Alleviation of salinity stress by ternary Se/CuO/ZnO nanocomposite synthesis by Urtica urens biomass and their antioxidant, cytotoxic properties

Selim , S., Alammari, B. S., Mohamed, M. Y. A., Alsalamah, S. A., Alsrhani, A., Kashmery, H. A., Saddiq, A. A., Baeshen, A. A., Aljeddani, G. S., and Amin, M. A.-A. (2026). "Alleviation of salinity stress by ternary Se/CuO/ZnO nanocomposite synthesis by Urtica urens biomass and their antioxidant, cytotoxic properties," BioResources 21(3), 7433–7453.

Abstract

Salinity stress is a major environmental constraint that adversely affects plant growth and their productivity. In this work, ternary selenium/copper oxide/zinc oxide nanocomposite (Se/CuO/ZnO NCs; TSCZ NCs) were synthesized for the first time using aqueous leaf extract of Urtica urens and evaluated for their ability to alleviate salinity stress in Eruca sativa plants. The synthesized nanoparticles were characterized using TEM, SAED, EDX, and XRD analyses, which confirmed the formation of predominantly spherical crystalline nanoparticles (average size of 58.71 ± 3.58 nm) and distinct elemental composition of Se, CuO, and ZnO. A pot experiment was conducted under normal and saline conditions (100 mM NaCl) using different concentrations of TSCZ NCs (up to 200 ppm). Salinity stress significantly reduced shoot and root growth, photosynthetic pigments, carbohydrate and protein contents, while increasing proline accumulation, antioxidant enzyme activities, and lipid peroxidation. Foliar application of TSCZ NCs partially mitigated these adverse effects, particularly at 100 ppm. The nanoparticles also modulated antioxidant defense enzymes in treated plants. In addition, TSCZ NCs exhibited strong antioxidant activity in the DPPH assay, reaching 89.4% scavenging at 1000 µg/mL, and demonstrated low cytotoxicity toward Vero normal cell lines. These findings suggest that Se/CuO/ZnO NCs may represent a promising nano-enabled strategy for improving salinity tolerance in plants.

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**Alleviation of Salinity Stress by Ternary Se/CuO/ZnO Nanocomposite Synthesis by Urtica urens Biomass and their Antioxidant, Cytotoxic Properties**

Samy Selim ,^a,* Badriah Saleh Alammari ,^bMarwa Yousry A. Mohamed,^bSulaiman A. Alsalamah,^b Abdullah Alsrhani,^aHeba A. Kashmery,^c Amna A. Saddiq ,^d Areej Ali Baeshen,^d Ghalia Saleem Aljeddani,^e Mohamed A. Amin ^f,*

DOI: 10.15376/biores.21.3.7433-7453

Keywords: Ternary nanoparticles; Urtica urens; Salinity; Antioxidant; Cytotoxicity activity

Contact information a: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka, Saudi Arabia; b: Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), P. O. Box: 90950, Riyadh 11623, Kingdom of Saudi Arabia; c: Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; d: Department of Biological Sciences, College of Science, University of Jeddah, Jeddah, Saudi Arabia; e: Department of Environmental Sciences, College of Science, University of Jeddah, Jeddah, Saudi Arabia; f: Botany and Microbiology Department, Faculty of Science (Boys), Al-Azhar University Cairo 11884, Egypt;

Corresponding authors: sabdulsalam@ju.edu.sa (S. S.), mamin7780@azhar.edu.eg (M.A.A.)

INTRODUCTION

The easiest way to appreciate the complexity of the abiotic global challenges in agricultural systems is to consider salinity (Devi et al. 2026). Water scarcity combined with the challenges of salinity in agricultural systems is a major constraint on productive capacity of agriculture (Petkova et al. 2019). The situation is critically important for crop productivity because non-salinized soil has been regarded as a prerequisite for irrigated land, and salinity is projected to affect more than 50% of irrigated land by 2050 (Khondoker et al. 2023). Salt can either directly or indirectly impact the growth and yield of crops in saline land. The first mechanism is that osmotic and water deficit salinity slows down the growth of crops as a result of slowing down the capacity of roots to take up water. The second mechanism is that the accumulation of salt in the leaf can negatively impact transpiration and increase the salinity level of the leaf, making leaf transpiration less active and thereby slowing down growth further (Hailu and Mehari 2021). Three major aspects of salt stress are: (a) reduction of water potential, (b) imbalance of ions resulting in perturbations of homeostasis, and (c) toxic ions (Arif et al. 2020). Salt stress impacts crops at different levels such as growth, biomass, and yield; it also impacts many physiological processes. Such damage leads to lower crop yields (Bhattacharya 2021; Balasubramaniam et al. 2023; Abid et al. 2026). Recent research has demonstrated that the effects of salt stress on different plant species can be mitigated by nanotechnology (Abid et al. 2026). Materials with dimensions between one nanometer and one hundred nanometers are referred to as nanoparticles. When compared to their bulk-sized counterparts, these materials are unique (Yadav et al. 2026). A nanocomposite is a multiphase solid in which nanoscale fillers (such as carbon nanotubes, clays, or metal oxides) are used to reinforce a bulk matrix (such as polymer, ceramic, or metal). These materials can significantly increase mechanical, thermal, electrical, and barrier properties at low filler loadings by utilizing fillers smaller than 100 nm (Amin et al. 2024).

The present study used nanocomposite of selenium/copper oxide/zinc oxide, which selenium is known to improve ROS scavenging and preserve membrane integrity (Liu et al. 2022), Plant-mediated reduction of selenium oxyanions (such as selenite or selenate) into elemental selenium (Se⁰) is known to result in selenium nanoparticles (Se NPs). Naturally adsorbed biological macromolecules (such proteins and polysaccharides) that connect via covalent thiol bonds and non-covalent forces significantly stabilize them (Zambonino et al. 2023). Zinc and copper are necessary cofactors for numerous antioxidant and photosynthetic enzymes (Osman et al. 2026). Excess Na⁺ causes oxidative burst and ionic imbalance in saline environments, which results in membrane lipid peroxidation, chlorophyll degradation, and metabolic inhibition. By boosting antioxidant defense systems (such as CAT, POX, and PPO), lowering ROS buildup and MDA formation, stabilizing photosynthetic pigments, and promoting osmolyte control such as proline metabolism (Chakraborty et al. 2018), foliar-applied ternary Se/CuO/ZnO nanocomposite may mitigate these impacts (Bhati et al. 2026). Furthermore, compared to traditional mineral salts or crude plant extracts, nanoparticles may show better leaf penetration and regulated micronutrient release, resulting in longer-lasting physiological advantages (Khan et al. 2026). The oxidation state of selenium is particularly important because, according to earlier research, partial surface oxidation to SeO_x may contribute to progressive selenium bioavailability, but soluble Se(IV) species are typically less phytotoxic and more stable than Se₃ nanoparticles (Burmistrov et al. 2025). The main component of selenium nanoparticles (Se NPs) is elemental selenium in the zero-oxidation state (Se⁰). Their distinct biocompatibility and antioxidant action are based on this zero-valent state, which offers substantially less toxicity than harmful inorganic forms like selenite (Se⁺⁴) or selenate (Sampath et al. (2024). A biogenic ZnO/CuO/Se nanocomposite with an average size of 40.9 to 50.2 nm demonstrated strong anti-H. pylori activity and strong antioxidant properties (Alharbi et al. 2025).

Among all leafy vegetables enjoyed by humans globally, rocket (Eruca sativa) has a prominent position (Tripodi et al. 2025). Salt is an important environmental constraint that limits both vegetative growth and productivity, and this species shows specific responses to its effects (Cavalcante et al. 2025). In the literature regarding the other crops, the responses to the application of Se, Cu, and Zn nanoparticles have drawn a lot of interest concerning their growth- stimulating and stress alleviating effects (Pérez-Labrada et al. 2019; Mogazy and Hanafy 2022; Mishra et al. 2025). In regard to salinity stress, little is known of the effects of the combination of Se/CuO/ZnO in the form of a combined nanoparticle preparation for use as a soil amendment for rocket plants. It has not been resolved sufficiently how such systems might regulate growth and photosynthetic capacity, osmotic regulation, and oxidative stress. By combining these actions, unrecognized routes of stress mitigation could be revealed, and the scope for using nanoscale solutions for the sustainable cultivation of vegetables could be expanded. There has been growing interest in the use of nanoscale solutions for the mitigation of abiotic stress; however, the application of selenium-based/copper/zinc oxide nanoparticles in saline environments remains largely unexplored (Al-Khayri et al. 2023; Ochoa-Chaparro et al. 2026). In particular, the responses of this system to growth, the control of photosynthesis, osmotic regulation, and oxidative stress in the species rocket (Eruca sativa) has not been studied in detail. Therefore, the current study focused on the use of Urtica urens leaf extract for the biosynthesis of ternary Se/CuO/ZnO nanocomposite (TSCZ NCs) and the exposure of these nanoparticles to salinity stress to study the effects of these nanoparticles on the morphology, physiology, biochemistry, and stress mitigation in terms of their antioxidant activity and cytotoxicity.

EXPERIMENTAL

Materials and their Preparation

Urtica urens leaves were gathered, rinsed three times with tap water to get rid of loose or water-soluble contaminants, left to air dry at room temperature, and then ground into a fine powder using a lab grinder to get uniform sizes. To create the extract, 10 g of the resultant powder was mixed with 100 mL of distilled water and shaken at 150 rpm for 60 minutes at 50 °C. After that, the mixture was centrifuged to create a transparent supernatant that was used as a catalyst to create ternary Se/CuO/ZnO NCs.

Green Synthesized TSCZ NCs

In order to create TSZC NCs, 5 mM of solutions (Na₂SeO₃, Zn (CH₃COO)₂·2H₂O, and Cu (CH₃COO)₂‧H₂O) were mixed at 1:1:1 proportion with the gathered aqueous extract (3:1, v/v) at pH 8 while stirring at 70 °C for 60 min done. During this, the plant extract is used to reduce metal precursors and create NPs, a reddish-brown hue appears. The evaporation of the resulting solution followed by a wash with deionized water and alcohol allowed to get rid of any contaminants, the residue was recovered. The collected the reddish-brown powder residue was then annealed for two hours at 200 °C. This powder was then kept in a falcon tube for the next procedures.

Characterization of TSCZ NCs

Utilizing transmission electron microscopy (JEOL-1010-TEM, Tokyo, Japan), the morphological characteristics (sizes and forms) of the produced NPs were identified. To identify the crystalline or amorphous structure, the SAED (Selected area electron diffraction) pattern was also gathered. Additional analysis using JSM6360LA energy dispersive X-ray diffraction (SEM-EDX, JEOL, Japan) with an acceleration voltage of 5 kv, revealed the chemical makeup of NPs. Also, XRD analysis was also performed.

Growth Conditions

This study employed rocket (Eruca sativa) plant seeds that had been thoroughly washed with sterile distilled water under aseptic circumstances after being sterilized on the surface for 15 min using 0.1% sodium hypochlorite (NaOCl). Eight treatment combinations with two salinity levels (0 and 100 mM NaCl) and four concentrations of selenium/copper oxide/zinc oxide nanocomposite (TSCZ NCs) at 0, 50, 100, and 200 ppm were used in the experiment. Five replications of each treatment were conducted. Each plastic pot held 6.0 kg of sandy loam soil and was filled with sterilized seeds. Each pot was seeded with ten seeds. After the seedlings, each pot was thinned and five healthy plants were kept. Forty-five days after seeding, morphological and biochemical investigations were performed.

Plant Analysis Techniques

Photosynthetic pigment determination

Fresh leaves were treated with 80% acetone to extract photosynthetic pigments, which were then measured spectrophotometrically at 470, 649, and 665 nm (Vernon and Seely 2014). The Smith (2013) method was used to calculate carotenoids and chlorophylls.

Metabolic contents estimation

The anthrone-sulfuric acid method (Umbriet et al. 1959) was used to measure total soluble carbohydrates, and the Bio-Rad test (Lowery 1951) was used to measure soluble proteins. A UV spectrophotometer was used for the measurements.

Free proline determination

In accordance with Bates et al. (1973), free proline was measured using the acid ninhydrin reaction and sulfosalicylic acid extraction. Following toluene phase separation, absorbance was measured at 520 nm.

Lipid peroxidation evaluation

Malondialdehyde (MDA) was measured using the thiobarbituric acid technique in order to evaluate lipid peroxidation (Zhang et al. 2015). MDA was reported as µmol g⁻¹ FW, and absorbance was measured at 450, 532, and 600 nm.

Enzymatic antioxidant activity assays

According to Mukherjee and Choudhuri (1983), the antioxidant enzymes were extracted from young leaves and terminal buds using phosphate buffer and centrifugation at low temperature. The supernatant served as the crude enzyme extract for enzyme assays.

Catalase

By tracking the breakdown of H₂O₂ at 240 nm for 60 seconds, catalase activity was determined. Under controlled reaction circumstances, activity was measured in accordance with Aebi (1983).

Peroxidase

The increase in absorbance at 470 nm in the presence of H₂O₂ was used to measure peroxidase activity. The assay was carried out in accordance with Bergmeyer et al. (1974).

Polyphenol oxidase

Using catechol as the substrate, polyphenol oxidase activity was assessed by measuring absorbance at 395 nm following incubation (Kar and Mishra 1976). The rate of change in optical density per gram of fresh weight was used to express enzyme activity.

DPPH test and cytotoxicity for TSCZ NCs

0.96 mL of the DPPH solution was combined with varying amounts of TSCZ NCs (1.95–1000 µg/mL). A UV-Vis spectrophotometer (PerkinElmer, Germany) was used to quantify the decrease in absorbance at 520 nm. Using the MTT assay method, the cytotoxicity of TSCZ NCs was examined in relation to Vero cells, a mammalian cell line.

Statistical Analysis

Data were analyzed using one-way ANOVA followed by Tukey’s test in SPSS software. Differences were considered significant at p ≤ 0.05 (Nowwar et al. 2023).

RESULTS AND DISCUSSION

Ternary selenium/copper oxide/zinc oxide nanocomposite (TSCZ NCs) mediated by Urtica urens mostly showed a spherical and semi-spherical particle morphology when viewed under TEM. The average size of the nanoparticles was 58.71±3.58 nm, as illustrated in Fig. 1a and 1b.

Fig. 1. Characterization of green-synthesized TSCZ NCs: The TEM and size distribution are displayed in A and B, and the SAED analysis for crystallinity detection is displayed in C.

The size of nanoparticles and morphology are critical parameters that define the physicochemical properties, biological activity, and scope of application. A comparable study used TEM to characterize spherical ternary Au/ZnO/CuO NPs (35 nm) prepared by aqueous extract of Verbena officinalis (Dobrucka et al. 2021).

The crystallinity of NPs can be assessed by the Selective Area Electron Diffraction (SAED) which is a technique used in electron microscopy. The concentric circular dark fringes observed in the SAED image of the TSCZ NCs, demonstrated the crystalline nature of the sample and the presence of the CuO, Se, and ZnO crystalline domains (Fig. 1c). Similar SAED results of the ternary ZnO/Se/CuO NPs synthesized by fungi revealed 8 concentric rings which were attributed to the crystalline structure of the tri-metallic elements (Hashem et al. 2023).

The elemental composition of the TSCZ NCs was confirmed by EDX spectroscopy, revealing the presence of selenium (Se) and the copper (Cu) and zinc (Zn) as well as oxygen (O). The main peaks for the TSCZ NCs, illustrated in Fig. 2, employees these elements. The Se, Cu, and Zn were found to be present in the decreasing order of Se> Cu>Zn. The atomic percent composition of TSCZ NCs showed Se, Cu, and Zn to be present at 14.2%, 10.3%, and 5.8 % respectively. The weight percent composition showed Se, Cu and Zn to be present at 34.7%, 20.2%, and 11.7%, respectively. A strong presence of oxygen was recorded at 58.9 % in atomic percent and at 29.2% in weight percent. This is tentatively attributed to the absorption of oxygen from the environment to the nanoparticles surface, along with the formation of cupric/copper and zinc oxides. The presence of carbon was attributed to the binding of proteins to the NCs surfaces, as these biomolecules are significant in stabilizing and capping the produced NCs (Kunwar et al. 2023).

Fig. 2. EDX analysis of TSCZ NCs

The XRD pattern and peak assignments for the synthesized TSCZ NCs are shown in Fig. 3 and Table 1. XRD analysis revealed the variation in crystallite size for the different structures. For the ZnO phase, which is based on of the (100), (002), and (101) reflections, the average crystallite size was 20.1 ± 3.2 nm. For the copper oxide phase, which consists of the (111) and (200) reflections, the average crystallite size was 16.4 ± 2.8 nm. The Se-phase crystallite size was the smallest and was measured to be 13.7 ± 3.5 nm. The observed order of Se < CuO < ZnO may suggest that each of these phases nucleate and grow in a different way during the synthesis. However, without supporting studies on kinetics and surface chemistry, such a hypothesis is entirely speculative. Line broadening studies for the different phases showed small micro-strain in the lattice. The primary contributors to the line broadening for each of the phases were crystallite size and instrumental broadening

Table 1. Properties of XRD Analysis of TSCZ NCs

Fig. 3. XRD analysis of TSCZ NCs

Growth Parameters of Rocket Plant Under Salinity Stress

Elements like Se, Cu, and Zn, which may be deemed beneficial or essential, can be embedded in nanoparticles. These particles can promote positive alterations to stress response mechanisms in plants (Al-Khayri et al. 2023). Under salinity, CuO and ZnO NPs improved the growth of soybean, rice, and lemon balm (Ghasemian et al. 2021; Mishra et al. 2025; Chen et al. 2026).

Table 2 illustrates how salinity stress significantly reduced shoot and root lengths, shoot weight (dry and fresh), and root lengths as compared to the non-saline control. In contrast, when stressed plants were treated with 50, 100, and 200 ppm ternary Se/CuO/ZnO NCs, all the growth parameters showed highly significant improvement. Similar results were reported by Alamri et al. (2026) for oats, Riaz et al. (2026) for wheat, and Taj et al. (2024) for spinach. Selim et al. (2025) also investigated the biostimulant effect of bimetallic Se/ZnO NPs at 0, 50, 100, and 200 ppm on faba bean plants, and they reported that the treatment of bimetallic Se/ZnO NPs at 100 ppm resulted in the best values for root and shoot lengths, as well as shoot fresh and dry weights compared to the control.

Table 2. Effects of Ternary Se/CuO/ZnO NCs on Morphological Parameters of Rocket Plant Under Salinity Stress

Data represents means ± standard error (n=10). Different lowercase letters in the same species within columns indicate significant differences (P ≤ 0.05). HSD is honestly significant difference by post hoc-Tukey’s test. S is salinity.

Photosynthetic Pigments

Rocket plants exposed to salt stress, unlike the control plants, showed significant depletion in total chlorophyll (a and b), total carotenoids. and total chlorophyll (b), as indicated in Fig. 4. From a mechanistic point of view, these losses are understood as at saline conditions, there is an increase in activity of chlorophyllase and destruction of chloroplasts (Fatahiyan et al. 2025). Similar results were reported in maize (Saravanan et al. 2026) and in lettuce (Kiremit et al. 2026).

In contrast to the stressed plants that were not treated, the application of TSCZ NCs increased total carotenoids, total pigments, and chlorophyll (a and b). When compared to the untreated stressed plants, the stressed rocket plants at 100 ppm of TSCZ NCs showed the greatest increase in total chlorophyll (a and b) and total carotenoids under salt. This increase in total chlorophylls and carotenoid may have resulted from the stability of the photosynthetic machinery by selenium and the activation of chlorophyll biosynthesis enzymes encouraged by copper and zinc (Rodríguez et al. 2018; Liu et al. 2022; Ochoa-Chaparro et al. 2026). Increased photosynthetic efficiency and biomass recovery are also made possible by pigment recovery. This was also noted in tomatoes (Hernández-Hernández et al. 2019) and rice (Mishra et al. 2025). Additionally, Alammari (2025) investigated the effects of exogenous Se NPs and Se/CuO NPs on salt-stressed maize and discovered that both enhanced the levels of carotenoids, chlorophyll (a), chlorophyll (b), and total chlorophyll (a + b) while reducing the detrimental effects of stress.

Fig. 4. The effect of Se/CuO/ZnO NCs on rocket plants under salt stress in terms of carotenoid levels (mg/g fresh weight) and chlorophylls (a, b, and a+b). The data shows the mean ± standard error (n=3). In the same bars, different lowercase letters denote significant differences (P < 0.05). According to the post hoc Tukey’s test, HSD is an honestly significant difference. Salinity is represented by S.

Metabolic Contents

Under salinity stress, Rocket plants had significantly fewer primary metabolites (Fig. 5). A significant response to osmotic stress was shown by an 184% increase in free proline and 80% and 79% drop in protein and carbohydrate levels, respectively. According to Dichio et al. (2025), proline functions as a buffer to preserve cell turgor in this way, demonstrating adaptation to osmotic stress. These data are in agreement with Singh et al. (2025) and Rizzoli et al. (2025).

On the other hand, use of TSCZ NCs at different concentrations improved the biochemical makeup of plants. Under stressful conditions, plant treated with 100 ppm Se/CuO/ZnO NCs showed the biggest increase in carbohydrate contents by approximately 50%. TSCZ NCs at 200ppm showed the highest increase in protein contents by about 39% more than salt stress plants. On the other hand, by lowering proline buildup under stress by almost 91% compared to salt stress plant, 50 ppm Se/CuO/ZnO NCs showed osmotic stress mitigation. These outcomes show that these treatments were effective in reducing osmotic stress. These results are consistent with the findings that the relationship between Zn and Se enhances protein and sugar metabolism under stress (Gao et al. 2025). In another study, copper and zinc have been connected to the control of carbohydrate and protein synthesis (Sherefu and Zewide (2021). These data are also in agreement with Seleiman et al. (2023) in maize; El-Badri et al. (2021) in rapeseed and González-García et al. (2021) in bell pepper plants.

Fig. 5. Effects of Se/CuO/ZnO NCs on carbohydrate, protein, and proline contents (mg/g dry weight) of rocket plant under salinity stress

Enzymes Activities

This study shows a marked increase in the CAT, POX, and PPO enzymes in the rocket plant salt stress experiments (Fig. 6). The increased activity of antioxidant enzymes occurs as a defensive measure to increased levels of reactive oxygen species (ROS) in stressful conditions (Sachdev et al. 2021). These results are very similar to the most recent research by Osman et al. (2026) on faba beans and Shafiei et al. (2026) on cabbages.

Fig. 6. Effects of Se/CuO/ZnO NCs on catalase (CAT), peroxidase (POX), and polyphenol oxidase (PPO) enzymes activities (unit/g. F.wt./hour) of rocket plant under salinity stress.

On the contrary, the Se/CuO/ZnO NCs, administered in the absence of salt stress, caused a small increase in antioxidant enzymes, which was not significantly different from the control. Within the different treatments, the Se/CuO/ZnO NCs caused the least amount of ROS of the salt-stressed plants, which suggests that the ROS levels in the plants should be very low. As evidenced in the works of Hasanuzzaman et al. (2024), Se, especially in nanoparticulate form, can enhance peroxidase enzyme activity as a control mechanism for ROS and assist in their detoxification. The results of the present experiments are similar to the previous experimental study which suggests that the Se, CuO, ZnO in nanoscale can control the enzyme activities and assist in the reduction of ROS in several varieties of crops exposed to salt stress as reported by Elsheery et al. (2025) on faba beans, Mishra et al. (2025) on rice, and Ghasemian et al. (2021) on Melissa officinalis plants.

Malondialdehyde Contents

Rocket leaves under salt stress collected a higher quantity of malondialdehyde (MDA) than the control (Fig. 7). Salt buildup in a cell’s cytoplasm is a sign of ionic imbalance and hyperosmotic stress, both of which encourage the production of reactive oxygen species (ROS). As a result, membranes undergo oxidative peroxidation, which raises the MDA concentration (Ahmad et al. 2019). Other researchers have reported similar findings on other plant species: Tebini et al. (2025) reported comparable findings in Amaranthus caudatus treated to similar saline stress, while Khan et al. (2026) discovered higher levels of MDA in salt-stressed soybean.

Fig. 7. Effects of TSCZ NCs on malondialdehyde (MDA) contents (μmols/g. F.wt.) of rocket plant under salinity stress. Data represents means ± standard error (n=3). Different lowercase letters in the same bars indicate significant differences (P ≤ 0.05). HSD is honestly significant difference by post hoc-Tukey’s test. S is salinity.

By lowering MDA and lipid peroxidation, TSCZ NCs helped salinized plants experience less oxidative stress. The rocket plants benefited from the application of TSCZ NCs because it also increased antioxidant enzyme activity, which quickly eliminated ROS and decreased oxidative and salinity stress on the plants, improving plant growth. These results were consistent with those of Mishra et al. (2025), who found that applying Se and ZnO NPs together greatly decreased salt stress in rice plants, enhancing their antioxidant defense and capacity to eliminate ROS. Lipid peroxidation also dramatically decreased during the trial. The use of Se, CuO, ZnO NPs in saline conditions also supported the findings of Talat et al. (2026) in mung bean, Alharbi et al. (2025) in cowpea, and Seleiman et al. (2023) in maize where the use of the nanoparticles reduced oxidative stress in the plants.

Antioxidant Properties of TSCZ NCs

Free radicals, often known as ROS, are unstable molecules that can function on their own. They are produced in the human body by oxidation processes or by exposing cells to harmful chemicals like radiation, toxins, and other compounds. The main constituents of cells, including DNA, amino acids, proteins, and lipids, are negatively impacted by this ROS. Finding new antioxidant molecules is therefore crucial to mitigating the negative effects of ROS. Here, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging, a common antioxidant assay technique, was used to assess the antioxidant capacity of biogenic TSCZ NCs. Instead of utilizing ascorbic acid, DPPH scavenging (%) was used to assess the antioxidant properties of TSCZ NCs (Table 3). With TSCZ NCs concentrations ranging from 1.95 to 1000 μg/mL, the DPPH radical efficiency rose dose-dependently from 17.7% to 89.4%. Another study on biogenic ternary Cu/Ag/Zn appeared that the scavenging rates were 68.3%, 37.3%, and 35.7% at concentrations of 1000, 500, and 50 µg mL^-1(Kunwar et al. 2023).

Table 3. DPPH Assessment of TZCS NCs

Cytotoxicity of TSCZ NCs against Vero Cells

MTT assays were performed on Vero cells in order to determine whether TSCZ NCs exhibit very low cytotoxicity against normal cell lines. Results are shown in Table 4 and Fig. 8. The proliferation of the Vero cell line showed an inverse correlation with the nanoparticle concentration: at low concentrations the proliferation increased, whereas the proliferation decreased at high concentrations. Even at the highest concentration of 1000 µg mL^–1, the cell viability was 89.49%, and significantly increased with decreasing concentrations to 99.72% and 96.23% at 125 µg mL^–1 and 250 µg mL^–1, respectively. These results, in the context of cytotoxicity, demonstrate that the synthesized TSCZ NCs did not affect normal cell line proliferation at the tested concentrations (250 and 500 µg mL^–1). In comparison, Amin et al. (2025) investigated the Se/ZnO/CuO NPs from Nitraria retusa and reported an IC50 on the WI38 normal cell line to be 294.9 ± 4.4 µg mL^–1.

Table 4. Cytotoxicity of TSCZ NCs against Vero Cells

Fig. 8. Cytotoxicity of TSCZ NCs against Vero Cells

CONCLUSIONS

1. Successful green synthesis of biogenic ternary Se/CuO/ZnO nanocomposite by the leaf extract of Urtica urens was achieved. Structural characterization confirmed the formation of mainly spherical crystalline nanoparticles containing selenium, copper, zinc and oxygen associated phases.

2. Salinity stress had a significant inhibitory effect on the growth, photosynthetic pigments, metabolic constituents and oxidative balance of rocket (E. sativa) plants. However, foliar application of TSCZ NCs partially alleviated these negative effects through enhancing plant growth, restoring chlorophyll and carotenoid contents, improving carbohydrate and protein accumulation and reducing lipid peroxidation under saline conditions.

3. The observed modulation of antioxidant enzyme activities further suggests that TSCZ NCs contributed to improved oxidative-stress management in salt-stressed plants. The concentration-dependent response was observed as 100 ppm generally caused the most consistent physiological improvements among the tested concentrations under salinity stress.

4. Furthermore, the synthesized nanoparticles exhibited strong antioxidant activity and low cytotoxicity against Vero normal cell lines in the tested concentration range.

5. The results confirm the potential use of biogenic nanocomposite as nano-enabled plant bio stimulants for enhancing salinity tolerance. Nevertheless, additional studies are still needed to clarify the nanoparticle uptake, long-term phytotoxicity, environmental safety, and applicability at field scale before large-scale agricultural implementation.

ACKNOWLEDGEMENTS

Funding

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2601).

REFERENCES CITED

Abid, H., Ahmad, K. S., Shehzad, M. A., Mehmood, A., Habib, D., and Nazir, S. (2026). “Alleviation of salt, drought, nutrient, and temperature stresses in plants through nanoparticles,” in: Emerging Nanotechnologies for Agroecosystem Management, pp. 127-163. Springer. https://doi.org/10.1007/978-981-95-0187-8_5

Aebi, H. E. (1983). “Catalase,” in: Methods of Enzym Analysis 105, 121-126.

Ahmad, R., Hussain, S., Anjum, M. A., Khalid, M. F., Saqib, M., Zakir, I., Hassan, A., Fahad, S., and Ahmad, S. (2019). “Oxidative stress and antioxidant defense mechanisms in plants under salt stress,” in: Plant Abiotic Stress Tolerance: Agronomic, Molecular and Biotechnological Approaches, 191-205. https://doi.org/10.1007/978-3-030-06118-0_8

Al-Khayri, J. M., Rashmi, R., Surya Ulhas, R., Sudheer, W. N., Banadka, A., Nagella, P., Aldaej, M. I., Rezk, A. A., Shehata, W. F., and Almaghasla, M. I. (2023). “The role of nanoparticles in response of plants to abiotic stress at physiological, biochemical, and molecular levels,” Plants 12(2), article 292. https://doi.org/10.3390/plants12020292

Alammari, B. S. (2025). “Physiological and biochemical processes in the exogenous administration of selenium nanoparticles and selenium/copper oxide nanocomposite to reduce salt stress in Zea mays L,” BioResources 20(4), 10487-10503. https://doi.org/10.15376/biores.20.4.10487-10503

Alamri, A., Abdel-Maksoud, M. A., Almutairi, S., Algarzae, N. K., Alhaj Hamoud, Y., and Maridueña-Zavala, M. G. (2026). “Selenium nanoparticles enhance innate immunity in aphid-stressed oat plants by modulating photosynthetic pigments, sugar, nitrogen and fatty acid metabolism, and antioxidant defense,” BMC Plant Biology 26, 233(2026). https://doi.org/10.1186/s12870-025-08036-x

Alharbi, K., Desoky, E.-S. M., Almuziny, M., Abuzaid, A. O., Abu-Elsaoud, A. M., Algopishi, U. B., Serag, A. M., El-Saadony, M. T., Mathew, B. T., and El-Tarabily, K. A. “et al.” (2025). “Foliar-applied selenium nanoparticles improve antioxidant defense and photosynthetic to enhance salt stress tolerance in cowpea (Vigna unguiculata L.),” Scientia Horticulturae 353, article 114454. https://doi.org/10.1016/j.scienta.2025.114454

Alharbi, M. T., Al-Rajhi, A. M., Almuhayawi, M. S., Gattan, H. S., Alruhaili, M. H., Ali, Y., and Selim, S. (2025). “Nanocomposites of ZnO/CuO/Se synthesized by Schinus terebinthifolia biomass and their antioxidant, cytotoxicity, anti-h. pylori, and anti-obesity properties,” BioResources 20(2), 4432-4449.‏ https://doi.org/10.15376/biores.20.2.4432-4449

Amin, M. A., Abdelghany, T. M., and Nowwar, A. I. (2024). “Silver nanocomposite for improved plant-soil system and underlying mechanism, plant response to silver nanoparticles,” in: Plant Growth, Development, Production, and Protection, Springer Nature, Singapore, pp. 93-114.‏ https://doi.org/10.1007/978-981-97-7352-7_6

Amin, M. A., Algamdi, N. A., Waznah, M. S., Bukhari, D. A., Alsharif, S. M., Alkhayri, F., Abdel-Nassar, M., and Fouda, A. (2025). “An insight into antimicrobial, antioxidant, anticancer, and antidiabetic activities of ternary Se/ZnO/CuO nanoalloys fabricated by aqueous extract of Nitraria retusa,” Journal of Cluster Science 36(1), article 19.‏ https://doi.org/10.1007/s10876-024-02742-6

Arif, Y., Singh, P., Siddiqui, H., Bajguz, A., and Hayat, S. (2020). “Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance,” Plant Physiology and Biochemistry 156, 64-77. https://doi.org/10.1016/j.plaphy.2020.08.042

Balasubramaniam, T., Shen, G., Esmaeili, N., and Zhang, H. (2023). “Plants’ response mechanisms to salinity stress,” Plants 12(12), article 2253. https://doi.org/10.3390/plants12122253

Bates, L. S., Waldren, R. P., and Teare, I. D. (1973). “Rapid determination of free proline for water-stress studies,” Plant and Soil 39(1), 205-207. https://doi.org/10.1007/BF00018060

Bergmeyer, H. U., Gawehn, K., and Grassl, M. (1974). in: Methods of Enzymatic Analysis, H. U. Bergmeter (ed), Academic Press, New York 3, pp. 1205-1215.

Bhati, V. S., Singh, P. K., Parmar, Y. J., and Rokins, P. G. (2026). “Foliar uptake and translocation of nanoparticles in the plants: A review,” Journal of Plant Nutrition 49(2), 246-283.‏

Bhattacharya, A. (2021). “Effect of soil water deficit on growth and development of plants: A review,” Soil Water Deficit and Physiological Issues in Plants 393–488. https://doi.org/10.1007/978-981-33-6276-5_5

Burmistrov, D. E., Shumeyko, S. A., Semenova, N. A., Dorokhov, A. S., and Gudkov, S. V. (2025). “Selenium nanoparticles (Se NPs) as agents for agriculture crops with multiple activity: A review,” Agronomy 15(7), article 1591.

Cavalcante, J. S. J., Ferreira Neto, M., Peixoto, T. D. C., Pereira, K. T. O., Umbelino, B. F., Rodrigues Filho, R. A., Moreira, R. C. L., Fernandes, P. D., da Silva Dias, N., and da Silva Ribeiro, J. E. (2025). “Production, growth, photosynthetic response, and ionic responses of arugula cultivars under salt stress elicitors,” Journal of Plant Growth Regulation 44(10), 6200-6216. https://doi.org/10.1007/s00344-025-11823-3

Chakraborty, K., Basak, N., Bhaduri, D., Ray, S., Vijayan, J., Chattopadhyay, K., and Sarkar, R. K. (2018). “Ionic basis of salt tolerance in plants: nutrient homeostasis and oxidative stress tolerance,” in: Plant Nutrients and Abiotic Stress Tolerance, Springer Singapore, pp. 325-362.

Chen, Y., Zhao, S., Ma, X., Ling, L., Du, P., Liu, X., Yao, Y., Ma, Q., Cheng, Y., Wang, Y. et al. (2026). “Selenium nanoparticles and glutathione synergistically enhances salt tolerance in soybean via the jasmonic acid pathway and arbutin-regulated rhizosphere microbiota,” aBIOTECH 2026, article 100022. https://doi.org/10.1016/j.abiote.2026.100022

Devi, C. A., Dawud, K. M., and Pandey, A. K. (2026). “Crop improvement approaches for the mitigation of abiotic stresses in mustard (Brassica spp.) crops,” in: Crop Improvement Strategies in Brassica Species: Stress Resistance Strategies, Springer, Volume 2, pp. 21-41. https://doi.org/10.1007/978-981-95-4713-5_2

Dichio, C., Sofo, A., Yaghoubi Khanghahi, M., Crecchio, C., and Addesso, R. (2025). “Physiological and biochemical mechanisms of waterlogging tolerance in plants,” Physiologia Plantarum 177(6), article e70663. https://doi.org/10.1111/ppl.70663

Dobrucka, R., Romaniuk-Drapała, A., and Kaczmarek, M. (2021): “Anti-leukemia activity of Au/CuO/ZnO nanoparticles synthesized used Verbena officinalis extract,” Journal of Inorganic and Organometallic Polymers and Materials 31(1), 191-202. https://doi.org/10.1007/s10904-020-01690-8

El-Badri, A. M., Batool, M., Wang, C., Hashem, A. M., Tabl, K. M., Nishawy, E., Kuai, J., Zhou, G., and Wang, B. (2021). “Selenium and zinc oxide nanoparticles modulate the molecular and morpho-physiological processes during seed germination of Brassica napus under salt stress,” Ecotoxicology and Environmental Safety 225, article 112695. https://doi.org/10.1016/j.ecoenv.2021.112695

Elsheery, N. I., Nosier, A. M., Maswada, H. F., Teiba, I. I., Elhamahmy, M., Abdelrazik, E. M., Ismaeil, R. A., El-Araby, H. G., Yi, G., and Li, L. (2025). “Alleviating the harmful effect of salinity on faba bean plants using selenium nanoparticles,” Plant Nano Biology 12, article 100158. https://doi.org/10.1016/j.plana.2025.100158

Fatahiyan, F., Najafi, F., and Shirkhani, Z. (2025). “Enhancing salt stress tolerance in Carthamus tinctorius L. through selenium soil treatment: Anatomical, biochemical, and physiological insights,” BMC Plant Biology 25(1), article 100. https://doi.org/10.1186/s12870-025-06078-9

Gao, S., Tang, X., Zhang, J., Zhou, Q., Peng, T., Liu, A., Xue, Y., Xu, F., and Li, P. (2025). “Zinc-selenium interaction regulates leaf photosynthesis and mediates grain sugar metabolism to improve the yield and quality of hybrid rice: A physiological perspective,” Plant Physiology and Biochemistry 221, article 109611. https://doi.org/10.1016/j.plaphy.2025.109611

Ghasemian, S., Masoudian, N., Saeid Nematpour, F., and Safipour Afshar, A. (2021). “Selenium nanoparticles stimulate growth, physiology, and gene expression to alleviate salt stress in Melissa officinalis,” Biologia 76(10), 2879-2888. https://doi.org/10.1007/s11756-021-00854-2

González-García, Y., Cárdenas-Álvarez, C., Cadenas-Pliego, G., Benavides-Mendoza, A., Cabrera-De-La-Fuente, M., Sandoval-Rangel, A., Valdés-Reyna, J., and Juárez-Maldonado, A. (2021). “Effect of three nanoparticles (Se, Si and Cu) on the bioactive compounds of bell pepper fruits under saline stress,” Plants 10(2), article 217. https://doi.org/10.3390/plants10020217

Hailu, B., and Mehari, H. (2021). “Impacts of soil salinity/sodicity on soil-water relations and plant growth in dry land areas: A review,” J. Nat. Sci. Res 12(3), 1-10. https://doi.org/10.7176/JNSR/12-3-01

Hasanuzzaman, M., Raihan, M. R. H., Siddika, A., Bardhan, K., Hosen, M. S., and Prasad, P. V. V. (2024). “Selenium and its nanoparticles modulate the metabolism of reactive oxygen species and morpho-physiology of wheat (Triticum aestivum L.) to combat oxidative stress under water deficit conditions,” BMC Plant Biology 24(1), article 578. https://doi.org/10.1186/s12870-024-05282-3

Hashem, A. H., Al-Askar, A. A., Haponiuk, J., Abd-Elsalam, K. A., and Hasanin, M. S. (2023) “Biosynthesis, characterization, and antifungal activity of novel ternary copper oxide-selenium- zinc oxide nanoparticles against some mucorales fungi,” Microorganisms 11(6), article 1380. https://doi.org/10.3390/microorganisms11061380

Hernández-Hernández, H., Quiterio-Gutiérrez, T., Cadenas-Pliego, G., Ortega-Ortiz, H., Hernández-Fuentes, A. D., Cabrera de la Fuente, M., Valdés-Reyna, J., and Juárez-Maldonado, A. (2019). “Impact of selenium and copper nanoparticles on yield, antioxidant system, and fruit quality of tomato plants,” Plants 8(10), article 355. https://doi.org/10.3390/plants8100355

Kar, M., and Mishra, D. (1976). “Catalase, peroxidase, and polyphenoloxidase activities during rice leaf senescence,” Plant Physiology 57(2), 315-319. https://doi.org/10.1104/pp.57.2.315

Khan, T. A., Saeed, T., Tran, L. S. P., and Gururani, M. A. (2026). “Coordinated physiological and molecular reprogramming by brassinosteroids improves soybean tolerance to combined salt and drought stress,” Plant Signaling and Behavior 21(1), article 2616539. https://doi.org/10.1080/15592324.2026.2616539

Khondoker, M., Mandal, S., Gurav, R., and Hwang, S. (2023). “Freshwater shortage, salinity increase, and global food production: A need for sustainable irrigation water desalination – A scoping review,” Earth 4(2), 223-240. https://doi.org/10.3390/earth4020012

Kiremit, M. S., Arslan, H., Bakirova, A., and Abdulrasaq, S. A. (2026). “Synergistic effects of humic and salicylic acids on water productivity, physiology, and antioxidant defense in salt-stressed lettuce,” Journal of Soil Science and Plant Nutrition 1–22. https://doi.org/10.1007/s42729-026-03015-z

Kunwar, S., Roy, A., Bhusal, U., Gacem, A., Abdullah, M. M., Sharma, P., Yadav, K. K., Rustagi, S., Chatterjee, N., Deshwal, V. K., “et al.” (2023). “Bio-fabrication of Cu/Ag/Zn nanoparticles and their antioxidant and dye degradation activities,” Catalysts 13(5), article 891.‏ https://doi.org/10.3390/catal13050891

Liu, H., Xiao, C., Qiu, T., Deng, J., Cheng, H., Cong, X., Cheng, S., Rao, S., and Zhang, Y. (2022). “Selenium regulates antioxidant, photosynthesis, and cell permeability in plants under various abiotic stresses: A review,” Plants 12(1), article 44. https://doi.org/10.3390/plants12010044

Lowery, G. H. (1951). A Quantitative Study of the Nocturnal Migration of Birds, Vol. 3 (Vol. 1). Library of Alexandria.

Mishra, M., Afzal, S., Yadav, R., Singh, N. K., and Zarbakhsh, S. (2025). “Salinity stress amelioration through selenium and zinc oxide nanoparticles in rice,” Scientific Reports 15(1), article 27554. https://doi.org/10.1038/s41598-025-12106-3

Mogazy, A. M., and Hanafy, R. S. (2022). “Foliar spray of biosynthesized zinc oxide nanoparticles alleviate salinity stress effect on Vicia faba plants,” Journal of Soil Science and Plant Nutrition 22(2), 2647-2662. https://doi.org/10.1007/s42729-022-00833-9

Mukherjee, S. P., and Choudhuri, M. A. (1983). “Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings,” Physiologia Plantarum 58(2), 166-170. https://doi.org/10.1111/j.1399-3054.1983.tb04162.x

Nowwar, A. I., Farghal, I. I., Ismail, M. A., and Amin, M. A. (2023). “Impact of irrigation with wastewater on accumulation of heavy metals in Phaseolus vulgaris L. and its remediation,” Journal of Soil Science and Plant Nutrition 23(1), 761-777. https://doi.org/10.1007/s42729-022-01080-8

Ochoa-Chaparro, E. H., Castruita-Esparza, L. U., and Sánchez, E. (2026). “Eco-physiological and molecular roles of zinc oxide nanoparticles (ZnO-NPs) in mitigating abiotic stress: A comprehensive review,” Plants 15(1), article 147. https://doi.org/10.3390/plants15010147

Osman, M. E., Nessem, A. A., Ismael, E. H., and Abo-Shanab, W. A. (2026). “Mitigation of the salinity stress in Vicia faba (l.) seedlings using green-synthesized selenium nanoparticles,” Journal of Soil Science and Plant Nutrition 1–12. https://doi.org/10.1007/s42729-025-02966-z

Pérez-Labrada, F., López-Vargas, E. R., Ortega-Ortiz, H., Cadenas-Pliego, G., Benavides-Mendoza, A., and Juárez-Maldonado, A. (2019). “Responses of tomato plants under saline stress to foliar application of copper nanoparticles,” Plants 8(6), article 151. https://doi.org/10.3390/plants8060151

Petkova, B., Kuzmova, K., and Berova, M. (2019). “The main abiotic stress factors limiting crop cultivation and production in Bulgaria. Climate changes, drought, water deficit and heat stress,” Agricultural Sciences/Agrarni Nauki 11(26), artilce 2. https://doi.org/10.22620/agrisci.2019.26.002

Riaz, A., Anwar-Ul-Haq, M., Murtaza, G., and Ahmad, E. (2026). “Evaluation of the impact of zinc and iron nanoparticles on growth of wheat (Triticum aestivum L.) under salt stress conditions,” Pak. J. Bot. 58, 3. http://dx.doi.org/10.30848/PJB2026-3(5)

Rizzoli, M., Lutzu, G. A., Usai, L., Fais, G., Dessì, D., Soto-Ramirez, R., Cosenza, B., and Concas, A. (2025). “Photoautotrophic batch cultivation of Limnospira (Spirulina) platensis: Optimizing biomass productivity and bioactive compound synthesis through salinity and pH modulation,” Marine Drugs 23(7), article 281. https://doi.org/10.3390/md23070281

Rodríguez, F. E., Laporte, D., González, A., Mendez, K. N., Castro-Nallar, E., Meneses, C., Huidobro-Toro, J. P., and Moenne, A. (2018). “Copper-induced increased expression of genes involved in photosynthesis, carotenoid synthesis and C assimilation in the marine alga Ulva compressa,” BMC Genomics 19(1), article 829. https://doi.org/10.1186/s12864-018-5226-4

Sachdev, S., Ansari, S. A., Ansari, M. I., Fujita, M., and Hasanuzzaman, M. (2021). “Abiotic stress and reactive oxygen species: Generation, signaling, and defense mechanisms,” Antioxidants 10(2), article 277. https://doi.org/10.3390/antiox10020277

Sampath, S., Sunderam, V., Manjusha, M., Dlamini, Z., and Lawrance, A. V. (2024). “Selenium nanoparticles: A comprehensive examination of synthesis techniques and their diverse applications in medical research and toxicology studies,” Molecules 29(4), article 801.‏ https://doi.org/10.3390/molecules29040801

Saravanan, K., Dhanapal, S., Punithavathi, G., Afrith, W., Kiruthiga, N., and Deepa Joan of Arc, C. (2026). “Exogenous arginine as a potential biostimulant for enhancing salinity stress tolerance in maize (Zea mays L.),” Russian Journal of Plant Physiology 73(1), 11. https://doi.org/10.1134/S1021443725605300

Seleiman, M. F., Ahmad, A., Alhammad, B. A., and Tola, E. (2023). “Exogenous application of zinc oxide nanoparticles improved antioxidants, photosynthetic, and yield traits in salt-stressed maize,” Agronomy 13(10), article 2645. https://doi.org/10.3390/agronomy13102645

Selim, S., Saddiq, A. A., Ashy, R. A., Baghdadi, A. M., Alzahrani, A. J., Mostafa, E. M., Al Jaouni, S. K., Elamir, M. Y. M., Amin, M. A., and Salah, A. M. (2025). “Bimetallic selenium/zinc oxide nanoparticles: biological activity and plant biostimulant properties,” AMB Express 15(1), 1. https://doi.org/10.1186/s13568-024-01808-y

Shafiei, L., Ranjbar, M., Yazdanpanahi, N., and ShahaniPour, K. (2026). “Comparative analysis of salinity stress on the molecular, biochemical, and phenotypic changes of different cabbage species (Brassica),” International Journal of Horticultural Science and Technology 13(3), 531-548. https://doi.org/10.22059/ijhst.2025.390150.1026

Sherefu, A. and Zewide, I. (2021). “Review paper on effect of micronutrients for crop production,” J. Nutrition Food Proc. 4(7). https://doi.org/10.31579/2637-8914/063

Singh, P., Kumar, V., and Sharma, A. (2025). “Assessment of yield and quality management with the exogenous application of silicon in contrasting wheat varieties under salinity stress,” Cereal Research Communications 53(1), 225-244. https://doi.org/10.1007/s42976-024-00522-6

Smith, K. C. (2013). The Science of Photobiology, Springer Science and Business Media.

Taj, H., Noreen, Z., Aslam, M., Usman, S., Shah, A. A., Rafique, M., Raja, V., and El-Sheikh, M. A. (2024). “Effects of SNP, MgSO₄, and MgO-NPs foliar application on Spinacia oleracea L. growth and physio-biochemical responses under cadmium stress,” Scientific Reports 14(1), article 26687. https://doi.org/10.1038/s41598-024-77221-z

Talat, F., Javed, M. T., Ali, Q., and Nawaz, M. (2026). “Biogenically synthesized zno-nps conferred drought tolerance in mung bean (Vigna radiata L. Wilczek) by modulating growth, physio-biochemical attributes, and antioxidant status,” Pak. J. Bot. 58, 5. http://dx.doi.org/10.30848/PJB2026-5(10)

Tebini, M., Chieb, M., Luu, D.-T., Dailly, H., Lutts, S., Ahmed, H. Ben, and Chalh, A. (2025). “Assessment of salt stress effects on antioxidant levels and membrane transport protein in Amaranthus caudatus,” Journal of Plant Growth Regulation 44, 3318–3329. https://doi.org/10.1007/s00344-024-11619-x

Tripodi, P., Macellaro, R., Baiamonte, I., Nardo, N., and Raffo, A. (2025). “Genomic diversity and nutritional composition of ready to eat rocket salad (Eruca sativa) germplasm panel representing the worldwide diversity,” European Food Research and Technology 251(8), 2485-2498.‏ https://doi.org/10.1007/s00217-025-04780-4

Umbriet, W. W., Burris, R. H., Stauffer, J. F., Cohen, P. P., Johnse, W. J., Leepage, G. A., Potten, V. R., and Schneider, W. C. (1959). Manometric Techniques. A Manual Describing Methods Applicable to the Study of Tissue Metabolism, Burgess, Minneapolis, p 239.

Vernon, L. P., and Seely, G. R. (2014). The Chlorophylls, Academic press.

Yadav, S., Tripathy, S., and Shimpi, N. G. (2026). “Nanomaterial’s: Types, synthesis and application environment remediation,” in: Advanced Nanotechnology Applications for Environmental Sustainability, E. Priyadarshini, A. Sanroman, A. P. Das, (eds) Springer, pp. 1-17. https://doi.org/10.1007/978-3-032-12933-8_1

Zambonino, M. C., Quizhpe, E. M., Mouheb, L., Rahman, A., Agathos, S. N., and Dahoumane, S. A. (2023). “Biogenic selenium nanoparticles in biomedical sciences: properties, current trends, novel opportunities and emerging challenges in theranostic nanomedicine,” Nanomaterials 13(3), 424.‏ https://doi.org/10.3390/nano13030424

Zhang, Z., Huber, D. J., Qu, H., Yun, Z., Wang, H., Huang, Z., Huang, H., and Jiang, Y. (2015). “Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols,” Food Chemistry 171, 191-199. https://doi.org/10.1016/j.foodchem.2014.09.001

Article submitted: March 17, 2026; Peer review completed: May 3, 2026; Revised version received: May 17, 2026; Further revised version received and accepted: June 14, 2026; Published: June 25, 2026.

DOI: 10.15376/biores.21.3.7433-7453