https://orcid.org/0000-0002-1555-0887
Figures
Abstract
Green synthesis, which creates nanoparticles from natural sources such as microorganisms or plant extracts, is a sustainable alternative to traditional chemical methods since it employs less hazardous chemicals and has fewer negative environmental consequences. In this study, the antioxidant, antibacterial, and toxicological properties of ZnO nanoparticles were investigated by synthesizing them using an aqueous extract of Catunaregam spinosa (C. spinosa) leaves. X-ray Powder Diffraction (XRD), Field Emission Scanning Electron Microscopy (FE-SEM), Energy Dispersive X-ray Analysis (EDX), Fourier Transform Infrared Spectroscopy (FTIR), and UV-vis spectroscopy were used to confirm the formation and properties of ZnO-NPs. FE-SEM investigation indicated a nearly spherical form, and XRD computed an average crystallite size of 12.39 ± 3.84 nm. The UV-vis spectrum showed a high absorption peak at 366 nm. FTIR indicated the existence of bioactive functional groups, which are important for nanoparticle capping and stability. ZnO-NPs showed antibacterial efficacy against Escherichia coli, Shigella sonnei, Klebsiella pneumoniae, and Staphylococcus aureus at 50 μg/mL. The inhibition zones were 9 mm, 13 mm, 16 mm, and 15 mm, respectively. Klebsiella pneumoniae and Staphylococcus aureus had the same minimum inhibitory concentration (MIC) of 6.25 mg/mL. Nanoparticles also demonstrated significant antioxidant activity in the DPPH experiment. Toxicity evaluation against Artemia salina yielded an LC50 of 55.20 ± 16.19 μg/mL, indicating dose-dependent effects. Overall, this study found that ZnO-NPs generated with Catunaregam spinosa leaf extract have promising antibacterial, antioxidant, and toxic characteristics, indicating potential biological applications.
Citation: Baraili R, Pathak I, Sharma S, Bhusal M, Sharma KR (2025) Green synthesis of zinc oxide nanoparticles using Catunaregam spinosa (Thunb.) triveng for biologicals applications. PLoS One 20(12): e0320475. https://doi.org/10.1371/journal.pone.0320475
Editor: Muthugounder Subramanian Shivakumar, Periyar University, INDIA
Received: February 19, 2025; Accepted: December 9, 2025; Published: December 31, 2025
Copyright: © 2025 Baraili et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are available at https://doi.org/10.5281/zenodo.16785370.
Funding: The author(s) received no specific funding for this work.
Competing interests: No conflict of interest on this research paper.
Introduction
The fundamental goals of the rapidly growing field of nanotechnology are the creation, modification, and application of materials ranging in size from 10 to 500 nm for a variety of medical procedures and drug delivery systems [1]. It is seen as a benefit to modern medicine since it has progressed to the point where new potential for nanoscience exists, particularly in nanodrug, gene transport, and biosensing [2,3]. Because of their surface plasmon resonance, solubility, adhesiveness, and chemical stability, metal and metal oxide (MO) nanoparticles (NPs) are expected to spread easily throughout the ecosystem [4–6]. The most recent studies on antibacterial, antioxidant, and anticancer activities have concentrated on ZnO, CuO, SnO2, Ag2O, Fe2O3, CaO, NiO, MgO, and AuNPs [7,8].
A wide range of chemical and physical techniques has been used to synthesize NPs, including thermal, sol-gel, hydrothermal, sonochemical, rapid precipitation, and microwave irradiation techniques [9]. Bioresource-based synthesis is a potential solution to the risk of using harmful chemicals in various synthesis processes, as it is less toxic, more ecologically friendly, and more adaptable [10]. Polyols, which are found in different plant parts such as peels, leaves, roots, fruits, seeds, and bark, can stabilize metallic nanoparticles by acting as chelating and capping agents during their rapid production [11,12]. It emphasizes the efficacy of “green synthesis,” which reduces synthesis costs and energy needs when compared to chemical or physical NP synthesis [13,14]. Economic interest in developing ecologically friendly ZnO nanoparticles is significant due to their exceptional durability, bioactivity, and biocompatibility [15].
Zinc oxide (ZnO) is a promising biomedical candidate with anti-inflammatory, antibacterial, antioxidant, biosensing, anti-cancer, and cell imaging properties [16,17]. It has also been successfully used to treat diabetes since zinc is known to preserve the structural integrity of insulin [18]. Furthermore, due to their exceptional brightness, ZnO-NPs are among the best options for bioimaging [19–21]. Although interest in ZnO’s exceptional antibacterial activity has grown, scientists are still working to figure out how ZnO-NPs develop their therapeutic properties.
C. spinosa (mountain pomegranate) is a medium-sized, perennial wild shrub in the Rubiaceae family [22]. It is a thorny shrub that may grow up to 4,000 feet above sea level and reach a height of 5 meters. It features oblong, glossy, pubescent leaves with honey-like fragrances from its white blossoms. Indigenous people utilized the plant’s stem and bark to heal muscle soreness, and its roots to treat dandruff. Aside from wound healing, C. spinosa leaves are used to treat fever, gastrointestinal issues, tumors, piles, snake bites, and diarrhea. Some investigations have found that C. spinosa has antihyperglycemic, antimicrobial, hepatoprotective, anti-inflammatory, and anticataleptic effects [23,24].
Although nanoparticles have been manufactured from a variety of plant extracts, very few investigations have been conducted on nanoparticles, particularly ZnO, derived from C. spinosa. There are very limited instances of green NP synthesis using C. spinosa fruits and leaves that have shown effective, eco-friendly, and unique medicinal properties, paving the way for the development of cutting-edge nanotechnology solutions [25,26]. To summarize, this work emphasizes the plant’s high phytochemical content for eco-friendly synthesis, which is defined by optimal synthesis, full characterization, and evaluation of antibacterial, antioxidant, and toxic capabilities.
C. spinosa leaves were chosen because they contain bioactive secondary metabolites and are readily available and regenerative, making them a viable and sustainable choice for green synthesis. Following optimization, ZnO-NPs were characterized by FTIR, EDX, FE-SEM, UV-Vis, and XRD studies. Furthermore, the plant’s role as a natural stabilizer and reducer in the synthesis of metal oxide nanoparticles was explored, and the ZnO-NPs’ cytotoxic, antibacterial, and antioxidant capabilities were assessed. The use of C. spinosa extract, which has received little attention for the production of green nanoparticles, in conjunction with a thorough assessment of their biological activity, is the most creative and powerful aspect of this work. Fig 1 shows some of the bio-medicinal applications of ZnO-NPs.
Materials and methods
Collection of plant material and extraction of metabolites
The healthy and fresh leaves of C. spinosa were collected in June 2023 from a community forest in Bardiya district (midwestern Nepal) at latitude 28° 9′ 8′′ N and longitude 81° 29′ 2′′ E. It’s 479 feet above sea level. The herbarium was recognized by the National Herbarium and Plant Laboratories in Godawari, Lalitpur, Nepal, and with the voucher code RB-002 (KATH). The specimen was stored in the herbarium at a temperature of 15–16 °C in a standard cabinet. Fig 2 shows an image of Catunaregam spinosa (Thunb.) Triveng. The leaves were washed, dried in the shade, and then pulverized into a fine powder. Next, 100 milliliters of deionized water and five grams of powder were put in glasses and spun for half an hour at 60° C. After filtering the mixture, the extract was refrigerated for later use. Because the sample is immersed in the solvent for a long time during the extraction process, a small amount of non-polar component may be extracted despite the solvent’s polarity, potentially aiding in the formation of nanoparticles.
Synthesis of ZnO-NPs
After preparing 500 mL of a 0.13 M zinc nitrate hexahydrate solution, 100 mL of the extract was mixed with 5 mL of zinc nitrate hexahydrate to produce ZnO-NPs. This mixture was then agitated for an hour at 60 °C. After centrifugation at 9000 rpm, the mixtures were calcined at 400 °C for two hours. Fig 3 depicts the schematic route for the synthesis of ZnO-NPs.
Fig 4 shows a proposed reaction pathway for the formation of ZnO-NPs using C. spinosa leaf extract, in which the zinc precursor and the extract’s functional components ligate. On the word of the literature evaluations, organic chemical groups such as phenolics and flavonoids found in plant extracts operate as ligands. These components, which contain hydroxy aromatic ring groups, interact with zinc ions to form complex ligands. Nanoparticles are created and stabilized through nucleation and shaping processes. When the organic mixture is heated at 400 °C, it directly breaks down and produces ZnO nanoparticles [27].
Characterization of ZnO-NPs
While the ZnO-NPs solution was being stirred, its UV-visible absorption spectra were measured using a UV-visible spectrophotometer (SPECORD 200 PLUS, An Endress+Hauser Company) in the scanning range of 300–600 nm, with deionized water as the blank. Bio-synthesized ZnO-NPs were placed on a potassium bromide pellet for FTIR analysis and scanned with a Bruker Tensor 27 in the 400 cm-1 to 4000 cm-1 wavelength range to determine their functional group. A D2 phaser (Bruker, NAST, Nepal), a powerful X-ray beam diffraction analyzer, was used to study the produced nanoparticles further and determine their crystal structure. The size and shape of ZnO nanoparticles (ZnO-NPs) were studied using FE-SEM at 10 kV using SU-70 equipment in Korea. Energy-dispersive X-ray (EDX) analysis with EDAX APEX software was used to determine the various elemental compositions of the nanoparticles.
Evaluation of antioxidant activity
The antioxidant potential (IC50) is the amount necessary to block and evaluate its DPPH scavenging ability by half [28]. The DPPH radical scavenging experiment was utilized to assess antioxidant activity [29,30]. Nanoparticles (3.75 μg/mL from 100 μg/mL) and aqueous C. Spinosa leaf extract (100 μL) were combined with a methanolic solution of DPPH (100 μL, 0.1 mM) at various doses (500, 250, 125, 62.5, 31.25, and 15.625 μg/mL). After 30 minutes of dark incubation, the absorbance at 517 nm was measured. The reference standard was quercetin (20 μg/mL to 0.625 μg/mL), and each test was conducted in triplicate. Eq (1) was used to calculate the percentage of scavenging.
Where, A sample = absorbance of sample, A control = absorbance of control
Evaluation of antimicrobial activity
The antimicrobial properties of ZnO-NPs were investigated in vitro using the Well diffusion method with reference antibiotic 1 mg/mL of neomycin following a specified procedure against three Gram-negative strains of Shigella sonnei (ATCC 25931), Klebsiella pneumoniae (ATCC 700603), and Escherichia coli (ATCC 25912), as well as one Gram-positive strain of Staphylococcus aureus (ATCC 43300) [31]. The testing pathogens were incubated at 37 °C after being injected into Muller-Hinton Broth (MHB). Upon bringing the turbidity down to the standard 0.5 McFarland, 1.5 × 108 CFU/mL was the final inoculum. A well holding 25 µL of the test sample (50 mg/mL) dissolved in 100% distilled water, a negative control (100% distilled water), and a positive control (1 mg/mL, neomycin) were all included in each experiment set. The contents were allowed to diffuse at room temperature for 15 minutes before being incubated at 37°C for 24 hours. The ZOI (mm) that surrounded the well was apparent on the plates following incubation. ZnO-NPs, controls, and crude extract were kept in separate wells in each plate for comparison. Thus, it is possible to compare and contrast whether the antibacterial activity is caused by nanoparticles, crude extracts, or controls. To create a strong enough inhibitory impact on the targeted bacteria to allow for clear observation of the antimicrobial agent’s efficacy, high concentrations, such as 50 mg/mL of the tested sample, were used in antimicrobial experiments. This experiment is merely the first step in determining nanoparticles’ antibacterial properties. MIC and MBC assays were used to further examine the lowest concentration of sample required for inhibition.
Estimation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The Resazurin Microtiter Method (RMM) was used to measure MIC [32]. In a 96-well plate filled with Muller-Hinton broth (MHB), the test samples were serially diluted to achieve various concentrations. After calibrating to a turbidity of 0.5 McFarland, 5 µL of bacterial suspension was injected into each well (except the negative control). The well-known medication neomycin was used as the positive control since it is an aminoglycoside antibiotic that has been extensively tested and demonstrated to be effective against a wide range of bacteria. Because of its predictable and continuous antibacterial effect, it is an excellent tool for comparing the performance of various antimicrobial medications or determining the susceptibility of bacterial strains. After 24 hours of incubation at 37 °C, the microtiter plates were then incubated for 4 hours with 0.003% resazurin added. The wells with bacterial growth turned pink, whereas those with none remained blue. The contents of the wells were streaked onto nutrient agar plates to calculate the MBC, which was then incubated at 37 °C for 24 hours.
Brine shrimp lethality assay
The toxicity was assessed using a modified brine shrimp lethality assay [33]. To create artificial saline water for the successful hatching of brine shrimp cysts, one liter of double-distilled water was mixed with 40.56 grams of salt. To maintain a pH between 8 and 8.5, 1N NaOH was utilized. Then, a 5 mL test solution comprising 4 mL of saline water and 1 mL of the test sample at different concentrations (1000, 500, 250, 125, 100, and 10 μL) was administered to 10 freshly hatched nauplii for a full day. After one day, the number of live nauplii in each test tube was recorded. The mortality percentage (%) was determined using Eq (2).
Nc and Nt reflect the number of live nauplii in the control and tested agents, respectively, after 24 hours, while M denotes the mortality rate.
Statistical analysis
GraphPad Prism 9.5.1 and MS Excel software were used to calculate the IC50 in the DPPH and LC50 in the brine shrimp lethality experiment, as well as to create the appropriate graphs. The tests were repeated three times, and the results were presented as mean ± standard deviation. Origin 2019b (64-bit) and High Score (Plus) were used to plot the FTIR, XRD, and UV-visible spectra. SEM pictures were analyzed using the ImageJ software.
Results
Structural and optical characteristics
When Zn (NO3)2.6H2O was added to the extract, it turned yellowish white, indicating that ZnO-NPs were formed. Following calcination, a white powder was generated, which is consistent with prior studies by [34]. Fig 5(a) depicts the effective synthesis of ZnO-NPs, with an absorbance peak for ZnO at 366 nm. Apart from indicating purity, the XRD peaks show the crystal structure of ZnO-NP, as shown in Fig 5(b). The peaks correspond to standard JCPDS card No. 01-079-0205 and are located at approximately 2θ values of 69.1°, 68.0°, 62.9°, 56.6°, 47.5°, 36.2°, 34.4°, and 31.7°. ZnO crystal planes (112), (200), (103), (110), (102), (101), (002), and (100) are shown by peaks with appropriate 2θ values. Strong, clear peaks with good crystallinity indicated that the ZnO-NPs had a well-organized crystalline structure with a crystallite size (D) of 12.39 ± 3.84 nm, as determined by Eq (3). The utilization of several peaks to calculate the average crystallite size, however, is most likely the reason for the significant standard deviation from the mean, as various peaks have varied full width at half maximum (FWHM) values and related Bragg angles.
The dimensionless form factor ( ̴1) is represented by k, the crystalline grain size by D, the full width at half maximum (FWHM) in radians by β, the Bragg’s angle, or half of the 2θ value of the selected peak, by θ, and the wavelength of x-ray radiation by λ.
Numerous bands in the 4000–400 cm-1 range revealed the samples’ organic composition, as illustrated in Fig 5(c). Aqueous extract peaks were found at 3676, 2978, 2902, 2322, 2169, 2017, 1658, 1398, 1238, 1064, 879, and 671 cm−1. FTIR peaks at 3676 cm-1 are often formed by stretching and bending vibrations of hydroxyl groups (O-H), which are frequently related to the presence of alcohols, phenols, or carboxylic acids in plant extracts. These groups can produce ZnO nanoparticles by transforming zinc ions (Zn²⁺) into zinc atoms (Zn⁰) via electron donation [35]. The stretching vibration of aliphatic C-H groups is related to the peaks at 2978 cm−1 and 2902 cm−1. Peaks at 2322 cm−1, 2017 cm−1, 1392 cm−1, and 1238 cm−1 may suggest the presence of functional groups like C-O or C-N, which can act as capping or stabilizing agents during nanoparticle production. A peak at 2169 cm-1 or 1658 cm-1 represents an alkene C = C bond, amide I (C = O stretching), or esterified carboxyl groups. A peak at 879 cm-1 is most likely associated with alkenes or alkynes, while a peak at 1064 cm-1 is associated with C-O stretching vibrations or aromatic ring vibrations. The FTIR spectra of ZnO-NPs showed similar peaks with varying intensities at 3674, 3414, 2976, 2902, 2326, 2169, 2017, 1647, 1535, 1392, 1244, 1062, 889, 671 cm−1, and 418 cm−1. The spectra revealed a clear Zn-O bonding peak around 671 cm-1 to 418 cm−1, similar to prior research [36].
Morphological and compositional characteristics
Fig 6(a–d) depicts the results of an FE-SEM analysis that examined the surface form and elemental composition of the produced ZnO-NPs. In a previous study [25], spherical ZnO-NPs measuring 37.49 nm were produced utilizing an aqueous fruit extract of Capparis spinosa L. In this instance, FE-SEM revealed that the average size of the nearly spherical ZnO-NPs was 22.08 ± 0.36 nm, which is less than 100 nm. This disagreement could be caused by differences in several variables, such as plant species, location, sample collection time, extract to metal salt ratio, pH utilized, and other laboratory conditions, as stated by [37]. Fig 7 shows the elemental composition of ZnO-NPS, as well as the color mapping established by EDX testing. Following Fig 8(a), the EDX peaks provide strong evidence that ZnO-NPs were manufactured with 70.4% zinc and 18.3% oxygen. The sample also contained trace amounts of carbon and nitrogen. The histogram seen in Fig 8(b) illustrates the particle size distribution.
Antioxidant potential of nanoparticles
Fig 9 (a and b) graphically shows the inhibitory potential of the aqueous extract and standard quercetin. Fig 9 (c) illustrates the NPs’ antioxidant capability. ZnO-NPs had a lower IC50 (94.83 ± 0.00 µg/mL) than the plant extract, indicating improved antioxidant action over the aqueous extract. As the hue of the DPPH solution changed over time, the maximum intensity for the samples at 517 nm gradually dropped. The IC50 values for produced ZnO-NPs, aqueous extracts, and standard quercetin are shown in Table 1.
Antimicrobial activity
Table 2 and Fig 10 show that, in comparison to other bacteria such as Shigella sonnei (13 mm) and Escherichia coli (9 mm), ZnO-NPs inhibited Klebsiella pneumoniae (16 mm) and Staphylococcus aureus (15 mm) with the biggest zone of inhibition. Escherichia coli samples were the least susceptible of the four strains examined. As particle size decreased, greater surface area permitted optimal NP dispersion in the media. This expedited the formation of surface oxygen species, eventually causing the pathogen to die by rupturing its membrane. The effectiveness of ZnO-NPs against bacteria may be explained by their direct interaction with pathogen cell walls, which disintegrates and releases antimicrobial ions (Zn2+) [38]. As seen in Fig 11, all of the pathogens’ vital bacterial functions are disrupted after these free ions connect to their proteins and polysaccharides. The results clearly show that the activity of the nanoparticles created is greater than that of the crude extract, whilst the negative control (water) has no effect on any of the microorganisms under investigation.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of nanoparticles
After testing against four bacterial strains, the nanoparticles showed the most potent antibacterial action against one Gram-positive and one Gram-negative pathogen. This motivated further research into the MIC and MBC for these bacteria. NPs suppressed both bacterial strains at 50 μg/mL. Table 3 shows that for each strain chosen, ZnO-NPs had MBC and MIC of 12.5 mg/mL and 6.25 mg/mL, respectively. Fig 12(a–c) demonstrates the minimal inhibitory concentration and nutritional agar plates with the lowest bactericidal concentration of ZnO-NPs against Staphylococcus aureus and Klebsiella pneumoniae, respectively.
Brine shrimp lethality assay
Over 24 hours, the average mortality rate of ZnO-NPs ranged from 40% (10 µg/mL) to 83.33% (1000 µg/mL). Similar to [39], the mortality rate rose with NP content. This indicates that ZnO-NPs were relatively toxic to Artemia nauplii throughout the course of a 24-hour exposure at the maximum test concentration. Table 4 shows the toxicity of ZnO-NPs produced from aqueous leaf extract, with an LC50 of 55.20 ± 16.19 µg/mL. Fig 13 depicts the concentration and mortality percentage of Artemia nauplii caused by ZnO-NPs.
Discussion
The physicochemical factors of nanoparticles (NPs), such as size, chemical composition, and surface charge, are widely accepted to influence their properties. One of the key properties of nanoparticles is their small size. As more molecules are exposed, the surface area available for interaction with biomolecules increases [40,41]. The aqueous extract of C. spinosa leaves was employed in this study to successfully produce zinc oxide nanoparticles (ZnO-NPs) in a cheap and environmentally friendly method.
Plants may have therapeutic qualities due to particular molecules or the synergistic interaction of numerous phytoconstituents that reduce Zn (NO3)2.6H2O into ZnO during the formation of ZnO-NPs. The direct observation of a change in color in the solution containing plant extract and metal salt precursor provided initial proof of the formation of nanoparticles utilizing C. spinosa leaf extract.
The structural characterization methods, such as XRD, FE-SEM, EDX, FTIR, and UV-vis spectroscopy, verified the development of ZnO-NPs. The nanoparticles have a 3.24 eV band gap energy and a clear absorption peak at 366 nm. Green-synthesized ZnO-NPs exhibited similar peaks at 334 and 374 nm [25,42]. The crystallite size of nanoparticles was determined using the Debye-Scherrer formula, and the result was 12.39 ± 3.84 nm, comparable to Artocarpus hirsutus [43] and El Salvador persica [44].
The interaction between ZnO-NPs and functional groups was found to be responsible for the small shift and change in the position and intensity of multiple connected peaks in the NPs’ FTIR spectra [45]. The FTIR spectrum of ZnO-NPs also includes a peak at a wavelength of 418 cm-1, suggesting vibrational strain absorption of the Zn-O connection and confirming the effective production of ZnO particles. The functional groups in C. spinosa leaf extract, including (O-H), (C ≡ N), and (C = O), can stabilize, reduce, and cap NPs during biosynthesis. Capping agents appear to use a variety of processes to stabilize NPs, including van der Waals forces, hydration force stabilization, steric stabilization, and electrostatic stability [46]. The results are similar to ZnO-NPs prepared with extracts from Acacia catechu, Artemisia vulgaris, and Cynodon dactylon previously described by [47].
The SEM image analysis revealed that the NPs were agglomerated and comparably spherical. A SEM histogram revealed an average particle size of 22.08 ± 0.36 nm. The EDX data validated the production of ZnO-NPs. Other elements, such as carbon and nitrogen, were present in the produced particles. Similar to the current study, [48] verified that the NPs were spherical with sizes of 25.14 ± 7.4 nm via SEM analysis.
ZnO-NPs had a high antioxidant activity, with an IC50 of 94.83 ± 0.00 µg/mL, as measured by a DPPH experiment. The antioxidant activity of ZnO-NPs rose as concentration increased; however, it remained below the recommended level, i.e., standard as in the research published [49,50]. Using the DPPH test, [50] also evaluated the ZnO-NPs’ capacity to scavenge radicals caused by Moringa oleifera extract, in which ZnO-NPs concentration was directly related to radical scavenging activity, peaking at 67% at 100 µg/mL.
The generated ZnO-NPs were efficient against Staphylococcus aureus and Klebsiella pneumoniae at ZOIs of 15 mm and 16 mm, respectively. The MIC and MBC values of ZnO-NPs against Staphylococcus aureus and Klebsiella pneumoniae were 6.25 mg/mL and 12.5 mg/mL, respectively. This suggests that the nanoparticles inhibit Gram-positive and Gram-negative bacteria at roughly the same rate. ZnO-NPs were found to have antibacterial activity against Aspergillus terreus (18.00 ± 0.50), Aspergillus niger (19.00 ± 1.00), and Staphylococcus aureus (11.00 ± 0.50) in a similar study [51]. In addition, [52] tested 28 isolates for MIC and MBC. The MIC and MBC of ZnO-NPs were 31.25 µg/mL and 62.5 µg/mL, respectively.
Artemia salina demonstrated enhanced catalase (CAT) activity in a study by [53], implying that NPs’ interaction may alter the equilibrium of aquatic species. They can withstand ambient levels of toxins, notably metal oxides, while less resistant species, such as zebrafish, may suffer if the concentration exceeds Artemia salina’s LC50 [54]. In the toxicity assay, NPs were found to be more effective, with an LC50 value of 55.20 ± 16.19 µg/mL. As said by [55], nanoparticles with an LC50 of less than 100 µg/mL are more potent. Furthermore, at different concentrations, ZnO-NPs demonstrated variable mortality rates against Artemia salina, which is compatible with the findings of [56]. Similarly, previous studies on ZnO-NPs biosynthesized from Ficus racemosa L. leaf extract showed reduced acute toxicity to brine shrimp after 48 hours of treatment [57]. In summary, these data reveal that ZnO-NP derived from C. spinosa aqueous leaf extract has excellent antibacterial, antioxidant, and toxic properties that could be tremendously beneficial in biomedicine. Because the chemistry of NPs is complex, further research is needed to properly understand their potential effects on human health and the environment.
Conclusions
This study describes the green synthesis of ZnO-NPs with C. spinosa leaf extract as a reducing agent. UV-vis, FTIR, and EDX characterizations were carried out to validate the formation of ZnO-NPs. XRD and FE-SEM were used to calculate the average particle size of the nanoparticles. Antioxidant assays revealed strong free radical scavenging activity at higher concentrations, whereas antibacterial studies revealed significant inhibitory effects, particularly against Klebsiella pneumoniae and Staphylococcus aureus, indicating that the nanoparticles inhibit both Gram-positive and Gram-negative bacteria. ZnO-NPs exhibited dose-dependent toxicity against Artemia salina, becoming more toxic at higher doses (1000 µg/mL) and less dangerous at lower levels (10 µg/mL). Certain findings may be comparable to the literature since the plant sample contains similar compounds, but the results vary from previous research for a variety of reasons, including plant species, geography, collection period, sample size, and other factors. All things considered, this study successfully connects plant bioactive chemicals to the creation and functionality of nanoparticles, thereby providing a sustainable and biologically relevant platform for future nanobiotechnology applications.
Future prospects
These findings demonstrate that biogenic ZnO-NPs derived from C. Spinosa have a wide range of potential biomedical uses, promoting green chemistry and sustainable nanotechnology. Thus, future studies should concentrate on increasing synthesis volume, improving reaction conditions, measuring cytotoxicity and in vivo effectiveness, and investigating more untapped therapeutic potentials for producing ecologically friendly nanoparticles.
Acknowledgments
We would like to express our sincere gratitude to the National Herbarium and Plant Laboratories, Godawari, Lalitpur, Nepal, for the identification of the plant. We are grateful to the Nepal Academy of Science and Technology (NAST), Lalitpur, Nepal, for the XRD data. The Institute of Biomolecule Reconstruction at Sun Moon University, Republic of Korea, for supplying the bacterial strains.
References
- 1. Abdelbaky AS, Abd El-Mageed TA, Babalghith AO, Selim S, Mohamed AMHA. Green synthesis and characterization of ZnO nanoparticles using Pelargonium odoratissimum (L.) Aqueous Leaf Extract and Their Antioxidant, Antibacterial and Anti-inflammatory Activities. Antioxidants (Basel). 2022;11(8):1444. pmid:35892646
- 2. Kamaraj C, Gandhi PR, Ragavendran C, Sugumar V, Kumar RCS, Ranjith R, et al. Sustainable development through the bio-fabrication of ecofriendly ZnO nanoparticles and its approaches to toxicology and environmental protection. Biomass Convers Biorefin. 2022;:1–17. pmid:36320445
- 3. Halwani AA. Development of pharmaceutical nanomedicines: From the bench to the market. Pharmaceutics. 2022;14(1):106. pmid:35057002
- 4. Sánchez-López E, Gomes D, Esteruelas G, Bonilla L, Lopez-Machado AL, Galindo R, et al. Metal-based nanoparticles as antimicrobial agents: An overview. Nanomaterials (Basel). 2020;10(2):292. pmid:32050443
- 5. Ahmad W, Kalra D. Green synthesis, characterization and anti microbial activities of ZnO nanoparticles using Euphorbia hirta leaf extract. Journal of King Saud University - Science. 2020;32(4):2358–64.
- 6. Anjum R, Maqsood H, Anwar A, Hussain S, Aleem K, Mohsin S, et al. Evaluation the hepatoprotective effect of quercetin against zinc oxide nanoparticles induced toxicity in mouse model. Biol Clin Sci Res J. 2023;2023(1):246.
- 7. Disha SA, Sahadat Hossain Md, Habib MdL, Ahmed S. Green Synthesis of Nano‐Sized Metal Oxides (Ag2O, CuO, ZnO, MgO, CaO, and TiO2) using plant extract for a sustainable environment. Nano Select. 2025;6(9).
- 8. Nipa ST, Akter R, Raihan A, Rasul SB, Som U, Ahmed S, et al. State-of-the-art biosynthesis of tin oxide nanoparticles by chemical precipitation method towards photocatalytic application. Environ Sci Pollut Res Int. 2022;29(8):10871–93. pmid:34997495
- 9. Akintelu SA, Folorunso AS, Folorunso FA, Oyebamiji AK. Green synthesis of copper oxide nanoparticles for biomedical application and environmental remediation. Heliyon. 2020;6(7):e04508. pmid:32715145
- 10.
Bhardwaj AK, Naraian R, Sundaram S, Kaur R. Biogenic and Non-Biogenic Waste for the Synthesis of Nanoparticles and Their Applications. 1st ed. CRC Press. 2022.
- 11. Hoag GE, Collins JB, Holcomb JL, Hoag JR, Nadagouda MN, Varma RS. Degradation of bromothymol blue by ‘greener’ nano-scale zero-valent iron synthesized using tea polyphenols. J Mater Chem. 2009;19(45):8671.
- 12. Tiwari AK, Jha S, Tripathi SK, Awasthi RR, Bhardwaj AK, Singh AK, et al. Antioxidant response of Calendula officinalis L. assisted synthesized zinc oxide nanoparticles. Mater Res Express. 2024;11(8):085005.
- 13. Koupaei MH, Shareghi B, Saboury AA, Davar F, Semnani A, Evini M. Green synthesis of zinc oxide nanoparticles and their effect on the stability and activity of proteinase K. RSC Adv. 2016;6(48):42313–23.
- 14. Raja A, Ashokkumar S, Pavithra Marthandam R, Jayachandiran J, Khatiwada CP, Kaviyarasu K, et al. Eco-friendly preparation of zinc oxide nanoparticles using Tabernaemontana divaricata and its photocatalytic and antimicrobial activity. J Photochem Photobiol B. 2018;181:53–8. pmid:29501725
- 15. Kamaraj C, Ragavendran C, Naveenkumar S, Al-Ghanim KA, Priyadharsan A, Vetrivel C. Green synthesized yttrium-doped ZnO nanoparticles: A multifaceted approach to mosquito control, antibacterial activity, cytotoxic properties of liver cancer cells, and photocatalytic properties. Inorg Chem Commun. 2025;176:114225.
- 16. Islam MF, Islam S, Miah MAS, Huq AKO, Saha AK, Mou ZJ, et al. Green synthesis of zinc oxide nano particles using Allium cepa L. waste peel extracts and its antioxidant and antibacterial activities. Heliyon. 2024;10(3):e25430. pmid:38333859
- 17. Sivasankarapillai VS, Krishnamoorthy N, Eldesoky GE, Wabaidur SM, Islam MA, Dhanusuraman R, et al. One-pot green synthesis of ZnO nanoparticles using Scoparia Dulcis plant extract for antimicrobial and antioxidant activities. Appl Nanosci. 2022;1–11. pmid:36120603
- 18. Jiang J, Pi J, Cai J. The advancing of zinc oxide nanoparticles for biomedical applications. Bioinorg Chem Appl. 2018;2018:1062562. pmid:30073019
- 19. Almoneef MM, Awad MA, Aldosari HH, Hendi AA, Aldehish HA, Merghani NM, et al. Exploring the multi-faceted potential: Synthesized ZnO nanostructure - Characterization, photocatalysis, and crucial biomedical applications. Heliyon. 2024;10(12):e32714. pmid:39022102
- 20. Dhobale S, Thite T, Laware SL, Rode CV, Koppikar SJ, Ghanekar R-K, et al. Zinc oxide nanoparticles as novel alpha-amylase inhibitors. J Appl Phys. 2008;104(9).
- 21. Sadhukhan P, Kundu M, Rana S, Kumar R, Das J, Sil PC. Microwave induced synthesis of ZnO nanorods and their efficacy as a drug carrier with profound anticancer and antibacterial properties. Toxicol Rep. 2019;6:176–85. pmid:30809470
- 22. Judkevich MD, Gonzalez AM, Salas RM. A new species of randia (Rubiaceae) and the taxonomic significance of foliar anatomy in the species of randia of the southern cone of America. Systematic Botany. 2020;45(3):607–19.
- 23. Liu J-X, Luo M-Q, Xia M, Wu Q, Long S-M, Hu Y, et al. Marine compound catunaregin inhibits angiogenesis through the modulation of phosphorylation of akt and eNOS in vivo and in vitro. Mar Drugs. 2014;12(5):2790–801. pmid:24824025
- 24. Saini H, Dwivedi J, Paliwal H, Kataria U, Chauhan P, Garg R. Anti-inflammatory, analgesic and antipyretic activity of Catunaregam spinosa (Thumb.) tirveng extracts. J Drug Delivery Ther. 2019;9(5):89–94.
- 25. Neamah SA, Albukhaty S, Falih IQ, Dewir YH, Mahood HB. Biosynthesis of zinc oxide nanoparticles using Capparis spinosa L. fruit extract: Characterization, biocompatibility, and antioxidant activity. Appl Sci. 2023;13(11):6604.
- 26. Thavamurugan S, Pavithra SK, Kavipriya MR, Prabha AL. Synthesis of silver nanoparticles using Catunaregam spinosa fruit extract for their biological activities. Biomass Conv Bioref. 2022;15(15):21719–32.
- 27. Lamichhane YR, Parajuli K, Pradhanang SS. Preparation of zinc oxide nanoparticles using Citrus sinensis fruit peel aqueous extract and its antimicrobial study. Sci World. 2024;17(17):99–105.
- 28. Bibi Sadeer N, Montesano D, Albrizio S, Zengin G, Mahomoodally MF. The versatility of antioxidant assays in food science and safety-chemistry, applications, strengths, and limitations. Antioxidants (Basel). 2020;9(8):709. pmid:32764410
- 29. Baliyan S, Mukherjee R, Priyadarshini A, Vibhuti A, Gupta A, Pandey RP, et al. Determination of antioxidants by DPPH radical scavenging activity and quantitative phytochemical analysis of ficus religiosa. Molecules. 2022;27(4):1326.
- 30. Gulcin İ, Alwasel SH. DPPH Radical Scavenging Assay. Processes. 2023;11(8):2248.
- 31. Daoud A, Malika D, Bakari S, Hfaiedh N, Mnafgui K, Kadri A, et al. Assessment of polyphenol composition, antioxidant and antimicrobial properties of various extracts of Date Palm Pollen (DPP) from two Tunisian cultivars. Arab. J Chem. 2019;12(8):3075–86.
- 32. Sarker SD, Nahar L, Kumarasamy Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods. 2007;42(4):321–4. pmid:17560319
- 33. Waghulde S, Kale MK, Patil V. Brine Shrimp lethality assay of the aqueous and ethanolic extracts of the selected species of medicinal plants. Proceedings. (2019);41(1): Article 1.
- 34. Khan I, Saeed K, Khan I. Nanoparticles: Properties, applications and toxicities. Arab J Chem. 2019;12(7):908–31.
- 35. Udayagiri H, Sana SS, Dogiparthi LK, Vadde R, Varma RS, Koduru JR, et al. Phytochemical fabrication of ZnO nanoparticles and their antibacterial and anti-biofilm activity. Sci Rep. 2024;14(1):19714. pmid:39181904
- 36. Tiwari AK, Jha S, Tripathi SK, Shukla R, Awasthi RR, Bhardwaj AK, et al. Spectroscopic investigations of green synthesized zinc oxide nanoparticles (ZnO NPs): Antioxidant and antibacterial activity. Discov Appl Sci. 2024;6(8).
- 37. Harikrishnan A, Ramalingam B, Nadeem A, Ramachandran B, Veena VK, Muthupandian S. Eco-friendly synthesis of zinc oxide nanoparticles (ZnOnps) from Piper betel leaf extract: spectral characterization and its application on plant growth parameters in maize, fenugreek and red gram. Mater Technol. 2023;39(1).
- 38. Masud S, Munir H, Irfan M, Tayyab M. Allium cepa-based zinc oxide nanoparticles: Characterization and biochemical potentials. Bioinspi Biomim Nan. 2023;12(2):82–93.
- 39. Hua J, Vijver MG, Richardson MK, Ahmad F, Peijnenburg WJGM. Particle-specific toxic effects of differently shaped zinc oxide nanoparticles to zebrafish embryos (Danio rerio). Environ Toxicol Chem. 2014;33(12):2859–68. pmid:25244315
- 40. Nabeshi H, Yoshikawa T, Matsuyama K, Nakazato Y, Arimori A, Isobe M, et al. Size-dependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells. Pharmazie. 2010;65(3):199–201. pmid:20383940
- 41. Wingett D, Louka P, Anders CB, Zhang J, Punnoose A. A role of ZnO nanoparticle electrostatic properties in cancer cell cytotoxicity. Nanotechnol Sci Appl. 2016;9:29–45. pmid:27486313
- 42. Vairamuthu S, Sampath S, Sravanthy G, Surya M, Aruliah R, Ali H. Alshehri M, et al. Clove mediated synthesis of Ag-ZnO doped fucoidan nanocomposites and their evaluation of antibacterial, antioxidant and cytotoxicity efficacy study. Results Chem. 2024;11:101783.
- 43. Sampath S, Sunderam V, Madhavan Y, Hariharan NM, Mohammed SSS, Muthupandian S, et al. Facile green synthesis of zinc oxide nanoparticles using Artocarpus hirsutus seed extract: spectral characterization and in vitro evaluation of their potential antibacterial-anticancer activity. Biomass Conv Bioref. 2023;15(4):4969–83.
- 44. Al Rahbi AS, Al Mawali AH, Al Rawahi SS, Al Dighishi RK, Al Abri FA, Ahmed A, et al. Green synthesis of zinc oxide nanoparticles from Salvadora persica leaf extract: Characterization and studying methyl orange removal by adsorption. Water Pract Technol. 2024;19(4):1219–31.
- 45. Alamdari S, Sasani Ghamsari M, Lee C, Han W, Park H-H, Tafreshi MJ, et al. Preparation and characterization of zinc oxide nanoparticles using leaf extract of sambucus ebulus. Appl Sci. 2020;10(10):3620.
- 46. C FC, T K. Advances in stabilization of metallic nanoparticle with biosurfactants- a review on current trends. Heliyon. 2024;10(9):e29773. pmid:38699002
- 47. Acharya R, Tettey F, Gupta A, Sharma KR, Parajuli N, Bhattarai N. Bioinspired synthesis and characterization of zinc oxide nanoparticles and assessment of their cytotoxicity and antimicrobial efficacy. Discov Appl Sci. 2024;6(3).
- 48. Ragavendran C, Kamaraj C, Alrefaei AF, Priyadharsan A, de Matos LP, Malafaia G, et al. Green-route synthesis of ZnO nanoparticles via Solanum surattense leaf extract: Characterization, biomedical applications and their ecotoxicity assessment of zebrafish embryo model. S Afr J Bot. 2024;167:643–62.
- 49. Islam F, Shohag S, Uddin MJ, Islam MR, Nafady MH, Akter A, et al. Exploring the Journey of Zinc Oxide Nanoparticles (ZnO-NPs) toward biomedical applications. Materials (Basel). 2022;15(6):2160. pmid:35329610
- 50. Sarwar K, Nazli Z-I-H, Munir H, Aslam M, Khalofah A. Biosynthesis of zinc oxide nanoparticles using Moringa oleifera leaf extract, probing antibacterial and antioxidant activities. Sci Rep. 2025;15(1):20413. pmid:40595209
- 51. Yagoub AEA, Al-Shammari GM, Al-Harbi LN, Subash-Babu P, Elsayim R, Mohammed MA, et al. Antimicrobial properties of zinc oxide nanoparticles synthesized from lavandula pubescens shoot methanol extract. Appl Sci. 2022;12(22):11613.
- 52. Kakian F, Mirzaei E, Moattari A, Takallu S, Bazargani A. Determining the cytotoxicity of the Minimum Inhibitory Concentration (MIC) of silver and zinc oxide nanoparticles in ESBL and carbapenemase producing Proteus mirabilis isolated from clinical samples in Shiraz, Southwest Iran. BMC Res Notes. 2024;17(1):40. pmid:38287416
- 53. Bhuvaneshwari M, Sagar B, Doshi S, Chandrasekaran N, Mukherjee A. Comparative study on toxicity of ZnO and TiO2 nanoparticles on Artemia salina: effect of pre-UV-A and visible light irradiation. Environ Sci Pollut Res Int. 2017;24(6):5633–46. pmid:28039626
- 54. Casiano-Muñiz IM, Ortiz-Román MI, Lorenzana-Vázquez G, Román-Velázquez FR. Synthesis, characterization, and ecotoxicology assessment of zinc oxide nanoparticles by in vivo models. Nanomaterials (Basel). 2024;14(3):255. pmid:38334526
- 55. R. Hamidi M, Jovanova B, Kadifkova Panovska T. Toxicological evaluation of the plant products using Brine Shrimp (Artemia salina L.) model. Maced Pharm Bull. 2014;60(01):9–18.
- 56. Gomes AR, de Matos LP, Guimarães ATB, Freitas ÍN, Luz TM da, Silva AM, et al. Plant-ZnO nanoparticles interaction: An approach to improve guinea grass (Panicum maximum) productivity and evaluation of the impacts of its ingestion by freshwater teleost fish. J Hazard Mater. 2023;451:131173. pmid:36924744
- 57. Ragavendran C, Kamaraj C, Jothimani K, Priyadharsan A, Anand Kumar D, Natarajan D, et al. Eco-friendly approach for ZnO nanoparticles synthesis and evaluation of its possible antimicrobial, larvicidal and photocatalytic applications. Sustain Mater Technol. 2023;36:e00597.