decomposed, leaving behind the final, thermally stable NiO product [ 22 ]. This final stage confirms the complete formation of the NiO NPs, highlighting their purity and thermal stability. 5.5. Enzymatic Inhibition Activity of Alpha-Amylase The inhibitory effect of NiO NPs on alpha-amylase was evaluated by pre-incubating various concentrations of NiO NPs (100–500 µg/mL) with 0.5 mL of phosphate buffer containing 500 µL of alpha-amylase enzyme (1 mg/mL) at 37°C for 10 min [ 23 ]. Subsequent to the pre-incubation phase, 1 mL of starch solution was introduced, and the resultant mixture was subjected to an additional incubation period for 5 min at 37°C. The enzymatic reaction was subsequently terminated by the addition of NaOH and DNS, followed by heating the mixture for 5 min at 100°C. Upon reaching room temperature, the absorbance of the solution was quantitatively measured at a wavelength of 540 nm as illustrated in Fig. 5(a). Furthermore, Acarbose was employed as a standard inhibitory agent, yielding an IC50 value of 0.029 µg/mL. The inhibition activity of NiO NPs was measured as the percentage of enzyme activity inhibited, with IC50 indicating the concentration required to inhibit 50% of enzyme activity, as illustrated in Fig. 5(b). The results showed that the inhibition of alpha-amylase by NiO NPs was directly proportional to their concentration: higher concentrations led to greater inhibition and lower absorbance values. The absorbance values of NiO NPs at 10, 20, 40, 60, 80, and 100 µg/mL were 0.094, 0.078, 0.044, 0.012, and 0.002, respectively. Corresponding percent inhibition activities were 67%, 72%, 84%, 95%, and 99%. All the parameters are summarized in Table 3 . The IC50 value for NiO NPs, determined from the data, was 1.18 µg/mL. This indicates that NiO NPs are effective inhibitors of alpha-amylase, with their inhibition potential increasing with concentration. Table 3 Percentage inhibition of NiO NPs No. Concentration(µg\mL) Absorbance %inhibition IC50 1 10 0.094 67.5 1.18 2 20 0.078 72.6 3 40 0.044 84.5 4 60 0.012 95.3 5 80 0.002 99.1 5.6. Antioxidant Antioxidants are substances capable of neutralizing free radicals and reducing their formation. The antioxidant activity of prepared NPs was evaluated using the ABTS free radical and results were compared with standard ascorbic acid as shown in Fig. 6 (a-b). As various concentrations of NiO NPs were tested for antioxidant activity against ABTS free radicals [ 24 ]. A clear trend emerged, as the concentration of NiO NPs increased from 20 to 80 µL, the ABTS radical scavenging activity also increases, ranging from 44–78%, respectively. This suggests that a higher concentration of NiO NPs provides a greater number of surface binding sites for free radicals, thereby enhancing their scavenging potential. A similar trend was observed with ascorbic acid, where at 80 µL it achieved a 58.5% scavenging activity compared to the 78.4% displayed by NiO NPs at the same concentration. These results highlight the superior ABTS •+ radical scavenging activity of NiO NPs over ascorbic acid. Additionally, the lower IC50 value and higher correlation coefficient further support the enhanced antioxidant potential of NiO NPs compared to the standard. All the calculated parameters are tabulated in Table 4 . Furthermore, the % of radical reduction (%RSA) was calculated based on the decolorization of the ABTS solution, which occurs due to the reaction between the radicals and the antioxidants in the nanoparticles. The IC50 value, indicating the concentration required to achieve 50% inhibition, was derived from these measurements. NiO NPs were tested at concentrations ranging from (20–100 µg/mL). Higher concentrations of NiO NPs resulted in a greater decrease in ABTS absorbance, reflecting increased radical neutralization activity [ 25 – 27 ]. The absorbance values for NiO NPs at different concentrations were 0.12, 0.090, 0.069, 0.046, and 0.019 for 20, 40, 60, 80, and 100 µg/mL, respectively. The corresponding percent reduction values were 44%, 58%, 68%, 78%, and 91%. The IC50 value for NiO NPs was calculated to be 28 µg/mL, indicating their effective concentration for 50% radical inhibition. Table 4 Absorbance %RSA and IC50 reading of NiO NPs No. Concentration (µg\mL) Absorbance %RSA (Å-A\Å) 100 IC50 1 20 0.12 44 28 2 40 0.090 58 3 60 0.069 68 4 80 0.046 78 5.7. Iron Chelating Activity The iron chelating activity of NiO NPs was evaluated using a method based on the reduction of the ferrous ion-ferrozine complex [ 25 ] and shown in Fig. 7 . Ferrozine forms a red-colored complex with Fe²⁺ ions and the reduction in this color measured by the absorbance at 517 nm, indicates the iron chelating activity of the NPs. The IC50 value for NiO NPs, representing the concentration required to achieve 50% inhibition of the ferrozine-Fe²⁺ complex formation, was calculated to be 23 µg/mL. All the calculated values at various conditions are given in Table 5 . Table 5 Fe + percentage inhibition of NiO NPs No. Concentration (µg\mL) Absorbance %Fe + 2 IC50 1 10 0.170 40 23 2 20 0.142 50 3 40 0.110 61 4 60 0.078 72 5 80 0.044 84 5.8. Antibacterial activity The antibacterial activity of NiO NPs was evaluated against both Gram-positive bacteria (S. pyogenes, S. aureus) and Gram-negative bacteria (P. aeruginosa, K. pneumoniae, S. marcescens). The results represented by zones of inhibition (in mm) are shown in Fig. 8 , while Table 6 enlists the inhibition zone measurements for both the NiO NPs and conventional antibiotics. It was observed that the synthesized NiO NPs were particularly effective against Gram-positive bacteria, showing inhibition zones of 18 mm for S. pyogenes , and 13 mm for S. aureus which were larger compared to those seen for Gram-negative pathogens. This indicates that Gram-negative bacteria exhibited greater resistance to NiO NPs than Gram-positive strains. The antibacterial activity of the nanoparticles depends on various factors, including the specific bacterial species, particle size, stability, and the concentration within the growth medium. The nanoscale pores in the outer membranes of bacterial cells allow for greater interaction between the NPs and pathogens. In this study, NiO NPs synthesized using Piper nigrum extract had a very small particle size which plays a critical role in their antibacterial efficacy. The antibacterial mechanism of the NiO NPs is likely attributed to electrostatic interactions between the positively charged nickel ions (Ni²⁺) and the negatively charged bacterial cell membranes. This interaction facilitates the release osf Ni²⁺ ions, which penetrate the bacterial cell wall, damaging essential biomolecules such as DNA and proteins, disrupting mitochondrial function, and interfering with electron transport, ultimately leading to cell death [ 28 – 30 ]. The calculated parameters are tabulated in Table 6 . Table 6 Antibacterial activity and inhibition zone of NiO NPs Bacteria Antibiotics Diameter(nm) of inhibition zones concentrations (5 mg/50 mL) 5 10 30 50 100 S.aru 30 ± 01 7.33 ± 1.247 27 ± 2.054 26.3 ± 2.867 15.3 ± 2.054 13 ± 2.449 S.pyo 20 ± 2.2 13 ± 2.054 14 ± 2.449 14 ± 2.449 16.3 ± 3.299 18.3 ± 2.867 S.mre 20 ± 4.1 8.33 ± 0.471 11 ± 1.635 11.3 ± 1.243 17.3 ± 1.249 16 ± 1.642 K.pne 28 ± 1.2 14 ± 1.381 14.3 ± 2.494 13 ± 2.449 14.6 ± 3.299 15.3 ± 3.681 P.se 30 ± 3.2 9.3 ± 2.054 2.6 ± 1.632 11.6 ± 1.247 17 ± 1.632 13.6 ± 1.246 6. Conclusion In this study, NiO NPs were successfully synthesized using Piper nigrum leaf extract via a green synthesis approach. The structural and morphological analysis confirmed the formation of cubic NiO NPs with high crystallinity and significant porosity, which contributes to their biological effectiveness. The synthesis process proved to be eco-friendly, utilizing plant-based extracts as reducing and stabilizing agents, making it a sustainable alternative to traditional methods that often involve toxic chemicals and high energy consumption. The biological activities of the synthesized NiO NPs were explored, demonstrating promising results. The NPs exhibited significant antioxidant potential, surpassing that of standard ascorbic acid, indicating their ability to neutralize free radicals effectively. Their alpha-amylase inhibition activity further confirmed their therapeutic potential, particularly in controlling glucose levels. Additionally, the NiO NPs showed notable antibacterial activity, especially against Gram-positive bacteria, highlighting their potential application in antibacterial coatings and infection control. Declarations Ethical Approva l Not applicable. This study did not involve any human or animal subjects. Funding Not applicable. References Harun-Ur-Rashid, I. Jahan, T. 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Ali, A. M. Yameen, A. Mezni, A. Hedfi, K. S. Haleem, & S. Haq, In-Vitro and In-Vivo Tolerance and Therapeutic Investigations of Phyto-Fabricated Iron Oxide Nanoparticles against Selected Pathogens, Toxics 9 105–120. D. D. Gültek A. A. Güngör, H. Önem, & A.Babag, A. Synthesis of Copper Nanoparticles Using a Different Method: Determination of Their Antioxidant and Antimicrobial Activity, Turk. Chem. Scociety 3 623–636 A. Fouda, D. Hassan, M.A. Abdo, & M. S. El-Gamal, Antimicrobial, Antioxidant and Larvicidal Activities of Spherical Silver Nanoparticles Synthesized by Endophytic Streptomyces spp, Biol. Trace Elem. Res. 195 707–724. 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5337394","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371153309,"identity":"0d7fa6d1-a5c5-45da-947f-2fb6cc724039","order_by":0,"name":"Hajra Akram","email":"","orcid":"","institution":"Women University of Azad Jammu \u0026 Kashmir","correspondingAuthor":false,"prefix":"","firstName":"Hajra","middleName":"","lastName":"Akram","suffix":""},{"id":371153311,"identity":"5783eeaa-deb3-49ff-af7d-2d37ce9122c7","order_by":1,"name":"Ammar Tariq","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYNCCChsgwdjA8AFIsbETpeVMmgRIC+MMkBZmYnQwth2WANHMPGCSgGr+aacTP1e2Mdfxzz7ceNvm1zZ5PmYGxg8fc3Brkbidu1nyzDk2CYlzic3WuX23DduYGZglZ27DY83t3A2SDWU8EgxnGNukc3tuMwK1sDHz4tEiD7TlZwPQEnmQFsue2/YEtRjczt0m2dBmIGEA0sLw43YiQS2GQC2WDWcSJDeeYWy27G24ndzGzNiM1y9yQIfdbKj4zy93hv3hjR9/btvOb28++OEjPu8jAwnGNhAFTAZEAwmGP8QrHgWjYBSMgpEDAD36UYKCQh/5AAAAAElFTkSuQmCC","orcid":"","institution":"University of Messina","correspondingAuthor":true,"prefix":"","firstName":"Ammar","middleName":"","lastName":"Tariq","suffix":""},{"id":371153314,"identity":"df6d1999-a413-4454-9388-f3c1a939309c","order_by":2,"name":"Ayesha Khalid","email":"","orcid":"","institution":"Women University of Azad Jammu \u0026 Kashmir","correspondingAuthor":false,"prefix":"","firstName":"Ayesha","middleName":"","lastName":"Khalid","suffix":""},{"id":371153315,"identity":"59ffa2ae-4772-4b01-a428-9256a4b3726a","order_by":3,"name":"Iqra Saddique","email":"","orcid":"","institution":"Government College University, Lahore","correspondingAuthor":false,"prefix":"","firstName":"Iqra","middleName":"","lastName":"Saddique","suffix":""},{"id":371153316,"identity":"07106e41-8ae7-4f7c-a935-332cdcbd24df","order_by":4,"name":"Sirajul Haq","email":"","orcid":"","institution":"University of Azad Jammu and Kashmir","correspondingAuthor":false,"prefix":"","firstName":"Sirajul","middleName":"","lastName":"Haq","suffix":""},{"id":371153317,"identity":"70cf817b-e26d-4d31-9946-c605d238b6a9","order_by":5,"name":"Ashfaq Ahmad khan","email":"","orcid":"","institution":"Women University of Azad Jammu \u0026 Kashmir","correspondingAuthor":false,"prefix":"","firstName":"Ashfaq","middleName":"Ahmad","lastName":"khan","suffix":""}],"badges":[],"createdAt":"2024-10-26 12:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5337394/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5337394/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67854789,"identity":"e924c4e8-615c-4893-876a-4f06a2c793fa","added_by":"auto","created_at":"2024-10-30 11:12:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249618,"visible":true,"origin":"","legend":"\u003cp\u003eXRD patterns of NiO\u003csub\u003e \u003c/sub\u003eNPs,\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/23e8b9c6110614fef25cb786.png"},{"id":67854791,"identity":"780ad789-6151-4e88-b4ab-b1871ae4a8e2","added_by":"auto","created_at":"2024-10-30 11:12:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":662364,"visible":true,"origin":"","legend":"\u003cp\u003eFESEM image of (a) NiO, (b)STEM image of NiO (c) EDX spectra of NiO NPs\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/400bd68bd70d3966550d7f11.png"},{"id":67854784,"identity":"9068f2c7-3c1c-45fa-a359-cfd90f58237f","added_by":"auto","created_at":"2024-10-30 11:12:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":222202,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR absorption spectrum of NiO NPs\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/259fe9a25c572ac9926e657c.png"},{"id":67854947,"identity":"5ac9f145-a6e6-4f4d-9689-f295ca980e31","added_by":"auto","created_at":"2024-10-30 11:20:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":157253,"visible":true,"origin":"","legend":"\u003cp\u003eTGA spectrum of NiO NPs\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/a634952a9442a068d47311c9.png"},{"id":67854948,"identity":"5b06b8ce-356d-49bd-88af-f22f2ff8f0b3","added_by":"auto","created_at":"2024-10-30 11:20:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":312076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Graphical representation of alpha amylase inhibitory activity, and \u003cstrong\u003e(b)\u003c/strong\u003e Calibration curve of acarbose for percent inhibition of α-amylase\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/624e0e0549f15e7a3c086c14.png"},{"id":67854785,"identity":"2e6d120e-9e64-415b-b413-2b3c533a6d6d","added_by":"auto","created_at":"2024-10-30 11:12:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":594877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Percentage activity ABTS scavenging activity for Ascorbic acid, and \u003cstrong\u003e(b)\u003c/strong\u003e Percentage activity of NiO NPs at different concentration\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/29a838ee0f2f2e54c66242a7.png"},{"id":67854787,"identity":"46e073de-6b6d-4cb7-8a2a-f74229bc5b52","added_by":"auto","created_at":"2024-10-30 11:12:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":101369,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curve of iron chelating activity for NiO NPs\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/e0fa840cb3e8bb9aca28d55e.png"},{"id":67854788,"identity":"3ad8110a-f348-43e9-952b-f5c8bfb85b9b","added_by":"auto","created_at":"2024-10-30 11:12:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":469994,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of NiO NPs, \u003cstrong\u003e(a)\u003c/strong\u003e S. aureus, \u003cstrong\u003e(b)\u003c/strong\u003e S. pyogens, \u003cstrong\u003e(c)\u003c/strong\u003e S. epidermis, \u003cstrong\u003e(d)\u003c/strong\u003e S. marcescom and \u003cstrong\u003e(e)\u003c/strong\u003e k,pneumonia.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/936f8056fd194ad8012d7a04.png"},{"id":67856253,"identity":"4e7545da-b253-4808-af12-02fc48acef27","added_by":"auto","created_at":"2024-10-30 11:36:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3519962,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5337394/v1/6ec7fe6e-2098-491b-a65b-a959eba39232.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biosynthesis of nickel oxide nanoparticles from piper nigrum leaf Extract and their enhanced antioxidant, antibacterial, and enzyme Inhibition Activities","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn the realm of biomedical applications, there is an escalating need for innovative materials that address persistent challenges such as bacterial resistance, toxicity, and inefficiency of current treatments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite advancements in conventional therapies, issues such as drug resistance and limited efficacy of antibacterial agents continue to challenge the field, underscoring the need for novel solutions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Researchers have increasingly turned to nanomaterials for their potential to overcome these limitations. Among the diverse array of nanomaterials, metal oxide nanoparticles (NPs) have gained prominent position due to their distinctive characteristics and multifunctional applicability [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Numerous metal oxides, for example zinc oxide (ZnO), nickel oxide (NiO), and titanium dioxide (TiO₂) have been subject to extensive research aimed at elucidating their photocatalytic, antimicrobial, antibacterial, and various other therapeutic functionalities. [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Traditional methods for synthesizing these metal oxide nanoparticles include techniques like sol-gel, hydrothermal, and microwave-assisted synthesis. These methods often involve hazardous chemicals, high energy consumption, and environmental concerns [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, green synthesis, utilizing biological systems for NPs production, offers a sustainable alternative. This approach leverages natural extracts as reducing and capping agents, reducing the environmental impact and enhancing the safety profile of the nanoparticles [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated the efficacy of various plant extracts in NPs synthesis, highlighting their role in creating materials with enhanced biological and catalytic properties. For instance, NiO NPs synthesized by using \u003cem\u003eE. heterophylla\u003c/em\u003e leaf extract exhibited a particle size of 12\u0026ndash;15 nm and an FCC structure. These NPs showed significant antibacterial activity, particularly against \u003cem\u003eE. coli\u003c/em\u003e, with inhibition zones increasing at higher concentrations (200, 400, and 600 \u0026micro;g/\u0026micro;L). The NPs also demonstrated notable cytotoxicity against A549 and HepG2 cancer cell lines. Their non-toxic nature on human erythrocytes and strong bactericidal properties makes them promising candidates for biomedical applications like antimicrobial coatings and cancer treatments [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similarly, Vijayakumar \u003cem\u003eet al\u003c/em\u003e., synthesized Ln-ZnO NPs by following green synthesis with leaf extract of the local plant \u003cem\u003eLaurus nobilis\u003c/em\u003e, yielding flower-like structures with average grain size of 45 nm. The antibacterial efficacy of these NPs was notably higher against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Gram-positive), exhibiting excellent inhibition zones diameter of 11.4, 12.6, and 14 mm at varying concentrations. In contrast, for Pseudomonas aeruginosa (Gram-negative), the observed inhibition zones were 9.8, 10.2, and 11.3 mm at corresponding concentrations. At a concentration of 75 mg/mL, this material effectively curtailed biofilm development, and at 80 mg/mL, they demonstrated cytotoxic effects on A549 lung cancer cells, while exhibiting no toxicity towards normal murine macrophage cells. These results underscore their promising potential for applications in the pharmaceutical and biomedical fields [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Ezhilarasi \u003cem\u003eet al\u003c/em\u003e., reported the environmentally friendly synthesis of NiO NPs using Moringa oleifera extract. These NPs were then characterized by XRD, FTIR, HRTEM, EDX, and PL spectroscopy. NiO NPs exhibited significant photoluminescence at 305.46 nm and 410 nm along with strong cytotoxic effects on HT-29 colon cancer cells and effective antibacterial properties [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, Subhapriya \u003cem\u003eet al\u003c/em\u003e., reported the biosynthesis of TiO₂ NPs using \u003cem\u003eTrigonella foenum-graecum\u003c/em\u003e leaf extract, producing spherical NPs ranging from 20\u0026ndash;90 nm. These TF-TiO₂ NPs exhibited significant antimicrobial activity, with inhibition zones of 10.6 mm for \u003cem\u003eYersinia enterocolitica\u003c/em\u003e, 10.8 mm for \u003cem\u003eEscherichia coli\u003c/em\u003e, 11.2 mm for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and 11.6 mm for \u003cem\u003eStreptococcus faecalis\u003c/em\u003e. The antimicrobial effect was stronger against Gram-positive bacteria because of their thicker cell walls. The bactericidal activity of TiO₂ NPs is due to reactive oxygen species that disrupt bacterial membranes and cause cell death, indicating its potential for antimicrobial treatments like infections and wound care [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy considering the above survey, our work focuses on the preparation of NiO NPs using Piper nigrum leaf extract, evaluating their biological applications in terms of antioxidant activity and antibacterial activity. Piper nigrum, commonly known as black pepper, is one such plant with substantial potential for green synthesis. The leaf extract of Piper nigrum contains bioactive compounds that can facilitate the preparation of NiO NPs. The use of Piper nigrum leaf extract, particularly from Muzaffarabad Azad Kashmir, Pakistan is novel and represents a notable advancement for green synthesis. By leveraging the natural properties of this extract, we aim to develop a more sustainable method for producing NiO NPs and to explore their potential in addressing critical biological challenges.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation of Leaf Extract\u003c/h2\u003e \u003cp\u003eInitially, Piper nigrum leaves were collected from Muzaffarabad, Azad Kashmir. The leaves were air-dried at room temperature for 8 h. Further in a clean beaker, 20 g of the dried leaves were washed twice with distilled water to remove surface contaminants. After washing, these leaves were dried further on filter paper to remove excess moisture. To prepare the leaf extract, 500 mL of deionized water was incorporated with the leaves, which were subsequently subjected to boiling to yield a concentrated extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of NiO NPs\u003c/h2\u003e \u003cp\u003eA 1 mM nickel sulfate solution was created by dissolving 1.3 g of NiSO₄6H₂O in 500 mL of deionized water to create a 10 mM solution, which was then diluted to 1 mM. The 1 mM nickel sulfate solution was added to the freshly prepared Piper nigrum leaf extract. The mixture was continuously stirred and heated to facilitate the reduction of nickel ions and the formation of NiO NPs. After the synthesis process, the resultant product underwent an extensive washing procedure utilizing distilled water in order to eliminate any residual solution, and the synthesized NPs were subsequently collected for further characterization.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Characterization","content":"\u003cp\u003eThe synthesized NiO NPs underwent characterization through an array of analytical methodologies to elucidate their structural, morphological, and chemical attributes. The crystal architecture of prepared material was scrutinized utilizing X-ray diffraction (XRD) spectroscopy, employing a Bruker D8 X-ray diffractometer, which yielded significant insights into the phase composition and crystallinity of the material. The morphological characteristics and elemental constituents were investigated through Field Emission Scanning Electron Microscopy (FESEM) in conjunction with Energy-Dispersive X-ray Spectroscopy (EDX), facilitating comprehensive examination of particle shape, size, and elemental distribution. Fourier Transform Infrared Spectroscopy (FTIR) was employed to ascertain the functional groups present and to validate the existence of nickel-oxygen bonding interactions. Thermogravimetric Analysis (TGA) was performed to examine the thermal stability profile of the NiO NPs.\u003c/p\u003e"},{"header":"4. Biological Activities","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Alpha Amylase Inhibition Activity\u003c/h2\u003e \u003cp\u003eThe alpha-amylase inhibition activity of NiO NPs was assessed by pre-incubating various concentrations (100\u0026ndash;500 \u0026micro;g/mL) of the nanoparticles with amylase enzyme and starch solution. After incubation, the reaction mixture was subjected to treatment with NaOH and DNS, followed by analysis of absorbance at λ\u0026thinsp;=\u0026thinsp;540 nm. The inhibition % was determined using \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:({A}_{c}-{A}_{s})/{A}_{c}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e. In this formula, ​\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{c}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{s}\\:\\)\u003c/span\u003e\u003c/span\u003erepresent the absorbance of the control sample and the absorbance in the presence of nanoparticles, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Antibacterial Activity\u003c/h2\u003e \u003cp\u003eThe antibacterial potential of Piper nigrum-derived NiO NPs was investigated by well-diffusion method against bacterial strains including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eSerratia marcescens\u003c/em\u003e, \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Antioxidant Activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of as prepared NPs was analyzed using the ABTS free radical neutralization method, with the percentage neutralization evaluated by comparing the absorbance of the control and NPs samples. Similarly, the metal chelating activity was determined by incubating the NPs with ferrous sulfate and ferrozine, and calculating the chelating percentage based on the absorbance at 517 nm. Both activities were quantified using: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:({A}_{o}-{A}_{i})/{A}_{o}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e where A\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e is the control absorbance and A\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the absorbance with NPs.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e5.1. Structural Analysis\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the XRD results of NPs within 2θ range of 20\u0026deg; to 70\u0026deg;. The spectrum reveals distinct diffraction peaks at 2θ values of 37.48\u0026deg;, 43.49\u0026deg;, and 62.94\u0026deg;. These prominent peaks correspond to the (111), (200), and (220) Brags planes, respectively. These diffraction peaks, corresponding to their respective \u003cem\u003ehkl\u003c/em\u003e planes, match the reference data from JCPDS card no. 01-073-1519, confirming the synthesized NiO NPs exhibit a cubic crystal structure, as shown in the inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The observed sharpness of the peaks indicates high crystallinity and well-defined crystal planes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The absence of any additional impurity peaks further supports the purity of the synthesized material, suggesting that the synthesis process was successful, and the NPs are free from contamination. The crystallite size of the NiO NPs yielded 22.37 nm calculated by Debye-Scherrer equation. This relatively small crystallite size indicates that the nanoparticles have a high degree of crystallinity and a well-defined structure. The small size of the crystallites is likely to enhance the biological activity of the NiO NPs due to the increased surface area and reactivity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. All lattice parameters are tabulated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Overall, these results indicate the successful preparation of high-quality NiO NPs with a cubic structure, which is expected to positively impact their biological activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLattice constant, crystallite size, and X-ray density of NiO\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLattice Constant (\u0026Aring;)\u003c/p\u003e \u003cp\u003e\u003cem\u003ea\u0026thinsp;=\u0026thinsp;b\u0026thinsp;=\u0026thinsp;c\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrystallite Size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX-ray Density (g/ cm\u003csup\u003e3)\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNiO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e5.2. Morphological Study\u003c/h2\u003e \u003cp\u003eThe morphology of the prepared sample was extensively studied by FESEM at 20,000x as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a). These visuals revealed that the NiO NPs exhibit significant agglomeration, with small particles forming spherical shapes with pronounced porosity within the sample, and numerous visible voids, indicating a high degree of porosity. To quantify the particle size, ImageJ software was employed, yielding an average grain size of 65 nm. This size estimation supports the observed morphology and confirms the presence of small, spherical particles with notable agglomeration and porosity. Further confirmation of the particle morphology was obtained through Scanning Transmission Electron Microscopy (STEM). The STEM analysis provided a clearer view of the spherical grains, confirming an average grain size of 40 nm as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b). The STEM images corroborated the FESEM findings, showing a high level of porosity and spherical particle shapes, enhancing the overall understanding of the material\u0026rsquo;s morphology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe EDX spectra of NiO NPs is depicted Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c). The analysis reveals that the primary elements are nickel and oxygen, confirming the nickel oxide composition. Prominent peaks for nickel and oxygen validate the material's identity. Small peaks at 0.25 and 0.5 keV are due to C and O, while the main peaks at 0.90, 7.5, and 8.3 keV correspond to nickel. Minor peaks at 1.1, 1.8, and 2.6 keV indicate the presence of sodium, silicon, and chlorine. The elemental composition is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Carbon, chlorine, silicon, and sodium are likely residuals from the synthesis using plant materials.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eElemental composition of prepared NPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWt.%\u003c/p\u003e \u003cp\u003eNi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWt.%\u003c/p\u003e \u003cp\u003eO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWt.%\u003c/p\u003e \u003cp\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWt.%\u003c/p\u003e \u003cp\u003eCl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWt.%\u003c/p\u003e \u003cp\u003eSi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWt.%\u003c/p\u003e \u003cp\u003eNa\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNiO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5.3. FTIR Analysis\u003c/h2\u003e \u003cp\u003eFTIR works by using IR photons to vibrate atoms within chemical bonds, causing vibrational transitions at specific energy levels. Molecules absorb infrared light at particular wavelengths, and the resulting absorption spectrum identifies functional groups and compounds. Contaminants also emit unique IR bands, allowing for impurity detection. Fourier transformation is used to decode individual frequencies, and a computer processes the data to provide spectral information for analysis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The FTIR spectrum of NiO NPs as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e exhibits multiple distinctive peaks. The prominent peak observed at 3701 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is ascribed to the presence of water molecules. A broad absorbance band spanning the range of 3565\u0026thinsp;\u0026minus;\u0026thinsp;3398 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating the stretching vibrations of (O-H) groups [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The peak around 1655.81 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is attributed to the bending vibration of O-H, indicating the existence of H\u003csub\u003e2\u003c/sub\u003eO molecules in the sample. The peak at 1383.31 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is due to the nitro group (NO₃), which originate from the precursor used in the synthesis process. Two prominent peaks at 1104.24 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1068.89 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are designated to the robust stretching vibrations of the Ni\u0026ndash;O bond, signifying the formation of nickel oxide. Additionally, the peak at 898.85 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represents the stretching vibrations of Ni\u0026ndash;O [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Further, the bands at 545.2 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 447.64 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to the bending and wagging vibrations of Ni\u0026ndash;O, confirming the presence of NiO in the sample [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5.4. Thermogravimetric Analysis\u003c/h2\u003e \u003cp\u003eThermogravimetric analysis (TGA) monitors a sample's mass change with temperature and time, providing accurate compositional data for quality and process control. However, it cannot identify volatile compounds without additional analytical tools. TGA can also determine oxidation induction time (OIT) by heating a small sample (typically\u0026thinsp;\u0026lt;\u0026thinsp;10 mg) in oxygen at an isothermal temperature, generally around 200\u0026deg;C, and observing the mass gain when oxidation occurs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The TGA curve of NiO NPs presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows two significant stages of weight loss. The first sharp weight loss occurs between 160\u0026deg;C and 275\u0026deg;C, which corresponds to the evaporation of physically absorbed water molecules. This is typical for samples synthesized using plant-based methods, where moisture remains after synthesis. The second stage, occurring between 276\u0026deg;C and 540\u0026deg;C, reflects a gradual weight loss due to the removal of chemically adsorbed water and the decomposition of organic compounds. These organic compounds are likely residuals from the plant extract used in the green synthesis process. After 540\u0026deg;C, the weight stabilizes, indicating that all volatile and organic components have been fully decomposed, leaving behind the final, thermally stable NiO product [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This final stage confirms the complete formation of the NiO NPs, highlighting their purity and thermal stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5.5. Enzymatic Inhibition Activity of Alpha-Amylase\u003c/h2\u003e \u003cp\u003eThe inhibitory effect of NiO NPs on alpha-amylase was evaluated by pre-incubating various concentrations of NiO NPs (100\u0026ndash;500 \u0026micro;g/mL) with 0.5 mL of phosphate buffer containing 500 \u0026micro;L of alpha-amylase enzyme (1 mg/mL) at 37\u0026deg;C for 10 min [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Subsequent to the pre-incubation phase, 1 mL of starch solution was introduced, and the resultant mixture was subjected to an additional incubation period for 5 min at 37\u0026deg;C. The enzymatic reaction was subsequently terminated by the addition of NaOH and DNS, followed by heating the mixture for 5 min at 100\u0026deg;C. Upon reaching room temperature, the absorbance of the solution was quantitatively measured at a wavelength of 540 nm as illustrated in Fig.\u0026nbsp;5(a). Furthermore, Acarbose was employed as a standard inhibitory agent, yielding an IC50 value of 0.029 \u0026micro;g/mL. The inhibition activity of NiO NPs was measured as the percentage of enzyme activity inhibited, with IC50 indicating the concentration required to inhibit 50% of enzyme activity, as illustrated in Fig.\u0026nbsp;5(b). The results showed that the inhibition of alpha-amylase by NiO NPs was directly proportional to their concentration: higher concentrations led to greater inhibition and lower absorbance values. The absorbance values of NiO NPs at 10, 20, 40, 60, 80, and 100 \u0026micro;g/mL were 0.094, 0.078, 0.044, 0.012, and 0.002, respectively. Corresponding percent inhibition activities were 67%, 72%, 84%, 95%, and 99%. All the parameters are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The IC50 value for NiO NPs, determined from the data, was 1.18 \u0026micro;g/mL. This indicates that NiO NPs are effective inhibitors of alpha-amylase, with their inhibition potential increasing with concentration.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage inhibition of NiO NPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration(\u0026micro;g\\mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsorbance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%inhibition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIC50\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.6. Antioxidant\u003c/h2\u003e \u003cp\u003eAntioxidants are substances capable of neutralizing free radicals and reducing their formation. The antioxidant activity of prepared NPs was evaluated using the ABTS free radical and results were compared with standard ascorbic acid as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e(a-b). As various concentrations of NiO NPs were tested for antioxidant activity against ABTS free radicals [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A clear trend emerged, as the concentration of NiO NPs increased from 20 to 80 \u0026micro;L, the ABTS radical scavenging activity also increases, ranging from 44\u0026ndash;78%, respectively. This suggests that a higher concentration of NiO NPs provides a greater number of surface binding sites for free radicals, thereby enhancing their scavenging potential. A similar trend was observed with ascorbic acid, where at 80 \u0026micro;L it achieved a 58.5% scavenging activity compared to the 78.4% displayed by NiO NPs at the same concentration. These results highlight the superior ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e radical scavenging activity of NiO NPs over ascorbic acid. Additionally, the lower IC50 value and higher correlation coefficient further support the enhanced antioxidant potential of NiO NPs compared to the standard. All the calculated parameters are tabulated in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Furthermore, the % of radical reduction (%RSA) was calculated based on the decolorization of the ABTS solution, which occurs due to the reaction between the radicals and the antioxidants in the nanoparticles. The IC50 value, indicating the concentration required to achieve 50% inhibition, was derived from these measurements. NiO NPs were tested at concentrations ranging from (20\u0026ndash;100 \u0026micro;g/mL). Higher concentrations of NiO NPs resulted in a greater decrease in ABTS absorbance, reflecting increased radical neutralization activity [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The absorbance values for NiO NPs at different concentrations were 0.12, 0.090, 0.069, 0.046, and 0.019 for 20, 40, 60, 80, and 100 \u0026micro;g/mL, respectively. The corresponding percent reduction values were 44%, 58%, 68%, 78%, and 91%. The IC50 value for NiO NPs was calculated to be 28 \u0026micro;g/mL, indicating their effective concentration for 50% radical inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAbsorbance %RSA and IC50 reading of NiO NPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration (\u0026micro;g\\mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsorbance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%RSA\u003c/p\u003e \u003cp\u003e\u003cem\u003e(\u0026Aring;-A\\\u0026Aring;)\u003c/em\u003e100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIC50\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e5.7. Iron Chelating Activity\u003c/h2\u003e \u003cp\u003eThe iron chelating activity of NiO NPs was evaluated using a method based on the reduction of the ferrous ion-ferrozine complex [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Ferrozine forms a red-colored complex with Fe\u0026sup2;⁺ ions and the reduction in this color measured by the absorbance at 517 nm, indicates the iron chelating activity of the NPs. The IC50 value for NiO NPs, representing the concentration required to achieve 50% inhibition of the ferrozine-Fe\u0026sup2;⁺ complex formation, was calculated to be 23 \u0026micro;g/mL. All the calculated values at various conditions are given in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFe\u003csup\u003e+\u003c/sup\u003e percentage inhibition of NiO NPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(\u0026micro;g\\mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsorbance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%Fe\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIC50\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5.8. Antibacterial activity\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of NiO NPs was evaluated against both Gram-positive bacteria (S. pyogenes, S. aureus) and Gram-negative bacteria (P. aeruginosa, K. pneumoniae, S. marcescens). The results represented by zones of inhibition (in mm) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e, while Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e enlists the inhibition zone measurements for both the NiO NPs and conventional antibiotics. It was observed that the synthesized NiO NPs were particularly effective against Gram-positive bacteria, showing inhibition zones of 18 mm for \u003cem\u003eS. pyogenes\u003c/em\u003e, and 13 mm for \u003cem\u003eS. aureus\u003c/em\u003e which were larger compared to those seen for Gram-negative pathogens. This indicates that Gram-negative bacteria exhibited greater resistance to NiO NPs than Gram-positive strains. The antibacterial activity of the nanoparticles depends on various factors, including the specific bacterial species, particle size, stability, and the concentration within the growth medium. The nanoscale pores in the outer membranes of bacterial cells allow for greater interaction between the NPs and pathogens. In this study, NiO NPs synthesized using Piper nigrum extract had a very small particle size which plays a critical role in their antibacterial efficacy. The antibacterial mechanism of the NiO NPs is likely attributed to electrostatic interactions between the positively charged nickel ions (Ni\u0026sup2;⁺) and the negatively charged bacterial cell membranes. This interaction facilitates the release osf Ni\u0026sup2;⁺ ions, which penetrate the bacterial cell wall, damaging essential biomolecules such as DNA and proteins, disrupting mitochondrial function, and interfering with electron transport, ultimately leading to cell death [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The calculated parameters are tabulated in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibacterial activity and inhibition zone of NiO NPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibiotics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eDiameter(nm) of inhibition zones concentrations (5 mg/50 mL)\u003c/p\u003e \u003cp\u003e5 10 30 50 100\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS.aru\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.867\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.449\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS.pyo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.867\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS.mre\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.635\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.642\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eK.pne\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e 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\u003cp\u003e9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.246\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this study, NiO NPs were successfully synthesized using Piper nigrum leaf extract via a green synthesis approach. The structural and morphological analysis confirmed the formation of cubic NiO NPs with high crystallinity and significant porosity, which contributes to their biological effectiveness. The synthesis process proved to be eco-friendly, utilizing plant-based extracts as reducing and stabilizing agents, making it a sustainable alternative to traditional methods that often involve toxic chemicals and high energy consumption. The biological activities of the synthesized NiO NPs were explored, demonstrating promising results. The NPs exhibited significant antioxidant potential, surpassing that of standard ascorbic acid, indicating their ability to neutralize free radicals effectively. Their alpha-amylase inhibition activity further confirmed their therapeutic potential, particularly in controlling glucose levels. Additionally, the NiO NPs showed notable antibacterial activity, especially against Gram-positive bacteria, highlighting their potential application in antibacterial coatings and infection control.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approva\u003c/strong\u003el\u003c/p\u003e\n\u003cp\u003eNot applicable. This study did not involve any human or animal subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHarun-Ur-Rashid, I. Jahan, T. Foyez, \u0026amp;A.B. 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Res. 195 707\u0026ndash;724.\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":"Nickel oxide nanoparticles, Antibacterial activity, Green synthesis, Piper nigrum","lastPublishedDoi":"10.21203/rs.3.rs-5337394/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5337394/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent years, the use of eco-friendly and sustainable methods for synthesizing nanoparticles has gained significant attention due to environmental concerns associated with conventional chemical approaches. This study explores the preparation of nickel oxide (NiO) NPs using Piper nigrum leaf extract sourced from Muzaffarabad Azad Kashmir, Pakistan a novel and environmentally benign approach. These NPs were studied using various techniques to determine their morphology, size, and structural properties. Furthermore, the biological activity of the freshly prepared NPs was evaluated, focusing on their inhibitory effect on alpha-amylase, a key enzyme related to diabetes management. The NPs exhibited excellent antioxidant properties, with a peak scavenging activity of 78% at 80 \u0026micro;g/mL, and an IC50 value of 28 \u0026micro;g/mL. In alpha-amylase inhibition assays, NiO NPs demonstrated significant enzyme inhibition with an IC50 of 1.18 \u0026micro;g/mL. Antibacterial tests revealed strong activity against Gram-positive bacteria, with peak inhibition zones of 18 mm for Streptococcus pyogenes and 13 mm for Staphylococcus aureus. These results highlight the NPs potential for biological applications.\u003c/p\u003e","manuscriptTitle":"Biosynthesis of nickel oxide nanoparticles from piper nigrum leaf Extract and their enhanced antioxidant, antibacterial, and enzyme Inhibition Activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-30 11:11:57","doi":"10.21203/rs.3.rs-5337394/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":"fce88282-1b4b-47a5-91cd-fcd413ce0e61","owner":[],"postedDate":"October 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-30T11:12:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-30 11:11:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5337394","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5337394","identity":"rs-5337394","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}